CN101448852A - Compositions and methods for producing a composition - Google Patents

Compositions and methods for producing a composition Download PDF

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Publication number
CN101448852A
CN101448852A CNA2006800531165A CN200680053116A CN101448852A CN 101448852 A CN101448852 A CN 101448852A CN A2006800531165 A CNA2006800531165 A CN A2006800531165A CN 200680053116 A CN200680053116 A CN 200680053116A CN 101448852 A CN101448852 A CN 101448852A
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China
Prior art keywords
ctla4
molecule
composition
average ratio
molar average
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Pending
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CNA2006800531165A
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Chinese (zh)
Inventor
K·J·莱斯特
E·J·夏菲尔
R·贝蒂斯
E·A·布伦后
D·M·帝狄欧
R·唐纳森
A·R·费雪
H·G·哈杰狄
D·H·可克利
J·M·塔柏
L·K·泰
P·桑玛纳
A·费勒尤翰
D·E·史慕林
R·J·罗素
T·凡丹布恩
J·施林舍尔
J·怀特赫德
D·布劳内尔
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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Application filed by Bristol Myers Squibb Co filed Critical Bristol Myers Squibb Co
Priority to CN201610350033.8A priority Critical patent/CN106589132B/en
Publication of CN101448852A publication Critical patent/CN101448852A/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The invention provides for mammalian cells capable of producing recombinant CTLA4-Ig and variants thereof. The invention also provides for compositions comprising CTLA4-Ig and formulations thereof. The invention further provides for methods for mass- producing CTLA4-Ig from mammalian cells capable of producing this recombinant protein, and for purifying the CTLA4-Ig, the mammalian cells can produce the recombinant protein.

Description

Composition and be used for the production method for compositions
Present patent application requires the right of priority of following patent: the U.S. serial of submitting on December 20th, 2,005 60/752,267, the U.S. serial of submitting on October 5th, 2,006 60/849,543 and the U.S. serial 60/752 submitted on December 20th, 2005,150, all described patents are incorporated herein by reference in this integral body.The application also will be incorporated herein by reference in the patent application integral body that on December 19th, 2006 submitted to, and the name of described patent application is called " StableProtein Formulations ", and proxy number is 10739 PCT.
All patents, patent application and publication that this paper quotes all are incorporated herein by reference in this integral body.
This patent disclosure comprises material protected by copyright.The copyright owner does not oppose that anyone duplicates the patent document or patent disclosure content that occurs as in United States Patent (USP) and trademark office's (U.S.Patent and Trademark Office) patent filing or record, but keeps any in other respects and all copyrights.
Background of invention
Cytotoxic T lymphocyte antigen 4 (CTLA4), the member of immunoglobulin superfamily is the molecule by the activated T cell expressing.CTLA4 is similar to T cell co-stimulatory molecules CD28, and 2 kinds of molecules all combine with B7-1 (CD80) and B7-2 (CD86) on the antigen presenting cell (APCs).Yet CTLA4 will suppress signal and pass to the T cell, and CD28 transmits stimulus signal.
The CTLA4-Ig molecule is the ligand binding domains of cytotoxic T lymphocyte antigen 4 (CTLA4) and the fusion rotein of immunoglobulin (Ig) (Ig) CH.This shla molecule is by its physiological action of following performance: combines with the lip-deep B7 antigen of various antigen presenting cells (APC) (CD80 and CD86), thus the functional interaction of the CD28 on blocking-up B7-1 and B7-2 and the T cell surface.This blocking-up causes the inhibition of T cell activation, and therefore suppresses immunne response.The method that the CTLA4-Ig molecule therefore can be provided for suppressing organizing and/or solid organ transplantation repels, and the disease or the illness that are used to relate in general to the immunne response of imbalance comprise autoimmune therepic use.For example, the CTLA4-Ig molecule can suppress the anti-dsDNA production of antibodies and reduce ephritis in the mouse that easily suffers from lupus; Can reduce the proteinuria in the mouse with ephritis in late period and prolong survival; And can improve clinical effectiveness about psoriasis and rheumatoid arthritis.
In order to improve the treatment validity of CTLA4-Ig molecule, importantly determine to prepare to strengthen the molecular changes of molecule as the effect of the inhibitor of T cytositimulation, for example, by increasing molecule for antigenic avidity of B7 and effectiveness.Increase in the avidity of CTLA4-Ig molecule and the effectiveness can allow to use the CTLA4-Ig molecule of reduction to reach required curative effect (that is using than low dosage) to the patient.The avidity of CTLA4-Ig molecule and render a service in increase can also reduce the dosage number used to the patient or dose frequency to reach required curative effect.
Summary of the invention
The composition that the present invention relates to improve and be used to produce the CTLA4-Ig method for compositions.The composition and being used for that the present invention relates to the CTLA4-Ig molecule, comprises the improvement of CTLA4-Ig molecule is produced the method for the improvement of (comprising mass production) CTLA4-Ig molecule and other recombinant proteins.
No matter the present invention includes any arrangement and/or the combination of any element described herein and feature, be to be described separately or with some combination or arrangement.
Cell: the invention provides clone's Chinese hamster ovary cell colony that can produce CTLA4-Ig.The invention provides clone's Chinese hamster ovary cell colony that can produce CTLA4-Ig, each cell comprises 30 of the CTLA4-Ig expression cassette or multiple copied more.The present invention also provides the clone's Chinese hamster ovary cell that can produce CTLA4-Ig colony, each cell comprises 30 of the CTLA4-Ig expression cassette or multiple copied more, wherein said 30 or more on the single site of multi-copy integration in the genome of each cell.The invention provides clone's Chinese hamster ovary cell colony that can produce CTLA4-Ig, wherein the CTLA4-Ig expression cassette is stable in about 105 times go down to posterity.In one embodiment, CTLA4-Ig is by the expression cassette coding that comprises nucleotide sequence, its by people such as KoduriR. ( Gene, 2001,280:87-95) and at U.S. Patent number 6,800, obtain describing in 457 and 6,521,419, described reference is incorporated herein by reference in this integral body.In another embodiment, CTLA4-Ig is by being incorporated into from the expression cassette on the specific gene seat in the cellular genome of cell colony coding, its by people such as KoduriR. ( Gene, 2001,280:87-95) and at U.S. Patent number 6,800, to describe in 457 and 6,521,419, described reference is incorporated herein by reference in this integral body.In one embodiment, colony comprises the subgroup of cell, and described cell comprises 33 of the CTLA4-Ig expression cassette or multiple copied more, wherein said 33 or more on the single site of multi-copy integration in the genome of each cell of subgroup.
The invention provides clone's Chinese hamster ovary cell colony that can produce CTLA4-Ig, wherein at least 75% cell colony has 30 of the CTLA4-Ig expression cassette or multiple copied more, wherein said 30 or more on the single site of multi-copy integration in the genome of each cell of 75% colony.The invention provides clone's Chinese hamster ovary cell colony that can produce CTLA4-Ig, wherein at least 85% cell colony has 30 of the CTLA4-Ig expression cassette or multiple copied more, wherein said 30 or more on the single site of multi-copy integration in the genome of each cell of 85% colony.The invention provides clone's Chinese hamster ovary cell colony that can produce CTLA4-Ig, wherein at least 95% cell colony has 30 of the CTLA4-Ig expression cassette or multiple copied more, wherein said 30 or more on the single site of multi-copy integration in the genome of each cell of 95% colony.In one embodiment, liquid culture can be produced greater than 0.5 or more gram CTLA4-Ig protein/be risen to cell colony, and wherein CTLA4-Ig demonstrates acceptable carbohydrate feature, wherein 1, the mol ratio of sialic acid and CTLA4-Ig is about 6-about 14 on 000L or the very big cultivation scale.In another embodiment, cell colony has adapted to serum-free, chemical ingredients knows substratum.In another embodiment, the optical extinction coefficient that has 1.00 ± 0.05AU mL cm-1mg-1 by the CTLA4-Ig of the culture production of cell colony.In another embodiment, when growing in culture, cell colony can be produced the CTLA4-Ig polypeptide, and wherein: (a) about 90% CTLA4-Ig polypeptide comprises the aminoacid sequence of the SEQ ID NO:2 that begins from the methionine(Met) of residue 27; (b) about 10% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that 26 L-Ala begins from residue; (c) about 4% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the Methionin at 383 places finishes with residue, and (d) about 96% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the glycine at 382 places finishes with residue; Randomly, (e) comprise the aminoacid sequence of numbering the SEQ ID NO:2 that 25 methionine(Met) begins from residue less than 1% CTLA4-Ig polypeptide approximately.
The invention provides the progeny cell of clone cell, wherein said progeny cell is produced CTLA4-Ig.In one embodiment, progeny cell obtains through at least 5 generations by cultivating clone's parental cell.In another embodiment, progeny cell by culturing cell through at least 10 generations, through at least 20 generations, through at least 40 generations, through at least 50 generations, obtain through at least 75 generations or through at least 100 generations.The invention provides the clone of producing by clone cell.In one embodiment, clone is cloned.The invention provides and to produce following clone: the aminoacid sequence (glycine at the methionine(Met) at amino acid position 27 places and amino acid position 382 places that (a) has SEQ ID NO:10; Figure 1A and 1B) the CTLA4-Ig fusion rotein; (b) has the aminoacid sequence (Methionin at the methionine(Met) at amino acid position 27 places and amino acid position 383 places of SEQ ID NO:7; Figure 1A and 1B) the CTLA4-Ig fusion rotein; (c) has the aminoacid sequence (glycine at the L-Ala at amino acid position 26 places and amino acid position 382 places of SEQ ID NO:9; Figure 1A and 1B) the CTLA4-Ig fusion rotein; (d) has the aminoacid sequence (Methionin at the L-Ala at amino acid position 26 places and amino acid position 383 places of SEQ ID NO:6; Figure 1A and 1B) the CTLA4-Ig fusion rotein; (e) has the aminoacid sequence (glycine at the methionine(Met) at amino acid position 25 places and amino acid position 382 places of SEQ ID NO:8; Figure 1A and 1B) the CTLA4-Ig fusion rotein; Or (f) has the aminoacid sequence (Methionin at the methionine(Met) at amino acid position 25 places and amino acid position 383 places of SEQ ID NO:5; Figure 1A and 1B) the CTLA4-Ig fusion rotein.In another embodiment, clone can be produced the CTLA4-Ig fusion rotein, and wherein: (a) about 90% CTLA4-Ig polypeptide comprises the aminoacid sequence of the SEQ ID NO:2 that begins from the methionine(Met) of residue 27; (b) about 10% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that 26 L-Ala begins from residue; (c) about 4% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the Methionin at 383 places finishes with residue, and (d) about 96% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the glycine at 382 places finishes with residue; Randomly, (e) comprise the aminoacid sequence of numbering the SEQ ID NO:2 that 25 methionine(Met) begins from residue less than 1% CTLA4-Ig polypeptide approximately.
In one embodiment, the CTLA4-Ig fusion rotein of being produced by culturing cell system has the optical extinction coefficient of 1.00 ± 0.05AU mL cm-1mg-1.The invention provides cell colony derived from cloned cell line.In embodiments, with initial cloned cell line comparison, cell colony is made up of at least a other hereditary change, and wherein the deutero-cell colony can be produced CTLA4-Ig.In another embodiment, compare with parental cell, cell colony is made up of at least 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 10 kinds, 15 kinds or 20 kinds of other hereditary changes at least at least at least at least at least at least at least at least at least, and wherein the deutero-cell colony can be produced CTLA4-Ig.In one embodiment, hereditary change be included in cellular genome or the coding CTLA4-Ig recombinant expression cassettes at least a non-conservative sudden change.In another embodiment, hereditary change comprises intracellular at least a other recombinant nucleic acid.In further embodiment, change the sudden change that comprises cellular genome.In another embodiment, change comprises to cellular genome interpolation nucleic acid or as trans nucleic acid, described nucleic acid encoding anti-apoptotic polypeptide.In another embodiment, the anti-apoptotic polypeptide relates to glycosylation.In another embodiment, hereditary change comprises at least a sudden change of the recombinant expression cassettes of cellular genome or coding CTLA4-Ig.
Composition: the CTLA4-Ig molecule colony that the invention provides sialic acids groups with about 6-about 18 and the molar average ratio of CTLA4-Ig dimer or molecule.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 8-about 18 and the molar average ratio of CTLA4-Ig dimer or molecule.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 11-about 18 and the molar average ratio of CTLA4-Ig dimer or molecule.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 12-about 18 and the molar average ratio of CTLA4-Ig dimer or molecule.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 13-about 18 and the molar average ratio of CTLA4-Ig dimer or molecule.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 14-about 18 and the molar average ratio of CTLA4-Ig dimer or molecule.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 15-about 17 and the molar average ratio of CTLA4-Ig dimer or molecule.The invention provides CTLA4-Ig molecule colony with about 16 sialic acids groups and molar average ratio of CTLA4-Ig dimer or molecule.The invention provides wherein that to surpass 95% molecule be the dimeric CTLA4-Ig molecule of CTLA4-Ig colony.In one embodiment, surpassing 98% molecule is the CTLA4-Ig dimer.In another embodiment, surpassing 99% molecule is the CTLA4-Ig dimer.In another embodiment, surpassing 99.5% molecule is the CTLA4-Ig dimer.In another embodiment, the molecule of about 95%-about 99.5% is the CTLA4-Ig dimer, and the molecule of about 0.5%-about 5% is the CTLA4-Ig tetramer or high molecular weight species.In another embodiment, about 98.6% molecule is the CTLA4-Ig dimer, and about 1.2% molecule is the CTLA4-Ig tetramer or high molecular weight species, and is the CTLA4-Ig monomer less than 0.7% molecule approximately.The invention provides the colony that forms by the CTLA4-Ig dimer.The invention provides wherein, colony is substantially free of the monomeric CTLA4-Ig molecule of CTLA4-Ig colony.The invention provides wherein, colony is substantially free of the tetrameric CTLA4-Ig molecule of CTLA4-Ig colony.The invention provides and be substantially free of CTLA4-Ig dimer and tetrameric CTLA4-Ig monomer molecule colony.In one embodiment, every kind of dimeric each monomer of CTLA4-Ig has at least 3 sialic acids groups.In another embodiment, every kind of dimeric each monomer of CTLA4-Ig has at least 3 sialic acids groups-at least 8 sialic acids groups.The invention provides the purifying colony of CTLA4-Ig tetramer molecule, described colony is substantially free of the CTLA4-Ig dimer, and randomly, wherein said colony comprises the amount that surpasses about 100 grams.The invention provides the purifying colony of CTLA4-Ig tetramer molecule, described colony is substantially free of the CTLA4-Ig monomer, and randomly, wherein said colony comprises the amount that surpasses about 100 grams.In one embodiment, each tetramer molecule comprises 2 pairs of CTLA4-Ig polypeptide, wherein each polypeptide has the aminoacid sequence that is selected from SEQ ID NOS:5-10, and wherein right each member and another member of polypeptide is covalently bound, and wherein 2 pairs of non-covalent each other combinations of polypeptide.In another embodiment, each tetramer molecule can combine with CD80 or CD86.In further embodiment, to compare with the CTLA4-Ig dimer molecule, each tetramer molecule has at least 2 times big avidity for CD80 or CD86.In another embodiment, compare with the CTLA4-Ig dimer molecule, each tetramer molecule has at least 2 times big T cell proliferation or activation inhibition.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said composition is included in advantage isoform visual on the isoelectrofocusing gel of CTLA4-Ig, and as measuring by isoelectrofocusing, described isoform has and is less than or equal to 5.1 iso-electric point, pI.In one embodiment, the invention provides the composition that comprises the CTLA4-Ig molecule, wherein said composition is included in advantage isoform visual on the isoelectrofocusing gel of CTLA4-Ig, as measuring by isoelectrofocusing, described isoform has and is less than or equal to 5.8 iso-electric point, pI.In one embodiment, pI handles the back at neuraminidase increases.In one embodiment, composition is included in advantage isoform visual on the isoelectrofocusing gel of CTLA4-Ig, as measuring by isoelectrofocusing, described isoform has and is less than or equal to 5.7,5.6,5.5,5.4,5.3,5.2,5.1,5.0,4.9,4.8,4.7,4.6 or 4.5 iso-electric point, pI.In another embodiment, as measuring by isoelectrofocusing, at least 40% CTLA4-Ig molecule demonstration is less than or equal to about 5.1 iso-electric point.In another embodiment, as measuring by isoelectrofocusing, at least 70% CTLA4-Ig molecule demonstration is less than or equal to about 5.1 iso-electric point.In another embodiment, as measuring by isoelectrofocusing, at least 90% CTLA4-Ig molecule demonstration is less than or equal to about 2.5 iso-electric point.The invention provides the CTLA4-Ig molecule colony of the pI with about 2.0 ± 0.2-about 5.0 ± 0.2.The invention provides the CTLA4-Ig molecule colony of the pI with about 4.0 ± 0.2-about 5.0 ± 0.2.The invention provides the CTLA4-Ig molecule colony of the pI with about 4.3 ± 0.2-about 5.0 ± 0.2.The invention provides the CTLA4-Ig molecule colony of the pI with about 3.3 ± 0.2-about 4.7 ± 0.2.The invention provides and be used to prepare method for compositions, described composition comprises the CTLA4-Ig molecule of the pI with about 2.0 ± 0.2-about 5.0 ± 0.2, described method comprises: (a) mixture of CTLA4-Ig molecule is implemented the isoelectrofocusing gel electrophoresis, wherein the single band on gel represents to have the CTLA4-Ig molecule colony of specific pI, (b) separate the CTLA4-Ig molecule colony of the pI with about 2.0 ± 0.2-about 5.0 ± 0.2, so that prepare composition.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that GlcNAc/ mole CTLA4-Ig dimer or its molar average ratio to the CTLA4-Ig molecule of about 15-about 35.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that GalNAc/ mole CTLA4-Ig dimer or its molar average ratio to the CTLA4-Ig molecule of about 1.7-about 3.6.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that semi-lactosi/mole CTLA4-Ig dimer or its molar average ratio to the CTLA4-Ig molecule of about 8-about 17.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that Fucose/mole CTLA4-Ig dimer or its molar average ratio to the CTLA4-Ig molecule of about 3.5-about 8.3.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that seminose/mole CTLA4-Ig dimer or its molar average ratio to the CTLA4-Ig molecule of about 7.2-about 22.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that sialic acid/mole CTLA4-Ig dimer or its molar average ratio to the CTLA4-Ig molecule of about 6-about 12.
The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc/ mole CTLA4-Ig dimer of about 15-about 35 or CTLA4-Ig molecule; (b) the molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig dimer or CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc/ mole CTLA4-Ig dimer of about 15-about 35 or CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc/ mole CTLA4-Ig dimer of about 1.7-about 3.6 or CTLA4-Ig molecule; (c) the molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig dimer or CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc/ mole CTLA4-Ig dimer of about 15-about 35 or CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc/ mole CTLA4-Ig dimer of about 1.7-about 3.6 or CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig dimer or CTLA4-Ig molecule; (d) the molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig dimer or CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc/ mole CTLA4-Ig dimer of about 15-about 35 or CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc/ mole CTLA4-Ig dimer of about 1.7-about 3.6 or CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig dimer or CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 3.5-about 8.3/mole CTLA4-Ig dimer or CTLA4-Ig molecule; (e) the molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig dimer or CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc/ mole CTLA4-Ig dimer of about 15-about 35 or CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc/ mole CTLA4-Ig dimer of about 1.7-about 3.6 or molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig dimer or molecule; (d) the molar average ratio of the Fucose of about 3.5-about 8.3/mole CTLA4-Ig dimer or molecule; (e) the molar average ratio of the seminose of about 7.2-about 22/mole CTLA4-Ig dimer or molecule; (f) the molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig dimer or CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, the NANA chromatogram peak of the NGNA chromatogram peak of the about 9.589+ of wherein said composition exhibiting/-0.3 and about 10.543+/-0.3.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule shows the carbohydrate overview as shown in Figure 68.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule display structure territory I-IV (for example, I-IV) carbohydrate overview, wherein structural domain I comprises the peak of the oligosaccharides of representing asialoization, domain II comprises the peak of the oligosaccharides of representing mono-sialylated, domain II I comprises the peak of the oligosaccharides of representing double-sialylated, and structural domain IV comprises the peak of representing three sialylated oligosaccharides.Structural domain V comprises the peak of representing four sialylated oligosaccharides.In one embodiment, the difference in the retention time of N connection oligosaccharides is about 28 minutes of about 22-between first peak among the structural domain I and the main peak in the domain II.The invention provides the composition that comprises the CTLA4-Ig dimer molecule, wherein at least 0.5% CTLA4-Ig dimer molecule is a cysteinylization.In one embodiment, at least 1.0% CTLA4-Ig dimer molecule is a cysteinylization.The invention provides CTLA4-Ig molecule colony, wherein said colony shows the mass spectroscopy overview as shown in Fig. 8 and 10.The invention provides the colony of CTLA4-Ig molecule, wherein said colony shows the capillary electrophoresis overview as shown in Figure 20 and 21.The invention provides the composition of sialic acids groups with about 6-about 18 and the CTLA4-Ig molecule of the dimeric molar average ratio of CTLA4-Ig.The invention provides the CTLA4-Ig composition that obtains by any method described herein.The invention provides CTLA4-Ig molecule colony, wherein said molecule is glycosylated on following residue: the amino acid asparagine residue at the 102nd place of SEQ ID NO:2, the amino acid asparagine residue at the 134th place of SEQ ID NO:2, the amino acid asparagine residue at the 233rd place of SEQ ID NO:2, the Serine amino-acid residue at the 155th place of SEQ ID NO:2, or the Serine amino-acid residue at the 165th place of SEQ ID NO:2.
The invention provides CTLA4-Ig molecule colony, wherein said molecule colony is characterised in that: (a) the molar average ratio of the GlcNAc/ mole CTLA4-Ig dimer of about 15-about 35 or CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc/ mole CTLA4-Ig dimer of about 1.7-about 3.6 or molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig dimer or molecule; (d) the molar average ratio of the Fucose of about 3.5-about 8.3/mole CTLA4-Ig dimer or molecule; (e) the molar average ratio of the seminose of about 7.2-about 22/mole CTLA4-Ig dimer or molecule; (f) the molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig dimer or molecule; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 2.4 ± 0.2-about 5.0 ± 0.2; (h) be less than or equal to the MCP-1 of 5ppm; (i) less than 3.0% the tetramer (for example, 2.5% the high molecular weight species or the tetramer, 2.0% the high molecular weight species or the tetramer; (j) less than 0.5% monomer; (k) have with SEQ ID NOS:2-8 in the CTLA4-Ig polypeptide of any one at least 95% amino acid whose colony that is equal to; (1) wherein intragroup CTLA4-Ig molecule can combine with CD80 and CD86.
Composition: the invention provides the CTLA4-Ig molecule of the present invention that comprises significant quantity and the composition of pharmaceutically acceptable carrier.The invention provides the composition that comprises as the vehicle of describing in the U. S. application submitted on December 20th, 2005 number 60/752,150.In one embodiment, composition comprises CTLA4-Ig.In one embodiment, composition further comprises pharmaceutically acceptable thinner, adjuvant or carrier.In another embodiment, composition further comprises maltose, sodium orthophosphate (monometallic) monohydrate, sodium-chlor, sodium hydroxide and sterilized water.In another embodiment, composition comprises sucrose, poloxamer, sodium orthophosphate (monometallic) monohydrate, anhydrous sodium orthophosphate dimetallic, sodium-chlor, sodium hydroxide and sterilized water.
Preparation and test kit: the invention provides freeze dried CTLA4-Ig mixture, it comprises at least 95% CTLA4-Ig dimer and is no more than 5% the CTLA4-Ig tetramer.In one embodiment, mixture comprises at least 98% CTLA4-Ig dimer and is no more than 2% the CTLA4-Ig high molecular weight species or the tetramer.In another embodiment, mixture comprises at least 99% CTLA4-Ig dimer and is no more than 1% the CTLA4-Ig high molecular weight species or the tetramer.In another embodiment, mixture comprises at least 8.0 moles of sialic acids/mole CTLA4-Ig dimer or molecule.In another embodiment, mixture comprises about 31 moles of GlcNAc/ mole CTLA4-Ig dimers of about 15.7-or molecule.In another embodiment, mixture comprises about 3.2 moles of GalNAc/ mole CTLA4-Ig dimers of about 1.6-or molecule.In another embodiment, mixture comprises the about 15.5 moles of semi-lactosis of about 9.3-/mole CTLA4-Ig dimer or molecule.In one embodiment, mixture comprises the about 7.9 moles of Fucoses of about 3.6-/mole CTLA4-Ig dimer or molecule.In one embodiment, mixture comprises about 9.7 moles of seminoses/mole CTLA4-Ig dimer or molecule.The present invention also provides pharmaceutical kit, and it comprises: the container that (a) comprises freeze dried CTLA4-Ig mixture of the present invention; (b) be used for freeze dried CTLA4-Ig mixture is reconstituted the specification sheets of the solution that is used to inject.
The methods of treatment of illustrative: the invention provides the method that is used for suppressor T cell propagation (or activation), described method comprises makes the T cell contact with the CTLA4-Ig composition of the present invention of significant quantity.The invention provides the method for the immunne response that is used for suppressing the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides and be used for inducing the method for experimenter at antigenic immunological tolerance, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the inflammation among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of rheumatoid arthritis, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the psoriasic method that is used for the treatment of among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the lupus among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides and be used for the treatment of or prevent allergic method among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of or prevents the graft versus host disease (GVH disease) among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of or prevents the transplant organ among the experimenter to repel, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the multiple sclerosis among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method for the CrohnShi disease that is used for the treatment of among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the type i diabetes among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the inflammatory bowel among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the ovaritis among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the brightic method that is used for the treatment of among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the allergic encephalitis among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the myasthenia gravis among the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged.
The CTLA4-Ig molecule colony that the invention provides sialic acids groups with about 6-about 18 and the molar average ratio of CTLA4-Ig dimer or molecule is used for the purposes of medicine of the treating and/or preventing property processing of immune disorders in preparation.The invention provides the purposes of CTLA4-Ig molecule colony in the agent of preparation resisting rheumatoid arthritis of sialic acids groups with about 6-about 18 and the molar average ratio of CTLA4-Ig dimer or molecule, described resisting rheumatoid arthritis agent together with about the specification sheets of its purposes in rheumatoid arthritis treatment in packing.
The combination treatment of illustrative: the invention provides the method that is used for suppressor T cell propagation (or activation), described method comprises makes the T cell contact with the CTLA4-Ig composition of the present invention of significant quantity, described composition and methotrexate combination.The invention provides the method for the immunne response that is used for suppressing the experimenter, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged, described composition and methotrexate combination.The invention provides and be used for inducing the method for experimenter at antigenic immunological tolerance, described method comprises the CTLA4-Ig composition of the present invention of using significant quantity to the experimenter that these needs are arranged, described composition and methotrexate combination.
Be used to produce the method for CTLA4-Ig: the invention provides the method that is used to produce recombinant protein, described method comprises: (a) mammalian cell of amplification secretion recombinant protein, wherein said amplification is from the inoculum to the liquid culture, and wherein recombinant protein concentration is at least 0.5 gram/L liquid culture; (b) separating recombinant proteins matter from liquid culture.Liquid culture can be at least 1,000L, at least 5,000L, at least 10,000L, at least 15,000L, at least 20,000L, at least 25,000L, at least 30,000L, at least 40,000L.In one embodiment, the amplification of step (a) comprises: (i) know in the substratum with at least 4 generations of cell cultures, so that obtain at least about 1.0 x 10 in serum-free, chemical ingredients 5The cell density of viable cell/mL, wherein each seed stage is with about 2 x 10 5/ ml begins and proceeds to 1-2 1,000,000 cells/ml; Cell is kept in culture to be enough to by the time of culture production at least about 0.5g/L.In one embodiment, protein is glycoprotein.In one embodiment, protein is CTLA4-Ig protein.In one embodiment, mammalian cell is the offspring that can produce the CHO cloned cell line of CTLA4-Ig fusion rotein, and wherein Chinese hamster ovary celI has at least 30 copies of the CTLA4-Ig expression cassette of stable integration in its genome.In one embodiment, time enough is that the viability of cell does not drop to the time below 30%.In another embodiment, time enough is that the viability of cell does not drop to the time below 40%.In another embodiment, time enough is that the viability of cell does not drop to the time below 50%.In another embodiment, time enough is that the viability of cell does not drop to the time below 60%.In another embodiment, time enough is that the viability of cell does not drop to the time below 70% or 80% or 90% or 95%.
In further embodiment, go down to posterity at least 4 times and comprise: (i) in the volume of culture of 50mL at least culturing cell until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml, (ii) in the volume of culture of 10L at least culturing cell until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml; (iii) in the volume of culture of 100L at least culturing cell until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml; (iv) in the volume of culture of 200L culturing cell until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml.In one embodiment, semi-lactosi is added serum-free, chemical ingredients knows in the substratum.In one embodiment, keep and comprise (i) and make the temperature of culture be reduced to 34 ± 2 ℃ from 37 ± 2 ℃; (ii) make the temperature of culture be reduced to 32 ± 2 ℃ from 34 ± 2 ℃.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 5 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 6 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 7 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 8 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 9 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 10 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 11 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 12 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 13 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 14 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 15 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 16 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 17 days.In another embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 18 days.In another embodiment, temperature maintenance is up to 18 days in 32 ± 2 ℃ scope.In another embodiment, temperature maintenance is about 30 x 10 until the cell density of culture in 32 ± 2 ℃ scope 5-Yue 79 x 10 5Cell/mL liquid culture.
The invention provides the method that is used to produce recombinant protein, described method comprises: (a) mammalian cell of amplification secretion recombinant protein from the inoculum to the liquid culture, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) separating recombinant proteins matter from liquid culture, wherein said separation only take place when liquid culture comprises more than or equal to about 6.0 moles of NANA/ mole CTLA4-Ig protein or dimer.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) mammalian cell of amplification secretion recombinant protein from the inoculum to the liquid culture, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) separating recombinant proteins matter from liquid culture, wherein said separation only take place when liquid culture has the cell density of about 79 x of about 33 x 105-105 cells/mL.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) mammalian cell of amplification secretion recombinant protein from the inoculum to the liquid culture, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) separating recombinant proteins matter from liquid culture, the wherein said cell survival that is separated in the liquid culture do not drop to about 20% or about 30% or about 38% and take place when following.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) from inoculum at least 10, the mammalian cell of the liquid culture amplification secretion recombinant protein of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) separating recombinant proteins matter from liquid culture, wherein said separation only take place when intracellular toxin is less than or equal to about 76.8EU/mL liquid culture.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) from inoculum at least 10, the mammalian cell of the liquid culture amplification secretion recombinant protein of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) from least 10, separating recombinant proteins matter in the liquid culture of 000L, wherein said separation only takes place during liquid culture less than 1 colony forming unit/mL at biological load.Liquid culture of the present invention can be at least 5,000L, at least 10,000L, at least 15,000L, at least 20,000L, at least 25,000L, at least 30,000L, at least 40,000L, at least 50,000L, at least 60, the volume of 000L.
The invention provides the method that is used to produce recombinant protein, described method comprises: (a) mammalian cell of amplification secretion recombinant protein from the inoculum to the liquid culture, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) separating recombinant proteins matter from liquid culture, wherein said separation only takes place during 2 in satisfying following condition at least: (i) liquid culture comprises more than or equal to about 6.0 moles of NANA/ mole protein, (ii) liquid culture has the cell density of about 79 x of about 33 x 105-105 cells/mL, (iii) the cell survival in the liquid culture does not drop to about 20% or about 30% or about below 38%, or (iv) the amount of the CTLA4-Ig in the culture greater than 0.5g/L.In one embodiment, separation comprises: (i) obtain cell culture supernatant; (ii) supernatant liquor is implemented anion-exchange chromatography, to obtain the protein of wash-out; (iii) the protein of step wash-out is (ii) implemented hydrophobic interaction chromatography, so that obtain the protein of enrichment; (iv) the protein of enrichment is implemented affinity chromatography, to obtain the protein of wash-out and enrichment; (v) the protein of (iv) wash-out and enrichment is implemented anion-exchange chromatography.In another embodiment, the protein of the enrichment that obtains in (iii) of step is characterised in that the per-cent of any high molecular weight contaminants is all less than 25 weight %.In another embodiment, step anion-exchange chromatography (ii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 75mM HEPES and about 360mM NaCl, and has about 8.0 pH.In another embodiment, step anion-exchange chromatography (ii) uses elution buffer to carry out, and described elution buffer comprises about 25mM HEPES and about 325mM NaCl, and has about 7.0 pH.In another embodiment, step hydrophobic interaction chromatography (iii) uses single lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM HEPES and about 850mM NaCl, and has about 7.0 pH.In another embodiment, step affinity chromatography (iv) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM Tris and about 250mM NaCl, and has about 8.0 pH.In another embodiment, step affinity chromatography (iv) uses elution buffer to carry out, and described elution buffer comprises about 100mM glycine, and has about 3.5 pH.In another embodiment, (anion-exchange chromatography v) uses lavation buffer solution to carry out to step, and described lavation buffer solution comprises about 25mM HEPES and the about 130mM NaCl of about 120mMNaCl-, and has about 8.0 pH.In another embodiment, (anion-exchange chromatography v) uses elution buffer to carry out to step, and described elution buffer comprises about 25mM HEPES and about 200mM NaCl, and has about 8.0 pH.In another embodiment, step anion-exchange chromatography (ii) uses the post with anionite-exchange resin to carry out, and described anionite-exchange resin has primary, secondary, uncle or quaternary amine functional group.In another embodiment, resin has quaternary amine functional group.In another embodiment, step hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin has phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.In another embodiment, functional group is a phenyl functional group.In another embodiment, step affinity chromatography is (iv) used and to be comprised the proteic post of A and carry out.
The invention provides the method that is used to prepare CTLA4-Ig, described method comprises purifying CTLA4-Ig from the liquid cell culture, thereby make the CTLA4-Ig (a) of purifying have about 38ngMCP-1/mg CTLA4-Ig dimer, (b) comprise and be less than 2.5 weight %CTLA4-Ig high molecular weight species (for example, the tetramer).The invention provides the method that is used to produce CTLA4-Ig, described method comprises: (a) amplification can be produced progeny cell or the Chinese hamster ovary celI of CTLA4-Ig, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, wherein CTLA4-Ig concentration is at least 0.5 gram/L liquid culture; (b) from least 10, separation of C TLA4-Ig in the liquid culture of 000L, wherein chromatography is on the post with hydrophobic interaction resin, described hydrophobic interaction resin has phenyl functional group at least, wherein said separation comprises uses single lavation buffer solution to carry out the step of hydrophobic interaction chromatography, described lavation buffer solution comprises about 25mMHEPES and about 850mM NaCl, and has about 7.0 pH.
The CTLA4-Ig molecule comprises the beta polypeptides molecule.CTLA4 A29YL104E-Ig is the beta polypeptides molecule.The present invention relates to be used for producing the method for compositions of (comprising mass production) beta polypeptides composition or beta polypeptides molecular composition and improvement.The composition and being used for that the present invention relates to the beta polypeptides molecule, comprises the improvement of beta polypeptides molecule is produced the method for the improvement of (comprising mass production) beta polypeptides molecule and other recombinant glycoproteins.
Be used to produce the method for beta polypeptides and other glycoprotein: the invention provides the method that is used to produce recombinant glycoprotein, described method comprises: (a) mammalian cell of amplification secretion recombinant glycoprotein, wherein said amplification is at least about 10 from inoculum, the liquid culture of 000L, wherein recombinant protein concentration is at least about 0.5 gram/L liquid culture; Wherein said amplification comprises: (i) know in the substratum with at least 4 generations of cell cultures, so that obtain at least about 1.0 x 10 in serum-free, chemical ingredients 5The cell density of viable cell/mL, wherein each seed stage is with about 2 x10 5/ ml begins and proceeds to 1-2 1,000,000 cells/ml, and wherein said cultivation comprises: (1) makes cell know in the inoculum substratum in serum-free, chemical ingredients and cultivated about 15 days-Yue 25 days; (2) know that in serum-free, chemical ingredients culturing cell is until reaching the cell density of at least 4 100 ten thousand cells/mL approximately in the basic medium subsequently; Cell is kept in culture to be enough to by the time of culture production at least about the recombinant protein of 0.5g/L; (b) from least about 10, separating recombinant proteins matter in the 000L liquid culture.
The invention provides the method that is used to produce recombinant glycoprotein, described method comprises: (a) mammalian cell of amplification secretion recombinant glycoprotein, wherein said amplification is at least about 10 from inoculum, the liquid culture of 000L, wherein recombinant protein concentration is at least about 0.5 gram/L liquid culture; Wherein said amplification comprises: (i) know in the substratum with at least 4 generations of cell cultures, so that obtain at least about 1.0 x 10 in serum-free, chemical ingredients 5The cell density of viable cell/mL, wherein each seed stage is with about 2 x 10 5/ ml begins and proceeds to 1-2 1,000,000 cells/ml; Cell is kept in culture to be enough to by the time of culture production at least about the recombinant protein of 0.5g/L, and wherein said keeping comprises: (1) makes the temperature of culture be reduced to 34 ± 2 ℃ from 37 ± 2 ℃; (2) in culture, add polyanionic compound; (b) from least about 10, separating recombinant proteins matter in the 000L liquid culture.
The invention provides the method that is used to produce recombinant glycoprotein, described method comprises: (a) mammalian cell of amplification secretion recombinant glycoprotein, wherein said amplification is at least about 10 from inoculum, the liquid culture of 000L, wherein recombinant protein concentration is at least about 0.5 gram/L liquid culture; (b) from least about 10, separating recombinant proteins matter in the 000L liquid culture; Wherein said separation comprises: the soluble fraction that (i) obtains the culture of step (a); (ii) soluble fraction is implemented affinity chromatography, to obtain the protein of wash-out; (iii) the protein of step wash-out is (ii) implemented anion-exchange chromatography, so that obtain the protein of wash-out and enrichment; (iv) the protein of enrichment is implemented hydrophobic interaction chromatography, to obtain the protein of enrichment.
In one embodiment of the invention, protein comprises CTLA4-Ig.In another embodiment, protein comprises beta polypeptides or beta polypeptides molecule.In another embodiment, protein comprise have SEQ ID NO:11,12,13,14,15 or 16 beta polypeptides.
In one embodiment of the invention, go down to posterity at least 4 times and comprise: (i) in the volume of culture of 50mL at least culturing cell until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml; (ii) in the volume of culture of 10L at least culturing cell until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml; (iii) in the volume of culture of 100L at least culturing cell until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml; (iv) in the volume of culture of 200L culturing cell until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml.
In one embodiment of the invention, separation comprises: the soluble fraction that (i) obtains the culture of step (a); (ii) soluble fraction is implemented affinity chromatography, to obtain the protein of wash-out; (iii) the protein of step wash-out is (ii) implemented anion-exchange chromatography, so that obtain the protein of wash-out and enrichment; (iv) the protein of enrichment is implemented hydrophobic interaction chromatography, to obtain the protein of enrichment.
In one embodiment, the protein of the enrichment that obtains in (iv) of step is characterised in that the polymeric per-cent of any high molecular is less than 25 weight %.In another embodiment, step anion-exchange chromatography (iii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 50mM HEPES and about 135mM NaCl, and has about 7 pH.In another embodiment, step anion-exchange chromatography (iii) uses elution buffer to carry out, and described elution buffer comprises about 50mM HEPES and about 200mM NaCl, and has about 7 pH.In another embodiment, step hydrophobic interaction chromatography (iv) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 50mM HEPES and about 1.2M (NH 4) 2SO 4, and have about 7 pH.In another embodiment, step affinity chromatography (ii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM NaH 2PO 4With about 150mM NaCl, and has about 7.5 pH.In another embodiment, step affinity chromatography (ii) uses elution buffer to carry out, and described elution buffer comprises about 250mM glycine and has about 3 pH.In another embodiment, step anion-exchange chromatography (iii) uses the post with anionite-exchange resin to carry out, and described anionite-exchange resin has primary, secondary, uncle or quaternary amine functional group.In another embodiment, resin has quaternary amine functional group.In another embodiment, step hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin has phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.In another embodiment, functional group is a phenyl functional group.In another embodiment, step affinity chromatography is (ii) used and to be comprised the proteic post of A and carry out.
In another embodiment, amplification comprises: (i) know in the substratum with at least 4 generations of cell cultures, so that obtain at least about 1.0 x 10 in serum-free, chemical ingredients 5The cell density of viable cell/mL, wherein each seed stage is with about 2 x 10 5/ ml begins and proceeds to 1-2 1,000,000 cells/ml; Cell is kept in culture to be enough to by the time of culture production at least about the recombinant protein of 0.5g/L.In another embodiment, cultivation comprises: cell is known in the inoculum substratum in serum-free, chemical ingredients cultivated about 15 days-Yue 25 days; (ii) know that in serum-free, chemical ingredients culturing cell is until reaching the cell density of at least 4 100 ten thousand cells/mL approximately in the basic medium subsequently.
In another embodiment, keep and comprise (i) and make the temperature of culture be reduced to 34 ± 2 ℃ from 37 ± 2 ℃; (ii) in culture, add polyanionic compound.In one embodiment, polyanionic compound is a T 500, and in the final concentration adding culture of wherein said T 500 with about 50mg/ml.In another embodiment, temperature maintenance is in 34 ± 2 ℃ of scopes at least 5 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 6 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 7 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 8 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 9 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 10 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 11 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 12 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 13 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 14 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 15 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 16 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 17 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 18 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 19 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 20 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 21 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 22 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 23 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 24 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 25 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 26 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 27 days.In another embodiment, temperature maintenance is in 34 ± 2 ℃ scope at least 28 days.In another embodiment, temperature maintenance is up to 28 days in 34 ± 2 ℃ scope.In another embodiment, temperature maintenance is about 30 x 10 until the cell density of culture in 34 ± 2 ℃ scope 5-Yue 79 x 10 5Cell/mL liquid culture.
In one embodiment, time enough is that the viability of cell does not drop to the time below 30%.In another embodiment, time enough is that the viability of cell does not drop to the time below 40%.In another embodiment, time enough is that the viability of cell does not drop to the time below 50%.In another embodiment, time enough is that the viability of cell does not drop to the time below 60%.In another embodiment, time enough is that the viability of cell does not drop to the time below 70% or 80% or 90% or 95%.
In one embodiment, semi-lactosi is added serum-free, chemical ingredients knows in the substratum.In another embodiment, be separated in when liquid culture comprises more than or equal to about 6 moles of sialic acids/mole protein and take place.In another embodiment, be separated in when liquid culture comprises about 7.6 moles of sialic acids of about 5.2-/mole protein and take place.In another embodiment, be separated in liquid culture and have about 33 x 10 5-Yue 79 x 10 5Take place during the cell density of cell/mL.In another embodiment, being separated in cell survival in the liquid culture does not drop to about 37% and takes place when following.In another embodiment, be separated in when intracellular toxin is less than or equal to about 4.8EU/mL liquid culture and take place.Take place when in another embodiment, being separated in biological load less than about 1 colony forming unit/mL (cfu/ml) liquid culture.Take place when in another embodiment, only separating at least 2 in satisfying following condition: (i) liquid culture comprises more than or equal to about 6.0 moles of sialic acids/mole protein, and (ii) liquid culture has about 33 x10 5-Yue 79 x 10 5The cell density of cell/mL, (iii) the cell survival in the liquid culture does not drop to approximately below 37%, or (iv) the amount of the glycoprotein in the culture is the about 0.71g/L of about 0.46g/L-.
In one embodiment, mammalian cell is the offspring of Chinese hamster ovary cloned cell line who produces any combination of beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises SEQ IDNO:11,12,13,14,15 or 16, and wherein said Chinese hamster ovary cell has at least 30 copies of the expression cassette of the expression SEQ ID NO:3 of stable integration in its genome separately.In one embodiment, liquid culture comprises the cell of production clone of the present invention or the progeny cell of described cell.
The invention provides by what obtain and comprise SEQ ID NO:11,12,13,14,15 or 16 beta polypeptides by method provided by the invention.The invention provides the composition that comprises beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises the SEQ ID NO:11,12,13,14,15 or 16 that obtains by by method provided by the invention.The invention provides the beta polypeptides that obtains by by method provided by the invention.
Cell: the clone's Chinese hamster ovary cell that the invention provides the nucleic acid that comprises coding beta polypeptides or beta polypeptides molecule.The invention provides the clone's Chinese hamster ovary cell colony that produces beta polypeptides or beta polypeptides molecule.In one embodiment, beta polypeptides comprises SEQ ID NO:11,12,13,14,15 or 16.The invention provides the clone's Chinese hamster ovary cell that comprises nucleic acid, described nucleic acid comprises the expression cassette of coding SEQ ID NO:11,12,13,14,15 or 16 aminoacid sequence.In one embodiment, expression cassette comprises SEQ ID NO:3.The invention provides the clone's Chinese hamster ovary cell colony that produces beta polypeptides or beta polypeptides molecule, wherein said beta polypeptides is expressed by the nucleotide sequence derived from the plasmid with ATCC registration number PTA-2104, described plasmid is specified in according to budapest treaty (Buda β est Treaty) and was preserved in American type culture collection (ATCC) on June 20th, 2000,10801 University Blvd., Manassas, VA, 20110.
The invention provides the clone's Chinese hamster ovary cell colony that produces beta polypeptides or beta polypeptides molecule, every kind of cell comprises 30 of the beta polypeptides expression cassette or multiple copied more.The invention provides the clone's Chinese hamster ovary cell colony that produces beta polypeptides or beta polypeptides molecule, every kind of cell comprises 30 of the beta polypeptides expression cassette or multiple copied more, wherein said 30 or more on the single site of multi-copy integration in the genome of each cell.The invention provides the clone's Chinese hamster ovary cell colony that produces beta polypeptides or beta polypeptides molecule, wherein the beta polypeptides expression cassette is stable in about 105 times go down to posterity.In one embodiment, beta polypeptides is by the expression cassette coding that is incorporated in the cellular genome.
The invention provides the clone's Chinese hamster ovary cell colony that produces beta polypeptides, wherein at least 75% cell colony has 30 of the beta polypeptides expression cassette or multiple copied/cell more, wherein said 30 or more on the single site of multi-copy integration in the genome of each cell of 75% colony.The invention provides the clone's Chinese hamster ovary cell colony that produces beta polypeptides, wherein at least 85% cell colony has 30 of the beta polypeptides expression cassette or multiple copied/cell more, wherein said 30 or more on the single site of multi-copy integration in the genome of each cell of 85% colony.The invention provides the clone's Chinese hamster ovary cell colony that produces beta polypeptides, wherein at least 95% cell colony has 30 of the beta polypeptides expression cassette or multiple copied/cell more, wherein said 30 or more on the single site of multi-copy integration in the genome of each cell of 95% colony.In one embodiment, expression cassette is derived from the plasmid as ATCC registration number PTA-2104 preservation.In another embodiment, expression cassette comprises the nucleic acid of the sequence with SEQ ID NO:3.In one embodiment, cell colony is produced at least about 0.5 gram beta polypeptides/L liquid culture, and wherein said beta polypeptides is 1, has the mol ratio of sialic acid and beta polypeptides dimer or the beta polypeptides molecule of about 5.5-about 8.5 on 000L or the bigger cultivation scale.In another embodiment, cell colony production at least 5, at least 10 or at least 20 gram beta polypeptides/L liquid cultures.In another embodiment, beta polypeptides is 1, has the mol ratio of sialic acid and beta polypeptides dimer or the beta polypeptides molecule of about 5-about 10 on 000L or the bigger cultivation scale.In another embodiment, cell colony has adapted to serum-free, chemical ingredients knows substratum.In another embodiment, the beta polypeptides by the culture production of cell colony has 1.0 ± 0.05AU mL cm -1Mg -1Optical extinction coefficient.In another embodiment, when growing in culture, cell colony is produced beta polypeptides, and wherein: (a) about 90% or about 80% beta polypeptides comprises the aminoacid sequence of the SEQ ID NO:4 that begins from the methionine(Met) of residue 27; (b) about 10% or about 20% beta polypeptides comprises the aminoacid sequence of numbering the SEQ ID NO:4 that 26 L-Ala begins from residue; (c) beta polypeptides of about 4%-about 8% comprises the aminoacid sequence of numbering the SEQID NO:4 that the Methionin at 383 places finishes with residue, and (d) beta polypeptides of about 92%-about 96% comprises the aminoacid sequence of SEQ ID NO:4 of numbering the glycine end at 382 places with residue; Randomly, (e) comprise the aminoacid sequence of numbering the SEQID NO:4 that 25 methionine(Met) begins from residue less than 1% beta polypeptides approximately.
The invention provides the progeny cell of cell colony of the present invention, wherein said progeny cell is produced beta polypeptides.In one embodiment, progeny cell is obtained through at least 5,10,20,40,50,75 generations by culturing cell at least at least at least at least at least.In another embodiment, progeny cell was obtained by 27 generations of culturing cell.
The invention provides the clone that produces by any cell provided by the invention.In one embodiment, clone is cloned.In one embodiment, clone production: (a) have the beta polypeptides of the aminoacid sequence (methionine(Met) at amino acid position 27 places of SEQ ID NO:4 and the glycine at amino acid position 382 places) of SEQ ID NO:16; (b) has the beta polypeptides of the aminoacid sequence (methionine(Met) at amino acid position 27 places of SEQ ID NO:4 and the Methionin at amino acid position 383 places) of SEQ IDNO:13; (c) has the beta polypeptides of the aminoacid sequence (L-Ala at amino acid position 26 places of SEQ ID NO:4 and the glycine at amino acid position 382 places) of SEQ ID NO:15; (d) has the beta polypeptides of the aminoacid sequence (L-Ala at amino acid position 26 places of SEQ ID NO:4 and the Methionin at amino acid position 383 places) of SEQ ID NO:12; (e) has the beta polypeptides of the aminoacid sequence (methionine(Met) at amino acid position 25 places of SEQ IDNO:4 and the Methionin at amino acid position 383 places) of SEQ ID NO:11; (f) has the beta polypeptides of the aminoacid sequence (methionine(Met) at amino acid position 25 places of SEQ ID NO:4 and the glycine at amino acid position 382 places) of SEQ ID NO:14; Or (g) its any combination.In one embodiment, clone is produced beta polypeptides or beta polypeptides molecule, and wherein: (a) about 90% or about 80% beta polypeptides comprises the aminoacid sequence of the SEQ ID NO:4 that begins from the methionine(Met) of residue 27; (b) about 10% or about 20% beta polypeptides comprises the aminoacid sequence of numbering the SEQ ID NO:4 that 26 L-Ala begins from residue; (c) beta polypeptides of about 4%-about 8% comprises the aminoacid sequence of numbering the SEQID NO:4 that the Methionin at 383 places finishes with residue; (d) beta polypeptides of about 92%-about 96% comprises the aminoacid sequence of numbering the SEQ ID NO:4 that the glycine at 382 places finishes with residue; Randomly, (e) comprise the aminoacid sequence of numbering the SEQID NO:4 that 25 methionine(Met) begins from residue less than 1% beta polypeptides approximately.In one embodiment, the beta polypeptides that is wherein produced by culturing cell system has the optical extinction coefficient of 1.0 ± 0.05AU mL cm-1 mg-1.
The invention provides cell colony derived from cell of the present invention.In one embodiment, compared by its deutero-cell of the present invention with colony, cell colony comprises at least a other hereditary change, and wherein said cells produce beta polypeptides.In another embodiment, compare by its deutero-cell of the present invention with colony, cell colony comprises at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds or at least 20 kinds of other hereditary changes, and wherein said cells produce beta polypeptides.In one embodiment, hereditary change be included in cellular genome or the coding beta polypeptides expression cassette at least a non-conservative sudden change.In another embodiment, hereditary change comprises intracellular at least a other recombinant nucleic acid.In another embodiment, hereditary change comprises the sudden change of cellular genome.In another embodiment, hereditary change comprises to cellular genome interpolation nucleic acid or as trans nucleic acid, and wherein said nucleic acid encoding anti-apoptotic polypeptide.In another embodiment, the anti-apoptotic polypeptide relates to glycosylation.In another embodiment, hereditary change comprises at least a sudden change of the expression cassette of cellular genome or coding beta polypeptides.In another embodiment, when in culture, growing, cell colony production: (a) have the beta polypeptides of the aminoacid sequence (methionine(Met) at amino acid position 27 places of SEQ ID NO:4 and the glycine at amino acid position 382 places) of SEQ IDNO:16; (b) has the beta polypeptides of the aminoacid sequence (methionine(Met) at amino acid position 27 places of SEQ ID NO:4 and the Methionin at amino acid position 383 places) of SEQ ID NO:7; (c) has the beta polypeptides of the aminoacid sequence (L-Ala at amino acid position 26 places of SEQ ID NO:4 and the glycine at amino acid position 382 places) of SEQ ID NO:15; (d) has the beta polypeptides of the aminoacid sequence (L-Ala at amino acid position 26 places of SEQ IDNO:4 and the Methionin at amino acid position 383 places) of SEQ ID NO:12; (e) has the beta polypeptides of the aminoacid sequence (methionine(Met) at amino acid position 25 places of SEQ ID NO:4 and the Methionin at amino acid position 383 places) of SEQ ID NO:11; (f) has the beta polypeptides of the aminoacid sequence (methionine(Met) at amino acid position 25 places of SEQ ID NO:4 and the glycine at amino acid position 382 places) of SEQ ID NO:14; Or (g) its any combination.
Composition: the isolating colony that the invention provides beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, has the sialic acids groups of about 5-about 10 and the molar average ratio of beta polypeptides dimer or beta polypeptides molecule.In one embodiment, sialic acids groups is about 5.5-about 8.5 with the molar average ratio of beta polypeptides dimer or beta polypeptides molecule.In one embodiment, sialic acids groups is about 5.2-about 7.6 with the molar average ratio of beta polypeptides dimer or beta polypeptides molecule.The invention provides the isolating colony of beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, has the molar average ratio of about 6 sialic acids groups and beta polypeptides dimer or beta polypeptides molecule.The invention provides the isolating colony of beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and surpasses 95% polypeptide and form dimer.In one embodiment, surpass 98%, surpass 99% or surpass 99.5% polypeptide and form dimer.In another embodiment, the polypeptide of about 95%-about 99.5% forms dimer, and the polypeptide of about 0.5%-about 5% forms the tetramer or high molecular weight species.In another embodiment, about 98.6% polypeptide forms dimer, and about 1.2% polypeptide forms the tetramer or high molecular weight species, and is monomer less than 0.7% polypeptide approximately.In another embodiment, about 95% polypeptide forms dimer, and about 4% polypeptide forms the tetramer or high molecular weight species, and about 1% polypeptide is the dimeric isolating colony of beta polypeptides, and wherein each polypeptide monomer comprises SEQ ID NO:11,12,13,14,15 or 16 sequence.In one embodiment, colony is substantially free of the beta polypeptides monomer.In another embodiment, colony is substantially free of the beta polypeptides tetramer.The invention provides and be substantially free of beta polypeptides dimer and the monomeric isolating colony of tetrameric beta polypeptides.In one embodiment, every kind of dimeric each monomer of beta polypeptides comprises SEQ ID NO:11,12,13,14,15 or 16 sequence and has at least 2.5 sialic acids groups.
The invention provides the isolating colony of beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, compare with the CTLA4-Ig standard, in conjunction with the effectiveness that has about 70%-about 130% in measuring, wherein said mensuration comprises the surface measurements plasma resonance at B7.The invention provides the isolating colony of beta polypeptides, wherein every peptide species comprises SEQID NO:11,12,13,14,15 or 16 sequence, compares with standard, has the effectiveness of about 50%-about 150% in people's cell IL-2 suppresses to measure.The invention provides the colony of the purifying of the beta polypeptides tetramer or high molecular weight species, wherein each polypeptide monomer comprises SEQ IDNO:11,12,13,14,15 or 16 sequence, described colony is substantially free of the beta polypeptides dimer, and randomly, wherein said colony comprises the amount that surpasses about 100 grams.The invention provides the colony of the purifying of the beta polypeptides tetramer or high molecular weight species, wherein each polypeptide monomer comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, described colony is substantially free of the beta polypeptides monomer, and randomly, wherein said colony comprises the amount that surpasses about 100 grams.In one embodiment, each tetramer molecule comprises 2 pairs of beta polypeptides, wherein each polypeptide monomer comprises SEQID NO:11,12,13,14,15 or 16 sequence, and wherein right each member and another member of polypeptide is covalently bound, and 2 pairs of polypeptide covalent attachment not each other wherein.In one embodiment, each tetramer molecule can combine with CD80 or CD86.In one embodiment, compare with the beta polypeptides dimer, each tetramer molecule has the big avidity at least 2 times of CD80 or CD86, and wherein dimeric each polypeptide monomer comprises SEQ IDNO:11,12,13,14,15 or 16 sequence.In another embodiment, compare with the CTLA4-Ig tetramer molecule of the sequence that comprises SEQ ID NO:2, each tetramer molecule has the big avidity at least 2 times of CD80 or CD86.In another embodiment, compare with the beta polypeptides dimer, each tetramer molecule has at least 2 times big T cell proliferation or activation to be suppressed, and wherein dimeric each polypeptide monomer comprises SEQ ID NO:11,12,13,14,15 or 16 sequence.In another embodiment, compare with the CTLA4-Ig tetramer molecule of the sequence that comprises SEQ IDNO:2, each tetramer molecule has at least 2 times big T cell proliferation or activation inhibition.
The invention provides the composition isolated that comprises beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said composition is included in advantage isoform visual on the isoelectrofocusing gel, as measuring by isoelectrofocusing, described advantage isoform has and is less than or equal to 5.5 iso-electric point, pI.In one embodiment, pI handles the back at neuraminidase increases.In one embodiment, as measuring by isoelectrofocusing, at least 40% beta polypeptides or the demonstration of beta polypeptides molecule are less than or equal to about 5.3 iso-electric point.In one embodiment, as measuring by isoelectrofocusing, at least 70% beta polypeptides or the demonstration of beta polypeptides molecule are less than or equal to about 5.3 iso-electric point.In one embodiment, as measuring by isoelectrofocusing, at least 90% beta polypeptides or the demonstration of beta polypeptides molecule are less than or equal to about 5.3 iso-electric point.The invention provides the beta polypeptides of the pI with about 2.0 ± 0.2-about 5.2 ± 0.2 or the segregating population of beta polypeptides molecule.The invention provides the beta polypeptides of the pI with about 4.5 ± 0.2-about 5.2 ± 0.2 or the segregating population of beta polypeptides molecule.The invention provides the beta polypeptides of the pI with about 4.7 ± 0.2-about 5.1 ± 0.2 or the segregating population of beta polypeptides molecule.The invention provides and be used to prepare method for compositions, described composition comprises beta polypeptides or the beta polypeptides molecule of the pI with about 2.0 ± 0.2-about 5.2 ± 0.2, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, described method comprises: (a) mixture of beta polypeptides is implemented the isoelectrofocusing gel electrophoresis, wherein the single band on gel represents to have the colony of beta polypeptides or the beta polypeptides molecule of specific pI, (b) separate beta polypeptides or the beta polypeptides molecule colony of the pI with about 2.0 ± 0.2-about 5.2 ± 0.2, so that prepare composition.
The invention provides the composition isolated that comprises beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that the G1cNAc/ mole beta polypeptides dimer of about 24-about 28 or the molar average ratio of beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that the GalNAc/ mole beta polypeptides dimer of about 2.7-about 3.6 or the molar average ratio of beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that the semi-lactosi/mole beta polypeptides dimer of about 11-about 13 or the molar average ratio of beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that the Fucose/mole beta polypeptides dimer of about 6.4-about 7.0 or the molar average ratio of beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that the seminose/mole beta polypeptides dimer of about 14-about 16 or the molar average ratio of beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said molecule is characterised in that the sialic acid/mole beta polypeptides dimer of about 5.5-about 8.5 or the molar average ratio of beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said molecule is characterised in that the sialic acid/mole beta polypeptides dimer of about 5-about 10 or the molar average ratio of beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the GlcNAc/ mole beta polypeptides dimer of about 24-about 28 or beta polypeptides molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said molecule is characterised in that: (a) the molar average ratio of the GlcNAc/ mole beta polypeptides dimer of about 24-about 28 or beta polypeptides molecule; (b) the molar average ratio of the GalNAc/ mole beta polypeptides dimer of about 2.7-about 3.6 or beta polypeptides molecule; (c) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said molecule is characterised in that: (a) the molar average ratio of the GlcNAc/ mole beta polypeptides dimer of about 24-about 28 or beta polypeptides molecule; (b) the molar average ratio of the GalNAc/ mole beta polypeptides dimer of about 2.7-about 3.6 or beta polypeptides molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (d) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the GlcNAc/ mole beta polypeptides dimer of about 24-about 28 or beta polypeptides molecule; (b) the molar average ratio of the GalNAc/ mole beta polypeptides dimer of about 2.7-about 3.6 or beta polypeptides molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (d) the molar average ratio of the Fucose of about 6.4-about 7.0/mole beta polypeptides dimer or beta polypeptides molecule; (e) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the GlcNAc/ mole beta polypeptides dimer of about 24-about 28 or beta polypeptides molecule; (b) the molar average ratio of the GalNAc/ mole beta polypeptides dimer of about 2.7-about 3.6 or beta polypeptides molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (d) the molar average ratio of the Fucose of about 6.4-about 7.0/mole beta polypeptides dimer or beta polypeptides molecule; (e) the molar average ratio of the seminose of about 14-about 16/mole beta polypeptides dimer or beta polypeptides molecule; (f) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the semi-lactosi of about 8-about 17/mole beta polypeptides dimer or beta polypeptides molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule; (c) the carbohydrate overview substantially the same with Fig. 8.The invention provides the composition isolated that comprises beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the semi-lactosi of about 8-about 17/mole beta polypeptides dimer or beta polypeptides molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule; (c) the carbohydrate overview substantially the same with Fig. 8; (d) less than about 5% beta polypeptides tetramer content.The invention provides the composition isolated that comprises beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule; (c) less than about 5% beta polypeptides tetramer content.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule; (b) the carbohydrate overview substantially the same with Fig. 8.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (b) the carbohydrate overview substantially the same with Fig. 8.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule; (b) less than about 5% the beta polypeptides tetramer or high molecular weight species content.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide is characterised in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (b) less than about 5% the beta polypeptides tetramer or high molecular weight species content.
The invention provides the composition isolated that comprises beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said polypeptide shows the carbohydrate overview substantially the same with Fig. 8.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and the carbohydrate overview of wherein said polypeptide display structure territory I-IV, wherein structural domain I comprises the peak of the oligosaccharides of representing asialoization, domain II comprises the peak of the oligosaccharides of representing mono-sialylated, domain II I comprises the peak of the oligosaccharides of representing double-sialylated, and structural domain IV comprises the peak of representing three sialylated oligosaccharides.In one embodiment, the difference in the retention time of N connection oligosaccharides is about 13 minutes of about 11-between first peak among the structural domain I and the main peak in the domain II.In one embodiment, the summation of domain II I and IV comprises the total carbohydrates overview of about 25%-about 36%.
The invention provides the composition isolated that comprises beta polypeptides dimer or beta polypeptides molecule, wherein each polypeptide monomer comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein at least 0.5% molecule is a cysteinylization.The invention provides the isolating colony of beta polypeptides, wherein each polypeptide monomer comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said colony shows mass spectroscopy overview as shown in Figure 11.The invention provides the isolating colony of beta polypeptides or beta polypeptides molecule, wherein every peptide species comprises SEQ IDNO:11,12,13,14,15 or 16 sequence, have the sialic acids groups of about 5.5-about 8.5 and the molar average ratio of beta polypeptides dimer or beta polypeptides molecule, wherein said beta polypeptides dimer or beta polypeptides molecule are by the cells produce of production clone.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, wherein said polypeptide is glycosylated on following residue: the amino acid asparagine residue at the 102nd place of SEQ ID NO:4, the amino acid asparagine residue at the 134th place of SEQ ID NO:4, the amino acid asparagine residue at the 233rd place of SEQ ID NO:4.The invention provides the composition isolated that comprises beta polypeptides, wherein every peptide species comprises SEQID NO:11,12,13,14,15 or 16 sequence, and wherein said molecule is characterised in that: (a) the molar average ratio of the GlcNAc/ mole beta polypeptides dimer of about 24-about 28 or beta polypeptides molecule; (b) the molar average ratio of the GalNAc/ mole beta polypeptides dimer of about 2.7-about 3.6 or beta polypeptides molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (d) the molar average ratio of the Fucose of about 6.4-about 7.0/mole beta polypeptides dimer or beta polypeptides molecule; (e) the molar average ratio of the seminose of about 14-about 16/mole beta polypeptides dimer or beta polypeptides molecule; (f) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 2.4 ± 0.2-about 5.2 ± 0.2; (h) be less than or equal to the MCP-1 of 5ppm; (i) less than 5% the tetramer or high molecular weight species; (j) less than 1% beta polypeptides monomer; (k) have with SEQ ID NOS:4,11,12,13,14,15 or 16 in the beta polypeptides or the beta polypeptides molecule of any one at least 95% amino acid whose colony that is equal to, wherein intragroup beta polypeptides can combine with CD80 and CD86.The invention provides the isolating colony of beta polypeptides, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said molecule colony is characterised in that: (a) the molar average ratio of the GlcNAc/ mole beta polypeptides dimer of about 24-about 28 or beta polypeptides molecule; (b) the molar average ratio of the GalNAc/ mole beta polypeptides dimer of about 2.7-about 3.6 or beta polypeptides molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13/mole beta polypeptides dimer or beta polypeptides molecule; (d) the molar average ratio of the Fucose of about 6.4-about 7.0/mole beta polypeptides dimer or beta polypeptides molecule; (e) the molar average ratio of the seminose of about 14-about 16/mole beta polypeptides dimer or beta polypeptides molecule; (f) the molar average ratio of the sialic acid of about 5.5-about 8.5/mole beta polypeptides dimer or beta polypeptides molecule; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 2.4 ± 0.2-about 5.2 ± 0.2; (h) be less than or equal to the MCP-1 of 5ppm; (i) less than 5% the tetramer or high molecular; (j) less than 1% monomer; (k) have with SEQ ID NOS:4,11,12,13,14,15 or 16 in the beta polypeptides of any one at least 95% amino acid whose colony that is equal to, wherein intragroup beta polypeptides molecule can combine with CD80 and CD86; Or its pharmacy Equivalent.
The invention provides the beta polypeptides of the present invention that comprises significant quantity and the composition of pharmaceutically acceptable carrier.The invention provides the composition that comprises as the vehicle of describing in the U. S. application number 60/752,150; Described U. S. application was submitted on December 20th, 2005.In one embodiment, composition comprises the beta polypeptides molecule.In one embodiment, composition further comprises pharmaceutically acceptable vehicle, adjuvant or carrier.In one embodiment, composition further comprises sucrose, sodium orthophosphate (monometallic) monohydrate, sodium-chlor, sodium hydroxide, hydrochloric acid and sterilized water.In another embodiment, composition further comprises sucrose, poloxamer, sodium orthophosphate (monometallic) monohydrate, anhydrous sodium orthophosphate dimetallic, sodium-chlor, sodium hydroxide and sterilized water.In one embodiment, composition is freeze dried.The invention provides the lyophilised compositions of the beta polypeptides of the present invention, sucrose, sodium orthophosphate (monometallic) monohydrate, sodium-chlor, sodium hydroxide and the hydrochloric acid that comprise significant quantity.
Preparation and test kit: the invention provides freeze dried beta polypeptides mixture, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, comprises at least 95% beta polypeptides dimer and is no more than the 5% beta polypeptides tetramer (high molecular weight species).In one embodiment, mixture comprises at least 98% beta polypeptides dimer and is no more than the 2% beta polypeptides tetramer (high molecular weight species).In one embodiment, mixture comprises at least 99% beta polypeptides dimer and is no more than the 1% beta polypeptides tetramer (high molecular weight species).In one embodiment, mixture comprises at least 5 moles of sialic acids/mole beta polypeptides dimer or beta polypeptides molecule.In one embodiment, mixture comprises the about 28 moles of GlcNAc/ mole beta polypeptides dimers of about 24-(high molecular weight species).In one embodiment, mixture comprises about 3.6 moles of GalNAc/ mole beta polypeptides dimers of about 2.7-or beta polypeptides molecule.In one embodiment, mixture comprises the about 13 moles of semi-lactosis of about 11-/mole beta polypeptides dimer or beta polypeptides molecule.In one embodiment, mixture comprises the about 7.0 moles of Fucoses of about 6.4-/mole beta polypeptides dimer or beta polypeptides molecule.In one embodiment, mixture comprises the about 16 moles of seminoses of about 14-/mole beta polypeptides dimer or beta polypeptides molecule.The present invention also provides pharmaceutical kit, and it comprises: the container that (a) comprises freeze dried beta polypeptides mixture of the present invention; (b) be used for freeze dried beta polypeptides mixture is reconstituted the specification sheets of the solution that is used to inject.
The methods of treatment of illustrative: be used for suppressor T cell propagation, activation or both methods, described method comprises makes the T cell contact with the beta polypeptides composition of the present invention of significant quantity.The invention provides the method for the immunne response that is used for suppressing the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the immune disorders among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides and be used for inducing the method for experimenter at antigenic immunological tolerance, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.Present method provides the method that is used for the treatment of the inflammation among the experimenter, and described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.Present method has supplied to be used for the treatment of the method for rheumatoid arthritis, and described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the psoriasic method that is used for the treatment of among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the lupus among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides and be used for the treatment of or prevent allergic method among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of or prevents the graft versus host disease (GVH disease) among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of or prevents the transplant organ among the experimenter to repel, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of or prevents the transplanted tissue among the experimenter to repel, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of or prevents the transplanted cells among the experimenter to repel, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.In one embodiment, transplanted cells is a medullary cell.In another embodiment, transplanted cells is an islet cells.In another embodiment, transplanted cells is the insulin production islet cells.The invention provides the method that is used for the treatment of the multiple sclerosis among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method for the CrohnShi disease that is used for the treatment of among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the type i diabetes among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the inflammatory bowel among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the ovaritis among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the brightic method that is used for the treatment of among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the allergic encephalitis among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.The invention provides the method that is used for the treatment of the myasthenia gravis among the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged.
The invention provides beta polypeptides or beta polypeptides molecule colony are used for the treating and/or preventing property processing of immune disorders in preparation the purposes of medicine, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said colony has the sialic acids groups of about 5-about 10 and the molar average ratio of beta polypeptides dimer or beta polypeptides molecule.The invention provides beta polypeptides or the beta polypeptides molecule colony purposes in the agent of preparation resisting rheumatoid arthritis, wherein every peptide species comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said colony has the sialic acids groups of about 5-about 10 and the molar average ratio of beta polypeptides dimer or beta polypeptides molecule, described resisting rheumatoid arthritis agent together with about the specification sheets of its purposes in rheumatoid arthritis treatment in packing.In one embodiment, colony has the sialic acids groups of about 5.5-about 8.5 and the molar average ratio of beta polypeptides dimer or beta polypeptides molecule.
The combination treatment of illustrative: the invention provides and be used for suppressor T cell propagation, activation or both methods, described method comprises makes the T cell contact with the beta polypeptides composition of the present invention of significant quantity, and described composition and methotrexate make up.The invention provides the method for the immunne response that is used for suppressing the experimenter, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged, described composition and methotrexate combination.The invention provides and be used for inducing the method for experimenter at antigenic immunological tolerance, described method comprises the beta polypeptides composition of the present invention of using significant quantity to the experimenter that these needs are arranged, described composition and methotrexate combination.
The accompanying drawing summary
Figure 1A-1B has presented about the nucleotide sequence of the part of the expression cassette of CTLA4-Ig molecule (SEQ ID NO:1).Also shown aminoacid sequence (SEQ ID NO:2) by this nucleic acid encoding.The molecule that can comprise aminoacid sequence: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 26-382, (the v) 25-382 of SEQ ID NO:2 and (the vi) 25-383 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 by the CTLA4-Ig molecule of this expression cassette production with following residue.Expression cassette comprises following zone: (a) oncostatin M signal sequence (the Nucleotide 11-88 of SEQ ID NO:1; The amino acid/11-26 of SEQ ID NO:2); (b) ectodomain of human CTLA 4 (the Nucleotide 89-463 of SEQ ID NO:1; The amino acid 27-151 of SEQ ID NO:2); (c) the modified part of human IgG1's constant region (the Nucleotide 464-1159 of SEQ ID NO:1; The amino acid/11 52-383 of SEQ ID NO:2), comprise modified hinge area (the Nucleotide 464-508 of SEQ ID NO:1; The amino acid/11 52-166 of SEQ ID NO:2), modified human IgG1 C H2 structural domains (the Nucleotide 509-838 of SEQ ID NO:1; The amino acid/11 67-276 of SEQ ID NO:2) and human IgG1 C H3 structural domains (the Nucleotide 839-1159 of SEQ ID NO:1; The amino acid 277-383 of SEQ ID NO:2).
Fig. 2 has presented corresponding CTLA4 A29YL104EThe nucleic acid of-Ig (top row) and amino acid (bottom line) sequence.The amino acid that aminoacid sequence comprises from the sequence shown in Fig. 1 changes, wherein with the sort of the comparing of SEQ ID NO:2, described variation is located at the 29th (A to Y) and the 104th (L to E), wherein methionine(Met) (M) beginning of the numbering of amino-acid residue from being labeled as "+1 ".CTLA4 A29YL104EThe nucleotide sequence of-Ig is shown among this figure, and the A that locates from the 79th (that is, under M by the position of "+1 " mark) begins up at the A at nucleotide position 1149 places (SEQ ID NO:3).Especially, coding CTLA4 A29YL104EThe nucleotide sequence of-Ig is from the Nucleotide at 1149 places of Nucleotide to the of the 79th, called after SEQ ID NO:3.Be shown in the full length nucleotide sequence called after SEQ ID NO:23 among Fig. 2, and comprise the nucleotide sequence of coding oncostatin M signal peptide.
Fig. 3 has presented the CTLA4 that comprises oncostatin M presequence (referring to the runic italics) A29YL104EThe aminoacid sequence of-Ig molecule (SEQ ID NO:4).That can produce is CTLA4 A29YL104EThe polypeptide of-Ig molecule comprises the molecule of the aminoacid sequence with following residue: (i) 26-383 of SEQ ID NO:4, (ii) 26-382, (iii) 27-383, (iv) 26-382, (the v) 25-382 of SEQID NO:4 and (the vi) 25-383 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4.
Fig. 4 is the CTLA4 that 2 amino-acid substitutions (L104E and A29Y) of carrying out in N linked glycosylation site (N76, N108 and N207), C120-C120 disulfide linkage and CTLA-4 structural domain show A29YL104E-Ig model.
Fig. 5 represents CTLA4 A29YL104EThe theoretical cDNA deutero-aminoacid sequence (SEQID NO:4) of-Ig.2 amino-acid substitutions carry out (L104E and A29Y) in the CTLA-4 ectodomain, to produce CTLA4 A29YL104E-Ig.The signal peptide of this Sequence Identification oncostatin M (presequence) is together with N linked glycosylation site.
Fig. 6 describes CTLA4 A29YL104EThe bonded graphic representation of the anti-human IgG Fc of-Ig sample and goat antibody.CTLA4 A29YL104EThe combination of-Ig sample is measured replying of obtaining and is detected by comparing with the sensor chip surface of unmodified on this surface.3 kinds of different CTLA4 of various batches of expressions A29YL104E-Ig sample.
Fig. 7 is the graphic representation that shows apparent molecular weight, described apparent molecular weight is corresponding to polymer, the tetramer or the dimer fraction of removing peak (cleaning peak) as the CTLA4-Ig HIC that passes through 2 post SEC overlay measurements, and described 2 post SEC cover with the retention time on dynamic light scattering detection (DSL) and the SEC.
Fig. 8 A (top) and 8B (bottom) show from HIC and remove the representative IEF gel that separates the peak with the fraction of the glycosylation CTLA4-Ig molecule (comprising SEQ ID NO:2 monomer) of purifying.The application of sample of top gel is in proper order: swimming lane 1, pI mark (Amersham); Swimming lane 2, CTLA4-Ig dimer standard; Swimming lane 3, A albumen eluate; Swimming lane 4, polymer; Swimming lane 5, the tetramer; Swimming lane 6, dimer.The application of sample of bottom gel is in proper order: swimming lane 1, pI mark (Amersham); Swimming lane 2, swimming lane 2, CTLA4-Ig dimer standard; Swimming lane 3, the tetramer; Swimming lane 4, the dissociated tetramer.This little figure shows that the tetramer is still less more sialylated than dimer.
Fig. 9 is presented at the relative quantity that comprises observed dominant carbohydrate structure and carbohydrate on the monomeric CTLA4-Ig dimer of SEQ ID NO:2.Amino-acid residue numbering among this figure is inconsistent with SEQ ID NO:2.In order to make the amino-acid residue numbering among this figure consistent with SEQID NO:2, numbering needs to increase by 26, i.e. N 76Be N 102
Figure 10 has a mind to leave a blank
Figure 11 demonstration comprises the SEQ ID NO:2 dimeric representative IEF gel of monomeric CTLA4-Ig (pH4.0-6.5 ).Swimming lane 1 and 5 shows calibration criterion, and swimming lane 2,3,4 shows 20 μ g/ μ l CTLA4-Ig dimers separately.
Figure 12 demonstration comprises the monomeric CTLA4 of SEQ ID NO:4 A29YL104EThe dimeric representative IEF gel of-Ig (pH4.0-6.5 ).Swimming lane 1 and 8 shows calibration criterion, and swimming lane 2-7 shows 10 μ g/ μ l CTLA4 separately A29YL104E-Ig dimer.
Figure 13 shows the N connection carbohydrate overview of the monomeric CTLA4-Ig molecule colony comprise SEQ ID NO:2.Use LC/MS PGC N connection oligosaccharides technology to collect carbohydrate and separation from glycopeptide.Chromatogram provides the colony's overview about each N connection attachment site.A) from the Asn of T5 peptide 76(the Asn of SEQ ID NO:2 102) carbohydrate and B) from the Asn of T7 peptide 108(the Asn of SEQ ID NO:2 134) carbohydrate, all be presented at the distribution in the single and how sialylated kind.C) from the Asn of T14 peptide 207(the Asn of SEQ ID NO:2 233) form by the kind of the ground asialoization of preponderating.D) show the distribution that joins carbohydrate about the N of CTLA4-Ig molecule.E) show main peak from the selected original spectrum of T5 peptide corresponding to the structure of described pair of feeler mono-sialylated.F) take off the main peak of sialic structure from the selected original spectrum demonstration of T14 corresponding to two feelers.G) the less important kind of the peak co-elute of selected original spectrum by with 64.23 minutes the time is formed, and described less important kind is corresponding to the structure of three feeler double-sialylated.Main kind in peak when H) selected original spectrum is disclosed in 64.23 minutes, described main kind is corresponding to the structure of two feeler double-sialylated.
Figure 14 A-B is presented at UV and the TIC arteries and veins mark that joins the oligosaccharides overview under the acid elution requirement (0.05% TFA) from the monomeric N of the CTLA4-Ig SEQ ID NO:2 of PGC chromatography.The arteries and veins mark of Figure 14 A is presented under the acid elution requirement (0.05% TFA) the negatively charged ion grand total (TIC) about the PGC chromatogram.The arteries and veins mark of Figure 14 B is presented under the acid elution requirement (0.05% TFA) about the PGC chromatogram at the UV at 206nm place arteries and veins mark.
Figure 15 A-B is presented at alkaline elution requirement (0.4%NH 4OH) UV and the TIC arteries and veins mark that join the oligosaccharides overview under from the monomeric N of the CTLA4-Ig SEQ ID NO:2 of PGC chromatography.The arteries and veins mark of Figure 15 A is presented at alkaline elution requirement (0.4%NH 4OH) under about the negatively charged ion grand total (TIC) of PGC chromatogram.The arteries and veins mark of Figure 15 B is presented at alkaline elution requirement (0.4%NH 4OH) under about the PGC chromatogram at the UV at 206nm place arteries and veins mark.
Figure 16 represents to comprise the CTLA4 of SEQ ID NO:4 A29YL104EThe comparison N connection oligosaccharides carbohydrate overview of-Ig molecule.Observe 4 kinds of oligosaccharide structure territories: structural domain I comprises the kind of asialoization, and domain II, III and IV comprise mono-sialylated, double-sialylated and three sialylated kinds respectively.With the chromatography mode separate oligosaccharides and by mass spectroscopy to its structural domain that carried out assay determination.
Figure 17 shows the HPAEC-PAD overview of the N connection oligosaccharides that comprises the monomeric CTLA4-Ig molecule of SEQ ID NO:2.Structural domain is to show about the cumulative order of the sialic acid content of oligosaccharides.Structural domain I, II, III and IV comprise respectively and have 0,1,2 and 3 sialic oligosaccharide structure.The peak mark is represented the specified oligosaccharide structure of HPAEC-PAD profile analysis by the peak of collecting from the PGC profile analysis.The structure of carbohydrate structure is identified consistent with previous mensuration.
Figure 18 A-B shows the PGC overview that comprises the monomeric CTLA4-Ig molecule of SEQ ID NO:2.This overview derives from the direct injection of the carbohydrate digestion mixture of preparation as described in example 3 above.Direct injection causes the detection of the structure P4144 of wash-out in the time of 130 minutes.Four sialylated structure P4144 do not observe in the isolating oligosaccharides overview before injection.
Figure 19 presents about the segmental LC/MS of the monomeric T9 of SEQ ID NO:2 and goes the positive electrospray spectrum of flatung (deconvoluted).This spectrum illustrates 3 kinds of main O and connects structure.This spectrum for example understands to have and O connection structure (GalNAc) 1(Gal) 1(NeuAc) 1The basic peptide of consistent sugar ladder.With respect to the non-bolded section of this spectrum, the bolded section of this spectrum has strengthened 10 times, and for example understands to have (GalNAc) 1(Gal) 1(NeuAc) 2(GalNAc) 1(GlcNAc) 1(Gal) 2(NeuAc) 22 kinds of other O connect structures.
Figure 20 shows the attachment point and relative colony of the O connection carbohydrate structure of the CTLA4-Ig strand with SEQ ID NO:2 sequence monomer.Relative quantity on each site shows the data that produced by 2 kinds or more quadrature technique and implements variability.The position of covalency cysteinylization is also described.
The figure of interstitial granules piLN-huCTLA4-Ig during Figure 21 describes.This plasmid has comprised the sequence (that is SEQ ID NO:1) of the human CTLA 4 of can encoding-Ig molecule (huCTLA4-Ig) of side joint restriction enzyme sites HindIII and XbaI.
Figure 22 describes the figure of plasmid pD16LEA29Y.This plasmid comprises the human CTLA 4 of can encoding A29YL104EThe sequence of-Ig molecule (that is SEQ ID NO:4).
Figure 23 is the photo of the southern blotting technique of the DNA that extracts from Chinese hamster ovary celI, and described expressing cho cell is derived from 1D5-100A1 (for example, clone's 17) CTLA4-Ig expression cassette.The gel swimming lane from left to right is: swimming lane M, dna molecular amount mark; Swimming lane N, the CHO DNA (5mg) of the untransfected of EcoRI/XbaI digestion; Swimming lane 1, CHO DNA (2.5 μ the g)+1ng pcSDhuCTLA4-Ig of the untransfected of EcoRI/XbaI digestion; Swimming lane 2, CHO DNA (2.5 μ the g)+0.5ng pcSDhuCTLA4-Ig of the untransfected of EcoRI/XbaI digestion; Swimming lane 3, CHO DNA (2.5 μ the g)+0.25ngpcSDhuCTLA4-Ig of the untransfected of EcoRI/XbaI digestion; Swimming lane 4, CHO DNA (2.5 μ the g)+0.125ng pcSDhuCTLA4-Ig of the untransfected of EcoRI/XbaI digestion; Swimming lane 5, CHO DNA (2.5 μ the g)+0.0625ng pcSDhuCTLA4-Ig of the untransfected of EcoRI/XbaI digestion; Swimming lane 6, CHO DNA (2.5 μ the g)+0.03125ng pcSDhuCTLA4-Ig of the untransfected of EcoRI/XbaI digestion; Swimming lane 7, the DNA:MCB (5.0 μ g) of EcoRI/XbaI digestion; Swimming lane 8, the DNA:EPCB lot number C20030618A-01 (5.0 μ g) of EcoRI/XbaI digestion; Swimming lane 9, the DNA:EPCB lot number C20030712A-01 (5.0 μ g) of EcoRI/XbaI digestion; Swimming lane 10, the DNA:EPCB lot number C20030801A-01 (5.0 μ g) of EcoRI/XbaI digestion; Swimming lane 11, the DNA:MCB (2.5 μ g) of EcoRI/XbaI digestion; Swimming lane 12, the DNA:EPCB lot number C20030618A-01 (2.5 μ g) of EcoRI/XbaI digestion; Swimming lane 13, the DNA:EPCB lot number C20030712A-01 (2.5 μ g) of EcoRI/XbaI digestion; Swimming lane 14, the DNA:EPCB lot number C20030801A-01 (2.5 μ g) of EcoRI/XbaI digestion; Swimming lane 15, the DNA:MCB (1.25 μ g) of EcoRI/XbaI digestion; Swimming lane 16, the DNA:EPCB lot number C20030618A-01 (1.25 μ g) of EcoRI/XbaI digestion; Swimming lane 17, the DNA:EPCB lot number C20030712A-01 (1.25 μ g) of EcoRI/XbaI digestion; Swimming lane 18, the DNA:EPCB lot number C20030801A-01 (1.25 μ g) of EcoRI/XbaI digestion.
Figure 24 describes the schema of production-scale cultural method.This method allows 25, and 000-L produces mass production recombinant protein in the bio-reactor.
Figure 25 shows the representative chromatography spectrum of NGNA and NANA system suitability criterion.Peak in the time of～9.7 minutes is NGNA, and the peak in the time of～10.7 minutes is NANA.
Figure 26 shows the representative chromatography spectrum of the CTLA4-Ig molecule that comprises the monomeric hydrolysis of SEQ ID NO:2.Peak in the time of～8.4 minutes is a solvent peak.Peak in the time of～9.6 minutes is NGNA.Peak in the time of～10.5 minutes is NANA.Peak in the time of～11.3 minutes is the NANA of degraded, is produced by hydrolysising condition.The planimeter array of the NANA of NANA and degraded is share in calculating the NANA mol ratio.
Figure 27 A, 27B and 27C show the MALDI spectrum of the peptide of CTLA4-Ig cysteinylization.Acquisition is for the Cys that comprises SEQ ID NO:2 146The MALDI spectrum of CTLA4-Ig trypsinase/chymotrypsin fragment.Figure 27 A shows the strand peptide spectrum that illustrates the cysteinyl modification.Figure 27 B shows the spectrum of the strand peptide after the reduction, and confirms to be modified at Cys 146The place takes place.Figure 27 C shows the alkanisation of reductive strand peptide, and this confirms that cysteinylization is at Cys 146The place takes place.
Figure 28 presents the clone's scheme that is used to produce carrier pcSD.PcDNA3 digests with restriction enzyme NaeI, comprises the 3.821Kb fragment of the replication orgin of CMV promotor, ampicillin resistance gene and intestinal bacteria (E.coli) with separation.PSV2-dhfr digests with restriction enzyme PvuII and BamHI, comprises the 1.93Kb fragment of SV40 promotor and dhfr gene with separation, and carry out flush endization subsequently.In order to produce pcSD, 2 fragments are connected.The figure of plasmid pcSD is shown in the bottom of this figure.
Figure 29 presents the clone's scheme that is used to produce expression vector pcSDhuCTLA4-Ig.PcSD digests with restriction enzyme EcoRV and XbaI.PiLN-huCTLA4-Ig digests with restriction enzyme HindIII, flush endization, and digest with restriction enzyme XbaI subsequently, to separate the 1.2KbhuCTLA4-Ig fragment.In order to produce pcSDhuCTLA4-Ig, the CTLA4-Ig fragment is connected to the pcSD carrier of digestion.The figure of plasmid pcSDhuCTLA4-Ig is shown in the bottom of this figure.With this plasmid linearization and transfection in the Chinese hamster ovary celI that does not contain functional dhfr gene.Because plasmid comprises functional dhfr gene, stable transfectant can be selected based on cell survival.PcSDhuCTLA4-Ig has and comprises following expression cassette: the sequence (that is SEQ ID NO:1) of CMV promotor, the human CTLA 4 of can encoding-Ig molecule (huCTLA4-Ig) and from the polyadenylic acid tailer sequence of BGH.
Figure 30 show be described as relative fluorescence unit (RFU) to the time (minute) the electrophorogram of the amino monose of system's suitability.
Figure 31 show be described as relative fluorescence unit (RFU) to the time (minute) the electrophorogram of the neutral monose of system's suitability.
Figure 32 represents to have the CTLA4 of the peptide of mark A29YL104EThe tryptic peptide figure of-Ig.Table 23 is corresponding with the peptide of mark.
Figure 33 shows CTLA4 A29YL104EThe RNA hybridization analysis of-Ig.Little figure A describes the painted sepharose of bromination second pyridine, and wherein swimming lane M is the RNA mark; Swimming lane 1 is total CHO RNA; Swimming lane 2 is total MCB RNA; With swimming lane 3 are total EPCB RNA.Little figure B is corresponding autoradiogram(ARGM), and wherein swimming lane M is the RNA mark; Swimming lane 1 is total CHO RNA; Swimming lane 2 is total MCB RNA; With swimming lane 3 are total EPCB RNA.
Figure 34 A-C describes the size exclusion chromatography spectrum, and it makes CTLA4 A29YL104E-Ig dimer distinguishes with high and low molecular weight species.
Figure 35 shows the CTLA4 with Coomassie blue stain A29YL104EThe SDS-PAGE of-Ig (reduction and non-reduced type) analyzes.Swimming lane 1 molecular weight marker application of sample; Swimming lane 2,7 and 12 is blank; Swimming lane 3-6 is CTLA4 A29YL104E-Ig sample (reduced form); Swimming lane 8-11 is CTLA4 A29YL104E-Ig sample (non-reduced type).
Figure 36 shows the silver-colored painted CTLA4 of enforcement A29YL104EThe SDS-PAGE of-Ig (reduction and non-reduced type) analyzes.Swimming lane 1 molecular weight marker application of sample; Swimming lane 2,7 and 12 is blank; Swimming lane 3-6 is CTLA4 A29YL104E-Ig sample (reduced form); Swimming lane 8-11 is CTLA4 A29YL104E-Ig sample (non-reduced type).
Figure 37 describes to use the non-reduced CTLA4 of trypsinase and Quimotrase combination digestion A29YL104EThe peptide figure of-Ig.
Figure 38 describes to use the non-reduced CTLA4 of trypsinase and elastoser combination digestion A29YL104EThe peptide figure of-Ig.
Figure 39 is a chart of describing to have the patient that anti-CTLA 4-Ig or anti-CTLA-4 reply.As described in example 32 above, use measuring A and B measures at complete CTLA4-Ig molecule (CTLA-4 and Ig part) and CTLA-4 antibody response partly only.
Figure 40 confirms that clearance rate by the immunogenicity state distributes and the synoptic diagram of the volume of central compartment.
Figure 41 is the graphic representation that confirms in the monkey of using the 10mg/kg medicine of producing by method of the present invention along with the overview of average (SD) CTLA4-Ig serum-concentration in past time.
Figure 42 is the graphic representation of size exclusion chromatography (SEC) chromatogram of the A albumen (MAbSelect) of purifying from the CTLA4-Ig material of contrast and depolymerization.
Figure 43 is the graphic representation of material that depolymerization the is handled N glycan analysis of (ii) comparing with contrast (i).
Figure 44 describes the graphic representation of average CTLA4-Ig serum-concentration [μ g/ml] to time (through 71 days).
Figure 45 show be depicted as relative fluorescence unit (RFU) to the time (minute) the electrophorogram of neutral monose.
Figure 46 show be depicted as relative fluorescence unit (RFU) to the time (minute) the electrophorogram of amino monose.
Figure 47 represents about comprising the CTLA4 of SEQ ID NO:4 A29YL104EThe comparison N connection oligosaccharides carbohydrate overview of-Ig molecule.Observe 4 kinds of oligosaccharide structure territories, wherein structural domain I comprises non-sialylated kind, and domain II, III and IV comprise mono-sialylated, double-sialylated and three sialylated kinds respectively.
Figure 48 is and CTLA4 A29YL104E-Ig1: the graphic representation of the capillary electrophoresis separation of 1 blended CTLA4-Ig.The main peak transition time is about 0.8 minute at interval.
Figure 49 is the CTLA4 of hydrolysis A29YL104EThe chromatogram of-Ig material, wherein the NANA peak was observed in the time of 3.4 minutes.
Figure 50 is depicted in several in the various N connection carbohydrate structures of finding in the mammalian proteins matter.All chains are shared the common core structure that comprises 2 GlcNAc and 3 mannose residues.
Figure 51 is a graphic representation of describing to expose as the CTLA4-Ig of the sialylated function of glycoprotein (NANA than) (AUC).
Figure 52 is a graphic representation of describing to expose as the CTLA4-Ig of the carbohydrate overview function of CTLA4-Ig (AUC).A large amount of peaks are produced by anionresin HPLC, and it resolves into 4 kinds or 5 kinds of structural domains. Structural domain 1 and 2 mainly is the structure of asialoization and mono-sialylated, and structural domain 3 and 4 mainly is two and three sialylated structures.
Figure 53 represents to have the CTLA4 of the peptide of mark A29YL104EThe tryptic peptide figure of-Ig.Table 56 is corresponding with the peptide of mark.
Figure 54 represents the comparison N connection oligosaccharides carbohydrate overview about the CTLA4-Ig molecule that comprises SEQ ID NO:2.Observe 4 kinds of oligosaccharide structure territories, wherein structural domain I comprises non-sialylated kind, and domain II, III and IV comprise mono-sialylated, double-sialylated and three sialylated kinds respectively.
Figure 55 A-D is the CTLA4-Ig of expression by HPAEC-PAD and the graphic representation of the oligosaccharides overview of peptide T5, T7 and T14.
Figure 56 is the graphic representation of oligosaccharides overview of mark of describing to derive from the CTLA4-Ig of PGC (Hypercarb) post.
Figure 57 describes the graphic representation of pharmacokinetic data, be presented on the Y-axis monkey AUC and as from the structural domain I of the carbohydrate overview on the X-axis and the per-cent of the N linked glycosylation as shown in the II.Referring to for example measuring the method that N joins the carbohydrate overview among the embodiment 3,44,22 and 37.Along with the per-cent increase (and the per-cent of domain II I, IV and V reduces) of structural domain I and II, clearance rate increases.Notice negative control, have low sialic CTLA4-Ig and removed very apace.Should be understood that the CTLA4-Ig variant, LEA (CTLA4-Ig A29YL104E-Ig) be included in this graphic representation.
The arteries and veins mark of the N connection carbohydrate chromatogram of N that Figure 58 A and 58B describe to discharge from CTLA4-Ig connection carbohydrate (as derive among the embodiment 3,44,22 and 37 for example those methods).Arteries and veins mark among Figure 58 A comes the comfortable analysis of not adding the CTLA4-Ig that produces in the culture method of other semi-lactosi in culture.Arteries and veins mark among Figure 58 B has added semi-lactosi really in culture.The percentages show of the N connection carbohydrate in each structural domain is in inserting table.
The arteries and veins mark of the N connection carbohydrate chromatogram of N that Figure 59 describes to discharge from CTLA4-Ig connection carbohydrate (as derive among the embodiment 3,44,22 and 37 for example those methods).When coming comfortable the 8th day, this in substratum, adds the analysis of the CTLA4-Ig that produces in the culture method of semi-lactosi.This arteries and veins mark comes the comfortable analysis of not adding the CTLA4-Ig that produces in the culture method of other semi-lactosi in culture.The percentages show of the N connection carbohydrate in each structural domain is in inserting table.
The arteries and veins mark of the N connection carbohydrate chromatogram of N that Figure 60 describes to discharge from CTLA4-Ig connection carbohydrate (as derive among the embodiment 3,44,22 and 37 for example those methods).When coming comfortable the 14th day, this in culture, adds the analysis of the CTLA4-Ig that produces in the culture method of semi-lactosi.This arteries and veins mark comes the comfortable analysis of not adding the CTLA4-Ig that produces in the culture method of other semi-lactosi in culture.The percentages show of the N connection carbohydrate in each structural domain is in inserting table.
The arteries and veins mark of the N connection carbohydrate chromatogram of N that Figure 61 A and 61B describe to discharge from CTLA4-Ig connection carbohydrate (as derive among the embodiment 3,44,22 and 37 for example those methods).Figure 61 A comes the comfortable analysis of not adding the CTLA4-Ig that produces in the culture method of semi-lactosi, and Figure 61 B is from the analysis of wherein adding semi-lactosi the 14th day the time in culture.This arteries and veins mark comes the comfortable analysis of not adding the CTLA4-Ig that produces in the culture method of other semi-lactosi in culture.The percentages show of the N connection carbohydrate in each structural domain is in inserting table.
The arteries and veins mark of the N connection carbohydrate chromatogram of N that Figure 62 describes to discharge from CTLA4-Ig connection carbohydrate (as derive among the embodiment 3,44,22 and 37 for example those methods).This arteries and veins mark derives from the CTLA4-Ig material that reclaims from the washing step of QFF post, produce the part (cut) with low sialic CTLA4-Ig material.Compare with the arteries and veins mark that shows among Figure 61,60 and 59, the relative quantity of structural domain I and II increases, and domain II I and Iv minimizing.The percentages show of the N connection carbohydrate in each structural domain is in inserting table.
Figure 63 shows the tryptic peptide figure of CTLA4-Ig, points out T8 wash-out when the anterior end of solvent, and T9 wash-out when the T27 shoulder.
Figure 64 is that expression is corresponding to the complete mass spectral graphic representation from the glycopeptide T8 of CTLA4-Ig.
Figure 65 is that expression is corresponding to the complete mass spectral graphic representation from the glycopeptide T9 of CTLA4-Ig.
Figure 66 is expression corresponding to the graphic representation from the MALDI-TOF data of the T9 peptide fragment of CTLA4-Ig.
Figure 67 A-B has described ion chromatography spectrum and the mass spectrum from the oxidation of CTLA4-Ig and natural tryptic peptide.
Figure 68 has described general N connection oligosaccharides overview (structural domain I, II, III, IV and V, and peak 1A and 1B in 5% batch mean value). Peak 1A, 1B and 1C represent that the sialic acid N that takes off of G0, G1 and G2 joins oligosaccharide structure.About the data of this overview under table in.Referring to embodiment 44.
The peak title RT Area The % area
1 Structural domain I 19.413 47807873 31.3
2 Domain II 29.076 50746179 33.2
3 Domain II I 42.819 36640805 24.0
4 Structural domain V 67.546 3421324 2.2
5 Structural domain IV 55.899 14331509 9.4
6 Peak 1A 19.413 11115168 7.3
7 Peak 1B 20.290 16331761 10.7
8 Peak 1C 21.032 13507144 8.8
9 Peak 2 21.925 4285962 2.8
10 22.685 2567838 1.7
11 29.076 2808537 1.8
12 30.763 27989176 18.3
13 31.577 19948466 13.0
14 42.819 4555254 3.0
15 Peak 3 43.823 22213064 14.5
16 46.626 9872487 6.5
17 55.899 3898179 2.5
18 Peak 4 57.368 6789516 4.4
19 60.333 3643813 2.4
20 67.546 3421324 2.2
Figure 69 describes the isoelectrofocusing gel of CTLA4-Ig.Band is characterised in that:
Swimming lane Describe Protein application of sample (microgram) The band numbering Accumulation % band intensity Relative band per-cent (%)
1 The IEF mark NA NA NA NA
2 The CTLA4-Ig material 20 16 100 NA
3 The CTLA4-Ig medicine 20 16 100 100
4 The dyeing contrast 1 NA NA NA
Figure 70 describes the representative isoelectrofocusing gel quantitative analysis report of CTLA4-Ig.Quantitative and the data of carrying out gel are as follows:
Figure A200680053116D00741
Figure 71 A is depicted in the general 20 μ L injection of the system's suitability criterion on the TOSO HAAS 3000 SWXL posts that are equipped with guard column.Figure 71 B is depicted in CTLA4-Ig on the TOSO HAAS3000 SWXL post that is equipped with guard column with reference to the 20 μ L injection of material.
Figure 72-CTLA4-Ig obtains the example of image by the numeral of the SDS-PAGE analysis of polyacrylamide (4-20) gel electrophoresis of Coomassie blue stain
Swimming lane Describe Protein application of sample (microgram) Irreducibility (NR)/reductibility (R) condition Per-cent (%) band intensity
1 The CTLA4-Ig medicine 10 NR 100
2 The CTLA4-Ig material 10 NR 99
3 Blank NA NR NA
4 The CTLA4-Ig medicine 10 R 100
?5 The CTLA4-Ig material 10 ?R 100
6 Molecular weight marker NA NA NA
7 The CTLA4-Ig medicament production 10 NR 99
8 The CTLA4-Ig material 10 NR 100
9 Blank NA NR NA
10 The CTLA4-Ig medicament production 10 R 99
11 The CTLA4-Ig material 10 R 99
12 Trypsin inhibitor dyeing contrast NA NA NA
Figure 73 describes the example about the quantitative analysis report of the SDS-PAGE of Coomassie blue stain.
Figure 74 shows the table of setting forth the quantitative analysis of painted SDS-PAGE gel among Figure 73.
Figure 75 describes the example of the enhancing image that the SDS-PAGE of the gel of CTLA4-Ig Coomassie blue stain analyzes, and is used to illustrate the migration position with respect to the minor band of the main and expection of molecular weight marker.
Figure 76 is the description for property N connection carbohydrate overview of CTLA4-Ig reference/standard material.This is the representative carbohydrate overview of moving in the Waters system.Retention time is that system is dependent.
Figure 77 is the description of representing stachyose system suitability chromatogram.
Figure 78 is the arteries and veins mark that the representative chromatography of the CTLA4-Ig material of hydrolysis is composed.
Figure 79 describes CTLA4 A29YL104EScanning (ccanned) gel images that the SDS-PAGE of the polyacrylamide of-Ig Coomassie blue stain (4-20%) gel electrophoresis Coomassie blue stain analyzes.
Figure A200680053116D00751
Figure 80 describes to gather in the crops the schema of step, referring to embodiment 28.
Figure 81 describes the electrophorogram of system's suitability of amino monose.Referring to embodiment 16.
Figure 82 describes the graphic representation of pharmacokinetic data, be presented on the Y-axis monkey AUC and as from the structural domain I of the carbohydrate overview on the X-axis and the per-cent of the N linked glycosylation as shown in the II.Referring to for example measuring the method that N joins the carbohydrate overview among the embodiment 3,44,22 and 37.Along with the per-cent increase (and the per-cent of domain II I, IV and V reduces) of structural domain I and II, AUC increases.Notice negative control, have low sialic CTLA4-Ig and removed very apace.Should be understood that mutant CTLA4-Ig molecule, CTLA4-Ig A29YL104E-Ig (called after LEA) is included in this graphic representation.
Figure 83 describes the graphic representation of pharmacokinetic data, is presented at the AUC on the Y-axis and the per-cent of the N linked glycosylation as shown in the domain II I on the X-axis and IV (as being measured by N connection carbohydrate overview).Along with the per-cent increase of domain II I and IV, AUC increases.Notice negative control, have low sialic CTLA4-Ig and removed very apace.Referring to for example measuring the method that N joins the carbohydrate overview among the embodiment 3,44,22 and 37.Should be understood that the CTLA4-Ig molecule of sudden change, CTLA4-Ig A29YL104E-Ig (called after LEA) is included in this graphic representation.
Figure 84 describes another graphic representation of pharmacokinetic data, be presented on the Y-axis AUC and as per-cent from the domain II I of the carbohydrate overview on the X-axis and the N linked glycosylation as shown in the IV.Along with the per-cent increase of domain II I and IV, AUC increases.Notice negative control, have low sialic CTLA4-Ig and removed very apace.Referring to for example measuring the method that N joins the carbohydrate overview among the embodiment 3,44,22 and 37.Should be understood that the CTLA4-Ig molecule of sudden change, CTLA4-Ig A29YL104E-Ig (called after LEA) is included in this graphic representation.
Figure 85 describes the trypsinase figure of CTLA4-Ig standard, distributes referring to the table when embodiment 65 finishes about the peak.The small peak that is labeled as T1+A is the T1 tryptic peptide that extends by the N-terminal alanine residue.The small peak that is labeled as T31+K is the T31 tryptic peptide that extends by the N-terminal lysine residue.
Figure 86 describes about added admixture 5 moles of % T6ox indicator peptides, the covering of trypsinase Figure 28 0nm data of the CTLA4-Ig standard of Met (O) 85 (84-93) of CTLA4-Ig standard.Referring to embodiment 65.
Figure 87 describes about CTLA4-Ig standard added admixture 5 moles of %T26deaml indicator peptides, the enlarged view of the 215nm data of the CTLA4-Ig standard trypsinase figure of isoAsp294 (281-302).Referring to embodiment 65.
Detailed Description Of The Invention
CTLA4Ig can be used for the treatment of various illnesss, comprises the illness such as autoimmunity and the allergy that relate to abnormal immune hyperplasia and immunoreactivity phenomenon. The invention provides and comprise for example CTLA4-Ig composition of CTLA4Ig colony, described CTLA4Ig colony have specific glycosylation modified, have particular carbon hydrate overview or feature, have specific poly structure and/or have specific antibodies parent antigenicity intensity. The same file that is incorporated herein by reference in this integral body of describing CTLA4Ig, its purposes and method comprises U.S. Patent number 5,434,131; 5,851,795; 5,885,796; 5,885,579; With 7,094,874.
The present invention also provides can be via the clone of a large amount of productions provided herein and a large amount of CTLA4Igs of cultivation production. A kind of concrete clone of the present invention is to be used for a large amount of cloned cell lines of producing CTLA4Ig, thereby so that it has specific glycosylation and carbohydrate overview. And have ATCC registration number 68629 (Referring toU.S. Patent number 5,434,131, it is incorporated herein by reference in this integral body) heterogeneous and non-clone cell colony compare, cloned cell line of the present invention can be secreted the CTLA4Ig colony with more consistent or more uniform glycosylation or carbohydrate overview. In addition, compare with the heterogeneous and non-clone cell colony with ATCC registration number 68629, cloned cell line of the present invention can be secreted more substantial CTLA4-Ig molecule, in part because this cloned cell line selects to have the CTLA4-Ig expression cassette in the Single locus in the genome that the high copy number purpose is incorporated into cell.
The invention provides the avidity of following discovery: CTLA4-Ig (SEQ ID NO:2) and render a service and can obtain increasing by carry out 2 amino acid replacements in the B7 land of CTLA-4 binding structural domain: (i) alanine at the 29th of SEQ ID NO:2 the place replaces with tyrosine (A29Y), and (ii) lysine at the 104th place of SEQ ID NO:2 replaces with glutamic acid (L104E). The invention provides the subgenus of the CTLA4Ig that comprises beta polypeptides, be called " beta polypeptides molecule ", it has B7 in conjunction with active and can comprise amino acid sequence (having the CTLA4 ectodomain that A29Y and L104E suddenly change) among the SEQ ID NO:24 that is connected with constant region for immunoglobulin or its part.
[SEQ ID NO：24]
MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI
YVIDPEPCPDSD
[SEQ ID NO:18]-CTLA4 ectodomain
MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI
YVIDPEPCPDSD
Term
As used herein, term " clone " refers to the cell colony by unicellular amplification. With regard to the cloned cell line or clone cell colony that can express CTLA4Ig, cloned cell line or colony be by the unicellular amplification that separates from cell colony, and described cell colony is with the expression vector transfection of coding CTLA4-Ig molecule. The colony of the transfection of cell can be heterogeneous population. The all cells that cloned cell line or colony can be considered as in colony is homogeneity from the meaning of single transfectant.
As used herein, term " B7-1 " refers to CD80; Term " B7-2 " refers to CD86; And term " B7 " refers to any or both among B7-1 and the B7-2 (CD80 and CD86). Term " B7-1-Ig " or " B7-1Ig " refer to CD80-Ig; Term " B7-2-Ig " or " B7-2Ig " refer to CD86-Ig.
As used herein, term " CTLA4-Ig " or " CTLA4Ig " or " CTLA4Ig molecule " or " CTLA4-Ig protein " or " CTLA4Ig protein " are used interchangeably, and refer to comprise at least protein molecule of CTLA4-Ig polypeptide, described CTLA4-Ig polypeptide has CTLA4 ectodomain and constant region for immunoglobulin or its part. In certain embodiments, for example, the CTLA4-Ig polypeptide comprises at least amino acid sequence of SEQ ID NO:18. In certain embodiments, CTLA4 ectodomain and constant region for immunoglobulin or its part can be wild types, or saltant or modification. The CTLA4-Ig polypeptide of sudden change is the CTLA4-Ig polypeptide that comprises the CTLA4 ectodomain of sudden change. The CTLA4Ig of sudden change comprises at least CTLA4-Ig polypeptide of sudden change. In certain embodiments, CTLA4 ectodomain and constant region for immunoglobulin or its part can be mammals, comprise people or mouse. In certain embodiments, the CTLA4 ectodomain of sudden change can have amino acid sequence, and the CTLA4 ectodomain at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% shown in itself and Fig. 1 or the SEQ ID NO:18 is equal to. In certain embodiments, the constant region for immunoglobulin of sudden change or its part can have and as shown in fig. 1 immunoglobulin (Ig) (g) constant region 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence that is equal at least. Polypeptide can further comprise other protein domain. CTLA4Ig can refer to the monomer of CTLA4-Ig polypeptide, and can also refer to the polymer form of polypeptide, such as (or other high molecular weight species) such as dimer, the tetramer and six aggressiveness. CTLA4Ig can also be combined with CD80 and/or CD86. CTLA4Ig comprises the CTLA4Ig of sudden change, for example " beta polypeptides molecule ", for example CTLA4A29YL104E-Ig. For example, CTLA4-Ig comprises CTLA4Ig, and CTLA4A29YL104E-Ig comprises beta polypeptides molecule (example of the CTLA4Ig of sudden change).
As used herein, term " CTLA4 ectodomain " refers to comprise all or part of protein domain of the amino acid sequence shown in the SEQ ID NO:18, itself and B7-1 (CD80) and/or B7-2 (CD86) combination. In certain embodiments, the CTLA4 ectodomain can comprise the polypeptide with amino acid sequence, the amino acid 27-150 at least 75%, 80%, 85%, 90%, 95%, 96%, 97% of described amino acid sequence and SEQ ID NO:2,98% or 99% is equal to, and this is identical with the amino acid shown in the SEQ ID NO:18. The amino acid/11 51 of SEQ ID NO:2 is joint amino acid.
As used herein, the CTLA4-Ig polypeptide that term " beta polypeptides " phalangeal process becomes, its (1) comprises the amino acid sequence of SEQ ID NO:18, wherein the amino acid mutation at the 29th place becomes tyrosine, and the amino acid mutation at the 104th place becomes glutamic acid, optional have various other modifications, and constant region for immunoglobulin or its part; (2) can be combined with CD80 and/or CD86. In certain embodiments, for example, beta polypeptides comprises at least CTLA4A29YL104EThe amino acid sequence of the ectodomain of-Ig (as shown in SEQ ID NO:24). The non-limitative example of beta polypeptides comprises Bei Latasai (belatace β t) and SEQ ID NOS:4 and 11-16. In certain embodiments, constant region for immunoglobulin or its part can be wild type or saltant or modification. In certain embodiments, constant region for immunoglobulin or its part can be mammiferous, comprise people or mouse. The other non-limitative example of beta polypeptides is included in constant region for immunoglobulin or its part and (for example comprises one or more amino acid mutations, the displacement of the cysteine 120 of SEQ ID NO:4) beta polypeptides, and the one or more places in the amino acid position 25,30,93,96,103 or 105 of SEQ ID NO:18 comprise the beta polypeptides of further sudden change. The beta polypeptides molecule comprises beta polypeptides. The beta polypeptides molecule can refer to the polymer form of monomer and the beta polypeptides of beta polypeptides, such as dimer, the tetramer and six aggressiveness etc. For example Bei Latasai comprises the beta polypeptides molecule. The beta polypeptides molecule is further to obtain describing in the U.S. Provisional Application submitted on October 5th, 2006 number 60/849,543, and described application is incorporated herein by reference in this integral body.
As used herein, term " glutamic acid (glutamate) " and " glutamic acid (glutamic acid) " are used interchangeably.
As used herein, term " dimer " refers to CTLA4-Ig protein or the CTLA4Ig by 2 CTLA4-Ig polypeptide that connect or combine or monomer composition. Key between the dimeric monomer can be non-covalent bond or interaction, covalent bond or interaction or both. The dimeric example of CTLA4-Ig is shown among Fig. 4. The CTLA4-Ig protein or the CTLA4Ig that are comprised of 2 same monomer are homodimers. The CTLA4-Ig homodimer also comprises such molecule, its be included in may be slightly different in the sequence 2 monomers. Homodimer comprises the dimer that the monomer that wherein links together has substantially the same sequence. The monomer that consists of homodimer is shared suitable macrostructure homology. For example, the difference in the sequence can be because the terminal processing of the N of monomer is modified.
As used herein, " conservative sudden change " refers to that (for example, a nonpolar amino acid is replaced another, for example isoleucine, valine, leucine or methionine with the change in another the nucleotide sequence of an amino acid replacement identical type; Or a polar amino acid replaces another, arginine displacement lysine for example, glutamic acid displacement aspartic acid or glutamine displacement asparagine).
As used herein, " non-conservative sudden change " refer in different types of another the nucleotide sequence of amino acid replacement change (for example, with an acidic amino acid aspartic acid or glutamic acid displacement basic amino acid, for example lysine, arginine or histidine for example). For example, based on size, electric charge, polarity, reactivity or amino acid whose other this features, amino acid can be different from another amino acid in biochemistry.
As used herein, " separation " refers to from its natural surroundings to take out and is in the molecule that is different from the naturally occurring environment of molecule wherein, from other components of its environment, partly or entirely reclaim or the material that separates (for example, protein), thereby so that material (for example, protein) be the dominant kind that exists in the set of resulting composition, mixture or component (for example, kinds of protein) (for example, it is abundanter than any other individual species in the composition on mole foundation). For example, preparation can by surpass about 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94 or or 95% CTLA4-Ig that separates form. " separation " do not get rid of CTLA4Ig with from the mixture of other CTLA4Igs of the naturally occurring environment of molecule wherein. " separation " do not get rid of the pharmaceutically acceptable excipient with the CTLA4-Ig combination, and wherein CTLA4-Ig reclaims from its environment such as cell culture, batch culture or bioreactor etc. As used herein, " separation " refers to implementation or the CTLA4Ig of method to obtain to separate.
As used herein, term " soluble CTL A 4 " mean the molecule that can circulate in vivo or not with the membrane-bound CTLA4 of cell. For example, soluble CTL A 4 can comprise CTLA4-Ig, and it comprises the extracellular region of the CTLA4 that is connected with Ig.
As used herein, term " soluble fraction of cell culture " refers to be different from or is substantially free of for example liquid part of the cell culture of cell, cell membrane and nuclear of insoluble, the particle of cell culture or solid constituent. Soluble fraction can be the centrifugal rear resulting supernatant of cell culture for example, or cell culture filter after resulting filtrate.
As used herein, term " expression cassette " (for example refers to have at least 5 ' regulatory region of being operably connected with the nucleotide sequence of coded polypeptide, promoter) and randomly the nucleic acid of 3 ' terminator of untranslated (for example, terminator codon and polyadenylation sequence). Under suitable condition, by the polypeptide of expression cassette coding by expression cassette production. Expression cassette can also have one or more nucleotide sequences (for example, referring to the people such as Koduri (2001) Gene 280:87-95) that make in the specific site of expression cassette targeted integration in the genome of host cell. For example, derived from preservation be the CTLA4 of the plasmid of ATCC registration number PTA-2104A29YL104E-Ig polypeptide expression box is coding CTLA4A29YL104EAn example of the expression cassette of-Ig.
As used herein, term " basically purifying " refers to comprise the composition of the CTLA4Ig colony of CTLA4Ig or selection, it (for example takes out from its natural surroundings, separate) and at least 90% do not contain, 91% do not contain, 92% do not contain, 93% do not contain, 94% do not contain, 95 % do not contain, 96% do not contain, 97% do not contain, 98% do not contain, 99% do not contain, 99.5% do not contain or 99.9 % do not contain other components of with it natural combination, for example cell material or culture medium. For example, with regard to the CTLA4-Ig protein molecule of recombinant production, term " basically purifying " can also refer to comprise the composition of CTLA4-Ig protein molecule, it takes out from production environment, thereby so that protein molecule at least 90% does not contain, 91% do not contain, 92% do not contain, 93% do not contain, 94% do not contain, 95% do not contain, 96% do not contain, 97% do not contain, 98% do not contain, 99% do not contain, 99.5% do not contain or 99.9% protein molecule that does not contain the mutant polypeptide that not is the polypeptide of purpose SEQ ID NO:2 or SEQ ID NO:2. " basically purifying " do not get rid of the mixture of CTLA4Ig (for example dimer) and other CTLA4Igs (for example tetramer). " basically purifying " do not get rid of pharmaceutically acceptable excipient or the carrier with the CTLA4Ig combination, and wherein CTLA4Ig takes out from its natural surroundings.
As used herein, term " large-scale methods " can with term " commercial scale method " Alternate. Term " culture vessel " can with " bioreactor ", " reactor " and " groove " Alternate.
" liquid culture " refers to grow at holder, or in liquid nutrient media the cell (for example, bacterium, plant, insect, yeast or zooblast) of suspension growth.
" inoculum " refers to the cell culture of growing for the culture medium that is used for the inoculation more volume. Inoculum can be used for the culture medium of inoculation more volume, with the cell number (for example, the cell of suspension growth) that increases and grow in cultivation.
As used herein, " cultivation " refers to make one or more cell in-vitro growths under restriction or controlled condition. The example of the condition of culture that can limit comprises temperature, admixture of gas, time and culture medium prescription.
As used herein, " amplification " refers to for the cultured cell that obtains big figure more and at one or more cells of in vitro culture.
As used herein, " colony " refers to it is characterized in that one or more can measure or the existence of detectability matter or non-existent 2 kinds or more polymolecular (" molecule colony ") or cell (" cell colony ") group. In homogeneous colony, the molecule in the colony or cell are characterised in that identical or substantially the same character (for example, the cell of cloned cell line). In heterogeneous population, molecule in the colony or cell are characterised in that identical or substantially the same at least a character, wherein cell or molecule can also show not identical character (for example, have basically similar average sialic acid content but have the CTLA4Ig colony of dissimilar mannose content).
As used herein, " HMW gathering thing " can with " high molecular weight species " Alternate, to refer to comprise the CTLA4Ig of at least 3 CTLA4-Ig monomers. For example, HMW gathering thing can be the tetramer, pentamer or six aggressiveness.
" percentage (%) output " refer to actual production divided by theoretical yield and with on duty with 100. Actual production can be used as to restrain or the weight of mole expression (for example, mole output) and providing. Theoretical yield can be used as the output of ideal or mathematical computations and provides.
As used herein, " amount of MCP-1 " refers to the MCP-1 (monocyte chemoattractant protein-1 that (1) is independent, hamster MCP-1 particularly) amount, or the amount of (2) " MCP-1 sample " protein, wherein " MCP-1 sample " protein comprises MCP-1, together with the fragment of the protein of MCP-1 homology, MCP-1 and/or with the fragment of the protein of MCP-1 homology (for example, in each of above-mentioned situation, as measuring cross reaction with for detection of MCP-1 with antibody (for example, polyclone ELISA)). Expected do not exist MCP-1 (and/or with the fragment of the protein of MCP-1 homology, MCP-1 and/or with the fragment of the protein of MCP-1 homology) situation, wherein with regard to the scope of the amount of MCP-1, do not provide lower limit.
As used herein, " glycosylation content " refers to the amount with the N of protein molecule covalent attachment connection or O connection saccharide residue, for example as the glycoprotein of CTLA4Ig.
As used herein, term " mol ratio of sialic acid and protein " calculates and provides as mole number/mole protein (CTLA4Ig) or the dimer of sialic acid molecule.
As used herein, term " glycoprotein " refers to by adding one or more carbohydrate, comprises and adds the protein that one or more saccharide residues are modified.
As used herein, protein showed in term " sialylated ", comprises that glycoprotein adds sialic acid residues.
As used herein, term " glycoprotein isoform " refers to be characterised in that the molecule of its carbohydrate and sialic acid content, as by being used for measuring via isoelectric focusing (IEF) gel electrophoresis of the different proteins of its molecular weight, electric charge and/or other feature differentiation mixtures or other suitable methods. For example, every kind of molecule that different band representatives have specific isoelectric point (pI) and therefore have identical clean total electrical charge observing at the IEF gel. The glycoprotein isoform can be the different bands of observing at the IEF gel, and wherein each band can be the molecule colony with specific pI.
" immune tolerance " refers to that the people is usually to the specific antigen of its response or antigen group's unresponsiveness state (for example, wherein the T cell no longer responds the state of antigen).
" effectiveness " refers to reply the measurement as the ligand concentration function. For example, activator is renderd a service quantitatively for producing half maximum effect (EC50) ligand concentration. The non-limiting pharmacology definition of rendeing a service comprises the component of affinity and effect, and wherein effect is that medicine is in case in conjunction with causing the ability of replying. Render a service relevantly with affinity, but render a service and affinity is pharmaceutically-active different measuring.
As used herein, " pharmaceutically acceptable carrier " refers to the medium for pharmacologically active agents. Carrier promotes bioactive agent delivery to deliver to target site and the function of reagent is stopped. The non-limitative example of the carrier of suitable form comprises solution, emulsifiable paste, gel, gel emulsion, gel, paste, lotion, ointment, spray, ointment, powder, solid mixture, aerosol, emulsion (for example, Water-In-Oil or oil-in-water), gel solution, the aqueous solution, suspension, liniment, tincture and is suitable for the patch of local application.
As used herein, phrase " pharmaceutically acceptable composition " (or " pharmaceutical composition ") refers to for for example giving the human acceptable composition of medicament administration. Except any or multiple actives, this kind composition can be included in the material (this kind level comprises the situation that does not have this kind impurity) of the impurity on the level of the acceptable level that is no more than medicament administration, and can comprise pharmaceutically acceptable excipient, medium, carrier and other non-activity compositions, for example be used for being easy to use to prepare this kind composition. For example, pharmaceutically acceptable CTLA4-Ig composition can comprise MCP-1 or DNA, as long as those materials are for giving the using on the acceptable level of people.
" medicine " is included in the active pharmaceutical ingredient in the pharmaceutical composition. Term " medicine " comprises the active pharmaceutical ingredient with solution and/or buffering form. " drug products " is to comprise the pharmaceutical composition that preparation is used for the medicine of medicament administration. For the mensuration that the embodiment that may relate to medicine and/or drug products and other places of this paper comprise, exemplary medicine and drug products can followingly be measured.
That the concentration that is dissolved in aqueous buffer solution (25mM sodium phosphate, 50mM sodium chloride, pH 7.5) is the CTLA4-Ig protein of 50mg/ml about the exemplary medicine that comprises SEQ ID NO:2,5,6,7,8,9,10 or 18 CTLA4Ig.
CTLA4-Ig protein, 500mg maltose, 17.2mg sodium orthophosphate and the 14.6mg sodium chloride of 250mg freeze-drying about the illustrative drug product that comprises SEQ ID NO:2,5,6,7,8,9,10 or 18 CTLA4Ig molecule, pH7.0-8.0; Or
The composition of the CTLA4-Ig protein of freeze-drying (250mg/ bottle) drug products
 
Component Amount (mg/ bottle)a
CTLA4-Ig protein 262.5
The maltose monohydrate 525
Sodium orthophosphate, monohydrateb 18.1
Sodium chlorideb 15.3
Hydrochloric acid Be adjusted to pH7.5
NaOH Be adjusted to pH7.5
Aqueous buffer solution (25mM sodium phosphate, 10mM sodium chloride, pH7.5).
About comprising the illustrative drug product of SEQ ID NO:4,11,12,13,14,15,16 or 24 CTLA4Ig:
Freeze dried CLTA4 A29YL104EThe composition of-Ig100mg/ bottle medicament production
Component Amount/bottle (mg)
CLTA4 A29YL104E-Ig 110
Sucrose 220
The sodium orthophosphate (monometallic) monohydrate 15.18
Sodium-chlor 2.55
1N sodium hydroxide Be adjusted to pH7.5
1N hydrochloric acid Be adjusted to pH7.5
As used herein, term " substratum " and " cell culture medium " and " charging substratum " and " fermention medium " refer to be used for growth and or keep the nutrient solution of cell, particularly mammalian cell.Unrestrictedly, these solution provide at least a component from one or more following kinds usually: (1) energy is generally for example form of glucose of carbohydrate; (2) all indispensable amino acids, and be generally 20 seed amino acids and add basic group of halfcystine; (3) VITAMIN and/or other organic compound that needs with lower concentration; (4) free fatty acids or lipid, for example linolic acid; (5) trace elements, wherein trace elements is defined as generally with extremely low concentration, is generally mineral compound or naturally occurring element that micro-molar range needs.Nutrient solution can selectivity be added one or more components from any following kind: (1) hormone and other somatomedins are serum, Regular Insulin, transferrin and Urogastron for example; (2) salt, for example magnesium, calcium and phosphoric acid salt; (3) damping fluid, for example HEPES; (4) for example adenosine, thymidine and xanthoglobulin of nucleosides and base; (5) protein and tissue water hydrolysis products for example can derive from the peptone or the peptone mixture of gelatin, vegetable material or the animal byproduct of purifying; (6) microbiotic, for example gentamicin; (7) cytoprotective, for example poly alcohol (pluronic polyol); (8) semi-lactosi.
As used herein, term " inoculation " refers to cell is added in the substratum with initial cultivation.
As used herein, refer to the wherein main splitted index cell growth time section (for example, logarithmic phase) fast of cell " vegetative period " of term cell cultures.In this phase process, the rate of increase in the viable cell density is higher than any other time point.
As used herein, " production phase " phalangeal cell growth fixation of term cell cultures or maintain time period near constant level.The density of viable cell keeps approximately constant in preset time in the section.The growth of logarithm cell stops, and protein production is the primary activity of production period.At this moment general supplemented medium is to support the successive protein production and to reach the desired sugars protein product.
As used herein, term " expression " is used in reference to transcribing and translating of taking place in cell.Product expression of gene level in the host cell can be based on the amount that is present in the corresponding mRNA in the cell or via the proteinic amount of the product genes encoding by cells produce, or both determine.
As used herein, " glycosylation " refers to add the composite oligosaccharide structure to protein on polypeptide intrachain specific site.The following process of the carbohydrate of proteinic glycosylation and interpolation can influence protein folding and structure, protein stability comprise protein transformation period and proteinic functional property.Protein Glycosylation Overview can rely on the sequence background of wherein modifying generation to be divided into 2 classes: O linked glycosylation and N linked glycosylation.O connection polysaccharide and hydroxyl are connected with the hydroxyl of Serine or threonine residues usually.The O glycan is not to add to each Serine and threonine residues.O connection oligosaccharides is normally single or two feelers, and promptly they comprise one or 2 branches (feeler) at the most, and comprise 1-4 the inhomogeneous saccharide residue that adds one by one.The amide nitrogen of N connection polysaccharide and l-asparagine adheres to.Having only 2 kinds of tripeptide sequences---the l-asparagine of the part of one of l-asparagine-X-Serine or l-asparagine-X-Threonine (wherein X is any amino acid except that proline(Pro)) is glycosylated target.N connection oligosaccharides can have 1-4 branch, is called single, double, three, four feelers.It is different with saccharide residue to join the structure of finding in the oligosaccharides at N and O.Although there is the sort of difference, the terminal residue in each branch of N and O connection polysaccharide can be modified by sialic acid molecule, and this modification is called sialic acid and adds cap.Sialic acid is the popular name of 9 carbon monose families of uniqueness, and it can be connected with other oligosaccharides.2 family members are N-n acetylneuraminic acid ns, are abbreviated as Neu5Ac or NANA and N-hydroxyacetylneuraminic acid, are abbreviated as Neu5Gc or NGNA.The most common form of sialic acid in the people is NANA.N-n acetylneuraminic acid n (NANA) is the main sialic acid kind that is present in the CTLA4-Ig molecule.Yet, but should be understood that N hydroxyacetylneuraminic acid (NGNA) less but detection level also is present in the CTLA4-Ig molecule.Therefore in addition, method described herein can be used to measure the sialic acid mole number for NANA and NGNA, and the level of NANA and NGNA is measured with regard to the CTLA4-Ig molecule and reported.N joins the branch that oligosaccharides has different numbers, the different positions number that it provides sialic acid molecule to adhere to it with O.N connection oligosaccharides can be provided for sialicly being up to 4 attachment positions, and O connection oligosaccharides can be provided for 2 sites that sialic acid adheres to.
As used herein, term " large-scale methods " can exchange with term " technical scale method " and use.Term " culture vessel " can exchange with " bio-reactor ", " reactor " and " groove " and use.
As used herein, phrase " working solution " refers to the solution that uses in method.The non-limitative example of working solution comprises damping fluid.
As used herein, " with reference to material " refers to the material of the standard that is used as in method.For example, can be used as the standard that laboratory sample will compare with it with reference to material.
Expected and the non-existent situation of material wherein with regard to the scope of this type of amount of substance, do not provide lower limit.
As used herein, refer to that about the described temperature of cell cultures the temperature on the instrument of the temperature of regulating bio-reactor sets.Certainly, the temperature of liquid culture self will adopt the temperature on the instrument of the temperature of regulating bio-reactor to set.When temperature referred to maintain cell cultures on the shelf in the incubator, temperature referred to the shelf temperature of incubator subsequently.
Non-limiting embodiments of the present invention
The CTLA4-Ig molecule that the invention provides the composition of CTLA4-Ig molecule and sudden change is CTLA4 for example A29YL104EThe composition of-Ig.The invention provides and have some combination of features thing, for example a certain amount of bacterial endotoxin of described feature, biological load, pI (or some the IEF band in certain limit pI) in the certain limit, a certain amount of monomer (strand), dimer or high molecular weight species are (for example, the tetramer), certain tryptic peptide overview, certain group of main band on the SDS-PAGE, certain dna content is no more than the amount of a certain peaked MCP-1, is no more than the amount of a certain peaked cell protein, be no more than the amount of a certain peaked TritonX-100, be no more than the proteic amount of a certain peaked A, a certain overview of N connection carbohydrate, certain amino monose is formed (GlcNac, GalNAc), certain neutral monose is formed (semi-lactosi, Fucose, seminose), a certain amount of B7 combination suppresses a certain amount of activity in the raji cell assay Raji at IL-2, and/or certain sialic acid is formed (NANA, NGNA), wherein said in each case a certain amount of can be one or more scopes.The invention provides and have any above-mentioned feature, or surpass a kind of above-mentioned feature, be up to all above-mentioned combination of features things that comprise with any and all possible arrangement or combination.The present invention includes separating or purified form basically, or be not to separate or all compositions of the present invention of purified form basically.The invention provides composition as pharmaceutical composition.
In one aspect, the present invention relates to be used to obtain method for compositions, described composition comprises the isolating colony from the CTLA4-Ig molecule of liquid nutrient medium, described substratum comprises the original population of CTLA4-Ig molecule, wherein the CTLA4-Ig molecule of (1) original population has one or more sialic acid residueses, (2) number of sialic acid residues/CTLA4-Ig molecule changes in original population, (3) original population comprises CTLA4-Ig dimer and high molecular gathering thing, and described method comprises the liquid nutrient medium of (a) results from the mammalian cell cultures of expressing the CTLA4-Ig molecule; (b) the CTLA4-Ig molecule is separated with cellular component; (c) making CTLA4-Ig dimer and CTLA4-Ig high molecular assemble thing separates; (d) the CTLA4-Ig molecule is divided into 2 or more multistage branch, wherein compare with at least one other fraction, at least one part has the mol ratio of bigger sialic acid and CTLA4-Ig molecule, and wherein step (b), (c) and (d) carry out simultaneously or with any order are so that obtain described composition.
In an embodiment of the inventive method, the results in the step (a) comprise the soluble fractions that obtains liquid culture.In another embodiment, the step of this method (c) and (d) comprise the use of column chromatography is so that obtain to have the fraction of the CTLA4-Ig molecule of different sialic acid contents.In the another one embodiment, this method further comprises the use of column chromatography, to reduce the MCP-1 content in the composition.
In some embodiments of the inventive method, the CTLA4-Ig molecule comprise have SEQ IDNO:2,5,6,7,8, one or more polypeptide of 9 or 10.In other embodiments, the CTLA4-Ig molecule comprise have SEQ ID NO:4, one or more polypeptide of 11,12,13,14,15 or 16.
In some embodiments of the inventive method, the fraction that has the bigger sialic acid and the mol ratio of CTLA4-Ig molecule in (d) shows the sialic acid of about 8-about 14 and the molar average ratio of CTLA4-Ig molecule.In specific embodiments, molar average is than, about 8-about 10 about 11 for about 8-or about 8-about 9.
The invention provides the method that is used for separation of C TLA4-Ig molecule, described method comprises: (i) acquisition comprises the soluble fraction of the liquid culture of mammalian cell, and described mammalian cell is produced the composition of CTLA4-Ig molecule; (ii) soluble fraction is implemented anion-exchange chromatography, comprise the composition of the wash-out of CTLA4-Ig molecule with acquisition; (iii) step composition is (ii) implemented hydrophobic interaction chromatography, so that acquisition comprises the composition of the enrichment of CTLA4-Ig molecule; (iv) composition is (iii) implemented affinity chromatography, comprise the composition of the further enrichment of CTLA4-Ig molecule with acquisition; (v) composition is (iv) implemented anion-exchange chromatography.In one embodiment, the (ii) middle composition that obtains of step is characterised in that: (a) average 6.0-10.1 mole NANA/ mole CTLA4Ig molecule; (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 25.7 area % as detecting by size exclusion chromatography and spectrophotometry.In another embodiment, the (iii) middle composition that obtains of step is characterised in that: (a) average 6.8-11.4 mole NANA/ mole CTLA4Ig molecule; (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.5 area % as detecting by size exclusion chromatography and spectrophotometry.In further embodiment, the (iv) middle composition that obtains of step is characterised in that: (a) average 8.0-11.0 mole NANA/ mole CTLA4-Ig molecule; (b) be less than or equal to the CTLA4-Ig high molecular weight species of 2.5 area %.In another embodiment, (composition that obtains v) is characterised in that step: (a) average 8.0-11.9 mole NANA/ mole CTLA4-Ig molecule; (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.0 area % as detecting (SPD) by size exclusion chromatography and spectrophotometry.In one embodiment, the example of SPD can be at the A280nm place.
The present invention also provides the method for compositions that is used for separation of C TLA4-Ig molecule, it comprises: (i) acquisition comprises the soluble fraction of the liquid culture of the mammalian cell of producing the CTLA4-Ig molecule, with with any order, (ii) soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iii) soluble fraction is implemented hydrophobic interaction chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iv) soluble fraction is implemented affinity chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (v) soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out.In yet another aspect, the invention provides and be used to separate the method for compositions that comprises the CTLA4-Ig molecule, described method comprises: the soluble fraction that (i) obtains to comprise the liquid culture of the mammalian cell of producing the CTLA4-Ig molecule; (ii) soluble fraction is implemented anion-exchange chromatography, comprise the composition of the wash-out of CTLA4-Ig molecule with acquisition; (iii) step protein is (ii) implemented hydrophobic interaction chromatography, so that acquisition comprises the composition of the enrichment of CTLA4-Ig molecule; (iv) protein is (iii) implemented affinity chromatography, comprise the composition of the further enrichment of CTLA4-Ig molecule with acquisition; (v) protein is (iv) implemented anion-exchange chromatography, so that separate the composition that comprises the CTLA4-Ig molecule.
In one embodiment, the composition that comprises the CTLA4-Ig molecule that the step of this method obtains in (ii) is characterised in that: (a) the molar average ratio of the NANA of 6.0-10.1 and CTLA4Ig molecule, (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.5 area % as detecting by size exclusion chromatography and spectrophotometry.In another embodiment, the (iii) middle composition that comprises the CTLA4-Ig molecule that obtains of step is characterised in that: (a) measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig high molecular weight species is less than about 2.5 area %, (b) cell protein less than about 95ng/ml and (c) MCP-1 less than about 5ppm.In other embodiments, the composition that comprises the CTLA4-Ig molecule that step obtains in (iii) is characterised in that: (a) the molar average ratio of the NANA of 6.8-11.4 and CTLA4-Ig molecule, (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.5 area % as detecting by size exclusion chromatography and spectrophotometry.In further embodiment, the composition that comprises the CTLA4-Ig molecule that step obtains in (iv) is characterised in that: (a) the molar average ratio of the NANA of 8.0-11.0 and CTLA4-Ig molecule, (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.5 area % as detecting by size exclusion chromatography and spectrophotometry.In the another one embodiment, the composition that step obtains in (iii) is characterised in that, measures as detecting by size exclusion chromatography and spectrophotometry, and the CTLA4-Ig high molecular weight species is less than 2.5% area percentage.In the another one embodiment, (protein composition that comprises the CTLA4-Ig molecule v) is characterised in that step: (a) the molar average ratio of the NANA of 8.0-11.9 and CTLA4-Ig molecule, (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.0 area % as detecting by size exclusion chromatography and spectrophotometry.
In yet another aspect, the present invention also provides the method for compositions that is used for separation of C TLA4-Ig molecule, it comprises: (i) acquisition comprises the soluble fraction of the liquid culture of the mammalian cell of producing the CTLA4-Ig molecule, with with any order, (ii) soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iii) soluble fraction is implemented hydrophobic interaction chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iv) soluble fraction is implemented affinity chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (v) soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out, wherein the (iii) middle composition that obtains of step is characterised in that the per-cent of CTLA4-Ig high molecular weight species is less than about 2.5 area %, cell protein is less than 95ng/ml, and MCP-1 is less than about 5ppm.
Aspect another one, the invention provides the method for compositions that is used for separation of C TLA4-Ig molecule, described method comprises: (i) acquisition comprises the soluble fraction of the liquid culture of the mammalian cell of producing the CTLA4-Ig molecule, with with any order, (ii) soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iii) soluble fraction is implemented hydrophobic interaction chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iv) soluble fraction is implemented affinity chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (v) soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out, wherein the (iii) middle composition that obtains of step is characterised in that the per-cent of CTLA4-Ig high molecular weight species is less than about 2.5 area %, cell protein is less than 95ng/ml, MCP-1 is less than about 5ppm, and the molar average of NANA and CTLA4-Ig molecule is than being about 8.0-about 12.
In one embodiment, the step of this method anion-exchange chromatography (ii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 75mM HEPES and about 360mMNaCl, and has about 8.0 pH.In another embodiment, step of the present invention anion-exchange chromatography (ii) uses elution buffer to carry out, and described elution buffer comprises about 25mMHEPES and about 850mM NaCl, and has about 7.0 pH.In other embodiments, the step of this method hydrophobic interaction chromatography (iii) uses single lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM HEPES and about 850mM NaCl, and has about 7.0 pH.In further embodiment, the step of this method affinity chromatography (iv) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM Tris and about 250mM NaCl, and has about 8.0 pH.In the another one embodiment, the step of this method affinity chromatography (iv) uses elution buffer to carry out, and described elution buffer comprises about 100mM glycine, and has about 3.5 pH.In the another one embodiment, (anion-exchange chromatography v) uses lavation buffer solution to carry out to the step of this method, and described lavation buffer solution comprises about 25mM HEPES and the about 130mM NaCl of about 120mM NaCl-, and has about 8.0 pH.In the another one embodiment, (anion-exchange chromatography v) uses elution buffer to carry out to the step of this method, and described elution buffer comprises about 25mM HEPES and about 200mMNaCl, and has about 8.0 pH.In the another one embodiment, the step of this method anion-exchange chromatography (ii) uses the post with anionite-exchange resin to carry out, and described anionite-exchange resin comprises primary, secondary, uncle or quaternary amine functional group.In specific embodiments, resin comprises quaternary amine functional group.In the another one embodiment, the step of this method hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin comprises phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.In specific embodiments, functional group comprises phenyl functional group.In the another one embodiment, the affinity chromatography use (iv) of the step of this method comprises the proteic affinity chromatography resin of A and carries out.
Aspect another one, the invention provides and be used to prepare the method for compositions that comprises the CTLA4-Ig molecule, it comprises purifying CTLA4-Ig molecule from the liquid cell culture, wherein the CTLA4-Ig composition of purifying comprises the MCP-1/mg CTLA4-Ig molecule of (a) pharmaceutically acceptable amount, (b) measure, less than the CTLA4-Ig high molecular weight species of 2.5 area % as detecting by size exclusion chromatography and spectrophotometry.In one embodiment, the MCP-1 of pharmaceutically acceptable amount comprises the about 0.5ng/mg CTLA4-Ig of about 40-molecule.In another embodiment, the MCP-1 of pharmaceutically acceptable amount comprises the about 0.5ng/mgCTLA4-Ig molecule of about 35-.In other embodiments, the MCP-1 of pharmaceutically acceptable amount comprises the about 0.5ng/mg CTLA4-Ig of about 10-molecule.In further embodiment, the step of this method affinity chromatography (iv) uses post to carry out, and described post comprises can reduce the resin of the MCP-1 in the protein of wash-out.In the another one embodiment, the step of this method hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and wherein said resin can (a) make the CTLA4-Ig dimer separate with the CTLA4-Ig high molecular weight species; (b) sialic acid content of the CTLA4-Ig molecule of increase wash-out; Or (c) (a) and (b) both.In the another one embodiment, step (ii) or step (iv) or both anion-exchange chromatographies use anionite-exchange resin to carry out, wherein said resin can (a) reduces the CTLA4-Ig high molecular of the composition of wash-out and assembles thing content; (b) sialic acid content of the composition of increase wash-out; Or (c) (a) and (b) both.
In yet another aspect, the invention provides and be used to separate the method for compositions that comprises the CTLA4-Ig molecule, described method comprises: (i) obtain to comprise the mammalian cell of producing the CTLA4-Ig molecule liquid culture soluble fraction and with any order; (ii) soluble fraction is implemented affinity chromatography, so that acquisition comprises the composition of the wash-out of CTLA4-Ig molecule; (iii) soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the wash-out of CTLA4-Ig molecule and the composition of enrichment; (iv) soluble fraction is implemented hydrophobic interaction chromatography, so that obtain to comprise the wash-out of CTLA4-Ig molecule and the composition of enrichment.In one embodiment, the affinity chromatography step is at first carried out.In another embodiment, the step of this method affinity chromatography is (ii) used and to be comprised the proteic resin of A and carry out.In other embodiments, step affinity chromatography (ii) uses the elution buffer that comprises guanidine to carry out.In further embodiment, step affinity chromatography (ii) uses the elution buffer that comprises urea to carry out.In the another one embodiment, step affinity chromatography (ii) causes comprising the dimeric increase of CTLA4-Ig in the wash-out composition of CTLA4-Ig molecule.
Aspect another one, the invention provides and be used for separating the method for compositions that comprises the CTLA4-Ig molecule from the liquid of mammalian cell cultures from results, wherein said cells produce CTLA4-Ig molecule, described method comprises: the soluble fraction that (i) obtains the liquid of results; (ii) soluble fraction is implemented affinity chromatography, comprise the composition of the wash-out of CTLA4-Ig molecule with acquisition; (iii) step composition is (ii) implemented anion-exchange chromatography, so that obtain to comprise the wash-out of CTLA4-Ig molecule and the composition of enrichment; (iv), comprise the composition of the further enrichment of CTLA4-Ig molecule with acquisition to implementing hydrophobic interaction chromatography from step composition (iii).In one embodiment, the (iv) middle composition that obtains of the step of this method is characterised in that: measure as detecting by size exclusion chromatography and spectrophotometry, the per-cent of high molecular weight species is less than about 2.5 area %, with the per-cent of cell protein less than the per-cent of about 95ng/ml and MCP-1 less than about 5ppm.In another embodiment, step anion-exchange chromatography (iii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 50mM HEPES and about 135mM NaCl, and has about 7 pH.In other embodiments, step anion-exchange chromatography (iii) uses elution buffer to carry out, and described elution buffer comprises about 50mM HEPES and about 200mM NaCl, and has about 7 pH.In specific embodiments, step hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin comprises phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.In further embodiment, step hydrophobic interaction chromatography (iv) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 50mM HEPES and about 1.2M (NH 4) 2SO 4, and have about 7 pH.In the another one embodiment, step affinity chromatography (ii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM NaH 2PO 4With about 150mM NaCl, and has about 7.5 pH.In the another one embodiment, step affinity chromatography (ii) uses elution buffer to carry out, and described elution buffer comprises about 250mM glycine and has about 3 pH.In another embodiment, step anion-exchange chromatography (iii) uses the post with anionite-exchange resin to carry out, and described anionite-exchange resin comprises primary, secondary, uncle or quaternary amine functional group.In specific embodiments, resin comprises quaternary amine functional group.
In one embodiment, step hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin comprises phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.In one embodiment, functional group comprises phenyl functional group.In one embodiment, step affinity chromatography is (ii) used and to be comprised the proteic resin of A and carry out.The invention provides the composition that comprises the CTLA4-Ig molecule that obtains by any method of the present invention.In one embodiment, composition comprise have SEQ ID NO:2, one or more polypeptide of 5,6,7,8,9 or 10.In one embodiment, composition comprise have SEQ ID NO:4, one or more polypeptide of 11,12,13,14,15 or 16.The invention provides the CTLA4-Ig expression plasmid of nucleotide sequence with SEQ ID NO:17.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid and the proteinic molar average ratio of CTLA4-Ig of about 5.5-about 18.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 5.5-about 9.5 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 5-about 10 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 6-about 18 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 8-about 18 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 8-about 12 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 8-about 11 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 7-about 12 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 7-about 11 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 11-about 18 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 12-about 18 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 13-about 18 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 14-about 18 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 15-about 17 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the molar average ratio of about 16 sialic acid and CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the molar average ratio of about 10 sialic acid and CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the molar average ratio of about 6 sialic acid and CTLA4-Ig molecule.In one embodiment, sialic acid is N-n acetylneuraminic acid n (NANA).The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the NANA of about 8-about 12 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the molar average ratio that is less than or equal to about 1.5 N-hydroxyacetylneuraminic acid (NGNA) and CTLA4-Ig molecule.
The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the NGNA of about 0.5-about 1.5 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the NGNA of about 1.0-about 1.5 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 6-about 18 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is characterised in that the molar average ratio of sialic acid/mole CTLA4-Ig molecule of about 6-about 12.
The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, wherein every peptide species of molecule comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein the CTLA4-Ig molecule is characterised in that the molar average ratio of sialic acid/mole CTLA4-Ig molecule of about 5.5-about 9.5.In one embodiment, the mol ratio of sialic acid/mole CTLA4-Ig molecule is measured by acid hydrolysis and HPLC.In one embodiment, the CTLA4-Ig molecule comprise have SEQ ID NO:2, one or more polypeptide of 5,6,7,8,9 or 10.
In one embodiment, the CTLA4-Ig molecule comprise have SEQ ID NO:4, one or more polypeptide of 11,12,13,14,15 or 16.The invention provides the composition of the purifying basically that comprises the CTLA4-Ig molecule, is the CTLA4-Ig dimer more than or equal to 95% CTLA4-Ig molecule wherein.In one embodiment, be the CTLA4-Ig dimer more than or equal to 98% CTLA4-Ig molecule.In one embodiment, be the CTLA4-Ig dimer more than or equal to 99% CTLA4-Ig molecule.
In one embodiment, be the CTLA4-Ig dimer more than or equal to 99.5% CTLA4-Ig molecule.In one embodiment, measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule of about 95%-about 99.5% is the CTLA4-Ig dimer, and the molecule of the about 5 area % of about 0.5 area %-is the CTLA4-Ig high molecular weight species.In one embodiment, measure as detecting by size exclusion chromatography and spectrophotometry meter, about 98.6% molecule is the CTLA4-Ig dimer, and the molecule of about 1.2 area % is the CTLA4-Ig high molecular weight species, and is the CTLA4-Ig monomer less than the molecule of 0.7 area % approximately.In one embodiment, be to comprise 5 or the monomeric polymer of more CTLA4-Ig less than about 0.3% molecule approximately.The invention provides the composition of forming by the CTLA4-Ig dimer basically.The invention provides basically by the molecular composition of CTLA4-Ig, wherein colony is substantially free of the CTLA4-Ig monomer.The invention provides basically by the molecular composition of CTLA4-Ig, wherein colony is substantially free of the CTLA4-Ig high molecular weight species.The invention provides the composition of being made up of the CTLA4-Ig monomer basically, it is substantially free of CTLA4-Ig dimer and high molecular weight species.In one embodiment, every kind of dimeric each monomer of CTLA4-Ig has at least 3 sialic acids groups.In one embodiment, every kind of dimeric each monomer of CTLA4-Ig has at least 2.5 sialic acids groups.In one embodiment, every kind of dimeric each monomer of CTLA4-Ig has at least 3 sialic acids groups-at least 8 sialic acids groups.
In one embodiment, every kind of dimeric each monomer of CTLA4-Ig has at least 2.5 sialic acids groups-at least 5 sialic acids groups.In one embodiment, every kind of dimer comprises 2 CTLA4-Ig polypeptide, and wherein each polypeptide has the aminoacid sequence that is selected from SEQ ID NOS:5-16.In one embodiment, composition comprise have SEQ ID NO:2, one or more polypeptide of 5,6,7,8,9 or 10.In one embodiment, composition comprise have SEQ ID NO:4, one or more polypeptide of 11,12,13,14,15 or 16.The invention provides and comprise the tetrameric composition isolated of CTLA4-Ig, it is substantially free of the CTLA4-Ig dimer.The invention provides and comprise the tetrameric composition isolated of CTLA4-Ig, it is substantially free of the CTLA4-Ig monomer.In one embodiment, composition exists as the amount greater than about 100 grams.In one embodiment, each tetramer comprises 2 pairs of CTLA4-Ig polypeptide, and wherein each polypeptide has the aminoacid sequence that is selected from SEQ ID NOS:5-10.In one embodiment, each tetramer comprises 2 pairs of CTLA4-Ig polypeptide, and wherein each polypeptide has the aminoacid sequence that is selected from SEQ ID NOS:11-16.In one embodiment, each tetramer can combine with CD80 or CD86.The invention provides the pharmaceutically acceptable composition that comprises the CTLA4-Ig molecule, wherein composition is substantially free of MCP-1.The invention provides the pharmaceutically acceptable composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is no more than about 25ppm MCP-1.In one embodiment, composition comprises and is no more than 10ppmMCP-1.In one embodiment, composition comprises the about 10ng/mlMCP-1 of about 0.2ng/ml MCP-1-.In one embodiment, the invention provides the pharmaceutically acceptable composition that comprises the CTLA4-Ig molecule, wherein composition comprises (a) about 10ng/mlMCP-1 of about 0.2ng/ml MCP-1-and (b) is no more than 25ng/ml CHO protein or be no more than 10ng/ml CHO protein.In one embodiment, composition comprises and is no more than about 20pg/ml DNA.
The invention provides the composition isolated that comprises the CTLA4-Ig molecule, wherein, when the intravenous dosages with about 10mg/kg was applied to the experimenter, the CTLA4-Ig molecule can show: the area under curve of about 44400 μ g.h/ml (AUC); The volume of distribution of about 0.09L/kg; The peak concentration of about 292 μ g/ml (Cmax); Clearance rate with about 0.23ml/h/kg.The invention provides the composition isolated that comprises the CTLA4-Ig molecule, wherein composition is included in the advantage isoform of CTLA4-Ig molecule visual on the isoelectrofocusing gel, as measuring by isoelectrofocusing, described isoform has and is less than or equal to 5.1 ± 0.2 iso-electric point, pI.In one embodiment, the average pI of composition handles the back at neuraminidase increases.In one embodiment, as measuring by isoelectrofocusing, at least 40% CTLA4-Ig molecule demonstration is less than or equal to about 5.1 ± 0.2 iso-electric point.In one embodiment, as measuring by isoelectrofocusing, at least 70% CTLA4-Ig molecule demonstration is less than or equal to about 5.1 ± 0.2 iso-electric point.In one embodiment, as measuring by isoelectrofocusing, at least 90% CTLA4-Ig molecule demonstration is less than or equal to about 5.1 ± 0.2 iso-electric point.The invention provides the composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 3.0 ± 0.2-about 5.0 ± 0.2.The invention provides the composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 4.3 ± 0.2-about 5.0 ± 0.2.
The invention provides the composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 3.3 ± 0.2-about 4.7 ± 0.2.In one embodiment, composition is a purifying basically.The invention provides and be used to prepare method for compositions, composition comprises the CTLA4-Ig molecule of the pI with about 3.0 ± 0.2-about 5.0 ± 0.2, described method comprises: (a) mixture of CTLA4-Ig molecule is implemented the isoelectrofocusing gel electrophoresis, wherein the single band on gel represents to have the CTLA4-Ig molecule colony of specific pI, (b) separate the CTLA4-Ig molecule colony of the pI with about 3.0 ± 0.2-about 5.0 ± 0.2, so that prepare composition.The invention provides the composition isolated that comprises the CTLA4-Ig molecule, wherein composition is included in advantage isoform visual on the isoelectrofocusing gel, and as measuring by isoelectrofocusing, described isoform has and is less than or equal to 5.5 ± 0.2 iso-electric point, pI.In one embodiment, the average pI of composition handles the back at neuraminidase increases.In one embodiment, as measuring by isoelectrofocusing, at least 40% CTLA4-Ig molecule demonstration is less than or equal to about 5.3 ± 0.2 iso-electric point.In one embodiment, as measuring by isoelectrofocusing, at least 70% CTLA4-Ig molecule demonstration is less than or equal to about 5.3 ± 0.2 iso-electric point.In one embodiment, as measuring by isoelectrofocusing, at least 90% CTLA4-Ig molecule demonstration is less than or equal to about 5.3 ± 0.2 iso-electric point.The invention provides the composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 3.0 ± 0.2-about 5.2 ± 0.2.
The invention provides the composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 4.5 ± 0.2-about 5.2 ± 0.2.The invention provides the composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 4.7 ± 0.2-about 5.1 ± 0.2.In one embodiment, composition is a purifying basically.
The invention provides and be used to prepare method for compositions, described composition comprises the CTLA4-Ig molecule of the pI with about 2.0 ± 0.2-about 5.2 ± 0.2, described method comprises: (a) mixture of CTLA4-Ig molecule is implemented the isoelectrofocusing gel electrophoresis, wherein the single band on gel represents to have the CTLA4-Ig molecule colony of specific pI, (b) separate the CTLA4-Ig molecule colony of the pI with about 3.0 ± 0.2-about 5.2 ± 0.2, so that prepare composition.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is characterised in that the GlcNAc of about 17-about 28 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is characterised in that the GlcNAc of about 17-about 25 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is characterised in that the GlcNAc of about 15-about 35 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein every peptide species of molecule comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein the CTLA4-Ig molecule is characterised in that the GlcNAc of about 24-about 28 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is characterised in that the GalNAc of about 1.7-about 3.6 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein every peptide species of molecule comprises SEQ IDNO:11,12,13,14,15 or 16 sequence, and wherein the CTLA4-Ig molecule is characterised in that the GalNAc of about 2.7-about 3.6 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is characterised in that the semi-lactosi of about 8-about 17 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein every peptide species of molecule comprises SEQ IDNO:11,12,13,14,15 or 16 sequence, and wherein the CTLA4-Ig molecule is characterised in that the semi-lactosi of about 11-about 13 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is characterised in that the Fucose of about 3.5-about 8.3 and the molar average ratio of CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein every peptide species of molecule comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein the CTLA4-Ig molecule is characterised in that the Fucose of about 6.4-about 7.0 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is characterised in that the seminose of about 7.7-about 22 and the molar average ratio of CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein every peptide species of molecule comprises SEQ IDNO:11,12,13,14,15 or 16 sequence, and wherein the CTLA4-Ig molecule is characterised in that the seminose of about 14-about 16 and the molar average ratio of CTLA4-Ig molecule.
In one embodiment, the mol ratio of GlcNAc and CTLA4-Ig molecule is measured by capillary electrophoresis.In one embodiment, the mol ratio of GalNAc and CTLA4-Ig molecule is measured by capillary electrophoresis.In one embodiment, the mol ratio of semi-lactosi and CTLA4-Ig molecule is measured by capillary electrophoresis.
In one embodiment, the mol ratio of Fucose and CTLA4-Ig molecule is measured by capillary electrophoresis.In one embodiment, the mol ratio of seminose and CTLA4-Ig molecule is measured by capillary electrophoresis.In one embodiment, the enzymatic of CTLA4-Ig molecule by one or more carbohydrate and molecule adheres to and obtains.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein molecule is included in the external carbohydrate residue that adheres to the molecule enzymatic.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; (b) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (d) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule; (e) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule; (e) the molar average ratio of the seminose of about 7.2-about 22 and CTLA4-Ig molecule; (f) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (d) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 6.4-about 7.0 and CTLA4-Ig molecule; (e) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 6.4-about 7.0 and CTLA4-Ig molecule; (e) seminose of about 14-about 16 and the proteinic molar average ratio of CTLA4-Ig; (f) sialic acid of about 5.5-about 9.5 and the proteinic molar average ratio of CTLA4-Ig.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is glycosylated on following residue: the amino acid asparagine residue at SEQ ID NO:2 or 4 the 102nd place, the amino acid asparagine residue at SEQ ID NO:2 or 4 the 134th place, the amino acid asparagine residue at SEQ ID NO:2 or 4 the 233rd place, the Serine amino-acid residue at SEQ ID NO:2 or 4 the 155th place, or the Serine amino-acid residue at the 165th place of SEQ ID NO:2 or 4.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule is glycosylated, and is the O linked glycosylation at least about 2% glycosylation total mass wherein.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the NGNA chromatogram peak of composition exhibiting about 9.6 ± 0.3 and about 10.5 ± 0.3 NANA chromatogram peak.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule shows the carbohydrate overview substantially the same with Figure 68.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule shows the carbohydrate overview as shown in Figure 68.The invention provides basically by the molecular composition of CTLA4-Ig, the carbohydrate overview of CTLA4-Ig molecule display structure territory I-IV wherein, wherein structural domain I comprises the peak of the oligosaccharides of representing asialoization, domain II comprises the peak of the oligosaccharides of representing mono-sialylated, domain II I comprises the peak of the oligosaccharides of representing double-sialylated, structural domain IV comprises the peak of representing three sialylated oligosaccharides, and structural domain V comprises the peak of representing four sialylated oligosaccharides, and wherein said overview is the chromatogram of the oligosaccharides that discharged by CTLA4-Ig.In one embodiment, the difference in the retention time of N connection oligosaccharides is about 12 minutes of about 10-between first peak among the structural domain I and the main peak in the domain II.In one embodiment, the difference in the retention time of N connection oligosaccharides is about 13 minutes of about 11-between first peak among the structural domain I and the main peak in the domain II.In one embodiment, as measuring by HPAEC, the glycosylation of domain II I and IV comprises the N linked glycosylation of about 25%-about 36%.In one embodiment, as measuring by HPAEC, the glycosylation of structural domain I comprises the N linked glycosylation of about 24.5%-about 35.2%.In one embodiment, as measuring by HPAEC, the glycosylation of domain II comprises the N linked glycosylation of about 26.3%-about 34.1%.In one embodiment, as measuring by HPAEC, the glycosylation of domain II I comprises the N linked glycosylation of about 21.9%-about 31.5%.In one embodiment, as measuring by HPAEC, the glycosylation of structural domain IV and structural domain V comprises the N linked glycosylation of about 7.9%-about 18.6%.
In one embodiment: (a) structural domain I shows the area percentage at least about 31; (b) domain II shows the area percentage at least about 33; (c) domain II I shows the area percentage at least about 24; (iv) structural domain IV shows the area percentage at least about 9.4; (v) structural domain V shows the area percentage at least about 67; Or wherein said area is measured according to the chromatogram of the oligosaccharides that is discharged by CTLA4-Ig.
In one embodiment: (a) structural domain I shows at least about 5 peaks; (b) domain II shows at least about 5 peaks; (c) domain II I shows at least about 5 peaks; (d) structural domain IV shows at least about 6 peaks, or (e) structural domain V shows at least about 6 peaks, and wherein said peak shows on chromatogram.Composition, wherein structural domain I shows at least 2 peaks, wherein first peak has about 4.5% minimum area and about 11.2% maximum area, and wherein second peak has about 8.7% minimum area and about 11.8% maximum value.
In one embodiment, as measuring by HPAEC, domain II I and IV show the area percentage of about 25%-about 36%.In one embodiment, as measuring by HPAEC, structural domain I shows the area percentage of about 24.5%-about 35.2%.In one embodiment, as measuring by HPAEC, domain II shows the area percentage of about 26.3%-about 34.1%.In one embodiment, as measuring by HPAEC, domain II I shows the area percentage of about 21.9%-about 31.5%.In one embodiment, as measuring by HPAEC, structural domain IV shows the area percentage of about 7.9%-about 18.6%.
