Purification method of antibody composition

Abstract

The present invention addresses the problem of providing a method for eliminating a glycosylation isomer from an antibody drug, etc. The present invention provides, for example, a purification method of an antibody composition, said purification method comprising loading the antibody composition on a conventional chromatography so as to allow the chromatography support to adsorb an antibody which has no sugar chain attached to a site other than the Fc region glycosylation consensus region, eluting the support with an elution solvent and thus eluting the antibody adsorbed on the support to thereby give a purified antibody composition.

Description

Method for purifying an antibody composition

[0001] The present invention relates to a method for producing an antibody composition, and more particularly to a method for purifying an antibody composition in which isomers in which sugar chains are added to sites other than the Fc region sugar chain binding consensus region are reduced.

[0002] Antibody drugs, which contain monoclonal antibodies as their active ingredients, are expected to serve as molecularly targeted drugs that take advantage of the high binding affinity and specificity of antibody molecules for antigens, and research and development has progressed accordingly. Antibody drugs have become essential for the treatment of various diseases, including cancer and autoimmune diseases, and nearly 100 products are currently approved and in use worldwide (Non-Patent Document 1, Non-Patent Document 2). Expectations for the development of new antibody drugs to satisfy unmet medical needs remain high, and it is expected that many new antibody drugs will continue to be researched and developed.

[0003] Antibodies (IgG) have an N-linked sugar chain at the 297th Asn residue (Asn297) in the glycosylation consensus region (Asn297-X-Ser / Thr, where X is any amino acid other than Pro) present in the Fc portion of their heavy chains. The sugar chains present in this glycosylation consensus region are known to contribute to the biological activity, blood kinetics, safety, and other aspects of antibody molecules (Non-Patent Documents 3 and 4). For example, it is known that removal of the Fuc residue (core fucose) attached to the N-acetylglucosamine (GlcNAc) residue at the reducing end of the N-linked sugar chain at Asn297 enhances antibody-dependent cellular cytotoxicity (ADCC) activity. Similarly, it has been found that the greater the number of galactose (Gal) residues at the non-reducing end of the sugar chain bound to the Fc portion, the stronger the binding to the first component of complement (C1q), resulting in enhanced complement-dependent cytotoxicity (CDC) activity (Non-Patent Document 5).

[0004] On the other hand, there are also well-known examples of antibodies (glycosylation isomers) in which sugar chains are bound to sites other than the glycosylation consensus region of the Fc region of the antibody. For example, it was confirmed that N-linked sugar chains are bound to the Fab portion of cetuximab, an anti-cancer drug produced in SP2 / 0 cells (Non-Patent Document 3). In addition to this, various reports on glycosylation isomers have been published (Non-Patent Documents 6, 7, and 8).

[0005] Glycosylation isomers are known to have the potential to affect various antibody properties, such as biological activity (Non-Patent Documents 10 and 11), immunogenicity (Non-Patent Documents 8 and 10), and blood half-life (Non-Patent Document 9). In particular, when a sugar chain is attached to the Fab region or near the CDR region, there is concern that biological activity may be reduced. Similarly, glycosylation isomers of antibodies to which a sugar chain is attached outside the glycosylation consensus region also raise safety concerns, such as immunogenicity.

[0006] If the addition of glycans outside the glycosylation consensus region significantly reduces biological activity or significantly increases immunogenicity, these glycosylation isomers will have undesirable negative effects on antibody pharmaceutical components, and are therefore considered to be product-derived impurities. Such product-derived impurities are undesirable as pharmaceutical components, and from a regulatory perspective, they should be removed as much as possible (Non-Patent Document 12).

[0007] When an antibody containing a consensus sequence to which a sugar chain may be added other than the glycosylation consensus region of the antibody's Fc region is identified as a candidate molecule for drug development, it is common to change the consensus sequence to a different amino acid sequence so that sugar chains will not bind to that site. In this case, the binding activity may be reduced compared to the performance of the original antibody, and it may not be possible to obtain an antibody with the desired therapeutic effect.

[0008] Glycosylation isomers in which glycans are attached to regions of an antibody other than the glycosylation consensus region, such as the Fab or CDR, can be evaluated and distinguished by separating the glycosylation isomers at a small-scale, high-performance analytical level. Methods that combine high-performance liquid chromatography and mass spectrometry to evaluate peptide fragments and methods that evaluate the glycans themselves cleaved from antibodies are known (Non-Patent Documents 6 and 9). There are also known cases in which antibodies with glycans attached to their Fab regions have been analyzed by hydrophobic interaction chromatography (HIC) (Non-Patent Document 13). However, these cases all use very small amounts of antibody for analytical purposes, and require methods with extremely high analytical performance, making the separated compositions unusable as antibody pharmaceuticals. Furthermore, a review of HIC analysis (Non-Patent Document 14) has disclosed the usefulness of the separation performance of many antibodies, but has not suggested that HIC purification methods can separate glycosylation isomers.

[0009] Thus, no method was known for separating and removing such glycosylation isomers and preparing an antibody composition that does not contain or has a sufficiently reduced amount of isomers with glycosylation outside the glycosylation consensus region. That is, with conventional techniques, glycosylation isomers can only be evaluated at the analytical level to determine the glycosylation status, and no method existed for separating and removing antibody glycosylation isomers and producing an antibody composition in an amount that can be provided as a pharmaceutical, nor were such antibody compositions known.

[0010] Therefore, when a glycosylation isomer is contained in an antibody Fc region other than the glycosylation consensus region, these components are not separated and are included as part of the antibody drug. The aforementioned Cetuximab has also been developed with a mixture composition in which glycosylation is added to both the glycosylation consensus region in the Fc region and the non-consensus region in the Fab (Non-Patent Document 3). If the glycosylation isomer does not have biological activity, it would undesirably result in an impurity derived from the target substance being included as a component of the drug.

[0011] Separation of glycosylated isomers by affinity chromatography using a lectin that specifically binds to a specific sugar chain has been reported (Non-Patent Documents 15 and 16). This method uses concanavalin A as a ligand and is known to have high separation properties, but because it is an affinity separation method and uses natural products, it is not suitable for the production of pharmaceuticals, and there are no examples of its application to the production of antibody pharmaceuticals.

[0012] Known techniques for separating glycan components other than antibodies include the separation of recombinant antithrombin by chromatography based on the difference in the number of glycans (Patent Document 1). Furthermore, recombinant erythropoietin and its derivatives have been separated by chromatography based on the difference in the number of sialic acids added (Patent Document 2). Similarly, there are cases in which ovalbumin and transferrin have been separated using isoelectric focusing chromatography (Patent Document 3). However, these techniques cannot be applied directly to antibodies, and no production methods for separating and removing glycosylated isomers from antibody compositions have been reported.

[0013] Another known technique for removing glycans is enzymatic treatment (Non-Patent Document 17). However, from a safety standpoint, the separation and removal of the enzyme and its origin are problematic, making it difficult to use this technique for manufacturing pharmaceuticals. When removing glycans from antibodies using enzymes, it is also impossible to distinguish between glycans in the glycosylation consensus region and those in the non-consensus region.

[0014] The anti-hDLK-1 antibody described in Patent Document 4 is an antibody that is expected to have antitumor activity.

[0015] International Publication No. WO 2008 / 120801 JP 2001-064300 A International Publication No. WO 2005 / 100379 International Publication No. WO 2014 / 054820

[0016] YAKUGAKU ZASSHI 137(7), 815-816 (2017) Bull. Natl. Inst. Health Sci. , 132, 36-46 (2014) CHROMATOGRAPHY, 34(2), 83-88 (2013) Protein Cell, 9, 63-73 (2018) Front. Immunol. , 8 (646), 1-8 (2017) J. Biol. Chem. , 284 (47), 32493-32506 (2009) Embo J. , 10, 2717-2723 (1991) J. Immunol. , 196, 1435-1441 (2016) Anal. Biochem. , 349, 197-207 (2006) Nature Review | Immunology, published online, 04 Feb, (2019) Biochem. J. , 338 (2), 529-538 (1999) ICH Q6 B Guideline Abs, 6 (4), 852-858 (2014) J. Pharm. Biomed. Anal. , 130, 3-18 (2016) J. Biol. Chem. , 285 (21), 16012-22 (2010) Oncotarget, 7 (21), 31166-76 (2016) J. Immunol. Methods, 467, 58-62 (2019)

[0017] Glycosylation sites other than the glycosylation consensus region of an antibody are undesirable from the viewpoints of biological activity and safety, and there is a need for the development of a technology that can remove glycosylation isomers contained in antibody pharmaceuticals to a sufficient level and that enables the easy preparation of homogeneous purified antibody compositions. Therefore, an objective of the present invention is to provide a method for removing glycosylation isomers from antibody pharmaceuticals.

[0018] In another aspect, an objective of the present invention is to obtain a purified composition of an anti-hDLK-1 antibody that is more effective and safer.

[0019] As shown by the above-mentioned conventional techniques, although there have been reports of separating glycosylation isomers by chromatography with precise and high resolution for the purpose of analytical evaluation, there have been no reports of manufacturing or preparation techniques capable of separating and removing glycosylation isomers of antibody pharmaceuticals by chromatography. As a result of intensive research to solve the above-mentioned problems, the present inventors surprisingly found that by using a conventional hydrophobic interaction chromatography support, and in particular by optimizing the adsorption and separation conditions for glycosylation isomers and target substances, it is possible to prepare purified antibody compositions with reduced glycosylation isomers.

[0020] Furthermore, the present inventors used their newly discovered method for reducing glycosylation isomers to separate glycosylation isomers and purify non-glycosylated isomers of the anti-hDLK-1 antibody described in Patent Document 4. Glycosylation isomers with glycans near the CDRs of an antibody may affect the binding activity of the antibody, but the degree of activity reduction is related to the size and binding position of the glycan, so it is possible that they do not affect activity. Surprisingly, the inventors found that the activity of the anti-hDLK-1 antibody described in Patent Document 4 is completely abolished by the glycosylation isomer, and therefore becomes an "impurity" in pharmaceuticals. Based on this, the present inventors succeeded in identifying glycosylation isomers as new impurities in crude anti-hDLK-1 antibody products, and by enabling their removal, they achieved the provision of a more effective and safer purified anti-hDLK-1 antibody composition.

