Patent Description:
Antibody drugs are the most prevalent biopharmaceutical products. Affinity chromatography, e.g., performed with a natural or engineered staphylococcal protein A ligand, is widely used as a capture method in the antibody drug manufacturing process to remove impurities and contaminants. Protein A binds the Fc-region of antibodies, with protein A columns considered to be selective for purification of monoclonal antibodies. Affinity chromatography with protein A typically involves a clean-in-place (CIP) step to clean and remove impurities that are bound to the column, such as precipitated or denatured substances. CIP is normally performed with a sodium hydroxide solution.

In addition to cleaning, sodium hydroxide solutions, or phosphoric acid solutions with benzyl alcohol, are used to reduce the number of microbes in a protein A chromatography matrix or column. Bacteria from the media used to culture monoclonal-antibody producing cells, as well as associated host cell proteins and DNA, can quickly increase the bioburden of a protein A column during use. The bioburden increases as such bacteria and microbes accumulate on the column. Column performance generally degrades as bioburden increases. Signs of such degradation include decrease in product purity, column packing deterioration, and increased backpressure.

Managing and reducing microbial bioburden on protein A columns is important because protein A columns are very expensive, with the packing and unpacking of such affinity columns being labor intensive. To avoid expenses in replacing the protein A column or adding process steps upstream of protein A column purification, there is a need to find agents that remove a significant amount of bioburden from the column quickly without negatively affecting the structure and function of protein A, and that have few downstream effects.

The importance of microbial bioburden reduction is not limited to protein A chromatography matrices but encompasses other chromatography matrices with proteinaceous ligands coupled to a support as well as matrices not involving proteinaceous ligands such as, e.g., various ion exchange chromatography matrices, hydrophobic interaction chromatography (HIC) matrices, mixed mode chromatography matrices, size exclusion chromatography matrices, etc..

Microbial bioburden reduction of chromatography matrices is particularly important in the context of a good manufacturing practice (GMP), or current good manufacturing practice (CGMP). Such practices must provide consistency in manufacturing steps and quality of product so as to meet requirements of regulatory bodies, such as the U. Food and Drug Administration. GMP and CGMP require a high degree of predictability and standardization in manufacturing processes, particularly with ensuring purity of the manufactured therapeutic biomolecules used in human patients. With the labor involved in growing cultures to produce biomolecules, great expense arises when a failure occurs. An excessive bioburden can decrease column performance, which can interfere with purifying the product in a standardized and predictable way and possibly cause other failure points to be triggered.

The agents presently known in the art to reduce bioburden have negative downstream effects. For example, solutions based on sodium hydroxide, and those based on phosphoric acid with benzyl alcohol, may be effective to kill microorganisms but also tend to denature the proteinaceious ligands (e.g., protein A) thus negatively affecting their function.

Further, oxidants and other components of such solutions can remain with the purified monoclonal antibodies and further degrade them downstream.

Thus, those of ordinary skill in the art appreciate that it is difficult to reduce bioburden in chromatography matrices, especially in matrices with proteinaceous ligands, such as protein A chromatography matrices.

The following documents are also mentioned:.

As specified above, 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, and especially those involving 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.

Accordingly, the present invention provides a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition, wherein said composition consists of:.

wherein the contacting step is performed for at least <NUM> hour.

In one aspect, the method for microbial bioburden reduction of a chromatography matrix comprises contacting the chromatography matrix with a composition consisting of from about <NUM> to about <NUM> acetic acid, wherein the contacting step is performed for at least about <NUM> hours.

In a related aspect, the method for microbial bioburden reduction of a chromatography matrix comprises contacting the chromatography matrix with a composition consisting of from about <NUM> to about <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol, wherein the contacting step is performed for at least about <NUM> hour.

In a related aspect, the method for microbial bioburden reduction of a chromatography matrix results in one or more of a reduction in the amount of spore forming bacteria by at least <NUM> log<NUM>, a reduction in the amount of gram positive bacteria by at least <NUM> log<NUM>, and a reduction in the amount of gram negative bacteria by at least <NUM> log<NUM> in the chromatography matrix.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising from about <NUM> to about <NUM> urea, wherein the contacting step is performed for at least about <NUM> minutes.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising from about <NUM> to about <NUM> urea, wherein the contacting step results in one or more of a reduction in the amount of spore forming bacteria by at least <NUM> log<NUM>, a reduction in the amount of gram positive bacteria by at least <NUM> log<NUM>, and a reduction in the amount of gram negative bacteria by at least <NUM> log<NUM>, in the chromatography matrix.

In one embodiment, the invention provides a method for microbial bioburden reduction of MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> acetic acid, wherein the contacting step is performed for at least about <NUM> hours.

In another embodiment, the invention provides a method for microbial bioburden reduction of MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> acetic acid and about <NUM>% ethanol, wherein the contacting step is performed for at least about <NUM> hours.

Also disclosed is a method for microbial bioburden reduction of MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> urea, wherein the contacting step is performed for at least about <NUM> hour.

Also disclosed is a method for microbial bioburden reduction of MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> urea and about <NUM>% ethanol, wherein the contacting step is performed for at least about <NUM> hour.

In one embodiment, the invention provides a method for reducing microbial load before applying a composition comprising a pharmaceutical agent for purification comprising (a) providing a chromatography matrix; (b) performing any of the above methods of the invention; and (c) applying the composition comprising the pharmaceutical agent to the chromatography matrix.

These and other aspects of the present invention will be apparent to those of ordinary skill in the art in the following description, claims and drawings.

The present invention provides a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition, wherein said composition consists of:.

In one aspect, the present invention provides a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting of from about <NUM> to about <NUM> acetic acid, wherein the contacting step is performed for at least about <NUM> hours. In various embodiments, the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours. In one embodiment, the contacting step is performed for at least about <NUM> hours. In various embodiments, the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours. In one embodiment, the composition consists of about <NUM> acetic acid and the contacting step is performed for at least about <NUM> hours. In one embodiment, the composition consists of about <NUM> acetic acid and the contacting step is performed for at least about <NUM> hours. In various embodiments, the composition consists of about <NUM> acetic acid and the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours.

In one embodiment, the composition consists of <NUM> to <NUM> acetic acid and <NUM>% benzyl alcohol. In one specific embodiment, the composition consists essentially of about <NUM> acetic acid and about <NUM>% ethanol.

