WO2022239704A1 - 抗体組成物の精製方法 - Google Patents

抗体組成物の精製方法 Download PDF

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WO2022239704A1
WO2022239704A1 PCT/JP2022/019548 JP2022019548W WO2022239704A1 WO 2022239704 A1 WO2022239704 A1 WO 2022239704A1 JP 2022019548 W JP2022019548 W JP 2022019548W WO 2022239704 A1 WO2022239704 A1 WO 2022239704A1
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antibody
carrier
chromatography
glycosylated
seq
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French (fr)
Japanese (ja)
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孝行 吉森
ステファン アイルソ
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Chiome Bioscience Inc
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Chiome Bioscience Inc
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Priority to KR1020237040492A priority Critical patent/KR20240005771A/ko
Priority to AU2022271741A priority patent/AU2022271741A1/en
Priority to CN202280033860.8A priority patent/CN117279929A/zh
Priority to IL308169A priority patent/IL308169A/en
Priority to EP22807406.8A priority patent/EP4353734A4/en
Priority to US18/289,914 priority patent/US20240383945A1/en
Priority to JP2023520995A priority patent/JPWO2022239704A1/ja
Priority to CA3219950A priority patent/CA3219950A1/en
Publication of WO2022239704A1 publication Critical patent/WO2022239704A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to a method for producing an antibody composition. More specifically, it relates to a method for purifying an antibody composition in which isomers with sugar chains added to other than the Fc region sugar chain-binding consensus region are reduced.
  • Antibody drugs that use monoclonal antibodies as active ingredients are expected to be one of the molecular target drugs that make use of the high binding affinity and binding specificity of antibody molecules to antigens, and research and development have progressed. Antibody drugs have become indispensable for the treatment of various diseases, including cancer and autoimmune diseases, and currently, nearly 100 products have been approved and used around the world (non-patented). Document 1, Non-Patent Document 2). There are still high expectations for the development of new antibody drugs that satisfy unmet medical needs, and it is expected that many new antibody drugs will be researched and developed in the future.
  • Antibodies have N at the 297th Asn residue (Asn297) in the glycosylation consensus region (Asn297-X-Ser/Thr, where X is an amino acid other than Pro) present in the Fc portion of the heavy chain. - It has a conjugated sugar chain.
  • the sugar chains present in this glycosylation consensus region are known to contribute to biological activity, blood dynamics, safety, etc. as antibody molecules (Non-Patent Documents 3 and 4). For example, removal of the Fuc residue (core fucose) attached to the N-acetylglucosamine (GlcNAc) residue at the reducing end of the N-linked sugar chain of Asn297 enhances antibody-dependent cellular cytotoxicity (ADCC) activity.
  • ADCC antibody-dependent cellular cytotoxicity
  • Non-Patent Document 5 Non-Patent Document 5
  • Non-Patent Document 3 Non-Patent Document 6
  • Non-Patent Document 8 Non-Patent Document 8
  • Glycosylation isomers have various properties such as biological activity (Non-Patent Document 10, Non-Patent Document 11), immunogenicity (Non-Patent Document 8, Non-Patent Document 10), blood half-life (Non-Patent Document 9), etc. It is known that it can affect the properties of antibodies. In particular, when a sugar chain is bound near the Fab region or the CDR region, there is concern that the biological activity may decrease. Similarly, glycosylated isomers of antibodies in which sugar chains are bound to sites other than the glycosylated consensus region have safety concerns such as immunogenicity.
  • glycosylation outside the glycosylation consensus region significantly reduces biological activity or significantly increases immunogenicity, it will have negative effects that are not desirable as a component of an antibody drug. Therefore, these glycosylated isomers are regarded as target substance-derived impurities. Such target-substance-derived impurities are not desirable to be included as components of pharmaceuticals, and it is desirable to remove them as much as possible from the viewpoint of regulation (Non-Patent Document 12).
  • the sugar chain will not bind to that site. Generally, such as changing the consensus sequence to another amino acid sequence. In that case, the binding activity may be lowered compared to the performance of the original antibody, and it may not be possible to obtain an antibody having the desired therapeutic effect.
  • Glycosylation isomers in which sugar chains are bound to regions other than the glycosylation consensus region of the antibody, such as Fab or CDR, are evaluated by separating glycosylation isomers at a small amount and at a high-performance analytical level. , can be discerned.
  • a method of evaluating a peptide fragment by combining high-performance liquid chromatography and mass spectrometry and a method of evaluating a sugar chain itself cleaved from an antibody are known (Non-Patent Document 6, Non-Patent Document 9).
  • HIC hydrophobic interaction chromatography
  • Non-Patent Document 14 discloses the usefulness of separation performance of many antibodies, but does not suggest that glycosylation isomers can be separated by a purification method using HIC.
  • Non-Patent Documents 15 and 16 There are reports on the separation of glycosylated isomers by affinity chromatography using a lectin that specifically binds to a given sugar chain. It uses Concanavalin A as a ligand and is known to have high separation characteristics, but since it is separation by affinity and uses natural products, it is not suitable for the production of pharmaceuticals, and is not suitable for the production of antibody pharmaceuticals. There are no applicable cases.
  • Patent Document 1 As a technology that has realized the separation of sugar chain components other than antibodies, there is a known case of separating recombinant antithrombin by chromatography based on the difference in the number of sugar chains (Patent Document 1). In addition, recombinant erythropoietin and its derivatives are separated by chromatography based on the difference in the number of sialic acid additions (Patent Document 2). Similarly, there is a case in which isoelectric focusing chromatography was used to separate ovalbumin and transferrin (Patent Document 3). However, these techniques cannot be directly applied to antibodies, and there is no report on a production method for separating and removing glycosylated isomers in antibody compositions.
  • Non-Patent Document 17 a method of removing sugar chains by enzymatic treatment is known (Non-Patent Document 17).
  • the isolation, removal, and origin of the enzyme are also problematic, making it difficult to use this method as a manufacturing technology for pharmaceuticals.
  • the sugar chains of antibodies are removed with an enzyme, it is also impossible to distinguish between the sugar chains of the glycosylated consensus region and the non-consensus region.
  • the anti-hDLK-1 antibody described in Patent Document 4 is expected to have anti-tumor activity.
  • an object of the present invention is to provide a method for removing glycosylated isomers of antibody drugs.
  • an object of the present invention is to obtain a more effective and safer purified anti-hDLK-1 antibody composition.
  • glycosylated isomers of antibody drugs cannot be separated by chromatography.
  • no removable manufacturing or preparation techniques have been reported.