[0021] That is, the present invention relates to the following (1) to (27): (1) A method for purifying an anti-hDLK-1 antibody composition, the heavy chain of which 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 the light chain of which has the amino acid sequence of SEQ ID NO: 10 or 12, the method comprising: loading a crude antibody product onto a conventional chromatography to adsorb an antibody that is not glycosylated at a site other than the Fc region glycosylation consensus region onto the chromatography carrier; and eluting the carrier with an elution solution to elute the antibody adsorbed to the carrier, thereby obtaining a purified antibody composition, wherein the content of a glycosylation isomer in which a glycosylation is added to a site other than the Fc region glycosylation consensus region in the obtained purified antibody composition is reduced compared to that of the crude antibody product. (2) The purification method according to (1), wherein the conventional chromatography support is a hydrophobic interaction chromatography support or a mixed-mode chromatography support. (3) The purification method according to (1) or (2), wherein the conventional chromatography support has an average particle size of 15 μm or more. (4) The purification method according to (1) or (2), wherein the conventional chromatography support has an average particle size of 20 to 100 μm. (5) The purification method according to any one of (1) to (4), wherein the conventional chromatography support has a benzyl group or a butyl group. (6) The purification method according to any one of (1) to (5), wherein the protein load per unit amount of the conventional chromatography support is 20 mg / mL or more. (7) The purification method according to any one of (1) to (6), wherein the carrier for conventional chromatography is a hydrophobic interaction chromatography carrier, and the method comprises washing the carrier with a washing solution after loading the crude antibody onto the carrier and before elution. (8) The purification method according to (7), wherein the salt concentration of the washing solution is 10 mM or more higher than the salt concentration of the elution solution. (9) The purification method according to (7) or (8), wherein the salt concentration of the washing solution is 0.5 M or more.(10) The purification method according to (7) or (8), characterized in that the salt concentration of the washing solution is 1.0 M or more. (11) The purification method according to any one of (7) to (10), characterized in that the pH of the washing solution is lower by 0.2 units or more than the pH of the elution solution and is within the range of pH 4 to 8. (12) The purification method according to any one of (7) to (11), characterized in that the washing is performed by passing a washing solution in an amount of at least two carrier volumes. (13) The purification method according to any one of (7) to (12), characterized in that washing and elution are performed by using a mobile phase whose pH and / or salt concentration changes stepwise or linearly. (14) The purification method according to any one of (7) to (13), characterized in that the elution solution has a salt concentration of 0.5 M or less or is salt-free and has a pH of 5 to 7. (15) The purification method according to any one of (1) to (14), characterized in that the yield is 20% or more. (16) The purification method according to any one of (1) to (15), wherein the ratio of glycosylation isomers to the total antibody in the purified antibody composition is 5% or less. (17) The purification method according to (16), wherein the ratio of glycosylation isomers to the total antibody in the crude antibody product is greater than 5%. (18) A method for purifying an anti-hDLK-1 antibody composition according to (1), comprising: loading a crude antibody product onto conventional chromatography using a carrier having a particle size of 20 to 100 μm and having a benzyl group or a butyl group; adsorbing an antibody that is not glycosylated at a site other than the Fc region glycosylation consensus region onto the carrier; removing glycosylation isomers by washing the carrier one or more times with a washing solution of pH 4 to 6; and eluting the antibody adsorbed onto the carrier with an elution solution of pH 5 to 7 having a salt concentration of 0.5 M or less or containing no salt to obtain a purified antibody composition, wherein the ratio of glycosylation isomers to total antibody in the purified antibody composition is reduced compared to that of the crude antibody product.(19) A method for purifying an antibody composition according to (1), comprising: loading the crude antibody product, adjusted to a salt concentration of 0.5 M or more, onto conventional chromatography using a carrier having a particle size of 20 to 100 μm and containing benzyl or butyl groups, to remove glycosylated isomers into a flow-through fraction; washing the carrier with a washing solution; and 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 to obtain a purified antibody composition, wherein the ratio of glycosylated isomers to total antibody in the purified antibody composition is reduced compared to that of the crude antibody product. (20) A method for purifying an antibody composition according to (1), comprising the steps of: loading the crude antibody product, adjusted to pH 4 to 6, onto conventional chromatography using a carrier having a particle size of 20 to 100 μm and having a benzyl group or a butyl group, to remove glycosylated isomers into a flow-through fraction; washing the carrier with a washing solution; and 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 to obtain a purified antibody composition, wherein the ratio of glycosylated isomers to total antibody in the purified antibody composition is reduced compared to the crude antibody product. (21) The method according to any one of (1) to (20), wherein the protein load per unit amount of carrier for the conventional chromatography is 20 g / L or more, and the washing is performed by passing a washing solution in an amount of 5 carrier volumes or more. (22) A method for producing an antibody composition, comprising the purification method according to any one of (1) to (21), in which the proportion of antibodies not glycosylated at sites other than the Fc region glycosylation consensus region is 95% or more relative to the total antibodies.(23) A method for producing a purified anti-hDLK-1 antibody composition, wherein 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 the light chain has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, comprising the steps of: loading a crude antibody onto conventional chromatography using a carrier having a particle size of 20 to 100 μm and having a benzyl group or a butyl group; adsorbing an antibody that is not glycosylated at a site other than the Fc region glycosylation consensus region onto the carrier; removing glycosylation isomers by washing the carrier one or more times with a washing solution containing 0.5 M or more of salt; and eluting the antibody adsorbed onto the carrier with an elution solution having a salt concentration of 0.5 M or less or a salt-free pH of 5 to 7 to obtain a purified antibody composition. (24) An antibody composition produced by the production method according to (22) or (23), wherein the proportion of glycosylation isomers relative to the total antibody in the purified antibody composition is reduced compared to that in the crude antibody product. (25) A method for removing glycosylation isomers in which a glycosylation site other than the Fc region glycosylation consensus region is attached from a crude anti-hDLK-1 antibody whose 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 whose light chain has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, the method comprising: loading the crude antibody onto a hydrophobic interaction chromatography carrier to adsorb an antibody that is not glycosylated at a site other than the Fc region glycosylation consensus region onto the carrier; washing the carrier with a washing solution; and eluting the antibody adsorbed to the carrier by eluting from the carrier with an elution solution to obtain a purified antibody composition. (26) An anti-hDLK-1 antibody composition, wherein 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 the light chain has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, wherein 95% or more of the total antibody is free of glycosylation at sites other than the Fc region glycosylation consensus region.(27) An anti-hDLK-1 antibody, wherein 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 the light chain has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, wherein no glycosylation occurs at a site other than the Fc region glycosylation consensus region.

[0022] The present invention makes it possible to reduce or remove glycosylation isomers in which a sugar chain is added to a region other than the glycosylation consensus region of an antibody.

[0023] A first effect of the present invention is to provide a medical antibody composition with a reduced proportion of glycosylation isomers, and thus a highly purified antibody composition with reduced glycosylation isomers purified by this method can be used as a pharmaceutical preparation with higher purity.

[0024] A second effect of the present invention is that it can provide a medical antibody composition from which glycosylation isomers have been removed. This allows for the provision of a composition with an extremely high purity of the active ingredient, i.e., a pharmaceutical composition with excellent efficacy and safety. In particular, the purified anti-hDLK-1 antibody composition purified in the present invention can be provided as a pharmaceutical composition with excellent efficacy and safety, since glycosylation isomers, which are inactive impurities, have been removed.

[0025]

[0023] Figure 1 is a graph showing the HIC-HPLC analysis pattern of a crude product obtained from the culture medium of cells producing a humanized anti-hDLK-1 monoclonal antibody. It is a photograph showing the results of SDS-PAGE analysis after Protein A purification of the culture supernatant of CHO cells transiently expressing a humanized anti-hDLK-1 monoclonal antibody. It is a graph showing the separation profile of an antibody composition using a Capto Butyl hydrophobic interaction chromatography support. It is a graph and a table showing the results of a purity analysis test by HIC-HPLC of a purified antibody composition separated using a Capto Butyl support. In the table, Peak 1 and Peak 2 represent the percentage (%) of glycosylated isomer antibodies (Peak 1 represents a glycosylated isomer antibody in which a glycosylated chain is bound to one CDR of the four polypeptide chains, and Peak 2 represents a glycosylated isomer antibody in which a glycosylated chain is bound to two CDRs), and Peak 3 represents the percentage (%) of an antibody in which a glycosylated chain is bound only to the glycosylation consensus region. Graph showing the separation profile of an antibody composition using a Capto Butyl carrier. Graph showing the separation profile of an antibody composition using a Poros Benzyl Ultra carrier. Graph showing the results of an experimental design analysis of the control of glycosylation isomer content by optimizing chromatography conditions using a POROS Benzyl Ultra carrier. Diagram showing the sequence of chromatography steps in the purification process of an antibody composition. Graphs showing the elution pattern, yield, and purity when the amount of washing solution in hydrophobic interaction chromatography is changed (top graph: conditions where washing solution 1 is 6 CV and washing solution 2 is 5 CV; middle graph: conditions where washing solution 1 is 8 CV and washing solution 2 is 5 CV; bottom graph: conditions where washing solution 1 is 15 CV and washing solution 2 is 5 CV). Analytical data of isolated components by precision fractionation of a crude antibody product using HIC-HPLC analysis are shown. (a) shows the results of HIC-HPLC analysis of a crude antibody product before isolation. (b) shows the results of HIC-HPLC analysis of the fraction collected as the main peak in HIC-HPLC of (a). (c) shows the results of HIC-HPLC analysis of the fraction collected as the pre-peak in HIC-HPLC of (a). Graphs showing the results of evaluating the ADCC activity of glycosylation isomers. The vertical axis shows fluorescence intensity indicating ADCC activity, and the horizontal axis shows antibody concentration.Circles indicate fractions collected as the main peak in Figure 9(b) (components not containing glycosylation isomers), squares indicate fractions collected as the front peak in Figure 9(c) (glycosylation isomers), and triangles indicate the results of the crude antibody product before isolation in Figure 9(a). The nucleotide sequence (SEQ ID NO: 37) and amino acid sequence (SEQ ID NO: 38) of the coding region for the H chain (γ1 chain) of HuBA-1-3D-1 are shown. Amino acids are represented by single letters, with the position of the termination codon indicated by a dot (•). The nucleotide sequence (SEQ ID NO: 41) and amino acid sequence (SEQ ID NO: 42) of the coding region for the H chain (γ1 chain) of HuBA-1-3D-2 are shown. Amino acids are represented by single letters, with the position of the termination codon indicated by a dot (•). The nucleotide sequence (SEQ ID NO: 69) and amino acid sequence (SEQ ID NO: 70) of the coding region for the L chain (κ chain) are shown. In the figure, amino acids are represented by single letters, and the position of the termination codon is indicated by a "." The nucleotide sequence (SEQ ID NO: 53) and amino acid sequence (SEQ ID NO: 54) of the coding region of the H chain (γ1 chain) of HuBA-1-3D-1 T73K are shown. Amino acids are represented by single letters, and the position of the termination codon is indicated by a "." In the deduced amino acid sequence (SEQ ID NO: 18) of the VH of HuBA-1-3D-1 T73K, the peptide consisting of 19 amino acids from the N-terminus is a signal peptide. The cDNA nucleotide sequence of the mature peptide of VH 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 nucleotide sequence (SEQ ID NO: 61) and amino acid sequence (SEQ ID NO: 62) of the coding region of the H chain (γ1 chain) of HuBA-1-3D-1 A24G / T73K are shown. Amino acids are represented by single letters, and the position of the termination codon is indicated by "." In the deduced amino acid sequence of VH of HuBA-1-3D-1 A24G / T73K (SEQ ID NO: 22), the peptide consisting of 19 amino acids from the N-terminus is a signal peptide. 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.

