Purification methods for antibodies with altered Fc regions

Abstract

A purification resin with sufficient binding affinity for Fc region alterations with reduced binding to protein A has been discovered. Specifically, a protein A alteration ligand comprising a structure replacing a C-domain amino acid is used as an Fc ligand to purify immunoglobulins containing Fc region alterations with reduced binding to protein A.

Description

Technical Field

[0001] The present invention relates to a method for purifying an antibody, and to a method for purifying an antibody having an Fc region modified with a specific amino acid variation. Prior Art

[0002] With the development of gene recombination technology, we can provide a stable supply of various protein preparations and develop various antibody pharmaceuticals.

[0003] When antibodies are produced using mammalian cells as hosts through genetic recombination techniques, the ability of Protein A or Protein G to bind to the Fc chain of IgG is exploited, and after treatment with Protein A or Protein G affinity column chromatography, purification is performed using various chromatographic methods. In particular, antibody purification using Protein A affinity column chromatography is most commonly used in the production of antibody pharmaceuticals, as a step for recovering antibodies from culture media.

[0004] For example, in Japanese Patent Application No. 5-504579 (Patent Document 1), an antibody-containing aqueous medium obtained from mammalian cell culture is applied to protein A or protein G column chromatography to adsorb the antibody to the column. The antibody is then eluted using an acidic solution (about 0.1 M citric acid, pH 3.0-3.5). The resulting acidic eluate is then applied sequentially to ion exchange column chromatography and particle size exclusion column chromatography for purification.

[0005] Meanwhile, technologies for controlling the isoelectric point (pI) of antibodies by amino acid substitution are known for the purpose of improving blood retention or in vivo dynamics. Specifically, these technologies modify the amino acid residues exposed on the antibody surface to control the pI of antibodies (WO 07 / 114319 (Patent Document 2), WO 2017 / 104783 (Patent Document 3)). Patent Document 2 discloses that modifying the pI of antibodies by modifying the amino acid residues of antibodies can improve plasma retention and half-life, leading to a reduction in the dosage or an extension of the dosing interval for antibodies used as pharmaceuticals. Furthermore, Patent Document 3 discloses that by inserting amino acid substitutions Q311R and P343R into the CH2 or CH3 regions of antibodies to increase the pI, the pI can be increased, thereby promoting antigen elimination from plasma when the antibodies are administered in vivo.

[0006] Protein A, used in antibody purification, is a protein found in the cell wall of Staphylococcus aureus and binds to the Fc region of immunoglobulins, particularly IgG. Staphylococcal Protein A proteins typically contain a repeating structure of five immunoglobulin-binding domains with similarities: the E domain, the D domain, the A domain, the B domain, and the C domain. Only one of each binding domain is capable of binding to an immunoglobulin. Recombinant proteins composed of immunoglobulin-binding domains with only partially modified amino acids, in addition to native Protein A, can also be used as affinity ligands for affinity chromatography. For example, a protein A column has been developed with the goal of improving antibody purification efficiency by modifying a staphylococcal protein A C domain variant in which glycine at position 29 in the C domain is substituted with alanine, or a staphylococcal protein A B domain variant in which glycine at position 29 in the B domain is substituted with alanine, by replacing one or more of the 4th, 7th, and 35th lysine residues in the Z domain with amino acids other than lysine (Patent Documents 4 and 5). [Prior Art Literature] [Patent Document]

[0007] [Patent Document 1] Japanese Patent Publication No. 5-504579 [Patent Document 2] WO07 / 114319 [Patent Document 3] WO2017 / 104783 [Patent Document 4] Japanese Patent Application Laid-Open No. 2007-252368 [Patent Document 5] WO2015 / 034000 Summary of the Invention

[0008] [Problems to be Solved by the Invention]

[0009] Protein A has long been used as a ligand for antibody purification. However, purification methods for antibodies with amino acid modifications that alter their pI (hereinafter referred to as pI-modified antibodies) have not been explored in detail, nor have the purification process been thoroughly investigated. Therefore, for example, it is unknown whether there are antibodies that cannot be purified using commonly used Protein A columns.

[0010] The inventors of this application have discovered that there are pI-modified antibodies that cannot be efficiently purified using commonly used Protein A columns. Furthermore, there is a need for an efficient purification method applicable to such antibodies. Specifically, the present invention aims to provide a highly efficient and economical purification method that can be produced on an industrial scale, even for pI-modified antibodies that cannot be efficiently purified using conventional Protein A columns. [Means for solving the problem]

[0011] After intensive research to achieve the above objectives, the inventors of this case discovered that even pI-modified antibodies that cannot be purified efficiently using commonly used Protein A columns can be purified more efficiently by using a resin with a specific modified Protein A ligand.

[0012] That is, the present invention provides the following [1]~

[20] . [1] A method for purifying an IgG antibody from a composition containing substitutions of amino acid residues including Q311R and P343R, comprising the following steps: (a) preparing an affinity column comprising a carrier immobilized with a modified protein A ligand, wherein the modified protein A ligand comprises a modified immunoglobulin binding domain having a modification in which any one or more of the 4th, 7th, and 35th lysine residues originally present in the C domain variant of Staphylococcus protein A defined by SEQ ID NO: 1 or the Z domain defined by SEQ ID NO: 2 are substituted with an amino acid residue other than lysine, or an aggregate of such modified immunoglobulin binding domains; (b) a step of filling the affinity column of step (a) with the composition containing the above-mentioned IgG antibody; and (c) a step of eluting and recovering IgG antibodies from the affinity column of step (b). [2] The method as described in [1], wherein the aggregate of the modified immunoglobulin binding domains is a dimer to a decamer, and at the first and / or second position from the N-terminus or C-terminus, there is an immunoglobulin binding domain in which at least one of the 40th, 43rd, 46th, 53rd, 54th, and 56th amino acid residues originally present in the C domain variant (sequence number 1) or the Z domain (sequence number 2) is replaced with a lysine residue. [3] The method as described in [1] or [2], wherein the substitution is a substitution of any one or more of the 4th, 7th, and 35th lysine residues originally present in the C domain variant or Z domain of protein A with any amino acid residue selected from the group consisting of an alanine residue (A), a glutamine residue (Q), an asparagine residue (D), a valine residue (V), a serine residue (S), a threonine residue (T), a histidine residue (H), a tyrosine residue (Y), an arginine residue (R), a glutamine residue (E), a phenylalanine residue (F), a leucine residue (L), an isoleucine residue (I), and a proline residue (P). [4] The method as described in any one of [1] to [3], wherein the above-mentioned altered immunoglobulin binding domain is (i) an altered immunoglobulin binding domain having the amino acid sequence shown in SEQ ID NO: 3 or 5, or (ii) an altered immunoglobulin binding domain comprising an amino acid sequence in which one or more amino acid residues other than amino acid residues 4, 7 and 35 in the amino acid sequence shown in SEQ ID NO: 3 or 5 are substituted, deleted, added and / or inserted. [5] The method as described in any one of [1] to [4], wherein the altered immunoglobulin binding domain has the ability to bind to an IgG antibody containing substitutions of the amino acid residues Q311R and P343R. [6] The method as described in any one of [1] to [5], wherein the above-mentioned protein A altered ligand is an altered ligand comprising an altered immunoglobulin binding domain formed by at least one amino acid sequence selected from the group consisting of sequence numbers 3, 4 and 5. [7] The method according to any one of [1] to [6], wherein the protein A-modified ligand is immobilized on the carrier by any means selected from the group consisting of the following (1)-(5): (1) A method for immobilizing a modified immunoglobulin binding domain on a carrier by further replacing one to six of the 40th, 43rd, 46th, 53rd, 54th and 56th amino acid residues with lysine residues in the C domain variant or Z domain of protein A. (2) A method of introducing cysteine ​​into the C-terminus of protein A and immobilizing it by binding it to a carrier with a disulfide bond or a thioether bond. (3) A method for immobilizing an amino-containing solidification support by cyaniding thiocyanate groups. (4) A method for immobilizing an aggregate of modified immunoglobulin binding domains having cysteine ​​residues on an amino-containing support using 4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC) as a cross-linking agent, and (5) A method of immobilizing a plurality of lysine residues added to the C-terminus of a modified immunoglobulin binding domain in which the 42nd, 49th, 50th, and 58th lysine residues of the C domain variant of protein A are replaced with amino acids other than lysine residues, or a modified immunoglobulin binding domain in which the 49th, 50th, and 58th lysine residues of the Z domain are replaced with amino acids other than lysine residues, on a carrier. [8] The method according to any one of [1] to [7], wherein the IgG antibody further comprises substitutions of one or more amino acid residues selected from the group consisting of M428L, N434A, Y436T, Q438R and S440E in the CH3 region of the IgG antibody. [9] The method of any one of [1] to [8], wherein the CH3 region of the IgG antibody is an IgG antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 11.

