Aseptic chromatography resin and its use in a production method

Abstract

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 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 a reduced-bioburden chromatography column comprising a reduced-bioburden chromatography resin, a composition comprising a chromatography resin and a liquid comprising at least one alcohol, a method for performing reduced-bioburden column chromatography using one of these reduced-bioburden chromatography columns, and an integrated, closed, continuous method for producing reduced-bioburden purified recombinant proteins. [Selected Figure] 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 on August 31, 2018; the entire content of which is incorporated herein by reference.

[0002] The present invention relates to biotechnology and biomanufacturing methods for recombinant proteins.

Background Art

[0003] Mammalian cells containing nucleic acids encoding recombinant proteins are often used to produce therapeutically or commercially important proteins. In the current environment regarding 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 strategy for efficiently isolating 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) within the system that can lead to product contaminants, reduced production yields, and decreased flow rate (or increased pressure) within the system. For example, increased bioburden within the system can cause a complete shutdown of that system.

Summary of the Invention

Means for Solving the Problems

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

[0005] A method for reducing the bioburden of a chromatography resin is provided, which includes exposing a container containing a composition comprising (i) a chromatography resin and (ii) a liquid containing 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 improve the loss of binding capacity of the chromatography resin after exposure to that dose of gamma irradiation.

[0006] In some embodiments, the method can further include placing the composition within the container prior to exposure.

[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 container is a packed chromatography column.

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

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

[0012] In some embodiments of any of the methods described herein, 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, at least one alcohol comprises benzyl alcohol.

[0014] In some embodiments of any of the methods described herein, the total combined concentration of 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 improve the loss of binding capacity of the chromatography resin upon exposure to a dose of gamma irradiation.

[0017] In some embodiments of any of the methods 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 acid, lidocaine, naringenin, fullerenes, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanols.

[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 desferrioxamine.

[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 an N-benzyl-N-methyl-ethanolamine group.

[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 exposure 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 exposure is carried out at a temperature of about 0 °C to about 25 °C.

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

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

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

[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 an anion exchange chromatography resin, a cation exchange chromatography resin, an affinity chromatography resin, a hydrophobic interaction chromatography resin, and a size exclusion chromatography resin.

[0037] In some embodiments, the chromatography resin comprises an affinity chromatography resin comprising 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 an N-benzyl-N-methyl-ethanolamine group.

[0041] Provided herein is a method for producing a chromatography column packed with a reduced bioburden, the method comprising the steps of preparing any of the chromatography resins of the reduced bioburden described herein, and packing the chromatography resin into a column of reduced bioburden in a sterilized environment.

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

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

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

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

[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 pseudo-affinity chromatography resin comprising 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 an N-benzyl-N-methyl-ethanolamine group.

[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 improve the loss of binding ability of the chromatography resin upon treatment with a dose of gamma irradiation 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, at least one alcohol includes benzyl alcohol.

[0056] In some embodiments of any of the compositions described herein, the total concentration of 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 improve the loss of binding capacity of the chromatography resin after exposure of the dose to gamma irradiation.

[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, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoid, phenolpropanoic acid, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanols.

[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 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 desferrioxamine.

[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] A method of performing column chromatography with a reduced bioburden is provided herein, the method comprising: (a) preparing any of the chromatography columns filled with the reduced bioburden described herein; and (b) performing column chromatography using a chromatography column filled with the reduced bioburden and a buffer of the reduced bioburden in a closed system.

[0069] In some embodiments, the column chromatography of the reduced bioburden using a chromatography column filled with the reduced bioburden is performed continuously for at least 4 days.

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

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

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

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

[0074] In some embodiments, the resin in the chromatography column filled with the reduced bioburden in (a) has a binding capacity ratio 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 chromatography column filled with the reduced bioburden 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.

[0076] In some embodiments, the resin comprises an affinity chromatography resin comprising 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 and closed continuous method for the production of a reduced bioburden of a purified recombinant protein, comprising: (a) preparing a liquid culture medium comprising a recombinant protein substantially free of cells; and (b) continuously feeding the liquid culture medium into a multi-column chromatography system (MCCS) comprising at least one of any of the chromatography columns filled with the reduced bioburden described herein, the method utilizing a buffer for the reduced bioburden, being integrated, and operating 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 unit operations of capturing a recombinant protein and inactivating a virus.

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

[0084] In some embodiments, the MCCS includes chromatography columns filled with at least two reduced bioburdens.

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

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

[0087] In some embodiments, the MCCS includes a column for affinity chromatography, and the affinity chromatography is performed in the method 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, the affinity chromatography is performed by a method with a Protein A binding capture mechanism, and the recombinant protein is an antibody or antibody fragment.

[0089] A closed, integrated, and continuous method for producing a reduced bioburden of a purified recombinant protein, the method comprising: (a) preparing a liquid culture medium containing a recombinant protein 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 containing the recombinant protein from MCCS1 and continuously feeding the eluate into a second multi-column chromatography system (MCCS2); (e) continuously feeding the recombinant protein from the eluate into MCCS2 and then eluting the recombinant protein to produce a purified recombinant protein, the method utilizing a buffer with reduced bioburden, being integrated, operating continuously from the liquid culture medium to the purified recombinant protein, and at least one column within MCCS1 and / or MCCS2 containing any of the chromatography columns filled with the reduced bioburden 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 unit operations of capturing a recombinant therapeutic protein and inactivating viruses.

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

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

[0095] In some embodiments, MCCS1 is a first periodic countercurrent 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 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.

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

[0099] In some embodiments, 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 method is performed continuously for a period of at least 4 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 run continuously for a period of at least 7 days.

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

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

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

[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 self-replicating biological contaminants present in a composition containing a chromatography resin or a packed chromatography resin (e.g., self-replicating biological contaminants present in the packed chromatography resin within a packed chromatography column). In other examples, bioburden can refer to self-replicating biological contaminants on the inner surface of a chromatography column and / or within the chromatography resin within the chromatography column (e.g., biological contaminants on the inner surface of the chromatography column and biological contaminants in the packed chromatography resin within the 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), mycobacterium, viruses (e.g., vesivirus, Cache Valley virus, parvovirus, herpesvirus, and bunyavirus), parasites, fungi, yeast, and protozoa. Representative 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 decrease (e.g., a detectable decrease) in 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). Non-limiting examples of methods for reducing the bioburden of a chromatographic resin (e.g., a packed chromatographic resin), a buffer, and / or a chromatographic column (e.g., a packed chromatographic 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 "chromatographic resin with reduced bioburden" means a chromatographic resin that has been treated to reduce the level of self-replicating biological contaminants present in the chromatographic resin (e.g., a detectable decrease in the level of self-replicating biological contaminants present in a composition containing the chromatographic resin, such as a slurry). For example, a chromatographic resin with reduced bioburden can be a resin that has been exposed to a dose of gamma irradiation sufficient to reduce the level of self-replicating biological contaminants in the chromatographic resin (e.g., a composition containing a chromatographic resin that has been exposed to a dose of gamma irradiation sufficient to reduce the level of self-replicating biological contaminants in the chromatographic resin). For example, a chromatographic resin with reduced bioburden can be a resin that has been exposed to gamma irradiation at a dose of about 1 kGy to about 15 kGy, about 1 kGy to about 20 kGy of gamma irradiation, about 1 kGy to about 25 kGy of gamma irradiation, about 1 kGy to about 30 kGy of gamma irradiation, or about 1 kGy to about 35 kGy. Representative methods for reducing the bioburden of a chromatographic resin are described herein. Additional methods for reducing the bioburden of a chromatographic resin are known in the art.

[0111] The term "chromatography column with reduced bioburden" means a chromatography column (e.g., a packed chromatography column) containing a processed 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 containing the unprocessed chromatography resin. For example, a chromatography column with reduced bioburden can contain a processed chromatography resin having a sterility assurance level of at least or about 1×10 -6 1×10 -7 1×10 -8 1×10 -9 or 1×10 -10 .

[0112] The term "buffer with reduced bioburden" is known in the art and means a processed (e.g., filtered, autoclaved, and / or gamma-irradiated) liquid (e.g., a processed buffer solution) having a level of self-replicating contaminating agents that is lower than the level of self-replicating contaminating agents found in the same unprocessed liquid. For example, a buffer with reduced bioburden can have a sterility assurance level of at least or about 1×10 -6 1×10 -7 1×10 -8 1×10 -9 or 1 or 10 -10 .

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

[0114] "Sterile" or "sterility" means about or 1.0×10-6 less than (e.g., about or 1.0×10 -7 less than, about or 1.0×10 -8 less than, about or 1.0×10 -9 or 1×10 -10 less than) and is used to describe a composition or method having a sterility assurance level. The determination of whether a composition or method is sterile can be tested using many established production methods known in the art. For example, a sterile composition or method can be completely free of viable 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 is known in the art).

[0115] The term "sterilize" means 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), which are indicator organisms of resistance, can be measured during the treatment process.

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

[0117] The term "sterilized" is used to describe a composition or method that does not contain self-replicating biological contaminants (e.g., any of the self-replicating biological contaminants described herein) that cause disease or cause symptoms. A sterilized composition or method can also be clean (as the term is known in the art).

[0118] The term "unit operation" is a technical term and refers to a functional step that can be performed in a method for purifying a recombinant protein from a liquid culture medium. For example, one unit of operation can be filtering (e.g., removing contaminants such as bacteria, yeast, viruses and / or mycobacteria, and / or particulate matter emerging from a fluid containing the recombinant protein), capturing, epitope tag removal, purifying, holding or storing, polishing, virus inactivation, adjusting the ionic concentration and / or pH of a fluid containing the recombinant protein, and removing unwanted salts.

[0119] The term "capturing" means a step performed to partially purify or isolate (e.g., at least or about 5% by weight, 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 or about 95% pure) a recombinant protein (e.g., a recombinant therapeutic protein) from one or more other components present in a liquid culture medium or a diluted liquid culture medium (e.g., a culture medium protein present in or secreted from mammalian cells or one or more other components (e.g., DNA, RNA, or other proteins)) and to concentrate it. Typically, capturing is performed using a chromatography resin to which the recombinant protein binds (e.g., by use of affinity chromatography). Non-limiting methods for capturing a recombinant protein from a liquid culture medium or a diluted liquid culture medium are described herein, and other methods are known in the art. The recombinant protein can be captured from the 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 "purify" means a process carried out to isolate a recombinant protein (e.g., a recombinant therapeutic protein) from one or more other impurities (e.g., bulky impurities) or components present in a fluid containing the recombinant protein (e.g., liquid culture medium proteins present in or secreted from mammalian cells or one or more other components (e.g., DNA, RNA, other proteins, endotoxins, viruses, etc.)). For example, purification can be carried out during or after an initial capture step. Purification can be carried out using a chromatography resin, membrane or any other solid support that binds to either the recombinant protein or 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 chromatography column and / or chromatography membrane (e.g., any of the chromatography columns or chromatography membranes described herein).

[0121] The term "polish" is a technical term and means 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) that is close to the desired final purity. For example, polishing can be carried out by passing a fluid containing the recombinant protein through a chromatography column(s) or membrane adsorbent(s) that selectively binds to the target recombinant protein or selectively binds to a small amount 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 adsorbent(s) contains the recombinant protein.

[0122] The term "filtering" means removing at least a portion (e.g., at least 80%, 90%, 95%, 96%, 97%, 98% or 99%) of unwanted biological contaminants (e.g., mammalian cells, bacteria, yeast cells, viruses or mycobacteria) and / or particulate matter (e.g., precipitated protein) 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 technical term and means a fluid discharged from a chromatography column or chromatography membrane containing a detectable amount of a recombinant protein (e.g., a recombinant therapeutic protein).

[0124] The term "integrated method" means a method carried out using structural elements that function in cooperation to achieve a particular result (e.g., purification of a recombinant protein from a liquid culture medium).

[0125] The term "continuous method" means a method of continuously supplying a fluid through at least a portion of a system. For example, a continuous method is a method of continuously supplying a liquid culture medium containing a recombinant protein from a bioreactor through an MCC. Another example of a continuous method is a method of continuously supplying a liquid culture medium containing a recombinant protein from a bioreactor through first and second MCCs (MCC1 and MCC2). Further examples include a method of continuously supplying a liquid culture medium containing a recombinant protein through an MCC, a method of continuously supplying a liquid culture medium containing a recombinant protein through MCC1 and MCC2, or a method of continuously supplying a fluid containing a recombinant protein through MCC2.

[0126] The term "closed method" is a technical term and refers to a method in which the components of a method of contacting a recombinant protein (e.g., chromatography resin and / or buffer) or a liquid containing the recombinant protein are not intentionally exposed to contaminants for a significant period of time (e.g., not intentionally exposed to air for a significant period of time).

[0127] The term "therapeutic protein drug substance" means a recombinant protein (e.g., immunoglobulin, protein fragment, artificially modified protein or enzyme) that is sufficiently purified or sufficiently isolated from proteins, lipids and nucleic acids that cause contamination (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 biological type contaminants (e.g., viral type contaminants and bacterial type contaminants), and can be formulated into a pharmaceutical product without using any further significant purification and / or contamination removal steps (s).

[0128] The term "multi-column chromatography system" or "MCCS" means a system made up of a total of two or more chromatography columns and / or chromatography membranes that are interconnected or will undergo switching processes. Non-limiting examples of multi-column chromatography systems are periodic countercurrent chromatography systems (PCCs) that include a total of two or more chromatography columns and / or chromatography membranes that are interconnected or will undergo switching processes. Further examples of multi-column chromatography systems are described herein and are also known in the art.

[0129] The term "substantially free of" means a composition (e.g., a liquid culture medium) that contains at least or about 90% less (e.g., at least or about 95%, 96%, 97%, 98%, or at least or about 99% less, or about 100% less) of a specified substance (e.g., mammalian cells or contaminating proteins, nucleic acids, carbohydrates, or lipids derived from mammalian cells).

[0130] The term "mammalian cell" means any cell made from or derived from any mammal (e.g., human, hamster, mouse, monkey, rat, pig, cow, or rabbit). For example, a 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" means the maintenance or growth of mammalian cells under a set of physical conditions under control.

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

[0133] The term "liquid culture medium" means a fluid containing nutrients sufficient to allow cells (e.g., mammalian cells) to grow or proliferate in vitro. For example, a 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 derived from a mammal. In some embodiments, the liquid culture medium does not contain serum or another extract derived from a mammal (a 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. A liquid culture medium may contain mammalian cells at any density. For example, as used herein, a certain volume of liquid culture medium withdrawn from a bioreactor may not substantially contain mammalian cells.

[0134] The term "animal-derived component-free liquid culture medium" means a liquid culture medium that does not contain any components (e.g., proteins or serum) derived from a mammal.

[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 technical term and means a liquid culture medium in which all of the 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 products typically contain complex mixtures of albumin and lipids.

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

[0139] The term "immunoglobulin" means a polypeptide comprising an amino acid sequence consisting of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids) related to an immunoglobulin protein (e.g., a variable domain sequence, a framework sequence and / or a constant domain sequence). Immunoglobulins can include, for example, a light chain immunoglobulin of at least 15 amino acids, for example, a heavy chain immunoglobulin of at least 15 amino acids. Immunoglobulins are isolated antibodies (e.g., IgG, IgE, IgD, IgA, or IgM), for example, subclasses of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). Immunoglobulins can be antibody fragments, for example, Fab fragments, F(ab’)2 fragments or scFv fragments. Immunoglobulins can be bispecific or trispecific antibodies, or dimeric, trimeric or multimeric antibodies, or diabodies, Affibody® or Nanobody®. Immunoglobulins can be artificially modified proteins (e.g., fusion proteins) containing at least one immunoglobulin domain. Non-limiting examples of immunoglobulins are described herein, and further examples of immunoglobulins are known in the art.

[0140] The term "protein fragment" or "polypeptide fragment" means a part of a polypeptide sequence that is at least or about 4 amino acids in length, at least or about 5 amino acids in length, at least or about 6 amino acids in length, at least or about 7 amino acids in length, at least or about 8 amino acids in length, at least or about 9 amino acids in length, at least or about 10 amino acids in length, at least or about 11 amino acids in length, at least or about 12 amino acids in length, at least or about 13 amino acids in length, at least or about 14 amino acids in length, at least or about 15 amino acids in length, at least or about 16 amino acids in length, at least or about 17 amino acids in length, at least or about 18 amino acids in length, at least or about 19 amino acids in length, 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" means a polypeptide that is not naturally encoded by endogenous nucleic acids present within an organism (e.g., a mammal). Examples of artificially modified proteins include enzymes (e.g., those that have undergone one or more amino acid substitutions, deletions, insertions, or additions that result in an increase in the 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 contain at least one recombinant scaffold sequence.

