Methods for regenerating chromatography resins

Abstract

To provide a method for cleaning and regenerating a chromatography resin.SOLUTION: A method comprises contacting a chromatography resin with a first buffer, which comprises acetic acid and benzyl alcohol, and a second buffer, which comprises sodium hydroxide, sodium citrate, and benzyl alcohol. The first buffer does not contain phosphoric acid. The first buffer comprises about 150-200 mM of acetic acid and about 1-3% (v / v) of benzyl alcohol. The second buffer comprises about 100-300 mM of sodium hydroxide, about 100-300 mM of sodium citrate, and about 0.5-1.5% (v / v) of benzyl alcohol.SELECTED DRAWING: Figure 1

Description

Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 835,049, filed April 17, 2019, which is incorporated herein by reference in its entirety. [Technical Field]

[0002] The object of the present invention generally relates to chromatography and cleaning of chromatography resins. [Background technology]

[0003] Chromatography resins are used to purify proteins of interest from other impurities in sample solutions. Chromatography resins are frequently used to purify biopharmaceuticals, such as monoclonal antibodies (MAbs) and other Fc-containing proteins, by affinity chromatography. Affinity chromatography utilizes protein-ligand interactions by attaching a ligand that specifically binds to the protein of interest to the resin, such as Protein A, which is a ligand that can bind to the Fc region of antibodies. Protein A affinity chromatography is a highly effective protein purification method, but the cost of Protein A resin is high and accounts for a significant portion of the raw resin cost in MAb production (Fahrner, RL, et al., Biotechnol. Appl. Biochem. 1999, 30, 121-128; Kelley, B., Biotechnol. Prog. 2007, 23:995-1008). The cost issue of Protein A resin is further exacerbated by the resin's typically short life cycle, with overall performance and binding capacity declining rapidly after only a few purification cycles. This requires large-scale protein purification operations to purchase and use new Protein A quite frequently, further increasing the overall cost of protein purification. The decline in resin performance and binding capacity is often due to resin fouling, which occurs as a result of the buildup of impurities from both the product and processing. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, there is a need for a method to extend the life cycle of a chromatography resin by maintaining its overall performance and binding capacity over time, for example, by regenerating and / or cleaning the chromatography resin while maintaining the integrity of the resin. [Means for solving the problem]

[0005] The present invention provides a method for cleaning and / or regenerating a chromatography resin, which may be cleaned and / or regenerated for use with the same product or for use with a different product. DETAILED DESCRIPTION OF THE INVENTION

[0006] In one embodiment, the present invention provides a method for washing a chromatography resin, comprising contacting the resin with a first buffer containing acetic acid and benzyl alcohol and a second buffer containing sodium hydroxide, sodium citrate, and benzyl alcohol. In one embodiment, the first buffer does not contain phosphate. In one embodiment, the first buffer contains about 150-200 mM acetic acid and about 1-3% (v / v) benzyl alcohol. In one embodiment, the second buffer contains about 100-300 mM sodium hydroxide, about 100-300 mM sodium citrate, and about 0.5-1.5% (v / v) benzyl alcohol. In one embodiment, the first buffer contains about 167 mM acetic acid. In one embodiment, the first buffer contains about 2% (v / v) benzyl alcohol. In one embodiment, the second buffer contains about 200 mM sodium hydroxide. In one embodiment, the second buffer contains about 200 mM sodium citrate. In one embodiment, the second buffer comprises about 1% (v / v) benzyl alcohol. In one embodiment, the first buffer comprises about 167 mM acetic acid and about 2% (v / v) benzyl alcohol, and the second buffer comprises about 200 mM sodium hydroxide, about 200 mM sodium citrate, and about 1% (v / v) benzyl alcohol.

[0007] In one embodiment, the first buffer comprises about 167 mM acetic acid and about 2% (v / v) benzyl alcohol, and the second buffer comprises about 100 mM sodium hydroxide, about 200 mM sodium citrate, and about 1% (v / v) benzyl alcohol.

[0008] In one embodiment, the neutralization buffer is passed over the chromatography resin after the first buffer and before the second buffer.

[0009] In one embodiment, the chromatography resin is kept in the first buffer for about 15 minutes or less. In one embodiment, the chromatography resin is kept in the second buffer for about 15 minutes or less. In one embodiment, the chromatography resin is kept in the second buffer for about 45 minutes or less. In one embodiment, the reuse occurs on the same day as the wash.

[0010] In one embodiment, the chromatography resin is present in a chromatography column.

[0011] In one embodiment, the chromatography resin is an affinity resin. In one embodiment, the chromatography resin comprises an affinity ligand bound to a support. In one embodiment, the affinity ligand is Staphylococcal protein A, or a portion or derivative thereof. In one embodiment, the affinity ligand is Staphylococcal protein G, or a portion or derivative thereof. In one embodiment, the resin or support comprises a polymer selected from the group consisting of polysaccharides, agarose, polyvinyl ether, polyvinyl alcohol, polymethacrylate, polyacrylate, polystyrene, polyacrylamide, polymethacrylamide, and polycarbonate, or a mixture thereof.

[0012] The present invention also provides a method for purifying a polypeptide, comprising applying the polypeptide to a chromatography resin and washing the chromatography resin using the methods described herein.

[0013] In one embodiment, the binding capacity of the washed chromatography resin is retained after washing the chromatography resin using the methods described herein, hi one embodiment, the binding capacity is maintained after at least 50, at least 100, at least 150, or at least 200 washes.

[0014] In one embodiment, the pressure of the chromatography resin does not increase during washing of the chromatography resin using the methods described herein, hi one embodiment, the pressure of the chromatography resin does not increase after 50 or more, 100 or more, 150 or more, or 200 or more washes.

[0015] In one embodiment, the polypeptide is an immunoglobulin comprising a CH2 domain and a CH3 domain. In one embodiment, the polypeptide is an antibody or an antigen-binding fragment thereof. In one embodiment, the antibody is an IgG monoclonal antibody. In one embodiment, the IgG monoclonal antibody is a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the polypeptide is an enzyme, a hormone, a fusion protein, an immunoconjugate, a cytokine, or an interleukin.

