Method for reducing host cell proteins in affinity chromatography

Abstract

To provide a method for purifying a protein for obtaining an antibody with reduced content of impurities (e.g., a host cell protein) in affinity chromatography.SOLUTION: Disclosed herein is a method for purifying an antibody by reducing the content of a host cell protein; in particular, a method for using a low conductivity aqueous solution in a wash step of protein A chromatography for reducing the content of a host cell protein, where the protein A chromatography is used to purify a human IgG1 or IgG4 isotype antibody, and where the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of polypeptide purification. In particular, the present invention relates to the reduction of host cell proteins such as phospholipase B-like 2 (PLBL2) or clusterin in solutions containing antibodies.

Background Art

[0002] Background of the Invention Proteins, especially immunoglobulins, play an important role in the modern medical portfolio. For human application, therapeutic proteins must meet certain clear criteria. To ensure the safety of biopharmaceuticals for humans, it is necessary, among other things, to remove by-products accumulated during the production process. To meet regulatory specifications, one or more purification steps are required after the manufacturing process. In particular, purity, throughput, and yield play important roles in determining an appropriate purification process.

[0003] For protein purification, various methods have been established and widely used, such as affinity chromatography (e.g., protein A or protein G affinity chromatography, single-chain Fv ligand affinity chromatography), ion exchange chromatography (e.g., cation exchange (sulfopropyl resin or carboxymethyl resin), anion exchange (aminoethyl resin) and mixed-mode ion exchange), thiophilic adsorption (e.g., by beta-mercaptoethanol ligand and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., by phenyl-sepharose, aza-areneophilic resin, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., by Ni(II)-affinity material and Cu(II)-affinity material), size exclusion chromatography, and electrophoresis (gel electrophoresis, capillary electrophoresis, etc.).

[0004] The purification of recombinant immunoglobulins often involves a combination of different column chromatography steps. During purification, host cell proteins and DNA, as well as non-immunoglobulin contaminants such as endotoxins and viruses, are depleted. Therefore, typically, one or more additional separation steps are performed after an affinity chromatography step, such as protein A affinity chromatography. Generally, it is stated that high-conductivity buffers are used in the washing step of affinity chromatography methods.

[0005] US 6,127,526 (Patent Document 1) describes a method for purifying a protein by protein A chromatography, comprising the steps of (a) adsorbing the protein onto protein A immobilized on a solid phase containing silica or glass, (b) removing impurities bound to the solid phase by washing the solid phase with a hydrophobic electrolyte solvent, and (c) recovering the protein from the solid phase.

[0006] WO2011 / 038894 (Patent Document 2) reports a protein A chromatography method that significantly depletes host cell proteins and DNA through a special washing step prior to the recovery of immunoglobulins from protein A chromatography material.

[0007] WO2013 / 177118 (Patent Document 3) reports a composition and method for isolating and purifying antibodies from a sample matrix.

[0008] WO2013 / 033517 (Patent Document 4) reports a method for isolating polypeptides of interest (such as antibodies) from viruses.

[0009] EP2583973 (Patent Document 5) reports a method for purifying proteins, comprising one or more chromatography processes in which an amino acid, or its dipeptide, oligopeptide, or polyamino acid, is contained in a buffer solution (equilibrium buffer, wash buffer, and elution buffer) used in at least one chromatography process, thereby purifying high-purity proteins with extremely small amounts of impurities (e.g., polymers or host cell proteins). [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] US 6,127,526 [Patent Document 2] WO2011 / 038894 [Patent Document 3] WO2013 / 177118 [Patent Document 4] WO2013 / 033517 [Patent Document 5] EP2583973 [Overview of the Initiative]

[0011] This specification reports a method for producing antibodies with reduced host cell protein content by purifying them using affinity chromatography.

[0012] More specifically, the present invention, which uses a low-conductivity aqueous solution in the affinity chromatography washing step prior to the recovery of antibodies from the chromatography material, has been found to reduce the content of certain host cell proteins in the antibody-containing solution. As a result, it was found that the content of phospholipases (particularly phospholipase B-like 2 (PLBL2)) can be reduced. The PLBL2 content was found to be reduced by 100 times or more when the antibody is of the IgG4 isotype.

[0013] One aspect reported herein is the use of low-conductivity aqueous solutions to reduce the host cell protein content in the washing step of protein A chromatography used to purify human IgG1 or human IgG4 isotype antibodies.

[0014] In one aspect of this aspect, the human IgG4 isotype antibody is an antibody against P-selectin, or a bispecific antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. In one aspect of this aspect, the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.

[0015] In one aspect of this situation, a low-conductivity aqueous solution has a conductivity value of approximately 0.5 mS / cm or less.

[0016] In one aspect of this situation, the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

[0017] In one aspect of this situation, the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 8 mM of tris.

[0018] In one aspect of this situation, the low-conductivity aqueous solution contains approximately 0.05 mM to approximately 2 mM calcium phosphate.

[0019] In one aspect of this situation, a low-conductivity aqueous solution has a pH of approximately 7 or higher.

[0020] In one aspect of this situation, the low-conductivity aqueous solution cleaning step is performed after or before the high-conductivity aqueous solution cleaning step.

[0021] In one aspect of this situation, the high-conductivity aqueous solution has a conductivity value of about 20 mS / cm or more.

[0022] In one aspect of this situation, an intermediate washing step with a medium-conductivity aqueous solution is performed between the low-conductivity aqueous solution washing step and the high-conductivity aqueous solution washing step.

[0023] In one aspect of this situation, the medium-conductivity aqueous solution has a conductivity value exceeding 0.5 mS / cm and less than 20 mS / cm.

[0024] In one aspect of this situation, the high (or medium)-conductivity aqueous solution contains histidine.

[0025] One aspect reported in this specification is (a) culturing a cell containing a nucleic acid encoding a human IgG4 isotype antibody or a human IgG1 isotype antibody, (b) recovering a human IgG4 isotype antibody or a human IgG1 isotype antibody from the cell or the culture medium, (c) contacting a human IgG4 isotype antibody or a human IgG1 isotype antibody with a protein A chromatography material, (d) washing the protein A chromatography material with a low-conductivity aqueous solution, (e) recovering a human IgG4 isotype antibody or a human IgG1 isotype antibody from the protein A chromatography material comprising thereby producing a human IgG4 isotype antibody or a human IgG1 isotype antibody, <第0000109号>A method for producing a human IgG4 isotype antibody or a human IgG1 isotype antibody. <000011-0号>

[0026] 号> 号>One aspect reported in this specification is <00001-13号>(a) preparing a sample containing a human IgG4 isotype antibody or a human IgG1 isotype antibody, 号>(b) A step of purifying a human IgG4 isotype antibody or a human IgG1 isotype antibody by a protein A chromatography method / step that includes washing the protein A chromatography material with a low-conductivity aqueous solution. This is a method for purifying human IgG4 isotype antibodies or human IgG1 isotype antibodies from a sample.

[0027] In one aspect of all aspects, the human IgG4 isotype antibody is an antibody against P-selectin, or a bispecific antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. In one aspect of all aspects, the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.

[0028] In one embodiment of all aspects, the low-conductivity aqueous solution has a conductivity value of approximately 0.5 mS / cm or less.

[0029] In one aspect of all aspects, the host cell protein content is reduced, and the (specific) host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

[0030] In one aspect of all aspects, the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 8 mM of tris.

[0031] In one embodiment of all aspects, the low-conductivity aqueous solution contains approximately 0.05 mM to approximately 2 mM potassium phosphate.

[0032] In one aspect of all aspects, the low-conductivity aqueous solution has a pH of approximately 7 or higher.

[0033] In one aspect of the method, the method further includes the step of washing affinity chromatography material with a high-conductivity aqueous solution and / or a medium-conductivity aqueous solution before or after washing protein A chromatography material with a low-conductivity aqueous solution.

[0034] In one embodiment of all aspects, the highly conductive aqueous solution has a conductivity value of approximately 20 mS / cm or higher.

[0035] In one aspect of all aspects, the medium conductivity aqueous solution has conductivity values ​​ranging from greater than 0.5 mS / cm to less than 20 mS / cm.

[0036] In one embodiment of all aspects, the highly conductive aqueous solution or the moderately conductive aqueous solution contains histidine. [Invention 1001] The use of a low-conductivity aqueous solution with a conductivity value of approximately 0.5 mS / cm or less to reduce the host cell protein content during the washing step of protein A chromatography used to purify human IgG4 isotype antibodies or human IgG1 isotype antibodies. [Invention 1002] Use of Invention 1001, wherein the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin. [Invention 1003] Use of any of invention 1001 to 1002, wherein the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 8 mM of tris. [Invention 1004] Use of any of invention 1001 to 1002, wherein the low-conductivity aqueous solution contains approximately 0.05 mM to approximately 2 mM potassium phosphate. [Invention 1005] Use of any of the low-conductivity aqueous solutions having a pH of approximately 7 or higher, as described in invention 1001 to 1004. [Invention 1006] Use of any of the invention 1001 to 1005, wherein the low-conductivity aqueous solution cleaning step is performed after or before the high-conductivity aqueous solution cleaning step. [Invention 1007] Use of the present invention 1006, in which the highly conductive aqueous solution has a conductivity value of approximately 20 mS / cm or higher. [Invention 1008] Use of any of the invention's 1006-1007, wherein the highly conductive aqueous solution contains histidine. [Invention 1009] Use of any of the inventions 1001 to 1008, wherein the human IgG4 isotype antibody is an antibody against P-selectin, or an antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. [Invention 1010] Use of any of the inventions 1001 to 1008, wherein the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3. [Invention 1011] (a) A step of culturing cells containing nucleic acids encoding human IgG4 isotype antibody or human IgG1 isotype antibody, (b) A step of recovering human IgG4 isotype antibody or human IgG1 isotype antibody from the cells or culture medium, (c) A step of contacting a human IgG4 isotype antibody or a human IgG1 isotype antibody with a protein A chromatography material. (d) A step of washing the protein A chromatography material with a low-conductivity aqueous solution having a conductivity value of approximately 0.5 mS / cm or less. (e) Recovering human IgG4 isotype antibody or human IgG1 isotype antibody from protein A chromatography material. Includes, This produces human IgG4 isotype antibodies or human IgG1 isotype antibodies. A method for producing human IgG4 isotype antibodies or human IgG1 isotype antibodies. [Invention 1012] (a) The step of preparing a sample containing a human IgG4 isotype antibody or a human IgG1 isotype antibody, (b) A step of purifying a human IgG4 isotype antibody or a human IgG1 isotype antibody by a protein A chromatography method / step that includes washing the protein A chromatography material with a low-conductivity aqueous solution having a conductivity value of approximately 0.5 mS / cm or less. A method for purifying human IgG4 isotype antibody or human IgG1 isotype antibody from a sample, including [specific antibody details]. [Invention 1013] A method according to any one of the present invention 1011 to 1012, wherein the amount of host cell protein is reduced, and the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin. [Invention 1014] A method according to any of the present invention 1011 to 1013, wherein the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 8 mM of tris. [Invention 1015] A method according to any of the present invention 1011 to 1014, wherein the low-conductivity aqueous solution contains approximately 0.05 mM to approximately 2 mM potassium phosphate. [Invention 1016] A method according to any of the present invention 1011 to 1015, wherein the low-conductivity aqueous solution has a pH of approximately 7 or higher. [Invention 1017] Any method of the present invention 1011 to 1016, further comprising the step of washing affinity chromatography material with a high-conductivity aqueous solution and / or a medium-conductivity aqueous solution before or after washing protein A chromatography material with a low-conductivity aqueous solution. [Invention 1018] The method of the present invention 1017, wherein the highly conductive aqueous solution has a conductivity value of approximately 20 mS / cm or higher. [Invention 1019] The method of the present invention 1017, wherein the aqueous solution with medium conductivity has a conductivity value ranging from more than 0.5 mS / cm to less than 20 mS / cm. [Invention 1020] A method according to any one of the present invention 1017 to 1019, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains histidine. [Invention 1021] The method according to any of items 1011 to 1020 of the present invention, wherein the human IgG4 isotype antibody is an antibody against P-selectin, or an antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. [Invention 1022] The method according to any of items 1011 to 1020 of the present invention, wherein the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3. [Modes for carrying out the invention]

[0037] Detailed description of the invention This specification reports improved affinity chromatography methods and applications, including the washing of affinity chromatography materials with low-conductivity aqueous solutions.