[0033] Figure 1 shows an amino acid sequence alignment of HuBA-1-3D-VH1, HuBA-1-3D-VH1 A24G, HuBA-1-3D-VH1 T73K, HuBA-1-3D-VH1 A24G / T73K, HuBA-1-3D-VH2, HuBA-1-3D-VH2 A24G, HuBA-1-3D-VH2 T73K, and HuBA-1-3D-VH2 A24G / T73K. The numbers in parentheses indicate the sequence numbers in the sequence listing.

[0026] The present invention is described in detail below. The scope of the present invention is not limited by these descriptions, and modifications other than those exemplified below can be made as appropriate without departing from the spirit of the present invention. This specification encompasses the entire specification of Japanese Patent Application No. 2021-079977 (filed May 10, 2021), from which priority is claimed. All publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference. As used herein, the term "glycosylation consensus sequence" or "glycosylation consensus sequence" refers to an amino acid sequence represented by Asn-X-Ser / Thr (where X is an amino acid other than Pro), regardless of the position of this sequence in the antibody or the position where Asn is present. As used herein, the terms "Fc region glycosylation consensus region," "glycosylation consensus region," or "glycosylation consensus region" refer to a glycosylation consensus sequence typically containing Asn297 present in the Fc portion of an antibody (typically, Asn297-X-Ser / Thr (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.

[0027] As used herein, the term "glycosylation isomer" refers to an antibody in which a glycosylation chain is attached to an amino acid other than the glycosylation consensus region. A glycosylation chain attached to an amino acid other than the glycosylation consensus region is referred to as a "non-consensus glycosylation chain." Regions to which non-consensus glycosylation chains are attached in glycosylation isomers include the Fab region, the Fv (variable) region which is the antigen-binding region, the complementary determining region (CDR) region, the Fc region other than the glycosylation consensus region, and the fusion sequence portion in a fusion antibody, and are typically the CDR region. The glycosylation mode of a non-consensus glycosylation chain to an amino acid other than the glycosylation consensus region may be an N-glycosylation chain to the glycosylation consensus sequence Asn-X-Ser / Thr (X is an amino acid other than Pro), or an O-glycosylation chain to Ser or Thr.

[0028] The structure of an antibody molecule is usually a heterotetramer, formed by the binding of two identical polypeptide chains in pairs. Therefore, theoretically, there are two binding sites for non-consensus glycans in each antibody molecule. The crude antibody product to be purified in the present invention may contain an antibody having one or more non-consensus glycans. The number of non-consensus glycans bound per antibody molecule may be one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. Furthermore, an antibody is composed of four polypeptide chains (usually two heavy chains and two light chains), and the number of non-consensus glycans bound to any one of the chains may be one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. The percentage of sugar chains bound to potential non-consensus sugar chain binding regions in a crude antibody product, i.e., the abundance of glycosylated isomers, may be 0.1% to 200% per antibody molecule, assuming that a non-consensus sugar chain is completely bound to one "heavy chain and light chain combination" or a corresponding structure in one antibody molecule as 100%, and is usually 1% to 50% when the method of the present invention is to be applied. Here, 200% means that a non-consensus sugar chain is bound to both (two sites) of the paired "heavy chain and light chain combination" in one antibody molecule.

[0029] As used herein, the term "antibody" includes not only full-length antibodies, but also antibody fragments, and fusions of full-length antibodies or antibody fragments with other substances, such as mouse antibodies, mouse-human chimeric antibodies, humanized antibodies, human antibodies, and amino acid variants, additions, deletions, substitutions, and glycosylation variants thereof. The immunoglobulin class of the antibody is not particularly limited, and may be any of the immunoglobulin classes (isotypes) of IgG, IgM, IgA, IgE, IgD, or IgY, with IgG being preferred. Furthermore, when the antibody of the present invention is an IgG, it may be any of the subclasses (IgG1, IgG2, IgG3, or IgG4). The antibody fragment is preferably an antigen-binding fragment, and is an 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 triplebodies, nanobodies, divalent VHHs, pentavalent VHHs, minibodies, (two-chain) diabodies, tandem diabodies, bispecific tribodies, bispecific bibodies, dual affinity retargeting molecules (DARTs), triabodies (or tribodies), tetrabodies (or [sc(Fv) ２ ] ２ , or (scFv-SA)4), disulfide-linked Fv (hereinafter referred to as "dsFv"), compact IgG, heavy chain antibody, or polymers thereof. Fusion products of antibody fragments with other substances include fusion proteins, particularly Fc fusion proteins.

[0030] In particular, the antibody referred to herein refers to an anti-hDLK-1 antibody whose 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 whose light chain has the amino acid sequence set forth in SEQ ID NO: 10 or 12. Preferably, the antibody is an anti-hDLK-1 antibody whose heavy chain has an amino acid sequence selected from SEQ ID NOs: 4, 8, 16, 20, 24, 28, 32, and 36, and whose light chain has the amino acid sequence set forth in SEQ ID NO: 12.

[0031] Herein, an antibody composition containing a glycosylation isomer to be purified is referred to as a "crudely purified antibody product." Any crude antibody product can be used as long as it contains a glycosylation isomer. Examples of crude antibody products include biologically derived compositions such as plasma or processed products thereof, and culture fluids (or culture supernatants; the same applies hereinafter) of transformed cells into which antibody genes have been incorporated or processed products thereof. Examples of biologically derived compositions include compositions containing antibodies obtained from transgenic non-human animals or plants. The transformed cells may be any cells capable of binding sugar chains. Specific examples include cell lines capable of binding sugar chains, such as animal cells, plant cells, or yeast cells. More specific examples include Chinese hamster ovary cells (CHO cells), mouse myeloma cells NS0 cells and SP2 / 0 cells, rat myeloma cells YB2 / 0 cells and IR983F cells, Syrian hamster kidney-derived BHK cells, human fetal kidney-derived 293 cells, human myeloma cells Namalwa cells, embryonic stem cells, and 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. For example, CHO cells such as CHO K1, CHO DG44, and CHO S strains, as well as derivatives thereof, can be used (Palsson et al., Nature Biotechnology 31(8), 759-765, 2013). These cells are cultured in a medium suitable for protein production to obtain a crude antibody product as a culture medium. The medium may be any medium, such as a serum-containing medium, a medium free of animal-derived components such as serum albumin or serum fractions, a serum-free medium, or a protein-free medium, but preferably a serum-free medium, a medium free of animal-derived materials, a protein-free medium, or a completely chemically synthesized medium.

[0032] Furthermore, the crude antibody product may be a biologically derived composition or culture medium that has been subjected to treatments such as filtration, salting out, one or more types of chromatography, pH adjustment, buffer exchange, concentration, dilution, or the like, or an intermediate composition obtained by subjecting a biologically derived composition or culture medium to purification or other procedures. The intermediate composition that has been subjected to a purification procedure may be a solution obtained after any unit operation in constructing a manufacturing process for an antibody pharmaceutical. For example, a composition obtained after Protein A affinity chromatography, cation exchange chromatography, anion exchange chromatography, buffer exchange, low pH treatment, or filtration is desirable.

[0033] In the case of N-glycans, the possibility of the presence of glycosylation isomers can be estimated from the amino acid sequence or gene sequence of the antibody. Furthermore, regardless of the type of glycan, the glycosylation site can be estimated by peptide mapping and mass spectrometry of the antibody. A simpler method is to compare an antibody treated with peptide-N-glycosidase (PNGase) with an untreated antibody by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), whereby glycosylation can be observed as a change in the protein migration band.

[0034] The types of antibodies are as described above, but this method is suitable as a purification method for the production of antibodies as antibody pharmaceuticals, including therapeutic, diagnostic, or prophylactic antibodies. This is because antibody pharmaceuticals require a consistent content of antibody isomers and impurities derived from the target substance must be reduced as much as possible (ICH Q6B guideline). Examples of therapeutic or prophylactic antibodies include antibodies that bind to a ligand and neutralize its activity, antibodies that bind to a cell surface receptor and neutralize ligand binding, and antibodies that bind to the cell surface and exhibit cell-cytotoxicity. Examples of diagnostic antibodies include antibodies that bind to a ligand or a cell surface receptor. Examples of cytotoxic activity include antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Furthermore, the technology of the present invention can be used for antibody derivatives having a sugar chain, such as chemically modified antibodies such as antibody-drug conjugates and radioisotope-labeled antibodies, fused antibodies with cytokines, etc., and multi-specific antibodies (Nature, 580(16), 330-338 (2020)).

[0035] More specifically, the present method can be used to purify antibodies against protein antigens (preferably protein antigens derived from humans) such as CD3, EGF receptor, CD20, respiratory syncytial virus, TNFα, CD25, IL-6 receptor, CD33, VEGF, IgE, complement C5, IL-12, IL-23, IL-1β, RANKL, CCR4, HER2, CD30, IL-5, IL-5 receptor, α4 integrin, α4β7 integrin, PD-1, CD52, IL-17, IL-17A, IL-17 receptor, CTLA-4, PCSK9, SLAMF7, BlyS, CD38, PD-L1, IL-4α receptor, CD22, CD23, factor IXa, factor X, CD19, sclerostin, and DLK-1. Examples of 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, etc. The present method can be used to purify any of these antibodies or fusion proteins.

