WO2015041303A1 - Fc結合性タンパク質、当該タンパク質の製造方法および当該タンパク質を用いた抗体吸着剤、ならびに当該吸着剤を用いた抗体の精製方法および識別方法 - Google Patents
Fc結合性タンパク質、当該タンパク質の製造方法および当該タンパク質を用いた抗体吸着剤、ならびに当該吸着剤を用いた抗体の精製方法および識別方法 Download PDFInfo
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- 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
- C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07—ORGANIC CHEMISTRY
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- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
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- the present invention relates to an Fc-binding protein having affinity for immunoglobulin, an antibody adsorbent using the protein, and a method for purifying and identifying an antibody using the adsorbent. More specifically, the present invention relates to an Fc-binding protein having higher stability against heat and acid than wild type, a method for producing the protein, and an antibody adsorbent obtained by immobilizing the protein on an insoluble carrier. In particular, the present invention relates to an adsorbent capable of specifically adsorbing an antibody having a sugar chain among antibodies, a method for purifying an antibody having a sugar chain using the adsorbent, and whether or not a sugar chain is added to the antibody. It relates to a method for identifying.
- Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Individual molecules recognize a single or the same group of immunoglobulin isotypes by a recognition domain on the Fc receptor by a recognition domain belonging to the immunoglobulin superfamily. This determines which accessory cells are driven in the immune response. Fc receptors can be further classified into several subtypes, such as Fc ⁇ receptors that are receptors for IgG (immunoglobulin G), Fc ⁇ receptors that bind to the Fc region of IgE, Fc ⁇ receptors that bind to the Fc region of IgA, etc. There is.
- Fc ⁇ receptors have been reported to have Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa, and Fc ⁇ RIIIb (Non-patent Document 1: Takai. T., Jpn. J. Clin. Immunol. , 28, 318-326, 2005).
- Fc ⁇ RIIIa is present on the surface of natural killer cells (NK cells) and macrophages, and is an important receptor involved in ADCC (antibody-dependent cellular cytotoxicity) activity, which is important in the human immune system. It is.
- NK cells natural killer cells
- ADCC antibody-dependent cellular cytotoxicity
- FIG. 1 shows a schematic diagram of the structure of human Fc ⁇ RIIIa.
- the amino acid numbers in FIG. 1 correspond to the amino acid numbers described in SEQ ID NO: 1. That is, the signal sequence (S) is from the first methionine (Met) to the 16th alanine (Ala) in SEQ ID NO: 1, and the extracellular region is from the 17th glycine (Gly) to the 208th glutamine (Gln).
- ADCC antibody-dependent cytotoxicity activity
- a medicine using the specificity of a monoclonal antibody
- ADCC antibody-dependent cytotoxicity
- ADCC activity is known to change depending on the difference in the N-type sugar chain added to the 297th asparagine residue in the Fc region. It has been reported that ADCC activity is improved by an antibody from which fucose is removed (Non-patent Document 3: Shinkawa. T., J. Biol. Chem., 278, 3466-3473, 2003). That is, in antibody medicine, the sugar chain structure possessed by an antibody has an important meaning.
- antibody drugs are usually produced using gene recombination techniques using animal cells as hosts, and it is difficult to control the sugar chains added to the antibodies in the host. In addition, it takes a lot of time and effort to analyze the sugar chain of the produced antibody.
- the first object of the present invention is to provide an Fc-binding protein having improved stability to heat and acid, a method for producing the protein, and an antibody adsorbent using the protein. Furthermore, the second object of the present invention is to provide a method capable of identifying the presence or absence of sugar chain addition to an antibody and a material used in the method.
- the present inventors have identified an amino acid residue involved in improving stability in human Fc ⁇ RIIIa, and substituted the amino acid residue with another amino acid residue.
- the body was found to have excellent stability to heat and acid, and the present invention was completed.
- the present inventors have used an adsorbent obtained by immobilizing Fc ⁇ RIIIa, which is one of the receptors for IgG (Fc ⁇ receptor), on an insoluble carrier, and thus the antibody
- Fc ⁇ receptor one of the receptors for IgG
- the present inventors have found that the presence or absence of sugar chain addition to can be identified, and have completed the present invention.
- the present application includes the embodiments described in the following (A) to (Z): (A) Of the amino acid sequence set forth in SEQ ID NO: 1, the amino acid residues from the 17th to the 192nd are included, and the amino acid residues from the 17th to the 192nd are the following (1) to (40) An Fc binding protein wherein at least one amino acid substitution has occurred.
- the amino acid sequence of SEQ ID NO: 1 comprises the 17th to 192nd amino acid residues, and at least the 35th tyrosine of SEQ ID NO: 1 is asparagine in the 17th to 192nd amino acid residues.
- the Fc-binding protein according to (A) which is substituted with any of acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, and histidine.
- the amino acid sequence of SEQ ID NO: 1 comprises the 17th to 192nd amino acid residues, and at least the 35th tyrosine of SEQ ID NO: 1 is asparagine in the 17th to 192nd amino acid residues.
- (F) The Fc-binding protein according to any one of (A) to (C), further comprising at least one amino acid substitution among the following (41) to (44).
- (41) The 66th leucine in SEQ ID NO: 1 is replaced with histidine or arginine (42)
- the 147th glycine of SEQ ID NO: 1 is replaced with aspartic acid (43)
- the 158th tyrosine of SEQ ID NO: 1 is replaced with histidine (44 )
- (M) A polynucleotide encoding the Fc-binding protein according to any one of (A) to (F).
- (T) human Fc ⁇ RIIIa contains at least amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence shown in SEQ ID NO: 1, and at least one of the amino acid residues is a residue of another amino acid.
- Human Fc ⁇ RIIIa contains at least amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence set forth in SEQ ID NO: 1, and the following amino acid residues from the 17th to the 192nd (
- the Fc-binding protein of the present invention is a protein having a binding property to the Fc region of an antibody, and at least of the extracellular region (EC region of FIG. 1) of human Fc ⁇ RIIIa comprising the amino acid sequence set forth in SEQ ID NO: 1.
- the amino acid substitution at the specific position is specifically Met18Arg in the amino acid sequence shown in SEQ ID NO: 1 (this notation indicates that the 18th methionine of SEQ ID NO: 1 is substituted with arginine, and so on.
- substitution of any of Tyr35Asp, Tyr35Gly, Tyr35Lys, Tyr35Leu, Tyr35Asn, Tyr35Pro, Tyr35Ser, Tyr35Thr, Tyr35His, and Glu121Gly is preferable because thermal stability is improved.
- wild-type human Fc ⁇ RIIIa is known to have a mutant in which substitution of Leu66His, Leu66Arg, Gly147Asp, Tyr158His or Val176Phe has occurred, but may contain these amino acid substitutions in addition to the amino acid substitution at the specific position. .
- the amino acid residue at a specific position may be substituted with an amino acid other than those described above as long as it has antibody binding activity.
- One example is a conservative substitution that substitutes between amino acids whose physical and / or chemical properties of both amino acids are similar. Conservative substitutions are not limited to Fc-binding proteins, and are generally known to those skilled in the art to maintain protein function between those with substitutions and those without substitutions. Examples of conservative substitutions include substitutions that occur between glycine and alanine, between aspartic acid and glutamic acid, between serine and proline, or between glutamic acid and alanine (protein structure and function, Medical Science International, 9 , 2005).
- the number of amino acids to be substituted is not particularly limited.
- Fc-binding proteins shown in the following (a) to (h) can be mentioned. These Fc-binding proteins are preferable in terms of improving heat and acid stability.
- (A) The amino acid substitution of Val27Glu and Tyr35Asn occurs in the amino acid residues from 17th to 192th in the amino acid sequence described in SEQ ID NO: 1 and from the 17th to 192nd amino acid residues.
- Fc binding protein (Fc binding protein containing the amino acid sequence from the 33rd position to the 208th position in the amino acid sequence described in SEQ ID NO: 27) (b) From the 17th position to the 192nd position in the amino acid sequence described in SEQ ID NO: 1
- Fc-binding protein comprising a column
- the amino acid sequence of SEQ ID NO: 1 comprises amino acid residues 17 to 192, and in the amino acid residues 17 to 192, Val27Glu, Fc binding protein (Fc binding protein containing the amino acid sequence from the 33rd to the 208th of the amino acid sequence described in SEQ ID NO: 33) in which amino acid substitution of Tyr35Asn, Phe75Leu and Glu121Gly occurs (d) in SEQ ID NO: 1 Amino acid substitution of Val27Glu, Tyr35Asn, Phe75Leu, Asn92Ser, and Glu121Gly is included in the amino acid residues from the 17th to the 192nd in the described amino acid sequence and the 17th to the 192nd amino acid residues Resulting Fc-binding protein (Fc-binding protein containing amino acid sequences from 33 to 208 in the amino acid sequence described in SEQ ID NO: 37) (e) From 17 in the amino acid sequence
- FIG. 1 shows a schematic diagram of the structure of human Fc ⁇ RIIIa.
- the amino acid numbers in FIG. 1 correspond to the amino acid numbers described in SEQ ID NO: 1.
- the signal sequence (S) is from the first methionine (Met) to the 16th alanine (Ala) in SEQ ID NO: 1, and the extracellular region is from the 17th glycine (Gly) to the 208th glutamine (Gln).
- EC from the 209th valine (Val) to the 229th valine (Val) is the transmembrane region (TM) and from the 230th lysine (Lys) to the 254th lysine (Lys) is the intracellular region (C ).
- the human Fc ⁇ RIIIa used as a ligand in the adsorbent of the present invention does not necessarily need to use the full length of human Fc ⁇ RIIIa (SEQ ID NO: 1), and at least the 192nd to 192nd glycine in the amino acid sequence described in SEQ ID NO: 1. Any polypeptide containing amino acid residues up to glutamine may be used.
- the amino acid sequence set forth in SEQ ID NO: 1 contains at least amino acid residues from the 17th glycine to the 192nd glutamine, and one or more of the amino acid residues are substituted with other amino acid residues, Even an inserted or deleted polypeptide is included in human Fc ⁇ RIIIa used as a ligand in the adsorbent of the present invention.
- the 66th leucine (Leu) is histidine (His) or arginine (Arg)
- the 147th glycine (Gly) is aspartic acid (Asp)
- the 158th tyrosine (Tyr) is included in human Fc ⁇ RIIIa used as a ligand in the adsorbent of the present invention.
- the 66th leucine (Leu) is histidine (His) or arginine (Arg)
- the 147th glycine (Gly) is aspartic acid (Asp)
- His histidine
- Val 176th valine
- Phe phenylalanine
- Human Fc ⁇ RIIIa which is a ligand of the Fc-binding protein or adsorbent of the present invention, may further be added with an oligopeptide useful for separation from a solution in the presence of contaminants on the N-terminal side or C-terminal side thereof.
- the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid and the like.
- cysteine-containing oligopeptide useful for immobilizing human Fc ⁇ RIIIa which is the ligand of the Fc-binding protein or adsorbent of the present invention, on a solid phase such as a chromatographic support, It may be further added to the N-terminal side or C-terminal side of human Fc ⁇ RIIIa which is a ligand of protein or adsorbent.
- the length of the oligopeptide added to the N-terminal side or C-terminal side of human Fc ⁇ RIIIa which is a ligand of Fc-binding protein or adsorbent is the IgG binding of human Fc ⁇ RIIIa which is the ligand of Fc-binding protein or adsorbent of the present invention.
- a polynucleotide encoding the oligopeptide is prepared and then genetic engineering is performed using methods well known to those skilled in the art.
- oligopeptide may be added to the N-terminal side or C-terminal side of Fc-binding protein of the present invention or human Fc ⁇ RIIIa. It may be added chemically bonded to the side.
- a signal peptide for promoting efficient expression in the host may be added to the N-terminal side of human Fc ⁇ RIIIa which is a ligand of the Fc-binding protein or adsorbent of the present invention.
- Examples of the signal peptide in the case where the host is Escherichia coli include PelB, DsbA, MalE (uniprot No. P0AEX9 amino acid sequence from the first to the 26th region), and the periplasm such as TorT.
- a signal peptide can be exemplified (Japanese Patent Laid-Open No. 2011-097898).
- an amino acid sequence of human Fc ⁇ RIIIa which is a ligand of the Fc-binding protein or adsorbent of the present invention, is converted into a nucleotide sequence, and a polynucleotide containing the nucleotide sequence is artificially converted.
- DNA amplification method such as PCR method from directly synthetically,
- a method of ligating the prepared polynucleotide with an appropriate method is an appropriate method.
- the conversion when converting from an amino acid sequence to a nucleotide sequence, the conversion is preferably performed in consideration of the frequency of codon usage in the host to be transformed.
- the host is Escherichia coli
- AGA / AGG / CGG / CGA is used for arginine (Arg)
- ATA is used for isoleucine (Ile)
- CTA is used for leucine (Leu)
- GGA is used for glycine (Gly).
- CCC is less frequently used in proline (Pro) (because it is a so-called rare codon)
- it may be converted so as to avoid those codons.
- Analysis of codon usage frequency can also be performed by using a public database (for example, Codon Usage Database on the homepage of Kazusa DNA Research Institute).
- an error-prone PCR method can be used.
- the reaction conditions in the error-prone PCR method are not particularly limited as long as a desired mutation can be introduced into a polynucleotide encoding human Fc ⁇ RI (or Fc-binding protein).
- deoxynucleotides which are substrates
- polynucleotides by making the concentration of dATP / dTTP / dCTP / dGTP polynucleotides by making the concentration of dATP / dTTP / dCTP / dGTP
- MnCl 2 polynucleotides by making the concentration of dATP / dTTP / dCTP / dGTP
- Mutations can be introduced.
- a polynucleotide containing a whole or partial sequence of human Fc ⁇ RI is contacted / acted with a mutagen agent or irradiated with ultraviolet rays to mutate the polynucleotide.
- the method of producing by introducing is mentioned.
- a drug used as a mutagen in this method a mutagenic drug commonly used by those skilled in the art such as hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, nitrous acid, sulfite, hydrazine may be used. .
- the polynucleotide of the present invention When transforming a host using the polynucleotide of the present invention, the polynucleotide of the present invention itself may be used, but an expression vector (for example, bacteriophage, cosmid, or the like commonly used for transformation of prokaryotic cells or eukaryotic cells) may be used. It is more preferable to use a plasmid or the like in which the polynucleotide of the present invention is inserted at an appropriate position.
- the expression vector is not particularly limited as long as it is stably present in the host to be transformed and can be replicated.
- pET plasmid vector When Escherichia coli is used as a host, pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, pCDF plasmid vector A pBBR plasmid vector can be exemplified.
- the appropriate position means a position where the replication function of the expression vector, a desired antibiotic marker, and a region related to transmissibility are not destroyed.
- promoter examples include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, ⁇ phage ⁇ PL promoter, ⁇ PR promoter and the like when the host is Escherichia coli.