[0016] The present invention also provides a method for washing an affinity chromatography column containing a chromatography resin, the method comprising washing the chromatography resin in one or more wash cycles, each wash cycle comprising contacting the chromatography resin with a first solution comprising acetic acid and benzyl alcohol for about 15 minutes or less, and a second solution comprising sodium hydroxide, sodium citrate, and benzyl alcohol for about 15 minutes or less or about 45 minutes or less. In one embodiment, the first solution does not contain phosphoric acid. In one embodiment, the method improves microbial disinfection. In one embodiment, the method reduces host cell protein fouling. In one embodiment, the method reduces yield loss. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the step yield of Protein A with increasing number of purification cycles for wash protocol 1 (-1-), wash protocol 2 (-2-), wash protocol 3 (-3-), and wash protocol 6 (-4-). [Figure 2] FIG. 2 shows the step yield of Protein A with increasing number of purification cycles for wash protocol 9 (-1-), wash protocol 10 (-2-), and wash protocol 11 (-3-). [Figure 3] FIG. 3 shows the step yield of Protein A with increasing number of purification cycles for wash protocol 2 (-1-), wash protocol 4 (-2-), wash protocol 5 (-3-) and wash protocol 7 (-4-). [Figure 4] FIG. 4 shows the delta column pressure with increasing number of purification cycles for cleaning protocol 1 (-1-), cleaning protocol 7 (-2-) and cleaning protocol 8 (-3-). [Figure 5] FIG. 5 shows the residual protein A (ppm) in the elution fractions at increasing numbers of purification cycles for wash protocol 10 (-1-) and wash protocol 11 (-2-). [Figure 6] FIG. 6 shows the residual Protein A (ng / mL) in the washes (Wash 1, Wash 2, and Wash 3), eluate, CIP, and neutralized fractions at cycle 171 for Wash Protocol 9 and Wash Protocol 11. [Figure 7] FIG. 7 shows the microbial killing ability of various cleaning solutions applied at various times. [Figure 8] FIG. 8 shows the step yield of Protein A for wash protocol 12 with a 20 minute CIP2 contact time and a 45 minute CIP2 contact time. [Figure 9] FIG. 9 shows the DBC curves of Protein A over 100 cycles for wash protocol 12 with a 20 minute CIP2 contact time (left) and a 45 minute CIP2 contact time (right). Detailed Description of the Invention

[0018] Provided herein are methods for cleaning or regenerating chromatography resins. The methods of the present invention can be used for small-scale and large-scale (e.g., manufacturing-scale) regeneration of chromatography resins. Currently recognized processes for cleaning chromatography resins include acidic stripping using phosphoric acid. The present invention is based on the surprising and unexpected discovery that chromatography resins can be cleaned without the use of harsh agents such as phosphoric acid. While phosphoric acid can clean fouling from systems containing silica- or methacrylic-based stationary phases (but not agarose-based stationary phases), the use of phosphoric acid can lead to increased pressure due to flow path blockage, potentially resulting in reduced yields.

[0019] (definition) As used herein, "chromatography" refers to a dynamic separation technique that can separate a target molecule of interest (e.g., an immunoglobulin or another Fc-containing protein) from other molecules in a mixture to isolate the target molecule. Generally, in chromatography, a liquid mobile phase moves a sample containing the target molecule of interest through or past a stationary phase (usually solid). Selected molecules temporarily bind to the stationary phase due to differences in partition coefficients or affinities for the stationary phase, while the mobile phase moves various molecules at different times.

[0020] As used herein, "affinity chromatography" refers to a chromatography format in which target molecules (e.g., protein molecules, such as Fc-containing proteins) to be separated interact with molecules (e.g., protein A-based ligands) immobilized on a chromatography resin via a "lock and key" mechanism. This specific interaction results in the binding of the desired molecules and the elution of undesired molecules. The target molecules are then eluted in high purity by altering the temperature, pH, or ionic strength of the mobile phase. In various embodiments described herein, affinity chromatography involves applying a sample containing the target molecules (e.g., immunoglobulins or other Fc-containing proteins) to a solid support (referred to as a protein A affinity chromatography medium or resin) bearing a ligand based on the C domain (or, in some cases, a modified B domain) of protein A. Other ligands used in affinity chromatography include protein G from Streptococcus pyogenes, which binds to the Fc region of immunoglobulins.

[0021] As used herein, "protein A affinity chromatography" refers to the separation or isolation of a substance using protein A, a protein present in the cell wall of the bacterium Staphylococcus aureus that can bind to the Fc region of immunoglobulin. Protein A is immobilized on a solid support and brought into contact with the target protein.

[0022] The term "solid support" generally refers to any resin (porous or non-porous) to which a ligand is attached. The attachment of the ligand to the solid support is either covalent (via ether, thioether, carbon-carbon or other bonds), as in the case of grafting, or by coating, adhesion, adsorption and similar mechanisms. Exemplary solid supports for use in the methods described herein include polysaccharides, agarose, polyvinyl ether, polyvinyl alcohol, polymethacrylate, polyacrylate, polystyrene, polyacrylamide, polymethacrylamide and polycarbonate.

[0023] Examples of Protein A affinity chromatography resins known in the art include those in which Protein A is immobilized on a controlled pore glass scaffold, such as PROSEP TM Media / resin (EMD MILLIPORE); Protein A immobilized on a polystyrene solid support, e.g., POROS TM MabCapture TM A Media / Resin (APPLIED BIOSYSTEMS INC, POROS TM MabCapture TM media / resin (APPLIED BIOSYSTEMS, INC.); and Protein A immobilized on an agarose solid support, e.g., rPROTEIN A SEPHAROSE FAST FLOW TM or MABSELECT TM Media or resin (GE HEALTHCARE).

[0024] The terms "affinity resin" or "affinity chromatography resin" or "affinity media," as used interchangeably herein, refer to an affinity chromatography ligand (e.g., based on Protein A) attached to a solid support, such as those described herein (e.g., resulting in Protein A affinity resin or Protein A resin). Generally, the terms "affinity resin" and "affinity media" are used interchangeably herein. Other examples of affinity chromatography resins include resins with Protein G from Streptococcus, which can bind to the Fc region of immunoglobulins. Also included are affinity resins with Protein L, which binds to immunoglobulins via kappa light chains. These proteins can be used in affinity chromatography to purify immunoglobulins or other proteins that specifically bind to these affinity resins.

