RU2805607C2 - Sterile chromatographic resin and its application in production methods - Google Patents

Abstract

FIELD: medical resins.

SUBSTANCE: methods for reducing the bioburden (for example, sterilization) of a chromatography resin. A composition is proposed for reducing the bioburden of an affinity chromatography resin containing a protein ligand after exposure to a dose of gamma radiation, containing (i) an affinity chromatography resin containing a protein ligand, and (ii) a liquid containing from about 15 mM to about 35 mM sodium ascorbate, from about 15 mM to about 35 mM methionine, from about 15 mM to about 35 mM mannitol, and from about 15 mM to about 35 mM histidine. Also a container for reducing the bioburden of an affinity chromatography resin containing a protein ligand after exposure to a dose of gamma radiation containing the specified composition is proposed.

EFFECT: reducing the bioburden of an affinity chromatography resin.

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Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit from U.S. Provisional Patent Application No. 61/928929, filed January 17, 2014, and U.S. Provisional Patent Application No. 62/001498, filed May 21, 2014, each of which is incorporated herein by reference in its entirety. .

TECHNICAL FIELD

The present invention relates to methods for biotechnology and biological production of recombinant proteins.

BACKGROUND OF THE ART

Mammalian cells containing a nucleic acid encoding a recombinant protein are often used to produce therapeutically or commercially important proteins. In the current environment of diversified product pipelines, biotechnology companies are increasingly under pressure to develop innovative solutions for highly flexible and cost-effective production of therapeutic protein drug substances. One strategy for efficiently isolating recombinant proteins is to use methods involving continuous chromatography (eg, using a closed system). One known limitation of continuous chromatography is the presence of contaminants in the system (eg, increased bioburden), resulting in contaminated product, reduced product yield, and reduced flow rate (or increased pressure) in the system. For example, increased bioload in a system can lead to complete system shutdown.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery that gamma irradiation of a chromatography resin reduces the binding capacity of the chromatography resin. In light of this discovery, the present invention provides methods for reducing the bioburden of a chromatography resin, comprising exposing a container comprising a composition comprising a chromatography resin and at least one antioxidant and/or chelating agent to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin wherein the at least one antioxidant and/or chelating agent is present in an amount sufficient to improve the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation. The invention also provides methods for reducing the bioburden of a chromatography resin, comprising exposing a container comprising a composition comprising a chromatography resin (and, optionally, at least one antioxidant and/or chelating agent in an amount sufficient to improve the loss of binding capacity of the chromatography resin after exposure to gamma irradiation) gamma irradiation at a rate of from about 0.1 kGy/h to about 6 kGy/h and/or at a temperature of about 4°C to about 25°C for a dose of gamma radiation sufficient to reduce the bioburden of the container and chromatography resin . The invention also relates to chromatography columns with reduced bioburden containing a chromatography resin with reduced bioburden obtained by any of the methods presented in the present description, compositions including a chromatography resin and at least one chelating agent and/or antioxidant, methods for performing chromatography on a reduced bioburden column bioburden using at least one of these reduced bioburden chromatography columns and integrated, closed or substantially closed, and continuous methods for producing purified recombinant protein with reduced bioburden, including the use of at least one of these chromatography columns with reduced bioburden. Any chromatography resins prepared by any of the methods presented herein, any of the packed chromatography columns prepared by any of the methods presented herein, any of the column chromatography methods, and any of the methods presented herein may be sterile , absolutely sterile, aseptic or with reduced bioburden. Any of the chromatography resins produced by any of the methods presented herein, any of the chromatography columns prepared by any of the methods presented herein, and any of the methods presented herein may be aseptic and sterile, absolutely sterile, aseptic or with reduced bioburden.

The present invention relates to methods for reducing the bioburden of a chromatography resin, comprising: exposing a container comprising a composition comprising a chromatography resin and at least one antioxidant and/or chelating agent to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein: at least one antioxidant and/or chelating agent is present in an amount sufficient to improve the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation. Some embodiments of these methods further include placing the composition in a container prior to exposure. In some examples, irradiation is performed at a power of from about 0.1 kGy/h to about 6 kGy/h and/or at a temperature of from about 4°C to about 25°C. In some examples of any of these methods, the container is a storage vessel, a chromatography column, or a packed chromatography column. In some examples of any of the methods presented herein, the composition is a suspension of a sedimented chromatography resin in a liquid comprising at least one antioxidant and/or chelating agent. In some examples, the liquid contains: (i) from 75 mM to about 125 mM (e.g., from 80 mM to about 120 mM, from about 85 mM to about 115 mM, from about 90 mM to about 110 mM, or from about 95 mM to approximately 105 mM) mannitol; (ii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) methionine (or alternatively cysteine or glutathione); (iii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) ascorbate sodium; (iv) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) histidine ; (v) 30 mM to about 70 mM (e.g., about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) methionine (or, alternatively, cysteine or glutathione) and about 30 mM to about 70 mM (eg, about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) histidine; (vi) about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) methionine (or, alternatively, cysteine or glutathione ), about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) histidine, and about 10 mM to about 50 mM mM (eg, about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) sodium ascorbate; or (vii) about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) sodium ascorbate, from about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) methionine (or, alternatively, cysteine or glutathione), about 5 mM to about 45 mM (e.g., from about 10 mM to about 40 mM, from about 15 mM to about 35 mM, or from about 20 mM to about 30 mM) mannitol; and from about 5 mM to about 45 mM (for example, from about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) histidine. In some examples, the liquid is a buffer solution (eg, a phosphate buffer solution, such as a sodium phosphate buffer solution, such as 50 mM sodium phosphate, pH 6.0).