- the expression vector of the present invention In order to transform a host using the expression vector inserted with the polynucleotide of the present invention (hereinafter referred to as the expression vector of the present invention) produced by the above-described method, a person skilled in the art may carry out the method.
- a microorganism belonging to the genus Escherichia E. coli JM109 strain, E. coli BL21 (DE3) strain, E. coli W3110 strain, etc.
- known literature eg, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992. And the like.
- a transformant capable of expressing the Fc-binding protein of the present invention (hereinafter referred to as the transformant of the present invention). ) Can be obtained.
- the host which expresses human FcgammaRIIIa which is the ligand of Fc binding protein or adsorption agent of this invention As an example, an animal cell (CHO cell, HEK cell, Hela cell, COS cell etc.), yeast (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, such as Schizosaccharomyces pombe), insect cells (Sf9, Sf21, etc.), E.
- an alkaline extraction method or a QIAprep Spin Miniprep kit (manufactured by Qiagen) is commercially available from the culture obtained by culturing the transformant of the present invention.
- the extraction kit may be used.
- the Fc-binding protein of the present invention can be produced by culturing the transformant of the present invention and recovering the Fc-binding protein of the present invention from the obtained culture.
- the culture includes not only the cultured cells of the transformant of the present invention itself but also a medium used for the culture.
- the transformant used in the protein production method of the present invention may be cultured in a medium suitable for culturing the target host.
- an LB (Luria-Bertani) medium supplemented with necessary nutrient sources is preferable.
- An example of the medium is given.
- a drug corresponding to the drug resistance gene contained in the vector it is preferable to add to the medium and culture.
- the vector contains a kanamycin resistance gene, kanamycin may be added to the medium.
- an appropriate nutrient source may be added to the medium, and if desired, the medium is selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol.
- One or more reducing agents may be included.
- a reagent that promotes protein secretion from the transformant to the culture solution such as glycine may be added.
- glycine is contained in the medium at 2% (w / v) or less. Addition is preferred.
- the culture temperature is generally 10 ° C. to 40 ° C., preferably 20 ° C.
- the pH of the medium is pH 6.8 to pH 7.4, preferably around pH 7.0.
- an inducible promoter included in the vector of the present invention, it is preferable that the induction be performed under conditions that allow the Fc-binding protein of the present invention to be expressed well. Examples of the inducer include IPTG (isopropyl- ⁇ -D-thiogalactopyranoside).
- the turbidity (absorbance at 600 nm) of the culture solution is measured, and when it reaches about 0.5 to 1.0, an appropriate amount of IPTG is added, followed by further culturing, thereby allowing Fc binding. Protein expression can be induced.
- the addition concentration of IPTG may be appropriately selected from the range of 0.005 to 1.0 mM, but is preferably in the range of 0.01 to 0.5 mM.
- Various conditions relating to the IPTG induction may be performed under conditions well known in the art.
- the Fc-binding protein of the present invention may be recovered by separating / purifying from the culture. For example, when expressed in the culture supernatant, the cells are separated by centrifugation, and the Fc-binding protein of the present invention may be purified from the obtained culture supernatant. In addition, when expressed in cells (including periplasm), after collecting the cells by centrifugation, the cells are disrupted by adding an enzyme treatment agent, a surfactant or the like, and the Fc of the present invention.
- Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and the like. By performing a purification operation by combining these chromatography, the Fc binding property of the present invention can be obtained. Proteins can be prepared with high purity.
- the binding activity to IgG is measured using the Enzyme-Linked ImmunoSorbent Assay (hereinafter referred to as ELISA) method or the surface plasmon resonance method.
- ELISA Enzyme-Linked ImmunoSorbent Assay
- the IgG used for the measurement of the binding activity is preferably human IgG, and human IgG1 and human IgG3 are particularly preferable.
- the adsorbent of the present invention can be produced by binding the Fc-binding protein of the present invention or human Fc ⁇ RIIIa to an insoluble carrier.
- the insoluble carrier is not particularly limited, and carriers made from polysaccharides such as agarose, alginate (alginate), carrageenan, chitin, cellulose, dextrin, dextran, starch, polyvinyl alcohol, polymethacrylate, poly (2- Examples thereof include a carrier made of a synthetic polymer such as hydroxyethyl methacrylate and polyurethane, and a carrier made of ceramics such as silica. Of these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers.
- the preferred carrier examples include polymethacrylate gels introduced with hydroxyl groups such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by GE Healthcare), and cellulose gels such as Cellufine (manufactured by JNC). .
- the shape of the insoluble carrier is not particularly limited, and may be granular or non-particulate, porous or non-porous.
- N-hydroxysuccinimide (NHS) activated ester group epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group
- An active group such as a group haloacetamide may be imparted and immobilized by covalently binding a human Fc-binding protein and an insoluble carrier via the active group.
- the carrier provided with the active group may be a commercially available carrier as it is, or may be prepared by introducing an active group on the surface of the carrier under appropriate reaction conditions.
- TOYOPEARL AF-Epoxy-650M TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated SepharoseFast-Effects4 Examples include 6B (both manufactured by GE Healthcare) and SulfoLink Coupling Resin (manufactured by Thermo Scientific).
- examples of the method for introducing an active group on the surface of the carrier include a method in which one of compounds having two or more active sites reacts with a hydroxyl group, an epoxy group, a carboxyl group, an amino group, etc. present on the surface of the carrier. it can.
- examples of the compound that introduces an epoxy group into the hydroxyl group or amino group on the surface of the carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.
- Examples of the compound that introduces an epoxy group on the carrier surface with the compound and then introduces a carboxyl group on the carrier surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3- Examples thereof include aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.
- Examples of the compound that introduces a maleimide group into the hydroxyl group, epoxy group, carboxyl group or amino group present on the surface of the carrier include N- ( ⁇ -maleimidocaproic acid) hydrazide, N- ( ⁇ -maleimidopropionic acid) hydrazide, 4- [ 4-N-maleimidophenyl] acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1- (4-aminophenyl) maleimide, 1- (3-aminophenyl) maleimide, 4- (maleimido) phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, (N- [ ⁇ -maleimidoacetoxy] succinimide ester), (m-maleimidobenzoyl) N-hydroxys
- Compounds that introduce a haloacetyl group into the hydroxyl group or amino group present on the surface of the carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, Iodoacetic anhydride, 2- (iodoacetamido) acetic acid-N-hydroxysuccinimide ester, 3- (bromoacetamido) propionic acid-N-hydroxysuccinimide ester, 4- (iodoacetyl) aminobenzoic acid-N-hydroxysuccinimide ester It can be illustrated.
- An example is a method in which ⁇ -alkenyl alkanglycidyl ether is reacted with a hydroxyl group or amino group present on the surface of the carrier, and then the ⁇ -alkenyl moiety is halogenated with a halogenating agent to activate.
- ⁇ -alkenyl alkanglycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether.
- halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide. it can.
- the method for introducing an active group on the surface of the carrier there is a method for introducing an activating group into the carboxyl group present on the surface of the carrier using a condensing agent and an additive.
- the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide, and carbonyldiimidazole.
- the additive include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenzotriazole.
- Buffers used when immobilizing the Fc-binding protein or human Fc ⁇ RIIIa of the present invention on an insoluble carrier include acetate buffer, phosphate buffer, MES (2-Morpholine ethansulfonic acid) buffer, HEPES (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) buffer, Tris buffer, borate buffer.
- the reaction temperature for immobilization may be appropriately set in the temperature range from 5 ° C. to 50 ° C. in consideration of the reactivity of the active group and the stability of the Fc-binding protein of the present invention or human Fc ⁇ RIIIa. It is preferably in the range of 10 ° C to 35 ° C.
- the adsorbent of the present invention In order to purify an antibody having a sugar chain using the adsorbent of the present invention obtained by immobilizing the Fc-binding protein of the present invention or human Fc ⁇ RIIIa on an insoluble carrier, for example, the adsorbent of the present invention is packed.
- a buffer solution containing an antibody having a sugar chain on a column is added using a liquid feeding means such as a pump to specifically adsorb the antibody having a sugar chain to the adsorbent of the present invention,
- the antibody having a sugar chain may be eluted by adding the eluate to the column.
- An antibody having a sugar chain that can be purified by the adsorbent of the present invention is an antibody that has at least the Fc region of an antibody having a sugar chain that has affinity for an Fc receptor such as Fc-binding protein or Fc ⁇ RIIIa. I just need it. Examples include chimeric antibodies, humanized antibodies, human antibodies, and amino acid substitutions thereof that are generally used as antibodies used in antibody pharmaceuticals. In addition, bispecific antibodies (bispecific antibodies), Fc regions of antibodies with sugar chains and fusion proteins of other proteins, Fc regions of antibodies with sugar chains and drugs (ADC), etc. Even an antibody whose structure has been artificially modified can be purified with the adsorbent of the present invention.
- the buffer solution include a buffer solution containing an inorganic salt as a component, such as a phosphate buffer solution, and the pH of the buffer solution is pH 3 to 10, preferably pH 5 to 8.
- the interaction between the antibody having a sugar chain and a ligand may be weakened.
- a ligand Fc-binding protein of the present invention or human Fc ⁇ RIIIa
- pH change by buffer solution, counter peptide, temperature change, salt concentration change can be exemplified.
- the eluate for eluting the antibody having a sugar chain adsorbed to the adsorbent of the present invention it is more acidic than the solution used for adsorbing the antibody having a sugar chain to the adsorbent of the present invention.
- Side buffer As a specific example of the eluate for eluting the antibody having a sugar chain adsorbed to the adsorbent of the present invention, it is more acidic than the solution used for adsorbing the antibody having a sugar chain to the adsorbent of the present invention.
- the buffer solution examples include a citrate buffer solution, a glycine hydrochloride buffer solution, and an acetate buffer solution having a buffer capacity on the acidic side.
- the pH of the buffer may be set within a range that does not impair the function of the antibody, preferably pH 2.5 to 6.0, more preferably pH 3.0 to 5.0, still more preferably pH 3.3 to 4. 0.
- the elution position (elution fraction) of the antibody varies depending on the sugar chain structure of the antibody. Therefore, by separating the antibody using the adsorbent of the present invention, the difference in the sugar chain structure of the antibody can be identified.
- the structure of the glycans There are no particular limitations on the structure of the glycans that can be identified.
- the adsorbent of the present invention can be separated based on the difference in the sugar chain structure of the antibody, it can also be used for separation of the sugar chain itself.
- the adsorbent of the present invention can identify the difference in the sugar chain structure of the antibody.
- an Fc receptor other than Fc ⁇ RIIIa Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIb, FcRn
- Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIb, FcRn an Fc receptor other than Fc ⁇ RIIIa
- the Fc-binding protein of the present invention is a protein in which an amino acid residue at a specific position in the extracellular region of human Fc ⁇ RIIIa is substituted with another amino acid residue.
- the Fc binding protein of the present invention has improved heat and acid stability compared to wild type human Fc ⁇ RIIIa. Therefore, the Fc binding protein of the present invention is useful as a ligand of an adsorbent for separating immunoglobulin.
- the present invention also relates to an invention related to an adsorbent obtained by immobilizing human Fc ⁇ RIIIa on an insoluble carrier, and the adsorbent specifically adsorbs an antibody having a sugar chain among antibodies.
- the presence or absence of sugar chain addition to the antibody, which has been difficult until now, can be easily identified.
- the antibody having a sugar chain can be specifically purified by using the adsorbent of the present invention, it is possible to efficiently produce an antibody having a sugar chain.
- FIG. 1 is a schematic diagram of human Fc ⁇ RIIIa.
- the numbers in the figure indicate the amino acid sequence numbers described in SEQ ID NO: 1.
- S represents a signal sequence
- EC represents an extracellular region
- TM represents a transmembrane region
- C represents an intracellular region.
- S represents a signal sequence
- EC represents an extracellular region
- TM represents a transmembrane region
- C represents an intracellular region.
- S represents a signal sequence
- EC represents an extracellular region
- TM represents a transmembrane region
- C represents an intracellular region.
- the wild type in the figure indicates an Fc binding protein without amino acid substitution.
- the wild type in the figure indicates an Fc binding protein without amino acid substitution.
- FIG. 5 is a diagram (SDS-PAGE) showing the results of antibody purification using FcR5a-immobilized gel.
- A shows the purification result of human IgG1
- B shows the purification result of human IgG3.
- FIG. 3 is a diagram comparing the molecular weights of human IgG1 having a sugar chain (human IgG1 with a sugar chain) and human IgG1 having a sugar chain removed (sugar chain-removed human IgG1) by SDS-PAGE.
- Lane (1) in the figure is a human IgG1 with a sugar chain
- lane (2) is a human IgG1 with a sugar chain removed. It is the figure which compared the binding property of human Fc ⁇ RIIIa and protein A to human IgG1 with sugar chain and human IgG1 with sugar chain removed.
- (A) shows the results for human Fc ⁇ RIIIa
- (B) shows the results for protein A.
- Example 1 Preparation of Fc Binding Protein or Human Fc ⁇ RIIIa Expression Vector
- SEQ ID NO: 1 the amino acid sequence from the 17th glycine (Gly) to the 192nd glutamine (Gln) Based on the DNAworks method (Nucleic Acids Res., 30, e43, 2002), a nucleotide sequence in which a codon was converted from a human type to an E. coli type was designed. The designed nucleotide sequence is shown in SEQ ID NO: 2.
- the DNA mix in Table 1 means a solution obtained by sampling a predetermined amount of each of 18 types of oligonucleotides having the sequences described in SEQ ID NOs: 3 to 20 and mixing them.
- the second-stage PCR uses FcRp1 synthesized in (2-1) as a template, SEQ ID NO: 21 (5′-TAGCCATGGGCATGCCGTACGAGAATCTGCCCGAAAGCTCTGTGGATGTCCCTGTGGGTAATCTGTG An oligonucleotide consisting of the sequence described in 1 was used as a PCR primer.
- a reaction solution having the composition shown in Table 2 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, the second step at 62 ° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step of 1.5 minutes at ° C. for 30 cycles.
- the polynucleotide obtained in (2) is purified, digested with restriction enzymes NcoI and HindIII, and then ligated to an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII.
- Escherichia coli BL21 strain (DE3) was transformed with the ligation product.
- the obtained transformant was cultured in an LB medium containing 50 ⁇ g / mL kanamycin, and then the expression vector pET-eFcR was extracted using QIAprep Spin Miniprep kit (manufactured by Qiagen).
- the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (manufactured by Life Technologies) based on the chain terminator method for the polynucleotide encoding human Fc ⁇ RIIIa and its surrounding region in the expression vector pET-eFcR prepared in (4)
- the nucleotide sequence was analyzed with a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Life Technologies).
- an oligonucleotide having the sequence described in SEQ ID NO: 23 (5′-TAATACGACTCACTATAGGGG ′) or SEQ ID NO: 24 (5′-TATGCTAGTTATTGCTCAG-3 ′) was used as a sequencing primer.
- the amino acid sequence of the polypeptide expressed by the expression vector pET-eFcR is shown in SEQ ID NO: 25, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 26, respectively.