[0036] One of the most desirable examples is the purification of anti-DLK-1 antibodies. Specifically, the humanized anti-human DLK-1 antibodies are those described in the aforementioned Patent Document 4 (WO2014 / 054820), such as antibodies comprising a heavy chain having 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 (particularly, heavy chains having these amino acid sequences as a variable region; the same applies hereinafter in this paragraph) and a light chain having an amino acid sequence set forth in SEQ ID NO: 10 or 12 (particularly, light chains having these amino acid sequences as a variable region; the same applies hereinafter in this paragraph), and preferably, antibodies comprising a heavy chain having an amino acid sequence selected from SEQ ID NOs: 4, 8, 16, 20, 24, 28, 32, and 36 and a light chain having the amino acid sequence set forth in SEQ ID NO: 12. Such a humanized anti-human DLK-1 monoclonal antibody has a glycosylation consensus sequence in the variable region of its light chain.

[0037] Tables 1 and 2 below contain the full-length sequences of the anti-human DLK-1 antibodies described in the aforementioned Patent Document 4 (WO 2014 / 054820) (the underlined portion indicates the signal sequence; the mature protein does not contain the underlined sequence). However, in these antibodies, for example, the N in the NSS sequence enclosed in a box is the glycosylation consensus sequence. Therefore, when this gene is expressed in animal cells or the like, there is a possibility that a glycosylation composition containing a glycosylation isomer will be produced. Similarly, DLK-1 antibodies having a sequence similar to that of the anti-DLK-1 antibody have the same possibility. This method is useful as a method for removing or reducing glycosylation isomers from crude antibody products containing such glycosylation isomers. The amino acid sequences of the HuBA-1-3D-1 H chain, HuBA-1-3D-2 H chain, HuBA-1-3D-1 A24G H chain, HuBA-1-3D-2 A24G H chain, HuBA-1-3D-1 T73K H chain, HuBA-1-3D-2 T73K H chain, HuBA-1-3D-1 A24G / T73K H chain, HuBA-1-3D-2 A24G / T73K H chain, and HuBA-1-3D L chain listed in the table below are listed in SEQ ID NOs: 38, 42, 46, 50, 54, 58, 62, 66, and 70, respectively. The amino acid sequences of the mature proteins obtained by removing the signal sequences from these sequences are set forth in SEQ ID NOs: 40, 44, 48, 52, 56, 60, 64, 68, and 72, respectively. In the present specification, "H chain" means a heavy chain, and "L chain" means a light chain.

[0038] 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), and the amino acid sequence of CDR3 is "GGLREYYYAMDY" (SEQ ID NO: 75). Furthermore, the amino acid sequence of CDR1 of HuBA-1-3D VL is "KSSQSLLNSSNQKNYLA" (SEQ ID NO: 76), the amino acid sequence of CDR2 is "FASTRES" (SEQ ID NO: 77), and the amino acid sequence of CDR3 is "QQHYSTPPT" (SEQ ID NO: 78). The antibodies of the present invention may have all or part of these CDRs. These CDR sequences were defined according to the definitions of Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). In the present specification, "VH" refers to a heavy chain variable region, and "VL" refers to a light chain variable region.

[0039]

[0040]

[0041] An antibody composition having a reduced content of glycosylation isomers compared to a crude antibody product (herein referred to as a "purified antibody composition") can be obtained by subjecting a crude antibody product containing glycosylation isomers to conventional chromatography as a starting material. Thus, the present invention relates to a method for purifying a crude antibody product, which comprises loading the crude antibody product onto conventional chromatography to adsorb antibodies that are not glycosylated at sites other than the Fc region glycosylation consensus region onto the carrier, and eluting the carrier with an elution solution to elute the antibodies adsorbed to the carrier, thereby obtaining a purified antibody composition, wherein the content of glycosylation isomers in which glycosylation isomers are added to sites other than the Fc region glycosylation consensus region in the obtained purified antibody composition is reduced compared to the crude antibody product before purification.

[0042] Carriers used in conventional chromatography can include hydrophobic interaction chromatography carriers, hydrophobic chromatography carriers, or mixed-mode chromatography carriers (also known as multi-mode chromatography carriers, preferably mixed-mode chromatography carriers having hydrophobic chromatographic properties). Examples of hydrophobic interaction chromatography carriers include those having hydrophobic functional groups such as methyl, ethyl, propyl, butyl, octyl, hexyl, propylene glycol, phenyl, alkylphenyl, benzyl, and alkylbenzyl groups bonded to a base substrate. Examples of mixed-mode chromatography carriers include those in which the hydrophobic functional groups and ion-exchange functional groups are mixed in any ratio. For example, N-benzyl-N-methylethanolamine can be used as the functional group. Representative examples of cation-exchange functional groups include CM (Carboxymethyl, -O-CH ２ -COOH), SP (Sulfopropyl, -OC ３ H ６ -SO ３ A typical anion exchanger is DEAE (Diethylaminoethylene, -O-C ２ H ４ -N-(C ２ H ５ ) ２ ), QAE (Quaternized aminoethyl or Dietyl-(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. Examples of the carrier base include cellulose, Sephadex, cross-linked agarose, polyacrylamide, methacrylate, and various synthetic polymers. The carrier can be used with or without micropores. Another form of mixed-mode chromatography carrier is hydroxyapatite (Ca10(PO ４ )６ (OH) ２ ) and fluoroapatite (Ca １０ (P.O. ４ ) ６ F ２ ) having a functional group such as calcium phosphate.

[0043] Chromatography carriers are commercially available and can be used. Specifically, Butyl-Sepharose (registered trademark) 4 Fast Flow (average particle size 90 μm), Butyl-S Sepharose 6 Fast Flow (average particle size 90 μm), Octyl Sepharose (registered trademark) 4 Fast Flow (average particle size 90 μm), Phenyl Sepharose (registered trademark) 6 Fast Flow (high sub) (average particle size 90 μm), Phenyl Sepharose (registered trademark) 6 Fast Flow (low sub) (average particle size 90 μm), Butyl Sepharose (registered trademark) High Performance (average particle size 90 μm), Phenyl Sepharose High Performance (average particle size 90 μm), SOURCE 15ETH (average particle size 15 μm), SOURCE 15ISO (average particle size 15 μm), SOURCE 15PHE (average particle size 15 μm), Capto Phenyl (High Sub) (average particle size 90 μm), Capto Butyl (average particle size 90 μm), Capto Octyl (average particle size 90 μm), Capto Phenyl ImpRes (average particle size 36-44 μm), Capto Butyl ImpRes (average particle size 36-44 μm), Capto adhere (average particle size 90 μm), Capto adhere ImpRes (average particle size 36-44 μm), Capto MMC (average particle size 90 μm), Capto MMC ImpRes (average particle size 36-44 μm), Capto Core 700 (average particle size 90 μm), ReadyToProcess Adsorber Phenyl (Cytiva, UK), TOYOPEARL (registered trademark) Butyl-600 (average particle size 40-90 μm), TOYOPEARL (registered trademark) Phenyl-600 (average particle size 40-90 μm), TOYOPEARL (registered trademark) PPG-600 (average particle size 40-90 μm), TOYOPEARL (registered trademark) Butyl-650 (average particle size 40-90 μm), TOYOPEARL (registered trademark) Phenyl-650 (average particle size 40-90 μm),TOYOPEARL (registered trademark) SuperButyl-550 (average particle size 40-90 μm), TOYOPEARL (registered trademark) Hexyl-650 (average particle size 50-150 μm), TOYOPEARL (registered trademark) Ether-650 (average particle size 40-90 μm) (all manufactured by Tosoh Corporation), POROS Ethyl (average particle size 50 μm), POROS Benzyl (average particle size 50 μm), POROS Benzyl Ultra (average particle size 50 μm) (all manufactured by Thermo Fisher), Macro-Prep t-Butyl HIC (average particle size 50 μm), Macro-Prep Methyl HIC (average particle size 50 μm), ceramic hydroxyapatite (average particle size 20-80 μm), ceramic fluoroapatite (average particle size 20-80 μm), BioGel HT (average particle size 20-80 μm) (all manufactured by Bio-Rad), QMA Spherosil (average particle size 50 μm), Methyl Ceramic Hyper D (average particle size 50 μm) (all manufactured by Pall Corporation), Fractogel EMD Phenyl (S) (average particle size 20-90 μm), Fractogel EMD Propyl (S) (average particle size 20-90 μm) (all manufactured by Merck), Cellufine MAX Phenyl (average particle size 40-130 μm), Cellufine MAX Phenyl Examples of carriers include Capto Butyl LS (average particle size 40-130 μm), Cellufine MAX Butyl (average particle size 40-130 μm) (all manufactured by JNC Corporation), butylated Chitopearl, and phenylated Chitopearl (all manufactured by Fujibo Holdings Co., Ltd.), and a more appropriate carrier is selected and used from these carriers. More preferred are carriers such as Capto Butyl, POROS Benzyl Ultra, and POROS Butyl Ultra.

[0044] The average particle size of the chromatography support used for the purification purpose of the present invention can be 15 μm or more, 20 μm or more, 30 μm or more, or 40 μm or more. The liquid volume of the crude antibody product that can be processed by the chromatography support can be 100 mL or more, 1 L or more, 10 L or more, or 100 L or more. Chromatography columns packed with this support can have a capacity of approximately 100 mL to 1,000 L (diameter approximately 5 cm to 2 m). The amount of 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, even more preferably 500 L or more, and up to approximately 20,000 L. Because such a large amount of crude antibody product must be processed, the linear flow rate 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 in terms of protein, preferably 100 g or more, more preferably 1 kg or more, and even more preferably 10 kg or more. The glycosylation isomers to be removed or reduced by this technology are contained in the first half of the antibody eluted by chromatography, and the desired antibody is eluted in the second half of the fraction, thereby separating and removing the glycosylation isomers.

