Method for the reduction of host cell proteins in affinity chromatography

Abstract

The present invention reports a method for purifying antibodies by reducing the host cell protein content. The method utilizes a washing step using a low-conductivity aqueous solution in affinity chromatography.

Description

Technology Field

[0001] The present invention relates to the field of polypeptide purification. The present invention is particularly related to the reduction of host cell proteins, such as phospholipase B-like 2 (PLBL2) or clusterin, in antibody-containing solutions. Background Technology

[0002] Proteins, and particularly immunoglobulins, play a significant role in the portfolio of modern medicine. For human application, all therapeutic proteins must meet specific criteria. To ensure the safety of biopharmaceutical preparations for humans, byproducts accumulated during the manufacturing process must be eliminated. To meet regulatory standards, one or more purification steps must follow the production process. Among other factors, purity, throughput, and yield play a critical role in determining the appropriate purification process.

[0003] Different methods 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 or carboxymethyl resin), anion exchange (aminoethyl resin) and mixed-method ion exchange), sulfur affinity adsorption (e.g., to beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., to phenyl-sepharose, aza-arenophilic resin or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., to Ni(II)- and Cu(II)-affinity substances), size exclusion chromatography, and electrophoretic methods (e.g., gel electrophoresis, capillary electrophoresis) are well established and widely used for protein purification.

[0004] For the purification of recombinant immunoglobulins, a combination of different column chromatography steps is often used. During purification, non-immunoglobulin contaminants, such as host cell proteins and host cell DNA, as well as endotoxins and viruses, are reduced. Therefore, an affinity chromatography step, such as protein A affinity chromatography, is generally followed by one or more additional separation steps. Generally, a high conductivity buffer is described as being used in the washing step of the affinity chromatography method.

[0005] US 6,127,526 describes a method for purifying a protein by protein A chromatography, comprising the following steps: (a) adsorbing a protein onto a protein A immobilized on a solid phase comprising silica or glass; (b) washing the solid phase with a hydrophobic electrolyte solvent to remove contaminants bound to the solid phase; and (c) recovering the protein from the solid phase.

[0006] WO2011 / 038894 reports a protein A chromatography in which host cell proteins and DNA are significantly reduced by a specific washing step before the recovery of immunoglobulin from the protein A chromatography material.

[0007] WO2013 / 177118 reports a composition and method for isolating and purifying antibodies from a sample matrix.

[0008] WO2013 / 033517 reports a method for isolating a polypeptide of interest (e.g., an antibody) from a virus.

[0009] A protein purification method is reported in EP2583973, comprising one or more chromatographic processes in which an amino acid; or its dipeptide, oligopeptide, or polyamino acid is included in a buffer solution (equilibration buffer, washing buffer, and elution buffer) used in one or more chromatographic processes, thereby purifying a high-purity protein having very small amounts of impurities (e.g., polymers or host cell proteins).

[0010] In this invention, a method is reported to produce an antibody having a reduced amount of host cell protein by purifying the antibody through an affinity chromatography step.

[0011] More specifically, it has been discovered that the content of a specific host cell protein in a solution containing an antibody can be reduced by the method of the present invention, which uses a low-conductivity aqueous solution in the washing step of affinity chromatography before recovering the antibody from the chromatographic material. Accordingly, it has been discovered that the content of phospholipase (particularly phospholipase B-like 2 (PLBL2)) can be reduced. It has been discovered that when the antibody is an IgG4 isoform, the PLBL2 content can be reduced by more than 100 times.

[0012] One aspect as reported herein is the use of a low-conductivity aqueous solution in the washing step of protein A chromatography to reduce the content of host cell proteins, wherein protein A chromatography is used to purify human IgG1 or human IgG4 isoform antibodies.

[0013] In one embodiment of this aspect, the human IgG4 homozygous 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 embodiment of this aspect, the human IgG1 homozygous 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.

[0014] In one embodiment of this aspect, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less.

[0015] In one embodiment of this aspect, the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

[0016] In one embodiment of this aspect, the low conductivity aqueous solution contains about 0.1 mM to about 8 mM Tris.

[0017] In one embodiment of this aspect, the low conductivity aqueous solution contains about 0.05 mM to about 2 mM potassium phosphate.

[0018] In one embodiment of this aspect, the low conductivity aqueous solution has a pH of about 7 or higher.

[0019] In one embodiment of this aspect, the low conductivity aqueous solution washing step follows or precedes the high conductivity aqueous solution washing step.

[0020] In one embodiment of this aspect, the high conductivity aqueous solution has a conductivity value of about 20 mS / cm or more.

[0021] In one embodiment of this aspect, the intermediate washing step is performed with an intermediate conductivity aqueous solution between the low conductivity aqueous solution washing step and the high conductivity aqueous solution washing step.

[0022] In one embodiment of this aspect, the medium conductivity aqueous solution has a conductivity value greater than 0.5 mS / cm and less than 20 mS / cm.

[0023] In one embodiment of this aspect, a high (or medium) conductivity aqueous solution contains histidine.

[0024] One aspect as reported herein is a method for producing human IgG4 or IgG1 homotype antibodies comprising the following steps:

[0025] a) a step of culturing cells containing nucleic acids encoding human IgG4 or IgG1 homozygous antibodies,

[0026] b) a step of recovering human IgG4 or IgG1 homozygous antibodies from cells or culture media,

[0027] c) a step of contacting a human IgG4 or IgG1 homozygous antibody with a protein A chromatographic material,

[0028] d) a step of washing the protein A chromatographic material with a low conductivity aqueous solution,

[0029] e) Recover human IgG4 or IgG1 isoform antibodies from protein A chromatographic material, and

[0030] The step of generating human IgG4 or IgG1 isoform antibodies thereby.

[0031] One aspect as reported herein is a method for purifying human IgG4 or IgG1 isoform antibodies from a sample, comprising the following steps:

[0032] a) a step of providing a sample containing human IgG4 or IgG1 homozygous antibodies,

[0033] b) a step of purifying human IgG4 or IgG1 isoform antibodies by a protein A chromatography method / step comprising washing the protein A chromatography material with a low conductivity aqueous solution.

[0034] In one embodiment of all aspects, the human IgG4 homozygous 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 embodiment of all aspects, the human IgG1 homozygous 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.

[0035] In one embodiment of all aspects, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less.

[0036] In one embodiment of all aspects, the content of the host cell protein is reduced, and the (specific) host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

[0037] In one embodiment of all aspects, the low conductivity aqueous solution contains about 0.1 mM to about 8 mM Tris.

[0038] In one embodiment of all aspects, the low conductivity aqueous solution contains about 0.05 mM to about 2 mM potassium phosphate.

[0039] In one embodiment of all aspects, the low conductivity aqueous solution has a pH of about 7 or higher.

[0040] In one embodiment of all method aspects, the method further comprises washing the affinity chromatographic material with a high conductivity aqueous solution and / or a medium conductivity aqueous solution before or after washing the protein A chromatographic material with a low conductivity aqueous solution.

[0041] In one embodiment of all aspects, the high conductivity aqueous solution has a conductivity value of about 20 mS / cm or more.

[0042] In one embodiment of all aspects, the medium conductivity aqueous solution has a conductivity value greater than 0.5 mS / cm and less than 20 mS / cm.

[0043] In one embodiment of all aspects, the high or medium conductivity aqueous solution contains histidine. Specific details for implementing the invention

[0044] Detailed description of the invention

[0045] An improved affinity chromatography method and its use are reported, which includes washing the affinity chromatography material with a low conductivity aqueous solution.

[0046] It has been found that when a washing step with a low conductivity aqueous solution is used in an affinity chromatography step, for example, in a protein A chromatography step, specific host cell proteins can be reduced by the washing step with a low conductivity aqueous solution. Affinity chromatography steps are used in methods for the purification or production of antibodies. The washing step with a low conductivity aqueous solution is particularly effective in reducing the content of phospholipase B-like 2 (PLBL2).

[0047] One aspect as reported herein is the use of a low-conductivity aqueous solution in the washing step of affinity chromatography to reduce the content of (specific) host cell proteins.

[0048] One aspect as reported herein is a method for producing human IgG isotype antibodies comprising the following steps:

[0049] a) a step of culturing cells containing nucleic acid encoding a human IgG isoform antibody,

[0050] b) A step of recovering human IgG isoform antibodies from cells or culture medium,

[0051] c) a step of contacting a human IgG isomorphic antibody (a solution containing it) with an affinity chromatography material,

[0052] d) a step of washing the affinity chromatography material with a low conductivity aqueous solution while ensuring that more than 90% of the bispecific antibodies are bound to the affinity chromatography material,

[0053] e) Recover human IgG isoform antibodies from the affinity chromatography material, and

[0054] The step of generating human IgG isomorphic antibodies therefrom.

[0055] One aspect as reported herein is a method for purifying human IgG isomorphic antibodies from a sample, comprising the following steps:

[0056] a) a step of providing a (buffered aqueous) sample containing human IgG isoform antibodies,

[0057] b) a step of purifying a human IgG isoform antibody by an affinity chromatography method / step, comprising washing the affinity chromatography material with a low conductivity aqueous solution.

[0058] The recombinant polypeptide generated in CHO cells can be purified according to the method described herein to remove or reduce the level of host cell proteins.

[0059] Exemplary recombinant polypeptides include therapeutic antibodies and immunoadhesions, e.g., but not limited to, antibodies including antibody fragments against one or more of the following antigens: 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, CDlla, CDllb, CDllc, CD18, ICAM, VLA-4, VCAM, IL-17A and / or F, IgE (e.g., omalizumab), DRS, CD40, Apo2L / TRAIL, EGFL7 (e.g., parsatuzumab), NRP1, integrin beta7 (e.g., etrolizumab), IL-13 (e.g., Lebrikizumab), Abeta (e.g., crenezumab, gantenerumab), P-selectin (e.g., inclakumab), IL-6R (e.g., tociluzumab), IFNa (e.g., lontalizumab), M1prime (e.g., quillizumab), mycogen-activated protein kinase (MAPK), OX40L, TSLP, Factor D (e.g., rampalizumab) and receptors e.g.: IL-9 receptor, IL-5 receptor, IL-4 receptor alpha, IL-13 receptor alpha1 and IL-13 receptor alpha2, OX40, TSLP-R, IL-7R alpha (co-receptor for TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2, FcepsilonRI and FcepsilonRII / CD23 (IgE Receptor for ).Other exemplary antibodies are, to a non-limiting extent, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD10 antibody, anti-CD12 antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, It includes those selected from 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-melanosomal 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.