- the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
- the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- Example 2 Mutation Introduction to Fc Binding Protein and Library Preparation Of the Fc binding protein expression vector pER-eFcR prepared in Example 1, the polynucleotide portion encoding the Fc binding protein was subjected to error-prone PCR. Mutation was randomly introduced. (1) Error prone PCR was performed using pET-eFcR prepared in Example 1 as a template. In error-prone PCR, after preparing a reaction solution having the composition shown in Table 3, the reaction solution is heat-treated at 95 ° C. for 2 minutes, the first step at 95 ° C. for 30 seconds, the second step at 60 ° C.
- the PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
- the reaction solution was introduced into E. coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 ⁇ g / mL kanamycin (18 hours at 37 ° C.), and then placed on the plate. The formed colonies were used as a random mutant library.
- Example 3 Screening of heat-stabilized Fc binding protein (1)
- the random mutant library (transformant) prepared in Example 2 was subjected to 2YT liquid medium containing 50 ⁇ g / mL kanamycin (peptone 16 g / L, yeast).
- (Extract 10 g / L, sodium chloride 5 g / L) was inoculated into 200 ⁇ L, and cultured with shaking at 30 ° C. overnight using a 96-well deep well plate.
- Blocking was performed with 20 mM Tris-HCl buffer (pH 7.4) containing SKIM MILK (BD) of% (w / v) and 150 mM sodium chloride. (4-2) Fc for evaluating antibody binding activity after washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w / v] Tween 20, 150 mM NaCl) A solution containing a binding protein was added to react the Fc binding protein with the immobilized gamma globulin (1 hour at 30 ° C.).
- Table 4 summarizes the amino acid substitution position and the remaining activity (%) after heat treatment of the Fc binding protein expressed by the transformant selected in (5) with respect to the wild type (without amino acid substitution) Fc binding protein.
- the amino acid residues from the 17th glycine to the 192nd glutamine and the 17th to 192nd amino acid residues are Met18Arg (this notation is SEQ ID NO: 1 represents that 18th methionine of 1 is substituted with arginine, the same applies below), Val27Glu, Phe29Leu, Phe29Ser, Leu30Gln, Tyr35Asn, Tyr35Asp, Tyr35Sr, Tyr35His, Lys46Thr, Lys46Thr, Lys46Thr, , Glu54Asp, Glu54Gly, Asn56Thr, Gln59Arg, Phe61Tyr, Glu64Asp, Ser6 Arg, Ala71Asp, Phe75Le
- FcR2 the Fc-binding protein with the highest residual activity of Val27Glu and Tyr35Asn in which the amino acid substitution occurred was named FcR2 and includes a polynucleotide encoding FcR2.
- the expression vector was named pET-FcR2.
- the amino acid sequence of FcR2 is shown in SEQ ID NO: 27, and the sequence of the polynucleotide encoding FcR2 is shown in SEQ ID NO: 28.
- the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
- the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- the amino acid sequence of FcR2 corresponding to the 17th to 192nd region of SEQ ID NO: 1
- the 209th to 210th glycine (Gly) Is a linker sequence
- histidines (His) from 211 to 216 are tag sequences.
- glutamic acid of Val27Glu is present at the 43rd position
- asparagine of Tyr35Asn is present at the 51st position.
- Example 4 Production of Amino Acid-Substituted Fc Binding Protein The stability was further improved by accumulating amino acid substitutions that were found in Example 3 and involved in improving the thermal stability of the Fc binding protein. Accumulation of substituted amino acids was mainly performed using PCR, and seven types of Fc binding proteins shown in (a) to (g) below were prepared.
- B FcR4 obtained by further performing amino acid substitution of Phe75Leu and Glu121Gly on FcR2.
- FcR3 Val27Glu, Tyr35Asn and Phe75Leu were selected from the amino acid substitutions involved in the improvement of thermal stability, which were revealed in Example 3, and these substitutions were accumulated in the wild-type Fc-binding protein.
- FcR3 was produced by introducing a mutation causing Phe75Leu into a polynucleotide encoding FcR2.
- A-1) PCR was performed using pET-FcR2 obtained in Example 3 as a template.
- primers for the PCR oligonucleotides having the sequences described in SEQ ID NO: 24 and SEQ ID NO: 29 (5′-AGCCAGGCGAGCAGCTACCCTTTATTGATGCG-3 ′) were used.
- PCR In PCR, after preparing a reaction solution having the composition shown in Table 5, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. 30 cycles of the reaction in which the third step for 1 minute was set to 1 cycle were performed by heat treatment at 72 ° C. for 7 minutes.
- the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m3F.
- A-2) Except that pET-FcR2 obtained in Example 3 was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 30 (5′-CCACCGTCGCCCGCATCAATAAGGTTAGCTGC-3 ′) was used as a PCR primer. , (A-1).
- the purified PCR product was designated as m3R.
- A-3) Two types of PCR products (m3F and m3R) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed, and a PCR product m3p in which m3F and m3R were linked was obtained.
- (A-4) PCR was performed using the PCR product m3p obtained in (a-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
- the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
- the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
- a polynucleotide encoding FcR3 in which an amino acid substitution at one site was introduced into FcR2 was prepared.
- the amino acid sequence of FcR3 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 31, and the sequence of the polynucleotide encoding the FcR3 is shown in SEQ ID NO: 32.
- the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
- the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- the amino acid sequence of FcR3 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences.
- glutamic acid of Val27Glu is present at the 43rd position, asparagine of Tyr35Asn is present at the 51st position, and leucine of Phe75Leu is present at the 91st position.
- FcR4 Val27Glu, Tyr35Asn, Phe75Leu, and Glu121Gly were selected from the amino acid substitutions involved in improving the stability of the Fc-binding protein revealed in Example 3, and these substitutions were determined as wild-type Fc binding properties.
- FcR4 accumulated in protein was produced. Specifically, FcR4 was produced by introducing a mutation that causes Phe75Leu and Glu121Gly to the polynucleotide encoding FcR2.
- B-1) A PCR product m3F was obtained in the same manner as (a-1).
- oligonucleotide comprising the sequences described in SEQ ID NO: 24 and SEQ ID NO: 29, which was obtained in Example 3, using the plasmid expressing Fc-binding protein (Table 4) containing amino acid substitutions of Ala71Asp, Phe75Leu and Glu121Gly as a template.
- PCR product m4R was obtained by PCR in the same manner as in (a-1) using as a PCR primer.
- B-2) After mixing the two types of PCR products (m3F, m4R) obtained in (b-1), PCR was performed in the same manner as in (a-3) to link m3F and m4R.
- the obtained PCR product was designated as m4p.
- (B-3) A method similar to (a-4), using the PCR product m4p obtained in (b-2) as a template and an oligonucleotide comprising the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR4.
- (B-4) After purification of the polynucleotide obtained in (b-3), digestion with restriction enzymes NcoI and HindIII and expression vector pETmalE previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli E. coli. E. coli BL21 (DE3) strain was transformed.
- the amino acid sequence of FcR4 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 33, and the sequence of a polynucleotide encoding the FcR4 is shown in SEQ ID NO: 34.
- SEQ ID NO: 33 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- FcR5a Val27Glu, Tyr35Asn, Phe75Leu, Asn92Ser, and Glu121Gly were selected from among the amino acid substitutions involved in improving the stability of the Fc-binding protein revealed in Example 3, and these substitutions were selected as wild-type Fc.
- FcR5a accumulated in the binding protein was produced. Specifically, FcR5a was prepared by introducing a mutation that caused Asn92Ser to the polynucleotide encoding FcR4 prepared in (b).
- C-2 except that pET-FcR4 prepared in (b) was used as a template, and an oligonucleotide having the sequence described in SEQ ID NO: 21 and SEQ ID NO: 36 (5′-GATCGCTCCAGGGTGCTCAGGCTGGTTCTGGC-3 ′) was used as a PCR primer, PCR was performed in the same manner as (a-1). The purified PCR product was designated as m5aR.
- C-3 After mixing the two kinds of PCR products (m5aF, m5aR) obtained in (c-1) and (c-2), PCR was performed in the same manner as in (a-3), and m5aF And m5aR were ligated.
- the obtained PCR product was designated as m5ap.
- C-4 A method similar to (a-4), using the PCR product m5ap obtained in (c-3) as a template and an oligonucleotide comprising the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR5a.
- C-5 After purifying the polynucleotide obtained in (c-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli E. coli. E.
- coli BL21 (DE3) strain was transformed.
- C-6 The obtained transformant was cultured in LB medium supplemented with 50 ⁇ g / mL kanamycin.
- C-7) The nucleotide sequence of pET-FcR5a was analyzed in the same manner as in Example 1 (5).
- the amino acid sequence of FcR5a added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 37, and the sequence of the polynucleotide encoding the FcR5a is shown in SEQ ID NO: 38.
- SEQ ID NO: 37 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- FcR5b Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, and Glu121Gly were selected from the amino acid substitutions involved in improving the stability of the Fc-binding protein revealed in Example 3, and these substitutions were selected as wild-type Fc.
- FcR5b accumulated in the binding protein was produced. Specifically, FcR5b was prepared by introducing a mutation that produces Glu54Asp into the polynucleotide encoding FcR4 prepared in (b).
- (D-2) Except that pET-FcR4 prepared in (b) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 21 and SEQ ID NO: 40 (5′-CACTGGGGTCGTTTATCATCCGGGCTATAC-3 ′) was used as a PCR primer, PCR was performed in the same manner as (a-1). The purified PCR product was designated as m5bR. (D-3) After mixing the two types of PCR products (m5bF, m5bR) obtained in (d-1) and (d-2), PCR was performed in the same manner as in (a-3), and m5bF and m5bR was ligated.
- the obtained PCR product was designated as m5 bp.
- D-4 A method similar to (a-4), using the PCR product m5bp obtained in (d-3) as a template and an oligonucleotide consisting of the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR5b.
- D-5 After purifying the polynucleotide obtained in (d-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli E. coli.
- E. coli BL21 (DE3) strain was transformed.
- D-6 The obtained transformant was cultured in an LB medium supplemented with 50 ⁇ g / mL kanamycin.
- a plasmid pET-FcR5b containing a polynucleotide encoding FcR5b which is a polypeptide obtained by substituting amino acids at five positions with respect to the wild-type Fc-binding protein, is obtained. It was.
- D--7 The nucleotide sequence of pET-FcR5b was analyzed in the same manner as in Example 1 (5).
- the amino acid sequence of FcR5b added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 41, and the sequence of the polynucleotide encoding the FcR5b is shown in SEQ ID NO: 42.
- SEQ ID NO: 41 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- FcR6a From the amino acid substitutions involved in improving the stability of the Fc-binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Asn92Ser and Glu121Gly are selected, and these substitutions are wild-type. FcR6a accumulated in the Fc-binding protein was prepared. Specifically, FcR6a was prepared by introducing a mutation that produces Glu54Asp into the polynucleotide encoding FcR5a prepared in (c).
- E-1 The same method as (a-1), except that pET-FcR5a prepared in (c) was used as a template, and an oligonucleotide consisting of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 39 was used as a PCR primer. PCR was performed. The purified PCR product was designated as m6aF.
- E-2 The same method as (a-1) except that pET-FcR4 prepared in (b) was used as a template, and an oligonucleotide consisting of the sequences of SEQ ID NO: 21 and SEQ ID NO: 40 was used as a PCR primer. PCR was performed. The purified PCR product was designated as m6aR.
- E-3) After mixing the two kinds of PCR products (m6aF, m6aR) obtained in (e-1) and (e-2), PCR was performed in the same manner as in (a-3), and m6aF and m6aR was ligated. The obtained PCR product was designated as m6ap.
- E-4) A method similar to (a-4), using the PCR product m6ap obtained in (e-3) as a template and an oligonucleotide comprising the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR6a.
- E-5 After purifying the polynucleotide obtained in (e-4), digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E. coli E. coli.
- E. coli BL21 (DE3) strain was transformed.
- E-6 The obtained transformant was cultured in an LB medium supplemented with 50 ⁇ g / mL kanamycin.
- the amino acid sequence of FcR6a to which a signal sequence and a polyhistidine tag are added is shown in SEQ ID NO: 43, and the sequence of the polynucleotide encoding the FcR6a is shown in SEQ ID NO: 44.
- the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
- the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- FcR6a There is an amino acid sequence of FcR6a from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the 17th to 192nd region of SEQ ID NO: 1), and the 209th to 210th glycine (Gly). ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence.
- glutamic acid of Val27Glu is position 43
- asparagine of Tyr35Asn is position 51
- aspartic acid of Glu54Asp is position 70
- leucine of Phe75Leu is position 91
- serine of Asn92Ser is position 108
- glycine of Glu121Gly is position 137.
- FcR6b From the amino acid substitutions involved in improving the stability of the Fc-binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Glu120Val and Glu121Gly are selected, and these substitutions are wild-type. FcR6b accumulated in the Fc-binding protein was prepared. Specifically, FcR6b was prepared by introducing a mutation that produces Glu120Val into the polynucleotide encoding FcR5b prepared in (d).
- the obtained PCR product was set to m6 bp.
- F-4 (a-4) except that the PCR product m6bp obtained in (f-3) was used as a template, and an oligonucleotide consisting of the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 was used as a PCR primer. PCR was performed in the same manner. This produced a polynucleotide encoding FcR6b.
- F-5 After purifying the polynucleotide obtained in (f-4), digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) And ligated to E.
- the amino acid sequence of FcR6b added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 47, and the sequence of the polynucleotide encoding the FcR6b is shown in SEQ ID NO: 48.
- SEQ ID NO: 47 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- FcR6b There is an amino acid sequence of FcR6b from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence.
- glutamic acid of Val27Glu is position 43
- aspartic acid of Tyr35Asn is position 51
- aspartic acid of Glu54Asp is position 70
- leucine of Phe75Leu is position 91
- valine of Glu120Val is position 136
- glycine of Glu121Gly is position 137.
- FcR7 From the amino acid substitutions involved in improving the stability of the Fc-binding protein revealed in Example 3, Val27Glu, Tyr35Asn, Glu54Asp, Phe75Leu, Asn92Ser, Glu120Val and Glu121Gly are selected and the substitutions are performed. FcR7 accumulated in the wild type Fc binding protein was produced. Specifically, FcR7 was prepared by introducing a mutation that produces Glu120Val into the polynucleotide encoding FcR6a prepared in (e).
- (G-1) The same method as (a-1) except that pET-FcR6a prepared in (e) was used as a template, and an oligonucleotide having the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 45 was used as a PCR primer. PCR was performed. The purified PCR product was designated as m7F.
- (G-2) The same method as (a-1) except that pET-FcR6a prepared in (e) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 21 and SEQ ID NO: 46 was used as a PCR primer. PCR was performed. The purified PCR product was designated as m7R.
- the amino acid sequence of FcR7 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 49, and the sequence of the polynucleotide encoding the FcR7 is shown in SEQ ID NO: 50.