[10] The method of any one of [1] to [9], wherein the heavy chain constant region of the IgG antibody is an IgG antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 57.

[11] The method of any one of [1] to

[10] , wherein the pI value of the antibody is 4.0-10.0.

[12] The method as described in any one of [1] to

[11] , wherein the amount of binding of the protein A to the Fc region of the IgG antibody is altered to be 5 times or more compared to the binding capacity of the unaltered protein A.

[13] The method as described in any one of [1] to

[12] , wherein before step (c), the method further comprises a step of washing the affinity column with a washing solution.

[14] The method as described in

[13] , wherein the washing solution is a combination of a buffer and a salt, comprising at least one selected from the group consisting of phosphoric acid, acetic acid, citric acid, glycine, and trishydroxymethylaminomethane as a buffer, and comprising at least one selected from the group consisting of arginine, sodium chloride, and sodium sulfate as a salt solution.

[15] The method of any one of [1] to

[14] , wherein after step (c), the method further comprises a purification step of the IgG antibody by at least one chromatography method selected from the group consisting of cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, multimodal chromatography, and hydroxyapatite chromatography.

[16] The method of any one of [1] to

[15] , wherein step (c) comprises the step of eluting the IgG antibody from the affinity column using a dissolution solution containing at least one selected from the group consisting of hydrochloric acid, acetic acid, citric acid, arginine, glycine or phosphoric acid.

[17] The method of any one of [1] to

[16] , wherein the antibody is a humanized antibody or a human antibody.

[18] The method of any one of [1] to

[17] , wherein the antibody is an anti-myostatin antibody, an anti-IL-6 receptor antibody, an anti-IL-6 antibody, an anti-IL-8 antibody or an anti-IL-31 receptor antibody.

[19] A use, which is the use of an affinity column comprising a carrier immobilized with a protein A modified ligand for purifying an IgG antibody containing substitutions of amino acid residues Q311R and P343R, wherein the protein A modified ligand comprises any one or both of the C domain variant and the Z domain of protein A with substitutions of amino acids, wherein the substitution is a substitution of any one or more of the 4th, 7th, and 35th lysine residues originally present in the C domain variant or the Z domain into an amino acid residue other than lysine, and the modified ligand has the ability to bind to the IgG antibody.

[20] A method for producing an IgG antibody having amino acid residue substitutions of Q311R and P343R, comprising the following steps: (i) providing a step of providing a composition comprising an IgG antibody having substitutions of amino acid residues including Q311R and P343R; (ii) preparing an affinity column comprising a carrier immobilized with a modified protein A ligand, wherein the modified protein A ligand comprises a modified immunoglobulin binding domain having a modification in which any one or more of the 4th, 7th, and 35th lysine residues originally present in the C domain variant of Staphylococcus protein A defined by SEQ ID NO: 1 or the Z domain defined by SEQ ID NO: 2 is substituted with an amino acid residue other than lysine, or an aggregate of such modified immunoglobulin binding domains; (iii) a step of filling the affinity column of step (ii) with the composition containing the above-mentioned IgG antibody; and (iv) a step of eluting and recovering IgG antibodies from the affinity column packed with the IgG antibody composition in step (iii). Alternatively, the present invention provides: [1'] A method for purifying an IgG antibody from a composition containing substitutions of amino acid residues including Q311R and P343R, the method comprising the following steps: (a) preparing an affinity column comprising a carrier immobilized with a protein A-modified ligand; (b) a step of filling the affinity column of step (a) with the composition containing the above-mentioned IgG antibody; and (c) a step of dissolving and recovering IgG antibodies from the affinity column of step (b), The modified protein A ligand comprises a C domain variant or a Z domain variant of protein A with amino acid substitutions in one or both of the C domain variant and the Z domain, wherein the substitutions are substitutions of at least one of the 4th, 7th, and 35th lysine residues originally present in the C domain variant or the Z domain with an amino acid residue other than lysine, and the modified protein A ligand has the ability to bind to the IgG antibody. [Effects of the Invention]

[0013] According to the present invention, even pI-modified antibodies that cannot be purified well on a conventional protein A column can be purified simply and efficiently. Simple diagram description

[0014] [Figure 1] Figure 1 shows the results of dynamic binding capacity (DBC) measurements of antibodies without modifications in the Fc region on various Protein A-immobilized resins. The vertical axis represents DBC (g / L resin) and the horizontal axis represents residence time (minutes). [Figure 2-1] shows the results (real-time binding curve) of evaluating the binding affinity between the AF-rProtein A HC-650F ligand (structure represented by formula (1')) and the antibody using the BLItz (registered trademark) (ForteBio) evaluation system. [Figure 2-2] shows the results (real-time binding curves) of evaluating the binding affinity between the MabSelect SuRe ligand and the antibody using the BLItz (registered trademark) evaluation system (ForteBio). Implementation Method

[0015] Hereinafter, the present invention will be described in detail. The present invention relates to a method for purifying a composition containing an antibody with an increased isoelectric point (pI). Specifically, the present invention relates to a method for purifying an IgG antibody from a composition containing amino acid substitutions including Q311R and P343R, comprising: (a) preparing an affinity column comprising a carrier immobilized with a modified protein A ligand, wherein the modified protein A ligand comprises a modified immunoglobulin binding domain having a modification in which any one or more of the 4th, 7th, and 35th lysine residues originally present in the C domain variant of Staphylococcus protein A defined by SEQ ID NO: 1 or the Z domain defined by SEQ ID NO: 2 is substituted with an amino acid residue other than lysine, or an aggregate of such modified immunoglobulin binding domains; (b) a step of filling the affinity column of step (a) with the composition containing the above-mentioned IgG antibody; and (c) A step of dissolving and recovering the IgG antibody filled in step (b).

[0016] IgG antibodies comprising the amino acid substitutions Q311R and P343R in the CH2 or CH3 regions of the present invention are those in which glutamine (Q) at position 311 in the CH2 or CH3 regions is replaced with arginine (R), and proline (P) at position 343 is replaced with arginine (R), as indicated by EU numbering in the CH2 or CH3 regions. Generally speaking, the CH2 region, in the hinge region, corresponds to EU numbers 231 to 340, and the CH3 region corresponds to EU numbers 341 to 447. The "Fc region" herein refers to the Fc region before the amino acid substitutions are introduced, as described herein. Preferred examples of Fc regions include those derived from natural antibodies. Antibodies can be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Natural antibodies may also contain naturally occurring variation. Multiple allotypes of IgG resulting from genetic polymorphism are described in "Sequences of Proteins of Immunological Interest," NIH Publication No. 91-3242, and any of these sequences may be used in the present invention. Specifically, for human IgG1, the amino acid sequence at positions 356 to 358 (EU numbering) may be either DEL or EEM. Preferred examples of affinity Fc regions include those derived from the heavy chain constant regions of human IgG1 (SEQ ID NO: 58), human IgG2 (SEQ ID NO: 59), human IgG3 (SEQ ID NO: 60), and human IgG4 (SEQ ID NO: 61). Another preferred example of an affinity Fc region is an Fc region derived from the heavy chain constant region SG1 (SEQ ID NO: 62). Furthermore, an affinity Fc region may be created by adding amino acid modifications other than those described herein to an Fc region derived from a natural antibody.

[0017] Furthermore, regarding the antibodies of the present invention, amino acid modifications performed for other purposes can be combined with the antibodies used in the present invention. For example, amino acid substitutions that enhance FcRn binding activity (Hinton et al., J. Immunol. 176(1):346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Petkova et al., Intl. Immunol. 18(12):1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159 (2010); WO2006 / 019447; WO2006 / 053301; and WO2009 / 086320), as well as amino acid substitutions to improve antibody heterogeneity or stability (WO2009 / 041613), can be added. Alternatively, amino acid modifications applicable to polypeptides with enhanced antigen clearance properties described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704, or WO2013 / 180201, polypeptides with target tissue-specific binding properties described in WO2013 / 180200, or polypeptides with repeated binding to multiple antigen molecules described in WO2009 / 125825, WO2012 / 073992, or WO2013 / 047752 may be combined with the antibodies used in the present invention. Alternatively, amino acid modifications disclosed in EP1752471 and EP1772465 may be combined with the antibodies used in the present invention for the purpose of conferring binding to other antigens. Alternatively, to increase plasma retention, amino acid modifications that lower the pI of the constant region (WO 2012 / 016227) can be combined with the antibodies used in the present invention. Alternatively, to enhance cellular uptake, amino acid modifications that increase the pI of the constant region (WO 2014 / 145159) can be combined. Alternatively, to promote elimination of the target molecule from plasma, amino acid modifications that increase the pI of the constant region (Japanese Patent Application Nos. 2015-021371 and 2015-185254) can be combined.