[0038] It has been found that using a washing step with a low-conductivity aqueous solution in the affinity chromatography stage, such as the protein A chromatography stage, can reduce the amount of specific host cell proteins. The affinity chromatography stage is used in antibody purification or antibody production methods. The low-conductivity aqueous solution washing step is particularly effective in reducing the content of phospholipase B-like 2 (PLBL2).

[0039] One aspect reported herein is the use of low-conductivity aqueous solutions to reduce the content of (specific) host cell proteins during the washing step of affinity chromatography.

[0040] One aspect reported herein is, (a) A step of culturing cells containing nucleic acids encoding human IgG isotype antibodies, (b) A step of recovering human IgG isotype antibodies from the cells or culture medium, (c) A step of contacting a human IgG isotype antibody (or a solution containing it) with an affinity chromatography material. (d) A step in which the affinity chromatography material is washed with a low-conductivity aqueous solution while maintaining that at least 90% of the bispecific antibodies are bound to the affinity chromatography material. (e) Recovery of human IgG isotype antibodies from affinity chromatography material. Includes, This produces human IgG isotype antibodies. This is a method for producing human IgG isotype antibodies.

[0041] One aspect reported herein is, (a) The step of preparing a sample containing human IgG isotype antibodies (buffered water), (b) A step of purifying human IgG isotype antibodies by affinity chromatography / steps, which includes washing affinity chromatography material with a low-conductivity aqueous solution. This is a method for purifying human IgG isotype antibodies, including [specific antibodies], from a sample.

[0042] Recombinant polypeptides produced in CHO cells can be purified according to the methods described herein to remove or reduce the levels of host cell proteins.

[0043] Examples of recombinant polypeptides include therapeutic antibodies and immunoadhesins, such as antibodies (including antibody fragments) against one or more of the following antigens, but not limited to: HER1 (EGFR), HER2 (e.g., trastuzumab, pertuzumab), HER3, HER4, VEGF (e.g., bevacizumab, ranibizumab), MET (e.g., onartuzumab), CD20 (e.g., rituximab, obinutuzumab, ocrelizumab), CD22, CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, VCAM, IL-17A and / or F, IgE (e.g., omalizumab), DRS, CD40, Apo2L / TRAIL, EGFL7 (e.g., parsatuzumab), NRP1, integrin beta 7 (e.g., etrolizumab), IL-13 (e.g., lebri). Kizumab), A-beta (e.g., crenezumab, gantenerumab), P-selectin (e.g., incrumab), IL-6R (e.g., tosilzumab), IFNa (e.g., rontalizumab), M1prime (e.g., quilizumab), mitogen-activated protein kinase (MAPK), OX40L, TSLP, factor D (e.g., lamparizumab), and receptors, e.g., IL-9 receptor, IL-5 receptor, IL-4 receptor alpha, IL-13 receptor alpha-1 and IL-13 receptor alpha-2, OX40, TSLP-R, IL-7R-alpha (co-receptor of TSLP), IL17RB (receptor of IL-25), ST2 (receptor of IL-33), CCR3, CCR4, CRTH2, Fc-epsilon RI and Fc-epsilon RII / CD23 (receptors of IgE).Other exemplary antibodies, though not limited to them, include anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P glycoprotein antibodies, anti-CEA antibodies, Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9 / p24 antibodies, anti-CD10 antibodies, anti-CD11c antibodies, anti-CD13 antibodies, anti-CD14 antibodies, anti-CD15 antibodies, anti-CD19 antibodies, anti-CD23 antibodies, anti-CD30 antibodies, and anti-CD3 antibodies. Examples include antibodies selected from 1 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate-specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn antigen antibody.

[0044] In some embodiments, exemplary antibodies include antibodies against A beta, antibodies against IL17 A / F, and antibodies against CMV. Exemplary anti-A beta antibodies and methods for producing such antibodies have been previously described, for example, in WO2008011348, WO2007068429, WO2001062801, and WO2004071408. Exemplary anti-IL17 A / F antibodies and methods for producing such antibodies have been previously described, for example, in WO2009136286 and U.S. Patent No. 8,715,669. Exemplary anti-CMV antibodies, including anti-CMV-MSL, and methods for producing such antibodies have been previously described, for example, in WO2012047732.

[0045] In some embodiments, affinity chromatography is used to purify human IgG isotype antibodies. In some embodiments, affinity chromatography is used to purify IgG4 antibodies. In one embodiment, the IgG4 isotype antibody is an antibody against P-selectin, or a (bispecific) antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. In some embodiments, affinity chromatography is used to purify IgG1 isotype antibodies. In one embodiment, the IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a (bispecific) antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.

[0046] One aspect reported herein is, (a) A step of culturing cells containing nucleic acids encoding human IgG4 isotype antibodies, (b) A step of recovering human IgG4 isotype antibodies from the cells or culture medium, (c) The step of contacting a human IgG4 isotype antibody with an affinity chromatography material. (d) A step in which the affinity chromatography material is washed with a low-conductivity aqueous solution. (e) Recovery of human IgG4 isotype antibodies from affinity chromatography material. Includes, This produces human IgG4 isotype antibodies. This is a method for producing human IgG4 isotype antibodies (containing solutions).

[0047] One aspect reported herein is, (a) A step of culturing cells containing nucleic acids encoding IgG4 isotype antibodies, (b) A step of recovering IgG4 isotype antibodies from the cells or culture medium, (c) The step of contacting the IgG4 isotype antibody with the affinity chromatography material. (d) A step in which the affinity chromatography material is washed with a low-conductivity aqueous solution. (e) Step of recovering IgG4 isotype antibodies from affinity chromatography material. Includes, This produces IgG4 isotype antibodies. This is a method for producing IgG4 isotype antibodies (containing solutions).

[0048] One aspect reported herein is, (a) The step of preparing a sample containing human IgG4 isotype antibodies, (b) A step of purifying human IgG4 isotype antibodies by an affinity chromatography method / step that includes washing affinity chromatography material with a low-conductivity aqueous solution. This is a method for purifying human IgG4 isotype antibodies, including [specific antibody names], from a sample.

[0049] One aspect reported herein is, (a) Steps to prepare a sample containing IgG4 isotype antibody, (b) A step of purifying IgG4 isotype antibodies by affinity chromatography / steps, which includes washing affinity chromatography material with a low-conductivity aqueous solution. This is a method for purifying IgG4 isotype antibodies, including [specific antibody names], from a sample.

[0050] It has been found that if the conductivity of the aqueous solution used in the washing stage is low, i.e., if a low-conductivity aqueous solution is used for washing, the content of host cell proteins can be reduced. In one aspect of all aspects, the low-conductivity aqueous solution has a conductivity value of about 1 mS / cm or less. In one preferred aspect of all aspects, the low-conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less. In one aspect, the low-conductivity aqueous solution has a conductivity value of about 0.03 μS / cm to about 0.5 mS / cm. In one aspect, the low-conductivity aqueous solution has a conductivity value of about 0.05 μS / cm to about 0.35 mS / cm. In one aspect of all aspects, the low-conductivity aqueous solution is deionized water. Deionized water is unsuitable for use in the washing stage for some applications. In some aspects, the low-conductivity aqueous solution is not deionized water.

[0051] Protein A affinity chromatography has been found to be usable for the purposes reported herein. In one preferred embodiment of all aspects, affinity chromatography is protein A affinity chromatography. In one embodiment, protein A affinity chromatography is selected from the group including MabSelectSure affinity chromatography, ProSep vA affinity chromatography, Poros Mab Capture A affinity chromatography, ProSep Ultra Plus affinity chromatography, MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl AF-rProtein A-650F; Toyopearl AF-rProtein A HC-650HF). In one embodiment, affinity chromatography is protein G affinity chromatography. In one embodiment, affinity chromatography is affinity chromatography using recombinant protein as a ligand, i.e., recombinant protein ligand affinity chromatography. In one embodiment, affinity chromatography is affinity chromatography using single-chain Fv as a ligand, i.e., single-chain Fv ligand affinity chromatography. In one embodiment, affinity chromatography includes a mutant protein A coupled to a chromatography matrix, or a fragment of protein A coupled to a chromatography matrix.

[0052] It was found that the content of (specific) host cell proteins could be reduced. In particular, the content of phospholipase B-like 2 (PLBL2) could be reduced. In one embodiment, the (specific) host cell protein is Chinese hamster ovary (CHO) host cell protein. In one preferred embodiment of all aspects, the (specific) host cell protein is phospholipase B-like 2 (PLBL2) or clusterin. In one embodiment, the (specific) host cell protein is phospholipase B-like 2 (PLBL2).

[0053] It has been found that low-conductivity aqueous solutions may contain certain buffering substances, such as small amounts of tris or potassium phosphate. In one embodiment, the low-conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (tris). In one embodiment, the low-conductivity aqueous solution contains about 0.1 mM to about 10 mM of tris. In one embodiment, the low-conductivity aqueous solution contains about 0.5 mM to about 6.5 mM of tris. In one embodiment, the low-conductivity aqueous solution contains about 2 mM of tris. In one embodiment, the low-conductivity aqueous solution contains potassium phosphate. In one embodiment, the low-conductivity aqueous solution contains about 0.05 mM to about 5 mM of potassium phosphate. In one embodiment, the low-conductivity aqueous solution contains about 0.05 mM to about 2 mM of potassium phosphate. In one embodiment, the low-conductivity aqueous solution contains about 0.5 mM of potassium phosphate.