[0060] In some embodiments, the exemplary antibodies include antibodies against Abeta, antibodies against IL17 A / F, and antibodies against CMV. Exemplary anti-Abeta antibodies and methods for producing such antibodies have been previously described, for example, in W02008011348, W02007068429, W02001062801, and W02004071408. Exemplary anti-IL17 A / F antibodies and methods for producing such antibodies have been previously described, for example, in WO 2009136286 and US 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 WO 2012047732.

[0061] In some embodiments, affinity chromatography is used to purify human IgG homozygous antibodies. In some embodiments, affinity chromatography is used to purify IgG4 antibodies. In one embodiment, the IgG4 homozygous 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 homozygous antibodies. In one embodiment, the IgG1 homozygous 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.

[0062] One aspect as reported herein is a method for producing a human IgG4 homozygous antibody (containing solution) comprising the following steps:

[0063] a) a step of culturing cells containing nucleic acid encoding a human IgG4 homozygous antibody,

[0064] b) A step of recovering human IgG4 homozygous antibodies from cells or culture medium,

[0065] c) a step of contacting a human IgG4 isoform antibody with an affinity chromatography material,

[0066] d) a step of washing the affinity chromatography material with a low conductivity aqueous solution,

[0067] e) Recover human IgG4 isoform antibodies from the affinity chromatography material, and

[0068] The step of generating human IgG4 isoform antibodies therefrom.

[0069] One aspect as reported herein is a method for producing an IgG4 homozygous antibody (containing solution) comprising the following steps:

[0070] a) a step of culturing cells containing nucleic acid encoding an IgG4 homozygous antibody,

[0071] b) a step of recovering IgG4 homozygous antibodies from cells or culture medium,

[0072] c) a step of contacting an IgG4 homozygous antibody with an affinity chromatography material,

[0073] d) a step of washing the affinity chromatography material with a low conductivity aqueous solution,

[0074] e) Recover IgG4 isoform antibodies from the affinity chromatography material, and

[0075] The step of generating IgG4 isoform antibodies therefrom.

[0076] One aspect as reported herein is a method for purifying human IgG4 isoform antibodies from a sample, comprising the following steps:

[0077] a) a step of providing a sample containing human IgG4 homozygous antibodies,

[0078] b) a step of purifying a human IgG4 isoform antibody by an affinity chromatography method / step, comprising washing the affinity chromatography material with a low conductivity aqueous solution.

[0079] One aspect as reported herein is a method for purifying IgG4 isoform antibodies from a sample, comprising the following steps:

[0080] a) a step of providing a sample containing an IgG4 homozygous antibody,

[0081] b) a step of purifying an IgG4 isoform antibody by an affinity chromatography method / step, comprising washing the affinity chromatography material with a low conductivity aqueous solution.

[0082] It has been found that if the conductivity of the aqueous solution used in the washing step is low, that is, if a low-conductivity aqueous solution is used for washing, the host cell protein content may be reduced. In one embodiment of all aspects, the low-conductivity aqueous solution has a conductivity value of about 1 mS / cm or less. In one preferred embodiment of all aspects, the low-conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less. In one embodiment, the low-conductivity aqueous solution has a conductivity value of about 0.03 μS / cm to about 0.5 mS / cm. In one embodiment, the low-conductivity aqueous solution has a conductivity value of about 0.05 μS / cm to about 0.35 mS / cm. In one embodiment of all aspects, the low-conductivity aqueous solution is deionized water. For some applications, deionized water is not suitable for use in the washing step. In some embodiments, the low-conductivity aqueous solution is not deionized water.

[0083] It has been discovered that protein A affinity chromatography can be used for purposes such as those reported herein. In one preferred embodiment of all aspects, the affinity chromatography is protein A affinity chromatography. In one embodiment, the protein A affinity chromatography is selected from the group comprising 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; and Toyopearl AF-rProtein A HC-650HF. In one embodiment, the affinity chromatography is protein G affinity chromatography. In one embodiment, the affinity chromatography is an affinity chromatography using a recombinant protein as a ligand, which means that the affinity chromatography is a recombinant protein ligand affinity chromatography. In one embodiment, the affinity chromatography is an affinity chromatography using a single-strand Fv as a ligand, which means that the affinity chromatography is a single-strand Fv ligand affinity chromatography. In one embodiment, the affinity chromatography comprises a mutant protein A coupled to a chromatography matrix or a fragment of protein A coupled to a chromatography matrix.

[0084] It was found that the content of (specific) host cell proteins may be reduced. In particular, it was found that the content of phospholipase B-like 2 (PLBL2) may be reduced. In one embodiment, the (specific) host cell protein is a 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).

[0085] It has been discovered that a low conductivity aqueous solution may contain a low amount of a specific buffering substance, e.g., 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 Tris. In one embodiment, the low conductivity aqueous solution contains about 0.5 mM to about 6.5 mM Tris. In one embodiment, the low conductivity aqueous solution contains about 2 mM 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 potassium phosphate. In one embodiment, the low conductivity aqueous solution contains about 0.05 mM to about 2 mM potassium phosphate. In one embodiment, the low conductivity aqueous solution contains about 0.5 mM potassium phosphate.

[0086] It has been discovered that when a low conductivity aqueous solution has a specific pH, the effect of reducing host cell protein content is manifested. In one embodiment, the low conductivity aqueous solution has a pH of about 7 or higher. In one embodiment, the low conductivity aqueous solution has a pH of about 7.5 or higher. In one embodiment, the low conductivity aqueous solution has a pH of about 7 to about 9.5. In one embodiment, the low conductivity aqueous solution has a pH of about 7.5 to about 8.5. In one embodiment, the low conductivity aqueous solution has a pH of about 8. In one embodiment, the low conductivity aqueous solution has a pH of about 9.

[0087] It was found that when the pH of the low conductivity aqueous solution is about 8.5 or higher and the low conductivity aqueous solution has a conductivity value of about 1.2 mS / cm or lower, the effect of reducing host cell protein content can also be achieved. In one embodiment, the low conductivity aqueous solution has a pH of about 8.5 or higher and the low conductivity aqueous solution has a conductivity value of about 1.2 mS / cm or lower. In one embodiment, the low conductivity aqueous solution has a pH of about 8.5 or higher and the low conductivity aqueous solution has a conductivity value of about 1 mS / cm or lower. In one embodiment, the low conductivity aqueous solution has a pH of about 8.5 or higher and the low conductivity aqueous solution contains about 55 mM or less of Tris. In one embodiment, the low conductivity aqueous solution has a pH of about 8.5 or higher and the low conductivity aqueous solution contains about 30 mM or less of Tris.

[0088] In one embodiment, the low conductivity aqueous solution has a pH range of pH 7 to less than pH 8.5 and has a conductivity value of about 0.5 mS / cm or less, and has a conductivity value of about 1.2 mS / cm or less at a pH of 8.5 or higher.

[0089] For example, it has been found that the content of host cell proteins, such as PLBL2, can be reduced to a specific level by the use and method as reported herein when compared to the loading amount of PLBL2 prior to a purification step, such as an affinity chromatography step. In one embodiment, the content of PLBL2 is reduced by more than 20 times. In one embodiment, the content of PLBL2 is reduced by more than 40 times. In one embodiment, the content of PLBL2 is reduced by more than 50 times. In one embodiment, the content of PLBL2 is reduced by more than 90 times. In one embodiment, the content of PLBL2 is reduced by more than 100 times. In some cases, the level of reduction is higher. In some embodiments, the content of PLBL2 is reduced by more than 200 times. In some embodiments, the content of PLBL2 is reduced by more than 250 times. In some embodiments, the content of PLBL2 is reduced by more than 300 times. In some embodiments, PLBL2 is reduced by more than 400 times. In some embodiments, the content of PLBL2 is reduced by more than 1,000 times. In one embodiment, the content of PLBL2 is reduced by at least 50%. In one embodiment, the content of PLBL2 is reduced by at least 66%. In one embodiment, the content of PLBL2 is reduced by at least 80%. In one embodiment, the content of PLBL2 is reduced by at least 90%. In one embodiment, the content of PLBL2 is reduced by at least 95%. In some embodiments, the content of PLBL2 is reduced to less than 10 ng per 1 mg of antibody. In some embodiments, the content of PLBL2 is reduced to less than 5 ng per 1 mg of antibody. In some embodiments, the content of PLBL2 is reduced to less than 2 ng per 1 mg of antibody.

[0090] In the methods and uses as reported herein, an additional washing step may be used with an intermediate and / or high conductivity aqueous solution. In one embodiment, a washing step with a low conductivity aqueous solution follows or precedes a washing step with a high conductivity aqueous solution. In one embodiment, the high conductivity aqueous solution has a conductivity value of about 20 mS / cm or greater. 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 washing step is performed with an intermediate conductivity aqueous solution between the washing step with a low conductivity aqueous solution and the washing step with a high conductivity aqueous solution. In one embodiment, the intermediate conductivity aqueous solution has a conductivity value greater than 0.5 mS / cm and less than 20 mS / cm.

[0091] It has been found that when a high or medium conductivity aqueous solution additionally contains amino acids, the host cell protein reduction effect may be improved. In one embodiment, the high or medium conductivity aqueous solution contains amino acids. In one embodiment, the high or medium conductivity aqueous solution contains histidine or arginine. In one embodiment, the high or medium conductivity aqueous solution contains histidine. In one embodiment, the high or medium conductivity aqueous solution contains histidine and Tris.