The invention provides the composition that comprises the CTLA4-Ig polypeptide, wherein: (a) about 80% polypeptide has two feeler N linked glycosylations; (b) about 14% polypeptide has three feeler N linked glycosylations; (c) about 6% polypeptide has four feeler N linked glycosylations.In one embodiment, the N linked glycosylation is measured by the high pH anion-exchange chromatography (HPEAC-PAD) with pulsed current detection.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule; (c) as detect by size exclusion chromatography and spectrophotometry measure CTLA4-Ig high molecular weight species area percentage less than about 3%.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule; (c) be less than or equal to the molar average ratio of about 1.5 NANA and CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule; (c) measure as detecting, assemble thing content less than the CTLA4-Ig high molecular of about 3 area % by size exclusion chromatography and spectrophotometry; (d) with the sort of substantially the same carbohydrate overview of Figure 68.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule; (c) measure as detecting, assemble thing content less than the CTLA4-Ig high molecular of about 3 area % by size exclusion chromatography and spectrophotometry; (d) as measuring, at least about domain II I, the IV of the about 50.1%N linked glycosylation of 29.8%-and the glycosylation content among the V by HPAEC.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule; (c) measure, less than the CTLA4-Ig high molecular weight species of about 3 area % as detecting by size exclusion chromatography and spectrophotometry.In one embodiment, molecule is further characterized in that the NANA of about 8-about 12 and the molar average ratio of CTLA4-Ig molecule.
In one embodiment, molecule is further characterized in that: (a) two feeler N linked glycosylations of about 80%; (b) about 14% three feeler N linked glycosylations; (c) about 6% four feeler N linked glycosylations.In one embodiment, molecule further comprises one or more any combination in following: (a) aminoacid sequence of SEQ ID NO:10 (methionine(Met) at amino acid position 27 places of SEQ ID NO:2 and the glycine at amino acid position 382 places); (b) aminoacid sequence of SEQ ID NO:7 (methionine(Met) at amino acid position 27 places of SEQ ID NO:2 and the Methionin at amino acid position 383 places); (c) aminoacid sequence of SEQ ID NO:9 (L-Ala at amino acid position 26 places of SEQ ID NO:2 and the glycine at amino acid position 382 places); (d) aminoacid sequence of SEQ ID NO:6 (L-Ala at amino acid position 26 places of SEQ ID NO:2 and the Methionin at amino acid position 383 places).In one embodiment, (a) about 90% molecule comprises the aminoacid sequence of the SEQID NO:2 that begins from the methionine(Met) of residue 27; (b) about 10% molecule comprises the aminoacid sequence of numbering the SEQ ID NO:2 that 26 L-Ala begins from residue; (c) about 4% molecule comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the Methionin at 383 places finishes with residue; (d) about 96% molecule comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the glycine at 382 places finishes with residue.The invention provides the composition that comprises the CTLA4-Ig polypeptide, wherein: (a) about 80% polypeptide has two feeler N linked glycosylations; (b) about 14% polypeptide has three feeler N linked glycosylations; (c) about 6% polypeptide has four feeler N linked glycosylations; (d) be less than or equal to the molar average ratio of 1.5 NGNA and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig polypeptide, wherein: (a) about 80% polypeptide has two feeler N linked glycosylations; (b) about 14% polypeptide has three feeler N linked glycosylations; (c) about 6% polypeptide has four feeler N linked glycosylations; (d) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig polypeptide, wherein: (a) about 80% polypeptide has two feeler N linked glycosylations; (b) about 14% polypeptide has three feeler N linked glycosylations; (c) about 6% polypeptide has four feeler N linked glycosylations; (d) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule; (c) measure, less than the CTLA4-Ig high molecular weight species of about 5 area % as detecting by size exclusion chromatography and spectrophotometry.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule; (c) measure as detecting, less than the CTLA4-Ig high molecular weight species of about 5 area % by size exclusion chromatography and spectrophotometry; (d) with the sort of substantially the same carbohydrate overview of Figure 68.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule; (c) measure as detecting, less than the CTLA4-Ig high molecular weight species of about 5 area % by size exclusion chromatography and spectrophotometry; (d) as measuring, at least about domain II I, the IV of the about 50.1%N linked glycosylation of 29.8%-and the glycosylation content among the V by HPAEC.
The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule; (c) measure, less than the CTLA4-Ig high molecular weight species content of about 5 area % as detecting by size exclusion chromatography and spectrophotometry.In one embodiment, molecule is further characterized in that: (a) two feeler N linked glycosylations of about 80%; (b) about 14% three feeler N linked glycosylations; (c) about 6% four feeler N linked glycosylations.In another embodiment, molecule further comprises one or more any combination in following: (a) aminoacid sequence of SEQ ID NO:16 (methionine(Met) at amino acid position 27 places of SEQ IDNO:4 and the glycine at amino acid position 382 places); (b) aminoacid sequence of SEQ ID NO:13 (methionine(Met) at amino acid position 27 places of SEQ ID NO:4 and the Methionin at amino acid position 383 places); (c) aminoacid sequence of SEQ ID NO:15 (L-Ala at amino acid position 26 places of SEQ ID NO:4 and the glycine at amino acid position 382 places); (d) aminoacid sequence of SEQ ID NO:12 (L-Ala at amino acid position 26 places of SEQ ID NO:4 and the Methionin at amino acid position 383 places).In another embodiment, (a) about 90% molecule comprises the aminoacid sequence of the SEQ ID NO:4 that begins from the methionine(Met) of residue 27; (b) about 10% molecule comprises the aminoacid sequence of numbering the SEQ ID NO:4 that 26 L-Ala begins from residue; (c) about 4% molecule comprises the aminoacid sequence of numbering the SEQ ID NO:4 that the Methionin at 383 places finishes with residue; (d) about 96% molecule comprises the aminoacid sequence of numbering the SEQ ID NO:4 that the glycine at 382 places finishes with residue.The invention provides the composition that comprises the CTLA4-Ig polypeptide, wherein: (a) about 80% polypeptide has two feeler N linked glycosylations; (b) about 14% polypeptide has three feeler N linked glycosylations; (c) about 6% polypeptide has four feeler N linked glycosylations; (d) the proteinic molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 24-about 28.The invention provides the composition that comprises the CTLA4-Ig polypeptide, wherein: (a) about 80% polypeptide has two feeler N linked glycosylations; (b) about 14% polypeptide has three feeler N linked glycosylations; (c) about 6% polypeptide has four feeler N linked glycosylations; (d) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule.In another embodiment, composition is the composition of purifying basically.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein being less than or equal to about 2.5% CTLA4-Ig molecule is oxidation.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein being less than or equal to about 2.0% CTLA4-Ig molecule is deacylated tRNA amine.The invention provides the composition that comprises the CTLA4-Ig dimer molecule, wherein at least 0.5% CTLA4-Ig dimer molecule is a cysteinylization.In one embodiment, at least 1.0% CTLA4-Ig dimer molecule is a cysteinylization.The invention provides the colony of CTLA4-Ig molecule, wherein colony's demonstration and Figure 64,65 or 67 substantially the same mass spectroscopy overviews.The invention provides the colony of CTLA4-Ig molecule, wherein colony shows the capillary electrophoresis overview substantially the same with Figure 48.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition is characterised in that: (a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule; (e) the molar average ratio of the seminose of about 7.2-about 22 and CTLA4-Ig molecule; (f) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 2.4 ± 0.2-about 5.0 ± 0.2; (h) be less than or equal to the MCP-1 of 3ppm; (i) measure as detecting, less than the high molecular weight species of 2.5 area % by size exclusion chromatography and spectrophotometry; (j) measure as detecting, less than the monomer of 0.5 area % by size exclusion chromatography and spectrophotometry; (k) have with SEQ ID NOS:5-10 in any one at least 95% amino acid whose CTLA4-Ig polypeptide that is equal to; (1) can with CD80 and CD86 bonded CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein molecule colony is characterised in that: (a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule; (e) the molar average ratio of the seminose of about 7.2-about 22 and CTLA4-Ig molecule; (f) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 3.4 ± 0.2-about 5.0 ± 0.2; (h) be less than or equal to the MCP-1 of 5ppm; (i) measure as detecting, less than the high molecular weight species of 2.5 area % by size exclusion chromatography and spectrophotometry; (j) measure as detecting, less than the monomer of 0.5 area % by size exclusion chromatography and spectrophotometry; (k) have with SEQ ID NOS:5-10 in any one at least 95% amino acid whose CTLA4-Ig polypeptide that is equal to; (1) can with CD80 and CD86 bonded CTLA4-Ig molecule; Or its pharmacy Equivalent.
The invention provides the composition isolated that comprises the CTLA4-Ig molecule, it has and is less than or equal to 7.4% immunogenicity incidence.In one embodiment, immunogenic incidence is about 2.1%-about 7.4%.In one embodiment, immunogenic incidence is less than or equal to 3.7%.In one embodiment, immunogenic incidence is less than or equal to 3.0%.In one embodiment, immunogenic incidence is about 2.8%-about 3.0%.The invention provides the composition isolated that comprises the CTLA4-Ig molecule, wherein, after using composition, in the people, take place to be less than or equal to 7.4% incidence with CTLA4-Ig molecule bonded production of antibodies to the people.In one embodiment, incidence is about 2.1%-about 7.4%.In one embodiment, incidence is less than or equal to 3.7%.In one embodiment, incidence is less than or equal to 3.0%.In one embodiment, incidence is about 2.8%-about 3.0%.The invention provides the composition isolated that comprises the CTLA4-Ig molecule, wherein, after using composition, in the people, take place to be less than or equal to 4.9% incidence with the CTLA4 part bonded production of antibodies of CTLA4-Ig molecule to the people.In one embodiment, incidence is about 0.5%-about 4.9%.In one embodiment, incidence is less than or equal to 1.2%.In one embodiment, incidence is less than or equal to 1.0%.In one embodiment, incidence is about 0.9%-about 1.0%.In one embodiment, incidence is measured in enzyme-linked immunosorbent assay (ELISA).In one embodiment, incidence is measured in (ECL) at electrochemiluminescence and is measured.
The invention provides the composition isolated that comprises the CTLA4-Ig molecule, wherein, after using composition to the people, in and the production of antibodies of CTLA4-Ig molecule in the people who has with the CTLA4 part bonded antibody of CTLA4-Ig molecule, take place to be less than or equal to 75% incidence.In one embodiment, incidence is 40-75%.In one embodiment, incidence is less than or equal to 40%.In one embodiment, incidence is measured in measuring based on the luciferase reporter gene of cell.
The invention provides and be used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, the wherein said liquid culture that is separated in shows more than or equal to about 6.0 moles of NANA/ mole CTLA4-Ig dimers or CTLA4-Ig and divides the period of the day from 11 p.m. to 1 a.m to take place, as what measure by the aliquots containig of assessment liquid culture.This method also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, the wherein said liquid culture that is separated in shows that the about 7.6 moles of sialic acids of about 5.2-/mole CTLA4-Ig dimer or CTLA4-Ig divide the period of the day from 11 p.m. to 1 a.m to take place, as what measure by the aliquots containig of assessment liquid culture.This method also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, the wherein said liquid culture that is separated in has about 33x 10 5Viable cell/mL liquid culture-Yue 79 x 10 5Take place during the cell density of cell/mL liquid culture.The present invention also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, the wherein said cell survival that is separated in the liquid culture are not less than generation in about 38% o'clock.The present invention also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, the wherein said cell survival that is separated in the liquid culture are not less than generation in about 37% o'clock.This method also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, wherein said being separated in when intracellular toxin is less than or equal to about 76.8EU/mL liquid culture taken place, as what measure by the aliquots containig of assessment liquid culture.This method also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, wherein said being separated in when intracellular toxin is less than or equal to about 4.8EU/mL liquid culture taken place, as what measure by the aliquots containig of assessment liquid culture.The present invention also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, wherein said separation only takes place during liquid culture less than 1 colony forming unit/mL at biological load, as what measure by the aliquots containig of assessment liquid culture.The present invention also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, wherein saidly take place when being separated at least 2 that satisfy in the following condition: (i) liquid culture comprises more than or equal to about 6.0 moles of NANA/ mole CTLA4-Ig dimers or CTLA4-Ig molecule; (ii) liquid culture has about 33 x 10 5Viable cell/mL liquid culture-Yue 79 x 10 5The cell density of viable cell/mL liquid culture; (iii) the cell survival in the liquid culture is not less than about 38%; Or (iv) the proteinic yield of CTLA4-Ig greater than about 0.5 gram CTLA4-Ig protein/L liquid culture, wherein the NANA concentration in (i) and (iv) in yield measure by the aliquots containig of assessing liquid culture.The present invention also provides and has been used to produce the CTLA4-Ig method of protein, described method comprises: (a) the proteinic mammalian cell of CTLA4-Ig is produced in amplification, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, until CTLA4-Ig protein with yield production, as what measure by the aliquots containig of assessment liquid culture at least about 0.5 gram CTLA4-Ig protein/L liquid culture; (b) from least 10, separation of C TLA4-Ig protein in the liquid culture of 000L, wherein saidly take place when being separated at least 2 that satisfy in the following condition: (i) liquid culture comprises the about 7.6 moles of sialic acids of about 5.2-/mole CTLA4-Ig dimer or CTLA4-Ig molecule; (ii) the cell survival in the liquid culture is not less than about 37%; Or (iii) the proteinic yield of CTLA4-Ig greater than about 0.5 gram CTLA4-Ig protein/L liquid culture, wherein the sialic acid content in (i) and (iii) in yield measure by the aliquots containig of assessing liquid culture.
Sequence:
SEQ ID NO:1[CTLA4-Ig nucleotide sequence is referring to Fig. 1]
SEQ ID NO:2[CTLA4-Ig aminoacid sequence is referring to Fig. 1]
SEQ ID NO:3[CTLA4 A29YL104E-Ig nucleotide sequence comprises the Nucleotide 79-1149 of nucleotide sequence shown in Fig. 2]
SEQ ID NO:23 is the full length nucleotide sequence shown in Fig. 2.This nucleotide sequence comprises the encoding sequence about presequence.
SEQ ID NO:4[CTLA4 A29YL104E-Ig aminoacid sequence, Fig. 3 does not contain presequence]
The amino acid 25-383 of SEQ ID NO:5[SEQ ID NO:2]
MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM
MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGT
QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK
The amino acid 26-383 of SEQ ID NO:6[SEQ ID NO:2]
AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM
GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI
YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK
The amino acid 27-383 of SEQ ID NO:7[SEQ ID NO:2]
MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY
VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK
The amino acid 25-382 of SEQ ID NO:8[SEQ ID NO:2]
MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM
MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGT
QTYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDLAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPG
The amino acid 26-382 of SEQ ID NO:9[SEQ ID NO:2]
AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM
GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI
YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPG
The amino acid 27-382 of SEQ ID NO:10[SEQ ID NO:2]
MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY
VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPG
The amino acid 25-383 of SEQ ID NO:11[SEQ ID NO:4]
MAMHVAQPAVVLASSRGIASF.V.CEYASP.GKYTEVRVTVLRQADSQVTEVCAATYM
MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGT
QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDLAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK
The amino acid 26-383 of SEQ ID NO:12[SEQ ID NO:4]
AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMM
GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI
YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK
The amino acid 27-383 of SEQ ID NO:13[SEQ ID NO:4]
MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY
VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK
The amino acid 25-382 of SEQ ID NO:14[SEQ ID NO:4]
MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYM
MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGT
QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPG
The amino acid 26-382 of SEQ ID NO:15[SEQ ID NO:4]
AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMM
GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI
YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPG
The amino acid 27-382 of SEQ ID NO:16[SEQ ID NO:4]
MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY
VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPG
SEQ?ID?NO：17
SEQ ID NO:18[CTLA4 ectodomain sequence]
MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI
YVIDPEPCPDSD
SEQ?ID?NO：19
5’-AGAAAAGGGGCTGGAGAGATGGCTCAGTGGTTAAGAGCA-3’
SEQ?ID?NOS：20-22
SEQ?ID?NO：20--5’-GTACTCAGG
SEQ?ID?NO：21--AGTCAGAGAC
SEQ?ID?NO：22--CGGCAGATCTCTGTGAGTTTGAGGCCAGCCTGG
TCTACAAAGCAAGTT-3’
CTLA4-Ig monomer and polymer
In certain embodiments, the invention provides clone with the expression cassette that comprises SEQ ID NO:1 (Figure 1A).This kind expression cassette is when comprising at mammalian cell when expressing in the Chinese hamster ovary celI, can cause the production of N and C-terminal variant, thereby make the aminoacid sequence that can have following residue by the protein of expression cassette production: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383 or (iv) 27-382 or randomly (the v) 25-382 of SEQ ID NO:2 or (the vi) 25-383 of SEQ ID NO:2 (Figure 1A) of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2.These protein can be called " SEQ ID NO:2 monomer " or " having SEQ ID NO:2 sequence " monomer in this article.These SEQ ID NO:2 monomers can dimerization, thereby makes the dimer combination for example can comprise: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); And, (vi) and (vi).These different dimers combinations can also be bonded to each other to form tetramer CTLA4-Ig molecule.These monomers, dimer, the tetramer and other polymers can be called " SEQ ID NO:2 protein " or " having SEQ ID NO:2 sequence " protein in this article.Although clone can tightly be produced these variants after translation, variant more generally can be the product of translation back effect in the cell.Clone is also secreted the CTLA4-Ig molecule.A Baxipu (Abatacept) refers to SEQ ID NO:2 protein.
The CTLA4-Ig molecule can comprise for example CTLA4-Ig protein of monomer, dimer, tripolymer, the tetramer, pentamer, six aggressiveness or other polymer forms.The CTLA4-Ig molecule can comprise the ectodomain at least with CTLA4 and the protein blend compound of constant region for immunoglobulin.The CTLA4-Ig molecule can have wild-type or mutant sequence, for example, and with respect to CTLA4 ectodomain and constant region for immunoglobulin sequence.Individually, or the CTLA4-Ig monomer of dimer, the tetramer or other polymer forms can be glycosylated.
In some embodiments, the invention provides the colony of the CTLA4-Ig molecule of dimer with certain percentage at least or other multimeric molecules.For example, the invention provides and surpass 90%, 95%, 96%, 97%, 98%, 99% or the dimeric CTLA4-Ig molecule of 99.5%CTLA4-Ig colony.In one embodiment, the invention provides and comprise about 99.5%CTLA4-Ig dimer of about 95%-and the tetrameric CTLA4-Ig molecule of the about 5%CTLA4-Ig of about 0.5%-colony.In another embodiment, CTLA4-Ig molecule colony comprises about 98% CTLA4-Ig dimer, about 1.5% the CTLA4-Ig tetramer and about 0.5% CTLA4-Ig monomer.
In one embodiment, the invention provides the colony that colony wherein is substantially free of the CTLA4-Ig molecule of CTLA4-Ig monomer molecule.Be substantially free of the CTLA4-Ig monomer molecule and can refer to have the colony that is less than 1%, 0.5% or 0.1% monomeric CTLA4-Ig molecule.
In one embodiment, the invention provides the colony that colony wherein is substantially free of the polymeric CTLA4-Ig molecule of CTLA4-Ig, described CTLA4-Ig polymer is greater than dimer, for example the tetramer, six aggressiveness etc. (for example, high molecular weight species).Be substantially free of the colony that can refer to have the CTLA4-Ig molecule that is less than 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% CTLA4-Ig polymer (for example, high molecular weight species) greater than dimeric CTLA4-Ig multimeric molecule greater than dimeric forms.
The CTLA4-Ig monomer molecule for example can have, following aminoacid sequence: (i) 26-383 of SEQ IDNO:2, (ii) 26-382, (iii) 27-383 or (iv) 27-382 or randomly (the v) 25-382 of SEQID NO:2 or (the vi) 25-383 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2.When the expression cassette of the nucleotide sequence that comprises SEQID NO:1 is expressed in Chinese hamster ovary celI, the dominant monomeric form of expressing has the N-terminal amino-acid residue (residue 27 of SEQ ID NO:2) of methionine(Met), the N-terminal amino-acid residue of its corresponding wild-type human CTLA 4.Yet, because SEQID NO:1 also comprises the encoding sequence of oncostatin M signal sequence (the Nucleotide 11-88 of SEQ ID NO:1), so comprise the oncostatin M signal sequence by SEQ ID NO:1 expressed protein.Signal sequence is being exported from cytoplasmic protein, or cuts with expressed protein during being secreted into extracellular process.But cutting can cause the N-terminal variant, cutting (the N-terminal that causes residue 26 between the amino-acid residue 25 and 26 of SEQ ID NO.2 for example, i.e. " Ala variant "), or the cutting (N-terminal that causes residue 25 between the amino- acid residue 24 and 25 of SEQ ID NO.2, i.e. " Met-Ala variant "), and the cutting between the amino-acid residue 26 and 27 of SEQ ID NO.2 (N-terminal that causes residue 27) compares.For example, the Met-Ala variant can be present in the mixture of CTLA4-Ig molecule with about 1%, and the Ala variant can be present in the mixture of CTLA4-Ig molecule with about 8-10%.In addition, because not exclusively processing can have the C-terminal variant by SEQ ID NO:1 expressed protein.Dominant C-terminal is the glycine at residue 382 places of SEQ ID NO:2.In the mixture of CTLA4-Ig molecule, the monomer with the Methionin (residue 383 of SEQ ID NO:2) at the C-terminal place can for example exist with about 4-5%.
In one embodiment, the CTLA4-Ig molecule has the aminoacid sequence (this amino acid 25-383 with SEQ ID NO:2 is identical) of following SEQ ID NO:5:
[SEQ?ID?NO：5]
MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM
MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGT
QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK；
In another embodiment, the CTLA4-Ig molecule has the aminoacid sequence (this amino acid 26-383 with SEQ ID NO:2 is identical) of following SEQ ID NO:6:
[SEQ?ID?NO：6]
AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM
GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI
YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK；
In another embodiment, the CTLA4-Ig molecule has the aminoacid sequence (this amino acid 27-383 with SEQ ID NO:2 is identical) of following SEQ ID NO:7:
[SEQ?ID?NO：7]
MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY
VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK；
In another embodiment, the CTLA4-Ig molecule has the aminoacid sequence (this amino acid 25-382 with SEQ ID NO:2 is identical) of following SEQ ID NO:8:
[SEQ?ID?NO：8]
MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM
MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGT
QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPG；
In one embodiment, the CTLA4-Ig molecule has the aminoacid sequence (this amino acid 26-382 with SEQ ID NO:2 is identical) of following SEQ ID NO:9:
[SEQ?ID?NO：9]
AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM
GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI
YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPG；
In one embodiment, the CTLA4-Ig molecule has the aminoacid sequence (this amino acid 27-382 with SEQ ID NO:2 is identical) of following SEQ ID NO:10:
[SEQ?ID?NO：10]
MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY
VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPG.
The CTLA4-Ig monomer molecule can comprise the ectodomain of human CTLA 4.In one embodiment, ectodomain can comprise the nucleotide sequence of the Nucleotide 89-463 of SEQ ID NO:1, the amino acid 27-151 of its coding SEQ ID NO:2.In another embodiment, ectodomain can comprise the mutant sequence of human CTLA 4.In another embodiment, the Nucleotide that ectodomain can comprise the Nucleotide 89-463 of SEQ ID NO:1 changes, thereby makes that carrying out conserved amino acid changes.In another embodiment, ectodomain can comprise the nucleotide sequence identical with the Nucleotide 89-463 at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO:1.
The CTLA4-Ig monomer molecule can comprise the constant region of human normal immunoglobulin.This constant region can be the part of constant region; This constant region can have wild-type or mutant sequence.Constant region can be from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE.Constant region can be from the light chain or the heavy chain of immunoglobulin (Ig).When constant region is divided the period of the day from 11 p.m. to 1 a.m from IgG, IgD or IgA, constant region can comprise one or more in the following constant region structural domain: C L, CH1, hinge, CH2 or CH3.When constant region during from IgM or IgE, constant region can comprise one or more in the following constant region structural domain: C L, C H1, C H2, C H3 or C H4.In one embodiment, constant region can comprise the one or more constant region structural domains from IgG, IgD, IgA, IgM or IgE.
In one embodiment, the CTLA4-Ig dimer comprises 2 monomers, wherein each monomer can have identical or different aminoacid sequence, and wherein sequence can be following aminoacid sequence: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:2 and (the vi) 25-383 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2.This kind CTLA4-Ig monomer can pass through the cysteine amino dimerization of the ectodomain of human CTLA 4 sequence via the 146th place of SEQ ID NO:2.
The CTLA4-Ig molecule can be by the interaction multimerization of IgM or IgA constant region structural domain and J chain protein.IgM and IgA usually as and other polypeptide chain---the polymer of J chain combination and producing.In five poly-IgM, monomer is cross-linked to each other by disulfide linkage in the CH3 structural domain, and crosslinked by CH4 structural domain and J chain.IgM also can form six aggressiveness that lack the J chain, and wherein multimerization is by reaching with separately disulfide linkage.In dimerization IgA, monomer has via its CH3 structural domain and J chain rather than disulfide linkage each other.Therefore, in one embodiment, the invention provides the CTLA4-Ig polymer, comprise dimer, pentamer and six aggressiveness, wherein Ig partly comprises IgM constant region or its part or IgA constant region or its part.This kind CTLA4-Ig polymer based on IgM or IgA can comprise the J chain.
In one embodiment, CTLA4-Ig monomer molecule (CTLA4 Genbank registration number I13253) comprises modified human IgG1's hinge area (Nucleotide 464-508 of SEQ ID NO:1; The amino acid/11 52-166 of SEQ ID NO:2), wherein the amino-acid residue 156,162 of SEQ ID NO:2 and the Serine at 165 places are undertaken engineered by the halfcystine that exists in the wild-type sequence.
In one embodiment, the CTLA4-Ig monomer molecule comprises modified human IgG1 CH2 district and wild-type CH3 district (modified human IgG1 C H2 structural domains have the Nucleotide 509-838 of SEQ ID NO:1 and the amino acid/11 67-276 of SEQ ID NO:2; Human IgG1 C H3 structural domains have the Nucleotide 839-1159 of SEQ ID NO:1 and the amino acid 277-383 of SEQ ID NO:2).
In one embodiment, CTLA4-Ig molecule colony comprise have as among Fig. 7,8 or 9 of publication number US2002/0182211 A1 laid-open U.S. Patents application any one or a plurality of, and as the sequence shown in publication number US20030083246 and the application of US20040022787 laid-open U.S. Patents, described patent application integral body separately is hereby incorporated by.
In one embodiment, CTLA4-Ig tetramer molecule comprises the dimer of 2 pairs of CTLA4-Ig polypeptide or 2 CTLA4-Ig polypeptide, and wherein each polypeptide has one of following aminoacid sequence: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:2 and (the vi) 25-383 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2.Polypeptide to or dimeric each member and another member covalently bound, thereby and the 2 pairs of polypeptide form the tetramer with non-covalent combination the each other.This kind tetramer molecule can combine with CD80 or CD86.In another embodiment, this kind tetramer molecule can combine with CD80 or CD86, and its avidity is a CTLA4-Ig dimer (monomer whose has one of above-mentioned aminoacid sequence) and at least 2 times of the avidity of CD80 or CD86.In another embodiment, this kind tetramer molecule can combine with CD80 or CD86, and its avidity is the binding affinity of wild-type CTLA4 and CD80 or CD86 or at least 2 times of avidity.The avidity that this kind is bigger can be facilitated treatment immune disorders and other diseases as described below, and suppresses the higher effect in tissue and/or the solid organ transplantation repulsion.In addition, avidity bigger or that improve can produce the result of higher pharmaceutical efficacy.For example, comprising the tetrameric therapeutic composition of CTLA4-Ig will have and have a higher avidity of the monomeric therapeutic composition of CTLA4-Ig, and therefore higher effectiveness than same amount.In another embodiment, compare with CTLA4-Ig dimer (monomer whose has one of above-mentioned aminoacid sequence), this kind tetramer molecule can have at least 2 times bigger inhibition to T cell proliferation.In another embodiment, compare with wild-type CTLA4 molecule, this kind tetramer molecule can have the bigger inhibition at least 2 times of T cell proliferations.
CTLA4 A29YL104E -Ig monomer, dimer and polymer
CTLA4 A29YL104E-Ig is CTLA4-Ig (Figure 1A; SEQ ID NOS:1-2) modified forms.Modification by the point mutation that causes 2 amino-acid substitutions (L104E and A29Y) as shown in Figure 2 (corresponding to the amino acid position among Fig. 3 55 and 130; SEQ ID NO:4) forms.With respect to CTLA4-Ig, CTLA4 A29YL104E-Ig (for example, SEQ ID NOS:5-10) with the avidity of about 2 times of increases in conjunction with CD80 (B7-1) and with the avidity of about 4 times of increases in conjunction with CD86 (B7-2).CTLA4 A29YL104EAspect-Ig kills and wounds in suppressor T cell propagation, cytokine production with by the CD28 dependency target cell of natural killer cell than CTLA4-Ig about 10 times more effective.CTLA4 A29YL104E-Ig causes the inhibition of the T cell proliferation appropriateness of B7-1 mediation, but obviously more effective than CTLA4-Ig aspect the T cell proliferation of blocking-up B7-2 mediation.The effectiveness that increases is comparable, no matter is to block B7-2 or blocking-up B7-1 and B7-2 separately, thus hint CTLA4 A29YL104EThe tuning active most probable of-Ig enhanced immunity belongs to the effectiveness of enhanced blocking-up B7-2.
CTLA4 A29YL104E-Ig is genetic engineering modified fusion rotein, and its Fc structural domain by the human normal immunoglobulin of the function binding domains of modified people CTLA-4 and IgG1 class is formed (Fig. 3 A-B).2 amino-acid substitutions carry out in the B7 of CTLA-4 structural domain calmodulin binding domain CaM (L104E and A29Y), to produce CTLA4 A29YL104E-Ig molecule.CTLA4 A29YL104E-Ig dimer comprises 2 glycosylated CTLA4 A29YL104E-Ig chain.It exists as the covalent dimer that connects by interchain disulfide bond.CTLA4 A29YL104EThe molecular model of-Ig molecule is shown among Fig. 4.CTLA4 A29YL104EIt is about 91 that-Ig molecule has, and the average quality of 800Da is as by (MALDI-TOF) mass spectrometric determination of substance assistant laser desorpted ionized flight time.
In certain embodiments, the invention provides clone with the expression cassette that comprises SEQ ID NO:3.This kind expression cassette is when when mammalian cell is for example expressed in the Chinese hamster ovary celI, can cause the production of N and C-terminal variant, thereby make the aminoacid sequence that can have following residue by the polypeptide of expression cassette production: (i) 26-383 of SEQ ID NO:4, (ii) 26-382, (iii) 27-383 or (iv) 27-382 or randomly (the v) 25-382 of SEQ ID NO:4 or (the vi) 25-383 of SEQ ID NO:4 of SEQID NO:4 of SEQ ID NO:4 of SEQID NO:4.These polypeptide can be called " SEQ ID NO:4 monomer " or " having SEQ ID NO:4 sequence " monomer in this article.
These SEQ ID NO:4 monomers can dimerization, thereby makes the dimer combination for example can comprise: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); And, (vi) and (vi).These different dimers combinations can also be bonded to each other to form tetramer CTLA4 A29YL104E-Ig molecule.These monomers, dimer, the tetramer and other polymers can be called " SEQID NO:4 polypeptide " or " having SEQ ID NO:4 sequence " polypeptide in this article.Although clone can tightly be produced these variants after translation, variant more generally can be the product of translation back effect in the cell.Dimer can be together covalently bound, non-covalent linking together, or both.The invention provides the composition of forming by covalent attachment dimer together basically.For example, the invention provides the composition that at least 50% CTLA4-Ig dimer wherein is made of covalently bound monomer.The composition that the present invention also provides at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% CTLA4-Ig dimer wherein to be made of covalently bound monomer.The present invention also provides the composition of CTLA4-Ig molecule, and wherein molecule is the dimerization form with preponderating, and dimer is formed by covalent linkage with preponderating.For example, the invention provides the covalently bound composition of wherein most of CTLA4-Ig dimers.The non-covalent connection of dimeric some fraction of CTLA4-Ig in the composition, this is possible.
CTLA4 A29YL104E-Ig molecule can comprise for example CTLA4 of monomer, dimer, tripolymer, the tetramer, pentamer, six aggressiveness or other polymer forms A29YL104E-Ig.CTLA4 A29YL104E-Ig molecule can comprise the ectodomain at least (SEQ ID NO:18) with modified CTLA4 and the protein blend compound of constant region for immunoglobulin.CTLA4 A29YL104E-Ig molecule can have the mutant sequence, for example, and with respect to modified CTLA4 ectodomain and constant region for immunoglobulin sequence.Individually, or the CTLA4 of dimer, the tetramer or other polymer forms A29YL104E-Ig monomer can be glycosylated.
In certain embodiments, the invention provides and have at least the dimer of certain percentage or the CTLA4 of other multimeric molecules A29YL104EThe colony of-Ig molecule.For example, the invention provides and surpass 90%, 95%, 96%, 97%, 98%, 99% or 99.5%CTLA4 A29YL104EThe dimeric CTLA4 of-Ig A29YL104E-Ig molecule colony.In one embodiment, the invention provides and comprise the about 99.5%CTLA4 of about 95%- A29YL104E-Ig dimer and the about 5%CTLA4 of about 0.5%- A29YL104EThe tetrameric CTLA4 of-Ig A29YL104E-Ig molecule colony.In further embodiment, the invention provides and comprise about 99.5% CTLA4 of about 95%- A29YL104E-Ig dimer, the about 2.5%CTLA4 of about 0.5%- A29YL104E-Ig monomer and the about 5%CTLA4 of about 0.5%- A29YL104EThe tetrameric CTLA4 of-Ig A29YL104E-Ig molecule colony.In another embodiment, CTLA4 A29YL104E-Ig molecule colony comprises about 96% CTLA4 A29YL104E-Ig dimer, about 2.5% CTLA4 A29YL104E-Ig the tetramer and about 0.5% CTLA4 A29YL104E-Ig monomer.
In one embodiment, the invention provides wherein, colony is substantially free of CTLA4 A29YL104EThe monomeric CTLA4 of-Ig A29YL104EThe colony of-Ig molecule.Be substantially free of CTLA4 A29YL104E-Ig monomer can refer to have and is less than 1%, 0.5% or 0.1% monomeric CTLA4 A29YL104EThe colony of-Ig molecule.
In another embodiment, the invention provides wherein, colony is substantially free of CTLA4 A29YL104EThe polymeric CTLA4 of-Ig A29YL104EThe colony of-Ig molecule, described CTLA4 A29YL104E-Ig polymer is greater than dimer, for example the tetramer, six aggressiveness etc.Be substantially free of greater than dimeric CTLA4 A29YL104E-Ig polymer can refer to have and is less than 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% greater than dimeric CTLA4 A29YL104EThe polymeric CTLA4 of-Ig A29YL104EThe colony of-Ig molecule.
CTLA4 A29YL104E-Ig monomer for example can have, following aminoacid sequence: (i) 26-383 of SEQID NO:4, (ii) 26-382, (iii) 27-383 or (iv) 27-382 or randomly (the v) 25-382 of SEQ ID NO:4 or (the vi) 25-383 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ IDNO:4 of SEQ ID NO:4.When the expression cassette of the nucleotide sequence that comprises SEQ ID NO:3 or 23 is expressed in Chinese hamster ovary celI, the dominant monomeric form of expressing has the N-terminal amino-acid residue (residue 27 of SEQ IDNO:4) of methionine(Met), and it is corresponding to the N-terminal amino-acid residue of human CTLA 4.Yet, because SEQ ID NO:23 also comprises the encoding sequence of oncostatin M signal sequence (the Nucleotide 11-88 of SEQ ID NO:23), so comprise the oncostatin M signal sequence by SEQ ID NO:23 expressed protein.
Signal sequence is being exported from cytoplasmic protein, or cuts with expressed protein during being secreted into extracellular process.But cutting can cause the N-terminal variant, cutting (the N-terminal that causes residue 26 between the amino-acid residue 25 and 26 of SEQ ID NO:4 for example, i.e. " Ala variant "), or the cutting (N-terminal that causes residue 25 between the amino- acid residue 24 and 25 of SEQ ID NO:4, i.e. " Met-Ala variant "), and the cutting between the amino-acid residue 26 and 27 of SEQ ID NO:4 (cause begin from the Met residue of amino acid position 27 N-terminal) compares.For example, the Met-Ala variant can be present in CTLA4 with about 1% A29YL104EIn the mixture of-Ig molecule, and the Ala variant is present in CTLA4 with about 10-20% A29YL104EIn the mixture of-Ig molecule.
In addition, because not exclusively processing, can have the C-terminal variant by the protein of the expression of nucleic acid that comprises SEQ ID NO:3.Dominant C-terminal is the glycine at residue 382 places of SEQ ID NO:4.At CTLA4 A29YL104EIn the mixture of-Ig molecule, the monomer with the Methionin (residue 383 of SEQ ID NO:4) at the C-terminal place can for example exist with about 4-8%.
In one embodiment, CTLA4 A29YL104E-Ig molecule comprises the aminoacid sequence (this amino acid 25-383 with SEQ ID NO:4 is identical) of following SEQ ID NO:11:
[SEQ?ID?NO：11]
MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYM
MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGT
QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK.
In another embodiment, CTLA4 A29YL104E-Ig molecule comprises the aminoacid sequence (this amino acid 26-383 with SEQ ID NO:4 is identical) of following SEQ ID NO:12:
[SEQ?ID?NO：12]
AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMM
GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI
YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK.
In further embodiment, CTLA4 A29YL104E-Ig molecule comprises the aminoacid sequence (this amino acid 27-383 with SEQ ID NO:4 is identical) of following SEQ IDNO:13:
[5EQ?ID?NO：13]
MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY
MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY
VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK.
In another embodiment, CTLA4 A29YL104E-Ig molecule comprises the aminoacid sequence (this amino acid 25-382 with SEQ ID NO:4 is identical) of following SEQ ID NO:14:
[SEQ?ID?NO：14]
MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYM
MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGT
QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPG.
In one embodiment, CTLA4 A29YL104E-Ig molecule comprises the aminoacid sequence (this amino acid 26-382 with SEQ ID NO:4 is identical) of following SEQ ID NO:15:
[SEQ?ID?NO：15]
AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMM
GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI
YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPG.
In further embodiment, CTLA4 A29YL104E-Ig molecule comprises the aminoacid sequence (this amino acid 27-382 with SEQ ID NO:4 is identical) of following SEQ IDNO:16:
[SEQ?ID?NO：16]
MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG
NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY
VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPG.
CTLA4 A29YL104E-Ig monomer can comprise the ectodomain of human CTLA 4, wherein carries out 2 amino-acid substitutions (L104E and A29Y) (Fig. 5) in the CTLA-4 structural domain.In one embodiment, ectodomain can comprise the nucleotide sequence of the Nucleotide 89-463 of SEQ ID NO:23, the amino acid 27-151 of its coding SEQ ID NO:4.In another embodiment, ectodomain can comprise the mutant nucleotide sequence (for example, the substance among the amino acid 27-151 of SEQ IDNO:4, dual and triple site mutation body) of human CTLA 4.In another embodiment, the Nucleotide that ectodomain can comprise the Nucleotide 89-463 of SEQ ID NO:23 changes, thereby makes that carrying out conserved amino acid changes.In further embodiment, the Nucleotide that ectodomain can comprise the Nucleotide 89-463 of SEQ ID NO:23 changes, thereby makes that carrying out non-conserved amino acid changes.In another embodiment, ectodomain can comprise the nucleotide sequence identical with the Nucleotide 89-463 at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO:23.
CTLA4 A29YL104E-Ig monomer can comprise the constant region of human normal immunoglobulin.This constant region can be the part of constant region.This constant region can also have wild-type or mutant sequence.Constant region can be from human IgG 1, IgG 2, IgG 3, IgG 4, IgM, IgA 1, IgA 2, IgD or IgE.Constant region can be from the light chain or the heavy chain of immunoglobulin (Ig).When constant region is divided the period of the day from 11 p.m. to 1 a.m from IgG, IgD or IgA, constant region can comprise one or more in the following constant region structural domain: C L, C H1, hinge, C H2 or C H3.When constant region during from IgM or IgE, constant region can comprise one or more in the following constant region structural domain: C L, C H1, C H2, C H3 or C H4.In one embodiment, constant region can comprise the one or more constant region structural domains from IgG, IgD, IgA, IgM or IgE.
In one embodiment, CTLA4 A29YL104E-Ig dimer comprises 2 monomers, wherein each monomer can have identical or different aminoacid sequence, and wherein sequence can be following aminoacid sequence: (i) 26-383 of SEQ ID NO:4, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:4 and (the vi) 25-383 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4.This kind CTLA4 A29YL104E-Ig monomer can pass through cysteine amino (or the cysteine amino at 120th place of Fig. 5) dimerization of the ectodomain of human CTLA 4 sequence via the 146th place of SEQ ID NO:4.
CTLA4 A29YL104E-Ig molecule can be by the interaction multimerization of IgM or IgA constant region structural domain and J chain protein.IgM and IgA usually as and other polypeptide chain---the polymer of J chain combination and producing.In five poly-IgM, monomer is at C HBe cross-linked to each other by disulfide linkage in 3 structural domains, and pass through C H4 structural domains and J chain are crosslinked.IgM also can form six aggressiveness that lack the J chain, and wherein multimerization is by reaching with separately disulfide linkage.In dimerization IgA, monomer has via its C H3 structural domains and J chain rather than disulfide linkage each other.Therefore, in one embodiment, the invention provides CTLA4 A29YL104E-Ig polymer comprises dimer, pentamer and six aggressiveness, and wherein Ig partly comprises IgM constant region or its part or IgA constant region or its part.This kind CTLA4 based on IgM or IgA A29YL104E-Ig polymer can comprise the J chain.
In one embodiment, CTLA4 A29YL104E-Ig monomer comprises modified human IgG1's hinge area (Nucleotide 464-508 of SEQ ID NO:23; The amino acid/11 52-166 of SEQ ID NO:4), wherein the serine residue at the 156th, 162 and 165 of SEQ ID NO:4 the place is undertaken engineered by the halfcystine that exists in the wild-type sequence.
In one embodiment, CTLA4 A29YL104E-Ig monomer comprises modified human IgG1 CH2 district and wild-type CH3 district (modified human IgG1 C H2 structural domains have the Nucleotide 509-838 of SEQ ID NO:1 and the amino acid/11 67-276 of SEQ ID NO:2; Human IgG1 C H3 structural domains have the Nucleotide 839-1159 of SEQ ID NO:1 and the amino acid 277-383 of SEQ ID NO:2).
In one embodiment, CTLA4 A29YL104E-Ig molecule colony comprises and has U.S. Patent Application Publication No. U.S.2002/0039577, U.S.2003/0007968, U.S.2004/0022787, U.S.2005/0019859 and U.S.2005/0084933, and U.S. Patent number 7,094, the monomer of the sequence shown in 874, described patent document integral body separately is hereby incorporated by.
In one embodiment, CTLA4 A29YL104E-Ig the tetramer comprises 2 couples of CTLA4 A29YL104E-Ig molecule or 2 CTLA4 A29YL104EThe dimer of-Ig molecule, wherein each polypeptide has one of following aminoacid sequence: (i) 26-383 of SEQ ID NO:4, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:4 and (the vi) 25-383 of SEQ IDNO:4 of SEQID NO:4 of SEQ ID NO:4 of SEQ ID NO:4.Polypeptide to or dimeric each member and another member covalently bound, thereby and the 2 pairs of polypeptide form the tetramer with non-covalent combination the each other.This kind tetramer molecule can combine with CD80 or CD86.In another embodiment, this kind tetramer molecule can combine with CD80 or CD86, and its avidity is CTLA4 A29YL104EAntigenic at least 2 times of the binding antibody parent of-Ig dimer (monomer whose has one of above-mentioned aminoacid sequence) and CD80 or CD86.
The avidity that this kind is bigger can be facilitated treatment immune disorders and other diseases as described below, and suppresses the higher effect in tissue and/or the solid organ transplantation repulsion.In addition, avidity bigger or that improve can produce the result of higher pharmaceutical efficacy.For example, comprise CTLA4 A29YL104EThe tetrameric therapeutic composition of-Ig will have the CTLA4 that has than same amount A29YL104EThe avidity that the monomeric therapeutic composition of-Ig is higher, and so higher effectiveness.In another embodiment, with CTLA4 A29YL104E-Ig dimer (monomer whose has one of above-mentioned aminoacid sequence) is compared, and this kind tetramer molecule can have the bigger inhibition at least 2 times of T cell proliferations.In another embodiment, compare with CTLA4-Ig tetramer molecule, this kind tetramer molecule can have the bigger inhibition at least 2 times of T cell proliferations.
CTLA4-Ig and CTLA4 A29YL104E The sign of-Ig molecule
T cell proliferation can use standard test known in the art to measure.For example, one of common methods of assessment T cell proliferation is to stimulate the T cell via antigen or at the antagonistic antibodies of TCR, and measure the tritiated thymidine that mixes in the proliferative T cell for example ( 3H-TdR) or be discharged into the amount of the cytokine in the culture via proliferative T cell.Therefore can measure CTLA4-Ig or CTLA4 A29YL104E-Ig molecule is to T cell activation or inhibition of proliferation effect.
The avidity of CTLA4-Ig molecule is that molecule and single part comprise CD80, CD86 or CD80Ig or CD86Ig fusion rotein bonded intensity.CTLA4-Ig or CTLA4 A29YL104EThe avidity of-Ig and part can for example be measured based on the binding interactions analysis (BIA) of surface plasma body technique by using.Except that measuring bonding strength, it allows The real time measure binding kinetics, for example combination and the rate constant of dissociating.Surface matrix with it covalent attachment, by the sensor chip of forming with the slide glass of thin metal film bag quilt, be CTLA4-Ig, CTLA4 with one of interactant (interactant) A29YL104EOne of-Ig or part wrap quilt.Allow to comprise the solution stream of another interactant through its surface.The opposite side of continuous light beam alignment surface, and measure its reflection angle.CTLA4-Ig or CTLA4 A29YL104EWhen-Ig combines with part, the resonance angle of light beam change (because it depends on the specific refractory power of the substratum of the reaction side that approaches transmitter, this successively with substratum in the positive correlation of dissolved concentration of material).It is analyzed by means of computer subsequently.
In one embodiment, CTLA4-Ig can pass through surface plasma body resonant vibration (SPR) and carries out on BIAcore instrument (BIAcore AG, Uppsala, Sweden) in conjunction with experiment.CTLA4-Ig can be by the carboxymethylated dextran matrix covalent coupling on primary amine group and the BIAcore sensor chip, thereby makes CTLA4-Ig be immobilized into sensor chip.Alternately, anti-constant region antibody can be used for making the CTLA4-Ig indirect securementization to sensor surface via the Ig fragment., part added in chip, to measure and part bonded CTLA4-Ig thereafter.Avidity is measured can be for example as van der Merwe, people such as P., J.Exp.Med.(1997) 185 (3): carry out described in the 393-404, described reference integral body is hereby incorporated by.In another embodiment, CTLA4 A29YL104E-Ig can use surface plasma body resonant vibration (SPR) technology to carry out (Fig. 6 in conjunction with experiment as mentioned above; Referring to embodiment 21).
Can also measure CTLA4-Ig or CTLA4 A29YL104EThe avidity of-Ig molecule.Avidity can be defined as 2 kinds of molecules or cell on a plurality of sites with the intensity summation that is bonded to each other.Avidity is different from avidity, and described avidity is 1 site and its part bonded intensity on the molecule.Not bound by theory, CTLA4-Ig or CTLA4 A29YL104EThe higher avidity of-Ig molecule can cause via CTLA4-Ig or CTLA4 A29YL104E-Ig molecule is to the effectiveness of the increase of T cell proliferation and activatory inhibition.Avidity can for example be measured by 2 class Solid-phase Assays: a) competitive inhibition is measured, and b) wash-out mensuration.In 2 kinds of mensuration, part and solid support adhere to.In competitive inhibition is measured, CTLA4-Ig or CTLA4 A29YL104E-Ig molecule subsequently in solution with fixed concentration, add together with the free ligand of different concns, and measure and make solid phase in conjunction with the amount that suppresses 50% part.The part that needs is few more, and avidity is strong more.In wash-out was measured, part added in solution.After obtaining equilibrium state, chaotropic agent or denaturing agent (for example lsothiocyanates, urea or diethylamine) add with different concns, to destroy CTLA4-Ig/ ligand interaction or CTLA4 A29YL104E-Ig/ ligand interaction.With ELISA measure the CTLA4-Ig or the CTLA4 that withstand wash-out thereafter A29YL104EThe amount of-Ig.Avidity is high more, a certain amount of CTLA4-Ig of wash-out or CTLA4 A29YL104EThe required chaotropic agent of-Ig is many more.CTLA4-Ig molecule or CTLA4 A29YL104EThe relative avidity of the heterogeneous mixture of-Ig can be expressed as avidity index (AI), equals wash-out 50% bonded CTLA4-Ig or CTLA4 A29YL104EThe eluant strength that-Ig molecule is required.The Accurate Analysis of data can be when being determined at the different concns of eluent the CTLA4-Ig or the CTLA4 of wash-out A29YL104E-Ig percentage recently carries out.
Phenyl sepharose 4 (the Phenyl Sepharose 4 Fast Flow) column chromatography that flows fast, hydrophobic interaction chromatography (HIC) method can be used for reducing the amount (referring to embodiment 15) of the CTLA4-Ig high molecular weight species of HIC purification step wash-out.Therefore, the removing peak from the HIC post is rich in CTLA4-Ig HMW kind.For example, preparation type list or columns in series SEC HPLC can be used for purifying is removed the peak material from HIC dimer, the tetramer and polymer subgroup.In one embodiment, the component of purifying is CTLA4-Ig dimer, the tetramer and six aggressiveness.HIC removes the sign of the high molecular weight component of the CTLA4-Ig that exists in the peak and can be undertaken by static and dynamic light scattering technology.Dimer, the tetramer and polymeric existence when the sample that obtains when hydrophobic interaction chromatography (HIC) method steps is followed the trail of is disclosed in each sampling spot.Six aggressiveness kinds can only detect in the sample corresponding to " removing the initial of peak " and " removing the maximum OD in peak ".Ten aggressiveness kinds only detect in " removing the maximum OD in peak ".Molar mass and hydrodynamic radius form can adopt with quasi-elastic light scattering (QELS) and detect the polygonal scattering of light of link coupled (MALS), measure via size exclusion chromatography (SEC) by fractional separation.
With regard to the CTLA4-Ig molecule of being produced by clone, SEC shows that A albumen eluate is the mixture of polymer, the tetramer and dimer component.The fractional separation of this mixture on preparation type series connection SEC post makes it possible to separate a large amount of polymers, the tetramer and dimer kind.Area percentage about every kind of component in the SEC analysis of isolating fraction reclaims the homogeneity that causes for every kind of fraction 93-98%.In one aspect, the purifying of individual components makes it possible to dimeric those the physicochemical property of the component of comparison CTLA4-Ig HMW material and CTLA4-Ig.Fig. 7 shows apparent molecular weight, and it is corresponding to polymer, the tetramer and the dimer fraction at CTLA4-Ig HIC removing peak, as what measure with dynamic light scattering detection (DSL) and the retention time on SEC by SEC.In one embodiment, from HIC remove the peak purified components biologic specificity in conjunction with active with combine can comparing that mensuration measured in conjunction with activity by immobilization B7-1Ig based on BIAcore.In yet another aspect, be 4.9-7.6 about the sialic acid mol ratio of removing isolating component the peak from HIC, and the sialic acid mol ratio of CTLA4-Ig molecule or dimer (not removing in the peak at HIC) is 8-10.Point out to compare with the dimeric migration of CTLA4-Ig by the IEF gel analysis, the movability of being removed the CTLA4-Ig isoform of peak purifying by HIC reduces.This with remove the peak observed lower sialic acid mol ratio of fraction consistent (Fig. 8) for CTLA4-Ig HIC.
The selection of cell culture condition can influence the strand (that is monomer) of recombinant protein product and the formation of high molecular weight species (that is, dimer, the tetramer etc.).Growth conditions also includes but not limited to the substratum composition, is the factor that can influence strand formation and cysteinyl level.This may be the result who causes the reagent existence of disulfide bond reduction.Give secretion CTLA4-Ig or CTLA4 A29YL104EThe substratum that the cell of-Ig is added halfcystine directly or comprised halfcystine can cause the quick formation of strand and high molecular weight species.The amount of the halfcystine of speed and interpolation is proportional.In another embodiment, with the compound of free sulfhydryl groups reaction---iodo-acid amide add CTLA4-Ig or the CTLA4 that blocking-up depends on disulfide linkage A29YL104EThe formation of-Ig high molecular weight species.
For example, iodo-acid amide susceptibility and non-sensibility high molecular approach emphasize can form among the CTLA4-Ig 2 kinds of main and visibly different mechanism of high molecular weight species.Add that high salt concentration (0.5M) causes continuing, high molecular rate of formation fast for CTLA4-Ig solution.EDTA, ConAcidSol II and yeast autolysis solution (yeastolates) moderately increase strand and form (referring to embodiment 5).
In certain embodiments, the invention provides the method that is used for production high molecular CTLA4-Ig colony, wherein comprise the CTLA4-Ig monomer or dimeric mixture has been added high salt with preponderating, thereby make mixture have to surpass about 0.3,0.4,0.45,0.5 or the salt concn of 0.6M.In one embodiment, this kind method produces the mixture comprise CTLA4-Ig colony, and described CTLA4-Ig colony has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%CTLA4-Ig tetramer molecule.
In one embodiment, the invention provides and be included in Cys 146On the colony of CTLA4-Ig strand kind of modification, thereby make that it is cysteinylization (referring to embodiment 4).Cysteinylization is posttranslational modification, and wherein polypeptide intrachain halfcystine is modified by adhere to another halfcystine via disulfide linkage.Proteinic cysteinylization has involved the modifying protein biological activity, comprises the immunogenicity and the antigenicity of the viral determinant of MHC I class restriction.In one embodiment, the invention provides the composition of the strand CTLA4-Ig molecule that comprises at least 1,5,10,15,20,25,50,75,90 or 95% cysteinylization.In another embodiment of the invention, CTLA4-Ig colony has and is no more than about 1%CTLA4-Ig monomer molecule, or in another embodiment, less than the 0.6%CTLA4-Ig monomer.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the sialic acid of about 5-about 18 and the molar average ratio of CTLA4-Ig molecule.In some embodiments, sialic acid is the about Y of about X-with the molar average ratio of CTLA4-Ig molecule, comprise X and Y, wherein X is about 4,5,6,7,8,9,10,11,12,13,14,15,16 or 17, and Y is about 5,6,7,8,9,10,11,12,13,14,15,16,17 or 18.In other embodiments, the molar average of sialic acid and CTLA4-Ig molecule comprises X and Y than being the about Y of about X-, and wherein X is about 4.0,4.5,5.0,5.5 or 6.0, and with Y be about 8.0,8.5,9.0,9.5 or 10.0.In other embodiments, the molar average of sialic acid and CTLA4-Ig molecule comprises X and Y than being the about Y of about X-, and wherein X is about 6.0,6.5,7.0,7.5,8.0,8.5 or 9.0, and Y is about 11.0,11.5,12.0,12.5 or 13.0.In other embodiments, the molar average of sialic acid and CTLA4-Ig molecule, about 8-about 12 more about 13 or about 9-about 11 than, about 7-about 14 for about 6-.In other embodiments, about 9, the about 6-about 10 of the molar average of sialic acid and CTLA4-Ig molecule, about 6-more about 9.5 or about 7-about 10 than, about 5.5-about 9 for about 5-.In other embodiments, sialic acid compares more than or equal to 5 or more than or equal to 8 with the molar average of CTLA4-Ig molecule.In certain embodiments, sialic acid is N n acetylneuraminic acid n (NANA).
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has and is less than or equal to 2.5, is less than or equal to 2.0, is less than or equal to 1.5, is less than or equal to 1.0 or be less than or equal to the molar average ratio of 0.5 N hydroxyacetylneuraminic acid (NGNA) and CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule is more than or equal to 93.0 area percentages, more than or equal to 93.5 area percentages, more than or equal to 94.0 area percentages, more than or equal to 94.5 area percentages, more than or equal to 95.0 area percentages, more than or equal to 95.5 area percentages, more than or equal to 96.0 area percentages, more than or equal to 96.5 area percentages, or more than or equal to the CTLA4-Ig dimer of 97.0 area percentages.In some embodiments, composition comprises the CTLA4-Ig molecule, wherein measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule is more than or equal to the CTLA4-Ig dimer of 95.0 area percentages and is less than or equal to the high molecular weight species of 4.0 area percentages.In some embodiments, composition comprises the CTLA4-Ig molecule, wherein measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule is more than or equal to the CTLA4-Ig dimer of 95.0 area percentages and is less than or equal to the high molecular weight species of 5.0 area percentages.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule has and is less than or equal to 2.0 area percentages, is less than or equal to 1.5 area percentages, is less than or equal to 1.0 area percentages, is less than or equal to the CTLA4-Ig monomer (that is strand) of 0.5 area percentage.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule has and is less than or equal to 5.0 area percentages, be less than or equal to 4.5 area percentages, be less than or equal to 4.0 area percentages, be less than or equal to 3.5 area percentages, be less than or equal to 3.0 area percentages, be less than or equal to 2.5 area percentages, be less than or equal to 2.0 area percentages, be less than or equal to 1.5 area percentages, be less than or equal to 1.0 area percentages, or be less than or equal to the CTLA4-Ig high molecular weight species (for example, the tetramer) of 0.5 area percentage.In some embodiments, those that particularly relate to the concentrate composition that comprises the CTLA4-Ig molecule (for example, be used for those of subcutaneous administration), measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule has and is less than or equal to 10 area percentages, is less than or equal to 9 area percentages, is less than or equal to 8 area percentages, is less than or equal to 7 area percentages, is less than or equal to the CTLA4-Ig high molecular weight species of 6 area percentages.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 50ppm, is less than or equal to 40ppm, is less than or equal to 38ppm, is less than or equal to 30ppm, is less than or equal to 20ppm, is less than or equal to 10ppm, 5ppm, is less than or equal to 4ppm, is less than or equal to 3ppm, is less than or equal to 2ppm or is less than or equal to the amount of MCP-1 or the MCP-1 sample material of 1ppm.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 50ng/mg CTLA4-Ig molecule, be less than or equal to 40ng/mg CTLA4-Ig molecule, be less than or equal to 38ng/mg CTLA4-Ig molecule, be less than or equal to 30ng/mg CTLA4-Ig molecule, be less than or equal to 20ng/mg CTLA4-Ig molecule, be less than or equal to 10ng/mg CTLA4-Ig molecule, be less than or equal to the 5ng/mgCTLA4-Ig molecule, be less than or equal to 4ng/mg CTLA4-Ig molecule, be less than or equal to the 3ng/mgCTLA4-Ig molecule, be less than or equal to 2ng/mg CTLA4-Ig molecule or be less than or equal to the MCP-1 or the MCP-1 sample material of 1ng/mg CTLA4-Ig molecule.The invention provides the composition of the amount (comprising not existing of MCP-1) that comprises CTLA4-Ig molecule and MCP-1, wherein said composition is pharmaceutically acceptable composition.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the semi-lactosi of about 6-about 19 and the molar average ratio of CTLA4-Ig molecule.In some embodiments, sialic acid is the about Y of about X-with the molar average ratio of CTLA4-Ig molecule, wherein X is about 6,7,8,9,10,11,12,13,14,15,16,17 or 18, and Y is about 7,8,9,10,11,12,13,14,15,16,17,18 or 19.In other embodiments, semi-lactosi is about X-Y with the molar average ratio of CTLA4-Ig molecule, comprise X and Y, wherein X is about 6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5 or 10.0, and Y is about 12.0,12.5,13.0,13.5,14.0,14.5,15.0,15.5 or 16.0.In other embodiments, semi-lactosi is the about Y of about X-with the molar average ratio of CTLA4-Ig molecule, comprise X and Y, wherein X is about 6.0,6.5,7.0,7.5 or 8.0, and Y is about 15.0,15.5,16.0,16.5,17.0,17.5,18.0,18.5 or 19.0.In other embodiments, the molar average of semi-lactosi and CTLA4-Ig molecule, about 9-about 13 more about 14 or about 10-about 12 than, about 8-about 15 for about 7-.In other embodiments, about 17, the about 9-about 16 of the molar average of semi-lactosi and CTLA4-Ig molecule, about 9-more about 17 or about 10-about 15 than, about 8-about 18 for about 7-.In other embodiments, semi-lactosi compares more than or equal to 8 with the molar average of CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the Fucose of about 0.5-about 12 and the molar average ratio of CTLA4-Ig molecule.In some embodiments, sialic acid is the about Y of about X-with the molar average ratio of CTLA4-Ig molecule, comprise X and Y, wherein X is about 0.5,1.0,1.5,2.0,2.5,3.0,3.5 or 4.5, and Y is about 7.0,7.5,8.0,8.5,9.0,9.5,10.0 or 10.5.In other embodiments, the molar average of Fucose and CTLA4-Ig molecule comprises X and Y than being about X-Y, and wherein X is about 2.9,3.1,3.3,3.5,3.7,3.9 or 4.1, and Y is about 7.9,8.1,8.3,8.5,8.7,8.9 or 9.1.In other embodiments, Fucose is about X-Y with the molar average ratio of CTLA4-Ig molecule, comprise X and Y, wherein X is about 1.0,1.5,1.7,1.9,2.1,2.3 or 2.5, and Y is about 8.7,8.9,9.1,9.3,9.6,9.9,10.1,10.3 or 10.5.In other embodiments, about 8.1, the about 3.9-about 7.9 of the molar average of Fucose and CTLA4-Ig molecule, about 3.7-more about 8.3 than, about 3.5-about 8.5 for about 3.3-.In other embodiments, the molar average of Fucose and CTLA4-Ig molecule, about 1.9-about 9.1 more about 9.3 or about 2.1-about 8.9 than, about 1.7-about 9.5 for about 1.5-.In other embodiments, Fucose compares more than or equal to 1.7 with the molar average of CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the seminose of about 5-about 25 and the molar average ratio of CTLA4-Ig molecule.In some embodiments, sialic acid is the about Y of about X-with the molar average ratio of CTLA4-Ig molecule, comprise X and Y, wherein X is about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 21, and Y is about 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 or 24.In other embodiments, seminose is about X-Y with the molar average ratio of CTLA4-Ig molecule, comprise X and Y, wherein X is about 6.5,7.0,7.5,7.7,7.9,8.1,8.3,8.5,9.0,9.5,10.0,10.5,11.0,11.5 or 12.0, and and Y be about 17.0,17.5,18.0,18.5,19.0,19.5,20.0,20.5,21.0,21.5,22.0,22.5,23,23.5 or 24.0.In other embodiments, the molar average of seminose and CTLA4-Ig molecule comprises X and Y than being about X-Y, and wherein X is about 8,8.5,9.0,9.510.0 or 11.0, and Y is about 17.0,17.5,18.0,18.5,19.0,19.5 or 20.0.In other embodiments, about 19, the about 11-about 19 of about 20, the about 10-of about 21, the about 9-of about 22, the about 8-of the molar average of seminose and CTLA4-Ig molecule, about 7.7-more about 22 and about 11-about 17 than, about 7-about 23 for about 6-.In other embodiments, the molar average of seminose and CTLA4-Ig molecule, about 10-about 17 more about 18 or about 11-about 16 than, about 9-about 19 for about 8-.In other embodiments, seminose compares more than or equal to 7 with the molar average of CTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has and is less than or equal to 5.0 area percentages, is less than or equal to 4.5 area percentages, is less than or equal to 4.0 area percentages, is less than or equal to 3.5 area percentages, is less than or equal to 3.0 area percentages, is less than or equal to 2.5 area percentages, is less than or equal to 2.0 area percentages, is less than or equal to 1.5 area percentages, is less than or equal to 1.0 area percentages or is less than or equal to the oxidation kind of 0.5 area percentage.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has and is less than or equal to 5.0 area percentages, is less than or equal to 4.5 area percentages, is less than or equal to 4.0 area percentages, is less than or equal to 3.5 area percentages, is less than or equal to 3.0 area percentages, is less than or equal to 2.5 area percentages, is less than or equal to 2.0 area percentages, is less than or equal to 1.5 area percentages, is less than or equal to 1.0 area percentages or is less than or equal to the deacylated tRNA amine kind of 0.5 area percentage.In certain embodiments, composition comprises the CTLA4-Ig molecule, and wherein the CTLA4-Ig molecule has oxidation kind that is less than or equal to 3.5 area percentages and the deamidation kind that is less than or equal to 2.5 area percentages.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 0.7EU/mg CTLA4-Ig molecule, be less than or equal to 0.6EU/mg CTLA4-Ig molecule, be less than or equal to 0.5EU/mg CTLA4-Ig molecule, be less than or equal to the 0.42EU/mgCTLA4-Ig molecule, be less than or equal to 0.4EU/mg CTLA4-Ig molecule, be less than or equal to 0.35EU/mg CTLA4-Ig molecule, be less than or equal to 0.3EU/mg CTLA4-Ig molecule, be less than or equal to 0.25EU/mg CTLA4-Ig molecule, be less than or equal to 0.20EU/mg CTLA4-Ig molecule, be less than or equal to 0.15EU/mg CTLA4-Ig molecule, or be less than or equal to the bacterial endotoxin LAL of 0.05EU/mgCTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 2CFU/10mL, is less than or equal to 1.5CFU/10mL, is less than or equal to 1CFU/10mL or is less than or equal to the biological load of 0.5CFU/10mL.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 25pg/mg CTLA4-Ig molecule, be less than or equal to 20pg/mg CTLA4-Ig molecule, be less than or equal to 15pg/mg CTLA4-Ig molecule, be less than or equal to the 10pg/mgCTLA4-Ig molecule, be less than or equal to 5.0pg/mg CTLA4-Ig molecule, be less than or equal to 4.0pg/mg CTLA4-Ig molecule, be less than or equal to 3.5pg/mg CTLA4-Ig molecule, be less than or equal to 3.0pg/mg CTLA4-Ig molecule, be less than or equal to 2.5pg/mg CTLA4-Ig molecule, be less than or equal to 1.5pg/mg CTLA4-Ig molecule, be less than or equal to 1.0pg/mg CTLA4-Ig molecule, or be less than or equal to 0.5pg/mg CTLA4-Ig molecule, or be less than or equal to the DNA of 0.20pg/mlCTLA4-Ig molecule.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 200ng/mg CTLA4-Ig molecule, be less than or equal to 150ng/mg CTLA4-Ig molecule, be less than or equal to 125ng/mg CTLA4-Ig molecule, be less than or equal to the 100ng/mgCTLA4-Ig molecule, be less than or equal to 90ng/mg CTLA4-Ig molecule, be less than or equal to 80ng/mg CTLA4-Ig molecule, 70ng/mg CTLA4-Ig molecule, be less than or equal to the 60ng/mgCTLA4-Ig molecule, be less than or equal to 50ng/mg CTLA4-Ig molecule, be less than or equal to 40ng/mg CTLA4-Ig molecule, be less than or equal to 30ng/mg CTLA4-Ig molecule, be less than or equal to 25ng/mg CTLA4-Ig molecule, be less than or equal to 20ng/mg CTLA4-Ig molecule, be less than or equal to 15ng/mg CTLA4-Ig molecule, be less than or equal to 10ng/mg CTLA4-Ig molecule, or be less than or equal to the cell protein (for example, CHO protein or CHOP) of 5ng/mg CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 200ppm, is less than or equal to 150ppm, is less than or equal to 125ppm, is less than or equal to 100ppm, is less than or equal to 90ppm, is less than or equal to 80ppm, 70ppm, is less than or equal to 60ppm, is less than or equal to 50ppm, is less than or equal to 40ppm, is less than or equal to 30ppm, is less than or equal to 25ppm, is less than or equal to 20ppm, is less than or equal to 15ppm, is less than or equal to 10ppm or is less than or equal to the cell protein of 5ppm.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 4.0ng/mg CTLA4-Ig molecule, is less than or equal to 3.5ng/mg CTLA4-Ig molecule, is less than or equal to 3.0ng/mg CTLA4-Ig molecule, is less than or equal to the 2.5ng/mgCTLA4-Ig molecule, is less than or equal to 2.0ng/mg CTLA4-Ig molecule, is less than or equal to 1.5ng/mg CTLA4-Ig molecule, is less than or equal to 1.0ng/mg CTLA4-Ig molecule or is less than or equal to the Triton-X (for example, Triton X-100) of 0.5ng/mg CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 4.0ppm, is less than or equal to 3.5ppm, is less than or equal to 3.0ppm, is less than or equal to 2.5ppm, is less than or equal to 2.0ppm, is less than or equal to 1.5ppm, is less than or equal to 1.0ppm or is less than or equal to the Triton-X of 0.5ppm.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 8.0ng/mg CTLA4-Ig molecule, be less than or equal to 7.5ng/mg CTLA4-Ig molecule, be less than or equal to 7.0ng/mg CTLA4-Ig molecule, be less than or equal to the 6.5ng/mgCTLA4-Ig molecule, be less than or equal to 6.0ng/mg CTLA4-Ig molecule, be less than or equal to 5.5ng/mg CTLA4-Ig molecule, be less than or equal to 5.0ng/mg CTLA4-Ig molecule, be less than or equal to 4.5ng/mg CTLA4-Ig molecule, be less than or equal to 4.0ng/mg CTLA4-Ig molecule, be less than or equal to 3.5ng/mg CTLA4-Ig molecule, be less than or equal to 3.0ng/mg CTLA4-Ig molecule, be less than or equal to 2.5ng/mg CTLA4-Ig molecule, be less than or equal to the 2.0ng/mgCTLA4-Ig molecule, be less than or equal to 1.5ng/mg CTLA4-Ig molecule, be less than or equal to 1.0ng/mg CTLA4-Ig molecule or be less than or equal to the A albumen of 0.5ng/mg CTLA4-Ig molecule.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein composition comprises and is less than or equal to 8.0ppm, is less than or equal to 7.5ppm, is less than or equal to 7.0ppm, is less than or equal to 6.5ppm, is less than or equal to 6.0ppm, is less than or equal to 5.5ppm, is less than or equal to 5.0ppm, is less than or equal to 4.5ppm, is less than or equal to 4.0ppm, is less than or equal to 3.5ppm, is less than or equal to 3.0ppm, is less than or equal to 2.5ppm, is less than or equal to 2.0ppm, is less than or equal to 1.5ppm, is less than or equal to 1.0ppm or is less than or equal to the A albumen of 0.5ppm.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the GlcNAc of about 10-about 40 and the molar average ratio of CTLA4-Ig molecule.In some embodiments, GlcNAc is the about Y of about X-with the molar average ratio of CTLA4-Ig molecule, comprises X and Y, and wherein X is any integer of 10-39, and Y is any integer of 11-40.In other embodiments, the molar average of GlcNAc and CTLA4-Ig molecule comprises X and Y than being about X-Y, and wherein X is about 12,14,14,15,16 or 17, and Y is about 32,33,34,35,36 or 37.In other embodiments, about 35, the about 15-about 35 of the molar average of GlcNAc and CTLA4-Ig molecule, about 14-more about 35 than, about 13-about 35 for about 12-.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein the CTLA4-Ig molecule has the GalNAc of about 0.5-about 7.0 and the molar average ratio of CTLA4-Ig molecule.In some embodiments, GalNAc is the about Y of about X-with the molar average ratio of CTLA4-Ig molecule, comprise X and Y, wherein X is 0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9 or 2.0, and Y is 3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5 .46,4.7,4.8,4.9,5.0,6.0,7.0 or 8.0.In other embodiments, the molar average of GalNAc and CTLA4-Ig molecule comprises X and Y than being about X-Y, and wherein X is about 0.6,0.7,0.8,0.9 or 1.0, and Y is about 3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1 or 4.2.In other embodiments, the molar average of GalNAc and CTLA4-Ig molecule, about 0.9-about 3.9 more about 4.0 or about 1.0-about 3.8 or about 1.1-about 3.7 than, about 0.8-about 4.1 for about 0.7-.In other embodiments, the molar average of GalNAc and CTLA4-Ig molecule, about 1.8-about 3.5 more about 3.6 or about 1.9-about 3.4 than, about 1.7-about 3.7 for about 1.6-.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein measure the band in the following pI scope of CTLA4-Ig composition exhibiting: about 10-Yue 22 bands in the pI scope of about 4.3-about 5.6 as going up at isoelectrofocusing gel (IEF gel); About 3 main bands in the accumulation band intensity of about 90%-about 110% in the pI scope of about 4.3-about 5.3 and the pI scope of about 4.5-about 5.2.In one embodiment, the band in about 5.6 scopes of about 4.3-is about 5-Yue 30, about 6-Yue 29, about 7-Yue 28, about 8-Yue 27, about 9-Yue 26, about 10-Yue 25, about 11-Yue 24, about 12-Yue 23, about 13-Yue 22, about 14-Yue 21, about 15-Yue 20, about 16-Yue 19, about 17-Yue 20, about 18-Yue 19.
Glycosylated CTLA4-Ig and CTLA4 A29YL104E -Ig molecule and colony thereof
Without stint, glycosylation can refer to add the composite oligosaccharide structure to protein on polypeptide intrachain specific site.The following process of the carbohydrate of proteinic glycosylation and interpolation can influence protein folding and structure, protein stability comprise protein transformation period and proteinic functional property.Protein Glycosylation Overview can rely on the sequence background of wherein modifying generation to be divided into 2 classes: O linked glycosylation and N linked glycosylation.O connection polysaccharide and hydroxyl are connected with the hydroxyl of Serine or threonine residues usually.The O glycan is not to add to each Serine and threonine residues.O connection oligosaccharides is normally single or two feelers, and promptly they comprise one or 2 branches (feeler) at the most, and comprise 1-4 the inhomogeneous saccharide residue that adds one by one.
The amide nitrogen of N connection polysaccharide and l-asparagine adheres to.Having only 2 kinds of tripeptide sequences---the l-asparagine of the part of one of l-asparagine-X-Serine or l-asparagine-X-Threonine (wherein X is any amino acid except that proline(Pro)) is glycosylated target.N connection oligosaccharides can have 1-4 branch, is called single, double, three, four feelers.It is different with saccharide residue to join the structure of finding in the oligosaccharides at N and O.Although there is the sort of difference, the terminal residue in each branch of N and O connection polysaccharide can be modified by sialic acid molecule, and this modification is called sialic acid and adds cap.Sialic acid is the popular name of 9 carbon monose families of uniqueness, and it can be connected with other oligosaccharides.2 family members are N-n acetylneuraminic acid ns, are abbreviated as Neu5Ac or NANA and N-hydroxyacetylneuraminic acid, are abbreviated as Neu5Gc or NGNA.
Modal sialic acid form is NANA in the people.N-n acetylneuraminic acid n (NANA) is the main sialic acid kind that is present in the CTLA4-Ig molecule.Yet, but should be understood that N hydroxyacetylneuraminic acid (NGNA) less but detection level also is present in the CTLA4-Ig molecule.Therefore in addition, method described herein can be used to measure the sialic acid mole number for NANA and NGNA, and the level of NANA and NGNA is measured with regard to the CTLA4-Ig molecule and reported.N joins the branch that oligosaccharides has different numbers, the different positions number that it provides sialic acid molecule to adhere to it with O.N connection oligosaccharides can be provided for sialicly being up to 4 attachment positions, and O connection oligosaccharides can be provided for 2 sites that sialic acid adheres to.
Wherein many glycosylated proteins of having produced by the recombinant DNA technology method (glycoprotein) have great interests as diagnosis and therapeutical agent.The many eucaryon transmembrane proteins and the excretory albumen that are oriented to cell surface carry out posttranslational modification, to mix N connection and O connection carbohydrate group.When the asparagus fern amide residues was the part of peptide motif Asn-X-Ser/Thr, N connection oligosaccharides and they were adhered to, and wherein X can be any amino acid except that proline(Pro).O connection oligosaccharides adheres to Serine or threonine residues.The structure and the middle separately saccharide residue of finding of N connection and O connection oligosaccharides can be different.A common class sugar of finding is N-n acetylneuraminic acid n (NANA on both; Be called sialic acid hereinafter).Usually, sialic acid is the terminal residue of N connection and O connection oligosaccharides.Because its negative charge, glycoprotein can show acidity.
Glycosylated protein it is said to be increased protein folding, is regulating cell sorting and transportation, prevention protein aggregation, mediated cell-cell adhesion and increase and work aspect the resistance of proteolysis.In eukaryote, by relating to the process that receptor-mediated born of the same parents hold and remove, glycosylated nature and extent can have profound influence to the circulating half-life and the biological activity of glycoprotein therapeutical agent.Receptor-mediated system is considered to play a major role in removing seroglycoid by the various sugar components of identification oligosaccharides.The terminal sialic acids groups of glycoprotein can influence absorption, transformation period and serum clearance rate.Therefore, keep the glycoprotein production strategy of the terminal sialic acid component of glycosylated protein, can increase proteinic bioavailability and serum half-life better.Having obtained research, particularly substratum about several production process parameters of recombinant glycoprotein synthetic forms and the effect of temperature variation in various production strategies.
The CTLA4-Ig dimer of being made up of the monomer of the aminoacid sequence with following residue can have about 78,000-about 79,000 daltonian prediction theory MW:(i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:2 or (the vi) 25-383 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2.Yet the dimeric MW of this kind that obtains by MALDI-TOF is about 91,000 dalton.About 13 among the MW, 000-14,000 daltonian this species diversity part at least is because glycosylation, in one embodiment, described glycosylation accounts for the quality of this specific CTLA4-Ig monomer molecule about 15%.Above specified monomer has 3 N linked glycosylation sites, and it confirms to take place on the l-asparagine at the residue 102,134 of SEQ ID NO:2 and 233 places by peptide mapping.Can use peptide N Glycosylase F (PNGase F) to carry out the selectivity cutting by the carbohydrate molecule that l-asparagine connects.In one case, the monomer of handling the sequence 27-383 with SEQ ID NO:2 with PNGase F causes MW about 80,200 daltonian kinds, and because this monomeric theoretical MW is about 80,200, thus handle that hint do not count 1,400 dalton (80,200-78,800=1,400) may be because the O linked glycosylation.Have numerous Serines and the threonine residues that becomes glycosylation site potential although exist, only identified 2 O connection sites: the Ser of SEQ ID NO:2 155And Ser 165In one embodiment, the dominant glycan that adheres to these 2 sites is HexNAc-Hex-NeuAc.
For example, Fig. 9 is presented on the full figure that N connection on the CTLA4-Ig molecule and O join carbohydrate structure, described CTLA4-Ig molecule comprises and has from the monomer of the sequence of SEQ ID NO:2 (promptly, monomer with one of following sequence: (i) 26-383 of SEQ ID NO:2, the (ii) 26-382 of SEQ ID NO:2, the (iii) 27-383 of SEQ ID NO:2, the (iv) 27-382 of SEQ ID NO:2), (the v) 25-382 of SEQ ID NO:2, or (the vi) 25-383 of SEQ ID NO:2, wherein in one embodiment, this kind molecule of the feature of carbohydrate shown in having is by clone of the present invention or its offspring, according to described in the embodiment 14-15 and the production method shown in Figure 10 produce.The main structure of listing about each site is based on quadrature technique (referring to this paper).For every kind of structure, exist in the estimation per-cent of the observed the sort of structure of these experimental sessions.These per-cents are represented the optimum estimate from quadrature technique.
The CTLA4 that forms by the monomer of aminoacid sequence with following residue A29YL104E-Ig dimer can have about 78,000-about 79,000 daltonian prediction theory MW:(i) 26-383 of SEQ IDNO:4, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:4 or (the vi) 25-383 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4.Yet the dimeric MW of this kind that obtains by MALDI-TOF is about 91,500 dalton.About 12 among the MW, 000-13,000 daltonian this species diversity part at least is because glycosylation.Above specified monomer has 3 N linked glycosylation sites, and it confirms to take place on the l-asparagine of the residue 102,134 of SEQ ID NO:4 and 233 places (N76 of Fig. 4, N108 and N207) by peptide mapping.Can use peptide N Glycosylase F (PNGase F) to carry out the selectivity cutting by the carbohydrate molecule that l-asparagine connects.Have numerous Serines and the threonine residues that becomes glycosylation site potential although exist, only identified 3 O connection sites: Ser149, the Ser155 of SEQ ID NO:4 and Ser165 (referring to the table 25 among the embodiment 22).In one embodiment, the dominant glycan that adheres to these sites is HexNAc-Hex-NeuAc.
In certain embodiments, CTLA4-Ig or CTLA4 A29YL104E-Ig molecule is can be by the glycoprotein of culture method production of the present invention.In one embodiment, CTLA4-Ig glycoprotein is modified with the oligosaccharides of representative about 15% (w/w) molecule.These oligosaccharides can be at CTLA4-Ig or CTLA4 A29YL104EPlay an important role in the pharmacokinetics of-Ig glycoprotein (PK) parameter.In addition, different oligosaccharides overviews can influence proteinic stability and degraded.For example, O connection oligosaccharides can come enhanced CT LA4 by the self-dissolving in the hinge area in epidemic prevention immunoglobulin constant district A29YL104EThe stability of-Ig molecule.
Because the complicacy of cell cultures and method is at CTLA4-Ig or CTLA4 A29YL104EIt can be heterogeneous in the character that oligosaccharides in the colony of-Ig molecule is distributed in.Because glycosylation site is occupied to fully and do not occupy and the following fact, can have heterogeneity: any specific site can be filled with the fact of many different oligosaccharide structures, and this can further show the variation in the pattern that sialic acid modifies.
In one embodiment, CTLA4-Ig or CTLA4 A29YL104EMain sialic acid on the-Ig molecule partly is that (NeuAc, NANA), and less important sialic acid partly is N hydroxyacetylneuraminic acid (NGNA) to the N n acetylneuraminic acid n.Sialic charged character and complicated contain sialic acid structure and can cause CTLA4-Ig or CTLA4 respectively A29YL104EThe multiple isoform of-Ig, wherein this kind isoform can be tangible in isoelectrofocusing (IEF) overview.For example, about the IEF overview of CTLA4-Ig referring to Figure 11 and embodiment 3.In addition, about CTLA4 A29YL104EThe IEF overview of-Ig is referring to Figure 12 and embodiment 22.
In one embodiment, the invention provides the CTLA4-Ig molecule colony with dominant CTLA4-Ig isoform, described CTLA4-Ig isoform has and is less than or equal to 5.1 or 5.0 iso-electric point (pI), and this can for example measure by IEF.In another embodiment, provide and had dominant CTLA4 A29YL104EThe CTLA4 of-Ig isoform A29YL104E-Ig molecule colony, described CTLA4 A29YL104E-Ig isoform has and is less than or equal to 5.5 iso-electric point (pI), and this can for example measure (Figure 12) by IEF.
In one embodiment, the invention provides have that about 4.2-is about 5.7, about 4.25-is about 5.5, the CTLA4-Ig molecule colony of the pI of about 4.3-about 5.3 or about 4.5-about 5.2.In another embodiment, the invention provides the CTLA4-Ig molecule colony of the pI with about 4.45-about 5.30.In further embodiment, the invention provides the CTLA4-Ig molecule colony of the pI with about 4.3-about 5.1.In specific embodiments, the invention provides the CTLA4-Ig molecule colony of the pI with about 4.45-about 5.0.In one embodiment, the invention provides the colony of CTLA4-Ig molecule, wherein as measuring, in the colony at least 40% by IEF, 50%, 60%, 70%, 80%, 90% or 95% molecule demonstration is less than or equal to about 5.7,5.6,5.5,5.4,5.3,5.2,5.1,5.0,4.9,4.8,4.7,4.6,4.5,4.4,4.3,4.2,4.1,4.0,3.9,3.8,3.7,3.6,3.5,3.4,3.3,3.2,3.1,3.0,2.9,2.8,2.7,2.6,2.5,2.4,2.3,2.2,2.1 or 2.1 iso-electric point (these values can have ± 0.2 standard deviation).In one embodiment, the invention provides the method for the colony that is used to prepare the CTLA4-Ig molecule, described CTLA4-Ig molecule has the pI of about 4.45-about 5.30 or about 4.45-about 5.1 or about 4.45-about 5.0, wherein said method comprises that the colony to the CTLA4-Ig molecule implements the IEF gel electrophoresis, wherein the single band on the gel represents to have the subgroup of the CTLA4-Ig molecule of specific pI, and by cutting band from gel and protein purification separates the CTLA4-Ig molecule with specific pI from the gel band that cuts subsequently subgroup.
In further embodiment, the invention provides the CTLA4 of the pI with about 4.5-about 5.2 A29YL104E-Ig molecule colony.In other embodiments, the invention provides the CTLA4 of the pI with about 4.7-about 5.1 A29YL104E-Ig molecule colony.In another embodiment, the invention provides the CTLA4 of the pI with about 2.0-about 5.2 A29YL104E-Ig molecule colony.In one embodiment, the invention provides CTLA4 A29YL104EThe colony of-Ig molecule, wherein as measuring by IEF, at least 40%, 50%, 60%, 70%, 80%, 90% or 95% molecule shows and is less than or equal to about iso-electric point of 5.5,5.4,5.3,5.2,5.1,5.0,4.9,4.8,4.7,4.6,4.5,4.4,4.3,4.2,4.1,4.0,3.9,3.8,3.7,3.6,3.5,3.4,3.3,3.2,3.1 or 3.0 (these values can have ± 0.2 standard deviation) in the colony.In one embodiment, the invention provides and be used to prepare CTLA4 A29YL104EThe method of the colony of-Ig molecule, described CTLA4-Ig molecule has about 4.5-about 5.2; About 4.7-about 5.1; The pI of about 2.0-about 5.2, wherein said method comprises CTLA4 A29YL104EThe colony of-Ig molecule implements the IEF gel electrophoresis, and wherein the single band on the gel represents to have the CTLA4 of specific pI A29YL104EThe subgroup of-Ig molecule, and by cutting band from gel and subsequently from the gel band that cuts protein purification separate CTLA4 with specific pI A29YL104EThe subgroup of-Ig molecule.
In certain embodiments, the invention provides the CTLA4-Ig molecule colony with mole sialic acids groups and following molar average ratio of mole CTLA4-Ig molecule: about 6-is about 32, about 8-is about 32, about 11-is about 30, about 12-is about 20, about 13-is about 19, about 14-is about 18, about 15-is about 17, about 6-is about 16, about 8-is about 16, about 8-is about 14, about 8-about 12.
In some embodiments ,≤25ppm characterizes the composition of CTLA4-Ig molecule to the maximum admissible CHO host cell proteins matter of≤10ng/mg.In another embodiment, the composition of CTLA4-Ig molecule is characterised in that the host cell DNA the to≤1.0pg/mg level at≤2.5pg/mg.In another embodiment, the composition of CTLA4-Ig molecule be characterised in that≤1.0ng/mg or≤Triton X-100 on the 1.0ppm level.The concentration of Triton X-100 can be by using Waters OASIS-HLB Solid-Phase Extraction, and extraction Triton X-100 washes with water subsequently to remove residual protein and measures.Bonded Triton X-100 is by removing with the acetonitrile wash-out.The acetonitrile eluate is analyzed by reversed phase chromatography, use SAS Hypersil 5 μ m posts with by acetonitrile: the advection that water (80: 20) is formed is mutually.Detection is via carrying out in the UV at 225nm place absorbancy.In one embodiment, the composition of CTLA4-Ig molecule is characterised in that≤oxidation of 2.5 area % and the deacylated tRNA amine of≤2.0 area %.In another embodiment, the composition of CTLA4-Ig molecule is characterised in that≤oxidation of 3.0 area % and the deacylated tRNA amine of≤2.5 area %.The tryptic peptide graphing method is used for quantitative oxidation and deacylated tRNA amine.Per-cent oxidation data are measured by utilizing the mapping of RP-HPLC trypsinase, and the Met85 in the quantitative CTLA4-Ig protein of described mensuration is to the area percentage oxidation of methionine sulfoxide.Per-cent oxidation in this method obtains by the UV peak area among the measure R P-HPLC trypsinase figure, and this UV peak is about T6 tryptic peptide that comprises the residue 84-93 that contains Met85 and the oxidation tryptic peptide T6ox that contains Met (O) 85 accordingly.The area percentage oxidation of Met85 to Met (O) 85 and the area percentage at T6ox peak are proportional: per-cent oxidation=100*AT6ox/ (AT6ox+AT6), wherein AT6=is about the T6 tryptic peptide, peak area (84-93).AT6ox=is about the T6ox tryptic peptide, the peak area of Met (O) 85 (84-93).Per-cent deacylated tRNA amine data obtain by the UV peak area among the measure R P-HPLC trypsinase figure, this UV peak is about comprising the T26 tryptic peptide of the residue 281-302 that contains Asn294, the tryptic peptide T26deam1 that contains the deacylated tRNA amine of isoAsp294 accordingly, described data are measured by the RP-HPLC trypsinase mapping of using quantitative area percentage oxidation and deacylated tRNA amine and are obtained.Subsequently, the area percentage deacylated tRNA amine of Asn294 to isoAsp294 and the area percentage at T26deam1 peak are proportional: wherein AT26=is about T26, peak area (281-302), AT26deam1=be about T26deam1, the peak area of isoAsp294 (281-302).AT26deam2=is about T26deam2, the peak area of Asp299 (281-302).AT26deam3=is about T26deam3, the peak area of Asp294 (281-302).AT26deam4=is about T26deam4, the peak area of Asu294 (281-302).
In another embodiment, the composition of CTLA4-Ig molecule is characterised in that the 15-35 mole: the mole proteinic N-acetyl-glucosamine of CTLA4-Ig (GlcNAc), or 1.7-3.6 mole: the mole proteinic N-acetylgalactosamine of CTLA4-Ig (GalNAc).Amino monose is undertaken quantitatively by capillary electrophoresis (CE) after discharging from protein via acid hydrolysis.The amino monose that discharges is re-acetylated, and carries out fluorescent mark with amino pyrene trisulfonic acid (APTS) and detect with quantitative to promote it.The N-acetylmannosamine is added in sample and the amino monose standard to serve as internal standard.The peak area of the amino monose in the sample uses internal standard to carry out stdn, and by carrying out quantitatively with its standardized separately amino monose peak area ratio in standard.Calculate the mol ratio of every kind of monose subsequently with respect to the CTLA4-Ig molecule.
In one embodiment, the composition of CTLA4-Ig molecule is characterised in that the explanation of following N connection oligosaccharides overview:
The explanation of N connection oligosaccharides overview
Figure A200680053116D01431
In one embodiment, the composition of CTLA4-Ig molecule is characterised in that composition wherein has the neutral monose of following approximately ratio:
Semi-lactosi: 8.0-17 mole: mole CTLA4-Ig protein
Fucose: 3.5-8.3 mole: mole CTLA4-Ig protein
Seminose: 7.7-22 mole: mole CTLA4-Ig protein, or
Semi-lactosi: 9.0-17 mole: mole CTLA4-Ig protein
Seminose: 11-19 mole: mole CTLA4-Ig protein.
The neutral monose composition of illustrative: the mole of semi-lactosi, Fucose and seminose: mole protein
Figure A200680053116D01441
Illustrative sialic acid (NANA: mole protein)
Figure A200680053116D01442
In another embodiment, about CTLA4 A29YL104EThe monose molar ratio range of-Ig composition is as follows: the about 10-20 moles/mole of seminose protein; The about 4.2-7.0 moles/mole of Fucose protein; With the about 9.2-17 moles/mole of semi-lactosi protein.In another embodiment, CTLA4 A29YL104E-Ig composition is characterised in that the proteinic NANA mol ratio of about 5.0-10.0 mole NANA/ mole.In another embodiment, CTLA4 A29YL104E-Ig composition is characterised in that＜1.5 moles of proteinic NGNA mol ratios of NGNA/ mole.In some embodiments, about the % deviation of sialic mol ratio be≤15% or≤20% or≤30%.
In one embodiment, the colony of CTLA4-Ig molecule can comprise the CTLA4-Ig monomer that has at least 3 sialic acids groups separately.In another embodiment, the colony of CTLA4-Ig molecule comprises the CTLA4-Ig monomer that has 3-8 sialic acids groups separately.
In one embodiment, the invention provides the colony of CTLA4-Ig molecule, wherein the demonstration of at least 40%, 50%, 60%, 70%, 80%, 90% or 95% molecule is less than or equal to about iso-electric point of 5.7,5.6,5.5,5.4,5.3,5.2,5.1,5.0,4.9,4.8,4.7,4.6,4.5,4.4,4.3,4.2,4.1,4.0,3.9,3.8,3.7,3.6,3.5,3.4,3.3,3.2,3.1 or 3.0 in the colony.
In some embodiments, the invention provides the CTLA4-Ig molecule colony with mole NANA and mole CTLA4-Ig molecule or dimeric following molar average ratio: about 6-is about 16, about 6-is about 14, about 6-is about 12, about 8-is about 12, about 8-is about 14, about 8-about 16.
In other embodiments, the invention provides and have the CTLA4-Ig molecule colony that is less than or equal to about 2,1.8,1.6,1.5,1.4,1.0,0.8 or 0.5 mole NGNA and mole CTLA4-Ig molecule or dimeric molar average ratio.
In specific embodiments, the invention provides mole sialic acids groups and mole CTLA4 with about 5.5-about 8.5 A29YL104EThe CTLA4 of-Ig molecule or dimeric molar average ratio A29YL104E-Ig molecule colony.In another embodiment, the invention provides mole sialic acids groups and mole CTLA4 with about 5-about 10 A29YL104EThe CTLA4 of-Ig molecule or dimeric molar average ratio A29YL104E-Ig molecule colony.
In one embodiment, CTLA4 A29YL104EThe colony of-Ig molecule can comprise the CTLA4 that has at least 2.5 sialic acids groups separately A29YL104E-Ig monomer.In another embodiment, CTLA4 A29YL104EThe colony of-Ig molecule comprises the CTLA4 that has 2.5-5 sialic acids groups separately A29YL104E-Ig monomer.
In other embodiments, the invention provides the colony of CTLA4-Ig molecule, its mole amino monose and/or neutral monose by colony and/or sialic acid and mole CTLA4-Ig molecule or dimeric molar average are than being distinguished.In specific embodiments, the invention provides CTLA4 A29YL104EThe colony of-Ig molecule, it is by the amino monose of mole and/or neutral monose and/or the sialic acid and the mole CTLA4 of colony A29YL104E-Ig molecule or dimeric molar average are than being distinguished.Amino monose comprises N-acetylgalactosamine (GalNAc) and N-acetyl-glucosamine (GlcNAc).Neutral monose comprises seminose, Fucose and semi-lactosi.Sialic acid comprises N-n acetylneuraminic acid n (NANA) and N-hydroxyacetylneuraminic acid (NGNA).
In one embodiment, the invention provides the colony of CTLA4-Ig molecule, it is characterized in that about 10-is about 40, about 15-is about 35, the mole GlcNAc/ mole CTLA4-Ig dimer of about 15-about 25 or about 15-about 20 or to the molar average ratio of CTLA4-Ig molecule.In another embodiment, the invention provides the colony of CTLA4-Ig molecule, wherein at least 40%, 50%, 60%, 70%, 80%, 90% or 95% molecule is characterised in that and is less than or equal to about 40,38,35,30,25,20,18 or 15 mole GlcNAc/ mole CTLA4-Ig dimer or to the molar average ratio of CTLA4-Ig molecule in the colony.
In another embodiment, the invention provides the colony of CTLA4-Ig molecule, it is characterized in that about 1.5-is about 8.5, about 1.7-is about 3.0, about 1.7-is about 4.0, about 1.7-is about 5.0, about 1.7-is about 6.0, about 1.7-is about 7.0, the mole GalNAc/ mole CTLA4-Ig dimer of about 1.7-about 8.0 or about 1.7-about 8.3 or to the molar average ratio of CTLA4-Ig molecule.In another embodiment, the invention provides the colony of CTLA4-Ig molecule, wherein at least 40%, 50%, 60%, 70%, 80%, 90% or 95% molecule is characterised in that and is less than or equal to about 8.5,8,7.5,7,6.5,6,5.5,5,4.5,4.0,3.8,3.6,3.5,3.0,2.5,2.0,1.7 or 1.5 mole GalNAc/ mole CTLA4-Ig dimer or to the molar average ratio of CTLA4-Ig molecule in the colony.
In further embodiment, the invention provides the colony of CTLA4-Ig molecule, it is characterized in that about 7.5-is about 20.0, about 8.0-is about 19.0, about 8-is about 18.0, about 8.0-is about 17.0, mole semi-lactosi/mole CTLA4-Ig dimer of about 8.5-about 17.0 or about 9.0-about 17.0 or to the molar average ratio of CTLA4-Ig molecule.In another embodiment, the invention provides the colony of CTLA4-Ig molecule, wherein at least 40%, 50%, 60%, 70%, 80%, 90% or 95% molecule is characterised in that and is less than or equal to mole semi-lactosi/mole CTLA4-Ig dimer of about 20.0,19.0,18.0,17.0,16.0,15.0,14.0,13.0,12.0,11.0,10.0,9.0,8.5,8.0 or 7.5 or to the molar average ratio of CTLA4-Ig molecule in the colony.
In further embodiment, the invention provides the colony of CTLA4-Ig molecule, it is characterized in that about 3-is about 8.5, about 3.5-is about 8.5, about 3.5-is about 8.3, about 3.5-is about 8.0, mole Fucose/mole CTLA4-Ig dimer of about 3.5-about 7.5 or about 3.5-about 7.0 or to the molar average ratio of CTLA4-Ig molecule.In another embodiment, the invention provides the colony of CTLA4-Ig molecule, wherein at least 40%, 50%, 60%, 70%, 80%, 90% or 95% molecule is characterised in that and is less than or equal to mole Fucose/mole CTLA4-Ig dimer of about 8.5,8.3,8.0,7.5,7.0,6.5,6.0,5.5,5.0,4.5,4.0,3.5,3.2 or 3.0 or to the molar average ratio of CTLA4-Ig molecule in the colony.
In further embodiment, the invention provides the colony of CTLA4-Ig molecule, it is characterized in that about 7-is about 23, about 7.5-is about 23, about 7.7-is about 23, about 7.7-is about 22.5, about 7.7-is about 22, about 7.7-is about 20, about 7.7-is about 18, about 7.7-is about 16, about 8.0-is about 16.0, about 9.0-is about 17.0, mole seminose/mole CTLA4-Ig dimer of about 10-about 19.0 or about 11-about 19.0 or to the molar average ratio of CTLA4-Ig molecule.In another embodiment, the invention provides the colony of CTLA4-Ig molecule, wherein at least 40%, 50%, 60%, 70%, 80%, 90% or 95% molecule is characterised in that and is less than or equal to about 23,22.5,22,21,20,19,18,17,16,15,14,13,12,11,10,9.5,9,8.5,8,7.7,7.5,7.3 or 7 mole seminose/mole CTLA4-Ig molecule or dimer or to the molar average ratio of CTLA4-Ig molecule in the colony.
In one embodiment, the invention provides glycosylated CTLA4-Ig colony, its show for example result from or as the PK value by the removing that from serum, the reduces increase that confirms of retains biological activity simultaneously, for example exposure of Zeng Jiaing is as passing through area under curve (AUC) measurement.In another embodiment, the invention provides glycosylated CTLA4 A29YL104E-Ig colony, it shows pharmacokinetics (PK) value as the increase that confirms by the removing while retains biological activity that reduces from serum.
In some embodiments, the invention provides the analogue of soluble CTL A 4-Ig molecule, it has other glycosylation site.In other embodiments, the invention provides soluble CTL A 4 A29YL104EThe analogue of-Ig molecule, it has other glycosylation site.Other glycosylation site provides that be used for can be by the attachment point of sialylated other carbohydrate structure.The sialic acid content that increases can cause the PK value that increases and/or the glycoprotein stability of increase.Higher sialic acid content is favourable.Can use enzyme to add method behind the other sialic external purifying, to produce CTLA4-Ig of the present invention or CTLA4 A29YL104EThe further embodiment of-Ig molecule.
Embodiment of the present invention comprise any one scope disclosed herein with any one or a plurality of scope combination disclosed herein.Embodiment of the present invention comprise any feature or the character with the CTLA4-Ig disclosed herein of any or various features of CTLA4-Ig disclosed herein or properties of combination.
Be used for analyzing and separation of C TLA4-Ig and CTLA4 A29YL104E The method of-Ig glycoprotein
Following method described herein can be used for distinguishing, identify or separate specific CTLA4-Ig or CTLA4 based on various sugared overviews A29YL104E-Ig molecule colony, described sugared overview includes but not limited to, the amino monose of mole and/or the neutral monose and/or the sialic acid/mole CTLA4-Ig or the CTLA4 of colony A29YL104E-Ig molecule or dimeric molar average ratio.
Can from substratum or supernatant liquor, separate by cultured cells excretory glycoprotein.When needing, when total cell cultures phase finishes, use as known in the art with practice or separates as described herein and purification process, by the glycoprotein of cell generation collect, reclaim, separation and/or purifying or purifying basically.In one embodiment, by cell expressing but can't help the glycoprotein of the present invention of emiocytosis and still can from cell, reclaim, for example, via preparing cell lysate and separating glycoprotein and/or use known in the art and practice and method as described further below.
The glycoprotein that is produced by cell culture processes of the present invention comprises the complex carbohydrates that can analyze by the various technology of carbohydrate analysis.For example, technology lectin trace for example well-known in the art discloses for example ratio of semi-lactosi of terminal seminose or other sugar., three or four feeler oligosaccharides single, double by the sialic acid end can be confirmed by following, use anhydrous hydrazine or enzymatic method from protein, to discharge sugar, and by ion exchange chromatography, size exclusion chromatography or additive method fractional separation oligosaccharides known in the art.
Exist in the glycosidic link of 2 main types finding in the glycoprotein, N and O connection.The N glycosylation generates by the amide nitrogen that makes glycan and asparagine residue is covalently bound.The O glycosidic link by making Serine, Threonine, hydroxylysine or oxyproline hydroxyl and glycan is covalently bound generates.The carbohydrate of glycoprotein partly relates to numerous molecular recognition phenomenons, comprises that host-pathogenic agent interacts, removes from serum and the target of different tissues.With regard to CTLA4-Ig and CTLA4 A29YL104E-Ig molecule, the carbohydrate part can influence CTLA4-Ig molecule and CD80 or CD86 at least, or CTLA4 A29YL104ECombining between-Ig molecule and CD80 or the CD86.
Carbohydrate structure generally is present on the expressed protein as N connection or O connection carbohydrate.The N connection joins carbohydrate mainly different aspect its core texture with O.The N linked glycosylation refers to that carbohydrate partly adheres to via the asparagine residue in GlcNAc and the peptide chain.In one embodiment, N connection carbohydrate all comprises common Man1-6 (Man1-3) Man β1-4Glc-NAc β1-4GlcNAc β-R core texture, wherein the R in this core texture represents asparagine residue.The proteinic peptide sequence that produces will comprise l-asparagine-X-Serine, l-asparagine-X-Threonine and l-asparagine-X-halfcystine, and wherein X is any amino acid except that proline(Pro).
By contrast, O connection carbohydrate is characterised in that the common core texture, and it comprises the GalNAc that the hydroxyl with Threonine or Serine adheres to.In N connection and O connection carbohydrate, the most important thing is compound N and O connection carbohydrate.This kind complex carbohydrates comprises several feeler structures.Single, double, sialic interpolation is important to three and four feeler structures for end.This kind outer chains structure provides the other site about specific sugar and key, and described sugar and key comprise the carbohydrate of protein.
Treatment glycoprotein uses the recombinant DNA cell culture technology to produce usually.Protein Glycosylation Overview distribution in the cell cultures can be subjected to the variable effect in pH, cell density, nutrient concentrations and the metabolite concentration.Glycan distributes and must monitor the glycan distribution carefully in product development and production period to the susceptibility of environmental activity is feasible, to guarantee to make repeatably product.
The exploitation that is used for the treatment of the recombinant chou deutero-glycoprotein of purposes has caused its carbohydrate structure is characterized cumulative needs with the method for profile analysis.The oligosaccharides mapping is used during the initial characterization of recombinant protein, for comparing with natural protein, the oligosaccharide structure that exists with evaluation, consistence with monitoring oligosaccharides composition, with the assessment variation that can cause, and identify because the variation in the glycosylation that expression takes place in different clones by the change in cell cultures or the production process.
Multiple technologies can be used to assess carbohydrate structure and distribute.These comprise the gel-filtration of detection technique link coupled, chromatography and electrophoretic separation technique with broad range.If sample size is limited, with fluorescent reagent for example derive so that improve detection usually by 2-benzaminic acid and 2-aminopyridine for glycoprotein so.Yet derive and the purifying of derivative can be time-consuming.When sample size was not problem, the direct assessment that carbohydrate structure distributes was possible.
The analysis of the oligosaccharide content of glycoprotein
Specific glycoprotein can show the heterogeneity of carbohydrate.Heterogeneity can found out on several levels: glycosylation site can not change between occupying fully, and any specific site can be filled with many different oligosaccharide structures, and wherein each structure can for example NANA or NGNA modify by sialic acid molecule.
Proteinic carbohydrate content of the present invention can be analyzed by methods known in the art, comprises the method for describing among this paper embodiment.Several method about the glycosylation analysis is known in the art and useful in background of the present invention.These methods provide about the oligosaccharides characteristic of adhering to the peptide of producing and the information of composition.Combine the method that is used for carbohydrate analysis with the present invention and include but not limited to, the lectin chromatography; Detect the high-efficiency anion displacement chromatography (HPAEC-PAD) of combination with pulsed current, described HPAEC uses high pH anion-exchange chromatography with based on the charge separation oligosaccharides; NMR; Mass spectroscopy; HPLC; Porous graphite carbon (GPC) chromatography.
The method that is used to discharge oligosaccharides is known.These methods comprise 1) enzymatic method, it uses peptide-N-Glycosylase F/ inscribe-alpha-galactosidase to carry out usually; 2) β null method uses harsh alkaline environment to connect structure with main release O; With 3) use anhydrous hydrazine to discharge the chemical process of N and O connection oligosaccharides.The method that is used to analyze can comprise the steps: 1. samples at deionized water dialysis to remove all buffering salts, freeze-drying subsequently.2. discharge complete oligonucleotide chain with anhydrous hydrazine.3. the oligonucleotide chain of using anhydrous methanol HCl processes complete is to discharge the indivedual monose as the O-methyl-derivatives.4. the N-acetylize of any primary amino.5. derivatize is to produce complete-O-trimethyl silyl methylglycoside.6. on the CP-SIL8 post, separate these derivatives by kapillary solution-air chromatography (GLC).7. compare with known standard, identify the individual sugars glycoside derivates by retention time from GLC and mass spectroscopy.By FID by the quantitative individual derivatives of internal standard (13-O-methyl D-glucose).
Existence neutral and aminosugar can be by using high-efficiency anion displacement chromatography (the HPAEC-PAD Carbohydrate System that detects combination with pulsed current; Dionex Corp.) measures.For example, sugar can be by obtaining discharging in 100 ℃ of hydrolysis in 20% (v/v) trifluoroacetic acid in 6 hours.Hydrolysate carries out drying by freeze-drying or with Speed-Vac (Savant Instruments) subsequently.Residue is dissolved in the 1% sodium acetate trihydrate solution subsequently, and on HPLC-A S6 post, analyze (as by people such as Anumula, 1991, Anal.Biochem., 195:269-280 describes).
Alternately, can carry out the immunoblotting carbohydrate analysis.In this program, the carbohydrate of protein bound uses commercial glycan detection system (Boehringer) to detect, described detection system backbone by people such as Haselbeck (1993, Glycoconjugate J., 7:63) the oxidation immunoblotting program of Miao Shuing.Abide by the dyeing rules by manufacturer recommendation, except protein transduction being moved to PVDF membrane rather than nitrocellulose membrane, and the sealing damping fluid comprises and is dissolved in the 10mM Tris damping fluid with 0.9% sodium-chlor, 5% bovine serum albumin(BSA) of pH7.4.Detection uses the anti-digoxigenin antibody (Boehringer) that is connected with the alkaline phosphate conjugate to carry out, dilution in 1: 1000 in the Tris buffer saline, use is dissolved in 100mM Tris damping fluid, the phosphatase substrate of pH9.5, chlorination 4-nitroblue tetrazolium(NBT), 0.03% (w/v) and 5-bromo-4-chloro-3-indyl (indoyl)-phosphoric acid salt 0.03% (w/v), described Tris damping fluid comprises 100mM sodium-chlor and 50mM magnesium chloride.The protein band that comprises carbohydrate manifested in the time of about 10-15 minute usually.
Also can cut with the carbohydrate of protein bound by digesting with Peptide N-glycosidase F.According to this program, residue is suspended in comprises in 0.18% SDS, 18mM beta-mercaptoethanol, 90mM phosphoric acid salt, the 3.6mM EDTA 14 μ L damping fluids under pH8.6, and in 100 ℃ of heating 3 minutes.After being cooled to room temperature, reaction mixture is divided into 2 parts that approximately equate.Not further 1 part of served as control handling.Another part is adjusted to about 1%NP-40 stain remover, adds the 0.2 Peptide N-glycosidase F of unit (Boehringer) subsequently.All heated 2 hours and analyzed by the SDS-polyacrylamide gel electrophoresis subsequently for 2 parts in 37 ℃.
The glycan mapping of glycoprotein becomes and is accepted day by day.Method described herein allows fast characterizing oligosaccharides aspect the glycan type on the non-reducing end of carbohydrate, sialylated degree and the number of branches.Therefore, in certain embodiments, the invention provides the CTLA4-Ig colony that is characterised in that specific oligosaccharides overview.The oligosaccharides profile analysis generally chromatographic separation by oligosaccharides detects subsequently with relative quantification and finishes.The alternative scheme of chromatography profile analysis is by ESI perfusion direct analysis oligosaccharides after the online desalination.
Oligosaccharides profile analysis by PGC can be used to characterize the N connection oligosaccharides from the CTLA4-Ig molecule.Existence comprises the structure kind that comprises the acetylizad sialic acids groups of O-by the oligosaccharides of 31 structure kinds of CTLA4-Ig molecule (SEQ ID NO:2) evaluation.The checking of structure kind reaches by using MS/MS and positive ion mode MS.The relative quantification of structure kind is possible by integration at the UV at 206nm place arteries and veins mark.From the subgroup overview in indivedual N connection site relatively be known, thereby disclose significant population difference between the N connection site.Use the oligosaccharides profile analysis of PGC to provide about more traditional profile analysis rule such as the informative easily alternative scheme of HPAEC.
The N that comprises in the monomeric CTLA4-Ig molecule of SEQ ID NO:2 connects structure
Every chain (promptly on CTLA4-Ig polymer or dimer, each monomer) there are 3 N linked glycosylation sites, wherein monomer has the sequence from SEQ ID NO:2, for example the 26-383 of (i) SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ IDNO:2 or (the vi) 25-383 of SEQ ID NO:2) of SEQ ID NO:2 of SEQID NO:2 of SEQ ID NO:2.Variation via the site in the glycosylation is analyzed by separate the peptide fragment that comprises N connection glycan from proteinic tryptic digestion.N linked glycosylation site on the protein is positioned at Asn 102, Asn 134And Asn 233On, be included in respectively in trypsinase fragment 5,7 and 14.The enzymatic of N connection oligosaccharides from isolating peptide fragment discharges, and is that the PGC profile analysis of the oligosaccharides of release causes the overview shown in Figure 13 subsequently.By from Asn 233(trypsinase fragment 14, T14) the overview visible of the middle glycan that discharges is rich in the oligosaccharides colony and takes off sialic acid structure (not containing sialic structure).From attached to Asn 102And Asn 134The oligosaccharides overview of the glycan on (T5 and T7) comprises a large amount of sialylated structures.
To be injected directly into by the isolating oligosaccharides that glycoprotein discharges in the porous graphite carbon LC/UV/MS system.Figure 14 and 15 shows the TIC and the UV chromatogram of the general PGC overview that produces by the acetonitrile gradient that comprises acid and alkaline additive.In most of the cases, comprise mass peak from the mass spectrum of single chromatographic peak about single oligosaccharides.30 oligosaccharide structure kinds are identified by the wash-out overview that comprises TFA.Have only 16 oligosaccharide structure kinds by comprising NH 4The wash-out overview of OH is identified.In each structure kind, exist to comprise displacement and the acetylizad variant structure of sialic acid in various degree that N-hydroxyacetylneuraminic acid (NGNA) replaces N-n acetylneuraminic acid n (NANA).Although relatively can only obtain qualitative information by the ion counting about the oligosaccharides kind, the interior separately primary structure kind of obvious 4 kinds of structural domains is P2100, P2111, P2122 and P3133.This integrated value with the UV arteries and veins mark that gets comfortable 206nm place is consistent.Further structure verification can derive from positive ion mass spectrum figure.Positive ion mode ionization promotes the source fracture of oligosaccharides, mainly on glycosidic link.Because, have the good separation of oligosaccharides, so from the spectrum analog positive ion MS/MS spectrum of the fracture of positive ion mode as measuring by negative ion mass spectrum.Domain II I (structure of double-sialylated) comprises the acetylizad structure P2122-Ac of O-of significant quantity.The O-acetylize of one of sialic acid on the positive ion m/s data underwork.The modal O-acetylize of sialic acid residues site in C-7 and C-9 position (Shi WX, Chammas R., Varki A., J.Biol.Chem.271 (1996) 15130-15188).During pH, the O-acetonyl ester on C-7 spontaneously moves to C-9 outside the physiology born of the same parents.Therefore most probable O-acetylize site is C-9.
N joins the analysis of oligosaccharide content: analytical technology can comprise by the column chromatography cutting and separate N connection oligosaccharides, and described column chromatography uses the Hypercarb post in non-limiting embodiments.The glycan of implementing the Hypercarb chromatography is separated, and can analyze by HPAEC-PAD, and the type of the carbohydrate of specific glycoprotein is modified in described assay determination.The analysis mode of N connection oligosaccharides characterizes and can also finish by liquid chromatography (LC)/mass spectroscopy (LC/MS), uses porous graphite carbon (PGC).Carbohydrate analysis can also comprise trypsinase, Asp-N and trypsinase/chymotryptic peptide mapping, comprises the peptide of carbohydrate structure with mensuration.
N connection oligosaccharide structure can use a series of quadrature mass spectroscopies and HPAEC-PAD technology analyze ( Referring toEmbodiment).These technology comprise that several endopeptidase cuttings are the LC/MS/MS analysis subsequently.About having CTLA4-Ig monomer, use LC/MS and LC/MS/MS electrospray ionization to characterize 3 main sites of N linked glycosylation, and be determined at the primary structure on each N connection site from the sequence of SEQ ID NO:2.These data are summarized among Fig. 9.At Asn 102, Asn 134And Asn 233Last at least 3 main attachment points that exist about N connection oligosaccharides.In addition, find Asn 233Comprise the colony that N that time of about 80% takes place connects structure, described N connects structure and does not comprise sialic acids groups.
Combine with the consensus sequence motif of Asn-X-Ser/Thr by the LC/MS of glycopeptide, the LC/MS of oligosaccharides and N connection oligosaccharide structure: the N connection carbohydrate of the CTLA4-Ig that HPAEC-PAD measures.This sequence occurs 3 times having on the CTLA4-Ig monomer chain of one of following sequence: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQID NO:2 and (the vi) 25-383 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2.The consensus sequence motif occurs on the following residue in SEQ ID NO:2: Asn 102Leu 103Thr 104Asn 134Gly 135Thr 136And Asn 233Ser 234Thr 235Based on consensus sequence, have 6 N connection carbohydrate site/dimer molecules, described dimer molecule is formed by any one or 2 in the following sequence monomer: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:2 and (the vi) 25-383 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2.
N connection carbohydrate can have 3 general mutation: high mannose, mixture (hybrid) and/or mixture.The LC/MS technology that is used for the glycopeptide analysis obtains exploitation.Monomer (have one of following sequence: (i) 26-383 of SEQ ID NO:2, (ii) SEQ ID NO:2 26-382, (iii) SEQ ID NO:2 27-383, (iv) SEQ ID NO:2 27-382, (v) the cutting of the trypsinase endoproteolysis of the 25-382 of SEQ ID NO:2 and (the vi) 25-383 of SEQ ID NO:2) causes comprising 3 kinds of peptides of N linked glycosylation.All 3 N connection sites all are filled with carbohydrate structure.Trypsinase fragment T5 corresponding to the amino acid 65-109 of SEQ ID NO:2 is included in Asn 102On glycosylation.Trypsinase fragment T7 corresponding to the amino acid/11 20-154 of SEQ ID NO:2 is included in Asn 134On glycosylation.Trypsinase fragment T14 corresponding to the amino acid 229-237 of SEQ ID NO:2 is included in Asn 233On glycosylation.
In order to measure the glycosylated particular type on each site, protein digestion and isolating grade are collected T5, T7 subsequently and the T14 peptide obtains carbohydrate from each specific site by increasing.Purpose tryptic peptide peak is handled and is processed with PNGase F and is used for analyzing on the Hypercarb post by LC/MS.The result is presented at that compound on each site is two, the heterogeneous population of three and four feeler structures.These can find out in Figure 13 that wherein chromatography makes sugar become 5 kinds of structural domains: take off sialic acid, single sialic acid, bifunctional sialyltransferase, three sialic acids and four sialic acid structures (being called structural domain I, II, III, IV and V).About Asn 102(T5) chromatogram of carbohydrate (the little figure A of Figure 13) illustrates a series of lists and bifunctional sialyltransferase structure on the site.About Asn 134(T7) chromatogram (the little figure B of Figure 13) illustrates 2 main bifunctional sialyltransferase structures with single sialic acid structure colony.About Asn 233(T14) chromatogram (the little figure C of Figure 13) illustrates seldom sialylated.For each N connection carbohydrate site, MS spectrum and corresponding construction (referring to Figure 13, little figure E, F, H) have been shown about the main peak in each chromatogram.At Figure 13, among the little figure D, total N connection carbohydrate overview of CTLA4-Ig is shown in the chromatogram.The quality and the structure at selected peak are recited in the table 1.Oligosaccharides LC/MS data obtain the support of the in-depth analysis of peptide figure.Asn 102(T5 peptide) has from two feelers, structure to four feeler of asialoization, the maximum carbohydrate heterogeneity of four sialylated structures.Asn 134(T7 peptide) mainly comprises two feeler structures.This site comprises compares Asn 102The heterogeneity of site much less.Asn 233(T14 peptide) site comprises seldom sialylated.Also using the third analytical technology HPAEC-PAD finds to support 2 quadrature LC/MS.
Table 1: use the observed main N of LC/MS method to connect structure and selected less important composite structure
Figure A200680053116D01541
*Taking off the sialic acid species detection is the TFA adducts.
The colony of total N connection carbohydrate uses HPAEC-PAD to analyze.The data list of Huo Deing is in table 2 and 3 by this method.In table 2, listed (the Asn of SEQ IDNO:2 in each site 102, Asn 134And Asn 233) the interior relative area per-cent that takes off sialic acid to three sialic acid structure territory.In table 3, oligosaccharide structure territory area percentage is listed as the mark of whole oligosaccharides colony.
Table 2: by the observed area percentage of HPAEC-PAD about every kind of structural domain
Figure A200680053116D01542
Table 3: the area hundred that is expressed as the weighted mean of table 2 data set about every kind of structural domain Proportion by subtraction.
Figure A200680053116D01551
Suppose complete glycosylation.
By the LC/MS of glycopeptide, the LC/MS of oligosaccharides and the CTLA4 that HPAEC-PAD measures A29YL104EN connection oligosaccharide structure: the N connection carbohydrate of-Ig molecule combines with the consensus sequence motif of Asn-X-Ser/Thr.This sequence has the CTLA4 of one of following sequence A29YL104EOccur on-Ig monomer the chain 3 times: (i) 26-383 of SEQ ID NO:4, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:4 and (the vi) 25-383 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4.The consensus sequence motif occurs on the following residue in SEQ ID NO:4: Asn 102Leu 103Thr 104Asn 134Gly 135Thr 136And Asn 233Ser 234Thr 235Based on consensus sequence, have 6 N connection carbohydrate site/dimer molecules, described dimer molecule is formed by any one or 2 in the following sequence monomer: (i) 26-383 of SEQ ID NO:4, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:4 and (the vi) 25-383 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4.
N connection carbohydrate can have 3 general mutation: high mannose, mixture and/or mixture.The LC/MS technology that is used for the glycopeptide analysis obtains exploitation.Monomer (have one of following sequence: (i) 26-383 of SEQ ID NO:4, (ii) SEQ ID NO:4 26-382, (iii) SEQ ID NO:4 27-383, (iv) SEQ ID NO:4 27-382, (v) the cutting of the trypsinase endoproteolysis of the 25-382 of SEQ ID NO:4 and (the vi) 25-383 of SEQ ID NO:4) causes comprising 3 kinds of peptides (referring to the table 25 among the embodiment 22) of N linked glycosylation.All 3 N connection sites all are filled with carbohydrate structure.Trypsinase fragment T5 corresponding to the amino acid 65-109 of SEQ ID NO:4 is included in Asn 102On glycosylation.Trypsinase fragment T7 corresponding to the amino acid/11 20-154 of SEQ ID NO:4 is included in Asn 134On glycosylation.Trypsinase fragment T14 corresponding to the amino acid 229-237 of SEQ ID NO:4 is included in Asn 233On glycosylation (referring to the table 25 among the embodiment 22).
In order to measure the glycosylated particular type on each site, protein digestion and isolating grade are collected T5, T7 subsequently and the T14 peptide obtains carbohydrate from each specific site by increasing.Purpose tryptic peptide peak is handled and is processed with PNGase F and is used for analyzing on the Hypercarb post by LC/MS.The result is presented at that compound on each site is two, the heterogeneous population of three and four feeler structures.These can find out in Figure 16 that wherein chromatography makes sugar become 4 kinds of structural domains: take off sialic acid, single sialic acid, bifunctional sialyltransferase and three sialic acid structures (being called structural domain I, II, III and IV).Can the glycosylation molecule comprise the colony of glycosylation molecule or composition between analyze and the feature of carbohydrate overview relatively comprise between peak area per-cent, structural domain area percentage, trough distance or peak separation from.
The LC/MS of CTLA4-IgN connection oligosaccharides characterizes
LC/MS porous graphite carbon (PGC) chromatography is the method that is used for carrying out profile analysis to N connection oligosaccharides, and the several advantages that surpass high pH anion-exchange chromatography (HPAEC) can be provided.In these advantages some comprise: by the direct profile analysis of digestion mixture, this makes sample forfeiture and degraded drop to minimum; Directly the MS interface provides the fast characterizing that is used for oligosaccharides and the method for analysis; The resolving power that increases by the PGC chromatography allows to compare and meticulousr structural domain inner analysis between structural domain.
LC/MS PGC method allows the quick profile analysis and the sign of the oligosaccharides with regard to the glycan type, and is determined at the sialylated and ramose degree on the non-reducing end of carbohydrate.Negative ion mode MS spectrum produces the data that are easy to explain subsequently, have MIN oligosaccharides fracture, and holotype ionization allows the checking of structure kind.Method described herein can be applied to the complete digestion mixture of glycoprotein, and previous isolating oligosaccharides sample and need not derivatize.The chromatographic flow of using allows to collect from the peak of overview mutually and is concentrated into drying, need not further processing and promptly can be used for more detailed sign.In one embodiment, this method is used to characterize CTLA4-Ig N connection oligosaccharides.Use LC/MS PGC method, 31 different types of oligosaccharides can identify on the CTLA4-Ig molecule that described CTLA4-Ig molecule comprises the sequence that has from SEQ ID NO:2, for example SEQID NO:5,6,7,8,9 or 10 monomer.
High pH anion-exchange chromatography (HPAEC) has been widely used in to the oligosaccharides that discharges from glycoprotein and has carried out profile analysis, and need not derivatize.The fact that the size of the saccharide residue type that the high resolving power of HPAEC and separation are existed, the type of key and glycan influences is the widely used reason of this technology.Principal element in the separation is an electric charge, and highly charged oligosaccharides is than the still less charged slower wash-out of glycan.The chromatography overview is divided into the structural domain that is limited by the charged species number on the glycan usually, and described charged species generally is sialic acid residues (Figure 17).
In order to obtain, can to collect HPAEC peak, desalination and characterize by MS and/or NMR about unknown oligosaccharide structure more information.A kind of Consideration of the HPAEC-PAD profile analysis that oligosaccharides distributes is a detecting pattern inherent variability.Electrochemical cell is aging to cause the overview variability with the electrode surface fouling.Reported that equally oligosaccharide structure and sialylated degree can cause the variability that detects in the cell when using the HPAEC (HPAEC-PAD) with pulsed current detection.This variability can influence be used for the effect that evaluation process changes or measure batch between conforming quantitatively and the relative quantification result.Because its speed and specificity, mass spectroscopy (MS) has obtained to popularize as the technology of the oligosaccharides profile analysis that is used to assess glycoprotein.Although the MS overview can't be used for directly measuring different configuration or branching pattern, the MS data can be used for identifying the structure kind and detect the qualitative variation that sugared shape distributes.
Porous graphite carbon (PGC) the chromatography profile analysis method that is used for the N connection oligosaccharides that enzymatic discharges uses ultraviolet ray (UV) and mass spectrum (MS) to detect, with to directly from the enzymatic digestion mixture or join oligosaccharides from the N of isolating oligosaccharides and carry out profile analysis and sign.This method can be used for the oligosaccharides that discharges from CTLA4-Ig glycoprotein is carried out profile analysis and sign.LC/MS PGC method can be assessed the oligosaccharides Uniformity of Distribution that is caused by production process, and modifies any variation in the oligosaccharides distribution that causes by process.In the chromatography trace analysis of the N of CTLA4-Ig molecule connection oligosaccharides, the N connection oligosaccharides that enzymatic discharges can easily separate by the PGC post, with cumulative sialylated and cumulative size sequence.The range of structures that exists and the relative quantity of every class formation are measured (embodiment 3) by the combination that MS and UV analyze.
In order to make LC/MS PGC method optimization, the optimization of mass spectrum condition can be useful.Optimization can comprise one group of surface mapping experiment, to assess the effect that solvent composition and MS ionization parameter detect oligosaccharides.The solvent composition parameter that is used to assess comprises per-cent acetonitrile (volume) and elutriant additive (trifluoroacetic acid and ammonium hydroxide).The MS ionization parameter of the evaluation that is used to assess comprises desolvation temperature, capillary voltage and awl voltage (conevoltage) setting about electrospray source.
The ionization parameter can play an important role in signal is replied.Measure the ionization parameter that the model that produces is used for setting the measurement in chromatography process by the surface mapping.Higher value about desolvation temperature and awl voltage causes bigger replying.Capillary voltage is just when depending on the elutriant additive, having the solvent systems that comprises TFA of higher slightly optimization capillary voltage and become.Factor with maximum effect is the volume percent of acetonitrile, and higher ethane nitrile content causes higher replying.
The porous graphite carbon-coating is analysed
Porous graphite carbon (PGC) has been used for the Solid-Phase Extraction desalination of oligosaccharides.PGC also is known as under acid and alkaline elution requirement and is used for the isolating effective chromatography media of oligosaccharides.The acidity of the oligosaccharides that discharges about enzymatic from the CTLA4-Ig molecule and the chromatography condition of alkaline profile analysis obtain exploitation, and described CTLA4-Ig molecule has the sequence monomer from SEQ ID NO:2.Every kind of condition is compatible with the MS detection with UV.As observed in the perfusion experiment, acid elution requirement causes the MS sensitivity higher than alkaline condition.MS about the neutral oligosaccharides that detects as the TFA adducts of wash-out under acidic conditions replys, be wash-out under alkaline condition respective peaks intensity 5-9 doubly.Difference during signal is replied is more not remarkable for acidic oligosaccharide, and 3 times of replying about the signal of mono-sialylated glycan of average out to and the signal that equates with the double-sialylated glycan are replied.With NH 4The chromatogram of OH wash-out (Figure 15 A-B) is compared, and the peak number order that increases in the chromatogram of TFA wash-out (Figure 14 A-B) is the isolating result of different capitiform formula of oligosaccharides.From the collection at indivedual peaks of TFA gradient elution with concentrate and cause unimodal 2 peaks that after being injected into again, are divided into equal in quality.Alkaline wash-out from the oligosaccharides of PGC post causes simpler overview (Figure 15 A-B).The alkalescence elution requirement does not cause different completely head to separate, yet, observe significant peak broadening.Peak resolving power can obtain increasing by increasing column temperature, described column temperature increase will speed up different capitiform formula exchange (people such as Itoh S., J.Chromatogr.A.2,002 968 (1-2), 89-100).Yet, compare with acid elution requirement, be still minimizing about the sensitivity (ion counting) of the oligosaccharides that detects.Reported salt for example the interpolation of ammonium acetate can increase sensitivity.(ChurmsSC， J.Chromatogr.A.500(1990)555-583.)
What the interpolation of ammonium acetate, trifluoroacetic acid ammonium or ammonium formiate caused increasing replys, but also causes asymmetric peak broadening.The potential interference that the salt pair UV of peak broadening that is produced and interpolation detects makes salt add becomes unappealing selection.Eliminating different isolating method alternately is to make oligosaccharides be reduced into corresponding sugar alcohol.
Higher sensitivity and chromatography resolving power make acid elution requirement useful for the oligosaccharides profile analysis.Specific profile analysis systems is by forming by 2 two-position 6 ports valves and Hypercarb 5 μ M posts (100 x 4.6mm) link coupled Luna C18 post.Hypercarb post and UV detector (Waters 2996 PDA) coupling, described UV detector is connected with the Q-ToFMicro with standard ESI probe (Micromass).By suitable on-off control, the CTLA4-Ig sample of prepurification can use the Hypercarb post to carry out profile analysis separately, or digestion mixture can by digestion mixture is injected directly into the placed in-line Luna C18 of Hypercarb post in carry out profile analysis.Usually overview derives from the N connection oligosaccharides that discharges from 10-20 nmole protein.
In certain embodiments, according to any one or a plurality of chromatogram at representative peak, the invention provides the colony of CTLA4-Ig molecule with chromatogram.Representative oligosaccharides overview chromatogram about the CTLA4-Ig molecule is shown among Figure 13, Figure 14 A-B, Figure 15 A-B (PGC), Figure 17 (HPAEC/PAD) and Figure 18 A-B, and described CTLA4-Ig molecule has the monomer from the sequence of SEQ ID NO:2.These chromatograms can resolve into 4 different structure territories that comprise oligosaccharide structure, and wherein sialylated degree is cumulative in the structural domain of wash-out after a while.The PGC chromatographic system allows the direct interface with mass detector.Even for the oligosaccharides that exists with low per-cent, mass resolution and signal to noise ratio also are acceptable.Compare with HPAEC, the chromatography resolving power of indivedual oligosaccharide structures seems bigger in the PGC chromatographic separation.
Collecting the peak from the HPAEC method needs desalination, and the high pH that uses introduces the possibility of stripping reaction, the precision architecture evaluation of described stripping reaction possibility Interference Peaks.Because the chromatography condition that uses with the PGC chromatography is saliferous not, so the oligosaccharides peak of wash-out can be collected and concentrate with MIN operation.The collection at the peak of this permission wash-out and concentrated is injected into the oligosaccharides of collecting in the HPAEC system subsequently.The oligosaccharides of collecting is injected into the structure that allows in the HPAEC-PAD system to be present in some peaks in the HPAEC overview again specifies (Figure 17).Because incomplete peak resolving power on anion-exchange column is not can map to the HPAEC overview in separative peak.
The overview that produces by the direct injection of digestion mixture and about from the isolating oligosaccharides of same protein quality sample those and inequality.The overview (Figure 18 A-B) that is produced by direct injection has different anomer ratios, thereby the oligosaccharides that hint is collected concentrated causes the different headization that increases.The more important thing is that the overview that is produced by direct injection comprises the peak, this is corresponding to four sialylated structures.This structure does not obtain identifying in the overview of collection and isolating oligosaccharides.Except that the minute that shortens, by avoid collecting with concentration process in Polyose degradation, directly can cause the expression that distributes of more accurate oligosaccharides from the profile analysis of digestion mixture.
Relative quantification
The volume percent that acetonitrile is pointed out in the surface mapping that the perfusion sample is carried out has remarkable effect to the ionization efficiency of wash-out oligosaccharides.Strength of signal is to the feasible retention time relative quantification oligosaccharides that depends on elution peak by MS of the dependence of the ethane nitrile content in the moving phase.Variation in the column condition can influence the elution time on the PGC post.For this reason, will be difficult to obtain to compose the consistent relative quantification of wash-out overview from ion chromatography.The influence that should not be subjected to solvent composition at the UV at 206nm place arteries and veins mark to the identical degree of ion arteries and veins mark.Relative quantification uses UV arteries and veins mark to carry out, and ion arteries and veins mark only is used for characterizing and qualitative comparison.For in quantitative 4 kinds of oligosaccharide structure territories each, the duplicate injection of isolating oligosaccharides causes the per-cent coefficient of variation (%RSD) less than 4% from single glycoprotein is criticized.
The O that comprises in the monomeric CTLA4-Ig molecule of SEQ ID NO:2 connects structure
Except that N connection carbohydrate, the CTLA4-Ig molecule can comprise O connection carbohydrate.O connection oligosaccharide structure can use a series of quadrature mass spectroscopy technology to analyze.These technology comprise that several endopeptidase cuttings are the LC/MS/MS analysis subsequently.
CTLA4-Ig molecule about being formed by the monomer that has from SEQ ID NO:5,6,7,8,9 or 10 sequence uses the exact mass electrospray ionization to characterize 2 main sites of O linked glycosylation, and is determined at the primary structure on each O connection site.These data are summarized among Fig. 9.It is consistent that 3 kinds of main O of data and existence connect structures: (GalNAc) 1(Gal) 1(NeuAc) 1(GalNAc) 1(Gal) 1(NeuAc) 2(GalNAc) 1(GlcNac) 1(Gal) 2(NeuAc) 2Every kind of structure is observed with the difference amount on each site.This tittle be relative quantification and expression derive from repeatedly the data of analyzing.O connection oligosaccharides is that CTLA4-Ig contributes quite a large amount of sialic acids.There are 2 main O connection oligosaccharides attachment point/chains.The main site that takes place about O connection oligosaccharides is Ser 165, this occupies for about 95% time.The less important site that takes place about O connection oligosaccharides is Ser 155/156, this occupies for 25% time.The orthogonal data that this paper presents provides the general introduction that is present in the dominant carbohydrate structure on this kind CTLA4-Ig molecule, and is summarized among Fig. 9.
Generally speaking, O connection carbohydrate has and is far longer than the structure heterogeneity that exists in the N connection carbohydrate.In addition, adhere to about O connection and do not have consensus sequence.Therefore, developed the structural characterization that a series of quadrature technique are used for O connection oligosaccharides: LC/MS complete analysis and LC/MS glycopeptide are analyzed.
Based on Edman degraded and MALDI, O connection site report is Ser 165(with regard to SEQ ID NO:2).In order to obtain about Ser 165The immediate data that glycosylation exists, MS/MS order-checking use b ' and the y ＂ ionization series (referring to table 4 and table 5) on the T9 peptide to carry out.Table 4 has been listed the ionization series about the T9 peptide in the glycosylation of 4 kinds of different states.In all 4 kinds of states, b ' ionization series, b1...b6 ion unanimity.Yet, b ' ionization series, b7...b MaxBy at b7 (Ser 165) on the different glycosylation state change.As confirmation, reported corresponding y ＂ ionization series.In all 4 kinds of y ＂ ionization seriess, the y1....y19 ion is in full accord.Yet, y ＂ ionization series, y20...ymax passes through at y20 (Ser 139) on the different glycosylation state change.B ' and y ＂ ionization series are supported following Edman order-checking hint: Ser together 139It is the main site of the O linked glycosylation on the T9 peptide.T9 is the peptide that comprises several Serines and threonine residues.
Table 4 presents about containing or do not contain (GalNAc) 1(Gal) 1(NeuAc) 1The LC/MS/MS b ' and the y of T9 peptide of O connection ladder " ion.B ' ionization series is identical for all spectrums, until b7, and wherein the O connection carbohydrate structure listed by each series above subsequently of spectrum and difference.Y ' ionization series is identical for all spectrums, until y19, and wherein the O connection carbohydrate structure listed by each series above subsequently of spectrum and difference.
Table 4: about the LC/MS/MS b ' and the y of T9 peptide " ion.
Figure A200680053116D01611
Table 5: O connection glycopeptide fragment with corresponding sequence number, aminoacid sequence and Theoretical Mass
At Ser 165On O connection carbohydrate structure represent the heterogeneous population of 3 main kinds.In Figure 19, the T9 glycopeptide is observed in going the flatung spectrum.Exist in the base peak at 2689.2amu place, this Theoretical Mass with this peptide 2689.11amu is consistent.This spectrum understands that for example 3 kinds of main O connect structure.This spectrum for example understands to have and O connection structure (GalNAc) 1(Gal) 1(NeuAc) 1The basic peptide of consistent sugar ladder.The bolded section that this spectrum is amplified has strengthened 10 times, and identifies and (GalNAc) 1(Gal) 1(NeuAc) 2(GalNAc) 1(GlcNAc) 1(Gal) 2(NeuAc) 22 kinds of consistent other O connect structure.
Mass spectroscopy is used to assess the relative abundance of each O connection kind.In Figure 19, (GalNAc) 1(Gal) 1(NeuAc) 1Glycan with (GalNAc) 1(Gal) 1(NeuAc) 2Glycan with the 10:1 ratio and with (GalNAc) 1(GlcNAc) 1(Gal) 2(NeuAc) 2Glycan is observed with the 30:1 ratio.Therefore in one embodiment, the invention provides the colony that comprises the CTLA4-Ig molecule, described CTLA4-Ig molecule has (GalNAc) 1(Gal) 1(NeuAc) 1Glycan with (GalNAc) 1(Gal) 1(NeuAc) 210: 1 ratios of glycan.In another embodiment, the invention provides the colony that comprises the CTLA4-Ig molecule, described CTLA4-Ig molecule has (GalNAc) 1(Gal) 1(NeuAc) 1Glycan with (GalNAc) 1(GlcNAc) 1(Gal) 2(NeuAc) 230: 1 ratios of glycan.(GalNAc) 1(Gal) 1(NeuAc) 2Glycan with (HexNAc) 2(Gal) 2(NeuAc) 2Glycan is observed with 20: 1 ratio.In another embodiment, the invention provides the colony that comprises the CTLA4-Ig molecule, described CTLA4-Ig molecule has (GalNAc) 1(Gal) 1(NeuAc) 2Glycan with (HexNAc) 2(Gal) 2(NeuAc) 220: 1 ratios of glycan.In another embodiment, the invention provides the colony of CTLA4-Ig molecule, described CTLA4-Ig molecule is included in all the described ratios in this paragraph.In addition, negative ion electrospray sprays these 3 kinds of dominant structures of spectrum confirmation; Relative abundance separately is shown among Fig. 9.
About comprising the monomeric CTLA4-Ig molecule of SEQ ID NO:2, remove Ser 165Outside the site, at Ser 155Or Ser 156On observe second O connection site.This site is called Ser 155/156Comprise Ser 155/156The D8 peptide produce by AspN digestion, and corresponding to the amino acid/11 50-156 of SEQ ID NO:2.This peptide separates by LC/MS and detects.Show the base peak of 790.2amu about the spectrum (this paper does not show) of D8O connection glycopeptide, this Theoretical Mass with 790.8amu is consistent.This spectrum is for example understood and structure (GalNAc) 1(Gal) 1(NeuAc) 1Consistent peptide ion and a series of ion.This peptide is not glycosylated with preponderating; Glycosylation in (GalNAc) 1(Gal) 1(NeuAc) 1Kind constitutes about 22% peak area.
The O connection oligosaccharide structure of CTLA4-Ig strand uses a series of quadrature mass spectroscopy technology to characterize.These technology comprise the endopeptidase cutting and LC/MS analysis in 2 dominant sites of O linked glycosylation, and utilize electrospray ionization to measure dominant structure on each O connection site.These data are summarized among Figure 20.Can exist 4 kinds of dominant O to connect structure: (GalNAc) 1(Gal) 1(NeuAc) 1(GalNAc) 1(Gal) 1(NeuAc) 2(HexNAc) 2(Gal) 2(NeuAc) 2(HexNAc) 2(Gal) 2(NeuAc) 3These structures detect with the difference amount on each site.CTLA4-Ig strand above 95% has at least (HexNAc) 2(Gal) 2(NeuAc) 2
Developed another kind of mensuration to confirm O connection carbohydrate and to seek more not general structure.This technology utilizes trypsinase and Quimotrase to digest altogether, to produce the peptide that is turned out to be THTSPPSPAPELL (the amino acid/11 59-171 of SEQ ID NO:2) by MS/MS.This peptide allows to identify 1 mono-sialylated, 2 double-sialylated and 1 three sialylated O connection kind.About three sialylated kinds really fixed structure do not illustrate yet, yet, 2 kinds of possibilities have been proposed: comprise the peptide that has 3 sialic core 2 structures or be present in 2 core 1 structures on 2 different amino-acid residues.
Complementary technology by the complete analysis of MS, is used to confirm the existence of the heterogeneous O linked glycosylation of CTLA4-Ig molecule.CTLA4-Ig dimer and CTLA4-Ig strand are handled with PNGase F, to remove N connection oligosaccharides.Molecule detects by mass spectrograph subsequently and corresponding ion goes flatung to become spectrum.In the strand material, dominant glycan composition is (HexNAc) 2 (Hex) 2 (NeuAc) 2, and reference is (HexNAc) 1 (Hex) 1 (NeuAc) 1 with preponderating.The glycosylation composition is with observed during the LC/MS peptide analysis those are consistent.The change in O linked glycosylation pattern, observe second kind of main modification.The quality of observing 113 ± 4u between non-reduced kind of strand and reduction CTLA4Ig standard moves.The quality of 113 ± 4u moves and is disappearing with DTT reduction back.In the dimer material, the ion that is produced is sealed flatung and is become the spectrum (this paper does not show) have in the main peaks at 79944amu place, and described main peaks is corresponding to 2 kinds (GalNAc) 1(Gal) 1(NeuAc) 1The existence of structure.Connect the combination of structure or maximum a kind of O of branch connection structures corresponding to 3 kinds of O at the next maximum peak at 80600amu place.The 3rd maximum peak connects structure or comprises the combination that maximum 2 kinds of O of branch connect structure corresponding to 4 kinds of O.
The mensuration of sialic acid content
Another aspect that glycoprotein characterizes is sialic mensuration.Sialic acid content can be the mark characteristic of glycoprotein.The sialic acid content of glycoprotein of the present invention can be assessed by ordinary method.For example, sialic acid can pass through direct colo(u)rimetry (people such as Yao, 1989, Anal.Biochem., 179:332-335) measure respectively, use in triplicate at least sample.The another kind of method that sialic acid is measured relates to thiobarbituricacid (thiobarbaturic acid) use (TBA), as by people such as Warren, and 1959, J.Biol.Chem., 234:1971-1975 describes.Another method relates to efficient chromatography, for example by people such as H.K.Ogawa, and 1993, J. ChromatograDhy, 612:145-149 describes.
In one embodiment, the method for the amount of mensuration N-n acetylneuraminic acid n (NANA) and N-hydroxyacetylneuraminic acid (NGNA) is to handle (for example, referring to embodiment 3) by the acid hydrolysis of purpose glycoprotein.In this method, NANA and NGNA cut open by acid hydrolysis and protein.In one embodiment, glycoprotein carries out purifying by the method that is suitable for its purifying basically.NANA that discharges and NGNA separate on Rezex MonosaccharideRHM post by HPLC, and detect by UV absorbancy (206nm).NANA and NGNA carry out quantitatively based on the NANA of operation simultaneously and the response factor of NGNA standard.The result can be reported as NANA and NGNA and proteinic mol ratio (MR) respectively.
The purpose of measuring the acid-hydrolysis method of sialic acid content is to measure CTLA4-Ig or CTLA4 A29YL104ETotal sialic acid (NANA and NGNA) and proteinic amount (mol ratio) in-the Ig sample.Yet important is to point out, these sialic acid mol ratios comprise bonded and free NANA and NGNA.Mol ratio result is based on CTLA4-Ig or CTLA4 from hydrolysis A29YL104EThe NANA of-Ig sample and NGNA obtain the NANA of non-hydrolysis and the peak area ratio of NGNA standard.Can also use the hydrolysis standard of NANA and NGNA.
For example, the CTLA4-Ig molecule for the aminoacid sequence with SEQ ID NO:2 obtains mol ratio.When not having hydrolysis, the purpose peak in the chromatogram of NANA and NGNA standard is as unimodal appearance.When NANA standard and the hydrolysis of CTLA4-Ig sample, the chromatogram that is produced shows that NANA is the subsequently and then little acromion of main peaks (＜10% a main peaks area; Be called " NANA of degraded "); Have and do not have the NANA standard of the same concentrations of hydrolysis to cause very approaching peak area, comprise degradation product (degradant).In the chromatogram of the NGNA kind of degrading, do not find out the peak significantly, although the about 8-9% of the visible minimizing of the area at the NGNA peak in the NGNA standard of hydrolysis counting." NGNA degradation product " in the NANA standard of mass spectroscopy (MS) experiment confirm hydrolysis and the CTLA4-Ig sample of hydrolysis produced by the loss from 18 dalton (water) of NANA.Therefore, this method suitably is included in the little acromion in the integration at NANA peak among the CTLA4-Ig of hydrolysis.Confirm also that by the MS experiment NGNA degrades after hydrolysis, its loss is 18 dalton.The NGNA degradation product is wash-out between NANA and NANA degradation product, thereby makes UV not detect it.Therefore in the CTLA4-Ig material, NGNA content is about 5% NANA content, and the co-elute of NGNA degradation product causes the NANA peak area less than 0.5% change, and this is in the variability scope of NANA peak area.This method can't be included in the area of the NGNA that degrades among the NGNA result; Therefore NGNA result can low＜10%, and is same in the variability of this method.
Because NGNA is considered to than NANA immunogenicity is arranged more, so exist clinical preferred for the reorganization therapeutical agent that comprises low NGNA mol ratio.In one embodiment of the invention, the sialic acid advantage in the colony of CTLA4-Ig molecule is NANA rather than NGNA, and wherein mole sialic acid/mole CTLA4-Ig molecule or dimeric mol ratio are about 5-about 18 in this colony.In another embodiment, CTLA4 A29YL104ESialic acid advantage in the colony of-Ig molecule is NANA rather than NGNA, wherein mole sialic acid/mole CTLA4 in this colony A29YL104E-Ig molecule or dimeric mol ratio are about 5.5-about 8.5.
CTLA4-Ig and CTLA4 A29YL104E -Ig expression cassette
The invention provides the nucleic acid of coding CTLA4-Ig molecule, it is expression cassette in one embodiment.The present invention also provides coding CTLA4 A29YL104EThe nucleic acid of-Ig molecule.In one embodiment, the nucleic acid of coding CTLA4-Ig molecule is included in the expression cassette.In another embodiment, the nucleic acid of coding CTLA4-Ig molecule is included in the expression cassette derived from the plasmid of the nucleotide sequence with SEQ ID NO:17.In further embodiment, coding CTLA4 A29YL104EThe nucleic acid of-Ig molecule is included in the expression cassette.In certain embodiments, coding CTLA4 A29YL104EIt is in the expression cassette of plasmid of ATCC registration number PTA-2104 that the nucleic acid of-Ig molecule is included in derived from preservation.
But nucleic acid of the present invention can be for example purpose cDNA, cDNA sample, DNA or the RNA nucleic acid molecule of expression cassette of expression-form, and it can be expressed by natural promoter or derivatives thereof or complete allogenic promotor.Alternately, purpose nucleic acid can encoding antisense RNA.Purpose nucleic acid can coded protein (for example, glycoprotein, for example CTLA4-Ig or CTLA4 A29YL104EAnd can comprise or not comprise intron-Ig glycoprotein).
In one embodiment, the nucleic acid of coding with the active peptide of CTLA4 can derive from the T cell genomic dna or be present in mRNA in the activated T lymphocyte.In another embodiment, coding CTLA4 A29YL104EThe nucleic acid of-Ig also can derive from the T cell genomic dna or be present in mRNA in the activated T lymphocyte.In another embodiment of the invention, coding target protein matter, for example CTLA4 or CTLA4 A29YL104EThe gene of-Ig can be cloned from genomic library or cDNA according to the standard schedule of those skilled in the art's practice.CTLA4 or CTLA4 for example encode A29YL104EThe cDNA of-Ig can obtain by separate total mRNA from suitable clone.Use methods known in the art, double-stranded cDNAs can and can insert in the suitable phage vector or plasmid subsequently by total mRNA preparation.The round pcr that gene can also use this area fully to establish is cloned.In one embodiment, coding CTLA4 or CTLA4 A29YL104EThe gene of-Ig can be via PCR according to being cloned by nucleotide sequence information provided by the invention.
In another embodiment, comprise CTLA4 or CTLA4 A29YL104EThe dna vector of-Ig cDNA can serve as template in PCR reaction, the Oligonucleolide primers in the purpose zone that wherein is designed to increase can be used to obtain to comprise that regional separated DNA fragment.In specific embodiments of the present invention, the purpose zone of target can be the ectodomain of CTLA4 among the CTLA4cDNA, comprises the ectodomain of human CTLA 4.In certain embodiments, CTLA4 A29YL104EAmong-Ig the cDNA purpose zone of target can be on the amino acid position 55 and 130 of SEQ ID NO:2, have a CTLA4 that amino acid changes ectodomain (for example, referring to SEQ ID NO:18), comprise ectodomain with human CTLA 4 that above-described amino acid changes.
For expressed fusion protein in background of the present invention, in one embodiment, (CTLA4-immunoglobulin (Ig) (CTLA4-Ig) fusion rotein or CTLA4 for example encode in the mosaic gene fusion A29YL104EThe gene of-Ig fusion rotein) comprises the nucleotide sequence of coded signal sequence, after mosaic gene is transcribed and translated, instruct synthetic fusion rotein secretion recently thus.In one embodiment, can use natural CTLA4 signal sequence (for example at Harper, K., wait the people (1991, J.Immunol.147, the human CTLA 4 signal sequence of describing among the 1037-1044) in alternative embodiment of the present invention, can use the allos signal sequence to instruct CTLA4-Ig or CTLA4 A29YL104E-Ig secretion (for example, the oncostatin M signal sequence (people such as MalikN., 1989, Mol Cell Biol9 (7), 2847-2853) or the immunoglobulin (Ig) signal sequence).The nucleotide sequence that those skilled in the art understand corresponding to signal sequence can insert in the mosaic gene fusion by the standard recombinant dna technology, for example by connecting in the frame that carries out signal sequence in nucleotide sequence 5 ' end of coding CTLA4.
According to the budapest treaty clause, coding was preserved in American type culture collection (ATCC), 10801 University Blvd., Manassas, VA, 20110 corresponding to the DNA of the aminoacid sequence of CTLA4-Ig fusion rotein on May 31st, 1991.It has specified ATCC registration number 68629.In addition, comprise coding corresponding to CTLA4 A29YL104EThe expression plasmid of the nucleotide sequence of the aminoacid sequence of-Ig is preserved in ATCC according to the budapest treaty clause on June 20th, 2000.The plasmid of preservation has been specified ATCC registration number PTA-2104.The preservation plasmid is also referred to as pD16LEA29Y and pD16L104EA29Y.CTLA4 A29YL104E-Ig is at U.S. Patent number 7,094, and 874 and common unsettled Application No. 09/579,927,60/287,576 and 60/214,065, and further described among International Patent Publication No. WO 01/923337 A2, described all patent documents are whole introduces the application as a reference.
Expression vector of the present invention can be used for transfectional cell, and eucaryon (for example, yeast, Mammals or insect cell) or protokaryon to produce by the nucleotide sequence coded protein of carrier (for example, for example CTLA4-Ig, CTLA4 of fusion rotein A29YL104E-Ig molecule etc.).Those skilled in the art understand the expression of desired protein product in prokaryotic organism and are everlasting most and carry out in the intestinal bacteria, wherein use the carrier that comprises composing type or inducible promoter.Some coli expression carriers (being also referred to as fusion vector in the art) are designed to add many amino-acid residues, normally give the N-terminal of the recombinant protein of expressing.Described fusion vector can be brought into play 3 kinds of functions: the solubility that 1) increases desired recombinant protein matter; 2) expression of increase purpose recombinant protein; With 3) help the recombinant protein purifying by the part that serves as in the affinity purification.Some examples of fusion expression vector include but not limited to: the pGEX (Amrad Corp, Melbourne, Australia) that glutathione s-transferase and desired protein are merged; B) pcDNA that 6x-His and purpose recombinant protein are merged TM3.1/V5-His A B ﹠amp; C (Invitrogen Corp, Carlsbad, CA); And c) make the conjugated protein pMAL that merges with the target recombinant protein of maltose E (New EnglandBiolabs, Beverly, Mass.).
Be suitable for to have the expression vector (construct) of introducing, for example plasmid etc. according to process of the present invention and method cultured cells.The expression vector establishment body can be via introducings such as transfection, fat transfection, conversion, injection, electroporation or infection.Expression vector can comprise coding and be used for proteinic encoding sequence or its part expressing and produce at cultural method.This kind expression vector can comprise for the encoding sequence that inserts transcribes and translates required component.Comprise the protein of coding production and the sequence of polypeptide, and the suitable expression vector of transcribing and translate controlling elements, can use those skilled in the art's method well-known and practice to produce.These methods comprise extracorporeal recombinant DNA technology, synthetic technology and vivo gene reorganization, it is people such as J.Sambrook, 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, people such as N.Y. and F.M.Ausubel, 1989, Current Protocols inMolecular Biology, John Wiley ﹠amp; Sons, New York obtains among the N.Y describing.
Selective marker can be used in recombinant expression vector (for example, plasmid), and wherein the carrier stable integration is in the genome of cell, to give resistance to the cell with carrier.This allows it to select in suitable selective medium.Can use many selective systems, include but not limited to, hypoxanthine-guanine phosphoribosyl transferase (HGPRT), herpes simplex virus thymidine kinase (HSV TK), (people such as Wigler, 1977, Cell, 11:223), (Szybalska and Szybalski, 1992, Proc Natl.Acad.Sci.USA, 48:202) and the fast cry of certain animals phosphoribosyltransferase of gland (APRT), (people such as Lowy, 1980, Cell, 22:817) gene, this can use in hgprt-, tk-or aprt-cell respectively.
The basis that the following non-limitative example that can be included in the marker gene in the expression vector also can be selected as the metabolic antagonist resistance: gpt, its give resistance at mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl.Acad.Sci.USA, 78:2072); Dhfr, its give resistance at methotrexate (people such as Wigler, 1980, Proc.Natl.Acad.Sci.USA, 77:357; And people such as O ' Hare, 1981, Proc.Natl.Acad.Sci.USA, 78:1527); Hygro, its give resistance at Totomycin (people such as Santerre, 1984, Gene, 30:147); And neo, its give at the resistance of aminoglycoside G418 ( Clinical Pharmacy, 12:488-505; Wu and Wu, 1991, Biotherapy, 3:87-95; Tolstoshev, 1993, Ann.Rev.Pharmacol.Toxicol., 32:573-596; Mulligan, 1993, Science, 260:926-932; Anderson, 1993, Ann.Rev. Biochem., 62:191-21; May, 1993, TIB Tech, 11 (5): 155-215).The common known recombinant DNA technology in this area can conventionally be used to select required recombinant cell clone.This kind technology is for example people such as Ausubel (editor), Current Protocols in MolecularBiology, John Wiley ﹠amp; Sons, NY (1993); Kriegler, 1990, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY; In the 12nd and 13 chapters, people such as Dracopoli (editor), Current Protocols in Human Genetics, John Wiley ﹠amp; Sons, NY (1994); People such as Colberre-Garapin, 1981. J.Mol. Biol., obtaining in 150: 1 describing, described reference integral body is incorporated herein by reference.
The expression level of expressed protein molecule can obtain via the amplification of expression vector increasing (about summary, referring to Bebbington and Hentschel, ＂ The use of vectors based ongene amplification for the expression of cloned genes in mammalian cellsin DNA cloning ＂, the 3rd volume, Academic Press, New York, 1987).When the mark in the expression vector system of expressing target protein matter be can increase the time, the increase in the inhibitor level that exists in the substratum of host cell will increase the copy number of marker gene.Because the zone of amplification combines with protein coding gene, thus protein production will increase concomitantly (people such as Crouse, 1983, Mol.Cell.Biol., 3:257).Carrier with nucleotide sequence of coding selective marker glutamine synthase (GS) or Tetrahydrofolate dehydrogenase (DHFR) can increase in the presence of medicine methionine(Met) sulfimide (methionine sulphoximine) or methotrexate respectively.The advantage of this kind carrier is the operability of clone, and for example rat bone marrow tumour cell is that NSO and Chinese hamster ovary Chinese hamster ovary celI are DG44, and it is respectively the negative and Tetrahydrofolate dehydrogenase feminine gender of glutamine synthase.
In one embodiment of the invention, coding soluble CTL A 4 or CTLA4 A29YL104EThe nucleotide sequence of-Ig fusion protein molecule can insert and be designed in the expression vector of expressing external sequence in eucaryon host.The adjusting component of carrier can become according to the eucaryon host of selecting to be used to use.Be used for expressing soluble CTL A 4 or CTLA4 at eukaryotic host cell A29YL104EThe carrier of-Ig can comprise the enhancer sequence that is used for the optimization protein expression.
Mammalian cell (for example bhk cell, VERO cell, Chinese hamster ovary celI etc.) can have expression vector via expression vector is introduced and (for example, comprise coding CTLA4-Ig fusion rotein or CTLA4 in the proper host cell A29YL104EThe expression carrier of-Ig fusion rotein).Therefore, the present invention comprises and contains coding CTLA4-Ig or CTLA4 A29YL104EThe expression vector of the nucleotide sequence of-Ig fusion rotein, and comprise and can this kind expression vector be introduced wherein host cell via methods known in the art.As described herein, expression vector of the present invention can comprise coding CTLA4-Ig or the CTLA4 that is connected with at least a adjusting sequence A29YL104EThe nucleotide sequence of-Ig fusion rotein, its mode allow nucleotides sequence to be listed in the host cell to express.For those skilled in the art, it is well-known regulating sequence, and can select to instruct target protein matter in proper host cell, to express, as Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press describes among the San Diego, Calif. (1990).Regulating sequence can comprise following: enhanser, promotor, polyadenylation signal and other expression controlling elementss.The practitioner in the art understands the design expression vector can depend on following factor, for example treats the selection of host cell of transfection and/or the type and/or the amount of desired protein to be expressed.
Clone and expression plasmid (for example, pcSD and piLN) can make up as described in example 11 above.In one embodiment of the invention, will be connected to from the separated DNA fragment of plasmid pSV2dhfr-in the pcDNA3 carrier main chain, thereby produce expression vector pcSD.Carrier pcSD comprises following characteristics: cytomegalovirus (CMV) promotor is multiple clone site (MCS) subsequently; Poly-polyadenylation signal of Trobest (BGH) and transcription termination sequence; Mouse dhfr cDNA sequence is used for selecting and amplification; Ampicillin resistance gene; Be used for selecting and keeping with the pUC replication orgin intestinal bacteria (Escherichia coli).Structure comprises the carrier piLN of cDNAs of the part of encoding amino acid sequence, described aminoacid sequence is corresponding to the fragment of the ectodomain of human CTLA 4 acceptor embodiment 11, the cDNA of the first seed amino acid sequence of wherein encoding is connected with the DNA of the coding second seed amino acid sequence, and described DNA changes the CTLA4 protein of expression (about the residue corresponding to CTLA4 born of the same parents' outside part and IgG1 constant region corresponding to passing through Referring toFig. 1 and about the summary of Fig. 1) the solubility IgC district that allows the CTLA4 acceptor gene to express.In one embodiment, the oncostatin M signal peptide sequence can with merge corresponding to the aminoacid sequence of CTLA4 ectodomain, its subsequently with corresponding to Ig structural domain (for example, people IgC γ 1Structural domain) the second seed amino acid sequence merges, as before described in Fig. 1.The oncostatin M signal sequence allow to produce CTLA4 gene (for example, the CTLA4-Ig) protein of soluble form.
In order to make up the pcSD expression vector of the gene that comprises coding CTLA4-domain-immunoglobulin fusion proteins, can use methods known in the art (for example, restriction site subclone).For one embodiment of the invention, starting material can be by the cloning vector piLN digestion of describing among the embodiment 11 and the dna fragmentation of cutting.In another embodiment, comprise the aminoacid sequence of oncostatin M signal sequence and CTLA4Ig fusion rotein by the dna fragmentation of described carrier cutting, wherein said dna fragmentation is connected with the pcSD carrier of digestion.Oncostatin M-CTLA4-IgDNA fragment can be inserted the CMV promotor and comprise between the box of BGH polyadenylation signal and transcription termination sequence.This makes the CTLA4-Ig gene product place (Figure 21 under the control of CMV promotor in the plasmid of called after pcSDhuCTLA4-Ig; SEQ ID NO:17).