[0045] Conditions for loading a crude antibody product containing a glycosylated isomer onto conventional chromatography must be such that at least the target component, an antibody with a glycosylation consensus region glycosylated only (a non-glycosylated isomer antibody), can be adsorbed to the chromatography support. Antibody adsorption occurs due to interactions between the antibody and the chromatography support, depending on the degree of hydrophobicity and hydrophilicity. Buffers commonly used in hydrophobic interaction chromatography, hydrophobic chromatography, and mixed-mode chromatography can be used. Any buffer that is stable to the antibody under chromatographic conditions can be used, including, for example, phosphate buffer, acetate buffer, citrate buffer, Tris buffer, glycine buffer, borate buffer, tartrate buffer, MES buffer, HEPES buffer, MOPS buffer, amino acid buffer, and mixtures thereof. The concentration of these buffers can be selected from a commonly used range of approximately 0.1 mM to 300 mM. The pH of the buffer solution can be selected arbitrarily within the range of pH 4 to 8, with pH 4 to 6 being preferred.

[0046] Salts such as sodium sulfate, ammonium sulfate, sodium chloride, and sodium citrate can be added to these buffers as needed, in an amount sufficient to prevent antibody precipitation, thereby enhancing the antibody's interaction with the chromatography support, thereby enabling adsorption to the chromatography support. One or more of the salts can be selected, and the salt concentration is set within the range of 300 mM to 2 M, at which the antibody is stable and sufficient for antibody adsorption to the chromatography support. A salt concentration of approximately 1 M is preferred, with low salt concentrations sufficient for antibody adsorption being preferred. The amount of antibody that can be adsorbed to a chromatography support per unit amount varies depending on the type of chromatography support and buffer conditions, but 10 mg or more of antibody can be adsorbed per mg of support, preferably 20 mg or more of antibody per mg of support. Chromatography can be performed at any temperature between 0°C and 40°C. Room temperature is preferred, and temperature-controlled conditions are even more desirable.

[0047] Under the above-mentioned conditions, a crude antibody product containing glycosylation isomers is loaded onto a chromatography column, followed by separation and purification by elution. Separation of glycosylation isomers by chromatography can be performed by adsorbing the antibody to a chromatography support, followed by decreasing the salt concentration, increasing the pH, or decreasing the conductivity of the buffer solution applied to the column, or by a combination of these, in a stepwise or continuous manner. After adsorbing the antibody to the chromatography support, the glycosylation isomers are primarily eluted first, followed by eluting the antibody with a glycosylation consensus region only (non-glycosylated antibody), thereby preparing the desired purified antibody composition. "Elution" refers to weakening the binding of antibody components bound to the chromatography support via hydrophobic interactions or the like, thereby removing the antibody components from the chromatography support. The conditions for eluting the glycosylated isomer are buffer conditions in which the interaction between the glycosylated isomer and the chromatography support is sufficiently weak compared to the interaction between the non-glycosylated isomer antibody and the chromatography support, and by washing the chromatography support with a sufficient amount of the buffer under these conditions, it is possible to elute and remove only the glycosylated isomer while leaving only the non-glycosylated isomer antibody, the antibody of interest, adsorbed to the chromatography support.

[0048] Any buffer can be used for eluting and removing (washing) glycosylated isomers as long as the difference in salt concentration between the buffer used to elute non-glycosylated isomer antibodies is 10 mM or more and / or the difference in pH between the buffer used to elute non-glycosylated isomer antibodies is 0.2 units or more. After non-glycosylated isomer antibodies are adsorbed onto a chromatography support at a high salt concentration (usually in the range of 300 mM to 2 M), the salt concentration of the washing solution used to wash the chromatography support can be the same or higher than the salt concentration used for elution. For example, the salt concentration can be the same as or in the range of from the same to 1 M higher, or in the range of from the same to 0.5 M higher, or the salt concentration can be 10 mM or higher than the salt concentration used for eluting the antibody, for example, 0.5 M or higher or 1.0 M or higher. The pH of the washing solution can be the same as or up to 2 units higher than the pH used for antibody adsorption, or the same as or up to 2 units lower than the pH used for elution. For example, the pH of the washing solution may be 0.2 units lower than the pH of the eluent. The pH of the washing solution may be within the range of pH 4 to 8. Furthermore, the amount of the buffer solution (washing solution) passed through to elute and remove glycosylated isomers may be 2 carrier volumes (CV) or more, 3 CV or more, 4 CV or more, 5 CV or more, 10 CV or more, 15 CV or more, or 20 CV or more, or 5 to 20 CV, 5 to 15 CV, 5 to 10 CV, or 10 to 20 CV relative to the volume of the chromatography carrier.

[0049] The change in pH and / or salt concentration from the loading of the crude antibody product to the washing may be stepwise (one or more steps, two or more steps, three or more steps, several steps, etc.) or continuous. The pH range from the loading of the crude antibody product to the washing is usually within the range of 4 to 6.

[0050] The above conditions of salt concentration, pH, and / or amount of washing solution can be appropriately set depending on the content of glycosylated isomers in the crude antibody product used as the starting material, and the properties of the antibody itself, such as the isoelectric point and amino acid sequence of the antibody.

[0051] The method for separating glycosylation isomers by chromatography herein may include flowing the glycosylation isomers through the chromatography support, followed by eluting antibodies glycosylated only at the glycosylation consensus region, thereby enabling the preparation of a purified antibody composition with a high purity of non-glycosylated isomer antibodies. Flowing through refers to the process of loading a crude antibody product onto a column packed with a chromatography support, and eluting the glycosylation isomers directly from the column without being adsorbed to the chromatography support. In this case, although glycosylation isomers in the crude antibody product may have a weak interaction with the chromatography support, components containing unwanted glycosylation isomers can be removed from the column by continuously or intermittently passing an equilibration buffer (preferably at least one column volume) through the column. As a buffer for specifically passing through a glycosylation isomer, a buffer is selected under conditions in which the interaction between the glycosylation isomer and the chromatography support is sufficiently weak compared to the interaction between the glycosylation isomer and the chromatography support and an antibody having a glycan bound only to the glycosylation consensus region. By using a sufficient amount of the buffer to pass through only the glycosylation isomer from the chromatography support, it is possible to adsorb only the target antibody to the chromatography support. Next, the bound antibody is eluted from the chromatography support using a buffer having different conditions, such as salt concentration, from the above buffer to prepare the desired purified antibody composition.

[0052] As a buffer solution that allows only glycosylated isomers to pass through, any buffer solution can be used as the pass-through wash solution as long as it has a salt concentration difference of 10 mM or more (a salt concentration 10 mM or more higher) and / or a pH difference of 0.2 units or more (a pH 0.2 or more lower) compared to the elution solution that elutes antibodies with glycans bound only to the glycosylation consensus region. The salt concentration of the buffer can be the same as or up to 1 M higher (preferably up to 0.5 M higher) than the salt concentration used to elute non-glycosylated isomer antibodies from the carrier. In this case, the salt concentration is usually 100% or more of the salt concentration used to elute the antibody. Alternatively, washing is performed with a wash solution whose pH is the same as or up to 2 units higher than the pH used for antibody adsorption, or whose pH is the same as or up to 2 units lower than the pH used for elution. The change in pH and / or salt concentration may be in one step, several steps, or continuous. Furthermore, the salt concentration and pH of the buffer solution are usually adjusted to an appropriate range before applying the starting crude antibody product to the chromatography support. The volume of the buffer solution (washing solution) passed through to allow glycosylated isomers to pass through is selected to be at least twice the volume of the chromatography support, preferably at least five times. The volume of the washing solution passed through is more preferably 10 to 20 times or more. The salt concentration, pH, and / or volume of the washing solution are appropriately set depending on the content of glycosylated isomers in the starting crude antibody product.

[0053] Since the load of antibody in a crude antibody product onto a chromatography support, i.e., the protein load, affects the separation ability of glycosylation isomers, the protein load per chromatography support is determined so that the resulting purified antibody composition has the desired yield and purity (glycosylation isomer content). Generally, in a chromatography operation, there is a limit to the amount of protein that can be adsorbed onto a support, and the chromatography operation is controlled using parameters such as the maximum dynamic binding capacity (DBC) as an indicator. Generally, by setting the protein load at or below the maximum dynamic binding capacity, the protein recovery rate and separation ability are improved, and the desired chromatographic separation effect can be expected. Furthermore, in the method of the present invention, a loading amount of at least a certain amount is preferred to prevent adsorption of unnecessary glycosylation isomers, and glycosylation isomers are separated and removed by loading at least 20 mg or more, 25 mg or more, 30 mg or more, or 35 mg or more of protein per unit amount of 1 g of chromatography support or per unit amount of 1 mL of chromatography support, but not more than the maximum dynamic adsorption capacity. The protein loading amount can be determined depending on the content of glycosylation isomers in the crude antibody product to be purified. That is, when the content of glycosylation isomers in the crude antibody product before chromatographic purification is high, a loading amount close to the maximum dynamic adsorption capacity can be used.

[0054] In one aspect, the method of the present invention includes the steps of selecting a conventional chromatography support to be used depending on the properties of the antibody to be separated, and optimizing the separation and removal of glycosylation isomers using the selected chromatography support.

[0055] The step of selecting a chromatography support can be carried out by first loading a crude antibody to be purified onto any two or more types of chromatography supports under the above-mentioned salt concentration and pH conditions, and selecting a chromatography support that adsorbs a large amount of antibody with a glycosylation consensus region. Furthermore, if necessary, elution is carried out with an eluate having a stepwise or continuous decrease in salt concentration, and the amount of glycosylation isomers and / or antibodies with a glycosylation consensus region in the eluate that has passed through the support is measured, and a chromatography support that gives an eluate with a small amount of glycosylation isomers and a large amount of antibody with a glycosylation consensus region can be selected as a chromatography support with better separation of glycosylation isomers.

[0056] The optimization step for separation and removal of glycosylated isomers using the selected chromatography support involves examining and selecting conditions such as the composition, concentration, and pH of the buffer solution used during loading, the type and concentration of salt added, the load amount of crude antibody product on the chromatography support, the composition, concentration, and pH of the buffer solution during washing, the number of washes and the amount of wash solution, the composition, concentration, and pH of the buffer solution during elution, the type and salt concentration of salt added and how to change these, and removal of glycosylated isomers by adsorption or flow-through. These optimization steps can be performed by optimizing each parameter one by one, or by using statistical analysis methods such as experimental design to select optimal conditions including the interaction of multiple parameters.