  • the inventors of the present invention have made intensive studies to solve the above problems, and found that, surprisingly, by using a conventional hydrophobic interaction chromatography carrier, in particular, glycosylation isomers and target substances can be adsorbed. And by optimizing the separation conditions, it was found that a purified antibody composition with reduced glycosylated isomers can be prepared.
  • the present inventors separated the glycosylated isomer and the purified. Glycosylated isomers having sugar chains near the CDRs of the antibody may affect the binding activity of the antibody. It is also possible that there is no effect. Surprisingly, it was found that in the anti-hDLK-1 antibody described in Patent Document 4, the glycosylated isomer completely lost its activity and therefore became an "impurity" in pharmaceuticals. As a result, the present inventors succeeded in identifying the glycosylation isomer as a new impurity in the crudely purified anti-hDLK-1 antibody, and by enabling its removal, it is more effective and safer. have achieved the provision of a purified composition of an anti-hDLK-1 antibody.
  • the present invention relates to the following (1) to (27).
  • the heavy chain has an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36;
  • the purified antibody composition obtained the content of glycosylated isomers in which sugar chains are added to a site other than the Fc region glycosylated consensus region is reduced compared to the crude antibody product. ing, the aforementioned method.
  • the carrier for conventional chromatography is a hydrophobic interaction chromatography carrier or a mixed mode chromatography carrier.
  • the carrier for conventional chromatography has an average particle size of 15 ⁇ m or more.
  • the carrier for conventional chromatography is a hydrophobic interaction chromatography carrier, and after loading the crude antibody product onto the carrier, washing the carrier with a washing solution prior to elution (1) The purification method according to any one of (6).
  • a method for purifying the antibody composition according to (1) The antibody crude product adjusted to a salt concentration of 0.5 M or more is subjected to conventional chromatography using a carrier having a particle size of 20 to 100 ⁇ m and having a benzyl group or a butyl group, and the glycosylated isomer is passed through. to remove to washing the carrier with a washing liquid; and obtaining a purified antibody composition by eluting the antibody adsorbed to the carrier with an elution solution having a salt concentration of 0.5 M or less or containing no salt and having a pH of 5 to 7;
  • the ratio of glycosylated isomers to the total antibody in the purified antibody composition is reduced compared to the crude antibody product.
  • a method for purifying the antibody composition according to (1) The crude antibody product adjusted to pH 4 to 6 is subjected to conventional chromatography using a carrier having a particle size of 20 to 100 ⁇ m and having a benzyl group or a butyl group to remove glycosylated isomers into a flow-through fraction.
  • a purified antibody composition by eluting the antibody adsorbed to the carrier with an elution solution having a salt concentration of 0.5 M or less or containing no salt and having a pH of 5 to 7, wherein in the purified antibody composition, The ratio of glycosylated isomers to the whole antibody is reduced compared to the crude antibody product, the aforementioned method.
  • the protein load per unit amount of the carrier for conventional chromatography is 20 g/L or more, and the washing is performed by flowing a washing solution of 5 carrier volumes or more, (1) to (20)
  • a method according to any one of (22) The ratio of the antibody, which does not have sugar chains added to sites other than the Fc region glycosylation consensus region, to the total antibody, which includes the purification method according to any one of (1) to (21), is 95. % or higher.
  • the heavy chain has an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36;
  • a method for producing a purified anti-hDLK-1 antibody composition wherein the light chain has the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, Applying the crudely purified antibody to conventional chromatography using a carrier having a particle size of 20 to 100 ⁇ m having a benzyl group or a butyl group, Adsorbing to the carrier an antibody having no sugar chain added to a site other than the Fc region glycosylation consensus region; removing glycosylated isomers by washing the carrier one or more times with a washing solution containing a salt of 0.5 M or more; Obtaining a purified antibody composition by eluting the antibody adsorbed to the carrier, Here, the ratio of glycosylated isomers to the total antibody in the purified antibody composition is reduced compared to the crude antibody product.
  • the heavy chain has an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; and a glycosylated isomer in which a sugar chain is added to a site other than the Fc region glycosylation consensus region from a crude anti-hDLK-1 antibody light chain having the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12
  • the heavy chain has an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; and an anti-hDLK-1 antibody composition in which the light chain has the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, and wherein no sugar chain is added to a site other than the Fc region glycosylation consensus region. , the antibody composition containing 95% or more of the total antibody.
  • the heavy chain has an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; and an anti-hDLK-1 antibody whose light chain has the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, wherein no sugar chain is added to a site other than the Fc region glycosylation consensus region.
  • the present invention makes it possible to reduce or eliminate glycosylated isomers in which sugar chains are added to non-glycosylated consensus regions of antibodies.
  • the first effect of the present invention it is possible to provide a medical antibody composition with a reduced proportion of glycosylated isomers.
  • a highly pure antibody composition purified by the method and having reduced glycosylated isomers can be made into a pharmaceutical preparation with higher purity.
  • the purified composition of the anti-hDLK-1 antibody purified in the present invention is a pharmaceutical composition with excellent efficacy and safety because glycosylation isomers, which are impurities having no activity, have been removed. can provide.
  • Fig. 3 is a graph showing the HIC-HPLC analysis pattern of the crude product obtained from the culture medium of cells producing humanized anti-hDLK-1 monoclonal antibody.
  • Fig. 10 is a photograph showing the results of SDS-PAGE analysis after protein A purification of the culture supernatant by CHO cells transiently expressing a humanized anti-hDLK-1 monoclonal antibody.
  • FIG. 10 is a graph showing the separation profile of antibody compositions on CaptoButyl hydrophobic interaction chromatography support.
  • FIG. 1 is a graph and a table showing the results of a purity analysis test by HIC-HPLC of a purified antibody composition separated on a CaptoButyl carrier.
  • Peak1 and Peak2 in the table are glycosylated isomer antibodies (Peak1 has a sugar chain attached to one CDR of four polypeptide chains, Peak2 has a sugar chain attached to two CDRs), Peak3 The ratio (%) of antibodies with sugar chains bound only to the glycosylation consensus region is shown.
  • FIG. 10 is a graph showing the separation profile of antibody compositions on CaptoButyl carrier.
  • FIG. 10 is a graph showing the separation profile of antibody compositions on Poros Benzyl Ultra carrier.
  • FIG. Fig. 4 is a graph showing the results of analysis by design of experiments on control of glycosylated isomer content by optimizing chromatography conditions using POROS Benzyl Ultra carrier.
  • (c) shows the results of HIC-HPLC analysis of the fraction collected as the pre-peak in HIC-HPLC of (a).
