Uses of sterile chromatography resins and their manufacturing methods

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 726,043, filed on 31 August 2018; the entire contents of that application are incorporated herein by reference.

[0002] This invention relates to biotechnology and biomanufacturing methods for recombinant proteins. [Background technology]

[0003] Mammalian cells containing nucleic acids encoding recombinant proteins are often used to produce proteins of critical importance for therapeutic or commercial purposes. In today's diverse product pipeline environment, biotechnology companies are increasingly driven to develop innovative solutions for the highly flexible and cost-effective production of therapeutic protein drug substances. One approach to efficiently isolating recombinant proteins involves processes encompassing continuous chromatography (e.g., using closed systems). One known limitation of continuous chromatography is the presence of contaminating agents in the system (e.g., increased bioburden), which can lead to contaminating products, reduced production yields, and decreased flow rates (or increased pressures) within the system. For example, increased bioburden in the system can cause a complete shutdown of the system. [Overview of the Initiative] [Means for solving the problem]

[0004] The present invention is at least in part based on the finding that gamma irradiation of a chromatographic resin reduces its binding capacity, and that irradiation in the presence of at least one alcohol may help prevent this reduction in binding capacity of the chromatographic resin caused by gamma irradiation. In view of this finding, the Spectral Invention provides a method for reducing the bioburden of a chromatographic resin, comprising exposing a container comprising a composition comprising (i) a chromatographic resin and (ii) a liquid comprising at least one alcohol to a dose of gamma irradiation sufficient to reduce the bioburden of the container and the 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 / at exposure to that dose of gamma irradiation. Also provided are a reduced bioburden chromatography resin prepared by any of the methods described herein, a reduced bioburden chromatography column containing a composition comprising (i) the chromatography resin and (ii) a liquid containing at least one alcohol, a method for performing reduced bioburden column chromatography using at least one of these reduced bioburden chromatography columns, and an integrated, closed or substantially closed continuous method for producing reduced bioburden of purified recombinant protein, comprising the use of at least one of these reduced bioburden chromatography columns. Any chromatography resin produced by any of the methods described herein, any packed chromatography column produced by any of the methods described herein, any method for performing column chromatography, and any of the methods described herein may be sterile, absolutely sterile, sterilized, or reduced bioburden. Any chromatography resin produced by any of the methods described herein, any chromatography column produced by any of the methods described herein, and any of the methods described herein may be sterilized and sterile, absolutely sterile, sterilized, or reduced bioburden.

[0005] Provided herein is a method for reducing bioburden of a chromatographic resin, comprising exposing a container comprising a composition comprising (i) a chromatographic resin and (i) a liquid comprising at least one alcohol to a dose of gamma irradiation sufficient to reduce 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 exposure to that dose of gamma irradiation.

[0006] In some embodiments, the method may further include placing the composition in a container before 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 filled chromatography column.

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

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

[0012] In some embodiments of any method 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, 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.

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

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

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

[0016] In some embodiments, the liquid contains at least one antioxidant and / or chelating agent in an amount sufficient to improve the loss of binding capacity of the chromatographic resin after exposure to gamma irradiation of that dose.

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

[0018] In some embodiments, the liquid contains 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 is (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) containing approximately 10 mM to 50 mM methionine, approximately 10 mM to 50 mM histidine, and approximately 10 mM to 50 mM sodium ascorbate; or (vii) containing approximately 5 mM to 45 mM sodium ascorbate, approximately 5 mM to 45 mM methionine, approximately 5 mM to 45 mM mannitol, and approximately 5 mM to 45 mM histidine.

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

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

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

[0024] In some embodiments, the composition comprises an affinity chromatography resin containing 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 contains an N-benzyl-N-methylethanolamine group.

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

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

[0030] In some embodiments, the doses are approximately 23 kGy and approximately 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] This specification provides chromatographic resins of reduced bioburden produced by any of the methods described herein.

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

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

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

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

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

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

[0040] In some embodiments, the anion exchange chromatography resin contains an N-benzyl-N-methylethanolamine group.

[0041] This specification provides a method for preparing a chromatography column packed with reduced bioburden, comprising the steps of preparing one of the reduced bioburden chromatography resins described herein, and packing the chromatography resin into a reduced bioburden column in a sterilized environment.

[0042] This specification provides chromatography columns packed with reduced bioburden, which are produced by any of the methods described herein.

[0043] This specification provides chromatography columns packed with reduced bioburden, which are produced by any of the methods described herein.

[0044] In some embodiments, the resin in the packed column is approximately 1 × 10⁻⁶ -8 ~Approx. 1×10 -5 It has a sterility assurance level (SAL).

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

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

[0047] In some embodiments, the resin comprises an affinity or pseudo-affinity chromatography resin containing a protein ligand.

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

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

[0050] In some embodiments, the anion exchange chromatography resin contains an N-benzyl-N-methylethanolamine group.

[0051] This specification provides a composition comprising (i) a chromatographic resin and (ii) a liquid containing at least one alcohol, wherein the at least one alcohol is present in an amount sufficient to improve the loss of binding capacity of the chromatographic resin when treated with gamma irradiation at a dose sufficient to reduce the bioburden of the composition.

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

[0055] In some embodiments, at least one alcohol comprises 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 approximately 0.01% v / v to approximately 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 chromatographic resin after exposure to gamma irradiation of that dose.

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

[0060] In some embodiments, the liquid contains 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 is (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 (vi) containing 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) containing about 5 mM to about 45 mM sodium ascorbate, about 5 mM to about 45 mM methionine, about 5 mM to about 45 mM mannitol, and about 5 mM to about 45 mM histidine.

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

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

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

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

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

[0068] This specification provides a method for performing reduced bioburden column chromatography, comprising the steps of (a) preparing one of the reduced bioburden-packed chromatography columns described herein, and (b) performing column chromatography using the reduced bioburden-packed chromatography column and a reduced bioburden buffer in a closed system.

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

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

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

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

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

[0074] In some embodiments, the resin in the chromatography column packed with the reduced bioburden in (a) has a 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 packed with reduced bioburden includes 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 containing a protein ligand.

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

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

[0079] This specification provides an integrated, closed, and continuous method for producing reduced bioburden of purified recombinant protein, comprising the steps of (a) preparing a liquid culture medium containing recombinant protein substantially free of cells, and (b) continuously supplying the liquid culture medium to a multi-column chromatography system (MCCS) comprising at least one chromatographic column packed with any of the reduced bioburdens described herein, wherein the method utilizes a buffer for the reduced bioburden, is integrated, and operates 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 switching columns.

[0082] In some embodiments, the MCCS performs unit operations that capture recombinant proteins and inactivate viruses.

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

[0084] In some embodiments, the MCCS includes a chromatography column packed with at least two reduced bioburdens.

[0085] In some embodiments, 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 by a capture mechanism selected from the group consisting of: a protein A binding capture mechanism, a substrate binding capture mechanism, an antibody or antibody fragment binding capture mechanism, an aptamer binding capture mechanism, and a cofactor binding capture mechanism.

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

[0089] This specification provides an integrated, closed, and continuous method for producing reduced bioburden of purified recombinant protein, comprising the steps of: (a) preparing a liquid culture medium containing recombinant protein substantially free of cells; (b) continuously supplying the liquid culture medium into a first multi-column chromatography system (MCCS1); (c) capturing recombinant protein in the liquid culture medium using MCCS1; (d) generating an eluate containing recombinant protein from MCCS1 and continuously supplying the eluate into a second multi-column chromatography system (MCCS2); and (e) continuously supplying recombinant protein from the eluate into MCCS2 and subsequently eluting the recombinant protein to produce purified recombinant protein, wherein the method utilizes a buffer of reduced bioburden, is integrated, operates continuously from liquid culture medium to purified recombinant protein, and at least one column in MCCS1 and / or MCCS2 houses one of the chromatography columns packed with reduced bioburden as described herein.

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

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

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

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

[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: protein A binding capture mechanisms, substrate binding capture mechanisms, antibody or antibody fragment binding capture mechanisms, aptamer binding capture mechanisms, and cofactor binding capture mechanisms.

[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 recombinant protein for therapeutic purposes.

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

[0102] In some embodiments of any of the methods described herein, the method is carried out continuously for a period of at least four days.

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

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

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

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

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

[0108] The term "bioburden" is commonly known in the industry 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 is self-replicating biological contaminants present in a composition containing chromatography resin or packed chromatography resin (e.g., packed chromatography in a packed chromatography column). Bioburden can refer to self-replicating biological contaminants present in the chromatography resin. In other examples, bioburden can refer to self-replicating biological contaminants on the inner surface of a chromatography column and / or in 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., buffers used in any of the methods or processes described herein). Non-limiting examples of self-replicating biological contaminants may be bacteria (e.g., Gram-positive or Gram-negative bacteria, or bacterial spores), mycobacteria, viruses (e.g., vesiviruses, Cache Valley viruses, parvoviruses, herpesviruses, and bunyaviruses), parasites, fungi, yeasts, and protists. Typical 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 industry and refers to a reduction (e.g., a detectable reduction) in the level of self-replicating biological contaminants present in a composition (e.g., solid or liquid) and / or on the surface of an article (e.g., external and / or internal surfaces). Non-limiting examples of methods for reducing bioburden in chromatography resins (e.g., packed chromatography resins), buffers, and / or chromatography columns (e.g., packed chromatography columns) are described herein. Additional methods for reducing bioburden in any of the compositions described herein are known in the art.

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

[0111] The term "chromatography column with reduced bioburden" means a processed chromatography column (e.g., a gamma-irradiated chromatography column) containing a processed chromatography resin that contains a lower level of self-replicating biological contaminants than the level of self-replicating biological contaminants present in the same chromatography column containing untreated 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 "reduced bioburden buffer" is well known in the industry and refers to a level of self-replicating contaminants lower than the level of self-replicating contaminants found in the same untreated liquid. This refers to a liquid (e.g., treated buffer solution) that has been processed (e.g., filtered, autoclaved, and / or gamma-irradiated) to a certain quality. For example, a reduced bioburden buffer contains at least or about 1 × 10⁻⁶ of a liquid. -6 , 1 x 10 -7 , 1 x 10 -8 , 1 x 10 -9 , or 1 or 10 -10 It can have a level of sterility assurance.

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

[0114] "Sterile" or "aseptic" is approximately 1.0 × 10 -6 Less than (for example, approximately or 1.0 × 10) -7 Less than, approximately, or 1.0 × 10 -8 Less than, approximately, or 1.0 × 10 -9 Or 1 x 10 -10 This term is used to describe a composition or method having a sterility assurance level of less than 1 / 2. Determining 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 may be completely free of living, self-replicating biological contaminants (e.g., any of the self-replicating biological contaminants described herein). A sterile composition or method may also be clean (as known in the art).

[0115] The term "sterilization" means an established method used to make a composition sterile (as defined herein). To determine whether sterility (as defined herein) has been achieved for a given composition, the inactivation rate of self-replicating biological contaminants (e.g., bacteria) that indicate resistance during the processing can be measured.

[0116] The term "Sterilization Assurance Level," or "SAL," is a commonly known industry term that refers to the confidence level at which absolute sterility is achieved in a single batch of processing units. This probability is typically calculated based on the results of inactivation studies performed during verification, and is 1 × 10⁻⁶. -n It is represented by a certain shape.

[0117] The term “sterilized” is used to describe a composition or method that is free from self-replicating biological contaminants that cause disease or symptoms (e.g., any of the self-replicating biological contaminants described herein). 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 that refers to a functional step that can be performed in a method for purifying recombinant proteins from liquid culture media. For example, a unit operation may be filtration (e.g., removal of contaminants such as bacteria, yeast, viruses and / or mycobacteria, and / or particulate matter coming out of a fluid containing recombinant proteins), capture, epitope tag removal, purification, retention or storage, polishing, virus inactivation, adjustment of the ionic concentration and / or pH of a fluid containing recombinant proteins, and removal of undesirable salts.

[0119] The term "capture" means partially purifying or isolating recombinant proteins (e.g., recombinant therapeutic proteins) from one or more other components present in liquid culture medium or diluted liquid culture medium (e.g., culture medium proteins present in or secreted from mammalian cells or one or more other components (e.g., DNA, RNA, or other proteins)) (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 This refers to a process performed to concentrate the recombinant protein (or approximately 95% pure). Typically, capture is performed using a chromatographic resin to which the recombinant protein binds (e.g., by the use of affinity chromatography). Non-limiting methods for capturing recombinant proteins from liquid culture media or diluted liquid culture media are described herein, and other methods are also known in the art. Recombinant proteins can be captured from liquid culture media using at least one chromatographic column and / or chromatographic membrane (e.g., any of the chromatographic columns and / or chromatographic membranes described herein).

[0120] The term “purify” means a process performed to isolate a recombinant protein (e.g., recombinant therapeutic protein) from one or more other impurities (e.g., bulky impurities) or components (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.)) present in a fluid containing the recombinant protein. For example, purification can be performed during or after an initial capture process. Purification can be performed using a chromatographic resin, membrane, or any other solid support that binds to either the recombinant protein or the impurities (e.g., by using affinity chromatography, hydrophobic interaction chromatography, anion exchange or cation exchange chromatography, or molecular sieve chromatography). Recombinant proteins can be purified from a fluid containing the recombinant protein using at least one chromatographic column and / or chromatographic membrane (e.g., any of the chromatographic columns or chromatographic membranes described herein).

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

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

[0123] The term "eluate / filtrate" is a technical term referring to the fluid drained from a chromatography column or chromatographic membrane containing a detectable amount of recombinant protein (e.g., recombinant therapeutic protein).

[0124] The term "integrated method" refers to a method performed using structural elements that work together to achieve a specific result (e.g., purification of recombinant proteins from liquid culture media).

[0125] The term "continuous method" refers to a method of continuously supplying fluid through at least a portion of a system. For example, a continuous method involves supplying a liquid culture medium containing recombinant proteins via a continuous flow. This method involves continuous supply from an osteoreactor via an MCCS. Another example of a continuous method is the continuous supply of a liquid culture medium containing recombinant protein from a bioreactor via first and second MCCSs (MCCS1 and MCCS2). Further examples include a method of continuously supplying a liquid culture medium containing recombinant protein via an MCCS, a method of continuously supplying a liquid culture medium containing recombinant protein via MCCS1 and MCCS2, or a method of continuously supplying a fluid containing recombinant protein via MCCS2.

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

[0127] The term "therapeutic protein active pharmaceutical ingredient" means recombinant proteins (e.g., immunoglobulins, protein fragments, artificially modified proteins or enzymes) that are sufficiently purified or isolated from contaminating proteins, lipids and nucleic acids (e.g., contaminating proteins, lipids and nucleic acids present in liquid culture media or derived from host cells (e.g., mammalian host cells, yeast host cells or bacterial host cells)), as well as biotype contaminants (e.g., viral and bacterial contaminants), and that can be incorporated into pharmaceuticals without any further extensive purification and / or decontamination steps.

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

[0129] The term "substantially free" means a composition (e.g., liquid culture medium) that is free of at least or about 90% (e.g., free of at least or about 95%, 96%, 97%, 98%, or at least or about 99%, or about 100%) the 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, green monkey, rat, pig, cow, or rabbit). For example, a mammalian cell may be an immortalized cell. In some embodiments, a mammalian cell is a differentiated cell. In some embodiments, a 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 terms "culture" or "cell culture" refer to the maintenance or proliferation of mammalian cells under a controlled set of physical conditions.

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

[0133] The term “liquid culture medium” refers to a fluid containing sufficient nutrients to enable 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., 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pirubate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, a liquid culture medium may contain mammalian serum. In some embodiments, a liquid culture medium may not contain mammalian serum or other extracts (defined as a liquid culture medium with an obvious composition). In some embodiments, a liquid culture medium may contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Another example of a liquid culture medium is a minimal medium (e.g., a medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture media are described herein. Further examples of liquid culture media are known in the art and are also commercially available. Liquid culture media may contain mammalian cells of any density. For example, when used herein, a certain volume of liquid culture medium withdrawn from a bioreactor may not substantially contain mammalian cells.

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

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

[0136] The term "serum-containing liquid culture medium" refers to a liquid culture medium containing mammalian serum.

[0137] The term "chemically-defined liquid culture medium" is a technical term referring to a liquid culture medium whose entire chemical composition is known. For example, a chemically-defined liquid culture medium does not contain fetal bovine serum, bovine serum albumin, or human serum albumin, although these 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 proteins (e.g., any detectable proteins).

[0139] The term "immunoglobulin" refers to 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 may include, for example, light chain immunoglobulins with at least 15 amino acids, or heavy chain immunoglobulins with 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 may be antibody fragments, for example, Fab fragments, F(ab')2 fragments, or scFv fragments. Immunoglobulins may be bispecific or trispecific antibodies, or dimeric, trimer, or multimeric antibodies, or Diabody, Affibody®, or Nanobody®. Immunoglobulins may also 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 described in this technical section. It is common knowledge in the field.

[0140] The terms "protein fragment" or "polypeptide fragment" mean a portion of a polypeptide sequence that is at least or approximately 4 amino acids long, at least or approximately 5 amino acids long, at least or approximately 6 amino acids long, at least or approximately 7 amino acids long, at least or approximately 8 amino acids long, at least or approximately 9 amino acids long, at least or approximately 10 amino acids long, at least or approximately 11 amino acids long, at least or approximately 12 amino acids long, at least or approximately 13 amino acids long, at least or approximately 14 amino acids long, at least or approximately 15 amino acids long, at least or approximately 16 amino acids long, at least or approximately 17 amino acids long, at least or approximately 18 amino acids long, at least or approximately 19 amino acids long, or at least or approximately 20 amino acids long, or longer than 20 amino acids long. Recombinant protein fragments can be prepared using any of the methods described herein.

[0141] The term "modified protein" refers to polypeptides that are not naturally encoded by endogenous nucleic acids present within an organism (e.g., a mammal). Examples of modified proteins include enzymes (e.g., those that have undergone one or more amino acid substitutions, deletions, insertions, or additions resulting in increased stability and / or catalytic activity of the 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 terms “secreted protein” or “secreted recombinant protein” refer to a protein (e.g., recombinant protein) in which at least one secretory signal sequence originally present is translated within a mammalian cell, and when the secretory signal sequence within the mammalian cell is at least partially cleaved by an enzyme, at least a portion of it is secreted into the extracellular space (e.g., liquid culture medium). Those skilled in the art will understand that, in order to be treated as a secreted protein, the “secreted” protein does not need to be completely detached from the cell.

[0143] The term “perfusion bioreactor” means a bioreactor containing multiple cells (e.g., mammalian cells) in a first liquid culture medium, where the culture of cells present in the bioreactor involves periodically or continuously withdrawing the first liquid culture medium and simultaneously or immediately thereafter adding substantially the same volume of a second liquid culture medium to the bioreactor. In some examples, there is an incremental change (e.g., increase or decrease) in the volume of the first liquid culture medium that is withdrawn and added over a period of time (e.g., a period of about 24 hours, a period between about 1 minute and about 24 hours, or a period of more than 24 hours) during the culture period (e.g., the culture medium resupply rate based on 1 day). The percentage of medium withdrawn and replaced daily may vary depending on the specific cells being cultured, the initial seeding density, and the cell density at a particular time. “RV” or “reactor volume” means the volume of culture medium present at the start of the culture process (e.g., the total volume of culture medium present after seeding).

[0144] The term "fed-batch bioreactor" is a technical term referring to a bioreactor containing multiple cells (e.g., mammalian cells) in a first liquid culture medium, wherein the culture of cells present in the bioreactor involves periodic or continuous addition of the second liquid culture medium to the first liquid culture medium without significant or substantial removal of the first or 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 cultures, the second liquid culture medium is the first liquid culture medium in a concentrated form. In this example, 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 bacterial or yeast cells (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free).

[0146] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. While methods and materials are described herein for use in the invention, other suitable methods and materials known in the art may also be used. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other reference materials mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification shall prevail, including its provisions.

