Compositions and methods for reducing bioburden in chromatography

Abstract

To provide a composition and a method for reducing bioburden in chromatography.SOLUTION: The present invention provides methods for microbial bioburden reduction of various chromatography matrices, including bioburden reduction in the context of large-scale protein A-based affinity chromatography columns. There is a need for new effective methods that can be used in the context of large-scale GMP and CGMP to reduce microbial bioburden of various chromatography matrices, especially chromatography matrices with proteinaceous ligands such as protein A. The present invention addresses this and other needs by providing compositions and methods for microbial bioburden reduction of chromatography matrices.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 452,140, ​​filed January 30, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention provides methods for the reduction of microbial bioburden of various chromatography matrices, including the reduction of bioburden in the context of large-scale Protein A-based affinity chromatography columns. [Background technology]

[0003] Background of the Invention Antibody drugs are the most widespread biopharmaceutical products. For example, affinity chromatography using natural or engineered Staphylococcus protein A ligands is widely used as a capture method to remove impurities and contaminants in the manufacturing process of antibody drugs. Protein A binds to the Fc region of antibodies, and protein A columns are considered selective for the purification of monoclonal antibodies. Affinity chromatography using protein A typically involves a clean-in-place (CIP) step to wash and remove column-bound impurities, such as precipitated or denatured substances. CIP is usually performed using sodium hydroxide solution.

[0004] In addition to washing, sodium hydroxide solution or phosphate solution containing benzyl alcohol is used to reduce the number of microorganisms in the Protein A chromatography matrix or column. Bacteria from the medium used to culture monoclonal antibody-producing cells and associated host cell proteins and DNA can rapidly increase the bioburden of a Protein A column during use. This increase in bioburden, as bacteria and microorganisms accumulate on the column, generally leads to a deterioration in column performance as the bioburden increases. Signs of such deterioration include a decrease in product purity, deterioration in column packing, and increased backpressure.

[0005] Control and reduction of microbial bioburden on Protein A columns is important because Protein A columns are very expensive and packing and unpacking such affinity columns is labor intensive. To avoid the expense of replacing Protein A columns or adding processing steps upstream of Protein A column purification, there is a need to discover agents that rapidly remove large amounts of bioburden from the column without negatively affecting Protein A structure and function, and that have little downstream effect.

[0006] The importance of reducing microbial bioburden is not limited to Protein A chromatography matrices, but also includes other chromatography matrices that use proteinaceous ligands linked to a support, as well as matrices without proteinaceous ligands, such as various ion exchange chromatography matrices, hydrophobic interaction chromatography (HIC) matrices, mixed-mode chromatography matrices, size exclusion chromatography matrices, and the like.

[0007] Reducing microbial bioburden in chromatography matrices is particularly important in the context of Good Manufacturing Practices (GMP) or Current Good Manufacturing Practices (CGMP). Such standards must provide consistency in manufacturing steps and product quality to meet the requirements of regulatory agencies such as the U.S. Food and Drug Administration. GMP and CGMP require a high degree of predictability and standardization in manufacturing processes, particularly to ensure the purity of therapeutic biomolecules produced for use in human patients. The labor involved in growing cultures to produce biomolecules results in significant costs when failures occur. Excessive bioburden can reduce column performance, which can interfere with product purification in a standardized and predictable manner and potentially introduce other points of failure.

[0008] Agents currently known in the art for reducing bioburden have negative downstream effects: for example, sodium hydroxide-based solutions and phosphoric acid-based solutions containing benzyl alcohol can be effective at killing microorganisms, but also tend to denature proteinaceous ligands (e.g., protein A), negatively affecting their function.

[0009] Additionally, oxidizing agents and other components of such solutions may remain with the purified monoclonal antibodies and further degrade them downstream.

[0010] Thus, those skilled in the art understand that it is difficult to reduce bioburden in chromatography matrices, particularly matrices with proteinaceous ligands, such as Protein A chromatography matrices. Summary of the Invention [Means for solving the problem]

[0011] Summary of the Invention As noted above, there is a need for novel, effective methods that can be used in large-scale GMP and CGMP settings to reduce the microbial bioburden of various chromatography matrices, particularly chromatography matrices with proteinaceous ligands such as Protein A. The present invention addresses this and other needs by providing compositions and methods for the reduction of microbial bioburden of chromatography matrices.

[0012] In one aspect, the invention provides a method for reducing microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.1 M to about 0.5 M acetic acid, wherein the contacting is performed for at least about 2 hours.

[0013] In a related aspect, the invention provides a method for reducing microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.5 M to about 1.0 M acetic acid, wherein the contacting is performed for at least about 1 hour.

[0014] In a related aspect, the invention provides a method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.1 M to about 1.0 M acetic acid, wherein the contacting step reduces the amount of spore-forming bacteria in the chromatography matrix by at least 3 logs. 10 reduction of the amount of gram-positive bacteria by at least 5 logs 10 and at least a 5 log reduction in the amount of Gram-negative bacteria. 10 The present invention provides a method for reducing one or more of the following:

[0015] In another aspect, the present invention provides a method for reducing microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 4.0 M to about 12.0 M urea, wherein the contacting is performed for at least about 30 minutes.

[0016] In a related aspect, the invention provides a method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 4.0 M to about 12.0 M urea, wherein the contacting reduces the amount of spore-forming bacteria in the chromatography matrix by at least 2 logs. 10 reduction of the amount of gram-positive bacteria by at least 5 logs 10 and at least a 5 log reduction in the amount of Gram-negative bacteria. 10 The present invention provides a method for reducing one or more of the following:

[0017] In one embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 0.5 M acetic acid, wherein the contacting is performed for at least about 4 hours.

[0018] In another embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 0.1 M acetic acid and about 20% ethanol, wherein the contacting is performed for at least about 4 hours.

[0019] In a further embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 8 M urea, wherein the contacting is performed for at least about 1 hour.

[0020] In yet another embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 8 M urea and about 20% ethanol, wherein the contacting is performed for at least about 1 hour.

[0021] In one embodiment, the present invention provides a method for reducing the microbial load of a composition comprising a pharmaceutical substance prior to application for purification, the method comprising the steps of: (a) providing a chromatography matrix; (b) performing any of the above methods of the present invention; and (c) applying a composition comprising the pharmaceutical substance to the chromatography matrix.

[0022] These and other aspects of the present invention will become apparent to those skilled in the art in the following description, claims and drawings. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 is a graph showing the results of a spike test with a solution containing 0.5 M acetic acid. The extent of bacterial kill is measured in solution without a chromatographic matrix. The solid bars represent the amount of Bacillus psuedofirmus on the MabSelect™ Xtra column before exposure to 0.5 M acetic acid (T=0) and after 1 hour of exposure to acetic acid (T=1 hour), and the shaded bars represent the amount of Microbacterium species.

[0024] [Figure 2] Figure 2 is a graph showing the results of killing Bacillus pseudofirmus in solution without a chromatographic matrix by spiking the solution with Bacillus pseudofirmus and measuring bacterial titer. The following agents were added to separate solutions: (a) water for injection (WFI), (b) 8 M urea, (c) 8 M urea and 20% ethanol, (d) 6 M guanidine hydrochloride, and (e) guanidine hydrochloride and 20% ethanol. For each, a spike confirmation measurement in PBS was performed, as well as measurements at 0, 30, and 60 minutes. Black bars represent WFI, horizontal bars represent 8 M urea, white bars represent 8 M urea and 20% ethanol, diagonal bars represent 6 M guanidine hydrochloride, and shaded bars represent 6 M guanidine hydrochloride and 20% ethanol.

[0025] [Figure 3] Figure 3 is a graph showing the results of killing Microbacterium species in solution without a chromatography matrix by spiking the solution with Bacillus pseudofirmus and measuring bacterial titer. The following agents were added: (a) water for injection (WFI), (b) 8 M urea, (c) 8 M urea and 20% ethanol, (d) 6 M guanidine hydrochloride, and (e) guanidine hydrochloride and 20% ethanol. Spike confirmation measurements in PBS were taken, as well as measurements at 0, 30, and 60 minutes. Black bars represent WFI; horizontal bars represent 8 M urea; white bars represent 8 M urea and 20% ethanol; diagonal bars represent 6 M guanidine hydrochloride; and shaded bars represent 6 M guanidine hydrochloride and 20% ethanol.

[0026] [Figure 4]Figure 4 is a graph showing the results of killing Stenotrophomonas maltophilia in solution without a chromatography matrix by spiking the solution with Bacillus pseudofirmus and measuring bacterial titer. The following agents were added: (a) water for injection (WFI), (b) 8 M urea, (c) 8 M urea and 20% ethanol, (d) 6 M guanidine hydrochloride, and (e) guanidine hydrochloride and 20% ethanol. Spike confirmation measurements in PBS were taken, as well as measurements at 0, 30, and 60 minutes. Black bars represent WFI; horizontal bars represent 8 M urea; white bars represent 8 M urea and 20% ethanol; diagonal bars represent 6 M guanidine hydrochloride; and shaded bars represent 6 M guanidine hydrochloride and 20% ethanol.