In one embodiment, the invention provides a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> to about <NUM> acetic acid, wherein the contacting step is performed for at least about <NUM> hours. In one specific embodiment, the contacting step is performed for at least about <NUM> hours. In various embodiments, the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours. In one specific embodiment, the composition consists essentially of about <NUM> acetic acid and the contacting step is performed for at least about <NUM> hours. In another specific embodiment, the composition consists essentially of about <NUM> acetic acid and the contacting step is performed for at least about <NUM> hours. In various embodiments, the composition consists of about <NUM> acetic acid and the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours.

In a related aspect, the invention provides a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting of from about <NUM> to about <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol, wherein the contacting step is performed for at least about <NUM> hour. In various embodiments, the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours. In one embodiment, the composition consists of about <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol, wherein the contacting step is performed for at least about <NUM> hour. In various embodiments, the composition consists of about <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol, wherein the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours.

In one specific embodiment, the composition consists essentially of about <NUM> acetic acid and about <NUM>% ethanol.

In one embodiment, the invention provides a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> to about <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol, wherein the contacting step is performed for at least about <NUM> hour. In various embodiments, the composition consists essentially of about <NUM> to about <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol, wherein the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours. In one specific embodiment, the composition consists essentially of about <NUM> acetic acid and the contacting step is performed for at least about <NUM> hour. In various embodiments, the composition consists essentially of about <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol, wherein the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours.

In a related aspect, the invention provides a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising from about <NUM> to about <NUM> acetic acid as described herein, wherein the contacting step results in one or more of a reduction in the amount of spore forming bacteria (e.g., Bacillus pseudofirmus) by at least <NUM> log<NUM>, a reduction in the amount of gram positive bacteria (e.g., Microbacterium spp. ) by at least <NUM> log<NUM>, and a reduction in the amount of gram negative bacteria (e.g., Stenotrophomonas maltophilia) by at least <NUM> log<NUM> in the chromatography matrix. In one specific 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, (<NUM>) a biofiltration assay, (<NUM>) microscopic bacterial staining, (<NUM>) IR/FTIR spectroscopy method, (<NUM>) a sterility test, or (<NUM>) a bacterial identification test. In various embodiments, the contacting step is performed for at least about <NUM> hour, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, for at least about <NUM> hours, for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours.

In one embodiment, the composition further comprises an alcohol, which may be <NUM>% ethanol or from about <NUM>% to about <NUM>% benzyl alcohol. In one specific embodiment, the composition consists essentially of about <NUM> acetic acid and about <NUM>% ethanol.

In one embodiment, the invention provides a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> to about <NUM> acetic acid as described herein, wherein the contacting step results in one or more of a reduction in the amount of spore forming bacteria (e.g., Bacillus pseudofirmus) by at least <NUM> log<NUM>, a reduction in the amount of gram positive bacteria (e.g., Microbacterium spp. ) by at least <NUM> log<NUM>, and a reduction in the amount of gram negative bacteria (e.g., Stenotrophomonas maltophilia) by at least <NUM> log<NUM>, in the chromatography matrix. In one specific 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, (<NUM>) a biofiltration assay, (<NUM>) microscopic bacterial staining, (<NUM>) IR/FTIR spectroscopy method, (<NUM>) a sterility test, or (<NUM>) a bacterial identification test. In various embodiments, the contacting step is performed for at least about <NUM> hour, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, at least about <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours.

In one embodiment of any of the above methods of the invention, the composition further comprises an acetate salt.

In one embodiment of any of the above methods of the invention, the composition has pH between about <NUM> and about <NUM>.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising from about <NUM> to about <NUM> urea, wherein the contacting step is performed for at least about <NUM> minutes. In one instance, the contacting step is performed for at least about <NUM> hour. In one embodiment, the composition comprises about <NUM> urea.

In one instance, the composition further comprises an alcohol, either about <NUM>% ethanol or from about <NUM>% to about <NUM>% benzyl alcohol. In one specific instance, the composition consists essentially of about <NUM> urea and about <NUM>% ethanol.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> to about <NUM> urea, wherein the contacting step is performed for at least about <NUM> minutes. In one specific instance, the contacting step is performed for at least about <NUM> hour. In one specific instance, the composition consists essentially of <NUM> urea.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising from about <NUM> to about <NUM> urea, wherein the contacting step results in one or more of a reduction in the amount of spore forming bacteria (e.g., Bacillus pseudofirmus) by at least <NUM> log<NUM>, a reduction in the amount of gram positive bacteria (e.g., Microbacterium spp. ) by at least <NUM> log<NUM>, and a reduction in the amount of gram negative bacteria (e.g., Stenotrophomonas maltophilia) by at least <NUM> log<NUM>, in the chromatography matrix. In one specific instance, 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, (<NUM>) a biofiltration assay, (<NUM>) microscopic bacterial staining, (<NUM>) IR/FTIR spectroscopy method, (<NUM>) a sterility test, or (<NUM>) a bacterial identification test.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> to about <NUM> urea, wherein the contacting step results in one or more of a reduction in the amount of spore forming bacteria (e.g., Bacillus pseudofirmus) by at least <NUM> log<NUM>, a reduction in the amount of gram positive bacteria (e.g., Microbacterium spp. ) by at least <NUM> log<NUM>, and a reduction in the amount of gram negative bacteria (e.g., Stenotrophomonas maltophilia) by at least <NUM> log<NUM>, in the chromatography matrix. In one specific instance, 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, (<NUM>) a biofiltration assay, (<NUM>) microscopic bacterial staining, (<NUM>) IR/FTIR spectroscopy method, (<NUM>) a sterility test, or (<NUM>) a bacterial identification test.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising from about <NUM> to about <NUM> guanidine hydrochloride, wherein the contacting step is performed for at least about <NUM> minutes. In one instance, the contacting step is performed for at least about <NUM> hour. In one instance, the composition comprises about <NUM> guanidine hydrochloride.

In one instance, the composition further comprises an alcohol, either about <NUM>% ethanol and from about <NUM>% to about <NUM>% benzyl alcohol. In one specific instance, the composition consists essentially of about <NUM> guanidine hydrochloride and about <NUM>% ethanol.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> to about <NUM> guanidine hydrochloride, wherein the contacting step is performed for at least about <NUM> minutes. In one specific instance, the contacting step is performed for at least about <NUM> hour. In one specific instance, the composition consists essentially of about <NUM> guanidine hydrochloride.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising from about <NUM> to about <NUM> guanidine hydrochloride, wherein the contacting step results in one or more of a reduction in the amount of spore forming bacteria (e.g., Bacillus pseudofirmus) by at least <NUM> log<NUM>, a reduction in the amount of gram positive bacteria (e.g., Microbacterium spp. ) by at least <NUM> log<NUM>, and a reduction in the amount of gram negative bacteria (e.g., Stenotrophomonas maltophilia) by at least <NUM> log<NUM>, in the chromatography matrix. In one specific instance, 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, (<NUM>) a biofiltration assay, (<NUM>) microscopic bacterial staining, (<NUM>) IR/FTIR spectroscopy method, (<NUM>) a sterility test, or (<NUM>) a bacterial identification test.