- SEQ ID NO: 49 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
- glutamic acid of Val27Glu is 43rd
- aspartic acid of Tyr35Asn is 51st
- aspartic acid of Glu54Asp is 70th
- leucine of Phe75Leu is 91st
- serine of Asn92Ser is 108th
- valine of Glu120Val is 136th
- Glu121Gly Glycine exists at the 137th position.
- Example 5 Thermal Stability Evaluation of Improved Fc Binding Protein
- Wild-type Fc binding protein prepared in Example 1, mutant Fc binding protein selected in Example 3 (FcR2), and Example 4 Transformants expressing the mutant Fc-binding proteins (FcR3, FcR4, FcR5a, FcR5b, FcR6a, FcR6b, FcR7) prepared in (1) above were inoculated into 3 mL of 2YT liquid medium each containing 50 ⁇ g / mL kanamycin, Pre-culture was performed by aerobically shaking culture at 37 ° C. overnight.
- the antibody binding activity of the wild-type Fc-binding protein and the mutant Fc-binding protein in the protein extract prepared in (4) was measured using the ELISA method described in Example 3 (4). .
- a calibration curve was prepared using the extracellular region of commercially available Fc ⁇ RIIIa (R & D Systems: 4325-FC-050), and the concentration was measured.
- the protein was diluted with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride so that the concentration of each protein was 5 ⁇ g / mL. This was divided into equal amounts, and one was heat-treated at 45 ° C. for 10 minutes using a thermal cycler (Eppendorf), and the other was not heat-treated.
- the antibody binding activity of the protein after the heat treatment or non-heat treatment was measured by the ELISA method described in Example 3 (4), and the antibody binding activity when the heat treatment was performed was the antibody binding activity when the heat treatment was not performed.
- the residual activity was calculated by dividing by.
- the results are shown in Table 8.
- the mutant Fc-binding proteins evaluated this time (FcR2, FcR3, FcR4, FcR5a, FcR5b, FcR6a, FcR6b, FcR7) have higher residual activity compared to the wild-type Fc-binding protein, and the mutant type It was confirmed that the thermal stability of the Fc-binding protein was improved.
- Example 6 Evaluation of Acid Stability of Improved Fc Binding Protein
- Improved Fc binding protein was prepared in the same manner as in Examples 5 (1) to (5).
- the protein was diluted with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride so that the concentration of each protein was 30 ⁇ g / mL.
- 60 ⁇ L of each diluted Fc-binding protein and 120 ⁇ L of 0.1 M glycine hydrochloride buffer (pH 3.0) were mixed and allowed to stand at 30 ° C. for 2 hours.
- the results are shown in Table 9.
- the mutant Fc-binding proteins evaluated this time (FcR2, FcR3, FcR4, FcR5a, FcR5b, FcR6a, FcR6b, FcR7) have higher residual activity compared to the wild-type Fc-binding protein, and the mutant type It was confirmed that the acid stability of the Fc-binding protein of 1 was improved.
- Example 7 Preparation of Fc-binding protein with amino acid substitution at one position Among the amino acid substitutions involved in improving the stability of the Fc-binding protein revealed in Example 3, the 27th valine (Val) of SEQ ID NO: 1, Regarding the 35th tyrosine (Tyr) and the 121st glutamic acid (Glu), Fc binding proteins substituted with other amino acids were prepared by the following methods, respectively.
- PCR was performed in the same manner as in Example 4 (a-1).
- the purified PCR product was 27 pR.
- A-3) After mixing the two PCR products (27pF, 27pR) obtained in (A-1) and (A-2), PCR was performed in the same manner as in Example 4 (a-3). 27pF and 27pR were ligated. The obtained PCR product was designated as 27p.
- Example 4 (a-4) and the PCR product 27p obtained in (A-3) as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed in the same manner.
- a polynucleotide encoding an Fc-binding protein in which the 27th valine of SEQ ID NO: 1 was substituted with an arbitrary amino acid was prepared.
- A-5) After purifying the polynucleotide obtained in (A-4), it is digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No.
- E. coli BL21 (DE3) strain was transformed.
- A-6 The obtained transformant was cultured in an LB medium supplemented with 50 ⁇ g / mL kanamycin.
- a plasmid was extracted from the collected microbial cells (transformants), and nucleotide sequence analysis was performed in the same manner as in Example 1 (5).
- PCR was performed in the same manner as in Example 4 (a-1).
- the purified PCR product was 35 pR.
- B-3 After mixing the two types of PCR products (35pF, 35pR) obtained in (B-1) and (B-2), PCR was performed in the same manner as in Example 4 (a-3). And 35 pF and 35 pR were ligated. The obtained PCR product was 35p.
- E. coli BL21 (DE3) strain was transformed.
- B-6 The obtained transformant was cultured in an LB medium supplemented with 50 ⁇ g / mL kanamycin.
- a plasmid was extracted from the collected microbial cells (transformants), and nucleotide sequence analysis was performed in the same manner as in Example 1 (5).
- Tyr35Cys (Y35C), Tyr35Asp (Y35D), Tyr35Phe (Y35F), Tyr35Gly (Y35G), Tyr35Lys (Y35K), Tyr35Leu (Y35L), Tyr35Asn (Y35N), Tyr35Tr35Yr, YrN ), Tyr35Thr (Y35T), Tyr35Val (Y35V), or Tyr35Trp (Y35W), a polynucleotide encoding an Fc-binding protein in which an amino acid substitution occurred was obtained.
- Example 2 Except that pET-eFcR prepared in Example 1 was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 56 (5′-AATCGGATCMNNCTCTTTGAACACCCCACCG-3 ′) was used as a PCR primer, PCR was performed in the same manner as in Example 4 (a-1). The purified PCR product was 121 pR. (C-3) After mixing the two kinds of PCR products (121pF, 121pR) obtained by (C-1) and (C-2), PCR was performed in the same manner as in Example 4 (a-3). In practice, 121pF and 121pR were ligated.
- the obtained PCR product was designated as 121p.
- C-4 The same as in Example 4 (a-4), using the PCR product 121p obtained in (C-3) as a template and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed.
- a polynucleotide encoding an Fc binding protein in which the 121st glutamic acid of SEQ ID NO: 1 was substituted with an arbitrary amino acid was prepared.
- Example 8 Evaluation of Antibody Binding Activity of 1 Amino Acid-Substituted Fc Binding Protein (1) Expression of wild-type Fc binding protein prepared in Example 1 and Fc binding protein substituted with one amino acid prepared in Example 7 The transformant was cultured in the same manner as in Example 3 (1) and (2), and wild type Fc binding protein and Fc binding protein substituted with 1 amino acid were expressed. (2) The binding activity of the expressed Fc binding protein substituted with one amino acid to the antibody was examined by the ELISA method described in Example 3 (4).
- the 35th tyrosine of SEQ ID NO: 1 is converted to aspartic acid (Y35D), phenylalanine (Y35F), glycine (Y35G), lysine (Y35K), leucine (Y35L), asparagine (Y35N), proline (Y35P), serine (Y35S).
- Y35D aspartic acid
- Y35F phenylalanine
- Y35G glycine
- Y35K lysine
- leucine Y35L
- asparagine Y35N
- proline Y35P
- serine Y35S
- Y35D, Y35G, Y35K, Y35L, Y35N, Y35P, Y35S, Y35T, and Y35W have significantly improved antibody binding activity compared to the wild-type Fc-binding protein.
- the 35th tyrosine of SEQ ID NO: 1 was substituted with cysteine (Y35C) or arginine (Y35R), the antibody binding activity was almost equivalent to that of the wild type Fc binding protein.
- E121G By replacing the 121st glutamic acid of SEQ ID NO: 1 with lysine (E121K), arginine (E121R), glycine (E121G), and histidine (E121H), the antibody binding activity was improved compared to the wild-type Fc-binding protein. . Among them, E121G showed significantly improved antibody binding activity as compared with wild-type Fc binding protein. On the other hand, when the glutamic acid at position 121 of SEQ ID NO: 1 was substituted with valine (E121V), the antibody-binding activity was almost equivalent to that of wild-type Fc binding protein, and when substituted with proline (E121P), wild-type Fc binding Antibody binding activity was reduced compared to the sex protein.
- E121K lysine
- E121R arginine
- E121G glycine
- E121H histidine
- Example 9 Thermal Stability Evaluation of 1 Amino Acid Substituted Fc Binding Protein
- heat treatment was performed in the same manner as in Example 3 (3). (45 ° C., 10 minutes), and the residual activity was calculated.
- the 35th tyrosine of SEQ ID NO: 1 is aspartic acid (Y35D), glycine (Y35G), lysine (Y35K), leucine (Y35L), asparagine (Y35N), proline (Y35P), serine (Y35S), threonine (Y35T).
- the Fc binding protein in which the 121st glutamic acid of SEQ ID NO: 1 was replaced with glycine (E121G) were significantly improved in thermal stability as compared to the wild type Fc binding protein. .
- Y35N and Y35P have greatly improved thermal stability compared to the wild type Fc binding protein.
- Example 10 Large-scale preparation of FcR5a (1) A transformant expressing FcR5a prepared in Example 4 (c) was added to 400 mL of 2YT liquid medium containing 50 ⁇ g / mL kanamycin (2 g of peptone 16 g / mL) in a 2 L baffle flask. L, yeast extract 10 g / L, sodium chloride 5 g / L), and precultured by aerobic shaking culture at 37 ° C. overnight.
- Glucose 10 g / L, yeast extract 20 g / L, trisodium phosphate dodecahydrate 3 g / L, disodium hydrogen phosphate dodecahydrate 9 g / L, ammonium chloride 1 g / L and kanamycin sulfate 50 mg 180 L of the culture medium (1) was inoculated into 1.8 L of a liquid medium containing / L, and main culture was performed using a 3 L fermenter (manufactured by Biot). The main culture was started under the conditions of a temperature of 30 ° C., a pH of 6.9 to 7.1, an aeration rate of 1 VVM, and a dissolved oxygen concentration of 30% saturation.
- the pH was controlled by using 50% phosphoric acid as the acid and 14% ammonia water as the alkali.
- the dissolved oxygen was controlled by changing the stirring speed, and the stirring speed was set at the lower limit of 500 rpm and the upper limit of 1000 rpm. .
- fed-batch medium glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L
- DO dissolved oxygen
- the cells were crushed with an output of about 150 W at 4 ° C. for about 10 minutes using an apparatus (Insonator 201M (trade name), manufactured by Kubota Corporation).
- the cell disruption solution was centrifuged twice at 10,000 rpm for 20 minutes at 4 ° C., and the supernatant was collected.
- (6) After adding imidazole to the disrupted solution obtained in (5) to a final concentration of 20 mM, equilibrate with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole in advance.
- XK 26/20 column GE Healthcare
- Ni Sepharose 6 Fast Flow GE Healthcare
- FcR5a was eluted using 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 0.5 M imidazole.
- FcR5a was eluted with 0.1 M glycine hydrochloride buffer (pH 3.0). The eluate was brought back to near neutrality by adding 1/4 volume of 1M Tris-HCl buffer (pH 7.0).
- Example 11 Preparation of FcR5a-immobilized gel
- FcR5a prepared in Example 10 was concentrated and buffer exchanged using an ultrafiltration membrane (Millipore: Amicon Ultra-15) to obtain 150 mM sodium chloride. The solution was concentrated to a concentration of 8.37 mg / mL in 20 mM Tris-HCl buffer (pH 7.4).
- An epoxy toyopearl gel was prepared by reacting 1,6-hexanediol diglycidyl ether with the hydroxyl group of a hydrophilic vinyl polymer (Tosoh Corporation: Toyopearl) as a carrier.
- a hydrophilic vinyl polymer Tosoh Corporation: Toyopearl
- the concentration of the protein contained in the solution collected in (5) and the washing solution was measured, and the amount of FcR5a immobilized on the gel was calculated to calculate the immobilization rate. As a result, 33.7% of the added FcR5a was obtained. Was immobilized on the gel.
- Example 12 Separation of antibody using FcR5a-immobilized gel
- 0.5 mL of FcR5a-immobilized gel prepared in Example 11 was packed in an HR16 / 5 column (manufactured by GE Healthcare) and AKTAprime plus (GE Healthcare) Made). Thereafter, equilibration was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.
- 20 mM Tris-HCl buffer solution (pH 7.4) containing 150 mM sodium chloride was flowed at a flow rate of 0.1 mL, and 1 mg with a 20 mM Tris-HCl buffer solution (pH 7.4) containing 150 mM sodium chloride.
- fraction A fraction 13
- fraction B fraction B
- Example 13 Sugar chain structure analysis of isolated antibody (1) The human IgG1 and elution fractions (FrA, FrB) before purification used in Example 12 were denatured by heat treatment at 100 ° C. for 10 minutes, and then glycoamidase A / pepsin and After sequentially treating with pronase, a sugar chain fraction was obtained through purification by gel filtration. (2) After concentrating and drying the sugar chain obtained in (1) with an evaporator, 2-aminopyridine and then dimethylamine borane are successively acted on in an acetic acid solvent to form a fluorescent labeled sugar chain. Purified.
- the fluorescence-labeled sugar chain obtained in (2) is monosialylated with a neutral sugar chain fraction on an anion exchange column (TSKgel DEAE-5PW, ⁇ 7.5 mm ⁇ 7.5 cm: manufactured by Tosoh Corporation). Separated into sugar chain fractions.
- the neutral sugar chain fraction and monosialylated sugar chain fraction obtained in (3) were isolated into individual sugar chains using an ODS column. After obtaining the molecular weight information of the sugar chain isolated by MALDI-TOF-MS analysis, the sugar chain structure was assigned in comparison with the retention time of the ODS column chromatograph.
- Table 10 shows the composition ratio of neutral sugar chains
- Table 11 shows the composition ratio of monosialylated sugar chains.
- the assigned sugar chain structures (N1 to N8 and M1 and M2) are shown in FIG. From the results shown in Table 10, antibodies having sugar chain structures N2 and N7 were detected before purification and with FrB, but not with FrA. That is, the antibodies having the two sugar chain structures (N2 and N7 in FIG. 5) were not detected in FrA, which was a fraction that eluted earlier, but were detected in FrB, which was a fraction that was eluted later. It was shown to bind strongly to FcR5a-immobilized gel as compared with the antibody having the sugar chain structure of. From the above results, it was found that the FcR5a-immobilized gel, which is one embodiment of the adsorbent of the present invention, can separate antibodies by the difference in sugar chain structure of the antibodies.
- Example 14 Antibody Purification with FcR5a Immobilized Gel Human IgG1 and human IgG3 were purified using the FcR5a immobilized gel prepared in Example 11.
- FIG. 6 shows the results of SDS-PAGE analysis of elution fractions containing human IgG1 or human IgG3.
- the elution fraction containing human IgG1 obtained by purifying the simulated culture solution added with human IgG1 shows the same position as human IgG1 added to the simulated culture solution, and the albumin found in the simulated culture solution Since no band is seen, it can be confirmed that human IgG1 is purified with high purity (FIG. 6A).