[0027] [Figure 5] Figure 5 shows the results of various product quality tests performed on Protein A-containing resins (MabSelect™ Xtra and MabSelect™ SuRe) exposed to 0.5 M acetic acid for different periods of time. The filled circles represent values ​​for MabSelect™ Xtra exposed to 0.5 M acetic acid for 375 hours or not at all. The crosses represent values ​​for MabSelect™ SuRe not exposed to 0.5 M acetic acid for 5, 10, 25, 200, or 400 hours, or not at all. [Figure 6] Figure 6 shows the results of various product quality tests performed on Protein A-containing resins (MabSelect™ Xtra and MabSelect™ SuRe) exposed to 0.5 M acetic acid for different periods of time. The filled circles represent values ​​for MabSelect™ Xtra exposed to 0.5 M acetic acid for 375 hours or not at all. The crosses represent values ​​for MabSelect™ SuRe not exposed to 0.5 M acetic acid for 5, 10, 25, 200, or 400 hours, or not at all. [Figure 7]Figure 7 shows the results of various product quality tests performed on Protein A-containing resins (MabSelect™ Xtra and MabSelect™ SuRe) exposed to 0.5 M acetic acid for different periods of time. The filled circles represent values ​​for MabSelect™ Xtra exposed to 0.5 M acetic acid for 375 hours or not at all. The crosses represent values ​​for MabSelect™ SuRe not exposed to 0.5 M acetic acid for 5, 10, 25, 200, or 400 hours, or not at all. [Figure 8] Figure 8 shows the results of various product quality tests performed on Protein A-containing resins (MabSelect™ Xtra and MabSelect™ SuRe) exposed to 0.5 M acetic acid for different periods of time. The filled circles represent values ​​for MabSelect™ Xtra exposed to 0.5 M acetic acid for 375 hours or not at all. The crosses represent values ​​for MabSelect™ SuRe not exposed to 0.5 M acetic acid for 5, 10, 25, 200, or 400 hours, or not at all. [Figure 9] Figure 9 shows the results of various product quality tests performed on Protein A-containing resins (MabSelect™ Xtra and MabSelect™ SuRe) exposed to 0.5 M acetic acid for different periods of time. The filled circles represent values ​​for MabSelect™ Xtra exposed to 0.5 M acetic acid for 375 hours or not at all. The crosses represent values ​​for MabSelect™ SuRe not exposed to 0.5 M acetic acid for 5, 10, 25, 200, or 400 hours, or not at all.

[0028] [Figure 10]10 shows an ANOVA analysis of various product quality tests performed on MabSelect™ Xtra Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 375 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. All of the post-acid treatment ranges overlap with the pre-acid treatment ranges. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid. [Figure 11] 11 shows an ANOVA analysis of various product quality tests performed on MabSelect™ Xtra Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 375 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. All of the post-acid treatment ranges overlap with the pre-acid treatment ranges. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid. [Figure 12] 12 shows an ANOVA analysis of various product quality tests performed on MabSelect™ Xtra Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 375 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. All of the post-acid treatment ranges overlap with the pre-acid treatment ranges. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid. [Figure 13]13 shows an ANOVA analysis of various product quality tests performed on MabSelect™ Xtra Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 375 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. All of the post-acid treatment ranges overlap with the pre-acid treatment ranges. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid. [Figure 14] 14 shows an ANOVA analysis of various product quality tests performed on MabSelect™ Xtra Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 375 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. All of the post-acid treatment ranges overlap with the pre-acid treatment ranges. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid.

[0029] [Figure 15] 15 shows an ANOVA analysis of various product quality tests performed on MabSelect™ SuRe Protein A resin. In the "after acid treatment" column, filled circles reflect values ​​from Table 1 for 400 hours of exposure to 0.5 M acetic acid. In the "before acid treatment" column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. The ranges are statistically significantly greater after acid exposure. [Figure 16]16 shows an ANOVA analysis of various product quality tests performed on MabSelect™ SuRe Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 400 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. The pre-acid treatment range overlaps with the post-acid treatment range. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid. [Figure 17] 17 shows an ANOVA analysis of various product quality tests performed on MabSelect™ SuRe Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 400 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. The pre-acid treatment range overlaps with the post-acid treatment range. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid. [Figure 18] 18 shows an ANOVA analysis of various product quality tests performed on MabSelect™ SuRe Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 400 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. The pre-acid treatment range overlaps with the post-acid treatment range. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid. [Figure 19]19 shows an ANOVA analysis of various product quality tests performed on MabSelect™ SuRe Protein A resin. In the post-acid treatment column, filled circles reflect values ​​from Table 1 for 400 hours of exposure to 0.5 M acetic acid. In the pre-acid treatment column, filled circles reflect values ​​from Table 1 for 0 hours of exposure to 0.5 M acetic acid. Diamonds indicate ranges based on 95% confidence intervals. The pre-acid treatment range overlaps with the post-acid treatment range. The ANOVA analysis shows no statistically significant negative effect on protein quality from long-term exposure of the resin to 0.5 M acetic acid. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description In one aspect, the invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.1 M to about 0.5 M acetic acid, wherein the contacting is performed for at least about 2 hours. In various embodiments, the contacting is performed for 2 to 5 hours, 2 to 10 hours, 2 to 25 hours, 2 to 200 hours, 2 to 375 hours, or 2 to 400 hours. In one embodiment, the contacting is performed for at least about 4 hours. In various embodiments, the contacting is performed for 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours. In one embodiment, the composition comprises about 0.1 M acetic acid and the contacting is performed for at least about 4 hours. In one embodiment, the composition comprises about 0.5 M acetic acid and the contacting is performed for at least about 4 hours. In various embodiments, the composition comprises about 0.5 M acetic acid, and the contacting step is carried out for 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours.

[0031] In one embodiment, the composition further comprises an alcohol. Non-limiting examples of alcohols that can be used include ethanol (e.g., about 20%) and benzyl alcohol (e.g., about 1% to about 2%). In one particular embodiment, the composition consists essentially of about 0.1 M acetic acid and about 20% ethanol.

[0032] In one embodiment, the present invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 0.1 M to about 0.5 M acetic acid, wherein the contacting is performed for at least about 2 hours. In one particular embodiment, the contacting is performed for at least about 4 hours. In various embodiments, the contacting is performed for 4-5 hours, 4-10 hours, 4-25 hours, 4-200 hours, 4-375 hours, or 4-400 hours. In one particular embodiment, the composition consists essentially of about 0.1 M acetic acid, and the contacting is performed for at least about 4 hours. In another particular embodiment, the composition consists essentially of about 0.5 M acetic acid, and the contacting is performed for at least about 4 hours. In various embodiments, the composition comprises about 0.5 M acetic acid, and the contacting is performed for 4-5 hours, 4-10 hours, 4-25 hours, 4-200 hours, 4-375 hours, or 4-400 hours.

[0033] In a related aspect, the invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.5 M to about 1.0 M acetic acid, wherein the contacting is performed for at least about 1 hour. In various embodiments, the contacting is performed for 1 to 5 hours, 1 to 10 hours, 1 to 25 hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours. In one embodiment, the composition comprises about 0.5 M acetic acid, and the contacting is performed for at least about 1 hour. In various embodiments, the composition comprises about 0.5 M acetic acid, and the contacting step is carried out for 1 to 5 hours, 1 to 10 hours, 1 to 25 hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours.

[0034] In one embodiment, the composition further comprises an alcohol. Non-limiting examples of alcohols that can be used include ethanol (e.g., about 20%) and benzyl alcohol (e.g., about 1% to about 2%). In one particular embodiment, the composition consists essentially of about 0.5 M acetic acid and about 20% ethanol.

[0035] In one embodiment, the invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 0.5 M to about 1.0 M acetic acid, wherein the contacting is performed for at least about 1 hour. In various embodiments, the composition consists essentially of about 0.5 M to about 1.0 M acetic acid, and the contacting is performed for 1 to 5 hours, 1 to 10 hours, 1 to 25 hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours. In one particular embodiment, the composition consists essentially of about 0.5 M acetic acid, and the contacting is performed for at least about 1 hour. In various embodiments, the composition consists essentially of about 0.5 M acetic acid, and the contacting step is carried out for 1 to 5 hours, 1 to 10 hours, 1 to 25 hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours.

[0036] In a related aspect, the invention provides a method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.1 M to about 1.0 M acetic acid, wherein the contacting reduces the amount of spore-forming bacteria (e.g., Bacillus pseudofirmus) in the chromatography matrix by at least 3 logs. 10 reduction of at least 5 logs of gram-positive bacteria (e.g., Microbacterium spp.) 10 and at least a 5 log reduction in the amount of gram-negative bacteria (e.g., Stenotrophomonas maltophilia). 10In one particular embodiment, the contacting step results in a reduction in the amount of one or more of spore-forming bacteria (e.g., Bacillus pseudofirmus), Gram-positive bacteria (e.g., Microbacterium spp.), and Gram-negative bacteria (e.g., Stenotrophomonas maltophilia) in the chromatography matrix to below the limit of detection as determined by an assay such as, for example, (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, or (5) a bacterial identification test. In various embodiments, the contacting step is carried out for at least about 1 hour, 1 to 5 hours, 1 to 10 hours, 1 to 25 hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, at least about 4 hours, 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours.

[0037] In one embodiment, the composition further comprises an alcohol. Non-limiting examples of alcohols that can be used include ethanol (e.g., about 20%) and benzyl alcohol (e.g., about 1% to about 2%). In one particular embodiment, the composition consists essentially of about 0.1 M acetic acid and about 20% ethanol. In one particular embodiment, the composition consists essentially of about 0.5 M acetic acid and about 20% ethanol.

[0038] In one embodiment, the present invention provides a method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 0.1 M to about 1.0 M acetic acid, wherein the contacting step reduces the amount of spore-forming bacteria (e.g., Bacillus pseudofirmus) in the chromatography matrix by at least 3 logs. 10 reduction of at least 5 logs of gram-positive bacteria (e.g., Microbacterium spp.) 10and at least a 5 log reduction in the amount of gram-negative bacteria (e.g., Stenotrophomonas maltophilia). 10 In one particular embodiment, the contacting step results in a reduction in the amount of one or more of spore-forming bacteria (e.g., Bacillus pseudofirmus), Gram-positive bacteria (e.g., Microbacterium spp.), and Gram-negative bacteria (e.g., Stenotrophomonas maltophilia) in the chromatography matrix to below the limit of detection as determined by an assay such as, for example, (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, or (5) a bacterial identification test. In various embodiments, the contacting step is carried out for at least about 1 hour, 1-5 hours, 1-10 hours, 1-25 hours, 1-200 hours, 1-375 hours, 1-400 hours, at least about 4 hours, 4-5 hours, 4-10 hours, 4-25 hours, 4-200 hours, 4-375 hours, or 4-400 hours.