In one instance, the composition further comprises an alcohol, either about <NUM>% ethanol or from about <NUM>% to about <NUM>% benzyl alcohol. In one specific instance, the composition consists essentially of about <NUM> guanidine hydrochloride and about <NUM>% ethanol.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> to about <NUM> guanidine hydrochloride, wherein the contacting step results in one or more of a reduction in the amount of spore forming bacteria (e.g., Bacillus pseudofirmus) by at least <NUM> log<NUM>, a reduction in the amount of gram positive bacteria (e.g., Microbacterium spp. ) by at least <NUM> log<NUM>, and a reduction in the amount of gram negative bacteria (e.g., Stenotrophomonas maltophilia) by at least <NUM> log<NUM>, in the chromatography matrix. In one specific instance, 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, (<NUM>) a biofiltration assay, (<NUM>) microscopic bacterial staining, (<NUM>) IR/FTIR spectroscopy method, (<NUM>) a sterility test, or (<NUM>) a bacterial identification test.

Also disclosed is a method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition comprising from about <NUM> to about <NUM> acetic acid and (i) from about <NUM> to about <NUM> urea and/or (ii) from about <NUM> to about <NUM> guanidine hydrochloride, wherein the contacting step is performed for at least about <NUM> hour. In one specific instance, the composition further comprises an alcohol. Alcohols that can be used are about <NUM>% ethanol and from about <NUM>% to about <NUM>% benzyl alcohol.

In one embodiment of any of the above methods of the invention, the contacting step is conducted at a temperature between <NUM> and <NUM>. In one specific embodiment, the contacting step is conducted at a temperature between <NUM> and <NUM>.

In one embodiment of any of the above methods of the invention, the composition is substantially free of oxidants.

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

In one embodiment of any of the above methods of the invention, the composition does not comprise a peroxide.

In one embodiment of any of the above methods of the invention, the composition does not comprise NaOH.

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

In one embodiment of any of the above methods of the invention, the chromatography matrix is packed in a chromatography column. In one specific embodiment, the chromatography column has an inner diameter between <NUM> and <NUM> and a bed height between <NUM> and <NUM>. In one specific embodiment, the chromatography column has an inner diameter of about <NUM> and a bed height of about <NUM>. In one specific embodiment, the chromatography column has an inner diameter between <NUM> and <NUM> meters and a bed height between <NUM> and <NUM>. In one specific embodiment, the chromatography column has an inner diameter of about <NUM> meters and a bed height of about <NUM>.

In one embodiment of any of the above methods of the invention, the chromatography matrix comprises a proteinaceous ligand coupled to a support. In one specific embodiment, the proteinaceous ligand comprises one or more immunoglobulin binding domains. In one specific embodiment, the proteinaceous ligand is Protein A or a fragment or a derivative thereof. In one specific 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 specific 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 <NUM> Fast Flow, Protein A Sepharose <NUM> Fast Flow, Protein A Mag Sepharose, Protein A Sepharose CL-4B, rmp Protein A Sepharose Fast Flow, rProtein A Sepharose <NUM> 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 <NUM> Fast Flow. In one specific 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 specific embodiment, the proteinaceous ligand is not measurably denatured after the method is performed.

In another embodiment, the invention provides a method for microbial bioburden reduction of MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> acetic acid and about <NUM>% ethanol, wherein the contacting step is performed for at least about <NUM> hours. In various embodiments, the contacting step is performed for at least about <NUM> hour, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, at least about <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours.

Also disclosed is a method for microbial bioburden reduction of MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> guanidine hydrochloride, wherein the contacting step is performed for at least about <NUM> hour.

Also disclosed is a method for microbial bioburden reduction of MabSelect™ Xtra chromatography matrix, comprising contacting the chromatography matrix with a composition consisting essentially of about <NUM> guanidine hydrochloride and about <NUM>% ethanol, wherein the contacting step is performed for at least about <NUM> hour.

It is to be understood that this invention is not limited to 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, since the scope of the present invention is defined by the claims.

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, a reference to "a method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.

The terms "about" and "approximately" are used interchangeably to mean within a statistically meaningful range of a value. Such a range can be within <NUM>%, more preferably within <NUM>%, still more preferably within <NUM>%, and even more preferably within <NUM>% of a given value or range.

As used herein, the terms "microbe" or "microorganism" encompass prokaryotic organisms including bacteria and archaea, and eukaryotic organisms, including fungi. These terms encompass both live cells and spores (for spore-forming organisms) as well as microbial products such as, e.g., endotoxins.

The terms "microbial bioburden reduction" and "microbe bioburden reduction" as used herein combines killing of microbes and some interference with the interaction between a microbe and a chromatography matrix. The reduction of microbial bioburden according to the present invention is not the same as any previously disclosed sanitization process that was designed to kill essentially all, or even at least <NUM>%, of the microbes that might exist on a chromatography column or matrix.

Rather, the present invention includes methods capable of killing less than <NUM>%, less than <NUM>%, less than <NUM>%, or <NUM>-<NUM>% of non-spore forming microbes. In such an embodiment, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>% or less than <NUM>%, or <NUM>-<NUM>% of the spore forming microbes, such as Bacillus, on a column would be killed by the methods of the present invention.

Even though the present invention includes methods that do not kill all of the bacteria on a column, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, about <NUM>%, about <NUM>% or about <NUM>% of the viable microbes capable of being detected by the biofiltration assay, or any other microbiological assay disclosed herein and known in the art are removed from the chromatography matrix after treatment. In some embodiments of the present invention, the level of microbial bioburden is reduced below GMP-acceptable levels in a pre-use flush sample, in an equilibration sample, in a load sample, or in all GMP samples from the load taken subsequently during that run. Even without killing all of the microbes, the present invention can reduce a microbial bioburden to below GMP alert levels because of interference between the interaction between a microbe and a chromatography matrix that also occurs during methods of the claimed invention. This interference may involve, but is not limited to mechanisms such as reducing the affinity, binding, or any other interaction between the microbe and the chromatography matrix. Such mechanisms are similar to the common strip step used on a chromatography column that impurities such as host cell proteins and DNA from a column. Accordingly, this interference could be detected after use of a method of the present invention, which does not kill all of the microbes, when the microbial bioburden has been reduced to levels consistent with the requirements of GMP production of a biologic pharmaceutical drug. By using one of the methods of the present invention that is not designed to kill all of the microbes on a column, the reagents are not necessarily as harsh and therefore are more favorable for approval from regulatory agencies, e.g., FDA or EMA, that have to approve the process and the product for market. Preferably, the same strip buffer components or at least the active agents that are used on a chromatography column during GMP production of a biologic pharmaceutical (acetic acid is common) can be used to reduce microbial bioburden when applied at a higher concentration (for example, 10X, 15X, or 20X) and for a longer contract time (for example, 3X, 4X, or 5X). Further, it may be more efficient for the same strip buffer to be used at a higher concentration for a longer time to reduce a microbial bioburden.

