Methods for enhancing impurity removal during Protein A chromatography

Abstract

Provided herein are methods for purifying polypeptides (e.g., antibodies) comprising an Fc region by Protein A chromatography; methods for using wash solutions comprising benzoate and / or benzyl alcohol during Protein A chromatography; and methods for conditioning the harvest using sodium benzoate prior to Protein A chromatography. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 609,214, filed December 21, 2017, and U.S. Provisional Patent Application No. 62 / 694,387, filed July 5, 2018, the contents of which are incorporated herein by reference in their entireties.

[0002] The present disclosure relates to methods for purifying polypeptides (e.g., antibodies) comprising an Fc region via Protein A chromatography. [Background technology]

[0003] Antibodies and other Fc region-containing proteins (such as immunoadhesins) have found widespread use in pharmaceutical and therapeutic applications. Their use (e.g., in human patients) requires careful purification from any contaminants / impurities that may arise during protein production. Purification of therapeutic proteins is often achieved using one or more chromatographic purification steps; a particularly useful type of chromatographic purification for proteins containing immunoglobulin Fc regions (e.g., antibodies) is Protein A chromatography. However, host cell proteins (HCPs) have been shown to co-elute with antibodies during traditional capture-mode protein chromatography (including Protein A chromatography), which can pose problems for downstream applications of these antibodies. Typically, one or more wash steps are employed after binding the product (e.g., a protein containing an immunoglobulin Fc region) to a chromatography resin prior to elution. Unfortunately, current wash formulations consisting of salts and buffer species may not be sufficient to disrupt the interactions of HCPs and other impurities with various monoclonal antibody (mAb) products. Therefore, improved purification methods (e.g., implementation of new detergent formulations) that reduce the concentration / number of impurities that co-purify with antibodies (e.g., during Protein A affinity chromatography) are needed.

[0004] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Means for solving the problem]

[0005] To meet these and other needs, disclosed herein are improved methods for purifying Fc region-containing polypeptides from one or more impurities. These methods involve contacting a Protein A chromatography matrix with a sample (e.g., a cell lysate) containing (i) a polypeptide comprising an Fc region and (ii) one or more impurities, and washing the matrix with a wash solution having a pH of about 4.0-10.0 and containing benzoate and / or benzyl alcohol. This disclosure is based, at least in part, on the surprising discovery that the use of benzoate (e.g., sodium benzoate) and / or benzyl alcohol in a wash solution at a pH of about 4.0-10.0 during Protein A chromatography provides superior clearance of impurities (e.g., host cell impurities) than currently utilized wash formulations (see Figure 1, Example 1). The present disclosure is also based, at least in part, on the discovery that the inclusion of one or more additional ingredients selected from benzenesulfonate (e.g., sodium benzenesulfonate), caprylic acid, hexylene glycol, and / or arginine can further improve the clearance of impurities when included in the cleaning solution (see Figures 2 and 3, Example 1).

[0006] Thus, in one aspect, provided herein is a method for purifying a polypeptide comprising an Fc region, the method comprising the steps of: (a) contacting a Protein A chromatography matrix with a sample containing (i) a polypeptide comprising an Fc region and (ii) one or more impurities under conditions in which the polypeptide comprising the Fc region binds to Protein A; and (b) washing the matrix with a wash solution, the wash solution comprising one or both of (i) benzoate at a concentration of about 0.1 M to about 1.0 M and (ii) benzyl alcohol at a concentration of about 0.5% to about 4% volume / volume (v / v), and the wash solution having a pH of about 4.0 to about 10.0. In some embodiments, the wash solution comprises (1) benzoate; (2) benzyl alcohol; or (3) benzoate and benzyl alcohol. In some embodiments, the benzoate is at a concentration of about 0.1 M to about 0.5 M. In some embodiments that may be combined with any of the preceding embodiments, the benzoate is an alkaline benzoate salt. In some embodiments that can be combined with any of the preceding embodiments, the benzoate is sodium benzoate. In some embodiments, the sodium benzoate is at a concentration of about 0.1 M to about 0.3 M. In some embodiments, the sodium benzoate is at a concentration of about 0.3 M. In some embodiments, the sodium benzoate is at a concentration of about 0.5 M. In some embodiments that can be combined with any of the preceding embodiments, the benzyl alcohol is at a concentration of about 1% to about 4% (v / v). In some embodiments that can be combined with any of the preceding embodiments, the benzyl alcohol is at a concentration of about 1% to about 2% (v / v). In some embodiments that can be combined with any of the preceding embodiments, the benzyl alcohol is at a concentration of about 2% (v / v). In some embodiments that can be combined with any of the preceding embodiments, the benzyl alcohol is at a concentration of about 4% (v / v).

[0007] In some embodiments that can be combined with any of the preceding embodiments, the wash solution further comprises a buffer. In some embodiments, the buffer is selected from phosphate, Tris, arginine, acetate, and citrate. In some embodiments, the buffer is at a concentration of about 10 mM to about 50 mM, or about 10 mM to about 500 mM. In some embodiments, the buffer is at a concentration of about 50 mM. In some embodiments, the buffer is at a concentration of about 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 that can be combined with any of the preceding embodiments, the cleaning solution further comprises sodium benzenesulfonate. In some embodiments, the sodium benzenesulfonate is at a concentration of about 0.1 M to about 0.5 M. In some embodiments that can be combined with any of the preceding embodiments, the cleaning solution further comprises caprylic acid. In some embodiments, the caprylic acid is at a concentration of about 10 mM to about 50 mM. In some embodiments that can be combined with any of the preceding embodiments, the cleaning solution further comprises hexylene glycol. In some embodiments, the hexylene glycol is at a concentration of about 1% to about 10% (v / v). In some embodiments that can be combined with any of the preceding embodiments, the cleaning solution further comprises creatine. In some embodiments, the creatine is at a concentration of about 10 mM to about 100 mM. In some embodiments that can be combined with any of the preceding embodiments, the cleaning solution further comprises arginine. In some embodiments, the arginine is at a concentration of about 0.1 M to about 1.0 M. In some embodiments, the arginine is at a concentration of about 0.5 M. In some embodiments, the arginine is arginine-HCl. In some embodiments, the wash solution containing arginine has a pH of about 4.0 to about 6.0. In some embodiments, the wash solution containing arginine has a pH of about 8.0 to about 10.0. In some embodiments that can be combined with any of the preceding embodiments, the wash solution further comprises one or more non-buffering salts. In some embodiments, the one or more non-buffering salts are selected from sodium chloride, sodium bromide, potassium chloride, potassium bromide, magnesium chloride, magnesium bromide, calcium chloride, calcium bromide, and any combination thereof. In some embodiments, the one or more non-buffering salts are sodium chloride and / or potassium chloride. In some embodiments, the one or more non-buffering salts are at a concentration of about 0.1 M to about 1.0 M.

[0009] In some embodiments that can be combined with any of the preceding embodiments, the cleaning solution is selected from the group consisting of: (i) a solution comprising sodium benzoate at a concentration of about 0.5 M and sodium bicarbonate at a concentration of about 50 mM, and having a pH of about 10.0; (ii) a solution comprising sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2%, arginine at a concentration of about 0.5 M, and sodium phosphate at a concentration of about 50 mM, and having a pH of about 9.0; (iii) a solution comprising sodium benzoate at a concentration of about 0.5 M and sodium bicarbonate at a concentration of about 2% (v / v). (iv) a solution containing sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and sodium chloride at a concentration of about 0.5 M, and having a pH of about 7.0; (v) a solution containing hexylene glycol at a concentration of about 10% (v / v), sodium benzoate at a concentration of about 0.5 M, and benzyl alcohol at a concentration of about 2% (v / v), and having a pH of about 7.0; (vi) a solution containing benzenesulfonate at a concentration of about 0.5 M, benzoic acid at a concentration of about 0.5 M. (vii) a solution containing caprylic acid at a concentration of about 50 mM, sodium benzoate at a concentration of about 0.5 M, arginine at a concentration of about 0.5 M, and sodium chloride at a concentration of about 0.5 M, and having a pH of about 7.0; (viii) a solution containing sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and arginine at a concentration of about 0.5 M, and having a pH of about 6.0; (ix) a solution containing caprylic acid at a concentration of about 50 mM, sodium benzoate at a concentration of about 0.5 M, arginine at a concentration of about 0.5 M, and sodium chloride at a concentration of about 0.5 M, and having a pH of about 6.0; The solution is selected from: (x) a solution containing sodium benzoate, benzyl alcohol at a concentration of about 2% (v / v), and arginine at a concentration of about 0.5 M, and having a pH of about 5.0; (xi) a solution containing benzyl alcohol at a concentration of about 4.0 (v / v), and having a pH of about 5.0 to about 10; (xii) a solution containing benzyl alcohol at a concentration of about 2% (v / v), and arginine at a concentration of about 0.5 M, and having a pH of about 5.0.

[0010] In some embodiments that can be combined with any of the preceding embodiments, the method further comprises washing the matrix with a first solution before washing the matrix with the wash solution as described above. In some embodiments, the first solution comprises a buffer selected from phosphate buffer, Tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combination thereof. In some embodiments, the first solution comprises a buffer at a concentration of about 10 mM to about 100 mM, or about 10 mM to about 500 mM. In some embodiments, the first solution is a phosphate buffer.

[0011] In some embodiments that can be combined with any of the preceding embodiments, the method further comprises washing the matrix with a second solution after washing the matrix with the wash solution as described above. In some embodiments, the second solution comprises a buffer selected from phosphate buffer, Tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combination thereof. In some embodiments, the second solution comprises a buffer at a concentration of about 10 mM to about 100 mM, or 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 second solution contains substantially low salt or no salt.

[0012] In some embodiments that can be combined with any of the preceding embodiments, the method further comprises, after one or more washing steps, contacting the Protein A chromatography matrix with an elution solution. In some embodiments, the method further comprises recovering an eluate comprising a polypeptide comprising an Fc region. In some embodiments, the method further comprises 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 that can be combined with any of the preceding embodiments, application of the methods described herein results in a polypeptide comprising an Fc region that is purified from one or more impurities to a greater extent than a corresponding method that lacks the step of washing the matrix with a wash solution. In some embodiments that can be combined with any of the preceding embodiments, the one or more impurities are host cell proteins (HCPs). In some embodiments, the one or more HCPs are selected from phospholipases (e.g., putative phospholipase B-like 2), clusterin, serine proteases, elongation factors, and any combination 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, the human Fc region comprises 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 comprises a mouse IgG1, IgG2, or IgG3 Fc region. In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprising an 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.

[0015] In some embodiments, any one of the above methods includes adjusting the harvest containing the polypeptide comprising an Fc region to achieve a final benzoate 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 produce a sample containing (i) the polypeptide comprising an Fc region and (ii) one or more impurities, prior to contacting the Protein A chromatography matrix with the sample containing (i) the polypeptide comprising an Fc region and (ii) one or more impurities. In some embodiments, the benzoate is an alkaline benzoate salt. In some embodiments, the benzoate is sodium benzoate. In some embodiments, the final concentration of benzoate in the harvest is about 0.4 M to about 0.5 M. In some embodiments, the pH of the harvest after adjustment is about 7.0 to about 8.0. In some embodiments, the pH of the harvest after adjustment is about 8.0 to about 9.0. In some embodiments, the harvest is generated from a culture comprising host cells engineered to express the polypeptide. In some embodiments, the host cells are eukaryotic host cells. In some embodiments, the eukaryotic host cells are Chinese hamster ovary (CHO) cells. In some embodiments, the harvest is clarified before conditioning. In some embodiments, the harvest is clarified after conditioning.