[0039] In one embodiment of any of the above methods of the invention, the composition further comprises acetate.

[0040] In one embodiment of any of the above methods of the invention, the composition has a pH of between about 2 and about 3.

[0041] In another aspect, the present invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 4.0 M to about 12.0 M urea, wherein the contacting is performed for at least about 30 minutes. In one embodiment, the contacting is performed for at least about 1 hour. In one embodiment, the composition comprises about 8 M urea.

[0042] In one embodiment, the composition further comprises an alcohol. Non-limiting examples of alcohols that can be used include ethanol (e.g., about 20%) and benzyl alcohol (e.g., about 1% to about 2%). In one particular embodiment, the composition consists essentially of about 8M urea and about 20% ethanol.

[0043] In one embodiment, the present invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 4.0 M to about 12.0 M urea, wherein the contacting is performed for at least about 30 minutes. In one particular embodiment, the contacting is performed for at least about 1 hour. In one particular embodiment, the composition consists essentially of 8 M urea.

[0044] In a related aspect, the invention provides a method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 4.0 M to about 12.0 M urea, wherein the contacting reduces the amount of spore-forming bacteria (e.g., Bacillus pseudofirmus) in the chromatography matrix by at least 2 logs. 10 reduction of at least 5 logs of gram-positive bacteria (e.g., Microbacterium spp.) 10 and at least a 5 log reduction in the amount of gram-negative bacteria (e.g., Stenotrophomonas maltophilia). 10In one particular embodiment, the contacting step results in a reduction in the amount of one or more of spore-forming bacteria (e.g., Bacillus pseudofirmus), Gram-positive bacteria (e.g., Microbacterium spp.), and Gram-negative bacteria (e.g., Stenotrophomonas maltophilia) in the chromatography matrix to below the limit of detection as determined by an assay such as, for example, (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, or (5) a bacterial identification test.

[0045] In one embodiment, the composition further comprises an alcohol. Non-limiting examples of alcohols that can be used include ethanol (e.g., about 20%) and benzyl alcohol (e.g., about 1% to about 2%). In one particular embodiment, the composition consists essentially of about 8M urea and about 20% ethanol.

[0046] In one embodiment, the present invention provides a method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 4.0 M to about 12.0 M urea, wherein the contacting step reduces the amount of spore-forming bacteria (e.g., Bacillus pseudofirmus) in the chromatography matrix by at least 2 logs. 10 reduction of at least 5 logs of gram-positive bacteria (e.g., Microbacterium spp.) 10 and at least a 5 log reduction in the amount of gram-negative bacteria (e.g., Stenotrophomonas maltophilia). 10In one particular embodiment, the contacting step results in a reduction in the amount of one or more of spore-forming bacteria (e.g., Bacillus pseudofirmus), Gram-positive bacteria (e.g., Microbacterium spp.), and Gram-negative bacteria (e.g., Stenotrophomonas maltophilia) in the chromatography matrix to below the limit of detection as determined by an assay such as, for example, (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, or (5) a bacterial identification test.

[0047] In a further aspect, the present invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 4.0 M to about 12.0 M guanidine hydrochloride, wherein the contacting is performed for at least about 30 minutes. In one embodiment, the contacting is performed for at least about 1 hour. In one embodiment, the composition comprises about 6 M guanidine hydrochloride.

[0048] In one embodiment, the composition further comprises an alcohol. Non-limiting examples of alcohols that can be used include ethanol (e.g., about 20%) and benzyl alcohol (e.g., about 1% to about 2%). In one particular embodiment, the composition consists essentially of about 6 M guanidine hydrochloride and about 20% ethanol.

[0049] In one embodiment, the present invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 4.0 M to about 12.0 M guanidine hydrochloride, wherein the contacting is performed for at least about 30 minutes. In one particular embodiment, the contacting is performed for at least about 1 hour. In one particular embodiment, the composition consists essentially of about 6 M guanidine hydrochloride.

[0050] In a related aspect, the invention provides a method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 4.0 M to about 12.0 M guanidine hydrochloride, wherein the contacting reduces the amount of spore-forming bacteria (e.g., Bacillus pseudofirmus) in the chromatography matrix by at least 2 logs. 10 reduction of at least 4 logs of gram-positive bacteria (e.g., Microbacterium spp.) 10 and at least a 2 log reduction in the amount of gram-negative bacteria (e.g., Stenotrophomonas maltophilia) 10 In one particular embodiment, the contacting step results in a reduction in the amount of one or more of spore-forming bacteria (e.g., Bacillus pseudofirmus), Gram-positive bacteria (e.g., Microbacterium spp.), and Gram-negative bacteria (e.g., Stenotrophomonas maltophilia) in the chromatography matrix to below the limit of detection as determined by an assay such as, for example, (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, or (5) a bacterial identification test.

[0051] In one embodiment, the composition further comprises an alcohol. Non-limiting examples of alcohols that can be used include ethanol (e.g., about 20%) and benzyl alcohol (e.g., about 1% to about 2%). In one particular embodiment, the composition consists essentially of about 6 M guanidine hydrochloride and about 20% ethanol.

[0052] In one embodiment, the present invention provides a method for the reduction of microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 4.0 M to about 12.0 M guanidine hydrochloride, wherein the contacting step reduces the amount of spore-forming bacteria (e.g., Bacillus pseudofirmus) in the chromatography matrix by at least 2 logs. 10 reduction of at least 4 logs of gram-positive bacteria (e.g., Microbacterium spp.) 10 and at least a 2 log reduction in the amount of gram-negative bacteria (e.g., Stenotrophomonas maltophilia) 10 In one particular embodiment, the contacting step results in a reduction in the amount of one or more of spore-forming bacteria (e.g., Bacillus pseudofirmus), Gram-positive bacteria (e.g., Microbacterium spp.), and Gram-negative bacteria (e.g., Stenotrophomonas maltophilia) in the chromatography matrix to below the limit of detection as determined by an assay such as, for example, (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, or (5) a bacterial identification test.

[0053] In one aspect, the present invention provides a method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.5 M to about 1.0 M acetic acid and (i) about 4.0 M to about 12.0 M urea and / or (ii) about 4.0 M to about 12.0 M guanidine hydrochloride, wherein the contacting is performed for at least about 1 hour. In one particular embodiment, the composition further comprises an alcohol. Non-limiting examples of alcohols that can be used include ethanol (e.g., about 20%) and benzyl alcohol (e.g., about 1% to about 2%).

[0054] In one embodiment of any of the above methods of the invention, the contacting step is carried out at a temperature between 15° C. and 30° C. In one particular embodiment, the contacting step is carried out at a temperature between 20° C. and 25° C.

[0055] In one embodiment of any of the above methods of the present invention, the composition is substantially free of an oxidizing agent.

[0056] In one embodiment of any of the above methods of the present invention, the composition does not comprise a peroxyacid.

[0057] In one embodiment of any of the above methods of the present invention, the composition does not contain peroxide.

[0058] In one embodiment of any of the above methods of the present invention, the composition does not include NaOH.

[0059] In one embodiment of any of the above methods of the invention, the contacting step is repeated at least once.

[0060] In one embodiment of any of the above methods of the invention, the chromatography matrix is ​​packed into a chromatography column. In one particular embodiment, the chromatography column has an internal diameter of between 0.5 cm and 1.5 cm and a bed height of between 15 cm and 30 cm. In one particular embodiment, the chromatography column has an internal diameter of about 1 cm and a bed height of about 20 cm. In one particular embodiment, the chromatography column has an internal diameter of between 40 cm and 1.6 meters and a bed height of between 15 cm and 30 cm. In one particular embodiment, the chromatography column has an internal diameter of about 1.4 meters and a bed height of about 20 cm.

[0061] In one embodiment of any of the above methods of the present invention, the chromatography matrix comprises a proteinaceous ligand linked to a support. In one particular embodiment, the proteinaceous ligand comprises one or more immunoglobulin-binding domains. In one particular embodiment, the proteinaceous ligand is protein A or a fragment or derivative thereof. In one particular embodiment, the proteinaceous ligand is selected from the group consisting of Staphylococcus protein A, Peptostreptococcus protein L, Streptococcus protein G, Streptococcus protein A, and fragments and derivatives thereof. In one particular embodiment, the chromatography matrix is ​​selected from the group consisting of MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, MabSelect™ SuRe pcc, MabSelect™ SuRe LX, MabCapture™ A, nProtein A Sepharose 4 Fast Flow, Protein A Sepharose 4 Fast Flow, Protein A Mag Sepharose, Protein A Sepharose CL-4B, rmp Protein A Sepharose Fast Flow, rProtein A Sepharose 4 Fast Flow, Capto™ L, ProSep™-A, ProSep Ultra Plus, AbSolute™, CaptivA™ PriMab™, Protein A Diamond, Eshmuno™ A, Toyopearl™ AF-rProtein A, Amsphere™ Protein A, KanCapA™, Protein G Mag Sepharose Xtra, and Protein G Sepharose 4 Fast Flow.In one particular embodiment, the chromatography matrix is ​​selected from the group consisting of MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, MabSelect™ SuRe PCC, and MabSelect™ SuRe LX. In one particular embodiment, the proteinaceous ligand is not measurably denatured after the method is performed.

[0062] In one embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 0.5 M acetic acid, wherein the contacting is performed for at least about 4 hours.

[0063] In another embodiment, the invention provides a method for reducing microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 0.1 M acetic acid and about 20% ethanol, wherein the contacting is performed for at least about 4 hours. In various embodiments, the contacting is performed for at least about 1 hour, 1-5 hours, 1-10 hours, 1-25 hours, 1-200 hours, 1-375 hours, 1-400 hours, at least about 4 hours, 4-5 hours, 4-10 hours, 4-25 hours, 4-200 hours, 4-375 hours, or 4-400 hours.

[0064] In a further embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 8 M urea, wherein the contacting is performed for at least about 1 hour.