In addition, clone and expression plasmid (for example, pD16LEA29Y) can be derived from Invitrogen plasmid pcDNA3.Carrier pD16LEA29Y (Figure 22) comprises following characteristics: the neomycin resistance gene from pcDNA3 is replaced with mouse dhfr gene, under the control of no enhanser (enhancerless) (weakening) SV40 promotor; Coding CTLA4 A29YL104EThe gene of-Ig by CMV promoter expression and polyadenylation signal from bovine growth hormone gene; Expression cassette side about goal gene is a transcription termination sequence, promptly 5 of promotor ' with in 3 of polyadenylation site '; This carrier comprises 2 kinds of different restriction site polylinkers, 13 of promotor ' be used to clone goal gene and 1 in 5 of promotor ' the be used for carrier linearizing before transfection; This carrier comprises ampicillin resistance gene and the ColE1 replication orgin is used for breeding at the plasmid of intestinal bacteria; CTLA4 A29YL104E-Ig sequence (SEQ ID NO:3) is after the oncostatin M signal peptide and be called in the expression vector of carrier pD16LEA29Y and assemble.
Structure comprises the carrier of cDNAs of the part of encoding amino acid sequence, described aminoacid sequence is corresponding to the fragment of the ectodomain of human CTLA 4 acceptor (SEQ ID NO:2), wherein the amino acid Ala at the 55th place is replaced by amino acid Tyr, and the amino acid Leu at the 130th place is replaced (Fig. 3) by amino acid Glu.These amino acid change at the CTLA4 with SEQ ID NO:4 A29YL104EObtain in-Ig the aminoacid sequence describing.The cDNA of the first seed amino acid sequence of encoding (for example, coding CTLA4 A29YL104EThe sequence of-Ig) DNA with the coding second seed amino acid sequence is connected, and described DNA is corresponding to the CTLA4 of the expression that has SEQ ID NO:3 by change A29YL104-Ig protein (about corresponding to the residue of modified CTLA4 born of the same parents' outside part and IgG1 constant region referring to Fig. 3 with about the summary of Fig. 3) solubility allow CTLA4 A29YL104The IgC district that-Ig acceptor gene is expressed.
In one embodiment, the oncostatin M signal peptide sequence can with merge corresponding to the aminoacid sequence of CTLA4 ectodomain, its subsequently with corresponding to Ig structural domain (for example, people IgC γ 1Structural domain) the second seed amino acid sequence merges, as before described in Fig. 3.The oncostatin M signal sequence allows to produce CTLA4 gene (for example, the CTLA4 of soluble form A29YL104E-Ig) protein.
Produce the stable transfection of clone
(for example, fusion constructs, glycoprotein etc. the carrier of) DNA can be transformed in the proper host cell (for example, bacterial cell), to produce a large amount of clones' DNA to comprise coding target protein matter.Some non-limitative examples of the bacterial cell that is used to transform comprise that bacterial cell is coli strain DH5 α or MC1061/p3 (Invitrogen Corp., San Diego, Calif.), this can use the standard program of this area practice to transform, and bacterium colony can screen with regard to suitable plasmid expression subsequently.
About eukaryotic cell for example the expression vector of mammalian cell can comprise promotor and the control sequence compatible with mammalian cell.In one embodiment of the invention, these regulatory elements can be the CMV promotors of for example finding in pcSD or pD16LEA29Y carrier, or are arranged in the avian sarcomata virus (ASV) of piLN carrier.Other early stage and late promoters commonly used include but not limited to, from simian virus 40 (SV40) (people such as Fiers, 1973, Nature273:113) those, or other viral promotors are for example derived from those of cow teats knurl, polyoma and adenovirus 2 viruses.Except that known in the art other, also can use adjustable promotor hMTII (people such as Karin, 1982, Nature299:797-802).About the recombinant protein expression in the insect cell of cultivating (for example, the SF9 cell), some baculovirus vectors of available comprise pVL series (Lucklow, V.A., and Summers, M.D., 1989, Virology170:31-39) and pAc series (people such as Smith, 1983, Mol.Cell Biol.3:2156-2165).It also is important in optimized expression that those skilled in the art also understand enhancing subarea (those sequences that promoter region upstream in the noncoding DNA district or downstream are found).Can use replication orgin in case of necessity, if for example use prokaryotic hosts to be used to introduce plasmid DNA from viral source.Yet chromosomal integration is the common mechanism about the dna replication dna in the eukaryote.
Although in embodiments of the invention, to use mammalian host cell (for example Chinese hamster ovary celI) and be used to express desired protein (for example, fusion rotein, glycoprotein etc.), other eukaryotes also can be used as the host.Can use the laboratory strain of budding yeast Saccharomyces cerevisiae (Saccharomycescerevisiae) (being also referred to as bread yeast or cereuisiae fermentum), and other yeast strains grain wine fragmentation sugar yeast (Schizosaccharomyces pombe) for example.Have coding target protein matter (for example, fusion constructs, glycoprotein etc., for example CTLA4-Ig or CTLA4 A29YL104E-Ig) the yeast vector of DNA can utilize Broach, Meth.EnzThe .101:307 2 μ replication orgin of (1983), or other replication orgin compatible with yeast (for example, people such as Stinchcomb, 1979, Nature282:39; People such as Tschempe, 1980, Gene10:157; With people such as Clarke, 1983, Meth.Enz.101:300).The regulatory element that comprises in the yeast vector can be the promotor that is used for synthetic glycolytic ferment (people such as Hess, 1968, J. Adv.Enzyme Reg.7:149; People such as Holland, 1978, Biochemistry17:4900).
Those skilled in the art can also utilize wherein growth conditions can regulate described other promotors of regulating gene transcription, and can comprise following non-limitative example: different cell pigment C (isocytochrome C), alcoholdehydrogenase 2, be responsible for enzyme, acid phosphatase and the degrading enzyme relevant with nitrogen metabolism of maltose and galactose utilization.Similar with mammalian expression system, the terminator sequence in the Yeast expression carrier also is required on 3 ' end of encoding sequence, and finds in 3 ' non-translational region behind the open reading-frame (ORF) in yeast deutero-gene.Be suitable for that (for example, in Saccharomyces cerevisiae some non-limitative examples of the yeast vector of) recombinant protein expression comprise, pMFa (Kurjan and Herskowitz, (1982) in the yeast Cell30:933-943), pJRY88 (people such as Schultz, 1987, Gene54:113-123), pYepSec1 (people such as Baldari, 1987, Embo J.6:229-234), pYES2 (Invitrogen Corporation, San Diego, Calif.), and those of pRS family that belong to yeast vector.
(clone of) DNA bacterial clone for example for example, fusion constructs, glycoprotein etc. subsequently can transfection for example in the mammalian cell, be used to express required product to proper host cell to the coding target protein matter that comprises that obtains as mentioned above.The standard technique that is suitable for described host cell that rotaring dyeing technology uses this area to establish is carried out, and wherein rotaring dyeing technology depends on the host cell of use.For example, the mammalian cell transfection can be used transfection, the CaPO of fat transfection, protoplastis fusion, the mediation of DEAE-dextran 4Co-precipitation, electroporation, directly microinjection and additive method known in the art are finished, and described additive method can comprise: scraping, directly picked-up, osmotic pressure or sucrose shock, N,O-Diacetylmuramidase merges or red corpuscle merges, microinjection is for example via the technology of red corpuscle mediation and/or by host cell is implemented electric current indirectly.Because be used for the other technologies that genetic information is introduced in the host cell will be obtained exploitation, so the above-mentioned tabulation of transfection method is not considered as exhaustively.
The special utilization (for example encoded target protein matter in background of the present invention, fusion constructs, glycoprotein etc.) DNA (for example derived from multicellular organism, the Mammals origin) expression (the Tissue Cultures in the eukaryotic host cell, Academic Press, Cruz and Patterson, editor (1973)).Host cell derived from multicellular organism has the ability that intron is fallen in montage, and therefore can be directly used in expressing gene group dna fragmentation.As mentioned before, useful host cell system includes but not limited to Chinese hamster ovary (CHO), bhk cell, monkey kidney (COS), VERO and HeLa cell.In the present invention, use the clone of stably express target protein matter (for example, fusion constructs, glycoprotein etc.).In one embodiment, mammal cell line (for example Chinese hamster ovary celI system) carries out transfection (for example passing through electroporation) with the expression vector (for example pcSDhuCTLA4-Ig, pD16LEA29Y etc.) of the dna sequence dna that comprises coding purpose glycoprotein.In one embodiment, purpose glycoprotein can be CTLA4-Ig protein, comprises one or more CTLA4-Ig protein with the aminoacid sequence that comprises among the SEQ ID NO:2, by the part coding of the nucleotide sequence among the SEQ ID NO:1.In another embodiment, purpose glycoprotein can be CTLA4 A29YL104E-Ig comprises the CTLA4 with the aminoacid sequence that comprises among the SEQ ID NO:4 A29YL104-Ig protein is by the part coding of the nucleotide sequence among the SEQ ID NO:23.
Recombinant protein is CTLA4-Ig or CTLA4 for example A29YL104E-Ig can for example express in mammalian cell (for example CHO, BHK, VERO or NSO cell), insect cell (for example, using baculovirus vector) or the yeast cell at eukaryotic host cell.Those skilled in the art can use other proper host cell, for example those that preamble is described in background of the present invention.In one embodiment, use recombination fusion protein (for example CTLA4-Ig or CTLA4 A29YL104E-Ig) eucaryon rather than prokaryotic expression.The eucaryon recombinant protein is human CTLA 4-Ig or CTLA4 for example A29YL104E-Ig eukaryotic cell for example the expression in the Chinese hamster ovary celI can cause part and/or glycosylation fully, and in the chain or the formation of interchain disulfide bond.For the instantaneous amplification and the expression of desired protein, have coding target protein matter (for example, for example CTLA4-Ig or CTLA4 such as fusion constructs, glycoprotein A29YL104E-Ig) the carrier of DNA is delivered in the eukaryotic cell by transfection method known in the art, but unconformability is in the genome of cell.The expression of gene of transfection can be measured in 16-96 hour.Mammalian cell (for example COS cell) can with carrier for example pCDM8 combine be used for the transient expression desired protein (Gluzman, Y., 1981, Cell23:175-182; Seed, B., 1987, Nature329:840).
The stable transfection for mammalian cell is understood in this area, and the cell of small portion can be incorporated into DNA in its genome, and successful integration can depend on the expression vector and the transfection method of utilization.For the stable amplification and the expression of desired protein, have coding target protein matter (for example, for example CTLA4-Ig or CTLA4 such as fusion constructs, glycoprotein A29YL104E-Ig) the carrier stable integration of DNA in the genome of eukaryotic cell (for example mammalian cell), thereby cause the stably express of the gene of transfection.In order to identify and select the clone of the gene of stably express coding target protein matter, the gene of coding selective marker (for example, to antibiotic resistance) can be introduced in the host cell together with goal gene.The selective marker of being used by those skilled in the art can be to give at medicine those of resistance of G418 and Totomycin for example.The gene of coding selective marker can be introduced in the host cell on the plasmid that separates, or can introduce on the plasmid identical with goal gene.The cell that comprises goal gene can identify that the cell that has wherein mixed selectable marker gene will be survived by medicament selection in the presence of described medicine, and the necrocytosis of not mixing selectable marker gene.Survivaling cell subsequently can be with regard to desired protein (for example, CTLA4-Ig protein or CTLA4 A29YL104E-Ig) production is screened.
As mentioned before, the Chinese hamster ovary celI of Tetrahydrofolate dehydrogenase (dhfr) genetic expression defective only can be survived by adding nucleosides.When described cell usefulness had the dna vector stable transfection of dhfr gene, cell can be produced essential nucleosides subsequently.By using dhfr as selective marker, those skilled in the art understand in the presence of the metabolic antagonist methotrexate, the goal gene of dhfr and transfection (for example, CTLA4-Ig or CTLA4 A29YL104E-Ig) gene amplification easily takes place.In one embodiment of the invention, mammalian cell for example CHO dhfr-cell for example pcSDhuCTLA4-Ig (embodiment 11-13) or pD16LEA29Y carry out transfection with expression vector, with produce can stablize amplification and can stably express desired protein product (for example, respectively, CTLA4-Ig or β pCTLA4 A29YL104E-Ig polypeptide) cell colony.In another embodiment, the dhfr-negative cells is that (Invitrogen Corp.Carlsbad CA) can be applied to stable transfection to DG44.In another embodiment of the invention, transfection can take place via electroporation.
Easily put into practice as this area, mammalian cells transfected (for example dhfr negative Chinese hamster ovary celI) maintained in the non-selective substratum that comprises serum in 1-2 days after the transfection.Cell is handled with trypsinase subsequently, and in comprising the substratum of serum, paves plate again under the existence of selective pressure (for example, medicine such as methotrexate).Cell is cultivated 2-3 week in comprising the selective medium of serum, frequently changes selective medium eliminating fragment and dead cell, until visible bacterium colony clearly.Indivedual bacterium colonies can carry out tryptic digestion subsequently, and place and be used for further propagation and amplification in the presence of selective medium in the porous plate, for example ELISAs or immunoprecipitation are identified the producer who expresses high-caliber desired protein (for example, fusion constructs, glycoprotein etc.) with the method for establishing via this area.In one embodiment of the invention, can carry out aforesaid method and be used for the negative Chinese hamster ovary celI (for example DG44 cell) of transfection dhfr-, establish to express purpose recombinant protein () stable cell lines (referring to for example, embodiment 12-13) for example, CTLA4-Ig protein.In another embodiment, establish expression CTLA4 A29YL104EThe stable cell lines of-Ig (referring to embodiment 23).
Stable CHO of the present invention is a stably express CTLA4-Ig protein molecule, as the CTLA4-Ig monomer with following sequence: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:2 and (the vi) 25-383 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ IDNO:2.This clone can be secreted the colony of the CTLA4-Ig molecule of the polymer form that can be used as (for example dimer, the tetramer etc.) existence, and wherein the polymer form can have the different monomers sequence of SEQID NO:2.The expression cassette that is incorporated in this clone comprises SEQ IDNO:1, and is included in the pcSDhuCTLA4-Ig.
The present invention also provides stably express CTLA4 A29YL104EThe stable CHO of-Ig is.In one embodiment, expression of cell lines has the CTLA4 of following sequence A29YL104-Ig monomer: (i) 26-383 of SEQ ID NO:4, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ IDNO:4 and (the vi) 25-383 of SEQ ID NO:4 of SEQ ID NO:4 of SEQID NO:4 of SEQ ID NO:4.In another embodiment, clone can be secreted the CTLA4 that the polymer form that can be used as (for example dimer, the tetramer etc.) exists A29YL104EThe colony of-Ig molecule, wherein the polymer form can have the different monomers sequence of SEQ IDNO:4.The expression cassette that is incorporated in this clone comprises SEQ ID NO:3, and is included in the pD16LEA29Y.
The subclone of celliferous clonal population
The cell that is accredited as the producer of desired protein (for example fusion constructs, glycoprotein etc.) separates from cell cultures, and subsequently therein substratum can comprise under the production condition of equivalence of serum and increase.Can use subclone method known in the art such as but not limited to, soft-agar cloning.The stable recombinant cell clone that obtains can further be bred under the condition of serum-free and animal product subsequently.According to the present invention, express desired protein product (for example, CTLA4-Ig, CTLA4 A29YL104E-Ig etc.) stabilized cell clone reaches via obtain recombinant cell clone from cell culture, and described cell culture is cultivated in comprising the substratum of serum and obtained after the reorganization initial cell clones and make cell to adapt to the substratum of serum-free and animal product again.In one embodiment, the cell clone of expressing CTLA4-Ig can continue to cultivate through at least 50 generations in the substratum of serum-free and animal product.In another embodiment of the invention, cell clone can continue to cultivate through at least 75 generations as mentioned above.According to the present invention, cell clone can also continue to cultivate through at least 100 generations in the substratum of serum-free and animal product.
In further embodiment, express CTLA4 A29YL104EThe cell clone of-Ig can continue to cultivate through at least 27 generations in the substratum of serum-free and animal product.In another embodiment, cell clone can continue to cultivate through at least 75 generations as mentioned above.In addition, cell clone can also continue to cultivate through at least 100 generations in the substratum of serum-free and animal product.
In one embodiment, the invention provides and produce the clone that comprises the monomeric CTLA4-Ig molecule of SEQ ID NO:2, wherein clone is stable surpassing in 100 generations, and wherein clone stability comprises: (1) doubling time when the 100th generation was less than about 24.5 ± 2.6 hours; (2) cell survival when the 100th generation surpasses 95%, (3) when the 100th generation in the 5-L bio-reactor production titre about CTLA4-Ig surpass 1.69mg/mL; (4) sialic acid and proteinic mol ratio are about 9.3-about 11.0 when the 105th generation.
Stable recombinant cell clone of the present invention exists with unpack format, wherein separates to take place according to the method (for example, soft-agar cloning or limited dilution cloning etc.) of this area practice.In the present invention, stable recombinant cell clone is derived from suspending or the recombinant mammalian cells (for example, Chinese hamster ovary celI) of adherent growth, and it comprises coding purpose recombinant protein (for example, fusion constructs, glycoprotein etc., for example CTLA4-Ig or CTLA4 A29YL104E-Ig) dna sequence dna.Recombinant protein by expression of cell lines of the present invention can be treatment glycoprotein, for example CTLA4-Ig or CTLA4 A29YL104E-Ig.According to the present invention, be useful derived from the stable recombinant cell clone of eukaryotic cell (for example negative Chinese hamster ovary celI of Mammals Chinese hamster ovary celI, DG44 cell or dhfr-), described recombinant cell clone comprises coding recombinant glycoprotein for example CTLA4-Ig or CTLA4 A29YL104EThe dna sequence dna of-Ig, and can the stably express recombinant glycoprotein in several generations.
In one embodiment of the invention, stably express target protein matter (for example, fusion constructs, glycoprotein etc., for example CTLA4-Ig or CTLA4 A29YL104E-Ig) the colony of mammalian host cell obtains via the cell of amplification stable transfection under the condition of serum-free and animal product.According to the present invention, recombinant cell clone can characterize subsequently, and wherein for example it is stable through at least 105 generations in the substratum of serum-free and no animal product.
In one embodiment of the invention, the clonal population of cell produces the CTLA4-Ig molecule.Some special characteristics of this kind of groups of CTLA4-Ig molecule are listed in following table 6.The colony of CTLA4-Ig molecule can comprise the CTLA4-Ig dimer molecule at least, and it comprises 2 monomer molecules that can have one of following sequence separately: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:2 and (the vi) 25-383 of SEQ IDNO:2 of SEQID NO:2 of SEQ ID NO:2 of SEQ ID NO:2.Therefore, the colony of CTLA4-Ig molecule can comprise preponderate ground homodimer or heterodimer.Colony can comprise homodimer and heterodimer.In one embodiment, the invention provides colony or its pharmacy Equivalent of CTLA4-Ig molecule with the feature shown in the table 6.As used herein, the pharmacy Equivalent is that wherein molecule colony has security and the effect overview of equal value with the original population (standard group) that is used for the treatment of the patient, as government organs for example FDA will understand.For example, CTLA4-Ig of the present invention colony can have the feature shown in the table 6.In another embodiment, CTLA4-Ig molecule of the present invention colony can have the feature shown in the table 6 or its Equivalent individually or with its any combination and permutation.
In another embodiment, the purpose clone can also characterize according to recombinant products and the biochemical characteristics (CTLA4-Ig that for example, has specific optical extinction coefficient value) thereof expressed.The optical extinction coefficient value (is also referred to as uptake factor value (a s)) can derive in theory or experimentally.At the 280nm place, the uptake factor value (a of CTLA4-Ig s) use people's such as Mach (Analytical Biochemistry, the 200th volume, 74-80 page or leaf, 1992) as detailed below method to be determined as 1.01mL mg -1Cm -1
Equation 1 is used to measure molar absorption coefficient (ε).
Equation 1: ε=[(disulfide linkage number x 134)+(tryptophan residue number x 5,540)+(tyrosine residues number x 1,480)]
CTLA4-Ig has 9 disulfide linkage, 8 tryptophan residues and 32 tyrosine residuess, with provide as shown in equation 2 92,886M -1Cm -1Molar absorption coefficient (ε).
Equation 2: ε=(9 x 134)+(8 x 5,540)+(32 x 1,480)]=92,886M -1Cm -1
Uptake factor constant (a s) by molar absorption coefficient (ε) is calculated divided by molecular weight, wherein molecular weight is measured by MALDI-TOF as shown in equation 3:
Equation 3:a s=ε/molecular weight=92,886M -1Cm -1/ 92,278Da=1.01mL mg -1Cm -1
Use amino acid analysis that 2 crowdes of CTLA4-Ig (comprising SEQ ID NO:2) material is carried out deutero-uptake factor value and the comparison of the uptake factor value of mensuration experimentally in theory.Experimentally the mean absorption coefficient constant of Ce Dinging is 1.00 ± 0.05mL mg -1Cm -1Experimental value confirms 1.01mL mg in the measuring error -1Cm -1Theoretical value.Therefore, in one embodiment, the invention provides the clone that produces the CTLA4-Ig molecule, described CTLA4-Ig molecule has about 1.00 ± 0.05mL mg -1Cm -1Uptake factor value or optical extinction coefficient.
According to the present invention, the purpose recombinant clone also can characterize according to the number of loci that is incorporated into the dna sequence dna in the host cell gene group, described dna sequence encoding target protein matter (for example, fusion constructs, glycoprotein etc. for example CTLA4-Ig).Those skilled in the art understand the standard DNA hybridization technique will allow this kind analysis.In one embodiment of the invention, the single hybridized fragment of about 1.2kb detects in by the EcoRI of the genomic dna of recombinant cell clone of the present invention preparation and XbaI restriction digestion separately, with consistent (the DNA hybrid of expection size of CTLA4-Ig gene; Figure 23).This figure describes consistent with the single integration site of plasmid, and does not have by detectable insertion of DNA hybridization analysis or disappearance in the CTLA4-Ig gene.
In one embodiment, the invention provides the Chinese hamster ovary celI colony that can produce the CTLA4-Ig molecule, wherein each cell of colony comprises at least 30 copies of the proteinic nucleic acid of coding CTLA4-Ig, wherein 30 or more multiple copied incorporate in series on the single site in the genome of cell, and wherein cell colony is cloned.In other embodiments, the Chinese hamster ovary celI colony that can produce the CTLA4-Ig molecule comprises in the colony wherein the colony that at least 75%, 80%, 85%, 90%, 95% or 99% cell comprises at least 30 copies of the proteinic nucleic acid of coding CTLA4-Ig.
In another embodiment, the invention provides when in condition, cultivating according to Figure 10 or embodiment 14, generation comprises the clone of the CTLA4-Ig molecule of SEQ ID NO:2, and the amount of described CTLA4-Ig molecule is at least 1.0,1.3,1.6,1.8,2.0 or 2.5 gram CTLA4-Ig molecule/L cell cultures when the production phase.
According to the present invention, produced the mammal cell line (for example negative Chinese hamster ovary celI of dhfr system) of expression desired protein (for example CTLA4-Ig protein), when growing in suspension culture, described mammal cell line can produce the molecule colony that is secreted in the culture supernatants.This molecule colony can have in the following characteristics of listing in the table 6 for example one or more or all.
The CTLA4-Ig feature of table 6. illustrative
Feature
1 The N-terminal sequence The aa 27 (Met) of aa 26 (Ala) the SEQ ID NO:2 of SEQ ID NO:2
2 The C-terminal sequence The aa 383 (Lys) of aa 382 (Gly) the SEQ ID NO:2 of SEQ ID NO:2
3 The B7 combination 70-130％
4 pI 4.3-5.6
5? Sialic acid is than NANA NGNA 〉=8.0 moles/mole CTLA4-Ig molecule 8.0-12.0 moles/mole CTLA4-Ig molecule≤1.5 moles/mole CTLA4-Ig molecules
6 Dimer ≥95％
7 HMW kind (for example tetramer) ≤4.0％
8 Low molecular weight species (for example monomer) ≥0.5％
9 Optical extinction coefficient 1.0±0.05ml/mg·cm
10 Free sulfhydryl groups ≤ 0.24 free mercaptan/molecule
11 Amino monose composition: GlcNAc 15-35 moles/mole CTLA4-Ig molecule
12 Amino monose composition: GalNAc 1.7-8.3 moles/mole CTLA4-Ig molecule
13 Neutral monose composition: semi-lactosi 8-17 moles/mole CTLA4-Ig molecule
14 Neutral monose composition: Fucose 3.5-8.3 moles/mole CTLA4-Ig molecule
15 Neutral monose composition: seminose 7.7-22 moles/mole CTLA4-Ig molecule
According to table 6, the per-cent of the dimeric per-cent of CTLA4-Ig, HMW kind (for example, CTLA4-Ig polymer for example the tetramer) and the per-cent of LMW kind (for example, CTLA4-Ig monomer) are with regard to the colony of CTLA4-Ig molecule.Sugar mole of describing in the table 6 and sialic acid mole are with regard to CTLA4-Ig molecule or dimer mole.The B7 bonded per-cent of finding in the table 6 is the CTLA4-Ig that carries out on the BIAcore instrument about the surface plasma body resonant vibration of describing by the preamble phase (SPR) in conjunction with experiment, and wherein per-cent is the comparison that contrasts bonded B7 with CTLA4-Ig.
In one embodiment, mammal cell line (for example negative Chinese hamster ovary celI of dhfr system) produces the CTLA4-Ig molecule colony of demonstration from the characteristic attribute of the numbering 1-5 of table 6.In another embodiment of the invention, the mammal cell line generation has the CTLA4-Ig molecule colony from the characteristic attribute of the numbering 1-10 of table 6.In other embodiments, mammal cell line produces the CTLA4-Ig molecule colony have from the characteristic attribute of the numbering 1-15 of table 6.In further embodiment, the amount of the free sulfhydryl groups on CTLA4-Ig is about≤0.20 free mercaptan/molecule.
Behind the cell culture supernatant liquid purifying that comprises by mammalian cells transfected (for example negative Chinese hamster ovary celI of dhfr) colony's excretory desired protein (for example CTLA4-Ig protein), molecule colony can have further feature.Except that those features of listing in the table 6, this molecule colony can have in the following characteristics for example one or more or all: the pH scope of about 7.0-8.0; Make the ability of the active 50-150% of inhibition of people's cell IL-2; The MCP (MCP-1) in the final purified product of being present at≤5ng/mgCTLA4-Ig dimer or CTLA4-Ig molecule; ≤ the dimeric DNA concentration that is present in the final purified product of 2.5pg/mg CTLA4-Ig; ≤ the dimeric CHO host cell proteins matter that is present in the final purified product of 50ng/mg CTLA4-Ig; The Triton X-100 concentration in final purified product at≤1.0ppm; ≤ the proteic amount of the dimeric A of 5ng/mg CTLA4-Ig; The amount of the bacterial endotoxin when≤0.3EU/mg CTLA4-Ig dimer in final purified product; Amount at the biological load in final purified product of≤3.0CFU/10ml.
In one embodiment, MCP (MCP-1) is present in the final purified product at≤3ng/mgCTLA4-Ig dimer or CTLA4-Ig molecule; Be present in the DNA concentration in the final purified product at≤1.0pg/mg CTLA4-Ig dimer; Be present in the CHO host cell proteins matter in the final purified product at≤10ng/mg CTLA4-Ig dimer; ≤ the proteic amount of 1ng/mg CTLA4-Ig dimer A; Amount at the≤0.15EU/mg CTLA4-Ig dimer bacterial endotoxin in final purified product; Amount at the biological load of≤1.0 CFU/10ml in final purified product; The pH scope of about 7.2-7.8.In specific embodiments, MCP (MCP-1) is present in the final purified product at≤1ng/mg CTLA4-Ig dimer or CTLA4-Ig molecule.In further embodiment, the CTLA4-Ig molecule makes the active 60-140% of inhibition of people's cell IL-2.
In another embodiment of the invention, the clonal population of cell produces CTLA4 A29YL104E-Ig molecule.CTLA4 A29YL104ESome special characteristics of this kind of groups of-Ig molecule are listed in table 7.CTLA4 A29YL104EThe colony of-Ig molecule can comprise CTLA4 at least A29YL104E-Ig dimer molecule, it comprises 2 monomer molecules that can have one of following sequence separately: (i) 26-383 of SEQ ID NO:4, (ii) 26-382, (iii) 27-383, (iv) 27-382, (the v) 25-382 of SEQ ID NO:4 and (the vi) 25-383 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4 of SEQ ID NO:4.Therefore, CTLA4 A29YL104EThe colony of-Ig molecule can comprise preponderate ground homodimer or heterodimer, or its any mixture.In one embodiment, the invention provides CTLA4 with the feature shown in the table 7 A29YL104EThe colony of-Ig molecule or its pharmacy Equivalent.As used herein, the pharmacy Equivalent is that wherein molecule colony has security and the effect overview of equal value with the original population (standard group) that is used for the treatment of the patient, as government organs for example FDA will understand.For example, CTLA4 of the present invention A29YL104E-Ig colony can have the feature shown in the table 7.In another embodiment, CTLA4 of the present invention A29YL104E-Ig molecule colony can have the feature shown in the table 7 or its Equivalent individually or with its any combination and permutation.
In one embodiment, the invention provides and to produce CTLA4 A29YL104EThe Chinese hamster ovary celI colony of-Ig molecule, wherein each cell of colony comprises coding CTLA4 A29YL104EAt least 30 copies of the proteinic nucleic acid of-Ig, wherein 30 or more multiple copied incorporate in series on the single site in the genome of cell, and wherein cell colony is cloned.In other embodiments, can produce CTLA4 A29YL104EThe Chinese hamster ovary celI colony of-Ig molecule comprises in the colony wherein at least 75%, 80%, 85%, 90%, 95% or 99% cell and comprises coding CTLA4 A29YL104EThe colony of at least 30 copies of the nucleic acid of-Ig.
In another embodiment, the invention provides when in condition, cultivating, produce the CTLA4 that comprises SEQ ID NO:4 according to Figure 24 or embodiment 19-20 A29YL104EThe clone of-Ig molecule, described CTLA4 when the production phase A29YL104EThe amount of-Ig molecule is at least 22,22.5,23,27.5 or 28 gram CTLA4 A29YL104E-Ig molecule/L cell culture.
According to the present invention, produced expression desired protein (CTLA4 for example A29YL104E-Ig) mammal cell line (for example dhfr negative Chinese hamster ovary celI system), when growing in suspension culture, described mammal cell line can produce the molecule colony that is secreted in the culture supernatants.This molecule colony can have in the following characteristics of listing in the table 7 for example one or more or all.
The CTLA4 of table 7. illustrative A29YL104E-Ig feature
Feature
1 The N-terminal sequence The aa 27 (Met) of aa 26 (Ala) the SEQ ID NO:4 of SEQ ID NO:4
2 The C-terminal sequence The aa 383 (Lys) of aa 382 (Gly) the SEQ ID NO:4 of SEQ ID NO:4
3 The B7 combination 70-130％
4 pI 4.5-5.5
5 The sialic acid ratio The total CTLA4-Ig protein of 〉=5.0 moles/mole
6 Dimer ≥95％
7 HMW kind (for example tetramer) ≤4％
8 Low molecular weight species (for example monomer) ≤1％
9 Amino monose composition: GlcNAc The total CTLA4-Ig protein of 24-28 moles/mole
10 Amino monose composition: GalNAc 2.7-3.6 the total CTLA4-Ig protein of moles/mole
11 Neutral monose composition: semi-lactosi The total CTLA4-Ig protein of 11-13 moles/mole
12 Neutral monose composition: Fucose 6.4-7.0 the total CTLA4-Ig protein of moles/mole
13 Neutral monose composition: seminose The total CTLA4-Ig protein of 14-16 moles/mole
According to table 7, CTLA4 A29YL104EThe dimeric per-cent of-Ig, HMW kind (for example, CTLA4 A29YL104E-Ig polymer is the tetramer for example) per-cent and LMW kind (for example, CTLA4 A29YL104E-Ig monomer) per-cent is with regard to CTLA4 A29YL104EThe colony of-Ig molecule.Sugar mole and the sialic acid mole described in the table 7 are with regard to CTLA4 A29YL104E-Ig molecule or dimer mole.The B7 bonded per-cent of finding in the table 7 is the CTLA4 that carries out on the BIAcore instrument about the surface plasma body resonant vibration of describing by preamble (SPR) A29YL104E-Ig is in conjunction with experiment, and wherein per-cent is and CTLA4 A29YL104EThe comparison of-Ig contrast bonded B7.
In one embodiment, mammal cell line (for example negative Chinese hamster ovary celI of dhfr system) produces the CTLA4 of demonstration from the characteristic attribute of the numbering 1-5 of table 7 A29YL104E-Ig molecule colony.In another embodiment of the invention, the mammal cell line generation has the CTLA4 from the characteristic attribute of the numbering 1-10 of table 7 A29YL104E-Ig molecule colony.In other embodiments, mammal cell line produces the CTLA4 have from the characteristic attribute of the numbering 1-13 of table 7 A29YL104E-Ig molecule colony.
Comprising by mammalian cells transfected (for example dhfr negative Chinese hamster ovary celI) colony's excretory desired protein (CTLA4 for example A29YL104EBehind-Ig) the cell culture supernatant liquid purifying, molecule colony can have further feature.Except that those features of listing in the table 7, this molecule colony can have in the following characteristics for example one or more or all: at≤5ng/mgCTLA4 A29YL104E-Ig dimer is present in the MCP (MCP-1) in the final purified product; ≤ 2.5pg/mg CTLA4 A29YL104E-Ig dimer is present in the DNA concentration in the final purified product; With at≤50ng/mg CTLA4 A29YL104E-Ig dimer is present in the CHO host cell proteins matter in the final purified product.
The general cultivation of clone
According to the present invention, to produce desired protein, comprise glycoprotein as the conventional known cultivation mammalian cell of those skilled in the art.The mammalian cell of expressing purpose glycoprotein should express or handle expressing suitable enzyme, thereby makes that maximally related posttranslational modification takes place in vivo to glycosylation under satisfied condition.Enzyme comprises the interpolation of N and O connection carbohydrate and finishes required those, for example about N connection oligosaccharides at Hubbard and Ivatt, Ann.Rev.Biochem., those that describe among the 50:555-583 (1981).Optional oligosaccharyl transferase, alpha-glucosidase I, alpha-glucosidase II, the ER α (1 of comprising of enzyme, 2) mannosidase, golgi body α-seminase (mannodase) I, N-acetyl-glucosamine transferring enzyme) I, golgi body α-seminase II, N-acetyl-glucosamine transferring enzyme) II, α (1,6) fucosyltransferase, β (1,4) galactosyltransferase and suitable sialytransferase.
Delay in the apoptosis (apoptosis) can have the effect that increases cell survival during cell cultivation process.Minimizing in the apoptosis and successively the increase in life-span of specific cells can increase protein production from cell culture.The apoptosis incident can be by (for example introducing cell with one or more anti-apoptotic protein, mammalian cell, insect cell or yeast cell) be suppressed in the inherent cell, described anti-apoptotic protein is at the apoptosis that suppresses on the accurate point of apoptotic pathways in the cell.Suppress apoptotic another kind of method and be to suppress in the cell release from mitochondrial short apoptosis molecule.The variant of short apoptosis protein matter known in the art, for example the dominant form of Caspase-9 can be used as apoptosis inhibitor in cell.This kind variant proteins can be introduced in the cell, with the delay routine cell death.Apoptotic inhibition prolongs specific cells successively and produces the proteinic time, thereby causes desired protein via the overall increase in the production of specific cells.Several genes (for example, the chain inhibitor of apoptosis X (XIAP) or its variant) or anti-apoptotic gene (for example, the Bcl-2 and the Bcl-x of coding Caspase inhibitor LOr its variant), can transfection in genetic engineering modified mammalian cell (for example Chinese hamster ovary celI, VERO cell, bhk cell etc.) (Sauerwald, people such as T., 2003, Biotechnol Bioeng.81:329-340; Sauerwald, people such as T., 2002, Biotechnol Bioeng.77:704-716; Mastrangelo, people such as A., 2000, Biotechnol Bioeng.67:544-564; Kim, people such as N., 2002, J Biotechnol.95:237-248; Figueroa, people such as B., 2001, Biotechnol Bioeng.73:211-222).
In one embodiment, the mammalian cell of producing recombinant protein can carry out transfection with the carrier that comprises anti-apoptotic gene (for example bcl-2).In another embodiment, recombinant mammalian cells can carry out transfection with plasmid, and described plasmid comprises that gene, the code book those skilled in the art of variant of the gene of coding Caspase inhibitor or the aforesaid short apoptosis molecule of encoding are known to have the active proteinic gene of anti-apoptotic or its an any combination.
In another embodiment, the overall product quality (for example enhanced glycosylation) of desired recombinant protein matter (for example treating protein) can be enhanced.In order to increase the glycosylation of recombinant protein, mammalian cell (for example Chinese hamster ovary celI, VERO cell, bhk cell etc.) can carry out transfection with the nucleic acid of one or more enzymes of coding, described enzyme (for example relates to proteinic glycosylation, α 2,3-sialytransferase, β 1,4-galactosyltransferase etc.) (people such as Weikert, 1999 Nature Biotechnol17:1116-21).In one embodiment, coding β 1, the plasmid of 4-galactosyltransferase can be introduced in the mammalian cell of expressing target protein matter.In another embodiment, coding for alpha 2, the plasmid of 3-sialytransferase can be introduced in the mammalian cell of expressing target protein matter.
Various culture parameters can be used with regard to the host cell of cultivating.About the suitable culture condition of mammalian cell be well-known in the art (people such as Cleveland, J. Immunol.Methods56:221-234 (1983)) or can determine by the technician (referring to, for example, Animal Cell Culture:A Practical Approach the 2nd edition, Rickwood, D. and Hames, B.D. edit (Oxford University Press:New York, 1992)), and according to the concrete host cell of selecting become.
Without stint, cell culture medium (for example inoculum substratum, charging substratum, basic medium etc.) can refer to be used for to cultivate and or keep the nutrient solution of cell, particularly mammalian cell.These solution provide at least a component from one or more following kinds usually: (1) energy is generally for example form of glucose of carbohydrate; (2) all indispensable amino acids, and be generally 20 seed amino acids and add basic group of halfcystine; (3) VITAMIN and/or other organic compound that needs with lower concentration; (4) free fatty acids or lipid, for example linolic acid; (5) trace elements, wherein trace elements is defined as generally with extremely low concentration, is generally mineral compound or naturally occurring element that micro-molar range needs.Nutrient solution can selectivity be added one or more components from any following kind: (1) hormone and other somatomedins are serum, Regular Insulin, transferrin and Urogastron for example; (2) salt, for example magnesium, calcium and phosphoric acid salt; (3) damping fluid, for example HEPES; (4) for example adenosine, thymidine and xanthoglobulin of nucleosides and base; (5) protein and tissue water hydrolysis products for example can derive from the peptone or the peptone mixture of gelatin, vegetable material or the animal byproduct of purifying; (6) microbiotic, for example gentamicin; (7) cytoprotective, for example poly alcohol; (8) semi-lactosi.The example of basic medium can be cell growth substrate basal culture medium (Cell Growth Basal Medium).The example of inoculum substratum can be inoculum cell growth substrate basal culture medium (Inoculum CellGrowth Basal Medium).The example of charging substratum can be to produce bio-reactor charging substratum (Production Bioreactor Feed Medium).
The substratum that is obtained commercially can for example utilize and comprise, and minimum essential medium (Minimal Essential Medium) (MEM, Sigma, St.Louis, Mo.); The Eagles substratum (Dulbecco ' s Modified Eagles Medium) that DulbeccoShi modifies (DMEM, Sigma); Ham ' s F10 substratum (Sigma); The HyClone cell culture medium (HyClone, Logan, Utah); RPMI-1640 substratum (Sigma); Know (CD) substratum with chemical ingredients, it is prepared for concrete cell type, for example, the CD-CHO substratum (Invitrogen, Carlsbad, Calif.).Any in these substratum can have been added previously defined component or the composition added as required, and be necessary or comprise with the suitable concentration or the optional components of amount when needing.Can in the substratum of the concrete cell that is suitable for cultivating, be prepared with the mammalian cell cultures that the present invention uses.In one embodiment, cell culture medium can be generally not contain one of aforementioned from the serum (for example, foetal calf serum (FBS)) in any Mammals source.In another embodiment of the invention, mammalian cell cultures can be in having added table 15 specified other component, the chemical ingredients that is obtained commercially knows in (CD)-CHO substratum grows.In further embodiment, mammalian cell cultures can be in adding table 20 or 21 be grown in the CD-CHO substratum of specified other component.
Method of the present invention comprises the cultivation of numerous cell types.In one embodiment of the invention, cell is an animal or mammiferous.In another embodiment, a large amount of desired proteins can be expressed and secrete to cell.In another embodiment of the invention, cell can express and a large amount of purpose glycoprotein are secreted in the substratum.Animal or mammalian cell also can carry out molecular modification to express and secretion target protein matter.Protein by host cell production can be endogenous or homologous for host cell.Protein also can be allogenic (for example external) for host cell, and the genetic information of the target protein matter of encoding is thus introduced in the host cell via the standard method (for example, by electroporation, transfection etc.) of this area.In one embodiment, Mammals glycoprotein can and be secreted in the substratum via Chinese hamster ovary (CHO) host cell production.
In some embodiments, the invention provides the production method of discussing via this paper, be included in the colony of the CTLA4-Ig molecule of the mass production method production of describing among the embodiment 14 and in Figure 10, showing.This method can cause the production (for example, referring to embodiment 14 and 15) of the CTLA4-Ig molecule of high molecular (HMW).In another embodiment, provide the production method of discussing via this paper, for example in embodiment 19 and 20, described and CTLA4 that the mass production method that shows in Figure 24 is produced A29YL104EThe colony of-Ig molecule.This method can cause the CTLA4 of high molecular (HMW) A29YL104The production of-Ig molecule (for example, referring to embodiment 19 and 20).In some embodiments, the HMW kind can be by production method, comprises that chemical ingredients knows molecule or dimeric about 15-25% that (CD)-CHO1 fermentation process is produced.In other embodiments, the invention provides be used to separate, CTLA4-Ig or CTLA4 that purifying and sign are produced by the CD-CHO1 fermentation process A29YL104EThe method of-Ig HMW component.CTLA4-Ig or CTLA4 A29YL104E-Ig HMW component is to have than CTLA4-Ig or CTLA4 A29YL104EThe polymer of the molecular weight that-Ig dimer is higher (for example, the tetramer, six aggressiveness etc.).
Can comprise, express and a large amount of purpose glycoprotein are secreted into the animal or the mammalian host cell that are used for later separation and/or purifying in the substratum and include but not limited to, Chinese hamster ovary cell (CHO), CHO-K1 (ATCC CCL-61) for example, DG44 (people such as Chasin, 1986 Som.Cell Molec.Genet, 12:555-556; People such as Kolkekar, 1997, Biochemistry, 36:10901-10909; With WO 01/92337A2), the negative Chinese hamster ovary celI of Tetrahydrofolate dehydrogenase (CHO/dhfr-, Urlaub and Chasin, 1980, Proc.Natl.Acad. Sci.USA is 77:4216) with dp12.CHO cell (U.S. Patent number 5,721,121); Monkey kidney CV1 cell (COS cell, COS-7, ATCC CRL-1651) by the SV40 conversion; HEKC (for example, 293 cells, or subclone is used for 293 cells of growing in suspension culture, people such as Graham, and 1977, J.Gen.Virol., 36:59); Baby hamster kidney cell (BHK, ATCC CCL-10); Monkey-kidney cells (CV1, ATCC CCL-70); African green monkey kidney cell (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); The mouse sustenticular cell (TM4, Mather, 1980, Biol.Reprod., 23:243-251); Human cervical carcinoma cell (HELA, ATCC CCL-2); Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL-34); Human pneumonocyte (W138, ATCC CCL-75); Human liver cancer cell (HEP-G2, HB 8065); MMT cell (MMT 060562, ATCC CCL-51); Buffalo rat hepatocytes (BRL3A, ATCC CRL-1442); The TRI cell (Mather, 1982, Annals NY Acad.Sci., 383:44-68); MCR 5 cells; The FS4 cell.In one aspect of the invention, utilize Chinese hamster ovary celI, particularly CHO/dhfr-and CHO DG44 cell.
The example of the Mammals glycoprotein that can produce by method of the present invention includes but not limited to, cytokine, cytokine receptor, somatomedin (for example, EGF, HER-2, FGF-α, FGF-β, TGF-α, TGF-β, PDGF, IGF-1, IGF-2, NGF, NGF-β); Growth factor receptors comprises and merging or chimeric protein.Other examples include but not limited to, tethelin (for example, human growth hormone, Trobest); Regular Insulin (for example, INSULIN A chain and insulin B chain), proinsulin; Erythropoietin (EPO); G CFS (for example, G-CSF, GM-CSF, M-CSF); Interleukin-(for example, IL-1 to IL-12); Vascular endothelial growth factor (VEGF) and acceptor (VEGF-R) thereof; Interferon, rabbit (for example, IFN-α, β or γ); Tumour necrosis factor (for example, TNF-α and TNF-β) and acceptor thereof, TNFR-1 and TNFR-2; Thrombopoietin (TPO); Zymoplasm; Brain natriuretic peptide (BNP); Thrombin (for example, Factor IX, factors IX, the von Willebrands factor etc.); Anticoagulin; Tissue plasminogen activator (TPA), for example urokinase or people urine or tissue-type TPA; Ovarian follicle generates plain (FSH); Lutropin (LH); Thyrocalcitonin; CD protein (for example, CD3, CD4, CD8, CD28, CD19 etc.); CTLA protein (for example, CTLA4); T cell and B-cell receptor protein; Delicious peptide (BNPs, for example, BMP-1, BMP-2, BMP-3 etc.); Neurotrophic factor, for example bone derived neurotrophic factor (BDNF); Neurotrophin, for example 3-6; Feritin; Rheumatoid factors, polyclonal; RANTES; White protein; Relaxin; Macrophage inhibitory protein (for example, MIP-1, MIP-2); Virus protein or antigen; Surface membrane protein matter; Ionophorous protein; Enzyme; Regulate protein; Antibody; Immunity tuning albumen (for example, HLA, MHC, B7 family); Homing receptor; Translocator matter; Superoxide-dismutase (SOD); G protein coupled receptor protein (GPCRs); Neuromodulation protein; Alzheimer related protein and peptide (for example, A-β), and known in the art other.Can include but not limited to any fragment in fusion rotein, polypeptide, chimeric protein and above-mentioned protein and the polypeptide or part or mutant, variant or analogue by suitable protein, polypeptide and the peptide that method of the present invention is produced.
Method of the present invention can also be used to produce the CTLA4-Ig molecule as SEQ ID NO:5,6,7,8,9 or 10 variant.In one embodiment, the CTLA4-Ig molecule can be included in the monomer that has one or more variations among residue 55 (A55Y) and 130 (L130E) (residue of mentioning is from SEQ ID NO:2).Referring to the variant of the CTLA4-Ig that describes among US publication US 2002/0182211 A1 and the description of mutant, described patent document is incorporated herein by reference in this integral body.In another embodiment, the CTLA4-Ig variant can comprise the sudden change that has in the CTLA-4 zone or the CTLA4-Ig molecule of the sudden change in the Ig zone or its any combination.In one embodiment, CTLA4-Ig variant molecule comprises the CTLA4-Ig molecule with aminoacid sequence, and described aminoacid sequence and SEQ ID NOS:5,6,7,8,9 or 10 are equal at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99%.In one embodiment, CTLA4-Ig variant molecule can combine with CD80 or CD86.In another embodiment, variant can form dimer.In further embodiment, variant shows and is similar to the sort of carbohydrate overview that is shown by nonmutationed CTLA4-Ig molecule colony.In one embodiment, CTLA4-Ig variant molecule has identical potential N connection and the O linked glycosylation site that is present among the SEQ IDNO:2.In another embodiment, CTLA4-Ig variant molecule has identical N connection and the O linked glycosylation site that is present among the SEQ ID NO:2, and has other glycosylation site.Sudden change can include but not limited to, nucleotide deletion, insertion, interpolation; Aminoacid deletion, displacement, interpolation; The nucleic acid frameshit; Displacement can be non-conservative (for example, replacing glycine with tryptophane) or conservative substitution (for example, leucine displacement Isoleucine).
CTLA4-Ig variant molecule includes but not limited to, CTLA4-L104EA29YIg (uses the residue numbering system according to SEQ ID NO:2, CTLA4-L104EA29YIg is referred to herein as CTLA4-L130EA55YIg), and those CTLA4-Ig variant molecules of describing in the following reference: U.S. Patent Application Serial 09/865,321 (US publication US2002/0182211), 60/214,065 and 60/287,576; WO 01/92337A2; U.S. Patent number 6,090,914,5,844,095 and 5,773,253; With as people such as R.J.Peach, 1994, J ExD Med, describe among the 180:2049-2058.In one embodiment, the CTLA4-Ig variant molecule of Sheng Chaning can be by emiocytosis in the method, and described cell comprises the expression vector of coding CTLA4-Ig variant proteins.
CTLA4-Ig variant L130EA55Yig is the genetic engineering modified fusion rotein that is similar to the CTLA4-Ig molecule in structure.L130EA55Y-Ig has the Fc structural domain of the human normal immunoglobulin of the outer binding domains of functional cell of modified people CTLA-4 and IgG1 class.Carrying out 2 seed amino acids in the B7 of CTLA4-Ig structural domain calmodulin binding domain CaM modifies, the leucine at the 104th place of L104EA29Y variant is to L-glutamic acid (L104E), this is corresponding to the 130th of SEQ ID NO:2, with the L-Ala at the 29th place of L104EA29Y variant to tyrosine (A29Y), this is corresponding to the 55th of SEQ ID NO:2, to produce L130EA55Y.L130EA55Y-Ig can comprise about separately 45,700 daltonian 2 homology glycosylated polypeptides chains, and described polypeptide chain combines by 1 interchain disulfide bond and noncovalent interaction.According to the budapest treaty clause, the coding L130EA55Y-Ig DNA on June 20th, 2000 as the coding L104EA29Y-Ig DNA be preserved in American type culture collection (ATCC).It has given ATCC registration number PTA-2104.L104EA29Y-Ig (corresponding to the L130EA55Y-Ig among the application) is in common unsettled U.S. Patent Application Serial 09/579,927,60/287,576 and 60/214,065 and 09/865,321 and WO/01/923337 A2 in further described, described all patent documents are whole introduces the application as a reference.
Because the CTLA4-Ig monomer that has Ala with amino acid position 55 places at SEQ ID NO:2 and have Leu at amino acid position 130 places is compared, recombinant protein L130EA55Y-Ig only locates different at 2 amino acid (Tyr at amino acid position 55 places and the Glu at amino acid position 130 places), and, can have overview identical or closely similar glycosylation overview with the colony that comprises wild-type CTLA4-Ig so comprise the CTLA4-Ig variant molecule colony of L130EA55Y-Ig because these 2 sudden changes do not influence N or O linked glycosylation.In addition, because the CTLA4-Ig monomer that has Ala with amino acid position 55 places at SEQ ID NO:2 and have Leu at amino acid position 130 places is compared, recombinant protein L130EA55Y-Ig only locates different at 2 amino acid (Tyr at amino acid position 55 places and the Glu at amino acid position 130 places), thus method of the present invention should be able to produce have to table 6 in the L130EA55Y-Ig of the similar property attribute described.
Method of the present invention can also be used to produce CTLA4 A29YL104E-Ig molecule, it is SEQ IDNOS:11,12,13,14,15 or 16 variant.In one embodiment, CTLA4 A29YL104E-Ig can be included in the monomer that has one or more variations among the SEQ ID NO:3.For example, other CTLA4 A29YL104E-Ig molecule be described in U.S. Patent Application Publication No. U.S.2002/0039577, U.S.2003/0007968, U.S.2004/0022787, U.S.2005/0019859 and U.S.2005/0084933, and U.S. Patent number 7,094, obtain in 8874 describing, described patent document is incorporated herein by reference in this integral body.
In one embodiment, CTLA4 A29YL104E-Ig is included in one or more sudden change or the sudden change in the Ig zone or its any combinations in the CTLA-4 zone (SEQ IDNO:18).In other embodiments, CTLA4 A29YL104E-Ig molecule comprises the CTLA4 with aminoacid sequence A29YL104E-Ig, described aminoacid sequence and SEQ ID NOS:11,12,13,14,15 or 16 are equal at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99%.In further embodiment, aforesaid CTLA4 A29YL104E-Ig molecule can combine with CD80 or CD86.In another embodiment, CTLA4 A29YL104E-Ig can form dimer.In further embodiment, CTLA4 A29YL104E-Ig demonstration is similar to by nonmutationed CTLA4 A29YL104EThe sort of carbohydrate overview that-Ig molecule colony shows.In other embodiments, CTLA4 A29YL104E-Ig molecule has identical potential N connection and the O linked glycosylation site that is present among the SEQ ID NO:4.In another embodiment, CTLA4 A29YL104E-Ig has identical N connection and the O linked glycosylation site that is present among the SEQ ID NO:4, and has other glycosylation site.Sudden change can include but not limited to, nucleotide deletion, insertion, interpolation; Aminoacid deletion, displacement, interpolation; The nucleic acid frameshit; Displacement can be non-conservative (for example, replacing glycine with tryptophane) or conservative substitution (for example, leucine displacement Isoleucine).
In one embodiment of the invention, the colony of CTLA4-Ig variant molecule can produce by the mammalian cell (for example negative Chinese hamster ovary celI of dhfr) according to bulk method suspension growth of the present invention, the gene of described mammalian cell expression coding desired protein (for example, L130EA55Y-Ig protein etc.).According to the present invention, can reclaim according to results parameter described herein by the recombinant C TLA4-Ig variant proteins of mammalian cell production.In other embodiments, the purification scheme of being described in can be according to the present invention by the recombinant C TLA4-Ig variant proteins of mammalian cell production is carried out purifying (embodiment 15).
In one embodiment of the invention, CTLA4 A29YL104EThe colony of-Ig molecule can produce described mammalian cell expression coding desired protein (for example, CTLA4 by the mammalian cell (for example negative Chinese hamster ovary celI of dhfr) according to bulk method suspension growth of the present invention A29YL104E-Ig protein etc.) gene.According to the present invention, by the recombinant C TLA4 of mammalian cell production A29YL104E-Ig can reclaim according to results parameter described herein.In other embodiments, the recombinant C TLA4 that produces by mammalian cell A29YL104EThe purification scheme that-Ig describes in can be according to the present invention is carried out purifying (embodiment 19-20).
Cell culture type and general cultural method
Target protein matter for example glycoprotein, fusion rotein etc. can be produced by the cell of culture expression desired protein product under the various kinds of cell culture condition.Those skilled in the art understand the cell culture and the cultivation operation that are used for protein production can include but not limited to 3 kinds of general types: cultured continuously, batch culture and fed batch cultivation.In the cultured continuously method, fresh medium supplement (for example, the charging substratum) is supplied to cell during the incubation time section, remove old substratum simultaneously.The product of producing during the cultured continuously also can be for example on the every day basis or gather in the crops continuously.As long as it is alive that cell is still, and keep environment and culture condition, cell just can in cultivation, keep with the cultured continuously method in needed the same of a specified duration.
In the batch culture method, cell is cultivated in substratum at first, and this substratum is neither replaced, does not also remove, also do not replenished.Cell before cultivating run duration or finishing newly substratum carry out " charging ", therefore cultivation is lasting exhausts until nutrition.Protein is gathered in the crops when cultivating end of run.
For the fed batch cultivation method, cultivate working time can by run duration with fresh culture once a day or repeatedly (or continuously) supplemental medium obtain increase.In this method, during the incubation time section, supply fresh culture, " charging substratum " to cell.Fed batch cultivation can comprise previously described various feed time table, for example every day, per 2 days, every other day wait; Above 1 time/day or less than 1 time/day, or the like.Fed batch cultivation can also be carried out continuously feeding with the charging substratum.When cultivation/production run finishes, gather in the crops target protein matter product subsequently.
Be used for protein by mammalian host cell production and comprise that the cell culture system of little or scale operation of glycoprotein is useful in background of the present invention.Those skilled in the art understand tissue culture ware, revolving bottle (spinner-flask) and T bottle and generally are used for cultural method on the laboratory scale.Can be used for going up cultured method and include but not limited to, hollow-fiber bioreactor, fluidized bed bio reactor, steel basin bioreactor system or roll flask culture more extensive (for example, 500L, 5000L, 10,000L, 20,000L etc.).Next 2 kinds of methods can together with or do not use together with microcarrier.
This system can with in batches, fed-batch or continuous mode operate.For production-scale cultivation, because its handiness, the steel basin bio-reactor is the system of selecting.These reactors can make cell keep suspension via stirring by stirring with bubble jet or turbomachine.The steel basin bio-reactor can increase in proportion big industrial scale volume (for example, 20,000L), and can operate with the different feeds pattern.These systems provide the high surface area that is used for cell growth and metabolic waste, oxygen and nutraceutical effective transfer, and keep the homogeneous environment that spreads all over reactor via the component in continuously stirring or the mixing reactor by preventing cell settlement to bottom.For the proteic production of desired sugars, the present invention comprises and maintains in the steel basin bio-reactor, with extensive, the fed-batch cell cultures of charging substratum charging every day that comprises the D-semi-lactosi.In another embodiment, the fed-batch cell cultures also can maintain in the steel basin bio-reactor, carry out charging every day with the charging substratum, described charging substratum comprises the restrictive cell cultivation nutrition important to Protein Glycosylation Overview of suitable concn, described nutrition is glucose and glutamine (people such as Chee for example, 2005 Biotechnol.Bioeng.89:164-177).
Producing the cell of the culture of target protein matter can breed according to any scheme or routine, the concrete mammalian host cell that described scheme or routine are suitable for expecting most and the concrete production program.Can the developing cells culture condition, in the vegetative period of cell cultures, increase or grow for this kind amplification and grow and reach the time period of maximum to strengthen mammalian host cell colony.Comprise wherein main splitted index cell vegetative period (for example, logarithmic phase) fast of cell the vegetative period of cell cultures.In this phase process, the increment rate in the density of viable cell is higher than any other time point.
Equally, can the developing cells culture condition, to strengthen protein production certain hour section during the production phase of cell cultures.It is stable or maintain time period near constant level to comprise cell growth the production phase of cell cultures.The density of viable cell keeps approximately constant in preset time in the section.Logarithm cell growth has stopped and protein production is a main activity in process production phase.At this moment general supplemental medium is to support the successive protein production and to reach the desired sugars protein product.
Culture condition is temperature, pH, dissolved oxygen (DO for example 2) to wait be those of using in cultivating mammalian host cell of being appreciated by those skilled in the art.Be used to cultivate mammalian host cell for example the suitable temperature range of Chinese hamster ovary celI be 30-40 ℃, and about in one embodiment 37 ℃.PH generally uses acid or alkali to be adjusted to the level of about 6.5-7.5.Suitable DO 2Air saturation for 5-90%.These culture condition can be used to promote produce the cultivation of the mammalian cell of desired protein or glycoprotein product.
Mammalian host cell colony can increase in cultivating vegetative period and grow, and wherein the cell inoculation that may take out from storage is to for promoting in growth and the acceptable substratum of high viability.Cell can be kept the suitable time period by add fresh substratum in the host cell culture subsequently in production phase.In the production phase process, can implement all temps change to strengthen protein production by pair cell.Be the patent application U.S. serial 10/742,564 that on December 18th, 2003 submitted in the multiple temperature variation cultural method and in the U.S. serial of submitting on December 18th, 2,003 10/740,645, obtain description.The content whole of these applications is incorporated herein by reference.In the present invention, comprise 2 times or the cell culture processes of more temperature variations can cause increasing the viable cell of number, described viable cell is survived in cultivation and is finished until method or production run.In production phase of cultivating in the process, the survival cells order is many more can to cause the amount of the protein produced or glycoprotein product many more, thus the amount of protein when the method that is increased in finishes.
Concrete aspect of the present invention comprise fed-batch, extensive (for example, 500L, 5000L, 10000L etc.) the mammalian cell cultivation, its every day or carry out charging with the charging substratum of describing in the table 14,15 that comprises the D-semi-lactosi is so that cells produce purpose glycoprotein.In order to increase in this embodiment the proteinic amount of producing, during the incubation time section, can adopt 2 times or more temperature variations surpass when use temperature change not so that the protein production phase prolongs, or take place when only using 1 temperature variation the sort of.In another embodiment, the present invention need fed-batch, extensive (for example, 500L, 5000L, 10000L etc.) the mammalian cell cultivation, it is with the charging substratum charging every day one or many of describing in the table 22 that comprises the D-semi-lactosi, so that cells produce purpose glycoprotein (for example, CTLA4 A29YL104E-Ig).During the incubation time section, also can adopt once or more temperature variations, surpass when use temperature does not change, take place the sort of so that the protein production phase prolongs, to increase the amount of glycoprotein.Alternately, follow temperature variation can in culture, add T 500.
The mass production of recombinant protein in bio-reactor
The invention provides and be used for eukaryotic conventional steel basin bioreactor culture () method for example, 20000 L cell culture volumes is used in particular for producing the desired protein product of planting cell expressing thus of extensive or commercial quantities.Cultural method is the eukaryotic fed batch cultivation method of suspension growth, follows the results of culture supernatants, and the eukaryotic cell of wherein expressing target protein matter for example mammalian cell is secreted into the desired protein product in the substratum.
The present invention has comprised the method that is used for the large scale culturing mammalian cell, is used in particular for producing a large amount of desired protein products of planting cell expressing thus.This method can be undertaken by comprising following step:
(i) seed cells into comprise serum free medium the seed culture container (for example, the T-175 bottle) in, and the breeding seed culture (for example, be used to inoculate the starter culture of more volume) reach MIN cross inoculation density until cell at least, density is the enough breeding required preset value of cell in follow-up volume of culture thus;
(ii) the inoculum with breeding is transferred to the bigger culture vessel (for example, rolling bottle or cell bags) that comprises the substratum that lacks the animal derived component, with the amplification cultivation thing;
(iii) the inoculum with amplification is transferred to the large scale culturing container that comprises serum free medium, with further propagated cell culture; With
The large scale culturing thing is maintained in the substratum that lacks the animal derived component, reach target density or show the particular organisms chemical feature until described cell at least.
In some embodiments, this method can comprise the step of (iv) gathering in the crops substratum and replacing the sort of substratum with fresh culture.
In other embodiments, the method that is used for the large scale culturing of mammalian cell can be undertaken by comprising following step:
(i) seed cells into and (for example comprise serum free medium, the inoculum substratum) seed culture container (for example, the T-175 bottle) in, and the breeding seed culture (for example, be used to inoculate the starter culture of more volume) reach MIN cross inoculation density until cell at least, density is the enough breeding required preset value of cell in follow-up volume of culture thus;
(ii) the inoculum of breeding is transferred to and comprises the substratum that lacks the animal derived component () bigger culture vessel (for example, rolling bottle or cell bags) for example, the inoculum substratum is with the amplification cultivation thing;
(iii) the inoculum with amplification is transferred to the large scale culturing container (for example, the 1000-L bio-reactor) that comprises serum free medium (for example, basic medium), with further propagated cell culture; With
The large scale culturing thing is maintained in the substratum (for example, the charging substratum) that lacks the animal derived component, reach target density or show the particular organisms chemical feature until described cell at least.
In some embodiments of the present invention, this method can comprise the steps:
(v) gather in the crops substratum and replace spent substratum with fresh culture.
The present invention can be applicable to any cell type in any substratum preparation that lacks the animal derived component, to produce the desired protein product of extensive amount, and can utilize any in following 2 kinds of methods or its variation: a) microcarrier method, or b) suspension cell method.The cell for example cultivation of mammalian cell can utilize at 2 different times---arbitrary method of operating in vegetative period and production phase.In another embodiment of the invention, (some non-limitative example is the production that bovine serum albumin(BSA) (BSA) or any substratum preparation FBS) also can be used for aforesaid large-scale protein quality to comprise the animal derived component.
Those skilled in the art understand the microcarrier method and are not limited to standard microcarrier method or perfusion microcarrier method, can be used for the cell cultures that wherein cell and macropore carrier adhere to and/or be fixed on macropore carrier.In standard microcarrier method, seed cells in the seed culture container that comprises serum free medium, and breed until cell and reach MIN inoculum density.Subsequently, the inoculum with breeding is transferred to the large scale culturing container that comprises serum free medium and microcarrier.In this vegetative period, cell is grown on microcarrier and is built the group fully until carrier, for example under the situation of the method for using macropore carrier by cell migration in carrier.
The substratum exchange can take place when microcarrier is settled down to bottom of culture vessel, takes out the long-pending and fresh culture that the corresponding per-cent cell body of interpolation amasss in container of cell body of predetermined percentage after this.Microcarrier is resuspended in the substratum.The technician understands the process that substratum takes out and replace can carry out repetition in for example per 24 hours with predetermined time interval, and the amount of the substratum of the Ti Huaning cell body that depends on cell density and can be generally 25%-80% amasss thus.When cell density reached the preset value that is suitable for protein expression, the tank culture base of 60-95% can be changed in per 24 hours in the groove.Those skilled in the art also use above-mentioned substratum exchange % value usually from start to finish in production phase.
In the production phase process, substratum can exchange by allowing microcarrier be settled down to trench bottom, and it is long-pending and to add corresponding % cell body in container long-pending to take out cell body of selected % after this, for example the fresh culture of aforesaid 60-95%.Microcarrier is resuspended in the substratum, and the process that substratum takes out and replaces can be carried out repetition every day.
Microcarrier method for filling similar standard microcarrier method and also operating in growth/amplification with in production phase.Main difference between 2 kinds of methods is the method that is used to change substratum.The cell body of limited amount is long-pending, and for example total cell body of 60-95% amasss in standard microcarrier method and changes suddenly, and substratum adds continuously in method for filling.Basically, the long-pending substratum of % cell body is progressively changed through scheduled time length, and microcarrier maintains in the container by using tripping device (or device for casting) simultaneously, and described tripping device allows substratum to leave container but microcarrier is retained in the groove.Vegetative period in this method is as describing for standard microcarrier method, except substratum exchange progressively.
2 kinds of non-limiting options of suspension cell method comprise suspension cell method for filling and suspension cell batch processes.In method for filling, the cell in the substratum freely suspends and is not immobilized in the carrier, and as the microcarrier method, can not operate in the same period (for example, vegetative period and production phase) at 2.In the vegetative period of suspension cell method for filling process, seed cells in the seed culture container that comprises serum free medium, and breed until cell and reach target cross inoculation density.The inoculum of breeding can be transferred to the large scale culturing container that comprises the substratum that lacks the animal derived component subsequently, and breed the predetermined cell density value that is suitable for protein expression until reaching.Carrying out culture vessel uses the continous pouring of fresh culture to carry out the substratum switching method.
In the suspension cell batch processes, cell cultures can be carried out via following non-limiting way: a) simple batch processes or b) fed-batch process.In simple batch processes, seed cells in the seed culture container that comprises the substratum that lacks the animal derived component, and breed until cell and reach predetermined cross inoculation density.Subsequently, the inoculum of breeding is transferred to the large scale culturing container that comprises serum free medium, and the nutrition that culture vessel is operated in substratum exhausts.In fed-batch process, the nutraceutical concentrated solution of charging (for example in groove, the charging substratum) can prolong nutrition supplement in the substratum of this cultural method, thereby prolong the treatment time and finally cause increase in the production of the desired protein in culture vessel.The method of adding the charging substratum can change.It can be used as monopulse bolus (every day 1 time, 2 times, 3 inferior) and adds or can carry out progressively charging from start to finish 24 hour time period.This charging allows cell to breed in the large scale culturing container, and comprises the substratum of excretory target protein matter product and can be when end of run become at any nutrition and gather in the crops before exhausting.Replace taking out from container all the elements thing, those skilled in the art will only take out part cell body long-pending (can be about 80%).
The optional aspect of fed-batch process is the use of temperature variation.In this method, the temperature that is used as service temperature in production phase in the process is lower than the temperature of using in the process in vegetative period.The described temperature range of the method for for example using in the present invention about fed-batch process can be by the initial growth phase under the temperature of cell line growth in being suitable for specific use, be the reduction composition in the service temperature when predetermined cell density subsequently.
In one embodiment, the method that is used for extensive fed batch cultivation method comprises following: (i) seed cells into comprise serum free medium the seed culture container (for example, the T-175 bottle) in, and the breeding seed culture reaches predetermined cross inoculation density until cell at least under the temperature that is suitable for growing; (ii) the inoculum with breeding is transferred to the bigger culture vessel (for example, rolling bottle or cell bags) that comprises the substratum that lacks the animal derived component, with amplification cultivation thing under suitable temperature; (iii) the inoculum with amplification is transferred to the large scale culturing container that comprises serum free medium, with further propagated cell culture under suitable temperature; (iv) under the temperature of the reduction that is suitable for protein expression, the large scale culturing thing is maintained in the substratum that lacks the animal derived component, follow replace the every day of fresh feed substratum, reach target density or marginal time length until described cell at least.
The step of replacing with fresh material substratum in (iv) can need to take out pre-determined volume, and for example 80% cell body is long-pending, and with the fresh feed substratum replacement of equal volume it.
In further embodiment, the method that is used for extensive fed batch cultivation method comprises following: (i) seed cells into comprise serum free medium the seed culture container (for example, the T-175 bottle) in, and the breeding seed culture reaches predetermined cross inoculation density until cell at least under the temperature that is suitable for growing; (ii) the inoculum with breeding is transferred to the bigger culture vessel (for example, rolling bottle or cell bags) that comprises the substratum that lacks the animal derived component, with amplification cultivation thing under suitable temperature; (iii) the inoculum with amplification is transferred to the large scale culturing container (for example, the 1000-L bio-reactor) that comprises serum free medium, with further propagated cell culture under suitable temperature; (iv) under the temperature of the reduction that is suitable for protein expression, the large scale culturing thing is maintained in the substratum that lacks the animal derived component, follow replace the every day of fresh feed substratum, reach target density or marginal time length until described cell at least.
The cell body that the step of replacing with the fresh feed substratum in (iv) can need to take out pre-determined volume for example about 80% amasss, and replaces it with the fresh feed substratum of equal volume.
In one embodiment of the invention, cultured cells is the mammalian cell of expressing the desired protein product, for example Chinese hamster ovary celI in fed-batch process.With mammalian cell be inoculated into comprise serum free medium for example CD-CHO substratum (embodiment 13) the seed culture container (for example, the T-175 bottle) in, and under the temperature that is suitable for growing, for example bred 3-4 days in about 35-39 ℃, reaching predetermined cross inoculation density until cell (for example, has 〉=6.0 x 10 6Viable cell, or final cultures viability 〉=80% wherein).Inoculum with breeding is transferred to the big culture vessel (for example, rolling bottle) that comprises the substratum that lacks the animal derived component subsequently, is used for that (for example, in about 35-39 ℃) increased about 3-4 days under suitable temperature.Make cell culture comprise serum free medium for example the CD-CHO substratum bigger culture vessel (for example, 20L cell bags, 100L cell bags etc.) in, under the temperature that is suitable for growing, for example further increased 3-4 days in about 35-39 ℃, reaching the target inoculum density until cell (for example, has 〉=1-2 x 10 6Viable cell/ml, or final cultures viability 〉=80% wherein).In one embodiment, the inoculum amplification comprises minimum 4 generations.In another embodiment of the invention, the inoculum amplification need be no more than for 20 generations.
The inoculum of amplification (for example can be used for large scale culturing groove that inoculation comprises serum free medium (for example CD-CHO substratum) subsequently, 1000L, 4000L bio-reactor etc.), with under suitable temperature for example in about 35-39 ℃ of further propagated cell culture about 3-6 days, reaching the target inoculum density until cell (for example, has 〉=1-2 x 10 6Viable cell/ml, or final cell cultivation thing viability 〉=80% wherein).The large scale culturing thing (for example in bio-reactor etc. 10,000L, 15,000L, 20, the 000L culture) maintain in the serum free medium subsequently, wherein substratum is charging substratum (for example eRDF substratum, embodiment 14), in protein expression that is suitable for the excretory protein and production, (for example be lower than under the temperature of growth temperature, in or about 33-35 ℃ 3-4 days and in or about 31-33 ℃ 6-8 days).The charging substratum is replaced with the fresh feed substratum every day, and the fresh feed substratum of groove replace to need takes out pre-determined volume for example 80% cell body is long-pending thus, and with the fresh feed substratum replacement groove of equal volume.The commercial size culture is kept the target value that reaches manufacturing parameter until described cell, described manufacturing parameter can be but be not limited to, time span, target cell density or biological chemistry protein characteristic (for example, aforesaid NANA mol ratio), wherein viable cell density can be 3.0-8.0 x 10 6Cell/ml; The NANA mol ratio can be 〉=6.0; Final cell cultivation thing viability can be 〉=30%; With final protein titre can for 〉=0.5g/L).
In specific embodiments of the present invention, cultured cells is to express desired protein product (for example, the CTLA4-Ig molecule) mammalian cell, for example Chinese hamster ovary celI in fed-batch process.With Chinese hamster ovary celI be inoculated into comprise serum free medium for example the CD-CHO substratum the seed culture container (for example, the T-175 bottle) in, and under the temperature that is suitable for growing,, reach predetermined cross inoculation density until cell and (for example, have 〉=10.0 x 10 for example in about 37 ℃ of breedings 3-4 days 6Viable cell, or final cultures viability 〉=84% wherein).Inoculum with breeding is transferred to the big culture vessel (for example, rolling bottle) that comprises the substratum that lacks the animal derived component subsequently, is used under suitable temperature (about 37 ℃) amplification about 4 days.Make cell culture comprise serum free medium for example the CD-CHO substratum bigger culture vessel (for example, 20L cell bags, 100L cell bags etc.) in, (for example in about 37 ℃) further increased 4 days under the temperature that is suitable for growing, reaching the target inoculum density until cell (for example, has 〉=1-2 x 10 6Viable cell/ml, or final cultures viability 〉=91% wherein).The inoculum amplification can comprise minimum 7 generations.
The inoculum of amplification (for example is used for large scale culturing groove that inoculation comprises serum free medium (for example CD-CHO substratum) subsequently, 4000L bio-reactor etc.), with under suitable temperature for example in about 37 ℃ of further propagated cell cultures about 5-6 days, reaching the target inoculum density until cell (for example, has 〉=1-2 x 10 6Viable cell/ml, or final cell cultivation thing viability 〉=86% wherein).The commercial size culture (for example in bio-reactor 20, the 000L culture) maintain in the serum free medium subsequently, wherein substratum is charging substratum (a for example eRDF substratum), (for example, protein expression CTLA4-Ig) and production, be lower than under the temperature of growth temperature being suitable for the excretory protein.The commercial size culture at first from about 37 ℃ reduce to about 34 ℃ 4 days, and implemented temperature variations for the second time in 8 days by making temperature reduce to about 32 ℃ subsequently from about 34 ℃.The charging substratum is replaced with fresh supplemented medium every day, and the charging substratum in the biological respinse tank replace to need takes out pre-determined volume for example 80% cell body is long-pending thus, and with the fresh feed substratum replacement of equal volume it.Commercial size is kept the target value that reaches following non-limiting manufacturing parameter until described Chinese hamster ovary celI and/or excretory protein: viable cell density 4.0-7.0 x 10 6Cell/ml; NANA mol ratio 〉=8.0; Final cell cultivation thing viability 〉=38%; With final protein titre 〉=0.6g/L.
In another embodiment of the invention, cultured cells is the mammalian cell of expressing the desired protein product, for example Chinese hamster ovary celI in fed-batch process.Mammalian cell is inoculated into comprises serum free medium for example in the seed culture container of CD-CHO substratum (embodiment 19) (for example, the T-175 bottle), and about 39 ℃ of breedings of for example about 35-about 3-4 days under the temperature that is suitable for growing; Or about 36 ℃-Yue 38 ℃ of breedings are up to about 4 days approximately, reach predetermined cross inoculation density until cell and (for example, have more than or equal to 1.5 x 10 6Cell density, or wherein the final cultures viability more than or equal to about 80%).Subsequently the inoculum of breeding is transferred to comprise the substratum that lacks the animal derived component big culture vessel (for example, roll bottle), be used under suitable temperature (for example, about 39 ℃ of about 35-, or about 36 ℃-Yue 38 ℃) amplification about 3-4 days or be up to about 4 days.Make cell culture comprise serum free medium for example the CD-CHO substratum bigger culture vessel (for example, 20L cell bags, 100L cell bags etc.) in, for example about 35-is about 39 ℃ under the temperature that is suitable for growing, or about 36 ℃-Yue 38 ℃ of further amplifications about 3-4 days or be up to about 4 days, reaching the target inoculum density until cell (for example, has at least about 1.5 x 10 6Viable cell/ml, or wherein the final cultures viability more than or equal to 80%).In one embodiment, the inoculum amplification comprises minimum 4 generations.In another embodiment of the invention, the inoculum amplification need be no more than for 20 generations.In some embodiments, the CD-CHO substratum is a CD-CHO inoculum substratum.
The inoculum of amplification can be used for inoculation subsequently and comprise serum free medium (CD-CHO substratum for example, for example CD-CHO inoculum substratum and/or CD-CHO basic medium) the large scale culturing groove (for example, 1000L, 4000L bio-reactor etc.), with under suitable temperature for example about 35 ℃-Yue 39 ℃, or about 6 days of about 36 ℃-Yue 38 ℃ of about 3-of further propagated cell culture or about 4 days-Yue 5 days or about 4.7 days or be less than or equal to about 113 hours, reach the target inoculum density until cell and (for example, have about 2.3 x 10 6Viable cell/ml, or wherein final cell cultivation thing viability at least about 88%).
The commercial size culture (for example in bio-reactor etc. 10,000L, 15,000L, 20,000L, 30, the 000L culture) in serum free medium, keeps subsequently, wherein substratum is charging substratum (for example eRDF substratum, embodiment 19), keeps about 3-about 6 days or about 4 days-Yue 5 days under the protein expression that is suitable for the excretory protein and production, about 35 ℃-Yue 39 ℃ temperature.Alternately, can be (for example with polyanionic compound, T 500) adds in the culture that maintains in the serum free medium, wherein substratum be as hereinafter with U.S. Patent Application Publication No. 2005/0019859 in charging substratum (the eRDF substratum for example described, embodiment 19), described patent document is incorporated herein by reference in this integral body.Can follow to culture implement the single step temperature reduce (for example, in or about 32 ℃ as for or about 304 hours of about 14 days of about 36 ℃ of about 3-or about 13 days of about 10-or about 234-.
In one embodiment of the invention, can also follow to culture implement the multistep temperature reduce (for example, in or about 33 ℃ as for or about 35 ℃ of about 3-6 days and in or about 31 ℃ as for or about 33 ℃ of about 6-8 days).Above-mentioned processing is suitable for the protein expression and the production of secretory protein product.
Charging substratum under above-described situation can every day (grade every days 1,2,3) or per a couple of days replace with the fresh feed substratum.Groove is replaced with the fresh feed substratum to be needed to take out pre-determined volume for example 80% cell body is long-pending, and with the fresh feed substratum replacement groove of equal volume.The commercial size culture is kept the target value that reaches manufacturing parameter until described cell, described manufacturing parameter can be but be not limited to, time span, target cell density or biological chemistry protein characteristic (for example, aforesaid NANA mol ratio), wherein viable cell density can be 3.0-8.0 x 10 6Cell/ml; The NANA mol ratio can be 〉=5.0, or about 6, or about 5.2-about 7.6; Final cell cultivation thing viability can be for more than or equal to about 30%, or more than or equal to about 37%; With final protein titre can be for the about 0.71g/L of about 0.46-, more than or equal to 0.5g/L, or more than or equal to 20g/L.
According to the present invention, provide the cell culture processes of the delay interpolation that comprises polyanionic compound.This method is included in the inoculation back some time (for example, in the vegetative period of cultural method process or in the production phase process) and adds polyanionic compound in cell culture.The cell survival that reaches increase is added in the delay of polyanionic compound.In one embodiment, the present invention relates to cell culture processes, it comprises the host cell of culture expression target protein matter and adds polyanionic compound in the some time after the inoculation in cell culture.
Polyanionic compound includes but not limited to, T 500 (can be from Sigma-Aldrich, St.Louis, Mo. obtains), heparin (can obtain) from Sigma-Aldrich, Suleparoid (can obtain) from Sigma-Aldrich, the sulfuric acid mannosans, chondroitin sulfate (can obtain) from Sigma-Aldrich, dermatan sulfate (can obtain) from Sigma-Aldrich, keratan sulfate (can obtain) from Sigma-Aldrich, hyaluronate (can obtain) from Sigma-Aldrich, poly-sulfuric acid vinyl ester (can obtain) from Sigma-Aldrich, κ-carrageenan (can obtain) from Sigma-Aldrich, and Suramine (can obtain) from Sigma-Aldrich.Compound can easily obtain from the source of listing, or easily obtains by method known to those skilled in the art.These compounds can obtain with the form of salt usually, include but not limited to sodium salt, but also can use with the form of non-salt.Polyanionic compound comprises its form of ownership, includes but not limited to salt form, for example sodium salt.
Useful especially, the non-limitative example of polyanionic compound of the present invention comprises poly-sulfation (poysulfated) compound: T 500, heparin, Suleparoid, sulfuric acid mannosans, chondroitin sulfate, dermatan sulfate, keratan sulfate, poly-sulfuric acid vinyl ester, κ-carrageenan and Suramine.In one embodiment, polyanionic compound is a T 500.T 500 can have 5,000-500, the molecular-weight average of 000Da.In another embodiment of the invention, use to have 5, the T 500 of the molecular weight of 000Da.
The method according to this invention, polyanionic compound can be during designated cell incubation time sections, and (for example, the some time after inoculation is for example in vegetative period or production phase process) adds 1 time, 2 times, 3 times or any number of times in cell culture.One or more polyanionic compounds can be used in combination.For example, any given single of polyanionic compound adds the interpolation that can comprise one or more other polyanionic compounds.Similarly, if exist surpassing once of polyanionic compound to add, so different polyanionic compounds can add when difference is added.Additional compounds and material comprise polyanionic compound, can be before the interpolation of polyanionic compound, simultaneously or afterwards, at the appointed time at the appointed time add in the culture during the section during the section or not.In specific embodiments, exist the single of polyanionic compound for example to add for 1 time.In another embodiment, add a kind of polyanionic compound.
Polyanionic compound can add in the cell culture with any means.The means of adding polyanionic compound include but not limited to, are dissolved in the water, are dissolved in the substratum, be dissolved in the charging substratum, be dissolved in the suitable medium and with its obtained form.Especially, polyanionic compound is dissolved in the water and adds.According to the present invention, add polyanionic compound so that the concentration in the culture reaches suitable level.As non-limitative example, add the concentration of polyanionic compound to 1-1000mg/L, 1-200mg/L, 1-100mg/L or 25-75mg/L.The useful especially polyanionic compound concentration that adds in the cell culture includes but not limited to about 25-200mg/L; About 25-100mg/L; With about 50-100mg/L.In one embodiment of the invention, the polyanionic compound concentration that adds in the culture is about 50mg/L.In another embodiment, the polyanionic compound concentration that adds in the culture is about 100mg/L.
Method of the present invention provides cultivation can move length any time after the interpolation of polyanionic compound.Cultivate and to determine by those skilled in the art working time, the level of the contaminative cell category (for example protein and DNA) in the supernatant liquor that produces based on for example callable proteinic amount of correlative factor and quality and by lysis, described contaminative cell category will make the recovery of target protein matter complicate.In some embodiments of cell culture processes, polyanionic compound some time (for example, in the vegetative period of cell culture processes process or the production phase of cell culture processes in the process) after inoculation adds.Polyanionic compound when vegetative period or when finishing approximately after the inoculation in the process some time add.Especially, polyanionic compound is in the production phase process, and the some time adds after the inoculation when for example begin production phase.
In specific embodiments of the present invention, cultured cells is to express desired protein product (for example, CTLA4 in fed-batch process A29YL104E-Ig molecule) mammalian cell, for example Chinese hamster ovary celI.With Chinese hamster ovary celI be inoculated into comprise serum free medium for example CD-CHO substratum (for example CD-CHO inoculum substratum) the seed culture container (for example, the T-175 bottle) in, and under the temperature that is suitable for growing, for example bred about 3-4 days in about 37 ℃, reaching predetermined cross inoculation density until cell (for example, has 〉=1.5 x 10 6Viable cell, or final cultures viability 〉=80% wherein).Inoculum with breeding is transferred to the big culture vessel (for example, rolling bottle) that comprises the substratum that lacks the animal derived component subsequently, is used under suitable temperature (in about 37 ℃) amplification about 4 days.Make cell culture comprise serum free medium for example CD-CHO substratum (for example CD-CHO inoculum substratum) bigger culture vessel (for example, 20L cell bags, 100L cell bags etc.) in, (for example in about 37 ℃) further increased about 4 days under the temperature that is suitable for growing, reaching the target inoculum density until cell (for example, has 〉=1.5 x 10 6Viable cell/ml, or final cultures viability 〉=80% wherein).The inoculum amplification can comprise minimum 7 generations.
The inoculum of amplification is used for inoculation subsequently and comprises serum free medium (CD-CHO substratum for example, CD-CHO basic medium for example) large scale culturing groove (for example, 1000-L, 4000-L bio-reactor etc.), with under suitable temperature for example in about 37 ℃ of further propagated cell cultures about 5-6 days, reach the target inoculum density until cell and (for example, have about 2.3 x 10 6Viable cell/ml, or final cell cultivation thing viability 〉=88% wherein).The commercial size culture (for example in bio-reactor 10,000L, 15,0000L, 20,000L culture etc.) maintain in the serum free medium subsequently, wherein substratum is charging substratum (a for example eRDF substratum), is being suitable for excretory protein (for example, CTLA4 A29YL104E-Ig) protein expression and production, be lower than under the temperature of growth temperature.
The commercial size culture for example from about 37 ℃ reduce to about 34 ℃ about 4 days.Polyanionic compound can be followed when temperature reduces and add in the culture.Alternately, the commercial size culture from about 35 ℃-37 ℃ reduce to about 32 ℃-Yue 36 ℃ about 12 days, and polyanionic compound can be followed when temperature reduces and adds in the culture.
Charging substratum under above-described situation can every day (grade every days 1,2,3) or per a couple of days replace with the fresh feed substratum.Groove is replaced with the fresh feed substratum to be needed to take out pre-determined volume for example 80% cell body is long-pending, and with the fresh feed substratum replacement groove of equal volume.In one embodiment, the charging substratum adds every day, about altogether 2-3 days, reduces to 1g/L until for example glucose concn.In another embodiment, for example, in case glucose concn has reached 1g/L, the charging substratum added with regard to per 8 hours.Commercial size is kept the target value that reaches following non-limiting manufacturing parameter until described Chinese hamster ovary celI and/or excretory protein: the NANA mol ratio is about 6.0, or about 5.2-about 7.6; Final cell cultivation thing viability 〉=37%; With the about 0.71g/L of the about 0.46-of final protein titre.
In one embodiment of the invention, cultured cells can be a mammalian cell, or the mammal cell line of establishing, and includes but not limited to CHO (for example, ATCC CCL61), HEK293 (for example, ATCC CRL1573; People such as Graham, J.Gen.Virol.36:59-72,1977), COS-1 (for example, ATCC CRL1650), DG44 (Chinese hamster ovary celI system) ( Cell, 33:405,1983 Hes Somatic Cell and Molecular Genetics12:555,1986) and young hamster kidney (BHK) clone.Other useful non-limitative examples are fusions of myelomatosis, 3T3 cell, Namalwa cell and myelomatosis and other cells.In some embodiments, cell can be sudden change or reconstitution cell, for example expresses the cell with the enzyme of its deutero-cell type different range, and the proteinic posttranslational modification of described enzyme catalysis (for example, processive enzyme is propetide or glycosylase for example, for example glycosyltransferase and/or Glycosylase).Of the present invention one concrete aspect, use the CHO/dhfr-cell especially.
The culture vessel that is used for the amplifying cells culture can be but be not limited to, and Erlenmeyer flask, T-175 bottle, rolls bottle and cell bags.The large scale culturing container can be for example wherein to stir the airlift reactor that obtains by means of from the container bottom introducing air, or wherein stir the conventional stirred tank reactor (CSTR) that obtains by means of conventional impeller-type.Temperature, pH and dissolved oxygen tension force (DOT) are arranged in the parameter in being controlled at specified limit.Temperature control medium in this system is a water, and can heat or cool off as required.Water can be through immersing coil pipe (piping coil) or the container chuck on every side in the cell culture medium.PH for example can be by adding alkali or by changing the CO in the head space gas when needed in cell culture medium 2Concentration is regulated.DOT can be kept by spraying pure oxygen or air or its mixture.
Therefore the present invention provides the method that is used to produce recombinant protein, this method comprises at least 2 steps: (a) make the secretion recombinant protein (promptly, mammalian cell is not expressed or the protein of overexpression usually, wherein recombinant protein is expressed in cell to expression vector or construct in cell or the cell parental via transfection) mammalian cell be expanded at least 10 from inoculum, the liquid culture of 000L, (b) from least 10, separating recombinant proteins matter in the 000L liquid culture.In one embodiment, this method can be used like this, thus make recombinant protein before protein purification from liquid culture with the concentration production of at least 0.5 gram/L liquid culture.In another embodiment, the method according to this invention can be used for producing and is at least about the recombinant protein of the about 0.71 gram/L liquid culture of 0.46-in concentration before the liquid culture protein purification.
In one embodiment, amplification step can comprise that (i) cultivated for 4 generations cell at least in serum free medium, so that obtain at least about 1.0 x 10 5The cell density of viable cell/mL and cell is kept be enough to produce time in cultivation at least about 0.5 recombinant protein.In one embodiment, the number that goes down to posterity was no more than for 36 generations.In another embodiment, the number that goes down to posterity can surpass for 36 generations, and wherein cell is stable in several generations with regard to copy number, cell survival and the doubling time of the nucleic acid of coding recombinant protein.
The time that is enough to produce at least about the about 1.3g/L recombinant protein of 0.5-can be to measure any time, as long as cell survival do not drop to 5%, 10%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, 95%, below 98%, and/or as long as cell algebraically was no more than for 50,75,100,105 or 125 generations.Keep step and can also comprise the temperature variation step, the temperature that for example makes culture is at first reduced to 34 ± 2 ℃ and in that the time is reduced to 32 ± 2 ℃ from 34 ± 2 ℃ subsequently from 37 ± 2 ℃.32 ± 2 ℃ temperature can be kept 5,6,7,8,9,10,11,12,13,14,15,16,17,18,20,30,50 or 100 days at least.32 ± 2 ℃ temperature can keep at least 20,50,75 or 100 cell generations.The cell density that 32 ± 2 ℃ temperature can be kept until culture is about 100 x 10 of about 30- 5Cell/mL liquid culture.
In other embodiments, the invention provides the method that is used to produce recombinant protein, this method comprises following at least step: (a) make the mammalian cell of secretion recombinant protein be expanded at least 10 from inoculum, the liquid culture of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) when following situation takes place in liquid culture, from least 10, separating recombinant proteins matter in the 000L liquid culture: (i) comprise more than or equal to about 6.0 moles of NANA/ mole protein (glycoprotein in this case); (ii) have about 79 x 10 of about 33- 5The cell density of cell/mL; (iii) the cell survival in the liquid culture be not less than about 38%, or more than or equal to about 38%; (iv) intracellular toxin is less than or equal to about 76.8EU/mL liquid culture; And/or (v) biological load is less than 1 colony forming unit/mL liquid culture.
In further embodiment, amplification step can comprise that (i) cultivated for 4 generations cell at least in serum free medium, so that obtain at least about 1.0 x 10 6The cell density of viable cell/mL and cell is kept be enough to the time of producing in cultivation at least about the about 0.71 grammes per square metre histone matter/L liquid culture of 0.46-.In one embodiment, the number that goes down to posterity was no more than for 36 generations.In another embodiment, the number that goes down to posterity can surpass for 36 generations, and wherein cell is stable in several generations with regard to copy number, cell survival and the doubling time of the nucleic acid of coding recombinant protein.
The time that is enough to produce the about 0.71g/L recombinant protein of about 0.46-can be amount any time, as long as cell survival do not drop to 5%, 10%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, 95%, below 98%, and/or as long as cell algebraically was no more than for 27,50,75,100,105 or 125 generations.Keep step and can also comprise the temperature variation step, for example make the temperature of culture at first reduce to 34 ± 2 ℃ from 37 ± 2 ℃.34 ± 2 ℃ temperature can be kept 5,6,7,8,9,10,11,12,13,14,15,16,17,18,20,30,50 or 100 days at least.Alternately, keep step and can be the temperature variation step, for example make the temperature of culture reduce to 34 ± 2 ℃ from 37 ± 2 ℃.
Polyanionic compound can add when temperature reduces beginning.The concentration that adds the polyanionic compound in the culture can be about 1mg/L, 5mg/L, 10mg/L, 12.5mg/L, 15mg/L, 25mg/L, 50mg/L, 75mg/L, 100mg/L, 200mg/L, 250mg/L, 500mg/L, 750mg/L or 1000mg/L.32 ± 2 ℃ temperature can be kept 5,6,7,8,9,10,11,12,13,14,15,16,17,18,20,30,50 or 100 days at least.34 ± 2 ℃ temperature can keep at least 20,27,50,75 or 100 cell generations.
In further embodiment, the invention provides the method that is used to produce recombinant protein, this method comprises following at least step: (a) make the mammalian cell of secretion recombinant protein be expanded at least 10 from inoculum, the liquid culture of 000L, thus make recombinant protein concentration be the about 0.71 gram/L liquid culture of 0.46-at least; (b) when following situation takes place in liquid culture, from least 10, separating recombinant proteins matter in the culture of 000L liquid culture: (i) comprise about 6 moles of NANA/ mole protein (glycoprotein in this case); (ii) the cell survival in the liquid culture is not less than about 37%; (iii) intracellular toxin is less than or equal to about 4.8EU/mL liquid culture; And/or (iv) biological load less than 1 colony forming unit/mL liquid culture.
The recombinant protein of producing by these methods of the present invention can be excretory protein, glycoprotein, cytokine, hormone, CTLA4-Ig protein or CTLA4 A29YL104E-Ig protein.In one embodiment, mammalian cell is offspring or the subclone by cell provided by the invention.In another embodiment, mammalian cell is offspring or the subclone derived from the cell of clone of the present invention.In further embodiment, mammalian cell comes the personal clonal population that comprises the expression cassette cells transfected of SEQ ID NO:1.In specific embodiments, mammalian cell comes the personal clonal population that comprises the expression cassette cells transfected of SEQ ID NO:3.
Be used for the general technology of purifying from the recombinant protein of culture
At the protein production after date of cell culture processes, target protein matter for example glycoprotein uses the technology of those skilled in the art's understanding to reclaim from cell culture medium.Especially, target protein matter reclaims from substratum as the excretory polypeptide, although it also can reclaim from the host cell lysate.Substratum or lysate carry out centrifugal to remove cell debris and particle at first.The following non-limiting purifying procedure that desired protein uses this area fully to establish subsequently carries out purifying: SDS-PAGE from contaminant dna, soluble protein and polypeptide; Ammonium sulfate precipitation; Ethanol sedimentation; The fractional separation on the affine or ion exchange column in immunity; Reversed-phase HPLC; At silicon-dioxide or the anionite-exchange resin chromatography on QAE or the DEAE for example; Chromatofocusing; Use for example Sephadex G-75 TMThe gel-filtration of post; With A Protein S epharose TMPost is to remove for example IgG of pollutent.The proteinase inhibitor for example interpolation of phenyl methyl sulfonic acid fluoride (PMSF) or protease inhibitor cocktail also can be used to be suppressed at proteolysis degraded during the purifying.Those skilled in the art will recognize that be suitable for target protein matter for example the purification process of glycoprotein may need to change, to solve the variation of in reconstitution cell is cultivated, expressing in the proteinic feature in back.
Purification technique and the method selected at the carbohydrate group of glycoprotein also have effectiveness in background of the present invention.For example, this kind technology comprises HPLC or uses positively charged ion or the ion exchange chromatography of anionite-exchange resin, wherein depends on the carbohydrate of selection, collects more alkaline or tart fraction more.The use of this kind technology also can cause the removal of following of pollutent.
In the present invention, can produce CTLA4-Ig or CTLA4 A29YL104EThe Chinese hamster ovary celI of-Ig fusion rotein grows to predetermined cell density as suspension in CHO specificity substratum.Express the Chinese hamster ovary celI of suspension growth in the substratum at serum-free and produce CTLA4-Ig or CTLA4 subsequently A29YL104E-Ig molecule, it is secreted in the substratum by Chinese hamster ovary celI.Cell suspending liquid can be via the centrifugal clarification that becomes, and the CTLA4-Ig molecule can separate with clarifying culture supernatants by the standard purification technology subsequently.Be used for obtaining alone or in combination CTLA4-Ig or CTLA4 A29YL104EThe bigger purity of-Ig and the non-limitative example of homogeneous suitable purifying procedure are: the affinity chromatography on agarose; Fractional separation on anion-exchange column (AEC); And hydrophobic interaction chromatography (HIC).
In some embodiments, CTLA4-Ig molecule or other protein (comprising glycoprotein) that separates from production method described herein can comprise the steps: at least that (i) obtains cell culture supernatant liquid; (ii) supernatant liquor is implemented anion-exchange chromatography to obtain the protein of wash-out; (iii) the protein of step wash-out is (ii) implemented hydrophobic interaction chromatography, so that obtain the protein of enrichment; (iv) the protein of enrichment is implemented affinity chromatography, to obtain the protein of wash-out and enrichment; (v) the protein of (iv) wash-out and enrichment is implemented anion-exchange chromatography.The feature of the protein of the enrichment that step obtains in (iii) for example can be it any HMW protein or contamination percentage less than 5,10,15 or 25%.Step anion-exchange chromatography (ii) can be for example by using lavation buffer solution to carry out, and described lavation buffer solution comprises about 25-100mM HEPES and about 300-900mMNaCl, and has the pH of about 7.0-8.0.Step hydrophobic interaction chromatography (iii) can for example have about 7.0 pH by use and comprise about 25mM HEPES and the single lavation buffer solution of about 850mM NaCl; Or the lavation buffer solution that has about 8.0 pH and comprise about 25mM Tris and about 250mM NaCl carries out.Step affinity chromatography (iv) can be for example by using elution buffer to carry out, and described elution buffer has about 3.5 pH, and comprises about 100mM glycine.(affinity chromatography v) can for example have about 8.0 pH by use and comprise the lavation buffer solution of about 25mM HEPES and the about 130mM NaCl of about 120mM NaCl-step, or has about 8.0 pH and comprise about 25mM HEPES and the lavation buffer solution of about 200mM NaCl carries out.Step anion-exchange chromatography (ii) can use the post with anionite-exchange resin to carry out, and described anionite-exchange resin has primary, secondary, uncle or quaternary amine functional group.Step hydrophobic interaction post (iii) can use the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin has phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.
In other embodiments, separation is from the CTLA4 of production method described herein A29YL104E-Ig molecule or other protein (comprising glycoprotein) can comprise the steps: at least that (i) obtains cell culture supernatant liquid; (ii) supernatant liquor is implemented affinity chromatography to obtain the protein of wash-out; (iii) the protein of step wash-out is (ii) implemented anion-exchange chromatography, so that obtain the protein of enrichment; (iv) the protein of enrichment is implemented hydrophobic interaction chromatography, with the wash-out of high molecular (HMW) protein complex that obtains to have minimizing and the protein of enrichment.The feature of the protein of the enrichment that step obtains in (iv) for example can be it any HMW protein or contamination percentage less than 5,10,15 or 25%.Step affinity chromatography (ii) can be for example by using elution buffer to carry out, and described elution buffer has about 3.0 pH, and comprises about 250mM glycine.Step affinity chromatography (ii) can be for example by using lavation buffer solution to carry out, and described elution buffer has about 7.5 pH, and comprises about 25mM NaH 2PO 4With about 150mM NaCl.Step anion-exchange chromatography (iii) can be for example by using lavation buffer solution to carry out, and described lavation buffer solution comprises about 50mM HEPES and about 135mM NaCl, and has about 7.0 pH.Step anion-exchange chromatography (iii) can be for example by using elution buffer to carry out, and described elution buffer comprises about 50mM HEPES and about 200mM NaCl, and has about 7.0 pH.Step hydrophobic interaction chromatography (iv) can be for example by using lavation buffer solution to carry out, and described lavation buffer solution has about 7.0 pH, and comprises about 50mM HEPES and about 1.2M (NH 4) 2SO 4Step anion-exchange chromatography (iii) can use the post with anionite-exchange resin to carry out, and described anionite-exchange resin has primary, secondary, uncle or quaternary amine functional group.Step hydrophobic interaction post (iv) can use the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin has phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.
In one embodiment, the invention provides the method that is used for from liquid cell culture purifying CTLA4-Ig molecule, thereby make the CTLA4-Ig of purifying be substantially free of MCP-1 (MCP-1).In one embodiment, the invention provides the pharmaceutically acceptable composition of CTLA4-Ig molecule, wherein composition comprises and is no more than 0.5ppm MCP-1,1ppm MCP-1,2ppm MCP-1,3ppm MCP-1,4ppm MCP-1,5ppmMCP-1,6ppm MCP-1,7ppm MCP-1,8ppm MCP-1,9ppm MCP-1 or 10ppm MCP-1.In another embodiment, in composition, the amount of MCP-1 cannot surpass the weight of the CTLA4-Ig of 1%, 0.5% or 0.1% purifying.In another embodiment, the composition of CTLA4-Ig molecule is substantially free of MCP-1, wherein exists in QFF eluate liquid less than 50,45,40,38,35,30,25,20,15,10,9,8,7,6,5,4,3,2 or 1ng/mL MCP-1.In another embodiment, the invention provides the method that is used for from liquid cell culture purifying CTLA4-Ig molecule, thereby make the CTLA4-Ig of purifying be substantially free of MCP-1 and comprise the tetramer less than 2.5%CTLA4-Ig.
The amount of the MCP 1 (MCP-1) in the composition can use the ELISA method to carry out quantitatively.Coated antibody is a goat anti-mouse MCP-1IgG antibody.Second antibody is the mouse MCP-1IgG of a rabbit Chinese People's Anti-Japanese Military and Political College antibody.Detecting the goat anti-rabbit igg antibody and the substrate TMB that use horseradish peroxidase to put together finishes.Horseradish peroxidase reagent produces the colorimetric reaction with the proportional colour developing of protein mass of catching.ELISA is with respect to the quantitative MCP-1 level of standard of materials curve.In one embodiment, MCP-1 carries out in composition quantitatively, and the MCP-1 level is 0-0.097ng/mg to 0.014 0.154ng/mg.
In another embodiment, the invention provides and be used for from liquid cell culture purifying CTLA4 A29YL104EThe method of-Ig molecule, thus the CTLA4 of purifying made A29YL104E-Ig is substantially free of MCP-1 (MCP-1).In one embodiment, the amount of MCP-1 cannot surpass the weight of the CTLA4-Ig of 1%, 0.5% or 0.1% purifying.In another embodiment, CTLA4 A29YL104E-Ig is substantially free of MCP-1, wherein exists in HIC eluate liquid less than 50,45,40,38,35 or 30ng/mL MCP-1.In further embodiment, the invention provides and be used for from liquid cell culture purifying CTLA4 A29YL104EThe method of-Ig molecule, thus the CTLA4 of purifying made A29YL104-Ig is substantially free of MCP-1 and comprises less than 2.5%CTLA4 A29YL104-Ig the tetramer.
From cell culture, reclaim glycoprotein and purifying
The invention describes and be used for from comprising purpose glycoprotein (for example CTLA4-Ig or CTLA4 A29YL104E-Ig) with in impure, the cell culture supernatant liquid of undesirable pollutent, the protein library separate glycoprotein (for example CTLA4-Ig or CTLA4 A29YL104E-Ig) series of steps.Impure, cell culture supernatant liquid can be used for purifying CTLA4-Ig or CTLA4 as starting material A29YL104E-Ig glycoprotein.
In one embodiment of the invention, impure, the cell culture supernatant liquid that will comprise CTLA4-Ig glycoprotein and undesirable pollutent is applied to anionic exchange medium.The CTLA4-Ig glycoprotein that is present in impure, the cell culture supernatant liquid combines with anionic exchange medium.Anionic exchange medium washs subsequently, to remove any unconjugated material from anionic exchange medium.CTLA4-Ig glycoprotein carries out wash-out after removing unconjugated material, and collects eluate.
In one embodiment of the invention, will comprise CTLA4 A29YL104EImpure, the cell culture supernatant liquid of-Ig glycoprotein and undesirable pollutent are applied to affinity chromatography medium.Be present in the CTLA4 in impure, the cell culture supernatant liquid A29YL104E-Ig glycoprotein combines with affinity chromatography medium.Affinity chromatography medium washs subsequently, to remove any unconjugated material from anionic exchange medium.CTLA4 A29YL104E-Ig glycoprotein carries out wash-out after removing unconjugated material, and collects eluate.
In specific embodiments of the present invention, for example use the Q-Sepharose anion-exchange chromatography (AEC) of Q-Sepharose XL post (GEHealthcare), be applied to make CTLA4-Ig glycoprotein and results material separation, and be used to reduce a large amount of pollutents.This post can be used for the cell culture medium of fractional separation results as from the early stage step in the CTLA4-Ig glycoprotein purifying of mammalian cell cultures.In another embodiment, the Q-Sepharose anion-exchange chromatography is Q-Sepharose Fast Flow (GE Healthcare) for example, can use after the affinitive layer purification step.The very high mobile character of anion-exchange column allows in follow-up chromatographic step for example before SP-Sepharose or the HIC, easily concentrate the cell culture medium of a large amount of CTLA4-Ig glycoprotein or results by regularization condition, thereby make CTLA4-Ig glycoprotein combine with post.For about pH5-9, the lavation buffer solution of about especially 8 pH, 75mM HEPES and 360mM NaCl concentration are useful.Usually, for about pH5-8, the elution buffer of about especially 7 pH, 25mM HEPES and 325mM NaCl concentration are useful.
The isolating appropriate resin of substratum that is used to make CTLA4-Ig glycoprotein and results is to have those of immobilized amine functional group.Quaternary amine functional group resin the most usefully, for example from the Q-Sepharose Fast Flow resin of GEHealthcare those, wherein the quaternary ammonium part combines with high porosity, Sepharose.Equally usefully primary, the second month in a season and tertiary amine functional group resin, for example from the DEAE Sepharose Fast Flow resin of GE Healthcare those, wherein uncle's diethyllaminoethyl part combines with high porosity, Sepharose.
In another embodiment of the invention, collect from the eluate that comprises CTLA4-Ig glycoprotein of anionic exchange medium, and contact with the hydrophobic interaction resin.As described below, the volume that comprises CTLA4-Ig glycoprotein is through the HIC post, and the storehouse of the collection that can be further purified combines with anionite-exchange resin subsequently.
HIC is used to make required CTLA4-Ig glycoprotein dimer to separate with other protein impurities from mammalian cell cultures with high molecular weight material.For example, the Chinese hamster ovary celI culture of expression CTLA4-Ig comprises the high molecular gathering thing of CTLA4-Ig glycoprotein.What also find in the mammalian cell substratum is the Chinese hamster ovary celI protein impurities.These undesirable products can produce unwanted antigen and reply and facilitate weak product quality or activity in the patient.HIC makes hydrophobic variant CTLA4-Ig glycoprotein dimer effectively separate with the CHO protein impurities with CTLA4-Ig glycoprotein h MW mixture, and it is via product and the HIC resin-bonded and the realization of CTLA4-Ig glycoprotein dimer process post of back.Therefore, can obtain to be substantially free of these kinds and be particularly suitable for for example CTLA4-Ig glycoprotein storehouse of anion-exchange chromatography of another chromatographic step.The source that is used for the CTLA4-Ig glycoprotein mixture that uses with HIC is a mammalian cell cultures, for example the Chinese hamster ovary celI culture.Especially, can implement foregoing at least one previous purification step to culture.
In another embodiment of the invention, the HIC method can be made amendment, to collect the storehouse of other glycoprotein (for example, CTLA4-Ig HMW mixture).The HMW aggregation can with HIC resin-bonded (for example, comprising the CTLA4-Ig tetramer etc.).These HMW mixtures have higher avidity and in vivo than independent more effectively combination of CTLA4-Ig dimer.Therefore, those skilled in the art can obtain the storehouse of CTLA4-Ig HMW aggregation by wash-out CTLA4-Ig storehouse from HIC.
The most useful HIC resin that is used for separation of C TLA4-Ig glycoprotein form is to have those of immobilized phenyl functional group.In phenyl-HIC resin, the PhenylSepharose Fast Flow HighSub of GE Healthcare (high displacement) is the most useful.The PhenylToyopearl medium of TosoHaas and TSK Phenyl 5PW are the non-limitative examples of operable other phenyl-HIC resin.Other HIC functional groups include but not limited to, propyl group, octyl group, alkoxyl group, butyl and isopentyl part.
Phenyl Sepharose 4 Fast Flow column chromatographies for example, hydrophobic interaction chromatography (HIC) method can be used for reducing CTLA4-Ig or the CTLA4 at HIC purification step wash-out A29YL104EThe amount of-Ig high molecular weight species (referring to embodiment 15 and embodiment 20).Therefore, the removing peak from the HIC post is rich in CTLA4-Ig or CTLA4 A29YL104E-Ig HMW kind.
Can to from the HIC purification step comprise CTLA4-Ig glycoprotein implement other purification process, affinity chromatography for example, and resulting eluate can be applied to anionic exchange medium subsequently in conjunction with fraction.CTLA4-Ig glycoprotein combines with anionite-exchange resin, and described anionite-exchange resin can wash subsequently to remove unconjugated protein.After removing unconjugated protein, CTLA4-Ig glycoprotein carries out wash-out from second kind of anionite-exchange resin.Eluate is collected and can be carried out further concentrating.
In another embodiment of the invention, affinity chromatography is rProtein A SepharoseFast Flow (GE Healthcare) for example, be applied to make the further enrichment of CTLA4-Ig glycoprotein, this can further be the anion-exchange chromatography step subsequently, for example Q-Sepharose Fast Flow (GE Healthcare).The affinity chromatography step can also reduce the level of CHO protein and MCP (MCP-1, chemokine) impurity.Affinity chromatography comprises fractionation by adsorption, and molecules of interest wherein to be purified is CTLA4-Ig glycoprotein for example, be immobilized in some matrix or resin on the ligand specificity and reversibly combine.Some non-limitative examples of affinity purification post comprise lectin; Affinity tag (for example, GST post or 6X-His post); Streptavidin; Heparin; Or antibody (for example, A albumen post or G albumen post).Especially, the present invention utilizes the A protein resin to be used for the glycoprotein in conjunction with CTLA4-Ig.For the about 5-9 of pH, more effective lavation buffer solution under pH about 8,25mM Tris and 250mM NaCl concentration are useful.For the about 2-5 of pH, more effective elution buffer under pH about 3.5, the 100mM glycine concentration is useful.The affinity chromatography eluate can neutralize and be loaded into subsequently on the anion-exchange chromatography post, and Q-Sepharose Fast Flow is the most useful.
In order further to reduce the level of A albumen, DNA and unwanted CTLA4-Ig glycoprotein kind in the product behind aforementioned recovery/initial purification step, another ion-exchange step can be mixed in the purifying procedure.The present invention can use the ion exchange column that is obtained commercially, for example from the Q-Sepharose Fast Flow post of GE Healthcare or equally from the DEAE Sepharose Fast Flow post of GE Healthcare.Determine that as this paper the isolating only resin of substratum that is used to make CTLA4-Ig glycoprotein and results is to have those of immobilized amine functional group.Other useful groups are quaternary amine functional group resins, and for example from the Q-Sepharose Fast Flow post of GE Healthcare those, wherein the quaternary ammonium part combines with high porosity, Sepharose.Equally usefully primary, the second month in a season and tertiary amine functional group resin, for example from the DEAE Sepharose Fast Flow post of GEHealthcare those, wherein uncle's diethyllaminoethyl part combines with high porosity, Sepharose.In specific embodiments of the present invention, utilize the post that uses strong anion exchanger, for example Q-Sepharose Fast Flow column post.
In one embodiment of the invention, CTLA4-Ig glycoprotein eluate is loaded on the anion-exchange column, for example Q-Sepharose Fast Flow.This post washs and CTLA4-Ig glycoprotein wash-out from anion-exchange column subsequently.For the about 5-9 of pH, in one embodiment, the lavation buffer solution of pH8,25mM HEPES and 100-140mM NaCl concentration are useful.For the about 5-9 of pH, or in another embodiment, the elution buffer of pH8,25mM HEPES and 200mM NaCl concentration are useful.The CTLA4-Ig glycoprotein of wash-out reclaims, concentrates and washing from anionic exchange medium, and it is realized by diafiltration or other appropriate method well known by persons skilled in the art, so that the CTLA4-Ig glycoprotein product of final purifying to be provided.CTLA4-Ig glycoprotein product according to method of the present invention preparation has high purity, for example comprises 〉=95% CTLA4-Ig dimer, comprises≤5% CTLA4-Ig HMW product and comprising≤1% CTLA4-Ig monomer.
Purification process may further include deactivation and/or removes virus and/or retroviral other step, and described virus and/or retrovirus may be present in the cell culture medium of mammal cell line potentially.A large amount of virus sweep steps are available, include but not limited to, handle with chaotropic agent, for example urea or guanidine, stain remover, other ultrafiltration/diafiltration step, the conventional separation, for example ion-exchange or size exclusion chromatography, pH is extreme, heat, proteolytic enzyme, organic solvent or its any combination.
In another embodiment of the invention, affinity chromatography for example MabSelect Protein ASepharose resin (GE Healthcare) is applied to catch CTLA4 A29YL104E-Ig glycoprotein, this can further be the anion-exchange chromatography step subsequently, for example Q-Sepharose Fast Flow (GE Healthcare).The affinity chromatography step can also reduce the level of CHO protein and MCP (MCP-1, chemokine) impurity.Affinity chromatography comprises fractionation by adsorption, and molecules of interest wherein to be purified is CTLA4 for example A29YL104E-Ig glycoprotein, be immobilized in some matrix or resin on the ligand specificity and reversibly combine.Some non-limitative examples of affinity purification post comprise lectin; Affinity labelling (for example, GST post or 6X-His post); Streptavidin; Heparin; Or antibody (for example, A albumen post or G albumen post).Especially, the present invention utilizes the A protein resin to be used in conjunction with CTLA4 A29YL104E-Ig glycoprotein.For the about 5-9 of pH, more effective lavation buffer solution under pH about 7.5,25mM Tris, 25mM NaH 2PO 4With 250mM NaCl concentration be useful.For the about 2-5 of pH, more effective elution buffer under pH about 3.5, the 100-300mM glycine concentration is useful.The affinity chromatography eluate can neutralize and be loaded into subsequently on the anion-exchange chromatography post, and Q-Sepharose Fast Flow is the most useful.
In order further to reduce A albumen, DNA and unwanted CTLA4 in the product behind aforementioned recovery/initial purification step A29YL104EThe level of-Ig glycoprotein kind can be mixed another ion-exchange step in the purifying procedure.The present invention can use the ion exchange column that is obtained commercially, for example from the Q-Sepharose Fast Flow post of GE Healthcare, Q-Sepharose XL post (GEHealthcare) or equally from the DEAE Sepharose Fast Flow post of GE Healthcare.Be used for separation of C TLA4 A29YL104EThe only resin of-Ig glycoprotein is to have those of immobilized amine functional group.Other useful groups are quaternary amine functional group resins, and for example from the Q-Sepharose Fast Flow post of GEHealthcare those, wherein the quaternary ammonium part combines with high porosity, Sepharose.Equally usefully primary, the second month in a season and tertiary amine functional group resin, for example from the DEAE Sepharose Fast Flow post of GE Healthcare those, wherein uncle's diethyllaminoethyl part combines with high porosity, Sepharose.In specific embodiments of the present invention, utilize the post that uses strong anion exchanger, for example Q-Sepharose Fast Flow column post.
In one embodiment of the invention, with CTLA4 A29YL104E-Ig glycoprotein eluate is loaded on the anion-exchange column, for example Q-Sepharose Fast Flow.This post washs and CTLA4 A29YL104E-Ig glycoprotein is wash-out from anion-exchange column subsequently.For the about 5-9 of pH, in one embodiment, the lavation buffer solution of pH7,25-55mM HEPES and 100-140mM NaCl concentration are useful.For the about 5-9 of pH, or in another embodiment, the elution buffer of pH7,25-50mM HEPES and 200mM NaCl concentration are useful.
In another embodiment of the invention, collect from the CTLA4 that comprises of anionic exchange medium A29YL104EThe eluate of-Ig glycoprotein, and contact with the hydrophobic interaction resin subsequently.HIC is used to make required CTLA4 A29YL104E-Ig glycoprotein dimer separates with other protein impurities from mammalian cell cultures with high molecular weight material.For example, express CTLA4 A29YL104EThe Chinese hamster ovary celI culture of-Ig comprises CTLA4 A29YL104EThe high molecular of-Ig glycoprotein is assembled thing.What also find in the mammalian cell substratum is the Chinese hamster ovary celI protein impurities.These undesirable products can produce unwanted antigen and reply and facilitate weak product quality or activity in the patient.
HIC makes hydrophobic variant CTLA4 A29YL104E-Ig glycoprotein dimer and CTLA4 A29YL104E-Ig glycoprotein h MW mixture effectively separates with the CHO protein impurities, and it is via product and the HIC resin-bonded and the CTLA4 of back A29YL104E-Ig glycoprotein dimer is realized through post.Therefore, can obtain to be substantially free of the CTLA4 of these kinds A29YL104E-Ig glycoprotein storehouse.Be used for the CTLA4 that uses with HIC A29YL104EThe source of-Ig glycoprotein mixture is a mammalian cell cultures, for example the Chinese hamster ovary celI culture.Especially, can implement foregoing at least one previous purification step to culture.
In another embodiment of the invention, the HIC method can be made amendment, to collect other glycoprotein (for example, CTLA4 A29YL104E-Ig HMW mixture) storehouse.The HMW aggregation can (for example, comprise CTLA4 with the HIC resin-bonded A29YL104E-Ig the tetramer etc.).These HMW mixtures have higher avidity and in vivo than independent CTLA4 A29YL104EThe more effectively combination of-Ig dimer.Therefore, those skilled in the art are by wash-out CTLA4 from HIC A29YL104E-Ig storehouse can obtain CTLA4 A29YL104EThe storehouse of-Ig HMW aggregation.
The CTLA4 of wash-out from the HIC medium A29YL104E-Ig glycoprotein reclaims, concentrates and washing, and it is realized by diafiltration or other appropriate method well known by persons skilled in the art, so that the CTLA4 of final purifying to be provided A29YL104E-Ig glycoprotein product.CTLA4 according to method preparation of the present invention A29YL104E-Ig glycoprotein product has high purity, for example comprises 〉=95% CTLA4 A29YL104E-Ig dimer comprises≤5% CTLA4 A29YL104EThe CTLA4 of-Ig HMW product and comprising≤1% A29YL104E-Ig monomer.
Be used for separation of C TLA4 A29YL104EThe most useful HIC resin of-Ig glycoprotein form is to have those of immobilized phenyl functional group.In phenyl-HIC resin, the Phenyl Sepharose Fast Flow High Sub of GE Healthcare (high displacement) is the most useful.The Phenyl Toyopearl medium of TosoHaas and TSK Phenyl 5PW are the non-limitative examples of operable other phenyl-HIC.Other HIC functional groups include but not limited to, propyl group, octyl group, alkoxyl group, butyl and isopentyl part.
Purification process may further include deactivation and/or removes virus and/or retroviral other step, and described virus and/or retrovirus may be present in the cell culture medium of mammal cell line potentially.A large amount of virus sweep steps are available, include but not limited to, handle with chaotropic agent, for example urea or guanidine, stain remover, other ultrafiltration/diafiltration step, the conventional separation, for example ion-exchange or size exclusion chromatography, pH is extreme, heat, proteolytic enzyme, organic solvent or its any combination.
In one aspect, the CTLA4-Ig molecule that has concentrated and implemented the purifying of diafiltration steps can be filled into 2-L
Figure A200680053116D02151
In bottle, 50-L biological processing bag or any other suitable containers.CTLA4-Ig molecule in this kind container can be in about 60 days of 2 ℃-8 ℃ storages before freezing.The CTLA4-Ig of purifying can cause the tetrameric ratio of CTLA4-Ig to increase in 2 ℃-8 ℃ prolongation storage.Therefore, for Long-term Storage, the CTLA4-Ig molecule can be frozen in-70 ℃ of pacts and preserve in-40 ℃ approximately before storage.Freezing temp can be from-50 ℃-Yue-90 ℃ variation approximately.Freezing time can change, and depends on the number of the container that loads in the volume of a container that comprises the CTLA4-Ig molecule and the refrigerator to a great extent.For example, in one embodiment, the CTLA4-Ig molecule is at 2-L
Figure A200680053116D02152
In the bottle.In refrigerator, load less than 4 2-L Bottle may need at least 18 hours freezing time of about 14-.The loading of at least 4 bottles may need at least 24 hours freezing time of about 18-.Container with refrigerated CTLA4-Ig molecule is preserved in about-35 ℃-Yue-55 ℃ temperature.
Storage time in about-35 ℃-Yue-55 ℃ temperature can change and may be as little to 18 hours.Refrigerated CTLA4-Ig molecule can thaw with control mode.Thawing of refrigerated CTLA4-Ig molecule is controlled, and can finish in about 20 ℃-Yue 24 ℃ temperature in incubator.The time length of the step of thawing is depended on the loading of incubator, wherein less than 4 2-L
Figure A200680053116D02161
The loading of bottle may be less than about 24 hours thawing time.4 2-L
Figure A200680053116D02162
The loading of bottle may need about 18 hours.The solution that thaws that comprises the CTLA4-Ig molecule can mix, to avoid possible concentration gradient.Therefore, thaw and can finish in the controlled temperature incubator, described controlled temperature incubator also allows to comprise the vibration of the container of CTLA4-Ig.Hunting speed can be the about 80rpm of about 40-.The CTLA4-Ig molecule that thaws can further mix other 5-10 minute with the speed of rotation of about 3rpm.The CTLA4-Ig molecule that thaws can be preserved in 2 ℃-8 ℃, carries out stratification (alequated) and freeze-drying at the production period of the pharmaceutical composition that comprises CTLA4-Ig.
The present invention can further be applied to the treatment glycoprotein of scale operation other, the purifying of non-limitative example.Method of the present invention can be applied to have the production of other glycoprotein that surpass a kind of glycosylation variant in mammalian cell cultures.It will be appreciated by those skilled in the art that in the process that is modified at the proteic production of example methodology adaptation different sugar and may become necessary.
Preparation and test kit
The present invention also provides any described CTLA4-Ig molecule as freeze-dried mixture.Comprise the preparation for the treatment of freeze dried CTLA4-Ig and can further comprise 3 kinds of basal components: one or more activeconstituentss that (1) is other, comprise other recombinant proteins or small molecules (for example immunosuppressor), (2) one or more vehicle and (3) one or more solvents.Vehicle comprises pharmaceutically acceptable reagent; with good lyophilized cake character (weighting agent) is provided and be provided at the proteinic cryoprotection of duration of storage (lyoprotection) and/or cryoprotection (" stablizer "), pH keep (buffer reagent) and proteinic correct conformation; thereby make that the basic reservation of biological activity (comprising activeconstituents stability, for example protein stability) is kept.About vehicle, the example of preparation can comprise one or more in one or more buffer reagents, one or more weighting agents, one or more protein stabilizing agents and one or more biocides.Sugar or polyvalent alcohol can be as the nonspecific proteins matter stablizers in solution and during freeze thawing and lyophilize.Polymkeric substance can be used for being stabilized in solution and the protein during freeze thawing and lyophilize.A kind of general polymkeric substance is a serum albumin, and this is as cryoprotection agent and cryoprotectant.In one embodiment, the invention provides and do not contain albuminised preparation.Various salt can be used as weighting agent.Illustrative salt weighting agent comprises for example NaCl, MgCl 2And CaCl 2Some amino acid can be used as cryoprotection agent and/or cryoprotectant and/or weighting agent.Operable amino acid includes but not limited to, glycine, proline(Pro), 4-Hydroxyproline, L-Serine, Sodium Glutamate, L-Ala, arginine and lysine hydrochloride.The many buffer reagents that comprise the wide pH value scope can be used for selecting in preparation.Buffer reagent for example comprises, acetate, Citrate trianion, glycine, Histidine, phosphoric acid salt (sodium or potassium), diethanolamine and Tris.Buffer reagent is included in and makes before the freeze-drying pH value of solution maintain those reagent in the tolerance interval.Obtain describing in the Application No. 60/752,150 that preparation before had been to submit on December 20th, 2005, described patent document is incorporated herein by reference in this integral body.
In one embodiment, the invention provides and comprise at least 90%, 95%, 99% or 99.5% the dimeric freeze dried CTLA4-Ig mixture of CTLA4-Ig.In one embodiment, the invention provides freeze dried CTLA4-Ig mixture, it comprises at least 90%, 95%, 99% or 99.5% CTLA4-Ig dimer and is no more than 5%, 4%, 3%, 2% or 1% the CTLA4-Ig tetramer.In another embodiment, the invention provides freeze dried CTLA4-Ig mixture, it comprises at least 90%, 95%, 99% or 99.5% CTLA4-Ig dimer, with be no more than 5%, 4%, 3%, 2% or 1% the CTLA4-Ig tetramer and be no more than 2%, 1.5%, 1.0%, 0.8%, 0.5% or 0.3% CTLA4-Ig monomer.In further embodiment, the invention provides freeze dried CTLA4-Ig mixture, it comprises at least 8.0 moles of sialic acids/mole CTLA4-Ig dimer or to the CTLA4-Ig molecule.In another embodiment, the invention provides freeze dried CTLA4-Ig mixture, it comprises: about 35 moles of GlcNac/ mole CTLA4-Ig molecules of about 15-or dimer; About 5 moles of GalNac/ mole CTLA4-Ig dimers of about 1-or to the CTLA4-Ig molecule; About 5 moles-Yue 20 moles of semi-lactosis/mole CTLA4-Ig dimer or to the CTLA4-Ig molecule; The about 10 moles of Fucoses of about 2-/mole CTLA4-Ig dimer or to the CTLA4-Ig molecule; And/or about 5-15 mole seminose/mole CTLA4-Ig dimer or to the CTLA4-Ig molecule.
CTLA4 A29YL104E-Ig medicine can be used as concentration with about 25mg/mL (22.5-27.5mg/mL) and is dissolved in 25mM sodium phosphate under pH～7.5 and the aqueous solution utilization in the 10mM sodium-chlor damping fluid.CTLA4 A29YL104E-Ig has the trend that forms high molecular weight species in the aqueous solution.Therefore, developed lyophilisation product, so that the level of the high molecular weight species that may form in the medicament production drops to is minimum.Various vehicle for example maltose, sucrose and amino acid for example the L-arginine monohydrochloride as CTLA4 A29YL104ECryoprotectant possible during the lyophilize of-Ig screens.Find that sucrose is the most effective cryoprotectant.Further observe increase sucrose and improve protein stability with the protein ratio.Select the sucrose of 2:1 (wt.:wt.): the protein ratio is used to treat cryodesiccated protein soln.Cryodesiccated medicament production has enough stability and satisfied structure behavior.
Methods of treatment
According to the present invention, can be by the disease of the interaction of T cell and B7 positive cell mediation by accepting CTLA4-Ig or CTLA4 A29YL104EThe pharmaceutically acceptable preparation of-Ig is treated.(for example, have coding CTLA4-Ig or CTLA4 by engineered mammal cell line A29YL104EThe negative Chinese hamster ovary line of the dhfr-of the DNA of-Ig) excretory CTLA4-Ig or CTLA4 A29YL104E-Ig molecule can be the molecule colony with specific glycosylation overview.As described herein, specific glycosylation overview can influence CTLA4-Ig or CTLA4 A29YL104E-Ig combines with CD80 and/or CD86's, thereby makes CTLA4-Ig or CTLA4 A29YL104E-Ig molecule can provide T cell activation and/or the bigger inhibition of propagation.As described herein, specific glycosylation overview can be subjected to the influence of clone and production method.Therefore, in certain embodiments of the invention, the invention provides the CTLA4-Ig or the CTLA4 that in production method described herein, produce by clone A29YL104E-Ig molecule, with cell-mediated disease or the illness of treatment T, it includes but not limited to, usually any T cell dependency lymphocytic hyperplasia disease or illness and any T cell dependency autoimmune disease or illness, and more specifically: t cell lymphoma, T cell acute lymphoblastic leukemia, testicular vessel centrality t cell lymphoma, optimum lymphocytic vasculitis, graft versus host disease (GVH disease) (GVHD), repel relevant immune disorders with graft transplantation, psoriasis, inflammation, transformation reactions, ovaritis, inflammatory bowel, glomerulonephritis, encephalomyelitis, the HashimotoShi thyroiditis, the GravesShi disease, the AddisonShi disease, the CrohnShi disease, the Sjogren Cotard, lupus erythematosus, the primary solid edema, pernicious anemia, the autoimmunity atrophic gastritis, rheumatoid arthritis, insulin-dependent diabetes, good pasture Cotard, myasthenia gravis, pemphigus, multiple sclerosis, sympathetic ophthalmia, the autoimmunity uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic active hepatitis, ulcerative colitis, scleroderma, polymyositis and MCTD.
The invention provides disclosed CTLA4-Ig or CTLA4 A29YL104EAny purposes in following method in the-Ig molecule: be used for suppressor T cell propagation or activation, the experimenter or in the vitro inhibition immunne response be used for the treatment of the immune disorders among the experimenter or in the experimenter, induce at antigenic immunological tolerance.Immunological tolerance is wherein to develop the type of Lymphoid tissue at the special non-reacted immunne response of specific antigen, and wherein under the situation that does not have tolerance, antigen can induce immune response.In one embodiment, CTLA4-Ig of the present invention or CTLA4 A29YL104E-Ig molecule and composition can be used for the treatment of has accepted the experimenter that transplants, to induce tolerance and to reduce the possibility of repelling.In another embodiment, transplanting is organ transplantation, tissue transplantation or Transplanted cells.In another embodiment, Transplanted cells comprises medullary cell or islet cells.
The invention provides disclosed CTLA4-Ig or CTLA4 A29YL104EAny in the-Ig molecule is used for the treatment of purposes in the medicine of any above-mentioned disease or illness in preparation.The present invention also provides disclosed CTLA4-Ig or CTLA4 A29YL104EAny and another kind of reagent in-the Ig molecule is used the purposes that is used for the treatment of above-mentioned disease or illness altogether.CTLA4-Ig of the present invention or CTLA4 A29YL104E-Ig molecule is intravenously, subcutaneous and/or be applied to the experimenter by suction for example.Can be applicable to the CTLA4-Ig or the CTLA4 of intravenously or subcutaneous administration A29YL104EObtain describing in the U.S. serial 60/752,150 that-Ig preparation is to submit on December 20th, 2005, described patent document is incorporated herein by reference in this integral body.CTLA4-Ig or CTLA4 A29YL104E-Ig preparation can also comprise the preparation based on liposome, and wherein liposome can be with CTLA4-Ig or CTLA4 A29YL104E-Ig molecule is delivered to target cell or tissue.CTLA4-Ig or CTLA4 A29YL104E-Ig molecule can also be delivered to target cell or tissue by using virus vector, and described virus vector comprises CTLA4-Ig or CTLA4 A29YL104E-Ig expression casette.CTLA4-Ig or CTLA4 A29YL104E-Ig molecule colony use with dosage at the U.S. Patent application that is disclosed as US20030083246 and US20040022787, and the pendent U.S. Patent Application Serial of submitting on April 6th, 2,005 60/668, obtain in 774 describing, described all patent documents are incorporated herein by reference in this integral body.
CTLA4-Ig described herein or CTLA4 A29YL104E-Ig molecule can be multiple formulation, and described formulation includes but not limited to, but the solution of liquor or suspension, tablet, pill, powder, suppository, polymerization microcapsule or microvesicle, liposome and injectable or infusion.Form depends on mode of administration and treatment application.Depend on the healthy of severity of disease and the course of disease, experimenter and to the judgement of replying and treating the doctor of treatment about effective mode of administration of molecule of the present invention and dosage.Therefore, the dosage of molecule is tackled indivedual experimenters and is carried out titration.Based on mg/m 2Surface-area about the dosage mutual relationship of the animals and human beings of all size and species by Freireich, E.J. wait people (Quantitative Comparison of Toxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man. Cancer Chemother, Rep., 50, No.4,219-244, May 1966) be described.Can carry out the adjustment in the dosage, suppress to reply with optimized growth.
Dosage can be with the basis of every day separately and use, or dosage can depend on situation and reduces in proportion.For example, broken dose can be used every day or every month several times, or dosage can be as the minimizing in proportion by concrete treatment situation indication.In one embodiment, using is every month, per season, every day, 1 day 2 times, per approximately 10 hours, per approximately 6 hours, per approximately 4 hours, per approximately 2 hours, about 1 hour 1 time.According to practice of the present invention, the significant quantity that is used for the treatment of the experimenter can be the about 10mg/kg experimenter's body weight of about 0.1-.Equally, significant quantity can be the amount of the about 10mg/kg experimenter's body weight of about 1-.CTLA4-Ig of the present invention or CTLA4 A29YL104E-Ig molecule also has clinical application in the body.They can be used for the monitoring that immunology changes after counting, leukemia and the lymphadenomatous phenotype analytical and the organ transplantation of the diagnosis of some immune deficiency situations or prognosis B7 positive cell.
Sending of composition described herein can mouthful be sent via injection, footpath, suction, subcutaneous injection, the intravenously of sprays or other particular dispersion are sent, local delivery, suppository, eye are sent, nose or mouth are sent and reached.Composition can be sent in liposome or other film sample delivery vehicle via tunicaization.Composition can be sent via blood or other fluids, described blood or other fluids before handled with composition and the experimenter that infuses subsequently in.
Sequence table
SEQ ID NO:17 is the nucleotide sequence of coding pcSDhuCTLA4Ig:
Figure A200680053116D02211
Figure A200680053116D02221
Figure A200680053116D02231
Figure A200680053116D02241
Figure A200680053116D02251
SEQ ID NO:18 is the aminoacid sequence of the ectodomain of human CTLA 4.
Further non-limiting embodiments:
The invention provides clone's Chinese hamster ovary cell colony that can produce CTLA4-Ig.In one embodiment, cell colony can be produced and surpass 0.5 or more gram CTLA4-Ig protein/L liquid culture, and wherein CTLA4-Ig is 1, shows sialic acid and the dimeric mol ratio of CTLA4-Ig of about 6-about 14 when 000L or bigger cultivation scale.In one embodiment, cell colony has adapted to serum-free, chemical ingredients knows substratum.In another embodiment, cultivate the CTLA4-Ig that produces by cell colony and have 1.00 ± 0.05AU mL cm -1Mg -1Optical extinction coefficient.In further embodiment, when growing in culture, cell colony can be produced the CTLA4-Ig polypeptide, and wherein: (a) about 90% CTLA4-Ig polypeptide comprises the aminoacid sequence of the SEQ ID NO:2 that begins from the methionine(Met) of residue 27; (b) about 10% CTLA4-Ig polypeptide comprises the aminoacid sequence of the SEQ ID NO:2 that begins from the L-Ala of residue 26; (c) about 4% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the Methionin at 383 places finishes with residue; (d) about 96% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the glycine at 382 places finishes with residue; Randomly, (e) comprise the aminoacid sequence of numbering the SEQ ID NO:2 that 25 methionine(Met) begins from residue less than 1% CTLA4-Ig polypeptide approximately.
The invention provides the progeny cell of above-described cell, wherein said progeny cell is produced CTLA4-Ig.In one embodiment, progeny cell is obtained through at least 5 generations by culturing cell.In another embodiment, progeny cell is obtained through at least 10 generations by culturing cell.In another embodiment, progeny cell is obtained through at least 20 generations by culturing cell.In another embodiment, progeny cell is obtained through at least 40 generations by culturing cell.In another embodiment, progeny cell is obtained through at least 50 generations by culturing cell.In another embodiment, progeny cell is obtained through at least 75 generations by culturing cell.In another embodiment, progeny cell is obtained through at least 100 generations by culturing cell.
The invention provides clone by any production in the above-described cell.In one embodiment, clone is cloned.In another embodiment, clone can be produced: the CTLA4-Ig fusion rotein that (a) has the aminoacid sequence (methionine(Met) at amino acid position 27 places of SEQ ID NO:2 and the glycine at amino acid position 382 places) of SEQ ID NO:8; (b) has the CTLA4-Ig fusion rotein of the aminoacid sequence (methionine(Met) at amino acid position 27 places of SEQ ID NO:2 and the Methionin at amino acid position 383 places) of SEQ ID NO:5; (c) has the CTLA4-Ig fusion rotein of the aminoacid sequence (L-Ala at amino acid position 26 places of SEQ ID NO:2 and the glycine at amino acid position 382 places) of SEQ ID NO:7; (d) has the CTLA4-Ig fusion rotein of the aminoacid sequence (L-Ala at amino acid position 26 places of SEQ ID NO:2 and the Methionin at amino acid position 383 places) of SEQ ID NO:4; (e) has the CTLA4-Ig fusion rotein of the aminoacid sequence (methionine(Met) at amino acid position 25 places of SEQ ID NO:2 and the Methionin at amino acid position 383 places) of SEQ ID NO:4; Or (f) has a CTLA4-Ig fusion rotein of the aminoacid sequence (methionine(Met) at amino acid position 25 places of SEQ ID NO:2 and the glycine at amino acid position 382 places) of SEQ ID NO:6.
In another embodiment, clone can be produced the CTLA4-Ig fusion rotein, and wherein: (a) about 90% CTLA4-Ig polypeptide comprises the aminoacid sequence of the SEQID NO:2 that begins from the methionine(Met) of residue 27; (b) about 10% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that 26 L-Ala begins from residue; (c) about 4% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the Methionin at 383 places finishes with residue, and (d) about 96% CTLA4-Ig polypeptide comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the glycine at 382 places finishes with residue; Randomly, (e) comprise the aminoacid sequence of numbering the SEQ ID NO:2 that 25 methionine(Met) begins from residue less than 1% CTLA4-Ig polypeptide approximately.
In one embodiment, the CTLA4-Ig fusion rotein of being produced by culturing cell system has the optical extinction coefficient of 1.00 ± 0.05AU mL cm-1 mg-1.In one embodiment, the invention provides cell colony derived from cell of the present invention.In one embodiment, compare with initial cells transfected, cell colony is made up of at least a other hereditary change, and wherein the deutero-cell colony can be produced CTLA4-Ig.In other embodiments, compare with initial cells transfected, cell colony by at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26 kind of other hereditary change form, and wherein the deutero-cell colony can be produced CTLA4-Ig.In one embodiment, hereditary change be included in cellular genome or the coding CTLA4-Ig recombinant expression cassettes at least a non-conservative sudden change.
In one embodiment, hereditary change comprises intracellular at least a other recombinant nucleic acid.In one embodiment, change the sudden change that comprises cellular genome.In one embodiment, change comprises to cellular genome interpolation nucleic acid or as trans nucleic acid, described nucleic acid encoding anti-apoptotic polypeptide.In one embodiment, the anti-apoptotic polypeptide relates to glycosylation.
In one embodiment, hereditary change comprises at least a sudden change of the recombinant expression cassettes of cellular genome or coding CTLA4-Ig.In one embodiment, when growing in culture, cell colony can be produced: the CTLA4-Ig fusion rotein that (a) has the aminoacid sequence (methionine(Met) at amino acid position 27 places of SEQ ID NO:2 and the glycine at amino acid position 382 places) of SEQ ID NO:8; (b) has the CTLA4-Ig fusion rotein of the aminoacid sequence (methionine(Met) at amino acid position 27 places of SEQ IDNO:2 and the Methionin at amino acid position 383 places) of SEQ ID NO:5; (c) has the CTLA4-Ig fusion rotein of the aminoacid sequence (L-Ala at amino acid position 26 places of SEQ IDNO:2 and the glycine at amino acid position 382 places) of SEQ ID NO:7; (d) has the CTLA4-Ig fusion rotein of the aminoacid sequence (L-Ala at amino acid position 26 places of SEQ IDNO:2 and the Methionin at amino acid position 383 places) of SEQ ID NO:4; (e) has the CTLA4-Ig fusion rotein of the aminoacid sequence (methionine(Met) at amino acid position 25 places of SEQ ID NO:2 and the Methionin at amino acid position 383 places) of SEQ ID NO:4; Or (f) has a CTLA4-Ig fusion rotein of the aminoacid sequence (methionine(Met) at amino acid position 25 places of SEQ IDNO:2 and the glycine at amino acid position 382 places) of SEQ ID NO:6.
The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 6-about 18 and the dimeric molar average ratio of CTLA4-Ig.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 8-about 18 and the dimeric molar average ratio of CTLA4-Ig.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 11-about 18 and the dimeric molar average ratio of CTLA4-Ig.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 12-about 18 and the dimeric molar average ratio of CTLA4-Ig.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 13-about 18 and the dimeric molar average ratio of CTLA4-Ig.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 14-about 18 and the dimeric molar average ratio of CTLA4-Ig.The invention provides the CTLA4-Ig molecule colony of sialic acids groups with about 15-about 17 and the dimeric molar average ratio of CTLA4-Ig.The invention provides CTLA4-Ig molecule colony with about 16 sialic acids groups and the dimeric molar average ratio of CTLA4-Ig.
The invention provides wherein that to surpass 95% molecule be the dimeric CTLA4-Ig molecule of CTLA4-Ig colony.In one embodiment, surpassing 98% molecule is the CTLA4-Ig dimer.In one embodiment, surpassing 99% molecule is the CTLA4-Ig dimer.In one embodiment, surpassing 99.5% molecule is the CTLA4-Ig dimer.In one embodiment, the molecule of about 95%-about 99.5% is the CTLA4-Ig dimer, and the molecule of about 0.5%-about 5% is the CTLA4-Ig tetramer.In one embodiment, about 98.6% molecule is the CTLA4-Ig dimer, and about 1.2% molecule is the CTLA4-Ig tetramer, and is the CTLA4-Ig monomer less than 0.7% molecule approximately.The invention provides the colony that forms by the CTLA4-Ig dimer.The invention provides wherein, colony is substantially free of the monomeric CTLA4-Ig molecule of CTLA4-Ig colony.The invention provides wherein, colony is substantially free of the tetrameric CTLA4-Ig molecule of CTLA4-Ig colony.The invention provides and be substantially free of CTLA4-Ig dimer and tetrameric CTLA4-Ig monomer molecule colony.In one embodiment, every kind of dimeric each monomer of CTLA4-Ig has at least 3 sialic acids groups.
In one embodiment, every kind of dimeric each monomer of CTLA4-Ig has at least 3 sialic acids groups-at least 8 sialic acids groups.The invention provides the purifying colony of CTLA4-Ig tetramer molecule, described colony is substantially free of the CTLA4-Ig dimer, and randomly, wherein said colony comprises the amount that surpasses about 100 grams.The invention provides the purifying colony of CTLA4-Ig tetramer molecule, described colony is substantially free of the CTLA4-Ig monomer, and randomly, wherein said colony comprises the amount that surpasses about 100 grams.In one embodiment, each tetramer molecule comprises 2 pairs of CTLA4-Ig polypeptide, wherein each polypeptide has the aminoacid sequence that is selected from SEQ ID NOS:3-8, and wherein right each member and another member of polypeptide is covalently bound, and wherein 2 pairs of non-covalent each other combinations of polypeptide.In one embodiment, each tetramer molecule can combine with CD80 or CD86.In one embodiment, compare with the CTLA4-Ig dimer molecule, each tetramer molecule has at least 2 times big avidity for CD80 or CD86.In one embodiment, compare with the CTLA4-Ig dimer molecule, each tetramer molecule has at least 2 times big T cell proliferation or activation inhibition.
The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said composition is included in advantage isoform visual on the isoelectrofocusing gel of CTLA4-Ig, and as measuring by isoelectrofocusing, described isoform has and is less than or equal to 5.1 iso-electric point, pI.In one embodiment, pI handles the back at neuraminidase increases.In one embodiment, as measuring by isoelectrofocusing, at least 40% CTLA4-Ig molecule demonstration is less than or equal to about 5.1 iso-electric point.In one embodiment, as measuring by isoelectrofocusing, at least 70% CTLA4-Ig molecule demonstration is less than or equal to about 5.1 iso-electric point.In one embodiment, as measuring by isoelectrofocusing, at least 90% CTLA4-Ig molecule demonstration is less than or equal to about 2.5 iso-electric point.The invention provides the CTLA4-Ig molecule colony of the pI with about 2.0 ± 0.2-about 5.0 ± 0.2.The invention provides the CTLA4-Ig molecule colony of the pI with about 4.3 ± 0.2-about 5.0 ± 0.2.The invention provides the CTLA4-Ig molecule colony of the pI with about 3.3 ± 0.2-about 4.7 ± 0.2.The invention provides and be used to prepare method for compositions, described composition comprises the CTLA4-Ig molecule of the pI with about 2.0 ± 0.2-about 5.0 ± 0.2, described method comprises: (a) mixture of CTLA4-Ig molecule is implemented the isoelectrofocusing gel electrophoresis, wherein the single band on gel represents to have the CTLA4-Ig molecule colony of specific pI, (b) separate the CTLA4-Ig molecule colony of the pI with about 2.0 ± 0.2-about 5.0 ± 0.2, so that prepare composition.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 17-about 25.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 15-about 35.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that the dimeric molar average ratio of the GalNAc/ mole CTLA4-Ig of about 1.7-about 3.6.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that the dimeric molar average ratio of semi-lactosi/mole CTLA4-Ig of about 8-about 17.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that the dimeric molar average ratio of Fucose/mole CTLA4-Ig of about 3.5-about 8.3.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that the dimeric molar average ratio of seminose/mole CTLA4-Ig of about 7.2-about 22.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is characterised in that the dimeric molar average ratio of sialic acid/mole CTLA4-Ig of about 6-about 12.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 15-about 35; (b) the dimeric molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 15-about 35; (b) the dimeric molar average ratio of the GalNAc/ mole CTLA4-Ig of about 1.7-about 3.6; (c) the dimeric molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 15-about 35; (b) the dimeric molar average ratio of the GalNAc/ mole CTLA4-Ig of about 1.7-about 3.6; (c) the dimeric molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig; (d) the dimeric molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 15-about 35; (b) the dimeric molar average ratio of the GalNAc/ mole CTLA4-Ig of about 1.7-about 3.6; (c) the dimeric molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig; (d) the dimeric molar average ratio of the Fucose of about 3.5-about 8.3/mole CTLA4-Ig; (e) the dimeric molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig.The invention provides the composition that comprises the CTLA4-Ig molecule, it is characterized in that: (a) the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 15-about 35; (b) the dimeric molar average ratio of the GalNAc/ mole CTLA4-Ig of about 1.7-about 3.6; (c) the dimeric molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig; (d) the dimeric molar average ratio of the Fucose of about 3.5-about 8.3/mole CTLA4-Ig; (e) the dimeric molar average ratio of the seminose of about 7.2-about 22/mole CTLA4-Ig; (f) the dimeric molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig.
The invention provides the composition that comprises the CTLA4-Ig molecule, the NANA chromatogram peak of the NGNA chromatogram peak of the about 9.589+ of wherein said composition exhibiting/-0.3 and about 10.543+/-0.3.The invention provides the composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule shows carbohydrate overview as shown in Figure 7.The invention provides the composition that comprises the CTLA4-Ig molecule, the carbohydrate overview of wherein said CTLA4-Ig molecule display structure territory I-IV, wherein structural domain I comprises the peak of the oligosaccharides of representing asialoization, domain II comprises the peak of the oligosaccharides of representing mono-sialylated, domain II I comprises the peak of the oligosaccharides of representing double-sialylated, and structural domain IV comprises the peak of representing three sialylated oligosaccharides.In one embodiment, the difference in the retention time of N connection oligosaccharides is about 28 minutes of about 22-between first peak among the structural domain I and the main peak in the domain II.The invention provides the composition that comprises the CTLA4-Ig dimer molecule, wherein at least 0.5% CTLA4-Ig dimer molecule is a cysteinylization.In another embodiment, at least 1.0% CTLA4-Ig dimer molecule is a cysteinylization.The invention provides the colony of CTLA4-Ig molecule, wherein said colony shows the mass spectroscopy overview as shown in Fig. 8 and 10.The invention provides the colony of CTLA4-Ig molecule, wherein said colony shows the capillary electrophoresis overview as shown in Figure 20 and 21.The invention provides the composition of sialic acids groups with about 6-about 18 and the CTLA4-Ig molecule of the dimeric molar average ratio of CTLA4-Ig, wherein the CTLA4-Ig dimer is by the cells produce of commercial clone.The invention provides the CTLA4-Ig composition that obtains by any method of the present invention.The invention provides the colony of CTLA4-Ig molecule, wherein said molecule is glycosylated on following residue: the amino acid asparagine residue at the 102nd place of SEQ IDNO:2, the amino acid asparagine residue at the 134th place of SEQ ID NO:2, the amino acid asparagine residue at the 233rd place of SEQ ID NO:2, the Serine amino-acid residue at the 155th place of SEQ ID NO:2, or the Serine amino-acid residue at the 165th place of SEQID NO:2.The invention provides the colony of CTLA4-Ig molecule, wherein said molecule colony is characterised in that: (a) the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 15-about 35; (b) the dimeric molar average ratio of the GalNAc/ mole CTLA4-Ig of about 1.7-about 3.6; (c) the dimeric molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig; (d) the dimeric molar average ratio of the Fucose of about 3.5-about 8.3/mole CTLA4-Ig; (e) the dimeric molar average ratio of the seminose of about 7.2-about 22/mole CTLA4-Ig; (f) the dimeric molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 2.4 ± 0.2-about 5.0 ± 0.2; (h) be less than or equal to the MCP-1 of 5ppm; (i) less than 2.5% the tetramer; (j) less than 0.5% monomer; (k) have with SEQ IDNOS:2-8 in the CTLA4-Ig polypeptide of any one at least 95% amino acid whose colony that is equal to; (1) wherein intragroup CTLA4-Ig molecule can combine with CD80 and CD86.The invention provides the colony of CTLA4-Ig molecule, wherein said molecule colony is characterised in that: (a) the dimeric molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 15-about 35; (b) the dimeric molar average ratio of the GalNAc/ mole CTLA4-Ig of about 1.7-about 3.6; (c) the dimeric molar average ratio of the semi-lactosi of about 8-about 17/mole CTLA4-Ig; (d) the dimeric molar average ratio of the Fucose of about 3.5-about 8.3/mole CTLA4-Ig; (e) the dimeric molar average ratio of the seminose of about 7.2-about 22/mole CTLA4-Ig; (f) the dimeric molar average ratio of the sialic acid of about 6-about 12/mole CTLA4-Ig; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 2.4 ± 0.2-about 5.0 ± 0.2; (h) be less than or equal to the MCP-1 of 5ppm; (i) less than 2.5% the tetramer; (j) less than 0.5% monomer; (k) have with SEQ ID NOS:2-8 in the CTLA4-Ig polypeptide of any one at least 95% amino acid whose colony that is equal to; (1) wherein intragroup CTLA4-Ig molecule can combine with CD80 and CD86; Or its pharmacy Equivalent.The invention provides the CTLA4-Ig molecule that comprises significant quantity and the composition of pharmaceutically acceptable carrier.The invention provides the composition of the CTLA4-Ig molecule that comprises significant quantity, wherein said composition further comprises a certain amount of maltose monohydrate.In one embodiment, composition further comprises pharmaceutically acceptable thinner, adjuvant or carrier.In one embodiment, composition further comprises maltose, sodium orthophosphate (monometallic) monohydrate, sodium-chlor, sodium hydroxide and sterilized water.In one embodiment, composition further comprises sucrose, poloxamer, sodium orthophosphate (monometallic) monohydrate, anhydrous sodium orthophosphate dimetallic, sodium-chlor, sodium hydroxide and sterilized water.
The invention provides freeze dried CTLA4-Ig mixture, it comprises at least 95% CTLA4-Ig dimer and is no more than 5% the CTLA4-Ig tetramer.In one embodiment, mixture comprises at least 98% CTLA4-Ig dimer and is no more than 2% the CTLA4-Ig tetramer.In one embodiment, mixture comprises at least 99% CTLA4-Ig dimer and is no more than 1% the CTLA4-Ig tetramer.In one embodiment, mixture comprises at least 8.0 moles of sialic acids/mole CTLA4-Ig dimer.In one embodiment, mixture comprises the about 31 moles of GlcNAc/ mole CTLA4-Ig dimers of about 15.7-.In one embodiment, mixture comprises the about 3.2 moles of GalNAc/ mole CTLA4-Ig dimers of about 1.6-.In one embodiment, mixture comprises the about 15.5 moles of semi-lactosis of about 9.3-/mole CTLA4-Ig dimer.In one embodiment, mixture comprises the about 7.9 moles of Fucoses of about 3.6-/mole CTLA4-Ig dimer.In one embodiment, mixture comprises about 9.7 moles of seminoses/mole CTLA4-Ig dimer.The present invention also provides pharmaceutical kit, and it comprises: the container that (a) comprises the freeze dried CTLA4-Ig mixture of claim 1; (b) be used for freeze dried CTLA4-Ig mixture is reconstituted the specification sheets of the solution that is used to inject.
The invention provides the method that is used for suppressor T cell propagation (or activation), described method comprises makes the T cell contact with the CTLA4-Ig composition of significant quantity.The invention provides the method for the immunne response that is used for suppressing the experimenter, described method comprises the composition of using significant quantity to the experimenter that these needs are arranged.The invention provides the present composition, be used for inducing the experimenter at antigenic immunological tolerance by the amount of giving experimenter's administering therapeutic disease or illness, inflammation among the treatment experimenter, the treatment rheumatoid arthritis, psoriasis among the treatment experimenter, lupus among the treatment experimenter, transformation reactions among treatment or the prevention experimenter, graft versus host disease (GVH disease) among treatment or the prevention experimenter, transplant organ among treatment or the prevention experimenter repels, multiple sclerosis among the treatment experimenter, type i diabetes among the treatment experimenter, inflammatory bowel among the treatment experimenter, ovaritis among the treatment experimenter, glomerulonephritis among the treatment experimenter, allergic encephalitis among the treatment experimenter, or the method for the myasthenia gravis among the treatment experimenter.Composition can with pharmaceutically acceptable carrier combinations.The CTLA4-Ig molecule colony that the invention provides sialic acids groups with about 6-about 18 and the dimeric molar average ratio of CTLA4-Ig is used for the purposes of medicine of the treating and/or preventing property processing of immune disorders in preparation.The invention provides the purposes of CTLA4-Ig molecule colony in the agent of preparation resisting rheumatoid arthritis of sialic acids groups with about 6-about 18 and the dimeric molar average ratio of CTLA4-Ig, described resisting rheumatoid arthritis agent together with about the specification sheets of its purposes in rheumatoid arthritis treatment in packing.The invention provides the method that is used for suppressor T cell propagation (or activation), described method comprises makes the T cell contact with the composition of the present invention of significant quantity, described composition and methotrexate combination.The invention provides the method for the immunne response that is used for suppressing the experimenter, described method comprises the composition of the present invention of using significant quantity to the experimenter that these needs are arranged, described composition and methotrexate combination.The invention provides and be used for inducing the method for experimenter at antigenic immunological tolerance, described method comprises composition, described composition and the methotrexate combination of using among the claim 1-64 of significant quantity each to the experimenter that these needs are arranged.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) mammalian cell of amplification secretion recombinant protein, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, wherein recombinant protein concentration is at least 0.5 gram/L liquid culture; (b) from least 10, separating recombinant proteins matter in the liquid culture of 000L.In one embodiment, the amplification of step (a) comprises: (i) know in the substratum with at least 4 generations of cell cultures, so that obtain at least about 1.0 x 10 in serum-free, chemical ingredients 5The cell density of viable cell/mL, wherein each seed stage is with about 2 x10 5/ ml begins and proceeds to 1-2 1,000,000 cells/ml; Cell is kept in culture to be enough to by the time of culture production at least about 0.5g/L.In one embodiment, protein is glycoprotein.In one embodiment, protein is CTLA4-Ig protein.In one embodiment, mammalian cell is a progeny cell.In one embodiment, mammalian cell is the offspring that can produce the CHO cloned cell line of CTLA4-Ig fusion rotein, and wherein Chinese hamster ovary celI has at least 30 copies of the CTLA4-Ig expression cassette of stable integration in its genome.In one embodiment, time enough is that the viability of cell does not drop to the time below 30%.In one embodiment, time enough is that the viability of cell does not drop to the time below 40%.In one embodiment, time enough is that the viability of cell does not drop to the time below 50%.In one embodiment, time enough is that the viability of cell does not drop to the time below 60%.In one embodiment, time enough is that the viability of cell does not drop to the time below 70% or 80% or 90% or 95%.In one embodiment, go down to posterity at least 4 times and comprise: (i) culturing cell in the volume of culture of 50mL at least, until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml, (ii) culturing cell in the volume of culture of 10L at least is until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml; (iii) culturing cell in the volume of culture of 100L at least is until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml; (iv) culturing cell in the volume of culture of 200L is until the cell density that reaches about 100 Wan-Yue 2.5 hundred ten thousand cells/ml.In one embodiment, semi-lactosi is added serum-free, chemical ingredients knows in the substratum.In one embodiment, keep and comprise (i) and make the temperature of culture reduce to 34 ± 2 ℃ from 37 ± 2 ℃; (ii) make the temperature of culture reduce to 32 ± 2 ℃ from 34 ± 2 ℃.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 5 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 6 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 7 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 8 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 9 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 10 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 11 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 12 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 13 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 14 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 15 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 16 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 17 days.In one embodiment, temperature maintenance is in 32 ± 2 ℃ scope at least 18 days.In one embodiment, temperature maintenance is up to 18 days in 32 ± 2 ℃ scope.In one embodiment, temperature maintenance is about 30 x 10 until the cell density of culture in 32 ± 2 ℃ scope 5-Yue 79 x 10 5Cell/mL liquid culture.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) from inoculum at least 10, the mammalian cell of the liquid culture amplification secretion recombinant protein of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) from least 10, separating recombinant proteins matter in the liquid culture of 000L, wherein said separation only takes place when liquid culture comprises more than or equal to about 6.0 moles of NANA/ mole protein.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) from inoculum at least 10, the mammalian cell of the liquid culture amplification secretion recombinant protein of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) from least 10, separating recombinant proteins matter in the liquid culture of 000L, wherein said separation only has about 33 x 10 at liquid culture 5-Yue 79 x 10 5Take place during the cell density of cell/mL.