  • 1 is a graph showing the results of evaluating the ADCC activity of glycosylated isomers. The vertical axis indicates fluorescence intensity indicating ADCC activity, and the horizontal axis indicates antibody concentration. Circles are the fractions fractionated as the main peak in FIG. 9(b) (components not containing the glycosylated isomer), isomers), and the triangles indicate the results of crude antibody purification before isolation in FIG. 9(a).
  • the base sequence (SEQ ID NO: 37) and amino acid sequence (SEQ ID NO: 38) of the coding region of the H chain ( ⁇ 1 chain) of HuBA-1-3D-1 are shown.
  • Amino acids are represented by single-letter code, and termination codon positions are indicated by " ⁇ ".
  • the base sequence (SEQ ID NO: 41) and amino acid sequence (SEQ ID NO: 42) of the coding region of the H chain ( ⁇ 1 chain) of HuBA-1-3D-2 are shown. Amino acids are represented by single-letter code, and termination codon positions are indicated by " ⁇ ”.
  • the nucleotide sequence (SEQ ID NO: 69) and amino acid sequence (SEQ ID NO: 70) of the coding region of the L chain ( ⁇ chain) are shown. In the figure, amino acids are represented by single-letter codes, and the positions of termination codons are indicated by " ⁇ ".
  • HuBA-1-3D-1 shows the nucleotide sequence (SEQ ID NO: 53) and amino acid sequence (SEQ ID NO: 54) of the H chain ( ⁇ 1 chain) coding region of T73K. Amino acids are represented by single-letter code, and termination codon positions are indicated by " ⁇ ".
  • SEQ ID NO: 18 In the deduced amino acid sequence of VH of HuBA-1-3D-1 T73K (SEQ ID NO: 18), a peptide consisting of 19 amino acids from the N-terminus is a signal peptide.
  • the cDNA nucleotide sequence of the VH mature peptide of HuBA-1-3D-1 T73K is shown in SEQ ID NO:19, and its deduced amino acid sequence is shown in SEQ ID NO:20.
  • the cDNA nucleotide sequence of the mature peptide of VH of HuBA-1-3D-1 A24G/T73K is shown in SEQ ID NO:23, and its deduced amino acid sequence is shown in SEQ ID NO:24.
  • the numbers in parentheses indicate the sequence numbers in the sequence listing.
  • Fc region glycosylation consensus region refers to a glycosylation consensus sequence usually present in the Fc portion of an antibody, including Asn297 ( Typically, it refers to Asn297-X-Ser/Thr (where X is an amino acid other than Pro)).
  • the glycosylation consensus sequence and the nucleotide sequence encoding the glycosylation consensus region include any sequence that encodes the amino acid sequence.
  • glycosylation isomer means an antibody in which sugar chains are added to amino acids other than the glycosylation consensus region.
  • a sugar chain attached to an amino acid other than the glycosylated consensus region is referred to as a “non-consensus sugar chain”.
  • the regions to which the non-consensus sugar chain is bound in the glycosylated isomer include the Fab region, the Fv (variable) region that binds to the antigen, the complementarity determining region (CDR) region, and regions other than the glycosylated consensus region. Included are Fc regions, and fusion sequence portions in fusion antibodies, typically CDR regions.
  • the N-type sugar chain to Asn-X-Ser/Thr (where X is an amino acid other than Pro), which is a glycosylation consensus sequence. or an O-type sugar chain to Ser or Thr.
  • a crude antibody purification product to be purified in the present invention may contain an antibody having one or more non-consensus sugar chains.
  • the number of non-consensus sugar chains bound per antibody molecule is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, Or it may be 10 or more.
  • an antibody consists of four polypeptide chains (usually two heavy chains and two light chains).
  • the binding ratio of sugar chains to the binding regions of non-consensus sugar chains in crude antibody preparations is determined by the ratio of the heavy and light chains in one antibody molecule.
  • the non-consensus sugar chain to the structure corresponding to "combination" or the case where the non-consensus sugar chain to the corresponding structure is completely bound is taken as 100%, it may be 0.1% or more and 200% or less per antibody molecule.
  • it is 1% or more and 50% or less.
  • 200% means that non-consensus sugar chains are bound to both (two sites) of the paired "combination of heavy chain and light chain" in one antibody molecule.
  • the term "antibody” includes full-length antibodies, antibody fragments, fusions of full-length antibodies or antibody fragments with other substances, such as mouse antibodies, mouse-human chimeric antibodies, humanized antibodies, human antibodies, It also includes amino acid variants, adducts, deletions, substitutions, sugar chain variants and the like of these.
  • the immunoglobulin class of the antibody is not particularly limited, and may be any immunoglobulin class (isotype) of IgG, IgM, IgA, IgE, IgD, or IgY, preferably IgG.
  • the antibody of the present invention is IgG, it may be of any subclass (IgG1, IgG2, IgG3, or IgG4).
  • Antibody fragments are preferably antigen-binding fragments such as F(ab') 2 , Fab', Fab, Fab 3 , single-chain Fv (hereinafter referred to as "scFv"), (tandem) bispecific single-chain Fv (sc(Fv) 2 ), single-chain triple body, nanobody, divalent VHH, pentavalent VHH, minibody, (double-stranded) diabody, tandem diabody, bispecific tribody, bispecific bibody, dual affinity retargeting molecules (DART), triabodies (or tribodies), tetrabodies (or [sc(Fv) 2 ] 2 or (scFv-SA) 4), disulfide-bonded Fv (hereinafter referred to as "dsFv”), compact IgG , heavy chain antibodies, or polymers thereof. Fusions of antibody fragments with other substances include fusion proteins, particularly Fc fusion proteins.
  • the antibodies herein have a heavy chain selected from and the light chain has the amino acid sequence set forth in SEQ ID NO: 10 or 12.
  • the anti-hDLK wherein the heavy chain has an amino acid sequence selected from SEQ ID NO: 4, 8, 16, 20, 24, 28, 32 and 36, and the light chain has the amino acid sequence set forth in SEQ ID NO: 12 -1 antibody.
  • an antibody composition containing a glycosylated isomer to be purified is referred to as a "crude antibody product".
  • Crude antibody product Any crude antibody product can be used as long as it contains a glycosylated isomer.
  • crude antibody products include a biological composition such as plasma or a processed product thereof, and a culture medium of transformed cells into which an antibody gene has been integrated (a culture supernatant may be used; the same shall apply hereinafter) or a processed product thereof. can give.
  • bio-derived compositions include compositions containing antibodies obtained from transgenic non-human animals or plants. Transformed cells may be any cells that can bind sugar chains.