[0065] In yet another embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 8 M urea and about 20% ethanol, wherein the contacting is performed for at least about 1 hour.

[0066] In a further embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 6 M guanidine hydrochloride, wherein the contacting is performed for at least about 1 hour.

[0067] In another embodiment, the present invention provides a method for the reduction of microbial bioburden of a MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about 6 M guanidine hydrochloride and about 20% ethanol, wherein the contacting is performed for at least about 1 hour.

[0068] In one embodiment, the present invention provides a method for reducing the microbial load of a composition comprising a pharmaceutical substance prior to application for purification, the method comprising the steps of: (a) providing a chromatography matrix; (b) performing any of the above methods of the present invention; and (c) applying a composition comprising the pharmaceutical substance to the chromatography matrix.

[0069] It is to be understood that the invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, and the scope of the present invention is defined by the claims.

[0070] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the kind described herein and / or that will be apparent to those skilled in the art upon reading this disclosure.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0072] The terms "about" and "approximately" are used interchangeably to mean within a statistically meaningful range of values. Such a range may be within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range.

[0073] As used herein, the terms "microbe" or "microorganism" include prokaryotes, such as bacteria and archaea, and eukaryotes, such as fungi. These terms include both living cells and spores (in the case of spore-forming organisms), as well as microbial products, such as endotoxins.

[0074] As used herein, the terms "microbial bioburden reduction" and "microbe bioburden reduction" combine killing of microorganisms and some interference with the interactions between microorganisms and a chromatography matrix. Microbial bioburden reduction according to the present invention is not the same as any previously disclosed disinfection process designed to kill essentially all, or at least 99%, of the microorganisms that may be present on a chromatography column or matrix.

[0075] Rather, the present invention includes methods that can kill less than 80%, less than 70%, less than 60%, or 40-60% of the non-spore-forming microorganisms. In such embodiments, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, or 10-20%, of the spore-forming microorganisms, e.g., Bacillus, on the column are killed by the methods of the present invention.

[0076] The present invention includes methods that do not kill all bacteria on the column, but remove at least 85%, at least 90%, at least 95%, at least 97%, about 98%, about 99%, or about 100% of viable microorganisms detectable by biofiltration assays or any other microbiological assays disclosed herein or known in the art from the chromatography matrix after processing. In some embodiments of the present invention, the level of microbial bioburden is reduced to below GMP acceptable levels in pre-use flush samples, equilibration samples, load samples, or all GMP samples from the load taken after the run. Even without killing all microorganisms, the present invention can reduce microbial bioburden to below GMP warning levels by interfering with interactions between microorganisms and the chromatography matrix, which also occur during the methods of the claimed invention. This interference can involve mechanisms such as, but not limited to, reduced affinity, binding, or any other interaction between microorganisms and the chromatography matrix. Such mechanisms are similar to the common stripping steps used on chromatography columns to remove impurities such as host cell proteins and DNA from the column. Thus, this interference may be detected after use of a method of the present invention that does not kill all microorganisms when the microbial bioburden is reduced to a level consistent with the requirements of GMP manufacturing of biological pharmaceuticals. By using one of the methods of the present invention that is not designed to kill all microorganisms on the column, the reagents are not necessarily harsh and are therefore more favorable for approval by regulatory agencies, such as the FDA or EMA, that must approve the process and product for market. Preferably, the same strip buffer components or at least the activator (acetic acid is common) used on the chromatography column during GMP manufacturing of biological pharmaceuticals can be used to reduce microbial bioburden when applied at higher concentrations (e.g., 10X, 15X, or 20X) and for longer contract times (e.g., 3X, 4X, or 5X). Furthermore, using the same strip buffer at higher concentrations for longer periods of time can be more effective for reducing microbial bioburden.

[0077] Any of the methods, aspects, and embodiments described herein can be used as part of Good Manufacturing Practices (GMP) or Current Good Manufacturing Practices (CGMP). Such standards must provide consistency in manufacturing steps and product quality to meet the requirements of regulatory agencies, such as the U.S. Food and Drug Administration. GMP and CGMP typically require a high degree of predictability and standardization in the manufacturing process, particularly to ensure the purity of the therapeutic biomolecules produced for use in human patients. There are many failure points in GMP or CGMP where parameters are detected that require the process to be stopped and / or the manufacturing batch to be discarded. Along with the labor involved in growing cultures to produce biomolecules, significant costs are incurred when failures occur.

[0078] If the bioburden or microbial load becomes too high in a chromatography column or matrix used in a separation, various unpredictable and / or undesirable effects can occur. A failure point can be triggered to stop the process so that microbially contaminated products are identified and further production is prevented. To prevent a failure point, a warning can be triggered upon detection of a bioburden of at least 5 CFU per 10 mL. A bioburden of at least 10 CFU per 10 mL can trigger an action, which can include performing one or more of the methods or embodiments described herein, alone or in combination, to reduce the bioburden.

[0079] For example, microorganisms may be introduced into the product, rendering it unacceptable for therapeutic use. Excessive bioburden may also reduce column performance, which may interfere with product purification in a standardized and predictable manner and may lead to the introduction of other failure points. Therefore, it is desirable to proactively use aspects and embodiments described herein that reduce bioburden to ensure compliance with GMP or CGMP and minimize the introduction of failure points and associated troubleshooting and downtime.

[0080] Any aspect or embodiment described herein may further include applying a small molecule- or biomolecule-containing (e.g., monoclonal antibody-containing) preparation for purification after the contacting step. Methods for reducing microbial load prior to applying a small molecule- or biomolecule-containing (e.g., monoclonal antibody-containing) preparation for purification may include the steps of any of the microbial bioburden reduction methods described herein. Alternatively, methods for purifying biomolecules may include performing the steps of any of the methods described herein, followed by applying a biomolecule-containing preparation to a chromatography matrix.

[0081] Any aspect or embodiment described herein may be performed after the chromatography matrix is ​​removed from storage and before applying a drug-containing, biomolecule-containing, or monoclonal antibody-containing preparation for purification. During long-term storage, small amounts of bacteria present in the chromatography matrix or column may grow and increase the bioburden.

[0082] Any aspect or embodiment described herein may be used as part of or to support a sterilization technique. The resulting reduction in bioburden in the chromatography matrix may be sufficient for the sterilization technique or may be used before or after other steps in the sterilization technique. The described methods of reducing bioburden may reduce the likelihood of inducing a failure point in the sterilization technique and may be used in response to the initiation of an impending failure point.

[0083] In another aspect, a MabSelect™ Xtra chromatography matrix is ​​subjected to a reduction in microbial bioburden by contacting the matrix with a composition consisting essentially of about 0.5 M acetic acid for at least 4 hours. In various embodiments, the contacting step is carried out for 4-5 hours, 4-10 hours, 4-25 hours, 4-200 hours, 4-375 hours, or 4-400 hours. In another aspect, a MabSelect™ Xtra chromatography matrix is ​​subjected to a reduction in microbial bioburden by contacting the matrix with a composition consisting essentially of 0.1 M acetic acid and about 20% ethanol for at least 4 hours.

[0084] A variety of chromatography matrices can be used. The chromatography matrix may comprise a proteinaceous ligand linked to a support. The proteinaceous ligand may then comprise one or more immunoglobulin binding domains. Other useful chromatography matrices include, but are not limited to, various ion exchange chromatography matrices, hydrophobic interaction chromatography (HIC) matrices, mixed-mode chromatography matrices, and size exclusion chromatography matrices.

[0085] The proteinaceous ligand of the chromatography matrix may be Protein A or a fragment or derivative thereof. Exemplary proteinaceous ligands include Staphylococcus Protein A, Peptostreptococcus Protein L, Streptococcus Protein G, Streptococcus Protein A, and fragments and derivatives of any of Staphylococcus Protein A, Peptostreptococcus Protein L, Streptococcus Protein G, and Streptococcus Protein A.

[0086] Staphylococcus protein A is a protein found in the bacterium Staphylococcus Protein A can be found on the cell wall of Staphylococcus aureus. Protein A can bind to antibodies in the Fc region between the CH2 and CH3 domains. Protein A can be cultured in Staphylococcus aureus or recombinantly produced in other bacteria, such as E. coli or Brevibacillus. Fragments or derivatives of Staphylococcus protein A can also bind to antibodies in the Fc region between the CH2 and CH3 domains.

[0087] Peptostreptococcus protein L can be found on the surface of Peptostreptococcus magnus and can bind to antibodies through interactions with antibody light chains. Unlike protein A, protein L can bind to single-chain variable fragments (scFv) and Fab fragments. Fragments or derivatives of protein L can also bind to antibody light chains, single-chain variable fragments (scFv), and Fab fragments.

[0088] Streptococcus protein G can be found on the cell wall of group G Streptococcus strains. Protein G can bind to antibodies at the Fab and Fc regions. Protein G can also be recombinantly produced in other bacteria, such as E. coli. Fragments or derivatives of protein G can also bind to antibodies at the Fab and Fc regions.

[0089] The chromatography matrix can be a resin that is part of a column. One suitable resin is MabSelect SuRe™ from GE Healthcare. An exemplary column suitable for small-scale purification is packed with MabSelect SuRe™ and is about 1.0 cm in diameter and about 20 cm in length. Larger columns, such as 1.4 m x 20 cm, can be used for production-scale purification.

[0090] More generally, the methods of the present invention can be used for chromatography columns of various sizes, including laboratory scale, large processing scale, and very large processing scale. In some embodiments, the chromatography column may have an internal diameter of between 0.5 cm and 1.5 cm and a bed height of between 15 and 30 cm (e.g., 20 cm). The internal diameter may be between 0.7 and 1.2 cm, or alternatively, 0.9 and 1.4 cm, 1.2 and 1.5 cm, 1.0 and 1.2 cm, or about 1 cm. In some embodiments, the chromatography column may have an internal diameter of between 40 cm and 1.6 meters (e.g., 60 cm, 80 cm, 1.0 meter, 1.2 meter, or 1.4 meter). The chromatography column may have a bed height of between 15 and 30 cm (e.g., 20 cm).

