Methods for purification of polypeptides using polysorbates

Abstract

To provide a method for purifying a protein from a sample comprising the protein and at least one impurity with hydrolytic activity.SOLUTION: The method comprises the following steps of: i) a) applying a sample comprising protein and at least one impurity with hydrolytic activity to a chromatography material, b) applying a solution which comprises a polysorbate to the chromatography material, and c) recovering the protein from the chromatography material; or ii) a) adding a polysorbate to the sample comprising the protein and at least one impurity with hydrolytic activity, b) applying the mixture of the step a) to a chromatography material, and c) recovering the protein from the chromatography material.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention generally relates to the field of polypeptide purification or protein purification.The present invention particularly relates to reducing hydrolytic activity in sample.This paper reports a method for purifying polypeptide or protein, by contacting the sample containing polypeptide with polysorbate (e.g. polysorbate 20 or polysorbate 80) or polysorbate-like detergent before and / or during purification step, such as chromatography step. [Background technology]

[0002] 2. Background of the Invention Surface-active agents or surfactants have become essential components of robust and stable biopharmaceutical formulations to prevent destabilization of active pharmaceutical ingredients (APIs). To this end, the majority of currently marketed biotherapeutic drugs, such as monoclonal antibodies (mABs), contain non-ionic detergents / surfactants, especially polysorbate 20 (PS20 or Tween® 20) or polysorbate 80 (PS80 or Tween® 80) [1]. It is known that polysorbates improve protein stability by minimizing the contact area of ​​APIs with surfaces [2-5] and air-liquid interfaces [5-7], as well as by disrupting protein-protein interactions [8, 9]. These advantageous attributes result in protecting APIs from aggregation, denaturation, and reduction in effective protein concentration.

[0003] Polysorbates may contain several impurities and degradation products that are present in neat materials and arise in biopharmaceutical formulations during quiescent storage, as they are susceptible to oxidation and hydrolysis leading to the formation of various polysorbate degradation products [1, 17, 18]. The oxidative pathway is mainly characterized by the presence of peroxides, aldehydes, ketones and short-chain esterified sorbitan / isosorbide-PEG species, whereas hydrolysis leads to increased levels of free fatty acids (FFAs) and non-esterified sorbitan / isosorbide-PEG species. In addition, it has been reported that the appearance of visible and subvisible particles in mAB formulations after prolonged storage is attributed to the accumulation of FFAs due to their low solubility in aqueous solutions [18, 19]. The loss of molecular integrity has been associated with a reduction in API stability by lowering the concentration of functional surfactants that provide a protective effect and by directly affecting the intact conformational structure [17, 20].

[0004] Polysorbate hydrolysis can occur enzymatically or by acid / base catalyzed mechanisms

[21] ; however, the latter is negligible under typical formulation conditions. In contrast, recent studies have revealed that it is difficult to remove CHO host cell protein (HCP) impurities that are likely involved in polysorbate hydrolysis in biopharmaceutical formulations. It has been reported that the presence of putative phospholipase B-like 2 (PLBL2) and lipoprotein lipase (LPL) or other types of lipases and esterases may be responsible for polysorbate degradation [22-24].

[0005] Recent findings have established that lipases associated with biopharmaceutical manufacturing are expressed in upstream processes. In general, HCPs can be removed in downstream purification processes (e.g., Protein A), but it has been shown that some HCPs can be difficult to remove due to specific interactions with antibodies (Vanderlaan, M., et al., Bioproc Int. 2015, 13:18-29), and therefore remain in trace amounts in drug substances and drug products (K.Lee, et al., A Chinese Hamster Ovary Cell Host Cell Protein That Impacts PS-80 Degradation.AccBio Conference(2015)). Therefore, it is important for storage of biopharmaceutical products to prevent and minimize the degradation of polysorbates and the formation of particles formed, for example, by the accumulation of free fatty acids. In particular, the enzymatic degradation of polysorbates needs to be addressed.

[0006] To address this challenge, there are ongoing efforts to identify lipases and remove them from protein drugs, for example by engineering cells with reduced lipase expression (WO2015 / 095568) and to find formulations with reduced polysorbate degradation (WO2017 / 117311). However, there remains a need to inhibit / reduce the enzymatic degradation of surfactants such as polysorbates, which remains a key challenge in biopharmaceutical development.

[0007] WO2016 / 057739 presents a process for reducing sub-visible particles in pharmaceutical formulations. Summary of the Invention

[0008] This document reports a method for purifying a protein or polypeptide (e.g., an antibody) using polysorbate in a purification step, e.g., in a chromatography step, to reduce hydrolytic activity in a sample or preparation. Also reported are methods for producing a protein or polypeptide and methods for producing a protein or polypeptide preparation.

[0009] Also reported herein is a method for purifying a protein or polypeptide (e.g., an antibody), using polysorbate in a purification step, e.g., in a chromatography step, to inhibit or reduce particle formation in pharmaceutical formulations.Also reported herein is a method for purifying a protein or polypeptide (e.g., an antibody), using polysorbate in a purification step, e.g., in a chromatography step, to inhibit or reduce polysorbate degradation in pharmaceutical formulations.

[0010] It has been found that the method and use of the present invention can significantly reduce hydrolytic activity in a sample. The sample containing the protein to be purified and impurities with hydrolytic activity is contacted or mixed with a non-ionic detergent such as polysorbate before loading the sample onto a chromatographic material, or while the sample is being purified in a chromatographic step, or both, before and during the chromatographic step. In all of these situations, hydrolytic activity, such as that which leads to polysorbate degradation, can be reduced.

[0011] It has been found that hydrolytic activity (especially with regard to polysorbate hydrolysis) is significantly reduced in samples that have been contacted or mixed with polysorbate during the purification procedure, for example compared to samples purified by chromatographic steps where no polysorbate is used before and / or during the chromatographic steps. Thus, it has been found that polysorbate degradation (by hydrolysis) in purified samples (e.g. antibody preparations / formulations) can be reduced by implementing a step in the preceding purification procedure that actually uses polysorbate as an additive. Thus, without being bound by theory, the reduction of impurities such as enzymes with hydrolytic activity, such as esterases, is performed / achieved in / during the purification procedure by adding a substrate or target of the impurity (which can later be subject to hydrolytic cleavage, for example during storage).

[0012] Thus, one aspect reported herein is a method for purifying a protein (from a sample containing the protein and at least one impurity with hydrolytic activity), comprising the steps of: i) a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a solution comprising polysorbate to the chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering the protein from the chromatographic material; or iii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material.

[0013] One aspect reported herein is a method for reducing hydrolytic activity in a sample containing a protein and at least one impurity with hydrolytic activity, the method comprising the steps of: i) a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a solution comprising polysorbate to the chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering the protein from the chromatographic material; or iii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material.

[0014] In a further embodiment of all aspects, the impurity is a hydrolase.

[0015] In a further embodiment of all aspects, the impurity is an esterase.

[0016] In a further embodiment of all aspects, the polysorbate is selected from the group comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, or polysorbate 80.

[0017] In a further embodiment of all aspects, the polysorbate is polysorbate 20 or polysorbate 80.

[0018] In a further embodiment of all aspects, the polysorbate is polysorbate 20.

[0019] In a further embodiment of all aspects, the polysorbate is polysorbate 80.

[0020] In a further embodiment of all aspects, the polysorbate is added to a final concentration of at least about 0.001% (w / v).

[0021] In a further embodiment of all aspects, polysorbate is added to a final concentration of about 0.001% (w / v) to about 10% (w / v).

[0022] In a further embodiment of all aspects, the chromatographic material is an affinity chromatographic material, a cation exchange chromatographic material, an anion exchange chromatographic material, a hydrophobic interaction chromatographic material, or a mixed mode chromatographic material.

[0023] In a further embodiment of all aspects, the chromatographic material is an affinity chromatographic material or a cation exchange chromatographic material.

[0024] In a further embodiment of all aspects, the chromatographic material is an affinity chromatographic material or a strong cation exchange chromatographic material.

[0025] In a further embodiment of all aspects the chromatography material is an affinity chromatography material selected from Protein A chromatography or a chromatography material in which a camelid-derived single domain antibody fragment is the ligand.

[0026] In a further embodiment of all aspects, the affinity chromatography material is CaptureSelect FcXL or MabSelect SuRe.

[0027] In a further embodiment of all aspects the cation exchange chromatography material is SP Sepharose Fast Flow or Poros XS or Poros 50 HS.

[0028] In a further embodiment of all aspects, the protein is a monoclonal antibody.

[0029] In a further embodiment of all aspects, the protein is a humanized type II anti-CD20 antibody or an anti-VEGF / Ang2 antibody or an anti-HER2 antibody.

[0030] One aspect reported herein is a liquid antibody preparation with reduced hydrolytic activity (improved polysorbate degradation stability / reduced polysorbate degradation) obtainable by the method reported herein.

[0031] One aspect reported herein is a liquid antibody preparation with improved stability obtained by the method reported herein.

[0032] One aspect reported herein is a liquid antibody preparation with reduced particle formation (due to free fatty acid aggregation) obtainable by the method reported herein.

[0033] One aspect reported herein is a liquid composition comprising an antibody and a polysorbate, wherein no more than 20% of the polysorbate is degraded per year during storage of the liquid composition.

[0034] In one embodiment, the antibody is a humanized type II anti-CD20 antibody or an anti-VEGF / Ang2 antibody or an anti-HER2 antibody. In one embodiment, the antibody is obinutuzumab, faricimab, trastuzumab or pertuzumab. [The present invention 1001] A method for purifying a protein from a sample containing said protein and at least one impurity with hydrolytic activity, comprising the steps of: i) a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a solution comprising polysorbate to the chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering the protein from the chromatographic material; or iii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material. [The present invention 1002] 1. A method for reducing hydrolytic activity in a sample containing a protein and at least one impurity having hydrolytic activity, comprising the steps of: i) a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a solution comprising polysorbate to the chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering the protein from the chromatographic material; or iii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein the impurity is a hydrolase. [The present invention 1004] The method according to any one of claims 1001 to 1003, wherein the impurity is an esterase. [The present invention 1005] The method according to any one of claims 1001 to 1004, wherein the polysorbate is selected from the group comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, or polysorbate 80. [The present invention 1006] The method according to any one of claims 1001 to 1005, wherein the polysorbate is polysorbate 20 or polysorbate 80. [The present invention 1007] The method of any of claims 1001 to 1006, wherein the polysorbate is added to a final concentration of at least about 0.001% (w / v). [The present invention 1008] The method according to any one of claims 1001 to 1007, wherein the polysorbate is added to give a final concentration of about 0.001% (w / v) to about 10% (w / v). [The present invention 1009] The method according to any one of claims 1001 to 1008, wherein said chromatography material is an affinity chromatography material, a cation exchange chromatography material, an anion exchange chromatography material, a hydrophobic interaction chromatography material or a mixed mode chromatography material. [The present invention 1010] The method according to any one of claims 1001 to 1009, wherein said chromatography material is an affinity chromatography material or a cation exchange chromatography material. [The present invention 1011] The method according to any one of claims 1001 to 1010, wherein said chromatography material is an affinity chromatography material selected from Protein A chromatography or a chromatography material having a camelid-derived single domain antibody fragment as a ligand. [The present invention 1012] The method according to any one of claims 1001 to 1011, wherein the affinity chromatography material is CaptureSelect FcXL or MabSelect SuRe. [The present invention 1013] The method according to any one of claims 1001 to 1010, wherein said cation exchange chromatography material is SP Sepharose Fast Flow or Poros 50HS or Poros XS. [The present invention 1014] The method according to any one of claims 1001 to 1013, wherein the protein is a monoclonal antibody. [The present invention 1015] The method according to any one of claims 1001 to 1014, wherein said protein is a humanized type II anti-CD20 antibody, an anti-VEGF / Ang2 antibody, or an anti-HER2 antibody. [The present invention 1016] A liquid antibody preparation having reduced hydrolytic activity obtainable by any of the methods of the invention. [The present invention 1017] 1. A method for producing a protein, comprising: a) culturing a mammalian cell containing a nucleic acid encoding the protein; b) recovering the protein from the cells or culture medium, and c) purifying the protein according to any one of the methods of the present inventions 1001 to 1015. thereby producing said protein. [The present invention 1018] A liquid composition comprising an antibody and a polysorbate, wherein the polysorbate is degraded by no more than 20% per year during storage of the liquid composition. [The present invention 1019] The liquid composition of the present invention 1018, wherein said antibody is a humanized type II anti-CD20 antibody, or an anti-VEGF / Ang2 antibody, or an anti-HER2 antibody. [The present invention 1020] The liquid composition of any one of claims 1018 to 1019, wherein the antibody is obinutuzumab, faricimab, trastuzumab, or pertuzumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description of the Invention Reported herein is a method for purifying proteins by contacting a sample containing the protein (and impurities with hydrolytic activity) with a polysorbate, such as polysorbate 20 (PS20), before and / or during the chromatographic step.

[0036] It has been found that proteins can be purified from samples by using polysorbates as additives in purification steps, such as chromatography steps, and in particular by reducing impurities with hydrolytic activity, such as hydrolases (e.g. lipases).

[0037] Thus, one aspect reported herein is a method for purifying a protein from a sample containing the protein and at least one impurity with hydrolytic activity, the method comprising the steps of: i) a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material (under conditions suitable for the protein to bind to the chromatographic material); b) applying a (washing) solution comprising a polysorbate / polysorbate-like non-ionic detergent to the chromatographic material (under conditions suitable for the protein to bind to the chromatographic material); and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the sample of step a) comprising the protein and at least one impurity with hydrolytic activity, and a solution comprising polysorbate) to a chromatographic material (under conditions suitable for the protein to bind to the chromatographic material); and c) recovering the protein from the chromatographic material; or iii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the sample of step a) comprising the protein and at least one impurity with hydrolytic activity, and a solution comprising polysorbate) to a chromatographic material (under conditions suitable for the protein to bind to the chromatographic material), c) applying a (washing) solution comprising polysorbate to the chromatographic material (under conditions suitable for the protein to bind to the chromatographic material); and d) recovering the protein from the chromatographic material.