The invention provides the method that is used to produce recombinant protein, described method comprises: (a) from inoculum at least 10, the mammalian cell of the liquid culture amplification secretion recombinant protein of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) from least 10, separating recombinant proteins matter in the liquid culture of 000L, the wherein said cell survival that is separated in the liquid culture does not drop to about 20% or about 30% or about 38% and takes place when following.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) from inoculum at least 10, the mammalian cell of the liquid culture amplification secretion recombinant protein of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) from least 10, separating recombinant proteins matter in the liquid culture of 000L, wherein said separation only takes place when intracellular toxin is less than or equal to about 76.8EU/mL liquid culture.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) from inoculum at least 10, the mammalian cell of the liquid culture amplification secretion recombinant protein of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) from least 10, separating recombinant proteins matter in the liquid culture of 000L, wherein said separation only takes place during liquid culture less than 1 colony forming unit/mL at biological load.The invention provides the method that is used to produce recombinant protein, described method comprises: (a) from inoculum at least 10, the mammalian cell of the liquid culture amplification secretion recombinant protein of 000L, thus make recombinant protein concentration be at least 0.5 gram/L liquid culture; (b) from least 10, separating recombinant proteins matter in the liquid culture of 000L, wherein said separation only takes place during 2 in satisfying following condition at least: (i) liquid culture comprises more than or equal to about 6.0 moles of NANA/ mole protein, and (ii) liquid culture has about 33 x 10 5-Yue 79 x 10 5The cell density of cell/mL, (iii) the cell survival in the liquid culture does not drop to about 20% or about below 38%, or (iv) the amount of the CTLA4-Ig in the culture greater than 0.5g/L.In one embodiment, separation comprises: (i) obtain cell culture supernatant liquid; (ii) supernatant liquor is implemented anion-exchange chromatography, to obtain the protein of wash-out; (iii) the protein of step wash-out is (ii) implemented hydrophobic interaction chromatography, so that obtain the protein of enrichment; (iv) the protein of enrichment is implemented affinity chromatography, to obtain the protein of wash-out and enrichment; (v) the protein of (iv) wash-out and enrichment is implemented anion-exchange chromatography.In one embodiment, the protein of the enrichment that obtains in (iii) of step is characterised in that the polymeric per-cent of any high molecular is less than 25 weight %.In one embodiment, step anion-exchange chromatography (ii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 75mM HEPES and about 360mMNaCl, and has about 8.0 pH.In one embodiment, step anion-exchange chromatography (ii) uses elution buffer to carry out, and described elution buffer comprises about 25mM HEPES and about 325mM NaCl, and has about 7.0 pH.In one embodiment, step hydrophobic interaction chromatography (iii) uses single lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM HEPES and about 850mM NaCl, and has about 7.0 pH.In one embodiment, step affinity chromatography (iv) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM Tris and about 250mM NaCl, and has about 8.0 pH.In one embodiment, step affinity chromatography (iv) uses elution buffer to carry out, and described elution buffer comprises about 100mM glycine, and has about 3.5 pH.In one embodiment, (anion-exchange chromatography v) uses lavation buffer solution to carry out to step, and described lavation buffer solution comprises about 25mM HEPES and the about 130mM NaCl of about 120mM NaCl-, and has about 8.0 pH.In one embodiment, (anion-exchange chromatography v) uses elution buffer to carry out to step, and described elution buffer comprises about 25mM HEPES and about 200mM NaCl, and has about 8.0 pH.In one embodiment, step anion-exchange chromatography (ii) uses the post with anionite-exchange resin to carry out, and described anionite-exchange resin has primary, secondary, uncle or quaternary amine functional group.In one embodiment, resin has quaternary amine functional group.In one embodiment, step hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin has phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.In one embodiment, functional group is a phenyl functional group.In one embodiment, step affinity chromatography is (iv) used and to be comprised the proteic post of A and carry out.In one embodiment for being used to prepare the method for CTLA4-Ig, described method comprises from liquid cell culture purifying CTLA4-Ig, thereby make the CTLA4-Ig (a) of purifying have about 38ng MCP-1/mg CTLA4-Ig dimer and (b) comprise and be less than the 2.5 weight %CTLA4-Ig tetramers.
In one embodiment, the liquid cell culture comprises cell of the present invention or progeny cell.The invention provides the method that is used to produce CTLA4-Ig, described method comprises: (a) amplification can be produced any commercial clone of CTLA4-Ig or the progeny cell of Chinese hamster ovary celI, wherein said amplification is at least 10 from inoculum, the liquid culture of 000L, wherein CTLA4-Ig concentration is at least 0.5 gram/L liquid culture; (b) from least 10, separation of C TLA4-Ig in the liquid culture of 000L, wherein chromatography is on the post with hydrophobic interaction resin, described hydrophobic interaction resin has at least a phenyl functional group, wherein said separation comprises uses single lavation buffer solution to carry out the step of hydrophobic interaction chromatography, described lavation buffer solution comprises about 25mM HEPES and about 850mM NaCl, and has about 7.0 pH.
Embodiments of the invention
Hereinafter provide many embodiment to promote understanding more fully of the present invention.Following embodiment for example understands preparation and puts into practice exemplary approach of the present invention.Yet scope of the present invention is not limited to disclosed specific embodiments among these embodiment, and it only is used for the illustrative purpose, because alternative method can be used to obtain similar results.
Following embodiment relates to one or more the CTLA4-Ig molecule of sequence that comprises among the SEQ ID NOS:2,4,5,6,7,8,9,10,11,12,13,14,15 or 16.It is restrictive that these embodiment are not intended to, and those skilled in the art understand that embodiment can expand and be fit to for example other CTLA4-Ig molecules, other glycoprotein and relate to or comprise other protein of the proteinic part of Ig superfamily.
Following table has been set forth and has been related to CTLA4-Ig and CTLA4 A29YL104EThe embodiment of-Ig.
The CTLA4-Ig protein characteristic Exemplary CTLA4-Ig protein numbering 1-have SEQ ID NO:1,2,5,6,7,8,9 or 10 CTLA4-Ig Exemplary CTLA4-Ig protein numbering 2-have SEQ ID NO:3 or 4 or the CTLA4 of 11-16 A29YL104E-Ig
With combining of B7-1; In conjunction with/dissociation rate; Render a service, tire Embodiment 6
Biological load Embodiment 49
Capillary electrophoresis Embodiment 38
Carbohydrate content, N connection HPEAC overview, structural domain Embodiment 3 embodiment 44 Embodiment 22 embodiment 37
The clone transfection Embodiment 12 Embodiment 23
CHO?DNA Embodiment 58 Embodiment 55
CHO host cell proteins matter Embodiment 60 Embodiment 52
Heredity characterizes Embodiment 24
Depolymerization Embodiment 5 embodiment 33
Intracellular toxin Embodiment 48 Embodiment 48
Final filling Embodiment 30
The CTLA4-Ig protein characteristic Exemplary CTLA4-Ig protein numbering 1-have SEQ ID NO:1,2,5,6,7,8,9 or 10 CTLA4-Ig Exemplary CTLA4-Ig protein numbering 2-have SEQ ID NO:3 or 4 or the CTLA4 of 11-16 A29YL104E-Ig
Preparation Embodiment
2 Embodiment 27
GalNAc, the GlcNAc mol ratio Embodiment 17, and embodiment 63 Embodiment 36
IEF Embodiment 50 Embodiment 22
The IL-2 biological assay Embodiment 45 Embodiment 40
Immunogenicity Embodiment 31
The healthy PK of single agent Embodiment 66
MALDI-TOF Embodiment 8
Seminose, Fucose, semi-lactosi mol ratio Embodiment 18 embodiment 64 Embodiment 35
Mass spectrum Embodiment 7
MCP-1 Embodiment 59 Embodiment 54
Substratum/cultivation Embodiment 9
Monkey PK Embodiment 32
Monomer Embodiment 4
NANA, the NGNA mol ratio Embodiment 3, and embodiment 16 Embodiment 39
The O connection Embodiment 46
Oxidation and deacylated tRNA amine Embodiment 47
The PK dependency Embodiment 42
Plasmid Embodiment 1, and embodiment 11, and embodiment 67
Produce Embodiment 14 embodiment 28 Embodiment 19
A albumen Embodiment 62 Embodiment 53
The CTLA4-Ig protein characteristic Exemplary CTLA4-Ig protein numbering 1-have SEQ ID NO:1,2,5,6,7,8,9 or 10 CTLA4-Ig Exemplary CTLA4-Ig protein numbering 2-have SEQ ID NO:3 or 4 or the CTLA4 of 11-16 A29YL104E-Ig
Purifying Embodiment
15 embodiment 29 Embodiment 20
Multi-agent RA PK Embodiment 34
SDS-PAGE Embodiment 51 Embodiment 26 embodiment 56
SEC-HMW, dimer, monomer, big or small homogeneity Embodiment 10 Embodiment 25
Combine (BIAcore) of SPR-and B7.1 Embodiment 21 Embodiment 41
The subclone of clone Embodiment 13 Embodiment 24
Triton-X100 Embodiment 61 Embodiment 57
The trypsinase mapping Embodiment 3, and embodiment 47, and embodiment 65 Embodiment 22
Abbreviation:
15N 15 nanometers
A 280Absorbancy at the 280nm place
CTLA4-Ig cytotoxic T lymphocyte antigen-4 immunoglobulin (Ig); CTLA-4Ig
The API active pharmaceutical ingredient
The AU absorbance unit
B7 CTLA-4 receptors ligand
The cfu colony forming unit
The CHO Chinese hamster ovary
CHOP Chinese hamster ovary host cell proteins matter
The CV column volume
Medicine filling drug concentration/diafiltration and filling step
The ELISA enzyme-linked immunosorbent assay
The EU endotoxin unit
The Fc antibody constant region
The GalNAc N-acetylgalactosamine
The GlcNAc N-acetyl-glucosamine
HEPES 4-(2-hydroxyethyl) piperazine-1-ethyl sulfonic acid
The HIC hydrophobic interaction chromatography; Phenyl Sepharose TMFast Flow
The HMW high molecular
The HPLC high performance liquid chromatography
IgGl G1 immunoglobulin like protein
Control in the IPC process
The LAL LAL
The main batch record of MBR (s) (s)
MCP-1 MCP 1
The MTX methotrexate
The MW molecular weight
N/A is unavailable
NANA N-n acetylneuraminic acid n; Sialic acid; SA
The NMWC nominal molecular weight is blocked
The OD optical density(OD)
PAR proves acceptable scope
Planova virus is removed strainer, aperture 15nm
PP (s) machined parameters
The PQ assessment of performance
Psi pound/square inch
Psid pressure reduction (pound/square inch)
Psig gauge pressure (pound/square inch)
QFF?Q?Sepharose TM?Fast?Flow
QXL?Q?Sepharose?Extreme?Load
RPA reorganization A albumen agarose is mobile (Recombinant Protein A SepharoseFast Flow) fast
The SA sialic acid; The N-n acetylneuraminic acid n; NANA
The SDS-PAGE SDS-PAGE
The SOP Standard operation procedure SOP
Tris three (methylol) aminomethane
Triton uncle X-100 octylphenoxy polyethoxy ethanol; Polyoxyethylene glycol
Uncle's octyl phenyl ether
The UF ultrafiltration
The UV ultraviolet ray
The v/v volumetric ratio
The VF virus filtration; Nanofiltration
The VI inactivation of virus
Embodiment 1: the confirmation of the CTLA4-Ig encoding sequence among the plasmid pcSDhuCTLA4-Ig
The note nucleotide sequence of the CTLA4-Ig gene that exists among the pcSDhuCTLA4-Ig and the CTLA4-Ig aminoacid sequence of corresponding derivation are shown among Fig. 1.The pcSDhuCTLA4-Ig nucleic acid transfection in Chinese hamster ovary celI, can be expressed stable transfection (referring to embodiment 12) of CTLA4-Ig molecule with generation.Transfectant screens, and some transfectant carries out subclone or amplification to produce cloned cell line.
What dna sequence data analysis confirmation produced during plasmid construction is connected to as design, and the correct oncostatin M signal sequence of the synthetic oligonucleotide primer deposits yields CTLA4-Ig sequence upstream of using in the polymerase chain reaction.Required halfcystine in the hinge area of fusion rotein to Serine changes (at the 156th, 162 and 165 places of SEQ ID NO:2) and is confirmed.These amino-acid residues indicate with runic and asterisk in Fig. 1.Also detect at the proline(Pro) at the 174th place of SEQ ID NO:2 other amino acid and change to Serine.This variation is at IgG 1CDNA was introduced by polymerase chain reaction between synthesis phase.This amino-acid residue also indicates with runic and asterisk in Fig. 1.
When with human CTLA 4 and human IgG 1When the disclosed nucleotide sequence of constant region compares, a kind of other difference of dna sequence data Analysis and Identification.Codon at amino acid/11 10 places of CTLA4 coding region is accredited as ACC (Threonine) rather than GCC (L-Ala).
Embodiment 2:CTLA4-Ig preparation
Be used to inject, the CTLA4-Ig composition of 250mg/ bottle is to be used for aseptic, the no pyrogeneous substance lyophile that intravenously (IV) is used.The colony of CTLA4-Ig molecule is packaged in the 15-cc I type flint tubing glass bottle.Each bottle clogs with the stopper of 20-mm Daikyo gray butyl D-21-7-S/B2-TR fluoro-resin bag quilt, and seals and seal with 20-mm aluminium, white, turn-up (flip-off).
The bottle that each single uses comprises 250mg CTLA4-Ig composition, and it is by sterile water for injection, and USP constitutes, and uses 0.9% sodium chloride injection in use, and USP further dilutes.Be used to inject, the CTLA4-Ig composition of 250mg/ bottle, and the function of every kind of component is listed in following table.
Table 8:CTLA4-Ig composition
Figure A200680053116D02421
bThese components are present in the CTLA4-Ig composition solution
The CTLA4-Ig composition comprises the 25mM sodium phosphate buffer agent that is dissolved under pH7.5 and about 50mg/mL CTLA4-Ig of 50mM sodium-chlor.Between development stage,, select the sort buffer system in early days based on the physics of the CTLA4-Ig that becomes according to pH, buffer reagent type, buffer concentration and sodium chloride concentration and the assessment of chemical stability.The stability of CTLA4-Ig solution is studied in pH scope 5-9.The result points out that the formation of high molecular weight species is that pH is dependent, and the pH scope of maximum stable is 7-8.In the mensuration of separating, the effect of assessment buffer reagent type and concentration finds that wherein the CTLA4-Ig composition is stable equally in the sodium phosphate of pH8 or tris buffer reagent.In addition, the buffer concentration of 10-100mM concentration to the stability of the CTLA4-Ig composition under 2 ℃-8 ℃ without any influence.Similarly, the existence of the sodium-chlor of 30-500mM concentration is to preserving the not influence of solution state stability in 2 ℃-8 ℃ CTLA4-Ig composition.
Based on these results, select to be dissolved in the 25mM sodium phosphate buffer agent under pH7.5 and the CTLA4-Ig composition of 50mM sodium-chlor with 10mg/mL, be used for preparation with 50mg/ bottle intensity.CTLA4-Ig concentration becomes 50mg/mL subsequently in identical combinations of buffers thing, with the CTLA4-Ig composition that allows exploitation to have 200-and 250mg/ bottle intensity.
The CTLA4-Ig that is used to inject adds that with maltose sodium phosphate buffer and sodium-chlor prepares.The function of the vehicle that uses in this product is listed in last table.
The inorganic salt for example existence of sodium-chlor and sodium phosphate buffer fluid component reduce the second-order transition temperature (Tg ') of refrigeration system.In addition, the sodium orthophosphate dimetallic that original position forms under pH7.5 experiences preferential crystallization between pool period, and this reduces the microenvironment pH of frozen soln.For these reasons, select the sodium-chlor and the sodium phosphate buffer of minimum, so that its influence to freeze-drying process drops to minimum.Maltose adds as stablizer, and it serves as very low temperature and cryoprotectant respectively during the freeze-drying of medicament production and during follow-up storage.Be used to inject, the CTLA4-Ig composition of 250mg/ bottle is that aseptic, the single that does not contain anti-microbial preservative uses bottle.
The carbohydrate content analysis of embodiment 3:CTLA4-Ig composition
The purpose of this method provides the chromatography overview of CTLA4-Ig N connection oligosaccharides.This program can be used for obtaining CTLA4-Ig sample N-n acetylneuraminic acid n (NANA) and N-hydroxyacetylneuraminic acid (NGNA) and proteinic mol ratio (overall bonded adds free).NANA and NGNA are sialic 2 kinds of forms.Glycosylation on the CTLA4-Ig protein comprises N connection oligosaccharides.For example, these oligosaccharides can be by obtaining discharging with the process of PNGase F enzymically hydrolyse through 22 hours.Free oligosaccharides uses high pH anion-exchange chromatography to utilize Electrochemical Detection to carry out profile analysis.Use high pH anion-exchange chromatography (HPAEC-PAD) to assess about the carbohydrate overview of N connection oligosaccharides with pulsed current detection.These overviews are confirmed, and structural information is used and MS link coupled porous graphite carbon (PGC) chromatography obtains.Technology and result obtain describing in this part.This program is for SEQ ID NO: CTLA4-Ig be illustrative.
N connection oligosaccharides separates: (N connection) oligosaccharides that l-asparagine connects and the cutting of CTLA4-Ig use PNGase F to carry out by enzymically hydrolyse.In order to carry out de-glycosylation, CTLA4-Ig at first reduces and sex change: the 1-2mg CTLA4-Ig that is dissolved in the 176 μ L5mM sodium phosphate buffers that comprise 0.5%SDS and 1% beta-mercaptoethanol allows to be cooled to envrionment temperature subsequently in 100 ℃ of heating 2 minutes.In the refrigerative mixture, add 16 μ L 10%NP-40.Make the sample thorough mixing, and add 40 μ L PNGase F (50,000U/mL is dissolved in the 50mM sodium phosphate buffer).Make the sample thorough mixing, and in 38 ℃ of incubations 24 hours.The oligosaccharides (glycan) that enzymatic discharges carries out purifying by RPHPLC (reversed-phase high-performance liquid chromatography) (HPLC), wherein uses and ThermoHyperCarb post (4.6 x 100mm; 5 μ L) link coupled Phenomenex Luna C18 post (4.6x 150mm; 5 μ L) with 1.0mL/ minute flow velocity; Being used for isolating chromatograph is the Waters Alliance 2695 that is equipped with the Waters2996 detector.Post at first uses 0.05% trifluoroacetic acid (TFA) to carry out balance.After the injected sample, initial acetonitrile gradient, the solvent compositions with the 0.05%TFA that is dissolved in 12% acetonitrile in the time of 15 minutes stops.Glycan is subsequently with the stepwise gradient wash-out from the HyperCarb post to the 0.05%TFA that is dissolved in 60% acetonitrile.Passing through when the UV at 206nm place absorbancy is monitored, to collect glycan, and be concentrated into drying in a vacuum.Before follow-up injection, the LunaC18 post cleans with the 0.05%TFA that is dissolved in 40% acetonitrile, 40% Virahol, 20% water.
NANA stock solution (N-n acetylneuraminic acid n) preparation (1mg/mL).Material particular: before weighing, allow the NANA standard to heat to room temperature.So do not do the water condensation that to cause in the standard.Only open the time that is enough to obtain aequum, make bottle tightly seal and send back to refrigerator once more subsequently, preserve with siccative.Weighing 3-10mg N-n acetylneuraminic acid n accurately.Be recorded near 0.1mg.If weighing uses pan paper/dish to finish, be transferred to the container of suitable size so.The HPLC grade water that adds q.s is to produce the solution of concentration 1mg/mL.With splash bar or by vortex mixed until dissolving.In polypropylene tube, preserve in 2-8 ℃ maximum 3 months.
NAGA stock solution (N-hydroxyacetylneuraminic acid) preparation (1mg/mL).Material particular: before weighing, allow the NAGA standard to heat to room temperature.So do not do the water condensation that to cause in the stoste.Only open the time that is enough to obtain aequum, make bottle tightly seal and send back to refrigerator once more subsequently, preserve with siccative.Accurately weighing 3-10mg (is recorded near the 0.1mgN-hydroxyacetylneuraminic acid.Be recorded near 0.1mg.If weighing uses pan paper/dish to finish, be transferred to the container of suitable size so.The HPLC grade water that adds q.s is to produce the solution of target level 1mg/mL.With splash bar or by vortex mixed until dissolving.In polypropylene tube, preserve in 2-8 ℃ maximum 3 months.
System's suitability solution.In suitable containers, 0.050mL1mg/mL NANA and NGNA stock solution are added in the 0.900mL HPLC grade water separately.Pass through vortex mixed.Preserve in 2-8 ℃ maximum 3 months.N-n acetylneuraminic acid n working solution (0.050mg/mL).Accurately measure in 0.050mL1mg/mL NANA stock solution and the adding 0.950mL HPLC grade water.Pass through vortex mixed.Prepare in use in duplicate.N-hydroxyacetylneuraminic acid working solution (0.050mg/mL).Accurately measure in 0.050mL1mg/mL NGNA stock solution and the adding 0.950mL HPLC grade water.Pass through vortex mixed.Prepare in use in duplicate.
Sample and the preparation of hydrolysis barren.According to the protein concn of analysis certificate (COA) acquisition about the CTLA4-Ig material.By 0.190mL HPLC grade water being added preparation hydrolysis barren single sample in the 1.5mL Eppendorf tube.Use HPLC grade water to prepare 2 kinds of 1mg/mL solution of sample and CTLA4-Ig reference material.Pass through vortex mixed.
Sample, CTLA4-Ig reference material and the hydrolysis of hydrolysis barren.Duplicate reference material and sample formulation in the 1.5mL Eppendorf tube are hydrolyzed.Annotate: use the Eppendorf tube that is fit to heat block fully very important.0.010mL 1 M H2SO4 is added in the 1mg/mL dilution and hydrolysis blank of 0.190mL sample and CTLA4-Ig reference material.By vortex mixed and by cover lock (lid-lock) or the fastening lid of adhesive tape.Eppendorf tube was placed 80 ℃ ± 2 ℃ heat blocks 1 hour ± 2 minutes.Behind the incubation, take-off pipe from heat block places microcentrifuge and rotation to impel the bottom of sample to pipe pipe.The hydrating solution aliquots containig is arrived in the automatic sampling bottle, and place automatic sampler to be used for injection.
Instrument condition.Prepare high performance liquid chromatography (HPLC) system.Following condition is set.Under the temperature of 0.6mL/ minute flow velocity and 40 ℃, made column equilibration at least 1 hour.
Moving phase A：5mM?H 2SO 4B:HPLC grade water (post washing)
Flow velocity 0.6mL/ minute
Working time
25 minutes
The detector wavelength 206nm
Column temperature
40℃
Automatic sampling actuator temperature volume injected 4℃?5μL
Retention time NGNA NANA ± 1 minute 9.8 (system is dependent) 10.8 ± 1 minutes (system is dependent)
Gradient condition Deng degree
System's suitability.Injection with moving phase begins to analyze with the evaluating system baseline as the instrument blank, and described baseline should be smooth and stable.If baseline is also uneven and stable, should prepare other blank injection so.Annotate: baseline does not move should surpass 0.25AU.6 duplicate injections of executive system suitability solution.Calculating resolution (R) and theoretical tray (N).
Use system's suitability injection for the first time, equation theory of computation stage number (N) below using: theoretical plate number → N = 16 ( t W ) 2
Wherein
N=theoretical tray counting
The retention time at t=NANA peak is with a minute expression
The NANA peak width at W=baseline place is with a minute expression
Calculate NANA and NGNA mole in CTLA4-Ig reference material and the sample.NANA and NGNA standard are injected when each run begins and finish.Reinjected area counting to NANA and NGNA is averaged.Use this area below in the equation:
NANA in A Baxipu reference material or the sample or
Figure A200680053116D02462
NANA in the working solution that X=calculates in part 7.3.1 or NGNA mole
Y=is about NANA or the NGNA area counting (annotate: the peak about NANA integrated referring to part 7.2 and Fig. 3) of every kind of preparation in A Baxipu reference material or sample
The average area of Z=multiple NANA or NGNA in working solution
In one embodiment, the peak-to-peak resolving power of NGNA and NANA is necessary〉1.3.The theoretical tray counting is necessary〉or=4000.The system's suitability injection circulation ratio of area counting %RSD that is evaluated as the NANA peak is necessary≤and 3%.Necessary 〉=4000 of theoretical tray counting.The system's suitability injection circulation ratio of area counting %RSD that is evaluated as the NANA peak is necessary≤and 3%.
By having high DH anion-exchange chromatography (HPAEC-PAD) that pulsed current detects N connection oligosaccharides profile analysis: the HPAEC of isolating oligosaccharides carries out on the chromatographic system of being made up of the Waters Alliance that is equipped with the Waters464 electrochemical detector, and it utilizes Dionex CarboPackPA1 (4 x 250mm) anion-exchange column and Dionex CarboPack guard column.The oligosaccharides sample uses the sodium acetate gradient be dissolved in 200mM sodium hydroxide to carry out wash-out (0mM of sodium acetate concentration during from injection increases to the 225mM in the time of 60 minutes).Electrochemical detector moves under pulse mode, uses pulsed voltage E1=0.05V (t1=0.4 second), S=0.75V (t 2=0.2 second), E3=-0.15V (t 3=0.4 second).Detection box (cell) is made electrode, stainless steel counter electrode and silver/silver chloride reference electrode by metal working and is formed.
This method has been described the program of HPAEC (high pH anion-exchange chromatography) the oligosaccharides overview of the N connection oligosaccharides that the protein measured from the CTLA4-Ig sample discharges.The purpose of this method provides the chromatography overview of CTLA4-Ig, for example can be used for the CTLA4-Ig medicine N connection oligosaccharides of the comparative analysis between the different compositions of CTLA4-Ig molecule.Glycosylation on the CTLA4-Ig protein comprises N connection oligosaccharides.These oligosaccharides are by (peptide: N-Glycosylase F) enzymically hydrolyse obtains discharging through 22 hours process with PNGase F.Free oligosaccharides uses high pH anion-exchange chromatography to utilize Electrochemical Detection to carry out profile analysis.
CTLA4-Ig medicine in bulk is dissolved in 25mM sodium phosphate, 10mM
NaCl, the CTLA4-Ig of pH=7.5
Waters Waters Corporation with bonded PTFE/ siloxanes partition, catalog number (Cat.No.) always reclaim bottle (Total Recovery Vials) 186000234
RapiGest SF Waters Corporation, catalog number (Cat.No.)
186001861
Be equipped with following Alliance HPLC system: automatic Waters Corporation
Sampler (refrigerated), eluent degassing module
(Eluent?Degas?Module)
Model 2465 electrochemical detectors
Post: CarboPac PA-1 4 x 250mm Dionex Corporation, catalog number (Cat.No.)
35391
Guard column: CarboPac PA-1 4 x 50mm Dionex Corporation, catalog number (Cat.No.)
43096
The Empower data gathering system
The oligosaccharides overview of medicine is assessed at the sample of the reference material of moving simultaneously.The percent deviation at the identical peak in the structural domain that the result is reported as selection and peak and the reference standard.
The chromatography condition of the oligosaccharides overview by anion-exchange chromatography
29 ℃ of column temperatures
Flow velocity 1mL/ minute
Moving phase and gradient condition gradient program
The 1:500mM NaOAc time
2:400mM NaOH (minute) %1 %2 %3
3:HPLC grade water initial 0 30 70
0.0 0 30 70
11.0 0 30 70
12.0 4 30 66
20.0 10 30 60
80.0 45 30 25
81.0 0 30 70
100 0 30 70
Waters 2465 is provided with
Mode pulse
Empower is provided with scope=5 μ A
E1＝+0.05V?E2＝+0.75V?E3＝-0.15V
T1=400 millisecond t2=200 millisecond t3=400 millisecond
Sample time (ts)=100 millisecond
Time constant (strainer) t=0.1 second
Scope skew=5%
Polarity+
Temperature=29 ℃
Annotate: injecting before, made post and detector balance about 2 hours with the analysis flow velocity with initial flow, or until baseline stability.
The automatic sampling actuator temperature is made as:
4℃
Volume injected 60 μ L
100 minutes working times
The potential hump that is dominant in each structural domain
About retention time (RT; Minute)
(referring to Fig. 1); Value can depend on system
The RT of suitability (SS) standard and becoming
About RTs (minute)
SS： 18.5
Peak 1A:20.0
Peak 1B:20.8
Peak 1C:21.4
Peak 1D:22.4
Peak 1E:23.1
Peak 2 31.5
Peak 3:44.8
Peak 4:58.5
Be used for the preparation of the moving phase of HPAEC oligosaccharides carbohydrate profile analysis
HPAEC eluent 1:500mM sodium acetate (NaOAc).Weighing 20.51 ± 0.05g sodium acetate (anhydrous) places in the 500mL graduated cylinder that comprises 400mL HPLC grade water.Make volume reach 500mL with HPLC grade water, and use plastics serology transfer pipet to stir 5 minutes until mixing fully.By 0.2 μ m NF filtering solution.Be transferred to the 1L eluant bottle.Sprayed 20 minutes with bottle loosely cover lid and with helium.Cover tight lid and make the bottle pressurization with helium.Solution was preserved under helium in maximum 3 weeks of room temperature.
HPAEC eluent 2:400mM sodium hydroxide (NaOH).Use the 1L graduated cylinder, measure 960mL HPLC grade water and be transferred to clean 1L eluant bottle.Use the serology plastic suction pipet, 40.0mL 10N NaOH is directly added in the eluant bottle, and by reverberating mixtures of eluents.Sprayed 20 minutes with bottle loosely cover lid and with helium.Cover tight lid and make the bottle pressurization with helium.Solution was preserved under helium in maximum 3 weeks of room temperature.
HPAEC eluent 3:HPLC grade water.Fill the 1L eluant bottle with about 1L HPLC grade water.Eluant bottle is placed in the system loosely cover lid and spraying about 20 minutes.Cover tight lid and make the bottle pressurization with helium.Solution was preserved under helium in maximum 3 weeks of room temperature.
The 50mM sodium phosphate buffer, 0.02% sodiumazide, pH=7.5.
NaH 2PO 4·H 2O 6.9g
Na?N 3 0.2g
H 2O 1.0L final volume
Weigh up 6.9g ± 0.1g NaH 2PO 4H 2O and 0.2g NaN 3, and be dissolved in 800mL HPLC grade H in the 1L reagent bottle 2Among the O, wherein use the continuous mixing of magnetic stirring bar.Use pH meter, use 10M NaOH that the pH of solution is adjusted to 7.5.Use the 1L graduated cylinder to make final volume reach 1.0L.Solution was preserved in room temperature maximum 6 months.
Be dissolved in the 50mM sodium phosphate buffer, 0.02% sodiumazide, the PNGase F enzyme working solution of pH=7.5.
The 50mM sodium phosphate buffer
0.02% sodiumazide, pH=7.5. 1.8mL
From test kit, the PNGase F 0.2mL of catalog number (Cat.No.) P0704L
With the 1.8mL50mM sodium phosphate buffer, 0.02% sodiumazide, pH=7.5 are pipetted in the 1.8mL profound hypothermia bottle.Interpolation is from the 0.2mL PNGase F and the thorough mixing of test kit.Solution is preserved in-20 ℃ or lower maximum 6 months.Solution can carry out aliquots containig before freezing.
The external system suitability criterion.Stachyose stock solution (1.25mg/mL).Weighing 0.125g stachyose on pan paper.Operational analysis sky chessboard and be transferred to the 100mL volumetric flask.Be fills up to mark and thorough mixing with HPLC grade water.Arrive in the Nalgene cryovial with 2mL part aliquots containig.Solution is preserved in-20 ℃ or lower maximum 6 months.
Stachyose system suitability criterion (12.5 μ g/mL).1mL 1.25mg/mL stoste is pipetted in the 100mL volumetric flask.Be fills up to mark and thorough mixing with HPLC grade water.Arrive in the 0.65mL Eppendorf tube with 200 μ L part aliquots containigs.Pipe is placed the storage container of suitable mark.System's suitability solution is preserved in-20 ℃ or lower maximum 6 months.
Standard and specimen preparation
The reference material preparation.In the bottle that comprises the freeze dried RapiGest SF of 1mg, add and comprise 0.02% sodiumazide, the 625 μ L 50mM sodium phosphate buffers of pH7.5.In the 0.65mLEppendorf pipe, add the RapiGest SF that 120 μ L comprise damping fluid.Add 40 μ L reference materials (～50mg/mL).RapiGest SF final concentration should be 0.12% w/v.Add 40 μ LPNGase F work stoste, thorough mixing is rotated down sample, and places 38 ± 2 ℃ 22 ± 2 hours (water-bath or Alliance automatic sampler compartments).Sample is pipetted in the microconYM-10 centrifugal filter, and 13 under the 000g centrifugal 30 minutes.200 μ LHPLC water are placed filter, and, be rinsed in the filtrate in centrifugal other 30 minutes under the 000g by 13.Make the filtrate vortex 15 seconds of combination, and made sample centrifugal 10 seconds.Use transfer pipet that resulting solution (～380 μ L) is transferred to HPLC and always reclaim automatic sampler bottle (clauses and subclauses 1.15).
Specimen preparation.In 0.65mL Eppendorf pipe, add the RapiGest SF that 120 μ L comprise damping fluid.Add 40 μ L protein examples (this volume should equal 1-2mgCTLA4-Ig).RapiGest SF final concentration should be 0.12%w/v.Add 40 μ L PNGase F work stoste, by 10 seconds thorough mixing of vortex.Sample is rotated down, and places 38 ± 2 ℃ 22 ± 2 hours (water-bath or Alliance automatic sampler compartments).Sample is pipetted in the microconYM-10 centrifugal filter, and 13 under the 000g centrifugal 30 minutes.200 μ L HPLC water are placed filter, and, be rinsed in the filtrate in centrifugal other 30 minutes under the 000g by 13.Make the filtrate vortex 15 seconds of combination, and made sample centrifugal 10 seconds.Use transfer pipet that resulting solution (～380 μ L) is transferred to total recovery HPLC automatic sampler bottle (clauses and subclauses 1.15).
System's suitability
Electrochemical detector box stabilization.Inject the outside stachyose system's suitability criterion (12.5 μ g/mL) of 30 μ L.Guarantee peak height 〉=800nA about stachyose.Guarantee not exist excessive electrical noise and baseline smooth from box.Representative system suitability chromatogram is shown among Fig. 2.If stachyose sensitivity or baseline are unacceptable, check the damping fluid composition so, cleaning electrode or replacement electrode.If there is excessive noise, check that so box is to guarantee to remove all bubbles.Make box stabilization and injection water threose standard once more again.
Theoretical tray (N).Use following formula to measure theoretical plate number (N) based on the stachyose peak.This finishes by the Empower data analysis system or can also manual finish.N=16 (t/W) 2, wherein
T: the retention time of when maximum height, measuring from inject time to the peak elution time
W: by the peak width of side to the baseline extrapolation.
N is necessary 〉=and 6000.If the plate counting, is adjusted the operation gradient so less than 6000 or is replaced post.
Tailing factor (T).Use following formula based on stachyose peak measuring column tailing factor (T).This finishes by the Empower data analysis system or can also manual finish.T=(W 0.05/ 2f), wherein:
W 0.05: the peak width during height 5% (0.05h).
F: from W 0.05The time the peak front edge to the measurement (width) of peak maximum.
T is necessary≤and 1.2.Form if tailing factor, is checked damping fluid so greater than 1.2, replace post or by indication cleaning post, and injecting systems suitability criterion once more.
The suitability criterion retention time checking of stachyose system.Retention time is that system is dependent.Stachyose system suitability criterion should demonstrate 18.5 ± 2.0 minutes retention time.The CTLA4-Ig standard material.Observe the carbohydrate overview of first kind of (bracketing) reference material of carrying out together of injecting before the comfortable injected sample.The carbohydrate overview should be similar to the sort of shown in Fig. 1.Be that system is dependent absolute retention time.Determine that first peak (peak 1A) among the structural domain I and the difference in the retention time between the main peak (peak 3) among the domain II I are 22 minutes to 28 minutes.If the profile at peak does not resemble obtain among Fig. 1 the sort of, take suitable action (for example, inspection apparatus function, cleaning post, inspection/replacement damping fluid, replacement post) and assessment once more so.Following program can be used to clean post: closing box and with 80 % eluent 1,20% eluent 2 post is cleaned 5 minutes, is 50 % eluent 1,50% eluent, 2 cleanings 10 minutes subsequently.Under starting condition, make post and box (box is opened) balance once more, and assessment once more.
Injection sequence
The following injection sequence that isolating oligosaccharides is set:
Stachyose standard (30 μ L)
Reference material (60 μ L)
One or more samples (60 μ L)
Reference material (60 μ L)
Move between the reference material injection of recommending≤5 kinds of samples together to carry out.
Data analysis
Handle chromatogram.In Empower, handle chromatogram about reference material and sample.Set integration parameters, thereby make peak profile and baseline be similar to the sort of shown in Figure 76, integrating line may need manual laying.Execution is about the relative structural domain area of demonstration and the calculating of relative peak area.If carry out duplicate injection, measure mean value so about these parameters of CTLA4-Ig material and every kind of sample.For reference material, measure about each multiple structural domain I, II, III, peak 1A and the relative deviation of 1B with regard to all multiple mean values.
The comparison of sample overview and reference material overview.Range estimation relatively.Whether working sample and reference material have the structural domain and the main peaks of similar number.Main peaks is those peaks ( peak 1A, 1B, 1C, 1D, 2,3 and 4) of mark among Figure 76.Relative quantification relatively.The relative area of comparative sample (structural domain I, II and III and peak 1A and 1B; If carry out the duplicate injection of sample, use its mean value so) with average relative area from the CTLA4-Ig injection of carrying out.Mensuration is from the relative different of these areas of average CTLA4-Ig material value.Calculating-% structural domain area (structural domain area relatively).Calculating is about the % structural domain area of the structural domain of the overview of reference material and sample.Schema reference Figure 76 about the structural domain area.According to the example among Figure 76, by using following information and formula computation structure territory percentage (retention time is the dependent and result of reflection among Figure 76 of system):
Structural domain I: the summation (peak 1A-1E) of the peak area in the time of about retention time 18-24 minute
Domain II: from the summation at 26-38 minute peak
Domain II I: from the summation at 39-50 minute peak
Structural domain IV: from the summation at 51-64 minute peak
Structural domain V: from the summation at 65-75 minute peak
Annotate: will change according to the variation in the every day chromatographic property about the retention time window of structural domain.Correspondingly adjust the time.
Figure A200680053116D02531
For structural domain I-III, if carry out duplicate injection, reference material injection that so same calculating is carried out together and the mean value in the sample.
% peak area (relative peak area).Calculating is about the % peak area of peak 1A, the 1B of the overview of reference material and sample, 1C and 3.Schema reference Fig. 1 about peak area; Retention time is that system is dependent.Calculate the peak percentage by using following information and formula:
Figure A200680053116D02541
For among peak 1A and the 1B each, if carry out duplicate injection, reference material injection that so same calculating is carried out together and the mean value in the sample.According to reference material mean value calculation percentage difference.Use following formula to compare the percentage difference in the average relative area of structures of samples territory I-III, peak 1A and 1B with reference material to calculate:
％Diff＝|RM-S|/RM?x?100
Wherein:
RM=is about the purpose relative area mean value of reference material
S=is about the purpose relative area mean value of sample
| |=absolute value
Example value.For acceptable operation, must meet example value, and all injections relevant with sample must take place successfully.In addition, for the reference material injection of at every turn carrying out together, about the % structural domain area of structural domain I, II and III and about the % peak area of peak 1A and 1B must its mean value 15% in.
Analyze by HPAEC-PAD: N connection oligosaccharides and CTLA4-Ig molecule cut and analyze by HPAEC-PAD.Become 4 kinds of structural domains based on the sialic amount oligosaccharides eluted that exists.Structural domain is determined based on the migration of oligosaccharides standard and is confirmed by MS.Structural domain I represents the kind of asialoization.Domain II, III and IV represent single, double respectively and three sialylated kinds.In order to be characterized in 3 oligosaccharide structures on the N connection site, individually isolated peptides T5, T7 and T14 (about the characteristic reference table 59 of these peptides).This uses the tryptic digestion of CTLA4-Ig, and the manual peak of collecting corresponding to these 3 kinds of peptides carries out.
Isolating peptide is handled to discharge oligosaccharides with PNGase F, and described oligosaccharides carries out purifying subsequently and analyzes by HPAEC-PAD.Because owing to its extreme hydrophobicity is difficult to purifying, so peptide T5 can't produce good overview.Because the low recovery of this peptide from the reversed phase chromatography step behind tryptic digestion, so the amount of the oligosaccharides of cutting is low.
The quality of table 59. expected in theory and observed CTLA4-Ig
Figure A200680053116D02551
Table 59 (continuing). the quality of expected in theory and observed CTLA4-Ig
Figure A200680053116D02561
NO does not detect
NE does not reckon with (these quality do not reckon with according to digestion, non-specific cutting) according to tryptic digestion.
aTryptic peptide T5, T7 and T14 have the N linked glycosylation.The quality of listing is peptide the sort of of sugar basedization.
bTryptic peptide T8 and 9 has the O linked glycosylation.The quality of listing is peptide the sort of of sugar basedization.
cObserve several different masies corresponding to the different sugar shape of glycosylated peptide.
The HPAEC-PAD overview that joins carbohydrate about all N from CTLA4-Ig and 3 kinds of peptides is shown among Figure 55 A-D.Little figure A shows the N connection oligosaccharides overview of CTLA4-Ig molecule, and little figure B, C and D show the overview about T5, T7 and T14 respectively.About the amount of the oligosaccharides of every kind of peptide injection because preparation method and difference.Detected most of oligosaccharides are made up of the oligosaccharides of list and double-sialylated on peptide T5.The overview of T7 mainly comprises single, double and some do not have and three sialylated glycan kinds.On T5, only can detect a small amount of three sialylated structures.Peptide 14 is made up of oligosaccharides and a small amount of oligosaccharides single and double-sialylated of the ground asialoization of preponderating.
The result who obtains by HPAEC-PAD provides the information about locus specificity N linked glycosylation.Comprise recently those sialylated kinds of vast scale more from the N connection oligosaccharides in the CTLA4 district of CTLA4-Ig from the Fc district of CTLA4-Ig.
The trypsinase of CTLA4-Ig, Asp-N and trypsinase/chymotryptic peptide mapping: CTLA4-Ig carries out sex change and reduction in the 50mM Tris damping fluid (pH8.0) that comprises 6M guanidine and 5mM dithiothreitol (DTT) (DTT).In 50 ℃ of incubations after 20 minutes, add iodo-acid amide (IAA) to the final concentration of 10mM so that the free mercaptan alkylation, and continued incubation in the dark other 20 minutes in 50 ℃.To reduce and alkylating mixture be loaded on the desalting column (Amersham NAP-5), use 50mM Tris subsequently, 10mM CaCl2, pH8.0 or 50mM sodium phosphate buffer, pH7.5 is eluted to void volume.After the desalination, reduction/alkylating CTLA4-Ig uses 2 kinds of different proteolytic enzyme to digest: trypsinase or Asp-N.Add pancreas milk reducing protease digesting for trypsinase, CTLA4-Ig neither reduces also not alkylation.
For tryptic digestion, add sequence grade trypsin Roche, 2%, w/w, enzyme/protein) and in 37 ℃ of incubations 4 hours.For Asp-N digestion, add sequence grade Asp-N (Roche, 4%, w/w, enzyme/protein) and in 37 ℃ of incubations 16 hours.For trypsinase/pancreas milk reducing protease digesting, add sequence grade trypsin Promega, 4%, w/w, enzyme/protein) and in 37 ℃ of incubations 4 hours, add a-Quimotrase (Sigma subsequently, 4%, w/w, enzyme/protein) and in 37 ℃ of incubations 16 hours.All samples is placed in the refrigerator in digestion.
Resulting peptide mixt passed through from Waters Atlantis with 0.120mL/ minute on Waters Alliance HPLC Workstation TGradient elution separates in the M dC18 post (2.1 x 250mm).Post and the Waters Micromass Q-Tof micro that is equipped with the ion injection source TThe M mass spectrograph directly connects and is used to collect mass spectrum.Peptide mixt is gone up with 0.7mL/ minute at Varian Polaris C18 post (4.6x 250mm) equally and is used identical HPLC workstation to separate.Post carries out balance with solvent orange 2 A (water-soluble 0.02%TFA), and peptide carries out wash-out by the cumulative concentration of solvent B (95% acetonitrile/0.02%TFA, water-soluble).The rear pillar diverter valve is used for 15% the direct traffic Q-Tof workstation with positive TOF pattern (m/z 100-2000) operation.The general ion injection electric that uses is 3000V.
CTLA4-Ig by MS analyzes: CTLA4-Ig 100mM Tris, 25mM NaCl, pH8 are diluted to the final concentration of 0.7mg/mL.PNGase F (New England Biolabs) uses 100mM Tris, 25mM NaCl, and pH8 dilutes 30 times of final concentrations to 17U/ μ L.The Glycosylase solution of the purification of fermentation sample of the dilution of equal-volume (each 60 μ L) and dilution mixed and in 37 ℃ of incubations 4 hours.
Resulting deglycosylated CTLA4-Ig (2 μ g) is loaded into the Waters based on polymkeric substance (multipolymer of polystyrene and poly N-vinyl-2-Pyrrolidone)
Figure A200680053116D0257155415QIETU
On the reversed phase extraction cylinder post (2.1 x 20mm).The post that loads is with 5% solvent B (solvent orange 2 A: 1% water-soluble formic acid, solvent B: 1% formic acid that is dissolved in acetonitrile) wash 5 minutes with desalination with 0.2mL/ minute flow velocity, wherein make eluent turn to refuse.When finishing in 5 minutes, gradient (5% solvent B to 95% solvent B in 10 minutes) begins wash-out CTLA4-Ig from post fast; This is in the shunting of flowing (chromatographic system of use is the Waters Alliance2695 that is equipped with Waters 2996 detectors) back and with 45 μ L/ minutes eluent is imported mass spectrograph (Waters Micromass Q-Tofmicro TM) in.
About Q-Tofmicro TThe capillary voltage of M is made as 3kV, and sample cone voltage is 40V.Scanning in per 0.9 second on average becomes 1 scanning; The scanning room time is 0.1 second.The Q-Tof analyser is scanned up to 2500 from m/z 800.Spectrum corresponding to the part that is higher than half maximum peak height (in the TIC chromatogram) is used Waters MassLynx TM software makes up.The spectrum of combination is implemented Waters MaxEntl remove flatung.Resolving power is made as the 1Da/ passage, and selects consistent Gauss (Gaussian) infringement model, and wherein half width of highly locating is made as 0.5-1Da.About the minimum strength at Zuo Feng and right peak than all being made as 50%.
Use the N connection of porous graphite carbon (PGC) by liquid chromatography (LC)/mass spectroscopy (LC/MS) Oligosaccharides is analyzed: isolating oligosaccharides is at porous graphite post (Thermo Hypercarb; 4.6 separate x 100mm), wherein use the Waters Alliance 2695 HPLC systems that are equipped with Waters 2996 photodiode array detectors.2 stage gradient that oligosaccharides separates the cumulative acetonitrile ratio that is dissolved in 0.05% TFA of use reach.In first stage of gradient, acetonitrile per-cent 24% during from 9.6% to 80 minute of when injection.In second stage of gradient, 60% during acetonitrile per-cent 24% to 110 minute during from 80 minutes.Use 0.2mL/ minute flow velocity from start to finish.Wash-out stream detects by the UV at the 206nm place and monitors and analyze by mass spectroscopy, wherein uses Waters MicroMass Q-Tof micro TM is used for quality evalution.
Carbohydrate analysis by LC/MS PGC method: (New England Biolabs, Beverly MA) carries out to use PNGase F by proteinic deglycosylated oligosaccharides separation by enzymically hydrolyse.For de-glycosylation, the 1-2mg glycoprotein that is dissolved in the 160 μ L 50mM sodium phosphate buffers that comprise 0.15% (w/v) Rapigest SF (WatersCorporation) is by carrying out sex change in 2 minutes in 100 ℃ of heating.Refrigerative solution is mixed, and (50,000U/mL is dissolved in the 50mM sodium phosphate buffer, pH7.5) to add 40 μ L PNGase F.Sample carries out vortex subsequently in 38 ℃ of incubations 24 hours.The oligosaccharides that enzymatic discharges carries out purifying by high performance liquid chromatography.Anti-phase liquid chromatography (LC) with Thermo HyperCarb 5 μ L (4.6 x 100mm, Phenomenex, Torrance, CA) link coupled Phenomenex Luna 5 μ L C18 post (4.6 x 150mm, Phenomenex, Torrance, CA) flow velocity of going up with 1.0mL/ minute carries out.Post carries out balance with 0.05% trifluoroacetic acid (TFA) before injection.After the sample injection (150 μ L digestion mixture), initial acetonitrile gradient stops in the time of 15 minutes with under the 0.05%TFA solvent composition that is dissolved in 12% acetonitrile.Glycan is subsequently by washing wash-out from the HyperCarb post with the 0.05%TFA that is dissolved in 60% acetonitrile.Glycan is by detecting in the UV at 206nm place absorbancy.The peak of collection wash-out from Hypercarb washing and be concentrated into drying in a vacuum.Before follow-up injection, Luna C18 post cleans with the 0.05%TFA that is dissolved in 40% acetonitrile, 40% Virahol, 20% water.
The isolating oligosaccharides profile analysis of PGC
The system that is used for the PGC chromatography of isolating oligosaccharides is made up of the Waters Alliance that is equipped with Waters 2996 photodiode array detectors, wherein uses Hypercarb post (2.1 x 100mm).The oligosaccharides sample uses acetonitrile gradient to carry out wash-out.
Acid moving phase wash-out: the acetonitrile gradient that is dissolved in 0.05% trifluoroacetic acid (TFA).2 stage gradient of cumulative acetonitrile are used for the chromatographic separation of oligosaccharides.Second gradient of the 60% acetonitrile increase acetonitrile volume percent when the 24% initial linear gradient that increases the acetonitrile volume percent during from 9.6% to 80 minute of when injection be during from 80 minutes 24% to 110 minute subsequently.Gradient is used 0.15ml/ minute flow velocity from start to finish.Wash-out stream is monitored with Waters UV detector at the 206nm place, is used for quality evalution for Micromass Q-Tof Micro subsequently.The following setting of ionization parameter about the EST probe: capillary voltage=3kV, awl voltage=45V, the desolvation temperature of 80 ℃ of source temperatures and 175 ℃.
Alkalescence moving phase wash-out: be dissolved in 0.4% ammonium hydroxide (NH 4OH) acetonitrile gradient is used for the chromatographic separation of oligosaccharides.28% linear gradient that increases the acetonitrile volume percent with 0.15mL/ minute flow velocity during from 10.4% to 150 minute of when injection is used to produce overview.Wash-out stream is monitored with Waters UV detector at the 206nm place, is used for quality evalution for Micromass Q-Tof Micro subsequently.The following setting of ionization parameter about the EST probe: capillary voltage=3kV, awl voltage=45V, the desolvation temperature of 80 ℃ of source temperatures and 175 ℃.
The oligosaccharides digestion mixture is with the direct profile analysis of PGC: (4.6 x 100mm, Waters Alliance Thermo) forms by assembling Luna C18 and Hypercarb porous graphite post to be used for the system of PGC chromatography of oligosaccharides digestion mixture.This system engages with Waters 2996 photodiode array detectors and Q-ToF Micro (Micromass).Proteinic de-glycosylation uses PNGase F to carry out by enzymically hydrolyse.For de-glycosylation, the 1-2mg glycoprotein that is dissolved in the 160 μ L 50mM sodium phosphate buffers that comprise 0.15 weight % Rapigest SF (Waters Corporation) is by carrying out sex change in 2 minutes in 100 ℃ of heating.Make refrigerative solution thorough mixing, and (50,000U/mL is dissolved in the 50mM sodium phosphate buffer, pH7.5) to add 40 μ L PNGase F.Sample mixes and subsequently in 38 ℃ of incubations 24 hours.The oligosaccharides that enzymatic discharges carries out profile analysis by high performance liquid chromatography.Anti-phase liquid chromatography (LC) is being carried out with the last flow velocity with 1.0mL/ minute of Thermo HyperCarb post 5 μ L (4.6 x 100mm) link coupled Phenomenex Luna 5|u C18 post (4.6x150mm).Post carries out balance with 0.05% trifluoroacetic acid (TFA).After the sample injection (150|i digestion mixture), initial acetonitrile gradient stops in the time of 11 minutes with under the 0.05%TFA solvent composition that is dissolved in 9% acetonitrile.The post switching is used to separate the hypercarb post, and glycan is used linear gradient wash-out from the Hypercarb post of cumulative acetonitrile per-cent subsequently.Use 36% during from 9% to 160 minute of when injection increases the linear gradient of acetonitrile per-cent.60% acetonitrile during when second gradient relates to from 160 minutes 36% to 170 minute increases the acetonitrile volume percent.Gradient is used 0.15mL/ minute flow velocity from start to finish.Glycan is by detecting in the UV at 206nm place absorbancy, and the mass range by MS scanning 400-3000m/z.The MS parameter is made as following value: kapillary 3kV, awl 45V.Before follow-up injection, Luna C18 post cleans with the 0.05%TFA that is dissolved in 40% acetonitrile, 40% Virahol, 20% water.
The analysis of analysing by the porous graphite carbon-coating: use from the structure of the oligosaccharides of structural domain I-IV and to analyse (PGC) with MS link coupled porous graphite carbon-coating and study.N connection oligosaccharides from CTLA4-Ig, separating and purifying described in HPAEC-PAD part formerly.Oligosaccharides uses Hypercarb (PGC) post with UV detector, analyzes for Q-TOF ESI/MS subsequently.The PGC overview that joins oligosaccharides from CTLA4-Ig about the N that discharges by PNGase F digestion is shown among Figure 56, and wherein structural domain is indicated.Elution order is with observed the sort of identical in HPAEC-PAD.The structure that obtains is shown in the table 60.
The nomenclature that is used for the oligosaccharides of name table 60 demonstration shows hereinafter:
The gray shade zone shows N connection oligosaccharides core texture, wherein P represents PNGase F digestion, and the numeral of back is described the number of seminose (circle), Fucose (triangle of Zhiing downwards), semi-lactosi (triangle that refers to) and sialic acid residues (star) respectively to the right.N-acetyl-glucosamine (GlcNAc) is by square expression.
Detected N connection oligosaccharide structure and quality among the table 60.CTLA4-Ig
Figure A200680053116D02611
Detected N connection oligosaccharide structure and quality among table 60 (continuing) .CTLA4-Ig
Figure A200680053116D02621
In structural domain I, 6 peaks have been identified, corresponding to the oligosaccharides (structure P2100, P2110, P2120) of 3 kinds of asialoizations.113 daltonian mass discrepancies are because the detection of trifluoroacetic acid (TFA) adducts between predict and the observed quality.Point out that corresponding to the different peaks of P2100 they may be different anomers with 1,463 equal in quality.
In domain II, identified 6 peaks, corresponding to the oligosaccharides (being respectively structure P2111, P2121 and P3131) of 3 kinds of two feelers and three feelers, each self-contained 1 sialic acid residues (the N-n acetylneuraminic acid n, NANA).
In domain II I, 6 peaks have been identified, corresponding to the oligosaccharides (being respectively structure P2122, P3132 and P4142) of 3 kinds of two feelers, three feelers and four feelers, each self-contained 2 sialic acid residues (NANA).
In structural domain IV, 2 peaks have been identified, corresponding to the oligosaccharides (structure P3133 and P4133) of 3 kind of three feeler and four feelers, each self-contained 3 sialic acid residues (NANA).
A mole sialic acid that acid hydrolysis by the CTLA4-Ig molecule is handled and a mole CTLA4-Ig branch Sub or dimeric mol ratio is measured(referring to Figure 25 and 26): in this method, NANA and NGNA cut by acid hydrolysis and protein.The NANA that discharges separates on Rezex Monosaccharide RHM post by HPLC with NGNA, and detects by UV absorbancy (206nm).NANA and NGNA carry out quantitatively based on the NANA of operation simultaneously and the response factor of NGNA standard.Test result is reported as NANA and NGNA and proteinic mol ratio (MR) respectively.This mensuration is determined combination and unconjugated sialic total moles.
The reagent that in mensuration, uses, instrument configuration and chromatography condition: 1M sulfuric acid H 2SO 4(stoste) and 5mM H 2SO 4The moving phase running buffer; 1mg/ml NANA standardized solution; The 1mg/mlNGNA standardized solution.Alliance chromatographic system-the have WatersCorporation 2695 Separations Module of the automatic sampler of integration; Rezex monose RHM post-8 micron, 7.8x300mm, Phenomenex is equipped with 7.8 x 50mm guard columns, Phenomenex; Detector---Waters Corporation 996 photodiode array detectors or WatersCorporation 2487 dual wavelength absorption photometric detectors.Chromatography parameter: flow---0.600mL/ minute; Moving phase---5mM H 2SO 4Volume injected---5 μ L; Target level---1mg/ml; Working time---25 minutes; Column temperature---40 ℃; The automatic sampling actuator temperature---4 ℃; Wavelength-206nm; Retention time NANA (system is dependent)-10.8 minute (+or-1 minute), retention time NGNA (system is dependent)-9.8 minute (+or-1 minute).
System's suitability criterion: with 900 μ L H in each 1mg/mL NANA of 50 μ L and the NGNA adding suitable containers 2O.Preserve in 4 ℃ maximum 3 months; N-n acetylneuraminic acid n working standard (0.05mg/mL); N-hydroxyacetylneuraminic acid working standard (0.05mg/mL).
The hydrolysis of sample and CTLA4-Ig standard material: sample and CTLA4-Ig standard material are at H 2Be diluted to 1mg/mL among the O and be used for hydrolysis.CTLA4-Ig sample and CTLA4-Ig standard material pass through 10 μ L1M H 2SO 4Add among the sample of 190 μ L1mg/mL dilution and the CTLA4-Ig and be hydrolyzed.Hydrolysis is carried out in the 1.5mL Eppendorf tube in duplicate.Lid obtains fastening by cover lock or adhesive tape.Pipe mixes by vortex and placed 80 ℃ of heat blocks 1 hour.Behind the incubation, take-off pipe from heat block in room temperature cooling 3 minutes, and places whizzer to be used for fast rotational, so that sample collection is to the bottom of pipe.From pipe with 50 μ L hydrating solution aliquots containigs in sample flasket, described sample flasket place the refrigerative automatic sampler be used for the injection.Add 10 μ L 1M H by 190 μ L water in the 1.5mL Eppendorf tube 2SO 4The hydrolysis blank is prepared as single preparation.Blank corresponds with a sample and processes.
System's suitability: for the check system suitability, 6 duplicate injections of injecting systems suitability criterion (each 5 μ L) are injected for 1 time of hydrolysis blank (5 μ L) subsequently.Use last 1 subsystem suitability criterion injection, difference calculating resolution (R), acceptable value is higher than 1.3, and theoretical tray (N), and acceptable theoretical tray counting is necessary at least 4000.Use last 5 subsystem suitabilities to repeat, calculate the circulation ratio of NANA counting, and assessment hydrolysis blank.
Resolving power: use last 1 subsystem suitability criterion injection, equation calculates peak resolving power: R=2 (T2-T1)/(W2+W1) below using, wherein R is a resolving power, T2 is the retention time at NANA peak (peak 2), T1 is the retention time at NGNA peak (peak 1), W2 is a width that describe and the baseline place tangent line in 2 sides, peak, and W1 is a width that describe and the baseline place tangent line in 1 side, peak.Figure 25 describes general system's suitability injection.
Theoretical tray (N): use last 1 subsystem suitability criterion injection, equation theory of computation column plate counting (N) below using: N=16 (RT 2/ W), wherein N is a theoretical tray counting, and RT is with minute retention time at the NANA peak of expression, and W is a width that describe and the baseline place tangent line in side, NANA peak.
Last 5 injections of system's suitability criterion are used to calculate average area counting and the standard deviation thereof about NANA.The coefficient of variation is equal to or less than 3%.The hydrolysis blank does not contain any remarkable peak of the retention time with NANA and NGNA.
Injection sequence: 1 injection of each of injection NANA and NGNA working standard, be the CTLA4-Ig specimen material of hydrolysis (bipartite sample) subsequently, be the CTLA4-Ig material of hydrolysis subsequently.After the CTLA4-Ig operation fully, 1 injection of each of injection NANA and NGNA working standard.
In order to measure NANA or the NGNA mole of injecting in the working standard, equation below using: mole NANA or NGNA=(C) be (I)/MW (P), wherein C is NANA and the NGNA concentration in the working standard, P is the purity of standard, I is a volume injected, MW is molecular weight (is the 309.2g/ mole and is the 325.3g/ mole about NGNA about NANA).
In order to measure NANA and the NGNA mole in the CTLA4-Ig sample, equation below using: mole NANA in the sample or NGNA=(X) (Y)/Z, wherein X is the mole number in the working standard of NANA and NGNA, Y is NANA in the CTLA4-Ig sample and the average counter of NGNA, and Z is the average area of bipartite NANA and NGNA in the working standard.According to the standard of duplicate injection, the area of NANA and NGNA standard counting must have less than 10%RSD.
In order to measure the amount of injecting in every kind of sample, equation below using: mole protein=(C) (D) (I)/MW, wherein C is the CTLA4-Ig dimer concentration of representing with g/ml (deriving from UV analyzes), D is the dilution (0.95) about hydrolysis, I is volume injected (0.005ml), with MW be as according to the dimeric molecular weight of the CTLA4-Ig of mass spectrometric determination (92,439g/mol).
The proteinic mol ratio of NANA or NGNA and CTLA4-Ig (MR) is calculated by following equation: MR=A/B, wherein A is that mole number and the B of NANA or NGNA are CTLA4-Ig molecule or dimeric mole number.
The proteinic mol ratio of sialic acid, NANA and NGNA and CTLA4-Ig (MR) is calculated by following equation: MR=(A+B)/C, and wherein A is the mole number of NANA, B is that mole number and the C of NGNA is CTLA4-Ig molecule or dimeric mole number.Bipartite CTLA4-Ig sample and CTLA4-Ig standard material must have less than 10%RSD in about the mol ratio of NANA.
The linearity of measuring by the hydrolysis method of measuring sialic acid content of replying: (with regard to the NANA normal concentration of the μ g/mL (200% nominal NANA standard) of～0.1% nominal NANA standard=NANA～QL)-98.7, the NANA RACK response acknowledge is linear with regard to 0.5 μ g/mL.For the NGNA normal concentration of 5.0 μ g/mL (10% nominal NGNA standard)-82.0 μ g/mL (160% nominal NGNA standard), the NGNA RACK response acknowledge is linear.
With regard to the protein concn of 0.25mg/mL (25% nominal CTLA4-Ig load)-2.0mg/mL CTLA4-Ig (200% nominal CTLA4-Ig load), be linear from the NANA RACK response acknowledge of the CTLA4-Ig material of hydrolysis.For 0.25mg/mL (25% nominal CTLA4-Ig load)-2.0mg/Mlctla4-Ig200% nominal CTLA4-Ig load) protein concn, be linearity from the NGNA RACK response acknowledge of the CTLA4-Ig material of hydrolysis.
Measure the accuracy of the hydrolysis method of sialic acid content: accuracy confirms for the CTLA4-Ig material (1mg/mL) of blending NANA or NGNA standard.
Measure the precision of the hydrolysis method of sialic acid content: confirmatory experiment confirms to cross over the accuracy of instrument (%RSD＜3%) of different sample formulation, different number of days, different instrument and different analysts (about % RSD＜6% of NANA, about % RSD＜12% of NGNA), about the repeatability (%RSD＜4%) and the circulation ratio of sample formulation.NANA and NGNA mol ratio are considered as in 10% and 20% scope of reporting the result respectively accurately.
Measure the scope of the hydrolysis method of sialic acid content: the working range about this mensuration is shown as 0.49mg/mL-3.87mg/mL CTLA4-Ig material.
Measure the limit of detection (DL) of the hydrolysis method of sialic acid content: use photodiode array detector (PDA; HPLC system 1) the indivedual DL values for NANA and NGNA standard are respectively 0.464 μ g/mL and 0.402 μ g/mL; Use dual wavelength detector (HPLC system 2) to be respectively 0.131 μ g/mL and 0.111 μ g/mL for indivedual DL values of NANA and NGNA.For 2 kinds of sialic acid kinds with based on the least use of sensitive detector, this method DL is 0.5 μ g/mL for NANA and NGNA.
Measure the quantitation limit (QL) of the hydrolysis method of sialic acid content: use photodiode array detector (PDA; HPLC system 1) the indivedual QL values for NANA and NGNA standard are respectively 1.68 μ g/mL and 1.52 μ g/mL; Use dual wavelength detector (HPLC system 2) to be respectively 0.48 μ g/mL and 0.41 μ g/mL for the QL value of NANA and NGNA.For 2 kinds of sialic acid kinds with based on the least use of sensitive detector, this method QL is 1.7 μ g/mL for NANA and NGNA.
Weather resistance/soundness: about use, different N ANA and NGNA batch use of 48 hours refrigerated stabilities of solution of sample, different posts and have ± use of the moving phase that 5% concentration changes, this method turns out to be firm.
The IEF gel electrophoresis: the DI of glycoprotein also can and measure handle with the removal sialic acid with neuraminidase before afterwards.Sialic existence on the glycoprotein is pointed out in the increase that neuraminidase is handled among the pI of back.The IEF gel can be used to measure the number of iso-electric point, CTLA4-Ig isoform.The appropriate system that is used to move the IEF gel is the IEF gel (Amersham Biosciences) of Multiphore II Electrophoresis System and pH4.0-6.5.Anode buffer liquid has following composition: the 0.1M L-glutamic acid that is dissolved in 0.5M phosphoric acid.The negative electrode damping fluid has following composition: the 0.1M Beta-alanine.The IEF gel carries out prefocus and reaches 〉=300V until voltage under firm power (25 watts) and electric current (25mAmps).Load the sample of IEF calibration standard and suitable concn, and gel operation under firm power (25 watts) and electric current (25mAmps), use maximum value 2000V, move 2.5 hours.After IEF gel sets and the Coomassie blue stain, protein band manifests by photodensitometer.The general IEF gel of CTLA4-Ig dimer preparation is shown among Figure 11.
The separation and the sign of the strand of embodiment 4:CTLA4-Ig (monomer)
The preparation of natural strand CTLA4-Ig: the sample of the CTLA4-Ig recombinant protein by method of the present invention preparation by non-sex change SEC use 2695 Alliance HPLC (Waters, Milford, MA), at placed in-line 2 21.5 x 300mm TSK
Figure A200680053116D02661
G3000SW XL(Tosoh Bioscience, Montgomery separate on PA) preparation type post.Sample separates under isocratic condition with 30 injections (each 1.0mL) of～50mg/mL, wherein uses the NaH by 0.2M 2PO 4, 0.9% NaCl, the moving phase that pH7.0 forms was with 1.0mL/ minute flow velocity.Sample uses Water ' s 2996 PDA detectors to monitor under the absorbancy of 280nm.Analyze and use Waters Millennium 4.0 TMAnd Empower
Figure A200680053116D0267160015QIETU
Software carries out.From 90 to 150 minutes collection fractions (each 1.0mL) on Foxy 200 automatization fraction collectors.Merge fraction 16-39 (begin and finish) and use the centricon thickener that ends to concentrate with 3500MW with 129mL with 105mL.
Sample (with the 2.0mL of～4mg/mL) uses HiLoad 26/60Superdex 200 preparation scale post (Amersham Biosciences under the sex change condition, Piscataway, NJ) carry out further chromatography with 2.0mL/ minute flow velocity such as degree such as grade, wherein use the 200mMNaH under pH6.0 2PO4, the conduct of 6.0M Guanidinium hydrochloride
Figure A200680053116D0267160057QIETU
(AmershamBiosciences) moving phase on.Collect fraction 12-16, merge, use HiPrep 26/10 desalting column (Amersham Biosciences) buffer exchange to be 200mM NaH 2PO 4, pH6.0, and final concentrating.
The preparation of inductive strand CTLA4-Ig: inductive strand CTLA4-Ig is prepared by sex change, reduction and the alkylation of CTLA4-Ig recombinant protein, and described CTLA4-Ig recombinant protein is prepared by method of the present invention.(0.684g) is weighed in the 1.5mLEppendorf centrifuge tube with Guanidinium hydrochloride, and adds 512 μ L200mM NaH 2PO 4, pH6.0 and carry out vortex dissolves fully until Guanidinium hydrochloride.The CTLA4-Ig recombinant protein by with 238 μ LCTLA4-Ig materials (concentration: 50mg/mL) add in the aforementioned tube and carry out sex change and carry out vortex, thereby cause being dissolved in the 6.0M Guanidinium hydrochloride～the CTLA4-Ig final concentration of 10.0mg/mL.The protein of sex change is by adding 2.6 μ L1.0M DTT and reducing in 90 minutes in 37 ℃ of incubations.Reductive protein carries out alkylation by following subsequently: 0.047g iodo-acid amide solid added in the sample mixture, and vortex subsequently, and in 37 ℃ of incubations 60 minutes in the dark.Sample (for the 2.0mL of per injection with～4.0mg/mL) uses HiLoad26/60Superdex200 preparation scale post to carry out chromatography with 2.0mL/ minute flow velocity such as degree such as grade under the sex change condition, wherein uses
Figure A200680053116D0267160136QIETU
Go up the 200mM NaH under pH6.0 2PO 4, the 6.0M Guanidinium hydrochloride.Collect resulting strand fraction (9-12), merge, buffer exchange is 200mM NaH on HiPrep 26/10 desalting column 2PO 4, pH6.0, and concentrate.
The MALDI-TOF analytical reagent composition of natural and inductive strand CTLA4-Ig: strand sample (20 μ L) carries out desalination and (Millipore, Billerica MA) concentrate, and carry out wash-out with 20 μ L, 80% acetonitrile that has by 0.1% saturated TFA of sinapinic acid subsequently with C4 ZipTips.On the hole of MALDI sample panel, and before placing mass spectrograph, allow it air-dry mixture (1.0 μ L) point.(Bruker Daltonics MA) goes up use nitrogen laser (337nm) and obtains the MALDI-MS spectrum at the OmniFlex mass spectrograph.Sample is analyzed by the extraction that postpones with reflection, positive ion mode, wherein uses the time of lag of acceleration voltage and the 200ns of 20kV.Add up 250 injection spectrums (single-shot spectia) altogether for every kind of sample.Use the mixture of trypsinogen (23982m/z), A albumen (44613m/z) and bovine albumin (66431m/z) to reach external calibration.
Use the strand analysis of sex change (guanidine HCl) size exclusion chromatography: the CTLA4-Ig supernatant liquor preparation of the growth of cell culture of collecting on the difference in the next comfortable time-histories process is used for HPLC and analyzes.Sample is prepared by following: weighing 0.114g Guanidinium hydrochloride (Mallinckrodt BakerInc.) in 0.65mL Eppendorf Eppendorf tube; Add 125uL time-histories CTLA4-Ig sample, and vortex so that guanidine HCl dissolve fully.Immediately add 1.8uL250mM iodo-acid amide and mixing, and in 37 ℃ of incubations 30 minutes.
Series connection with TSK post protection (SWXL, 6.0mm ID x 4.0cm)
Figure A200680053116D0268160225QIETU
The strand SEC that G3000SWXL size exclusion post (7.8mm ID x 30cm) is used for carrying out on Waters 2695 separation modules with 2996 photodiode array detectors analyzes.Every kind of sample of 25 μ L is expelled to 200mM sodium phosphate, 6.0M Guanidinium hydrochloride pH6.0 as on the moving phase equilibrated post.Protein flow velocity with 0.5mL/ minute on post separates, and resulting chromatogram is collected through 60 minutes windows.The integration at indivedual peaks (monomer, strand, etc.) and quantitatively use Empower Pro software to carry out.Correctly work in order to ensure the HPLC system, injection also moving phase, protein example damping fluid, system's suitability criterion and sample is injected before and present CTLA4-Ig material afterwards carries out.Calculating is about the peak resolving power and the plate counting of system's suitability criterion chromatogram.
The CTLA4-Ig strand is by the cysteinyl fractional analysis of LC/MS peptide analysis: CTLA4-Ig carries out sex change and reduction in the 50mM Tris damping fluid (pH8.0) that comprises 6M guanidine and 5mM dithiothreitol (DTT) (DTT).In 50 ℃ of incubations after 20 minutes, add iodo-acid amide (IAM) to the final concentration of 10mM so that the free mercaptan alkylation, and continued incubation in the dark other 20 minutes in 50 ℃.To reduce and alkylating mixture be loaded into desalting column (Amersham, NAP-5) on, use 50mM Tris subsequently, 10mM CaCl2, pH8.0 or 50mM sodium phosphate buffer, pH7.5 is eluted to void volume.After the desalination, reduction/alkylating CTLA4-Ig uses 2 kinds of different proteolytic enzyme to digest: trypsinase or Asp-N.Also do not implement trypsinase/pancreas milk reducing protease digesting to having reduction and alkylating CTLA4-Ig material.
For tryptic digestion, add sequence grade trypsin Roche, 2%, w/w, enzyme/protein) and mixture in 37 ℃ of incubations 4 hours.For Asp-N digestion, add sequence grade Asp-N (Roche, 2%, w/w, enzyme/protein), and mixture was in 37 ℃ of incubations 16 hours.For trypsinase/pancreas milk reducing protease digesting, add sequence grade trypsin Promega, 4%, w/w, enzyme/protein), and mixture was in 37 ℃ of incubations 4 hours, add a-Quimotrase (Sigma, 4%, w/w subsequently, enzyme/protein), and mixture in 37 ℃ of incubations 16 hours.All samples carries out freezing (20 ℃) after digestion.
Resulting peptide mixt passed through from Waters Atlantis with 0.12mL/ minute on Waters Alliance HPLC Workstation TGradient elution separates in the M dC18 post (2.1 x 250mm).Post and the Waters Micromass Q-Tof micro that is equipped with the ion injection source TThe M mass spectrograph directly connects and is used to collect mass spectrum.Peptide mixt is gone up with 0.70mL/ minute at Varian Polaris C18 post (4.6x 250mm) equally and is used identical HPLC workstation to separate.Post carries out balance with solvent orange 2 A (water-soluble 0.02%TFA), and peptide carries out wash-out by the cumulative concentration of solvent B (95% acetonitrile/0.02%TFA, water-soluble).The rear pillar diverter valve is used for 15% the direct traffic Q-Tof workstation with positive TOF (flight time) pattern (m/z 100-2000) operation.The general ion injection electric that uses is 3000V.
The loss of 113 ± 4u hint exists proteinic covalent disulfide bonds modification after reduction.Change about the cysteinyl prediction is 119.14u.Yet, in the actual mass loss of strand kind reduction back expection 111.14u.119.14u loss result from the removal of halfcystine, and the acquisition of 8u results from the halfcystine (Cys of SEQ ID NO:2 in 8 chains 47,74,92,118,197, 157,303,361) reduction back 8 protons interpolation.Therefore, the other 113 ± 4u on full-quality is corresponding to the cysteinylization with cysteine amino acids.The halfcystine that most probable is modified is Cys 146, because based on complete MALDI data, interchain disulfide bond does not exist in the strand kind.Use the LC/MS peptide analysis to comprise Cys with inspection 146Peptide, find retention time and quality that reduction is different with the strand material with the demonstration of non-reduced material.In order to confirm cysteinylization, collection comprises Cys 146The strand peptide and analyze with MALDI.
That collects comprises Cys 146The peak have the quality of 1787.48u, and for having Cys 146The 1787.51u uniform quality (Figure 27, little figure A) of peptide prediction of cysteinylization.This peptide is implementing reduction back loss 119.11u, and is consistent with the prediction loss of 119.14u; The losing of halfcystine (Figure 27, little figure B).This material is further handled with iodo-acid amide subsequently, produces the change of 56.99u, obtains consistent (Figure 27, little figure C) with the 57.03u quality of prediction.
Embodiment 5: handle monomer or polymer CTLA4-Ig preparation
Substratum and the agonism of nutrient media components to the strand preparation: the agonism of different substratum and nutrient media components forms with regard to strand and measures.The CTLA4-Ig molecule can be under 37 ℃ with various substratum and indivedual substratum moiety incubations time period through 60 hours, and form with regard to strand by columns in series sex change SEC and to analyze.Find to reply after in the preparation damping fluid, adding the 10mM halfcystine about the overwhelming agonist that strand forms.The fast rise that exists strand to form, this adds the back at halfcystine and reached the peak in about 6 hours.This replying in residue gradually reduced in 56 hours.In addition ,+the 30%gal charging influences strand to be increased more progressively but still relatively fast in forming.+ 30%gal charging is the composition of semi-lactosi and 117E.Add this mixture every day with the cell of feeding.Although in this experiment, halfcystine does not rely on artificial a large amount of introducing of 117E, needs to determine which kind of 117E component can relate to strand and form.
The specific components of 117E and other substratum is with the time period of CTLA4Ig incubation through 60 hours, and forms with regard to strand by columns in series sex change SEC and to analyze.This Study on culture medium concentrates on possible disulfide bond reduction component and/or inhibitor, and this will realize that strand forms based on previous experiment, show that interchain disulfide bond does not exist.Test the known moiety that disulfide linkage is had the 117E of some reduction influences; Thioctic Acid, Gelucystine, halfcystine, methionine(Met) and gsh.Once more, in the preparation damping fluid, find to reply behind the interpolation halfcystine about the overwhelming agonist that strand forms.Have the rapid answer of strand in forming, this adds the back at halfcystine and reached the peak in about 6 hours.This replying in residue gradually reduced in 56 hours.Main strand forms with the substratum that comprises halfcystine and takes place: halfcystine, yeast autolysis solution and fermention medium.Other sulfur component for example methionine(Met) and gsh form strand and do not have to very little influence.Do not observe the influence of ammonium chloride.
Therefore the present invention comprises and is used to provide proteinic strand: the method for dimeric forms ratio, this proteinoid can exist with dimer and single stranded form, comprising that step (1) provides and/or keeps (for example during step (2)) liquid cell culture medium is used for the described proteinic cell of culture expression, wherein select to reduce or to suppress the concentration of the reagent (for example halfcystine) that dimer forms and cultivate described cell to express described protein so that described ratio and (2) to be provided.This kind reagent (for example in interpolation and/or the increase liquid cell culture medium, halfcystine) concentration provides this kind protein higher strand: the dimeric forms ratio, and the concentration of removing, reduce or eliminate this kind reagent in the liquid cell culture medium (for example, halfcystine) reduces described proteinic strand: the dimeric forms ratio.
A specific embodiments of this method is that wherein said protein is to form dimeric glycoprotein, CTLA4-Ig molecule for example of the present invention by forming interchain disulfide bond.
The agonism that high salt pair high molecular forms: during purge process, make CTLA4-Ig be exposed to the various time quantums of high salt concentration.The influence of high salt concentration forms with regard to high molecular and measures.CTLA4-Ig (with～50mg/mL) at the 500mM sodium phosphate, pH=6.0 carries out incubation under 37 ℃ the existence.When high concentrations of phosphoric acid sodium, there is exciting influence; Observing the high molecular form in 100 hours time period mainly is persistent in the tetramer, increase fast.
Therefore the present invention comprises and is used to reduce proteinic aggregation: the method for dimeric forms ratio, during the proteinic processing of this kind (for example during the purifying), this kind protein can exist with aggregation and dimeric forms, comprises the use as one or more liquid of non-aggregation salts solution." non-aggregation salts solution " refers to comprise therein the liquid of dissolved salt concn, and with respect to the same liquid of this kind salt that comprises greater concn, its excitability in aggregation forms is less.
A specific embodiments of this method is that wherein said protein is to form dimeric glycoprotein, CTLA4-Ig molecule for example of the present invention by forming interchain disulfide bond.
Antagonistic action to strand formation: previous modeling experiment confirm comprises halfcystine by interpolation component forms great and influence fast to strand.Halfcystine is the amino acid that comprises free sulfhydryl groups.If sulfydryl relates to the formation of strand, it should be blocked by using antagonist compound so.A kind of this kind compound is an iodo-acid amide.Iodo-acid amide is to react to form the water-soluble cpds of irreversible thioether bond with immediate mode and any free sulfhydryl groups.CTLA4-Ig is with various substratum, halfcystine and the iodo-acid amide incubation time period through 60 hours, and forms with regard to strand by columns in series sex change SEC and to analyze and to analyze with regard to HMW by the non-sex change SEC of columns in series.Iodoacetamide is not only blocked strand formation and is also blocked aggregation formation in CTLA4-Ig composition and high salt.Iodo-acid amide is not blocked aggregation formation in low sialic acid monomer.Yet the amount of the aggregation that forms in low sialic acid material is comparable to the amount that forms in the CTLA4-Ig composition.
This model provides the understanding to the mechanism that does not before fully understand as yet.Seem in the CTLA4-Ig method of having identified, to exist the main path of at least 2 kinds of aggregations formation.First kind of approach produces a large amount of aggregations, and the free hydroxyl group halfcystine serves as agonist and is used to form the strand kind.The excitement influence of halfcystine can be blocked by adding iodo-acid amide.Surprisingly, to be not only that strand forms still be the antagonist that high molecular forms to iodo-acid amide.Should be understood that this method is designed to produce and the fermentation during downstream purification is compared, comprise the sialic composition of increasing amount.In second kind of approach of the aggregation that produces much less, with regard to the formation of strand or aggregation, comprise the influence that a small amount of sialic subspecies are not subjected to iodo-acid amide.
Therefore the present invention comprises and is used to reduce proteinic strand: the method for dimeric forms ratio, this kind protein can exist with dimer and single stranded form, comprising that step (1) provides and/or keeps (for example during step (2)) liquid cell culture medium is used for the described proteinic cell of culture expression, this kind substratum comprises cultivates described cell to express described protein to the reagent (for example iodo-acid amide) of strand formation antagonism and (2).
Therefore the present invention comprises and is used to reduce proteinic aggregation: the method for dimeric forms ratio, this kind protein can exist with aggregation and dimeric forms, comprising that step (1) provides and/or keeps (for example during step (2)) liquid cell culture medium is used for the described proteinic cell of culture expression, this kind substratum comprises to aggregation chain formation antagonism and/or to reagent (for example iodo-acid amide) and (2) of strand formation antagonism cultivates described cell to express described protein.
A specific embodiments of this method is that wherein said protein is to form dimeric glycoprotein, CTLA4-Ig molecule for example of the present invention by forming interchain disulfide bond.
Based on these data, at least 2 kinds of approach that form about aggregation not hard to imagine are induced in CTLA4-Ig.In main path, strand relates to the formation of aggregation by the mechanism of understanding fully not yet.The second kind of minor path that does not rely on strand formation relates to the formation of aggregation.These approach can help to explain the high molecular weight species that has at least 3 kinds of forms that can chromatographic separation why.These are models and must test in the real attenuation method, to determine the magnitude of actual influence (if present) and influence.Should consider not only to test the fermentation of present fermentation process but also test shortage halfcystine based on this data.
The embodiment 6:CTLA4-Ig dimer and the tetramer
Be bonded to the CTLA4-Ig dimer and the tetramer of B7-1Ig
The invention provides and be used for assessment and B7-1Ig bonded CTLA4-Ig dimer and tetrameric method.Physical features (for example, spread coefficient, molecular weight, combination are tired) and Instrument operation parameter (for example flow velocity, chip density) can influence the Biacore measurement result.Under the mass transfer restriction; For same molar ratio, tetrameric association rate is than dimer about slowly 20%.Under given flow rate, to compare with dimer, tetramer molecule may not penetrate matrix more effectively.In high-density B7-1Ig immobilization chip, the tetramer and dimeric competitiveness are in conjunction with show comparable inhibition curve.This point out the tetramer (4) to the theory of dimer (2) tire to the not influence that combines of B7-1Ig.Tetrameric volumetric molar concentration can be calculated based on the dimer typical curve.Make in this way, find to compare with dimer, the tetramer has the dose-dependently that equates with the combination of B7-1Ig and replys.
The tetramer and dimeric influence in conjunction with the unit of measure of rendeing a service that relatively is used for preparation standard and sample.The tetramer and dimer sample can compare when the concentration of 2000ng/mL, on quality (ng/mL) basis, use each kind of typical curve of identical type to show about 100% combination effectiveness.Yet tetrameric combination is renderd a service and is reduced by half when measuring on the dimer typical curve, and dimeric combination is tired above twice when measuring on tetramer typical curve.Because the Biacore instrument is based on the quality examination interaction of molecules, so should be identical with dimeric signal resonance units (RU) from the tetramer of same concentrations (ng/mL).Although detection system is the function of quality, the interaction between the molecule takes place on mole foundation.Therefore, the tetrameric volumetric molar concentration of CTLA4-Ig dimer and CTLA4-Ig is respectively 21.6nM and 10.8nM when 2000ng/mL.Use volumetric molar concentration, CTLA4-Ig dimer and CTLA4-Ig tetramer sample are show comparable in conjunction with rendeing a service on the dimer typical curve.The same molar ratio method of use on CTLA4-Ig tetramer typical curve compared with tetramer sample, and CTLA4-Ig dimer sample is presented in conjunction with other 30% increase in rendeing a service.This observation is owing to the minimizing in the slope of tetramer typical curve when high density, thereby causes the concentration for the higher calculating of given initial association rate.A kind of explanation about this observation is the effect of mass transfer.
Mass transfer restriction experiment points out that for the similar number molecule the tetrameric initial association rate of CTLA4-Ig is than about slowly 20% (being that tetrameric association rate and dimer differ the factor 0.8) of CTLA4-Ig dimer.This observed difference is owing to the molecular weight of 2 kinds and to the effect of molecular diffusion to chip surface.The spread coefficient of molecule and the cubic root of molecular weight are inversely proportional to.Lower spread coefficient moves more slowly with indication molecule.Based on dividing other molecular weight, the spread coefficient that the CTLA4-Ig tetramer calculates is the sort of 0.8 times of CTLA4-Ig dimer, or dimer is the sort of 1.25 times of the tetramer on the contrary.Experimental data and CTLA4-Ig dimer wherein show that this observations of 133% effectiveness is consistent, as by the calculating of CTLA4-Ig tetramer typical curve use volumetric molar concentration.Mass transfer on superchip is limited in low flow velocity and is more remarkable during than the harmonic analysis substrate concentration.Along with flow velocity increases, to compare with the tetramer, dimer shows initial faster association rate.Therefore, the molecular weight and the lower spread coefficient of tetramer increase are facilitated the initial association rate difference of comparing with dimer.
The CTLA4-Ig tetramer and dimer combine with the competitiveness of B71Ig points out that the tetramer and dimer show that under the mass transfer restricted condition similar combination tires.The other tetramer is to pointing out the low potentiality that combine with the effect of dimer or tetramer bonded B7-1Ig chip at first.This observes the binding site operability of restriction no thanks to because other dimer can with combine the tetrameric B7-1Ig chips incorporate of CTLA4-Ig dimer or CTLA4-Ig at first.Limited penetrating into can be explained observed minimizing in the tetramer combination in the matrix.
The wherein property effect result's of binding molecule explanation on Biacore.Owing to the difference in its molecular size, the tetramer and dimer molecule spread with different rates under the mass transfer restricted condition.In addition, penetrate into matrix at the follow-up molecule of the lip-deep sterically hindered influence of superchip.
When on Biacore, carrying out concentration analysis, must consider for example combination of molecular weight, spread coefficient and each kind of physical features.The standard that is used for comparison goal analysis thing should be same material.Yet, when considering molecular size, if standard and sample at the enterprising line display of mole foundation, the tetramer still can be analyzed at the dimer standard so.The data that present point out that the CTLA4-Ig dimer and the CTLA4-Ig tetramer are comparable with combining of B7-1Ig.
The CTLA4-Ig dimer demonstrates similar association rate (k with the tetramer On).Yet the tetramer demonstrates slower dissociation rate (k oFf), this is owing to the avidity owing to the increase in the binding site number.
The 2 kinds of protein for example binding kinetics analysis between part and the acceptor can carry out on Biacore, wherein uses the chip of the low density part of the 600-800RUs that fixedly has an appointment, thereby makes maximum binding capacity (R MaX) be 50-150RU.The purpose of low density chip is to make the effect of avidity and mass transportation drop to minimum.Because when dissociating of indivedual binding sites taken place, available part closely approaching when the maintenance of multivalence analyte combines with chip surface, observed avidity.When in the surface of chip and the analyte concentration between the bulk solution, having significant difference, observe mass transportation.
In one embodiment, the CTLA4-Ig molecule is by 2 dimers that single chain molecule is formed that connect by single interchain disulfide bond, and comprises 2 binding sites about the B7 molecule.In another embodiment, as confirming that by scattering of light, SEC and SDS-PAGE the tetrameric formation of CTLA4-Ig causes having the molecule of 4 binding sites potentially.The monomer of purifying and dimeric binding kinetics and CTLA4-Ig dimer material carry out statistics relatively.At k OnThere is not significant difference (p value〉0.05) in the speed.Tetrameric k oFf speed and dimeric k oFf speed is significantly different.Remove the dimeric k of peak purifying by HIC oThe k of ff speed and CTLA4-Ig dimer material oFf speed does not have remarkable difference.Therefore, the tetramer dissociates more slowly than dimer, thereby points out because the avidity effect of the binding site number/molecule that increases.
The tetramer can be handled to separate and fold and be dissociated into dimer by guanidine.Analyze the CTLA4-Ig dimer that this guanidine is handled, and the result point out its binding kinetics feature class be similar to the CTLA4-Ig that under physiological condition, forms dimeric those.This observe to support following hypothesis: avidity in binding kinetics, work situation in, the k between the dimer and the tetramer oIn the ff speed observed difference tire with the combination of molecule and the inherent nature of particular B iacore method relevant.
Folder is removed the affinity purification of the CTLA4-Ig material at peak from HIC:
A albumen-agarose affinity chromatography: 500mL HIC removes the peak by 0.22 micron 1L filter system (Corning, Corning, NY, numbering of part 430517) filters and be loaded on the rProtein A-Sepharose post (5cm x 15cm), described post is used in the phosphate-buffered saline (Sigma under the pH7.4, St.Louis, MO P-4417) carries out pre-equilibration.This post 700mL PBS, pH7.4 washs, and uses the 100mM glycine, and pH3.5 carries out wash-out.By add 0.5mL 2.0M tris in collection tube, pH10 obtains neutralization to the fraction of each 50mL in collection process.Fraction is measured, is merged at 280nm absorbancy place and uses centriprepYM-3 cylinder (Millipore Corporation, Bedford, MA, numbering of part 4203) to concentrate.The protein soln of purifying is preserved in-70 ℃.
PROSEP-rA (reorganization A protein) affinity chromatography: 500mL HIC removes the peak by 0.22 micron 1L filter system (Corning, Corning, NY, numbering of part 430517) filters, and with 25mL/ minute flow velocity be loaded into PROSEP-rA High Capacity (Millipore Corporation, Bedford is MA) on the post (25cm x 28cm), the described post 25mM Tris that comprises 250mM sodium-chlor, pH8.0 carries out pre-equilibration.The Waters PrepLC system that is equipped with Waters2767 Sample Manager and Waters 2996 photodiode array detectors (Photodiode Array Detector) is used for this chromatography.This post with 25mL/ minute flow velocity washing 30 minutes, and is used the 100mM acetate with 25 level pads, and pH3.0 was with 25mL/ minute flow velocity wash-out 30 minutes.The fraction of each 10mL surpasses 50ul 2.0M tris by collecting before to have added in the pipe in elution process, pH10 obtains neutralization.Having fraction at the high absorbance at 280nm place merges and uses centriprepYM-3 cylinder (Millipore Corporation, Bedford, MA, numbering of part 4203) to concentrate.The protein soln of purifying is preserved in-70 ℃.
Size exclusion chromatography: size exclusion chromatography carries out on Waters Alliance 2695 separation modules, and described module is equipped with Waters 2996 photodiode array detectors, and (Milford is MA) with by Millennium 32The Foxy 200 fraction collectors of version 3 .20 or Empower software control.(Montgomery, PA) TSK G3000 SW (21.5mm x 300mm) and series connection TSK G3000 SWxL (7.8mm x 300mm) post are respectively applied for preparation type and analysis mode SEC to series connection TOSOH BIOSCIENCE.The protein of wash-out is by monitoring in the UV at 280nm place absorbancy.
The SEC that dimer, the tetramer and polymeric preparation type separate by the HIC removing peak material of A protein purification reaches.13 kinds of samples of A albumen eluate (respectively～10mg) are expelled in the preparation type series connection SEC post, wherein use the moving phase that comprises the 100mM sodium orthophosphate (monometallic) pH of buffer 7.0 of 0.9% sodium-chlor with 1mL/ minute flow velocity.For each of 13 kinds of injections, per minute was collected fraction in 90-160 minute.Be 180 minutes the working time about single injection.
Every kind of fraction is checked by columns in series analysis mode size exclusion HPLC.Merge fraction 13-15 (comprising polymer), fraction 22 and 23 (comprising the tetramer) and fraction 43-39 (comprising dimer).The polymer of purifying, the tetramer and dimer fraction detect (DSL) with dynamic light scattering and check on analysis mode 2 post SEC.
HIC removes CTLA4-Ig component and the component of purifying and the life of immobilized B7-1Ig at peak Thing specificity binding analysis: the BIAcore C biosensor (BIAcore, AB, Piscataway NJ) that CTLA4-Ig is used in combination based on SPR with the biologic specificity of immobilized B7-1Ig (on the CM5 chip) is measured.The CTLA4-Ig material is used to produce typical curve.B7-1Ig is with 5000-10, and (density of RU ' s) is immobilized on the activatory CM5 sensor chip 000 resonance units.CTLA4-Ig reference standard, quality contrast and sample are expelled on the B7-1Ig sensor chip surface to produce sensing figure with 20 μ L/ minutes flow velocity.CTLA4-Ig is measuring on high-density B7-1Ig surface under the diffusional limitation condition in the initial association rate (RU/s) on the immobilized B7-1Ig, and this is directly related with the active concentration of CTLA4-Ig in the sample.Standard, quality control sample and unknown sample concentration push away in being undertaken by typical curve, and described typical curve is by producing RU ' s to the CTLA4-Ig plotted against concentration in the 125-8000ng/mL nominal range.Net result is expressed as per-cent in conjunction with (mean concns of the unknown/sample/2000) x 100.
The mensuration of molar mass and hydrodynamic radius: the SEC separation is carried out with TSK3000 SWXL post and corresponding protection post.Moving phase is by 25mM HEPES, 850mM NaCl, and pH7.0 forms, for the use of the wash-out 0.8mL/ minute under isocratic condition.HPLC analyzes and carries out at ambient temperature, and sample is maintained at 4 ℃ during analyzing.Molar mass is measured and is mixed WyattDawn EOS, utilizes 15 different scattering angle to measure the angle variation about the scattering of light of each kind.Zimm drawing form is used for molar mass to be measured, and wherein averages for molar mass about the section of each kind.Concrete index increment (dn/dc) value that is used to calculate absolute molar mass is 0.189, it uses a) and Optilab DSP interferometer (RI obtains.Hydrodynamic radius (Rh) is measured and is carried out with Photon Correlator QELS detector axial (in-line), and described detector is to place at the about 90 ℃ angle of flow cell.The translation diffusion constant is measured according to sort signal, and R hUse einstein-stokes (Einstein-Stokes) relation to calculate.Data analysis is used and is finished from the Astra software version 4.90 of Wyatt Technology.
Discovery is respectively 86-91kDa and 172-199kDa about the molecular weight and the hydrodynamic radius value of dimer and tetramer kind.Observe by molecular weight and to remove the peak sample and comprise other HMW kind corresponding to six aggressiveness and ten aggressiveness.Hydrodynamic radius scope about the dimer kind is 3.8-4.7nm.The hydrodynamic radius scope of heterogeneous tetramer kind is 5.7 to 6.2-6.3nm.
Use the CTLA4-Ig dimer of surface plasma body resonant vibration and the knot of the tetramer and B7-1Ig Close: concentration analysis: (Biacore, Piscataway carry out on NJ), according to Method 7441-4 the concentration analysis of various CTLA4-Ig kinds and B7-1Ig at Biacore 3000 instruments 2Follow minor modifications.Modification comprises following: use Biacore 3000 rather than Biacore C.Flow velocity is 10 μ L/ minutes rather than 20 μ L/ minutes.Opposite with 15 seconds, sample injection 60 seconds.Sensor chip surface was regenerated by following with 30 μ L/ minutes: comprise the 10mM Trisodium Citrate of 100mM NaCl, 3 short 30-second (15 μ L) pulse of pH4.0 (regeneration damping fluid) is 1 30-pulse per second (PPS) of water subsequently.
The CM5 sensor chip is dissolved in acetate buffer in order to the concentration of 20 μ g/mL, and the B7-1Ig of pH5.0 fixes, at the target density of 6000-12000 resonance units (RU).Concentration and dimer to the concentration of 125-16000ng/mL (0.675-86.3nM) of standard by the CTLA4-Ig material series being diluted to 62.5-8000ng/mL (0.675-86.3nM) in the HBS-EP damping fluid is prepared.To be diluted to by the specimen that monomer or dimer are formed～target level of 2000ng/mL and on Biacore, analyzing.Concentration is used CTLA4-Ig material (〉 98% monomer by BIAevaluation software (edition 4 .0.1)) or the dimeric typical curve of purifying measure.Be calculated as the concentration on Biacore, measured per-cent in conjunction with rendeing a service divided by the concentration of measuring by A280nm.
The association rate on molecule and given surface is the function of concentration, and this allows to measure unknown concentration.Compare with the CTLA4-Ig tetramer, the CTLA4-Ig dimer shows the higher association rate as concentration (ng/mL) function.
CTLA4-Ig dimer and tetrameric combination are renderd a service and are summarized in the table 7.Based on ng/mL, the combination of dimer sample is renderd a service and is calculated by the dimer typical curve, and is found to be 99.5%; And the concentration of equal value of tetramer sample is 47.2%.On the contrary, the combination of dimer sample is renderd a service and is calculated by tetramer typical curve, and is found to be 266%; And the concentration of equal value of tetramer sample is 103%.Yet, when dimer and the tetramer are expressed as volumetric molar concentration (nM), 2 kinds be comparable in conjunction with rendeing a service.Be respectively 99.4% and 94.3% by the dimer of dimer typical curve calculating and the combination effectiveness of tetramer sample.On tetramer typical curve, it is respectively 133% and 103%.Based on volumetric molar concentration, dimer and tetrameric typical curve are comparable.
Table 9:CTLA4-Ig dimer and tetrameric in conjunction with rendeing a service.
In conjunction with tiring: (before μ L mixture is expelled to the density of 9392 RU on the immobilized B7-1Ig chip with 10 μ L/ minutes flow velocity, make the CTLA4-Ig dimer (25-1600nM) or the tetramer (25-400nM) and B7-1Ig 30 with various mol ratio pre-mixings 3 minutes.Chip by 3 30-pulse per second (PPS)s of regeneration damping fluid, is regenerated for 1 30-pulse per second (PPS) of water after per injection subsequently.RU ' the s that will obtain when per injection finishes compares.
In theory, to tire be 2 in the combination of dimer molecule; Every strand is made up of 1 binding site.Tetramer molecule is made up of 2 dimer molecules, and therefore has in conjunction with tiring 4.In order to measure dimer and tetrameric apparent combination is tired, design competition mensuration and on Biacore3000, carrying out.In experiment, be injected into B7-1Ig chip (9392 RU) before last 1 minute at mixture with 10 μ L/ minutes flow velocity, make analyte and B7-1Ig with various mol ratio pre-mixings 3 minutes.Table 10 shows dimer or the tetramer per-cent with the B7-1Ig competitive inhibition of cumulative molar weight.When the mol ratio of 1.25 (B7-1Ig) and 1 (dimer or the tetramer), in competitive inhibition, observe significant difference, wherein monomer is 96.1%, and dimer is 84.9%.Yet the dimer and the tetramer show similarly inhibition curve, and this hint is tired and approximately equated.In addition, use more low-density chip, the same similarly inhibition overview that shows of the dimer and the tetramer.
Table 10: suppress the dimer and the tetramer with B7-1Ig
Figure A200680053116D02791
B7-1Ig chip saturated: the CTLA4-Ig dimer or the tetramer (200,1000 or 8000ng/mL) to high-density B7-1Ig chip (6738RU) last 1 minute, are the injection in a series of 7 times 1 minute with monomer or dimer (200,1000 or 8000ng/mL) with 10 μ L/ minute initial injection subsequently.Chip injection by 4 times 25 μ L regeneration damping fluids after each condition is that the injection of 25 μ L water is regenerated subsequently.RU ' the s that will obtain when per injection finishes compares.
Dimer or tetramer duplicate injection to wrapping in advance on the B7-1Ig surface of quilt with the dimer or the tetramer, and there is not surface regeneration.Do not hinder the dimeric combination of injection subsequently with tetrameric initial the combination, yet, to compare with the dimer injection, other tetramer injection causes the obviously association rate of minimizing.Combining at first with dimer is that dimeric follow-up injection causes the combination towards saturated increase subsequently.Follow-up injection shows gradually reducing in the combination for the tetramer of the pre-envelope chip of dimer, and dissociating and lacking the tetramer of hint molecule and chip penetrates in the matrix.Initial injection with 200ng/mL or 8000ng/mL CTLA4-Ig molecule is observed similar results.
The CTLA4-Ig tetramer has for the higher avidity of B7-1Ig acceptor: the CTLA4-Ig kind comprises that the part that discards of purification column for example carries out purifying in the removing peak of HIC and QFF post, and its binding kinetics is analyzed on Biacore.
Remove the CTLA4-Ig kind at peak from HIC: the HIC post is used to remove for example DNA of high molecular weight species in the CTLA4-Ig method.CTLA4-Ig kind from the removing peak of HIC post can be used for subsequent purification and dynamic analysis.Make HIC remove the peak, to catch all CTLA4-Ig kinds and to remove other impurity that may exist through A albumen post.The eluate of being made up of the mixture of all CTLA4-Ig kinds (that is, dimer, the tetramer, six aggressiveness polymers) from A albumen post shows the apparent k that is comparable to CTLA4-Ig dimer standard OnAnd k oFf speed.A albumen eluate causes 3 kinds of CTLA4-Ig kinds by the separation of 2 post SEC: dimer, the tetramer and six aggressiveness/polymer, wherein k OnAnd k oFf speed is summarized in the table 11.Compare with the CTLA4-Ig dimer, the dimeric sialic acid content of purifying of removing the peak from HIC is low.
In table 11, the sample concentration of 75-200nM is tested on B7-1Ig chip (694RU).Compare with the CTLA4-Ig reference, the CTLA4-Ig kind of being removed the peak purifying by HIC shows lower combination.The tetramer of depolymerization provides and the comparable k of CTLA4-Ig reference OnAnd k oFf speed.
Table 11: the dynamic analysis of removing the CTLA4-Ig kind at peak from HIC
Kind k onx?10 5 (1/Ms) k off?x?10 3 (1/s) K A?x?10 7 (1/M) K D(nM)
CLTA4-Ig dimer standard 4.03 9.65 4.18 23.9
A albumen eluate 3.77 9.73 3.87 25.8
Dimer 1.52 5.88 2.59 38.7
The tetramer 1.65 8.07 2.04 48.9
Six aggressiveness 1.54 12.3 1.25 79.9
The tetramer of dimerization 3.12 9.88 3.16 31.7
The statistical analysis use Si Shi T of data checks and carries out, based on 7 observations of CTLA4-Ig dimer standard and dimer and tetrameric 14 observations of purifying.The dimer or the tetramer that compare CTLA4-Ig dimer standard and purifying are at k OnNot there are differences in the speed.Yet, when comparing with reference to the tetramer with purifying, k oFf speed and K DOn the statistics remarkable (table 12).Compare the tetramer of purifying and the dimer of purifying, k OnAnd k oFf speed and K DDifferent on the statistics.Although should be understood that packet is the dimer and the tetramer, indivedual classification of sample (for example, front, rear portion etc.) can have different slightly features, and described feature can influence its binding kinetics.For dimer, k OnAnd k oFf speed is average out to 3.3 ± 1.0 x 10 respectively 5M-1s -1With 8.8 ± 3.5 x 10 -3s- 1Tetrameric k OnAnd k oFf speed is respectively 2.6 ± 0.8 x 10 5M-1s- 1With 3.1 ± 1.4 x 10 -3s- 1
In table 12, the dimer (n=14) and the tetramer (n=14) and CTLA4-Ig dimer standard (n=7) have been analyzed.
The statistical analysis of table 12:CTLA4-Ig kind.
Figure A200680053116D02811
Remove the CTLA4-Ig kind at peak from OFF:The QFF post is the final purification post that is used to remove from the residual impurity of product.The CTLA4-Ig kind uses 2 post SEC methods to separate from the removing peak of QFF post, and analyzes on Biacore 3000.The binding kinetics of data presentation this " QFF removes the peak " sample is similar to CTLA4-Ig dimer standard.In addition, and compared by those of composition purifying, the dimer and the tetramer of being removed the peak purifying by QFF provide similar binding kinetics.In addition, the sialic acid content of the monomer fraction of purifying the sort of greater than CTLA4-Ig dimer standard.
Guanidine is handled (tetrameric dimerization): the tetramer can be transformed into dimer by carrying out dialysis with the guanidine processing subsequent in the phosphate buffered saline buffer, and confirms to exist as dimer by analysis mode SEC.Remove its k of " dimerization " tetrameric dynamic analysis demonstration at peak from HIC OnAnd k OffSpeed be similar to CTLA4-Ig dimeric those.
Embodiment 7: by the complete analysis of MS electrospray ionization (ESI)
CTLA4-Ig 100mM Tris, 25mM NaCl, pH8 are diluted to the final concentration of 0.7mg/mL.PNGase F (New England Biolabs) uses 100mM Tris, 25mMNaCl, and pH8 dilutes 30 times of final concentrations to 17 units/μ L.The Glycosylase solution of the sample of the dilution of equal-volume (each 60 μ L) and dilution mixed and in 37 ℃ of incubations 4 hours.
Resulting deglycosylated CTLA4-Ig (2 μ L) is loaded into Waters
Figure A200680053116D0281162148QIETU
On the reversed phase extraction cylinder post (2.1 x 20mm).The post that loads is with 5% solvent B (solvent orange 2 A: 1% water-soluble formic acid, solvent B: 1% formic acid that is dissolved in acetonitrile) wash 5 minutes with desalination with 0.2mL/ minute flow velocity, wherein make eluent turn to refuse.When finishing in 5 minutes, gradient (5% solvent B to 95% solvent B in 10 minutes) begins wash-out CTLA4-Ig from post fast; This is in the shunting of flowing (chromatographic system of use is the WatersAlliance 2695 that is equipped with the Waters2996 detector) back and with 45 μ L/ minutes eluent is imported mass spectrograph (Waters MicromassQ-T of micro TM) in.
About Q-T of micro TThe capillary voltage of M is made as 3kV, and sample cone voltage is 40V.Scanning (per 0.9 second) on average becomes 1 scanning; The scanning room time is 0.1 second.The Q-Tof analyser is scanned up to 2500 from m/z800.Spectrum corresponding to the part that is higher than half maximum peak height (in the TIC chromatogram) is used Waters MassLynx TM software makes up.The spectrum of combination is implemented Waters MaxEntl remove flatung.Resolving power is made as the 1Da/ passage, and selects consistent Gauss to damage model, and wherein half width of highly locating is made as 0.5-1Da.About the minimum strength at Zuo Feng and right peak than all being made as 50%.
Embodiment 8: substance assistant laser desorpted ionized-flight time (MALDI-TOF)
The MALDI-MS spectrum uses nitrogen laser (337nm) at OmniFlex TM(BrukerDaltonics MA) goes up acquisition.Use need not the protein example of desalination or desalination, use with
Figure A200680053116D02821
The Solid-Phase Extraction of transfer pipet end (Millipore, Bedford MA) form.The transfer pipet end is with acetonitrile-water (1: 1v/v) wetting, and carry out balance with 0.1% trifluoroacetic acid (TFA) before use.The transfer pipet end is subsequently by extracting and discharge 10 μ L samples and load for 3 times from transfer pipet.The sample that loads washs 3 times with 10 μ L0.1% TFA.The protein example of desalination is with 10 μ L acetonitriles and water (1: 1v/v) wash-out from transfer pipet.Carry out point sample on the stainless steel target by 1 μ L sample (desalination or comprise damping fluid) being mixed with 1 μ L matrix solution and 1 μ L mixture being placed.Matrix solution is to be dissolved in 1: 1 water comprising 0.1% TFA and the sinapinic acid saturated solution of acetonitrile (v/v).To mix object point on the MALDI sample panel, and before placing mass spectrograph, allow it air-dry.The all proteins sample is analyzed by the extraction that postpones with linear, positive ion mode, wherein uses acceleration voltage and the time of lag of 200 nanoseconds of 20kV.Accumulate 400 injections spectrums altogether for every kind of sample.The standard protein mixture that use comprises trypsinogen (23982m/z), A albumen (44613m/z) and bovine albumin (66431m/z) reaches external calibration.
The MALDI-TOF MS of peptide analyzes
Peptide mixt separates by reversed phase chromatography, and collects from the fraction of chromatographic peak and be evaporated to drying.Sample is reconstructed in 50 μ L 25mM phosphate buffered saline buffer pH7.5.The final concentration that adds DTT to 5mM, and fraction was in 50 ℃ of incubations 20 minutes.After the reduction, add the final concentration of IAM to 10mM and in the dark in other 20 minutes of 50 ℃ of incubations.
The MALDI-MS spectrum is used nitrogen laser (337nm), and (BrukerDaltonics MA) goes up acquisition at OmniFlex.By being mixed with 1 μ L matrix solution, 1 μ L sample prepares sample.Matrix solution is to be dissolved in 1 couple, 1 water with 0.1% TFA: the a-cyano group of acetonitrile-4-hydroxycinnamic acid saturated solution.To mix object point on the hole of MALDI sample panel, and before placing mass spectrograph, allow it air-dry.All peptides are analyzed by the extraction that postpones with reflection, positive ion mode, wherein use acceleration voltage and the time of lag of 200 nanoseconds of 20kV.Accumulate 100 injections spectrums altogether for every kind of sample.Use the standard peptide mixture to reach external calibration, described standard peptide mixture comprises Angiotensin II (1046.54m/z), angiotensin I (1296.68m/z), P material (1347.74m/z), bombesin (1619.82m/z), ACTH clip 1-17 (2093.09m/z), ACTH clip 18-39 (2465.20m/z) and Somatostatin (3147.47m/z).
Embodiment 9: substratum and substratum moiety are to CTLA4-Ig strand and polymer kind The analysis of the effect of class
Monomeric species and the purifying of preparation CTLA4-Ig dimer, low sialic acid subfraction, high sialic acid subfraction, monomer front and CTLA4-Ig.These samples are used for a series of modeling experiments, and described modeling experimental design is for measuring substratum and substratum moiety through the influence of at least 60 hours time of 0-to strand and high molecular formation.The influence independent and combination of preparation damping fluid, iodo-acid amide, sodium phosphate, ammonium chloride, basic medium, fermented liquid, substratum 177e, the spissated acid solution I of substratum, the spissated acid solution II of substratum, Regular Insulin, EDTA, halfcystine, Thioctic Acid, gsh, methionine(Met) and yeast autolysis solution is tested.
Embodiment 10: by dimension analysis CTLA4-Ig molecule
Use size exclusion chromatography (SEC) method of sex change condition can be used for the quantitatively kinds of protein of different sizes.In one embodiment, use the series connection SEC method of sex change condition can be applied to quantitative CTLA4-Ig strand kind.Strand CTLA4-Ig can be the kind that lacks interchain disulfide bond.Isolating strand CTLA4-Ig kind is called natural strand CTLA4-Ig during purge process.The strand of the purifying that produces by the dimeric reduction of CTLA4-Ig and alkylation is called the inductive strand.Natural and inductive strand CTLA4-Ig has same characteristic features.
Material:
Generation potassiumphosphate (KH 2PO 4) the ACS grade
Potassium hydroxide (KOH) 45%w/w solution A CS grade
Sodium-chlor (NaCl) ACS grade
The adjustable single pipettor of calibration, 100_L
Rainin, (catalog number (Cat.No.) P-100)
Water (H 2O) HPLC grade
2.0mL profound hypothermia bottle Nalgene, (catalog number (Cat.No.) 5000-0020)
Concentrated hydrochloric acid (HCl) Fisher (catalog number (Cat.No.) A144-212)
Sodium hydroxide (NaOH) 10N solution
J.T.Baker, (catalog number (Cat.No.) 5674-02)
1000mL filter unit 0.22mm Coming, (catalog number (Cat.No.) 430517)
Polypropylene 15mL test tube Falcon, (catalog number (Cat.No.) 352097)
Sodiumazide (NaN3) ACS grade
Sodium orthophosphate (monometallic), monohydrate (NaH2PO4H2O)
Potassium hydroxide (KOH) ball (Pellets)
Instrument configuration and condition
The Waters of HPLC system 2695 separation modules
Post Toso Haas 5 μ TSK 3000 SWXL, 300mm x 7.8mm I.D. (numbering of part 08541)
Guard column Toso Haas 5 μ TSK 3000 SWXL, 40mm x 6.0mm I.D. (numbering of part 08543)
Detector Waters 2487 dual wavelength detector wavelength 280nm flow velocitys 1mL/ minute
Integration system Empower
Volume injected 20 μ L
Measure target level 10mg/mL
Moving phase 0.2M KH2PO4,0.9% NaCl, pH6.8 contains KOH
Measure 20 minutes working times
The column temperature environment
4 ℃ of sample temperatures
Retention time monomer 8.7 minutes ± 1.0 minutes, HMW kind be in 7.5 minutes ± 1.0 minutes, if exist, the MW kind will be behind the monomer peak wash-out.
Reagent
4N potassium hydroxide (4N KOH) (100mL)
One of following preparation of use as described:
40mL HPLC grade water and 11.6mL 45%w/w KOH solution are added in the 100mL volumetric flask.Make volume reach 100mL with HPLC grade water.
In the 100mL volumetric flask, add 80mLHPLC grade water, weighing 22.4 gram KOH balls, and magnetic stirs until dissolving fully.Make volume reach 100mL with HPLC grade water.
Solution is transferred in the 250mL glass reagent bottle.By the reversing thorough mixing.Preserved in room temperature maximum 1 year.
Moving phase (0.2M KH 2PO 4, 0.9%NaCl, pH6.8)
Weigh up 27.2 gram KH 2PO 4With 9.0 gram NaCl, add in the 1000mL beaker.Use is blended in dissolved solids in the 800mL HPLC grade water continuously by magnetic stirring bar.
Use pH meter, use 4N KOH solution that the pH of solution is adjusted to 6.8.If pH surpasses 6.8, adjust it with dense HCl so.
Use the 1000mL graduated cylinder to make final volume reach 1L.By 0.22 μ m filter unit filtering solution.
Transfer in the 1000mL glass reagent bottle and supersound process vacuum outgas simultaneously 5+/-1 minute.Before each the use, outgas.
Preserved in room temperature maximum 1 month.
2N sodium hydroxide (2N NaOH)
20mL10N NaOH is transferred in the 100mL glass cylinder.
Make volume reach 100mL with HPLC water.
Solution is transferred in the 250mL glass reagent bottle.By the reversing thorough mixing.Preserved in room temperature maximum 1 year.
Weigh up 3.45 gram NaH 2PO 4H 2O and 2.92 gram NaCl, and in 900mL HPLC grade water, dissolve by mixing with stirring rod.Use pH meter, use 2N NaOH that the pH of solution is adjusted to 7.4.If pH surpasses 7.4, adjust it with dense HCl so.
Use the 1000mL graduated cylinder to make final volume reach 1L.By 0.22 μ m filter unit filtering solution.
Preserve in 2-8 ℃ maximum 6 months.
Post storage damping fluid (water-soluble 0.05%w/v NaN 3)
Weighing 50 ± 5mg sodiumazide, and with magnetic stirring bar it is added in the 1000mL beaker.
In beaker, add 500mL HPLC grade water, and stir until dissolving fully.
Water makes volume reach 1L.In 0.22 μ m filter unit filtering solution and impouring 1000mL HPLC reagent bottle.
Preserved in room temperature maximum 6 months.
The preparation of high molecular weight species and system's suitability criterion
This will provide the 3 times of dilutions and the 15%-30% high molecular weight species area percentage amount of the CTLA4-Ig reference material that is heated to not heating.In 15mL Falcon test tube, use about 3mL reference material of CTLA4-Ig dilution buffer liquid preparation 10.0 ± 1.0mg/mL.The initial concentration of CTLA4-Ig is from COA.By that value preparation 10.0 ± 1.0mg/mL dilution.Shift the reference material of 1.0mL dilution, use the 1mL pipettor that it is added in the 2.0mL cryovial, and it was heated 45 ± 2 minutes in 67 ± 2 ℃ in water-bath.The heating reference material of using the 1mL pipettor to prepare shifts back 15mL in vitro.Move down the in vitro tolerant of liquid 1mL volume gently, 10 times altogether.This is system's suitability criterion.Holding conditions: 150 μ L aliquots containigs of preparation system suitability criterion in the 2.0mL cryovial are used for being up to 1 year-80 ± 5 ℃ of following storages.Obtain the initial concentration of reference material, and by that value preparation 10.0 ± 1.0mg/mL dilution.The dilution buffer liquid that uses bottom line 100 μ l reference materials and appropriate amount is to reach the final concentration of 10.0 ± 1.0mg/mL.About diluting with reference to equation
Figure A200680053116D02861
For the CTLA4-Ig medicine, by protein concn preparation 10.0 ± 1.0mg/mL dilution, the dilution buffer liquid that wherein uses the sample of bottom line 100 μ l aliquots containigs and appropriate amount is to reach the final concentration of 10.0 ± 1.0mg/mL.About diluting with reference to equation:
Figure A200680053116D0286162611QIETU
In case be diluted to 10.0 ± 1.0mg/mL, sample just can be in maximum 24 hours of 2-8 ℃ of storage.For the CTLA4-Ig medicine, by protein concn preparation 10.0 ± 1.0mg/mL dilution, the dilution buffer liquid that wherein uses the sample of bottom line 200 μ l aliquots containigs and appropriate amount is to reach the final concentration of 10.0 ± 1.0mg/mL.About diluting with reference to equation:
Figure A200680053116D0286162633QIETU
Place 1 solvent reservoir and HPLC grade water to place another moving phase.Supersound process and vacuum outgas before operation.Open detector and allow and before operation, heated 15 minutes.Before new or present post is used for analyzing, washing post at least 20 minutes with HPLC grade water, is moving phase damping fluid balance at least 20 minutes subsequently.Use 1.0mL/ minute flow velocity.System's suitability criterion of the 150 μ L aliquots containigs of thawing will place automatic sampler in its adding automatic sampler bottle and with bottle.Injection 20 μ L standards under condition.
The per-cent of the high molecular weight species of wash-out when measuring about 7.5 minutes according to following formula: (in following formula, in fact monomer refers to dimer.)
Figure A200680053116D02871
Wherein:
The peak area of A=high molecular weight species
B=monomer peak area
The area percentage high molecular weight species should be less than 15%.If it is less than 15%, so other spissated high molecular weight species is added in the said system suitability criterion.Resolving power (R) is measured and the retention time assessment.Inject 20 μ Lindenmayer system suitability criterion.Use the resolving power between equation calculating high molecular weight species (about 7.5 minutes of retention time) and monomer peak (about 8.7 minutes of the retention time): (in following formula, " monomer " in fact refers to dimer.)
Figure A200680053116D02872
Wherein:
t 1The retention time of=high molecular weight species
t 2=monomeric retention time
W 1The peak width of=high molecular weight species
W 2=monomeric peak width
Peak width is sentenced in the bottom at peak after straight relatively side, peak is extrapolated to baseline and minute is measured.Retention time and peak width are measured with same units.In one embodiment, (R) necessary 〉=1.2 and should be 8.7 ± 1.0 minutes about the retention time at peak.
Theoretical plate number is measured (N)
According to system's suitability criterion chromatogram, the efficient of coming measuring column by theory of computation stage number (N) according to equation:
N = 16 ( t w ) 2
Wherein
The monomeric retention time of t=(minute)
W=by the side, peak be extrapolated to the width that baseline obtains at monomeric baseline place (minute)
To carry out six (6) the inferior injections altogether of CTLA4-Ig material.The 10mg/mL reference material aliquots containig of 200 μ L in the automatic sampler bottle, and is placed automatic sampler with bottle and injects 6 times.Handle chromatogram, and calculate dimer peak area about each chromatograph.Totalling is from the dimer peak area of 6 chromatographs and according to equation calculating mean value, standard deviation and %RSD:
X 1 + X 2 + X 3 . . . . X n n x
Wherein:
X 1,2,3......The designated value of=data centralization
n x=one class value (x)
5.4.4.2 following base of calculation is poor:
nΣ x 2 - ( Σx ) 2 n ( n - 1 )
Wherein:
The value of n=data centralization (x) number
1 value of x=data centralization
5.4.4.3 following calculating % coefficient of variation (RSD):
The example of injection sequence:
Sample Injection
The dilution buffer liquid air is white 1 injection
System's suitability 1 injection
Reference material
6 injections
Sample #
1 2 injections
Sample #
2 2 injections
Sample #
3 2 injections
Reference material
1 injection
The dilution buffer liquid air is white 1 injection
The integration at peak
All peak areas of 5.5-11.8 minute in the integration chromatogram.The baseline that amplifies chromatogram is included in the integration with the total area of guaranteeing all LMW and HMW kind.Ignorance is corresponding to the peak in the sample at the peak in the contrast.Including volume (inclusion volume) peak (11.8-13.5 minute) and peak does not afterwards take in this calculates.Yet, if when including volume area be the total area 0.1% or bigger, it should be pointed out so.The dimer peak is wash-out in the time of 8.7 ± 1.0 minutes, and the high molecular weight species peak is wash-out in the time of 7.5 ± 1.0 minutes, and low molecular weight species (for example, monomer, if present) will be behind the dimer peak wash-out.Have the 280nm absorbancy that surpasses 0.1 area % of total peak area, any peak except that high molecular weight species, dimer or low molecular weight species should be pointed out.Following reference area per-cent: (mention that in this example " A Baxipu monomer " refers to the CTLA4-Ig dimer.)
Figure A200680053116D02891
Figure A200680053116D02892
Long-pending % monomer=100-(area %HMW+ area %LMW)
Wherein:
A=A Baxipu monomer peak area.
The B=retention time is less than the total area at monomeric all peaks of A Baxipu.
The C=retention time is greater than the total area (eliminating includes volume) at all peaks at A Baxipu monomer peak.
Sample separates by size exclusion chromatography, wherein uses 2 7.8x300mm TSK Gel G3000SWXL that place in series connection TMPost (Tosoh Biosep, Montgomery, PA) Water ' s on
Figure A200680053116D02901
2695 (Milford, MA), it adopts 6.0 x 40mm guard columns.Inject the sample of every kind of purifying of 25 microlitres and under isocratic condition, separate, wherein use 0.1M Na 2HPO 4, 0.1M Na 2SO4, pH6.8 was with 1.0mL/ minute.Sample uses 996 PDA (photodiode array) detector, and (Waters, Milford MA) detect, and use Millennium 4.0 at the 280nm place TM(Waters, Milford MA) analyze chromatography software.
Show from the covering chromatogram of the natural and inductive strand material of sex change analysis mode series connection size exclusion chromatography and to be respectively 27.96 ± 0.02 and 27.99 ± 0.02 average retention time (6 repetitions).Discovery is 495525.0 ± 9589.6 about the average peak area of natural strand, and is 463311.8 ± 7997.2 (tables 13) about the inductive strand.Covering MALDI-TOF spectrum natural and inductive strand material has quite wide peak, and its baseline width is about 15000 mass units.Because glycosylated heterogeneity in 2 kinds of samples, this expects.The summit at strand peak is used to calculate average quality in every kind of mass spectrum.Based on 6 multiple analyses, natural and average quality inductive CTLA4-Ig strand is respectively 45694.426 ± 297.735 and 45333.086 ± 264.778 mass units (table 14).Natural strand quality expection is higher than the sort of of inductive strand, because there is the Cys of SEQ ID NO:2 on the natural strand 146On extra halfcystine (residue quality 103 dalton), and the inductive strand single interchain disulfide bond that is selective reduction is subsequently for adding the alkylating result of ethanoyl (quality 58 dalton).
The natural analysis via series connection sex change SEC chromatography and MALDI-TOF with the inductive material of these data presentation produces equivalent result.These results confirm the comparability between natural and the inductive CTLA4-Ig strand material.
Table 13: the HPLC SEC data of natural and inductive strand
Figure A200680053116D02911
Table 14: the MALDI-TOF mass spectroscopy data of natural and inductive strand
Repeat Inductive SC Natural SC quality
1 45397.896 45597.475
2 45432.199 45621.300
3 45256.929 45543.839
4 45433.849 45555.893
5 45381.812 45634.620
6 45348.376 45340.712
Mean value 45375.177 45548.973
Stdev 66.265 108.043
％RSD 0.15 0.24
The existence of halfcystine allows the formation of disulfide linkage in the polypeptide chain, and described disulfide linkage can be an intramolecularly or intermolecular, thereby causes the formation of dimer or polymer protein mixture.CTLA4-Ig is as the C that passes through on every chain 146Between the dimer of 1 disulfide bonds have (wherein dimer is made of any one 2 monomers that have in the following sequence: (i) 26-383 of SEQ ID NO:2, (ii) 26-382, (iii) 27-383, the (iv) 27-382 of SEQ ID NO:2 of SEQ ID NO:2 of SEQ ID NO:2), (the v) 25-382 of SEQ ID NO:2 and (the vi) 25-383 of SEQ ID NO:2.The reduction of this disulfide linkage can cause the formation of 2 equivalent protein chains combining by non-covalent electrostatic force.When enforcement overwhelmed or surpass the sex change condition that attracts electrostatic force, this kind CTLA4-Ig can dissociate fully, thereby causes 2 identical protein structures of about 46kDa.Resulting structure is called strand or monomer.The existence of strand can be monitored by the columns in series size exclusion chromatography that moves under the sex change condition.
The subgroup of CTLA4-Ig molecule is included in Cys 146On modification: the disulfide linkage that is present in most of colonies becomes free cysteine amino acid (being called cysteinylization).The CTLA4-Ig dimer mainly is the protein with molecular weight of about 92,000.It comprises by at Cys 146On 1 interchain disulfide bond and 2 glycosylated polypeptides chains (being also referred to as CTLA4-Ig " dimer ") of combining of noncovalent interaction.The protein of purifying exists and comprises modification for example glycosylation on N and C-terminal and variation as heterogeneous population.
The different groups that have the CTLA4-Ig molecule that lacks interchain disulfide bond.The colony of this non-covalent connection is present in the dimer peak, front that produces by size exclusion chromatography.Find that the front dimer lacks interchain disulfide bond.The CTLA4-Ig kind that lacks interchain disulfide bond is passed through at Cys 146On cysteinylization modify, based on the ESI-MS partial data, this modification betides〉on 99% the strand kind.The Cys of O connection carbohydrate 146Cysteinylization and enrichment are the main modifications of 2 kinds on CTLA4-Ig strand kind.The front dimer is implemented the sex change size exclusion chromatography, thereby cause the separation at CTLA4-Ig strand peak.The front monomer material of purifying is with use and do not use the CTLA4-Ig of Solid-Phase Extraction (SPE) to compare, and analyzes on MALDI.The front monomer of purifying comprises 2 dominant species: about SPE handle at the 47005u place or about untreated main kind at the 46897u place; About SPE handle at the 95070u place or about untreated less important kind at the 96172u place.The CTLA4-Ig material also comprises 2 dominant species: about SPE handle at the 91518u place or about untreated main kind at the 91143u place; About SPE handle at the 45660u place or about untreated less important kind at the 46014u place.
In one embodiment, the CTLA4-Ig composition has following characteristics about size homogeneity (HPLC) analysis of dimer, high MW kind and low MW kind (monomer, strand):
〉=97.0% dimer
≤ 2.0%HMW kind
≤ 0.5%LMW kind (for example, monomer, strand)
In another embodiment, the CTLA4-Ig composition has the following characteristics amount of each kind:
〉=95.5% dimer
≤ 3.0%HMW kind
≤ 0.5%LMW kind (for example, monomer, strand)
The summary that the SE-HPLC that method CD-CHO1 criticizes analyzes
Figure A200680053116D02931
For the medicine of using method CD-CHO1 preparation, the per-cent monomer is 98.4-99.8%, and its mean value is 99.4%.Per-cent HMW kind is 0.2-1.6%.Mean value is 0.6%, and CV is 45.7%.95% tolerance interval is 〉=98.7% for dimer, and is≤1.3% for the HMW kind.The per-cent HMW kind of criticizing is 2.1% variation from minimum value 0.4% to maximum value.Average percent HMW kind is 0.8%, and %CV is 40%.In all cases, LMW or monomeric species are lower than limit of detection (DL=0.1%).For CTLA4-Ig via method CD-CHO1 preparation, about 95% tolerance interval (so that the covering about 99 area % of colony to be provided) of HMW kind be respectively≤1.3 and≤1.8%.For from the dimer in the medicine of method CD-CHO1,95% tolerance interval is respectively 98.7% and 96.5%.
Summary about the data splitting of CTLA4-Ig
Figure A200680053116D02941
Last table show percent HMW kind is 0.2-2.1%, and its mean value is 0.6%.Dimer is 94.8%-99.8%, its mean value be 99.3% and precision be 0.3%.Be changed to 0.3-0.5% between dimeric site, in the site and total site.Be changed to 26.4% between site for HMW.For being respectively 44.2 and 51.4% with the variation of total site in the site of per-cent HMW kind.For 95% tolerance interval of dimer (97.3%) and HMW kind (1.8%) in specification sheets.
Embodiment 11: vector construction
The structure of pcSD expression vector: as shown in Figure 28, (Invitrogen, Carlsbad CA) make up expression vector pcSD by the pcDNA3 carrier that is obtained commercially.The neomycin resistance gene box is by taking out from plasmid pcDNA3 with restriction endonuclease Nae I digestion.Restriction endonuclease Nae I produces flush end.Dna fragmentation separates by agarose gel electrophoresis, and 3.821kb pcDNA3 carrier main chain carries out purifying from gel.The dna fragmentation that comprises the gene of encoding murine Tetrahydrofolate dehydrogenase (dhfr) and SV40 promotor is by separating from plasmid pSV2-dhfr with BamH I digested plasmid with restriction endonuclease PvuII.1.93kb fragment corresponding to the dhfr box gene is separated and purifying by agarose gel electrophoresis.Fill up by the terminal Klenow fragment of dna polymerase i that uses of 3-' notch that BamH I digestion produces, to produce flush end.This isolating fragment is connected with the 3.8kb pcDNA3 carrier main chain of flush end, to produce expression vector pcSD.This expression vector has following characteristics: cytomegalovirus (CMV) promotor subsequently for multiple clone site, Trobest (BGH) polyadenylation signal and transcription termination sequence, be used for selecting and the mouse dhfrcDNA sequence that increases, be used for ampicillin resistance gene and the pUC replication orgin selecting and keep intestinal bacteria.
The structure of pcSDhuCTLA4-Ig expression vector: 1.2 kilobase (kb) dna fragmentation that comprises the proteinic sequence of coding CTLA4-Ig is by digestion separates from plasmid pCDM8-CTLA4-Ig with Xba I with restriction enzyme Hind III.1.2-kbHind III/Xba I fragment is connected in the previous carrier piLN with restriction enzyme Hind III and Xba I digestion.Resulting plasmid construction body called after piLN-huCTLA4-Ig is shown among Figure 21.The piLN-huCTLA4-Ig plasmid is used to make up final expression vector pcSDhuCTLA4-Ig as the source of CTLA4-Ig encoding sequence.
The final carrier that is used to express the CTLA4-Ig gene makes up as shown in Figure 29.The 1.2kb dna fragmentation that comprises the CTLA4-Ig gene separates from plasmid piLN-huCTLA4-Ig by 2 step restriction digest programs.Plasmid piLN-huCTLA4-Ig at first digests with restriction enzyme Hind III.Resulting 3-' notch end is filled up by handling with the Klenow fragment of dna polymerase i.Plasmid digests with restriction enzyme XbaI subsequently, comprises the 1.2kb fragment of CTLA4-Ig gene with release.This fragment is carried out purifying and is connected with Xba I fragment with isolating EcoR V from the restriction digest of pcSD.In the multiple clone site of EcoR V and the pcSD of Xba I restriction site between CMV promotor and box, described box comprises Trobest polyadenylation signal and transcription termination sequence.This places under the control of CMV promotor the CTLA4-Ig gene fragment.This plasmid called after pcSDhuCTLA4-Ig, and comprise SEQ ID NO:1.
The transfection of embodiment 12:CTLA4-Ig expression vector is to obtain stable clone
This embodiment and embodiment 13 have described the colony of cells transfected recently by its selection and the indivedual clones of amplification, and the clone of therefore amplification is different from the cell that is preserved in ATCC as registration number CRL-10762.Previous Chinese hamster ovary celI cording has and comprises the expression vector of coding corresponding to the DNA (DNA with ATCC registration number 68629) of the aminoacid sequence of CTLA4-Ig, is described in U.S. Patent number 5,434, in 131.In brief, the expression plasmid that comprises cDNA of 68629 times preservations of ATCC registration number (for example, pCDM8), use standard program by the fat transfection transfection in the negative Chinese hamster ovary celI of dhfr, to obtain the clone of stably express CTLA4-Ig.Use immunostaining in substratum, to cause expressing stable transfection of CTLA4-Ig in conjunction with the positive Chinese hamster ovary celI of screening active ingredients B7 system with regard to B7.This heterogeneous population called after Chinese hamster ovary line CTLA4-Ig-24 of cells transfected, and be preserved in ATCC on May 31st, 1991 according to budapest treaty, have ATCC registration number CRL-10762.
Chinese hamster ovary line DG44 comprises the disappearance of the gene of the Tetrahydrofolate dehydrogenase of encoding.Expression plasmid pcSDhuCTLA4-Ig comprises the copy of dihydrofolate reductase gene (dhfr).To cause having complementary functions of dhfr disappearance in the plasmid pcSDhuCTLA4-Ig insertion DG44 genome.This having complementary functions can be used for selecting transfectant and amplification dhfr and contiguous gene in the presence of methotrexate (MTX).
As shown in Figure 29, human CTLA 4-Ig secretory cell is that 1D5 is by making up with pcSDhuCTLA4-Ig expression plasmid transfectional cell series DG44.Plasmid DNA uses standard program known in the art to introduce in the DG44 cell by electroporation.Transfectant uses the minimum essential medium (MEM of the foetal calf serum of adding 5% (v/v) dialysis; JRH Biosciences, Inc. Kansas) selects.From the culture supernatants of transfectant use the sandwich ELISA method with regard to human IgG production screen.The anti-human IgG of Fc specificity goat is as capture antibody.The anti-human IgG antibody of goat who puts together with horseradish peroxidase is used to detect human IgG.The transfectant of selecting to express higher levels of human CTLA 4-Ig gene is used for further amplification.
The gene amplification of selected transfectant is by finishing in the final concentration adding substratum of MTX with 100nM.MTX is the folacin that serves as the competitive inhibitor of Tetrahydrofolate dehydrogenase.The transfectant that MTX is added in the substratum Tetrahydrofolate dehydrogenase of the multiple copied that allows to select to comprise the dhfr gene and elevated levels.Use human IgG specific ELISA method to identify the transfectant of the multiple copied that also comprises contiguous CTLA4-Ig gene.Select the CTLA4-Ig production clone of called after 1D5 to be used for further developing in the part that embodiment 13 describes.
Embodiment 13: the subclone of the cell of stable transfection
Pair cell is that 1D5 implements soft-agar cloning.Derived from the subclone of soft-agar cloning use the ELISA method with regard to human IgG production analyze.Selected subclone produces with regard to CTLA4-Ig and growth properties is assessed.Select the leading subclone (leadsubclone) of called after 1D5-100A1.
(JRHBiosciences, Inc., Kansas adapt to the chemical ingredients that is called CD-CHO and know substratum (table 15) 1D5-100A1 clone from the raw-material DE substratum that comprises animal-origin.The CD-CHO substratum is by Invitrogen Corporation, and that Carlsbad, CA make is patented, the substratum of no animal ingredient.
Table 15: the composition of method Y substratum
Clone 1D5-100A1 cultivates in the DE substratum according to normal structure cultivation rules and goes down to posterity.Subsequently with cell transfer to the substratum of forming by 50%DE substratum and 50% CD-CHO substratum.After this substratum goes down to posterity several times, with cell transfer to the T bottle that comprises the 100%CD-CHO substratum.The cell several generations of in 100% CD-CHO substratum, growing.By limiting dilution the cell that adapts to is implemented the clone subsequently.
The cell of the 1D5-100A1 clone that adapts to from CD-CHO uses serum free medium to clone by limiting dilution.The 1D5-100A1 cell is inoculated in the 96 hole microtiter plates that comprise the MCDB substratum of adding with the target of 1 cells/well.MCDB is by Sigma-Aldrich, St.Louis, and the chemical ingredients of MO distribution knows the substratum preparation.The MCDB substratum has been added 4mM glutamine, 500 μ g/mL recombinant human insulins, 100nM MTX and 10% conditioned medium.Conditioned medium is that described 1D5-100A1 clone is grown in the MCDB substratum from the supernatant liquor of the filtration sterilization of the culture of the 1D5-100A1 clone of CD-CHO adaptation.
Evaluation comprises the hole of single colony, and uses the ELISA method to produce the assessment clone with regard to CTLA4-Ig.The selected clone 6 porocyte culture plates that from 96 hole microtiter plates, increase.The culture 25cm that further increases 2T bottle and rolling in the bottle subsequently.
With regard to CTLA4-Ig titre, CTLA4-Ig sialic acid content and growth assessment roller bottle culture.Select 3 clones to be used in further assessment and further sign of bio-reactor.Preserve in the cryovial research original seed of-80 ℃ cloned cell line 1D5-100A1.17 and be used to produce cell bank.
Embodiment 14: produce CTLA4-Ig via fed-batch process in bio-reactor
Expressing the commercial size of the suspension mammalian cell of CTLA4-Ig cultivates: this embodiment has described by the negative Chinese hamster ovary celI production of the dhfr of suspension culture and has comprised the monomeric CTLA4-Ig molecule of SEQ ID NO:2.The method of describing among this embodiment can be fit to and expand be used to produce other recombinant proteins, include but not limited to, excretory albumen for example cytokine and other hormones, as the Ig superfamily member or comprise the excretory albumen of the proteinic part of Ig superfamily and any protein of usually in Chinese hamster ovary celI, expressing.Process flow sheet about the CTLA4-Ig culturing step is shown among Figure 10.
Culturing bottle (for example, T-175 and Erlenmeyer flask), roll bottle and cell bags is used for the inoculum amplification step of CTLA4-Ig cultural method, with from the continuous propagated cell of cryovial with viable cell that enough numbers are provided to inoculate 20, the 000-L bio-reactor.
The cell of single bottle takes out from the vapour phase of liquid nitrogen storage refrigerator and thaws in 37 ℃ in water-bath.Transfer in the conical centrifuge tube of aseptic 15-mL the entire content of bottle is aseptic.Add the CD-CHO substratum so that final volume reaches 10mL.Cell suspending liquid is centrifugal, abandoning supernatant and cell precipitation is resuspended in the 10mL CD-CHO cell culture medium.With the cell transfer of resuspension to the T-175 bottle that comprises 10mL CD-CHO substratum.Measure the viable cell density and the per-cent viability of the culture in the T-175 bottle.Establishment is in the per-cent viability standard of this step time 〉=84%.In the T-175 bottle, add the CD-CHO substratum to reach 1.7-2.6x 10 5The target viable cell density of cell/mL.
Make the T-175 bottle in 37 ℃ of maximum 4 days of incubations in the atmosphere of 6% carbonic acid gas, to reach 〉=6 x 10 6The final target cell number of viable cell.As shown in Figure 10, after the T-175 bottle step, use a series of bottle, 1-L and 2-L of shaking to roll a bottle amplification cultivation thing.When going down to posterity, make cell with 2.0 x 10 at every turn 5The inoculation of the target density of viable cell/mL, wherein the culture target has 〉=80% final cultures cell survival.Culture in the CD-CHO substratum in 37 ℃ of maximum 4 days of incubations in the atmosphere of 6% carbonic acid gas.
The amplification of culture takes place in a series of cell bags (20-L, 100-L and 200-L), with further inoculation 1000-L bio-reactor.Merging is rolled bottle cell culture material of inoculum amplification step from 2-L, to press 2.0 x 10 5The target inoculum density inoculation 20-L cell bags of viable cell/mL.Establishment is 1.0-2.0 x 10 when rolling bottle inoculum amplification step at 2-L 6The condition of the final viable cell density of cell/mL and 〉=80% lowest percentage cell survival.After the inoculation, make 20-L cell bags culture in the CD-CHO substratum in 37 ℃ of maximum 4 days of incubations in the atmosphere of 6% carbonic acid gas.For each follow-up going down to posterity (100-L and 200-L cell bags), cell is with 2.0 x 10 5The inoculation of the target density of viable cell/mL, wherein the culture target has 〉=80% final cultures cell survival.Make culture in the CD-CHO substratum in 37 ℃ of maximum 4 days of incubations in the atmosphere of 6% carbonic acid gas.Establishment is about 1.0-2.0 x 10 when 20-L, 100-L and the 200-L cell bags inoculum amplification step 6The condition of the final viable cell density of cell/mL and 〉=80% lowest percentage cell survival.These example values guarantee that the viable cell of enough numbers is used to inoculate the 1000-L bio-reactor.
The purpose of the 1000-L of CTLA4-Ig method and 4000-L seed bio-reactor inoculum amplification step provides the viable cell of enough numbers, and to inoculate 20,000-L produces bio-reactor.
The seed bio-reactor uses the CD-CHO cell culture medium to operate with batch mode.Temperature, pH, dissolved oxygen, pressure, stirring and be subjected to the control of distribution control system (DCS) about the airflow rate of air, oxygen and carbon dioxide, and the condition about culture optimum growh in the seed bio-reactor is provided.The seed bio-reactor is in 37 ℃ of operations.From the seed bio-reactor, take out culture samples, be used for determining viable cell density, per-cent viability and metabolite concentration.
Use from the inoculum of 200-L cell bags amplification step and inoculate 1000-L seed bio-reactor to 1.0-3.0 x 10 5The initial viable cell density of the target of viable cell/mL.Culture in the CD-CHO substratum in maximum 5 days of 37 ℃ of incubations.Example values about the final viable cell density when the 1000-L seed bio-reactor inoculum amplification step is 1.0-2.0 x 10 6Cell/mL and lowest percentage cell survival are 〉=80%.
Use from the inoculum of 1000-L seed bio-reactor amplification step and inoculate 4000-L seed bio-reactor to 1.0-3.0 x 10 5The initial target viable cell density of viable cell/mL.Culture in the CD-CHO substratum in maximum 6 days of 37 ℃ of incubations.Example values about the final viable cell density when the 4000-L seed bio-reactor inoculum amplification step is 1.0-2.0 x 10 6Cell/mL and lowest percentage cell survival are 〉=80%.These example values guarantee that the viable cell of enough numbers is used to inoculate 20, and 000-L produces bio-reactor.
Use the inoculum inoculation 20 from 4000-L seed bio-reactor amplification step, 000-L seed bio-reactor is to 1.0-1.8 x 10 5The initial target viable cell density of viable cell/mL.Culture in the CD-CHO substratum in maximum 6 days of 37 ℃ of incubations.About 20, the example values of the final viable cell density during 000-L seed bio-reactor inoculum amplification step is 1.0-2.0 x10 6Cell/mL and lowest percentage cell survival are 〉=80%.These example values guarantee initial 20, and 000-L produces the viable cell that uses enough numbers before production phase in the bio-reactor.
The commercial mass production of CTLA4-Ig: 20,000-L produces and produces a large amount and high-quality CTLA4-Ig protein the production phase of the present invention of taking place in the bio-reactor, and it relates to the cultivation operation with the change of 2 Buwen's degree.The 6th day when finishing (logarithmic phase) in the CD-CHO substratum in 20 of 37 ℃ of incubations maximum 6 days (as mentioned above), the 000L culture is implemented the temperature variation (T change) from 37 ℃ to 34 ℃.After 37 ℃ to 34 ℃ the temperature variation 12 hours, the CD-CHO substratum was with eRDF charging substratum (Invitrogen Corp., Carlsbad, the CA of improvement; Table 16,17) add, and this charging is supplied to the production reactor every day as bolus (1%w/w).
The composition of table 16:eRDF charging substratum
Figure A200680053116D03001
Table17 : the composition of eRDF-1 substratum
Table17 : the composition of eRDF-1 substratum
20, the 000L culture was added in the CD-CHO substratum of eRDF charging substratum in maximum 4 days of 34 ℃ of incubations in every day.In the time of the 10th day, to 20, the 000L culture is implemented the T change second time from 34 ℃ to 32 ℃.In producing bio-reactor 20,000L produce culture be maintained at 32 ℃ maximum 8 days.At the 18th day, culture samples was analyzed with regard to following example values: 20, the viable cell density during 000-L seed bio-reactor production stage is 3.0-8.0 x 10 6Cell/mL; The lowest percentage cell survival is 〉=38%; Final sialic acid mol ratio (elsewhere description) is 〉=6; With final CTLA4-Ig protein titre be 0.5-1.3g/L.These example values guarantee that enough the protein of product quality and quantity is produced via recombinaant CHO cell system, and 20, the 000-L mammalian cell cultures will soon be gathered in the crops.
ERDF substratum (the table 16 of the following use improvement of the culture in the bio-reactor in the production phase process, 17) give the bolus charging every day: starting temperature change (37 ℃-34 ℃) beginning in back 12 hours, bottom line 1% volume of culture is added as the charging substratum; If glucose level drops to below the 3g/L, add volume calculated so so that glucose level returns to 3g/L.
During 000L scale have 18 day time length 20 production phase.Obtaining sample every day from produce bio-reactor is used for analyzing.For example, (Sigma, St.Louis Mo.) dye with Trypan Blue to be used for Cytometric sample.Cell counting and cell survival are measured and are used hematimeter to carry out, with the viable cell that dyes at the microscopically counting.To the analysis of dried metabolite, other sample aliquot under 2000rpm (4 ℃) centrifugal 20 minutes with sedimentation cell.Technology that put into practice as usual supernatant liquor use this area and rules are with regard to protein titre, sialic acid, glucose, lactic acid, glutamine, L-glutamic acid, pH, pO 2, pCO 2, ammonia and LDH analyze.
Embodiment 15: the purifying of recombinant C TLA4-Ig
The QXL that is used for the CTLA4-Ig purifying goes through ion exchange chromatography: the anion-exchange chromatography step of CTLA4-Ig method is used Q Sepharose Extreme Load (QXL) anion-exchange chromatography resin.This resin is by GE Healthcare, Waukesha, and WI (AmershamBiosciences in the past) provides.The QXL chromatographic step is from being used for the processing of further downstream from (in-process) material capture and concentrated CTLA4-Ig dimer the carrying out of results operation steps.
1.0-2.0m the internal diameter post is filled to the height of 17-30cm with the QXL resin, the volume of the about 643L-1018L of expression.By measuring the height equivalent to a theoretical plate (HE-TP) (HETP) and the asymmetry (A of packed column s) post qualified being used for used.0.02-0.08cm HETP and the A of 0.8-1.2 sBe used for the qualification evaluation of QXL post.
The QXL column operation carries out at ambient temperature.Clarifying cell culture liquid nutrient medium is loaded on the equilibrated QXL post.The Peak Flow Rate that the QXL chromatographic step was used 99.4L/ minute carries out.Column inlet pressure maintains under the 35psig.Maximum CTLA4-Ig protein load about the QXL post is 28 gram CTLA4-Ig/L resins.
The QXL chromatography column at first carries out sanitary measure with the 1N sodium hydroxide solution.Sanitary measure uses the 1N sodium hydroxide solution of 2-4 column volume (CV) to carry out.When the electroconductibility of effluent equaled 169 ± 33mS/cm and post and keeps 60-120 minute, sanitary measure was finished.
Behind the sanitising step, post 75mM HEPES, 360mM sodium-chlor, the pH8.0 damping fluid carries out balance.When bottom line 3CV level pad has been 8.0 ± 0.2 and the electroconductibility of effluent when being 13.4 ± 1.0mS/cm through the pH of post and effluent, balance is finished.
To be loaded on the QXL post from material in the carrying out of results operation steps.(75mM HEPES, 360mM NaCl pH8.0) washs post, and effluent is at 280nm (A with bottom line 10CV lavation buffer solution 280) absorbancy located measures when washing step finishes.CTLA4-Ig uses 25mM HEPES subsequently, 325mM NaCl or 850mM NaCl, pH7.0 damping fluid wash-out from post.Work as A 280When increasing to the AU value that surpasses when washing step finishes 〉=0.02 absorbance unit (AU), make eluate turn to collection container.Collect the A that trail edge of eluate until elution peak 280Reduce to≤value of 1.0AU.
Collect a mole sialic acid and mole CTLA4-Ig dimer product of the proteinic mol ratio of CTLA4-Ig 〉=8, and CTLA4-Ig high molecular weight material storehouse exists with≤25.7%.Comprising that tetrameric CTLA4-Ig high molecular weight material can be further purified is used for being used for facture described herein as separate substance.
The phenyl sepharose FF HIC that is used for the CTLA4-Ig purifying: hydrophobic interaction chromatography (HIC) step is used mobile fast (the Phenyl Sepharose Fast Flow) resin (GEHealthcare of phenyl sepharose, Waukesha, WI (Amersham Biosciences in the past)).The HIC step reduces the level of the CTLA4-Ig high molecular weight material that exists in the QXL product storehouse.The CTLA4-Ig dimer under the loading conditions that is used for the HIC step not with the HIC resin-bonded.
1.0-2.0m the internal diameter post is filled to the height of 18-22cm with the quick stir-in resin of phenyl sepharose, the volume of the about 680L-852L of expression.By measuring the HETP and the A of packed column sPost qualified being used for used.0.02-0.08cm HETP and the A of 0.8-1.2 sBe used for the qualification evaluation of HIC post.
The HIC column operation carries out at ambient temperature.Need not further processing will be loaded into from the eluate storehouse of QXL post step on the equilibrated HIC post.The HIC step with 65.4L/ minute Peak Flow Rate and≤working pressure of 13psig operates.The maximum CTLA4-Ig protein load that is applied to the HIC post is 10.0g CTLA4-Ig protein/rise resin.Based on the proteinic amount of CTLA4-Ig that is present in the QXL eluate storehouse, can use the Multiple Cycle of HIC step.
The HIC post at first carries out sanitary measure with the 1N sodium hydroxide solution.When the 1N of 2-4CV sodium hydroxide solution had passed through post, sanitary measure was finished.Post keeps 60-120 minute subsequently to guarantee sanitary measure.
Behind the sanitising step, post 75mM HEPES, 2.55mM sodium-chlor, the pH7.0 damping fluid carries out balance.When bottom line 3CV level pad has been 7.0 ± 0.3 and electroconductibility when being 71.5-75.5mS/cm through the pH of post and effluent, balance is finished.
To be applied to equilibrated HIC post from the eluate of QXL step.Post washs with the tracking level pad subsequently, until the A of effluent 280Reduce to 0.8-1.0AU.Comprising the proteinic effluent of CTLA4-Ig from each round-robin of HIC step is filled in the common stainless steel collection container by 0.2 μ m rhodia strainer.This HIC product storehouse remains in 2-8 ℃ in collection container.Maximum retention time in collection container is 3 days.
Collect a mole sialic acid and mole CTLA4-Ig dimer product of the proteinic mol ratio of CTLA4-Ig 〉=8, and CTLA4-Ig high molecular weight material storehouse exists with≤2.5%.
The reorganization A albumen affinity chromatography that is used for the CTLA4-Ig purifying: mobile fast (Protein A Sepharose FastFlow) the affine resin (rPA) of reorganization A albumen agarose that uses in downstream CTLA4-Ig production method derives from GE Healthcare (Waukesha, WI (Amersham Biosciences in the past)).RPA column chromatography step is further purified CTLA4-Ig protein.This step is removed DNA and host cell proteins matter, comprises MCP 1 (MCP-1).
80-140m internal diameter post is filled to the height of 18-25cm with the rPA resin, the volume of the about 339L-372L of expression.By measuring the HETP and the A of packed column sPost qualified being used for used.0.02-0.08cm HETP and the A of 0.8-1.2 sBe used for the qualification evaluation of post.The maximum access times of establishing in research resin life that are used for the rPA resin are 60.
The rPA column operation carries out at ambient temperature.Inactivation of virus product storehouse is loaded on the equilibrated rPA post.The rPA step with 26.7L/ minute Peak Flow Rate and≤working pressure of 13psig operates.The maximum CTLA4-Ig protein load that is applied to the rPA post is 25g CTLA4-Ig protein/rise resin.
RPA post 25mM Tris, 250mM NaCl, the pH8.0 damping fluid carries out balance.When bottom line 3CV level pad was respectively 7.8-8.2 and 23.0-27.0mS/cm through the pH of post and effluent and electroconductibility value, balance was finished.
Inactivation of virus step products storehouse is applied to equilibrated rPA post.The rPA chromatographic step comprises 2 washing steps.First washing step uses bottom line 5CV 25mM Tris, 250mMNaCl, and 0.5% Triton X-100, the pH8.0 damping fluid carries out, to remove weak bonded material from the rPA post.Second washing step uses 25mM Tris, 250mM NaCl, and the pH8.0 damping fluid carries out.Second washing step uses bottom line 5CV to remove residual Triton X-100 from the rPA post.
CTLA4-Ig protein uses the 100mM glycine, and the pH3.5 damping fluid is wash-out from the rPA chromatography column.Work as A 280Increase to when surpassing baseline 〉=0.2AU, eluate is turned to collection container.Effluent is filled in the collection container that is equipped with agitator by 0.2 μ m rhodia strainer.Collect the A that trail edge of eluate until elution peak 280Reduce to≤value of 0.2AU.The pH in eluate storehouse uses 2M HEPES, and the pH8.0 damping fluid is adjusted to pH7.5 ± 0.2.RPA chromatographic step product storehouse remain in 2-8 ℃ maximum 3 days.
Collect a mole sialic acid and mole CTLA4-Ig dimer product of the proteinic mol ratio of CTLA4-Ig 〉=8; CTLA4-Ig high molecular weight material storehouse exists with≤2.5%; And the MCP-1 storehouse of existence≤38ng/mL.
The QFF that is used for the CTLA4-Ig purifying goes through ion exchange chromatography: the anion-exchange chromatography step of downstream CTLA4-Ig production method is used Q agarose mobile fast (QFF) anion-exchange chromatography resin (GE Healthcare (Waukesha, WI (AmershamBiosciences in the past).The purpose of QFF chromatographic step is to reduce residual A protein level, and the other minimizing from the host cell DNA in virus filtration step products storehouse is provided.QFF post step also is used to control the sialic acid and the CTLA4-Ig protein mol ratio in QFF chromatographic step product storehouse, and the other control of carrying out middle CTLA4-Ig HMW material horizontal is provided.The reference mark of mainly carrying out that is used for reducing the CTLA4-IgHMW material is the HIC step.
60-140m internal diameter post is filled to the height of 28-35cm with the QFF resin, the volume of the about 536L-667L of expression.By measuring the HETP and the A of packed column sPost qualified being used for used.0.02-0.08cm HETP and the A of 0.8-1.2 sBe used for the qualification evaluation of post.
The QFF column operation carries out at ambient temperature.Virus filtration step products storehouse is loaded on the equilibrated QFF post.The QFF step with 38.7L/ minute Peak Flow Rate and≤working pressure of 35psig operates.The maximum CTLA4-Ig protein load that is applied to the QFF post is 25g CTLA4-Ig protein/rise resin.
The QFF chromatography column at first carries out sanitary measure with the 1N sodium hydroxide solution.Sanitary measure uses the 1N sodium hydroxide solution of 2-4CV to carry out.When the electroconductibility of effluent equaled 136-202mS/cm and post and keeps 60-120 minute, sanitary measure was finished.
Behind the sanitising step, post 25mM HEPES, 100mM sodium-chlor, the pH8.0 damping fluid carries out balance.When bottom line 4CV level pad has been 7.7-8.3 and electroconductibility when being 10.5-12.9mS/cm through the pH of post and effluent, balance is finished.
The virus filtration step products storehouse that comprises in the biological processing bag is transferred in the aseptic stainless steel collection container.
Virus filtration step products storehouse is applied to equilibrated QFF post.The QFF chromatographic step comprises 2 washing steps.First washing step uses bottom line 5.0CV 25mM HEPES, 120mM NaCl, and the pH8.0 damping fluid carries out.Second washing step uses bottom line 5.0CV25mM HEPES, 130mM NaCl, and the pH8.0 damping fluid carries out.
The CTLA4-Ig dimer uses 25mM HEPES, 200mM NaCl, and the pH8.0 damping fluid is wash-out from the QFF chromatography column.A when effluent 280When beginning to increase, initial eluate is collected.During wash-out, effluent is filled in the stainless steel collection container by 0.2 μ m rhodia strainer.Collecting eluate reduces to above baseline≤0.2AU until the absorbancy of trailing the edge of elution peak.Make collection container be cooled to 2-8 ℃ subsequently.Is 3 days about QFF chromatographic step product storehouse in 2-8 ℃ maximum retention time.
Collect a mole sialic acid and mole CTLA4-Ig dimer product of the proteinic mol ratio of CTLA4-Ig 〉=8; CTLA4-Ig high molecular weight material storehouse exists with≤2.5%; CTLA4-Ig low molecular weight material (for example CTLA4-Ig monomer) storehouse exists with≤0.5%; And the MCP-1 storehouse of existence≤9.5ng/mL.
Pall Filtron TFF system is used for concentrating and diafiltration steps of downstream CTLA4-Ig production method.The purpose of this step is the elution buffer that makes QFF chromatographic step product storehouse be concentrated into 45-55g/L and be used by the final buffer-exchanged QFF chromatographic step that is used for the CTLA4-Ig composition.Spissated CTLA4-Ig protein storehouse is shifted and is entered in the 50-L biological processing bag by 0.2 μ m poly(vinylidene fluoride) strainer.
Embodiment 16: the CTLA4-Ig-mol ratio of amino monose is measured
This embodiment provides the method for amino monose (N-acetylgalactosamine, N-acetyl-glucosamine) and proteinic mol ratio in the acquisition CTLA4-Ig sample.
Instrument configuration: capillary electrophoresis system Beckman P/ACE MDQ CE system; The fluorescence (Laser-Induced-Fluorescence) of detector Beckman induced with laser is detection system (with P/ACE MDQ coupling) (LIF); Kapillary (the i.d.25 μ m that the end applies; O.d.360 μ m), the 27-31cm length overall is to hold P/ACE MDQ or 5510 PolyMicro Technologies, catalog number (Cat.No.) TSP025375; Maxi-Mix mixing tank Thermolyne, (VWR catalog number (Cat.No.) 58810-185).
Reagent:
Hydrating solution (the 4N HCl aqueous solution
160mL 6N HCl and 80mL HPLC grade water are added in the 250mL vial.
Stirring is with thorough mixing.
Preserve in 2-8 ℃ maximum 6 months.
Derivatize solution I (the 0.1M APTS aqueous solution)
192 μ LHPLC grade water are added in the 10mg APTS powder in the vial.
Make bottle vortex 5-10 second to dissolve APTS fully.
Preserve in 2-8 ℃ maximum 1 year.
Derivatize solution II (1M acetate and 0.25M NaBH 3 CN)
In the 0.4mL centrifuge tube, dilute 20 μ L acetate (17 times of dilutions), with preparation 1M acetic acid solution with 320 μ LHPLC grade water.
With 2.0 ± 0.5mg NaBH 3CN is weighed in the profound hypothermia bottle.
Use following formula, the 1M acetic acid solution that adds suitable volumes is with preparation 0.25M NaBH 3CN.Volume (μ L)=10 3* (the NaBH that represents with mg 3CN weight)/(62.84g/mol * 0.25m0l/L)
. sodium cyanoborohydride (NaBH 3CN) should preserve in moisture eliminator in the dark.Recommend following reagent is assigned to again to be used for storage in a series of 2.0mL cryovials, to avoid the initial reagent bottle of repeated open:. 1.0g ± 0.2mg sodium cyanoborohydride is weighed in the 2.0mL cryovial.By this way will be from the entire contents aliquots containig of the sodium cyanoborohydride in the initial bottle.Cover lid and together with reagent name, lot number and 6 months validity period orders (1,2,3 etc.) mark cryovial tightly.Bottle is used the Parafilm sealing to avoid moisture.Weighing up sodium cyanoborohydride from identical cryovial is used for the derivatize solution II and is no more than 3 times.On the laboratory work table, indicate this point and cryovial sequence number (SN).Observed reagent peak or weak mark may be after the cryovial repeated open or are taken place with the sodium cyanoborohydride of the sort of particular batch in the CE overview.If this influences the result, discard the cryovial of use so and from cryovial or from the new lot sodium cyanoborohydride, weigh up reagent with next sequence number (SN).
Again the acetylize damping fluid (the 25mM sodium bicarbonate, pH9.5)
0.210 ± 0.02g sodium bicarbonate is weighed in the clean glass beaker of clean 100mL.
Add 90mL HPLC grade water, and on agitating plate, mix and dissolve fully until salt.
With 10N NaOH pH is adjusted to 9.5 ± 0.1.
Add HPLC grade water to obtain the final volume of 100mL.Filter (step 1.26) solution and preserved in room temperature maximum 3 months.
Running buffer (60 ± 5mM sodium tetraborate, pH925)
1.21 ± 0.02g sodium tetraborate is weighed in the clean glass beaker of 100mL.
Add 90mL HPLC grade water, and on agitating plate, mix and dissolve fully until salt.
With 10N NaOH pH is adjusted to 9.25 ± 0.10.
For the final concentration of 60 ± 5mM, add HPLC grade water to obtain the final volume of 100mL.
For 55mM solution, weighing 1.11g (± 0.02) sodium tetraborate and be used for according to the above description the dissolving and titration.
For 65mM solution, weighing 1.31g (± 0.02) sodium tetraborate and be used for according to the above description the dissolving and titration.
Preserved in room temperature maximum 3 months.If peak resolving power influenced (R value＜1.0) prepares fresh damping fluid so.
Choose wantonly: (± 5mM) final concentration is by adding dilution tetraborate buffered soln (MicroSolv) in the 80mL150mM sodium tetraborate damping fluid with the 120mL ultrapure water for 60mM.With 10N NaOH titration so that pH value of solution reaches 9.25 (± 0.1).
For 55mM tetraborate solution, 66mL150mM tetraborate damping fluid is diluted in the 114mL ultrapure water.As above titration.
For 65mM tetraborate solution, the slow middle liquid of 78mL150mM tetraborate is diluted in the 102mL ultrapure water.As above titration.
Solution was preserved in room temperature maximum 3 months.