[0054] The effect of reducing the host cell protein content was found to be particularly pronounced when the low-conductivity aqueous solution has a specific pH. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 7 or higher. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 7.5 or higher. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 7 to approximately 9.5. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 7.5 to approximately 8.5. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 8. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 9.

[0055] It was found that the effect of reducing the host cell protein content can also be achieved when the pH of the low-conductivity aqueous solution is approximately 8.5 or higher and the low-conductivity aqueous solution has a conductivity value of approximately 1.2 mS / cm or less. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 8.5 or higher and a conductivity value of approximately 1.2 mS / cm or less. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 8.5 or higher and a conductivity value of approximately 1 mS / cm or less. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 8.5 or higher and contains approximately 55 mM or less of Tris. In one embodiment, the low-conductivity aqueous solution has a pH of approximately 8.5 or higher and contains approximately 30 mM or less of Tris.

[0056] In one embodiment, the low-conductivity aqueous solution has a conductivity value of approximately 0.5 mS / cm or less in the pH range from pH 7 to less than pH 8.5, and a conductivity value of approximately 1.2 mS / cm or less at pH values ​​of 8.5 or higher.

[0057] The uses and methods reported herein have shown that the content of host cell proteins such as PLBL2 can be reduced to certain levels compared to the PLBL2 load before a purification step, such as an affinity chromatography step. In one embodiment, the PLBL2 content is reduced to at least 1 / 20th. In one embodiment, the PLBL2 content is reduced to at least 1 / 40th. In one embodiment, the PLBL2 content is reduced to at least 1 / 50th. In one embodiment, the PLBL2 content is reduced to at least 1 / 90th. In one embodiment, the PLBL2 content is reduced to at least 1 / 100th. In some cases, the level of reduction is even higher. In some embodiments, the PLBL2 content is reduced to at least 1 / 200th. In some embodiments, the PLBL2 content is reduced to at least 1 / 250th. In some embodiments, the PLBL2 content is reduced to at least 1 / 300th. In some embodiments, the PLBL2 content is reduced to at least 1 / 400th. In some embodiments, the PLBL2 content is reduced to at least 1 / 1000th. In one embodiment, the PLBL2 content is reduced by at least 50%. In one embodiment, the PLBL2 content is reduced by at least 66%. In one embodiment, the PLBL2 content is reduced by at least 80%. In one embodiment, the PLBL2 content is reduced by at least 90%. In one embodiment, the PLBL2 content is reduced by at least 95%. In some embodiments, the PLBL2 content is reduced to less than 10 ng per mg of antibody. In some embodiments, the PLBL2 content is reduced to less than 5 ng per mg of antibody. In some embodiments, the PLBL2 content is reduced to less than 2 ng per mg of antibody.

[0058] In the methods and uses reported herein, a further cleaning step with a medium-conductivity aqueous solution and / or a high-conductivity aqueous solution may be used. In one embodiment, the low-conductivity aqueous solution cleaning step is performed after or before the high-conductivity aqueous solution cleaning step. In one embodiment, the high-conductivity aqueous solution has a conductivity value of about 20 mS / cm or more. In one embodiment, the high-conductivity aqueous solution has a conductivity value of about 20 mS / cm to about 100 mS / cm. In one embodiment, an intermediate cleaning step with a medium-conductivity aqueous solution is performed between the low-conductivity aqueous solution cleaning step and the high-conductivity aqueous solution cleaning step. In one embodiment, the medium-conductivity aqueous solution has a conductivity value of more than 0.5 mS / cm to less than 20 mS / cm.

[0059] It has been found that the host cell protein reduction effect can be improved if the highly conductive or moderately conductive aqueous solution further contains amino acids. In one embodiment, the highly conductive or moderately conductive aqueous solution contains amino acids. In one embodiment, the highly conductive or moderately conductive aqueous solution contains histidine or arginine. In one embodiment, the highly conductive or moderately conductive aqueous solution contains histidine. In one embodiment, the highly conductive or moderately conductive aqueous solution contains histidine and tris.

[0060] The methods and uses reported herein may include one or more further chromatographic steps. In one embodiment, at least one additional chromatographic method / step is performed. In one embodiment, an additional ion exchange chromatography method / step is performed. In one embodiment, an additional anion exchange chromatography method / step is performed. In one embodiment, an additional anion exchange chromatography method / step and an additional cation exchange chromatography method / step are performed.

[0061] It has been found that the use of the hydrophobic interaction chromatography step can be omitted. In one embodiment, this use or method does not involve hydrophobic interaction chromatography / step.

[0062] One aspect reported herein is the use of a low-conductivity aqueous solution having a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher to reduce the content of PLBL2 or clusterin in the washing step of protein A chromatography used to purify IgG4 isotype antibodies or IgG1 isotype antibodies, such as human IgG4 antibodies or human IgG1 antibodies.

[0063] One aspect is the use of a low-conductivity aqueous solution having a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher to reduce the content of PLBL2 or clusterin in the washing step of protein A chromatography used to purify human IgG4 isotype antibodies or human IgG1 isotype antibodies. In some embodiments, the antibody is an IgG4 isotype antibody, e.g., an antibody against P-selectin, or a bispecific antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. In some embodiments, the antibody is an IgG1 isotype antibody, e.g., an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.

[0064] In certain circumstances, this disclosure is relevant. (a) A step of culturing cells containing nucleic acids encoding human IgG4 isotype antibody or human IgG1 isotype antibody, (b) A step of recovering human IgG4 isotype antibody or human IgG1 isotype antibody from the cells or culture medium, (c) A step of contacting a human IgG4 isotype antibody or a human IgG1 isotype antibody with a protein A affinity chromatography material. (d) Washing the protein A affinity chromatography material with a low-conductivity aqueous solution. (e) The step of recovering human IgG4 isotype antibody or human IgG1 isotype antibody from affinity chromatography material. Includes, This produces human IgG4 isotype antibodies or human IgG1 isotype antibodies. A method for producing human IgG4 isotype antibodies or human IgG1 isotype antibodies, The present invention provides a method for which a low-conductivity aqueous solution has a conductivity value of approximately 0.5 mS / cm or less and a pH of approximately 7 or higher.

[0065] In certain circumstances, this disclosure is relevant. (a) A step of culturing cells containing nucleic acids encoding human IgG4 isotype antibody or human IgG1 isotype antibody, (b) A step of recovering human IgG4 isotype antibody or human IgG1 isotype antibody from the cells or culture medium, (c) A step of contacting a human IgG4 isotype antibody or a human IgG1 isotype antibody with a protein A affinity chromatography material. (d) Washing the protein A affinity chromatography material with a low-conductivity aqueous solution. (e) The step of recovering human IgG4 isotype antibody or human IgG1 isotype antibody from affinity chromatography material. Includes, This produces human IgG4 isotype antibodies or human IgG1 isotype antibodies. A method for producing human IgG4 isotype antibodies or human IgG1 isotype antibodies, Low conductivity aqueous solutions have a conductivity value of approximately 0.5 mS / cm or less and a pH of approximately 7 or higher. Human IgG4 isotype antibodies are antibodies against P-selectin, or bispecific antibodies against factor IXa and factor X, or antibodies against IL-13, or antibodies against amyloid beta. Human IgG1 isotype antibodies are antibodies against influenza B, or antibodies against VEGF-A, or antibodies against CD22, or bispecific antibodies against HER3 and EGFR, or antibodies against amyloid beta, or antibodies against Her2, or bispecific antibodies against Ang2 and VEGF-A, or bispecific antibodies against carcinoembryonic antigen (CEA) and CD3. The above method is provided.

[0066] In certain circumstances, this disclosure is relevant. (a) The step of preparing a sample containing a human IgG4 isotype antibody or a human IgG1 isotype antibody, (b) A step of purifying a human IgG4 isotype antibody or a human IgG1 isotype antibody by a protein A affinity chromatography method / step, which includes the step of washing the protein A affinity chromatography material with a low-conductivity aqueous solution. A method for purifying a human IgG4 isotype antibody or a human IgG1 isotype antibody from a sample, comprising: The present invention provides a low-conductivity aqueous solution having a conductivity value of approximately 0.5 mS / cm or less and a pH of approximately 7 or higher.

[0067] In certain circumstances, this disclosure is relevant. (a) The step of preparing a sample containing a human IgG4 isotype antibody or a human IgG1 isotype antibody, (b) A step of purifying a human IgG4 isotype antibody or a human IgG1 isotype antibody by a protein A affinity chromatography method / step, which includes the step of washing the protein A affinity chromatography material with a low-conductivity aqueous solution. A method for purifying a human IgG4 isotype antibody or a human IgG1 isotype antibody from a sample, comprising: Low conductivity aqueous solutions have a conductivity value of approximately 0.5 mS / cm or less and a pH of approximately 7 or higher. Human IgG4 isotype antibodies are antibodies against P-selectin, or against factor IXa and factor X, or against IL-13, or against amyloid beta; human IgG1 isotype antibodies are antibodies against influenza B, or against VEGF-A, or against CD22, or against HER3 and EGFR, or against amyloid beta, or against Her2, or bispecific antibodies against Ang2 and VEGF-A, or bispecific antibodies against carcinoembryonic antigen (CEA) and CD3. The above method is provided.

[0068] In certain circumstances, this disclosure is relevant. (a) A step of culturing cells containing nucleic acids encoding human IgG4 isotype antibodies, (b) A step of recovering human IgG4 isotype antibodies from the cells or culture medium, (c) The step of contacting a human IgG4 isotype antibody with a protein A affinity chromatography material. (d) Washing the protein A affinity chromatography material with a low-conductivity aqueous solution. (e) Recovery of human IgG4 isotype antibodies from affinity chromatography material. Includes, This produces human IgG4 isotype antibodies. A method for producing human IgG4 isotype antibodies, Low conductivity aqueous solutions have a conductivity value of approximately 0.5 mS / cm or less and a pH of approximately 7 or higher. Human IgG4 isotype antibodies are antibodies against factor IXa and factor X. The above method is provided.

[0069] In certain circumstances, this disclosure is relevant. (a) The step of preparing a sample containing human IgG4 isotype antibodies, (b) A step of purifying human IgG4 isotype antibodies by a protein A affinity chromatography method / step that includes the step of washing the protein A affinity chromatography material with a low-conductivity aqueous solution, A method for purifying human IgG4 isotype antibodies from a sample, including, Low conductivity aqueous solutions have a conductivity value of approximately 0.5 mS / cm or less and a pH of approximately 7 or higher. Human IgG4 isotype antibodies are antibodies against factor IXa and factor X. The above method is provided.

[0070] The terms “anti-P-selectin antibody” and “antibody that binds to P-selectin” or “antibody against P-selectin” refer to antibodies that have the ability to bind to P-selectin with sufficient affinity so that they can serve as diagnostic and / or therapeutic agents in targeting P-selectin. In one embodiment, the degree of binding of an anti-P-selectin antibody to unrelated non-P-selectin proteins is less than about 10% of the binding of that antibody to P-selectin, as measured, for example, by ELISA or surface plasmon resonance. In a particular embodiment, an anti-P-selectin antibody binds to P-selectin epitopes that are conserved among P-selectins from different species. The above also applies to terms such as “antibodies against factor IXa and factor X” or “antibodies against IL-13” or “antibodies against amyloid beta.”

