RU2773852C2 - Methods for improved impurity removal during chromatography based on binding with protein a - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to methods for purification of polypeptide containing Fc-region (for example, an antibody) by chromatography based on binding with protein A. In the method for purification of polypeptide containing Fc-region, a washing solution is used, containing benzoate salt and/or benzyl alcohol, during chromatography based on binding with protein A. A method for purification of polypeptide containing Fc-region is also proposed, in which collected material, which contains polypeptide containing Fc-region, is corrected using sodium benzoate before chromatography based on binding with protein A.

EFFECT: methods allow for improvement of polypeptide purification from impurities.

85 cl, 8 dwg, 5 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority over U.S. Provisional Application Serial Number 62/609214, filed December 21, 2017, and U.S. Provisional Application Serial Number 62/694387, filed July 5, 2018, the contents of each incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present invention relates to methods for purifying a polypeptide containing an Fc region (e.g., antibodies), by protein-A binding chromatography.

BACKGROUND OF THE INVENTION

[0003] Antibodies and other proteins containing an Fc region (such as immunoadhesins) have found widespread use in pharmaceutical/therapeutic applications. The use of these molecules (for example, in human patients) requires careful cleaning of any contaminants/impurities that may occur during protein production. Purification of therapeutic proteins is often accomplished using one or more chromatographic purification steps; a particularly applicable type of chromatographic purification of proteins that contain the immunoglobulin Fc region (for example, antibody) is protein A binding chromatography. However, host cell proteins (HCPs) have been shown to co-elute with antibodies during conventional protein capture chromatography (including protein A binding chromatography) such that may present a problem for the subsequent use of these antibodies. Typically one or more wash steps are used after product binding (e.g. protein containing the Fc region of the immunoglobulin) with a chromatographic resin before elution. Unfortunately, current wash formulations made from salt and buffers may not be suitable for disrupting the interaction of HCP and other contaminants with various monoclonal antibody (mAb) products. Therefore, there is a need for improved purification methods (for example, when introducing new flushing compositions) that reduce the concentration/amount of impurities (for example, HCP) when co-purified with antibodies (e.g. during affinity chromatography based on binding to protein A).

[0004] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB/Swiss-Prot access numbers, are incorporated herein by reference in their entirety, as if each individual reference were specifically and separately listed for inclusion by reference.

BRIEF DESCRIPTION OF THE INVENTION

[0005] To meet the above and other needs, this document discloses improved methods for purifying polypeptides containing an Fc region from one or more impurities. These methods involve contacting a protein A binding chromatography matrix with a sample (e.g., cell lysate) containing (i) a polypeptide containing an Fc region and (ii) one or more contaminants, and washing the matrix with a wash solution having a pH of about 4.0 to 10.0 and containing a benzoate salt and /or benzyl alcohol. The present invention is based at least in part on the unexpected discovery that the use of a benzoate salt (for example, sodium benzoate) and/or benzyl alcohol in a wash solution at a pH of from about 4.0 to 10.0 during protein A binding chromatography provides excellent removal of impurities (e.g., host cell contaminants) compared to currently used wash formulations (see fig. 1, example 1). The present invention is also based at least in part on the discovery that the inclusion of one or more additional components selected from benzenesulfonate (for example, sodium benzenesulfonate), caprylic acid, hexylene glycol, and/or arginine, can further improve the removal of impurities when included in the wash solution (see fig. 2 and 3, example 1).

[0006] Therefore, in one aspect, this document provides a method for purifying a polypeptide containing an Fc region, the method comprising the steps of: (a) contacting a protein A binding chromatography matrix with a sample containing (i) a polypeptide , containing the Fc region, and (ii) one or more impurities, provided that the polypeptide containing the Fc region binds to protein A; and (b) washing the matrix with a wash solution, where the wash solution contains one of (i) a benzoate salt at a concentration of from about 0.1 M to about 1.0 M and (ii) benzyl alcohol at a concentration of from about 0.5% to about 4% v/v (v/v), or both, and wherein the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution contains: (1) a benzoate salt; (2) benzyl alcohol; or (3) benzoate salt and benzyl alcohol. In some embodiments, the benzoate salt is present at a concentration of from about 0.1 M to about 0.5 M. In some embodiments, which may be combined with any of the previous embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, which may be combined with any of the previous embodiments, the benzoate salt is sodium benzoate. In some embodiments, sodium benzoate is present at a concentration of from about 0.1 M to about 0.3 M. In some embodiments, sodium benzoate is present at a concentration of about 0.3 M. In some embodiments, sodium benzoate is present at a concentration of of about 0.5 M. In some embodiments, which may be combined with any of the previous embodiments, the benzyl alcohol is present at a concentration of from about 1% to about 4% (v/v). In some embodiments, which may be combined with any of the previous embodiments, the benzyl alcohol is present at a concentration of from about 1% to about 2% (v/v). In some embodiments, which may be combined with any of the previous embodiments, the benzyl alcohol is present at a concentration of approximately 2% (v/v). In some embodiments, which may be combined with any of the previous embodiments, the benzyl alcohol is present at a concentration of approximately 4% (v/v).

[0007] In some embodiments, which may be combined with any of the previous embodiments, the wash solution further comprises a buffering agent. In some embodiments, the buffering agent is selected from phosphate, tris, arginine, acetate, and citrate. In some embodiments, the buffering agent is present at a concentration of about 10 mM to about 50 mM, or about 10 mM to about 500 mM. In some embodiments, the buffering agent is present at a concentration of approximately 50 mM. In some embodiments, the buffering agent is present at a concentration of approximately 500 mM. In some embodiments, the wash solution has a pH of about 5.0 to about 10.0. In some embodiments, the wash solution has a pH of about 5.0 to about 9.0. In some embodiments, the wash solution has a pH of about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, or about 10.0.

[0008] In some embodiments, which may be combined with any of the previous embodiments, the wash solution further comprises sodium benzenesulfonate. In some embodiments, the sodium benzenesulfonate is present at a concentration of from about 0.1 M to about 0.5 M. In some embodiments, which may be combined with any of the previous embodiments, the wash solution further contains caprylic acid. In some embodiments, caprylic acid is present at a concentration of from about 10 mM to about 50 mM. In some embodiments, which may be combined with any of the previous embodiments, the wash solution further comprises hexylene glycol. In some embodiments, hexylene glycol is present at a concentration of from about 1% to about 10% (v/v). In some embodiments, which may be combined with any of the previous embodiments, the wash solution further comprises creatine. In some embodiments, the creatine is present at a concentration of from about 10 mM to about 100 mM. In some embodiments, which may be combined with any of the previous embodiments, the wash solution further comprises arginine. In some embodiments, the arginine is present at a concentration of about 0.1 M to about 1.0 M. In some embodiments, the arginine is present at a concentration of about 0.5 M. In some embodiments, the arginine is arginine-HCl. In some embodiments, the arginine-containing wash solution has a pH of about 4.0 to about 6.0. In some embodiments, the arginine-containing wash solution has a pH of about 8.0 to about 10.0. In some embodiments, which may be combined with any of the previous embodiments, the wash solution further comprises one or more non-buffer salts. In some embodiments, the one or more non-buffer salts are selected from sodium chloride, sodium bromide, potassium chloride, potassium bromide, magnesium chloride, magnesium bromide, calcium chloride, calcium bromide, and any combinations thereof. In some embodiments, the one or more non-buffer salts are sodium chloride and/or potassium chloride. In some embodiments, one or more non-buffer salts are present at a concentration of from about 0.1 M to about 1.0 M.

[0009] In some embodiments, which may be combined with any of the previous embodiments, the wash solution is a solution selected from: (i) a solution containing sodium benzoate at a concentration of approximately 0.5 M and sodium bicarbonate at a concentration of approximately 50 mm, characterized by a pH of approximately 10.0; (ii) a solution containing sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2%, arginine at a concentration of approximately 0.5 M, and sodium phosphate at a concentration of approximately 50 mM, characterized by a pH of approximately 9.0; (iii) a solution containing sodium benzoate at a concentration of approximately 0.5 M and benzyl alcohol at a concentration of approximately 2% (v/v), characterized by a pH of approximately 7.0; (iv) a solution containing sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and sodium chloride at a concentration of approximately 0.5 M, characterized by the index a pH of approximately 7.0; (v) a solution containing hexylene glycol at a concentration of approximately 10% (v/v), sodium benzoate at a concentration of approximately 0.5 M, and benzyl alcohol at a concentration of approximately 2% (v/v). ), characterized by a pH of approximately 7.0; (vi) a solution containing benzenesulfonate at a concentration of approximately 0.5 M, sodium benzoate at a concentration of approximately 0.5 M, and benzyl alcohol at a concentration of approximately 2% (v/v), characterized by pH , which is approximately 7.0; (vii) a solution containing caprylic acid at a concentration of approximately 50 mM, sodium benzoate at a concentration of approximately 0.5 M, arginine at a concentration of approximately 0.5 M, and sodium chloride at a concentration of approximately 0.5 M, characterized by a pH of approximately 7.0; (viii) a solution containing sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and arginine at a concentration of approximately 0.5 M, characterized by pH , which is approximately 6.0; (ix) a solution containing sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and arginine at a concentration of approximately 0.5 M, characterized by pH , which is approximately 5.0; (x) a solution containing benzyl alcohol at a concentration of about 4% (v/v) having a pH of about 5.0 to about 10; (xi) a solution containing benzyl alcohol at a concentration of about 4% (v/v) having a pH of about 9.0; and (xii) a solution containing benzyl alcohol at a concentration of approximately 2% (v/v) and arginine at a concentration of approximately 0.5 M, characterized by a pH of approximately 5.0.

[0010] In some embodiments, which may be combined with any of the previous embodiments, the method further comprises the step of washing the matrix with the first solution prior to washing the matrix with the wash solution described above. In some embodiments, the first solution contains a buffer selected from phosphate buffer, Tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combinations thereof. In some embodiments, the first solution contains a buffer at a concentration of from about 10 mM to about 100 mM, or from about 10 mM to about 500 mM. In some embodiments, the first solution is a phosphate buffer.

[0011] In some embodiments, which may be combined with any of the previous embodiments, the method further comprises the step of washing the matrix with a second solution after washing the matrix with the wash solution described above. In some embodiments, the second solution contains a buffer selected from phosphate buffer, tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combinations thereof. In some embodiments, the implementation of the second solution contains a buffer at a concentration of from about 10 mm to about 100 mm or from about 10 mm to about 500 mm. In some embodiments, the second solution has a pH of about 5.0 to about 7.0. In some embodiments, the implementation of the second solution contains essentially a low amount of salt or does not contain salt.

[0012] In some embodiments, which may be combined with any of the previous embodiments, the method further comprises the step of contacting the protein A binding chromatography matrix with the elution solution after one or more washing steps. In some embodiments, the method further comprises the step of collecting an eluate containing a polypeptide containing an Fc region. In some embodiments, the method further comprises the step of filtering the eluate by depth filtration. In some embodiments, the eluate contains less than about 500 parts per million (ppm) of one or more impurities.

[0013] In some embodiments, which may be combined with any of the previous embodiments, the use of the methods described herein results in a polypeptide containing an Fc region purified from one or more impurities to a greater extent than provided by a corresponding method in which there is no step of washing the matrix with a wash solution. In some embodiments, which may be combined with any of the previous embodiments, one or more impurities are host cell proteins (HCPs). In some embodiments, one or more HCPs are selected from phospholipases (eg, protein 2 like the putative phospholipase B), clusterin, serine proteases, elongation factors, and any combinations thereof. In some embodiments, the host cell is a mammalian host cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0014] In some embodiments, the Fc region is a human Fc region. In some embodiments, a human Fc region provides for a human IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a mouse Fc region. In some embodiments, the mouse Fc region provides for a mouse IgG1, IgG2, or IgG3 Fc region. In some embodiments, which may be combined with any of the previous embodiments, the Fc region-containing polypeptide is an antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a bispecific antibody or a trispecific antibody.

[0015] In some embodiments, any of the above methods further comprises, prior to contacting the protein A binding chromatography matrix, with a sample comprising (i) a polypeptide containing an Fc region, and (ii) one or more impurities, adjusting a harvested material containing an Fc region containing polypeptide to achieve a final benzoate salt concentration of about 0.1 M to about 0.5 M and a pH of about 7.0 to about 9.0, e.g. , to obtain a sample containing (i) a polypeptide containing an Fc region, and (ii) one or more impurities. In some embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, the benzoate salt is sodium benzoate. In some embodiments, the final concentration of the benzoate salt in the collected material is from about 0.4 M to about 0.5 M. In some embodiments, the pH of the collected material after adjustment is from about 7.0 to about 8.0. In some embodiments, the pH of the collected material after adjustment is from about 8.0 to about 9.0. In some embodiments, the harvested material is from a culture containing a host cell engineered to express the polypeptide. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the eukaryotic host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the collected material is clarified prior to correction. In some embodiments, the collected material is clarified after adjustment.

[0016] In a related aspect, a method is provided for purifying an Fc region containing polypeptide, the method comprising the steps of: (A) adjusting the harvested material containing the Fc region containing polypeptide to achieve a final benzoate salt concentration of from about 0.1 M to about 0.5 M, and a pH of about 7.0 to about 9.0, for example, to obtain a sample containing (i) a polypeptide containing an Fc region, and (ii) one or more impurities; and (B) contacting the sample with at least one chromatography matrix. In some embodiments, at least one chromatography matrix provides an affinity chromatography matrix. In some embodiments, the affinity chromatography matrix is a Protein A binding chromatography matrix or a Protein G binding chromatography matrix. In some embodiments, the method further comprises the step of contacting at least one chromatography matrix with to at least one wash solution. In some embodiments, the method further comprises the step of contacting at least one chromatography matrix with the elution solution. In some embodiments, the method further comprises the step of collecting an eluate containing a polypeptide containing an Fc region. In some embodiments, the method further comprises the step of filtering the eluate by depth filtration. In some embodiments, the eluate contains less than about 500 parts per million (ppm) of one or more impurities.

[0017] In some embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, the benzoate salt is sodium benzoate. In some embodiments, the final concentration of the benzoate salt in the collected material is from about 0.4 M to about 0.5 M. In some embodiments, the pH of the collected material after adjustment is from about 7.0 to about 8.0. In some embodiments, the pH of the collected material after adjustment is from about 8.0 to about 9.0. In some embodiments, the harvested material is from a culture containing a host cell engineered to express the polypeptide. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the eukaryotic host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the collected material is clarified prior to correction. In some embodiments, the collected material is clarified after adjustment. In some embodiments, the method results in a polypeptide containing an Fc region purified from one or more contaminants to a higher degree than provides a corresponding method for obtaining a sample, in which there is no step of correcting the collected material containing the polypeptide containing the Fc region, with receiving a sample. In some embodiments, one or more impurities are host cell proteins (HCPs). In some embodiments, one or more HCPs are selected from the group consisting of phospholipases, clusterin, serine proteases, elongation factors, and any combinations thereof. In some embodiments, the implementation of the HCP is a protein 2, like the hypothetical phospholipase B (PLBL2). In some embodiments, the Fc region is a human Fc region. In some embodiments, a human Fc region provides for a human IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a mouse Fc region. In some embodiments, the mouse Fc region provides for a mouse IgG1, IgG2, or IgG3 Fc region. In some embodiments, the implementation of the polypeptide containing the Fc region is an antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a bispecific antibody or a trispecific antibody.

[0018] It should be understood that one, more or all of the features of the various embodiments described herein can be combined to obtain other embodiments of the present invention. These and other aspects of the present invention will be apparent to a person skilled in the art. These and other embodiments of the present invention are further disclosed in the following detailed description.

BRIEF DESCRIPTION OF GRAPHICS

[0019] FIG. 1 shows the protein contaminant concentration (HCP) of Chinese Hamster Ovary (CHO) host cells in antibody samples eluted from Protein A columns after washing with the specified control or test wash solutions.

[0020] FIG. 2A-B show specific HCP (HCP-A) concentrations in antibody samples eluted from Protein A columns . FIG. 2A shows the concentration of HCP-A in antibody samples eluted from protein A columns after washing with 2% benzyl alcohol ± 0.5 M sodium benzoate and/or 0.5 M arginine, compared to a control wash as assessed by ELISA. In FIG. 2B shows the concentration of HCP-A in antibody samples eluted from Protein A columns after washing with various washes at pH 9.0 or 10.0 compared to a control wash as assessed by ELISA.

[0021] In FIG. 3 shows the concentration of HCP-A in antibody samples eluted from Protein A columns after washing with the indicated wash solutions containing additional test compounds, as assessed by ELISA.

[0022] Onfig. 4A - B shows the concentrations of total HCP and PLBL2 in antibody samples eluted from protein A columns. On thefig. 4A shows the concentration of total HCP in antibody samples eluted from protein A columns after washing with 0.5 M arginine, 0.5 M sodium benzoate, or 4% benzyl alcohol compared to the control washing process as assessed by ELISA. On thefig. 4B shows the concentration of total PLBL2 in antibody samples eluted from protein A columns after washing with 0.5 M arginine, 0.5 M sodium benzoate, or 4% benzyl alcohol compared to the control washing process as assessed by ELISA.