If peak resolving power influenced (R value＜1.0) prepares fresh damping fluid so.
Capillary douche solution
N NaOH solution
The adding of 1mL 10N NaOH solution is comprised in the 15mL scale plastics tubing of 9mL HPLC grade water.By vortex 5-10 thorough mixing second.
Solution was preserved in room temperature maximum 6 months.
N HCl solution:
The adding of 1mL6N HCl solution is comprised in the 15mL scale plastics tubing of 5mL HPLC grade water.By vortex 5-10 thorough mixing second.
Solution was preserved in room temperature maximum 6 months.
3.6.3 80% methanol solution
The adding of 8mL HPLC grade methyl alcohol is comprised in the 15mL scale plastics tubing of 2mL HPLC grade water.By vortex 5-10 thorough mixing second.
Solution was preserved in room temperature maximum 6 months.
Monose standard stock solution
N-acetyl-glucosamine (GalNAc)
5 ± 1mg GalNAc accurately is weighed in the 2.0mL profound hypothermia bottle.
Add 1mL HPLC grade water and pass through the vortex thorough mixing until dissolving.
The accurate concentration (mg/mL) of recording solution.
N-acetylgalactosamine (GlcNAc)
5 ± 1mg GlcNAc accurately is weighed in the 2.0mL profound hypothermia bottle.
Add 1mL HPLC grade water and pass through the vortex thorough mixing until dissolving.
The accurate concentration (mg/mL) of recording solution.
N-acetylmannosamine (ManNAc)
5 ± 1mg ManNAc accurately is weighed in the 2.0mL profound hypothermia bottle.
Add 1mL HPLC grade water and pass through the vortex thorough mixing until dissolving.
The accurate concentration (mg/mL) of recording solution.
Preserve in-20 ℃ monose standard stock solution maximum 1 year.
Monose working solution I: internal standard working solution
Use HPLC grade water that the stock solution of ManNAc is diluted 100 times in the 2mL profound hypothermia bottle by 20 μ L ManNAc stock solutions are added, described profound hypothermia bottle has comprised 1980 μ L HPLC grade water.The about 5-10 of vortex second.
Preserve in 2-8 ℃ the internal standard working solution maximum 6 months.
Monose working solution II: amino hybrid standard working solution
In the 2.0mL profound hypothermia bottle that comprises 1960 μ L HPLC grade water, add the stock solution of 20 μ LGalNAc and GlcNAc respectively.The about 5-10 of vortex second.
Preserve in 2-8 ℃ amino hybrid standard working solution maximum 6 months.
Sample and reference material solution.
In the 2-8 ℃ of refrigerated protein example that thaws, and by being inverted mixing gently.
With HPLC grade water sample and reference material are diluted to about 1.0mg/mL.Indicate concentration to 3 significant figure.
The CE operational conditions
Running buffer (step 2.5) 60mM sodium tetraborate, pH9.25
25 ℃ of capillary column body temperature degree
Voltage 25-30kV, holotype
Detector condition LIF detector excites: 488nm, emission: 520nm.
Sample injection pressure injection pattern, 20s when 0.5PSI
10 minutes working times
10 ℃ of sample storages
Program
Annotate: use 10 μ L pipettors and little tip (micro tips) with shift 10 μ L sample volumes and suitably the pipettor of size to shift other reagent (referring to the scope of step 2.10 in 2.14).
Hydrolysis
In the 0.65mL centrifuge tube, add 10 μ L ManNAc working solutions and 200 μ L 4N hydrating solutions (step 3.1).This serves as system's blank.
In the 0.65mL centrifuge tube, add the amino mixed standard solution (step 3.9) of 10 μ L ManNAc working solutions and 10 μ L.Further add 200 μ L4N hydrating solutions.This serves as and is used for quantitatively and the monose standard of system's suitability.Prepare in duplicate.
In the 0.65mL centrifuge tube, add 10 μ L ManNAc working solutions and 10 μ LCTLA4-Ig reference material solution (about 1mg/mL).
Further add 200 μ L 4N HCl solution.Prepare in duplicate.
In the 0.65mL centrifuge tube, add 10 μ L ManNAc working solutions and 10 μ L sample solutions (about 1mg/mL).Further add 200 μ L 4N HCl solution.Prepare in duplicate.
Make about 10 seconds of sample vortex and centrifugal about 5-10 second.Sample placed the little bottle stand in 96 positions and at baking box in 95 ℃ of incubations 6 hours.
After the hydrolysis, with the sample of hydrolysis place-20 ℃ 10 minutes with cooling.
Make that the sample of hydrolysis is of short duration centrifugally to be forced to bottom (in high speed time 5-10 second) to pipe until any condensation product.In SpeedVac, make sample evaporation to dry.
Annotate: close the SpeedVac heating, and vaporator rate is made as " low ".
With 100 μ L HPLC grade every kind of samples of water reconstruct and vortex 10-15 second.In SpeedVac, make sample evaporation to dry.
Annotate: close the SpeedVac heating, and vaporator rate is made as " low ".
Acetylize again
With 10 μ L M6 more every kind of sample of acetylize damping fluid reconstruct and vortex 10-15 second with thorough mixing.With 4 μ L M3 again acetylation reagent add in each pipe.The about 5-10 of vortex second.Incubation on ice 30 minutes.
Annotate: acetylize damping fluid (M6) and reagent (M3) can be used the 25mM NaHCO of inner (in house) preparation respectively again 3(adding 20 μ L) and diacetyl oxide (adding 4 μ L) are replaced.
In SpeedVac, make sample evaporation to dry.
Annotate: close the SpeedVac heating, and vaporator rate is made as " low ".
With 100 μ LHPLC grade every kind of samples of water reconstruct and vortex 10-15 second.In SpeedVac, make sample evaporation to dry.
Annotate: close the SpeedVac heating, and vaporator rate is made as " low ".
Derivatize
Eppendorf centrifuge is placed the oven temperature of baking box with balance to 55 ℃.
With 10 μ L derivatize solution I (0.1M APTS solution, step 3.2) every kind of samples of reconstruct.The about 5-10 of vortex second.
Add 5 μ L derivatize solution II (1M HAc and 0.25M NaBH 3CN, step 3.3).Vortex about 5-10 second and centrifugal.
Sample flasket is loaded in the whizzer of heating in advance fast, and whizzer is put back in 55 ℃ of baking boxs.3 hours whiles of incubation are centrifugal under 2000rpm.This prevents solvent condensation on little bottle surface.
The instrument configuration preparation
New kapillary is installed, is used following step to wash with high pressure mode (80PSI):
1N NaOH 20 minutes.
HPLC grade water 10 minutes.
60mM sodium tetraborate damping fluid 10 minutes.
Operation
Before each operation, operation wash/rinse order is with the flushing kapillary.
Operational system suitability criterion (monose standard) is fit to guarantee system subsequently.
Use 1N NaOH may etching from the inside capillaceous of different vendor, and cause the change that moves in the transition time from start to finish.If this transition time that causes postpeak (GlcNAc) surpasses 10.0 minutes,, may replace 1N NaOH with 0.1N NaOH or HPLC grade water so for step 2 flushing.
When using kapillary of equal value and above-mentioned washing operation enough, may use 80% methyl alcohol and/or 1N HCl so that postpeak (GlcNAc) in 10.0 minutes example values.
Preparation with dry injection
Behind the derivatize, make sample be cooled to room temperature.At room temperature centrifugal about 10 seconds, be forced to bottom to pipe until condensation product.
In each pipe, add 85 μ L HPLC grade water so that the final volume of every kind of sample reaches 100 μ L.Vortex 5-10 second.
From each pipe, shift 10 μ L samples in CE trace bottle (micro vial), and in each pipe, add 190 μ LHPLC grade water.Vortex 5-10 second.
Rinse step and injection sequence:
Annotate: for per 4 injections, with the CE runtime buffer fluid exchange CE running buffer (because ion depletion action) of prepared fresh.Carry out capillary douche with 40psi.
Figure A200680053116D03131
Figure A200680053116D03141
System's suitability
Annotate: except as otherwise noted, the first time of system's suitability value using system suitability criterion, injection was measured.
The electrophorogram of first kind of system's suitability should be similar to the sort of shown in Figure 81, and wherein peak 1 is GalNAc; Peak 2 is ManNAc; Peak 3 is GlcNAc.
Annotate: when the CE instrument that uses except that Beckman PACE MDO, owing to keep the various configurations of the cylinder capillaceous that separates, length capillaceous can be different from this method specified the sort of.This will cause the variation in analyte transition time and the peak intensity.
Calculate 2 peak-to-peak resolving power of vicinity according to equation by instrument about first kind of system's suitability criterion:
R = 2 ( t 2 - t 1 ) ( W 1 + W 2 )
Wherein:
R-resolving power
t 2, t 1-2 adjacent peaks are divided other transition time
W 1, W 2-2 adjacent peaks are respectively in the peak width at baseline place
The R value is necessary 〉=and 1.0.If R＜1.0 are so with wash/rinse order flushing kapillary; If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.For final system suitability injection, use the postpeak (GlcNAc) of following formula must have＜1.4 tailing factor:
T＝W 0.05/2f
Wherein:
T-tailing factor
W 0.05-in the peak width in 5% when height
F-the width of front, peak when peak maximum
If T 〉=1.4 are so with wash/rinse order flushing kapillary; If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.
Duplicate injection shows following example values:
■ GlcNAc is to the peak area ratio of MaNAc: RSD≤10% (calculating in step 7.1)
The transition time of ■ GlcNAc was answered≤10.0 minutes
The ■ overview should be equivalent to Figure 81, and wherein observing 3 peaks and internal standard (ManNAc) is the 2nd peak.
If before specimen, do not reach any one in the above-mentioned example values,, at first increase voltage if the transition time of GlcNAc was greater than 10.0 minutes so.Secondly, if peak area ratio〉10%, determine its pH or replace kapillary thereby prepare fresh CE damping fluid so.After adjusting instrument, the injection of duplicated system suitability.When analyzing the peak overview, if the remarkable reduction in the peak height of generation ManNac is checked so to guarantee to enter the not misalignment of fiberoptic cable in the LIF module.
Recently measure the monose standard percentage by the peak area that compares internal standard and monose standard ingredient and compare RSD.Will be about the peak area of each monosaccharide component divided by peak area about the internal standard of each monose standard injection.Calculating is about the GalNAc of 2 kinds of standards of carrying out together and the per-cent RSD of GlcNAc.RSD answers≤10%.If do not meet this example average, kapillary should as above wash or replace so.
Calculate
Calculate GalNAc and GlcNAc peak area ratio with respect to internal standard (ManNAc).The duplicate injection of using the forth day of a lunar month kind system suitability criterion is so that meet above-mentioned example values, and all of before the sample and back injection are carried out together, system's suitability criterion carries out identical calculations.
Peak area ratio=will (GlcNAc, peak area GalNAc) be divided by the peak area about the internal standard (ManNAc) of each system suitability criterion injection about every kind of monosaccharide component.
Figure A200680053116D03161
Calculating in system's suitability criterion about the mean value of the peak area ratio of GlcNAc and GalNAc.Also base of calculation poor (S.D.) and per-cent coefficient of variation (%RSD)
Example values: about RSD≤10% of the peak area ratio of GlcNAc.
2 kinds of the injection of before the sample and back, carry out together, system's suitability criterion: about per-cent RSD≤10% of the peak area ratio of GlcNAc and GalNAc.
If do not meet this example average (RSD〉10%), kapillary must wash once more with cleaning procedure so, and those samples and the monose standard of carrying out together need operation once more.If still do not meet example average, replace kapillary and flushing so.Move sample once more and carry out the monose standard together.
Figure A200680053116D03171
Wherein:
Measurement number in the n-sample
Ce Liang not for x-
Calculate the proteinic mol ratio of GalNAc/:
Figure A200680053116D03173
Wherein:
R GalNAc=GalNAc is to proteinic mol ratio
A GalNAcThe peak area of GalNAc in the=sample (μ V second)
A ManNAcThe peak area of ManNAc in the=sample (μ V second)
A ManNAcOThe peak area of ManNAc in the=monose standard (μ V second) mean value
A GalNAcOThe peak area of GalNAc in the=monose standard (μ V second) mean value
V GalNAcO=be used for the GalNAc volume (representing) that the monose working solution of hydrolysis comprises with μ L
C GalNAcO=be used for the GalNAc concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=is used for the volume (representing with μ L) of the protein example of hydrolysis
Cp=is used for the concentration (representing with mg/mL) of the protein example of hydrolysis
MW A Baxipu=according to the molecular weight of the A Baxipu reference material of analysis certificate (COA)
MW GlcNAcThe molecular weight of=GalNAc (221.2 dalton)
Standard is carried out together
When calculating the mol ratio of CTLA4-Ig material and sample, use all 8 kinds system's suitability criterion of carrying out together.The mean value of asking peak area is to be included in this equation.This will be used for preceding 3 kinds of samples.For every other sample, use the average peak area of ensuing 4 kinds of monose standards of carrying out together and preceding 4 kinds of monose standards of carrying out together to be used for mol ratio calculating always.
Calculate the proteinic mol ratio of GIcNAc/
R GalNAc = A GlcNAc x A ManNAc 0 x V GlcNAc 0 x C GlcNAc 0 x MW CTLA 4 - Ig A ManNAc x A GlcNAc 0 xVpxCpx MW GlcNAc
Wherein:
R GlcNAc=GlcNAc is to proteinic mol ratio
A GlcNAcThe peak area of GlcNAc in the=sample (μ V second)
A ManNAcThe peak area of ManNAc in the=sample (μ V second)
A ManNAcOThe peak area of ManNAc in the=monose standard (μ V second) mean value
A GlcNAcOThe peak area of GlcNAc in the=monose standard (μ V second) mean value
V GlcNAcO=be used for the GlcNAc volume (representing) that the monose working solution of hydrolysis comprises with μ L
C GlcNAcO=be used for the GlcNAc concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=is used for the volume (representing with μ L) of the protein example of hydrolysis
Cp=is used for the concentration (representing with mg/mL) of the protein example of hydrolysis
MW CTLA4-IgThe molecular weight of=CTLA4-Ig reference material
MW GlcNAcThe molecular weight of=GlcNAc (221.2 dalton)
Example values.Should not surpass 10% about 2 kinds, per-cent RSD that carry out together, amino system suitability criterion peak area ratio.About the molar average ratio of the amino monose in the reference material should under in the table in the specified scope.For every kind of component, about the 4 kinds of results' duplicate injection of preparation (duplicate) %RSD necessary＜/=25%.
The molar ratio range of table-CTLA4-Ig reference material
Monose Scope
GalNAc 2.0-3.2
GlcNAc 18-32
Embodiment 17: measure amino monose (GalNAc and GlcNAc) by capillary electrophoresis (CE) Mol ratio
In one embodiment, the CTLA4-Ig composition has about 15-35 mole GlcNAc/ mole protein and the proteinic feature of about 1.7-3.6 mole GalNAc/ mole.Following example has been described the method for measuring these mol ratios.
Reagent: hydrating solution (4N HCl); Derivatize solution I (0.1M8- amino 1,3,6-trisulfonic acid, trisodium salt (APTS) aqueous solution); The derivatize solution II (is dissolved in the 0.25MNaBH of 1M acetate 3CN); Again the acetylize damping fluid (the 25mM sodium bicarbonate, pH9.5); Running buffer (60 ± 5mM sodium tetraborate, pH9.25); Capillary douche solution (1N NaOH; 1NHCl; 80% methyl alcohol); Concentration is GalNAc, the GlcNAc of 5mg/ml and the monose standard stock solution of ManNAc; Monose working solution I: the internal standard working solution is 100 times of dilutions of ManNAc stock solution; Monose working solution II: amino hybrid standard working solution, 100 times of dilutions of GalNAc and GlcNAc stock solution.
Instrument configuration: the CE system is a Beckman P/ACE MDQ CE system; Detector: with P/ACE MDQ) (LIF) detection system of link coupled Beckman induced with laser; Uncoated kapillary (i.d.25 μ m, o.d.360 μ m) 27-31cm length overall is to hold P/ACE MDQ.
The capillary electrophoresis operational conditions: running buffer (the 60mM sodium tetraborate, pH9.25); Capillary column body temperature degree: 25 ℃; Voltage: 25-30kV, holotype; The detector condition: the LIF detector, excite at the 488nm place, launch at the 520m place; Sample injection: pressure injection pattern, 20s when 0.5PSI; Working time: 10 minutes; Sample storage: 10 ℃.
Hydrolysis: 10 μ LManNAc working solutions and 200 μ L4N HCl are mixed, with the preparation system blank.The amino mixed standard solution of 10 μ L ManNAc working solutions and 10 μ L is mixed, with preparation monose standard with 200 μ L4N HCl.10 μ L ManNAc working solutions and 10 μ LCTLA4-Ig dimers (about 1mg/ml) are mixed, with the preparation specimen with 200 μ L 4N HCl.With all pipe vortexs 10 seconds, and centrifugal 10 seconds, subsequently in 95 ℃ of incubations 6 hours.Behind the hydrolysing step, sample is placed-20 ℃ 10 minutes with the cooling.Make sample be rotated down 10 seconds and in SpeedVac, be evaporated to drying.
Acetylize again: with 100 water reconstruct hydrolysis of μ L HPLC grade and exsiccant samples.The sample of reconstruct carries out acetylize again by following: add 10 μ L M6 N-acetylize damping fluid (Glyko) and 4 μ L M3 acetylation reagent (Glyko) more again, mix subsequently and at incubation (30 minutes) on ice.Make sample be rotated down 10 seconds and in SpeedVac, be evaporated to drying.
Derivatize: 55 ℃ of balances of the sample of reconstruct (100 μ L HPLC grade water), add 10 μ L derivatize solution I subsequently, of short duration mixing, and add 5 μ L derivatize solution II.Sample is loaded in the whizzer of heating in advance and simultaneously centrifugal under 2000rpm in 55 ℃ of incubations 3 hours.
CE injection: make by adding HPLC grade water that the final volume of sample reaches 100 μ L behind the derivatize, and with 10 μ L sample transfer to CE trace bottle with 190 μ LHPLC grade water.Before the sample injection, the CE cylinder washes (1-3 minute working time) widely with HPLC grade water, subsequently with running buffer balance flushing (5 minute working time).After the initial flush, monose standard that will be used for analyzing and sample are expelled to CE cylinder (10 minute working time).After the injection operation of every kind of standard or specimen, the CE cylinder is with HPLC grade water and running buffer washes and balance.The electrophorogram of system's suitability should be similar to Figure 30.
Calculate: calculate GalNAc and GLCNAc peak area ratio with respect to internal standard ManNAc.
Peak area ratio=monose peak area (GalNAc or GlcNAc)/ManNAc peak area,
Wherein the coefficient of variation (RSD) about peak area ratio is equal to or less than 10%.
Calculate monose (for example GalNAc) and the proteinic ratio of CTLA4-Ig:
Ratio GalNAc=(A GalNAcX A ManNAcOX V GalNAcOX C GalNAcOX MW CTLA4-Ig two Aggressiveness)/(A ManNAcX A GalNAcOX Vp x Cp x MW GalNAc)
Ratio GalNAc=GalNAc is to proteinic mol ratio
A GalNAcPeak area in the=GalNAc sample (μ V second)
A ManNAcPeak area in the=ManNAc sample (μ V second)
A ManNAcOThe peak area of ManNAc in the=monose standard (μ V second) mean value
A GalNAcOThe peak area of GalNAc in the=monose standard (μ V second) mean value
V GalNAcO=be used for the GalNAc volume (representing) that the monose working solution of hydrolysis comprises with μ L
C GalNAcO=be used for the GalNAc concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=is used for the volume (representing with μ L) of the protein example of hydrolysis
Cp=is used for the concentration (representing with mg/mL) of the protein example of hydrolysis
MW CTLA4-IgThe dimeric molecular weight of=CTLA4-Ig
MW GalNAc=221.2 dalton
Table 18: monose and CTLA4-Ig molecule or dimeric molar average ratio
Monose Scope
GalNAc 2.0-3.2
GlcNAc 18-32
Embodiment 18: measure neutral monose (seminose, rock algae by capillary electrophoresis (CE) Sugar and semi-lactosi) mol ratio
Reagent: hydrating solution (2M trifluoroacetic acid (TFA)); Derivatize solution I (0.1M8- amino 1,3,6-trisulfonic acid, trisodium salt (APTS) aqueous solution); The derivatize solution II (is dissolved in the 0.25M NaBH of 1M acetate 3CN); Running buffer (60 ± 5mM sodium tetraborate, pH9.25); Capillary douche solution (1N NaOH; 1N HCl; 80% methyl alcohol); Concentration is the monose standard stock solution of seminose (Man), Fucose (Fuc), semi-lactosi (Gal) and the wood sugar (Xyl) of 5mg/ml; Monose working solution I: the internal standard working solution is 100 times of dilutions of Xyl stock solution; Monose working solution II: neutral hybrid standard working solution, 100 times of dilutions of Man, Fuc and Gal stock solution.
Instrument configuration: the CE system is a Beckman P/ACE MDQ CE system; Detector: with P/ACE MDQ) (LIF) detection system of link coupled Beckman induced with laser; Uncoated kapillary (i.d.25 μ m, o.d.360 μ m) 27-31cm length overall is to hold P/ACE MDQ.
The capillary electrophoresis operational conditions: running buffer (the 60mM sodium tetraborate, pH9.25); Capillary column body temperature degree: 25 ℃; Voltage: 25-30kV, holotype; The detector condition: the LIF detector, excite at the 488nm place, launch at the 520m place; Sample injection: pressure injection pattern, 20s when 0.5PSI; Working time: 10 minutes; Sample storage: 10 ℃.
Hydrolysis: 10 μ L wood sugar working solutions and 200 μ L 2M TFA are mixed, with the preparation system blank.The neutral mixed standard solution of 10 μ L wood sugar working solutions and 10 μ L is mixed, with preparation monose standard with 200 μ L 2MTFA.10 μ L wood sugar working solutions and 10 μ L CTLA4-Ig dimers (about 1mg/ml) are mixed, with the preparation specimen with 200 μ L 2M TFA.With all pipe vortexs 10 seconds, and centrifugal 10 seconds, subsequently in 95 ℃ of incubations 6 hours.Behind the hydrolysing step, sample is placed-20 ℃ 10 minutes with the cooling.Make sample be rotated down 10 seconds and in SpeedVac, be evaporated to drying.
Derivatize: with 100 μ LHPLC grade water reconstruct sample and 55 ℃ of balances, add 10 μ L derivatize solution I subsequently, of short duration mixing, and add 5 μ L derivatize solution II.Sample is loaded in the whizzer of heating in advance and simultaneously centrifugal under 2000rpm in 55 ℃ of incubations 3 hours.
CE injection: make by adding HPLC grade water that the final volume of sample reaches 100 μ L behind the derivatize, and with 10 μ L sample transfer to CE trace bottle with 190 μ L HPLC grade water.Before the sample injection, the CE cylinder washes (1-3 minute working time) widely with HPLC grade water, subsequently with running buffer balance flushing (5 minute working time).After the initial flush, monose standard that will be used for analyzing and sample are expelled to CE cylinder (10 minute working time).After the injection operation of every kind of standard or specimen, the CE cylinder is with HPLC grade water and running buffer washes and balance.The electrophorogram of system's suitability should be similar to Figure 31.
Calculate: calculate Man, Gal and Fuc peak area ratio with respect to the internal standard wood sugar.
Peak area ratio=monose peak area (Gal, Fuc or Man)/wood sugar peak area,
Wherein the coefficient of variation (RSD) about peak area ratio is equal to or less than 10%.
Calculate monose (for example Man) and the proteinic ratio of CTLA4-Ig:
Ratio Man=(A ManX A XylOX V ManOX C ManOX MW The CTLA4-Ig dimer)/(A XylXA ManOX Vp x Cp x MW Man)
Ratio Man=Man is to proteinic mol ratio
A ManThe peak area of Man in the=sample (μ V second)
A XylThe peak area of Xyl in the=sample (μ V second)
A XylOThe peak area of Xyl in the=monose standard (μ V second) mean value
A ManOThe peak area of Man in the=monose standard (μ V second) mean value
V ManO=be used for the seminose volume (representing) that the monose working solution of hydrolysis comprises with μ L
C ManO=be used for the mannose concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=the be used for volume (representing) of the protein example of hydrolysis with μ L
Cp=the be used for concentration (representing) of the protein example of hydrolysis with mg/mL
MW CTLA4-IgThe dimeric molecular weight of=CTLA4-Ig
MW Man=180.2 dalton.
Table 19: monose and CTLA4-Ig molecule or dimeric molar average ratio
Monose Scope
Seminose 10-20
Fucose 4.2-7.0
Semi-lactosi 9.2-17
Embodiment 19:CTLA4 A29YL104E The production of-Ig
CTLA4 A29YL104E-Ig is genetic engineering modified fusion rotein, and its Fc structural domain by the human normal immunoglobulin of the function binding domains of modified people CTLA-4 and IgG1 class is formed.In the B7 of CTLA-4 structural domain calmodulin binding domain CaM, carry out 2 amino-acid substitutions (L104E and A29Y), to produce this molecule.It comprises each 357 amino acid whose 2 glycosylated polypeptides chain.It exists as the covalent dimer that connects by interchain disulfide bond.As by substance assistant laser desorpted-ionization flight time (MALDI-TOF) mass spectrometric determination, CTLA4 A29YL104EIt is about 91 that-Ig has, the average quality of 800Da.
CTLA4 A29YL104E-Ig is the modified forms of CTLA4-Ig.Modification is made up of the point mutation that causes 2 amino-acid substitutions (L104E and A29Y).With respect to CTLA4-Ig, CTLA4 A29YL104E-Ig with the avidity of～2 times of increases in conjunction with CD80 (B7-1) and with the avidity of～4 times of increases in conjunction with CD86 (B7-2).CTLA4 A29YL104EIt is about 10 times that A Baxipu renders a service aspect-Ig kills and wounds in suppressor T cell propagation, cytokine production with via the CD28 dependency target cell of natural killer cell.CTLA4 A29YL104E-Ig causes that the appropriateness of the T cell proliferation of B7-1 mediation suppresses, but obviously more effective aspect the T cell proliferation of blocking-up B7-2 mediation.This embodiment has described the CTLA4 that comprises SEQ ID NO:4 A29YL104EThe production of-Ig molecule.The method of describing among this embodiment can be fit to and expand be used to produce other recombinant proteins, include but not limited to, excretory albumen for example cytokine and other hormones, as the Ig superfamily member or comprise the excretory albumen of the proteinic part of Ig superfamily and any protein of usually in Chinese hamster ovary celI, expressing.
About CTLA4 A29YL104EThe process flow sheet of-Ig culturing step is shown among Figure 24.CTLA4 A29YL104E-Ig produces in the bio-reactor at the 5000-L with about 4000L working volume and produces.The 1 batch of medicine is produced by the single production bio-reactor derived from the single bottle of cell bank.Production method comprises 3 stages being made up of inoculum amplification, production cell culture and downstream purification.The inoculum amplification stage uses the substratum of no animal ingredient to carry out.Produce the cell culture stage and also in the substratum of no animal ingredient, carry out, except using the D-semi-lactosi.
Cell culture medium.All substratum are prepared in the clean medium container of suitable size and sterilize via filtration.The composition that is used for the substratum of inoculum amplification is presented in following table.
Inoculum cell growth substrate basal culture medium
Figure A200680053116D03241
Seed and production bio-reactor cell growth substrate basal culture medium
Figure A200680053116D03242
Produce bio-reactor charging substratum
Figure A200680053116D03251
The inoculum amplification
Under controlled temperature, thaw and centrifugal from the cryovial of cell bank to remove cryoprotection agent medium.Cell is resuspended in the inoculum substratum and in the T bottle reclaims.80% minimum cell survival is an example values after thawing.Temperature and carbonic acid gas are controlled during T bottle incubation step.T bottle incubation is until obtaining 1.0 x 10 7The viable cell number of cell, and content transferred to shake in the bottle.Culture increases to reach required inoculum volume by a series of bottles that shake.About shaking a bottle inoculum density scope that goes down to posterity is 1.0-3.0 x 10 5Viable cell/mL.Temperature, carbonic acid gas and shaking table speed are controlled during shaking bottle incubation step.Reaching 1.5-3.0 x 10 6After the viable cell density scope of cell/mL, the shake-flask culture thing is merged in the aseptic inoculum transfer vessel.To transfer to 140-L seed bio-reactor from about 20L of last shaking flask inoculation kind thing amplification step, to reach 0.2-1.0 x 10 6The initial cell density range of viable cell/mL.
The seed bioreactor operation
140-L seed bio-reactor with about 90L working volume is operated with batch mode.Temperature in the 140-L seed bio-reactor, pH, pressure and dissolved oxygen concentration use distribution control system (DCS) to monitor and control.From 140-L seed bio-reactor, obtain sample every day with the growth of monitoring cell.The inoculum density scope of 140-L seed bio-reactor is 0.2-1.0 x 10 6Viable cell/mL.When reaching 〉=1-5 x 10 6During the viable cell density of cell/mL, 140-L seed bioreactor culture thing is used to inoculate 1100-L seed bio-reactor.The time length of 140-L seed bio-reactor step is about 3 days.Primary target viable cell density in the 1100-L seed bio-reactor is 0.4-1.5 x 10 6Viable cell/mL.
1100-L seed bio-reactor comprises the starting culture volume of 260L.1100-L seed bio-reactor is operated with batch mode.Temperature in the 1100-L seed bio-reactor, pH, pressure and dissolved oxygen concentration use DCS to monitor and control.When viable cell density reaches 〉=1.5x 10 6During cell/mL, the volume of culture uses basic medium to increase to 900L.From 1100-L seed bio-reactor, obtain sample every day with the growth of monitoring cell.When reaching 〉=2.0 x 10 6During the viable cell density of cell/mL, 1100-L seed bioreactor culture thing is used to inoculate 5000-L and produces bio-reactor.The time length of 1100-L seed bio-reactor step is about 4 days.The primary target viable cell density that 5000-L produces in the bio-reactor is 0.4-1.5 x 10 6Viable cell/mL.
The production bioreactor operation
5000-L produces the starting culture volume that bio-reactor comprises 3000L.5000-L produces bio-reactor and operates with fed-batch mode, and wherein temperature, pH, pressure and dissolved oxygen concentration use DCS to monitor and control.In the time of about 72 hours, in culture, add the T 500 bolus.In the operating period of producing bio-reactor, in the time of 144 ± 8 hours, the culture temperature setting point is from 37 ℃ of changes to 34 ℃.Carry out temperature variation and T 500 and add, to prolong the time length that 5000-L produces high cell survival in the bio-reactor step.From bio-reactor, obtain sample, with monitoring cell growth and viability, glucose, lactic acid and ammonia concentration.Sample is also with regard to CTLA4 A29YL104E-Ig concentration and sialic acid and CTLA4 A29YL104E-Ig protein mol ratio is tested.The charging substratum is added in the bio-reactor to keep required glucose concn.Main results standard about the production bio-reactor is sialic acid and CTLA4 A29YL104E-Ig protein mol ratio.Produce bio-reactor at 〉=6 target sialic acid and CTLA4 A29YL104EGather in the crops during-Ig protein mol ratio.The time length that 5000-L produces the bio-reactor step is about 14 days.The results volume that 5000-L produces bio-reactor is about 4000L.
Cell takes out and product concentrates
Cell uses 0.65 μ m film to take out from the culture fluid substratum by the tangential flow microfiltration.The microfiltration penetrant uses the 30kDa nominal molecular weight to concentrate by (NMWCO) film by cross-flow ultrafiltration.Transmembrane pressure and flow velocity are controlled during microfiltration and ultrafiltration step.Make enriched material through a series of membrane filters subsequently, the wherein final filter that uses by the 0.2LLm single that filters.The pH of enriched material is adjusted to 8.0 by adding 0.5M Tris solution.Microfiltration and ultra filtration filter are nonexpondable.The microfiltration strainer purifies and preserves in phosphoric acid with clorox and TritonX-100.The ultra filtration filter purifies and preserves in sodium hydroxide subsequently with clorox and sodium hydroxide.
Embodiment 20: recombinant C TLA4 A29YL104E The purifying of-Ig
Embodiment 20-A:CTLA4 A29YL104EThe example of-Ig purification process is shown in the following flow diagram, and the description of purification process is provided by this embodiment.
Figure A200680053116D03271
Inactivation of virus
The pH of clarifying concentrated results material is adjusted to 8.0 by adding 0.5M Tris solution.The external viral agent of potential carries out deactivation by the final concentration that adds 20%Triton X-100 to 0.5% (v/v).The protein soln that Triton X-100 handles mixed 〉=2 hours.
Affinity chromatography
Use the affinity chromatography of MabSelect A protein resin (GE Healthcare was called AmershamBiosciences in the past) post to be used for from carrying out material capture CTLA4 from the inactivation of virus step A29YL104E-Ig protein, and Bei Latasai protein is separated with most of impurity.
MabSelect A albumen post 25mM NaH 2PO 4, 150mM NaCl, the pH7.5 damping fluid carries out balance.When 350cm/ hour linear velocity, the dynamic binding capacity of affine resin is 25g CTLA4 A29YL104E-Ig protein/rise resin.157-L post bed can be in conjunction with about 3.9kgCTLA4 A29YL104E-Ig protein.
Material is applied to MabSelect A albumen post in the carrying out that Triton X-100 is handled, and post washs with the level pad of bottom line 3 column volumes (CV), to remove the impurity of weak reservation.These impurity comprise cytokine MCP-1 (MCP-1) and TritonX-100.CTLA4 A29YL104E-Ig protein is used the 250mM glycine subsequently, pH3.0 damping fluid wash-out from post.CTLA4 A29YL104E-Ig protein is as narrow peak wash-out and collect and comprise 2M HEPES in about 2-3CV elution buffer, in the groove of pH7.5 damping fluid, with quick increase pH and therefore make the formation of Bei Latasai high molecular (HMW) kind drop to minimum.
Anion-exchange chromatography
The use Q-agarose anion-exchange chromatography of mobile (QFF) resin (GE Healthcare) fast is mainly used in enrichment CTLA4 A29YL104EThe amount of the kind of the proteinic more high sialic acidization of-Ig.The Bei Latasai product storehouse of adjusting from the pH of MabSelect A albumen post before being applied to the QFF post with about 2 times of water for injection (WFI) dilution.
QFF post 50mM HEPES, 50mM NaCl, the pH7.0 damping fluid carries out balance.The MabSelect A albumen step products storehouse of pH and electroconductibility adjustment is applied to the QFF post, and post washs with the level pad of bottom line 3CV, to remove weak bonded impurity.Post is used 50mM HEPES subsequently, 140mM NaCl, and the pH7.0 damping fluid washs, and has the CTLA4 of low sialic acid content with removal A29YL104E-Ig kinds of protein.CTLA4 A29YL104E-Ig protein more the kind of high sialic acidization uses≤the 50mM HEPES of 5CV 200mMNaCl, pH7.0 damping fluid wash-out from post subsequently.
Hydrophobic interaction chromatography
Use the hydrophobic interaction chromatography (HIC) of Toyopearl Phenyl 650M resin (Tosoh Biosciences) to be mainly used in minimizing from the CTLA4 in the product storehouse of QFF chromatographic step A29YL104EThe amount of-Ig HMW kind.Before being applied to the HIC post, QFF chromatographic step product storehouse 50mM HEPES, pH7.0 damping fluid and 50mM HEPES, 3.6M ammonium sulfate, the pH7.0 damping fluid dilutes, with the electroconductibility that reaches about 135mS/cm in the QFF product storehouse and≤CTLA4 of 1g/L A29YL104E-Ig concentration.
HIC post 50mM HEPES, 1.2M ammonium sulfate, the pH7.0 damping fluid carries out balance.CTLA4 with concentration and electroconductibility adjustment A29YL104E-Ig QFF chromatographic step product storehouse is applied to post.Post is used 50mM HEPES subsequently, 1.2M ammonium sulfate, and the pH7.0 damping fluid washs, to remove weak bonded impurity.CTLA4 A29YL104E-Ig protein uses 50mM HEPES, 0.55M ammonium sulfate, and the pH7.0 damping fluid is wash-out from the HIC post.
Virus filtration
CTLA4 from the HIC step A29YL104EConcentrate and the diafiltration in-Ig product storehouse reach by ultrafiltration (UF).The UF step is utilized 30-kDa NMWCO film and 25mM NaH 2PO 4, 10mM NaCl, pH7.5 damping fluid.The UF step is subsequently for using the virus filtration step of 15-nm Planova film (Asahi Kasei).CTLA4 A29YL104E-Ig protein storehouse uses 30-kDa NMWCO film to be adjusted to the protein concn of 25g/L by UF subsequently.
Post sanitary measure and storage
MabSelect A protein chromatographic post uses 0.1N NaOH solution to carry out sanitary measure, uses 25mM NaH 2PO 4, 150mM NaCl, pH7.5 damping fluid wash with reduction pH, and preserve in 20% ethanol in 2-8 ℃ subsequently.The QFF chromatography column carries out sanitary measure with 1N NaOH solution, and at room temperature preserves in 0.1N NaOH solution.The HIC post carries out sanitary measure with 0.1N NaOH solution, washs with 20% ethanol, and at room temperature preserves in 20% ethanol.
Embodiment 20-B: the further example of this kind purification process is as follows:
Inactivation of virus
The pH of clarifying concentrated results material is adjusted to 8.0 by adding 0.5M Tris solution.The external viral agent of potential carries out deactivation by the final concentration that adds 20% Triton X-100 to 0.5% (v/v).The protein soln that Triton X-100 handles mixed 〉=2 hours.
Be used for CTLA4 A29YL104EThe A albumen affinity chromatography of-Ig purifying: use the affinity chromatography of MabSelect A protein resin (GE Healthcare was called Amersham Biosciences in the past) post to be used for from carrying out material capture CTLA4 from the inactivation of virus step A29YL104E-Ig, and make CTLA4 A29YL104E-Ig separates with most of impurity.
140cm internal diameter post is filled to the height of 18-25cm with MabSelect PrA resin, the volume of the about 339L-372L of expression.By measuring the HETP and the A of packed column sPost qualified being used for used.0.02-0.08cm HETP and the A of 0.8-1.2 sBe used for the qualification evaluation of HIC post.
MabSelect PrA column operation carries out at ambient temperature.Inactivation of virus product storehouse is loaded on the equilibrated MabSelect PrA post.MabSelect PrA step 26.7L/ minute Peak Flow Rate and≤operate under the working pressure of 13psip.When 350cm/ hour linear velocity, be applied to the maximum CTLA4 of MabSelect PrA post A29YL104E-Ig protein load is 25 gram CTLA4 A29YL104E-Ig protein/rise resin.The post bed can be in conjunction with about 3.9kgCTLA4 A29YL104E-Ig protein.
MabSelect PrA post 25mM NaH 2PO 4, 150mM NaCl, the pH7.5 damping fluid carries out balance.When bottom line 3CV level pad was respectively 7.3-7.7 and 14.5-17.5mS/cm through the pH of post and effluent and electroconductibility value, balance was finished.
The middle material of carrying out that Triton X-100 is handled is applied to equilibrated MabSelect PrA post.Post bottom line 3CV25mM NaH 2PO 4, 150mM NaCl, 0.5%Triton X-100, the pH7.5 damping fluid washs, to remove the weak impurity that keeps from MabSelect PrA post.These impurity comprise cytokine MCP-1 (MCP-1) and Triton X-100.The subsequent wash step is used 25mM NaH 2PO 4, 150mM NaCl, the pH7.5 damping fluid carries out, to remove residual Triton X-100 from MabSelect PrA post.
CTLA4 A29YL104E-Ig uses the 250mM glycine, and the pH3.0 damping fluid is wash-out from the MabSelectPrA chromatography column.Work as A 280Increase to when surpassing baseline 〉=0.2AU, make eluate turn to collection container.Effluent is filled in the collection container that is equipped with agitator by 0.2 μ m rhodia strainer.Collect the A that trail edge of eluate until elution peak 280Reduce to≤value of 0.2AU.CTLA4 A29YL104E-Ig eluate is as narrow peak wash-out in about 2-3CV elution buffer.Therefore use 2M HEPES, the pH7.5 damping fluid is adjusted to pH7.5 ± 0.2 with the pH in eluate storehouse, with quick increase pH and make CTLA4 A29YL104EThe formation of-Ig high molecular (HMW) kind drops to minimum.MabSelect PrA chromatographic step product storehouse kept maximum 5 days at ambient temperature.The product storehouse can be cooled off and is used for storage; CTLA4 A29YL104EThe stable overview of-Ig is identical down with 22 ℃ at 5 ℃.Product can be preserved maximum 5 days.
Collect about 6 or about 5.2-about 7.6 the mole sialic acid with the mole CTLA4 A29YL104EThe CTLA4 of-Ig protein mol ratio A29YL104E-Ig dimer product.
Be used for CTLA4 A29YL104EThe QFF anion-exchange chromatography of-Ig purifying: the use Q-agarose anion-exchange chromatography of mobile (QFF) resin (GE Healthcare) fast is mainly used in enrichment CTLA4 A29YL104EThe amount of the kind of the more high sialic acidization of-Ig and reduce residual A protein level.The CTLA4 that adjusts from the pH of MabSelect A albumen post A29YL104E-Ig product storehouse is diluted about 2 times with water for injection (WFI) before being applied to the QFF post.
80cm internal diameter post is filled to the height of 27-35cm with the QFF resin, the volume of the about 136L-176L of expression.By measuring the HETP and the A of packed column sPost qualified being used for used.0.02-0.08cm HETP and the asymmetry (A of 0.8-1.2 s) be used for the qualification evaluation of post.
The QFF column operation carries out at ambient temperature.QFF post 50mM HEPES, 50mMNaCl, the pH7.0 damping fluid carries out balance.The MabSelect A albumen step products storehouse of pH and electroconductibility adjustment is applied to the QFF post.The QFF step is operated under the maximum operating pressure of the Peak Flow Rate of 16.4L/ minute (196cm/h) and 35psig.
Post carries out sanitary measure with the back with 1N NaOH solution before use.The sodium hydroxide solution that makes bottom line 2 column volumes is through post.Post kept static 60-120 minute subsequently.Acceptable electroconductibility scope about solution and effluent is 136-202mS/cm.
The post 50mM HEPES of bottom line 5 column volumes, 50mM sodium-chlor, the pH7.0 damping fluid carries out balance.PH and electroconductibility scope about sort buffer liquid are respectively 6.8-7.2 and 5.0-7.0mS/cm.These scopes also are used for determining whether balance of post.
The MabSelect A albumen step products storehouse of pH and electroconductibility adjustment is applied to the QFF post, and post washs with the level pad of bottom line 3CV, to remove weak bonded impurity.Post is used 50mM HEPES subsequently, 135mM NaCl, and the pH7.0 damping fluid washs, and has the CTLA4 of low sialic acid content with removal A29YL104E-Ig kind.
CTLA4 A29YL104E-Ig more the kind of high sialic acidization uses 50mM HEPES, 200mM NaCl, and the pH7.0 damping fluid is wash-out from the QFF chromatography column.When elution buffer at first was applied to post, initial eluate was collected.During wash-out, effluent is filled in the collection container by 0.2 μ m filter.Collecting eluate reduces to above baseline≤0.2AU until the absorbancy of trailing the edge of elution peak.The 50mM HEPES of use≤5CV, 200mM NaCl, the pH7.0 damping fluid is wash-out CTLA4 from post A29YL104E-Ig.Make collection container be cooled to 2-8 ℃ subsequently.Is 3 days about QFF chromatographic step product storehouse in 2-8 ℃ maximum retention time.
Collect about 6 or about 5.2-about 7.6 the mole sialic acid with the mole CTLA4 A29YL104EThe CTLA4 of-Ig protein mol ratio A29YL104E-Ig dimer product.
Be used for CTlA4 A29YL104EThe phenyl sepharose FF HIC of-Ig purifying: use the hydrophobic interaction chromatography (HIC) of ToyopearlPhenyl 650M resin (Tosoh Biosciences) to be mainly used in minimizing from the CTLA4 in the product storehouse of QFF chromatographic step A29YL104EThe amount of-Ig HMW kind.
100cm internal diameter post is filled to the height of 18-22cm with phenyl sepharose Phenyl 650M resin, the volume of the about 141-173L of expression.By measuring the HETP and the A of packed column sPost qualified being used for used.0.02-0.08cm HETP and the A of 0.8-1.2 sBe used for the qualification evaluation of HIC post.
The HIC column operation carries out at ambient temperature.Before being applied to the HIC post, QFF chromatographic step product storehouse 50mM HEPES, pH7.0 damping fluid and 50mM HEPES, 3.6M ammonium sulfate, the pH7.0 damping fluid dilutes, with the electroconductibility that reaches about 135mS/cm in the QFF product storehouse and≤CTLA4 of 1g/L A29YL104E-Ig concentration.The HIC step is operated under the maximum operating pressure of the Peak Flow Rate of 22.7L/ minute (173cm/h) and 45psi.Based on the CTLA4 that is present in the QXL eluate storehouse A29YL104EThe amount of-Ig can adopt the Multiple Cycle of HIC step.
The HIC post at first carries out sanitary measure with the 1N sodium hydroxide solution.When the 1N of 2-4CV sodium hydroxide solution had passed through post, sanitary measure was finished.Post keeps 60-120 minute subsequently to guarantee sanitary measure.
Behind the sanitising step, HIC post 50mM HEPES, 1.2M ammonium sulfate, the pH7.0 damping fluid carries out balance.When bottom line 3CV level pad has been 7.0 ± 0.3 and electroconductibility during for about 135mS/cm through the pH of post and effluent, balance is finished.
CTLA4 with concentration and electroconductibility adjustment A29YL104E-Ig QFF chromatographic step product storehouse is applied to post.Post is used 50mM HEPES subsequently, 1.2M ammonium sulfate, and the pH7.0 damping fluid washs, to remove weak bonded impurity.CTLA4 A29YL104E-Ig uses 50mM HEPES, 0.55M ammonium sulfate, and the pH7.0 damping fluid is wash-out from the HIC post.This HIC product storehouse remains in 2-8 ℃ in collection container.Maximum retention time in collection container is 3 days.
About 6 or about 5.2-about 7.6 the mole sialic acid with the mole CTLA4 A29YL104EThe CTLA4 of-Ig protein mol ratio A29YL104E-Ig dimer product; CTLA4 A29YL104E-Ig high molecular weight material storehouse exists with≤2.5%; CTLA4 A29YL104E-Ig low molecular weight material (for example, CTLA4 A29YL104E-Ig monomer) storehouse exists with≤0.5%; And the MCP-1 storehouse of existence≤9.5ng/mL.
Virus filtration.CTLA4 from the HIC step A29YL104EConcentrate and the diafiltration in-Ig product storehouse reach by ultrafiltration (UF).The UF step is utilized 30-kDa NMWCO film and 25mM NaH 2PO 4, 10mM NaCl, pH7.5 damping fluid.The UF step is subsequently for using the virus filtration step of 15-nmPlanova film (Asahi Kasei).CTLA4 A29YL104E-Ig product storehouse uses 30-kDa NMWCO film to be adjusted to the protein concn of 25g/L by UF subsequently.
Pall Filtron TFF system is used for downstream CTLA4 A29YL104EConcentrating and diafiltration steps of-Ig production method.The purpose of this step is to make HIC chromatographic step product storehouse be concentrated into 45-55g/L and by being used for CTLA4 A29YL104EThe elution buffer that uses in the final buffer-exchanged HIC chromatographic step of-Ig composition.Spissated CTLA4 A29YL104E-Ig product storehouse is transferred in the 50-L biological processing bag by 0.2 μ m poly(vinylidene fluoride) strainer.
Embodiment 21: biological activity-decide CTLA4 by surface plasmon resonance measurement A29YL104E -Ig Combine with the biologic specificity of B-7IG coreceptor
Surface plasma body resonant vibration (B7 combination)
This method is by surface plasmon resonance measurement amount CTLA4 A29YL104E-Ig combines with representative B7 coreceptor.B7Ig is immobilized into activatory CM5 sensor chip surface with high-density via primary amino.CTLA4 A29YL104EContrast of-Ig material, quality and diluted sample are to the concentration of 0.125-8ng/mL and be expelled on the B7Ig surface, to produce in conjunction with sensing figure.CTLA4 A29YL104E-Ig and immobilized B7Ig bonded initial velocity (slope) are being measured under mass transfer (diffusion) restricted condition on this B7Ig surface.The initial association rate of expression is directly related with active concentration with resonance units/second (RU/s).The association rate of sample uses the reference standard curve to be transformed into active concentration, wherein CTLA4 A29YL104EThe association rate of-Ig material is drawn at concentration.Net result is expressed as sample with respect to CTLA4 A29YL104EThe per-cent combination of-Ig material.
CTLA4 A29YL104EThe existence in human IgG1 Fc district uses surface plasma body resonant vibration (SPR) to detect among-the Ig.SPR makes it possible to measure in real time biologic specificity and interacts.Antibody fragment (the goat F special to the Fc district of human IgG (ab ') 2Anti-human IgG Fc) covalent immobilization is on the surface of sensor chip.CTLA4 A29YL104EThe combination of-Ig sample is compared with the sensor chip surface of unmodified by measuring, and replying of obtaining on this surface detects.As shown in Fig. 6 and table 23, be comparable about the result of method B, method C and concurrent mixture bonded resonance units.
Table 23. uses SPR at CTLA4 A29YL104EThe detection of human IgG Fc in the-Ig lot
Figure A200680053116D03341
People's cell IL2 suppresses to measure
This method is based on when with anti-CD3 and B cytositimulation, via CTLA4 A29YL104E-Ig suppresses to produce from the IL-2 of T cell.The Jurkat T cell that is used in the luciferase genes transfection under the control of IL-2 promotor is at various concentration C TLA4 A29YL104EThe existence of-Ig stimulates altogether with Daudi B cell and anti-CD3 down.Stimulate altogether and activate the IL-2 promotor, it produces luciferase protein matter successively.Resulting luminous signal uses the luciferase assay system to measure.In this system, CTLA4 A29YL104E-Ig produces the minimizing of dose-dependently in the luciferase activity.
As shown in Table 24, be comparable about the result of method B batch 000929-278, method C batch 224818-2004-007 and concurrent mixture batch 55128-162.EC50 value, slope factor and upper and lower asymptotic line are similar for all 3 kinds of samples, in 1 standard deviation.This points out from method C with from the CTLA4 of method B A29YL104E-Ig is performance comparably in external titration.
The comparison of table 24. luciferase activity of human IL-2's promotor mediation in the external biological assay of tiring.Dose response curve parameter about method of the present invention
Figure A200680053116D03342
Material:
-sensor chip CM5, regular grade Biacore (catalog number (Cat.No.) BR-1000-13)
-HBS-EP buffer B IA Certified 10mM HEPES pH7.4,150mM NaCl, 3.4mM EDTA .005%v/v
-tensio-active agent P20 Biacore (catalog number (Cat.No.) BR-1001-88)
-amine coupling reagent kit (Amine Coupling Kit) BIA Certified 115mg N-hydroxy-succinamide (NHS), 750mg 1-ethyl-3-(3 dimethylamino-propyl) carbodiimide hydrochloride (EDC), 10.5mL thanomin HCl Biacore (catalog number (Cat.No.) BR-1000-50)
-have the Biacore C instrument of PC compatible computer Biacore, (a catalog number (Cat.No.) BR-1100-51)
-Biacore C control software the Biacore that provides with Biacore C instrument, version 1.0.1
Amine coupling reagent kit BIA Certified: this test kit respectively comprises 1 bottle 115mg NHS, 750mg EDC and 10.5mL thanomin.Prepare each bottle according to manufacturer specification.With the NHS of 200 μ L volumes and EDC solution aliquots containig in indivedual plastic/glass bottles with suitable size and lid.When preserving in-20 ℃, these solution were stable for 2 months.With 200 μ L thanomin aliquots containigs in indivedual plastic/glass bottles with suitable size and lid.Preserve this solution in 2-8 ℃ and be stable according to the specification sheets of manufacturers.
In order to ensure with the flow cell good combination, flow cell will use 1 week or 286 injections, whichsoever at first take place.New flow cell will carry out immobilization when beginning weekly.Be used for the preparation immobilization B7.1Ig of sample test.Annotate: all solution aliquots containigs of 200 μ L are used for analyzing in the 7mmBiacore pipe.1 bottle that comprises B7.1Ig is thawed.Use 10mM acetate pH5.0 damping fluid (1.7) dilution B7.1Ig, to reach the surface quality of 3000-9000 resonance units (RU).Each 1 bottle (200 μ L) of EDC and NHS is thawed to envrionment temperature.From refrigerator, take out thanomin HCl and allow to be warming up to room temperature.According to Biacore software: open the open project " B7 IgImmobilization " that is selected from " Immobilization Wizard ".Open open file " B7 Immob.blw ".Progressively carrying out and pass through to click " Next " by guide confirms or changes to select.Select flow cell down and in " Notebook " label, provide experiment information at " User Information ".As described reagent and part bottle are placed specimen holder.Check instruction.Temporary file is saved as: B7 Immob BIOQC# Date InitialsChip # Flow cell #.blw.By clicking " Start " beginning immobilization.Destination file is saved as: B7 Immob BIOQC# Date Initial chip # flow cell #.blr.When mensuration is finished, print Wizard result and sensing figure.
Embodiment 22:CTLA4 A29YL104E The carbon of-Ig composition, trypsinase peptide mapping and IEF The hydrate content analysis
The tryptic digest peptide mapping
In this tryptic digestion method, CTLA4 A29YL104E-Ig sample uses guanidine-HCl to carry out sex change, and uses DTT and IAA reduces and alkylation.Sample uses the NAP-5 post to carry out desalination and digests with trypsinase.Digestion mixture separate by anti-phase (C18) chromatography and the peak by detecting in the UV at 215nm place absorbancy.
Reagent: mobile phase A solution (water-soluble 0.02% trifluoroacetic acid (TFA) is (v/v)); Mobile phase B solution (being dissolved in the 0.02%TFA (v/v) of 95%ACN (acetonitrile) and 5% water); Alkylating agent (200mM iodo-acid amide (IAA)); Dilution buffer liquid (100mM Tris, 25mMNaCl, pH8.0); The sex change damping fluid (the 8M guanidine, 50mM TRIS, pH8.0); Digestion damping fluid (50mM TRIS, 10mM CaCl 2, pH8.0); Reductive agent (100mM DTT).
Instrument configuration: (can use instrument configuration) NAP-5 post (Amersham, catalog number (Cat.No.) 17-0853-02); HPLC post well heater; Water ' sAlliance HPLC system with post well heater and UV detector.
Program is summarized
Figure A200680053116D03361
Reduction and alkanisation: sample (for example, CTLA4 A29YL104E-Ig etc.) be diluted to 10mg/ml (1mg) by adding the final volume that adds water to 100 μ L.560 μ L sex change damping fluids and 35 μ L reductive agents (100mM DTT) are added in the 100 μ L samples, mix, and in Eppendorf centrifuge, be rotated down 3 seconds.Sample is subsequently in 50 ℃ of incubations 20 minutes ± 2 minutes.Subsequently 35 μ L alkylating agents (200mM IAA) are added in every kind of sample, and mix once more, and in Eppendorf centrifuge, be rotated down 3 seconds.Sample covers with aluminium foil and subsequently in 50 ℃ of incubations 20 minutes ± 2 minutes.The NAP-5 post is by after pouring into 3 column volumes (about 7-8mL) digestion damping fluid and carrying out balance, the mixture of 500 μ L reduction and alkanisation is poured on the NAP-5 post, thereby permission liquid discharged by post.Subsequently via from the NAP-5 post, collecting sample with 1mL digestion damping fluid elution samples from post.
Digestion: sample is with 20 μ L trypsin, 0.5 μ g/ μ L) 4 hours (± 0.5 hour) of digestion in 38 ℃ of water-baths.After digestion was finished, sample carried out acidifying with 2.5 μ LTFA.Subsequently sample is placed in the automatic sampler bottle and be used for subsequent analysis.
Instrumental method: instrumental method is shown in hereinafter:
Time (minute) (mL/ minute) mobile phase A Mobile phase B flows
0 0.7 100 0
17 0.7 83 17
27 0.7 78 22
42 0.7 73 27
58 0.7 65 35
74 0.7 52 48
79 0.7 0 100
84 0.7 100 0
88 0.7 100 0
Before injection for the first time, post was with 100% mobile phase A damping fluid balance 25 minutes.The UV absorbancy is monitored at the 215nm place, makes column temperature be maintained at 37 ℃ and make the automatic sampler temperature maintenance in 4 ℃ simultaneously.Mobile phase A damping fluid blank is moved before first kind of system's suitability criterion, is thereafter the single 50 μ L injection of every kind of sample.The reference material injection should be carried out together in per 6 samples injection.
Theoretical plate number: the column efficiency as the theoretical plate number assessment can use retention time and peak width to carry out quantitative measurment according to equation:
N = 16 ( t W ) 2
Wherein:
" w " is by straight relatively side being extrapolated to the peak width at the baseline place of base measurement, and " t " is the retention time at the peak measured of the elution time from inject time to the peak maximum.
If N＜50000 make post balance again so.
Resolving power: 2 peaks for example as shown in Figure 32 peak T2 and the mensuration of the resolving power (R) between the peak T12 can use equation to measure:
R = 2 ( t 2 - t 1 ) ( w 1 + w 2 )
Wherein:
t 1, t 2=be respectively the retention time of fragment peak T2 and peak T12
w 1, w 2=have retention time t respectively 1And t 2The peak width that limits of the tangent line at peak base place.
If R＜1.5, post balance again so, and if problem exist, post should be replaced so.
Figure 32 and table 25 demonstration derive from CTLA4 A29YL104EThe peptide fragment of the tryptic digestion of-Ig.Incomplete treatments of the sample is reflected in the zone of show peptide T7 and T9 sometimes in the time of～50 minutes, and the peak can show the character that every day is all different; Yet all samples demonstrates comparability in operation.
Table 25:CTLA4 A29YL104EThe trypsinase peptide fragment of-Ig
Figure A200680053116D03391
Figure A200680053116D03401
Figure A200680053116D03411
aPeptide with N connection carbohydrate
bPeptide with O connection carbohydrate
cAbout the quality of T5, T7, T9 and T14 is the quality of carbohydrate containing part not.
dObserve many quality corresponding to glycosylated peptide
Isoelectrofocusing
Isoelectrofocusing (IEF) is used for evaluate drugs and medicament production CTLA4 A29YL104EThe iso-electric point of the various isoforms of-Ig (pI).This method is used the Pharmacia Biotech when the pH of 4.0-6.5 gradient PAGplates and Multiphore II FlatbedElectrophoresis System.Sample (for example, CTLA4 A29YL104E-Ig etc.) in Milli-Q water, dilute and use the sample application bar directly to be loaded on the gel.Gel focuses on 2.5 hours under increasing voltage, wherein use the cathode strip of 100mM Beta-alanine immersion and the anode strap of 100mM L-glutamic acid/500mM phosphoric acid dip.After the focusing, gel uses sulphosalicylic acid/trichoroacetic acid(TCA) to fix, and uses the Coomassie blue stain system to dye subsequently.After the dyeing, use photodensitometer wet gel to be scanned in the digital image file with 50 or 100 μ m spatial resolution and the optical density(OD) resolving power that is up to 4096 levels based on laser.CTLA4 A29YL104E-Ig focuses in 10-15 the interior band of pI4.5-5.5.
As shown in Figure 12, for method C batch 224818-2004-007, method B batch 000929-287 and concurrent mixture batch 55128-162, natural CTLA4 A29YL104EThe isoelectrofocusing of-Ig on gel (pH4.0-6.5) produces the similar band pattern in the pI scope 4.6-5.5.This programdisplay is when analyzing on identical IEF gel, and method B and C material are comparable.
The isoelectrofocusing standard should easily distinguish (referring to Figure 12) with background.
The protein standard PI
LcA 8.65
8.45
H-Mb 7.35
6.85
Conalbumin 5.90
Lactoglobulin 5.20
Trypsin inhibitor SBTI 4.55
Amyloglucosidase 3.50
CTLA4 A29YL104E-Ig is accredited as a plurality of bands (〉 10 of the pI scope with about 4.5-about 5.5) (Figure 32).
CTLA4 A29YL104E-Ig is modified ligand binding domains and the human IgG by cytotoxic T lymphocyte antigen 4 (CTLA4) 1The s-generation CTLA4-Ig fusion glycoprotein that the constant region of heavy chain is formed.This novel molecular has as the treatment of immunosuppressor to be used.CTLA4 A29YL104E-Ig comprises the multiple charge isoform that can separate by isoelectrofocusing (IEF).Be used to analyze CTLA4 A29YL104EThe IEF method of-Ig medicine and medicament production has obtained exploitation.This method is used for
Figure A200680053116D03421
Check CTLA4A29YL104E-Ig in the PAG plate pH4.0-6.5 Multiphore II flatbed electrophoresis system.CTLA4 A29YL104E-Ig medicine, medicament production and reference material are diluted in Milli-Q water and directly are loaded on the gel.Gel focuses on 2.5 hours under increasing voltage, wherein use the cathode strip of 100mM Beta-alanine immersion and the anode strap of 100mM L-glutamic acid/500mM phosphoric acid dip.After the focusing, Coomassie blue stain is fixed and used to gel.Stained gel scans by the laser light densitometry and the semi-quantitative analysis of gel band carries out on digital image file.
Material:
Ampholine PAG slab gel GE Healthcare (catalog number (Cat.No.) 80-1124-81)
pH4.0-6.5
IEF electrode strip 6 x 280mm GE Healthcare (catalog number (Cat.No.) 80-1004-40)
Sample application sheet GE Healthcare (catalog number (Cat.No.) 80-1129-46)
Equipment:
Multiphor II electrophoresis system GE Healthcare (catalog number (Cat.No.) 18-1018-06)
Cooling plate 125 x 260mm GE Healthcare (catalog number (Cat.No.) 80-1106-54)
Power supply NOVEX (model Basic3540)
BioRad (model PAC3000)
Constant temperature circulator VWR (model 13271-074/
1160S1160A)
Orbital shaker IKA (model KS250/260)
Individual photodensitometer SI GE Healthcare (model 375)
ImageQuantTL software GE Healthcare
Reagent preparation:
Anode buffer solution (100mL): the 0.1M L-glutamic acid that is dissolved in 0.5M phosphoric acid; 3.4mL85% phosphoric acid; 1.47 ± 0.02g L-glutamic acid; Milli-Q water.L-glutamic acid is added in the 50mL Milli-Q water.Add 85% phosphoric acid and Q.S. to 100mL, stir to mix.Specify 6 months validity period and preserve in 4 ℃.
Negative electrode buffered soln (100mL): 0.1M Beta-alanine, 0.9 ± 0.02g Beta-alanine, Milli-Q water., stir to 100mL with Milli-Q water Q.S. reagent to mix.Specify 6 months validity period and preserve in 4 ℃.
Fixed solution (2000mL): be dissolved in the 3.5%5-sulphosalicylic acid of 12% trichoroacetic acid(TCA), 240 ± 5.0g trichoroacetic acid(TCA), 70 ± 2.0g5-sulphosalicylic acid, Milli-Q water.Composite reagent is also used Milli-Q water Q.S. to 2000mL.Specify 3 months validity period and preserve in room temperature.
Device and preparing gel.The chill station of Multiphore II electrophoretic cell is connected with Multi-Temp constant temperature circulator and temperature is made as 10 ℃.Allow circulator to reach 10 ± 2 ℃.From refrigerator, take out gel.Use scissors, along all 4 side cuttings of big envelope, guarantee not cut in gel/gel upholder carefully.About 1.0mL Milli-Q water is added to 1 edge of chill station.1 edge of gel/gel upholder is placed in the water, thereby make water move to cross the whole edge of gel.Carefully gel is crossed chill station and use, avoid forming bubble.Surface removal transparent film from gel.Soak each electrode strip with the suitable solution electrode of about 3.0mL (following table).Use electrode strip from the top and the about 10mm of bottom margin of gel.Cathode strip is placed near (+) mark near placement of (-) mark on the chill station and anode strap.After electrode strip had been used, cutting rod to be being fit to gel, thereby avoided contacting with the gel upholder.
Solution electrode and electrophoresis parameter setting
The pH scope Anodic dissolution Cathode solution Voltage (V) Electric current (mA) Power (W) Time (h)
4.0-6.5 Be dissolved in 0.5M H 3PO 40.1M L-glutamic acid 0.1M Beta-alanine 2000 25 25 2.5
The sample application sheet is applied to the about 10mm in cathode strip top.The electrophoresis parameter that limits in the table directly over the use makes gel prefocus reach 300V until voltage.
IEF pI mark and dyeing contrast preparation.With 100 μ L Milli-Q water reconstruct IEF pI marks.With 1000 μ L Milli-Q water reconstruct carbonic anhydrase II dyeing contrast, with preparation 1.0mg/mL stock solution.10 μ L stock solutions (1.0mg/mL) are added the final loading concentration that is used for 0.10mg/mL in the 90 μ L Milli-Q water.
Specimen preparation.With CTLA4 A29YL104E-Ig reference material and diluted sample are to the concentration of 2mg/mL.Example: if CTLA4 A29YL104E-Ig sample has the concentration of 25mg/mL, uses the final loading concentration of following dilution with preparation 2mg/mL so:
10 μ L (25mg/mL)+115 μ L Milli-Q water=2mg/mL
Sample concentration≤2.0mg/mL annotates: if need not dilution and load sample so.
Gel loads.Load gel to promote sample survey based on operational mode.Do not load gel symmetrically.As under load IEF pI mark, dyeing contrast, CTLA4 described in the table A29YL104E-Ig reference material and CTLA4 A29YL104E-Ig sample.On the sample application sheet, load all samples.
The gel loading pattern
Swimming lane Describe Load concentration (μ g/ μ L) Load volume (μ L) Protein load (μ g)
1 IEF pI mark * - 10.0 -
2 IEF pI mark - 10.0 -
3 CTLA4 A29YL104E-Ig reference material 2.0 10.0 20
4 Sample 1 2.0 10.0 20
5 The dyeing contrast 0.10 10.0 1.0
6 Sample 2 2.0 10.0 20
7 CTLA4 A29YL104E-Ig reference material 2.0 10.0 20
8 IEF pI mark - 10.0 -
*It is that qualification gel direction is necessary that IEF pI mark in the swimming lane 1 loads.Beginning loading pattern and in swimming lane 2 for other sample repetition loading pattern.IEF pI mark must load per at least the 10th swimming lane (example: MRS 1S 2S 3S 4S 5S 6RM; The M-mark; The R-reference material, S X-sample).
Gel processing.Place the electrode fixer on the Multiphor II unit and make electrode strip center-aligned on electrode and the gel.Make 2 electrodes of self-electrode fixer to be connected and relief cover is positioned over the appropriate location with base unit.Hole in the use self adhesive tape covering relief cover is to prevent gel drying.Electrode is connected with power supply.Under suitable voltage, electric current and power, move electrophoresis.When electrophoresis is finished, turn off power supply and remove relief cover and electrode fixer.From gel, take out electrode strip and sample application sheet carefully.From cooling plate, take out whole gel and gel upholder, and place 280 x, the 180 x 40mm Pyrex that comprise the 200mL fixed solution TMIn the ware.Cover ware and placed on the orbital shaker in room temperature minimum 20 minutes with the plastic wraps thing.Annotate: gel should be fixed maximum 1 hour.When fixedly finishing, with about 200mL Milli-Q water with gel detergent 3 times, respectively 5 minutes.By bottle is put upside down GelCode Blue staining reagent solution is mixed.Dyed blended reagent is important before distribution, to guarantee to use the even sample of reagent.About 200mL staining reagent is added in the ware.Cover ware and place on the orbital shaker with the plastic wraps thing in room temperature 18-20 hour, to reach best band colour developing.When dyeing is finished, come detergent gel by replacing staining reagent with about 200mL Milli-Q water.For optimum, in 1-2 hour time period, carry out minimum 3 water and change.
Gel scanning and analysis.The sweep parameter that limits in the table directly over the use scans gel.The analysis of gel is carried out on the image file of scanning.
Gel scanning and analytical parameters
Sweep parameter Be provided with
The scanning element size 100
Scanning digital resolving power 12 bits
The band detect parameters
Minimum slope Initial 100
Noise reduces Initial 10
The % maximum peak Initial 0
Swimming lane % width Be made as 90%
Annotate: table 3 has been summarized and has been used for the general guide that gel images is analyzed.About the details of the suitable adjustment of every kind of band detect parameters, with reference to (on-screen) instruction on ImageQuant TL (v2003.03) handbook and the screen.
Open among the ImageQuantTL from＜1D Gel Analysis〉gel images file (raw data of scanning).Forward on the toolbar＜Contrast, and reduction＜Image Histogram parameter is high-visible until all bands.Selection＜Lane Creation〉and selected＜Manual, to be analyzed to set＜the swimming lane number 〉.The wide motherwort of adjustment＜swimming lane %〉cover the gel swimming lane until the highest 100%.Single swimming lane in case of necessity correctly aligns.Use＜Rolling Ball〉method is with background correction.Analysis is not crucial for the IEF gel images for this.Initial＜minimum slope of listing in the use table 3 〉,＜noise reduces〉and＜the % maximum peak〉test strip is set.The adjustment of these values is to identify that accurately band is necessary.Manual band and the wrong band of identifying of proofreading and correct any omission.For the pH/pI4.0-6.5 gel, calculate band pI value by using from the standard pI mark of listing in system's suitability part through the mark of mark.Do not carry out calibration and standardised step.The data that comprise in the measurement window are outputed in the Excel table, be used for further calculating and report.The Excel data are input in the spreadsheet of checking, with the quantitative analysis that is used to report the result.
System's suitability.Isoelectrofocusing standard (pI mark) must easily distinguish with background, and the range estimation of the gel images by scanning show limited distortion (about the pI mark listed referring under table).
The isoelectrofocusing standard
Protein The pI value
Trypsinogen 9.30
LcA, alkalescence 8.65
LcA, the centre 8.45
LcA, acidity 8.15
Myohaemoglobin, alkalescence 7.35
Myohaemoglobin, acidity 6.85
CAB (people) 6.55
CAB (ox) 5.85
B-lactoglobulin A 5.20
Trypsin inhibitor SBTI 4.55
Methyl red (dyestuff) 3.75
Amyloglucosidase 3.50
Annotate: be not that all isoelectrofocusing standards all will manifest on gel, because the pH/pI scope of gel is 4.0-6.5.PI mark at 3.50,4.55,5.20 and 5.85 places will obtain identifying and mark on gel.
CTLA4 A29YL104EThe band pattern of-Ig reference material and test article should show limited distortion by the range estimation of the gel images of scanning.The dyeing contrast of the carbonic anhydrase II (pI5.4) of low-level protein load (1.0 μ g) is used to confirm the gel-colored of unanimity.Band must easily distinguish with background by the range estimation of the gel images of scanning.CTLA4 A29YL104E-Ig reference material must be included in 8-15 the band that has band intensity 〉=1.0% in the pI scope of 4.5-5.6.CTLA4 in the pI of 4.5-5.6 scope A29YL104E-Ig reference material band must have 〉=95% cumulative percentage specific tenacity.
Data computation.Equation is used to calculate CTLA4 A29YL104E-Ig sample is with respect to the cumulative percentage specific tenacity of reference material:
Figure A200680053116D03471
Example: have 100% % band intensity (pI4.5-5.6) if sample has 95% % band intensity (pI4.5-5.6) and reference material, accumulating % intensity so will be 95%.
In one embodiment, CTLA4 A29YL104E-Ig material will have the band of relative band intensity 〉=1.0% in the pI of 4.5-5.6 scope.CTLA4 A29YL104E-Ig material has with respect to CTLA4 in the pI of 4.5-5.6 scope A29YL104EThe sort of cumulative percentage specific tenacity of-Ig reference material.
Embodiment 23: the transfection of clone and generation
Before the electroporation, expression vector pD16LEA29Y carries out linearizing with the BstBI enzyme, to produce compatible 4bp overhang.Smart carrier DNA of the Pacific herring of linearizing carrier and shearing (as carrier) and ethanol co-precipitation, and be used for electroporation in the DG44 cell in the aseptic PF of the being resuspended to CHO substratum (JRH Biosciences).
Behind the electroporation, allow cell in non-selective substratum, to reclaim.Cell is inoculated in 96 orifice plates in the PFCHO selective medium subsequently, and described PF CHO selective medium comprises 500ng/mL Recombulin (Gibco), 4mM L-glutaminate (Gibco) and methotrexate (ICN).
Use the following progress of methotrexate (MTX) concentration that adds in the substratum to select from this CTLA4 that paves plate A29YL104E-Ig production clone is used for expressing amplification:
20 nM ⇒ 50 nM ⇒ 100 nM ⇒ 250 nM ⇒ 500 nM ⇒ 1 μM MTX .
With complete CTLA4 A29YL104E-Ig expression plasmid is incorporated in the cell genome.
Production clone is selected
2 of the main aperture clone of optimal representation, amplification is separated final production clone GF1.1.9 after taking turns limited dilution cloning.The selection of clone GF1.1.9 comprises the high molecular weight component of reduction and the product of high sialic acid content more based on growth pattern, titre and with respect to materials of being produced by other clones.
Embodiment 24:CTLA4 A29YL104E The heredity of-Ig characterizes
The genome stability study
Separated DNA and RNA are used for DNA and RNA hybridization analysis from the cell in derived cell storehouse, and are used for CTLA4 A29YL104EThe cDNA order-checking of-Ig encoding sequence.The result with derive from CTLA4 A29YL104EThe result of-Ig compares.
Be presented in hereinafter about RNA hybridization analysis and cDNA order-checking results estimated.
The RNA hybridization analysis
Use culture from the cell inoculation of cell bank to increase and be used for isolation of RNA and be used for the RNA hybridization analysis.The culture representative of preparation surpasses CTLA4 A29YL104EThe cell in about 27 generations at cell in vitro age of using in-Ig the production method.From derived from CTLA4 A29YL104EThe cell of-Ig cell bank and the CTLA4 of amplification A29YL104EExtract total RNA in the cell of-Ig cell.Be used to also use in these experiments of contrast from total RNA of parental generation Chinese hamster ovary celI system.Under the sex change condition, the total RNA of about 5 μ g is implemented agarose gel electrophoresis.With the RNA trace in the gel to nylon membrane, and with comprise CTLA4 A29YL104E-Ig gene 32The 1.2kbHindIII/XbaI dna fragmentation hybridization of P mark.The 1.2kb HindIII/XbaI dna fragmentation that is used for probe separates from plasmid pD16LEA29Y.
As shown in Figure 33, in total RNA sample, detect and CTLA4 from cell bank A29YL104EThe mRNA kind of about 1.7 kilobase of-Ig gene probe hybridization.Little figure A that shows among Figure 33 and little figure B represent the sepharose and the corresponding autoradiogram(ARGM) of ethidium bromide staining respectively.
These results point out derived from CTLA4 A29YL104EExpress coding CTLA4 in the culture in-Ig expanded cells storehouse A29YL104EUnique a kind of transcript of-Ig.In addition, compare, in these samples, do not observe CTLA4 with the result who uses cell bank to obtain A29YL104EDetectable variation in the-Ig mRNA transcript.
Embodiment 25: size exclusion chromatography
The size exclusion method has been developed and has been used to analyze CTLA4 A29YL104E-Ig composition wherein uses the 7.8mm x 300mm TosoHaas TSK-3000 SWXL post that is equipped with guard column, detects at the 280nm place.CTLA4 A29YL104E-Ig assesses with regard to the product homogeneity, comprises monomer (strand), dimer or high molecular weight species (for example, the tetramer).This method is presented at～good precision (＜2%) during the nominal concentration of 10mg/mL, and from～0.5-15mg/mL is linear (r 2=0.999).DL (limit of detection) is that～2.26 Φ g/mL and QL (quantitation limit) are～7.53 Φ g/mL.The fusion rotein of using as the potential treatment of immunosuppressor is formed, had to these soluble CTL A 4s-Ig molecule by the ligand binding domains of cytotoxic T lymphocyte antigen 4 (CTLA4) and the constant region of human IgG1's heavy chain.These compounds are brought into play its physiological action by combining with the lip-deep B7 antigen of various antigen presenting cells (APC) (CD80 and CD86), thereby the function of the CD28 on blocking-up B7.1 and B7.2 and the T cell surface interacts.This blocking-up causes the inhibition of T cell activation, thereby the inhibition of immunne response.Although LEA29Y is only at 2 amino-acid residue Leu 104-glu and Ala 29Be different from CTLA4Ig on the-Try, but this molecule has at B7.1 and the remarkable different avidity of B7.2 antigen.With parent CTLA4Ig relatively, LEA29Y shows the people's form 5-10 big avidity doubly for B7.2 (CD86), with for the similar avidity of people B7.1 (CD80).
Use TSK-3000SWXL post (7.8mm x 300mm) that is equipped with guard column and the size exclusion chromatography that detects at the 280nm place to be used for regard to homogeneous assay CTLA4 A29YL104E-Ig medicine.Distinguish CTLA4 A29YL104E-Ig dimer, high molecular (HMW) and lower molecular weight (LMW) kind.
Size exclusion chromatography (SEC) is used for regard to product homogeneity assessment CTLA4 A29YL104E-Ig.Figure 34 A-C shows the CTLA4 about method B, method C and concurrent mixture batch A29YL104EThe SEC chromatogram of-Ig.CTLA4 A29YL104EThe SEC of-Ig points out that method C material is 99.8 area percentage dimers, 0.2 area percentage HMW kind and does not have detectable LMW kind.These results and method B material (dimer 97.4 area percentages, HMW2.6 area percentage and LMW＜DL) can compare.
Reagent: 4N KOH (100mL); System's suitability criterion (being dissolved in the molecular weight marker in the HPLC grade water); Moving phase running buffer (0.2M KH 2PO 4, 0.9%NaCl, pH6.8); 4N NaOH; Dilution buffer liquid (25mM NaH 2PO 4-H 2O, 10mM NaCl, pH7.5)
Instrument configuration and condition-instrument configuration of equal value can replace:
Pump type Waters model 600
Post Toso Haas 5:m TSK 3000 SWXL; 300mm x 7.8m I.D.Hewlett Packard; (catalog number (Cat.No.) 79912S3-597); 5:m TSK 3000 SWXL are equipped with; 40mm x 6.0mm I.D. guard column; Hewlett Packard, (catalog number (Cat.No.) 79912S3-527)
Detector Waters model 486.Allow heating in 15 minutes
Wavelength 280nm
Flow velocity 1mL/ minute
Integrating system VG Multichrom
Injecting systems Waters model 717 adds automatic sampler, is equipped with
Be refrigerated to 4 ℃
Volume injected 20mL
Measure target level 10mg/mL
Moving phase 0.2M KH 2PO 4, 0.9%NaCl, pH6.8 has KOH
Measure 20 minutes working times
The column temperature environment
Retention time CTLA4 A29YL104E-Ig～8.5 minute ± 0.5 minute, high molecular weight species is in the time of～7.5 minutes ± 0.5 minute
Standard and sample (10mg/ml) are prepared as the 50ml volume in the automatic sampler bottle of mark.Sample is prepared in duplicate.
Calculate
Resolving power (R) is measured and the retention time assessment: injection 20mL system suitability criterion, (for example, 1 peak has～peak 1 of 8.5 minutes retention time to use equation to calculate 2 peaks from the chromatogram that uses this kind standard to produce, with second peak, have～peak 2 of 10 minutes retention time) between resolving power:
Figure A200680053116D03511
Wherein:
t 1The retention time at=peak 1
t 2The retention time at=peak 2
W 1The peak width at=peak 1
W 2The peak width at=peak 2
Figure A200680053116D03521
= 2.12 ∃ 1.3
Peak width equals the width (with a minute expression) that straight relatively side, peak is extrapolated to place, baseline postpeak bottom.Retention time and peak width are measured with same units.
R is necessary 1.3 and should be about the retention time at peak
Figure A200680053116D0352173138QIETU
Minute.
Theoretical plate number is measured: according to system's suitability criterion chromatogram, according to equation by theory of computation stage number can measuring column efficient:
N = 16 ( t w ) 2
Wherein:
(t)=retention time at peak 2 (with a minute expression)
(w)=as shown in Figure 1, the width (with a minute expression) at 2 baseline place that is extrapolated to by the side, peak that baseline obtains at the peak.
N should
Figure A200680053116D0352173229QIETU
2500.
The integration at peak: the peak area in the integration chromatogram (for example, Figure 34 A-C).CTLA4 A29YL104E-Ig dimer peak is when approximately～8.5 minutes time the and high molecular weight species peak was at～7.4 minutes.
Area percentage can calculate according to following formula:
Area % dimer=100-(area % high molecular weight species+area % low molecular weight species)
Figure A200680053116D03524
Wherein:
A=CTLA4 A29YL104E-Ig dimer peak area
B=has less than CTLA4 A29YL104EThe total area at all peaks of the dimeric retention time of-Ig
C=has greater than CTLA4 A29YL104EThe total area (eliminating includes volume) at all peaks of the retention time at-Ig dimer peak.
Measure the %RSD of total area counting (eliminating includes volume).The %RSD of total area counting is necessary for 2% or littler.If area＜2707 areas counting, the result is reported as #DL so, (limit of detection) (～2.26 μ g/mL).If the area counting is 2707-9014, the result is reported as #QL so, (quantitation limit) (～7.53 μ g/mL).If the area counting is
Figure A200680053116D0352173229QIETU
9014, the result reports to nearest 1/10th of per-cent so.
Embodiment 26:SDS-PAGE and disulfide linkage
SDS-PAGE
Be used to analyze CTLA4 A29YL104EThe SDS-PAGE of-Ig (SDS-PAGE) program is as purity test.In the having (reduction) or do not exist under (non-reduced) of dithiothreitol (DTT) (DTT), sample is prepared in Tris-HCl (pH6.8), SDS, sucrose and tetrabromophenol sulfonphthalein sample buffer.Sample was placed 80 ℃ of water-baths 2 minutes and carry out electrophoresis, wherein use Tris-glycine SDS running buffer at ready-formed, gradient (4-20%) polyacrylamide sds gel.Behind the electrophoresis, gel is fixed and is used the dyeing of Coomassie blue or silver-colored coloring system.Non-reducing CTLA4 A29YL104E-Ig as have～1 big band of the apparent approximate molecular weight of 104kD observes.CTLA4 A29YL104E-Ig as have～1 main band of the apparent approximate molecular weight of 53kD observes.
The sample of method B batch of 000929-278, method C batch 224818-2004-007 and concurrent mixture batch 551218-162 uses 4-20% gradient SDS-PAGE gel to carry out electrophoretic separation under reduction and non-reduced condition.As shown in Figure 35 and Figure 36, gel separates dyeing with coomassie or silver dyeing.Non-reducing CTLA4 A29YL104EThe SDS-PAGE of-Ig is presented at the main band at about 104kDa place, represents complete monomer.3 minor band that on electronic reproduction, are not easy to see also～200,65 and the 53kDa place observe.CTLA4 A29YL104E-Ig goes back the main band that raw sample is presented at about 53kDa place, expression single stranded form and in the minor band at～150kDa place.This relatively demonstration is when analyzing on identical gel, and the material of 3 kinds of tests is comparable.
Disulfide linkage
Disulfide linkage uses a batch 224818-2004-007 to characterize about method C medicine.CTLA4 A29YL104EEvery chain of-Ig comprises 9 halfcystines.These are Cys21, Cys48, Cys66, Cys92, Cys120, Cys171, Cys231, Cys277 and Cys335.Have reduction and non-reducing CTLA4 A29YL104EThe peptide mapping of online (on-line) LC/MS/MS of-Ig is used to identify CTLA4 A29YL104EThe intramolecularly among the-Ig and the site of intermolecular disulfide bond.Derive from non-reducing CTLA4 A29YL104EThe peptide tabulation of the peptide mapping of-Ig is shown in the table 27 together with expection and observed MW.
Among the non-reducing peptide figure among the disappearance at some peak and the reductive peptide figure appearance at new peak the evidence of the peptide that 3 kinds of disulfide linkage are connected is provided: T2-T6, T11-T17 and T25-T30, it is corresponding to disulfide linkage Cys21-Cys92, Cys171-Cys231 and Cys277-Cys335.Peptide T5 has relative high molecular weight with T7 and comprises N connection carbohydrate, and this makes and is difficult to locate disulfide linkage.In order to produce shorter and peptide carbohydrate, CTLA4 A29YL104E-Ig digests with the mixture of trypsinase and Quimotrase.As the result of other Quimotrase cutting, T7 foreshortens to the fifteen amino acid peptide from 35 amino acid peptides, called after T7 '-T7 ', and wherein N connection carbohydrate is removed.(referring to Figure 37) appears in peptide T7 '-T7 ' that disulfide linkage connects in non-reducing figure.Confirm its sequence and at the interchain disulfide bond at Cys120-Cys120 place about the MS/MS of T7 '-T7 '.
CTLA4 under the comfortable non-reduced condition of table 27. A29YL104EThe peptide sequence and the MW of the peptide that the disulfide linkage of the tryptic digestion of-Ig connects
Figure A200680053116D03541
aBecause the heterogeneity of N linked glycosylation, peptide T5 and T7-T7 produce several quality, thereby make and be difficult to locate disulfide linkage.
Mixture process with trypsinase and Quimotrase also causes forming fragment, the peptide that described fragment connects corresponding to other disulfide linkage of short-form more, and this observes by independent trypsin hydrolyzing CTLA4A29YL104E-Ig.These are shown in the table 28.
Table 28. digests CTLA4 with (trypsinase and Quimotrase) A29YL104EThe peptide sequence and the MW of the peptide that the disulfide linkage of-Ig connects
Mixture digestion CTLA4 with trypsinase and Quimotrase A29YL104E-Ig has established the disulphide pairing among the T7-T7, and confirms by independent tryptic digestion visible disulfide linkage.Yet, this kind of enzyme mixture to the peptide T5 that is similarly glycopeptide without any effect.In order from T5, to remove N connection carbohydrate, as shown in Figure 38, CTLA4 A29YL104E-Ig digests with the mixture of trypsinase and elastoser.As shown in Table 29, this kind of enzyme mixture makes T5 hydrolysis on 4 different loci, produces the shorter peptide of called after (T5 '-T5 ").The peptide of this generation has the prospective quality of 1259Da and comprises disulfide linkage corresponding to Cys46-Cys66.Derive from the non-reduced CTLA4 of mixture hydrolysis by trypsinase and elastoser A29YL104EThe peptide figure overview of-Ig is shown among Figure 38, and peptide T5 '-T5 " sequence in table 29.
Table 29: with the mixture digestion CTLA4 of trypsinase and elastoser A29YL104EPeptide T5 '-T5 that-Ig obtains " peptide sequence and MW
Figure A200680053116D03552
The result points out CTLA4 A29YL104E-Ig has 4 intramolecular disulfide bonds locating at position Cys21-Cys92 (T2-T6), Cys48-Cys66 (corresponding to a kind of single peptide T5), Cys171-Cys231 (T11-T17) and Cys277-Cys335 (T25-T30) and 1 interchain disulfide bond locating at position Cys120-Cys120 (T7-T7).Data interpretation all 18 cysteine residues.Do not observe wrong pairing.
Embodiment 27:CTLA4 A29YL104E -Ig preparation
Be used to inject, the CTLA4 of 100mg/ bottle A29YL104E-Ig is aseptic no pyrogeneous substance lyophile.The composition of medicament production provides in table 30.It is clog with the gray butyl stopper and I type vial with alumiseal thing sealing in the white that provides to canescence, integral body or the cake that ruptures.This product comprises that 10% overfill is to solve bottle, pin and syringe hold-up.
Before using, be used to inject, the CTLA4 of 100mg/ bottle A29YL104E-Ig 4.2mL sterile water for injection, USP makes up, to produce the concentration of 25mg/mL.It can use 5% glucose injection, USP or 0.9% sodium chloride injection, and USP further is diluted to the concentration that is low to moderate 1mg/mL.Make up and the solution of dilution is clarification, colourless and be substantially free of particulate matter when range estimation.
Table 30. is used to inject, the CTLA4 of 100mg/ bottle A29YL104EThe composition of-Ig
Figure A200680053116D03561
aEach bottle comprises bottle, pin and the syringe hold-up that 10% overfill is used for the solution of reconstruct.
bDuring freeze-drying, remove
The second-order transition temperature for the treatment of cryodesiccated frozen soln is determined as-28.9 ℃.Lyophilize research is carried out under various storage temperatures, with the highest permissible possible storage temperature during the mensuration preliminarily dried, and does not damage product quality.Based on these research, select-20 ℃ storage temperature to be used for CTLA4 A29YL104EPreliminarily dried step during the-Ig lyophilize.When lyophilization cycle finished, bottle under reduced pressure clogged.
Production method relates to the freezing bottle that comprises the bulk solution for the treatment of freeze-drying (having suitable vehicle) in the lyophilizer chamber, and refrigerated water subsequently distils under controlled temperature and pressure.Temperature and pressure condition in the chamber is carried out optimization, does not damage product quality to have effective distillation.
The consistency of research solution and various product surface in contact and packing composition.Find solution and stainless steel 316L, siloxanes tube of material, Acrodisc TM, HT Tuffryn (polysulfones), MilliporePVDF (poly-1,2-vinylidene difluoride fluorochemical) filter membrane is compatible with selected container closure system.
Be used to inject, the CTLA4 of 100mg/ bottle A29YL104E-Ig is packaged in the 15cc I type flint tubing glass bottle, and clogs with the stopper of 20mm Daikyo gray butyl D-21-7-S/B2-TR fluoro-resin bag quilt, and seals with 20mm aluminium turn-up and to seal.
The CTLA4 that is used to inject A29YL104EThe bottle of-Ig is selected based on the packing volume of 5.5mL guaranteeing effective lyophilize, and the stopper of 20mm Daikyo gray butyl D-21-7-S/B2-TR fluoro-resin bag quilt is selected based on compatibility data.
Carried out widely Study on Compatibility duration of service.When being configured to 25mg/mL with sterile water for injection, the CTLA4 that is used to inject A29YL104E-Ig 100mg/mL can preserve in following 24 hours of the ambient room temperature of 15-25 ℃ (59 °-77 ℉) and room light.When with 0.9% sodium chloride injection (physiological saline/NS) or with 5% glucose injection (D5W) further be diluted to 1mg/mL or 10mg/mL, and when preserving in PVC or Intra Via non-PVC bag under the ambient room temperature of 15-25 ℃ (59 °-77 ℉) and the room light, the solution of structure showed through time period of 24 hours not to be increased in loss or the high molecular weight species in rendeing a service.The solution of dilution must filter by 0.2 μ m or 1.2 μ m blended Mierocrystalline cellulose/acetate filters before using.The solution of dilution and 0.2 μ m or 1.2 μ m blended Mierocrystalline cellulose/the acetate filter is compatible.
Product and siloxanes are incompatible.It and siloxanes interact to form visible particle.Therefore, should avoid with the surface of siloxane treated for example the syringe of siliconization contact.
Embodiment 28: The CTLA4-Ig production method
CTLA4-Ig uses Chinese hamster ovary (CHO) clone to produce in mass cell is cultivated as excretory albumen.The CTLA4-Ig production method uses a series of bottles and seed bio-reactor inoculum amplification step to come initial.The content of last seed bio-reactor is used to inoculate 5000-L and produces bio-reactor.Producing the cell culture of gathering in the crops the bio-reactor from 5000-L purifies and concentrates by microfiltration and ultrafiltration.Acellular results material is used for downstream processing with regard to pH and electroconductibility adjustment in preparation.CTLA4-Ig uses a series of chromatographies and filtration step to carry out purifying.Downstream CTLA4-Ig production method comprises 2 anion-exchange chromatography steps, 1 hydrophobic interaction chromatography step and 1 affinity chromatography step.The purpose of these steps is a purifying CTLA4-Ig protein, removes the sialic acid content of high molecular CTLA4-Ig material and control CTLA4-Ig medicine.The downstream procedure of processing also comprises inactivation of virus step and virus filtration step, to remove the external viral agent of potential.The CTLA4-Ig medicine of purifying is filled in the 2-L polycarbonate bottles, and carries out freezing preserving before-40 ℃ target temperature target temperature in-70 ℃.The refrigerated medicine is thawed.
N-n acetylneuraminic acid n (NANA) is the main sialic acid kind that is present in the CTLA4-Ig medicine.This part mentions that from start to finish sialic acid specifically refers to this kind.The N-hydroxyacetylneuraminic acid (NGNA) of less level also is present in the CTLA4-Ig medicine.Measure the level of NANA and NGNA for final CTLA4-Ig medicine.Process flow sheet about the CTLA4-Ig production method is shown in hereinafter.
Figure A200680053116D03591
CTLA4-Ig produces in the bio-reactor at the 5000-L with about 4300L working volume and produces.The 1 batch of CTLA4-Ig medicine is prepared by the single production bio-reactor derived from the single bottle of cell bank.Upstream cell culture production method is used from the cell of the single bottle of cell bank initial.Bottle thaws and entire content is used to inoculate the T bottle that comprises the cell cultures growth medium.Cell increases in a series of revolving bottles subsequently.Bottle from last revolving bottle inoculum amplification step is used to inoculate 140-L seed bio-reactor.140-L seed bio-reactor has the working volume of about 100L.The content of 140-L seed bio-reactor is used to inoculate 1100-L seed bio-reactor.1100-L seed bio-reactor has the working volume of about 600L.The content of 1100-L seed bio-reactor is used to inoculate 5000-L and produces bio-reactor.Cell is produced in the bio-reactor at 5000-L and was cultivated about 14 days.After producing the bio-reactor step, will be used for further processing to gathering in the crops container from the cell cultures liquid media transfer of single bio-reactor.Tangential flow microfiltration (MF) unit is used to make excretory CTLA4-Ig protein to separate with cell debris with host cell.Comprise the proteinic MF penetrant of CTLA4-Ig and concentrate by ultrafiltration (UF) subsequently, and in preparation, be used for first chromatographic step with regard to pH and electroconductibility adjustment.
About the purifying of CTLA4-Ig medicine and downstream procedure of processing by anion-exchange chromatography, hydrophobic interaction chromatography (HIC), inactivation of virus, affinity chromatography, tangential flow UF concentrates and diafiltration, virus filtration, anion-exchange chromatography and UF concentrates and diafiltration is formed for the second time.The amount that depends on CTLA4-Ig to be processed, the HIC step utilizes Multiple Cycle/CTLA4-Ig to criticize.To merge the inactivation of virus step that is used for subsequently from material in the carrying out of multiple HIC step cycle.Nonjoinder is carried out middle material from what difference was criticized.A plurality of viral filters can parallel use be criticized CTLA4-Ig with work sheet.Behind the virus filtration, filtrate merging is used for further processing.
The every crowd of CTLA4-Ig be filled in 2-L polycarbonate (PC) bottle by 0.2 μ m filter and interim storage in 2-8 ℃.The 2-L PC bottle of CTLA4-Ig carries out freezing and preserves target temperature in-40 ℃ subsequently in-70 ℃ target temperature.The bottle of medicine thaws and is cooled to 2-8 ℃ in 22-24 ℃ before shipment in incubator.
Produce
CTLA4-Ig uses Chinese hamster ovary (CHO) clone to produce in mass cell is cultivated.CTLA4-Ig production upstream method is by initial from thawing of the cryovial of cell bank.Culture is bred in a series of revolving bottles are cultivated in the T bottle subsequently.These cultures are transferred to 140-L seed bio-reactor.To be transferred to 1100-L seed bio-reactor from the culture of 140-L seed bio-reactor.Culture from 1100-L seed bio-reactor is used to inoculate 5000-L production bio-reactor.Producing bio-reactor mainly gathers in the crops based on target sialic acid and CTLA4-Ig protein mol ratio.The cell culture results use the combination of microfiltration (MF) and ultrafiltration (UF) to purify and concentrate.At last, spissated acellular results material is adjusted, and specified electroconductibility and pH are used for downstream processing in the preparation to reach.
Cell cultures and charging medium preparation
Solid and liquid nutrient medium component are carried out weighing and measurement.In method, use 2 kinds of cell culture mediums.Substratum 127-G is used for T bottle, revolving bottle, seed bio-reactor and produces the bio-reactor step.Substratum 117-E is used as the charging substratum in producing the bio-reactor step.In showing under the composition of substratum 127-G is shown in.
Figure A200680053116D03611
The composition of substratum 117-E is shown in hereinafter.
Figure A200680053116D03612
E-RDF-1 substratum composed as follows:
Figure A200680053116D03621
Figure A200680053116D03631
The 127-G cell culture medium that is used for T bottle and revolving bottle in method is used to mix the medium container that is used for stereometry with the scale visor and is prepared being equipped with agitator.The batch size of the substratum 127-G that uses in T bottle and revolving bottle inoculum amplification step is 75L.Substratum 127-G uses water for injection (WFI) to be prepared.Solid and liquid nutrient medium component are added among the WFI.After adding every kind of component, make substratum mixing required time section.Add WFI so that substratum reaches the finally volume in batches of 75L.From final substratum preparation, take out sample, and the glucose concn of measure sample, pH and Osmolality, the standard of accepting of qualification met to guarantee substratum.Substratum filters and is assigned in aseptic dibasic alcohol modification polyethylene terephthalate (PETG) bottle by 0.2 μ m filter.The substratum 127-G that preparation is used for T bottle and revolving bottle inoculum amplification step preserves in 2-8 ℃ maximum 42 days.The substratum 127-G that is used for 140-L and 1100-L seed bio-reactor step is used for the blended container at the outfit agitator and is prepared.The batch size that is used for the substratum 127-G of 140-L seed bio-reactor step is 120L.The container that is used to prepare the substratum that is used for 140-L seed bio-reactor is equipped with the scale visor and is used for stereometry.The batch size that is used for the substratum 127-G of 1100-L seed bio-reactor step is 600kg.The container that is used to prepare the substratum that is used for 1100-L seed bio-reactor is equipped with and divides pressure transmitter to be used for weight determination.
By successive 0.2 ∝ m and 0.1 μ m filter volume required substratum 127-G is transferred to 140-L and 1100-L seed bio-reactor.The substratum 127-G that preparation is used for 140-L and 1100-L seed bio-reactor step can keep maximum 48 hours at 37 ℃.Substratum can keep other 84 hours at 4 ℃.
Be used for 5000-L and produce the 127-G of bio-reactor step and 117-E cell culture medium Preparation.
The substratum 127-G that is used for 5000-L production bio-reactor step is prepared at the medium preparation container that is equipped with agitator and divide pressure transmitter to be used for weight determination.The batch size that is used for the substratum 127-G of 5000-L production bio-reactor step is 2900kg.By successive 0.2 μ m and 0.1 μ m filter volume required substratum 127-G is transferred to 5000-L and produces bio-reactor.The substratum 127-G that preparation is used for 5000-L production bio-reactor step can keep maximum 48 hours at 37 ℃.Substratum can keep other 84 hours at 4 ℃.
Charging substratum 117-E is used for mixing and divide the medium preparation container that pressure transmitter is used for weight determination and is prepared being equipped with agitator.The batch size that is used for the substratum 117-E of 5000-L production bio-reactor step is 1800kg.Substratum 117-E component is added among the WFI of specified wt in the medium preparation container.After adding every kind of component, make substratum mixing required time section.Add WFI so that substratum reaches the appointment final weight.From final substratum preparation, take out sample, and the glucose concn of measure sample, pH and Osmolality, the standard of accepting of qualification met to guarantee substratum.By successive 0.2 μ m and 0.1 ∝ m filter volume required substratum 117-E is transferred to charging substratum receiving tank.The substratum 117-E that preparation is used for 5000-L production bio-reactor step can keep maximum 2 days at 37 ℃.Substratum can keep other 4 days at 4 ℃.
Inoculum amplification step in T bottle and the revolving bottle inoculum amplification step
The purpose of the T bottle of CTLA4-Ig production method and revolving bottle inoculum amplification step is from the continuous propagated cell of cell bank bottle, with viable cell that enough numbers are provided with inoculation 140-L seed bio-reactor.From the vapour phase of liquid nitrogen storage refrigerator, take out and in water-bath, thaw from the single bottle of cell bank in 37 ℃.Transfer in the conical centrifuge tube of aseptic 15-mL the entire content of bottle is aseptic.Add substratum 127-G so that final volume reaches 10mL.Cell suspending liquid is centrifugal, abandoning supernatant and cell precipitation is resuspended in the 10mL127-G cell culture medium.With the cell transfer of resuspension to the T-175 bottle that comprises 10mL substratum 127-G.Measure the viable cell density and the per-cent viability of the culture in the T-175 bottle.Determine per-cent viability at this step time 〉=84%.In the T-175 bottle, add substratum 127-G to reach 2.1 x 10 5The target viable cell density of cell/mL.
Make the T-175 bottle in 37 ℃ of maximum 4 days of incubations in the atmosphere of 6% carbonic acid gas, to reach 1.80 x 10 7The final target cell number of viable cell.After the T-175 bottle step, culture uses a series of 0.25-L, 1-L and 3-L revolving bottle step to increase.When going down to posterity, cell is with 2.0 x 10 at every turn 5The target density inoculation of viable cell/mL.The revolving bottle culture carries out incubation in 37 ℃ in the atmosphere of 6% carbonic acid gas.
To be incorporated in the aseptic 20-L inoculum transfer vessel from the cell culture material of final 3-L revolving bottle inoculum amplification step.Determine 1.0-2.0 x 10 when 3-L revolving bottle inoculum amplification step 6The final viable cell density of cell/mL and 〉=80% lowest percentage cell survival.These example values guarantee that the viable cell of enough numbers is used to inoculate 140-L seed bio-reactor.The cell culture that merges from the cumulative volume 12L-18L of final 3-L revolving bottle inoculum amplification step is used to inoculate 140-L seed bio-reactor.
140-L and 1100-L seed bio-reactor inoculum amplification step
The purpose of the 140-L of CTLA4-Ig production method and 1100-L seed bio-reactor inoculum amplification step provides the viable cell of enough numbers and produces bio-reactor with inoculation 5000-L.The seed bio-reactor uses cell culture medium 127-G to operate with batch mode.Temperature, pH, dissolved oxygen, pressure, stirring and be subjected to the control of distribution control system (DCS) about the airflow rate of air, oxygen and carbon dioxide, and the condition about culture optimum growh in the seed bio-reactor is provided.The seed bio-reactor is in 37 ℃ of operations.From the seed bio-reactor, take out culture samples, be used for determining viable cell density, per-cent viability and metabolite concentration.
140-L seed bio-reactor uses the merging inoculum from 3-L revolving bottle inoculum amplification step to be seeded to 2.0 x 10 5The initial viable cell density of the target of cell/mL.Determine 1.0-2.5 x 10 when 1100-L seed bio-reactor inoculum amplification step 6The final viable cell density of cell/mL and 〉=80% minimum cell per-cent viability.These are accepted standard and guarantee that the viable cell of enough numbers is used to inoculate 5000-L and produces bio-reactor.To be transferred to 5000-L from the cell culture of 1100-L seed bio-reactor and produce bio-reactor, to reach 1.5 x 10 5The initial viable cell density of the target of cell/mL.
Produce the bio-reactor step
The purpose that 5000-L produces the bio-reactor step is the number of amplification viable cell and produces CTLA4-Ig protein.The time length of producing the bio-reactor step is about 14 days.To be inoculated into the 5000-L that comprises cell culture medium 127-G from the inoculum of 1100-L seed bio-reactor produces in the bio-reactor.Producing bio-reactor operates with batch mode.Temperature, pH, dissolved oxygen, pressure, stirring and be subjected to the control of DCS about the airflow rate of air, oxygen and carbon dioxide, and provide about the optimum growh of culture and the condition of CTLA4-Ig protein production in producing bio-reactor.
During producing the bio-reactor step, use 5000-L three phase temperature control strategies to produce with growth of optimization cell and CTLA4-Ig.The initial heated culture temperature of producing bio-reactor is controlled at 37 ℃ to reach the optimum cell growth.Reach 4.0 x 10 when producing in the bio-reactor 6The viable cell density of cell/mL or when 144 hours whens inoculation distance whichsoever takes place earlier, cools the temperature to 34 ℃.Temperature is reduced to 32 ℃ and maintain 32 ℃ until results in the time of 240 hours.Obtain sample every day from 5000-L production bio-reactor, with the growth of monitoring cell, cell survival, metabolite concentration, CTLA4-Ig titre and sialic acid and CTLA4-Ig protein mol ratio.
Substratum 117-E gives the charging of producing bio-reactor initial between 12-24 hour when the distance inoculation.Add substratum 117-E every day reaching the target of charging substratum and culture volume 1% (v/v), or the charging substratum 117-E of enough volumes is so that glucose concn reaches 3g/L.This charging strategy provides the glucose and other nutrition of enough levels to culture, with the proteinic production of CTLA4-Ig during the support production bio-reactor step.
Substratum 117-E adds the glycosylation of D-semi-lactosi to promote that CTLA4-Ig protein increases.Semi-lactosi is added the increase that causes in the CTLA4-Ig sialic acid content of proteinic end last.Sialic acid and CTLA4-Ig protein mol ratio are the important results standards in the CTLA4-Ig production method.
Three-stage policy also is used for controlling the dissolved oxygen and the stir speed (S.S.) of producing bio-reactor.The initial stir speed (S.S.) of 30rpm is guaranteed the uniformity coefficient of physical condition and is prevented cell sedimentation in 5000-L produces bio-reactor.40% initial dissolved oxygen setting point is guaranteed the operability of the dissolved oxygen of enough levels, to support the growth of culture in producing bio-reactor.When 96 hours whens inoculation distance, increase to 50% and 40rpm respectively about the setting point of dissolved oxygen and stir speed (S.S.).When 120 hours whens inoculation distance, further increase to 60% and 50rpm respectively about the setting point of dissolved oxygen and stir speed (S.S.).This strategy is guaranteed the dissolved oxygen of enough levels, to maintain the cell cultures of producing during the bio-reactor step.The proteinic titre of CTLA4-Ig increases in producing the bio-reactor step process.This step process is monitored the culture viability from start to finish.From 6 days whens inoculation distance when the results, monitor 2 sialic acids and CTLA4-Ig protein mol ratio every day.Sialic acid and CTLA4-Ig protein mol ratio reach about 10 vertex when about 8 days of when inoculation distance, and gradually reduce through the rest part of producing the bio-reactor step.Main results standard about the production bio-reactor is sialic acid and CTLA4-Ig protein mol ratio.Producing bio-reactor gathers in the crops when 8.0 sialic acid and CTLA4-Ig protein target mol ratio.
Also determined 38% minimum cell survival value for the results of culture.The consistence of gathering in the crops material during these results standards are guaranteed to carry out is used for the downstream and is processed as the CTLA4-Ig medicine.The initial total algebraically of cell until the results of producing bio-reactor from the inoculum amplification in the CTLA4-Ig production upstream method is about 38 generations.The clone of in method, using in the clone stability study, confirm for 105 generations be stable.
The results operation steps
The purpose of results operation steps is to remove cell and cell debris from the results material, and make comprise that CTLA4-Ig is proteinic and carry out in stream concentrate and be used for the processing of further downstream.MF and UF system carry out sanitary measure before processing results material.MF and UF system wash with peracetic acid solution.MF and UF handle with liquid lime chloride and sodium hydroxide solution respectively subsequently.At last, MF and UF system wash in reaching retention (retentate) and penetrant≤electroconductibility of 3 μ S/cm with WFI.To be transferred to the results container from the cell culture liquid nutrient medium that 5000-L produces bio-reactor.Tangential flow MF with 0.65 μ m poly(vinylidene fluoride) film is used for removing cell and cell debris the material from comprising proteinic results of CTLA4-Ig.With acellular MF permeate collection in permeant container.Acellular MF penetrant concentrates by the poly (ether sulfone) film that tangential flow UF uses 30 kilodaltons (kDa) nominal molecular weight to end simultaneously.The UF penetrant is used for the MF method steps as filtration media.0.1N phosphoric acid solution is used to preserve the MF system.0.1N sodium hydroxide solution is used to preserve the UF system.At MF and UF operating period monitoring and controlled temperature, penetrant and retention flow velocity and transmembrane pressure.Flow velocity is measured by being present in the axial flow velocity meter that filters on the guide rail.Transmitter is used for measuring stress and temperature.Determine 163-235L/ minute MF retention flow velocity and≤the MF transmembrane pressure of 3.8psig.These values guarantee to gather in the crops the consistence in the operation steps performance.
Final step in the results operation is the pH and the electroconductibility adjustment of results material in clarification and spissated the carrying out.The pH and the electroconductibility adjustment of results material are used for catching CTLA4-Ig protein during first downstream chromatography procedure of processing in spissated the carrying out.PH is adjusted to 8.0 by adding 2M Tris solution, and the electroconductibility of concentrated penetrant is reduced to 10mS/cm by adding WFI.Concentrate with the carrying out of adjusting in the results material filter by comprising parallel and 1 the successive filter cover of 3 of 0.2 μ m disposable filter subsequently, and be transferred to dashpot (surge tank) in the downstream purification zone.
The composition of table 31:CD-CHO substratum
Figure A200680053116D03681
The composition of table 32:eRDF charging substratum
Figure A200680053116D03682
Table 33: the composition of modified 50%CD-CHO substratum
Figure A200680053116D03691
Embodiment 29-CTLA4-Ig purification process
At first use the anion-exchange chromatography step to process from the final pH of the results operation steps of describing among the embodiment 28 and the material of electroconductibility adjustment.Use the hydrophobic interaction chromatography step to process subsequently from the product storehouse of this first anion-exchange chromatography step.The material that comprises CTLA4-Ig is handled with the external viral agent of deactivation potential with Triton X-100 subsequently.The material that TritonX-100 handles uses reorganization A albumen affinity chromatography step to process.Product storehouse from reorganization A protein chromatographic step concentrates and diafiltration.Carry out the virus filtration step subsequently and be used to remove the external viral agent of potential.Filtrate uses second anion-exchange chromatography step to be further purified.At last, the CTLA4-Ig protein of purifying concentrate and diafiltration in the final medicine damping fluid.
CTLA4-Ig is genetic engineering modified fusion rotein, and its Fc structural domain by the human monoclonal immunoglobulin (Ig) of the function binding domains of people's cytotoxic T lymphocyte antigen 4 and IgG1 class is formed.The CTLA4-Ig dimer comprises 2 glycosylated polypeptide chains of homology by the covalently bound about 46kDa separately of single disulfide linkage.The process flow sheet that has shown the downstream procedures that is used for the CTLA4-Ig production method.CTLA4-Ig uses Chinese hamster ovary (CHO) clone to produce bio-reactor at 5000-L and produces.Chromatography and filtration step in the CTLA4-Ig production method of downstream carry out at ambient temperature.Carrying out material preserves between procedure of processing in 2-8 ℃.Downstream processes is initial by accepting from results material in the carrying out of results operation steps.This material at first uses Q Sepharose by the anion-exchange chromatography post TMExtreme Load (QXL) resin is processed.The QXL post is used to catch the CTLA4-Ig protein from the results material.The step of catching the QXL post realizes that also the volume minimizing is used for the processing of further downstream.
Make QXL product storehouse through utilizing hydrophobic interaction chromatography (HIC) post of the quick stir-in resin of phenyl sepharose.During this step, CTLA4-Ig high molecular (HMW) material and Chinese hamster ovary host cell proteins matter (CHOP) combine with post.The CTLA4-Ig protein dimer is not with the HIC resin-bonded and through post.After the HIC step, use
Figure A200680053116D0370173929QIETU
The inactivation of virus of X-100 stain remover (VI) is with the external viral agent of deactivation potential.Mobile fast (rPA) resin affinity column of next procedure utilization reorganization A albumen agarose.In this chromatographic step, reduce the level of CHOP and MCP 1 (MCP-1).The rPA step is defined as the virus sweep step.After the affinity chromatography step, CTLA4-Ig protein uses 30kDa ultrafiltration (UF) film to concentrate and dialyses, and through Planova TMThe processing of 15nm filter is to remove the external reagent of potential.After virus filtration (VF) step was finished, CTLA4-Ig protein used the Q agarose QFF that flows fast on second anion-exchange column) resin processes.The QFF chromatographic step reduces residual reorganization A albumen and dna level.The QFF step is defined as the virus sweep step.At last, CTLA4-Ig protein uses 30kDa UF film at the 25mM sodium phosphate, 50mMNaCl, and the solution of pH7.5 concentrates and diafiltration.Before final filling step, the CTLA4-Ig medicine filters by 0.2 μ m filter subsequently.
Method related impurities CHOP, MCP-1, residual reorganization A albumen, DNA and TritonX-100 reduce when the procedure of processing of the specific downstream of CTLA4-Ig production method.In mainly carrying out, determine to have the PPs of effect boundary during the reference mark about every kind of impurity.3 product storehouse CHOP and MCP-1 are accredited as the PPs about the rPA chromatographic step.The residual reorganization in product storehouse A protein ligands, DNA and Triton X-100 are accredited as the PPs about the QFF chromatographic step.Known structure and chromatography based on Regular Insulin keep, and based on interactional orthogonal modes, HIC, rPA and QFF step should provide the remarkable removing of Regular Insulin.In addition, the Regular Insulin with molecular weight of 5.8kDa should concentrate by 3 30kDa in results and downstream processes/diafiltration steps obtain removing.The rPA step provides methotrexate (MTX)〉removing of 3.0 log10.In addition, the MTX with molecular weight of 0.455kDa should concentrate by 3 30kDa in results and downstream processes/diafiltration steps obtain removing.Regular Insulin and MTX add in the fermention medium with fixed amount, and before the processing of downstream during the 5000-L fermentation process function as cellular metabolism consume.Measure on Regular Insulin and the MTX a plurality of points in downstream processes.Regular Insulin on each of these points and MTX level are lower than the quantitative level of the past operation of finishing.
Damping fluid: the purpose of buffer preparation is to produce the downstream processing buffer liquid that meets example values, effect boundary and alarm limit.Consistent damping fluid quality is to guarantee that reproducible chromatographic property is necessary.The downstream procedures of CTLA4-Ig method CD-CHO1 needs 17 kinds of damping fluids and solution.Damping fluid and solution be prepared and be used for batch in the particular process step.Be considered as the important method damping fluid with the damping fluid that contacts of material during CTLA4-Ig carries out.Product contact damping fluid comprises balance, washing, wash-out and product storehouse adjustment damping fluid.The damping fluid and the solution that are used for removing with the function test of ultraviolet ray (UV) detector of sanitising step and chromatography guide rail have stated limit widely.Because the extensive stated limit of these damping fluids and solution and do not exist product to contact is not specified PPs to these damping fluids and solution.PPs and corresponding example values that 10 kinds of product contact damping fluids are limited have been summarized.Maximum retention time boundary about these damping fluids is 3 days, and keeps research 9 derived from buffer container, and obtains the support of damping fluid stability study.Product contact damping fluid also has specified PPs and corresponding alarm limit.PPs and corresponding alarm limit have been presented about these damping fluids.Do not have and have alarm or act on the specified PPs of boundary and presented with the damping fluid that contact of material during CTLA4-Ig carries out and solution.Non-product contact damping fluid and solution are not tested intracellular toxin, because they are sanitary measure solution or chromatographic resin cleaning buffer or solution before the post sanitising step.
Q SeDharose Extreme Load anion-exchange chromatography step.
Anion-exchange chromatography uses the QXL resin from GE Healthcare (AmershamBiosciences in the past) to carry out.1.0m the internal diameter post is filled to the height of 17-24cm with the QXL resin, the volume of expression 133-188L.By measuring the height equivalent to a theoretical plate (HE-TP) (HETP) and the asymmetry (A of packed column s) post qualified being used for used.Need the HETP of 0.02-0.08cm and the A of 0.8-1.2 sBe used for the qualification evaluation of QXL post.The QXL post is used for catching the CTLA4-Ig protein from gathering in the crops material.The step of catching QXL realizes that also the volume minimizing is used for the processing of further downstream.The QXL column operation carries out at ambient temperature.Clarifying cell culture liquid nutrient medium is loaded on the equilibrated QXL post.The Peak Flow Rate that the QXL chromatographic step was used 28L/ minute carries out.Column inlet pressure maintains under the 35psig.Maximum A Baxipu protein load about the QXL post is 28 gram A Baxipu/L resins.Post passes through to use 25mMHEPES, 100mM NaCl, and pH 8.0 damping fluid balances are prepared.Behind the column equilibration, will gather in the crops material and be loaded on the post, and follow the monitoring continuously effluent to absorb at the UV at 280nm place.After loading application, post 25mM HEPES, 120mM NaCl, the pH8.0 damping fluid washs.CTLA4-Ig protein 25mM HEPES, 850mM NaCl, pH7.0 damping fluid wash-out from post.Under table show process parameter about Q Sepharose Extreme Load chromatographic step.
Parameter Setting point/target value The effect boundary Accept standard
The protein load a，b N/A c N/A ≤28g/L Resin
Product storehouse biological load a N/A N/A <1cfu/mL
Product storehouse intracellular toxin b，d N/A N/A ≤5.0EU/mL
The results hold-time is guaranteed the process consistence with the alarm limit of determining.To pre-filtering product storehouse biological load parameter specify respectively＜10cfu/mL and＜middle the alarm of 100cfu/mL and act on boundary.6 kinds of QXL PPs that limited by the effect boundary are post height, flow velocity, lavation buffer solution electroconductibility, elution peak terminal point optical density(OD) (OD), step yield and load biological load.Determine the post altitude range with resin that enough volumes are provided to catch product from the results material.Effect boundary about this parameter is determined by data.Flow velocity is an important factor of guaranteeing the consistence and the performance of chromatographic step.It is defined as the PP with maximum effect boundary for the QXL step.Determine that lavation buffer solution electroconductibility is to remove weak bonded impurity from the QXL resin.It is to keep the conforming important factor of QXL step that this parameter is measured in the research of reduction scope in proportion.Limit elution peak terminal point OD so that in the product storehouse level of CTLA4-Ig HMW material drop to minimum.Wash-out when the CTLA4-IgHMW material finishes at elution peak.Step yield effect boundary is guaranteed the process consistence about the QXL step.Determine effect boundary about flow velocity, elution peak terminal point OD and step yield PPs.To loading the effect boundary of biological load appointment＜1cfu/mL.As present, 12 kinds of QXL PP are limited by alarm limit.
PH of buffer and electroconductibility value in the monitoring boundary.The damping fluid that does not meet pH and electroconductibility example values in the preparation is excluded and damping fluid batch is discarded.For the QXL step, limit level pad electroconductibility and pH, lavation buffer solution pH and elution buffer electroconductibility and pH.Column inlet pressure is guaranteed the consistence during combination and elution chromatography step.The QXL step is carried out with the peak pressure boundary of 35psig.According to the specification sheets of manufacturers, the pressure limit of using 35psig is to prevent the compression of QXL resin.Product storehouse electroconductibility is the PP with alarm limit.To product storehouse electroconductibility specified alarm boundary, to guarantee CTLA4-Ig HMW material in the QXL product storehouse and CHOP and subsequently effective combination of HIC post.Determine the alarm limit of 58.5-69.1mS/cm.To product storehouse titre, sialic acid (SA) N-n acetylneuraminic acid n (NANA) mol ratio, CTLA4-Ig HMW material and CHOP specified alarm boundary, to guarantee the process consistence.Determine product storehouse titre and SA alarm limit.
To be loaded on the QXL post from material in the carrying out of results operation steps.(25mM HEPES, 120mM NaCl pH8.0) washs this post, and measures at the 280nm of effluent (A when washing step finishes with bottom line 10CV lavation buffer solution 280) absorbancy located.A Baxipu uses 25mM HEPES subsequently, and 850mM NaCl, pH7.0 damping fluid carry out wash-out from post.Work as A 280When increasing to the AU value that surpasses when washing step finishes 〉=0.02 absorbance unit (AU), make eluate turn to collection container.Collect the A that trail edge of eluate until elution peak 280Reduce to≤value of 1.0AU.Elution buffer is directly added in the eluate collection container to reach the eluate target weight of 600 ± 10kg.The stir speed (S.S.) of 30 ± 5rpm is used to guarantee that content obtains thorough mixing in the eluate collection container.After 〉=5 minutes the mixing time section, the content of collection container arrives by 0.2 μ m rhodia filter direct filtration and keeps in the container.Other ε 50kg elution buffer is added in the collection container, and arrive in the identical maintenance container by 0.2 μ m rhodia filter direct filtration subsequently.Keep the content of container to preserve in 2-8 ℃ maximum 72 hours.
Table 5. is about the process parameter of Q Sepharose Extreme Load chromatographic step
Parameter a Setting point/target value Alarm limit The effect boundary
The results hold-time b N/A c ≤ 10 hours ≤ 24 hours
Pre-filtering product storehouse biological load d N/A <10cfu/mL <100cfu/mL
The post height e N/A N/A 17-24cm
Flow velocity 15-28L/ minute N/A ≤ 28L/ minute
Lavation buffer solution electroconductibility f N/A N/A 11.0-15.0mS/cm
Elution peak terminal point OD 1.00AU g N/A 0.97-1.03AU h
The step yield N/A N/A 69-107％
Load biological load i N/A N/A <1cfu/mL
Level pad electroconductibility N/A 10.7-12.7mS/cm N/A
Level pad pH N/A 7.8-8.2 N/A
Lavation buffer solution pH N/A 7.7-8.3 N/A
Elution buffer electroconductibility N/A 69.8-77.1mS/cm N/A
Elution buffer pH N/A 6.7-7.3 N/A
Loading time j N/A ≤ 6 hours N/A
Column inlet pressure N/A ≤35psig N/A
Product storehouse electroconductibility k N/A 58.5-69.1mS/cm N/A
Product storehouse titre N/A ≥2.0g/L N/A
The mobile fast hydrophobic interaction chromatography step of phenyl sepharose
The main purpose of HIC step is to reduce the level of the CTLA4-Ig high molecular weight species (for example, the tetramer) that exists in the QXL product storehouse.The CTLA4-Ig tetramer under the loading conditions that is used for the HIC step not with the HIC resin-bonded.
The HIC step is used and is carried out from the quick stir-in resin of the phenyl sepharose of GE Healthcare.The HIC post is in conjunction with CTLA4-Ig HMW material and CHOP, thereby reduces its concentration in the CTLA4-Ig protein stream.The HIC post passes through to use 25mM HEPES, 850mM NaCl, and pH 7.0 damping fluid balances are prepared.To be applied to the equilibrated post from the CTLA4-Ig product storehouse of QXL step.After loading application, with 25mM HEPES, 850mM NaCl, the pH7.0 damping fluid is applied to post.CTLA4-Ig is collected by post in circulation and post tracking fraction.Depend on the total mass of CTLA4-Ig in the QXL product storehouse, the HIC post is operated with work sheet with Multiple Cycle and is criticized CTLA4-Ig.Post cleans and sanitary measure in circulation with between criticizing.
HIC loads biological load PP and is limited by alarm and effect boundary.HIC is loaded biological load PP specify respectively＜10cfu/mL and＜middle the alarm of 100cfu/mL and act on boundary.5 kinds of HIC values that limit for post are post height, flow velocity, tracking terminal point OD, step yield and load the hold-time.The measuring column height to be providing enough resin volumes, with 1,2 or 3 circulation/batch in process wash-out storehouse from the QXL post.Effect boundary about this parameter is determined by data.Flow velocity is an important factor of guaranteeing the consistence and the performance of chromatographic step.It is defined as the process parameter with maximum effect boundary for the HIC step.Limit to follow the trail of terminal point OD so that in the product storehouse level of CTLA4-Ig HMW material drop to minimum.CTLA4-Ig HMW material wash-out when elution peak finishes.The method steps yield is guaranteed the process consistence about the HIC step.Determine effect boundary about flow velocity, tracking terminal point OD and step yield PPs.Guarantee the process consistence about the effect boundary of loading the hold-time.The effect boundary of≤5 days loading hold-time PP obtains the support of research of product container hold-time and biological chemistry stability study.As present, 9 kinds of HIC PPs are limited by alarm limit.PH of buffer in the downstream processes and electroconductibility parameter are accredited as the PPs with alarm limit.The damping fluid that does not meet pH and electroconductibility example values in the preparation is excluded and damping fluid batch is discarded.For the HIC step, the electroconductibility and the pH of balance/tracking damping fluid are defined as the PPs with alarm limit.
Column inlet pressure is guaranteed the consistence during chromatographic step.The HIC step is carried out with the peak pressure boundary of 13psig.According to the specification sheets of manufacturers, the pressure limit of using 13psig is to prevent the compression of HIC resin.Product storehouse titre and SA alarm limit are guaranteed the process consistence and are determined in PAR report 12.To product storehouse DNA, CHOP and MCP-1 specified alarm boundary when this step, with promote that it removes in rPA step subsequently quantitatively.
Table 7. is about flow the fast process parameter of hydrophobic interaction chromatography step of phenyl sepharose
Parameter a Setting point/target value Alarm limit The effect boundary
Load biological load b N/A c <10cfu/mL <100cfu/mL
The post height d N/A N/A 18-22cm
Flow velocity 7.6-18L/ minute N/A ≤ 18L/ minute
Follow the trail of terminal point OD 1.0AU e N/A 0.8-1.0AU e
The step yield N/A N/A 55-79％
Load the hold-time N/A N/A ≤ 5 days
Balance/tracking damping fluid electroconductibility N/A 71.5-75.5mS/cm N/A
Balance/tracking pH of buffer N/A 6.7-7.3 N/A
The loading chute temperature 22℃ 19-25℃ N/A
Column inlet pressure N/A ≤13psig N/A
Product storehouse titre N/A ≥1.0g/L N/A
Product storehouse SA NANA mol ratio N/A 6.8-11.4 N/A
Product storehouse CHOP f N/A ≤6600ng/mL N/A
Product storehouse DNA f N/A ≤45,000,000pg/mL N/A
Product storehouse MCP-1 g N/A ≤5600ng/mL N/A
aInformation derives from the PAR final report: purifying 12In table 2.
The inactivation of virus step:Deactivation from the external viral agent of potential in the product storehouse of HIC step reaches by the final concentration that adds 20%Triton X-100 to 0.5% (v/v).Before advancing to next procedure, the solution of detergent-treatment mix and remain in 22 ± 3 ℃ 1-4 hour.5 kinds of PPs that the VI step is limited have been presented.Be limited to 3.8% on the 20%Triton X-100 interpolation parameter.Referring under table, it shows the process parameter about the inactivation of virus step.
Parameter Setting point/target value The effect boundary Accept standard
20%Triton X-100 adds a，b 2.5% (the volume % in HIC storehouse) N/A c 1.3-3.8% (the volume % in HIC storehouse)
Mixed duration d N/A N/A c 〉=20 minutes
Stir speed (S.S.) d 30rpm 25-35 ≥20rpm
Product storehouse biological load
Product storehouse intracellular toxin b，e N/A N/A ≤5.0EU/mL
aInformation derives from virus sweep research, PQ-58-virus sweep-BMS 188667-001 23
Table 9. is about the process parameter of inactivation of virus step
Parameter Setting point/target value Alarm limit The effect boundary
Groove temperature when Triton X-100 adds a，b? 22℃ 19-25℃ 2-25℃
Load the hold-time c N/A d N/A ≤ 5 days
The Triton X-100 incubation time a，b N/A N/A 1-4 hour
The step yield a N/A N/A 94-108％
aInformation derives from the PAR final report: purifying 12In table 3.
The fast mobile affine step of reorganization A albumen agar.
Affinity chromatography is used and is carried out from GE Healthcare fixed rPA resin.The rPA chromatographic step is further purified CTLA4-Ig protein by the level that reduces the external viral agent of CHOP, MCP-1 and potential.Affinity column 25mM Tris, 250mM NaCl, the pH8.0 damping fluid carries out balance.Behind the column equilibration, the material that will handle from the Triton X-100 of VI step is applied to affinity column.This post is at first used 25mM Tris, 250mM NaCl, and 0.5% TritonX-100, the pH8.0 damping fluid washs, subsequently for to use 25mM Tris, 250mM NaCl, the washing second time of pH8.0 damping fluid.CTLA4-Ig protein 100mM glycine, pH3.5 damping fluid carry out wash-out from post.From the pH 2M HEPES in the product storehouse of affinity column, the pH8.0 damping fluid is adjusted to 7.5 ± 0.2.4 kinds of PPs that the rPA step limits are presented in the table 10.The rPA chromatographic step is accredited as the virus sweep step, thereby the post bed height is defined as new PP, and its example values is 21-25cm.Product storehouse CHOP and product storehouse MCP-1 before be accredited as the PPs about the rPA step.These impurity redefine to having the PPs of effect boundary.Referring under table, it shows the process parameter about the quick flow chromatographic step of reorganization A albumen agarose.
Parameter a Setting point/target value The effect boundary Accept standard
The post height b N/A c N/A 21-25cm
The protein load N/A N/A ≤25g/L Resin
Product storehouse biological load N/A N/A <1cfu/mL
Intracellular toxin d
Information derives from the PAR final report (FR): the table 4 of purifying.
As present, limit by alarm and effect boundary about 3 kinds of PPs of rPA step.Load the hold-time guarantee with the PAR report in the process consistence of the alarm limit determined.Guarantee the process consistence about the effect boundary of loading the hold-time.The effect boundary of≤48 hours loading hold-time PP obtains the support of research of product container hold-time and biological chemistry stability study.Scope research identifies that elution buffer pH is for influencing the important factor of the level of CTLA4-Ig HMW material in the rPA product storehouse.Determine alarm limit about elution buffer pH.Effect boundary about elution buffer pH is determined by the scope that reduces in proportion.RPA is loaded biological load PP specify respectively＜10cfu/mL and＜middle the alarm of 100cfu/mL and act on boundary.
7 kinds of rPA PPs that limited by the effect boundary are column inlet pressure, flow velocity, step yield, the initial pH in product storehouse, product storehouse HMW, product storehouse CHOP and product storehouse MCP-1.According to the specification sheets of manufacturers, the pressure limit of employing≤13psig is to prevent the compression of rPA resin.Flow velocity is an important factor of guaranteeing the consistence and the performance of chromatographic step.It is defined as the PP with maximum effect boundary for the rPA step.Guarantee process consistence about the effect boundary of step yield and the initial pH in product storehouse about the rPA step.Determine effect boundary about flow velocity and the initial pH in product storehouse.The potential formation of CTLA4-Ig HMW material necessitates the qualification about the effect boundary of this parameter≤2.5% during the peak wash-out.Use is determined the effect compass of 66-108% from the mean value ± 3 standard deviation data of single batch of rPA resin.This scope is consistent with observed step yield in research resin life.Process related impurities CHOP and MCP-1 before be accredited as the PPs about the rPA step.In this report, these impurity redefine to having the PPs of effect boundary.Product storehouse CHOP and MCP-1 are defined as the CQAs with example values, to guarantee the control of these process related impuritieses.As present, 12 kinds of rPA PPs are limited by alarm limit.PH of buffer in the downstream processes and electroconductibility parameter are accredited as the PPs with alarm limit.The damping fluid that does not meet pH and electroconductibility example values in the preparation is excluded and damping fluid batch is discarded.For the rPA step, balance/washing 2 damping fluid electroconductibility and pH, washing 1 damping fluid electroconductibility and pH and elution buffer electroconductibility are defined as the PPs with alarm limit.
Table 11. is about the process parameter of the quick flow chromatographic step of reorganization A albumen
Parameter a Setting point/target value Alarm limit The effect boundary
Load the hold-time b N/A c ≤ 43 hours ≤ 48 hours
Elution buffer pH N/A 3.4-3.7 3.2-3.8
Load biological load d N/A <10cfu/mL <100cfu/mL
Column inlet pressure N/A N/A ≤13psig
Flow velocity 6.7-9.6L/ minute N/A ≤ 9.6L/ minute
The step yield N/A N/A 66-108％
The initial pH in product storehouse N/A N/A ≥5.8
Product storehouse HMW N/A N/A ≤2.5％
Product storehouse CHOP N/A N/A ≤380ng/mL
Product storehouse MCP-1 N/A N/A ≤38ng/mL
Balance/washing 2 damping fluid electroconductibility N/A 23.0-27.0mS/cm N/A
Balance/washing 2 pH of buffer N/A 7.8-8.2 N/A
Wash
1 damping fluid electroconductibility N/A 22.2-27.4mS/cm N/A
Wash
1 pH of buffer N/A 7.7-8.3 N/A
Elution buffer electroconductibility N/A 0.5-1.5mS/cm N/A
Product storehouse final pH 7.5 7.3-7.7 N/A
Product storehouse titre N/A ≥6.0g/L N/A
Product storehouse SA NANA mol ratio N/A 8.0-11.0 N/A
The adjusted product of pH storehouse volume e N/A 127-294kg N/A
Product storehouse DNA N/A ≤47000pg/mL N/A
The residual reorganization in product storehouse A albumen f N/A ≤160ng/mL N/A
Product storehouse Triton X-100 g N/A ≤4.0μg/mL N/A
aInformation derives from the PAR final report: purifying 12In table 4.
Concentrate/diafiltration and virus filtration step.Behind the wash-out, the product storehouse concentrates to reach the target volume in the CTLA4-Ig concentration limit from the rPA post.Subsequently enriched material is implemented to use 25mMHEPES, 100mM NaCl, the diafiltration of pH8.0 damping fluid is wherein used to have the UF system of 30kDa nominal molecular weight by (NMWCO) film.After the diafiltration, use the filter string to remove the external virion of potential.The filter string is made up of 0.2 μ m filter, 0.1 μ m filter and 15nm membrane filter (Planova 15N filter).The filter that uses in the VF step (0.2 μ m, 0.1 μ m and 15nm) is the filter that single uses.Based on the upper limit of the virus sweep increase of using these conditions to confirm about CTLA4-Ig concentration behind the UF and Planova dividing potential drop scope.See table, it shows the process parameter about the virus filtration step.
Parameter Setting point/target value The effect boundary Accept standard
A Baxipu concentration behind the UF a，b N/A c N/A 6.0-22g/L
Load volume and surface area ratio a ≤36kg/m 2 N/A ≤100kg/m 2
The Planova dividing potential drop N/A N/A ≤14psid
Biological load e N/A N/A <1cfu/mL
Product storehouse intracellular toxin e，f N/A N/A ≤0.50EU/mL
aInformation derives from virus sweep research, PQ-58 virus sweep-BMS188667-001 23, BD-2004-001.0 31And BD-2005-832.0 36
Table 13. is about the process parameter of virus filtration step
Parameter a Setting point/target value Alarm limit The effect boundary
UF I product storehouse biological load b N/A c <10cfu/mL <100cfu/mL
The diafiltration volume N/A N/A ≥5.0
The step yield N/A N/A 86-114％
Load the hold-time d N/A N/A ≤ 5 days
UF eFeed pressure N/A ≤35psig N/A
UF eThe retention flow N/A 2.5-7.5L/ minute N/A
Filtrate electroconductibility N/A 10.7-12.7mS/cm N/A
aInformation derives from the PAR final report: purifying 12In table 5.
bInformation derives from BD-2005-706.0 15
The mobile fast anion-exchange chromatography step of Q agarose.
The purpose of QFF chromatographic step is to reduce residual A protein level, and the other minimizing from the host cell DNA in virus filtration step products storehouse is provided.QFF post step also is used to control sialic acid and the CTLA4-Ig molecule or the protein mol ratio in QFF chromatographic step product storehouse, and the other control of HMW material horizontal is provided.QFF anion-exchange chromatography step can also be removed residual reorganization A albumen, host cell DNA, Triton X-100 and the external viral agent of potential.
Anion-exchange chromatography uses the QFF resin from GE Healthcare to carry out.QFF post 25mM HEPES, 100mM NaCl, the pH8.0 damping fluid carries out balance.Behind the column equilibration, Planova filtrate is applied to the QFF post.This post is at first used 25mM HEPES, 120mMNaCl, and the pH8.0 damping fluid washs, subsequently for to use 25mM HEPES, 130mM NaCl, the washing second time of pH8.0 damping fluid.CTLA4-Ig protein 25mM HEPES, 200mM NaCl, pH8.0 damping fluid carry out wash-out from post.
Parameter Setting point/target value The effect boundary Accept standard
The post height a，b N/A c N/A 28-35cm
The protein load a N/A N/A ≤25g/L Resin
Product storehouse biological load a N/A N/A <1cfu/mL
Intracellular toxin a，d
aInformation is from the PAR final report (FR): purifying 12Table 6
QFF load the biological load value be respectively＜10cfu/mL or＜100cfu/mL.By 12 kinds of QFF PPs limiting of effect boundary is the level of A albumen, DNA, Triton X-100, SA, HMW, CHOP and MCP-1 residual in flow velocity, washing 2 damping fluid electroconductibility, elution buffer electroconductibility, loading hold-time, step yield and the QFF product storehouse.Flow velocity is to guarantee the factor considered in the consistence of chromatographic step and the performance.It is defined as the process parameter of determining with maximum effect boundary in the PAR report for the QFF step.The electroconductibility value of washing 2 and elution buffer is the PPs with effect boundary, because they are important in determination step yield and product quality.Effect boundary about these parameters is measured during the QFF scope research that reduces in proportion.Guarantee the process consistence about the effect boundary of loading the hold-time.The effect boundary of≤5 days loading hold-time PP obtains the support of research of product container hold-time and biological chemistry stability study.The step yield is guaranteed the process consistence about the QFF step.Q factor, product storehouse SA, HMW, CHOP and MCP-1 must closely be controlled in final chromatographic step.Determine effect boundary about the level of SA, CHOP in step yield and the QFF product storehouse and MCP-1.Residual A albumen, DNA and the Triton X-100 of process related impurities before had been accredited as the PPs about the QFF step.These impurity redefine to having the PPs of effect boundary.Residual A albumen, DNA and Triton X-100 are defined as the CQAs with example values, to guarantee the control of these process related impuritieses.In this example, about the effect boundary of product storehouse HMW from≤2.5 be revised as≤2.0%.
PH of buffer in the downstream processes and electroconductibility parameter are accredited as the PPs with alarm limit.The damping fluid that does not meet pH and electroconductibility example values in the preparation is excluded and damping fluid batch is discarded.For the QFF step, the electroconductibility of level pad and pH, washing 1 damping fluid electroconductibility and pH, washing 2 pH of buffer and elution buffer pH are defined as the PPs with alarm limit.Determine these boundaries.Column inlet pressure is guaranteed the consistence during combination and elution chromatography step.According to the specification sheets of manufacturers, the QFF step is carried out with the peak pressure boundary of 35psig.Parameter is washed 1 damping fluid volume, washing 2 damping fluid volumes, product storehouse titre and product storehouse volume specified alarm boundary 12 to guarantee the process consistence.
Table 15. is about the process parameter of the quick flow chromatographic step of Q agarose
Parameter a Setting point/target value Alarm limit The effect boundary
Load biological load b N/A c <10cfu/mL <100cfu/mL
Flow velocity d 4.8-8.7L/ minute N/A ≤ 8.7L/ minute
Wash 2 damping fluid electroconductibility e N/A N/A 12.8-15.2mS/cm
Elution buffer electroconductibility e N/A N/A 18.5-20.9mS/cm
Load the hold-time f N/A N/A ≤ 5 days
The step yield N/A N/A 65-104％
The reorganization A albumen that the product storehouse is residual N/A N/A ≤9.5ng/mL
Product storehouse DNA g N/A N/A ≤20pg/mL
Product storehouse TritonX-100 h N/A N/A ≤4.0μg/mL
Product storehouse SA NANA mol ratio N/A N/A 8.0-11.9
Product storehouse HMW N/A N/A ≤2.0％
Product storehouse CHOP N/A N/A ≤95ng/mL
Product storehouse MCP-1 N/A N/A ≤9.5ng/mL
Level pad electroconductibility N/A 10.5-12.9mS/cm N/A
Level pad pH N/A 7.7-8.3 N/A
Wash 1 damping fluid electroconductibility N/A 124-14.4mS/cm N/A
Wash 1 pH of buffer N/A 7.7-8.3 N/A
Wash 2 pH of buffer N/A 7.7-8.3 N/A
Elution buffer pH N/A 7.7-8.3 N/A
Column inlet pressure N/A ≤35psig N/A
Wash 1 damping fluid volume N/A 5.0-5.3CV N/A
Wash 2 damping fluid volumes N/A 5.0-5.4CV N/A
Product storehouse titre N/A ≥0.65g/L N/A
Product storehouse volume N/A ≤800kg N/A
aInformation derives from the PAR final report: purifying 12In table 6.
Concentrate/diafiltration and filling step.Product storehouse from QFF anion-exchange chromatography step concentrates, and implements to use the 25mM sodium phosphate, 50mM NaCl, and the diafiltration of pH7.5 damping fluid is wherein used to have the UF system of 30kDa NMWC film.The enriched material of diafiltration is filled in the sterile chamber by 0.2 μ m filter and preserves in 2-8 ℃.If need then medicine to carry out freezing and preserve at-70 ℃ in-40 ℃.Presented single PP for concentrated/diafiltration and filling step qualification.Referring under table, it shows about medicine and concentrates/process parameter of diafiltration and filling step.
Parameter Setting point/target value The effect boundary Accept standard
Final concentration a，b 50g/L 48-52g/L 45-55g/L
To UF II product storehouse biological load PP specify respectively＜10cfu/mL and＜middle the alarm of 50cfu/mL and act on boundary.As present, about concentrate/6 kinds of PPs that diafiltration and filling step are limited by the effect boundary are diafiltration volume, filtrate electroconductibility and pH, step yield, loading hold-time and method yield.Determine the lower limit of diafiltration volume, to guarantee that the proteinic complete buffer exchange of CTLA4-Ig becomes final medicine damping fluid before filling step.Filtrate electroconductibility is further guaranteed consistent medicine preparation with filtrate pH.The step yield is guaranteed the process consistence about concentrated/diafiltration and filling step.Determine effect boundary about diafiltration volume, filtrate electroconductibility and pH and step yield.Guarantee the process consistence about the effect boundary of loading the hold-time.≤ 5 days UF II loading hold-time obtains the support of research of product container hold-time and biological chemistry stability study.Recommend the method yield effect boundary of 20-62%.As present, 2 kinds of PPs are limited to guarantee the process consistence about step by alarm limit.
Table 17. concentrates/process parameter of diafiltration and filling step about medicine
Parameter a Setting point Alarm limit The effect boundary
UF II product storehouse biological load b N/A c <10cfu/mL <50cfu/mL
The diafiltration volume N/A N/A ≥5.0
Filtrate electroconductibility N/A N/A 8.3-10.3mS/cm
Filtrate pH N/A N/A 7.3-7.7
The step yield N/A N/A 73-110％
Load the hold-time d N/A N/A ≤ 5 days
The method yield e N/A N/A 20-62％
The UF feed pressure N/A ≤35psig N/A
UF retention flow N/A 2.5-7.5L/ minute N/A
aInformation derives from the PAR final report: purifying 12In table 7.
Embodiment 30: the final filling step of medicine
After concentrate and the diafiltration II step of CTLA4-Ig finished, the CTLA4-Ig medicine was filled in pre-sterilized 2-L and the 10-LBiotainer PC bottle in 300-L biological processing bag in Class 100 environment.
The outside of each bottle carries out disinfection with 70% aqueous isopropanol.Be removed and bottle tares before filling from the sealer around each bottle cap.Record calculates guaranteeing that filling weight is 500 grams-1950 grams for the 2-L bottle in batch record, and is 7500 grams-10200 grams for the 10-L bottle.Medicine uses peristaltic pump to be assigned in the Biotainer bottle by 0.45/0.2 μ m filter and the filling cone that single uses.Cover tightly to specifying the torque setting with the bottle cap upper cover and with lid.The lid of each filling bottle seals with band, and band carries out initial signature and dates.Each bottle be marked with authentication information and fill date, filling bottle batch in sequence number (SN) and operator's initial signature.
During filling process, carry out monitoring of air and surface microorganism and grain count.Drug sample obtains during stuffing operation.Before first bottle filling, obtain 1 sample and be used for the intracellular toxin test.The other sample that is used for intracellular toxin test obtains in the middle of filling process and when finishing.During filling process, obtain sample and be used for medicine release test.
Embodiment 31: immunogenicity research
CTLA4-Ig is a soluble fusion protein, and its ectodomain by people's cytotoxic t lymphocyte-associated antigen 4 (CTLA-4) that the Fc modified with human normal immunoglobulin (Ig) G1 (hinge, CH2 and CH3 structural domain) part is connected is formed.It is that first approval is used for the treatment of rheumatoid arthritis (RA) in the new kind reagent, and its selectivity is regulated the CD80/CD86:CD28 costimulatory signal that is used for complete T cell activation.In RA, suppose that unknown antigen presents via main histocompatibility complex and activate autoreactive T cell in the presence of costimulatory signal.Subsequently, the downstream immunocyte is recruited and activated to the activated T cell, cooperates and make the cell processes that causes inflammation and destruction of joint keep (Choy and Panayi (2001) NEngl J Med, 344 (12): 907-916).
Treatment reorganization biology agent for example CTLA4-Ig can be immunogenic and therefore have the possibility that causes antibody response.Immunogenicity at the biology agent can influence security, effect and pharmacokinetics in theory.The antibody-mediated removing of biology therapy can cause the minimizing in the levels of drugs, thereby causes the effect that reduces.Antibody response can also prevent that medicine from combining with its pharmacology target, and this also will reduce effect.This can cause needs-so-called ' the dosage creep ' along with time dosage expansion in the past.For anti-TNF antibodies, the prolongation of English husband monoclonal antibody in the RA treatment used and reported dosage creep (Anderson (2005) Semin Arthritis Rheum, 34 (5 Supp1l): 19-22), and recently pointed out that this is the result of anti-English husband antibody mab, it causes (the people such as Haraoui of the minimizing in the clinical efficacy among some patients, (2006) JRheumatol, 33 (1): 31-36).
Checked the formation of anti-CTLA 4-Ig and anti-CTLA-4 antibody and herein, to the latent effect of CTLA4-Ig therapeutic efficiency and security.Because based on its stimulate the active of conditioning agent altogether as selectivity and in non-clinical model observed replying, CTLA4-Ig may suppress the immunne response at himself, so also checked 1-2 agent or the therapy discontinued effect to the immunogenicity level that lacks.At last, with regard to neutralizing antibody active testing seropositivity sample.
Materials and methods
The research of assessment
For measure CTLA4-Ig whether in suffering from the patient of RA induction of immunity originality reply, through multiple II and III clinical trial phase assessment antibody response, be included in 2,6 DB and 4 OL search time sections among 237 RA patients, described patient comprises that methotrexate (MTX) or anti-TNF therapy are had the insufficient patient who replys.CTLA4-Ig is used for RA as monotherapy treatment (table 40) is also assessed in a research (IIa phase).Sample generally before research, treatment from start to finish and last agent time back 56 and/or 85 days assess with the time that allows CTLA4-Ig to remove.
Table 40. is included in the summary of the research in this assessment.
Figure A200680053116D03871
Figure A200680053116D03891
Figure A200680053116D03901
The RA=rheumatoid arthritis; The MTX=methotrexate; The DB=double blinding; The TNF=tumour necrosis factor; DMARD=is alleviated the moist medicine of wind resistance of disease; ETAN=etanercept (etanercept); The OL=open label; The PK=pharmacokinetics
*OL, unmatchful be those the subgroup of finishing DB, placebo-controlled study time period according to the experimenter in the time period
In the patient that replys at the positive antibody of CTLA4-Ig or CTLA-4 of development, check (peri-infusional) adverse events (AEs) around the preassigned infusion, overall AEs and serious AEs (SAEs) and the incidence and the type of interrupting.Also in having the patient that positive antibody replys, reply the effect of immunogenicity of checking to effect by assessment American College of Rheumatology (ACR) 20 and HAQ (Health Assessment Questionnaire).
Immunogenicity determining
Basic mensuration form: because at human serum, the particularly height of the Fc of CTLA4-Ig part, the cross reactivity that is pre-existing in the RA colony, the enzyme-linked immunosorbent assay of 2 kinds of direct forms (ELISAs) is used to assess antibody response.Anti-CTLA 4-Ig measures the antibody response of metering needle to all parts of molecule, but has lower susceptibility.Anti-CTLA-4 measures measurement only at the antibody response of CTLA-4 part, has removed Ig zone and so given bigger susceptibility.2 kinds of mensuration are used with terminal point titre (EPT) form (measuring A) or screening form (measuring B).
Measure the A form: the clinical immunogenicity determining method of II phase double blinding
Anti-CTLA 4-Ig mensuration and the anti-CTLA-4 mensuration used at II phase RA duration of test are referred to as mensuration A.In measuring A, CTLA4-Ig or CTLA-4 are adsorbed on the 96 hole microtiter plates, described 96 hole microtiter plates are subsequently with test sera (3 times of serial dilutions that begin with 1:10) incubation.(Southern Biotech, Birmingham US) detects anti-people's mixtures of antibodies that bonded antibody use alkaline phosphatase is puted together, and uses p-nitrophenyl phosphate (PNPP) substrate to manifest.Because there be not available people anti-CTLA 4-Ig antibody or positive control serum,, these use the CTLA4-Ig specific antisera that in macaque, produces to verify so measuring.Result from every kind of mensuration is expressed as the EPT value.When (the 1st day) EPT before the administration individual with respect to that he/when her EPT increased by 2 or more serial dilutions (〉=9 times), the patient was considered as seroconversion.
Measure the B form: the clinical immunogenicity determining method of III phase and II phase open label
Test and 2 years II phase OL time periods for the III phase, anti-CTLA 4-Ig and anti-CTLA-4 measure and make amendment reducing non-specific background, to improve susceptibility, and measure the male method and change and be referred to as and measure B.These are measured also to use by the CTLA4-Ig specific antibody of macaque antiserum(antisera) purifying and verify.For every kind of ELISA, with 96 hole microtiter plates of CTLA4-Ig (0.25 μ g/mL) or CTLA-4 (0.5 μ g/mL) bag quilt with the test sera of dilution in 1: 400 in 22 ± 5 ℃ (incubation of anti-CTLA 4-Ig) 2 hours, or with the test sera of diluting at 1: 25 in 32-40 ℃ of (anti-CTLA-4) incubation 2 hours.Behind the preliminary incubation, anti-people's mixtures of antibodies that bonded antibody uses horseradish peroxidase (HRP) to put together detects for the tetramethyl benzidine substrate subsequently.
The result who measures about anti-CTLA 4-Ig is expressed as back/preceding ratio, its by the administration that will on same plate, analyze after sample OD value divided by administration accordingly before sample OD value calculate.The positive cutoff of determining based on the RA patient's sample that uses placebo treatment.If ratio is less than ending, sample is considered as feminine gender and be reported as＜400 titre value so.Surpass this any value of ending and be considered as the condition male.
The result who measures about anti-CTLA-4 is expressed as that ' than 1 ' value, its average patient serum sample OD by will be on same plate calculates divided by the average OD of negative control.Positive in using the value of determining from as the RA patient's of the placebo treatment of negative control pooled serum.If this value is ended less than appointment, sample is considered as feminine gender and be reported as＜25 titre value so.Surpass this any value of ending and be considered as the condition male.
Validating analysis
The condition positive of identifying in every kind of mensuration (anti-CTLA 4-Ig and anti-CTLA-4) and every kind of mensuration form (measuring A and B) is assessed in immunodepletion mensuration, the specificity of replying with mensuration.Anti-CTLA 4-Ig positive and about 40 μ g/mL CTLA4-Ig (the CTLA-4 part of molecule), another kind of irrelevant Ig fusion rotein (CD40Ig) or irrelevant protein (ovalbumin) preincubation, with identify anti-CTLA 4-Ig reactivity may at molecular moiety (CTLA-4, Ig or connecting zone).Anti-CTLA-4 positive and CTLA4-Ig, CTLA-4 part or ovalbumin preincubation similarly are to confirm the reactive specificity of anti-CTLA-4.After the preincubation, all samples is analyzed in identical as mentioned above original assay method form once more.Wherein to compare with untreated sample, preincubation causes among the OD of preincubation sample 〉=and 30% sample that reduces is considered as confirming male.If be confirmed, the titration sample causes equaling the specifically serum dilution of the ratio that ends of mensuration with evaluation so, and this value reporting is EPT.
The active assessment of neutralizing antibody
Carry out that biological assay has with assessment that patient's sample at the drug specificity antibody of CTLA-4 suppresses or in and the active ability of CTLA4-Ig (suppress with CD80/86 combine), it combines realization with CD80/86 on the T cell surface by prevention for it.Be expressed in stable Jurkat T cell transfecting of the luciferase genes under the control of interleukin-(IL)-2 promotor, in the presence of anti-cd 3 antibodies, carry out common stimulation with Daudi B cell.Stimulate altogether and the anti-cd 3 antibodies combination by this of the mediation of the interaction between the CD80/86 on CD28 on the Jurkat T cell and the Daudi B cell, activate the IL-2 promotor, thereby the luciferase genes that causes increasing is transcribed, and the luciferase protein matter that therefore increases is expressed.Luminous signal uses the luciferase assay system to measure.Because CTLA4-Ig blocking-up CD80/86:CD28 interacts,, and will recover to stimulate interaction altogether with neutralizing antibody preincubation and cause increase in luminous so CTLA4-Ig added in the cell mixture this IL-2 promotor activation of blocking-up and reduce luminously.
CTLA4-Ig neutralizing antibody activity is assessed by following: the CTLA4-Ig when measuring concentration 0.1,0.25 or 0.5 μ g/mL in the presence of the seropositivity serum after the administration in 1: 25 in biological assay replys, and statistically with itself and the comparison of replying in the presence of its corresponding the 1st day sample.In biological assay, have among the CTLA4-Ig in service in each analysis as positive control with active anti-people CTLA-4 mouse monoclonal antibody (11D4).Because inherent limitation in the biological assay method of testing, can only assess sample after the administration of existing level≤1 μ g/mL of CTLA4-Ig, because higher levels of drugs disturbs neutralization to reply, and further diluted sample will reduce the mensuration susceptibility.
The pharmacokinetics assessment
The DB time period of testing from the II/III phase is confirmed that wherein the patient's of positive immunne response serum sample data carry out colony's pharmacokinetics (POPPK) and analyze.The POPPK model of checking is applied to individual patient serum-concentration data, and obtains maximum a posteriori Bayes (Bayesian) estimation of indivedual PK parameter values.About intravital medicine minimum concentration (C after these patients' clearance rate, volume estimation, stable state area under curve (AUC) value and the administration Min) distribution of value, compare with the distribution of these values in the patient's of not developing immunne response from identical test the more large data sets.
The result
The incidence that anti-CTLA 4-Ig and anti-CTLA-4 reply
2,237 patients have before the baseline and the back serum sample altogether, and are qualified for assessment.As use and measure A or B measures, in these, 62 (2.8%) individual patients have the evidence (Figure 39) that anti-CTLA 4-Ig or anti-CTLA-4 reply.There is not the patient to confirm immunne response at Fc and the CTLA-4 structural domain of CTLA4-Ig.3 patients have replying at connecting zone.When using more sensitive to measure B, in 60 (3.0%) among 1,990 patient, detect antibody response (Figure 39) at CTLA4-Ig.
Among the patient of assessment (n=1,764), 203 interrupt the CTLA4-Ig treatment in DB or OL time period process, or do not enter OL search time section subsequently in studying in the III phase, and have no progeny in treatment and collected serum in 56 and/or 85 days.In 203 patients, 15 (7.4%) has (the whole molecule at CTLA4-Ig; N=5,2.5%) or CTLA-4 (n=10,4.9%; Table 42) immune positive response.Finish the III phase DB time period and continuing to enter among all the other 1,561 RA patients of OL treatment, 40 (2.6%) has positive antibody and replys in DB or OL time period process: 33 (2.1%) at CTLA4-Ig and 7 (0.4%) at CTLA-4.What is interesting is in CTLA4-Ig studied as the IIa phase of monotherapy, do not have the CTLA-4 part seroconversion of patient for CTLA4-Ig or this molecule; Yet, use be more insensitive mensuration A form.
191 patients participate in DB and OL at it and have between time period and surpass 30 days no CTLA4-Ig time period altogether.In these, 3 (1.6%) patients have at the positive antibody of CTLA4-Ig and reply and 1 (0.5%) patient has at the positive antibody of CTLA-4 and replys (table 41) in OL time period process.Also analyzed the serum from 587 RA patients, described RA patient misses on research 1-2 agent of pharmacotherapy and any point during studying and restarts.In these patients, 15 (2.6%) confirms to reply and 7 (1.2%) have at the positive antibody of CTLA-4 and reply (table 41) at the positive antibody of CTLA4-Ig.
Table 41. has the seropositivity patient number (%) of the interruption use of CTLA4-Ig
Figure A200680053116D03941
CTLA-4=cytotoxic t lymphocyte-associated antigen-4; The DB=double blinding; The OL=open label
The methotrexate that follows is to immunogenic effect
2451 patients MTX and 493 patients of accepting to follow do not accept altogether.In a word, having patient's per-cent of replying at the positive antibody of CTLA4-Ig generally is similarly, the MTX no matter whether they accept to follow (2.3% couple 1.4%) (table 42).
Table 42. is accepted or the methotrexate of not accepting to follow has patient's number (%) that anti-CTLA 4-Ig or anti-CTLA-4 reply.
Figure A200680053116D03951
CTLA-4=cytotoxic t lymphocyte-associated antigen-4
Immunogenicity is to the security of CTLA4-Ig and the influence of effect
Assessment is about the ratio of AEs around AEs, the SAEs of all positive patients, the infusion, and do not observe the association between immunogenicity and the security.Similarly, do not notice association between immunogenicity and the effect; Yet, because patient's number of seroconversion is limited, so the explanation of these data is restricted.
The neutralization activity of anti-CTLA-4 antibody
24 parts of serum samples from 20 patients confirm for anti-CTLA-4 reactivity it is male in anti-CTLA-4 antibody screening is measured.In these, 14 duplicate samples (from 13 patients, collecting) meet in and the example values (≤1 μ g/mLCTLA4-Ig) that is used to assess in the biological assay.In this 13 duplicate samples, 1 part is that male and 10 parts are male the 85th day the time after administration the 56th day the time after administration.9 parts in 14 duplicate samples (deriving from 8 patients) show the neutralizing antibody activity.The septicemia in 1 patient, the medically significant AEs of report not during during seroconversion or near seroconversion in these patients, it is considered as relevantly with treatment among these 8 patients maybe may being correlated with.(in had no progeny 56 and 85 days) collects during the time period that efficacy data is had no progeny under study for action, and this is the time period that dominant sample number is suitable for assessing neutralizing antibody.Equally, can not assess the influence of neutralizing antibody to effect.
The pharmacokinetics assessment
Pharmacokinetic parameter is assessed for 31 among 32 patients, has positive antibody in the DB time period process that described 32 patients tested in the II/III phase and replys.The serum sample that is used for the PK analysis was not necessarily collected on the same day that positive immunne response obtains proving.From the PK of the colony modeling hint of the patient data of DB search time section, the PK parameter of predicting in 31 immune positive patients is comparable to those in the bigger patient colony (n=386) of no positive immunne response.The paddy serum-concentration on the research same day when seroconversion obtains proving in DB time period process is 1.16-24.21 μ g/mL, and wherein most of serum-concentrations are 5-20 μ g/mL.Seroconversion seems not influence serum paddy level.The volume of clearance rate and central compartment is shown among Figure 40 via the distribution of immunogenicity state.
The MSD electrochemiluminescence is measured(drug tolerance) improved in the effort of binding immunoassay originality susceptibility of measuring and the ability that detects antibody in the presence of medicine, by the immunogenicity determining of new generation that utilized Meso-Scale Discovery (MSD) technological development, with the anti-drug antibodies of monitoring at CTLA4-Ig.This new technology is used mark luminous after electrochemical stimulation initial on the electrode surface of microtest plate.Compare with the ELISA form, the MSD form has been presented at susceptibility that has improvement under the existence of medicine and the more Canon power that detects antibody.This solution phase technology allows the medicine of mark and the medicine in the serum more effectively to compete, and the surperficial capacity that has the dynamicrange bigger above the ELISA form, signal to noise ratio and increase.Different with the CTLA4 part or the whole molecule that use molecule as the present ELISAs of capture agent, new mensuration is to utilize the bridging of the CTLA4-Ig molecule of biotinylation and ruthenium mark to measure, described CTLA4-Ig molecule before the MSD plate that adds avidin bag quilt with patient's sample incubation.The electrochemiluminescence signal that is sent by the ruthenium mark uses the MSD instrument to measure.Positive based on the mensuration cut point of verifying will further be assessed in MSD measures with CTLA4-Ig, CTLA4-T or CD40Ig by immunodepletion, confirming the positive and to show which part of immunogenic response, and limit the terminal point titre at the CTLA4-Ig molecule.
Embodiment 32: the pharmacokinetic parameter in the monkey
6 female macaque/groups are used the single intravenously 10-mg/kg dosage of the CTLA4-Ig that produces by the CD-CHO1 method.The control group of 6 female monkeys is accepted salt solution (1ml/kg).In order to assess the biological activity of CTLA4-Ig, all monkeys T cell dependence antigen keyhole limpet hemocyanin (KLH) with the 10mg/ animal in preceding 30 minutes of administration carries out the intramuscular immunity.