[0016] In a related aspect, a method for purifying a polypeptide comprising an Fc region is provided, comprising: (A) adjusting a harvest containing a polypeptide comprising an Fc region to achieve a final benzoate 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 produce a sample containing (i) the polypeptide comprising an Fc region and (ii) one or more impurities; and (B) contacting the sample with at least one chromatography matrix. In some embodiments, the at least one chromatography matrix comprises an affinity chromatography matrix. In some embodiments, the affinity chromatography matrix is ​​a Protein A chromatography matrix or a Protein G chromatography matrix. In some embodiments, the method further comprises contacting the at least one chromatography matrix with at least one wash solution. In some embodiments, the method further comprises contacting the at least one chromatography matrix with an elution solution. In some embodiments, the method further comprises collecting an eluate containing the polypeptide comprising an Fc region. In some embodiments, the method further comprises 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 is alkaline benzoate. In some embodiments, the benzoate is sodium benzoate. In some embodiments, the final concentration of benzoate in the harvest is about 0.4 M to about 0.5 M. In some embodiments, the pH of the harvest after adjustment is about 7.0 to about 8.0. In some embodiments, the pH of the harvest after adjustment is about 8.0 to about 9.0. In some embodiments, the harvest is generated from a culture comprising host cells engineered to express a polypeptide. In some embodiments, the host cells are eukaryotic host cells. In some embodiments, the eukaryotic host cells are Chinese hamster ovary (CHO) cells. In some embodiments, the harvest is clarified before conditioning. In some embodiments, the harvest is clarified after conditioning. In some embodiments, the method results in a polypeptide comprising an Fc region that is purified from one or more impurities to a greater extent than a corresponding method lacking the step of conditioning a harvest comprising a polypeptide comprising an Fc region to produce a sample. In some embodiments, the one or more impurities are host cell proteins (HCPs). In some embodiments, the one or more HCPs are selected from the group consisting of phospholipases, clusterin, serine proteases, elongation factors, and any combination thereof. In some embodiments, the HCP is putative phospholipase B-like 2 (PLBL2). In some embodiments, the Fc region is a human Fc region. In some embodiments, the human Fc region comprises 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 comprises a mouse IgG1, IgG2, or IgG3 Fc region. In some embodiments, the polypeptide comprising an 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, some, or all of the features of the various embodiments described above and herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further explained in the detailed description that follows. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the concentration of Chinese hamster ovary (CHO) host cell protein (HCP) impurities in antibody samples eluted from a Protein A column after washing with the indicated control or test wash solutions. [Figure 2A] Figure 2A shows the concentration of a specific HCP (HCP-A) in antibody samples eluted from a Protein A column 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. [Figure 2B] Figure 2B shows the concentration of a specific HCP (HCP-A) in antibody samples eluted from a Protein A column. Figure 2B shows the concentration of HCP-A in antibody samples eluted from a Protein A column after washing with various wash solutions at pH 9.0 or 10.0 compared to a control wash, as assessed by ELISA. [Figure 3] FIG. 1 shows the concentration of HCP-A in antibody samples eluted from a Protein A column after washing with the indicated wash solutions containing additional test compounds, as assessed by ELISA. [Figure 4]Figure 4A shows the concentration of generic HCP and PLBL2 in antibody samples eluted from a Protein A column. Figure 4A shows the concentration of generic HCP in antibody samples eluted from a Protein A column after washing with 0.5 M arginine, 0.5 M sodium benzoate, or 4% benzyl alcohol compared to the process control wash, as assessed by ELISA. Figure 4B shows the concentration of generic PLBL2 in antibody samples eluted from a Protein A column after washing with 0.5 M arginine, 0.5 M sodium benzoate, or 4% benzyl alcohol compared to the process control wash, as assessed by ELISA. [Figure 5] FIG. 1 shows the improved visual clarity of antibody samples eluted from a Protein A column washed with an intermediate wash containing 2% benzyl alcohol and 0.5 M sodium benzoate. [Figure 6] Figure 6A shows the decrease in off-column yield and PLBL2 removal when loading the Protein A column above 40 g / L. Figure 6A shows that the percentage off-column yield decreases linearly from 93.1% to 78.1% as the loading density of the Protein A column increases from 40 g / L to 60 g / L. Figure 6B shows that the level of PLBL2 washed off the Protein A column decreases from 32.1 ppm to 17 ppm as the loading density of the Protein A column increases from 40 g / L to 60 g / L. [Figure 7] Figure 1 shows that harvest adjustment to 0.5 M sodium benzoate and pH 7.2 or to 0.5 M sodium benzoate and pH 9 prior to Protein A purification improved removal of PLBL2 and HCP impurities. Harvest adjustment to 0.5 M sodium benzoate at pH 9.0 showed the lowest levels of PLBL2 and HCP impurities and demonstrated a greater log of PLBL2 clearance compared to pH adjustment alone. [Figure 8] 1 shows that the relationship between PLBL2 content and sodium benzoate concentration is approximately sigmoidal. For concentrations above 0.4 M sodium benzoate, a decrease in PLBL2 clearance was observed. DETAILED DESCRIPTION OF THE INVENTION

[0020] Described herein are methods for reducing the number of co-purified impurities (e.g., host cell protein impurities) during Protein A-based isolation of Fc region-containing proteins. The disclosed methods employ an intermediate wash step using a novel wash solution containing benzoate and / or benzyl alcohol, which has been shown to significantly reduce the levels of host cell protein impurities in the eluate collected during Protein A affinity chromatography (see Examples 1 and 2). Inclusion of one or more additives (e.g., benzenesulfonate, caprylic acid, hexylene glycol, creatine, and / or arginine) in this novel wash solution further improves the clearance of impurities from the protein eluate containing the Fc region-containing protein after capture and elution from the Protein A matrix.

[0021] I. Definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which may, of course, vary, and that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0022] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes a combination of two or more such molecules, and the like.

[0023] As used herein, the term "about" refers to the normal error range for each value, which is readily known to those skilled in the art. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to the value or parameter itself.

[0024] It is understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0025] As used herein, phrases such as the term "and / or," "A and / or B," etc. are intended to include both A and B; A or B; A alone; and B alone. Similarly, as used herein, phrases such as the term "and / or," "A, B, and / or C," etc. are intended to encompass 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 alone; B alone; and C alone.

[0026] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The terms also encompass amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety or toxin. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0027] The term "antibody" is used herein in the broadest sense 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 having one or more CDRs or CDR-derived sequences, so long as the polypeptide exhibits the desired activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. Generally, antibodies are considered to be Igs with defined or recognized specificity. Thus, while antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. The antibodies of the present disclosure may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.). "Type" and "class," as well as "subtype" and "subclass," are used interchangeably herein. Natural or wild-type (obtained from non-artificially engineered members of a population) antibodies and immunoglobulins are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at one end followed by a number 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 can be human, humanized, non-human animal (e.g., mouse, rat, hamster, rabbit, camel, etc.) or chimeric.

[0028] The term "variable," in the context of antibody variable domains, can refer to certain portions of the related molecule that differ extensively in sequence between and within antibodies and are used in the specific recognition and binding of, or targeting, a particular antibody to its particular target. However, variability is not uniformly distributed throughout the variable domains of antibodies. In both the light- and heavy-chain variable domains, variability is concentrated in three segments called complementarity-determining regions (CDRs), also known as hypervariable regions. The more highly conserved portions of the variable domains are called framework (FR) regions or sequences. Naturally occurring heavy- and light-chain variable domains each contain four FR regions, primarily adopting a β-sheet structure, connected by three CDRs, which form loops connecting and, in some cases, forming part of the β-sheet structure. The CDRs of each chain are often held in close proximity by FR regions and, together with the CDR2 of the other chain, contribute to the formation of the antibody's target (epitope or determinant) binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, immunoglobulin amino acid residue numbering is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. One CDR may have the ability to specifically bind to its cognate epitope.

[0029] As used herein, the term "hinge" or "hinge region" can refer to a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.

[0030] The term "bispecific antibody" can refer to a molecule that combines the antigen-binding sites of two antibodies in a single molecule. Thus, a bispecific antibody can simultaneously bind to two different antigens.

[0031] The term "monoclonal antibody," as used herein, can refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they retain the desired activity.

[0032] As used herein, the term "multivalent antibody" or "polyvalent antibody" can refer to an antibody that contains two or more antigen-binding sites and is therefore capable of simultaneously binding to two or more antigens that can have the same or different structures. The term "bivalent" means that the antibody contains two antigen-binding sites. The term "tetravalent" means that the antibody contains four antigen-binding sites.

[0033] As used herein, the term "antigen-binding site" can refer to the portion of an antibody that contains the area that specifically binds to and is complementary to part or all of an antigen. If the antigen is large, the antibody can only bind to a specific portion of the antigen, which is called an epitope. The antigen-binding domain is provided by one or more antibody variable domains and is created by the association of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0034] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain sequences derived from non-human immunoglobulins, compared to human antibodies. Generally, humanized antibodies contain substantially all of one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being from a human immunoglobulin template sequence. Humanized antibodies also typically contain at least a portion of an immunoglobulin constant region, which is from a selected human immunoglobulin template. Generally, the goal is to have an antibody molecule that is minimally immunogenic in humans. Therefore, one or more amino acids in one or more CDRs can also be changed to be less immunogenic in a human host without substantially minimizing the specific binding function of the one or more CDRs to their target. Alternatively, the FRs can be non-human, but their most immunogenic amino acids are replaced with less immunogenic amino acids. Nevertheless, CDR grafting (as described above) is not the only way to obtain humanized antibodies. For example, modifying the CDR regions may be insufficient because framework residues often play a role in determining the three-dimensional structure of the CDR loops and the overall affinity of the antibody for its ligand. Thus, any means can be taken to modify the non-human parent antibody molecule to make it less immunogenic to humans, and extensive sequence identity with human antibodies is not necessarily required.

[0035] The term "impurity" can refer to any foreign or undesired molecule present in a solution (such as a sample containing a polypeptide comprising an Fc region). Impurities can be biological molecules (e.g., macromolecules) such as DNA, RNA, or proteins that are also present in a sample containing the protein of interest. Impurities include undesired protein variants (e.g., aggregated proteins, misfolded proteins, disulfide-bonded proteins, fragments, etc.), other proteins from host cells, components from cell culture media, molecules that are part of the absorbent used in affinity chromatography (e.g., Protein A), endotoxins, nucleic acids, viruses, etc.

[0036] II. Methods for isolating and / or purifying FC region-containing polypeptides Overview Certain aspects of the present disclosure relate to methods for purifying a polypeptide (e.g., an antibody) comprising an Fc region via Protein A chromatography. In some embodiments, the method includes contacting a Protein A chromatography matrix or resin with a sample containing (1) a polypeptide (e.g., an antibody) comprising an Fc region and (2) one or more impurities (e.g., host cell impurities) under conditions in which the polypeptide (e.g., an antibody) comprising the Fc region binds to Protein A; and washing the matrix with a wash solution containing benzoate and / or benzyl alcohol. In some embodiments, the wash solution contains benzoate at a concentration of about 0.1 M to about 1.0 M. In some embodiments, the wash solution contains benzyl alcohol at a concentration of about 0.5% to about 4% volume / volume (v / v). In some embodiments, the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution comprises one or more additives (e.g., one or more of benzenesulfonate, caprylic acid, hexylene glycol, non-buffering salts (such as sodium chloride), creatine, and / or arginine). In some embodiments, the wash solution further comprises a buffering agent. In some embodiments, the harvest containing a polypeptide comprising an Fc region is adjusted to achieve a final benzoate concentration of about 0.1 M to 0.5 M and a pH of about 7 to about 9 to produce a sample containing (1) a polypeptide (e.g., an antibody) comprising an Fc region, and (2) one or more impurities (e.g., host cell impurities).

[0037] Contact of sample with Protein A matrix or resin Certain aspects of the present disclosure relate to methods for purifying a polypeptide (e.g., an antibody) comprising an Fc region via Protein A chromatography. In some embodiments, the method comprises contacting a Protein A chromatography matrix or resin with a sample comprising (1) a polypeptide (e.g., an antibody) comprising an Fc region, and (2) one or more impurities (e.g., host cell impurities) under conditions under which the polypeptide (e.g., an antibody) comprising the Fc region binds to Protein A.

[0038] In some embodiments, the present disclosure relates to methods for purifying a polypeptide (e.g., an antibody, immunoadhesin, fusion protein, etc.) comprising an Fc region from a sample (e.g., a cell lysate sample, a cell culture supernatant sample, etc.). In some embodiments, the sample is a cell culture supernatant (e.g., a supernatant from cells, such as CHO cells, engineered to produce and secrete the polypeptide) or is derived from a cell culture supernatant (e.g., a partially purified cell culture supernatant sample). In some embodiments, the polypeptide comprising 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 human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226 or Pro230 to the carboxyl terminus (the numbering of residues in the Fc region is in the EU index as in Kabat). The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3, and optionally comprises a CH4 domain. In some embodiments, the Fc region is an Fc region obtained from any suitable immunoglobulin, such as an IgG1, IgG2, IgG3, or IgG4 subtype, IgA, IgE, IgD, or IgM. In some embodiments, the polypeptide comprises an Fc region having the amino acid sequence of a human Fc region, the amino acid sequence of a non-human animal Fc region (e.g., mouse, rat, rabbit, hamster, etc.), or any combination thereof. In some embodiments, the Fc region is a mouse Fc region. In some embodiments, the mouse Fc region comprises a mouse IgG1, IgG2, or IgG3 Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, the human Fc region comprises a human IgG1, IgG2, and / or IgG4 Fc region.

[0039] In some embodiments, the polypeptide comprising an Fc region is an antibody. In some embodiments, the term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multivalent antibodies (e.g., bivalent, trivalent, tetravalent, etc.), and multispecific antibodies (e.g., bispecific, trispecific, etc.). Antibodies can be derived from any origin, including, for example, humans, non-human primates, rodents (e.g., mice, rats, hamsters, etc.), rabbits, camels, sharks, and / or recombinantly produced antibodies. 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 antibody and / or a multivalent antibody. In some embodiments, the antibody is a bispecific antibody or a trispecific antibody.

[0040] In some embodiments, a sample (e.g., a cell lysate sample, a cell culture supernatant sample, etc.) comprising a polypeptide comprising an Fc region further comprises one or more impurities. In some embodiments, the one or more impurities are present in the sample due to a process used to produce the polypeptide comprising an Fc region (e.g., a process for producing a secreted antibody). In some embodiments, the 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 can be any host cell known in the art that is suitable for producing polypeptides comprising an Fc region, including, for example, prokaryotic cells (such as E. coli cells, A. niger cells), eukaryotic cells (such as yeast cells, plant cells, insect cells (e.g., S1 cells), and / or mammalian (such as mouse, rat, hamster, rabbit, human, non-human primate) cells (e.g., hybridoma, CHO cells, 293T cells, PER.C6 cells, NS0 cells, etc.). In some embodiments, the one or more impurities are one or more host cell proteins (HCPs). In some embodiments, HCPs refer to non-purified proteins produced by host cells during cell culture or fermentation. In some embodiments, In some embodiments, the one or more impurities are one or more (e.g., one or more, two or more, three or more, four or more, etc.) host cell proteins (HCPs) selected from phospholipase, clusterin, serine protease, elongation factor, and / or any combination thereof. In some embodiments, the host cell is a CHO cell. In some embodiments, the one or more impurities are one or more CHO cell HCPs. In some embodiments, the one or more CHO cell HCPs are one or more of phospholipase, clusterin, serine protease, elongation factor, and / or any combination thereof.