[0023] FIG. 5 shows the improvement in visual clarity of antibody samples eluted from Protein A columns washed with an intermediate wash containing 2% benzyl alcohol and 0.5 M sodium benzoate.

[0024] In FIG. 6A-B show the reduction in post-column yield and removal of PLBL2 when loading columns with protein A greater than 40 g/L. In FIG. 6A shows that the post-column yield decreases linearly from 93.1% to 78.1% as the protein A column loading density increases from 40 g/L to 60 g/L. In FIG. 6B shows that the level of PLBL2 eluted from the Protein A column decreases from 32.1 ppm to 17 ppm as the loading density of the Protein A columns increases from 40 g/L to 60 g/L.

[0025] FIG. 7 shows that adjusting the harvest to 0.5 M sodium benzoate and pH 7.2 or adjusting the harvest to 0.5 M sodium benzoate and pH 9 prior to purification based on protein A binding resulted in improved removal of PLBL2 and HCP. Correction of the collected material to 0.5 M sodium benzoate at pH 9.0 showed the lowest level of impurities PLBL2 and HCP and showed a degree of purification from PLBL2, which is an order of magnitude higher than that of the correction of pH alone.

[0026] FIG. 8 shows that the relationship between PLBL2 content and sodium benzoate concentration is almost sigmoidal. Decreased gain in PLBL2 clearance was observed for sodium benzoate concentrations greater than 0.4 M.

DETAILED DESCRIPTION

[0027] This document describes methods for reducing the amount of impurities (for example, host cell protein contaminants) during copurification to isolate Fc region containing proteins based on protein A binding. which has been shown to significantly reduce levels of host cell protein contaminants in eluates collected from protein A binding affinity chromatography (see examples 1 and 2). The inclusion of one or more additives (for example, benzenesulfonate, caprylic acid, hexylene glycol, creatine and/or arginine) into this new wash solution further improves the decontamination of the protein eluate containing the Fc region containing protein after capture and elution from the protein A matrix.

I. Definitions

[0028] In considering the detailed description of the present invention, it should be appreciated that the present invention is not limited to specific compositions or biological systems, which, of course, may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0029] As used herein and in the appended claims, the singular forms are intended to refer to the plural unless the context clearly indicates otherwise. Thus, for example, reference to the term “molecule” does not necessarily include a combination of two or more such molecules, etc.

[0030] As used herein, the term "approximately" refers to the usual range of errors for the corresponding value, well known to a person skilled in the art. Reference to "about" in relation to a value or parameter in this document includes (and describes) embodiments that are directed to a given value or a given parameter per se .

[0031] The aspects and embodiments of the present invention described herein are intended to include the terms "comprising", "consisting of", and "consisting essentially of" the aspects and embodiments.

[0032] As used herein, the term "and/or" in a phrase such as "A and/or B" means the inclusion of both A and B; A or B; A (separate) and B (separate). Similarly, as used herein, the term “and/or” in a phrase such as “A, B and/or C” encompasses each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (separately); B (separate) and C (separate).

[0033] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be separated by non-amino acid compounds. The terms also cover a polymer of amino acids that has been modified naturally or through intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation to a labeling moiety or toxin. Also included within the scope of this definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids etc.), as well as other modifications known in the art.

[0034] As used herein, the term “antibody” broadly and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies etc.), antibody fragments or synthetic polypeptides bearing one or more CDR sequences or derived from CDRs, provided that the polypeptides exhibit the desired activity. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. In general, antibodies are considered to be Ig with defined or recognized specificity. Thus, while antibodies exhibit binding specificity for a particular target, immunoglobulins include both antibodies and other antibody-like molecules that do not have target specificity. The antibodies of the present invention may be of any class (for example, IgG, IgE, IgM, IgD, IgA etc.) or subclass (for example, IgG1, IgG2, IgG2a, gG3, IgG4, IgA1, IgA2 etc.). The terms "type", "class", "subtype", and "subclass" are used interchangeably herein. Antibodies and immunoglobulins, whether native or wild-type (obtained from a non-artificially manipulated member of the population), are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a series of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The antibodies described herein may be human antibodies, humanized antibodies, non-human animal antibodies (e.g., mouse, rat, hamster, camel rabbit etc.), or chimeric antibodies.

[0035] The term "variable" in the context of an antibody variable domain can refer to certain portions of the corresponding molecule that differ greatly in sequence among antibodies and are used for specific recognition and binding, or a particular antibody in relation to its particular target. However, variability is not evenly distributed across the variable domains of antibodies. Variability is concentrated in three segments called complementarity determining regions (CDRs), also known as hypervariable regions, in both the light chain and heavy chain variable domains. The most highly conserved portions of variable domains are called framework (FR) regions or sequences. Each of the native heavy and light chain variable domains contains four FR regions, mostly in the β-sheet configuration, connected by three CDRs that form loops that integrate the β-sheet structure and in some cases form part of it. The CDRs in each strand are held together, often in close proximity to the FR regions, and together with CDR2 from the other strand, contribute to the formation of an antibody target (epitope or determinant) binding site (see Kabatet al. Sequences of Proteins of Immunological Interest, Nation Institute of Health, Bethesda, MD (1987)). As used herein, the numbering of amino acid residues of immunoglobulin is in accordance with the numbering system of amino acid residues of immunoglobulin according to Kabatet al., unless otherwise specified. One CDR may be characterized by the ability to specifically bind to a genetically related epitope.

[0036] As used herein, the term "hinge" or "hinge region" may refer to a flexible polypeptide containing an amino acid between the first and second constant domains of an antibody.

[0037] The term "bispecific antibodies" may refer to molecules that combine the antigen-binding sites of two antibodies in one molecule. Thus, a bispecific antibody is capable of simultaneously binding to two different antigens.

[0038] As used herein, the term “monoclonal antibody” may refer to an antibody derived from a population of substantially homogeneous antibodies, i.e. the individual antibodies that make up a population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies as used herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain (chains) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they retain the required activity.

[0039] As used herein, the term "multivalent antibody" or "multivalent antibody" may refer to an antibody containing two or more antigen-binding sites, thus capable of simultaneously binding two or more antigens, which may have the same or different structure. The term "bivalent" means that the antibody contains two antigen-binding sites. The term "quadrivalent" means that the antibody contains four antigen-binding sites.

[0040] As used herein, the term “antigen binding site” may refer to the portion of an antibody that contains a site that specifically binds to and is complementary to part or all of the antigen. When the antigen is large, the antibody can only bind to a specific part of the antigen, which is called an epitope. An antigen binding domain may be provided by one or more antibody variable domains and may consist of an association of an antibody light chain (VL) variable domain and an antibody heavy chain (VH) variable domain.

[0041] “Humanized” forms of non-human antibodies (e.g., murine), are chimeric immunoglobulins, immunoglobulin chains or fragments thereof, which contain sequences derived from a non-human immunoglobulin, as opposed to a human antibody. In general, a humanized antibody will contain substantially all of one, and typically two, variable domains wherein all or substantially all of the CDRs correspond to such non-human immunoglobulin regions and all or substantially all of the FRs are as in the human immunoglobulin template sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region, typically a selected human immunoglobulin template. Typically, the goal is to obtain an antibody molecule that has minimal immunogenicity in the human body. Thus, it is possible that one or more amino acids in one or more CDRs can also be substituted for an amino acid that is less immunogenic to the human host without substantially minimizing the specific binding function of one or more CDRs to a target. Alternatively, the FR may be different from human, but the most immunogenic amino acids are replaced with less immunogenic ones. However, CDR grafting (as described above) is not the only way to obtain a humanized antibody. For example, modifying only the CDR regions may not be sufficient, since framework residues often play a role in determining the three-dimensional structure of the CDR loops and the overall affinity of an antibody for its ligand. Therefore, any means can be used, so the non-human parent antibody molecules are modified to be less immunogenic in humans, and general sequence identity with a human antibody is not always a necessary condition.

[0042] The term "impurity" can refer to any foreign or unwanted molecule that is present in solution (such as a sample containing a polypeptide containing an Fc region). The impurity can be a biological (molecule) (for example, macromolecule), such as DNA, RNA or protein, which is also present in the sample containing the protein of interest. Impurities may include unwanted protein variants (for example, aggregated proteins, misfolded proteins, proteins with reduced disulfide bonds, fragments etc.), other proteins from host cells, components of the cell culture medium, molecules that are part of the absorbent used for affinity chromatography (e.g., protein A), endotoxins, nucleic acids, viruses, etc.

II. Methods for isolating and/or purifying polypeptides containing an FC region

Review

[0043] Some aspects of the present invention relate to a method for purifying a polypeptide containing an Fc region (for example, antibodies), via protein A binding chromatography. In some embodiments, the method comprises the steps of contacting a protein A binding chromatography matrix or resin with a sample comprising (1) a polypeptide containing an Fc region (e.g., antibody), and (2) one or more impurities (for example, impurities of the host cell), provided that the polypeptide containing the Fc region is bound (for example, antibodies), with protein A and washing the matrix with a wash solution containing a benzoate salt and/or benzyl alcohol. In some embodiments, the wash solution contains a benzoate salt at a concentration of from about 0.1 M to about 1.0 M. In some embodiments, the wash solution contains benzyl alcohol at a concentration of from about 0.5% to about 4% by volume. /volume (vol./vol.). In some embodiments, the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution contains one or more additives (for example, one or more of benzenesulfonate, caprylic acid, hexylene glycol, non-buffered salt (such as sodium chloride), creatine and/or arginine). In some embodiments, the wash solution further comprises a buffering agent. In some embodiments, a harvested material that contains a polypeptide containing an Fc region is adjusted to achieve a final benzoate salt concentration of about 0.1M to 0.5M and a pH of about 7 to about 9, to obtain a sample containing (1) a polypeptide containing an Fc region (for example, antibody), and (2) one or more impurities (for example, host cell impurities).

Contacting the Sample with a Matrix or Resin Containing Protein A

[0044] Some aspects of the present invention relate to methods for purifying a polypeptide containing an Fc region (for example, antibodies), via protein A binding chromatography. In some embodiments, the method comprises the step of contacting a protein A binding chromatography matrix or resin with a sample comprising (1) a polypeptide containing an Fc region (e.g., antibody), and (2) one or more impurities (for example, impurities of the host cell), provided that the polypeptide containing the Fc region is bound (for example, antibodies), with protein A.

[0045] In some embodiments, the present invention relates to methods for isolating a polypeptide containing an Fc region (e.g., antibody, immunoadhesin, fusion protein etc.), from a sample (for example, cell lysate sample, cell culture supernatant sample etc.). In some embodiments, the sample is a cell culture supernatant (e.g., supernatant of cells such as CHO cells engineered to produce and secrete the polypeptide), or derived from cell culture supernatant (e.g., sample of partially purified cell culture supernatant). In some embodiments, the implementation of the polypeptide containing the Fc region is a secreted polypeptide. In some embodiments, the Fc region is the C-terminal region of an immunoglobulin heavy chain and may include native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is usually defined as the stretch from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus (numbering of residues in the Fc region is an index to the EU, as well as to Kabat). The Fc region of an immunoglobulin typically contains two constant domains, CH2 and CH3, and optionally contains a CH4 domain. In some embodiments, the Fc region is an Fc region derived from any suitable immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4, IgA, IgE, IgD, or IgM subtypes. In some embodiments, the polypeptide comprises an Fc region with the amino acid sequence of a human Fc region, a non-human animal Fc region (e.g., mice, rats, rabbits, hamsters etc.), or any combination of them. In some embodiments, the Fc region is a mouse Fc region. In some embodiments, the mouse Fc region provides for a mouse IgG1, IgG2, or IgG3 Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, a human Fc region provides for a human IgG1, IgG2, and/or IgG4 Fc region.

[0046] In some embodiments, the implementation of the polypeptide containing the Fc region is an antibody. In some embodiments, the term “antibody” is used herein in its broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multivalent antibodies (e.g., divalent, trivalent, tetravalent etc.) and multispecific antibodies (for example, bispecific, trispecific etc.). Antibodies can be of any origin, including, for example, derived from humans, non-human primates, rodents (e.g., mice, rats, hamsters etc.), rabbits, camels, sharks, and/or can be produced recombinantly. In some embodiments, the antibody is a human antibody, a humanized antibody, and/or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a multispecific and/or multivalent antibody. In some embodiments, the antibody is a bispecific antibody or a trispecific antibody.

[0047] In some embodiments, the implementation of the sample (for example, cell lysate sample, cell culture supernatant sample etc.), containing a polypeptide containing an Fc region, additionally contains one or more impurities. In some embodiments, one or more impurities are present in the sample due to the method used to produce the polypeptide containing the Fc region (e.g., method for producing secreted antibodies). In some embodiments, one or more impurities are one or more impurities derived from a host cell (e.g., one or more host cell proteins, one or more host cell nucleic acids, one or more host cell lipids etc.). The host cell may be any host cell known in the art suitable for the production of a polypeptide containing an Fc region, including, for example, prokaryotic cells (such as cellsE. coli,, cellsA.niger etc.), eukaryotic cells (such as yeast cells, plant cells, insect cells (for example, S1 cells) and/or mammalian cells (mouse, rat, hamster, rabbit, human, non-human primate etc.) (for example, hybridomas, CHO cells, 293T cells, PER.C6 cells, NS0 cells etc.). In some embodiments, one or more impurities are one or more host cell proteins (HCPs). In some embodiments, HCP refers to a non-product protein produced by a host cell during cell culture or fermentation. In some embodiments, one or more impurities are one or more (for example, one or more, two or more, three or more, four or more, etc..) host cell proteins (HCPs) selected from phospholipases, clusterin, serine proteases, elongation factors and/or any combination thereof. In some embodiments, the host cell is a CHO cell. In some embodiments, one or more contaminants are one or more CHO cell HCPs. In some embodiments, one or more of the CHO cell HCPs is one or more of phospholipases, clusterin, serine proteases, elongation factors, and/or any combinations thereof.

[0048] In some embodiments, the present invention relates to methods for purifying a polypeptide containing an Fc region from one or more impurities in a sample by protein A binding chromatography. In some embodiments, the sample is contacted with a matrix or resin containing protein A. In some embodiments, the sample is contacted with a matrix or resin containing protein A under conditions suitable for binding the polypeptide containing the Fc region in the sample to protein A. Methods and suitable conditions for contacting and binding containing The Fc region of a polypeptide with a matrix or resin containing Protein A is well known to the person skilled in the art (e.g., methods described in the protocol of the manufacturer of commercially available matrix or resin containing protein A). Any suitable matrix or resin containing Protein A known in the art may be used in the methods of the present invention, including, for example: MabSelect, MabSelect Xtra, MabSelect Sure, MabSelect Sure LX Protein A, MabSelect pcc, MabSelect PrismA , rProtein A Sepharose CL-4B and nProtein A Sepharose 4 FF (GE Healthcare); EshmunoA, ProSep A, ProSep-vA High Capacity, ProSep-vA Ultra and ProSep-vA UltraPlus (Millipore); Poros A and Mabcapture A (Poros); IPA-300, IPA-400 and IPA-500 (RepliGen Corp.); Protein A Affigel and Protein A Affiprep (Bio-Rad); MABsorbent A1PP and MABsorbent A2P (Affinity Chromatography Ltd.); Protein A Ceramic Hyper D F (Pall Corp.); Protein A Ultralink Immobilized and Protein A Agarose (PIERCE); Protein A Cellthru 300 and Protein A Ultraflow (Bioseparation); Amsphere A3 (JSR) and/or Toyopearl AF-rProtein A HC-650F (Tosoh Biosciences). In some embodiments, a protein A matrix or resin is used in a column chromatography format. In some embodiments, one or more parameters for the Protein A matrix or resin (such as pH, ionic strength, temperature, addition of other substances) are adjusted prior to contacting the Protein A matrix or resin with the sample. In some embodiments, the Protein A matrix or resin is rinsed, washed, equilibrated, stripped, and/or decontaminated before and/or after the Protein A matrix or resin is contacted with the sample. In some embodiments, the Protein A matrix or resin is equilibrated and/or washed prior to contacting the Protein A matrix or resin with the sample. Any suitable equilibration and/or wash buffer known in the art may be used. In some embodiments, the protein A matrix or resin is decontaminated, stripped, and/or regenerated prior to use.

Washing the Matrix or Resin Containing Protein A with a Wash Solution

[0049] Certain aspects of the present invention relate to methods for purifying a polypeptide containing an Fc region by protein A binding chromatography by washing a protein A matrix or resin bound to a polypeptide containing an Fc region (e.g., antibody), wash solution containing benzoate salt and/or benzyl alcohol. In some embodiments, the method includes the step of contacting a Protein A binding chromatography matrix or resin with a sample containing (1) a polypeptide containing an Fc region (e.g., antibody), and (2) one or more impurities (for example, impurities of the host cell), provided that the polypeptide containing the Fc region is bound (for example, antibodies), with Protein A, and washing the matrix or resin with a wash solution containing a benzoate salt at a concentration of from about 0.1 M to about 1.0 M, and/or benzyl alcohol at a concentration of from about 0.5% to about 4% v/v (v/v), wherein the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution contains a benzoate salt. In some embodiments, the wash solution contains benzyl alcohol. In some embodiments, the wash solution contains a benzoate salt and benzyl alcohol.