Animal was observed for 6 weeks after treatment.Blood sample is before administration; After the administration 3 and 30 minutes the time; 1,2,4,8,24 and 48 hour the time; Obtain during with the 4th, 8,11,15,22,29,36 and 43 day, to measure and comparative drug dynamic metabolism overview.In the pharmacokinetics report, these research fates correspond respectively to the 0th, 1,2,3,7,10,14,21,28,35 and 42 day.Serum sample is analyzed with regard to CTLA4-Ig by the ELISA method.Comparable blood sample is collected from control animal when identical fate, and is used to assess anti-KLH antibody response suitably the time.To before CTLA4-Ig treatment zooscopy and the serum that obtains weekly thereafter carry out the assessment of the formation of CTLA4-Ig specific antibody.The serum sample mensuration KLH specific antibody that the preceding and immune back of immunity is derived from approximately all animals weekly forms, and experiences for 4 weeks.The other example values that is used to assess comprises survival, clinical symptom, physical examination (comprising neuroscience, respiration rate and auscultation assessment), body weight, body temperature and food consumption.
Clinical pathology is evaluated at before the research and the 45th day the time carries out.All animals are sent storage back to after research is finished.
Table 43. is by the pharmacokinetic parameter of the CTLA4-Ig of method production of the present invention.
When merely hitting at the 29th day or drug specificity antibody response (table 43 takes place thereafter with 3 in 6 monkeys of CD-CHO1 method material treatment; Figure 41).In with the monkey of CD-CHO1 method material treatment in the blood urea nitrogen (BUN) minimizing (14%) in MIN increase (20%) and the serum potassium on the physiology or nonsensical on the toxicology, because value is only slightly outside the historical control scope, and, there is not the increase of following in the serum creatinine for BUN.Do not notice other variations in the clinical pathology mathematic(al) parameter.In the monkey of using CD-CHO1 method material, observe the remarkable inhibition (〉=94% peak contrast is replied) of KLH antibody response.Immunogenicity obviously postpones, and until at the 29th day the time or thereafter the CTLA4-Ig serum level drops under the immunosuppression level of about 1 μ g/mL.
Clinical pathology.Before administration, after (the-13 days) and the last pharmacokinetics bloodletting (the 45th day), from the femoral vein of fasting animal, collect blood sample.The urine that (the 45th day) after (the-13 days) before studying and the last pharmacokinetics sampling collected through 18 hour time period carries out urinalysis.Measure the following analysis parameter: hematology: measure the assessment of morphocytology in oxyphorase, hematocrit, red blood cell count(RBC), average corpusculum volume, average corpusculum oxyphorase, average corpusculum hemoglobin concentration, reticulocyte count, summation difference white blood cell count(WBC), platelet count and the peripheral blood film.Solidify: measure prothrombin time, activated partial thromboplastin time and plasma fibrinogen.Serum chemistry: measure blood urea nitrogen, creatinine, glucose, total cholesterol, gross protein, white protein, sphaeroprotein, white protein/sphaeroprotein ratio, gpt, glutamic-oxal(o)acetic transaminase, alkaline phosphatase, total bilirubin, triglyceride level, γ glutamyl transferase, sodium, potassium, calcium, muriate and phosphorus.Urinalysis: measure the qualitative test of output, proportion, pH, color and outward appearance and glucose, protein, ketone, bilirubin, occult blood and urobilinogen.The urine settling is checked with microscope.
Specific antibody is replied.In the time of the 29th day or thereafter, but the drug specificity antibody response of comparative quantity level takes place merely hitting with 3 in 6 monkeys of CD-CHO1 method material treatment.As expection, in the time of the 29th day or thereafter, obviously postpone about the immunogenicity of method, drop to until the CTLA4-Ig serum level under the immunosuppression level of about 1 μ g/mL.Reply about the average CTLA4-Ig specific antibody of the monkey that is given CD-CHO1 method material and to become the positive the 36th day the time, and (the final time point of assessment) peaks in the time of the 43rd day.The peak antibody titers that is given in indivedual monkeys of CD-CHO1 method material is 23-10,448.
Embodiment 33: Depolymerization on the post
The collecting process of separating through the affinity chromatography resin is useful, and has for all applicabilitys based on the recombinant molecule of IgG-Fc of producing in mammalian cell.Chaotropic agent can be used to destroy high molecular weight protein or material.This depolymerization can be finished on post for any this kind protein.This embodiment provides and has been used for exemplary materials: promptly CTLA4-Ig carries out the method for separating collecting process.
By in solution (batch mode) or combine (pattern on the post) with the affinity chromatography step and use chaotropic agent, produce CTLA4-Ig high molecular (HMW) material of producing in the bio-reactor and can partly be transformed into function CTLA4-Ig dimer.This process is called " depolymerization ".Based on the analysis and characterization research of the CTLA4-Ig material of depolymerization, the material of depolymerization seems to be comparable to the control material of not implementing the depolymerization process on biological chemistry.
In the fermentation process of producing CTLA4-Ig molecule of the present invention, the resulting CTLA4-Ig protein of about 20-25% can be the form of HMW material (that is aggregation).Therefore the part of this material reclaims as the function dimer has〉10% overall process yield enhanced possibility.
Batch mode is separated collecting process
Depolymerization in batch mode (in solution) be confirmed by following, with the chaotropic agent of intermediate concentration for example Guanidinium hydrochloride or urea handle, subsequently rapid dilution in the less salt refolding damping fluid to help molecule to obtain its native conformation.
Using the CTLA4-Ig of A albumen (resin uses MAbSelect) purifying to be adjusted to～final concentration of 4-5mg/ml is with the starting material that act on these batch experiments.This contacts from the liquid damping fluid with 2x is spissated with 1: 1 volume ratio subsequently, to reach the final chaotropic agent concentration of 1-3M (for Guanidinium hydrochloride) and 2-7M (for urea).2M concentration of guanidine hydrochloride and=urea concentration of 4M is effective causing aspect the depolymerization of CTLA4-Ig HMW material.The moving phase that is used for these experiments is the phosphatebuffer buffer system in the pH of 6.5-7.0 scope.Depolymerization reaction arrives by 50mM Tris by rapid dilution (with 1: 5 volume ratio), and 25mM NaCl carries out quencher in the refolding damping fluid that pH8.5 forms.In the experiment of depolymerization in batches, the HMW material of 50-60% with 95% step yield is transformed into the CTLA4-Ig dimer.Minimizing after the depolymerization step in the observed HMW material horizontal is shown among Figure 42.
Separate collecting process on the post
In order to overcome during separating collecting process and enlarging in proportion the potential groove and to mix limitation in batches, assessment makes A albumen catch the method for step and the combination of depolymerization step.This method relate to use from liquor as being used for the elution buffer of A albumen step, subsequently the wash-out storehouse is collected in refolding/dilution buffer liquid.
Use in batches with post on process observe similar performance in the depolymerization efficient.Separate collecting process on the post and will have the distinct advantages that reduces the procedure of processing number.Be summarized in the following table about the experimental detail of using the A albumen step of depolymerization step on the post.The resin that uses: MAbSelect (from GEHealthcare); Post bed height: 20-25cm
Step Damping fluid The damping fluid volume Residence time (minute)
Balance 25mM phosphoric acid salt, 150 mM NaCl, pH7.5 3CV waits to continue until effluent pH and electroconductibility near those of level pad, or in the pH of the column volume that makes progress separately for the damping fluid that uses and electroconductibility, further do not change 5
Column load The cell culture fluid of results The 30g/l resin 5
Washing 25mM phosphoric acid salt, 150 mM NaCl, pH7.5 3CV waits to continue to get back to＜0.2AU until absorbancy 5
Wash-out 25mM phosphoric acid salt, 2.65M GdHCl, pH 6.5 (± 0.2) When absorbancy reached on the baseline 0.2AU, initial peak was collected when absorbancy is got back on the baseline 0.2AU, stops the peak collection 10
The peak dilution 50mM Tris，25mM?NaCl，?pH8.5 Elution peak is tightly collected in the dilution buffer liquid with 1: 5 volume ratio N/A
Post cleans 0.1N?NaOH ～3CV 5
The post storage 20% ethanol ～3CV 5
Mix the feasibility in the downstream processes
Carry out sample 3 column purification strings to produce the final method material that uses and do not use A albumen depolymerization step on the post.The chromatographic step yield that derives from 2 purifying strings provides in table 44.
Table 44. is about containing and do not contain the chromatography yield of 3 column methods of depolymerization
Figure A200680053116D04011
Mix the depolymerization step in sample 3 column methods and cause in the method yield about 10% improvement.Analysis is from the product storehouse of 2 kinds of method orders, to assess the biological chemistry comparability of resulting CTLA4-Ig material.
2 kinds of product storehouses are shown among Figure 43 via the N glycan analysis of MALDI-TOF.Based on this analysis, material looks like comparable.This MALDI-TOF result uses the N glycan assay method based on HPLC to confirm.HPLC analyzes between the final method material be presented at contrast and depolymerization in two feeler sialic acid peaks＜1.7% difference.
Also the deacylated tRNA amine that exists in the material of trypsinase peptide mapping with quantitative purifying and the per-cent of oxidation peptide are carried out in 2 kinds of product storehouses.Result's (being summarized in the table 45) confirms about 2 kinds of comparable deacylated tRNA amine of sample and oxidation level.
Table 45. peptide figure result
Sample T26 deacylated tRNA amine site (% area) T6 oxidation site (% area)
Contrast 3 column methods 0.76 0.35
3 column methods that contain the depolymerization step 0.69 0.33
Standard material (5g/L) 0.94 0.37
In addition, B7 is respectively 101% and 98% in conjunction with measurement result for depolymerization material on contrast and the post.
Be used to make recombinant molecule based on IgG-Fc, the method of those depolymerization of producing by mammalian cell for example, comprise the step that the composition that makes this kind molecule that comprises aggregated forms contacts with chaotropic agent (for example Guanidinium hydrochloride or urea), its amount and time be enough to make this kind gathering molecule to the small part depolymerization, optional subsequently for making the part of described molecule depolymerization contact (for example, by rapid dilution in the less salt refolding damping fluid to help this kind molecule acquisition native conformation) with refolding and/or quencher.Contact with chaotropic agent can be for example with in batches, semi-batch and continuous mode carry out, and for example in solution (for example in batch mode) or for example combine during the affinity chromatography step (pattern on the post) with chromatographic step and carry out.
Make purifying on the post for example the A albumen method of catching step and the combination of above-mentioned depolymerization can strengthen group method efficient, and be another embodiment of the invention.Therefore, the present invention has expected and has been used to make recombinant molecule based on IgG-Fc, the method of those depolymerization of producing by mammalian cell for example, comprise the step that the composition that makes this kind molecule that comprises aggregated forms contacts with chaotropic agent (for example Guanidinium hydrochloride or urea), its amount and time be enough to make described gathering molecule to the small part depolymerization, wherein said contact takes place on chromatography column, for example wherein said chaotropic agent uses at the solution that is used for the described post of wash-out (elution buffer that for example is used for A albumen post), optional subsequently for making the part of described molecule depolymerization contact (for example, in less salt refolding damping fluid, obtaining native conformation) with refolding and/or quencher with the help molecule by rapid dilution.
The composition for the treatment of to plant therewith the chaotropic agent contact can comprise the recombinant molecule based on IgG-Fc of the form (for example single stranded form or dimer) except that aggregated forms, adds the described molecule that comprises aggregated forms.
Exemplary molecule based on IgG-Fc can comprise glycoprotein CTLA4-Ig molecule for example of the present invention.
Embodiment 34: pharmacokinetics
2B phase, multicenter, randomization, double blinding, placebo-controlled study are used to assess security and the clinical efficacy of CTLA4-Ig that intravenously is applied to 2 kinds of various dose of experimenter, described experimenter's tool suffers from active rheumatoid arthritis and accepts methotrexate simultaneously): in this research, the experimenter accepts CTLA4-Ig or the placebo with 2 kinds of various dose of MTX combination (2 and 10mg/kg).CTLA4-Ig the method according to this invention is produced, and supplies in comprising indivedual bottles of 200mgCTLA4-Ig.CTLA4-Ig in the time of the 1st, 15 and 30 day and per thereafter 30 days IV be applied to the experimenter, experience 1 year.The serum-concentration that multi-agent PK obtained from the experimenter who participates in locus specificity PK and study derived from the administration time interim between the 60th and 90 day is to time data.For the experimenter in the PK research, blood sample is collected at following time point: the PK overview that begins during before the administration in the 60th day with for the 60th day (finished corresponding to the CTLA4-Ig infusion) in the time of 30 minutes, when infusion begins back 4 hours and thereafter once in a week until the 90th day.90 experimenters register and participate in PK research altogether.Yet the complete PK overview the from the 60th to 90 day the administration time interval derives from 29 experimenters, and (15 experimenters are with the 2mg/kg administration; 14 experimenters are with the 10mg/kg administration).
The summary of PK parameter is presented in the table 46.Result from this research shows that Cmax and AUC (TAU) are to increase wherein TAU=30 days with the proportional mode of dosage.For the nominal standard dose that increases with the ratio of 1:5, the geometrical mean of Cmax increases with 1: 5.2 ratio, and increases for the geometrical mean of AUC (TAU) ratio with 1: 5.0.In addition, T-HALF, CLT and Vss value seem not rely on dosage.In these RA experimenters, average T-HALF, CLT and Vss value be respectively about 13 days ,～0.2mL/h/kg and～0.07L/kg.Little Vss points out that the CTLA4-Ig major limitation is in extracellular fluid volume.During based on 2 and 4 weeks behind first time infusion, the dosage regimen of 1 administration in every month thereafter subsequently reaches when the 3rd every month single administration about the steady state conditions of CTLA4-Ig.Equally, as the result of dosage regimen, serum-concentration surpasses stable state paddy concentration during preceding 2 months of treatment.Paddy (Cmin) value pointed out relatively that CTLA4-Ig seemed not accumulate behind the single administration at every month in the time of the 60th, 90 and 180 day.Average Cmin steady-state value about all experimenters of 2 and the 10mg/kg CTLA4-Ig that accept every month IV dosage is respectively 4.4-6.7 μ g/mL and 22.0-28.7 μ g/mL.
The summary of table 46. multiple PK research in the rheumatoid arthritis experimenter
In RA patient, repeatedly behind the intravenous infusion, the pharmacokinetics of CTLA4-Ig shows C MaxWith the proportional increase of AUC in the dosage range of 2mg/kg to 10mg/kg.When 10mg/kg, serum-concentration reaches stable state when seeming by the 60th day, and its average (scope) paddy concentration is 24 (1-66) mcg/mL.The general accumulation of CTLA4-Ig does not take place after the treatment with 10mg/kg continuously repeatedly with timed interval of every month in RA patient.
Colony's pharmacokinetics analysis among the RA patient discloses and has the trend of cumulative body weight towards the higher clearance rate of CTLA4-Ig of following.Age and sex (when carrying out timing for body weight) do not influence clearance rate.The methotrexate that follows (MTX), non-steroidal anti-inflammatory drug (NSAIDs), reflunomide and TNF blocker do not influence the CTLA4-Ig clearance rate.
Embodiment 35: the mol ratio of measuring seminose, Fucose and semi-lactosi via CE
Capillary electrophoresis has been developed and has been used for LEA2CTLA4 A29YL104EThe quantitative analysis of the neutral contents of monosaccharides the among-Ig.Neutral monose comprise seminose, Fucose and semi-lactosi by the acidic hydrolysis under hot conditions (2M trifluoroacetic acid, in 95 ℃ 6 hours) from CTLA4 A29YL104EObtain in-Ig the sample discharging.The neutral monose that discharges is subsequently as the acetate of catalyzer with as the NaBH of reductive agent 3The existence of CN is carried out fluorescent mark (67mMAPTS, 330mM HAc, 83mM NaBH with amino pyrene trisulfonic acid (APTS) down 3CN, in 55 ℃ 3 hours).Add wood sugar in every kind of sample and serve as internal standard.The peak area of every kind of neutral monose is used for quantitative at the sort of ratio of internal standard.
Reagent: hydrating solution (2M trifluoroacetic acid (TFA)); Derivatize solution I (0.1M8-amino-1,3,6 trisulfonic acids, trisodium salt (APTS) aqueous solution); The derivatize solution II (is dissolved in the 0.25M NaBH of 1M acetate 3CN); Running buffer (60 ± 5mM sodium tetraborate, pH9.25); Capillary douche solution (1N NaOH; 1N HCl; 80% methyl alcohol); Concentration is the monose standard stock solution of seminose (Man), Fucose (Fuc), semi-lactosi (Gal) and the wood sugar (Xyl) of 10mg/ml; Monose working solution I: the internal standard working solution is 100 times of dilutions of Xyl stock solution; Monose working solution II: neutral hybrid standard working solution, 100 times of dilutions of Man, Fuc and Gal stock solution.
Instrument configuration: the CE system is a Beckman P/ACE MDQ CE system; Detector: with P/ACE MDQ) (LIF) detection system of link coupled Beckman induced with laser; Uncoated kapillary (i.d.25 μ m, o.d.360 μ m) 27-31cm length overall is to hold P/ACE MDQ.
The capillary electrophoresis operational conditions: running buffer (the 60mM sodium tetraborate, pH9.25); Capillary column body temperature degree: 25 ℃; Voltage: 25-30kV, holotype; Testing conditions: the LIF detector, excite at the 488nm place, launch at the 520m place; Sample injection: pressure injection pattern, 20s when 0.5PSI; Working time: 10 minutes; Sample storage: 10 ℃.
Hydrolysis: 10 μ L wood sugar working solutions and 200 μ L 2M TFA are mixed, with the preparation system blank.The neutral mixed standard solution of 10 μ L wood sugar working solutions and 10 μ L is mixed, with preparation monose standard with 200 μ L 2M TFA.Make 10 μ L wood sugar working solutions and 10 μ L sample (for example, CTLA4 A29YL104E-Ig, about 1mg/ml) mix with 200 μ L 2M TFA, with the preparation specimen.With all pipe vortexs 10 seconds, and centrifugal 10 seconds, subsequently in 95 ℃ of incubations 6 hours.Behind the hydrolysing step, sample is placed-20 ℃ 10 minutes with the cooling.Make sample be rotated down 10 seconds and in SpeedVac, be evaporated to drying.
Derivatize: with 10 μ L derivatize solution I reconstruct samples.Make the of short duration mixing of sample, and add 5 μ L derivatize solution II.Sample is loaded in the whizzer of heating in advance and simultaneously centrifugal under 2000rpm in 55 ℃ of incubations 3 hours.
CE injection: make by adding HPLC grade water that the final volume of sample reaches 100 μ L behind the derivatize, and with 10 μ L sample transfer to CE trace bottle with 190 μ LHPLC grade water.Before the sample injection, the CE cylinder washes (1-3 minute working time) widely with HPLC grade water, subsequently with running buffer balance flushing (5 minute working time).After the initial flush, monose standard that will be used for analyzing and sample are expelled to CE cylinder (15 minute working time).After the injection operation of every kind of standard or specimen, the CE cylinder is with HPLC grade water and running buffer washes and balance (table 51).The electrophorogram of system's suitability should be similar to Figure 45, and wherein peak 1 is a seminose; Peak 2 is wood sugars; Peak 3 is Fucoses; And peak 4 is semi-lactosis.
Table 51. instrumental method
Time Incident Value Time length Summarize Describe
Flushing-pressure 40.0 psi? 3.00 minute Forward The water flushing
Flushing-pressure 40.0 psi? 5.00 minute Forward The running buffer flushing
Injection-pressure 0.5psi 20.00 second Override (override), forward Injection
0.00 minute Separation-voltage 30kV 15.00 minute 0.17 minute oblique ascension, normal polarity Separate
0.05 minute Auto zero
15.00 minute Stop data
15.00 minute Finish
System's suitability:
The electrophorogram of system's suitability should be similar to show among Figure 45 the sort of, and wherein peak 1 is a seminose; Peak 2 is wood sugars; Peak 3 is Fucoses; And peak 4 is semi-lactosis.
When the CE instrument that uses except that Beckman MDQ system, length capillaceous can be different from this method specified the sort of.This can cause the variation in analyte transition time and the peak intensity.But the peak pattern of monose analyte should keep identical.
Can calculate 2 peak-to-peak resolving power of vicinity according to equation about first kind of system's suitability criterion:
R＝2(t 2-t 1)/(W 1+W 2)
Wherein,
R: resolving power
t 2, t 1: 2 adjacent peaks are divided other transition time
W 1, W 2: 2 adjacent peaks are respectively in the peak width at baseline place
The R value is necessary 〉=and 1.0.If R＜1.0 are so with wash/rinse order flushing kapillary.If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.
For the suitability injection of last system, use the last peak (semi-lactosi) of following formula must have＜1.4 tailing factor:
T＝W 0.05/2f
Wherein: T: tailing factor
W 0.05: the peak width when 5% height
F: the width of front, peak when peak maximum
If T 〉=1.4 are so with wash/rinse order flushing kapillary; If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.The peak area ratio of semi-lactosi and wood sugar must have≤10% RSD.The transition time of semi-lactosi needs≤15.0 minutes.The electrophorogram overview should be equivalent to Figure 45.
Can measure the monose standard percentage than RSD by the peak area ratio of internal standard and monose standard ingredient relatively, it is via realizing divided by the peak area about the internal standard of each monose standard injection about the peak area of each monosaccharide component.Per-cent RSD can calculate for seminose, Fucose and semi-lactosi.RSD answers≤10%.
The mensuration of neutral monose and proteinic mol ratio
Neutral monose (for example, Man, Gal and Fuc) can calculate according to formula provided below with respect to the peak area ratio of internal standard wood sugar, to measure every kind of neutral monose and proteinic mol ratio.For example, peak area ratio equals monose peak area (Gal, Fuc or Man) divided by the wood sugar peak area, and wherein the coefficient of variation (RSD) about peak area ratio is equal to or less than 10%.Equation can be used to calculate following:
For seminose/proteinic mol ratio:
R man = A man &times; A xyl 0 &times; V man 0 &times; C man 0 &times; MW LEA 29 Y A xyl &times; A man 0 &times; V p &times; C p &times; 180.2
Wherein,
R Man: seminose is to proteinic mol ratio
A Man: the peak area of seminose in the sample (μ V second)
A Xyl: the peak area of wood sugar in the sample (μ V second)
A Xyl0: the peak area of wood sugar in the monose standard (μ V second) mean value
A Man0: the peak area of seminose in the monose standard (μ V second) mean value
V Man0: be used for the seminose volume (representing) that the monose working solution of hydrolysis comprises with μ L
C Man0: be used for the mannose concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
V p: the volume (representing) that is used for the protein example of hydrolysis with μ L
C p: the concentration (representing) that is used for the protein example of hydrolysis with mg/mL
MW LEA29Y: LEA29Y (or CTLA4 A29YL104E-Ig) molecular weight (91,232Da)
The MW:180.2 dalton of seminose
For Fucose/proteinic mol ratio:
R fiic = A fiic &times; A xyl 0 &times; V fiic 0 &times; C fiic 0 &times; MW LEA 29 Y A xyl &times; A fiic 0 &times; V p &times; C p &times; 164.2
Wherein,
R Fc: Fucose is to proteinic mol ratio
A Fuc: the peak area of Fucose in the sample (μ V second)
A Xyl: the peak area of wood sugar in the sample (μ V second)
A Xyl0: the peak area of wood sugar in the monose standard (μ V second) mean value
A Fuc0: the peak area mean value of Fucose in the monose standard (μ V second)
V Fuc0: be used for the Fucose volume (representing) that the monose working solution of hydrolysis comprises with μ L
C Fuc0: be used for the Fucose concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
V p: the volume (representing) that is used for the protein example of hydrolysis with μ L
C p: the concentration (representing) that is used for the protein example of hydrolysis with mg/mL
MW LEA29Y: LEA29Y (or CTLA4 A29YL104E-Ig) molecular weight (91,232Da)
The MW:164.2 dalton of Fucose
For semi-lactosi/proteinic mol ratio:
R gal = A gal &times; A xyl 0 &times; V gal 0 &times; C gal 0 &times; MW LEA 29 Y A xyl &times; A gal 0 &times; V p &times; C p &times; 180.2
Wherein,
R Gal: semi-lactosi is to proteinic mol ratio
A Gal: the peak area of semi-lactosi in the sample (μ V second)
A Xyl: the peak area of wood sugar in the sample (μ V second)
A Xyl0: the peak area of wood sugar in the monose standard (μ V second) mean value
A Gal0: the peak area of semi-lactosi in the monose standard (μ V second) mean value
V Gal0: be used for the semi-lactosi volume (representing) that the monose working solution of hydrolysis comprises with μ L
C Gal0: be used for the galactose concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
V p: the volume (representing) that is used for the protein example of hydrolysis with μ L
C p: the concentration (representing) that is used for the protein example of hydrolysis with mg/mL
MW LEA29Y: LEA29Y (or CTLA4 A29YL104E-Ig) molecular weight (91,232Da)
MW semi-lactosi: 180.2 dalton
Table 52: monose and CTLA4 A29YL104EThe proteinic molar average ratio of-Ig
Monose Scope
Seminose 11-23
Fucose 4.2-7.5
Semi-lactosi 9.2-18
Embodiment 36: the mol ratio of measuring GalNAc and GlcNAc via CE
Capillary electrophoresis has been developed and has been used for CTLA4 A29YL104EThe quantitative analysis of the amino contents of monosaccharides the among-Ig, described CTLA4 A29YL104E-Ig is the glycoprotein with 6 N linked glycosylation sites and at least 1 O linked glycosylation site.Amino monose comprises that N-acetylgalactosamine (GalNAc) and N-acetyl-glucosamine (GlcNAc) pass through acidic hydrolysis (4N HCl, in 95 ℃ 6 hours) under hot conditions from CTLA4 A29YL104EObtain in-Ig the sample discharging.The amino monose that discharges is by incubation experiences acetylize step again half an hour on ice with diacetyl oxide.They are subsequently as the acetate of catalyzer with as the NaBH of reductive agent 3The existence of CN is carried out fluorescent mark (67mM APTS, 330mM HAc, 83mM NaBH with amino pyrene trisulfonic acid (APTS) down 3CN, in 55 ℃ 3 hours).Add the N-acetylmannosamine in every kind of sample and serve as internal standard.The peak area of every kind of amino monose is used for quantitative at the sort of ratio of internal standard.
Reagent: hydrating solution (4N HCl); Derivatize solution I (0.1M8- amino 1,3,6 trisulfonic acids, trisodium salt (APTS) aqueous solution); The derivatize solution II (is dissolved in the 0.25MNaBH of 1M acetate 3CN); Again the acetylize damping fluid (the 25mM sodium bicarbonate, pH9.5); Running buffer (60 ± 5mM sodium tetraborate, pH9.25); Capillary douche solution (1N NaOH; 1NHCl; 80% methyl alcohol); Concentration is GalNAc, the GlcNAc of 10mg/ml and the monose standard stock solution of ManNAc; Monose working solution I: the internal standard working solution is 100 times of dilutions of ManNAc stock solution; Monose working solution II: amino hybrid standard working solution, 100 times of dilutions of GalNAc and GlcNAc stock solution.
Instrument configuration: the CE system is a Beckman P/ACE MDQ CE system; Detector: with P/ACE MDQ) (LIF) detection system of link coupled Beckman induced with laser.
The capillary electrophoresis operational conditions: running buffer (the 60mM sodium tetraborate, pH9.25); Capillary column body temperature degree: 25 ℃; Voltage: 25-30kV, holotype; Testing conditions: the LIF detector, excite at the 488nm place, launch at the 520m place; Sample injection: pressure injection pattern, 20s when 0.5PSI; Working time: 10 minutes; Sample storage: 10 ℃.
Hydrolysis: 10 μ L ManNAc working solutions and 200 μ L 4N HCl are mixed, with the preparation system blank.The amino mixed standard solution of 10 μ L ManNAc working solutions and 10 μ L is mixed, with preparation monose standard with 200 μ L4N HCl.Make 10 μ L ManNAc working solutions and 10 μ L sample (for example, CTLA4 A29YL104E-Ig sample etc.; About 1mg/ml) mixes with 200 μ L 4N HCl, with the preparation specimen.With all pipe vortexs 10 seconds, and centrifugal 10 seconds, subsequently in 95 ℃ of incubations 6 hours.Behind the hydrolysing step, sample is placed-20 ℃ 10 minutes with the cooling.Make sample be rotated down 10 seconds and in SpeedVac, be evaporated to drying.
Acetylize again:, mix subsequently and at incubation (30 minutes) on ice with 20 μ L acetylize damping fluid and 4 reconstruct hydrolysis of μ L diacetyl oxide and exsiccant samples again.Make sample be rotated down 10 seconds and in SpeedVac, be evaporated to drying.Each personal 100 μ L HPLC grade water reconstruct of sample and in SpeedVac, be evaporated to drying.
Derivatize: the sample (10 μ L derivatize solution I HPLC) to reconstruct provides 5 μ L derivatize solution II.After the mixing, sample is loaded in the whizzer of heating in advance and in 55 ℃ of incubations 3 hours, simultaneously centrifugal under 2000rpm.
CE injection: make by adding HPLC grade water that the final volume of sample reaches 100 μ L behind the derivatize, and with 10 μ L sample transfer to CE trace bottle with 190 μ LHPLC grade water.Before the sample injection, the CE cylinder washes (1-3 minute working time) widely with HPLC grade water, subsequently with running buffer balance flushing (5 minute working time).After the initial flush, monose standard that will be used for analyzing and sample are expelled to CE cylinder (10 minute working time).After the injection operation of every kind of standard or specimen, the CE cylinder is with HPLC grade water and running buffer washes and balance.The electrophorogram of system's suitability should be similar to Figure 46, and wherein peak 1 is GalNAc; Peak 2 is ManNAc; And peak 3 is GlcNAc.
Table 53. instrumental method
Time Incident Value Time length Summarize Describe
Flushing-pressure 40.0 psi? 3.00 minute Forward The water flushing
Flushing-pressure 40.0 psi? 5.00 minute Forward The running buffer flushing
Injection-pressure 0.5psi 20.00 second Override, forward Injection
0.00 minute Separation-voltage 30kV 10.00 minute 0.17 minute oblique ascension, normal polarity Separate
0.05 minute Auto zero
10.00 minute Stop data
10.00 minute Finish
System's suitability:
The electrophorogram of system's suitability should be similar to show among Figure 46 the sort of, and wherein peak 1 is GalNAc; Peak 2 is ManNAc; And peak 3 is GlcNAc.
When the CE instrument that uses except that Beckman MDQ system, length capillaceous can be different from this method specified the sort of.This can cause the variation in analyte transition time and the peak intensity.But the peak pattern of monose analyte should keep identical.
Can calculate 2 peak-to-peak resolving power of vicinity according to equation about first kind of system's suitability criterion:
R＝2(t 2-t 1)/(W 1+W 2)
Wherein,
R: resolving power
t 2, t 1: 2 adjacent peaks are divided other transition time
W 1, W 2: 2 adjacent peaks are respectively in the peak width at baseline place
The R value is necessary 〉=and 1.0.If R＜1.0 are so with wash/rinse order flushing kapillary.If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.
For the suitability injection of last system, use the last peak (GlcNAc) of following formula must have＜1.4 tailing factor:
T＝W 0.05/2f
Wherein: T: tailing factor
W 0.05: the peak width when 5% height
F: the width of front, peak when peak maximum
If T 〉=1.4 are so with wash/rinse order flushing kapillary; If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.The peak area ratio of GlcNAc and ManNAc must have≤10% RSD.The transition time of GlcNAc is necessary≤and 10.0 minutes.The electrophorogram overview should be equivalent to Figure 46.
Can measure the monose standard percentage than RSD by the peak area ratio of internal standard and monose standard ingredient relatively, via realizing divided by peak area about the peak area of each monosaccharide component about the internal standard of each monose standard injection.Per-cent RSD can calculate for GalNAc and GlcNAc.RSD answers≤10%.
The mensuration of amino monose and proteinic mol ratio
Amino monose (for example, GalNAc and GlcNAc) can calculate according to formula provided below with respect to the peak area ratio of internal standard ManNAc, to measure every kind of amino monose and proteinic mol ratio.For example, peak area ratio equals monose peak area (GalNAc and GlcNAc) divided by the ManNAc peak area, and wherein the coefficient of variation (RSD) about peak area ratio is equal to or less than 10%.Equation can be used to calculate following:
For the proteinic mol ratio of GalNAc/:
R GalNAc = A GalNAc &times; A ManNAc 0 &times; V GalNAc 0 &times; C GalNac 0 &times; MW LEA 29 Y A ManNAc &times; A GalNAc 0 &times; V p &times; C p &times; 221.2
Wherein,
R GalNAc: GalNAc is to proteinic mol ratio
A GalNAc: the peak area of GalNAc in the sample (μ V second)
A ManNAc: the peak area of ManNAc in the sample (μ V second)
A ManNAcO: the peak area of ManNAc in the monose standard (μ V second) mean value
A GalNAcO: the peak area of GalNAc in the monose standard (μ V second) mean value
V GalNAc0: be used for the GalNAc volume (representing) that the monose working solution of hydrolysis comprises with μ L
C GalNAcO: be used for the GalNAc concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
V p: the volume (representing) that is used for the protein example of hydrolysis with μ L
C p: the concentration (representing) that is used for the protein example of hydrolysis with mg/mL
MW LEA29Y: LEA29Y (or CTLA4 A29YL104E-Ig) molecular weight (91,232Da)
MW GalNAc:221.2 dalton
For the proteinic mol ratio of GlcNAc/:
R GlcNAc = A GlcNAc &times; A ManNAc 0 &times; V GlcNAc 0 &times; C GlcNac 0 &times; MW LEA 29 Y A ManNAc &times; A GlcNAc 0 &times; V p &times; C p &times; 221.2
Wherein,
R GlcNAc: GlcNAc is to proteinic mol ratio
A GlcNAc: the peak area of GlcNAc in the sample (μ V second)
A ManNAc: the peak area of ManNAc in the sample (μ V second)
A ManNAcO: the peak area of ManNAc in the monose standard (μ V second) mean value
A GlcNAc0: the peak area of GlcNAc in the monose standard (μ V second) mean value
V GlcNAc0: be used for the GlcNAc volume (representing) that the monose working solution of hydrolysis comprises with μ L
C GlcNAc0: be used for the GlcNAc concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
V p: the volume (representing) that is used for the protein example of hydrolysis with μ L
C p: the concentration (representing) that is used for the protein example of hydrolysis with mg/mL
MW LEA29Y: LEA29Y (or CTLA4 A29YL104E-Ig) molecular weight (91,232Da)
MW GlcNAc:221.2 dalton
Table 54: mole monose and mole CTLA4 A29YL104EThe proteinic molar average ratio of-Ig.
Monose Scope
GalNAc 2.0-3.2
GlcNAc 18-32
Embodiment 37:CTLA4 A29YL104E -Ig is via the N connection oligosaccharides of high-efficiency anion displacement chromatography The carbohydrate profile analysis
The carbohydrate structure that exists on the glycoprotein can influence clearance rate in its function and the body.Therefore the structural integrity of carbohydrate of monitoring the recombinant production batch of glycoprotein is important.Herein, monitoring CTLA4 A29YL104EN connection (l-asparagine connects) carbohydrate of the last existence of-Ig.In this method, oligosaccharides is by (peptide: N-Glycosylase F) enzymatic digestion is cut, and (HPAEC) separates by the high-efficiency anion displacement chromatography, and monitors by Electrochemical Detection (amperometry of integration) with PNGase F.The chromatogram that produces is N connection carbohydrate overview, wherein CTLA4 A29YL104EThe overview of-Ig sample should be similar to this kind.
The reagent that is used to separate the moving phase of oligosaccharides by anti-phase and graphite carbon HPLC: eluent 1 (being dissolved in 0.05% trifluoroacetic acid (TFA) of HPLC grade water); Eluent 2 (be dissolved in 60% acetonitrile (ACN), 40%HPLC grade water (60: 40, ACN: H 2O) 0.05%TFA; Eluent 3: be dissolved in 40% acetonitrile, 40% Virahol (IPA), 20%HPLC grade water (40: 40: 20, ACN:IPA:H 2O) 0.05%TFA).
Be used to prepare the reagent of the moving phase that is used for HPAEC oligosaccharides carbohydrate profile analysis: eluent 1:500mM sodium acetate (NaOAc); Eluent 2:400mM sodium hydroxide (NaOH); Milli-Q water; 4M sodium hydroxide (about 4M NaOH); The 50mM sodium phosphate buffer, 0.02% sodiumazide, pH=7.5; Be dissolved in the 50mM sodium phosphate buffer, 0.02% sodiumazide, the PNGase F enzyme working solution of pH=7.5; Stachyose stock solution (1.25mg/mL); Stachyose system suitability criterion (12.5 μ g/mL).
Instrument configuration and condition-(instrument configuration of equal value can replace.)
Instrument configuration:
Alliance HPLC system is equipped with Waters Corporation
Scale temperature control automatic sampler
(37 ℃), Rheodyne switches
Valvegear and UV detector
Synergi6 column selector Phenomenex, (catalog number (Cat.No.)
AV0-6080)
Post 1:Luna5 μ, C18 (2) 4.6 Phenomenex, (catalog number (Cat.No.)
x150mm 00F-4252-E0)
Post 2:HyperCarb 5 μ 4.6mm Phenomenex, (catalog number (Cat.No.)
x100mm CH0-3301)
The Dionex Corporation of Dionex DX-500HPLC system
Comprise:
The GP50 gradient pump
The AS50 automatic sampler
(refrigerated)
Eluent degassing module
Liquid chromatography (LC)
Module
The ED40 detector
DX-LAN
On suitable computer system
PeakNet software (version 5.1 or
Upgraded version)
Post: CarboPac PA-1 Dionex Corporation, (order
4x250mm record numbers 35391)
Guard column: CarboPac PA-14x Dionex Corporation, (order
50mm record numbers 43096
Millennium data gathering system version 3 .2
Specimen preparation: add in 1.7mL Eppendorf pipe and comprise 0.02% sodiumazide (NaAzide), the 80 μ L 50mM sodium phosphate buffers of pH7 and 80 μ L samples are (for 2mg CTLA4 altogether A29YL104E-Ig etc.～25 μ g/ μ L), be 16 μ L10X sex change damping fluids (5% SDS, 10% beta-mercaptoethanol) subsequently.Make sample thorough mixing and subsequent boiling 2 minutes so that protein denaturation.Make bottle be cooled to room temperature, add 16 μ L NP40 (10%) and 40 μ L PNGase F working solutions and mixing subsequently subsequently.Sample, is transferred to them in the HPLC automatic sampler bottle after 24 ± 2 hours in 37 ℃ of incubations, prepares injection on the HPLC instrument.
Be used for the isolating chromatography condition of oligosaccharides:
Column temperature environment (22-25 ℃)
The first arrival of flow velocity program 30.0 minutes 1.0mL/ minute
30.0-35.0 minute, 1.0-2.0 "
35.0-40.0 minute, 2.0-1.0 "
40.0-50.0 minute, 1.0 "
50.0-60.0 minute, 1.0-0.1 "
Moving phase and gradient condition gradient program
The 1:0.05%TFA time
2: be dissolved in ACN/H 2O (60:40) (divide %1 %2 %3
0.05%TFA Clock)
3: be dissolved in ACN/IPA/H 2O
(40/40/20) 0.05%TFA
Initial 100 00
15.00 80 20 0
15.01 0 100 0
25.00 0 100 0
30.00 0 0 100
35.00 0 0 100
40.00 100 0 0
50.00 100 0 0
60.01 100 0 0
37 ℃ of automatic sampling actuator temperatures
Volume injected 100 μ L
50 minutes working times
Data collection time 50 minutes
About 6 post switching valves and Waters time-event function
Alliance Rheodyne device (minute)
The post handover event
Initial 1 pass of switching
Initial switching 2 is opened
Initial switching 3 is opened
Initial 4 passes of switching
11.0 switching 4 opens
30.0 switching 4 closes
30.0 switching 1 opens
30.0 switching 2 closes
40.0 switching 2 opens
40.0 switching 1 closes
The chromatography condition that is used for oligomer (Oligo) overview via anion-exchange chromatography
Column temperature environment (22-25 ℃)
Flow velocity 1mL/ minute
Moving phase and gradient condition gradient program
*Point out the degree that gradient can be revised about second value of some gradient steps hereinafter, with the retention time of Adjustment System suitability criterion
The 1:500mM NaOAc time
2:400mM NaOH (minute) %1 %2 %3
3:Milli-Q water
Initial 0 50-35 50-65
0.0 0 50-35 50-65
1.0 0 50-35 50-65
2.0 4 50-40 46-56
60.0 45 50 5
61.0 0 50 50
80.0 0 50 50
The ED40 detector is provided with
Mode integrated amperometry
The current potential of using Time Pot. Integ.
0.00 +0.05
0.20+0.05 beginning
0.40+0.05 finishes
0.41 +0.75
0.60 +0.75
0.61 -0.15
1.00 -0.15
Scope 200nC
Simulation output is provided with output=skew
Zero-bit=5% full scale
Volt full scale=1.0v
Rise time=1 second
Polarity=+
4 ℃ of automatic sampler temperature
Injection progradation 30 μ L
80 minutes working times
According to system's suitability (SS) standard
The approximate retention time (RT of RT;
Minute) approximate retention time (minute)
SS： 9.6
Peak 1A:10.5
Peak 1B:11.5
Peak 1C:12.5
Peak 1D:13.5
Peak 1E:15.0
Peak 2:24.5
Peak 3:37.5
CTLA4 A29YL104E-Ig sample can have the carbohydrate overview of describing in the chromatogram of Figure 47.As identifying among Figure 47, the retention time of each structural domain should be similar to:
Structural domain I (peak 1A, 1B, 1C, 1D and 1E) 10-17 minute
Domain II: 21-29 minute
Domain II I:33-43 minute
Structural domain IV:48-56 minute
Retention time is that system is dependent and should move similarly with stachyose.
Calculate
Theoretical tray (N): theoretical plate number (N) can use following formula to measure based on the stachyose peak.This finishes by the Millennium data analysis system or can also manual finish.
N＝16(t/W) 2
Wherein:
T: the retention time of when maximum height, measuring from when injection to the peak elution time
W: the peak width that is extrapolated to baseline by the side.
N is necessary 〉=and 6000.If the plate counting should be replaced post so less than 6000.
Tailing factor (T): post tailing factor (T) can use following formula to calculate based on the stachyose peak.This finishes by the Millennium data analysis system or can also manual finish.
T＝(W 0.05/2f)
Wherein:
W 0.05: the peak width during 5% height (0.05h).
F: during maximum height from W 0.05The time the peak front edge measure (width) to the peak intermediary.
T is necessary≤and 1.2.Form if tailing factor, should be assessed damping fluid so greater than 1.2, should replace or clean post.
% structural domain area:
Structural domain I: summation (the peak 1A-1E of the peak area in the time of about retention time 10-15 minute; For example, Figure 47)
Domain II: from the summation at 21-27 minute peak
Domain II I: from the summation at 34-40 minute peak
Structural domain IV: for the peak area at the peak 45-56 minute the time
Figure A200680053116D04201
Per-cent main peak area: can use equation to calculate: peak 1A, 1B, 1C, 2 (2 undivided kinds), 3 (2 undivided kinds) and 4 for following about 5 main peaks % peak area separately.
Figure A200680053116D04202
Embodiment 38: CTLA4 in medicine and the medicament production A29YL104E The capillary electrophoresis mirror of-Ig Fixed
CTLA4 A29YL104E-Ig is the water-soluble sugar albumen with immunosuppressive activity.Use the capillary electrophoresis of uncoated fused-silica capillary tubes to be used to identify CTLA4 A29YL104E-Ig.Sample (for example, CTLA4 A29YL104E-Ig etc.), and detect by the UV that is set in the 241nm place subsequently and analyze immediately to confirm evaluation in 70 ℃ of heating 5 minutes.
In addition, with CTLA4 A29YL104E1: 1 mixture of-Ig and CTLA4-Ig material mixes and injects, and can separate and be distinguished to confirm 2 kinds of protein.This method is by comparing CTLA4 A29YL104EThe transition time of-Ig material and sample can be distinguished CTLA4 A29YL104E-Ig and CTLA4-Ig.
Equipment (equivalent device can replace):
Capillary electrophoresis instrument Beckman P/ACE MDQ
Detector module UV is at the 214nm place
Kapillary Polymicro Technologies Inc., 360 μ m o.d./75 μ m i.d., no coating, (part numbers 2000019, TSP075375)
Capillary window of tube producer MicroSolve Technology, (catalog number (Cat.No.) 07200-S)
Reagent and solution: running buffer (14.4mM Sodium Tetraborate; 17.3mM sodium lauryl sulphate; 2.0% acetonitrile); Phosphoric acid salt dilution buffer liquid (22.3mM sodium orthophosphate dimetallic; 4.2mM sodium orthophosphate (monometallic); 53.4mM sodium-chlor); Borate dilution buffer liquid (83.5mM NaBO 2, pH9.6); Reference or sample working solution (being dissolved in 10 ± 1mg/mL sample of phosphoric acid salt dilution buffer liquid); Reference or sample injection solution (10.0 μ L samples+50 μ L borate dilution buffer liquid); Sample 1:1CTLA4-Ig-CTLA4 A29YL104E-Ig mixing solutions (10.0 μ L CTLA4-Ig sample+10.0 μ LCTLA4 A29YL104E-Ig sample+50 μ L borate dilution buffer liquid); Injection solution.
Operational conditions:
UV detector wavelength 214nm
Data rate 4Hz
Peak width (point) 16-25
Inject 10 seconds (0.5psi)
Length overall *～60cm
The kapillary useful length *～50cm
15 ℃ of capillary column body temperature degree
Voltage 19kV-23kV
15 minutes working times
*Length overall: from the capillary inlet to the outlet
*Useful length: from the capillary inlet to the detection window
The CTLA4-Ig sample transition time of main peak is about 11.0 ± 0.4 minutes.The CTLA4 of main peak A29YL104E-Ig sample transition time is about 12.0 ± 0.4 minutes.Main peak transition time between every kind of sample should be separated by at least 0.6 minute (Figure 48).
Embodiment 39: about CTLA4 A29YL104E The N-n acetylneuraminic acid n of-Ig and N-hydroxyl acetyl The hydrolysis of neuraminic acid assay and HPLC analyze
Degree sialylated in the recombinant protein can influence pharmacokinetics and clearance rate.CTLA4 A29YL104E-Ig is the recombinant protein with N and O connection carbohydrate site.The glycan that occupies these sites has the sialylated of variable pitch.Except its sialylated structure heterogeneity, indivedual sialic content can be criticized variation from criticizing to.Therefore obtain the overall measurement of sialic acid and proteinic ratio.
Check CTLA4 A29YL104EN-n acetylneuraminic acid n (NANA) that-Ig presents and N-hydroxyacetylneuraminic acid (NGNA) content.Sialic acid obtains discharging and carrying out fluorescent mark with DMB subsequently from protein by acid hydrolysis.The sialic acid of mark separates on RP-HPLC C-18 post, and is undertaken quantitatively by the response factor of the sialic acid standard of moving simultaneously.Specific data analysis and report value in this illustrative methods (as NANA or NGNA and proteinic mol ratio).This embodiment has described and has been used to measure CTLA4 A29YL104EThe method of the N-n acetylneuraminic acid n (NANA) that exists among-the Ig and the amount of N-hydroxyacetylneuraminic acid (NGNA).Sialic acid obtains discharging from protein by acid hydrolysis.The NANA and the NGNA that discharge use 1,2 subsequently ,-diamino-4, and 5-anisole (DMB) carries out fluorescent mark.The sialic acid of mark separates on the RP-HPLCC-18 post, and (Ex=373nm Em=448nm) detects by fluoroscopic examination.NANA and NGNA carry out quantitatively based on the NANA of operation simultaneously and the response factor of NGNA standard.Test result is reported as NANA and NGNA and proteinic mol ratio (MR) respectively.
Reagent and solution: 1.0M H 2SO 450mM H 2SO 4Fluorescent labeling reagent (18mM V-Brite B (Na 2S 2O 4), 7%2-mercaptoethanol, 7mM1,2 ,-diamino-4,5-methylene radical-dioxy benzene dihydrochloride (DMB)); Moving phase running buffer A (20% methyl alcohol); Moving phase running buffer B (70% methyl alcohol); N-n acetylneuraminic acid n standardized solution (1mg/mL); N-hydroxyacetylneuraminic acid standardized solution (1mg/mL); System's suitability criterion (being dissolved in 50 μ L NANA or NGNA solution in the 900 μ L water); Sample solution (for example, 2mg/mlCTLA4 A29YL104E-Ig etc.); The NANA stoste standardized solution (making stoste be diluted to 50 μ g/mL) of working; The NGNA stoste standardized solution (making stoste be diluted to 50 μ g/mL) of working.
Hydrolysis: with each 20 μ LNANA standard, NGNA standard, CTLA4 A29YL104E-Ig and system's suitability criterion solution aliquots containig arrive divides in other 1.5mL centrifuge tube, and adds 200 μ L 50mM sulphuric acid solns in each bottle.Content mixed gently and in 80EC incubation 1 hour
Figure A200680053116D0423181821QIETU
Minute.When hydrolysis is finished, make sample centrifugal fast.
Fluorescent mark: add in every kind of sample fluorescent labeling reagent (200 μ L) and thorough mixing.Sample is subsequently in the dark in the 80EC incubation
Figure A200680053116D0423181830QIETU
And with postcooling.
Instrument configuration and chromatography condition (instrument configuration of equal value can replace):
Ternary pump system Hewlett Packard model 1090
RP C-18HPLC post, Jones Chromatography, (catalog number (Cat.No.) 4M5313)
4.6?x?50mm，3μ
Fluorimetric detector Hewlett Packard model 1046A
Automatic sampler Hewlett Packard model 1090 is equipped with and is refrigerated to 4 ℃
Integrating system VG/Multichrom
HPLC?Chemstation Hewlett?Packard
(DOS series)
The chromatography parameter
Flow: 1.0mL/ minute
Mobile phase A: 20%MeOH/ water
Mobile phase B: 70%MeOH/ water
Linear gradient: Time %A %B
0.01 98 2.0
1.0 98 2.0
4.0 90 1.0
4.01 98 2.0
6.00 98 2.0
Volume injected: 10 μ L
Working time: 6 minutes
Column temperature: room temperature
Retention time (NANA):
Figure A200680053116D0424182106QIETU
Minute
Retention time (NGNA):
Figure A200680053116D0424182118QIETU
Minute
Peak pressure: 300 crust
Excitation wavelength: 373nm
Emission wavelength: 448nm
PMT gain: 8
HPLC system: Waters 2690/2695 separation module or Equivalent.
Fluorimetric detector: Waters 2475 multi-wavelength fluorimetric detector or Equivalents.
Data are obtained: Waters Millennium 32 or Empower.
Post: Luna5 μ, C18,100A, 150x4.6mm, Phenomenex, (catalog number (Cat.No.) 00F-4252-E0)
Numeral heat block WVR, (catalog number (Cat.No.) 13259-056) or Equivalent.
Compact centrifuge WVR, (Model No.C-1200) or Equivalent.
Mobile phase A: 10% (v/v) MeOH/90% water
Mobile phase B: 70% (v/v) MeOH/30% water
Flow velocity: 1mL/ minute
Volume injected: 10 μ L
Working time: 30 minutes
Column temperature: room temperature
Excitation wavelength: 373nm
Emission wavelength: 448nm
Gain: 1
Gradient:
Time Flow %A %B Curve
Initial 1.0 85.0 15.0 *
20.00 1.0 85.0 15.0 6
20.50 1.0 0.0 100.0 6
25.00 1.0 0.0 100.0 6
25.50 1.0 85.0 15.0 6
30.00 1.0 85.0 15.0 6
35.00 0.05 0.0 100.0 11
The sialic acid standard preparation (～5mM).The N-n acetylneuraminic acid n (NANA, MW=309.3) standard (～5mM).Accurately weighing 154.5 ± 1.0mg N-n acetylneuraminic acid n adds in the 100mL volumetric flask.With DI water dissolution and Q.S. to volume, thorough mixing.The solution aliquots containig is arrived in the 2mL profound hypothermia bottle.
Conc . ( mM ) = Wt ( mg ) xP 309.3 x 100 ( mL )
The purity of P=NANA-from the COA of manufacturer (that is, 99%=0.99).
The N-hydroxyacetylneuraminic acid (NGNA, MW=325.7) standard (～0.25mM).
Accurately weighing 8.0 ± 1.0mg N-hydroxyacetylneuraminic acid adds in the 100mL volumetric flask.With DI water dissolution and Q.S. to volume, thorough mixing.The solution aliquots containig is arrived in the 2mL profound hypothermia bottle.
Conc . ( mM ) = Wt ( mg ) xP 325.7 x 100 ( mL )
The purity of P=NGNA-from the COA of manufacturer (that is, 99%=0.99).The sialic acid standard of aliquots containig can be preserved in-20 ℃ maximum 6 months.
The preparation of sialic acid standard mixture.Be used for system's suitability and quantitative sialic acid standard mixture (50 μ M NANA, 1 μ M NGNA).With 1mL5mM NANA, 400 μ l 0.25mMNGNA add in the 100mL volumetric flask.With DI water Q.S. to volume and thorough mixing.Sialic acid standard mixture aliquots containig is arrived in the 2mL profound hypothermia bottle.The sialic acid standard mixture of aliquots containig can be preserved in-20 ℃ maximum 6 months.
Sample and reference material preparation.In the 2-8 ℃ of refrigerated protein example that thaws, thorough mixing.Sample and reference material are diluted to about 0.5mg/mL CTLA4 A29YL104E-Ig (for example protein concn=25.0mg/mL adds 50.0 μ L protein in the 2450 μ L water).With the specimen of dilution and reference material 10, under the 000rpm centrifugal 5 minutes to remove particle.
Hydrolysis.Blank: as in the 2.0mL centrifuge tube, to add 50 μ L DI water and 200 μ L 50mM sulfuric acid.This serves as blank.Be used for system's suitability and quantitative sialic acid standard.In the 2.0mL centrifuge tube, add 50 μ L sialic acid standard mixtures and 200 μ L 50mM sulfuric acid.Prepare in duplicate.Be labeled as Std1 and Std2.Reference material: the CTLA4 that in the 2.0mL centrifuge tube, adds 50 μ L dilution A29YL104E-Ig reference material (～0.5mg/mL) and 200 μ L 50mM sulfuric acid.Prepare in duplicate, be labeled as RM1 and RM2.Specimen: the CTLA4 that in the 2.0mL centrifuge tube, adds 50 μ L dilution A29YL104E-Ig medicine (～0.5mg/mL) and 200 μ L 50mM sulfuric acid.Prepare in duplicate.Be labeled as S1-1, S1-2; S2-1, S2-2; S3-1, S3-2; Deng.Make about 5 seconds of sample vortex and centrifugal about 5-10 second.Sample placed heat block and in 80 ℃ ± 5 ℃ incubations 1 hour ± 5 minutes.Allow the sample of hydrolysis to be cooled to room temperature.Make the sample of hydrolysis of short duration centrifugal with force condensation product enter in the pipe (with at a high speed～10 seconds).
Derivatize.Make heat block be preheated to 80 ℃ ± 5 ℃.200 μ L fluorescent labeling reagents are added in the sample of every kind of hydrolysis.About 5 seconds of vortex and centrifugal～10 second.Sample was placed 80 ℃ ± 5 ℃ heat blocks 40 ± 5 minutes.Cover heat block with aluminium foil, because label solution is light activated.Make the sample of derivatize be cooled to room temperature.Make sample vortex and centrifugal about 10 seconds to force condensation product to enter in the pipe.
Injection preparation.Guarantee that post carries out balance with moving phase before analysis.(100-200 μ L) transfers in the automatic sampler bottle with limited inset from each centrifuge tube with enough samples.The general automatic sampler that is used for 10 sample injections loads as follows:
Sample # Describe Injection #
1 Blank 1
2 Std1 2
3 Std1 2
4 Std2 2
5 RM1 1
6 RM2 1
7 S1-1 1
8 S1-2 1
9 S2-1 1
10 S2-2 1
11 S3-1 1
12 S3-2 1
13 S4-1 1
14 S4-2 1
15 Std1 1
16 Std2 1
Wherein Std1 and Std2 are the preparations of sialic acid standard mixture solution; RM1 and RM2 are the preparations about control sample; And S is the sample injection.Preceding 4 times (sample #2 and 3) sialic acid standards (Std1) injection will be used for system's suitability purposes.4 injections of sample #3 (Std1) and sample #4 (Std2) will be used for calculating.Sample injection for other repeats automatic sampler and loads 5-16.
System's suitability.The tomographic map overview of system's suitability sample should be similar to the chromatogram shown in Figure 49.For injection first time of system's suitability criterion (Std1), about the USP resolving power (R) of NGNA and NANA necessary 〉=1.5.4 injections of system's suitability criterion (Std1) must meet following example values: the RSD about the peak area of NANA is necessary≤and 5%.RSD about the peak area of NGNA is necessary≤and 10%.The transition time at NGNA peak must carry out wash-out in the time of 11.3 ± 2.0 minutes.The transition time at NANA peak must carry out wash-out in the time of 16.0 ± 2.5 minutes.The RSD of the peak area of 4 standards injection (Std1, sample #3) and (Std2, sample #4) is necessary≤and 10%.The RSD of peak area from all standard injections that carry out together of order is necessary≤and 15%.
The preparation of HPLC system: post with 98% buffer A and 2% buffer B with 1mL/ minute balance 15 minutes.The fluorescently-labeled system of 10 μ L suitability injection of solution is advanced in the system.Peak resolving power and theoretical plate number can use equation to calculate subsequently:
R = 2 ( T 2 - T 1 ) W 2 + W 1
Wherein: R=resolving power
The retention time of T1=N-hydroxyacetylneuraminic acid (minute)
The retention time of T2=N-n acetylneuraminic acid n (minute)
W1=the peak width at the baseline place of N-hydroxyacetylneuraminic acid (minute)
W2=the peak width at the baseline place of NANA (minute)
Resolution value is necessary 1.5.
Theoretical plate number can use equation to calculate:
N＝16(T2/W2) 2
Wherein:
The N=theoretical plate number
The retention time of T2=N-n acetylneuraminic acid n (minute)
The peak width at baseline line place of W2=N-n acetylneuraminic acid n (minute)
Theoretical plate number is necessary for 2000.CTLA4 A29YL104E-Ig hydrolysis overview chromatogram should be similar to Figure 49.
Sample is analyzed in the following sequence by reversed-phase HPLC (RP-HPLC): at first inject NANA and NGNA standard, injected sample subsequently, duplicate (for example, CTLA4 when needing A29YL104E-Ig etc.).Behind the sample analysis, post washed 20 minutes with 0.5mL/ minute with Mobile phase B.When needing, post can reverse.
The mensuration of sialic acid (NANA or NGNA) and proteinic mol ratio (MR)
Sialic acid and proteinic mol ratio can be calculated by Millennium or Empower software.
Dilution factor:
Figure A200680053116D04291
Wherein,
V ProteinThe volume (μ l) of the protein stock solution of=interpolation,
V WaterThe volume (μ l) of the water of=interpolation
Protein working solution (protein that is used for hydrolysis) concentration (μ M):
Figure A200680053116D04292
Wherein,
C ProteinThe concentration of=protein working solution (μ M),
C A280The A of=protein stock solution 280Concentration (mg/ml),
MW CTLA4A29YL104E-IgThe molecular weight of=CTLA4A29YL104E-Ig, 91232Da.
Sialic acid concentration in the protein working solution (μ M):
Figure A200680053116D04293
Wherein,
C UnknownSialic acid in the=unknown sample (NANA or NGNA) concentration
C StdSialic acid in the=standard (NANA or NGNA) concentration (μ M)
A uSialic acid in the=unknown sample (NANA or NGNA) peak area
A StdSialic acid in the=standard (NANA or NGNA) peak area
Sialic acid (NANA or NGNA) and proteinic mol ratio (M.R.):
Figure A200680053116D04294
The calculating of sialic acid and proteinic total mol ratio (TSA):
TSA=NANA mol ratio+NGNA mol ratio
The coefficient of variation who counts about the area of 2 NANA standards of carrying out together answers＜10%.The coefficient of variation who counts about the area of 2 NGNA standards of carrying out together answers＜10%.The coefficient of variation who counts about the area of 2 independent hydrolysates answers＜10%.
In one embodiment, CTLA4 A29YL104EAmong-the Ig sialic molar average ratio must under in the table in the specified scope.
CTLA4 A29YL104EThe molar ratio range of-Ig material
Monose Scope
NANA 5.0-10.0
NGNA <1.5
% deviation about sialic mol ratio in the reference material of 2 kinds of sample formulation and the sample is necessary≤15% ,≤20% ,≤25% ,≤30% or≤35%.
Embodiment 40: about CTLA4 A29YL104E The external biological assay of-Ig based on cell
The T cell needs 2 kinds of signals to be used for activation and follow-up propagation.First kind of signal provided by the interaction of antigen peptide and TCR-CD3 mixture.Second kind of costimulatory signal followed CD28 on the T cell and the interaction between the B7 protein on the antigen presenting cell and taken place.After these 2 kinds of signals are accepted, T emiocytosis cytokine interleukin II (IL-2).The release of IL-2 causes cell activation and propagation.Solubility, immunosuppressive compounds CTLA4 A29YL104E-Ig also with antigen presenting cell on the B7 protein bound, thereby blocking-up interacts with the function of CD28 and stops IL-2 to produce required costimulatory signal.
In this method, the Jurkat T cell that is used in the luciferase genes transfection under the IL-2 promotor control in the presence of anti-CD3 with Daudi B cell co-stimulatory.Sting activating signal activation IL-2 promotor altogether, this produces luciferase protein matter successively.Resulting luminous signal uses the luciferase assay system to measure.In this system, CTLA4 A29YL104E-Ig produces the minimizing of dose-dependently in the luciferase activity.
This method is checked CTLA4 A29YL104E-Ig produces the effect of required costimulatory signal to IL-2.Soluble CTL A 4 A29YL104EThe existence of-Ig stops the signal conduction between T cell and the antigen presenting cell.If there is not sort signal, do not produce IL-2 so, thereby stop the clonal expansion of T cell.Carrier with luciferase genes uses the IL-2 promotor to produce.Jurkat T cell carries out transfection with this report carrier subsequently.Select positive colony Jurkat.CA and be used for this method.
This biological assay relates to the T cell (Jurkat.CA) that stimulates transfection with anti-CD3 and B cell (Daudi).The common stimulation that is provided by the B cell is subjected to CTLA4 A29YL104EThe inhibition that-Ig adds.With Jurkat.CA and Daudi cell inoculation in 96 holes, white, opaque, flat-floored hole, and at the CTLA4 of different concns A29YL104EThe existence of-Ig stimulates with anti-CD3 down.After 37 ℃ of incubation 16-20 hours, the hole is measured with regard to luciferase activity.Stimulate via CTLA4 altogether A29YL104EAs seen the inhibition of-Ig reduces as dose-dependently in the luciferase activity.
Reagent: Daudi cell culture medium (being dissolved in 10% foetal calf serum of RPMI 1640, the 1%MEM Sodium.alpha.-ketopropionate); Jurkat.CA cell culture medium (being dissolved in 10% calf serum of RPMI 1640,1%MEM Sodium.alpha.-ketopropionate, 400 μ g/mL Geneticins); Biological assay substratum (being dissolved in the 0.2 μ g/mL anti-cd 3 antibodies and the 1% penicillin-Streptomycin sulphate solution of Daudi cell culture medium); Bright-Glo luciferase solution (Promega, catalog number (Cat.No.) E2620) from the system of mensuration.
Instrument configuration: Nikon, Diaphot 200 inverted microscopes; Packard TopCount NXT luminometer; Tecan Genesis liquid processor; Coulter Vi-Cell cell counter; ZymarkRapidPlate-96.
Program
Figure A200680053116D04321
The preparation of working solution: be dissolved in the 3mL CTLA4 in the biological assay substratum A29YL104E-Ig solution (5000ng/mL)
The Daudi cell culture medium.300mL RPMI 1640 is added in the 1L Corning filter unit.Add 100mL foetal calf serum and 10mL MEM Sodium.alpha.-ketopropionate subsequently.Add enough RPMI 1640 to obtain 1L.Filter and preserve in 4 ℃ maximum 1 month.
The Jurkat.CA cell culture medium.300mL RPMI 1640 is added in the 1L Corning filter unit.Add the 8mL Geneticin (final concentration is 400 μ g/mL) of 100mL calf serum, 10mL MEM Sodium.alpha.-ketopropionate and 50mg/mL subsequently.Add enough RPMI 1640 to obtain 1L.Filter and preserve in 4 ℃ maximum 1 month.
The biological assay substratum.100mL Daudi cell culture medium (2.1) is added in the 100mL medium bottle.The concentration and the 1mL penicillin-Streptomycin sulphate solution (1.11) that add anti-cd 3 antibodies to 0.2 μ g/mL subsequently.Be no more than 8 hours by reversing to mix gently and preserve in room temperature.
Bright-Glo luciferase solution.Described in system (1.21), prepare solution, mix by measuring in the damping fluid adding luciferase substrate and by reversing.This reagent should use in 2 hours or preserve in-20 ℃ and lucifuge protects maximum 4 weeks.
Clone is kept.Measure cell number/mL for Jurkat.CA and Daudi clone by counting with cell counter.Cell should be 1 x 10 5-1.5 x 10 6Cell/mL.With 12 x 10 6Jurkat.CA cell and 12 x 10 6The Daudi groups of cells is combined in the aseptic centrifuge tube.Make cell under～125xg centrifugal 10 minutes in room temperature.By move liquid gently repeatedly with the serology transfer pipet cell fully is resuspended in the 9mL Daudi cell culture medium (2.1), until can't see cell mass, to produce 2.7 x 10 6The concentration of cell/mL.By the checking of counting cells on cell counter cell concn.The cell of resuspension is inoculated into 75mL/ hole (200,000 cells/well) in the hole of 96 orifice plates (1.3).At 37 ℃, 5% CO 2Incubation plate during with the incubator setting point of 85% humidity, preparation standard, quality control sample simultaneously.The CTLA4 of preparation nominal concentration A29YL104E-Ig is used for standard, quality contrast and sample 1 and 2.In biological assay substratum (2.3), prepare 3mL CTLA4 with 5000ng/mL A29YL104E-Ig material working solution is used for using at typical curve.In biological assay substratum (2.3), prepare 3mLCTLA4 with 5000ng/mL A29YL104E-Ig material working solution is used for using in the quality control curve.2 kinds of CTLA4A29YL104E-Ig sample working solutions that prepare each 3mL with 5000ng/mL in biological assay substratum (2.3) are used for using at the sample curve.(about CTLA4 A29YL104EThe approximate concentration of-Ig sample can be used to prepare 8 point curves, but and when the concentration time spent of measuring, the relative potency value can be proofreaied and correct described in 5.5)
As shown in following table 55,5000,200,100,50,25,10,5 and 0.1ng/mLCTLA4 A29YL104EPreparation is about 8 point curves of standard, quality contrast and sample during the concentration of-Ig, and being diluted in the plate final concentration of back in mensuration at 2 times is 2500,100,50,25,12.5,5,2.5 and 0.05ng/mL.
Table 55. is used to produce the dilution of typical curve.
Curve point Typical curve The quality contrast Sample 1 Sample 2
1 5000ng/mL 5000ng/mL 5000?ng/mL 5000?ng/mL
2 200 200 200 200
3 100 100 100 100
4 50 50 50 50
5 25 25 25 25
6 10 10 10 10
7 5 5 5 5
8 0.1 0.1 0.1 0.1
Can use 2 kinds of plate figure.Plate figure need use liquid processor to be used for being provided with at random.In order plate figure has contiguous in triplicate, and adds with orderly in turn design about each curve point that detects article.For plate figure at random, as shown in plate figure hereinafter, every kind of solution of 75 μ L (4.8) are added in the appropriate bore of the plate that comprises cell (4.5).For orderly plate figure, as shown in plate figure hereinafter, every kind of solution of 75 μ L (4.8) are added in the appropriate bore of 2 blocks of plates that comprise cell (4.5).
With TopSeal-A (1.22) sealing plate.Guarantee that sealer tightly in position goes up.The clearance does not have.At 37 ℃, 5%CO 2Made the plate incubation 16-20 hour during with the incubator setting point of 85% humidity.Make plate and Bright-Glo luciferase solution (2.4) balance to instrument temperature.In each hole, add 150 μ L Bright-Glo luciferase solution and mixing simultaneously.Plate tightly is placed among the TopCount NXT with balance in the dark 10 minutes in mixing.In TopCountNXT, use 1 second integrations/hole or the luminometer of the particular type of use is suitably measured luminous signal.Record is from the output signal of TopCount NXT.When using orderly plate figure, will write down 2 blocks of plates.The 1st block of plate (axial) will be with the hole A1 record in the left hand corner up.Second flat board (inverted) will with below hole A1 record in the right hand corner.
Biological assay plate figure at random is provided with:
1 2 3 4 5 6 7 8 9 10 11 12
A QC 0.05 Smp112.5 Stnd0.05 Stnd2500 QC 12.5 Stnd100? Stnd5 Smp25 Smp212.5 Stnd2.5? Smp112.5 Stnd25
B Stnd5 Smp225 Smp1 Smp10.05 Smp112.5 QC?2.5 Stnd0.05 QC 2500 Smp125 QC25 QC5? Smp2100?
C Smp25 Stnd100? QC5? Stnd2.5? QC 0.05 QC5? Smp2100? Smp10.05 QC?100 Smp1100? QC50 Stnd5
D Smp1100? Smp212.5 Smp150 QC50 Smp12500 Smp22500 Smp12.5? Stnd25 Smp25 QC 2500 Stnd100? Smp150
E QC?100 Smp2100? QC25 Smp22500 Smp212.5 Smp250 Stnd2500 Smp125 Smp15 Smp12.5? Smp10.05 Smp22.5?
F QC 2500 Stnd12.5 Smp12.5? Stnd25 Stnd12.5 Smp15 Stnd2.5? Smp22.5? Smp20.05 Stnd2500 QC?2.5 Smp250
G QC?2.5 Smp250 Smp20.05 Smp125 Smp20.05 QC50 Smp1100? QC?100 Stnd0.05 QC 0.05 Smp12500 Smp22500
H Stnd50 Smp12500 Smp22.5? QC 12.5 QC25 Smp225 Smp150 Stnd50 Stnd50 QC 12.5 Smp225 Stnd12.5
Stnd: the standard of 2500-0.05ng/mL final concentration in the hole
QC: the quality of 2500-0.05ng/mL final concentration contrast in the hole
Smp1, Smp2: the sample 1-2 of 2500-0.05ng/mL final concentration in the hole
The orderly plate figure of biological assay is provided with:
1 2 3 4 5 6 7 8 9 10 11 12
A Stnd2500 Stnd2500 Stnd2500 Stnd100? Stnd100? Stnd100? Stnd50 Stnd50 Stnd50 Stnd25 Stnd25 Stnd25
B Stnd12.5 Stnd12.5 Stnd12.5 Stnd5 Stnd5 Stnd5 Stnd2.5? Stnd2.5? Stnd2.5? Stnd0.05 Stnd0.05 Stnd0.05
C QC 2500 QC 2500 QC 2500 QC?100 QC?100 QC?100 QC50 QC50 QC50 QC25 QC25 QC25
D QC 12.5 QC 12.5 QC 12.5 QC5? QC5? QC5? QC?2.5 QC?2.5 QC?2.5 QC 0.05 QC 0.05 QC 0.05
E Smp12500 Smp12500 Smp12500 Smp1100? Smp1100? Smp1100? Smp150 Smp150 Smp150 Smp125 Smp125 Smp125
F Smp112.5 Smp112.5 Smp112.5 Smp15 Smp15 Smp15 Smp12.5? Smp12.5? Smp12.5? Smp10.05 Smp10.05 Smp10.05
G Smp22500 Smp22500 Smp22500 Smp2100? Smp2100? Smp2100? Smp250 Smp250 Smp250 Smp225 Smp225 Smp225
H Smp212.5 Smp212.5 Smp212.5 Smp25 Smp25 Smp25 Smp22.5? Smp22.5? Smp22.5? Smp20.05 Smp20.05 Smp20.05
Stnd: the standard of 2500-0.05ng/mL final concentration in the hole
QC: the quality of 2500-0.05ng/mL final concentration contrast in the hole
Smp1, Smp2: the sample 1-2 of 2500-0.05ng/mL final concentration in the hole.
Each hole of 96 orifice plates adds 200,000 cells and at 37 ℃, 5%CO 2Carry out incubation during with 85% humidity.With 12 x 10 6Jurkat.CA cell and 12 x 10 6The Daudi groups of cells is combined in the aseptic centrifuge tube.Make cell under～125xg centrifugal 10 minutes, and cell fully is resuspended in the 9mL Daudi cell culture medium, until can't see cell mass, to produce 2.7 x 10 by move liquid gently repeatedly with the serology transfer pipet in room temperature 6The concentration of cell/mL.To add in the appropriate bore of the plate that comprises cell from every kind of solution of 75 μ L of table 55.Plate seals with TopSeal-A subsequently, and at 37 ℃, 5%CO 2Incubation is 16-20 hour during with 85% humidity.Plate and Bright-Glo luciferase solution equilibria add 150 μ L Bright-Glo luciferase solution and mixing simultaneously in each hole to instrument temperature.Plate tightly is placed in mixing and was used in the dark balance among the TopCount NXT 10 minutes subsequently.In TopCount NXT, use 1 second integrations/hole subsequently or the luminometer of the particular type of use is suitably measured luminous signal.
Record reads in the standard analyzer from the output of TopCount NXT, and data transform by getting its logarithm (end 10).As shown in following equation, 4 parameter logarithmic models are carried out match from the conversion data of each article:
Equation 1: Log 10 ( y jk ) = D + ( A - D ) 1 + ( x j C ) B
Wherein:
A is the level ground, top (pleteau) of curve, and D is the level ground, bottom of curve, and B is a slope factor, and C is the concentration that produces the effect of the mean value that equals A and D.
Can calculate R for each article 2Statistic and shortage match F check.Can also calculate the ratio of test article with respect to minimum value, maximum value and the slope of standard material.In addition, can also calculate fiducial interval about ratio.
The relative potency of each article is measured by making single equation pair and the data fitting from the purpose article from the data combination of reference article.
Log 10 ( y ijk ) = D + ( A - D ) 1 + ( x ij C A * ( C R C A ) I ) B
Wherein
A, B and D parameter are that reference and test article are total, and C RBe reference parameter, C ABe test article parameters, and compare C R/ C AIt is relative potency.Subscript I is the indication variable.If data are from the purpose article, it is made as and equals 1 so, and if data from standard material, it is 0 so.
Change relative potency of each test article into the per-cent grade, and relative potency is as providing from the output of program.
The adjustment of the relative potency value that obtains with approximate concentration: because the time lag between the accurate protein concn is accepted and obtained to sample, sample can be tested with approximate concentration in mensuration, and adjusts the result when accurate concentration is determined.This adjustment uses equation 2 to carry out, and the relative potency of wherein measuring in mensuration multiply by the CTLA4 that is used to be provided with mensuration A29YL104EThe CTLA4 of-Ig concentration and mensuration A29YL104EThe ratio of-Ig sample concentration.
Equation 2:
Figure A200680053116D04371
Example:
Sample protein concn with 25mg/mL in mensuration is tested.
The relative potency of measuring is 105%.
The CTLA4 that measures A29YL104E-Ig concentration determination is 25.5mg/mL
Reportable relative potency=(105*25)/25.5=103%.
Standard material: about the EC of standard of output 50Value should be 5-35ng/mL.Difference between 2500ng/mL and the 0.05ng/mL concentration standard (scope) should be 〉=40,000 counting/seconds (CPS).R square value about reference should be greater than 0.95.
Test article relative potency value in the table 9 is necessary for the 25-175% of reference standard, and this is the scope of measuring.If the relative potency value is outside this scope, sample must dilute or concentrate dropping in this scope so, and sample is analyzed once more.
Daudi B clone:
The source: the Daudi cell derives from ATCC.Generation comprises the master library of 64 bottles.The work storehouse produces after being gone down to posterity for 4 times by the master library bottle.(annotate: in the 0th generation, be considered as thawing, and before the work storehouse produces, carry out other 3 times subsequently go down to posterity).
Substratum: the Daudi cell is grown in the RPMI1640 substratum of adding 10% foetal calf serum and 1% Sodium.alpha.-ketopropionate (comprising HEPES and L-glutaminate).
The incubator condition: cell is at 37 ℃, 5%CO 2Maintain during with 70-90% humidity in the airy T bottle.
The rules of thawing: the bottle cell takes out from the liquid nitrogen freezing machine and thaws in 37 ℃ of water-baths.Content is mixed with the 10mL substratum.Cell is counted and subsequently by collecting in centrifugal 10 minutes under 125xg.After centrifugal.Remove supernatant liquor and make cell with 3 x 10 5Viable cell/mL is suspended in the fresh culture.Cell this time be defined as for the 0th generation.
Growth properties: this clone suspension growth.
The cultivation of going down to posterity: culture is kept for 2 times by going down to posterity weekly, wherein is no more than 5 days between going down to posterity.Cell is at 0.5 x 10 5-2 x 10 5Viable cell/mL goes down to posterity in having the airy T bottle of fresh culture.Cell should not reach and surpass 1.5 x 10 6The density of cell/mL.As assessing by trypan blue staining, cell should surpass 80% survival.Date and passage number should be in the back of going down to posterity in the enterprising row labels of T bottle.
Doubling time: the doubling time is 18-26 hour.
The restriction of going down to posterity: the cell from the work storehouse should go down to posterity before being used for biological assay 3 times, and promptly they should be in the 3rd generation or higher.Cell should only be kept in cultivation for 20 generations.New work bottle should thaw at that time.
Freezing rules: cell is with 5-10 x 10 6Cell/mL is frozen in the cryovial.Profound hypothermia protective material substratum is prepared by adding complete cell culture medium with 5% (v/v) DMSO.Cell carries out freezingly reaching liquid nitrogen temperature (～190 ℃) until them with 1 ℃/minute speed.
Jurkat.CA T clone:
The source: Jurkat T cell carries out transfection with the plasmid of coding CTLA4-Ig molecule.The work storehouse produces after being gone down to posterity for 3 times by the master library bottle.(annotate: in the 0th generation, be considered as thawing, and before the work storehouse produces, carry out other 2 times subsequently go down to posterity).
Substratum: the Jurkat.CA cell is being added 10% calf serum and 1% Sodium.alpha.-ketopropionate, is being added growth in the RPMI1640 substratum (comprising HEPES and L-glutaminate) of Geneticin (G418 vitriol) of final concentration 400 μ g/mL.
The incubator condition: cell is at 37 ℃, 5%CO 2Maintain during with 70-90% humidity in the airy T bottle.
The rules of thawing: the bottle cell takes out from the liquid nitrogen freezing machine and thaws in 37 ℃ of water-baths.Content is mixed with the 10mL substratum.Cell is counted and subsequently by collecting in centrifugal 10 minutes under 125xg.After centrifugal.Remove supernatant liquor and make cell with 3 x 10 5Viable cell/mL is suspended in the fresh culture.Cell this time be defined as for the 0th generation.
Growth properties: this clone suspension growth.
The cultivation of going down to posterity: culture is kept for 2 times by going down to posterity weekly, wherein is no more than 5 days between going down to posterity.Cell is at 0.5 x 10 5-2 x 10 5Viable cell/mL goes down to posterity in having the airy T bottle of fresh culture.Cell should not reach and surpass 1.5 x 10 6The density of cell/mL.As assessing by trypan blue staining, cell should surpass 80% survival.Date and passage number should be in the back of going down to posterity in the enterprising row labels of T bottle.
Doubling time: the doubling time is 18-26 hour.
The restriction of going down to posterity: the cell from the work storehouse should go down to posterity before being used for biological assay 3 times, and promptly they should be in the 3rd generation or higher.Cell should only be kept in cultivation for 20 generations.New work bottle should thaw at that time.
Freezing rules: cell is with 5-10 x 10 6Cell/mL is frozen in the cryovial.Profound hypothermia protective material substratum is prepared by adding complete cell culture medium with 5% (v/v) DMSO.Cell carries out freezingly reaching liquid nitrogen temperature (～190 ℃) until them with 1 ℃/minute speed.
Embodiment 41: measure CTLA4 via surface plasma body resonant vibration (BIAcore) A29YL104E -Ig Combine with the biologic specificity of B7.1-Ig acceptor
CTLA4 A29YL104E-Ig sample uses the BIAcore instrument to measure with the relative combination of B7.1Ig acceptor by surface plasma body resonant vibration.In this mensuration, CTLA4 A29YL104E-Ig combines with B7.1Ig domain-immunoglobulin fusion proteins derived from APC cell membrane protein B7.1.After the B7.1Ig acceptor is immobilized on the activatory sensor chip surface with high-density, CTLA4 A29YL104E-Ig material, quality contrast and sample dilute to produce in conjunction with sensing figure.CTLA4 A29YL104EThe initial association rate (slope) on-Ig and immobilized B7.1Ig surface/bonded resonance units (RU) is measured on this high-density B7.1Ig surface under mass transfer (diffusion) restricted condition.So that resonance units/second, (RU ' s) the initial association rate of expression was directly related with biological activity concentration.The association rate of sample uses the reference standard curve calculation to become active concentration, CTLA4 in described reference standard curve A29YL104EThe association rate of-Ig material is drawn at concentration.Net result by sample with respect to CTLA4 A29YL104EThe per-cent of-Ig material is in conjunction with expression or be expressed as concentration.
Reagent: the amine coupling reagent kit BIA (Amine Coupling Kit BIACertified) through identifying (test kit comprise each 115mg N-hydroxy-succinamide (NHS), 750mgN-ethyl-N '-(3-dimethyl) (EDC) and 1 bottle of thanomin); The regeneration damping fluid (the 10mM Trisodium Citrate, 100mM NaCl, pH4.0);
Instrument configuration: BIAcore C instrument (BIAcore (catalog number (Cat.No.) BR-1100-51)) with PC compatible computer; BIAcore C control software version 1.0.2; BIAcore assessment software 1.0; BIAcore 96 hole microtiter plate U type BIAcore (catalog number (Cat.No.) BR-1005-03); BIAcore 96 hole microtest plate paper tinsel plate sealers (catalog number (Cat.No.) BR-1005-78).
The method general introduction:
Figure A200680053116D04401
Material
Sensor chip (Sensor Chip) CM5, BIAcoreAB (catalog number (Cat.No.)
Regular grade BR-1000-12)
Figure A200680053116D04402
C instrument handbook BIAcore (catalog number (Cat.No.) BR-1005-11)
The 10mM BIAcore (catalog number (Cat.No.) BR1001-88) of HBS buffer B IA through identifying
HEPES?pH?7.4，150mM?NaCl，3.4
MM EDTA 0.005%v/v tensio-active agent
P20
BIA stdn solution B IAcore AB (catalog number (Cat.No.)
(40% glycerine solution) BR-1002-22)
Amine coupling reagent kit BIA BIAcore (catalog number (Cat.No.) BR-1000-50) through identifying
115mg N-hydroxyl succinyl-Asia
Amine 750mg N-ethyl-N '-(3-
Dimethyl)
Sodium-chlor (NaCl) Sigma (Catalog No.S-9888)
Acetate buffer pH5.0 BIAcore (catalog number (Cat.No.) BR-1003-51)
Trisodium Citrate (C 6H 3Na 3O) Sigma (catalog number (Cat.No.) S-4641)
Hydrochloric acid (HCl) Fisher Scientific (catalog number (Cat.No.)
A144-212)
BIAcore keeps test kit BIAcore (catalog number (Cat.No.) BR-1006-67)
Reagent
Amine coupling reagent kit BIA through identifying.This test kit comprises 1 bottle of each 115mg N-hydroxy-succinamide, 750mg N-ethyl-N '-(3-dimethyl) and thanomin.According to every kind of solution of manufacturer specification aliquots containig and as described below the storage:
11.5mg/ml the aliquots containig of N-hydroxy-succinamide (NHS) is preserved in-20 ℃.This refrigerated aliquots containig lost efficacy from preparing at 2 months on date.The aliquots containig of 75mg/ml1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) is preserved in-20 ℃.This refrigerated aliquots containig lost efficacy from preparing at 2 months on date.1.0M the aliquots containig of thanomin-HCl pH8.5 is preserved in-20 ℃.This refrigerated aliquots containig lost efficacy from preparing at 2 months on date.The regeneration damping fluid (the 10mM Trisodium Citrate, 100mM NaCl, pH4.0).Weigh up 1.5 ± 0.1g Trisodium Citrate and 2.9 ± 0.1g NaCl by analysis.Add in the 500mL Milli-Q water and pH is adjusted to 4.0 with 1N HCl.With 0.22 μ m filter filtering solution, arrive in the 45mL/50mL conical tube with the 500mL aliquots containig subsequently.When preserving in 2-8 ℃, solution lost efficacy from preparing at 6 months on date.
Instrument configuration
BIAcore (catalog number (Cat.No.) BR-1100-51) with PC compatible computer
BIAcore C instrument
BIAcore C control software: BIAcore is as providing version 1.0.2 with BIAcore C instrument
Assessment software BIAcore is as providing version 1.0 with the BIAcoreC instrument
PH meter ORION, model 720A+
The preparation of sensor chip.In the box mouth on the detector cell of new sensor chip CM5 insertion instrument.Use starting system described in HBS-EP damping fluid such as the BIAcore C handbook.
Be used for the operation of the BIAcore C instrument of methodological function.BIAcore C instrument is controlled by BIAcore C control software under Microsoft Windows environment from the PC computer of compatibility.Using and keeping, reference work specification sheets, " Operation ofBIAcore C Instrument " and " Calibration and Maintenance of BIAcore CInstrument " about BIAcore C instrument.
The process for fixation of B7.1Ig
The preparation of B7.1Ig: the bottle that comprises B7.1Ig wherein uses 10mM acetate pH5.0 damping fluid as thinner in 22.5 ± 5 ℃ of concentration of thawing and being diluted to immobilization 3000-9000RU.The bottle of EDC, NHS and thanomin is being thawed by the amine coupling reagent kit as described separately.Reagent and part bottle be the specimen holder that places as being indicated by the BIAcore program subsequently, and according to BIAcore instrument specification sheets initial securedization.
CTLA4 A29YL104E The preparation of-Ig typical curve
Standard and sample (for example, CTLA4 A29YL104E-Ig etc.) at room temperature thaw.The standard that will be used to produce typical curve is diluted to the target level of typical curve (250,500,1000,2000,4000 and 8000ng/mL).
For standard model (CTLA4 for example A29YL104E-Ig material stoste) dilution and concentration are 25mg/mL, and people can carry out following dilution:
I. by 20 μ L being added 1/50 dilution (500 μ g/mL) among the 980 μ L HBS-EP
Ii. by being added among the 908 μ L HBS-EP, 30 μ L500 μ g/mL are diluted to 16 μ g/mL
Iii. with 1/2 step (the previous dilution of 500 μ L+500 μ L HBS-EP) serial dilution downwards to 250ng/mL
Each standard can be analyzed with bipartite injection, and this will cause 2 slope value.
Quality control sample.CTLA4 A29YL104EThe quality of-Ig contrast (QC) sample in the HBS-EP damping fluid with 750,2500 and 3 target level levels of 5000ng/mL be prepared, and under-80 ℃, be frozen in the 7mm plastic jar as 200 μ L aliquots containigs.CTLA4 in the QC sample A29YL104E-Ig concentration makes in this way and independently measures in the concentration analysis experiment at 3 times.In each experiment, all QC sample injections must be acceptable, detect in ± 20% nominal concentration.Qualified and refrigerated QC sample lost efficacy in preparation in back 6 months.Testing the same day each 1 bottle of the 3 kinds of QC samples that at room temperature thaw.Described in method guide, the QC sample placed phase specimen holder position, and analyze every kind of QC with in triplicate injection.Alternately, QCs can analyze prepared fresh on the same day.
CTLA4 A29YL104E-Ig specimen.CTLA4 A29YL104E-Ig sample must be diluted to the concentration (750-5000ng/mL) in the measurement range.Has known approximate CTLA4 A29YL104EThe sample of-Ig concentration should be diluted to the target level of 2000ng/mL.After sample at room temperature thaws, use the HBS-EP damping fluid that they are diluted to the target level of 2000ng/mL.Can prepare the diluted sample thing is used for analyzing through polypropylene test tube or the 96 hole microtiter plates identified at BIAcore.Following is about CTLA4 A29YL104EAn example of-Ig specimen dilution:
After sample at room temperature thaws, CTLA4 A29YL104EThe concentration of-Ig diluted sample to the measurement range (for example, 750-5000ng/mL).Has known approximate CTLA4 A29YL104EThe sample of-Ig concentration should use the HBS-EP damping fluid BIAcore through identifying the polypropylene test tube or 96 hole microtiter plates in be diluted to the target level of 2000ng/mL.
Following is about CTLA4 A29YL104E-Ig sample (example of the dilution of concentration=25.0mg/mL):
I. by 20 μ L being added 1/50 dilution (500 μ g/mL) among the 980 μ LHBS-EP
Ii. by 30 μ L, 500 μ g/mL being added 1/25 dilution (20 μ g/mL) among the 720 μ L HBS-EP
Iii. by 100 μ L, 20 μ g/mL being added 1/10 dilution (2 μ g/mL) among the 900 μ L HBS-EP
Make every kind of dilution with moderate speed's vortex 2-4 second.Sample is prepared (from 3 kinds of independent dilutions of sample stock solution) in triplicate, and every kind of dilution is analyzed with 1 injection.Sample is prepared (from 3 kinds of independent dilutions of sample stock solution) in triplicate, and every kind of dilution is analyzed with 1 injection.
Use BIAcore C software to begin to analyze: to open BIAcore C control software and select File-〉Project-〉Published-〉8164-2-〉Concentration Analysis Wizard, click the open template that " Next " and selection are suitable for pending analysis, for example " CTLA4 A29YL104E-Ig Sample Concentration Analysis.blw ".Input is about the maximum sample number of the analysis of plan.This sample number comprises repetition, therefore for example in order to analyze in triplicate 8 duplicate samples, should import number 24.Select the immobilized flow cell of B7.1Ig part (1-4) thereon.Click ID ' s, dilution factor and the repetition number of " Next " and input sample.Click " Next " and check the bottle position.The position of bottle can move to required location on this aspect.Click " Next " and setting and the selection of affirmation on " Preparation for Analysis " screen about measuring.Click " Next " with downward rolling.Standard, QC ' s, regeneration soln and specimen are placed suitable position.Click " Start " to begin analysis.
Data evaluation.Begin BIAcore C software and open the BIAcore file of generation * *.blr.From " View " menu, select " Wizard Results " with assessment data subsequently.
Example values about the B7.1Ig surface
The quality representation that is immobilized in the B7.1Ig on the activation flow cell is RU ' s (resonance units).For the mensuration performance of the best, surface quality should be 3000-9000 RU ' s.If surface quality does not meet this example values, can carry out so soak time (EDC/NHS injection) or B7.1Ig concentration adjustment and can another flow cell of immobilization.
Example values about baseline wander
The baseline (" absolute response value ") that the baseline wander of each run is calculated as between each circulation changes with respect to the per-cent of the immobilization surface quality of part.Largest percentage between any 2 circulations of operation changes necessary≤5.0%.For example: if at baseline=13500RU of 20 o'clock of circulation, at baseline=13650RU of 23 o'clock of circulation, and B7.1Ig surface density=6000RU, the x 100=2.5% of baseline wander so=(13650-13500)/6000.
Example values about standard
Example values is applied to when 500ng/mL or surpasses the typical curve concentration of 500ng/mL.The variation coefficient (%CV) of the value on each slope value when being used for each normal concentration of bioassay standard curve must be in ± 10%.The mean intensity value of when each normal concentration, calculating (ng/mL) must target (nominal) value 15% in.Concentration and the difference between the target (nominal concentration) calculated can and multiply by 100 divided by target (nominal) concentration.For example, when standard 500ng/mL, the concentration that BIAcore C calculates is 510ng/mL.The % deviation is with following calculating: (510ng/mL-500ng/mL)/and 500ng/mL x 100%=2.0%.
Example values about the QC sample: the %CV about the concentration value of the triplicate calculating of each QC sample target level must be in ± 10%.In measuring for 9 QC at least 7 times, the QC sample result must its respectively the target value ± 15% in.Difference in the slope value between QC injects for the first time and for the last time when the 2500ng/mL level must be in ± 5.0%.For example, first slope value is 10.366RU/s, and last slope value is 10.230RU/s, and percentage difference is with as follows:
(10.366-10.230)/10.366?x?100％＝1.3％
Example values about specimen
The %CV of the triplicate observation that obtains for each specimen target level must be in ± 20%.Must be in the scope of this method about the slope value of sample: the average gradient of the average gradient≤quality control sample 3 of the average gradient≤sample during in quality control sample 1.
Sample result is calculated
In order to measure specimen with respect to CTLA4 A29YL104EThe per-cent combination of-Ig material is calculated by typical curve with ng/mL about the concentration value of each specimen.The guide as a result of BIAcore instrument configuration is based on calculating CTLA4 in the undiluted sample for the dilution factor of every kind of sample input with mg/mL A29YL104E-Ig concentration.
Measure the per-cent combination: the mean intensity value of calculating for each sample of testing can multiply by 100, and divided by the absorbancy (A as passing through to measure at the 280nm place 280) protein concn of the sampling report measured.Value can be used as with respect to the per-cent of standard model in conjunction with report subsequently to immediate integer.For example, sample dilutes (the sample protein matter concentration of about 25mg/mL) by 12,500 dilution factor.Average CTLA4 by the in triplicate injection calculation of sample A29YL104E-Ig concentration is 25.3mg/mL.A about sample 280Be 24.2mg/mL.Can be as follows with respect to the per-cent combination that standard model calculates: 25.3mg/mL/24.2mg/mL x100%=104.545%.
CTLA4 A29YL104E-Ig immobilization guide template
The immobilization injection parameters
EDC/NHS B7.1-Ig Thanomin Citrate trianion
Duration of contact (minute) 7 7 6 2
Flow velocity (μ L/ minute) 5 5 5 10
Volume injected (μ L) 35 35 30 20
The immobilization reporting point
ID Time (s) Front/rear Beginning/end Injection Type With respect to Window (s) Report
Baseline
10 Before Beginning Injection for the first time AbsResp --- 5 Not
Activation
10 Before Beginning Injection for the second time RelResp Baseline 5 Not
The immobilization level 85 After Finish Injection for the third time RelResp Baseline 5 Be
The carbohydrate analysis data of embodiment 42:CTLA4-Ig and pharmacokinetics number According between association
Carbohydrate structure on the CTLA4-Ig plays an important role in the pharmacokinetics (PK) of CTLA4-Ig therapeutic composition.Several analytical methods have been developed and have been used to characterize these carbohydrate structures.2 kinds of analytical parameters are well related with clearance rate: sialic acid (NANA) and CTLA4-Ig protein than and the per-cent domain II I and the IV that come the self-carbon water compound overview.The third parameter (from the semi-lactosi and the seminose ratio of monose analysis) seems also good related.For example, the detailed description about these parameters can be:
NANA:CTLA4-Ig protein is than 〉=8.0
Carbohydrate overview domain II I and IV 〉=25% (method 1)
Semi-lactosi: seminose is than 〉=0.65
Important step in the CTLA4-Ig fermentation process can be the selection of results parameter, and described results parameter will make the yield (titre) of the final product that comprises this paper specific characteristic reach maximum (referring to table 6).A parameter (NANA: the protein ratio) enough gather in the crops one of parameter to be used as fast and accurately.About 8.0 NANA can guarantee that with the proteinic target mol ratio of CTLA4-Ig most of cuttings will have〉7.0 NANA ratio.Enhancing during purifying can produce subsequently has NANA than 9.0 CTLA4-Ig molecule.
CTLA4-Ig is the glycoprotein with several N connection and O linked glycosylation site.N connection carbohydrate structure generally is two or three feelers, and if fully sialylated, finish with carbohydrate NANA-Gal-GlcNAc so.Most of molecules are that only part is sialylated, and comprise some carbohydrate chains that finish with Gal or GlcNAc.Not existing of terminal sialic acid (NANA) residue is the factor that causes from the exposure of blood flow increase.In this example, the data that present point out that terminal galactose also provides some that be not subjected to remove fast to protect.
Being used to assess glycosylated significant parameter is NANA:CTLA4-Ig protein mol ratio.Monkey PK research has shown that acceptable removing can use the CTLA4-Ig molecule with NANA ratio of 6.9 to reach.First method Y CTLA4-Ig material (deriving from the CTLA4-Ig composition of Y method) of testing in monkey has 7.1 NANA ratio.Surprisingly, find that it removes with 2 times of speed of CTLA4-Ig molecule with NANA ratio of 6.9.The summary of the monose analysis of 2 kinds of samples points out that X method material has than the obviously more semi-lactosi of CD-CHO method material.Developed the method (CD-CHO1) that in charging, comprises semi-lactosi.The CTLA4-Ig that is produced by this method has than higher levels of semi-lactosi of Y method material and sialic acid.For from the analysis of the material of CD-CHO1 method with the PK data are discussed hereinafter and compare with method Y and method X material.
Analytical method
The carbohydrate profile analysis is removed by the enzymatic of complete carbohydrate structure and is formed by the anion-exchange chromatography separation.The carbohydrate peak is divided into 4 or 5 bunches (" structural domain ") based on the sialic acid residues number in every kind of structure to a great extent.Structural domain I mainly is an asialoization, and domain II is a mono-sialylated, and domain II I is a double-sialylated, and structural domain IV is three sialylated, and structural domain V is four sialylated.The total area per-cent under all peaks can be measured and be reported as to area under each peak or the structural domain.
Clearance rate is measured by following: inject monkey in triplicate with 10mg/kg CTLA4-Ig, follow the tracks of subsequently through the minimizing in 28 day time period serum-concentration.Clearance rate is relevant with " area under curve " or the AUC of measurement.AUC is relevant with removing, and higher clearance rate is relevant with littler AUC, and lower clearance rate is relevant with bigger AUC value.The higher slower removing of value representation CTLA4-Ig.
Several in many N connection carbohydrate structures that Figure 50 has described to find in mammalian proteins matter.All chains are shared the common core structure that comprises 2 GlcNAc and 3 mannose residues.From this core, stretch out the 2-4 bar chain of forming by one of following 3 kinds of structures :-GlcNAc-Gal-NANA ,-GlcNAc-Gal or-GlcNAc (Figure 50, structure (1), (2) and (3)).Suppose the removing of terminal sialic acid (NANA) responsible minimizing protein from blood flow.Although NANA is than keeping identical, the clearance rate of CTLA4-Ig doubles in monkey PK research (table 56), this be surprising (table 56-in Figure 51-52 sample CTLA4-Ig S1 and CTLA4-Ig (-) Gal) respectively.Observed 1 species diversity is semi-lactosi and protein mol ratio between the sample for preparing in different substratum, this in by the sample of method X preparation apparently higher than method Y (CTLA4-Ig).This hint clearance rate is mainly by terminal GlcNAc residue decision, and terminal galactose may provide some protections.In order to increase the galactosylation of CTLA4-Ig, semi-lactosi is added the charging that is used for the Y method.Novel method (CD-CHO1; CTLA4-Ig S2 among Figure 51-52) significantly increases semi-lactosi and the NANA mol ratio of the CTLA4-Ig in the bio-reactor.
Further monkey PK research is carried out.These have comprised CTLA4-Ig product (method X, method Y and the CD-CHO1 method of being produced by 3 kinds of different methods; CTLA4-Ig S1, CTLA4-Ig (-) Gal and CTLA4-Ig S2 respectively in Figure 51-52).In addition, the CTLA4-Ig with low-down NANA ratio (reclaims from the washing step of purifying procedure; Referring to Figure 62) test.From the analysis and the PK data sink of all samples of test are organized in the table 56 up to now.In nearest PK research (table 56), assessed the CTLA4-Ig (CTLA4-Ig S3) that produces by the method Y fermentation operation that prolongs.
Table 51 shows about the NANA ratio of all samples in 4 kinds of research and the association between the monkey PK AUC value.Show strong association between these parameters by the sample of Y method (referring to CTLA4-Ig (-) Gal among Figure 51) and CD-CHO1 method (the CTLA4-Ig S2 among Figure 51) preparation, thereby point out that NANA comparison CTLA4Ig clearance rate has remarkably influenced.Point out when the NANA=9 about the analysis of the Trendline of these points, by the CTLA4-Ig of Y (CTLA4-Ig (-) Gal) or the preparation of CD-CHO1 (CTLA4-Ig S2) method will with method X material C TLA4-Ig S1) approximately identical speed removes.That AUC among the monkey PK is reduced is about 25% than being reduced to 8 to make NANA, makes AUC increase about 25% and make this ratio increase to 10.Although under Y (CTLA4-Ig (-) Gal) and CD-CHO1 (CTLA4-Ig S2) method background, between NANA and AUC, exist strong relatedly, remember that it is important that NANA=7 about X material (CTLA4-Ig S1) has the clearance rate more identical than the CD-CHO1 material (CTLA4-Ig S2) that is 9 with NANA.Therefore, NANA is than the clearance rate that is not individual responsibility decision CTLA4-Ig.
The carbohydrate assessment of table 56.CTLA4-Ig.M-1 points out that CTLA4-Ig material using method 1 analyzes, and M-2 points out that the CTLA4-Ig material uses slightly diverse ways 2 to analyze." PA " points out that material is the sample from the A protein purification of fermented liquid
Figure A200680053116D04501
Figure A200680053116D04521
Carry out another monkey PK research (table 56) to compare the clearance rate of the CTLA4Ig that produces by 3 kinds of different methods (method X, method Y and CD-CHO1 method).In addition, the CTLA4-Ig (reclaiming from the washing step of purifying procedure (PA)) with low-down NANA ratio is included in this research.The CTLA4Ig that is prepared by the CD-CHO method in 50L or 5000L bio-reactor has the NANA mol ratio near X method material, and in PK research, the both has the method X value AUC value of half approximately.The CTLA4Ig that is produced by the CD-CHO1 method has the AUC value of the higher NANA of ratio method X material than (9.9 pairs 6.9) and high about 30%, thereby points out slower clearance rate.Detergent (NANA=2.3) weak sialylated and galactosylation is removed (AUC=2337hr-μ g/ml is to 15753 of method X material) as quick as thought.
Another monkey PK research (table 56) is compared 5, the CTLA4Ig that is prepared and gathered in the crops when different number of days by the CD-CHO1 method in the 000L bio-reactor.In the fermentation operational process, NANA peaks in the time of about the 8th day than generally, progressively descends subsequently.From 2 operations, aliquots containig was taken out in the time of the 12nd, 14 and 16 day, carried out purifying subsequently.Behind the purifying, NANA is than being 8.8-10.0.The the 12nd and 14 day sample (being respectively NANA=9.8 and 10.0) have the AUC value of the high 20-30% of ratio method X material.The 16th day sample (NANA=8.8) have and method X material AUC value much at one.
Glycosylated another analysis tool of assessment CTLA4-Ig is the carbohydrate overview.Complete N connection carbohydrate structure is removed by enzymatic and is separated by anionresin HPLC.The a large amount of peaks (referring to Figure 58-62) that are divided into 4 or 5 structural domains have been produced.Structural domain I and II mainly are the structures of asialoization and mono-sialylated, and domain II I and IV and V mainly are two and three and four sialylated structures.
Observe by rule of thumb for all samples and comprise method X material, general overview per-cent and AUC good related (Figure 52) among domain II I and the IV.Most of structure expections in these structural domains are fully sialylated and galactosylation.Use is from the data of M-1 group, and about 29% domain II I should have and the identical clearance rate of method X material with IV per-cent.Domain II I and IV data from method 2 are generally hanged down about 4% (21% pair 25%) than the data that produced by method 1.
In addition, for all samples, also observe general overview per-cent and AUC good related (Figure 57) among structural domain I and the II.Most of structure expections in these structural domains mainly are the structures of asialoization and mono-sialylated.Figure 57 demonstration is compared with those samples of structural domain I with higher per-cent and II, and clearance rate is higher in the sample of structural domain I with lower per-cent and II.Structural domain I is related with the existence of glycosylated peptide supposition increase among domain II I and the IV with the galactosylation that II reduces.
Although sialic acid and protein mol ratio generally have been used to predict the clearance rate of CTLA4Ig, the different fermentations substratum has been created in has identical NANA ratio but the molecule of different clearance rates in the monkey PK research.In order to develop the more best method of prediction clearance rate, assess 2 groups other analytical data (monose analysis and carbohydrate overview) and compare with monkey PK data.The analytical parameters that predictability is arranged most is the galactosylation degree of CTLA4Ig.In order to reduce the analytical variance of this mensuration, the semi-lactosi mol ratio is carried out stdn to the seminose mol ratio.Comprise method X material for all samples, resulting Gal:Man is than well related with the AUC result who studies from monkey PK.This result is mainly consistent by the model of the terminal GlcNAc residue number decision that exposes on the molecule with the clearance rate of CTLA4Ig.If this model is correct, the degree of galactosylation should be than the sialylated clearance rate of predicting better so.In order to ensure pharmacokinetics comparability (from the AUC of the material of X method〉75%), recommend Gal:Man for a large amount of CTLA4-Ig medicines (BDS) of purifying〉0.65 the semi-lactosi and the specific requirement of seminose ratio.
When the material only analyzed by the preparation of Y method or CD-CHO1 method, NANA and the protein mol ratio clearance rate that can calculate to a nicety.Yet this association is not suitable for the material by the preparation of X method.In order can to compare with X method material, the NANA that must be had high 2 units by the CTLA4-Ig of Y method or the preparation of CD-CHO1 method is than (can compare about the NANA=9 of CD-CHO1 and NANA=7 about X method material).Because sialic acid is measured to be accurate and to have the turnover time fast, so its is useful as analysis tool in carrying out with the quality of monitor fermentation run duration CTLA4Ig.
In order to keep comparable pharmacokinetics (AUC of reference〉75%), recommend NANA for the BDS of purifying〉8 specific requirement.This value also represents to be used to gather in the crops the reasonable target of fermentation operation.Because purification process can make sialic acid than increasing at least 2 units, be made as NANA=8 and will guarantee the actual cut value of NANA=7 at least so will gather in the crops target.Purification process can make this value be increased to NANA=9 at least, and this and method X material can compare and far surpass the minimum specific requirement of above recommending of BDS.
When the percentage area under domain II I and the IV and AUC compared, for method X material and method Y material, the carbohydrate overview was predicted monkey PK result equally well.Domain II I and IV mainly are made up of fully sialylated and carbohydrate structure galactosylation.
Can further present the data in the table 56, so that the association between the percent of total of the AUC summation of demonstration AUC value, NANA value, Ga1 value and domain II and IV.Vide infra:
AUC NANA Gal The summation of domain II I and IV
2,337 2.3 4.1 9.7
8,832 7.1 8.1 19.7
7,266 7.3 11.1 22.0
9,425 7.9 11.5 22.0
7,765 6.9 9.1 23.4
15,779 8.8 11.4 26.8
18,750 10 13.0 28.2
17,06015,75315,459 6.9 13.1 28.5
17,739 10.3 12.6 29.7
20,445 9.9 15.8 33.4
20,707 9.8 12.8 36.2
Existence is related between the summation that last table is presented at domain II I (and IV) and the PK result of composition.The AUC of domain II I and IV and V directly relates to the mol ratio of NANA and Gal and CTLA4-Ig protein mole.Therefore, the invention provides composition, it is characterized in that its carbohydrate overview comprises domain II I and the summation of IV or the summation of domain II I, IV and V of the about 37AUC% of 18-.In one embodiment, about 29, the about 28AUC% of about 27-of about 30, the about 26-of about 31, the about 25-of about 32, the about 24-of about 33, the about 23-of about 34, the about 22-of about 35, the about 21-of the summation of domain II, IV and V, about 20-about 36 for about 19-.In one embodiment, the invention provides the CTLA4-Ig composition, it is characterized in that domain II I has 19 ± 4 AUC% altogether; And structural domain IV has 7 ± 4 AUC% altogether.
The trypsinase peptide mapping of embodiment 43:CTLA4-Ig
CTLA4-Ig derived from Chinese hamster ovary (CHO) cell of transfection is the glycoprotein with about 92500 daltonian molecular weight.Peptide mapping is to be used for measuring the super-sensitive method of proteinic primary structure characteristic and in that to detect posttranslational modification useful.This protein uses guanidine-HCl to carry out sex change, use DTT and IAA reduces and alkylation.This protein uses the NAP-5 post to carry out desalination and digestion mixture is analyzed by anti-phase (C18) chromatography.The peak detects by finishing in the UV at 215nm place absorbancy.
Reagent: mobile phase A solution (water-soluble 0.02% trifluoroacetic acid (TFA) is (v/v)); Mobile phase B solution (being dissolved in the 0.02%TFA (v/v) of 95%ACN (acetonitrile) and 5% water); Alkylating agent (200mM iodo-acid amide (IAA)); Dilution buffer liquid (100mM Tris, 25mMNaCl, pH8.0); The sex change damping fluid (the 8M guanidine, 50mM TRIS, pH8.0); Digestion damping fluid (50mM TRIS, 10mM CaCl 2, pH8.0); Reductive agent (100mM DTT).
Instrument configuration: (can use instrument configuration of equal value) NAP-5 post (Amersham, catalogue #17-0853-02); HPLC post well heater; Water ' sAlliance HPLC system with post well heater and UV detector.
Reduction and alkanisation: sample (for example, CTLA4 A29YL104E-Ig, standard etc.) be diluted to 10mg/ml by adding the final volume (1mg) that adds water to 100 μ L.560 μ l sex change damping fluids and 35 μ L reductive agents (100mM DTT) are added in the 100 μ l samples, mix, and in Eppendorf centrifuge, be rotated down 3 seconds.Sample is subsequently in 50 ℃ of incubations 20 minutes ± 2 minutes.Subsequently 35 μ L alkylating agents (200mM IAA) are added in every kind of sample, and make sample mix once more, and in Eppendorf centrifuge, be rotated down 3 seconds.Sample is subsequently in 50 ℃ of incubations 20 minutes ± 2 minutes in the dark.The NAP-5 post is by after pouring into 3 column volumes (about 7-8mL) digestion damping fluid and carrying out balance, the mixture of 500 μ l reduction and alkanisation is poured on the NAP-5 post, thereby allows liquid to discharge through post.Subsequently via collecting sample from the NAP-5 post with 1mL digestion damping fluid elution samples from post.
Digestion: sample is with 20 μ L trypsin, 0.5 μ g/ μ L) 4 hours (± 0.5 hour) of digestion in 38 ℃ of water-baths.After digestion is finished.Sample carries out acidifying with 2.5 μ LTFA.Subsequently sample is placed in the automatic sampler bottle and be used for subsequent analysis.
Instrumental method: instrumental method is shown in hereinafter:
Time (minute) flow (mL/ minute) mobile phase A Mobile phase B
0 0.7 100 0
17 0.7 83 17
27 0.7 78 22
42 0.7 73 27
58 0.7 65 35
74 0.7 52 48
79 0.7 0 100
84 0.7 100 0
88 0.7 100 0
Before injection for the first time, post was with 100% mobile phase A damping fluid balance 25 minutes.The UV absorbancy is monitored at the 215nm place, and simultaneously column temperature maintains 37 ℃ and automatic sampler temperature maintenance at 4 ℃.Mobile phase A damping fluid blank is moved before first system's suitability criterion, is thereafter the single 50 μ L injection of every kind of sample subsequently.The reference material injection should be carried out together in per 6 samples injection.Describe by Figure 53 for the peptide figure chromatogram that the CTLA4-Ig sample produces.Retention time difference about peak T2, T3, T15 and T19 (Figure 53 and table 57) between the reference material chromatogram that initial sum is carried out together is necessary for ± and 0.5 minute.
The table 57. theory expectation fragment of the CTLA4-Ig of tryptic digestion
Segment number The residue numbering Single isotopic mass of planting Average quality Sequence
T1 1-14 1464.8 1465.7 MHVAQPAVVLASSR
T2 15-28 1484.7 1485.6 GIASFVCEYASPGK
T3 29-33 5743 574.6 ATEVR
T4 34-38 586.4 586.7 VTVLR
T5 * 39-83 4895.2 48983 QADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLR
T6. 84-93 1170.5 1171.4 AMDTGLYICK
T7 * 94-128 3993.9 3996.4 VELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPK
T8 ** 129-132 435.2 435.4 SSDK
T9 ** 133-158 2687.4 2689.1 THTSPPSPAPELLGGSSVFLFPPKPK
T10 159-165 834.4 835.0 DTLMISR
T11 166-184 2138.0 2139.3 TPEVTCVVVDVSHEDPEVK
T12 185-198 1676.8 1677.8 FNWYVDGVEVHNAK
T13 199-202 500.3 500.6 TKPR
T14 * 203-211 1188.5 1189.2 EEQYNSTYR
T15 212-227 1807.0 1808.1 VVSVLTVLHQDWLNGK
T16 228-230 438.2 438.5 EYK
T17 231-232 306.1 306.3 CK
T18 233-236 446.2 446.5 VSNK
T19 237-244 837.5 838.0 ALPAPIEK
T20 245-248 447.3 447.5 TISK
T21 249-250 217.1 217.3 AK
T22 251-254 456.2 456.5 GQPR
T23 255-265 1285.7 1286.5 EPQVYTLPPSR
T24 266-270 604.3 604.7 DELTK
T25 271-280 1160.6 1161.4 NQVSLTCLVK
T26 281-302 2543.1 2544.7 GFYPSDIAVEWESNGQPENNYK
T27 303-319 1872.9 1874.1 TTPPVLDSDGSFFLYSK
T28 320-324 574.3 574.7 LTVDK
T29 325-326 261.1 261.3 SR
T30 327-349 2800.3 2802.1 WQQGNVFSCSVMHEALHNHYTQK
T31 350-356 659.3 659.7 SLSLSPG
*Comprise N connection carbohydrate *Comprise O connection carbohydrate
Theoretical plate number: the post effect as the theoretical plate number assessment can use retention time and peak width to carry out quantitative measurment according to equation:
N = 16 ( t w ) 2
Wherein:
" w " is by straight relatively side being extrapolated to the peak width at the baseline place of base measurement, and " t " is the retention time at the peak measured of the elution time from inject time to the peak maximum.
If N＜50000 make post balance again so.
Resolving power: 2 peaks for example as shown in Figure 53 peak T30 and the resolving power (R) between the peak T12 can use equation to measure:
R = 2 ( t 2 - t 1 ) ( w 1 + w 2 )
Wherein:
t 1, t 2=be respectively the retention time of fragment peak T30 and peak T12
w 1, w 2=have retention time t respectively 1And t 2The peak width that limits of the tangent line at peak base place.
If R＜1.5, post balance again so, and if problem exist, post should be replaced so.
Example values: about the difference between the relative peak area of peak T3, T15 in test article and the reference material and T19 necessary≤10.0%.The relative peak area at peak is defined as the peak area of representing as the peak area per-cent of peak T2.Difference between the relative peak area of test article and the reference of starter system suitability obtains shown in hereinafter.Relative peak area (R SX) can calculate separately for peak T3, T15 and T19 in the chromatogram of test article by using following formula:
R SX＝(As X/A S2)*100
Wherein
R SXThe relative peak area of peak X in the=chromatogram
A SXIn=the sample area of peak X and
A S2The area of peak T2 in the=sample.
Similarly, relative peak area (R RX) can calculate separately for peak T3, T15 and the T19 in the chromatogram of standard.Difference between the relative peak area in sample and the standard (DX) can be calculated by using following formula subsequently:
D X＝[(R SX-R RX)/(R RX)]*100。
If there is single other peak in the sample, compare with peak T11 so, can measure by using following formula about the relative peak height at the sort of peak:
Relative peak height R T=(H T/ H 11) * 100 wherein
H T=have a peak height of retention time t minute
H 11=peak T 11Height, the climax in the chromatogram.
In one embodiment, if relative peak height≤5.0% at new peak, this overview is considered as consistent with the overview of CTLA4-Ig standard so.If the relative peak height at new peak〉5.0%, this overview is considered as with the overview of CTLA4-Ig standard material inconsistent so.
Per-cent oxidation data obtain by using the mapping of RP-HPLC trypsinase to measure, and Met85 is to the area percentage oxidation of methionine sulfoxide in the described mensuration quantitative protein.Per-cent oxidation in this method obtains by the UV peak area about the tryptic peptide T6ox of the T6 tryptic peptide that comprises the residue 84-93 that contains Met85 and the corresponding oxidation that comprises Met (O) 85 among the measure R P-HPLC trypsinase figure.The area percentage oxidation of Met85 to Met (O) 85 and the area percentage at T6ox peak are proportional:
Per-cent oxidation=100*A T6ox/ (A T6ox+ A T6)
Wherein,
A T6=about the T6 tryptic peptide, peak area (84-93).
A T6ox=about the T6ox tryptic peptide, Met (O) 85Peak area (84-93).
Per-cent deacylated tRNA amine data obtain by using the mapping of RP-HPLC trypsinase to measure, the area percentage oxidation of deacylated tRNA amine in the described mensuration quantitative assay, it is by obtaining about T26 tryptic peptide that comprises the residue 281-302 that contains Asn294 and the UV peak area that comprises the corresponding deacylated tRNA amine tryptic peptide T26deam1 of isoAsp294 among the measure R P-HPLC trypsinase figure.Subsequently, the area percentage at the area percentage deacylated tRNA amine of Asn294 to isoAsp294 and T26deam1 peak is proportional:
Wherein,
A T26=about T26, peak area (281-302)
A T26deam1=about T26deam1, isoAsp 294Peak area (281-302).
A T26deam2=about T26deam2, Asp 299Peak area (281-302).
A T26deam3=about T26deam3, Asp 294Peak area (281-302).
A T26deam4=about T26deam4, Asu 294Peak area (281-302).
Embodiment 44: the high-efficiency anion displacement chromatography by having Electrochemical Detection CTLA4-IgN connection oligosaccharides carbohydrate profile analysis
The carbohydrate structure that exists on the glycoprotein can influence in its function and the body to be removed.Therefore the structural integrity of carbohydrate of monitoring the recombinant production batch of glycoprotein is important.CTLA4-Ig is the recombinant protein that comprises N connection and O connection (Serine is connected) glycosylation site.Herein, monitoring CTLA4-Ig goes up N connection (l-asparagine connects) carbohydrate that exists.In this method, oligosaccharides is by (peptide: N-Glycosylase F) enzymatic digestion is cut with PNGase F, separate in 2 column systems by reversed-phase HPLC subsequently, separate by high-efficiency anion displacement chromatography (HPAEC), and monitor by Electrochemical Detection (amperometry of integration).The chromatogram that produces is a N connection carbohydrate overview, and wherein the overview of CTLA4-Ig sample should be similar to this kind.
This method has been described the program of the HPAEC oligosaccharides overview of measuring the N connection oligosaccharides that discharges from the CTLA4-Ig sample.The purpose of this method provides the chromatography overview of CTLA4-Ig medicine N connection oligosaccharides, the comparative analysis between this can be used for.Glycosylation on the CTLA4-Ig comprises N connection oligosaccharides.These oligosaccharides are by obtaining discharging with the process of PNGase F enzymically hydrolyse through 22 hours.The free oligosaccharides uses and utilizes the high pH anion-exchange chromatography of Electrochemical Detection to carry out profile analysis.The oligosaccharides overview of medicine is assessed at the sample of the reference material of moving simultaneously.The percent deviation at the identical peak in the structural domain that the result is reported as selection and peak and the reference standard.
％Diff Percentage difference
％RSD The per-cent relative deviation
HPAEC High pH anion-exchange chromatography
HPLC High performance liquid chromatography
NaOAc Sodium acetate
NaOH Sodium hydroxide
PNGase?F Peptide: N-Glycosylase F
Material.Except as otherwise noted, equivalent material can substitute.
Has bonded PTFE/ siloxanes Waters Corporation, catalog number (Cat.No.)
The Waters of partition always reclaims bottle 186000234
Microcon YM 10 centrifugal filter Millipore, catalog number (Cat.No.) 42407
Device
RapiGest SF Waters Corporation, catalog number (Cat.No.)
186001861
Instrument configuration and condition.Except as otherwise noted, can use instrument configuration of equal value.
Instrument configuration.
The Waters Corporation of Alliance HPLC system
Be equipped with: automatic sampler is (freezing
), eluent degassing module
Model 2465 electrochemical detectors
Post: CarboPac PA-14 x 250mm Dionex Corporation, catalogue
Numbers 35391
Guard column: CarboPac PA-14 x 50 Dionex Corporation, catalogue
Mm numbers 43096
Empower data gathering system version 3 .2 or the BMS version of verifying at present
The chromatography condition of the oligosaccharides overview by anion-exchange chromatography
29 ℃ of column temperatures
Flow velocity 1mL/ minute
Moving phase and gradient condition gradient program
The 1:500mM NaOAc time
2:400mM NaOH (divide %1 %2 %3
3:HPLC grade water Clock)
Initial 0 30 70
0.0 0 30 70
11.0 0 30 70
12.0 4 30 66
20.0 10 30 60
80.0 45 30 25
81.0 0 30 70
100 0 30 70
Waters 2465 is provided with
Mode pulse
Empower is provided with scope=5 μ A
E1＝+0.05V?E2＝+0.75V?E3
＝-0.15V
T1=400 millisecond t2=200 millisecond t3
=400 milliseconds
Sample time (ts)=100 millisecond
Time constant (strainer) t=0.1 second
Scope skew=5%
Polarity+
Temperature=29 ℃
Annotate: injecting before, made post and detector balance about 2 hours with the analysis flow velocity with initial flow, or until baseline stability.
The automatic sampling actuator temperature is made as:
4℃
Volume injected 60 μ L
100 minutes working times
The potential hump that is dominant in each structural domain
About retention time (RT; Minute)
(referring to Fig. 1); Value can rely on
In system's suitability (SS) standard
RT and becoming
About RTs (minute)
SS： 18.5
Peak 1A:20.0
Peak 1B:20.8
Peak 1C:21.4
Peak 1D:22.4
Peak 1E:23.1
Peak 2 31.5
Peak 3:44.8
Peak 4:58.5
Electrode cleaning (Waters 2465).Abide by the cleaning explanation in the detector handbook.Use the diamond slurry that provides in the flow cell test kit to polish the surface of electrode.If polishing does not produce acceptable result, replace electrode with new flow cell test kit so.Use new spacer (50 μ m) to rebuild flow cell.
Reagent.Annotate: according to department's program mark and write down all reagent preparation.
Be used for the preparation of the moving phase of HPAEC oligosaccharides carbohydrate profile analysis
HPAEC eluent 1:500mM sodium acetate (NaOAc).Weighing 20.51 ± 0.05g sodium acetate (anhydrous), adding comprises in the 500mL graduated cylinder of 400mL HPLC grade water.Make volume reach 500mL with HPLC grade water, and use plastics serology transfer pipet to stir 5 minutes until mixing fully.By 0.2 μ m NF filtering solution.Be transferred to the 1L eluant bottle.Sprayed 20 minutes with bottle loosely cover lid and with helium.Cover tight lid and make the bottle pressurization with helium.Solution was preserved under helium in maximum 3 weeks of room temperature.
HPAEC eluent 2:400mM sodium hydroxide (NaOH).Use the 1L graduated cylinder, measure 960mL HPLC grade water and be transferred to clean 1L eluant bottle.Use the serology plastic suction pipet, 40.0mL 10N NaOH is directly added in the eluant bottle, and by reverberating mixtures of eluents.Sprayed 20 minutes with bottle loosely cover lid and with helium.Cover tight lid and make the bottle pressurization with helium.Solution was preserved under helium in maximum 3 weeks of room temperature.
HPAEC eluent 3:HPLC grade water.Fill the 1L eluant bottle with about 1LHPLC grade water.Eluant bottle is placed in the system loosely cover lid and spraying about 20 minutes.Cover tight lid and make the bottle pressurization with helium.Solution was preserved under helium in maximum 3 weeks of room temperature.
The 50mM sodium phosphate buffer, 0.02% sodiumazide, pH=7.5.Sodiumazide (NaN 3) care should be used to handle with avoid sucking (poisonous) and with skin contact (stimulation).About other requirement consulting MSDS table.NaN 3After the weighing, the balance district should thoroughly clean.
NaH 2PO 4·H 2O 6.9g
Na?N 3 0.2g
H 2O 1.0L final volume
Weigh up 6.9g ± 0.1g NaH 2PO 4H 2O and 0.2g NaN 3, and be dissolved in 800mL HPLC grade H in the 1L reagent bottle 2Among the O, wherein use magnetic stirring bar to mix continuously.Use pH meter, use 10M NaOH that the pH of solution is adjusted to 7.5.Use the 1L graduated cylinder to make final volume reach 1.0L.Solution was preserved in room temperature maximum 6 months.Be dissolved in the 50mM sodium phosphate buffer, 0.02% sodiumazide, the PNGase F enzyme working solution of pH=7.5.
The 50mM sodium phosphate buffer
0.02% sodiumazide, pH=7.5. 1.8mL
From the PNGase F of test kit, catalog number (Cat.No.) P0704L 0.2mL
With 1.8mL 50mM sodium phosphate buffer, 0.02% sodiumazide, pH=7.5 are pipetted in the 1.8mL profound hypothermia bottle.Interpolation is from the 0.2mL PNGase F and the thorough mixing of test kit.Solution is preserved in-20 ℃ or lower maximum 6 months.Solution can carry out aliquots containig before freezing.
The external system suitability criterion.Stachyose stock solution (1.25mg/mL).Weighing 0.125g stachyose on pan paper.Operational analysis sky chessboard and be transferred to the 100mL volumetric flask.Fill to mark and thorough mixing with HPLC grade water.Arrive in the Nalgene cryovial with 2mL part aliquots containig.Solution is preserved in-20 ℃ or lower maximum 6 months.
Stachyose system suitability criterion (12.5 μ g/mL).1mL1.25mg/mL stoste is pipetted in the 100mL volumetric flask.Fill to mark and thorough mixing with HPLC grade water.Arrive in the 0.65mL Eppendorf tube with 200 μ L part aliquots containigs.Pipe is placed the storage box of suitable mark.System's suitability solution is preserved in-20 ℃ or lower maximum 6 months.
Standard and specimen preparation
The reference material preparation.In the bottle that comprises the freeze dried RapiGest SF of 1mg, add and comprise 0.02% sodiumazide, the 625 μ L 50mM sodium phosphate buffers of pH7.5.Annotate: the single library that should use the damping fluid that comprises RapiGest SF for all samples in the sample sets.A few bottle RapiGest SF can be reconstructed and make up so that enough volumes to be provided.In 0.65mL Eppendorf pipe, add the damping fluid that 120 μ L comprise RapiGest SF.Add 40 μ L reference materials (～50mg/mL).RapiGest SF final concentration should be 0.12%w/v.Add 40 μ L PNGase F work stoste, thorough mixing is rotated down sample, and places 38 ± 2 ℃ 22 ± 2 hours (water-bath or Alliance automatic sampler compartments).Sample is pipetted in the microcon YM-10 centrifugal filter, and 13 under the 000g centrifugal 30 minutes.200 μ L HPLC water are placed filter, and, be flushed in the filtrate in centrifugal other 30 minutes under the 000g by 13.Make the filtrate vortex 15 seconds of combination, and made sample centrifugal 10 seconds.Use transfer pipet that resulting solution (～380 μ L) is transferred to HPLC and always reclaim the automatic sampler bottle.
Specimen preparation.In 0.65mL Eppendorf pipe, add the damping fluid that 120 μ L comprise RapiGest SF.Add 40 μ L protein examples (this volume should equal 1-2mg CTLA4-Ig).RapiGest SF final concentration should be 0.12%w/v.Add 40 μ L PNGase F work stoste, by 10 seconds thorough mixing of vortex.Sample is rotated down, and places 38 ± 2 ℃ 22 ± 2 hours (water-bath or Alliance automatic sampler compartments).Sample is pipetted in the microcon YM-10 centrifugal filter, and 13 under the 000g centrifugal 30 minutes.200 μ LHPLC water are placed filter, and, be flushed in the filtrate in centrifugal other 30 minutes under the 000g by 13.Make the filtrate vortex 15 seconds of combination, and made sample centrifugal 10 seconds.Use transfer pipet that resulting solution (～380 μ L) is transferred to total recovery HPLC automatic sampler bottle.
Electrochemical detector pond stability: inject the outside stachyose system's suitability criterion (12.5 μ g/mL) of 30 μ L.Guarantee peak height 〉=800nA about stachyose.Guarantee not exist excessive electrical noise and baseline smooth from the pond.If stachyose sensitivity or baseline are unacceptable, check the damping fluid composition so, cleaning electrode or replacement electrode.If there is excessive noise, check that so the pond is to guarantee to remove all bubbles.Make pond stabilization and injection water threose standard once more once more.If problem exists, take other suitable actions or contact management person so.
Theoretical tray (N).Use following formula to measure theoretical plate number (N) based on the stachyose peak.This finishes by the Empower data analysis system or can also manual finish.
N＝16(t/W) 2，
Wherein
T: the retention time of when maximum height, measuring from inject time to the peak elution time
W: the peak width that is extrapolated to baseline by the side.
N is necessary 〉=and 6000.If the plate counting, is adjusted the operation gradient so less than 6000 or is replaced post.
Tailing factor (T).Use following formula based on stachyose peak measuring column tailing factor (T).This finishes by the Empower data analysis system or can also manual finish.
T＝(W 0.05/2f)，
Wherein:
W 0.05: the peak width during 5% height (0.05h).
F: from W 0.05The time the peak front edge to the measurement (width) of peak maximum.
T is necessary≤and 1.2.Form if tailing factor, is checked damping fluid so greater than 1.2, replace post or cleaning post, and injecting systems suitability criterion once more.
The suitability criterion retention time checking of stachyose system: retention time is that system is dependent.Stachyose system suitability criterion should demonstrate 18.5 ± 2.0 minutes retention time.
CTLA4-Ig reference material-observation comes the carbohydrate overview of first kind of reference material of carrying out together of the preceding injection of comfortable sample injection.The carbohydrate overview should be similar to the sort of shown in Figure 68.Be that system is dependent absolute retention time.Guarantee that first peak (peak 1A) among the structural domain I and the difference in the retention time between the main peak (peak 3) among the domain II I are 22 minutes to 28 minutes.If the profile at peak does not resemble obtain among Figure 68 the sort of, take suitable action (for example, inspection apparatus function, cleaning post, inspection/replacement damping fluid, replacement post) and assessment once more so.Following program can be used to clean post: turning off the pond and with 80 % eluent 1,20% eluent 2 post is cleaned 5 minutes, is 50 % eluent 1,50% eluent, 2 cleanings 10 minutes subsequently.Under starting condition, make post and pond (open in the pond) balance once more, and assessment once more.
Injection sequence:
The following injection sequence that isolating oligosaccharides is set:
Stachyose standard (30 μ L)
Reference material (60 μ L)
One or more samples (60 μ L)
Reference material (60 μ L)
Move between the reference material injection of recommending≤5 kinds of samples together to carry out.
Data analysis: handle chromatogram.In Empower, handle chromatogram about reference material and sample.Set integration parameters, thereby make peak profile and baseline be similar to the sort of shown in Figure 68, integrating line may need manual laying.Execution is about the calculating of relative structural domain area and relative peak area (table that comprises when finishing referring to the summary of Figure 68 and this embodiment).If carry out duplicate injection, measure mean value so about these parameters of reference material and every kind of sample.
For reference material, measure about each multiple structural domain I, II, III, peak 1A and the relative deviation of 1B with regard to all multiple mean values.
The comparison of sample overview and reference material overview.
Range estimation relatively.Whether working sample and reference material have the structural domain and the main peaks of similar number.Main peaks is those peaks ( peak 1A, 1B, 1C, 1D, 2,3 and 4) of mark during Figure 68 describes.Relative quantification relatively.The relative area of comparative sample (structural domain I, II and III and peak 1A and 1B; If carry out the duplicate injection of sample, use its mean value so) with average relative area from the reference material injection of carrying out.Mensuration is from the relative different of these areas of average reference material value.
Calculate.% structural domain area (structural domain area relatively): calculating is about the % structural domain area of the structural domain of the overview of reference material and sample.Schema reference Figure 68 about the structural domain area.According to the example among Figure 68, by use following information and formula computation structure territory per-cent ratio (retention time is the dependent and result of reflection among Figure 68 of system:
Structural domain I: the summation of the peak area when about retention time 18-24 minute (peak 1A-1E)
Domain II: from the summation at 26-38 minute peak
Domain II I: from the summation at 39-50 minute peak
Structural domain IV: from the summation at 51-64 minute peak
Structural domain V: from the summation at 65-75 minute peak
Annotate: will change according to the variation in the every day chromatographic property about the retention time window of structural domain.Correspondingly adjust the time.
For structural domain I-III,, so also calculate the reference material injection carry out together and the mean value in the sample if carry out duplicate injection.
% peak area (relative peak area).Calculating is about the % peak area of peak 1A, the 1B of the overview of reference material and sample, 1C and 3.Schema reference Figure 68 about peak area; Retention time is that system is dependent.Calculate peak per-cent ratio by using following information and formula:
For among peak 1A and the 1B each,, so also calculate the reference material injection carried out together and the mean value in the sample if carry out duplicate injection.
Percentage difference from reference material mean value is calculated.Use following formula to compare the percentage difference in the average relative area of structures of samples territory I-III, peak 1A and 1B with reference material to calculate:
％Diff＝|RM-S|/RM?x?100
Wherein:
RM=is about the purpose relative area mean value of reference material
S=is about the purpose relative area mean value of sample
| |=absolute value
The result: result to be reported be about structural domain I, domain II, domain II, peak 1A and peak 1B that calculate with percentage difference reference material.Comprise representative chromatography spectrum about reference material and sample integration.Comprise the structural domain I-III of sample and reference material and the relative area per-cent of peak 1A and peak 1B (mean value of the injection of carrying out together), to 1/10th of per-cent.In addition, for the reference material injection of at every turn carrying out together, about the % structural domain area of structural domain I, II and III and about the % peak area of peak 1A and 1B should its mean value 15% in.
Figure 57-62,68,76 and 82-84 show as described herein by the resulting data of N connection oligosaccharides overview.Figure 68 described general N connection oligosaccharides overview (structural domain I, II, III, IV and V and peak 1A and peak 1B batch mean value 5% in). Peak 1A, 1B and 1C represent that the sialic acid N that takes off of G0, G1 and G2 joins oligosaccharide structure.
Figure 68 shows the General N connection carbohydrate overview about the CTLA4-Ig composition.
Structural domain/peak Retention time (minute) Area percentage Peak height with respect to the climax The area percentage of parent's structural domain Structural domain is formed
Structural domain I 19.413 31.3 . . 5 peaks
Domain II 29.076 33.2 . 5 peaks
Domain II I 42.819 24 5 peaks
Structural domain IV 55.899 94 6 peaks
Structural domain V 67.546 2.2 6 peaks
Peak
1A 19.413 7.3 89.8 23.3
Peak 1B 20.29 10.7 100 34.2
Peak 1C 21.032 8.8 94.3 28.1
Peak 1D 21.925 28 27.5 8.95
Peak 1E 22.685 1.7 11.8 5.43
Peak 2 30.763 18.3 88.9 55.1
Peak 3 43.823 14.5 57.8 60.4
Peak 4 57.368 4.4 20.1 46.8
Directly over table show data about the tabulation of the N connection oligosaccharides overview of CTLA4-Ig.
Under table show observed CTLA4-Ig scope.
Structural domain/peak Minimum area % Maximum area %
Structural domain I 24.5 35.2
Domain II 26.3 34.1
Domain II I 21.9 31.5
Structural domain IV+V 7.9 18.6
Peak 1A 4.5 11.2
Peak 1B 8.7 11.8
Embodiment 45: about the external biological assay based on cell of CTLA4-Ig
The T cell needs 2 kinds of signals to be used for activation and follow-up propagation.First kind of signal provided by the interaction of antigen peptide and TCR-CD3 mixture.Second kind of costimulatory signal followed CD28 on the T cell and the interaction between the B7 protein on the antigen presenting cell and taken place.After these 2 kinds of signals are accepted, T emiocytosis cytokine interleukin II (IL-2).The release of IL-2 causes cell activation and propagation.Solubility, immunosuppressive compounds CTLA4-Ig also with antigen presenting cell on the B7 protein bound, thereby blocking-up interacts with the function of CD28 and stops IL-2 to produce required costimulatory signal.
In this method, the Jurkat T cell that is used in the luciferase genes transfection under the IL-2 promotor control in the presence of anti-CD3 with Daudi B cell co-stimulatory.Sting activating signal activation IL-2 promotor altogether, this produces luciferase protein matter successively.Resulting luminous signal uses the luciferase assay system to measure.In this system, CTLA4-Ig produces the minimizing of dose-dependently in the luciferase activity.
This method checks that CTLA4-Ig produces the effect of required costimulatory signal to IL-2.The existence of soluble CTL A 4-Ig stops the signal conduction between T cell and the antigen presenting cell.If there is not sort signal, do not produce IL-2 so, thereby stop the clonal expansion of T cell.Carrier with luciferase genes uses the IL-2 promotor to produce.Jurkat T cell carries out transfection with this report carrier subsequently.Select positive colony Jurkat.CA and be used for this method.
This biological assay relates to the T cell (Jurkat.CA) that stimulates transfection with anti-CD3 and B cell (Daudi).The inhibition that the common stimulation that is provided by the B cell is added by CTLA4-Ig.Jurkat.CA and Daudi cell inoculation in 96 holes, white, opaque, flat-floored hole, and are stimulated with anti-CD3 in the presence of the CTLA4-Ig of different concns.After 37 ℃ of incubation 16-20 hours, the hole is measured with regard to luciferase activity.Stimulate inhibition to reduce as seen altogether as dose-dependently in the luciferase activity via CTLA4-Ig.
Reagent: Daudi cell culture medium (being dissolved in 10% foetal calf serum of RPMI1640, the 1%MEM Sodium.alpha.-ketopropionate); Jurkat.CA cell culture medium (being dissolved in 10% calf serum of RPMI1640,1%MEM Sodium.alpha.-ketopropionate, 400 μ g/mL Geneticins); Biological assay substratum (being dissolved in the 0.2 μ g/mL anti-cd 3 antibodies and the 1% penicillin-Streptomycin sulphate solution of Daudi cell culture medium); Bright-Glo luciferase solution (Promega, catalog number (Cat.No.) E2620) from the system of mensuration.
Instrument configuration: Nikon, Diaphot 200 inverted microscopes; Packard TopCount NXT luminometer; Tecan Genesis liquid processor; Coulter Vi-Cell cell counter; ZymarkRapidPlate-96.
Programflow chart:
Figure A200680053116D04711
The preparation of working solution: be dissolved in the 3mL CTLA4-Ig solution (5000ng/mL) in the biological assay substratum
As shown in following table 58, preparation is about 8 point curves of standard, quality contrast and sample when the concentration of 100,4,2,1,0.5,0.2,0.1 and 0.002 μ g/mLCTLA4-Ig, and the final concentration after 2 times are diluted in the plate in mensuration is 50,2,1,0.5,0.25,0.1,0.05 and 0.001 μ g/mL.
Table 58. is used to produce the dilution of typical curve.
Curve point Typical curve The quality contrast Sample 1 Sample 2
1 100μg/mL 100μg/mL 100μg/mL 100μg/mL
2 4 4 4 4
3 2 2 2 2
4 1 1 1 1
5 0.5 0.5 0.5 0.5
6 0.2 0.2 0.2 0.2
7 0.1 0.1 0.1 0.1
8 0.002 0.002 0.002 0.002
Each hole of 96 orifice plates adds 200,000 cells and at 37 ℃, 5% CO 2Carry out incubation during with 85% humidity.With 12 x 10 6Jurkat.CA cell and 12 x 10 6The Daudi groups of cells is combined in the aseptic centrifuge tube.Make cell under～125xg centrifugal 10 minutes, and cell fully is resuspended in the 9mL Daudi cell culture medium, until can't see cell mass, to produce 2.7 x 10 by move liquid gently repeatedly with the serology transfer pipet in room temperature 6The concentration of cell/mL.To add in the appropriate bore of the plate that comprises cell from every kind of solution of 75 μ L of table 58.Plate seals with TopSeal-A subsequently, and at 37 ℃, 5% CO 2Incubation is 16-20 hour during with 85% humidity.Plate and Bright-Glo luciferase solution equilibria add 150 μ L Bright-Glo luciferase solution and mixing simultaneously in each hole to instrument temperature.Plate tightly is placed in mixing and was used in the dark balance among the TopCount NXT 10 minutes.In TopCount NXT, use 1 second integrations/hole subsequently or the luminometer of the particular type of use is suitably measured luminous signal.
Record reads in the standard analyzer from the output of TopCount NXT, and data transform by getting its logarithm (end 10).As shown in following equation, 4 parameter logarithmic models are carried out match from the conversion data of each article:
Equation 1: Log 10 ( y jk ) = D + ( A - D ) 1 + ( x j C ) B
Wherein:
A is the level ground, top of curve, and D is the level ground, bottom of curve, and B is a slope factor, and C is the concentration that produces the effect of the mean value that equals A and D.
Can calculate R for each article 2Statistic and shortage match F check.Can also calculate the ratio of test article with respect to minimum value, maximum value and the slope of standard material.In addition, can also calculate fiducial interval about ratio.
The relative potency of each article is measured by making single equation pair and the data fitting from the purpose article from the data combination of reference article.
Equation 2: Lo g 10 ( y ijk ) = D + ( A - D ) 1 + ( x ij C A * ( C R C A ) I ) B
Wherein
A, B and D parameter are that reference and test article are total, and C RBe reference parameter, C ABe test article parameters, and compare C R/ C AIt is relative potency.Subscript I is the indication variable.If data are from the purpose article, it is made as and equals 1 so, and if data from the CTLA4-Ig material, it is 0 so.
Change relative potency of each test article into the per-cent grade, and relative potency is as providing from the output of program.
8 relative potency results from the output of each 8 data set of analyzing can average, and standard deviation can use equation 3 and 4 to calculate respectively.Average result is reported as " the per-cent relative potency " that is rounded to nearest integer.
Equation 3:
Figure A200680053116D04732
Equation 4:
Figure A200680053116D04733
Wherein:
The 8th, the number of tiring and measuring
Ce Liang not for x=
The adjustment of the relative potency value that obtains with approximate concentration: because the time lag between the accurate protein concn is accepted and obtained to sample, sample can be tested with approximate concentration in mensuration, and adjusts the result when accurate concentration is determined.This adjustment uses equation 5 hereinafter to carry out, and the relative potency of wherein measuring in mensuration multiply by and is used to be provided with the CTLA4-Ig concentration of mensuration and the ratio of the CTLA4-Ig sample concentration of mensuration.
Equation 5:
Figure A200680053116D04741
Example:
Sample protein concn with 25mg/mL in mensuration is tested.
The relative potency of measuring is 105%.
The CTLA4-Ig concentration determination of measuring is 25.5mg/mL
Reportable relative potency=(105*25)/25.5=103%.
Test article relative potency value is necessary for the 25-175% of reference standard, and this is the scope of measuring.If the relative potency value is outside this scope, sample must dilute or concentrate dropping in this scope so, and sample is analyzed once more.
The structural characterization of embodiment 46:O connection oligosaccharides
The O linked glycosylation of CTLA4-Ig characterizes for ESI-MS/MS and MALDI-TOF subsequently by peptide mapping.
Analyze T8 and T9 by peptide mapping and ESI-MS/MS
Carry out the modified manual form of tryptic digestion method, it has the mass spectrometric axial ESI-MS/MS of the Finnigan ion trap (Ion Trap) of use, to characterize O connection glycopeptide T8 and T9 (about peptide feature reference table 59).
Figure 63 shows the tryptic peptide figure of CTLA4-Ig, wash-out when it points out that T8 finishes in the solvent front, and T9 is at the shoulder wash-out of T27.
The full mass spectrum of peptide T8 is shown among Figure 64, and wherein peak 436.2 is corresponding to the expection MW of the peptide T8 of single charged unmodified, and peak 1092.2 is corresponding to the MW of single charged glycosylated T8.Quality and structure (T8-HexNAc-Hex-NeuAc) thereof corresponding to main peak are shown in the table 61.
Table 61
Table 3.2.S.3.1.4.1.T03: the oligosaccharide structure of peptide T8
The full mass spectrum of peptide T9 is shown among Figure 65, the two and three charged ions of glycopeptide wherein occur, corresponding to a series of heterogeneous sugared shapes.Main peak 1115.9 is corresponding to the MW with the charged T9 of HexNAc-Hex-NeuAc glycosylated three.Peak 1213.0 and 1334.8 corresponds respectively to HexNAc (NeuAc)-Hex-NeuAc and (HexNAc-Hex-NeuAc) 2The molecular weight (table 62) of glycosylated three charged T9.Dominant sugared shape is the T9-HexNAc-Hex-NeuAc of mono-sialylated, and other sugared shapes exist with much lower abundance.
Table 62
Table 3.2.S.3.1.4.1.T04: the oligosaccharide structure of peptide T9
Figure A200680053116D04752
In order to measure O connection site, described in previous embodiment 4, carry out the peptide mapping of CTLA4-Ig, and collect fraction T8-9 (because by tryptic incomplete digestion) and implement the Edman order-checking.Sequencing data is presented in first circulation, the extra peak except that Ser occurs.The retention time at extra peak is the sort of all inconsistent with any standard amino acid, thereby the Ser (Ser 129) at 1st place of hint in T8 is modified and comprises O and joins glycan.The appearance at Ser and extra peak points out that Ser is that part is modified.This with about the MS data consistent of T8.The O connection site that the order-checking experiment also discloses among the T9 is Ser 139.In a word, 2 O linked glycosylation sites have been identified at amino-acid residue Serine 129 and Serine 139 places.The dominant glycan that adheres to 2 sites is HexNAc-Hex-NeuAc.
The MALDI-TOF of T9 peptide analyzes
The MALDI-TOF of T9 peptide analyzes exist (Figure 66) that confirms several sugared shapes.Peak with MW of 2690.8 is consistent with the T9 fragment.It is relevant that peak with MW of 2893.7 and T9 add HexNAc.It is relevant that peak with MW of 3055.7 and T9 add HexNAc-Hex.Peak indication T9 with MW of 3345.8 adds HexNAc-Hex-NANA.In T9, detect semi-lactosi and N-acetylgalactosamine based on the monose analysis, so the main O connection kind among the T9 is assumed to GalNAc-Gal-NANA.Because the low yield that reclaims, the MALDI-TOF that does not carry out peptide T8 analyzes.
Oxidation among the embodiment 47:CTLA4-Ig and deacylated tRNA amine variant
Oxidation and deacylated tRNA amine are the common product variations of peptide and protein.They can take place during fermentation, the clarification of results/cell, purifying, sample/medicament production duration of storage and sample analysis.
The protein oxidation general feature is that one or more Sauerstoffatoms add proteinic chemistry.Compare with other natural amino acids, a few seed amino acid Met, Cys, Tyr, His and Trp are easier to oxidation.The amino acid that has at the susceptibility of the top of oxidation is methionine(Met).The most protein oxidation of Jian Dinging up to now is the variant that methionine(Met) is oxidizing to sulfoxide.Oxidation in the protein can be caused by several different mechanisms.The common mechanism of oxidation is exposed by light or is transition metal-catalyzed and take place.
Deacylated tRNA amine is NH 3From protein, lose, thereby form the succinimide intermediate that can experience hydrolysis.The succinimide intermediate causes the quality of parent's peptide 17u to reduce.Hydrolysis subsequently causes the quality of 18u to increase.Because the unstable under aqueous conditions, the separation of succinimide intermediate is difficult.Equally, the quality increase that generally can be used as 1u of deacylated tRNA amine detects.The deacylated tRNA amine of l-asparagine causes aspartic acid or different aspartic acid.The parameter that influences deacylated tRNA amine speed comprises pH, temperature, solvent specific inductivity, ionic strength, primary sequence, local polypeptide conformation and tertiary structure.Amino-acid residue with the Asn vicinity in the peptide chain influences deacylated tRNA amine speed.Gly in the protein sequence behind the Asn and Ser cause the susceptibility higher to deacylated tRNA amine.
Materials and methods
Sample: the CTLA4-Ig standard
The trypsinase of CTLA4-Ig/AsD-N/ trypsinase and chymotryptic peptide mapping: protein carries out sex change and reduction in the 50mM Tris damping fluid (pH8.0) that comprises 6M guanidine and 5mM dithiothreitol (DTT) (DTT).In 50 ℃ of incubations after 20 minutes, add the final concentration of iodo-acid amide (IAM), and make sample in the dark in other 20 minutes of 50 ℃ of incubations to 10mM.The mixture of reduction and alkanisation is loaded on the NAP-5 post, and uses 50mM Tris, 10mMCaCl subsequently 2, the pH8.0 wash-out goes out.Add sequence grade trypsin 2%, w/w, enzyme: protein) and in 37 ℃ of incubations 4 hours.Under the situation of Asp-N digestion, add sequence grade Asp-N (4%, protein) and make sample w/w, enzyme: in 37 ℃ of incubations 16 hours.
Under the situation of trypsinase and pancreas milk reducing protease digesting, protein is at the 50mM sodium phosphate buffer, among the pH7.5.Protein) and make sample add sequence grade trypsin 4%, w/w, enzyme: in 37 ℃ of incubations 4 hours.Add Quimotrase (4%, protein) and make sample w/w, enzyme: in 37 ℃ of incubations 16 hours.All samples is preserved after digestion in-20 ℃.
(Waters, Milford MA) upward separated by gradient elution from Atlantis C18 post (2.1 x 250mm) with 0.120mL/ minute peptide mixt at Waters Alliance HPLC Workstation.(Waters, Milford MA) directly connect and collect mass spectrum for post and the Q-T of micro that is equipped with the electrospray ionization injection source.Under the situation of Asp-N peptide mapping, peptide mixt uses identical HPLC workstation upward to separate with 0.7mL/ minute at Varian C18 post (4.6 x 250mm).Post carries out balance with solvent orange 2 A (water-soluble 0.02%TFA), and peptide carries out wash-out by the cumulative concentration of solvent B (95% acetonitrile/0.02%TFA, water-soluble).Diverter valve is used for 15% direct traffic Q-T of micro behind the post.Instrument moves with holotype (m/z100-2000).Capillary voltage is made as 3000V.
The MS/MS of peptide analyzes: collect to be filled in the Q-Tofmicro from the fraction of anti-phase chromatography and with 20 μ L/ minutes flow velocity.The MS/MS spectrum is used and is obtained for the optimized collision energy of indivedual peptides (25-42eV).
The result
The oxidation of CTLA4-Ig: CTLA4-Ig has 7 methionine(Met)/strand: Met 1, Met 53, Met 54, Met 85, Met 97, Met 162And Met 338Peptide mapping is used for identifying the oxidation products variant of these sites on each.Met 1, Met 85And Met 162Oxidation use the tryptic peptide plotting technique to identify (Figure 67 A-B).There is not detected Met 338Oxidation.Comprise Met 53, Met 54Or Met 97The trypsinase fragment be the big peptide that comprises heterogeneous N connection carbohydrate.These peptides are difficult for accepting the evaluation and the relative quantification of oxidation.Therefore, carry out the proteolysis that uses plurality of enzymes, described enzyme produces shorter, nonglycosylated peptide.Asp-N peptide EVCAATYMMGN (46-56) and trypsinase/chymotryptic peptide MYPPPY (97-102) is used to measure Met 53, Met 54And Met 97Oxidation.The relative quantification of methionine(Met) oxidation calculates according to the peak area per-cent of the ion chromatography spectrum of extracting.The relative quantity of oxidation peptide is listed in table 63.Detect the oxidation on 6 in 7 CTLA4-Ig methionine(Met)s of every strand.Peak A and B are single oxidised forms at peptide EVCAATYMMGN (46-56) (peak 3).Peptide from peak 2A and 2B is isobaric, and compares with the peptide quality of unmodified, and the quality with 16u increases.Each peak is illustrated in the oxidation on the different Met.The degree of oxidation is different on 2 sites.
The oxidation of Met among the table 63.CTLA4-Ig
Met Peptide Prospective quality is non-oxide Observed quality is non-oxide The prospective quality oxidation Observed quality oxidation Oxidation per-cent
1 AT1：AMHVAQPAVVLASSR 768.9(+2)? 768.9 776.91(+2) 776.9 0.9
1 T1：MHVAQPAVVLASSR 733.4(+2)? 733.4 741.4(+2)? 741.4 2.4
53 D5：EVCAATYMMGN 1246.5 1246.6 1262.5 1262.5 34
54 D5：EVCAATYMMGN 1246.5 1246.6 1262.5 1262.6 4.3
85 T6：AMDTGLYICK 1171.6 1171.5 1187.6 1187.5 1.0
97 MYPPPY 767.3 767.9 783.3 783.9 1.5
162 T10：DTLMISR 835.4 835.4 851.4 851.4 1.7
338 T30： WQQGDVFSCSVMHEALHNHYTQK 1401.1(+2) 1401.1 1409.1(+2)
*Because the less contribution of AT1 oxidation, the per-cent of AT1 oxidation are not included in the calculating of estimated value of CTLA4-Ig oxidation.
The overall estimated value of CTLA4-Ig methionine(Met) oxidation is calculated as 2.0% for CTLA4-Ig.This is by calculating with the total oxidation per-cent addition on each site and divided by methionine(Met) overall number (it is 7).Oxidation of listing in the his-and-hers watches 63 and native peptides carry out MS/MS and analyze.
All peptides except that D5 use MS/MS to check order.The amino acid of oxidation is measured by the mass discrepancy in b and the y ionization series.The MS/MS spectrum of oxidation and natural T1 peptide makes and needs the precursor ion of m/z741.4 and 733.4 place's double-electrics.Mass discrepancy is 8u (for the double charge state), is 16u when carrying out timing for state of charge.Tryptic peptide T1 (1-14) comprises Met 1Residue.Native peptides and oxidized derivatives thereof are to carry out isolating baseline by reversed phase chromatography.Double-electric ionic ion chromatography spectrum about T1 and derivative thereof is shown among Figure 67 A-B.B and y ionization series are the dominant ions that produces in the dissociating of collision-induced.The y6-y13 ion has identical modified and mass of ion unmodified.The high 16u of corresponding b2 ion of the b2 ion ratio native peptides of oxidation peptide.B and y ionization series identify that together with the peptide quality methionine(Met) 1 is modified amino acid in the T1 peptide.In the same manner, identified Met oxidation in AT1, T6, T10 and MYPPPY (97-102) peptide.
The deacylated tRNA amine of CTLA4-Ig: CTLA4-Ig has 15 l-asparagine/strands.3 l-asparagines are known to be adhered to N connection carbohydrate structure.Peptide mapping is used to identify the deacylated tRNA amine that takes place with relative quantification on other 12 Asn residues.Asn 186, Asn 225, Asn 271, Asn 294And Asn 344Deacylated tRNA amine identify (table 64) by LC/MS tryptic peptide figure.The relative quantification of l-asparagine deacylated tRNA amine calculates according to the peak area per-cent of the ion chromatography spectrum of extracting.Taking off the relative quantity of amidated peptide lists in table 64.The overall estimated value of CTLA4-Ig l-asparagine deacylated tRNA amine is calculated as 0.3% for CTLA4-Ig.This is by calculating with the total deacylated tRNA amine per-cent addition on each site and divided by 15.Deacylated tRNA amine of listing in the his-and-hers watches 64 and native peptides carry out MS/MS and analyze.
The deacylated tRNA amine of Asn among the table 64.CTLA4-Ig
Asn Peptide The non-deacylated tRNA amine of prospective quality The non-deacylated tRNA amine of observed quality Prospective quality deacylated tRNA amine Observed quality deacylated tRNA amine Deacylated tRNA amine per-cent
186 T12： FNWYVDGVEVHNAK 839.4(+2)? 839.4 839.9(+2)? 839.9 0.9
225 T15： VVSVLTVLHQDWLNGK 904.5(+2)? 904.5 905.0(+2)? 905.0/905.0? 1.5/0.8
271 T25：NQVSLTCLVK 1161.6 1161.7 1162.6 1162.7 0.3
294 T26： GFYPSDIAVEWESNGQPENNYK 1272.6(+2) 1272.6 1273.1 (+2)? 1273.1 1.2
344 T30： WQQGNVFSCSVMHEALHNHYTQK 1401.1(+2) 1401.2 1401.6 (+2)? 1401.7 0.2
The amino acid of deacylated tRNA amine is measured by the mass discrepancy in b and the y ionization series.For example, if having 2 deacylated tRNA amine peaks then quality is 905.0u for peptide 15.Peak 1 is wash-out before natural peak; Peak 3 is wash-out behind natural peak.Tryptic peptide and deacylated tRNA amine form thereof are to carry out isolating baseline on reversed-phase column.Tryptic peptide is carried out MS/MS to be analyzed and takes off amidated peptide and list in table 64.Peak 1-3 comprises identical y1 and y2 ion, thereby points out that C-terminal amino acid is all identical for all 3 peaks; Yet, from the y3-y14 ion ratio of peak 1 and 3 the high 1u of corresponding ion from peak 2.Mass discrepancy between y2 and the y3 is 114u for peak 2; This is corresponding to the Asn residue.Comparing with Asn residue quality, is that the quality of 1u increases about the 115u of peak 1 and 3; This is corresponding to aspartic acid or different aspartic acid.In the same manner, identified the deacylated tRNA amine among T12, T25, T26 and the T30, and fragment ions is identified decorating site.
L-asparagine deacylated tRNA amine and methionine(Met) oxidation are measured by the LC/MS and the LC/MS/MS analysis of the endopeptidase cutting of CTLA4-Ig.Modification is identified by the variation of quality between the peptide modified and unmodified.Modified amino acid is identified by the MS/MS order-checking.6 methionine(Met) oxidations and 5 l-asparagine deacylated tRNA amine/strands are present in the CTLA4-Ig material with little per-cent.Find CTLA4-Ig Met 1, Met 53, Met 54, Met 85, Met 97And Met 162It all is oxidation.These oxidations that discovery is measured by CTLA4-Ig are less than 2.5% of all CTLA4-Ig methionine(Met)s.Find CTLA4-Ig Asn 186, Asn 225, Asn 271, Asn 294And Asn 344All to carry out deacylated tRNA amine on a small quantity.These deacylated tRNA amine that discovery is measured by CTLA4-Ig are less than 0.5% of all CTLA4-Ig l-asparagines.
Embodiment 48-is about CTLA4-Ig and CTLA4 A29YL104E The bacterial endotoxin of-Ig is measured
In one embodiment, CTLA4-Ig composition medicine has the bacterial endotoxin that is less than or equal to 0.15EU/mg.CTLA4-Ig is based on USP＜85〉use LAL (LAL) gel grumeleuse technology to measure with regard to the existence of bacterial endotoxin.In the preparation of measuring and using, observe the precaution in the sample preparation, to avoid total microbial contamination; Handle all containers or the utensil used and may be present in its lip-deep external intracellular toxin, for example in xeothermic baking box, use the circulation heating of checking to destroy.
LAL reagent: (Associates of Cape Cod, Inc.-or Equivalent) freeze dried king crab (Limulus)
Amebocyte lysate (LAL) reagent for example Pyrotell should be preserved according to the specification sheets of manufacturers.The LAL reagent of reconstruct can keep freezing and be no more than 1 month, and should not thaw or freezing surpassing 1 time.
The intracellular toxin standard: the control standard endotoxin (CSE) that uses in (Associates of Cape Cod, Inc.-or Equivalent) this test must be traceable, and carries out stdn at reference standard intracellular toxin (RSE).The intracellular toxin container of reconstruct is not preserved in refrigerator; In case reconstruct, they just can remain in 2-8 ℃ and be no more than 14 days, unless show suitable reactivity about the checking of longer time section.
The test of production batch
The product inhibition of LAL program or enhanced lack to tackle in every kind of pharmaceutical preparation to be verified.The test of product batch uses representative to produce beginning, 3 kinds of individual elements groups middle and that finish are carried out.These unit can separately or merge operation.Must comprise representative positive products contrast for Validity Test with CSE (or RSE) sample that inoculate, test concentrations of 2 times of amounts of solute marking sensitivity.Use aseptic no endotoxic HCl, NaOH or suitable damping fluid to adjust pH, thereby make that final product/solute solution is 6.0-8.0.In some cases, use buffer reagent for example Pyrosol may be useful as the replacement scheme that pH adjusts.About the explanation of concrete use with reference to manufacturers.
Be used for the use of the buffer reagent of solute reconstruct
Buffer reagent for example Pyrosol (Associates of Cape Cod Inc.) is used for the solute that reconstruct is used to test, and is designed to enlarge the surge capability of solute.The solute of Pyrosol reconstruct can be used for test may be needed to adjust pH with acid or alkali in other cases, or sedimentary sample or diluted sample thing take place when pH adjusts.When making up with specimen, the solute of Pyrosol reconstruct produces pink, to indicate pH in being used for the scope of Validity Test.If outside scope, color will be yellow or purple so; In this case, specimen will need other pH to adjust.Concrete grammar will indicate Pyrosol to be used for the purposes of solute reconstruct.