[0142] The term "secreted protein" or "secreted recombinant protein" means a protein (e.g., a recombinant protein) in which at least one originally included secretion signal sequence is translated inside mammalian cells and at least partially cleaved by an enzyme by the secretion signal sequence in the mammalian cells, and at least a part thereof is secreted into the extracellular space (e.g., a liquid culture medium). It will be understood by those skilled in the art that for a "secreted" protein to be treated as a secreted protein, it need not be completely separated from the cell.

[0143] The term "perfusion bioreactor" means a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, where the culturing of the cells present in the bioreactor includes periodically or continuously withdrawing the first liquid culture medium and simultaneously or shortly thereafter adding a second liquid culture medium of substantially the same volume to the bioreactor. In some examples, there is a change (e.g., an increase or decrease) in the increment of the volume of the first liquid culture medium in which the withdrawal and addition are carried out over an increment period (e.g., a period of about 24 hours, a period between about 1 minute and about 24 hours, or a period longer than 24 hours) during the culturing period (e.g., the culture medium resupply ratio based on one day). The ratio of the medium withdrawn and replaced daily 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" means the volume of the culture medium present at the start of the culturing process (e.g., the total volume of the culture medium present after seeding).

[0144] The term "fed-batch bioreactor" is a technical term and means a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, where the culturing of the cells present in the bioreactor includes periodically or continuously adding a second liquid culture medium to the first liquid culture medium without substantially or significantly withdrawing the first liquid culture medium or the second liquid culture medium from the cell culture. The second liquid culture medium may be the same as the first liquid culture medium. In some examples of fed-batch culturing, the second liquid culture medium is the first liquid culture medium in a concentrated form. In some examples of fed-batch culturing, the second liquid culture medium is added as a dry powder.

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

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

[0147] Other features and advantages of the present invention will become apparent from the following detailed description, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0148]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0149] A method for reducing the bioburden of a chromatographic resin, the method comprising exposing a container containing a composition comprising (i) a chromatographic resin and (ii) a liquid comprising at least one (e.g., two, three, four, or five) alcohol to a gamma radiation dose sufficient to reduce the bioburden of the container and the chromatographic resin, wherein at least one alcohol is present in an amount sufficient to improve the loss of binding capacity of the chromatographic resin after / during exposure to that dose of gamma radiation. Also provided is a chromatographic column with reduced bioburden containing a chromatographic resin with reduced bioburden prepared by any of the methods described herein, a composition comprising (i) a chromatographic resin and (ii) a liquid comprising at least one (e.g., two, three, four, or five) alcohol, a method for performing column chromatography with reduced bioburden using at least one of these chromatographic columns with reduced bioburden, and an integrated, closed or substantially closed continuous method for the production of a purified recombinant protein with reduced bioburden comprising the use of at least one of these chromatographic columns with reduced bioburden. Non-limiting aspects of these methods and processes are described below. As will be appreciated in the art, the various aspects described below can be used in any combination, without limitation.

[0150] A composition containing a chromatographic resin and at least one alcohol The present specification provides 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) alcohol (e.g., any of the representative alcohols described herein or known in the art), wherein at least one alcohol is present in an amount sufficient to improve the loss of binding capacity of the chromatography resin upon treatment with a dose of gamma irradiation 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 pseudo-affinity 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 having 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 the deposited chromatography resin. In some examples, the composition can be a mixture of wet or moist solids. In some examples, the composition is a chromatography resin filled with 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 composition, the total concentration of 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 12% v / v, from about 0.01% v / v to about 11% v / v, from about 0.01% v / v to about 10% v / v, from about 0.01% v / v to about 9% v / v, from about 0.01% v / v to about 8% v / v, from about 0.01% v / v to about 7% v / v, from about 0.01% v / v to about 6% v / v, from about 0.01% v / v to about 5% v / v, from about 0.01% v / v to about 4.5% v / v, from about 0.01% v / v to about 4.0% v / v, from about 0.01% v / v to about 3.5% v / v, from about 0.01% v / v to about 3.0% v / v, from about 0.01% v / v to about 2.5% v / v, from about 0.01% v / v to about 2.2% v / v, from about 0.01% v / v to about 2.0% v / v, from about 0.01% v / v to about 1.8% v / v, from about 0.01% v / v to about 1.6% v / v, from about 0.01% v / v to about 1.4% v / v, from about 0.01% v / v to about 1.2% v / v, from about 0.01% v / v to about 1.0% v / v, from about 0.01% v / v to about 0.8% v / v, from about 0.01% v / v to about 0.6% v / v, from about 0.01% v / v to about 0.4% v / v, from about 0.01% v / v to about 0.2% v / v, from about 0.01% v / v to about 0.1% v / v, from about 0.01% v / v to about 0.05% v / v, from about 0.05% v / v to about 20% v / v, from about 0.05% v / v to about 19% v / v, from about 0.05% v / v to about 18% v / v, from about 0.05% v / v to about 17% v / v, from about 0.05% v / v to about 16% v / v, from about 0.05% v / v to about 15% v / v, from about 0.05% v / v to about 14% v / v, from about 0.05% v / v to about 13% v / v, from about 0.05% v / v to about 12% v / v, from about 0.05% v / v to about 11% v / v, from about 0.05% v / v to about 10% v / v, from about 0.05% v / v to about 9% v / v, from about 0.05% v / v to about 8% v / v, from about 0.05% v / v to about 7% v / v, from about 0.05% v / v to about 6% v / v, from about 0.05% v / v to about 5% v / v, from about 0.05% v / v to about 4.5% v / v, from about 0.05% v / v to about 4.0% v / v, from about 0.05% v / v to about 3.5% v / v, from about 0.05% v / v to about 3.0% v / v, from about 0.05% v / v to about 2.5% v / v, from about 0.05% v / v to about 2.2% v / v, from about 0.05% v / v to about 2.0% v / v, from about 0.05% v / v to about 1.8% v / v, from about 0.05% v / v to about 1.6% v / v, from about 0.05% v / v to about 1.4% v / v, from about 0.05% v / v to about 1.2% v / v, from about 0.05% v / v to about 1.0% v / v, from about 0.05% v / v to about 0.8% v / v, from about 0.05% v / v to about 0.6% v / v, from about 0.05% v / v to about 0.4% v / v, from about 0.05% v / v to about 0.2% v / v, from about 0.05% v / v to about 0.1% v / v, from about 0.1% v / v to about 20% v / v, from about 0.1% v / v to about 19% v / v, from about 0.1% v / v to about 18% v / v, from about 0.1% v / v to about 17% v / v, from about 0.1% v / v to about 16% v / v, from about 0.1% v / v to about 15% v / v, from about 0.1% v / v to about 14% v / v, from about 0.1% v / v to about 13% v / v, from about 0.1% v / v to about 12% v / v, from about 0.1% v / v to about 11% v / v, from about 0.1% v / v to about 10% v / v, from about 0.1% v / v to about 9% v / v, from about 0.1% v / v to about 8% v / v, from about 0.1% v / v to about 7% v / v, from about 0.1% v / v to about 6% v / v, from about 0.1% v / v to about 5% v / v, from about 0.1% v / v to about 4.5% v / v, from about 0.1% v / v to about 4.0% v / v, from about 0.1% v / v to about 3.5% v / v, from about 0.1% v / v to about 3.0% v / v, from about 0.1% v / v to about 2.5% v / v, from about 0.1% v / v to about 2.2% v / v, from about 0.1% v / v to about 2.0% v / v, from about 0.1% v / v to about 1.8% v / v, from about 0.1% v / v to about 1.6% v / v, from about 0.1% v / v to about 1.4% v / v, from about 0.1% v / v to about 1.2% v / v, from about 0.1% v / v to about 1.0% v / v, from about 0.1% v / v to about 0.8% v / v, from about 0.1% v / v to about 0.6% v / v, from about 0.1% v / v to about 0.4% v / v, from about 0.1% v / v to about 0.2% v / v, from about 0.2% v / v to about 20% v / v, from about 0.2% v / v to about 19% v / v, from about 0.2% v / v to about 18% v / v, from about 0.2% v / v to about 17% v / v, from about 0.2% v / v to about 16% v / v, from about 0.2% v / v to about 15% v / v, from about 0.2% v / v to about 14% v / v, from about 0.2% v / v to about 13% v / v, from about 0.2% v / v to approximately 12% v / v, approximately 0.2% v / v to approximately 11% v / v, approximately 0.2% v / v to approximately 10% v / v, approximately 0.2% v / v to approximately 9% v / v, approximately 0.2% v / v to approximately 8% v / v, approximately 0.2% v / v to approximately 7% v / v, approximately 0.2% v / v to approximately 6% v / v, approximately 0.2% v / v to approximately 5% v / v, approximately 0.2% v / v to approximately 4.5% v / v, approximately 0.2% v / v to approximately 4.0% v / v, approximately 0.2% v / v to approximately 3.5% v / v, approximately 0.2% v / v to approximately 3.0% v / v, approximately 0.2% v / v to approximately 2.5% v / v, approximately 0.2% v / v to approximately 2.2% v / v, approximately 0.2% v / v to approximately 2.0% v / v, approximately 0.2% v / v to approximately 1.8% v / v, approximately 0.2% v / v to approximately 1.6% v / v, approximately 0.2% v / v to approximately 1.4% v / v, approximately 0.2% v / v to approximately 1.2% v / v, approximately 0.2% v / v to approximately 1.0% v / v, approximately 0.2% v / v to approximately 0.8% v / v, approximately 0.2% v / v to approximately 0.6% v / v, approximately 0.2% v / v to approximately 0.4% v / v, approximately 0.4% v / v to approximately 20% v / v, approximately 0.4% v / v to approximately 19% v / v, approximately 0.4% v / v to approximately 18% v / v, approximately 0.4% v / v to approximately 17% v / v, approximately 0.4% v / v to approximately 16% v / v, approximately 0.4% v / v to approximately 15% v / v, approximately 0.4% v / v to approximately 14% v / v, approximately 0.4% v / v to approximately 13% v / v, approximately 0.4% v / v to approximately 12% v / v, approximately 0.4% v / v to approximately 11% v / v, approximately 0.4% v / v to approximately 10% v / v, approximately 0.4% v / v to approximately 9% v / v, approximately 0.4% v / v to approximately 8% v / v, approximately 0.4% v / v to approximately 7% v / v, approximately 0.4% v / v to approximately 6% v / v, approximately 0.4% v / v to approximately 5% v / v, approximately 0.4% v / v to approximately 4.5% v / v, approximately 0.4% v / v to approximately 4.0% v / v, approximately 0.4% v / v to approximately 3.5% v / v, approximately 0.4% v / v to approximately 3.0% v / v, approximately 0.4% v / v to approximately 2.5% v / v, approximately 0.4% v / v to approximately 2.2% v / v, approximately 0.4% v / v to approximately 2.0% v / v, approximately 0.4% v / v to approximately 1.8% v / v, approximately 0.4% v / v to approximately 1.6% v / v, approximately 0.4% v / v to approximately 1.4% v / v, approximately 0.4% v / v to approximately 1.2% v / v, approximately 0.4% v / v to approximately 1.0% v / v, approximately 0.4% v / v to approximately 0.8% v / v, approximately 0.4% v / v to approximately 0.6% v / v, approximately 0.6% v / v to approximately 20% v / v, approximately 0.6% v / v to approximately 19% v / v, approximately 0.6% v / v to approximately 18% v / v, approximately 0.6% v / v to approximately 17% v / v, approximately 0.6% v / v to approximately 16% v / v, approximately 0.6% v / v to approximately 15% v / v, approximately 0.6% v / v to approximately 14% v / v, approximately 0.6% v / v to approximately 13% v / v, approximately 0.6% v / v to approximately 12% v / v, approximately 0.6% v / v to approximately 11% v / v, approximately 0.6% v / v to approximately 10% v / v, approximately 0.6% v / v to approximately 9% v / v, approximately 0.6% v / v to approximately 8% v / v, approximately 0.6% v / v to approximately 7% v / v, approximately 0.6% v / v to approximately 6% v / v, approximately 0.6% v / v to approximately 5% v / v, approximately 0.6% v / v to approximately 4.5% v / v, approximately 0.6% v / v to approximately 4.0% v / v, approximately 0.6% v / v to approximately 3.5% v / v, approximately 0.6% v / v to approximately 3.0% v / v, approximately 0.6% v / v to approximately 2.5% v / v, approximately 0.6% v / v to approximately 2.2% v / v, approximately 0.6% v / v to approximately 2.0% v / v, approximately 0.6% v / v to approximately 1.8% v / v, approximately 0.6% v / v to approximately 1.6% v / v, approximately 0.6% v / v to approximately 1.4% v / v, approximately 0.6% v / v to approximately 1.2% v / v, approximately 0.6% v / v to approximately 1.0% v / v, approximately 0.6% v / v to approximately 0.8% v / v, approximately 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 to approximately 17% v / v, approximately 1.2% v / v to approximately 16% v / v, approximately 1.2% v / v to approximately 15% v / v, approximately 1.2% v / v to approximately 14% v / v, approximately 1.2% v / v to approximately 13% v / v, approximately 1.2% v / v to approximately 12% v / v, approximately 1.2% v / v to approximately 11% v / v, approximately 1.2% v / v to approximately 10% v / v, approximately 1.2% v / v to approximately 9% v / v, approximately 1.2% v / v to approximately 8% v / v,. About 1.2% v / v to about 7% v / v, about 1.2% v / v to about 6% v / v, about 1.2% v / v to about 5% v / v, about 1.2% v / v to about 4.5% v / v, about 1.2% v / v to about 4.0% v / v, about 1.2% v / v to about 3.5% v / v, about 1.2% v / v to about 3.0% v / v, about 1.2% v / v to about 2.5% v / v, about 1.2% v / v to about 2.2% v / v, about 1.2% v / v to about 2.0% v / v, about 1.2% v / v to about 1.8% v / v, about 1.2% v / v to about 1.6% v / v, about 1.2% v / v to about 1.4% v / v, about 1.4% v / v to about 20% v / v, about 1.4% v / v to about 19% v / v, about 1.4% v / v to about 18% v / v, about 1.4% v / v to about 17% v / v, about 1.4% v / v to about 16% v / v, about 1.4% v / v to about 15% v / v, about 1.4% v / v to about 14% v / v, about 1.4% v / v to about 13% v / v, about 1.4% v / v to about 12% v / v, about 1.4% v / v to about 11% v / v, about 1.4% v / v to about 10% v / v, about 1.4% v / v to about 9% v / v, about 1.4% v / v to about 8% v / v, about 1.4% v / v to about 7% v / v, about 1.4% v / v to about 6% v / v, about 1.4% v / v to about 5% v / v, about 1.4% v / v to about 4.5% v / v, about 1.4% v / v to about 4.0% v / v, about 1.4% v / v to about 3.5% v / v, about 1.4% v / v to about 3.0% v / v, about 1.4% v / v to about 2.5% v / v, about 1.4% v / v to about 2.2% v / v, about 1.4% v / v to about 2.0% v / v, about 1.4% v / v to about 1.8% v / v, about 1.4% v / v to about 1.6% v / v, about 1.6% v / v to about 20% v / v, about 1.6% v / v to about 19% v / v, about 1.6% v / v to about 18% v / v, about 1.6% v / v to about 17% v / v, about 1.6% v / v to about 16% v / v, about 1.6% v / v to about 15% v / v, about 1.6% v / v to about 14% v / v, about 1.6% v / v to about 13% v / v, about 1.6% v / v to about 12% v / v, about 1.6% v / v to about 11% v / v, about 1.6% v / v to about 10% v / v, about 1.6% v / v to about 9% v / v, about 1.6% v / v to about 8% v / v, about 1.6% v / v to about 7% v / v, about 1.6% v / v to about 6% v / v, about 1.6% v / v to about 5% v / v, about 1.6% v / v to about 4.5% v / v, about 1.6% v / v to about 4.0% v / v, about 1.6% v / v to about 3.5% v / v, about 1.6% v / v to about 3.0% v / v, 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, approximately 2.0% v / v to approximately 2.5% v / v, approximately 2.0% v / v to approximately 2.2% v / v, approximately 2.2% v / v to approximately 20% v / v, approximately 2.2% v / v to approximately 19% v / v, approximately 2.2% v / v to approximately 18% v / v, approximately 2.2% v / v to approximately 17% v / v, approximately 2.2% v / v to approximately 16% v / v, approximately 2.2% v / v to approximately 15% v / v, approximately 2.2% v / v to approximately 14% v / v, approximately 2.2% v / v to approximately 13% v / v, approximately 2.2% v / v to approximately 12% v / v, approximately 2.2% v / v to approximately 11% v / v, approximately 2.2% v / v to approximately 10% v / v, approximately 2.2% v / v to approximately 9% v / v, approximately 2.2% v / v to approximately 8% v / v, approximately 2.2% v / v to approximately 7% v / v, approximately 2.2% v / v to approximately 6% v / v, approximately 2.2% v / v to approximately 5% v / v, approximately 2.2% v / v to approximately 4.5% v / v, approximately 2.2% v / v to approximately 4.0% v / v, approximately 2.2% v / v to approximately 3.5% v / v, approximately 2.2% v / v to approximately 3.0% v / v, approximately 2.2% v / v to approximately 2.5% v / v, approximately 2.5% v / v to approximately 20% v / v, approximately 2.5% v / v to approximately 19% v / v, approximately 2.5% v / v to approximately 18% v / v, approximately 2.5% v / v to approximately 17% v / v, approximately 2.5% v / v to approximately 16% v / v, approximately 2.5% v / v to approximately 15% v / v, approximately 2.5% v / v to approximately 14% v / v, approximately 2.5% v / v to approximately 13% v / v, approximately 2.5% v / v to approximately 12% v / v, approximately 2.5% v / v to approximately 11% v / v, approximately 2.5% v / v to approximately 10% v / v, approximately 2.5% v / v to approximately 9% v / v, approximately 2.5% v / v to approximately 8% v / v, approximately 2.5% v / v to approximately 7% v / v, approximately 2.5% v / v to approximately 6% v / v, approximately 2.5% v / v to approximately 5% v / v, approximately 2.5% v / v to approximately 4.5% v / v, approximately 2.5% v / v to approximately 4.0% v / v, approximately 2.5% v / v to approximately 3.5% v / v, approximately 2.5% v / v to approximately 3.0% v / v, approximately 3.0% v / v to approximately 20% v / v, approximately 3.0% v / v to approximately 19% v / v, approximately 3.0% v / v to approximately 18% v / v, approximately 3.0% v / v to approximately 17% v / v, approximately 3.0% v / v to approximately 16% v / v, approximately 3.0% v / v to approximately 15% v / v, approximately 3.0% v / v to approximately 14% v / v, approximately 3.0% v / v to approximately 13% v / v, approximately 3.0% v / v to approximately 12% v / v, approximately 3.0% v / v to approximately 11% v / v, approximately 3.0% v / v to approximately 10% v / v, approximately 3.0% v / v to approximately 9% v / v, approximately 3.0% v / v to approximately 8% v / v, approximately 3.0% v / v to approximately 7% v / v, approximately 3.0% v / v to approximately 6% v / v, approximately 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, approximately 5% v / v to approximately 20% v / v, approximately 5% v / v to approximately 19% v / v, approximately 5% v / v to approximately 18% v / v, approximately 5% v / v to approximately 17% v / v, approximately 5% v / v to approximately 16% v / v, approximately 5% v / v to approximately 15% v / v, approximately 5% v / v to approximately 14% v / v, approximately 5% v / v to approximately 13% v / v, approximately 5% v / v to approximately 12% v / v, approximately 5% v / v to approximately 11% v / v, approximately 5% v / v to approximately 10% v / v, approximately 5% v / v to approximately 9% v / v, approximately 5% v / v to approximately 8% v / v, approximately 5% v / v to approximately 7% v / v, approximately 5% v / v to approximately 6% v / v, approximately 6% v / v to approximately 20% v / v, approximately 6% v / v to approximately 19% v / v, approximately 6% v / v to approximately 18% v / v, approximately 6% v / v to approximately 17% v / v, approximately 6% v / v to approximately 16% v / v, approximately 6% v / v to approximately 15% v / v, approximately 6% v / v to approximately 14% v / v, approximately 6% v / v to approximately 13% v / v, approximately 6% v / v to approximately 12% v / v, approximately 6% v / v to approximately 11% v / v, approximately 6% v / v to approximately 10% v / v, approximately 6% v / v to approximately 9% v / v, approximately 6% v / v to approximately 8% v / v, approximately 6% v / v to approximately 7% v / v, approximately 7% v / v to approximately 20% v / v, approximately 7% v / v to approximately 19% v / v, approximately 7% v / v to approximately 18% v / v, approximately 7% v / v to approximately 17% v / v, approximately 7% v / v to approximately 16% v / v, approximately 7% v / v to approximately 15% v / v, approximately 7% v / v to approximately 14% v / v, approximately 7% v / v to approximately 13% v / v, approximately 7% v / v to approximately 12% v / v, approximately 7% v / v to approximately 11% v / v, approximately 7% v / v to approximately 10% v / v, approximately 7% v / v to approximately 9% v / v, approximately 7% v / v to approximately 8% v / v, approximately 8% v / v to approximately 20% v / v, approximately 8% v / v to approximately 19% v / v, approximately 8% v / v to approximately 18% v / v, approximately 8% v / v to approximately 17% v / v, approximately 8% v / v to approximately 16% v / v, approximately 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,It is 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., two, three, four, or five) 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., two, three, four, or five) antioxidant and / or chelating agent in an amount sufficient to improve the loss of binding ability of the chromatographic resin after exposure of that dose to gamma irradiation.