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

[0148] [Figure 1]Figure 1 is a graph showing the binding capacity percentage of MabSelect® SuRe® (Protein A chromatography resin) in multiple chromatography cycles after irradiation at 40-49 kGy in the presence of one of the following buffers: (i) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, and 25 mM mannitol in 50 mM sodium phosphate buffer ("SMM'H"); (ii) 2% v / v benzyl alcohol ("2% BA"); and (iii) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, and 2% v / v benzyl alcohol in 50 mM sodium phosphate buffer ("SMM'H+2% BA") (compared to an unirradiated chromatography resin loaded with the same material). [Figure 2] Figure 2 is a graph showing the binding capacity percentage of Capto Adhere chromatography resins in multiple chromatography cycles (compared to unirradiated chromatography resins loaded with the same material) in the presence of one of the following buffers: (i) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, and 25 mM mannitol in 50 mM sodium phosphate buffer ("SMM'H"); or (ii) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, and 2% v / v benzyl alcohol in 50 mM sodium phosphate buffer ("SMM'H+2%BA"). [Modes for carrying out the invention]

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

[0150] A composition containing a chromatography resin and at least one alcohol. This specification provides a composition comprising (i) a chromatographic resin (e.g., any chromatographic resin described herein or known in the art) and (ii) a liquid containing at least one (e.g., two, three, four, or five) alcohols (e.g., any representative alcohol 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 chromatographic resin upon treatment with gamma irradiation at a dose sufficient to reduce the bioburden of the composition. For example, the chromatographic resin may be at least one of anion exchange chromatographic resins, cation exchange chromatographic resins, affinity or pseudoaffinity chromatographic resins, hydrophobic interaction chromatographic resins, and size exclusion chromatographic resins, or any combination thereof. In some examples, the chromatographic resin is a resin containing a protein or peptide ligand (e.g., an affinity chromatographic resin with a protein or peptide ligand, e.g., a protein A or protein G chromatographic resin).

[0151] The composition may be, for example, a slurry of deposited chromatography resin. In some examples, the composition may be a mixture of wet or moist solids. In some examples, the composition may be chromatography resin filled in a liquid.

[0152] In some examples of any composition, 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, 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, at least one alcohol can be benzyl alcohol.

[0153] In some examples of any of the compositions, the total total concentration of one or more alcohols in the liquid or composition is approximately 0.01% v / v to approximately 20% v / v, approximately 0.01% v / v to approximately 19% v / v, approximately 0.01% v / v to approximately 18% v / v, approximately 0.01% v / v to approximately 17% v / v, approximately 0.01% v / v to approximately 16% v / v, approximately 0.01% v / v to approximately 15% v / v, approximately 0.01% v / v to approximately 14% v / v, approximately 0.01% v / v to approximately 13% v / v, and approximately 0. 01%v / v ~ approx. 12%v / v, approx. 0.01%v / v ~ approx. 11%v / v, approx. 0.01%v / v ~ approx. 10%v / v, approx. 0.01%v / v ~ approx. 9%v / v, approx. 0.01%v / v ~ approx. 8%v / v, approx. 0.01%v / v ~ approx. 7%v / v, about 0.01%v / v to about 6%v / v, about 0.01%v / v to about 5%v / v, about 0.01%v / v to about 4.5%v / v, about 0.01%v / v to about 4.0%v / v, about 0.01%v / v to about 3.5%v / v, about 0.01%v / v ~ approx. 3.0% v / v, approx. 0.01% v / v ~ approx. 2.5% v / v, approx. 0.01% v / v ~ approx. 2.2% v / v, approx. 0.01% v / v ~ approx. 2.0% v / v, approx. 0.01% v / v ~ approx. 1.8% v / v, approx. 0.01% v / v ~ approx. 1.6% v / v, approx. 0.01% v / v ~ approx. 1.4% v / v, approximately 0.01%v / v to approximately 1.2%v / v, approximately 0.01%v / v to approximately 1.0%v / v, approximately 0.01%v / v to approximately 0.8%v / v, approximately 0.01%v / v to approximately 0.6%v / v, approximately 0.01%v / v to approximately 0.4%v / v, approximately 0.01%v / v to approximately 0.2%v / v, approximately 0.01%v / v ~ approx. 0.1% v / v, approx. 0.01% v / v ~ approx. 0.05% v / v, approx. 0.05% v / v ~ approx. 20% v / v, approx. 0.05% v / v ~ approx. 19% v / v, approx. 0.05% v / v ~ approx. 18% v / v, approx. 0.05% v / v ~ approx. 17% v / v, approx. 0.05% v / v ~ approx. 16% v / v, approx. 0 0.05%v / v ~ approx. 15%v / v, approx. 0.05%v / v ~ approx. 14%v / v, approx. 0.05%v / v ~ approx. 13%v / v, approx. 0.05%v / v ~ approx. 12%v / v, approx. 0.05%v / v ~ approx. 11%v / v, approx. 0.05%v / v ~ approx. 10%v / v, approx. 0.05%v / v ~ approx. 9%v / v, approx. 0.05%v / v ~ approx. 8%v / v, approx. 0.05%v / v ~ approx. 7%v / v, approx. 0.05%v / v ~ approx. 6%v / v, approx. 0.05%v / v ~ approx. 5%v / v, approx. 0.05%v / v ~ approx. 4.5%v / v, approx. 0.05%v / v ~ approx. 4.0%v / v, approx. 0.05%v / v ~ approx. 3.5%v / v Approximately 0.05% v / v to approximately 3.0% v / v, approximately 0.05% v / v to approximately 2.5% v / v, approximately 0.05% v / v to approximately 2.2% v / v, approximately 0.05% v / v to approximately 2.0% v / v, approximately 0.05% v / v to approximately 1.8% v / v, approximately 0.05% v / v to approximately 1.6% v / v, approximately 0.05% v / v ~approx. 1.4% v / v, approx. 0.05% v / v ~ approx. 1.2% v / v, approx. 0.05% v / v ~ approx. 1.0% v / v, approx. 0.05% v / v ~ approx. 0.8% v / v, approx. 0.05% v / v ~ approx. 0.6% v / v, approx. 0.05% v / v ~ approx. 0.4% v / v, approx. 0.05% v / v ~ approx. 0.2% v / v Approximately 0.05% v / v to approximately 0.1% v / v, approximately 0.1% v / v to approximately 20% v / v, approximately 0.1% v / v to approximately 19% v / v, approximately 0.1% v / v to approximately 18% v / v, approximately 0.1% v / v to approximately 17% v / v, approximately 0.1% v / v to approximately 16% v / v, approximately 0.1% v / v to approximately 15% v / v, approximately 0.1% v / v ~ approx. 14% v / v, approx. 0.1% v / v ~ approx. 13% v / v, approx. 0.1% v / v ~ approx. 12% v / v, approx. 0.1% v / v ~ approx. 11% v / v, approx. 0.1% v / v ~ approx. 10% v / v, approx. 0.1% v / v ~ approx. 9% v / v, approx. 0.1% v / v ~ approx. 8% v / v, approx. 0.1% v / v ~ Approximately 7% v / v, approximately 0.1% v / v to approximately 6% v / v, approximately 0.1% v / v to approximately 5% v / v, approximately 0.1% v / v to approximately 4.5% v / v, approximately 0.1% v / v to approximately 4.0% v / v, approximately 0.1% v / v to approximately 3.5% v / v, approximately 0.1% v / v to approximately 3.0% v / v, approximately 0.1% v / v to approximately 2.5% v / v. %v / v, approximately 0.1%v / v to approximately 2.2%v / v, approximately 0.1%v / v to approximately 2.0%v / v, approximately 0.1%v / v to approximately 1.8%v / v, approximately 0.1%v / v to approximately 1.6%v / v, approximately 0.1%v / v to approximately 1.4%v / v, approximately 0.1%v / v to approximately 1.2%v / v, approximately 0.1%v / v to approximately 1 0.0% v / v, approximately 0.1% v / v to approximately 0.8% v / v, approximately 0.1% v / v to approximately 0.6% v / v, approximately 0.1% v / v to approximately 0.4% v / v, approximately 0.1% v / v to approximately 0.2% v / v, approximately 0.2% v / v to approximately 20% v / v, approximately 0.2% v / v to approximately 19% v / v, approximately 0.2% v / v to approximately 1 8% v / v, approximately 0.2% v / v to approximately 17% v / v, approximately 0.2% v / v to approximately 16% v / v, approximately 0.2% v / v to approximately 15% v / v, approximately 0.2% v / v to approximately 14% v / v, approximately 0.2% v / v to approximately 13% v / v, approximately 0.2% v / v to approximately 12% v / v, approximately 0.2% v / v to approximately 11% v / 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 ~ approx. 4.0% v / v, approx. 0.2% v / v ~ approx. 3.5% v / v, approx. 0.2% v / v ~ approx. 3.0% v / v, approx. 0.2% v / v ~ approx. 2.5% v / v, approx. 0.2% v / v ~ approx. 2.2% v / v, approx. 0.2% v / v ~ approx. 2.0% v / v, approx. 0.2% v / v ~ approx. 1.8% v / v, approx. 0.2% v / v ~ approx. 1.6% v / v, approx. 0.2% v / v ~ approx. 1.4% v / v, approx. 0.2% v / v ~ approx. 1.2% v / v, approx. 0.2% v / v ~ approx. 1.0% v / v, approx. 0.2% v / v ~ approx. 0.8% v / v, approx. 0.2% v / v ~ approx. 0.6% v / v, approx. 0.2% v / v ~ approx. 0.4% v / v, approx. 0.4%v / v~20%v / v、0.4%v. / v ~ approx. 19% v / v, approx. 0.4% v / v ~ approx. 18% v / v, approx. 0.4% v / v ~ approx. 17% v / v, approx. 0.4% v / v ~ approx. 16% v / v, approx. 0.4% v / v ~ approx. 15% v / v, approx. 0.4% v / v ~ approx. 14% v / v, approx. 0.4% v / v ~ approx. 13% v / v, approx. 0.4% v / v ~ approx. 12% v / v %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 ~ approx. 4.5% v / v, approx. 0.4% v / v ~ approx. 4.0% v / v, approx. 0.4% v / v ~ approx. 3.5% v / v, approx. 0.4% v / v ~ approx. 3.0% v / v, approx. 0.4% v / v ~ approx. 2.5% v / v, approx. 0.4% v / v ~ approx. 2.2% v / v, approx. 0.4% v / v ~ approx. 2.0% v / v, approx. 0.4% v / v ~ approx. 1.8% v / v, approx. 0.4% v / v ~ approx. 1.6% v / v, approx. 0.4% v / v ~ approx. 1.4% v / v, approx. 0.4% v / v ~ approx. 1.2% v / v, approx. 0.4% v / v ~ approx. 1.0% v / v, approx. 0.4% v / v ~ approx. 0.8% v / v, approx. 0.4% v / v ~ approx. 0.6% v / v, approx. 0.6% v / v %v / v ~ approx. 20%v / v, approx. 0.6%v / v ~ approx. 19%v / v, approx. 0.6%v / v ~ approx. 18%v / v, approx. 0.6%v / v ~ approx. 17%v / v, approx. 0.6%v / v ~ approx. 16%v / v, approx. 0.6%v / v ~ approx. 15%v / v, approx. 0.6%v / v ~ approx. 14%v / v, approx. 0.6%v / v ~ approx. 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 ~ approx. 5% v / v, approx. 0.6% v / v ~ approx. 4.5% v / v, approx. 0.6% v / v ~ approx. 4.0% v / v, approx. 0.6% v / v ~ approx. 3.5% v / v, approx. 0.6% v / v ~ approx. 3.0% v / v, approx. 0.6% v / v ~ approx. 2.5% v / v, approx. 0.6% v / v ~ approx. 2.2% v / v, approx. 0.6 %v / v ~ approx. 2.0%v / v, approx. 0.6%v / v ~ approx. 1.8%v / v, approx. 0.6%v / v ~ approx. 1.6%v / v, approx. 0.6%v / v ~ approx. 1.4%v / v, approx. 0.6%v / v ~ approx. 1.2%v / v, approx. 0.6%v / v ~ approx. 1.0%v / v, approx. 0.6%v / v ~ approx. 0.8%v / v, approx. 0.8% v / v ~ approx. 20% v / v, approx. 0.8% v / v ~ approx. 19% v / v, approx. 0.8% v / v ~ approx. 18% v / v, approx. 0.8% v / v ~ approx. 17% v / v, approx. 0.8% v / v ~ approx. 16% v / v, approx. 0.8% v / v ~ approx. 15% v / v, approx. 0.8% v / v ~ approx. 14% v / v, approx. 0.8% v / v ~ 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 0.8% v / v ~ approx. 5% v / v, approx. 0.8% v / v ~ approx. 4.5% v / v, approx. 0.8% v / v ~ approx. 4.0% v / v, approx. 0.8% v / v ~ approx. 3.5% v / v, approx. 0.8% v / v ~ approx. 3.0% v / v, approx. 0.8% v / v ~ approx. 2.5% v / v, approx. 0.8% v / v ~ approx. 2.2% v / v, approx. 0. 8% v / v ~ approx. 2.0% v / v, approx. 0.8% v / v ~ approx. 1.8% v / v, approx. 0.8% v / v ~ approx. 1.6% v / v, approx. 0.8% v / v ~ approx. 1.4% v / v, approx. 0.8% v / v ~ approx. 1.2% v / v, approx. 0.8% v / v ~ approx. 1.0% v / v, approx. 1.0% v / v ~ approx. 20% v / v, approx. 1. 0% v / v ~ approx. 19% v / v, approx. 1.0% v / v ~ approx. 18% v / v, approx. 1.0% v / v ~ approx. 17% v / v, approx. 1.0% v / v ~ approx. 16% v / v, approx. 1.0% v / v ~ approx. 15% v / v, approx. 1.0% v / v ~ approx. 14% v / v, approx. 1.0% v / v ~ approx. 13% v / v, approx. 1.0% v / v ~ 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 ~ approx. 4.5% v / v, approx. 1.0% v / v ~ approx. 4.0% v / v, approx. 1.0% v / v ~ approx. 3.5% v / v, approx. 1.0% v / v ~ approx. 3.0% v / v, approx. 1.0% v / v ~ approx. 2.5% v / v, approx. 1.0% v / v ~ approx. 2.2% v / v, approx. 1.0% v / v ~ approx. 2.0% v / v, approx. 1 0% v / v ~ approx. 1.8% v / v, approx. 1.0% v / v ~ approx. 1.6% v / v, approx. 1.0% v / v ~ approx. 1.4% v / v, approx. 1.0% v / v ~ approx. 1.2% v / v, approx. 1.2% v / v ~ approx. 20% v / v, approx. 1.2% v / v ~ approx. 19% v / v, approx. 1.2% v / v ~ approx. 18% v / v, approx. 1.2%. %v / v ~ approx. 17%v / v, approx. 1.2%v / v ~ approx. 16%v / v, approx. 1.2%v / v ~ approx. 15%v / v, approx. 1.2%v / v ~ approx. 14%v / v, approx. 1.2%v / v ~ approx. 13%v / v, approx. 1.2%v / v ~ approx. 12%v / v, approx. 1.2%v / v ~ approx. 11%v / v, approx. 1.2%v / v ~ approx. 10%v / v, approx. 1.2%v / v ~ approx. 9%v / v, approx. 1.2%v / v ~ approx. 8%v / v Approximately 1.2% v / v to approximately 7% v / v, approximately 1.2% v / v to approximately 6% v / v, approximately 1.2% v / v to approximately 5% v / v, approximately 1.2% v / v to approximately 4.5% v / v, approximately 1.2% v / v to approximately 4.0% v / v, approximately 1.2% v / v to approximately 3.5% v / v, approximately 1.2% v / v to approximately 3.0% v / v, approximately 1.2 %v / v ~ approx. 2.5%v / v, approx. 1.2%v / v ~ approx. 2.2%v / v, approx. 1.2%v / v ~ approx. 2.0%v / v, approx. 1.2%v / v ~ approx. 1.8%v / v, approx. 1.2%v / v ~ approx. 1.6%v / v, approx. 1.2%v / v ~ approx. 1.4%v / v, approx. 1.4%v / v ~ approx. 20%v / v, approx. 1.4%v / v %v / v ~ approx. 19%v / v, approx. 1.4%v / v ~ approx. 18%v / v, approx. 1.4%v / v ~ approx. 17%v / v, approx. 1.4%v / v ~ approx. 16%v / v, approx. 1.4%v / v ~ approx. 15%v / v, approx. 1.4%v / v ~ approx. 14%v / v, approx. 1.4%v / v ~ approx. 13%v / v, approx. 1.4%v / v ~ approx. 12% v / v, approximately 1.4% v / v to approximately 11% v / v, approximately 1.4% v / v to approximately 10% v / v, approximately 1.4% v / v to approximately 9% v / v, approximately 1.4% v / v to approximately 8% v / v, approximately 1.4% v / v to approximately 7% v / v, approximately 1.4% v / v to approximately 6% v / v, approximately 1.4% v / v to approximately 5% v / v, approximately 1.4% v / v %v / v ~ approx. 4.5%v / v, approx. 1.4%v / v ~ approx. 4.0%v / v, approx. 1.4%v / v ~ approx. 3.5%v / v, approx. 1.4%v / v ~ approx. 3.0%v / v, approx. 1.4%v / v ~ approx. 2.5%v / v, approx. 1.4%v / v ~ approx. 2.2%v / v, approx. 1.4%v / v ~ approx. 2.0%v / v, approx. 1. 4% v / v ~ approx. 1.8% v / v, approx. 1.4% v / v ~ approx. 1.6% v / v, approx. 1.6% v / v ~ approx. 20% v / v, approx. 1.6% v / v ~ approx. 19% v / v, approx. 1.6% v / v ~ approx. 18% v / v, approx. 1.6% v / v ~ approx. 17% v / v, approx. 1.6% v / v ~ approx. 16% v / v, approx. 1.6% v / v v ~ approx. 15% v / v, approx. 1.6% v / v ~ approx. 14% v / v, approx. 1.6% v / v ~ approx. 13% v / v, approx. 1.6% v / v ~ approx. 12% v / v, approx. 1.6% v / v ~ approx. 11% v / v, approx. 1.6% v / v ~ approx. 10% v / v, approx. 1.6% v / v ~ approx. 9% v / v, approx. 1.6% v / v ~ approx. 8% v / v v, approximately 1.6% v / v to approximately 7% v / v, approximately 1.6% v / v to approximately 6% v / v, approximately 1.6% v / v to approximately 5% v / v, approximately 1.6% v / v to approximately 4.5% v / v, approximately 1.6% v / v to approximately 4.0% v / v, approximately 1.6% v / v to approximately 3.5% v / v, approximately 1.6% v / v to approximately 3.0% v / v, approximately 1.6% v / v ~ approx. 2.5% v / v, approx. 1.6% v / v ~ approx. 2.2% v / v, approx. 1.6% v / v ~ approx. 2.0% v / v, approx. 1.6% v / v ~ approx. 1.8% v / v, approx. 1.8% v / v ~ approx. 20% v / v, approx. 1.8% v / v ~ approx. 19% v / v, approx. 1.8% v / v ~ approx. 18% v / v, approx. 1.8% v / v ~ approx. 17% v / v, approx. 1.8% v / v ~ approx. 16% v / v, approx. 1.8% v / v ~ approx. 15% v / v, approx. 1.8% v / v ~ approx. 14% v / v, approx. 1.8% v / v ~ approx. 13% v / v %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 0.0% 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 ~ approx. 12% v / v, approx. 2.0% v / v ~ approx. 11% v / v, approx. 2.0% v / v ~ approx. 10% v / v, approx. 2.0% v / v ~ approx. 9% v / v, approx. 2.0% v / v ~ approx. 8% v / v, approx. 2.0% v / v ~ approx. 7% v / v, approx. 2.0% v / v ~ approx. 6% v / v, approx. 2.0% v / v ~ approx. 5% v / v, approx. 2.0% v / v ~ approx. 4.5% v / v, approx. 2.0% v / v ~ approx. 4.0% v / v, approx. 2.0% v / v ~ approx. 3.5% v / v, approx. 2.0% v / v ~ approx. 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 0.2% v / v ~ approx. 15% v / v, approx. 2.2% v / v ~ approx. 14% v / v, approx. 2.2% v / v ~ approx. 13% v / v, approx. 2.2% v / v ~ approx. 12% v / v, approx. 2.2% v / v ~ approx. 11% v / v, approx. 2.2% v / v ~ approx. 10% v / v, approx. 2.2% v / v ~ approx. 9% v / v, approx. 2.2% v / v v ~ approx. 8% v / v, approx. 2.2% v / v ~ approx. 7% v / v, approx. 2.2% v / v ~ approx. 6% v / v, approx. 2.2% v / v ~ approx. 5% v / v, approx. 2.2% v / v ~ approx. 4.5% v / v, approx. 2.2% v / v ~ approx. 4.0% v / v, approx. 2.2% v / v ~ approx. 3.5% v / v, approx. 2.2% v / v ~ approx. 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 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 ~ approx. 7%v / v, approx. 2.5%v / v ~ approx. 6%v / v, approx. 2.5%v / v ~ approx. 5%v / v, approx. 2.5%v / v ~ approx. 4.5%v / v, approx. 2.5%v / v ~ approx. 4.0%v / v, approx. 2.5%v / v ~ approx. 3.5%v / v, approx. 2.5%v / v ~ approx. 3.0%v / v, approx. 3.0%v / v v ~ approx. 20% v / v, approx. 3.0% v / v ~ approx. 19% v / v, approx. 3.0% v / v ~ approx. 18% v / v, approx. 3.0% v / v ~ approx. 17% v / v, approx. 3.0% v / v ~ approx. 16% v / v, approx. 3.0% v / v ~ approx. 15% v / v, approx. 3.0% v / v ~ approx. 14% v / v, approx. 3.0% v / v ~ approx. 1 3% 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 ~ approx. 5% v / v, approx. 3.0% v / v ~ approx. 4.5% v / v, approx. 3.0% v / v ~ approx. 4.0% v / v, approx. 3.0% v / v ~ approx. 3.5% v / v, approx. 3.5% v / v ~ approx. 20% v / v, approx. 3.5% v / v ~ approx. 19% v / v, approx. 3.5% v / v ~ approx. 18% v / v, approx. 3.5% v / v ~ approx. 17% v / v, approx. 3.5% v / v ~ approx. 16% v / v, approx. 3.5% v / v ~ approx. 15% v / v, approx. 3.5% v / v ~ approx. 14% v / v, approx. 3.5% v / v ~ approx. 13% v / v, approx. 3.5% v / v ~ approx. 12% v / v, approx. 3.5% v / v v ~ approx. 11% v / v, approx. 3.5% v / v ~ approx. 10% v / v, approx. 3.5% v / v ~ approx. 9% v / v, approx. 3.5% v / v ~ approx. 8% v / v, approx. 3.5% v / v ~ approx. 7% v / v, approx. 3.5% v / v ~ approx. 6% v / v, approx. 3.5% v / v ~ approx. 5% v / v, approx. 3.5% v / v ~ approx. 4.5% v / v, approx. 3.5% v / v ~ approx. 4.0% v / v, approx. 4.0% v / v ~ approx. 20% v / v, approx. 4.0% v / v ~ approx. 19% v / v, approx. 4.0% v / v ~ approx. 18% v / v, approx. 4.0% v / v ~ approx. 17% v / v, approx. 4.0% v / v ~ approx. 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~ 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 ~ approx. 5% v / v, approx. 5% v / v ~ approx. 20% v / v, approx. 5% v / v ~ approx. 19% v / v, approx. 5% v / v ~ approx. 18% v / v, approx. 5% v / v ~ approx. 17% v / v, approx. 5% v / v ~ approx. 16% v / v, approx. 5% v / v ~ approx. 15% v / v, approx. 5. %v / v ~ approx. 14%v / v, approx. 5%v / v ~ approx. 13%v / v, approx. 5%v / v ~ approx. 12%v / v, approx. 5%v / v ~ approx. 11%v / v, approx. 5%v / v ~ approx. 10%v / v, approx. 5%v / v ~ approx. 9%v / v, approx. 5%v / v ~ approx. 8%v / v, approx. 5%v / v ~ approx. 7%v / v, approx. 5%v / v ~ approx. 6%v / v, approx. 6%v / v ~ approx. 20%v / v, approx. 6%v / v ~ approx. 19%v / v %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 ~ approx. 8% v / v, approx. 6% v / v ~ approx. 7% v / v, approx. 7% v / v ~ approx. 20% v / v, approx. 7% v / v ~ approx. 19% v / v, approx. 7% v / v ~ approx. 18% v / v, approx. 7% v / v ~ approx. 17% v / v, approx. 7% v / v ~ approx. 16% v / v, approx. 7% v / v ~ approx. 15% v / v, approx. 7% v / v ~ approx. 14% v / v, approx. 7% v / v ~ approx. 13% v / v, approx. 7% v / v ~ approx. 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 Approximately 8% v / v ~ approximately 14% v / v, approximately 8% v / v ~ approximately 13% v / v, approximately 8% v / v ~ approximately 12% v / v, approximately 8% v / v ~ approximately 11% v / v, approximately 8% v / v ~ approximately 10% v / v, approximately 8% v / v ~ approximately 9% v / v, approximately 9% v / v ~ approximately 20% v / v, approximately 9% v / v ~ approximately 19% v / v, approximately 9% v / v ~ approximately 18% v / v, approximately 9% v / v ~ approximately 17% v / v, approximately 9% v / v ~ approximately 16% v / v, approximately 9% v / v ~ approximately 15% v / v, approximately 9% v / v ~ approximately 14% v / v, approximately 9% v / v ~ approximately 13% v / v, approximately 9% v / v ~ approximately 12% v / v, approximately 9% v / v ~ approximately 11% v / v, approximately 9% v / v ~ approximately 10% v / v, approximately 10% v / v to approximately 20% v / v, approximately 10% v / v to approximately 19% v / v, approximately 10% v / v to approximately 18% v / v, approximately 10% v / v to approximately 17% v / v, approximately 10% v / v to approximately 16% v / v, approximately 10% v / v to approximately 15% v / v, approximately 10% v / v to approximately 14% v / v, approximately 10% v / v ~ approx. 13% v / v, approx. 10% v / v ~ approx. 12% v / v, approx. 10% v / v ~ approx. 11% v / v, approx. 11% v / v ~ approx. 20% v / v, approx. 11% v / v ~ approx. 19% v / v, approx. 11% v / v ~ approx. 18% v / v, approx. 11% v / v ~ approx. 17% v / v, approx. 11% v / v ~ approx. 16% v / v, approx. 11% v / v ~ approx. 15% v / v, approx. 11% v / v ~ approx. 14% v / v, approx. 11% v / v ~ approx. 13% v / v, approx. 11% v / v ~ approx. 12% v / v, approx. 12% v / v ~ approx. 20% v / v, approx. 12% v / v ~ approx. 19% v / v, approx. 12% v / v ~ approx. 18% v / v, approx. 12% v / v ~ approx. 17% v / v, approximately 12% v / v to approximately 16% v / v, approximately 12% v / v to approximately 15% v / v, approximately 12% v / v to approximately 14% v / v, approximately 12% v / v to approximately 13% v / v, approximately 13% v / v to approximately 20% v / v, approximately 13% v / v to approximately 19% v / v, approximately 13% v / v to approximately 18% v / v, approximately 13% v / v~ Approximately 17% v / v, approximately 13% v / v to approximately 16% v / v, approximately 13% v / v to approximately 15% v / v, approximately 13% v / v to approximately 14% v / v, approximately 14% v / v to approximately 20% v / v, approximately 14% v / v to approximately 19% v / v, approximately 14% v / v to approximately 18% v / v, approximately 14% v / v to approximately 17% v / v, approximately 14% v / v v / v ~ approx. 16% v / v, approx. 14% v / v ~ approx. 15% v / v, approx. 15% v / v ~ approx. 20% v / v, approx. 15% v / v ~ approx. 19% v / v, approx. 15% v / v ~ approx. 18% v / v, approx. 15% v / v ~ approx. 17% v / v, approx. 15% v / v ~ approx. 16% v / v, approx. 16% v / v ~ approx. 20% v / vThe humidity levels are approximately 16% v / v to 19% v / v, 16% v / v to 18% v / v, 16% v / v to 17% v / v, 17% v / v to 20% v / v, 17% v / v to 19% v / v, 17% v / v to 18% v / v, 18% v / v to 20% v / v, 18% v / v to 19% v / v, or 19% v / v to 20% v / v.