The dextran that is used for solute reconstruct suppresses the use of damping fluid
Glycan suppress damping fluid for example Glucashield (Associates of Cape Cod Inc.) is used for the solute that reconstruct is used to test, and is designed to block the potential dextran and disturbs.The solute of Glucashield reconstruct can be used to test the sample or the diluted sample thing that may comprise the dextran pollutent.Interference from the dextran pollutent can produce false positive in some test material, therefore use the solute of Glucashield reconstruct can block or reduce interference, thereby allows to reduce false-positive number.Concrete grammar will indicate Glucashield to be used for the purposes of solute reconstruct.
Diluted sample
If must in aseptic no intracellular toxin water, prepare the suitable serial dilution of sample so near the endotoxin concns level in the sample.Vortex and in each of 2 aseptic no intracellular toxin 10x75mm glass test tubees, add 0.1mL every kind of preparation to be tested.
Be used for the preparation of the intracellular toxin standardized solution of typical curve
The specification sheets endotoxic bottle of no intracellular toxin water reconstruct according to manufacturers.Bottle was acutely mixed 30 minutes off and on.Enriched material is stored in refrigerator was no more than for 4 weeks under 2-8 ℃.Use violent mixing of turbine mixer to be no less than 3 minutes before use.With reference to the analysis certificate of the manufacturers concentration with checking intracellular toxin stoste, and preparation is designed to include the standard intracellular toxin dilution series of terminal point, for example in following example:
0.5mL (1000EU/mL)+9.5mL do not have intracellular toxin water=50EU/mL
5.0mL (50EU/mL)+5.0mL do not have intracellular toxin water=25EU/mL
1.0mL (25EU/mL)+9.0mL do not have intracellular toxin water=2.5EU/mL
1.0mL (2.5EU/mL)+9.0mL do not have intracellular toxin water=0.25EU/mL
5.0mL (0.25EU/mL)+5.0mL do not have intracellular toxin water=0.125EU/mL
5.0mL (0.125EU/mL)+5.0mL do not have intracellular toxin water=0.06EU/mL
5.0mL (0.06EU/mL)+5.0mL do not have intracellular toxin water=0.03EU/mL
5.0mL (0.03EU/mL)+5.0mL do not have intracellular toxin water=0.015EU/mL
Before advancing to next time dilution, guarantee to make every kind of violent vortex of dilution at least 30 seconds.Comprise the negative water contrast of the thinner of use.Vortex and add 0.125EU/mL, 0.06EU/mL, 0.03EU/mL, 0.015EU/mL concentration (or alternative curve) and the contrast of negative water of each 0.1mL in each of 2 aseptic no intracellular toxin 10x75mm glass test tubees is to provide 2 repetition curves.
The preparation of LAL reagent solution
From refrigerator, take out freeze dried LAL reagent.Aseptic interpolation 5.0mL does not have intracellular toxin water (unless using the concrete grammar of reconstruct damping fluid that explanation is arranged in addition) in bottle.Bottle is reverberated or roll with solubilising reagent.
The preparation of positive products contrast
All products must be tested with the positive products contrast.About the explanation of preparation positive control, with reference to the available concrete grammar.If do not furnish an explanation, prepare the positive products contrast so, to be included in the intracellular toxin spike (spike) on the solute sensitivity 2X level, with the product combination on its testing level.When test injection water or high-quality process water, use the dilution of the intracellular toxin standard of reconstruct, thereby make that when in the adding sample, endotoxin concns will be the sensitivity of 2X LAL solution.For example, if solute sensitivity=0.06EU/mL, in the 1.9mL specimen, add so 0.1mL 2.5 EU/mL endotoxin solutions (with add the identical form of LAL solution)=0.125EU/mL.The volume of endotoxin solution is no more than 0.1mL, and overall dilution is not less than 1:20.
Test procedure
1. be assigned in each test specimens QC and the intracellular toxin standard pipe carefully not crossed contamination between pipe with 0.1mL LAL reagent solution is aseptic.Annotate: any residue reagent is being made an appointment with-10 ℃--25 ℃ place refrigerator.
2. the mixing about product-solute mixture illustrates, with reference to concrete solute inset.
3. each pipe is placed the incubation device for example water-bath or the heat block that maintain 37 ± 1 ℃.The temperature of record incubation time opening and water-bath or heat block.
4. make each pipe ground without disturbance incubation 60 ± 2 minutes.
5. behind the incubation, the record incubation concluding time and by make pipe reverse gently 180 ° observe each pipe.Positive findings keeps firm firm gel indication by careful when reversing, and negative findings is characterised in that and does not have this kind gel, maybe can't keep the formation of the viscous gel of its integrity.Handle carefully and manage and avoid to cause the vibration that false negative is observed its enforcement.
Assessment
Each minimum concentration that repeats generation positive findings in the series is called terminal point.Calculate geometrical mean terminal point about test by following program: the antilogarithm of the logarithm summation of geometrical mean=gelling terminal point is divided by repeating the endpoint determination number.Test is effectively, prerequisite be the geometrical mean terminal point in 2 times of dilutions of the solute sensitivity of mark, the positive products contrast is a male, and the contrast of negative water is negative.Solute sensitivity by mark relatively and positive or negative result measure the approximate bacterial endotoxin level in the test event or on it, the dilution factor coupling of the project of described positive or negative result and test.If do not form firm gel on the specified level of endotoxin in indivedual feature articles or specification sheets, article meet the requirement of test so.
About CTLA4 A29YL104E The method of-Ig
This method is used for using dynamically than turbid LAL standard measure CTLA4 A29YL104EBacterial endotoxin in the sample of-Ig medicine and medicament production.The result is reported as EU/mL and EU/mg medicament production Equivalent.
Term:
The LAL-LAL
The CSE-control standard endotoxin
EU-American Pharmacopeia endotoxin unit
EU/mg-American Pharmacopeia endotoxin unit/milligram
EU/mL-American Pharmacopeia endotoxin unit/milliliter
The LRW-LAL reagent water
LAL is than turbid
Figure A200680053116D04841
Solution.Allow
Figure A200680053116D04842
With Bottle before opening, heat to room temperature 30 minutes.Removal from
Figure A200680053116D04844
Metallic seal thing and aseptic removal stopper.Use 5.0mL
Figure A200680053116D04845
The reconstruct of reconstruct damping fluid
Figure A200680053116D04846
Bottle is reverberated gently to mix until dissolving fully.Reconstruct damping fluid before use tightly.Reconstruct
Figure A200680053116D04847
Can be maintained at 2-8 ℃ and be no more than 24 hours.With
Figure A200680053116D04848
The top of layer covering container.
Control standard endotoxin solution.Allow the bottle of CSE (1.2) before opening, to heat to room temperature 30 minutes.Removal is from the metallic seal thing and the aseptic removal stopper of bottle.Mention stopper carefully and only be enough to allow air admission, thereby destroy vacuum.With 5.0mL LRW (1.4) reconstruct control standard endotoxin (CSE).With
Figure A200680053116D04849
Sealing.At room temperature, experience the 30-60 minute timed interval with violent vortex of 5-10 minute the timed interval 1 minute.CSE is ready-made subsequently available.For obtain with the USP-EU/ bottle represent at
Figure A200680053116D048410
Concrete batch control standard endotoxin (CSE) is tired, with reference to the analysis certificate of manufacturers.Calculate the USP-EU/mL of the CSE in the bottle.The CSE of reconstruct can be maintained at 2-8 ℃ 30 days.After each the use, with new Sealed vessel.
Example:
For with 5.0mL LRW reconstruct have 6, the bottle of tiring of 000USP-EU/ bottle, tiring can be 1,200USP-EU/mL.
Figure A200680053116D048412
Be provided with
Figure A200680053116D048413
Location parameter in the software.General parameters.Parameter in the general test information is set as shown in following table.
General test information
Parameter Value
The significant temp stated range minimum 36.5
Significant temp scope maximum value 37.5
Reclaim stated range minimum about spike 50
Reclaim the scope maximum value about spike 200
Threshold value OD (mAbs) 20
The maximum OD (mAbs) of storage 100
The full test time (minute) 120
Baseline Adj. activity Check
Automatically finish Do not check
Check negative control Do not check
Option
Parameter Value
The mark relation conefficient Check
Show relation conefficient Unchecked
The mark variation coefficient Unchecked
Extrapolation exceeds Unchecked
Automatic test I D Unchecked
Demonstration is not by passing through the result Check
Automatically print Unchecked
Pipe appointment-example
The pipe numbering Sample description Standard/spike concentration Unit The sample dilution *
1 Negative control EU/mL
2 Negative control EU/mL
3 Standard 0.064 EU/mL
4 Standard 0.064 EU/mL
5 Standard 0.032 EU/mL
6 Standard 0.032 EU/mL
7 Standard 0.016 EU/mL
8 Standard 0.016 EU/mL
9 Standard 0.008 EU/mL
10 Standard 0.008 EU/mL
11 Standard 0.004 EU/mL
12 Standard 0.004 EU/mL
13 Standard 0.002 EU/mL
14 Standard 0.002 EU/mL
15 Sample 1 EU/mL 1：1
16 Sample 1 EU/mL 1：1
17 Sample 1 spike 0.016 EU/mL 1：1
18 Sample 1 spike 0.016 EU/mL 1：1
19 Sample 2 EU/mL 1：1
20 Sample 2 EU/mL 1：1
21 Sample 2 spikes 0.016 EU/mL 1：1
22 Sample 2 spikes 0.016 EU/mL 1：1
23 Sample 3 EU/mL 1：1
24 Sample 3 EU/mL 1：1
25 Sample 3 spikes 0.016 EU/mL 1：1
26 Sample 3 spikes 0.016 EU/mL 1：1
27 Sample 4 EU/mL 1：1
28 Sample 4 EU/mL 1：1
29 Sample 4 spikes 0.016 EU/mL 1：1
30 Sample 4 spikes 0.016 EU/mL 1：1
31 Positive water contrast 0.016 EU/mL
32 Positive water contrast 0.016 EU/mL
Preparation standard curve concentration.Control standard endotoxin (CSE, 2.2) the work stock solution of preparation 4USP-EU/mL.The CSE working solution of tiring of preparation 4EU/mL.Use equation 1 to calculate the required LRW volume of dilution.Add 20 μ LCSE solution (2.2) among the LRW (1.4) of appropriate amount in polystyrene tube (1.9) (equation 1).Made the dilution vortex 30 seconds.
Example:
For 1, the CSE solution of tiring of 200EU/mL adds 20 μ L CSE solution in 5,980 μ LLRW.
The CSE work stock solution of preparation 0.64EU/mL.The CSE working solution of tiring for preparing 0.64USP-EU/mL by the 1.6mL CSE stock solution that adds 4USP-EU/mL among the 8.4mLLRW in polystyrene tube.Vortex 30 seconds.
The preparation standard stock solution.The standard stock solution by the CSE working solution in the polystyrene tube with 0.128,0.064,0.032,0.016,0.008 and 0.004EU/mL be prepared.After dilution, make each pipe vortex 30 seconds.Dilution scheme is shown in the following table.
Dilution scheme about the standard stock solution
Volume (μ L) concentration (EU/mL) LRW?(mL) Final original liquid concentration (EU/mL) *
2mL0.64EU/mL solution 8 0.128
4mL0.128EU/mL solution 4 0.064
4mL0.064EU/mL solution 4 0.032
4mL0.032EU/mL solution 4 0.016
4mL0.016EU/mL solution 4 0.008
4mL0.008EU/mL solution 4 0.004
*Finally 2 times are diluted in the reaction tubes and take place.
Specimen preparation.The drug sample preparation.Prepare diluted sample thing to 0.25mg/mL.Use equation to calculate the required LRW volume of dilution.Take out the top of sampling receptacle carefully and 50 μ L samples are added the LRW of the appropriate amount (equation) in the polystyrene tube, with preparation 0.25mg/mL solution.Made the sample vortex 30 seconds.With
Figure A200680053116D04881
The top of layer covering sampling receptacle.
Example:
For sample, in 4.89mL water, add 50 μ L (0.05mL) samples with 24.7mg/mL concentration
Equation 2:
Figure A200680053116D04882
The specimen preparation of medicament production lyophile.Annotate: single medicament production batch is made up of 3 kinds of samples that separate-" beginning ", " centre " and " end ".Identify the bottle of (PI) specific requirement according to product with LAL reagent water reconstruct medicament production.The diluted sample that makes reconstruct is to 0.25mg/mL.Use equation 2 to calculate the required LRW volume of dilution.Take out the top of sampling receptacle carefully and 50 μ L samples are added the LRW of the appropriate amount (equation 2) in the polystyrene tube, with preparation 0.25mg/mL solution.Made the sample vortex 30 seconds.With
Figure A200680053116D04883
Cover the top of sampling receptacle.
Medicament production i.e. the specimen preparation of usefulness.Make diluted sample to 0.25mg/mL.Use equation 3 to calculate the required LRW volume of dilution.Take out the top of sampling receptacle carefully and 10 μ L samples are added the LRW of the appropriate amount (equation 3) in the polystyrene tube, with preparation 0.25mg/mL solution.Made the sample vortex 30 seconds.With
Figure A200680053116D04884
Cover the top of sampling receptacle.
Example:
For sample, in 4.99mL water, add 10 μ L (0.01mL) samples with 125.0mg/mL concentration
Equation 3:
Figure A200680053116D04885
Medicament production i.e. the placebo of usefulness.Make placebo (just as its nominal medicament production protein concn) be diluted to 0.25mg/mL at 125mg/mL.This is equivalent to 1: 500 dilution.Use equation 3 to calculate the required LRW volume of dilution.Take out the top of sampling receptacle carefully and 10 μ L samples are added the LRW of the appropriate amount (equation 3) in the polystyrene tube, with preparation 0.25mg/mL solution of equal value.
Positive water contrast.Positive control is prepared by the typical curve that 100 μ L, 0.032 EU/mL standard is added among the 100 μ LLRW in the reaction tubes.
Reaction tubes setting-example
The pipe numbering Describe Standard original liquid concentration (EU/mL) Standard/sample (μ L) LRW (μL) Spike Soln. (μ L) Final concentration (EU/mL)
1 Negative control - 0 200 0 0.000
2 Negative control - 0 200 0 0.000
3 Std.1 0.004 100 100 0 0.002
4 Std.1 0.004 100 100 0 0.002
5 Std.2 0.008 100 100 0 0.004
6 Std.2 0.008 100 100 0 0.004
7 Std.3 0.016 100 100 0 0.008
8 Std.3 0.016 100 100 0 0.008
9 Std.4 0.032 100 100 0 0.016
10 Std.4 0.032 100 100 0 0.016
11 Std.5 0.064 100 100 0 0.032
12 Std.5 0.064 100 100 0 0.032
13 Std.6 0.128 100 100 0 0.064
14 Std.6 0.128 100 100 0 0.064
15 Sample 1 - 100 100 0 Unknown
16 Sample 1 - 100 100 0 Unknown
17 Sample 1 spike - 100 0 100 Form+0.016
18 Sample 1 spike - 100 0 100 Form+0.016
19 Sample 2 - 100 100 0 Unknown
20 Sample 2 - 100 100 0 Unknown
21 Sample 2 spikes - 100 0 100 Form+0.016
22 Sample 2 spikes - 100 0 100 Form+0.016
23 Sample 3 - 100 100 0 Unknown
24 Sample 3 - 100 100 0 Unknown
25 Sample 3 spikes - 100 0 100 Form+0.016
26 Sample 3 spikes - 100 0 100 Form+0.016
27 Sample 4 - 100 100 0 Unknown
28 Sample 4 - 100 100 0 Unknown
29 Sample 4 spikes - 100 0 100 Form+0.016
30 Sample 4 spikes - 100 0 100 Form+0.016
31 Positive water contrast 0.032 0 100 100 0.016
32 Positive water contrast 0.032 0 100 100 0.016
Add
Figure A200680053116D04901
Reagent.In each reaction tubes, add 50 μ L with repetitive pipettor
Figure A200680053116D04902
Solution (negative control, standard, sample, spiked sample and the contrast of positive water).Make each pipe vortex 1-2 second, and be inserted in the designation hole in the Pyros Kinetix instrument (table 1).
Data analysis.
Analyze.
Figure A200680053116D04903
Software will be carried out the automatic analysis of all standards, contrast and sample, and the sample result that report is represented with EU/mL when operation is finished.Every kind of bipartite sampling report is 2 a mean value.If one of pipe of bipartite paired value via
Figure A200680053116D04904
Software does not detect, and can get rid of that pipe so and do further data analysis, and recomputate the result.Spike recovery (positive contrast) will add the intracellular toxin amount (0.016EU/mL) of mixing in the sample based on the intracellular toxin amount in the sample and calculate.
The medicine of dilution or the EU/mL value of medicament production sample are transformed into the EU/mg value
Raw data produces as EU/mL, and as proteinic EU/mg report.For EU/mL is transformed into EU/mg, with intracellular toxin value (EU/mL) divided by protein concn (0.125mg/mL).Result's report is to 1 significant figure.
Example:
For the sample that has 0.23EU/mL in mensuration, reportable EU/mg value will be 2EU/mg (1.8 are rounded to 1 significant figure).
( 0.23 EU / mL 0.125 mg / mL ) = 1.84 EU / mg = 2 EU / mg
Result about drug sample.This result is determined as 1 significant figure.The QL that measures is 0.02EU/mg.If sample≤0.02EU/mg, so reportable result is [＜QL, (QL=0.02EU/mg)].Result about the medicament production sample.The mean value of 3 samples (" beginning ", " centre " and " end ") of every batch of medicament production representing with EU/mg is for the reportable result of medicament production sample with 1 significant figure.For the situation of wherein batch one or more sample＜QL (0.02EU/mg), the value of 0.02EU/mg will be used for calculating mean value.If average reportable result is≤0.02EU/mg that so reportable result is [＜QL, (QL=0.02EU/mg)].
Example:
Sample from medicament production batch Value EU/mg
Beginning <QL＝0.02EU/mg
Middle 0.023
Finish 0.031
Reportable value (mean value) 0.02EU/mg
Example hereto, the medicament production placebo will be reported as: 3EU/mL is equivalent to the 0.02EU/mg when the nominal medicament production concentration of 125mg/mL.
System's suitability.The medicament production sample should be contained in the polystyrene container in 2-8 ℃.If sample is contained in the different vessels under the differing temps, they can not use in mensuration so.The coefficient of determination (r about typical curve 2) necessary 〉=0.99.If r 2Value＜0.99, this mensuration is invalid and must carries out repetition so.Average endotoxin concns about the measurement of negative control is necessary＜0.002EU/mL.If negative control 〉=0.002EU/mL, this mensuration is invalid and must carries out repetition so.The sample value of spike must drop in the 50-200% scope of desired value.If the sample value of spike be desired value≤49% or 〉=201%, this mensuration is invalid and must carries out repetition so.Average endotoxin concns about the measurement of positive water contrast must drop in the 50-200% scope of the same concentrations in the typical curve.If positive water control value be desired value≤49% or 〉=201%, this mensuration is invalid and must carries out repetition so.Intracellular toxin value about sample must drop on (0.002-0.064 USP-EU/mL) in the intracellular toxin standard curve range.If sample＜0.002EU/mL, they are lower than the QL of mensuration and report according to part 5.4 so.If sample〉0.064 USP-EU/mL, sample must further be diluted in the scope of mensuration so.
Embodiment 49-is about the microbial limit test (biological load) of CTLA4-Ig
This method provides the test procedure that is used to assess the survival aerobic microorganism number of existence and is used to exempt the prescribed microorganism species.In one embodiment, the CTLA4-Ig composition should have≤biological load of 1CFU/10mL level.In the preparation and application of test, when handling sample, note the aseptic item of observing.Term " growth " is defined as the propagation of the existence and the supposition of live microorganism.The checking of test result relies on them to be applied to the confirmation of its specimen from the microorganism growth that does not suppress to exist to a great extent under test condition.This confirmation should comprise that the survival culture that separates with suitable attack biology attacks the suitably prepd sample of material to be tested, will not suppress the growth of these microbe species to guarantee to test, if they are present in the material of test.That part of any beta-lactam product that is used to test must be handled with the penicillinase of appropriate amount according to the program of checking.
Diluted fluid and substratum
1. the preparation of substratum and diluted fluid.Can prepare substratum and diluted fluid, maybe can use the substratum of dehydration, prerequisite be when as during the reconstruct instructed by manufacturers or dealer, they have similar composition and/or produce and derive from those comparable substratum of the prescription that this paper provides.By in the formulation substratum, soluble solids is dissolved in the water, use heating in case of necessity, dissolve fully with realization, and add the hydrochloric acid or the sodium hydroxide solution of q.s, to produce the required pH in the time spent substratum in sight.Substratum uses the sterilizing program of checking to sterilize in autoclave.PH is measured in the sterilization back in the time of 25 ± 2 ℃.
Growth
A) according to the condition that hereinafter describes in detail by using bipartite test container and incubation, with regard to every batch of autoclaved substratum of its growth aptitude tests less than every kind of substratum of 100 kinds of microbial inoculants.The substratum that biological distance is received at first by ATCC can not surpass for 5 generations.
B) if occurred the growth evidence for bacterium in 5 days in 48-72 hour and for fungi, test media is gratifying so.Growth test can be carried out simultaneously with the purposes that test media is used for testing of materials.Yet, if this growth test is unsuccessful, so testing of materials be considered as invalid.
Be used for test microbes in the growth test use of substratum
Figure A200680053116D04931
Figure A200680053116D04941
*Other non-pathogenic Salmonellas species are also suitable.
About total aerobic microorganism counting or the mould of total combination and the program of yeast counts
A) specimen preparation.Unless concrete grammar has explanation in addition, be prepared as follows sample and be used for test.Depend on and carry out which kind of test, about relating to the other information of substratum and incubation program, with reference to hereinafter suitable part.
I) loose powder and starting material-with 10 gram or 10mL sample dissolution or be suspended in the 90mL sterile phosphate pH of buffer 7.2.Thorough mixing and 1.0mL transferred in 2 sterile petri dish each.
Ii) capsule-with 2 capsule shell and content is aseptic transfers among the aseptic phosphate buffered saline buffer pH7.2 of 20mL.In water-bath (about 45 ℃), heated about 10 minutes.Thermal agitation becomes evenly until suspension, and 1.0mL is transferred in 2 sterile petri dish each.
Be used to iii) that the powder that suspends-explanation uses aseptic phosphate buffered saline buffer pH7.2 as thinner reconstruct sample according to label.Fully vibration and 1.0mL transferred in 2 sterile petri dish each.
Iv) solution/suspension-1.0mL is transferred in 2 sterile petri dish each.
V) tablet-4 tablets (hard tablet should at first with aseptic mortar and pestle pulverizing) are transferred to the aseptic phosphate buffered saline buffer pH7.2 of 20mL.Fully vibration is decomposed fully or is dissolved until tablet, and 1.0mL is transferred in 2 sterile petri dish each.
Vi) capsule shell-25 capsule shell are transferred to the aseptic phosphate buffered saline buffer pH7.2 of 100mL, and in water-bath (about 45 ℃), heated about 15 minutes, vibration is to dissolving off and on.Fully vibration and 1.0mL transferred in 2 sterile petri dish each.
Vii) ointment-in sterile chamber merges the about 5mL and the mixing of crossing in batch 3 samples that obtain each.With in each of sample transfer to 10 culture dish of this merging of 0.1mL.By means of aseptic curved glass rod (the bar-shaped thing of hockey) sample is coated on the media surface.Use aseptic rod separately, incubation described in " total aerobic counting " for every block of plate.
B) membrane filtration.As pouring into the alternative scheme of paving the plate program, can use membrane filtration test procedure suitable, checking.This may be particularly useful for the product that comprises inhibitory substance.
C) test again.In order to confirm suspicious result by any following program, test can use the original sample size of two and 1/2nd times (bottom lines) to carry out again, follows suitable dilution adjustment.
Test about total aerobic microorganism counting
1. the sample to be tested of preparation as described.Add the aseptic TSA of 15-20mL in every block of plate, described TSA has melted and has been cooled to about 45 ℃.Cover each ware, and ware is tilted gently or reverberate, and allow content at room temperature to solidify with biased sample and agar.Be inverted plate and in 30-35 ℃ of incubation 48-72 hour.
2. behind the incubation, use for example growth of Quebec colony counter inspection plate of multiplying arrangement, counting bacterium colony number, and as specified based on unit (every tablet, capsule, mL, gram etc.) calculation result in the material explanation, and acceptable at material explanation assessment.
3. further characterize bacterial contamination by gramstaining and micromorphology.Gram negative bacterium and gram-positive cocci are implemented biochemical test (or alternative suitable authentication method).
Annotate: when the counting bacterium colony, in order to promote the differentiation from the colony growth of material to be tested, use chlorination 2,3,2% solution of 5-triphenyltetrazolium (TTC) is desirable as the toughener that is used to observe microorganism growth sometimes.TTC is colourless oxidation-reduction indicator, reddens during the reducing sugar hydrogenation found in by viable cell, thereby makes bacterium colony become scarlet.For using TTC, covered with the Petri plate, and make plate in about 2 hours of 30-35 ℃ of incubation with 2% solution of about 1mL.Microbe colony will obviously protrude in the other materials that exists on the plate and can more easily count.
C. about the mould of total combination and the test of yeast counts.1. the sample to be tested of preparation as described.Add the aseptic SDA of 15-20mL in every block of plate, described SDA has melted and has been cooled to about 45 ℃.Cover each ware, and ware is tilted gently or reverberate, and allow content at room temperature to solidify with biased sample and agar.Be inverted plate and in 20-25 ℃ of incubation 5-7 days.[annotating: between incubation period, do not disturb plate ,] because mould can produce the counting higher than physical presence at the plate internal diffusion.
2. behind the incubation, use for example growth of Quebec colony counter inspection plate of multiplying arrangement, counting bacterium colony number, and as specified based on unit (every tablet, capsule, mL, gram etc.) calculation result in the material explanation, and acceptable at material explanation assessment.
3. further characterize fungal contamination by both macro and micro morphology.In the time of suitably, yeast is implemented biochemical test (or alternative suitable authentication method).
About testing disagreeable biological non-existent program
Unless a) specimen preparation-concrete grammar has explanation in addition, be used for testing about preparing sample described in the mould of total aerobic and total combination and the zymic specimen preparation partly as mentioned.Depend on and carry out which kind of test, about other information, with reference to hereinafter suitable part.
B) membrane filtration.As the alternative scheme of pre-concentration program, can use membrane filtration test procedure suitable, checking.This may be particularly useful for the product that comprises inhibitory substance.
C) test again.In order to confirm suspicious result by any following program, test can use the original sample size of two and 1/2nd times (bottom lines) to carry out again, follows suitable dilution adjustment.
B. about streptococcus aureus and the non-existent test of Pseudomonas aeruginosa.In sample, add TSB to obtain 100mL, mix and in 30-35 ℃ of incubation 24-48 hour.Check the growth of substratum, and use transfering loop that part is scoring to selective medium if present, the feature (identification kit that is obtained commercially can replace indivedual reaction tests) that incubation and inspection are hereinafter listed:
The feature of streptococcus aureus:
Figure A200680053116D04961
*With the representative suspicious colony lift of self-selectively agar to containing don't bother about of 0.5mL Mammals (preferred rabbit or horse) blood plasma, in 37 ℃ of incubations, in 3-4 hour and be up to 24 hours and check in the suitable timed interval subsequently and solidify.
The feature of Pseudomonas aeruginosa:
Figure A200680053116D04971
*For pigment test, with the suspicious bacterium colony of representativeness from the Centrimide agar streak to PSF with the PSP ware.Cover and in minimum 3 days of 35 ± 2 ℃ of incubations.Inspection plate under UV light.
*For the oxydase test, use N by being transferred to, the bar or the ware of N-dimethyl-p-phenylenediamine dihydrochloride dipping confirm any suspicious bacterium colony; If there is not pink colour to become the development of purple, so samples met about Pseudomonas aeruginosaNon-existent requirement.Should be understood that to use and confirmed can accept the test kit that is obtained commercially that form is carried out.
If do not observe growth, if or the bacterium colony of finding none meet the feature group of listing in showing, samples met is about the non-existent test request of the sort of biology so.
About Salmonellas species and the non-existent test of intestinal bacteria
With sample transfer to comprising the sterile chamber of 100mL fluid lactose medium altogether, and in 30-35 ℃ of incubation 24-48 hour.Vibration and inspection growth gently.(identification kit that is obtained commercially can replace indivedual reaction tests.If) a) salmonella-growth be present in the fluid lactose medium:
1. 1.0mL partly is transferred to the 10mL pipe of fluid selenite Gelucystine substratum and fluid tetrathionic acid salt culture medium, mixes and in 30-35 ℃ of incubation 24-48 hour.
2. by means of transfering loop, part is scoring on brilliant green agar substratum, wood sugar-Methionin-desoxycholate agar substratum and the bismuth sulfite agar medium from 2 kinds of substratum.Cover, be inverted and in 30-35 ℃ of incubation 24-48 hour, and check the morphological feature of hereinafter listing:
The feature of salmonella:
Figure A200680053116D04981
Further evaluation can by with suspicious colony lift extremely at the bottom of the inclined-plane of triple-sugar-iron-nutrient agar inclined-plane (buttslant) pipe carry out, it is by the surface of the slant culture of at first ruling, and subsequently wire is fully stung through under the surface.In 30-35 ℃ of incubation 24-48 hour and inspection.The evidence of (contain or do not contain the inclined-plane of following at the bottom of blackening) at the bottom of if pipe shows alkalescence (redness) inclined-plane and acidity (yellow) inclined-plane, samples met is about the non-existent test request of Salmonella so.
B) if intestinal bacteria-growth is present in the fluid lactose medium: 1., will partly rule to the MacConkey nutrient agar by means of transfering loop.Cover, be inverted and in 30-35 ℃ of incubation 24-48 hour.
If the bacterium colony that is produced none show silicon red appearance (having be settled out biliary may the surrounding area band) and be gram negative bacillus (coccobacillus) that samples met is about the non-existent test request of intestinal bacteria so.
3., so they are transferred to the Levine eosin methylene blue agar medium if bacterium colony meets this description.Cover ware, be inverted and in 30-35 ℃ of incubation 24-48 hour.If none demonstrates peculiar metalluster under reflected light and the black-and-blue outward appearance under transmitted light bacterium colony, samples met is about the non-existent test request of intestinal bacteria so.
Culture medium prescription
1. phosphate buffered saline buffer pH7.2
A) stock solution: generation potassiumphosphate 34.0g; Water (distillation or deionized) 1000mL; Sodium hydroxide TS 175mL.34 gram generation potassiumphosphates are dissolved in the about 500mL water that is included in the 1000-mL volumetric flask.Be adjusted to pH7.1-7.3 by adding sodium hydroxide TS (about 175mL), add adding water to volume and mixing.Sterilization and refrigeration (2-8 ℃) down storage until use.
B) working solution.For use, water dilutes stock solution and sterilization with 1 to 800 ratio.
2. TSA(tryptone beans peptone agar/soybean-casein digesting agar).Caseic pancreas digest 15.0g; Soyflour papoid (papaic) digest 5.0g; Sodium-chlor 5.0g; Agar 15.0g; Water 1000mL; PH:7.3 after the sterilization ± 0.2.
3. TSB(trypticase soy broth/soybean-casein digest liquid medium).Caseic pancreas digest 17.0g; Soyflour papain digestion thing 5.0g; Sodium-chlor 5.0g; Two generation potassiumphosphate 2.5g; Glucose 2.5g; Water 1000mL; PH:7.3 after the sterilization ± 0.2; (4.SDA Sabouraud agar glucose); Glucose 40g; Animal tissues's gastric enzyme digest of equal portions and caseic pancreas digest mixture 10.0g; Agar 15.0g; Water 1000mL; Mix and boil and dissolve with realization; PH:5.6 after the sterilization ± 0.2.
5. FLM (fluid lactose medium)Beef extract 3.0g; The pancreas digest 5.0g of gelatin; Lactose 5.0g; Water 1000mL; Cooling as quickly as possible after the sterilization.PH:7.1 after the sterilization ± 0.2.
The isoelectrofocusing gel analysis of embodiment 50-CTLA4-Ig
The purpose of this embodiment is to measure iso-electric point, isoform number and the microheterogeneity of CTLA4-Ig.
Material
IEF calibrates test kit (pH3-10), (Amersham Pharmacia, catalog number (Cat.No.) 17-0471-01) or (pH2.5-6.5), (Amersham Pharmacia, catalog number (Cat.No.) 17-0472-01).
Ampholine PAGplate gel: pH4.0-6.5, (Amersham Pharmacia, catalog number (Cat.No.) 80-1124-81).
IEF sample administration device, (Amersham Pharmacia, catalog number (Cat.No.) 80-1129-46).
The IEF electrode strip, (Amersham Pharmacia, catalog number (Cat.No.) 80-1104-40).
Phosphoric acid (85%), (EMD, catalog number (Cat.No.) PX0995-6).
Equipment
Multiphor II electrophoresis system, (GE Healthcare, catalog number (Cat.No.) 18-1018-06).
The preparation of reagent
Anode buffer solution (being dissolved in the 0.1M L-glutamic acid in the 0.5M phosphoric acid) (placing dull and stereotyped (+) side) with this core that soaks into.
Example:
3.4mL 85% phosphoric acid.
1.47g ± 0.02g L-glutamic acid.
HPLC grade water.
Mentioned reagent is combined in the 100mL graduated cylinder, reaches 100mL with HPLC grade water, cover lid and inversion are several times to mix.
Solution is preserved in 2-8 ℃ at the most 6 month.
Negative electrode buffered soln (0.1M Beta-alanine) (placing dull and stereotyped (-) side) with this core that soaks into.
Example:
0.9g ± 0.02g Beta-alanine.
HPLC grade water.
Mentioned reagent is combined in the 100mL graduated cylinder, reaches 100mL with HPLC grade water, cover lid and inversion are several times to mix.
Solution is preserved in 2-8 ℃ maximum 6 months.
Fixed solution (being dissolved in the 3.5%5-sulphosalicylic acid in 12% trichoroacetic acid(TCA)).
Example:
240g ± 5.0g trichoroacetic acid(TCA).
70g ± 2.0g5-sulphosalicylic acid.
2000mL HPLC water.
Mentioned reagent is made up to supply the 2000mL volume.
Solution was preserved in room temperature maximum 3 months.
Dyeing solution:
By bottle being reversed gently and reverberating several times, tightly mix GelCode Blue reagent solution before use.
Annotate: dyed blended reagent before pouring into or distributing importantly, to guarantee to use uniform reagent sample.
Dyeing contrast preparation:
With HPLC grade water reconstruct carbonic anhydrase II with preparation 1.0mg/mL stock solution.
10 μ L stock solutions are mixed with 90 μ LHPLC grade water to obtain 0.10 μ g/ μ L working solution.
10 μ L0.10 μ g/ μ L solution are loaded into (1.0 μ g loading) on the gel.
Program
Diluted sample
20 μ g/10 μ L solution of preparation sample and reference material in HPLC water.
Figure A200680053116D0501093045QIETU
Example:
Figure A200680053116D05012
(adding 192 μ l HPLC water)
Device and preparing gel
The cooling plate of Multiphore II electrophoretic cell is connected with constant temperature circulator and temperature is made as 10 ± 2 ℃.
Annotate: allow circulator to reach said temperature at least 20 minutes.
From refrigerator, take out gel.Along the side cutting of big envelope, guarantee not cut to gel/gel upholder carefully.
About 1.0mL:HPLC water is added to 1 edge of chill station.
1 edge of gel/gel upholder is placed in the water, thereby make capillary action carry the whole edge that water strides across gel.Lentamente gel is striden across chill station and use, guarantee not catch bubble.Can use other HPLC grade water as needs.
Surface removal transparent film from gel.
With 1 electrode of anode buffer solution soaking, and place on the gel edge near chill station (+) mark.
In negative electrode buffered soln, soak 1 electrode, and place on the gel opposite side near (-) mark on the chill station.
After electrode strip has been used, use new slasher to cut unnecessary amount carefully, thereby make bar finish in the edge of gel rather than gel upholder.
The sample application sheet is applied to guarantee well contact between sample application sheet and the gel on the side near (-) mark on the chill station.Annotate: guarantee that IEF sample administration device does not contact negative electrode damping fluid wetted bar, do not suct the negative electrode damping fluid, and fully separate to guarantee that each sample moves dividually.The sample administration device should firmly be placed on the slab gel.
Place the unitary electrode fixer of Multiphor II, and the electrode strip centrally aligned electrode on the gel.Make 2 electrodes of self-electrode fixer to be connected and relief cover is placed on the appropriate location with base unit.Hole in the use self adhesive tape covering relief cover is to prevent gel drying.Electrode is connected with power supply.
Make gel prefocus with the power supply that is set at following setting, reach 〉=300V until voltage.
Table 1: make the prefocusing power supply setting of IEF gel.
Operating parameter Be provided with
Voltage (varying parameter) Maximum 2000 volts
Electric current (constant parameter) 25mAmps
Power (constant parameter) 25 watts
Gel loads
Gel is after the prefocus, powered-down, removes relief cover, makes electrode separately and from the MultiphorII element take out the electrode fixer.
Sample is loaded on the sample administration device with particular order.Program guarantees that (-) negative electrode damping fluid bar side approaches analyte most hereto.
Sample loads from right to left.At first carry out 2 or more application of IEF calibration standard (swimming lane 1 and 2), 1 reference material (swimming lane 3), 1 specimen #1 (swimming lane 4), 1 dyeing control formulation (swimming lane 5), 1 specimen #2 (swimming lane 6), 1 reference material (swimming lane 7) and 1 IEF calibration standard (swimming lane 8).
By following interpolation the 3rd and 4 kind of sample, 1 application (swimming lane 11), 1 application (swimming lane 12) of specimen # 4,1 application (swimming lane 13) of reference material and 1 application (swimming lane 14) of IEF calibration standard of 1 application (swimming lane 9) of application reference material, 1 application (swimming lane 10) of specimen #3, dyeing control formulation.The 5th uses use swimming lane 15-20 with 6 kinds of samples by repeating the pattern identical with sample 3 and 4.The sample loading pattern is shown in the table 2.
Table 2-diluted sample and gel loading pattern
Swimming lane Describe Working concentration (μ g/ μ L) Load volume (μ L) Protein load (μ g)
1 IEF pI mark * - 10 -
2 IEF pI mark - 10 -
3 Reference material 2.0 10 20
4 Sample 1 2.0 10 20
5 The dyeing contrast 0.10 10 1.0
6 Sample 2 2.0 10 20
7 Reference material 2.0 10 20
8 IEF pI mark - 10 -
9 Reference material 2.0 10 20
10 Sample 3 2.0 10 20
11 The dyeing contrast 0.10 10 1.0
12 Sample 4 2.0 10 20
13 Reference material 2.0 10 20
14 IEF pI mark - 10 -
15 Reference material 2.0 10 20
16 Sample 5 2.0 10 20
17 The dyeing contrast 0.10 10 1.0
18 Sample 6 2.0 10 20
19 Reference material 2.0 10 20
20 IEF pI mark - 10 -
Electrophoresis.When gel loads, place the unitary electrode fixer of Multiphor II, and the electrode strip centrally aligned electrode on the gel.Make 2 electrodes of self-electrode fixer to be connected and relief cover is placed on the appropriate location with base unit.Hole in the use self adhesive tape covering relief cover is to prevent gel drying.Electrode is connected with power supply.Set suitable voltage, electric current and power setting and bring into operation.
Table 3: the power supply setting of operation IEF gel.
Operating parameter Be provided with
Voltage (varying parameter) Maximum 2000 volts
Electric current (constant parameter) 25mAmps
Power (constant parameter) 25 watts
Time (constant parameter) 2.5 hour
After gel has moved, powered-down, remove relief cover, make electrode take out the electrode fixer separately and from the MultiphorII unit.
Take out electrode strip and sample administration device from gel carefully.
Annotate: can directly be placed on slab gel in the fixed solution and electrode strip and application sheet are floated from gel.
Take out gel/gel upholder from cooling plate, and place the plate (step 3.3) of the fixed solution that comprises enough volumes so that gel keeps moistening.Cover and place on the track platform and incubation 20-60 minute with the plastic wraps thing.Start-of-record and stand-by time on the IEF worksheet.
Annotate: the IEF gel is placed the delamination sometimes of fixing agent long time.This is avoided by making the set time be limited to about 1 hour.
4.5 dyeing gel.
After fixing, with HPLC grade water flushing gel 3x5 minute of enough volumes so that the gel maintenance is moistening.Start-of-record and stand-by time on the IEF worksheet.
Make GelCode Blue staining reagent solution mix back (step 3.4) before the use, the staining agent that gel is placed enough volumes is to keep gel moistening.In the container of closely sealing incubation 15-24 hour, to prevent reagent evaporation.Start-of-record and stand-by time on the IEF worksheet.
Stained gel is decoloured by replacing staining fluid with HPLC grade water.At least change water through 1-2 hour time period and wash gel 3 times.Start-of-record and stand-by time on the IEF worksheet.After the decolouring, gel promptly is used for scanning.
Annotate: may need other washing to reduce the background dyeing on the IEF gel.
System's suitability
The isoelectrofocusing standard should easily distinguish with background.The protein standard that migrates to the pI value between the 4.0-6.5 on gel as seen.Protein standard with this extraneous pIs be can't see on gel.PI mark at 3.50,4.55,5.20 and 5.85 places obtains identifying and obtaining mark on gel images.
The component of table 4:IEF calibration standard.
The protein standard pI
Trypsinogen 9.30
LcA alkalescence band 8.65
The LcA intermediate strap 845
The acid band of LcA 8.15
Myohaemoglobin alkalescence band 7.35
The acid band of myohaemoglobin 6.85
People's CAB 6.55
BCA B 5.85
Beta-lactoglobulin A 5.20
Trypsin inhibitor SBTI 4.55
Amyloglucosidase 3.50
The dyeing contrast that low-level protein loads the carbonic anhydrase II standard (pI5.4) of (1.0 μ g) is used to manifest gel-colored consistence.This band must easily distinguish by the visual inspection and the background of gel images of scanning.
The CTLA4-Ig reference material should be enumerated with 10-22 band in the pI of 4.3-5.6 scope.
The cumulative percentage specific tenacity of the most remarkable band of CTLA4-Ig reference material should be in the pI of 4.3-5.3 scope 〉=and 90%.
For reference material, confirm between pI mark 4.5-5.2, to exist the main band (about main band pattern referring to figure) of 3 focusing by visual inspection.
Gel scanning and analysis
After electrophoresis and the dyeing, all gels use photodensitometer to scan.Image file is stored on the local hard disk drive/network of computer and via local area network and files.The analysis of the gel images of scanning uses ImageQuant TL software (v2003.03) to carry out.Scanning and analytical parameters are listed in table 5.Scanning use department program produces and is quantitative.
Table 5-gel scanning and analytical parameters
Sweep parameter Be provided with
The scanning element size 100
Scanning digital resolving power 12 bits
The band detect parameters
Minimum slope Initial 100
Noise reduces Initial 10
The % maximum peak Initial 0
Swimming lane % width Be set at 90%
Annotate: said procedure is provided for analyzing the basic step of gel images.Sweep parameter in the table obtains limiting.Band detect parameters hint in the table is initial setting.Because change in physical of gel (for example, dyeing/decolouring back gel shrinks) and gel strips belt shape and the change of moving, the adjustment of band detect parameters may be to identify that accurately band is necessary.Manual any omission or the wrong band of identifying proofreaied and correct.
The sweep parameter that limits in the use table scans gel.All analyses of gel and assessment are undertaken by the image of scanning.Open among the ImageQuantTL from＜1D Gel Analysis〉gel images file (raw data of scanning).Forward on the toolbar＜Contrast, and reduction＜ImageHistogram parameter is high-visible until all bands.Selection＜Lane Creation〉and selected＜Manual, to be analyzed to set＜the swimming lane number 〉.Adjustment＜swimming lane % width〉cover the gel swimming lane until the highest 100%.Single swimming lane in case of necessity correctly aligns.Use＜Rolling Ball〉method is with background correction.Initial＜minimum slope of listing in the use table 3 〉,＜noise reduces 〉,＜the % maximum peak〉test strip is set.The adjustment of these values is to identify that accurately band is essential.For the pH/pI4.0-6.5 gel, calculate band pI value by using from the standard pI mark of listing in system's suitability part through the mark of mark.Skip and proofread and correct and standardised step.Be set forth in the band number in the 4.3-5.6pI scope.The result is outputed in the Excel table, be used for further documentation and report.Sample is with respect to the calculating of the cumulative percentage specific tenacity of reference material.Annotate: about the cumulative percentage specific tenacity of sample be with swimming lane in all bands of existing 100% compare the band per-cent that in the pI of 4.3-5.3 scope, moves.Equation should be used for the cumulative percentage specific tenacity of calculation sample with respect to reference material:
Figure A200680053116D05071
Annotate:, should be used for calculating near the reference material of sample with reference to the gel loading pattern.If reference material has 100% accumulated value, and sample has 95% value, and the sample relative percentage is 95/100*100=95% so.If with reference to having 95% accumulated value, and sample has 100% value, and the sample relative percentage is 100/95*100=105% so.
Report the result
Be reported in the band number of enumerating in the pI of scope 4.3-5.6.Report is with respect to the sort of cumulative percentage specific tenacity (example of the report of the quantitative IEF gel analysis that about this method in limit referring to figure) of CTLA4-Ig reference material in the pI of 4.3-5.3 scope.Confirm between pI mark 4.5-5.2, to exist the main band (about main band pattern referring to Fig. 1) of 3 focusing by visual inspection, and be reported in the interior observed main band number of pI mark 4.5-5.2 with respect to reference material.Confirm between pI mark 4.5-5.2, do not have new remarkable band by visual inspection with respect to reference material.
In certain embodiments, the result of this method will be presented at the interior band of pI scope of 4.3-5.6 or 4.3-5.3, wherein identify 10-22 band, and the accumulation band intensity be 90-110%.In another embodiment, the pI scope is 4.5-5.2, has 3 main bands.In another embodiment, the pI scope is 4.3-5.6, has 10-22 main band.In another embodiment, the pI scope is 4.3-5.3, and the accumulation band intensity is 95-105%.In another embodiment, the pI scope is 4.5-5.2, has 2 remarkable bands.
The SDS-PAGE of embodiment 51:CTLA4-Ig
This embodiment shown via SDS-PAGE the reduction and non-reduced condition under check CTLA4-Ig.
Material
Tris-glycine (Tris-Gly) SDS sample buffer 2X (Invitrogen, catalog number (Cat.No.) LC2676).
NuPAGE " sample reductive agent 10X (Invitrogen, catalog number (Cat.No.) NP0004).
4-20% Tris-glycine gels-1.0mm x 12 holes (Invitrogen, catalog number (Cat.No.) EC60252BOX).
Tris-glycine SDS running buffer 10X (Invitrogen, catalog number (Cat.No.) LC2675).
Mark12 TMThe undyed standard of broad range (Invitrogen, catalog number (Cat.No.) LC5677).
GelCode Blue dyeing relates to (Coomassie blue), (Pierce, catalog number (Cat.No.) 24590:500mL catalog number (Cat.No.) 24592:3.5L).
Staining kit II (silver dyeing) (Pierce, catalog number (Cat.No.) 24612).
Instrument configuration:
Xcell SureLock Mini Cell (Invitrogen, catalog number (Cat.No.) EI0001).
About electrophoretic power supply (OWL Separation Systems, catalog number (Cat.No.) OSP-300).
Reagent:
The fixed solution (being dissolved in 50% methyl alcohol and 7% acetate of HPLC grade water) that is used for Coomassie blue stain.
Example:
In the container suitable size that comprises stirring rod, graduated:
Add 500mL methyl alcohol.
Add 70mL acetate.
With HPLC grade water volume is adjusted to 1000mL.
Preserved in room temperature maximum 6 months.
Coomassie blue stain agent (GelCode Blue).
Directly use from container.Add q.s to cover gel, for the about 50mL of 1 minigel in little pallet (10 x 10cm).
Preserve in 2-8 ℃ maximum 1 year.
Silver dyeing fixed solution (being dissolved in 30% ethanol and 10% acetate in the HPLC grade water).
In the container suitable size that comprises stirring rod, graduated: add 300mL ethanol.Add 100mL acetate.With HPLC grade water volume is adjusted to 1000mL.Solution is mixed and solution was preserved in room temperature maximum 6 months.Gel detergent solution (10% ethanol).
In the 50mL centrifuge tube: add 1.0mL toughener (silver-colored staining kit).Add 50mL silver staining agent (silver-colored staining kit).To manage cover lid and mix second by vortex 3-5 gently.The photographic developer working solution.Tightly preparation before use.
In the 50mL centrifuge tube: add 1mL toughener (silver-colored staining kit).Add 50mL photographic developer (silver-colored staining kit).To manage cover lid and mix second by vortex 3-5 gently.1X Tris-glycine SDS running buffer.When using the same day, prepare.In graduated cylinder: add 900mL HPLC grade water.Add 100mL Tris-glycine-SDS10X running buffer.With mentioned reagent combination, with Parafilm cover and reversing several times to mix.
The dyeing contrastIn the bottle that comprises 2mg trypsin inhibitor (dyeing contrast), add 1mL HPLC grade water.This will produce at-200 ℃ of following 6 months 2 stable μ g/ μ L stock solutions.In order to prevent because the repeatedly degraded of freeze-thaw cycle, to transfer to 50 μ L aliquots containigs in the tubule and preserve in-200 ℃.25 μ L stock solutions are added in the 75 μ L HPLC grade water to obtain 0.5 μ g/ μ L solution.40 μ L, 0.5 μ g/ μ L solution is added in the 160 μ L HPLC grade water to obtain the concentration of 0.1 μ g/ μ L.The 0.1 μ g/ μ L solution, 50 μ L 2X TrisGly sample buffers and the 40 μ L HPLC grade water that in Eppendorf tube, add 10 μ L.The final concentration of trypsin inhibitor dyeing contrast is 0.01 μ g/ μ L.Analyze and with the sample of Coomassie blue stain concentration loading with regard to discharging with 10mg/10 μ L.
For the Coomassie blue gel, in HPLC grade water with the concentration of reference material and diluted sample to 10 μ g/ μ L.Example: add 80 μ L, 50 μ g/ μ L reference material sample solution+320 μ LHPLC grade water.For non-reduced sample, 10 μ L, 10 μ g/ μ L strength of solution are added in the 50 μ L 2XTris-Gly sample buffers and 40 μ L HPLC grade water are added in the Eppendorf tube.For the reductive sample, 10 μ L, 10 μ g/ μ L solution are added in 50 μ L 2X Tris-Gly sample buffers, 30 μ L HPLC grade water and the 10 μ L 10X reductive agents.For silver-colored stained gel, in HPLC grade water, 10 μ g/ μ L solution further are diluted to 1 μ g/ μ L.Example: add 40 μ L, 10 μ g/ μ L solution+360 μ L HPLC grade water.For non-reduced sample, 10 μ L, 1 μ g/ μ L solution is added in the 50 μ L 2X TrisGly sample buffers and 40 μ L HPLC grade water in the Eppendorf tube.For the reductive sample, in Eppendorf tube in 10 μ L, 1 μ g/ μ L solution, 50 μ L 2X TrisGly samples, 30 μ LHPLC grade water and the adding of 10 μ L 10X reductive agents.For blank, 50 μ L 2X TrisGly sample buffers and 50 μ LHPLC grade water are combined in the Eppendorf tube.
Table 6: about being present in molecular weight (kDa) scope of the less protein band of expection in reduction and the non-reduced A Baxipu sample
Band is described Non-reducing (kDa) Reductive (kDa)
Minor band NA 15-45
Minor band 30-70 NA
Minor band NA 80-155
Minor band 175-230 175-200
Gel is taken out from its wrap, and outside with the flushing of HPLC grade water to remove the polyacrylamide sheet.Take out the hole comb carefully, guarantee that loading the tip with gel in case of necessity makes Kong Bianzhi.With HPLC grade water filling orifice and flick, thereby make water from the hole, be removed.The hole flushing is repeated 2 times again.5.2 gel is inserted in the XCell device, thereby makes minor plate face towards inner room.If only use 1 clotting glue, so the plexiglass plate is inserted on the offside.Gel is tightly wedged, thereby formed interior and mistress.Fill inner room with 1X Tris-glycine SDS running buffer.Check seepage, fill the mistress with 1X Tris-glycine SDS running buffer subsequently.All gels must comprise at least 1 blank swimming lane and 1 molecular weight marker swimming lane.Only the gel for Coomassie blue stain adds the dyeing contrast.It is most advanced and sophisticated to use gel to load.Between reduction and non-reduced sample, load at least one " blank ".With go back the same molecular weight marker of handling of raw sample.This will help prevent non-reducing sample owing to the leaching of reductive agent is reduced.The top of Xcell device is adhered to and electrode is connected with power supply.Electric current is adjusted to the 25mAmps/ gel, and make voltage (v) and power (w) be made as maximum value.If move 2 clotting glue, then electric current be made as 50mAmps.When during a plurality of Xcell device of operation, adjusting electric current at operation 2 clotting glue on the device or on same power supplies.Electrophoresis 60 ± 5 minutes or move at least 80% of available migration distance until the damping fluid front.Start-of-record time and stand-by time on worksheet.By prying open 2 plastic plates that hold gel are separated with the gel cutter.Gel is followed about painted program after separating.
Coomassie blue stain-gel was placed 50mL Coomassie blue fixed solution (50% methyl alcohol and 7% acetate) 15 minutes.With～100mL HPLC grade water with gel flushing 3 times 5 minutes, totally 15 minutes.Directly add the Coomassie blue stain agent in the gel and incubation 15-24 hour.Stained gel is decoloured by replacing Coomassie blue stain reagent with 100mL HPLC grade water.Decolour after 1 hour, gel promptly is used for scanning.
Gel scanning and analysisAfter electrophoresis and the dyeing, gel uses photodensitometer to scan.Image file is stored on local hard disk drive of computer and/or the network and via local area network and files.The analysis of the gel images of scanning uses ImageQuant TL software (v2003.03) to carry out.Scanning use department program produces and is quantitative.
Table 4: gel scanning and analytical parameters
Sweep parameter Be provided with
The scanning element size 100
Scanning digital resolving power 12 bits
The band detect parameters
Minimum slope Initial 100
Noise reduces Initial 10
The % maximum peak Initial 0
Swimming lane % width Be set at 90%
By visual inspection, the main band of reductive CTLA4-Ig must be as migrating near 55, and the wide band of the position of 400Da molecular weight marker (glutamate dehydrogenase) occurs.
Embodiment 52-measures CTLA4 by ELISA A29YL104E Chinese hamster in the-Ig medicine Ovary (CHO) host cell proteins matter impurity
This embodiment has described the enzyme-linked immunosorbent assay (ELISA) of the pollution level of the residual host cell proteins matter (HCP) of CD-CHO1 in the quantitative test sample.At first the anti-CD CHO1 of rabbit polyclonal HCP IgG is coated on the microtiter plate.HCP reference standard, quality contrast and CTLA4 A29YL104E-Ig drug sample and the anti-CD CHO1 of bonded rabbit HCP IgG incubation.Behind the washing microtiter plate, add the anti-CD CHO1 of multi-clone rabbit HCP vitamin H IgG antibody, its with initial step during the HCP that catches combine.The washing microtiter plate is to remove any unconjugated polyclonal antibody.Add streptavidin-horseradish peroxidase and wash microtiter plate once more, to remove any unconjugated antibody of puting together.Add the TMB chromogen subsequently to produce colorimetric reaction.This reaction stops with sulfuric acid and cultivates in the reader in 450nm place measurement absorbancy at 96 holes trace.Color and proportional the manifesting of HCP amount of catching.Sample concentration is measured based on typical curve, and described typical curve produces the HCP concentration in the 4.11ng/mL-3000ng/mL scope by marking and drawing absorbancy.
Chinese hamster ovary (CHO) clone (DG44) is used for CTLA4 A29YL104EThe production of-Ig.For the production of CD-CHO1 protein (HCP), the DG44 cell carries out stable transfection with recombinant vectors pD16, and grows in adding the CD-CHO1 substratum of semi-lactosi and Recombulin.The polyclonal antibody that is used for the ELISA of this form produces in the New Zealand white rabbit with the immunity of CD-CHO1 HCP material enriched material.Rabbit antibody carries out affinity purification (A albumen), carries out dialysis in the phosphate-buffered saline subsequently and concentrates.The IgG fraction of the anti-CD-CHO1 antibody of about 50mg rabbit uses N-hydroxyl sulfosuccinimide esterification to carry out biotinylation.The anti-CHO1 antibody of the rabbit of unmodified is used for bag by 96 hole microtiter plates.It catches CD-CHO1 HCP, and it detects by the anti-CD-CHO1 antibody of biotinylated rabbit.The streptavidin horseradish peroxidase conjugate combines with vitamin H, and is used for quantitative CD-CHO1 HCP with the colorimetric reaction of TMB chromogenic.
This method is designed to residual level by ELISA quantitative assay CD-CHO1 host cell proteins matter to be used for CTLA4 A29YL104EThe release test of-Ig drug material.
The method general introduction
Figure A200680053116D05131
The anti-CD-CHO1 antibody of rabbit uses the biotinylation test kit to carry out biotinylation, uses sulfosuccinimide base-6 (biotin amido group) capronate as biotinylation reagent.Can use biotinylation reagent with sulfo group-NHS-LC-vitamin H from PIERCE (product #21335).Antibody carries out mark according to the suggestion that manufacturers provides with biotinylation reagent in handbook.Mark is measured vitamin H and is mixed and make sample to be frozen in-70 ℃ or lower with 50 μ L or littler aliquots containig with after separating on the size exclusion post that provides.The vitamin H of final product/IgG ratio should be 2-4.Preserve in-70 ℃ or lower.
Plate bag quilt.Preparation is used for bag by the 8 μ g/mL solution of the anti-CD-CHO1 HCP of the rabbit IgG of the purifying of microtiter plate (every microtiter plate needs 12mL solution) in carbonate buffer solution.Use the multichannel pipettor of calibration that this solution of 100 μ L is added in each hole of Immulon 4 microtiter plates.Cover microtiter plate and in 4 ℃ of incubations 18 ± 2 hours with Parafilm.
Plate washing and sealing.Use plate washer instrument plate to be washed 3 times with lavation buffer solution.Use the multichannel pipettor of calibration that 300 μ L SeaBlock are added in each hole.Make dull and stereotyped in 22.5 ± 5 ℃ of incubations 1 hour.The concentration of preparation CD-CHO1 protein reference standard and quality control sample in the graduated aseptic polypropylene tube of 15mL.Use Teknova thinner (1.21) dilution reference material and quality control sample.Normal concentration was prepared with the concentration of listing in the example hereinafter during the same day in test:
About typical curve dilution of sample (example that is used for preparation every day)
The proteinic original liquid concentration of CD-CHO1 is 5.7mg/mL
Thinner A:26.3 μ L (5.7mg/mL)+4.973mL PTB thinner=30 μ g/ml
Diluent B: 0.6mL (30 μ g/mL)+5.4mL thinner 3000ng/mL
2mL (3000ng/mL)+4mL thinner 1000ng/mL
2mL (1000ng/mL)+4mL thinner 333.3ng/mL
2mL (333.3ng/mL)+4mL thinner 111.1ng/mL
2mL (111.1ng/mL)+4mL thinner 37.0ng/mL
2mL (37.0ng/mL)+4mL thinner 12.3ng/mL
2mL (12.3ng/mL)+4mL thinner 4.11ng/mL
0+4mL thinner 0ng/mL
The QC concentration analysis, preserve and expire
Prepared fresh and use quality contrast (QC) samples of 3 kinds of different target levels (25,100 and 700ng/mL) when analyzing the same day, or preserve in-70 ℃ or lower with bigger amount preparation and with aliquots containig.The refrigerated aliquots containig is analyzed in 3 independent experiments.Average result from 3 experiments is reported in 3 kinds of QC samples each in analysis certificate (COA).During the same day, refrigerated QC sample is thawed and as described below the analysis in experiment.After thawing, every kind of QC sample is with moderate speed's vortex 2-4 second.Refrigerated QC sample expired at a preparation day after date in 6 months.They use with its nominal concentration of reporting on COA.The QC of prepared fresh expired in preparation in back 24 hours.
About quality contrast (QC) dilution of sample (example)
Dilution A:26.3 μ L (5.7mg/mL)+4.973mL thinner=30 μ g/mL
233 μ L (30 μ g/mL)+9.767mL thinner=700ng/mL (QC1)
1.43mL (700ng/mL)+8.57mL thinner=100ng/mL (QC2)
2.5mL (100ng/mL)+7.5mL thinner=25ng/mL (QC3)
Specimen preparation.Drug sample is diluted to about 12.5,6.25 and 3.125mg/mL.
Dilution A:400 μ L sample (～25mg/mL)+400 μ L thinner=12.5mg/mL
Dilution B:400 μ L (～12.5mg/mL)+400 μ L thinner=6.25mg/mL
Dilution C:400 μ L (～6.25mg/mL)+400 μ L thinner=3.125mg/mL
The plate washing.Use the plate washer plate to be washed 3 times with lavation buffer solution.In the plate of sealing and washing, add the every kind of normal concentration in 100 μ L/ holes, sample and QC sample in triplicate.Every kind of QC concentration is added 6 hole/plates extremely altogether 2 times.About the placement of suggestion, referring to the plate figure in the method annex.In 22.5 ± 5 ℃ of incubations 1 hour.Washing step is repeated 5 times.In the Teknova damping fluid, the anti-CD-CHO1 HCP of rabbit vitamin H is diluted to 2 μ g/mL.Make solution with the about 2-4 of moderate speed's vortex second.Add 100 μ L/ holes.In 22.5 ± 5 ℃ of incubations 1 hour.Washing step is repeated 5 times.In the Teknova damping fluid, suitably dilute streptavidin-HRP (SA-HRP) (example: 1/20,000 dilution causes acceptable absorbancy reading usually).In each hole, add 100 μ L SA-HRP dilutions and in 22.5 ± 5 ℃ of incubations 1 hour.From refrigerator, take out the TMB chromogen and go in the suitable containers with bottom line 10mL/ plate decant.Place dark place and allow to reach room temperature.Washing step is repeated 5 times.In each hole, add 100 μ LTMB chromogens and in about 2 minutes of 22.5 ± 5 ℃ of incubations.By in each hole, adding 100 μ L 1N H 2SO 4Stop the chromogen reaction.In plate and hole, add stop bath with identical sequence, to guarantee the chromogen reaction times identical in each hole with enzyme with the chromogen interpolation.On 96 suitable orifice plate readers, use the absorbancy of the reference wavelength measurement of 630nm at the 450nm place.
Data analysis.Set up Softmax process template " CHO1 Elisa template.ppr " with the concentration that produces mean value, standard deviation, %CVs, calculating, curve fitting parameter etc.Typical curve.The reference standard data use 4 parametric line fitting functions that typical curve is carried out match:
Y＝ ((A-D)/(1+(X/C＾B))+D
Wherein:
Y=absorbance (A 450-A 630)
A=is corresponding to the asymptotic absorbance of minimum
D=is corresponding to the asymptotic absorbance of maximum
C=is corresponding to the absorbancy (flex point) of half antipode between the minimum and maximum asymptotic line value.
B=is at the slope at point of inflexion on a curve place
The concentration of X=CD-CHO1 HCP
Softmax process template " CHO1 Elisa template.ppr " uses the relation conefficient (R of the mean value mensuration of calculating about the tropic of standard 2).
Example values.Whether mensuration meets the example values of hereinafter listing about the result of standard, QC and sample.Example values about standard.Relation conefficient (R about typical curve 2) necessary 〉=0.99.Average background about the 0ng/mL normal concentration is necessary≤0.2 absorbance unit.If in 7 nominal concentration of typical curve 2 or more, get rid of 0 and be lower than the concentration (12.3ng/mL) of QL, ineligible 6.1.4 and 6.1.5, this mensuration is considered as invalid and must carries out repetition so.The mean value of the value of calculating when being used for each normal concentration of bioassay standard curve (ng/mL) gets rid of 0 and be lower than the concentration (12.3ng/mL) of QL, must target (nominal) value 20% in.The variation coefficient (%CV) of in triplicate absorbance when each normal concentration, eliminating 0 and the concentration (12.3ng/mL) that is lower than QL must be less than 20%.Can be used for calculating in order to ensure at least 2 suitable data points; Standard, quality contrast and sample are loaded in the in triplicate hole.Separately analyze each in triplicate value.Abandon being positioned at from target value farthest.Recomputate curve and analysis examples value once more.
Example:
Target value (ng/mL) Actual value (ng/mL)
25 12
24
26
Target value single value farthest from 25ng/mL is 12ng/mL.By get rid of the 12ng/mL value from triplicate, the mean value of residual value shi meets all example values.Still do not meet example values if show the mean value of 2 values of residue, get rid of a single point so and recomputate curve.
Example:
Target value (ng/mL) Actual value (ng/mL)
25 5.0
5.5
10
Mean value is apart from target〉20%, no matter which value is eliminated, therefore, a single point is abandoned from curve and curve will recomputate.
Example values about the QC sample.The QC sample is one group of 6 hole when prescribed concentration.About at least 4 in 6 holes of QC sample must about the nominal of acceptable QC sample 20% in.All 3 QC samples all must be acceptable.If these example values do not meet, this mensuration must be carried out repetition so.
Example values about specimen.Absorbance about the specimen measured must be less than the highest QC.If value surpasses the sort of of the highest QC, so specimen fully dilution so that obtain mean value between the 700-12.3ng/mL.For reportable result, at least a in 3 kinds of diluted sample things (12.5,6.25 and 3.125mg/mL) must be dropped in the scope of typical curve, unless be lower than the QL of mensuration about the absorbancy of all dilutions.Under the sort of situation, specimen is reported as＜QL.Should show CV greater than QL and the mean value that drops on the triplicate absorbance of the diluted sample thing in the measurement range less than 20%.
The calculating of CD-CHO1 HCP concentration and reporting the result in the sample.The calculating of CD-CHO1HCP concentration in the sample.For every kind of dilution, the running sample result be multiply by suitable dilution factor (promptly 2,4 and 8), with the CD CHO1 HCP concentration in the undiluted sample that obtains to represent with ng/mL.Mensuration is about the result's that drops on those dilutions in the measurement range mean value.With the CTLA4 of result divided by report A29YL104E-Ig protein concn (mg/mL) is with the CTLA4 that obtains to represent with ng/mg A29YL104EThe CD CHO1 HCP concentration of-Ig.
Example calculates:
The running sample result: 235ngCD-CHO1 HCP/mL=9.4ng/mg
Protein concn: 25.0mg/mL
Annotate: ng CD-CHO1 HCP/mg product (ng/mg) is equivalent to 1,000,000/(ppm).
Embodiment 53: measure CTLA4 by ELISA A29YL104E The A protein level of-Ig
Enzyme-linked immunosorbent assay (ELISA) is CTLA4 quantitatively A29YL104EThe proteic pollution level of A in the-Ig specimen.At first the anti-A albumen of rabbit is coated on the microtiter plate.A albumen reference standard, quality contrast, recovery contrast and CTLA4 A29YL104E-Ig sample and the anti-A protein I of bonded rabbit gG incubation.Behind the washing microtiter plate, add biotinylated monoclonal anti rabbit A protein I gG antibody, the A protein binding of catching during itself and the initial step.The washing microtiter plate is to remove any unconjugated monoclonal antibody.Add streptavidin-horseradish peroxidase after 1 hour subsequently at incubation; Wash microtiter plate once more, to remove any unconjugated antibody of puting together.Add the TMB chromogen subsequently to produce colorimetric reaction.This reaction stop with sulfuric acid and in 96 hole microtest plate readers in 450nm place measuring light density.Color and proportional the manifesting of A protein content of catching.Sample concentration is measured based on typical curve, and described typical curve produces the A protein concentration in the 0.188ng/mL-12ng/mL scope by marking and drawing optical density(OD).
The method general introduction
The reference of A albumen, sample and quality contrast are added trace
In the titer plate, incubation 1 hour
Figure A200680053116D05191
Carrying out plate with capture antibody applies.Be cushioned 1 μ g/mL solution of the anti-A protein antibodies of preparation rabbit in the liquid at bag, and 100 μ L solution added in each hole of Costar microtiter plate.In 4 ℃ of incubations 18 ± 2 hours.
Plate washing and sealing.Use the plate washer plate to be washed 3 times with lavation buffer solution.With 200 μ LSeaBlock TMAdd in each hole.Make microtiter plate incubation 60 minutes at ambient temperature.The preparation of reference standard is mixed into by the A albumen reference material with appropriate amount in the acetate buffer of suitable volumes and prepares reference standard.Make solution with moderate speed's vortex 2-4 second.Before adding microtiter plate, make reference standard, quality contrast and reclaim control sample incubation 10 minutes at ambient temperature.
Example: reference material A albumen, storage concentration 2.3mg/mL
1:200 10 μ L (2.3mg/mL)+1990 μ L (acetate buffer)=11500ng/mL
1:479 10 μ L (11500ng/mL)+4780 μ L (acetate buffer)=24ng/mL
2mL (24ng/mL)+2mL (acetate buffer)=12ng/mL
2mL (12ng/mL)+2mL (acetate buffer)=6ng/mL
2mL (6ng/mL)+2mL (acetate buffer)=3ng/mL
2mL (3ng/mL)+2mL (acetate buffer)=1.5ng/mL
2mL (1.5ng/mL)+2mL (acetate buffer)=0.75ng/mL
2mL (0.75ng/mL)+2mL (acetate buffer)=0.375ng/mL
2mL (0.375ng/mL)+2mL (acetate buffer)=0.188ng/mL
0mL+2mL (acetate buffer)=0ng/mL
Each normal concentration is analyzed in triplicate.Described in the method annex, sample is placed on the microtiter plate.The preparation of quality control sample.The proteic quality of A contrast (QC) sample in acetate buffer with 0.5,2 and 3 target level levels of 5ng/mL be prepared.They are in experiment prepared fresh during the same day, or with bigger amount preparation and with 750 μ L aliquots containigs preserve in≤-70 ℃.A protein concentration in the freezing QC sample makes in this way and independently carries out preliminary assay in the experiment of A protein ELISA at 3 times, and the mean concns result is reported as about " nominal " QC concentration in the analysis certificate of every kind of QC sample.
During the same day, each 1 bottle of 3 kinds of QC samples is at room temperature thawed in experiment.Every kind of QC concentration analysis 2 times (with 2 in triplicate analysis/concentration).Described in the method annex, the QC sample is placed on the microtiter plate.
Reference material A albumen, 2.3mg/mL
1:200 10 μ L (2.3mg/mL)+1990 μ L (acetate buffer)=11500ng/mL
1:479 10 μ L (11500ng/mL)+4780 μ L (acetate buffer)=24ng/mL
833.3 μ L (24ng/mL)+3.166mL (acetate buffer)=5ng/mL (QC1)
1.600mL (5ng/mL)+2.4mL (acetate buffer)=2ng/mL (QC2)
1mL (2ng/mL)+3mL (acetate buffer)=0.5ng/mL (QC3)
The preparation of specimen.Use acetate buffer (pH3.5) in polypropylene tube, to prepare the CTLA4 of concentration as 2.5mg/mL, 1.25mg/mL and 0.625mg/mL A29YL104E-Ig specimen.Before adding microtiter plate, make specimen about 10 minutes of incubation at ambient temperature.
The plate washing.Use the plate washer plate to be washed 3 times with washing soln.The plate washer should be made as with 300 μ L lavation buffer solution filling orifices, zero soak time.With the contrast of each 100 μ L reference standard, quality, reclaim contrast and specimen adds in each hole, and incubation 1 hour at ambient temperature.Repeating step.With the Teknova thinner biotinylated anti-A protein antibodies is diluted to as the desired concn by each new lot optimization is indicated.For example, prepare 1:64 for monoclonal anti A Biotin conjugate, 000 dilution adds 100 μ L in each hole with moderate speed's vortex and use multichannel pipettor.Incubation is 1 hour at ambient temperature.
With the Teknova thinner streptavidin-horseradish peroxidase is diluted to as the desired concn by each new lot optimization is indicated.For example, prepare 1:80,000 dilution for streptavidin-horseradish peroxidase.Add in each hole with moderate speed's vortex and with 100 μ L, and incubation 30 minutes at ambient temperature.Repeating step but wash 5 times.100 μ L TMB chromogens are added in each hole.At room temperature incubation is about 2 minutes.Optical density(OD) about the maximum concentration of typical curve should be 0.980-1.400.By adding 100 μ L/ hole 1NH 2SO 4Stop the chromogen reaction.In plate and hole, add stop bath to add identical order, to guarantee the chromogen reaction times identical in each hole with enzyme with chromogen.On 96 suitable orifice plate readers, use the absorbancy of the reference wavelength measurement of 630nm at the 450nm place.
Data analysis.About the A protein ELISA with reference to the Softmax process template, because it produces mean value, standard deviation and %CVs etc.All calculating of carrying out in the data analysis part will use the A protein ELISA template.ppr among the SoftMax Pro to carry out.
Ask mean value for the in triplicate absorbance (Abs) of each reference measured and sample concentration acquisition.Use not weighting 4 parametric regressions that the data about the A protein standard are carried out modeling:
Abs = min - max I + ( C / ED 50 ) B + max
Wherein:
The Abs=absorbancy
Min=is corresponding to the asymptotic absorbance of minimum
Max=is corresponding to the asymptotic absorbance of maximum
ED 50=corresponding to the absorbancy of half antipode between the minimum and maximum asymptotic line value
B=is at the slope at the flex point place of fitting of a curve
The proteic concentration of C=A
Example values.Example values about standard.Be applied to locate or those values on it about the example values of standard, because be lower than the ultimate value that the value of QL only is used to help definite curve in quantitative boundary (QL).Relation conefficient (R about typical curve 2) answer 〉=0.99.Average background about the 0ng/mL standard should≤0.08 absorbance units.Be used to measure except that 0 and QL each normal concentration of typical curve the time value (ng/mL) calculated mean value must target (nominal) value 15% in.The mean value of the triplicate absorbance of the QL of typical curve must show less than 20% %CV and target 20% in.
Embodiment 54-is used to measure CTLA4 A29YL104E MCP-1 sample among the-Ig is proteinic ELISA
Carry out this ELISA to measure CTLA4 A29YL104EMCP-1 sample protein concn among the-Ig.The concentration of typical curve (0.4-25.6ng/mL), quality contrast and specimen is applied to the microtiter plate that goat anti-mouse MCP-1 absorbs, and descended incubation 60 minutes in envrionment temperature (22.5 ± 5 ℃).Wash plate adds second antibody (the mouse MCP-1IgG of the rabbit Chinese People's Anti-Japanese Military and Political College), and 22.5 ± 5 ℃ of following incubations 60 minutes.Wash plate adds the goat anti-rabbit igg horseradish peroxidase in each hole, and 22.5 ± 5 ℃ of following incubations 30 minutes.Wash plate and adding TMB chromogen are to produce colorimetric reaction.Use 1N H 2SO 4After coming termination reaction, in 450nm place measuring light density, and data use 4 parametric regression curves to carry out modeling in 96 hole microtest plate readers.The proteinic concentration of MCP-1 sample is calculated for each specimen with respect to the MCP-1 reference material subsequently.Concentration with ng MCP-1 sample protein/mg (1,000,000/, sampling report ppm), it is by the realization of being divided by of the result that will obtain with respect to typical curve and undiluted sample concentration.This method allows to measure MCP-1 sample impurity in cell cultures deutero-biological sample, described biological sample comprise carry out in, the medicine and the medicament production of purifying.MCP-1 sample protein may reside in the biological sample that produces in Chinese hamster ovary (CHO) cell cultures.Identified and 2 kinds of polyclonal antibodies of MCP-1 sample impurity bonded by the Chinese hamster ovary celI purifying.Antibody is at mouse and rat MCP-1 (complete), and the both really with MCP-1 sample protein cross reaction from Chinese hamster ovary celI, this is confirmed in this report.
Plate bag quilt.For the bag quilt, be cushioned liquid with bag and make goat anti-mouse MCP-1 antibody dilution to 5 μ g/mL.Wrap by plate with 100 μ L/ holes.With plate sealer bag by plate and at 22.5 ± 5 ℃ of incubation 12-18 hours.The plate washing.Use the plate washer plate to be washed 3 times with washing soln.Washer should be made as 3 washings, 300 μ L/ holes and zero soak time.Alternately, plate can carry out hand washing.Use the multichannel pipettor of calibration that 300 μ L solution are added in each hole of every block of plate.In groove, come evacuation apertures by tip-tap, and on paper handkerchief, blot gently.Repeat 3 times.
The plate sealing.Using the multichannel pipettor of calibrating that 300 μ L bag is stabilized agent and seals damping fluid adds in each hole.Make plate at 22.5 ± 5 ℃ of following incubation 1-2 hours.Plate washing and storage.As described in 4.2 times, plate is washed 3 times with washing soln.Be cushioned liquid/hole infill panel with 300 μ L bag, with the covering of plate sealer and under 2-8 ℃, preserve and in dark, be up to for 1 week.
The preparation of standard.In PTB, prepare 25.6ng/mL solution by MCP-1 sample protein stoste, and use PTB to be diluted to 12.8,6.4,3.2,1.6,0.8,0.4 and 0ng/mL with serial dilution step (1: 2) thus as thinner.Alternately, the refrigerated standard can be used by preparing a large amount of standards.Aliquots containig and preserving in-70 ℃--80 ℃.Avoid repeatedly freezing/melt.The refrigerated standard 3 times independently ELISA need in service carry out quantitatively at quality contrast.Refrigerated standard and QC must be acceptable in each run.After 3 acceptable operations are finished, the issue analysis certificate.The refrigerated standard will be used with its nominal concentration.They expire from preparing at 3 months on date.
About original liquid concentration is the example of the MCP-1 reference of 0.97mg/mL:
The 5200ng/mL of preparation 2.5mL in PTB:
13.4mL stoste+2.487mL thinner
B) 25.6ng/mL of preparation 160mL in PTB:
788mL stoste a+160mL thinner
Reverse gently subsequently second and to mix every kind of solution for 2-3 time by managing the about 2-3 of vortex.
The quality contrast.Quality contrast (QCs) is with the MCP-1 sample protein of the nominal concentration preparation of 17.7ng/mL, 5.3ng/mL and 1.2ng/mL in PTB.Prepare a large amount of QCs, aliquots containig and preserve in-70 ℃--80 ℃ are maximum 3 months.Avoid repeatedly freezing/melt.3 independent ELISA are in service to carry out quantitatively at typical curve quality to impinging upon.QC must be acceptable in each run.After 3 acceptable operations were finished, report was about the average result of each acceptable QC and send analysis certificate.QC will use with its calculating concentration.They expire from preparing at 3 months on date.About by original liquid concentration being the example of the MCP-1 of 0.97mg/mL with reference to preparation refrigerated QC dilution:
A) 5200ng/mL of preparation 2.5mL in PTB:
13.4mL stoste+2.487mL thinner
B) be prepared as follows the final dilution of MCP-1 sample protein quality control sample:
The concentration of MCP-1 (ng/mL) The 5200 ng/mLMCP-1 sample protein (μ L) that add The amount (mL) of the PTB damping fluid that adds Cumulative volume (mL)
17.7 409 119.6 120
5.3 122 119.9 120
1.2 28 120 120
Reverse gently subsequently second by vortex 2-3 and to mix every kind of solution for 2-3 time.Alternately, QC can be when analyzing the same day prepared fresh.
Specimen.Specimen is prepared among the 200 μ LPTB and vortex 2-4 second by 200 μ L specimen are added.With reference standard, quality contrast (x2) and specimen in triplicate, add in the entering plate to 100 μ L/ holes, and at 22.5 ± 5 ℃ of following incubations 1 hour (not using external holes).With the mouse MCP-1 of the prepared at concentrations rabbit Chinese People's Anti-Japanese Military and Political College second antibody of 2 μ g/mL, its volume enough is used for the plate that uses in mensuration in the PTB damping fluid.Make the about 2-4 of solution vortex second.Repeated washing, but wash 5 times.In each hole, add 100 μ L second antibody solution and 22.5 ± 5 ℃ of following incubations 60 minutes.Use PTB as thinner preparation three grades of goat antirabbit HRP conjugate solution (for example 1: 20,000 dilution or suitable dilution).Make the about 2-4 of solution vortex second.Repeated washing.In each hole, add 100 μ LHRP conjugates and incubation 30 minutes in the dark under 22.5 ± 5 ℃.Repeated washing.In each hole, add 100 μ LTMB, and under 22.5 ± 5 ℃ incubation 3-6 minute or manifest until suitable color in the dark.By adding 100 μ L1N H to add the identical sequence of carrying out with chromogen 2SO 4Stop the chromogen reaction.On 96 suitable orifice plate readers, read in the optical density(OD) at 450nm place with the reference wavelength of 630nm.
Data reduction
Use Softmax TMSoftware makes up typical curve with rules file MCP-1.ppr with not weighting 4 parametric regressions that use following formula.
Figure A200680053116D05241
Wherein:
A=is corresponding to the asymptotic light absorption ratio of minimum 450Value.
D=is corresponding to the asymptotic light absorption ratio of maximum 450Value.
C=is corresponding to the concentration of half antipode between the minimum and maximum asymptotic line value.
The approximate slope of the linear portion of B=curve.
The concentration of X=reference standard.
Only report about having the result of the sample that can accept data for typical curve, quality contrast and specimen.
Example values about standard.Be applied to locate or those values on it about the example values of standard, because be lower than the ultimate value that the value of QL only is used to help to determine curve at QL (0.8ng/mL).Average background (0ng/mL standard) is necessary≤0.1 absorbance units.When being used for each normal concentration of bioassay standard curve average counterplot calculate (back-calculated) MCP-1 concentration (ng/mL) must target (nominal) value 15% in.Triplicate A when being used for each normal concentration of bioassay standard curve 450The value the variation coefficient (%CV) necessary≤15%.Triplicate A when the QL of typical curve 450The mean value of value must show≤20% %CV and counterplot calculate target 20% (nominal concentration, 0.8ng/mL) in.In triplicate each value that is used for calculating mean value will separately be analyzed.Be positioned at from mean value value farthest and will be abandoned, recomputate curve and analysis examples value once more.Still do not meet example values if show the mean value of 2 values of residue, eliminate a single point so and recomputate curve.
Example values about the QC sample.The QC sample is defined as one group of 3 hole when prescribed concentration, therefore have 6 QC samples altogether for specified 3 kinds of nominal concentration in this method.For the QC sample can be accepted, about at least 2 in 3 holes of QC sample counterplots calculate target level (referring to COA) that concentration must formerly measure 20% in.At least 4 in 6 QC samples must be acceptable; In 6 QC samples 2 (when being not same concentrations 2) can be unacceptable, and are no more than 6 target levels that can depart from separately in 18 QC sample wells and surpass 20%.If these example values do not meet, this mensuration must be carried out repetition so.
Example values about specimen.The MCP-1 concentration of average computation must be less than the concentration of the highest QC.If the MCP-1 concentration 〉=0.8ng/mL (QL) of average computation, the so triplicate %CV that measures is necessary≤and 20%.If this condition does not meet, sample result will be invalid and must carry out repetition so.If this standard meets, so average MCP-1 concentration is used to calculate net result.If the MCP-1 concentration＜0.8ng/mL (QL) that calculates, so sample be reported as＜QL and value "＜0.8ng/mL " be used to calculate net result.
Embodiment 55-detects CTLA4 by quantitative polyase chain reaction A29YL104E -Ig and CHO DNA among the CTLA4-Ig
Develop this program and detect CHO DNA residual in the drug sample to use real-time quantitative polymerase chain reaction (qPCR) to measure.PCR is duplicating of DNA mixture.This mensuration uses fluorescent probe with detection specificity CHO PCR product, because it gathers between test period.The amount of the initiate dna that exists in the amplification rate of PCR product and the sample is directly proportional.
The DNA value is transformed into pik/milligram.With the pg/mL value divided by by sample concentration at the absorbance measurement at 280nm place.Calculate about protein concn with by the example of the sample of the value=0.67fg/ μ L of Bioreliance report with 50mg/mL.Step 1: be transformed into pg/mL=0.67pg/mL.Step 2: be transformed into pg/mg=(0.67pg/mL/50.0mg/mL)=0.013pg/mg.
Embodiment 56: analyze CTLA4 by SDS-PAGE A29YL104E -Ig
This embodiment has described and has been used to assess CTLA4 A29YL104EThe CTLA4 of the purity of-Ig medicine and medicament production A29YL104E-Ig analyzes.CTLA4 A29YL104E-Ig is engineered fusion rotein, and it is made up of the modified ligand binding domains of cytotoxic T lymphocyte antigen 4 (CTLA4) and Fc γ 1 district of human IgG.CTLA4 A29YL104E-Ig has～molecular weight of 92 kilodaltons and the apparent molecular weight of 97 kilodaltons (non-reducing) or 55 kilodaltons (reductive).CTLA4 A29YL104EThe electrophoresis of-Ig on 4-20% gradient SDS-polyacrylamide gel makes the principal monomer kind and more high-molecular weight kind (polymer of aggregation, dimer and higher category) and any low molecular weight species (degradation fragment) are separated.The densitometric scan of the gel of Coomassie blue stain and ImageQuantTL TMQuantitatively produce the measurement of lipidated protein.The result is reported as CTLA4 A29YL104EThe per-cent purity of-Ig under reduction and non-reduced condition.
Reagent
Annotate: all reagent can be replaced with equivalent alternative.1X Tris-glycine-SDS running buffer.100mL Tris-glycine-SDS 10X running buffer is added in the 1L graduated cylinder.With Milli-Q or HPLC grade water Q.S. to 1L.Cover, reverse several times to mix.This reagent should be prepared when measuring the same day.Annotate: then prepare other volume as needs.
Coomassie blue stain reagent.By making bottle reversing or tilt and reverberate several times gently, tightly mix GelCode Blue staining reagent solution before use.When use had the 3.5LGelCode container (catalog number (Cat.No.) 24592) of manual pump, this kind mixing was even more important.Do not shake bottle with mixing solutions.Annotate: dyed blended reagent before distribution importantly is uniform to guarantee solution.
Fixed solution: 50% water-soluble methyl alcohol/7% acetate.500mL methyl alcohol and 70mL acetic acid group are combined in the 1L graduated cylinder.With Milli-Q water Q.S. to 1L and mixing.Fixed solution was at room temperature stablized maximum 6 months.Annotate: fixed solution should be prepared in the chemical smoke stink cupboard.
Dyeing contrast preparation.Use Milli-Q water reconstruct trypsin inhibitor with preparation 2mg/mL stock solution.Prepare 50 μ L aliquots containigs and-30 ± 10 ℃ freezing maximum 6 months down.Use following dilution plan so that the protein stock solution is diluted to working concentration:
25 μ L stock solutions+75 μ L Milli-Q water=0.5 μ g/ μ L
40 μ L0.5 μ g/ μ L+160 μ L Milli-Q water=100ng/ μ L
Table is with preparation dyeing contrast filling solution under the use.
Specimen preparation about the dyeing contrast
Figure A200680053116D05271
Load the protein load that 10 μ L dyeing control formulation will produce 100ng.
Standard and specimen preparation
Loading pattern about fixing means, GLP/GMP medicine, medicament production or stability sample.Dilution about the Coomassie blue stain gel.The working concentration that will test article and reference material be diluted to 1.0 μ g/ μ L, and as under load reduction with molecular weight marker and non-reducing described in the table.
Diluted sample and gel about the Coomassie blue stain gel load
Figure A200680053116D05281
1Blank=loading 10 μ L 1 X NR sample buffer
Annotate: dyeing contrast band be included in be used for visual inspection the scanning gel images with the evaluating system suitability.Dyeing contrast band does not exist in the gel images of pruning, to keep the consistence of gel report.
Specimen preparation and electrophoresis.Diluted sample about 10 μ g/ swimming lane protein load.The sample that is used for 10 μ g/10 μ L loading according to method preparation hereinafter.Use Milli-Q water as thinner, make specimen and reference material be diluted to 10mg/mL CTLA4A29YL104E-Ig.Approximate concentration can be used for calculating.For example, if the sample of CTLA4A29YL104E-Ig medicine has the concentration of 25mg/mL, prepare 1: 2.5 dilution (40 μ L samples are added in the 60 μ L Milli-Q water) so.According under table preparation be used for electrophoretic final sample.Use Eppendorf tube for these dilutions.
The dilution of specimen
Figure A200680053116D05291
Annotate: the volume of adjusting protein soln and Milli-Q water in case of necessity is to reach the final volume of 100 μ L.Annotate: if sample concentration＜10mg/mL prepares final sample according to last table so.Adjust the volume of protein soln and water so that the protein on the gel is loaded to such an extent that reach maximum.
The sample heating.Behind the preparation diluted sample thing, shut Eppendorf tube and make the pipe vortex with mixing solutions.In 80 ± 5 ℃ of water-baths, make sample heating 2.0 ± 0.5 minutes (using the timing register of calibration).Pine for taking out sample and allow it to be cooled to room temperature from adding.Make the pipe reversing several times to remove from pipe top and lateral condensation.
Device and preparing gel.From its packing, take out gel and take out comb carefully, thereby guarantee that hole wall is straight.Make Kong Bianzhi if need then can to load the tip with gel.Gel is inserted in the electrophoretic cell, thereby make short sheet glass towards inner room.If only move the monolithic gel, so the plexiglass spacer is inserted on the offside.Gel is tightly wedged, so that inner room and mistress's sealing.With 1X Tris-glycine SDS running buffer completely filled inner room.Check seepage, fill the bottom of mistress with 1X Tris-glycine SDS running buffer subsequently to the hole.Use transfer pipet with 1X Tris-glycine-SDS running buffer gently flushing port to remove any residual acrylamide.The repeating hole flushing is fully clear and definite until the hole.
Sample loads.It is most advanced and sophisticated to use gel to load, and loads each hole with 10 μ L samples.Fill all blank swimming lanes with the non-reduced sample buffer of 10 μ L1X.This will help prevent non-reduced sample owing to the leaching of reductive agent is reduced, and will keep and stride across salt concn like the whole gel phase.
Electrophoresis.Adhere to the gel lid, and electrode is connected with power supply.Electric current is adjusted to 25mAmps/ gel (mA), and make voltage (v) and power (w) be made as maximum value.Annotate: power supply setting can not waited from the manufacturer to the manufacturer.Adjust and be provided with to reach the 25mA/ gel.Make gel electrophoresis 60 minutes or just in time reach the bottom of gel until sample buffer dyestuff front.Powered-down, make lead separately and from device take out gel.Pry open plastic plate carefully.Make plastic plate and the gel that adheres to be placed on the suitable stationary solution that is used for staining technique.Gel is immersed in the solution leaves plastic plate until gel.
Gel sets.Annotate: at room temperature follow shaking gently on orbital shaker in steps.Annotate: in the container of tight seal, dye to prevent the reagent evaporation.Annotate: although can use the volume of quoting, necessary is gel all covers in steps fully in institute.In measuring the volume that needs, must consider the size of gel and staining tray.Behind the electrophoresis, added 50mL fixed solution (50% methyl alcohol/7% acetic acid solution) 15 minutes.With～100mL Milli-Q water is with gel flushing 3 times, and each is 5 minutes.Mix coomassie staining reagent solution before use, and add 50mL for the 8x10cm minigel.If use bigger pallet, may need other reagent so.Use orbital shaker that pallet was vibrated 20 ± 1 hours gently.For consistence, in identical all gel-colored identical time length that make in service.Decolour by replacing the coomassie staining reagent with 100mL Milli-Q water.Decolour after 1 hour, gel promptly is used for scanning.
Gel scanning and analysis
After electrophoresis and the dyeing, all gels use photodensitometer to scan and use ImageQuantTL TMSoftware is analyzed.Image file is stored on the local hard disk drive of computer and via local area network and files.Scanning and analytical parameters under list in showing.
Gel scanning and analytical parameters
Figure A200680053116D05301
Annotate: the sweep parameter in this table must not change in scan period.Band detect parameters, swimming lane % width (being set at 75%) and background correction (being set at 200 radius) (any change is all write down need) are recommended in gel images analysis for all scannings.Because the difference in the shape of variation in the physical properties of gel (for example, the gel after the dyeing/decolouring shrinks) or gel strips belt shape, minimum slope, noise reduce and the adjustment of % maximum peak parameter may be to identify that accurately band is necessary.Manual any omission or the wrong band of identifying proofreaied and correct.About the other information of band detect parameters, with reference to instructing on ImageQuantTL (v2003.03) handbook and the screen.
The sweep parameter of listing in the table in the use scans gel.All analyses of gel and assessment should be undertaken by the image of scanning.Open in the ImageQuantTL software from＜1D Gel Analysis〉gel images file (image of scanning).Forward on the toolbar＜Contrast, and general＜ImageHistogram-High parameter is made as 0.3, is used for clear all bands that manifest to strengthen gel images.Annotate: this step is to be used for following analysis and quantitative result is not had influence in order to manifest gel images easily.For the purpose of the visual assessment of gel band or gel report, do not use the enhanced gel images.Selection＜Lane Creation〉and selected＜Manual, to be analyzed to set＜the swimming lane number 〉.General＜swimming lane % width〉be made as 75%.Single swimming lane in case of necessity correctly aligns.Use＜RollingBall〉method is with background correction.In order accurately to explain background, general＜Radius〉be made as 200.Initial＜minimum slope of listing in the use table 4 〉,＜noise reduces 〉,＜the % maximum peak〉test strip is set.The adjustment of these values may be to identify that accurately band is necessary.Manual any omission of assessment or the wrong band of identifying.Measure the band molecular weight by using the molecular weight marker of hereinafter listing.Skip and proofread and correct and standardised step.The result is comprised that molecular weight, band volume and band % output in the Excel worksheet, are used for further documentation and report.11 kinds in 12 kinds of molecular weight standards listing in the table 5 should easily distinguish (Figure 79) with background.Annotate: insulin B chain (3,500Da) and the INSULIN A chain (2,500Da) can be used as that single wide band occurs or the INSULIN A chain can be on gel visual identification not.
12 kinds of molecular weight standards of mark
Figure A200680053116D05321
In this example, in will be on the gel identical relative position of the main band of the test article of non-reduced (monomer) and reduction (strand) with the CTLA4A29YL104E-Ig reference material.Referring to Figure 79.(21,500Da) dyeing of standard contrast must be on the gel images of scanning visible (Figure 79, swimming lane 12) with Trypsin inhibitor SBTI that 100ng/ loads.Except the minor band under non-reduced condition, described minor band approaches 55 usually, and the 400Da molecular weight marker is observed (strand), and the relative percentage intensity of any other band in the gel of Coomassie blue stain answers≤2% for reference material.Annotate: because its non-Gaussian distribution, the molecular weight of main band is estimated and can't accurately be measured.The visual inspection of reductive reference material produces and migrates near 55 the single wide band (referring to Figure 79) of the position of 400Da molecular weight marker.Per-cent purity about the main band of reductive is necessary 〉=and 97%.The visual inspection of the main band of non-reducing reference material must produce and migrate near 97,400Da and 116, the single wide band of the position of 300Da molecular weight marker (referring to Figure 79, swimming lane 2).The per-cent purity of the main band of reference material is necessary 〉=and 97%.The visual inspection of the main band of non-reducing reference material must produce and migrate near 97,400Da and 116, the single wide band of the position of 300Da molecular weight marker (referring to Figure 79, swimming lane 2).The per-cent purity of the main band of reference material is necessary 〉=and 97%.
Embodiment 57-is used for detection by quantitative CTLA4 A29YL104E TRITON X-100 among the-Ig The HPLC method
Triton X-100 by HPLC at CTLA4 A29YL104EIn the protein example of-Ig with low 1,000,000/(ppm) levels (＜10ppm) detect.This method relates on the Solid-Phase Extraction medium extraction Triton X-100, washes with water subsequently to remove residual protein and with acetonitrile wash-out Triton X-100.The acetonitrile eluate uses Phenomenex Hypersil C1 post and by acetonitrile: the moving phase that water (80:20) is formed is carried out chromatography.Detect through UV at the 225nm place.This method is linear between 1-22ppm, and wherein limit of detection is 0.25ppm.Potential immunosuppressor CTLA4 A29YL104E-Ig is a s-generation fusion rotein, is made up of the ligand binding domains of cytotoxic T lymphocyte antigen 4 (CTLA4) and the constant region of human IgG1's heavy chain.Nonionic surface active agent Triton X-100 is at CTLA4 A29YL104EBe used for inactivation of virus in the purifying of-Ig.Although Triton X-100 is removed, for product quality and adjusting purpose, needs are determined from the residual level of proteinic tensio-active agent or are not existed.For this reason, developed and to have detected and the quantitative method of the Triton X-100 of trace level.For the analysis via HPLC, Triton X-100 extracts from protein on the SPE medium and carries out wash-out with acetonitrile.
Standard fabrication
Blank.But any sample or the reference standard of before having analyzed and found to comprise the Triton X-100 of detection level by this method can be as blank.Protein concn in blank and the sample should be similar.Blank should be moved together with sample.
The standard stock solution.Accurately weighing
Figure A200680053116D05331
Triton X-100 adds in the 100mL volume, and is diluted with water to volume and mixing.Annotate: Triton X-100 is slowly dissolving in water.Check the dissolving fully (generally after 15 minutes) of solution before use.Triton X-100 is more more viscous than water, therefore undissolved amount in the presence of water as seen.
Standard operation solution.25 μ L Triton X-100 standard stock solutions are mixed 0.5mLCTLA4 A29YL104EIn-Ig the blank.By vortex or other suitable manner thorough mixing.This standard operation solution comprises about 5ppm (or 5 μ g/mL wt.vol.) Triton X-100.Annotate: standardized solution is answered prepared fresh every day.
Specimen preparation.Sample uses by present appearance.Blank should be moved together with sample.Extraction Triton X-100 from standard and sample solution.Following extraction step is carried out under the vacuum of 3-5 inch Hg.
The activation of Solid-Phase Extraction (SPE) medium.The lid of vacuum ' manifold is mentioned, and the test tube in the frame is placed in the manifold.These are " refuse " test tubes.Lid is resetted and the SPE pipe is placed on the vacuum ' manifold, thereby guarantee under each SPE pipe, to exist " refuse " test tube.In each pipe, add the 1mL acetonitrile, and pipe is used vacuum passed through media bed until all acetonitriles.Repeating step.Derive from Triton X-100 concentration on the SPE medium of standard/sample substrate.Standard operation solution, sample and the blank of in separating activatory SPE pipe, moving each 0.5mL of liquid.The SPE pipe is used vacuum to be passed through media bed until solution fully.From the SPE bed, remove residual protein.In each SPE pipe, add 1mL water, and pipe is used vacuum passed through media bed until water.Repeating step.Wash-out Triton X-100 from the SPE bed.Close vacuum to manifold so that the unit reaches normal pressure.The lid of manifold is mentioned gently, standard and sample SPE pipe is still adhered to.Replace " refuse " test tube with the test tube (for standard, blank and sample) of one group of preliminary making, be collected in the following step may be from the SPE bed any Triton X-100 of wash-out.These are eluate test tubes.The lid of manifold is resetted, thereby guarantee that every kind of sample, blank and standard SPE bed have other eluate test tube of branch under it.In each SPE pipe, add the 0.50mL acetonitrile, and pipe is used vacuum passed through media bed until all acetonitriles.Close vacuum, and mention lid to reclaim the eluate test tube to manifold.Standard, sample and barren acetonitrile eluate placed in the automatic sampler bottle be used to be injected in the chromatographic system.
System's suitability
Before start injection, use about 1 hour of mobile mutual-assistance post/system balancing.Obtain the chromatogram of standardized solution.Retention time about Triton X-100 should be
Figure A200680053116D0534094704QIETU
Minute.When calculating, necessary as the post effect about Triton X-100 of theoretical plate number N assessment according to equation〉2000 plates/post:
N = 16 ( t w ) 2
Wherein:
T is the retention time at Triton X-100 peak, and w is by the peak side being extrapolated to the width at Triton X-100 peak base place that baseline obtains.Carry out at least 3 injections of standardized solution.The per-cent RSD of the area counting of last 3 injections should not surpass 3.0%.
From standard and sample chromatogram, deduct blank chromatogram and carry out with following calculating: the concentration of TritonX-100 (ppm)=
Figure A200680053116D05342
Wherein:
Figure A200680053116D05351
Embodiment 58-is used to measure the mensuration of CTLA4-Ig composition Chinese hamster ovary celI dna content
Develop this program and detect CHO DNA residual in the CTLA4-Ig drug sample to use real-time quantitative polymerase chain reaction (qPCR) to measure.PCR is duplicating of DNA mixture.This mensuration uses fluorescent probe with detection specificity CHO PCR product, because it gathers between test period.The amount of the initiate dna that exists in the amplification rate of PCR product and the sample is directly proportional.Purpose is the cho genomic dna amount that detects in the sample of CTLA4-Ig composition.
Sample is analyzed via the CHO DNA in the detection of biological sample by the quantitative polyase chain reaction analysis.Calculate and result report to being 2 significant figure of unit with pg/mg.If the result is less than the quantitation limit of measuring, the result is reported as＜Q.L so, and the Q.L of record mensuration.The DNA value is transformed into pik/milligram.With the pg/mL value divided by by sample concentration at the absorbance measurement at 280nm place.Calculate about protein concn with by the example of the sample of the value=0.67fg/ μ L of Bioreliance report with 50mg/mL.Step 1: be transformed into pg/mL=0.67pg/mL.Step 2: be transformed into pg/mg=(0.67pg/mL/50.0mg/mL)=0.013pg/mg.
Embodiment 59-measures the MCP-1 protein in biological sample and the CTLA4-Ig composition
This embodiment sets forth and is used for quantitative biological sample and the residual MCP-1 sample method of protein of CTLA4-Ig sample.
Material:
Goat anti-mouse MCP-1 (anti-mouse R﹠amp; D Systems, (catalog number (Cat.No.)
JE[MCP-1]) neutralizing antibody AB-479-NA), preserve in-20 ℃
The mouse MCP-1 PeproTech of the rabbit Chinese People's Anti-Japanese Military and Political College, (catalog number (Cat.No.)
500-P76), preserve in-20 ℃
Goat antirabbit SouthernBiotech (the catalog number (Cat.No.) that HRP puts together
IgG (H+L) 4050-05 preserves in 2-8 ℃
Reagent
Phosphate-buffered saline (PBS, the 10mM phosphate buffered saline buffer, 137mM NaCl, 2.7mMKCl, pH7.3-7.5).Guidance preparation according to the manufacturers on the bottle.In the container of enough sizes, add HPLC grade water, PBS agglomerate and stirring rod.On agitating plate, mix until agglomerate and salt dissolving.Adjust pH7.3-7.5 with sodium hydroxide or hydrochloric acid in case of necessity.Stirring is until thorough mixing.Filter by disposable 0.22 μ m filter unit.Solution was preserved maximum 30 days down at 2-8 ℃ from preparing the date.
MCP-1 (MCP-1) recruits the human protein that works to damage and the sites of infection at monocyte.Based on homology and cross reactivity at the antibody of MCP-1, protein can be considered as the MCP-1 sample.Because the antibody that uses in ELISA is only discerned the part of target protein, thus the MCP-1 of clipped form can in assay method, react, although they are not technical active.So any variant of the hamster MCP-1 that will quantitatively in mensuration, react, thereby make this mensuration detect total length MCP-1 and any variant that comprises the MCP-1 of correct epi-position.Should be understood that and more may will propose many epi-positions by using the polyclonal antibody mixture.
Washing soln (the 1.0mM phosphate buffered saline buffer that comprises 0.01% v/v Tween 20,137mM NaCl, 0.27mM KCl, pH7.3-7.5).In 4.0L distillation or HPLC grade water, add stirring rod, 2 PBS tablets, 28.8g NaCl and 0.4mL Tween 20.Mix gently until all the elements thing and dissolve.Adjust pH7.3-7.5 with sodium hydroxide or hydrochloric acid in case of necessity.Preserved maximum 30 days down at 2-8 ℃ from preparing the date.
Thinner (PBS that comprises 1%w/v BSA and 0.05%v/v Tween 20, pH7.3 to 7.5) (should point out the replacement scheme that this is to use the thinner that is obtained commercially).In the 4L phosphate-buffered saline, add stirring rod, 40g BSA and 2mL Tween 20.Mix gently until all the elements thing and dissolve.Filter by 0.22 μ m filter.When storage unpacking from preparation date not 2-8 ℃ of maximum 30 days of storage down, or opened back 7 days.
The plate bag is by reagent.According to a few bottle goat anti-mouse of specification sheets reconstruct MCP-1 antibody of manufacturers, by reversing mixing several times and combination gently.Preparation aliquots containig and preserving in-20 ℃ maximum 1 year (being no more than the expiration date of manufacturers).Avoid repeatedly freezing melting.Alternately, freeze dried sample flasket can be preserved in-20 ℃ above 6 months.After the reconstruct, antibody can preserve in 2-4 ℃ 1 month.
Secondary reagent.According to the specification sheets reconstruct 1 mouse MCP-1 of the bottle rabbit Chinese People's Anti-Japanese Military and Political College antibody of manufacturers, by reversing mixing several times gently.This solution was preserved in 2-8 ℃ of maximum 4 weeks.Alternately, freeze dried sample flasket can be stable down above 1 year at-20 ℃.
Three grades of reagent.Merge a few bottle goat anti-rabbit igg (H+L) HRP and pass through the mixing several times of reversing gently.Preparation aliquots containig and preserving in-20 ℃ maximum 2 years (being no more than the expiration date of manufacturers).Avoid repeatedly freezing melting.In case thaw, bottle just can be preserved maximum 30 days down at 2-8 ℃.
The preparation of standard.In thinner, prepare 25.6ng/mL solution by MCP-1 sample protein stoste, and be diluted to 12.8,6.4,3.2,1.6,0.8 and 0.4 with serial dilution (2 times) step thus.The 0ng/mL standard is undiluted thinner.
About using the example of original liquid concentration as the MCP-1 reference material preparation standard of 0.97mg/mL:
A) (solution A) prepares the 5200ng/mL of 2.5mL in thinner:
13.4uL stoste+2.487mL thinner
B) (solution B) prepares the 25.6ng/mL of 3.9mL in thinner:
19.2uL stoste a+3.881mL thinner
Use the volume that hereinafter limits to prepare remaining standard:
Final concentration
Working solution volume (mL) thinner (mL) (ng/mL)
25.6ng/mL 1mL 0 25.6ng/mL
25.6ng/mL 1mL 1mL 12.8ng/mL
12.8ng/mL 1mL 1mL 6.4ng/mL
6.4ng/mL 1mL 1mL 3.2ng/mL
3.2ng/mL 1mL 1mL 1.6ng/mL
1.6ng/mL 1mL 1mL 0.8ng/mL
0.8ng/mL 1mL 1mL 0.4ng/mL
By being set the following about 2-3 of vortex second and reversing every kind of solution of 2-3 mixing subsequently gently medium.
The preparation of quality control sample.In thinner, contrast (QC) sample by MCP-1 sample protein bottle preparation quality.Preparation 520ng/mL stock solution concentration prepares following quality control sample by it and is used for freezing: 11.0ng/mL, 5.3ng/mL and 1.2ng/mL.Described in following table, dilute.
About using original liquid concentration to prepare the example of QCs as the MCP-1 reference material of 0.97mg/mL:
A) (solution A) prepares the 5200ng/mL of 2.5mL in thinner:
13.4uL stoste+2.487mL thinner
B) (solution B) prepares the 520ng/mL of 5.0mL in thinner:
500uL stoste a+4.500mL thinner
C) be prepared as follows the final dilution of MCP-1 sample protein QC sample:
The QC sign The concentration of MCP-1 (ng/mL) The QC solution B (μ L) that adds The amount (mL) of the thinner that adds Cumulative volume (mL)
QC1 11.0 106 4.894 5.0
QC2 5.3 53 5.147 5.2
QC3 1.2 12 5.188 5.2
By being set the following about 2-3 of vortex second and reversing every kind of solution of 2-3 mixing subsequently gently medium.
The preparation of specimen.Every kind of specimen is made up of the test article of dilution.Specimen is prepared by 200 μ L specimen are added in the 200 μ L thinners.By with moderate speed's vortex about 2-3 second and reverse gently subsequently and mix every kind of solution for 2-3 time.
Program.Plate bag quilt.With PBS with goat anti-mouse MCP-1 antibody dilution to 5 μ g/mL.By being set the following about 2-3 of vortex second and reversing 2-3 mixed antibody and PBS solution subsequently gently medium.Use multichannel pipettor, in each hole, add this solution of 100 μ L.With plate sealer wrapper plate and at room temperature incubation 12-18 hour.The plate washing.Use the plate washer plate to be washed 3 times with washing soln.Washer is set at 3 washings, 300 μ L/ holes and zero soak time.Alternately, plate can carry out hand washing.Use multichannel pipettor that 300 μ L washing solns are added in each hole of every block of plate.In groove, come evacuation apertures by tip-tap, and on paper handkerchief, blot gently.Repeat 3 times.The plate sealing.Use multichannel pipettor that 300 μ LCSBB are added in each hole.Make plate about 60 ± 10 minutes of incubation at room temperature.Sample adds.Triplicate in plate, ground, 100 μ L/ holes adds reference standard, quality contrast (every kind concentration 2 groups) and specimen, and about 60 ± 10 minutes of incubation at room temperature.Do not use external holes.The preparation rabbit mouse MCP-1 of Chinese People's Anti-Japanese Military and Political College second antibody.The mouse MCP-1 of rabbit Chinese People's Anti-Japanese Military and Political College antibody stoste is diluted to 2 μ g/mL or suitable dilution (according to batch a test of equal value or a COA) in thinner, its volume enough is used for the plate that uses in mensuration.Make solution with the about 2-4 of moderate speed's vortex second.The repeat plate washing, but wash 5 times.Use multichannel pipettor that 100 μ L second antibody solution are added in each hole, and incubation 60 ± 10 minutes at room temperature.Use thinner preparation three grades of goat antirabbit HRP conjugate solution (0.5 μ g/mL or suitable dilution).By being set the following about 2-3 of vortex second and reversing mixing HRP conjugate subsequently gently 2-3 time medium.Be diluted to the concentration of determining according to the SOP of department and be used for the reagent qualification.The plate washing is repeated 5 times.Use multichannel pipettor, in each hole, add 100 μ L HRP conjugates and about 30 ± 5 minutes of incubation in the dark at room temperature.The plate washing is repeated 5 times.Use multichannel pipettor, in each hole, add 100 μ L TMB and in the dark the about 2.5-4 of incubation minute at ambient temperature.Use multichannel pipettor, by adding 100 μ L 1.0N H to add the identical sequence of carrying out with TMB 2SO 4Stop the TMB reaction.On 96 orifice plate readers, read in the optical density(OD) at 450nm place with the reference wavelength of 630nm.
Example values.Be applied to (0.8ng/mL) locate or those values on it about the example values of standard, because be lower than the ultimate value that the value of QL only is used to help definite curve in quantitative boundary (QL).Use the average background (0ng/mL standard) of following calculating necessary≤0.1 absorbance units.
X 1 + X 2 + X 3 . . . . . . X n N
Wherein: X 1,2,3Particular value in=one group of data
The number of the value in one group of data of N=
Each normal concentration that is used for the bioassay standard curve gets rid of 0,0.4 and QL (0.8ng/mL), must target (nominal) value 15% in.Triplicate AB when being used for each normal concentration of bioassay standard curve 450The variation coefficient (%CV) of value, eliminating 0,0.4 and QL (0.8ng/mL), necessary≤15%.Answer≤11.0ng/mL for the average MCP-1 concentration that the specimen of CTLA4-Ig composition is calculated.If the average MCP-1 concentration 〉=0.8ng/mL that calculates (measuring QL), calculate so triplicate %CV that measures and example values necessary≤20%.If example values meets, so average MCP-1 concentration is used to calculate ppm.If the MCP-1 concentration≤0.8ng/mL that calculates (measuring QL), so sample be reported as＜QL and value "＜0.8ng/mL " be used to calculate ppm.The triplicate %CV that measures is not considered.
The QC sample is defined as one group of 3 hole when prescribed concentration, therefore have 6 QC samples altogether for specified 3 kinds of nominal concentration in this method.For the QC sample can be accepted, about at least 2 concentration in 3 holes of QC sample must specified nominal concentration 20% in.In 18 QC sample determinations at least 12 must its separately the target value 20% in.The nominal value that in 18 QC samples 6 (when being not same concentrations 3) can depart from separately surpasses 20%.At least 4 in 6 QC samples must be acceptable.2 allow is unacceptable, and prerequisite is that they are not when same concentrations 2.
Data evaluation.Use Softmax TMSoftware and rules file MCP-1.ppr make up typical curve with not weighting 4 parametric regressions that use following formula.
Wherein:
A=is corresponding to the asymptotic light absorption ratio of minimum 450Value.
D=is corresponding to the asymptotic light absorption ratio of maximum 450Value.
C=is corresponding to the concentration of half antipode between the minimum and maximum asymptotic line value.
The approximate slope of the linear portion of b=curve.
The concentration of x=reference standard.
Calculating about MCP-1 sample protein concn.The proteinic amount of MCP-1 sample in the specimen can use Softmax plate reader software to calculate.How example hereinafter can report net result if illustrating.Measure 2 times of dilutions of every kind of sample.The concentration of calculating be multiply by suitable dilution factor (2) to be created in the concentration in the undiluted test article.
Example:
1) Xi Shi specimen is 10.0ng/mL.
MCP-1 sample protein concn in the undiluted test article is:
10ng/mL?x?2＝20ng/mL?MCP-1
2) Xi Shi sample result is "＜0.8ng/mL ".
MCP-1 sample protein concn in the undiluted test article is:
“<0.8ng/mL”x2＝“<1.6ng/mL”MCP-1
For with ng MCP-1 sample protein/mg sample (ppm) report net result, with the result that above obtains divided by undiluted sample concentration.
Example:
1) specimen protein concn=50mg/mL
MCP-1 sample protein concn=200ng/mL:
200ng/mL?MCP-1/50mg/mL＝4ng/mg
Sampling report is 4ppm (1,000,000/)
2) specimen protein concn=50mg/mL
MCP-1 sample protein concn="＜1.6ng/mL ":
“<1.6ng/mL”MCP-1/50mg/mL＝“<0.032ng/mg”
Sampling report is "＜QL, (＜0.032ppm) "
Embodiment 60: the release test for the CTLA4-Ig drug material is used for surveying by ELISA Decide the mensuration of the proteinic residual level of CD-CHO1
Carbonate buffer solution.In the suitable vessel that comprises stirring rod, add 200mL HPLC grade water, 2 capsular contents of carbonate buffer solution.Use agitating plate to mix until material in solution.Use 1N NaOH or H in case of necessity 2SO 4With pH meter pH is adjusted to 9.6.Use agitating plate that solution was mixed minimum 5 minutes.By 0.22 μ m filter filtering solution.Solution is maximum 30 days of 2-8 ℃ of following storage and according to department's program mark.
Lavation buffer solution (PBS that comprises 0.05%v/v Tween 20, pH7.4).In 4L HPLC grade water bottle, add stirring rod, 20 PBS tablets, add 2mL Tween 20.Use agitating plate to mix until material in solution.Use 1N NaOH or 1N HCl pH to be adjusted to 7.4 in case of necessity with pH meter.Use agitating plate that solution was mixed minimum 5 minutes.Solution is maximum 30 days of 2-8 ℃ of following storage and according to department's program mark.
Streptavidin-HRP.In the bottle of streptavidin-HRP, add 0.5mL HPLC grade water.In order to mix, give bottle cover lid and about 10 seconds of vortex gently.0.5mL glycerine is added in the bottle of streptavidin-HRP.Mix.By solution being drawn into the clarity of checking solution in the clean pasteur pipet.If solution is not clarified, clarify until solution according to 3.3.2 mixing and repetition 3.3.5 so.Be identified for streptavidin HRP batch suitable dilution scheme according to department's program.With 20 μ L volume aliquots containigs in 0.5mL screw socket pipe.Cover lid and place the cell storage box.Solution is maximum 365 days of-20 ℃ of following storages and according to department's program mark.
Stop bath (1N H 2SO 4).In the smog stink cupboard, suitable containers is placed on the agitating plate.Add stirring rod.Add 485.6mL HPLC grade water.Open the stirring of agitating plate with beginning water.Slowly add the dense H of 14.4mL 2SO 4When at room temperature preserving, solution was stable for 90 days.According to department's program label solution.
Program.Plate bag quilt.At the 8 μ g/mL solution (every microtiter plate needs 10mL solution) that are used for wrapping by the anti-CD CD of the rabbit CHO1 antibody of the carbonate buffer solution of microtiter plate preparation purifying.Use multichannel pipettor that this solution of 100 μ L is added in each hole of Immulon4 microtiter plate.Cover microtiter plate and in 2-8 ℃ of incubation 18 ± 2 hours with Parafilm.
Plate washing and sealing.Use plate washer instrument plate to be washed 3 times with 300 μ L lavation buffer solutions.(alternately, plate can use multichannel pipettor to carry out hand washing.) after the last washing.Plate should be put upside down and pat at the paper handkerchief that is placed on the crust.
The plate sealing.Use multichannel pipettor that 300 μ L SeaBlock are added in each hole.Make plate incubation 1 hour (± 6 minutes) in the dark at room temperature.Plate can wrap up or place cabinet or drawer with aluminium foil.According to dilution scheme hereinafter, use Teknova thinner preparation standard curve sample in the graduated aseptic polypropylene tube of 15mL.Annotate: dilution scheme hereinafter is used for 5 blocks of plates.Adjust volume for the plate number in measuring in case of necessity.Obtain the protein concn of CD-CHO1 protein reference standard (Ref Std) by analysis certificate (COA).The 30 μ g/mL solution of preparation CD-CHO1 protein Ref Std.Calculate and obtain the required CD-CHO1 protein Ref Std volume of 30 μ g/mL solution.Minimum transfer volume about CD-CHO1 protein Ref Std should be 10 μ L.
Formula:
Annotate: guarantee that unit is compatible.
Example: 30 &mu;g / mLx 10 mL 25 mg / mL = 12 &mu;L
Calculate the volume of thinner by the volume that needs that from volume required, deducts CD-CHO1 protein Ref Std.
Formula: (volume required)-(Ref Std volume)=(diluent volume)
Example: the 10.0mL-0.012mL=9.988mL thinner
The CD-CHO1 protein Ref Std volume that calculates is added in the 15mL sterile tube of the diluent volume that comprises calculating.Give the pipe cover lid and be set vortex 2-4 second down.Prepare remaining standard.Following table shows as an example:
Working concentration (ng/mL) Volume (mL) Thinner (mL) Cumulative volume (mL) Concentration (ng/mL)
30,000 0.6 5.4 6.0 3,000
3,000 2.0 4.0 6.0 1,000
1,000 2.0 4.0 6.0 333.3
333.3 2.0 4.0 6.0 111.1
111.1 2.0 4.0 6.0 37.0
37.0 2.0 4.0 6.0 12.3
12.3 4.0 2.0 6.0 8.2
NA NA 4.0 4.0 0
Give the pipe cover lid after each dilution step, and before advancing to next dilution step vortex 2-4 second gently.
According to dilution scheme hereinafter, use Teknova thinner preparation quality contrast (QC) solution in the graduated aseptic polypropylene tube of 15mL.Obtain the protein concn of CD-CHO1 protein reference material (Ref Mat) by analysis certificate (COA).30 μ g/mL solution of preparation CD-CHO1 protein reference standard (Ref Mat).Give the pipe cover lid and between setting under vortex 2-4 second.Preparation concentration is 700,100 and the QC solution of 25ng/mL.The example dilution scheme is shown in hereinafter:
Working concentration (ng/mL) Volume (mL) Thinner (mL) Cumulative volume (mL) Concentration (ng/mL)
QC1 30,000 0.233 9.767 10.0 700
QC2 700 1.430 8.570 10.0 100
QC3 100 2.500 7.500 10.0 25
Give about the pipe cover lid of every kind of QC solution and vortex 2-4 second gently.The quality contrast solution can be when measuring the same day prepared fresh or they can carry out five equilibrium and freezing.In 3 independent experiments, measure the actual concentrations of 3 kinds of QC solution.Ask about every kind of QC solution from the mean value of 3 result of experiment and send about each COA in 3 kinds of QC solution.When to the execution analysis of CTLA4-Ig sample, these QC concentration of experimentally measuring will be as the target value.QC solution is preserved in-70 ℃ with the i.e. aliquots containig of usefulness.QC solution expired in preparation in back 6 months.When measuring the same day, from storage, take out QC solution and at room temperature thaw.Make the QC solution that thaws with moderate speed's vortex 2-4 second before use.
Specimen preparation.By 250 μ L drug samples are added in the 750 μ L thinners, preparation is about about 12.5mg/mL solution of every kind of CTLA4-Ig sample to be analyzed in thinner.Give pipe cover lid and vortex 2-4 second gently.Comprise in the pipe of 400 μ L thinners by 40012.5mg/mL solution is added, preparation is about the 6.25mg/mL solution of every kind of CTLA4-Ig sample to be analyzed.Give pipe cover lid and vortex 2-4 second gently.Comprise in the pipe of 400 μ L thinners by 4006.25mg/mL solution is added, preparation is about the 3.125mg/mL solution of every kind of CTLA4-Ig sample to be analyzed.Give pipe cover lid and vortex 2-4 second gently.Come wash plate by the repeated washing step.In the plate of sealing and washing, add every kind of normal concentration, sample and the QC solution in 100 μ L/ holes in triplicate.Every kind of QC solution adds 6 hole/plates extremely altogether 2 times.Incubation 1 hour (± 6 minutes) at room temperature in the dark.Repeated washing 5 times.From refrigerator, take out streptavidin-HRP and allow to reach room temperature.At the Teknova damping fluid the anti-CDCHO1-biotin antibody of rabbit is diluted to 2 μ g/mL.Make solution with the about 2-4 of moderate speed's vortex second.Use multichannel pipettor to add 100 μ L/ holes.Incubation 1 hour (± 6 minutes) at room temperature in the dark.According to department's program, streptavidin-HRP is diluted to the concentration that is identified in mensuration, using based on the qualification of reagent lot.Cover lid and make solution with the about 2-4 of moderate speed's vortex second.Use multichannel pipettor in each hole, to add 100 μ L streptavidin-HRP dilutions.Incubation 1 hour (± 6 minutes) at room temperature in the dark.Repeat.Use multichannel pipettor in each hole, to add 100 μ LTMB chromogens.Incubation 2 minutes (± 12 seconds) at ambient temperature.Use multichannel pipettor to add 100 μ L/ hole stop bath (1N H 2SO 4).In plate and hole, add stop bath with identical sequence, to guarantee the chromogen reaction times identical in each hole with enzyme with the chromogen interpolation.Use SpectraMax Plus plate reader, on 96 suitable orifice plate readers, use the absorbancy of the reference wavelength measurement of 630nm at the 450nm place.
Data evaluation.With reference to the Softmax process template, because it produces mean value, standard deviation and %CVs etc.Use is measured each example values for the in triplicate absorbance that every kind of reference measuring, QC and diluted sample thing obtain.Produce typical curve.4 parametric regressions that use following formula are to the reference standard data modeling.
AB = min - max 1 + ( C ED 50 ) B
Wherein:
AB=is in the light absorption ratio of 450 nanometers
A=is corresponding to the asymptotic light absorption ratio value of minimum
D=is corresponding to the asymptotic light absorption ratio value of maximum
C=is corresponding to the concentration of half antipode between the minimum and maximum asymptotic line value
The approximate slope of the linear portion of B=curve
The concentration of C=CD-CHO1 reference material
Use following formula to use the coefficient of determination (R of the mean value mensuration of calculating about the tropic of standard 2).CD-CHO1 concentration in the calculation sample.The running sample result be multiply by suitable dilution factor (promptly 4,8 and 16), with the CD-CHO1 concentration in the primary sample that obtains to represent with ng/ml.With the CTLA4-Ig protein concn (mg/ml) of result, with the CD-CHO1 concentration of the CTLA4-Ig that obtains to represent with ng/mg divided by report.Measure all results' mean value, this drops in the scope of typical curve.By using the CD-CHO1 protein concn in the dilution factor calculating undiluted sample.In order to measure CD-CHO1 protein concn, divided by undiluted concentration with respect to the CTLA4-Ig sample concentration.
Example calculates:
Figure A200680053116D05451
Annotate: ng CD CHO1mg product (ng/mg) is equivalent to 1,000,000/(ppm).
Example values.Example values about standard.The coefficient of determination (R about typical curve 2) answer 〉=0.99.Average background about the 0ng/mL standard should≤0.10 absorbance units.As measuring by software, the mean value of the value of calculating when being used for each normal concentration of bioassay standard curve (ng/mL) gets rid of 0 and be lower than the concentration (12.3ng/mL) of QL, must target (nominal) value 20% in.As measuring by software, the variation coefficient (%CV) of in triplicate absorbance when being used for each normal concentration of bioassay standard curve gets rid of 0 and be lower than the concentration (12.3ng/mL) of QL, must be less than 20%.Can be used for calculating in order to ensure at least 2 suitable data points, standard, quality contrast and sample are loaded in the in triplicate hole.Separately analyze each in triplicate value.Abandon being positioned at from target value farthest.
Example:
Target value (ng/mL) Actual value (ng/mL)
50 25
48
49
The target value single value farthest that is positioned at from 50ng/mL is 25ng/mL.By get rid of the 25ng/mL value from triplicate, the mean value of residual value shi meets all example values.
Still do not meet example values if show the mean value of 2 values of residue, get rid of a single point so and recomputate curve.
Example:
Target value (ng/mL) Actual value (ng/mL)
50 10
10.5
25
Mean value is apart from target〉20%, no matter which value is excluded, therefore, a single point is abandoned from curve and curve will recomputate.On typical curve, only can get rid of 2 points.
Example values about the QC sample.The QC sample is defined as one group of 3 hole when prescribed concentration, therefore have 6 QC samples altogether for specified 3 kinds of nominal concentration in this method.For the QC sample can be accepted, about at least 2 in 3 holes of QC sample must the nominal of QC sample 20% in.As by computed in software, in 6 QC samples at least 4 must its separately target level 20% in; In 6 QC samples 2 (when being not same concentrations 2) can surpass 20% and depart from nominal.Be no more than 6 target levels that can depart from separately in 18 QC sample wells and surpass 20%.
Example values about specimen.Mean light absorbency about the specimen measured must be less than the vertex on the typical curve.If the mean light absorbency of sample surpasses mean light absorbency 3000ng/mL standard, fully dilution of specimen so is so that obtain 3000 to 12.3ng/mL mean light absorbency.For reportable result, at least a mean light absorbency in 3 kinds of diluted sample things (12.5,6.25 and 3.125mg/mL) must drop in the scope of typical curve, unless all be lower than the mean light absorbency of QL (12.3ng/mL standard) about the mean light absorbency of all dilutions.Under the sort of situation, specimen is reported as＜QL.As measuring, must show CV less than 20% greater than QL and the mean value that drops on the triplicate absorbance of the diluted sample thing in the scope of typical curve by software.If back CV is eliminated and recomputated to one of triplicate absorbancy still〉20%, if or 2 diluted sample degree have absorbancy greater than CV ' s of 20% and drop in the scope of typical curve, the analysis of so this sample must be carried out repetition.
Embodiment 61-is about the mensuration of Triton X-100 amount residual in the CTLA4-Ig composition
Material
HPLC bottle Waters, Total Recovery Vial Kit has the screw socket (catalogue 186000385) of bonded preslit PTFE/ siloxanes partition
Annotate: require vial
Solid phase extraction tube Waters OASIS HLB, 30mg/lcc,
(catalog number (Cat.No.) WAT094225)
Post Thermo Electron Corp.SAS Hypersil, 5 μ, 4.6 x 250mm (part 30505-254630)
Instrument configuration
Liquid chromatography (LC) Waters 2695 Separations modules
Detector Waters 2487 Dual Wavelength UV detectors
SPE post treater JT Baker Vacuum Manifold, model Spe-21
Analytical balance is any balance of weighing 0.01mg accurately
Integration Waters Empower data system
Reagent preparation
The moving phase preparation.: acetonitrile: water (80: 20).For 1L, with stirring rod thorough mixing 800mL acetonitrile and 200mL purifying or HPLC grade water.Filter by 0.2 μ m NF.Use for example Alliance system or use helium to spray to make the moving phase degassing of axial de-gassing vessel.Every day prepared fresh.
2N?NaOH。Weighing and quantitatively shift 80 ± 1g solid NaOH to 1L bottle.Reach volume with purifying or HPLC grade water.Filter with the stirring rod thorough mixing and by 0.2 μ m filter for installation.Alternately, serial dilution 10N NaOH solution.At room temperature preserved maximum 6 months.
The medicine damping fluid.Weighing and quantitatively shift 3.45g NaH 2PO 4H20 and 2.92g NaCl to 1L bottle.Add about 800mL purifying or HPLC grade water.Reach volume with the stirring rod thorough mixing and with purifying or HPLC grade water.Make pH be adjusted to 7.5 with 2N NaOH.Filter by 0.2 μ m filter for installation.Preserved maximum 4 months down at 4 ℃.
The preparation of standard model barren.But any CTLA4-Ig sample or the reference material of before having analyzed and found to comprise the Triton X-100 of detection level can be used as sample blank.Sample blank protein should move together with sample.Triton X-100 is slowly dissolving in water.Check the dissolving fully (generally after 15 minutes) of solution before use.Triton X-100 is more more viscous than water, therefore undissolved amount in the presence of water as seen.
Prepare 10.0 μ g/mL Triton X-100 stoste standard #1.Accurately be weighed to 10.0 ± 1.0mg TritonX-100 in the 100mL volumetric flask and be diluted with water to volume; And mix gently with stirring rod.Be labeled as TX100 stoste standard A.10mL TX100 stoste standard A is put into the 100mL volumetric flask.Be diluted with water to volume.Be labeled as TX100 stoste standard #1.Every day prepared fresh.
Prepare 10.0 μ g/mL Triton X-100 stoste standard #2.Accurately be weighed to 10.0 ± 1.0mg TritonX-100 in the 100mL volumetric flask and be diluted with water to volume; And mix gently with stirring rod.Be labeled as TX100 stoste standard B.B puts into the 100mL volumetric flask with 10mL TX100 stoste standard.Be diluted with water to volume.Be labeled as TX100 stoste standard #2.Every day prepared fresh.
The preparation of the suitability assessment solution #1 of TX100 system (SS#1).By with 300 μ L dilution in acetonitrile, 300 μ L TX100 stoste standard #1, prepare 5.0 μ g/mLTX100 systems suitability assessment solution by TX-100 stoste standard #1.By on move down the liquid thorough mixing.
The preparation of the suitability assessment solution #2 of TX100 system (SS#2).By with 300 μ L dilution in acetonitrile, 300 μ L TX100 stoste standard #1, prepare 5.0 μ g/mLTX100 systems suitability assessment solution by TX-100 stoste standard #2.By on move down the liquid thorough mixing.
TX100 is through the preparation of contrast, limitation standard, failure contrast.#1 prepares the solution of identifying in the following table by TX stoste standard.
The preparation of sample.Use the sample that does not have concentrating or dilute.Program: extraction Triton X-100 from standard and sample solution.Attention: following extraction step is carried out under the vacuum of 3-3.5 inch Hg.
The activation of Solid-Phase Extraction (SPE) medium.The lid of vacuum ' manifold is mentioned, and the 12 x 75mm test tubes of the sky in the frame are placed in the manifold.These are " refuse " test tubes.Lid is resetted and the SPE cylinder is placed on the vacuum ' manifold, thereby guarantee under each SPE pipe, to exist " refuse " test tube.In each SPE cylinder, add 1000 μ L acetonitriles, and use vacuum and passed through media bed until all acetonitriles.Repeat with other 1000 μ L acetonitriles.In each SPE cylinder, add 500 μ L purifying or HPLC grade water, and use vacuum and passed through media bed until all water.Repeat with other 500 μ L purifying or HPLC grade water.
Concentrate about the Triton X-100 on the SPE medium of limitation standard and contrast.With the limitation standard of each 500 μ L, be pipetted into separately in the activatory SPE cylinder through contrast, failure contrast blank and sample.The SPE pipe is used vacuum to be passed through media bed until every kind of solution fully.
From the SPE bed, remove residual protein.In each SPE cylinder, add 1000 μ L water, and pipe is used vacuum passed through media bed until water.With other 1000 μ L water repeating steps.
Wash-out Triton X-100 from the SPE bed.Close the vacuum of manifold extremely zero with releasing unit pressure.The lid of manifold is mentioned gently, the SPE cylinder is still adhered to." eluate " test tube or automatic sampler bottle (limitation standard of preliminary making, process contrast, failure contrast, blank and sample) with one group of preliminary making are replaced " refuse " test tube, to collect any Triton X-100 of wash-out from the SPE bed.The lid of manifold is resetted, thereby guarantee that every kind of limitation standard, process contrast, failure contrast, blank and sample SPE cylinder have eluate test tube or automatic sampler bottle separately under it.In each SPE cylinder, add 500 μ L acetonitriles, and pipe is used vacuum passed through media bed until all acetonitriles.Close vacuum, and mention lid to reclaim eluate test tube or automatic sampler bottle to manifold.If use the eluate test tube, the acetonitrile eluate of then limitation standard, process are contrasted, fail contrast, blank and sample places in the automatic sampler bottle and is used to be expelled in the chromatographic system.If use the automatic sampler bottle to collect eluate, so bottle directly placed in the chromatographic system.
Operational conditions
Wavelength 225nm
Sensitivity 0.1AUFS
Moving phase acetonitrile: water (80: 20)
Flow velocity 0.8mL/ minute
Volume injected 25 μ L
Column temperature environment (20-25 ℃)
Approximate retention time Triton X-100:5.0 ± 1 minute
Total run time 10 minutes
10 ± 4 ℃ of automatic sampling actuator temperatures
System's suitability.Set up chromatographic system; Before analysis, allow lamp to add gentle system and use moving phase balance at least 1 hour.SS#1 injects least four times.Except as otherwise noted, use SS#1 injection for the second time for all system's suitability analyses.Retention time about Triton X-100 should be 5.0 ± 1 minutes.When calculating according to equation, as the post effect about Triton X-100 of theoretical plate number (N) assessment necessary 〉=2000 plates/post:
N = 16 ( t w ) 2
Wherein:
The retention time at the Triton X-100 peak that the elution time of t=from inject time to the peak maximum measured.
W=is by the side, peak being extrapolated to the width at the Triton X-100 peak that baseline records.
Resolving power between Triton X-100 peak and the most contiguous peak (if present) is necessary 〉=and 1.
R = 2 ( t 2 - t 1 ) W 1 + W 2
Wherein:
The Triton X-100 peak in the t=standard and the retention time of adjacent peaks.
W=is by the respective width of the peak base that the side, peak is extrapolated to baseline and obtains.
Use equation to calculate the response factor of the injection of last 3 SS#1:
RF = A W
Wherein:
The area at A=Triton X-100 peak
The Triton X-100 weight (mg) that W=uses in the preparation of corresponding TX100 stoste standardized solution
The response factor of last 3 injections of SS#1 must have≤10% coefficient of variation (RSD).Use equation to calculate %RSD:
%RSD=standard deviation/mean value * 100
Figure A200680053116D05511
Wherein:
Measurement number in the n=sample
Ce Liang not for x=
The average response factor of the response factor of the single injection of comparison SS#2 and last 3 injections of SS#1.Percentage difference between the average response factor of SS#2 response factor and 3 SS#1 injection is necessary≤and 10%.Use equation to calculate percentage difference.
Figure A200680053116D05512
Wherein:
RF1=is about the average response factor of 3 SS#1 injections
The response factor of RF2=SS#2
Carry out the single injection of sample blank behind the SPE.If find Triton X-100 level, implement 2 injections of sample blank so again.If there is Triton X-100, discards the CTLA4-Ig medicine so, but and prepare new limitation standard and contrast with the new lot of the CTLA4-Ig medicine of the TritonX-100 that before analyzed and found not comprise detection level.If above-mentioned example values does not meet, make the balance of post prolong another hour and injection standard solution once more so.If example values does not still meet, carry out following step so.Check seepage, thereby guarantee that all pipe connection are firm.If the balance that prolongs is inoperative, adjusts the organic content of moving phase and/or the new lot of preparation moving phase so, and prepare SS#1 and SS#2 again.If the balance and/or the moving phase adjustment that prolong do not cause example values to meet, change post so.Repeat forward horizontal stand at least 1 hour.
Injection sequence.The rough balance of HPLC system and above-mentioned successful operation.Carry out the single injection of medicine damping fluid behind the SPE, with reference to above part is blank as proofreading and correct.Observe and do not exist Triton X-100 to reply.If when the retention time at Triton X-100 peak, there is the peak, continues the injection blank so and do not reply until recording.Carry out the single injection of CTLA4-Ig medicine behind the SPE, with reference to part above as sample blank.Observe and do not exist Triton X-100 to reply.Before carrying out data processing, this chromatogram will be deducted from standard, contrast and sample chromatogram.Pass through the duplicate injection of contrast, limitation standard and failure contrast.Carry out the single injection of medicine damping fluid, and must not record triton X-100 and reply.Carry out the duplicate injection of sample.The sample injection is carried out with the duplicate injection of limitation standard and 1 injection of medicine damping fluid, is no more than 10 samples injections thereby exist between feasible limitation standard that carries out and the medicine injection together.The end of operation must be finished with duplicate injection and 1 injection of medicine damping fluid barren of limitation standard.
Data processing.Before carrying out data processing, the chromatogram of deduction sample blank injection from all standards, contrast and sample chromatogram.Guarantee in limitation standard, contrast and sample chromatogram suitably integration of Triton X-100 peak.If the Triton X-100 peak in the sample exists, then must be in the retention time window identical with limitation standard.Ask the mean value of the peak area of every group of duplicate injection.Duplicate injection must have in the peak area≤10% % difference.If limitation standard, do not meet this standard through the duplicate injection of contrast or failure contrast, whole service is considered as invalid and need is carried out repetition so.If the duplicate injection of sample does not meet this standard, sample is considered as invalid and need is carried out repetition so.Every other sample, contrast and standard are considered as effectively, as long as they meet≤10% standard.Average peak area about the Triton X-100 peak in all limitation standards that carry out together comprises last injection, must the initial average peak area of limitation standard 10% in.If any intermediate limitation standard fails to meet 10% relatively requirement, so in the end all samples by the limitation standard post analysis is considered as invalid and must reanalyses.
The assessment of limitation standard, process contrast, failure control sample.Compare about limitation standard, through the average T riton X-100 peak area that contrasts and failure contrasts.The peak area of failure control sample must be greater than limitation standard the sort of.Through the peak area of control sample must be less than limitation standard the sort of.If 2 conditions all meet, continue the assessment of sample so.
The assessment of sample.The average T riton X-100 peak area of limitation standard must carry out following correction, with the quantity of material of weighing in the preparation of explaining Triton X-100 stoste standard #1:
A L＝A LSX(10.00mg/Wt.)
Wherein:
A L=proofread and correct to the peak area of 0.5 μ g/mL restriction
A LSThe average T riton X-100 peak area of=limitation standard that carries out together
Annotate: A LIt is gauged area and will be used for comparison with sample when 0.5 μ g/mL limits.
Compare average T riton X-100 peak area and A from sample LIf peak area≤A L, sample is by specific requirement so.If peak area〉A L, sample is by specific requirement so.To be reported as "＜0.50ppm " by the result of specific requirement, or be not reported as "〉0.50ppm by specific requirement ", or as by other required modes of report convention.
The mensuration of the residual A protein content in the quantitative CTLA4-Ig drug material of embodiment 62-.
Material
The anti-A protein antibodies of rabbit Sigma, (catalog number (Cat.No.) P3775)
Biotinylated anti-A protein monoclonal antibody Sigma, (catalog number (Cat.No.) B3150)
Reagent
The carbonate bag is cushioned liquid.In suitable containers, add; Add stirring rod.Add 500mLHPLC grade or Millipore water.Add 5 capsular contents of carbonate.Stirring is until thorough mixing.Inspection and use NaOH (1.16) or HCl (1.23) make pH be adjusted to 9.6 ± 0.1.In solution impouring 500mL filtration sterilization system.Use vacuum so that the solution filtration sterilization.Under aseptic condition, take out filter unit and give the bottle cap upper cover.When preserving down for 2-8 ℃, solution was stable for 30 days.Solution is labeled as " the carbonate bag is cushioned liquid ".Lavation buffer solution: (PBS+0.05%Tween20): in 4L HPLC grade or Millipore water bottle, add; Add stirring rod.Add 20 PBS tablets.Add 2.0mL Tween 20.Inspection and use NaOH or HCl make pH be adjusted to 7.4 ± 0.1.Use agitating plate to stir until thorough mixing.When preserving down for 2-8 ℃, solution was stable for 30 days.Solution is labeled as " lavation buffer solution ".Stop buffer (1N H 2SO 4) or use 1.000 equivalent sulfuric acid from VWR, need not dilution.In the smog stink cupboard, suitable containers is placed on the agitating plate: add stirring rod.Add 485.6mL HPLC grade or Millipore water.Open the stirring of agitating plate with beginning water.Slowly add the dense H of 14.4mL 2SO 4When at room temperature preserving, solution was stable for 30 days.With Container Tag is " stop bath ".
(0.1%Tween 20, pH3.5) for 0.5M acetate, 0.1M sodium-chlor for acetate buffer.In the smog stink cupboard, suitable containers is placed on the agitating plate: add stirring rod.Add 400mL HPLC grade or Millipore water.Open the stirring of agitating plate with beginning water.Slowly add the dense Glacial acetic acid of 14.8mL.Stirring is until thorough mixing.Add 2.9 gram sodium-chlor.Stirring is until thorough mixing.Inspection and use NaOH or HCl make pH be adjusted to 3.5 ± 0.1.Add 0.5mLTween 20.Stirring is until thorough mixing.Be adjusted to the final volume of 500mL with HPLC grade or Millipore water.Stirring is until thorough mixing.When preserving down for 2-8 ℃, solution was stable for 30 days.With Container Tag is " acetate buffer ".
The anti-A protein antibodies of rabbit.From refrigerator, take out bottle and allow to reach room temperature.Add 2.0mLHPLC grade or Millipore water.With 20 μ L volume aliquots containigs in 0.5mL screw socket pipe.Cover lid and place the cell storage box.Solution was stable for 365 days during storage down at-20 ℃.Biotinylated anti-A protein antibodies takes out bottle and allows to reach room temperature from refrigerator.Add 1.0mL HPLC grade or Millipore water.With 60 μ L volume aliquots containigs in 2.0mL screw socket pipe.Cover lid and place the cell storage box.Solution was stable for 365 days during storage down at-20 ℃.
Streptavidin-HRP.In the bottle of streptavidin-HRP, add 0.5mL HPLC grade or Millipore water.In order to mix, give bottle cover lid and about 10 seconds of vortex gently.0.5mL glycerine is added in the bottle of streptavidin-HRP.Give the bottle cover lid and bottle is reversed several times gently.By solution being drawn into the clarity of checking solution in the clean pasteur pipet.With 20 μ L volume aliquots containigs in 0.5mL screw socket pipe.Cover lid and place the cell storage box.When preserving down for-20 ℃, solution was stable for 365 days.
The preparation of standard.Obtain the protein concn of A albumen reference material (ref mat) by analysis certificate (COA).Prepare 11 of A albumen ref mat, 500ng/mL solution by the A albumen stock solution that at room temperature thaws.Calculate and obtain 11, the A albumen ref mat volume that 500ng/mL solution is required.Minimum transfer volume should be 10 μ L.
Formula:
Figure A200680053116D05541
Annotate: guarantee that unit is compatible.
Example: 2.3 &mu;g / mLx 10 mL 0 . 25 mg / mL = 92 &mu;L
Calculate the volume of acetate buffer (3.4) by the volume that needs that from volume required, deducts A albumen ref mat.
Formula: (volume required)-(Ref Std volume)=(diluent volume)
Example: the 10.0mL-0.920mL=9.180mL thinner
The A albumen ref mat volume that calculates is added in the 15mL sterile tube of the acetate buffer volume that comprises calculating.Give the pipe cover lid.Vortex (on Vortex Genie 2 be provided with 4) 2-4 second gently.Use the volume that hereinafter limits to prepare remaining standard:
Working concentration (ng/mL) Working concentration volume (mL) Acetate buffer (mL) Cumulative volume (mL) Final concentration (ng/mL)
2,300，000(2.3mg/mL) 0.010 1.990 2.0 11,500
11,500 0.010 4.780 4.790 24.0
24.0 2.0 2.0 4.0 12.0
12.0 2.0 2.0 4.0 6.00
6.00 2.0 2.0 4.0 3.00
3.00 2.0 2.0 4.0 1.50
1.50 2.0 2.0 4.0 0.75
0.75 2.0 2.0 4.0 0.375
0.375 2.0 2.0 4.0 0.188
N/A N/A 2.0 2.0 0
Give the pipe cover lid after each dilution step, and before advancing to next dilution step vortex 2-4 second gently.Before adding microtiter plate, at room temperature make reference standard and quality control sample incubation 10 minutes.Each normal concentration is analyzed in triplicate hole.
The preparation of specimen.Use acetate buffer in polypropylene tube, to prepare 5mg/mL, 2.5mg/mL and the 1.25mg/mL concentration of CTLA4-Ig specimen.The CTLA4-Ig sample at room temperature thaws before being used to prepare dilution.Calculate and obtain the required specimen volume of 5.0mg/mL solution.Minimum transfer volume should be 10 μ L.
Formula:
Figure A200680053116D05551
Example: 5.0 mg / mLx 1.0 mL 25 mg / mL = 200 &mu;L
5.1.3 calculate the volume of acetate buffer by the volume that needs that from volume required, deducts specimen.
Formula: (volume required)-(specimen volume)=(diluent volume)
Example: the 10.0mL-0.200mL=0.800mL thinner
The specimen volume that calculates is added in the 15mL sterile tube of the acetate buffer volume that comprises calculating.Give pipe cover lid and vortex (on Vortex Genie 2 be provided with 4) 2-4 second gently.Specimen incubation 10 minutes at room temperature before adding microtiter plate.
The preparation of quality control sample.Obtain the protein concn of A albumen reference material by COA.11 of preparation A albumen refmat, 500ng/mL solution.Preparation quality contrast (QC) sample described in following table.
Working concentration (ng/mL) Working concentration volume (mL) Acetate buffer (mL) Cumulative volume (mL) Final concentration (ng/mL)
2,300,000?(2.3mg/mL) 0.010 1.990 2.0 11,500
11,500 0.010 4.780 4.790 24.0
24.0 0.8333 3.166 4.0 5.0(QC1)
5.0 1.6 2.4 4.0 2.0(QC1)
2.0 1.0 3.0 4.0 0.5(QC1)
With 700 μ L volume aliquots containigs in 2.0mL screw socket pipe.Cover lid and place the cell storage box.When-70 ℃ or following storage, solution was stable for 180 days.A protein concentration in the QC sample is measured in advance by following: make and carry out independently A protein ELISA mensuration in this way 3 times.18 results (3 times independent mensuration taken advantage of 6 hole/mensuration) will average.To the A protein concentration of every kind of preparation appointment about each QC sample.When testing the same day, each 1 bottle QC sample that at room temperature thaws, or by the proteic stoste bottle of A prepared fresh QC sample.Vortex (on Vortex Genie 2 be provided with 4) 2-4 second gently.
Program.With the capture antibody bag by plate.Obtain the protein concn of the anti-A protein antibodies of rabbit by the COA of manufacturers.Calculate and obtain the required anti-A protein antibodies of the rabbit volume of 10mL 100 μ g/mL solution.Minimum transfer volume should be 10 μ L.
Formula:
Figure A200680053116D05561
Annotate: guarantee that unit is compatible.
Example: 100 &mu;g / mLx 3 mL 25 mg / mL = 12 &mu;L
Calculate the volume of thinner by the volume that needs that from volume required, deducts the anti-A protein antibodies of rabbit.
Formula: (volume required)-(antibody volume)=(diluent volume)
Example: the 3.0mL-0.012mL=2.988mL thinner
The anti-A protein antibodies of the rabbit volume that calculates is added in the 15mL sterile tube of the diluent volume that comprises calculating.Give pipe cover lid and vortex 2-4 second gently.The use formula is cushioned the 1 μ g/mL solution for preparing the anti-A protein antibodies of rabbit in the liquid at bag.100 μ L solution are added in each hole of Immulon IV microtiter plate.2-8 ℃ of following incubation 18 ± 2 hours.Use the plate washer plate to be washed 3 times with washing soln with 300 μ L lavation buffer solutions and zero soak time.Alternately, plate can use multichannel pipettor to wash.Use multichannel pipettor with 200 μ LSuperBlock TMAdd in each hole.Microtiter plate is incubation 60 minutes in the dark at room temperature.Repeated washing.In assay plate, add each 100 μ L reference standard, quality contrast and specimen.Microtiter plate is incubation 60 minutes ± 10 minutes in the dark at room temperature.Repeated washing.Obtain the protein concn of the anti-A protein antibodies of vitamin H by the COA of manufacturers.The anti-A protein antibodies of preparation 1.0mL1.0mg/mL vitamin H solution in thinner.With moderate speed's vortex.50 μ L 1mg/mL solution (7.9.1) are added in the 0.950mL thinner.With moderate speed's vortex.150 μ L, 50 μ g/mL solution (7.9.3) are added in the 14.850mL thinner.Use multichannel pipettor in each hole, to add 100 μ L.At room temperature incubation is 60 minutes ± 10 minutes.Repeated washing.Following dilution streptavidin-HRP: 10 μ L streptavidin-HRP are added in the 0.990 μ L thinner, to produce 0.01mg/mL streptavidin-HRP.With moderate speed's vortex.80 μ L 0.01mg/mL streptavidins-HRP is added in the 39.920mL thinner.With moderate speed's vortex.Use multichannel pipettor in each hole, to add 100 μ L.At room temperature incubation is 30 minutes ± 5 minutes.From refrigerator, take out TMB and bottom line 10mL/ plate TMB decant is gone in the suitable containers.Place dark place and allow to reach room temperature.Repeated washing, but wash 5 times.In each hole, add 100 μ L TMB chromogens.At room temperature incubation is about 2 minutes ± 1 minute.Stop the chromogen reaction by adding 100 μ L/ hole stop baths.In plate and hole, add stop bath with identical sequence, to guarantee the chromogen reaction times identical in each hole with enzyme with the chromogen interpolation.Measure absorbancy (AB) with the reference wavelength of 630nm at the 450nm place.
Example values.Example values about typical curve.Be applied to locate or those values on it about the example values of standard, because be lower than the ultimate value that the value of QL only is used to help definite curve in quantitative boundary (QL).As measuring, about the coefficient of determination (R of typical curve by SoftMax PRO software 2) answer 〉=0.99.Average background about the 0ng/mL standard should≤0.08 absorbance units.As measuring by software, be used for each normal concentration of bioassay standard curve (except that 0 and QL) time calculating value (ng/mL) mean value must target (nominal) value 15% in.As measure triplicate AB when being used for each normal concentration of bioassay standard curve (eliminating 0 and QL) by software 450The value the variation coefficient (%CV) necessary≤15%.As measuring by software, the mean value of the triplicate absorbance of the QL of typical curve must show less than 20% %CV and in 20% target.Can be used for calculating in order to ensure at least 2 suitable data points, standard, quality contrast and sample are loaded in the in triplicate hole.In triplicate each value that is used for calculating mean value will separately be analyzed.
For example:
Target value (ng/mL) Actual value (ng/mL)
2.5 1.2
2.3
2.5
The target value single value farthest that is positioned at from 2.5ng/mL is 1.2ng/mL.By get rid of the 1.2ng/mL value from triplicate, the mean value of residual value shi meets all example values.If after recomputating, another point does not meet example values, this mensuration will be invalid and must carry out again so.Still do not meet example values if show the mean value of 2 values of residue, get rid of a single point (all 3 holes) so and recomputate curve.
Example values about the QC sample.The QC sample is defined as one group of 3 hole when prescribed concentration, therefore have 6 QC samples (18 holes altogether) altogether for specified 3 kinds of nominal concentration in this method.For the QC sample can be accepted, about at least 2 in 3 holes of QC sample must nominal 20% in.At least 4 in 6 QC samples must be acceptable; Be no more than 6 target levels that can depart from separately in 6 QC samples 2 (when being not same concentrations 2) and 18 the QC sample wells and surpass 20%.
Example values about specimen.As measuring by software, the mean value that in triplicate each value of the specimen of mensuration must be when that concentration 20% in.If 2 or more AB 450Mean value can't calculate, the vertex because it is above standard on the curve, and sample must redeterminate with higher dilution so, and at least 2 in 3 values are dropped on the typical curve.As measuring by software, the %CV of the triplicate observation that obtains for every kind of specimen target level is necessary≤and 20%.
Calculate.About the A protein ELISA, with reference to the Softmax process template, because it produces mean value, standard deviation and %CVs.Ask mean value in triplicate absorbancy (AB) value of each reference measured and sample concentration acquisition.Not weighting 4 parametric regressions that use following formula are to the data modeling about the A protein standard.
AB = min - max 1 + ( C ED 50 ) B
Wherein:
Min=is corresponding to the asymptotic AB value of minimum
Max=is corresponding to the asymptotic AB value of maximum
ED 50=corresponding to the AB of half antipode between the minimum and maximum asymptotic line value
The approximate slope of the linear portion of B=curve
The proteic concentration of C=A
The calculating of the A protein concentration in the sample.The running sample result be multiply by suitable dilution factor (promptly 2,4 and 8), with the A protein concentration in the primary sample that obtains to represent with ng/mL.With the CTLA4-Ig protein concn (mg/mL) of result, with the A protein concentration among the CTLA4-Ig that obtains to represent with ng/mg divided by report.
Example calculates:
Figure A200680053116D05592
Annotate: ngA albumen/mg CTLA4-Ig (ng/mg) is equivalent to 1,000,000/(ppm).
Calculating about the A protein concentration in the Ng/mL sample.A protein content in the specimen uses present SoftMax software to use regression equation to calculate.Measure 3 dilutions of each sample.The concentration of calculating multiply by suitable dilution factor to produce the concentration in the initial specimen.
Example:
Concentration in the specimen is 50mg/mL.
Diluted sample Dilution factor
5mg/mL 10
2.5mg/mL 20
1.25mg/mL 40
5mg/mL sample-ELISA data determination A protein concentration is 10ng/mL
100ng/mL A albumen in 10ng/mL x 10 (dilution factor)=specimen
2.5mg/mL sample-ELISA data determination A protein concentration is 5.0ng/mL
100ng/mL A albumen in 5ng/mL x 20 (dilution factor)=specimen
1.25mg/mL sample-ELISA data determination A protein concentration is 2.5ng/mL
2.5ng/mL the 100ng/mL A albumen in x 40 (dilution factor)=specimen
Calculating is from the mean concns of 3 values.
Result's report.The result can report with the form of " %w/w total protein, ng A albumen/mg total protein ", or be called " ppm " (w/w).
Example:
0.0001％w/w＝1ng/mg＝1ppm(w/w)
Example calculates:
Figure A200680053116D05601
Cause AB than QL (0.188ng/mL) 450Little AB 450The sample of value should be reported as " less than QL ".Cause AB than QL (0.188ng/mL) 450Big AB 450The sample of value should be as 1,000,000/(ppm) reports to nearest integer.
Example:
Sample concentration is 50mg/mL.The highest sample concentration of analyzing is 5mg/mL (1/10 dilution).The AB of sample 450Less than QL.
QL＝0.188ng/mL
≤0.188ng/5mg
≤0.04ng/mg
Be reported as "≤, QL=0.04ppm "
Embodiment 63: obtain amino monose (the N-acetyl semi-lactosi in the CTLA4-Ig drug sample Amine, N-acetyl-glucosamine) with the method for proteinic mol ratio
Reagent:
Hydrating solution (the 4N HCl aqueous solution).160mL 6N HCl and 80mL HPLC grade water are added in the 250mL vial.Stirring is with thorough mixing.Preserve in 2-8 ℃ maximum 6 months.Derivatize solution I (the 0.1M APTS aqueous solution).192 μ L HPLC grade water are added in the 10mg APTS powder in the vial.Make bottle vortex 5-10 second to dissolve APTS fully.Preserve in-20 ℃ maximum 1 year.
Derivatize solution II (1M acetate and 0.25M NaBH 3CN).In the 0.4mL centrifuge tube, dilute 20 μ L acetate (17 times of dilutions), with preparation 1M acetic acid solution with 320 μ L HPLC grade water.With 2.0 ± 0.5mg NaBH 3CN is weighed in the profound hypothermia bottle.Use following formula, the 1M acetic acid solution that adds suitable volumes is with preparation 0.25M NaBH 3CN.Volume (μ L)=10 3* (the NaBH that represents with mg 3The weight of CN)/(62.84g/mol * 0.25mol/L).Annotate: tightly preparation before use.Sodium cyanoborohydride (NaBH 3CN) should preserve in moisture eliminator in the dark.Recommend following reagent is assigned to again to be used for storage in a series of 2.0mL cryovials, to avoid the original reagent bottle of repeated open: 1.0g ± 0.2mg sodium cyanoborohydride is weighed in the 2.0mL cryovial.By this way will be from the entire contents aliquots containig of the sodium cyanoborohydride in the original bottle.Cover lid and together with reagent name, lot number and 6 months validity period mark cryovial such as (1,2,3) in turn tightly.Bottle is used the Parafilm sealing to avoid moisture.Weighing up sodium cyanoborohydride from identical cryovial is used for the derivatize solution II and is no more than 3 times.On the laboratory work table, indicate this point and cryovial sequence number (SN).Observed reagent peak or weak mark may take place after the cryovial repeated open or behind the sodium cyanoborohydride with the sort of particular batch in the CE overview.If this influences the result, discard the cryovial of use so and from cryovial or from the new lot sodium cyanoborohydride, weigh up reagent with next sequence number (SN).
Again the acetylize damping fluid (the 25mM sodium bicarbonate, pH9.5).0.210 ± 0.02g sodium bicarbonate is weighed in the clean glass beaker of clean 100mL.Add 90mL HPLC grade water, and on agitating plate, mix and dissolve fully until salt.With 10N NaOH pH is adjusted to 9.5 ± 0.1.Add HPLC grade water to obtain the final volume of 100mL.Filtering solution and preserving in room temperature maximum 3 months.Running buffer (60 ± 5mM sodium tetraborate, pH9.25).1.21 ± 0.02g sodium tetraborate is weighed in the clean glass beaker of 100mL.Add 90mL HPLC grade water, and on agitating plate, mix and dissolve fully until salt.With 10N NaOH pH is adjusted to 9.25 ± 0.10.For the final concentration of 60 ± 5mM, add HPLC grade water to obtain the final volume of 100mL.For 55mM solution, weighing 1.11g (± 0.02) sodium tetraborate and be used for according to the above description the dissolving and titration.For 65mM solution, weighing 1.31g (± 0.02) sodium tetraborate and be used for according to the above description the dissolving and titration.Preserved in room temperature maximum 3 months.If peak resolving power (as definition in system's suitability part) influenced (R value＜1.0) prepares fresh damping fluid so.Choose wantonly: (± 5mM) final concentration is by adding dilution tetraborate buffered soln (MicroSolv) in the 80mL 150mM sodium tetraborate damping fluid with the 120mL ultrapure water for 60mM.With 10N NaOH titration so that pH value of solution reaches 9.25 (± 0.1).For 55mM tetraborate solution, 66mL 150mM sodium tetraborate damping fluid is diluted in the 114mL ultrapure water.As above titration.For 65mM tetraborate solution, 78mL150mM sodium tetraborate salt buffer is diluted in the 102mL ultrapure water.As above titration.Solution was preserved in room temperature maximum 3 months.If peak resolving power (as definition in system's suitability part) influenced (R value＜1.0) prepares fresh damping fluid so.
Capillary douche solution.
1N NaOH solution: the adding of 1mL 10N NaOH solution is comprised in the graduated plastics tubing of 15mL of 9mL HPLC grade water.By vortex 5-10 thorough mixing second.Solution was preserved in room temperature maximum 6 months.
1N HCl solution: the adding of 1mL 6N HCl solution is comprised in the graduated plastics tubing of 15mL of 5mL HPLC grade water.By vortex 5-10 thorough mixing second.Solution was preserved in room temperature maximum 6 months.80% methanol solution: the adding of 8mL HPLC grade methyl alcohol is comprised in the graduated plastics tubing of 15mL of 2mL HPLC grade water.By vortex 5-10 thorough mixing second.Solution was preserved in room temperature maximum 6 months.
Monose standard stock solution:
N-acetyl-glucosamine (GalNAc).5 ± 1mg GalNAc accurately is weighed in the 2.0mL profound hypothermia bottle.Add 1mL HPLC grade water and pass through the vortex thorough mixing until dissolving.The accurate concentration (mg/mL) of recording solution.
N-acetylgalactosamine (GlcNAc): 5 ± 1mg GlcNAc accurately is weighed in the 2.0mL profound hypothermia bottle.Add 1mL HPLC grade water and pass through the vortex thorough mixing until dissolving.The accurate concentration (mg/mL) of recording solution.
N-acetylmannosamine (ManNAc): 5 ± 1mg ManNAc accurately is weighed in the 2.0mL profound hypothermia bottle.Add 1mL HPLC grade water and pass through the vortex thorough mixing until dissolving.The accurate concentration (mg/mL) of recording solution.