[0050] In some embodiments, the present invention relates to a wash solution containing a benzoate salt and/or benzoic acid (e.g., with adjusted pH). Any suitable source or form of the benzoate salt (for example, alkali metal salts) and/or benzoic acid known in the art can be used in the wash solutions of the present invention, including, for example, sodium benzoate, potassium benzoate, lithium benzoate, calcium benzoate, magnesium benzoate, beryllium benzoate, barium benzoate, strontium benzoate, rubidium benzoate, cesium benzoate and/or any combination thereof. In some embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, the benzoate salt is sodium benzoate or potassium benzoate. In some embodiments, the benzoate salt is sodium benzoate.

[0051] In some embodiments, the benzoate salt (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of from about 0.1 M to about 1.0 M. For example, the benzoate salt (for example, sodium benzoate) and/or benzoic acid may be present in the wash solution at a concentration of from about 0.1 M to about 1.0 M, from about 0.1 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.1 M to about 0.7 M, from about 0.1 M to about 0.6 M, from about 0.1 M to about 0.5 M, from about 0.1 M to about 0.4 M, from about 0.1 M to about 0.3 M, from about 0.1 M to about 0.2 M, from about 0.2 M to about 1.0 M, from about 0 .2 M to about 0.9 M, from about 0.2 M to about 0.8 M, from about 0.2 M to about 0.7 M, from about 0.2 M to about 0.6 M, from about 0.2 M to about 0.5 M, about 0.2 M to about 0.4 M, about 0.2 M to about 0.3 M, about 0.3 M to about 1.0 M , from about 0.3M to about 0.9M, about 0.3M to about 0.8M, about 0.3M to about 0.7M, about 0.3M to about 0.6M , from about 0.3 M to about 0.5 M, from about 0.3 M to about 0.4 M, from about 0.4 M to about 1.0 M, from about 0.4 M to about 0, 9 M, about 0.4 M to about 0.8 M, about 0.4 M to about 0.7 M, about 0.4 M to about 0.6 M, about 0.4 M to about 0.5 M, from about 0.5 M to about 1.0 M, from about 0.5 M to about 0.9 M, from about 0.5 M to about 0.8 M, from about 0.5 M to about 0.7 M, from about 0.5 M to about 0.6 M, from about 0.6 M to about 1.0 M, from about 0.6 M to about 0.9 M, from about 0, 6 M to approx. 0.8 M, from approx. 0.6 M to about 0.7 M, from about 0.7 M to about 1.0 M, from about 0.7 M to about 0.9 M, from about 0.7 M to about 0.8 M, from about 0, 8 M to about 1.0 M, about 0.8 M to about 0.9 M, or about 0.9 M to about 1.0 M. In some embodiments, the benzoate salt (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of from about 0.1M to about 0.5M. In some embodiments, the benzoate salt (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of from about 0.1 M to about 0.3 M.

[0052] In some embodiments, the benzoate salt (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at any one of about 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0, 4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0, 9 M, 0.95 M, or 1.0 M. In some embodiments, the implementation of the benzoate salt (for example, sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of approximately 0.5 M. In some embodiments, the benzoate salt (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of about 0.1 M or less about 0.1 M, about 0.3 M or less than about 0.3 M, about 0.5 M or less than about 0.5 M, about 0.75 M, or less than about 0.75 M, or about 1.0 M, or less than about 1.0 M. In some embodiments, the benzoate salt (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of about 0.5M or less than about 0.5M.

[0053] In some embodiments, the present invention relates to a wash solution containing benzyl alcohol. Any suitable source or form of benzyl alcohol known in the art can be used in the wash solutions of the present invention.

[0054] In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of from about 0.5% to about 4.0% v/v (v/v). For example, benzyl alcohol may be present in the wash solution at a concentration of from about 0.5% to about 4%, from about 1% to about 4%, from about 1.5% to about 4%, from about 2% to about 4%, about 2.5% to about 4%, about 3% to about 4%, about 3.5% to about 4%, about 0.5% to about 3.5%, about 1 % to about 3.5%, from about 1.5% to about 3.5%, from about 2% to about 3.5%, from about 2.5% to about 3.5%, from about 3% to about 3.5%, about 0.5% to about 3%, about 1% to about 3%, about 1.5% to about 3%, about 2% to about 3%, about 2, 5% to about 3%, about 0.5% to about 2.5%, about 1% to about 2.5%, about 1.5% to about 2.5%, about 2% to about 2.5%, about 0.5% to about 2%, about 1% to about 2%, about 1.5% to about 2%, from about 0.5% to about 1.5%, from about 1% to about 1.5%, or from about 0.5% to about 1% (v/v). In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of from about 1% to about 4% v/v (v/v). In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of from about 1% to about 2% v/v (v/v).

[0055] In some embodiments, benzyl alcohol is present in the wash solution at any one of about 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75% or about 4% (v/v). In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 2% (v/v). In some embodiments, benzyl alcohol is present in the wash solution at a concentration of about 1%, or less than about 1%, about 2%, or less than about 2%, about 3%, or less than about 3%, or about 4%, or less than about 4%. In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 4% or less than about 4% (v/v). In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 4% or less than about 2% (v/v).

Additives

[0056] In some embodiments, the wash solution of the present invention further comprises one or more (e.g., one or more, two or more, three or more, four or more, or all five) of the following additives: benzenesulfonate, caprylic acid, hexylene glycol, non-buffered salt, and/or creatine at any of the concentrations described herein. In some embodiments, the wash solution containing one or more additives has a pH of about 4.0 to about 10.0. In some embodiments, the inclusion of one or more additives in the wash solution further enhances the purification of the polypeptide containing the Fc region from one or more impurities (e.g., host cell contaminants) when carrying out the methods described herein.

[0057] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol and one of benzenesulfonate, caprylic acid, hexylene glycol, a non-buffered salt, and/or creatine at a pH of about 4.0 to about 10 ,0. For example, the wash solution may contain a benzoate salt and/or benzyl alcohol and benzenesulfonate; benzoate salt and/or benzyl alcohol and caprylic acid; benzoate salt and/or benzyl alcohol and hexylene glycol; a benzoate salt and/or benzyl alcohol and a non-buffered salt; or a benzoate salt and/or benzyl alcohol and creatine at a pH of about 4.0 to about 10.0.

[0058] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol and two of benzenesulfonate, caprylic acid, hexylene glycol, a non-buffered salt, and/or creatine at a pH of about 4.0 to about 10 ,0. For example, the wash solution may contain a benzoate salt and/or benzyl alcohol, benzenesulfonate and caprylic acid; benzoate salt and/or benzyl alcohol, benzenesulfonate and hexylene glycol; benzoate salt and/or benzyl alcohol, benzenesulfonate and non-buffered salt; benzoate salt and/or benzyl alcohol, benzenesulfonate and creatine; benzoate salt and/or benzyl alcohol, caprylic acid and hexylene glycol; a benzoate salt and/or benzyl alcohol, caprylic acid and a non-buffered salt; benzoate salt and/or benzyl alcohol, caprylic acid and creatine; benzoate salt and/or benzyl alcohol, hexylene glycol and non-buffered salt; benzoate salt and/or benzyl alcohol, hexylene glycol and creatine; or a benzoate salt and/or benzyl alcohol, a non-buffered salt, and creatine at a pH of about 4.0 to about 10.0.

[0059] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol and three of benzenesulfonate, caprylic acid, hexylene glycol, a non-buffered salt, and/or creatine at a pH of about 4.0 to about 10 ,0. For example, the wash solution may contain a benzoate salt and/or benzyl alcohol, benzenesulfonate, caprylic acid and hexylene glycol; benzoate salt and/or benzyl alcohol, benzenesulfonate, caprylic acid and non-buffered salt; benzoate salt and/or benzyl alcohol, benzenesulfonate, caprylic acid and creatine; benzoate salt and/or benzyl alcohol, benzenesulfonate, hexylene glycol and non-buffered salt; benzoate salt and/or benzyl alcohol, benzenesulfonate, hexylene glycol and creatine; benzoate salt and/or benzyl alcohol, benzenesulfonate, non-buffered salt and creatine; benzoate salt and/or benzyl alcohol, caprylic acid, hexylene glycol and non-buffered salt; benzoate salt and/or benzyl alcohol, caprylic acid, hexylene glycol and creatine; or a benzoate salt and/or benzyl alcohol, caprylic acid, a non-buffered salt and creatine; a benzoate salt and/or benzyl alcohol, hexylene glycol, a non-buffered salt, and creatine at a pH of about 4.0 to about 10.0.

[0060] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol and four of benzenesulfonate, caprylic acid, hexylene glycol, a non-buffered salt, and/or creatine at a pH of about 4.0 to about 10 ,0. For example, the wash solution may contain a benzoate salt and/or benzyl alcohol, benzenesulfonate, caprylic acid, hexylene glycol, and a salt that is not a buffer; benzoate salt and/or benzyl alcohol, benzenesulfonate, caprylic acid, hexylene glycol and creatine; benzoate salt and/or benzyl alcohol, benzenesulfonate, caprylic acid, non-buffered salt and creatine; benzoate salt and/or benzyl alcohol, benzenesulfonate, hexylene glycol, non-buffered salt and creatine; or a benzoate salt and/or benzyl alcohol, caprylic acid, hexylene glycol, a non-buffered salt, and creatine at a pH of about 4.0 to about 10.0.

[0061] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol and all five of benzenesulfonate, caprylic acid, hexylene glycol, a non-buffered salt, and creatine at a pH of about 4.0 to about 10, 0.

[0062] In some embodiments, the implementation of the present invention relates to a wash solution containing benzenesulfonate. Any suitable form or source of benzenesulfonate known in the art may be used in the wash solutions of the present invention, including, for example, the benzenesulfonate salt (e.g., alkali metal salt), such as sodium benzenesulfonate or potassium benzenesulfonate, benzenesulfonic acid, and/or any combinations thereof. In some embodiments, the benzenesulfonate is sodium benzenesulfonate.

[0063] In some embodiments, the benzenesulfonate (e.g., sodium benzenesulfonate) is present in the wash solution at a concentration of from about 0.1M to about 0.5M. For example, sodium benzenesulfonate may be present in the wash solution at a concentration of from about 0.1M to about 0.5M, from about 0.1 M to about 0.4 M, from about 0.1 M to about 0.3 M, from about 0.1 M to about 0.2 M, from about 0.2 M to about 0.5 M, from about 0.2 M to about 0.4 M, from about 0.2 M to about 0.3 M, from about 0.3 M to about 0.5 M, from about 0.3 M to about 0 .4 M, or from about 0.4 M to about 0.5 M. In some embodiments, sodium benzenesulfonate is present in the wash solution at a concentration of from about 0.1 M to about 0.3 M.

[0064] In some embodiments, sodium benzenesulfonate is present in the wash solution at any one of about 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0 .4 M, 0.45 M, or 0.5 M. In some embodiments, sodium benzenesulfonate is present in the wash solution at a concentration of about 0.5 M. In some embodiments, sodium benzene sulfonate is present in the wash solution at a concentration of about 0 ,1 M or less than about 0.1 M, about 0.3 M or less than about 0.3 M or about 0.5 M or less than about 0.5 M. In some embodiments, sodium benzenesulfonate is present in the wash solution at a concentration of about 0.5 M or less than about 0.5 M.

[0065] In some embodiments, the implementation of the present invention relates to a wash solution containing caprylic acid. Any suitable form or source of caprylic acid known in the art may be used in the wash solutions of the present invention.

[0066] In some embodiments, caprylic acid is present in the wash solution at a concentration of from about 1 mM to about 50 mM. For example, caprylic acid may be present in the wash solution at a concentration of from about 1 mM to about 50 mM, from about 10 mM to about 50 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, from about 40 mM to about 50 mM, about 1 mM to about 40 mM, about 10 mM to about 40 mM, about 20 mM to about 40 mM, about 30 mM to about 40 mM, about 1 mM to about 30 mM, about 10 mM to about 30 mM, about 20 mM to about 30 mM, about 1 mM to about 20 mM, about 10 mM to about 20 mM, or about 1 mM to about 10 mM. In some embodiments, caprylic acid is present in the wash solution at a concentration of from about 10 mM to about 50 mM.

[0067] In some embodiments, caprylic acid is present in the wash solution at any concentration of about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mm. In some embodiments, caprylic acid is present in the wash solution at a concentration of approximately 50 mM. In some embodiments, caprylic acid is present in the wash solution at a concentration of about 10 mM or less than about 10 mM, about 30 mM or less than about 30 mM, or about 50 mM or less than about 50 mM. In some embodiments, caprylic acid is present in the wash solution at a concentration of about 50 mM or less than about 50 mM.

[0068] In some embodiments, the implementation of the present invention relates to a wash solution containing hexylene glycol. Any suitable form or source of hexylene glycol known in the art can be used in the wash solutions of the present invention.

[0069] In some embodiments, hexylene glycol is present in the wash solution at a concentration of from about 0.5% to about 10% (v/v). For example, hexylene may be present in the wash solution at a concentration of from about 0.5% to about 10%, from about 1% to about 10%, from about 2% to about 10%, from about 4% to about 10%, about 6% to about 10%, about 8% to about 10%, about 9% to about 10%, about 0.5% to about 9%, about 1% to about 9%, about 2 % to about 9%, about 4% to about 9%, about 6% to about 9%, about 8% to about 9%, about 0.5% to about 8%, about 1% to about 8%, about 2% to about 8%, about 4% to about 8%, about 6% to about 8%, about 0.5% to about 6%, about 1% to about 6%, from approx. 2% to approx. 6%, about 4% to about 6%, about 0.5% to about 4%, about 1% to about 4%, about 2% to about 4%, about 0.5% to about 2%, about 1% to about 2%, or about 0.5% to about 1% (v/v). In some embodiments, hexylene glycol is present in the wash solution at a concentration of from about 1% to about 10% (v/v).

[0070] In some embodiments, hexylene glycol is present in the wash solution at any concentration from about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% (vol. /about.). In some embodiments, hexylene glycol is present in the wash solution at a concentration of about 10% (v/v). In some embodiments, hexylene glycol is present in the wash solution at a concentration of about 1% or less than about 1%, about 2% or less than about 2%, about 4% or less than about 4%, about 6% or less than about 6%, about 8% or less about 8% or about 10% or less than about 10% (v/v). In some embodiments, hexylene glycol is present in the wash solution at a concentration of about 10% or less than about 10% (v/v).

[0071] In some embodiments, the present invention relates to a wash solution containing one or more (for example, one, two or more, three or more etc.) non-buffer salts. Any suitable form or source of non-buffered salt known in the art can be used in the wash solutions of the present invention. Non-buffer salts may include halogen salts (such as those containing Cl or Br), in particular alkali metal (such as Na or K) or alkaline earth (such as Ca or Mg) containing halogen salts. In some embodiments, the non-buffer salt is sodium chloride or potassium chloride. In some embodiments, the non-buffered salt is sodium chloride.

[0072] In some embodiments, a non-buffered salt (e.g., sodium chloride) is present in the wash solution at a concentration of from about 0.1 M to about 1.0 M. For example, a salt that is not a buffer (for example, sodium chloride) may be present in the wash solution at a concentration of from about 0.1 M to about 1.0 M, from about 0.1 M to about 0.8 M, from about 0.1 M to about 0.6 M, from about 0.1 M to about 0.5 M, from about 0.1 M to about 0.4 M, from about 0.1 M to about 0.2 M, from about 0.2 M to about 1 .0 M, from about 0.2 M to about 0.8 M, from about 0.2 M to about 0.6 M, from about 0.2 M to about 0.5 M, from about 0.2 M to about 0.4 M, from about 0.4 M to about 1.0 M, from about 0.4 M to about 0.8 M, from about 0.4 M to about 0.6 M, from about 0.4 M to about 0.5 M, from about 0.5 M to about 1.0 M, from about 0.5 M to about 0.8 M, from about 0.5 M to about 0.6 M, from about 0 .6 M to about 1.0 M, from about 0.6 M to about 0.8 M, or from about 0.8 M to about 1.0 M. In some embodiments, a non-buffered salt (e.g., sodium chloride) is present in the wash solution at a concentration of from about 0.1M to about 0.5M. In some embodiments, the non-buffer salt (e.g., sodium chloride) is present in the wash solution at a concentration of from about 0.5 M to about 1.0 M.