[0025] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acids of any length. The polymers may be linear or branched, and may contain modified amino acids or may be interrupted by non-amino acids. The terms also include polymers of amino acids that are modified naturally or by intervening modifications, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulations or modifications, such as conjugation with a labeling component. The definition also includes polypeptides containing, for example, one or more analogs of an amino acid (e.g., unnatural amino acids), as well as other modifications known in the art. As used herein, the terms "polypeptide" and "protein" specifically encompass antibodies and Fc domain-containing polypeptides (e.g., immunoadhesins).

[0026] The terms "target protein" or "protein of interest," used interchangeably herein, refer to any protein that can be purified using an affinity resin, e.g., an Fc-containing molecule of Protein A. In various embodiments, the target protein is an Fc-containing protein, e.g., an immunoglobulin or an Fc-fusion protein.

[0027] The terms "immunoglobulin," "Ig," or "antibody" (used interchangeably herein) refer to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In certain antibodies, e.g., naturally occurring IgG antibodies, the heavy chain constant region is composed of a hinge and three domains: CH1, CH2, and CH3. In certain antibodies, e.g., naturally occurring IgG antibodies, each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one domain (abbreviated herein as CL). The VH and VL regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs) and highly conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified, amino acids in the variable region are numbered using the Kabat numbering system, and amino acids in the constant region are numbered using the EU system. "Antibody" includes, for example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and totally synthetic antibodies.

[0028] Immunoglobulins or antibodies may be monoclonal or polyclonal and may exist, for example, in monomeric or polymeric form (e.g., IgM antibodies that exist as pentamers and / or IgA antibodies that exist as monomers, dimers, or multimers). As used herein, the term "fragment," when used in conjunction with "antibody," refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained by chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. Examples of fragments include Fab, Fab', F(ab')2, Fc, and / or Fv fragments. The antibody or fragment to be purified can be human, humanized, or chimeric.

[0029] It is understood that target proteins purified using the methods described herein contain an Fc region and can therefore be purified with Protein A or Protein G from Steptococci. As used herein, the term "Fc region" or "Fc" refers to the amino acid residues of an immunoglobulin molecule that interact with Protein A. The Fc region is the crystallizable tail region of an antibody, which interacts with cell surface receptors called Fc receptors.

[0030] As used herein, the terms "Fc-binding," "binds to the Fc portion," or "binding to the Fc portion" refer to the ability of an affinity ligand described herein to bind to the constant domain (Fc) of an antibody. In one embodiment, the ligand of the invention has at least 10 -7 M or at least 10 -8 M or at least 10 -9 The Fc portion of an antibody (e.g., human IgG1, IgG2, or IgG4) having an affinity of M.

[0031] As used herein, the term "fragment" may refer to a portion of a full-length Fc-containing protein, such as an immunoglobulin. Examples of fragments include Fab fragments, single-chain antibody molecules, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0032] Reference herein to "about" a value or parameter includes (and describes) variations on that value or parameter itself, and a statement referring to "about X" includes a statement of "X."

[0033] As used in this specification and the appended claims, the singular articles "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. References to aspects and variations of the invention described herein are understood to include "comprising," "containing," and / or "consisting essentially of" aspects and variations.

[0034] (Method of the present invention) Chromatography resin cleaning The present invention provides a method for efficiently regenerating or cleaning affinity chromatography resins. Use of the method of the present invention results in a more efficient cleaning process, thereby extending the life cycle of the resin material. The more efficient cleaning method of the present invention can prolong the performance of affinity resins used in the large-scale production of protein products.

[0035] As used herein, the term "washing" refers to a step in the purification process for target proteins (e.g., immunoglobulins or other Fc-containing proteins) to remove impurities and contaminants remaining on affinity chromatography resins (e.g., Protein A columns) and maintain resin performance. Washing removes impurities from the resin, but ideally should have minimal impact on resin integrity, as measured by binding capacity (the amount of target protein that the resin can retain in purity) and resolution (the ability of the resin to separate the target protein from undesired substances). Often, commercially available affinity chromatography resins, such as those using Staphylococcus Protein A or its derivatives, are washed with a solution containing phosphoric acid or an alkaline solution using NaOH, e.g., MabSelect SuRe. TM Protein A columns are washed with diluted NaOH. TM Protein A columns are typically washed with phosphoric acid.

[0036] "Binding capacity" refers to the amount of molecules that will bind to a specified volume of packed resin or media under specified conditions during a column run (analytical process). Binding capacity can be measured as static or dynamic binding capacity. Static binding capacity is the amount of molecules that bind to a specified volume of resin when exposed to unrestricted contact. Static binding capacity is the maximum amount of target molecule that a resin can bind. In practice, this value is often obtained by exposing an excess amount of target molecule to the resin for at least four hours at minimum or no flow. Dynamic binding capacity, on the other hand, is the amount of target molecule that a resin can bind per volume of resin at a set flow rate. For any given resin, dynamic binding capacity varies significantly depending on the operating conditions. Generally, the slower the flow rate, the higher the dynamic binding capacity. As the flow rate approaches zero, the binding capacity approaches the static binding capacity, which is the maximum available capacity. Without proper cleaning and sterilization, the binding capacity of affinity resins typically decreases from its initial value during repeated binding and elution cycles. If the binding capacity drops below a certain value, the resin may be eluted with the flow-through fraction containing impurities, leading to product loss. Proper cleaning with appropriate chemicals can maintain the resin's binding capacity for extended periods.

[0037] Protein A affinity resins are typically washed after each cycle to ensure stable purification throughout their lifecycle, i.e., to maintain the resin's binding capacity. Washing Protein A affinity chromatography resins is particularly important for two reasons: (1) Protein A resins have a higher initial cost compared to ion exchange or hydrophobic interaction (HIC) resins; and (2) Protein A chromatography resins are often subjected to clarified cell culture media, which contains many impurities. Therefore, some residual impurities bind to the Protein A resin, resulting in a reduced binding capacity or increased impurities in the elution pool upon reuse. This is highly undesirable in manufacturing environments, as it can lead to reduced productivity or reduced product purity (due to reduced binding capacity). Therefore, regular washing after each Protein A binding and elution cycle is crucial to maintain consistent resin performance, thereby maintaining consistent product purity and process throughput.