[0092] Methods and uses as reported herein may include one or more additional chromatography steps. In one embodiment, one or more additional chromatography methods / steps are performed. In one embodiment, additional ion exchange chromatography methods / steps are performed. In one embodiment, additional anion exchange chromatography methods / steps are performed. In one embodiment, additional anion exchange chromatography methods / steps and additional cation exchange chromatography methods / steps are performed.

[0093] It was found that the use of a hydrophobic interaction chromatography step can be omitted. In one embodiment, the use or method does not have a hydrophobic interaction chromatography method / step.

[0094] One aspect as reported herein is the use of a low-conductivity aqueous solution in the washing step of protein A chromatography to reduce PLBL2 or clusterin content, wherein protein A chromatography is used to purify IgG4 or IgG1 isoforms, e.g., human IgG4 or IgG1, antibodies, and the low-conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher.

[0095] One aspect is the use of a low-conductivity aqueous solution in the washing step of protein A chromatography to reduce PLBL2 or clusterin content, wherein protein A chromatography is used to purify human IgG4 or IgG1 homotype antibodies, and the low-conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher. In some embodiments, the antibody is an IgG4 homotype 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 homozygous 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.

[0096] In one aspect, the present disclosure provides a method for producing human IgG4 or IgG1 homotype antibodies comprising the following steps:

[0097] a) a step of culturing cells containing nucleic acids encoding human IgG4 or IgG1 homozygous antibodies,

[0098] b) a step of recovering human IgG4 or IgG1 homozygous antibodies from cells or culture media,

[0099] c) a step of contacting a human IgG4 or IgG1 homozygous antibody with a protein A affinity chromatography material,

[0100] d) a step of washing the protein A affinity chromatographic material with a low conductivity aqueous solution,

[0101] e) Recover human IgG4 or IgG1 isoform antibodies from the affinity chromatography material, and

[0102] The step of generating human IgG4 or IgG1 isoform antibodies,

[0103] Here, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher.

[0104] In one aspect, the present disclosure provides a method for producing human IgG4 or IgG1 homotype antibodies comprising the following steps:

[0105] a) a step of culturing cells containing nucleic acids encoding human IgG4 or IgG1 homozygous antibodies,

[0106] b) a step of recovering human IgG4 or IgG1 homozygous antibodies from cells or culture media,

[0107] c) a step of contacting a human IgG4 or IgG1 homozygous antibody with a protein A affinity chromatography material,

[0108] d) a step of washing the protein A affinity chromatographic material with a low conductivity aqueous solution,

[0109] e) Recover human IgG4 or IgG1 isoform antibodies from the affinity chromatography material, and

[0110] The step of generating human IgG4 or IgG1 isoform antibodies,

[0111] Here, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher, and

[0112] Human IgG4 homozygous 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, and human IgG1 homozygous 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.

[0113] In one aspect, the present disclosure provides a method for purifying human IgG4 or IgG1 isotype antibodies from a sample, comprising the following steps:

[0114] a) a step of providing a sample containing human IgG4 or IgG1 homozygous antibodies,

[0115] b) a step of purifying a human IgG4 or IgG1 isoform antibody by a protein A affinity chromatography method / step comprising washing the protein A affinity chromatography material with a low conductivity aqueous solution,

[0116] Here, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher.

[0117] In one aspect, the present disclosure provides a method for purifying human IgG4 or IgG1 isotype antibodies from a sample, comprising the following steps:

[0118] a) a step of providing a sample containing human IgG4 or IgG1 homozygous antibodies,

[0119] b) a step of purifying a human IgG4 or IgG1 isoform antibody by a protein A affinity chromatography method / step comprising washing the protein A affinity chromatography material with a low conductivity aqueous solution,

[0120] Here, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher, and

[0121] Human IgG4 homozygous antibodies are antibodies against P-selectin, or antibodies against factor IXa and factor X, or antibodies against IL-13, or antibodies against amyloid beta, and human IgG1 homozygous antibodies are antibodies against influenza B, or antibodies against VEGF-A, or antibodies against CD22, or 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.

[0122] In one aspect, the present disclosure provides a method for producing a human IgG4 homozygous antibody comprising the following steps:

[0123] a) a step of culturing cells containing nucleic acid encoding a human IgG4 homozygous antibody,

[0124] b) A step of recovering human IgG4 homozygous antibodies from cells or culture medium,

[0125] c) a step of contacting a human IgG4 homozygous antibody with a protein A affinity chromatography material,

[0126] d) a step of washing the protein A affinity chromatographic material with a low conductivity aqueous solution,

[0127] e) Recover human IgG4 isoform antibodies from the affinity chromatography material, and

[0128] The step of generating human IgG4 isoform antibodies,

[0129] Here, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher, and

[0130] Human IgG4 isoform antibodies are antibodies against factor IXa and factor X.

[0131] In one aspect, the present disclosure provides a method for purifying a human IgG4 isoform antibody from a sample, comprising the following steps:

[0132] a) a step of providing a sample containing human IgG4 homozygous antibodies,

[0133] b) a step of purifying a human IgG4 isoform antibody by a protein A affinity chromatography method / step, comprising washing the protein A affinity chromatography material with a low conductivity aqueous solution,

[0134] Here, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less and a pH of about 7 or higher, and

[0135] Human IgG4 isoform antibodies are antibodies against factor IXa and factor X.

[0136] The terms "anti-P-selectin antibody," "antibody binding to P-selectin," or "antibody against P-selectin" refer to antibodies capable of binding to P-selectin with sufficient affinity and are useful as diagnostic agents and / or therapeutic agents in targeting P-selectin. In one embodiment, the degree of binding of the anti-P-selectin antibody to an unrelated, non-P-selectin protein is less than about 10% of the antibody binding to P-selectin, as measured, for example, by ELISA or surface plasmon resonance. In a specific embodiment, the anti-P-selectin antibody binds to an epitope of P-selectin that is conserved among P-selectins from different species. The foregoing also applies to the terms "antibody against Factor IXa and Factor X," "antibody against IL-13," or "antibody against amyloid beta," etc.

[0137] In some embodiments, the antibody against P-selectin is inclakumab (IgG4 isoform) as described in WO 2005 / 100402 or SEQ ID NO: 07–12. In some embodiments, the antibody is a bispecific antibody against factor IXa and factor X, e.g., an anti-FIXa / X antibody (IgG4 isoform) as described in WO 2012 / 067176. In some embodiments, the antibody is an antibody against Her2, e.g., trastuzumab (IgG1 isoform) as described in WO 1992 / 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 isoform) as described in WO 2011 / 117329 or SEQ ID NO: 01 to 04. In some embodiments, the antibody is an antibody against amyloid beta, e.g., gantenerumab (IgG1 isoform) or crenezumab (IgG4 isoform) as described in WO 2003 / 070760 or SEQ ID NO: 05 to 06. In some embodiments, the antibodies are 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 or VEGF-A may be used interchangeably herein.

[0138] 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 process (SPR, BIAcore, GE-Healthcare Uppsala, Sweden). The affinity of binding is denoted by the terms ka (rate constant for the binding of an antibody to an antibody / antigen complex), kd (Dissociation constant) and K D (k d / k a It is defined by ). 'Combining' or 'specifically combining' is 10 -7 Binding affinity of mol / L or less (K D It means ).

[0139] The term “antibody” is used in the broadest sense herein and includes various antibody structures, e.g., monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit the desired antigen-binding activity.

[0140] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an 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. A Fab fragment is an antibody fragment obtained by papain digestion of a (whole / full) antibody.

[0141] "Bispecific antibody" is an antibody having two different antigen-binding specificities. As used herein, the term "bispecific" antibody refers to an antibody having two or more binding sites that bind to different epitopes.

[0142] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or type, while the remaining heavy chain and / or light chain originates from a different source or type.

[0143] The "class" of an antibody indicates the type of constant domain or constant part retained by the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further differentiated into subclasses (homogeneous), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively.

[0144] The term "human IgG isoform antibody" refers to an antibody containing a constant derived from a human wild-type IgG isoform, that is, for example, it may contain a constant derived from a human IgG isoform having a mutation, for example, the P329G mutation (numbered according to Kabat).

[0145] The term "human IgG4 homozygous antibody" refers to an antibody containing a constant derived from a human wild-type IgG4 homozygous, that is, for example, it may contain a constant derived from a human IgG4 homozygous having a mutation, for example, the P329G mutation and / or the S228P, L235E mutation (numbering according to Kabat).

[0146] In this document, the term “Fc-site” is used to define a C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. The term includes native sequence Fc-sites and variant Fc-sites. In one embodiment, the human IgG heavy chain Fc-site extends from Cys226 or Pro230 to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or C-terminal glycyl-lysine dipeptide (Gly446Lys447) of the Fc-site may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc-region or constant region follows the EU numbering system, also referred to as the EU indicator, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0147] "Backbone" or "FR" refers to variable domain residues other than the hypervariability (HVR) residues. The FRs of the variable domain typically consist of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0148] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to a cell into which exogenous nucleic acid is introduced, including progeny of the cell. The host cell includes “transformers” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom regardless of passage number. The progeny may not be exactly the same as the parent cell in terms of nucleic acid content but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected from the original transformed cell is included herein. The term “cell” includes a cell used for the expression of nucleic acid. 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 an HEK 293 cell, or an HEK 293 EBNA 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 expression “cell” includes the target cell and its progeny.

[0149] The term "washing" refers to applying a solution to an affinity chromatographic material to remove non-specifically bound polypeptides and non-polypeptide compounds from the chromatographic material, in particular to remove host cell proteins and host cell DNA. The term "washing" does not include the elution of bound materials from the affinity chromatographic material.

[0150] Different methods are well established and widely used for protein recovery and purification {e.g., 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-method exchange), sulfur affinity adsorption (e.g., using beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., using phenyl-sepharose, aza-arenophilic resin or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., using Ni(II)- and Cu(II)-affinity materials), size exclusion chromatography and electrophoretic methods (e.g., gel electrophoresis, capillary electrophoresis)}. These methods can be independently combined in different embodiments as reported herein.

[0151] The term "Protein A" refers to a Protein A polypeptide obtained from natural sources or produced synthetically.

[0152] The term "protein A chromatographic material" refers to an inert solid phase in which protein A is covalently linked.