[0073] In some embodiments, a non-buffered salt (e.g., sodium chloride), present in the wash solution at any concentration of approximately 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0 .45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0 .95 M or 1.0 M. In some embodiments, a non-buffered salt (e.g., sodium chloride) is present in the wash solution at a concentration of approximately 0.5 M. In some embodiments, a non-buffer salt (e.g., sodium chloride) is present in the wash solution at a concentration of approximately 1.0 M. In some embodiments, a non-buffer salt (e.g., sodium chloride), present in the wash solution at a concentration of about 0.1 M or less than about 0.1 M, about 0.2 M or less than about 0.2 M, about 0.4 M or less than about 0.4 M , about 0.5 M or less about 0.5 M, about 0.6 M or less about 0.6 M, about 0.8 M or less about 0.8 M or about 1.0 M or less about 1, 0 M. In some embodiments, a salt that is not a buffer (for example, sodium chloride) is present in the wash solution at a concentration of about 0.5M or less than about 0.5M. In some embodiments, a non-buffer salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 1.0 M or less than about 1.0 M.

[0074] In some embodiments, the present invention relates to a wash solution containing creatine. Any suitable form or source of creatine known in the art may be used in the wash solutions of the present invention, including, for example, creatine-HCl, esters of creatine, creatine pyruvate, creatine phosphate, creatine alpha-ketoglutarate, creatine citrate and/or any combination thereof. In some embodiments, the creatine is creatine-HCl.

[0075] In some embodiments, creatine is present in the wash solution at a concentration of from about 1 mM to about 100 mM. For example, creatine may be present in the wash solution at a concentration of from about 1 mM to about 100 mM, from about 10 mM to about 100 mM, from about 25 mM to about 100 mM, from about 50 mM to about 100 mM, from about 75 mM to about 100 mM, about 1 mM to about 75 mM, about 10 mM to about 75 mM, about 25 mM to about 75 mM, about 50 mM to about 75 mM, about 1 mM to about 50 mM, from about 10 mM to about 50 mM, from about 25 mM to about 50 mM, from about 1 mM to about 25 mM, from about 10 mM to about 25 mM, or from about 1 mM to about 10 mM. In some embodiments, creatine is present in the wash solution at a concentration of from about 10 mM to about 100 mM. In some embodiments, creatine is present in the wash solution at a concentration of from about 10 mM to about 50 mM. In some embodiments, creatine is present in the wash solution at any concentration of about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM , 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM. In some embodiments, creatine is present in the wash solution at a concentration of approximately 50 mM.

Arginine

[0076] In some embodiments, the implementation of the present invention relates to a wash solution additionally containing arginine and/or an arginine derivative. In some embodiments, the inclusion of arginine and/or an arginine derivative in the wash solution further enhances the purification of the polypeptide containing the Fc region from one or more impurities (e.g., host cell contaminants) when carrying out the methods described herein. Any suitable form or source of arginine and/or an arginine derivative known in the art may be used in the wash solutions of the present invention, including, for example, arginine, arginine-HCl, acetylarginine, agmatine, arginic acid, N-alpha -butyroyl-L-arginine, N-alpha-pivaloylarginine and/or any combination thereof. The arginine and/or arginine derivative may be L-arginine and/or D-arginine and derivatives thereof. In some embodiments, the arginine and/or arginine derivative is arginine-HCl.

[0077] In some embodiments, the present invention relates to the use of arginine and/or an arginine derivative (e.g., arginine-HCl) in a wash solution containing benzoate salt and/or benzyl alcohol. In some embodiments, the wash solution contains a benzoate salt and arginine and/or an arginine derivative (e.g., arginine-HCl). In some embodiments, the wash solution contains benzyl alcohol and arginine and/or an arginine derivative (e.g., arginine-HCl). In some embodiments, the wash solution contains a benzoate salt, benzyl alcohol, and arginine and/or an arginine derivative (e.g., arginine-HCl). In some embodiments, the wash solution further comprises one or more (e.g., one or more, two or more, three or more, four or more, or all five) of benzenesulfonate, caprylic acid, hexylene glycol, non-buffered salt, and/or creatine at any of the concentrations described herein. In some embodiments, the wash solution containing arginine and/or an arginine derivative has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution containing arginine and/or an arginine derivative has a pH of about 4.0 to about 6.0. In some embodiments, the wash solution containing arginine and/or an arginine derivative has a pH of about 4.0 to about 5.0. In some embodiments, the wash solution containing arginine and/or an arginine derivative has a pH of about 8.0 to about 10.0. In some embodiments, the wash solution containing arginine and/or an arginine derivative has a pH of about 8.0 to about 9.0.

[0078] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol, arginine, and/or an arginine derivative (e.g., arginine-HCl) and one of benzenesulfonate, caprylic acid, hexylene glycol, non-buffered salt and/or creatine. For example, the wash solution may contain a benzoate salt and/or benzyl alcohol, arginine and benzenesulfonate; benzoate salt and/or benzyl alcohol, arginine and caprylic acid; benzoate salt and/or benzyl alcohol, arginine and hexylene glycol; benzoate salt and/or benzyl alcohol, arginine and non-buffered salt; or a benzoate salt and/or benzyl alcohol, arginine and creatine.

[0079] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol, arginine, and/or an arginine derivative (e.g., arginine-HCl) and two from benzenesulfonate, caprylic acid, hexylene glycol, non-buffered salt and/or creatine. For example, the wash solution may contain a benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate and caprylic acid; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate and hexylene glycol; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate and non-buffered salt; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate and creatine; benzoate salt and/or benzyl alcohol, arginine, caprylic acid and hexylene glycol; benzoate salt and/or benzyl alcohol, arginine, caprylic acid and non-buffered salt; benzoate salt and/or benzyl alcohol, arginine, caprylic acid and creatine; benzoate salt and/or benzyl alcohol, arginine, hexylene glycol and non-buffered salt; benzoate salt and/or benzyl alcohol, arginine, hexylene glycol and creatine; or a benzoate salt and/or benzyl alcohol, arginine, a non-buffered salt, and creatine.

[0080] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol, arginine, and/or an arginine derivative (e.g., arginine-HCl) and three of benzenesulfonate, caprylic acid, hexylene glycol, non-buffered salt and/or creatine. For example, the wash solution may contain a benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, and hexylene glycol; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, caprylic acid and non-buffered salt; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, caprylic acid and creatine; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, hexylene glycol and non-buffered salt; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, hexylene glycol and creatine; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, non-buffered salt and creatine; benzoate salt and/or benzyl alcohol, arginine, caprylic acid, hexylene glycol and non-buffered salt; benzoate salt and/or benzyl alcohol, arginine, caprylic acid, hexylene glycol and creatine; benzoate salt and/or benzyl alcohol, arginine, caprylic acid, non-buffered salt and creatine; or a benzoate salt and/or benzyl alcohol, arginine, hexylene glycol, a non-buffered salt and creatine.

[0081] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol, arginine, and/or an arginine derivative (e.g., arginine-HCl) and four of benzenesulfonate, caprylic acid, hexylene glycol, non-buffered salt and/or creatine. For example, the wash solution may contain a benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, hexylene glycol, and a non-buffered salt; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, hexylene glycol and creatine; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, non-buffered salt and creatine; benzoate salt and/or benzyl alcohol, arginine, benzenesulfonate, hexylene glycol, non-buffered salt and creatine; benzoate salt and/or benzyl alcohol, arginine, caprylic acid, hexylene glycol, non-buffered salt, and creatine.

[0082] In some embodiments, the wash solution contains a benzoate salt and/or benzyl alcohol, arginine, and/or an arginine derivative (e.g., arginine-HCl) and all five of benzenesulfonate, caprylic acid, hexylene glycol, non-buffered salt, and creatine.

[0083] In some embodiments, arginine and/or an arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of from about 0.1 M to about 1.0 M. For example, arginine and/or an arginine derivative (for example, arginine-HCl) may be present in the wash solution at a concentration of from about 0.1 M to about 1.0 M, from about 0.1 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.1 M to about 0.7 M, from about 0.1 M to about 0.6 M, from about 0.1 M to about 0.5 M, from about 0.1 M to about 0 .4 M, from about 0.1 M to about 0.3 M, from about 0.1 M to about 0.2 M, from about 0.2 M to about 1.0 M, from about 0.2 M to about 0.9 M, from about 0.2 M to about 0.8 M, from about 0.2 M to about 0.7 M, from about 0.2 M to about 0.6 M, from about 0.2 M to about 0.5 M, from about 0.2 M to about 0.4 M, from about 0.2 M to about 0.3 M, from about 0.3 M to about 1.0 M, from about 0 .3 M to at about 0.9 M, from about 0.3 M to about 0.8 M, from about 0.3 M to about 0.7 M, from about 0.3 M to about 0.6 M, from about 0.3 M to about 0.5 M, from about 0.3 M to about 0.4 M, from about 0.4 M to about 1.0 M, from about 0.4 M to about 0.9 M, from about 0 .4 M to about 0.8 M, from about 0.4 M to about 0.7 M, from about 0.4 M to about 0.6 M, from about 0.4 M to about 0.5 M, from about 0.5 M to about 1.0 M, about 0.5 M to about 0.9 M, about 0.5 M to about 0.8 M, about 0.5 M to about 0.7 M , from about 0.5 M to about 0.6 M, from about 0.6 M to about 1.0 M, from about 0.6 M to about 0.9 M, from about 0.6 M to about 0, 8 M, from about 0.6 M to about 0.7 M, about t about 0.7 M to about 1.0 M, from about 0.7 M to about 0.9 M, from about 0.7 M to about 0.8 M, from about 0.8 M to about 1.0 M, from about 0.8 M to about 0.9 M, or from about 0.9 M to about 1.0 M. In some embodiments, arginine and/or an arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of from about 0.1M to about 0.5M. In some embodiments, arginine and/or an arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of from about 0.1 M to about 0.3 M.

[0084] In some embodiments, arginine and/or an arginine derivative (e.g., arginine-HCl) is present in the wash solution at any concentration from about 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0 .45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0 .95 M or 1.0 M. In some embodiments, the implementation of arginine and/or an arginine derivative (for example, arginine-HCl) is present in the wash solution at a concentration of approximately 0.5 M. In some embodiments, arginine and/or an arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.1 M or less than about 0.1 M, about 0.2 M or less than about 0.2 M, about 0.3 M or less than about 0.3 M , about 0.4 M or less about 0.4 M, about 0.5 M or less about 0.5 M, about 0.75 M or less about 0.75 M, or about 1.0 M or less about 1 .0 M. In some embodiments, the implementation of arginine and/or an arginine derivative (for example, arginine-HCl) is present in the wash solution at a concentration of about 0.5 M or less than about 0.5 M.

pH value

[0085] In some embodiments, the present invention provides a wash solution having a pH of about 4.0 to about 10.0. For example, the wash solution may have a pH of about 4.0 to about 10.0, about 5.0 to about 10.0, about 6.0 to about 10.0, about 6.5 to about 10.0, about 7.0 to about 10.0, about 7.5 to about 10.0, about 8.0 to about 10.0, about 9.0 to about 10.0, from 4 0 to about 9.0, about 5.0 to about 9.0, about 6.0 to about 9.0, about 6.5 to about 9.0, about 7.0 to about 9, 0, about 7.5 to about 9.0, about 8.0 to about 9.0, 4.0 to about 8.0, about 5.0 to about 8.0, about 6.0 to about 8.0, from about 6.5 to about 8.0, from about 7.0 to about 8.0, from about 7.5 to about 8.0, 4.0 to about 7.5, about 5.0 to about 7.5, about 6.0 to about 7.5, about 6.5 to about 7.5, about 7 0 to about 7.5, about 4.0 to about 7.0, about 5.0 to about 7.0, about 6.0 to about 7.0, about 6.5 to about 7, 0, 4.0 to about 6.5, about 5.0 to about 6.5, about 6.0 to about 6.5, 4.0 to about 6.0, about 5.0 to about 6.0, or from about 4.0 to about 5.0. In some embodiments, the wash solution has a pH of about 5.0 to about 9.0. In some embodiments, the wash solution has a pH of about 4.0 to about 6.0. In some embodiments, the wash solution has a pH of about 4.0 to about 5.0. In some embodiments, the wash solution has a pH of about 8.0 to about 10.0. In some embodiments, the wash solution has a pH of about 8.0 to about 9.0.

[0086] In some embodiments, the wash solution has any pH of about 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0 , 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9 .25, 9.5, 9.75 or 10.0. In some embodiments, the wash solution has a pH of about 4.0. In some embodiments, the wash solution has a pH of about 5.0. In some embodiments, the wash solution has a pH of about 6.0. In some embodiments, the wash solution has a pH of about 6.5. In some embodiments, the wash solution has a pH of about 7.0. In some embodiments, the wash solution has a pH of about 7.5. In some embodiments, the wash solution has a pH of about 9.0. In some embodiments, the wash solution has a pH of about 10.0.

Buffer

[0087] In some embodiments, the wash solution of the present invention further comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more etc.) buffer funds. Any suitable buffer agent known in the art may be used in the wash solutions of the present invention, including, for example, phosphate, tris(tris(hydroxymethyl)methylamine), bis-tris, bis-trispropane, arginine, histidine, triethanolamine, diethanolamine, formate, acetate, carbonate MES (2-(N-morpholino)ethanesulfonic acid), citrate, HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid) , TAPS (3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), bicine (N,N-bis(2-hydroxyethyl)glycine), tricine (N-tris(hydroxymethyl)methylglycine), TES (2-{ [tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), PIPES (piperazine-N, N'-bis(2-ethanesulfonic acid), cacodylate (dimethylarsic acid), SSC (sodium citrate saline) and/or any combinations thereof. In some embodiments, the buffering agent is one or more of phosphate, tris, arginine, acetate, and/or citrate.

[0088] In some embodiments, the buffer means (for example, phosphate, tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of from about 1 mM to about 100 mM, or from about 1 mM to about 500 mM. For example, a buffer (for example, phosphate, tris, arginine, acetate, and/or citrate) may be present in the wash solution at a concentration of about 1 mM to about 500 mM, 10 mM to about 500 mM, 50 mM to about 500 mM, 100 mM to approximately 500 mM, 150 mM to approximately 500 mM, 200 mM to approximately 500 mM, 250 mM to approximately 500 mM, 300 mM to approximately 500 mM, 350 mM to approximately 500 mM, 400 mM to approximately 500 mM, 450 mM to about 500 mM, 1 mM to about 450 mM, 1 mM to about 400 mM, 1 mM to about 350 mM, 1 mM to about 300 mM, 1 mM to about 250 mM, 1 mM to about 200 mM, 1 mM to about 150 mM, 1 mM to about 100 mM, about 10 mM to about 100 mM, about 25 mM to about 100 mM, about 40 mM to about 100 mM , from about 50 mM to about 100 mM, from about 60 mM to about 100 mM, about 75 mM to about 100 mM, about 1 mM to about 75 mM, about 10 mM to about 75 mM, about 40 mM to about 75 mM, about 50 mM to about 75 mM, about 60 mM to about 75 mM, about 1 mM to about 60 mM, about 10 mM to about 60 mM, about 25 mM to about 60 mM, about 40 mM to about 60 mM, from about 50 mM to about 60 mM, about 1 mM to about 50 mM, about 10 mM to about 50 mM, about 25 mM to about 50 mM, about 40 mM to about 50 mM, about 1 mM to about 40 mM, from about 10 mM to about 40 mM, from about 25 mM to about 40 mM, from about 1 mM to about 25 mM, from about 10 mM d about 25 mm or from about 1 mm to about 10 mm. In some embodiments, the buffer means (for example, phosphate, tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of about 10 mM to about 50 mM, or about 10 mM to about 500 mM.

[0089] In some embodiments, the buffer means (for example, phosphate, tris, arginine, acetate and/or citrate) is present in the wash solution at any concentration of approximately 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM. Alternatively, a buffering agent (for example, phosphate, tris, arginine, acetate, and/or citrate) is present in the wash solution at any concentration of about 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. In some embodiments, the buffer means (for example, phosphate, tris, arginine, acetate and/or citrate) is present in the wash solution at a concentration of approximately 500 mM. In some embodiments, the buffer means (for example, phosphate, tris, arginine, acetate and/or citrate) is present in the wash solution at a concentration of approximately 50 mM. In some embodiments, the buffer means (for example, phosphate, tris, arginine, acetate and/or citrate) is present in the wash solution at a concentration of about 10 mM or less about 10 mM, about 25 mM or less about 25 mM, about 50 mM or less about 50 mM, about 75 mM or less than about 75 mM, or about 100 mM, or less than about 100 mM, or about 500 mM, or less than about 500 mM. In some embodiments, the buffer means (for example, phosphate, tris, arginine, acetate and/or citrate) is present in the wash solution at a concentration of about 50 mM or less than about 50 mM. In some embodiments, the buffer means (for example, phosphate, tris, arginine, acetate and/or citrate) is present in the wash solution at a concentration of about 500 mM or less than about 500 mM.

Illustrative Wash Solutions

[0090] In some embodiments, the wash solution of the present invention contains sodium benzoate and/or benzyl alcohol and has a pH of about 7.0. In some embodiments, the wash solution contains about 0.5 M sodium benzoate and/or about 2% (v/v) benzyl alcohol and has a pH of about 7.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M and benzyl alcohol at a concentration of approximately 2% (v/v) and has a pH of approximately 7.0. In some embodiments, the wash solution further comprises a phosphate buffer (e.g., at a concentration of approximately 50 mM).