[0038] One aspect reported herein is a method for purifying a protein from a sample containing the protein and at least one impurity with hydrolytic activity, the method comprising the steps of: i) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the solution of step a) comprising the sample containing the protein and at least one impurity with hydrolytic activity, and polysorbate) to a chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the solution of step a) comprising the sample containing the protein and at least one impurity with hydrolytic activity, and polysorbate) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material.

[0039] One aspect reported herein is a method for purifying a protein from a sample containing the protein and at least one impurity with hydrolytic activity, the method comprising the steps of: i) a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a (washing) solution comprising polysorbate to the chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the solution of step a) comprising the sample containing the protein and at least one impurity with hydrolytic activity, and polysorbate) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material.

[0040] One aspect reported herein is a method for purifying a protein from a sample containing the protein and at least one impurity with hydrolytic activity, the method comprising the steps of: a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a (washing) solution comprising polysorbate to the chromatographic material; and c) recovering said protein from said chromatographic material.

[0041] One aspect reported herein is a method for purifying a protein from a sample containing the protein and at least one impurity with hydrolytic activity, the method comprising the steps of: a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the solution of step a) comprising the sample containing the protein and at least one impurity with hydrolytic activity, and polysorbate) to a chromatographic material; and c) recovering said protein from said chromatographic material.

[0042] One aspect reported herein is a method for purifying a protein from a sample containing the protein and at least one impurity with hydrolytic activity, the method comprising the steps of: a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the solution of step a) comprising the sample containing the protein and at least one impurity with hydrolytic activity, and polysorbate) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material.

[0043] It has been found that by adding impurity substrates or targets (which may subsequently undergo hydrolytic cleavage during storage) in / during the purification procedure, the hydrolytic activity in the sample can be reduced. The hydrolytic activity can have activity against polysorbates. The reduced hydrolytic activity increases the stability of polysorbates.

[0044] One aspect reported herein is a method for reducing hydrolytic activity (e.g., towards polysorbate) in a sample containing protein and at least one impurity with hydrolytic activity (improving the degradation stability of polysorbate), comprising the steps of: i) a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a (washing) solution comprising polysorbate to the chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the solution of step a) comprising the sample containing the protein and at least one impurity with hydrolytic activity, and polysorbate) to a chromatographic material; and c) recovering the protein from the chromatographic material; or iii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) (i.e. the solution of step a) comprising the sample containing the protein and at least one impurity with hydrolytic activity, and polysorbate) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material.

[0045] Hydrolytic activity may be due to hydrolytic enzymes, which can be reduced by the methods reported herein.

[0046] One aspect reported herein is a method for reducing the amount of hydrolytic enzymes in a sample containing proteins and hydrolytic enzymes, the method comprising the steps of: a) adding polysorbate to the sample comprising proteins and hydrolytic enzymes prior to applying the sample comprising proteins and hydrolytic enzymes to the chromatographic material, and / or b) applying the sample containing proteins and hydrolytic enzymes to the chromatographic material followed by washing with a solution containing polysorbate.

[0047] It has been found that the methods reported herein allow the production of recombinant proteins, such as antibodies, with, for example, improved storage stability or reduced hydrolytic activity.

[0048] One aspect reported herein is a method for producing a (recombinant) protein, comprising the following steps: a) culturing mammalian cells containing a nucleic acid encoding a (recombinant) protein; b) recovering the (recombinant) protein from the cells or the culture medium, and c) purifying the (recombinant) protein by the methods reported herein thereby producing a (recombinant) protein.

[0049] One aspect reported herein is a method for producing an antibody, comprising the steps of: a) culturing mammalian cells containing nucleic acid encoding the antibody; b) recovering the antibody from the cells or culture medium, and c) purifying the antibody by the method reported herein thereby producing an antibody.

[0050] It is understood that samples containing proteins can vary from very small volumes to very large volumes, and can expressly include preparative samples in large scale manufacturing of biotherapeutics.

[0051] Using the methods reported herein, protein or antibody preparations can also be prepared that have reduced hydrolytic activity and therefore improved stability.

[0052] The practitioners have found that the methods and uses reported herein make it possible to produce protein or antibody preparations with reduced hydrolytic activity and therefore with higher degradation stability. As polysorbates are themselves added to liquid antibody preparations to improve the stability of proteins in the preparation or formulation, the methods and uses reported herein are likewise suitable for improving the stability of liquid antibody preparations / formulations.

[0053] One aspect reported herein is a liquid antibody preparation (containing hydrolytically cleavable surfactants / polysorbates) with reduced hydrolytic activity (improved polysorbate degradation stability) obtainable by the methods reported herein.

[0054] One aspect reported herein is a liquid antibody preparation with improved stability obtained by the method reported herein.

[0055] Since particle formation in protein or antibody preparations can be promoted by the aggregation of free fatty acids that can arise by the degradation or cleavage of polysorbates, reducing hydrolytic activity with the methods reported herein can reduce particle formation in these preparations.

[0056] For example, it could be shown that a liquid antibody preparation comprising Pertuzumab formulated with polysorbate, after incubation with polysorbate, application to a cation exchange chromatography material and recovery, did not show any visible particles after 36 months of storage at 5°C.

[0057] Thus, one aspect reported herein is a liquid antibody preparation with reduced particle formation (formed by free fatty acids) obtainable by the method reported herein.

[0058] A further aspect reported herein is a liquid antibody composition with low polysorbate degradation during storage. One aspect of the present invention is a liquid composition comprising an antibody / protein and a polysorbate, wherein the polysorbate degrades 20% or less per year during the storage / shelf life of the liquid composition (in one embodiment 15% or less, in one embodiment 12% or less, in one embodiment 10% or less, in one embodiment 9% or less, in one embodiment 8% or less, in one embodiment 7% or less, in one embodiment 6% or less, in one embodiment 5% or less, in one embodiment 4% or less, in one embodiment 3% or less, in one embodiment 2% or less, in one embodiment 1% or less). In one embodiment, the polysorbate degrades 10% or less per year during storage of the liquid composition.

[0059] Another aspect is a liquid composition comprising an antibody and a polysorbate, wherein after one year, the polysorbate is present in the composition at a concentration of at least 80% (and in one embodiment at least 85%, and in one embodiment at least 88%, and in one embodiment at least 90%, and in one embodiment at least 91%, and in one embodiment at least 92%, and in one embodiment at least 93%, and in one embodiment at least 94%, and in one embodiment at least 95%, and in one embodiment at least 96%, and in one embodiment at least 97%, and in one embodiment at least 98%, and in one embodiment at least 99%) of the initial concentration, which is the concentration at the time the antibody was formulated or at the start of storage in the liquid composition.

[0060] In one embodiment of all aspects of the liquid composition of the present invention, the antibody is a therapeutic antibody. In one embodiment, the antibody is a therapeutic antibody of IgG1 isotype. In one embodiment, the antibody is a humanized type II anti-CD20 antibody or an anti-VEGF / Ang2 antibody or an anti-HER2 antibody. In one embodiment, the antibody specifically binds to VEGF. In one embodiment, the antibody is obinutuzumab, faricimab, trastuzumab, or pertuzumab. In one embodiment, the antibody is obinutuzumab. In one embodiment, the antibody is faricimab. In one embodiment, the antibody is trastuzumab. In one embodiment, the antibody is pertuzumab. In one embodiment, the antibody is formulated for subcutaneous injection. In one embodiment, the antibody concentration in the liquid composition is at least 50 mg / ml. In one embodiment, the antibody concentration in the liquid composition is at least 80 mg / ml. In one embodiment, the antibody concentration in the liquid composition is at least 100 mg / ml. In one embodiment, the antibody concentration in the liquid composition is at least 120 mg / ml. In one embodiment, the antibody concentration in the liquid composition is at least 150 mg / ml. In one embodiment, the antibody concentration in the liquid composition is at least 200 mg / ml.

[0061] It has been found that the use of polysorbate prior to or in one or more purification steps can reduce hydrolytic activity in a sample.

[0062] One aspect reported herein is the use of polysorbates to reduce hydrolytic activity in a sample containing proteins (improving the degradation stability of the polysorbates), Here, the sample containing the protein is i) polysorbate is added to the protein-containing sample before application to the chromatographic material; and / or ii) A sample containing proteins is applied to a chromatographic material which is then washed with a solution containing polysorbate.

[0063] Below, embodiments of the aspects reported herein are set forth, with the understanding that this list encompasses any combination of the individual embodiments.

[0064] Impurities arise during pharmaceutical manufacturing processes, inter alia, due to the release of host cell proteins from the host cells used, such as Chinese Hamster Ovary (CHO) cells. These HCPs may have hydrolytic activity. Exemplary HCPs belong to the enzyme class of hydrolases, including lipases, such as lipoprotein lipase (LPL), which hydrolyzes ester bonds in triglycerides.

[0065] A "hydrolytically active impurity" or "hydrolytically active impurity" (used interchangeably herein) has the ability to hydrolyze a chemical bond, i.e., to break a chemical bond by hydrolysis, i.e., by the addition of water. An example of acid-base hydrolysis is the hydrolysis of an amide or ester. Hydrolysis occurs when a nucleophile attacks the carbon of the carbonyl group of an ester or amide. Hydrolysis can be catalyzed by enzymes such as proteases or esterases.

[0066] In one embodiment, the impurity (with hydrolytic activity) is an enzyme. In a further embodiment, the impurity is a hydrolase. In a further embodiment, the impurity is an esterase. In a further embodiment, the impurity is a lipase.

[0067] "Polysorbates" are substances derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids (i.e., polyoxyethylene sorbitan esters). Polysorbates are a type of emulsifier (also known as surfactants), and polysorbates are non-ionic surfactants. Polysorbates are surfactants that can be cleaved by hydrolysis. Examples of polysorbates are, for example, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 65 (polyoxyethylene (20) sorbitan tristearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). The number 20 following "polyoxyethylene" refers to the total number of oxyethylene -(CH2CHO)- groups found in the molecule. The number following "polysorbate" refers to the type of fatty acid attached to the polyoxyethylene sorbitan portion of the molecule. Monolaurate is designated 20, monopalmitate 40, monostearate 60, and monooleate 80. Common trade names for polysorbates include Scattics, Alkest, Canarcel, and Tween; for example, Tween 20 is the trade name for polysorbate 20. "Polysorbate-like nonionic surfactants" are surfactants that exhibit chemical characteristics or behavior similar to those of polysorbates.

[0068] In a further embodiment, the polysorbate is selected from the group comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 or polysorbate 120, or a combination thereof. In a further embodiment, the polysorbate is selected from the group comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, or a combination thereof. In a further embodiment, the polysorbate is selected from the group comprising polysorbate 20 or polysorbate 80, or a combination thereof. In a further embodiment, the polysorbate is polysorbate 20. In a further embodiment, the polysorbate is polysorbate 80.

[0069] In a further embodiment, polysorbate is added to a final concentration of at least about 0.001% (w / v) and / or the washing solution containing polysorbate has a final concentration of at least about 0.001% (w / v). In a further embodiment, polysorbate is added to a final concentration of at least about 0.005% (w / v) and / or the washing solution containing polysorbate has a final concentration of at least about 0.005% (w / v). In a further embodiment, polysorbate is added to a final concentration of at least about 0.007% (w / v) and / or the washing solution containing polysorbate has a final concentration of at least about 0.007% (w / v). In a further embodiment, polysorbate is added to a final concentration of at least about 0.01% (w / v) and / or the washing solution containing polysorbate has a final concentration of at least about 0.01% (w / v). In a further embodiment, polysorbate is added to a final concentration of at least about 0.04% (w / v) and / or the wash solution containing polysorbate has a final concentration of at least about 0.04% (w / v). In a further embodiment, polysorbate is added to a final concentration of about 0.001% (w / v) to about 10% (w / v) and / or the wash solution containing polysorbate has a final concentration of at least about 0.001% (w / v) to about 10% (w / v). In a further embodiment, polysorbate is added to a final concentration of about 0.001% (w / v) to about 5% (w / v) and / or the wash solution containing polysorbate has a final concentration of at least about 0.001% (w / v) to about 5% (w / v). In a further embodiment, polysorbate is added to a final concentration of about 0.001% (w / v) to about 3% (w / v) and / or the wash solution containing polysorbate has a final concentration of at least about 0.001% (w / v) to about 3% (w / v).In a further embodiment, polysorbate is added to a final concentration of about 0.001% (w / v) to about 2% (w / v) and / or the wash solution containing polysorbate has a final concentration of at least about 0.001% (w / v) to about 2% (w / v).In a further embodiment, polysorbate is added to a final concentration of about 0.001% (w / v) to about 1% (w / v), and / or the washing solution containing polysorbate has a final concentration of at least about 0.001% (w / v) to about 1% (w / v). It is understood that after adding polysorbate to a sample containing a protein / polypeptide and at least one impurity with hydrolytic activity, the mixture is incubated for a certain period of time. The same can be done when using a polysorbate-containing solution in the washing step, for example by washing at a low flow rate or by stopping the flow for a certain period of time.

[0070] Common chromatographic techniques and their uses are known to those of skill in the art. See, for example, Heftmann, E. (ed.), Chromatography, 5 thFor reference, see edition, Part A: Fundamentals and Techniques, Elsevier Science Publishing Company, New York (1992), Deyl, Z. (ed.), Advanced Chromatography and Electromigration Methods in Biosciences, Elsevier Science BV, Amsterdam, The Netherlands (1998), Poole, C.F., and Poole, S.K., Chromatography Today, Elsevier Science Publishing Company, New York (1991), Scopes, Protein Purification: Principles and Practice, Springer Verlag (1982), Sambrook, J., et al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) or Ausubel, F.M., et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1987 - 1994).

