Sanitization Compositions and Methods

Abstract

The present technology relates to a novel sanitization method for chromatography media and supporting equipment, which involves treatment with a sanitizing / disinfecting solution containing acetic acid and hexylene glycol.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 185,786, filed May 7, 2021. The contents of the above-mentioned provisional application are incorporated herein by reference in their entirety. [Background technology]

[0002] The high commercial demand for biopharmaceuticals has resulted in pharmaceutical companies placing emphasis on maximizing productivity and product quality while controlling the costs associated with manufacturing. This drive towards improved efficiency has allowed affinity chromatography to come to the forefront, as it provides highly specific binding and reduces the overall number of steps required to purify the analyte of interest from a crude mixture. Affinity chromatography resins offer improved yields and purity standards when compared to traditional purification techniques.

[0003] Any bioprocess chromatography application, including affinity chromatography, requires a high degree of control over the removal of contaminants and impurities. These contaminants include, but are not limited to, proteins, carbohydrates, lipids, lipopolysaccharides (e.g., endotoxins), lipoproteins, nucleic acids, and / or microbial species. Macromolecular impurities, such as proteins, carbohydrates, lipids, nucleic acids, etc., are often addressed by utilizing various intermolecular forces to separate the target analytes from the impurities during the wash and elution stages of the chromatographic operation. Summary of the Invention [Problem to be solved by the invention]

[0004] However, microbial contamination poses an exceptional threat to any chromatography operation due to its ability to grow during the extended resin life associated with modern manufacturing. In fact, microbial contamination poses an even higher risk in processes where the chromatography step is integrated into a bioreactor for continuous capture. Traditionally, decontamination methods for chromatography resins include treatment with sodium hydroxide and / or gamma irradiation. However, affinity resins (e.g., resins conjugated to peptide-based ligands) are not very stable when exposed to sodium hydroxide or gamma irradiation. [Means for solving the problem]

[0005] In one aspect, the disclosure provides a method for sanitizing or sterilizing chromatography media and / or supporting equipment, the method comprising contacting the chromatography media and / or supporting equipment with a sanitizing or sterilizing solution comprising a carboxylic acid and about 20% hexylene glycol, wherein the concentration of the carboxylic acid is about 40 mM to about 200 mM.

[0006] In another aspect, the disclosure provides a method for eluting a target analyte bound to a chromatographic medium, comprising contacting the chromatographic medium with an elution buffer solution comprising a carboxylic acid and about 20% hexylene glycol, wherein the concentration of the carboxylic acid is about 40 mM to about 200 mM.

[0007] In another aspect, the disclosure provides a method for sanitizing or sterilizing chromatography media and / or support equipment, comprising contacting the chromatography media and / or support equipment with a sanitizing or sterilizing solution comprising a carboxylic acid and hexylene glycol such that the pH of the solution is 3.5 or less, resulting in high levels of bacteria, spores, and / or mold inactivation or kill within one hour of treatment with the solution.

[0008] In another aspect, the disclosure provides a method for eluting a target analyte bound to a chromatographic medium, comprising contacting the chromatographic medium with an elution buffer solution comprising a carboxylic acid and hexylene glycol such that the pH of the solution is 3.5 or less, resulting in improved product yield and peak sharpness.

[0009] In another aspect, the disclosure provides a method for increasing the lifespan of a chromatography medium, comprising sanitizing or sterilizing the chromatography medium with a solution comprising about 65 mM acetic acid and about 20% hexylene glycol, which allows the lifespan of the chromatography medium to be increased by at least about 10% compared to sanitizing or sterilizing the chromatography medium with at least one of: (i) gamma irradiation; or (ii) a buffer comprising sodium hydroxide.

[0010] In yet another aspect, the disclosure provides a solution comprising 65 mM acetic acid and 20% hexylene glycol.

[0011] In some embodiments, the carboxylic acid has the formula R 1 -C(=O)-OH (in the formula, R 1 is substituted or unsubstituted C1-C 12 In some embodiments, the carboxylic acid is acetic acid. In some embodiments, the concentration of acetic acid is 40-200 mM. In some embodiments, the concentration of acetic acid is 65 mM. In some embodiments, the concentration of hexylene glycol is 8-80%. In some embodiments, the concentration of hexylene glycol is 20%.

[0012] In some embodiments, the chromatography is affinity chromatography.

[0013] In some embodiments, the affinity ligand is based on Protein A or any variant thereof. In some embodiments, the affinity ligand is based on natural Protein A. In some embodiments, the affinity ligand is based on recombinant Protein A. In some embodiments, the affinity ligand is based on engineered Protein A. In some embodiments, the affinity ligand is based on artificial Protein A.

[0014] In some embodiments, the affinity ligand is based on Protein G or any variant thereof. In some embodiments, the affinity ligand is based on Protein A / G or any variant thereof. In some embodiments, the affinity ligand is based on Protein L or any variant thereof.

[0015] In some embodiments, the pH of the sanitizing solution is between 3.0 and 3.5, hi some embodiments, the pH of the sanitizing solution is 3.1.

[0016] In some embodiments, inactivation of bacteria, spores, and / or mold is achieved within 40 minutes of treatment with the sanitization solution.

[0017] In some embodiments, the sanitization method is used for purification of a polypeptide. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody.

[0018] In some embodiments, the polypeptide is a recombinant enzyme. In some embodiments, the recombinant enzyme is a human recombinant enzyme. In some embodiments, the recombinant enzyme is a lysosomal glycogen-specific enzyme. In some embodiments, the recombinant enzyme is the human enzyme acid alpha-glucosidase (GAA). In some embodiments, the recombinant enzyme is Myozyme®. [Brief description of the drawings]

[0019] [Figure 1]FIG. 1 shows a plot depicting the effect of various sanitization methods on the binding capacity of an affinity chromatography resin. [Diagram 2] FIG. 1 shows a plot depicting the effect of various sanitization methods on resin life. [Figure 3A] FIG. 1 shows a plot depicting the effect of a solution containing 65 mM acetic acid and 20% hexylene glycol (AAH sanitization solution) on resin life. [Figure 3B] FIG. 1 shows a plot depicting the effect of a solution containing 65 mM acetic acid and 20% hexylene glycol (AAH sanitization solution) on resin life. [Figure 4] 1 is a table showing the results of the microbial kill study described in Example 4. [Diagram 5] FIG. 1 shows plots representing the elution profiles of Myozyme® in elution buffer containing no ethylene glycol, 20% ethylene glycol, or 20% hexylene glycol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The configuration, object, and advantages of the present technology will be apparent in the following detailed description. However, it should be understood that the detailed description, while illustrating embodiments and aspects of the present technology, is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present technology will become apparent to those skilled in the art from the detailed description.

[0021] Definitions of certain terms as used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0022] definition The moieties below may be substituted or unsubstituted. "Substituted" refers to the replacement of a hydrogen atom of a molecule or R group with one or more additional R groups, such as deuterium, halogen, alkyl, haloalkyl, alkenyl, alkoxy, alkoxyalkyl, alkylthio, trifluoromethyl, acyloxy, hydroxy, hydroxyalkyl, mercapto, carboxy, cyano, acyl, aryloxy, aryl, arylalkyl, heteroaryl, amino, aminoalkyl, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, nitro, phosphine, phosphinate, phosphonate, sulfate, =O, =S, or other R groups. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group. The combinations of substituents contemplated herein are preferably those that result in the formation of a stable (e.g., substantially unchanged for more than one week when maintained at a temperature of 40°C or less in the absence of moisture or other chemically reactive conditions) or chemically feasible compound.