[0091] Exemplary chromatography matrices include MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, MabSelect™ SuRe pcc, MabSelect™ SuRe LX, nProtein A Sepharose 4 Fast Flow, Protein A Sepharose 4 Fast Flow, Protein A Mag Sepharose, Protein A Sepharose CL-4B, rmp Protein A Sepharose Fast Flow, rProtein A Sepharose 4 Fast Flow, Capto™ L, ProSep™-A, ProSep Ultra Plus, AbSolute™, CaptivA™ PriMab™, Protein A Diamond, Eshmuno™ A, Toyopearl™ AF-rProtein A, Amsphere™ Protein A, KanCapA™, Protein G Mag Sepharose Xtra, and Protein G Sepharose 4 Fast Flow.

[0092] MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, and MabSelect™ SuRe LX have recombinant Protein A ligand produced in E. coli attached to a highly cross-linked agarose matrix.

[0093] Reducing microbial bioburden can restore the performance of the chromatography matrix so that it can be used for additional purification rather than being replaced. Thus, in any of the methods described herein, reducing microbial bioburden can be performed after the chromatography matrix has been used. In such cases, bacteria and other microorganisms that may be introduced into the chromatography matrix from a cell culture broth containing the monoclonal antibody of interest can be removed. Significant cost savings can be achieved when using a chromatography matrix containing a proteinaceous ligand, such as protein A.

[0094] Furthermore, using an acetic acid-containing solution instead of the commonly used sodium hydroxide-containing solution can result in less denaturation of the protein ligand and less damage to the chromatography matrix. Denaturation of the protein ligand and / or damage to the chromatography matrix can be measured indirectly by assaying various performance characteristics of the chromatography matrix, such as by assaying the purity and amount of the product eluted from the matrix and by assaying the residual elution of a component of the matrix (e.g., Protein A). For example, if the protein ligand is Protein A, the purity and amount of a monoclonal antibody are assayed. Exemplary assays include size exclusion chromatography (e.g., SE-HPLC and SE-UPLC), capillary electrophoresis (e.g., CE-SDS), and capillary isoelectric focusing (iCIEF), which may optionally include whole-column imaging. Residual Protein A from the column can also be measured (e.g., by assays including ELISA).

[0095] Microbial bioburden reduction achieved according to the methods described herein can be a cost-effective way to maintain the performance of columns containing Protein A or other proteinaceous ligands by removing bacteria without damaging Protein A. For example, exposure of Protein A-containing matrices to acetic acid-containing solutions (e.g., 0.5 M acetic acid) for 375 or 400 hours does not lead to any statistically significant loss of performance of columns containing Protein A. See, e.g., Example 6, Table 10, and Figures 5-19. For example, there is no statistically significant loss of purity of monoclonal antibodies eluted by size exclusion chromatography, capillary electrophoresis under reducing or non-reducing conditions, or capillary isoelectric focusing.

[0096] The reduction of microbial bioburden by the methods described herein can remove nearly all of the bacteria without killing all of the bacteria. Without wishing to be bound by theory, acetic acid may interfere with the affinity between bacteria and proteinaceous ligands, such as protein A. Microbial-contaminated matrices or columns can have microorganisms reduced according to the methods described herein by disrupting the interaction between the microorganisms and the resin. Bacteria tend to persist in the acetic acid-containing solution. Bacterial removal can be further enhanced by repeating the contacting step with acetic acid or by additional flushing of the chromatography matrix with an acetic acid-containing solution.

[0097] In some embodiments, the method removes spore-forming bacteria from the chromatography matrix. Spore-forming bacteria have the ability to switch to an endospore form. The endospore form is a minimal, dormant form to which bacteria can reduce themselves. Endospore formation is usually induced by a lack of nutrients or harsh conditions, such as an acidic or alkaline environment. Endospores allow bacteria to remain dormant for long periods of time even under unfavorable conditions. When the environment becomes more favorable, endospores can be reactivated to a vegetative state. Examples of bacteria that can form endospores include Bacillus and Clostridium. It is believed that these spore-forming bacteria can form endospores under normal operating conditions in the chromatography purification process due to the presence of unfavorable conditions for these spore-forming bacteria in the manufacturing process. There are many methods that can be used to detect spore-forming bacteria, including, but not limited to, microscopic bacterial staining, IR / FTIR spectroscopy, sterility testing, and bacterial identification tests (e.g., biochemical reactions, 16S rRNA sequencing, or taxon-specific sequencing).

[0098] In some embodiments, the method removes Gram-positive bacteria from the chromatography matrix. In some embodiments, the method can remove Gram-negative bacteria from the chromatography matrix. In some embodiments, the method removes spore-forming bacteria, Gram-positive bacteria, and Gram-negative bacteria from the chromatography matrix. The contacting step can result in a reduction in the amount of one or more of spore-forming bacteria, Gram-positive bacteria, and Gram-negative bacteria in the chromatography matrix to below the detection limit of an assay selected from the group consisting of: (1) biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) sterility testing, and (5) bacterial identification tests (e.g., biochemical reactions, 16S rRNA sequencing, or taxon-specific sequencing). Biofiltration assays are described in Chapter 71 of the United States Pharmacopeia, titled "Sterility Tests." In a biofiltration assay, the eluate from the column is passed through a filter that selectively binds bacteria. The filter is then placed on agar containing an appropriate medium for bacterial growth, incubated, and the bacteria are counted. Dilution of the column eluate can be performed as needed.

[0099] Microscopic bacterial staining is described in the United States Pharmacopeia, Chapter 61, titled "Microbial examination of nonsterile products: microbial enumeration tests." IR / FTIR spectroscopy is described in BRUKER Application Note AN#405, Current Research, Technology and Education Topics in Applied Microbiology Biotechnology, edited by A. Mendez-Vilas. Microbiological testing is described in Reynolds, J. et al., "Differential staining of bacteria: endospore stain," Curr Proc. Microbiol. 2009, Supplement 3:Supplement 3J.

[0100] In some embodiments, the concentration of acetic acid in the composition is about 0.1 M to about 1.0 M. In some embodiments, the concentration of acetic acid in the composition is about 0.2 M to about 0.8 M. In some embodiments, the concentration of acetic acid in the composition is about 0.4 M to about 0.7 M. In some embodiments, the concentration of acetic acid in the composition is about 0.5 M. In some embodiments, the concentration of acetic acid in the composition is about 0.1 M to about 0.5 M. In some embodiments, the concentration of acetic acid in the composition is about 0.1 M.

[0101] In some embodiments, the concentration of urea in the composition is about 4 M to about 12 M. In some embodiments, the concentration of urea in the composition is about 6 M to about 10 M. In some embodiments, the concentration of urea in the composition is about 6 M to about 8 M. In some embodiments, the concentration of urea in the composition is about 8 M.

[0102] In some embodiments, the concentration of guanidine hydrochloride in the composition is about 3 M to about 10 M. In some embodiments, the concentration of guanidine hydrochloride in the composition is about 4 M to about 8 M. In some embodiments, the concentration of guanidine hydrochloride in the composition is about 5 M to about 7 M. In some embodiments, the concentration of guanidine hydrochloride in the composition is about 6 M.

[0103] In some embodiments, the concentration of guanidine hydrochloride in the composition is about 3 M to about 10 M. In some embodiments, the concentration of guanidine hydrochloride in the composition is about 4 M to about 8 M. In some embodiments, the concentration of guanidine hydrochloride in the composition is about 5 M to about 7 M. In some embodiments, the concentration of guanidine hydrochloride in the composition is about 6 M.

[0104] In some embodiments, the pH of the solution is at least 2.0. The pH may be between 2.0 and 7.0. The pH may be between 2.5 and 6.5, between 3.0 and 6.0, between 4.0 and 7.0, between 2.0 and 5.0, between 3.5 and 5.5, between 3.0 and 4.0, or about 4.0. The pH may be approximately any of the following: 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0105] In some embodiments, the composition includes ethanol. The composition may include 1-40% ethanol, 5-35% ethanol, 10-25% ethanol, 15-30% ethanol, 18-24% ethanol, about 20% ethanol, or 20% ethanol. In some embodiments, the composition includes about 0.1 M acetic acid and about 20% ethanol. In some embodiments, the composition consists essentially of about 0.1 M acetic acid and about 20% ethanol. Due to toxicity or adverse effects in humans that can result from the presence of benzyl alcohol, it is advantageous to use ethanol while minimizing the amount of benzyl alcohol used or avoiding benzyl alcohol.

[0106] In some embodiments, the composition further comprises an acetate salt. The acetate salt may act as a buffer for the acetic acid-containing composition. For example, the composition may include sodium acetate in addition to acetic acid, such that the composition is a buffer. The composition may include 0.1 M to 1.0 M sodium acetate, 0.2-0.8 M sodium acetate, 0.4-0.7 M sodium acetate, about 0.5 M sodium acetate, or 0.5 M sodium acetate. Buffering the acetic acid with sodium acetate or another acetate salt can be effective for maintaining the pH of the solution in the chromatography matrix. The pH may be at least 2.0, about 2-3, 2.0-3.0, 2.0-7.0, 2.5-6.5, 3.0-6.0, 4.0-7.0, 2.0-5.0, 3.5-5.5, 3.0-4.0, or about 4.0. The pH may be approximately any of the following: 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0107] In some embodiments, the composition includes acetic acid, the contacting step is carried out for at least 1 hour, alternatively 1-4 hours, or alternatively at least 2 hours. In some embodiments, the contacting step is carried out for 2-4 hours. In some embodiments, the contacting step is carried out for at least 4 hours. In various embodiments, the contacting step is carried out for 90 minutes to 6 hours, 2 hours to 5 hours, 4 hours to 5 hours, 4 hours to 6 hours, 90 minutes to 3 hours, 5 hours to 6 hours, 4 hours to 10 hours, 4 hours to 25 hours, 4 hours to 200 hours, 4 hours to 375 hours, or 4 hours to 400 hours.