[0041] In some embodiments, the present disclosure relates to methods for purifying a polypeptide comprising an Fc region from one or more impurities in a sample via Protein A chromatography. In some embodiments, the sample is contacted with a Protein A matrix or resin. In some embodiments, the sample is contacted with the Protein A matrix or resin under conditions suitable for binding of polypeptides comprising an Fc region in the sample to Protein A. Methods and suitable conditions for contacting and binding Fc region-containing polypeptides to a Protein A matrix or resin are readily understood by those of skill in the art (e.g., methods described in the manufacturer's protocols for commercially available Protein A matrices or resins).Any suitable protein A matrix or resin known in the art can be used in the disclosed methods, 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.); Affigel protein A and Affiprep protein A (Bio-Rad); MABsorbent A1PP and MABsorbent A2P (Affinity Chromatography Ltd.); Protein A Ceramic Hyper DF (Pall Corp.); Ultralink Examples of suitable agarose gels include Immobilized Protein A and Agarose Protein A (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, the Protein A matrix or resin is used in a column chromatography format. In some embodiments, one or more parameters of the Protein A matrix or resin (e.g., pH, ionic strength, temperature, addition of other substances, etc.) are adjusted before 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 sanitized before and / or after contacting the Protein A matrix or resin with a sample. In some embodiments, the Protein A matrix or resin is equilibrated and / or washed before contacting the Protein A matrix or resin with a sample. Any suitable equilibration and / or wash buffer known in the art can be used. In some embodiments, the Protein A matrix or resin is sanitized, stripped, and / or regenerated between uses.

[0042] Washing the Protein A matrix or resin with a wash solution Certain aspects of the present disclosure relate to methods for purifying a polypeptide comprising an Fc region via Protein A chromatography by washing a Protein A matrix or resin bound to the polypeptide (e.g., an antibody) with a wash solution containing benzoate and / or benzyl alcohol. In some embodiments, the method includes contacting a Protein A chromatography matrix or resin with a sample containing (1) a polypeptide (e.g., an antibody) comprising an Fc region and (2) one or more impurities (e.g., host cell impurities) under conditions under which the polypeptide (e.g., the antibody) binds to Protein A; and washing the matrix or resin with a wash solution containing benzoate at a concentration of about 0.1 M to about 1.0 M and / or benzyl alcohol at a concentration of about 0.5% to about 4% volume / volume (v / v), where the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution contains benzoate. In some embodiments, the wash solution contains benzyl alcohol. In some embodiments, the cleaning solution comprises a benzoate and benzyl alcohol.

[0043] In some embodiments, the present disclosure relates to a cleaning solution (e.g., pH adjusted) comprising benzoate and / or benzoic acid. Any suitable source or form of benzoate (e.g., alkali salt) and / or benzoic acid known in the art can be used in the cleaning solution of the present disclosure, 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 is an alkali benzoate. In some embodiments, the benzoate is sodium benzoate or potassium benzoate. In some embodiments, the benzoate is sodium benzoate.

[0044] In some embodiments, the benzoate (e.g., sodium benzoate) and / or benzoic acid is present in the wash solution at a concentration of about 0.1 M to about 1.0 M. For example, the benzoate (e.g., sodium benzoate) and / or benzoic acid may be present at a concentration of about 0.1 M to about 1.0 M, about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.1 M to about 0.2 M, about 0. 2M~about 1.0M, about 0.2M~about 0.9M, about 0.2M~about 0.8M, about 0.2M~about 0.7M, about 0.2M~about 0.6M, about 0.2M~about 0.5M, about 0.2M ~0.4M, 0.2M~0.3M, 0.3M~1.0M, 0.3M~0.9M, 0.3M~0.8M, 0.3M~0.7M, 0.3M~0 .6M, about 0.3M to about 0.5M, about 0.3M to about 0.4M, about 0.4M to about 1.0M, about 0.4M to about 0.9M, about 0.4M to about 0.8M, about 0.4M to about 0.7 M, about 0.4M to about 0.6M, about 0.4M to about 0.5M, about 0.5M to about 1.0M, about 0.5M to about 0.9M, about 0.5M to about 0.8M, about 0.5M to about 0.7M, about The benzoate may be present in the wash solution at a concentration of about 0.5 M to about 0.6 M, about 0.6 M to about 1.0 M, about 0.6 M to about 0.9 M, about 0.6 M to about 0.8 M, about 0.6 M to about 0.7 M, about 0.7 M to about 1.0 M, about 0.7 M to about 0.9 M, about 0.7 M to about 0.8 M, 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 (e.g., sodium benzoate) and / or benzoic acid is present in the wash solution at a concentration of about 0.1 M to about 0.5 M. In some embodiments, the benzoate (e.g., sodium benzoate) and / or benzoic acid is present in the wash solution at a concentration of about 0.1 M to about 0.3 M.

[0045] In some embodiments, benzoate (e.g., sodium benzoate) and / or benzoic acid are present in the wash solution at a concentration of about any of 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M, 0.55 M, 0.6 M, 0.65 M, 0.7 M, 0.75 M, 0.8 M, 0.85 M, 0.9 M, 0.95 M, or 1.0 M. In some embodiments, benzoate (e.g., sodium benzoate) and / or benzoic acid are present in the wash solution at a concentration of about 0.5 M. In some embodiments, benzoate (e.g., sodium benzoate) and / or benzoic acid is present in the wash solution at a concentration of about or less than 0.1 M, about or less than 0.3 M, about or less than 0.5 M, about or less than 0.75 M, or about or less than 1.0 M. In some embodiments, benzoate (e.g., sodium benzoate) and / or benzoic acid is present in the wash solution at a concentration of about or less than 0.5 M.

[0046] In some embodiments, the present disclosure relates to cleaning solutions comprising benzyl alcohol. Any suitable source or form of benzyl alcohol known in the art can be used in the cleaning solutions of the present disclosure.

[0047] In some embodiments, benzyl alcohol is present in the wash solution at a concentration of about 0.5% to about 4.0% volume / volume (v / v). For example, benzyl alcohol may be present in the wash solution at a concentration of about 0.5% to about 4%, about 1% to about 4%, about 1.5% to about 4%, about 2% to about 4%, about 2% to about 4%, 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%, about 1.5% to about 3.5%, about 2% to about 3.5%, about 2.5% to about 3.5%, about 3% to about 3.5%, about 0.5% to about 3%, or about Benzyl alcohol may be present in the wash solution at a concentration of 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%, about 0.5% to about 1.5%, about 1% to about 1.5%, or about 0.5% to about 1% (v / v). In some embodiments, benzyl alcohol is present in the wash solution at a concentration of about 1% to about 4% volume / volume (v / v). In some embodiments, benzyl alcohol is present in the wash solution at a concentration of about 1% to about 2% volume / volume (v / v).

[0048] In some embodiments, benzyl alcohol is present in the wash solution at a concentration 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, 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, about 2% or less, about 3% or less, or about 4% or less. In some embodiments, benzyl alcohol is present in the wash solution at a concentration of about 4% or less (v / v). In some embodiments, benzyl alcohol is present in the wash solution at a concentration of about 2% or less (v / v).

[0049] additives In some embodiments, wash solutions of the present disclosure further comprise 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-buffering salts, and / or creatine, at any of the concentrations described herein. In some embodiments, wash solutions containing one or more additives have 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 improves the purification of a polypeptide comprising an Fc region from one or more impurities (e.g., host cell impurities) by the methods described herein.

[0050] In some embodiments, the cleaning solution comprises one of benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, hexylene glycol, a non-buffering salt, and / or creatine at a pH of about 4.0 to about 10.0. For example, the cleaning solution may comprise benzoate and / or benzyl alcohol, and benzenesulfonate; benzoate and / or benzyl alcohol, and caprylic acid; benzoate and / or benzyl alcohol, and hexylene glycol; benzoate and / or benzyl alcohol, and a non-buffering salt; or benzoate and / or benzyl alcohol, and creatine.

[0051] In some embodiments, the cleaning solution comprises benzoate and / or benzyl alcohol and two of benzenesulfonate, caprylic acid, hexylene glycol, non-buffering salts, and / or creatine at a pH of about 4.0 to about 10.0. For example, the cleaning solution may contain benzoate and / or benzyl alcohol, benzenesulfonate, and caprylic acid; benzoate and / or benzyl alcohol, benzenesulfonate, and hexylene glycol; benzoate and / or benzyl alcohol, benzenesulfonate, and a non-buffered salt; benzoate and / or benzyl alcohol, benzenesulfonate, and creatine; benzoate and / or benzyl alcohol, caprylic acid, and hexylene glycol; benzoate and / or benzyl alcohol, caprylic acid, and a non-buffered salt; benzoate and / or benzyl alcohol, caprylic acid, and creatine; benzoate and / or benzyl alcohol, hexylene glycol, and a non-buffered salt; benzoate and / or benzyl alcohol, hexylene glycol, and creatine; or benzoate and / or benzyl alcohol, a non-buffered salt, and creatine, at a pH of about 4.0 to about 10.0.

[0052] In some embodiments, the cleaning solution comprises three of benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, hexylene glycol, a non-buffering salt, and / or creatine at a pH of about 4.0 to about 10.0. For example, the cleaning solution may comprise three of benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, and hexylene glycol; benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, and a non-buffering salt; benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, and creatine; benzoate and / or benzyl alcohol, benzenesulfonate, hexylene glycol, and a non-buffering salt ... xylene glycol, and creatine; benzoate and / or benzyl alcohol, benzenesulfonate, unbuffered salts, and creatine; benzoate and / or benzyl alcohol, caprylic acid, hexylene glycol, and unbuffered salts; benzoate and / or benzyl alcohol, caprylic acid, hexylene glycol, and creatine; or benzoate and / or benzyl alcohol, caprylic acid, unbuffered salts, and creatine; benzoate and / or benzyl alcohol, hexylene glycol, unbuffered salts, and creatine.

[0053] In some embodiments, the cleaning solution comprises four of benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, hexylene glycol, an unbuffered salt, and / or creatine at a pH of about 4.0 to about 10.0. For example, the cleaning solution may comprise benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, hexylene glycol, and an unbuffered salt; benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, hexylene glycol, and creatine; benzoate and / or benzyl alcohol, benzenesulfonate, caprylic acid, an unbuffered salt, and creatine; benzoate and / or benzyl alcohol, benzenesulfonate, hexylene glycol, an unbuffered salt, and creatine; or benzoate and / or benzyl alcohol, caprylic acid, hexylene glycol, an unbuffered salt, and creatine at a pH of about 4.0 to about 10.0.

[0054] In some embodiments, the cleaning solution comprises all five of benzoate and / or benzyl alcohol, as well as benzenesulfonate, caprylic acid, hexylene glycol, non-buffering salts, and creatine at a pH of about 4.0 to about 10.0.

[0055] In some embodiments, the present disclosure relates to a cleaning solution comprising a benzenesulfonate salt. Any suitable form or source of benzenesulfonate known in the art can be used in the cleaning solution of the present disclosure, including, for example, benzenesulfonate salts (e.g., alkali salts), such as sodium benzenesulfonate or potassium benzenesulfonate, benzenesulfonic acid, and / or any combination thereof. In some embodiments, the benzenesulfonate salt is sodium benzenesulfonate.

[0056] In some embodiments, benzenesulfonate (e.g., sodium benzenesulfonate) is present in the cleaning solution at a concentration of about 0.1 M to about 0.5 M. For example, sodium benzenesulfonate may be present in the cleaning solution at a concentration of about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.1 M to about 0.2 M, about 0.1 M to about 0.2 M, 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 0.5 M, about 0.3 M to about 0.4 M, or about 0.4 M to about 0.5 M. In some embodiments, sodium benzenesulfonate is present in the cleaning solution at a concentration of about 0.1 M to about 0.3 M.

[0057] In some embodiments, sodium benzenesulfonate is present in the wash solution at a concentration 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 benzenesulfonate is present in the wash solution at a concentration of about or less than 0.1 M, about or less than 0.3 M, or about or less than 0.5 M. In some embodiments, sodium benzenesulfonate is present in the wash solution at a concentration of about or less than 0.5 M.

[0058] In some embodiments, the present disclosure relates to a cleaning solution comprising caprylic acid. Any suitable form or source of caprylic acid known in the art can be used in the cleaning solution of the present disclosure.

[0059] In some embodiments, caprylic acid is present in the wash solution at a concentration of about 0.1 mM to about 50 mM. For example, caprylic acid may be present in the wash solution at a concentration of about 1 mM to about 50 mM, about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 30 mM to about 50 mM, 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 about 10 mM to about 50 mM.

[0060] In some embodiments, caprylic acid is present in the wash solution at a 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 about 50 mM. In some embodiments, caprylic acid is present in the wash solution at a concentration of about 10 mM or less, about 30 mM or less, or about 50 mM or less. In some embodiments, caprylic acid is present in the wash solution at a concentration of about 50 mM or less.

[0061] In some embodiments, the present disclosure relates to cleaning solutions comprising hexylene glycol. Any suitable form or source of hexylene glycol known in the art can be used in the cleaning solutions of the present disclosure.

[0062] In some embodiments, hexylene glycol is present in the cleaning solution at a concentration of about 0.5% to about 10% (v / v). For example, hexylene may be present in the cleaning solution at a concentration of about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, about 4% to about 10%, about 6% to about 10%, about 8% to about 10%, about 9% to about 10%, 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%, or about 1% to about 1%. Hexylene glycol may be present in the cleaning solution at a concentration of 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%, about 2% to about 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 cleaning solution at a concentration of about 1% to about 10% (v / v).

[0063] In some embodiments, hexylene glycol is present in the wash solution at a concentration of about 0.5%, 1%, 1.5%, 2.5%, 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% (v / v). 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, about 2% or less, about 4% or less, about 6% or less, about 8% or less, or about 10% or less (v / v). In some embodiments, hexylene glycol is present in the wash solution at a concentration of about 10% or less (v / v).