[0071] A variety of methods are established and widely used for protein recovery and purification, including affinity chromatography with microbial proteins (e.g., Protein A or Protein G or Protein L affinity chromatography), affinity chromatography with recombinant proteins as ligands (e.g., single-chain Fvs as ligands, e.g., Kappa select), affinity chromatography with camelid-derived single domain antibody fragments as ligands (e.g., Capture Select FcXL), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin), and mixed-mode exchange), thiophilic adsorption (e.g., with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., Ni(II)- and Cu(II)-affinity materials), size exclusion chromatography, and electrophoretic methods (e.g., gel electrophoresis, capillary electrophoresis). These methods can be combined independently in various embodiments reported herein.

[0072] The term "applying to" refers to a partial step of the purification method in which the solution is contacted with a chromatographic material or a material for chromatography (these terms can be used interchangeably). This refers either to a) adding the solution to a chromatographic device containing the chromatographic material or to b) adding the chromatographic material to the solution. In the case of a), the solution passes through the device, allowing an interaction between the chromatographic material and the substances contained in the solution. Depending on the conditions, such as pH, conductivity, salt concentration, temperature and / or flow rate, some substances of the solution will bind to the chromatographic material and can be recovered from the chromatographic material in a further step. Substances that remain dissolved can be found in the flow-through fraction. The "flow-through fraction" refers to the solution obtained after passing through the device, which can be either the applied solution or the buffer solution used to wash the column or to cause elution of substances bound to the chromatographic material.

[0073] The skilled artisan will understand how to recover proteins from a given chromatographic material, for example this can be by applying a solution with a low pH value or an increased salt concentration, such that the protein is no longer bound by the chromatographic material and is found in this solution (e.g. the elution fraction obtained by applying an elution buffer).

[0074] The device can be, for example, a column or a cassette. In case b), the chromatographic material can be added, for example as a solid, to a solution containing, for example, the substance of interest to be purified, allowing an interaction between the chromatographic material and the substances in the solution. After the interaction, the chromatographic material is removed, for example by filtration, and with it the substances bound to the chromatographic material are also removed from the solution, while the substances not bound to the chromatographic material remain dissolved. If a (washing) solution containing polysorbate is implemented as a step of the procedure, the conditions of the chromatographic step are chosen such that the protein of interest remains bound to the chromatographic material.

[0075] As used herein, a "material" for chromatography, e.g., a cation exchange material or affinity material, refers to a stationary or solid phase. It can also be a resin or a matrix. A "material" in this context provides a matrix to which a component of a mixture, e.g., a protein of interest, can bind. The material can be or include a column, e.g., an expanded bed column or a packed bed column. The material can be in the form of discrete particles or discrete beads. The material can be in the form of a membrane. The material can be in the form of a porous monolithic material. The material can be in the form of a functionalized fiber, a functionalized fleece or a functionalized mesh. The material can be in the form of any other solid support capable of carrying functional groups or exhibiting chromatographic properties. A ligand capable of interacting with the protein to be purified can be covalently attached to the solid phase. For example, a "material for protein A chromatography" refers to an inert solid phase to which protein A is covalently attached.

[0076] In a further embodiment, the chromatographic material is an affinity chromatographic material, a cation exchange chromatographic material, an anion exchange chromatographic material, a hydrophobic interaction chromatographic material or a mixed mode chromatographic material. In a further embodiment, the chromatographic material is an affinity chromatographic material, a cation exchange chromatographic material or an anion exchange chromatographic material. In a further embodiment, the chromatographic material is an affinity chromatographic material or a cation exchange chromatographic material. In a further embodiment, the chromatographic material is an affinity chromatographic material or a strong cation exchange chromatographic material.

[0077] In a further embodiment, the chromatographic material is an affinity chromatographic material. In a further embodiment, the chromatographic material is an affinity chromatographic material selected from the group comprising Protein A or Protein G or Protein L affinity chromatography, single chain Fv ligand affinity chromatography, metal chelate affinity chromatography, or affinity chromatographic material with a camelid-derived single domain antibody fragment as a ligand (FcXL). In a further embodiment, the chromatographic material is an affinity chromatographic material selected from Protein A chromatography or a chromatography material with a camelid-derived single domain antibody fragment as a ligand. In a further embodiment, the affinity chromatography material is a material having a matrix of aldehyde-activated agarose and a CaptureSelect FcXL affinity ligand, which is a single domain antibody fragment of camelid origin, that specifically binds to the CH3 domain of all human IgG subclasses, where the ligand is coupled to the matrix by aldehyde coupling via the NH2 residue of the ligand (CaptureSelect™ FcXL), or a material having a matrix of cross-linked agarose and an alkali-stabilized Protein A-derived ligand (MabSelect™ SuRe™). In a further embodiment, the affinity chromatography material is a material with a matrix of aldehyde-activated agarose, an average particle size of 65 μm and a CaptureSelect™ FcXL affinity ligand, which is a single domain antibody fragment of camelid origin that specifically binds to the CH3 domain of all human IgG subclasses, where the ligand is coupled to the matrix by aldehyde coupling via the NH2 residue of the ligand (CaptureSelect™ FcXL), or a material with a matrix of cross-linked agarose, an alkali-stabilized Protein A-derived ligand and an average particle size of 85 μm (MabSelect™ SuRe™).In a further embodiment, the affinity chromatography material is CaptureSelect™ FcXL or MabSelect™ SuRe™.

[0078] In a further embodiment, the chromatographic material is a cation exchange chromatographic material. In a further embodiment, the chromatographic material is a strong cation exchange chromatographic material. A "strong" ion exchanger does not lose the charge on its matrix once the column is equilibrated, and therefore a wide range of pH buffers can be used. In a further embodiment, the cation exchange chromatographic material is a material with a matrix of cross-linked agarose and sulfopropyl as a ligand (e.g., SP Sepharose® Fast Flow). In a further embodiment, the cation exchange chromatographic material is a material with a matrix of cross-linked agarose (6%) with a particle size of 45-165 μm (average 90 μm) and sulfopropyl as a ligand (SP Sepharose® Fast Flow). In a further embodiment, the cation exchange chromatographic material is SP Sepharose® Fast Flow. In a further embodiment, the cation exchange chromatographic material is a material with a matrix of cross-linked poly(styrene divinylbenzene) and sulfopropyl as a ligand (e.g., Poros XS or Poros 50 HS). In a further embodiment, the cation exchange chromatography material is Poros XS or Poros 50 HS. In a further embodiment, the cation exchange chromatography material is Poros 50 HS.

[0079] The terms "protein" and "polypeptide" refer to a polymer of amino acid residues, without any restriction regarding the minimum length of the polymer. Preferably, a protein or polypeptide has at least amino acids. This definition includes full-length proteins and fragments thereof, as well as modifications thereof (e.g., glycosylation, phosphorylation, deletions, additions and substitutions). Preferably, the protein or polypeptide is an antibody.

[0080] The term "antibody" is used in the broadest sense herein and includes various antibody structures, such as, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fusion proteins and antibody fragments, as long as they exhibit the desired antigen-binding activity. This term also includes molecules such as fusion proteins in which additional effector moieties are bound to the antibody. Also includes antibody drug conjugates (ADCs).

[0081] The term "effector moiety" as used herein refers to a polypeptide, e.g., a protein or glycoprotein, that affects the activity of a cell, e.g., via a signal transduction pathway or other cellular pathway. Thus, the effector moieties of the present invention may be involved in receptor-mediated signaling, which transmits a signal from outside the cell membrane to modulate a response in a cell bearing one or more receptors for the effector moiety. The effector moiety may also induce a cytotoxic response in a cell bearing one or more receptors for the effector moiety. The effector moiety may also induce a proliferative response in a cell bearing one or more receptors for the effector moiety. The effector moiety may induce differentiation in a cell bearing a receptor for the effector moiety. The effector moiety may also alter (i.e., upregulate or downregulate) the expression of an endogenous cellular protein in a cell bearing a receptor for the effector moiety. Non-limiting examples of effector moieties include cytokines, growth factors, hormones, enzymes, substrates and cofactors.

[0082] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2 These include, but are not limited to, diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments (e.g., multispecific Fab). Fab fragments are antibody fragments obtained by papain digestion of a (full length / complete) antibody.

[0083] A "multispecific antibody" is an antibody that has two or more different antigen-binding specificities. A "bispecific antibody" is an antibody that has two different antigen-binding specificities. As used herein, the term "bispecific" antibody refers to an antibody that has at least two binding sites, each of which binds to a different epitope.

[0084] Various anti-HER2 antibodies are known in the prior art. Such antibodies are preferably monoclonal antibodies. They can be either so-called chimeric antibodies, humanized antibodies or fully human antibodies. They can be either full-length anti-HER2 antibodies, anti-HER2 antibody fragments with the same biological activity, including amino acid sequence variants and / or glycosylation variants of such antibodies or fragments. Examples of humanized anti-HER2 antibodies are known under the INN names trastuzumab and pertuzumab. Another suitable anti-HER2 antibody is T-DM1, an antibody-toxin conjugate consisting of huMAb4D5-8 (Herceptin™) and maytansinoid (i.e. DM1=N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine; a highly potent anti-microtubule agent), which conjugate (with MCC linker) is currently being developed for metastatic breast cancer.Tagliabue et al., Int.J.Cancer, 47:933-937(1991);McKenzie et al., Oncogene, 4:543-548(1989);Cancer Res., 51:5361-5369(1991);Bacus et al., Molecular Carcinogenesis, 3:350-362(1990);Stancovski et al., PNAS(USA), 88:8691-8695(1991);Bacus et al., Cancer Research, 52:2580-2589(1992);Xu et al., Int.J.Cancer, 53:401-408(1993);WO94 / 00136;Kasprzyk et al., Cancer Research, 52:2771-2776(1992); Hancock et al., Cancer Res., 51:4575-4580(1991); Shawver et al., Cancer Res., 54:1367-1373(1994); Arteaga et al., Cancer Res., 54:3758-3765(1994); Harwerth et al., J.Biol.Chem., 267:15160-15167(1992); U.S. Patent No. 5,783,186; and Klapper et al., Oncogene, 14:2099-2109(1997) describe other HER2 antibodies with different properties. The most successful therapeutic anti-HER2 antibody is trastuzumab, sold under the trade name Herceptin™ by Genentech Inc. and F. Hoffmann-La Roche Ltd. Further details regarding the HER2 antigen and antibodies thereto are found in many patent and non-patent literature (for a suitable overview see U.S. Pat. No. 5,821,337 and WO 2006 / 044908).

[0085] The terms "trastuzumab", "pertuzumab" and "T-DM1" encompass all corresponding anti-HER2 antibodies that meet the requirements necessary to obtain marketing authorization as an identical or biosimilar product in a country or region selected from the group of countries consisting of the United States, Europe and Japan. Trastuzumab has the CDR regions defined in EP-B-590058. Pertuzumab has the CDR regions defined in WO 01 / 00245. The activity of trastuzumab in the BT-474 antiproliferative assay [Nahta, R.et al., "The HER-2-targeting antibodies trastuzumab and pertuzumab synergistically inhibit the survival of breast cancer cells", Cancer Res.2004;64:2343.2346] ranges from 0.7 to 1.3 × 10 4 U / mg. T-DM1 is described in WO 2005 / 117986.

[0086] Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 as described in Table 3 of U.S. Pat. No. 5,821,337; i.e., trastuzumab (HERCEPTIN™); humanized 520C9 (WO 93 / 21319) and humanized 2C4 antibodies, such as pertuzumab, as further described herein below.

[0087] As used herein, "trastuzumab," "HERCEPTIN™," and "huMAb4D5-8" refer to anti-HER2 antibodies directed against the 4D5 epitope. Such antibodies preferably comprise light and heavy chain amino acid sequences as disclosed, for example, in FIG. 14 of WO 2006 / 044908.

[0088] "Epitope 4D5" is the region in the extracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463) and trastuzumab bind. This epitope is proximal to the transmembrane domain of HER2 and is within domain IV of HER2. To screen for antibodies that bind to the 4D5 epitope, a routine cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the region from about residue 529 to about residue 625 (inclusive) of HER2). "Epitope 7C2 / 7F3" is the region in the amino terminus, domain I, of the extracellular domain of HER2 to which the 7C2 and / or 7F3 antibodies bind. To screen for antibodies that bind to the 7C2 / 7F3 epitope, a routine cross-blocking assay, such as that described in "Antibodies, A Laboratory Manual" (Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)), can be performed. Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 7C2 / 7F3 epitope of HER2 (e.g., any one or more residues in the region from about residue 22 to about residue 53 of HER2).

[0089] As used herein, "pertuzumab" and "rhuMAb 2C4" refer to an antibody that binds to the 2C4 epitope and preferably comprises the variable light and variable heavy chain amino acid sequences disclosed in WO 2006 / 044908, more specifically humanized 2C4 version 574 as disclosed in Figure 2 of WO 2006 / 044908.

[0090] "Epitope 2C4" is the region in the extracellular domain of HER2 that antibody 2C4 binds to. To screen for antibodies that bind to the 2C4 epitope, a standard cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be performed to evaluate whether an antibody binds to the 2C4 epitope of HER2. Epitope 2C4 includes residues from domain II in the extracellular domain of HER2. 2C4 and pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II and III (Franklin et al. Cancer Cell 5:317-328 (2004)).

[0091] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein. The term "cell" includes cells used for expression of a nucleic acid. The host cell may be 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. As used herein, the term "cell" includes the subject cell and its progeny.