[0071] In some embodiments, the antibody against P-selectin is incrumab (IgG4 isotype) as described in WO2005 / 100402 or SEQ ID NO:07~12. In some embodiments, the antibody is a bispecific antibody against factor IXa and factor X, e.g., the anti-FIXa / X antibody (IgG4 isotype) as described in WO2012 / 067176. In some embodiments, the antibody is an antibody against Her2, e.g., trastuzumab (IgG1 isotype) as described in WO1992 / 022653. In some embodiments, the antibody is a bispecific antibody against angiopoietin 2 (Ang2) and vascular endothelial growth factor A (VEGF-A), e.g., vanucizumab (IgG1 isotype) as described in WO2011 / 117329 or SEQ ID NO:01~04. In some embodiments, the antibody is an antibody against amyloid beta, e.g., gantenerumab (IgG1 isotype) or crenezumab (IgG4 isotype) as described in WO2003 / 070760 or SEQ ID NO:05~06. In some embodiments, the antibody is an antibody against CD22, an antibody against IL13 (e.g., lebrikizumab), a bispecific antibody against Her3 and EGFR (e.g., duligotuzumab), an antibody against VEGF-A (e.g., bevacizumab), and an antibody against influenza B. The terms VEGF and VEGF-A may be used interchangeably herein.

[0072] As used herein, the terms “binding” or “specifically binding” refer to the binding of an antibody to an antigen epitope in an in vitro assay, preferably in a surface plasmon resonance assay (SPR, BIAcore, GE-Healthcare, Uppsala, Sweden). The affinity of binding is expressed in terms of ka (rate constant for antibody association of antibody / antigen complex), k d (Dissociation constant), and K D (k d / k a ) is defined by ). To bind or specifically bind means 10 -7Binding affinity (K) less than mol / L D ) means.

[0073] The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0074] An "antibody fragment" refers to a molecule other than the intact antibody itself, which contains the portion that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Fab fragments are antibody fragments obtained by papain digestion of (full-length / complete) antibodies.

[0075] A "bispecific antibody" is an antibody that has two different antigen-binding specificities. As used herein, the term "bispecific antibody" refers to an antibody that has at least two binding sites, each of which binds to a different epitope.

[0076] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining portion of the heavy chain and / or light chain originates from a different source or species.

[0077] The "class" of an antibody refers to the type of constant domain or constant region held in its heavy chain. Antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0078] The term "human IgG isotype antibody" refers to an antibody that contains a constant region derived from a human wild-type IgG isotype. That is, it may contain a constant region derived from a human IgG isotype that has, for example, a mutation, such as the P329G mutation (numbering follows Kabat).

[0079] The term "human IgG4 isotype antibody" refers to an antibody that contains a constant region derived from the human wild-type IgG4 isotype. That is, it may contain a constant region derived from the human IgG4 isotype that has mutations, e.g., the P329G mutation and / or the S228P, L235E mutation (numbering follows Kabat).

[0080] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term encompasses both native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 of the heavy chain to the carboxyl terminus. However, the C-terminal lysine (Lys447) or C-terminal glycyl-lysine dipeptide (Gly446Lys447) of the Fc region may or may not be present. Unless otherwise specified, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat, EA et al, Sequences of Proteins of Immunological Interest, 5th edition, National Institutes of Health, Public Health Service, Bethesda, Maryland (1991), NIH Publication 91-3242.

[0081] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains, namely FR1, FR2, FR3, and FR4. Therefore, the HVR sequence and FR sequence generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0082] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and the offspring of such cells are also included in these terms. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring may not have nucleic acid content that is exactly the same as that of the parent cell and may contain mutations. Mutant offspring that have the same function or biological activity as those screened or selected in the original transformed cell are included here. The term “cell” includes cells used for nucleic acid expression. In one embodiment, the host cell is a CHO cell (e.g., CHO K1, CHO DG44), or a BHK cell, or an NS0 cell, or an SP2 / 0 cell, or a HEK293 cell, or a HEK293EBNA cell, or a PER.C6® cell, or a COS cell. In another embodiment, the cell is a CHO cell, or a BHK cell, or a PER.C6® cell. As used herein, the term "cell" includes the cell in question and its offspring.

[0083] The term "wash" refers to applying a solution to affinity chromatography material to remove nonspecifically bound polypeptides and non-polypeptide compounds from the chromatography material, particularly host cell proteins and host cell DNA. The term "wash" does not include the elution of bound materials from the affinity chromatography material.

[0084] Various methods have been established and are widely used for protein recovery and purification, including affinity chromatography using microbial proteins (e.g., protein A or protein G affinity chromatography), affinity chromatography using recombinant proteins as ligands (e.g., single-chain Fv as ligand, e.g., Kappa select), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin), and mixed-mode exchange), thiophilic adsorption (e.g., with beta-mercaptoethanol ligand and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., with phenyl-sepharose, aza-alenophilic resin, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., with Ni(II)-affinity materials and Cu(II)-affinity materials), size exclusion chromatography, and electrophoresis (gel electrophoresis, capillary electrophoresis, etc.). These methods can be independently combined in various embodiments, as reported herein.

[0085] The term "protein A" refers to protein A polypeptides that are obtained from natural sources or produced synthetically.

[0086] The term "protein A chromatography material" refers to an inert solid phase to which protein A is covalently bonded.

[0087] In one embodiment, the protein A chromatography material is selected from MabSelectSure, ProSep vA, Mab Capture A, ProSep Ultra Plus, Mab Select, Mab Select Xtra, Poros A, or ProSep A.

[0088] The term "high-conductivity aqueous solution" refers to an aqueous solution with a high conductivity value. The conductivity value can be approximately 20 mS / cm or higher.

[0089] The term "medium conductivity aqueous solution" refers to an aqueous solution with an intermediate conductivity value. The conductivity value can range from over 0.5 mS / cm to less than 20 mS / cm.

[0090] The term "low conductivity aqueous solution" refers to an aqueous solution with a low conductivity value. The conductivity value can be approximately 0.5 mS / cm or less. If the pH is approximately 8.5 or higher, the conductivity value can be approximately 1.2 mS / cm or less. The conductivity value can be measured by standard methods known to those skilled in the art.

[0091] The following examples and sequences are provided to aid in understanding the present invention, but the true scope of the invention is described in the claims of this application. It is understood that the described procedures can be modified without departing from the spirit of the invention.