[0154] In some examples of any of the compositions described herein, the liquid may further contain at least one (e.g., two, three, four, or five) antioxidants and / or chelating agents. Alternatively, a chelating agent may be further included in an amount sufficient to improve the loss of binding capacity of the chromatographic resin after exposure to gamma-ray irradiation of that dose.

[0155] In some examples of any of the compositions described herein, the liquid may contain at least one antioxidant (e.g., two, three, four, or five) selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoids, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoids, phenolpropanoid acids, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanol. In some examples of any of the compositions described herein, the liquid may contain at least one antioxidant (e.g., two, three, or four) 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 may 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 is (i) mannitol in a concentration of 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) mannitol in a concentration of 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) Methionine (or cysteine ​​or glutathione); (iii) Sodium ascorbate in concentrations of 75 mM to approximately 125 mM (e.g., approximately 80 mM to approximately 120 mM, approximately 85 mM to approximately 115 mM, approximately 90 mM to approximately 110 mM, or approximately 95 mM to approximately 105 mM); (iv) 75 mM to approximately 125 mM (e.g., approximately 80 mM to approximately 120 mM, approximately 85 mM to approximately 115 mM, approximately 90 mM to approximately 110 mM, or approximately 95 mM to approximately 105 mM (M) histidine; (v) methionine (or cysteine ​​or glutathione) in approximately 30 mM to 70 mM (e.g., approximately 35 mM to 65 mM, approximately 40 mM to 60 mM, or approximately 45 mM to 55 mM) and histidine in approximately 30 mM to 70 mM (e.g., approximately 35 mM to 65 mM, approximately 40 mM to 60 mM, or approximately 45 mM to 55 mM); (vi) approximately 10 mM to 50 mM (e.g., approximately 15 mM to 45 mM) Methionine (or cysteine ​​or glutathione) in approximately 20 mM to 40 mM, or approximately 25 mM to 35 mM; histidine in approximately 10 mM to 50 mM (e.g., approximately 15 mM to 45 mM, approximately 20 mM to 40 mM, or approximately 25 mM to 35 mM); and sodium ascorbate in approximately 10 mM to 50 mM (e.g., approximately 15 mM to 45 mM, approximately 20 mM to 40 mM, or approximately 25 mM to 35 mM);Or (vii) it may contain approximately 5 mM to approximately 45 mM (e.g., approximately 10 mM to approximately 40 mM, approximately 15 mM to approximately 35 mM, or approximately 20 mM to approximately 30 mM) of sodium ascorbate, approximately 5 mM to approximately 45 mM (e.g., approximately 10 mM to approximately 40 mM, approximately 15 mM to approximately 35 mM, or approximately 20 mM to approximately 30 mM) of methionine (or cysteine ​​or glutathione), approximately 5 mM to approximately 45 mM (e.g., approximately 10 mM to approximately 40 mM, approximately 15 mM to approximately 35 mM, or approximately 20 mM to approximately 30 mM) of mannitol, and approximately 5 mM to approximately 45 mM (e.g., approximately 10 mM to approximately 40 mM, approximately 15 mM to approximately 35 mM, or approximately 20 mM to approximately 30 mM) of histidine. In some examples of any of the compositions described herein, the liquid may be a buffer solution (e.g., a phosphate buffer solution, e.g., a sodium phosphate buffer solution, e.g., 50 mM sodium phosphate, pH 6.0).

[0157] In some embodiments of any of the compositions described herein, the liquid may further contain at least one (e.g., two, three, four, or five) chelating agents (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 desferoxamine).

[0158] Also provided herein is a container (e.g., a storage vessel, e.g., a plastic container, or a chromatography column) containing a composition (e.g., any of the representative compositions described herein) comprising (i) a chromatography resin (e.g., any of the chromatography resins described herein or known in the art) and (ii) a liquid containing at least one alcohol (e.g., any of the representative alcohols described herein or known in the art), wherein the at least one alcohol is present in an amount sufficient to improve the binding capacity of the chromatography resin when treated with gamma irradiation at a dose sufficient to reduce the bioburden of the composition. For example, a container (e.g., a storage container, e.g., a plastic container, or a chromatography column) may 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 may have an internal volume of approximately 1 mL to 500 mL, 1 mL to 50 mL, 5 mL to 500 mL, 5 mL to 400 mL, 5 mL to 350 mL, 5 mL to 300 mL, 5 mL to 250 mL, 5 mL to 200 mL, 5 mL to 150 mL, 5 mL to 100 mL, or 5 mL to 50 mL. In some examples, the chromatographic resin in the container is a slurry of deposited chromatographic resin in a liquid.In some examples, the container contains a filled chromatography resin (e.g., filled in a liquid).

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

[0160] Any chelating agent that can be included in any of the compositions provided herein is a redox-active metal (e.g., Cu) with high affinity (e.g., less than about 1 μM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 250 nM, less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 80 nM, less than about 60 nM, less than about 40 nM, less than about 20 nM, or less than about 1 nm). 2+ and Fe 2+ It may have the ability to bind to ). Non-limiting examples of chelating agents that may 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 concentrations of each chelating agent and / or antioxidant that may be included in any of the compositions provided herein range from approximately 0.1 mM to approximately 150 mM (for example, approximately 0.1 mM to approximately 150 mM, approximately 0.1 mM to approximately 125 mM, approximately 0.1 mM to approximately 100 mM, approximately 0.1 mM to approximately 80 mM, approximately 0.1 mM to approximately 60 mM, approximately 0.1 mM to approximately 50 mM, approximately 0.1 mM). M ~ about 40mM, about 0.1mM - about 30mM, about 0.1mM - about 25mM, about 0.1mM - about 20mM, about 0.1mM - about 10mM, about 0.1mM - about 5.0mM, about 0.5 mM to about 150mM, about 0.5mM to about 100mM, about 0.5mM to about 50mM, about 0.5mM to about 25mM, about 0.5mM to about 15mM, about 0.5mM to about 10mM, about 0.5mM to about 5mM, about 1mM to about 125mM, about 1mM to about 120mM, about 1mM to about 100mM, about 1mM to about 80mM, about 1mM to about 60mM, about 1mM to about 50mM, about 1mM to about 40mM, about 1m M ~ about 30mM, about 1mM - about 25mM, about 5mM - about 150mM, about 5mM - about 125mM, about 5mM - about 100mM, about 5mM - about 80mM, about 5mM - about 60mM, about 5mM - about 50mM, about 5mM ~40mM, approximately 5mM ~ approximately 30mM, approximately 5mM ~ approximately 25mM, approximately 10mM ~ approximately 150mM, approximately 10mM ~ approximately 125mM, approximately 1mM ~ approximately 100mM, approximately 10mM ~ approximately 80mM, approximately 10mM ~ approximately 60mM, approximately 10mM to about 50mM, about 10mM to about 40mM, about 10mM to about 30mM, about 10mM to about 25mM, about 20mM to about 150mM, about 20mM to about 125mM, about 20mM to about 100mM, about 20mM to about 80mM, about 20mM to about 60mM, about 20mM to about 50mM, about 20mM to about 40mM, about 20mM to about 30mM, about 30mM to about 150mM, about 30mM to about 125mM, about 30mM to about 100mM, Approximately 30mM to approximately 80mM, approximately 30mM to approximately 60mM, approximately 30mM to approximately 50mM, approximately 30mM to approximately 40mM, approximately 40mM to approximately 150mM, approximately 40mM to approximately 125mM, approximately 40mM to approximately 100mM, approximately 40mM to It can be approximately 90mM, approximately 40mM to 80mM, approximately 40mM to 70mM, approximately 40mM to 60mM, approximately 50mM to 150mM, approximately 50mM to 125mM, approximately 50mM to 100mM, approximately 50mM to 80mM, approximately 50mM to 60mM, approximately 80mM to 150mM, approximately 80mM to 125mM, approximately 80mM to 100mM, approximately 100mM to 150mM, or approximately 100mM to 125mM.

[0162] In some examples, the compositions provided herein contain 5 mM to about 150 mM mannitol. Thor (for example, approximately 10mm to 150mm, approximately 20mm to 150mm, approximately 30mm to 150mm, approximately 40mm to 150mm, approximately 50mm to 150mm, approximately 60mm to 140mm, approximately 70mm to 130mm, approximately 80mm to 120mm, approximately 90mm to 110mm, approximately 95mm to 105mm, approximately 5mm to 50mm, approximately 5mm to 45mm, approximately 5mm to 40mm, approximately 5mm to 35mm, approximately 10mm to 35mm, approximately 15mm to 35mm, or approximately 20 mM to approximately 30 mM mannitol); 5 mM to approximately 150 mM (for example, approximately 10 mM to approximately 150 mM, approximately 20 mM to approximately 150 mM, approximately 30 mM to approximately 150 mM, approximately 40 mM to approximately 150 mM, approximately 50 mM to approximately 150 mM, approximately 60 mM to approximately 140 mM, approximately 70 mM to approximately 130 mM, approximately 80 mM to approximately 120 mM, approximately 90 mM to approximately 110 mM, approximately 95 mM to approximately 105 mM, approximately 30 mM to approximately 70 mM, approximately 35 mM to approximately 65 mM, approximately 40 mM to approximately 60 mM, approximately 45 mM to approximately 55 mM, approximately 2 Methionine (or cysteine ​​or glutathione) in concentrations of 0mM to approximately 50mM, approximately 25mM to approximately 45mM, approximately 30mM to approximately 40mM, approximately 30mM to approximately 35mM, approximately 5mM to approximately 45mM, approximately 10mM to approximately 40mM, approximately 15mM to approximately 35mM, approximately 20mM to approximately 30mM, or approximately 20mM to approximately 25mM); sodium ascorbate in concentrations of 5mM to 150mM (e.g., approximately 10mM to approximately 150mM, approximately 20mM to approximately 150mM, approximately 30mM to approximately 150mM, approximately 40mM to approximately 150mM) M, sodium ascorbate in concentrations of approximately 50mM to 150mM, 60mM to 140mM, 70mM to 130mM, 80mM to 120mM, 90mM to 110mM, 95mM to 105mM, 10mM to 50mM, 15mM to 45mM, 20mM to 40mM, 25mM to 35mM, 30mM to 35mM, 5mM to 45mM, 10mM to 40mM, 15mM to 35mM, 20mM to 30mM, and 20mM to 25mM);and 5mm to approximately 150mm (for example, approximately 10mm to approximately 150mm, approximately 20mm to approximately 150mm, approximately 30mm to approximately 150mm, approximately 40mm to approximately 150mm, approximately 50mm to approximately 150mm, approximately 60mm to approximately 140mm, approximately 70mm to approximately 130mm, approximately 80mm to approximately 120mm, approximately 90mm to approximately 110mm, approximately 95mm to approximately 105mm, approximately 30mm to approximately 70mm, approximately 35mm to approximately It may contain one or more histidines in concentrations of 65 mM, approximately 40 mM to 60 mM, approximately 45 mM to 55 mM, approximately 20 mM to 50 mM, approximately 25 mM to 45 mM, approximately 30 mM to 40 mM, approximately 30 mM to 35 mM, approximately 5 mM to 45 mM, approximately 10 mM to 40 mM, approximately 15 mM to 35 mM, approximately 20 mM to 30 mM, or approximately 20 mM to 25 mM.

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

[0164] The following are non-limiting doses of gamma irradiation sufficient to reduce the bioburden of any of the compositions provided herein. 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, percentages, and / or temperatures (any combination) for gamma irradiation as described herein. The bioburden of a composition can be determined, for example, by taking a sample from a composition that would contain self-replicating biological contaminants present in the composition, for example by stomaching, sonication, shaking, vortex mixing, flushing, blending, or scraping with a cotton swab, and qualitatively or quantitatively determining the level of self-replicating biological contaminants present in the sample (for example, by placing the sample in a growth medium that allows the biological contaminants to self-replicate, for example by flattening the sample in a Petri dish or by penetrating the sample with a membrane).

[0165] The amounts of at least one alcohol, at least one antioxidant, and / or at least one chelating agent sufficient to reduce the bioburden of the composition and to improve the binding capacity of the chromatographic resin during treatment can be determined, for example, by the method described in the examples. For example, the level of reduction in binding capacity of a chromatographic resin treated by gamma irradiation in the presence of at least one alcohol (and optionally, at least one further antioxidant and / or chelating agent) can be compared to the level of reduction in binding capacity of a chromatographic resin treated by the same dose of gamma irradiation in the absence of at least one alcohol (and optionally, at least one antioxidant and / or chelating agent). Here, the reduction in the level of reduction in binding capacity of a chromatographic resin treated by gamma irradiation in the presence of at least one alcohol (and optionally, at least one further antioxidant and / or chelating agent) compared to a chromatographic resin treated by gamma irradiation in the absence of at least one alcohol (and optionally, at least one further antioxidant and / or chelating agent) indicates that at least one alcohol (and optionally, antioxidant and / or chelating agent) was present in a sufficient amount to improve the dose of binding capacity of the chromatographic resin during gamma irradiation. Typical methods for determining the binding capacity of a chromatographic resin are described in the examples. Additional examples of methods for determining the binding capacity of chromatography resins are available here. It is publicly known in the technical field.

[0166] Method for reducing bioburden in chromatography resins This specification provides a method for reducing bioburden of a chromatographic resin. The method comprises the steps of exposing a container containing a composition comprising (i) a chromatographic resin and (ii) a liquid containing at least one alcohol (for example, any representative composition comprising a chromatographic resin and a liquid containing at least one alcohol as described herein) to a dose of gamma irradiation sufficient to reduce 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 the dose of gamma irradiation.