[0153] 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.

[0154] The term "high conductivity aqueous solution" refers to an aqueous solution having a high conductivity value. The conductivity value can be about 20 mS / cm or higher.

[0155] The term "medium conductivity aqueous solution" refers to an aqueous solution having a medium conductivity value. The conductivity value may be greater than 0.5 mS / cm and less than 20 mS / cm.

[0156] The term "low conductivity aqueous solution" refers to an aqueous solution having a low conductivity value. The conductivity value may be about 0.5 mS / cm or less. When the pH is about 8.5 or higher, the conductivity value may be about 1.2 mS / cm or less. The conductivity value may be measured by standard methods known to those skilled in the art.

[0157] The following examples and procedures are provided to aid in understanding the invention, and the true scope thereof is indicated by the appended claims. It is understood that modifications to the procedures described may be made without departing from the spirit of the invention.

[0158] Specific embodiments of the present invention

[0159] 1. Use of a low-conductivity aqueous solution in the washing step of affinity chromatography to reduce host cell protein content.

[0160] 2. In Embodiment 1, the affinity chromatography is used for purifying human IgG isoform antibodies.

[0161] 3. In Embodiment 2, the affinity chromatography is used to purify human IgG4 isoform antibodies or human IgG1 isoform antibodies.

[0162] 4. In Embodiment 3, the affinity chromatography is used to purify a human IgG4 isoform antibody or a human IgG1 isoform antibody having no glycosylated sites in its Fab fragment / exactly one glycosylated site (at position Asn 297 (numbered according to Kabat)).

[0163] 5. In Embodiment 4, the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less.

[0164] 6. In Embodiment 5, the low conductivity aqueous solution has a conductivity value of about 0.03 μS / cm to about 0.5 mS / cm.

[0165] 7. In Embodiment 5, the low conductivity aqueous solution has a conductivity value of about 0.05 μS / cm to about 0.35 mS / cm.

[0166] 8. In any one of embodiments 5 to 7, the low conductivity aqueous solution is used for purposes other than deionized water.

[0167] 9. In any one of the above embodiments, the affinity chromatography is protein A affinity chromatography or protein G affinity chromatography or single-chain Fv ligand (KappaSelect) affinity chromatography.

[0168] 10. In Embodiment 9, the affinity chromatography is used as protein A affinity chromatography.

[0169] 11. In Embodiment 10, the use of the protein A affinity chromatography is selected from the group comprising 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.

[0170] 12. In any one of the above embodiments, the host cell protein is a Chinese hamster ovary (CHO) host cell protein.

[0171] 13. In Embodiment 12, the host cell protein is phospholipase.

[0172] 14. In Embodiment 13, the host cell protein is phospholipase A, phospholipase B, phospholipase C, or phospholipase D.

[0173] 15. In Embodiments 12, 13, or 14, the host cell protein is phospholipase B-like 2 (PLBL2).

[0174] 16. In Embodiment 12, the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

[0175] 17. In any one of the above embodiments, the low conductivity aqueous solution is used for a purpose in which it contains tris(hydroxymethyl)aminomethane (Tris).

[0176] 18. In Embodiment 17, the use of a low conductivity aqueous solution comprising about 0.1 mM to about 10 mM Tris.

[0177] 19. In Embodiment 18, the use of a low conductivity aqueous solution comprising about 0.1 mM to about 8 mM Tris.

[0178] 20. In Embodiment 19, the use of a low conductivity aqueous solution comprising about 0.5 mM to about 6.5 mM Tris.

[0179] 21. In Embodiment 20, the low conductivity aqueous solution contains about 2 mM Tris.

[0180] 22. In any one of embodiments 17 to 21, the low conductivity aqueous solution is used for a purpose in which potassium phosphate is contained.

[0181] 23. In Embodiment 22, the low conductivity aqueous solution comprises about 0.05 mM to about 5 mM potassium phosphate.

[0182] 24. In Embodiment 23, the low conductivity aqueous solution comprises about 0.05 mM to about 2 mM potassium phosphate.

[0183] 25. In Embodiment 24, the low conductivity aqueous solution comprises about 0.5 mM potassium phosphate.

[0184] 26. In any one of the above embodiments, the low conductivity aqueous solution has a pH of about 7 or higher.

[0185] 27. In Embodiment 26, the low conductivity aqueous solution has a pH of about 7.5 or higher.

[0186] 28. In Embodiment 27, the low conductivity aqueous solution has an approx. pH of about 7 to about 9.5.

[0187] 29. In Embodiment 28, the low conductivity aqueous solution has a pH of about 7.5 to about 8.5.

[0188] 30. In Embodiment 29, the low conductivity aqueous solution has a pH of about 8.

[0189] 31. An application in which, in any one of the above embodiments, the low conductivity aqueous solution washing step follows or precedes the high conductivity aqueous solution washing step.

[0190] 32. In Embodiment 31, the use in which the low conductivity aqueous solution washing step follows the high conductivity aqueous solution washing step.

[0191] 33. In Embodiment 31, the high conductivity aqueous solution has a conductivity value of about 20 mS / cm or more.

[0192] 34. In Embodiment 33, the high conductivity aqueous solution has a conductivity value of about 20 mS / cm to about 100 mS / cm.

[0193] 35. An application in which, in Embodiment 31, the intermediate washing step is performed with an intermediate conductivity aqueous solution between the low conductivity aqueous solution washing step and the high conductivity aqueous solution washing step.

[0194] 36. In Embodiment 35, the medium conductivity aqueous solution has a conductivity value greater than 0.5 mS / cm and less than 20 mS / cm.

[0195] 37. In any one of embodiments 33 to 36, the use of a high or medium conductivity aqueous solution containing an amino acid.

[0196] 38. In Embodiment 37, the use of a high or medium conductivity aqueous solution containing histidine.

[0197] 39. In Embodiment 37, the use of a high or medium conductivity aqueous solution comprising histidine and Tris.

[0198] 40. An application in which one or more additional chromatography methods / steps are performed in any one of the above embodiments.

[0199] 41. An application in which an additional ion exchange chromatography method / step is performed in Embodiment 40.

[0200] 42. An application in which an additional anion exchange chromatography method / step is performed in Embodiment 41.

[0201] 43. An application in which an additional cation exchange chromatography method / step is performed in Embodiment 40.

[0202] 44. An application in which an additional anion exchange chromatography method / step and an additional cation exchange chromatography method / step are performed in Embodiment 40.

[0203] 45. An application that does not have a hydrophobic interaction chromatography method / step in Embodiment 40.

[0204] 46. ​​In any one of the above embodiments, the human IgG4 homozygous 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.

[0205] 47. In any one of embodiments 1 to 45, the human IgG1 homozygous 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.

[0206] 48. A method for producing human IgG isotype antibodies comprising the following steps:

[0207] a) a step of culturing cells containing nucleic acid encoding a human IgG isoform antibody,

[0208] b) A step of recovering human IgG isoform antibodies from cells or culture medium,

[0209] c) a step of contacting a human IgG isoform antibody with an affinity chromatography material,

[0210] d) a step of washing the affinity chromatography material with a low conductivity aqueous solution,

[0211] e) Recover human IgG isoform antibodies from the affinity chromatography material, and

[0212] The step of generating human IgG isomorphic antibodies therefrom.

[0213] 49. A method for producing human IgG4 isoform antibodies comprising the following steps:

[0214] a) a step of culturing cells containing nucleic acid encoding a human IgG4 homozygous antibody,

[0215] b) A step of recovering human IgG4 homozygous antibodies from cells or culture medium,

[0216] c) a step of contacting a human IgG4 isoform antibody with an affinity chromatography material,

[0217] d) a step of washing the affinity chromatography material with a low conductivity aqueous solution,

[0218] e) Recover human IgG4 isoform antibodies from the affinity chromatography material, and

[0219] The step of generating human IgG4 isoform antibodies therefrom.

[0220] 50. In Embodiment 48, the human IgG isoform antibody is a human IgG4 isoform antibody or a human IgG1 isoform antibody.

[0221] 51. A method according to Embodiment 48, wherein the human IgG isoform antibody does not have a glycosylated glycosylated site in its Fab fragment / is exactly one glycosylated site (at position Asn 297 (numbered according to Kabat)) and is a human IgG4 isoform antibody or a human IgG1 isoform antibody.

[0222] 52. A method according to Embodiment 48 in which a low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less.

[0223] 53. A method according to Embodiment 52 in which the low conductivity aqueous solution has a conductivity value of about 0.03 μS / cm to about 0.5 mS / cm.

[0224] 54. A method according to Embodiment 53, wherein the low conductivity aqueous solution has a conductivity value of about 0.05 μS / cm to about 0.35 mS / cm.

[0225] 55. In Embodiment 54, the low conductivity aqueous solution is not deionized water.

[0226] 56. A method in any one of embodiments 48 to 55, wherein the affinity chromatography is protein A affinity chromatography or protein G affinity chromatography or single-chain Fv ligand (KappaSelect) affinity chromatography.

[0227] 57. In Embodiment 56, a method wherein the affinity chromatography is protein A affinity chromatography.

[0228] 58. A method according to Embodiment 56, wherein the protein A affinity chromatography is selected from the group comprising 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; and Toyopearl AF-rProtein A HC-650HF.

[0229] 59. A method in which the content of host cell protein is reduced in Embodiment 48 or 49.

[0230] 60. A method in which, in Embodiment 59, the host cell protein is a Chinese hamster ovary (CHO) host cell protein.

[0231] 61. A method in which the host cell protein is phospholipase in Embodiment 60.

[0232] 62. In Embodiment 61, the method wherein the host cell protein is phospholipase A, phospholipase B, phospholipase C, or phospholipase D.

[0233] 63. In Embodiment 62, the host cell protein is phospholipase B-like 2 (PLBL2).

[0234] 64. A method according to Embodiment 60 in which the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

[0235] 65. A method in any one of embodiments 48 to 64, wherein the low conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (Tris).

[0236] 66. A method according to Embodiment 65, wherein the low conductivity aqueous solution comprises about 0.1 mM to about 10 mM Tris.