Any of the methods, aspects, and embodiments described herein can be used as part of a good manufacturing practice (GMP), or current good manufacturing practice (CGMP). Such practices must provide consistency in manufacturing steps and quality of product so as to meet requirements of regulatory bodies, such as the U. Food and Drug Administration. GMP and CGMP typically require a high degree of predictability and standardization in manufacturing processes, particularly with ensuring purity of the manufactured therapeutic biomoleculesused in human patients. There are many failure points during a GMP or CGMP, in which a parameter is detected that requires stopping the process and/or scrapping the production batch. With the labor involved in growing cultures to produce biomolecules, great expense arises when a failure occurs.

If the bioburden or microbial load gets too high in a chromatography column, or matrix used in separation, various unpredictable and/or undesirable effects can arise. A failure point could be triggered so as to stop the process so that product contaminated with microbes is identified and not further produced. To prevent failure points, detection of a bioburden of at least <NUM> CFU per <NUM> can trigger an alert. A bioburden of at least <NUM> CFU per <NUM> can trigger action, which can include undertaking one or more of the methods or embodiments described herein, alone or in combination, to reduce the bioburden.

For example, microbes can be introduced into the product, rendering it unacceptable for therapeutic use. An excessive bioburden can also decrease column performance, which can interfere with purifying the product in a standardized and predictable way and possibly cause other failure points to be triggered. Therefore, it is desirable to use the herein described aspects and embodiments of reducing bioburden preemptively to ensure compliance with a GMP or CGMP, and to minimize failure point triggering, and the associated troubleshooting and downtime.

Any of the aspects or embodiments described herein may further comprise a step of applying a small molecule-containing or biomolecule-containing (e.g., monoclonal antibody-containing) preparation for purification after the contacting step. A method for reducing microbial load before applying a small molecule-containing or biomolecule-containing (e.g., monoclonal antibody-containing) preparation for purification can comprise the steps of any of the methods of microbe bioburden reduction described herein. Alternatively, a method for purifying a biomolecule can comprise conducting the steps of any methods described herein and then applying a preparation comprising the biomolecule to the chromatography matrix.

Any of the aspects or embodiments described herein may be performed after the chromatography matrix is removed from storage but before application of a drug-containing, biomolecule-containing, or monoclonal antibody-containing preparation for purification. During long term storage, a small amount of bacteria present in a chromatography matrix or column may grow and increase bioburden.

Any of the aspects or embodiments described herein may be used as part of an aseptic technique, or to support an aseptic technique. The resulting reduction in bioburden on the chromatography matrix can be sufficient for an aseptic technique, or can be used before or after other steps in an aseptic technique. The described methods of reducing bioburden can reduce the odds that an aseptic technique failure point would be triggered and can be used in response to an impending failure point trigger.

In another aspect, a MabSelect™ Xtra chromatography matrix undergoes microbe bioburden reduction by contacting the matrix with a composition consisting essentially of about <NUM> acetic acid, for at least <NUM> hours. In various embodiments, the contacting step is performed for <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, <NUM> to <NUM> hours, or <NUM> to <NUM> hours. In another aspect, a MabSelect™ Xtra chromatography matrix undergoes microbe bioburden reduction by contacting the matrix with a composition consisting essentially of <NUM> acetic acid and about <NUM>% ethanol, for at least <NUM> hours.

Various chromatography matrices may be used. The chromatography matrix may comprise a proteinaceous ligand coupled to a support. The proteinaceous ligand, in turn, may comprise one or more immunoglobulin binding domains. Other useful chromatography matrices include, without limitation, various ion exchange chromatography matrices, hydrophobic interaction chromatography (HIC) matrices, mixed mode chromatography matrices, and size exclusion chromatography matrices.

The proteinaceous ligand of the chromatography matrix may be Protein A or a fragment or a 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, Streptococcus Protein A.

Staphylococcus Protein A may be found on the cell wall of the bacteria Staphylococcus aureus. Protein A may bind antibodies in the Fc region, between the CH2 and CH3 domains. Protein A may be cultured in Staphylococcus aureus or produced recombinantly in other bacteria, for example, E. coli or Brevibacillus. Fragments or derivatives of Staphylococcus Protein A may also bind to antibodies in the Fc region, between the CH2 and CH3 domains.

Peptostreptococcus Protein L may be found on the surface of Peptostreptococcus magnus and can bind to antibodies via an interaction with the antibody light chain. Unlike Protein A, Protein L may bind to single chain variable fragments (scFv) and Fab fragments. Fragments or derivatives of Protein L may also bind to the light chain of antibodies, single chain variable fragments (scFv) and Fab fragments.

Streptococcus Protein G may be found on the cell wall of group G Streptococcal strains. Protein G may bind antibodies in the Fab and Fc regions. Protein G may be produced recombinantly in other bacteria, for example, E. Fragments or derivatives of Protein G may also bind to antibodies in the Fab and Fc regions.

The chromatography matrix may 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 purifications is packed with MabSelect SuRe™, is about <NUM> in diameter, and about <NUM> long. A larger column, such as <NUM> x <NUM>, can be used for manufacturing scale purifications.

More generally, the methods of the present invention can be used for chromatography columns of various sizes, including laboratory scale, large process scale, and very large process scale. In some embodiments, the chromatography column may have an inner diameter between <NUM> to <NUM> and a bed height of between <NUM> to <NUM> (e.g., <NUM>). The inner diameter may be between <NUM> to <NUM>, alternatively <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, or about <NUM>. In some embodiments, the chromatography column may have an inner diameter of between <NUM> to <NUM> meters (e.g., <NUM>, <NUM>, <NUM> meter, <NUM> meters, or <NUM> meters). The chromatography column may have a bed height of between <NUM> to <NUM> (e.g., <NUM>).