[0092] In a further embodiment, the protein is a recombinant protein. In a further embodiment, the protein is an antibody. In a further embodiment, the protein is a monoclonal antibody. In a further embodiment, the protein is a multispecific, bispecific or monospecific antibody. In a further embodiment, the protein is a humanized type II anti-CD20 antibody, an anti-VEGF / Ang2 antibody or an anti-HER2 antibody. In a further embodiment, the protein is a humanized type II anti-CD20 antibody. In a further embodiment, the protein is an anti-VEGF / Ang2 antibody. In a further embodiment, the protein is an anti-HER2 antibody. In a further embodiment, the protein is a humanized type II anti-CD20 antibody comprising (a) a heavy chain variable region of SEQ ID NO:01 and (b) a light chain variable region of SEQ ID NO:02. In a further embodiment, the protein is an anti-VEGF / Ang2 antibody comprising SEQ ID NO:3 to SEQ ID NO:6 as variable regions. In a further embodiment, the protein is obinutuzumab, faricimab, trastuzumab, or pertuzumab. In a further embodiment, the protein is obinutuzumab, faricimab, or trastuzumab. In a further embodiment, the protein is obinutuzumab. In a further embodiment, the protein is faricimab. In a further embodiment, the protein is trastuzumab. In a further embodiment, the protein is pertuzumab.

[0093] It has been found that the degree of purification is particularly pronounced if the solution containing the polypeptide / protein is incubated / conditioned with polysorbate / polysorbate-like surfactants prior to chromatography and, in addition, the solution containing the polypeptide / protein is washed with a solution containing a polysorbate-like surfactant during chromatography.

[0094] The term "about" refers to a range of ±20% of the numerical value that follows it. In one embodiment, the term about refers to a range of ±10% of the numerical value that follows it. In one embodiment, the term about refers to a range of ±5% of the numerical value that follows it.

[0095] Procedures and methods for converting an amino acid sequence, e.g., the amino acid sequence of a polypeptide, into a corresponding nucleic acid sequence that encodes that amino acid sequence are well known to those of skill in the art. Thus, a nucleic acid is characterized not only by its nucleic acid sequence, made up of individual nucleotides, but also by the amino acid sequence of the polypeptide that it encodes.

[0096] Methods of making and storing antibody formulations or liquid antibody preparations are known to those of skill in the art. For example, for longer storage periods, liquid antibody preparations may be stored at low temperatures, for example at temperatures below 8° C., for example at 5° C.

[0097] Methods for measuring the amount of polysorbate in a given sample are also known to those skilled in the art. The amount of polysorbate can be evaluated, for example, by the method reported in Example 7. Thus, knowing the starting / initial polysorbate concentration in a sample, those skilled in the art can also determine the rate of decomposition of polysorbate for a given period of time.

[0098] Specific embodiments of the invention 1. A method for purifying a protein / polypeptide from a sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, comprising the steps of: i) a) applying said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity to a chromatographic material, b) applying a (washing) solution comprising a polysorbate / polysorbate-like non-ionic surfactant to the chromatographic material; and c) recovering said protein / polypeptide from said chromatographic material, or ii) a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; and c) recovering said protein / polypeptide from said chromatographic material, or iii) a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering said protein / polypeptide from said chromatographic material.

[0099] 2. A method for purifying a protein / polypeptide from a sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, comprising the steps of: i) a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; and c) recovering said protein / polypeptide from said chromatographic material, or ii) a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering said protein / polypeptide from said chromatographic material.

[0100] 3. A method for purifying a protein / polypeptide from a sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, comprising the steps of: i) a) applying said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity to a chromatographic material, b) applying a (washing) solution comprising polysorbate to the chromatographic material; and c) recovering said protein / polypeptide from said chromatographic material, or ii) a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering said protein / polypeptide from said chromatographic material.

[0101] 4. A method for purifying a protein / polypeptide from a sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, comprising the steps of: a) applying said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity to a chromatographic material, b) applying a (washing) solution comprising polysorbate to the chromatographic material; and c) recovering said protein / polypeptide from said chromatographic material.

[0102] 5. A method for purifying a protein / polypeptide from a sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, comprising the steps of: a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; and c) recovering said protein from said chromatographic material.

[0103] 6. A method for purifying a protein / polypeptide from a sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, comprising the steps of: a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering said protein / polypeptide from said chromatographic material.

[0104] 7. A method for reducing hydrolytic activity (improving the degradation stability of polysorbates) in a sample containing a protein / polypeptide and at least one impurity with hydrolytic activity, comprising the steps of: i) a) applying said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity to a chromatographic material, b) applying a (washing) solution comprising polysorbate to the chromatographic material; and c) recovering said protein / polypeptide from said chromatographic material, or ii) a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; and c) recovering said protein / polypeptide from said chromatographic material, or iii) a) adding polysorbate to said sample containing said protein / polypeptide and at least one impurity with hydrolytic activity, b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering said protein / polypeptide from said chromatographic material.

[0105] 8. The method according to any one of aspects 1 to 7, wherein the impurity (having hydrolytic activity) is an enzyme.

[0106] 9. The method of any one of aspects 1 to 8, wherein the impurity is a hydrolase.

[0107] 10. The method of any one of aspects 1 to 9, wherein the impurity is an esterase.

[0108] 11. The method of any one of aspects 1 to 10, wherein the impurity is a lipase.

[0109] 12. The method of any one of aspects 1 to 11, wherein the polysorbate is selected from the group comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, or polysorbate 120.

[0110] 13. The method of any one of aspects 1 to 12, wherein the polysorbate is selected from the group comprising polysorbate 20 or polysorbate 80.

[0111] 14. The method of any one of aspects 1 to 13, wherein the polysorbate is polysorbate 20.

[0112] 15. The method of any one of aspects 1 to 13, wherein the polysorbate is polysorbate 80.

[0113] 16. The method of any one of aspects 1 to 15, wherein the polysorbate is added to a final concentration of at least about 0.001% (w / v) and / or the wash solution comprising polysorbate has a final concentration of at least about 0.001% (w / v).

[0114] 17. The method of any one of aspects 1 to 16, wherein the polysorbate is added to a final concentration of at least about 0.005% (w / v) and / or the wash solution comprising polysorbate has a final concentration of at least about 0.005% (w / v).

[0115] 18. The method of any one of aspects 1 to 17, wherein the polysorbate is added to a final concentration of at least about 0.007% (w / v) and / or the wash solution comprising polysorbate has a final concentration of at least about 0.007% (w / v).

[0116] 19. The method of any one of aspects 1 to 18, wherein the polysorbate is added to a final concentration of at least about 0.01% (w / v) and / or the wash solution comprising polysorbate has a final concentration of at least about 0.01% (w / v).

[0117] 20. The method of any one of aspects 1 to 19, wherein the polysorbate is added to a final concentration of at least about 0.04% (w / v) and / or the wash solution comprising polysorbate has a final concentration of at least about 0.04% (w / v).

[0118] 21. The method of any one of aspects 1 to 20, wherein the polysorbate is added to a final concentration of about 0.001% (w / v) to about 10% (w / v) and / or the wash solution containing polysorbate has a final concentration of at least about 0.001% (w / v) to about 10% (w / v).

[0119] 22. The method of any one of aspects 1 to 21, wherein the chromatography material is an affinity chromatography material, a cation exchange chromatography material, an anion exchange chromatography material, a hydrophobic interaction chromatography material, or a mixed-mode chromatography material.

[0120] 23. The method of any one of aspects 1 to 22, wherein the chromatography material is an affinity chromatography material, a cation exchange chromatography material, or an anion exchange chromatography material.

[0121] 24. The method of any one of aspects 1 to 23, wherein the chromatography material is an affinity chromatography material or a cation exchange chromatography material.

[0122] 25. The method of any one of aspects 1 to 24, wherein the chromatography material is an affinity chromatography material.

[0123] 26. The method according to any one of aspects 1 to 25, wherein the chromatography material is an affinity chromatography material selected from the group comprising Protein A or Protein G or Protein L affinity chromatography, single chain Fv ligand affinity chromatography, metal chelate affinity chromatography, or an affinity chromatography material with a camelid-derived single domain antibody fragment as the ligand (FcXL).

[0124] 27. The method of any one of aspects 1 to 26, wherein the chromatography material is an affinity chromatography material selected from Protein A chromatography or a chromatography material in which a camelid-derived single domain antibody fragment is a ligand.

[0125] 28. A method according to any of aspects 1 to 27, wherein the affinity chromatography material is a material having a matrix of aldehyde-activated agarose and a CaptureSelect FcXL affinity ligand, which is a single domain antibody fragment of camelid origin, that specifically binds to the CH3 domain of all human IgG subclasses, which is coupled to the matrix via the NH2 residue of the ligand by aldehyde coupling (CaptureSelect™ FcXL), or a material having a matrix of cross-linked agarose and an alkali-stabilized Protein A-derived ligand (MabSelect™ SuRe™).

[0126] 29. A method according to any of aspects 1 to 28, wherein the affinity chromatography material is a material having a matrix of aldehyde-activated agarose, an average particle size of 65 μm and a CaptureSelect™ FcXL affinity ligand, which is a single domain antibody fragment of camelid origin, that specifically binds to the CH3 domain of all human IgG subclasses, which is coupled to the matrix via the NH2 residue of the ligand by aldehyde coupling (CaptureSelect™ FcXL), or a material having a matrix of cross-linked agarose, an alkali-stabilized Protein A derived ligand and an average particle size of 85 μm (MabSelect™ SuRe™).

[0127] 30. A method according to any one of aspects 1 to 29, wherein the affinity chromatography material is CaptureSelect™ FcXL or MabSelect™ SuRe™.

[0128] 31. The method of any one of aspects 1 to 24, wherein the chromatographic material is a cation exchange chromatographic material.

[0129] 32. A method according to any one of aspects 1 to 24 and 31, wherein the cation exchange chromatography material is a material having a matrix of cross-linked agarose and sulfopropyl as a ligand (SP Sepharose® Fast Flow).

[0130] 33. A method according to any one of aspects 1 to 24 and aspects 31 to 32, wherein the cation exchange chromatography material is a material having a matrix of cross-linked agarose (6%) with a particle size of 45 to 165 μm (average 90 μm) and sulfopropyl as a ligand (SP Sepharose® Fast Flow).

[0131] 34. A method according to any one of aspects 1 to 24 and aspects 31 to 33, wherein the cation exchange chromatography material is SP Sepharose® Fast Flow.

[0132] 35. A method according to any one of aspects 1 to 34, wherein the protein / polypeptide is a recombinant protein / polypeptide.

[0133] 36. A method according to any one of aspects 1 to 35, wherein the protein is an antibody.

[0134] 37. A method according to any one of aspects 1 to 36, wherein the protein / polypeptide is a monoclonal antibody.

[0135] 38. A method according to any one of aspects 1 to 37, wherein the protein / polypeptide is a multispecific antibody, a bispecific antibody, or a monospecific antibody.

[0136] 39. A method according to any one of aspects 1 to 38, wherein the protein / polypeptide is a humanized type II anti-CD20 antibody.

[0137] 40. The protein / polypeptide is (a) the heavy chain variable region of SEQ ID NO:01; (b) the light chain variable region of SEQ ID NO:02; 40. The method according to any one of aspects 1 to 39, wherein the antibody is a humanized type II anti-CD20 antibody comprising the steps of:

[0138] 41. The method of any one of aspects 1 to 40, wherein the protein / polypeptide is obinutuzumab.

[0139] 42. Use of a polysorbate to reduce hydrolytic activity in a sample containing protein (improving the degradation stability of the polysorbate), comprising: The sample containing the protein, i) polysorbate is added to the sample containing the protein before it is applied to a chromatographic material; and / or ii) applying the sample containing the protein to a chromatographic material which is then washed with a solution containing polysorbate; use.

[0140] 43. The use according to embodiment 42, wherein the polysorbate is selected from the group comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, or polysorbate 120.

[0141] 44. The use according to any one of aspects 42 to 43, wherein the polysorbate is selected from the group comprising polysorbate 20 or polysorbate 80.

[0142] 45. The use described in any of aspects 42 to 44, wherein the polysorbate is polysorbate 20.

[0143] 46. ​​The use described in any of aspects 42 to 44, wherein the polysorbate is polysorbate 80.

[0144] 47. The use of any one of aspects 42 to 46, wherein the polysorbate is added to a final concentration of at least about 0.001% (w / v).

[0145] 48. The use according to any one of aspects 42 to 47, wherein the polysorbate is added to a final concentration of at least about 0.005% (w / v).

[0146] 49. The use according to any one of aspects 42 to 48, wherein the polysorbate is added to a final concentration of at least about 0.007% (w / v).

[0147] 50. The use according to any one of aspects 42 to 49, wherein the polysorbate is added to a final concentration of at least about 0.01% (w / v).

[0148] 51. The use according to any one of aspects 42 to 50, wherein the polysorbate is added to a final concentration of at least about 0.04% (w / v).

[0149] 52. The use according to any one of aspects 42 to 51, wherein the polysorbate is added to a final concentration of about 0.001% (w / v) to about 1% (w / v) or about 0.001% (w / v) to about 10% (w / v).

[0150] 53. The use according to any one of aspects 42 to 52, wherein the chromatography material is an affinity chromatography material, a cation exchange chromatography material, an anion exchange chromatography material, a hydrophobic interaction chromatography material, or a mixed-mode chromatography material.

[0151] 54. The use according to any one of aspects 42 to 53, wherein the chromatography material is an affinity chromatography material, a cation exchange chromatography material, or an anion exchange chromatography material.

[0152] 55. The use according to any one of aspects 42 to 54, wherein the chromatography material is an affinity chromatography material or a cation exchange chromatography material.

[0153] 56. The use according to any one of aspects 42 to 55, wherein the chromatography material is an affinity chromatography material.

[0154] 57. The use according to any of aspects 42 to 56, wherein the chromatography material is an affinity chromatography material selected from the group comprising Protein A or Protein G or Protein L affinity chromatography, single chain Fv ligand affinity chromatography, metal chelate affinity chromatography, or an affinity chromatography material with a camelid-derived single domain antibody fragment as the ligand (FcXL).