Monose standard stock solution is preserved in-20 ℃ maximum 1 year:
Monose working solution I: internal standard working solution.By 20 μ L ManNAc stock solutions are added in the 2mL profound hypothermia bottle, use HPLC grade water that the stock solution of ManNAc is diluted 100 times, described profound hypothermia bottle has comprised 1980 μ L HPLC grade water.The about 5-10 of vortex second.The internal standard working solution is preserved in 2-8 ℃ maximum 6 months.
Monose working solution II: amino hybrid standard working solution.In the 2.0mL profound hypothermia bottle that comprises 1960 μ L HPLC grade water, add the stock solution of 20 μ L GalNAc and GlcNAc respectively.The about 5-10 of vortex second.Amino hybrid standard working solution is preserved in 2-8 ℃ maximum 6 months.
Sample and reference material solution.In the 2-8 ℃ of refrigerated protein example that thaws, and by being inverted mixing gently.With HPLC grade water sample and reference material are diluted to about 1.0mg/mL.Indicate concentration to 3 significant figure.
The CE operational conditions
Running buffer (step 2.5) 60mM sodium tetraborate, pH9.25
25 ℃ of capillary column body temperature degree
Voltage 25-30kV, holotype
Detector condition LIF detector excites: 488nm,
Emission: 520nm
Sample injection pressure injection pattern is at 0.5PSI
The time 20s
10 minutes working times
10 ℃ of sample storages
Program
Hydrolysis.In the 0.65mL centrifuge tube, add 10 μ L ManNAc working solutions and 200 μ L4N hydrating solutions.This serves as system's blank.In the 0.65mL centrifuge tube, add the amino mixed standard solution of 10 μ L ManNAc working solutions and 10 μ L.Further add 200 μ L 4N hydrating solutions.This serves as and is used for quantitatively and the monose standard of system's suitability.Prepare in duplicate.In the 0.65mL centrifuge tube, add 10 μ L ManNAc working solutions and 10 μ L CTLA4-Ig reference material solution (about 1mg/mL).Further add 200 μ L 4N HCl solution.Prepare in duplicate.In the 0.65mL centrifuge tube, add 10 μ L ManNAc working solutions and 10 μ L sample solutions (about 1mg/mL).Further add 200 μ L 4N HCl solution.Prepare in duplicate.Make about 10 seconds of sample vortex and centrifugal about 5-10 second.Sample placed the little bottle stand in 96 positions and at baking box in 95 ℃ of incubations 6 hours.After the hydrolysis, with the sample of hydrolysis place-20 ℃ 10 minutes with cooling.Make that the sample of hydrolysis is of short duration centrifugally to be forced to bottom (in high speed time 5-10 second) to pipe until all condensation products.In Speed Vac, make sample evaporation to dry.With 100 μ L HPLC grade every kind of samples of water reconstruct and vortex 10-15 second.In SpeedVac, make sample evaporation to dry.
Acetylize again.With 10 μ L M6 more every kind of sample of acetylize damping fluid reconstruct and vortex 5-10 second with thorough mixing.With 4 μ L M3 again acetylation reagent add in each pipe.The about 5-10 of vortex second.Incubation on ice 30 minutes.In SpeedVac, make sample evaporation to dry.With 100 μ L HPLC grade every kind of samples of water reconstruct and vortex 10-15 second.In SpeedVac, make sample evaporation to dry.
Derivatize.Eppendorf centrifuge is placed the oven temperature of baking box with balance to 55 ℃.With 10 μ L derivatize solution I (0.1M APTS solution) every kind of samples of reconstruct.The about 5-10 of vortex second.Add 5 μ L derivatize solution II (1M HAc and 0.25M NaBH 3CN).Vortex about 5-10 second and centrifugal.Sample flasket is loaded in the whizzer of heating in advance fast, and whizzer is put back in 55 ℃ of baking boxs.3 hours whiles of incubation are centrifugal under 2000rpm.This prevents solvent condensation on little bottle surface.
The instrument configuration preparation.
5.4.1 new kapillary is installed, is used following step to wash: 1NNaOH 20 minutes with high pressure mode (80PSI). HPLC grade water 10 minutes.60mM sodium tetraborate damping fluid 10 minutes.
Operation every day
Before operation every day, operation wash/rinse order is with the flushing kapillary.
Operational system suitability criterion (monose standard) is fit to guarantee system subsequently.
But use the capillaceous inside of 1N NaOH etching, and cause the change that moves in the transition time from start to finish from different vendor.If this transition time that causes postpeak (GlcNAc) surpasses 10.0 minutes,, may replace 1N NaOH with 0.1N NaOH or HPLC grade water so for step 2 flushing.
When using kapillary of equal value and above-mentioned washing procedure enough, may use 80% methyl alcohol and/or 1N HCl so that postpeak (G1cNAc) in 10.0 minutes example values.
The preparation that is used to inject
Behind the derivatize, make sample be cooled to room temperature.At room temperature centrifugal about 10 seconds, be forced to bottom to pipe until condensation product.
In each pipe, add 85 μ L HPLC grade water so that the final volume of every kind of sample reaches 100 μ L.Vortex 5-10 second.
From each pipe, shift 10 μ L samples in CE trace bottle, and in each pipe, add 190 μ LHPLC grade water.Vortex 5-10 second.
Rinse step and injection sequence:
Annotate: for per 4 injections, with the CE runtime buffer fluid exchange CE running buffer (because ion depletion action) of prepared fresh.Carry out capillary douche with 40psi.
Figure A200680053116D05651
Figure A200680053116D05661
*Annotate: carry out for maximum 3 kinds of sample repetitive sequences and with 2 injections of every kind of preparation of monose standard in duplicate.Use all 8 kinds of system's suitability criterion injections for the sample that places 3 group.If operation surpasses 3 kinds of samples, so as mentioned shown in the order from the 19th row other sample that brings into operation.Finish order by operational system suitability (Mono Std) as shown in the 47th to 53 row in the table.
*Sample is carried out with 2 injections of every kind of preparation of CTLA4-Ig reference material.
System's suitability is annotated: except as otherwise noted, system's suitability value uses system's suitability criterion of injection for the first time to measure.The electrophorogram of first kind of system's suitability should be similar to the sort of shown in Fig. 1, and wherein peak 1 is GalNAc; Peak 2 is ManNAc; Peak 3 is GlcNAc.Annotate: when the CE instrument that uses except that Beckman PACE MDO, owing to keep the various configurations of the cylinder capillaceous that separates, length capillaceous can be different from this method specified the sort of.This will cause the variation in analyte transition time and the peak intensity.
Calculate 2 adjacent peak-to-peak resolving power according to equation by instrument about first kind of system's suitability criterion:
R = 2 ( t 2 - t 1 ) ( W 1 + W 2 )
Wherein:
R=resolving power
t 2, t 1=2 adjacent peaks transition time separately
W 1, W 2The peak width at=2 each comfortable baseline places of adjacent peak
The R value is necessary 〉=and 1.0.If R＜1.0 are so with wash/rinse order flushing kapillary; If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.
For the suitability injection of last system, use the last peak (GlcNAc) of following formula must have＜1.4 tailing factor:
T＝W 0.05/2f
Wherein:
The T=tailing factor
W 0.05=in the peak width in 5% when height
The width of f=front, peak when peak maximum
If T 〉=1.4 are so with wash/rinse order flushing kapillary; If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.
6.1 the duplicate injection of system's suitability criterion must meet following example values:
■ GalNAc is to the peak area ratio of MaNAc: RSD≤10% (calculating in step 7.1)
The transition time of ■ GalNAc was answered≤10.0 minutes
The ■ overview should be equivalent to Fig. 1, and wherein observing 3 peaks and internal standard (ManNAc) is the 2nd peak.
If any one before specimen in the above-mentioned example values do not meet, so if the transition time of GlcNAc greater than 10.0 minutes, then at first increases voltage.Secondly, if peak area ratio〉10%, determine its pH or replace kapillary thereby prepare fresh CE damping fluid so.After adjusting instrument, the injection of duplicated system suitability.When analyzing the peak overview, if the remarkable reduction in the peak height of generation ManNac checks that so the fiberoptic cable to determine to enter in the LIF module is not misalignment.
Recently measure the monose standard percentage by the peak area that compares internal standard and monose standard ingredient and compare RSD.Will be about the peak area of each monosaccharide component divided by peak area about the internal standard of each monose standard injection.Calculating is about the GalNAc of 2 kinds of standards of carrying out together and the per-cent RSD of GlcNAc.RSD answers≤10%.If this example average does not meet, kapillary should as above wash or replace so.
Calculate
Calculate GalNAc and GlcNAc peak area ratio with respect to internal standard (ManNAc).Be used in the duplicate injection of the forth day of a lunar month kind system suitability criterion so that meet example values, and all of before the sample and back injection are carried out together, system's suitability criterion carries out identical calculations.
Peak area ratio=will (GlcNAc, peak area GalNAc) be divided by the peak area about the internal standard (ManNAc) of each system suitability criterion injection about every kind of monosaccharide component.
Figure A200680053116D05681
Calculating in system's suitability criterion about the mean value of the peak area ratio of GlcNAc and GalNAc.Also base of calculation poor (S.D.) and per-cent coefficient of variation (%RSD)
Example values: about RSD≤10% of the peak area ratio of GlcNAc.
2 kinds of the injection of before the sample and back, carry out together, system's suitability criterion: about per-cent RSD≤10% of the peak area ratio of GlcNAc and GalNAc.
If this example average does not meet (RSD〉10%), kapillary needs wash once more with cleaning procedure so, and those samples and the monose standard of carrying out together need to move once more.If example average does not still meet, so as described replacement kapillary and flushing.The monose standard of moving sample once more and carrying out together.
Figure A200680053116D05691
Wherein:
Measurement number in the n=sample
Ce Liang not for x=
Calculate the proteinic mol ratio of GalNAc/:
R GalNAc = A GalNAc x A ManNAc 0 x V GalNAc 0 x C GalNAc 0 x MW CTLA 4 - Ig A ManNAc x A GalNAc 0 xVpxCpx MW GlcNAC
Wherein:
R GalNAc=GalNAc is to proteinic mol ratio
A GalNAcThe peak area of GalNAc in the=sample (μ V second)
A ManNAcThe peak area of ManNAc in the=sample (μ V second)
A ManNAc0The peak area of ManNAc in the=monose standard (μ V second) mean value
A GalNAcOThe peak area of GalNAc in the=monose standard (μ V second) mean value
V GalNAcO=be used for the GalNAc volume (representing) that the monose working solution of hydrolysis comprises with μ L
C GalNAcO=be used for the GalNAc concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=the be used for volume (representing) of the protein example of hydrolysis with μ L
Cp=the be used for concentration (representing) of the protein example of hydrolysis with mg/mL
MW CTLA4-IgThe molecular weight of=CTLA4-Ig reference material
MW GlcNAcThe molecular weight of=GalNAc (221.2 dalton)
Standard is carried out together.When calculating the mol ratio of CTLA4-Ig reference material and sample, use all 8 kinds system's suitability criterion of carrying out together.Ask the mean value that is included in the peak area in this equation.This will be used for preceding 3 kinds of samples.For every other sample, use the average peak area of ensuing 4 kinds of monose standards of carrying out together and preceding 4 kinds of monose standards of carrying out together to be used for mol ratio calculating always.
Calculate the proteinic mol ratio of GIcNAc/
R GalNAc = A GlcNAc &times; A ManNAc 0 &times; V GlcNAc 0 &times; C GlcNAc 0 &times; ME CTLA 4 - Ig A ManNAc &times; A GlcNAc 0 &times; VpxCp &times; MW GlcNAC
Wherein:
R GlcNAc=GlcNAc is to proteinic mol ratio
A GlcNAcThe peak area of GlcNAc in the=sample (μ V second)
A ManNAcThe peak area of ManNAc in the=sample (μ V second)
A ManNAcOThe peak area of ManNAc in the=monose standard (μ V second) mean value
A GlcNAcOThe peak area of GlcNAc in the=monose standard (μ V second) mean value
V GlcNAcO=be used for the GlcNAc volume (representing) that the monose working solution of hydrolysis comprises with μ L
C GlcNAcO=be used for the GlcNAc concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=the be used for volume (representing) of the protein example of hydrolysis with μ L
Cp=the be used for concentration (representing) of the protein example of hydrolysis with mg/mL
MW CTLA4-Ig=according to the molecular weight of the CTLA4-Ig reference material of COA
MW GlcNAcThe molecular weight of=GlcNAc (221.2 dalton)
Example values.Should not surpass 10% about 2 kinds, per-cent RSD that carry out together, amino system suitability criterion peak area ratio.Molar average ratio about the amino monose in the reference material must be in specified scope.For every kind of component, about the 4 kinds of results' duplicate injection of preparation (duplicate) %RSD necessary＜/=25%.
The molar ratio range of CTLA4-Ig reference material
Monose Scope
GalNAc 2.0-3.2
GlcNAc 18-32
Report the result.Average result is reported as GalNAc molecule number/CTLA4-Ig molecule and GlcNAc molecule number/CTLA4-Ig molecule.Mol ratio result's report is to 2 significant figure.For every kind of component, about the 4 kinds of results' duplicate injection of preparation (duplicate) %RSD necessary＜/=25%.
Embodiment 64: obtain neutral monose (seminose, rock algae in the CTLA4-Ig drug sample Sugar, semi-lactosi) with the method for proteinic mol ratio
Reagent
Hydrating solution (the 2M TFA aqueous solution) adds 148 μ L TFA and 852 μ L HPLC grade water in 1.7 Eppendorf tubes.Vortex 5-10 second.Enlarge in proportion in case of necessity.Tightly prepare solution before use.
Derivatize solution I (the 0.1M APTS aqueous solution).192 μ L HPLC grade water are added in the 10mg APTS powder in the vial.Make bottle vortex 5-10 second to dissolve APTS fully.Preserve in-20 ℃ maximum 1 year.
Derivatize solution II (1M acetate and 0.25M NaBH 3CN).In the 0.4mL centrifuge tube, dilute 20 μ L acetate (17 times of dilutions), with preparation 1M acetic acid solution with 320 μ L HPLC grade water.With 2.0 ± 0.5mg NaBH 3CN is weighed in the profound hypothermia bottle.Use following formula, the 1M acetic acid solution that adds suitable volumes is with preparation 0.25M NaBH 3CN.
Volume (μ L)=10 3* (the NaBH that represents with mg 3CN weight)/62.84g/mol * 0.25mol/L)
Sodium cyanoborohydride (NaBH 3CN) should preserve in moisture eliminator in the dark.
Recommend following reagent is assigned to again to be used for storage in a series of 2.0mL cryovials, to avoid the initial reagent bottle of repeated open:
1g ± 0.2mg sodium cyanoborohydride is weighed in the 2.0mL cryovial.By this way will be from the entire contents aliquots containig of the sodium cyanoborohydride in the initial bottle.
Cover lid and together with reagent name, lot number and 6 months validity period mark cryovial such as (1,2,3) in turn tightly.
Bottle is used the Parafilm sealing to avoid moisture.
Weighing up sodium cyanoborohydride from identical cryovial is used for the derivatize solution II and is no more than 3 times.On the laboratory work table, indicate this point and cryovial sequence number (SN).
Observed reagent peak or weak mark may take place after the cryovial repeated open or behind the sodium cyanoborohydride with the sort of particular batch in the CE overview.If this influences the result, discard the cryovial of use so and from cryovial or from the new lot sodium cyanoborohydride, weigh up reagent with next sequence number (SN).
Running buffer (60 ± 5mM sodium tetraborate, pH9.25)
1.21 ± 0.02g sodium tetraborate is weighed in the clean vial of 100mL.
Add 90mL HPLC grade water, and on agitating plate, mix and dissolve fully until salt.
With 10N NaOH pH is adjusted to 9.25 ± 0.10.
(± 5mM) final concentration adds HPLC grade water to obtain the final volume of 100mL for 60mM.
For 55mM solution, weighing 1.11g (± 0.02) sodium tetraborate and be used for according to the above description the dissolving and titration.
For 65mM solution, weighing 1.31g (± 0.02) sodium tetraborate and be used for according to the above description the dissolving and titration.
Preserved in room temperature maximum 3 months.If peak resolving power (as definition in system's suitability part) influenced (R value＜1.0) prepares fresh damping fluid so.
Choose wantonly: (± 5mM) final concentration is by adding dilution tetraborate buffered soln (MicroSolv) in the 80mL 150mM sodium tetraborate damping fluid with the 120mL ultrapure water for 60mM.With 10N NaOH titration so that pH value of solution reaches 9.25 (± 0.1).
For 55mM tetraborate solution, 66mL150mM sodium tetraborate damping fluid is diluted in the 114mL ultrapure water.As above titration.
For 65mM tetraborate solution, 78mL 150mM sodium tetraborate damping fluid is diluted in the 102mL ultrapure water.As above titration.
Solution was preserved in room temperature maximum 3 months.If peak resolving power (as definition in system's suitability part) influenced (R value＜1.0) prepares fresh damping fluid so.
Capillary douche solution
1N NaOH solution
The adding of 1mL 10N NaOH solution is comprised in the graduated plastics tubing of 14mL of 9mL HPLC grade water.By vortex 5-10 thorough mixing second.
Solution was preserved in room temperature maximum 6 months.
1N HCl solution:
The adding of 1mL 6N HCl solution is comprised in the graduated plastics tubing of 15mL of 5mL HPLC grade water.By vortex 5-10 thorough mixing second.
Solution was preserved in room temperature maximum 6 months.
80% methanol solution:
The adding of 8mL HPLC grade methyl alcohol is comprised in the graduated plastics tubing of 15mL of 2mL HPLC grade water.By vortex 5-10 thorough mixing second.
Solution was preserved in room temperature maximum 6 months.
Monose standard stock solution
Seminose (Man)
5 ± 1mg seminose accurately is weighed in the 2.0mL profound hypothermia bottle.
Add 1mL HPLC grade water and pass through vortex 5-10 thorough mixing second.
The accurate concentration (mg/mL) of recording solution.
Fucose (Fuc)
5 ± 1mg Fucose accurately is weighed in the 2.0mL profound hypothermia bottle.
Add 1mL HPLC grade water and pass through vortex 5-10 thorough mixing second.
The accurate concentration (mg/mL) of recording solution.
Semi-lactosi (Gal)
5 ± 1mg semi-lactosi accurately is weighed in the 2.0mL profound hypothermia bottle.
Add 1mL HPLC grade water and pass through vortex 5-10 thorough mixing second.
The accurate concentration (mg/mL) of recording solution.
Wood sugar (Xyl)
5 ± 1mg wood sugar accurately is weighed in the 2.0mL.
Add 1mL HPLC grade water and pass through vortex 5-10 thorough mixing second.
The accurate concentration (mg/mL) of recording solution.
Monose standard stock solution is preserved in-20 ℃ maximum 1 year.
Monose working solution I: internal standard working solution.Be preparation internal standard working solution, by 20 μ L wood sugar stock solutions (3.6.4) are added in the 2mL profound hypothermia bottle, use HPLC grade water that the stock solution of wood sugar is diluted 100 times, described profound hypothermia bottle has comprised 1980 μ L HPLC grade water.The about 5-10 of vortex second.This internal standard working solution is preserved in 2-8 ℃ maximum 6 months.
Monose working solution II: neutral hybrid standard working solution.In the 2.0mL profound hypothermia bottle that comprises 1940 μ L HPLC grade water, add the stock solution of 20 μ L seminoses, Fucose and semi-lactosi respectively.The about 5-10 of vortex second.This internal standard working solution is preserved in 2-8 ℃ maximum 6 months.
Sample and reference material solution.In the 2-8 ℃ of refrigerated protein example that thaws, and by being inverted mixing gently.With the HPLC grade sample and reference material are diluted to about 1.0mg/mL.
The CE operational conditions
Running buffer 60mM sodium tetraborate, pH9.25
25 ℃ of capillary column body temperature degree
Voltage 25-30kV, holotype
Detector condition LIF detector excites: 488nm, and emission:
520nm
Sample injection pressure injection pattern, 20s when 0.5 PSI
15 minutes working times
10 ℃ of sample storages
Program
Hydrolysis: in the 0.65mL centrifuge tube, add 10 μ L wood sugar working solutions and 200 μ L2MTFA solution.This serves as system's blank.In the 0.65mL centrifuge tube, add the neutral hybrid standard working solution of 10 μ L wood sugar working solutions and 10 μ L.Further add 200 μ L 2M TFA solution and vortex 3-4 second.This serves as and is used for quantitatively and the monose standard of system's suitability.Prepare in duplicate.In the 0.65mL centrifuge tube, add 10 μ L wood sugar working solutions and 10 μ L CTLA4-Ig reference material solution (about 1mg/mL).Further add 200 μ L 2M TFA solution and vortex 3-4 second.Prepare in duplicate.In the 0.65mL centrifuge tube, add 10 μ L wood sugar working solutions and 10 μ L sample solutions (about 1mg/mL).Further add 200 μ L 2M TFA solution and vortex 3-4 second.Prepare in duplicate.Make about 20 seconds of sample vortex and centrifugal about 5-10 second.Sample placed the little bottle stand in 96 positions and at baking box in 95 ℃ of incubations 6 hours.After the hydrolysis, sample is placed-20 ℃ 10 minutes with the cooling.Make that the sample of hydrolysis is of short duration centrifugally to be forced to bottom (in high speed time 5-10 second) to pipe until all condensation products.In SpeedVac, make sample evaporation to dry.With 100 μ LHPLC grade every kind of samples of water reconstruct and vortex 10-15 second.In SpeedVac, make sample evaporation to dry.
Derivatize: Eppendorf centrifuge is placed the oven temperature of baking box with balance to 55 ℃.With 10 μ L derivatize solution I (0.1M APTS solution) every kind of samples of reconstruct.The about 5-10 of vortex second.Add 5 μ L derivatize solution II (1M HAc and 0.25M NaBH 3CN).Vortex about 5-10 second and centrifugal.Sample flasket is loaded in the whizzer of heating in advance fast, and whizzer is put back in 55 ℃ of baking boxs.3 hours whiles of incubation are centrifugal under 2000rpm.This prevents solvent condensation on little bottle surface.
Instrument configuration preparation: new kapillary is installed, is used following step to wash with high pressure mode (80PSI):
1N NaOH20 minute.
HPLC grade water 10 minutes.
60mM sodium tetraborate damping fluid 10 minutes.
Operation wash/rinse order is with the flushing kapillary.Operational system suitability criterion (monose standard) is fit to guarantee system subsequently.But use the capillaceous inside of 1N NaOH etching, and cause the change that moves in the transition time from start to finish from different vendor.If this transition time that causes postpeak (semi-lactosi) surpasses 15.0 minutes,, may replace 1N NaOH with 0.1N NaOH or HPLC grade water so for step 2 flushing.When using kapillary of equal value and above-mentioned washing procedure enough, may use 80% methyl alcohol and/or 1N HCl so that postpeak (semi-lactosi) in 15.0 minutes example values.
The preparation that is used to inject: behind the derivatize, make sample be cooled to room temperature.At room temperature centrifugal about 10 seconds, be forced to bottom to pipe until condensation product.In each pipe, add 85 μ L HPLC grade water so that the final volume of every kind of sample reaches 100 μ L.Vortex 5-10 second.From each pipe, shift 10 μ L samples in CE trace bottle, and in each pipe, add 190 μ L HPLC grade water.Vortex 5-10 second.
Rinse step and injection sequence:
Figure A200680053116D05761
Figure A200680053116D05771
*Carry out with 2 injections for maximum 3 kinds of sample repetitive sequences in duplicate with every kind of preparation of monose standard.Use all 8 kinds of system's suitability criterion injections for the sample that places 3 group.If operation surpasses 3 kinds of samples, so as mentioned shown in the order from the 19th row other sample that brings into operation.
*Sample is carried out with 2 injections of every kind of preparation of CTLA4-Ig reference material.
System's suitability.The electrophorogram of first kind of system's suitability should be similar to wherein, and peak 1 is a seminose; Peak 2 is wood sugars; Peak 3 is Fucoses; With peak 4 are semi-lactosis.Annotate: when the CE instrument that uses except that BeckmanPACE MDQ, owing to keep the various configurations of the cylinder capillaceous that separates, length capillaceous can be different from this method specified the sort of.This will cause the variation in analyte transition time and the peak intensity.
Calculate 2 adjacent peak-to-peak resolving power according to equation by instrument about first kind of system's suitability criterion:
R = 2 ( t 2 - t 1 ) ( W 1 + W 2 )
Wherein:
R=resolving power
t 2, t 1=2 adjacent peaks transition time separately
W 1, W 2The peak width at=2 each comfortable baseline places of adjacent peak
The R value is necessary 〉=and 1.0.If R＜1.0 are so with wash/rinse order flushing kapillary; If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.
For the suitability injection of last system, use the last peak (semi-lactosi) of following formula must have＜1.4 tailing factor:
T＝W 0.05/2f
Wherein:
The T=tailing factor
W 0.05=in the peak width in 5% when height
The width of f=front, peak when peak maximum
If T 〉=1.4 are so with wash/rinse order flushing kapillary; If problem exists, replace old damping fluid or replace kapillary with the running buffer of prepared fresh so.
The duplicate injection of kind system suitability criterion of the forth day of a lunar month must meet following example values:
Semi-lactosi is to the peak area ratio of wood sugar: RSD≤10%.
The transition time of semi-lactosi needs≤15.0 minutes
Overview should be equivalent to Fig. 1, and wherein observing 4 peaks and internal standard (wood sugar) is the 2nd peak.
If any one in the above-mentioned example values do not meet, so if the transition time of semi-lactosi greater than 15.0 minutes, then at first increases voltage.Secondly, if peak area ratio〉10%, determine its pH or replace kapillary thereby prepare fresh CE damping fluid so.
After adjusting instrument, the injection of duplicated system suitability.When analyzing the peak overview, if the remarkable reduction in the peak height of generation wood sugar checks that so the fiberoptic cable to determine to enter in the LIF module is not misalignment.Recently measure the monose standard percentage by the peak area that compares internal standard and monose standard ingredient and compare RSD.Will be about the peak area of each monosaccharide component divided by peak area about the internal standard of each monose standard injection.Calculating is about the per-cent RSD of seminose, Fucose and the semi-lactosi of 2 kinds of standards of carrying out together.RSD answers≤10%.If this example average does not meet, kapillary should as above wash or replace so.Sample and the monose standard of carrying out together need be carried out repetition.
Calculate.Calculate Man, Fuc and Gal peak area ratio with respect to internal standard (wood sugar).The duplicate injection that is used in the forth day of a lunar month kind system suitability criterion is so that meet example values, and all of before the sample and back injection are carried out together, system's suitability criterion carries out identical calculations.Peak area ratio=will be about the peak area of every kind of monosaccharide component (Man, Fuc and Gal) divided by peak area about the internal standard (wood sugar) of each system suitability criterion injection.
Figure A200680053116D05791
Calculating in system's suitability criterion about the mean value of the peak area ratio of Man, Fuc and Gal.Also base of calculation poor (S.D.) and per-cent coefficient of variation (%RSD).Example values: about RSD≤10% of the peak area ratio of semi-lactosi.2 kinds of the injection of before the sample and back, carry out together, system's suitability criterion:
Per-cent RSD≤10% about the peak area ratio of Man, Fuc and Gal.
If this example average does not meet (RSD〉10%), kapillary needs wash once more with cleaning procedure so, and those samples and the monose standard of carrying out together need to move once more.If example average does not still meet, replace kapillary and flushing so.The monose standard of moving sample once more and carrying out together.
Figure A200680053116D05801
Wherein:
Measurement number in the n=sample
Ce Liang not for x=
Figure A200680053116D05802
Calculate seminose/proteinic mol ratio:
R Man = A Man x A Xy 10 x V Man 0 x C Man 0 x MW CTLA 4 - Ig A Xy 1 x A Man 0 x V p x C p x MW Man
Wherein:
R Man=seminose (Man) is to proteinic mol ratio
A ManThe peak area of Man in the=sample (μ V second)
A XylThe peak area of wood sugar in the=sample (Xyl) (μ V second)
A Xyl0The peak area of Xyl in the=monose standard (μ V second) mean value
A Man0The peak area of Man in the=monose standard (μ V second) mean value
V Man0=be used for the Man volume (representing) that the monose working solution of hydrolysis comprises with μ L
C Man0=be used for the Man concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=is used for the volume (representing with μ L) of the protein example of hydrolysis
Cp=is used for the concentration (representing with mg/mL) of the protein example of hydrolysis
MW CTLA4-Ig=according to the molecular weight of the CTLA4-Ig reference material of analysis certificate (COA)
MW ManThe molecular weight of=seminose (180.2 dalton).
Standard is carried out together.When calculating the mol ratio of CTLA4-Ig reference material and sample, use all 8 kinds system's suitability criterion of carrying out together.Ask the mean value that is included in the peak area in this equation.This will be used for preceding 3 kinds of samples.For every other sample, use the average peak area of ensuing 4 kinds of monose standards of carrying out together and preceding 4 kinds of monose standards of carrying out together to be used for mol ratio calculating always.
Calculate Fucose/proteinic mol ratio:
R Fuc = A Fuc x A Xy 10 x V Fuc 0 x C Fuc 0 x MW CTLA 4 - Ig A Xy 1 x A Fuc 0 x V p x C p x MW Fuc
Wherein:
R Fuc=Fucose (Fuc) is to proteinic mol ratio
A FucThe peak area of Fuc in the=sample (μ V second)
A XylThe peak area of wood sugar in the=sample (Xyl) (μ V second)
A Xyl0The peak area of Xyl in the=monose standard (μ V second) mean value
A Fuc0The peak area of Fuc in the=monose standard (μ V second) mean value
V Fuc0=be used for the Fuc volume (representing) that the monose working solution of hydrolysis comprises with μ L
C Fuc0=be used for the Fuc concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=is used for the volume (representing with μ L) of the protein example of hydrolysis
Cp=is used for the concentration (representing with mg/mL) of the protein example of hydrolysis
MW CTLA4-Ig=according to the molecular weight of the CTLA4-Ig reference material of analysis certificate (COA)
MW FucThe molecular weight of=Fucose (164.2 dalton)
Calculate semi-lactosi/proteinic mol ratio:
R Gal = A Gal &times; A Xy 10 &times; V Gal 0 &times; C Gal 0 &times; MW CTLA 4 - Ig A Xy 1 &times; A Gal 0 &times; V p &times; C p &times; MW Gal
Wherein:
R Gal=semi-lactosi (Gal) is to proteinic mol ratio
A GalThe peak area of Gal in the=sample (μ V second)
A XylThe peak area of wood sugar in the=sample (Xyl) (μ V second)
A Xyl0The peak area of Xyl in the=monose standard (μ V second) mean value AGal 0
V Gal0=be used for the Gal volume (representing) that the monose working solution of hydrolysis comprises with μ L
C Gal0=be used for the Gal concentration (representing) that the monose working solution of hydrolysis comprises with mg/mL
Vp=is used for the volume (representing with μ L) of the protein example of hydrolysis
Cp=is used for the concentration (representing with mg/mL) of the protein example of hydrolysis
MW CTLA4-Ig=according to the molecular weight of the CTLA4-Ig reference material of analysis certificate (COA)
MW GalThe molecular weight of=Gal (180.2 dalton)
Annotate: when calculating the mol ratio of CTLA4-Ig reference material and sample, use last system's suitability criterion and following a kind of system's suitability criterion preparation that carries out together.Ask the mean value that is included in the peak area in this equation.This will be used for preceding 6 kinds of samples.For every other sample, use the average peak area of 2 kinds of monose standards of carrying out together to be used for mol ratio calculating always.
Example values.Should not surpass 10% about 2 kinds, per-cent RSD that carry out together, neutralized system suitability criterion peak area ratio.Molar average ratio about the neutral monose in the reference material can be in the specified scope of following table.For every kind of component, about the 4 kinds of results' duplicate injection of preparation (duplicate) %RSD necessary＜/=25%.
The molar ratio range of CTLA4-Ig reference material
Monose Scope
Seminose 11-18
Fucose 4.2-7.5
Semi-lactosi 9.2-18
Report the result.Average result is reported as mannose molecules number/CTLA4-Ig molecule, Fucose molecule number/CTLA4-Ig molecule and galactose molecule number/CTLA4-Ig molecule.Mol ratio result's report is to 2 significant figure.For every kind of component, about the 4 kinds of results' duplicate injection of preparation (duplicate) %RSD necessary＜/=25%.
The trypsinase mapping of embodiment 65-CTLA4-Ig oxidation and deacylated tRNA amine quantitatively
The purpose of this method is to use manual tryptic peptide plotting program with the specific detection of methionine(Met) oxidation and l-asparagine deacylated tRNA amine with quantitatively monitor consistence between CTLA4-Ig batch.Peptide mapping relates to proteinic proteolysis or other rupture to produce well-defined peptide fragment group, and it is analyzed via HPLC subsequently usually.Chromatogram or peptide figure in the proteinic chemical structure in addition minimum variation all very responsive, and therefore for detecting and to characterize posttranslational modification useful.With before the proteolytic ferment tryptic digestion, the CTLA4-Ig protein example carries out sex change in 8M guanidine-HCl damping fluid, and the halfcystine disulfide linkage reduces with dithiothreitol (DTT) and carries out the S-alkanisation with iodo-acid amide.Resulting trypsinase peptide mixt subsequently by RPHPLC (reversed-phase high-performance liquid chromatography) (RP-HPLC) with 215 and the UV at 280nm place detect and analyze.Some abbreviations are listed hereinafter:
Asn L-asparagine
Asp Aspartic acid
Asu Amino succinimide
isoAsp Different aspartic acid
Met(O) Methionine sulfoxide
T26 (281-302) tryptic peptide
T26deam1 IsoAsp294 (281-302) tryptic peptide
T26deam2 Asp299 (281-302) tryptic peptide
T26deam3 Asp294 (281-302) tryptic peptide
T26deam4 Asu294 (281-302) tryptic peptide
T6 (84-93) tryptic peptide
T6ox Met (O) 85 (84-93) tryptic peptide
Pharmaceutical chemicals and reagent
Mobile phase A-the be dissolved in 0.1%TFA of HPLC grade water
The entire content of 1mL ampoule TFA is transferred to 1000mL HPLC grade water, and thorough mixing is with preparation 0.1%TFA (mobile phase A).0.1%TFA can preserve in room temperature maximum 2 months.
Mobile phase B-the be dissolved in 0.1%TFA of 80%ACN and 20%HPLC grade water
The entire content of 1mL ampoule TFA is transferred to 800mL acetonitrile and 200mL HPLC grade water, and thorough mixing is dissolved in the 0.1%TFA (Mobile phase B) of 80%ACN with preparation, it can be preserved in room temperature maximum 2 months.
Dilution buffer liquid-100mM TRIS, 25mM NaCl, pH7.6
By stirred solution on magnetic stirring plate, 14.0g Trizma Pre-Set Crystal pH7.6 and 1.46g NaCl are dissolved in the 1000mL HPLC grade water.By 0.2 μ m filter unit filtering solution.Solution is preserved in 2-8 ℃ maximum 2 months.
Sex change damping fluid-8M guanidine, 50mM TRIS, pH8.0
By stirred solution on magnetic stirring plate, 152.8g Guanidinium hydrochloride and 1.4g TrizmaPre-Set Crystal pH8 are dissolved in the 90mL HPLC grade water.With HCl or NaOH pH is adjusted to 8.0, and reaches final volume 200mL with HPLC grade water.By 0.2 μ m filter filtering solution.Solution was preserved in room temperature maximum 6 months.
Digestion damping fluid-50mM TRIS, 10mM CaCl 2, pH7.6
By stirred solution on magnetic stirring plate, with 7.0g Trizma Pre-Set Crystal pH7.6 and 1.47g CaCl 2Be dissolved in the 1000mL HPLC grade water.By 0.2 μ m strainer filtering solution.Solution is preserved in 2-8 ℃ maximum 2 months.
Reductive agent-200mM dithiothreitol (DTT) (DTT)
Tightly in 30.8 ± 0.2mg DTT, add 1000 μ L water and vortex before use until dissolving.Solution expired from 24 hours whens preparation.
Alkylating agent-400mM iodo-acid amide (IAM)
Tightly in 74.0 ± 0.5mg iodo-acid amide, add 1000 μ L water and vortex before use until dissolving.Solution expired from 24 hours whens preparation.
1.0M?HCl
With HPLC grade water the 8.7mL concentrated hydrochloric acid is diluted to 100mL.Solution was preserved in room temperature maximum 2 months.
Standard and contrast
T6ox peptide standard, 30 μ M
T6ox tryptic peptide synthetic standards is Ala-Met (O)-Asp-Thr-Gly-Leu-Tyr-Ile-Cys-Lys2TFA, and FW 1358.4 ,～95 weight % purity.The solid tight seal is preserved in-20 ℃, and always moisture eliminator heat to room temperature to prevent moisture absorption.Weigh up 1.0 ± 0.1mg T6ox, the record exact weight, and be dissolved in the 1.50mL digestion damping fluid.Add 40uL 200mM DTT and place 37 ℃ 20 minutes.Be cooled to room temperature, add 48 μ L 400mM iodo-acid amides, and alkanisation 30 minutes in the dark at room temperature.The final volume that is diluted to 24.5mL with the digestion damping fluid is to produce 30 ± 3 μ M standardized solution.Preserve in-70 ℃ the 1mL aliquots containig of 30 μ M T6ox standards maximum 24 months.
T26deam1 peptide standard, 30 μ M
T26deam1 tryptic peptide synthetic standards is Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-Ser-isoA sp-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys2TFA, and FW 2773.7 ,～85 weight % purity.The solid tight seal is preserved in-20 ℃, and always moisture eliminator heat to room temperature to prevent moisture absorption.Weigh up 1.0 ± 0.1mg T26deam1, the record exact weight, and be dissolved in 1mL and be dissolved in the 30%v:v acetonitrile of digestion in the damping fluid.The final volume that is diluted to 10.7mL is to produce 30 ± 3 μ M standardized solution.Preserve in-70 ℃ the 1mL aliquots containig of 30 μ M T26deam1 standards maximum 24 months.
The preparation of standard and sample
Reduction and alkanisation
Protein concn scope about peptide mapping is about 20mg/mL.If protein concn〉20mg/mL, use dilution buffer liquid with the final concentration of diluted sample to about 20mg/mL so.The solution of preparation at least 100 μ L dilution.In the 1.7mL centrifuge tube, 100 μ L20mg/mL (2mg) CTLA4-Ig solution (sample or reference material) are added in the 500 μ L sex change damping fluids.Add 35 μ L200mM reductive agents, make pipe vortex 3-5 second, centrifugal subsequently about 3 seconds.Make pipe in 37 ℃ of incubations 20 ± 2 minutes.In each pipe, add 38.5 μ L 400mM alkylating agents, vortex 3-5 second, centrifugal subsequently about 3 seconds.Make sample incubation 20 ± 2 minutes in the dark at room temperature.The NAP-5 post is placed shelf.Use 1 post/sample.When sample in IAM during incubation, with 7.5mL digestion damping fluid balance NAP-5 post.Discard effluent according to the site program.The mixture of 500 μ L reduction and alkanisation is added on the NAP-5 post, thereby allows liquid to discharge by post.Discard effluent according to the site program.1.0mL is digested damping fluid add in the NAP-5 post, and collect effluent in the 1.7mL centrifuge tube and the effluent of mixed collection gently.
Digestion.For every mL sample and reference material to be digested, with 1 trypsinase bottle of every kind of trypsinase damping fluids of 86 μ L (supplying) reconstruct (20 μ g), add other 1 trypsinase bottle, to produce 0.25 μ g/ μ L with enzyme.The content of trypsinase bottle is merged in 1 bottle together.With the trypsin solution/mL sample of the above-mentioned merging of 80 μ L under 37 ℃ with every kind of treatments of the sample 120 ± 12 minutes.After digestion is finished, come acidified sample with 40 μ L 1.0M HCl/mL samples, and vortex 3-5 second.The digest of each 100 μ L sample and reference material is pipetted in the automatic sampler bottle.Preparation comprises the other system suitability contrast with 5 μ L, 30 μ M T6ox peptide standards and 5 μ L30 μ M T26deam1 peptide standard blended, 95 μ L reference material digests, to produce the digest with 5%T6ox and 5%T26deam1 spike.Placing the little device of automatic sampler to be used for HPLC under 5 ± 5 ℃ all bottles analyzes.All residue sample digestions are preserved in-70 ℃.
Chromatography condition
Following table shows flow velocity and chromatography gradient.
Time (minute) Flow (mL/ minute) Mobile phase A Mobile phase B
0 0.25 100 0
4 0.25 100 0
10 0.25 92 8
72 0.25 72 28
84 0.25 60 40
92 0.25 0 100
94 0.40 0 100
95 0.40 100 0
109 0.40 100 0
110 0.25 100 0
Before injection for the first time, 55 ℃ down with 100% mobile phase A with column equilibration at least 25 minutes.The UV absorbancy at monitoring 215nm and 280nm place.Pipeline from the post to the detector should have≤0.01 " internal diameter so that the diffusion band broadening drops to minimum.Make column temperature be maintained at 55 ± 2 ℃.Make the automatic sampler temperature maintenance in 5 ± 5 ℃.Precession phase A is as blank injection.Sample (at every turn being no more than 10) is carried out with the reference material injection.Following table shows the injection sequence that is used for chromatographic analysis.Should be understood that all volume injected are 25 μ L, except by the control sample of forming with the reference material of 5%T6ox and 5%T26deam1 peptide standard spike, it injects 28 μ L:
Bottle # Injection The sample title Volume injected (μ L)
1 1 Blank (mobile phase A 25
2 1 Reference material 25
3 1 Sample 1 25
4 1 Sample 2 25
5 1 Sample 3 25
?6 ?1 Reference material with 5% T6ox and 5% T26deam1 spike 28
2 1 Reference material 25
Example values.
Example values.With regard to regard to remarkable peak number order, relative size and the elution order of 215nm and 280nm trace, about the peptide figure overview of reference material must be visually can with the chromatogram that presents among Fig. 1 relatively.Retention time difference about peak T4, T25 and T27 in the reference material that initial sum is carried out together should be no more than ± and 0.5 minute.Theoretical plate number is necessary 〉=and 100,000.If N＜100,000 make post balance again so.If problem exists, replace post so.For T2 and T12 peak, resolving power (R) is necessary 〉=and 1.5.If R＜1.5 make post balance again so.If problem exists, replace post so.As shown in Figure 85, must show increase about the T6ox peak of wash-out～33.0 minutes the time with the 280nm chromatogram of the reference material of 5%T6ox and T26deam1 spike.As shown in Figure 87, must show increase about the T26deam1 peak of wash-out～66.5 minutes the time with the 215nm chromatogram of the reference material of 5%T6ox and T26deam1 spike.
The sampling value.As pointing out about marker peak among Figure 85, with regard to remarkable peak number order, relative size and the elution order of 215nm and 280nm trace, the chromatogram of reference material injection for the first time and sample must be visually of equal value, except the oxidation and/or deacylated tRNA amine peak about T6ox and T26deam of separately report.Must be in retention time ± 0.5 of the respective peaks of the reference material injection first time minute in the sample about the retention time of peak T4, T25 and T27.
Calculate.Annotate: except as otherwise noted, calculate use 215nm data for these.The retention time (Figure 85) of the operating peak T4 of the reference material of carrying out, T25 and T27 should not differ and surpass 0.5 minute (Figure 85) together.
Theoretical plate number.Post effect as theoretical plate number (N) assessment uses retention time and the width (Figure 85) of the peak T27 that moves from reference material to calculate according to equation:
N = 16 ( t w ) 2
Wherein:
W=is by being extrapolated to straight relatively side the peak width at the baseline place of base measurement
The retention time of the peak T27 that the elution time of t=from inject time to the peak maximum measured
Resolving power.Resolving power (R) between peak T12 and the peak T2 (Figure 85) uses equation to calculate:
R = 2 ( t 2 - t 1 ) ( w 1 + w 2 )
Wherein:
t 1, t 2=be respectively the retention time of peak T12 and peak T2
w 1, w 2=have retention time t respectively 1And t 2The peak width that limits of the tangent line at peak base place.
For all samples and standard, following per-cent oxidation by 280nm peak area data computation Met85:
Oxidation per-cent=100*A T6ox/ (A T6ox+ A T6)
Wherein:
A T6=in the 280nm trace about T6, peak area (84-93)
A T6ox=in the 280nm trace about T6ox, Met (O) 85Peak area (84-93)
For all samples and standard, according to the per-cent deacylated tRNA amine of the Asn294 of the 215nm of data computation shown in Figure 87 peak area:
Figure A200680053116D05881
Wherein:
A T26=in the 215nm trace, about T26, peak area (281-302)
A T26deam1=in the 215nm trace, about T26deam1, isoAsp 294Peak area (281-302).
A T26deam2=in the 215nm trace, about T26deam2, Asp 299Peak area (281-302).
A T26deam3=in the 215nm trace, about T26deam3, Asp 294Peak area (281-302).
A T26deam4=in the 215nm trace, about T26deam4, Asu 294Peak area (281-302).
The theory expectation fragment (with reference to Figure 85) of CTLA4-Ig tryptic digestion thing
The fragment residue sequence
T1 1-14 MHVAQPAVVLASSR
T2 15-28 GIASFVCEYASPGK
T3 29-33 ATEVR
T4 34-38 VTVLR
T5 ＊ 39-83 QADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQ
VNLTIQGLR
T6 84-93 AMDTGLYICK
T7 ＊ 94-128 VELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPK
T8 ＊＊ 129-132 SSDK
T9 ＊＊ 133-158 THTSPPSPAPELLGGSSVFLFPPKPK
T10 159-165 DTLMISR
T11 166-184 TPEVTCVVVDVSHEDPEVK
T12 185-198 FNWYVDGVEVHNAK
T13 199-202 TKPR
T14 ＊ 203-211 EEQYNSTYR
T15 212-227 VVSVLTVLHQDWLNGK
T16 228-230 EYK
T17 231-232 CK
T18 233-236 VSNK
T19 237-244 ALPAPIEK
T20 245-248 TISK
T21 249-250 AK
T22 251-254 GQPR
T23 255-265 EPQVYTLPPSR
T24 266-270 DELTK
T25 271-280 NQVSLTCLVK
T26 281-302 GFYPSDIAVEWESNGQPENNYK
T27 303-319 TTPPVLDSDGSFFLYSK
T28 320-324 LTVDK
T29 325-326 SR
T30 327-349 WQQGNVFSCSVMHEALHNHYTQK
T31 350-356 SLSLSPG
*Comprise N connection carbohydrate.
*Comprise O connection carbohydrate.
Single agent people research of embodiment 66-health
Unit point, randomization, single agent research are used for assessing the pharmacokinetics of CTLA4-Ig (being produced by CD-CHO1) method the health volunteer.Reach ten three (13) the individual experimenters that comprise and get rid of example values and enter clinical research unit, and accept the CTLA4-Ig through 30 minutes single intravenous infusions by method CD-CHO1 generation as 10mg/kg.Each experimenter observed in clinical research unit 24 hours behind the infusion.Be used for the quantitative of CTLA4-Ig collecting blood sample to the highest 71 days at the appointed time after the administration.The experimenter assesses with regard to pharmacokinetics: about each experimenter's Cmax, Tmax, AUC (INF), T-HALF, CLT and Vss derived from serum-concentration to time data.CTLA4-Ig supplies as 200mg/ bottle preparation.Use the 10mg/kg CTLA4-Ig of 30 minutes IV infusions to the health volunteer.PK mensuration, security and immunogenicity determining are being assessed on the 71st day fixed time point after the administration.
Statistical method:
Sample size: 13 experimenters' sample size provides 90% degree of confidence, wherein for CTLA4-Ig, the assessment of geometrical mean ratio will about the true value of Cmax 15% in and about the true value of AUC (INF) 10% in.Statistical analysis: summarized experimenter consensus data, physical examination, laboratory test data and vital sign.The incidence of adverse events is tabulated by body system and severity.For Cmax and the AUC (INF) of CTLA4-Ig, by 90% fiducial interval of calculating about the The results of analysis of variance of log (Cmax) and log (AUC) about colony's geometrical mean ratio of method CD-CHO1.
Pharmacokinetics result: pharmacokinetics result uses the non-compartment routine analyzer of checking to measure.Must use by oneself 13 experimenters of method CD-CHO1CTLA4-Ig administration of pharmacokinetic parameter.Following table has been listed in the health volunteer pharmacokinetic parameter about CTLA4-Ig.Following table has shown the summary cartogram about the CTLA4-Ig pharmacokinetic parameter.
Pharmacokinetic parameter (N＝3)
Cmax (μ g/mL) geometrical mean (CV%) 284.7?(23％)
AUC (INF) (μ g.h/mL) geometrical mean (CV%) 44403.0(18％)?
Tmax (h) intermediate value (min, max) 0.50 (0.50，2.00)
T-HALF (my god) mean value (SD) 16.68?(3.24)
CLT (mL/h/kg) mean value 0.23
(SD) (0.04)
Vss (L/kg) mean value (SD) 0.09 (0.02)
CTLA4-Ig has about 17 days average T-HALF value in the health volunteer, consistent with the transformation period that obtains in psoriasis experimenter (10-18 days) and patient with rheumatoid arthritis (about 13 days).The average Vss value of observed 0.09-0.10L/kg point out the CTLA4-Ig major limitation in vascular system and and be distributed to indistinctively in the blood vessel external space.
Following table has shown serum-concentration (ng/ml)/experimenter.
Figure A200680053116D05911
Figure A200680053116D05912
Cmax AUC (INF) Tmax T-Half clearance rate VSS
The treatment statistic (mog/mL) (mog.h/mL) (h) (my god) (mL/h/Kg) (L/Kg)
---------?----------?------------?------------?------------?------------?------------?------------
CDCHOl N 13 13 13 13 13 13
Mean value 291.84 45068.89 0.92 16.68 0.23 0.09
S.D. 67.29 8085.22 0.64 3.24 0.04 0.02
Geometrical mean 284.71 44403.04 0.77 16.40 0.23 0.09
C.V. 23 18 69 19 18 23
Intermediate value 286.47 46276.20 0.50 15.97 0.22 0.09
MIN 174.57 33477.26 0.50 12.42 0.16 0.06
MAX 426.97 61322.42 2.00 23.45 0.30 0.13
Determination of serum about CTLA4-Ig
Analyze for 25 times altogether in service by enzyme-linked immunosorbent assay (ELISA) with regard to CTLA4-Ig serum analysis sample.All analytical resultss meet the example values of determining before the sample analysis, thereby point out that the ELISA method quantitatively is accurately with accurately for the CTLA4-Ig in the study sample.Typical curve parameter and average QC data summary about the CTLA4-Ig in the serum are presented in the table 48.Be respectively 4.5% and 3.5%CV about variability between the operation of the analysis QCs of CTLA4-Ig and in the operation.Analyze the observed mean concns of QC sample and depart from nominal value less than ± 8.9% (table 48).
Table 48: the quality contrasting data about the mensuration of CTLA4-Ig in the human serum is summarized
Nominal concentration Low (3.000ng/mL) In (12.500ng/mL) High (24.000ng/mL)
Observed mean concns 2.866 13.608 24.526
％Dev -4.5 8.9 2.2
Precision between operation (%CV) 4.5 2.8 3.0
Precision (%CV) in the operation 24 3.5 2.9
Total variation (%CV) 5.1 4.5 4.2
n 75 75 75
The operation number 25 25 25
The pharmacokinetics of CTLA4-Ig
Be presented in the following table for the mean value and the standard deviation of all experimenters' CTLA4-Ig serum-concentration via method.Average CTLA4-Ig serum-concentration through 71 days is presented among Figure 44 the time overview.
About the average serum concentration of CTLA4-Ig (ng/ml) to time data
My god Hour Minute N Mean value SD ％CV
. . 0 15 0.42 0.87 207.21
. . 15 15 135475.0 28811.82 21.27
. . 30 15 273867.9 71406.26 26.07
. 1 0 15 253311.5 43221.58 17.06
. 2 0 15 254479.4 39611.12 15.57
. 6 0 15 219082.5 44894.29 20.49
. 12 0 15 191885.0 45180.00 23.55
1 0 0 15 161732.2 28740.25 17.77
3 0 0 15 101411.4 18615.59 18.36
7 0 0 15 59375.96 13598.10 22.90
14 0 0 15 33676.97 8148.64 24.20
21 0 0 15 21909.72 5226.77 23.86
28 0 0 15 17193.52 5145.61 29.93
42 0 0 15 8828.95 3246.22 36.77
56 0 0 15 5244.51 2621.36 49.98
70 0 0 15 2970.32 1811.64 60.99
Summary statistics about pharmacokinetic parameter (Cmax, AUC (INF), CLT, Vss, Tmax and T-HALF) is presented in the table 50.The result points out to have the average T-HALF value of about 17 days (scope 7-25 days) by the CTLA4-Ig that method of the present invention produces.The Vss value of observed 0.09-0.10L/kg points out that the CTLA4-Ig major limitation is in extracellular fluid volume.
Table 50: about the summary cartogram of the pharmacokinetic parameter of the CTLA4-Ig that produces by method of the present invention
Preparation Cmax (μ g/mL) geometrical mean (CV%) AUC (INF) (μ g.h/mL) geometrical mean (CV%) Clearance rate (mL/h/kg) mean value (SD) Vss (L/kg) mean value (SD) Tmax (h) intermediate value (min, max) T-HALF (my god) mean value (SD)
Process (n=13) 284.71(23％) 44403.04(18％) 023 (0.04) 0.09 (0.02) 0.50 (0.50，2.00) 16.68?(3.24)
The result points out that the average T-HALF about the CTLA4-Ig that is produced by method of the present invention is about 17 days.Clearance rate and volume of distribution value also are presented in the table 50.
Behind the single 10mg/kg intravenous infusion normal adults experimenter and repeatedly behind the 10mg/kg intravenous infusion CTLA4-Ig pharmacokinetics among the RA patient in table 47, list.
Table 47. is the pharmacokinetic parameter (mean value, scope) among health volunteer and the RA patient behind the 10mg/kg intravenous infusion
The PK parameter Health volunteer's (after single agent of 10mg/kg) n=13 RA patient is (at the 10mg/kg multi-agent aThe back) n=14
Peak concentration (C max)[mcg/mL] 292(175-427) 295(171-398)
Terminal transformation period (t 1/2) [my god] 16.7(12-23) 13.1(8-25)
System Cleaning rate (CL) [mL/h/kg] 0.23(0.16-0.30) 0.22(0.13-0.47)
Volume of distribution (Vss) [L/kg] 0.09(0.06-0.13) 0.07(0.02-0.13)
aRepeatedly intravenous infusion was used at the 1st, 15,30 day and every month thereafter.
The dna sequence dna of embodiment 67-plasmid pcSDhuCTLA4Ig
BglII
～～～～～
1 GATCTCCCGA?TCCCCTATGG?TCGACTCTCA?GTACAATCTG?CTCTGATGCC?GCATAGTTAA
CTAGAGGGCT?AGGGGATACC?AGCTGAGAGT?CATGTTAGAC?GAGACTACGG?CGTATCAATT
61 GCCAGTATCT?GCTCCCTGCT?TGTGTGTTGG?AGGTCGCTGA?GTAGTGCGCG?AGCAAAATTT
CGGTCATAGA?CGAGGGACGA?ACACACAACC?TCCAGCGACT?CATCACGCGC?TCGTTTTAAA
121 AAGCTACAAC?AAGGCAAGGC?TTGACCGACA?ATTGCATGAA?GAATCTGCTT?AGGGTTAGGC
TTCGATGTTG?TTCCGTTCCG?AACTGGCTGT?TAACGTACTT?CTTAGACGAA?TCCCAATCCG
----------------the CMV promotor-------------------
181 GTTTTGCGCT?GCTTCGCGAT?GTACGGGCCA?GATATACGCG?TTGACATTGA?TTATTGACTA
CAAAACGCGA?CGAAGCGCTA?CATGCCCGGT?CTATATGCGC?AACTGTAACT?AATAACTGAT
-----------------------------------------------------------------
241 GTTATTAATA?GTAATCAATT?ACGGGGTCAT?TAGTTCATAG?CCCATATATG?GAGTTCCGCG
CAATAATTAT?CATTAGTTAA?TGCCCCAGTA?ATCAAGTATC?GGGTATATAC?CTCAAGGCGC
-----------------------------------------------------------------
301 TTACATAACT?TACGGTAAAT?GGCCCGCCTG?GCTGACCGCC?CAACGACCCC?CGCCCATTGA
AATGTATTGA?ATGCCATTTA?CCGGGCGGAC?CGACTGGCGG?GTTGCTGGGG?GCGGGTAACT
-----------------------------------------------------------------
361 CGTCAATAAT?GACGTATGTT?CCCATAGTAA?CGCCAATAGG?GACTTTCCAT?TGACGTCAAT
GCAGTTATTA?CTGCATACAA?GGGTATCATT?GCGGTTATCC?CTGAAAGGTA?ACTGCAGTTA
-----------------------------------------------------------------
421 GGGTGGACTA?TTTACGGTAA?ACTGCCCACT?TGGCAGTACA?TCAAGTGTAT?CATATGCCAA
CCCACCTGAT?AAATGCCATT?TGACGGGTGA?ACCGTCATGT?AGTTCACATA?GTATACGGTT
-----------------------------------------------------------------
481 GTADGCCCCC?TATTGACGTC?AATGACGGTA?AATGGCCCGC?CTGGCATTAT?GCCCAGTACA
CATGCGGGGG?ATAACTGCAG?TTACTGCCAT?TTACCGGGCG?GACCGTAATA?CGGGTCATGT
NooI
～～～
-----------------------------------------------------------------
541 TGACCTTATG?GGACTTTCCT?ACTTGGCAGT?ACATCTACGT?ATTAGTCATC?GCTATTACCA
ACTGGAATAC?CCTGAAAGGA?TGAACCGTCA?TGTAGATGCA?TAATCAGTAG?CGATAATGGT
NooI
～～～
--------------------------CMV promotor--------------------------
601 TGGTGATGCG?GTTTTGGCAG?TACATCAATG?GGCGTGGATA?GCGGTTTGAC?TCACGGGGAT
ACCACTACGC?CAAAACCGTC?ATGTAGTTAC?CCGCACCTAT?CGCCAAACTG?AGTGCCCCTA
-----------------------------------------------------------------
661 TTCCAAGTCT?CCACCCCATT?GACGTCAATG?GGAGTTTGTT?TTGGCACCAA?AATCAACGGG
AAGGTTCAGA?GGTGGGGTAA?CTGCAGTTAC?CCTCAAACAA?AACCGTGGTT?TTAGTTGCCC
-----------------------------------------------------------------
721 ACTTTCCAAA?ATGTCGTAAC?AACTCCGCCC?CATTGACGCA?AATGGGCGGT?AGGGGTGTAC
TGAAAGGTTT?TACAGCATTG?TTGAGGCGGG?GTAACTGCGT?TTACCCGCCA?TCCGCACATG
-----------------------------------------------------------------
781 GGTGGGAGGT?CTATATAAGC?AGAGCTCTCT?GGCTAACTAG?AGAACCCACT?GCTTACTGGC
CCACCCTCCA?GATATATTCG?TCTCGAGAGA?CCGATTGATC?TCTTGGGTGA?CAATGACCG
HindIII BamHI
-----------> ～～～～ ～～～
841 TTATCGAAAT?TAATACGACT?CACTATAGGG?AGACCCAAGC?TTGGTACCGA?GCTCGGATCC
AATAGCTTTA?ATTATGCTGA?GTGATATCCC?TCTGGGTTCG?AACCATCGCT?CGAGCCTAGG
PatI
EcoRI?～～～～～～ ？-huCTLA-4lg-
～～～～～～～ M G V L
901 ACTAGTAACG?GCCGCCAGTG?TGCTGGAATT?CTGCAGATAG?CTTCACCAAT?GGGTGTACTG
TGATCATTGC?CGGCGGTCAC?ACGACCTTAA?GACGTCTATC?GAAGTGGTTA?CCCACATGAC
-----------------------------------------------------------------
L T Q R T L L S L V L A L L F P S M A S
961 CTCACACAGA?GGACGCTGCT?CAGTCTGGTC?CTTGCACTCC?TGTTTCCAAG?CATGGCGAGC
GAGTGTGTCT?CCTGCGACGA?GTCAGACCAG?GAACGTGAGG?ACAAAGGTTC?GTACCGCTCG
-----------------------------------------------------------------
M A M H V A Q P A V V L A S S R G I A S
1021 ATGGCAATGC?ACGTGGCCCA?GCCTGCTGTG?GTACTGGCCA?GCAGCCGAGG?CATCGCCAGC
TACCGTTACG?TGCACCGGGT?CGGACGACAC?CATGACCGGT?CGTCGGCTCC?GTAGCGGTCG
-----------------------------------------------------------------
F V C E Y A S P G K A T E V R V T V L R
1031 TTTGTGTGTG?AGTATGCATC?TCCAGGCAAA?GCCACTGAGG?TCCGGGTGAC?AGTGCTTCGG
AAACACACAC?TCATACGTAG?AGTCCGTTT?CGGTGACTCC?AGGCCCACTG?TCACGAAGCC
----------------------------huCTLA-4lg---------------------------
Q A D S Q V T E V C A A T Y M M G N E L
1141 CAGGCTGACA?GCCAGGTGAC?TGAAGTCTGT?GCGGCAACCT?ACATGATGGG?GAATGAGTTG
GTCCGACTGT?CGGTCCACTG?ACTTCAGAGA?CGCCGTTGGA?TGTACTACCC?CTTACTCAAC
-----------------------------------------------------------------
T F L D D S I C T G T S S G N Q V N L T
1201 ACCTTCCTAG?ATGATTCCAT?CTGCACGGGC?ACCTCCAGTG?GAAATCAAGT?GAACCTCACT
TGGAAGGATC?TACTAAGGTA?GACGTGCCCG?TGGAGGTCAC?CTTTAGTTCA?CTTGGAGTGA
NooI
～～～～～～～
-----------------------------------------------------------------
I Q G L R A M D T G L Y I C K V E L M Y
1261 ATCCAAGGAC?TGAGGGCCAT?GGACACGGGA?CTCTACATCT?GCAAGGTGGA?GCTCATGTAC
TAGGTTCCTG?ACTCCCGGTA?CCTGTGCCCT?GAGATGTAGA?CGTTCCACCT?CGAGTACATG
-----------------------------------------------------------------
P P P Y Y L G I G N G T Q I Y V I D P B
1321 CCACCGCCAT?ACTACCTGGG?CATAGGCAAC?GGAACGCAGA?TTTATGTAAT?TGATCCAGAA
GGTGGCGGTA?TGATGGACCC?GTATCCGTTG?CCTTGGGTCT?AAATACATTA?ACTAGGTCTT
-----------------------------------------------------------------
P C P D S D Q B P K S S D K T H T S P P
1381 CCGTGCCCAG?ATTCTGATCA?GGAGCCCAAA?TCTTCTGACA?AAACTCACAC?ATCCCCACCG
GGCACGGGTC?TAAGACTAGT?CCTCGGGTTT?AGAAGACTGT?TTTGAGTGTG?TAGGGGTGGC
-----------------------------------------------------------------
S P A P E L L G G S S V F L F P P K P K
1441 TCCCCAGCAC?CTGAACTCCT?GGGGGGATCG?TCAGTCTTCC?TCTTCCCCCC?AAAACCCAAG
AGGGGTCGTG?GACTTGAGGA?CCCCCCTAGC?AGTCAGAAGG?AGAAGGGGGG?TTTTGGGTTC
-----------------------------------------------------------------
D T L M I S R T P E V T C V V V D V S H
1501 GACACCCTCA?TGATCTCCCG?GAGCCCTGAG?GTCACATGCG?TGGTGGTGGA?CGTGAGCCAC
CTGTGGGAGT?ACTAGAGGGC?CTGGGGACTC?CAGTGTACGC?ACCACCACCT?GCACTCGGTG
-----------------------------------------------------------------
E D P E V K F N W Y V D G V E V H N A K
1561 GAAGACCCTG?AGGTCAAGTT?CAACTGGTAC?GTGGACGGCG?TGGAGGTGCA?TAATGCCAAG
CTTCTGGGAC?TCCAGTTCAA?GTTGACCATG?CACCTGCCGC?ACCTCCACGT?ATTACGGTTC
---------------------------huCTLA-4lg---------------------------
T K P R E E Q Y N S T Y R V V S V L T V
1621 ACAAAGCCGC?GGGAGGAGGA?GTACAACAGC?ACGTAGCGTG?TGGTCAGCGT?CCTCACCGTC
TGTTTCGGCG?CCCTCCTCGT?CATGTTGTCG?TGCATGGCAC?ACCAGTCGCA?GGAGTGGCAG
-----------------------------------------------------------------
L H Q D W L N G K E Y K C K V S N K A L
1681 CTGCACCAGG?AGTGGCTGAA?TGGCAAGGAG?TACAAGTGCA?AGGTCTCCAA?CAAAGCCCTC
GACGTGGTCC?TGACCGACTT?ACCGTTCCTC?ATGTTCACGT?TCCAGAGGTT?GTTTCGGGAG
-----------------------------------------------------------------
P A P I E K T I S K A K G Q P R E P Q V
1741 CCAGCCCCCA?TCGAGAAAAC?CATCTCCAAA?GCCAAAGGGC?AGCCCCGAGA?ACCACAGGTG
GGTCGGGGGT?AGCTCTTTTG?GTAGAGGTTT?CGGTTTCCCG?TCGGGGCTCT?TGGTGTCCAC
SmaI
～～～～～～～
-----------------------------------------------------------------
Y T L P P S R D E L T K N Q V S L T C L
1801 TACACCCTGC?CCCCATCCCG?GGATGAGCTG?ACCAAGAACC?AGGTCAGCCT?GACCTGCCTG
ATGTGGGACG?GGGGTAGGGC?CCTACTCGAC?TGGTTCTTGG?TCCAGTCGGA?CTGGACGGAC
-----------------------------------------------------------------
V K G F Y P S D I A V E W E S N G Q P E
1861 GTCAAAGGCT?TCTATCCCAG?CGACATCGCC?GTGGAGTGGG?AGAGCAATGG?GCAGCCGGAG
CAGTTTCCGA?AGATAGGGTC?GCTGTAGCGG?CACCTCACCC?TCTCGTTACC?CGTCGGCCTC
-----------------------------------------------------------------
N N Y K T T P P V L D S D G S F F L Y S
1921 AACAACTACA?AGACCACGCC?TCCCGTGCTG?GACTCCGACG?GCTCCTTCTT?CCTCTACAGC
TTGTTGATGT?TCTGGTGCGG?AGGGCACGAC?CTGAGGCTGC?CGAGGAAGAA?GGAGATGTCG
-----------------------------------------------------------------
K L T V D K S R W Q Q G N V F S C S V M
1981 AAGCTCACCG?TGGACAAGAG?CAGGTGGCAG?CAGGGGAACG?TCTTCTCATG?CTCCGTGATG
TTCGAGTGGC?ACCTGTTCTC?GTCCACCGTC?GTCCCCTTGC?AGAAGAGTAC?GAGGCACTAC
------------------------------------------------------------->
H E A L H N H Y T Q K S L S L S P G K ＊
2041 CATGAGGCTC?TGCACAACCA?CTACACGCAG?AAGAGCCTCT?CCCTGTCTCC?GGGTAAATGA
GTACTCCGAG?ACGTGTTGGT?GATGTGCGTC?TTCTCGGAGA?GGGACAGAGG?CCCATTTACT
SmaI XbaI
～～～～～～～ ～～～～
2101 GTGCGACGGC?CGGCAAGCCC?CCGCTCCCCG?GGCTCTCGCG?GTCGCACGAG?GATGCTTCTA
CACGCTGCCG?GCCGTTCGGG?GGCGAGGGGC?CCGAGAGCGC?CAGCGTGCTC?CTACGAAGAT
XbaI
～～?----BGH polyadenylation signal-----
2161 GAGGGCCCTA?TTCTATAGTG?TCACCTAAAT?GCTAGAGCTG?GCTGATCAGC?CTCGACTGTG
CTCCCGGGAT?AAGATATCAC?AGTGGATTTA?CGATCTCGAG?CGACTAGTCG?GAGCTGACAC
-----------------------------------------------------------------
2221 CCTTCTAGTT?GCCAGCCATC?TGTTGTTTGC?CCCTCCCCCG?TGCCTTCCTT?GACCCTGGAA
GGAAGATCAA?CGGTCGGTAG?ACAACAAACG?GGGAGGGGGC?ACGGAAGGAA?CTGGGACCTT
-----------------------------------------------------------------
2281 GGTGCCACTC?CCACTGTCCT?TTCCTAATAA?AATGAGGAAA?TTGCATCGCA?TTGTCTGAGT
CCACGGTGAG?GGTGACAGGA?AAGGATTATT?TTACTCGTTT?AACGTAGCGT?AACAGACTCA
-----------------------------------------------------------------
2341 AGGTGTCATT?CTATTCTGGG?GGGTGGGGTG?GGGCAGGACA?GCAAGGGGGA?GGATTGGGAA
TCCACAGTAA?GATAAGACCC?CCCACCCCAC?CCCGTCCTGT?CGTTCCCCCT?CCTAACCCTT
------------------------>
2401 GACAATAGCA?GGCATGCTGG?GGATGCGGTG?GGCTCTATGG?CTTCTGAGGC?GGAAAGAACC
CTGTTATCGT?CCGTACGACC?CCTACGCCAC?CCGAGATACC?GAAGACTCCG?CCTTTCTTGG
2461 AGCTGGGGCT?CTAGGGGGTA?TCCCCACGCG?CCCTGTAGCG?GCGCATTAAG?CGCGGCGGGT
TCGACCCCGA?GATCCCCCAT?AGGGGTGCGC?GGGACATCGC?CGCGTAATTC?GCGCCGCCCA
2521 GTGGTGGTTA?CGCGCAGCGT?GACCGCTACA?CTTGCCAGCG?CCCTAGCGCC?CGCTCCTTTC
CACCACCAAT?GCGCGTCGCA?CTGGCGATGT?GAACGGTCGC?GGGATCGCGG?GCGAGGAAAG
2581 GCTTTCTTCC?CTTCCTTTCT?CGCCACGTTC?GCCCTGTGGA?ATGTGTGTCA?GTTAGGGTGT
CGAAAGAAGG?GAAGGAAAGA?GCGGTGCAAG?CGGGACACCT?TACACACAGT?CAATCCCACA
2641 GGAAACTCCC?CAGGCTCCCC?AGCAGGCAGA?AGTATGCAAA?GCATGCATCT?CAATTAGTCA
CCTTTCAGGG?GTCCGAGGGG?TCGTCCGTCT?TCATACGTTT?CGTACGTAGA?GTTAATCAGT
2701 GCAACCAGGT?GTGGAAAGTC?CCCAGGCTCC?CCAGCAGGCA?GAAGTATGCA?AAGCATGCAT
CGTTGGTCCA?CACCTTTCAG?GGGTCCGAGG?GGTCGTCCGT?CTTCATACGT?TTCGTACGTA
2761 CTCAATTAGT?CAGCAACCAT?AGTCCCGCCC?CTAACTCCGC?CCATCCCGCC?CCTAACTCCG
GAGTTAATCA?GTCGTTGGTA?TCAGGGCGGG?GATTGAGGCG?GGTAGGGCGG?GGATTGAGGC
NcoI
～～～～～～?------the SV40 promotor---------
2821 CCCAGTTCCG?CCCATTCTCC?GCCCCATGGC?TGACTAATTT?TTTTTATTTA?TGCAGAGGCC
GGGTCAAGGC?GGGTAAGAGG?CGGGGTACCG?ACTGATTAAA?AAAAATAAAT?ACGTCTCCGG
-------------------------------------------------------------->
2881 GAGGCCGCCT?CGGCCTCTGA?GCTATTCCAG?AAGTAGTGAG?GAGGCTTTTT?TGGAGGCCTA
CTCCGGCGGA?GCCGGAGACT?CGATAAGGTC?TTCATCACTC?CTCCGAAAAA?ACCTCCGGAT
HindIII
～～～～～～
2941 GGCTTTTGCA?AAAAGCTTGG?ACAGCTGAGG?GCTGCGATTT?CGCGCCAAAC?TTGACGGCAA
CCGAAAACGT?TTTTCGAACC?TGTCGACTCC?CGACGCTAAA?GCGCGGTTTG?AACTGCCGTT
-------dhfr--------
3001 TCCTAGCGTG?AAGGCTGGTA?GGATTTTATC?CCCGCTGCCA?TCATGGTTCG?ACCATTGAAC
AGGATCGCAC?TTCCGACCAT?CCTAAAATAG?GGGCGACGGT?AGTACCAAGC?TGGTAACTTG
-----------------------------------------------------------------
3061 TGCATCGTCG?CCGTGTCCCA?AGATATGGGG?ATTGGCAAGA?ACGGAGACCT?ACCCTGGCCT
ACGTAGCAGC?GGCACAGGGT?TCTATACCCC?TAACCGTTCT?TGCCTCTGGA?TGGGACCGGA
-----------------------------------------------------------------
3121 CCGCTCAGGA?ACGAGTTCAA?GTACTTCCAA?AGAATGACCA?CAACCTCTTC?AGTGGAAGGT
GGCGAGTCCT?TGCTCAAGTT?CATGAAGGTT?TCTTACTGGT?GTTGGAGAAG?TCACCTTCCA
-----------------------------------------------------------------
3181 AAACAGAATC?TGGTGATTAT?GGGTAGGAAA?ACCTGGTTCT?CCATTCCTGA?GAAGAATCGA
TTTGTCTTAG?ACCACTAATA?CCCATCCTTT?TGGACCAAGA?GGTAAGGACT?CTTCTTAGCT
-----------------------------------------------------------------
3241 CCTTTAAAGG?ACAGAATTAA?TATAGTTCTC?AGTAGAGAAC?TCAAAGAACC?ACCACGAGGA
GGAAATTTCC?TGTCTTAATT?ATATCAAGAG?TCATCTCTTG?AGTTTCTTGG?TGGTGCTCCT
-----------------------------dhfr--------------------------------
3301 GCTCATTTTC?TTGCCAAAAG?TTTGGATGAT?GCCTTAAGAC?TTATTGAACA?ACCGGAATTG
CGAGTAAAAG?AACGGTTTTC?AAACCTACTA?CGGAATTCTG?AATAACTTGT?TGGCCTTAAC
-----------------------------------------------------------------
3361 GCAAGTAAAG?TAGACATGGT?TTGGATAGTC?GGAGGCAGTT?CTGTTTACCA?GGAAGCCATG
CGTTCATTTC?ATCTGTACCA?AACCTATCAG?CCTCCGTCAA?GACAAATGGT?CCTTCGGTAC
-----------------------------------------------------------------
3421 AATCAACCAG?GCCACCTCAG?ACTCTTTGTG?ACAAGGATCA?TGCAGGAATT?TGAAAGTGAC
TTAGTTGGTC?CGGTGGAGTC?TGAGAAACAC?TGTTCCTAGT?ACGTCCTTAA?ACTTTCACTG
-----------------------------------------------------------------
3481 ACGTTTTTCC?CAGAAATTGA?TTTGGGGAAA?TATAAACTTC?TCCCAGAATA?CCCAGGCGTC
TGCAAAAAGG?GTCTTTAACT?AAACCCCTTT?ATATTTGAAG?AGGGTCTTAT?GGGTCCGCAG
-----------------------------------------------------------------
3541 CTCTCTGAGG?TCCAGGAGGA?AAAAGGCATC?AAGTATAAGT?TTGAAGTCTA?CGAGAAGAAA
GAGAGACTCC?AGGTCCTCCT?TTTTCCGTAG?TTCATATTCA?AACTTCAGAT?GCTCTTCTTT
-->
3601 GACTAACAGG?AAGATGCTTT?CAAGTTCTCT?GCTCCCCTCC?TAAAGCTATG?CATTTTTATA
CTGATTGTCC?TTCTACGAAA?GTTCAAGAGA?CGAGGGGAGG?ATTTCGATAC?GTAAAAATAT
NcoI BglII
～～～～～～ ～～～～～～～
3661 AGACCATGGG?ACTTTTGCTG?GCTTTAGATC?TTTGTGAAGG?AACCTTACTT?CTGTGGTGTG
TCTGGTACCC?TGAAAACGAC?CGAAATCTAG?AAACACTTCC?TTGGAATGAA?GACACCACAC
3721 ACATAATTGG?ACAAACTACC?TACAGAGATT?TAAAGCTCTA?AGGTAAATAT?AAAATTTTTA
TGTATTAACC?TGTTTGATGG?ATGTCTCTAA?ATTTCGAGAT?TCCATTTATA?TTTTAAAAAT
3781 AGTGTATAAT?GTGTTAAACT?ACTGATTCTA?ATTGTTTGTG?TATTTTAGAT?TCCAACCTAT
TCACATATTA?CACAATTTGA?TGACTAAGAT?TAACAAACAC?ATAAAATCTA?AGGTTGGATA
3841 GGAACTGATG?AATGGGAGCA?GTGGTGGAAT?GCCTTTAATG?AGGAAAACCT?GTTTTGCTCA
CCTTGACTAC?TTACCCTCGT?CACCACCTTA?CGGAAATTAC?TCCTTTTGGA?CAAAACGAGT
3901 GAAGAAATGC?CATCTAGTGA?TGATGAGGCT?AGTGCTGACT?CTCAACATTC?TACTCCTCCA
CTTCTTTACG?GTAGATCACT?ACTACTCCGA?TGACGACTGA?GAGTTGTAAG?ATGAGGAGGT
3961 AAAAAGAAGA?GAAAGGTAGA?AGACCCCAAG?GACTTTCCTT?CAGAATTGCT?AAGTTTTTTG
TTTTTCTTCT?CTTTCCATCT?TCTGGGGTTC?CTGAAAGGAA?GTCTTAACGA?TTCAAAAAAC
4021 AGTCATGCTG?TGTTTAGTAA?TAGAACTCTT?GCTTGCTTTG?CTATTTACAC?CACAAAGGAA
TCAGTACGAC?ACAAATCATT?ATCTTGAGAA?CGAACGAAAC?GATAAATGTG?GTGTTTCCTT
4081 AAAGCTGCAC?TGCTATACAA?GAAAATTATG?GAAAAATATT?CTGTAACCTT?TATAAGTAGG
TTTCGACGTG?ACGATATGTT?CTTTTAATAC?CTTTTTATAA?GACATTGGAA?ATATTCATCC
4141 CATAACAGTT?ATTATCATAA?CATACTGTTT?TTTCTTACTC?CACACAGGCA?TAGAGTGTCT
GTATTGTCAA?TATTAGTATT?GTATGACAAA?AAAGAATGAG?GTGTGTCCGT?ATCTCACAGA
4201 GCTATTAATA?ACTATGCTCA?AAAATTGTGT?ACCTTTAGCT?TTTTAATTTG?TAAAGGGGTT
CGATAATTAT?TGATACGAGT?TTTTAACACA?TGGAAATCGA?AAAATTAAAC?ATTTCCCCAA
4261 AATAAGGAAT?ATTTGATGTA?TAGTGCCTTG?ACTAGAGATC?ATAATCAGCC?ATACCACATT
TTATTCCTTA?TAAACTACAT?ATCACGGAAC?TGATCTCTAG?TATTAGTCGG?TATGGTGTAA
4321 TGTAGAGGTT?TTACTTGCTT?TAAAAAACCT?CCCACACCTC?CCCCTGAACC?TGAAACATAA
ACATCTCCAA?AATGAACGAA?ATTTTTTGGA?GGGTGTGGAG?GGGGACTTGG?ACTTTGTATT
4381 AATGAATGCA?ATTGTTGTTG?TTAACTTGTT?TATTGCAGCT?TATAATGGTT?ACAAATAAAG
TTACTTACGT?TAACAACAAC?AATTGAACAA?ATAACGTCGA?ATATTACCAA?TGTTTATTTC
4441 CAATAGCATC?ACAAATTTCA?CAAATAAAGC?ATTTTTTTCA?CTGCATTCTA?GTTGTGGTTT
GTTATCGTAG?TGTTTAAAGT?GTTTATTTCG?TAAAAAAAGT?GACGTAAGAT?CAACACCAAA
4501 GTCCAAACTC?ATCAATGTAT?CTTATCATGT?CTGGATCGGC?TGGATGATCC?TCCAGCGCGG
CAGGTTTGAG?TAGTTACATA?GAATAGTACA?GACCTAGCCG?ACCTACTAGG?AGGTCGCGCC
4561 GGATCTCATG?CTGGAGTTCT?TCGCCCACCC?CAACTTGTTT?ATTGCAGCTT?ATAATGGTTA
CCTAGAGTAC?GACCTCAAGA?AGCGGGTGGG?GTTGAACAAA?TAACGTCGAA?TATTACCAAT
4621 CAAATAAAGC?AATAGCATCA?CAAATTTCAC?AAATAAAGCA?TTTTTTTCAC?TGCATTCTAG
GTTTATTTCG?TTATCGTAGT?GTTTAAAGTG?TTTATTTCGT?AAAAAAAGTG?ACGTAAGATC
4681 TTGTGGTTTG?TCCAAACTCA?TCAATGTATC?TTATCATGTC?TGTATACCGT?CGACCTCTAG
AACACCAAAC?AGGTTTGAGT?AGTTACATAG?AATAGTACAG?ACATATGGCA?GCTGGAGATC
4741 CTAGAGCTTG?GCGTAATCAT?GGTCATAGCT?GTTTCCTGTG?TGAAATTGTT?ATCCGCTCAC
GATCTCGAAC?CGCATTAGTA?CCAGTATCGA?CAAAGGACAC?ACTTTAACAA?TAGGCGAGTG
4801 AATTCCACAC?AACATACGAG?CCGGAAGCAT?AAAGTGTAAA?GCCTGGGGTG?CCTAATGAGT
TTAAGGTGTG?TTGTATGCTC?GGCCTTCGTA?TTTCACATTT?CGGACCCCAC?GGATTACTCA
4861 GAGCTAACTC?ACATTAATTG?CGTTGCGCTC?ACTGCCCGCT?TTCCAGTCGG?GAAACCTGTC
CTCGATTGAG?TGTAATTAAC?GCAACGCGAG?TGACGGGCGA?AAGGTCAGCC?CTTTGGACAG
4921 GTGCCAGCTG?CATTAATGAA?TCGGCCAACG?CGCGGGGAGA?GGCGGTTTGC?GTATTGGGCG
CACGGTCGAC?GTAATTACTT?AGCCGGTTGC?GCGCCCCTCT?CCGCCAAACG?CATAACCCGC
4981 CTCTTCCGCT?TCCTCGCTCA?CTGACTCGCT?GCGCTCGGTC?GTTCGGCTGC?GGCGAGCGGT
GAGAAGGCGA?AGGAGCGAGT?GACTGAGCGA?CGCGAGCCAG?CAAGCCGACG?CCGCTCGCCA
5041 ATCAGCTCAC?TCAAAGGCGG?TAATACGGTT?ATCCACAGAA?TCAGGGGATA?ACGCAGGAAA
TAGTCGAGTG?AGTTTCCGCC?ATTATGCCAA?TAGGTGTCTT?AGTCCCCTAT?TGCGTCCTTT
？------------------------
Figure A200680053116D0602143304QIETU
-----------------------------
5101 GAACATGTGA?GCAAAAGGCC?AGCAAAAGGC?CAGGAACCGT?AAAAAGGCCG?CGTTGCTGGC
CTTGTACACT?CGTTTTCCGG?TCGTTTTCCG?GTCCTTGGCA?TTTTTCCGGC?GCAACGACCG
----------------------------------------------------------------
5161 GTTTTTCCAT?AGGCTCCGCC?CCCCTGACGA?GCATCACAAA?AATCGACGCT?CAAGTCAGAG
CAAAAAGGTA?TCCGAGGCGG?GGGGACTGCT?CGTAGTGTTT?TTAGCTGCGA?GTTCAGTCTC
-----------------------------------------------------------------
5221 GTGGCGAAAC?CCGACAGGAC?TATAAAGATA?CCAGGCGTTT?CCCCCTGGAA?GCTCCCTCGT
CACCGCTTTG?GGCTGTCCTG?ATATTTCTAT?GGTCCGCAAA?GGGGGACCTT?CGAGGGAGCA
---------------------------
Figure A200680053116D0602143304QIETU
-------------------------------
5281 GCGCTCTCCT?GTTCCGACCC?TGCCGCTTAC?CGGATACCTG?TCCGCCTTTC?TCCCTTCGGG
CGCGAGAGGA?CAAGGCTGGC?ACGGCGAATG?GCCTATGGAC?AGGCGGAAAG?AGGGAAGCCC
-----------------------------------------------------------------
5341 AAGCGTGGCG?CTTTCTCAAT?GCTCACGCTG?TAGGTATCTC?AGTTCGGTGT?AGGTCGTTCG
TTCGCACCGC?GAAAGAGTTA?CGAGTGCGAG?ATCCATAGAG?TCAAGCCACA?TCCAGCAAGC
ApaLI
～～～～～～～
-----------------------------------------------------------------
5401 CTCCAAGCTG?GGCTGTGTGC?ACGAACCCCC?CGTTCAGCCC?GACCGCTGCG?CCTTATCCGG
GAGGTTCGAC?CCGACACACG?TGCTTGGGGG?GCAAGTCGGG?CTGGCGACGC?GGAATAGGCC
-----------------------------------------------------------------
5461 TAACTATCGT?CTTGAGTCCA?ACCCGGTAAG?ACACGACTTA?TCGCCACTGG?CAGCAGCCAC
ATTGATAGCA?GAACTCAGGT?TGGGCCATTC?TGTGCTGAAT?AGCGGTGACC?GTCGTCGGTG
-----------------------------------------------------------------
5521 TGGTAACAGG?ATTAGCAGAG?CGAGGTATGT?AGGCGGTGCT?ACAGAGTTCT?TGAAGTGGTG
ACCATTGTCC?TAATCGTCTC?GCTCCATACA?TCCGCCACGA?TGTCTCAAGA?ACTTCACCAC
-----------------------------------------------------------------
5581 GCCTAACTAC?GGCTACACTA?GAAGGACAGT?ATTTGGTATC?TGCGCTCTGC?TGAAGCCAGT
CGGATTGATG?CCGATGTGAT?CTTCCTGTCA?TAAACCATAG?ACGCGAGACG?ACTTCGGTCA
-----------------------------------------------------------------
5641 TACCTTCGGA?AAAAGACTTG?GTAGCTCTTG?ATCCGGCAAA?CAAACCACCG?CTGGTAGCGG
ATGGAAGCCT?TTTTCTCAAC?CATCGAGAAC?TAGGCCGTTT?GTTTGGTGGC?GACCATCGCC
-----------------------------------------------------------------
5701 TGGTTTTTTT?GTTTGCAAGC?AGCAGATTAC?GCGCAGAAAA?AAAGGATCTC?AAGAAGATCC
ACCAAAAAAA?CAAACGTTCG?TCGTCTAATG?CGCGTCTTTT?TTTCCTAGAG?TTCTTCTAGG
------------------？
5761 TTTGATCTTT?TCTACGGGGT?CTGACGCTCA?GTGGAACGAA?AACTCACGTT?AAGGGATTTT
AAACTAGAAA?AGATGCCCCA?GACTGCGAGT?CACCTTGCTT?TTGAGTGCAA?TTCCCTAAAA
5821 GGTCATGAGA?TTATCGAAAA?GGATCTTCAC?CTAGATCCTT?TTAAATTAAA?AATGAAGTTT
CCAGTACTCT?AATAGTTTTT?CCTAGAAGTG?GATCTAGGAA?AATTTAATTT?TTACTTCAAA
<-----ampR-----
5881 TAAATCAATC?TAAAGTATAT?ATGAGTAAAC?TTGGTCTGAC?AGTTACCAAT?GCTTAATCAG
ATTTAGTTAG?ATTTCATATA?TACTCATTTG?AACCAGACTG?TCAATGGTTA?CGAATTAGTC
-----------------------------------------------------------------
5941 TGAGGCACCT?ATCTCAGCGA?TCTGTCTATT?TCGTTCATCC?ATAGTTGCCT?GACTCCCCGT
ACTCCGTGGA?TAGAGTCGCT?AGACAGATAA?AGCAAGTAGG?TATCAACGGA?CTGAGGGGCA
-----------------------------------------------------------------
6001 CGTGTAGATA?ACTACGATAC?GGGAGGGCTT?ACCATCTGGC?CCCAGTGCTG?CAATGATACC
GCACATCTAT?TGATGCTATG?CCCTCCCGAA?TGGTAGACCG?GGGTCACGAC?GTTACTATGG
-----------------------------------------------------------------
6061 GCGAGACCCA?CGCTCACCGG?CTCCAGATTT?ATCAGCAATA?AACCAGCCAG?CCGGAAGGGC
CGCTCTGGGT?GCGAGTGGCC?GAGGTCTAAA?TAGTCGTTAT?TTGGTCGGTC?GGCCTTCCCG
-----------------------------------------------------------------
6121 CGAGCGCAGA?AGTGGTCCTG?CAACTTTATC?CGCCTCCATC?CAGTCTATTA?ATTGTTGCCG
GCTCGCGTCT?TCACCAGGAC?GTTGAAATAG?GCGGAGGTAG?GTCAGATAAT?TAACAACGGC
-----------------------------------------------------------------
6181 GGAAGCTAGA?GTAAGTAGTT?CGCCAGTTAA?TAGTTTGCGC?AACGTTGTTG?CCATTGCTAC
CCTTCGATCT?CATTCATCAA?GCGGTCAATT?ATCAAACGCG?TTGCAACAAC?GGTAACGATG
-----------------------------------------------------------------
6241 AGGCATCGTG?GTGTCACGCT?CGTCGTTTGG?TATGGCTTCA?TTCAGCTCCG?GTTCCCAACG
TCCGTAGCAC?CACAGTGCGA?GCAGCAAACC?ATACCGAAGT?AAGTCGAGGC?CAAGGGTTGC
-----------------------------------------------------------------
6301 ATCAAGGCGA?GTTACATGAT?CCCCCATGTT?GTGCAAAAAA?GCGGTTAGCT?CCTTCGGTCC
TAGTTCCGCT?CAATGTACTA?GGGGGTACAA?CACGTTTTTT?CGCCAATCGA?GGAAGCCAGG
PvuI
～～～～～
------------------------------ampR------------------------------
6361 TCCGATCGTT?GTCAGAAGTA?AGTTGGCCGC?AGTGTTATCA?CTCATGGTTA?TGGCAGCACT
AGGCTAGCAA?CAGTCTTCAT?TCAACCGGCG?TCACAATAGT?GAGTACCAAT?ACCGTCGTGA
-----------------------------------------------------------------
6421 GCATAATTCT?CTTACTGTCA?TGCCATCCGT?AAGATGCTTT?TCTGTGACTG?GTGAGTACTC
CGTATTAAGA?GAATGACAGT?ACGGTAGGCA?TTCTACGAAA?AGACACTGAC?CACTCATGAG
-----------------------------------------------------------------
6481 AACCAAGTCA?TTCTGAGAAT?AGTGTATGCG?GCGACCGAGT?TGCTCTTGCC?CGGCGTCAAT
TTGGTTCAGT?AAGACTCTTA?TCACATACGC?CGCTGGCTCA?ACGAGAACGG?GCCGCAGTTA
-----------------------------------------------------------------
6541 ACGGGATAAT?ACCGCGCCAC?ATAGCAGAAC?TTTAAAAGTG?CTCATCATTG?GAAAACGTTC
TGCCCTATTA?TGGCGCGGTG?TATCGTCTTG?AAATTTTCAC?GAGTAGTAAC?CTTTTGCAAG
-----------------------------------------------------------------
6601 TTCGGGGCGA?AAACTCTCAA?GGATCTTACC?GCTGTTGAGA?TCCAGTTCGA?TGTAACCCAC
AAGCCCCGCT?TTTGAGAGTT?CCTAGAATGG?CGACAACTCT?AGGTCAAGCT?ACATTGGGTG
ApaLI
～～～～～～
-----------------------------------------------------------------
6661 TCGTGCACCC?AACTGATCTT?CAGCATCTTT?TACTTTCACC?AGCGTTTCTG?GGTGAGCAAA
AGCACGTGGG?TTGACTAGAA?GTCGTAGAAA?ATGAAAGTGG?TCGCAAAGAC?CCACTCGTTT
-----------------------------------------------------------------
6721 AACAGGAAGG?CAAAATGCCG?CAAAAAAGGG?AATAAGGGCG?ACACGGAAAT?GTTGAATACT
TTGTCCTTCC?GTTTTACGGC?GTTTTTTCCC?TTATTCCCGC?TGTGCCTTTA?CAACTTATGA
--？
6781 CATACTCTTC?CTTTTTCAAT?ATTATTGAAG?CATTTATCAG?GGTTATTGTC?TCATGAGCGG
GTATGAGAAG?GAAAAAGTTA?TAATAACTTC?GTAAATAGTC?CCAATAACAG?AGTACTCGCC
6841 ATACATATTT?GAATGTATTT?AGAAAAATAA?ACAAATAGGG?GTTCCGCGCA?CATTTCCCCG
TATGTATAAA?CTTACATAAA?TCTTTTTATT?TGTTTATCCC?CAAGGCGCGT?GTAAAGGGGC
BglII
～
6901 AAAAGTGCCA?CCTGACGTCG?ACGGATCGGG?A
TTTTCACGGT?GGACTGCAGC?TGCCTAGCCC?T
Embodiment 1 above, 2,3,4,5,6,7,8,9,10,11,12,13,14,16,17,18,21,28,29,30,31,32,33,34,42,44,45,46,47,48,49,50,51,58,59,60,61,62,63,64,65,66,67 are particularly related to and have SEQ ID NO:1,2,5,6,7,8,9,10 or 18 CTLA4-Ig protein, and embodiment above 19,20,22,23,24,25,26,27,35,36,37,38,39,40,41,52,53,54,55,56,57 are particularly related to and have SEQ IDNO:4,11,12,13,14,15 or 16 CTLA4-Ig protein.As described in this manual, relating to these method of protein among the embodiment and uses thereof is illustrative for relating to other CTLA4-Ig method of protein of the present invention and uses thereof.
Exemplary and the non-limiting composition that comprises CTLA4-Ig molecule of the present invention comprises this kind composition, wherein:
(1) the CTLA4-Ig molecule comprises one or more among the SEQ ID NO:2,5,6,7,8,9,10 or 18, and as what measured by size exclusion chromatography and spectrophotometry detection (measuring method about it is set forth in embodiment 10), the CTLA4-Ig molecule is less than or equal to the CTLA4-Ig high molecular weight species (or CTLA4-Ig tetramer) of about 5.0 area percentages.More specifically, the invention provides this kind composition that further has one or more following characteristics:
Be no more than the bacterial endotoxin maximum (it can be not existing of bacterial endotoxin) of 0.35EU/mg CTLA4-Ig molecule or 76.8EU/mL; Be used for measuring the method for this feature in embodiment 48 elaborations;
Be no more than the maximum biological load (it can be not existing of biological load) of 1CFU/10mL or 1CFU/mL; Be used for measuring the method for this feature in embodiment 49 elaborations;
Provide (as the CTLA4-Ig molecule or as described composition) (a) to have an about 10-22 band of the pI scope of about 4.3-about 5.6, the accumulation band intensity of 90%-110% when the pI scope of about 4.3-about 5.3, with about 3 the main bands when the pI scope of about 4.5-about 5.2, or (b) have a CTLA4-Ig isoform of preponderating that is less than or equal to 5.1 pI, and at least 90% CTLA4-Ig molecule shows and is less than or equal to about 5.3 pI; Be used for measuring the method for this feature in embodiment 50 elaborations;
The CTLA4-Ig molecule that is less than or equal to 3.5 area percentages is its oxidation kind, and the CTLA4-Ig molecule that is less than or equal to 2.5 area percentages is its deacylated tRNA amine kind; Be used for measuring the method for these features in embodiment 47 elaborations;
Measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule is the CTLA4-Ig dimer more than or equal to 95.0 area percentages; Be used for measuring the method for this feature in embodiment 10 elaborations;
Measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule is the CTLA4-Ig high molecular weight species (or CTLA4-Ig tetramer) less than 5.0 area percentages; Be used for measuring the method for this feature in embodiment 10 elaborations;
Measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule is the low molecular weight species (or CTLA4-Ig monomer) that is less than or equal to 0.5 area percentage, or as measuring, less than the low molecular weight species (or CTLA4-Ig monomer) of 0.5 area percentage by size exclusion chromatography and spectrophotometry detection; Be used for measuring the method for this feature in embodiment 10 elaborations;
Be no more than the dimeric DNA maximum of 2.5 piks/mg CTLA4-Ig molecule or 2.5 piks/mg CTLA4-Ig (it can be not existing of DNA); Be used for measuring the method for this feature in embodiment 58 elaborations;
Be no more than the total CTLA4-Ig molecule of 3.0ng/mg, 5ppm or the dimeric MCP-1 maximum of 5ng/mg CTLA4-Ig (it can be not existing of MCP-1); Be used for measuring the method for this feature in embodiment 59 elaborations;
Be no more than the dimeric host cell proteins matter of 25ng/mg CTLA4-Ig molecule or 50ng/mg CTLA4-Ig (being also referred to as cell protein) maximum (it can be not existing of host cell proteins matter or cell protein); Be used for measuring the method for this feature in embodiment 60 elaborations;
Be no more than the Triton X-100 maximum (it can be not existing of Triton X) of 1.0ng/mg CTLA4-Ig molecule; Be used for measuring the method for this feature in embodiment 61 elaborations;
Be no more than the A albumen maximum (its can be that A is proteic do not exist) of 5.0ng/mg CTLA4-Ig molecule; Be used for measuring the method for this feature in embodiment 62 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 63 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 63 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the semi-lactosi of about 8.0-about 17 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 64 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 64 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the seminose of about 7.7-about 22 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 64 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, more than or equal to 8.0, and the molar average ratio of the sialic acid of for example about 8.0-about 12.0 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 16 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, more than or equal to 8.0, and the molar average ratio of the NANA of for example about 8.0-about 12.0 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 16 elaborations;
The CTLA4-Ig molecule has the N linked glycosylation, thereby make structural domain I show the area percentage of about 24.5%-about 35.2%, or domain II shows the area percentage of about 26.3%-about 34.1%, or the area percentage of domain II I demonstration about 21.9%-about 31.5%, or the area percentage of structural domain IV and structural domain V demonstration about 7.9%-about 18.6%; Be used for measuring the method for this feature in embodiment 44 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, is less than or equal to the molar average ratio of 1.5 NGNA and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 16 elaborations.
The invention provides as separating or this kind composition of purifying basically.The invention provides this kind composition as pharmaceutical composition or pharmaceutically acceptable composition.The invention provides any arrangement with these features or the composition of combination.
The invention provides as separating or this kind composition of purifying basically.The invention provides this kind composition as pharmaceutical composition or pharmaceutically acceptable composition.The invention provides any arrangement with these features or the composition of combination:
(2) the CTLA4-Ig molecule comprises the one or more (CTLA4 for example among the SEQ ID NO:4,11,12,13,14,15,16 or 24 A29YL104E-Ig), and as what measured by size exclusion chromatography and spectrophotometry detection (measuring method about it is set forth in embodiment 25), the CTLA4-Ig molecule is less than or equal to the CTLA4-Ig high molecular weight species (or CTLA4-Ig tetramer) of about 5.0 area percentages.More specifically, the invention provides this kind composition that further has one or more following characteristics:
Measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule is the CTLA4-Ig dimer more than or equal to 95.0 area percentages; Be used for measuring the method for this feature in embodiment 25 elaborations;
Measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig molecule is the CTLA4-Ig low molecular weight species (or CTLA4-Ig monomer) that is less than or equal to 1.0 area percentages; Be used for measuring the method for this feature in embodiment 25 elaborations;
Provide about 8-15 band of pI scope that (as the CTLA4-Ig molecule or as described composition) have about 4.5-about 5.6 and the accumulation band intensity of 95%-105% when the pI scope of about 4.5-about 5.6; Be used for measuring the method for this feature in embodiment 22 elaborations;
Be no more than the DNA maximum of about 2.5pg/mg CTLA4-Ig molecule; Be used for measuring the method for this feature in embodiment 55 elaborations;
Be no more than the A albumen maximum of 5ng/mg CTLA4-Ig molecule; Be used for measuring the method for this feature in embodiment 53 elaborations;
Be no more than the MCP-1 maximum of 5ng/mg CTLA4-Ig molecule; Be used for measuring the method for this feature in embodiment 54 elaborations;
Be no more than host cell proteins matter (the being also referred to as cell protein) maximum of 50ng/mg CTLA4-Ig molecule; Be used for measuring the method for this feature in embodiment 52 elaborations;
Be no more than the bacterial endotoxin maximum of 0.42EU/mg CTLA4-Ig molecule; Be used for measuring the method for this feature in embodiment 48 elaborations;
Be no more than the maximum biological load of 1CFU/mL; Be used for measuring the method for this feature in embodiment 49 elaborations;
Be no more than the Triton X-100 maximum of 2ppm; Be used for measuring the method for this feature in embodiment 57 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, more than or equal to 5.0, and the molar average ratio of the sialic acid of for example about 5.0-about 9.0 or about 5.0-about 10.0 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 39 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, more than or equal to 5.0, and the molar average ratio of the NANA of for example about 5.0-about 9.0 or about 5.0-about 10.0 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 39 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the GalNAc of about 0.8-about 4.0 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 36 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the GlcNAc of about 14-about 35 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 36 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the semi-lactosi of about 8.0-about 14 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 35 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the Fucose of about 1.7-about 9.3 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 35 elaborations;
The CTLA4-Ig molecule has the moles/mole of being expressed as protein, the molar average ratio of the seminose of about 9-about 18 and CTLA4-Ig molecule (or with CTLA4-Ig dimer); Be used for measuring the method for this feature in embodiment 35 elaborations;
The invention provides and separate or this kind composition of purifying basically.The invention provides any arrangement with these features or the composition of combination.
Embodiment 1,2,3,4,5,6,7,8,9,10,11,12,13,14,16,17,18,21,28,29,30,31,32,33,34,42,44,45,46,47,48,49,50,51,58,59,60,61,62,63,64,65,66,67 above is particularly related to above the CTLA4-Ig protein of (1), although, as described in this manual, relating to this kind method of protein among these embodiment and uses thereof is illustrative for relating to other CTLA4-Ig method of protein of the present invention and uses thereof.
Embodiment 19,20,22,23,24,25,26,27,35,36,37,38,39,40,41,52,53,54,55,56,57 above is particularly related to above the CTLA4-Ig protein of (2), although, as described in this manual, relating to this kind method of protein among these embodiment and uses thereof is illustrative for relating to other CTLA4-Ig method of protein of the present invention and uses thereof.

Claims (296)

1. be used to obtain to comprise method for compositions from the isolating CTLA4-Ig molecule colony of liquid nutrient medium, described substratum comprises the original population of CTLA4-Ig molecule, wherein the CTLA4-Ig molecule of (1) described original population has one or more sialic acid residueses, (2) number of described sialic acid residues/CTLA4-Ig molecule changes in described original population, (3) described original population comprises CTLA4-Ig dimer and high molecular gathering thing, and described method comprises:
(a) results are from the described liquid nutrient medium of the mammalian cell cultures of expressing the CTLA4-Ig molecule;
(b) described CTLA4-Ig molecule is separated with cellular component;
(c) making CTLA4-Ig dimer and CTLA4-Ig high molecular assemble thing separates; With
(d) described CTLA4-Ig molecule is divided into 2 or more multistage branch, wherein compares with at least one other fraction, at least one fraction has the mol ratio of bigger sialic acid and CTLA4-Ig molecule,
Wherein step (b), (c) and (d) carry out simultaneously or with any order are so that obtain described composition.
2. the process of claim 1 wherein that the described results in the step (a) comprise the soluble fraction that obtains described liquid culture.
3. the process of claim 1 wherein step (c) and (d) comprise the use of column chromatography, so that obtain to have the fraction of the CTLA4-Ig molecule of different sialic acid contents.
4. the method for claim 1, it further comprises the use of column chromatography, to reduce the MCP-1 content in the described composition.
5. the process of claim 1 wherein described CTLA4-Ig molecule comprise have SEQ IDNO:2,5,6,7,8, one or more polypeptide of 9 or 10.
6. the process of claim 1 wherein described CTLA4-Ig molecule comprise have SEQ IDNO:4,11,12,13,14, one or more polypeptide of 15 or 16.
7. the process of claim 1 wherein and have bigger sialic acid and the sialic acid of described fraction demonstration about 8-about 14 of the mol ratio of CTLA4-Ig molecule and the molar average ratio of CTLA4-Ig molecule in (d).
8. the method for claim 7, wherein said molar average is than, about 8-about 10 about 11 for about 8-or about 8-about 9.
9. one kind is used to separate the method for compositions that comprises the CTLA4-Ig molecule, and described method comprises:
(i) obtain to comprise the mammalian cell of producing the CTLA4-Ig molecule liquid culture soluble fraction and with any order,
(ii) described soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out;
(iii) described soluble fraction is implemented hydrophobic interaction chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out;
(iv) described soluble fraction is implemented affinity chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; With
(v) described soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out.
10. one kind is used to separate the method for compositions that comprises the CTLA4-Ig molecule, and described method comprises:
(i) acquisition comprises the soluble fraction of the liquid culture of the mammalian cell of producing the CTLA4-Ig molecule;
(ii) described soluble fraction is implemented anion-exchange chromatography, comprise the composition of the wash-out of CTLA4-Ig molecule with acquisition;
(iii) step protein is (ii) implemented hydrophobic interaction chromatography, so that acquisition comprises the composition of the enrichment of CTLA4-Ig molecule;
(iv) protein is (iii) implemented affinity chromatography, comprise the composition of the further enrichment of CTLA4-Ig molecule with acquisition; With
(v) protein is (iv) implemented anion-exchange chromatography, so that separate the composition that comprises the CTLA4-Ig molecule.
11. the method for claim 9 or 10, wherein the described composition that comprises the CTLA4-Ig molecule that obtains in (ii) in step is characterised in that: (a) the molar average ratio of the NANA of 6.0-10.1 and CTLA4Ig molecule, (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 25.7 area % as detecting by size exclusion chromatography and spectrophotometry.
12. the method for claim 9 or 10, wherein the described composition that comprises the CTLA4-Ig molecule that obtains in (iii) in step is characterised in that: (a) measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig high molecular weight species is less than about 2.5 area %, (b) cell protein less than about 6600ng/ml and (c) MCP-1 less than about 5600ppm.
13. the method for claim 9 or 10, wherein the described composition that comprises the CTLA4-Ig molecule that obtains in (iii) in step is characterised in that: (a) the molar average ratio of the NANA of 6.8-11.4 and CTLA4-Ig molecule, (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.5 area % as detecting by size exclusion chromatography and spectrophotometry.
14. the method for claim 9 or 10, wherein the described composition that comprises the CTLA4-Ig molecule that obtains in (iv) in step is characterised in that: (a) the molar average ratio of the NANA of 8.0-11.0 and CTLA4-Ig molecule, (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.5 area % as detecting by size exclusion chromatography and spectrophotometry.
15. the method for claim 9 or 10, wherein the composition of the described enrichment that obtains in (iii) in step is characterised in that, measure as detecting by size exclusion chromatography and spectrophotometry, the CTLA4-Ig high molecular weight species is less than 2.5% area percentage.
16. the method for claim 9 or 10, wherein (the described protein composition that comprises the CTLA4-Ig molecule v) is characterised in that: (a) the molar average ratio of the NANA of 8.0-11.9 and CTLA4-Ig molecule in step, (b) measure, be less than or equal to the CTLA4-Ig high molecular weight species of 2.0 area % as detecting by size exclusion chromatography and spectrophotometry.
17. method for compositions that is used for separation of C TLA4-Ig molecule, described method comprises: (i) acquisition comprises the soluble fraction of the liquid culture of the mammalian cell of producing the CTLA4-Ig molecule, with with any order, (ii) described soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iii) described soluble fraction is implemented hydrophobic interaction chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iv) described soluble fraction is implemented affinity chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (v) described soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out, wherein (composition that obtains v) is characterised in that: the per-cent of CTLA4-Ig high molecular weight species is less than about 2.0 area % in step, cell protein is less than about 95ng/ml, and MCP-1 is less than about 9.55ng/ml.
18. method for compositions that is used for separation of C TLA4-Ig molecule, described method comprises: (i) acquisition comprises the soluble fraction of the liquid culture of the mammalian cell of producing the CTLA4-Ig molecule, with with any order, (ii) described soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iii) described soluble fraction is implemented hydrophobic interaction chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (iv) described soluble fraction is implemented affinity chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out; (v) described soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the enrichment of CTLA4-Ig molecule and the composition of wash-out, wherein the composition that obtains in (iii) in step is characterised in that: the per-cent of CTLA4-Ig high molecular weight species is less than about 2.5 area %, cell protein is less than 95ng/ml, MCP-1 and MCP-1 sample material be less than about 5ppm, and the molar average of NANA and CTLA4-Ig molecule is than being about 8.0-about 12.
19. the method for claim 9 or 10, wherein step described anion-exchange chromatography (ii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 75mM HEPES and about 360mM NaCl, and has about 8.0 pH.
20. the method for claim 9 or 10, wherein step described anion-exchange chromatography (ii) uses elution buffer to carry out, and described elution buffer comprises about 25mM HEPES and about 850mM NaCl, and has about 7.0 pH.
21. the method for claim 9 or 10, wherein step described hydrophobic interaction chromatography (iii) uses single lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM HEPES and about 850mM NaCl, and has about 7.0 pH.
22. the method for claim 9 or 10, wherein step described affinity chromatography (iv) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM Tris and about 250mMNaCl, and has about 8.0 pH.
23. the method for claim 9 or 10, wherein step described affinity chromatography (iv) uses elution buffer to carry out, and described elution buffer comprises about 100mM glycine, and has about 3.5 pH.
24. the method for claim 9 or 10, wherein (described anion-exchange chromatography v) uses lavation buffer solution to carry out to step, and described lavation buffer solution comprises about 25mM HEPES and the about 130mM NaCl of about 120mM NaCl-, and has about 8.0 pH.
25. the method for claim 9 or 10, wherein (described anion-exchange chromatography v) uses elution buffer to carry out to step, and described elution buffer comprises about 25mM HEPES and about 200mM NaCl, and has about 8.0 pH.
26. the method for claim 9 or 10, wherein step described anion-exchange chromatography (ii) uses the post with anionite-exchange resin to carry out, and described anionite-exchange resin comprises primary, secondary, uncle or quaternary amine functional group.
27. the method for claim 26, wherein said resin comprises quaternary amine functional group.
28. the method for claim 9 or 10, wherein step described hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin comprises phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.
29. the method for claim 28, wherein said functional group comprises phenyl functional group.
30. the method for claim 9 or 10, wherein step described affinity chromatography is (iv) used and is comprised the proteic affinity chromatography resin of A and carry out.
31. one kind is used to prepare the method for compositions that comprises the CTLA4-Ig molecule, described method comprises purifying CTLA4-Ig molecule from the liquid cell culture, the CTLA4-Ig composition of wherein said purifying comprises the MCP-1 and the MCP-1 sample material/mg CTLA4-Ig molecule of (a) pharmaceutically acceptable amount, (b) measure, less than the CTLA4-Ig high molecular weight species of 2.5 area % as detecting by size exclusion chromatography and spectrophotometry.
32. the method for claim 31, the MCP-1 of wherein said pharmaceutically acceptable amount and MCP-1 sample material comprise the about 0.5ng/mg CTLA4-Ig of about 40-molecule.
33. the method for claim 31, the MCP-1 of wherein said pharmaceutically acceptable amount and MCP-1 sample material comprise the about 0.5ng/mg CTLA4-Ig of about 35-molecule.
34. the method for claim 31, the MCP-1 of wherein said pharmaceutically acceptable amount and MCP-1 sample material comprise the about 0.5ng/mg CTLA4-Ig of about 10-molecule.
35. the method for claim 9 or 10, wherein step described affinity chromatography (iv) uses post to carry out, and described post comprises the MCP-1 that can reduce in the protein of described wash-out and the resin of MCP-1 sample material.
36. the method for claim 9 or 10, wherein step described hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and wherein said resin can (a) make the CTLA4-Ig dimer separate with the CTLA4-Ig high molecular weight species; (b) sialic acid content of the CTLA4-Ig molecule of the described wash-out of increase; Or (c) (a) and (b) both.
37. the method for claim 9 or 10, wherein step (ii) or step (v) or both described anion-exchange chromatographies use anionite-exchange resin to carry out, wherein said resin can (a) reduces the CTLA4-Ig high molecular of the composition of described wash-out and assembles thing content; (b) sialic acid content of the composition of the described wash-out of increase; Or (c) (a) and (b) both.
38. one kind is used to separate the method for compositions that comprises the CTLA4-Ig molecule, described method comprises:
(i) obtain to comprise the mammalian cell of producing the CTLA4-Ig molecule liquid culture soluble fraction and with any order;
(ii) described soluble fraction is implemented affinity chromatography, so that acquisition comprises the composition of the wash-out of CTLA4-Ig molecule;
(iii) described soluble fraction is implemented anion-exchange chromatography, so that obtain to comprise the wash-out of CTLA4-Ig molecule and the composition of enrichment; With
(iv) described soluble fraction is implemented hydrophobic interaction chromatography, so that obtain to comprise the wash-out of CTLA4-Ig molecule and the composition of enrichment.
39. the method for claim 38, wherein said affinity chromatography step is at first carried out.
40. the method for claim 38, wherein step described affinity chromatography is (ii) used and is comprised the proteic resin of A and carry out.
41. the method for claim 38, wherein step described affinity chromatography (ii) uses the elution buffer that comprises guanidine to carry out.
42. the method for claim 38, wherein step described affinity chromatography (ii) uses the elution buffer that comprises urea to carry out.
43. the method for claim 38, wherein step described affinity chromatography (ii) causes comprising the dimeric increase of CTLA4-Ig in the described wash-out composition of CTLA4-Ig molecule.
44. one kind is used for comprising the method for compositions of CTLA4-Ig molecule from results from the liquid separation of mammalian cell cultures, wherein said cells produce CTLA4-Ig molecule, and described method comprises:
(i) soluble fraction of the liquid of the described results of acquisition;
(ii) described soluble fraction is implemented affinity chromatography, comprise the composition of the wash-out of CTLA4-Ig molecule with acquisition;
(iii) step described composition is (ii) implemented anion-exchange chromatography, so that obtain to comprise the wash-out of CTLA4-Ig molecule and the composition of enrichment; With
(iv), comprise the composition of the further enrichment of CTLA4-Ig molecule with acquisition to implementing hydrophobic interaction chromatography from step described composition (iii).
45. the method for claim 44, wherein the (iv) middle described composition that obtains of step is characterised in that: measure as detecting by size exclusion chromatography and spectrophotometry, the per-cent of high molecular weight species is less than about 2.5 area %, and the per-cent of cell protein is less than about 95ng/ml, and the per-cent of MCP-1 is less than about 5ppm.
46. the method for claim 44, wherein step described anion-exchange chromatography (iii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 50mM HEPES and about 135mM NaCl, and has about 7 pH.
47. the method for claim 44, wherein step described anion-exchange chromatography (iii) uses elution buffer to carry out, and described elution buffer comprises about 50mM HEPES and about 200mM NaCl, and has about 7 pH.
48. the method for claim 44, wherein step described hydrophobic interaction chromatography (iv) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 50mM HEPES and about 1.2M (NH 4) 2SO 4, and have about 7 pH.
49. the method for claim 44, wherein step described affinity chromatography (ii) uses lavation buffer solution to carry out, and described lavation buffer solution comprises about 25mM NaH 2PO 4With about 150mMNaCl, and has about 7.5 pH.
50. the method for claim 44, wherein step described affinity chromatography (ii) uses elution buffer to carry out, and described elution buffer comprises about 250mM glycine and has about 3 pH.
51. the method for claim 44, wherein step described anion-exchange chromatography (iii) uses the post with anionite-exchange resin to carry out, and described anionite-exchange resin comprises primary, secondary, uncle or quaternary amine functional group.
52. the method for claim 51, wherein said resin comprises quaternary amine functional group.
53. the method for claim 47, wherein step described hydrophobic interaction chromatography (iii) uses the hydrophobic interaction resin to carry out, and described hydrophobic interaction resin comprises phenyl, octyl group, propyl group, alkoxyl group, butyl or isopentyl functional group.
54. the method for claim 53, wherein said functional group comprises phenyl functional group.
55. the method for claim 44, wherein step described affinity chromatography is (ii) used and is comprised the proteic resin of A and carry out.
56. a composition, it comprises the CTLA4-Ig molecule that obtains by each method among the claim 1-56.
57. a CTLA4-Ig composition that obtains by each method among the claim 1-56, wherein said composition comprise have SEQ ID NO:2, one or more polypeptide of 5,6,7,8,9 or 10.
58. a CTLA4-Ig composition that obtains by each method among the claim 1-56, wherein said composition comprise have SEQ ID NO:4, one or more polypeptide of 11,12,13,14,15 or 16.
59. a CTLA4-Ig expression plasmid, it has the nucleotide sequence of SEQ ID NO:17.
60. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid and the proteinic molar average ratio of CTLA4-Ig of about 5.5-about 18.
61. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 5.5-about 9.5 and the molar average ratio of CTLA4-Ig molecule.
62. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 5-about 10 and the molar average ratio of CTLA4-Ig molecule.
63. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 6-about 18 and the molar average ratio of CTLA4-Ig molecule.
64. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 8-about 18 and the molar average ratio of CTLA4-Ig molecule.
65. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 8-about 12 and the molar average ratio of CTLA4-Ig molecule.
66. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 8-about 11 and the molar average ratio of CTLA4-Ig molecule.
67. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 7-about 12 and the molar average ratio of CTLA4-Ig molecule.
68. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 7-about 11 and the molar average ratio of CTLA4-Ig molecule.
69. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 11-about 18 and the molar average ratio of CTLA4-Ig molecule.
70. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 12-about 18 and the molar average ratio of CTLA4-Ig molecule.
71. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 13-about 18 and the molar average ratio of CTLA4-Ig molecule.
72. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 14-about 18 and the molar average ratio of CTLA4-Ig molecule.
73. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 15-about 17 and the molar average ratio of CTLA4-Ig molecule.
74. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the molar average ratio of about 16 sialic acid and CTLA4-Ig molecule.
75. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the molar average ratio of about 10 sialic acid and CTLA4-Ig molecule.
76. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the molar average ratio of about 6 sialic acid and CTLA4-Ig molecule.
77. each composition among the claim 60-76, wherein said sialic acid are N-n acetylneuraminic acid n (NANA).
78. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the NANA of about 8-about 12 and the molar average ratio of CTLA4-Ig molecule.
79. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the molar average ratio that is less than or equal to about 1.5 N-hydroxyacetylneuraminic acid (NGNA) and CTLA4-Ig molecule.
80. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the NGNA of about 0.5-about 1.5 and the molar average ratio of CTLA4-Ig molecule.
81. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the NGNA of about 1.0-about 1.5 and the molar average ratio of CTLA4-Ig molecule.
82. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule have the sialic acid of about 6-about 18 and the molar average ratio of CTLA4-Ig molecule.
83. a composition that comprises the purifying basically of CTLA4-Ig molecule, wherein said CTLA4-Ig molecule are characterised in that the molar average ratio of sialic acid/mole CTLA4-Ig molecule of about 6-about 12.
84. composition that comprises the purifying basically of CTLA4-Ig molecule, every peptide species of wherein said molecule comprises SEQ ID NO:11,12,13,14,15 or 16 described sequence, and described CTLA4-Ig molecule is characterised in that the molar average ratio of sialic acid/mole CTLA4-Ig molecule of about 5.5-about 9.5.
85. each composition among the claim 78-84, the mol ratio of wherein said sialic acid/mole CTLA4-Ig molecule is measured by acid hydrolysis and HPLC.
86. each composition among the claim 78-84, wherein said CTLA4-Ig molecule comprise have SEQ ID NO:2, one or more polypeptide of 5,6,7,8,9 or 10.
87. each composition among the claim 78-84, wherein said CTLA4-Ig molecule comprise have SEQ ID NO:4, one or more polypeptide of 11,12,13,14,15 or 16.
88. a composition that comprises the purifying basically of CTLA4-Ig molecule is wherein measured as detecting by size exclusion chromatography and spectrophotometry, is the CTLA4-Ig dimer more than or equal to the described CTLA4-Ig molecule of 95 area %.
89. the composition of claim 88 is the CTLA4-Ig dimer more than or equal to 98% described CTLA4-Ig molecule wherein.
90. the composition of claim 88 is the CTLA4-Ig dimer more than or equal to 99% described CTLA4-Ig molecule wherein.
91. the composition of claim 88 is the CTLA4-Ig dimer more than or equal to 99.5% described CTLA4-Ig molecule wherein.
92. the composition of claim 88, wherein measure as detecting by size exclusion chromatography and spectrophotometry, the described CTLA4-Ig molecule of about 95%-about 99.5% is the CTLA4-Ig dimer, and the described molecule of the about 5 area % of about 0.5 area %-is the CTLA4-Ig high molecular weight species.
93. the composition of claim 88, wherein measure as detecting by size exclusion chromatography and spectrophotometry, about 98.6% described molecule is the CTLA4-Ig dimer, and the described molecule of about 1.2 area % is the CTLA4-Ig high molecular weight species, and is the CTLA4-Ig monomer less than the described molecule of 0.7 area % approximately.
94. the composition of claim 88 is to comprise 5 or the monomeric polymer of more CTLA4-Ig less than about 0.3% described molecule approximately wherein.
95. a composition, it is made up of the CTLA4-Ig dimer basically.
96. one kind basically by the molecular composition of CTLA4-Ig, wherein said colony is substantially free of the CTLA4-Ig monomer.
97. one kind basically by the molecular composition of CTLA4-Ig, wherein said colony is substantially free of the CTLA4-Ig high molecular weight species.
98. a composition of being made up of the CTLA4-Ig monomer basically, it is substantially free of CTLA4-Ig dimer and high molecular weight species.
99. each composition among the claim 95-98, wherein every kind of dimeric each monomer of CTLA4-Ig has at least 3 sialic acids groups.
100. each composition among the claim 95-98, wherein every kind of dimeric each monomer of CTLA4-Ig has at least 2.5 sialic acids groups.
Each composition among the claim 95-98, wherein every kind of dimeric each monomer of CTLA4-Ig has at least 3 sialic acids groups-at least 8 sialic acids groups.
Each composition among the claim 95-98, wherein every kind of dimeric each monomer of CTLA4-Ig has at least 2.5 sialic acids groups-at least 5 sialic acids groups.
Each composition among the claim 95-98, wherein every kind of dimer comprises 2 CTLA4-Ig polypeptide, and wherein each polypeptide has the aminoacid sequence that is selected from SEQ ID NOS:5-16.
Each composition among the claim 95-98, wherein said composition comprise have SEQ ID NO:2, one or more polypeptide of 5,6,7,8,9 or 10.
Each composition among the claim 95-98, wherein said composition comprise have SEQ ID NO:4, one or more polypeptide of 11,12,13,14,15 or 16.
A kind of tetrameric composition isolated of CTLA4-Ig that comprises, it is substantially free of the CTLA4-Ig dimer.
A kind of tetrameric composition isolated of CTLA4-Ig that comprises, it is substantially free of the CTLA4-Ig monomer.
Claim 106 or 107 composition, wherein said composition exists as the amounts greater than about 100 grams.
Claim 106 or 107 the tetrameric composition of CTLA4-Ig, wherein each tetramer comprises 2 pairs of CTLA4-Ig polypeptide, and wherein each polypeptide has the aminoacid sequence that is selected from SEQ ID NOS:5-10.
110. the tetrameric composition of the CTLA4-Ig of claim 106 or 107, wherein each tetramer comprises 2 pairs of CTLA4-Ig polypeptide, and wherein each polypeptide has the aminoacid sequence that is selected from SEQ ID NOS:11-16.
111. the composition of claim 106 or 107, wherein each tetramer can combine with CD80 or CD86.
112. a pharmaceutically acceptable composition that comprises the CTLA4-Ig molecule, wherein said composition is substantially free of MCP-1.
113. comprising, a pharmaceutically acceptable composition that comprises the CTLA4-Ig molecule, wherein said composition be no more than about 25ppm MCP-1.
114. comprising, the composition of claim 113, wherein said composition be no more than 10ppmMCP-1.
115. the composition of claim 113, wherein said composition comprise the about 10ng/ml MCP-1 of about 0.2ng/mlMCP-1-.
116. a pharmaceutically acceptable composition that comprises the CTLA4-Ig molecule, wherein said composition comprise the about 10ng/ml MCP-1 of (a) about 0.2ng/ml MCP-1-and (b) are no more than 25ng/ml CHO protein or be no more than 10ng/ml CHO protein.
117. comprising, the composition of claim 116, wherein said composition be no more than about 20pg/ml DNA.
118. a composition isolated that comprises the CTLA4-Ig molecule, wherein, when the intravenous dosages with about 10mg/kg was applied to the experimenter, described CTLA4-Ig molecule can show:
(a) area under curve of about 44400 μ g.h/ml (AUC);
(b) volume of distribution of about 0.09L/kg;
(c) peak concentration of about 292 μ g/ml (Cmax); With
(d) clearance rate of about 0.23ml/h/kg.
119. composition isolated that comprises the CTLA4-Ig molecule, wherein said composition is included in the advantage isoform of CTLA4-Ig molecule visual on the isoelectrofocusing gel, as measuring by isoelectrofocusing, described isoform has and is less than or equal to 5.1 ± 0.2 iso-electric point, pI.
120. the composition of claim 119, the average pI of wherein said composition handles the back at neuraminidase to be increased.
121. the composition of claim 119, wherein as measuring by isoelectrofocusing, at least 40% described CTLA4-Ig molecule demonstration is less than or equal to about 5.1 ± 0.2 iso-electric point.
122. the composition of claim 119, wherein as measuring by isoelectrofocusing, at least 70% described CTLA4-Ig molecule demonstration is less than or equal to about 5.1 ± 0.2 iso-electric point.
123. the composition of claim 119, wherein as measuring by isoelectrofocusing, at least 90% described CTLA4-Ig molecule demonstration is less than or equal to about 5.1 ± 0.2 iso-electric point.
124. a composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 3.0 ± 0.2-about 5.0 ± 0.2.
125. a composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 4.3 ± 0.2-about 5.0 ± 0.2.
126. a composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 3.3 ± 0.2-about 4.7 ± 0.2.
127. claim 124,125 or 126 composition, wherein said composition are purifying basically.
128. one kind is used to prepare method for compositions, described composition comprises the CTLA4-Ig molecule of the pI with about 3.0 ± 0.2-about 5.0 ± 0.2, described method comprises: (a) mixture of CTLA4-Ig molecule is implemented the isoelectrofocusing gel electrophoresis, wherein the single band on described gel represents to have the CTLA4-Ig molecule colony of specific pI, (b) separation has the CTLA4-Ig molecule colony of the pI of about 3.0 ± 0.2-about 5.0 ± 0.2, so that prepare described composition.
129. a composition isolated that comprises the CTLA4-Ig molecule, wherein said composition are included in advantage isoform visual on the isoelectrofocusing gel, as measuring by isoelectrofocusing, described isoform has and is less than or equal to 5.5 ± 0.2 iso-electric point, pI.
130. the composition of claim 129, the average pI of wherein said composition handles the back at neuraminidase to be increased.
131. the composition of claim 129, wherein as measuring by isoelectrofocusing, at least 40% described CTLA4-Ig molecule demonstration is less than or equal to about 5.3 ± 0.2 iso-electric point.
132. the composition of claim 129, wherein as measuring by isoelectrofocusing, at least 70% described CTLA4-Ig molecule demonstration is less than or equal to about 5.3 ± 0.2 iso-electric point.
133. the composition of claim 129, wherein as measuring by isoelectrofocusing, at least 90% described CTLA4-Ig molecule demonstration is less than or equal to about 5.3 ± 0.2 iso-electric point.
134. a composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 3.0 ± 0.2-about 5.2 ± 0.2.
135. a composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 4.5 ± 0.2-about 5.2 ± 0.2.
136. a composition isolated that comprises the CTLA4-Ig molecule, it has the pI of about 4.7 ± 0.2-about 5.1 ± 0.2.
137. claim 134,135 or 136 composition, wherein said composition are purifying basically.
138. one kind is used to prepare method for compositions, described composition comprises the CTLA4-Ig molecule of the pI with about 2.0 ± 0.2-about 5.2 ± 0.2, described method comprises: (a) mixture of CTLA4-Ig molecule is implemented the isoelectrofocusing gel electrophoresis, wherein the single band on described gel represents to have the CTLA4-Ig molecule colony of specific pI, (b) separation has the CTLA4-Ig molecule colony of the pI of about 3.0 ± 0.2-about 5.2 ± 0.2, so that prepare described composition.
139. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule are characterised in that the GlcNAc of about 17-about 28 and the molar average ratio of CTLA4-Ig molecule.
140. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule are characterised in that the GlcNAc of about 17-about 25 and the molar average ratio of CTLA4-Ig molecule.
141. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule are characterised in that the GlcNAc of about 15-about 35 and the molar average ratio of CTLA4-Ig molecule.
142. composition that comprises the CTLA4-Ig molecule, every peptide species of wherein said molecule comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said CTLA4-Ig molecule is characterised in that the GlcNAc of about 24-about 28 and the molar average ratio of CTLA4-Ig molecule.
143. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule are characterised in that the GalNAc of about 1.7-about 3.6 and the molar average ratio of CTLA4-Ig molecule.
144. composition that comprises the CTLA4-Ig molecule, every peptide species of wherein said molecule comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said CTLA4-Ig molecule is characterised in that the GalNAc of about 2.7-about 3.6 and the molar average ratio of CTLA4-Ig molecule.
145. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule are characterised in that the semi-lactosi of about 8-about 17 and the molar average ratio of CTLA4-Ig molecule.
146. composition that comprises the CTLA4-Ig molecule, every peptide species of wherein said molecule comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said CTLA4-Ig molecule is characterised in that the semi-lactosi of about 11-about 13 and the molar average ratio of CTLA4-Ig molecule.
147. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule are characterised in that the Fucose of about 3.5-about 8.3 and the molar average ratio of CTLA4-Ig molecule.
148. composition that comprises the CTLA4-Ig molecule, every peptide species of wherein said molecule comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said CTLA4-Ig molecule is characterised in that the Fucose of about 6.4-about 7.0 and the molar average ratio of CTLA4-Ig molecule.
149. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule are characterised in that the seminose of about 7.7-about 22 and the molar average ratio of CTLA4-Ig molecule.
150. composition that comprises the CTLA4-Ig molecule, every peptide species of wherein said molecule comprises SEQ ID NO:11,12,13,14,15 or 16 sequence, and wherein said CTLA4-Ig molecule is characterised in that the seminose of about 14-about 16 and the molar average ratio of CTLA4-Ig molecule.
151. each composition among the claim 139-150, wherein the described mol ratio of GlcNAc and CTLA4-Ig molecule is measured by capillary electrophoresis.
152. each composition among the claim 139-150, wherein the described mol ratio of GalNAc and CTLA4-Ig molecule is measured by capillary electrophoresis.
153. each composition among the claim 139-150, wherein the described mol ratio of semi-lactosi and CTLA4-Ig molecule is measured by capillary electrophoresis.
154. each composition among the claim 139-150, wherein the described mol ratio of Fucose and CTLA4-Ig molecule is measured by capillary electrophoresis.
155. each composition among the claim 139-150, wherein the described mol ratio of seminose and CTLA4-Ig molecule is measured by capillary electrophoresis.
156. adhering to, each composition among the claim 139-150, the wherein said CTLA4-Ig molecule enzymatic by one or more carbohydrate and described molecule obtains.
157. a composition that comprises the CTLA4-Ig molecule, wherein said molecule are included in the external carbohydrate residue that adheres to the molecule enzymatic.
158. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; With
(b) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.
159. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule; With
(c) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.
160. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule;
(c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; With
(d) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.
161. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule;
(c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule;
(d) the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule; With
(e) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.
162. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule;
(c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule;
(d) the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule;
(e) the molar average ratio of the seminose of about 7.2-about 22 and CTLA4-Ig molecule; With
(f) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule.
163. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule; With
(b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.
164. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule; With
(c) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.
165. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule;
(c) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; With
(d) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.
166. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule;
(c) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule;
(d) the molar average ratio of the Fucose of about 6.4-about 7.0 and CTLA4-Ig molecule; With
(e) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.
167. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule;
(c) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule;
(d) the molar average ratio of the Fucose of about 6.4-about 7.0 and CTLA4-Ig molecule;
(e) seminose of about 14-about 16 and the proteinic molar average ratio of CTLA4-Ig; With
(f) sialic acid of about 5.5-about 9.5 and the proteinic molar average ratio of CTLA4-Ig.
168. composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is glycosylated on following residue: the amino acid asparagine residue at SEQ ID NO:2 or 4 the 102nd place, the amino acid asparagine residue at SEQ ID NO:2 or 4 the 134th place, the amino acid asparagine residue at SEQ ID NO:2 or 4 the 233rd place, the Serine amino-acid residue at SEQ ID NO:2 or 4 the 155th place, or the Serine amino-acid residue at the 165th place of SEQ ID NO:2 or 4.
169. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule is glycosylated, and is the O linked glycosylation at least about 2% glycosylation total mass wherein.
170. a composition that comprises the CTLA4-Ig molecule, the NGNA chromatogram peak of wherein said composition exhibiting about 9.6 ± 0.3 and about 10.5 ± 0.3 NANA chromatogram peak.
171. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule shows the carbohydrate overview substantially the same with Figure 68.
172. a composition that comprises the CTLA4-Ig molecule, wherein said CTLA4-Ig molecule shows the carbohydrate overview as shown in Figure 68.
173. one kind basically by the molecular composition of CTLA4-Ig, the carbohydrate overview of wherein said CTLA4-Ig molecule display structure territory I-IV, wherein structural domain I comprises the peak of the oligosaccharides of representing asialoization, domain II comprises the peak of the oligosaccharides of representing mono-sialylated, domain II I comprises the peak of the oligosaccharides of representing double-sialylated, structural domain IV comprises the peak of representing three sialylated oligosaccharides, and structural domain V comprises the peak of representing four sialylated oligosaccharides, and wherein said overview is the chromatogram of the oligosaccharides that discharged by CTLA4-Ig.
174. the composition of claim 172, wherein the difference in the retention time of N connection oligosaccharides is about 12 minutes of about 10-between first peak among the structural domain I and the main peak in the domain II.
175. the composition of claim 172, wherein the difference in the retention time of N connection oligosaccharides is about 13 minutes of about 11-between first peak among the structural domain I and the main peak in the domain II.
176. the composition of claim 172, wherein as measuring by HPAEC, the glycosylation of domain II I and IV comprises the N linked glycosylation of about 25%-about 36%.
177. the composition of claim 172, wherein as measuring by HPAEC, the glycosylation of structural domain I comprises the N linked glycosylation of about 24.5%-about 35.2%.
178. the composition of claim 172, wherein as measuring by HPAEC, the glycosylation of domain II comprises the N linked glycosylation of about 26.3%-about 34.1%.
179. the composition of claim 172, wherein as measuring by HPAEC, the glycosylation of domain II I comprises the N linked glycosylation of about 21.9%-about 31.5%.
180. the composition of claim 172, wherein as measuring by HPAEC, the glycosylation of structural domain IV and structural domain V comprises the N linked glycosylation of about 7.9%-about 18.6%.
181. the composition of claim 172, wherein:
(a) structural domain I shows the area percentage at least about 31%;
(b) domain II shows the area percentage at least about 33%;
(c) domain II I shows the area percentage at least about 24%;
(iv) structural domain IV shows the area percentage at least about 9.4%; Or
(v) structural domain V shows the area percentage at least about 6.7%;
Wherein said area is measured according to the chromatogram of the oligosaccharides that is discharged by CTLA4-Ig.
182. the composition of claim 172, wherein:
(a) structural domain I shows at least about 5 peaks;
(b) domain II shows at least about 5 peaks;
(c) domain II I shows at least about 5 peaks;
(d) structural domain IV shows at least about 6 peaks,
(e) structural domain V shows at least about 6 peaks; Or
(f) structural domain I shows at least 3 peaks,
Wherein the peak shows on chromatogram.
183. a composition that comprises the CTLA4-Ig polypeptide, wherein:
(a) about 80% described polypeptide has two feeler N linked glycosylations;
(b) about 14% described polypeptide has three feeler N linked glycosylations; With
(c) about 6% described polypeptide has four feeler N linked glycosylations.
184. the composition of claim 183, wherein said N linked glycosylation is measured by the high pH anion-exchange chromatography (HPEAC-PAD) with pulsed current detection.
185. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; With
(b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule.
186. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule;
(b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule; With
(c) as detect by size exclusion chromatography and spectrophotometry measure CTLA4-Ig high molecular weight species area percentage less than about 3%.
187. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule;
(b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule; With
(c) be less than or equal to the molar average ratio of about 1.5 NGNA and CTLA4-Ig molecule.
188. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule;
(b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule;
(c) measure as detecting, assemble thing content less than the CTLA4-Ig high molecular of about 3 area % by size exclusion chromatography and spectrophotometry; With
(d) with the sort of substantially the same carbohydrate overview of Figure 68.
189. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule;
(b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule;
(c) measure as detecting, assemble thing content less than the CTLA4-Ig high molecular of about 3 area % by size exclusion chromatography and spectrophotometry; With
(d) as measuring, at least about domain II I, the IV of the about 50.1%N linked glycosylation of 29.8%-and the glycosylation content among the V by HPAEC.
190. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule;
(b) the molar average ratio of the NANA of about 6-about 12 and CTLA4-Ig molecule; With
(c) measure as detecting, less than the CTLA4-Ig high molecular weight species of about 3 area % by size exclusion chromatography and spectrophotometry.
191. each composition among the claim 185-190, wherein said molecule are further characterized in that the NANA of about 8-about 12 and the molar average ratio of CTLA4-Ig molecule.
192. each composition among the claim 185-190, wherein said molecule is further characterized in that:
(a) two feeler N linked glycosylations of about 80%;
(b) about 14% three feeler N linked glycosylations; With
(c) about 6% four feeler N linked glycosylations.
193. each composition in the claim 192, wherein said molecule further comprise one or more any combination in following:
(a) aminoacid sequence of SEQ ID NO:10 (methionine(Met) at amino acid position 27 places of SEQ ID NO:2 and the glycine at amino acid position 382 places);
(b) aminoacid sequence of SEQ ID NO:7 (methionine(Met) at amino acid position 27 places of SEQ ID NO:2 and the Methionin at amino acid position 383 places);
(c) aminoacid sequence of SEQ ID NO:9 (L-Ala at amino acid position 26 places of SEQ ID NO:2 and the glycine at amino acid position 382 places); With
(d) aminoacid sequence of SEQ ID NO:6 (L-Ala at amino acid position 26 places of SEQ ID NO:2 and the Methionin at amino acid position 383 places).
194. each composition among the claim 185-190, wherein
(a) about 90% described molecule comprises the aminoacid sequence of the SEQ IDNO:2 that begins from the methionine(Met) of residue 27;
(b) about 10% described molecule comprises the aminoacid sequence of numbering the SEQID NO:2 that 26 L-Ala begins from residue;
(c) about 4% described molecule comprises the aminoacid sequence of numbering the SEQID NO:2 that the Methionin at 383 places finishes with residue; With
(d) about 96% described molecule comprises the aminoacid sequence of numbering the SEQ ID NO:2 that the glycine at 382 places finishes with residue.
195. a composition that comprises the CTLA4-Ig polypeptide, wherein:
(a) about 80% described polypeptide has two feeler N linked glycosylations;
(b) about 14% described polypeptide has three feeler N linked glycosylations;
(c) about 6% described polypeptide has four feeler N linked glycosylations; With
(d) be less than or equal to the molar average ratio of 1.5 NGNA and CTLA4-Ig molecule.
196. a composition that comprises the CTLA4-Ig polypeptide, wherein:
(a) about 80% described polypeptide has two feeler N linked glycosylations;
(b) about 14% described polypeptide has three feeler N linked glycosylations;
(c) about 6% described polypeptide has four feeler N linked glycosylations; With
(d) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule.
197. a composition that comprises the CTLA4-Ig polypeptide, wherein:
(a) about 80% described polypeptide has two feeler N linked glycosylations;
(b) about 14% described polypeptide has three feeler N linked glycosylations;
(c) about 6% described polypeptide has four feeler N linked glycosylations; With
(d) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule.
198. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; With
(b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule.
199. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule;
(b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule; With
(c) measure as detecting, less than the CTLA4-Ig high molecular weight species of about 5 area % by size exclusion chromatography and spectrophotometry.
200. a composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule;
(b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule;
(c) measure as detecting, less than the CTLA4-Ig high molecular weight species content of about 5 area % by size exclusion chromatography and spectrophotometry; With
(d) with the sort of substantially the same carbohydrate overview of Figure 68.
A kind of composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule;
(b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule;
(c) measure as detecting, less than the CTLA4-Ig high molecular weight species of about 5 area % by size exclusion chromatography and spectrophotometry; With
(d) as measuring, at least about domain II I, the IV of the about 50.1%N linked glycosylation of 29.8%-and the glycosylation content among the V by HPAEC.
A kind of composition that comprises the CTLA4-Ig molecule is characterized in that:
(a) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule;
(b) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule; With
(c) measure as detecting, less than the CTLA4-Ig high molecular weight species content of about 5 area % by size exclusion chromatography and spectrophotometry.
Each composition among the claim 195-202, wherein said molecule is further characterized in that:
(a) two feeler N linked glycosylations of about 80%;
(b) about 14% three feeler N linked glycosylations; With
(c) about 6% four feeler N linked glycosylations.
Each composition among the claim 195-202, wherein said molecule further comprise one or more any combination in following:
(a) aminoacid sequence of SEQ ID NO:16 (methionine(Met) at amino acid position 27 places of SEQ ID NO:4 and the glycine at amino acid position 382 places);
(b) aminoacid sequence of SEQ ID NO:13 (methionine(Met) at amino acid position 27 places of SEQ ID NO:4 and the Methionin at amino acid position 383 places);
(c) aminoacid sequence of SEQ ID NO:15 (L-Ala at amino acid position 26 places of SEQ ID NO:4 and the glycine at amino acid position 382 places); With
(d) aminoacid sequence of SEQ ID NO:12 (L-Ala at amino acid position 26 places of SEQ ID NO:4 and the Methionin at amino acid position 383 places).
Each composition among the claim 195-202, wherein
(a) about 90% described molecule comprises the aminoacid sequence of the SEQ IDNO:4 that begins from the methionine(Met) of residue 27;
(b) about 10% described molecule comprises the aminoacid sequence of numbering the SEQID NO:4 that 26 L-Ala begins from residue;
(c) about 4% described molecule comprises the aminoacid sequence of numbering the SEQID NO:4 that the Methionin at 383 places finishes with residue; With
(d) about 96% described molecule comprises the aminoacid sequence of numbering the SEQ ID NO:4 that the glycine at 382 places finishes with residue.
A kind of composition that comprises the CTLA4-Ig polypeptide, wherein:
(a) about 80% described polypeptide has two feeler N linked glycosylations;
(b) about 14% described polypeptide has three feeler N linked glycosylations;
(c) about 6% described polypeptide has four feeler N linked glycosylations; With
(d) the proteinic molar average ratio of the GlcNAc/ mole CTLA4-Ig of about 24-about 28.
A kind of composition that comprises the CTLA4-Ig polypeptide, wherein:
(a) about 80% described polypeptide has two feeler N linked glycosylations;
(b) about 14% described polypeptide has three feeler N linked glycosylations;
(c) about 6% described polypeptide has four feeler N linked glycosylations; With
(d) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule.
Each composition among claim 157-172,195-202 or the 206-207, wherein said composition are the compositions of purifying basically.
A kind of composition that comprises the CTLA4-Ig molecule, wherein being less than or equal to about 2.5% described CTLA4-Ig molecule is oxidation.
210. a composition that comprises the CTLA4-Ig molecule, wherein being less than or equal to about 2.0% described CTLA4-Ig molecule is deacylated tRNA amine.
211. a composition that comprises the CTLA4-Ig dimer molecule, wherein at least 0.5% described CTLA4-Ig dimer molecule is a cysteinylization.
212. the composition of claim 211, wherein at least 1.0% described CTLA4-Ig dimer molecule is a cysteinylization.
213. the colony of a CTLA4-Ig molecule, wherein said colony shows the mass spectroscopy overview substantially the same with Figure 49.
214. the colony of a CTLA4-Ig molecule, wherein said colony shows the capillary electrophoresis overview substantially the same with Figure 48.
215.CTLA4-Ig composition, it obtains by any method among the claim 1-55.
216. a composition that comprises the CTLA4-Ig molecule, wherein said composition is characterised in that:
(a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule;
(c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule;
(d) the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule;
(e) the molar average ratio of the seminose of about 7.2-about 22 and CTLA4-Ig molecule;
(f) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule;
(g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 2.4 ± 0.2-about 5.0 ± 0.2;
(h) be less than or equal to the MCP-1 of 3ppm;
(i) measure as detecting, less than the high molecular weight species of 2.5 area % by size exclusion chromatography and spectrophotometry;
(j) measure as detecting, less than the monomer of 0.5 area % by size exclusion chromatography and spectrophotometry;
(k) have with SEQ ID NOS:5-10 in the CTLA4-Ig polypeptide of any one at least 95% aminoacid sequence that is equal to;
(1) can with CD80 and CD86 bonded CTLA4-Ig molecule.
217. a composition that comprises the CTLA4-Ig molecule, wherein said molecule colony is characterised in that: (a) the molar average ratio of the GlcNAc of about 15-about 35 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 1.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 8-about 17 and CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 3.5-about 8.3 and CTLA4-Ig molecule; (e) the molar average ratio of the seminose of about 7.2-about 22 and CTLA4-Ig molecule; (f) the molar average ratio of the sialic acid of about 6-about 12 and CTLA4-Ig molecule; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 3.4 ± 0.2-about 5.0 ± 0.2; (h) be less than or equal to the MCP-1 of 5ppm; (i) measure as detecting, less than the high molecular weight species of 2.5 area % by size exclusion chromatography and spectrophotometry; (j) measure as detecting, less than the monomer of 0.5 area % by size exclusion chromatography and spectrophotometry; (k) have with SEQ ID NOS:5-10 in the CTLA4-Ig polypeptide of any one at least 95% aminoacid sequence that is equal to; (1) can with CD80 and CD86 bonded CTLA4-Ig molecule; Or its pharmacy Equivalent.
218. a composition that comprises the CTLA4-Ig molecule, wherein said molecule colony is characterised in that:
(a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule;
(b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule;
(c) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule;
(d) the molar average ratio of the Fucose of about 6.4-about 7.0 and CTLA4-Ig molecule;
(e) the molar average ratio of the seminose of about 14-about 16 and CTLA4-Ig molecule;
(f) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule;
(g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 2.4 ± 0.2-about 5.2 ± 0.2;
(h) be less than or equal to the MCP-1 of 5ppm;
(i) measure as detecting, less than the high molecular weight species of 5 area % by size exclusion chromatography and spectrophotometry;
(j) measure as detecting, less than the monomer of 1 area % by size exclusion chromatography and spectrophotometry;
(k) have with SEQ ID NOS:11-16 in the CTLA4-Ig polypeptide of any one at least 95% aminoacid sequence that is equal to;
(1) can with CD80 and CD86 bonded CTLA4-Ig molecule.
219. a composition that comprises the CTLA4-Ig molecule, wherein said molecule colony is characterised in that: (a) the molar average ratio of the GlcNAc of about 24-about 28 and CTLA4-Ig molecule; (b) the molar average ratio of the GalNAc of about 2.7-about 3.6 and CTLA4-Ig molecule; (c) the molar average ratio of the semi-lactosi of about 11-about 13 and CTLA4-Ig molecule; (d) the molar average ratio of the Fucose of about 6.4-about 7.0 and CTLA4-Ig molecule; (e) the molar average ratio of the seminose of about 14-about 16 and CTLA4-Ig molecule; (f) the molar average ratio of the sialic acid of about 5.5-about 9.5 and CTLA4-Ig molecule; (g) as by visual mensuration on the isoelectrofocusing gel, the pI of about 3.4 ± 0.2-about 5.2 ± 0.2; (h) be less than or equal to the MCP-1 of 5ppm; (i) measure as detecting, less than the high molecular weight species of 5 area % by size exclusion chromatography and spectrophotometry; (j) measure as detecting, less than the monomer of 1 area % by size exclusion chromatography and spectrophotometry; (k) have with SEQ ID NOS:11-16 in the CTLA4-Ig polypeptide of any one at least 95% aminoacid sequence that is equal to; (1) can with CD80 and CD86 bonded CTLA4-Ig molecule; Or its pharmacy Equivalent.
220. each composition among the claim 216-219, it further comprises pharmaceutically acceptable carrier.
221. each composition among the claim 216-219, it further comprises a certain amount of maltose monohydrate.
222. each composition among the claim 216-219, it further comprises pharmaceutically acceptable thinner, adjuvant or carrier.
223. each composition among the claim 216-219, it further comprises maltose, sodium orthophosphate (monometallic) monohydrate, sodium-chlor, sodium hydroxide and sterilized water.
224. each composition among the claim 216-219, it further comprises sucrose, poloxamer, sodium orthophosphate (monometallic) monohydrate, anhydrous sodium orthophosphate dimetallic, sodium-chlor, sodium hydroxide and sterilized water.
225. each composition among the claim 216-219, it further comprises sucrose, sodium orthophosphate (monometallic) monohydrate, sodium-chlor, sodium hydroxide, hydrochloric acid and sterilized water.
226. each composition among the claim 216-219, wherein said composition is freeze dried.
227. a lyophilised compositions, it comprises among the claim 216-219 of significant quantity each composition, maltose, sodium orthophosphate (monometallic) monohydrate, sodium-chlor, sodium hydroxide and hydrochloric acid.
228. a lyophilised compositions, it comprises among the claim 216-219 of significant quantity each composition, sucrose, sodium orthophosphate (monometallic) monohydrate, sodium-chlor, sodium hydroxide and hydrochloric acid.
229. the lyophilised compositions of a CTLA4-Ig molecule, its comprise as detect by size exclusion chromatography and spectrophotometry measures the CTLA4-Ig dimer of at least 95 area % and be no more than the CTLA4-Ig high molecular weight species of 5 area %.
230. the composition of claim 229, wherein said composition comprise as detect by size exclusion chromatography and spectrophotometry measure the CTLA4-Ig dimer of at least 98 area % and be no more than the CTLA4-Ig high molecular weight species of 2 area %.
231. the composition of claim 229, wherein said composition comprise as detect by size exclusion chromatography and spectrophotometry measure the CTLA4-Ig dimer of at least 99 area % and be no more than the CTLA4-Ig high molecular weight species of 1 area %.
232. the freeze dried CTLA4-Ig protein composition of claim 229, wherein said composition comprise at least 8.0 moles of sialic acids/mole CTLA4-Ig protein.
233. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 31 moles of GlcNAc/ mole CTLA4-Ig protein of about 15.7-.
234. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 3.2 moles of GalNAc/ mole CTLA4-Ig protein of about 1.6-.
235. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 15.5 moles of semi-lactosis of about 9.3-/mole CTLA4-Ig protein.
236. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 7.9 moles of Fucoses of about 3.6-/mole CTLA4-Ig protein.
237. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 22 moles of seminoses of about 7.7-/mole CTLA4-Ig protein.
238. the freeze dried CTLA4-Ig protein composition of claim 229, wherein said composition comprise at least 5.0 moles of sialic acids/mole CTLA4-Ig protein.
239. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 28 moles of GlcNAc/ mole CTLA4-Ig protein of about 24-.
240. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 3.6 moles of GalNAc/ mole CTLA4-Ig protein of about 2.7-.
241. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 13 moles of semi-lactosis of about 11-/mole CTLA4-Ig protein.
242. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 7.0 moles of Fucoses of about 6.4-/mole CTLA4-Ig protein.
243. the freeze dried CTLA4-Ig composition of claim 229, wherein said composition comprise the about 16 moles of seminoses of about 14-/mole CTLA4-Ig protein.
244. a pharmaceutical kit, it comprises: the container that (a) comprises the freeze dried CTLA4-Ig composition of claim 229; (b) be used for described freeze dried CTLA4-Ig composition is reconstituted the specification sheets of the solution that is used to inject.
245. a method that is used for suppressor T cell propagation or T cell activation, described method comprise that each composition contacts among claim 56-58,60-127,129-137 and the 139-244 that makes T cell and significant quantity.
246. a method that is used for suppressing experimenter's immunne response, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
247. a method that is used for the treatment of the immune disorders among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
248. one kind is used for inducing the method for experimenter at antigenic immunological tolerance, described method comprises the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
249. a method that is used for the treatment of the inflammation among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
250. a method that is used for the treatment of rheumatoid arthritis, it comprises the pharmaceutical composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
251. a method that is used for the treatment of rheumatism, it comprises the pharmaceutical composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
252. a method that is used for the treatment of ulcerative colitis, it comprises the pharmaceutical composition of using in the claim of significant quantity each to the experimenter that these needs are arranged.
253. a psoriasic method that is used for the treatment of among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
254. a method that is used for the treatment of the lupus among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
255. an allergic method that is used for the treatment of or prevents among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
256. a method that is used for the treatment of or prevents the graft versus host disease (GVH disease) among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
257. the method that the transplant organ that is used for the treatment of or prevents among the experimenter repels, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
258. the method that the transplanted tissue that is used for the treatment of or prevents among the experimenter repels, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
259. the method that the transplanted cells that is used for the treatment of or prevents among the experimenter repels, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
260. a method that is used for the treatment of the multiple sclerosis among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
261. a method that is used for the treatment of the CrohnShi disease among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
262. a method that is used for the treatment of the type i diabetes among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
263. a method that is used for the treatment of the inflammatory bowel among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
264. a method that is used for the treatment of the ovaritis among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
265. a brightic method that is used for the treatment of among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
266. a method that is used for the treatment of the allergic encephalitis among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
267. a method that is used for the treatment of the myasthenia gravis among the experimenter, described method comprise the composition of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
268. the sialic acids groups with about 5-about 18 and the CTLA4-Ig molecule colony of the proteinic molar average ratio of CTLA4-Ig are used for the purposes of medicine of the treating and/or preventing property processing of immune disorders in preparation.
269. have the purposes of CTLA4-Ig molecule colony in the agent of preparation resisting rheumatoid arthritis of the sialic acids groups of about 5-about 18 and the proteinic molar average ratio of CTLA4-Ig, described resisting rheumatoid arthritis agent together with about the specification sheets of its purposes in rheumatoid arthritis treatment in packing.
270. a method that is used for suppressor T cell propagation or T cell activation, described method comprise that each composition contacts among claim 56-58,60-127,129-137 and the 139-244 that makes T cell and significant quantity, described composition and methotrexate make up.
271. method that is used for suppressing experimenter's immunne response, described method comprises composition, described composition and the methotrexate combination of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
272. one kind is used for inducing the method for experimenter at antigenic immunological tolerance, described method comprises composition, described composition and the methotrexate combination of using among claim 56-58,60-127,129-137 and the 139-244 of significant quantity each to the experimenter that these needs are arranged.
273. a composition isolated that comprises the CTLA4-Ig molecule, it has and is less than or equal to 7.4% immunogenicity incidence.
274. the composition isolated of claim 273, wherein said immunogenic incidence is about 2.1%-about 7.4%.
275. the composition isolated of claim 273, wherein said immunogenic incidence is less than or equal to 3.7%.
276. the composition isolated of claim 273, wherein said immunogenic incidence is less than or equal to 3.0%.
277. the composition isolated of claim 273, wherein said immunogenic incidence is about 2.8%-about 3.0%.
278. a composition isolated that comprises the CTLA4-Ig molecule wherein, after giving people's applying said compositions, takes place to be less than or equal to 7.4% incidence in the people with described CTLA4-Ig molecule bonded production of antibodies.
279. the composition isolated of claim 278, wherein said incidence is about 2.1%-about 7.4%.
280. the composition isolated of claim 278, wherein said incidence is less than or equal to 3.7%.
281. the composition isolated of claim 278, wherein said incidence is less than or equal to 3.0%.
282. the composition isolated of claim 278, wherein said incidence is about 2.8%-about 3.0%.
283. a composition isolated that comprises the CTLA4-Ig molecule wherein, after giving people's applying said compositions, takes place to be less than or equal to 4.9% incidence in the people with the described CTLA4 part bonded production of antibodies of described CTLA4-Ig molecule.
284. the composition isolated of claim 283, wherein said incidence is about 0.5%-about 4.9%.
285. the composition isolated of claim 283, wherein said incidence is less than or equal to 1.2%.
286. the composition isolated of claim 283, wherein said incidence is less than or equal to 1.0%.
287. the composition isolated of claim 283, wherein said incidence is about 0.9%-about 1.0%.
288. each composition isolated among the claim 273-287, wherein said incidence is measured in enzyme-linked immunosorbent assay (ELISA).
289. each composition isolated among the claim 273-287, wherein said incidence are measured in (ECL) at electrochemiluminescence and are measured.
290. composition isolated that comprises the CTLA4-Ig molecule, wherein, after giving people's applying said compositions, the production of antibodies of the described CTLA4-Ig molecule that neutralizes takes place to be less than or equal to 75% incidence in the people who has with the described CTLA4 part bonded antibody of described CTLA4-Ig molecule.
291. the composition isolated of claim 290, wherein said incidence are 40-75%.
292. the composition isolated of claim 291, wherein said incidence is less than or equal to 40%.
293. each composition isolated among the claim 290-292, wherein said incidence is measured in measuring based on the luciferase reporter gene of cell.
294. a composition that comprises the CTLA4-Ig molecule is characterized in that as measuring by HPAEC, among the structural domain I at least about the N linked glycosylation content of the about 35.2 area % of 24.5 area %-.
295. the composition of claim 294, wherein said structural domain I comprises 3 peaks, and wherein said first peak comprises about 7.3 area %, and described second peak comprises about 10.7 area %, and described the 3rd peak comprises about 8.8 area %, just like measuring by HPAEC.
296. a composition that comprises the CTLA4-Ig molecule is characterized in that as measuring by HPAEC, in the domain II at least about the N linked glycosylation content of the about 34.1 area % of 26.3 area %-.
CNA2006800531165A 2005-12-20 2006-12-19 Compositions and methods for producing a composition Pending CN101448852A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104520316A (en) * 2012-06-29 2015-04-15 百时美施贵宝公司 Methods for reducing glycoprotein aggregation
CN109187940A (en) * 2018-07-27 2019-01-11 南京谱利健生物技术有限公司 The preparation and application of a kind of isotopic tag reagent for Analysis of polysaccharides
CN111398308A (en) * 2020-03-27 2020-07-10 上海健康医学院 Automatic detection method and system for packaging quality of aluminum-plastic bubble caps of tablets and capsules
CN112948761A (en) * 2019-12-10 2021-06-11 中国科学院地质与地球物理研究所 River nitrogen pollutant quantitative source analysis system
CN113176920A (en) * 2021-04-29 2021-07-27 上海云扩信息科技有限公司 Universal RPA element selector management system
CN114340653A (en) * 2019-09-05 2022-04-12 分子免疫中心 Human recombinant low-sialylated erythropoietin, method for purifying same and therapeutic use thereof
CN114574075A (en) * 2022-04-12 2022-06-03 江苏中基复合材料有限公司 Aluminum foil for resin-coated aluminum foil cover plate and preparation method thereof

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104520316A (en) * 2012-06-29 2015-04-15 百时美施贵宝公司 Methods for reducing glycoprotein aggregation
CN104520316B (en) * 2012-06-29 2021-07-06 百时美施贵宝公司 Method for reducing glycoprotein aggregation
CN109187940A (en) * 2018-07-27 2019-01-11 南京谱利健生物技术有限公司 The preparation and application of a kind of isotopic tag reagent for Analysis of polysaccharides
CN114340653A (en) * 2019-09-05 2022-04-12 分子免疫中心 Human recombinant low-sialylated erythropoietin, method for purifying same and therapeutic use thereof
CN114340653B (en) * 2019-09-05 2023-01-31 分子免疫中心 Human recombinant low-sialylated erythropoietin, method for purifying same, and therapeutic use thereof
CN112948761A (en) * 2019-12-10 2021-06-11 中国科学院地质与地球物理研究所 River nitrogen pollutant quantitative source analysis system
CN111398308A (en) * 2020-03-27 2020-07-10 上海健康医学院 Automatic detection method and system for packaging quality of aluminum-plastic bubble caps of tablets and capsules
CN113176920A (en) * 2021-04-29 2021-07-27 上海云扩信息科技有限公司 Universal RPA element selector management system
CN114574075A (en) * 2022-04-12 2022-06-03 江苏中基复合材料有限公司 Aluminum foil for resin-coated aluminum foil cover plate and preparation method thereof

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