[0018] In the present invention, amino acid changes refer to any substitution, deletion, addition, insertion, modification, or any combination thereof. In the present invention, amino acid changes may also be referred to as amino acid variations.

[0019] The antibodies used in the present invention are preferably IgG antibodies that further comprise substitutions of one or more amino acid residues selected from the group consisting of M428L, N434A, Y436T, Q438R, and S440E in the CH3 region. More preferably, they are IgG antibodies that comprise substitutions of two or more amino acid residues selected from the group consisting of M428L, N434A, Y436T, Q438R, and S440E in the CH3 region. More preferred examples include IgG antibodies that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 11 in the CH3 region, and IgG antibodies that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 57 in the heavy chain constant region.

[0020] The antibodies used in the present invention are generally not particularly limited as long as they bind to the desired antigen, and can be polyclonal antibodies or monoclonal antibodies.

[0021] The monoclonal antibodies used in the present invention include not only those derived from humans, mice, rats, hamsters, rabbits, sheep, camels, monkeys, and other animals, but also artificially modified recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. Furthermore, recombinant antibodies with artificial alterations to the constant regions of antibodies, for example, may be prepared by modifying their physical properties (specifically, altering their isoelectric point (pI) or Fc receptor affinity) to improve their blood retention or in vivo dynamics.

[0022] Furthermore, the immunoglobulin class of the antibodies used in the present invention is not particularly limited and may be IgG1, IgG2, IgG3, IgG4, etc. Preferred IgGs in the present invention, especially when the Fc region is of human origin, are IgG1, IgG2, and IgG4.

[0023] The antibodies used in the present invention can also be used as pharmaceutical compositions. When parenteral, intrapulmonary, or nasal administration is desired, or when local treatment is desired, administration can be performed by any conventional means, including intralesional administration. Parenteral injections include intramuscular, intravenous, intraperitoneal, or subcutaneous administration.

[0024] When the antibodies used in the present invention are used as pharmaceutical compositions, a product can be provided, comprising the pharmaceutical composition and a device useful for treatment, prevention, and / or diagnosis. The product comprises a container and a label or accessories associated with the container. Preferred containers include, for example, bottles, vials, syringes, and IV solution bags. Containers can be made of various materials, such as glass or plastic, and silicone-free syringes can also be used.

[0025] The antibodies used in the present invention can be produced using methods generally known to those skilled in the art. Hybridomas producing monoclonal antibodies can be produced using generally known techniques as follows. Specifically, the desired antigen and cells expressing the desired antigen are used as sensitizing antigens, and immunization is performed according to conventional immunization methods. The resulting immune cells are then fused with conventional mother cells using conventional cell fusion methods. Monoclonal antibody-producing cells (hybridomas) are then screened using conventional screening methods to produce the antibody. Hybridomas can be produced using methods such as those described by Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). If the antigen has low immunogenicity, it can be conjugated to an immunogenic macromolecule such as albumin before immunization.

[0026] Alternatively, antibody genes can be cloned from hybridomas, then inserted into appropriate vectors for introduction into a host, allowing the production of recombinant antibodies using genetic recombination techniques (e.g., Carl, A.K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA encoding the variable region (V region) of the antibody is synthesized from the mRNA of the hybridoma using reverse transcriptase. After obtaining the DNA encoding the desired antibody V region, it is ligated with DNA encoding the desired antibody constant region (C domain) and then inserted into an expression vector. Alternatively, the antibody V region encoding DNA can be inserted into an expression vector containing the antibody C region DNA. Expression regulatory regions are incorporated into the expression vector to enable expression under the control of, for example, enhancers and promoters. Subsequently, this expression vector is transformed into host cells for antibody expression.

[0027] In the present invention, artificially altered recombinant antibodies, such as chimeric and humanized antibodies, may also be used to reduce xenoantigenicity to humans. These altered antibodies can be produced using known methods. Chimeric antibodies are antibodies formed by combining the variable regions of the heavy and light chains of non-human mammals, such as mice, with the constant regions of the heavy and light chains of human antibodies. Chimeric antibodies can be produced by linking the DNA encoding the variable regions of the mouse antibody with the DNA encoding the constant regions of the human antibody, embedding the resulting DNA into an expression vector, and then introducing it into a host for production.

[0028] Humanized antibodies, also known as reshaped human antibodies, are constructed by transplanting the complementarity determining regions (CDRs) of non-human mammalian antibodies, such as mouse antibodies, onto the complementarity determining regions (CDRs) of human antibodies. General genetic recombination techniques are well-known. Specifically, a DNA sequence designed to link the CDRs of a mouse antibody with the framework regions (FRs) of a human antibody is synthesized using PCR from several oligonucleotides that have overlapping ends. This DNA is then ligated with DNA encoding the constant regions of a human antibody, then embedded in an expression vector and introduced into a host for production (see EP 239400 and WO 96 / 02576). The FRs of human antibodies linked via CDRs can be selected so that the complementarity determining regions form an appropriate antigen-binding site. If necessary, amino acids in the framework regions of the antibody variable regions can be substituted so that the complementarity determining regions of the reconstructed human antibody can form an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0029] Technologies for replacing amino acids in antibodies to improve their activity, physical properties, pharmacodynamics, safety, etc., such as those described below, are also known. The antibodies used in the present invention also include antibodies with such amino acid substitutions (including deletions or additions).

[0030] As technologies for performing amino acid substitutions on the variable regions of IgG antibodies, there have been reports on humanization (Tsurushita N, Hinton PR, Kumar S., Design of humanized antibodies: from anti-Tac to Zenapax., Methods. 2005 May;36(1):69-83.), affinity maturation by amino acid substitutions in the complementarity determining regions (CDRs) for enhancing binding activity (Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc Natl Acad Sci USA. 2005 Jun 14;102(24):8466-71.), and improvement of physicochemical stability by amino acid substitutions in the framework (FRs) (Ewert S, Honegger A, Pluckthun A., Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering., Methods. 2004 Oct;34(2):184-99. Review). Furthermore, as a technique for performing amino acid substitutions on the Fc region of IgG antibodies, techniques for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity and complement-dependent cellular cytotoxicity (CDC) activity are known (Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31;20(1):17-29. Review.).Furthermore, there are reports of amino acid substitutions in the Fc region that not only enhance effector function but also increase the half-life of antibodies in the blood (Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1;176(1):346-56., Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 Jul;15(7):637-40.). Furthermore, there is also a known technique for substituting various amino acids in the constant region for the purpose of further improving the physical properties of antibodies (WO 09 / 41613). .

[0031] Furthermore, methods for obtaining human antibodies are also known. For example, human lymphocytes can be sensitized in vitro with a desired antigen or cells expressing the desired antigen, and the sensitized lymphocytes can be fused with human myeloma cells, such as U266, to obtain the desired human antibodies with antigen-binding activity (see Japanese Patent Publication No. 1-59878). Alternatively, transgenic animals harboring a complete repertoire of human antibody genes can be immunized with an antigen to obtain the desired human antibodies (see WO 93 / 12227, WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO 96 / 34096, and WO 96 / 33735). Furthermore, techniques for obtaining human antibodies using panning of human antibody libraries are also known. For example, the variable regions of human antibodies can be displayed on the surface of phages in the form of single-chain antibodies (scFvs) using phage display, and phages that bind to the antigen can be selected. If the genes of the selected phages can be analyzed, the DNA sequence encoding the variable regions of the human antibodies that bind to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is known, an appropriate expression vector containing this sequence can be prepared to obtain the human antibody. These methods are well known and can be found in WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, and WO 95 / 15388. The antibodies used in the present invention also include such human antibodies.

[0032] Once the antibody gene is isolated and introduced into a suitable host to produce the antibody, a combination of a suitable host and an expression vector can be used. When using eukaryotic cells as hosts, animal cells, plant cells, and fungal cells can be used. Known animal cells include (1) mammalian cells, such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, and Vero, (2) amphibian cells, such as Xenopus oocytes, or (3) insect cells, such as sf9, sf21, and Tn5. Known plant cells include Nicotiana, such as cells derived from Nicotiana tabacum, which can be cultured using callus tissue. Known fungal cells include yeast, such as Saccharomyces, such as Saccharomyces serevisiae, and filamentous fungi, such as Aspergillus, such as Aspergillus niger. When using prokaryotic cells, bacterial cell strains can be used. Known bacterial cells include Escherichia coli (E. coli) and Bacillus subtilis. Antibodies can be obtained by transforming these cells with the desired antibody gene and culturing the transformed cells in vitro.