[0155] 58. The use according to any one of aspects 42 to 57, wherein the chromatography material is an affinity chromatography material selected from Protein A chromatography or a chromatography material in which a camelid-derived single domain antibody fragment is the ligand.

[0156] 59. Use according to any of aspects 42 to 58, wherein the affinity chromatography material is a material having a matrix of aldehyde-activated agarose and a CaptureSelect FcXL affinity ligand, which is a single domain antibody fragment of camelid origin, that specifically binds to the CH3 domain of all human IgG subclasses, which is coupled to the matrix via the NH2 residue of the ligand by aldehyde coupling (CaptureSelect™ FcXL), or a material having a matrix of cross-linked agarose and an alkali-stabilized Protein A-derived ligand (MabSelect™ SuRe™).

[0157] 60. Use according to any of aspects 42 to 59, wherein the affinity chromatography material is a material having a matrix of aldehyde-activated agarose, an average particle size of 65 μm and a CaptureSelect FcXL affinity ligand, which is a single domain antibody fragment of camelid origin, that specifically binds to the CH3 domain of all human IgG subclasses, which ligand is coupled to the matrix by aldehyde coupling via the NH2 residue of the ligand (CaptureSelect™ FcXL), or a material having a matrix of cross-linked agarose, an alkali-stabilized Protein A-derived ligand and an average particle size of 85 μm (MabSelect™ SuRe™).

[0158] 61. The use of any of aspects 42 to 55, wherein the affinity chromatography material is CaptureSelect™ FcXL or MabSelect™ SuRe™.

[0159] 62. The use of any one of aspects 42 to 55, wherein the chromatography material is a cation exchange chromatography material.

[0160] 63. The use according to any one of aspects 42 to 55 and aspect 62, wherein the cation exchange chromatography material is a material having a matrix of cross-linked agarose and sulfopropyl as ligand (SP Sepharose® Fast Flow).

[0161] 64. The use according to any one of aspects 42 to 55 and aspects 62 to 63, wherein the cation exchange chromatography material is a material having a matrix of cross-linked agarose (6%) with a particle size of 45 to 165 μm (average 90 μm) and sulfopropyl as a ligand (SP Sepharose® Fast Flow).

[0162] 65. The use described in any one of aspects 42 to 55 and aspects 62 to 64, wherein the cation exchange chromatography material is SP Sepharose® Fast Flow.

[0163] 66. The use according to any one of aspects 42 to 65, wherein the protein is a recombinant protein.

[0164] 67. The use according to any one of aspects 42 to 66, wherein the protein is an antibody.

[0165] 68. The use of any one of aspects 42 to 67, wherein the protein is a monoclonal antibody.

[0166] 69. The use of any of aspects 42 to 68, wherein the protein is a multispecific antibody, a bispecific antibody, or a monospecific antibody.

[0167] 70. The use according to any one of aspects 42 to 69, wherein the protein is a humanized type II anti-CD20 antibody.

[0168] 71. The protein, (a) the heavy chain variable region of SEQ ID NO:01; (b) the light chain variable region of SEQ ID NO:02; 71. The use according to any of aspects 42 to 70, wherein the antibody is a humanized type II anti-CD20 antibody comprising:

[0169] 72. The use of any one of aspects 42 to 71, wherein the protein is obinutuzumab.

[0170] 73. A liquid antibody preparation (containing polysorbate) having reduced hydrolytic activity (improved degradation stability of polysorbate), obtainable by a method according to any one of aspects 1 to 41 or aspects 77 to 107.

[0171] 74. The liquid antibody preparation of embodiment 73, wherein the antibody is a humanized type II anti-CD20 antibody.

[0172] 75. The antibody, (a) the heavy chain variable region of SEQ ID NO:01; (b) the light chain variable region of SEQ ID NO:02; 75. The liquid antibody preparation according to any of aspects 73-74, which is a humanized type II anti-CD20 antibody comprising:

[0173] 76. The liquid antibody preparation of any of aspects 73-75, wherein the antibody is obinutuzumab.

[0174] 77. A method for reducing the amount of hydrolases in a sample containing proteins and hydrolases, comprising the steps of: a) adding polysorbate to the sample comprising proteins and hydrolytic enzymes before applying said sample comprising proteins and hydrolytic enzymes to a chromatographic material, and / or b) applying said sample containing proteins and hydrolytic enzymes to a chromatographic material followed by washing with a solution containing polysorbate.

[0175] 78. The method of embodiment 77, wherein the polysorbate is selected from the group including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, or polysorbate 120.

[0176] 79. The method of any one of aspects 77 to 78, wherein the polysorbate is selected from the group including polysorbate 20 or polysorbate 80.

[0177] 80. The method of any one of aspects 77 to 79, wherein the polysorbate is polysorbate 20.

[0178] 81. The method of any one of aspects 77 to 79, wherein the polysorbate is polysorbate 80.

[0179] 82. A method according to any one of aspects 77 to 81, wherein the polysorbate is added to a final concentration of at least about 0.001% (w / v) and / or the washing solution comprising polysorbate has a final concentration of at least about 0.001% (w / v).

[0180] 83. A method according to any one of aspects 77 to 82, wherein the polysorbate is added to a final concentration of at least about 0.005% (w / v) and / or the washing solution comprising polysorbate has a final concentration of at least about 0.005% (w / v).

[0181] 84. A method according to any one of aspects 77 to 83, wherein the polysorbate is added to a final concentration of at least about 0.007% (w / v) and / or the washing solution comprising polysorbate has a final concentration of at least about 0.007% (w / v).

[0182] 85. A method according to any one of aspects 77 to 84, wherein the polysorbate is added to a final concentration of at least about 0.01% (w / v) and / or the washing solution comprising polysorbate has a final concentration of at least about 0.01% (w / v).

[0183] 86. A method according to any one of aspects 77 to 85, wherein the polysorbate is added to a final concentration of at least about 0.04% (w / v) and / or the washing solution comprising polysorbate has a final concentration of at least about 0.04% (w / v).

[0184] 87. A method according to any one of aspects 77 to 86, wherein the polysorbate is added to a final concentration of about 0.001% (w / v) to about 1% (w / v) and / or the washing solution containing polysorbate has a final concentration of about 0.001% (w / v) to about 1% (w / v) or about 0.001% (w / v) to about 10% (w / v).

[0185] 88. The method of any one of aspects 77 to 87, wherein the chromatography material is an affinity chromatography material, a cation exchange chromatography material, an anion exchange chromatography material, a hydrophobic interaction chromatography material, or a mixed-mode chromatography material.

[0186] 89. The method of any one of aspects 77 to 88, wherein the chromatography material is an affinity chromatography material, a cation exchange chromatography material, or an anion exchange chromatography material.

[0187] 90. The method of any one of aspects 77 to 89, wherein the chromatography material is an affinity chromatography material or a cation exchange chromatography material.

[0188] 91. The method of any one of aspects 77 to 90, wherein the chromatography material is an affinity chromatography material.

[0189] 92. The method of any of aspects 77 to 91, wherein the chromatography material is an affinity chromatography material selected from the group comprising Protein A or Protein G or Protein L affinity chromatography, single chain Fv ligand affinity chromatography, metal chelate affinity chromatography, or an affinity chromatography material with a camelid-derived single domain antibody fragment as the ligand (FcXL).

[0190] 93. The method of any one of aspects 77 to 92, wherein the chromatography material is an affinity chromatography material selected from Protein A chromatography or a chromatography material in which a camelid-derived single domain antibody fragment is a ligand.

[0191] 94. A method according to any of aspects 77 to 93, wherein the affinity chromatography material is a material having a matrix of aldehyde-activated agarose and a CaptureSelect FcXL affinity ligand (which is a single domain antibody fragment of camelid origin) that specifically binds to the CH3 domain of all human IgG subclasses, the ligand being coupled to the matrix via the NH2 residue of the ligand by aldehyde coupling (CaptureSelect™ FcXL), or a material having a matrix of cross-linked agarose and an alkali-stabilized Protein A-derived ligand (MabSelect™ SuRe™).

[0192] 95. A method according to any of aspects 77 to 94, wherein the affinity chromatography material is a material having a matrix of aldehyde-activated agarose, an average particle size of 65 μm and a CaptureSelect FcXL affinity ligand, which is a single domain antibody fragment of camelid origin, that specifically binds to the CH3 domain of all human IgG subclasses, wherein the ligand is coupled to the matrix via the NH2 residue of the ligand by aldehyde coupling (CaptureSelect™ FcXL), or a material having a matrix of cross-linked agarose, an alkali-stabilized Protein A-derived ligand and an average particle size of 85 μm (MabSelect™ SuRe™).

[0193] 96. A method according to any one of aspects 77 to 95, wherein the affinity chromatography material is CaptureSelect™ FcXL or MabSelect™ SuRe™.

[0194] 97. The method of any one of aspects 77 to 90, wherein the chromatographic material is a cation exchange chromatographic material.

[0195] 98. A method according to any one of aspects 77 to 90 and aspect 97, wherein the cation exchange chromatography material is a material having a matrix of cross-linked agarose and sulfopropyl as a ligand (SP Sepharose® Fast Flow).

[0196] 99. A method according to any one of aspects 77 to 90 and aspects 97 to 98, wherein the cation exchange chromatography material is a material having a matrix of cross-linked agarose (6%) with a particle size of 45 to 165 μm (average 90 μm) and sulfopropyl as a ligand (SP Sepharose (registered trademark) Fast Flow).

[0197] 100. A method according to any one of aspects 77 to 90 and aspects 97 to 99, wherein the cation exchange chromatography material is SP Sepharose® Fast Flow.

[0198] 101. The method of any one of aspects 77 to 100, wherein the protein is a recombinant protein.

[0199] 102. A method according to any one of aspects 77 to 101, wherein the protein is an antibody.

[0200] 103. The method of any one of aspects 77 to 102, wherein the protein is a monoclonal antibody.

[0201] 104. The method of any one of aspects 77 to 103, wherein the protein is a multispecific antibody, a bispecific antibody, or a monospecific antibody.

[0202] 105. The method of any one of aspects 77 to 104, wherein the protein is a humanized type II anti-CD20 antibody.

[0203] 106. The protein, (a) the heavy chain variable region of SEQ ID NO:01; (b) the light chain variable region of SEQ ID NO:02; The method according to any of aspects 77 to 105, wherein the antibody is a humanized type II anti-CD20 antibody comprising:

[0204] 107. The method of any one of aspects 77 to 106, wherein the protein is obinutuzumab.

[0205] 108. (Recombinant) A method for producing a protein comprising the steps of: a) culturing mammalian cells containing a nucleic acid encoding a (recombinant) protein; b) recovering the (recombinant) protein from the cells or culture medium, and c) purifying the (recombinant) protein by a method according to any one of aspects 1 to 41 thereby producing said (recombinant) protein.

[0206] 109. A liquid antibody preparation with improved stability, obtainable by a method according to any one of aspects 1 to 41 or aspects 77 to 107.

[0207] 110. A liquid antibody preparation with reduced particle formation (formed by free fatty acid aggregation), obtainable by a method according to any of aspects 1 to 41 or aspects 77 to 107.

[0208] 111. A method according to any one of aspects 1 to 41 or aspects 77 to 107, wherein the polysorbate is added to a final concentration of about 0.001% (w / v) to about 5% (w / v) and / or the washing solution containing polysorbate has a final concentration of at least about 0.001% (w / v) to about 5% (w / v).

[0209] 112. A method according to any one of aspects 1 to 41 or aspects 77 to 107 or aspect 111, wherein the polysorbate is added to a final concentration of about 0.001% (w / v) to about 3% (w / v) and / or the washing solution comprising polysorbate has a final concentration of at least about 0.001% (w / v) to about 3% (w / v).

[0210] 113. A method according to any one of aspects 1 to 41, or aspects 77 to 107, or aspects 111 to 112, wherein the polysorbate is added to a final concentration of about 0.001% (w / v) to about 2% (w / v) and / or the washing solution comprising polysorbate has a final concentration of at least about 0.001% (w / v) to about 2% (w / v).

[0211] 114. A method according to any one of aspects 1 to 41, or aspects 77 to 107, or aspects 111 to 113, wherein the polysorbate is added to a final concentration of about 0.001% (w / v) to about 1% (w / v) and / or the washing solution comprising polysorbate has a final concentration of at least about 0.001% (w / v) to about 1% (w / v).

[0212] 115. The method of any one of embodiments 1 to 41, or embodiments 77 to 107, or embodiments 111 to 114, wherein the chromatographic material is in a column.

[0213] 116. The method of any one of embodiments 1 to 41, or embodiments 77 to 107, or embodiments 111 to 114, wherein the chromatographic material is suspended in the solution / sample.

[0214] 117. The method according to any of embodiments 1 to 38 or any of embodiments 77 to 104 or any of embodiments 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is a humanized type II anti-CD20 antibody or an anti-VEGF / Ang2 antibody or an anti-HER2 antibody.

[0215] 118. The method according to any of embodiments 1 to 38 or embodiment 117 or embodiment 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is an anti-VEGF / Ang2 antibody.

[0216] 119. The method according to any of embodiments 1 to 38 or embodiments 117 to 118 or embodiments 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is an anti-VEGF / Ang2 antibody comprising SEQ ID NO:3 to SEQ ID NO:6 as variable region.

[0217] 120. The method according to any of embodiments 1 to 38 or embodiment 117 or embodiment 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is an anti-HER2 antibody.

[0218] 121. The method according to any of embodiments 1 to 40 or embodiments 117 to 120 or embodiments 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is obinutuzumab or faricimab or trastuzumab or pertuzumab.

[0219] 122. The method according to any of embodiments 1 to 40 or any of embodiments 117 to 121 or any of embodiments 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is obinutuzumab or faricimab or trastuzumab.

[0220] 123. The method according to any of embodiments 1 to 38 or embodiments 117 to 119 or embodiments 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is faricimab.