- the elution fraction containing human IgG3 obtained by purifying the simulated culture solution added with human IgG3 also shows the same position as human IgG3 added to the simulated culture solution, and is found in the simulated culture solution.
- Example 15 Preparation of sugar chain-removed human IgG1 N-type sugar chains were removed from human IgG1 by the method shown below.
- (1) It was diluted with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride so that the concentration of human IgG1 (Fitzgerald: 31-AI17) was 3 mg / mL.
- (2) 100 ⁇ L of 1 M Tris-HCl buffer (pH 8.6) was added to 100 ⁇ L of the diluted solution of (1), 10 ⁇ L of N-glycosidase F (500 mU / ⁇ L (Takara Bio Inc .: 4450) was added, and then 37 ° C.
- the N-type sugar chain of IgG1 was removed by allowing to stand for 24 hours.
- Human IgG1 from which the type sugar chain was removed (hereinafter simply referred to as human IgG1 from which the sugar chain was removed) was purified. The eluate was neutralized by adding 1/4 volume of 1M Tris-HCl buffer (pH 8.0). (5) Equal amounts of sample buffer (2 (w / v)% sodium dodecyl sulfate, 6 (w / v)% ⁇ -mercaptoethanol, (W / v)% glycerin and 0.005 (w / v)% bromophenol blue-containing 50 mM Tris-HCl buffer (pH 6.8)) and heat-treated, thereby removing human IgG1 from which sugar chains were removed. Reduced.
- sample buffer (2 (w / v)% sodium dodecyl sulfate, 6 (w / v)% ⁇ -mercaptoethanol, (W / v)% glycerin and 0.005 (w / v)% bromophenol blue
- Human IgG1 was separated by electrophoresis using a 5 to 20% gradient SDS-PAGE gel (Ato). For comparison, an aqueous solution (concentration: 0.5 mg / mL) of human IgG1 that has not been subjected to a sugar chain treatment (hereinafter referred to as human IgG1 with a sugar chain) was subjected to the same reduction treatment as described in (5), and SDS- Separated by PAGE.
- Example 16 Large-scale preparation of human Fc ⁇ RIIIa (1) A transformant capable of expressing human Fc ⁇ RIIIa obtained in Example 1 was added to a 400 mL 2YT liquid medium containing 50 ⁇ g / mL kanamycin (16 g of peptone) in a 2 L baffle flask. / L, yeast extract 10 g / L, sodium chloride 5 g / L), and precultured by aerobic shaking culture at 37 ° C. overnight.
- the pH was controlled by using 50% phosphoric acid as the acid and 14% ammonia water as the alkali.
- the dissolved oxygen was controlled by changing the stirring speed, and the stirring speed was set at the lower limit of 500 rpm and the upper limit of 1000 rpm. .
- fed-batch medium glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L
- DO dissolved oxygen
- the cells were crushed with a power of about 150 W at 4 ° C. for about 10 minutes using a netter 201M (trade name, manufactured by Kubota Corporation).
- the cell disruption solution was centrifuged twice at 10,000 rpm for 20 minutes at 4 ° C., and the supernatant was collected.
- (6) After adding imidazole to the disrupted solution obtained in (5) to a final concentration of 20 mM, equilibrate with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole in advance.
- XK 26/20 column GE Healthcare
- Ni Sepharose 6 Fast Flow GE Healthcare
- human Fc ⁇ RIIIa was eluted using 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 0.5 M imidazole.
- human Fc ⁇ RIIIa was eluted with 0.1 M glycine hydrochloride buffer (pH 3.0). The eluate was returned to near neutrality by adding 1/4 volume of 1M Tris-HCl buffer (pH 8.0).
- Example 17 Measurement of binding of human Fc ⁇ RIIIa to antibody (1)
- Human Fc ⁇ RIIIa prepared in Example 16 was treated with phosphate buffer (137 mM NaCl, 8.1 M Na 2 HPO 4, 2.68 mM KCl and 1.47 mM KH 2 PO 4.
- the buffer solution was exchanged by dialysis against a buffer solution (pH 7.4 containing pH 7.4), and the concentration of human Fc ⁇ RIIIa was measured from the absorbance at 280 nm.
- protein A manufactured by Protenova
- CM5 manufactured by GE Healthcare
- Example 18 Preparation of human Fc ⁇ RIIIa-immobilized gel
- Human Fc ⁇ RIIIa prepared in Example 16 was concentrated and buffer exchanged using an ultrafiltration membrane (Millipore: Amicon Ultra-15). The solution was concentrated to a concentration of 2.6 mg / mL in 20 mM Tris-HCl buffer (pH 7.4) containing sodium chloride.
- An epoxy toyopearl gel was prepared by reacting 1,6-hexanediol diglycidyl ether with the hydroxyl group of a hydrophilic vinyl polymer (Tosoh Corporation: Toyopearl) as a carrier.
- Example 19 Antibody Separation Using Human Fc ⁇ RIIIa Immobilized Gel
- the human Fc ⁇ RIIIa immobilized gel prepared in Example 18 was packed into an HR16 / 5 column (manufactured by GE Healthcare), and the column was subjected to liquid chromatography. It connected to AKTAprime (made by GE Healthcare).
- (2) The column prepared in (1) was equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, and prepared with human IgG1 (Fitzgerald: 31-AI17) or Example 2.
- 0.1 mL of the sugar chain-removed human IgG1 (the solution concentration was 1 mg / mL) was added at a flow rate of 0.1 mL / min. After washing with the buffer used for equilibration, elution was performed with 0.1 M glycine hydrochloride buffer (pH 3.5).
- the adsorbent obtained by immobilizing human Fc ⁇ RIIIa on an insoluble carrier has the ability to specifically adsorb an antibody having a sugar chain, and this ability can be used to add a sugar chain to the antibody. It can be seen that the presence or absence can be identified.
- the adsorbent of the present invention specifically adsorbs an antibody having a sugar chain, the antibody having a sugar chain can be separated and purified with high purity. Therefore, the adsorbent of the present invention can be used for antibody drug production and quality control.
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Abstract
Description
(A)配列番号1に記載のアミノ酸配列のうち17番目から192番目までのアミノ酸残基を含み、かつ当該17番目から192番目までのアミノ酸残基において以下の(1)から(40)のうち少なくともいずれか1つのアミノ酸置換が生じている、Fc結合性タンパク質。
(1)配列番号1の18番目のメチオニンがアルギニンに置換
(2)配列番号1の27番目のバリンがグルタミン酸に置換
(3)配列番号1の29番目のフェニルアラニンがロイシンまたはセリンに置換
(4)配列番号1の30番目のロイシンがグルタミンに置換
(5)配列番号1の35番目のチロシンがアスパラギン酸、グリシン、リジン、ロイシン、アスパラギン、プロリン、セリン、スレオニン、ヒスチジンのいずれかに置換
(6)配列番号1の46番目のリジンがイソロイシンまたはスレオニンに置換
(7)配列番号1の48番目のグルタミンがヒスチジンまたはロイシンに置換
(8)配列番号1の50番目のアラニンがヒスチジンに置換
(9)配列番号1の51番目のチロシンがアスパラギン酸またはヒスチジンに置換
(10)配列番号1の54番目のグルタミン酸がアスパラギン酸またはグリシンに置換
(11)配列番号1の56番目のアスパラギンがスレオニンに置換
(12)配列番号1の59番目のグルタミンがアルギニンに置換
(13)配列番号1の61番目のフェニルアラニンがチロシンに置換
(14)配列番号1の64番目のグルタミン酸がアスパラギン酸に置換
(15)配列番号1の65番目のセリンがアルギニンに置換
(16)配列番号1の71番目のアラニンがアスパラギン酸に置換
(17)配列番号1の75番目のフェニルアラニンがロイシン、セリン、チロシンのいずれかに置換
(18)配列番号1の77番目のアスパラギン酸がアスパラギンに置換
(19)配列番号1の78番目のアラニンがセリンに置換
(20)配列番号1の82番目のアスパラギン酸がグルタミン酸またはバリンに置換
(21)配列番号1の90番目のグルタミンがアルギニンに置換
(22)配列番号1の92番目のアスパラギンがセリンに置換
(23)配列番号1の93番目のロイシンがアルギニンまたはメチオニンに置換
(24)配列番号1の95番目のスレオニンがアラニンまたはセリンに置換
(25)配列番号1の110番目のロイシンがグルタミンに置換
(26)配列番号1の115番目のアルギニンがグルタミンに置換
(27)配列番号1の116番目のトリプトファンがロイシンに置換
(28)配列番号1の118番目のフェニルアラニンがチロシンに置換
(29)配列番号1の119番目のリジンがグルタミン酸に置換
(30)配列番号1の120番目のグルタミン酸がバリンに置換
(31)配列番号1の121番目のグルタミン酸がアスパラギン酸またはグリシンに置換
(32)配列番号1の151番目のフェニルアラニンがセリンまたはチロシンに置換
(33)配列番号1の155番目のセリンがスレオニンに置換
(34)配列番号1の163番目のスレオニンがセリンに置換
(35)配列番号1の167番目のセリンがグリシンに置換
(36)配列番号1の169番目のセリンがグリシンに置換
(37)配列番号1の171番目のフェニルアラニンがチロシンに置換
(38)配列番号1の180番目のアスパラギンがリジン、セリン、イソロイシンのいずれかに置換
(39)配列番号1の185番目のスレオニンがセリンに置換
(40)配列番号1の192番目のグルタミンがリジンに置換
(41)配列番号1の66番目のロイシンがヒスチジンまたはアルギニンに置換
(42)配列番号1の147番目のグリシンがアスパラギン酸に置換
(43)配列番号1の158番目のチロシンがヒスチジンに置換
(44)配列番号1の176番目のバリンがフェニルアラニンに置換
(i)配列番号1の66番目のロイシンがヒスチジンまたはアルギニンに置換
(ii)配列番号1の147番目のグリシンがアスパラギン酸に置換
(iii)配列番号1の158番目のチロシンがヒスチジンに置換
(iv)配列番号1の176番目のバリンがフェニルアラニンに置換
(1)配列番号1に記載のヒトFcγRIIIaアミノ酸配列のうち、17番目のグリシン(Gly)から192番目のグルタミン(Gln)までのアミノ酸配列を基に、DNAworks法(Nucleic Acids Res.,30,e43,2002)を用いて、コドンをヒト型から大腸菌型に変換したヌクレオチド配列を設計した。設計したヌクレオチド配列を配列番号2に示す。
(2)配列番号2に記載の配列を含むポリヌクレオチドを作製するために、配列番号3から20に記載の配列からなるオリゴヌクレオチドを合成し、前記オリゴヌクレオチドを用いて、下記に示す二段階PCRを行なった。