[0155] In some examples of any of the compositions described herein, the liquid can comprise at least one (e.g., two, three, four, or five) antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoid, phenolpropanoic acid, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanols. In some examples of any of the compositions described herein, the liquid can comprise 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 is (i) mannitol at 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); (ii) methionine (or cysteine or glutathione) at 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); (iii) sodium ascorbate at 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); (iv) histidine at 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); (v) methionine (or cysteine or glutathione) at 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) and histidine at 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); (vi) methionine (or cysteine or glutathione) at 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 at 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), and sodium ascorbate at 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);or (vii) can contain sodium ascorbate at 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) at 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 at 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), and histidine at 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). 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 can further contain at least one (e.g., two, three, four, or five) chelating agent (e.g., 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 desferrioxamine).

[0158] Also provided 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 exemplary 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 exemplary alcohols described herein or known in the art), wherein the at least one alcohol is present in an amount sufficient to improve the dose of the binding capacity of the chromatography resin upon treatment by gamma irradiation at a dose sufficient to reduce the bioburden of the composition. For example, the container (e.g., a storage container, e.g., a plastic container, or a chromatography column) can have an internal volume of, for example, 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, from about 1 mL to about 500 mL, from about 1 mL to about 50 mL, from about 5 mL to about 500 mL, from about 5 mL to about 400 mL, from about 5 mL to about 350 mL, from about 5 mL to about 300 mL, from about 5 mL to about 250 mL, from about 5 mL to about 200 mL, from about 5 mL to about 150 mL, from about 5 mL to about 100 mL, or from about 5 mL to about 50 mL. In some examples, the chromatography resin in the container is a slurry of the chromatography resin deposited in the liquid.In some examples, the container contains a filled chromatography resin (e.g., filled with 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 antioxidant 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 group, carbonate group, superoxide anion, peroxyl group, 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, phenolpropanoic acids, lidocaine, naringenin, fullerenes, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanols. 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 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 contain one or more substrates for the enzyme. Antioxidants can be confirmed using, for example, spin traps, redox-sensitive dyes, and some methods known in the art including chemiluminescence methods.

[0160] Any chelating agent that can be included in any of the compositions provided herein can bind to a redox-active metal having a 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), such as 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 chelating agent and / or antioxidant that can be included in any of the compositions provided herein is from about 0.1 mM to about 150 mM (e.g., from about 0.1 mM to about 150 mM, from about 0.1 mM to about 125 mM, from about 0.1 mM to about 100 mM, from about 0.1 mM to about 80 mM, from about 0.1 mM to about 60 mM, from about 0.1 mM to about 50 mM, from about 0.1 mM to about 40 mM, from about 0.1 mM to about 30 mM, from about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, from about 0.1 mM to about 5.0 mM, from about 0.5 mM to about 150 mM, from about 0.5 mM to about 100 mM, from about 0.5 mM to about 50 mM, from about 0.5 mM to about 25 mM, from about 0.5 mM to about 15 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to approximately 5 mM, approximately 1 mM to approximately 125 mM, approximately 1 mM to approximately 120 mM, approximately 1 mM to approximately 100 mM, approximately 1 mM to approximately 80 mM, approximately 1 mM to approximately 60 mM, approximately 1 mM to approximately 50 mM, approximately 1 mM to approximately 40 mM, approximately 1 mM to approximately 30 mM, approximately 1 mM to approximately 25 mM, approximately 5 mM to approximately 150 mM, approximately 5 mM to approximately 125 mM, approximately 5 mM to approximately 100 mM, approximately 5 mM to approximately 80 mM, approximately 5 mM to approximately 60 mM, approximately 5 mM to approximately 50 mM, approximately 5 mM to approximately 40 mM, approximately 5 mM to approximately 30 mM, approximately 5 mM to approximately 25 mM, approximately 10 mM to approximately 150 mM, approximately 10 mM to approximately 125 mM, approximately 1 mM to approximately 100 mM, approximately 10 mM to approximately 80 mM, approximately 10 mM to approximately 60 mM, approximately 10 mM to approximately 50 mM, approximately 10 mM to approximately 40 mM, approximately 10 mM to approximately 30 mM, approximately 10 mM to approximately 25 mM, approximately 20 mM to approximately 150 mM, approximately 20 mM to approximately 125 mM, approximately 20 mM to approximately 100 mM, approximately 20 mM to approximately 80 mM, approximately 20 mM to approximately 60 mM, approximately 20 mM to approximately 50 mM, approximately 20 mM to approximately 40 mM, approximately 20 mM to approximately 30 mM, approximately 30 mM to approximately 150 mM, approximately 30 mM to approximately 125 mM, approximately 30 mM to approximately 100 mM, approximately 30 mM to approximately 80 mM, approximately 30 mM to approximately 60 mM, approximately 30 mM to approximately 50 mM, approximately 30 mM to approximately 40 mM, approximately 40 mM to approximately 150 mM, approximately 40 mM to approximately 125 mM, approximately 40 mM to approximately 100 mM, approximately 40 mM to approximately 90 mM, approximately 40 mM to approximately 80 mM, approximately 40 mM to approximately 70 mM, approximately 40 mM to approximately 60 mM, approximately 50 mM to approximately 150 mM, approximately 50 mM to approximately 125 mM, approximately 50 mM to approximately 100 mM, approximately 50 mM to approximately 80 mM, approximately 50 mM to approximately 60 mM, approximately 80 mM to approximately 150 mM, approximately 80 mM to approximately 125 mM, approximately 80 mM to approximately 100 mM, approximately 100 mM to approximately 150 mM, or approximately 100 mM to approximately 125 mM). It can be such that.

[0162] In some examples, the compositions provided herein include mannitol at 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 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 to about 30 mM mannitol); methionine (or cysteine or glutathione) at 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 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); and sodium ascorbate at 5 mM to 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 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 can contain one or more of histidine at 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 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 composition are: (i) mannitol at 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) (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0); (ii) methionine (or cysteine or glutathione) at 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) (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0); (iii) sodium ascorbate at 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) (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0); (iv) histidine at 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) (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0); (v) methionine (or cysteine or glutathione) at 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) and histidine at 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) (e.g., in a buffer solution, e.g., a phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.(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) of 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) of 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, about 25 mM to about 35 mM, or about 30 mM to about 35 mM) of sodium ascorbate (e.g., in a buffer solution, e.g., 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) of 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) of 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) of 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) of histidine (e.g., in a buffer solution, e.g., phosphate buffer, e.g., 50 mM sodium phosphate, pH 6.0).

[0164] The 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) for performing the gamma irradiation described herein. The bioburden of a composition can be determined, for example, by sampling the composition, which would contain self-replicating biological contaminants present in the composition, by, for example, stomaching, sonication, shaking, vortex mixing, flushing, blending, or scraping with a swab, and (e.g., placing the sample in a growth medium that allows the self-replicating biological contaminants to replicate themselves, e.g., plating the sample in a Petri dish or passing a membrane through the sample) quantifying or qualifying the level of self-replicating biological contaminants present in the sample.

[0165] The amount of at least one alcohol, at least one antioxidant and / or at least one chelating agent sufficient to improve the dose of the binding capacity of the chromatographic resin during the treatment 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 decrease in the binding capacity of a chromatographic resin treated by gamma irradiation in the presence of an amount of at least one alcohol (and optionally, further in the presence of at least one antioxidant and / or chelating agent) can be compared with the level of decrease in the binding capacity of a chromatographic resin treated by gamma irradiation at the same dose in the absence of at least one alcohol (and optionally, at least one antioxidant and / or chelating agent), where the decrease in the level of decrease in the binding capacity of the chromatographic resin irradiated with gamma rays in the presence of at least one alcohol (and optionally, further in the presence of at least one antioxidant and / or chelating agent) compared to the chromatographic resin irradiated with gamma rays in the absence of at least one alcohol (and optionally, antioxidant and / or chelating agent) indicates that at least one alcohol (and optionally, antioxidant and / or chelating agent) was present in an amount sufficient to improve the dose of the binding capacity of the chromatographic resin during the treatment by gamma irradiation. Representative methods for determining the binding capacity of the chromatographic resin are described in the examples. Additional examples of methods for determining the binding capacity of the chromatographic resin are known in the art.

[0166] Method for reducing the bioburden of a chromatographic resin A method for reducing the bioburden of a chromatographic resin is provided herein. The method includes exposing a container containing a composition (e.g., any of the exemplary compositions containing a chromatographic resin and a liquid containing at least one alcohol as described herein) comprising (i) a chromatographic resin and (ii) a liquid containing at least one alcohol to a dose of gamma irradiation sufficient to reduce the bioburden of the container and the chromatographic resin, wherein the at least one alcohol is present in an amount sufficient to improve the loss of binding capacity of the chromatographic resin after (or during) exposure to that dose of gamma irradiation.

[0167] Also provided is a method for reducing the bioburden of a chromatography resin, comprising exposing a composition (e.g., any composition comprising a chromatography resin described herein and a liquid comprising at least one alcohol) comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol in an amount sufficient to improve the loss of binding capacity of the chromatography resin after / upon exposure to gamma-ray irradiation, to gamma-ray irradiation at a dose sufficient to reduce the bioburden of the container and the chromatography resin at a rate of about 0.1 kGy / hour to about 6 kGy / hour (e.g., about 0.1 kGy / hour to about 5.5 kGy / hour, about 0.1 kGy / hour to about 5.0 kGy / hour, about 0.1 kGy / hour to about 4.5 kGy / hour, about 0.1 kGy / hour to about 4.0 kGy / hour, about 0.1 kGy / hour to about 3.5 kGy / hour, about 0.1 kGy / hour to about 3.0 kGy / hour, about 0.1 kGy / 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 / hour, about 0.5 kGy / hour to about 3.0 kGy / hour, about 0.5 kGy / hour to about 2.5 kGy / hour, about 0.5 kGy / hour to about 2.0 kGy / hour) 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, a container or composition comprising a chromatographic resin and a liquid comprising at least one alcohol (e.g., any container or any composition comprising a chromatographic resin and a liquid comprising at least one alcohol as described herein) for 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 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 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,a period of 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 week 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 8 months, about 8 months to about 1 year, about 8 months to about 11 months, about 8 months to about 10 months, about 8 months to about 9 months, about 9 months to about 1 year, about 9 months to about 11 months, about 9 months to about 10 months, about 10 months to about 1 year, about 10 months to about 11 months, or about 11 months to about 1 yearFor example, 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°C, about 4°C to about 6°C, about 6°C to about 40°C, about 6°C to about 35°C, about 6°C to about 30°C, about 6°C to about 28°C, about 6°C to about 26°C, about 6°C to about 24°C, about 6°C to about 22°C, about 6°C to about 20°C, about 6°C to about 18°C, about 6°C to about 16°C, about 6°C to about 14°C, about 6°C to about 12°C, about 6°C to about 10°C, about 6°C to about 8°C, about 28°C to about 40°C, about 8°C to about 35°C, about 8°C to about 30°C, about 8°C to about 28°C, about 8°C to about 26°C, about 8°C to about 24°C, about 8°C to about 22°C, about 8°C to about 20°C, about 8°C to about 18°C, about 8°C to about 16°C, about 8°C to about 14°C, about 8°C to about 12°C, about 8°C to about 10°C, 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 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°C to about 30°C, about 14°C to about 28°C, about 14°C to about 26°C, about 14°C to about 24°C, about 14°C to about 22°C, about 14°C to about 20°C, about 14°C to about 18°C, about 14°C to about 16°C, about 16°C to about 40°C, about 16°C to about 35°C, about 16°C to about 30°C, about 16°C to about 28°C, about 16°C to about 26°C, about 16°C to about 24°C, about 16°C to 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°CIt can include storing at a temperature of 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 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 the binding ability of the gamma-irradiated chromatography resin produced by these methods is higher than the level of the binding ability of the gamma-irradiated chromatography resin irradiated with gamma rays at a rate greater than 6.1 kGy / hour and / or at a temperature higher than 25°C for one or both.