[0023] Unless otherwise specified, the term “carboxylic acid” as used herein refers to a compound of the formula R 1 -C(=O)-OH (in the formula, R 1 is hydrogen, deuterium, halo, amino, hydroxy, cyano, formyl, furyl, nitro, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, acyloxy, alkoxy, haloalkoxy, thioalkoxy, halothioalkoxy, alkanoyl, haloalkanoyl, thioalkanoyl, halothioalkanoyl, carboxy, carbonyloxy, halocarbonyloxy, carbonylthio, halocarbonylthio, thiocarbonyloxy, halothiocarbonyloxy, thiocarbonylthio, halothiocarbonylthio), and -S(O) n R 11 (n=0-2, R 11 is directly linked to S, where R 11refers to a compound selected from the group consisting of hydrogen, deuterium, halo, amino, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, acyloxy, alkoxy, haloalkoxy, thioalkoxy, halothioalkoxy, alkanoyl, haloalkanoyl, thioalkanoyl, halothioalkanoyl, carboxy, carbonyloxy, halocarbonyloxy, carbonylthio, halocarbonylthio, thiocarbonyloxy, halothiocarbonyloxy, thiocarbonylthio, and halothiocarbonylthio. In some embodiments, the carboxylic acid is selected from the group consisting of acetic acid, citric acid, succinic acid, and formic acid.

[0024] Unless otherwise specified, the term “glycol” as used herein refers to a compound having the formula (R 2 )(R 3 )-C(OH)-C(OH)-(R 4 )(R 5 )(wherein, R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, deuterium, halo, amino, hydroxy, cyano, formyl, furyl, nitro, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, acyloxy, alkoxy, haloalkoxy, thioalkoxy, halothioalkoxy, alkanoyl, haloalkanoyl, thioalkanoyl, halothioalkanoyl, carboxy, carbonyloxy, halocarbonyloxy, carbonylthio, halocarbonylthio, thiocarbonyloxy, halothiocarbonyloxy, thiocarbonylthio, halothiocarbonylthio), and -S(O) n R 11 (n=0-2, R 11 is directly linked to S, where R 11refers to a compound selected from the group consisting of hydrogen, deuterium, halo, amino, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, acyloxy, alkoxy, haloalkoxy, thioalkoxy, halothioalkoxy, alkanoyl, haloalkanoyl, thioalkanoyl, halothioalkanoyl, carboxy, carbonyloxy, halocarbonyloxy, carbonylthio, halocarbonylthio, thiocarbonyloxy, halothiocarbonyloxy, thiocarbonylthio, and halothiocarbonylthio.

[0025] The term "sanitization" as used herein refers to a process of reducing and / or inactivating microbial contamination in a given environment. In some embodiments, sanitization according to the present disclosure may include inactivating microorganisms that may contaminate chromatographic media and / or supporting equipment. In some embodiments, the methods described herein are considered microbistatic.

[0026] The term "sterilization" as used herein refers to the process of destroying or eliminating all microorganisms in a given environment. In some embodiments, sterilization is the complete elimination of microorganisms that may contaminate chromatographic media and / or supporting equipment. In some embodiments, sterilization is a greater than 6 log reduction of microorganisms that may contaminate chromatographic media and / or supporting equipment. In some embodiments, the methods described herein are considered microbiocidal.

[0027] The term "inactivation" as used herein refers to any process that reduces or inhibits the replication of a microorganism.

[0028] The term "kill" as used herein refers to any method that causes a permanent end to vital cellular processes such that cells can no longer survive or reproduce. The term "complete kill" as used herein refers to the situation where when a culture is inoculated into a germicidal solution, incubated (where die-off occurs), and then plated in an environment favorable for microbial growth, nothing grows, thereby indicating that the organisms in the original spike did not survive exposure to the germicidal solution.

[0029] The term "variant" as used herein encompasses any form of a particular protein that is recombinantly expressed in a host cell or a non-native host cell. In some embodiments, the term "variant" refers to a protein that is recombinantly expressed from its native DNA sequence. In some embodiments, the term "variant" refers to a protein that is recombinantly expressed from a codon-optimized DNA sequence. In some embodiments, the term "variant" refers to a recombinantly expressed full-length protein. In some embodiments, the term "variant" refers to a truncated form of a recombinantly expressed protein. In some embodiments, the term "variant" refers to a recombinantly expressed mutant protein. In some embodiments, a mutant protein contains point mutations at one or more positions in its amino acid sequence. In some embodiments, the term "variant" refers to a recombinantly expressed engineered protein, e.g., a genetically engineered protein. In some embodiments, the term "variant" refers to a recombinantly expressed engineered protein.

[0030] Sanitization / disinfection solution In some embodiments, the present technology relates to a sanitizing or disinfecting solution comprising a carboxylic acid and a glycol.

[0031] In some embodiments, the present technology relates to a sanitizing or disinfecting solution comprising acetic acid. In some embodiments, the solution comprises about 40-200 mM acetic acid. In some embodiments, the solution comprises about 40-200 mM, about 50-190 mM, about 60-180 mM, about 70-170 mM, about 80-160 mM, about 90-150 mM, about 100-140 mM, or about 110-130 mM acetic acid. In some embodiments, the solution comprises about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 (or any number between any two of the aforementioned values) mM acetic acid. In some embodiments, the solution comprises about 65 mM acetic acid.

[0032] In some embodiments, the present technology relates to a sanitizing or disinfecting solution comprising hexylene glycol. In some embodiments, the solution comprises about 8-80% hexylene glycol. In some embodiments, the solution comprises about 10-70%, about 12-60%, about 14-50%, about 16-40%, about 18-30%, or about 20-25% hexylene glycol. In some embodiments, the solution comprises about 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% hexylene glycol. In some embodiments, the solution comprises about 20% hexylene glycol.

[0033] In some embodiments, the technology relates to a sanitizing or disinfecting solution comprising acetic acid and hexylene glycol. In some embodiments, the technology relates to a sanitizing or disinfecting solution comprising about 40-200 mM acetic acid and about 8-80% hexylene glycol, about 50-190 mM acetic acid and about 10-70% hexylene glycol, about 60-180 mM acetic acid and about 12-60% hexylene glycol, about 70-170 mM acetic acid and about 14-50% hexylene glycol, about 80-160 mM acetic acid and about 16-40% hexylene glycol, about 90-150 mM acetic acid and about 18-30% hexylene glycol, about 100-140 mM acetic acid and about 20-25% hexylene glycol, or about 110-130 mM acetic acid and about 20-25% hexylene glycol.

[0034] In some embodiments, the present technology provides a method for preparing a glycerol solution containing about 40 mM acetic acid and about 8% hexylene glycol, about 50 mM acetic acid and about 10% hexylene glycol, about 60 mM acetic acid and about 12% hexylene glycol, about 70 mM acetic acid and about 14% hexylene glycol, about 80 mM acetic acid and about 16% hexylene glycol, about 90 mM acetic acid and about 18% hexylene glycol, about 100 mM acetic acid and about 20% hexylene glycol, about 110 mM acetic acid and about 25% hexylene glycol, about 120 mM acetic acid and about 30% hexylene glycol, or a mixture thereof. The present technology relates to a sanitizing or disinfecting solution comprising about 65 mM acetic acid and about 20% hexylene glycol, about 130 mM acetic acid and about 40% hexylene glycol, about 140 mM acetic acid and about 50% hexylene glycol, about 150 mM acetic acid and about 60% hexylene glycol, about 160 mM acetic acid and about 70% hexylene glycol, about 170 mM acetic acid and about 80% hexylene glycol, about 180 mM acetic acid and about 80% hexylene glycol, about 190 mM acetic acid and about 80% hexylene glycol, or about 200 mM acetic acid and about 80% hexylene glycol. In some embodiments, the present technology relates to a sanitizing or disinfecting solution comprising about 65 mM acetic acid and about 20% hexylene glycol.

[0035] In some embodiments, the technology relates to a sanitizing or disinfecting solution having a pH of about 3.5. In some embodiments, the solution has a pH of about 3.4, about 3.3, about 3.2, or about 3.1, about 3.0, or about 2.9. In some embodiments, the solution has a pH of about 3.0.

[0036] In some embodiments, the solution comprises about 65 mM acetic acid and about 20% hexylene glycol and has a pH of about 3.0.

