Sterile chromatography resin and use thereof in manufacturing methods

Abstract

To provide methods of reducing bioburden of a chromatography resin.SOLUTION: Provided herein are methods of reducing bioburden of a chromatography resin that include exposing a container including a composition including (i) a chromatography resin and (ii) a liquid including at least one alcohol to a dose of gamma-irradiation sufficient to reduce the bioburden of the container and the chromatography resin, where the at least one alcohol is present in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin after / upon exposure to the dose of gamma-irradiation. Also provided are reduced bioburden chromatography columns including the reduced bioburden chromatography resin, compositions including a chromatography resin and a liquid including at least one alcohol, and methods of performing reduced bioburden column chromatography using one of these reduced bioburden chromatography columns.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 726,043, filed Aug. 31, 2018; the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to biotechnology and biomanufacturing methods relating to recombinant proteins. [Background technology]

[0003] Mammalian cells containing nucleic acids encoding recombinant proteins are often used to produce proteins of therapeutic or commercial importance. In the current environment of diverse product pipelines, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective production of therapeutic protein drug substances. One approach to efficiently isolate recombinant proteins is by a process involving continuous chromatography (e.g., using a closed system). One known limitation of continuous chromatography is the presence of contaminating agents (e.g., increased bioburden) in the system, which can result in contaminating products, reduced production yields, and reduced flow rates (or increased pressure) in the system. For example, increased bioburden in a system can cause a complete shutdown of the system. Summary of the Invention [Means for solving the problem]

[0004] The present invention is based, at least in part, on the discovery that gamma irradiation of a chromatography resin reduces the binding capacity of the chromatography resin, and irradiation in the presence of at least one alcohol can help prevent this reduction in binding capacity of the chromatography resin caused by gamma irradiation. In light of this discovery, provided herein is a method of reducing the bioburden of a chromatography resin, comprising exposing a container containing a composition comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol, to a dose of gamma irradiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein the at least one alcohol is present in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin after / during exposure to the dose of gamma irradiation. Also provided are reduced bioburden chromatography resins prepared by any of the methods described herein, reduced bioburden chromatography columns containing a composition comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol, a method of performing reduced bioburden column chromatography using at least one of these reduced bioburden chromatography columns, and an integrated, closed or substantially closed, continuous method for the production of reduced bioburden of purified recombinant proteins, comprising the use of at least one of these reduced bioburden chromatography columns. Any of the chromatography resins produced by any of the methods described herein, any of the packed chromatography columns produced by any of the methods described herein, any of the methods of performing column chromatography, and any of the methods described herein can be sterile, absolutely sterile, sterilized, or reduced bioburden. Any of the chromatography resins produced by any of the methods described herein, any of the chromatography columns produced by any of the methods described herein, and any of the methods described herein can be sterilized and sterile, absolutely sterile, sterilized, or reduced bioburden.

[0005] Provided herein is a method for reducing the bioburden of a chromatography resin, comprising exposing a container containing a composition comprising: (i) a chromatography resin; and (i) a liquid comprising at least one alcohol, to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein the at least one alcohol is present in an amount sufficient to ameliorate loss of binding capacity of the chromatography resin following exposure to the dose of gamma radiation.

[0006] In some embodiments, the method can further include placing the composition in a container prior to the exposing.

[0007] In some embodiments, the container is a storage vessel.

[0008] In some embodiments, the container is a chromatography column.

[0009] In some embodiments, the vessel is a packed chromatography column.

[0010] In some embodiments, the composition is a slurry of the deposited chromatography resin.

[0011] In some embodiments, the composition is a wet solid mixture.

[0012] In some embodiments of any of the methods described herein, the at least one alcohol is selected from benzyl alcohol, cyclohexanol, isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol, propan-2-ol, propan-1-ol, butan-1-ol, pentan-1-ol, hexadecan-1-ol, 2-phenylethanol, sec-phenylethanol, 3-phenyl-1-propanol, 1-phenyl-1-propanol, 2-phenyl-1-propanol, 2-phenyl-2-propanol, 1-phenyl-2-butanol, 2-phenyl-1-butanol, 3-phenyl-1-butanol, 4-phenyl-2-butanol, dl-1-phenyl-2-pentanol, 5-phenyl-1-pentanol, and 4-phenyl-1-butanol.

[0013] In some embodiments, the at least one alcohol comprises benzyl alcohol.

[0014] In some embodiments of any of the methods described herein, the total total concentration of the one or more alcohols in the liquid is from about 0.01% v / v to about 10% v / v.

[0015] In some embodiments of any of the methods described herein, the liquid can further comprise at least one antioxidant and / or chelating agent.

[0016] In some embodiments, the liquid comprises at least one antioxidant and / or chelating agent in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin following exposure to a dose of gamma radiation.

[0017] In some embodiments of any of the methods described herein, the liquid is selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoids, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoids, phenolpropanoid acid, It contains at least one antioxidant selected from the group consisting of lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanol.

[0018] In some embodiments, the liquid comprises at least one antioxidant selected from the group consisting of mannitol, sodium ascorbate, histidine, and methionine.

[0019] In some embodiments, the liquid comprises mannitol, sodium ascorbate, histidine, and methionine.

[0020] In some embodiments of any of the methods described herein, the liquid comprises: (i) 75 mM to about 125 mM mannitol; (ii) 75 mM to about 125 mM methionine; (iii) 75 mM to about 125 mM sodium ascorbate; (iv) 75 mM to about 125 mM histidine; (v) 30 mM to about 70 mM methionine and about 30 mM to about 70 mM histidine; (vi) about 10 mM to about 50 mM methionine, about 10 mM to about 50 mM histidine, and about 10 mM to about 50 mM sodium ascorbate; or (vii) about 5 mM to about 45 mM sodium ascorbate, about 5 mM to about 45 mM methionine, about 5 mM to about 45 mM mannitol, and about 5 mM to about 45 mM histidine.

[0021] In some embodiments of any of the methods described herein, the liquid is a buffer solution.

[0022] In some embodiments of any of the methods described herein, the liquid comprises at least one chelating agent selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium 2,3-dimercapto-1-propanesulfonate (DMPS), dimercaptosuccinic acid (DMSA), metallothionein, and desferoxamine.

[0023] In some embodiments of any of the methods described herein, the chromatography resin is selected from the group consisting of anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin.

[0024] In some embodiments, the composition comprises an affinity chromatography resin comprising a protein ligand.

[0025] In some embodiments, the protein ligand is Protein A.

[0026] In some embodiments, the composition comprises an anion exchange chromatography resin.

[0027] In some embodiments, the anion exchange chromatography resin comprises N-benzyl-N-methyl-ethanolamine groups.

[0028] In some embodiments of any of the methods described herein, the dose is from about 15 kGy to about 45 kGy.

[0029] In some embodiments, the dose is from about 20 kGy to about 30 kGy.

[0030] In some embodiments, the dose is about 23 kGy and about 27 kGy.

[0031] In some embodiments of any of the methods described herein, the exposing is carried out at a temperature of about -25°C to about 0°C.

[0032] In some embodiments of any of the methods described herein, the exposing is carried out at a temperature of about 0°C to about 25°C.

[0033] Provided herein is a reduced bioburden chromatography resin produced by any of the methods described herein.

[0034] In some embodiments, the resin is about 1×10 -8 ~Approx. 1×10 -5 It has a sterility assurance level (SAL).

[0035] In some embodiments, the resin is about 1×10 -7 ~Approx. 1×10 -6 It has a sterility assurance level (SAL).

[0036] In some embodiments of any of the resins described herein, the chromatography resin comprises at least one resin selected from the group consisting of anion exchange chromatography resins, cation exchange chromatography resins, affinity chromatography resins, hydrophobic interaction chromatography resins, and size exclusion chromatography resins.

[0037] In some embodiments, the chromatography resin comprises an affinity chromatography resin that comprises a protein ligand.

[0038] In some embodiments, the protein ligand is Protein A.

[0039] In some embodiments, the chromatography resin comprises an anion exchange chromatography resin.

[0040] In some embodiments, the anion exchange chromatography resin comprises N-benzyl-N-methyl-ethanolamine groups.

[0041] Provided herein is a method of making a reduced bioburden packed chromatography column, the method comprising providing any of the reduced bioburden chromatography resins described herein, and packing the chromatography resin into a reduced bioburden column in a sterile environment.

[0042] Provided herein is a chromatography column packed with reduced bioburden produced by any of the methods described herein.

[0043] Provided herein is a chromatography column packed with reduced bioburden produced by any of the methods described herein.

[0044] In some embodiments, the resin in the packed column is about 1×10 -8 ~Approx. 1×10 -5 It has a sterility assurance level (SAL).

[0045] In some embodiments, the resin is about 1×10 -7 ~Approx. 1×10 -6 It has a sterility assurance level (SAL).

[0046] In some embodiments of any of the resins described herein, the resin in the packed column comprises at least one resin selected from the group consisting of anion exchange chromatography resins, cation exchange chromatography resins, affinity chromatography resins, hydrophobic interaction chromatography resins, and size exclusion chromatography resins.

[0047] In some embodiments, the resin comprises an affinity or pseudoaffinity chromatography resin that contains a protein ligand.

[0048] In some embodiments, the ligand is Protein A.

[0049] In some embodiments, the resin comprises an anion exchange chromatography resin.

[0050] In some embodiments, the anion exchange chromatography resin comprises N-benzyl-N-methyl-ethanolamine groups.

[0051] Provided herein is a composition comprising: (i) a chromatography resin; and (ii) a liquid comprising at least one alcohol, wherein the at least one alcohol is present in an amount sufficient to ameliorate loss of binding capacity of the chromatography resin upon treatment with gamma radiation at a dose sufficient to reduce the bioburden of the composition.

[0052] In some embodiments, the composition is a slurry of the deposited chromatography resin.

[0053] In some embodiments, the composition is a wet solid mixture.

[0054] In some embodiments of any of the compositions described herein, the at least one alcohol is selected from the group consisting of benzyl alcohol, cyclohexanol, isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol, propan-2-ol, propan-1-ol, butan-1-ol, pentan-1-ol, hexadecan-1-ol, 2-phenylethanol, sec-phenylethanol, 3-phenyl-1-propanol, 1-phenyl-1-propanol, 2-phenyl-1-propanol, 2-phenyl-2-propanol, 1-phenyl-2-butanol, 2-phenyl-1-butanol, 3-phenyl-1-butanol, 4-phenyl-2-butanol, dl-1-phenyl-2-pentanol, 5-phenyl-1-pentanol, and 4-phenyl-1-butanol.

[0055] In some embodiments, the at least one alcohol comprises benzyl alcohol.

[0056] In some embodiments of any of the compositions described herein, the total concentration of the one or more alcohols in the liquid is from about 0.01% v / v to about 10% v / v.

[0057] In some embodiments of any of the compositions described herein, the liquid further comprises at least one antioxidant and / or chelating agent.

[0058] In some embodiments, the liquid further comprises at least one antioxidant and / or chelating agent in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin following exposure to a dose of gamma radiation.

[0059] In some embodiments of any of the compositions described herein, the liquid comprises at least one antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoids, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoids, phenolpropanoid acids, lidocaine, naringenin, fullerenes, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanol.

[0060] In some embodiments, the liquid comprises at least one antioxidant selected from the group consisting of mannitol, sodium ascorbate, histidine, and methionine.

[0061] In some embodiments, the liquid comprises mannitol, sodium ascorbate, histidine, and methionine.

[0062] In some embodiments of any of the compositions described herein, the liquid comprises: (i) 75 mM to about 125 mM mannitol; (ii) 75 mM to about 125 mM methionine; (iii) 75 mM to about 125 mM sodium ascorbate; (iv) 75 mM to about 125 mM histidine; (v) 30 mM to about 70 mM methionine and about 30 mM to about 70 mM glycerol; histidine; (vi) about 10 mM to about 50 mM methionine, about 10 mM to about 50 mM histidine, and about 10 mM to about 50 mM sodium ascorbate; or (vii) about 5 mM to about 45 mM sodium ascorbate, about 5 mM to about 45 mM methionine, about 5 mM to about 45 mM mannitol, and about 5 mM to about 45 mM histidine.

[0063] In some embodiments of any of the compositions described herein, the liquid is a buffer solution.

[0064] In some embodiments of any of the compositions described herein, the composition comprises at least one chelating agent selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium 2,3-dimercapto-1-propanesulfonate (DMPS), dimercaptosuccinic acid (DMSA), metallothionein, and desferoxamine.

[0065] In some embodiments of any of the compositions described herein, the chromatography resin comprises at least one resin selected from the group consisting of anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin.

[0066] In some embodiments of any of the compositions described herein, the resin comprises an affinity chromatography resin comprising a protein ligand.

[0067] In some embodiments, the protein ligand is Protein A.

[0068] Provided herein is a method of performing reduced bioburden column chromatography, the method comprising: (a) providing any of the reduced bioburden packed chromatography columns described herein; and (b) performing column chromatography using the reduced bioburden packed chromatography column and reduced bioburden buffer in a closed system.

[0069] In some embodiments, the reduced bioburden column chromatography using a reduced bioburden packed chromatography column is performed continuously for a period of at least four days.

[0070] In some embodiments, the reduced bioburden column chromatography using a reduced bioburden packed chromatography column is performed continuously for a period of at least 5 days.

[0071] In some embodiments, the reduced bioburden column chromatography using a reduced bioburden packed chromatography column is performed continuously for a period of at least 7 days.

[0072] In some embodiments, the reduced bioburden column chromatography using a reduced bioburden packed chromatography column is performed continuously for a period of at least 14 days.

[0073] In some embodiments, the reduced bioburden column chromatography using a reduced bioburden packed chromatography column is performed continuously for a period of at least 28 days.

[0074] In some embodiments, the resin in the chromatography column packed with the reduced bioburden in (a) has a percent binding capacity of about 75% to about 100% compared to the same resin that has not been treated with gamma irradiation.

[0075] In some embodiments, the resin in the reduced bioburden packed chromatography column comprises at least one resin selected from the group consisting of anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin.

[0076] In some embodiments, the resin comprises an affinity chromatography resin that comprises a protein ligand.

[0077] In some embodiments, the protein ligand is Protein A.

[0078] In some embodiments, the resin comprises an anion exchange chromatography resin.

[0079] Provided herein is an integrated, closed, continuous method for the production of reduced bioburden of purified recombinant protein, comprising the steps of: (a) providing a liquid culture medium comprising the recombinant protein that is substantially free of cells; and (b) continuously feeding the liquid culture medium to a multi-column chromatography system (MCCS) comprising at least one of any of the reduced bioburden packed chromatography columns described herein, wherein the method utilizes a reduced bioburden buffer and is integrated and runs continuously from the liquid culture medium to the eluate from the MCCS, which is the purified recombinant protein.

[0080] In some embodiments, the MCCS performs at least two different unit operations.

[0081] In some embodiments, the method includes column switching.

[0082] In some embodiments, the MCCS performs the unit operations of capturing recombinant proteins and inactivating viruses.

[0083] In some embodiments, the MCCS performs the unit operations of capturing and purifying the recombinant protein.

[0084] In some embodiments, the MCCS comprises at least two reduced bioburden packed chromatography columns.

[0085] In some embodiments, the MCCS is a cyclic countercurrent chromatography system.

[0086] In some embodiments, the MCCS comprises multiple columns for affinity or pseudoaffinity chromatography, cation exchange chromatography, anion exchange chromatography, or size exclusion chromatography, or any combination thereof.

[0087] In some embodiments, the MCCS comprises a column for affinity chromatography, which in the method is performed by a capture mechanism selected from the group consisting of: a protein A-binding capture mechanism, a substrate-binding capture mechanism, an antibody or antibody fragment-binding capture mechanism, an aptamer-binding capture mechanism, and a cofactor-binding capture mechanism.

[0088] In some embodiments, affinity chromatography is performed by a Protein A binding capture mechanism and the recombinant protein is an antibody or an antibody fragment.

[0089] Provided herein is an integrated, closed, continuous method for the production of reduced bioburden of purified recombinant protein, comprising: (a) providing a liquid culture medium comprising the recombinant protein that is substantially free of cells; and (b) continuously feeding the liquid culture medium into a first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein in the liquid culture medium using MCCS1; (d) generating an eluate comprising the recombinant protein from MCCS1 and continuously feeding the eluate into a second multi-column chromatography system (MCCS2); and (e) continuously feeding the recombinant protein from the eluate into MCCS2 and thereafter eluting the recombinant protein to produce a purified recombinant protein, wherein the method utilizes a reduced bioburden buffer and is integrated and runs continuously from the liquid culture medium to the purified recombinant protein, and at least one column in MCCS1 and / or MCCS2 contains any of the reduced bioburden packed chromatography columns described herein.

[0090] In some embodiments, MCCS1 and / or MCCS2 perform at least two different unit operations.

[0091] In some embodiments, the method includes column switching.

[0092] In some embodiments, MCCS1 performs the unit operations of capturing recombinant therapeutic proteins and inactivating viruses.

[0093] In some embodiments, MCCS2 performs the unit operations of purifying and polishing the recombinant protein.

[0094] In some embodiments, MCCS1 and / or MCCS2 comprise at least two chromatography columns.

[0095] In some embodiments, the MCCS1 is a first periodic counter-current chromatography system (PCCS1).

[0096] In some embodiments, capture is performed using affinity chromatography, cation exchange chromatography, anion exchange chromatography, or size exclusion chromatography, or any combination thereof.

[0097] In some embodiments, affinity chromatography is performed with a capture mechanism selected from the group consisting of: a protein A-binding capture mechanism, a substrate-binding capture mechanism, an antibody or antibody fragment-binding capture mechanism, an aptamer-binding capture mechanism, and a cofactor-binding capture mechanism.

[0098] In some embodiments, the affinity chromatography implements a Protein-A binding capture mechanism and the recombinant protein is an antibody or an antibody fragment.

[0099] In some embodiments, the MCCS2 is a second periodic countercurrent (PCCS2) chromatography system.

[0100] In some embodiments of any of the methods described herein, the recombinant protein is a therapeutic recombinant protein.

[0101] In some embodiments of any of the methods described herein, the method further comprises formulating the purified therapeutic recombinant protein into a pharmaceutical composition.

[0102] In some embodiments of any of the methods described herein, the methods are performed for a period of at least 4 consecutive days.

[0103] In some embodiments, the method is performed continuously for a period of at least 5 days.

[0104] In some embodiments, the method is performed continuously for a period of at least 7 days.

[0105] In some embodiments, the method is performed continuously for a period of at least 14 days.

[0106] In some embodiments, the method is performed continuously for a period of at least 28 days.

[0107] As used herein, the word "a" before a noun refers to one or more of that noun. For example, the phrase "reduced bioburden chromatography columns" refers to "one or more reduced bioburden chromatography columns."

[0108] The term "bioburden" is known in the art and refers to the level of self-replicating biological contaminants present in a composition (e.g., solid or liquid) and / or on the surface of an article (e.g., external and / or internal surfaces). For example, bioburden can refer to the level of self-replicating biological contaminants present in a chromatography resin or a composition containing packed chromatography resin (e.g., the packed chromatographic resin in a packed chromatography column). In another example, bioburden can refer to self-replicating biological contaminants present on the interior surface of a chromatography column and / or in a chromatography resin packed within a chromatography column (e.g., biological contaminants on the interior surface of a chromatography column and biological contaminants in a packed chromatography resin within a chromatography column). Bioburden can also refer to self-replicating biological contaminants present in a liquid (e.g., a buffer used in any of the methods or processes described herein). Non-limiting examples of self-replicating biological contaminants can be bacteria (e.g., gram-positive or gram-negative bacteria, or bacterial spores), mycobacteria, viruses (e.g., vesivirus, Cache Valley virus, parvovirus, herpes virus, and bunyavirus), parasites, fungi, yeast, and protozoa. Exemplary methods for determining bioburden are described herein. Additional methods for determining bioburden are known in the art.

[0109] The term "reducing bioburden" is known in the art and refers to a reduction (e.g., a detectable reduction) in the level of self-replicating biological contaminants present in a composition (e.g., solid or liquid) and / or on the surface (e.g., external and / or internal surfaces) of an article. Non-limiting examples of methods for reducing the bioburden of a chromatography resin (e.g., packed chromatography resin), buffer, and / or chromatography column (e.g., packed chromatography column) are described herein. Additional methods for reducing the bioburden of any of the compositions described herein are known in the art.

[0110] The term "reduced bioburden chromatography resin" refers to a chromatography resin that has been treated to reduce the level of self-replicating biological contaminants present in the chromatography resin (e.g., a detectable reduction in the level of self-replicating biological contaminants present in a composition containing the chromatography resin, e.g., a slurry). For example, a reduced bioburden chromatography resin can be a resin that has been exposed to a sufficient dose of gamma radiation to reduce the level of self-replicating biological contaminants in the chromatography resin (e.g., a composition containing a chromatography resin that has been exposed to a sufficient dose of gamma radiation to reduce the level of self-replicating biological contaminants in the chromatography resin). For example, the reduced bioburden chromatography resin can be a resin that has been exposed to a dose of gamma radiation of about 1 kGy to about 15 kGy, about 1 kGy to about 20 kGy, about 1 kGy to about 25 kGy, about 1 kGy to about 30 kGy, or about 1 kGy to about 35 kGy. Exemplary methods for reducing the bioburden of a chromatography resin are described herein. Additional methods for reducing the bioburden of a chromatography resin are known in the art.

[0111] The term "reduced bioburden chromatography column" refers to a chromatography column (e.g., a packed chromatography column) that includes a treated chromatography resin (e.g., a gamma irradiated chromatography resin) that contains a level of self-replicating biological contaminants that is lower than the level of self-replicating biological contaminants present in the same chromatography column that includes an untreated chromatography resin. For example, a reduced bioburden chromatography column has a level of at least or about 1×10 -6 , 1×10 -7 , 1×10 -8 , 1×10 -9 , or 1 × 10 -10 The present invention can include a treated chromatography resin having a sterility assurance level of 0.1 to 100%.

[0112] The term "reduced bioburden buffer" is known in the art and refers to a buffer that contains a level of self-replicating contaminating agents that is lower than the level of self-replicating contaminating agents found in the same untreated liquid. By "bioburden" is meant a treated (e.g., filtered, autoclaved, and / or gamma irradiated) liquid (e.g., a treated buffer solution) having a reduced bioburden of at least or about 1×10 -6 , 1×10 -7 , 1×10 -8 , 1×10 -9 , or 1 or 10 -10 The sterility assurance level can be as high as 100%.

[0113] "Absolutely sterile" or "absolutely sterile" is a term used to describe a composition or method that is completely free of self-replicating biological contaminants. For example, the term can apply to gamma-irradiated chromatography resins, the interior and contents of a chromatography column (e.g., chromatography resins), and / or buffers. An absolutely sterile composition or method can be clean (as known in the art).

[0114] "Germ-free" or "sterile" is approximately 1.0 x 10 -6 Less than (e.g., about or 1.0 × 10 -7 Less than, about, or 1.0 x 10 -8 Less than, about, or 1.0 x 10 -9 Or 1×10 -10 Sterile is a term used to describe a composition or method that has a sterility assurance level of less than 100%. Determining whether a composition or method is sterile can be tested using a number of validated production methods known in the art. For example, a sterile composition or method can be completely free of living, self-replicating biological contaminants (e.g., any of the self-replicating biological contaminants described herein). A sterile composition or method can also be clean (as known in the art).

[0115] The term "sterilization" refers to an established method used to render a composition sterile (as defined herein). To determine whether sterility (as defined herein) has been achieved for a composition, the inactivation rate of self-replicating biological contaminants (e.g., bacteria) of resistant indicator bacteria during the treatment process can be measured.

[0116] The term "sterility assurance level" or "SAL" is known in the industry and refers to the confidence level of achieving absolute sterility in a batch of processing units. This probability is usually calculated based on the results of inactivation studies performed during validation and is in the range of 1 x 10 -n It is expressed in the form:

[0117] The term "sterile" is used to describe a composition or method that is free of disease-causing or symptom-causing self-replicating biological contaminants (e.g., any of the self-replicating biological contaminants described herein). A sterile composition or method can also be clean (as that term is known in the art).

[0118] The term "unit operation" is a term of art and refers to a functional step that can be performed in a method of purifying a recombinant protein from a liquid culture medium. For example, a unit operation can be filtering (e.g., removing contaminating bacteria, yeast, viruses and / or mycobacteria, and / or particulate matter from a fluid containing the recombinant protein), capturing, epitope tag removal, purifying, retaining or storing, polishing, viral inactivation, adjusting the ion concentration and / or pH of a fluid containing the recombinant protein, and removing undesirable salts.

[0119] The term "capture" refers to partially purifying or isolating (e.g., at least or about 5%, e.g., at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 10% by weight of a recombinant protein (e.g., a recombinant therapeutic protein) from one or more other components present in the liquid culture medium or diluted liquid culture medium (e.g., culture medium proteins or one or more other components (e.g., DNA, RNA, or other proteins) present in or secreted from a mammalian cell). "Capture" refers to a step performed to enrich the recombinant protein in a culture medium (e.g., about 95% pure). Typically, capture is performed using a chromatography resin to which the recombinant protein binds (e.g., by using affinity chromatography). Non-limiting methods of capturing recombinant protein from liquid culture medium or diluted liquid culture medium are described herein, and other methods are known in the art. Recombinant protein can be captured from liquid culture medium using at least one chromatography column and / or chromatography membrane (e.g., any of the chromatography columns and / or chromatography membranes described herein).

[0120] The term "purifying" refers to a step carried out to isolate a recombinant protein (e.g., a recombinant therapeutic protein) from one or more other impurities (e.g., bulk impurities) or components (e.g., liquid culture medium proteins or one or more other components (e.g., DNA, RNA, other proteins, endotoxins, viruses, etc.) present in a fluid containing the recombinant protein (e.g., a liquid culture medium protein present in or secreted from a mammalian cell). For example, purifying can be carried out during or after an initial capturing step. Purification can be carried out using chromatographic resins, membranes or any other solid support that bind either the recombinant protein or the contaminants (e.g., by use of affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography, or molecular sieve chromatography). The recombinant protein can be purified from a fluid containing the recombinant protein using at least one chromatographic column and / or chromatographic membrane (e.g., any of the chromatographic columns or chromatographic membranes described herein).

[0121] The term "polishing" is a technical term and refers to a process carried out to remove trace or small amounts of residual contaminants or impurities from a fluid containing a recombinant protein (e.g., a recombinant therapeutic protein) close to the desired final purity. For example, polishing can be carried out by passing the fluid containing the recombinant protein through a chromatography column(s) or membrane-type adsorbent(s) that selectively binds to the targeted recombinant protein or to small amounts of contaminants or impurities present in the fluid containing the recombinant protein. In such an example, the eluate / filtrate of the chromatography column(s) or membrane-type adsorbent(s) contains the recombinant protein.

[0122] The term "filtering" refers to removing at least a portion (e.g., at least 80%, 90%, 95%, 96%, 97%, 98% or 99%) of undesirable biotype contaminants (e.g., mammalian cells, bacteria, yeast cells, viruses or mycobacteria) and / or particulate matter (e.g., precipitated proteins) from a liquid (e.g., a liquid culture medium or fluid present in any of the methods described herein).

[0123] The term "eluate / filtrate" is a term of art and refers to the fluid exiting a chromatography column or chromatographic membrane that contains a detectable amount of recombinant protein (eg, a recombinant therapeutic protein).

[0124] The term "integrated process" refers to a process carried out using structural elements that function in concert to achieve a particular result (eg, purification of a recombinant protein from a liquid culture medium).

[0125] The term "continuous method" refers to a method in which a fluid is continuously fed through at least a portion of a system. For example, a continuous method may involve feeding liquid culture medium containing a recombinant protein through a bioreactor. Another example of a continuous method is a method of continuously feeding liquid culture medium containing a recombinant protein from a bioreactor through a first and second MCCS (MCCS1 and MCCS2). Further examples include a method of continuously feeding liquid culture medium containing a recombinant protein through an MCCS, a method of continuously feeding liquid culture medium containing a recombinant protein through MCCS1 and MCCS2, or a method of continuously feeding a fluid containing a recombinant protein through MCCS2.

[0126] The term "closed process" is a term of art and refers to a process that is carried out such that components of the process that come into contact with the recombinant protein (e.g., chromatography resins and / or buffers) or fluids containing the recombinant protein are not intentionally exposed to contaminating agents for a significant period of time (e.g., are not intentionally exposed to air for a significant period of time).

[0127] The term "therapeutic protein drug substance" means a recombinant protein (e.g., an immunoglobulin, protein fragment, artificially modified protein or enzyme) that has been sufficiently purified or isolated from contaminating proteins, lipids and nucleic acids (e.g., contaminating proteins, lipids and nucleic acids present in a liquid culture medium or derived from a host cell (e.g., derived from a mammalian host cell, a yeast host cell or a bacterial host cell) and organism-type contaminants (e.g., viral and bacterial-type contaminants) such that it can be incorporated into a pharmaceutical product without any further significant purification and / or decontamination step(s).