[0170] The chromatography resin can be exposed to gamma irradiation using methods known in the art. For example, isotopes such as cobalt-60 or cesium-137 can be used as gamma ray sources. The chromatography resin can be exposed to gamma irradiation 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 irradiation at a dose 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 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. The chromatography resin is about 1×10 -6 or less, about 1×10 -7 or less, about 10×10 -8 or less, about 1×10-11 or less than, or about 1×10 -12 or less than, or about 1×10 -6 ~ about 1×10 -12 , about 1×10 -6 ~ about 1×10 -11 , about 1×10 -6 ~ about 1×10 -10 , about 1×10 -6 ~ about 1×10 -9 , about 1×10 -6 ~ about 1×10 -8 , 1×10 -6 ~ about 1×10 -7 , about 1×10 -7 ~ about 1×10 -12 , about 1×10 -7 ~ about 1×10 -11 , about 1×10 -7 ~ about 1×10 -10 , about 1×10 -7 ~ about 1×10 -9 , about 1×10 -7 ~ about 1×10 -8 , about 1×10 -8 ~ about 1×10 -12 , about 1×10 -8 ~ about 1×10 -11 , about 1×10 -8 ~ about 1×10 -10 , or about 1×10 -8 ~ about 1×10 -9 such that it can be exposed to a dose of gamma irradiation sufficient to result in a sterility assurance level of about 1×10

[0171] The dosage of gamma-ray irradiation sufficient to reduce the bioburden of a chromatographic resin can be determined using methods known in the art. For example, the bioburden level of a chromatographic resin treated with a certain dosage of gamma-ray irradiation can be compared with the bioburden level of an untreated (e.g., control, non-gamma-ray irradiated) chromatographic resin, and a decrease in the bioburden level of the gamma-ray irradiated chromatographic resin when compared to the untreated chromatographic resin indicates that the dosage of gamma-ray irradiation is sufficient to reduce the bioburden of the chromatographic resin. Representative methods for determining the bioburden level of a composition (e.g., chromatographic resin) are described herein. Additional methods for determining the bioburden level of a composition (e.g., chromatographic 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 protein or peptide ligands (e.g., from about 5 amino acids to about 100 amino acids, from about 5 amino acids to about 90 amino acids, from about 5 amino acids to about 80 amino acids, from about 5 amino acids to about 70 amino acids, from about 5 amino acids to about 60 amino acids, from about 5 amino acids to about 50 amino acids, from about 5 amino acids to about 40 amino acids, from about 5 amino acids to about 30 amino acids, from about 5 amino acids to about 25 amino acids, or from about 5 amino acids to about 20 amino acids), small molecule substrates or cofactors of enzymes, aptamers, inhibitors (e.g., competitive protein inhibitors), or metals. 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., an anion exchange chromatography resin and a hydrophobic interaction chromatography resin). The chromatography resin can be an anion exchange chromatography resin (e.g., an anion exchange chromatography resin containing an N-benzyl-N-methyl-ethanolamine group).

[0173] The container containing the chromatography resin can be a plastic container (e.g., a cylindrical tube, a sealed or fixed box, or a sealed bag). Non-limiting examples of containers used in these methods include storage vessels or chromatography columns. For example, a composition (e.g., any of the representative compositions described herein) containing (i) a chromatography resin and (ii) a liquid containing 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 placed within a blister pack. The container (e.g., a storage vessel or a chromatography column) can have an internal total volume of from about 1 mL to about 1 L (e.g., from about 1 mL to about 900 mL, from about 1 mL to about 800 mL, from about 1 mL to about 700 mL, from about 1 mL to about 600 mL, from about 1 mL to about 500 mL, from about 1 mL to about 450 mL, from about 1 mL to about 400 mL, from about 1 mL to about 350 mL, from about 1 mL to about 300 mL, from about 1 mL to about 250 mL, from about 1 mL to about 200 mL, from about 1 mL to about 150 mL, from about 1 mL to about 100 mL, from about 1 mL to about 75 mL, from about 1 mL to about 50 mL, from about 1 mL to about 40 mL, from about 1 mL to about 30 mL, or from about 1 mL to about 20 mL).

[0174] A composition (e.g., any of the representative compositions described herein) containing (i) a chromatographic resin and (ii) a liquid containing at least one alcohol contained within a container can exist as a wet or moist solid mixture. For example, the container can contain a slurry of chromatographic resin deposited in the liquid. In some embodiments, the container can contain a filled chromatographic resin. For example, a container containing a composition (e.g., any of the compositions described herein) containing (i) a chromatographic resin and (ii) a liquid containing at least one alcohol is a filled chromatographic column (e.g., when the resin is filled with a liquid containing at least one alcohol). Some embodiments include placing, prior to exposure, a composition (e.g., any of the compositions described herein) containing (i) a chromatographic resin and (ii) a liquid containing at least one alcohol into a container.

[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 contain at least one alcohol 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, hexadecane-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 can further contain at least one antioxidant (e.g., at least one antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoid, phenolpropanoic acid, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanols) 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 desferrioxamine).

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

[0177] Also provided herein is a chromatography resin with reduced bioburden produced by any of the methods described herein (e.g., a chromatography resin with reduced bioburden provided in a storage container, e.g., a sealed storage container). The chromatography resin with reduced bioburden produced using any of the methods described herein has about 1×10 -6 or less, about 1×10 -7 or less, about 10×10 -8 or less, about 1×10 -11 or less, or about 1×10 -12 or less, or about 1×10 -6 ~ about 1×10 -12 、about 1×10 -6 ~ about 1×10 -11 、about 1×10 -6 ~ about 1×10 -10 、about 1×10 -6 ~ about 1×10 -9 、about 1×10 -6 ~ about 1×10 -8 、1×10 -6 ~ about 1×10 -7 、about 1×10 -7 ~ about 1×10 -12 、about 1×10 -7 ~ about 1×10 -11 、about 1×10 -7 ~ about 1×10 -10 、about 1×10 -7 ~ about 1×10 -9 、about 1×10 -7~about 1×10 -8 、about 1×10 -8 ~about 1×10 -12 、about 1×10 -8 ~about 1×10 -11 、about 1×10 -8 ~about 1×10 -10 、or about 1×10 -8 ~about 1×10 -9 and can have a sterility assurance level. The reduced bioburden chromatography resin produced by any of the methods described herein, when using the same protein to test the binding capacity of both the chromatography resin produced by the methods described herein and the untreated control chromatography resin, is 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 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% - 95%, about 74% - about 95%, about 76% - about 95%, at least about 78% - about 95%, about 80% - about 95%, or about 74% - about 90%, about 76% - about 90%, about 78% - about 90%, or about 80% - about 90% of the binding capacity of the same untreated (e.g., not gamma irradiated) chromatography resin for reducing its bioburden.

[0178] Method for making a chromatography column filled with reduced bioburden Also provided herein is a method of producing a reduced-packed chromatography column, comprising the steps of preparing a reduced-bioburden chromatography resin produced by any of the methods described herein, and packing the chromatography resin into a column with reduced bioburden in a sterilized or reduced-bioburden environment. In some embodiments, a chromatography column packed with reduced bioburden can be produced by exposing a column containing a packed chromatography resin and a liquid comprising at least one alcohol (e.g., optionally further comprising at least one antioxidant and / or chelating agent, e.g., any of the exemplary liquids described herein) to a dose of gamma irradiation sufficient to reduce the bioburden of the column and the packed chromatography resin, wherein the at least one alcohol is present in an amount sufficient to improve the loss of binding capacity of the packed chromatography resin after exposure to that dose of gamma irradiation.

[0179] Also provided herein is a chromatography column 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 have about 1×10 -6 or less, about 1×10 -7 or less, about 10×10 -8 or less, about 1×10 -11 or less, or about 1×10 -12 or less, or about 1×10 -6 ~ about 1×10 -12 、about 1×10 -6 ~ about 1×10 -11 、about 1×10 -6 ~ about 1×10 -10 、about 1×10 -6 ~ about 1×10 -9 、about 1×10 -6 ~ about 1×10 -8 、1×10 -6 ~ about 1×10 -7 、about 1×10-7 ~ about 1×10 -12 、 about 1×10 -7 ~ about 1×10 -11 、 about 1×10 -7 ~ about 1×10 -10 、 about 1×10 -7 ~ about 1×10 -9 、 about 1×10 -7 ~ about 1×10 -8 、 about 1×10 -8 ~ about 1×10 -12 、 about 1×10 -8 ~ about 1×10 -11 、 about 1×10 -8 ~ about 1×10 -10 、 or about 1×10 -8 ~ about 1×10 -9 can have a sterility assurance level. Any of the chromatography columns filled with a reduced bioburden generated by the methods described herein can contain at least one chromatography resin selected from the group consisting of anion exchange chromatography resins, cation exchange chromatography resins, affinity chromatography resins (e.g., any of the affinity chromatography resins described herein or known in the art), hydrophilic interaction chromatography resins, and size exclusion chromatography resins. For example, any of the chromatography columns filled with a reduced bioburden described herein can contain an affinity chromatography resin containing a protein ligand (e.g., Protein A). The chromatography columns filled with a reduced bioburden described herein can contain an anion exchange chromatography resin (e.g., an anion exchange chromatography resin containing an N-benzyl-N-methyl-ethanolamine group).

[0180] Method for Performing Chromatography with Reduced Bioburden The methods described herein include the use of a chromatography column filled with a reduced bioburden provided herein, and the methods described herein include the use of one or two MCCS including at least one chromatography column filled with a reduced bioburden provided herein. The gamma-irradiated chromatography resin can be any of the types of resins described herein (or any of the types of chromatography resins known in the art).

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

[0182] A chromatography column filled with a reduced bioburden for use in any of the methods described herein has from about 1×10 -3 to about 1×10 -12 ; from about 1×10 -4 to about 1×10 -12 ; 1×10 -5 to about 1×10 -11 ; from about 1×10 -5 to about 1×10 -10 ; from about 1×10 -5 to about 1×10 -9 ; from about 1×10 -6 to about 1×10 -9 ; or from about 1×10 -6 to about 1×10 -8 sterility assurance level (SAL).

[0183] Buffers with reduced bioburden The methods and processes described herein can be carried out using one or more buffers with reduced bioburden. As will be understood in the art, a buffer with reduced bioburden can be any type of buffer used in a chromatography cycle (e.g., a buffer used in any step of a chromatography cycle or a unit operation described herein). Representative methods for reducing the bioburden of a buffer include filtration (filtration through a 0.2 μm pore size filter), autoclaving, and gamma irradiation. Further methods for reducing the bioburden of a buffer are known in the art. A buffer with reduced bioburden has from about 1×10 -3 to about 1×10 -12 ; from about 1×10 -4 to about 1×10 -12 ; 1×10 -5 to about 1×10 -11 ; from about 1×10 -5 to about 1×10 -10 ; from about 1×10 -5 to about 1×10 -9 ; from about 1×10 -6 to about 1×10 -9 ; or from about 1×10 -6 to about 1×10 -8It can have a sterility assurance level (including both ends).

[0184] Recombinant therapeutic protein 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., galactosidase (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 can be an artificially modified antigen-binding polypeptide that includes at least one multifunctional recombinant protein scaffold (e.g., see Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent Application Publication No. 2012 / 0164066, which is incorporated herein by reference in its entirety, for antigen-binding recombinant proteins). Non-limiting examples of recombinant therapeutic proteins that are antibodies include panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afucosumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, apolizumab, atinumab, tocilizumab, basiliximab, bectumomab, belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab, etrolizumab, golimumab, infliximab, natalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab and trastuzumab.Further examples of recombinant therapeutic antibodies that can be produced by the methods described herein are known in the art. Further non-limiting examples of recombinant therapeutic proteins that can be produced / purified by the methods include alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, coagulation factor IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa, and alteplase.

[0185] Secreted soluble recombinant therapeutic proteins can be recovered from a liquid culture medium (e.g., a first liquid culture medium and / or a 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 the liquid culture medium from the cells (e.g., mammalian cells) are known in the art and include, for example, centrifugation, filtration, pipetting, and / or aspiration. The secreted recombinant therapeutic protein can then be recovered and further purified from the liquid culture medium using a variety of biochemical techniques including, for example, 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 chromatography cycle 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., a recombinant therapeutic protein). For example, an affinity chromatography column can include steps of loading a fluid containing the target recombinant protein onto the affinity chromatography column, washing the column to remove unwanted biological materials (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 where the target recombinant protein binds to the chromatography resin during the loading step can include steps of loading a fluid containing the target protein onto the column, washing the column to remove unwanted biological materials, eluting the target recombinant protein bound to the column, and re-equilibrating the column. In other examples, a chromatography cycle using a cation and / or anion exchange chromatography column where unwanted biological materials bind to the chromatography resin during the loading step but the target recombinant protein does not can include steps of loading a fluid containing the target protein onto the column, collecting the target recombinant protein in the flow-through fraction, 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 in any combination, for example, 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 the number and type of buffer(s) used in each step.

[0187] Method for performing column chromatography with reduced bioburden Provided herein are methods of performing chromatography with reduced bioburden. These methods include preparing a chromatography column filled with reduced bioburden generated using any of the methods described herein, and performing column chromatography using the chromatography column filled with reduced bioburden. The chromatography column filled with reduced bioburden can comprise at least one of any of the chromatography resins described herein in any combination. For example, the chromatography resin present within the chromatography column filled with reduced bioburden can be an affinity resin comprising a protein ligand (e.g., Protein A), or can comprise an anion exchange chromatography resin. The chromatography column filled with reduced bioburden can have any of the representative internal volumes described herein. The chromatography column filled with reduced bioburden can have any shape (e.g., cylindrical, substantially cylindrical, or elliptical) described herein or known in the art. The column chromatography performed by these methods can be used to purify or isolate a recombinant protein (e.g., any recombinant therapeutic protein described herein or known in the art). In some examples, the chromatography column filled with reduced bioburden is part of a multi-column chromatography system (MCCS), and can be part of, for example, a periodic countercurrent chromatography system (PCCS).

[0188] The column chromatography performed can include at least one cycle of chromatography described herein or known in the art. For example, at least one cycle of chromatography can include: capturing a recombinant protein by exposing a liquid containing the recombinant protein to a chromatography resin; washing the chromatography resin by exposing the chromatography resin to a washing 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., a liquid culture medium collected from perfusion or batch culture) or a diluted liquid culture medium (e.g., a culture medium diluted in a buffer).

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

[0190] Column chromatography can include chromatography of 2 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. In some examples, column chromatography is performed continuously over 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 a chromatography column filled with a reduced bioburden has a binding capacity percentage 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 80% 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 100%, 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%) compared to the same resin that has not been treated with gamma irradiation (when testing the binding ability of both resins using the same protein) (e.g., evaluated immediately after exposure to gamma irradiation).

[0192] An integrated, closed or substantially closed, continuous method for producing a recombinant protein Provided herein are integrated, closed or substantially closed, continuous methods for producing a purified recombinant protein (e.g., a recombinant therapeutic protein). These methods include preparing a liquid culture medium containing a substantially cell-free recombinant protein (e.g., a recombinant therapeutic protein).

[0193] Some methods involve continuously supplying a liquid culture medium to a multi-column chromatography system (MCCS) that includes at least one chromatography column filled with a reduced bioburden provided herein, where these methods utilize a buffer of the reduced bioburden, are integrated, and operate continuously up to the eluate from the MCCS that is a recombinant protein (e.g., a therapeutic protein drug substance) purified from the liquid culture medium.

[0194] Some methods involve continuously supplying a liquid culture medium to a first MCCS (MCCS1), capturing a recombinant protein from the liquid culture medium using MCCS1, generating an eluate containing the recombinant protein from MCCS1, continuously supplying the eluate to a second MCCS (MCCS2), continuously supplying the recombinant protein from the eluate to MCCS2, and subsequently eluting the recombinant protein to produce a purified recombinant protein, where at least one column within MCCS1 and / or MCCS2 is a chromatography column filled with a reduced bioburden provided herein, the method utilizes a buffer of the reduced bioburden, is integrated, and operates continuously up to the recombinant protein purified from the liquid culture medium.