[0108] In some embodiments, where the composition includes urea, the contacting step is carried out for at least 30 minutes, alternatively at least 1 hour, or alternatively at least 2 hours. In some embodiments, the contacting step is carried out for 1-2 hours. In some embodiments, the contacting step is carried out for at least 2 hours. In various embodiments, the contacting step is carried out for 30 minutes to 4 hours, 1 hour to 3 hours, or 90 minutes to 2 hours.

[0109] In some embodiments, where the composition comprises guanidine hydrochloride, the contacting step is carried out for at least 30 minutes, alternatively at least 1 hour, or alternatively at least 2 hours. In some embodiments, the contacting step is carried out for 1-2 hours. In some embodiments, the contacting step is carried out for at least 2 hours. In various embodiments, the contacting step is carried out for 30 minutes to 4 hours, 1 hour to 3 hours, or 90 minutes to 2 hours.

[0110] In some embodiments, where the composition comprises urea or guanidine hydrochloride, the contacting step is carried out for at least 30 minutes, alternatively at least about 1 hour, alternatively 1-4 hours, alternatively at least 2 hours.

[0111] In some embodiments, the contacting step can be repeated. For example, the contacting step may be performed for about 1 hour and then repeated multiple times. In some embodiments, the contacting step is repeated 2, 3, 4, 5, or 6 times. Repeating the microbial bioburden reduction may expose the column to additional acetic acid, which may result in additional destruction of bacteria and microorganisms from the chromatography matrix. Repeating the contacting step may lead to more flushing or removal of bacteria and microorganisms from the chromatography matrix, e.g., Protein A ligand, and column. The microbial bioburden reduction process may result in greater bioburden reduction when performed multiple times in succession.

[0112] The effectiveness of any of the microbial bioburden reduction methods described herein can be monitored using any number of bioburden assays. One such assay is a filtration assay, in which a volume of eluate is passed through a filter membrane, which captures bacteria present in the eluate. Bacterial titer can be determined by placing the filter membrane on an agar plate so that the bacteria on the filter membrane form colonies on the plate. The agar plate may contain trypticase soy agar (TSA). Incubation can be performed for 3 to 7 days at a temperature between 25°C and 37°C. The number of colonies is then counted. If too many colonies are formed, dilution of the eluate can be performed.

[0113] In some embodiments, at least 1.5 logs of the amount of spore-forming bacteria 10 For example, if a chromatography matrix is ​​contaminated with Bacillus pseudofirmus, contacting such a matrix with an 8 M urea solution, an 8 M urea / 20% ethanol solution, a 6 M guanidine hydrochloride solution, or a 6 M guanidine hydrochloride / 20% ethanol solution for at least 1 hour reduces the number of Bacillus pseudofirmus by at least 1.5 log. 10 It can be reduced.

[0114] In some embodiments, when a method for reducing microbial bioburden is performed, the binding capacity of the chromatography matrix is ​​preserved for 10 or more cycles. In other embodiments, the binding capacity is preserved for 50 or more cycles. In some other embodiments, the binding capacity is preserved for 100 or more cycles. In some embodiments, the binding capacity is preserved for 200 or more cycles.

[0115] In some embodiments, there is no substantial degradation of the chromatography matrix during or over multiple contacting steps where the exposure to the composition is at least 5 hours, at least 10 hours, at least 25 hours, at least 200 hours, at least 375 hours, or at least 400 hours. In some embodiments, there is no measurable degradation of the chromatography matrix during or over multiple contacting steps where the exposure to the composition is at least 5 hours, at least 10 hours, at least 25 hours, at least 200 hours, at least 375 hours, or at least 400 hours. In some embodiments, degradation of the chromatography matrix is ​​measured by the protein quality of proteins that bind to the matrix (e.g., monoclonal antibodies that bind to a Protein A matrix).

[0116] In some embodiments, the proteinaceous ligand is not measurably denatured, which can be determined using functional assays such as, for example, measuring column performance, product yield and / or quality, proteinaceous ligand leachable from the matrix, denaturation of the proteinaceous ligand, etc.

[0117] In some embodiments, there is no measurable leaching of the proteinaceous ligand or Protein A during the contacting step or across multiple contacting steps. In some embodiments, there is no statistically significant measurable leaching of the proteinaceous ligand or Protein A after exposure of the chromatography matrix to the composition (e.g., 0.5 M acetic acid) for 5, 10, 25, 200, 375, or 400 hours. Measuring leaching of Protein A is one such exemplary assay. Denaturation of Protein A can alter its identity so that it no longer interacts with the beads or other solid support in the chromatography matrix. The denatured Protein A then leaches from the beads or solid support into the liquid phase. Detection of the proteinaceous ligand or Protein A in the affinity column eluate or liquid phase can therefore indicate measurable denaturation. Denaturation of other proteinaceous ligands, in addition to Protein A, can also lead to their leaching from the chromatography matrix. In some embodiments, measuring leaching of Protein A and / or other proteinaceous ligands involves ELISA. Exemplary assays are described in Example 6 and Figures 8, 13, and 18. [Example]

[0118] The following examples describe various aspects and embodiments described above. However, the use of these and other examples elsewhere in this specification is merely illustrative and in no way limits the scope and meaning of any of the present disclosure or any exemplified subject matter. Similarly, the subject matter of any claim is not limited to any particular preferred embodiment described herein. In fact, many modifications and variations may become apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the aspects and embodiments disclosed herein. Therefore, the subject matter of any claim is limited only by the subject matter of those claims, along with the full scope of equivalents to which such claims are entitled.

[0119] Example 1 Acetic acid solution reduces bioburden in Protein A columns Microbial reduction was assessed in three packed 1 cm MabSelect™ Xtra columns by measuring bioburden after spiking the columns with specific bacteria and again after reducing the microorganisms with a 0.5 M acetic acid solution.

[0120] First, the column was flushed with 2 column volumes of water for injection (WFI). A 20 mL sample was collected, which served as a negative control for the amount of bacteria in the column.

[0121] For each of the three MabSelect™ Xtra columns, a representative microorganism was added to the column by adding the microorganism to the WFI to form a spiked WFI, with the microorganisms being approximately 10 5 The samples were spiked with spore-forming bacteria, specifically Bacillus psuedofirmus. One column was spiked with gram-positive bacteria, specifically Microbacterium spp. The third column was spiked with gram-negative bacteria, specifically Stenotrophomonas maltophilia.

[0122] The spiked WFI was then loaded onto each column. The columns were then flushed with 14 column volumes of WFI at 229 cm / hr and collected. Each column was held for 1 hour and then flushed again with the spiked WFI. A 20 mL sample was collected from each column as a positive control, and the amount of Gram-negative, Gram-positive, and spore-forming bacteria in the sample was then assayed.

[0123] Next, two column volumes of a 0.5 M acetic acid microbial reduction solution were applied to each column at 229 cm / hr. Each column was held for 1 hour and then flushed with two column volumes of WFI at 229 cm / hr. For Microbacterium spp. and Stenotrophomonas maltophilia, 1.5 column volumes of WFI were flushed through the column, after which 1.5 column volumes of effluent were collected. For Bacillus psuedofirmus, two column volumes of equilibration buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.2) were flushed through the column, after which 1.5 column volumes of effluent were collected.

[0124] The above experiment was repeated, except that the column was held for 4 hours instead of 1 hour after applying the microbial reduction solution of 0.5M acetic acid to the column.

[0125] A filtration-based bioburden assay was performed. Agar plates with TSA medium were prepared.

[0126] All of the above samples from the chromatography column were placed into conical tubes, inverted 10 times, and then passed through a filter manifold connected to a sterile disposable filter funnel with a 0.45 micron filter membrane, or through a Milliflex® Plus pump with a Milliflex® filter funnel unit with a 0.45 micron filter membrane. Aseptic technique was used in handling the filter manifold or filter funnel unit to avoid introducing additional bacteria not found in the chromatography sample. Each membrane was then placed on an agar plate prepared with TSA medium. The plates were incubated at 30–35°C for 5–7 days. The number of colony-forming units was then counted and recorded.

[0127] Prepare negative control plates by passing 100 mL of sterile PBS through separate filter manifolds or filter funnel units with 0.45 micron filter membranes. Next, place each membrane on an agar plate prepared with TSA medium. Incubate the plates at 30-35°C for 5-7 days. Then, count the number of colony-forming units and calculate the logarithm. 10 It is expressed in the form of

[0128] The table below shows the colony forming units before and after reduction for each of the three bacterial types. [Table 1] [Table 2] [Table 3]

[0129] For Bacillus pseudofirmus, a microbial bioburden reduction of 0.4 log was observed when a 0.5 M acetic acid solution was held in the column for 1 hour. 10 A 3.4 log reduction was observed when the 0.5 M acetic acid microbial bioburden reduction solution was held in the column for 4 hours. 10 A reduction in was observed.

[0130] For Microbacterium spp. bacteria, a microbial bioburden reduction of 1.4 log was observed when a 0.5 M acetic acid solution was held in the column for 1 hour. 10 A reduction of 5.6 log was observed after 4 hours of holding. 10 A reduction in was observed.

[0131] For Stenotrophomonas maltophila bacteria, a microbial bioburden reduction of 5.5 log was observed when a 0.5 M acetic acid solution was held in the column for 1 hour. 10 A reduction of 6.2 log was observed after 4 hours of holding. 10 A reduction in was observed.

[0132] 0.5M acetic acid is effective in removing a wide range of microorganisms, including spore-forming bacteria, when retained in a Protein A column for 4 hours.