[0167] Furthermore, a method for reducing bioburden of a chromatographic resin, comprising (i) a chromatographic resin and (ii) a liquid containing at least one alcohol in an amount sufficient to improve the loss of binding capacity of the chromatographic resin after / during exposure to gamma irradiation (for example, any composition containing the chromatographic resin and the liquid containing at least one alcohol as described herein), and a container being subjected to gamma irradiation at a dose sufficient to reduce bioburden of the container and the chromatographic resin for approximately 0.1 kGy / hour to approximately 6 kGy / hour (for example, approximately 0.1 kGy / hour to approximately 5.5 kGy / hour, approximately 0.1 kGy / hour to approximately 5.0 kGy / hour, approximately 0.1 kGy / hour to approximately 4.5 kGy / hour, approximately 0.1 kGy / hour to approximately 4.0 kGy / hour, approximately 0.1 kGy / hour to approximately 3.5 kGy / hour, approximately 0.1 kGy / hour to approximately 3.0 kGy / hour, approximately 0.1 kG kGy / hour ~ approximately 2.5 kGy / hour, approximately 0.1 kGy / hour ~ approximately 2.0 kGy / hour, approximately 0.1 kGy / hour ~ approximately 1.5 kGy / hour, approximately 0.1 kGy / hour ~ approximately 1.0 kGy / hour, approximately 0.5 kGy / hour ~ approximately 6 kGy / hour, approximately 0.5 kGy / hour ~ approximately 5.5 kGy / hour, approximately 0.5 kGy / hour ~ approximately 5.0 kGy / hour, approximately 0.5 kGy / hour ~ approximately 4.5 kGy / hour, approximately 0.5 kGy / hour ~ approximately 4.0 kGy / hour, approximately 0.5 kGy / hour Methods are also provided that include exposure to gamma ray irradiation at a rate of approximately 3.5 kGy / hour, approximately 0.5 kGy / hour to approximately 3.0 kGy / hour, approximately 0.5 kGy / hour to approximately 2.5 kGy / hour, and approximately 0.5 kGy / hour to approximately 2.0 kGy / hour, and / or at a temperature of approximately 4°C to approximately 25°C (for example, approximately 4°C to approximately 20°C, approximately 4°C to approximately 15°C, approximately 4°C to approximately 10°C, approximately 10°C to approximately 25°C, approximately 10°C to approximately 20°C, approximately 10°C to approximately 15°C, or approximately 15°C to approximately 25°C).

[0168] Some embodiments of any of these methods involve, before and / or after the exposure step, a container or composition containing a liquid comprising a chromatographic resin and at least one alcohol (e.g., any container or any composition containing a liquid comprising a chromatographic resin and at least one alcohol as described herein) being exposed for approximately 1 hour to approximately 1 year, approximately 1 hour to approximately 11 months, approximately 1 hour to approximately 10 months, approximately 1 hour to approximately 9 months, approximately 1 hour to approximately 8 months, approximately 1 hour to approximately 7 months, approximately 1 hour to approximately 6 months, approximately 1 hour to approximately 5 months, approximately 1 hour to approximately 4 months, approximately 1 hour to approximately 3 months, approximately 1 hour to approximately 2 months, approximately 1 hour to approximately 1 month, approximately 1 hour to approximately 2 weeks, approximately 1 hour to approximately 1 week, approximately 1 hour to approximately 5 days, approximately 1 hour to approximately 2 days, approximately 1 hour to approximately 1 day, approximately 1 hour to approximately 12 hours, approximately 1 hour to approximately 6 hours, approximately 6 hours to approximately 1 year, approximately 6 hours to approximately 11 months, approximately 6 hours to approximately 10 months, approximately 6 hours to approximately 9 months, approximately 6 hours to approximately 8 months, approximately 6 hours to approximately 7 months, approximately 6 hours to approximately 6 months, approximately 6 hours to approximately 5 months, approximately 6 hours to approximately 4 months, approximately 6 hours to approximately 3 months, approximately 6 hours to approximately 2 months, approximately 6 hours to approximately 1 month, approximately 6 hours to approximately 2 weeks, approximately 6 hours to approximately 1 week, approximately 6 hours to approximately 5 days, approximately 6 hours to approximately 2 days, approximately 6 hours to approximately 1 day, approximately 6 hours to approximately 12 hours, approximately 12 hours to approximately 1 year, approximately 12 hours to approximately 11 months, approximately 12 hours to approximately 10 months, approximately 12 hours to approximately 9 months, approximately 12 hours to approximately 8 months, approximately 12 hours to approximately 7 months, approximately 12 hours to approximately 6 months, approximately 12 hours to approximately 5 months, approximately 12 hours to approximately 4 months, approximately 12 hours to approximately 3 months, approximately 12 hours to approximately 2 months, approximately 12 hours to approximately 1 month, approximately 12 hours to approximately 2 weeks, approximately 12 hours to approximately 1 week, approximately 12 hours to approximately 5 days, approximately 12 hours to approximately 2 days, approximately 12 hours to approximately 1 day, approximately 1 day to approximately 1 year, approximately 1 day to approximately 11 months, approximately 1 day to approximately 10 months, approximately 1 day to approximately 9 months, approximately 1 day to approximately 8 months, approximately 1 day to approximately 7 months, approximately 1 day to approximately 6 months, approximately 1 day to approximately 5 months, approximately 1 day to approximately 4 months, approximately 1 day to approximately 3 months, approximately 1 day to approximately 2 months, approximately 1 day to approximately 1 month, approximately 1 day to approximately 2 weeks, approximately 1 day to approximately 1 week, approximately 1 day to approximately 5 days, approximately 1 day to approximately 2 days, approximately 2 days to approximately 1 year, approximately 2 days to approximately 11 months, approximately 2 days to approximately 10 months, approximately 2 days to approximately 9 months, approximately 2 days to approximately 8 months, approximately 2 Days to approximately 7 months, approximately 2 days to approximately 6 months, approximately 2 days to approximately 5 months, approximately 2 days to approximately 4 months, approximately 2 days to approximately 3 months, approximately 2 days to approximately 2 months, approximately 2 days to approximately 1 month, approximately 2 days to approximately 2 weeks, approximately 2 days to approximately 1 week, approximately 2 days to approximately 5 days, approximately 5 days to approximately 1 year, approximately 5 days to approximately 11 months, approximately 5 days to approximately 10 months, approximately 5 days to approximately 9 months, approximately 5 days to approximately 8 months, approximately 5 days to approximately 7 months, approximately 5 days to approximately 6 months, approximately 5 days to approximately 5 months, approximately 5 days to approximately 4 months, approximately 5 days to approximately 3 months, approximately 5 days to approximately 2 months, approximately 5 days to approximately 1 month, approximately 5 days to approximately 2 weeks, approximately 5 days to approximately 1 week, approximately 1 week to approximately 1 year, approximately 1 week to approximately 11 months, approximately 1 week to approximately 10 months, approximately 1 week to approximately 9 months, approximately 1 Weeks to approximately 8 months, approximately 1 week to approximately 7 months, approximately 1 week to approximately 6 months, approximately 1 week to approximately 5 months, approximately 1 week to approximately 4 months, approximately 1 week to approximately 3 months, approximately 1 week to approximately 2 months, approximately 1 week to approximately 1 month, approximately 1 week to approximately 2 weeks, approximately 2 weeks to approximately 1 year, approximately 2 weeks to approximately 11 months, approximately 2 weeks to approximately 10 months, approximately 2 weeks to approximately 9 months, approximately 2 weeks to approximately 8 months, approximately 2 weeks to approximately 7 months, approximately 2 weeks to approximately 6 months, approximately 2 weeks to approximately 5 months, approximately 2 weeks to approximately 4 months, approximately 2 weeks to approximately 3 months, approximately 2 weeks to approximately 2 months, approximately 2 weeks to approximately 1 month, approximately 1 month to approximately 1 year, approximately 1 month to approximately 11 months, approximately 1 month to approximately 10 months, approximately 1 month to approximately 9 months, approximately 1 month to approximately 8 months, approximately 1 month to approximately 7 months, approximately 1 month to approximately 6 months, approximately 1 month to approximately 5 months, approximately 1 month to approximately 4 months, approximately 1 month to approximately 3 months, approximately 1 month to approximately 2 months, approximately 2 months to approximately 1 year, approximately 2 months to approximately 11 months, approximately 2 months to approximately 10 months, approximately 2 months to approximately 9 months, approximately 2 months to approximately 8 months, approximately 2 months to approximately 7 months, approximately 2 months to approximately 6 months, approximately 2 months to approximately 5 months, approximately 2 months to approximately 4 months, approximately 2 months to approximately 3 months, approximately 3 months to approximately 1 year, approximately 3 months to approximately 11 months, approximately 3 months to approximately 10 months, approximately 3 months to approximately 9 months, approximately 3 months to approximately 8 months, approximately 3 months to approximately 7 months, approximately 3 months to approximately 6 months, approximately 3 months to approximately 5 months, approximately 3 months to approximately 4 months, approximately 4 months to approximately 1 year,Approximately 4 months to 11 months, approximately 4 months to 10 months, approximately 4 months to 9 months, approximately 4 months to 8 months, approximately 4 months to 7 months, approximately 4 months to 6 months, approximately 4 months to 5 months, approximately 5 months to 1 year, approximately 5 months to 11 months, approximately 5 months to 10 months, approximately 5 months to 9 months, approximately 5 months to 8 months, approximately 5 months to 7 months, approximately 5 months to 6 months, approximately 6 months to 1 year, approximately 6 months to 11 months, approximately 6 months to 10 months, approximately 6 months to 9 months, approximately 6 months to 8 months, approximately 6 months to 7 months, approximately 7 months to 1 year, approximately 7 months to 11 months, approximately 7 months to 10 months, approximately 7 months to 9 months, approximately 7 months For example, periods ranging from 1 month to approximately 8 months, 8 months to approximately 1 year, 8 months to approximately 11 months, 8 months to approximately 10 months, 8 months to approximately 9 months, 9 months to approximately 1 year, 9 months to approximately 11 months, 9 months to approximately 10 months, 10 months to approximately 1 year, 10 months to approximately 11 months, or approximately 11 months to approximately 1 year, such as approximately 4°C to approximately 40°C, approximately 4°C to approximately 35°C, approximately 4°C to approximately 30°C, approximately 4°C to approximately 28°C, approximately 4°C to approximately 26°C, approximately 4°C to approximately 24°C, approximately 4°C to approximately 22°C, approximately 4°C to approximately 20°C, approximately 4°C to approximately 18°C, approximately 4°C to approximately 16°C, approximately 4°C to approximately 14°C, approximately 4°C to approximately 12°C, approximately 4°C to approximately 10°C, approximately 4°C to approximately 8°C, and approximately 4°C. ~6°C, 6°C~40°C, 6°C~35°C, 6°C~30°C, 6°C~28°C, 6°C~26°C, 6°C~24°C, 6°C~22°C, 6°C~20°C, 6°C~18°C, 6°C~16°C, 6°C~14°C, 6°C~12°C, 6°C~10°C, 6°C ℃~approx. 8℃, approx. 28℃~approx. 40℃, approx. 8℃~approx. 35℃, approx. 8℃~approx. 30℃, approx. 8℃~approx. 28℃, approx. 8℃~approx. 26℃, approx. 8℃~approx. 24℃, approx. 8℃~approx. 22℃, approx. 8℃~approx. 20℃, approx. 8℃~approx. 18℃, approx. 8℃~approx. 16℃, approx. 8℃~approx. 14℃, approx. 8℃~approx. 12℃, approx. 8℃~approx. 10℃, approx. 10℃ to approximately 40℃, approximately 10℃ to approximately 35℃, approximately 10℃ to approximately 30℃, approximately 10℃ to approximately 28℃, approximately 10℃ to approximately 26℃, approximately 10℃ to approximately 24℃, approximately 10℃ to approximately 22℃, approximately 10℃ to approximately 20℃, approximately 10℃ to approximately 18℃, approximately 10℃ to approximately 16℃, approximately 10℃ to approximately 14℃, approximately 10℃ to approximately 12℃, approximately 12℃ to Approximately 40°C, approximately 12°C to 35°C, approximately 12°C to 30°C, approximately 12°C to 28°C, approximately 12°C to 26°C, approximately 12°C to 24°C, approximately 12°C to 22°C, approximately 12°C to 20°C, approximately 12°C to 18°C, approximately 12°C to 16°C, approximately 12°C to 14°C, approximately 14°C to 40°C, approximately 14°C to 35°C,Approximately 14℃ to approximately 30℃ °C, approximately 14°C to 28°C, approximately 14°C to 26°C, approximately 14°C to 24°C, approximately 14°C to 22°C, approximately 14°C to 20°C, approximately 14°C to 18°C, approximately 14°C to 16°C, approximately 16°C to 40°C, approximately 16°C to 35°C, approximately 16°C to 30°C, approximately 16°C to 28°C, approximately 16°C to 26°C, approximately 16°C to 24°C, approximately 16°C ~22℃, approx. 16℃~approx. 20℃, approx. 16℃~approx. 18℃, approx. 18℃~approx. 40℃, approx. 18℃~approx. 35℃, approx. 18℃~approx. 30℃, approx. 18℃~approx. 28℃, approx. 18℃~approx. 26℃, approx. 18℃~approx. 24℃, approx. 18℃~approx. 22℃, approx. 18℃~approx. 20℃, approx. 20℃~approx. 40℃, approx. 20℃~approx. 35℃, approx. 20℃~approx. 30℃, Approximately 20°C to 28°C, approximately 20°C to 26°C, approximately 20°C to 24°C, approximately 20°C to 22°C, approximately 22°C to 40°C, approximately 22°C to 35°C, approximately 22°C to 30°C, approximately 22°C to 28°C, approximately 22°C to 26°C, approximately 22°C to 24°C, approximately 24°C to 40°C, approximately 24°C to 35°C, approximately 24°C to 30°C, approximately 24°C to 40°C This may include storage at temperatures of 28°C, approximately 24°C to 26°C, approximately 26°C to 40°C, approximately 26°C to 35°C, approximately 26°C to 30°C, approximately 26°C to 28°C, approximately 28°C to 40°C, approximately 28°C to 35°C, approximately 28°C to 30°C, approximately 30°C to 40°C, approximately 30°C to 35°C, or approximately 35°C to 40°C.

[0169] In the methods described in this paragraph, the binding capacity of the gamma-irradiated chromatographic resin produced by these methods is higher than that of the gamma-irradiated chromatographic resin irradiated at a rate greater than 6.1 kGy / hour, or at a temperature higher than 25°C.

[0170] Chromatographic resins 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. Chromatographic resins can be exposed to gamma ray irradiation at temperatures of approximately -25°C to approximately 0°C, or approximately 0°C to approximately 25°C. Chromatographic resins can be exposed to gamma ray irradiation at temperatures of approximately 0.1 kGy to approximately 100 kGy, approximately 1 kGy to approximately 100 kGy, approximately 1 kGy to approximately 90 kGy, approximately 1 kGy to approximately 80 kGy, approximately 1 kGy to approximately 70 kGy, approximately 1 kGy to approximately 65 kGy, approximately 5 kGy to approximately 65 kGy, approximately 10 kGy to approximately 60 kGy, approximately 10 kGy to approximately 55 kGy, approximately 10 kGy to approximately 50 kGy, and approximately 10 kGy to approximately 50 kGy. The chromatographic resin can be exposed to gamma ray irradiation at doses of approximately 45 kGy, approximately 10 kGy to approximately 40 kGy, approximately 10 kGy to approximately 35 kGy, approximately 10 kGy to approximately 30 kGy, approximately 15 kGy to approximately 50 kGy, approximately 15 kGy to approximately 45 kGy, approximately 15 kGy to approximately 40 kGy, approximately 15 kGy to approximately 35 kGy, approximately 20 kGy to approximately 30 kGy, or approximately 23 kGy to approximately 27 kGy. The chromatographic resin can be exposed to gamma ray irradiation at doses of approximately 1 × 10⁻¹⁶ of the chromatographic resin. -6 Or less, approximately 1 × 10 -7 Or less, approximately 10 x 10 -8 Or less, approximately 1 × 10 -11 Or less, or about 1 × 10 -12 Or less, or about 1 × 10 -6 ~Approx. 1×10 -12 , about 1×10 -6 ~Approx. 1×10 -11 , about 1×10 -6 ~Approx. 1×10 -10 , about 1×10 -6 ~Approx. 1×10 -9 , about 1×10 -6 ~Approx. 1×10 -8 , 1 x 10 -6 ~Approx. 1×10 -7 , about 1×10 -7 ~Approx. 1×10 -12 , about 1×10 -7 ~Approx. 1×10 -11 , about 1×10 -7 ~Approx. 1×10 -10 , about 1×10 -7 ~Approx. 1×10 -9, about 1×10 -7 ~Approx. 1×10 -8 , about 1×10 -8 ~Approx. 1×10 -12 , about 1×10 -8 ~Approx. 1×10 -11 , about 1×10 -8 ~Approx. 1×10 -10 , or approximately 1 x 10 -8 ~Approx. 1×10 -9 It can be exposed to gamma ray irradiation in a dose sufficient to produce a level of sterility assurance.

[0171] The dose of gamma 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 dose of gamma irradiation can be compared to the bioburden level of an untreated (e.g., control, unirradiated) chromatographic resin, and the reduction in the bioburden level of the gamma-irradiated chromatographic resin compared to the untreated chromatographic resin indicates that the dose of gamma irradiation is sufficient. This demonstrates that it is sufficient to reduce the bioburden of chromatography resins. Typical methods for determining the level of bioburden in a composition (e.g., chromatography resin) are described herein. Additional methods for determining the level of bioburden in a composition (e.g., chromatography resin) are known in the art.

[0172] In any of these methods, the chromatography resin may be anion exchange chromatography resin, cation exchange chromatography resin, size exclusion chromatography resin, hydrophobic interaction chromatography resin, or affinity chromatography resin, or any combination thereof. Non-limiting examples of affinity chromatography resins may include protein or peptide ligands (e.g., about 5 to about 100 amino acids, about 5 to about 90 amino acids, about 5 to about 80 amino acids, about 5 to about 70 amino acids, about 5 to about 60 amino acids, about 5 to about 50 amino acids, about 5 to about 40 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, or about 5 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 contains a protein ligand (e.g., protein A). Examples of additional affinity chromatography resins include cofactor ligands, substrate ligands, metal ligands, product ligands, or aptamer ligands. In some examples, the chromatography resin is a biomodal chromatography resin (e.g., anion exchange chromatography resin and hydrophobic interaction chromatography resin). The chromatography resin can also be anion exchange chromatography resin (e.g., anion exchange chromatography resin containing an N-benzyl-N-methylethanolamine group).

[0173] The container containing the chromatography resin may 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 comprising (i) a chromatography resin and (ii) a liquid containing at least one alcohol (e.g., any of the representative compositions described herein) may be present in a sealed container (e.g., a slurry in a sealed container or a filled chromatography resin in a sealed container (e.g., a chromatography column)). The container used in the methods described herein may be a disposable chromatography column. In some embodiments, the container used in the methods described herein is a disposable chromatography column in a blister pack. A container (e.g., a storage vessel or chromatography column) can have an internal total volume of approximately 1 mL to approximately 1 L (e.g., approximately 1 mL to 900 mL, approximately 1 mL to 800 mL, approximately 1 mL to 700 mL, approximately 1 mL to 600 mL, approximately 1 mL to 500 mL, approximately 1 mL to 450 mL, approximately 1 mL to 400 mL, approximately 1 mL to 350 mL, approximately 1 mL to 300 mL, approximately 1 mL to 250 mL, approximately 1 mL to 200 mL, approximately 1 mL to 150 mL, approximately 1 mL to 100 mL, approximately 1 mL to 75 mL, approximately 1 mL to 50 mL, approximately 1 mL to 40 mL, approximately 1 mL to 30 mL, or approximately 1 mL to 20 mL).

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

[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 may 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 may further contain at least one antioxidant (e.g., at least one antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoids, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoids, phenolpropanoid acids, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and dimethylmethoxychromanol) and / or at least one chelating agent (e.g., at least one chelating agent selected from the group consisting of EDTA, DMPS, DMSA, metallothionein, and desferoxamine).

[0176] This specification describes typical methods for determining / confirming the amounts 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 chromatographic resin during treatment with gamma irradiation at a dose sufficient to reduce the bioburden of the composition. Additional methods for determining / confirming the amounts of at least one antioxidant and / or chelating agent sufficient to improve the loss of binding capacity of a chromatographic resin during treatment with gamma irradiation at a dose sufficient to reduce the bioburden of the composition are known in the art.