Exemplary chromatography matrices include MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, MabSelect™ SuRe pcc, MabSelect™ SuRe LX, nProtein A Sepharose <NUM> Fast Flow, Protein A Sepharose <NUM> Fast Flow, Protein A Mag Sepharose, Protein A Sepharose CL-4B, rmp Protein A Sepharose Fast Flow, rProtein A Sepharose <NUM> 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 <NUM> Fast Flow.

MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, and MabSelect™ SuRe LX have a recombinant protein A ligand, produced in E. , that is attached to a highly cross-linked agarose matrix.

Microbe bioburden reduction can restore performance of a chromatography matrix so that it can be used for additional purification rather than being replaced. Thus, in any of the methods described herein, microbe bioburden reduction can be conducted after the chromatography matrix has been used. In such scenarios, bacteria and other microorganisms that may be introduced into the chromatography matrix from cell culture broths comprising a monoclonal antibody of interest, can be removed. Substantial expense can be saved when using chromatography matrices comprised of proteinaceous ligands, such as Protein A.

Furthermore, using acetic acid-containing solutions instead of commonly used sodium hydroxide-containing solutions 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, e.g., by assaying the purity and abundance of the product that elutes from the matrix and by assaying residual elution of a component of the matrix (e.g., Protein A). For example, if the proteinaceous ligand is Protein A, the purity and abundance of monoclonal antibodies would be assayed. Exemplary assays include size exclusion chromatography (e.g., SE-HPLC and SE-UPLC), capillary electrophoresis (e.g., CE-SDS), capillary isoelectric focusing (iCIEF) that can optionally include whole column imaging. Also, residual Protein A from the column can be measured (e.g., by an assay comprising ELISA).

Microbe bioburden reduction undertaken according to the methods described herein can be a cost-effective way to maintain performance of a column comprising Protein A or other proteinaceous ligands, by removing bacteria without damaging the Protein A. For example, exposure of a Protein A-containing matrix to an acetic acid-comprising solution (e.g., <NUM> acetic acid) for <NUM> or <NUM> hours does not lead to any statistically significant loss of performance of a column comprising Protein A. See, e.g., Example <NUM>, Table <NUM> and <FIG>. For example, there is no statistically significant loss of purity of eluted monoclonal antibody by size exclusion chromatography, capillary electrophoresis under reducing or non-reducing conditions, or by capillary isoelectric focusing.

Microbe bioburden reduction according to 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 can interfere with the affinity between bacteria and the proteinaceous ligand, e.g., protein A. A microbe-contaminated matrix or column can have microbes reduced according to methods described herein by disrupting interactions between microbial organisms and the resin. The bacteria would tend to remain in the acetic acid-containing solution. Removal of the bacteria may be further enhanced by repeating the contacting steps with acetic acid or undertaking additional flushing of the chromatography matrix with acetic acid-containing solutions.

In some embodiments, the method removes spore-forming bacteria from the chromatography matrix. Spore-forming bacteria have the ability to switch to endospore form. Endospore form is a stripped-down, dormant form to which the bacterium can reduce itself. Endospore formation is usually triggered by a lack of nutrients or harsh conditions, such as acidic or basic environment. Endospores enable bacteria to lie dormant for extended periods even in unfavorable conditions. When the environment becomes more favorable, the endospore can reactivate to the vegetative state. Examples of bacteria that can form endospores include Bacillus and Clostridium. These spore-forming bacteria are thought to be able to form endospore under normal operating conditions in chromatography purification processes due to the existence 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 and those include but not limited to microscopic bacterial staining method, IR/FTIR spectroscopy method, sterility tests, and bacterial identification tests (e.g., biochemical reactions, <NUM> rRNA sequence determination, or taxa-specific sequence determinations).

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 may 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 limit of detection of an assay selected from the group consisting of (<NUM>) a biofiltration assay, (<NUM>) microscopic bacterial staining, (<NUM>) IR/FTIR spectroscopy method, (<NUM>) a sterility test, and (<NUM>) a bacterial identification test (e.g., a biochemical reaction, <NUM> rRNA sequence determination, or a taxa-specific sequence determination). Biofiltration assays are described in the U. Pharmacopeia Chapter <<NUM>>, titled "Sterility Tests. " In biofiltration assays, elutions from the column are passed through a filter that selectively binds bacteria. The filter is then plated on agar with appropriate media to grow bacteria, incubated, and the bacteria counted. Dilutions of the column elution can be performed as needed.

Microscopic bacteria staining is described in the U. Pharmacopeia Chapter <<NUM>>, titled "Microbial examination of nonsterile products: microbial enumeration tests". IR/FTIR spectroscopy methods are described in <NPL>. ) Microbiological tests are described in <NPL>.

The concentration of acetic acid in the composition is from about <NUM> to about <NUM>. In some embodiments, the concentration of acetic acid in the composition is from about <NUM> to about <NUM>. In some embodiments, the concentration of acetic acid in the composition is from about <NUM> to about <NUM>. In some embodiments, the concentration of acetic acid in the composition is about <NUM>. In some embodiments, the concentration of acetic acid in the composition is from about <NUM> to about <NUM>. In some embodiments, the concentration of acetic acid in the composition is about <NUM>. In particular, the composition consists of: (a) <NUM> to <NUM> acetic acid; or (b) <NUM> to <NUM> acetic acid and benzyl alcohol; or (c) <NUM> acetic acid and <NUM> % ethanol; or (d) <NUM> to <NUM> acetic acid and <NUM>% benzyl alcohol; or (e) <NUM> to <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol.

Also disclosed is a composition wherein the concentration of urea in the composition is from about <NUM> to about <NUM>. In some instances, the concentration of urea in the composition is from about <NUM> to about <NUM>. In some instances, the concentration of urea in the composition is from about <NUM> to about <NUM>. In some instances, the concentration of urea in the composition is about <NUM>.

Also disclosed is a composition wherein the concentration of guanidine hydrochloride in the composition is from about <NUM> to about <NUM>. In some instances, the concentration of guanidine hydrochloride in the composition is from about <NUM> to about <NUM>. In some instances, the concentration of guanidine hydrochloride in the composition is from about <NUM> to about <NUM>. In some instances, the concentration of guanidine hydrochloride in the composition is about <NUM>.

In some embodiments, the pH of the solution is at least <NUM>. The pH may be from <NUM> to <NUM>. The pH may be from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, or about <NUM>. The pH may be about any of the following: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,or <NUM>.

In some embodiments, the composition comprises <NUM>% ethanol. In some embodiments, the composition consists essentially of about <NUM> acetic acid and about <NUM>% ethanol. There are advantages to using ethanol, while and minimizing the amount of benzyl alcohol used, or avoiding benzyl alcohol due to toxicity in humans, or adverse effects in humans, that may arise from presence of benzyl alcohol.