(2-1)一段階目のPCRは、表1に示す組成の反応液を調製し、当該反応液を98℃で5分熱処理後、98℃で10秒間の第1ステップ、62℃で5秒間の第2ステップ、72℃で90秒間の第3ステップを1サイクルとする反応を10サイクル繰り返すことでポリヌクレオチドを合成し、これをFcRp1とした。なお表1中のDNAミックスとは、配列番号3から20に記載の配列からなる18種類のオリゴヌクレオチドをそれぞれ一定量サンプリングし混合した溶液を意味する。
(4)得られた形質転換体を50μg/mLのカナマイシンンを含むLB培地にて培養後、QIAprep Spin Miniprep kit(キアゲン社製)を用いて、発現ベクターpET-eFcRを抽出した。
(5)(4)で作製した発現ベクターpET-eFcRのうち、ヒトFcγRIIIaをコードするポリヌクレオチドおよびその周辺の領域について、チェーンターミネータ法に基づくBig Dye Terminator Cycle Sequencing ready Reaction kit(ライフテクノロジーズ社製)を用いてサイクルシークエンス反応に供し、全自動DNAシークエンサーABI Prism 3700 DNA analyzer(ライフテクノロジーズ社製)にてヌクレオチド配列を解析した。なお当該解析の際、配列番号23(5’-TAATACGACTCACTATAGGG-3’)または配列番号24(5’-TATGCTAGTTATTGCTCAG-3’)に記載の配列からなるオリゴヌクレオチドをシークエンス用プライマーとして使用した。
実施例1で作製したFc結合性タンパク質発現ベクターpER-eFcRのうち、Fc結合性タンパク質をコードするポリヌクレオチド部分に、エラープローンPCRによりランダムに変異導入を施した。
(1)鋳型として実施例1で作製したpET-eFcRを用いてエラープローンPCRを行なった。エラープローンPCRは、表3に示す組成の反応液を調製後、当該反応液を95℃で2分間熱処理し、95℃で30秒間の第1ステップ、60℃で30秒間の第2ステップ、72℃で90秒間の第3ステップを1サイクルとする反応を35サイクル行ない、最後に72℃で7分間熱処理することで行なった。前記エラープローンPCRによりFc結合性タンパク質をコードするポリヌクレオチドに良好に変異が導入され、その平均変異導入率は1.26%であった。
(3)ライゲーション反応終了後、反応液をエレクトロポレーション法により大腸菌BL21(DE3)株に導入し、50μg/mLのカナマイシンを含むLBプレート培地で培養(37℃で18時間)後、プレート上に形成したコロニーをランダム変異体ライブラリーとした。
(1)実施例2で作製したランダム変異体ライブラリー(形質転換体)を、50μg/mLのカナマイシンを含む2YT液体培地(ペプトン16g/L、酵母エキス10g/L、塩化ナトリウム5g/L)200μLに接種し、96穴ディープウェルプレートを用いて、30℃で一晩振とう培養した。
(2)培養後、5μLの培養液を500μLの0.05mMのIPTG(isopropyl-β-D-thiogalactopyranoside)、0.3%のグリシンおよび50μg/mLのカナマイシンを含む2YT液体培地に植え継ぎ、96穴ディープウェルプレートを用いて、さらに20℃で一晩振とう培養した。
(3)培養後、遠心操作によって得られた培養上清を150mMの塩化ナトリウムを含む20mMのトリス塩酸緩衝液(pH7.4)で2倍に希釈した。希釈した溶液を45℃で10分間熱処理を行なった。
(4)(3)の熱処理を行なったときのFc結合性タンパク質の抗体結合活性と、(3)の熱処理を行なわなかったときのFc結合性タンパク質の抗体結合活性を、それぞれ下記に示すELISA法にて測定し、熱処理を行なった時のFc結合性タンパク質の抗体結合活性を、熱処理を行なわなかったときのFc結合性タンパク質の抗体結合活性で除することで、残存活性を算出した。
(4-1)ヒト抗体であるガンマグロブリン製剤(化学及血清療法研究所製)を、96穴マイクロプレートのウェルに1μg/wellで固定化し(4℃で18時間)、固定化終了後、2%(w/v)のSKIM MILK(BD社製)および150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)によりブロッキングした。
(4-2)洗浄緩衝液(0.05%[w/v]のTween 20、150mMのNaClを含む20mM Tris-HCl緩衝液(pH7.4))で洗浄後、抗体結合活性を評価するFc結合性タンパク質を含む溶液を添加し、Fc結合性タンパク質と固定化ガンマグロブリンとを反応させた(30℃で1時間)。
(4-3)反応終了後、前記洗浄緩衝液で洗浄し、100ng/mLに希釈したAnti-6His抗体(Bethyl Laboratories社製)を100μL/wellで添加した。
(4-4)30℃で1時間反応させ、前記洗浄緩衝液で洗浄した後、TMB Peroxidase Substrate(KPL社製)を50μL/wellで添加した。1Mのリン酸を50μL/wellで添加することで発色を止め、マイクロプレートリーダー(テカン社製)にて450nmの吸光度を測定した。
(5)(4)の方法で約2700株の形質転換体を評価し、その中から野生型(アミノ酸置換のない)Fc結合性タンパク質と比較して熱安定性が向上したFc結合性タンパク質を発現する形質転換体を選択した。前記選択した形質転換体を培養し、QIAprep Spin Miniprep kit(キアゲン社製)を用いて発現ベクターを調製した。
(6)得られた発現ベクターに挿入されたFc結合性タンパク質をコードするポリヌクレオチド領域の配列を実施例1(5)の記載と同様の方法によりヌクレオチド配列を解析し、アミノ酸の変異箇所を特定した。
実施例3で判明した、Fc結合性タンパク質の熱安定性向上に関与するアミノ酸置換を集積することで、さらなる安定性向上を図った。置換アミノ酸の集積は、主にPCRを用いて行ない、以下の(a)から(g)に示す7種類のFc結合性タンパク質を作製した。
(a)FcR2に対し、さらにPhe75Leuのアミノ酸置換を行なったFcR3
(b)FcR2に対し、さらにPhe75LeuおよびGlu121Glyのアミノ酸置換を行なったFcR4
(c)FcR4に対し、さらにAsn92Serのアミノ酸置換を行なったFcR5a
(d)FcR4に対し、さらにGlu54Aspのアミノ酸置換を行なったFcR5b
(e)FcR5aに対し、さらにGlu54Aspのアミノ酸置換を行なったFcR6a
(f)FcR5bに対し、さらにGlu120Valのアミノ酸置換を行なったFcR6b
(g)FcR6aに対し、さらにGlu120Valのアミノ酸置換を行なったFcR7以下、各Fc結合性タンパク質の作製方法を詳細に説明する。
(a-1)実施例3で取得した、pET-FcR2を鋳型としてPCRを実施した。当該PCRにおけるプライマーは、配列番号24および配列番号29(5’-AGCCAGGCGAGCAGCTACCTTATTGATGCG-3’)に記載の配列からなるオリゴヌクレオチドを用いた。PCRは、表5に示す組成の反応液を調製後、当該反応液を98℃で5分間熱処理し、98℃で10秒間の第1ステップ、55℃で5秒間の第2ステップ、72℃で1分間の第3ステップを1サイクルとする反応を30サイクル行ない、最後に72℃で7分間熱処理することで行なった。増幅したPCR産物をアガロースゲル電気泳動に供し、そのゲルからQIAquick Gel Extraction kit(キアゲン社製)を用いて精製した。精製したPCR産物をm3Fとした。
(a-3)(a-1)および(a-2)で得られた2種類のPCR産物(m3F、m3R)を混合し、表6に示す組成の反応液を調製した。当該反応液を98℃で5分間熱処理後、98℃で10秒間の第1ステップ、55℃で5秒間の第2ステップ、72℃で1分間の第3ステップを1サイクルとする反応を5サイクル行なうPCRを行ない、m3Fとm3Rを連結したPCR産物m3pを得た。
(a-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出することで、野生型Fc結合性タンパク質に対して3箇所アミノ酸置換したポリペプチドである、FcR3をコードするポリヌクレオチドを含むプラスミドpET-FcR3を得た。
(a-7)pET-FcR3のヌクレオチド配列の解析を、実施例1(5)と同様の方法で行なった。
(b-1)(a-1)と同様の方法でPCR産物m3Fを得た。また実施例3で取得した、Ala71Asp、Phe75LeuおよびGlu121Glyのアミノ酸置換を含んだFc結合性タンパク質(表4)を発現するプラスミドを鋳型とし、配列番号24および配列番号29に記載の配列からなるオリゴヌクレオチドをPCRプライマーとして、(a-1)と同様の方法でPCRを行なうことでPCR産物m4Rを得た。
(b-2)(b-1)により得られた2種類のPCR産物(m3F、m4R)を混合後、(a-3)と同様の方法にてPCRを行ない、m3Fとm4Rを連結した。得られたPCR産物をm4pとした。
(b-3)(b-2)で得られたPCR産物m4pを鋳型とし、配列番号23および配列番号24に記載の配列からなるオリゴヌクレオチドをPCRプライマーとして、(a-4)と同様の方法でPCRを行なった。これによりFcR4をコードするポリヌクレオチドを作製した。
(b-4)(b-3)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、あらかじめ制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(b-5)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出することで、野生型Fc結合性タンパク質に対して4箇所アミノ酸置換したポリペプチドである、FcR4をコードするポリヌクレオチドを含むプラスミドpET-FcR4を得た。
(b-6)pET-FcR4のヌクレオチド配列の解析を、実施例1(5)と同様の方法で行なった。
(c-1)(b)で作製した、pET-FcR4を鋳型とし、配列番号22および配列番号35(5’-GAATATCGTTGCCAGACCAGCCTGAGCACC-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm5aFとした。
(c-2)(b)で作製したpET-FcR4を鋳型とし、配列番号21および配列番号36(5’-GATCGCTCAGGGTGCTCAGGCTGGTCTGGC-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm5aRとした。
(c-3)(c-1)および(c-2)で得られた2種類のPCR産物(m5aF、m5aR)を混合後、(a-3)と同様の方法にてPCRを行ない、m5aFとm5aRを連結した。得られたPCR産物をm5apとした。
(c-4)(c-3)で得られたPCR産物m5apを鋳型とし、配列番号21および配列番号22に記載の配列からなるオリゴヌクレオチドをPCRプライマーとして、(a-4)と同様の方法でPCRを行なった。これによりFcR5aをコードするポリヌクレオチドを作製した。
(c-5)(c-4)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、あらかじめ制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(c-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出することで、野生型Fc結合性タンパク質に対して5箇所アミノ酸置換したポリペプチドである、FcR5aをコードするポリヌクレオチドを含むプラスミドpET-FcR5aを得た。
(c-7)pET-FcR5aのヌクレオチド配列の解析を、実施例1(5)と同様の方法で行なった。
(d-1)(b)で作製した、pET-FcR4を鋳型とし、配列番号22および配列番号39(5’-CAGGGCGCGTATAGCCCGGATGATAACAGC-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm5bFとした。
(d-2)(b)で作製したpET-FcR4を鋳型とし、配列番号21および配列番号40(5’-CACTGGGTGCTGTTATCATCCGGGCTATAC-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm5bRとした。
(d-3)(d-1)および(d-2)で得られた2種類のPCR産物(m5bF、m5bR)を混合後、(a-3)と同様の方法でPCRを行ない、m5bFとm5bRを連結した。得られたPCR産物をm5bpとした。
(d-4)(d-3)で得られたPCR産物m5bpを鋳型とし、配列番号21および配列番号22に記載の配列からなるオリゴヌクレオチドをPCRプライマーとして、(a-4)と同様の方法でPCRを行なった。これによりFcR5bをコードするポリヌクレオチドを作製した。
(d-5)(d-4)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、あらかじめ制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(d-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出することで、野生型Fc結合性タンパク質に対して5箇所アミノ酸置換したポリペプチドである、FcR5bをコードするポリヌクレオチドを含むプラスミドpET-FcR5bを得た。
(d-7)pET-FcR5bのヌクレオチド配列の解析を、実施例1(5)と同様の方法で行なった。
(e-1)(c)で作製したpET-FcR5aを鋳型とし、配列番号22および配列番号39に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm6aFとした。
(e-2)(b)で作製したpET-FcR4を鋳型とし、配列番号21および配列番号40に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm6aRとした。
(e-3)(e-1)および(e-2)で得られた2種類のPCR産物(m6aF、m6aR)を混合後、(a-3)と同様の方法でPCRを行ない、m6aFとm6aRを連結した。得られたPCR産物をm6apとした。
(e-4)(e-3)で得られたPCR産物m6apを鋳型とし、配列番号21および配列番号22に記載の配列からなるオリゴヌクレオチドをPCRプライマーとして、(a-4)と同様の方法でPCRを行なった。これによりFcR6aをコードするポリヌクレオチドを作製した。
(e-5)(e-4)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、あらかじめ制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(e-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出することで、野生型Fc結合性タンパク質に対して6箇所アミノ酸置換したポリペプチドである、FcR6aをコードするポリヌクレオチドを含むプラスミドpET-FcR6aを得た。
(e-7)pET-FcR6aのヌクレオチド配列の解析を、実施例1(5)と同様の方法で行なった。
(f-1)(d)で作製したpET-FcR5bを鋳型とし、配列番号22および配列番号45(5’-GTGTTCAAAGTGGGGGATCCGATTCATCTG-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm6bFとした。
(f-2)(d)で作製したpET-FcR5bを鋳型とし、配列番号21および配列番号46(5’-AATCGGATCCCCCACTTTGAACACCCACCG-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm6bRとした。
(f-3)(f-1)および(f-2)で得られた2種類のPCR産物(m6bF、m6bR)を混合後、(a-3)と同様の方法でPCRを行ない、m6bFとm6bRを連結した。得られたPCR産物をm6bpとした。
(f-4)(f-3)で得られたPCR産物m6bpを鋳型とし、配列番号21および配列番号22に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-4)と同様の方法でPCRを行なった。これによりFcR6bをコードするポリヌクレオチドを作製した。
(f-5)(f-4)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、あらかじめ制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(f-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出することで、野生型Fc結合性タンパク質に対して6箇所アミノ酸置換したポリペプチドである、FcR6bをコードするポリヌクレオチドを含むプラスミドpET-FcR6bを得た。
(f-7)pET-FcR6bのヌクレオチド配列の解析を、実施例1(5)と同様の方法で行なった。
(g-1)(e)で作製したpET-FcR6aを鋳型とし、配列番号22および配列番号45に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm7Fとした。
(g-2)(e)で作製したpET-FcR6aを鋳型とし、配列番号21および配列番号46に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-1)と同様の方法でPCRを行なった。精製したPCR産物をm7Rとした。
(g-3)(g-1)および(g-2)で得られた2種類のPCR産物(m7F、m7R)を混合後、(a-3)と同様の方法にてPCRを行ない、m7Fとm7Rを連結した。得られたPCR産物をm7pとした。