[0128] The term "multi-column chromatography system" or "MCCS" refers to a system made up of a total of two or more chromatography columns and / or chromatographic membranes that are interconnected or undergo a switching process. A non-limiting example of a multi-column chromatography system is a periodic countercurrent chromatography system (PCC), which includes a total of two or more chromatography columns and / or chromatographic membranes that are interconnected or undergo a switching process. Further examples of multi-column chromatography systems are described herein and are also known in the art.

[0129] The term "substantially free" refers to a composition (e.g., liquid culture medium) that is at least or about 90% free (e.g., at least or about 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100% free) of a specified substance (e.g., a mammalian cell or a contaminating protein, nucleic acid, carbohydrate, or lipid derived from a mammalian cell).

[0130] The term "mammalian cell" refers to any cell made from or derived from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow, or rabbit). For example, the mammalian cell may be an immortalized cell. In some embodiments, the mammalian cell is a differentiated cell. In some embodiments, the mammalian cell is an undifferentiated cell. Non-limiting examples of mammalian cells are described herein. Further examples of mammalian cells are known in the art.

[0131] The term "culture" or "cell culture" refers to the maintenance or growth of mammalian cells under a controlled set of physical conditions.

[0132] The term "mammalian cell culture" means a liquid culture medium containing a plurality of mammalian cells maintained or grown under a controlled set of physical conditions.

[0133] The term "liquid culture medium" refers to a fluid that contains sufficient nutrients to allow cells (e.g., mammalian cells) to grow or proliferate in vitro. For example, the liquid culture medium may contain one or more of the following: amino acids (e.g., the 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, the liquid culture medium may contain serum from a mammal. In some embodiments, the liquid culture medium does not contain serum or another extract from a mammal (defined liquid culture medium). In some embodiments, the liquid culture medium may contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Another example of a liquid culture medium is a minimal medium (e.g., a medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture media are described herein. Further examples of liquid culture media are known in the art and are also commercially available. Liquid culture media can contain mammalian cells at any density. For example, as used herein, a volume of liquid culture medium removed from a bioreactor can be substantially free of mammalian cells.

[0134] The term "animal-derived component-free liquid culture medium" refers to a liquid culture medium that does not contain any components derived from mammals (eg, proteins or serum).

[0135] The term "serum-free liquid culture medium" means a liquid culture medium that does not contain mammalian serum.

[0136] The term "serum-containing liquid culture medium" means a liquid culture medium that contains mammalian serum.

[0137] The term "chemically-defined liquid culture medium" is a term of art and refers to a liquid culture medium whose entire chemical composition is known. For example, a chemically-defined liquid culture medium does not contain fetal bovine serum, bovine serum albumin, or human serum albumin, although such preparations typically contain a complex mixture of albumin and lipids.

[0138] The term "protein-free liquid culture medium" means a liquid culture medium that does not contain any proteins (eg, any detectable proteins).

[0139] The term "immunoglobulin" refers to a polypeptide that includes an amino acid sequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids) for an immunoglobulin protein (e.g., variable domain sequence, framework sequence, and / or constant domain sequence). An immunoglobulin can, for example, include a light chain immunoglobulin, e.g., of at least 15 amino acids, and a heavy chain immunoglobulin, e.g., of at least 15 amino acids. An immunoglobulin is an isolated antibody (e.g., IgG, IgE, IgD, IgA, or IgM), e.g., a subclass of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). An immunoglobulin can be an antibody fragment, e.g., a Fab fragment, a F(ab')2 fragment, or a scFv fragment. The immunoglobulin may be a bispecific or trispecific antibody, or a dimeric, trimeric or multimeric antibody, or a diabody, Affibody® or Nanobody®. The immunoglobulin may be an artificially engineered protein (e.g., a fusion protein) that includes at least one immunoglobulin domain. Non-limiting examples of immunoglobulins are described herein, and further examples of immunoglobulins can be found in the art. It is well known in the field.

[0140] The term "protein fragment" or "polypeptide fragment" refers to a portion of a polypeptide sequence that is at least or about 4 amino acids, at least or about 5 amino acids, at least or about 6 amino acids, at least or about 7 amino acids, at least or about 8 amino acids, at least or about 9 amino acids, at least or about 10 amino acids, at least or about 11 amino acids, at least or about 12 amino acids, at least or about 13 amino acids, at least or about 14 amino acids, at least or about 15 amino acids, at least or about 16 amino acids, at least or about 17 amino acids, at least or about 18 amino acids, at least or about 19 amino acids, or at least or about 20 amino acids in length, or more than 20 amino acids in length. Recombinant protein fragments can be produced using any of the methods described herein.

[0141] The term "artificially modified protein" refers to a polypeptide that is not naturally encoded by an endogenous nucleic acid present within an organism (e.g., a mammal). Examples of artificially modified proteins include enzymes (e.g., that have undergone one or more amino acid substitutions, deletions, insertions, or additions that result in increased stability and / or catalytic activity of the artificially modified enzyme), fusion proteins, antibodies (e.g., bivalent antibodies, trivalent antibodies, or diabodies), and antigen binding proteins that include at least one engineered scaffold sequence.

[0142] The term "secreted protein" or "secreted recombinant protein" refers to a protein (e.g., a recombinant protein) that is at least partially secreted into the extracellular space (e.g., liquid culture medium) when at least one originally contained secretory signal sequence is translated inside a mammalian cell and the secretory signal sequence in the mammalian cell is at least partially cleaved by an enzyme. It will be understood by those skilled in the art that a "secreted" protein need not be completely free of the cell to be considered a secreted protein.

[0143] The term "perfusion bioreactor" refers to a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, where culturing the cells present in the bioreactor includes periodically or continuously withdrawing a first liquid culture medium and simultaneously or shortly thereafter adding a substantially equal volume of a second liquid culture medium to the bioreactor. In some examples, there is an incremental change (e.g., increase or decrease) in the volume of the first liquid culture medium that is withdrawn and added over an incremental period (e.g., about a 24 hour period, a period between about 1 minute and about 24 hours, or a period greater than 24 hours) during the culture period (e.g., culture medium refeed rate on a daily basis). The percentage of medium withdrawn and replaced each day can vary depending on the particular cells being cultured, the initial seeding density, and the cell density at a particular time. "RV" or "reactor volume" refers to the volume of culture medium present at the start of the culture process (e.g., the total volume of culture medium present after seeding).

[0144] The term "fed-batch bioreactor" is a term of art and refers to a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, where culturing the cells present in the bioreactor involves periodic or continuous addition of a second liquid culture medium to the first liquid culture medium without significant or significant withdrawal of the first liquid culture medium or the second liquid culture medium from the cell culture. The second liquid culture medium can be the same as the first liquid culture medium. In some examples of fed-batch cultures, the second liquid culture medium is the first liquid culture medium in a concentrated form. In some examples of fed-batch cultures, the second liquid culture medium is the first liquid culture medium in a concentrated form. In one example, the second liquid culture medium is added as a dry powder.

[0145] The term "clarified liquid culture medium" refers to a liquid culture medium obtained from a bacterial or yeast cell culture that is substantially free of bacterial or yeast cells (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% free).

[0146] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in the present invention, but other suitable methods and materials known in the art can also be used.The materials, methods and examples are only for illustration and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries and other reference materials mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including provisions, will control.

[0147] Other features and advantages of the invention will become apparent from the following detailed description, drawings, and claims. [Brief description of the drawings]

[0148] [Figure 1]FIG. 1 is a graph showing the percent binding capacity of MabSelect™ SuRe™ (Protein A chromatography resin) over multiple cycles of chromatography after irradiation to 40-49 kGy in the presence of one of the following buffers: (i) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, and 25 mM mannitol in 50 mM sodium phosphate buffer ("SMM'H"); (ii) 2% v / v benzyl alcohol ("2% BA"); and (iii) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, and 2% v / v benzyl alcohol in 50 mM sodium phosphate buffer ("SMM'H+2% BA"). [Diagram 2] FIG. 2 is a graph showing the percent binding capacity of Capto Adhere chromatography resin (compared to unirradiated chromatography resin loaded with the same material) over multiple cycles of chromatography after irradiation to 28-34 kGy or 40-49 kGy (as described in Example 2) in the presence of one of the following buffers: (i) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, and 25 mM mannitol in 50 mM sodium phosphate buffer ("SMM'H"); or (ii) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, and 2% v / v benzyl alcohol in 50 mM sodium phosphate buffer ("SMM'H+2% BA"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0149] Provided herein is a method of reducing the bioburden of a chromatography resin, comprising exposing a container containing a composition comprising (i) a chromatography resin and (ii) a liquid comprising at least one (e.g., 2, 3, 4, or 5) alcohols to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein the at least one alcohol is present in an amount sufficient to ameliorate loss of binding capacity of the chromatography resin after / during exposure to the dose of gamma radiation. Also provided are reduced bioburden chromatography columns containing reduced bioburden chromatography resins prepared by any of the methods described herein; compositions comprising (i) a chromatography resin and (ii) a liquid comprising at least one (e.g., 2, 3, 4, or 5) alcohols; and the reduced bioburden chromatography columns containing reduced bioburden chromatography resins prepared by any of the methods described herein. Also provided are methods for performing reduced bioburden column chromatography using at least one of the reduced bioburden chromatography columns, and integrated, closed or substantially closed, continuous methods for the production of reduced bioburden purified recombinant proteins comprising the use of at least one of these reduced bioburden chromatography columns. Non-limiting aspects of these methods and processes are described below. As can be understood in the art, the various aspects described below can be used in any combination without limitation.

[0150] Compositions containing a chromatography resin and at least one alcohol Provided herein is a composition comprising (i) a chromatography resin (e.g., any chromatography resin described herein or known in the art) and (ii) a liquid comprising at least one (e.g., two, three, four, or five) alcohols (e.g., any of the representative alcohols described herein or known in the art), wherein the at least one alcohol is present in an amount sufficient to ameliorate loss of binding capacity of the chromatography resin upon treatment with gamma radiation at a dose sufficient to reduce the bioburden of the composition. For example, the chromatography resin can be at least one of an anion exchange chromatography resin, a cation exchange chromatography resin, an affinity or pseudoaffinity chromatography resin, a hydrophobic interaction chromatography resin, and a size exclusion chromatography resin, or any combination thereof. In some examples, the chromatography resin is a resin comprising a protein or peptide ligand (e.g., an affinity chromatography resin with a protein or peptide ligand, e.g., a protein A or protein G chromatography resin).

[0151] The composition can be, for example, a slurry of deposited chromatography resin. In some instances, the composition can be a mixture of wet or moist solids. In some instances, the composition is a chromatography resin packed in a liquid.

[0152] In some examples of any of the compositions, at least one (e.g., two, three, four, or five) alcohols can be selected from the group consisting of benzyl alcohol, cyclohexanol, isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol, propan-2-ol, propan-1-ol, butan-1-ol, pentan-1-ol, hexadecan-1-ol, 2-phenylethanol, sec-phenylethanol, 3-phenyl-1-propanol, 1-phenyl-1-propanol, 2-phenyl-1-propanol, 2-phenyl-2-propanol, 1-phenyl-2-butanol, 2-phenyl-1-butanol, 3-phenyl-1-butanol, 4-phenyl-2-butanol, dl-1-phenyl-2-pentanol, 5-phenyl-1-pentanol, and 4-phenyl-1-butanol. In some examples, at least one alcohol can be benzyl alcohol.

[0153] In some examples of any of the compositions, the total concentration of the one or more alcohols in the liquid or composition is from about 0.01% v / v to about 20% v / v, from about 0.01% v / v to about 19% v / v, from about 0.01% v / v to about 18% v / v, from about 0.01% v / v to about 17% v / v, from about 0.01% v / v to about 16% v / v, from about 0.01% v / v to about 15% v / v, from about 0.01% v / v to about 14% v / v, from about 0.01% v / v to about 13% v / v, from about 0.01% v / v to about 15% v / v, from about 0.01% v / v to about 16% v / v, from about 0.01% v / v to about 17% v / v, from about 0.01% v / v to about 18% v / v, from about 0.01% v / v to about 19% v / v, from about 0.01% v / v to about 20% v / v, from about 0.01% v / v to about 20% v / v, from about 0.01% v / v to about 21% v / v, from about 0.01% v / v to about 22% v / v, from about 0.01% v / v to about 23% v / v, from about 0.01% v / v to about 24% v / v, from about 0.01% v / v to about 25% v / v, from about 0.01% v / v to about 26% v / v, from about 0.01% v / v to about 27% v / v, from about 0.01% v / v to about 28% v / v, from about 01%v / v~Approx. 12%v / v, Approx. 0.01%v / v~Approx. 11%v / v, Approx. 0.01%v / v~Approx. 10%v / v, Approx. 0.01%v / v~Approx. 9%v / v, Approx. 0.01%v / v~Approx. 8%v / v, Approx. 0.01%v / v~Approx. 7%v / v, about 0.01%v / v to about 6%v / v, about 0.01%v / v to about 5%v / v, about 0.01%v / v to about 4.5%v / v, about 0.01%v / v to about 4.0%v / v, about 0.01%v / v to about 3.5%v / v, about 0.01%v / v ~ about 3.0% v / v, about 0.01% v / v to about 2.5% v / v, about 0.01% v / v to about 2.2% v / v, about 0.01% v / v to about 2.0% v / v, about 0.01% v / v to about 1.8% v / v, about 0.01% v / v to about 1.6% v / v, about 0.01% v / v to about 1.4% v / v, about 0.01% v / v to about 1.2% v / v, about 0.01% v / v to about 1.0% v / v, about 0.01% v / v to about 0.8% v / v, about 0.01% v / v to about 0.6% v / v, about 0.01% v / v to about 0.4% v / v, about 0.01% v / v to about 0.2% v / v, about 0.01% v / v to about 0.1% v / v, about 0.01% v / v to about 0.05% v / v, about 0.05% v / v to about 20% v / v, about 0.05% v / v to about 19% v / v, about 0.05% v / v to about 18% v / v, about 0.05% v / v to about 17% v / v, about 0.05% v / v to about 16% v / v, about 0.05% v / v to about 15% v / v, about 0.05% v / v to about 14% v / v, about 0.05% v / v to about 13% v / v, about 0.05% v / v to about 12% v / v, about 0.05% v / v to about 11% v / v, about 0.05% v / v to about 10% v / v, about 0.05% v / v to about 9% v / v, about 0.05% v / v to about 8% v / v, about 0.05% v / v to about 7% v / v, about 0.05% v / v to about 6% v / v, about 0.05% v / v to about 5% v / v, about 0.05% v / v to about 4.5% v / v, about 0.05% v / v to about 4.0% v / v, about 0.05% v / v to about 3.5% v / v, about 0.05% v / v to about 3.0% v / v, about 0.05% v / v to about 2.5% v / v, about 0.05% v / v to about 2.2% v / v, about 0.05% v / v to about 2.0% v / v, about 0.05% v / v to about 1.8% v / v, about 0.05% v / v to about 1.6% v / v, about 0.05% v / v to about 1.4% v / v, about 0.05% v / v to about 1.2% v / v, about 0.05% v / v to about 1.0% v / v, about 0.05% v / v to about 0.8% v / v, about 0.05% v / v to about 0.6% v / v, about 0.05% v / v to about 0.4% v / v, about 0.05% v / v to about 0.2% v / v, about 0.05% v / v to about 0.1% v / v, about 0.1% v / v to about 20% v / v, about 0.1% v / v to about 19% v / v, about 0.1% v / v to about 18% v / v, about 0.1% v / v to about 17% v / v, about 0.1% v / v to about 16% v / v, about 0.1% v / v to about 15% v / v, about 0.1% v / v to approximately 14% v / v, 0.1% v / v to approximately 13% v / v, 0.1% v / v to approximately 12% v / v, 0.1% v / v to approximately 11% v / v, 0.1% v / v to approximately 10% v / v, 0.1% v / v to approximately 9% v / v, 0.1% v / v to approximately 8% v / v, 0.1% v / v to approximately 7% v / v, 0.1% v / v to approximately 6% v / v, 0.1% v / v to approximately 5% v / v, 0.1% v / v to approximately 4.5% v / v, 0.1% v / v to approximately 4.0% v / v, 0.1% v / v to approximately 3.5% v / v, 0.1% v / v to approximately 3.0% v / v, 0.1% v / v to approximately 2.5% v / v, 0.1% v / v to approximately 2.2% v / v, 0.1% v / v to approximately 2.0% v / v, 0.1% v / v to approximately 1.8% v / v, 0.1% v / v to approximately 1.6% v / v, 0.1% v / v to approximately 1.4% v / v, 0.1% v / v to approximately 1.2% v / v, 0.1% v / v to approximately 1.0% v / v, 0.1% v / v to approximately 0.8% v / v, 0.1% v / v to approximately 0.6% v / v, 0.1% v / v to approximately 0.4% v / v, 0.1% v / v to approximately 0.2% v / v, 0.2% v / v to approximately 20% v / v, 0.2% v / v to approximately 19% v / v, 0.2% v / v to approximately 18% v / v, 0.2% v / v to approximately 17% v / v, 0.2% v / v to approximately 16% v / v, 0.2% v / v to approximately 15% v / v, 0.2% v / v to approximately 14% v / v, 0.2% v / v to approximately 13% v / v, 0.2% v / v to approximately 12% v / v, 0.2% v / v to approximately 11% v / v, 0.2% v / v to approximately 10% v / v, 0.2% v / v to approximately 9% v / v, 0.2% v / v to approximately 8% v / v, 0.2% v / v to approximately 7% v / v, 0.2% v / v to approximately 6% v / v, 0.2% v / v to approximately 5% v / v, 0.2% v / v to approximately 4.5% v / v, 0.2% v / v to approximately 4.0% v / v, 0.2% v / v to approximately 3.5% v / v, 0.2% v / v to approximately 3.0% v / v, 0.2% v / v to approximately 2.5% v / v, 0.2% v / v to approximately 2.2% v / v, 0.2% v / v to approximately 2.0% v / v, 0.2% v / v to approximately 1.8% v / v, 0.2% v / v to approximately 1.6% v / v, 0.2% v / v to approximately 1.4% v / v, 0.2% v / v to approximately 1.2% v / v, 0.2% v / v to approximately 1.0% v / v, 0.2% v / v to approximately 0.8% v / v, 0.2% v / v to approximately 0.6% v / v, 0.2% v / v to approximately 0.4% v / v, 0.4%v / v~20%v / v, 0.4%v. / v~about 19% v / v, about 0.4% v / v~about 18% v / v, about 0.4% v / v~about 17% v / v, about 0.4% v / v~about 16% v / v, about 0.4% v / v~about 15% v / v, about 0.4% v / v~about 14% v / v, about 0.4% v / v~about 13% v / v, about 0.4% v / v~about 12% v / v, about 0.4% v / v~about 11% v / v, about 0.4% v / v~about 10% v / v, about 0.4% v / v~about 9% v / v, about 0.4% v / v~about 8% v / v, about 0.4% v / v~about 7% v / v, about 0.4% v / v~about 6% v / v, about 0.4% v / v~about 5% v / v, about 0.4% v / v~about 4.5% v / v, about 0.4% v / v~about 4.0% v / v, about 0.4% v / v~about 3.5% v / v, about 0.4% v / v~about 3.0% v / v, about 0.4% v / v~about 2.5% v / v, about 0.4% v / v~about 2.2% v / v, about 0.4% v / v~about 2.0% v / v, about 0.4% v / v~about 1.8% v / v, about 0.4% v / v~about 1.6% v / v, about 0.4% v / v~about 1.4% v / v, about 0.4% v / v~about 1.2% v / v, about 0.4% v / v~about 1.0% v / v, about 0.4% v / v~about 0.8% v / v, about 0.4% v / v~about 0.6% v / v, about 0.6% v / v~about 20% v / v, about 0.6% v / v~about 19% v / v, about 0.6% v / v~about 18% v / v, about 0.6% v / v~about 17% v / v, about 0.6% v / v~about 16% v / v, about 0.6% v / v~about 15% v / v, about 0.6% v / v~about 14% v / v, about 0.6% v / v~about 13% v / v, about 0.6% v / v~about 12% v / v, about 0.6% v / v~about 11% v / v, about 0.6% v / v~about 10% v / v, about 0.6% v / v~about 9% v / v, about 0.6% v / v~about 8% v / v, about 0.6% v / v~about 7% v / v, about 0.6% v / v~about 6% v / v, about 0.6% v / v~about 5% v / v, about 0.6% v / v~about 4.5% v / v, about 0.6% v / v~about 4.0% v / v, about 0.6% v / v~about 3.5% v / v, about 0.6% v / v~about 3.0% v / v, about 0.6% v / v~about 2.5% v / v, about 0.6% v / v~about 2.2% v / v, about 0.6% v / v~about 2.0% v / v, about 0.6% v / v~about 1.8% v / v, about 0.6% v / v~about 1.6% v / v, about 0.6% v / v~about 1.4% v / v, about 0.6% v / v~about 1.2% v / v, about 0.6% v / v~about 1.0% v / v, about 0.6% v / v~about 0.8% v / v, about 0.8% v / v to approximately 20% v / v, approximately 0.8% v / v to approximately 19% v / v, approximately 0.8% v / v to approximately 18% v / v, approximately 0.8% v / v to approximately 17% v / v, approximately 0.8% v / v to approximately 16% v / v, approximately 0.8% v / v to approximately 15% v / v, approximately 0.8% v / v to approximately 14% v / v, approximately 0.8% v / v to approximately 13% v / v, approximately 0.8% v / v to approximately 12% v / v, approximately 0.8% v / v to approximately 11% v / v, approximately 0.8% v / v to approximately 10% v / v, approximately 0.8% v / v to approximately 9% v / v, approximately 0.8% v / v to approximately 8% v / v, approximately 0.8% v / v to approximately 7% v / v, approximately 0.8% v / v to approximately 6% v / v, approximately 0.8% v / v to approximately 5% v / v, approximately 0.8% v / v to approximately 4.5% v / v, approximately 0.8% v / v to approximately 4.0% v / v, approximately 0.8% v / v to approximately 3.5% v / v, approximately 0.8% v / v to approximately 3.0% v / v, approximately 0.8% v / v to approximately 2.5% v / v, approximately 0.8% v / v to approximately 2.2% v / v, approximately 0.8% v / v to approximately 2.0% v / v, approximately 0.8% v / v to approximately 1.8% v / v, approximately 0.8% v / v to approximately 1.6% v / v, approximately 0.8% v / v to approximately 1.4% v / v, approximately 0.8% v / v to approximately 1.2% v / v, approximately 0.8% v / v to approximately 1.0% v / v, approximately 1.0% v / v to approximately 20% v / v, approximately 1.0% v / v to approximately 19% v / v, approximately 1.0% v / v to approximately 18% v / v, approximately 1.0% v / v to approximately 17% v / v, approximately 1.0% v / v to approximately 16% v / v, approximately 1.0% v / v to approximately 15% v / v, approximately 1.0% v / v to approximately 14% v / v, approximately 1.0% v / v to approximately 13% v / v, approximately 1.0% v / v to approximately 12% v / v, approximately 1.0% v / v to approximately 11% v / v, approximately 1.0% v / v to approximately 10% v / v, approximately 1.0% v / v to approximately 9% v / v, approximately 1.0% v / v to approximately 8% v / v, approximately 1.0% v / v to approximately 7% v / v, approximately 1.0% v / v to approximately 6% v / v, approximately 1.0% v / v to approximately 5% v / v, approximately 1.0% v / v to approximately 4.5% v / v, approximately 1.0% v / v to approximately 4.0% v / v, approximately 1.0% v / v to approximately 3.5% v / v, approximately 1.0% v / v to approximately 3.0% v / v, approximately 1.0% v / v to approximately 2.5% v / v, approximately 1.0% v / v to approximately 2.2% v / v, approximately 1.0% v / v to approximately 2.0% v / v, approximately 1.0% v / v to approximately 1.8% v / v, approximately 1.0% v / v to approximately 1.6% v / v, approximately 1.0% v / v to approximately 1.4% v / v, approximately 1.0% v / v to approximately 1.2% v / v, approximately 1.2% v / v to approximately 20% v / v, approximately 1.2% v / v to approximately 19% v / v, approximately 1.2% v / v to approximately 18% v / v, approximately 1.2... % v / v ~ about 17% v / v, about 1.2% v / v ~ about 16% v / v, about 1.2% v / v ~ about 15% v / v, about 1.2% v / v ~ about 14% v / v, about 1.2% v / v ~ about 13% v / v, about 1.2% v / v ~ about 12% v / v, about 1.2% v / v ~ about 11% v / v, about 1.2% v / v ~ about 10% v / v, about 1.2% v / v ~ about 9% v / v, about 1.2% v / v ~ about 8% v / v, From about 1.2% v / v to about 7% v / v, from about 1.2% v / v to about 6% v / v, from about 1.2% v / v to about 5% v / v, from about 1.2% v / v to about 4.5% v / v, from about 1.2% v / v to about 4.0% v / v, from about 1.2% v / v to about 3.5% v / v, from about 1.2% v / v to about 3.0% v / v, from about 1.2% v / v to about 2.5% v / v, from about 1.2% v / v to about 2.2% v / v, from about 1.2% v / v to about 2.0% v / v, from about 1.2% v / v to about 1.8% v / v, from about 1.2% v / v to about 1.6% v / v, from about 1.2% v / v to about 1.4% v / v, from about 1.4% v / v to about 20% v / v, from about 1.4% v / v to about 19% v / v, from about 1.4% v / v to about 18% v / v, from about 1.4% v / v to about 17% v / v, from about 1.4% v / v to about 16% v / v, from about 1.4% v / v to about 15% v / v, from about 1.4% v / v to about 14% v / v, from about 1.4% v / v to about 13% v / v, from about 1.4% v / v to about 12% v / v, from about 1.4% v / v to about 11% v / v, from about 1.4% v / v to about 10% v / v, from about 1.4% v / v to about 9% v / v, from about 1.4% v / v to about 8% v / v, from about 1.4% v / v to about 7% v / v, from about 1.4% v / v to about 6% v / v, from about 1.4% v / v to about 5% v / v, from about 1.4% v / v to about 4.5% v / v, from about 1.4% v / v to about 4.0% v / v, from about 1.4% v / v to about 3.5% v / v, from about 1.4% v / v to about 3.0% v / v, from about 1.4% v / v to about 2.5% v / v, from about 1.4% v / v to about 2.2% v / v, from about 1.4% v / v to about 2.0% v / v, from about 1.4% v / v to about 1.8% v / v, from about 1.4% v / v to about 1.6% v / v, from about 1.6% v / v to about 20% v / v, from about 1.6% v / v to about 19% v / v, from about 1.6% v / v to about 18% v / v, from about 1.6% v / v to about 17% v / v, from about 1.6% v / v to about 16% v / v, from about 1.6% v / v to about 15% v / v, from about 1.6% v / v to about 14% v / v, from about 1.6% v / v to about 13% v / v, from about 1.6% v / v to about 12% v / v, from about 1.6% v / v to about 11% v / v, from about 1.6% v / v to about 10% v / v, from about 1.6% v / v to about 9% v / v, from about 1.6% v / v to about 8% v / v, from about 1.6% v / v to about 7% v / v, from about 1.6% v / v to about 6% v / v, from about 1.6% v / v to about 5% v / v, from about 1.6% v / v to about 4.5% v / v, from about 1.6% v / v to about 4.0% v / v, from about 1.6% v / v to about 3.5% v / v, from about 1.6% v / v to about 3.0% v / v, from about 1.6% v / v to approximately 2.5% v / v, approximately 1.6% v / v to approximately 2.2% v / v, approximately 1.6% v / v to approximately 2.0% v / v, approximately 1.6% v / v to approximately 1.8% v / v, approximately 1.8% v / v to approximately 20% v / v, approximately 1.8% v / v to approximately 19% v / v, approximately 1.8% v / v to approximately 18% v / v, approximately 1.8% v / v to approximately 17% v / v, approximately 1.8% v / v to approximately 16% v / v, approximately 1.8% v / v to approximately 15% v / v, approximately 1.8% v / v to approximately 14% v / v, approximately 1.8% v / v to approximately 13% v / v, approximately 1.8% v / v to approximately 12% v / v, approximately 1.8% v / v to approximately 11% v / v, approximately 1.8% v / v to approximately 10% v / v, approximately 1.8% v / v to approximately 9% v / v, approximately 1.8% v / v to approximately 8% v / v, approximately 1.8% v / v to approximately 7% v / v, approximately 1.8% v / v to approximately 6% v / v, approximately 1.8% v / v to approximately 5% v / v, approximately 1.8% v / v to approximately 4.5% v / v, approximately 1.8% v / v to approximately 4.0% v / v, approximately 1.8% v / v to approximately 3.5% v / v, approximately 1.8% v / v to approximately 3.0% v / v, approximately 1.8% v / v to approximately 2.5% v / v, approximately 1.8% v / v to approximately 2.2% v / v, approximately 1.8% v / v to approximately 2.0% v / v, approximately 2.0% v / v to approximately 20% v / v, approximately 2.0% v / v to approximately 19% v / v, approximately 2.0% v / v to approximately 18% v / v, approximately 2.0% v / v to approximately 17% v / v, approximately 2.0% v / v to approximately 16% v / v, approximately 2.0% v / v to approximately 15% v / v, approximately 2.0% v / v to approximately 14% v / v, approximately 2.0% v / v to approximately 13% v / v, approximately 2.0% v / v to approximately 12% v / v, approximately 2.0% v / v to approximately 11% v / v, approximately 2.0% v / v to approximately 10% v / v, approximately 2.0% v / v to approximately 9% v / v, approximately 2.0% v / v to approximately 8% v / v, approximately 2.0% v / v to approximately 7% v / v, approximately 2.0% v / v to approximately 6% v / v, approximately 2.0% v / v to approximately 5% v / v, approximately 2.0% v / v to approximately 4.5% v / v, approximately 2.0% v / v to approximately 4.0% v / v, approximately 2.0% v / v to approximately 3.5% v / v, approximately 2.0% v / v to approximately 3.0% v / v,... From about 2.0% v / v to about 2.5% v / v, from about 2.0% v / v to about 2.2% v / v, from about 2.2% v / v to about 20% v / v, from about 2.2% v / v to about 19% v / v, from about 2.2% v / v to about 18% v / v, from about 2.2% v / v to about 17% v / v, from about 2.2% v / v to about 16% v / v, from about 2.2% v / v to about 15% v / v, from about 2.2% v / v to about 14% v / v, from about 2.2% v / v to about 13% v / v, from about 2.2% v / v to about 12% v / v, from about 2.2% v / v to about 11% v / v, from about 2.2% v / v to about 10% v / v, from about 2.2% v / v to about 9% v / v, from about 2.2% v / v to about 8% v / v, from about 2.2% v / v to about 7% v / v, from about 2.2% v / v to about 6% v / v, from about 2.2% v / v to about 5% v / v, from about 2.2% v / v to about 4.5% v / v, from about 2.2% v / v to about 4.0% v / v, from about 2.2% v / v to about 3.5% v / v, from about 2.2% v / v to about 3.0% v / v, from about 2.2% v / v to about 2.5% v / v, from about 2.5% v / v to about 20% v / v, from about 2.5% v / v to about 19% v / v, from about 2.5% v / v to about 18% v / v, from about 2.5% v / v to about 17% v / v, from about 2.5% v / v to about 16% v / v, from about 2.5% v / v to about 15% v / v, from about 2.5% v / v to about 14% v / v, from about 2.5% v / v to about 13% v / v, from about 2.5% v / v to about 12% v / v, from about 2.5% v / v to about 11% v / v, from about 2.5% v / v to about 10% v / v, from about 2.5% v / v to about 9% v / v, from about 2.5% v / v to about 8% v / v, from about 2.5% v / v to about 7% v / v, from about 2.5% v / v to about 6% v / v, from about 2.5% v / v to about 5% v / v, from about 2.5% v / v to about 4.5% v / v, from about 2.5% v / v to about 4.0% v / v, from about 2.5% v / v to about 3.5% v / v, from about 2.5% v / v to about 3.0% v / v, from about 3.0% v / v to about 20% v / v, from about 3.0% v / v to about 19% v / v, from about 3.0% v / v to about 18% v / v, from about 3.0% v / v to about 17% v / v, from about 3.0% v / v to about 16% v / v, from about 3.0% v / v to about 15% v / v, from about 3.0% v / v to about 14% v / v, from about 3.0% v / v to about 13% v / v, from about 3.0% v / v to about 12% v / v, from about 3.0% v / v to about 11% v / v, from about 3.0% v / v to about 10% v / v, from about 3.0% v / v to about 9% v / v, from about 3.0% v / v to about 8% v / v, from about 3.0% v / v to about 7% v / v, from about 3.0% v / v to about 6% v / v, from about 3.0% v / v to approximately 5% v / v, approximately 3.0% v / v to approximately 4.5% v / v, approximately 3.0% v / v to approximately 4.0% v / v, approximately 3.0% v / v to approximately 3.5% v / v, approximately 3.5% v / v to approximately 20% v / v, approximately 3.5% v / v to approximately 19% v / v, approximately 3.5% v / v to approximately 18% v / v, approximately 3.5% v / v to approximately 17% v / v, approximately 3.5% v / v to approximately 16% v / v, approximately 3.5% v / v to approximately 15% v / v, approximately 3.5% v / v to approximately 14% v / v, approximately 3.5% v / v to approximately 13% v / v, approximately 3.5% v / v to approximately 12% v / v, approximately 3.5% v / v to approximately 11% v / v, approximately 3.5% v / v to approximately 10% v / v, approximately 3.5% v / v to approximately 9% v / v, approximately 3.5% v / v to approximately 8% v / v, approximately 3.5% v / v to approximately 7% v / v, approximately 3.5% v / v to approximately 6% v / v, approximately 3.5% v / v to approximately 5% v / v, approximately 3.5% v / v to approximately 4.5% v / v, approximately 3.5% v / v to approximately 4.0% v / v, approximately 4.0% v / v to approximately 20% v / v, approximately 4.0% v / v to approximately 19% v / v, approximately 4.0% v / v to approximately 18% v / v, approximately 4.0% v / v to approximately 17% v / v, approximately 4.0% v / v to approximately 16% v / v, approximately 4.0% v / v to approximately 15% v / v, approximately 4.0% v / v to approximately 14% v / v, approximately 4.0% v / v to approximately 13% v / v, approximately 4.0% v / v to approximately 12% v / v, approximately 4.0% v / v to approximately 11% v / v, approximately 4.0% v / v to approximately 10% v / v, approximately 4.0% v / v to approximately 9% v / v, approximately 4.0% v / v to approximately 8% v / v, approximately 4.0% v / v to approximately 7% v / v, approximately 4.0% v / v to approximately 6% v / v, approximately 4.0% v / v to approximately 5% v / v, approximately 4.0% v / v to approximately 4.5% v / v, approximately 4.5% v / v to approximately 20% v / v, approximately 4.5% v / v to approximately 19% v / v, approximately 4.5% v / v to approximately 18% v / v, approximately 4.5% v / v to approximately 17% v / v, approximately 4.5% v / v to approximately 16% v / v, approximately 4.5% v / v to approximately 15% v / v, approximately 4.5% v / v to approximately 14% v / v, approximately 4.5% v / v to approximately 13% v / v, approximately 4.5% v / v to approximately 12% v / v, approximately 4.5% v / v to approximately 11% v / v, approximately 4.5% v / v to approximately 10% v / v, approximately 4.5% v / v to approximately 9% v / v, approximately 4.5% v / v to approximately 8% v / v, approximately 4.5% v / v to approximately 7% v / v, approximately 4.5% v / v to approximately 6% v / v, approximately 4.5% v / v to approximately 5% v / v, 5% v / v to approximately 20% v / v, 5% v / v to approximately 19% v / v, 5% v / v to approximately 18% v / v, 5% v / v to approximately 17% v / v, 5% v / v to approximately 16% v / v, 5% v / v to approximately 15% v / v, 5. % v / v to approximately 14% v / v, 5% v / v to approximately 13% v / v, 5% v / v to approximately 12% v / v, 5% v / v to approximately 11% v / v, 5% v / v to approximately 10% v / v, 5% v / v to approximately 9% v / v, 5% v / v to approximately 8% v / v, 5% v / v to approximately 7% v / v, 5% v / v to approximately 6% v / v, 6% v / v to approximately 20% v / v, 6% v / v to approximately 19% v / v, 6% v / v to approximately 18% v / v, 6% v / v to approximately 17% v / v, 6% v / v to approximately 16% v / v, 6% v / v to approximately 15% v / v, 6% v / v to approximately 14% v / v, 6% v / v to approximately 13% v / v, 6% v / v to approximately 12% v / v, 6% v / v to approximately 11% v / v, 6% v / v to approximately 10% v / v, 6% v / v to approximately 9% v / v, 6% v / v to approximately 8% v / v, 6% v / v to approximately 7% v / v, 7% v / v to approximately 20% v / v, 7% v / v to approximately 19% v / v, 7% v / v to approximately 18% v / v, 7% v / v to approximately 17% v / v, 7% v / v to approximately 16% v / v, 7% v / v to approximately 15% v / v, 7% v / v to approximately 14% v / v, 7% v / v to approximately 13% v / v, 7% v / v to approximately 12% v / v, 7% v / v to approximately 11% v / v, 7% v / v to approximately 10% v / v, 7% v / v to approximately 9% v / v, 7% v / v to approximately 8% v / v, 8% v / v to approximately 20% v / v, 8% v / v to approximately 19% v / v, 8% v / v to approximately 18% v / v, 8% v / v to approximately 17% v / v, 8% v / v to approximately 16% v / v, 8% v / v to approximately 15% v / v, about 8% v / v to about 14% v / v, about 8% v / v to about 13% v / v, about 8% v / v to about 12% v / v, about 8% v / v to about 11% v / v, about 8% v / v to about 10% v / v, about 8% v / v to about 9% v / v, about 9% v / v to about 20% v / v, about 9% v / v to about 19% v / v, about 9% v / v to about 18% v / v, about 9% v / v to about 17% v / v, about 9% v / v to about 16% v / v, about 9% v / v to about 15% v / v, about 9% v / v to about 14% v / v, about 9% v / v to about 13% v / v, about 9% v / v to about 12% v / v, about 9% v / v to about 11% v / v, about 9% v / v to about 10% v / v, about 10% v / v to about 20% v / v, about 10% v / v to about 19% v / v, about 10% v / v to about 18% v / v, about 10% v / v to about 17% v / v, about 10% v / v to about 16% v / v, about 10% v / v to about 15% v / v, about 10% v / v to about 14% v / v, about 10% v / v to about 13% v / v, about 10% v / v to about 12% v / v, about 10% v / v to about 11% v / v, about 11% v / v to about 20% v / v, about 11% v / v to about 19% v / v, about 11% v / v to about 18% v / v, about 11% v / v to about 17% v / v, about 11% v / v to about 16% v / v, about 11% v / v to about 15% v / v, about 11% v / v to about 14% v / v, about 11% v / v to about 13% v / v, about 11% v / v to about 12% v / v, about 12% v / v to about 20% v / v, about 12% v / v to about 19% v / v, about 12% v / v to about 18% v / v, about 12% v / v to about 17% v / v, about 12% v / v to about 16% v / v, about 12% v / v to about 15% v / v, about 12% v / v to about 14% v / v, about 12% v / v to about 13% v / v, about 13% v / v to about 20% v / v, about 13% v / v to about 19% v / v, about 13% v / v to about 18% v / v, about 13% v / v to about 17% v / v, about 13% v / v to about 16% v / v, about 13% v / v to about 15% v / v, about 13% v / v to about 14% v / v, about 14% v / v to about 20% v / v, about 14% v / v to about 19% v / v, about 14% v / v to about 18% v / v, about 14% v / v to about 17% v / v, about 14% v / v to about 16% v / v, about 14% v / v to about 15% v / v, about 15% v / v to about 20% v / v, about 15% v / v to about 19% v / v, about 15% v / v to about 18% v / v, about 15% v / v to about 17% v / v, about 15% v / v to about 16% v / v, about 16% v / v to about 20% v / v,about 16% v / v to about 19% v / v, about 16% v / v to about 18% v / v, about 16% v / v to about 17% v / v, about 17% v / v to about 20% v / v, about 17% v / v to about 19% v / v, about 17% v / v to about 18% v / v, about 18% v / v to about 20% v / v, about 18% v / v to about 19% v / v, or about 19% v / v to about 20% v / v.