[0221] 124. The method according to any of embodiments 1 to 38 or embodiment 117 or embodiment 120 to 122 or embodiment 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is trastuzumab or pertuzumab.

[0222] 125. The method according to any of embodiments 1 to 38 or embodiment 117 or embodiment 120 to 122 or embodiment 124 or embodiment 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is trastuzumab.

[0223] 126. The method according to any of embodiments 1 to 38 or embodiment 117 or embodiment 120 to 122 or embodiment 124 or embodiment 111 to 116 or the use according to any of embodiments 42 to 69 or the liquid antibody preparation according to embodiment 73 or any of embodiments 109 to 110, wherein said protein / polypeptide is pertuzumab.

[0224] 127. A method for purifying faricimab from a sample containing faricimab and at least one impurity having hydrolytic activity, the method comprising the steps of: i) a) adding polysorbate to said sample containing faricimab and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering faricimab from the chromatographic material; or ii) a) adding polysorbate to said sample containing faricimab and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering faricimab from the chromatographic material.

[0225] 128. A method for purifying obinutuzumab from a sample containing obinutuzumab and at least one impurity with hydrolytic activity, comprising the steps of: i) a) adding polysorbate to said sample containing obinutuzumab and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering obinutuzumab from the chromatographic material; or ii) a) adding polysorbate to said sample containing obinutuzumab and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) recovering obinutuzumab from the chromatographic material.

[0226] 129. A method for purifying trastuzumab from a sample containing trastuzumab and at least one impurity with hydrolytic activity, comprising the steps of: i) a) adding polysorbate to said sample containing trastuzumab and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering trastuzumab from the chromatographic material; or ii) a) adding polysorbate to said sample containing trastuzumab and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a (washing) solution comprising polysorbate to the chromatographic material; and d) Recovering trastuzumab from the chromatographic material.

[0227] 130. A liquid composition comprising an antibody and a polysorbate, wherein no more than 20% of the polysorbate is degraded per year during storage of the liquid composition.

[0228] 131. The liquid composition of embodiment 130, wherein the antibody is a humanized type II anti-CD20 antibody, or an anti-VEGF / Ang2 antibody, or an anti-HER2 antibody.

[0229] 132. The liquid composition of either embodiment 130 or embodiment 131, wherein the antibody is obinutuzumab, faricimab, trastuzumab, or pertuzumab.

[0230] 133. The liquid composition of either embodiment 130 or embodiment 131, wherein the antibody is obinutuzumab.

[0231] 134. The liquid composition of either embodiment 130 or embodiment 131, wherein the antibody is faricimab.

[0232] 135. The liquid composition of either embodiment 130 or embodiment 131, wherein the antibody is trastuzumab.

[0233] 136. The liquid composition of either embodiment 130 or embodiment 131, wherein the antibody is pertuzumab.

[0234] 137. A liquid antibody preparation with improved stability, obtainable by a method according to any one of aspects 1 to 41 or aspects 111 to 129.

[0235] 138. A liquid antibody preparation with reduced particle formation (formed by free fatty acid aggregation), obtainable by a method according to any of aspects 1 to 41 or aspects 111 to 129.

[0236] References TIFF2025081536000001.tif139143TIFF2025081536000002.tif235143TIFF2025081536000003.tif224143TIFF2025081536000004.tif30142

[0237] The following examples, sequences and figures are provided for the purpose of understanding the present invention, the true scope of which is set forth in the appended claims. It will be understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0238] Description of sequence listing SEQ ID NO:01 Amino acid sequence of the heavy chain variable region (VH) of humanized type II anti-CD20 antibody (obinutuzumab). SEQ ID NO:02 Amino acid sequence of the light chain variable region (VL) of humanized type II anti-CD20 antibody (obinutuzumab). SEQ ID NO:03 Amino acid sequence of heavy chain variable region 1 (VH-1) of anti-VEGF / Ang2 antibody (faricimab). SEQ ID NO:04 Amino acid sequence of the light chain variable region 1 (VL-1) of the anti-VEGF / Ang2 antibody (faricimab). SEQ ID NO:05 Amino acid sequence of the heavy chain variable region 2 (VH-2) of the anti-VEGF / Ang2 antibody (faricimab). SEQ ID NO:06 Amino acid sequence of the light chain variable region 2 (VL-2) of the anti-VEGF / Ang2 antibody (faricimab). [Brief description of the drawings]

[0239] [Figure 1]Schematic outline of the application of downstream processing workflows performed on an anti-CD20 antibody (obinutuzumab) including an optional pre-incubation step (conditioning HCCF load / load SP with polysorbate 20 (PS20)), washing of bound mAb with a PS20-containing solution, or a combination of both steps. The hydrolytic activity (measured as remaining PS20 content as a function of time) of the corresponding elution fractions (eluate: PS20 conditioning, eluate: PS20 wash, and eluate: PS20 conditioning / wash) was compared to a reference eluate using standard chromatographic conditions. [Diagram 2] Residual PS20 content in MabSelect SuRe elution fractions of an anti-CD20 antibody (obinutuzumab) after 238 h (A) and 317 h (B) incubation times at a PS20 concentration of 0.04% (w / v) used for downstream processing, relative to the relative PS20 content of the corresponding reference elution. A higher residual PS20 content indicates reduced hydrolytic activity. [Diagram 3] Residual PS20 content in CaptureSelect FcXL elution fractions of an anti-CD20 antibody (obinutuzumab) after 86 h (A) and 163 h (B) incubation times at a PS20 concentration of 0.04% (w / v) used for downstream processing, relative to the relative PS20 content of the corresponding reference elution. Higher residual PS20 content indicates reduced hydrolytic activity. [Figure 4] Residual PS20 content of CaptureSelect™ FcXL elution fractions of an anti-CD20 antibody (obinutuzumab) after 86 hours (A) and 163 hours (B) of incubation time at a PS20 concentration of 0.28% (w / v) used for downstream processing, relative to the relative PS20 content of the corresponding reference elution. Higher residual PS20 content indicates reduced hydrolytic activity. [Diagram 5]Residual PS20 content in SP Sepharose Fast Flow elution fractions of an anti-CD20 antibody (obinutuzumab) after 238 h (A) and 317 h (B) incubation times at a PS20 concentration of 0.04% (w / v) used for downstream processing, relative to the relative PS20 content of the corresponding reference eluate. A higher residual PS20 content indicates reduced hydrolytic activity. [Figure 6] The sum of free lauric and free myristic acids in the MabSelect SuRe elution fraction of an anti-CD20 antibody (obinutuzumab) after 141 h incubation time with a PS20 concentration of 0.04% (w / v) used for downstream processing, relative to the amount of the corresponding reference elution. A lower amount of free fatty acids indicates reduced hydrolytic activity. [Figure 7] The sum of free lauric and free myristic acids in the SP Sepharose Fast Flow elution fractions of an anti-CD20 antibody (obinutuzumab) after 141 h incubation time with a PS20 concentration of 0.04% (w / v) used for downstream processing, relative to the amount of the corresponding reference elution. A lower amount of free fatty acids indicates reduced hydrolytic activity. [Figure 8] Residual PS20 content in CaptureSelect FcXL elution fractions of an anti-VEGF / ANG2 antibody (faricimab) after 87 h (A) and 159 h (B) incubation times at a PS20 concentration of 0.28% (w / v) used for downstream processing, relative to the relative PS20 content of the corresponding reference eluate. Higher residual PS20 content indicates reduced hydrolytic activity. [Figure 9] The combined amount of free lauric and free myristic acids in CaptureSelect FcXL elution fractions of an anti-VEGF / ANG2 antibody (faricimab) after 116 (A) and 162 (B) hours of incubation at a PS20 concentration of 0.28% (w / v) used for downstream processing, relative to the corresponding reference elution amounts. Lower amounts of free fatty acids indicate reduced hydrolytic activity. [Figure 10]Total amount of free lauric and free myristic acids in SP Sepharose Fast Flow elution fractions of anti-HER2 antibody (trastuzumab) after 168 h incubation time with PS20 concentration of 0.28% (w / v) used for downstream processing, based on the relative amount of the corresponding reference eluate. Blank subtraction was performed for all samples using a buffer control (0.1 mM Tris / HCl, 10 mM methionine, pH 8.0). Lower amounts of free fatty acids indicate reduced hydrolytic activity. [Figure 11] Total free lauric and myristic acids in anti-HER2 antibody (Trastuzumab) samples processed to bulk levels without conditioning, including optional downstream PS20 conditioning and washing steps (using a PS20 concentration of 0.28% (w / v) before / during purification by cation exchange chromatography (SP Sepharose)) after 140 (A) and 235 (B) incubation times, relative to the relative amounts at the corresponding reference conditions. Blank subtractions were performed for all samples using a buffer control (0.1 mM Tris / HCl, 10 mM methionine, pH 8.0). Lower amounts of free fatty acids indicate reduced hydrolytic activity. EXAMPLES

[0240] General method Protein Purity Measurement Protein purity was measured by size-exclusion high performance liquid chromatography (SE-HPLC) using a Dionex UltiMate 3000 HPLC system (Thermo Scientific). Protein separation was performed on a TSKGEL G3000SWXL 7.8X300 column (Tosoh Bioscience LLC) with 0.2MK. 2 HPO 4 / KH 2 PO 4 The running buffer was 0.25 M KCl, pH 7.0, and the flow rate was 0.5 ml / min.

[0241] Measurement of protein concentration Protein concentrations were measured by UV spectroscopy using a Cary® 50 UV-Vis spectrophotometer (Varian). Protein samples were diluted in their respective buffers and measured in duplicate. Concentrations were determined according to the following equation derived from the Lambert-Beer law: c = (A 280nm -A 320nm ) / ε·d·F, where c is the protein concentration [mg / ml], A is the absorbance, ε is the extinction coefficient [ml / (mg·cm)], d is the cell length [cm], and F is the dilution factor. The specific extinction coefficient of obinutuzumab is 1.49 ml / (mg·cm).

[0242] antibody The invention is illustrated using exemplary antibodies such as the humanized type II anti-CD20 antibody (obinutuzumab) described in WO 2005 / 044859, which comprises SEQ ID NO:01 to SEQ ID NO:02 as variable regions, or the bispecific antibody against VEGF and Ang2 (anti-VEGF / Ang2 antibody; faricimab) described in WO 2014 / 009465, which comprises SEQ ID NO:3 to SEQ ID NO:6 as variable regions, or an antibody against HER2 (anti-HER2 antibody; trastuzumab), e.g. as described in WO 2011 / 012637.

[0243] Refining Overview A schematic overview of a downstream processing application is depicted in FIG.

[0244] Example 1 Purification of anti-CD20 antibodies by Protein A chromatography (MabSelect SuRe) Loading preparation: The following steps were carried out in sequence: 1) Untreated HCCF containing obinutuzumab (anti-CD20) were thawed and filtered using a 0.22 μm pore size filter unit (Merck Millipore). 2) HCCF was divided into four equal parts. For PS20 conditioning, PS20 was added to the HCCF at a final concentration of 0.04% (w / v). The conditioned solutions were homogenized by stirring (300 rpm, 10 min, room temperature) and incubated at 4°C for 24 h without stirring. All parts were stored at -20°C.

[0245] Chromatography: Chromatographic protein purification was performed using a MabSelect™ SuRe™ column (φ1 cm, h=25.5 cm, V=20.3 ml) (GE Healthcare) on an AEKTA Explorer 100 system (GE Healthcare) at a constant flow rate of 440 cm / h for all steps. The experimental details are listed as follows:

[0246] Test 1 (baseline): Load density: 38.2g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Road HCCF 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 0.7M Tris / HCl, pH 7.2 5. Wash 3 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 6. Elute with 30 mM acetic acid (pool 0.25AU-0.25AU (1 cm UV cell))

[0247] Experiment 2 (conditioned HCCF containing 0.04% (w / v) PS20): Load density: 37.45g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Loaded HCCF Conditioned HCCF containing 0.04% (w / v) PS20 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 0.7M Tris / HCl, pH 7.2 5. Wash 3 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 6. Elute with 30 mM acetic acid (pool 0.25AU-0.25AU (1 cm UV cell))

[0248] Experiment 3 (additional wash-stripping with 0.04% (w / v) PS20): Load density: 38.2g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Road HCCF 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 0.04% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3 0.7M Tris / HCl, pH 7.2 6. Wash 4 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 7. Elute with 30 mM acetic acid (pool 0.25AU-0.25AU (1 cm UV cell))

[0249] Experiment 4 (HCCF with conditioning containing 0.04% (w / v) PS20 + additional wash-stripping with 0.04% (w / v) PS20): Load density: 37.45g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Loaded HCCF Conditioned HCCF containing 0.04% (w / v) PS20 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 0.04% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3 0.7M Tris / HCl, pH 7.2 6. Wash 4 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 7. Elute with 30 mM acetic acid (pool 0.25AU-0.25AU (1 cm UV cell))

[0250] Pooling overview of MSS experiments (see Figures 2 and 6): The MabSelect SuRe® eluate (containing obinutuzumab) was adjusted to a pH value of 6.0±0.2 with 1.5 M Tris-amino and subsequently filtered using Minisart® filter units (Sartorius Stedim) with pore sizes of 0.45 μm and 0.22 μm. The filtered eluate was frozen at −20° C. and served as starting material for subsequent analysis. TIFF2025081536000005.tif71138

[0251] Example 2 Purification of anti-CD20 antibodies by anti-IgG Fc chromatography / chromatography using camelid-derived single domain antibody fragment ligand material (CaptureSelect™ FcXL) Loading preparation: The following steps were carried out in sequence: 1) Untreated HCCF containing obinutuzumab (anti-CD20) were thawed and filtered using a 0.22 μm pore size filter unit (Merck Millipore). 2) HCCF was divided into seven equal parts. For PS20 conditioning, PS20 was added to the HCCF at a final concentration of 0.04% (w / v) or 0.28% (w / v). The conditioned solutions were homogenized by stirring (300 rpm, 10 min, room temperature) and incubated at 4°C for 24 h without stirring. All parts were stored at -20°C.