[0038] As used herein, the terms "cycle," "affinity cycle," or "Protein A affinity chromatography purification cycle" refer to a multistep process that begins with equilibrating a chromatography column using an affinity resin with a neutral buffer, followed by loading the column with clarified cell culture feed (the clarified cell culture medium containing the target protein to be purified). In the case of Protein A affinity resin, the target protein is an Fc-containing protein (e.g., a monoclonal antibody) to be purified. After washing the column with one or more (often three) different buffers to remove loosely bound impurities that do not interfere with the binding of the target protein to a ligand (or, for example, the Fc-containing protein to the Protein A resin), an elution buffer (e.g., pH 2.5-4.5) is used to elute the target protein from the affinity resin, e.g., the Fc-containing protein from the Protein A resin. This multistep process of equilibration, loading, washing, and elution constitutes one cycle, or binding and elution cycle. After each cycle, there is usually a washing step to remove trace levels of impurities from the column before proceeding to the next cycle.

[0039] In some embodiments described herein, the resin is washed with a first wash buffer containing acetic acid and benzyl alcohol and a second wash buffer containing sodium hydroxide, sodium citrate, and benzyl alcohol. As used herein with respect to washing, the term "wash buffer" refers to the buffer passed over the solid support (e.g., containing immobilized Protein A) after elution of the target protein.

[0040] In some embodiments, the chromatography resin is contacted with both the first buffer and the second buffer after each cycle. In other embodiments, the chromatography resin is contacted with either the first buffer or the second buffer after each cycle, and the first buffer and the second buffer are alternated throughout the purification process. In some embodiments, the chromatography resin is contacted with the second buffer and then with the first buffer.

[0041] The present invention provides buffer solutions for use in the methods of the present invention. The first buffer solution of the present invention does not contain phosphate. In one embodiment, the first buffer solution may contain, for example, about 100 to 300 mM acetic acid, about 125 to 250 mM acetic acid, about 150 to 200 mM acetic acid, or about 167 mM acetic acid. The first buffer solution may also contain about 2 to 4% (v / v) benzyl alcohol, about 2.5 to 3.5% (v / v) benzyl alcohol, about 2 to 3% (v / v) benzyl alcohol, about 1.5 to 2.5% (v / v) benzyl alcohol, about 1 to 2% (v / v) benzyl alcohol, or about 2% (v / v) benzyl alcohol. In one embodiment, the first buffer solution contains 167 mM acetic acid and about 2% (v / v) benzyl alcohol.

[0042] In another embodiment, the present invention provides a second buffer solution that can be used to reduce the burden of microbial contamination and appropriately preserve the resin. The second buffer solution of the present invention may contain, for example, about 50 to 400 mM, about 100 to 350 mM, about 150 to 300 mM, about 100 to 300 mM, or about 200 mM sodium hydroxide. The second buffer solution may also contain about 50 to 400 mM, about 100 to 350 mM, about 150 to 300 mM, about 100 to 300 mM, or about 200 mM sodium citrate. The second buffer solution may also contain about 0.5 to 3% (v / v), about 0.5 to 1.5% (v / v), about 1 to 2% (v / v), about 1.5 to 2.5% (v / v), or about 1% (v / v) benzyl alcohol. A non-limiting example of a second buffering agent is about 200 mM sodium hydroxide, about 200 mM sodium citrate, and 1% (v / v) benzyl alcohol.

[0043] In some embodiments, the chromatography resin is present in a chromatography column. In some embodiments, the column is washed with at least 2, 3, 4, or 5 column volumes. In some embodiments, the column is washed with the first buffer and the second buffer until no further impurities, or substantially no impurities, elute from the column.

[0044] In some embodiments of any of the processes described herein, the flow rate is less than about 50, 40, or 30 material volumes per hour. The flow rate may be between 5 and 50, 10 and 40, or between 18 and 36 material volumes per hour. In some embodiments, the flow rate is between about 9, 18, 25, 30, 36, or 40 material volumes per hour.

[0045] In some embodiments, the flow rate is less than about 90 column volumes (CV) / hr, about 80 CV / hr, about 70 CV / hr, about 60 CV / hr, about 50 CV / hr, about 40 CV / hr, or about 30 CV / hr. The flow rate may be between about 5 CV / hr and about 50 CV / hr, between about 10 CV / hr and about 40 CV / hr, or between about 18 CV / hr and about 36 CV / hr. In some embodiments, the flow rate is between about 9 CV / hr, about 18 CV / hr, about 25 CV / hr, about 30 CV / hr, about 36 CV / hr, or about 40 CV / hr. In some embodiments of any of the methods described herein, the flow rate is less than 100 cm / hr, less than 75 cm / hr, or less than 50 cm / hr. The flow rate may be from about 25 cm / hr to about 150 cm / hr, from about 25 cm / hr to about 100 cm / hr, from about 50 cm / hr to about 100 cm / hr, or from about 65 cm / hr to about 85 cm / hr.

[0046] Furthermore, the methods of the present invention reduce the time required for column regeneration: in some embodiments, contact time with wash buffer is typically reduced from 75 minutes to 30 minutes, or even to about 9 minutes, allowing for multiple additional purifications using the same resin in a short period of time.

[0047] contaminants In some embodiments of the methods described herein, the at least one contaminant can be any one or more of host cell material, eluted Protein A, nucleic acid, a variant, fragment, aggregate or derivative of the polypeptide of interest, another polypeptide, endotoxin, viral contaminant, cell culture media component, carboxypeptidase B, gentamicin, etc. In some examples, the contaminant can be, for example, but not limited to, host cell proteins (HCPs) from bacterial cells, such as E. coli cells, insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells, etc.

[0048] Eluted Protein A refers to Protein A that has been desorbed or washed from the solid phase to which it is bound. For example, eluted Protein A can be eluted from a Protein A chromatography material. The amount of Protein A can be measured, for example, using an enzyme-linked immunosorbent assay (ELISA). In some embodiments of any of the methods described herein, the amount of eluted Protein A is reduced by more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. The amount of eluted Protein A can be reduced by any of about 10% to 99%, about 30% to 95%, about 30% to 99%, about 50% to 95%, about 50% to 99%, about 75% to 99%, or about 85% to 99%. In some embodiments, the amount of eluted Protein A is reduced by any of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, this reduction is determined by comparing the amount of eluted Protein A in the composition recovered from the purification step(s) to the amount of eluted Protein A in the composition before the purification step(s).

[0049] Host cell proteins (HCPs) are proteins derived from cells that produced a polypeptide. The amount of HCPs can be measured by ELISA or Meso Scale Discovery ("MSO"). In some embodiments of any of the methods described herein, the amount of HCPs in the eluate is minimal in mock elution. In some embodiments, the levels of host cell proteins in the eluate from mock elution are compared with and without a wash method or before and after a wash method.