[0237] 67. A method according to Embodiment 66, wherein the low conductivity aqueous solution comprises about 0.1 mM to about 8 mM Tris.

[0238] 68. A method according to Embodiment 67, wherein the low conductivity aqueous solution comprises about 0.5 mM to about 6.5 mM Tris.

[0239] 69. A method according to Embodiment 68, wherein the low conductivity aqueous solution comprises about 2 mM Tris.

[0240] 70. A method in any one of embodiments 65 to 69, wherein the low conductivity aqueous solution contains potassium phosphate.

[0241] 71. A method according to Embodiment 70, wherein the low conductivity aqueous solution comprises about 0.2 mM to about 5 mM potassium phosphate.

[0242] 72. A method according to Embodiment 71, wherein the low conductivity aqueous solution comprises about 0.05 mM to about 2 mM potassium phosphate.

[0243] 73. A method according to Embodiment 72, wherein the low conductivity aqueous solution comprises about 0.5 mM potassium phosphate.

[0244] 74. A method in any one of embodiments 48 to 73, wherein the low conductivity aqueous solution has a pH of about 7 or higher.

[0245] 75. A method according to Embodiment 74 in which the low conductivity aqueous solution has a pH of about 7.5 or higher.

[0246] 76. A method according to Embodiment 75, wherein the low conductivity aqueous solution has a pH of about 7 to about 9.5.

[0247] 77. A method according to Embodiment 76, wherein the low conductivity aqueous solution has a pH of about 7.5 to about 8.5.

[0248] 78. A method according to Embodiment 77 in which the low conductivity aqueous solution has a pH of about 8.

[0249] 79. A method according to Embodiment 48 or 49, further comprising 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.

[0250] 80. A method according to Embodiment 79, further comprising washing the affinity chromatography material with a high conductivity aqueous solution before washing the affinity chromatography material with a low conductivity aqueous solution.

[0251] 81. A method according to Embodiment 79, further comprising washing the affinity chromatography material with a high conductivity aqueous solution and / or an intermediate conductivity aqueous solution before or after washing the affinity chromatography material with a low conductivity aqueous solution.

[0252] 82. A method according to Embodiment 79, further comprising washing the affinity chromatography material with a high conductivity aqueous solution and / or an intermediate conductivity aqueous solution before washing the affinity chromatography material with a low conductivity aqueous solution.

[0253] 83. A method in any one of embodiments 79 to 82, wherein the high conductivity aqueous solution has a conductivity value of about 20 mS / cm or more.

[0254] 84. A method according to Embodiment 83, wherein the high conductivity aqueous solution has a conductivity value of about 20 mS / cm to about 100 mS / cm.

[0255] 85. A method according to Embodiment 81 or 82, wherein the medium conductivity aqueous solution has a conductivity value greater than 0.5 mS / cm and less than 20 mS / cm.

[0256] 86. A method in any one of embodiments 79 to 85, wherein a high or medium conductivity aqueous solution comprises an amino acid.

[0257] 87. A method according to Embodiment 86, wherein a high or medium conductivity aqueous solution contains histidine.

[0258] 88. A method according to Embodiment 86 or 87, wherein a high or medium conductivity aqueous solution comprises histidine and Tris.

[0259] 89. A method according to Embodiment 48 or 49, wherein one or more additional chromatography methods / steps are performed after step e).

[0260] 90. In Embodiment 89, an additional ion exchange chromatography method / step is performed after step e).

[0261] 91. In Embodiment 90, an additional anion exchange chromatography method / step is performed after step e).

[0262] 92. In Embodiment 90, an additional cation exchange chromatography method / step is performed after step e).

[0263] 93. In Embodiment 90, an additional anion exchange chromatography method / step and an additional cation exchange chromatography method / step are performed after step e).

[0264] 94. A method of Embodiment 48 or 49 that does not have a hydrophobic interaction chromatography method / step.

[0265] 95. A method in any one of embodiments 49 to 94, wherein the human IgG4 homozygous 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.

[0266] 96. A method in any one of embodiments 48 or 50 to 94, wherein the human IgG1 homozygous 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.

[0267] 97. A method for purifying human IgG isoform antibodies from a sample, comprising the following steps:

[0268] a) a step of providing a sample containing human IgG isoform antibodies,

[0269] b) a step of purifying a human IgG isoform antibody by an affinity chromatography method / step, comprising washing the affinity chromatography material with a low conductivity aqueous solution.

[0270] 98. In Embodiment 97, the human IgG isoform antibody is a human IgG4 isoform antibody or a human IgG1 isoform antibody.

[0271] 99. In Embodiment 98, the human IgG isoform antibody is a human IgG4 isoform antibody or a human IgG1 isoform antibody having no glycosylated glycosylated sites in its Fab fragment / exactly one glycosylated site (at position Asn 297 (numbered according to Kabat)).

[0272] 100. A method in any one of embodiments 97 to 99, wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less.

[0273] 101. A method according to Embodiment 100, wherein the low conductivity aqueous solution has a conductivity value of about 0.03 μS / cm to about 0.5 mS / cm.

[0274] 102. A method according to Embodiment 101, wherein the low conductivity aqueous solution has a conductivity value of about 0.05 μS / cm to about 0.35 mS / cm.

[0275] 103. In Embodiment 102, the low conductivity aqueous solution is not deionized water.

[0276] 104. A method in any one of embodiments 97 to 104, wherein the affinity chromatography is protein A affinity chromatography or protein G affinity chromatography or single-chain Fv ligand (KappaSelect) affinity chromatography.

[0277] 105. A method according to Embodiment 104, wherein the affinity chromatography is protein A affinity chromatography.

[0278] 106. A method according to Embodiment 105, wherein the protein A affinity chromatography is selected from the group comprising 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; and Toyopearl AF-rProtein A HC-650HF.

[0279] 107. A method in which the content of host cell protein is reduced in any one of embodiments 97 to 106.

[0280] 108. A method in which, in Embodiment 107, the host cell protein is a Chinese hamster ovary (CHO) host cell protein.

[0281] 109. A method in which the host cell protein in Example 108 is phospholipase.

[0282] 110. A method according to Embodiment 109, wherein the host cell protein is phospholipase A, phospholipase B, phospholipase C, or phospholipase D.

[0283] 111. A method according to Example 110 in which the host cell protein is phospholipase B-like 2 (PLBL2).

[0284] 112. A method according to Embodiment 107 in which the host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

[0285] 113. A method in any one of embodiments 97 to 112, wherein the low conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (Tris).

[0286] 114. A method according to Embodiment 113, wherein the low conductivity aqueous solution comprises about 0.1 mM to about 10 mM Tris.

[0287] 115. A method according to Embodiment 114, wherein the low conductivity aqueous solution comprises about 0.1 mM to about 8 mM Tris.

[0288] 116. A method according to Embodiment 115, wherein the low conductivity aqueous solution comprises about 0.5 mM to about 6.5 mM Tris.

[0289] 117. A method according to Embodiment 116, wherein the low conductivity aqueous solution comprises about 2 mM Tris.

[0290] 118. A method in any one of embodiments 113 to 117, wherein the low conductivity aqueous solution contains potassium phosphate.

[0291] 119. A method according to Embodiment 118, wherein the low conductivity aqueous solution comprises about 0.2 mM to about 5 mM potassium phosphate.

[0292] 120. A method according to Embodiment 119, wherein the low conductivity aqueous solution comprises about 0.2 mM to about 2 mM potassium phosphate.

[0293] 121. A method according to Embodiment 120, wherein the low conductivity aqueous solution comprises about 0.5 mM potassium phosphate.

[0294] 122. A method in any one of embodiments 97 to 121, wherein the low conductivity aqueous solution has a pH of about 7 or higher.

[0295] 123. A method according to Embodiment 122 in which the low conductivity aqueous solution has a pH of about 7.5 or higher.

[0296] 124. A method according to Embodiment 123, wherein the low conductivity aqueous solution has a pH of about 7 to about 9.5.

[0297] 125. A method according to Embodiment 124, wherein the low conductivity aqueous solution has a pH of about 7.5 to about 8.5.

[0298] 126. A method according to Embodiment 125 in which the low conductivity aqueous solution has a pH of about 8.

[0299] 127. A method in any one of embodiments 97 to 126, further comprising 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.

[0300] 128. A method according to Embodiment 127, further comprising washing the affinity chromatography material with a high conductivity aqueous solution before washing the affinity chromatography material with a low conductivity aqueous solution.

[0301] 129. A method according to Embodiment 127, further comprising washing the affinity chromatography material with a high conductivity aqueous solution and / or an intermediate conductivity aqueous solution before or after washing the affinity chromatography material with a low conductivity aqueous solution.

[0302] 130. A method according to Embodiment 127, further comprising washing the affinity chromatography material with a high conductivity aqueous solution and / or an intermediate conductivity aqueous solution before washing the affinity chromatography material with a low conductivity aqueous solution.

[0303] 131. A method in any one of embodiments 127 to 130, wherein the high conductivity aqueous solution has a conductivity value of about 20 mS / cm or more.

[0304] 132. A method according to Embodiment 131, wherein the high conductivity aqueous solution has a conductivity value of about 20 mS / cm to about 100 mS / cm.

[0305] 133. A method according to Embodiment 129 or 130, wherein the medium conductivity aqueous solution has a conductivity value greater than 0.5 mS / cm and less than 20 mS / cm.

[0306] 134. A method in any one of embodiments 127 to 133, wherein a high or medium conductivity aqueous solution comprises an amino acid.

[0307] 135. A method according to Embodiment 134 in which a high or medium conductivity aqueous solution contains histidine.

[0308] 136. A method according to Embodiment 134, wherein a high or medium conductivity aqueous solution comprises histidine and Tris.

[0309] 137. A method according to Embodiment 97 or 98, wherein one or more additional chromatography methods / steps are performed after step b).

[0310] 138. In Embodiment 137, an additional ion exchange chromatography method / step is performed after step b).

[0311] 139. In Embodiment 138, an additional anion exchange chromatography method / step is performed after step b).