[0064] In some embodiments, the present disclosure relates to a cleaning solution comprising one or more (e.g., one or more, two or more, three or more, etc.) non-buffering salts. Any suitable form or source of non-buffering salts known in the art can be used in the cleaning solutions of the present disclosure. The non-buffering salts can include halogen salts (e.g., salts containing Cl or Br), particularly halogen salts containing alkali metals (e.g., Na or K) or alkaline earth metals (e.g., Ca or Mg). In some embodiments, the non-buffering salt is sodium chloride or potassium chloride. In some embodiments, the non-buffering salt is sodium chloride.

[0065] In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.1 M to about 1.0 M. For example, the non-buffering salt (e.g., sodium chloride) may be present at a concentration of about 0.1 M to about 1.0 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.6 M, 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.2 M, about 0.2 M to about 1.0 M, about 0.2 M to about 0.8 M, about 0.2 M to about 0.6 M, about 0.2 M to about 0.5 M, about 0.2 M to about 0.2 The non-buffering salt (e.g., sodium chloride) may be present in the wash solution at a concentration of about 0.1 M to about 0.5 M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.5 M to about 1.0 M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.5 M to about 1.0 M.

[0066] In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration 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, 0.5 M, 0.55 M, 0.6 M, 0.65 M, 0.7 M, 0.75 M, 0.8 M, 0.85 M, 0.9 M, 0.95 M, or 1.0 M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.5 M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 1.0 M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 1.0 M. In some embodiments, a non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about or less than 0.1 M, about or less than 0.2 M, about or less than 0.4 M, about or less than 0.5 M, about or less than 0.6 M, about or less than 0.8 M, or about or less than 1.0 M. In some embodiments, a non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about or less than 0.5 M. In some embodiments, a non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about or less than 1.0 M.

[0067] In some embodiments, the present disclosure relates to a cleaning solution containing creatine.Any suitable form or source of creatine known in the art can be used in the cleaning solution of the present disclosure, including, for example, creatine-HCl, creatine ester, creatine pyruvate, creatine phosphate, creatine alpha-ketoglutarate, creatine citrate, and / or any combination thereof.In some embodiments, the creatine is creatine-HCl.

[0068] In some embodiments, creatine is present in the wash solution at a concentration of about 1 mM to about 100 mM. For example, creatine may be present in the wash solution at a concentration of about 1 mM to about 100 mM, about 10 mM to about 100 mM, about 25 mM to about 100 mM, about 50 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 25 mM to about 75 mM, about 50 mM to about 75 mM, about 1 mM to about 50 mM, about 10 mM to about 50 mM, about 25 mM to about 50 mM, about 1 mM to about 25 mM, about 10 mM to about 25 mM, or about 1 mM to about 10 mM. In some embodiments, creatine is present in the wash solution at a concentration of about 10 mM to about 100 mM. In some embodiments, creatine is present in the wash solution at a concentration of about 10 mM to about 50 mM. In some embodiments, creatine is present in the wash solution at a 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 about 50 mM.

[0069] Arginine In some embodiments, the present disclosure relates to a wash solution further comprising arginine and / or an arginine derivative. In some embodiments, the inclusion of arginine and / or an arginine derivative in the wash solution further improves the purification of a polypeptide comprising an Fc region from one or more impurities (e.g., host cell impurities) by the methods described herein. Any suitable form or source of arginine and / or an arginine derivative known in the art can be used in the wash solution of the present disclosure, 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 an arginine derivative can be L-arginine and / or D-arginine, as well as derivatives thereof. In some embodiments, the arginine and / or an arginine derivative is arginine-HCl.

[0070] In some embodiments, the present disclosure relates to the use of arginine and / or an arginine derivative (e.g., arginine-HCl) in a cleaning solution comprising benzoate and / or benzyl alcohol. In some embodiments, the cleaning solution comprises benzoate and arginine and / or an arginine derivative (e.g., arginine-HCl). In some embodiments, the cleaning solution comprises benzyl alcohol and arginine and / or an arginine derivative (e.g., arginine-HCl). In some embodiments, the cleaning solution comprises benzoate, benzyl alcohol, and arginine and / or an arginine derivative (e.g., arginine-HCl). In some embodiments, the cleaning 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, a non-buffering salt, and / or creatine, in any of the concentrations described herein. In some embodiments, a cleaning solution containing arginine and / or an arginine derivative has a pH of about 4.0 to about 10.0. In some embodiments, a cleaning solution containing arginine and / or an arginine derivative has a pH of about 4.0 to about 6.0. In some embodiments, a cleaning solution containing arginine and / or an arginine derivative has a pH of about 4.0 to about 5.0. In some embodiments, a cleaning solution containing arginine and / or an arginine derivative has a pH of about 8.0 to about 10.0. In some embodiments, a cleaning solution containing arginine and / or an arginine derivative has a pH of about 8.0 to about 9.0.

[0071] In some embodiments, the cleaning solution includes one of benzoate and / or benzyl alcohol, arginine and / or an arginine derivative (e.g., arginine-HCl), and benzenesulfonate, caprylic acid, hexylene glycol, non-buffering salts, and / or creatine. For example, the cleaning solution can include benzoate and / or benzyl alcohol, arginine, and benzenesulfonate; benzoate and / or benzyl alcohol, arginine, and caprylic acid; benzoate and / or benzyl alcohol, arginine, and hexylene glycol; benzoate and / or benzyl alcohol, arginine, and non-buffering salts; or benzoate and / or benzyl alcohol, arginine, and creatine.

[0072] In some embodiments, the cleaning solution includes two of benzoate and / or benzyl alcohol, arginine and / or an arginine derivative (e.g., arginine-HCl), and benzenesulfonate, caprylic acid, hexylene glycol, a non-buffering salt, and / or creatine. For example, the cleaning solution may include two of benzoate and / or benzyl alcohol, arginine, benzenesulfonate, and caprylic acid; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, and hexylene glycol; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, and a non-buffering salt; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, and creatine; benzoate and / or benzyl alcohol, arginine, caprylic acid, and benzoate and / or benzyl alcohol, arginine, caprylic acid, and unbuffered salts; benzoate and / or benzyl alcohol, arginine, caprylic acid, and creatine; benzoate and / or benzyl alcohol, arginine, hexylene glycol, and unbuffered salts; benzoate and / or benzyl alcohol, arginine, hexylene glycol, and creatine; or benzoate and / or benzyl alcohol, arginine, unbuffered salts, and creatine.

[0073] In some embodiments, the cleaning solution includes three of benzoate and / or benzyl alcohol, arginine and / or an arginine derivative (e.g., arginine-HCl), and benzenesulfonate, caprylic acid, hexylene glycol, a non-buffering salt, and / or creatine. For example, the cleaning solution may include three of benzoate and / or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, and hexylene glycol; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, and a non-buffering salt; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, and creatine; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, hexylene glycol, and a non-buffering salt .... benzoate and / or benzyl alcohol, arginine, benzenesulfonate, unbuffered salts, and creatine; benzoate and / or benzyl alcohol, arginine, caprylic acid, hexylene glycol, and unbuffered salts; benzoate and / or benzyl alcohol, arginine, caprylic acid, hexylene glycol, and creatine; benzoate and / or benzyl alcohol, arginine, caprylic acid, unbuffered salts, and creatine; or benzoate and / or benzyl alcohol, arginine, hexylene glycol, unbuffered salts, and creatine.

[0074] In some embodiments, the cleaning solution includes four of benzoate and / or benzyl alcohol, arginine and / or an arginine derivative (e.g., arginine-HCl), and benzenesulfonate, caprylic acid, hexylene glycol, an unbuffered salt, and / or creatine. For example, the cleaning solution may include benzoate and / or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, hexylene glycol, and an unbuffered salt; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, hexylene glycol, and creatine; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, caprylic acid, an unbuffered salt, and creatine; benzoate and / or benzyl alcohol, arginine, benzenesulfonate, hexylene glycol ....

[0075] In some embodiments, the cleaning solution includes all five of benzoate and / or benzyl alcohol, arginine and / or an arginine derivative (e.g., arginine-HCl), and benzenesulfonate, caprylic acid, hexylene glycol, a non-buffering salt, and creatine.

[0076] 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 to about 1.0 M. For example, arginine and / or an arginine derivative (e.g., arginine-HCl) may be present at a concentration of about 0.1 M to about 1.0 M, about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, or about 0.1 M to about 0. 2M, about 0.2M to about 1.0M, about 0.2M to about 0.9M, about 0.2M to about 0.8M, about 0.2M to about 0.7M, about 0.2M to about 0.6M, about 0.2M to about 0.5M, Approximately 0.2M to approximately 0.4M, approximately 0.2M to approximately 0.3M, approximately 0.3M to approximately 1.0M, approximately 0.3M to 0.9M, approximately 0.3M to approximately 0.8M, approximately 0.3M to approximately 0.7M, approximately 0.3M ~0.6M, approx. 0.3M~0.5M, approx. 0.3M~0.4M, approx. 0.4M~1.0M, approx. 0.4M~0.9M, approx. 0.4M~0.8M, approx. 0.4M~0 .7M, about 0.4M to about 0.6M, about 0.4M to about 0.5M, about 0.5M to about 1.0M, about 0.5M to about 0.9M, about 0.5M to about 0.8M, about 0.5M to about 0.7M The arginine and / or arginine derivative (e.g., arginine-HCl) may be present in the wash solution at a concentration of about 0.5 M to about 0.6 M, about 0.6 M to about 1.0 M, about 0.6 M to about 0.9 M, about 0.6 M to about 0.8 M, about 0.6 M to about 0.7 M, about 0.7 M to about 1.0 M, about 0.7 M to about 0.9 M, about 0.7 M to about 0.8 M, 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 arginine and / or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.1 M to about 0.5 M. In some embodiments, the arginine and / or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.1 M to about 0.3 M.

[0077] In some embodiments, arginine and / or an arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about any of 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M, 0.55 M, 0.6 M, 0.65 M, 0.7 M, 0.75 M, 0.8 M, 0.85 M, 0.9 M, 0.95 M, or 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 about 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 or less than 0.1 M, about or less than 0.2 M, about or less than 0.3 M, about or less than 0.4 M, about or less than 0.5 M, about or less than 0.75 M, or about or less than 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 about or less than 0.5 M.

[0078] pH In some embodiments, the present disclosure relates to a cleaning solution having a pH of about 4.0 to about 10.0. For example, the cleaning 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, 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, or about 6.0 to about The cleaning solution may have a pH of about 8.0, about 6.5 to about 8.0, about 7.0 to about 8.0, 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 about 4.0 to about 5.0. In some embodiments, the cleaning solution has a pH of about 5.0 to about 9.0. In some embodiments, the cleaning 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.

[0079] In some embodiments, the wash solution has a 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.

[0080] buffer In some embodiments, the cleaning solutions of the present disclosure further comprise one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) buffering agents. Any suitable buffering agent known in the art can be used in the cleaning solutions of the present disclosure, such as, for example, phosphate, Tris (tris(hydroxymethyl)methylamine, bis-tris, bis-trispropane, arginine, histidine, triethanolamine, diethanolamine, formate, carbonate, MES (2-(N-morpholino)ethanesulfonic acid), citrate, HEPES (4-2-hydroxyethyl-1-piperazinesulfonic 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), cacodylic acid (dimethylarsinic acid), SSC (saline sodium citrate), and / or any combination thereof. In some embodiments, the buffering agent is one or more of phosphate, Tris, arginine, acetate, and / or citrate.

[0081] In some embodiments, the buffering agent (e.g., phosphate, Tris, arginine, acetate, and / or citrate) is present in the wash solution at a concentration of about 1 mM to about 100 mM, or about 1 mM to about 500 mM. For example, the buffering agent (e.g., phosphate, Tris, arginine, acetate, and / or citrate) may be present 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 about 500 mM, 150 mM to about 500 mM, 200 mM to about 500 mM, 250 mM to about 500 mM, 300 mM to about 500 mM, 350 mM to about 500 mM, or 400 mM to about 500 mM. M~500mM, 400mM~500mM, 450mM~500mM, 1mM~450mM, 1mM~400mM, 1mM~350mM, 1mM~30 0mM, 1mM to about 250mM, 1mM to about 200mM, 1mM to about 150mM, 1mM to about 100mM, about 10mM to about 100mM, about 25mM to about 100mM, about 4 0mM to about 100mM, about 50mM to about 100mM, about 60mM to about 100mM, about 75mM to about 100mM, about 1mM to about 75mM, about 10mM to about 75mM, about 4 0mM to about 75mM, about 50mM to about 75mM, about 60mM to about 75mM, about 1mM to about 60mM, about 10mM to about 60mM, about 25mM to about 60mM, about 40mM to about In some embodiments, a buffer (e.g., phosphate, Tris, arginine, acetate, and / or citrate) may be present in the wash solution at a concentration of about 10 mM to about 50 mM, or about 10 mM to about 500 mM.

[0082] In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and / or citrate) is present in the wash solution at a 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. Alternatively, the buffer (e.g., phosphate, Tris, arginine, acetate, and / or citrate) is present in the wash solution at a concentration of about 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. In some embodiments, the buffering agent (e.g., phosphate, Tris, arginine, acetate, and / or citrate) is present in the wash solution at a concentration of about 500 mM. In some embodiments, the buffering agent (e.g., phosphate, Tris, arginine, acetate, and / or citrate) is present in the wash solution at a concentration of about 50 mM. In some embodiments, the buffering agent (e.g., phosphate, Tris, arginine, acetate, and / or citrate) is present in the wash solution at a concentration of about or less than 10 mM, about or less than 25 mM, about or less than 50 mM, about or less than 75 mM, or about or less than 100 mM, or about or less than 500 mM. In some embodiments, a buffering agent (e.g., phosphate, Tris, arginine, acetate, and / or citrate) is present in the wash solution at a concentration of about 50 mM or less, hi some embodiments, a buffering agent (e.g., phosphate, Tris, arginine, acetate, and / or citrate) is present in the wash solution at a concentration of about 500 mM or less.