[0050] Methods for measuring DNA, e.g., host cell DNA, are known to those of skill in the art. In some embodiments of the methods described herein, the amount of DNA is reduced by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The amount of DNA may be reduced by any of about 10% to 99%, about 30% to 95%, about 30% to 99%, about 50% to 95%, about 50% to 99%, about 75% to 99%, or about 85% to 99%. The amount of DNA may be reduced by any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, this reduction is determined by comparing the amount of DNA in a composition recovered from a purification step(s) to the amount of DNA in the composition prior to the purification step(s).

[0051] The fragment polypeptide can be a low molecular weight (LMW) protein. In some embodiments, the fragmented polypeptide is a fragment of a polypeptide of interest. Examples of LMW proteins include, but are not limited to, a Fab (fragment antigen-binding) region, an Fc (fragment, crystallizable) region, or a combination of both, or any randomly fragmented portion of an antibody of interest. Methods for measuring fragmented proteins (e.g., LMW proteins) are known in the art and are described in the Examples section. In some embodiments of any of the methods described herein, the amount of LMW protein is reduced by more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. The amount of LMW protein can be reduced by any of about 10%-99%, about 30%-95%, about 30%-99%, about 50%-95%, about 50%-99%, about 75%-99%, or about 85%-99%. The amount of LMW protein can be reduced by any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, this reduction is determined by comparing the amount of fragmented protein (e.g., LMW protein) in a composition recovered from a purification step(s) to the amount of fragmented protein (e.g., LMW protein) in the composition prior to the purification step(s).

[0052] The aggregated polypeptide may be a high molecular weight (HMW) protein. In some embodiments, the aggregated polypeptide is a multimer of the polypeptide of interest. The HMW protein may be a dimer, up to eight times more monomers of the polypeptide of interest, or more monomers. Methods for measuring aggregated proteins (e.g., HMW proteins) are known in the art. In some embodiments, the level of HMW in the mock eluate is minimal, e.g., less than about 5 ppm, less than about 4 ppm, less than about 3 ppm, less than about 2 ppm, or less than about 1 ppm. In some embodiments of any of the methods described herein, the amount of aggregated protein is reduced by more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. The amount of aggregated protein may be reduced by about 10% to 99%, about 30% to 95%, about 30% to 99%, about 50% to 95%, about 50% to 99%, about 75% to 99%, or about 85% to 99%. The amount of aggregated protein may be reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, this reduction is determined by comparing the amount of aggregated protein (e.g., HMW protein) in a composition recovered from a purification step(s) to the amount of aggregated protein (e.g., HMW protein) in the composition prior to the purification step(s).

[0053] Cell culture medium components refer to components present in cell culture medium. The cell culture medium may be the cell culture medium at the time of cell harvesting. In some embodiments, the cell culture medium component is gentamicin. The amount of gentamicin may be measured by ELISA. In some embodiments of any of the methods described herein, the amount of the cell culture medium component is reduced by more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. The amount of the cell culture medium component may be reduced by any of about 10% to 99%, about 30% to 95%, about 30% to 99%, about 50% to 95%, about 50% to 99%, about 75% to 99%, or about 85% to 99%. In some embodiments, the amount of the cell culture media component is reduced by any of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98%. In some embodiments, this reduction is determined by comparing the amount of the cell culture media component in the composition recovered from the purification step(s) to the amount of the cell culture media component in the composition before the purification step(s).

[0054] Polypeptides The methods of the invention may be used to wash chromatography materials used in the purification of multiple polypeptide preparations. In some embodiments, the chromatography material is used in large-scale production of polypeptides, such as antibodies or fragments thereof; e.g., manufacturing-scale production. In some embodiments, the chromatography material is used to purify a first polypeptide (e.g., a first antibody), the material is then washed by the methods of the invention, and the chromatography material can be used to purify a second polypeptide (e.g., a second antibody). In some embodiments, this washing is effective to render the preparation containing the second purified polypeptide substantially free of the first polypeptide. In some embodiments, the preparation containing the second purified polypeptide (e.g., a second antibody) contains less than 1 ppm of the first polypeptide (e.g., the first antibody). In some embodiments, the second purified polypeptide contains less than 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, or 100 ppm of the first polypeptide.

[0055] In some embodiments, the methods of the present invention are used to recycle chromatographic materials used to purify a therapeutic polypeptide. In some embodiments, the polypeptide is an antagonist. In some embodiments, the polypeptide is an agonist. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide is an epitope tag. In some embodiments, the polypeptide retains biological and / or immunological activity. In some embodiments, the polypeptide is an antagonist. In some embodiments, the polypeptide initiates complement-dependent cytotoxicity. In some embodiments, the polypeptide is an antibody or immunoadhesin.

[0056] Polypeptides purified using washed regenerated chromatography material according to the methods described herein are generally produced using recombinant techniques. Methods for producing recombinant proteins are described, for example, in U.S. Patent Nos. 5,534,615 and 4,816,567, specifically incorporated herein by reference.

[0057] Polypeptides can be recombinantly produced in eukaryotic or prokaryotic cells. Proteins can be obtained from genetically engineered plants, transgenic animals, or secreted by production cells adapted to grow in cell culture. Production cells can be bacterial (e.g., Escherichia coli, Streptomyces, and Bacillus), fungal (e.g., Aspergillus), invertebrate (e.g., insect), or mammalian. Examples of mammalian cells commonly used in the art include CHO, VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (especially murine), PC12, and W138 cells. A particularly preferred host cell is Chinese hamster ovary (CHO) cell, which has been widely used to produce several complex recombinant proteins (e.g., cytokines, coagulation factors, and antibodies) (Brasel et al., 1996, Blood 88:2004-2012; Kaufman et al., 1988, J. Biol Chem 263: 6352-6362; McKinnon et al., 1991, J. Mol Endocrinol 6: 231-239; Wood et al., 1990, J. Immunol 145: 3011-3016). DXB11 and DG-44, dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), are preferred CHO host cell lines. This is because recombinant proteins can be expressed at high levels in these cells due to efficient DHFR-selectable and amplifiable gene expression systems (Kaufman RJ, 1990, Meth Enzymol 185:527-566). Furthermore, these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells and recombinant proteins expressed in CHO cells have been extensively characterized and approved by regulatory authorities for use in clinical manufacturing.