In some embodiments, the composition further comprises an acetate salt. The acetate salt may serve as a buffer for compositions comprising acetic acid. For example, the composition may comprise sodium acetate in addition to acetic acid such that the composition is a buffer. The composition may comprise from <NUM> sodium acetate to <NUM> sodium acetate, <NUM> to <NUM> sodium acetate, <NUM> to <NUM> sodium acetate, about <NUM> sodium acetate, or <NUM> sodium acetate. Buffering the acetic acid with sodium acetate or another acetate salt may be effective to maintain the pH of the solution in the chromatography matrix. The pH may be at least <NUM>, from about <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, or about <NUM>. The pH may be about any of the following: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,or <NUM>.

In some embodiments in which the composition comprises acetic acid, the contacting step is performed for at least one hour, alternatively for <NUM> to <NUM> hours, alternatively for at least <NUM> hours. In some embodiments, the contacting step is performed for <NUM> to <NUM> hours. In some embodiments, the contacting step is performed for at least <NUM> hours. In various embodiments, the contacting step is performed for <NUM> minutes to <NUM> hours, <NUM> hours to <NUM> hours, <NUM> hours to <NUM> hours, <NUM> hours to <NUM> hours, <NUM> minutes to <NUM> hours, <NUM> hours to <NUM> hours, <NUM> hours to <NUM> hours, <NUM> hours to <NUM> hours, <NUM> hours to <NUM> hours, <NUM> hours to <NUM> hours, or <NUM> hours to <NUM> hours.

In some instances in which a composition comprises urea, the contacting step is performed for at least <NUM> minutes, alternatively at least one hour, alternatively for at least two hours. In some instances, the contacting step is performed for one to two hours. In some instances, the contacting step is performed for at least two hours. In various instances, the contacting step is performed for <NUM> minutes to <NUM> hours, <NUM> hour to <NUM> hours, or <NUM> minutes to <NUM> hours.

In some instances in which the composition comprises guanidine hydrochloride, the contacting step is performed for at least <NUM> minutes, alternatively at least one hour, alternatively for at least two hours. In some instances, the contacting step is performed for one to two hours. In some instances, the contacting step is performed for at least two hours. In various embodiments, the contacting step is performed for <NUM> minutes to <NUM> hours, <NUM> hour to <NUM> hours, or <NUM> minutes to <NUM> hours.

In some instances in which the composition comprises urea or guanidine hydrochloride, the contacting step is performed for at least <NUM> minutes, alternatively for at least about <NUM> hour, alternatively for <NUM> to <NUM> hours, alternatively for at least <NUM> hours.

In some embodiments, the contacting step can be repeated. For example, the contacting step may be performed for about one hour and then repeated multiple times. In some embodiments, the contacting step is repeated <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> times. By repeating the microbe bioburden reduction, the column may be exposed to additional acetic acid, which can result in additional disruption of bacteria and microorganisms from the chromatography matrix. Repeating the contacting step can lead to more flushing, or removal, of bacteria and microorganisms from the chromatography matrix, e.g., a Protein A ligand, and the column. The microbe bioburden reduction process may reduce bioburden more when conducted multiple times in succession.

The effectiveness of any of the microbe bioburden reduction methods described herein can be monitored by using any number of bioburden assays. One such assay is a filtration assay where a volume of eluate is passed through a filter membrane that traps the bacteria present in the eluate. The bacteria titer can be determined by placing the filter membrane on agar plates such that the bacteria on the filter membrane form colonies on the plates. The agar plates may contain trypticase soy agar (TSA). Culturing may occur for <NUM> to <NUM> days at temperatures between <NUM> and <NUM>. The number of colonies is then counted. If there are too many colonies formed, dilutions of the eluate can be undertaken.

In some embodiments, there is a reduction in the amount of spore forming bacteria by at least <NUM> log<NUM>. For example, when a chromatography matrix is contaminated with Bacillus pseudofirmus, contacting such matrix with an <NUM> urea solution, <NUM> urea / <NUM>% ethanol solution, a <NUM> guanidine hydrochloride solution, or a <NUM> guanidine hydrochloride / <NUM>% ethanol solution, for at least one hour can reduce the number of Bacillus pseudofirmus by at least <NUM> log<NUM>.

In some embodiments, the binding capacity of the chromatography matrix is preserved over <NUM> or more cycles when the method for microbial bioburden reduction is undertaken. In other embodiments, the binding capacity is preserved over <NUM> or more cycles. In some other embodiments, the binding capacity is preserved over <NUM> or more cycles. In some embodiments, the binding capacity is preserved over <NUM> or more cycles.

In some embodiments, there is no substantial degradation of the chromatography matrix during the contacting step or over multiple contacting steps wherein the exposure to the composition is for at least <NUM> hours, at least <NUM> hours, at least <NUM> hours, at least <NUM> hours, at least <NUM> hours, or at least <NUM> hours. In some embodiments, there is no measurable degradation of the chromatography matrix during the contacting step or over multiple contacting steps wherein the exposure to the composition is for at least <NUM> hours, at least <NUM> hours, at least <NUM> hours, at least <NUM> hours, at least <NUM> hours, or at least <NUM> hours. In some embodiments, the degradation of the chromatography matrix is measured by the protein quality of a protein that binds to the matrix (e.g., a monoclonal antibody that binds to a Protein A matrix).

In some embodiments, the proteinaceous ligand is not measurably denatured. Denaturation can be determined using functional assays such as, e.g., measuring column performance, product yield and/or quality, leachable proteinaceous ligand from the matrix, denaturation of the proteinaceous ligand, etc..

In some embodiments, there is no measurable leaching of the proteinaceous ligand, or Protein A during the contacting step or over 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., <NUM> acetic acid) for <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours or <NUM> hours. Measurement of leaching of Protein A is one such exemplary assay. Denaturation of Protein A can change its confirmation such that it no longer interacts with beads or other solid phase support in the chromatography matrix. The denatured Protein A then would leach off of the beads or solid support and enter the liquid phase. Detection of the proteinaceous ligand, or Protein A, in the eluate or liquid phase of the affinity column can thus indicate measurable denaturation. Denaturation of other proteinaceous ligands, besides Protein A, may also lead to their leaching from the chromatography matrix. In some embodiments, the measurement of leaching of Protein A and/or other proteinaceous ligands comprises ELISA. An exemplary assay is described in Example <NUM> and <FIG>, <FIG> and <FIG>.