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

[0196] The method described in this specification provides continuous and time-efficient production of purified recombinant proteins from a liquid culture medium containing recombinant proteins. For example, the elapsed time from the supply of a liquid culture medium 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 (including both ends), for example, 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, 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 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 (including both ends). In other examples, the elapsed time from the supply of a liquid culture medium containing a recombinant protein to MCCs or MCCs1 to the elution of the recombinant protein from MCCs or MCCs2, respectively, can be, for example, greater than about 4 hours to less than about 40 hours (including both ends), for example, greater 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 (including both ends).

[0197] Non-limiting aspects of MCCs (MCCs, MCC1, and / or MCC2) that can be used in any of these methods are described in U.S. Provisional Patent Application 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 the recombinant protein for a total period of, for example, from about 5 minutes to less than about 6 hours (including both ends), for example, from about 5 minutes to about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes (including both ends).

[0199] Some methods utilize 1, 2, 3, 4, 5, or 6 reservoirs (e.g., holding tanks), and can have a volume of, for example, from 1 mL to about 300 mL (including both ends), such as from 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 (including both ends). Any reservoir(s) (e.g., holding tank(s)) used to hold fluid (by any of the methods described herein) prior to being supplied to the MCCs or MCCs1 can have a volume of, for example, from 1 mL to about 100% (including both ends) of the loading volume of the first column of the MCCs, i.e., MCCs1, such as 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% (including both ends). A reservoir(s) (e.g., holding tank(s)) can be used to hold the eluate from MCCs1 prior to the eluate entering MCCs2, and this reservoir can have a volume of, for example, from 1 mL to about 100% (including both ends) of the loading volume of the first column of MCCs2, such as 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% (including both ends).

[0200] Various further aspects of these methods are described in detail below and can be used in any combination without limitation in the methods provided herein. Representative aspects of the methods provided are described below, but as will be understood by those skilled in the art, additional steps can be added to the methods described herein and any of the steps of the methods described herein can be performed using other materials.

[0201] Liquid culture medium A liquid culture medium containing a substantially cell-free recombinant protein (e.g., a recombinant therapeutic protein) can be derived from any source. For example, the liquid culture medium can be obtained 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 bacteria or yeast cells that secrete a recombinant protein.

[0202] The liquid culture medium obtained from a recombinant cell culture can be filtered or clarified to obtain a liquid culture medium substantially free of cells and / or viruses. Methods for filtering or clarifying the liquid culture medium to remove cells are known in the art (e.g., 0.2-μm filtration and filtration using an Alternating Tangential Flow (ATF TM ) system). The recombinant cells can also be removed from the liquid culture medium using centrifugation to remove the supernatant, which is a liquid culture medium substantially free of cells, or by sedimenting the cells at the gravitational bottom of a container (e.g., a bioreactor) containing the liquid culture medium and removing the liquid culture medium that is far from the sedimented recombinant cells (a liquid culture medium substantially free of cells).

[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 a recombinant protein (e.g., a 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: a liquid culture medium free of animal-derived components, a serum-free liquid culture medium, a liquid culture medium containing serum, a liquid culture medium of known composition, and a protein-free liquid culture medium. In any of the methods described herein, the liquid culture medium obtained from the culture can be diluted by the addition of a second fluid (e.g., a buffer) prior to being supplied to the MCCs or MCCs1.

[0205] A liquid culture medium containing a recombinant protein substantially free of cells can be stored 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) at a temperature of less than about 15 °C (e.g., less than about 10 °C, less than about 4 °C, less than about 0 °C, less than about -20 °C, less than about -50 °C, less than about -70 °C, or less than about -80 °C) prior to being supplied to the MCCs or MCCs1. Alternatively, in some examples, the liquid culture medium is supplied directly from the bioreactor to the MCCs or MCCs1 (e.g., 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 chromatography columns, two or more chromatography membranes, or a combination of at least one chromatography column and at least one chromatography membrane. In non-limiting examples, an MCCS (e.g., an MCCS in any of the methods herein, MCCS1 and / or MCCS2) can include four chromatography columns, three chromatography columns and one chromatography membrane, three chromatography columns, two chromatography columns, two chromatography membranes, and a combination of two chromatography columns and one chromatography membrane. For further examples of combinations of chromatography columns and / or chromatography membranes, one of ordinary skill in the art can envision, without limitation, use in an MCCS (e.g., an MCCS in any of the methods described herein, MCCS1 and / or MCCS2). The individual chromatography columns and / or chromatography membranes present within an MCCS can be the same (e.g., having the same shape, volume, resin, capture mechanism, and unit operation) or different (e.g., having one or more of different shapes, volumes, resins, capture mechanisms, and / or unit operations). The individual chromatography column(s) and / or chromatography membrane(s) present within an MCCS (e.g., an MCCS in any of the methods described herein, MCCS1 and / or MCCS2) can perform the same unit operation (e.g., a unit operation of capturing, purifying, or polishing) or different unit operations (e.g., different unit operations selected from the group consisting of, for example, capturing, purifying, polishing, inactivating a virus, adjusting the ionic concentration and / or pH of a fluid containing a recombinant protein, and filtering).For example, in an example of the method described in this specification, at least one chromatographic column and / or chromatographic membrane within MCCS or MCCS1 performs a unit operation of capturing a recombinant protein.

[0207] One or more chromatography columns (plural available) that may be present within the MCCS (e.g., may be present within MCCS, MCCS1, and / or MCCS2) may have a resin volume between about 1 mL and about 2 mL, including the upper and lower limits, 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. One or more chromatography columns (plural available) that may be present within the MCCS (e.g., may be present within MCCS, MCCS1, and / or MCCS2) may have a resin volume 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 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 columns (plural available) 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 columns (plural available) within the MCCS (e.g., MCCS, MCCS1, and / or MCCS2) may 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 (plural available) within the MCCS (e.g., within MCCS, MCCS1, and / or MCCS2) may have substantially the same shape or may have substantially different shapes. For example, one or more chromatography columns (plural available) within the MCCS (e.g., within MCCS, MCCS1, and / or MCCS2) may have substantially the same shape that is circular cylinder-shaped or may have substantially the same shape that is oval cylinder-shaped.

[0209] One or more chromatography membranes (which may be present within, for example, MCCS, MCCS1 and / or MCCS2) that can be present within MCCS can have a bed volume, for example, between about 1 mL and about 500 mL (for example, 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, 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 2 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., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) different types of reduced bioburden buffers can be used during the use of MCCs, MCCs1, and / or MCCs2 in any of the methods described herein. As is known in the art, one or more types of the reduced bioburden buffers used for MCCs, MCCs1, and / or MCCs2 in the methods described herein depend on the resin present in the chromatography column(s) and / or chromatography membrane(s) of MCCs, MCCs1, and / or MCCs2, the biophysical properties of the recombinant protein, and the unit operations (e.g., any of the representative unit operations described herein) performed by the specific chromatography column(s) and / or chromatography membrane(s) of MCCs, MCCs1, and / or MCCs2. 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 ordinary 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 the recombinant protein, activity of the recombinant protein, level of purity of the recombinant protein, and removal of bioburden contaminants from the fluid containing the recombinant protein (e.g., liquid culture medium) (e.g., absence of active virus, mycobacteria, yeast, bacteria, or mammalian cells).

[0211] MCCS, MCCS1, and / or MCCS2 may be a periodic countercurrent chromatography system (PCCS). The PCCS may include, for example, two or more chromatography columns (e.g., three columns or four columns) that are switched to enable continuous elution of recombinant proteins from two or more chromatography columns. The PCCS may include two or more chromatography columns, may include two or more chromatography membranes, or may include at least one chromatography column and at least one chromatography membrane. The column operation (cycle) generally consists of a loading step, a washing step, an elution step, and a regeneration step. In the PCCS, multiple columns are used to operate the same steps separately and continuously in a cyclic manner. Since the columns operate in series, the flow-through fraction and the washing agent coming out of one column are captured by another column. Such a characteristic peculiar to the PCCS enables the loading of a resin that is closer to the static binding capacity rather than the dynamic binding capacity, similar to that typical during chromatography in batch mode. As a result of continuous cyclic operation and elution, the fluid entering the PCCS is continuously processed, and an eluate containing recombinant protein is continuously produced.

[0212] Column switching strategies are used to proceed from one step to another in the PCCS cycle. Examples of column switching that can be used in PCCS are described in U.S. Provisional Patent Application Nos. 61 / 775,060 and 61 / 856,390. For example, the column switching method can use two automated switching operations per column: the first is related to breakthrough of the first product, and the second corresponds to column saturation. The decision of when to initiate the column switching operation can be determined by monitoring the concentration of the recombinant protein in the eluate exiting each chromatography column present within 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 using a feedback control. The PAT tool is capable of real-time in-line measurement of recombinant protein concentration using a feedback control. Similar to what is known in the art, column switching can also be designed based on the time or amount of fluid (e.g., buffer) passing through one or more chromatography columns and / or chromatography membranes within MCCS, MCCS1, and / or MCCS2.

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

[0214] One or more unit operations that can be performed by the MCCs or MCC1 and / or MCC2 used in the methods described herein include, for example, capturing a recombinant protein, inactivating a virus present in a fluid containing the recombinant protein, purifying the recombinant protein, polishing the recombinant protein, holding a fluid containing the recombinant protein (e.g., using any of the exemplary holding tanks described herein), filtering or removing particulate material and / or cells emerging from the fluid containing the recombinant protein, and adjusting the ionic concentration and / or pH of the fluid containing the recombinant protein.

[0215] In some embodiments, the MCCs or MCC1 comprises at least one chromatography column and / or chromatography membrane that performs the unit operation of capturing a recombinant protein. The unit operation of capturing can be performed using, for example, at least one chromatography column and / or chromatography resin that utilizes a capture mechanism. Non-limiting examples of capture mechanisms include a protein A binding-based capture mechanism, an antibody binding-based or antibody fragment binding-based capture mechanism, a substrate binding-based capture mechanism, an aptamer binding-based capture mechanism, a tag binding-based capture mechanism (e.g., a capture mechanism based on a polyHis tag), and a cofactor binding-based capture mechanism. Capture can also be performed using a resin that can be used to perform cation exchange or anion exchange chromatography, molecular sieve chromatography, or hydrophobic interaction chromatography. Non-limiting resins that can be used to capture a recombinant protein are described herein. Further examples of resins that can be used to capture a recombinant protein are known in the art.

[0216] The unit operation of inactivating a virus present in a fluid containing a recombinant protein can be carried out using MCCS, MCCS1, and / or MCCS2 (for example, including a chromatography column, a chromatography membrane, or a holding tank) by incubating the fluid containing the recombinant protein at a pH between about 3.0 and 5.0 (for example, 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 (for example, a period between about 30 minutes and 1.5 hours, a period between about 30 minutes and 1.25 hours, a period between about 0.75 hours and 1.25 hours, or a period of about 1 hour).

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

[0218] The unit operations for polishing recombinant proteins can be performed using one or more MCCSs (e.g., MCCS, MCCS1 and / or MCCS2), including, for example, chromatography columns or chromatography membranes, containing resins 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 for polishing recombinant proteins are described herein. Further examples of resins that can be used for polishing recombinant proteins are known in the art.

[0219] The unit operation of holding a fluid containing a recombinant protein can be carried out using an MCCS (e.g., MCCS, MCCS1, and / or MCCS2), including at least one reservoir (e.g., a holding tank), or in combination with MCCS or MCCS1 and MCCS2, including a maximum of one, two, three, four, or five reservoirs (e.g., holding tanks (plural)). For example, the reservoir(s) (e.g., holding tank(s)) that can be used to achieve the above-described holding unit operation can each have a volume 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 capacity, for example, between 1 mL and about 300 mL, including the upper and lower limits, for example, between 1 mL and 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, including the upper and lower limits. Any reservoir(s) (e.g., holding tank(s)) used to hold the fluid until it enters the MCCS or MCCS1 (in any of the methods described herein) can have a capacity, for example, between 1 mL and about 100% of the loading capacity of the first column of the first MCCS, including the upper and lower limits, 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 loading capacity of the first column of the MCCS or MCCS1, including the upper and lower limits.Any reservoir(s) (e.g., holding tank(s)) used to hold the eluate from MCC S1 (including the recombinant protein) before the eluate enters MCC S2 can have a volume of, for example, 1 mL to about 100% (including both ends), such as 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% (including both ends) of the loading volume of the first column of MCC S2.

[0220] Each reservoir(s) (e.g., holding tank(s)) can 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., holding tank(s)) holds only the recombinant protein for a period of, for example, a total time between about 5 minutes and less than about 6 hours, including the upper and lower limits, such as between about 5 minutes and about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes, including the upper and lower limits. The reservoir(s) (e.g., holding tank(s)) can be used to perform both the holding of the fluid containing the recombinant protein and refrigeration (e.g., at a temperature below 25°C, below 15°C, or below 10°C). The reservoir can have any shape, including a circular cylinder, an oval cylinder, or a substantially rectangular and sealed impermeable bag.

[0221] The unit operation of filtering a fluid containing a recombinant protein can be performed using an MCC (e.g., MCC, MCC1, and / or MCC2) that includes, for example, a chromatography column or a chromatography membrane that includes a filter or 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) can remove any precipitated material and / or cells (e.g., precipitated unfolded protein; precipitated unwanted host cell protein; precipitated lipid; bacteria; yeast cells; fungal cells; mycobacteria; and / or mammalian cells). Filters having a pore size of about 0.2 μm or less are known to effectively remove bacteria from a fluid containing a recombinant protein. As is known in the art, a chromatography column or a chromatography membrane that includes a molecular sieve resin can also be used within an MCC (e.g., MCC, MCC1, and / or MCC2) to perform the unit operation of filtering a fluid containing a recombinant protein.

[0222] Unit operations for adjusting the ion concentration and / or pH of a fluid containing a recombinant protein can be carried out using an MCC (e.g., MCC, MCC1, and / or MCC2) that includes a buffer adjustment reservoir (e.g., an in-line buffer adjustment reservoir) and that is utilized. This buffer adjustment reservoir adds a fresh buffer solution into the fluid containing the recombinant protein (e.g., between columns within an MCC, MCC1, and / or MCC2, or after the last column within the penultimate MCC (e.g., MCC1) until the fluid containing the recombinant protein is fed into the first column of the next MCC (e.g., MCC2)). As can be appreciated in the art, the in-line buffer adjustment reservoir can be of any size (e.g., greater than 100 mL) and can contain any buffer (e.g., a buffer having one or more of the following: a pH that increases or decreases compared to the fluid containing the recombinant protein, an ion (e.g., salt) concentration that increases or decreases compared to the fluid containing the recombinant protein, and / or an agent whose concentration increases or decreases by competing with the recombinant protein in binding to a resin present in at least one chromatographic column or at least one chromatographic membrane within an MCC (e.g., MCC, MCC1, and / or MCC2)).

[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 a virus present in a fluid containing the recombinant protein; capturing a recombinant protein, inactivating a virus present in a fluid containing the recombinant protein, and adjusting the ionic concentration and / or pH of a liquid containing the recombinant protein; purifying a recombinant protein and polishing the recombinant protein; purifying a recombinant protein, polishing the recombinant protein, and filtering a fluid containing the recombinant protein or removing precipitate and / or specific substances from a fluid containing the recombinant protein; and purifying a recombinant protein, polishing the recombinant protein, filtering a fluid containing the recombinant protein or removing precipitate and / or specific particulate matter from a fluid containing the recombinant protein, and adjusting the ionic concentration and / or pH of a liquid containing the recombinant protein.

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

[0225] The liquid culture medium can be supplied (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 and about 25.0 mL / min, between about 1.0 mL / min and 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 supplying it into the MCCS or MCCS1. Any of the typical means of filtering the liquid culture medium or fluid containing the recombinant protein described herein, or any filtering means known in the art, can be used to filter the liquid culture medium containing the recombinant protein before supplying it into the MCCS or MCCS1.

[0227] In the method described herein, capture of the recombinant protein from the liquid culture medium is performed using MCCs or MCCs1. As can be appreciated in the art, to achieve capture of the recombinant protein, 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 a resin 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 (wherein the capture antigen is specifically recognized by the recombinant antibody or antibody fragment). If the recombinant protein is an enzyme, the capture mechanism can be an antibody or antibody fragment that specifically binds to the enzyme to capture the recombinant enzyme, the substrate of the enzyme to capture the recombinant enzyme, the cofactor of 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 the recombinant protein are described herein, and additional resins that can be used to capture the recombinant protein 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), and Kaneka KanCap A (Osaka, Japan).