[0083] Exemplary Cleaning Solutions In some embodiments, the cleaning solution of the present disclosure comprises sodium benzoate and / or benzyl alcohol and has a pH of about 7.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M and / or benzyl alcohol at a concentration of about 2% (v / v), and has a pH of about 7.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M and benzyl alcohol at a concentration of about 2% (v / v), and has a pH of about 7.0. In some embodiments, the cleaning solution further comprises a phosphate buffer (e.g., at a concentration of about 50 mM).

[0084] In some embodiments, the cleaning solution of the present disclosure comprises sodium benzoate, benzyl alcohol benzoate, and / or sodium chloride, and has a pH of about 7.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and / or sodium chloride at a concentration of about 0.5 M, and has a pH of about 7.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and sodium chloride at a concentration of about 0.5 M, and has a pH of about 7.0. In some embodiments, the cleaning solution further comprises a phosphate buffer (e.g., at a concentration of about 50 mM).

[0085] In some embodiments, the wash solution of the present disclosure comprises sodium benzoate, benzyl alcohol, arginine, and / or sodium chloride, and has a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), arginine at a concentration of about 0.5 M, and / or sodium chloride at a concentration of about 0.5 M, and has a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), arginine at a concentration of about 0.5 M, and sodium chloride at a concentration of about 0.5 M, and has a pH of about 7.0. In some embodiments, the wash solution further comprises a phosphate buffer (e.g., at a concentration of about 50 mM).

[0086] In some embodiments, the cleaning solution of the present disclosure comprises sodium benzoate, benzyl alcohol, phosphate buffer, and / or arginine, and has a pH of about 9.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), phosphate buffer at a concentration of about 50 mM, and / or arginine at a concentration of about 0.5 M, and has a pH of about 9.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), phosphate buffer at a concentration of about 50 mM, and arginine at a concentration of about 0.5 M, and has a pH of about 9.0.

[0087] In some embodiments, the cleaning solution of the present disclosure comprises sodium benzoate, benzyl alcohol, and / or arginine, and has a pH of about 6.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and / or arginine at a concentration of about 0.5 M, and has a pH of about 6.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and arginine at a concentration of about 0.5 M, and has a pH of about 6.0.

[0088] In some embodiments, the cleaning solution of the present disclosure comprises hexylene glycol, sodium benzoate, and / or benzyl alcohol and has a pH of about 7.0. In some embodiments, the cleaning solution comprises hexylene glycol at a concentration of about 10% (v / v), sodium benzoate at a concentration of about 0.5 M, and / or benzyl alcohol at a concentration of about 2% (v / v), and has a pH of about 7.0. In some embodiments, the cleaning solution comprises hexylene glycol at a concentration of about 10% (v / v), sodium benzoate at a concentration of about 0.5 M, and benzyl alcohol at a concentration of about 2% (v / v), and has a pH of about 7.0.

[0089] In some embodiments, the cleaning solution of the present disclosure comprises benzenesulfonate, sodium benzoate, and / or benzyl alcohol and has a pH of about 7.0. In some embodiments, the cleaning solution comprises benzenesulfonate at a concentration of about 0.5 M, sodium benzoate at a concentration of about 0.5 M, and / or benzyl alcohol at a concentration of about 2% (v / v), and has a pH of about 7.0. In some embodiments, the cleaning solution comprises benzenesulfonate at a concentration of about 0.5 M, sodium benzoate at a concentration of about 0.5 M, and benzyl alcohol at a concentration of about 2% (v / v), and has a pH of about 7.0.

[0090] In some embodiments, the cleaning solution of the present disclosure comprises sodium benzoate, benzyl alcohol, and / or arginine (e.g., arginine-HCl) and has a pH of about 5.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and / or arginine (e.g., arginine-HCl) at a concentration of about 0.5 M, and has a pH of about 5.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and arginine (e.g., arginine-HCl) at a concentration of about 0.5 M, and has a pH of about 5.0.

[0091] In some embodiments, the cleaning solution of the present disclosure comprises sodium benzoate, benzyl alcohol, and / or arginine (e.g., arginine-HCl) and has a pH of about 6.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and / or arginine (e.g., arginine-HCl) at a concentration of about 0.5 M, and has a pH of about 6.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, benzyl alcohol at a concentration of about 2% (v / v), and arginine (e.g., arginine-HCl) at a concentration of about 0.5 M, and has a pH of about 6.0.

[0092] In some embodiments, the cleaning solution of the present disclosure comprises benzyl alcohol and / or arginine (e.g., arginine-HCl) and has a pH of about 5.0. In some embodiments, the cleaning solution comprises benzyl alcohol at a concentration of about 2% (v / v) and / or arginine (e.g., arginine-HCl) at a concentration of about 0.5 M and has a pH of about 5.0. In some embodiments, the cleaning solution comprises benzyl alcohol at a concentration of about 2% (v / v) and arginine (e.g., arginine-HCl) at a concentration of about 0.5 M and has a pH of about 5.0.

[0093] In some embodiments, the cleaning solution of the present disclosure comprises 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 cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, arginine (e.g., arginine-HCl) at a concentration of about 0.5 M, caprylic acid at a concentration of about 50 mM, and / or sodium chloride at a concentration of about 0.5 M, and has a pH of about 9.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, arginine (e.g., arginine-HCl) at a concentration of about 50 mM, caprylic acid at a concentration of about 0.5 M, and sodium chloride at a concentration of about 9.0.

[0094] In some embodiments, the cleaning solution of the present disclosure comprises 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 cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, arginine (e.g., arginine-HCl) at a concentration of about 0.5 M, caprylic acid at a concentration of about 50 mM, and / or sodium chloride at a concentration of about 0.5 M, and has a pH of about 7.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M, arginine (e.g., arginine-HCl) at a concentration of about 50 mM, caprylic acid, and sodium chloride at a concentration of about 0.5 M, and has a pH of about 7.0.

[0095] In some embodiments, the cleaning solution of the present disclosure comprises sodium benzoate and / or sodium bicarbonate and has a pH of about 10.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M and / or sodium bicarbonate at a concentration of about 50 mM and has a pH of about 10.0. In some embodiments, the cleaning solution comprises sodium benzoate at a concentration of about 0.5 M and sodium bicarbonate at a concentration of about 50 mM and has a pH of about 10.0.

[0096] In some embodiments, the cleaning solution of the present disclosure comprises 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 cleaning solution comprises benzyl alcohol at a concentration of about 4% (v / v) and has a pH of about 9.0.

[0097] Preparing the collection containing the Fc region-containing polypeptide prior to chromatography In one aspect, a method for purifying a polypeptide comprising an Fc region is provided, comprising: (A) adjusting a harvest containing a polypeptide comprising an Fc region to achieve a final benzoate concentration of about 0.1 M to about 0.5 M and a pH of about 7.0 to about 9.0 to produce a sample containing (i) the polypeptide comprising an Fc region and (ii) one or more impurities; and (B) contacting the sample with at least one chromatography matrix. In some embodiments, the at least one chromatography matrix is ​​an affinity chromatography matrix, e.g., a Protein A chromatography matrix and / or a Protein G chromatography matrix. In some embodiments, the method further comprises contacting the at least one chromatography matrix with at least one wash solution. In some embodiments, the method further comprises contacting the at least one chromatography matrix with an elution solution. In some embodiments, the method further comprises collecting an eluate containing the polypeptide comprising an Fc region.

[0098] In some embodiments, the term "harvest" refers to a fluid present at the end of or after cell culture, e.g., a cell lysate sample or a cell culture supernatant sample (e.g., supernatant from cells, such as CHO cells, engineered to produce and secrete a polypeptide). In some embodiments, the harvest contains intact host cells and / or cell debris. In some embodiments, the harvest does not contain intact host cells and / or cell debris. For example, in some embodiments, the fluid present at the end of or after cell culture is subjected to one or more centrifugation and / or filtration steps to achieve a final benzoate concentration of about 0.1 M to about 0.5 M and a pH of about 7.0 to about 9.0 before conditioning. In some embodiments, the harvest is derived from a fluid present at the end of or after cell culture. For example, in some embodiments, the fluid present at the end of or after cell culture is subjected to one or more pretreatment steps to optimize the cell separation and / or purification of polypeptides comprising an Fc region.

[0099] In a related aspect, any one of the methods for purifying a polypeptide comprising an Fc region described herein further comprises adjusting the harvest comprising the polypeptide comprising an Fc region to achieve a final benzoate concentration of 0.1 M to about 0.5 M, and a pH of about 7.0 to about 9.0, to produce a sample comprising (i) the polypeptide comprising an Fc region, and (ii) one or more impurities.

[0100] In some embodiments, the benzoate is an alkaline benzoate, hi some embodiments, the benzoate is sodium benzoate. In some embodiments, the harvest is adjusted to achieve a final concentration of about one of any of 0.025M, 0.05M, 0.075M, 0.1M, 0.125M, 0.15M, 0.175M, 0.2M, 0.225M, 0.25M, 0.275M, 0.3M, 0.325M, 0.35M, 0.375M, 0.4M, 0.425M, 0.45M, 0.475M, 0.5M, 0.525M, 0.55M, 0.575M, 0.6M, 0.625M, 0.65M, 0.675M, 0.7M, 0.725M, 0.75M, 0.775M, or 0.8M, including any ranges between these values. In some embodiments, the pH of the harvest is adjusted to about one of 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 ranges between these values. In some embodiments, the harvest is clarified before conditioning (e.g., adding sodium benzoate and adjusting the pH). In some embodiments, the harvest is clarified after conditioning (e.g., adding sodium benzoate and adjusting the pH).

[0101] In some embodiments, the method includes adjusting the harvest to achieve a final sodium benzoate concentration of about 0.5 M and a pH of about 7. In some embodiments, the method includes adjusting the harvest to achieve a final sodium benzoate concentration of about 0.1 M and a pH of about 9. In some embodiments, the method includes adjusting the harvest to achieve a final sodium benzoate concentration of about 0.2 M and a pH of 9. In some embodiments, the method includes adjusting the harvest to achieve a final sodium benzoate concentration of about 0.3 M and a pH of 9. In some embodiments, the method includes adjusting the harvest to achieve a final sodium benzoate concentration of about 0.4 M and a pH of 9. In some embodiments, the method includes adjusting the harvest to achieve a final sodium benzoate concentration of about 0.5 M and a pH of 9.

[0102] In some embodiments, conditioning the harvest (e.g., adding sodium benzoate and adjusting the pH) results in a polypeptide comprising an Fc region that is purified from one or more impurities to a greater extent than a corresponding method lacking the step of conditioning the harvest containing the polypeptide comprising an Fc region to produce a sample. In some embodiments, the one or more impurities are host cell proteins such as phospholipases, clusterin, serine proteases, elongation factors, and any combination thereof. In some embodiments, the HCP is putative phospholipase B-like 2 (PLBL2).

[0103] In some embodiments, the conditioned harvest (e.g., sodium benzoate has been added to achieve a final concentration described herein, and the pH has been adjusted as described herein) is a sample that contains or comprises (i) a polypeptide comprising an Fc region, and (ii) one or more impurities. In some embodiments, the sample is contacted with at least one chromatography matrix (e.g., the sample is subjected to at least one chromatography step). In some embodiments, the at least one chromatography matrix comprises one or more of an affinity chromatography matrix, a mixed-mode chromatography matrix (e.g., a multimode chromatography matrix), a hydrophobic interaction (HIC) chromatography matrix, an anion exchange chromatography matrix, a cation exchange chromatography matrix, a size-exclusion chromatography matrix, a ceramic hydroxyapatite (CHT) chromatography matrix, and / or a hydrophilic interaction liquid chromatography matrix (HILIC), in any order. In some embodiments, the sample is contacted with an affinity chromatography matrix, e.g., a Protein A matrix or a Protein G matrix. In some embodiments, the method further comprises contacting the at least one chromatography matrix with at least one wash solution. In some embodiments, the method further comprises contacting at least one chromatography matrix with an elution solution, hi some embodiments, the method further comprises recovering the eluate comprising the polypeptide comprising the Fc region.

[0104] Impurity removal Certain aspects of the present disclosure relate to methods for purifying a polypeptide comprising an Fc region via Protein A chromatography by washing a Protein A matrix bound to the polypeptide (e.g., an antibody) comprising an Fc region with a wash solution comprising benzoate and / or benzyl alcohol to improve purification of the polypeptide from one or more impurities. In some embodiments, the method includes contacting the Protein A chromatography matrix with a sample comprising (1) a polypeptide (e.g., an antibody) comprising an Fc region and (2) one or more impurities (e.g., host cell impurities) under conditions under which the polypeptide (e.g., the antibody) comprising the Fc region binds to Protein A; and washing the matrix with a wash solution comprising benzoate at a concentration of about 0.1 M to about 1.0 M and / or benzyl alcohol at a concentration of about 0.5% to about 4% volume / volume (v / v), wherein the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, washing the Protein A matrix with a wash solution results in a polypeptide comprising an Fc region that is purified from one or more impurities to a greater extent than a corresponding method (described above) that lacks the step of washing the matrix with a wash solution.