[0091] In some embodiments, the wash solution of the present invention contains sodium benzoate, benzyl alcohol, and/or sodium chloride and has a pH of about 7.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and/or sodium chloride at a concentration of approximately 0.5 M , and has a pH of approximately 7.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and sodium chloride at a concentration of approximately 0.5 M, and characterized by a pH of approximately 7.0. In some embodiments, the wash solution further comprises a phosphate buffer (e.g., at a concentration of approximately 50 mM).

[0092] In some embodiments, the wash solution of the present invention contains sodium benzoate, benzyl alcohol, arginine, and/or sodium chloride and has a pH of about 7.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), arginine at a concentration of approximately 0.5 M, and/or sodium chloride at a concentration of approximately 0.5 M and has a pH of approximately 7.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), arginine at a concentration of approximately 0.5 M, and sodium chloride. at a concentration of approximately 0.5 M and has a pH of approximately 7.0. In some embodiments, the wash solution further comprises a phosphate buffer (e.g., at a concentration of approximately 50 mM).

[0093] In some embodiments, the wash solution of the present invention contains sodium benzoate, benzyl alcohol, phosphate buffer and/or arginine and has a pH of about 9.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), phosphate buffer at a concentration of approximately 50 mM, and/or arginine at a concentration of approximately 0.5 M and has a pH of approximately 9.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), phosphate buffer at a concentration of approximately 50 mM, and arginine at a concentration of , which is approximately 0.5 M, and is characterized by a pH of approximately 9.0.

[0094] In some embodiments, the implementation of the wash solution of the present invention contains sodium benzoate, benzyl alcohol and/or arginine and has a pH of approximately 6.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and/or arginine at a concentration of approximately 0.5 M, and is characterized by a pH of approximately 6.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and arginine at a concentration of approximately 0.5 M, and is characterized a pH of approximately 6.0.

[0095] In some embodiments, the wash solution of the present invention contains hexylene glycol, sodium benzoate, and/or benzyl alcohol and has a pH of about 7.0. In some embodiments, the wash solution contains hexylene glycol at a concentration of approximately 10% (v/v), sodium benzoate at a concentration of approximately 0.5 M, and/or benzyl alcohol at a concentration of approximately 2% (v/v). /vol.), and is characterized by a pH of approximately 7.0. In some embodiments, the wash solution contains hexylene glycol at a concentration of approximately 10% (v/v), sodium benzoate at a concentration of approximately 0.5 M, and benzyl alcohol at a concentration of approximately 2% (v/v). .), and has a pH of approximately 7.0.

[0096] In some embodiments, the wash solution of the present invention contains benzenesulfonate, sodium benzoate, and/or benzyl alcohol and has a pH of about 7.0. In some embodiments, the wash solution contains benzenesulfonate at a concentration of approximately 0.5 M, sodium benzoate at a concentration of approximately 0.5 M, and/or benzyl alcohol at a concentration of approximately 2% (v/v), and is characterized by a pH of approximately 7.0. In some embodiments, the wash solution contains benzenesulfonate at a concentration of approximately 0.5 M, sodium benzoate at a concentration of approximately 0.5 M, and benzyl alcohol at a concentration of approximately 2% (v/v) and is characterized a pH of approximately 7.0.

[0097] In some embodiments, the wash solution of the present invention contains sodium benzoate, benzyl alcohol, and/or arginine (e.g., arginine-HCl) and has a pH of approximately 5.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and/or arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M has a pH of approximately 5.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M has a pH of approximately 5.0.

[0098] In some embodiments, the wash solution of the present invention contains sodium benzoate, benzyl alcohol, and/or arginine (e.g., arginine-HCl) and has a pH of approximately 6.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and/or arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M has a pH of approximately 6.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, benzyl alcohol at a concentration of approximately 2% (v/v), and arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M has a pH of approximately 6.0.

[0099] In some embodiments, the wash solution of the present invention contains benzyl alcohol and/or arginine (e.g., arginine-HCl) and has a pH of approximately 5.0. In some embodiments, the wash solution contains benzyl alcohol at a concentration of approximately 2% (v/v) and/or arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M and has a pH of approximately 5.0. In some embodiments, the wash solution contains benzyl alcohol at a concentration of approximately 2% (v/v) and arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M and has a pH of approximately 5.0.

[0100] In some embodiments, the wash solution of the present invention contains sodium benzoate, arginine (e.g., arginine-HCl), caprylic acid and/or sodium chloride and has a pH of about 9.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M, caprylic acid at a concentration of approximately 50 mM, and/or sodium chloride at a concentration of approximately 0.5 M, has a pH of approximately 9.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M, caprylic acid at a concentration of approximately 50 mM, and sodium chloride at a concentration of approximately 0.5 M, and has a pH of approximately 9.0.

[0101] In some embodiments, the wash solution of the present invention contains sodium benzoate, arginine (e.g., arginine-HCl), caprylic acid and/or sodium chloride and has a pH of about 7.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M, caprylic acid at a concentration of approximately 50 mM, and/or sodium chloride at a concentration of approximately 0.5 M, and has a pH of approximately 7.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M, arginine (e.g., arginine-HCl) at a concentration of approximately 0.5 M, caprylic acid at a concentration of approximately 50 mM, and sodium chloride at a concentration of approximately 0.5 M, and has a pH of approximately 7.0.

[0102] In some embodiments, the wash solution of the present invention contains sodium benzoate and/or sodium bicarbonate and has a pH of about 10.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M and/or sodium bicarbonate at a concentration of approximately 50 mM and has a pH of approximately 10.0. In some embodiments, the wash solution contains sodium benzoate at a concentration of approximately 0.5 M and sodium bicarbonate at a concentration of approximately 50 mM and has a pH of approximately 10.0.

[0103] In some embodiments, the wash solution of the present invention contains benzyl alcohol at a concentration of about 4% (v/v) and has a pH of about 5.0 to about 10. In some embodiments, the wash the solution contains benzyl alcohol at a concentration of approximately 4% (v/v) and has a pH of approximately 9.0.

Correction of the collected material, which contains the polypeptide containing the Fc region, prior to chromatography

[0104] According to one aspect, a method is provided for purifying a polypeptide containing an Fc region, comprising the steps of: (A) adjusting (A) adjusting the collected material containing a polypeptide containing an Fc region, to achieve a final benzoate salt concentration of from about 0, 1 M to about 0.5 M, and a pH of about 7.0 to about 9.0, to obtain a sample containing (i) a polypeptide containing an Fc region, and (ii) one or more impurities; and (B) contacting the sample with at least one chromatography matrix. In some embodiments, at least one chromatography matrix is an affinity chromatography matrix, e.g., a protein A binding chromatography matrix and/or a protein G binding chromatography matrix. In some embodiments, the method further comprises the step of contacting at least one chromatography matrix with at least one wash solution. In some embodiments, the method further comprises the step of contacting at least one chromatography matrix with the elution solution. In some embodiments, the method further comprises the step of collecting an eluate containing a polypeptide containing an Fc region.

[0105] In some embodiments, the term “collected material/collected material” refers to the liquid present at the end of cell culture or after cell culture, for example, to a cell lysate sample or a cell culture supernatant sample (for example, cell supernatant, such as CHO cells engineered to produce and secrete the polypeptide). In some embodiments, the collected material contains intact host cells and/or cellular debris. In some embodiments, the collected material does not contain intact host cells and/or cellular debris. For example, in some embodiments, the liquid present at the end of cell culture or after cell culture is subjected to one or more centrifugation and/or filtration steps until adjusted to achieve a final benzoate salt concentration of about 0.1 M to about 0.5 M , and a pH of about 7.0 to about 9.0. In some embodiments, the collected material is obtained from the fluid present at the end of the cell culture or after the cell culture. For example, in some embodiments, the liquid present at the end of cell culture or after cell culture is subjected to one or more pretreatment steps to optimize cell separation and/or purification of the Fc region-containing polypeptide.

[0106] According to a related aspect, any of the methods for purifying a polypeptide containing an Fc region described herein further includes the step of adjusting the harvested material that contains a polypeptide containing an Fc region to achieve a final benzoate salt concentration of between 0.1 M to about 0.5 M, and a pH of about 7.0 to about 9.0, to obtain a sample containing (i) a polypeptide containing an Fc region, and (ii) one or more impurities.

[0107] In some embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, the benzoate salt is sodium benzoate. In some embodiments, the collected material is adjusted to achieve a final benzoate salt concentration (e.g., sodium benzoate) of any of about 0.025 M, 0.05 M, 0.075 M, 0.1 M, 0.125 M, 0.15 M, 0.175 M 0.2 M, 0.225 M, 0.25 M, 0.275 M, 0.3 M, 0.325 M, 0.35 M .5 M, 0.525 M, 0.55 M, 0.575 M, 0.6 M, 0.625 M, 0.65 M, 0.675 M, 0.7 M, 0.725 M, 0.75 M, 0.775 M or 0.8 M, including any range between these values. In some embodiments, the pH of the collected material is adjusted to any of about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 , 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9 .1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, including any range between these values. In some embodiments, the collected material is clarified prior to adjustment (eg, addition of sodium benzoate and pH adjustment). In some embodiments, the collected material is clarified after adjustment (eg, by adding sodium benzoate and adjusting the pH).

[0108] In some embodiments, the method includes adjusting the collected material to achieve a final concentration of sodium benzoate of about 0.5 M and a pH of about 7. In some embodiments, the method includes adjusting the collected material to achieve a final concentration of sodium benzoate, about 0.1 M and a pH of about 9. In some embodiments, the method includes adjusting the collected material to achieve a final sodium benzoate concentration of about 0.2 M and a pH of about 9. In some embodiments, the method involves adjusting the collected material to achieve a final concentration of sodium benzoate of approximately 0.3 M and a pH of 9. In some embodiments, the method includes adjusting the collected material salt to achieve a final concentration of sodium benzoate of approximately 0.4 M and a pH of 9. In some embodiments, the method includes adjusting the collected material to achieve a final concentration of sodium benzoate of approximately 0.5 M and a pH of 9.

[0109] In some embodiments, adjusting the harvested material (e.g., by adding sodium benzoate and adjusting the pH) results in a polypeptide containing an Fc region purified from one or more impurities to a greater extent than the corresponding method provides, in which lacks the step of correcting the collected material containing the polypeptide containing the Fc region, obtaining a sample. In some embodiments, one or more impurities are host cell proteins (HCPs), such as phospholipases, clusterin, serine proteases, elongation factors, and any combinations thereof. In some embodiments, the implementation of the HCP is a protein 2, like the hypothetical phospholipase B (PLBL2).

[0110] In some embodiments, the adjusted harvest (e.g., to which sodium benzoate has been added to the final concentration described herein and whose pH has been adjusted as described herein) contains or is a sample containing (i) a polypeptide containing an Fc region, and (ii) one or more impurities. In some embodiments, the sample is contacted with at least one chromatography matrix (eg, the sample is subjected to at least one chromatography step). In some embodiments, the at least one chromatography matrix comprises any one or more of an affinity chromatography matrix, a mixed mode chromatography matrix (e.g., a multimodal separator matrix), a hydrophobic interaction chromatography (HIC) matrix, anion exchange chromatography matrices, cation exchange chromatography matrices, size exclusion chromatography matrices, ceramic hydroxyapatite chromatography (CHT) matrices and/or hydrophilic interaction liquid chromatography (HILIC) matrices, etc. in any order. In some embodiments, the sample is contacted with an affinity chromatography matrix, e.g., a matrix containing protein A or a matrix containing protein G. In some embodiments, the method further comprises the step of contacting at least one chromatography matrix with at least at least one wash solution. In some embodiments, the method further comprises the step of contacting at least one chromatography matrix with the elution solution. In some embodiments, the method further comprises the step of collecting an eluate containing a polypeptide containing an Fc region.

Impurity removal

[0111] Certain aspects of the present invention relate to methods for purifying a polypeptide containing an Fc region by protein A binding chromatography by washing a matrix containing protein A associated with a polypeptide containing an Fc region (e.g., antibody), a wash solution containing a benzoate salt and/or benzyl alcohol, to improve the purification of the polypeptide from one or more impurities. In some embodiments, the method comprises the steps of contacting a protein A binding chromatography matrix with a sample containing (1) a polypeptide containing an Fc region (e.g., antibody), and (2) one or more impurities (for example, impurities of the host cell), provided that the polypeptide containing the Fc region is bound (for example, antibodies), with Protein A, and washing the matrix with a wash solution containing a benzoate salt at a concentration of from about 0.1 M to about 1.0 M, and/or benzyl alcohol at a concentration of from about 0.5% to about 4 % v/v (v/v), where the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, washing a matrix containing Protein A with a wash solution results in a polypeptide containing an Fc region purified from one or more contaminants to a higher degree than provides a corresponding method (described above) in which there is no step of washing the matrix with a wash solution. solution.

[0112] The standard protein A coupling method typically provides a product purity of approximately 95% without the use of the washing step described herein. The largest proportions of impurities in the product are due to high molecular weight aggregates (HMW) and/or fragments of a low molecular weight product (LMW). These variants of the product are considered impurities due to their ability to separate from the product, which is due to various parameters (for example, charge, hydrophobicity, differences in size etc.). These HMW and LMW impurities comprise approximately 4-5% of the protein A pool. In addition, the standard protein A binding method, which does not use the washing step described herein, also typically results in inclusion of impurities of the host cell protein (HCP) of the order of 1000 ppm or ~0.1% of the product pool. However, due to specifications established for mAb-based injectable products (see, for example, FDA guidelines), reducing and/or completely removing this 0.1% HCP contaminant is of great importance. Inclusion of the wash solution step described herein can reduce the amount of HCP present in the pool to ~100-10 ppm (10-100 fold reduction in HCP compared to the same protein A binding method that lacks washing step described in this document), which is 90-99% relative improvement. Methods for measuring the purity and/or impurity levels of a protein sample (for example, by ELISA assay) are commonly known to those skilled in the art. An exemplary purification of a monoclonal antibody (mAb) from one or more host cell proteins using a standard method compared to any of the methods described herein is shown intable A below.

Table A. Exemplary Cleaning Method

Composition (after Protein A) HCP mAb Condition: ppm: % % Standard 1000 0.10 94.90 Including flushing step (described in this document) 100 0.01 94.99 Change (%) 90 90 0.09

[0113] In some embodiments, the methods described herein produce a pool of proteins containing a polypeptide containing an Fc region (e.g., monoclonal antibody), after eluting Protein A that contains less than about 500 ppm (parts per million) HCP (e.g., one or more HCPs from a CHO cell). For example, a protein pool containing a polypeptide containing an Fc region produced by the methods described herein may contain less than about 500 ppm, less than about 450 ppm, less than about 400 ppm, less than about 350 ppm, less than about 300 ppm, less than about 250 ppm, less than about 200 ppm, less than about 150 ppm, less than about 100 ppm, less than about 75 ppm, less than about 50 ppm, less than about 25 ppm, less than about 10 ppm, or less than about 1 ppm HCP (e.g., one or more HCPs from a CHO cell). In some embodiments, the protein pool containing the Fc region containing polypeptide obtained by the methods described herein contains less than about 100 ppm HCP (e.g., one or more HCPs from a CHO cell). In some embodiments, the protein pool containing the Fc region containing polypeptide obtained by the methods described herein contains less than about 10 ppm HCP (e.g., one or more HCPs from a CHO cell).

[0114] In some embodiments, the methods described herein produce a pool of proteins containing a polypeptide containing an Fc region (e.g., monoclonal antibody), after eluting Protein A that contains less than about 0.1% HCP (e.g., one or more HCPs from a CHO cell). For example, a pool of proteins containing a polypeptide containing an Fc region obtained by the methods described herein may contain less than about 0.1%, less than about 0.09%, less than about 0.08%, less than about 0.07% , less than about 0.06%, less than about 0.05%, less than about 0.04%, less than about 0.03%, less than about 0.02%, or less than about 0.01% HCP (e.g., one or more HCPs from a CHO cell). In some embodiments, the protein pool containing the Fc region-containing polypeptide produced by the methods described herein contains less than about 0.05% HCP (e.g., one or more HCPs from a CHO cell). In some embodiments, the protein pool containing the Fc region containing polypeptide obtained by the methods described herein contains less than about 0.01% HCP (e.g., one or more HCPs from a CHO cell).