[0057] Antibody purification methods involve various steps. Many antibody purification processes are carried out using two or more different chromatography modes, but typically, a three-step purification process is used. For example, a combination of two or more of the following chromatography methods is used: protein A affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed-mode chromatography, and hydrophobic interaction chromatography. The method of the present invention can be incorporated into any of the conventional chromatography steps in these antibody purification processes. Specifically, in a purification process using protein A affinity chromatography as the first step, cation exchange chromatography as the second step, and hydrophobic interaction chromatography as the third step, the hydrophobic interaction chromatography step can be used as the method of the present invention. Furthermore, in a purification process using Protein A affinity chromatography in the first step, mixed-mode chromatography in the second step, and hydrophobic interaction chromatography in the third step, the hydrophobic interaction chromatography in the third step can be considered a method of the present invention. Furthermore, in a purification process using Protein A affinity chromatography in the first step, hydrophobic interaction chromatography in the second step, and mixed-mode chromatography in the third step, the hydrophobic interaction chromatography in the second step can be considered a method of the present invention. Furthermore, in a purification process using 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 can be considered a method of the present invention. Furthermore, in these chromatography sequences, the mixed-mode chromatography step may be used in addition to or instead of hydrophobic interaction chromatography.

[0058] The methods of the present invention efficiently remove glycosylation isomers and can provide purified antibody compositions containing glycosylation isomers at a desired reduced level. The ratio of the amount of glycosylation isomers to the total antibody amount in the purified antibody composition after purification by the methods of the present invention can be 10% or less, more desirably 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or 0.2% or less. Furthermore, the ratio of the amount of glycosylation isomers contained in a crude antibody product before purification to the total antibody amount may be 50% or more, 20% or more, 10% or more, or 5% or more. The methods of the present invention may also be methods for obtaining antibodies with reduced glycosylation isomer amounts at a step yield of 20% or more, 40% or more, or 50% or more.

[0059] The content and proportion of glycosylation isomers in the final purified antibody composition or the antibody composition after purification, as well as the purification yield, can be achieved by adjusting the aforementioned chromatographic parameters, which are preferably optimized so that glycosylation isomers are reduced to a desired level and a purification yield acceptable for antibody production is obtained.

[0060] The removal status of glycosylated isomers can be confirmed by analytical high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC). These analyses use a carrier with an average particle size of 15 μm or less, and allow separation of glycosylated isomers by chromatography at an ultra-high flow rate and ultra-high pressure. For example, TSKgel Butyl-NPR column (average particle size 2.5 μm, Tosoh Corporation), TSKgel Phenyl-5PR column (average particle size 10 or 13 μm, Tosoh Corporation), TSKgel Ether-5PW column (average particle size 10 μm, Tosoh Corporation), TSKgel BioAssist Phenyl column (average particle size 10 μm, Tosoh Corporation), Protein-Pak Hi Res HIC column (Waters Corporation), BioPro HIC column (average particle size 2.3 or 4 μm, YMC Corporation), Proteomix HIC column (average particle size 1.7 or 5 μm, MS Equipment Co., Ltd.), AdvanceBio HIC column (average particle size 3.5 μm, Agilent Corporation), MAbPac Suitable columns include an HIC-10 LC column (average particle size 5 μm, ThermoFisher), an MAbPac HIC-20 LC column (average particle size 5 μm, ThermoFisher), an MAbPac HIC-Butyl LC column (average particle size 5 μm, ThermoFisher), a Shim-pack Bio-HIC column (average particle size 4 μm, Shimadzu Corporation), a Shodex HIC PH-814 (average particle size 10 μm, Shoko Tsusho), and a COSMOSIL HIC (average particle size 5 μm, Nacalai Tesque).

[0061] The purified antibody composition purified by the method of the present invention can be used as an active ingredient in antibody pharmaceuticals, as therapeutic, preventive or diagnostic agents for various diseases in humans and animals.

[0062] The present invention will be described in more detail below using examples, but the scope of the present invention is not limited thereto.

[0063] Preparation of crude antibody product (culture supernatant) with sugar chains bound to sites other than the glycosylation consensus region of the antibody and evaluation of the composition ratio As a glycosylated antibody, a humanized anti-human DLK-1 monoclonal antibody (hereinafter referred to as "anti-hDLK-1 antibody") having an H chain and an L chain (H chain in the present application: SEQ ID NO: 64, L chain: SEQ ID NO: 72) described in WO 2014 / 054820 was used. Since this antibody has a glycosylation consensus sequence in the variable region of its light chain, when this gene is expressed in animal cells, etc., there is a possibility that a sugar chain composition containing a glycosylation isomer will be generated and become contaminated.

[0064] The CHO cell line DG44 was transformed with an expression vector carrying a gene encoding the amino acid sequence of the anti-hDLK-1 antibody, and a stable expression cell line pool, DGC8-R-T11-14.2d, was prepared. This cell line pool was used to culture in a 1 L-scale DASGIP® Parallel Bioreactor System (Eppendorf) bioreactor. Culture was carried out using a serum-free, chemically defined medium using a fed-batch method for 13 days at pH 7 and 34°C to 37°C. The culture supernatant was purified by Protein A chromatography (MabSelect SuRe carrier (Cytiva)) to obtain an antibody composition.

[0065] <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) Analytical column: TSKgel Butyl-NPR column (Tosoh, 0014947; 4.6 mm × 3.5 cm, average particle size 2.5 μm) 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 × 10 μL 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

[0066] As shown in Figure 1, three peaks were separated, with a main peak (Peak 3) detected at an elution time of approximately 13 minutes, and Peaks 2 and 1 detected at elution times of approximately 12.5 and 12.0 minutes, respectively. Peak 3 was thought to represent an antibody in which a glycosylation chain was bound only to the glycosylation consensus region, Peak 2 was thought to represent a glycosylation isomer in which a glycosylation chain was bound to an additional site on the antibody in Peak 3, and Peak 1 was thought to represent a glycosylation isomer in which a glycosylation chain was bound to two additional sites on the antibody in Peak 3 (see Example 2). This analysis confirmed that the crude antibody product obtained by culturing the above pooled cell line contained approximately 10% glycosylation isomers.

[0067] Preparation of a crude antibody product with a sugar chain bound to a site other than the glycosylation consensus region of the antibody (culture supernatant) and confirmation of glycosylation status. ExpiCHO cells were transformed with an expression vector (pFUSE) containing gene sequences encoding the amino acid sequences of the heavy and light chains of an anti-hDLK-1 antibody, and an expression vector (pFUSE) encoding the heavy chain amino acid sequence and a mutant sequence (Asn-Ser-Ala) in which a point mutation was introduced at the third amino acid of the glycosylation consensus sequence (Asn-Ser-Ser) of the light chain of the antibody, and transient expression of the antibody protein was performed. The culture supernatant was purified by protein A affinity chromatography, and the resulting crude antibody product was treated with or without peptide-N-glycosidase F (PNGase F) and analyzed by reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (silver staining). The anti-hDLK-1 antibody before mutation is referred to as the NSS antibody, and the anti-hDLK-1 antibody after mutation of the light chain glycosylation consensus sequence is referred to as the NSA antibody.

[0068] As a result, as shown in Figure 2, in the NSS antibody, a band with a molecular weight greater than 25 kDa was observed above the 25 kDa light chain band when the antibody was not treated with PNGase F, but this band disappeared after treatment with PNGase F. This confirmed that in the NSS antibody, a sugar chain is bound to a light chain having a consensus sequence other than the glycosylation consensus region of the antibody. On the other hand, this band was not observed in the NSA, regardless of whether or not the antibody was treated with PNGase F. These results demonstrated that the crudely purified product of the transiently expressed humanized anti-DLK-1 monoclonal antibody (NSS antibody) contains a glycosylated isomer in which a sugar chain is also attached to the light chain.

[0069] Separation of glycosylated isomers using a hydrophobic interaction chromatography support 1. The culture supernatant containing the prepared anti-hDLK-1 antibody was purified by Protein A affinity chromatography to obtain a crude antibody product containing approximately 10% glycosylated isomers in the same manner as in Example 1. Sodium chloride was added to this crude antibody product to a final concentration of 1.0 M, and the pH was adjusted to 5.0 to prepare a sample for loading onto the chromatography support.

[0070] Using this crude antibody product, the adsorption characteristics on a hydrophobic interaction chromatography column (carrier volume 5 mL) packed with Capto Butyl (average particle size 90 μm, manufactured by Cytiva) were evaluated in adsorption-desorption mode. The sample load on the column was 15 mg / mL in terms of 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-15 mM Tris buffer (pH 5.0) containing 1 M sodium chloride. Mobile phase B: 20 ​​mM sodium citrate-15 mM Tris buffer (pH 5.0). 1. Equilibration: 100% mobile phase A; 10 column volumes (CV). 2. Load: crude antibody product sample. 3. Wash: 100% mobile phase A; 10 CV. 4. Linear gradient elution: 0% mobile phase B to 100% mobile phase B in 40 CV. 5. Column wash: 1 M sodium hydroxide; 5 CV.

[0071] The elution chromatogram (HIC389) is shown in Figure 3. This result confirms the adsorption of antibody components to the carrier under linear gradient conditions using the Capto Butyl carrier. The sample loaded onto the column, the flow-through fraction from the Capto Butyl column, and the adsorbed fraction were evaluated by the HIC-HPLC analysis method described in Example 1. Figure 4 shows the results. Glycosylation isomers, present at 2.1% (Peak 1) and 12.1% (Peak 2) in the loaded sample, were removed from the flow-through fraction (HIC389 Flow-Through), and the adsorbed fraction (HIC389 Elute) from the column yielded a 100% pure antibody composition (antibody with no glycosylation outside the glycosylation consensus region) (Peak 3) from which glycosylation isomers had been removed.

[0072] These results demonstrate that glycosylation isomers can be separated by using a hydrophobic interaction chromatography support (Capto Butyl). Using the selected chromatography support, glycosylation isomers were efficiently removed into the flow-through fraction, and a highly purified antibody composition was obtained in the adsorption fraction.