[0154] In some examples of any of the compositions described herein, the liquid can further comprise at least one (e.g., 2, 3, 4, or 5) antioxidant and / or chelating agent. In some examples of any of the compositions described herein, the liquid can further comprise at least one (e.g., 2, 3, 4, or 5) antioxidant and / or chelating agent. Or, a chelating agent may further be included in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin after exposure to that dose of gamma radiation.

[0155] In some examples of any of the compositions described herein, the liquid may include at least one (e.g., two, three, four, or five) antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoids, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoids, phenolpropanoid acid, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanol. In some examples of any of the compositions described herein, the liquid may include at least one (e.g., two, three, or four) antioxidant selected from the group consisting of mannitol, sodium ascorbate, histidine, and methionine.

[0156] In some examples of any of the compositions described herein, the liquid can contain at least one (e.g., one, two, three, or four) of methionine (or cysteine ​​or glutathione), sodium ascorbate, histidine, and mannitol. In some examples of any of the compositions described herein, the liquid can contain methionine (or cysteine ​​or glutathione), sodium ascorbate, histidine, and mannitol. In some examples of any of the compositions described herein, the liquid may contain (i) 75 mM to about 125 mM (e.g., 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) mannitol; (ii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) mannitol. (iii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) sodium ascorbate; (iv) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) sodium ascorbate; (v) about 30 mM to about 70 mM (e.g., about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) methionine (or cysteine ​​or glutathione) and about 30 mM to about 70 mM (e.g., about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) histidine; (vi) about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM , about 20 mM to about 40 mM, or about 25 mM to about 35 mM) methionine (or cysteine ​​or glutathione), about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) histidine, and about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) sodium ascorbate;or (vii) about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) sodium ascorbate, about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) methionine (or cysteine ​​or glutathione), about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) mannitol, and about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) histidine. In some examples of any of the compositions described herein, the liquid can be a buffer solution (e.g., a phosphate buffer solution, e.g., a sodium phosphate buffer solution, e.g., 50 mM sodium phosphate, pH 6.0);

[0157] In some embodiments of any of the compositions described herein, the liquid may further comprise at least one (e.g., two, three, four, or five) chelating agents (e.g., at least one chelating agent selected from the group of ethylenediaminetetraacetic acid (EDTA), sodium 2,3-dimercapto-1-propanesulfonate (DMPS), dimercaptosuccinic acid (DMSA), metallothionein, and desferoxamine).

[0158] Also provided herein is a container (e.g., a storage vessel, e.g., a plastic container, or a chromatography column) containing a composition (e.g., any of the representative compositions described herein) comprising: (i) a chromatography resin (e.g., any of the chromatography resins described herein or known in the art); and (ii) a liquid comprising at least one alcohol (e.g., any of the representative alcohols described herein or known in the art), wherein the at least one alcohol is present in an amount sufficient to improve the binding capacity of the chromatography resin upon treatment with a dose of gamma radiation sufficient to reduce the bioburden of the composition. For example, a container (e.g., a storage container, e.g., a plastic container, or a chromatography column) can have an internal volume of, e.g., at least about 1 mL, 5 mL, at least about 10 mL, at least about 20 mL, at least about 30 mL, at least about 40 mL, at least about 50 mL, at least about 60 mL, at least about 70 mL, at least about 80 mL, at least about 90 mL, at least about 100 mL, at least about 110 mL, at least about 120 mL, at least about 130 mL, at least about 140 mL, at least about 150 mL, at least about 160 mL, at least about 170 mL, at least about 180 mL, at least about 190 mL, at least about 200 mL, at least about 210 mL, at least about 220 mL, at least about 230 mL, at least about 240 mL, at least about 250 mL, at least 300 mL, at least 350 mL, at least 400 mL, or at least 500 mL. For example, the container can have an internal volume of, for example, about 1 mL to about 500 mL, about 1 mL to about 50 mL, about 5 mL to about 500 mL, about 5 mL to about 400 mL, about 5 mL to about 350 mL, about 5 mL to about 300 mL, about 5 mL to about 250 mL, about 5 mL to about 200 mL, about 5 mL to about 150 mL, about 5 mL to about 100 mL, or about 5 mL to about 50 mL. In some examples, the chromatography resin in the container is a slurry of deposited chromatography resin in a liquid.In some instances, the vessel contains a packed chromatography resin (eg, packed in a liquid).

[0159] In any of the compositions provided herein, the liquid can further contain at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) antioxidant and / or at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) chelating agent. Any of the antioxidants that can be included in any of the compositions provided herein can have the ability to quench one or more of the following reactive oxygen and / or nitrogen species: hydroxyl groups, carbonate groups, superoxide anions, peroxyl groups, peroxynitrite, nitrogen dioxide, and nitric oxide. Non-limiting examples of antioxidants that can be included in any of the compositions provided herein include: reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoids, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoids, phenolpropanoid acids, lidocaine, naringenin, fullerenes, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanol. Additional non-limiting examples of antioxidants include antioxidant enzymes (e.g., superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase, and thioredoxin reductase). Additional examples of antioxidants that can be included in any of the compositions provided herein include mannitol, sodium ascorbate, methionine, and and histidine. Further examples of antioxidants include cysteine, taurine, mercaptopropionylglycine, N-acetylcysteine, garlic oil, diallyl sulfide, dihydrolipoic acid, and diallyl trisulfide. Some embodiments that include an antioxidant enzyme as an antioxidant can further include one or more substrates for the enzyme. Antioxidants can be identified using several methods known in the art, including, for example, spin traps, redox-sensitive dyes, and chemiluminescence methods.

[0160] Any of the chelators that can be included in any of the compositions provided herein are those that have high affinity (e.g., about 1 μM or less, about 800 nM or less, about 700 nM or less, about 600 nM or less, about 500 nM or less, about 400 nM or less, about 300 nM or less, about 250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 80 nM or less, about 60 nM or less, about 40 nM or less, about 20 nM or less, or about 1 nM or less) to chelate redox-active metals (e.g., Cu 2+ and Fe 2+ ). Non-limiting examples of chelating agents that can be included in any of the compositions provided herein include ethylenediaminetetraacetic acid (EDTA), sodium 2,3-dimercapto-1-propanesulfonate (DMPS), dimercaptosuccinic acid (DMSA), metallothionein, and desferoxamine.

[0161] The concentration of each of the chelating agents and / or antioxidants that can be included in any of the compositions provided herein is about 0.1 mM to about 150 mM (e.g., about 0.1 mM to about 150 mM, about 0.1 mM to about 125 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about 15 ...00 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about 150 mM, about M ~ about 40mM, about 0.1mM - about 30mM, about 0.1mM - about 25mM, about 0.1mM - about 20mM, about 0.1mM - about 10mM, about 0.1mM - about 5.0mM, about 0.5 mM to about 150mM, about 0.5mM to about 100mM, about 0.5mM to about 50mM, about 0.5mM to about 25mM, about 0.5mM to about 15mM, about 0.5mM to about 10mM, about 0.5mM to about 5mM, about 1mM to about 125mM, about 1mM to about 120mM, about 1mM to about 100mM, about 1mM to about 80mM, about 1mM to about 60mM, about 1mM to about 50mM, about 1mM to about 40mM, about 1m M ~ about 30mM, about 1mM - about 25mM, about 5mM - about 150mM, about 5mM - about 125mM, about 5mM - about 100mM, about 5mM - about 80mM, about 5mM - about 60mM, about 5mM - about 50mM, about 5mM ~about 40mM, about 5mM to about 30mM, about 5mM to about 25mM, about 10mM to about 150mM, about 10mM to about 125mM, about 1mM to about 100mM, about 10mM to about 80mM, about 10mM to about 60mM, about 10mM to about 50mM, about 10mM to about 40mM, about 10mM to about 30mM, about 10mM to about 25mM, about 20mM to about 150mM, about 20mM to about 125mM, about 20mM to about 100mM, about 20mM to about 80mM, about 20mM to about 60mM, about 20mM to about 50mM, about 20mM to about 40mM, about 20mM to about 30mM, about 30mM to about 150mM, about 30mM to about 125mM, about 30mM to about 100mM, Approximately 30mM to approximately 80mM, approximately 30mM to approximately 60mM, approximately 30mM to approximately 50mM, approximately 30mM to approximately 40mM, approximately 40mM to approximately 150mM, approximately 40mM to approximately 125mM, approximately 40mM to approximately 100mM, approximately 40mM to It can be about 90 mM, about 40 mM to about 80 mM, about 40 mM to about 70 mM, about 40 mM to about 60 mM, about 50 mM to about 150 mM, about 50 mM to about 125 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 50 mM to about 60 mM, about 80 mM to about 150 mM, about 80 mM to about 125 mM, about 80 mM to about 100 mM, about 100 mM to about 150 mM, or about 100 mM to about 125 mM).

[0162] In some examples, the compositions provided herein contain between 5 mM and about 150 mM mannitol. to about 150 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, about 60 mM to about 140 mM, about 70 mM to about 130 mM, about 80 mM to about 120 mM, about 90 mM to about 110 mM, about 95 mM to about 105 mM, about 5 mM to about 50 mM, about 5 mM to about 45 mM, about 5 mM to about 40 mM, about 5 mM to about 35 mM, about 10 mM to about 35 mM, about 15 mM to about 35 mM, or about 20 mM mannitol at 5 mM to about 30 mM; 5 mM to about 150 mM (for example, about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, about 60 mM to about 140 mM, about 70 mM to about 130 mM, about 80 mM to about 120 mM, about 90 mM to about 110 mM, about 95 mM to about 105 mM, about 30 mM to about 70 mM, about 35 mM to about 65 mM, about 40 mM to about 60 mM, about 45 mM to about 55 mM, about 0 mM to about 50 mM, about 25 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 5 mM to about 45 mM, about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 20 mM to about 25 mM) methionine (or cysteine ​​or glutathione); 5 mM to 150 mM sodium ascorbate (for example, about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM) M, about 50 mM to about 150 mM, about 60 mM to about 140 mM, about 70 mM to about 130 mM, about 80 mM to about 120 mM, about 90 mM to about 110 mM, about 95 mM to about 105 mM, about 10 mM to about 50 mM, about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM, about 30 mM to about 35 mM, about 5 mM to about 45 mM, about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM sodium ascorbate);and 5 mM to about 150 mM (e.g., about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, about 60 mM to about 140 mM, about 70 mM to about 130 mM, about 80 mM to about 120 mM, about 90 mM to about 110 mM, about 95 mM to about 105 mM, about 30 mM to about 70 mM, about 35 mM to about It may contain one or more of histidines at a concentration of about 65 mM, about 40 mM to about 60 mM, about 45 mM to about 55 mM, about 20 mM to about 50 mM, about 25 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 5 mM to about 45 mM, about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 20 mM to about 25 mM);

[0163] Non-limiting examples of any of the compositions include: (i) about 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) mannitol (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0); (ii) about 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM , about 90 mM to about 110 mM, or about 95 mM to about 105 mM) methionine (or cysteine ​​or glutathione) (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0); (iii) about 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) sodium ascorbate (iv) about 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) histidine (e.g., in a buffer solution, for example, a phosphate buffer, for example, 50 mM sodium phosphate, pH 6.0); (v) about 30 mM to about 70 mM (e.g., about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) methionine (or cysteine ​​or glutathione) and about 30 mM to about 70 mM (e.g., about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) histidine (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0); (vi) about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM, or about 30 mM to about 35 mM) methionine (or cysteine ​​or glutathione), about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM, or about 30 mM to about 35 mM) histidine, and about 10 mM (vii) about 5 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM, or about 30 mM to about 35 mM) sodium ascorbate (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0); or (vii) about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 23 mM to about 27 mM) sodium ascorbate, about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 23 mM to about 27 mM) methionine (or cysteine ​​or glutathione), about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 23 mM to about 27 mM) 5 mM, about 20 mM to about 30 mM, or about 23 mM to about 27 mM) mannitol, and about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 23 mM to about 27 mM) histidine (e.g., in a buffer solution, such as a phosphate buffer, for example 50 mM sodium phosphate, pH 6.0).

[0164] Non-limiting doses of gamma irradiation sufficient to reduce the bioburden of any of the compositions provided herein are described below. Additional doses of gamma irradiation sufficient to reduce the bioburden of any of the compositions provided herein are known in the art. For example, any of the compositions described herein can be gamma irradiated at any of the doses, any of the rates of gamma irradiation, and / or any of the temperatures (any combination) described herein for performing gamma irradiation. The bioburden of a composition can be determined, for example, by taking a sample from the composition that may contain the self-replicating biological contaminant present in the composition, for example, by stomaching, sonicating, shaking, vortex mixing, flushing, blending, or swabbing, and qualifying or quantifying the level of the self-replicating biological contaminant present in the sample (for example, by placing the sample in a growth medium that allows the biological contaminant to self-replicate, and for example, by plating the sample on a Petri dish or piercing the sample through a membrane).

[0165] The amount of at least one alcohol, at least one antioxidant, and / or at least one chelating agent sufficient to improve the binding capacity of a chromatography resin upon treatment with sufficient to reduce the bioburden of the composition can be determined, for example, using the methods described in the Examples. For example, the level of reduction in binding capacity of a chromatography resin treated by gamma-irradiation in the presence of an amount of at least one alcohol (and optionally at least one antioxidant and / or chelating agent) can be compared to the level of reduction in binding capacity of a chromatography resin treated by the same dose of gamma-irradiation in the absence of at least one alcohol (and optionally at least one antioxidant and / or chelating agent), where a reduction in the level of reduction in binding capacity of a chromatography resin treated by gamma-irradiation in the presence of at least one alcohol (and optionally at least one antioxidant and / or chelating agent) compared to a chromatography resin treated by gamma-irradiation in the absence of at least one alcohol (and optionally at least one antioxidant and / or chelating agent) indicates that at least one alcohol (and optionally at least one antioxidant and / or chelating agent) was present in an amount sufficient to improve the dose of the binding capacity of the chromatography resin upon treatment by gamma-irradiation. A representative method for determining the binding capacity of a chromatography resin is described in the Examples. Additional examples of methods for determining the binding capacity of a chromatography resin can be found in It is well known in the art.

[0166] Methods for reducing bioburden on chromatography resins Provided herein is a method for reducing the bioburden of a chromatography resin, comprising exposing a container containing a composition comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol (e.g., any of the exemplary compositions comprising a chromatography resin and a liquid comprising at least one alcohol described herein) to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein the at least one alcohol is present in an amount sufficient to ameliorate loss of binding capacity of the chromatography resin after (or during) exposure to the dose of gamma radiation.

[0167] Also provided is a method for reducing bioburden on a chromatography resin, comprising: (i) administering to the chromatography resin a composition comprising (i) the chromatography resin and (ii) a liquid comprising at least one alcohol in an amount sufficient to ameliorate loss of binding capacity of the chromatography resin after / during exposure to gamma radiation (e.g., any composition comprising a chromatography resin and a liquid comprising at least one alcohol as described herein) and exposing the composition to a dose of gamma radiation sufficient to reduce bioburden on the container and the chromatography resin at a rate of about 0.1 kGy / hr to about 6 kGy / hr (e.g., about 0.1 kGy / hr to about 5.5 kGy / hr, about 0.1 kGy / hr to about 5.0 kGy / hr, about 0.1 kGy / hr to about 4.5 kGy / hr, about 0.1 kGy / hr to about 4.0 kGy / hr, about 0.1 kGy / hr to about 3.5 kGy / hr, about 0.1 kGy / hr to about 3.0 kGy / hr, about 0.1 kGy / hr to about 5.5 kGy / hr, about 0.1 kGy / hr to about 5.0 kGy / hr, about 0.1 kGy / hr to about 4.5 kGy / hr, about 0.1 kGy / hr to about 4.0 kGy / hr, about 0.1 kGy / hr to about 5.5 kGy / hr, about 0.1 kGy / hr to about 5 ... y / hour to about 2.5 kGy / hour, about 0.1 kGy / hour to about 2.0 kGy / hour, about 0.1 kGy / hour to about 1.5 kGy / hour, about 0.1 kGy / hour to about 1.0 kGy / hour, about 0.5 kGy / hour to about 6 kGy / hour, about 0.5 kGy / hour to about 5.5 kGy / hour, about 0.5 kGy / hour to about 5.0 kGy / hour, about 0.5 kGy / hour to about 4.5 kGy / hour, about 0.5 kGy / hour to about 4.0 kGy / hour, about 0.5 kGy / hour to about 3.5 kGy / hr, about 0.5 kGy / hr to about 3.0 kGy / hr, about 0.5 kGy / hr to about 2.5 kGy / hr, about 0.5 kGy / hr to about 2.0 kGy / hr) and / or at a temperature of about 4° C. to about 25° C. (e.g., about 4° C. to about 20° C., about 4° C. to about 15° C., about 4° C. to about 10° C., about 10° C. to about 25° C., about 10° C. to about 20° C., about 10° C. to about 15° C., or about 15° C. to about 25° C.).