[0115] In some embodiments, the methods described herein provide for a reduction in the amount and/or concentration (e.g., in parts per million) of one or more impurities (for example, one or more HCPs, such as one or more HCPs from a CHO cell) when co-purified with an Fc-containing polypeptide by at least about 10% compared to the amount of one or more impurities when co-purified with an Fc-containing polypeptide an area cleaned in an appropriate manner without the step of washing the matrix containing protein A with a wash solution. For example, the methods described herein reduce the amount and/or concentration (for example, in parts per million) of one or more impurities (for example, one or more HCPs, such as one or more HCPs from a CHO cell) when co-purified with a polypeptide containing an Fc region of at least about 10%, at least about 15%, at least about 20%, at least at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% compared to the amount of one or more impurities when copurified with an Fc region containing polypeptide purified in an appropriate manner without a washing step matrix containing protein A, washing solution. In some embodiments, the methods described herein reduce the amount and/or concentration (e.g., in parts per million) of one or more impurities (for example, one or more HCPs, such as one or more HCPs from a CHO cell) when co-purified with a polypeptide containing an Fc region, at least about 1.5 times the amount of one or more impurities when co-purified with a polypeptide containing Fc region purified in an appropriate manner without the step of washing the matrix containing protein A with a wash solution. For example, the methods described herein reduce the amount and/or concentration (for example, in parts per million) of one or more impurities (for example, one or more HCPs, such as one or more HCPs from a CHO cell) when co-purified with an Fc region-containing polypeptide by at least about 1.5 times, at least about 2 times, at least about 2.5 times times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least at least about 9 times, at least about 9.5 times, at least about 10 times, at least about 50 times, or at least about 100 times the amount of one or more impurities when combined local purification with a polypeptide containing an Fc region purified in an appropriate manner without the step of washing the matrix containing protein A with a wash solution.

Additional stages

[0116] In some embodiments, the methods described herein additionally include one or more additional washing steps. In some embodiments, the methods described herein additionally include one or more elution steps. In some embodiments, the methods described herein further include one or more washing steps and one or more elution steps.

[0117] In some embodiments, the present invention relates to washing the matrix containing Protein A with the first solution prior to washing the matrix with the wash solution. In some embodiments, the matrix is washed with the first solution, one or more (for example, one or more, two or more, three or more, four or more, five or more etc.) times before washing the matrix with wash solution. In some embodiments, the matrix is washed once with the first solution before the matrix is washed with the wash solution. In some embodiments, the first solution contains a buffer. Any suitable buffer known in the art may be used in the first solution, including, for example, phosphate, tris(tris(hydroxymethyl)methylamine), acetate, carbonate, citrate, bis-tris, bis-trispropane, arginine, histidine , triethanolamine, diethanolamine, formate, MES (2-(N-morpholino)ethanesulfonic acid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TAPS (3 -{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), bicine (N,N-bis(2-hydroxyethyl)glycine), tricine (N-tris(hydroxymethyl)methylglycine), TES (2-{[tris(hydroxymethyl )methyl]amino}ethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), cacodylate (dimethylarsic acid), SSC (sodium citrate saline), and/or any combinations thereof. In some embodiments, implementation the first solution contains a phosphate buffer, a tris buffer, an acetate buffer, a carbonate buffer, and/or a citrate buffer.In some embodiments, the first solution solution contains a phosphate buffer. In some embodiments, the first solution contains one or more additional components (for example, a benzoate salt, benzyl alcohol, one or more of the additives described herein, etc.). In some embodiments, the first solution has a pH of about 5.0 to about 10.0 (e.g., about 6.0 to about 10.0, about 6.0 to about 9.0, about 7.0 to about 10.0, about 7.0 to about 9.0, about 8.0 to about 10.0, about 8.0 to about 9.0, about 9.0 to about 10.0, about 5.0 to about 8.0, about 6.0 to about 8.0, from about 7.0 to about 8.0, about 5.0 to about 7.0, about 6.0 to about 7.0, or about 5.0 to about 6.0). In some embodiments, the first solution has a pH of approximately 7.0.

[0118] In some embodiments, the implementation of the first solution contains a buffer at a concentration of from about 10 mm to about 100 mm or from about 10 mm to about 500 mm. For example, the first solution may contain a buffer at a concentration of from about 10 mM to about 500 mM, from about 100 mM to about 500 mM, from about 150 mM to about 500 mM, from about 200 mM to about 500 mM, from about 250 mM to about 500 mM, from about 300 mM to about 500 mM, from about 350 mM to about 500 mM, from about 400 mM to about 500 mM, from about 450 mM to about 500 mM, from about 10 mM to about 450 mM , from about 10 mM to about 400 mM, from about 10 mM to about 350 mM, from about 10 mM to about 300 mM, from about 10 mM to about 250 mM, from about 10 mM to about 200 mM, from about 10 mM up to about 150 mM, from about 10 mM to about 100 mM, from about 25 mM to about 100 mM, from approx. 40 mM to about 100 mM, about 50 mM to about 100 mM, about 60 mM to about 100 mM, about 75 mM to about 100 mM, about 10 mM to about 75 mM, about 25 mM to about 75 mM, about 40 mM to about 75 mM, about 50 mM to about 75 mM, about 60 mM to about 75 mM, about 10 mM to about 60 mM, about 25 mM to about 60 mM, from about 40 mM to about 60 mM, about 50 mM to about 60 mM, about 10 mM to about 50 mM, about 25 mM to about 50 mM, about 40 mM to about 50 mM, about 10 mM to about 40 mM, from about 25 mM to about 40 mM, or from about 10 mM to about 25 mM. In some embodiments, the first solution contains a buffer at a concentration of from about 10 mM to about 50 mM, or from about 10 mM to about 500 mM.

[0119] In some embodiments, the first solution contains a buffer at any concentration of about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM. Alternatively, the first solution contains buffer at any concentration of about 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. In some embodiments, the implementation of the first solution contains a buffer at a concentration of approximately 500 mm. In some embodiments, the first solution contains a buffer at a concentration of approximately 50 mM. In some embodiments, the implementation of the first solution contains a phosphate buffer at a concentration of approximately 500 mm. In some embodiments, the implementation of the first solution contains a phosphate buffer at a concentration of approximately 50 mm.

[0120] In some embodiments, the implementation of the first solution contains a phosphate buffer (for example, sodium phosphate) and sodium chloride. In some embodiments, the first solution contains a phosphate buffer (for example, sodium phosphate) and sodium chloride and has a pH of approximately 7.0. In some embodiments, the first solution contains a phosphate buffer (for example, sodium phosphate) at a concentration of approximately 50 mM; and sodium chloride at a concentration of approximately 0.5 M. In some embodiments, the first solution contains a phosphate buffer (e.g., sodium phosphate) at a concentration of approximately 50 mm, and sodium chloride at a concentration of approximately 0.5 M, and has a pH of approximately 7.0.

[0121] In some embodiments, the present invention relates to washing the matrix containing Protein A with a second solution after washing the matrix with the wash solution. In some embodiments, the matrix is washed with the second solution one or more (for example, one or more, two or more, three or more, four or more, five or more etc.) times after washing the matrix with wash solution. In some embodiments, the matrix is washed once with the second solution after the matrix is washed with the wash solution. In some embodiments, the implementation of the second solution contains a buffer. Any suitable buffer known in the art may be used in the second solution, including, for example, phosphate, tris(tris(hydroxymethyl)methylamine), acetate, carbonate, citrate, bis-tris, bis-trispropane, arginine, histidine , triethanolamine, diethanolamine, formate, MES (2-(N-morpholino)ethanesulfonic acid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TAPS (3 -{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), bicine (N,N-bis(2-hydroxyethyl)glycine), tricine (N-tris(hydroxymethyl)methylglycine), TES (2-{[tris(hydroxymethyl )methyl]amino}ethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), cacodylate (dimethylarsic acid), SSC (sodium citrate saline), and/or any combinations thereof. In some embodiments, implementation the second solution contains a phosphate buffer, a tris buffer, an acetate buffer, a carbonate buffer, and/or a citrate buffer. the solution contains a phosphate buffer. In some embodiments, the implementation of the second solution contains essentially a low amount of salt or does not contain salt. In some embodiments, the second solution has a pH of about 4.0 to about 8.0 (e.g., about 5.0 to about 8.0, about 6.0 to about 8.0, about 7.0 to about 8.0, about 4.0 to about 7.0, about 5.0 to about 7.0, about 6.0 to about 7.0, about 4.0 to about 6.0, about 5.0 to about 6.0, or about 4.0 to about 5.0). In some embodiments, the second solution has a pH of about 5.0 to about 7.0. In some embodiments, the second solution has a pH of approximately 7.0. In some embodiments, the implementation of the second solution contains essentially a low amount of salt. In some embodiments, the implementation of the second solution does not contain salt.

[0122] In some embodiments, the implementation of the second solution contains a buffer at a concentration of from about 10 mm to about 100 mm or from about 10 mm to about 500 mm. For example, the second solution may contain buffer at a concentration of from about 10 mM to about 500 mM, from about 100 mM to about 500 mM, from about 150 mM to about 500 mM, from about 200 mM to about 500 mM, from about 250 mM to about 500 mM, from about 300 mM to about 500 mM, from about 350 mM to about 500 mM, from about 400 mM to about 500 mM, from about 450 mM to about 500 mM, from about 10 mM to about 450 mM , from about 10 mM to about 400 mM, from about 10 mM to about 350 mM, from about 10 mM to about 300 mM, from about 10 mM to about 250 mM, from about 10 mM to about 200 mM, from about 10 mM up to about 150 mM, from about 10 mM to about 100 mM, from about 25 mM to about 100 mM, from approx. 40 mM to about 100 mM, about 50 mM to about 100 mM, about 60 mM to about 100 mM, about 75 mM to about 100 mM, about 10 mM to about 75 mM, about 25 mM to about 75 mM, about 40 mM to about 75 mM, about 50 mM to about 75 mM, about 60 mM to about 75 mM, about 10 mM to about 60 mM, about 25 mM to about 60 mM, from about 40 mM to about 60 mM, about 50 mM to about 60 mM, about 10 mM to about 50 mM, about 25 mM to about 50 mM, about 40 mM to about 50 mM, about 10 mM to about 40 mM, from about 25 mM to about 40 mM, or from about 10 mM to about 25 mM. In some embodiments, the implementation of the second solution contains a buffer at a concentration of from about 10 mm to about 50 mm or from about 10 mm to about 500 mm.

[0123] In some embodiments, the second solution contains a buffer at any concentration of about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM. Alternatively, the second solution contains buffer at any concentration of about 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. In some embodiments, the implementation of the second solution contains a buffer at a concentration of approximately 500 mm. In some embodiments, the implementation of the second solution contains a buffer at a concentration of approximately 50 mm.

[0124] In some embodiments, the implementation of the second solution contains a phosphate buffer (for example, sodium phosphate). In some embodiments, the second solution contains a phosphate buffer (for example, sodium phosphate) and has a pH of approximately 7.0. In some embodiments, the second solution contains a phosphate buffer (for example, sodium phosphate) at a concentration of approximately 50 mm. In some embodiments, the second solution contains a phosphate buffer (for example, sodium phosphate) at a concentration of about 50 mM and has a pH of about 7.0.

[0125] In some embodiments, the methods of the present invention relate to washing the matrix containing protein A with a wash solution and do not include the step of washing the matrix with the first solution (before the wash solution) or the second solution (after the wash solution). In some embodiments, the methods of the present invention relate to washing a matrix containing protein A with a first solution followed by washing the matrix with a wash solution and do not include the step of washing the matrix with a second solution (after the wash solution). In some embodiments, the methods of the present invention relate to washing a matrix containing protein A with a wash solution followed by washing the matrix with a second solution and do not include the step of washing the matrix with a first solution (before the wash solution). In some embodiments, the methods of the present invention involve washing a matrix containing Protein A with a first solution, followed by washing the matrix with a wash solution, and then washing the matrix with a second solution.

[0126] In some embodiments, a matrix containing protein A is contacted with an elution solution by one or more (e.g., one or more, two or more, three or more, four or more, five or more etc.) times after one or more washing steps. In some embodiments, the implementation of the matrix once brought into contact with the elution solution. Any solution known in the art suitable for eluting a polypeptide bound to a matrix containing Protein A can be used as an elution solution in the methods of the present invention (e.g., eluting solution containing 40 mM sodium acetate and having a pH of approximately 3.1). In some embodiments, the eluent solution further comprises one or more additional components (e.g., arginine at any of the concentrations described in this document). In some embodiments, the eluate containing the polypeptide containing the Fc region is collected after contacting the matrix with the elution solution. In some embodiments, two or more eluates containing an Fc region containing polypeptide are collected after contacting the matrix with the elution solution two or more times. In some embodiments, two or more eluates are combined after elution. In some embodiments, the eluate(s) are filtered. Any suitable eluate filtration method known in the art may be used, including, for example, depth filtration. In some embodiments, the eluate(s) are filtered by depth filtration.

[0127] In some embodiments, eluates obtained from a protein A matrix as described herein may be further processed and/or purified (e.g., using an additional chromatography and/or filtration step (for example, by using one or more of ion exchange chromatography, mixed mode chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, size exclusion chromatography, diafiltration, ultrafiltration and/or filtering with virus removal)), and/or compiled (for example, to obtain a pharmaceutical composition suitable for administration to a subject in need (such as a human subject)).

[0128] The above description is considered sufficient to enable a person skilled in the art to carry out the present invention. The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

EXAMPLES

Example 1 Wash Solutions for Improved or Enhanced Impurity Removal during Antibody Purification

[0129] The following example describes the use of various combinations of sodium benzoate and benzyl alcohol in intermediate wash solutions for improved/enhanced removal of impurities during protein A binding chromatography.

Materials and methods

Getting Samples

[0130] Two separate human monoclonal antibody harvests were prepared for protein A binding chromatography as follows: The harvest was generated in a suspension culture of recombinant Chinese Hamster Ovary (CHO) cells engineered to constitutively express either antibody. The recombinant product was secreted into the culture medium, which was then centrifuged and clarified by depth filtration for further processing. The clarified collected material was filtered with a 0.22 µm polyethersulfone (PES) filter before loading onto the Protein A column.

Protein A binding chromatography

[0131] Protein A resin/columns were prepared as follows: MabSelect Sure LX Protein A Binding Chromatography Resin (GE Healthcare Life Sciences; Cat # 17-5474) was subjected to an exchange reaction by gravity settling with 0.5 M sodium chloride solution. Columns were filled using AKTA Pure or AKTA Avant (GE Healthcare Life Sciences) using either (A) 1.0 cm column (Essential Life Solutions 10/250 Snap Column; Cat # S10/250-PPSL-OE-FP10) or (B) 0.66 cm columns (Omnifit ^TM 6.6/100; p/n 006BCC0610FF). The resin was filled to a bed height of 20 cm ± 10% for column (A) or to a bed height of 5 cm ± 10% for column (B). Column quality was evaluated using 1% column volume injection of 1.0 M sodium chloride solution into the column equilibrated in 0.1 M sodium chloride solution, and trace conductivity was analyzed using Unicorn Evaluation software. The column efficiency should be at least 775 theoretical plates per meter and should exhibit a skewness of 0.8-1.8.

[0132] Prior to loading the collected material, the columns were washed with back osmosis deionized water to remove the storage buffer of 20% ethanol. The column was then washed with 0.5 M acetic acid to ensure that any associated materials were removed before equilibration. The column was equilibrated with 50 mM phosphate buffer with 0.5 M sodium chloride until the pH of the column was >6.5.

[0133] The prepared samples were then loaded onto Protein A columns. The columns were loaded with either mAb A (IgG1 subtype) or mAb B (IgG4 subtype) to a target of 40 g/L resin. The loaded columns were washed as described above with a phosphate buffer solution containing 50 mM phosphate and 0.5 M sodium chloride. The columns were then washed with the test wash solution followed by a salt-free buffer solution at pH 7. Finally, the antibody was eluted from the column using a solution containing 40 mM sodium acetate at pH 3.1. ATtable 1 the following is an exemplary chromatography method.

Table 1. Description of the method of column chromatography

Stage buffer/solution Flow direction Hold time (minutes) Volume (CV) Washing Water for injection (WFI) downstream eight 2.5 Stripping 0.5 M acetic acid downstream 5 2 Balancing 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 downstream 5 3 Loading Collected mAb material filtered through a 0.22 µm filter downstream 5 Wash 1 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 downstream 5 3 Flushing 2 Test solution downstream 5 2 Flushing 3 50 mM sodium phosphate, pH 7.0 downstream 5 3 Elution 40 mM sodium acetate, pH 3.1 downstream 5 2 Stripping 0.5 M acetic acid downstream 5 3 Subsequent balancing 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 updraft 5 3 Disinfection 0.5 M sodium hydroxide updraft 5 3 Subsequent balancing 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 updraft 5 2 Storage 20% ethanol updraft eight 2

Host cell protein detection

[0134] After elution, antibody samples were tested for the presence of host cell protein (HCP) contaminants using the ^3rd generation CHO HCP ELISA kit (Cygnus Technologies) according to the manufacturer's protocol. HCP ELISA was performed at dilutions from 1:400 to 1:800 and absorbance read at 450/600 nm using a Spectramax Plus 384 plate reader.