[0105] Standard Protein A processes typically result in approximately 95% product purity without the use of the wash steps described herein. The largest proportion of impurities in the product are due to high molecular weight (HMW) aggregates and / or low molecular weight (LMW) fragments of the product. These product variants are considered impurities due to their ability to be separated from the product based on various parameters (e.g., differences in charge, hydrophobicity, size, etc.). These HMW and LMW impurities account for approximately 4-5% of the Protein A pool. Furthermore, standard Protein A processes lacking the use of the wash steps described herein also typically result in the inclusion of host cell protein (HCP) impurities on the order of approximately 1000 ppm, or approximately 0.1%, of the product pool. However, due to the specifications set for injectable mAb products (see, e.g., FDA guidelines), the reduction and / or complete removal of this 0.1% HCP impurity is critical. Inclusion of a step applying the wash solution described herein can reduce the amount of HCPs present in the pool to approximately 100-10 ppm (a 10- to 100-fold reduction in HCPs compared to the same Protein A process lacking the wash step described herein), accounting for a relative improvement of 90-99%. Methods for measuring sample protein purity and / or impurity levels (e.g., by ELISA assay) are generally known to those of skill in the art. An exemplary purification of a monoclonal antibody (mAb) from one or more host cell proteins using either standard methods versus the methods described herein is shown in Table A below.

[0106] [Table 1]

[0107] In some embodiments, the methods described herein produce a protein pool containing a polypeptide (e.g., a monoclonal antibody) comprising an Fc region after Protein A elution containing less than about 500 parts per million (ppm) of HCPs (e.g., one or more HCPs from CHO cells). For example, a protein pool containing a polypeptide comprising 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 of HCPs (e.g., one or more HCPs from CHO cells). In some embodiments, a protein pool containing a polypeptide comprising an Fc region produced by the methods described herein contains less than about 100 ppm of HCPs (e.g., one or more HCPs from CHO cells). In some embodiments, a protein pool containing a polypeptide comprising an Fc region produced by the methods described herein contains less than about 10 ppm of HCPs (e.g., one or more HCPs from CHO cells).

[0108] In some embodiments, the methods described herein produce a protein pool containing polypeptides comprising an Fc region (e.g., monoclonal antibodies) after Protein A elution that contains less than about 0.1% HCPs (e.g., one or more HCPs from CHO cells). For example, a protein pool containing polypeptides comprising an Fc region produced 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% HCPs (e.g., one or more HCPs from CHO cells). In some embodiments, a protein pool containing polypeptides comprising an Fc region produced by the methods described herein contains less than about 0.05% HCPs (e.g., one or more HCPs from CHO cells). In some embodiments, a protein pool containing a polypeptide comprising an Fc region produced by the methods described herein contains less than about 0.01% HCPs (e.g., one or more HCPs from CHO cells).

[0109] In some embodiments, the methods described herein reduce the amount and / or concentration (e.g., parts per million) of one or more impurities (e.g., one or more HCPs, such as one or more HCPs from CHO cells) co-purified with a polypeptide comprising an Fc region by at least about 10% compared to the amount of one or more impurities co-purified with a polypeptide comprising an Fc region purified by a corresponding method lacking the step of washing the Protein A matrix with a wash solution. For example, the methods described herein reduce the amount and / or concentration (e.g., parts per million) of one or more impurities (e.g., one or more HCPs, such as one or more HCPs from CHO cells) co-purified with a polypeptide comprising an Fc region by at least about 10%, at least about 15%, at least about 20%, 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 co-purified with a polypeptide comprising an Fc region purified by a corresponding method lacking the step of washing the Protein A matrix with a wash solution. In some embodiments, the methods described herein reduce the amount and / or concentration (e.g., parts per million) of one or more impurities (e.g., one or more HCPs, such as one or more HCPs from CHO cells) co-purified with a polypeptide comprising an Fc region by at least 1.5-fold compared to the amount of one or more impurities co-purified with a polypeptide comprising an Fc region purified by a corresponding method lacking the step of washing the Protein A matrix with a wash solution.For example, the methods described herein reduce the amount and / or concentration (e.g., parts per million) of one or more impurities (e.g., one or more HCPs, such as one or more HCPs from CHO cells) co-purified with a polypeptide comprising an Fc region, compared to the amount of one or more impurities co-purified with a polypeptide comprising an Fc region purified by a corresponding method lacking the step of washing the Protein A matrix with a wash solution, by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 50-fold, or at least about 100-fold.

[0110] Additional processes In some embodiments, the methods described herein further comprise one or more additional washing steps. In some embodiments, the methods described herein further comprise one or more elution steps. In some embodiments, the methods described herein further comprise one or more washing steps and one or more elution steps.

[0111] In some embodiments, the present disclosure relates to washing a Protein A matrix with a first solution before washing the matrix with a wash solution. In some embodiments, before washing the matrix with a wash solution, the matrix is ​​washed one or more times (e.g., one or more times, two or more times, three or more times, four or more times, five or more times, etc.) with the first solution. In some embodiments, before washing the matrix with a wash solution, the matrix is ​​washed once with the first solution. In some embodiments, the first solution comprises a buffer. Any suitable buffer known in the art may be used in the first solution, such as, 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)methyl) In some embodiments, the first solution comprises a phosphate buffer, a Tris buffer, an acetate buffer, a carbonate buffer, and / or a citrate buffer. In some embodiments, the first solution comprises a phosphate buffer. In some embodiments, the first solution comprises one or more additional components (e.g., a benzoate, benzyl alcohol, one or more 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, 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 about 7.0.

[0112] In some embodiments, the first solution contains a buffer solution at a concentration of about 10 mM to about 100 mM, or about 10 mM to about 500 mM. For example, the first solution may contain a buffer solution at a concentration of about 10 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 200 mM to about 500 mM, about 250 mM to about 500 mM, about 300 mM to about 500 mM, about 350 mM to about 500 mM, about 400 mM to about 500 mM, about 450 mM to about 500 mM, or about 500 mM. 0mM to about 500mM, about 10mM to about 450mM, about 10mM to about 400mM, about 10mM to about 350mM, about 10mM to about 300mM , about 10mM to about 250mM, about 10mM to about 200mM, about 10mM to about 150mM, about 10mM to about 100mM, about 25mM to about 100 The solution may contain a buffer solution with a concentration of about 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, 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, about 25 mM to about 40 mM, or about 10 mM to about 25 mM. In some embodiments, the first solution comprises a buffer at a concentration of about 10 mM to about 50 mM, or about 10 mM to about 500 mM.

[0113] In some embodiments, the first solution comprises a buffer solution at a 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 comprises a buffer solution at a concentration of about 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. In some embodiments, the first solution comprises a buffer solution at a concentration of about 500 mM. In some embodiments, the first solution comprises a buffer solution at a concentration of about 50 mM. In some embodiments, the first solution comprises a phosphate buffer solution at a concentration of about 500 mM. In some embodiments, the first solution comprises a phosphate buffer at a concentration of about 50 mM.

[0114] In some embodiments, the first solution comprises a phosphate buffer (e.g., sodium phosphate) and sodium chloride. In some embodiments, the first solution comprises a phosphate buffer (e.g., sodium phosphate) and sodium chloride, and has a pH of about 7.0. In some embodiments, the first solution comprises a phosphate buffer (e.g., sodium phosphate) at a concentration of about 50 mM and sodium chloride at a concentration of about 0.5 M. In some embodiments, the first solution comprises a phosphate buffer (e.g., sodium phosphate) at a concentration of about 50 mM, sodium chloride at a concentration of about 0.5 M, and has a pH of about 7.0.

[0115] In some embodiments, the present disclosure relates to washing the Protein A matrix with a second solution after washing the matrix with a wash solution. In some embodiments, after washing the matrix with the wash solution, the matrix is ​​washed one or more times (e.g., one or more times, two or more times, three or more times, four or more times, five or more times, etc.) with the second solution. In some embodiments, after washing the matrix with the wash solution, the matrix is ​​washed once with the second solution. In some embodiments, the second solution comprises a buffer. Any suitable buffer known in the art may be used in the second solution, such as, 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}ethyl In some embodiments, the second solution comprises a phosphate buffer, a Tris buffer, an acetate buffer, a carbonate buffer, and / or a citrate buffer. In some embodiments, the second solution comprises a phosphate buffer. In some embodiments, the second solution comprises substantially low salt or no 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 about 7.0. In some embodiments, the second solution is substantially low in salt. In some embodiments, the second solution is salt-free.

[0116] In some embodiments, the second solution contains a buffer solution at a concentration of about 10 mM to about 100 mM, or about 10 mM to about 500 mM. For example, the second solution may contain a buffer solution at a concentration of about 10 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 200 mM to about 500 mM, about 250 mM to about 500 mM, about 300 mM to about 500 mM, about 350 mM to about 500 mM, about 400 mM to about 500 mM, about 450 mM to about 500 mM, or about 500 mM. 0mM to about 500mM, about 10mM to about 450mM, about 10mM to about 400mM, about 10mM to about 350mM, about 10mM to about 300mM , about 10mM to about 250mM, about 10mM to about 200mM, about 10mM to about 150mM, about 10mM to about 100mM, about 25mM to about 100 The solution may contain a buffer solution with a concentration of about 100 mM, about 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, 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, about 25 mM to about 40 mM, or about 10 mM to about 25 mM. In some embodiments, the second solution comprises a buffer at a concentration of about 10 mM to about 50 mM, or about 10 mM to about 500 mM.

[0117] In some embodiments, the second solution comprises a buffer solution at a 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 comprises a buffer solution at a concentration of about 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. In some embodiments, the second solution comprises a buffer solution at a concentration of about 500 mM. In some embodiments, the second solution comprises a buffer solution at a concentration of about 50 mM.

[0118] In some embodiments, the second solution comprises a phosphate buffer (e.g., sodium phosphate). In some embodiments, the second solution comprises a phosphate buffer (e.g., sodium phosphate) and has a pH of about 7.0. In some embodiments, the second solution comprises a phosphate buffer (e.g., sodium phosphate) at a concentration of about 50 mM. In some embodiments, the second solution comprises a phosphate buffer (e.g., sodium phosphate) at a concentration of about 50 mM and has a pH of about 7.0.

[0119] In some embodiments, the disclosed methods involve washing a Protein A matrix with a wash solution and do not include washing the matrix with a first solution (before the wash solution) or a second solution (after the wash solution). In some embodiments, the disclosed methods involve washing a Protein A matrix with a first solution, then washing the matrix with a wash solution, and do not include washing the matrix with a second solution (after the wash solution). In some embodiments, the disclosed methods involve washing a Protein A matrix with a wash solution, then washing the matrix with a second solution, and do not include washing the matrix with the first solution (before the wash solution). In some embodiments, the disclosed methods involve washing a Protein A matrix with a first solution, then washing the matrix with a wash solution, and then washing the matrix with a second solution.

[0120] In some embodiments, the Protein A matrix is ​​contacted with the elution solution one or more times (e.g., one or more times, two or more times, three or more times, four or more times, five or more times, etc.) after one or more washing steps. In some embodiments, the matrix is ​​contacted with the elution solution once. Any solution known in the art suitable for eluting polypeptides bound to a Protein A matrix can be used as the elution solution in the disclosed methods (e.g., an elution solution comprising 40 mM sodium acetate having a pH of about 3.1). In some embodiments, the elution solution further comprises one or more additional components (e.g., arginine at any of the concentrations described herein). In some embodiments, an eluate comprising a polypeptide comprising an Fc region is collected after contacting the matrix with the elution solution. In some embodiments, two or more eluates comprising a polypeptide comprising an Fc region are collected after contacting the matrix with the elution solution two or more times. In some embodiments, the two or more eluates are combined after elution. In some embodiments, the eluate(s) are filtered. Any suitable method of filtering the eluate known in the art can be used, including, for example, via depth filtration. In some embodiments, the eluate(s) is filtered via depth filtration.

[0121] In some embodiments, the eluate from a Protein A matrix described herein is further processed and / or purified (e.g., using additional chromatography and / or filtration steps (e.g., 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 viral removal filtration)), and / or formulated (e.g., to prepare a pharmaceutical formulation suitable for administration to a subject (such as a human subject) in need thereof).

[0122] The foregoing written description is deemed sufficient to enable one skilled in the art to practice the present disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, various modifications of the present disclosure, 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. [Example]

[0123] Washing solutions to improve or accelerate the removal of impurities during antibody purification The following examples describe the use of various combinations of sodium benzoate and benzyl alcohol in intermediate wash solutions to improve / accelerate the removal of impurities during Protein A chromatography.

[0124] material and method Sample preparation Two separate human monoclonal antibody harvest materials were prepared for Protein A chromatography as follows: the harvests were generated in suspension cultures of recombinant Chinese hamster ovary (CHO) cells engineered to constitutively express one of the antibodies. The recombinant product was secreted into the culture medium, then centrifuged and clarified by depth filtration for downstream processing. The clarified harvest material was filtered through a 0.22 μm polyethersulfone (PES) filter before loading onto the Protein A column.

[0125] Protein A chromatography Protein A resin / columns were prepared as follows: MabSelect Sure LX Protein A chromatography resin (GE Healthcare Life Sciences; catalog number 17-5474) was exchanged with 0.5 M sodium chloride solution by gravity settling. Columns were packed using either (A) a 1.0 cm diameter column (Essential Life Solutions 10 / 250 Snap column; catalog number S10 / 250-PPSL-OE-FP10) or (B) a 0.66 cm diameter column (Omnifit™ 6.6 / 100; catalog number 006BCC0610FF) in an AKTA Pure or AKTA Avant (GE Healthcare Life Sciences). The resin was packed to a bed height of 20 cm ± 10% for column (A) or a bead height of 5 cm ± 10% for column (B). Column qualification was performed using a 1% column volume injection of 1.0 M sodium chloride solution onto a column equilibrated with 0.1 M sodium chloride solution, and the conductivity trace was analyzed using Unicorn Evaluation software. Column efficiency was required to be at least 775 theoretical plates per meter and exhibit asymmetry of 0.8 to 1.8.