[0092] Specific aspects of the present invention 1. Use of low-conductivity aqueous solutions to reduce the host cell protein content during the washing stage of affinity chromatography. 2. Use of Embodiment 1, wherein affinity chromatography is used to purify human IgG isotype antibodies. 3. Use of aspect 2, wherein affinity chromatography is used to purify human IgG4 isotype antibody or human IgG1 isotype antibody. 4. Use of Embodiment 3, in which affinity chromatography is used to purify a human IgG4 isotype antibody or a human IgG1 isotype antibody that does not have a glycosylated glycosylation site in its Fab fragment / has just one glycosylation site (at position Asn297 in Kabat numbering). 5. Use of Embodiment 4, wherein the low-conductivity aqueous solution has a conductivity value of approximately 0.5 mS / cm or less. 6. Use of Embodiment 5, wherein the low-conductivity aqueous solution has a conductivity value of approximately 0.03 μS / cm to approximately 0.5 mS / cm. 7. Use of embodiment 5, wherein the low-conductivity aqueous solution has a conductivity value of approximately 0.05 μS / cm to approximately 0.35 mS / cm. 8. Use of any one embodiment of embodiments 5 to 7, wherein the low-conductivity aqueous solution is not deionized water. 9. Use of any one of the above embodiments, wherein the affinity chromatography is protein A affinity chromatography, protein G affinity chromatography, or single-chain Fv ligand (KappaSelect) affinity chromatography. 10. Use of embodiment 9, wherein the affinity chromatography is protein A affinity chromatography. 11. Use of Embodiment 10, wherein protein A affinity chromatography is selected from the group including MabSelect Sure affinity chromatography, ProSep vA affinity chromatography, Poros Mab Capture A affinity chromatography, ProSep Ultra Plus affinity chromatography, MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl AF-rProtein A-650F; Toyopearl AF-rProtein A HC-650HF). 12. Use of any one embodiment of the above, wherein the host cell protein is Chinese hamster ovary (CHO) host cell protein. 13. Use of embodiment 12, wherein the host cell protein is a phospholipase. 14. Use of embodiment 13, wherein the host cell protein is phospholipase A, phospholipase B, phospholipase C, or phospholipase D. 15. Use according to embodiment 12, 13, or 14, wherein the host cell protein is phospholipase B-like 2 (PLBL2). 16. Use of embodiment 12, wherein the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin. 17. Use of any one embodiment of the above, wherein the low-conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (tris). 18. Use of Embodiment 17, wherein the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 10 mM of tris. 19. Use of embodiment 18, wherein the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 8 mM of tris. 20. Use of Embodiment 19, wherein the low-conductivity aqueous solution contains approximately 0.5 mM to approximately 6.5 mM of tris. 21. Use of embodiment 20, wherein the low-conductivity aqueous solution contains approximately 2 mM tris. 22. Use of any one embodiment of embodiments 17 to 21, wherein the low-conductivity aqueous solution contains potassium phosphate. 23. Use of Embodiment 22, wherein the low-conductivity aqueous solution contains approximately 0.05 mM to approximately 5 mM potassium phosphate. 24. Use of Embodiment 23, wherein the low-conductivity aqueous solution contains approximately 0.05 mM to approximately 2 mM potassium phosphate. 25. Use of embodiment 24, wherein the low-conductivity aqueous solution contains approximately 0.5 mM potassium phosphate. 26. Use of any one of the above embodiments, wherein the low-conductivity aqueous solution has a pH of approximately 7 or higher. 27. Use of embodiment 26, wherein the low-conductivity aqueous solution has a pH of approximately 7.5 or higher. 28. Use of the low conductivity aqueous solution having a pH of approximately 7 to approximately 9.5, as described in Embodiment 27. 29. Use of embodiment 28, wherein the low-conductivity aqueous solution has a pH of approximately 7.5 to approximately 8.5. 30. Use of the low conductivity aqueous solution having a pH of approximately 8, as described in Embodiment 29. 31. Use of any one of the above embodiments, wherein the low-conductivity aqueous solution cleaning step is performed after or before the high-conductivity aqueous solution cleaning step. 32. Use of embodiment 31, wherein the low-conductivity aqueous solution cleaning step is performed after the high-conductivity aqueous solution cleaning step. 33. Use of embodiment 31, wherein the high-conductivity aqueous solution has a conductivity value of approximately 20 mS / cm or higher. 34. Use of embodiment 33, wherein the high-conductivity aqueous solution has a conductivity value of approximately 20 mS / cm to approximately 100 mS / cm. 35. The use of embodiment 31, wherein an intermediate cleaning step with a medium-conductivity aqueous solution is performed between the low-conductivity aqueous solution cleaning step and the high-conductivity aqueous solution cleaning step. 36. Use of embodiment 35, wherein the aqueous solution with medium conductivity has conductivity values ​​ranging from more than 0.5 mS / cm to less than 20 mS / cm. 37. Use of any one embodiment of embodiments 33 to 36, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains an amino acid. 38. Use of embodiment 37, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains histidine. 39. Use of embodiment 37, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains histidine and tris. 40. Use of any one of the above embodiments, wherein at least one additional chromatography method / step is performed. 41. Use of embodiment 40, wherein an additional ion exchange chromatography method / step is performed. 42. Use of embodiment 41, in which an additional anion exchange chromatography method / step is performed. 43. Use of embodiment 40, wherein an additional cation exchange chromatography method / step is performed. 44. Use of embodiment 40, wherein additional anion exchange chromatography / steps and additional cation exchange chromatography / steps are performed. 45. Use of embodiment 40 without the step of hydrophobic interaction chromatography. 46. ​​Use of any one embodiment of the above, wherein the human IgG4 isotype antibody is an antibody against P-selectin, or an antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. 47. Use of any one embodiment of Embodiments 1 to 45, wherein the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or an antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or an antibody against Ang2 and VEGF-A, or an antibody against carcinoembryonic antigen (CEA) and CD3. 48. (a) A step of culturing cells containing nucleic acids encoding human IgG isotype antibodies, (b) A step of recovering human IgG isotype antibodies from the cells or culture medium, (c) The step of contacting a human IgG isotype antibody with an affinity chromatography material. (d) A step in which the affinity chromatography material is washed with a low-conductivity aqueous solution. (e) Recovery of human IgG isotype antibodies from affinity chromatography material. Includes, This produces human IgG isotype antibodies. A method for producing human IgG isotype antibodies. 49. (a) A step of culturing cells containing nucleic acids encoding human IgG4 isotype antibodies, (b) A step of recovering human IgG4 isotype antibodies from the cells or culture medium, (c) The step of contacting a human IgG4 isotype antibody with an affinity chromatography material. (d) A step in which the affinity chromatography material is washed with a low-conductivity aqueous solution. (e) Recovery of human IgG4 isotype antibodies from affinity chromatography material. Includes, This produces human IgG4 isotype antibodies. A method for producing human IgG4 isotype antibodies. 50. The method of embodiment 48, wherein the human IgG isotype antibody is a human IgG4 isotype antibody or a human IgG1 isotype antibody. 51. The method of aspect 48, wherein the human IgG isotype antibody is a human IgG4 isotype antibody or a human IgG1 isotype antibody that does not have a glycosylated glycosylation site in its Fab fragment / has just one glycosylation site (at position Asn297 in Kabat numbering). 52. The method of embodiment 48, wherein the low-conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less. 53. The method according to embodiment 52, wherein the low-conductivity aqueous solution has a conductivity value of approximately 0.03 μS / cm to approximately 0.5 mS / cm. 54. The method of embodiment 53, wherein the low-conductivity aqueous solution has a conductivity value of approximately 0.05 μS / cm to approximately 0.35 mS / cm. 55. The method of embodiment 54, wherein the low-conductivity aqueous solution is not deionized water. 56. A method according to any one embodiment of embodiments 48 to 55, wherein the affinity chromatography is protein A affinity chromatography, protein G affinity chromatography, or single-chain Fv ligand (KappaSelect) affinity chromatography. 57. The method according to embodiment 56, wherein the affinity chromatography is protein A affinity chromatography. 58. The method of embodiment 56, wherein protein A affinity chromatography is selected from the group including MabSelectSure affinity chromatography, ProSep vA affinity chromatography, Poros Mab Capture A affinity chromatography, ProSep Ultra Plus affinity chromatography, MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl AF-rProtein A-650F; Toyopearl AF-rProtein A HC-650HF). 59. The method according to embodiment 48 or 49, wherein the host cell protein content is reduced. 60. A method according to any one embodiment of embodiment 59, wherein the host cell protein is Chinese hamster ovary (CHO) host cell protein. 61. The method according to embodiment 60, wherein the host cell protein is a phospholipase. 62. The method according to embodiment 61, wherein the host cell protein is phospholipase A, phospholipase B, phospholipase C, or phospholipase D. 63. A method according to any one embodiment of embodiment 62, wherein the host cell protein is phospholipase B-like 2 (PLBL2). 64. The method according to embodiment 60, wherein the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin. 65. A method according to any one embodiment of embodiments 48 to 64, wherein the low-conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (tris). 66. The method of embodiment 65, wherein the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 10 mM of tris. 67. The method of embodiment 66, wherein the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 8 mM of tris. 68. The method of embodiment 67, wherein the low-conductivity aqueous solution contains approximately 0.5 mM to approximately 6.5 mM of tris. 69. The method of embodiment 68, wherein the low-conductivity aqueous solution contains approximately 2 mM tris. 70. A method according to any one embodiment of embodiments 65 to 69, wherein the low-conductivity aqueous solution contains potassium phosphate. 71. The method according to embodiment 70, wherein the low-conductivity aqueous solution contains approximately 0.2 mM to approximately 5 mM potassium phosphate. 72. The method according to embodiment 71, wherein the low-conductivity aqueous solution contains approximately 0.05 mM to approximately 2 mM potassium phosphate. 73. The method according to embodiment 72, wherein the low-conductivity aqueous solution contains about 0.5 mM potassium phosphate. 74. A method according to any one embodiment of embodiments 48 to 73, wherein the low-conductivity aqueous solution has a pH of approximately 7 or higher. 75. The method according to embodiment 74, wherein the low-conductivity aqueous solution has a pH of approximately 7.5 or higher. 76. The method according to embodiment 75, wherein the low-conductivity aqueous solution has a pH of about 7 to about 9.5. 77. The method according to embodiment 76, wherein the low-conductivity aqueous solution has a pH of approximately 7.5 to approximately 8.5. 78. The method of embodiment 77, wherein the low-conductivity aqueous solution has a pH of about 8. 79. The method of embodiment 48 or 49, further comprising the step of washing the affinity chromatography material with a high-conductivity aqueous solution before or after washing the affinity chromatography material with a low-conductivity aqueous solution. 80. The method of embodiment 79, further comprising the step of washing the affinity chromatography material with a high-conductivity aqueous solution before washing the affinity chromatography material with a low-conductivity aqueous solution. 81. The method of embodiment 79, further comprising the step of washing the affinity chromatography material with a high-conductivity aqueous solution and / or a medium-conductivity aqueous solution before or after washing the affinity chromatography material with a low-conductivity aqueous solution. 82. The method of embodiment 79, further comprising the step of washing the affinity chromatography material with a high-conductivity aqueous solution and / or a medium-conductivity aqueous solution before washing the affinity chromatography material with a low-conductivity aqueous solution. 83. A method according to any one embodiment of embodiments 79 to 82, wherein the highly conductive aqueous solution has a conductivity value of approximately 20 mS / cm or more. 84. The method of embodiment 83, wherein the highly conductive aqueous solution has a conductivity value of approximately 20 mS / cm to approximately 100 mS / cm. 85. A method according to any one embodiment of embodiments 81 to 82, wherein the aqueous solution with medium conductivity has a conductivity value ranging from more than 0.5 mS / cm to less than 20 mS / cm. 86. A method according to any one embodiment of embodiments 79 to 85, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains an amino acid. 87. The method of embodiment 86, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains histidine. 88. The method of embodiment 86 or 87, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains histidine and tris. 89. The method of aspect 48 or 49, wherein at least one additional chromatography method / step is performed after step (e). 90. The method of embodiment 89, wherein an additional ion exchange chromatography method / step is performed after step (e). 91. The method of embodiment 90, wherein an additional anion exchange chromatography method / step is performed after step (e). 92. The method of embodiment 90, wherein an additional cation exchange chromatography method / step is performed after step (e). 93. The method of embodiment 90, wherein an additional anion exchange chromatography method / step and an additional cation exchange chromatography method / step are performed after step (e). 94. Hydrophobic interaction chromatography method / Method according to embodiment 48 or 49, without a step. 95. A method according to any one embodiment of embodiments 49 to 94, wherein the human IgG4 isotype antibody is an antibody against P-selectin, or an antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. 96. A method according to any one embodiment of 48 or 50-94, wherein the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or an antibody against carcinoembryonic antigen (CEA) and CD3. 97. (a) Step of preparing a sample containing human IgG isotype antibodies, (b) A step of purifying human IgG isotype antibodies by affinity chromatography / steps, which includes washing affinity chromatography material with a low-conductivity aqueous solution. A method for purifying human IgG isotype antibodies, including [specific antibodies], from a sample. 98. The method of embodiment 97, wherein the human IgG isotype antibody is a human IgG4 isotype antibody or a human IgG1 isotype antibody. 99. The method of aspect 98, wherein the human IgG isotype antibody is a human IgG4 isotype antibody or a human IgG1 isotype antibody that does not have a glycosylated glycosylation site in its Fab fragment / has just one glycosylation site (at position Asn297 in Kabat numbering). 100. A method according to any one embodiment of embodiments 97 to 99, wherein the low-conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less. 101. The method of embodiment 100, wherein the low-conductivity aqueous solution has a conductivity value of approximately 0.03 μS / cm to approximately 0.5 mS / cm. 102. The method according to embodiment 101, wherein the low-conductivity aqueous solution has a conductivity value of approximately 0.05 μS / cm to approximately 0.35 mS / cm. 103. The method of embodiment 102, wherein the low-conductivity aqueous solution is not deionized water. 104. A method according to any one embodiment of embodiments 97 to 104, wherein the affinity chromatography is protein A affinity chromatography, protein G affinity chromatography, or single-chain Fv ligand (KappaSelect) affinity chromatography. 105. The method of embodiment 104, wherein the affinity chromatography is protein A affinity chromatography. 106. The method of embodiment 105, wherein protein A affinity chromatography is selected from the group including MabSelect Sure affinity chromatography, ProSep vA affinity chromatography, Poros Mab Capture A affinity chromatography, ProSep Ultra Plus affinity chromatography, MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl AF-rProtein A-650F; Toyopearl AF-rProtein A HC-650HF). 107. A method according to any one embodiment of embodiments 97 to 106, wherein the host cell protein content is reduced. 108. The method according to embodiment 107, wherein the host cell protein is Chinese hamster ovary (CHO) host cell protein. 109. The method according to embodiment 108, wherein the host cell protein is a phospholipase. 110. The method according to embodiment 109, wherein the host cell protein is phospholipase A, phospholipase B, phospholipase C, or phospholipase D. 111. A method according to any one embodiment of embodiment 110, wherein the host cell protein is phospholipase B-like 2 (PLBL2). 112. The method according to embodiment 107, wherein the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin. 113. A method according to any one embodiment of embodiments 97 to 112, wherein the low-conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (tris). 114. The method according to embodiment 113, wherein the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 10 mM of tris. 115. The method according to embodiment 114, wherein the low-conductivity aqueous solution contains approximately 0.1 mM to approximately 8 mM of tris. 116. The method according to embodiment 115, wherein the low-conductivity aqueous solution contains approximately 0.5 mM to approximately 6.5 mM of tris. 117. The method of embodiment 116, wherein the low-conductivity aqueous solution contains about 2 mM tris. 118. A method according to any one embodiment of embodiments 113 to 117, wherein the low-conductivity aqueous solution contains potassium phosphate. 119. The method of embodiment 118, wherein the low-conductivity aqueous solution contains approximately 0.2 mM to approximately 5 mM potassium phosphate. 120. The method according to embodiment 119, wherein the low-conductivity aqueous solution contains approximately 0.2 mM to approximately 2 mM potassium phosphate. 121. The method of embodiment 120, wherein the low-conductivity aqueous solution contains approximately 0.5 mM potassium phosphate. 122. A method according to any one embodiment of embodiments 97 to 121, wherein the low-conductivity aqueous solution has a pH of about 7 or higher. 123. The method according to embodiment 122, wherein the low-conductivity aqueous solution has a pH of approximately 7.5 or higher. 124. The method according to embodiment 123, wherein the low-conductivity aqueous solution has a pH of about 7 to about 9.5. 125. The method according to embodiment 124, wherein the low-conductivity aqueous solution has a pH of approximately 7.5 to approximately 8.5. 126. The method according to embodiment 125, wherein the low-conductivity aqueous solution has a pH of approximately 8. 127. A method according to any one embodiment of aspects 97 to 126, further comprising the step of washing the affinity chromatography material with a high-conductivity aqueous solution before or after washing the affinity chromatography material with a low-conductivity aqueous solution. 128. The method of embodiment 127, further comprising the step of washing the affinity chromatography material with a high-conductivity aqueous solution before washing the affinity chromatography material with a low-conductivity aqueous solution. 129. The method of embodiment 127, further comprising the step of washing the affinity chromatography material with a high-conductivity aqueous solution and / or a medium-conductivity aqueous solution before or after washing the affinity chromatography material with a low-conductivity aqueous solution. 130. The method of embodiment 127, further comprising the step of washing the affinity chromatography material with a high-conductivity aqueous solution and / or a medium-conductivity aqueous solution before washing the affinity chromatography material with a low-conductivity aqueous solution. 131. A method according to any one embodiment of embodiments 127 to 130, wherein the highly conductive aqueous solution has a conductivity value of approximately 20 mS / cm or more. 132. The method of embodiment 131, wherein the highly conductive aqueous solution has a conductivity value of approximately 20 mS / cm to approximately 100 mS / cm. 133. The method according to embodiment 129 or 130, wherein the aqueous solution with medium conductivity has a conductivity value ranging from more than 0.5 mS / cm to less than 20 mS / cm. 134. A method according to any one embodiment of embodiments 127 to 133, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains an amino acid. 135. The method of embodiment 134, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains histidine. 136. The method of embodiment 134, wherein the highly conductive aqueous solution or the moderately conductive aqueous solution contains histidine and tris. 137. The method of embodiment 97 or 98, wherein at least one additional chromatography method / step is performed after step (b). 138. The method of embodiment 137, wherein an additional ion exchange chromatography method / step is performed after step (b). 139. The method of embodiment 138, wherein an additional anion exchange chromatography method / step is performed after step (b). 140. The method of embodiment 138, wherein an additional cation exchange chromatography / step is performed after step (b). 141. The method of embodiment 138, wherein an additional anion exchange chromatography / step and an additional cation exchange chromatography / step are performed after step (b). 142. Hydrophobic interaction chromatography method / A method according to any one embodiment of embodiments 97 to 141, without a step. 143. The method according to embodiment 97 or 98, wherein the human IgG4 isotype antibody is an antibody against P-selectin, or an antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta. 144. The method of embodiment 97 or 98, wherein the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a (bispecific) antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3. 145. Use of a low-conductivity aqueous solution with a conductivity value of approximately 0.5 mS / cm or less to reduce the host cell protein content in the washing step of protein A chromatography used to purify human IgG4 isotype antibodies or human IgG1 isotype antibodies. 146. (a) A step of culturing cells containing nucleic acids encoding human IgG4 isotype antibody or human IgG1 isotype antibody, (b) A step of recovering human IgG4 isotype antibody or human IgG1 isotype antibody from the cells or culture medium, (c) A step of contacting a human IgG4 isotype antibody or a human IgG1 isotype antibody with a protein A chromatography material. (d) A step of washing the protein A chromatography material with a low-conductivity aqueous solution having a conductivity value of approximately 0.5 mS / cm or less. (e) Recovering human IgG4 isotype antibody or human IgG1 isotype antibody from protein A chromatography material. Includes, This produces human IgG4 isotype antibodies or human IgG1 isotype antibodies. A method for producing human IgG4 isotype antibodies or human IgG1 isotype antibodies. 147. (a) The step of preparing a sample containing a human IgG4 isotype antibody or a human IgG1 isotype antibody, (b) A step of purifying a human IgG4 isotype antibody or a human IgG1 isotype antibody by a protein A chromatography method / step that includes washing the protein A chromatography material with a low-conductivity aqueous solution having a conductivity value of approximately 0.5 mS / cm or less. A method for purifying human IgG4 isotype antibody or human IgG1 isotype antibody from a sample, including [specific antibody details]. 148. Use of any one embodiment of embodiments 5 to 7, wherein the low-conductivity aqueous solution is deionized water. 149. The method according to embodiment 54, wherein the low-conductivity aqueous solution is deionized water.