(g-4)(g-3)で得られたPCR産物m7pを鋳型とし、配列番号21および配列番号22に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、(a-4)と同様のPCRを行なった。これによりFcR7をコードするポリヌクレオチドを作製した。
(g-5)(g-4)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、あらかじめ制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(g-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出することで、野生型Fc結合性タンパク質に対して7箇所アミノ酸置換したポリペプチドである、FcR7をコードするポリヌクレオチドを含むプラスミドpET-FcR7を得た。
(g-7)pET-FcR7のヌクレオチド配列の解析を、実施例1(5)と同様の方法で行なった。
(1)実施例1で作製した野生型Fc結合性タンパク質、実施例3で選択した変異型のFc結合性タンパク質(FcR2)、および実施例4で作製した変異型のFc結合性タンパク質(FcR3、FcR4、FcR5a、FcR5b、FcR6a、FcR6b、FcR7)を発現する形質転換体を、それぞれ50μg/mLのカナマイシンを含む3mLの2YT液体培地に接種し、37℃で一晩、好気的に振とう培養することで前培養を行なった。
(2)50μg/mLのカナマイシンを添加した20mLの2YT液体培地(ペプトン16g/L、酵母エキス10g/L、塩化ナトリウム5g/L)に前培養液を200μL接種し、37℃で好気的に振とう培養を行なった。
(3)培養開始1.5時間後、培養温度を20℃に変更して30分間振とう培養した。その後、終濃度0.01mMとなるようIPTGを添加し、引き続き20℃で一晩、好気的に振とう培養した。
(4)培養終了後、遠心分離により集菌し、BugBuster Protein extraction kit(タカラバイオ社製)を用いてタンパク質抽出液を調製した。
(5)(4)で調製したタンパク質抽出液中の野生型Fc結合性タンパク質および変異型のFc結合性タンパク質の抗体結合活性を、実施例3(4)に記載のELISA法を用いて測定した。この時、市販のFcγRIIIaの細胞外領域(R&D Systems社:4325-FC-050)を用いて検量線を作製し、濃度測定を行なった。
(6)各タンパク質の濃度が5μg/mLになるよう150mMの塩化ナトリウムを含んだ20mMのトリス緩衝液(pH7.4)で希釈した。これを等量に分け、一方はサーマルサイクラー(エッペンドルフ社製)を用いて45℃で10分間加熱処理を行ない、もう一方は熱処理をしなかった。前記熱処理後、または非熱処理のタンパク質の抗体結合活性を実施例3(4)に記載のELISA法によって測定し、熱処理を行なった場合の抗体結合活性を、熱処理を行なわなかったときの抗体結合活性で除することで、残存活性を算出した。
(1)実施例5(1)から(5)と同様の方法で改良Fc結合性タンパク質を調製した。
(2)各タンパク質の濃度が30μg/mLになるよう150mMの塩化ナトリウムを含んだ20mMのトリス緩衝液(pH7.4)で希釈した。希釈した各Fc結合性タンパク質60μLと0.1Mのグリシン塩酸緩衝液(pH3.0)120μLを混合し、30℃で2時間静置した。
(3)実施例3(4)に記載のELISA法によって、グリシン塩酸緩衝液(pH3.0)による酸処理を行なった後のタンパク質の抗体結合活性と、前記酸処理を行なわなかったときのタンパク質の抗体結合活性を測定した。その後、酸処理を行なった場合の抗体結合活性を、酸処理を行なわなかったときの抗体結合活性で除することで、残存活性を算出した。
実施例3で明らかになったFc結合性タンパク質の安定性向上に関与するアミノ酸置換のうち、配列番号1の27番目のバリン(Val)、35番目のチロシン(Tyr)および121番目のグルタミン酸(Glu)について、他のアミノ酸に置換したFc結合性タンパク質を、それぞれ下記の方法で作製した。
(A-1)実施例1で作製したpET-eFcRを鋳型とし、配列番号24および配列番号51(5’-CTGCCGAAAGCGNNKGTGTTTCTGGAACCG-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、実施例4(a-1)と同様の方法でPCRを行なった。精製したPCR産物を27pFとした。
(A-2)実施例1で作製したpET-eFcRを鋳型とし、配列番号23および配列番号52(5’-TTCCAGAAACACMNNCGCTTTCGGCAGATC-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、実施例4(a-1)と同様の方法でPCRを行なった。精製したPCR産物を27pRとした。
(A-3)(A-1)および(A-2)で得られた2種類のPCR産物(27pF、27pR)を混合後、実施例4(a-3)と同様の方法でPCRを行ない、27pFと27pRを連結した。得られたPCR産物を27pとした。
(A-4)(A-3)で得られたPCR産物27pを鋳型とし、配列番号23および配列番号24に記載の配列からなるオリゴヌクレオチドをPCRプライマーとして、実施例4(a-4)と同様の方法でPCRを行なった。これにより配列番号1の27番目のバリンを任意のアミノ酸に置換したFc結合性タンパク質をコードするポリヌクレオチドを作製した。
(A-5)(A-4)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、あらかじめ制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(A-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出し、実施例1(5)と同様の方法でヌクレオチド配列解析を行なった。
(B-1)実施例1で作製したpET-eFcRを鋳型とし、配列番号24および配列番号53(5’-AACCGCAGTGGNNKCGCGTGCTGGAGAAAG-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、実施例4(a-1)と同様の方法でPCRを行なった。精製したPCR産物を35pFとした。
(B-2)実施例1で作製したpET-eFcRを鋳型とし、配列番号23および配列番号54(5’-AGCACGCGMNNCCACTGCGGTTCCAGAAAC-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、実施例4(a-1)と同様の方法でPCRを行なった。精製したPCR産物を35pRとした。
(B-3)(B-1)および(B-2)で得られた2種類のPCR産物(35pF、35pR)を混合後、実施例4(a-3)と同様の方法にてPCRを行ない、35pFと35pRを連結した。得られたPCR産物を35pとした。
(B-4)(B-3)で得られたPCR産物35pを鋳型とし、配列番号23および配列番号24に記載の配列からなるオリゴヌクレオチドをPCRプライマーとして、実施例4(a-4)と同様の方法でPCRを行なった。これにより配列番号1の35番目のチロシンを任意のアミノ酸に置換したFc結合性タンパク質をコードするポリヌクレオチドを作製した。
(B-5)(B-4)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、あらかじめ制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(B-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出し、実施例1(5)と同様の方法でヌクレオチド配列解析を行なった。
(C-1)実施例1で作製したpET-eFcRを鋳型とし、配列番号24および配列番号55(5’-GTGTTCAAAGAGNNKGATCCGATTCATCTG-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、実施例4(a-1)と同様の方法でPCRを行なった。精製したPCR産物を121pFとした。
(C-2)実施例1で作製したpET-eFcRを鋳型とし、配列番号23および配列番号56(5’-AATCGGATCMNNCTCTTTGAACACCCACCG-3’)に記載の配列からなるオリゴヌクレオチドをPCRプライマーとした他は、実施例4の(a-1)と同様の方法でPCRを行なった。精製したPCR産物を121pRとした。
(C-3)(C-1)および(C-2)により得られた2種類のPCR産物(121pF、121pR)を混合後、実施例4(a-3)と同様の方法にてPCRを行ない、121pFと121pRを連結した。得られたPCR産物を121pとした。
(C-4)(C-3)で得られたPCR産物121pを鋳型とし、配列番号23および配列番号24に記載の配列からなるオリゴヌクレオチドをPCRプライマーとして実施例4(a-4)と同様のPCRを行なった。これにより配列番号1の121番目のグルタミン酸が任意のアミノ酸に置換されたFc結合性タンパク質をコードするポリヌクレオチドを作製した。
(C-5)(C-4)で得られたポリヌクレオチドを精製後、制限酵素NcoIとHindIIIで消化し、制限酵素NcoIとHindIIIで消化した発現ベクターpETMalE(特開2011-206046号公報)にライゲーションし、これを用いて大腸菌E.coli BL21(DE3)株を形質転換した。
(C-6)得られた形質転換体を50μg/mLのカナマイシンを添加したLB培地で培養した。回収した菌体(形質転換体)からプラスミドを抽出し、実施例1(5)と同様の方法でヌクレオチド配列解析を行なった。
(1)実施例1で作製した野生型Fc結合性タンパク質、および実施例7で作製した1箇所アミノ酸置換したFc結合性タンパク質を発現する形質転換体を、実施例3(1)および(2)と同様の方法でそれぞれ培養を行ない、野生型Fc結合性タンパク質および1アミノ酸置換したFc結合性タンパク質を発現させた。
(2)発現した1アミノ酸置換したFc結合性タンパク質を実施例3(4)に記載のELISA法にて抗体との結合活性を調べた。
実施例8で評価した1アミノ酸置換したFc結合性タンパク質の熱安定性を比較するため、実施例3(3)と同様の方法で熱処理を行ない(45℃、10分間)、残存活性を算出した。
(1)実施例4(c)で作製したFcR5aを発現する形質転換体を2Lのバッフルフラスコに入った50μg/mLのカナマイシンを含む400mLの2YT液体培地(ペプトン16g/L、酵母エキス10g/L、塩化ナトリウム5g/L)に接種し、37℃で一晩、好気的に振とう培養することで前培養を行なった。
(2)グルコース10g/L、酵母エキス20g/L、リン酸三ナトリウム十二水和物3g/L、リン酸水素二ナトリウム十二水和物9g/L、塩化アンモニウム1g/Lおよび硫酸カナマイシン50mg/Lを含む液体培地1.8Lに、(1)の培養液180mLを接種し、3L発酵槽(バイオット社製)を用いて本培養を行なった。温度30℃、pH6.9から7.1、通気量1VVM、溶存酸素濃度30%飽和濃度の条件に設定し、本培養を開始した。pHの制御には酸として50%リン酸、アルカリとして14%アンモニア水をそれぞれ使用し、溶存酸素の制御は撹拌速度を変化させることで制御し、撹拌回転数は下限500rpm、上限1000rpmに設定した。培養開始後、グルコース濃度が測定できなくなった時点で、流加培地(グルコース248.9g/L、酵母エキス83.3g/L、硫酸マグネシウム七水和物7.2g/L)を溶存酸素(DO)により制御しながら加えた。
(3)菌体量の目安として600nmの吸光度(OD600nm)が約150に達したところで培養温度を25℃に下げ、設定温度に到達したことを確認した後、終濃度が0.5mMになるようIPTGを添加し、引き続き25℃で培養を継続した。
(4)培養開始から約48時間後に培養を停止し、培養液を4℃で8000rpm、20分間の遠心分離により菌体を回収した。
(5)(4)で回収した菌体の一部を150mMの塩化ナトリウムを含む20mMのトリス塩酸緩衝液(pH7.4)に5mL/1g(菌体)となるように懸濁し、超音波発生装置(インソネーター201M(商品名)、久保田商事製)を用いて、4℃で約10分間、約150Wの出力で菌体を破砕した。菌体破砕液は4℃で20分間、10000rpmの遠心分離を2回行ない、上清を回収した。
(6)(5)で得られた破砕液に終濃度で20mMとなるようイミダゾールを添加後、あらかじめ150mMの塩化ナトリウムおよび20mMのイミダゾールを含む20mMのトリス塩酸緩衝液(pH7.4)で平衡化したNi Sepharose 6 Fast Flow(GEヘルスケア社製)50mLを充填したXK 26/20カラム(GEヘルスケア社製)にアプライした。平衡化に用いた緩衝液で洗浄後、150mMの塩化ナトリウムおよび0.5Mのイミダゾールを含む20mMのトリス塩酸緩衝液(pH7.4)を用いてFcR5aを溶出した。
(7)(6)で得られた溶出液を、あらかじめ150mMの塩化ナトリウムを含む20mMのトリス塩酸緩衝液(pH7.4)で平衡化したIgGセファロース(GEヘルスケア社製)30mLを充填したHR 16/10カラム(GEヘルスケア社製)にアプライした。平衡化に用いた緩衝液で洗浄後、0.1Mのグリシン塩酸緩衝液(pH3.0)でFcR5aを溶出した。なお溶出液は、溶出液量の1/4量の1Mトリス塩酸緩衝液(pH7.0)を加えることでpHを中性付近に戻した。
(1)実施例10で調製したFcR5aを、限外ろ過膜(ミリポア社製:アミコンウルトラ-15)を用いて濃縮・緩衝液交換を行ない、150mMの塩化ナトリウムを含む20mMのトリス塩酸緩衝液(pH7.4)に8.37mg/mLの濃度まで濃縮した。
(2)担体である親水性ビニルポリマー(東ソー社製:トヨパール)の水酸基に1,6-ヘキサンジオールジグリシジルエーテルを反応させてエポキシトヨパールゲルを調製した。
(3)(2)で調製したエポキシトヨパールゲル100μLを入れたスピンカラム(バイオラッド社製)を5本用意し、0.5Mの塩化ナトリウムを含んだ0.1Mのホウ酸緩衝液(pH9.0)0.5mLで3回洗浄した。
(4)(1)で調製したFcR5a溶液0.3mLと0.5Mの塩化ナトリウムを含んだ0.1Mのホウ酸緩衝液(pH9.0)0.45mLとを混合した溶液を、(3)に記載のゲルを充填したスピンカラムにそれぞれ添加し、35℃で3時間振とうした。
(5)ゲルに加えたFcR5a溶液と0.5Mの塩化ナトリウムを含んだ0.1Mのホウ酸緩衝液の混合溶液を回収した後、0.1Mのグリシン塩酸緩衝液(pH3.0)0.2mLで3回洗浄した。その後、150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)0.5mLで3回洗浄することでpHを中性付近に戻し、FcR5a固定化ゲル0.5mLを調製した。
(1)実施例11で作製したFcR5a固定化ゲル0.5mLをHR16/5カラム(GEヘルスケア社製)に充填し、AKTAprime plus(GEヘルスケア社製)につなげた。その後、150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)で平衡化した。
(2)150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)を0.1mLの流速で流し、150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)で1mg/mLに調製したヒトIgG1(Sigma社製:I5154-1MG)溶液0.5mLをアプライし、ヒトIgG1をFcR5a固定化ゲルに吸着させた。その後、20mMの酢酸緩衝液(pH5.0)を流すことで平衡化し、20mMのグリシン塩酸緩衝液(pH3.0)によるpHグラジエントで、吸着したヒトIgG1を溶出した。溶出したヒトIgG1は0.5mLごとにフラクションを回収した。
(1)実施例12で使用した精製前のヒトIgG1および溶出フラクション(FrA、FrB)を100℃、10分の熱処理により変性後、グリコアミダーゼA/ペプシンおよびプロナーゼで順次処理し、ゲルろ過法による精製操作を経て糖鎖画分を取得した。
(2)(1)で得られた糖鎖をエバポレーターにて濃縮・乾燥後、酢酸溶媒下、2-アミノピリジン、次いでジメチルアミンボランを順次作用させて蛍光ラベル化糖鎖とし、ゲルろ過法により精製した。
(3)(2)で得られた蛍光ラベル化糖鎖を陰イオン交換カラム(TSKgel DEAE-5PW、φ7.5mm×7.5cm:東ソー社製)にて、中性糖鎖画分とモノシアリル化糖鎖画分に分離した。
(4)(3)で得られた中性糖鎖画分とモノシアリル化糖鎖画分をODSカラムを用いて、個々の糖鎖に単離した。MALDI-TOF-MS分析により単離した糖鎖の分子量情報を取得後、ODSカラムクロマトグラフのリテンションタイムと照らし合わせて糖鎖構造を帰属した。
実施例11で作製したFcR5a固定化ゲルを用いて、ヒトIgG1およびヒトIgG3の精製を行なった。
(1)実施例11と同様の方法でFcR5a固定化ゲル0.2mLを作製した。作製したFcR5a固定化ゲル0.1mLをスピンカラムに充填した。
(2)(1)で作製したFcR5a固定化ゲルを充填したカラムを150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)0.5mLで3回洗浄した。
(3)動物細胞用の培地であるDMEM/F12培地(ライフテクノロジー社製)に10%のウシ胎児血清(FCS)を添加した培地に対し、ヒトIgG1(シグマ社製:I5154-1MG)またはヒトIgG3(シグマ社製:I4639-1MG)を0.3mg/mLとなるように添加することで模擬培養液を調製した。