[0073] Separation of glycosylated isomers using a hydrophobic interaction chromatography support 2 A culture supernatant containing the prepared humanized anti-human DLK-1 monoclonal antibody was purified by Protein A affinity chromatography to obtain a crude antibody product containing approximately 10% glycosylated isomers in the same manner as in Example 1. Sodium chloride was added to this crude antibody product to a final concentration of 1.0 M, and the pH was adjusted to 4.5, 4.7, or 5.0 and the conductivity to 70 mS / cm or less to prepare a sample to be loaded onto the chromatography support.

[0074] Using this crude antibody product, the adsorption characteristics of each hydrophobic interaction chromatography column (carrier volume 5 mL) was evaluated in adsorption-desorption mode: Capto Butyl (average particle size 90 μm, manufactured by Cytiva) and POROS Benzyl Ultra (average particle size 50 μm, manufactured by Thermo Fisher). The protein sample was loaded onto each column at a protein amount per carrier of 20 mg / mL, 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. Equilibration: 100% mobile phase A; 5 CV. 2. 1. Load: Sample solution 2. Wash: 100% mobile phase A; 8 CV 3. Wash 1: 100% mobile phase A; 5 CV 4. Wash 2: 100% mobile phase B + 0.8 M sodium chloride; 5 CV 5. Elution: 100% mobile phase B; 10 CV 6. Column wash: Water (appropriate amount) 7. Column wash: 1 M sodium hydroxide (appropriate amount)

[0075] The elution chromatograms for Capto Butyl and POROS Benzyl Ultra are shown in Figures 5A and 5B, respectively. Under all pH conditions, the antibody composition was adsorbed to the Capto Butyl carrier and subsequently eluted. Good adsorption and elution were also observed with POROS Benzyl Ultra. Specifically, in the graphs of Figures 5A and 5B, the peaks near 0 to 50 mL on the horizontal axis represent antibodies (glycosylation isomers) that were not adsorbed and passed through the column, while the peaks eluted between 50 and 300 mL represent antibodies with no glycosylation outside the glycosylation consensus region that were gradually eluted from the column. With Capto Butyl, antibodies were eluted throughout the entire wash / elution range, regardless of pH. This demonstrates that highly pure antibodies with no glycosylation outside the glycosylation consensus region can be obtained by recovering the intermediate layer of elution. Furthermore, with Poros Benzyl Ultra, 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, approximately 80% of the antibody is not adsorbed and elutes during washing), demonstrating that the eluted components can be controlled depending on the pH. These results demonstrate that glycosylation isomers can be separated using either Capto Butyl or POROS Benzyl Ultra, demonstrating the feasibility of separating glycosylation isomers using a hydrophobic interaction chromatography support. Furthermore, these results demonstrate that antibody compositions can be adsorbed and glycosylation isomers can be removed by optimizing the pH, salt concentration, and volume of the wash / elution solutions in hydrophobic interaction chromatography.

[0076] Investigation of glycosylated isomer removal efficiency (purity and yield) in hydrophobic interaction chromatography supports depending on salt concentration during loading, salt concentration and pH during washing, and type of elution buffer. Capto Butyl and POROS Benzyl Ultra chromatography supports were investigated for their ability to remove glycosylated isomers by stepwise elution. The loading solution used was the crude antibody product of Example 1 purified by Protein A affinity chromatography, adjusted to pH 4.5 or pH 5.0 with 0.5 M Tris. The salt concentration of the sample loading solution, the pH and salt concentration of the washing solutions (washing solution I and washing solution II), and the type and pH of the elution buffer were investigated as parameters, and the antibody yield and non-glycosylated isomer antibody purity (purity of antibodies with no glycosylation outside the glycosylation consensus region) (glycosylated isomer content by HIC-HPLC analysis) of the purified fraction were measured. The test results are shown in Table 3.

[0077]

[0078] The purification yields using the Capto Butyl carrier under each condition were 23% to 73%, and the purity of the purified antibody composition (antibody component not containing glycosylated isomers) by HIC-HPLC analysis was 97.1% to 99.3%. On the other hand, although the purification yields using the POROS Benzyl Ultra carrier were somewhat lower (33% to 67%), the purity of the purified antibody composition by HIC-HPLC analysis was always 100%. In particular, when using the POROS Benzyl Ultra carrier, the optimal conditions in terms of purity and yield were loading a sample containing 0.8 M sodium chloride, washing with wash solution I containing 0.8 M sodium chloride and wash solution II containing 0.6 M sodium chloride in that order, and elution with sodium citrate buffer at pH 6.

[0079] Furthermore, a series of tests revealed the following: (i) Increasing the pH of the washing solution (washing solution I and / or washing solution II) improves purity. (ii) Increasing the pH of the washing solution (washing solution I and / or washing solution II) reduces yield. (iii) Increasing the pH of the elution solution improves purity. (iv) Increasing the pH of the elution solution reduces yield. (v) Reducing the salt concentration of the loading solution and / or washing solution reduces yield. (vi) Conditions can be established that enable the antibody composition to achieve a purity of 95% or more, ideally 99% or more.

[0080] As shown by the above results, it was found that in hydrophobic interaction chromatography supports (e.g., Capto Butyl and POROS Benzyl Ultra), the yield and the amount of contaminating glycosylated isomers can be optimized by optimizing the salt concentration and pH of the loading solution, the salt concentration and pH of the washing solution, the volume of the washing solution, the number of washings, the pH during elution, and the type of buffer.

[0081] Optimization of Washing Conditions for Controlling the Contamination Rate of Glycosylated Isomers and the Purity and Yield of the Target Substance Culture supernatant containing an anti-hDLK-1 antibody prepared in the same manner as in Example 1 was purified by Protein A affinity chromatography to obtain a crude antibody product containing approximately 10% glycosylated isomers. Sodium chloride was added to this crude antibody product to a final concentration of 1.0 M, and the pH and conductivity were adjusted appropriately to prepare a sample for loading onto a chromatography support. Using this crude antibody product and a POROS Benzyl Ultra support, the yield and purity, which are output parameters, were evaluated by HIC-HPLC analysis, with the sample load, the volumes of Wash Solution I and Wash Solution II as input parameters.

[0082] The results showed that by adjusting the protein load per unit amount of chromatography support to 25 g / L or more and adjusting wash solution I and wash solution II to 5 to 15 column volumes (CV), it was possible to control the yield to 40 to 80% with a purity of 97 to 100% (Table 4). In particular, the purity of the purified antibody composition obtained tended to be higher when the protein load was 30 g / L or 35 g / L compared to 25 g / L. Furthermore, increasing the wash solution volume tended to decrease the yield of the purified antibody composition but improve its purity. These results indicated that, under low protein load conditions, increasing the wash solution volume could increase the purity of the purified antibody composition. These results demonstrated that the content of glycosylated isomers could be reduced and controlled from approximately 10% to a range of 3 to 0% by maintaining a certain protein load or higher in chromatography and / or by appropriately controlling the wash solution volume. The results of the analysis of the results by design of experiment (DoE) are shown in Figure 6. The influence of the volume of cleaning solution I and cleaning solution II on the yield and purity is shown as a contour map, and it is clear that the desired yield and purity can be controlled by the volume of the cleaning solution.

[0083]

[0084] Effect of the position of the glycosylation isomer removal step within the purification process and the amount of wash solution on purity A culture supernatant containing an anti-hDLK-1 antibody prepared in the same manner as in Example 1 was purified by Protein A affinity chromatography to obtain a crude antibody product containing approximately 10% glycosylation isomers. This crude antibody product was neutralized by maintaining it at pH 3 to 4 for a certain period of time and used as a sample to be loaded onto the chromatography support.

[0085] This crude antibody product was purified using the two flow charts shown in Figure 7, either by hydrophobic interaction chromatography followed by mixed-mode chromatography, or by mixed-mode chromatography followed by hydrophobic interaction chromatography. POROS Benzyl Ultra was used as the hydrophobic interaction chromatography support, and Capto MMC was used as the mixed-mode chromatography support. The hydrophobic interaction chromatography conditions were as follows: ID Nos. HIC446, HIC457, and HIC463 were subjected to hydrophobic chromatography followed by mixed-mode chromatography, and ID No. HIC447 was subjected to mixed-mode chromatography followed by hydrophobic chromatography.

[0086] <Conditions for hydrophobic interaction chromatography> Carrier: POROS Benzyl Ultra Column size: 260 mL, bed height 13.3 cm Flow rate: 300 cm / h Protein load per carrier: 30 g / L Chromatography conditions: 1. Equilibration: 20 mM sodium citrate (pH 5.0) containing 0.8 M sodium chloride; 5 CV 2. Load: Crude antibody sample 3. Wash 1: 20 mM sodium citrate (pH 5.0) containing 0.8 M sodium chloride; 6 CV (HIC446 / HIC447), 8 CV (HIC457), or 15 CV (HIC463) 4. Wash 2: 20 mM sodium citrate (pH 5.0) containing 0.6 M sodium chloride; 5 CV 5. 6. Elution: 20 mM sodium citrate (pH 6.0); 6.3 CV 6. Column washing: 1 M sodium hydroxide aqueous solution; 3 CV

[0087] The yield and purity of the purified antibody composition obtained in the hydrophobic interaction chromatography step are shown in Table 5 and Figure 8. In all purification flows, the yield was 60% or higher, and the purity (indicating the amount of contaminating glycosylation isomers) obtained by HIC-HPLC analysis was 99% or higher. It was also revealed that the yield and purity could be controlled depending on the volume of wash 1 (6 to 15 CV). That is, it was found that a composition with an antibody purity of approximately 90% was improved to approximately 98% by 5 CV washing, to approximately 99% by 8 CV washing, and to approximately 99.5% by 15 CV washing.

[0088]

[0089] Purification process including a glycosylation isomer removal step (POROS Benzyl Ultra) Approximately 200 L of culture supernatant containing an anti-hDLK-1 antibody was obtained in the same manner as in Example 1. It was confirmed that the glycosylation isomer was contained in an amount of approximately 10% in the crude antibody product.

[0090] The culture supernatant was filtered and subjected to steps such as Protein A affinity chromatography (MabSelect SuRe 10 L; Cytiva), low-pH virus inactivation, hydrophobic interaction chromatography using POROS Benzyl Ultra carrier (Thermo Fisher, 10 L), mixed-mode chromatography using Capto adhere carrier (Cytiva, 10 L), virus filtration, substitution with a formulation buffer by tangential flow filtration (TFF), and sterile filtration to obtain a highly purified antibody composition.