  • cell lines that have the property of binding sugar chains such as animal cells, plant cells or yeast cells, and more specifically cell lines that can bind sugar chains.
  • Chinese hamster ovary cells CHO cells
  • mouse myeloma cells NS0 cells, SP2/0 cells, rat myeloma cells YB2/0 cells, IR983F cells Syrian hamster kidney-derived BHK cells
  • human embryonic kidney-derived 293 cells human myeloma cells
  • human myeloma cells such as Namalwa cells, embryonic stem cells, or cells into which an antibody gene has been introduced, such as fertilized egg cells.
  • Various subspecies derived from primary immortalized cell lines can also be used as the aforementioned cell lines.
  • CHO K1 strain for CHO cells, CHO K1 strain, CHO DG44 strain, CHO S strain, and derivatives thereof can be used (Palsson et al., Nature Biotechnology 31(8), 759-765, 2013).
  • a crude antibody product By culturing these cells in a medium suitable for protein production, a crude antibody product can be obtained as a culture medium.
  • Any medium can be used, for example, serum-containing medium, medium containing no animal-derived components such as serum albumin or serum fractions, serum-free medium or protein-free medium, but preferably serum-free.
  • a medium, an animal-derived raw material-free medium, a protein-free medium, or a completely chemically defined medium is used.
  • the above crudely purified antibody includes a bio-derived composition or culture solution that has been subjected to filtration, salting out, one or more types of chromatography, pH adjustment, buffer replacement, concentration, dilution, etc., and a bio-derived composition. It is also possible to use a composition such as an intermediate composition obtained by performing an operation such as purification on a culture solution. As an intermediate composition that has undergone a purification operation, any solution after any unit operation for constructing the manufacturing process of an antibody drug can be used. For example, a composition after Protein A affinity chromatography, cation exchange chromatography, anion exchange chromatography, buffer exchange, low pH treatment, and filtration is desired.
  • the possibility of the presence of glycosylated isomers can be estimated from the amino acid sequence or gene sequence of the antibody.
  • the sugar chain binding site can be estimated by peptide mapping and mass spectrometry of the antibody.
  • a peptide-N-glycosidase (PNGase)-treated antibody and an untreated antibody are compared by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to determine changes in protein migration zone.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • the types of antibodies are as described above, and this method is suitable as a purification method in the production of antibodies as antibody pharmaceuticals, including therapeutic, diagnostic or prophylactic antibodies. This is because antibody drugs are required to have a constant content of antibody isomers contained therein, and it is necessary to reduce impurities derived from the target substance as much as possible (ICH Q6B guideline).
  • therapeutic or prophylactic antibodies include antibodies that bind to ligands and neutralize the activity of ligands, antibodies that bind to cell surface receptors and neutralize ligand binding, antibodies that bind to cell surface and Antibodies exhibiting cytotoxic activity can be mentioned.
  • diagnostic antibodies include antibodies that bind to ligands or cell surface receptors.
  • Cell-damaging activity includes antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, etc. mentioned.
  • chemically modified antibodies such as antibody-drug conjugates, radioisotope-labeled antibodies, fusion antibodies with cytokines, etc., multi-specific antibodies (Nature, 580 ( 16), 330-338 (2020)), the technology of the present invention can be used for antibody derivatives having sugar chains.
  • Fusion proteins include soluble TNF receptor Fc fusion proteins, CTLA4 modified Fc fusion proteins, Fc-TPOR agonist peptide fusion proteins, VEGF receptor-Fc fusion proteins, and the like. This method can be used to purify any of these antibodies or fusion proteins.
  • Tables 1 and 2 below contain the full-length sequences of the anti-human DLK-1 antibodies described in Patent Document 4 (WO2014/054820) supra (the underline indicates the signal sequence. In the mature protein, the underlined sequence is not included), but in the antibody, for example, the N of the NSS sequence surrounded by a square frame is the sugar chain binding consensus sequence. Therefore, when this gene is expressed in animal cells or the like, a sugar chain composition containing glycosylated isomers may be produced. Similarly, DLK-1 antibodies with sequences similar to those of the anti-DLK-1 antibody have similar potential. This method is useful as a method for removing or reducing glycosylated isomers from a crude antibody containing such glycosylated isomers.
  • the amino acid sequences of the H chain of A24G/T73K and the L chain of HuBA-1-3D are listed in SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, 66, and 70, respectively .
  • amino acid sequences of the mature proteins obtained by removing the signal sequence from these sequences are listed in SEQ ID NOs: 40, 44, 48, 52, 56, 60, 64, 68, and 72, respectively.
  • H chain means heavy chain
  • L chain means light chain.
  • the amino acid sequence of CDR1 of HuBA-1-3D VH1 and HuBA-1-3D VH2 is "DYAMH” (SEQ ID NO: 73)
  • the amino acid sequence of CDR2 is "VISTYYGNTNYNQKFKG” (SEQ ID NO: 74)
  • the amino acid sequence of CDR3 is "GGLREYYYAMDY”. ” (SEQ ID NO: 75).
  • the amino acid sequence of CDR1 of HuBA-1-3DVL is "KSSQSLLNSSNQKNYLA” (SEQ ID NO: 76)
  • the amino acid sequence of CDR2 is “FASTRES” (SEQ ID NO: 77)
  • the amino acid sequence of CDR3 is "QQHYSTPPT” (SEQ ID NO: 78).
  • Antibodies of the present invention may be antibodies having all or part of these CDRs. These CDR sequences were defined by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991).
  • VH means a heavy chain variable region
  • VL means a light chain variable region.
  • An antibody composition in which the content of the glycosylated isomer is reduced compared to the crude antibody is a crude antibody containing the glycosylated isomer. It can be obtained by subjecting it to conventional chromatography as a starting material. Therefore, the present invention provides a method for purifying a crude antibody product, wherein the crude antibody product is subjected to conventional chromatography to obtain an antibody having no sugar chain added to a site other than the Fc region glycosylation consensus region.
  • the present invention relates to a method in which the content of glycosylated isomers in which sugar chains are added to sites other than the region glycosylation consensus region is reduced compared to the crude antibody product before purification.
  • hydrophobic interaction chromatography carriers include hydrophobic functional groups such as methyl, ethyl, propyl, butyl, octyl, hexyl, propylene glycol, phenyl, alkylphenyl, benzyl and alkylbenzyl groups. to the base material can be used.
  • Examples of mixed-mode chromatography carriers include those obtained by mixing the above-described hydrophobic functional groups and ion-exchange functional groups in an arbitrary ratio.