[0133] Example 2 Acetic acid / ethanol solution reduces bioburden in Protein A columns The steps of Example 1 above were followed, except that a solution of 0.1 M acetic acid and 20% ethanol was used instead of 0.5 M acetic acid. As in Example 1, the 0.1 M acetic acid and 20% ethanol solution was held for 1 hour in one set of experiments and for 4 hours in another set of experiments. The results below also show a significant reduction in the amount of bacteria after holding the microbial bioburden reduction solution in the column for 1 hour or 4 hours. [Table 4] [Table 5-1] [Table 5-2] [Table 6]

[0134] For Bacillus pseudofirmus, a microbial bioburden reduction solution of 0.1 M acetic acid and 20% ethanol was retained in the column for 1 hour, resulting in a 0.4 log 10 A 2.5 log reduction was observed when a microbial bioburden reduction solution of 0.1 M acetic acid and 20% ethanol was held in the column for 4 hours. 10 A reduction in was observed.

[0135] For Microbacterium spp. bacteria, a microbial bioburden reduction solution of 0.1 M acetic acid and 20% ethanol was retained in the column for 1 hour, resulting in a 4.5 log 10A reduction of 5.1 log was observed after 4 hours of holding. 10 A reduction in was observed.

[0136] For Stenotrophomonas maltophila bacteria, a microbial bioburden reduction solution of 0.1 M acetic acid and 20% ethanol was retained in the column for 1 hour, resulting in a 5.3 log 10 A reduction of 5.1 log was observed after 4 hours of holding. 10 A reduction in was observed. Example 3 Urea solution reduces bioburden in Protein A columns

[0137] The steps of Example 1 above were followed, except that a solution of 8 M urea and a solution of 8 M urea / 20% ethanol were used instead of 0.5 M acetic acid, and only a 1-hour retention was measured. The results in Table 7 below show a significant reduction in bacteria after 1 hour of retention of the microbial bioburden reduction solution in the column. The reduction of spore-forming B. psuedofirmus, especially after only 1 hour of treatment, was large and unexpected. [Table 7] Example 4 Guanidine hydrochloride solution reduces bioburden in Protein A columns

[0138] The steps of Example 1 above were followed, except that a solution of 6 M guanidine hydrochloride and a solution of 6 M guanidine hydrochloride / 20% ethanol were used instead of 0.5 M acetic acid, and only a 1 hour retention was measured. The results in Table 8 below show a significant reduction in bacteria after 1 hour of retention of the microbial bioburden reduction solution in the column. The reduction of spore-forming B. psuedofirmus, especially after only 1 hour of treatment, was large and unexpected. [Table 8]

[0139] Example 5 Testing of microbial bioburden reducers in solution Solution spike tests were performed using 0.5 M acetic acid to measure the extent of killing of Bacillus psuedofirmus and Microbacterium species in solution without a chromatographic matrix. The data are shown in Figure 1. After 1 hour, little killing of Bacillus psuedofirmus was observed, but there was some killing of Microbacterium species. These data indicate that killing alone is not responsible for the reduction of microbial bioburden of these bacteria on a chromatographic matrix with 0.5 M acetic acid. Disruption of the interaction between the chromatographic matrix and Bacillus psuedofirmus and Microbacterium species leads to increased bioburden reduction beyond that expected from killing.

[0140] Additional solution spike tests were performed using other agents to measure the extent of killing of Bacillus psuedofirmus, Microbacterium species, and Stenotrophomonas maltophilia in solution. The following agents were added: (a) water for injection (WFI), (b) 8 M urea, (c) 8 M urea and 20% ethanol, (d) 6 M guanidine hydrochloride, and (e) 6 M guanidine hydrochloride and 20% ethanol. Spike confirmation measurements in PBS were also performed, as well as measurements at 0, 30, and 60 minutes. The data are shown in Figures 2–4, where blue bars represent WFI, yellow bars represent 8 M urea, gray bars represent 8 M urea and 20% ethanol, red bars represent 6 M guanidine hydrochloride, and green bars represent 6 M guanidine hydrochloride and 20% ethanol.

[0141] For Bacillus pseudofirmus, bioburden reduction is achieved with at least the following solutions: 0.5 M acetic acid, 8 M urea and 8 M urea / 20% ethanol, 6 M guanidine hydrochloride and 6 M guanidine hydrochloride / 20% ethanol, by a combination of killing and disrupting the interaction between the microorganism and the chromatography resin.

[0142] For Microbacterium species, a combination of killing and disruption of interactions may result in bioburden reduction for 8M urea and 0.5M acetic acid, solutions that do not kill 100% of Microbacterium species. However, killing may largely be responsible for bioburden reduction for 8M urea / 20% ethanol, 6M guanidine hydrochloride, and 6M guanidine hydrochloride / 20% ethanol, which were observed to kill 100% of Microbacterium species in solution. Similarly, for Stenotrophomonas maltophilia, 8M urea and 8M urea / 20% ethanol were able to kill 100% of Stenotrophomonas maltophilia in solution.

[0143] In summary, Examples 1-5 demonstrate that 0.5 M acetic acid with a 4 hour hold, 8 M urea with a 1 hour hold, 8 M urea / 20% ethanol with a 1 hour hold, 6 M guanidine hydrochloride with a 1 hour hold, and 6 M guanidine hydrochloride / 20% ethanol with a 1 hour hold are found to be effective methods of reducing microbial bioburden in the production of packed MabSelect™ Xtra columns. As described in Examples 1-5, these agents are effective through a combination of killing the microorganisms and disrupting the interactions between the microorganisms and the chromatography resin, or through 100% killing of the microorganisms.

[0144] Furthermore, exposure of MabSelect™ Xtra resin to 0.5 M acetic acid had minimal effect on the Protein A resin.

[0145] Example 6 Affinity columns maintain performance after long-term exposure to acetic acid The performance characteristics of two different chromatographic affinity resins, MabSelect™ Xtra and MabSelect™ SuRe, were evaluated after the resins were soaked in 0.5 M acetic acid for various periods of time. Specifically, a portion of MabSelect™ Xtra (used to capture mAb A) was soaked in 0.5 M acetic acid for 375 hours. As a negative control, another portion of MabSelect™ Xtra was not soaked in 0.5 M acetic acid. Five different portions of MabSelect™ SuRe (used to capture mAb B) were soaked in 0.5 M acetic acid for 5, 10, 25, 200, or 400 hours, respectively. As a control, another portion of MabSelect™ SuRe was not soaked in 0.5 M acetic acid.

[0146] Each of the above portions was then subjected to five different experiments to evaluate performance: Size exclusion chromatography (SE-HPLC and SE-UPLC) was performed to evaluate the mAb purity after purification for each of the above chromatographic affinity resins. [Table 9]

[0147] Two different capillary electrophoresis experiments were performed. CE-SDS was performed for mAb A, and PICO Microchip CE-Electrophoresis (PICO MCE-SDS) was performed for mAb B. Capillary electrophoresis was performed in an SDS-containing gel-filled capillary (CE-SDS) to measure the molecular weight distribution and relative abundance of the monoclonal antibody light and heavy chains. These proteins were separated based on size and electrophoretic mobility. The relative abundance of total light and heavy chains was measured under reducing and non-reducing conditions. CE-SDS was performed using an IgG Purity Analysis Kit (Beckman Coulter, A10663) with a bare fused silica capillary (capillary length 57 cm, effective length 50 cm). PICO MCE-SDS was performed using a Protein Express LabChip, LabChip® GXII, or LabChip® GXII Touch HT (Perkin Elmer, 760499 or 760528). Relative migration times were normalized using an internal standard.

[0148] To assay the effect of acetic acid on Protein A-containing matrices, eluates from MabSelect™ Xtra and MabSelect™ SuRe columns were analyzed and quantified for residual Protein A by high-throughput ELISA.

[0149] Capillary isoelectric focusing with whole-column imaging (iCIEF) was performed to quantify the amount of complementarity-determining region 2 (CDR2) in monoclonal antibody samples. The relative amount of CDR2 in each electropherogram was calculated by integrating the area under the observed isoelectric point (pi) distribution peak from each sample and calculating the percentage attributable to CDR2. The iCIEF region 2 reported is the major peak of neutral species and corresponds to the largest protein peak in the internal reference standard.

[0150] The results of each of the above analyses are shown in Table 10 below. [Table 10-1] [Table 10-2]

[0151] The data from the above tables are shown in each of Figures 5-9. Visual inspection of these figures shows no negative correlation between performance characteristics and duration of exposure to 0.5 M acetic acid.

[0152] Analysis of variance (ANOVA) of product quality data was performed to assess statistically significant differences between resins before and after prolonged exposure to 0.5 M acetic acid using three chromatographic runs per resin condition. Figures 10-14 show the protein quality of the mAb A pool obtained after MabSelect Xtra purification.