Identification of specific “HCP-A”

[0135] Specific HCP (HCP-A) concentration was quantified in eluted antibody samples using a commercially available Hamster (CHO) ELISA kit (ICL Labs) according to the manufacturer's protocol. ELISA of HCP-A was performed at dilutions from 1:100 to 1:800 and absorbance was read at 450 nm using a Spectramax Plus 384 plate reader.

results

[0136] Host cell proteins (HCPs) have been shown to co-elute with monoclonal antibodies (mAbs), which may present a problem for subsequent use of these antibodies. To identify potential wash additives capable of reducing the amount of HCP contaminants that co-elute with the mAb of interest, a sample containing a secreted human IgG1 monoclonal antibody (mAb A) assembled from CHO cells that were both centrifuged and clarified by depth filtration prior to further processing ( see Table 1 ). The columns were first washed with phosphate buffer solution. The columns were then washed with one of a number of test washes containing 50 mM phosphate and additive, either alone or in combination with sodium benzoate, benzyl alcohol, and arginine, at pH 7.0 ( Figure 1 ). Control runs did not include any wash additive. After the washes tested, the columns were washed with salt-free 50 mM phosphate, pH 7.0. Finally, the monoclonal antibody was eluted from the column and the pH was adjusted to pH 6.0 using 2 M Tris base. After adjustment, the eluate pools were filtered using a 0.22 µm PES filter and tested for the presence of HCP impurities ( Figure 1 ). Interestingly, washes containing 2% benzyl alcohol and/or 0.5 M sodium benzoate effectively reduced the levels of HCP contaminants in the eluted antibody samples. Also noted that the inclusion of arginine in the wash improves the clearance of HCP.

[0137] The specific presence of HCP-A in the antibodies eluted from the Protein A columns was then examined. The test and control washings used in this experiment are shown in Table 2 below.

Table 2 Wash Solutions Tested

Test solution: Components: pH: Control No extra flush 7.0 one 2% benzyl alcohol; 0.5 M sodium benzoate 7.0 2 2% benzyl alcohol; 0.5 M sodium benzoate; 0.5 M arginine 6.0 3 0.5 M benzenesulfonate; 0.5 M sodium benzoate; 2% benzyl alcohol 7.0 four 50 mM caprylic acid; 0.5 M sodium benzoate; 0.5 M arginine, 0.5 M sodium chloride 9.0 5 10% hexylene glycol; 2% benzyl alcohol; 0.5 M sodium benzoate 7.0 6 2% benzyl alcohol; 0.5 M sodium benzoate; 0.5 M arginine 6.0 7 2% benzyl alcohol; 0.5 M arginine 5.0 eight 0.5 M sodium benzoate, 50 mM sodium bicarbonate 10.0 9 2% benzyl alcohol, 0.5 M sodium benzoate, 0.5 M arginine, 40 mM sodium phosphate 9.0

[0138] Having demonstrated the ability of a combination of benzyl alcohol and sodium benzoate to remove HCP impurities, additional formulations were tested to target HCP-A removal. An IgG4 antibody (mAb B) with known high expression of HCP-A was used to specifically increase the loading of HCP-A on the column. Table 1 provides a summary of the steps that were carried out during the purification process. Briefly, after equilibration to load pH and conductivity, a 15.7 ml protein A binding chromatography column was loaded with a sample containing a secreted human monoclonal antibody collected from CHO cells by centrifugation and clarification by depth filtration before further processing. After reaching a load of 40 g/l (resin) the column was re-equilibrated using a solution of phosphate buffer. Then, one of the wash solutions (test solutions 1, 2 and 7) shown in Table 2 was applied as 2 column volumes (CV) followed immediately by 3 CV of a solution of saline-free phosphate buffer at pH 7.0. After elution with 40 mM sodium acetate, the eluate pools were adjusted to pH 5.5 using 2 M Tris base. The eluate pools were then filtered through either a 0.22 μm PES filter or a Millipore C0HC depth filter and tested for HCP-A content by ELISA ( FIG. 2A ). Wash solutions under all three conditions (test solutions 1, 2 and 7) were able to clear additional HCP-A compared to control (294 ppm). The results also showed that the effect of HCP-A removal was cumulative, as the combination of benzyl alcohol, sodium benzoate, and arginine removed almost 90% of HCP-A compared to control. In addition, depth filtration provided a subsequent additional removal of HCP-A by 15-25% for eluates in the wash condition.

[0139] Following a procedure similar to that described above, the effectiveness of the high pH wash solutions was then tested by washing the columns at pH 9.0 or 10.0. An IgG4 antibody (mAb B) was generated in a cell line with a known high expression of HCP-A to specifically increase the loading of HCP-A on the protein A column. Table 1 provides a summary of the steps that were carried out during the purification method. Briefly, after equilibration to load pH and conductivity, a 15.7 ml protein A binding chromatography column was loaded with a sample containing secreted human monoclonal antibody harvested from CHO cells harvested by centrifugation and depth filtration clarification prior to further processing. After reaching a load of 60 g/l (resin), the column was re-equilibrated using a solution of phosphate buffer. One of the test solutions 8 or 9 ( Table 2 ) was used for two CVs and immediately followed by three CVs of saline-free phosphate buffer at pH 7.0. After elution with 40 mM sodium acetate, the eluate pools were adjusted to pH 5.5 using 2 M Tris base. The eluate pools were then filtered through a 0.22 μm PES filter. As shown in FIG. 2B , 0.5 M buffered sodium benzoate at pH 10.0 is highly effective in removing HCP-A (92% reduction) compared to control. In addition, the addition of benzyl alcohol and arginine was again complementary and improved HCP-A clearance at pH 9.0. Taken together, the results shown in FIG. 2A-B show the stable pH range that provided significant clearance from HCP-A.

[0140] Finally, additional additives were screened to further improve washing with a solution of 2% benzyl alcohol and 0.5 M sodium benzoate. The same IgG4 mAb (mAb B) was used on a column with a scale value of 1.7 ml. Table 1 provides a summary of the steps that were carried out during the purification process. Briefly, the column was loaded up to 40 g/l after equilibration. Then one of the four wash solutions (test solutions 3-6) shown in Table 2 was applied to the column. After the elution samples were adjusted to pH 5.5 with 2 M Tris base and filtered using a 0.22 μm PES filter. The content of HCP-A was then measured using the HCP-A-specific ELISA ( Figure 3 ). Washes with test solutions showed a cumulative response in removing HCP-A due to the addition of arginine, caprylic acid, benzenesulfonate and hexylene glycol compared to the combination of 2% benzyl alcohol and 0.5 M sodium benzoate (201.3 ppm). A wash solution containing 2% benzyl alcohol, 0.5 M sodium benzoate and a component selected from hexylene glycol, sodium benzenesulfonate, caprylic acid, or arginine was very effective in removing HCP-A.

[0141] Taken together, the data presented in this example demonstrate that an intermediate wash step comprising sodium benzoate and/or benzyl alcohol is capable of providing excellent purification of host cell protein contaminants during purification of a human monoclonal antibody with protein A. More In addition, the inclusion of one or more additives selected from benzenesulfonate, caprylic acid, hexylene glycol and/or arginine in the wash solution further improved the purification of host cell protein contaminants during protein A purification of the target monoclonal antibody.

Example 2 Identification of specific host cell proteins present after protein A binding chromatography, development of washes for improved or enhanced removal of contaminants during antibody purification, and evaluation of the interaction of protein 2, like a hypothetical phospholipase B, with a human monoclonal antibody

[0142] The following example describes the identification of specific host cell proteins (HCPs) in a purified antibody eluate after protein A binding chromatography. The following example describes the use of sodium benzoate and benzyl alcohol in intermediate washes for improved/enhanced HCP removal including protein 2 like the putative phospholipase B (PLBL2) during protein A binding chromatography. Finally, the example describes the effect of loading conditions on the performance of protein A binding chromatography.

Materials and methods

Getting Samples

[0143] Collected human monoclonal antibody materials for protein A binding chromatography were prepared as described in Example 1. Briefly, the harvested material was prepared in a suspension culture of recombinant CHO cells engineered to constitutively express any of the human monoclonal antibodies. The recombinant product was secreted into the culture medium, which was then centrifuged and clarified by depth filtration for further processing. The clarified collected material was filtered with a 0.22 µm polyethersulfone (PES) filter before loading onto the Protein A column.

Protein A binding chromatography

[0144] Protein A resin/columns were prepared as described in Example 1. Briefly, MabSelect Sure LX Protein A Binding Chromatography Resin (GE Healthcare Life Sciences; Cat # 17-5474) was subjected to an exchange reaction by gravity settling with 0.5 M sodium chloride solution. Columns were filled using AKTA Pure or AKTA Avant (GE Healthcare Life Sciences) using either (A) 1.0 cm column (Essential Life Solutions 10/250 Snap Column; Cat # S10/250-PPSL-OE-FP10) or (B) 0.66 cm columns (Omnifit ^TM 6.6/100; p/n 006BCC0610FF). The resin was filled to a bed height of 20 cm ± 10% for column (A) or to a bed height of 5 cm ± 10% for column (B). Column quality was evaluated using 1% column volume injection of 1.0 M sodium chloride solution into the column equilibrated in 0.1 M sodium chloride solution, and trace conductivity was analyzed using Unicorn Evaluation software. The column efficiency should be at least 775 theoretical plates per meter and should exhibit a skewness of 0.8-1.8.

[0145] Before loading the collected material, the columns were washed with back osmosis deionized water to remove the storage buffer from 20% ethanol. The column was then washed with 0.5 M acetic acid to ensure that any associated materials were removed before equilibration. The column was equilibrated with 50 mM phosphate buffer with 0.5 M sodium chloride until the pH of the column was >6.5.

[0146] The prepared samples were then loaded into Protein A columns. Typically, the columns were loaded to a 40 g/L resin target with either human monoclonal antibody A (IgG1 subtype) or human monoclonal antibody B (IgG4 subtype). The loaded columns were washed as described above with a phosphate buffer solution containing 50 mM phosphate and 0.5 M sodium chloride. The columns were then washed with the test wash solution, followed by washing with a salt-free buffer solution at pH 7. However, the control treatments were not washed with the test wash solution. Finally, the antibody was eluted from the column using a solution containing 40 mM sodium acetate at pH 3.1. ATtable 1 an exemplary chromatography method is presented.

Detection of HCP using mass spectrometry

[0147] After purification of the protein A column and elution under standard conditions, the human monoclonal antibody eluates were analyzed by mass spectrometry to determine the relative amount of each HCP. In particular, the relative amount of clusterin and putative phospholipase B-like protein 2 (PLBL2) was identified in protein A-purified human monoclonal antibody samples. Mass spectroscopy was performed using an Acquity H-Class Xevo G2-XS Q-Tof and a 2.1×150 mm ACQUITY UPLC column with 1.7 μm CSH C18. Samples were first denatured with 0.05% Rapigest in 50 mM ammonium bicarbonate and then reduced and alkylated with 20 mM DTT (dithiothreitol) and 40 mM IAA (iodoacetamide). Enzymatic digestion was carried out with 2% LysC overnight and then for 3 hours with 4% trypsin. Rapigest was removed by centrifugation and the samples were acidified using formic acid. An internal standard was then added with the addition of 2.5 fmol/µl E. coli ClpB to compare the relative amounts of HCP and PLBL2. Fixed mass calibration was performed at approximately 785.8426 mass/charge. MS ^E was used for ion data analysis and HCP identification.

Detection of HCP using ELISA

[0148] After elution, the presence of total HCPs in human monoclonal antibody samples was assessed as described in Example 1. Briefly, antibody samples were tested for the presence of HCP contaminants using a ^3rd generation CHO HCP ELISA kit (Cygnus Technologies) according to the manufacturer's protocol. HCP ELISA was performed at dilutions from 1:400 to 1:800 and absorbance read at 450/600 nm using a Spectramax Plus 384 plate reader.

PLBL2 detection by ELISA

[0149] PLBL2 concentration was quantified in eluted antibody samples using a commercially available Hamster (CHO) ELISA kit (ICL Labs, E-65PLB) according to the manufacturer's protocol. PLBL2 ELISA was performed at dilutions from 1:100 to 1:800 and absorbance was read at 450 nm using a Spectramax Plus 384 plate reader.

results

Detection of HCP using mass spectrometry

[0150] It has been shown that after purification with protein A, HCP co-elutes with a human monoclonal antibody, which can be a problem for subsequent use of these antibodies. To identify specific HCPs present in the purified human monoclonal antibody solution after protein A purification, a sample containing a secreted human IgG1 monoclonal antibody (mAb A) harvested from CHO cells was loaded into a 1.7 ml protein A chromatography column. was both centrifuged and clarified by depth filtration before further processing (see table 1). The columns were first washed with phosphate buffer solution. The columns were then washed with salt-free 50 mM phosphate, pH 7.0. Finally, the monoclonal antibody was eluted from the column and the pH was adjusted to pH 6.0 using 2 M Tris base. After adjustment, the eluate pools were filtered using a 0.22 μm PES filter and analyzed by mass spectrometry to identify relative amounts of HCP. Of note, clusterin and PLBL2 were the two most abundant HCPs present in the human monoclonal antibody eluate.

Identification of intermediate flush conditions for improved HCP/PLBL2 removal

[0151] The mass spectrometry analysis detailed above provided additional evidence that HCPs including PLBL2 co-elute with human monoclonal antibodies after protein A purification. ELISA screens were then developed to identify intermediate wash solutions that could be used to further improve and enhance the removal of HCP and PLBL2 ( FIGS. 4A and 4B ). Interestingly, intermediate washes containing 4% benzyl alcohol or 0.5 M sodium benzoate effectively reduced the levels of HCP and PLBL2 contaminants in the antibody eluate samples. Wash solutions containing only 0.5 M arginine did not reduce the level of total HCP, but did reduce the level of contaminant PLBL2.

[0152] PLBL2 ELISA screens further show that depth filtration combined with intermediate washes including 1) 2% benzyl alcohol and 0.5 M arginine, pH 5.0, 2) 2% benzyl alcohol and 0 .5 M sodium benzoate, pH 7.0, or 3) 2% benzyl alcohol, 0.5 M sodium benzoate, 0.5 M arginine, pH 6.0, effectively removed PLBL2 during protein A purification. , washing solutions with elevated pH levels (0.5 M sodium benzoate and 50 mM sodium bicarbonate pH 10.0 or 2% benzyl alcohol, 0.5 M sodium benzoate, 0.5 M arginine and 50 mM sodium phosphate pH 9.0 ) were highly effective in removing PLBL2 during purification with protein A. Indeed, the sodium benzoate wash solution with 50 mM sodium bicarbonate, pH 10.0, was able to remove more than 92% of PLBL2 compared to the control treatment. Finally, wash solutions comprising 1) 0.5 M sodium benzoate, 2% benzyl alcohol and 0.5 M benzenesulfonate, pH 7.0, 2) 0.5 M sodium benzoate, 50 mM caprylic acid, 0.5 M arginine and 0.5 M sodium chloride, pH 7.0, 3) 0.5 M sodium benzoate, 2% benzyl alcohol and 10% hexylene glycol, pH 7.0, or 4) 0.5 M sodium benzoate, 2% benzyl alcohol and 0.5 M arginine, pH 6.0, showed consistent ability to remove PLBL2 compared to control wash solutions.

[0153] In addition, a visual comparison of the antibody eluate after both experimental and control washes showed that samples washed with 2% benzyl alcohol and 0.5 M sodium benzoate had improved clarity compared to samples washed with control wash solutions ( Fig. 5 ).

[0154] The PLBL2 ELISAs detailed above demonstrate that an intermediate wash comprising benzyl alcohol and sodium benzoate can effectively reduce the level of PLBL2 in protein A-purified antibody eluate. This result was then further confirmed orthogonally by mass spectrometry. . Mass spectrometry results show that the test wash solution with 0.5 M sodium benzoate, 0.5 M arginine, 50 mM caprylic acid, 0.5 M NaCl, pH 9.0 removed almost 93% of PLBL2 from the eluate relative to the control treatment .

Evaluation of the effect of column loading on PLBL2 yield and removal

[0155] The above experiments demonstrate that the host cell PLBL2 protein is present in the protein A purified antibody eluate and can be effectively removed using intermediate washes containing benzyl alcohol and sodium benzoate. The effect of protein A column loading on post-column yield and PLBL2 removal was then evaluated. To identify ideal loading conditions to maximize both post-column yield and PLBL2 removal, Protein A columns were loaded in the ranges of 40 to 60 g/L ( FIGS. 6A and 6B ). The results show that increasing the column loading above 40 g/l reduces both the post-column yield and the removal of PLBL2.

[0156] Taken together, the data presented in this example indicate that PLBL2 is among the host cell proteins that co-elute with the human monoclonal antibody after protein A purification. sodium benzoate and/or benzyl alcohol, is capable of providing excellent purification of host cell proteins and PLBL2 contaminants during protein A purification of a human monoclonal antibody. Moreover, the data show that excessive column loading with protein A resin reduces post-column yield and removal of PLBL2.

Example 3 Evaluation of the Effect of pH and Benzoate Salt Concentration in the Collected Material Containing an Antibody on the Removal of Impurities During Antibody Purification

[0157] The following example describes experiments that were performed to determine whether adjusting the concentration of sodium benzoate and pH of collected material containing a monoclonal antibody improves or enhances the removal of impurities during antibody purification.