[0177] Furthermore, this specification also provides reduced bioburden chromatographic resins produced by any of the methods described herein (e.g., reduced bioburden chromatographic resins provided in storage containers, e.g., sealed storage containers). The reduced bioburden chromatographic resin produced using any of the methods described herein is approximately 1 × 10⁻⁶ -6 Or less, approximately 1 × 10 -7 Or less, approximately 10 x 10 -8 Or less, approximately 1 × 10 -11 Or less, or about 1 × 10 -12 Or less, or about 1 × 10 -6 ~Approx. 1×10 -12 , about 1×10 -6 ~Approx. 1×10 -11 , about 1×10 -6 ~Approx. 1×10 -10 , about 1×10 -6 ~Approx. 1×10 -9 , about 1×10 -6 ~Approx. 1×10 -8 , 1 x 10 -6 ~Approx. 1×10 -7 , about 1×10 -7 ~Approx. 1×10 -12 , about 1×10 -7 ~Approx. 1×10 -11 , about 1×10 -7 ~Approx. 1×10 -10 , about 1×10 -7 ~Approx. 1×10 -9 , about 1×10 -7~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. When the same protein is used to test the binding capacity of both the chromatography resin with reduced bioburden produced by any of the methods described herein and the untreated control chromatography resin, 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 chromatography resin having a reduced bioburden ​Also provided herein is a method for producing a reduced-packed chromatography column, comprising the steps of preparing a reduced-bioburden chromatography resin generated 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 generated 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 generated by the methods described herein. Any of the chromatography columns packed with reduced bioburden generated 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 ~Approx. 1×10 -12 , about 1×10 -7 ~Approx. 1×10 -11 , about 1×10 -7 ~Approx. 1×10 -10 , about 1×10 -7 ~Approx. 1×10 -9 , about 1×10 -7 ~Approx. 1×10 -8 , about 1×10 -8 ~Approx. 1×10 -12 , about 1×10 -8 ~Approx. 1×10 -11 , about 1×10 -8 ~Approx. 1×10 -10 , or approximately 1 x 10 -8 ~Approx. 1×10 -9 It can have a level of sterility assurance. Any chromatography column packed with reduced bioburden produced by the method described herein can accommodate 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 affinity chromatography resin described herein or known in the art), hydrophilic interaction chromatography resins, and size exclusion chromatography resins. For example, any chromatography column packed with reduced bioburden described herein may contain an affinity chromatography resin containing a protein ligand (e.g., protein A). A chromatography column packed with reduced bioburden described herein may contain an anion exchange chromatography resin (e.g., an anion exchange chromatography resin containing an N-benzyl-N-methyl-ethanolamine group).

[0180] How to perform chromatography on reduced bioburden The methods described herein include the use of a chromatography column packed with reduced bioburden provided herein, and the methods described herein include the use of one or two MCCSs including at least one chromatography column packed with reduced bioburden provided herein. The gamma-irradiated chromatography resin may be any type of resin described herein (or any type of chromatography resin known in the art).

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

[0182] A chromatography column packed with reduced bioburden used in any of the methods described herein is approximately 1 × 10⁶ -3 ~Approx. 1×10 -12 , about 1×10 -4 ~Approx. 1×10 -12 , 1 x 10 -5 ~Approx. 1×10 -11 , about 1×10 -5 ~Approx. 1×10 -10 , about 1×10 -5 ~Approx. 1×10 -9 , about 1×10 -6 ~Approx. 1×10 -9 , or approximately 1 x 10 -6 ~Approx. 1×10 -8 It can have a sterility assurance level (SAL).

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

[0184] Recombinant therapeutic proteins The recombinant proteins described herein may be recombinant therapeutic proteins. Non-limiting examples of recombinant therapeutic proteins that may be produced by the methods provided herein include immunoglobulins (including light-chain 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). Recombinant therapeutic proteins may be artificially modified antigen-binding polypeptides comprising at least one multifunctional recombinant protein scaffold (see, for example, Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent Application Publication No. 2012 / 0164066, incorporated herein by reference in its entirety). Non-exclusive examples of recombinant therapeutic proteins that are antibodies include panitumumab, omalizumab, avagobomab, absiximab, actoxumab, adalimumab, adekatumumab, aferimomab, aftuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatsuximab, anatumomab, apolizumab, atinumab, tocilizumab, and basil Examples include izimab, bectumomab, belimumab, bevacizumab, bisilomab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, efangumab, ertumaxomab, etalacizumab, 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 method include alglucosidase alpha, laronidase, abatacept, galsulfase, lutropin alpha, antihemophilic factor, agalsidase beta, interferon beta-1a, derbepoetin alpha, tenecteplase, etanercept, coagulation factor IX, follicle-stimulating hormone, interferon beta-1a, imiglucerase, dorunase alpha, epoetin alpha, and alteplase.

[0185] The secreted soluble recombinant therapeutic proteins can be recovered from liquid culture media (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). Various different methods for removing liquid culture media from cells (e.g., mammalian cells) are known in the art and include, for example, centrifugation, filtration, pipetting, and / or aspiration. The secreted recombinant therapeutic proteins can then be recovered from the liquid culture media and further purified using various biochemical techniques, including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography, anion exchange chromatography, or hydrophobic interaction chromatography, or any combination thereof) and / or filtration (e.g., molecular weight cutoff 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., recombinant therapeutic protein). For example, an affinity chromatography column may include steps of loading a fluid containing the target recombinant protein into the affinity chromatography column, washing the column to remove undesirable biological material (e.g., contaminating proteins and / or small molecules), eluting the target recombinant protein bound to the column, and re-equilibriumizing the column. A chromatography cycle using a cation and / or anion exchange chromatography column in which the target recombinant protein is bound to the chromatography resin during the loading step may include steps of loading a fluid containing the target protein into the column, washing the column to remove undesirable biological material, eluting the target recombinant protein bound to the column, and re-equilibriumizing the column. In another example, a chromatography cycle using a cation and / or anion exchange chromatography column in which undesirable biological material binds to the chromatography resin during the loading process but the target recombinant protein does not bind may include the steps of loading a fluid containing the target protein into the column, collecting the target recombinant protein in the pass-through fraction, and re-equilibriumating the column. As is well known in the art, any single step in a chromatography cycle may include a single buffer or multiple buffers (e.g., two or more buffers), and any one or more single steps in a chromatography cycle may include a buffer gradient.Any combination of various well-known aspects of a single chromatography cycle can be used in these methods, for example, by varying different chromatography resins, flow rates, buffers, column void volumes, column bed volumes, the volume of buffer used in each step, the volume of fluid containing the target protein, and the number and type of buffer used in each step, in any combination.

[0187] How to perform column chromatography with reduced bioburden This specification provides methods for performing chromatography of reduced bioburden. These methods include the steps of preparing a chromatography column packed with reduced bioburden, which is produced using one of the methods described herein, and performing column chromatography using the chromatography column packed with reduced bioburden. The chromatography column packed with reduced bioburden may contain at least one of the chromatography resins described herein in any combination. For example, the chromatography resin present in the chromatography column packed with reduced bioburden may be an affinity resin containing a protein ligand (e.g., protein A), or it may contain an anion exchange chromatography resin. The chromatography column packed with reduced bioburden may have one of the representative volumes described herein. The chromatography column packed with reduced bioburden may have one of the shapes described herein or known in the art (e.g., cylindrical, substantially cylindrical, or elliptical). Column chromatography performed using these methods can be used to purify or isolate recombinant proteins (e.g., recombinant therapeutic proteins described herein or known in the art). In some cases, a chromatography column packed with reduced bioburden is part of a multi-column chromatography system (MCCS), which can be part of a periodic countercurrent chromatography system (PCCS), for example.

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

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

[0190] Column chromatography can include two or more (for example, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, thirty or more, thirty-five or more, forty or more, forty-five or more, fifteen or more, fifteen or more, sixteen or more, sixteen or more, seventeen or more, seventeen or more, eightteen or more, eightteen or more, nineteen or more, nineteen or more, or one hundred or more) cycles of chromatography. In some cases, column chromatography is performed continuously for a period of at least 3 days (for example, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 days).

[0191] In some embodiments, the chromatographic resin in a chromatographic column packed with reduced bioburden showed a binding capacity of approximately 75% to 100% (e.g., approximately 76% to 98%, 76% to 96%, 76% to 94%, 76% to 92%, 76% to 90%, 78% to 100%, 78% to 98%, 78% to 96%, 78% to 94%, 78% to 92%, 78% to 90%, and 8% compared to the same resin that had not been treated with gamma irradiation (when testing the binding capacity of both resins using the same protein). 0%~approx. 100%, approx. 80%~approx. 98%, approx. 80%~approx. 96%, approx. 80%~approx. 94%, approx. 80%~approx. 92%, approx. 80%~approx. 90%, approx. 82%~approx. 100%, approx. 82%~approx. 98%, approx. 82%~approx. 96%, approx. 82%~approx. 94%, approx. 82%~approx. 92%, approx. 82%~approx. 90%, approx. 84%~approx. 100%, approx. 84%~approx. 98%, approx. 84%~approx. 96%, approx. 84%~approx. 94%, approx. 84%~approx. 92%, approx. 84%~approx. 90%, approx. 86%~approx. 100%, approx. 86%~approx. 98%, approx. 86%~approx. 96%, approx. 86%~approx. 94%, approx. 86%~approx. 92%, approx. 88%~approx. 10 It has binding capacity ratios of 0%, approximately 88% to 98%, approximately 88% to 96%, approximately 88% to 94%, approximately 90% to 100%, approximately 90% to 98%, approximately 90% to 96%, approximately 92% to 100%, or approximately 92% to 98% (e.g., evaluated immediately after exposure to gamma radiation).

[0192] Integrated, closed or substantially closed, continuous method for producing recombinant proteins This specification provides integrated, closed or substantially closed, continuous methods for producing purified recombinant proteins (e.g., recombinant therapeutic proteins). These methods include preparing a liquid culture medium containing substantially cell-free recombinant proteins (e.g., recombinant therapeutic proteins).

[0193] Some methods involve continuously supplying liquid culture medium to a multi-column chromatography system (MCCS) comprising at least one chromatography column packed with reduced bioburden provided herein, wherein these methods utilize the reduced bioburden buffer and operate continuously from the liquid culture medium to the eluate from the MCCS, which is a purified recombinant protein (e.g., a therapeutic protein drug).

[0194] Several methods include continuously supplying liquid culture medium to a first MCCS (MCCS1), capturing 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 purified recombinant protein, wherein at least one column in MCCS1 and / or MCCS2 is a chromatography column packed with reduced bioburden provided herein, and the method utilizes and integrates the reduced bioburden buffer and operates continuously from liquid culture medium to purified recombinant protein.

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

[0196] The method described herein provides continuous and time-efficient production of purified recombinant proteins from liquid culture media containing recombinant proteins. For example, the elapsed time from supplying liquid culture media containing therapeutic proteins to MCCS or MCCS1 to elution of recombinant proteins from MCCS or MCCS2, respectively, is, for example, about 4 hours to about 48 hours (inclusive), 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, It can be approximately 6 to 20 hours, approximately 6 to 18 hours, approximately 6 to 14 hours, approximately 8 to 16 hours, approximately 8 to 14 hours, approximately 8 to 12 hours, approximately 10 to 20 hours, approximately 10 to 18 hours, approximately 10 to 16 hours, approximately 10 to 14 hours, approximately 12 to 14 hours, approximately 10 to 40 hours, approximately 10 to 35 hours, approximately 10 to 30 hours, approximately 10 to 25 hours, approximately 15 to 40 hours, approximately 15 to 35 hours, approximately 15 to 30 hours, approximately 20 to 40 hours, approximately 20 to 35 hours, or approximately 20 to 30 hours (including both ends). In other examples, from supplying the liquid culture medium containing recombinant protein to MCCS or MCCS1, M The time elapsed to the elution of recombinant protein from CCS or MCCS2 is, for example, more than approximately 4 hours but less than approximately 40 hours (inclusive), for example, more than approximately 4 hours but less than approximately 39 hours, approximately 38 hours, approximately 37 hours, approximately 36 hours, approximately 35 hours, approximately 34 hours, approximately 33 hours, approximately 32 hours, approximately 31 hours, approximately 30 hours, approximately 29 hours, approximately 28 hours, approximately 27 hours, approximately 26 hours, approximately 25 hours, approximately 24 hours, approximately 23 hours, approximately 22 hours, approximately 21 hours, approximately 20 hours, approximately 19 hours, approximately 18 hours, approximately 17 hours, approximately 16 hours, approximately 15 hours, approximately 14 hours, approximately 13 hours, approximately 12 hours, approximately 11 hours, approximately 10 hours, approximately 9 hours, approximately 8 hours, approximately 7 hours, approximately 6 hours, approximately 5 hours, or approximately 4.5 hours (inclusive).

[0197] Non-limiting aspects of MCCS (MCCS, MCCS1, and / or MCCS2) that can be used in any of these methods are described in U.S. Provisional Patent Applications No. 61 / 775,060 and No. 61 / 856,390 (incorporated herein by reference, respectively).

[0198] Some representative methods do not utilize a retention step (for example, do not use a reservoir (e.g., a break tank) throughout the entire method). Other methods use up to one, two, three, four, or five reservoirs (e.g., break tanks) throughout the entire method. Any of the methods described herein may utilize up to one, two, three, four, or five reservoirs (e.g., break tanks) throughout the entire method, where each break tank retains only recombinant proteins for an entire period, for example, about 5 minutes to less than 6 hours (inclusive), for example, about 5 minutes to about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes (inclusive).

[0199] Several methods utilize one, two, three, four, five, or six reservoirs (e.g., stagnant tanks) and can have capacities such as 1 mL to approximately 300 mL (including both ends), 1 mL to approximately 280 mL, approximately 260 mL, approximately 240 mL, approximately 220 mL, approximately 200 mL, approximately 180 mL, approximately 160 mL, approximately 140 mL, approximately 120 mL, approximately 100 mL, approximately 80 mL, approximately 60 mL, approximately 40 mL, approximately 20 mL, or approximately 10 mL (including both ends). Any reservoir(s) (e.g., stagnant tank(s)) used (in any of the methods described herein) to hold fluid before supplying it to the MCCS or MCCS1 may have a capacity of, for example, 1 mL to about 100% (including both ends) of the loading volume of the first column of the MCCS, i.e., MCCS1, for example, 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., a stagnant tank(s)) may be used to hold the eluate from MCCS1 before it enters MCCS2, and this reservoir may have a capacity of, for example, 1 mL to about 100% (including both ends) of the loading volume of the first column of MCCS2, for example, 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 provided herein, and any step of the methods provided herein can be performed using other materials.

[0201] Liquid culture medium Liquid culture media containing recombinant proteins that are substantially free of cells (e.g., recombinant therapeutic proteins) can be derived from any source. For example, liquid culture media can be obtained from recombinant cell cultures (e.g., recombinant bacteria, yeast, or mammalian cell cultures). Liquid culture media can be obtained from fed-batch cell (e.g., mammalian cell) cultures (e.g., a fed-batch bioreactor containing a culture of recombinant protein-secreting mammalian cells) or perfusion cell (e.g., mammalian cell) cultures (e.g., a perfusion bioreactor containing a culture of recombinant protein-secreting mammalian cells). Liquid culture media can also be clarified liquid culture media from cultures of recombinant protein-secreting bacterial or yeast cells.

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

[0203] Liquid culture media can be obtained from cultures of recombinant cells (e.g., recombinant bacteria, yeast, or mammalian cells) that produce any of the recombinant proteins described herein or known in the art (e.g., recombinant therapeutic proteins). Some examples of any of the methods described herein may further include the step of culturing recombinant cells (e.g., recombinant bacteria, yeast, or mammalian cells) that produce recombinant proteins (e.g., recombinant therapeutic proteins).

[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: liquid culture media free of animal-derived components, liquid culture media free of serum, liquid culture media containing serum, liquid culture media of known composition, and liquid culture media free of protein. In any of the methods described herein, the liquid culture medium obtained from the culture can be diluted by adding a second fluid (e.g., buffer) before being supplied to the MCCS or MCCS1.

[0205] Liquid culture media containing recombinant proteins that are substantially free of cells can be stored for at least one day (e.g., at least about two days, at least about five days, at least about ten days, at least about fifteen days, at least about twenty days, or at least about thirty days) before supplying the liquid culture media to the MCCS or MCCS1 (e.g., at a temperature below about 15°C (e.g., below about 10°C, below about 4°C, below about 0°C, below about -20°C, below about -50°C, below about -70°C, or below about -80°C)). Alternatively, in some examples, the liquid culture media may be supplied directly from the bioreactor to the MCCS or MCCS1 (e.g., supplied directly from the bioreactor to the MCCS or MCCS1 after a filtration or clarification step).

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

[0207] One or more chromatographic columns that may be present within the MCCS (for example, within MCCS, MCCS1 and / or MCCS2) may have resin volumes between approximately 1 mL and 2 mL, approximately 5 mL, approximately 10 mL, approximately 15 mL, approximately 20 mL, approximately 25 mL, approximately 30 mL, approximately 35 mL, approximately 40 mL, approximately 45 mL, approximately 50 mL, approximately 55 mL, approximately 60 mL, approximately 65 mL, approximately 70 mL, approximately 75 mL, approximately 80 mL, approximately 85 mL, approximately 90 mL, approximately 95 mL, or approximately 100 mL, including upper and lower limits. One or more chromatography columns that may be present in MCCS (for example, MCCS, MCCS1 and / or MCCS2) are available in sizes of approximately 2 mL to 100 mL, 2 mL to 90 mL, 2 mL to 80 mL, 2 mL to 70 mL, 2 mL to 60 mL, 2 mL to 50 mL, 5 mL to 50 mL, 2 mL to 45 mL, and 5 mL. The resin volumes may range from approximately 45 mL, from approximately 2 mL to approximately 40 mL, from approximately 5 mL to approximately 40 mL, from approximately 2 mL to approximately 35 mL, from approximately 5 mL to approximately 35 mL, from approximately 2 mL to approximately 30 mL, from approximately 5 mL to approximately 30 mL, from approximately 2 mL to approximately 25 mL, from approximately 5 mL to approximately 25 mL, from approximately 15 mL to approximately 60 mL, from approximately 10 mL to approximately 60 mL, from approximately 10 mL to approximately 50 mL, and from approximately 15 mL to approximately 50 mL. One or more chromatographic columns in an 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 chromatographic columns in an MCCS (e.g., MCCS, MCCS1 and / or MCCS2) may be, for example, between approximately 0.2 mL / min and approximately 25 mL / min (e.g., between approximately 0.2 mL / min and approximately 20 mL / min, between approximately 0.5 mL / min and approximately 20 mL / min, between approximately 0.2 mL / min and approximately 15 mL / min, between approximately 0.5 mL / min and approximately 15 mL / min, between approximately 0.5 mL / min and approximately 10 mL / min, between approximately 0.5 mL / min and approximately 14 mL / min, between approximately 1.0 mL / min and approximately 25.0 mL / min, or between approximately 1.0 mL / min and approximately 15.0 mL / min).

[0208] Even if one or more chromatography columns within an MCCS (for example, within MCCS, MCCS1 and / or MCCS2) have substantially the same shape, They may or may have substantially different shapes. For example, one or more chromatography columns within an MCCS (e.g., within MCCS, MCCS1 and / or MCCS2) may have substantially the same shape, being circular cylinders or substantially the same shape, being oval cylinders.

[0209] One or more chromatographic membranes that may be present within the MCCS (for example, within MCCS, MCCS1 and / or MCCS2) may be between approximately 1 mL and 500 mL (for example, between approximately 1 mL and 475 mL, approximately 1 mL to 450 mL, approximately 1 mL to 425 mL, approximately 1 mL to 400 mL, approximately 1 mL to 375 mL, approximately 1 mL to 350 mL, approximately 1 mL to 325 mL, approximately 1 mL to 300 mL, approximately 1 mL to 275 mL, approximately 1 mL to 250 mL, approximately 1 mL to 225 mL, approximately 1 mL to 200 mL, approximately 1 mL to 175 mL, approximately 1 mL to 150 mL, approximately 1 mL to 125 mL, approximately 1 mL to 100 mL). It may have a bed volume of approximately 2 mL to 100 mL, approximately 5 mL to 100 mL, approximately 1 mL to 80 mL, approximately 2 mL to 80 mL, approximately 5 mL to 80 mL, approximately 1 mL to 60 mL, approximately 2 mL to 60 mL, approximately 5 mL to 60 mL, approximately 1 mL to 40 mL, approximately 2 mL to 40 mL, approximately 5 mL to 40 mL, approximately 1 mL to 30 mL, approximately 2 mL to 30 mL, approximately 5 mL to 30 mL, between approximately 1 mL and 25 mL, between approximately 2 mL and 25 mL, between approximately 1 mL and 20 mL, between approximately 2 mL and 20 mL, between approximately 1 mL and 15 mL, between approximately 2 mL and 15 mL, between approximately 1 mL and 10 mL, or between approximately 2 mL and 10 mL.