[0093] Explanation of the sequence list SEQ ID NO:01 <vegf>Variable heavy chain domain VH SEQ ID NO:02 <vegf>Variable light chain domain (VL) SEQ ID NO:03 <ang-2>Variable heavy chain domain VH SEQ ID NO:04 <ang-2>Variable light chain domain (VL) SEQ ID NO:05 Variable heavy chain domain VH of anti-amyloid beta antibody (IgG1 isotype) SEQ ID NO:06 Variable light chain domain (VL) of anti-amyloid beta antibody (IgG1 isotype) SEQ ID NO:07 Variable heavy chain domain VH1 of anti-P-selectin antibody SEQ ID NO:08 Variable heavy chain domain VH2 of anti-P-selectin antibody SEQ ID NO:09 Variable heavy chain domain VH3 of anti-P-selectin antibody SEQ ID NO:10 Variable light chain domain VL1 of anti-P-selectin antibody SEQ ID NO:11 Variable light chain domain VL2 of anti-P-selectin antibody SEQ ID NO:12 Variable light chain domain VL3 of anti-P-selectin antibody [Examples]

[0094] Example 1 material and method antibody The present invention is illustrated by several exemplary antibodies, including: an antibody against P-selectin as described in WO 2005 / 100402 or SEQ ID NO:07~SEQ ID NO:12 (anti-P-selectin antibody; incrumab; IgG4 isotype); a bispecific antibody against factor IXa and factor X as described in WO 2012 / 067176 (anti-FIXa / X antibody; IgG4 isotype); an antibody against Her2; a bispecific antibody against Ang2 and VEGF-A as described in WO 2011 / 117329 or SEQ ID NO:01~SEQ ID NO:04 (anti-Ang2 / VEGF-A antibody; vanucizumab; IgG1 isotype); and an antibody against amyloid beta as described in WO 2003 / 070760 or SEQ ID NO:05~SEQ ID NO:06 (anti-amyloid beta antibody; gantenerumab; IgG1 isotype). As shown in the following examples, multiple IgG1 and IgG4 antibodies are also included herein.

[0095] Methods for detecting total host cell proteins (HCP), phospholipase B-like protein 2 (PLBL2), and clusterin. (a) CHO HCP assay The residual CHO HCP content in process samples is measured by electrochemiluminescence immunoassay (ECLIA) using a cobas e 411 immunoassay analyzer (Roche Diagnostics).

[0096] This assay is based on the sandwich principle using polyclonal anti-CHO HCP antibodies from sheep.

[0097] First incubation: Chinese hamster ovary host cell protein (CHO HCP) from 15 μL of sample (neat and / or diluted) forms a sandwich complex with biotin-conjugated polyclonal CHO HCP-specific antibody, which then binds to streptavidin-coated microparticles through the interaction of biotin and streptavidin.

[0098] Second incubation: After adding a polyclonal CHO HCP-specific antibody labeled with a ruthenium complex (tris(2,2'-bipyridyl)ruthenium(II) complex), a ternary complex is formed on the microparticles.

[0099] The reaction mixture is drawn into a measurement cell. In this cell, fine particles are magnetically trapped on the surface of the electrodes. Next, any unbound material is removed in a washing step. Then, a voltage is applied to the electrodes to induce chemiluminescence, which is measured by a photomultiplier tube.

[0100] Finally, the concentration of CHO HCP in the test sample is calculated from a standard curve of CHO HCP with known concentrations.

[0101] (b) CHO PLBL2 assay The residual Chinese hamster ovary (CHO) phospholipase B-like 2 protein (PLBL2) content in process samples is measured by electrochemiluminescence immunoassay (ECLIA) using a cobas e 411 immunoassay analyzer (Roche Diagnostics).

[0102] This assay is based on the sandwich principle using monoclonal anti-CHO PLBL2 antibodies from mice.

[0103] In the first incubation stage, CHO PLBL2 from 30 μL of sample (neat and / or diluted), biotin-labeled monoclonal CHO PLBL2-specific antibody, and monoclonal CHO PLBL2-specific antibody labeled with a ruthenium complex (tris(2,2'-bipyridyl)ruthenium(II) complex) form a sandwich complex.

[0104] In the second stage, after the addition of streptavidin-coated microparticles, the ternary complex becomes bound to the solid phase through the interaction of biotin and streptavidin.

[0105] The reaction mixture is drawn into a measurement cell. In this cell, fine particles are magnetically trapped on the surface of the electrodes. Next, any unbound material is removed in a washing step. Then, chemiluminescence is induced by applying a voltage to the electrodes, which is measured by a photomultiplier tube.

[0106] Finally, the CHO PLBL2 concentration in the test sample is calculated from a standard curve of CHO PLBL2 with known concentrations.

[0107] (c) Clusterin assay The residual clusterin content in the process samples was measured using a commercially available assay from Merck Millipore (GyroMark HT Kit GYRCLU-37K). This commercially available assay was used according to the manufacturer's instructions.

[0108] Simply put, this assay is performed sequentially. (1) Conjugation of rat clustering biotinylation capture antibody to a streptavidin-coated affinity column of Bioaffy 1000nL CD. (2) Capture of rat clusterin molecules from the sample by anti-clusterin antibody, (3) Binding of the second dye-labeled anti-clusterin detection antibody to the captured molecule, (4) Quantitative determination of rat clusterin using Gyrolab Evaluator This is a sandwich ELISA based on [a specific method / technology].