  • hydrophobic functional groups for example, N-benzyl-N-methylethanolamine or the like can be used as the functional group.
  • Typical examples of cation-exchangeable functional groups include CM (Carboxymethyl, —O—CH 2 —COOH), SP (Sulfopropyl, —O—C 3 H 6 —SO 3 H) and the like.
  • exchangeability examples include DEAE (Diethylaminoethy, -O-C 2 H 4 -N-(C 2 H 5 ) 2 ), QAE (Quaternized aminoethyl or Diethyl-(2-hydroxypropyl)-aminoethyl, -O- C 2 H 4 —N—(C 2 H 5 ) 2 (CH 2 —CH(OH)—CH 2 )) and the like can be used.
  • Carrier bases include cellulose, Sephadex, cross-linked agarose, polyacridamides, methacrylates and various synthetic polymers. Either porous or non-porous supports can be used.
  • mixed-mode chromatography support includes those having functional groups such as calcium phosphate, such as hydroxyapatite ( Ca10 ( PO4) 6 (OH) 2 ) and fluoroapatite ( Ca10 (PO4) 6F2 ). is mentioned.
  • the chromatography carrier can be obtained and used as a commercial product. Specifically, Butyl-Sepharose (registered trademark) 4 Fast Flow (average particle diameter 90 ⁇ m), Butyl-S Sepharose 6 Fast Flow (average particle diameter 90 ⁇ m), Octyl Sepharose (registered trademark) 4 Fast Flow (average particle diameter 90 ⁇ m) ), Phenyl Sepharose (registered trademark) 6 Fast Flow (high sub) (average particle diameter 90 ⁇ m), Phenyl Sepharose (registered trademark) 6 Fast Flow (low sub) (average particle diameter 90 ⁇ m), Butyl Sepharose (registered trademark) High Performance (average particle size 90 ⁇ m), Phenyl Sepharose High Performance (average particle size 90 ⁇ m), SOURCE 15ETH (average particle size 15 ⁇ m), SOURCE 15 ISO (average particle size 15 ⁇ m), SOURCE 15PHE (average particle size 15 ⁇ m), Capto Phenyl (High Performance) ) (average particle size
  • the average particle size of the chromatography carrier used for the purification purpose of the present invention can be 15 ⁇ m or more, 20 ⁇ m or more, 30 ⁇ m or more, or 40 ⁇ m or more.
  • the liquid volume of the crude antibody product that can be processed by the chromatography carrier can be 100 mL or more, 1 L or more, 10 L or more, or 100 L or more.
  • the chromatography column packed with this carrier can have a capacity of about 100 mL to 1,000 L (diameter of about 5 cm to 2 m).
  • the amount of the crude antibody product subjected to chromatographic purification is at least 1 L or more, preferably 10 L or more, more preferably 100 L or more, still more preferably 500 L or more, and up to about 20,000 L.
  • the linear flow velocity in chromatography is 1,000 cm/hr or less, preferably 500 cm/hr or less.
  • the amount of antibody to be purified is 10 g or more, preferably 100 g or more, more preferably 1 kg or more, and still more preferably 10 kg or more in terms of protein amount.
  • the glycosylated isomers to be removed or reduced by this technique are contained in the first half fraction of the antibody eluted by chromatography, and the desired antibody of interest is eluted in the second half fraction to obtain a sugar chain. It separates and removes addition isomers.
  • an antibody non-glycosylated isomer antibody
  • glycosylated chains added only to at least the glycosylated consensus region, which is the target component
  • Conditions that allow adsorption to the chromatography carrier are required. Adsorption of antibodies is caused by interaction between the degree of hydrophobicity and hydrophilicity of antibodies and chromatography carriers.
  • buffers commonly used for hydrophobic interaction chromatography, hydrophobic chromatography, and mixed mode chromatography can be used.
  • Any antibody that is stable under chromatographic conditions may be used, such as phosphate buffer, acetate buffer, citrate buffer, Tris buffer, glycine buffer, borate buffer, tartrate buffer, MES buffer. , HEPES buffers, MOPS buffers, amino acid buffers, and mixed buffers thereof.
  • concentration of these buffer solutions can be arbitrarily selected within the generally used range of about 0.1 mM to 300 mM.
  • the pH of the buffer solution can be arbitrarily selected within the range of pH 4-8, but pH 4-6 is desirable.
  • Salts such as sodium sulfate, ammonium sulfate, sodium chloride, sodium citrate, etc., are added to these buffers in an appropriate amount as necessary to the extent that the antibody does not form a precipitate, thereby enhancing the interaction with the chromatography carrier. , adsorption to chromatographic supports.
  • One or more of the above salts can be arbitrarily selected, and the salt concentration used is such that the antibody can be stably present in the range of 300 mM to 2 M and the antibody can be sufficiently adsorbed to the chromatography carrier.
  • a salt concentration of about 1 M is preferred, and a low salt concentration that allows the antibody to be adsorbed is preferred.
  • the amount of antibody that can be adsorbed to the chromatography carrier per unit amount varies depending on the type of chromatography carrier and the conditions of the buffer solution, but 10 mg or more of antibody per 1 mg of carrier, preferably 20 mg or more of antibody per 1 mg of carrier should be adsorbed. can be done. Chromatographic operations can be performed at any temperature between 0°C and 40°C. Operation at room temperature is desirable, and operation under temperature-controlled conditions is more desirable.
  • the crude antibody containing glycosylated isomers is loaded on the chromatography, and then separated and purified by elution. Separation of glycosylated isomers by chromatography is carried out by adsorbing an antibody to a chromatography carrier, and then stepwise, continuously, or a combination of both to lower the salt concentration or raise the pH of the buffer flowing through the column. , reduced conductivity, or a combination thereof. After the antibody is adsorbed on the chromatography carrier, the glycosylated isomer is first eluted, and then the antibody with the sugar chain added only to the glycosylated consensus region (non-glycosylated isomer antibody) is eluted.
  • the term “elution” refers to weakening the binding of an antibody component bound to a chromatographic carrier by hydrophobic interaction or the like to the carrier and discharging the antibody component from the chromatographic carrier.
  • Conditions for elution of glycosylated isomers include buffer conditions where the interaction between the glycosylated isomer and the chromatography carrier is sufficiently weaker than the interaction between the non-glycosylated isomer antibody and the chromatography carrier. is.