[0126] Prior to loading the harvest material, the column was flushed with reverse osmosis deionized water to remove the 20% ethanol storage buffer. The column was then flushed with 0.5 M acetic acid to ensure removal of any entities bound prior to equilibration. The column was equilibrated with 50 mM phosphate buffer containing 0.5 M sodium chloride until the column pH was >6.5.

[0127] The prepared sample was then loaded onto a Protein A column. The column was loaded with either mAb A (IgG1 subtype) or mAb B (IgG4 subtype) to a target of 40 g / L resin. The loaded column was washed as described above with a phosphate buffer containing 50 mM phosphate and 0.5 M sodium chloride. The column was then washed with the test wash solution, followed by a salt-free wash using a pH 7 buffer. Finally, the antibody was eluted from the column using a solution containing 40 mM sodium acetate at pH 3.1. Table 1 below provides an exemplary chromatography process.

[0128] [Table 2]

[0129] Host cell protein detection After elution, antibody samples were tested for the presence of host cell protein (HCP) impurities using a third-generation CHO HCP ELISA kit (Cygnus Technologies) according to the manufacturer's protocol. HCP ELISA was performed at dilutions between 1:400 and 1:800, and absorbance was read at 450 / 600 nm using a Spectramax Plus 384 plate reader.

[0130] Detection of specific "HCP-A" The concentration of specific HCP (HCP-A) was quantified in the eluted antibody samples using a commercially available hamster (CHO) ELISA kit (ICL Labs) according to the manufacturer's protocol. HCP-A ELISA was performed at dilutions between 1:100 and 1:800, and absorbance was read at 450 nm using a Spectramax Plus 384 plate reader.

[0131] result Host cell proteins (HCPs) have been shown to co-elute with monoclonal antibodies (mAbs), which can be problematic for downstream applications of these antibodies. To identify potential wash additives that can reduce the amount of contaminating HCPs co-eluting with the mAb of interest, a 1.7 mL Protein A chromatography column was loaded with a sample containing a secreted IgG1 human monoclonal antibody (mAb A) recovered from CHO cells and subjected to both centrifugation and depth filtration clarification prior to downstream processing (see Table 1). The column was first washed with phosphate buffer. Next, the column was washed with one of several test wash solutions containing 50 mM phosphate and additives, either individually or in combination with sodium benzoate, benzyl alcohol, and arginine, at pH 7.0 (Figure 1). A control experiment did not incorporate any additive wash solution. After the test wash, the column was 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 6.0 using 2 M Tris base. After conditioning, the eluate pool was filtered using a 0.22 μm PES filter and tested for the presence of HCP impurities (Figure 1). Interestingly, wash solutions containing 2% benzyl alcohol and / or 0.5 M sodium benzoate effectively reduced the levels of contaminating HCPs in the eluted antibody samples. The inclusion of arginine in the wash solution was also observed to improve HCP clearance.

[0132] The specific presence of HCP-A in the antibodies eluted from the Protein A column was then examined. The test and control wash solutions used in this experiment are shown in Table 2 below.

[0133] [Table 3]

[0134] Having demonstrated the ability of the 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 HCP-A expression was used to specifically increase HCP-A loading on the column. Table 1 provides an overview of the steps performed during the purification process. Briefly, after equilibration for loading pH and conductivity, a 15.7 mL Protein A chromatography column was loaded with a sample containing secreted human monoclonal antibodies recovered from CHO cells by both centrifugation and depth filtration clarification prior to downstream processing. Upon reaching a load of 40 g / L (of resin), the column was re-equilibrated using phosphate buffer. Two column volumes (CV) of one of the wash solutions (Test Solutions 1, 2, and 7) listed in Table 2 were then applied, immediately followed by 3 CV of salt-free phosphate buffer at pH 7.0. After elution with 40 mM sodium acetate, the eluate pool was adjusted to pH 5.5 using 2 M Tris base. The eluate pool was then filtered through either a 0.22 μm PES filter or a Millipore COHC depth filter and tested for HCP-A content by ELISA (Figure 2A). All three wash conditions (test solutions 1, 2, and 7) were able to remove additional HCP-A compared to the control (294 ppm). The results also suggested that the effect of HCP-A removal was cumulative, as the combination of benzyl alcohol, sodium benzoate, and arginine removed nearly 90% of HCP-A compared to the control. Furthermore, depth filtration subsequently resulted in an additional 15–25% HCP-A removal relative to the wash condition eluate.

[0135] Following a similar procedure as above, the effectiveness of a high-pH wash solution was then tested by washing the column at a pH of 9.0 or 10.0. An IgG4 antibody (mAb B) was produced in a cell line highly expressing a known HCP-A to specifically increase HCP-A loading onto the Protein A column. Table 1 provides an overview of the steps performed during the purification process. Briefly, after equilibration for loading pH and conductivity, a 15.7 mL Protein A chromatography column was loaded with a sample containing secreted human monoclonal antibody recovered from CHO cells, which had been harvested by both centrifugation and depth filtration clarification prior to downstream processing. Upon reaching a load of 60 g / L (of resin), the column was re-equilibrated using phosphate buffer. Then, 2 CV of either Test Solution 8 or 9 (Table 2) was applied, immediately followed by 3 CV of salt-free phosphate buffer at pH 7.0. After elution with 40 mM sodium acetate, the elution pool was adjusted to pH 5.5 using 2 M Tris base. The eluate pool was then filtered through a 0.22 μm PES filter. As shown in Figure 2B, 0.5 M sodium benzoate with a buffer salt of pH 10.0 was highly effective in removing HCP-A (92% reduction) compared to the control. Furthermore, the addition of benzyl alcohol and arginine, again complementary, increased HCP-A clearance at pH 9.0. Taken together, the results shown in Figures 2A-B demonstrate a robust pH range that allows significant clearance of HCP-A.

[0136] Finally, additional additives were screened to further enhance the wash with 2% benzyl alcohol and 0.5 M sodium benzoate. The same IgG4 mAb (mAb B) was used in a 1.7 mL-scale column. Table 1 provides an overview of the steps performed during the purification process. Briefly, after equilibration, the column was loaded at 40 g / L. Next, one of the four washes (test solutions 3–6) listed in Table 2 was applied to the column. The eluted sample was then adjusted to pH 5.5 using 2 M Tris base and filtered using a 0.22 μm PES filter. HCP-A content was then measured using an HCP-A-specific ELISA (Figure 3). Washes with the test solutions demonstrated a cumulative response of HCP-A removal with 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 highly effective in removing HCP-A.

[0137] In summary, the data provided in this example demonstrate that intermediate wash steps containing sodium benzoate and / or benzyl alcohol could provide superior clearance of host cell protein impurities during Protein A purification of human monoclonal antibodies. Furthermore, the inclusion of one or more additives selected from benzenesulfonate, caprylic acid, hexylene glycol, and / or arginine in the wash solution further improved the clearance of host cell protein impurities during Protein A purification of the target monoclonal antibody. [Example]

[0138] Identification of specific host cell proteins present after Protein A chromatography, development of wash solutions to improve or accelerate removal of impurities during antibody purification, and evaluation of putative phospholipase B-like 2 interactions with human monoclonal antibodies The following example describes the identification of specific host cell proteins (HCPs) in purified antibody eluates after Protein A chromatography. This example further describes how sodium benzoate and benzyl alcohol in intermediate wash solutions can be used to improve / accelerate the removal of HCPs, including putative phospholipase B-like 2 (PLBL2), during Protein A chromatography. Finally, this example describes the impact of loading conditions on the efficiency of Protein A chromatography.

[0139] material and method Sample preparation Human monoclonal antibody harvest material was prepared for Protein A chromatography as described in Example 1. Briefly, harvest was generated in suspension cultures of recombinant CHO cells engineered to constitutively express one of the human monoclonal antibodies. The recombinant product was secreted into the culture medium and then centrifuged and clarified by depth filtration for downstream processing. The clarified harvest material was filtered through a 0.22 μm polyethersulfone (PES) filter before loading onto a Protein A column.

[0140] Protein A chromatography Protein A resin / column was prepared as described in Example 1. Briefly, MabSelect Sure LX Protein A chromatography resin (GE Healthcare Life Sciences; catalog number 17-5474) was exchanged with 0.5 M sodium chloride solution by gravity settling. Columns were packed using either (A) a 1.0 cm diameter column (Essential Life Solutions 10 / 250 Snap column; catalog number S10 / 250-PPSL-OE-FP10) or (B) a 0.66 cm diameter column (Omnifit™ 6.6 / 100; catalog number 006BCC0610FF) using an AKTA Pure or AKTA Avant (GE Healthcare Life Sciences). The resin was packed to a bed height of 20 cm ± 10% for column (A) or a bead height of 5 cm ± 10% for column (B). Column qualification was performed using a 1% column volume injection of 1.0 M sodium chloride solution onto a column equilibrated with 0.1 M sodium chloride solution, and the conductivity trace was analyzed using Unicorn Evaluation software. Column efficiencies were required to be at least 775 theoretical plates per meter and exhibit asymmetry between 0.8 and 1.8.

[0141] Prior to loading the harvest material, the column was flushed with reverse osmosis deionized water to remove the 20% ethanol storage buffer. The column was then flushed with 0.5 M acetic acid to ensure removal of any entities bound prior to equilibration. The column was equilibrated with 50 mM phosphate buffer containing 0.5 M sodium chloride until the column pH was >6.5.

[0142] The prepared sample was then loaded onto a Protein A column. Typically, the column was loaded with either human monoclonal antibody A (IgG1 subtype) or human monoclonal antibody B (IgG4 subtype) to a target of 40 g / L resin. The loaded column was washed as described above with a phosphate buffer containing 50 mM phosphate and 0.5 M sodium chloride. Next, the column was washed with the test wash solution, followed by a salt-free wash using a pH 7 buffer. However, the control treatment did not wash 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. Table 1 provides an exemplary chromatography process.

[0143] HCP detection by mass spectrometry After Protein A column purification and elution under standard conditions, the human monoclonal antibody eluate was analyzed by mass spectrometry to determine the relative abundance of each HCP. Specifically, the relative amounts of clusterin and putative phospholipase B-like 2 (PLBL2) were identified in the Protein A-purified human monoclonal antibody samples. Mass spectrometry was performed using an Acquity H-Class Xevo G2-XS Q-Tof and a 2.1 x 150 mm ACQUITY UPLC 1.7 μm CSH C18 column. The sample was first denatured using 0.05% Rapigest in 50 mM ammonium bicarbonate, then reduced and alkylated using 20 mM DTT (dithiothreitol) and 40 mM IAA (iodoacetamide). Enzymatic digestion was performed overnight with 2% LysC, followed by 3 hours with 4% trypsin. The Rapigest was removed by centrifugation, and the sample was acidified using formic acid. A spike-in internal standard of 2.5 fmol / μl ClpB E. coli was then added to compare the relative amounts of HCP and PLBL2. Lock mass calibration was performed around m / z 785.8426. MS E was used to analyze the ion data and identify the HCPs.

[0144] HCP detection by ELISA After elution, the presence of generic HCPs in human monoclonal antibody samples was assessed as described in Example 1. Briefly, antibody samples were tested for the presence of HCP impurities using a 3rd generation CHO HCP ELISA kit (Cygnus Technologies) according to the manufacturer's protocol. HCP ELISA was performed at dilutions between 1:400 and 1:800, and absorbance was read at 450 / 600 nm using a Spectramax Plus 384 plate reader.

[0145] PLBL2 detection by ELISA The concentration of PLBL2 was quantified in the 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 between 1:100 and 1:800, and absorbance was read at 450 nm using a Spectramax Plus 384 plate reader.

[0146] result Detection of HCPs by mass spectrometry After Protein A purification, HCPs have been shown to co-elute with human monoclonal antibodies, which can be problematic for downstream applications of these antibodies. To identify the specific HCPs present in the purified human monoclonal antibody solution after Protein A purification, a 1.7 mL Protein A chromatography column was loaded with a sample containing a secreted IgG1 human monoclonal antibody (mAb A) recovered from CHO cells and clarified by both centrifugation and depth filtration prior to downstream processing (see Table 1). The column was first washed with phosphate buffer. Next, the column was 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 6.0 using 2 M Tris base. After adjustment, the eluate pool was filtered using a 0.22 μm PES filter and analyzed by mass spectrometry to identify the relative amounts of HCPs. Notably, clusterin and PLBL2 were the two most abundant HCPs present in the human monoclonal antibody eluate.

[0147] Identification of intermediate wash conditions to improve HCP / PLBL2 removal Mass spectrometry analysis, as detailed above, provided further evidence that HCPs, including PLBL2, co-elute with human monoclonal antibodies after Protein A purification. Next, an ELISA screen was developed to identify intermediate wash solutions that could be used to further improve and accelerate the removal of HCPs and PLBL2 (Figure 4A and 4B). Interestingly, intermediate wash solutions containing 4% benzyl alcohol or 0.5 M sodium benzoate effectively reduced the levels of contaminating HCPs and PLBL2 in antibody eluate samples. Washes containing only 0.5 M arginine did not reduce generic HCP levels but did reduce contaminating PLBL2 levels.

[0148] The PLBL2 ELISA screen further demonstrates that depth filtration combined with intermediate wash solutions containing 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, and 0.5 M arginine pH 6.0 effectively removed PLBL2 during Protein A purification. Furthermore, wash 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 Protein A purification. Indeed, the sodium benzoate wash solution containing 50 mM sodium bicarbonate pH 10.0 was able to remove over 92% of PLBL2 compared to the control treatment. Finally, wash solutions containing 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 demonstrated robust ability to remove PLBL2 compared to the control wash.