[0033] Antibodies conjugated to various molecules, such as polyethylene glycol (PEG) and cytotoxic agents, can also be used as modified antibodies (Farmaco. 1999 Aug 30;54(8):497-516, Cancer J. 2008 May-Jun;14(3):154-69). Antibodies used in the present invention also include such modified antibodies. Such modified antibodies can be obtained by chemically modifying antibodies. Such methods are well established in this field.

[0034] Examples of antibodies used in the present invention include, but are not limited to, anti-tissue factor antibodies, anti-IL-6 receptor antibodies, anti-IL-6 antibodies, HM1.24 antigen monoclonal antibodies, anti-parathyroid hormone-related peptide antibodies (anti-PTHrP antibodies), anti-glypican-3 antibodies, anti-ganglioside GM3 antibodies, anti-TPO receptor agonist antibodies, coagulation factor VIII functional replacement antibodies, anti-IL31 receptor antibodies, anti-HLA antibodies, anti-AXL antibodies, anti-CXCR4 antibodies, anti-NR10 antibodies, and bispecific antibodies recognizing factor IX (a) and factor X.

[0035] Furthermore, the pI value of the antibodies used in the present invention is preferably 4.0-10.0, more preferably 5.0-9.5, and even more preferably 6.0-9.0. These pI values ​​are increased compared to the pI values ​​of IgG antibodies before amino acid modification. Furthermore, the isoelectric point of IgG antibodies can be assessed using conventional analytical methods such as isoelectric electrophoresis.

[0036] The amount of binding of the modified Protein A ligand of the present invention to the Fc region of the IgG antibody to be purified is preferably 5-fold or greater, and more preferably 10-fold or greater, compared to the amount of binding of the unmodified Protein A. While the method for determining the binding amount is not particularly limited herein, examples thereof include the method for determining dynamic binding capacity described in the Examples of this invention.

[0037] Specific examples of commonly used protein A columns include HiTrap MabSelect SuRe (manufactured by GE Healthcare, trade name), Amsphere A3 (manufactured by JSR Life Sciences, registered trademark), MiniChrom Column Eshmuno A (manufactured by Merck Millipore, registered trademark), MabSpeed ​​rP202 (manufactured by Mitsubishi Chemical, registered trademark), and KanCap Pre-packaged Column (manufactured by KANEKA, trade name).

[0038] As a protein A affinity column used in the present invention, an affinity column containing a carrier immobilized with a protein A modified ligand can be used. The protein A modified ligand comprises a modified immunoglobulin binding domain having a modification in which one or more of the 4th, 7th, and 35th lysine residues originally present in the C domain variant or the Z domain are substituted with an amino acid residue other than lysine, and has the ability to bind to IgG antibodies containing the amino acid residue substitutions Q311R and P343R, or a collection of such modified immunoglobulin binding domains. The present protein A modified ligand is characterized by a modification in which the glycine residue at position 29 of the amino acid sequence of the protein A C domain is substituted with an alanine residue in a C domain variant (SEQ ID NO. 1), or in which at least one of the 4th, 7th, and 35th lysine residues in the Z domain (SEQ ID NO. 2) is substituted with an amino acid residue other than lysine. This modification allows for a targeted improvement in affinity for immunoglobulins, compared to the pre-modified molecule, when immobilized on an insoluble carrier via its own amine groups.

[0039] In the present invention, an "immunoglobulin binding domain" refers to a functional unit of a polypeptide that possesses immunoglobulin-binding activity alone, and an "altered immunoglobulin binding domain" refers to a modification of an already existing immunoglobulin binding domain. A "ligand" is a molecule that binds to a specific molecule with specific affinity. In the present invention, it refers to an immunoglobulin-binding protein that selectively binds to an immunoglobulin. A "protein A-altered ligand" is an immunoglobulin-binding protein that comprises an altered immunoglobulin binding domain that has been modified from a protein A-binding domain. Throughout this specification, "altered immunoglobulin binding domain" and "protein A-altered ligand" are collectively referred to as "altered protein."

[0040] The modified immunoglobulin binding domain used in the present invention may comprise an amino acid sequence in which at least one of the 4th, 7th, and 35th lysine residues in the C domain variant (SEQ ID NO. 1) or the additional Z domain (SEQ ID NO. 2) is substituted with other amino acid residues. For example, it is desirable to substitute at least two of the 4th, 7th, and 35th lysine residues with other amino acid residues, preferably three lysine residues.

[0041] The type of amino acid after substitution of any one or more of the 4th, 7th, and 35th amino acids in the C domain variant (SEQ ID NO. 1) or the Z domain (SEQ ID NO. 2) is not particularly limited, but is preferably alanine, glutamine, asparagine, valine, serine, threonine, histidine, tyrosine, or arginine, with alanine, threonine, or arginine being particularly preferred.

[0042] More specifically, the type of amino acid after the fourth substitution in the C domain variant (SEQ ID NO. 1) or the Z domain (SEQ ID NO. 2) is preferably glutamine, isoleucine, arginine, alanine, valine, serine, threonine, or histidine, with alanine being more preferred.

[0043] Furthermore, the amino acid type after the seventh substitution in the C domain variant (SEQ ID NO: 1) or the Z domain (SEQ ID NO: 2) is preferably tyrosine, phenylalanine, glutamine, leucine, isoleucine, proline, threonine, alanine, valine, serine, arginine, or histidine, with threonine being more preferred.

[0044] Furthermore, the amino acid type after the substitution at position 35 of the C domain variant (SEQ ID NO: 1) or the Z domain (SEQ ID NO: 2) is preferably arginine, glutamine, asparagine, or tyrosine, with arginine being more preferred.

[0045] Furthermore, in addition to the aforementioned modifications, the C domain variant (SEQ ID NO: 1) or Z domain (SEQ ID NO: 2) may also have one or more amino acid substitutions, as long as the variant has binding ability to IgG antibodies containing the amino acid residue substitutions Q311R and P343R. In particular, in the C domain variant (SEQ ID NO: 1) or Z domain (SEQ ID NO: 2), it is preferred that the number of constituent amino acids contained is low. In addition to the aforementioned modifications, it is desirable to substitute one to four, preferably three or four, and more preferably four, of the original lysine residues at positions 42, 49, 50, and 58 with amino acid residues other than lysine.

[0046] The type of amino acid after substitution of any one or more of amino acids 42, 49, 50, and 58 in the C domain variant (SEQ ID NO. 1) or the Z domain (SEQ ID NO. 2) is not particularly limited, but is preferably alanine, glutamine, asparagine, valine, serine, threonine, histidine, tyrosine, or arginine, with alanine or arginine being more preferred.

[0047] More specifically, when the 42nd lysine residue originally present in the C domain variant (SEQ ID NO. 1) or the Z domain (SEQ ID NO. 2) is substituted with another amino acid residue, the type of amino acid after the substitution is preferably alanine, valine, serine, threonine, or histidine, with alanine being more preferred.

[0048] Furthermore, when the lysine residue originally present at position 49 in the C domain variant (SEQ ID NO: 1) or the Z domain (SEQ ID NO: 2) is substituted with another amino acid residue, the amino acid after the substitution is preferably arginine, glutamine, asparagine, or tyrosine, with arginine being more preferred.

[0049] Furthermore, when the 50th lysine residue originally present in the C domain variant (SEQ ID NO: 1) or the Z domain (SEQ ID NO: 2) is substituted with another amino acid residue, the amino acid after the substitution is preferably arginine, glutamine, asparagine, or tyrosine, with arginine being more preferred.

[0050] Furthermore, when the lysine residue originally present at position 58 in the C domain variant (SEQ ID NO: 1) or the Z domain (SEQ ID NO: 2) is substituted with another amino acid residue, the amino acid after the substitution is preferably arginine, glutamine, asparagine, or tyrosine, with arginine being more preferred.

[0051] Furthermore, in addition to the aforementioned modifications, the modified immunoglobulin-binding domains of the present invention may also include substitution of the 37th aspartic acid residue in the C domain variant (SEQ ID NO: 1) or the Z domain (SEQ ID NO: 2) with an amino acid residue other than aspartic acid. This modification further enhances the chemical stability of the molecule under acidic pH conditions compared to the unmodified molecule.

[0052] When the aspartic acid residue originally present at position 37 of the C domain variant (SEQ ID NO: 1) or the Z domain (SEQ ID NO: 2) is substituted with another amino acid residue, the type of the substituted amino acid is not particularly limited, but alanine, glutamine, serine, threonine, leucine, or isoleucine is preferred.