[0037] Sanitization / Disinfection Methods In some embodiments, the present disclosure provides a sanitization or sterilization method for chromatography media and / or supporting equipment, comprising treatment with a sanitization or sterilization solution comprising a carboxylic acid and a glycol. Potential microbial contaminants addressed by the present technology include, without limitation, viruses, bacteria, fungi, and parasites. In some embodiments, the present methods result in the inactivation or killing of a high degree of bacteria, spores, and / or molds.

[0038] In some embodiments, the microbial contaminant is a virus, e.g., a DNA virus, an RNA virus, an enveloped virus, or a non-enveloped virus. Non-limiting examples of viral contaminants include human immunodeficiency virus (HIV), hepatitis virus, human herpes virus, cytomegalovirus, Epstein-Barr virus, and West Nile virus. In some embodiments, the microbial contaminant is a bacterium, e.g., a gram-negative bacterium, a gram-positive bacterium, and / or a biofilm-forming bacterium. Non-limiting examples of bacterial contaminants include Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trachomatis, Streptococcus pyogenes, Mycobacterium tuberculosis, Brucella melitensis, Ehrlichia spp., Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. In some embodiments, the microbial contaminant is a fungus. Non-limiting examples of fungal contaminants include Aspergillus spp., Penicillium spp., Fusarium spp., and Alternaria spp. In some embodiments, the microbial contaminant is a parasite. Non-limiting examples of parasite contaminants include Amoeba, Plasmodium, Trypanosoma cruzi, and Babesia microti.

[0039] In some embodiments, the present disclosure relates to a method of sanitizing or sterilizing a chromatographic medium and / or supporting equipment by contacting the medium and / or equipment with a sanitizing or sterilizing solution described in the present disclosure, hi some embodiments, the sanitizing or sterilizing solution comprises a carboxylic acid and a glycol.

[0040] In some embodiments, the present technology relates to a method of sanitizing or sterilizing a chromatographic medium and / or supporting equipment by contacting the medium and / or equipment with a sanitizing or sterilizing solution comprising acetic acid. In some embodiments, the solution comprises about 40-200 mM acetic acid. In some embodiments, the solution comprises about 40-200 mM, about 50-190 mM, about 60-180 mM, about 70-170 mM, about 80-160 mM, about 90-150 mM, about 100-140 mM, or about 110-130 mM acetic acid. In some embodiments, the solution comprises about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 (or any number between any two of the aforementioned values) mM acetic acid. In some embodiments, the solution comprises about 65 mM acetic acid.

[0041] In some embodiments, the present technology relates to a method of sanitizing or sterilizing a chromatographic medium and / or supporting equipment by contacting the medium and / or equipment with a sanitizing or sterilizing solution comprising hexylene glycol. In some embodiments, the solution comprises about 8-80% hexylene glycol. In some embodiments, the solution comprises about 10-70%, about 12-60%, about 14-50%, about 16-40%, about 18-30%, or about 20-25% hexylene glycol. In some embodiments, the solution comprises about 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% hexylene glycol. In some embodiments, the solution comprises about 20% hexylene glycol.

[0042] In some embodiments, the present technology relates to a method of sanitizing or sterilizing a chromatographic media and / or supporting equipment by contacting the media and / or equipment with a sanitizing or sterilizing solution comprising acetic acid and hexylene glycol. In some embodiments, the solution comprises about 40-200 mM acetic acid and about 8-80% hexylene glycol, about 50-190 mM acetic acid and about 10-70% hexylene glycol, about 60-180 mM acetic acid and about 12-60% hexylene glycol, about 70-170 mM acetic acid and about 14-50% hexylene glycol, about 80-160 mM acetic acid and about 16-40% hexylene glycol, about 90-150 mM acetic acid and about 18-30% hexylene glycol, about 100-140 mM acetic acid and about 20-25% hexylene glycol, or about 110-130 mM acetic acid and about 20-25% hexylene glycol.

[0043] In some embodiments, the technology provides for the preparation of a medium and / or device with about 40 mM acetic acid and about 8% hexylene glycol, about 50 mM acetic acid and about 10% hexylene glycol, about 60 mM acetic acid and about 12% hexylene glycol, about 70 mM acetic acid and about 14% hexylene glycol, about 80 mM acetic acid and about 16% hexylene glycol, about 90 mM acetic acid and about 18% hexylene glycol, about 100 mM acetic acid and about 20% hexylene glycol, about 110 mM acetic acid and about 25% hexylene glycol, about 120 mM acetic acid and about 30% hexylene glycol, about 130 mM acetic acid and about 25% hexylene glycol, about 140 mM acetic acid and about 25% hexylene glycol, about 150 mM acetic acid and about 25% hexylene glycol, about 160 mM acetic acid and about 25% hexylene glycol, about 170 mM acetic acid and about 25% hexylene glycol, about 180 mM acetic acid and about 25% hexylene glycol, about 190 mM acetic acid and about 30% hexylene glycol, about 200 mM acetic acid and about 30% hexylene glycol, about 210 mM acetic acid and about 30% hexylene glycol, about 220 mM acetic acid and about 30% hexylene glycol, about 230 mM acetic acid and about 30% hexylene glycol, about 240 mM acetic acid and about 30% hexylene glycol, about 250 mM acetic acid and about 30% hexylene glycol, about 260 mM acetic acid and about 30% hexylene glycol, about 270 mM acetic acid and about 30% hexylene glycol, about 280 mM acetic acid and about 30% hexylene glycol, The present invention relates to a method of sanitizing or sterilizing a chromatographic medium and / or supporting equipment by contacting the medium with a sanitizing or sterilizing solution comprising an acid and about 40% hexylene glycol, about 140 mM acetic acid and about 50% hexylene glycol, about 150 mM acetic acid and about 60% hexylene glycol, about 160 mM acetic acid and about 70% hexylene glycol, about 170 mM acetic acid and about 80% hexylene glycol, about 180 mM acetic acid and about 80% hexylene glycol, about 190 mM acetic acid and about 80% hexylene glycol, or about 200 mM acetic acid and about 80% hexylene glycol. In some embodiments, the solution comprises about 65 mM acetic acid and about 20% hexylene glycol.

[0044] In some embodiments, the present technology relates to a method of sanitizing or sterilizing a chromatographic medium and / or supporting equipment by contacting the medium and / or equipment with a sanitizing or sterilizing solution having a pH of about 3.5. In some embodiments, the solution has a pH of about 3.4, about 3.3, about 3.2, or about 3.1, about 3.0, or about 2.9. In some embodiments, the solution has a pH of about 3.0.

[0045] In some embodiments, the sanitizing solution of the present disclosure inactivates all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms (e.g., viruses, bacteria, fungi, parasites, etc.) within about 10 hours of treatment with the solution. In some embodiments, the sanitizing solution of the present disclosure inactivates all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms within about 0-10 hours, about 0-9 hours, about 0-8 hours, about 0-7 hours, about 0-6 hours, about 0-5 hours, about 0-4 hours, about 0-3 hours, about 0-2 hours, or about 0-1 hours of treatment with the solution. In some embodiments, the sanitizing solution of the present disclosure inactivates all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms within about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour of treatment with the solution. In some embodiments, the sanitization solution of the present disclosure inactivates all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms within about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes of treatment with the solution. In some embodiments, the sanitization solution of the present disclosure inactivates all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms within about 40 minutes of treatment with the solution. In some embodiments, the sanitization method is sufficient to allow the sanitized media and / or equipment to be subsequently utilized in the detection, purification, and / or preparation of materials for therapeutic administration.

[0046] In some embodiments, the germicidal solutions of the present disclosure kill all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms (e.g., viruses, bacteria, fungi, parasites, etc.) within about 10 hours of treatment with the solution. In some embodiments, the germicidal solutions of the present disclosure kill all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms within about 0-10 hours, about 0-9 hours, about 0-8 hours, about 0-7 hours, about 0-6 hours, about 0-5 hours, about 0-4 hours, about 0-3 hours, about 0-2 hours, or about 0-1 hours of treatment with the solution. In some embodiments, the germicidal solutions of the present disclosure kill all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms within about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour of treatment with the solution. In some embodiments, the germicidal solutions of the present disclosure kill all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms within about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes of treatment with the solution. In some embodiments, the germicidal solutions of the present disclosure kill all vegetative, biofilm-forming, spore-forming, and / or mold-forming microorganisms within about 40 minutes of treatment with the solution. In some embodiments, the germicidal methods are sufficient to allow the sterilized media and / or equipment to be subsequently utilized in detection, purification, and / or preparation of materials for therapeutic administration.