[0058] Polypeptides purified using the washed, regenerated chromatography material described herein can be recovered from culture medium or host cell lysate. Cells used to express the polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents. If the polypeptide is produced intracellularly, the first step is to remove particulate debris, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for isolating polypeptides secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. When the polypeptide is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available polypeptide concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Either of these steps may include a protease inhibitor, such as PMSF, to inhibit proteolysis, and / or an antibiotic to prevent the growth of adventitious contaminants.

[0059] Examples of polypeptides that can be purified using reusable chromatographic materials washed according to the methods described herein include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, fusion proteins, Fe-containing proteins, and immunoconjugates.

[0060] The resulting expressed polypeptide can then be purified or partially purified from such cultures or components (e.g., culture medium, cell extracts, or body fluids) using a combination of the methods of the present invention and known methods. "Partially purified" means that, despite some fractionation method or procedure, more polypeptide species (at least 10%) than the protein of interest are present. "Purified" means that the protein is substantially homogeneous, i.e., less than 1% of contaminating proteins are present. Fractionation methods include, but are not limited to, one or more steps of filtration, centrifugation, precipitation, phase separation, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction chromatography (HIC: using resins such as phenyl ether, butyl ether, propyl ether, etc.), or HPLC, or a combination thereof. Any of the above chromatography resins can be cleaned and regenerated using the methods of the present invention.

[0061] The final target level of purity varies depending on the intended use of the polypeptide. For example, when the polypeptide is administered in vivo, a relatively high degree of purity is desired. In such cases, the polypeptide is purified so that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Those skilled in the relevant art will understand that if multiple bands corresponding to the polypeptide are observed upon SDS-PAGE, these may be due to different glycosylation, different post-translational processes, etc. Most preferably, the polypeptide of the present invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. This polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radioactively labeled) autoradiography.

[0062] The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all documents cited in this application are hereby incorporated by reference. [Example]

[0063] Resin cleaning is a key factor in maintaining a long and appropriate life cycle for Protein A columns used in mAb purification, and because Protein A resin is one of the most expensive consumables used in downstream processes, efficient resin cleaning is also important for maintaining sustainable material costs.

[0064] The effects of fouling affect the column life cycle by reducing binding capacity due to spatial limitations and overall interparticle porosity, resulting in further poor mAb adsorption with increasing fouling concentration. Increasing the concentration of sodium hydroxide solution has been shown to remove deposited fouling material, but at the expense of capacity due to increased ligand hydrolysis. Additives in the sodium hydroxide solution, such as ethylene glycol, propylene glycol, sodium sulfate, and sodium citrate, have been shown to have a small but significant effect in slowing the rate of ligand hydrolysis.

[0065] New formulations of wash solutions are needed to improve overall productivity and effectiveness using next-generation sodium hydroxide-stable Protein A resins.

[0066] material and method [Table 1]

[0067] Resin and Loading Materials The Protein A column used in the experiments was a 0.8 cm internal diameter x 5.0 cm bed height column packed with Mabselect SuRe LX resin (GE Healthcare, Uppsala, Sweden) on MiniChrom hardware (Repligen, Waltham, MA). Cleaning experiments were performed on an AEKTA Avant 150 System (GE Healthcare) using a clarified cell culture harvest with a titer of 4.1–4.6 g / L as the load material. Separate experiments were performed on an AEKTA Avant 150 System (GE Healthcare) using the same resin packed in an Omnifit glass column (Kinesis) with a 1 cm internal diameter x 10 cm bed height and clarified cell culture with a titer of 4.1–4.6 g / L as the load material.

[0068] analysis Product concentrations for capture runs were determined by UV absorbance at 280 nm using a DropSense96 UV-Vis Spectrophotometer (Trinean, Gentbrugge, Belgium). High-throughput ELISAs to quantify residual CHO-HCP and rProA were performed using a Tecan Liquid Handling System (Morrisville, NC). HCP levels were quantified using a CHO HCP 3rd Generation Kit (Cygnus Technologies, Southport, NC), and rProA was quantified using a Repligen Protein-A ELISA kit (Repligen Corporation, Waltham, MA), according to the manufacturer's protocol. Residual CHO DNA (rDNA) in the samples was measured using real-time quantitative PCR (RT-qPCR). Size-exclusion chromatography (SEC) was performed using a Waters Acquity H-Class Bio UPLC to measure the content of monomers and aggregates in the product pool. Quantification of monomers, low molecular weight species, and high molecular weight species was performed using Empower Software (Waters Corp.).

[0069] Chromatography The MabSelect SuRe LX Protein A column was run in bind-and-elute mode. For each wash protocol, multiple cycles of Protein A were performed until termination criteria were met: yield (<80%) and delta column pressure (≥3.00 MPa). A summary of the steps, residence times, and buffer column volumes (CV) required for the MabSelect SuRe LX Protein A chromatography run is shown in Table 2. [Table 2]

[0070] The Protein A column was equilibrated with 3 CV of 20 mM sodium phosphate, 150 mM sodium chloride buffer (pH 7.2). Then, 1 M sodium chloride was added to the column, and equilibration continued with 2 CV of 20 mM sodium phosphate, 150 mM sodium chloride buffer (pH 7.2). After equilibration, the clarified bulk was loaded onto the column at 2°C-8°C using a loading target of 45 g / L resin. The column was then washed with 2 CV of 20 mM sodium phosphate, 150 mM sodium chloride buffer (pH 7.2), followed by 5 CV of 50 mM carbonate, 100 mM sodium chloride, and 0.5% polysorbate-80 buffer (pH 10.0), and then 5 CV of 20 mM citrate phosphate buffer (pH 5.1). The column was eluted with 20 mM citrate phosphate buffer (pH 3.4). A total of 5 CV of elution was collected, with each cycle's eluate collected in a separate container. After elution, the Protein A column was washed in place with 3 CV of CIP1 buffer, neutralized with 2 CV of 20 mM sodium phosphate, 150 mM sodium chloride buffer (pH 7.2), and sanitized with 3 CV of CIP2 buffer. If CIP1 was not present in a given wash protocol, CIP1 and Neutralization 1 were omitted from the chromatographic method. Similarly, if CIP2 was not present in a given wash protocol, both CIP2 and Neutralization 2 were omitted from the chromatographic method. The Protein A column was re-equilibrated a second time with 3 CV of 20 mM sodium phosphate, 150 mM sodium chloride buffer (pH 7.2) for recycling, followed by subsequent steps leading to loading, elution, cleaning in place, and sanitization. After the final CIP step, an equilibration step was performed with 3 CV of 20 mM sodium phosphate, 150 mM sodium chloride buffer (pH 7.2), followed by preserving the column with 3 CV of 2% (v / v) benzyl alcohol.