  • the buffer for elution removal (washing) of the glycosylated isomers should have a salt concentration difference of 10 mM or more and/or 0.5 mM between the buffer for eluting the non-glycosylated isomer antibodies. Any buffer with a pH difference of 2 units or more can be used. After the non-glycosylated isomer antibody is adsorbed on the chromatography carrier at a high salt concentration (usually in the range of 300 mM to 2 M), the washing solution used to wash the chromatography carrier has a salt concentration equal to or higher than the elution salt concentration.
  • the salt concentration can be 10 mM or more higher than the concentration of 0.5 M or more, or 1.0 M or more, for example.
  • the pH of the washing solution can be in the range equal to or up to 2 units higher than the pH at which the antibody is adsorbed, or in the range equal to or up to 2 units lower than the pH at the time of elution. can.
  • the pH of the wash solution may be 0.2 units below the pH of the elution solution.
  • the pH of the wash solution may be in the range of pH 4-8.
  • the amount of buffer solution (washing solution) for eluting and removing glycosylated isomers is 2 CV or more, 3 CV or more, 4 CV or more, 5 CV or more, or 10 CV or more with respect to the chromatography carrier volume. , 15 CV or more, or 20 CV or more, or 5-20 CV, 5-15 CV, 5-10 CV, or 10-20 CV.
  • the change in pH and/or salt concentration from loading of the crude antibody product to washing may be stepwise (one or more steps, two or more steps, three or more steps, several steps, etc.). , may be continuous.
  • the pH range from loading to washing of antibody crudes is usually in the range of 4-6.
  • the conditions of salt concentration, pH and/or amount of washing solution are determined according to the content of the glycosylated isomer in the crude antibody used as a raw material, and the properties of the antibody itself such as the isoelectric point and amino acid sequence of the antibody. can be set as appropriate.
  • the method for separating glycosylated isomers by chromatography is to allow the glycosylated isomers to pass through the chromatography carrier (flow-through), and then separate the antibody having a glycosylated chain added only to the glycosylated consensus region. Elution may also be included, which allows the preparation of a purified antibody composition with high purity of the non-glycosylated isomeric antibody.
  • Flow-through refers to elution of glycosylated isomers out of the column without being adsorbed onto the chromatography carrier when the crude antibody product is loaded onto a column packed with the chromatography carrier.
  • the glycosylated isomer in the crude antibody may have a weak interaction with the chromatography carrier, but the equilibration buffer (preferably 1 column volume or more) is passed continuously or intermittently.
  • the equilibration buffer preferably 1 column volume or more
  • the interaction between the glycosylated isomer and the chromatographic carrier should be such that the interaction between the glycosylated isomer and the chromatographic carrier is such that the antibody and the chromatographic carrier have sugar chains bound only to the glycosylated consensus regions. Select a buffer with sufficiently weak conditions compared to the interaction.
  • a buffer solution with conditions such as salt concentration different from the above buffer solution is used to elute the bound antibody from the chromatography carrier to prepare a desired purified antibody composition.
  • the buffer that allows only the glycosylated isomers to pass through should have a salt concentration difference of 10 mM or more (10 mM or more higher salt concentration ), and/or any buffer solution with a pH difference of 0.2 units or more (0.2 or more lower pH) can be used as a wash-through solution.
  • the salt concentration of the buffer should be equal to or 1M higher (preferably 0.5M higher) than the salt concentration at which the non-glycosylated isomer antibody is eluted from the carrier. can be done.
  • the salt concentration at this time is usually in the range of 100% or more of the salt concentration for eluting the antibody.
  • the change in pH or/and salt concentration may be in one step or in several steps, and may be continuous.
  • the salt concentration and pH of the buffer solution are usually adjusted to an appropriate range before the crude antibody as a raw material is applied to the chromatography carrier.
  • the amount of the buffer solution (washing solution) to allow the glycosylated isomer to pass through is selected to be two times or more, preferably five times or more, the amount of the chromatography carrier.
  • the flow rate of the cleaning liquid More desirably, it is 10 to 20 times or more as the flow rate of the cleaning liquid.
  • the above salt concentration, pH and/or the amount of washing solution are appropriately set according to the content of glycosylated isomers in the crude antibody used as a raw material.
  • the amount of loading of the antibody in the crude antibody purification product affects the ability to separate glycosylated isomers, so that the resulting purified antibody composition has the desired yield and purity (
  • the amount of protein loaded per chromatography carrier is determined so that the content of glycosylated isomers).
  • chromatographic operations there is a limit to the amount of protein that can be adsorbed onto a carrier, and chromatographic operations are managed using parameters such as the maximum dynamic binding capacity (DBC) as indices.
  • DBC maximum dynamic binding capacity
  • the loading amount it is preferable to set the loading amount to a certain amount or more so as not to adsorb unnecessary glycosylated isomers.
  • the glycosylated isomers are separated and removed by loading 20 mg or more, 25 mg or more, 30 mg or more, or 35 mg or more and not more than the maximum dynamic adsorption capacity.
  • the amount of protein to be loaded can be determined according to the content of glycosylated isomers in the crude antibody product to be purified. That is, when the content of glycosylated isomers in the crude antibody product before chromatographic purification is high, the loading amount can be close to the maximum dynamic adsorption capacity.
  • the method of the present invention comprises a step of selecting a conventional chromatography carrier to be used according to the properties of the antibody to be separated, and a step of optimizing the separation and removal of glycosylated isomers by the selected chromatography carrier.
  • any two or more types of chromatography carriers are loaded with a crude antibody to be purified under the salt concentration and pH conditions described above, and sugar chains are added only to the glycosylation consensus region.
  • a chromatographic carrier with a large amount of adsorbed antibody to which is attached can be selected.
  • elution is carried out with an eluate whose salt concentration is reduced stepwise or continuously, so that only glycosylated isomers and/or glycosylated consensus regions in the eluate that has passed through the carrier are subjected to sugar chain addition.
  • the amount of the chain-attached antibody is measured, and a chromatography carrier that provides an eluate containing a small amount of glycosylated isomers and a large amount of the antibody having a sugar chain added only to the glycosylation consensus region is selected as a glycosylated carrier. It can be selected as a chromatographic support with better separation of adduct isomers.
  • Optimization steps for the separation and removal of glycosylated isomers by the selected chromatography carrier include the composition, concentration, and pH of the buffer solution used during loading, the type and concentration of salt to be added, and the crude antibody purification onto the chromatography carrier. Loading amount, composition, concentration, pH, number of washings and amount of washing solution during washing, composition, concentration and pH of buffer during elution, type and concentration of salt to be added, and method for changing them, sugar Examine and select conditions such as adsorption or straight-through removal of chain addition isomers. These optimization steps can be optimized for each parameter one by one, or the optimum conditions can be selected including the interaction of multiple parameters using statistical analysis methods such as design of experiments. can also
  • purification processes are usually constructed using three-step chromatography.