[0047] In some embodiments, the chromatographic medium and / or support equipment is exposed to the sanitizing or sterilizing solution of the present disclosure at a temperature of about 0° C. to about 40° C. In some embodiments, the chromatographic medium and / or support equipment is exposed to the solution at a temperature of about 0° C. to about 25° C. In some embodiments, the chromatographic medium and / or support equipment is exposed to the solution at a temperature of about 0° C. to about 15° C. In some embodiments, the chromatographic medium and / or support equipment is exposed to the solution at a temperature of about 0° C. to about 10° C. In some embodiments, the temperature is about 0° C., about 5° C., about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., or about 40° C. In some embodiments, the temperature is about 20° C. In some embodiments, the temperature is about 4° C.

[0048] In some embodiments, the disclosure relates to a method of sanitizing or sterilizing a chromatographic medium and / or support equipment by contacting the medium and / or support equipment with a solution comprising about 65 mM acetic acid and about 20% hexylene glycol, at a pH of about 3, for at least about 1 hour at a temperature of about 20° C. In some embodiments, the disclosure relates to a method of sanitizing or sterilizing a chromatographic medium and / or support equipment by contacting the medium and / or support equipment with a solution comprising about 65 mM acetic acid and about 20% hexylene glycol, at a pH of about 3, for at least about 40 minutes at a temperature of about 20° C.

[0049] By way of example and not limitation, in some embodiments, chromatographic media refers to any material packed into a column. In some embodiments, the material is a resin or a particle. In some embodiments, the resin is a polymeric support or base matrix. In some embodiments, the polymeric support or base matrix is ​​coupled to an affinity ligand. In some embodiments, the polymeric support or base matrix may include, without limitation, agarose, cellulose, sepharose, polymethacrylate, or polyvinyl ether. In some embodiments, the chromatographic medium is designed for use in immobilized metal affinity chromatography (IMAC), ion exchange chromatography (IEX), such as cation exchange chromatography (CEX) or anion exchange chromatography (AEX), gel filtration chromatography (also known as size exclusion chromatography (SEC)), hydrophobic interaction chromatography (HIC), supercritical fluid chromatography (SFC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), high turbulence liquid chromatography (HTLC), normal phase chromatography (NPC), reversed phase chromatography (RPC), capillary liquid chromatography, electrochromatography, membrane chromatography, monolith chromatography, and nano- or capillary liquid chromatography.

[0050] By way of example and not limitation, in some embodiments the support equipment is one or more selected from chromatographic columns, pumps, injectors, interconnecting tubing, detectors, sample collectors, mixers, flow restrictors, in-line filters, valves, bubble traps, and any other liquid contacting surface.

[0051] In some embodiments, the technology does not impair the function of the chromatography resin and does not shorten the life performance of the resin. Additionally or alternatively, in some embodiments, the sanitizing or disinfecting solution of the present disclosure has low toxicity, is non-flammable, does not cause protein aggregation, and / or is hypoallergenic to peptide affinity ligands. In some embodiments, the sanitizing or disinfecting solution of the present disclosure penetrates biofilms. In some embodiments, the sanitizing or disinfecting solution of the present disclosure has high wettability, which allows efficient distribution throughout the chromatography media itself, i.e., the chromatography beads and bead pores.

[0052] Sample preparation In some embodiments, the technology is applied to the purification and / or detection of one or more analytes of interest from any source sample, such as a biological sample or an environmental sample. In some embodiments, the biological sample may be derived from any living organelle, such as human, animal, plant, microorganism, or cell and tissue culture, tissue biopsy, whole blood, dried blood spot, plasma, deproteinized plasma, serum, deproteinized serum, ascites, semen, sputum, urine, stool, sweat, saliva, bile, tears, cerebrospinal fluid, swabs from body sites, skin, and hair. In some embodiments, the environmental sample may be an air sample, a soil sample, a water sample, a food sample, and any material sample. In some embodiments, the source sample is obtained from a cell culture. In some embodiments, the source sample is obtained from a cell culture supernatant. In some embodiments, the source sample is obtained from a cell lysate.

[0053] In some embodiments, the analyte of interest can be a small molecule, such as, for example, a drug substance, as well as a macromolecule, such as a polypeptide, a peptide, a nucleic acid, a lipid or fatty acid, a carbohydrate, a lipoprotein, a lipopolysaccharide (e.g., endotoxin), a hormone, a vitamin, a steroid, and a metabolite. In some embodiments, the analyte of interest is a polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is an enzyme or a recombinant enzyme. In some embodiments, the recombinant enzyme is a human recombinant enzyme. By way of example and not limitation, in some embodiments, the recombinant enzyme is a lysosomal glycogen-specific enzyme, the human enzyme acid alpha-glucosidase (GAA), alglucosidase alpha, avalglucosidase alpha (neoGAA), Myozyme®, Lumizyme®, Fabrazyme®, Cerezyme®, tissue plasminogen activator (tPA), factor VIII (FVIII), factor IX (FIX), or acid sphingomyelinase (ASM). In some embodiments, the polypeptide is a non-enzymatic protein, such as a structural protein (e.g., collagen), a transport protein (e.g., hemoglobin), a regulatory protein (e.g., a peptide hormone), a motor protein (e.g., myosin), or an immune protein (e.g., an antibody). In some embodiments, the polypeptide is an antibody, such as a monoclonal antibody (mAb), a polyclonal antibody (pAb), a bispecific antibody (BsAb), a trispecific antibody (TsAb), an antigen-binding fragment thereof, or an antibody fusion protein. In some embodiments, the antibody is a recombinant monoclonal antibody. The term "antigen-binding fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to the same antigen as the whole antibody from which the portion is derived. Examples of "antigen-binding fragments" include, without limitation, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated complementarity determining regions (CDRs), scFvs, and diabodies.

[0054] In some embodiments, most of the contaminants and interfering substances are removed before applying chromatographic methods to the source sample. In some embodiments, the analytes of interest are enriched and isolated by filtration, precipitation, centrifugation, extraction, dilution, or a combination thereof. In some embodiments, the analytes of interest are enriched from the source sample by solid phase extraction (SPE). SPE enriches the analytes of interest by using a sample preparation cartridge. The SPE extract containing the analytes is dried and reconstituted in a solvent system compatible with the chromatographic system.

[0055] In some embodiments, analytes of interest are extracted from a source sample by liquid-liquid extraction (LLE). LLE is used to separate analytes based on their relative solubility in two immiscible or partially miscible liquids, usually a polar solvent such as water and a non-polar organic solvent. The target analytes are first partitioned by the solvent and then extracted, concentrated, and diluted.

[0056] In some embodiments, the analytes of interest are extracted from the source sample by solid-supported liquid-liquid extraction (SLE). In SLE, an aqueous solution of the source sample is loaded onto a support comprising diatomaceous earth. After sample absorption into the support, the support is washed several times with an organic extraction solvent such as methyl tert-butyl ether. The analytes of interest are partitioned into the organic phase and then concentrated by drying before being reconstituted in a solvent compatible with the chromatographic system.

[0057] In some embodiments where the analytes of interest are proteins, they are enriched from the source sample by protein precipitation extraction (PPE). Protein precipitation methods may include desalting, isoelectric precipitation, and organic solvent extraction. As an example, the source sample is prepared by desalting for loading into a chromatographic system. This protein precipitation technique relies on proteins being "salted out" of solution in response to increasing concentrations of a neutral salt, such as ammonium sulfate. In some embodiments, the source sample is prepared by isoelectric precipitation; this method is used to precipitate contaminant proteins rather than the target protein. The isoelectric point (pI) is the pH at which the net primary charge of a protein is zero. For most proteins, the pI is in the pH range of 4-6. In some embodiments, inorganic acids such as hydrochloric acid and sulfuric acid are used as precipitants. A potential drawback of isoelectric precipitation is the irreversible denaturation caused by inorganic acids.