[0053] Preferred examples of amino acid sequences for altering the immunoglobulin binding domain used in the present invention include the amino acid sequences set forth in SEQ ID NOs. 3 or 5. The amino acid sequence set forth in SEQ ID NO. 3 is an amino acid sequence in which the 4th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an alanine residue, the 7th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an arginine residue, the 35th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 5 is an amino acid sequence in which the 4th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an alanine residue, the 7th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an arginine residue, the 42nd amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an alanine residue, the 49th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an arginine residue, the 50th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 5 is an amino acid sequence in which the 4th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an alanine residue, the 7th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an arginine residue, the 58th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 5 is an amino acid sequence in which the 4th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an alanine residue, the 7th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 1 is substituted with an arginine residue, the 50th amino acid residue in the amino acid sequence set forth in SEQ ID NO. 5 is an amino acid sequence in which Alternatively, in addition to the above, the following amino acid sequences can also be cited as preferred examples of amino acid sequences of the modified immunoglobulin binding domain of the present invention: [1] In the C domain variant defined by SEQ ID NO: 1, the 35th lysine is replaced by glutamine or arginine; [2] In the C domain variant defined by SEQ ID NO: 1, amino acid residues 40, 43, 46, and 53 are substituted with lysine, and lysine residue 35 is substituted with arginine or valine; [3] In the amino acid sequence of [2], the seventh lysine is additionally replaced with tyrosine, phenylalanine, threonine, arginine, glutamine, valine, leucine, isoleucine, histidine, alanine, or proline; [4] In the amino acid sequence of [2] or [3], the fourth lysine is additionally replaced by alanine; [5] In the C domain variant defined by SEQ ID NO: 1, amino acid residues 40, 43, 46, and 53 are substituted with lysine, and the fourth lysine is substituted with valine, isoleucine, glutamine, or arginine; [6] In the C domain variant defined by SEQ ID NO: 1, the 42nd lysine is substituted with alanine, and the 49th, 50th, and 58th lysines are substituted with arginine, and the 4th lysine is further substituted with valine, isoleucine, glutamine, or arginine; [7] In the amino acid sequence of [5] or [6], the 7th and 35th lysines are additionally replaced by arginines.

[0054] In the case of a collection of immunoglobulin-binding domains as constituent units of a protein A-modified ligand, it suffices to include at least one immunoglobulin-binding domain containing the aforementioned modification. The number of immunoglobulin-binding domain units contained in the collection is, for example, 2 to 10, preferably 2 to 8, more preferably 4 to 7, and even more preferably 6. As long as the collection includes at least one immunoglobulin-binding domain containing the aforementioned modification, immunoglobulin-binding domains not containing the aforementioned modification may also be included. In the collection, the proportion of immunoglobulin-binding domains containing the aforementioned modification relative to the total number of immunoglobulin-binding domains contained as constituent units is preferably 50% or more, and more preferably 100% (i.e., all immunoglobulin-binding domains contained as constituent units have the aforementioned modification).

[0055] In the case of a protein A modified ligand assembly comprising immunoglobulin-binding domains as constituent units, the first and / or second immunoglobulin-binding domains located at the N-terminus or C-terminus of the assembly may be substituted with amino acid residues capable of covalently binding to a carrier (e.g., lysine residues) to facilitate immobilization to a carrier. For example, in the first and / or second C-domain variants (SEQ ID NO. 1) or (SEQ ID NO. 2) located at the N-terminus or C-terminus of the assembly, at least one of the original amino acid residues at positions 40, 43, 46, 53, 54, and 56, preferably 2 to 6, more preferably 3 or 4, and even more preferably 4, may be substituted with lysine residues, thereby facilitating immobilization to a carrier.

[0056] Furthermore, in the first and / or second immunoglobulin-binding domains located at the N-terminus or C-terminus of the assembly, the C-domain variant (SEQ ID NO. 1) or (SEQ ID NO. 2) may have at least one of the 4th, 7th, and 35th lysine residues substituted with other amino acid residues, or at least one of the lysine residues substituted with other amino acid residues. An example of the first and / or second immunoglobulin-binding domains located at the N-terminus or C-terminus of the assembly is the amino acid sequence set forth in SEQ ID NO. 4. The amino acid sequence set forth in SEQ ID NO. 4 is an amino acid sequence in which the 4th residue is substituted with an alanine residue, the 7th residue is substituted with a threonine residue, the 35th residue is substituted with an arginine residue, the 40th residue is substituted with a lysine residue, the 43rd residue is substituted with a lysine residue, the 46th residue is substituted with a lysine residue, and the 53rd residue is substituted with a lysine residue.

[0057] As an example of the above-mentioned aggregate, there can be mentioned the modified protein A ligand represented by the following formula (1). (R1) n-(R2) m, or (R2) m-(R1) n(1)

[0058] In formula (1), the left end is the N end and the right end is the C end.

[0059] In formula (1), n ​​is an integer from 1 to 9, preferably from 1 to 7, more preferably from 3 to 6, and more preferably 5. m is an integer from 1 to 2, preferably 1. The total number of domains n+m is from 2 to 10, preferably from 2 to 8, more preferably from 4 to 7, and more preferably 6.

[0060] In formula (1), (R1) is a modified immunoglobulin-binding domain in which any one or more (preferably all) of the 4th, 7th, and 35th amino acid residues of the C domain variant (SEQ ID NO. 1) or the Z domain (SEQ ID NO. 2) are substituted with amino acid residues other than lysine residues. In addition to the aforementioned substitutions at positions 4, 7, and 35, (R1) preferably has any one or more (preferably all) of positions 42, 49, 50, and 58 substituted with amino acid residues other than lysine residues. The N (R1)s may all have the same amino acid sequence or may be different amino acid sequences.

[0061] In formula (1), (R2) is an immunoglobulin-binding domain in which any one or more (preferably all) of the 40th, 43rd, 46th, 53rd, 54th, and 56th residues of the C domain variant (SEQ ID NO. 1) or the Z domain (SEQ ID NO. 2) are substituted with lysine residues. (R2) preferably has any one or more (preferably all) of the 4th, 7th, and 35th residues substituted with amino acid residues other than lysine, in addition to the substitutions of any one or more of the 40th, 43rd, 46th, 53rd, 54th, and 56th residues. When m is 2, the two (R2) residues may be identical or different amino acid sequences.

[0062] As a preferred example of the modified protein A ligand represented by the above formula (1), n ​​is 5; m is 1; (R1) is the amino acid sequence represented by SEQ ID NO: 5 (in SEQ ID NO: 1, the 4th amino acid is substituted with an alanine residue, the 7th amino acid is substituted with a threonine residue, the 35th amino acid is substituted with an arginine residue, the 42nd amino acid is substituted with an alanine residue, the 49th amino acid is substituted with an arginine residue, the 50th amino acid is substituted with an arginine residue, and the 58th amino acid is substituted with an arginine residue). (R2) is an altered immunoglobulin binding domain formed by the amino acid sequence shown in SEQ ID NO: 4 (an amino acid sequence in which the 4th amino acid residue is substituted with an alanine residue, the 7th amino acid residue is substituted with a threonine residue, the 35th amino acid residue is substituted with an arginine residue, the 40th amino acid residue is substituted with a lysine residue, the 43rd amino acid residue is substituted with a lysine residue, the 46th amino acid residue is substituted with a lysine residue, and the 53rd amino acid residue is substituted with a lysine residue).

[0063] The modified Protein A proteins of the present invention can be produced using conventional genetic recombination techniques, such as those described in Current Protocols in Molecular Biology by Frederick M. Ausbel et al. Specifically, by transforming an expression vector containing a nucleic acid sequence encoding the desired modified protein into a host such as Escherichia coli and culturing the cells in an appropriate liquid medium, large quantities of the modified protein can be economically obtained from the cultured cells. Specifically, because the immunoglobulin-binding domain of Protein A is a small protein composed of approximately 60 amino acids, the desired expression vector can be obtained by, for example, fragmenting the DNA encoding the desired amino acid sequence into synthetic oligonucleotides consisting of several dozen bases, synthesizing these oligonucleotides, ligating them using a DNA ligase reaction, and inserting them into a plasmid vector.