[0252] Chromatography: Chromatographic protein purification was performed using a CaptureSelect FcXL column (φ1 cm, h=20.5 cm, V=16.1 ml) (Thermo Fisher) on an AEKTA AVANT 150 system (GE Healthcare) at a constant flow rate of 200 cm / h for all steps. The experimental details are listed as follows.

[0253] Test 1 (baseline): Load density: 19.81g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Road HCCF 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 Purified water (Type II) 5. Wash 3 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 6. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0254] Experiment 2 (conditioned HCCF containing 0.04% (w / v) PS20): Load density: 19.75g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Loaded HCCF Conditioned HCCF containing 0.04% (w / v) PS20 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 Purified water (Type II) 5. Wash 3 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 6. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0255] Experiment 3 (additional wash-stripping with 0.04% (w / v) PS20): Load density: 19.81g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Road HCCF 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 0.04% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3: Purified water (Type II) 6. Wash 4 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 7. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0256] Experiment 4 (HCCF with conditioning containing 0.04% (w / v) PS20 + additional wash-stripping with 0.04% (w / v) PS20) Load density: 19.77g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Loaded HCCF Conditioned HCCF containing 0.04% (w / v) PS20 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 0.04% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3: Purified water (Type II) 6. Wash 4 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 7. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0257] Experiment 5 (conditioned HCCF containing 0.28% (w / v) PS20): Load density: 19.82g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Loaded HCCF Conditioned HCCF containing 0.28% (w / v) PS20 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 Purified water (Type II) 5. Wash 3 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 6. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0258] Experiment 6 (additional wash-stripping with 0.28% (w / v) PS20): Load density: 19.81g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Road HCCF 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 0.28% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3: Purified water (Type II) 6. Wash 4 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 7. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0259] Experiment 7 (HCCF with conditioning containing 0.28% (w / v) PS20 + additional wash-stripping with 0.28% (w / v) PS20) Load density: 19.86g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.0 2. Loaded HCCF Conditioned HCCF containing 0.28% (w / v) PS20 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 4. Wash 2 0.28% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3: Purified water (Type II) 6. Wash 4 25 mM Tris / HCl, 25 mM NaCl, pH 7.0 7. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0260] Pooling overview for anti-IgG-Fc (CaptureSelect™ FcXL) experiments (see Figures 3 and 4): The CaptureSelect™ FcXL eluate (containing obinutuzumab) was adjusted to a pH value of 6.0±0.2 with 1.5 M Tris-amino and subsequently filtered using Minisart® filter units (Sartorius Stedim) with pore sizes of 0.45 μm and 0.22 μm. The filtered eluate was frozen at −20° C. and served as starting material for subsequent analysis. TIFF2025081536000006.tif99135

[0261] Example 3 Purification of anti-CD20 antibodies by cation exchange chromatography (SP-Sepharose Fast Flow) Loading preparation: The following steps were carried out in sequence: 1) Two unprocessed SP loads containing different concentrations of obinutuzumab ("Probe 4.1 Load SPFF"; G002.01E) were thawed and filtered separately using 0.22 μm pore size filter units (Merck Millipore). 2) The first SP load fraction was divided into three equal parts (used for experiments 1-3), and the second SP load was used for experiment 4. For PS20 conditioning, PS20 was added to the SP load at a final concentration of 0.04% (w / v). The conditioned solutions were homogenized by stirring (300 rpm, 10 min, room temperature) and incubated at 4°C for 24 h without stirring. All parts were stored at -20°C.

[0262] Chromatography: Chromatographic protein purification was performed using an SP Sepharose Fast Flow column (φ1 cm, h=27.3 cm, V=21.4 ml) (GE Healthcare) on an AEKTA Explorer system (GE Healthcare) at a constant flow rate of 160 cm / h for all steps. The experimental details are listed as follows:

[0263] Test 1 (baseline): Load density: 51.04g / L 樹脂 Chromatography steps: 1. Equilibrate 25mM Tris / acetic acid pH5.0 2. Road SPFF 3. Wash 1 10mM Tris / Acetic Acid pH 9.0 4. Wash 2 25mM Tris / Acetic Acid pH 5.0 5. Elution: 25 mM Tris / Acetic acid, 150 mM NaCl, pH 5.0 (Pooling 0.25AU-0.88AU (1cm UV cell))

[0264] Experiment 2 (SP loading with conditioning containing 0.04% (w / v) PS20): Load density: 49.02g / L 樹脂 Chromatography steps: 1. Equilibrate 25mM Tris / acetic acid pH5.0 2. Load SPFF containing 0.04% (w / v) PS20 3. Wash 1 10mM Tris / Acetic Acid pH 9.0 4. Wash 2 25mM Tris / Acetic Acid pH 5.0 5. Elution: 25 mM Tris / Acetic acid, 150 mM NaCl, pH 5.0 (Pooling 0.25AU-0.88AU (1cm UV cell))

[0265] Experiment 3 (additional wash-stripping with 0.04% (w / v) PS20): Load density: 49.68g / L 樹脂 Chromatography steps: 1. Equilibrate 25mM Tris / acetic acid pH5.0 2. Road SPFF 3. Wash 1 10mM Tris / Acetic Acid pH 9.0 4. Wash 2 0.04% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3 25mM Tris / Acetic Acid pH 5.0 6. Elution: 25 mM Tris / Acetic acid, 150 mM NaCl, pH 5.0 (Pooling 0.25AU-0.88AU (1cm UV cell))

[0266] Experiment 4 (SP load with conditioning containing 0.04% (w / v) PS20 + additional wash-stripping with 0.04% (w / v) PS20) Load density: 28.93g / L 樹脂 Chromatography steps: 1. Equilibrate 25mM Tris / acetic acid pH5.0 2. Load SPFF containing 0.04% (w / v) PS20 3. Wash 1 10mM Tris / Acetic Acid pH 9.0 4. Wash 2 0.04% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3 25mM Tris / Acetic Acid pH 5.0 6. Elution: 25 mM Tris / Acetic acid, 150 mM NaCl, pH 5.0 (Pooling 0.25AU-0.88AU (1cm UV cell))

[0267] Pooling overview of SP-Sepharose Fast Flow experiments (see Figures 5 and 7) The SP-Sepharose Fast Flow eluate (containing obinutuzumab) was frozen at -20°C and served as starting material for subsequent analysis. TIFF2025081536000007.tif84137

[0268] Example 4 Purification of an anti-VEGF / Ang2 antibody (Farisimab) by anti-IgG Fc chromatography / chromatography using camelid-derived single domain antibody fragment ligand material (CaptureSelect™ FcXL) Loading preparation: The following steps were carried out in sequence: 1) Untreated HCCF containing fasilimab (anti-VEGF / Ang2) were thawed and filtered using a 0.22 μm pore size filter unit (Merck Millipore). 2) HCCF was divided into two halves. For PS20 conditioning, PS20 was added to the HCCF at a final concentration of 0.28% (w / v). The conditioned solution was homogenized by stirring (300 rpm, 10 min, room temperature) and incubated at 4°C for 24 h without stirring. All portions were stored at -20°C.

[0269] Chromatography: Chromatographic protein purification was performed using a CaptureSelect™ FcXL column (φ1 cm, h=21.3 cm, V=16.73 ml) (Thermo Fisher) on an AEKTA Avant 150 system (GE Healthcare) at a constant flow rate of 200 cm / h for all steps. The experimental details are listed as follows:

[0270] Test 1 (baseline): Load density: 29.47g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.2 2. Road HCCF 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.2 4. Wash 2 Purified water (Type II) 5. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0271] Experiment 2 (HCCF with conditioning containing 0.28% (w / v) PS20 + additional wash-stripping with 0.28% (w / v) PS20) Load density: 28.8g / L 樹脂 Chromatography steps: 1. Equilibration 25mM Tris / HCl, 25mM NaCl, pH7.2 2. Loaded HCCF Conditioned HCCF containing 0.28% (w / v) PS20 3. Wash 1 25 mM Tris / HCl, 25 mM NaCl, pH 7.2 4. Wash 2 0.28% (w / v) PS20 (5 CV total: 4 CV, 30 min hold, 1 CV) 5. Wash 3: Purified water (Type II) 6. Elute with 30 mM acetic acid (pool 2.5AU-2.5AU (1 cm UV cell))

[0272] Pooling overview of anti-IgG-Fc (CaptureSelect™ FcXL) experiments (see Figures 8 and 9) The CaptureSelect™ FcXL eluate (containing Faricimab) was adjusted to a pH value of 6.0±0.2 with 1.5 M Tris-amino and subsequently filtered using Minisart® filter units (Sartorius Stedim) with pore sizes of 0.45 μm and 0.22 μm. The filtered eluate was frozen at −20° C. and served as starting material for subsequent analysis. TIFF2025081536000008.tif71143

[0273] Example 5 Purification of anti-HER2 antibody (trastuzumab) by cation exchange chromatography (SP-Sepharose Fast Flow) Loading preparation: The following steps were carried out in sequence: 1) Untreated SP load containing trastuzumab ("Probe 2.2 Load SP-FF"; G299.00P1) was thawed and filtered using a 0.22 μm pore size filter unit (Merck Millipore). 2) The SP load was divided into three equal parts. In all cases of PS20 conditioning, PS20 was added to the SP load at a final concentration of 0.28% (w / v). The conditioned solutions were homogenized by stirring (300 rpm, 10 min, room temperature) and incubated at 4°C for 24 h without stirring. All parts were stored at -70°C.

[0274] Chromatography: Chromatographic protein purification was performed using an SP Sepharose Fast Flow column (φ1.0 cm, h=36.80 cm, V=28.90 ml) (GE Healthcare) on an AEKTA Explorer system (GE Healthcare) at a constant flow rate of 150 cm / h for all steps. The experimental details are listed as follows.

[0275] Test 1 (baseline): Load density: 30.00g / L 樹脂 Chromatography steps: 1. Equilibration 30mM MES, 45mM NaCl, pH5.6 2. Road SP 3. Wash 1 30 mM MES, 45 mM NaCl, pH 5.6 4. Wash 2 Equilibration buffer / Elution buffer gradient (21-71% Elution buffer) 5. Elute in 30 mM MES, 95 mM NaCl, pH 5.6 (pool 0.6AU-0.5AU (1 cm UV cell))

[0276] Experiment 2 (loaded SP with conditioning containing 0.28% (w / v) PS20) Load density: 30.10g / L 樹脂 Chromatography steps: 1. Equilibration 30mM MES, 45mM NaCl, pH5.6 2. Load SP HCCF with conditioning containing 0.28% (w / v) PS20 3. Wash 1 30 mM MES, 45 mM NaCl, pH 5.6 4. Wash 2 30 mM MES, 45 mM NaCl, pH 5.6 5. Wash 3 Equilibration buffer / Elution buffer gradient (21-71% Elution buffer) 6. Elute in 30 mM MES, 95 mM NaCl, pH 5.6 (pool 0.6AU-0.5AU (1 cm UV cell))

[0277] Experiment 3 (Load SP with conditioning containing 0.28% (w / v) PS20 + additional wash-stripping with 0.28% (w / v) PS20) Load density: 30.10g / L 樹脂 Chromatography steps: 1. Equilibration 30mM MES, 45mM NaCl, pH5.6 2. Load SP HCCF with conditioning containing 0.28% (w / v) PS20 3. Wash 1 30 mM MES, 45 mM NaCl, pH 5.6 4. Wash 2 0.28% (w / v) PS20 (5 CV total; 4 CV, 30 min hold, 1 CV) 5. Wash 3 30 mM MES, 45 mM NaCl, pH 5.6 6. Wash 4 Equilibration buffer / Elution buffer gradient (21-71% Elution buffer) 7. Elute in 30 mM MES, 95 mM NaCl, pH 5.6 (pool 0.6AU-0.5AU (1 cm UV cell))

[0278] Pooling overview of SP-Sepharose Fast Flow experiment (see Figure 10) : The SP eluate (containing trastuzumab) was frozen at -20°C and served as starting material for subsequent analysis. TIFF2025081536000009.tif61141

[0279] Example 6 Purification of anti-Her2 antibody (trastuzumab) processed to bulk levels without conditioning Processing steps: TIFF2025081536000010.tif157128

[0280] Load preparation for experiment 2: The following steps were carried out in sequence: 1) Untreated SP load containing trastuzumab ("Probe 2.2 Load SP-FF"; G403.00P1) was thawed and filtered using a 0.22 μm pore size filter unit (Sartorius, Sartopore-2). 2) Conditioning of SP load with PS20: PS20 was added to the SP load at a final concentration of 0.28% (w / v). The conditioned solution was homogenized by stirring (300 rpm, 10 min, room temperature) and incubated at 4°C for 24 h without stirring. All portions were stored at -20°C.

[0281] Chromatography SP Sepharose Fast Flow Experiment 2: Chromatographic protein purification was performed using an SP Sepharose Fast Flow column (φ1.6 cm, h=37.0 cm, V=74.39 ml) (GE Healthcare) on an AEKTA Avant 150 system (GE Healthcare) at a constant flow rate of 102 cm / h for all steps. The experimental details are listed as follows:

[0282] Experiment 2 (Load SP with conditioning containing 0.28% (w / v) PS20 + additional wash-stripping with 0.28% (w / v) PS20) Load density: 35.3g / L 樹脂 Chromatography steps: 1. Equilibration 30mM MES, 45mM NaCl, pH5.6 2. Load SP HCCF with conditioning containing 0.28% (w / v) PS20 3. Wash 1 30 mM MES, 45 mM NaCl, pH 5.6 4. Wash 2 0.28% (w / v) PS20 (5 CV total; 4 CV, 30 min hold, 1 CV) 5. Wash 3 30 mM MES, 45 mM NaCl, pH 5.6 6. Wash 4 Equilibration buffer / Elution buffer gradient (21-72% Elution buffer) 6. Elute in 30 mM MES, 95 mM NaCl, pH 5.6 (pool 0.6AU-0.5AU (1 cm UV cell))

[0283] Pooling overview of experiments at bulk level without conditioning (see Figure 11) : The SP eluate (containing trastuzumab) was frozen at -20°C and served as starting material for subsequent analysis. TIFF2025081536000011.tif67161

[0284] Example 7 Measurement of hydrolytic activity A) Incubation of the eluted fractions with PS20 for subsequent quantification of the remaining intact PS20 content and measurement of free fatty acids by mass spectrometry. Stability studies were performed with material from different purification setups: for example, in the "Protein A (MabSelect SuRe)" setup, elution fractions after Protein A chromatography were evaluated; in the "Downscale model" setup, elution fractions after several purification steps leading to the bulk level without conditioning were evaluated, where the sample was purified to the final level.