[0168] Some embodiments of any of these methods include, before and / or after the exposing step, exposing a container or composition comprising the chromatography resin and a liquid comprising at least one alcohol (e.g., any container or any composition comprising the chromatography resin and a liquid comprising at least one alcohol as described herein) to about 1 hour to about 1 year, about 1 hour to about 11 months, about 1 hour to about 10 months, about 1 hour to about 9 months, about 1 hour to about 8 months, about 1 hour to about 7 months, about 1 hour to about 6 months, about 1 hour to about 5 months, about 1 hour to about 4 months, about 1 hour to about 3 months, about 1 hour to about 2 months, about 1 hour to about 1 month, about 1 hour to about 2 weeks, about 1 hour to about 1 week, about 1 hour to about 5 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 2 days ... 1 hour to about 12 hours, about 1 hour to about 6 hours, about 6 hours to about 1 year, about 6 hours to about 11 months, about 6 hours to about 10 months, about 6 hours to about 9 months, about 6 hours to about 8 months, about 6 hours to about 7 months, about 6 hours to about 6 months, about 6 hours to about 5 months, about 6 hours to about 4 months, about 6 hours to about 3 months, about 6 hours to about 2 months, about 6 hours to about 1 month, about 6 hours to about 2 weeks, about 6 hours to about 1 week, about 6 hours to about 5 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 6 hours to about 12 hours, about 12 hours to about 1 year, about 12 hours to about 11 months, about 12 hours to about 10 months, about 12 hours to about 9 months, about 12 hours to about 8 months, about 12 hours to about 7 months, about 12 hours to about 6 months, about 12 hours to about 5 months, about 12 hours to about 4 months, about 12 hours to about 3 months, about 12 hours to about 2 months, about 12 hours to about 1 month, about 12 hours to about 2 weeks, about 12 hours to about 1 week, about 12 hours to about 5 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 1 year, about 1 day to about 11 months, about 1 day to about 10 months, about 1 day to about 9 months, about 1 day to about 8 months, about 1 day to about 7 months, about 1 day to about 6 months, about 1 day to about 5 months, about 1 day to about 4 months, about 1 day to about 3 months, about 1 day to about 2 months, about 1 day to about 1 month, about 1 day to about 2 weeks, about 1 day to about 1 week, about 1 day to about 5 days, about 1 day to about 2 days, about 2 days to about 1 year, about 2 days to about 11 months, about 2 days to about 10 months, about 2 days to about 9 months, about 2 days to about 8 months, about 2 days to about 1 day days to about 7 months, about 2 days to about 6 months, about 2 days to about 5 months, about 2 days to about 4 months, about 2 days to about 3 months, about 2 days to about 2 months, about 2 days to about 1 month, about 2 days to about 2 weeks, about 2 days to about 1 week, about 2 days to about 5 days, about 5 days to about 1 year, about 5 days to about 11 months, about 5 days to about 10 months, about 5 days to about 9 months, about 5 days to about 8 months, about 5 days to about 7 months, about 5 days to about 6 months, about 5 days to about 5 months, about 5 days to about 4 months, about 5 days to about 3 months, about 5 days to about 2 months, about 5 days to about 1 month, about 5 days to about 2 weeks, about 5 days to about 1 week, about 1 week to about 1 year, about 1 week to about 11 months, about 1 week to about 10 months, about 1 week to about 9 months, about 1 weeks to about 8 months, about 1 week to about 7 months, about 1 week to about 6 months, about 1 week to about 5 months, about 1 week to about 4 months, about 1 week to about 3 months, about 1 week to about 2 months, about 1 week to about 1 month, about 1 week to about 2 weeks, about 2 weeks to about 1 year, about 2 weeks to about 11 months, about 2 weeks to about 10 months, about 2 weeks to about 9 months, about 2 weeks to about 8 months, about 2 weeks to about 7 months, about 2 weeks to about 6 months, about 2 weeks to about 5 months, about 2 weeks to about 4 months, about 2 weeks to about 3 months, about 2 weeks to about 2 months, about 2 weeks to about 1 month, about 1 month to about 1 year, about 1 month to about 11 months, about 1 month to about 10 months, about 1 month to about 9 months, about 1 month to about 8 months, about 1 month to about 7 months, about 1 month to about 6 months, about 1 month to about 5 months, about 1 month to about 4 months, about 1 month to about 3 months, about 1 month to about 2 months, about 2 months to about 1 year, about 2 months to about 11 months, about 2 months to about 10 months, about 2 months to about 9 months, about 2 months to about 8 months, about 2 months to about 7 months, about 2 months to about 6 months, about 2 months to about 5 months, about 2 months to about 4 months, about 2 months to about 3 months, about 3 months to about 1 year, about 3 months to about 11 months, about 3 months to about 10 months, about 3 months to about 9 months, about 3 months to about 8 months, about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 4 months to about 1 year,About 4 months to about 11 months, about 4 months to about 10 months, about 4 months to about 9 months, about 4 months to about 8 months, about 4 months to about 7 months, about 4 months to about 6 months, about 4 months to about 5 months, about 5 months to about 1 year, about 5 months to about 11 months, about 5 months to about 10 months, about 5 months to about 9 months, about 5 months to about 8 months, about 5 months to about 7 months, about 5 months to about 6 months, about 6 months to about 1 year, about 6 months to about 11 months, about 6 months to about 10 months, about 6 months to about 9 months, about 6 months to about 8 months, about 6 months to about 7 months, about 7 months to about 1 year, about 7 months to about 11 months, about 7 months to about 10 months, about 7 months to about 9 months, about 7 months to about 9 months, about 7 months to about 7 months, about 7 months to about 1 year, about 7 months to about 11 months, about 7 months to about 10 months, about 7 months to about 9 months, about 7 months to about 7 months, about 7 months to about 1 year for a period of about 4°C to about 40°C, about 4°C to about 35°C, about 4°C to about 30°C, about 4°C to about 28°C, about 4°C to about 26°C, about 4°C to about 24°C, about 4°C to about 22°C, about 4°C to about 20°C, about 4°C to about 18°C, about 4°C to about 16°C, about 4°C to about 14°C, about 4°C to about 12°C, about 4°C to about 10°C, about 4°C to about 8 ... ~6°C, ~6°C to ~40°C, ~6°C to ~35°C, ~6°C to ~30°C, ~6°C to ~28°C, ~6°C to ~26°C, ~6°C to ~24°C, ~6°C to ~22°C, ~6°C to ~20°C, ~6°C to ~18°C, ~6°C to ~16°C, ~6°C to ~14°C, ~6°C to ~12°C, ~6°C to ~10°C, ℃ to about 8℃, about 28℃ to about 40℃, about 8℃ to about 35℃, about 8℃ to about 30℃, about 8℃ to about 28℃, about 8℃ to about 26℃, about 8℃ to about 24℃, about 8℃ to about 22℃, about 8℃ to about 20℃, about 8℃ to about 18℃, about 8℃ to about 16℃, about 8℃ to about 14℃, about 8℃ to about 12℃, about 8℃ to about 10℃, about 10°C to about 40°C, about 10°C to about 35°C, about 10°C to about 30°C, about 10°C to about 28°C, about 10°C to about 26°C, about 10°C to about 24°C, about 10°C to about 22°C, about 10°C to about 20°C, about 10°C to about 18°C, about 10°C to about 16°C, about 10°C to about 14°C, about 10°C to about 12°C, about 12°C to about About 40°C, about 12°C to about 35°C, about 12°C to about 30°C, about 12°C to about 28°C, about 12°C to about 26°C, about 12°C to about 24°C, about 12°C to about 22°C, about 12°C to about 20°C, about 12°C to about 18°C, about 12°C to about 16°C, about 12°C to about 14°C, about 14°C to about 40°C, about 14°C to about 35°C,About 14℃~about 30℃, ℃, about 14℃ to about 28℃, about 14℃ to about 26℃, about 14℃ to about 24℃, about 14℃ to about 22℃, about 14℃ to about 20℃, about 14℃ to about 18℃, about 14℃ to about 16℃, about 16℃ to about 40℃, about 16℃ to about 35℃, about 16℃ to about 30℃, about 16℃ to about 28℃, about 16℃ to about 26℃, about 16℃ to about 24℃, about 16℃ ~ about 22°C, about 16°C to about 20°C, about 16°C to about 18°C, about 18°C ​​to about 40°C, about 18°C ​​to about 35°C, about 18°C ​​to about 30°C, about 18°C ​​to about 28°C, about 18°C ​​to about 26°C, about 18°C ​​to about 24°C, about 18°C ​​to about 22°C, about 18°C ​​to about 20°C, about 20°C to about 40°C, about 20°C to about 35°C, about 20°C to about 30°C, About 20°C to about 28°C, about 20°C to about 26°C, about 20°C to about 24°C, about 20°C to about 22°C, about 22°C to about 40°C, about 22°C to about 35°C, about 22°C to about 30°C, about 22°C to about 28°C, about 22°C to about 26°C, about 22°C to about 24°C, about 24°C to about 40°C, about 24°C to about 35°C, about 24°C to about 30°C, about 24°C to about This can include storing at a temperature of 28°C, about 24°C to about 26°C, about 26°C to about 40°C, about 26°C to about 35°C, about 26°C to about 30°C, about 26°C to about 28°C, about 28°C to about 40°C, about 28°C to about 35°C, about 28°C to about 30°C, about 30°C to about 40°C, about 30°C to about 35°C, or about 35°C to about 40°C.

[0169] In the methods described in this paragraph, the level of binding capacity of the gamma irradiated chromagraphy resin produced by these methods is higher than the level of binding capacity of gamma irradiated chromagraphy resin that has been gamma irradiated at one or both rates of greater than 6.1 kGy / hr and / or at a temperature greater than 25°C.

[0170] The chromatography resin can be exposed to gamma radiation using methods known in the art. For example, isotopes such as cobalt-60 or cesium-137 can be used as the gamma radiation source. The chromatography resin can be exposed to gamma radiation at a temperature of about -25°C to about 0°C, or about 0°C to about 25°C. The chromatography resin can be exposed to gamma radiation at a temperature of about 0.1 kGy to about 100 kGy, about 1 kGy to about 100 kGy, about 1 kGy to about 90 kGy, about 1 kGy to about 80 kGy, about 1 kGy to about 70 kGy, about 1 kGy to about 65 kGy, about 5 kGy to about 65 kGy, about 10 kGy to about 60 kGy, about 10 kGy to about 55 kGy, about 10 kGy to about 50 kGy, about 10 kGy to about 10 kGy. The chromatography resin can be exposed to a gamma radiation dose of about 45 kGy, about 10 kGy to about 40 kGy, about 10 kGy to about 35 kGy, about 10 kGy to about 30 kGy, about 15 kGy to about 50 kGy, about 15 kGy to about 45 kGy, about 15 kGy to about 40 kGy, about 15 kGy to about 35 kGy, about 20 kGy to about 30 kGy, or about 23 kGy to about 27 kGy. -6 or less, approximately 1 × 10 -7 or less, approximately 10 x 10 -8 or less, approximately 1 × 10 -11 or less, or about 1 × 10 -12 or less, or about 1 × 10 -6 ~Approx. 1×10 -12 , about 1×10 -6 ~Approx. 1×10 -11 , about 1×10 -6 ~Approx. 1×10 -10 , about 1×10 -6 ~Approx. 1×10 -9 , about 1×10 -6 ~Approx. 1×10 -8 , 1×10 -6 ~Approx. 1×10 -7 , about 1×10 -7 ~Approx. 1×10 -12 , about 1×10 -7 ~Approx. 1×10 -11 , about 1×10 -7 ~Approx. 1×10 -10 , about 1×10 -7 ~Approx. 1×10 -9, about 1×10 -7 ~Approx. 1×10 -8 , about 1×10 -8 ~Approx. 1×10 -12 , about 1×10 -8 ~Approx. 1×10 -11 , about 1×10 -8 ~Approx. 1×10 -10 , or approximately 1 × 10 -8 ~Approx. 1×10 -9 The container can be exposed to a dose of gamma radiation sufficient to produce a sterility assurance level of 100 μg / ml.

[0171] A dose of gamma radiation sufficient to reduce the bioburden of a chromatography resin can be determined using methods known in the art. For example, the bioburden level of a chromatography resin treated with a dose of gamma radiation can be compared to the bioburden level of an untreated (e.g., control, non-gamma irradiated) chromatography resin, and a reduction in the level of bioburden of the gamma irradiated chromatography resin compared to the untreated chromatography resin indicates that the dose of gamma radiation is sufficient to reduce the bioburden of the chromatography resin. The results show that the level of bioburden of a composition (e.g., a chromatography resin) is sufficient to reduce the bioburden of the composition. Exemplary methods for determining the level of bioburden of a composition (e.g., a chromatography resin) are described herein. Additional methods for determining the level of bioburden of a composition (e.g., a chromatography resin) are known in the art.

[0172] In any of these methods, the chromatography resin can be an anion exchange chromatography resin, a cation exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography resin, or an affinity chromatography resin, or any combination thereof. Non-limiting examples of affinity chromatography resins can include a protein or peptide ligand (e.g., about 5 amino acids to about 100 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, or about 5 amino acids to about 20 amino acids), a small molecule substrate or cofactor for an enzyme, an aptamer, an inhibitor (e.g., a competitive protein inhibitor), or a metal. In some embodiments, the affinity chromatography resin includes a protein ligand (e.g., Protein A). Additional examples of affinity chromatography resins include cofactor ligands, substrate ligands, metal ligands, product ligands, or aptamer ligands. In some examples, the chromatography resin is a biomodal chromatography resin (e.g., anion exchange chromatography resin and hydrophobic interaction chromatography resin). The chromatography resin can be an anion exchange chromatography resin (e.g., an anion exchange chromatography resin containing N-benzyl-N-methyl-ethanolamine groups).

[0173] The container containing the chromatography resin can be a plastic container (e.g., a cylindrical tube, a sealed or stationary box, or a sealed bag). Non-limiting examples of containers used in these methods include a storage vessel or a chromatography column. For example, a composition (e.g., any of the representative compositions described herein) comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol can be present in a sealed container (e.g., a slurry in a sealed container or a packed chromatography resin in a sealed container (e.g., a chromatography column)). The container used in the methods described herein can be a disposable chromatography column. In some embodiments, the container used in the methods described herein is a disposable chromatography column contained in a blister pack. The container (e.g., a storage vessel or chromatography column) can have a total internal volume of about 1 mL to about 1 L (e.g., about 1 mL to about 900 mL, about 1 mL to about 800 mL, about 1 mL to about 700 mL, about 1 mL to about 600 mL, about 1 mL to about 500 mL, about 1 mL to about 450 mL, about 1 mL to about 400 mL, about 1 mL to about 350 mL, about 1 mL to about 300 mL, about 1 mL to about 250 mL, about 1 mL to about 200 mL, about 1 mL to about 150 mL, about 1 mL to about 100 mL, about 1 mL to about 75 mL, about 1 mL to about 50 mL, about 1 mL to about 40 mL, about 1 mL to about 30 mL, or about 1 mL to about 20 mL).

[0174] The composition (e.g., any of the representative compositions described herein) containing (i) a chromatography resin and (ii) a liquid comprising at least one alcohol contained within the container can exist as a mixture of wet or moist solids. For example, the container can contain a slurry of deposited chromatography resin in a liquid. In some embodiments, the container can contain packed chromatography resin. For example, the composition can contain (i) a chromatography resin and (ii) a liquid comprising at least one alcohol. The container containing the composition (e.g., any of the compositions described herein) comprising a liquid comprising at least one alcohol is a packed chromatography column (e.g., where the resin is packed in a liquid comprising at least one alcohol). Some embodiments include placing a composition (e.g., any of the compositions described herein) comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol in the container prior to exposing.

[0175] Any of the alcohols, antioxidants, and / or chelating agents described herein can be used in any combination, using any combination of the representative concentrations described herein. For example, the liquid can include at least one alcohol selected from the group of benzyl alcohol, cyclohexanol, isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol, propan-2-ol, propan-1-ol, butan-1-ol, pentan-1-ol, hexadecan-1-ol, 2-phenylethanol, sec-phenylethanol, 3-phenyl-1-propanol, 1-phenyl-1-propanol, 2-phenyl-1-propanol, 2-phenyl-2-propanol, 1-phenyl-2-butanol, 2-phenyl-1-butanol, 3-phenyl-1-butanol, 4-phenyl-2-butanol, dl-1-phenyl-2-pentanol, 5-phenyl-1-pentanol, and 4-phenyl-1-butanol. In some examples, the liquid may further comprise at least one antioxidant (e.g., at least one antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoids, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoids, phenolpropanoid acids, lidocaine, naringenin, fullerenes, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanol) and / or at least one chelating agent (e.g., at least one chelating agent selected from the group consisting of EDTA, DMPS, DMSA, metallothionein, and desferoxamine).

[0176] Described herein are exemplary methods for determining / confirming the amount of at least one alcohol, at least one antioxidant, and / or at least one chelating agent sufficient to ameliorate the loss of binding capacity of a chromatographic resin upon treatment with a dose of gamma radiation sufficient to reduce the bioburden of the composition. Additional methods are known in the art for determining / confirming the amount of at least one antioxidant and / or chelating agent sufficient to ameliorate the loss of binding capacity of a chromatographic resin upon treatment with a dose of gamma radiation sufficient to reduce the bioburden of the composition.

[0177] Also provided herein is a reduced bioburden chromatography resin (e.g., a reduced bioburden chromatography resin provided in a storage container, e.g., a sealed storage container) produced by any of the methods described herein. The reduced bioburden chromatography resin produced using any of the methods described herein has a concentration of about 1×10 -6 or less, approximately 1 × 10 -7 or less, approximately 10 x 10 -8 or less, approximately 1 × 10 -11 or less, or about 1 × 10 -12 or less, or about 1 × 10 -6 ~Approx. 1×10 -12 , about 1×10 -6 ~Approx. 1×10 -11 , about 1×10 -6 ~Approx. 1×10 -10 , about 1×10 -6 ~Approx. 1×10 -9 , about 1×10 -6 ~Approx. 1×10 -8 , 1×10 -6 ~Approx. 1×10 -7 , about 1×10 -7 ~Approx. 1×10 -12 , about 1×10 -7 ~Approx. 1×10 -11 , about 1×10 -7 ~Approx. 1×10 -10 , about 1×10 -7 ~Approx. 1×10 -9 , about 1×10 -7~Approx. 1×10 -8 , about 1×10 -8 ~Approx. 1×10 -12 , about 1×10 -8 ~Approx. 1×10 -11 , about 1×10 -8 ~Approx. 1×10 -10 , or approximately 1 × 10 -8 ~Approx. 1×10 -9 The reduced bioburden chromatography resin produced by any of the methods described herein can have a sterility assurance level of 0.1 to 1.0 μg / ml. ... When the same protein is used to test the binding capacity of both a control untreated chromatography resin and a control untreated chromatography resin, the binding capacity of the control untreated chromatography resin that has not been treated to reduce its bioburden (e.g., not gamma irradiated) should be at least 74% (e.g., at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least In some embodiments, the binding capacity of the antibody may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) or about 74% to 95%, about 74% to about 95%, about 76% to about 95%, at least about 78% to about 95%, about 80% to about 95%, or about 74% to about 90%, about 76% to about 90%, about 78% to about 90%, or about 80% to about 90%.

[0178] Method for producing a chromatography column packed with reduced bioburden - Patent Application 20070229633 Also provided herein is a method of making a reduced packed chromatography column, comprising providing a reduced bioburden chromatography resin produced by any of the methods described herein, and packing the chromatography resin into a reduced bioburden column under a sterilized or reduced bioburden environment. In some embodiments, a reduced bioburden packed chromatography column can be produced by exposing a column comprising a packed chromatography resin and a liquid comprising at least one alcohol (e.g., any of the representative liquids described herein, which may optionally further comprise at least one antioxidant and / or chelating agent) to a dose of gamma radiation sufficient to reduce the bioburden of the column and the packed chromatography resin, where the at least one alcohol is present in an amount sufficient to ameliorate the loss of binding capacity of the packed chromatography resin following exposure to that dose of gamma radiation.

[0179] Also provided are chromatography columns packed with reduced bioburden produced by the methods described herein. Any of the chromatography columns packed with reduced bioburden produced by the methods described herein has a concentration of about 1×10 -6 or less, approximately 1 × 10 -7 or less, approximately 10 x 10 -8 or less, approximately 1 × 10 -11 or less, or about 1 × 10 -12 or less, or about 1 × 10 -6 ~Approx. 1×10 -12 , about 1×10 -6 ~Approx. 1×10 -11 , about 1×10 -6 ~Approx. 1×10 -10 , about 1×10 -6 ~Approx. 1×10 -9 , about 1×10 -6 ~Approx. 1×10 -8 , 1×10 -6 ~Approx. 1×10 -7 , about 1×10-7 ~Approx. 1×10 -12 , about 1×10 -7 ~Approx. 1×10 -11 , about 1×10 -7 ~Approx. 1×10 -10 , about 1×10 -7 ~Approx. 1×10 -9 , about 1×10 -7 ~Approx. 1×10 -8 , about 1×10 -8 ~Approx. 1×10 -12 , about 1×10 -8 ~Approx. 1×10 -11 , about 1×10 -8 ~Approx. 1×10 -10 , or approximately 1 × 10 -8 ~Approx. 1×10 -9 Any of the reduced bioburden packed chromatography columns produced by the methods described herein can contain at least one chromatography resin selected from the group of anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin (e.g., any of the affinity chromatography resins described herein or known in the art), hydrophilic interaction chromatography resin, and size exclusion chromatography resin. For example, any of the reduced bioburden packed chromatography columns described herein can contain an affinity chromatography resin that includes a protein ligand (e.g., Protein A). The reduced bioburden packed chromatography columns described herein can contain an anion exchange chromatography resin (e.g., an anion exchange chromatography resin that includes N-benzyl-N-methyl-ethanolamine groups).

[0180] Method for performing reduced bioburden chromatography The methods described herein include the use of a chromatography column packed with a reduced bioburden as provided herein, and the methods described herein include the use of one or two MCCSs that include at least one reduced bioburden packed chromatography column as provided herein. The gamma irradiated chromatography resin can be any type of resin described herein (or any type of chromatography resin known in the art).

[0181] A chromatography column packed with reduced bioburden can be prepared using any of the methods described herein. For example, a chromatography column packed with reduced bioburden can be produced by packing a chromatography column with a composition (e.g., any of the compositions described herein) comprising a chromatography resin and a liquid comprising at least one alcohol, and exposing the packed column to gamma irradiation (e.g., using the exposures and conditions described herein). In another example, a chromatography column packed with reduced bioburden can be produced by exposing a container containing a chromatography resin and a liquid comprising at least one alcohol (e.g., any of the representative liquids described herein, which may optionally further comprise at least one antioxidant and / or at least one chelating agent) to a dose of gamma irradiation, and packing the resulting reduced bioburden chromatography resin into a chromatography column. In such a method, the chromatography resin (present in the container during exposure to gamma irradiation) can be present in the container as a slurry, and the chromatography column is packed in a reduced bioburden hood. In other methods, the chromatography resin present in a container with a liquid (e.g., any liquid described herein, which may optionally further comprise at least one antioxidant and / or at least one chelating agent) comprising at least an alcohol can be exposed to gamma irradiation as a wet or moist solid mixture in the container, and the resulting reduced bioburden chromatography resin slurry can be prepared with a reduced bioburden buffer (e.g., prepared in a reduced bioburden hood), and the resulting slurry is used to pack a chromatography column in a reduced bioburden hood. In some of these examples, the chromatography column can be treated (e.g., autoclaved, gamma irradiated, or exposed to ethylene oxide) to reduce the bioburden prior to packing.

[0182] The reduced bioburden packed chromatography column used in any of the methods described herein has a bioburden of about 1×10 -3 ~Approx. 1×10 -12 , about 1×10 -4 ~Approx. 1×10 -12 , 1×10 -5 ~Approx. 1×10 -11 , about 1×10 -5 ~Approx. 1×10 -10 , about 1×10 -5 ~Approx. 1×10 -9 , about 1×10 -6 ~Approx. 1×10 -9 , or approximately 1 × 10 -6 ~Approx. 1×10 -8 The sterility assurance level (SAL) may be

[0183] Reduced bioburden buffers The methods and processes described herein can be carried out using one or more reduced bioburden buffers. As understood in the art, the reduced bioburden buffer can be any type of buffer used in a chromatography cycle (e.g., a buffer used in a chromatography cycle or in any step of a unit operation described herein). Exemplary methods for reducing the bioburden of a buffer include filtration (0.2 μm pore size filtration), autoclaving, and gamma irradiation. Additional methods for reducing the bioburden of a buffer are known in the art. A reduced bioburden buffer has a bioburden of about 1×10 -3 ~Approx. 1×10 -12 , about 1×10 -4 ~Approx. 1×10 -12 , 1×10 -5 ~Approx. 1×10 -11 , about 1×10 -5 ~About 1× 10 -10 , about 1×10 -5 ~Approx. 1×10 -9 , about 1×10 -6 ~Approx. 1×10 -9 , or approximately 1 × 10 -6 ~Approx. 1×10 -8(inclusive) can have a sterility assurance level of

[0184] Recombinant Therapeutic Proteins The recombinant proteins described herein can be recombinant therapeutic proteins. Non-limiting examples of recombinant therapeutic proteins that can be produced by the methods provided herein include immunoglobulins (including light and heavy chain immunoglobulins), antibodies or antibody fragments (e.g., any of the antibody fragments described herein), enzymes (e.g., galactosidases (e.g., alpha-galactosidase), Myozyme® or Cerezyme®), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF), or interferon alpha or beta), or immunogenic or antigenic proteins or protein fragments (e.g., proteins for use in vaccines). The recombinant therapeutic protein may be an artificially engineered antigen-binding polypeptide that comprises at least one multifunctional recombinant protein scaffold (see, e.g., the antigen-binding recombinant proteins described in Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent Application Publication No. 2012 / 0164066, which are incorporated by reference in their entireties). Non-limiting examples of recombinant therapeutic proteins that are antibodies include panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, apolizumab, atinumab, tocilizumab, basiliximab, and the like. izimab, bectumomab, belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, efangumab, ertumaxomab, etaracizumab, golimumab, infliximab, natalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, and trastuzumab.Further examples of recombinant therapeutic antibodies that can be produced by the method described herein are known in the art.Further non-limiting examples of recombinant therapeutic proteins that can be produced / purified by the method include alglucosidase alpha, laronidase, abatacept, galsulfase, lutropin alpha, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alpha, tenecteplase, etanercept, coagulation factor IX, follicle-stimulating hormone, interferon beta-1a, imiglucerase, dornase alpha, epoetin alpha and alteplase.

[0185] The secreted soluble recombinant therapeutic protein can be recovered from the liquid culture medium (e.g., the first liquid culture medium and / or the second liquid culture medium) by removing or physically separating the liquid culture medium from the cells (e.g., mammalian cells). A variety of different methods for removing liquid culture medium from cells (e.g., mammalian cells) are known in the art, including, for example, centrifugation, filtration, pipetting and / or aspiration. The secreted recombinant therapeutic protein can then be recovered from the liquid culture medium and further purified using a variety of biochemical techniques, including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography, anion exchange chromatography, or hydrophobic interaction chromatography, or any combination thereof) and / or filtration (e.g., molecular weight cut-off filtration).

[0186] Chromatography cycle As is well known in the art, the steps in a cycle of chromatography can vary depending on the chromatography resin, the buffer used to perform each step in the cycle, and the biophysical properties of the target recombinant protein (e.g., recombinant therapeutic protein). For example, an affinity chromatography column can include the steps of loading a fluid containing the target recombinant protein onto the affinity chromatography column, washing the column to remove undesired biological material (e.g., contaminating proteins and / or small molecules), eluting the target recombinant protein bound to the column, and re-equilibrating the column. A chromatography cycle using a cation and / or anion exchange chromatography column, in which the target recombinant protein binds to the chromatography resin in the loading step, can include the steps of loading a fluid containing the target protein onto the column, washing the column to remove undesired biological material, eluting the target recombinant protein bound to the column, and re-equilibrating the column. In another example, a chromatography cycle using a cation and / or anion exchange chromatography column in which undesired biological material but not the target recombinant protein binds to the chromatography resin during a loading step can include loading a fluid containing the target protein onto the column, collecting the target recombinant protein in the flow-through, and re-equilibrating the column. As is well known in the art, any single step in a chromatography cycle can include a single buffer or multiple buffers (e.g., two or more buffers), and any one or more of the single steps in a chromatography cycle can include a buffer gradient.Any combination of various well-known aspects of a single cycle of chromatography can be used in these methods, e.g., different chromatography resin(s), flow rate(s), buffer(s), column void volume(s), column bed volume(s), volume(s) of buffer used in each step, volume(s) of fluid containing the target protein, and number and type of buffer(s) used in each step, in any combination.