[0058] Chromatography In some embodiments, once the source sample has been processed, for example by centrifugation and / or filtration, the clarified sample is loaded onto a chromatography system, for example a liquid chromatography system.

[0059] Liquid chromatography (LC) is a process that selectively retains one or more components of a fluid solution (mobile phase) as it permeates a column of fine material (stationary phase) by pumping, pressure, and / or gravity, achieving diffusion in and through the pores of the chromatographic medium. The retention of selective components in the fluid solution by the stationary phase is due to the higher affinity of the components to the stationary phase than to the mobile phase. In some embodiments, the liquid chromatography used is affinity chromatography (AC), ion exchange chromatography (IEX), size exclusion chromatography (SEC), supercritical fluid chromatography (SFC), high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UHPLC), high turbulence liquid chromatography (HTLC), normal phase chromatography (NPC), reversed phase chromatography (RPC), capillary liquid chromatography, electrochromatography, membrane chromatography, monolith chromatography, nano- or capillary liquid chromatography. In some embodiments, the liquid chromatography system used in this technology is affinity chromatography (AC).

[0060] In some embodiments, the analytes of interest are retained by the stationary phase and then eluted. In some embodiments, the analytes of interest flow through the stationary phase without being retained. In some embodiments, the analytes in the eluate or effluent are monitored by various means including UV, fluorescence, refractive index, light scattering, and electrical conductivity based on retention time, peak intensity, and peak area. In some embodiments, further detailed analysis of the analytes is performed using techniques such as mass spectrometry.

[0061] In some embodiments, LC solvents include organic solvents such as, without limitation, water, methanol, ethanol, acetonitrile, trifluoroacetic acid, heptafluorobutyric acid, ether, hexane, hexylene glycol, ethyl acetate, and hydrocarbon solvents (e.g., aliphatic and aromatic solvents), oxygenated solvents (e.g., alcohols, glycols, ketones, aldehydes, glycol ethers, esters, and glycol ether esters), and halogenated solvents (e.g., chlorinated and brominated hydrocarbons). In some embodiments, LC solvents are buffered and may contain various salts and buffers routinely used in the art, such as sodium acetate, ammonium acetate, ammonium formate, ammonium bicarbonate, acetic acid, trifluoroacetic acid, formic acid, trimethylamine, triethylamine, and the like. In some embodiments, LC solvents also include surfactants such as Tween, SDS, and the like.

[0062] Affinity Chromatography (AC) In some embodiments, the liquid chromatography used is affinity chromatography. Affinity chromatography utilizes specific biological interactions between molecules. The types of biological interactions commonly utilized in affinity chromatography include, without limitation, antigen-antibody interactions, protein-immunoglobulin interactions, enzyme-substrate / cofactor / inhibitor interactions, nucleic acid-nucleic acid binding protein interactions, lectin-polysaccharide / glycoprotein interactions, avidin-biotin interactions, calmodulin-calmodulin binding partner interactions, glutathione-GST fusion protein interactions, metal ion-polyhistidine fusion protein interactions, and receptor-hormone interactions.

[0063] In some embodiments, biospecific ligands (affinity ligands) are chemically immobilized on a solid support (e.g., cellulose, agarose, or polyacrylamide) in the column, such that those molecules with specific binding affinity for the ligand (target analytes) are adsorbed as the crude extract passes through the column. After other contaminants are washed away, the bound analyte is eluted from the support, leading to its purification from the original sample. In some embodiments, the affinity chromatography used is immunoaffinity chromatography (IAC), protein A, protein G, or protein L affinity chromatography, lectin affinity chromatography, dye-ligand affinity chromatography, immobilized metal affinity chromatography (IMAC), or boronic acid affinity chromatography. In some embodiments, the affinity chromatography used is protein A chromatography.

[0064] Affinity chromatography resins include polymeric supports with chemically coupled affinity ligands. Affinity ligands include biological and synthetic ligands. In some embodiments, affinity ligands are biological ligands, such as peptides, polypeptides (proteins), nucleotides, oligonucleotides (nucleic acids), coenzymes, vitamins, lectins, and antibodies. In some embodiments, affinity ligands are synthetic ligands. Synthetic ligands are generated either by de novo synthesis or by modification of existing molecular structures (e.g., triazinyl nucleotide mimics, purine and pyrimidine derivatives, non-natural peptides, triazinyl dyes, other triazine-based ligands, oligosaccharides, and boronic acid analogs).

[0065] In some embodiments, the affinity ligand binds to a peptide, a small molecule, a protein, or an enzyme. In some embodiments, the enzyme is a recombinant enzyme. In some embodiments, the recombinant enzyme is a human recombinant enzyme. By way of example and not limitation, in some embodiments, the recombinant enzyme is lysosomal glycogen specific enzyme, human enzyme acid alpha-glucosidase (GAA), alglucosidase alpha, avalglucosidase alpha (neoGAA), Myozyme®, Lumizyme®, Fabrazyme®, Cerezyme®, tissue plasminogen activator (tPA), factor VIII (FVIII), factor IX (FIX), or acid sphingomyelinase (ASM).

[0066] In some embodiments, the affinity ligand is based on Protein A or a variant thereof. In some embodiments, the affinity ligand is based on Protein G or a variant thereof. In some embodiments, the affinity ligand is based on Protein A / G or a variant thereof. In some embodiments, the affinity ligand is based on Protein L or a variant thereof.

[0067] Protein A affinity chromatography Protein A affinity chromatography is widely used for the purification of monoclonal antibodies (mAbs), polyclonal antibodies (pAbs), their antigen-binding fragments, and antibody fusion proteins. It uses a protein A affinity resin containing a protein A ligand crosslinked to a base matrix as the stationary phase to capture one or more antibodies of interest from the mobile phase. The term "protein A ligand" refers to an affinity ligand based on native protein A or any variant thereof. Staphylococcal protein A (SpA), a 42 kDa cell surface protein, uses its five homologous immunoglobulin-binding domains (E, D, A, B, and C) to bind to the Fc portion of immunoglobulins (e.g., immunoglobulin G or IgG).

[0068] Protein G affinity chromatography Protein G affinity chromatography is widely used for the purification of monoclonal antibodies (mAb), polyclonal antibodies (pAb), their antigen-binding fragments, and antibody fusion proteins. It uses a protein G affinity resin containing a protein G ligand cross-linked to a base matrix as the stationary phase to capture one or more antibodies of interest from the mobile phase. The term "protein G ligand" refers to an affinity ligand based on native protein G or any variant thereof. Streptococcus protein G, a cell wall protein containing two (or three) GA domains and two (or three) B domains, binds to the Fc and Fab portions of immunoglobulins (e.g., immunoglobulin G or IgG). However, the interaction between protein G and Fab is much weaker than its interaction with Fc.

[0069] Protein A / G affinity chromatography Protein A / G affinity chromatography is widely used for the purification of monoclonal antibodies (mAb), polyclonal antibodies (pAb), their antigen-binding fragments, and antibody fusion proteins. It uses a Protein A / G affinity resin containing Protein A / G ligand crosslinked to a base matrix as the stationary phase to capture one or more antibodies of interest from the mobile phase. The term "Protein A / G ligand" refers to an affinity ligand based on recombinant Protein A / G or any variant thereof. Protein A / G is a recombinant fusion protein of approximately 51 kDa that combines the antibody binding domains of Staphylococcus protein A and Streptococcus protein G. Protein A / G contains four Fc-binding domains from Protein A and two Fc-binding domains from Protein G. Protein A / G is used to purify polyclonal or monoclonal antibodies from a variety of species.