[0071] Formula for calculating Protein A step yield:

number

[0072] Delta column pressure or differential column pressure is the pressure drop across the packed bed of a column, i.e., the pressure at the column inlet [MPa] minus the pressure at the column outlet [MPa].

[0073] [Table 3] An additional replicate was performed using a 1 cm ID x 10 cm bed height MabSelect SuRe LX column using cleaning protocol 12 in Table 3. In this cleaning method, the residence time for each CIP step (CIP1 and CIP2) was 5 minutes instead of 3 minutes, resulting in a 15 minute contact time for each CIP solution under dynamic flow conditions. Two parallel replicates were performed with either a 5 minute or 30 minute rest period after the CIP2 step, resulting in a cumulative contact time of 20 minutes and 45 minutes for the CIP2 solution, respectively.

[0074] Results and Discussion This study revealed that inadequate removal of fouling materials, as well as inappropriate solution components such as phosphoric acid, impact yield and delta column pressure, exacerbating fouling and affecting delta column pressure. The mechanism of this interaction is not fully understood. Figure 1 shows the process yields for cleaning protocols 1, 2, 3, and 6 during the indicated purification cycles. Cleaning protocol 1, the control case, shows a gradual decline in yield, followed by a sharp drop in yield around cycle 65. In this case, the use of phosphoric acid in the CIP1 step and low concentrations of sodium hydroxide in the CIP2 step likely led to insufficient removal of fouling materials and column degradation. This case terminated due to column overpressurization, likely due to the low pH environment provided by the CIP1 conditions. Omission of the CIP2 step (cleaning protocol 2), which completely omits the CIP2 clean and does not perform any column cleans other than the phosphoric acid / acetic acid conditions of CIP1, also leads to poorer process performance, with a similar sharp drop in yield around cycle 60. Cleaning Protocol 3, which lacks benzyl alcohol in the CIP solution used in Cleaning Protocol 1, demonstrates more sustained process performance until a significant drop in yield occurs around cycle 120. This suggests that benzyl alcohol may also have a negative impact on column cleaning efficiency and overall yield. Organic solutions are known to precipitate peptides, proteins, and nucleic acids, which may affect columns where protein-containing fouling materials remain due to insufficient removal. Cleaning Protocol 6 shows a gradual decline in yield, but no sudden drop is observed even after 170 cycles. Although benzyl alcohol remains in the CIP solution used here, removing phosphoric acid and increasing the sodium hydroxide content from 20 mM to 200 mM significantly improved process yield. High-concentration hydroxide cleaning of fouling materials is important for maintaining process performance.

[0075] An alternative cleaning protocol (cleaning protocol 12 in Table 3) using 167 mM acetic acid and 2% (v / v) benzyl alcohol for the CIP1 step and 100 mM sodium hydroxide, 200 mM sodium citrate, and 1% (v / v) benzyl alcohol for the CIP2 step was also evaluated using a 1 cm i.d. x 10 cm bed height MabSelect SuRe LX column. This formulation used a moderate concentration of sodium hydroxide (100 mM) to adequately clean the stationary phase while minimizing the effects of ligand degradation. Cyclic tests were performed with cumulative contact times of 20 and 45 minutes with the CIP2 solution to evaluate the extent of resin cleaning and ligand degradation with this cleaning strategy.

[0076] Figure 8 shows the yield trends for cleaning protocol 12 performed over 91 cycles of Protein A. The overall trend is a nearly linear decrease for both 20- and 45-minute contact times. Note that these cases involve a longer overall contact time with the CIP2 solution than cleaning protocols 1 through 11, which only have a 9-minute overall contact time with each CIP solution (i.e., 3-minute residence time for three column volumes = 9 minutes). This linear decrease is likely related to the degradation of ligands on the stationary phase due to exposure to the harsh cleaning solution. However, the use of 100 mM sodium hydroxide provides sufficient cleaning to remove fouling materials, including proteins.

[0077] Figure 9 shows dynamic binding capacity (DBC) curves performed separately on the column used to test Cleaning Protocol 12. These curves were generated using pre-purified mAb from a process intermediate, with the column loaded at over 100 g / L resin. The general left shift in these flow-through curves indicates a decrease in binding capacity, which may be associated with ligand degradation and therefore a reduced affinity for the mAb product. The distinctive shape of the curves for the 20 min contact time indicates more fouling on this column, but the observed impact on yield is negligible compared to the overall process yield for both the 20 min and 45 min contact times.

[0078] While cleaning protocol 6 demonstrated that a 9-minute contact time was sufficient, longer contact times are typically required in manufacturing environments to fully exploit the bactericidal and antifungal effects of cleaning reagents. The longer contact times considered in the case of cleaning protocol 12 are more realistic in this respect, and this is why 100 mM sodium hydroxide was chosen as an intermediate value to mitigate the effect of the sodium hydroxide solution on decomposing Protein A ligands.

[0079] The microbial kill potential (LRV) of different solutions applied for various periods of time (contact time) was measured (Figure 7). 167 mM acetic acid, 2% (v / v) benzyl alcohol (CIP1 buffer) and 200 mM sodium hydroxide, 200 mM sodium citrate, 1% (v / v) benzyl alcohol (CIP2 buffer) were found to be the most effective of the solutions tested at the preferred contact time of 15 minutes.