[0210] One or more (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelfth, twelve, thirteen, fourteenth, fif The volume and type of buffer used in the use of MCCS, MCCS1 and / or MCCS2 in any of the methods described herein can also be determined by those skilled in the art (for example, discussed in more detail below). For example, the volume and type(s) of buffer used in 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): the overall yield of the recombinant protein, the activity of the recombinant protein, the level of purity of the recombinant protein, and the removal of biotype contaminants from the fluid (e.g., liquid culture medium) containing the recombinant protein (e.g., absence of active viruses, mycobacteria, yeast, bacteria, or mammalian cells).

[0211] MCCS, MCCS1, and / or MCCS2 may be periodic countercurrent chromatography systems (PCCS). A PCCS may include, for example, two or more chromatography columns (e.g., three or four columns) that are switched to allow for the continuous elution of recombinant proteins from two or more chromatography columns. A PCCS may include two or more chromatography columns, two or more chromatography membranes, or at least one chromatography column and at least one chromatography membrane. Column operation (cycle) generally consists of loading, washing, elution, and regeneration processes. In PCCS, multiple columns are used to operate the same process separately and continuously in a cyclical manner. Because the columns operate in series, the pass-through fraction and washing agent coming out of one column are captured by another column. This unique feature of PCCS allows for the loading of resins with static binding capacity rather than dynamic binding capacity, similar to what is typical in batch mode chromatography. As a result of continuous cycling and elution, the fluid entering the PCCS is continuously processed, and eluates containing recombinant proteins are continuously produced.

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

[0213] In PCCS, the residence time (RT) of recombinant proteins on each chromatography column and / or chromatography membrane within the PCCS can be shortened without increasing the size of the column / membrane, because breakthrough from the first column / membrane can be captured by another column / membrane within the PCCS. For a continuous system, the equation is: V=D * By varying the column / membrane volume (V) and RT using RT, it is possible to design a system to process liquid culture media at any perfusion rate (D).

[0214] One or more unit operations that can be performed by the MCCS or MCC1 and / or MCCS2 used in the method described herein include, for example, capturing recombinant proteins, inactivating viruses present in a fluid containing recombinant proteins, purifying recombinant proteins, polishing recombinant proteins, retaining a fluid containing recombinant proteins (for example, using any exemplary retaining tank(s) described herein), filtering or removing particulate matter and / or cells coming out of a fluid containing recombinant proteins, and adjusting the ion concentration and / or pH of a fluid containing recombinant proteins.

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

[0216] The unit operation to inactivate viruses present in a fluid containing recombinant proteins can be performed using MCCS, MCCS1 and / or MCCS2 (for example, including a chromatography column, chromatography membrane or retention tank) which allows the fluid containing recombinant proteins to be incubated at a pH between approximately 3.0 and 5.0 (for example, between approximately 3.5 and 4.5, between approximately 3.5 and 4.25, between approximately 3.5 and 4.0, between approximately 3.5 and 3.8, or between approximately 3.75) for a period of at least 30 minutes (for example, a period between approximately 30 minutes and 1.5 hours, a period between approximately 30 minutes and 1.25 hours, a period between approximately 0.75 hours and 1.25 hours, or a period of approximately 1 hour).

[0217] The unit operation for purifying recombinant proteins can be performed using one or more MCCSs (e.g., MCCS, MCCS1 and / or MCCS2), including, for example, a chromatography column or chromatography membrane containing a resin that utilizes a capture system. Non-limiting examples of capture mechanisms include protein A-binding capture mechanisms, antibody-binding or antibody-fragment-binding capture mechanisms, substrate-binding capture mechanisms, aptamer-binding capture mechanisms, tag-binding capture mechanisms (e.g., polyHis-tagged capture mechanisms), and cofactor-binding capture mechanisms. Purification can also be performed 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 to purify recombinant proteins are described herein. Further examples of resins that can be used to purify recombinant proteins are known in the art.

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

[0219] Unit operations for holding a fluid containing recombinant proteins can be performed using an MCCS (e.g., MCCS, MCCS1 and / or MCCS2) which includes at least one reservoir (e.g., a stagnant tank), or which includes up to one, two, three, four, or five reservoirs (e.g., stagnant tanks) in total, or MCCS or MCCS1 and MCCS2 combined. For example, each reservoir(s) (e.g., stabling tank(s)) that can be used to achieve the above-mentioned holding unit operation may have a volume between approximately 1 mL and approximately 1 L (e.g., between approximately 1 mL and approximately 800 mL, between approximately 1 mL and approximately 600 mL, between approximately 1 mL and approximately 500 mL, between approximately 1 mL and approximately 400 mL, between approximately 1 mL and approximately 350 mL, between approximately 1 mL and approximately 300 mL, between approximately 10 mL and approximately 250 mL, between approximately 10 mL and approximately 200 mL, between approximately 10 mL and approximately 150 mL, or between approximately 10 mL and approximately 100 mL). The reservoir(s) used in the method described herein (for example, the stagnant tank(s)) may range from 1 mL to about 300 mL, including upper and lower limits, for example 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, including upper and lower limits. The capacity may be between approximately 120 mL, approximately 100 mL, approximately 80 mL, approximately 60 mL, approximately 40 mL, approximately 20 mL, or approximately 10 mL. Any reservoir(s) (e.g., stabling tanks) used to hold the fluid until it enters the MCCS or MCCS1 (in any of the methods described herein) may have a capacity between approximately 1 mL, including upper and lower limits, and approximately 100% of the load on the first column of the first MCCS, or between approximately 1 mL, including upper and lower limits, and approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 10%, or approximately 5% of the load on the first column of the MCCS or MCCS1. Any of the reservoirs (e.g., retention tanks) used to hold the eluate from MCCS1 (containing recombinant protein) before it enters MCCS2 may have a volume of, for example, 1 mL to about 100% (including both ends) of the loading volume of the first column of MCCS2, for example, 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).

[0220] Each reservoir (or reservoir) (e.g., detention tank) can hold a fluid containing 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, a reservoir (or reservoir) (e.g., detention tank) can hold only recombinant protein for a period of time, for example, from about 5 minutes to less than 6 hours, including upper and lower limits, or for a total time of, 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 upper and lower limits. A reservoir (or reservoir) (e.g., detention tank) can be used to perform both the holding of a fluid containing recombinant protein and refrigeration (e.g., at temperatures below 25°C, below 15°C, or below 10°C). Reservoirs can have any shape, including circular cylinders, oval cylinders, or nearly rectangular, sealed, impermeable bags.

[0221] Unit operations for filtering fluids containing recombinant proteins can be performed using MCCS (e.g., MCCS, MCCS1 and / or MCCS2), which include, for example, a chromatography column or chromatography membrane containing a filter or molecular sieve resin. As is well known in the art, a wide variety of submicron filters (e.g., filters with pore sizes 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) are available in the art that can remove any precipitated material and / or cells (e.g., precipitated unfolded proteins; precipitated undesirable host cell proteins; precipitated lipids; bacteria; yeast cells; fungal cells; mycobacteria; and / or mammalian cells). Filters with pore sizes of about 0.2 μm or less than 0.2 μm are known to effectively remove bacteria from fluids containing recombinant proteins. As is well known in the art, chromatography columns or chromatography membranes containing molecular sieve resins can also be used within MCCS (e.g., MCCS, MCCS1 and / or MCCS2) to perform unit operations for filtering fluids containing recombinant proteins.

[0222] Unit operations to adjust the ion concentration and / or pH of a recombinant protein-containing fluid can be performed using an MCCS (e.g., MCCS, MCCS1 and / or MCCS2) that includes and utilizes a buffer adjustment reservoir (e.g., an in-line buffer adjustment reservoir), which adds a new buffer solution to the recombinant protein-containing fluid (e.g., between columns within MCCS, MCCS1 and / or MCCS2, or after the last column in the second-to-last MCCS (e.g., MCCS1) before the recombinant protein-containing fluid is fed into the first column of the next MCCS (e.g., MCCS2)). As can be understood, the in-line buffer conditioning 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 a fluid containing a recombinant protein, an ion (e.g., salt) concentration that increases or decreases compared to a fluid containing a recombinant protein, and / or an agent whose concentration increases or decreases in competing with a recombinant protein for binding to a resin present in at least one chromatography column or at least one chromatography membrane in an MCC (e.g., MCC, MCC1, and / or MCC2)).

[0223] MCC, MCC1, and / or MCC2 can perform two or more unit operations. For example, each of MCC, MCC1, and / or MCC2 can 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 ion 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 a precipitate and / or a specific substance 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 a precipitate and / or a specific particulate substance from a fluid containing the recombinant protein, and adjusting the ion concentration and / or pH of a liquid containing the recombinant protein.

[0224] Capture a recombinant protein This 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 various 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 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 MCCS or MCCS1. Any of the typical means of filtering a 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 MCCS or MCCS1.

[0227] In the method described herein, the capture of the recombinant protein from the liquid culture medium is MCC The process is carried out using S or MCCS1. As can be recognized in the art, in order to achieve the capture of recombinant proteins, at least one chromatographic column or at least one chromatographic membrane of MCCS or MCCS1 must contain a resin that utilizes a capture mechanism (e.g., any of the typical capture mechanisms described herein) or a resin that can perform cation exchange, anion exchange, 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 (in which case the captured antigen is specifically recognized by the recombinant antibody or antibody fragment). If the recombinant protein is an enzyme, the capture mechanism can use an antibody or antibody fragment that specifically binds to the enzyme to capture the recombinant enzyme, a substrate of the enzyme to capture the recombinant enzyme, a 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 recombinant proteins are described herein, and additional resins that can be used to capture recombinant proteins are known in the art. One non-limiting example of a resin that utilizes a protein A binding and 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] Typical non-limiting sizes and shapes of chromatography columns or chromatography membranes present within MCCS or MCCS1 that can be used to capture recombinant proteins are described herein. The liquid culture medium supplied (loaded) into MCCS or MCCS1 may contain recombinant protein in concentrations of approximately 0.05 mg / mL to 100 mg / mL (e.g., recombinant protein in concentrations of approximately 0.1 mg / mL to 90 mg / mL, 0.1 mg / mL to 80 mg / mL, 0.1 mg / mL to 70 mg / mL, 0.1 mg / mL to 60 mg / mL, 0.1 mg / mL to 50 mg / mL, 0.1 mg / mL to 40 mg / mL, 0.1 mg / mL to 30 mg / mL, 0.1 mg / mL to 20 mg / mL, 0.5 mg / mL to 20 mg / mL, 0.1 mg / mL to 15 mg / mL, 0.5 mg / mL to 15 mg / mL, 0.1 mg / mL to 10 mg / mL, or approximately 0.5 mg / mL to 10 mg / mL). The average time required for recombinant proteins to bind to the resin used to perform the capture unit operation can be, for example, about 5 seconds to about 10 minutes (e.g., about 10 seconds to about 8 minutes, about 10 seconds to about 7 minutes, about 10 seconds to about 6 minutes, about 10 seconds to about 5 minutes, about 30 seconds to about 5 minutes, about 1 minute to about 5 minutes, about 10 seconds to about 4 minutes, about 30 seconds to about 4 minutes, or about 1 minute to about 4 minutes).

[0229] As can be recognized in the art, in order to capture recombinant proteins using a chromatography column or chromatography membrane located within an MCCS or MCCS1, a series of chromatography steps must be performed to load, wash, elute, and regenerate the chromatography column or chromatography membrane located within the MCCS or MCCS1. Any of the typical flow rates, buffer volumes, and / or time lengths assigned to each of the series of chromatography steps described herein can be used in one or more of these different series of chromatography steps (e.g., one or more of the series of chromatography steps to load, wash, elute, and regenerate the chromatography column or chromatography membrane located within the MCCS or MCCS1 used to capture recombinant proteins). The non-limiting flow rates, buffer volumes, and / or time lengths to be allocated to each sequential chromatography step that can be used to capture tography columns and / or chromatography membranes are provided below. In addition, typical buffers that can be used with MCCS and / or MCCS1 are listed below.

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

[0231] After loading recombinant proteins onto at least one chromatography column or chromatography membrane in an MCCS or MCCS1 containing a resin capable of performing the unit operation of capture, the at least one chromatography column or chromatography membrane is washed with at least one washing buffer. As can be recognized in the art, at least one (e.g., two, three, or four) washing buffers means that all or most of the non-recombinant proteins are eluted from at least one chromatography column or chromatography membrane without disrupting the interaction between the recombinant proteins and the resin.

[0232] The washing buffer can pass through at least one chromatography column or chromatography membrane at a flow rate of approximately 0.2 mL / min to approximately 25 mL / min (for example, approximately 0.2 mL / min to approximately 20 mL / min, approximately 0.5 mL / min to approximately 20 mL / min, approximately 0.2 mL / min to approximately 15 mL / min, approximately 0.5 mL / min to approximately 15 mL / min, approximately 0.5 mL / min to approximately 10 mL / min, approximately 0.5 mL / min to approximately 14 mL / min, approximately 1.0 mL / min to approximately 25.0 mL / min, and approximately 1.0 mL / min to approximately 15.0 mL / min). The volume of washing buffer used (for example, the total volume of washing buffer when using more than 1 volume of washing buffer) can be, for example, about 1X column volume (CV) to about 15XCV (for example, about 1XCV to about 14XCV, about 1XCV to about 13XCV, about 1XCV to about 12XCV, about 1XCV to about 11XCV, about 2XCV to about 11XCV, about 3XCV to about 11XCV, about 4XCV to about 11XCV, about 5XCV to about 11XCV, or about 5XCV to about 10XCV). The total 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, or about 30 minutes to about 1 hour).

[0233] After washing at least one chromatography column or chromatography membrane in the MCCS or MCCS1 containing a resin capable of performing the unit capture operation, the recombinant protein is eluted by passing the elution buffer through at least one chromatography column or chromatography membrane in the MCCS or MCCS1 containing a resin capable of performing the unit capture operation. The elution buffer is used to elute the chromatographic membrane. The elution buffer can pass through at least one chromatographic column or chromatographic membrane containing a resin capable of performing the unit operation to capture at a flow rate of approximately 0.2 mL / min to approximately 25 mL / min (e.g., approximately 0.2 mL / min to approximately 20 mL / min, approximately 0.5 mL / min to approximately 20 mL / min, approximately 0.2 mL / min to approximately 15 mL / min, approximately 0.5 mL / min to approximately 15 mL / min, approximately 0.5 mL / min to approximately 10 mL / min, between approximately 0.5 mL / min and approximately 6.0 mL / min, between approximately 1.0 mL / min and approximately 5.0 mg / min, between approximately 0.5 mL / min and approximately 14 mL / min, between approximately 1.0 mL / min and approximately 25.0 mL / min, or between approximately 1.0 mL / min and approximately 15.0 mL / min). The volume of elution buffer used to elute recombinant proteins from each of at least one chromatography column or chromatography membrane containing a resin capable of performing the unit operation to be purified may be, for example, about 1X column volume (CV) to about 15XCV (e.g., about 1XCV to about 14XCV, about 1XCV to about 13XCV, about 1XCV to about 12XCV, about 1XCV to about 11XCV, about 2XCV to about 11XCV, about 3XCV to about 11XCV, about 4XCV to about 11XCV, about 5XCV to about 11XCV, or about 5XCV to about 10XCV). The total 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 recombinant protein. For example, the elution buffer may contain different concentrations of salt (e.g., increased salt concentration), different pH (e.g., increased or decreased salt concentration), or molecules that compete with the recombinant protein for binding to the resin on which the unit operation to be captured can be performed. Examples of such elution buffers for each of the typical capture mechanisms described herein are well known in the art.

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

[0235] In some of the methods described herein, MCCS or MCCS1 maintains a fluid containing recombinant protein at a low pH (e.g., pH less than 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, for example, about 1 minute to 1.5 hours (e.g., about 1 hour) and is present in the fluid containing recombinant protein. It includes a reservoir for inactivating the virus present. An example of a reservoir that can be used to perform the unit operation of inactivating the virus is a stirring flask (e.g., a 500-mL stirring flask) or a programmed stirring plate (e.g., a programmed stirring plate) that can hold the fluid containing the recombinant protein for, for example, about 1 minute to 1.5 hours before supplying the fluid containing the recombinant protein into the MCCS2. The reservoir used to perform the unit operation of virus inactivation may be a 500-mL stirring flask with a programmed stirring plate (e.g., a stirring plate programmed to mix the fluid in the reservoir, for example, every 4 hours (e.g., regularly)). Another example of a reservoir that can be used to perform the unit operation of virus inactivation is a plastic bag (e.g., a 500-mL plastic bag) that can hold the fluid containing the recombinant protein for, for example, about 1 minute to 1.5 hours before supplying the fluid containing the recombinant protein into the MCCS2. In some examples, the fluid containing the recombinant protein may already have a low pH (e.g., pH less than 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 supplied into the reservoir used to perform the unit operation of virus inactivation. As can be recognized by those skilled in the art, various other means can be used to perform the unit operation of virus inactivation. For example, UV irradiation of a fluid containing recombinant proteins can also be used to perform unit operations to inactivate viruses. Non-limiting examples of reservoirs that can be used to perform unit operations to inactivate viruses present in a fluid containing recombinant proteins are described herein.

[0236] An MCCS or MCCS1 may include a PCCS comprising four chromatographic columns, where at least three of the four chromatographic columns are used to perform a unit operation to capture recombinant proteins from liquid culture medium, using an MCCS comprising at least one chromatographic column containing a resin capable of performing a unit operation to capture (e.g., any of those described herein). In these examples, the fourth column of the PCC may perform a unit operation to inactivate viruses in a fluid containing recombinant proteins (e.g., any of the typical columns described herein that can be used to achieve inactivation of viruses in a fluid containing recombinant proteins).

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

[0238] Some of the methods described herein may further include a 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 may further include a step of holding or storing (and optionally refrigerating) the eluate from MCCS1 before supplying it to MCCS2. As described herein, this holding or storage step may be carried out using one of the reservoirs described herein (e.g., a backup tank).

[0240] The method described herein may also include a step of filtering the eluate from MCCS1 before supplying it to MCCS2. Either the filtration method described herein or a typical filter can be used to filter the eluate from MCCS1 before supplying it to 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, MCCS2 can be used to perform an operation for purifying and polishing recombinant proteins, and the eluate from MCCS2 is the protein drug substance. MCCS, MCCS1, and / or MCCS2 may include at least one (e.g., two, three, or four) chromatography columns or chromatography membranes that can be used to perform unit operations for purifying recombinant proteins, and at least one (e.g., two, three, or four) chromatography columns or chromatography membranes 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 may 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 may include a resin that can be used to perform anion exchange, cation exchange, molecular sieve chromatography, or hydrophobic interaction chromatography (e.g., any of the typical resins for performing anion exchange, cation exchange, molecular sieve chromatography, or hydrophobic interaction chromatography described herein or known in the art).

[0243] The size, shape, and The size and volume, and / or the size and shape of at least one chromatographic membrane that can be used to perform the unit operation for polishing recombinant proteins, may be any combination of typical sizes, shapes, and volumes of chromatographic columns or chromatographic membranes described herein. As will be apparent to those skilled in the art, the process of purifying or polishing recombinant proteins may include, for example, the steps of loading, washing, eluting, and equilibrating at least one chromatographic column or chromatographic membrane used to perform the unit operation for purifying or polishing recombinant proteins. Typically, the elution buffer coming out of the chromatographic column or chromatographic membrane used to perform the unit operation for purification contains recombinant proteins. Typically, the loading and / or washing buffer coming out of the chromatographic column or chromatographic membrane used to perform the unit operation for polishing contains recombinant proteins.