[0070] Protein L affinity chromatography Protein L affinity chromatography is widely used for the purification of monoclonal antibodies (mAbs), polyclonal antibodies (pAbs), their antigen-binding fragments, and antibody fusion proteins. It uses a Protein L affinity resin containing Protein L ligand cross-linked to a base matrix as the stationary phase to capture one or more antibodies of interest from the mobile phase. Protein L is a cell surface immunoglobulin-binding protein of approximately 95 kDa originally isolated from Peptococcus magnus. The term "Protein L Ligand" refers to an affinity ligand based on recombinant Protein L or any variant thereof recombinantly expressed in E. coli or any other non-native host cell.

[0071] Target analyte purification method In some embodiments, the present disclosure relates to the purification of macromolecular analytes (e.g., antibodies, enzymes, hormones, growth factors, DNA / RNA, lectins, therapeutic non-enveloped viruses, etc.) from cell cultures. In some embodiments, the target analyte is an antibody and the purification process comprises the following steps: (i) Sanitization Step - All chromatography media and supporting equipment are pre-sanitized with AAH sanitization solution, and the sanitization method includes contacting the media and equipment with the AAH sanitization solution at 0-40°C, preferably 15-25°C, for at least 1 hour. (ii) Harvesting step - Cells, cell debris, and other impurities are separated from the cell culture supernatant by centrifugation, depth filtration, microfiltration, and / or alternating tangential flow. (ii) Affinity chromatography step - target antibodies are captured from cell culture supernatants on a pre-sanitized and equilibrated affinity chromatography resin at neutral pH, washed and eluted at acidic pH. (iii) Viral inactivation step - If the targeting antibody is stable at the test pH, low pH retroviral inactivation is typically performed at low pH (3.3-3.6) with a hold time of 60 minutes or more. (iv) Cation Exchange Chromatography (CEX) step - Host cell proteins (HCPs), antibody aggregates, and antibody fragments are removed by passing the sample through a pre-sanitized cation exchange column. (v) Anion Exchange Chromatography (AEX) step - DNA, any leached Protein A, and other trace contaminants are removed by passing the sample through a pre-sanitized anion exchange column. (vi) Small Virus Retentive Filtration Step - The sample is subjected to a virus clearance step to remove any contaminating viruses. (vii) Ultrafiltration step - the target antibody is concentrated to a desired concentration and buffer exchanged into the desired formulation buffer.

[0072] In some embodiments, the target antibody is captured from the cell culture supernatant onto a Protein A affinity chromatography resin that includes a Protein A ligand chemically conjugated to a polymeric support, bead, or membrane. In some embodiments, the Protein A ligand is based on natural Protein A. In some other embodiments, the Protein A ligand is based on artificial Protein A. For example, artificial Protein A may include unnatural amino acid residues. In some embodiments, the Protein A ligand is based on natural Protein A extracted from Staphylococcus aureus. In some other embodiments, the Protein A ligand is based on recombinantly expressed Protein A or any variant thereof in Escherichia coli or Brevibacillus cochinensis. In some embodiments, the Protein A ligand is based on engineered Protein A (e.g., an alkaline-tolerant Protein A or Protein A variant containing repeat units derived from the B or C domain). In some embodiments, the Protein A ligand is based on mutant Protein A (e.g., a Protein A variant containing point mutations in the B and C domains). In some embodiments, the Protein A ligand is based on truncated Protein A.

[0073] In some embodiments, the target antibody is captured from the cell culture supernatant on a protein G affinity chromatography resin that includes a protein G ligand chemically conjugated to a polymeric support. In some embodiments, the protein G ligand is based on a natural protein G. In some other embodiments, the protein G ligand is based on an artificial protein G. For example, the artificial protein G may include a non-natural amino acid residue. In some other embodiments, the protein G ligand is based on a natural protein G isolated from group C or group G streptococcus. In some embodiments, the protein G ligand is based on a recombinantly expressed protein G in E. coli or any variant thereof. In some embodiments, the protein G ligand is based on a genetically engineered protein G (e.g., a non-albumin-binding form of protein G). In some embodiments, the protein G ligand is based on a mutant protein G. In some embodiments, the protein G ligand is based on a truncated protein G.

[0074] In some embodiments, the target antibody is captured from the cell culture supernatant onto a Protein A / G affinity chromatography resin comprising a Protein A / G ligand chemically conjugated to a polymeric support. In some embodiments, the Protein A / G ligand is based on Protein A / G recombinantly expressed in E. coli or any variant thereof. In some embodiments, the Protein A / G ligand is based on engineered Protein A / G. In some embodiments, the Protein A / G ligand is based on mutant Protein A / G. In some embodiments, the Protein A / G ligand is based on truncated Protein A / G. In some embodiments, the Protein A / G ligand is based on artificial Protein A / G.

[0075] In some embodiments, the target antibody is captured from the cell culture supernatant onto a protein L affinity chromatography resin that includes a protein L ligand chemically conjugated to a polymeric support. In some embodiments, the protein L ligand is based on a native protein L. In some other embodiments, the protein L ligand is based on an artificial protein L. For example, an artificial protein L may include unnatural amino acid residues. In some other embodiments, the protein L ligand is based on a native protein L isolated from Peptococcus magnus. In some embodiments, the protein L ligand is based on a recombinantly expressed protein L in E. coli or any variant thereof. In some embodiments, the protein L ligand is based on a genetically engineered protein L. In some embodiments, the protein L ligand is based on a mutant protein L. In some embodiments, the protein L ligand is based on a truncated protein L.

[0076] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, shall control. Overall, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Furthermore, singular terms shall include the plural and plural terms shall include the singular, unless the context requires otherwise. Throughout this specification and the embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises", or "comprising", are understood to imply the inclusion of a referenced thing or group of things, but not the exclusion of any other thing or group of things. Although several documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0077] Chromatography Buffers In some embodiments, the technology relates to a chromatography buffer solution comprising a carboxylic acid and a glycol. In some embodiments, the chromatography buffer is a wash buffer used to wash unbound or loosely bound (e.g., via non-specific interactions) proteins and / or other contaminants from the surface of the chromatography medium. In some embodiments, the chromatography buffer is an elution buffer used to remove bound target analytes (e.g., antigens, antibodies, enzymes, etc.) from the surface of the chromatography medium.

[0078] In some embodiments, the technology relates to an elution buffer or wash buffer comprising acetic acid. In some embodiments, the buffer comprises about 40-200 mM acetic acid. In some embodiments, the buffer comprises about 40-200 mM, about 50-190 mM, about 60-180 mM, about 70-170 mM, about 80-160 mM, about 90-150 mM, about 100-140 mM, or about 110-130 mM acetic acid. In some embodiments, the buffer comprises about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 (or any number between any two of the aforementioned values) mM acetic acid. In some embodiments, the buffer comprises about 65 mM acetic acid.

[0079] In some embodiments, the technology relates to an elution buffer or wash buffer comprising hexylene glycol. In some embodiments, the buffer comprises about 8-80% hexylene glycol. In some embodiments, the buffer comprises about 10-70%, about 12-60%, about 14-50%, about 16-40%, about 18-30%, or about 20-25% hexylene glycol. In some embodiments, the buffer comprises about 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% hexylene glycol. In some embodiments, the buffer comprises about 20% hexylene glycol.

[0080] In some embodiments, the technology relates to an elution buffer or wash buffer comprising acetic acid and hexylene glycol. In some embodiments, the technology relates to an elution buffer comprising about 40-200 mM acetic acid and about 8-80% hexylene glycol, about 50-190 mM acetic acid and about 10-70% hexylene glycol, about 60-180 mM acetic acid and about 12-60% hexylene glycol, about 70-170 mM acetic acid and about 14-50% hexylene glycol, about 80-160 mM acetic acid and about 16-40% hexylene glycol, about 90-150 mM acetic acid and about 18-30% hexylene glycol, about 100-140 mM acetic acid and about 20-25% hexylene glycol, or about 110-130 mM acetic acid and about 20-25% hexylene glycol.