(4)(2)で洗浄したカラムに(3)で調製した模擬培養液を0.4mL加え、25℃で2時間振とうした。
(5)模擬培養液を取り除き、150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)0.5mLで4回洗浄後、500μLの0.1Mのグリシン塩酸緩衝液(pH3.0)で溶出し、100μLごとにフラクションを回収した。
(6)模擬培養液、溶出フラクションをそれぞれ2×サンプルバッファー(2(w/v)%ドデシル硫酸ナトリウム、6(w/v)%β-メルカプトエタノール、10(w/v)%グリセリンおよび0.005(w/v)%ブロモフェノールブルーを含む50mMのトリス塩酸緩衝液(pH6.8))を添加し加熱処理した。その後、処理したサンプルを5から20%のグラジエントSDS-PAGE用ゲル(アトー社製)を用いた電気泳動にて分離した。なお比較として、模擬培養液に添加したヒトIgG1およびヒトIgG3(濃度:0.2mg/mL)についても同様の処理を行ない、SDS-PAGEで分析した。
以下に示す方法で、ヒトIgG1からN型糖鎖の除去を行なった。
(1)ヒトIgG1(Fitzgerald社製:31-AI17)の濃度が3mg/mLとなるよう、150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)で希釈した。
(2)(1)の希釈液100μLに1Mのトリス塩酸緩衝液(pH8.6)を100μL加え、N-glycosidase F(500mU/μL(タカラバイオ社製:4450)を10μL加えた後、37℃で24時間静置することでIgG1のN型糖鎖を除去した。
(3)あらかじめ150mM塩化ナトリウムを含んだ20mMトリス塩酸緩衝液(pH7.4)で平衡化した、Toyopearl AF-rProtein A-650F(東ソー社製:22803)を100μL充填したオープンカラム(バイオラッド社製)に、(2)の処理液をアプライした。
(4)500μLの150mM塩化ナトリウムを含んだ20mMトリス塩酸緩衝液(pH7.4)で3回洗浄後、100μLの0.1Mグリシン塩酸緩衝液(pH3.0)で7回溶出することで、N型糖鎖を除去したヒトIgG1(以下単に、糖鎖除去したヒトIgG1という)を精製した。なお当該溶出液は、1Mのトリス塩酸緩衝液(pH8.0)を溶出液の1/4量加えることで中和した。
(5)(4)で得られた糖鎖除去したヒトIgG1溶液に対し、等量のサンプルバッファー(2(w/v)%ドデシル硫酸ナトリウム、6(w/v)%β-メルカプトエタノール、10(w/v)%グリセリンおよび0.005(w/v)%ブロモフェノールブルーを含む50mMのトリス塩酸緩衝液(pH6.8))を添加し加熱処理することで、糖鎖除去したヒトIgG1を還元処理した。
(6)5から20%のグラジエントSDS-PAGE用ゲル(アトー社製)を用いた電気泳動にてヒトIgG1を分離した。なお比較として、糖鎖処理をしていないヒトIgG1(以下、糖鎖ありヒトIgG1という)水溶液(濃度:0.5mg/mL)についても(5)の記載と同様な還元処理を行ない、SDS-PAGEで分離した。
(1)実施例1で得られたヒトFcγRIIIaを発現可能な形質転換体を2Lのバッフルフラスコに入った50μg/mLのカナマイシンを含む400mLの2YT液体培地(ペプトン16g/L、酵母エキス10g/L、塩化ナトリウム5g/L)に接種し、37℃で一晩、好気的に振とう培養することで前培養を行なった。
(2)グルコース10g/L、酵母エキス20g/L、リン酸三ナトリウム十二水和物3g/L、リン酸水素二ナトリウム十二水和物9g/L、塩化アンモニウム1g/Lおよび硫酸カナマイシン50mg/Lを含む液体培地1.8Lに、(1)の培養液180mLを接種し、3L発酵槽を用いて本培養を行なった。温度30℃、pH6.9から7.1、通気量1VVM、溶存酸素濃度30%飽和濃度の条件に設定し、本培養を開始した。pHの制御には酸として50%リン酸、アルカリとして14%アンモニア水をそれぞれ使用し、溶存酸素の制御は撹拌速度を変化させることで制御し、撹拌回転数は下限500rpm、上限1000rpmに設定した。培養開始後、グルコース濃度が測定できなくなった時点で、流加培地(グルコース248.9g/L、酵母エキス83.3g/L、硫酸マグネシウム七水和物7.2g/L)を溶存酸素(DO)により制御しながら加えた。
(3)菌体量の目安として600nmの吸光度(OD600nm)が約150に達したところで培養温度を25℃に下げ、設定温度に到達したことを確認した後、終濃度が0.5mMになるようIPTG(イソプロピル-β-チオガラクトピラノシド)を添加し、引き続き25℃で培養を継続した。
(4)培養開始から約48時間後に培養を停止し、培養液を8000rpm、20分間の遠心分離により菌体を回収した。
(5)(4)で回収した菌体を150mMの塩化ナトリウムを含む20mMのトリス塩酸緩衝液(pH7.4)に5mL/1g(菌体)となるように懸濁し、超音波発生装置(インソネーター201M(商品名)、久保田商事製)を用いて、4℃で約10分間、約150Wの出力で菌体を破砕した。菌体破砕液は4℃で20分間、10000rpmの遠心分離を2回行ない、上清を回収した。
(6)(5)で得られた破砕液に終濃度で20mMとなるようイミダゾールを添加後、あらかじめ150mMの塩化ナトリウムおよび20mMのイミダゾールを含む20mMのトリス塩酸緩衝液(pH7.4)で平衡化したNi Sepharose 6 Fast Flow(GEヘルスケア社製)50mLを充填したXK 26/20カラム(GEヘルスケア社製)にアプライした。
(7)平衡化に用いた緩衝液で洗浄後、150mMの塩化ナトリウムおよび0.5Mのイミダゾールを含む20mMのトリス塩酸緩衝液(pH7.4)を用いてヒトFcγRIIIaを溶出した。
(8)(7)で得られた溶出液を、あらかじめ150mMの塩化ナトリウムを含む20mMのトリス塩酸緩衝液(pH7.4)で平衡化したIgGセファロース(GEヘルスケア社製)10mLを充填したHR 16/10カラム(GEヘルスケア社製)にアプライした。平衡化に用いた緩衝液で洗浄後、0.1Mのグリシン塩酸緩衝液(pH3.0)でヒトFcγRIIIaを溶出した。なお溶出液は、溶出液量の1/4量の1Mトリス塩酸緩衝液(pH8.0)を加えることでpHを中性付近に戻した。
(1)実施例16で調製したヒトFcγRIIIaをリン酸緩衝液(137mMのNaCl、8.1MのNa2HPO4、2.68mMのKClおよび1.47mMのKH2PO4を含むpH7.4の緩衝液)にて透析することで緩衝液交換を行ない、280nmの吸光度からヒトFcγRIIIaの濃度を測定した。
(2)(1)で濃度を測定したヒトFcγRIIIaを20mMの酢酸緩衝液(pH5.5)で10μg/mLに希釈後、アミンカップリングキット(GEヘルスケア社製)を用いてセンサーチップCM5(GEヘルスケア社製)に固定化し、Biacore T-100(GEヘルスケア社製)を用いてヒトFcγRIIIa固定化量を測定した。結果、ヒトFcγRIIIaの固定化量は488.2RU(1RU=1pg/mm2)であった。またプロテインA(プロテノバ社製)についても同様に、20mMの酢酸緩衝液(pH5.5)で10μg/mLに希釈し、CM5(GEヘルスケア社製)に固定化した。Biacore T-100で固定化量を測定した結果、固定化量は290.0RUであった。
(3)糖鎖ありヒトIgG1および実施例15で調製した糖鎖除去ヒトIgG1をHBS-EP(+)(10mMのHEPES、150mMのNaCl、3mMのEDTAおよび0.005(v/v)%のSurfactant P20(GEヘルスケア社製)を含む、pH7.4の溶液)にて128μg/mL、64μg/mL、32μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mLに希釈した。
(4)(2)で作製したタンパク質固定化チップのうち、ヒトFcγRIIIa固定化チップの場合は4μg/mLから128μg/mLの糖鎖ありヒトIgG1または糖鎖除去ヒトIgG1を流速30μL/分で流し、プロテインA固定化チップの場合は1μg/mLから16μg/mLの糖鎖ありヒトIgG1または糖鎖除去ヒトIgG1を流速30μL/分で流して、ヒトIgG1とチップに固定化したタンパク質とを結合させた後、Biacore T-100にて接触時間210秒、解離時間400秒の条件で測定することで、ヒトIgG1とチップに固定化したタンパク質との結合性を測定した。
(1)実施例16で調製したヒトFcγRIIIaを、限外ろ過膜(ミリポア社製:アミコンウルトラ-15)を用いて濃縮・緩衝液交換を行ない、150mMの塩化ナトリウムを含む20mMのトリス塩酸緩衝液(pH7.4)に2.6mg/mLの濃度まで濃縮した。
(2)担体である親水性ビニルポリマー(東ソー社製:トヨパール)の水酸基に1,6-ヘキサンジオールジグリシジルエーテルを反応させてエポキシトヨパールゲルを調製した。
(3)(2)で調製したエポキシトヨパールゲル70μLをスピンカラム(バイオラッド)に入れ、0.5Mの塩化ナトリウムを含んだ0.1Mのホウ酸緩衝液(pH9.0)0.5mLで3回洗浄した。
(4)(1)で調製したヒトFcγRIIIa溶液0.4mLと0.5Mの塩化ナトリウムを含んだ0.1Mのホウ酸緩衝液(pH9.0)0.6mLとを混合した溶液を、(2)のゲルに添加し、35℃で3時間振とうした。
(5)(4)で調製したゲルを0.1Mのグリシン塩酸緩衝液(pH3.0)0.2mLで3回洗浄した後、150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)0.5mLで3回洗浄することでpHを中性付近に戻し、ヒトFcγRIIIa固定化ゲル0.2mLを調製した。
(6)(2)のゲルに添加した溶液および洗浄液中に含まれるタンパク質濃度を測定し、ゲルに固定化されたヒトFcγRIIIa量を算出することで固定化率を計算したところ、添加したヒトFcγRIIIaの84%がゲルに固定化されていた。
(1)実施例18で作製したヒトFcγRIIIa固定化ゲルをHR16/5カラム(GEヘルスケア社製)に充填し、当該カラムを液体クロマトグラフィー装置AKTAprime(GEヘルスケア社製)に接続した。
(2)(1)で作製したカラムを150mMの塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)で平衡化し、ヒトIgG1(Fitzgerald社製:31-AI17)または実施例2で調製した糖鎖除去ヒトIgG1(溶液濃度はともに1mg/mL)を、流速0.1mL/minで0.1mL添加した。平衡化に用いた緩衝液で洗浄後、0.1Mのグリシン塩酸緩衝液(pH3.5)で溶出した。
Claims (19)
- 配列番号1に記載のアミノ酸配列のうち17番目から192番目までのアミノ酸残基を含み、かつ当該17番目から192番目までのアミノ酸残基において以下の(1)から(40)のうち少なくともいずれか1つのアミノ酸置換が生じている、Fc結合性タンパク質。
(1)配列番号1の18番目のメチオニンがアルギニンに置換
(2)配列番号1の27番目のバリンがグルタミン酸に置換
(3)配列番号1の29番目のフェニルアラニンがロイシンまたはセリンに置換
(4)配列番号1の30番目のロイシンがグルタミンに置換
(5)配列番号1の35番目のチロシンがアスパラギン酸、グリシン、リジン、ロイシン、アスパラギン、プロリン、セリン、スレオニン、ヒスチジンのいずれかに置換
(6)配列番号1の46番目のリジンがイソロイシンまたはスレオニンに置換
(7)配列番号1の48番目のグルタミンがヒスチジンまたはロイシンに置換
(8)配列番号1の50番目のアラニンがヒスチジンに置換
(9)配列番号1の51番目のチロシンがアスパラギン酸またはヒスチジンに置換
(10)配列番号1の54番目のグルタミン酸がアスパラギン酸またはグリシンに置換
(11)配列番号1の56番目のアスパラギンがスレオニンに置換
(12)配列番号1の59番目のグルタミンがアルギニンに置換
(13)配列番号1の61番目のフェニルアラニンがチロシンに置換
(14)配列番号1の64番目のグルタミン酸がアスパラギン酸に置換
(15)配列番号1の65番目のセリンがアルギニンに置換
(16)配列番号1の71番目のアラニンがアスパラギン酸に置換
(17)配列番号1の75番目のフェニルアラニンがロイシン、セリン、チロシンのいずれかに置換
(18)配列番号1の77番目のアスパラギン酸がアスパラギンに置換
(19)配列番号1の78番目のアラニンがセリンに置換
(20)配列番号1の82番目のアスパラギン酸がグルタミン酸またはバリンに置換
(21)配列番号1の90番目のグルタミンがアルギニンに置換
(22)配列番号1の92番目のアスパラギンがセリンに置換
(23)配列番号1の93番目のロイシンがアルギニンまたはメチオニンに置換
(24)配列番号1の95番目のスレオニンがアラニンまたはセリンに置換
(25)配列番号1の110番目のロイシンがグルタミンに置換
(26)配列番号1の115番目のアルギニンがグルタミンに置換
(27)配列番号1の116番目のトリプトファンがロイシンに置換
(28)配列番号1の118番目のフェニルアラニンがチロシンに置換
(29)配列番号1の119番目のリジンがグルタミン酸に置換
(30)配列番号1の120番目のグルタミン酸がバリンに置換
(31)配列番号1の121番目のグルタミン酸がアスパラギン酸またはグリシンに置換
(32)配列番号1の151番目のフェニルアラニンがセリンまたはチロシンに置換
(33)配列番号1の155番目のセリンがスレオニンに置換
(34)配列番号1の163番目のスレオニンがセリンに置換
(35)配列番号1の167番目のセリンがグリシンに置換
(36)配列番号1の169番目のセリンがグリシンに置換
(37)配列番号1の171番目のフェニルアラニンがチロシンに置換
(38)配列番号1の180番目のアスパラギンがリジン、セリン、イソロイシンのいずれかに置換
(39)配列番号1の185番目のスレオニンがセリンに置換
(40)配列番号1の192番目のグルタミンがリジンに置換 - 配列番号1に記載のアミノ酸配列のうち17番目から192番目までのアミノ酸残基を含み、かつ当該17番目から192番目までのアミノ酸残基において少なくとも配列番号1の35番目のチロシンがアスパラギン酸、グリシン、リジン、ロイシン、アスパラギン、プロリン、セリン、スレオニン、ヒスチジンのいずれかに置換した、請求項1に記載のFc結合性タンパク質。
- 配列番号1に記載のアミノ酸配列のうち17番目から192番目までのアミノ酸残基を含み、かつ当該17番目から192番目までのアミノ酸残基において少なくとも配列番号1の35番目のチロシンがアスパラギンまたはプロリンに置換した、請求項2に記載のFc結合性タンパク質。
- 配列番号1に記載のアミノ酸配列のうち17番目から192番目までのアミノ酸残基を含み、かつ当該17番目から192番目までのアミノ酸残基において少なくとも27番目のバリンがグルタミン酸に、35番目のチロシンがアスパラギンにそれぞれ置換した、請求項3に記載のFc結合性タンパク質。
- 配列番号27、配列番号31、配列番号33、配列番号37、配列番号41、配列番号43、配列番号47、配列番号49のいずれかに記載のアミノ酸配列のうち33番目から208番目までのアミノ酸残基を含む、請求項4に記載のFc結合性タンパク質。
- 配列番号27、配列番号31、配列番号33、配列番号37、配列番号41、配列番号43、配列番号47、配列番号49のいずれかに記載のアミノ酸配列からなる、請求項5に記載のFc結合性タンパク質。
- さらに、以下の(41)から(44)のうち少なくともいずれか1つのアミノ酸置換が生じている、請求項1から6のいずれかに記載のFc結合性タンパク質。(41)配列番号1の66番目のロイシンがヒスチジンまたはアルギニンに置換(42)配列番号1の147番目のグリシンがアスパラギン酸に置換(43)配列番号1の158番目のチロシンがヒスチジンに置換(44)配列番号1の176番目のバリンがフェニルアラニンに置換
- 請求項1から7のいずれかに記載のFc結合性タンパク質を不溶性担体に固定化して得られる吸着剤。
- 請求項8に記載の吸着剤を用いた抗体の精製方法。
- 糖鎖を有した抗体を含む溶液を請求項8に記載の吸着剤に添加して当該吸着剤に吸着させる工程と、
前記吸着剤に吸着した糖鎖を有した抗体を溶出液を用いて溶出させる工程
とを含む、糖鎖を有した抗体の精製方法。 - 請求項8に記載の吸着剤を用いて抗体を分離することで、抗体が有する糖鎖構造の違いを識別する方法。
- 請求項9又は10に記載の精製方法で得られる抗体。
- 請求項8に記載の吸着剤を用いた糖鎖の分離方法。
- 請求項13に記載の分離方法で得られる糖鎖。
- 請求項1から7のいずれかに記載のFc結合性タンパク質をコードするポリヌクレオチド。
- 請求項15に記載のポリヌクレオチドを含む発現ベクター。
- 請求項16に記載の発現ベクターで宿主を形質転換して得られる形質転換体。
- 宿主が大腸菌である、請求項17に記載の形質転換体。
- 請求項17または18に記載の形質転換体を培養することによりFc結合性タンパク質を発現させ、その培養物から発現されたFc結合性タンパク質を回収する、Fc結合性タンパク質の製造方法。
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Cited By (13)
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WO2015199154A1 (ja) * | 2014-06-27 | 2015-12-30 | 東ソー株式会社 | 改良Fc結合性タンパク質、当該タンパク質の製造方法、当該タンパク質を用いた抗体吸着剤および当該吸着剤を用いた抗体の分離方法 |
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