[0285] To monitor the (residual) hydrolytic activity in each elution fraction from Examples 1-6, samples were adjusted to the same protein concentration and prepared for PS20 stability testing using stock solutions of PS20 or Super-refined PS20 (SR-PS20) and the corresponding elution buffer system or TRIS buffer (pH 8) as specified in the table below. In addition, a stock solution of methionine (100 mM) was added as an effective antioxidant to inhibit oxidative degradation of PS20 during the time course of the experiment. Samples were removed at various time points (e.g., as shown in the figure legend and sample preparation scheme below) for later quantification of the remaining intact PS20 content (see Example 7B) and for measurement of free fatty acids by mass spectrometry (see Example 7C).

[0286] 1) Protein A (MabSelect SuRe); anti-CD20 antibody; obinutuzumab: TIFF2025081536000012.tif167147

[0287] Samples were removed after 238 and 317 hours of incubation for later analysis of the content of remaining intact PS20, while samples for free fatty acid measurements were removed after 141 hours of incubation (see Figures 2 and 6).

[0288] 2) Anti-IgG-Fc (CaptureSelect(TM) FcXL); anti-CD20 antibody; obinutuzumab: TIFF2025081536000013.tif252143

[0289] Samples were removed after 86 and 163 hours of incubation for later analysis of the content of remaining intact PS20 (see Figures 3 and 4).

[0290] 3) CEX (SP Sepharose); anti-CD20 antibody; obinutuzumab: TIFF2025081536000014.tif156143

[0291] Samples were removed after 238 and 317 hours of incubation for later analysis of the content of remaining intact PS20, while samples for free fatty acid measurements were removed after 141 hours of incubation (see Figures 5 and 7).

[0292] 4) Anti-IgG-Fc (CaptureSelect(TM) FcXL); Anti-VEGF / Ang2 antibody; Faricimab: TIFF2025081536000015.tif94146

[0293] Samples were removed after 87 and 159 hours of incubation for later analysis of the content of remaining intact PS20, while samples for free fatty acid measurements were removed after 116 hours of incubation (see Figures 8 and 9).

[0294] 5) Cation exchange chromatography (SP-Sepharose); Anti-HER2 antibody, trastuzumab: TIFF2025081536000016.tif124156

[0295] Samples for free fatty acid measurements were removed after 168 hours of incubation (see FIG. 10).

[0296] 6) Downscale model; anti-HER2 antibody, trastuzumab: TIFF2025081536000017.tif94156

[0297] Samples for free fatty acid measurements were removed after 140 and 235 hours of incubation (see Figure 11).

[0298] All reaction mixtures were incubated in a Thermomixer at 37° C. or 40° C. with shaking at 300 rpm. Samples were drawn after designated time points (e.g., 238 and 317 hours of incubation for Protein A chromatography purification, as indicated in the respective figure legends) and stored at −80° C. until further analysis.

[0299] B) Quantification of remaining intact PS20 content The PS20 content was quantified using a method similar to that described by Hewitt et al. (Journal of chromatography.A 2011, 1218, 2138-2145) and Lippold et al. (Journal of pharmaceutical and biomedical analysis 2017, 132, 24-34). Samples were separated by mixed-mode HPLC using an UltiMate™ 3000 RS (Thermo Fisher) equipped with an Oasis MAX Cartridge column (30 μm, 2.1 × 20 mm, Waters) and an LPG-3400XRS Extended Pressure Range Quaternary Pump, an autosampler, a temperature-controlled column chamber, and a Corona™ Veo™ RS charged aerosol detector (CAD) (Thermo Fisher). The CAD settings were as follows: 50 psi internal nitrogen pressure provided by an in-house source, power function setting 1.00, and default range 200 pA. A post-column switch valve redirected the flow to the CAD from 2.4 to 6.5 minutes and to waste at all other times. For the analysis, 25 μL of sample with a nominal PS20 concentration of 0.4 mg / ml was injected and the PS20 content was determined by a calibration curve established with injection volumes ranging from 5 μL (2 μg) to 40 μL (16 μg). The column temperature was set at 30°C. Solvent A (2% formic acid in purified water) and solvent B (2% formic acid in methanol) were used as the mobile phase with a flow rate of 1.25 ml / min, with the following gradient: TIFF2025081536000018.tif108143

[0300] The PS20 concentration obtained for each sample (in mg / mL) was related to the corresponding t=0 value, which represents the amount of polysorbate spiked into each sample at the start of the experiment (relative PS20 concentration). All samples were incubated for a predefined period of time to allow hydrolysis of polysorbate to occur. For graphical representation, the reference eluate (set to 100%) was related to the relative PS20 concentration in the samples treated by the method reported herein (PS20 treatment) (see Figures 2-5 and 8). In this way, the remaining PS20 content at a given time point can be correlated with the remaining hydrolytic activity. That is, the more PS20 remaining in the elution fraction sample (containing the protein of interest together with hydrolytically active impurities) after incubation, the less remaining hydrolytic activity was present in the sample, i.e. the more effective was the reduction of hydrolytically active impurities during purification. In line with the finding that the hydrolytic activity was reduced in the elution fractions after treatment with polysorbate, a higher hydrolytic activity could also be detected in each wash fraction.

[0301] Complementary analyses were performed to detect free fatty acids resulting from the hydrolytic degradation of PS (see below). Similar to the results shown for the residual PS content, these results revealed that lower amounts of free fatty acids (degradation products) were detected in samples in which polysorbate treatment was performed during purification.

[0302] The degree of purification is most pronounced if the protein-containing solution is incubated / conditioned with polysorbate prior to the chromatography step and, in addition, washed with a solution containing polysorbate during chromatography. The combination of these treatments leads to better results than either treatment alone.

[0303] C) Measurement of free fatty acids by mass spectrometry To quantify the amount of lauric and myristic acids, the main degradation products of PS20 hydrolysis, a method equivalent to that described in M. Honemann, et al., Journal of Chromatography B, https: / / doi.org / 10.1016 / j.jchromb.2019.03.030 was used. Samples from the elution pool were taken and incubated with PS20 spiked therein. At different time points (see Example 7A for details), 50 μL was withdrawn from each sample, which was then transferred to a reaction tube. 200 μL of free fatty acid solvent solution (500 ng / mL dissolved in acetonitrile) was added to the reaction tube. 2 d 23 -Lauric acid and 500ng / mL 13 C 14 Myristic acid) was added and the mixture was vortexed briefly. Samples were centrifuged at 14000 rpm for 5 min and transferred to HPLC vials for MS analysis. Separation of fatty acids was achieved by reversed-phase chromatography using an ACQUITY UPLC Peptide BEH C18 column (1.7 μm 2.1 × 150 mm and 300 Å) on a Thermo Scientific Vanquish UHPLC system. The injection volume was set at 5 μL and the column chamber was maintained at 60°C. Solvent A (0.1% ammonium hydroxide in purified water) and solvent B (100% acetonitrile) were used as the mobile phase for the following gradient at a flow rate of 0.3 ml / min: TIFF2025081536000019.tif76128

[0304] The mass spectrometer (Triple TOF6600, AB Sciex) was operated in negative ionization mode with an ion spray voltage of -4500 V. The source temperature was set at 450 °C, and the time-of-flight (TOF) mass range was 100–1000 m / Z. The declustering potential was set at -120 V, and the collision energy was set at -10 V. The contents of lauric and myristic acids were determined by subtracting the peak areas of fatty acids from their respective internally labeled standards ( 2 d 23 -Lauric acid and 13 C 14Myristic acid) was determined by: In the formula, the peak area Σ of FFA and the peak area Σ of internal standard refer to the sum of the monoisotopic peak and +1 / +2 or -1 / -2 isotopic peaks of the extracted ion chromatogram (XIC), respectively. All measurements were performed in technical duplicate experiments.

[0305] For graphical representation, the reference eluate (set to 100%) was related to the total amount of lauric and myristic acids in the samples treated by the method reported herein (PS20 treatment) (see Figures 6, 7, 9, 10 and 11). Thus, the total amount of lauric and myristic acids at a given time point can be correlated with the residual hydrolytic activity. That is, the lower the amount of free fatty acids in the elution fraction sample (containing the protein of interest together with hydrolytically active impurities) after incubation, the lower the residual hydrolytic activity present in the sample, i.e., the more effective the reduction of hydrolytically active impurities during purification was. For Examples 5 and 6, an additional buffer control subtraction was applied using the buffer composition of the respective sample, since the (intrinsic) hydrolytic activity increases at high pH.

[0306] Sequence information SEQUENCE LISTING <110> F. Hoffmann-La Roche AG <120> Methods for purification of polypeptides using polysorbates <150> EP18169762.4 <151> 2018-04-27 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial <220> <223> amino acid sequences of variable region of the heavy chain (VH) of humanized B-Ly1 antibody (B-HH6) <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 2 <211> 115 <212> PRT <213> Artificial <220> <223> amino acid sequences of variable region of the light chain (VL) of humanized B-Ly1 antibody B-KV1 <400> 2 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val 115 <210> 3 <211> 123 <212> PRT <213> Artificial <220> <223> heavy chain variable domain VH, <vegf> <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 107 <212> PRT <213> Artificial <220> <223> light chain variable domain VL, <vegf> <400> 4 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 5 <211> 129 <212> PRT <213> Artificial <220> <223> heavy chain variable domain VH, <ang-2> <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120 125 Ser <210> 6 <211> 110 <212> PRT <213> Artificial <220> <223> light chain variable domain VL, <ang-2> <400> 6 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser 100 105 110 < / vegf> < / vegf>

Claims

1. A method for purifying a protein from a sample containing said protein and at least one impurity with hydrolytic activity, comprising the steps of: i） a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a solution comprising polysorbate to said chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering the protein from the chromatographic material; or iii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material.

2. 1. A method for reducing hydrolytic activity in a sample containing a protein and at least one impurity with hydrolytic activity, comprising the steps of: i） a) applying the sample containing the protein and at least one impurity with hydrolytic activity to a chromatographic material; b) applying a solution comprising polysorbate to said chromatographic material; and c) recovering the protein from the chromatographic material; or ii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; and c) recovering the protein from the chromatographic material; or iii) a) adding polysorbate to said sample containing said protein and at least one impurity with hydrolytic activity; b) applying the mixture of step a) to a chromatographic material; c) applying a solution comprising polysorbate to the chromatographic material; and d) recovering the protein from the chromatographic material.

3. 3. The method of claim 1 or 2, wherein the impurity is a hydrolase.

4. The method according to any one of claims 1 to 3, wherein the impurity is an esterase.

5. 5. The method of any one of claims 1 to 4, wherein the polysorbate is selected from the group comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, or polysorbate 80.

6. The method of any one of claims 1 to 5, wherein the polysorbate is polysorbate 20 or polysorbate 80.

7. 7. The method of any one of claims 1 to 6, wherein the polysorbate is added to a final concentration of at least about 0.001% (w / v).

8. 8. The method of any one of claims 1 to 7, wherein the polysorbate is added to a final concentration of about 0.001% (w / v) to about 10% (w / v).

9. The method according to any one of claims 1 to 8, wherein the chromatographic material is an affinity chromatographic material, a cation exchange chromatographic material, an anion exchange chromatographic material, a hydrophobic interaction chromatographic material or a mixed mode chromatographic material.

10. The method according to any one of claims 1 to 9, wherein the chromatographic material is an affinity chromatographic material or a cation exchange chromatographic material.

11. The method according to any one of claims 1 to 10, wherein the chromatography material is an affinity chromatography material selected from Protein A chromatography or a chromatography material in which a camelid-derived single domain antibody fragment is a ligand.

12. The method of any one of claims 1 to 11, wherein the affinity chromatography material is CaptureSelect FcXL or MabSelect SuRe.

13. The method according to any one of claims 1 to 10, wherein the cation exchange chromatography material is SP Sepharose Fast Flow or Poros 50HS or Poros XS.

14. The method according to any one of claims 1 to 13, wherein the protein is a monoclonal antibody.

15. The method according to any one of claims 1 to 14, wherein the protein is a humanized type II anti-CD20 antibody or an anti-VEGF / Ang2 antibody or an anti-HER2 antibody.

16. A liquid antibody preparation with reduced hydrolytic activity obtainable by the method according to any one of the preceding claims.

17. 1. A method for producing a protein, comprising: a) culturing a mammalian cell containing a nucleic acid encoding the protein; b) recovering the protein from the cells or culture medium, and c) purifying said protein by the method according to any one of claims 1 to 15. thereby producing said protein.

18. A liquid composition comprising an antibody and a polysorbate, wherein no more than 20% of the polysorbate is degraded per year during storage of the liquid composition.

19. The liquid composition of claim 18, wherein the antibody is a humanized type II anti-CD20 antibody, an anti-VEGF / Ang2 antibody, or an anti-HER2 antibody.

20. 20. The liquid composition of claim 18 or 19, wherein the antibody is obinutuzumab, faricimab, trastuzumab, or pertuzumab.

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