[0312] 140. In Embodiment 138, an additional cation exchange chromatography method / step is performed after step b).

[0313] 141. In Embodiment 138, an additional anion exchange chromatography method / step and an additional cation exchange chromatography method / step are performed after step b).

[0314] 142. A method in any one of embodiments 97 to 141 that does not have a hydrophobic interaction chromatography method / step.

[0315] 143. A method according to Embodiment 97 or 98, wherein the human IgG4 homozygous 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.

[0316] 144. A method according to Embodiment 97 or 98, wherein the human IgG1 homozygous 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.

[0317] 145. Use of a low conductivity aqueous solution in the washing step of protein A chromatography to reduce host cell protein content, wherein protein A chromatography is used to purify human IgG4 or IgG1 isoform antibodies and the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less.

[0318] 146. A method for producing human IgG4 or IgG1 homozygous antibodies comprising the following steps:

[0319] a) a step of culturing cells containing nucleic acids encoding human IgG4 or IgG1 homozygous antibodies,

[0320] b) a step of recovering human IgG4 or IgG1 homozygous antibodies from cells or culture media,

[0321] c) a step of contacting a human IgG4 or IgG1 homozygous antibody with a protein A chromatographic material,

[0322] d) a step of washing the protein A chromatographic material with a low conductivity aqueous solution (wherein the low conductivity aqueous solution has a conductivity value of approximately 0.5 mS / cm or less),

[0323] e) Recover human IgG4 or IgG1 isoform antibodies from protein A chromatographic material, and

[0324] The step of generating human IgG4 or IgG1 isoform antibodies thereby.

[0325] 147. A method for purifying human IgG4 or IgG1 homozygous antibodies from a sample, comprising the following steps:

[0326] a) a step of providing a sample containing human IgG4 or IgG1 homozygous antibodies,

[0327] b) a step of purifying a human IgG4 or IgG1 isoform antibody by a protein A chromatography method / step, comprising washing the protein A chromatography material with a low conductivity aqueous solution (wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS / cm or less).

[0328] 148. In any one of embodiments 5 to 7, the use of the low conductivity aqueous solution being deionized water.

[0329] 149. A method in which the low conductivity aqueous solution of Embodiment 54 is deionized water.

[0330] Listing of sequences

[0331] SEQ ID NO: 01 <vegf>The variable heavy chain domain VH

[0332] SEQ ID NO: 02 <vegf>The variable light chain domain VL

[0333] SEQ ID NO: 03 <ang-2>The variable heavy chain domain VH

[0334] SEQ ID NO: 04 <ang-2>The variable light chain domain VL

[0335] SEQ ID NO: 05 Variable heavy chain domain VH of anti-amyloid beta antibody (IgG1 isoform)

[0336] SEQ ID NO: 06 Variable light chain domain VL of anti-amyloid beta antibody (IgG1 isoform)

[0337] SEQ ID NO: 07 Variable heavy chain domain VH1 of anti-P-selectin antibody

[0338] SEQ ID NO: 08 VH2 variable heavy chain domain of anti-P-selectin antibody

[0339] SEQ ID NO: 09 VH3, the variable heavy chain domain of the anti-P-selectin antibody

[0340] SEQ ID NO: 10 VL1, the variable light chain domain of the anti-P-selectin antibody

[0341] SEQ ID NO: 11 VL2, the variable light chain domain of the anti-P-selectin antibody

[0342] SEQ ID NO: 12 VL3, the variable light chain domain of the anti-P-selectin antibody

[0343] Example 1

[0344] Materials and Methods

[0345] antibodies

[0346] Antibodies against P-Selectin (anti-P-Selectin antibody; inclakumab; IgG4 isoform) (as described in WO 2005 / 100402 or SEQ ID NO: 07–SEQ ID NO: 12); bispecific antibodies against Factor IXa and Factor X (anti-FIXa / X antibody; IgG4 isoform) (as described in WO 2012 / 067176); antibodies against Her2; bispecific antibodies against Ang2 and VEGF-A (anti-Ang2 / VEGF-A antibody; vanucizumab; IgG1 isoform) (as described in WO 2011 / 117329 or SEQ ID NO: 01–SEQ ID NO: 04); The present invention is illustrated using a number of exemplary antibodies comprising antibodies against amyloid beta (anti-amyloid beta antibodies; gantenerumab; IgG1 isoforms) (as described in WO 2003 / 070760 or SEQ ID NO: 05 to SEQ ID NO: 06). A number of IgG1 antibodies and IgG4 antibodies as described in the following examples are also included herein.

[0347] Detection method for whole host cell protein (HCP), phospholipase B-like protein 2 (PLBL2), and clusterin

[0348] a) CHO HCP test

[0349] The residual CHO HCP content in the sample is measured by electrochemiluminescence immunoassay (ECLIA) on a cobas e 411 immunoassay analyzer (Roche Diagnostics).

[0350] The assay is based on the sandwich principle using polyclonal anti-CHO HCP antibodies from sheep.

[0351] First incubation: Chinese hamster ovary host cell protein (CHO HCP) and biotin-conjugated polyclonal CHO HCP-specific antibody from 15 μL of sample (neat and / or diluted) form a sandwich complex, which binds to streptavidin-coated microparticles through the interaction of biotin and streptavidin.

[0352] Second incubation: After adding a polyclonal CHO HCP-specific antibody labeled with a ruthenium complex (tris(2,2'-bipyridyl)ruthenium(II)-complex), a ternary sandwich complex is formed on the microparticle.

[0353] The reaction mixture is drawn into a measurement cell where microparticles are magnetically captured on the electrode surface. Unbound material is removed in a subsequent washing step. The application of voltage to the electrode induces chemiluminescence radiation, which is measured by a photomultiplier tube.

[0354] The concentration of CHO HCP in the test sample is finally calculated from the standard curve of known concentrations of CHO HCP.

[0355] b) CHO PLBL2 test

[0356] The residual Chinese hamster ovary (CHO) phospholipase B-like protein 2 (PLBL2) content in the sample is measured by electrochemiluminescence immunoassay (ECLIA) on a cobas e 411 immunoassay analyzer (Roche Diagnostics).

[0357] The assay is based on the sandwich principle using a monoclonal anti-CHO PLBL2 antibody from mice.

[0358] In the first incubation step, CHO PLBL2 from 30 μL of sample (pure 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.

[0359] In the second step after the addition of streptavidin-coated microparticles, the ternary complex is bound to the solid phase through the interaction of biotin and streptavidin.

[0360] The reaction mixture is drawn into a measurement cell where microparticles are magnetically captured on the electrode surface. Unbound material is removed in a subsequent washing step. The application of voltage to the electrode induces chemiluminescence, which is measured by a photomultiplier tube.

[0361] The concentration of CHO PLBL2 in the test sample is finally calculated from the standard curve of CHO PLBL2 at known concentrations.

[0362] c) Clusterin test

[0363] The residual clusterin content in the sample is measured by a commercially available test (GyroMark HT Kit GYRCLU-37K) from Merck Millipore, used according to the manufacturer's instructions.

[0364] Briefly, this test is a sandwich ELISA based on sequential execution:

[0365] 1) Rat clusterin biotinylated capture antibody was conjugated to a streptavidin-coated affinity column of Bioaffy 1000nL CD,

[0366] 2) Capture rat clusterin molecules from the sample by anti-clusterin antibodies,

[0367] 3) Binding a second dye-labeled anticlusterin detection antibody to the captured molecule,

[0368] 4) Quantified rat clusterin using the Gyrolab Evaluator.

[0369] Example 2

[0370] Purification of anti-P-selectin antibody (IgG4 isoform) by protein A chromatography

[0371] Antibody: Anti-P-Selectin

[0372] General chromatography conditions

[0373] Column Resin: Protein A Substance "Mab Select SuRe" (GE-Healthcare) Ø 1 cm, H: 20.1 cm, CV: 15.79 ml

[0374] Equipment: Akta Avant 150

[0375] Flow rate: 300 cm / hour during all stages

[0376] A solution containing the anti-P-selectin antibody was applied to the protein A affinity column after column equilibration (Step 1). Initial loading of PLBL2 measured in the solution containing the anti-P-selectin antibody: 335 ng PLBL2 / mg (antibody). Initial loading of clusterin measured in the solution containing the anti-P-selectin antibody: 2874.8 ng clusterin / mg (antibody). Initial loading of CHOP measured in the solution containing the anti-P-selectin antibody: 100971 ng CHOP / mg (antibody).

[0377] The chromatography step was performed according to the following general schematic:

[0378] Step 1: Equilibration

[0379] Step 2: Loading of antibody-containing solution

[0380] Step 3: Wash I

[0381] Step 4: Wash II

[0382] Step 5: Wash III

[0383] Step 6: Wash IV (Additional Wash)

[0384] Step 7: Yongli

[0385] After elution from the protein A affinity column, the protein was measured by size exclusion chromatography (SEC) and spectrophotometric (OD) analysis.