[0187] Method for performing reduced bioburden column chromatography Provided herein are methods of performing reduced bioburden chromatography. These methods include providing a reduced bioburden packed chromatography column produced using any of the methods described herein, and performing column chromatography using the reduced bioburden packed chromatography column. The reduced bioburden packed chromatography column can include at least one of any of the chromatography resins described herein in any combination. For example, the chromatography resin present in the reduced bioburden packed chromatography column can be an affinity resin that includes a protein ligand (e.g., Protein A) or can include an anion exchange chromatography resin. The reduced bioburden packed chromatography column can have any of the representative internal volumes described herein. The reduced bioburden packed chromatography column can have any shape (e.g., cylindrical, approximately cylindrical, or elliptical) described herein or known in the art. Column chromatography performed in these methods can be used to purify or isolate recombinant proteins (e.g., any recombinant therapeutic protein described herein or known in the art). In some instances, the chromatography column packed with reduced bioburden can be part of a multi-column chromatography system (MCCS), for example, part of a cyclic countercurrent chromatography system (PCCS).

[0188] The column chromatography carried out may be any of the methods described herein or in the art. The method can include at least one cycle of chromatography, known in the art. For example, at least one cycle of chromatography can include: capturing the recombinant protein by exposing the chromatography resin to a liquid containing the recombinant protein; washing the chromatography resin by exposing the chromatography resin to a wash buffer, eluting the recombinant protein by exposing the chromatography resin to an elution buffer; and regenerating the chromatography resin by exposing the chromatography resin to a regeneration buffer. In some examples, the liquid containing the recombinant protein is a liquid culture medium (e.g., liquid culture medium collected from a perfusion or batch culture) or a diluted liquid culture medium (e.g., culture medium diluted in a buffer).

[0189] Column chromatography can be performed using a closed, integrated system (e.g., any of the exemplary closed, integrated systems described herein or known in the art). For example, column chromatography can be performed using a closed, integrated system in which the buffer is a reduced bioburden buffer. As is known in the art, reduced bioburden buffers can be produced using a variety of different methods (e.g., produced by filtration, autoclaving, or heat treatment).

[0190] The column chromatography can comprise two or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, or 100 or more) cycles of chromatography. In some examples, the column chromatography is performed continuously for a period of at least 3 days (e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, or at least 100 days).

[0191] In some embodiments, the chromatography resin in the chromatography column packed with reduced bioburden has a binding capacity of about 75% to about 100% (e.g., about 76% to about 98%, about 76% to about 96%, about 76% to about 94%, about 76% to about 92%, about 76% to about 90%, about 78% to about 100%, about 78% to about 98%, about 78% to about 96%, about 78% to about 94%, about 78% to about 92%, about 78% to about 90%, about 8% to about 95 ... 0% to about 100%, about 80% to about 98%, about 80% to about 96%, about 80% to about 94%, about 80% to about 92%, about 80% to about 90%, about 82% to about 100%, about 82% to about 98%, about 82% to about 96%, about 82% to about 94%, about 82% to about 92%, about 82% to about 90%, about 84% to about 100%, about 84% to about 98%, about 84% to about 96%, about 84% to about 94%, about 84% to about 92%, about 84% to about 90%, about 86% to about 100%, about 86% to about 98%, about 86% to about 96%, about 86% to about 94%, about 86% to about 92%, about 88% to about 10 0%, about 88% to about 98%, about 88% to about 96%, about 88% to about 94%, about 90% to about 100%, about 90% to about 98%, about 90% to about 96%, about 92% to about 100%, or about 92% to about 98% (e.g., assessed immediately after exposure to gamma radiation).

[0192] Integrated, closed or substantially closed, continuous process for producing recombinant proteins Provided herein are integrated, closed or substantially closed, continuous methods for producing purified recombinant proteins (e.g., recombinant therapeutic proteins), which include providing a liquid culture medium containing the recombinant protein (e.g., recombinant therapeutic protein) that is substantially cell-free.

[0193] Some methods include continuously feeding liquid culture medium to a multi-column chromatography system (MCCS) that includes at least one reduced bioburden packed chromatography column as provided herein, where the methods utilize a reduced bioburden buffer and operate continuously from the liquid culture medium to the eluate from the MCCS being the purified recombinant protein (e.g., a therapeutic protein drug substance).

[0194] Some methods include producing a purified recombinant protein by continuously feeding liquid culture medium to a first MCCS (MCCS1), capturing a recombinant protein from the liquid culture medium using MCCS1, generating an eluate from MCCS1 comprising the recombinant protein, continuously feeding the eluate to a second MCCS (MCCS2), continuously feeding the recombinant protein from the eluate to MCCS2, and subsequently eluting the recombinant protein, wherein at least one column in MCCS1 and / or MCCS2 is a reduced bioburden packed chromatography column provided herein, and the methods utilize a reduced bioburden buffer and are integrated and run continuously from the liquid culture medium to the purified recombinant protein.

[0195] In some examples, each of the chromatography columns used in MCCS, MCCS1, and / or MCCS2 is a chromatography column packed with a reduced bioburden as provided herein. Some embodiments further include formulating the purified recombinant protein into a pharmaceutical composition.

[0196] The methods described herein provide for continuous, time-efficient production of purified recombinant proteins from liquid culture media containing the recombinant proteins. For example, the time elapsed from the supply of liquid culture media containing a therapeutic protein to MCCS or MCCS1 to the elution of the recombinant protein from MCCS or MCCS2, respectively, can be, for example, about 4 hours to about 48 hours (inclusive), e.g., about 4 hours to about 40 hours, about 4 hours to about 35 hours, about 4 hours to about 30 hours, about 4 hours to about 28 hours, about 4 hours to about 26 hours, about 4 hours to about 24 hours, about 4 hours to about 22 hours, about 4 hours to about 20 hours, about 4 hours to about 18 hours, about 4 hours to about 16 hours, about 4 hours to about 14 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, The incubation time can be about 6 hours to about 20 hours, about 6 hours to about 18 hours, about 6 hours to about 14 hours, about 8 hours to about 16 hours, about 8 hours to about 14 hours, about 8 hours to about 12 hours, about 10 hours to about 20 hours, about 10 hours to about 18 hours, about 10 hours to about 16 hours, about 10 hours to about 14 hours, about 12 hours to about 14 hours, about 10 hours to about 40 hours, about 10 hours to about 35 hours, about 10 hours to about 30 hours, about 10 hours to about 25 hours, about 15 hours to about 40 hours, about 15 hours to about 35 hours, about 15 hours to about 30 hours, about 20 hours to about 40 hours, about 20 hours to about 35 hours, or about 20 hours to about 30 hours (inclusive). In another example, the incubation time can be about 6 hours to about 20 hours, about 6 hours to about 18 hours, about 6 hours to about 14 hours, about 8 hours to about 16 hours, about 8 hours to about 14 hours, about 8 hours to about 12 hours, about 10 hours to about 20 hours, about 10 hours to about 25 hours, about 15 hours to about 40 hours, about 15 hours to about 35 hours, about 15 hours to about 30 hours, about 20 hours to about 40 hours, about 20 hours to about 35 hours, or about 20 hours to about 30 hours (inclusive). The time elapsed until the recombinant protein is eluted from CCS or MCCS2 is, for example, more than about 4 hours to less than about 40 hours (inclusive), for example, more than about 4 hours to less than about 39 hours, about 38 hours, about 37 hours, about 36 hours, about 35 hours, about 34 hours, about 33 hours, about 32 hours, about 31 hours, about 30 hours, about 29 hours, about 28 hours, about 27 hours, about 26 hours, about 25 hours, about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, or about 4.5 hours (inclusive).

[0197] Non-limiting aspects of MCCS (MCCS, MCCS1, and / or MCCS2) that can be used in any of these methods are described in U.S. Provisional Patent Applications Nos. 61 / 775,060 and 61 / 856,390, each of which is incorporated herein by reference.

[0198] Some representative methods do not utilize a holding step (e.g., do not use a reservoir (e.g., a break tank) throughout the method). In other methods, up to 1, 2, 3, 4, or 5 reservoirs (e.g., break tanks) are used throughout the method. Any of the methods described herein can utilize up to 1, 2, 3, 4, or 5 reservoirs (e.g., break tanks) throughout the method, where each break tank holds only recombinant protein for a total period of, e.g., from about 5 minutes to less than about 6 hours, inclusive, e.g., from about 5 minutes to about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes, inclusive.

[0199] Some methods utilize 1, 2, 3, 4, 5, or 6 reservoirs (e.g., holding tanks) and can have a volume that is, for example, 1 mL to about 300 mL (inclusive), e.g., 1 mL to about 280 mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL, about 40 mL, about 20 mL, or about 10 mL (inclusive). Any reservoir(s) (e.g., holding tank(s)) used (in any of the methods described herein) to hold fluid before being delivered to the MCCS or MCCS1 can have a capacity that is, for example, from 1 mL to about 100% (inclusive) of the loading volume of the first column of the MCCS, i.e., MCCS1, for example, from 1 mL to about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% (inclusive). A reservoir(s) (e.g., holding tank(s)) can be used to hold the eluate from MCCS1 before it enters MCCS2, and the reservoir can have a capacity that is, for example, from 1 mL to about 100% (inclusive) of the loading volume of the first column of MCCS2, for example, from 1 mL to about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% (inclusive).

[0200] Various additional aspects of these methods are described in detail below and can be used in any combination without limitation in the methods provided herein. Although representative aspects of the methods provided are described below, one of skill in the art will understand that additional steps can be added to the methods described herein and other materials can be used to carry out any of the steps of the methods described herein.

[0201] Liquid culture medium The substantially cell-free liquid culture medium containing the recombinant protein (e.g., a recombinant therapeutic protein) can be from any source, for example, the liquid culture medium can be derived from a recombinant cell culture (e.g., a recombinant bacterial, yeast, or mammalian cell culture). The liquid culture medium can be obtained from a fed-batch cell (e.g., mammalian cell) culture (e.g., a fed-batch bioreactor containing a culture of mammalian cells that secrete a recombinant protein) or a perfusion cell (e.g., mammalian cell) culture (e.g., a perfusion bioreactor containing a culture of mammalian cells that secrete a recombinant protein). The liquid culture medium can also be a clarified liquid culture medium from a culture of bacterial or yeast cells that secrete a recombinant protein.

[0202] Liquid culture media from recombinant cell cultures can be filtered or clarified to obtain liquid culture media that is substantially free of cells and / or viruses. Methods for filtering or clarifying liquid culture media to remove cells are known in the art (e.g., 0.2-μm filtration and Alternating Tangential Flow (ATF) filtration). TM (Filtration using a filtration system). Recombinant cells can also be removed from liquid culture medium using centrifugation and removing a supernatant, which is substantially cell-free liquid culture medium, or by allowing the cells to settle to the gravitational bottom of a vessel (e.g., a bioreactor) containing the liquid culture medium and removing the liquid culture medium distal to the settled recombinant cells (the substantially cell-free liquid culture medium).

[0203] The liquid culture medium can be obtained from a culture of recombinant cells (e.g., recombinant bacteria, yeast, or mammalian cells) that produce any of the recombinant proteins (e.g., recombinant therapeutic proteins) described herein or known in the art. Some examples of any of the methods described herein can further include culturing recombinant cells (e.g., recombinant bacteria, yeast, or mammalian cells) that produce the recombinant protein (e.g., recombinant therapeutic protein).

[0204] The liquid culture medium can be any type of liquid culture medium described herein or known in the art. For example, the liquid culture medium can be selected from the group consisting of: animal-derived component-free liquid culture medium, serum-free liquid culture medium, serum-containing liquid culture medium, chemically defined liquid culture medium, and protein-free liquid culture medium. In any of the methods described herein, the liquid culture medium obtained from the culture can be diluted by adding a second fluid (e.g., a buffer) before being fed to the MCCS or MCCS1.

[0205] The liquid culture medium containing the recombinant protein, substantially free of cells, can be stored (e.g., at a temperature below about 15° C. (e.g., below about 10° C., below about 4° C., below about 0° C., below about −20° C., below about −50° C., below about −70° C., or below about −80° C.) for at least 1 day (e.g., at least about 2 days, at least about 5 days, at least about 10 days, at least about 15 days, at least about 20 days, or at least about 30 days) before the liquid culture medium is fed to the MCCS or MCCS1. Alternatively, in some examples, the liquid culture medium is fed directly from the bioreactor to the MCCS or MCCS1 (e.g., fed directly from the bioreactor to the MCCS or MCCS1 after a filtration or clarification step).

[0206] Multi-column Chromatography System The methods described herein include the use of an MCCS or two or more (e.g., two, three, four, five, or six) multi-column chromatography systems (MCCSs) (e.g., MCCS1 and MCCS2). An MCCS can include two or more chromatographic columns, two or more chromatographic membranes, or a combination of at least one chromatographic column and at least one chromatographic membrane. In a non-limiting example, an MCCS (e.g., the MCCS, MCCS1, and / or MCCS2 in any of the methods herein) can include four chromatographic columns, three chromatographic columns and one chromatographic membrane, three chromatographic columns and one chromatographic membrane, and one chromatographic membrane. The combination of chromatography columns and / or chromatographic membranes may include one chromatography column, two chromatography columns, two chromatography membranes, and two chromatography columns and one chromatography membrane. Further examples of combinations of chromatography columns and / or chromatographic membranes can be envisioned by those skilled in the art without limitation for use in an MCCS (e.g., MCCS, MCCS1 and / or MCCS2 in any of the methods described herein). The individual chromatography columns and / or chromatographic membranes present within an MCCS may be identical (e.g., may have the same shape, volume, resin, capture mechanism and unit operation) or different (e.g., may have one or more of different shapes, volumes, resins, capture mechanisms and / or unit operations). The individual chromatographic column(s) and / or chromatographic membrane(s) present within an MCCS (e.g., MCCS, MCCS1 and / or MCCS2 in any of the methods described herein) may perform the same unit operation (e.g., a capture unit operation, a purification unit operation or a polishing unit operation) or may perform different unit operations (e.g., a different unit operation selected from the group consisting of, for example, capture, purification, polishing, virus inactivation, adjusting the ionic concentration and / or pH of a fluid containing the recombinant protein, and filtration). For example, in the exemplary methods described herein, at least one chromatographic column and / or chromatographic membrane within the MCCS or MCCS1 performs the unit operation of capturing the recombinant protein.

[0207] One or more chromatography column(s) that may be present within an MCCS (e.g., may be present within MCCS, MCCS1 and / or MCCS2) may have a resin volume of, for example, between about 1 mL and about 2 mL, inclusive, about 5 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL, about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about 95 mL, or about 100 mL. The one or more chromatography column(s) that may be present in the MCCS (e.g., may be present in MCCS, MCCS1 and / or MCCS2) may have a volume of between about 2 mL and about 100 mL, between about 2 mL and about 90 mL, between about 2 mL and about 80 mL, between about 2 mL and about 70 mL, between about 2 mL and about 60 mL, between about 2 mL and about 50 mL, between about 5 mL and about 50 mL, between about 2 mL and about 45 mL, between about 5 ... The MCCS may have a resin volume of between about 2 mL and about 45 mL, between about 2 mL and about 40 mL, between about 5 mL and about 40 mL, between about 2 mL and about 35 mL, between about 5 mL and about 35 mL, between about 2 mL and about 30 mL, between about 5 mL and about 30 mL, between about 2 mL and about 25 mL, between about 5 mL and about 25 mL, between about 15 mL and about 60 mL, between about 10 mL and about 60 mL, between about 10 mL and about 50 mL, and between about 15 mL and about 50 mL. One or more chromatography column(s) within the MCCS (e.g., MCCS, MCCS1 and / or MCCS2) used in any of the methods described herein may have substantially the same resin volume or may have different resin volumes.The flow rate used for one or more chromatography column(s) within the MCCS (e.g., MCCS, MCCS1 and / or MCCS2) can be, for example, between about 0.2 mL / min and about 25 mL / min (e.g., between about 0.2 mL / min and about 20 mL / min, between about 0.5 mL / min and about 20 mL / min, between about 0.2 mL / min and about 15 mL / min, between about 0.5 mL / min and about 15 mL / min, between about 0.5 mL / min and about 10 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, or between about 1.0 mL / min and about 15.0 mL / min).

[0208] One or more chromatography columns within an MCCS (e.g., within MCCS, MCCS1, and / or MCCS2) may have substantially the same geometry. For example, one or more chromatography columns within an MCCS (e.g., within MCCS, MCCS1, and / or MCCS2) may have substantially the same shape, such as a circular cylinder, or may have substantially the same shape, such as an oval cylinder.

[0209] The one or more chromatographic membrane(s) that may be present in the MCCS (e.g., may be present in the MCCS, MCCS1 and / or MCCS2) may have a volume of, for example, between about 1 mL and about 500 mL (e.g., between about 1 mL and about 475 mL, about 1 mL to about 450 mL, about 1 mL to about 425 mL, about 1 mL to about 400 mL, about 1 mL to about 375 mL, about 1 mL to about 350 mL, about 1 mL to about 325 mL, about 1 mL to about 300 mL, about 1 mL to about 275 mL, about 1 mL to about 250 mL, about 1 mL to about 225 mL, about 1 mL to about 200 mL, about 1 mL to about 175 mL, about 1 mL to about 150 mL, about 1 mL to about 125 mL, about 1 mL to about 100 mL, etc. L, about 2 mL to about 100 mL, about 5 mL to about 100 mL, about 1 mL to about 80 mL, about 2 mL to about 80 mL, about 5 mL to about 80 mL, about 1 mL to about 60 mL, about 2 mL to about 60 mL, about 5 mL to about 60 mL, about 1 mL to about 40 mL, about 2 mL to about 40 mL, about 5 mL to about 40 mL, about 1 mL to about 30 mL, about 2 mL to about 30 mL, about 5 mL to about 30 mL, between about 1 mL and about 25 mL, between about 1 mL and about 20 mL, between about 2 mL and about 20 mL, between about 1 mL and about 15 mL, between about 2 mL and about 15 mL, between about 1 mL and about 10 mL, or between about 2 mL and about 10 mL.

[0210] One or more (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, or twenty-four) different types of reduced bioburden buffers may be used during the use of MCCS, MCCS1, and / or MCCS2 in any of the methods described herein. As is known in the art, the type or types of reduced bioburden buffers used with MCCS, MCCS1, and / or MCCS2 in the methods described herein will depend on the resin present in the MCCS, MCCS1, and / or MCCS2 chromatographic column(s) and / or chromatographic membrane(s), the biophysical properties of the recombinant protein, and the unit operation (e.g., any of the representative unit operations described herein) performed by the particular MCCS, MCCS1, and / or MCCS2 chromatographic column(s) and / or chromatographic membrane. The volume and type of buffer used during the use of MCCS, MCCS1 and / or MCCS2 in any of the methods described herein can also be determined by one of skill in the art (e.g., as discussed in more detail below). For example, the volume and type(s) of buffer used during the use of MCCS, MCCS1 and / or MCCS2 in any of the methods described herein can be selected to optimize one or more of the following in the purified recombinant protein (e.g., recombinant protein drug product): overall yield of recombinant protein, activity of recombinant protein, level of purity of recombinant protein, and removal of organism-type contaminants (e.g., absence of active virus, mycobacteria, yeast, bacteria or mammalian cells) from a fluid (e.g., liquid culture medium) containing the recombinant protein.

[0211] The MCCS, MCCS1 and / or MCCS2 may be a periodic countercurrent chromatography system (PCCS). The PCCS may, for example, comprise two or more chromatography columns (e.g., three or four columns) that are switched to allow for sequential elution of recombinant protein from two or more chromatography columns. The PCCS may comprise two or more chromatography columns, may comprise two or more chromatography membranes, or may comprise at least one chromatography column and at least one chromatography membrane. A column operation (cycle) generally consists of a loading step, a washing step, an elution step, and a regeneration step. In a PCCS, multiple columns are used to cycle and run the same steps separately and consecutively. As the columns operate in series, the flow-through and wash from one column is captured by another column. This unique feature of the PCCS allows for loading of the resin closer to its static binding capacity rather than its dynamic binding capacity, as is typical during chromatography in batch mode. As a result of the continuous cycling and elution, the fluid entering the PCCS is continuously processed and an eluate containing the recombinant protein is continuously produced.

[0212] A column switching strategy is used to proceed from one step to another in the PCCS cycle. Examples of column switching that can be used in the PCCS are described in US Provisional Patent Applications 61 / 775,060 and 61 / 856,390. For example, the column switching method can use two automated switching operations per column: the first of which is related to the breakthrough of the first product and the second corresponds to column saturation. The decision of when to cause a column switching operation can be determined by monitoring the recombinant protein concentration in the eluate coming out of each chromatographic column present in the PCCS (e.g., monitoring performed by UV monitoring). For example, column switching can be determined by any PAT tool capable of in-line measurement of recombinant protein concentration with feedback control. The PAT tool is capable of real-time in-line measurement of recombinant protein concentration with feedback control. As is known in the art, column conversion can also be designed based on the time or amount of fluid (e.g., buffer) passing through one or more chromatographic column(s) and / or chromatographic membranes within MCCS, MCCS1 and / or MCCS2.

[0213] In a PCCS, the residence time (RT) of a recombinant protein on each chromatographic column and / or chromatographic membrane present in the PCCS can be shortened without increasing the size of the column / membrane because the breakthrough from the first column / membrane can be captured by another column / membrane in the PCCS. A system in a continuous manner can be calculated according to the formula: V=D * By varying the column / membrane volume (V) and RT with RT, it can be designed to process liquid culture medium at any perfusion rate (D).

[0214] One or more unit operations that may be performed by the MCCS or MCC1 and / or MCCS2 used in the methods described herein include, for example, capturing the recombinant protein, inactivating viruses present in a fluid containing the recombinant protein, purifying the recombinant protein, polishing the recombinant protein, retaining a fluid containing the recombinant protein (e.g., using any of the exemplary holding tank(s) described herein), filtering or removing particulate material and / or cells emerging from a fluid containing the recombinant protein, and adjusting the ionic concentration and / or pH of a fluid containing the recombinant protein.

[0215] In some embodiments, the MCCS or MCCS1 comprises at least one chromatography column and / or chromatography membrane that performs the unit operation of capturing the recombinant protein. The unit operation of capturing can be performed, for example, using at least one chromatography column and / or chromatography resin that utilizes a capture mechanism. Non-limiting examples of capture mechanisms include protein A-binding capture mechanisms, antibody- or antibody fragment-binding capture mechanisms, substrate-binding capture mechanisms, aptamer-binding capture mechanisms, tag-binding capture mechanisms (e.g., poly-His tag-based capture mechanisms), and cofactor-binding capture mechanisms. Capture can also be performed using cation- or anion-exchange chromatography, molecular sieve chromatography, or hydrophobic interaction chromatography. The capture of recombinant proteins can also be performed using resins that can be used to perform the capture of recombinant proteins. Non-limiting resins that can be used to capture recombinant proteins are described herein. Further examples of resins that can be used to capture recombinant proteins are known in the art.

[0216] The unit operation of inactivating viruses present in a fluid comprising a recombinant protein can be performed using an MCCS, MCCS1 and / or MCCS2 (e.g., comprising a chromatography column, chromatography membrane or holding tank, capable of incubating the fluid comprising the recombinant protein at a pH of between about 3.0 and 5.0 (e.g., between about 3.5 and about 4.5, between about 3.5 and about 4.25, between about 3.5 and about 4.0, between about 3.5 and about 3.8, or about 3.75) for a period of at least 30 minutes (e.g., a period of between about 30 minutes and 1.5 hours, a period of between about 30 minutes and 1.25 hours, a period of between about 0.75 hours and 1.25 hours, or a period of about 1 hour).

[0217] The unit operation of purifying recombinant proteins can be carried out using one or more MCCSs (e.g., MCCS, MCCS1 and / or MCCS2), including, for example, a chromatography column or a chromatography membrane that includes a resin that utilizes a capture system. Non-limiting examples of capture mechanisms include protein A-binding capture mechanisms, antibody- or antibody fragment-binding capture mechanisms, substrate-binding capture mechanisms, aptamer-binding capture mechanisms, tag-binding capture mechanisms (e.g., poly-His tag-based capture mechanisms) and cofactor-binding capture mechanisms. Purifying can also be carried out using resins that can be used to carry out cation or anion exchange chromatography, molecular sieve chromatography or hydrophobic interaction chromatography. Non-limiting resins that can be used to purify recombinant proteins are described herein. Further examples of resins that can be used to purify recombinant proteins are known in the art.

[0218] The unit operation of polishing a recombinant protein can be performed using one or more MCCSs (e.g., MCCS, MCCS1 and / or MCCS2) that include, for example, a chromatography column or a chromatography membrane that contains a resin that can be used to perform, for example, cation exchange chromatography, anion exchange chromatography, molecular sieve chromatography or hydrophobic interaction chromatography. Non-limiting resins that can be used to polish a recombinant protein are described herein. Further examples of resins that can be used to polish a recombinant protein are known in the art.

[0219] Unit operations that hold fluids containing recombinant proteins can be performed using an MCCS (e.g., MCCS, MCCS1 and / or MCCS2) that includes at least one reservoir (e.g., a holding tank), or up to one, two, three, four or five reservoir(s) (e.g., holding tank(s)) with MCCS or MCCS1 and MCCS2 combined. For example, the reservoir(s) (e.g., holding tank(s)) that can be used to accomplish the above-mentioned holding unit operations can each have a volume of between about 1 mL and about 1 L (e.g., between about 1 mL and about 800 mL, between about 1 mL and about 600 mL, between about 1 mL and about 500 mL, between about 1 mL and about 400 mL, between about 1 mL and about 350 mL, between about 1 mL and about 300 mL, between about 10 mL and about 250 mL, between about 10 mL and about 200 mL, between about 10 mL and about 150 mL, or between about 10 mL and about 100 mL). The reservoir(s) (e.g., holding tank(s)) used in the methods described herein can have a volume of, for example, between 1 mL and about 300 mL, inclusive, such as, for example, between 1 mL and about 280 mL, inclusive, such as, for example, about 260 mL, about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL, about 140 mL, inclusive. , about 120 mL, about 100 mL, about 80 mL, about 60 mL, about 40 mL, about 20 mL, or about 10 mL. Any reservoir(s) (e.g., hold tank(s)) used (in any of the methods described herein) to hold the fluid until it enters the MCCS or MCCS1 may have a capacity that is, for example, between 1 mL and about 100% of the load volume of the first column of the first MCCS, inclusive, between about 1 mL and about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of the load volume of the first column of the MCCS or MCCS1, inclusive. Any of the reservoir(s) (e.g., holding tank(s)) used to hold the eluate from MCCS1 (including the recombinant protein) before the eluate enters MCCS2 can have a capacity of, for example, from 1 mL to about 100% (inclusive) of the loading volume of the first column of MCCS2, for example, from about 1 mL to about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% (inclusive).

[0220] The reservoir(s) (e.g., hold tank(s)) can each hold the fluid containing the recombinant protein for at least 10 minutes (e.g., at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, or at least 6 hours). In other examples, the reservoir(s) (e.g., hold tank(s)) holds only the recombinant protein for a period of time, e.g., between about 5 minutes and less than about 6 hours, inclusive, e.g., between about 5 minutes and about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes, inclusive. The reservoir(s) (e.g., hold tank(s)) can be used to both hold the fluid containing the recombinant protein and perform refrigeration (e.g., at temperatures below 25° C., below 15° C., or below 10° C.). The reservoirs can have any shape, including a circular cylinder, an oval cylinder, or a roughly rectangular, sealed, impermeable bag.

[0221] The unit operation of filtering a fluid containing a recombinant protein can be carried out using an MCCS (e.g., MCCS, MCCS1 and / or MCCS2), for example, a chromatography column or a chromatography membrane containing a filter or containing a molecular sieve resin. As is known in the art, a wide variety of submicron filters (e.g., filters having a pore size of less than 1 μm, less than 0.5 μm, less than 0.3 μm, about 0.2 μm, less than 0.2 μm, less than 100 nm, less than 80 nm, less than 60 nm, less than 40 nm, less than 20 nm or less than 10 nm) that can remove any precipitated material and / or cells (e.g., precipitated unfolded proteins; precipitated undesired host cell proteins; precipitated lipids; bacteria; yeast cells; fungal cells; mycobacteria; and / or mammalian cells) are available in the art. Filters having a pore size of about 0.2 μm or less than 0.2 μm are known to effectively remove bacteria from a fluid containing a recombinant protein. As is known in the art, chromatography columns or chromatographic membranes containing molecular sieving resins can also be used within the MCCS (e.g., MCCS, MCCS1 and / or MCCS2) to perform unit operations that filter fluids containing recombinant proteins.