[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 recombinant proteins may have a volume of, for example, about 2.0 mL to about 200 mL (e.g., about 2.0 mL to about 180 mL, about 2.0 mL to about 160 mL, about 2.0 mL to about 140 mL, about 2.0 mL to about 120 mL, about 2.0 mL to about 100 mL, about 2.0 mL to about 80 mL, about 2.0 mL to about 60 mL, about 2.0 mL to about 40 mL, about 5.0 mL to about 40 mL, about 2.0 mL to about 30 mL, about 5.0 mL to about 30 mL, or about 2.0 mL to about 25 mL). The flow rate of the fluid containing recombinant protein when loaded onto at least one chromatographic column or at least one chromatographic membrane, which can be used to perform unit operations for purifying recombinant proteins, 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 0.1 mL / min to about 2 mL / min, or about 0.2 mL / min to about 4 mL / min). The concentrations of recombinant protein in the fluid loaded onto at least one chromatography column or chromatography membrane, which can be used to perform unit operations for purifying recombinant proteins, are, for example, approximately 0.05 mg / mL to approximately 100 mg / mL of recombinant protein (e.g., approximately 0.1 mg / mL to approximately 90 mg / mL, approximately 0.1 mg / mL to approximately 80 mg / mL, approximately 0.1 mg / mL to approximately 70 mg / mL, approximately 0.1 mg / mL to approximately 6 mg / mL). It can be a recombinant protein with a concentration of 0 mg / mL, approximately 0.1 mg / mL to approximately 50 mg / mL, approximately 0.1 mg / mL to approximately 40 mg / mL, approximately 0.1 mg / mL to approximately 30 mg / mL, approximately 0.1 mg / mL to approximately 20 mg / mL, approximately 0.5 mg / mL to approximately 20 mg / mL, approximately 0.1 mg / mL to approximately 15 mg / mL, approximately 0.5 mg / mL to approximately 15 mg / mL, approximately 0.1 mg / mL to approximately 10 mg / mL, or approximately 0.5 mg / mL to approximately 10 mg / mL.The resin within at least one chromatography column or chromatography membrane used to perform a purification unit operation can be a resin capable of performing anion exchange or cation exchange chromatography. The resin within at least one chromatography column or chromatography membrane used to perform a purification unit operation can be a cation exchange resin (e.g., Capto-S resin, GE Healthcare Life Sciences, Piscataway, NJ).

[0245] After loading a recombinant protein onto at least one chromatography column or chromatography membrane that can be used to perform a purification unit operation for 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 elution of the recombinant protein in terms of the interaction between the recombinant protein and the resin or other aspects.

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

[0247] After washing at least one chromatography column or chromatography membrane used to perform the unit operation for purifying recombinant proteins, the recombinant proteins are eluted from at least one chromatography column or chromatography membrane by passing an elution buffer through the same chromatography column or chromatography membrane used to perform the unit operation for purifying recombinant proteins. The elution buffer can be passed through at least one chromatographic column or chromatographic membrane that can be used to perform unit operations for purifying recombinant proteins at a flow rate of approximately 0.2 mL / min to approximately 25 mL / min (e.g., approximately 0.2 mL / min to approximately 20 mL / min, approximately 0.5 mL / min to approximately 20 mL / min, approximately 0.2 mL / min to approximately 15 mL / min, approximately 0.5 mL / min to approximately 15 mL / min, approximately 0.5 mL / min to approximately 10 mL / min, between approximately 0.5 mL / min and approximately 6.0 mL / min, between approximately 1.0 mL / min and approximately 5.0 mg / min, between approximately 0.5 mL / min and approximately 14 mL / min, between approximately 1.0 mL / min and approximately 25.0 mL / min, or between approximately 1.0 mL / min and approximately 15.0 mL / min). The volume of elution buffer used to elute recombinant proteins from each of at least one chromatographic column or chromatographic membrane that can be used to perform unit operations for purifying recombinant proteins may be, for example, about 1X column volume (CV) to about 25XCV (e.g., about 1XCV to about 20XCV, about 15XCV to about 25XCV, about 1XCV to about 14XCV, about 1XCV to about 13XCV, about 1XCV to about 12XCV, about 1XCV to about 11XCV, about 2XCV to about 11XCV, about 3XCV to about 11XCV, about 4XCV to about 11XCV, about 5XCV to about 11XCV, or about 5XCV to about 10XCV). The total elution time can be, for example, approximately 2 minutes to approximately 3 hours (e.g., approximately 2 minutes to approximately 2.5 hours, approximately 2 minutes to approximately 2.0 hours, approximately 2 minutes to approximately 1.5 hours, approximately 2 minutes to approximately 1.5 hours, approximately 2 minutes to approximately 1.25 hours, approximately 2 minutes to approximately 1.25 hours, approximately 2 minutes to approximately 1 hour, between approximately 2 minutes and approximately 40 minutes, between approximately 10 minutes and approximately 40 minutes, between approximately 20 minutes and approximately 40 minutes, or between approximately 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 may contain different concentrations of salt (e.g., increased salt concentration), different pH (e.g., increased or decreased salt concentration), or molecules that compete with the recombinant protein for binding to the resin. Such elution buffers are used for each of the typical capture mechanisms described herein. Examples of this are well known in the field of technology.

[0248] After elution of recombinant proteins from at least one chromatography column or chromatography membrane used to perform the unit operation for purifying recombinant proteins, and before the next volume of the fluid containing recombinant proteins can be loaded onto that chromatography column or chromatography membrane, that 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 chromatographic column or chromatographic membrane used to perform unit operations for purifying recombinant proteins 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 unit operations for purifying recombinant proteins can be, for example, about 1X column volume (CV) to about 15XCV (e.g., about 1XCV to about 14XCV, about 1XCV to about 13XCV, about 1XCV to about 12XCV, about 1XCV to about 11XCV, about 2XCV to about 11XCV, about 3XCV to about 11XCV, about 2XCV to about 5XCV, about 2.5XCV ​​to about 7.5XCV, about 4XCV to about 11XCV, about 5XCV to about 11XCV, or about 5XCV to about 10XCV).The concentration of recombinant protein in the eluate of at least one chromatography column or chromatography membrane used to perform the unit operation for purifying recombinant protein is, for example, about 0.05 mg / mL to about 100 mg / mL of recombinant protein (e.g., about 0.1 mg / mL to about 90 mg / mL, about 0.1 mg / mL to about 80 mg / mL, about 0.1 mg / mL to about 70 mg / mL, about 0.1 mg / mL to about 60 mg / mL, about 0.1 mg / mL). This can be a recombinant protein with concentrations of approximately 50 mg / mL, 0.1 mg / mL to 40 mg / mL, 2.5 mg / mL to 7.5 mg / mL, 0.1 mg / mL to 30 mg / mL, 0.1 mg / mL to 20 mg / mL, 0.5 mg / mL to 20 mg / mL, 0.1 mg / mL to 15 mg / mL, 0.5 mg / mL to 15 mg / mL, 0.1 mg / mL to 10 mg / mL, or 0.5 mg / mL to 10 mg / mL.

[0249] The unit operation for polishing recombinant proteins may include a resin that can be used to perform cation exchange, anion exchange, or molecular sieve chromatography on at least one chromatography column or chromatography membrane. As can be recognized in the art, polishing recombinant proteins using at least one chromatography column or chromatography membrane that can be used to perform the unit operation for polishing recombinant proteins may include, for example, the steps of loading, chasing, and regenerating the at least one chromatography column or chromatography membrane that can be used to perform the unit operation for polishing recombinant proteins. For example, when polishing is performed using the loading, chasing, and regenerating steps, the recombinant protein does not bind to the resin in the at least one chromatography column or chromatography membrane used to perform the unit operation for polishing recombinant proteins, the recombinant protein is eluted from the at least one chromatography column or chromatography membrane in the loading and chasing steps, and the regenerating step is performed before additional fluid containing recombinant protein can be loaded onto the at least one chromatography column or chromatography membrane. It is used to remove all impurities. Typical flow rates and buffer volumes used in each of the loading, tracking, and regeneration processes are listed 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 recombinant proteins, and / or the size and shape of at least one chromatography membrane that can be used to perform a unit operation for polishing recombinant proteins, may be any combination of typical sizes, shapes, and volumes of chromatography columns or chromatography membranes described herein. For example, the size of at least one chromatographic column or chromatographic membrane that can be used to perform a unit operation for polishing recombinant proteins may 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 chromatographic column or chromatographic membrane, which can be used to perform unit operations for polishing the recombinant protein, can be, for example, about 0.1 mL / min to about 25 mL / min (e.g., about 0.1 mL / min to about 12.5 mL / min, about 0.1 mL / min to about 10.0 mL / min, about 0.1 mL / min to about 8.0 mL / min, about 0.1 mL / min to about 6 mL / min, about 0.1 mL / min to about 4 mL / min, about 0.1 mL / min to about 3 mL / min, about 2 mL / min to about 6 mL / min, about 0.1 mL / min to about 2 mL / min, or about 0.2 mL / min to about 4 mL / min).The total volume of the fluid containing recombinant protein loaded onto at least one chromatography column or chromatography membrane, which can be used to perform a unit operation to polish recombinant protein, can be, for example, about 1.0 mL to about 250 mL (e.g., about 1.0 mL to about 225 mL, about 1.0 mL to about 200 mL, about 1.0 mL to about 175 mL, about 1.0 mL to about 150 mL, about 100 mL to about 125 mL, about 100 mL to about 150 mL, about 1.0 mL to about 150 mL, about 1.0 mL to about 125 mL, about 1.0 mL to about 100 mL, about 1.0 mL to about 75 mL, about 1.0 mL to about 50 mL, or about 1.0 mL to about 25 mL). The resin in at least one chromatography column or chromatography membrane used to perform the polishing can be anion exchange or cation exchange resin. The resin in at least one chromatographic column or chromatographic membrane used to perform the polishing unit operation may be a cation exchange resin (e.g., Sartobind® Q resin, Sartorius, Goettingen, Germany).

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

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

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

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

[0255] Some embodiments of this example may further include the step of adjusting the ion concentration and / or pH of the eluates from the three chromatography columns in the PCCS before supplying them into the chromatography membrane in the PCCS. As described herein, the ion concentration and / or pH of the eluates from the three chromatography columns in the PCCS can be adjusted by adding a buffer to the eluates from the three chromatography columns in the PCCS (for example, by using an in-line buffer adjustment reservoir) before supplying these eluates into the chromatography membrane in the PCCS. The buffer can be added to the eluates 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).

[0256] These examples may further include a step of holding or storing the eluates from the three chromatography columns in the PCCS before supplying them into a chromatography membrane (a chromatography membrane that can be used to perform the unit operation of polishing recombinant proteins). As described herein, this holding or storage step can be performed using one of the reservoirs described herein (e.g., a backup tank).

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

[0258] As can be recognized in this technical field, purified recombinant proteins are The MCCS or MCCS2 can be periodically eluted using any of the methods described herein. For example, any of the methods described herein can elute purified recombinant proteins for durations 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), depending on the chromatographic column and / or chromatographic membrane used for the MCCS or MCCS1 and MCCS2, at frequencies 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 about 30 minutes).

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

[0260] cell Cells cultured by some of the methods described herein may be bacteria (e.g., Gram-negative bacteria), yeasts (e.g., Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, or Arxula adeninivorans)), or mammalian cells. Mammalian cells may be cells growing 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 meidian cells. Madin-Darby canine (Cocker Spaniel) kidney epithelial (MDCK) cells are available. In some examples of culturing adherent cells, the culture may also contain multiple microcarriers (e.g., microcarriers containing one or more pores). Additional mammalian cells that can be cultured by any of the methods described herein are known in the art.

[0261] Mammalian cells may contain recombinant nucleic acids (e.g., nucleic acids stably integrated into the genome of mammalian cells) that encode recombinant proteins (e.g., recombinant proteins). A non-limiting example of a typical recombinant nucleic acid encoding a recombinant protein is described below, along with the recombinant proteins that can be produced using the methods described herein. In some examples, mammalian cells cultured in a bioreactor (e.g., any of the bioreactors described herein) were derived from a larger culture.

[0262] Nucleic acids encoding recombinant proteins can be introduced into mammalian cells using a wide variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (e.g., lipofection), transduction (e.g., lentivirus, adenovirus, or retrovirus infection), and electroporation. In some cases, nucleic acids encoding recombinant proteins are not stably incorporated into the chromosomes of mammalian cells (transient transfection), while in others, the nucleic acids are incorporated. Alternatively, or in addition, nucleic acids encoding recombinant proteins can reside within plasmids and / or within mammalian artificial chromosomes (e.g., human artificial chromosomes). Alternatively, or in addition, nucleic acids can be introduced into cells using viral vectors (e.g., lentivirus, retrovirus, or adenovirus vectors). Nucleic acids can be functionally bound to promoter sequences (e.g., strong promoters such as the β-actin promoter and CMV promoter, or inductive promoters). Optionally, vectors containing nucleic acids can also contain selectable markers (e.g., genes conferring hygromycin, puromycin, or neomycin resistance to mammalian cells).

[0263] In some cases, recombinant proteins are secretory proteins released by mammalian cells into extracellular media (e.g., first and / or second liquid culture media). For example, a nucleic acid sequence encoding a soluble recombinant protein may contain a sequence encoding a secretory signal peptide at the N- or C-terminus of the recombinant protein, which is cleaved by enzymes present in mammalian cells and subsequently released into extracellular media (e.g., first and / or second liquid culture media).

[0264] Culture medium Liquid culture media are known in the art. Liquid culture media (e.g., first and / or second tissue culture media) can be supplemented with mammalian serum (e.g., fetal bovine serum and bovine serum), and / or growth hormones or growth factors (e.g., insulin, transferrin, and epidermal growth factor). Alternatively, or in addition, liquid culture media (e.g., first and / or second liquid culture media) can be liquid culture media of known composition, liquid culture media free of animal-derived components, serum-free liquid culture media, or serum-containing liquid culture media. 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 energy sources (e.g., carbohydrates such as glucose), essential amino acids (e.g., the basic set of 20 amino acids + cysteine), vitamins and / or other organic compounds required in low concentrations, free fatty acids, and / or trace elements. Liquid culture media (e.g., first and / or second liquid culture media) may optionally contain, for example, Mammalian hormones or growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts and buffers (e.g., calcium, magnesium, and phosphates), nucleosides and bases (e.g., adenosine, thymidine, and hypoxanthine), proteins and tissue hydrolysates, and / or any combination thereof may be supplemented.

[0266] A wide variety of different liquid culture media are known in the art that can be used to culture cells (e.g., mammalian cells) by any of the methods described herein. Similarly, medium components that may be useful in this method include, but are not limited to, hydrolyzed known composition (CD) products, e.g., CD peptones, CD polypeptides (2 or more amino acids), and CD growth factors. Examples of liquid tissue culture media and additions of medium components are known in the art.

[0267] As will be recognized by experienced practitioners, the first liquid culture medium and the second liquid culture medium described herein may be the same type of medium or different types of medium.

[0268] Additional features of a typical bioreactor The inner surface of any of the bioreactors described herein may have at least one coating (e.g., at least one coating of gelatin, collagen, poly-L-ornithine, polystyrene, and laminin), as well as one or more ports for sparging O2, CO2, and N2 into the liquid culture medium, and a stirring mechanism for stirring the liquid culture medium, as known in the art. The bioreactor can incubate cell cultures in a controlled humidification 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 may also be equipped with a mechanical device that can withdraw a certain volume of liquid culture medium from the bioreactor, and optionally within that mechanical device, a filter (e.g., an ATF system or cell filtration system described in U.S. Provisional Patent Application No. 61 / 878,502) for removing cells from the liquid culture medium during the process of transferring the liquid culture medium from the bioreactor.

[0269] temperature The mammalian cell culture process can be carried out at a temperature of approximately 31°C to 40°C. As experienced practitioners recognize, the temperature can be changed at specific points in time during the culture process, for example, on an hourly or daily basis. For example, the temperature can change or shift (e.g., increase or decrease) at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 days, or approximately 20 days or more after the initial seeding of cells (e.g., mammalian cells) in the bioreactor. For example, the temperature can shift upward (e.g., by a maximum or approximately 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 approximately 20°C change). For example, the temperature can shift downwards (e.g., by a maximum or approximately 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 approximately 20°C change).

[0270] CO2 The culture process described herein further involves the liquid culture medium in the bioreactor being maximized. This may include exposure to an atmosphere containing approximately 15% CO2 (for example, 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 culture steps described herein can be carried out using a perfusion bioreactor. Culturing cells (e.g., mammalian cells) in a perfusion bioreactor involves removing a first volume of a first liquid culture medium (e.g., a first volume of a first liquid culture medium containing any concentration of mammalian cells, or substantially cell-free) from the bioreactor, and adding a second volume of a second liquid culture medium to the first liquid culture medium. The removal and addition can be carried out simultaneously, sequentially, or in combination. Furthermore, removal and addition are (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 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%). The removal and replacement of a volume (or between 4% and 30%) over a given time period (e.g., over a 24-hour period, over increasing time periods of approximately 1 hour to approximately 24 hours, or over increasing time periods exceeding 24 hours) 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 any combination thereof. When performed regularly, the volume removed or replaced (for example, within a period of about 24 hours, within an increasing period of about 1 hour to about 24 hours, or within an increasing period of more than 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 (for example, 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 each 24-hour period (or, instead, over increasing time periods of about 1 hour to about 24 hours or over increasing time periods exceeding 24 hours) for the entire or partial period of the culture. As is known in the art, the rate at which the first volume of the first liquid culture medium is removed (volume / unit time) and the rate at which the second volume of the second liquid culture medium is added (volume / unit time) can be varied. The rate at which the first volume of the first liquid culture medium is removed (volume / unit time) and the rate at which the second volume of the second liquid culture medium is added (volume / unit time) can be approximately the same or different.

[0272] Alternatively, the volume removed and added can vary (e.g., gradually increasing) over each 24-hour period during the culture period (or, instead, over increasing periods of 1 hour to approximately 24 hours or over increasing periods exceeding 24 hours). For example, the volume of the first liquid culture medium removed and the volume of the second liquid culture medium added during each 24-hour period during the culture period (or, over increasing periods of approximately 1 hour to over 24 hours or over increasing periods exceeding 24 hours) can increase over the culture period from a volume of 0.5% to approximately 20% of the bioreactor volume or the first liquid culture medium volume to approximately 25% to approximately 150% of the bioreactor volume or the first liquid culture medium volume (e.g., gradually or by alternating increases).

[0273] As experienced practitioners will recognize, the first liquid culture medium and the second liquid culture medium can be of the same type. In other examples, the first liquid culture medium and the second liquid The culture medium can be different.

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

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

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

[0277] Fedbatch bioreactor The culture steps described herein can be carried out using a fed-batch bioreactor. Cell culture in a fed-batch bioreactor involves adding a second volume of a second liquid culture medium to a first liquid culture medium (e.g., periodic or continuous addition) for most of the culture period. The second liquid culture medium may be added continuously (for example, 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 volume of the bioreactor or the volume of the first liquid culture medium over a given time period (e.g., over a 24-hour period, over increasing time periods of about 1 hour to about 24 hours, or over increasing time periods 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 any combination thereof. When performed regularly, the volume added (e.g., within a period of approximately 24 hours, within an increasing period of approximately 1 hour to approximately 24 hours, or within an increasing period of more than 24 hours) can be, for example, 0.1% to 300% (e.g., 1% to 200%, 1% to 100%, 100% to 200%, 5% to 150%, 10% to 50%, 15% to 40%, 8% to 80%, or 4% to 30%) of the volume of the bioreactor or the volume of the first liquid culture medium. The second volume of the second liquid culture medium added can, in some examples, be kept approximately the same over each 24-hour period (or, instead, over an increasing period of approximately 1 hour to approximately 24 hours or over an increasing period of more than 24 hours) for the entire or partial culture period. As is known in the art, the rate (volume / unit time) of adding the second volume of the second liquid culture medium can be varied for the entire or partial culture period. For example, the volume of the second liquid culture medium added can vary (e.g., gradually increase) over each 24-hour period during the culture period (or alternatively, over increasing time periods of 1 hour to approximately 24 hours or over increasing time periods exceeding 24 hours).For example, during the culture period, the volume of the second liquid culture medium added within each 24-hour period (or, alternatively, increasing time periods of approximately 1 hour to over 24 hours or increasing time periods exceeding 24 hours) can be increased throughout the culture period (e.g., by gradual or alternating increases) from a volume of 0.5% to approximately 20% of the bioreactor volume or the first liquid culture medium volume to approximately 25% to approximately 150% of the bioreactor volume or the first liquid culture medium volume. The rate (volume / unit time) at which the second liquid culture medium is added in a volume of 2 can be approximately the same throughout or part of the culture period.

[0278] As experienced practitioners will recognize, the first and second liquid culture media can be of the same type. In other examples, the first and second liquid culture media 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 injection pump.

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

[0280] As experienced practitioners will recognize, any combination of various culture parameters (e.g., container, volume, frequency of culture volume exchange, stirring frequency, temperature, medium, and CO2 concentration) can be used to carry out these methods. Furthermore, any mammalian cell described herein or known in the art can be used to produce recombinant proteins.