[0228] The typical non-limiting sizes and shapes of chromatography columns or membranes present within MCCs or MCC1 that can be used to capture recombinant proteins are described herein. The liquid culture medium supplied (loaded) into MCCs or MCC1 can contain, 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, from about 0.1 mg / mL to about 50 mg / mL, from about 0.1 mg / mL to about 40 mg / mL, from about 0.1 mg / mL to about 30 mg / mL, from about 0.1 mg / mL to about 20 mg / mL, from about 0.5 mg / mL to about 20 mg / mL, from about 0.1 mg / mL to 15 mg / mL, from about 0.5 mg / mL to about 15 mg / mL, from about 0.1 mg / mL to about 10 mg / mL, or from 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 unit operation of capture 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 membrane present within an MCCS or MCCS1, a continuous chromatography process of loading, washing, eluting, and regenerating the chromatography column or membrane present within the MCCS or MCCS1 must be performed. Any of the typical flow rates, buffer volumes, and / or lengths of time assigned to each of the continuous chromatography processes described herein can be used in one or more of these different continuous chromatography processes (e.g., one or more of the continuous chromatography processes of loading, washing, eluting, and regenerating the chromatography column or membrane present within the MCCS or MCCS1 used to capture the recombinant protein). Non-limiting flow rates, buffer volumes, and / or lengths of time assigned to each of the continuous chromatography processes that can be used to capture a chromatography column and / or membrane with an MCCS or MCCS1 (e.g., a PCCS or PCCS1) are provided below. In addition, typical buffers that can be used with an 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 unit operation of capture (e.g., any of the exemplary resins that can be used for capture described herein) can be loaded with a liquid culture medium containing a 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 unit operation of capture 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 containing a resin capable of performing a unit operation of capture in series, the time required to load the two chromatography columns continuously 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 chromatography membrane within an MCCS or MCCS1 comprising a resin capable of performing a unit operation of capture, the at least one chromatography column or chromatography membrane is washed with at least one washing buffer. As can be appreciated in the art, at least one (e.g., 2, 3, or 4) washing buffer means that all or most of the proteins that are not the recombinant protein are eluted from the at least one chromatography column or chromatography membrane while not disturbing the interaction between the recombinant protein and the resin.

[0232] The washing 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 washing buffer used (e.g., the total combined volume of the washing buffer when using more than 1 washing buffer) can be, for example, about 1X column volume (CV) to about 15X CV (e.g., about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 4X CV to about 11X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV). The total time of washing 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 chromatography membrane in an MCCS or MCCS1 containing a resin capable of performing a unit operation of capture, the recombinant protein is eluted from at least one chromatography column or chromatography membrane by passing an elution buffer through the at least one chromatography column or chromatography membrane in the MCCS or MCCS1 containing a resin capable of performing a unit operation of capture. The elution buffer can pass through at least one chromatography column or chromatography membrane containing a resin capable of performing a unit operation of capture 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 mg / 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 elution buffer used to elute the recombinant protein from each of at least one chromatography column or chromatography membrane containing a resin capable of performing a unit operation of purification can be, for example, about 1X column volume (CV) to about 15X CV (e.g., about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 4X CV to about 11X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV). The total elution 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 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 1.25 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 the recombinant protein.For example, the elution buffer can include salts at different concentrations (e.g., increasing salt concentration), different pHs (e.g., increasing or decreasing salt concentration), or molecules that compete with the recombinant protein for binding to a resin capable of performing a unit operation of capture. Examples of such elution buffers for each of the typical capture mechanisms described herein are well known in the art.

[0234] After eluting the recombinant protein from at least one chromatography column or chromatography membrane within an MCCS or MCCS1 that includes a resin capable of performing a unit operation of capture, before the next volume of liquid culture medium can be loaded onto the at least one chromatography column or chromatography membrane, the at least one chromatography column or chromatography membrane must be equilibrated with a regeneration buffer. The regeneration buffer can be passed through at least one chromatography column or chromatography membrane that includes a resin capable of performing a unit operation of capture 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 mg / 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 the regeneration buffer used to equilibrate at least one chromatography column or chromatography membrane that includes a resin capable of performing a unit operation of capture can be, for example, about 1X column volume (CV) to about 15X CV (e.g., about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 2X CV to about 5X CV, about 4X CV to about 11X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV).

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

[0236] MCCS or MCCCS1 can include a PCCS that includes four chromatography columns, where at least three of the four chromatography columns are (e.g., any of at least one chromatography column that includes a resin capable of performing a unit operation of capturing (e.g., any of those described herein)) using an MCCS to perform a unit operation of capturing a recombinant protein from a liquid culture medium. In these examples, the fourth column of the PCC can perform a unit operation of inactivating viruses in a fluid containing a recombinant protein (e.g., any of the exemplary columns described herein that can be used to achieve virus inactivation of a fluid containing a recombinant protein).

[0237] In some examples, a fluid containing a recombinant protein is continuously eluted from MCCs1 (e.g., PCCs1) and continuously supplied to MCCs2 (e.g., PCCs2). The percentage (%) of the recombinant protein recovered in the eluate from 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 supplied into MCCs2 (e.g., PCCs2) using various means (e.g., piping) known in the art. The eluate from MCCs1 (e.g., PCCs1) can be supplied into 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 mg / min, between about 0.5 mL / min and about 14 mL / min, between about 1.0 mL / min and about 25.0 mL / min, about 5.0 mL / min to about 15.0 mL / min, about 15 mL / min to about 25 mL / min, or between about 1.0 mL / min and about 15.0 mL / min).

[0238] Some of the methods described herein can further include the step of adjusting the ion concentration and / or pH of the eluate from MCCS1 (e.g., PCCS1) before the eluate is supplied into MCCS2 (e.g., PCCS2). As described herein, the ion concentration and / or pH of the eluate from MCCS1 (e.g., PCCS1) can be adjusted by adding a buffer to the eluate (e.g., by using an in-line buffer adjustment reservoir) before the eluate is supplied into MCCS2. This buffer can 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 before supplying the eluate from MCCS1 into MCCS2. As described herein, this step of holding or storing can be carried out using any of the reservoirs described herein (e.g., backup tank).

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

[0241] Polish and purify recombinant proteins MCCS, MCCS1, and / or MCCS2 can be used to perform unit operations for purifying and polishing recombinant proteins. For example, an operation of purifying and polishing a recombinant protein can be performed using MCCS2, and the eluate from MCCS2 is the protein drug substance. MCCS, MCCS1, and / or MCCS2 can include at least one (e.g., two, three, or four) chromatography column or chromatography membrane that can be used to perform unit operations for purifying recombinant proteins, and at least one (e.g., two, three, or four) chromatography column or chromatography membrane that can be used to perform unit operations for polishing recombinant proteins.

[0242] At least one chromatography column or chromatography membrane that can be used to perform unit operations for purifying recombinant proteins 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 chromatography column or chromatography membrane that can be used to perform unit operations for polishing recombinant proteins can include a resin (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) that can be used to perform anion exchange, cation exchange, molecular sieve chromatography, or hydrophobic interaction chromatography.

[0243] The size, shape, and volume of at least one chromatography column or chromatography membrane that can be used to perform unit operations for purifying a recombinant protein, and / or the size and shape of at least one chromatography membrane that can be used to perform unit operations for polishing a recombinant protein, can be any of the typical combinations of size, shape, and volume of the chromatography columns or chromatography membranes described herein. As would be recognized by one of ordinary skill in the art, the process of purifying or polishing a recombinant protein can include, for example, steps of loading, washing, eluting, and equilibrating at least one chromatography column or chromatography membrane used to perform unit operations for purifying or polishing the recombinant protein. Typically, the elution buffer emerging from the chromatography column or chromatography membrane used to perform unit operations for purification contains the recombinant protein. Typically, the loading and / or washing buffer emerging from the chromatography column or chromatography membrane used to perform unit operations for 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 for purifying a recombinant protein can have a volume of, for example, about 2.0 mL to about 200 mL (such as 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 the fluid containing the recombinant protein when loaded onto at least one chromatography column or at least one chromatography membrane that can be used to perform a unit operation for purifying a recombinant protein can be, for example, about 0.1 mL / min to about 25 mL / min (such as 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 0.1 mL / min to about 2 mL / min, or about 0.2 mL / min to about 4 mL / min). The concentration of the recombinant protein in the fluid loaded onto at least one chromatography column or chromatography membrane that can be used to perform a unit operation for purifying a recombinant protein can be, for example, about 0.05 mg / mL to about 100 mg / mL of recombinant protein (such as 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 resin within at least one chromatography column or chromatography membrane used to perform the unit operation of purification can be a resin that can be used to perform anion exchange or cation exchange chromatography. The resin within at least one chromatography column or chromatography membrane used to perform the unit operation of purification can be a cation exchange resin (e.g., Capto-S resin, GE Healthcare Life Sciences, Piscataway, NJ).

[0245] After loading the recombinant protein onto at least one chromatography column or chromatography membrane that can be used to perform the unit operation of purifying the recombinant protein, the at least one chromatography column or chromatography membrane is washed with at least one washing buffer. As can be appreciated in the art, the at least one (e.g., two, three, or four) washing buffers elute any protein that is not the recombinant protein from the at least one chromatography column or chromatography membrane, while not disturbing the interaction between the recombinant protein and the resin or otherwise eluting the recombinant protein.

[0246] The washing buffer can pass through at least one chromatography column or chromatography membrane at a flow rate of about 0.2 mL / min to about 25 mL / min (for example, 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 washing buffer used (for example, the total combined volume of the washing buffer when using more than one washing buffer) can be, for example, about 1X column volume (CV) to about 15X CV (for example, about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 4X CV to about 11X CV, about 2.5X CV to about 5.0X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV). The total washing time can be, for example, about 2 minutes to about 3 hours (for example, 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 at least one chromatography column or chromatography membrane used to perform a unit operation for purifying a recombinant protein, the recombinant protein is eluted from at least one chromatography column or chromatography membrane by passing an elution buffer through the at least one chromatography column or chromatography membrane used to perform a unit operation for purifying the recombinant protein. The elution buffer can be passed through at least one chromatography column or chromatography membrane used to perform a unit operation for purifying 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 mg / 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 elution buffer used to elute the recombinant protein from each of the at least one chromatography column or chromatography membrane that can be used to perform a unit operation for purifying the recombinant protein can be, for example, about 1X column volume (CV) to about 25X CV (e.g., about 1X CV to about 20X CV, about 15X CV to about 25X CV, about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 4X CV to about 11X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV). The total elution 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 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 1.25 hours, about 2 minutes to about 1 hour, between about 2 minutes and about 40 minutes, between about 10 minutes and about 40 minutes, 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 recombinant protein. For example, the elution buffer can include different concentrations of salts (e.g., increasing salt concentration), different pHs (e.g., increasing or decreasing salt concentration), or molecules that compete with the recombinant protein for binding to the resin. Examples of such elution buffers for each of the typical capture mechanisms described herein are well known in the art.

[0248] After eluting the recombinant protein from at least one chromatography column or chromatography membrane used to perform a unit operation for purifying the recombinant protein, and before the next volume of the fluid containing the recombinant protein can be loaded onto at least one chromatography column or chromatography membrane, the at least one chromatography column or chromatography membrane must be equilibrated with a regeneration buffer. The regeneration buffer can be passed through the at least one chromatography column or chromatography membrane containing a resin that can be used to perform a unit operation for purifying 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 mg / 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 the 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 for purifying the recombinant protein can be, for example, about 1X column volume (CV) to about 15X CV (e.g., about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 2X CV to about 5X CV, about 2.5X CV to about 7.5X CV, about 4X CV to about 11X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV).The concentration of the recombinant protein in the eluate of at least one chromatography column or chromatography membrane used to perform unit operations for purifying the recombinant protein can be, 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, from about 0.1 mg / mL to about 50 mg / mL, from about 0.1 mg / mL to about 40 mg / mL, from about 2.5 mg / mL to about 7.5 mg / mL, from about 0.1 mg / mL to about 30 mg / mL, from about 0.1 mg / mL to about 20 mg / mL, from about 0.5 mg / mL to 20 mg / mL, from about 0.1 mg / mL to about 15 mg / mL, from about 0.5 mg / mL to about 15 mg / mL, from about 0.1 mg / mL to about 10 mg / mL, or from about 0.5 mg / mL to about 10 mg / mL of recombinant protein).

[0249] Unit operations for polishing a recombinant protein can include at least one chromatography column or chromatography membrane that can contain a resin used to perform cation exchange, anion exchange, or size exclusion chromatography. As can be appreciated in the art, polishing a recombinant protein using at least one chromatography column or chromatography membrane that can be used to perform unit operations for polishing a recombinant protein can include, for example, steps of loading, chasing, and regenerating at least one chromatography column or chromatography membrane that can be used to perform unit operations for polishing a recombinant protein. For example, when performing polishing using the steps of loading, chasing, and regenerating, the recombinant protein does not bind to the resin in at least one chromatography column or chromatography membrane used to perform unit operations for polishing the recombinant protein, the recombinant protein is eluted from at least one chromatography column or chromatography membrane in the loading and chasing steps, and the regeneration step is used to remove any impurities from at least one chromatography column or chromatography membrane before additional fluid containing the recombinant protein can be loaded onto at least one chromatography column or chromatography membrane. Typical flow rates and buffer volumes used in each of the loading, chasing, and regeneration steps are described below.

[0250] The size, shape, and volume of at least one chromatography column or chromatography membrane that can be used to perform a unit operation for polishing a recombinant protein, and / or the size and shape of at least one chromatography membrane that can be used to perform a unit operation for polishing a recombinant protein, can be any of the typical combinations of size, shape, and volume of the chromatography columns or chromatography membranes described herein. For example, the size of at least one chromatography column or chromatography membrane that can be used to perform a unit operation for 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 a unit operation for polishing a 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 a recombinant protein loaded onto at least one chromatography column or chromatography membrane that can be used to perform a unit operation for polishing the recombinant protein can be, for example, from about 1.0 mL to about 250 mL (e.g., from about 1.0 mL to about 225 mL, from about 1.0 mL to about 200 mL, from about 1.0 mL to about 175 mL, from about 1.0 mL to about 150 mL, from about 100 mL to about 125 mL, from about 100 mL to about 150 mL, from about 1.0 mL to about 150 mL, from about 1.0 mL to about 125 mL, from about 1.0 mL to about 100 mL, from about 1.0 mL to about 75 mL, from about 1.0 mL to about 50 mL, or from about 1.0 mL to about 25 mL). The resin in at least one chromatography column or chromatography membrane used to perform the unit operation for polishing can be an anion exchange or cation exchange resin. The resin in at least one chromatography column or chromatography membrane used to perform the unit operation for polishing can be a cation exchange resin (e.g., Sartobind® Q resin, Sartorius, Goettingen, Germany).

[0251] After the loading step, a tracking step is performed (e.g., passing a tracking buffer through at least one chromatography column or chromatography membrane to collect recombinant proteins that do not substantially bind to the at least one chromatography column or chromatography membrane). In these examples, the tracking buffer can be passed through the at least one chromatography column or chromatography 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 the tracking buffer used can be, for example, about 1X column volume (CV) to about 100X CV (e.g., about 1X CV to about 90X CV, about 1X CV to about 80X CV, about 1X CV to about 70X CV, about 1X CV to about 60X CV, about 1X CV to about 50X CV, about 1X CV to about 40X CV, about 1X CV to about 30X CV, about 1X CV to about 20X CV, about 1X CV to about 15X CV, about 5X CV to about 20X CV, or about 5X CV to about 30X CV, about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 4X CV to about 11X CV, about 2.5X CV to about 5.0X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV). The total time of tracking can be, for example, about 1 minute to about 3 hours (e.g., about 1 minute to about 2.5 hours, about 1 minute to about 2.0 hours, about 1 minute to about 1.5 hours, about 2 minutes to about 1.5 hours, about 1 minute to about 1.25 hours, about 2 minutes to about 1.25 hours, about 1 minute to about 5 minutes, about 1 minute to about 10 minutes, about 2 minutes to about 4 minutes, about 30 minutes to about 1 hour, about 2 minutes to about 10 minutes, about 2 minutes to about 15 minutes, or about 2 minutes to about 30 minutes).The combined concentration of the recombinant protein present in the eluate coming through the column in the loading step and the tracking step 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, between about 2.5 mg / mL and about 7.5 mg / mL, from about 0.1 mg / mL to about 30 mg / mL, from about 0.1 mg / mL to about 20 mg / mL, from about 0.5 mg / mL to 20 mg / mL, from about 0.1 mg / mL to about 15 mg / mL, from about 0.5 mg / mL to about 15 mg / mL, from about 0.1 mg / mL to about 10 mg / mL, from about 0.5 mg / mL to about 10 mg / mL, or from about 1 mg / mL to about 5 mg / mL of recombinant protein).