[0222] Unit operations that adjust the ionic concentration and / or pH of a fluid containing a recombinant protein can be performed using an MCCS (e.g., MCCS, MCCS1 and / or MCCS2) that includes and utilizes a buffer adjustment reservoir (e.g., an in-line buffer adjustment reservoir) that adds fresh buffer solution to the fluid containing the recombinant protein (e.g., between columns within the MCCS, MCCS1 and / or MCCS2, or after the last column in the penultimate MCCS (e.g., MCCS1) and before the fluid containing the recombinant protein is pumped into the first column of the next MCCS (e.g., MCCS2)). As can be appreciated, the in-line buffer conditioning reservoir may be of any size (e.g., greater than 100 mL) and may contain any buffer (e.g., a buffer having one or more of the following: an increased or decreased pH compared to the fluid containing the recombinant protein, an increased or decreased ion (e.g., salt) concentration compared to the fluid containing the recombinant protein, and / or an agent whose concentration increases or decreases in competition with the recombinant protein for binding to a resin present in at least one chromatography column or at least one chromatographic membrane within the MCCS (e.g., MCCS, MCCS1 and / or MCCS2)).

[0223] MCCS, MCCS1 and / or MCCS2 can perform two or more unit operations. For example, MCCS, MCCS1 and / or MCCS2 can each perform at least the following unit operations: capturing a recombinant protein and inactivating viruses present in a fluid containing the recombinant protein; capturing a recombinant protein, inactivating viruses present in a fluid containing the recombinant protein, and adjusting the ion concentration and / or pH of a liquid containing the recombinant protein; purifying a recombinant protein and polishing a recombinant protein; purifying a recombinant protein, polishing a recombinant protein, and filtering a fluid containing a recombinant protein or removing precipitates and / or specific material from a fluid containing a recombinant protein; and purifying a recombinant protein, polishing a recombinant protein, filtering a fluid containing a recombinant protein or removing precipitates and / or specific particulate material from a fluid containing a recombinant protein, and adjusting the ion concentration and / or pH of a liquid containing a recombinant protein.

[0224] Capturing recombinant proteins The method includes a step of capturing recombinant protein using MCCS or MCCS1. As can be recognized in the art, liquid culture medium containing recombinant protein can be continuously fed onto MCCS or MCCS1 using a variety of different means. For example, liquid culture medium can be actively pumped into MCCS or MCCS1, or liquid culture medium can be fed into MCCS or MCCS1 using gravity. Liquid culture medium can be stored in a reservoir (e.g., a holding tank) before being fed into MCCS or MCCS1, or liquid culture medium can be actively pumped into MCCS or MCCS1 from a bioreactor containing a culture of cells (e.g., mammalian cells that secrete recombinant protein into the medium).

[0225] The liquid culture medium can be fed (loaded) into the MCCS or MCCS1 at a flow rate of about 0.2 mL / min to about 25 mL / min (e.g., about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, about 0.5 mL / min to about 14 mL / min, between about 1.0 mL / min to about 25.0 mL / min, between about 1.0 mL / min to about 15.0 mL / min). The liquid culture medium containing the recombinant protein can be derived from any of the typical sources described herein or known in the art.

[0226] Some examples further include an optional step of filtering the liquid culture medium before feeding it into the MCCS or MCCS1. Any of the typical means for filtering liquid culture medium or fluids containing recombinant proteins described herein, or any filtering means known in the art, can be used to filter the liquid culture medium containing recombinant proteins before feeding it into the MCCS or MCCS1.

[0227] In the methods described herein, the capture of recombinant proteins from liquid culture medium is carried out using MCC The capture of recombinant proteins is carried out using MCCS or MCCS1. As can be appreciated in the art, to achieve the capture of recombinant proteins, at least one chromatography column or at least one chromatography membrane of MCCS or MCCS1 must contain a resin that utilizes a capture mechanism (e.g., any of the exemplary capture mechanisms described herein) or that can perform cation exchange, anion exchange, or molecular sieve chromatography or hydrophobic interaction chromatography. For example, if the recombinant protein is an antibody or antibody fragment, the capture system can be a protein A-binding capture mechanism or an antigen-binding capture mechanism (where the capture antigen is specifically recognized by the recombinant antibody or antibody fragment). If the recombinant protein is an enzyme, the capture mechanism can use an antibody or antibody fragment that specifically binds to the enzyme to capture the recombinant enzyme, a substrate for the enzyme to capture the recombinant enzyme, a cofactor for the enzyme to capture the recombinant enzyme, or, if the recombinant enzyme contains a tag, a protein, metal chelate, or antibody (or antibody fragment) that specifically binds to the tag present in the recombinant enzyme. Non-limiting resins that can be used to capture recombinant proteins are described herein, and additional resins that can be used to capture recombinant proteins are known in the art. One non-limiting example of a resin that utilizes a Protein A binding capture mechanism is Mab Select SuRe™ resin (GE Healthcare, Piscataway, NJ), JSR LifeSciences Amsphere ProA JWT203 (Sunnyvale, CA). CA), and Kaneka KanCap A (Osaka, Japan).

[0228] Exemplary, non-limiting sizes and shapes of chromatography columns or chromatography membranes present within the MCCS or MCCS1 that can be used to capture recombinant proteins are described herein. The liquid culture medium supplied (loaded) into the MCCS or MCCS1 can contain, for example, about 0.05 mg / mL to about 100 mg / mL of recombinant protein (e.g., about 0.1 mg / mL to about 90 mg / mL, about 0.1 mg / mL to about 80 mg / mL, about 0.1 mg / mL to about 70 mg / mL, about 0.1 mg / mL to about 60 mg / mL, about 0.1 mg / mL to about 50 mg / mL, about 0.1 mg / mL to about 40 mg / mL, about 0.1 mg / mL to about 30 mg / mL, about 0.1 mg / mL to about 20 mg / mL, about 0.5 mg / mL to about 20 mg / mL, about 0.1 mg / mL to 15 mg / mL, about 0.5 mg / mL to about 15 mg / mL, about 0.1 mg / mL to about 10 mg / mL, or about 0.5 mg / mL to about 10 mg / mL of recombinant protein). The average time required for the recombinant protein to bind to the resin used to perform the capturing unit operation can be, for example, from about 5 seconds to about 10 minutes (e.g., from about 10 seconds to about 8 minutes, from about 10 seconds to about 7 minutes, from about 10 seconds to about 6 minutes, from about 10 seconds to about 5 minutes, from about 30 seconds to about 5 minutes, from about 1 minute to about 5 minutes, from about 10 seconds to about 4 minutes, from about 30 seconds to about 4 minutes, or from about 1 minute to about 4 minutes).

[0229] As can be appreciated in the art, in order to capture a recombinant protein using a chromatography column or a chromatography membrane present in an MCCS or MCCS1, sequential chromatography steps must be performed to load, wash, elute, and regenerate the chromatography column or the chromatography membrane present in the MCCS or MCCS1. Any of the typical flow rates, buffer volumes, and / or lengths of time assigned to each of the sequential chromatography steps described herein can be used in one or more of these different sequential chromatography steps (e.g., one or more of the sequential chromatography steps to load, wash, elute, and regenerate the chromatography column or the chromatography membrane present in the MCCS or MCCS1 used to capture the recombinant protein). Non-limiting flow rates, buffer volumes, and / or lengths of time allotted to each successive chromatographic step that can be used to capture the chromatographic column and / or chromatographic membrane are provided below. In addition, exemplary buffers that can be used for MCCS and / or MCCS1 are described below.

[0230] An MCCS or MCCS1 comprising at least one chromatography column and / or chromatography membrane comprising a resin capable of performing a capture unit operation (e.g., any of the exemplary resins that can be used for capture described herein) can be loaded with liquid culture medium comprising the recombinant protein using any of the loading flow rates (feed rates) described above. In some examples, a single chromatography column or a single chromatography membrane containing a resin capable of performing a capture unit operation is loaded, for example, in about 10 minutes to about 90 minutes (e.g., between about 15 minutes and about 90 minutes, between about 20 minutes and about 80 minutes, between about 30 minutes and about 80 minutes, between about 40 minutes and about 80 minutes, between about 50 minutes and about 80 minutes, and between about 60 minutes and about 80 minutes). In some examples where the MCCS or MCCS1 comprises at least two chromatography columns in series containing resins capable of performing a capture unit operation, the time required to sequentially load the two chromatography columns is, for example, about 50 minutes to about 180 minutes (e.g., between about 60 minutes and about 180 minutes, between about 70 minutes and about 180 minutes, between about 80 minutes and about 180 minutes, between about 90 minutes and about 180 minutes, between about 100 minutes and about 180 minutes, between about 110 minutes and 150 minutes, and between about 125 minutes and about 145 minutes).

[0231] After loading the recombinant protein onto at least one chromatography column or chromatographic membrane in the MCCS or MCCS1 containing a resin capable of performing a unit operation of capturing, the at least one chromatography column or chromatographic membrane is washed with at least one wash buffer. As can be appreciated in the art, at least one (e.g., two, three, or four) wash buffers means that all or most of the proteins that are not recombinant proteins are eluted from the at least one chromatography column or chromatographic membrane while not disrupting the interaction of the recombinant protein with the resin.

[0232] The wash buffer can be passed through at least one chromatography column or chromatography membrane at a flow rate of about 0.2 mL / min to about 25 mL / min (e.g., about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, about 0.5 mL / min to about 14 mL / min, about 1.0 mL / min to about 25.0 mL / min, about 1.0 mL / min to about 15.0 mL / min). The volume of the wash buffer used (e.g., the total total volume of the wash buffer when more than one wash buffer is used) can be, for example, about 1X column volume (CV) to about 15XCV (e.g., about 1XCV to about 14XCV, about 1XCV to about 13XCV, about 1XCV to about 12XCV, about 1XCV to about 11XCV, about 2XCV to about 11XCV, about 3XCV to about 11XCV, about 4XCV to about 11XCV, about 5XCV to about 11XCV, or about 5XCV to about 10XCV). The total time of the wash can be, for example, about 2 minutes to about 3 hours (e.g., about 2 minutes to about 2.5 hours, about 2 minutes to about 2.0 hours, about 5 minutes to about 1.5 hours, about 10 minutes to about 1.5 hours, about 10 minutes to about 1.25 hours, about 20 minutes to about 1.25 hours, or about 30 minutes to about 1 hour).

[0233] After washing at least one chromatography column or chromatographic membrane in the MCCS or MCCS1 that contains a resin capable of performing a capture unit operation, the recombinant protein is eluted from the at least one chromatography column or chromatographic membrane by passing an elution buffer through at least one chromatography column or chromatographic membrane in the MCCS or MCCS1 that contains a resin capable of performing a capture unit operation. The elution buffer can be passed through at least one chromatography column or chromatography membrane comprising a resin capable of performing a capturing unit operation at a flow rate of about 0.2 mL / min to about 25 mL / min (e.g., about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min and about 6.0 mL / min, between about 1.0 mL / min and about 5.0 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, or between about 1.0 mL / min and about 15.0 mL / min). The volume of elution buffer used to elute the recombinant protein from each of at least one chromatography column or chromatography membrane comprising a resin capable of performing a purification unit operation can be, for example, from about 1X column volumes (CV) to about 15XCV (e.g., from about 1XCV to about 14XCV, from about 1XCV to about 13XCV, from about 1XCV to about 12XCV, from about 1XCV to about 11XCV, from about 2XCV to about 11XCV, from about 3XCV to about 11XCV, from about 4XCV to about 11XCV, from about 5XCV to about 11XCV, or from about 5XCV to about 10XCV). The total time for elution can be, for example, about 2 minutes to about 3 hours (e.g., about 2 minutes to about 2.5 hours, about 2 minutes to about 2.0 hours, about 2 minutes to about 1.5 hours, about 2 minutes to about 1.5 hours, about 2 minutes to about 1.25 hours, about 2 minutes to about 1 hour, between about 2 minutes and about 40 minutes, between about 10 minutes and about 40 minutes, or between about 20 minutes and about 40 minutes). Non-limiting examples of elution buffers that can be used in these methods depend on the capture mechanism and / or recombinant protein. For example, the elution buffer can contain a different concentration of salt (e.g., increased salt concentration), a different pH (e.g., increased or decreased salt concentration), or a molecule that competes with the recombinant protein for binding to a resin capable of performing the capture unit operation. Examples of such elution buffers for each exemplary capture mechanism described herein are well known in the art.

[0234] After elution of the recombinant protein from the at least one chromatography column or chromatographic membrane in the MCCS or MCCS1 containing the resin capable of performing the capturing unit operation, the at least one chromatography column or chromatographic membrane must be equilibrated with a regeneration buffer before the next volume of liquid culture medium can be loaded onto the at least one chromatography column or chromatographic membrane. The regeneration buffer can be passed through at least one chromatography column or chromatography membrane comprising a resin capable of performing a capture unit operation at a flow rate of, for example, about 0.2 mL / min to about 25 mL / min (e.g., about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min and about 6.0 mL / min, between about 1.0 mL / min and about 5.0 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, between about 5.0 mL / min and about 15.0 mL / min, or between about 1.0 mL / min and about 15.0 mL / min). The volume of regeneration buffer used to equilibrate at least one chromatography column or chromatography membrane containing a resin capable of performing a capturing unit operation can be, for example, from about 1X column volumes (CV) to about 15XCV (e.g., from about 1XCV to about 14XCV, from about 1XCV to about 13XCV, from about 1XCV to about 12XCV, from about 1XCV to about 11XCV, from about 2XCV to about 11XCV, from about 3XCV to about 11XCV, from about 2XCV to about 5XCV, from about 4XCV to about 11XCV, from about 5XCV to about 11XCV, or from about 5XCV to about 10XCV).

[0235] In some of the methods described herein, the MCCS or MCCS1 may be used to maintain the fluid containing the recombinant protein at a low pH (e.g., pH less than 4.6, 4.4, 4.2, 4.0, 3.8, 3.6, 3.4, 3.2, or 3.0), for example, for about 1 minute to 1.5 hours (e.g., about 1 hour), to detect any toxic or harmful compounds present in the fluid containing the recombinant protein. An example of a reservoir that can be used to perform a unit operation to inactivate viruses is a stir flask (e.g., a 500-mL stir flask, e.g., a programmed stir plate) that can hold a fluid containing a recombinant protein, e.g., for about 1 minute to 1.5 hours, before feeding the fluid containing the recombinant protein into the MCCS2. The reservoir used to perform the viral inactivation unit operation can be a 500-mL stir flask with a programmed stir plate (e.g., a stir plate programmed to mix (e.g., periodically mix) the fluid in the reservoir, e.g., every 4 hours). Another example of a reservoir that can be used to perform the viral inactivation unit operation is a plastic bag (e.g., a 500-mL plastic bag) in which the recombinant protein-containing fluid can be held, e.g., for about 1 minute to 1.5 hours, before the recombinant protein-containing fluid is fed into the MCCS2. In some examples, the recombinant protein-containing fluid can already have a low pH (e.g., pH less than 4.6, 4.4, 4.2, 4.0, 3.8, 3.6, 3.4, 3.2, or 3.0) when fed into the reservoir used to perform the viral inactivation unit operation. As can be appreciated by those skilled in the art, a variety of other means can be used to perform the viral inactivation unit operation. For example, UV irradiation of a fluid containing a recombinant protein can also be used to perform a unit operation of inactivating viruses. Non-limiting examples of reservoirs that can be used to perform the unit operation of inactivating viruses present in a fluid containing a recombinant protein are described herein.

[0236] The MCCS or MCCS1 can include a PCCS that includes four chromatography columns, where at least three of the four chromatography columns perform a unit operation of capturing recombinant protein from liquid culture medium using an MCCS that includes at least one chromatography column (e.g., any of those described herein) that includes a resin capable of performing a capturing unit operation. In these examples, the fourth column of the PCC can perform a unit operation of inactivating viruses in a fluid that includes the recombinant protein (e.g., any of the exemplary columns described herein that can be used to achieve viral inactivation of a fluid that includes a recombinant protein).

[0237] In some examples, a fluid containing recombinant protein is continuously eluted from MCCS1 (e.g., PCCS1) and continuously fed to MCCS2 (e.g., PCCS2). The percentage (%) of recombinant protein recovered in the eluate of MCCS or MCCS1 (e.g., PCCS or PCCS1) can be, for example, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98%. The eluate from MCCS1 (e.g., PCCS1) can be fed into MCCS2 (e.g., PCCS2) using various means (e.g., piping) known in the art. The eluate of MCCS1 (e.g., PCCS1) can be supplied to MCCS2 (e.g., PCCS2) at a flow rate of, for example, about 0.2 mL / min to about 25 mL / min (e.g., about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min and about 6.0 mL / min, between about 1.0 mL / min and about 5.0 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, between about 5.0 mL / min to about 15.0 mL / min, between about 15 mL / min to about 25 mL / min, or between about 1.0 mL / min and about 15.0 mL / min).

[0238] Some methods described herein may further include adjusting the ion concentration and / or pH of the eluate from MCCS1 (e.g., PCCS1) before the eluate is fed into MCCS2 (e.g., PCCS2). As described herein, the ion concentration and / or pH of the eluate from MCCS1 (e.g., PCCS1) may be adjusted by adding a buffer to the eluate (before the eluate is fed into MCCS2) (e.g., by using an in-line buffer adjustment reservoir). The buffer may be added to the eluate from MCCS1 at a flow rate of, for example, about 0.1 mL / min to about 15 mL / min (e.g., about 0.1 mL / min to about 12.5 mL / min, about 0.1 mL / min to about 10.0 mL / min, about 0.1 mL / min to about 8.0 mL / min, about 0.1 mL / min to about 6 mL / min, about 0.1 mL / min to about 4 mL / min, or about 0.5 mL / min to about 5 mL / min).

[0239] The methods described herein can further include the step of holding or storing (and optionally refrigerating) the eluate from MCCS1 prior to providing the eluate into MCCS2. As described herein, this holding or storing can be performed using any of the reservoirs described herein (e.g., a back-up tank).

[0240] The methods described herein can also include filtering the eluate from MCCS1 before feeding it into MCCS2. Any of the filtration methods or exemplary filters described herein can be used to filter the eluate from MCCS1 before feeding it into MCCS2.

[0241] Polishing and purifying recombinant proteins MCCS, MCCS1, and / or MCCS2 can be used to perform the unit operation of purifying and polishing a recombinant protein. For example, MCCS2 can be used to perform the operation of purifying and polishing a recombinant protein, and the eluate from MCCS2 is a protein drug substance. MCCS, MCCS1, and / or MCCS2 can include at least one (e.g., two, three, or four) chromatography columns or chromatographic membranes that can be used to perform the unit operation of purifying a recombinant protein, and at least one (e.g., two, three, or four) chromatography columns or chromatographic membranes that can be used to perform the unit operation of polishing a recombinant protein.

[0242] At least one chromatographic column or chromatographic membrane that can be used to perform the unit operation of purifying a recombinant protein can include a resin that utilizes a capture mechanism (e.g., any of the capture mechanisms described herein or known in the art) or a resin that can be used to perform anion exchange, cation exchange, molecular sieve chromatography, or hydrophobic interaction chromatography. At least one chromatographic column or chromatographic membrane that can be used to perform the unit operation of polishing a recombinant protein can include a resin that can be used to perform anion exchange, cation exchange, molecular sieve chromatography, or hydrophobic interaction chromatography (e.g., any of the typical resins for performing anion exchange, cation exchange, molecular sieve chromatography, or hydrophobic interaction chromatography described herein or known in the art).

[0243] The size, shape, and composition of at least one chromatography column or chromatography membrane that can be used to perform the unit operation of purifying a recombinant protein. The size and volume, and / or size and shape of at least one chromatographic membrane that can be used to perform a unit operation of polishing a recombinant protein can be any combination of typical sizes, shapes, and volumes of chromatographic columns or chromatographic membranes described herein. As can be appreciated by those skilled in the art, the process of purifying or polishing a recombinant protein can include, for example, loading, washing, eluting, and equilibrating at least one chromatographic column or chromatographic membrane used to perform a unit operation of purifying or polishing a recombinant protein. Typically, the elution buffer exiting from the chromatographic column or chromatographic membrane used to perform the unit operation of purifying contains the recombinant protein. Typically, the loading and / or washing buffer exiting from the chromatographic column or chromatographic membrane used to perform the unit operation of polishing contains the recombinant protein.

[0244] For example, the size of at least one chromatography column or chromatography membrane that can be used to perform a unit operation of purifying a recombinant protein can have a volume of, for example, about 2.0 mL to about 200 mL (e.g., about 2.0 mL to about 180 mL, about 2.0 mL to about 160 mL, about 2.0 mL to about 140 mL, about 2.0 mL to about 120 mL, about 2.0 mL to about 100 mL, about 2.0 mL to about 80 mL, about 2.0 mL to about 60 mL, about 2.0 mL to about 40 mL, about 5.0 mL to about 40 mL, about 2.0 mL to about 30 mL, about 5.0 mL to about 30 mL, or about 2.0 mL to about 25 mL). The flow rate of a fluid containing a recombinant protein when loaded onto at least one chromatography column or at least one chromatographic membrane that can be used to perform a unit operation of purifying the recombinant protein can be, for example, from about 0.1 mL / min to about 25 mL / min (e.g., from about 0.1 mL / min to about 12.5 mL / min, from about 0.1 mL / min to about 10.0 mL / min, from about 0.1 mL / min to about 8.0 mL / min, from about 0.1 mL / min to about 6 mL / min, from about 0.1 mL / min to about 4 mL / min, from about 0.1 mL / min to about 3 mL / min, from about 0.1 mL / min to about 2 mL / min, or from about 0.2 mL / min to about 4 mL / min). The concentration of recombinant protein in a fluid that is loaded onto at least one chromatography column or chromatography membrane that can be used to perform a unit operation to purify a recombinant protein can range, for example, from about 0.05 mg / mL to about 100 mg / mL of recombinant protein (e.g., from about 0.1 mg / mL to about 90 mg / mL, from about 0.1 mg / mL to about 80 mg / mL, from about 0.1 mg / mL to about 70 mg / mL, from about 0.1 mg / mL to about 60 mg / mL). The concentration of the recombinant protein can be about 0 mg / mL, about 0.1 mg / mL to about 50 mg / mL, about 0.1 mg / mL to about 40 mg / mL, about 0.1 mg / mL to about 30 mg / mL, about 0.1 mg / mL to about 20 mg / mL, about 0.5 mg / mL to about 20 mg / mL, about 0.1 mg / mL to 15 mg / mL, about 0.5 mg / mL to about 15 mg / mL, about 0.1 mg / mL to about 10 mg / mL, or about 0.5 mg / mL to about 10 mg / mL of the recombinant protein.The resin in at least one chromatography column or chromatographic membrane used to perform a purification unit operation can be a resin that can be used to perform anion exchange or cation exchange chromatography. The resin in at least one chromatography column or chromatographic membrane used to perform a purification unit operation can be a cation exchange resin (e.g., Capto-S resin, GE Healthcare Life Sciences, Piscataway, NJ).

[0245] After loading of the recombinant protein onto at least one chromatography column or chromatographic membrane that can be used to perform a unit operation of purifying the recombinant protein, the at least one chromatography column or chromatographic membrane is washed with at least one wash buffer. As can be appreciated in the art, the at least one (e.g., two, three, or four) wash buffers can be: This means that any protein that is not a recombinant protein is eluted from at least one chromatography column or chromatography membrane, while not perturbing the interaction of the recombinant protein with the resin or otherwise the elution of the recombinant protein.

[0246] The wash buffer can be passed through at least one chromatography column or chromatography membrane at a flow rate of about 0.2 mL / min to about 25 mL / min (e.g., about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, or between about 1.0 mL / min and about 15.0 mL / min). The volume of the wash buffer used (e.g., the total total volume of the wash buffer when more than one type of wash buffer is used) can be, for example, from about 1X column volume (CV) to about 15XCV (e.g., about 1XCV to about 14XCV, about 1XCV to about 13XCV, about 1XCV to about 12XCV, about 1XCV to about 11XCV, about 2XCV to about 11XCV, about 3XCV to about 11XCV, about 4XCV to about 11XCV, about 2.5XCV ​​to about 5.0XCV, about 5XCV to about 11XCV, or about 5XCV to about 10XCV). The total washing time can be, for example, about 2 minutes to about 3 hours (e.g., about 2 minutes to about 2.5 hours, about 2 minutes to about 2.0 hours, about 5 minutes to about 1.5 hours, about 10 minutes to about 1.5 hours, about 10 minutes to about 1.25 hours, about 20 minutes to about 1.25 hours, about 30 minutes to about 1 hour, between about 2 minutes and about 10 minutes, between about 2 minutes and about 15 minutes, or between about 2 minutes and about 30 minutes).

[0247] After washing the at least one chromatographic column or chromatographic membrane used to perform the unit operation of purifying the recombinant protein, the recombinant protein is eluted from the at least one chromatographic column or chromatographic membrane by passing an elution buffer through the at least one chromatographic column or chromatographic membrane used to perform the unit operation of purifying the recombinant protein. The elution buffer can be passed through at least one chromatography column or chromatographic membrane that can be used to perform a unit operation to purify the recombinant protein at a flow rate of about 0.2 mL / min to about 25 mL / min (e.g., about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min and about 6.0 mL / min, between about 1.0 mL / min and about 5.0 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, or between about 1.0 mL / min and about 15.0 mL / min). The volume of elution buffer used to elute the recombinant protein from each of at least one chromatography column or chromatography membrane that can be used to perform a unit operation to purify the recombinant protein can be, for example, from about 1X column volumes (CV) to about 25XCV (e.g., from about 1XCV to about 20XCV, from about 15XCV to about 25XCV, from about 1XCV to about 14XCV, from about 1XCV to about 13XCV, from about 1XCV to about 12XCV, from about 1XCV to about 11XCV, from about 2XCV to about 11XCV, from about 3XCV to about 11XCV, from about 4XCV to about 11XCV, from about 5XCV to about 11XCV, or from about 5XCV to about 10XCV). The total elution time can be, for example, from about 2 minutes to about 3 hours (e.g., from about 2 minutes to about 2.5 hours, from about 2 minutes to about 2.0 hours, from about 2 minutes to about 1.5 hours, from about 2 minutes to about 1.5 hours, from about 2 minutes to about 1.25 hours, from about 2 minutes to about 1.25 hours, from about 2 minutes to about 1 hour, between about 2 minutes and about 40 minutes, between about 10 minutes and about 40 minutes, between about 20 minutes and about 40 minutes, or between about 30 minutes and 1.0 hour).Non-limiting examples of elution buffers that can be used in these methods depend on the biophysical properties of the resin and / or the recombinant protein. For example, the elution buffer can contain a different concentration of salt (e.g., increased salt concentration), a different pH (e.g., elevated or decreased salt concentration), or a molecule that competes with the recombinant protein for binding to the resin. Such elution buffers for each of the exemplary capture mechanisms described herein. Examples of are well known in the art.

[0248] After elution of the recombinant protein from at least one chromatographic column or chromatographic membrane used to perform a unit operation to purify the recombinant protein, and before the next volume of fluid containing the recombinant protein can be loaded onto the at least one chromatographic column or chromatographic membrane, the at least one chromatographic column or chromatographic membrane must be equilibrated with a regeneration buffer. The regeneration buffer can be passed through at least one chromatography column or chromatographic membrane used to perform a unit operation to purify the recombinant protein at a flow rate of, for example, about 0.2 mL / min to about 25 mL / min (e.g., about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min and about 6.0 mL / min, between about 1.0 mL / min and about 5.0 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, between about 5.0 mL / min and about 15.0 mL / min, or between about 1.0 mL / min and about 15.0 mL / min). The volume of regeneration buffer used to equilibrate at least one chromatography column or chromatography membrane containing a resin that can be used to perform a unit operation to purify a recombinant protein can be, for example, from about 1X column volumes (CV) to about 15XCV (e.g., from about 1XCV to about 14XCV, from about 1XCV to about 13XCV, from about 1XCV to about 12XCV, from about 1XCV to about 11XCV, from about 2XCV to about 11XCV, from about 3XCV to about 11XCV, from about 2XCV to about 5XCV, from about 2.5XCV ​​to about 7.5XCV, from about 4XCV to about 11XCV, from about 5XCV to about 11XCV, or from about 5XCV to about 10XCV).The concentration of recombinant protein in the eluate of at least one chromatography column or chromatography membrane used to perform a unit operation of purifying a recombinant protein can be, for example, about 0.05 mg / mL to about 100 mg / mL of recombinant protein (e.g., about 0.1 mg / mL to about 90 mg / mL, about 0.1 mg / mL to about 80 mg / mL, about 0.1 mg / mL to about 70 mg / mL, about 0.1 mg / mL to about 60 mg / mL, about 0.1 mg / mL to about 100 mg / mL of recombinant protein). The recombinant protein can be present in a concentration of about 0.1 mg / mL to about 50 mg / mL, about 0.1 mg / mL to about 40 mg / mL, about 2.5 mg / mL to about 7.5 mg / mL, about 0.1 mg / mL to about 30 mg / mL, about 0.1 mg / mL to about 20 mg / mL, about 0.5 mg / mL to 20 mg / mL, about 0.1 mg / mL to about 15 mg / mL, about 0.5 mg / mL to about 15 mg / mL, about 0.1 mg / mL to about 10 mg / mL, or about 0.5 mg / mL to about 10 mg / mL.