[0281] Typical 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 No. 61 / 775,060 and No. 61 / 856,390 (incorporated by reference). In these representative systems, at least one (e.g., at least two, three, four, five, or six) chromatographic columns packed with reduced bioburden provided herein are present in the MCCS or in MCCS1 and / or MCCS2. For example, the entire system may include a total of two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, twelve, thirteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty chromatographic columns packed with reduced bioburden provided herein. For example, MCCS, MCCS1, and / or MCCS2 may include (or each may include) chromatographic columns packed with reduced bioburden provided herein in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

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

[0283] Some representative systems do not include a dwelling tank. In other examples, the system (for instance, each dwelling tank) treats the entire period, for example, from about 5 minutes to about 6 hours (including both ends). (If only holding proteins for use) The entire system may include up to 1, 2, 3, 4, or 5 stabling tanks. Each stabling tank can have a capacity of 1 mL to approximately 300 mL (including both ends). Any stabling tanks positioned in the system so that the fluid enters the stabling tank(s) before it enters MCCS1 or MCCS may each have a capacity of 1 mL to approximately 100% (including both ends) of the loading volume of the first column of MCCS1 or MCCS. Any stabling tanks positioned in the system so that the fluid enters the stabling tank(s) before it enters MCCS2 (and after it leaves MCCS1) may have a capacity of, for example, 1 mL to approximately 100% (including both ends) of the loading volume of the first column of MCCS2.

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

[0285] An MCCS or MCCS1 may 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. An MCCS or MCCS1 may be any of the representative MCCSs described herein, or may have one or more of the representative features (any combination) of the MCCSs described herein. The chromatography columns and / or chromatography membranes present in the MCCS or MCCS1 may have one or more of the representative shapes, sizes, volumes (bed volumes), and / or unit operations described herein.

[0286] Chromatographic columns (or more) and / or chromatographic membranes (or more) present in MCCS or MCCS1 may contain one or more representative resins described herein or known in the art. For example, a resin contained in one or more of the chromatographic columns (or more) and / or chromatographic membranes (or more) present in MCCS or MCCS1 may be a resin that utilizes a certain 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). A resin contained in one or more of the chromatographic columns (or more) and / or chromatographic membranes (or more) of MCCS or MCCS1 may be a cation exchange resin, anion exchange resin, molecular sieve resin, or 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 may be included in one or more chromatographic columns and / or chromatographic membranes present in MCCS or MCCS1. The chromatographic column(s) and / or chromatographic membrane present within the MCCS or MCCS1 may contain the same and / or different resins (e.g., any of the resins described herein or known in the art for use in the purification of recombinant proteins).

[0287] Two or more chromatography columns and / or chromatography resins present within MCCS or MCCS1 are used in one or more unit operations (e.g., a set The MCCS or MCCS1 can perform operations such as capturing recombinant proteins, purifying recombinant proteins, polishing recombinant proteins, inactivating viruses, adjusting the ion concentration and / or pH of a fluid containing recombinant proteins, or filtering a fluid containing recombinant proteins. In non-limiting examples, the MCCS or MCCS1 can perform unit operations such as capturing recombinant proteins from a fluid (e.g., liquid culture medium) and inactivating viruses present in a fluid containing recombinant proteins. The MCCS or MCCS1 can perform any combination of two or more unit operations described herein or known in the art.

[0288] Chromatographic columns and / or chromatographic membranes present within the MCCS or MCCS1 can be linked to or moved by a switching mechanism (e.g., a column switching mechanism). The MCCS or MCCS1 may also include one or more (e.g., two, three, four, or five) pumps (e.g., automated peristaltic pumps). Column switching events can be triggered by the detection of a certain level of recombinant protein in the fluid passing through the MCCS or MCCS1 (e.g., inputs to one or more chromatographic columns and / or chromatographic membranes within the MCCS or MCCS1 and / or elutes from them), a certain volume of liquid (e.g., buffer), or a certain level of recombinant protein detected over a certain period of time. Column switching generally refers to a mechanism that allows at least two different chromatographic columns and / or chromatographic membranes within MCCS or MCCS1 (e.g., two or more different chromatographic columns and / or chromatographic membranes present within 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 MCCS1 can be a periodic countercurrent chromatography system (PCCS). For example, a PCCS that is MCCS or MCCS1 (i.e., PCCS or PCCS1, respectively) can include four chromatography columns, where the first three columns perform the unit operation of capturing recombinant proteins from a fluid (e.g., liquid culture medium), and the fourth column of the PCCS performs the unit operation of inactivating viruses in the fluid containing recombinant proteins. A PCCS that is MCCS or MCCS1 can utilize a column switching mechanism. The PCC system can utilize an improved AKTA system (GE Healthcare, Piscataway, NJ) that can operate, for example, four, five, six, seven, or eight columns, or even more.

[0290] MCCS or MCCS1 may 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 MCCS1 may also be equipped with an operating system that utilizes software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ) to detect when a column switch should occur (e.g., based on UV absorbance, liquid volume, or elapsed time) and to influence (initiate) the column switch event.

[0291] MCCS or MCCS1 is a reservoir with 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 / Alternatively, a buffer reservoir may be further included. In other examples, the MCCS or MCCS1 may include one or more (e.g., two, three, four, five, or six) stagnant tanks capable of holding fluids that cannot easily pass through one or more chromatography columns and / or chromatography membranes within the MCCS or MCCS1. The systems described herein may include one or more stagnant tanks (e.g., the stagnant tanks described herein) within the MCCS, MCCS1, and / or MCCS2. Other examples of the systems described herein do not include stagnant tanks within the MCCS, MCCS1, or MCCS2, or do not include stagnant tanks in the entire system. Other examples of the systems described herein include up to one, two, three, four, or five stagnant tanks (e.g., any(s) of the stagnant tanks described herein) in the entire system.

[0292] Second MCCS A second MCCS (MCCS2) in a typical 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 typical MCCSs described herein, or it can have any one or more of the typical features of the MCCSs described herein (in any combination). The chromatography columns(s) and / or chromatography membranes(s) present in MCCS2 can have any one or more of the shapes, sizes, volumes (bed volumes), and / or unit operations described herein. The chromatography columns(s) and / or chromatography membranes(s) can contain any of the typical resins described herein or known in the art. For example, the resin contained in one or more chromatography columns and / or chromatography membranes present in 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 chromatographic column(s) and / or chromatographic membrane present in the MCCS2 may contain the same and / or different resins (e.g., any of the resins described herein or known in the art for use in the purification of recombinant proteins).

[0293] Chromatographic columns and / or chromatographic membranes present in MCCS2 can perform one or more unit operations (e.g., any of the unit operations described herein or any combination thereof). In an unspecified example, MCCS2 can perform the unit operations of purifying recombinant proteins from a fluid and polishing recombinant proteins present in a fluid containing recombinant proteins. In another unspecified example, MCCS2 can perform the unit operations of purifying recombinant proteins present in a fluid, polishing recombinant proteins present in a fluid, and filtering a fluid containing recombinant proteins. In yet another example, MCCS2 can perform the unit operations of purifying recombinant proteins present in a fluid, polishing recombinant proteins present in a fluid, filtering a fluid containing recombinant proteins, and adjusting the ion concentration and / or pH of a fluid containing recombinant proteins. MCCS2 can perform any combination of two or more unit operations described herein or known in the art.

[0294] Chromatography columns (multiple possible) and / or chromatographs located within MCCS2 Graph membranes(s) can be linked to or moved relative to each other by a switching mechanism (e.g., a column switching mechanism). The MCCS2 may also include one or more (e.g., two, three, four, or five) pumps (e.g., automated peristaltic pumps). Column switching events can be triggered by the detection of a certain level of recombinant protein in the fluid passing through the MCCS2 (e.g., inputs to one or more chromatography columns and / or chromatography membranes within the MCCS2 and / or eluates therefrom), by the UV absorbance corresponding to a certain level of recombinant protein, by a certain volume of liquid (e.g., buffer), or by the detection of a certain level of recombinant protein detected over a certain period of time.

[0295] MCCS2 can be a periodic countercurrent chromatography system (i.e., PCCS2). For example, a PCCS2 may include three columns that perform the unit operation of purifying recombinant proteins from a fluid, and a chromatography membrane that performs the unit operation of polishing recombinant proteins present in the fluid. For example, the three columns that perform the unit operation of purifying recombinant proteins from a fluid may include, for example, cation exchange resins, and the chromatography membrane that performs the unit operation of polishing may also include cation exchange resins. A column switching mechanism can be utilized in a PCCS2. A PCCS2 can utilize an improved AKTA system (GE Healthcare, Piscataway, NJ) that can operate with, for example, four, five, six, seven, or eight columns, or more.

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

[0297] The MCCS2 may 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) inline buffer preparation reservoirs and / or buffer reservoirs. In other examples, the MCCS2 may include one or more (e.g., 2, 3, 4, 5, or 6) stagnant tanks (e.g., any of the stagnant tanks described herein) that can hold fluids that cannot easily pass through one or more chromatography columns and / or chromatography membranes within the MCCS2.

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

[0299] The systems described herein may also include a fluid tube positioned between MCCS1 and MCCS2. Any of the fluid tubes described herein may be tubes made of, for example, polyethylene, polycarbonate, or plastic. The fluid tube positioned between MCCS1 and MCCS2 may further include: one or more inline buffer conditioning reservoirs, which are in fluid communication with the fluid tube and are positioned so that buffers stored in the inline buffer conditioning reservoir(s) are added to the fluid present in the fluid tube; a stagnant tank (for example, any of the stagnant tank(s) described herein), which is in fluid communication with the fluid tube and is positioned to hold any excess fluid present in the fluid tube that cannot be readily supplied into MCCS2; and one or more filters, which are positioned in the fluid tube to filter the fluid present in the fluid tube (for example, to remove bacteria). Any of the inline buffer preparation reservoirs can contain, for example, a volume of buffer solution ranging from approximately 0.5 L to 50 L (at temperatures such as 25°C, 15°C, or 10°C or below).

[0300] The system described herein may optionally include a fluid tube positioned between the final chromatography column or chromatography membrane in the MCCS2 and the outlet. The system described herein may further include one or more filters fluidly connected to the fluid tube positioned between the final chromatography column or chromatography membrane in the MCCS2 and the outlet, so that the filters can remove, for example, precipitated material, particulate matter, or bacteria from the fluid present in the fluid tube positioned between the final chromatography column or chromatography membrane in the MCCS2 and the outlet.

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

[0302] Some examples of the systems provided herein also include pump systems. A pump system may include one or more of the following: one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pumps (e.g., any of the pumps described herein or known in the art), one or more (e.g., 2, 3, 4, or 5) filters (e.g., any of the filters described herein or known in the art), one or more (e.g., 2, 3, 4, or 5) UV detectors, and one or more (e.g., 2, 3, 4, or 5) stagnant tanks (e.g., any of the stagnant tanks described herein). Some examples of the systems provided herein may further include fluid tubing (e.g., any of the typical fluid tubing described herein or known in the art) positioned between the pumps and the inlet of the MCCS or MCCS1. In some examples, this particular fluid tube may include one or more (e.g., two, three, or four) pumps (e.g., any of the pumps described herein or known in the art) and / or one or more (e.g., two, three, or four) stagnant tanks (e.g., any of the typical stagnant tanks described herein), where these pumps and / or stagnant tanks are fluid-connected to the fluid present in the fluid tube.

[0303] Some examples of the systems described herein further include a further fluid tube connected to the fluid tube between the pump and the inlet, one end of which is fluidly connected to the bioreactor and the other end is fluidly connected to the fluid tube between the pump and the inlet. This further fluid tube is a filter that can remove cells from the liquid culture medium removed from the bioreactor. For example, it may include an ATF cell retention system.

[0304] The system provided herein enables the continuous production of purified recombinant proteins (e.g., therapeutic protein APIs). As is known in the art, the system may enable periodic elution of purified recombinant proteins (e.g., therapeutic protein APIs). The system described herein may also yield purified recombinant proteins (e.g., therapeutic protein APIs) in net yields of at least about 5 g / day, at least about 10 g / day, at least about 15 g / day, at least about 25 g / day, at least about 30 g / day, at least about 40 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 70 days, at least about 80 days, at least about 90 days, or at least about 100 days, in net yields 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 g / day, at least about 30 g / day, or at least about 40 g / day over a continuous period of 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] Methods for reducing bioburden in chromatography resins, including the use of substantially dry chromatography resins. Furthermore, this specification provides a method for reducing the bioburden of a chromatographic resin, comprising (a) exposing a container containing a substantially dried chromatographic resin to a dose of gamma irradiation sufficient to reduce the bioburden of the container and the chromatographic resin, wherein the substantially dried chromatographic resin contains a liquid containing 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 chromatographic resin after exposure to that dose of gamma irradiation. Some embodiments of this method further include drying the chromatographic resin to substantially remove the liquid (but not all of the liquid) from the chromatographic resin prior to step (a). Drying of the chromatographic resin can be done using heat treatment (e.g., an oven) or a desiccator. Additional methods for drying chromatographic resins are known in the art.

[0306] Any of the conditions and doses for gamma irradiation described herein can be used in these methods. For example, the dose of gamma irradiation can be about 15 kGy to about 45 kGy (e.g., about 20 kGy to about 30 kGy). Any of the containers, chromatographic resins, and liquids containing at least one alcohol described herein can be used in these methods. For example, the container may be a storage vessel or a chromatographic column. The chromatographic resin in these methods may contain a protein ligand (e.g., protein A or protein G). In some examples, the chromatographic resin may contain anion-exchange chromatographic resin (e.g., a chromatographic resin containing an N-benzyl-N-methyl-ethanolamine group). In some examples, the chromatographic resin is covalently bonded to the surface of an article (e.g., a tip, membrane, or cassette). In some embodiments, the substantially dry chromatographic resin does not contain significant amounts of antioxidants or significant amounts of chelating agents. Reduced bioburden chromatographic resins produced by any of the methods described herein are also provided.

[0307] In some cases, the reduced chromatographic resin of the resulting bioburden is approximately 1 × 10⁶ -8 ~Approx. 1×10 -5 The sterility assurance level (SAL) (for example, approximately 1 × 10⁻⁶) -7 ~Approx. 1×10 -6 It has SAL. The reduced chromatography resin produced may 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 reduced chromatography resin produced is tan The invention includes an affinity chromatography resin containing a protein ligand (e.g., protein A). In some examples, the reduced chromatography resin produced includes an anion exchange chromatography resin (e.g., an anion exchange chromatography resin containing an N-benzyl-N-methyl-ethanolamine group). Also provided is a method for preparing a chromatography column packed with reduced bioburden, comprising the steps of preparing a chromatography resin of reduced bioburden produced by any of the methods described herein, and packing the chromatography resin into a column of reduced bioburden under a sterilized environment. Also provided is a chromatography column packed with reduced bioburden produced by any of the methods described herein.

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

[0309] Method for producing reduced bioburden membranes, resins, coatings, chips, and cassettes. This specification also provides a method for producing a film, resin, coating, chip, or cassette with reduced bioburden, comprising: (i) a film, resin, coating, chip, or cassette (e.g., a film, resin, coating, chip, or cassette based on cellulose, agarose, or sugars); and (ii) a liquid containing at least one alcohol (e.g., and optionally at least one antioxidant and / or chelating agent); exposing the container and the film, resin, coating, chip, or cassette to a dose of gamma irradiation sufficient to reduce bioburden, wherein at least one alcohol is present in an amount sufficient to improve damage to the film, 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 representative resins described herein or known in the art). In some embodiments, the film, resin, coating, chip, or cassette contains a protein A or protein G ligand covalently bound to at least one or part of its surface. In some embodiments, the composition contains the film, resin, coating, chip, or cassette and at least an alcohol (e.g.) For example, and optionally, a liquid comprising at least one antioxidant and / or at least one chelating agent. Any of the typical combinations and concentrations of alcohols, antioxidants, and / or chelating agents described herein can be used in any of these methods. Any of the typical liquids described herein can be used in any of these methods. In some embodiments, the composition is a wet or humid dry material. Also provided herein are reduced bioburden membranes, resins, coatings, chips, or cassettes produced using any of the methods described herein. In some examples, the container is a sealed storage container or vessel.

[0310] Also provided herein are methods for producing reduced bioburden films, resins, coatings, chips, and cassettes, comprising exposing a container containing a substantially dry film, resin, coating, chip, or cassette (e.g., a cellulose, agarose, or sugar-based film, resin, coating, chip, or cassette) to a dose of gamma irradiation sufficient to reduce the bioburden in the container and the film, resin, coating, chip, or cassette. Some examples further include a step of drying the film, resin, coating, chip, or cassette before the exposure step. In some embodiments, the film, resin, coating, chip, or cassette contains a covalently bound protein A or protein G ligand on its surface. In some examples, the cassette is a cassette containing a resin (e.g., any of the representative resins described herein or known in the art). Also provided herein are reduced bioburden films, resins, coatings, chips, or cassettes produced using any of the methods described herein. [Examples]

[0311] The present invention will be further illustrated by the following embodiments, which do not limit the scope of the invention as described in the claims. [Examples]

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

[0313] After irradiation, the chromatography resins were packed into separate chromatography columns and cycled using cell culture harvesting and a 6-minute residence time. The breakthrough binding capacity of the irradiated resins was recorded and compared to naive (unirradiated) MabSelect® SuRe® (Protein A chromatography) resins.

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

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

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

[0317] [Table 1]

[0318] This data indicates that irradiation of chromatographic resins in a liquid containing alcohol (e.g., benzyl alcohol) protects the resins from subsequent loss of binding ability over multiple chromatographic cycles. [Examples]

[0319] Protective effect of alcohol on gamma-irradiated Capto(trademark)Adhere chromatography resins GE Capto(trademark)adhere (a multimode functional anion exchange chromatography resin) was irradiated with gamma rays at doses of 28-49 kGy in three different buffer solutions with standard irradiation rates: (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, and 25 mM mannitol in 50 mM sodium phosphate buffer (40-49 kGy at dose rates >7.5 kGy / hour); and (C) 25 mM sodium ascorbate in 50 mM sodium phosphate buffer, 25 mM Methionine, 25 mM histidine, 25 mM mannitol, 2% v / v benzyl alcohol (40-49 kGy and higher dose rates >7.5 kGy / hour).

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

[0321] The data from this experiment is shown in Figure 2. The data in Figure 2 shows that no noticeable change in bonding ability 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 before gamma irradiation.

[0322] Table 2 below shows that irradiation of the resin in the presence of benzyl alcohol did not have a noticeable effect on other quality attributes of the resin's performance during protein purification.

[0323] [Table 2]

[0324] Other Embodiments The present invention has been described in detail, but it should be understood that the above description is for illustrative purposes only and does not limit the scope of the invention as defined by the attached claims. Other aspects, advantages, and modifications fall within the scope of the following claims.

Claims

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

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

3. The method according to claim 1 or 2, wherein the liquid comprises at least one chelating agent in an amount sufficient to improve the loss of binding capacity of the chromatographic resin after exposure to a dose of gamma-ray irradiation.

4. The method according to any one of claims 1 to 3, wherein the liquid comprises mannitol, sodium ascorbate, histidine, and methionine.

5. The aforementioned liquid is (i) 30 mM to 70 mM methionine and 30 mM to 70 mM histidine; (ii) 10 mM to 50 mM methionine, 10 mM to 50 mM histidine, and 1 0 mM to 50 mM sodium ascorbate; (iii) The method according to any one of claims 1 to 3, comprising 5 mM to 45 mM sodium ascorbate, 5 mM to 45 mM methionine, 5 mM to 45 mM mannitol, and 5 mM to 45 mM histidine.

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

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

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

9. The method according to claim 8, wherein the protein ligand is protein A.

10. The method according to claim 7, wherein the chromatography resin is an anion exchange chromatography resin.

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

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

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

14. The method according to claim 13, wherein the recombinant protein for therapeutic purposes is an enzyme.

15. The method according to claim 13, wherein the recombinant protein for therapeutic use is an antibody.

16. An integrated and closed process for the production of reduced bioburden from purified recombinant proteins, (a) Expose a packed chromatography column, comprising a composition including (i) a chromatography resin for purifying recombinant proteins and (ii) a liquid containing benzyl alcohol and at least two antioxidants selected from mannitol, sodium ascorbate, histidine, and methionine, to a gamma irradiation dose sufficient to reduce the bioburden of the packed chromatography column and chromatography resin, wherein the benzyl alcohol and at least two antioxidants are present in amounts sufficient to improve the loss of binding capacity of the chromatography resin after exposure to the dose of gamma irradiation, thereby producing a packed chromatography column with reduced bioburden; (b) A step of preparing a liquid containing recombinant protein; (c) A packed chromatography column with reduced bioburden of (a) The process of performing Lamb chromatography; and (d) The step of recovering the eluate from a packed chromatography column with reduced bioburden, wherein the eluate contains the recombinant protein, The process is integrated using a reduced bioburden buffer.

17. The process according to claim 16, wherein the recombinant protein is a recombinant protein for therapeutic purposes.

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

19. The process according to claim 18, wherein the recombinant protein for therapeutic purposes is an enzyme.

20. The process according to claim 17, wherein the recombinant protein for therapeutic use is an antibody.

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