  • two or more of protein A affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography and the like are used in combination.
  • the method of the invention can be incorporated into any step of antibody chromatography using conventional chromatography during the purification process of these antibodies. Specifically, in the purification process using protein A affinity chromatography in the first step, cation exchange chromatography in the second step, and hydrophobic interaction chromatography in the third step, the hydrophobic interaction chromatography in the third step is performed.
  • the hydrophobic interaction chromatography in the third step is performed by the method of the present invention.
  • the hydrophobic interaction chromatography in the second step is performed by the method of the present invention.
  • the purification process uses protein A affinity chromatography in the first step, anion exchange chromatography in the second step, and hydrophobic interaction chromatography in the third step, the hydrophobic interaction chromatography in the third step of the present invention.
  • the step of mixed mode chromatography together with or without hydrophobic interaction chromatography may be included in the method of the present invention.
  • glycosylated isomers are efficiently removed, and a purified antibody composition containing glycosylated isomers in an amount reduced to the desired content can be provided.
  • the ratio of the amount of the glycosylated isomer to the amount of the total antibody in the purified antibody composition after purification by the method of the present invention can be 10% or less, more preferably 5% or less, 4% or less, or 3%. 2% or less, 1% or less, 0.5% or less, or 0.2% or less.
  • the ratio of the amount of glycosylated isomer contained in the crude antibody product before purification to the total amount of antibody may be 50% or more, 20% or more, 10% or more, or 5% or more.
  • the method of the present invention may be a method of obtaining an antibody with a reduced amount of glycosylated isomers at a step yield of 20% or more, 40% or more, or 50% or more.
  • the content and content ratio of the glycosylated isomer and the purification yield in the final purified antibody composition or the antibody composition after purification can be achieved by adjusting the above-described chromatographic parameters, preferably , glycosylation isomers are reduced to any amount and optimized to give an acceptable purification yield for antibody production.
  • glycosylated isomers can be confirmed by high performance liquid chromatography (HPLC: High Performance Liquid Chromatography) or ultra high performance liquid chromatography (UHPLC: Ultra High Performance Liquid Chromatography) for analytical evaluation.
  • HPLC High Performance Liquid Chromatography
  • UHPLC Ultra High Performance Liquid Chromatography
  • a carrier having an average particle size of 15 ⁇ m or less is used, and glycosylation isomers can be separated by chromatography at ultrahigh flow rate and ultrahigh pressure.
  • TSKgel Butyl-NPR column (average particle size 2.5 ⁇ m, Tosoh), TSKgel Phenyl-5PR column (average particle size 10 or 13 ⁇ m, Tosoh), TSKgel Ether-5PW column (average particle size 10 ⁇ m, Tosoh)
  • TSKgel BioAssist Phenyl column (average particle size 10 ⁇ m, Tosoh)
  • Protein-Pak Hi Res HIC column Waters
  • BioPro HIC column average particle size 2.3 or 4 ⁇ m, YMC
  • Proteomix HIC column (average Particle size 1.7 or 5 ⁇ m, MS equipment), AdvanceBio HIC column (average particle size 3.5 ⁇ m, Agilent)
  • MAbPac HIC-10LC column average particle size 5 ⁇ m, ThermoFisher
  • MAbPac HIC-20LC column average Particle size 5 ⁇ m, ThermoFisher
  • MAbPac HIC-Butyl LC column (average
  • a purified antibody composition purified by the method of the present invention can be used as an active ingredient of an antibody drug, as a therapeutic, preventive, or diagnostic agent for various diseases in humans and animals.
  • the CHO cell line DG44 was transformed with an expression vector having a gene encoding the amino acid sequence of the anti-hDLK-1 antibody to prepare a stable expression cell line pool DGC8-R-T11-14.2d.
  • a stable expression cell line pool DGC8-R-T11-14.2d.
  • culturing was carried out in a 1 L-scale DASGIP (registered trademark) Parallel Bioreactor System (manufactured by Eppendorf) bioreactor. Cultivation was carried out using a serum-free completely chemically defined medium under the conditions of pH 7 and 34° C. to 37° C. by fed-batch method for 13 days. This culture supernatant was purified by Protein A chromatography (MabSelect SuRe carrier (Cytiva)) to obtain an antibody composition.
  • DASGIP registered trademark
  • Parallel Bioreactor System manufactured by Eppendorf
  • HIC-HPLC analysis of antibody composition The obtained antibody composition was subjected to HIC-HPLC analysis under the following conditions.
  • HPLC apparatus Prominence HPLC System (Shimadzu Corporation) Analysis column: TSKgel Butyl-NPR column (Tosoh, 0014947; 4.6 mm ⁇ 3.5 cm, average particle size 2.5 ⁇ m)
  • Mobile phase A 0.1 M sodium phosphate buffer (pH 7.0) containing 2.3 M ammonium sulfate
  • Mobile phase B 0.1 M sodium phosphate buffer (pH 7.0) Analysis conditions: protein concentration 2 mg/mL x 10 ⁇ L injection, gradient 0-3 min; 0% B, 3-15 min; 0-100% B, 15-20 min; 100% B, flow rate 0.5 mL/min, detection wavelength 220 nm or 280 nm
  • Peak 3 is an antibody with a sugar chain attached only to the glycosylation consensus region
  • peak 2 is a glycosylation isomer with a sugar chain attached to the antibody in peak 3 at another site
  • peak 1 is a sugar chain in the antibody in peak 3.
  • the culture supernatant was purified by Protein A affinity chromatography, and the obtained antibody crude product was treated with peptide-N-glycosidase F (PNGase F) or not treated with PNGase F, and then subjected to reducing SDS polyacrylamide gel electrophoresis (SDS -PAGE) (silver staining).
  • PNGase F peptide-N-glycosidase F
  • SDS -PAGE SDS polyacrylamide gel electrophoresis
  • FIG. 4 shows the results of evaluation by the HIC-HPLC analysis method of Example 1 for the sample loaded onto the column, the fraction passed through the CaptoButyl column, and the fraction adsorbed onto the same column.
  • the 2.1% (Peak 1) and 12.1% (Peak 2) of the glycosylated isomers contained in the load sample were removed in the flow-through fraction (HIC389 Flow-through) and transferred to the column adsorption fraction (HIC389 Elute).
  • An antibody (Peak3) composition with 100% purity from which glycosylated isomers were removed was obtained.