[0081] In some embodiments, the present technology provides a method for preparing a glycerol solution containing about 40 mM acetic acid and about 8% hexylene glycol, about 50 mM acetic acid and about 10% hexylene glycol, about 60 mM acetic acid and about 12% hexylene glycol, about 70 mM acetic acid and about 14% hexylene glycol, about 80 mM acetic acid and about 16% hexylene glycol, about 90 mM acetic acid and about 18% hexylene glycol, about 100 mM acetic acid and about 20% hexylene glycol, about 110 mM acetic acid and about 25% hexylene glycol, about 120 mM acetic acid and about 30% hexylene glycol, or a mixture thereof. The elution or washing buffer may comprise about 130 mM acetic acid and about 40% hexylene glycol, about 140 mM acetic acid and about 50% hexylene glycol, about 150 mM acetic acid and about 60% hexylene glycol, about 160 mM acetic acid and about 70% hexylene glycol, about 170 mM acetic acid and about 80% hexylene glycol, about 180 mM acetic acid and about 80% hexylene glycol, about 190 mM acetic acid and about 80% hexylene glycol, or about 200 mM acetic acid and about 80% hexylene glycol. In some embodiments, the technology relates to an elution buffer comprising about 65 mM acetic acid and about 20% hexylene glycol.

[0082] In some embodiments, the technology relates to an elution buffer or a wash buffer having a pH of about 3.5. In some embodiments, the buffer has a pH of about 3.4, about 3.3, about 3.2, or about 3.1, about 3.0, or about 2.9. In some embodiments, the buffer has a pH of about 3.0.

[0083] In some embodiments, the elution or wash buffer comprises about 65 mM acetic acid and about 20% hexylene glycol and has a pH of about 3.0.

[0084] In order that this technology may be better understood, the following examples are presented, which are for illustrative purposes only and should not be construed as limiting the scope of the technology in any way. EXAMPLES

[0085] Studies to evaluate the effect of sanitization methods on resin stability This example describes a batch binding study to evaluate the initial and ongoing impact of various sanitization methods on the functionality and longevity / durability of chromatographic media.

[0086] method Chemicals and Reagents The chromatographic medium comprises an affinity ligand chemically cross-linked to an agarose-based matrix by an epoxy bond.

[0087] Time course assay A time course was established by buffer exchanging a source pool of chromatography resin into a 50% slurry under the desired sanitization conditions, including treatment with sanitization buffer and exposure to gamma radiation. The composition of the various sanitization buffers used in this study is described in Table 1 below. At the designated times, 1 mL of resin was removed from the source pool, buffer exchanged into equilibration buffer, and then mixed with alglucosidase alfa drug substance to test for binding at a target binding capacity of 15 mg / mL resin. The binding capacity of the resin at the set time points was normalized to the binding capacity at T0 and plotted over the time course (Figure 1). Because the irradiation was a single exposure to doses of 2, 7, or 25 kGy / hr of gamma radiation, the gamma irradiated resin was evaluated only at T0 and compared to untreated resin.

[0088] [Table 1]

[0089] result This study revealed that exposure to gamma irradiation resulted in an initial 20% decrease in the binding capacity of the resin. Furthermore, prolonged exposure of the affinity ligand to sodium hydroxide buffer resulted in a decrease in binding capacity of 20% or more, depending on the duration of exposure and the concentration of sodium hydroxide. Surprisingly, the affinity ligand was much more stable in acidic buffer (pH=1.7) and showed a maintenance of binding capacity over the time course comparable to the control (100 mM sodium acetate, pH 5.6). EXAMPLES

[0090] Studies to evaluate the impact of sanitization methods on resin life. This example describes a cycling study to evaluate the effect of various sanitization methods on the lifetime performance of chromatographic media.

[0091] method Cycling studies were performed to investigate the effect of sanitization methods on resin lifetime. Under continuous operating conditions, a 1 cm column was cycled 25 times after gamma irradiation to 25 kGy at T0. In addition, a 0.66 cm column was cycled 100 times after treatment with sanitization buffer containing sodium hydroxide and / or tween at T0.

[0092] result Cycling of the gamma irradiated column showed a 50% decrease in binding capacity when compared to untreated virgin resin. Cycling of the gamma irradiated resin was stopped after 25 cycles due to a rapid decline in column performance suggesting severe structural damage to the resin itself.

[0093] Cycling the tween treated resin showed a 23% decrease in binding capacity when compared to untreated virgin resin. The resin was cycled 100 times to represent the expected lifetime of the affinity ligand. The 23% decrease in binding capacity is likely due in part to loss of column performance over the long lifetime, but also due to hydroxide exposure. To extend the resin lifetime, a minimum hydroxide exposure was explored in this lifetime study. Any increase in hydroxide exposure would further decrease the binding capacity. The dynamic binding capacity (DBC) of the column after cycling is shown in Figure 2. EXAMPLES

[0094] Studies to evaluate the effect of AAH sanitization buffers on resin life. This example describes a pilot-scale study to evaluate the effect of a sanitization buffer containing acetic acid and hexylene glycol on the lifetime performance of chromatography media.

[0095] method Two pilot-scale studies were conducted to investigate the effect of sanitization buffer containing 65 mM acetic acid and 20% hexylene glycol (AAH sanitization buffer) on the lifetime performance of affinity chromatography resins. Briefly, resin performance in a 10 cm column was evaluated after cycling the column 70 times (approximately 40 min / cycle) such that in each cycle the column was exposed to 2 CV of sanitization buffer.

[0096] result When operated at set-point conditions, there was no change in recovery following exposure to AAH sanitization buffer for up to 46 hours (for 70 cycles). This study demonstrated that AAH sanitization buffer had no detrimental effect on affinity ligand lifetime performance (Figures 3A and 3B).

[0097] This data indicates that the solutions of the present technology, including acetic acid and hexylene glycol, do not impair the function of the affinity ligand. Thus, the solutions of the present technology are useful in sanitization or disinfection methods for sanitizing, regenerating, and / or disinfecting chromatography media and / or supporting equipment. EXAMPLES

[0098] Studies to evaluate the microbiocidal efficacy of AAH sanitizing solutions. This example describes a microbial kill study to evaluate the microbiocidal efficacy of a sanitizing solution containing acetic acid and hexylene glycol.

[0099] method A screening batch kill study was conducted using AAH sanitization solution and other potential sanitization solutions. Briefly, a microbial spiking solution (10 8A total of 1000 samples (cells / mL) were prepared. These microorganisms included E. coli (ATCC10536; Gram-negative), S. aureus (ATCC6538; Gram-positive), O. anthropi (in-house isolate; Gram-negative), B. cereus (in-house isolate; Gram-negative) and B. thuringiensis (in-house isolate; spore-forming). Different tubes containing different sanitization solutions were spiked and sampled after predefined time points (T20, T40, T60, and T24 hours). Samples were then analyzed for bioburden concentration and the results were transformed into log values. Log10 reduction was calculated based on the T0 PBS control.

[0100] Additionally, pilot-scale studies were performed to detect the presence of endotoxins in the sample eluates. Briefly, a 10 cm column was operated as an open system and treated with 2 CV of AAH sanitization solution per cycle over the course of 73 cycles. As part of the run, every 4 column cycles were pooled into a single eluate (usually over the course of 24 hours). Endotoxins were measured using the Charles River Endosafe nexgen-PTS endotoxin test kit. In addition to sampling the eluate for endotoxins, the end-of-life (EoL) resin was exchanged into equilibration buffer (100 mM sodium acetate, pH 5.6) after completion of the life cycle and held at room temperature for 1 week. At the end of the hold, the effluent was tested for endotoxins.