[0064] In this case, for the purpose of efficient protein expression in Escherichia coli, it is generally practiced by those skilled in the art to use a nucleic acid sequence that uses the optimal codons for Escherichia coli. Furthermore, as the amino acid sequence of the immunoglobulin binding domain before modification, any domain of protein A may be used, but it is preferred to use the C domain, which has many amino acid residues after the 39th among the five domains originally present. Alternatively, the Z domain sequence, which has many examples of use as an affinity ligand for immunoglobulins, may also be used, but it is best to use the sequence of a C domain variant (shown in SEQ ID NO: 1 in the sequence listing) in which the 29th glycine residue is substituted with an alanine residue (Nilsson B. et. Al, Protein Engineering, 1(2), pp. 107-113), which is known to increase chemical stability. To achieve mutation of the DNA sequence with the desired amino acid substitution, the desired site can be easily introduced using methods such as overlap extension, where synthetic oligoDNAs containing mismatched base pairs are used as primers for polymerase chain reaction, or cassette mutagenesis, using the pre-altered cloned DNA as a template. Furthermore, when using immunoglobulin-binding proteins derived from Protein A as ligands for immunoglobulin affinity chromatography, aggregate proteins containing two or more, preferably about four, immunoglobulin-binding domains can be prepared and used according to conventional methods. Regarding the immunoglobulin-binding proteins obtained according to the present invention, aggregate proteins containing two or more, preferably 2-10, more preferably 4-7, and even more preferably 6, immunoglobulin-binding domains can be prepared and used. cDNAs encoding such aggregate proteins can be easily generated by tandemly linking only the desired number of cDNAs encoding a single immunoglobulin-binding domain. By inserting the thus generated cDNA into an appropriate expression plasmid and utilizing it, an aggregate protein of units linking two or more immunoglobulin binding domains can be easily produced.

[0065] As expression vectors for the present invention, any vectors, such as plasmids, phages, and viruses, that can replicate in host cells can be used. Examples of commercially available expression vectors include pQE vectors (QIAGEN), pDR540, pRIT2T (GE Healthcare Bio-Sciences), and pET vectors (Merck). The expression vector can be used in an appropriate combination with the host cell. For example, when using Escherichia coli as the host cell, a combination of a pET vector and the BL21 (DE3) E. coli strain, or a combination of the pDR540 vector and the JM109 E. coli strain, is preferred.

[0066] The modified protein of the present invention is obtained by collecting cultured cells by centrifugation, disrupting them using ultrasound or a French press, and recovering the soluble fraction. The modified protein can be purified by appropriately combining conventional separation and purification techniques. Specifically, in addition to separation techniques such as salting out, dialysis, and microfiltration, purification methods such as hydrophobic chromatography, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and reverse phase chromatography can also be used.

[0067] Insoluble supports for binding the modified protein of the present invention as an affinity ligand for immunoglobulins include, for example, natural polymers such as chitosan and polydextrose, and synthetic polymers such as vinyl polymers, highly cross-linked agarose, and polyimide. Alternatively, inorganic supports such as silica may be used. Typically, the ligand protein is immobilized on the support using coupling agents such as cyanogen bromide, epichlorohydrin, N-hydroxysuccinimide, tosyl / tresyl chloride, carbodiimide, glutaraldehyde, hydrazine, or carboxyl- or thiol-activated supports. This type of coupling reaction is well known in the art and is widely documented in the literature (e.g., Jansson, JC and Ryden, L., "Protein Purification," 2nd ed., pp. 375-442, ISBN 0-471-18626-0). The ligand protein of the present invention is characterized by being bound to a support via multiple amino groups arranged in a manner that spatially controls the orientation of the ligand. For protein immobilization, supports having active groups, such as trityl, epoxy, carboxyl, and formyl groups, that react with protein amino groups to form covalent bonds can be used. Examples of commercially available carriers include TOYOPEARL AF-Tresyl-650, TOYOPEARL AF-Epoxy-650, TOYOPEARL AF-Carboxy-650, TOYOPEARL AF-Formyl-650 (all from TOSOH Co., Ltd.), NHS-activated sepharose, cyanogen bromide-activated sepharose, and epoxy-activated sepharose (all from GE Healthcare Bio-Sciences Co., Ltd.).

[0068] In the protein A affinity column used in the present invention, the protein A modified ligand can be immobilized by any means, and can also be immobilized by the following means, for example. (1) In a C domain variant or Z domain of protein A, one to six of the 40th, 43rd, 46th, 53rd, 54th, and 56th amino acid residues are further substituted with lysine residues, and the substituted lysine residues are immobilized on a carrier. (2) A method of introducing cysteine ​​into the C-terminus of protein A and immobilizing the carrier by binding it to a disulfide bond or a thioether bond. (3) A method for immobilizing an amino-containing solidification support by cyaniding thiocyanate groups. (4) A method for immobilizing an aggregate of modified immunoglobulin binding domains having cysteine ​​residues on an amino-containing support using 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) as a cross-linking agent, and (5) A method of immobilizing a plurality of (e.g., 5) lysine residues added to the C-terminus of a modified immunoglobulin binding domain in which the 42nd, 49th, 50th, and 58th lysine residues of the C domain variant of protein A are replaced with amino acids other than lysine residues, or a modified immunoglobulin binding domain in which the 49th, 50th, and 58th lysine residues of the Z domain are replaced with amino acids other than lysine residues, on a carrier. The above-mentioned immobilization method can be performed by a commonly performed method.

[0069] Preferably, in the C domain variant or Z domain of protein A, one to six of the 40th, 43rd, 46th, 53rd, 54th, and 56th amino acid residues are further substituted with lysine residues, and the substituted lysine residues are immobilized on the carrier.

[0070] The protein A affinity column used in the present invention can be specifically exemplified by affinity resin AF-rProtein A HC-650F (manufactured by TOSOH) in which an Fc-binding ligand (recombinant modified protein A) is bound to a synthetic polymer support TOYOPEARL HW-56.

[0071] The protein A-modified ligand-immobilized support prepared in the above manner is loaded onto a column, which can serve as an affinity column (step a). Next, the prepared affinity column is loaded with a composition containing an IgG antibody with an altered amino acid sequence in the CH2 or CH3 region (step b). In the present invention, the composition containing IgG antibodies may refer to, for example, a culture of IgG antibody-expressing cells or its supernatant. Generally speaking, a culture medium is composed not only of the various nutrients necessary for culture but also of a complex composition composed of various components, such as cellular metabolic products. In order to purify the target IgG antibody to the required purity for pharmaceutical raw materials, it is necessary to determine purification conditions suitable for the target IgG antibody. According to the present invention, it is understood that for the purification of IgG antibodies containing the amino acid residue substitutions Q311R and P343R in the CH2 or CH3 region, variants of the C domain of protein A or Z domain in which at least one of the 4th, 7th, and 35th lysine residues originally present is substituted with an amino acid residue other than lysine, and which have the ability to bind to such IgG antibodies, are preferred purification tools.

[0072] The composition containing IgG antibodies may be pretreated by filtration or centrifugation before loading onto the affinity column. The composition containing IgG antibodies can be loaded onto the affinity column using a standard liquid chromatography system at an appropriate pressure and flow rate, depending on the column size and capacity, or the size of the carrier. The amount of IgG antibody composition loaded onto the affinity column is preferably equal to the IgG antibody binding capacity of the affinity column. For example, the IgG antibody binding capacity can be determined by monitoring the concentration of IgG antibodies eluting from the affinity column packed with the composition. Specifically, when the level of IgG antibodies eluting from the affinity column is equal to the IgG antibody concentration in the loaded composition, it can be determined that the IgG antibody binding capacity of the affinity column is close to that of the affinity column. Subsequently, when the IgG antibodies are eluted from the affinity column (step c), efficient purification of the IgG antibodies can be expected.

[0073] The purification method of the present invention may further comprise, before step (c), a step of washing the affinity column with a washing solution.

[0074] Although the cleaning solution is not particularly limited, it may be combined with a buffer and a salt. A solution containing at least one selected from the group consisting of phosphoric acid, acetic acid, citric acid, glycine, and tris(hydroxymethyl)aminomethane as a buffer and at least one selected from the group consisting of arginine, sodium chloride, and sodium sulfate as a salt can be used.

[0075] After washing the affinity column as needed, the purified IgG antibodies adsorbed to the affinity column can be recovered (step c). The method for dissolving the IgG antibodies adsorbed to the protein A-modified ligand can be appropriately selected from known conditions. For example, a solution containing at least one selected from the group consisting of hydrochloric acid, acetic acid, citric acid, arginine, glycine, or phosphoric acid can be used. The concentration of the solution used to dissolve the IgG antibodies from the affinity column can be adjusted according to the desired purpose. For example, if acetic acid is used, the concentration can be 20 to 500 mM, typically 50 to 200 mM, and if hydrochloric acid is used, the concentration can be 1 to 5 mM. When dissolving the IgG antibodies from the affinity column, the dissolution of the IgG antibodies can also be tracked by monitoring the protein concentration during dissolution.