[0080] (Conclusion) Of all the cleaning protocols evaluated, Cleaning Protocol 6 was determined to be the most effective. Cleaning Protocol 6 maintained a high yield (92.9%) for 170 cycles (Figure 1). This indicates adequate cleaning without significant pressure buildup, significant fouling, or Protein A elution. In contrast, Cleaning Protocol 1 only achieved 69 cycles before significant problems occurred. At this point, the yield dropped to 84.1% (Figure 1), and the delta column pressure rose to 0.3 MPa, indicating insufficient cleaning (Figure 4). All other cleaning protocols using phosphoric acid as one of the cleaning conditions showed significant decreases in yield or increases in delta column pressure (Figures 1 and 4), except for the case where a high concentration of sodium hydroxide was present in CIP2 (Clean Protocol 8; Figure 4). Additionally, runs without CIP2 (sodium hydroxide solution) tended to foul earlier than runs with CIP2 (Figure 3). None of these runs reached 100 cycles, whereas six of the seven runs using CIP2 conditions reached 100 cycles (Figures 1, 2, and 4). For wash protocols 10 and 11, the elution pool was tested for ligand hydrolysis every 10 cycles (Figure 5). The final cycles of wash protocols 9 and 11 included all of the recovery and regeneration steps, which were then tested for ligand hydrolysis (Figure 6). Residual protein A levels in the elution pool were within the normal range in both runs (Figure 5). However, the CIP and neutralization steps in the final cycles of both runs had significantly higher residual protein A levels (Figure 6). The high residual protein A levels and significant yield loss suggested that ligand hydrolysis was due to the high sodium hydroxide concentration in CIP2 conditions.

Claims

1. contacting the chromatographic resin with a first buffer solution comprising acetic acid and benzyl alcohol and a second buffer solution comprising sodium hydroxide, sodium citrate and benzyl alcohol; 1. A method for cleaning a chromatography resin, comprising:

2. 10. The method of claim 1, wherein the first buffer is phosphate-free.

3. 3. The method of claim 1, wherein the first buffer comprises about 150 to 200 mM acetic acid and about 1 to 3% (v / v) benzyl alcohol.

4. 4. The method of claim 1, wherein the second buffer solution comprises about 100 to 300 mM sodium hydroxide, about 100 to 300 mM sodium citrate, and about 0.5 to 1.5% (v / v) benzyl alcohol.

5. 5. The method of claim 1, wherein the first buffer comprises about 167 mM acetic acid.

6. The method of any one of claims 1 to 5, wherein the first buffer solution comprises about 2% (v / v) benzyl alcohol.

7. The method of any one of claims 1 to 6, wherein the second buffer comprises about 200 mM sodium hydroxide.

8. The method of any one of claims 1 to 7, wherein the second buffer comprises about 200 mM sodium citrate.

9. The method of any one of claims 1 to 8, wherein the second buffer solution comprises about 1% (v / v) benzyl alcohol.

10. 10. The method of claim 1, wherein the first buffer comprises about 167 mM acetic acid and about 2% (v / v) benzyl alcohol, and the second buffer comprises about 200 mM sodium hydroxide, about 200 mM sodium citrate, and about 1% (v / v) benzyl alcohol.

11. 10. The method of claim 1, wherein the first buffer comprises about 167 mM acetic acid and about 2% (v / v) benzyl alcohol, and the second buffer comprises about 100 mM sodium hydroxide, about 200 mM sodium citrate, and about 1% (v / v) benzyl alcohol.

12. 12. The method according to any one of claims 1 to 11, characterized in that a neutralizing buffer is passed over the chromatography resin after the first buffer and before the second buffer.

13. 13. The method of any one of claims 1 to 12, wherein the chromatography resin is retained in the first buffer solution for about 15 minutes or less.

14. The method of any one of claims 1 to 13, wherein the chromatography resin is retained in the second buffer solution for no more than about 15 minutes.

15. The method of any one of claims 1 to 13, wherein the chromatography resin is retained in the second buffer solution for no more than about 45 minutes.

16. The method of any one of claims 1 to 15, which can be reused on the day of cleaning.

17. The method of any one of claims 1 to 16, wherein the chromatography resin is present in a chromatography column.

18. The method according to any one of claims 1 to 17, wherein the chromatography resin is an affinity resin.

19. 20. The method of claim 18, wherein the chromatography resin comprises an affinity ligand bound to a support.

20. 20. The method of claim 19, wherein the affinity ligand is Staphylococcal protein A, or a part or derivative thereof.

21. 20. The method of claim 19, wherein the affinity ligand is Staphylococcal protein G, or a part or derivative thereof.

22. 22. The method of any one of claims 17 to 21, wherein the resin or support comprises a polymer selected from the group consisting of polyvinyl ether, polyvinyl alcohol, polymethacrylate, polyacrylate, polystyrene, polyacrylamide, polymethacrylamide, and polycarbonate.

23. A method for purifying a polypeptide, comprising the steps of applying the polypeptide to a chromatography resin and washing the chromatography resin using the method of any one of claims 1 to 22.

24. 24. The method of claim 23, wherein the binding capacity of the chromatography resin is retained after washing the chromatography resin at least 50 times, at least 100 times, at least 150 times, or at least 200 times using the method of any one of claims 1 to 20.

25. 24. The method of claim 23, wherein the pressure of the chromatography resin does not increase while washing the chromatography resin 50 or more, 100 or more, 150 or more, or 200 or more times using the method of any one of claims 1 to 20.

26. 24. The method of claim 23, wherein the polypeptide is an immunoglobulin comprising a CH2 domain and a CH3 domain.

27. 27. The method of claim 26, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

28. 28. The method of claim 27, wherein the antibody is an IgG monoclonal antibody.

29. 29. The method of claim 28, wherein the IgG monoclonal antibody is a chimeric antibody, a humanized antibody, or a human antibody.

30. 24. The method of claim 23, wherein the polypeptide is an enzyme, hormone, fusion protein, immunoconjugate, cytokine, or interleukin.

31. 1. A method for washing an affinity chromatography column containing a chromatography resin, comprising washing the chromatography resin with one or more wash cycles, wherein each wash cycle comprises contacting the chromatography resin with a first solution comprising acetic acid and benzyl alcohol for about 15 minutes or less, and contacting the chromatography resin with a second solution comprising sodium hydroxide, sodium citrate, and benzyl alcohol for about 15 minutes or less.

32. 1. A method for washing an affinity chromatography column containing a chromatography resin, comprising washing the chromatography resin with one or more wash cycles, each wash cycle comprising contacting the chromatography resin with a first solution comprising acetic acid and benzyl alcohol for about 15 minutes or less, and contacting the chromatography resin with a second solution comprising sodium hydroxide, sodium citrate, and benzyl alcohol for about 45 minutes or less.

33. 33. The method of claim 32, wherein the first solution does not contain phosphoric acid.

34. The method according to any one of claims 1 to 33, wherein the microbial killing activity is increased.

35. 34. The method of any one of claims 1 to 33, wherein host cell protein fouling is reduced.

36. 34. The method of any one of claims 1 to 33, which reduces yield loss.

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