[0101] result Figure 4 below shows the microbiocidal effectiveness of the AAH Sanitizing Solution in killing several representative bacteria, spores, and / or molds in comparison to sodium hydroxide and other acidic sanitizing agents. As shown in Figure 4, the AAH Sanitizing Solution was the only solution to achieve complete kill of all tested species in less than one hour. Caustic sanitizing agents such as 0.5M NaOH were not effective against spore-forming microbial contaminants. This complete kill in less than one hour proves that the AAH Sanitizing Solution is surprisingly superior to the current Protein A acidic sanitizing agent (2% PAB; Merck-Millipore), which operates under harsher conditions (pH 1.7) and at a slightly reduced temperature, and takes a minimum of 10 hours to kill the spore-forming species tested. Thus, the AAH Sanitizing Solution killed all tested microbial species in surprisingly little time (less than one hour) and under much milder conditions. Endotoxin was not detected in any of the eluate samples tested (all endotoxin levels were reported below the limit of detection). This suggests that the sanitization method achieved control over gram-negative bacteria. This data indicates that the solution of the present technology, which includes acetic acid and hexylene glycol, has microbiocidal properties. Thus, the solution of the present technology is useful in methods for sanitizing or disinfecting chromatography media and / or supporting equipment. EXAMPLES

[0102] Studies to evaluate the elution efficiency of AAH solutions This example describes two studies to evaluate the elution efficiency of solutions containing acetic acid and hexylene glycol.

[0103] method Various elution buffer formulations were tested to investigate the elution efficiency of buffers containing acetic acid and glycols (e.g., hexylene glycol). The composition of the elution buffers used in this study, the eluate protein concentrations, and the activity recovery of the target analyte are listed in Table 2 below. The target analyte used in this study was Myozyme®. In addition, a second study was conducted to compare the effect of ethylene glycol with the effect of hexylene glycol on the elution efficiency of the elution buffer. An elution buffer without glycol was used as a control in this study.

[0104] result As shown in Table 2, all buffer formulations tested in the first study resulted in product elution from the column. Figure 5 demonstrates that the elution buffer containing 20% ​​hexylene glycol resulted in better product yield and peak sharpness than elution buffers with no glycol or 20% ethylene glycol.

[0105] Thus, a solution containing acetic acid and hexylene glycol can also function as an elution buffer. The ability to integrate the AAH solution as a process step, combined with its microbicidal capabilities, further enhances its value for bioprocessing.

[0106] [Table 2]

Claims

1. 1. A method for sanitizing or sterilizing a chromatography medium and / or supporting equipment, comprising contacting the chromatography medium and / or supporting equipment with a sanitizing or sterilizing solution comprising a carboxylic acid and about 20% hexylene glycol, wherein the concentration of the carboxylic acid is from about 40 mM to about 200 mM.

2. 1. A method for eluting a target analyte bound to a chromatographic medium, comprising contacting the chromatographic medium with an elution buffer comprising a carboxylic acid and about 20% hexylene glycol, wherein the concentration of the carboxylic acid is from about 40 mM to about 200 mM.

3. Carboxylic acids are represented by the formula R １ -C(=O)-OH (wherein R １ is substituted or unsubstituted C １ ~C １２ 3. The method of claim 1 or 2, wherein the carboxylic acid is a carboxylic acid of a formula (I) of which the carboxylic acid is an alkyl, alkenyl, or alkynyl.

4. The method of claim 3, wherein the carboxylic acid is acetic acid.

5. 5. The method of claim 4, wherein the concentration of acetic acid is about 65 mM.

6. 1. A method for sanitizing or sterilizing chromatography media and / or support equipment, comprising contacting the chromatography media and / or support equipment with a sanitizing or sterilizing solution comprising a carboxylic acid and hexylene glycol such that the pH of the solution is 3.5 or less, resulting in high levels of bacteria, spores, and / or mold inactivation or kill within one hour of treatment with the solution.

7. 1. A method for eluting a target analyte bound to a chromatographic medium, comprising contacting the chromatographic medium with an elution buffer comprising a carboxylic acid and hexylene glycol such that the pH of the buffer is 3.5 or less, resulting in improved product yield and peak sharpness.

8. Carboxylic acids are represented by the formula R １ -C(=O)-OH (wherein R １ is substituted or unsubstituted C １ ~C １２ 8. The method according to claim 6 or 7, wherein the carboxylic acid is a carboxylic acid of the formula (I) which is an alkyl, alkenyl, or alkynyl group.

9. The method of claim 8, wherein the carboxylic acid is acetic acid.

10. 10. The method of claim 9, wherein the concentration of acetic acid is from about 40 mM to about 200 mM.

11. 11. The method of claim 10, wherein the concentration of acetic acid is about 65 mM.

12. The method of claim 6 or 7, wherein the concentration of hexylene glycol is from about 8% to about 80%.

13. 8. The method of any one of claims 1, 6 and 7, wherein the concentration of hexylene glycol is about 20%.

14. 8. The method of any one of claims 1, 6 and 7, wherein the chromatography is affinity chromatography.

15. 15. The method of claim 14, wherein the affinity chromatography comprises an affinity ligand based on Protein A or any variant thereof.

16. The method of claim 15, wherein the affinity ligand is based on native Protein A.

17. The method of claim 15, wherein the affinity ligand is based on recombinant Protein A.

18. The method of claim 15, wherein the affinity ligand is based on engineered Protein A.

19. The method of claim 15, wherein the affinity ligand is based on an artificial Protein A.

20. 15. The method of claim 14, wherein the affinity chromatography comprises an affinity ligand based on Protein G or any variant thereof.

21. 15. The method of claim 14, wherein the affinity chromatography comprises an affinity ligand based on Protein A / G or any variant thereof.

22. 15. The method of claim 14, wherein the affinity chromatography comprises an affinity ligand based on Protein L or any variant thereof.

23. The method of claim 1 or 6, wherein the pH of the sanitizing or disinfecting solution is from about 3.0 to about 3.

5.

24. 24. The method of claim 23, wherein the pH of the sanitizing or disinfecting solution is about 3.

1.

25. The method of claim 2 or 7, wherein the pH of the elution buffer solution is from about 3.0 to about 3.

5.

26. 26. The method of claim 25, wherein the pH of the elution buffer solution is about 3.

1.

27. Inactivation of bacteria, spores, and / or mold occurs within 4 days of treatment with a sanitizing or disinfecting solution. The method of claim 6, wherein the method is accomplished in 0 minutes or less.

28. The method of claim 1 or 6, wherein the sanitization method is used in a process which comprises a step for the purification of a polypeptide.

29. The method according to claim 2 or 7, wherein the elution method is used in a process which comprises a step for the purification of a polypeptide.

30. A method for increasing the lifespan of a chromatography medium, comprising sanitizing or sterilizing the chromatography medium with a solution comprising about 65 mM acetic acid and about 20% hexylene glycol, the method enabling the lifespan of the chromatography medium to be increased by at least about 10% compared to sanitizing or sterilizing the chromatography medium with at least one of: (i) gamma irradiation; or (ii) a buffer comprising sodium hydroxide.

31. 31. The method of claim 30, wherein the chromatography medium comprises at least one of an affinity resin, a resin based on Protein A or a variant thereof, or a resin based on Protein G or a variant thereof.

32. 32. The method of claim 30 or 31, wherein the sanitizing or sterilizing comprises a step within an integrated, continuous biomanufacturing process for the purification of the polypeptide.

33. 30. The method of claim 28, wherein the polypeptide is an antibody.

34. The method of claim 33, wherein the antibody is a monoclonal antibody.

35. 29. The method of claim 28, wherein the polypeptide is a recombinant enzyme.

36. 36. The method of claim 35, wherein the recombinant enzyme is a human recombinant enzyme.

37. 36. The method of claim 35, wherein the recombinant enzyme is a lysosomal glycogen-specific enzyme.

38. 36. The method of claim 35, wherein the recombinant enzyme is the human enzyme acid alpha-glucosidase (GAA).

39. 36. The method of claim 35, wherein the recombinant enzyme is Myozyme®.

40. 36. The method of claim 35, wherein the recombinant enzyme is avalglucosidase alpha (neoGAA).

41. A solution containing 65 mM acetic acid and 20% hexylene glycol.

42. 42. The solution of claim 41 for use in a method for sanitizing or disinfecting chromatography media and / or supporting equipment.

43. 42. The solution of claim 41 for use in a method for eluting a target analyte bound to a chromatographic medium.

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