[0249] The at least one chromatographic column or chromatographic membrane used to perform the unit operation of polishing the recombinant protein can include a resin that can be used to perform cation exchange, anion exchange, or molecular sieve chromatography. As can be appreciated in the art, polishing a recombinant protein using at least one chromatographic column or chromatographic membrane that can be used to perform the unit operation of polishing the recombinant protein can include, for example, steps of loading, chasing, and regenerating the at least one chromatographic column or chromatographic membrane that can be used to perform the unit operation of polishing the recombinant protein. For example, when polishing is performed using the steps of loading, chasing, and regenerating, the recombinant protein does not bind to the resin in the at least one chromatographic column or chromatographic membrane used to perform the unit operation of polishing the recombinant protein, the recombinant protein is eluted from the at least one chromatographic column or chromatographic membrane in the steps of loading and chasing, and the regeneration step removes the recombinant protein from the at least one chromatographic column or chromatographic membrane before additional fluid containing the recombinant protein can be loaded onto the at least one chromatographic column or chromatographic membrane. The typical flow rates and buffer volumes used in each of the loading, chase, and regeneration steps are given below.

[0250] The size, shape, and volume of the at least one chromatography column or chromatographic membrane that can be used to perform the unit operation of polishing a recombinant protein, and / or the size and shape of the at least one chromatographic membrane that can be used to perform the unit operation of polishing a recombinant protein, can be any combination of typical sizes, shapes, and volumes of chromatography columns or chromatographic membranes described herein. For example, the size of at least one chromatography column or chromatography membrane that can be used to perform the unit operation of polishing a recombinant protein can have a volume of, for example, about 0.5 mL to about 200 mL (e.g., about 0.5 mL to about 180 mL, about 0.5 mL to about 160 mL, about 0.5 mL to about 140 mL, about 0.5 mL to about 120 mL, about 0.5 mL to about 100 mL, about 0.5 mL to about 80 mL, about 0.5 mL to about 60 mL, about 0.5 mL to about 40 mL, about 5.0 mL to about 40 mL, about 0.5 mL to about 30 mL, about 5.0 mL to about 30 mL, about 0.5 mL to about 25 mL, about 0.2 mL to about 10 mL, or about 0.2 mL to about 5 mL). The flow rate of the fluid containing the recombinant protein when loaded onto at least one chromatography column or chromatography membrane that can be used to perform the unit operation of polishing the recombinant protein can be, for example, about 0.1 mL / min to about 25 mL / min (e.g., about 0.1 mL / min to about 12.5 mL / min, about 0.1 mL / min to about 10.0 mL / min, about 0.1 mL / min to about 8.0 mL / min, about 0.1 mL / min to about 6 mL / min, about 0.1 mL / min to about 4 mL / min, about 0.1 mL / min to about 3 mL / min, about 2 mL / min to about 6 mL / min, about 0.1 mL / min to about 2 mL / min, or about 0.2 mL / min to about 4 mL / min).The total volume of fluid containing the recombinant protein that is loaded onto at least one chromatography column or chromatography membrane that can be used to perform the unit operation of polishing the recombinant protein can be, for example, about 1.0 mL to about 250 mL (e.g., about 1.0 mL to about 225 mL, about 1.0 mL to about 200 mL, about 1.0 mL to about 175 mL, about 1.0 mL to about 150 mL, about 100 mL to about 125 mL, about 100 mL to about 150 mL, about 1.0 mL to about 150 mL, about 1.0 mL to about 125 mL, about 1.0 mL to about 100 mL, about 1.0 mL to about 75 mL, about 1.0 mL to about 50 mL, or about 1.0 mL to about 25 mL). The resin in the at least one chromatography column or chromatography membrane used to perform the polish can be an anion exchange or cation exchange resin. The resin in at least one chromatography column or chromatographic membrane used to perform the polishing unit operation can be a cation exchange resin (e.g., Sartobind® Q resin, Sartorius, Goettingen, Germany).

[0251] After the loading step, a chase step is performed (e.g., passing a chase buffer through at least one chromatography column or chromatographic membrane to collect recombinant protein that is not substantially bound to the at least one chromatography column or chromatographic membrane). In these examples, the chase buffer can be passed through at least one chromatography column or chromatographic membrane at a flow rate of about 0.2 mL / min to about 50 mL / min (e.g., about 1 mL / min to about 40 mL / min, about 1 mL / min to about 30 mL / min, about 5 mL / min to about 45 mL / min, about 10 mL / min to about 40 mL / min, about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, or between about 1.0 mL / min and about 15.0 mL / min). The volume of chase buffer used can range, for example, from about 1X column volume (CV) to about 100XCV (e.g., from about 1XCV to about 9 The amino acid sequence may be 0XCV, about 1XCV to about 80XCV, about 1XCV to about 70XCV, about 1XCV to about 60XCV, about 1XCV to about 50XCV, about 1XCV to about 40XCV, about 1XCV to about 30XCV, about 1XCV to about 20XCV, about 1XCV to about 15XCV, about 5XCV to about 20XCV, or about 5XCV to about 30XCV, about 1XCV to about 14XCV, about 1XCV to about 13XCV, about 1XCV to about 12XCV, about 1XCV to about 11XCV, about 2XCV to about 11XCV, about 3XCV to about 11XCV, about 4XCV to about 11XCV, about 2.5XCV ​​to about 5.0XCV, about 5XCV to about 11XCV, or about 5XCV to about 10XCV. The total tracking time can be, for example, from about 1 minute to about 3 hours (e.g., from about 1 minute to about 2.5 hours, from about 1 minute to about 2.0 hours, from about 1 minute to about 1.5 hours, from about 2 minutes to about 1.5 hours, from about 1 minute to about 1.25 hours, from about 2 minutes to about 1.25 hours, from about 1 minute to about 5 minutes, from about 1 minute to about 10 minutes, from about 2 minutes to about 4 minutes, from about 30 minutes to about 1 hour, from about 2 minutes to about 10 minutes, from about 2 minutes to about 15 minutes, or from about 2 minutes to about 30 minutes). The combined concentration of recombinant protein present in the eluate coming through the column in the loading and chasing steps can be, for example, from about 0.1 mg / mL to about 100 mg / mL of recombinant protein (e.g., from about 0.1 mg / mL to about 90 mg / mL, from about 0.1 mg / mL to about 80 mg / mL, from about 0.1 mg / mL to about 70 mg / mL, from about 0.1 mg / mL to about 60 mg / mL, from about 0.1 mg / mL to about 50 mg / mL, from about 0.1 mg / mL to about 40 mg / mL). mL, between about 2.5 mg / mL and about 7.5 mg / mL, about 0.1 mg / mL to about 30 mg / mL, about 0.1 mg / mL to about 20 mg / mL, about 0.5 mg / mL to 20 mg / mL, about 0.1 mg / mL to about 15 mg / mL, about 0.5 mg / mL to about 15 mg / mL, about 0.1 mg / mL to about 10 mg / mL, about 0.5 mg / mL to about 10 mg / mL, or about 1 mg / mL to about 5 mg / mL of recombinant protein).

[0252] After the tracing step, and before the next volume of fluid containing the recombinant protein can be loaded onto the at least one chromatography column or chromatographic membrane that can be used to perform the polishing unit operation, the at least one chromatography column or chromatographic membrane must be regenerated with a regeneration buffer. The regeneration buffer can be passed through at least one chromatography column or chromatographic membrane that can be used to perform a unit operation to polish the recombinant protein at a flow rate of, for example, about 0.2 mL / min to about 50 mL / min (e.g., about 1 mL / min to about 40 mL / min, about 1 mL / min to about 30 mL / min, about 5 mL / min to about 45 mL / min, about 10 mL / min to about 40 mL / min, about 0.2 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, or between about 1.0 mL / min and about 15.0 mL / min).The volume of regeneration buffer used to regenerate at least one chromatography column or chromatography membrane that can be used to perform a polishing unit operation can range, for example, from about 1X column volume (CV) to about 500XCV (e.g., from about 1XCV to about 450XCV, from about 1XCV to about 400XCV, from about 1XCV to about 350XCV, from about 1XCV to about 300XCV, from about 1XCV to about 250XCV, from about 1XCV to about 200XCV, from about 1XCV to about 150XCV, from about 1XCV to about 100XCV, from about 1XCV to about 90XCV, from about 1XCV to about 80XCV, or from about 1XCV to about 100XCV). CV to about 70XCV, about 1XCV to about 60XCV, about 1XCV to about 50XCV, about 1XCV to about 40XCV, about 1XCV to about 30XCV, about 1XCV to about 20XCV, about 1XCV to about 15XCV, about 5XCV to about 20XCV, about 5XCV to about 30XCV, about 1XCV to about 14XCV, about 1XCV to about 13XCV, about 1XCV to about 12XCV, about 1XCV to about 11XCV, about 2XCV to about 11XCV, about 3XCV to about 11XCV, about 4XCV to about 11XCV, about 2.5XCV ​​to about 5.0XCV, about 5XCV to about 11XCV, or about 5XCV to about 10XCV).

[0253] In other examples, one or more chromatography columns and / or chromatographic membranes used to perform the polishing unit operation contain a resin that selectively binds or retains impurities present in the fluid containing the recombinant protein, and once the binding capacity of the resin of the one or more columns and / or membranes is reached or substantially equivalent to being reached, instead of regenerating the one or more columns and / or membranes, the one or more columns and / or membranes are replaced (e.g., replaced with a substantially similar column and / or membrane).

[0254] In some examples of these methods described herein, the MCCS2 includes a PCCS that includes, for example, three chromatography columns and one chromatography membrane, where the three chromatography columns in the PCCS perform a unit operation of purifying a recombinant protein (e.g., with at least one chromatography column that can be used to perform a unit operation of purifying a protein), and the chromatography membrane in the PCCS performs a unit operation of polishing the recombinant protein. In these examples, the chromatography membrane in the PCCS that can be used to perform a unit operation of polishing a therapeutic protein can be any of the exemplary chromatography membranes described herein that can be used to perform a unit operation of polishing a recombinant protein. Any of the column switching methods described herein can be used to determine when the first three chromatography columns and chromatography membranes in the PCCS in this example can be switched.

[0255] Some embodiments of this example can further include adjusting the ionic concentration and / or pH of the eluate from the three chromatography columns in the PCCS before feeding the eluate into the chromatography membrane in the PCCS. As described herein, the ionic concentration and / or pH of the eluate from the three chromatography columns in the PCCS (in this example, before feeding the eluate into the chromatography membrane in the PCCS) can be adjusted (e.g., by use of an in-line buffer adjustment reservoir) by adding a buffer to the eluate of the three chromatography columns in the PCCS. The buffer can be added to the eluate at a flow rate of, for example, about 0.1 mL / min to about 15 mL / min (e.g., about 0.1 mL / min to about 12.5 mL / min, about 0.1 mL / min to about 10.0 mL / min, about 0.1 mL / min to about 8.0 mL / min, about 0.1 mL / min to about 6 mL / min, about 0.1 mL / min to 4 mL / min, or about 0.5 mL / min to about 5 mL / min).

[0256] These examples can further include a step of holding or storing the eluates from, in this example, the three chromatography columns in the PCCS prior to feeding the eluates into a chromatography membrane that can be used to perform a unit operation to polish the recombinant protein. As described herein, this holding or storing step can be performed using any of the reservoirs described herein (e.g., backup tanks).

[0257] These examples can also include filtering the eluate from a chromatographic membrane in a typical PCCS system that can be used to perform a unit operation to polish a recombinant protein. Any of the exemplary filters or methods for filtration described herein can be used to filter the eluate from a chromatographic membrane in this typical PCCS that can be used to perform a unit operation to polish a recombinant protein.

[0258] As can be appreciated in the art, purified recombinant proteins are Any of the methods described herein can be used to periodically elute from MCCS or MCCS2. For example, any of the methods described herein can elute purified recombinant protein for a duration of, for example, about 30 seconds to about 5 hours (e.g., between about 1 minute and about 4 hours, between about 1 minute and about 3 hours, between about 1 minute and about 2 hours, between about 1 minute and about 1.5 hours, between about 1 minute and about 1 hour, or between about 1 minute and about 30 minutes), for example, at a frequency of about 1 minute to about 6 hours (e.g., between about 1 minute and about 5 hours, between about 1 minute and about 4 hours, between about 1 minute and about 3 hours, between about 1 minute and about 2 hours, between about 1 minute and about 1 hour, or between about 1 minute and 30 minutes), depending, for example, on the chromatographic column and / or chromatographic membrane used for MCCS or MCCS1 and MCCS2.

[0259] How to culture Some of the methods described herein further include culturing cells (e.g., recombinant mammalian cells) that secrete the recombinant protein in a bioreactor (e.g., a perfusion or fed-batch bioreactor) containing a liquid culture medium, where a volume of the liquid culture medium substantially free of the cells (e.g., mammalian cells) is continuously or periodically removed from the bioreactor (e.g., a perfusion bioreactor) and fed into the MCCS or MCCS1. The bioreactor can have a volume of, for example, about 1 L to about 10,000 L (e.g., about 1 L to about 50 L, about 50 L to about 500 L, about 500 L to about 1000 L, 500 L to about 5000 L, about 500 L to about 10,000 L, about 5000 L to about 10,000 L, between about 1 L and about 10,000 L, between about 1 L and about 8,000 L, between about 1 L and about 6,000 L, between about 1 L and about 5,000 L, between about 100 L and about 5,000 L, between about 10 L and about 100 L, between about 10 L and about 4,000 L, between about 10 L and about 3,000 L, between about 10 L and about 2,000 L, or between about 10 L and about 1,000 L). The amount of liquid culture medium present in the bioreactor can be, for example, about 0.5 L to about 5,000 L (e.g., about 0.5 L to about 25 L, about 25 L to about 250 L, about 250 L to about 500 L, 250 L to about 2500 L, about 250 L to about 5,000 L, about 2500 L to about 5,000 L, between about 0.5 L and about 5,000 L, between about 0.5 L and about 4,000 L, between about 0.5 L and about 3,000 L, between about 0.5 L and about 2,500 L, between about 50 L and about 2,500 L, between about 5 L and about 50 L, between about 5 L and about 2,000 L, between about 5 L and about 1,500 L, between about 5 L and about 1,000 L, or between about 5 L and about 500 L). The culturing of cells can be carried out, for example, using a fed-batch bioreactor or a perfusion bioreactor. Non-limiting examples and various aspects of culturing cells (e.g., culturing mammalian cells) are described below and can be used in any combination.

[0260] cell The cells cultured in some of the methods described herein can be bacteria (e.g., gram-negative bacteria), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, or Arxula adeninivorans), or mammalian cells. The mammalian cells can be cells that grow in suspension or as adherent cells. Non-limiting examples of mammalian cells that can be cultured by any of the methods described herein include: Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells or CHO-K1s cells), Sp2.0, myeloma cells (e.g., NS / 0), B-cells, hybridoma cells, T-cells, human embryonic kidney (HEK) cells (e.g., HEK 293E and HEK 293F), African green monkey kidney epithelial (Vero) cells, and Mayde cells. and Madin-Darby Canine (Cocker Spaniel) Kidney Epithelial (MDCK) cells. In some instances of culturing adherent cells, the culture can also include a plurality of microcarriers (e.g., microcarriers containing one or more pores). Additional mammalian cells that can be cultured by any of the methods described herein are known in the art.

[0261] The mammalian cell can include a recombinant nucleic acid (e.g., a nucleic acid stably integrated into the genome of the mammalian cell) encoding a recombinant protein (e.g., a recombinant protein). Non-limiting examples of recombinant nucleic acids encoding exemplary recombinant proteins are described below, along with recombinant proteins that can be produced using the methods described herein. In some examples, the mammalian cells cultured in a bioreactor (e.g., any of the bioreactors described herein) were derived from a larger culture.

[0262] Nucleic acids encoding recombinant proteins can be introduced into mammalian cells using a wide variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (e.g., lipofection), transduction (e.g., lentiviral, adenoviral, or retroviral infection), and electroporation. In some instances, the nucleic acid encoding the recombinant protein is not stably integrated into the chromosome of the mammalian cell (transient transfection), while in other cases the nucleic acid is integrated. Alternatively, or in addition, the nucleic acid encoding the recombinant protein can be present in a plasmid and / or in a mammalian artificial chromosome (e.g., a human artificial chromosome). Alternatively, or in addition, the nucleic acid can be introduced into the cell using a viral vector (e.g., a lentiviral, retroviral, or adenoviral vector). The nucleic acid can be operably linked to a promoter sequence (e.g., a strong promoter such as the β-actin promoter and the CMV promoter, or an inducible promoter). Optionally, the vector containing the nucleic acid can also include a selectable marker (e.g., a gene that confers hygromycin, puromycin, or neomycin resistance to mammalian cells).

[0263] In some instances, the recombinant protein is a secreted protein and is released by the mammalian cell into the extracellular medium (e.g., the first and / or second liquid culture medium). For example, a nucleic acid sequence encoding a soluble recombinant protein can include a sequence at the N- or C-terminus of the recombinant protein that encodes a secretory signal peptide, which is cleaved by an enzyme present in the mammalian cell and then released into the extracellular medium (e.g., the first and / or second liquid culture medium).

[0264] Culture medium Liquid culture media are known in the art. The liquid culture media (e.g., the first and / or second tissue culture media) can be supplemented with mammalian serum (e.g., fetal bovine serum and bovine serum), and / or growth hormones or growth factors (e.g., insulin, transferrin, and epidermal growth factor). Alternatively, or in addition, the liquid culture media (e.g., the first and / or second liquid culture media) can be chemically defined, animal-derived component-free, serum-free, or serum-containing liquid culture media. Non-limiting examples of chemically defined, animal-derived component-free, serum-free, and serum-containing liquid culture media are commercially available.

[0265] The liquid culture medium typically contains an energy source (e.g., a carbohydrate such as glucose), essential amino acids (e.g., a basic set of 20 amino acids plus cysteine), vitamins and / or other organic compounds required in low concentrations, free fatty acids, and / or trace elements. The liquid culture medium (e.g., the first and / or second liquid culture medium) can optionally contain, for example, They may be supplemented with mammalian hormones or growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts and buffers (e.g., calcium, magnesium, and phosphate), nucleosides and bases (e.g., adenosine, thymidine, and hypoxanthine), protein and tissue hydrolysates, and / or any combination of these additives.

[0266] A wide variety of different liquid culture media are known in the art that can be used to culture cells (e.g., mammalian cells) in any of the methods described herein. Medium components that may also be useful in the present methods include, but are not limited to, chemically defined (CD) hydrolysates, such as CD peptones, CD polypeptides (two or more amino acids), and CD growth factors. Additional examples of liquid tissue culture media and medium components are known in the art.

[0267] As will be appreciated by the skilled practitioner, the first and second liquid culture media described herein can be the same type of medium or different media.

[0268] Additional Features of a Typical Bioreactor The inner surface of any of the bioreactors described herein may have at least one coating (e.g., at least one coating of gelatin, collagen, poly-L-ornithine, polystyrene, and laminin), as well as one or more ports for sparging O2, CO2, and N2 into the liquid culture medium, and a stirring mechanism for agitating the liquid culture medium, as known in the art. The bioreactor can incubate cell cultures in a controlled humidified atmosphere (e.g., at humidity levels greater than 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or at 100% humidity). The bioreactor can also be equipped with a mechanical device capable of removing a volume of liquid culture medium from the bioreactor, and optionally a filter (e.g., an ATF system or cell filtration system described in U.S. Provisional Patent Application No. 61 / 878,502) that removes cells from the liquid culture medium during the process of transferring the liquid culture medium from the bioreactor.

[0269] temperature The mammalian cell culture process can be carried out at a temperature of about 31° C. to about 40° C. As will be appreciated by the skilled practitioner, the temperature can be altered at certain times during the culture process, e.g., on an hourly or daily basis. For example, the temperature can be changed or shifted (e.g., increased or decreased) at about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or about 20 days or more after the bioreactor is initially seeded with cells (e.g., mammalian cells). For example, the temperature can be shifted upward (e.g., a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about a 20° C. change). For example, the temperature can be shifted downward (e.g., a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about a 20° C. change).

[0270] CO2 The culturing process described herein further comprises the step of: culturing the liquid culture medium in the bioreactor at or above can include exposing the culture to an atmosphere containing about 15% CO2 (e.g., up to or about 14% CO2, 12% CO2, 10% CO2, 8% CO2, 6% CO2, 5% CO2, 4% CO2, 3% CO2, 2% CO2, or up to or about 1% CO2).

[0271] Perfusion Bioreactor The culturing process described herein can be carried out using a perfusion bioreactor. Culturing cells (e.g., mammalian cells) in a perfusion bioreactor includes removing a first volume of a first liquid culture medium (e.g., a first volume of a first liquid culture medium containing any concentration of mammalian cells, e.g., substantially free of cells) from the bioreactor, and adding a second volume of a second liquid culture medium to the first liquid culture medium. The removal and addition can be carried out simultaneously or sequentially, or a combination of the two. Further, removals and additions may be between 0.1% and 800% (e.g., between 1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%, between 1% and 350%, between 1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 8 ... or between 4% and 30%) over a given time period (e.g., over a 24 hour period, over increasing time periods of about 1 hour to about 24 hours, or over increasing time periods of more than 24 hours)), or periodically (e.g., once every 3 days, once every 2 days, once a day, twice a day, three times a day, four times a day, or five times a day), or any combination thereof. When performed periodically, the volume removed or replaced (e.g., within about a 24 hour period, within increasing time periods of about 1 hour to about 24 hours, or within increasing time periods of more than 24 hours) can be, for example, 0.1% to 800% (e.g., between 1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%, between 1% and 300%, between 1% and 200%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and 30%) of the volume of the bioreactor or the volume of the first liquid culture medium.The first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added can, in some instances, be kept approximately the same over each 24 hour period (or alternatively, increasing time periods from about 1 hour to about 24 hours or increasing time periods greater than 24 hours) during the entire or part of the culture period. As is known in the art, the rate at which the first volume of the first liquid culture medium is removed (volume / unit time) and the rate at which the second volume of the second liquid culture medium is added (volume / unit time) can vary. The rate at which the first volume of the first liquid culture medium is removed (volume / unit time) and the rate at which the second volume of the second liquid culture medium is added (volume / unit time) can be approximately the same or can be different.

[0272] Alternatively, the volumes removed and added can vary (e.g., gradually increase) over each 24 hour period (or alternatively, increasing time periods of from about 1 hour to about 24 hours or increasing time periods greater than 24 hours) during the culture period. For example, the volume of the first liquid culture medium removed and the volume of the second liquid culture medium added within each 24 hour period (or increasing time periods of from about 1 hour to more than 24 hours or increasing time periods greater than 24 hours) during the culture period can increase (e.g., gradually or by staggered increments) from a volume that is 0.5% to about 20% of the bioreactor volume or first liquid culture medium volume to about 25% to about 150% of the bioreactor volume or first liquid culture medium volume during the culture period.

[0273] As will be appreciated by the skilled practitioner, the first liquid culture medium and the second liquid culture medium can be the same type of medium. The culture medium can be different.

[0274] The first volume of the first liquid culture medium can be removed, for example, by a mechanical system capable of removing the first volume of the first liquid culture medium from the bioreactor (e.g., removing the substantially cell-free first volume of the first liquid culture medium from the bioreactor). Alternatively, or in addition, the first volume of the first liquid culture medium can be removed by seepage or gravity flow of the first volume of the first liquid culture medium through a sterile membrane having a molecular weight cutoff that excludes cells (e.g., mammalian cells).

[0275] The second volume of the second liquid culture medium can be added automatically, for example by an infusion pump, to the first liquid culture medium.

[0276] In some examples, the removal of the first volume of the first liquid culture medium (e.g., the first volume of the first liquid culture medium that is substantially free of mammalian cells) and the addition of the second volume of the second liquid culture medium to the first liquid culture medium does not occur within at least 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 14 hours, within 16 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours, within 96 hours, or within 96 hours) after seeding the bioreactor with the mammalian cells.

[0277] Fed-batch Bioreactor The culturing steps described herein can be carried out using a fed-batch bioreactor. Cultivating cells in a fed-batch bioreactor involves adding (e.g., periodically or continuously) a second volume of a second liquid culture medium to a first liquid culture medium for most of the culture period. The addition of the second liquid culture medium can be performed continuously (e.g., at a rate of adding 0.1% to 300% (e.g., 1% to 250%, 1% to 100%, 100% to 200%, 5% to 150%, 10% to 50%, 15% to 40%, 8% to 80%, or 4% to 30%) of the volume of the bioreactor or the volume of the first liquid culture medium over a given time period (e.g., over a 24 hour period, over increasing time periods of about 1 hour to about 24 hours, or over increasing time periods of more than 24 hours)), or periodically (e.g., once every 3 days, once every 2 days, once a day, twice a day, three times a day, four times a day, or five times a day), or any combination thereof. When performed periodically, the volume added (e.g., within a period of about 24 hours, within increasing time periods of about 1 hour to about 24 hours, or within increasing time periods of more than 24 hours) can be, for example, 0.1% to 300% (e.g., 1% to 200%, 1% to 100%, 100% to 200%, 5% to 150%, 10% to 50%, 15% to 40%, 8% to 80%, or 4% to 30%) of the volume of the bioreactor or the volume of the first liquid culture medium. The second volume of the second liquid culture medium added can, in some instances, be kept about the same over each 24 hour period (or, alternatively, over increasing time periods of about 1 hour to about 24 hours or over increasing time periods of more than 24 hours) for all or a portion of the culture period. As is known in the art, the rate (volume / unit time) of adding the second volume of the second liquid culture medium can be varied for all or a portion of the culture period. For example, the volume of second liquid culture medium added can be varied (e.g., gradually increased) over each 24 hour period (or alternatively, increasing time periods from 1 hour to about 24 hours or increasing time periods greater than 24 hours) during the culture period.For example, during the culture period, the volume of the second liquid culture medium added within each 24 hour period (or alternatively, increasing time periods of about 1 hour to greater than 24 hours or increasing time periods greater than 24 hours) can be increased (e.g., by gradual or staggered increases) throughout the culture period from a volume that is 0.5% to about 20% of the bioreactor volume or the first liquid culture medium volume to about 25% to about 150% of the bioreactor volume or the first liquid culture medium volume. The rate (volume / unit time) of addition of the two volumes of the second liquid culture medium can be about the same over all or part of the culture period.

[0278] As will be appreciated by a skilled practitioner, the first liquid culture medium and the second liquid culture medium can be the same type of medium. In other examples, the first liquid culture medium and the second liquid culture medium can be different. The volume of the second liquid culture medium can be added automatically, for example, by an infusion pump, to the first liquid culture medium.

[0279] In some examples, the addition of the second volume of the second liquid culture medium to the first liquid culture medium does not occur within at least 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 14 hours, within 16 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours, within 96 hours, or within 96 hours) from the seeding of the mammalian cells into the bioreactor. The cell culture medium in the fed-batch culture is typically harvested at the end of the culture period and used in any of the methods described herein. However, the cell culture medium of the fed-batch culture can also be harvested at one or more time points during the culture period and used in any of the methods described herein.

[0280] As will be appreciated by the skilled practitioner, any of a variety of culture parameters (e.g., vessel, volume, frequency rate of culture volume exchange, agitation frequency, temperature, medium, and CO2 concentration) can be used in any combination to carry out these methods. Additionally, any of the mammalian cells described herein or known in the art can be used to produce recombinant proteins.

[0281] Representative biological production systems Examples of biological manufacturing systems useful for carrying out the methods described herein, including MCCS or MCCS1 and MCCS2, are described in U.S. Provisional Patent Application Nos. 61 / 775,060 and 61 / 856,390 (incorporated by reference). In these representative systems, at least one (e.g., at least 2, 3, 4, 5, or 6) reduced bioburden packed chromatography column provided herein is present in the MCCS or in MCCS1 and / or MCCS2. For example, the entire system can include a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reduced bioburden packed chromatography columns provided herein. For example, MCCS, MCCS1, and / or MCCS2 can include (or each can include) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 chromatography columns packed with reduced bioburden as provided herein.