[0158] The human monoclonal antibody harvest was prepared as described in Example 1. The clarified and depth filtered harvest was adjusted to varying pH and additive concentrations to screen effects on HCP and PLBL2 clearance. Sodium benzoate, previously shown to be effective in removing PLBL2 as part of an additional wash step, was added as a solid in amounts sufficient to achieve the target final concentration for a given volume of material collected. After the addition, the target pH was reached by adding 2 M Tris base. Sodium benzoate was added to the first material collected to reach a final concentration of 0.5 M sodium benzoate and the pH was adjusted to 7.2. Sodium benzoate was added to the second harvest to reach a final concentration of 0.5 M sodium benzoate and the pH was adjusted to 9. The third harvest was adjusted to pH 9 (no sodium benzoate added). See table 3.

Table 3

Additives to collected material pH value Correction with Tris base only 9.0 0.5 M sodium benzoate 7.2 0.5 M sodium benzoate 9.0

[0159] After each harvest was corrected and filtered through a 0.22 μm filter, each of the three Protein A columns was loaded with the corrected harvest to a target of 50 g/L resin. The loaded columns were washed with a phosphate buffer solution containing 50 mM phosphate and 0.5 M sodium chloride. The columns were then washed with reverse osmotic deionized (RODI) water. Finally, the antibody was eluted from the column using a solution containing 40 mM sodium acetate at pH 3.1. Table 4 below shows an exemplary chromatography method.

Table 4

Stage buffer/solution Flow direction Retention time (minutes) Volume (CV) Washing Water for injection (WFI) downstream eight 2.5 Stripping 0.5 M acetic acid downstream 5 2 Balancing 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 downstream 5 3 Loading Adjusted mAb harvest filtered through a 0.22 µm filter downstream 5 Wash 1 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 downstream 5 3 Flushing 2 Reverse osmosis deionized (RODI) water downstream 5 2 Elution 40 mM sodium acetate, pH 3.1 downstream 5 2 Stripping 0.5 M acetic acid downstream 5 3 Subsequent balancing 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 updraft 5 3 Disinfection 0.5 M sodium hydroxide updraft 5 3 Subsequent balancing 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 updraft 5 2 Storage 20% ethanol updraft eight 2

[0160] Each of the three protein A eluates was then tested for the presence of host cell protein (HCP) contaminants using an ELISA as described in Example 1 and for the presence of PLBL2 using a custom designed ELISA. As shown in FIG. 7 , using 0.5 M sodium benzoate at pH 9.0 exhibited the lowest levels of PLBL2 and HCP impurities and exhibited an order of magnitude greater PLBL2 clearance compared to pH adjustment alone. When 0.5 M sodium benzoate was added to the collected material at pH 7.2, only HCP clearance improved. Influence on PLBL2 required adjustment of the harvested material for both pH and sodium benzoate. The adjustment had no effect on HMW (not shown). Compared to uncorrected harvest with RODI wash alone, during the protein A binding step, HCP was reduced by 65% while PLBL2 was reduced by approximately 79%.

[0161] Sodium benzoate was then added to the collected materials to achieve a final concentration of 0.1 M, 0.2 M, 0.3 M, 0.4 M or 0.5 M and adjusted to pH 9 before purification with protein A, as described above. Cm. table 5.

Table 5

Additives to collected material pH value 0.1 M sodium benzoate 9.0 0.2 M sodium benzoate 9.0 0.3 M sodium benzoate 9.0 0.4 M sodium benzoate 9.0 0.5 M sodium benzoate 9.0

[0162] Each Protein A eluate was then tested for the presence of PLBL2 using a specially designed ELISA and the antibody yield in each eluate was assessed. In FIG. 8 shows that the relationship between PLBL2 content and sodium benzoate concentration is almost sigmoidal. Decreased gain in PLBL2 clearance was observed for sodium benzoate concentrations greater than 0.4 M. Antibody yield decreased slightly with increasing sodium benzoate concentration. From 0.1 M to 0.5 M sodium benzoate, the antibody yield decreased from 94.8% to 89.4%. The highest yield and highest purification from PLBL2 was observed when the collected material was adjusted to 0.4 M sodium benzoate and pH 9.0 prior to protein A purification. account for a slightly reduced antibody yield. All samples with 0.1 M sodium benzoate had comparable HCP clearance to about 250 ppm. At 0.1 M sodium benzoate and below, the HCP was approximately 500 ppm. The concentration of sodium benzoate from 0.2 M to 0.5 M did not affect the charge dispersion profile, and all eluates were within the specified limits.

[0163] Taken together, these data suggest that the presence of sodium benzoate in solution makes the association of host cell contaminants with the mAb highly unfavorable at high pH. These conditions can be achieved either by a separate wash step after the mAb has been bound to the protein A resin, or in solution before or during the column loading phase. This provides more flexibility in the operation of the protein A cleanup step and further supports the unique properties of sodium benzoate as a wash additive.

[0164] Although the above invention has been described in more detail by way of illustration and example for clarity and understanding, the present descriptions and examples should not be construed as limiting the scope of the present invention.

Claims (99)

1. A method for purifying a polypeptide containing an Fc region, the method comprising the steps of: (a) contacting a protein A binding chromatography matrix with a sample containing (i) an Fc region containing polypeptide and (ii) one or more impurities, provided that the Fc region containing polypeptide binds with protein A; and (b) washing the matrix with a wash solution, where the wash solution contains one of (i) a benzoate salt at a concentration of 0.1 M to 1.0 M, and (ii) benzyl alcohol at a concentration of 0.5 to 4 % v/v (v/v), or both, and wherein the wash solution has a pH of 4.0 to 10.0. 2. The method according to p. 1, where the wash solution contains: 1) benzoate salt; 2) benzyl alcohol; or 3) benzoate salt and benzyl alcohol. 3. The method according to claim 1 or 2, where the benzoate salt is present in a concentration of from 0.1 M to 0.5 M. 4. The method according to any one of paragraphs. 1-3, where the benzoate salt is an alkali metal benzoate salt. 5. The method according to any one of paragraphs. 1-4, where the benzoate salt is sodium benzoate. 6. The method according to claim 5, wherein sodium benzoate is present in a concentration of 0.1 M to 0.3 M. 7. The method according to claim 5, where sodium benzoate is present at a concentration of 0.3 M. 8. The method according to p. 5, where sodium benzoate is present at a concentration of 0.5 M. 9. The method according to any one of paragraphs. 1-8, where benzyl alcohol is present in a concentration of 1 to 4% (v/v). 10. The method according to any one of paragraphs. 1-8, where benzyl alcohol is present in a concentration of 1 to 2% (v/v). 11. The method according to any one of paragraphs. 1-8, wherein the benzyl alcohol is present at a concentration of 2% (v/v) to 4% (v/v). 12. The method according to any one of paragraphs. 1-11, where the wash solution additionally contains a buffer agent. 13. The method of claim 11 wherein the buffering agent is selected from the group consisting of phosphate, tris, arginine, acetate and citrate. 14. The method according to claim 11 or 13, wherein the buffering agent is present at a concentration of 10 mM to 500 mM. 15. The method of claim 11 or 13 wherein the buffering agent is present at a concentration of 50 mM or 500 mM. 16. The method according to any one of paragraphs. 1-15, where the wash solution has a pH of 5.0 to 10.0. 17. The method according to any one of paragraphs. 1-16, where the wash solution has a pH of 5.0 to 9.0. 18. The method according to any one of paragraphs. 1-16, where the wash solution has a pH of 5.0, 6.0, 7.0, 9.0, or 10.0. 19. The method according to any one of paragraphs. 1-18, where the wash solution additionally contains sodium benzenesulfonate. 20. The method according to p. 19, where sodium benzenesulfonate is present in a concentration of from 0.1 M to 0.5 M. 21. The method according to any one of paragraphs. 1-20, where the wash solution further contains caprylic acid. 22. The method of claim 21 wherein caprylic acid is present at a concentration of 10 mM to 50 mM. 23. The method according to any one of paragraphs. 1-22, where the wash solution further contains hexylene glycol. 24. The method of claim 23, wherein the hexylene glycol is present at a concentration of 1 to 10% (v/v). 25. The method according to any one of paragraphs. 1-24, where the wash solution additionally contains creatine. 26. The method of claim 25, wherein the creatine is present at a concentration of 10 mM to 100 mM. 27. The method according to any one of paragraphs. 1-26, where the wash solution additionally contains arginine. 28. The method according to p. 27, where arginine is present in a concentration of from 0.1 M to 1.0 M. 29. The method according to claim 27, where arginine is present at a concentration of 0.5 M. 30. The method according to any one of paragraphs. 27-29, where arginine is arginine-HCl. 31. The method according to any one of paragraphs. 27-30, where the wash solution containing arginine has a pH of 4.0 to 6.0. 32. The method according to any one of paragraphs. 27-30, where the wash solution containing arginine has a pH of 8.0 to 10.0. 33. The method according to any one of paragraphs. 1-32, where the wash solution additionally contains one or more non-buffer salts. 34. The method of claim 33 wherein the one or more non-buffer salts are selected from the group consisting of sodium chloride, sodium bromide, potassium chloride, potassium bromide, magnesium chloride, magnesium bromide, calcium chloride, calcium bromide, and any of their combinations. 35. The method of claim 33 wherein the one or more non-buffer salts are sodium chloride and/or potassium chloride. 36. The method according to any one of paragraphs. 33-35, wherein one or more non-buffer salts are present at a concentration of 0.1 M to 1.0 M. 37. The method according to any one of paragraphs. 1-36, where the wash solution is a solution selected from the group consisting of: (i) a solution containing sodium benzoate at a concentration of 0.5 M and sodium bicarbonate at a concentration of 50 mm, characterized by a pH of 10.0; (ii) a solution containing sodium benzoate at a concentration of 0.5 M, benzyl alcohol at a concentration of 2%, arginine at a concentration of 0.5 M, and sodium phosphate at a concentration of 50 mM, characterized by a pH value, constituting 9.0; (iii) a solution containing sodium benzoate at a concentration of 0.5 M and benzyl alcohol at a concentration of 2% (v/v), characterized by a pH of 7.0; (iv) a solution containing sodium benzoate at a concentration of 0.5 M, benzyl alcohol at a concentration of 2% (v/v), and sodium chloride at a concentration of 0.5 M, characterized by a pH value of 7.0; (v) a solution containing hexylene glycol at a concentration of 10% (v/v), sodium benzoate at a concentration of 0.5 M and benzyl alcohol at a concentration of 2% (v/v), characterized by a pH value of 7.0; (vi) a solution containing benzenesulfonate at a concentration of 0.5 M, sodium benzoate at a concentration of 0.5 M, and benzyl alcohol at a concentration of 2% (v/v), characterized by a pH of 7 .0; (vii) a solution containing caprylic acid at a concentration of 50 mM, sodium benzoate at a concentration of 0.5 M, arginine at a concentration of 0.5 M, and sodium chloride at a concentration of 0.5 M, characterized by the index pH of 7.0; (viii) a solution containing sodium benzoate at a concentration of 0.5 M, benzyl alcohol at a concentration of 2% (v/v), and arginine at a concentration of 0.5 M, characterized by a pH of 6 .0; (ix) a solution containing sodium benzoate at a concentration of 0.5 M, benzyl alcohol at a concentration of 2% (v/v), and arginine at a concentration of 0.5 M, characterized by a pH of 5 .0; and (x) a solution containing benzyl alcohol at a concentration of 2% (v/v) and arginine at a concentration of 0.5 M, characterized by a pH of 5.0. 38. The method according to any one of paragraphs. 1-37, further comprising the step of washing the matrix with the first solution prior to washing the matrix with the wash solution from step (b) according to paragraph 1. 39. The method of claim 38, wherein the first solution contains a buffer selected from the group consisting of phosphate buffer, tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combinations thereof. 40. The method of claim 39, wherein the first solution contains a buffer at a concentration of 10 mM to 100 mM. 41. The method according to any one of paragraphs. 38-40, where the first solution is a phosphate buffer. 42. The method according to any one of paragraphs. 1-41, further comprising the step of washing the matrix with the second solution after washing the matrix with the wash solution from step (b) according to paragraph 1. 43. The method of claim 42, wherein the second solution contains a buffer selected from the group consisting of phosphate buffer, tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combinations thereof. 44. The method of claim 43, wherein the second solution contains a buffer at a concentration of 10 mM to 100 mM. 45. The method according to any one of paragraphs. 42-44, where the second solution has a pH of 5.0 to 7.0. 46. The method according to any one of paragraphs. 42-45, where the second solution contains essentially a low amount of salt or does not contain salt. 47. The method according to any one of paragraphs. 1-46 further comprising the step of contacting the protein A binding chromatography matrix with the elution solution after one or more washing steps. 48. The method of claim 47, further comprising the step of collecting an eluate containing a polypeptide containing an Fc region. 49. The method of claim 48, further comprising the step of filtering the eluate by depth filtration. 50. The method of claim 48 or 49 wherein the eluate contains less than 500 parts per million (ppm) of one or more impurities. 51. The method according to any one of paragraphs. 1-50, where the Fc region is a human Fc region. 52. The method of claim 51 wherein the human Fc region comprises a human IgG1, IgG2 or IgG4 Fc region. 53. The method according to any one of paragraphs. 1-50, where the Fc region is the Fc region of the mouse. 54. The method of claim 53, wherein the mouse Fc region comprises a mouse IgG1, IgG2, or IgG3 Fc region. 55. The method according to any one of paragraphs. 1-54, where the polypeptide containing the Fc region is an antibody. 56. The method of claim 55, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody. 57. The method of claim 55 or 56, wherein the antibody is a monoclonal antibody. 58. The method of claim 55 or 56 wherein the antibody is a bispecific antibody or a trispecific antibody. 59. The method according to any one of paragraphs. 1-58, further comprising, before step (a), adding a benzoate salt to the collected material containing the polypeptide containing the Fc region, achieving a final concentration of the benzoate salt of 0.1 M to 0.5 M, and adjusting the pH of the collected material containing a polypeptide containing an Fc region, until a final pH of 7.0 to 9.0 is reached, to obtain a sample containing (i) a polypeptide containing an Fc region, and (ii) one or more impurities. 60. A method for purifying a polypeptide containing an Fc region, the method comprising the steps of: (A) adding the benzoate salt to the harvested material containing the Fc-containing polypeptide to achieve a final concentration of the benzoate salt of 0.1M to 0.5M and adjusting the pH of the harvested material containing the Fc-containing polypeptide region until a final pH of 7.0 to 9.0 is reached to obtain a sample containing (i) a polypeptide containing an Fc region and (ii) one or more impurities; and (B) contacting the sample with at least one chromatography matrix. 61. The method of claim 59 or 60, wherein the benzoate salt is an alkali metal benzoate salt. 62. The method of claim 61 wherein the benzoate salt is sodium benzoate. 63. The method according to any one of paragraphs. 59-62, where the final concentration of the benzoate salt in the collected material is from 0.4 M to 0.5 M. 64. The method according to any one of paragraphs. 59-63, where the collected material has a pH of 7.0 to 8.0. 65. The method according to any one of paragraphs. 59-63, where the collected material has a pH of 8.0 to 9.0. 66. The method according to any one of paragraphs. 59-61, where the harvested material is from a culture containing a host cell engineered to express the polypeptide. 67. The method of claim 66 wherein the host cell is a eukaryotic host cell. 68. The method of claim 67, wherein the eukaryotic host cell is a Chinese hamster ovary (CHO) cell. 69. The method according to any one of paragraphs. 59-68 where the collected material is clarified prior to correction. 70. The method according to any one of paragraphs. 59-68 where the collected material is clarified after adjustment. 71. The method according to any one of paragraphs. 60-70, wherein at least one chromatography matrix provides an affinity chromatography matrix. 72. The method of claim 71 wherein the affinity chromatography matrix is a protein A binding chromatography matrix or a protein G binding chromatography matrix. 73. The method according to any one of paragraphs. 59-72, further comprising the step of contacting at least one chromatography matrix with at least one wash solution. 74. The method according to any one of paragraphs. 59-73, further comprising the step of contacting at least one chromatography matrix with the elution solution. 75. The method of claim 74, further comprising the step of collecting an eluate containing a polypeptide containing an Fc region. 76. The method of claim 75, further comprising the step of filtering the eluate by depth filtration. 77. The method of claim 75 or 76 wherein the eluate contains less than 500 parts per million (ppm) of one or more impurities. 78. The method according to any one of paragraphs. 60-77, where the Fc region is a human Fc region. 79. The method of claim 76 wherein the human Fc region comprises a human IgG1, IgG2 or IgG4 Fc region. 80. The method according to any one of paragraphs. 60-79, where the Fc region is the Fc region of the mouse. 81. The method of claim 80 wherein the mouse Fc region comprises a mouse IgG1, IgG2, or IgG3 Fc region. 82. The method according to any one of paragraphs. 60-81, where the polypeptide containing the Fc region is an antibody. 83. The method of claim 82, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody. 84. The method of claim 82 or 83, wherein the antibody is a monoclonal antibody. 85. The method of claim 82 or 83, wherein the antibody is a bispecific antibody or a trispecific antibody.

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