[0091] For POROS Benzyl Ultra chromatography, the protein loading per unit amount of carrier was adjusted to 35 g / L, 30 g / L, and 16 g / L, and three chromatography runs were performed. The conditions and results of the POROS Benzyl Ultra chromatography step are shown in Tables 6 and 7, respectively.

[0092]

[0093]

[0094] The loading amounts for Run 1 and Run 2 were within the appropriate range, exceeding the specified amount, but the loading amount for Run 3 was low at 16 g / L. As a result, the yield was 77%, but the HIC-HPLC purity, which indicates the amount of glycosylated isomer contamination, was a low 89.74%. Therefore, only the fractions from Run 1 and Run 2 were combined and subjected to the next chromatography step.

[0095] The purified antibody composition finally obtained after all steps had a total yield of 46%. Furthermore, the purity of the purified antibody composition as determined by HIC-HPLC analysis was 100%, and it contained no glycosylation isomers. Thus, even under antibody chromatography conditions, it was possible to obtain a purified antibody composition with sufficiently reduced glycosylation isomers. It was also demonstrated that the protein load on the chromatography support is important for the removal rate of glycosylation isomers.

[0096] Purification process including a glycosylation isomer removal step (Capto Butyl) A culture supernatant containing an anti-hDLK-1 antibody was obtained in the same manner as in Example 1. It was confirmed that the glycosylation isomer was contained in an amount of approximately 10% in the crude antibody product.

[0097] This culture medium was used for purification in the following order: Protein A affinity chromatography step (carrier: MabSelect SuRe), low pH virus inactivation step, hydrophobic interaction chromatography step (Capto Butyl carrier), and mixed-mode chromatography step (Capto adhere).

[0098] As shown in Table 8, the hydrophobic chromatography step improved the rate of non-content of glycosylation isomers to 99.8% purity, and a purified antibody composition was obtained with an overall yield of 25%. These results demonstrate that a highly purified antibody composition can also be obtained using a hydrophobic interaction chromatography support other than the POROS Benzyl Ultra support.

[0099]

[0100] Fractionation of glycosylated isomers and measurement of biological activity A crude anti-hDLK-1 antibody containing approximately 10% glycosylated isomers obtained in the same manner as in Example 1 was precisely fractionated into each peak using the HIC-HPLC analysis system described in Example 1. Fractionation was carried out approximately 10 times, and after combining the peaks, the buffer was exchanged with an isotonic phosphate buffer. The mobile phase conditions for the HIC-HPLC used for fractionation were changed as follows: Mobile phase A: 50 mM phosphate buffer containing 2.3 M ammonium sulfate, pH 7 Mobile phase B: 50 mM phosphate buffer, pH 7

[0101] The crude antibody product before fractionation (a) and the purified antibody compositions after fractionation (b, c) were analyzed using HIC-HPLC as described in Example 1, and the results are shown in FIG.

[0102] These samples were diluted 3-fold from 10 μg / mL and assayed for ADCC activity using the ADCC Reporter Bioassay kit (Promega, G7010) at an E:T ratio of 6:1 (target cells were a HEK293 cell line highly expressing DLK1) for 6 hours. The results are shown in Figure 10. The crude antibody product before fractionation and the main peak after fractionation (Figure 9 (b)) exhibited nearly identical sigmoid curves, confirming their equivalent biological activity. On the other hand, the glycosylation isomer isolated by fractionation (Figure 9 (c)) showed almost no signal at any concentration, indicating its lack of biological activity. These results confirmed that glycosylation isomers are impurities that must be removed as much as possible from biopharmaceuticals.

[0103] According to the present invention, a purified antibody composition in which glycosylation isomers other than those in the Fc region glycosylation consensus region are reduced can be prepared and utilized.

Claims

1. 1. A method for purifying an anti-hDLK-1 antibody composition, wherein 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 the light chain has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, comprising: loading the crude antibody product onto conventional chromatography to adsorb the antibody having no glycosylation at a site other than the Fc region glycosylation consensus region onto a support for said chromatography; and and eluting the carrier with an elution solution to elute the antibody adsorbed on the carrier to obtain a purified antibody composition. wherein the content of glycosylation isomers in which a glycochain is added to a site other than the Fc region glycosylation consensus region in the obtained purified antibody composition is reduced compared to that of the crude antibody product. The method.

2. The purification method according to claim 1 , wherein the conventional chromatography support is a hydrophobic interaction chromatography support or a mixed mode chromatography support.

3. 2. The purification method according to claim 1, wherein the carrier for conventional chromatography has an average particle size of 15 μm or more.

4. 2. The purification method according to claim 1, wherein the average particle size of the carrier for conventional chromatography is 20 to 100 μm.

5. The purification method according to claim 1 , wherein the support for conventional chromatography has a benzyl group or a butyl group.

6. The purification method according to claim 1 , wherein the protein loading per unit amount of the carrier for conventional chromatography is 20 mg / mL or more.

7. 2. The purification method according to claim 1, wherein the carrier for conventional chromatography is a hydrophobic interaction chromatography carrier, and the method comprises washing the carrier with a washing solution after loading the crude antibody product onto the carrier and before elution.

8. 8. The purification method according to claim 7, wherein the salt concentration of the washing solution is at least 10 mM higher than the salt concentration of the elution solution.

9. 8. The purification method according to claim 7, wherein the washing solution has a salt concentration of 0.5 M or more.

10. 8. The purification method according to claim 7, wherein the washing solution has a salt concentration of 1.0 M or more.

11. 8. The purification method according to claim 7, wherein the pH of the washing solution is at least 0.2 units lower than the pH of the elution solution and is within the range of pH 4 to 8.

12. The purification method according to claim 7 , wherein the washing is carried out by flowing a washing solution having a volume of at least two carrier volumes.

13. 8. The purification method according to claim 7, wherein washing and elution are carried out by using a mobile phase in which the pH and / or salt concentration is changed stepwise or linearly.

14. The purification method according to claim 7, wherein the eluent has a salt concentration of 0.5 M or less or is salt-free and has a pH of 5 to 7.

15. The purification method according to claim 1, wherein the yield is 20% or more.

16. The purification method according to claim 1 , wherein the percentage of glycosylation isomers relative to the total antibody in the purified antibody composition is 5% or less.

17. The method according to claim 16, wherein the proportion of glycosylation isomers relative to the total antibody in the crude antibody preparation is greater than 5%.

18. A method for purifying the anti-hDLK-1 antibody composition according to claim 1, comprising: loading the crude antibody product onto conventional chromatography using a carrier having a particle size of 20 to 100 μm and having a benzyl group or a butyl group; Adsorbing an antibody having no glycosylation site other than the Fc region glycosylation consensus region onto the carrier; removing glycosylated isomers by washing the carrier one or more times with a wash solution having a pH of 4 to 6; and eluting the antibody adsorbed on the carrier with an elution solution having a salt concentration of 0.5 M or less or a salt-free pH of 5 to 7 to obtain a purified antibody composition; wherein the ratio of glycosylation isomers to the total antibody in the purified antibody composition is reduced compared to that in the crude antibody product. The method.

19. A method for purifying the antibody composition according to claim 1, comprising: loading the crude antibody product, the salt concentration of which has been adjusted to 0.5 M or more, onto conventional chromatography using a carrier having a particle size of 20 to 100 μm and containing a benzyl group or a butyl group, to remove glycosylated isomers into a pass-through fraction; Washing the carrier with a washing solution; and eluting the antibody adsorbed on 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 to obtain a purified antibody composition; wherein the ratio of glycosylation isomers to the total antibody in the purified antibody composition is reduced compared to that in the crude antibody product. The method.

20. A method for purifying the antibody composition according to claim 1, comprising: loading the crude antibody product adjusted to pH 4 to 6 onto conventional chromatography using a carrier having a particle size of 20 to 100 μm and containing a benzyl group or a butyl group, and removing glycosylation isomers into a pass-through fraction; Washing the carrier with a washing solution; and and eluting the antibody adsorbed on the carrier with an elution solution having a salt concentration of 0.5 M or less or containing no salt and a pH of 5 to 7 to obtain a purified antibody composition. wherein the ratio of glycosylation isomers to the total antibody in the purified antibody composition is reduced compared to that in the crude antibody product. The method.

21. The method according to any one of claims 1 to 20, wherein the protein load per unit amount of the carrier for conventional chromatography is 20 g / L or more, and the washing is performed by passing a washing solution of 5 carrier volumes or more.

22. A method for producing an antibody composition, in which the ratio of antibodies not having a glycosylation site other than the Fc region glycosylation consensus region to the total antibodies is 95% or more, the method comprising the purification method according to any one of claims 1 to 20.

23. 1. A method for producing a purified anti-hDLK-1 antibody composition, wherein 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 the light chain has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, loading the crude antibody product onto conventional chromatography using a carrier having a particle size of 20 to 100 μm and having a benzyl group or a butyl group; Adsorbing an antibody having no glycosylation site other than the Fc region glycosylation consensus region onto the carrier; removing glycosylated isomers by washing the carrier one or more times with a washing solution containing 0.5 M or more salt; and eluting the antibody adsorbed on the carrier with an elution solution having a salt concentration of 0.5 M or less or a salt-free pH of 5 to 7 to obtain a purified antibody composition; wherein the ratio of glycosylation isomers to the total antibody in the purified antibody composition is reduced compared to that in the crude antibody product. The method.

24. An antibody composition produced by the method of claim 22.

25. A method for removing glycosylation isomers in which a glycochain is added to a site other than the Fc region glycosylation consensus region from a crude anti-hDLK-1 antibody having 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 in its heavy chain and an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, comprising: loading a crude antibody product onto a hydrophobic interaction chromatography carrier, and allowing an antibody having no glycosylation at a site other than the Fc region glycosylation consensus region to be adsorbed onto the carrier; Washing the carrier with a washing solution; and and eluting the antibody adsorbed on the carrier from the carrier with an elution solution to obtain a purified antibody composition. The method.

26. An anti-hDLK-1 antibody composition, the heavy chain of which 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 the light chain of which has the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, wherein 95% or more of the total antibody of the antibody composition is free of glycosylation at a site other than the Fc region glycosylation consensus region.

27. An anti-hDLK-1 antibody, wherein 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 the light chain has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, wherein no glycosylation is performed at a site other than the Fc region glycosylation consensus region.

28. An antibody composition produced by the manufacturing method described in claim 23.