[0109] Example 2 Purification of anti-P-selectin antibodies (IgG4 isotype) in protein A chromatography Antibody: Anti-P-selectin General chromatography conditions Column resin: Protein A material "Mab Select SuRe" (GE-Healthcare), diameter: 1 cm, height: 20.1 cm, CV: 15.79 ml Equipment: Akta Avant 150 Flow velocity: 300 cm / hour at all stages

[0110] After equilibration of the protein A affinity column (step 1), a solution containing anti-P-selectin antibody was applied to the column. Initial loading of PLBL2 measured in the solution containing anti-P-selectin antibody: 335 ng PLBL2 / mg antibody. Initial loading of clusterin measured in the solution containing anti-P-selectin antibody: 2874.8 ng clusterin / mg antibody. Initial loading of CHOP measured in the solution containing anti-P-selectin antibody: 100971 ng CHOP / mg antibody.

[0111] The chromatography step was carried out according to the following general scheme. Stage 1: Equilibrium: Stage 2: Loading of antibody-containing solution Stage 3: Cleaning I Stage 4: Cleaning II Stage 5: Cleaning III Stage 6: Cleaning IV (Additional Cleaning) Stage 7: Elution

[0112] After elution from the Protein A affinity column, the proteins were measured by size exclusion chromatography (SEC) and spectrophotometric (OD) analysis.

[0113] SEC: Resin: TSK 3000(Tosoh) Column: 300 x 7.8 mm Flow rate: 0.5ml / min Buffer solution: 200 mM potassium phosphate containing 250 mM potassium chloride, adjusted to pH 7.0. Wavelength: 280nm OD: Ratio coefficient: 1.54 Wavelength: 280nm - 320nm

[0114] Specific buffer conditions for protein A chromatography (anti-P-selectin antibody) (a) Control (washing with equilibration buffer only) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 5: Cleaning III: --- Stage 6: Cleansing IV: --- Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0115] (b) Low conductivity washing (using Tris buffer only) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0116] (c) Low conductivity cleaning (using potassium phosphate (KP) only) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 0.5 mM potassium phosphate, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0117] (d) High conductivity washing (using Tris buffer solution only) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 700 mM Tris, pH 7.2 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Cleansing IV: --- Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0118] (e) Low conductivity washing (using Tris buffer only; pH ​​6.0) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 6.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0119] (f) High conductivity washing (only with histidine (His) / Tris buffer solution) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Wash II: 200mM His / 1000mM Tris, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Cleansing IV: --- Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0120] (g) Low conductivity tris + High conductivity histidine (His) / tris Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Wash II: 200mM His / 1000mM Tris, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0121] (h) Low conductivity potassium phosphate (KP) + high conductivity histidine (His) / tris Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Wash II: 200mM His / 1000mM Tris, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 0.5 mM potassium phosphate, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0122] (i) Low conductivity Tris + High conductivity Tris Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 700 mM Tris, pH 7.2 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0123] (j) Low conductivity Tris; pH 6.0+ High conductivity Tris Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 700 mM Tris, pH 7.2 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 6.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0124] result: TIFF0007887230000001.tif125147

[0125] Example 3 Purification of anti-amyloid beta antibodies (IgG1 isotype) by protein A chromatography The general conditions followed those described in Example 2. Antibody: Anti-amyloid beta. Initial loading of PLBL2 measured in a solution containing anti-amyloid-beta antibody: 2019.7 ng PLBL2 / mg antibody. Initial loading of CHOP measured in a solution containing anti-amyloid-beta antibody: 578908 ng CHOP / mg antibody.

[0126] Specific buffer conditions for Protein A chromatography (a) Control (washing with equilibration buffer only) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 5: Cleaning III: --- Stage 6: Cleansing IV: --- Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0127] (b) Low conductivity washing (using Tris buffer only) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0128] (c) High conductivity washing (using Tris buffer solution only) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 700 mM Tris, pH 7.2 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Cleansing IV: --- Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0129] (d) Low conductivity tris + high conductivity histidine (His) / tris Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Wash II: 200mM His / 1000mM Tris, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 8.0 Stage 7: Elution in 50 mM acetic acid, pH 4.0

[0130] TIFF0007887230000002.tif61147

[0131] Example 4 Purification of anti-Her2 antibody (IgG1 isotype) by protein A chromatography The general conditions followed those described in Example 2. Antibody: Anti-Her2 Initial loading amount of PLBL2 measured in a solution containing anti-Her2 antibody: 1662.5 ng PLBL2 / mg antibody. Initial loading amount of CHOP measured in a solution containing anti-Her2 antibody: 727070 ng CHOP / mg antibody.

[0132] Specific buffer conditions for protein A chromatography (a) Control (washing with only equilibration buffer) Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Loading Step 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Washing II: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 5: Washing III: --- Step 6: Washing IV: --- Step 7: Elution: 50 mM acetic acid, pH 4.0

[0133] (b) Low conductivity washing (with only Tris buffer) Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Loading Step 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Washing II: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Washing IV: 2 mM Tris, pH 8.0 Step 7: Elution: 50 mM acetic acid, pH 4.0

[0134] (c) High conductivity washing (with only Tris buffer) Step 1: Equilibration: 2� mM Tris, 25 mM NaCl, pH 7.0 Step 2: Loading Step 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 700 mM Tris, pH 7.2 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Cleansing IV: --- Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0135] (d) Low conductivity tris + high conductivity histidine (His) / tris Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Wash II: 200mM His / 1000mM Tris, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0136] TIFF0007887230000003.tif61147

[0137] Example 5 Purification of bispecific anti-Ang2 / VEGF-A antibodies (IgG1 isotype) in protein A chromatography The general conditions followed those described in Example 2. Antibody: Anti-Ang2 / VEGF-A Initial loading of PLBL2 measured in a solution containing bispecific anti-Ang2 / VEGF-A antibody: 919.7 ng PLBL2 / mg antibody. Initial loading of CHOP measured in a solution containing anti-Ang2 / VEGF-A antibody: 682304 ng CHOP / mg antibody.

[0138] Specific buffer conditions for Protein A chromatography (a) Control (washing with equilibration buffer only) Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Loading Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 5: Wash III: --- Step 6: Wash IV: --- Step 7: Elution: 50 mM acetic acid, pH (删除多余的冒号)4.0

[0139] (b) Low conductivity wash (only with Tris buffer) Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Loading Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV: 2 mM Tris, pH 8.0 Step 7: Elution: 50 mM acetic acid, pH 4.0

[0140] (c) High conductivity wash (only with Tris buffer) Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Loading Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 700 mM Tris, pH 7.2 [[ID=...]](这里原文可能有错误,推测是ID=44开始,为了保持格式一致,继续翻译) Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV: --- Step 7: Elution: 50 mM acetic acid, pH 4.0

[0141] (d) Low conductivity Tris + high conductivity histidine (His) / Tris<(0000983)(这里原文标签格式有误,推测是<这里少了右括号,翻译时修正为 )Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Wash II: 200mM His / 1000mM Tris, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0142] TIFF0007887230000004.tif61147

[0143] Example 6 Purification of bispecific anti-FIXa / X antibodies (IgG4 isotype) in protein A chromatography The purification of anti-FIXa / X antibodies was tested using two different chromatography settings.

[0144] Setting 1 The general conditions followed those described in Example 2. Antibody: Anti-FIXa / X Initial loading of PLBL2 measured in a solution containing anti-FIXa / X antibody: 557 ng PLBL2 / mg antibody. Initial loading of CHOP measured in a solution containing anti-FIXa / X antibody: 387377 ng CHOP / mg antibody.

[0145] Specific buffer conditions for Protein A chromatography (a) High conductivity washing (using Tris buffer solution only) Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Washing II: 700 mM Tris, pH 7.2 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Cleansing IV: --- Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0146] (b) Low conductivity tris + high conductivity histidine (His) / tris Stage 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 2: Loading Stage 3: Washing I: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 4: Wash II: 200mM His / 1000mM Tris, pH 7.0 Stage 5: Washing III: 25 mM Tris, 25 mM NaCl, pH 7.0 Stage 6: Washing IV: 2 mM Tris, pH 8.0 Stage 7: Elution: 50 mM acetic acid, pH 4.0

[0147] TIFF0007887230000005.tif39147

[0148] Setting 2 General chromatography conditions Column resin: Protein A material "Mab Select SuRe" (GE-Healthcare), diameter: 1 cm, height: 20.1 cm, CV: 15.79 ml Equipment: Akta Avant 150 Flow velocity: 300 cm / hour at all stages

[0149] After equilibrating the protein A affinity column (step 1), a solution containing anti-FIXa / X antibody was applied to the column.

[0150] Initial loading of PLBL2 measured in a solution containing anti-FIXa / X antibody: 557 ng PLBL2 / mg antibody.

[0151] The chromatography step was carried out according to the following general scheme. Stage 1: Equilibrium: Stage 2: Loading of antibody-containing solution Stage 3: Cleaning I Stage 4: Cleaning II Stage 5: Cleaning III (Additional Cleaning) Stage 6: Elution

[0152] Specific buffer conditions for Protein A chromatography (a) High conductivity washing (only with NaSO4 buffer) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 450mM NaSO4, 20mM NaAc, pH 4.8 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Cleaning III: --- Stage 6: Elution: 35 mM acetic acid, pH 4.0

[0153] (b) Low conductivity cleaning (Tris 1 mM) + High conductivity cleaning (using NaSO4) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 450mM NaSO4, 20mM NaAc, pH 4.8 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 1 mM Tris, pH 8.0 Stage 6: Elution: 50 mM acetic acid, pH 4.0

[0154] (c) Low conductivity cleaning (Tris 2mM) + High conductivity cleaning (using NaSO4) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 450mM NaSO4, 20mM NaAc, pH 4.8 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 2 mM Tris, pH 8.0 Stage 6: Elution: 35 mM acetic acid, pH 4.0

[0155] (d) Low conductivity cleaning (Tris 4mM) + high conductivity cleaning (using NaSO4) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 450mM NaSO4, 20mM NaAc, pH 4.8 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 4 mM Tris, pH 8.0 Stage 6: Elution: 50 mM acetic acid, pH 4.0

[0156] (e) Low conductivity cleaning (Tris 6mM) + High conductivity cleaning (using NaSO4) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 450mM NaSO4, 20mM NaAc, pH 4.8 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 6 mM Tris, pH 8.0 Stage 6: Elution: 50 mM acetic acid, pH 4.0

[0157] (f) Low conductivity cleaning (Tris 4 mM, pH 7.8) + High conductivity cleaning (using NaSO4) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 450mM NaSO4, 20mM NaAc, pH 4.8 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 4 mM Tris, pH 7.8 Stage 6: Elution: 50 mM acetic acid, pH 4.0

[0158] (g) Low conductivity cleaning (Tris 4 mM, pH 8.2) + High conductivity cleaning (using NaSO4) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 450mM NaSO4, 20mM NaAc, pH 4.8 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 4 mM Tris, pH 8.2 Stage 6: Elution: 50 mM acetic acid, pH 4.0