[0386] SEC:

[0387] Suzy: TSK 3000 (Tosoh)

[0388] Column: 300 x 7.8 mm

[0389] Flow rate: 0.5 ml / min

[0390] Buffer: 200 mM potassium phosphate containing 250 mM potassium chloride adjusted to pH 7.0

[0391] Wavelength: 280 nm

[0392] OD:

[0393] Specific coefficient: 1.54

[0394] Wavelength: 280 nm - 320 nm

[0395] Specific buffer conditions for protein A chromatography (anti-P-selectin Antibody)

[0396] a) Control group (washed only with equilibrating buffer)

[0397] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0398] Step 2: Loading

[0399] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0400] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0401] Step 5: Washing III: ---

[0402] Step 6: Wash IV : ---

[0403] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0404] b) Low conductivity washing (Tris buffer only)

[0405] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0406] Step 2: Loading

[0407] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0408] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0409] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0410] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0411] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0412] c) Low conductivity washing (with potassium phosphate (KP) only)

[0413] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0414] Step 2: Loading

[0415] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0416] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0417] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0418] Step 6: Wash IV: 0.5 mM potassium phosphate, pH 8.0

[0419] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0420] d) High conductivity washing (Tris buffer only)

[0421] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0422] Step 2: Loading

[0423] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0424] Step 4: Wash II: 700 mM Tris, pH 7.2

[0425] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0426] Step 6: Wash IV: ---

[0427] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0428] e) Low conductivity wash (Tris buffer only; pH ​​6.0)

[0429] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0430] Step 2: Loading

[0431] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0432] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0433] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0434] Step 6: Wash IV: 2 mM Tris, pH 6.0

[0435] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0436] f) High conductivity washing (histidine (His) / Tris buffer only)

[0437] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0438] Step 2: Loading

[0439] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0440] Step 4: Wash II: 200 mM His / 1000 mM Tris, pH 7.0

[0441] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0442] Step 6: Wash IV: ---

[0443] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0444] g) Low conductivity Tris + High conductivity histidine (His) / Tris

[0445] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0446] Step 2: Loading

[0447] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0448] Step 4: Wash II: 200 mM His / 1000 mM Tris, pH 7.0

[0449] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0450] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0451] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0452] h) Low conductivity potassium phosphate (KP) + high conductivity histidine (His) / Tris

[0453] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0454] Step 2: Loading

[0455] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0456] Step 4: Wash II: 200 mM His / 1000 mM Tris, pH 7.0

[0457] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0458] Step 6: Wash IV: 0.5 mM potassium phosphate, pH 8.0

[0459] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0460] i) Low conductivity Tris + High conductivity Tris

[0461] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0462] Step 2: Loading

[0463] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0464] Step 4: Wash II: 700 mM Tris, pH 7.2

[0465] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0466] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0467] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0468] j) Low conductivity Tris; pH 6.0 + high conductivity Tris

[0469] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0470] Step 2: Loading

[0471] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0472] Step 4: Wash II: 700 mM Tris, pH 7.2

[0473] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0474] Step 6: Wash IV: 2 mM Tris, pH 6.0

[0475] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0476] result:

[0477]

[0478] Example 3

[0479] Purification of anti-amyloid beta antibodies (IgG1 isoforms) by protein A chromatography

[0480] The general conditions followed the conditions described in Example 2.

[0481] Antibody: Anti-amyloid beta.

[0482] Initial loading of PLBL2 measured in solution containing anti-amyloid beta antibody: 2019.7 ng PLBL2 / mg (antibody). Initial loading of CHOP measured in solution containing anti-amyloid beta antibody: 578908 ng CHOP / mg (antibody).

[0483] Specific buffer conditions for protein A chromatography

[0484] a) Control group (washed only with equilibrating buffer)

[0485] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0486] Step 2: Loading

[0487] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0488] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0489] Step 5: Washing III: ---

[0490] Step 6: Wash IV: ---

[0491] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0492] b) Low conductivity washing (Tris buffer only)

[0493] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0494] Step 2: Loading

[0495] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0496] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0497] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0498] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0499] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0500] c) High conductivity washing (Tris buffer only)

[0501] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0502] Step 2: Loading

[0503] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0504] Step 4: Wash II: 700 mM Tris, pH 7.2

[0505] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0506] Step 6: Wash IV: ---

[0507] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0508] d) Low conductivity Tris + High conductivity histidine (His) / Tris

[0509] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0510] Step 2: Loading

[0511] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0512] Step 4: Wash II: 200 mM His / 1000 mM Tris, pH 7.0

[0513] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0514] Step 6: Wash IV : 2 mM Tris, pH 8.0

[0515] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0516]

[0517] Example 4

[0518] Purification of anti-Her2 antibody (IgG1 isoform) by protein A chromatography

[0519] The general conditions followed the conditions described in Example 2.

[0520] Antibody: Anti-Her2

[0521] Initial loading of PLBL2 measured in solution containing anti-Her2 antibody: 1662.5 ng PLBL2 / mg (antibody). Initial loading of CHOP measured in solution containing anti-Her2 antibody: 727070 ng CHOP / mg (antibody).

[0522] Specific buffer conditions for protein A chromatography

[0523] a) Control group (washed only with equilibrating buffer)

[0524] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0525] Step 2: Loading

[0526] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0527] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0528] Step 5: Washing III: ---

[0529] Step 6: Wash IV: ---

[0530] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0531] b) Low conductivity washing (Tris buffer only)

[0532] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0533] Step 2: Loading

[0534] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0535] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0536] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0537] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0538] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0539] c) High conductivity washing (Tris buffer only)

[0540] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0541] Step 2: Loading

[0542] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0543] Step 4: Wash II: 700 mM Tris, pH 7.2

[0544] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0545] Step 6: Wash IV: ---

[0546] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0547] d) Low conductivity Tris + High conductivity histidine (His) / Tris

[0548] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0549] Step 2: Loading

[0550] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0551] Step 4: Wash II: 200 mM His / 1000 mM Tris, pH 7.0

[0552] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0553] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0554] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0555]

[0556] Example 5

[0557] Purification of bispecific anti-Ang2 / VEGF-A antibody (IgG1 isoform) by Protein A chromatography

[0558] The general conditions followed the conditions described in Example 2.

[0559] Antibody: Anti-Ang2 / VEGF-A

[0560] Initial loading of PLBL2 measured in solution containing bispecific anti-Ang2 / VEGF-A antibody: 919.7 ng PLBL2 / mg (antibody). Initial loading of CHOP measured in solution containing anti-Ang2 / VEGF-A: 682304 ng CHOP / mg (antibody).

[0561] Specific buffer conditions for protein A chromatography

[0562] a) Control group (washed only with equilibrating buffer)

[0563] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0564] Step 2: Loading

[0565] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0566] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0567] Step 5: Washing III: ---

[0568] Step 6: Wash IV: ---

[0569] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0570] b) Low conductivity washing (Tris buffer only)

[0571] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0572] Step 2: Loading

[0573] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0574] Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0

[0575] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0576] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0577] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0578] c) High conductivity washing (Tris buffer only)

[0579] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0580] Step 2: Loading

[0581] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0582] Step 4: Wash II: 700 mM Tris, pH 7.2

[0583] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0584] Step 6: Wash IV: ---

[0585] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0586] d) Low conductivity Tris + High conductivity histidine (His) / Tris

[0587] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0588] Step 2: Loading

[0589] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0590] Step 4: Wash II: 200 mM His / 1000 mM Tris, pH 7.0

[0591] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0592] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0593] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0594]

[0595] Example 6

[0596] Purification of bispecific anti-FIXa / X antibody (IgG4 isoform) by Protein A chromatography

[0597] The purification of the anti-FIXa / X antibody was tested in the following two different chromatography settings.

[0598] Setting 1

[0599] The general conditions followed the conditions described in Example 2.

[0600] Antibody: Anti-FIXa / X

[0601] Initial loading of PLBL2 measured in solution containing anti-FIXa / X antibody: 557 ng PLBL2 / mg (antibody). Initial loading of CHOP measured in solution containing anti-FIXa / X: 387377 ng CHOP / mg (antibody).

[0602] Specific buffer conditions for protein A chromatography

[0603] a) High conductivity washing (Tris buffer only)

[0604] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0605] Step 2: Loading

[0606] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0607] Step 4: Wash II: 700 mM Tris, pH 7.2

[0608] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0609] Step 6: Wash IV: ---

[0610] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0611] b) Low conductivity Tris + High conductivity Histidine (His) / Tris

[0612] Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0

[0613] Step 2: Loading

[0614] Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0

[0615] Step 4: Wash II: 200 mM His / 1000 mM Tris, pH 7.0

[0616] Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

[0617] Step 6: Wash IV: 2 mM Tris, pH 8.0

[0618] Step 7: Elution: 50 mM acetic acid, pH 4.0

[0619]

[0620] Settings 2

[0621] General chromatography conditions

[0622] Column Resin: Protein A Substance "Mab Select SuRe" (GE-Healthcare) Ø 1 cm, H: 20.1 cm, CV: 15.79 ml

[0623] Equipment: Akta Avant 150

[0624] Flow rate: 300 cm / hour during all stages

[0625] A solution containing anti-FIXa / X antibody was applied to the protein A affinity column after column equilibration (step 1).

[0626] Initial loading of PLBL2 measured in solution containing anti-FIXa / X antibody: 557 ng PLBL2 / mg (antibody).

[0627] The chromatography step was performed according to the following general schematic:

[0628] Step 1: Equilibration:

[0629] Step 2: Loading of antibody-containing solution

[0630] Step 3: Wash I

[0631] Step 4: Wash II

[0632] Step 5: Wash III (Additional Wash)

[0633] Step 6: Yungri

[0634] Specific buffer conditions for protein A chromatography

[0635] a) High conductivity washing (with NaSO4 buffer only)

[0636] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0637] Step 2: Loading

[0638] Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

[0639] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0640] Step 5: Wash III: ---

[0641] Step 6: Elution: 35 mM acetic acid, pH 4.0

[0642] b) Low conductivity wash (Tris 1 mM) + high conductivity wash (with NaSO4)

[0643] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0644] Step 2: Loading

[0645] Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

[0646] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0647] Step 5: Wash III: 1 mM Tris, pH 8.0

[0648] Step 6: Elution: 50 mM acetic acid, pH 4.0

[0649] c) Low conductivity wash (Tris 2 mM) + high conductivity wash (with NaSO4)

[0650] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0651] Step 2: Loading

[0652] Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

[0653] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0654] Step 5: Wash III: 2 mM Tris, pH 8.0

[0655] Step 6: Elution: 35 mM acetic acid, pH 4.0

[0656] d) Low conductivity wash (Tris 4 mM) + high conductivity wash (with NaSO4)

[0657] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0658] Step 2: Loading

[0659] Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

[0660] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0661] Step 5: Wash III: 4 mM Tris, pH 8.0

[0662] Step 6: Elution: 50 mM acetic acid, pH 4.0

[0663] e) Low conductivity wash (Tris 6 mM) + high conductivity wash (with NaSO4)

[0664] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0665] Step 2: Loading

[0666] Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

[0667] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0668] Step 5: Wash III: 6 mM Tris, pH 8.0