  • glycosylation isomers can be separated by using a hydrophobic interaction chromatography carrier (Capto Butyl). It was found that when the selected chromatography carrier is used, glycosylated isomers are efficiently removed to the flow-through fraction, and a highly purified antibody composition can be obtained in the adsorbed fraction.
  • a hydrophobic interaction chromatography carrier Capto Butyl
  • Example 2 Separation of Glycosylation Isomers by Hydrophobic Interaction Chromatography Support 2
  • the culture supernatant containing the prepared humanized anti-human DLK-1 monoclonal antibody was purified by Protein A affinity chromatography, and a crude product of the same antibody containing about 10% glycosylated isomer was obtained. Obtained.
  • the pH was adjusted to 4.5, 4.7, or 5.0
  • the conductivity was adjusted to 70 mS / cm or less, and the product was transferred to a chromatography carrier. was used as a sample for loading.
  • Capto Butyl (average particle size 90 ⁇ m, manufactured by Cytiva) and POROS Benzyl Ultra (average particle size 50 ⁇ m, manufactured by Thermo Fisher) Hydrophobic interaction chromatography columns (carrier capacity 5 mL) ) was evaluated in the adsorption-desorption mode.
  • the amount of protein sample loaded onto each column was 20 mg/mL as protein amount per carrier, and the flow rate was 300 cm/h. Chromatography was performed under the following mobile phase conditions.
  • Mobile phase A 20 mM sodium citrate buffer (pH 4.5, 4.7, or 5.0) containing 1 M sodium chloride
  • Mobile phase B 20 mM sodium citrate buffer (pH 4.5, 4.7, or 5.0) 1.
  • the antibody is eluted throughout the entire washing/elution phase regardless of pH, and by collecting the middle layer of the elution, it is possible to obtain a high-purity antibody with no glycosylation other than the glycosylation consensus region. was shown.
  • the peak fluctuates depending on the pH (at pH 4.5, the antibody is well adsorbed and eluted from the column in the latter half of the elution, but at pH 5.0 about 80% is not adsorbed and during washing Antibodies are eluted), indicating that elution components can be controlled according to pH.
  • the salt concentration of the sample loading solution, the pH and salt concentration of the washing solutions (washing solution I, washing solution II), and the type and pH of the elution buffer are examined, and the antibody yield and non-glycosylation of the purified fraction are examined.
  • Isomeric antibody purity purity of antibody with no glycosylation other than the glycosylation consensus region
  • content of glycosylated isomers by HIC-HPLC analysis was measured. Table 3 shows the test results.
  • the yield is 40 to 40 with a purity of 97 to 100%. It was found to be controllable to 80% (Table 4).
  • the purity of the purified antibody composition tended to be higher with a protein loading of 30 g/L or 35 g/L rather than 25 g/L.
  • increasing the amount of washing fluid tended to improve the purity, although the yield of the resulting purified antibody composition decreased. This result indicated that the purity of the purified antibody composition could be increased by increasing the amount of washing solution under the condition of low protein load.
  • FIG. 6 shows the result of analyzing the results by the design of experiment (DoE).
  • DoE design of experiment
  • the position of the glycosylation isomer removal step in the purification process and the effect of the amount of washing solution on purity The culture supernatant containing the anti-hDLK-1 antibody prepared in the same manner as in Example 1 was purified by Protein A affinity chromatography. to obtain a crude product of the same antibody containing about 10% glycosylated isomer. This crude antibody product was kept at pH 3-4 for a certain period of time and neutralized, and used as a sample for loading onto a chromatography carrier.
  • This antibody crude product was purified in the order of hydrophobic interaction chromatography followed by mixed mode chromatography or mixed mode chromatography followed by hydrophobic interaction chromatography in the two types of flow shown in FIG. POROS Benzyl Ultra was used as the hydrophobic interaction chromatography carrier, and Capto MMC was used as the mixed mode chromatography carrier.
  • the conditions for hydrophobic interaction chromatography were as follows. ID No. HIC446, HIC457, and HIC463 were subjected to mixed-mode chromatography after hydrophobic chromatography, and ID No. HIC447 was subjected to mixed mode chromatography followed by hydrophobic chromatography.
  • Table 5 and Figure 8 show the yield and purity of the purified antibody composition obtained in the hydrophobic interaction chromatography step.
  • the yield was 60% or more, and the purity (indicating the amount of glycosylated isomer contaminant) by HIC-HPLC analysis was 99% or more. It was also found that the yield and purity can be controlled depending on the liquid volume (6 to 15 CV) of washing 1. That is, it was found that a composition with an antibody purity of about 90% improved to about 98% after 5 CV washing, about 99% after 8 CV washing, and about 99.5% after 15 CV washing.
  • the culture supernatant was filtered and subjected to Protein A affinity chromatography (MabSelect SuRe 10L; Cytiva), low pH virus inactivation, hydrophobic interaction chromatography using POROS Benzyl Ultra carrier (Thermo Fisher, 10L), Captoadhere carrier ( Cytiva, 10L), virus filtration, replacement with formulation buffer by tangential flow filtration (TFF), sterilization filtration, etc., to obtain a highly purified antibody composition.
  • Protein A affinity chromatography MobSelect SuRe 10L; Cytiva
  • POROS Benzyl Ultra carrier Thermo Fisher, 10L
  • Captoadhere carrier Cytiva, 10L
  • virus filtration replacement with formulation buffer by tangential flow filtration (TFF), sterilization filtration, etc.
  • the purified antibody composition finally obtained through all steps had a total yield of 46%.
  • the purity of the purified antibody composition was 100% by HIC-HPLC analysis, and did not contain glycosylated isomers.
  • glycosylation isomer removal step (Capto Butyl) A culture supernatant containing an anti-hDLK-1 antibody prepared in the same manner as in Example 1 was obtained. It was confirmed that about 10% of the glycosylated isomer was contained in the crude antibody product.
  • Protein A affinity chromatography step carrier: MabSelect SuRe
  • low pH virus inactivation step low pH virus inactivation step
  • hydrophobic interaction chromatography step Capto Butyl carrier
  • mixed mode chromatography step Capto adhere
  • the hydrophobic chromatography step improved the glycosylation isomer-free rate to 99.8% purity, and a purified antibody composition could be obtained with an overall yield of 25%. As shown in these results, it was found that a highly purified antibody composition can be obtained even by using a hydrophobic interaction chromatography carrier other than the POROS Benzyl Ultra carrier.
  • FIG. 9 shows the results of analyzing the crude antibody product (a) before fractionation and the purified antibody composition (b, c) after fractionation using HIC-HPLC shown in Example 1.

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