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

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

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

[0255] Some embodiments of this example can further include a step of adjusting the ionic concentration and / or pH of the eluate from the three chromatography columns in the PCCS before supplying 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 can be adjusted (in this example, before supplying this eluate into the chromatography membrane in the PCCS) (e.g., by using an in-line buffer adjustment reservoir) by adding 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 eluate from the three chromatography columns in the PCCS before feeding the eluate into a chromatography membrane (a chromatography membrane that can be used to perform a unit operation for polishing the recombinant protein). As described herein, this step of holding or storing can be carried out using any of the reservoirs described herein (e.g., a backup tank).

[0257] These examples can also include a step of filtering the eluate from the chromatography membrane (the eluate of a chromatography membrane that can be used to perform a unit operation for polishing the recombinant protein) in a typical PCCS system. The eluate from the chromatography membrane in this typical PCCS can be filtered using any of the typical filters or methods for filtration described herein (the eluate of a chromatography membrane that can be used to perform a unit operation for polishing the recombinant protein).

[0258] As can be appreciated in the art, the purified recombinant protein can be periodically eluted from the MCCs or MCCs2 using any of the methods described herein. For example, any of the methods described herein can elute the purified recombinant protein, depending on, for example, the chromatography columns and / or chromatography membranes used in the MCCs or MCCs1 and MCCs2, 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), at a frequency of, for example, 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).

[0259] Method of culturing Some of the methods described herein further include culturing cells (e.g., recombinant mammalian cells) that secrete a 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 cells (e.g., mammalian cells) is continuously or periodically removed from the bioreactor (e.g., a perfusion bioreactor) and supplied 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, about 1 L to 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 the 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 the cells can be carried out, for example, using a fed-batch bioreactor or a perfusion bioreactor. Non-limiting examples and various aspects of the culturing of cells (e.g., culturing of mammalian cells) are described below and can be used in any combination.

[0260] cells Cells cultured by 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. 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 Madin-Darby canine (Cocker Spaniel) kidney epithelial (MDCK) cells. In some examples of culturing adherent cells, the culture can also include a plurality of microcarriers (e.g., microcarriers that contain one or more pores). Additional mammalian cells that can be cultured by any of the methods described herein are known in the art.

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

[0262] Nucleic acids encoding recombinant proteins can be introduced into mammalian cells using a variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (e.g., lipofection), transduction (e.g., lentivirus, adenovirus, or retrovirus infection), and electroporation. In some examples, 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 within a plasmid and / or within 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 lentivirus, retrovirus, or adenovirus 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 contain a selectable marker (e.g., a gene conferring hygromycin, puromycin, or neomycin resistance to mammalian cells).

[0263] In some instances, the recombinant protein is a secreted protein and is released by mammalian cells 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 encoding a secretion signal peptide at the N- or C-terminus of the recombinant protein, which is cleaved by an enzyme present within 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 medium (e.g., the first and / or second tissue culture medium) 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 medium (e.g., the first and / or second liquid culture medium) can be a liquid culture medium of known composition, a liquid culture medium free of animal-derived components, a serum-free liquid culture medium, or a serum-containing liquid culture medium. Non-limiting examples of liquid culture media of known composition, liquid culture media free of animal-derived components, serum-free liquid culture media, and serum-containing liquid culture media are commercially available.

[0265] Liquid culture media typically contain an energy source (e.g., a carbohydrate such as glucose), essential amino acids (e.g., the basic set of 20 amino acids + cysteine), vitamins and / or other organic compounds required at low concentrations, free fatty acids, and / or trace elements. The liquid culture medium (e.g., the first and / or second liquid culture medium) can be supplemented, if desired, with, for example, 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), proteins and tissue hydrolysates, and / or any combination of these additives.

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

[0267] As will be appreciated by those skilled in the art, the first liquid culture medium and the second liquid culture medium 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 can have at least one coating (e.g., at least one coating of gelatin, collagen, poly-L-ornithine, polystyrene, and laminin), as well as, as known in the art, 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. The bioreactor can incubate the cell culture in a controlled humidified atmosphere (e.g., at a humidity higher than 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or at 100% humidity). The bioreactor can also have a mechanical device for removing a volume of the liquid culture medium from the bioreactor, and optionally, within that mechanical device, a filter for removing cells from the liquid culture medium during the transfer process of the liquid culture medium from the bioreactor (e.g., the ATF system or cell filtration system described in U.S. Provisional Patent Application No. 61 / 878,502).

[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 recognized by those skilled in the art, the temperature can be varied at specific times during the culture process, for example, on a hourly or daily basis. For example, the temperature can change or shift (e.g., increase or decrease) 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 initially seeding the cells (e.g., mammalian cells) into the bioreactor. For example, the temperature can shift upward (e.g., by a maximum 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 a maximum or about 20°C change). For example, the temperature can shift downward (e.g., by a maximum 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 a maximum or about 20°C change).

[0270] CO2 The culture process described herein can further include exposing the liquid culture medium within the bioreactor to an atmosphere containing up to or 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 in this specification can be carried out using a perfusion bioreactor. Culturing of cells (e.g., mammalian cells) in a perfusion bioreactor involves removal of a first volume of a first liquid culture medium (e.g., a first volume of a first liquid culture medium containing mammalian cells at any concentration, e.g., substantially cell-free) from the bioreactor, and addition of 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 in a combination of these two. Further, the removal and addition can be carried out continuously, 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 in any combination of these, at a rate of removing and replacing (e.g., 0.1% to 800% of the volume of the bioreactor or the volume of the first liquid culture medium, 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 80%, or between 4% and 30%) a volume over a given time period (e.g., over a 24-hour period, over an increasing time period of about 1 hour to about 24 hours, or over an increasing time period exceeding 24 hours). When carried out periodically, the volume removed or replaced (e.g., within a period of about 24 hours, within an increasing time period of about 1 hour to about 24 hours, or within an increasing time period exceeding 24 hours) can be, for example, 0.1% to 800% of the volume of the bioreactor or the volume of the first liquid culture medium (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%).The first volume of the first liquid culture medium to be removed and the second volume of the second liquid culture medium to be added can, in some examples, be kept approximately the same over the entire or a portion of the culture period, for each 24-hour period (or alternatively, an increasing time period from about 1 hour to about 24 hours or an increasing time period greater than 24 hours). As is known in the art, the rate (volume / unit time) at which the first volume of the first liquid culture medium is removed and the rate (volume / unit time) at which the second volume of the second liquid culture medium is added can be varied. The rate (volume / unit time) at which the first volume of the first liquid culture medium is removed and the rate (volume / unit time) at which the second volume of the second liquid culture medium is added can be approximately the same or can be different.

[0272] Alternatively, the volumes removed and added can vary (e.g., increase gradually) over each 24-hour period (or alternatively, an increasing time period from 1 hour to about 24 hours or an increasing time period 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 an increasing time period from about 1 hour to greater than 24 hours or an increasing time period greater than 24 hours) during the culture period can increase 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 (e.g., gradually or in alternating increasing amounts).

[0273] As will be appreciated by those skilled in the art, the first liquid culture medium and the second liquid culture medium can be of the same type of medium. In other examples, the first liquid culture medium and the second liquid 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 first volume of the first liquid culture medium substantially free of cells from the bioreactor). Alternatively, or in addition, the first volume of the first liquid culture medium can be removed by bleeding or gravity flow of the first volume of the first liquid culture medium through a sterile membrane having a molecular weight cut-off that excludes cells (e.g., mammalian cells).

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

[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 substantially free of mammalian cells) and the addition of the second volume of the second liquid culture medium to the first liquid culture medium do 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 after 96 hours) after seeding mammalian cells in the bioreactor.

[0277] Fed-batch bioreactor The culturing process described in this specification can be carried out using a fed-batch bioreactor. Culturing of cells in a fed-batch bioreactor involves the addition (e.g., periodic or continuous addition) of a second volume of a second liquid culture medium to a first liquid culture medium over most of the culturing period. The addition of the second liquid culture medium can be carried out continuously (e.g., at a rate of adding a volume of 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 bioreactor volume or the first liquid culture medium volume over a given time period (e.g., over a 24-hour period, over an increasing time period from about 1 hour to about 24 hours, or over an increasing time period exceeding 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 in any combination thereof. When carried out periodically, the volume added (e.g., within a period of about 24 hours, within an increasing time period from about 1 hour to about 24 hours, or within an increasing time period exceeding 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 bioreactor volume or the first liquid culture medium volume. The second volume of the second liquid culture medium added can, in some examples, be kept substantially the same over each 24-hour period (or, alternatively, an increasing time period from about 1 hour to about 24 hours or an increasing time period exceeding 24 hours) during all or part of the culturing period. As is known in the art, the rate (volume / unit time) at which the second volume of the second liquid culture medium is added can be varied during all or part of the culturing period. For example, the volume of the second liquid culture medium added can vary (e.g., increase gradually) over each 24-hour period (or, alternatively, an increasing time period from 1 hour to about 24 hours or an increasing time period exceeding 24 hours) during the culturing period.For example, during the cultivation period, the volume of the second liquid culture medium added within each 24-hour period (or alternatively, an increasing time period of about 1 hour to more than 24 hours or an increasing time period of more than 24 hours) can increase from a volume that is 0.5% to about 20% of the bioreactor volume or the volume of the first liquid culture medium to about 25% to about 150% of the bioreactor volume or the volume of the first liquid culture medium (e.g., by gradual or alternating increases) during the entire cultivation period. The rate (volume / unit time) at which the second volume of the second liquid culture medium is added can be substantially the same throughout the whole or a part of the cultivation period.

[0278] As will be appreciated by those skilled in the art, the first liquid culture medium and the second liquid culture medium can be of 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 automatically added to the first liquid culture medium, for example, by an infusion pump.

[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 after 96 hours) from the seeding of mammalian cells into the bioreactor. The cell culture medium in fed-batch culture is typically harvested at the end of the cultivation period and used in any of the methods described herein. However, the cell culture medium in fed-batch culture can also be harvested at one or more time points during the cultivation period and can also be used in any of the methods described herein.

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

[0281] Exemplary biological manufacturing 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 Applications Nos. 61 / 775,060 and 61 / 856,390 (incorporated by reference). In these exemplary systems, at least one (e.g., at least 2, 3, 4, 5, or 6) chromatography columns filled with a reduced bioburden provided herein are present within the MCCS or within 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 chromatography columns filled with a reduced bioburden provided herein. For example, the MCCS, MCCS1, and / or MCCS2 can each include (or can each include) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 chromatography columns filled with a reduced bioburden provided herein.

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

[0283] Some representative systems do not include holding tanks. In other examples, the system can include up to 1, 2, 3, 4, or 5 holding tanks in total (e.g., where each holding tank only holds the therapeutic protein for the entire period of, for example, from about 5 minutes to about 6 hours (including both ends)). The holding tank(s) can have a volume of from 1 mL to about 300 mL (including both ends). Any holding tank(s) placed in the system such that the fluid enters the holding tank(s) before entering MCCS1 or the MCCS can each have a volume that is from 1 mL to about 100% (including both ends) of the loaded volume of the first column of MCCS1 or the MCCS. Any holding tank(s) placed in the system such that the fluid enters the holding tank(s) before entering MCCS2 (and after exiting MCCS1) can each have a volume that is, for example, from 1 mL to about 100% (including both ends) of the loaded volume of the first column of MCCS2.

[0284] Structure and characteristics of additional representative systems MCCS or MCCS1 can include an inlet through which a fluid (e.g., a substantially cell-free liquid culture medium) can pass to enter the MCCS or MCCS1, respectively. The inlet can be of any structure known in the art for such purposes. The fluid tube can be inserted such that the fluid passes through the inlet and enters the MCCS or MCCS1 without significant leakage of the fluid from the inlet after insertion of the fluid tube into the inlet, and can include, for example, threads, ribs, or seals. Non-limiting inlets that can be used in the present system are known and will be understood by those skilled in the art.

[0285] MCCS or MCCS1 can have at least two chromatography columns, at least two chromatography membranes, or at least one chromatography column and at least one chromatography membrane, as well as an inlet. MCCS or MCCS1 can be any of the representative MCCS described herein, or can have any one or more of the representative features (in any combination) of the MCCS described herein. The chromatography column(s) and / or chromatography membrane(s) present within 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 chromatography column(s) and / or chromatography membrane(s) present within the MCCs or MCCs1 can comprise any one or more of representative resins described herein or known in the art. For example, the resin(s) included in one or more of the chromatography column(s) and / or chromatography membrane(s) present within the MCCs or MCCs1 can be a resin that utilizes 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 resin(s) included in one or more of the chromatography column(s) and / or chromatography membrane(s) of the MCCs or MCCs1 can be a cation exchange resin, an anion exchange resin, a molecular sieve resin, or a hydrophobic interaction resin, or any combination thereof. Further examples of resins that can be used to purify recombinant proteins are known in the art and can be included in one or more chromatography columns and / or chromatography membranes present within the MCCs or MCCs1. The chromatography column(s) and / or chromatography membrane(s) present within the MCCs or MCCs1 can comprise the same and / or different resins (e.g., any of the resins described herein or known in the art to be used in the purification of recombinant proteins).

[0287] Two or more chromatography columns and / or chromatography resins present within an MCCS or MCCS1 can perform one or more unit operations (e.g., capture of a recombinant protein, purification of a recombinant protein, polishing of a recombinant protein, inactivation of a virus, adjustment of the ionic concentration and / or pH of a fluid containing a recombinant protein, or filtration of a fluid containing a recombinant protein). In non-limiting examples, an MCCS or MCCS1 can perform unit operations of capturing a recombinant protein from a fluid (e.g., a liquid culture medium) and inactivating a virus present in a fluid containing the recombinant protein. An MCCS or MCCS1 can perform any combination of two or more unit operations described herein or known in the art.

[0288] Chromatography column(s) and / or chromatography membrane(s) present within an MCCS or MCCS1 can be connected to or moved relative to each other by a switching mechanism (e.g., a column switching mechanism). An MCCS or MCCS1 can also include one or more (e.g., 2, 3, 4, or 5) pumps (e.g., automated, e.g., an automated peristaltic pump). A column switching event can be initiated by detection of a specific level of UV absorbance corresponding to a recombinant protein in a fluid passing through the MCCS or MCCS1 (e.g., an input to and / or an eluate from one or more chromatography columns and / or chromatography membranes within the MCCS or MCCS1), a specific volume of liquid (e.g., a buffer), or a certain level of recombinant protein detected over a specific elapsed time. Column switching generally refers to a mechanism that allows at least two different chromatography columns and / or chromatography membranes within an MCCS or MCCS1 (e.g., two or more different chromatography columns and / or chromatography membranes present within an 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] MCCS or MCCSl can be a periodic countercurrent chromatography system (PCCS). For example, a PCCS that is MCCS or MCCSl (i.e., PCCS or PCCSl, respectively) can include four chromatography columns, where the first three columns perform the unit operation of capturing recombinant protein from a fluid (e.g., a liquid culture medium), and the fourth column of the PCCS performs the unit operation of inactivating viruses in the fluid containing the recombinant protein. A PCCS that is MCCS or MCCSl can utilize a column switching mechanism. The PCC system can utilize an improved AKTA system (GE Healthcare, Piscataway, NJ) that can operate, for example, 4, 5, 6, 7, or 8 columns, or even more columns.

[0290] MCCS or MCCSl can be equipped with one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) UV monitoring devices, 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. MCCS or MCCSl can also be equipped with an operating system that utilizes software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ) to detect when column switching should occur (e.g., based on UV absorbance, liquid volume, or elapsed time) and to affect (initiate) the column switching event.

[0291] MCCS or MCCS1 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 regulation reservoirs and / or buffer reservoirs. In other examples, MCCS or MCCS1 can include one or more (e.g., 2, 3, 4, 5, or 6) hold-up tanks that can hold fluids that do not readily pass through one or more chromatography columns and / or chromatography membranes within MCCS or MCCS1. The systems described herein can include one or more hold-up tanks (e.g., the hold-up tanks described herein) within MCCS, MCCS1, and / or MCCS2. Other examples of the systems described herein do not include a hold-up tank within MCCS, MCCS1, or MCCS2, or do not include a hold-up tank throughout the system. Other examples of the systems described herein include up to 1, 2, 3, 4, or 5 hold-up tanks (e.g., any of the hold-up tanks (plural) described herein) throughout the system.