[0076] After step (c), the recovered IgG antibodies may be further purified as needed. For example, the purification method of the present invention may further comprise a purification step of the IgG antibodies by at least one chromatographic method selected from the group consisting of cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, multimodal chromatography, and hydroxyapatite chromatography. Through the above steps, in a preferred embodiment of the present invention, IgG antibodies can be isolated from host cells or extracellularly (in culture medium, etc.) and purified into substantially pure and homogeneous IgG antibodies. Specifically, the present invention provides a method for producing purified IgG antibodies comprising the following steps: (i) providing a composition comprising an IgG antibody comprising amino acid substitutions of Q311R and P343R; (ii) preparing an affinity column comprising a carrier immobilized with a modified protein A ligand, wherein the modified protein A ligand comprises a modified immunoglobulin binding domain in which any one or more of the 4th, 7th, and 35th lysine residues originally present in the C domain variant of Staphylococcus protein A defined by SEQ ID NO: 1 or the Z domain defined by SEQ ID NO: 2 is substituted with an amino acid residue other than lysine, or a collection of such modified immunoglobulin binding domains; (iii) a step of filling the composition containing the above-mentioned IgG antibody into the affinity column of step (ii); and (iv) a step of eluting and recovering IgG antibodies from the affinity column packed with the composition containing IgG antibodies in step (iii). The present invention also includes highly purified IgG antibodies using the purification method.

[0077] Although the present invention is described in more detail below with reference to examples, the present invention is not limited to these examples. [Example 1]

[0078] Antibody A is an antibody with an Fc region identified as SEQ ID NO: 10, in which amino acid residues Q311R and P343R are substituted in the CH2 or CH3 regions to improve pharmacodynamics, thereby increasing its isoelectric point (pI) and enhancing its affinity for Fc receptor IIb (FcRIIb) or neonatal Fc receptor (FcRn). This substitution results in a decrease in the binding affinity of Antibody A for Protein A. While Antibody A improves its pharmacodynamics and thus its usefulness as a pharmaceutical, it also presents new manufacturing challenges.

[0079] The binding affinity of antibodies to Protein A is often used in purification procedures (affinity purification). Specifically, the process of adsorbing the antibody to a column immobilized with Protein A, washing it, and then eluting and recovering it is a widely used method for antibody purification. Because Protein A binds to the Fc region of antibodies, it can be used for a wide range of antibodies regardless of their antigenic specificity. Furthermore, various Protein A columns are commercially available, varying the method for immobilizing Protein A, the resin, or the Protein A itself.

[0080] To identify a protein A immobilized resin suitable for affinity purification of antibody A, the affinity of the antibody to the following commercially available protein A immobilized resins was compared: HiTrap MabSelect SuRe (manufactured by GE Healthcare, trade name); ToyoScreen AF-rProtein A HC-650F (manufactured by TOSOH, trade name); Amsphere A3 (manufactured by JSR Life Sciences, registered trademark); MiniChrom Column Eshmuno A (Merck Millipore, registered trademark); MabSpeed ​​rP202 (manufactured by Mitsubishi Chemical, registered trademark); KanCap Pre-packaged Column (manufactured by KANEKA, trade name).

[0081] (1) Dynamic binding capacity (DBC) of each column for Antibody A Purified Antibody A was dissolved in equilibration buffer and loaded onto a column packed with each Protein A-immobilized resin. The protein concentration of the buffer eluting from the column was tracked using UV light to identify the 5% breakthrough point. The DBC per liter of resin was then determined using the following formula. The 5% breakthrough point represents the amount of protein loaded into the column at which the protein concentration of the eluate exceeds 5% of the protein concentration of the antibody solution loaded into the column: Antibody A concentration (g / L) x volume of liquid filled (5% breakthrough point) (L) DBC= ――――――――――――――――――――――――――――――――― Column capacity (L) Similarly, for comparison, the DBC of each column was also determined for a humanized antibody containing no alterations in the Fc region of human IgG1.

[0082] (2) Dynamic binding capacity; DBC [Table 1]

[0083] F-rProtein A HC-650F, which binds the most Antibody A, also exhibited high DBCs of 46.6 and 45.2 when evaluated using two resins from different production batches. Amsphere A3, which exhibited the second-highest DBC after AF-rProtein A HC-650F, had a DBC of 13.6. The DBCs of the other resins ranged from 1.6 to 6.4, which were quite low. Meanwhile, for antibodies with unchanged Fc regions, as shown in Figure 1, the DBCs of each resin ranged from 20 to 70, which are sufficient for antibody purification.

[0084] F-rProtein A HC-650F (manufactured by TOSOH) is an affinity resin that binds an Fc-binding ligand (a recombinant modified protein A) to the synthetic polymer support TOYOPEARL HW-65. The ligand bound to AF-rProtein A HC-650F has the following structure (1'): (R1) 5-(R2) 1(1') In the above formula (1'), the left end is the N-terminus and the right end is the C-terminus. In the above formula (1'), (R2) represents a modified immunoglobulin-binding domain (SEQ ID NO: 4) in which the amino acid sequence of the C domain of the immunoglobulin-binding domain of protein A from Staphylococcus aureus is partially substituted, wherein the amino acid sequence of the C domain of the C domain of protein A from Staphylococcus aureus is partially substituted, wherein the 4th amino acid sequence is substituted with an alanine residue, the 7th amino acid sequence is substituted with a threonine residue, the 35th amino acid sequence is substituted with an arginine residue, the 40th amino acid sequence is substituted with a lysine residue, the 43rd amino acid sequence is substituted with a lysine residue, the 46th amino acid sequence is substituted with a lysine residue, and the 53rd amino acid sequence is substituted with a lysine residue. The five (R1) residues located on the N-terminal side of the modified immunoglobulin-binding domain (SEQ ID NO. 5) comprise a sequence in which the amino acid sequence of the C domain variant has the 4th residue substituted with an alanine residue, the 7th residue substituted with a threonine residue, and the 35th residue substituted with an arginine residue, and the amino acid sequence in which the 42nd residue is substituted with an alanine residue, the 49th residue substituted with an arginine residue, the 50th residue substituted with an arginine residue, and the 58th residue substituted with an arginine residue. In other words, formula (1') represents a ligand formed by linking the five amino acid residues of SEQ ID NO. 5 and the one amino acid residue of SEQ ID NO. 4 from the N-terminal side. In this variant, the immunoglobulin binding domain is modified into a hexamer, further enhancing binding to the Fc region. AF-rProtein A HC-650F utilizes this structural variant to significantly improve Fc binding and is an alkali-resistant affinity resin. The affinity resins used in this comparative test were all optimized for the structure of Protein A itself and its binding pattern to the carrier, enhancing binding to the Fc region and enhancing alkali resistance. While the Fc region remained unchanged, the resins exhibited excellent antibody binding. However, modifications to the Fc region significantly altered the binding properties of the affinity resin. [Example 2]

[0085] To evaluate the ligand binding performance of the pI-modified antibody containing the substitutions of the amino acid residues Q311R and P343R, the KD value of the BLItz (registered trademark) (ForteBio) evaluation system was measured for AF-rProtein A HC-650F and MabSelect SuRe used in Example 1. The ligands for AF-rProtein A HC-650F (structure shown in Formula (1')) and MabSelect SuRe were labeled with biotin (Lys:Biotin = 1:1) and immobilized on the sensor chip surface. After equilibration with PBS, the antibody-containing solution was diluted with PBS (+0.1% BSA) buffer, and PBS (+BSA) was further added to measure the dissociation reaction. In addition to Antibody A, the humanized anti-interleukin-6 (IL-6) receptor antibody tocilizumab (hPM-1 or MRA: see International Patent Application Publication No. WO92-19759) was used as a comparative example. Unlike Antibody A, tocilizumab does not contain the amino acid substitutions Q311R and P343R that are used to alter the pI (non-pI-altered antibody). The test results are summarized in Figure 2 and Table 2. Tocilizumab binds to the AF-rProtein A HC-650F ligand with a KD value of 1.55×10-9 M. Furthermore, while Antibody A has a KD value of 184×10-9 M, showing a weaker affinity than tocilizumab, binding is confirmed. Meanwhile, tocilizumab binds to the MabSelect SuRe ligand with a KD value of 8.11×10-9 M, no binding is confirmed.

[0086] [Table 2] [Example 3]

[0087] To evaluate the relationship between substitutions in the ligand's amino acid residues and the binding affinity to the pI-modified antibody, ligand monomers containing the 4th, 7th, and 35th mutations of the C domain variant (SEQ ID NO: 1) were used, and the KD value of Antibody A was measured using the same method as in Example 2 (but changing the Lys:Biotin ratio to 12:1). From the results in Table 3, it is believed that the affinity for Antibody A is increased by replacing the 35th lysine residue (K) of the C domain variant with a glutamine residue (Q) or an arginine residue (R).

[0088] [Table 3] * C domain variants indicate no substitutions (4th: K, 7th: K, 35th: K).