[0282] For example, a useful system can include an MCCS1 with an inlet and an MCCS2 with an outlet, or an MCCS with an inlet and an outlet. In some embodiments, the MCCS1 and MCCS2 are in fluid communication with each other. These systems can also be configured to allow fluid to enter the inlet, pass through the MCCS1 and MCCS2, and exit the manufacturing system through the outlet. These systems allow for continuous, time-efficient production of therapeutic drug substances from liquid culture media. For example, the elapsed time from feeding a fluid (e.g., liquid culture media) containing a therapeutic protein to the MCCS1 to elution of the purified recombinant protein (e.g., therapeutic protein drug substance) from the outlet of the MCCS2 can be, for example, about 4 hours to about 48 hours, inclusive.

[0283] Some exemplary systems do not include a holding tank. In other examples, the system (e.g., each holding tank holds the treatment for a total period of time, e.g., from about 5 minutes to about 6 hours, inclusive) The entire system may include up to 1, 2, 3, 4, or 5 hold-up tanks (if only holding protein for MCCS1). The hold-up tank(s) may have a volume of 1 mL to about 300 mL, inclusive. Any hold-up tank(s) positioned in the system such that fluid enters the hold-up tank(s) before entering MCCS1 or MCCS, respectively, may have a volume that is 1 mL to about 100% (inclusive) of the loading volume of MCCS1 or the first column of MCCS. Any hold-up tank(s) positioned in the system such that fluid enters the hold-up tank(s) before entering MCCS2 (and after leaving MCCS1) may have a volume that is, for example, 1 mL to about 100% (inclusive) of the loading volume of the first column of MCCS2.

[0284] Additional Representative System Structures and Features The MCCS or MCCS1 may include an inlet through which fluid (e.g., substantially cell-free liquid culture medium) can pass to enter the MCCS or MCCS1, respectively. The inlet may be of any structure known in the art for such purposes. It may include, for example, threads, ribs, or seals through which a fluid tube may be inserted such that fluid enters the MCCS or MCCS1 through the inlet without significant leakage of fluid from the inlet after insertion of the fluid tube into the inlet. Non-limiting inlets that may be used in the present system are known and would be understood by one of ordinary skill in the art.

[0285] The MCCS or MCCS1 can have at least two chromatographic columns, at least two chromatographic membranes, or at least one chromatographic column and at least one chromatographic membrane, and an inlet. The MCCS or MCCS1 can be any of the representative MCCSs described herein or can have any one or more of the representative features (in any combination) of the MCCSs described herein. The chromatographic column(s) and / or chromatographic membrane(s) present in the MCCS or MCCS1 can have any one or more of the representative shapes, sizes, volumes (bed volumes), and / or unit operations described herein.

[0286] The chromatographic column(s) and / or chromatographic membrane(s) present in the MCCS or MCCS1 may comprise any one or more of the representative resins described herein or known in the art. For example, the resins present in one or more of the chromatographic column(s) and / or chromatographic membrane(s) present in the MCCS or MCCS1 may be resins that utilize a capture mechanism (e.g., protein A-binding capture mechanism, protein G-binding capture mechanism, antibody or antibody fragment-binding capture mechanism, substrate-binding capture mechanism, cofactor-binding capture mechanism, aptamer-binding capture mechanism, and / or tag-binding capture mechanism). The resins present in one or more of the chromatographic column(s) and / or chromatographic membrane(s) of the MCCS or MCCS1 may be cation exchange resins, anion exchange resins, molecular sieve resins, or hydrophobic interaction resins, or any combination thereof. Further examples of resins that can be used to purify recombinant proteins are known in the art and may be included in one or more chromatographic columns and / or chromatographic membranes present in the MCCS or MCCS1. The chromatography column(s) and / or chromatographic membranes present within the MCCS or MCCS1 can contain the same and / or different resins (e.g., any of the resins described herein or known in the art to be used for purification of recombinant proteins).

[0287] Two or more chromatography columns and / or chromatography resins present within an MCCS or MCCS1 may serve as a single unit operation (e.g., a combination The MCCS or MCCS1 may perform the unit operations of capturing a recombinant protein from a fluid (e.g., liquid culture medium) and inactivating viruses present in the fluid containing the recombinant protein). The MCCS or MCCS1 may perform any combination of two or more unit operations described herein or known in the art.

[0288] The chromatographic column(s) and / or chromatographic membrane(s) present in the MCCS or MCCS1 can be connected or moved to each other by a switching mechanism (e.g., a column switching mechanism). The MCCS or MCCS1 can also include one or more (e.g., 2, 3, 4, or 5) pumps (e.g., automated, e.g., automated peristaltic pumps). A column switching event can be triggered by detection of a certain level of recombinant protein detected by UV absorbance corresponding to a certain level of recombinant protein in a fluid passing through the MCCS or MCCS1 (e.g., input to and / or elution from one or more chromatographic columns and / or chromatographic membranes in the MCCS or MCCS1), a certain volume of liquid (e.g., buffer), or a certain elapsed time. Column switching generally refers to a mechanism that allows at least two different chromatographic columns and / or chromatographic membranes within an MCCS or MCCS1 (e.g., two or more different chromatographic columns and / or chromatographic membranes present in MCCS1 or MCCS2) to pass through different steps (e.g., equilibration, loading, elution, or washing) substantially simultaneously during at least a portion of the method.

[0289] The MCCS or MCCS1 can be a periodic countercurrent chromatography system (PCCS). For example, the PCCS that is the MCCS or MCCS1 (i.e., PCCS or PCCS1, respectively) can include four chromatography columns, where the first three columns perform the unit operation of capturing a recombinant protein from a fluid (e.g., liquid culture medium) and the fourth column of the PCCS performs the unit operation of inactivating viruses in the fluid containing the recombinant protein. The MCCS or MCCS1 PCCS can utilize a column switching mechanism. The PCC system can utilize, for example, a modified AKTA system (GE Healthcare, Piscataway, NJ) that can run up to 4, 5, 6, 7, or 8 columns, or more.

[0290] The MCCS or MCCS1 can be equipped with: one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) UV monitors, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) valves, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pH meters, and / or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) conductivity meters. The MCCS or MCCS1 can also be equipped with an operating system that utilizes software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ) to detect when a column switch should occur (e.g., based on UV absorbance, volume of liquid, or elapsed time) and to affect (trigger) the column switch event.

[0291] The MCCS or MCCS1 may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-1, 20-2, 20-3, or 20-4) in-line buffer conditioning reservoirs and / or or a buffer reservoir. In other examples, the MCCS or MCCS1 can include one or more (e.g., 2, 3, 4, 5, or 6) hold tanks that can hold fluids that cannot easily pass through one or more chromatographic columns and / or chromatographic membranes in the MCCS or MCCS1. The systems described herein can include one or more hold tanks (e.g., hold tanks described herein) in the MCCS, MCCS1, and / or MCCS2. Other examples of the systems described herein do not include a hold tank in the MCCS, MCCS1, or MCCS2, or do not include a hold tank in the entire system. Other examples of the systems described herein include up to 1, 2, 3, 4, or 5 hold tanks (e.g., any hold tank(s) described herein) in the entire system.

[0292] Second MCCS The second MCCS (MCCS2) in the exemplary system includes at least two chromatographic columns, at least two chromatographic membranes, or at least one chromatographic column(s) and at least one chromatographic membrane(s), and an outlet. MCCS2 can be any of the exemplary MCCSs described herein, or can have any one or more of the exemplary features of the MCCSs described herein (in any combination). The chromatographic column(s) and / or chromatographic membrane(s) present in MCCS2 can have any one or more of the shapes, sizes, volumes (bed volumes), and / or unit operations described herein. The chromatographic column(s) and / or chromatographic membrane(s) can include any of the exemplary resins described herein or known in the art. For example, the resins included in one or more chromatographic columns and / or chromatographic membranes present in MCCS2 can be resins that utilize a capture mechanism (e.g., a protein A-binding capture mechanism, a protein G-binding capture mechanism, an antibody or antibody fragment-binding capture mechanism, a substrate-binding capture mechanism, a cofactor-binding capture mechanism, a tag-binding capture mechanism, and / or an aptamer-binding capture mechanism). Useful resins include, for example, cation exchange resins, anion exchange resins, molecular sieve resins, and hydrophobic interaction resins. Further examples of resins are known in the art. The chromatography column(s) and / or chromatography membranes present in MCCS2 can contain the same and / or different resins (e.g., any of the resins described herein or known in the art for use in purifying recombinant proteins).

[0293] The chromatography column(s) and / or chromatography membrane(s) present in the MCCS2 can perform one or more unit operations (e.g., any of the unit operations described herein or any combination of the unit operations described herein). In a non-limiting example, the MCCS2 can perform the unit operations of purifying a recombinant protein from a fluid and polishing a recombinant protein present in a fluid containing the recombinant protein. In another non-limiting example, the MCCS2 can perform the unit operations of purifying a recombinant protein present in a fluid, polishing a recombinant protein present in a fluid, and filtering a fluid containing the recombinant protein. In another example, the MCCS2 can perform the unit operations of purifying a recombinant protein present in a fluid, polishing a recombinant protein present in a fluid, filtering a fluid containing the recombinant protein, and adjusting the ion concentration and / or pH of a fluid containing the recombinant protein. The MCCS2 can perform any combination of two or more of the unit operations described herein or known in the art.

[0294] The chromatography column(s) and / or chromatograph present in the MCCS2 The chromatographic membrane(s) can be coupled or moved relative to each other by a switching mechanism (e.g., a column switching mechanism). The MCCS2 can also include one or more (e.g., 2, 3, 4, or 5) pumps (e.g., automated, e.g., automated peristaltic pumps). A column switching event can be triggered by detection of a certain level of recombinant protein detected by UV absorbance, a certain volume of liquid (e.g., buffer), or a certain elapsed time corresponding to a certain level of recombinant protein in a fluid passing through the MCCS2 (e.g., input to and / or elution from one or more chromatographic columns and / or chromatographic membranes in the MCCS2).

[0295] The MCCS2 can be a periodic countercurrent chromatography system (i.e., PCCS2). For example, the PCCS2 can include three columns that perform the unit operation of purifying a recombinant protein from a fluid, and a chromatographic membrane that performs the unit operation of polishing the recombinant protein present in the fluid. For example, the three columns that perform the unit operation of purifying a recombinant protein from a fluid can include, for example, a cation exchange resin, and the chromatographic membrane that performs the unit operation of polishing can include a cation exchange resin. The PCCS2 can utilize a column switching mechanism. The PCCS2 can utilize, for example, a modified AKTA system (GE Healthcare, Piscataway, NJ) that can run up to 4, 5, 6, 7, or 8 columns, or more.

[0296] The MCCS2 can be equipped with: one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) UV monitors, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) valves, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pH meters, and / or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) conductivity meters. The MCCS2 can also be equipped with an operating system that utilizes software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ) to detect when a column switching event should occur (e.g., based on UV absorbance, volume of liquid, or elapsed time) and affect the column switching event.

[0297] The MCCS2 can further include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-1, 20-2, 20-3, or 20-4) in-line buffer conditioning reservoirs and / or buffer reservoirs. In other examples, the MCCS2 can include one or more (e.g., 2, 3, 4, 5, or 6) hold-up tanks (e.g., any of the hold-up tanks described herein) that can hold fluids that cannot readily pass into one or more chromatographic columns and / or chromatographic membranes within the MCCS2.

[0298] The MCCS2 includes an outlet through which the therapeutic protein drug substance can exit the system. The outlet can include, for example, a thread, rib, or seal into which a fluid tube can be inserted, or a vial designed to hold or store the purified recombinant protein (e.g., therapeutic protein drug substance). The outlet can include a surface that can be used to seal a reduced bioburden vial or other such storage container to the outlet, such that the purified recombinant protein (e.g., therapeutic protein drug substance) can flow directly into the reduced bioburden vial or storage container. Non-limiting outlets that can be used in the present system are known and will be understood by those of skill in the art.

[0299] The system described herein may also include a fluid tube disposed between MCCS1 and MCCS2. Any of the fluid tubes described herein may be, for example, a tube made of, for example, polyethylene, polycarbonate, or plastic. The fluid tube disposed between MCCS1 and MCCS2 may further include, in any combination, one or more of the following: one or more in-line buffer adjustment reservoirs in fluid communication with the fluid tube and positioned so that the buffer stored in the in-line buffer adjustment reservoir(s) is added to the fluid present in the fluid tube; a hold-up tank (e.g., any of the hold-up tank(s) described herein) in fluid communication with the fluid tube and positioned to hold any excess fluid present in the fluid tube that cannot be readily provided in MCCS2; and one or more filters disposed in the fluid tube to filter (e.g., remove bacteria) the fluid present in the fluid tube. Any of the in-line buffer conditioning reservoirs can contain, for example, a volume of about 0.5 L to 50 L of buffer (eg, at a temperature below 25° C., 15° C., or 10° C.).

[0300] The systems described herein can optionally include a fluid conduit disposed between a final chromatographic column or chromatographic membrane in the MCCS2 and an outlet. The systems described herein can further include one or more filters in fluid communication with the fluid conduit disposed between a final chromatographic column or chromatographic membrane in the MCCS2 and an outlet, such that the filters can remove, for example, precipitated matter, particulate matter, or bacteria from fluid present in the fluid conduit disposed between the final chromatographic column or chromatographic membrane in the MCCS2 and an outlet.

[0301] Some examples of the systems provided herein also include a bioreactor in fluid communication with the inlet of the MCCS or MCCS1. Any of the representative bioreactors described herein or known in the art can be used in the systems.

[0302] Some examples of the systems provided herein also include a pump system. The pump system can include one or more of the following: one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pumps (e.g., any of the pumps described herein or known in the art), one or more (e.g., 2, 3, 4, or 5) filters (e.g., any of the filters described herein or known in the art), one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) UV detectors, and one or more (e.g., 2, 3, 4, or 5) hold-up tanks (e.g., any of the hold-up tanks described herein). Some examples of the systems provided herein further include a fluid line (e.g., any of the representative fluid lines described herein or known in the art) disposed between the pump and the inlet of the MCCS or MCCS1. In some examples, this particular fluid line can include one or more (e.g., two, three, or four) pumps (e.g., any of the pumps described herein or known in the art) and / or one or more (e.g., two, three, or four) holding tanks (e.g., any of the representative holding tanks described herein), where the pump(s) and / or holding tank(s) are in fluid connection with the fluid present in the fluid line.

[0303] Some examples of the systems described herein further include an additional fluid line coupled to the fluid line between the pump and the inlet, with one end of the additional fluid line fluidly connected to the bioreactor and the other end fluidly connected to the fluid line between the pump and the inlet. The additional fluid line may include a filter ( For example, the ATF cell retention system.

[0304] The systems provided herein allow for continuous production of purified recombinant protein (e.g., therapeutic protein drug substance). As known in the art, the systems may allow for periodic elution of purified recombinant protein (e.g., therapeutic protein drug substance). The systems described herein may also provide a net yield of at least about 5 g / day, at least about 10 g / day, at least about 15 g / day, at least about 20 g / day, at least about 30 g / day, or at least about 40 g / day of purified recombinant protein (e.g., therapeutic protein drug substance) for a continuous period of at least about 5 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, or at least about 100 days.

[0305] Method for reducing bioburden on a chromatography resin, comprising the use of a substantially dry chromatography resin Also provided herein is a method of reducing the bioburden of a chromatography resin, comprising (a) exposing a container containing a substantially dried chromatography resin to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein the substantially dried chromatography resin comprises a liquid comprising at least one alcohol, and the at least one alcohol is present in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin after exposure to the dose of gamma radiation. Some embodiments of this method further comprise drying the chromatography resin prior to step (a) to substantially remove the liquid (but not all of the liquid) from the chromatography resin. Drying the chromatography resin can be performed using heat treatment (e.g., an oven) or a desiccator. Additional methods of drying chromatography resins are known in the art.

[0306] Any of the conditions and doses for gamma irradiation described herein can be used in these methods. For example, the dose of gamma irradiation can be about 15 kGy to about 45 kGy (e.g., about 20 kGy to about 30 kGy). Any of the containers, chromatography resins, and liquids comprising at least one alcohol described herein can be used in these methods. For example, the container can be a storage vessel or a chromatography column. The chromatography resin in these methods can comprise a protein ligand (e.g., Protein A or Protein G). In some examples, the chromatography resin can comprise an anion exchange chromatography resin (e.g., a chromatography resin comprising N-benzyl-N-methyl-ethanolamine groups). In some examples, the chromatography resin is covalently attached to a surface of an article (e.g., a chip, membrane, or cassette). In some embodiments, the substantially dry chromatography resin does not contain a significant amount of an antioxidant or a significant amount of a chelating agent. Also provided is a reduced bioburden chromatography resin produced by any of the methods described herein.

[0307] In some instances, the reduced bioburden produced by chromatography resin is about 1×10 -8 ~Approx. 1×10 -5 Sterility assurance level (SAL) (e.g., approximately 1 × 10 -7 ~Approx. 1×10 -6 The resulting reduced chromatography resin can comprise at least one resin selected from the group consisting of anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin. In some examples, the resulting reduced chromatography resin can comprise at least one resin selected from the group consisting of tannins, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin. The reduced bioburden chromatography resin may include an affinity chromatography resin that includes a protein ligand (e.g., Protein A). In some examples, the reduced chromatography resin produced includes an anion exchange chromatography resin (e.g., an anion exchange chromatography resin that includes N-benzyl-N-methyl-ethanolamine groups). Also provided is a method of making a reduced bioburden packed chromatography column, comprising providing a reduced bioburden chromatography resin produced by any of the methods described herein, and packing the chromatography resin into a reduced bioburden column in a sterile environment. Also provided is a reduced bioburden packed chromatography column produced by any of the methods described herein.

[0308] Also provided is an integrated, closed, continuous process for the production of reduced bioburden of purified recombinant protein, comprising: (a) providing a liquid culture medium comprising the recombinant protein that is substantially free of cells; (b) continuously feeding the liquid culture medium to a multi-column chromatography system (MCCS) comprising at least one reduced bioburden packed chromatography column produced by any of the methods provided herein, wherein the process utilizes a reduced bioburden buffer and is integrated and operates continuously from the liquid culture medium to the eluate from the MCCS being the purified recombinant protein. Also provided is an integrated, closed, continuous method for the production of reduced bioburden of purified recombinant protein, comprising: (a) providing a liquid culture medium comprising the recombinant protein that is substantially free of cells; (b) continuously feeding the liquid culture medium into a first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein in the liquid culture medium using MCCS1; (d) generating an eluate comprising the recombinant protein from MCCS1 and continuously feeding the eluate into a second multi-column chromatography system (MCCS2); (e) producing a purified recombinant protein by continuously feeding the recombinant protein from the eluate into MCCS2 and thereafter eluting the recombinant protein, wherein the method utilizes a reduced bioburden buffer and is integrated and operates continuously from the liquid culture medium to the purified recombinant protein, and at least one column in MCCS1 and / or MCCS2 contains a chromatography column packed with reduced bioburden produced by any of the methods provided herein. Also, any of the representative aspects of the integrated, closed, continuous processes for the production of reduced bioburden of purified recombinant proteins described herein can be used in these processes.

[0309] Methods for Producing Reduced Bioburden Membranes, Resins, Coatings, Chips, and Cassettes Also provided herein is a method of producing a reduced bioburden membrane, resin, coating, chip, or cassette, comprising exposing a container containing a composition comprising (i) a membrane, resin, coating, chip, or cassette (e.g., a cellulose, agarose, or sugar-based membrane, resin, coating, chip, or cassette) and (ii) a liquid comprising at least one alcohol (e.g., and optionally at least one antioxidant and / or chelating agent) to a dose of gamma radiation sufficient to reduce the bioburden of the container and the membrane, resin, coating, chip, or cassette, wherein the at least one alcohol is present in an amount sufficient to ameliorate damage to the membrane, resin, coating, chip, and cassette after exposure to the dose of gamma radiation. In some examples, the cassette is a cassette containing a resin (e.g., any of the representative resins described herein or known in the art). In some embodiments, the membrane, resin, coating, chip, or cassette comprises a Protein A or Protein G ligand covalently bound to at least one or a portion of its surface. In some embodiments, the composition comprises a membrane, resin, coating, chip, or cassette and at least an alcohol (e.g. For example, the composition may comprise a liquid containing at least one alcohol, antioxidant, and / or at least one chelating agent, and optionally at least one antioxidant and / or at least one chelating agent. Any of the exemplary combinations and concentrations of alcohol, antioxidant, and / or chelating agent described herein may be used in any of these methods. Any of the exemplary liquids described herein may be used in any of these methods. In some embodiments, the composition is a moist or moist dry material. Also provided herein is a reduced bioburden membrane, resin, coating, chip, or cassette produced using any of the methods described herein. In some examples, the container is a sealed storage container or vessel.

[0310] Also provided herein is a method of producing reduced bioburden membranes, resins, coatings, chips, and cassettes, comprising exposing a container containing a substantially dried membrane, resin, coating, chip, or cassette (e.g., a cellulose, agarose, or sugar-based membrane, resin, coating, chip, or cassette) to a dose of gamma radiation sufficient to reduce the bioburden of the container and the membrane, resin, coating, chip, or cassette. Some examples further include a step of drying the membrane, resin, coating, chip, or cassette prior to the exposing step. In some embodiments, the membrane, resin, coating, chip, or cassette contains a Protein A or Protein G ligand covalently bound to its surface. In some examples, the cassette is a cassette containing a resin (e.g., any of the representative resins described herein or known in the art). Also provided herein is a reduced bioburden membrane, resin, coating, chip, or cassette produced using any of the methods described herein. EXAMPLES

[0311] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0312] Protective effect of alcohols on gamma-irradiated affinity chromatography resins. In the first set of experiments, MabSelect™ SuRe™ (Protein A affinity chromatography resin) was irradiated with doses of 40-49 kGy at standard irradiation rates in three different buffers: (1) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol in 50 mM sodium phosphate buffer (40-49 kGy at dose rates of >7.5 kGy / hr); (2) 2% v / v benzyl alcohol (40-49 kGy at a dose rate of >7.5 kGy / hour); and (3) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, 2% benzyl alcohol in 50 mM sodium phosphate buffer - higher radiation doses (40-49 kGy and higher dose rates >7.5 kGy / hr)

[0313] After irradiation, the chromatography resins were packed into separate chromatography columns and cycled with cell culture harvest and a residence time of 6 minutes. The breakthrough binding capacity was recorded for the irradiated resins and compared to naive (unirradiated) MabSelect™ SuRe™ (Protein A chromatography) resin.

[0314] The data from this experiment are shown in FIG.

[0315] The data in Figure 1 show that buffer (3) provides an additive effect of the combination of both buffers (1) and (2) to preserve binding capacity. In this example, 2% v / v vector in the buffer Benzylic alcohol acts as a preservative and also provides some protective properties when combined with the buffer (1).

[0316] Table 1 shows that all of the measured product quality attributes did not change significantly (very) based on the use of buffers (1)-(3), but were similar to expected values ​​for naive (unirradiated) MabSelect™ SuRe™ (Protein A chromatography resin).

[0317] [Table 1]

[0318] This data demonstrates that irradiation of a chromatography resin in a liquid containing an alcohol (eg, benzyl alcohol) protects the resin from subsequent loss of binding capacity over multiple cycles of chromatography. EXAMPLES

[0319] Protective effect of alcohols on gamma-irradiated Capto Adhere chromatography resin. GE Capto™ adhere (a multi-mode functional anion exchange chromatography resin) was gamma irradiated at doses of 28-49 kGy at standard exposure rates in three different buffers: (A) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, and 25 mM mannitol in 50 mM sodium phosphate buffer (28-34 kGy at a dose rate of 2-3 kGy / h); (B) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol in 50 mM sodium phosphate buffer (40-49 kGy at a dose rate of >7.5 kGy / hr); and (C) 25 mM sodium ascorbate in 50 mM sodium phosphate buffer, 25 mM Methionine, 25 mM histidine, 25 mM mannitol, 2% v / v benzyl alcohol (40-49 kGy and higher dose rates >7.5 kGy / hr).

[0320] After irradiation, the chromatography resins were packed into separate chromatography columns and cycled with cell culture harvest and a residence time of 6 minutes. The breakthrough binding capacity of the irradiated resins was recorded and compared to that of naive (unirradiated) GE Capto™ adhere (a multi-modal functional anion exchange chromatography resin).

[0321] The data from this experiment are shown in Figure 2. The data in Figure 2 indicate that no appreciable change in binding capacity was observed when the resin was irradiated in the presence of benzyl alcohol, and that the presence of benzyl alcohol provides the benefit of increased storage time prior to gamma irradiation.

[0322] Table 2 below shows that irradiation of the resin in the presence of benzyl alcohol did not appreciably affect other quality attributes of the resin's performance in protein purification.

[0323] [Table 2]

[0324] Other embodiments Although the present invention has been described in conjunction with a detailed description thereof, it is to be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for purifying a recombinant protein, comprising: (a) exposing a packed chromatography column containing a composition comprising (i) a chromatography resin and (ii) a liquid comprising benzyl alcohol and at least two antioxidants selected from mannitol, sodium ascorbate, histidine, and methionine to a dose of gamma radiation sufficient to reduce bioburden on the packed chromatography column and the chromatography resin, wherein the benzyl alcohol and the at least two antioxidants are present in an amount sufficient to ameliorate loss of binding capacity of the chromatography resin after exposure to the dose of gamma radiation, thereby producing a packed chromatography column with reduced bioburden; (b) providing a liquid containing the recombinant protein; (c) performing column chromatography on the liquid using a packed chromatography column with reduced bioburden; and (d) recovering an eluate from the packed chromatography column with reduced bioburden, wherein the eluate comprises the recombinant protein.

2. 2. The method of claim 1, wherein the concentration of benzyl alcohol in the liquid is 0.01% v / v to 10% v / v.

3. 3. The method of claim 1 or 2, wherein the liquid comprises at least one chelating agent in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation.

4. 4. The method of claim 1, wherein the liquid comprises mannitol, sodium ascorbate, histidine, and methionine.

5. The liquid is (i) 30 mM to 70 mM methionine and 30 mM to 70 mM histidine; (ii) 10 mM to 50 mM methionine, 10 mM to 50 mM histidine, and 1 0 mM to 50 mM sodium ascorbate; 4. The method of claim 1, comprising (iii) 5 mM to 45 mM sodium ascorbate, 5 mM to 45 mM methionine, 5 mM to 45 mM mannitol, and 5 mM to 45 mM histidine.

6. 4. The method of claim 3, wherein the at least one chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium 2,3-dimercapto-1-propanesulfonate (DMPS), dimercaptosuccinic acid (DMSA), metallothionein, and desferoxamine.

7. 7. The method of any one of claims 1 to 6, wherein the chromatography resin is selected from the group consisting of anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin.

8. 8. The method of claim 7, wherein the chromatography resin is an affinity chromatography resin containing a protein ligand.

9. The method of claim 8, wherein the protein ligand is Protein A.

10. 8. The method of claim 7, wherein the chromatography resin is an anion exchange chromatography resin.

11. The method according to any one of claims 1 to 10, wherein the dose is between 15 kGy and 45 kGy.

12. The method according to any one of claims 1 to 11, wherein the recombinant protein is a therapeutic recombinant protein.

13. 13. The method of claim 12, wherein the therapeutic recombinant protein is an antibody or antibody fragment, an enzyme, an artificially modified protein, or an immunogenic protein or protein fragment.

14. 14. The method of claim 13, wherein the therapeutic recombinant protein is an enzyme.

15. 14. The method of claim 13, wherein the therapeutic recombinant protein is an antibody.

16. 1. An integrated, closed process for the production of reduced bioburden of purified recombinant proteins, comprising: (a) exposing a packed chromatography column containing a composition comprising (i) a chromatography resin and (ii) a liquid comprising benzyl alcohol and at least two antioxidants selected from mannitol, sodium ascorbate, histidine, and methionine to a dose of gamma radiation sufficient to reduce bioburden on the packed chromatography column and the chromatography resin, wherein the benzyl alcohol and the at least two antioxidants are present in an amount sufficient to ameliorate loss of binding capacity of the chromatography resin after exposure to the dose of gamma radiation, thereby producing a packed chromatography column with reduced bioburden; (b) providing a liquid containing the recombinant protein; (c) performing column chromatography on the packed chromatography column of (a) with reduced bioburden; and (d) recovering an eluate from the reduced bioburden packed chromatography column, wherein the eluate comprises the recombinant protein; The process utilizes and integrates a reduced bioburden buffer.

17. 17. The process of claim 16, wherein the recombinant protein is a therapeutic recombinant protein.

18. 18. The process of claim 17, wherein the therapeutic recombinant protein is an antibody or antibody fragment, an enzyme, an artificially modified protein, or an immunogenic protein or protein fragment.

19. 20. The process of claim 18, wherein the therapeutic recombinant protein is an enzyme.

20. 18. The process of claim 17, wherein the therapeutic recombinant protein is an antibody.

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