[0159] (h) Low conductivity cleaning (Tris 2mM) + High conductivity cleaning (using histidine (His) / Tris 1M) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 200mM His / 1000mM Tris, pH 7.0 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 2 mM Tris, pH 8.0 Stage 6: Elution: 35 mM acetic acid, pH 4.0

[0160] (i) Low conductivity cleaning (Tris 2mM) + High conductivity cleaning (Histidine (His) / Tris 0.85M) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 200mM His / 850mM Tris, pH 7.0 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 2 mM Tris, pH 8.0 Stage 6: Elution: 50 mM acetic acid, pH 4.0

[0161] (j) Low conductivity cleaning (Tris 2mM) + High conductivity cleaning (Histidine (His) / Tris 0.7M) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 200mM His / 700mM Tris, pH 7.0 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 2 mM Tris, pH 8.0 Stage 6: Elution: 50 mM acetic acid, pH 4.0

[0162] (k) Low conductivity cleaning (Tris 2mM) + High conductivity cleaning (Histidine (His) / Tris 0.55M) Stage 1: Equilibration: 20 mM NaPO4, pH 7.5 Stage 2: Loading Stage 3: Washing I: 200mM His / 550mM Tris, pH 7.0 Stage 4: Washing II: 20 mM NaPO4, pH 7.5 Stage 5: Washing III: 2 mM Tris, pH 8.0 Stage 6: Elution: 50 mM acetic acid, pH 4.0

[0163] TIFF0007887230000006.tif136147

[0164] Example 7 General Procedures / Conditions: Mock cell culture medium Null harvested cell culture fluid was produced using non-transfected CHO-DP12 cells cultured in serum-free medium. Fermentation was carried out on a 2L scale using a typical cell culture process. At the end of 14 days of fermentation, the cell culture fluid was harvested by centrifugation and sterile filtration. This harvested cell culture fluid (HCCF) was then stored at -70°C until the experiment.

[0165] Purified PLBL2 Recombinant CHO PLBL2 with a C-terminal hexahistidine tag was expressed by transient transfection on a 35L scale, and purified from the harvested cell culture medium as previously described (Vanderlaan et al, 2015). Next, the purified PLBL2 was formulated in PBS solution and stored at -70°C until the experiment.

[0166] purified antibody Recombinant humanized antibodies were expressed in CHO cells and purified using column chromatography to ensure that the PLBL2 concentration was less than 20 ng / mg. Prior to the start of each study, each antibody was buffered in PBS using a PD-10 desalting column (GE Healthcare).

[0167] Preparation of loading materials for Protein A chromatography To standardize the population and abundance of host cell proteins in protein A loadings across antibodies, purified antibodies were diluted to the same concentration with PBS and spiked into HCCF from non-producing cell lines to obtain a final antibody titer of 5 g / L. A control was also prepared by adding PBS instead of the purified antibody to evaluate nonspecific host cell protein binding to protein A resin in the absence of the antibody.

[0168] Packed bed column chromatography All packed-bed column chromatography experiments were performed using a MabSelect SuRe (GE Healthcare) Protein A resin column with an inner diameter of 0.66 cm and a bed height of 20 cm. For each purification, the column was first equilibrated over 3 column volumes (CV) with 25 mM Tris, 25 mM NaCl, pH 7.7 (equilibrium buffer). Next, the Protein A load was applied to the target loading density of 30 g antibody / L resin, and the column was washed with 3 CV of equilibration buffer, 3 CV of different types of washing buffer, and then again with 3 CV of equilibration buffer. Next, the antibody was eluted at a low pH using 0.1–0.15 M acetic acid, and the collection of the eluate pool was started at 0.5 OD at the start of the elution peak and pooling was stopped after 2.8 CV. In the control experiment using PBS spike-null HCCF, a mock eluate pool of 2.8 CV was obtained by starting at 1 CV after the start of the elution phase and going up to 3.8 CV. Next, at the end of each experiment, each protein A eluate was titrated to pH 5.0 using 1.5 M Tris base. Then, the column was purified with 0.1 M sodium hydroxide solution. The volumetric flow rate was 20 CV / hour for all phases except the loading phase, the first equilibration washing phase, and the elution phase, which had a flow rate of 15 CV / hour.

[0169] (A) Purification of exemplary antibody (IgG4 isotype) and antibody A in protein A chromatography. Specific washing buffer conditions for purifying antibody A (IgG4 isotype) using the general procedure of Example 7 (outlined above): (a) 0.4M potassium phosphate, pH 7.0 (b) 25 mM Tris, 25 mM NaCl, pH 7.7 (c) 0.75M Arg-HCl, pH 7.0 (d) 0.6M NaCl, pH 7.0 (e) Deionized water

[0170] result: TIFF0007887230000007.tif89128

[0171] (B) Purification of exemplary antibody (IgG1 isotype) and antibody B in protein A chromatography. Specific washing buffer conditions for purifying antibody B (IgG1 isotype) using the general procedure of Example 7: (a) Deionized water

[0172] result: TIFF0007887230000008.tif46128

[0173] (C) Exemplary antibody (IgG4 isotype) and purification of antibody C in protein A chromatography. Specific washing buffer conditions for purifying antibody C (IgG4 isotype) using the general procedure of Example 7: (a) 0.4M potassium phosphate, pH 7.0 (b) 25 mM Tris, 25 mM NaCl, pH 7.7 (c) 0.75M Arg-HCl, pH 7.0 (d) 0.6M NaCl, pH 7.0 (e) Deionized water

[0174] result: TIFF0007887230000009.tif89128

[0175] (D) Purification of exemplary antibody (IgG1 isotype) and antibody D in protein A chromatography. Specific washing buffer conditions for purifying antibody D (IgG1 isotype) using the general procedure of Example 7: (a) Deionized water

[0176] TIFF0007887230000010.tif46128

[0177] (E) Exemplary antibody (IgG1 isotype) and purification of antibody E in protein A chromatography. Specific washing buffer conditions for purifying antibody E (IgG1 isotype) using the general procedure of Example 7: (a) 0.4M potassium phosphate, pH 7.0 (b) 31 mM Tris, pH 8.5 (c) 55 mM Tris, pH 9.0 (d) Deionized water

[0178] result: TIFF0007887230000011.tif72128

[0179] (F) Purification of exemplary antibody (IgG1 isotype) and antibody F in protein A chromatography. Specific washing buffer conditions for purifying antibody F using the general procedure of Example 7: (a) 25 mM Tris, pH 9.0

[0180] result: TIFF0007887230000012.tif39128

[0181] Sequence information SEQUENCE LISTING <110> F. Hoffmann-La Roche AG Genentech, Inc. <120> Method for the reduction of host cell proteins in affinity Chromatography <150> US 62 / 208,523 <151> 2015-08-21 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> one two three <212> PRT <213> Artificial <220> <223> variable heavy chain domain VH of <vegf>bevacizumab <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial <220> <223> variable light chain domain VL of <vegf>bevacizumab <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 128 <212> PRT <213> Artificial <220> <223> variable heavy chain domain VH of <ang-2>E6Q <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120 125 <210> 4 <211> 108 <212> PRT <213> Artificial <220> <223> variable light chain domain VL of < ANG-2> E6Q <400> 4 Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 <210> 5 <211> 126 <212> PRT <213> Homo sapiens <400> 5 Gln Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ala Ser Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr 100 105 110 Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 108 <212> PRT <213> Homo sapiens <400> 6 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ile Tyr Asn Met Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 7 <211> 124 <212> PRT <213> Homo sapiens <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Thr Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ile Ser Met Asp Arg Gly Val Lys Asn Asn Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 124 <212> PRT <213> Homo sapiens <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Ala Ala Gly Asp Ile Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Tyr Ser Gly Ser Gly Ser Tyr Tyr Asn Asp Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 124 <212> PRT <213> Homo sapiens <400> 9 Gln Pro Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Asn Thr Leu Thr Glu Leu 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asn Gly Glu Ala Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Leu Ala Gly Gly Ser Asp Phe Tyr Tyr Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 107 <212> PRT <213> Homo sapiens <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 107 <212> PRT <213> Homo sapiens <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 107 <212> PRT <213> Homo sapiens <400> 12 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 < / vegf> < / vegf> < / vegf> < / vegf>

Claims

1. The use of a highly conductive aqueous solution having a conductivity value of 20 mS / cm or higher to reduce the content of host cell protein in the washing step of protein A chromatography used to purify human IgG1 isotype antibodies, wherein the host cell protein is phospholipase B-like 2 (PLBL2), The low-conductivity aqueous solution washing step is performed after the high-conductivity aqueous solution washing step. The low-conductivity aqueous solution has a conductivity value of 0.5 mS / cm or less, and The highly conductive aqueous solution contains histidine. The use.

2. The use according to claim 1, wherein the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.

3. (a) A step of culturing cells containing nucleic acids encoding human IgG1 isotype antibodies, (b) A step of recovering human IgG1 isotype antibodies from the cells or culture medium, (c) The step of contacting the human IgG1 isotype antibody with the protein A chromatography material. (d) A step in which the protein A chromatography material is washed with a highly conductive aqueous solution having a conductivity value of 20 mS / cm or more, wherein the amount of host cell protein is reduced, and the host cell protein is phospholipase B-like 2 (PLBL2), The low-conductivity aqueous solution washing step is performed after the high-conductivity aqueous solution washing step. The low-conductivity aqueous solution has a conductivity value of 0.5 mS / cm or less, and The high-conductivity aqueous solution contains histidine, step, (e) Step of recovering human IgG1 isotype antibodies from protein A chromatography material. Includes, This produces human IgG1 isotype antibodies. A method for producing human IgG1 isotype antibodies.

4. (a) A step of preparing a sample containing a human IgG1 isotype antibody, wherein the sample contains host cell proteins, (b) A step of purifying a human IgG1 isotype antibody by a protein A chromatography method / step that includes washing a protein A chromatography material with a highly conductive aqueous solution having a conductivity value of 20 mS / cm or more, wherein the amount of host cell protein is reduced and the host cell protein is phospholipase B-like 2 (PLBL2), The low-conductivity aqueous solution washing step is performed after the high-conductivity aqueous solution washing step. The low-conductivity aqueous solution has a conductivity value of 0.5 mS / cm or less, and The highly conductive aqueous solution contains histidine, step A method for purifying human IgG1 isotype antibodies, including [specific antibody names], from a sample.

5. The method further includes the step of washing the affinity chromatography material with a medium conductivity aqueous solution before or after washing the protein A chromatography material with a high conductivity aqueous solution, The aqueous conductivity solution has conductivity values ​​ranging from more than 0.5 mS / cm to less than 20 mS / cm. The method according to claim 3 or 4.

6. The method according to claim 5, wherein the aqueous solution with medium conductivity contains histidine.

7. The method according to any one of claims 3 to 6, wherein the human IgG1 isotype antibody is an antibody against influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.

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