The following example describes the various aspects and embodiments described above. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of any of the disclosure or of any exemplified term. Likewise, any claimed subject matter is not limited to any particular preferred embodiments described here.

Reduction of microbes in three packed <NUM> MabSelect™ Xtra columns was assessed by measuring the bioburden after the column is spiked with a particular bacterium and then again after reducing microbes with a <NUM> acetic acid solution.

First, the column was flushed with two column volumes of water for injection (WFI). A <NUM> sample was collected and served as a negative control with respect to the amount of bacteria in the column.

In each of the three MabSelect™ Xtra columns, a representative microorganism was added to the column by adding the microorganism to WFI to form spiked WFI, wherein the microorganism is present in a titer of approximately <NUM><NUM> cfu/mL. One column was spiked with spore-forming bacteria, particularly Bacillus psuedofirmus. One column was spiked with Gram positive bacteria, particularly Microbacterium spp. The third column was spiked with Gram negative bacteria, particularly Stenotrophomonas maltophilia.

Each spiked WFI was then loaded onto each column. The columns were then flushed with <NUM> column volumes of WFI at <NUM>/hour, which were collected. Each column was held for one hour and then flushed again with the spiked WFI. A <NUM> sample was collected from each column as a positive control, with the amount of each of the Gram negative bacteria, Gram positive bacteria and spore-forming bacteria in the sample subsequently assayed.

Two column volumes of a microbe reducing solution of <NUM> acetic acid were then applied to each column at <NUM>/hour. Each column was held for one hour and then flushed with two column volumes of WFI at <NUM>/hour. For Microbacterium spp. and Stenotrophomonas maltophilia, <NUM> column volumes of WFI were flushed through the column, then <NUM> column volumes of effluent were collected. For Bacillus psuedofirmus, <NUM> column volumes of an equilibration buffer (<NUM> Sodium Phosphate, <NUM> Sodium Chloride, pH7. <NUM>) were flushed through the column, then <NUM> column volumes of effluent are collected.

The above experiment was repeated, with the columns held for four hours instead of one hour after the <NUM> acetic acid microbe reducing solution was applied to the column.

A filtration-based bioburden assay was performed. Agar plates are prepared that have TSA media.

All of the above samples from the chromatography column were placed into a conical tube, inverted <NUM> times, and then passed through a filter manifold connected to sterile single use filter funnels having a <NUM> micron filter membrane, or a Milliflex® plus pump with a Milliflex® filter funnel unit having a <NUM> micron filter membrane. Sterile technique was used in handling the filter manifold or filter funnel unit so as not to introduce additional bacteria not found in the chromatography sample. Each membrane was then placed on top of the agar plate prepared with TSA media. The plates were incubated for <NUM>-<NUM> days at <NUM>-<NUM>. The number of colony forming units was then counted and recorded.

Negative control plates are prepared by passing <NUM> of sterile PBS into a separate filter manifold or filter funnel unit with a <NUM> micron filter membrane. Each membrane is then placed on top of the agar plate prepared with TSA media. The plates were incubated for <NUM>-<NUM> days at <NUM>-<NUM>. The number of colony forming units was then counted and expressed in a log<NUM> format.

The tables below show the colony forming units pre-reduction and post-reduction for each of the three bacteria types.

For the Bacillus pseudofirmus, a <NUM> log<NUM> reduction was observed when the <NUM> acetic acid microbe bioburden reduction solution was held in the column for one hour. A <NUM> log<NUM> reduction was observed when the <NUM> acetic acid microbe bioburden reduction solution was held in the column for four hours.

For the Microbacterium spp. bacteria, a <NUM> log<NUM> reduction was observed when the <NUM> acetic acid microbe bioburden reduction solution was held in the column for one hour, and a <NUM> log<NUM> reduction was observed when held for four hours.

For the Stenotrophomonas maltophila bacteria, a <NUM> log<NUM> reduction was observed when the <NUM> acetic acid microbe bioburden reduction solution was held in the column for one hour, and a <NUM> log<NUM> reduction was observed when held for four hours.

<NUM> acetic acid is effective to remove a wide range of microorganisms, including spore-forming bacteria, when held in a protein A column for four hours.

The steps in Example <NUM> above were undertaken, except that instead of <NUM> acetic acid, a solution of <NUM> acetic acid and <NUM>% ethanol was used. As with Example <NUM>, the <NUM> acetic acid and <NUM>% ethanol solution was held for one hour in one set of experiments and for four hours in another set of experiments. The results below also show a substantial decrease in the amount of bacteria after the microbe bioburden reduction solution was held in the column for either one or four hours.

For the Bacillus pseudofirmus, a <NUM> log<NUM> reduction was observed when the <NUM> acetic acid and <NUM>% ethanol microbe bioburden reduction solution was held in the column for one hour. A <NUM> log<NUM> reduction was observed when the <NUM> acetic acid and <NUM>% ethanol microbe bioburden reduction solution was held in the column for four hours.

For the Microbacterium spp. bacteria, a <NUM> log<NUM> reduction was observed when the <NUM> acetic acid and <NUM>% ethanol microbe bioburden reduction solution was held in the column for one hour, and a <NUM> log<NUM> reduction was observed when held for four hours.

For the Stenotrophomonas maltophila bacteria, a <NUM> log<NUM> reduction was observed when the <NUM> acetic acid and <NUM>% ethanol microbe bioburden reduction solution was held in the column for one hour, and a <NUM> log<NUM> reduction was observed when held for four hours.

The steps in Example <NUM> above were undertaken, except that instead of <NUM> acetic acid, a solution of <NUM> urea and a solution of <NUM> urea/<NUM>% ethanol were used and only a one hour hold was measured. The results in Table <NUM> below show a substantial decrease in bacteria after the microbe bioburden reduction solution was held in the column for one hour. The reduction in spore-forming B. psuedofirmus was extensive and unexpected, particularly after only one hour of treatment.

The steps in Example <NUM> above were undertaken, except that instead of <NUM> acetic acid, a solution of <NUM> guanidine hydrochloride and a solution of <NUM> guanidine hydrochloride/<NUM>% ethanol were used and only a one hour hold was measured. The results in Table <NUM> below show a substantial decrease in bacteria after the microbe bioburden reduction solution was held in the column for one hour. The reduction in spore-forming B. psuedofirmus was extensive and unexpected, particularly after only one hour of treatment.