[0153] Figures 15-19 show the protein quality of the mAb B pool obtained after MabSelect SuRe purification. ANOVA analysis of protein quality shows no statistical significance (p<0.05) except in the SE-UPLC percent purity of the mAb B MabSelect SuRe pool. However, the post-acid treatment pool purity is higher than the pre-acid treatment pool purity. Therefore, there is no negative effect on the mAb B pool after purification with MabSelect SuRe with prolonged exposure to 0.5 M acetic acid. The claimed subject matter is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the claimed subject matter in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims. All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein. The present invention provides, for example, the following items. (Item 1) 1. A method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.1 M to about 0.5 M acetic acid, wherein the contacting is performed for about 1 hour to about 4 hours. (Item 2) 2. The method of claim 1, wherein the contacting step is carried out for at least about 4 hours. (Item 3) 10. The method of claim 1, wherein the composition comprises about 0.1 M acetic acid and the contacting step is carried out for at least about 4 hours. (Item 4) 10. The method of claim 1, wherein the composition comprises about 0.5 M acetic acid and the contacting step is carried out for at least about 4 hours. (Item 5) 5. The method of any one of items 1 to 4, wherein the composition further comprises an alcohol. (Item 6) Item 7. The method according to Item 5, wherein the alcohol is ethanol or benzyl alcohol. 7. The method of claim 6, wherein the composition comprises about 20% ethanol. (Item 8) 7. The method of claim 6, wherein the composition comprises about 1% to about 2% benzyl alcohol. (Item 9) 1. A method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 0.1 M to about 1.0 M acetic acid, wherein the contacting step reduces the amount of spore-forming bacteria in the chromatography matrix by at least 3 logs. 10 reduction of the amount of gram-positive bacteria by at least 5 logs 10 and at least a 5 log reduction in the amount of Gram-negative bacteria. 10 The method results in one or more of a reduction in: (Item 10) 10. The method of claim 9, wherein the composition further comprises an alcohol. (Item 11) 11. The method of claim 10, wherein the alcohol is ethanol or benzyl alcohol. (Item 12) 12. The method of claim 11, wherein the composition comprises about 20% ethanol. (Item 13) 12. The method of claim 11, wherein the composition comprises about 1% to about 2% benzyl alcohol. (Item 14) 10. The method of claim 9, wherein the contacting step results in a reduction in the amount of one or more of spore-forming bacteria, gram-positive bacteria, and gram-negative bacteria in the chromatography matrix to below the limit of detection as determined by an assay selected from the group consisting of: (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, and (5) a bacterial identification test. (Item 15) 15. The method of claim 14, wherein the spore-forming bacterium is Bacillus pseudofirmus, the Gram-positive bacterium is Microbacterium spp., and the Gram-negative bacterium is Stenotrophomonas maltophilia. (Item 16) 10. The method of claim 1 or 9, wherein the composition further comprises an acetate salt. (Item 17) 17. The method of any one of items 1 to 16, wherein the composition has a pH between about 2 and about 3. (Item 18) 1. A method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 4.0 M to about 12.0 M urea, wherein the contacting is performed for at least about 30 minutes. (Item 19) 20. The method of claim 18, wherein the contacting step is carried out for at least about 1 hour. (Item 20) 20. The method of claim 18 or 19, wherein the composition comprises about 8M urea. (Item 21) 21. The method of any one of items 18 to 20, wherein the composition further comprises an alcohol. (Item 22) 22. The method of claim 21, wherein the alcohol is ethanol or benzyl alcohol. (Item 23) 23. The method of claim 22, wherein the composition comprises about 20% ethanol. (Item 24) 23. The method of claim 22, wherein the composition comprises about 1% to about 2% benzyl alcohol. (Item 25) 1. A method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising about 4.0 M to about 12.0 M urea, wherein the contacting step reduces the amount of spore-forming bacteria in the chromatography matrix by at least 2 log 10 reduction of the amount of gram-positive bacteria by at least 5 logs 10 and at least a 5 log reduction in the amount of Gram-negative bacteria. 10 The method results in one or more of a reduction in: (Item 26) 26. The method of claim 25, wherein the composition further comprises an alcohol. (Item 27) 27. The method of claim 26, wherein the alcohol is ethanol or benzyl alcohol. (Item 28) 28. The method of claim 27, wherein the composition comprises about 20% ethanol. (Item 29) 28. The method of claim 27, wherein the composition comprises about 1% to about 2% benzyl alcohol. (Item 30) 26. The method of claim 25, wherein the contacting step results in a reduction in the amount of one or more of spore-forming bacteria, gram-positive bacteria, and gram-negative bacteria in the chromatography matrix to below the limit of detection as determined by an assay selected from the group consisting of: (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, and (5) a bacterial identification test. (Item 31) 31. The method of claim 30, wherein the spore-forming bacterium is Bacillus pseudofirmus, the Gram-positive bacterium is Microbacterium spp., and the Gram-negative bacterium is Stenotrophomonas maltophilia. (Item 32) 32. The method of any one of items 1 to 31, wherein the contacting step is carried out at a temperature between 15°C and 30°C. (Item 33) Item 33. The method of item 32, wherein the contacting step is carried out at a temperature between 20°C and 25°C. (Item 34) The chromatography matrix is ​​selected from the group consisting of MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, MabSelect™ SuRe pcc, MabSelect™ SuRe LX, MabCapture™ A, nProtein A Sepharose 4 Fast Flow, Protein A Sepharose 4 Fast Flow, Protein A Mag Sepharose, Protein A Sepharose CL-4B, rmp Protein A Sepharose Fast Flow, rProtein A Sepharose 4 Fast Flow, Capto™ L, ProSep™-A, ProSep Ultra Plus, AbSolute™, CaptivA™ PriMab™, Protein A Diamond, Eshmuno™ A, Toyopearl™ AF-rProtein A, Amsphere™ Protein A, KanCapA™, Protein G Mag Sepharose Xtra, and Protein G Sepharose 4 Fast Flow. 2. The method of claim 1, wherein the compound is selected from the group consisting of: (Item 35) 1. A method for reducing the microbial load of a composition containing a pharmaceutical agent prior to application for purification, comprising: providing a chromatography matrix; Carrying out the method described in item 1, and applying the composition containing the pharmaceutical agent to the chromatography matrix. A method comprising:

Claims

1. 1. A method for the reduction of microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising 0.1 M to 0.5 M acetic acid, wherein said contacting is performed for 4 hours, said composition does not comprise alcohol, guanidine hydrochloride or urea, and said method does not comprise contacting the chromatography matrix with a sodium hydroxide solution.

2. 10. The method of claim 1, wherein the composition comprises 0.1 M acetic acid and the contacting step is carried out for 4 hours.

3. 10. The method of claim 1, wherein the composition comprises 0.5 M acetic acid and the contacting step is carried out for 4 hours.

4. 1. A method for reducing the microbial bioburden of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising 0.1 M to 1.0 M acetic acid, wherein said contacting reduces the amount of spore-forming bacteria in the chromatography matrix by at least 3 log １０ reduction of at least 5 log of the amount of gram-positive bacteria １０ and at least 5 log reduction in the amount of gram-negative bacteria. １０ wherein the composition does not comprise alcohol, guanidine hydrochloride or urea, and the method does not include contacting the chromatography matrix with a sodium hydroxide solution.

5. 5. The method of claim 4, wherein the contacting step is capable of reducing the amount of one or more of spore-forming bacteria, gram-positive bacteria, and gram-negative bacteria in the chromatography matrix to below the limit of detection as determined by an assay selected from the group consisting of: (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, and (5) a bacterial identification test.

6. 6. The method of claim 5, wherein the spore-forming bacterium is Bacillus pseudofirmus, the Gram-positive bacterium is Microbacterium spp., and the Gram-negative bacterium is Stenotrophomonas maltophilia.

7. The method of claim 1 , wherein the composition further comprises an acetate salt.

8. The method of claim 4 , wherein the composition further comprises an acetate salt.

9. The method of claim 1, wherein the composition has a pH between 2 and 3.

10. 10. The method of claim 1 or claim 4, wherein the contacting step is carried out at a temperature between 15°C and 30°C.

11. 11. The method of claim 10, wherein the contacting step is carried out at a temperature between 20°C and 25°C.

12. 10. The method of claim 1 or claim 4, wherein the chromatography matrix comprises a proteinaceous ligand linked to a support.

13. The method of claim 4, wherein the composition has a pH between 2 and 3.

14. 1. A method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting of 0.1 M acetic acid and 20% ethanol, wherein the contacting is performed for 4 hours.

15. The contacting step may be performed to increase the amount of spore-forming bacteria in the chromatography matrix by at least 3 log １０ reduction of at least 5 log of the amount of gram-positive bacteria １０ and at least 5 log reduction in the amount of gram-negative bacteria. １０ 15. The method of claim 14, wherein the method can result in one or more of the following reductions:

16. 16. The method of claim 15, wherein the contacting step is capable of reducing the amount of one or more of spore-forming bacteria, gram-positive bacteria, and gram-negative bacteria in the chromatography matrix to below the limit of detection as determined by an assay selected from the group consisting of: (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, and (5) a bacterial identification test.

17. 17. The method of claim 16, wherein the spore-forming bacterium is Bacillus pseudofirmus, the Gram-positive bacterium is Microbacterium spp., and the Gram-negative bacterium is Stenotrophomonas maltophilia.

18. The method of claim 14, wherein the composition has a pH between 2 and 3.

19. 15. The method of claim 14, wherein the contacting step is carried out at a temperature between 15°C and 30°C.

20. 20. The method of claim 19, wherein the contacting step is carried out at a temperature between 20°C and 25°C.

21. 15. The method of claim 14, wherein the chromatography matrix comprises a proteinaceous ligand linked to a support.

22. 1. A method for reducing microbial bioburden on a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising 0.1 M acetic acid and 20% ethanol, wherein the contacting is performed for 4 hours.

23. 23. The method of claim 22, wherein the composition further comprises benzyl alcohol.

24. 24. The method of claim 23, wherein the composition further comprises 1% to 2% benzyl alcohol.

25. 23. The method of claim 22, wherein the composition further comprises an acetate salt.

26. The contacting step may be performed to increase the amount of spore-forming bacteria in the chromatography matrix by at least 3 log １０ reduction of at least 5 log of the amount of gram-positive bacteria １０ and at least 5 log reduction in the amount of gram-negative bacteria. １０ 23. The method of claim 22, wherein the method can result in one or more of a reduction in:

27. 23. The method of claim 22, wherein the contacting step is capable of reducing the amount of one or more of spore-forming bacteria, gram-positive bacteria, and gram-negative bacteria in the chromatography matrix to below the limit of detection as determined by an assay selected from the group consisting of: (1) a biofiltration assay, (2) microscopic bacterial staining, (3) IR / FTIR spectroscopy, (4) a sterility test, and (5) a bacterial identification test.

28. 28. The method of claim 27, wherein the spore-forming bacterium is Bacillus pseudofirmus, the Gram-positive bacterium is Microbacterium spp., and the Gram-negative bacterium is Stenotrophomonas maltophilia.

29. 23. The method of claim 22, wherein the composition has a pH between 2 and 3, or the contacting step is carried out at a temperature between 15°C and 30°C.

30. 30. The method of claim 29, wherein the contacting step is carried out at a temperature between 20°C and 25°C.

31. 23. The method of claim 22, wherein the chromatography matrix comprises a proteinaceous ligand linked to a support.

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