[0669] Step 6: Elution: 50 mM acetic acid, pH 4.0

[0670] f) Low conductivity wash (Tris 4 mM, pH 7.8) + High conductivity wash (with NaSO4)

[0671] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0672] Step 2: Loading

[0673] Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

[0674] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0675] Step 5: Wash III: 4 mM Tris, pH 7.8

[0676] Step 6: Elution: 50 mM acetic acid, pH 4.0

[0677] g) Low conductivity wash (Tris 4 mM, pH 8.2) + high conductivity wash (with NaSO4)

[0678] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0679] Step 2: Loading

[0680] Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

[0681] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0682] Step 5: Wash III: 4 mM Tris, pH 8.2

[0683] Step 6: Elution: 50 mM acetic acid, pH 4.0

[0684] h) Low conductivity wash (Tris 2 mM) + high conductivity wash (with histidine (His) / Tris 1 M)

[0685] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0686] Step 2: Loading

[0687] Step 3: Wash I: 200 mM His / 1000 mM Tris, pH 7.0

[0688] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0689] Step 5: Wash III: 2 mM Tris, pH 8.0

[0690] Step 6: Elution: 35 mM acetic acid, pH 4.0

[0691] i) Low conductivity wash (Tris 2 mM) + high conductivity wash (histidine (His) / Tris 0.85 M)

[0692] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0693] Step 2: Loading

[0694] Step 3: Wash I: 200 mM His / 850 mM Tris, pH 7.0

[0695] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0696] Step 5: Wash III: 2 mM Tris, pH 8.0

[0697] Step 6: Elution: 50 mM acetic acid, pH 4.0

[0698] j) Low conductivity wash (Tris 2 mM) + high conductivity wash (histidine (His) / Tris 0.7 M)

[0699] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0700] Step 2: Loading

[0701] Step 3: Wash I: 200 mM His / 700 mM Tris, pH 7.0

[0702] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0703] Step 5: Wash III: 2 mM Tris, pH 8.0

[0704] Step 6: Elution: 50 mM acetic acid, pH 4.0

[0705] k) Low conductivity wash (Tris 2 mM) + high conductivity wash (histidine (His) / Tris 0.55 M)

[0706] Step 1: Equilibration: 20 mM NaPO4, pH 7.5

[0707] Step 2: Loading

[0708] Step 3: Wash I: 200 mM His / 550 mM Tris, pH 7.0

[0709] Step 4: Wash II: 20 mM NaPO4, pH 7.5

[0710] Step 5: Wash III: 2 mM Tris, pH 8.0

[0711] Step 6: Elution: 50 mM acetic acid, pH 4.0

[0712]

[0713] Example 7

[0714] General Procedures / Conditions:

[0715] Mock cell culture medium

[0716] Null harvested cell cultures were produced using non-transfected CHO-DP12 cells cultured in serum-free medium. Fermentation was performed at a 2 L scale using a representative cell culture method. At the end of day 14 of fermentation, the cell cultures were harvested by centrifugation and sterile filtration. These harvested cell cultures (HCCF) were stored at -70°C until subsequent experiments.

[0717] Purified PLBL2

[0718] As previously described, recombinant CHO PLBL2 with a C-terminal hexahistidine-tag was expressed in a 35 L-scale transient transfection and purified from the harvested cell culture (Vanderlaan et al., 2015). Then, the purified PLBL2 was formulated in PBS solution and stored at -70°C until the experiment.

[0719] purified antibodies

[0720] 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. Before starting each study, each antibody was buffer-exchanged with PBS using a PD-10 desalting column (GE Healthcare).

[0721] Preparation of loading materials for protein A chromatography

[0722] To normalize the population and abundance of host cell proteins at protein A loading across the entire antibody, the purified antibody was diluted to the same concentration with PBS and spiked into HCCF from a non-producing cell line to provide a final antibody titer of 5 g / L. A control with PBS added instead of the purified antibody was also prepared to evaluate non-specific host cell proteins binding to the protein A resin in the absence of the antibody.

[0723] Packed-bed column chromatography

[0724] All packed-bed column chromatography experiments were performed using a 0.66 cm inner diameter, 20 cm layer height MabSelect SuRe (GE Healthcare) Protein A resin column. For each purification, the column was first equilibrated to 3 column volumes (CV) with 25 mM tris, 25 mM NaCl, pH 7.7 (equilibration buffer). Then, a Protein A loading was applied at a target loading density of 30 g antibody / L resin, followed by washing the column with 3 CV of equilibration buffer, 3 CV of a different type of wash buffer, and 3 CV of equilibration buffer again. Subsequently, the antibody was eluted with 0.1 to 0.15 M acetic acid at a low pH, and the eluate pool was collected starting at 0.5 OD at the beginning of the elution peak; pooling was terminated after 2.8 CV. For the control run on PBS-spiked null HCCF, a 2.8 CV mock elution pool was generated starting at 1 CV after the start of the elution phase and up to 3.8 CV. At the end of each run, each Protein A eluent was titrated to pH 5.0 using 1.5 M tris base. The column was then washed with 0.1 M sodium hydroxide solution. Except for the loading, first equilibration wash, and the elution phase (which had a flow rate of 15 CV / hour), all phases had a volumetric flow rate of 20 CV / hour.

[0725] A) Exemplary antibody (IgG4 isomorphism) in Protein A chromatography, purification of antibody A

[0726] Specific wash buffer conditions for the purification of antibody A (IgG4 isoform) using the general procedure of Example 7 (as schematically shown above):

[0727] a) 0.4 M potassium phosphate, pH 7.0

[0728] b) 25mM Tris, 25mM NaCl, pH 7.7

[0729] c) 0.75 M Arg-HCl, pH 7.0

[0730] d) 0.6 M NaCl, pH 7.0

[0731] e) Deionized water

[0732] result:

[0733]

[0734] B) Purification of exemplary antibody (IgG1 isomorphism) and antibody B in protein A chromatography

[0735] Specific wash buffer conditions for the purification of antibody B (IgG1 isomorphic) using the general procedure of Example 7:

[0736] a) Deionized water

[0737] result:

[0738]

[0739] C) Purification of exemplary antibody (IgG4 isomorphism) and antibody C in protein A chromatography

[0740] Specific wash buffer conditions for the purification of antibody C (IgG4 isomorphic) using the general procedure of Example 7:

[0741] a) 0.4 M potassium phosphate, pH 7.0

[0742] b) 25mM Tris, 25mM NaCl, pH 7.7

[0743] c) 0.75 M Arg-HCl, pH 7.0

[0744] d) 0.6 M NaCl, pH 7.0

[0745] e) Deionized water

[0746] result:

[0747]

[0748] D) Purification of exemplary antibody (IgG1 isomorphism) and antibody D in protein A chromatography

[0749] Specific wash buffer conditions for the purification of antibody D (IgG1 isomorphic) using the general procedure of Example 7:

[0750] a) Deionized water

[0751]

[0752] E) Purification of exemplary antibody (IgG1 isomorphism) and antibody E in protein A chromatography

[0753] Specific wash buffer conditions for the purification of antibody E (IgG1 isomorphic) using the general procedure of Example 7:

[0754] a) 0.4 M potassium phosphate, pH 7.0

[0755] b) 31 mM Tris, pH 8.5

[0756] c) 55 mM Tris, pH 9.0

[0757] d) Deionized water

[0758] result:

[0759]

[0760] F) Purification of exemplary antibody (IgG1 isomorphic) and antibody F in protein A chromatography

[0761] Specific wash buffer conditions for the purification of antibody F using the general procedure of Example 7:

[0762] a) 25 mM Tris, pH 9.0

[0763] result:

[0764] < / vegf> < / vegf>

Claims

Claim 1 A method for generating a human IgG1 isoform antibody comprising the following steps: a) culturing a cell containing a nucleic acid encoding a human IgG1 isoform antibody; b) recovering the human IgG1 isoform antibody from the cell or culture medium; c) contacting the human IgG1 isoform antibody with a protein A chromatographic material; d) washing the protein A chromatographic material with a low conductivity aqueous solution to reduce the amount of host cell protein (wherein the low conductivity aqueous solution has a conductivity value of 0.5 mS / cm or less), wherein the host cell protein is phospholipase B-like 2 (PLBL2) and the low conductivity aqueous solution is deionized water or has a pH of 7 or higher; e) recovering the human IgG1 isoform antibody from the protein A chromatographic material and thereby generating the human IgG1 isoform antibody. Claim 2 A method for purifying a human IgG1 isoform antibody from a sample, comprising the following steps: a) providing a sample containing a human IgG1 isoform antibody; b) purifying the human IgG1 isoform antibody by a protein A chromatography method to reduce the amount of host cell protein, comprising washing the protein A chromatography material with a low conductivity aqueous solution (wherein the low conductivity aqueous solution has a conductivity value of 0.5 mS / cm or less), wherein the host cell protein is phospholipase B-like 2 (PLBL2), and the low conductivity aqueous solution is deionized water or has a pH of 7 or higher. Claim 3 A method according to claim 1 or 2, wherein the low conductivity aqueous solution comprises 0.1 mM to 8 mM Tris. Claim 4 A method according to claim 1 or 2, wherein the low conductivity aqueous solution comprises 0.05 mM to 2 mM potassium phosphate. Claim 5 A method according to claim 1 or 2, wherein the low conductivity aqueous solution has a pH of 7 or higher. Claim 6 A method according to claim 1 or 2, further comprising washing the affinity chromatography material with one or more of a high conductivity aqueous solution and an intermediate conductivity aqueous solution before or after washing the protein A chromatography material with a low conductivity aqueous solution. Claim 7 In claim 6, a method in which a high-conductivity aqueous solution has a conductivity value of 20 mS / cm or more. Claim 8 In claim 6, a method wherein an aqueous solution of medium conductivity has a conductivity value greater than 0.5 mS / cm and less than 20 mS / cm. Claim 9 In claim 6, a method in which a high conductivity aqueous solution or a medium conductivity aqueous solution contains histidine. Claim 10 A method according to claim 1 or 2, wherein the human IgG1 homozygous 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. Claim 11 A method according to claim 1 or 2, wherein the human IgG1 homozygous 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. Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete

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