[0292] Second MCCS The second MCCS (MCCS2) in a representative system includes at least two chromatography columns, at least two chromatography membranes, or at least one chromatography column(s) and at least one chromatography membrane(s), as well as an outlet. MCCS2 can be any of the representative MCCS described herein, or can have any one or more of the representative features of the MCCS described herein (in any combination). The chromatography column(s) and / or chromatography membrane(s) present within MCCS2 can have any one or more of the shapes, sizes, volumes (bed volumes), and / or unit operations described herein. The chromatography column(s) and / or chromatography membrane(s) can include any of the representative resins described herein or known in the art. For example, the resin included in one or more chromatography columns and / or chromatography membranes present within MCCS2 can be a resin that utilizes 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, tag binding capture mechanism, and / or 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 membrane(s) present within MCCS2 can include the same and / or different resins (e.g., any of the resins described herein or known to be used in the purification of recombinant proteins in the art).

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

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

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

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

[0297] 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 adjustment reservoirs and / or buffer reservoirs. In other examples, 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 through one or more chromatography columns and / or chromatography membranes within MCCS2.

[0298] MCCS2 includes an outlet through which a therapeutic protein drug substance can exit the system. The outlet can include, for example, a screw, rib, or seal into which a fluid tube can be inserted, or a vial designed to hold or store a purified recombinant protein (e.g., a therapeutic protein drug substance). The outlet can include a surface that can be used to seal a vial or other such storage container with a reduced bioburden to the outlet such that a purified recombinant protein (e.g., a therapeutic protein drug substance) can flow directly into the vial or storage container with a reduced bioburden. 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 can also include a fluid tube disposed between MCCS1 and MCCS2. Any of the fluid tubes described herein can be, for example, a tube made of polyethylene, polycarbonate, or plastic. The fluid tube disposed between MCCS1 and MCCS2 can further include, in any combination, one or more of the following: one or more in-line buffer adjustment reservoirs that are in fluid communication with the fluid tube and are positioned such that buffer stored within the in-line buffer adjustment reservoir(s) is added to the fluid present within the fluid tube; a holding tank (e.g., any of the holding tanks (plural) described herein) that is in fluid communication with the fluid tube and is positioned such that it can hold any excess fluid present within the fluid tube that cannot be easily supplied into MCCS2; and one or more filters disposed within the fluid tube such that the fluid present within the fluid tube can be filtered (e.g., to remove bacteria). Any of the in-line buffer adjustment reservoirs can contain, for example, a buffer having a volume of about 0.5 L to 50 L (e.g., at a temperature of 25 °C, 15 °C, or 10 °C or less).

[0300] The system described herein can optionally include a fluid tube disposed between the final chromatography column or chromatography membrane within MCCS2 and the outlet. The system described herein can further include one or more filters that are in fluid connection with the fluid tube disposed between the final chromatography column or chromatography membrane within MCCS2 and the outlet, such that the filter can remove, for example, precipitated material, particulate matter, or bacteria from the fluid present within the fluid tube disposed between the final chromatography column or chromatography membrane within MCCS2 and the 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 representative bioreactor described herein or known in the art can be used in this system.

[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 pump described herein or known in the art), one or more (e.g., 2, 3, 4, or 5) filters (e.g., any filter 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) holding tanks (e.g., any holding tank described herein). Some examples of the systems provided herein further include a fluid pipe (e.g., any representative fluid pipe 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 pipe can include one or more (e.g., 2, 3, or 4) pumps (e.g., any pump described herein or known in the art) and / or one or more (e.g., 2, 3, or 4) holding tanks (e.g., any representative holding tank described herein), where these pumps (s) and / or holding tanks (s) are in fluid connection with the fluid present in the fluid pipe.

[0303] Some examples of the systems described herein further include an additional fluid pipe connected to the fluid pipe between the pump and the inlet, with one end of this additional fluid pipe in fluid connection with the bioreactor and the other end in fluid connection with the fluid pipe between the pump and the inlet. This additional fluid pipe can include a filter (e.g., an ATF cell retention system) capable of removing cells from the liquid culture medium removed from the bioreactor.

[0304] The systems provided herein enable the continuous production of purified recombinant proteins (e.g., therapeutic protein drug substances). As is known in the art, the systems may enable the periodic elution of purified recombinant proteins (e.g., therapeutic protein drug substances). The systems described herein can also provide purified recombinant proteins (e.g., therapeutic protein drug substances) at 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 over 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 the bioburden of a chromatography resin, including the use of a substantially dry chromatography resin Also provided herein is a method for reducing the bioburden of a chromatography resin, the method comprising (a) exposing a container comprising a substantially dry chromatography resin to a dose of gamma irradiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein the substantially dry chromatography resin comprises a liquid comprising at least one alcohol, and the at least one alcohol is present in an amount sufficient to improve the loss of binding capacity of the chromatography resin after exposure to that dose of gamma irradiation. Some embodiments of this method further comprise drying the chromatography resin prior to step (a) to substantially remove liquid (but not all liquid) from the chromatography resin. Drying of the chromatography resin can be accomplished using heat treatment (e.g., an oven) or a desiccator. Additional methods for drying chromatography resins are known in the art.

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

[0307] In some examples, the produced chromatography resin with a reduced bioburden is about 1×10 -8 to about 1×10 -5 sterility assurance level (SAL) (e.g., about 1×10 -7 to about 1×10 -6It has a reduced bioburden (SAL). The resulting reduced chromatography resin can include 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. In some examples, the resulting reduced chromatography resin includes an affinity chromatography resin containing a protein ligand (e.g., Protein A). In some examples, the resulting reduced chromatography resin includes an anion exchange chromatography resin (e.g., an anion exchange chromatography resin containing an N-benzyl-N-methyl-ethanolamine group). Also provided is a method of making a chromatography column filled with a reduced bioburden, the method including the step of preparing a chromatography resin of a reduced bioburden produced by any of the methods described herein, and the step of packing the chromatography resin into a column of the reduced bioburden in a sterile environment. Also provided is a chromatography column filled with a reduced bioburden produced by any of the methods described herein.

[0308] Also provided is an integrated and closed continuous method for the production of a reduced bioburden of a purified recombinant protein, the method comprising: (a) preparing a liquid culture medium containing a recombinant protein substantially free of cells; (b) continuously supplying the liquid culture medium to a multi-column chromatography system (MCCS) comprising at least one chromatography column filled with a reduced bioburden produced by any of the methods provided herein, the method utilizing a buffer of reduced bioburden, being integrated, and operating continuously from the liquid culture medium to the eluate from the MCCS, which is a purified recombinant protein. Also provided is an integrated and closed continuous method for the production of a reduced bioburden of a purified recombinant protein, the method comprising: (a) preparing a liquid culture medium containing a recombinant protein substantially free of cells; (b) continuously supplying 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 containing the recombinant protein from MCCS1 and continuously supplying the eluate into a second multi-column chromatography system (MCCS2); (e) continuously supplying the recombinant protein from the eluate into MCCS2 and then eluting the recombinant protein to generate a purified recombinant protein, the method utilizing a buffer of reduced bioburden, being integrated, and operating continuously from the liquid culture medium to the purified recombinant protein, and at least one column within MCCS1 and / or MCCS2 containing a chromatography column filled with a reduced bioburden produced by any of the methods provided herein. Also provided are any of the representative aspects of the integrated and closed continuous method for the production of a reduced bioburden of a purified recombinant protein described herein that can be used in these methods.

[0309] Method for generating membranes, resins, coatings, chips, and cassettes with reduced bioburden A method of producing a membrane, resin, coating, chip, or cassette with reduced bioburden, comprising exposing a container containing a composition comprising: (i) a membrane, resin, coating, chip, or cassette (e.g., a membrane, resin, coating, chip, or cassette based on cellulose, agarose, or saccharides), 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 irradiation 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 improve damage to the membrane, resin, coating, chip, and cassette after exposure to that dose of gamma irradiation. In some examples, the cassette is a cassette containing a resin (e.g., any of the exemplary 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 liquid comprising a membrane, resin, coating, chip, or cassette and at least an alcohol (e.g., and optionally at least one antioxidant and / or at least one chelating agent). Any of the exemplary combinations and concentrations of alcohols, antioxidants, and / or chelating agents described herein can be used in any of these methods. Any of the exemplary liquids described herein can be used in any of these methods. In some embodiments, the composition is a moist or humid dry material. Also provided herein is a membrane, resin, coating, chip, or cassette with reduced bioburden 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 for producing a reduced bioburden membrane, resin, coating, chip, and cassette, the method comprising exposing a container containing a substantially dry membrane, resin, coating, chip, or cassette (e.g., a membrane, resin, coating, chip, or cassette based on cellulose, agarose, or saccharides) to a gamma radiation dose 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 comprises 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 exemplary 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.

Example

[0311] The present invention is further illustrated by the following examples which do not limit the scope of the invention as claimed.

Example

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

[0313] After irradiation, the chromatographic resin was packed into separate chromatography columns and cycled using cell culture harvest and a 6-minute residence time. The breakthrough binding capacity was recorded for the irradiated resin and compared to naïve (unirradiated) MabSelect™ SuRe™ (protein A chromatography) resin.

[0314] The data from this experiment are shown in Figure 1.

[0315] The data in Figure 1 show that buffer (3) provides an additive effect of both buffers (1) and (2) and maintains the binding capacity. In this example, 2% v / v benzyl alcohol in the buffer acts as a preservative and also provides some protective properties when combined with 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 the expected values for naïve (unirradiated) MabSelect™ SuRe™ (protein A chromatography resin).

[0317]

Table 1

[0318] This data shows that irradiation of chromatographic resin in a liquid containing alcohol (e.g., benzyl alcohol) protects the resin from subsequent loss of binding capacity over multiple cycles of chromatography.

Example

[0319] Protective Effect of Alcohol on Gamma-Irradiated Capto™ Adhere Chromatography Resin Gamma irradiation of GE Capto™ adhere (a multimodal functional anion-exchange chromatography resin) was performed at doses of 28 - 49 kGy at standard irradiation 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 / hour); (B) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol in 50 mM sodium phosphate buffer (> 7.5 kGy / hour at 40 - 49 kGy); and (C) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, 2% v / v benzyl alcohol (> 7.5 kGy / hour at 40 - 49 kGy and higher dose rates).

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

[0321] The data from this experiment are shown in Figure 2. The data in Figure 2 show that no perceptible change in binding capacity was observed when the resin was irradiated in the presence of benzyl alcohol, indicating 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 have a perceptible effect on other quality attributes of the resin's performance in protein purification.

[0323]

Table 2

[0324] Other embodiments Although the present invention has been described with its detailed description, it should be understood that the above description is intended to be illustrative and not to limit the scope of the present invention as defined by the appended claims. Other aspects, advantages, and modifications fall within the scope of the following claims.

Claims

1. A method for reducing the bioburden of a chromatographic resin, comprising: (i) a chromatographic resin selected from an anion exchange chromatographic resin, a cation exchange chromatographic resin, or an affinity chromatographic resin for isolating a recombinant protein; and (ii) a liquid comprising benzyl alcohol and at least two antioxidants selected from the group consisting of mannitol, sodium ascorbate, histidine, and methionine, exposing a container containing the composition to a dose of gamma irradiation sufficient to reduce the bioburden of the container and the chromatographic resin, wherein the benzyl alcohol and the at least two antioxidants are present in an amount sufficient to improve the loss of binding capacity of the chromatographic resin after exposure to the dose of gamma irradiation.

2. The method according to claim 1, wherein the container is a storage vessel.

3. The method according to claim 1, wherein the container is a packed chromatographic column.

4. The method according to claim 1, wherein the composition is a slurry of deposited chromatographic resin.

5. The method according to any one of claims 1 to 4, wherein the total concentration of benzyl alcohol in the liquid is 0.01% v / v to 10% v / v.

6. The method according to any one of claims 1 to 5, wherein the liquid further comprises at least one chelating agent in an amount sufficient to further improve the loss of binding capacity of the chromatographic resin after exposure to the dose of gamma irradiation.

7. The method according to claim 1, wherein the liquid comprises mannitol, sodium ascorbate, histidine, and methionine.

8. The liquid comprises: (i) 30 mM to 70 mM of methionine and 30 mM to 70 mM of histidine; (ii) 10 mM to 50 mM of methionine, 10 mM to 50 mM of histidine, and 10 mM to 50 mM of sodium ascorbate; or (iii) 5 mM to 45 mM of sodium ascorbate, 5 mM to 45 mM of methionine, 5 mM to 45 mM of mannitol, and 5 mM to 45 mM of histidine.

9. The method according to claim 6, wherein the liquid contains at least two chelating agents selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium 2,3-dimercapto-1-propanesulfonate (DMSP), dimercaptosuccinic acid (DMSA), metallothionein, and desferrioxamine.

10. The method according to claim 1, wherein the composition contains an affinity chromatography resin containing a protein ligand.

11. The method according to claim 10, wherein the protein ligand is Protein A.

12. The method according to claim 1, wherein the composition contains an anion exchange chromatography resin.

13. The method according to any one of claims 1 to 12, wherein the dose is 15 kGy to 45 kGy.

14. A chromatography resin with a reduced bioburden produced by the method according to claim 2.

15. A method for producing a chromatography column filled with a chromatography resin with a reduced bioburden, comprising: a step of preparing the chromatography resin with a reduced bioburden according to claim 14, and a step of packing the chromatography resin into a column with a reduced bioburden in a sterilized environment. The above method.

16. A chromatography column filled with a chromatography resin with a reduced bioburden produced by the method according to claim 3 or 15.

17. A composition comprising (i) an anion exchange chromatography resin, a cation exchange chromatography resin, or an affinity chromatography resin, which is a chromatography resin for isolating a recombinant protein, and (ii) benzyl alcohol and a liquid containing at least two antioxidants selected from the group consisting of mannitol, sodium ascorbate, histidine, and methionine, wherein benzyl alcohol and at least two antioxidants are present in an amount sufficient to improve the loss of binding capacity of the chromatography resin during treatment by gamma-ray irradiation at a dose sufficient to reduce the bioburden of the composition.

18. A method for performing column chromatography with a reduced bioburden, comprising: (a) a step of preparing a chromatography column filled with a chromatography resin with a reduced bioburden according to claim 16, and Step of performing column chromatography using a chromatography column filled with a reduced bioburden and a buffer of the reduced bioburden in a closed system Said method comprising the same. **Claim 19** An integrated, closed, continuous method for the production of a reduced bioburden of a purified recombinant protein, comprising: (a) preparing a liquid culture medium containing at least 90% cell-free recombinant protein; and (b) continuously supplying the liquid culture medium to a multi-column chromatography system (MCCS) comprising at least one chromatography column filled with the reduced bioburden as described in claim 16 The method utilizes a buffer of the reduced bioburden, is integrated, and operates continuously from the liquid culture medium to the eluate from the MCCCS, which is a purified recombinant protein. **Claim 20** An integrated, closed, continuous method for the production of a reduced bioburden of a purified recombinant protein, comprising: (a) preparing a liquid culture medium containing at least 90% cell-free recombinant protein; (b) continuously supplying the liquid culture medium into a first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein in the liquid culture medium using MCCCS1; (d) generating an eluate containing the recombinant protein from MCCCS1 and continuously supplying the eluate into a second multi-column chromatography system (MCCS2); (e) continuously supplying the recombinant protein from the eluate into MCCCS2 and then eluting the recombinant protein to generate a purified recombinant protein The method utilizes a buffer of the reduced bioburden, is integrated, operates continuously from the liquid culture medium to the purified recombinant protein, and at least one column within MCCCS1 and / or MCCCS2 contains a chromatography column filled with the reduced bioburden as described in claim 16. **Claim 21** A method for reducing the bioburden of a chromatography resin, comprising: A composition comprising (i) an anion exchange chromatography resin, a cation exchange chromatography resin, or an affinity chromatography resin, which is a chromatography resin for isolating a recombinant protein, and (ii) a liquid containing benzyl alcohol, mannitol, sodium ascorbate, histidine, and methionine, in a container, including the step of exposing the container and the chromatography resin to a sufficient dose of gamma-ray irradiation to reduce the bioburden of the chromatography resin, wherein benzyl alcohol, mannitol, sodium ascorbate, histidine, and methionine are present in an amount sufficient to improve the loss of binding ability of the chromatography resin after exposure to a dose of gamma-ray irradiation, said method.

22. A composition comprising (i) an anion exchange chromatography resin, a cation exchange chromatography resin, or an affinity chromatography resin, which is a chromatography resin for isolating a recombinant protein, and (ii) a liquid containing benzyl alcohol, mannitol, sodium ascorbate, histidine, and methionine, wherein benzyl alcohol, mannitol, sodium ascorbate, histidine, and methionine are present in an amount sufficient to improve the loss of binding ability of the chromatography resin during treatment with a sufficient dose of gamma-ray irradiation to reduce the bioburden of the composition, said composition.

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