A solution spike study was performed using <NUM> acetic acid, where the extent of killing of Bacillus psuedofirmus and Microbacterium species was measured in solution, without chromatography matrix present. The data are illustrated in <FIG>. There was little killing of Bacillus psuedofirmus observed after one hour, while there was some killing of Microbacterium species. These data illustrate that killing is not solely responsible for the microbial bioburden reduction of these bacteria, on a chromatography matrix, with <NUM> acetic acid. Disruption of an interaction between the chromatography matrix and Bacillus psuedofirmus and Microbacterium species leads to increased reduction of bioburden than what would be expected from killing.

Additional solution spike studies were performed using other agents, where the extent of killing of Bacillus psuedofirmus, Microbacterium species, and Stenotrophomonas maltophilia were measured in solution. The following agents were added: (a) water for injection (WFI), (b) <NUM> urea, (c) <NUM> urea and <NUM>% ethanol, (d) <NUM> guanidine hydrochloride, (e) <NUM> guanidine hydrochloride with <NUM>% ethanol. A spike confirmation measurement in PBS was taken, as well as measurements at the <NUM> minute, <NUM> minute, and <NUM> minute time points. The data are shown in <FIG>, where the blue bar is for WFI, the yellow bar is for <NUM> urea, the gray bar is for <NUM> urea and <NUM>% ethanol, the red bar is for <NUM> guanidine hydrochloride and the green bar is for <NUM> guanidine hydrochloride and <NUM>% ethanol.

For Bacillus pseudofirmus, the bioburden reduction is achieved by a combination of killing and disrupting of interactions between microbes and chromatography resin with at least the following solutions: <NUM> acetic acid, <NUM> urea and <NUM> urea/<NUM>% ethanol, <NUM> guanidine hydrochloride and <NUM> guanidine hydrochloride/<NUM>% ethanol.

For Microbacterium species, bioburden reduction may occur by a combination of killing and disrupting interactions for <NUM> urea and <NUM> acetic acid, which are solutions that are not able to kill <NUM>% of Microbacterium species. However, killing may be largely responsible for bioburden reduction with <NUM> urea/<NUM>% ethanol, <NUM> guanidine hydrochloride and <NUM> guanidine hydrochloride/<NUM>% ethanol, which were observed to kill <NUM>% of the Microbacterium in solution. Similarly for Stenotrophomonas maltophilia, <NUM> urea, <NUM> urea/<NUM>% ethanol were able to kill <NUM>% Stenotrophomonas maltophilia in solution.

Examples <NUM>-<NUM> in summary show that <NUM> acetic acid with a <NUM>-hour hold, <NUM> urea with a <NUM>-hour hold and <NUM> urea/<NUM>% ethanol with a <NUM>-hour hold, <NUM> guanidine hydrochloride with <NUM>-hour hold and <NUM> guanidine hydrochloride/<NUM>% ethanol with <NUM>-hour hold were discovered to be an effective microbial bioburden reduction method for packed MabSelect™ Xtra column in manufacturing. As described in Examples <NUM>-<NUM>, these agents are effective through the combination of killing the microbes and disrupting the interaction between microbial organisms and chromatography resin, or <NUM>% to killing microbial organisms.

Additionally, MabSelect™ Xtra resin exposure to <NUM> acetic acid resulted in minimal impact on Protein A resin.

The performance characteristics of two different chromatography affinity resins, MabSelect™ Xtra and MabSelect™ SuRe, were assessed after the resins were soaked in <NUM> acetic acid for various lengths of time. Specifically, one fraction of MabSelect™ Xtra (used to capture mAb A) was soaked in <NUM> acetic acid for <NUM> hours. As a negative control, another fraction of MabSelect™ Xtra was not soaked in <NUM> acetic acid. Five different fractions of MabSelect™ SuRe (used to capture mAb B) were soaked in <NUM> acetic acid for each of <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, or <NUM> hours. As a control, another fraction of MabSelect™ SuRe was not soaked in <NUM> acetic acid.

Five different experiments were then conducted on each of the above fractions above to assess performance. Size exclusion chromatography (SE-HPLC and SE-UPLC) was performed to assess mAb purity after purification on each of the above chromatography affinity resins.

Two different capillary electrophoresis experiments were performed. CE-SDS was conducted for mAb A and PICO Microchip CE-Electrophoresis (PICO MCE-SDS) was conducted for mAb B. Capillary electrophoresis was conducted in SDS-containing gel-filled capillaries (CE-SDS) to measure the molecular weight distribution and relative abundance of light and heavy chain from monoclonal antibodies. These proteins were separated based on their size and electrophoretic mobility. The relative abundance of total light chain and heavy chain was conducted under reducing and non-reducing conditions. CE-SDS was performed using the IgG Purity Analysis Kit (Beckman Coulter, A10663) with a Bare Fused Silica Capillary (capillary length <NUM>, effective length <NUM>). PICO MCE-SDS was performed using Protein Express LabChip, LabChip® GXII, or LabChip® GXII Touch HT (Perkin Elmer, <NUM> or <NUM>). Internal standards were used to calibrate the relative migration time.

To assay for effect of acetic acid on the Protein-A containing matrix, a residual protein A analysis was performed on eluates from the MabSelect™ Xtra and MabSelect™ SuRe columns by high throughput ELISA and quantified.

Capillary isoelectric focusing with whole-column imaging (iCIEF) was performed to quantify the amount of complementarity determining region <NUM> (CDR2) in a monoclonal antibody sample. Relative abundance of the CDR2 was calculated in each electropherogram by integrating the area under each of the sample-derived isoelectric point (pi) distribution peaks observed and calculating the percentage attributable to CDR2. The reported iCIEF Region <NUM> is the principal peak of neutral species and corresponds to the largest protein peak in the internal reference standard.

The results of each of the above analyses are shown in the following Table <NUM>.

The data from the above table are shown in each of <FIG>. Visual inspection of these figures shows no negative correlation between the performance characteristic and the length of time exposed to <NUM> acetic acid.

Analysis of variance (ANOVA) for the product quality data was performed to assess for statistically significant differences between the resins before and after prolonged exposure to <NUM> acetic acid using three chromatography runs per resin condition. <FIG> show protein quality of the resultant mAb A pool after MabSelect Xtra purifications.

Claim 1:
A method for microbial bioburden reduction of a chromatography matrix, comprising contacting the chromatography matrix with a composition, wherein said composition consists of:
(a) <NUM> to <NUM> acetic acid; or
(b) <NUM> to <NUM> acetic acid and benzyl alcohol; or
(c) <NUM> acetic acid and <NUM> % ethanol; or
(d) <NUM> to <NUM> acetic acid and <NUM>% benzyl alcohol; or
(e) <NUM> to <NUM> acetic acid and <NUM>% to <NUM>% benzyl alcohol
wherein the contacting step is performed for at least <NUM> hour.