[0149] Furthermore, visual comparison of the antibody eluates after both experimental and control washes revealed that samples washed with 2% benzyl alcohol and 0.5 M sodium benzoate had improved clarity compared to samples washed with the control (Figure 5).

[0150] The PLBL2 ELISA detailed above demonstrates that an intermediate wash solution containing benzyl alcohol and sodium benzoate can effectively reduce the levels of PLBL2 in Protein A-purified antibody eluates. This result was then further confirmed orthogonally by mass spectrometry. Mass spectrometry results indicate that the test wash solution, 0.5 M sodium benzoate, 0.5 M arginine, 50 mM caprylic acid, 0.5 M NaCl pH 9.0, removed nearly 93% of PLBL2 from the eluate compared to the control treatment.

[0151] Evaluation of the effect of column loading on yield and PLBL2 removal The above experiments demonstrate that the host cell protein PLBL2 is present in Protein A-purified antibody eluates and can be effectively removed using an intermediate wash solution containing benzyl alcohol and sodium benzoate. Next, we evaluated the effect of Protein A column loading level on off-column yield and PLBL2 removal. To identify ideal loading conditions for maximizing both off-column yield and PLBL2 removal, the Protein A column was loaded at levels ranging from 40 to 60 g / L (Figure 6A and 6B). The results show that increasing the column load beyond 40 g / L decreases both off-column yield and PLBL2 removal.

[0152] Taken together, the data provided in this example demonstrate that PLBL2 is among the host cell proteins that co-elute with human monoclonal antibodies after Protein A purification. Furthermore, the data demonstrate that intermediate wash steps containing sodium benzoate and / or benzyl alcohol could provide superior clearance of host cell protein impurities and PLBL2 during Protein A purification of human monoclonal antibodies. Furthermore, the data demonstrate that overloading a Protein A resin column can reduce off-column yields and PLBL2 removal. [Example]

[0153] Evaluating the effect of pH and benzoate concentration in antibody-containing harvests on impurity removal during antibody purification The following example describes experiments conducted to determine whether adjusting the sodium benzoate concentration and pH of a harvest containing a monoclonal antibody would improve or accelerate the removal of impurities during antibody purification.

[0154] Human monoclonal antibody harvests were prepared as described in Example 1. The clarified and depth-filtered harvests were adjusted to various pHs and additive concentrations to screen for their effect on HCP and PLBL2 clearance. Sodium benzoate, previously shown to be effective in PLBL2 removal, was added as a solid in an amount sufficient to reach the target final concentration for a given volume of harvest as part of an additional wash step. After addition, 2 M Tris base was added to reach the target pH. The first harvest was supplemented with sodium benzoate to achieve a final concentration of 0.5 M sodium benzoate and adjusted to pH 7.2. The second harvest was supplemented with sodium benzoate to achieve a final concentration of 0.5 M sodium benzoate and adjusted to pH 9. The third harvest was adjusted to pH 9 (no sodium benzoate added). See Table 3.

[0155] [Table 4]

[0156] Once each harvest was adjusted and 0.22 μm filtered, three Protein A columns were each loaded with the adjusted harvest to a target of 50 g / L resin. The loaded columns were washed with a phosphate buffer containing 50 mM phosphoric acid and 0.5 M sodium chloride. The columns were then washed with reverse osmosis deionized (RODI) water. Finally, the antibody was eluted from the columns using a solution containing 40 mM sodium acetate at pH 3.1. Table 4 below provides an exemplary chromatography process.

[0157] [Table 5] Each of the three Protein A eluates was then tested for the presence of host cell protein (HCP) impurities by ELISA as described in Example 1 and for the presence of PLBL2 by a custom ELISA. As shown in Figure 7, 0.5 M sodium benzoate at pH 9.0 resulted in the lowest levels of PLBL2 and HCP impurities and demonstrated a greater logarithm of PLBL2 clearance compared to pH adjustment alone. Addition of 0.5 M sodium benzoate at pH 7.2 to the harvest improved only HCP clearance. The effect on PLBL2 required both pH and sodium benzoate adjustment to the harvest. HMW was unaffected by the adjustment (not shown). Compared to the unadjusted harvest using only the RODI wash during the Protein A step, HCP was reduced by 65%, while PLBL2 was reduced by approximately 79%.

[0158] The harvest was then supplemented with sodium benzoate to achieve a final concentration of 0.1M, 0.2M, 0.3M, 0.4M, or 0.5M and adjusted to pH 9 before Protein A purification as described above. See Table 5.

[0159] [Table 6]

[0160] Each Protein A eluate was then tested for the presence of PLBL2 via a custom ELISA to assess antibody yield in each eluate. Figure 8 shows that the relationship between PLBL2 content and sodium benzoate concentration was approximately sigmoidal. A decrease in PLBL2 clearance was observed at concentrations above 0.4 M sodium benzoate. Antibody yield decreased slightly with increasing sodium benzoate concentration. From 0.1 M to 0.5 M sodium benzoate, antibody yield decreased from 94.8% to 89.4%. The highest yield and highest PLBL2 clearance were observed when the harvest was adjusted to 0.4 M sodium benzoate and pH 9.0 before Protein A purification. Increasing the sodium benzoate concentration beyond 0.4 M reduced the clearance increase at the expense of a slight decrease in antibody yield. All samples at 0.1 M sodium benzoate had comparable HCP clearance of approximately 250 ppm. At 0.1 M sodium benzoate and below, the HCP was approximately 500 ppm. Sodium benzoate concentrations between 0.2 M and 0.5 M did not affect the charge fluctuation profile, and all eluates were within the specified limits.

[0161] Taken together, these data suggest that the presence of sodium benzoate in solution strongly disfavors the association of host cell impurities with the mAb at high pH. These conditions can be achieved by a separate wash step after the mAb binds to the Protein A resin, or in solution before or during the column loading step. This allows for greater operational flexibility in the Protein A purification process and further supports the unique properties of sodium benzoate as a wash additive.

[0162] Although the foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding, the illustrations and examples should not be construed as limiting the scope of the present disclosure.

Claims

1. 1. A method for purifying a polypeptide comprising an Fc region, the method comprising: (a) contacting a Protein A chromatography matrix with (i) the polypeptide comprising the Fc region, and (ii) a sample containing one or more impurities, under conditions such that the polypeptide comprising the Fc region binds to Protein A; and (b) washing the Protein A chromatography matrix to which the Fc region-containing polypeptide is bound with a wash solution, the wash solution comprises one or both of (i) benzoate at a concentration between 0.1 M and 1.0 M, and (ii) benzyl alcohol at a concentration between 0.5% volume / volume (v / v) and 4% volume / volume (v / v), and the wash solution has a pH between pH 4.0 and pH 10.0; The method comprising:

2. 10. The method of claim 1, wherein the cleaning solution comprises: 1) a benzoate; 2) benzyl alcohol; or 3) a benzoate and benzyl alcohol.

3. (i) the benzoate is at a concentration between 0.1 M and 0.5 M; and / or (ii) the benzoate is an alkali benzoate; and / or (iii) the benzoate is sodium benzoate; and / or (iv) the benzyl alcohol is at a concentration between 1% and 4% (v / v), or at a concentration between 1% and 2% (v / v), or at a concentration between 2% (v / v) and 4% (v / v); The method according to claim 1 or 2.

4. the benzoate is at a concentration between 0.1M and 0.3M, or at a concentration between 0.3M and 0.5M; The method according to any one of claims 1 to 3.

5. The cleaning solution comprises: (i) further comprising a buffer; and / or (ii) has a pH of (a) 5.0 to 10.0, or (b) 5.0 to 9.0, or (c) 5.0, 6.0, 7.0, 9.0, or 10.0; and / or (iii) further comprising: (a) sodium benzenesulfonate; and / or (b) caprylic acid; and / or (c) hexylene glycol; and / or (d) creatine; and / or (e) arginine, and / or (iv) further comprising one or more non-buffering salts; The method according to any one of claims 1 to 4.

6. the wash solution further comprises a buffering agent, the buffering agent being selected from the group consisting of phosphate, Tris, arginine, acetate, and citrate; and / or the buffering agent is at a concentration between 10 mM and 500 mM, or at a concentration between 50 mM and 500 mM; The method of claim 5.

7. the cleaning solution further comprises sodium benzenesulfonate, the sodium benzenesulfonate being at a concentration between 0.1M and 0.5M; 7. The method according to claim 5 or 6.

8. the wash solution further comprises caprylic acid, the caprylic acid being at a concentration of between 10 mM and 50 mM; The method according to any one of claims 5 to 7.

9. the cleaning solution further comprises hexylene glycol, the hexylene glycol being at a concentration between 1% (v / v) and 10% (v / v); The method according to any one of claims 5 to 8.

10. the wash solution further comprises creatine, the creatine being at a concentration of between 10 mM and 100 mM. The method according to any one of claims 5 to 9.

11. the wash solution further comprises arginine, the arginine being at a concentration between 0.1 M and 1.0 M, or the arginine being at a concentration of 0.5 M, and / or the arginine being arginine-HCl, and / or the wash solution comprising arginine having a pH between pH 4.0 and pH 6.0, or a pH between pH 8.0 and pH 10.0; The method according to any one of claims 5 to 10.

12. the wash solution further comprises one or more non-buffering salts, wherein the one or more non-buffering salts are at a concentration between 0.1 M and 1.0 M; and / or the one or more non-buffering 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 combination thereof; or the one or more non-buffering salts are sodium chloride and / or potassium chloride. The method according to any one of claims 5 to 10.

13. The cleaning solution comprises: (i) a solution containing sodium benzoate at a concentration of 0.5 M and sodium bicarbonate at a concentration of 50 mM, the solution having 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, having a pH of 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), having 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, and having a pH 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), and having a pH 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), having a pH of 7.0; (vii) a solution comprising 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, having a 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, and having 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, and having 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, and having a pH of 5.0; A solution selected from the group consisting of The method according to any one of claims 1 to 12.

14. The method of any one of claims 1 to 13, further comprising one or more of the following: (i) washing the Protein A chromatography matrix with a first solution prior to washing the matrix with the wash solution of step (b) of claim 1; (ii) after washing the Protein A chromatography matrix with the wash solution of step (b) of claim 1, washing the matrix with a second solution; and (iii) after one or more washing steps, contacting the Protein A chromatography matrix with an elution solution.

15. the first solution comprises a buffer selected from the group consisting of phosphate buffer, Tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combination thereof; 15. The method of claim 14.

16. the first solution comprises a buffer solution at a concentration between 10 mM and 100 mM; 16. The method of claim 15.

17. 17. The method of claim 15 or 16, wherein the first solution is a phosphate buffer solution.

18. the second solution comprises a buffer selected from the group consisting of phosphate buffer, Tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combination thereof; 15. The method of claim 14.

19. the second solution comprises a buffer solution at a concentration between 10 mM and 100 mM; 19. The method of claim 14 or 18.

20. 20. The method of claim 14, 18 or 19, wherein the second solution has a pH of from 5.0 to 7.

0.

21. 21. The method of any one of claims 14 and 18 to 20, wherein the second solution is substantially low in salt or salt-free.

22. The method of claim 14, further comprising the step of recovering an eluate containing the polypeptide comprising the Fc region.

23. 23. The method of claim 22, further comprising filtering the eluate by depth filtration and / or wherein the eluate contains less than 500 parts per million (ppm) of one or more impurities.

24. (i) the method results in a purified Fc region-containing polypeptide of higher purity compared to a method lacking the step of washing the matrix with the wash solution; and / or (ii) the one or more impurities are host cell proteins (HCPs); The method according to any one of claims 1 to 23.

25. the one or more HCPs are selected from the group consisting of phospholipases, clusterins, serine proteases, elongation factors, and any combination thereof, and / or the HCP is a putative phospholipase B-like 2 (PLBL2), and / or the host cell is a mammalian host cell, and / or the host cell is a Chinese hamster ovary (CHO) cell; 25. The method of claim 24.

26. (i) the Fc region is a human Fc region or a murine Fc region; and / or (ii) the polypeptide comprising the Fc region is an antibody; The method according to any one of claims 1 to 25.

27. The Fc region a human Fc region, including a human IgG1 Fc region, a human IgG2 Fc region, or a human IgG4 Fc region; or a murine Fc region, including a murine IgG1 Fc region, an IgG2 Fc region, or an IgG3 Fc region; 27. The method of claim 26.

28. The polypeptide comprising an Fc region is a human antibody, a humanized antibody, or a chimeric antibody, and / or the polypeptide comprising an Fc region is a monoclonal antibody, or a bispecific or trispecific antibody.

27. The method of claim 26.

29. 29. The method of any one of claims 1 to 28, further comprising, prior to step (a), adjusting the harvest containing the polypeptide comprising the Fc region to a final benzoate concentration of between 0.1 M and 0.5 M and a pH of between 7.0 and 9.0 to produce a sample containing (i) the polypeptide comprising the Fc region, and (ii) one or more impurities. The method described.

30. The method described in claim 1, wherein the cleaning solution contains sodium benzoate at a concentration of 0.3M to 0.7M, benzyl alcohol at a concentration of 1% to 3%, and has a pH of 7.0 to 8.0, and wherein the cleaning solution further contains Tris at a concentration of 10mM to 100mM.

31. The method described in claim 30, wherein the cleaning solution contains sodium benzoate at a concentration of 0.5 M, benzyl alcohol at a concentration of 2%, and Tris at a concentration of 50 mM, and has a pH of 7.5.

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