[0089] In addition, for C domain variants, ligand monomers in which amino acid residues 40, 43, 46, and 53 were substituted with lysine residues (K) (hereinafter referred to as "R2' structure ligand monomers") were introduced at the 4th, 7th, and 35th residues, and the KD value of antibody A was measured using the same method as in Example 2 (but changing the Lys:Biotin ratio to 12:1). Based on the results in Table 4, it is believed that the affinity for Antibody A is increased by replacing the 35th lysine residue (K) in the R2' structure ligand monomer with an arginine residue (R) or a valine residue (V), particularly the arginine residue (R). Furthermore, in addition to the 35th substitution, it is believed that the affinity for Antibody A is increased by replacing the 7th lysine residue (K) with any of the following: a tyrosine residue (Y), a phenylalanine residue (F), a threonine residue (T), an arginine residue (R), a glutamine residue (Q), a valine residue (V), a leucine residue (L), an isoleucine residue (I), a histidine residue (H), an alanine residue (A), or a proline residue (P), particularly a tyrosine residue (Y) or a phenylalanine residue (F).

[0090] [Table 4] * C domain variants indicate no substitutions (4th: K, 7th: K, 35th: K, 40th: V, 43rd: E, 46th: A, 53rd: D). [Example 4]

[0091] To evaluate the relationship between substitution of ligand amino acid residues and the binding affinity of the pI-modified antibody, the KD value of Antibody A was measured for multiple ligand dimers using the same method as in Example 2 (but changing the ratio of Lys:Biotin to 12:1). The ligand dimers tested were ligand monomers with an R2' structure in which amino acid residues 40, 43, 46, and 53 were substituted with lysine residues (K) for the C domain variant, and ligand monomers with amino acid residues 42 and 49, 50, and 58 with arginine residues (R) for the C domain variant (hereinafter referred to as "R1' structure ligand monomers"), with further mutations at the 4th, 7th, and 35th positions. Based on the results in Table 5, it is believed that dimers in which the fourth lysine residue (K) is replaced with any of the following: valine residue (V), isoleucine residue (I), glutamine residue (E), or arginine residue (R) all have increased affinity for antibody A.

[0092] [Table 5] *C domain variants indicate no substitutions (40th: V, 43rd: E, 46th: A, 53rd: D, 42nd: K, 49th: K, 50th: K, 58th: K). [Example 5]

[0093] To evaluate the relationship between the addition of polylysine residues (K) to the ligand monomer and the binding affinity of the pI-modified antibody, the KD value of Antibody A was measured using the same method as in Example 2 (but with a Lys:Biotin ratio of 12:1). The ligand monomer under investigation has an R1' structure in which the 42nd, 49th, 50th, and 58th lysine residues are replaced with amino acids other than lysine, and five lysine residues (K) are added to the C-terminus of the ligand monomer for carrier immobilization. Based on the results in Table 6, it is believed that the ligand monomer in which the 42nd, 49th, 50th, and 58th lysine residues were substituted with alanine residues (A) and arginine residues (R) other than lysine, and multiple lysine residues (K) were added to the C-terminus, has increased affinity for antibody A by substituting the 35th residue with an arginine residue.

[0094] [Table 6] [Industrial Applicability]

[0095] Antibodies with altered pI, which cannot be purified using conventional protein A columns, can be purified efficiently and simply using the specific protein A affinity columns of the present invention. This invention can be used in the industrial production of stable and efficient antibody pharmaceuticals.

[0096] none

Claims

1. A method for purifying an IgG antibody from a composition containing an IgG antibody with amino acid residues including Q311R and P343R, comprising the following steps: (a) preparing an affinity column containing a carrier immobilized with a protein A altered ligand, the protein A altered ligand comprising an aggregate of altered immunoglobulin binding domains; (b) filling the affinity column of step (a) with the composition containing the IgG antibody; and (c) dissolving and recovering the IgG antibody from the affinity column of step (b), wherein the IgG antibody comprises a heavy chain constant region having an amino acid sequence selected from the group consisting of sequence numbers 12 to 57, the aggregate of altered immunoglobulin binding domains being an aggregate of altered immunoglobulin binding domains having the ability to bind to IgG antibodies with amino acid residues including Q311R and P343R, and represented by the following formula (1) with the left side being the N-terminus and the right side being the C-terminus. (R1)n-(R2)m, or (R2)m-(R1)n (1), where, (A) R1 and R2 are respectively the altered C domain of Staphylococcus protein A defined by sequence number 1 or the Z domain defined by sequence number 2, (i) containing the 35th lysine residue replaced by an arginine residue or a glutamine residue. (ii) The fourth lysine residue is replaced with an alanine residue, a valine residue, an isoleucine residue, an arginine residue, or a glutamic acid residue, and the 35th lysine residue is replaced with an arginine residue, a glutamine residue, or a valine residue; or (iii) The fourth lysine residue is replaced with an alanine residue, a valine residue, an isoleucine residue, an arginine residue, or a glutamic acid residue, the seventh lysine residue is replaced with a tyrosine residue, a phenylalanine residue, a glutamine residue, a leucine residue, an isoleucine residue, a proline residue, a threonine residue, an alanine residue, a valine residue, an arginine residue, or a histidine residue, and the 35th lysine residue is replaced with an arginine residue, a glutamine residue, or a valine residue, (B) n is an integer of 1 to 9, (C) m is an integer of 1 or 2, (D) the total of n and m is 2 to 10. (E) n R1 fields can be sequences that are the same or different from each other, and (F) m R2 fields can be sequences that are the same or different from each other.

2. The method as described in claim 1, wherein R1 and R2 respectively comprise the replacement of the fourth lysine residue with an alanine residue, the seventh lysine residue with a threonine residue, and the 35th lysine residue with an arginine residue in the C domain alteration of Staphylococcus protein A as defined by sequence number 1 or the Z domain as defined by sequence number 2.

3. The method as described in claim 1, wherein R1 and R2 further comprise substitutions of 1 to 4 of the following: 42nd lysine residue replaced with an alanine residue; 49th lysine residue replaced with an arginine residue; 50th lysine residue replaced with an arginine residue; and 58th lysine residue replaced with an arginine residue.

4. The method as claimed in claim 1, wherein R1 and R2 further comprise the replacement of the 42nd lysine residue with an alanine residue, the 49th lysine residue with an arginine residue, the 50th lysine residue with an arginine residue, and the 58th lysine residue with an arginine residue.

5. The method as claimed in claim 1, wherein the first and / or second R1 and / or R2 from the N-terminal or C-terminal side further comprises at least one amino acid residue from the 40th, 43rd, 46th, 53rd, 54th, and 56th originally present amino acid residues replaced with lysine residues.

6. The method as described in claim 5, wherein the first and / or second R1 and / or R2 from the N-terminal or C-terminal side further comprises the replacement of the 40th, 43rd, 46th and 53rd original amino acid residues with lysine residues.

7. The method as described in claim 1, wherein the aforementioned aggregate of altered immunoglobulin binding domains is an aggregate of altered immunoglobulin binding domains in which R1 is formed by the amino acid sequence shown in sequence number 5 and R2 is formed by the amino acid sequence shown in sequence number 4.

8. The method as described in claim 1, wherein the above-mentioned collection of altered immunoglobulin binding domains is n = 5 and m = 1.

9. The method described in any one of claims 1 to 8, wherein the aforementioned protein A altered ligand is immobilized on the carrier by any means selected from the group consisting of (1)-(5) below: (1) an altered immunoglobulin binding domain immobilization method by further replacing one to six of the 40th, 43rd, 46th, 53rd, 54th and 56th amino acid residues with lysine residues in the C-domain altered or Z-domain of protein A, (2) a method of introducing cysteine ​​at the C-terminus of protein A and immobilizing it by binding the carrier to a disulfide bond or a thioether bond, (3) a method of immobilizing an amino-containing immobilized carrier by means of cyanothiol group, (4) A method of immobilizing an aggregate of altered immunoglobulin binding domains having cysteine ​​residues onto an amino-containing carrier by using 4-(N-cis-butenediaminomethyl)cyclohexane-1-carboxylic acid ester (SMCC) as a crosslinking agent, and (5) A method of immobilizing an aggregate of altered immunoglobulin binding domains having cysteine ​​residues onto an amino-containing carrier by replacing the 42nd, 49th, 50th, and 58th lysine residues of the altered immunoglobulin binding domain in the C domain of protein A with amino acids other than lysine residues, or by replacing the 49th, 50th, and 58th lysine residues of the altered immunoglobulin binding domain in the Z domain with amino acids other than lysine residues at the C-terminus of the altered immunoglobulin binding domain.

10. The method as described in any one of claims 1 to 8, wherein the pI value of the antibody is 4.0 to 10.0.

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