In some examples of any of the methods presented herein, the composition is a solid mixture. In some embodiments of any of the methods presented herein, the composition includes at least one antioxidant selected from the group of: reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, urinary acid, lipoic acid, flavonoid, phenylpropionic acid, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl and dimethylmethoxychromanol. In some embodiments of any of the methods presented herein, the composition includes at least one (one, two, three or four) antioxidant selected from mannitol, sodium ascorbate, methionine (or alternatively cysteine or glutathione) and histidine. In some embodiments, the composition includes mannitol, sodium ascorbate, methionine (or alternatively cysteine or glutathione) and histidine. In some embodiments of any of the methods presented herein, the composition includes at least one chelating agent selected from the group: ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanesulfonic acid sodium salt (DMPS), dimercaptosuccinic acid (DMSA), metallothionein and deferoxamine.

In some examples of any of the methods presented herein, the composition includes at least one chromatography resin selected from the group of: anion exchange chromatography resin, cation exchange chromatography resin, affinity or pseudoaffinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin ( or any combination thereof). In some examples of any of the methods presented herein, the chromatography resin may be a multimodal (e.g., bimodal) chromatography resin (e.g., a chromatography resin having anion exchange groups and hydrophobic interaction groups, or a chromatography resin having cation exchange groups and hydrophobic interaction groups) . In some examples of any of the methods presented herein, the composition includes an affinity chromatography resin including a protein ligand (eg, protein A or protein G). In some examples of any of the methods presented herein, the composition includes an anion exchange chromatography resin (eg, an anion exchange chromatography resin including N-benzyl-N-methyl-ethanolamine groups).

In some examples of any of the methods presented herein, the dose is from about 2 kGy to about 45 kGy (e.g., from about 20 kGy to about 30 kGy, from about 23 kGy to about 27 kGy, from about 2 kGy to about 40 kGy, from about 2 kGy to about 35 kGy, from about 2 kGy to about 30 kGy, from about 2 kGy to about 25 kGy, from about 10 kGy to about 45 kGy, from about 10 kGy to about 40 kGy, from about 10 kGy to about 35 kGy, from about 10 kGy to about 30 kGy, or from about 10 kGy to about 25 kGy). In some embodiments of any of the methods presented herein, exposure is carried out at a temperature of from about -25°C to about 0°C, inclusive, or from about 0°C to about 25°C, inclusive (for example, from about 4°C to approximately 25°C).

The present invention also relates to a chromatography resin with reduced bioburden, obtained by any of the methods presented in the present description. In some examples, any of the reduced bioburden chromatography resins provided herein have a Sterility Assurance Level (SAL) of about 1x10 ^-8 to about 1x10 ^-5 (e.g., about 1x10 ^-7 to about 1 ×10 ^-6 ). For example, the chromatography resin produced by any of the methods presented herein may have reduced bioburden, be sterile, be aseptic, or be completely sterile. In some embodiments, the chromatography resin produced by any of the methods presented herein may be aseptic and have reduced bioburden, be sterile, or be completely sterile. Some examples of any reduced bioburden chromatography resins provided herein include at least one resin selected from the group of anion exchange chromatography resin, cation exchange chromatography resin, affinity or pseudoaffinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin. In some embodiments, the reduced bioburden chromatography resin provided herein includes an affinity chromatography resin including a protein ligand (eg, protein A or protein G). In some embodiments, the reduced bioburden chromatography resin provided herein includes an anion exchange chromatography resin (eg, an anion exchange chromatography resin including N-benzyl-N-methyl-ethanolamine groups). In some embodiments, the reduced bioburden chromatography resin provided herein is a multimodal chromatography resin (eg, a bimodal chromatography resin).

The present invention also relates to methods for producing a packed chromatography column with reduced bioburden, including: producing any chromatography resins with reduced bioburden obtained by any of the methods presented in the present description; and loading the chromatography resin into a column with reduced bioburden under aseptic conditions. The present invention also relates to packed chromatography columns with reduced bioburden obtained by gamma irradiation of a composition comprising a chromatography resin for a packed column and at least one antioxidant and/or chelating agent (for example, any of the example compositions presented herein), included in the chromatographic column. Any of the packed reduced bioburden chromatography columns provided herein may have a Sterility Assurance Level (SAL) of about 1×10 ^-8 to about 1×10 ^-5 (e.g., about 1×10 ^-7 to about 1× 10 ^-6 ). Any of the chromatography columns produced by any of the methods presented herein may have reduced bioburden, be sterile, be completely sterile, or be aseptic. For example, any of the chromatography columns produced by any of the methods presented herein may be aseptic and may have reduced bioburden, be sterile, or be completely sterile.

In some embodiments, any of the packed reduced bioburden chromatography columns provided herein, the resin in the packed column comprises at least one resin selected from the group of: anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic chromatography resin interactions and size exclusion chromatography resin. In some embodiments, any of the packed reduced bioburden chromatography columns provided herein, the resin includes an affinity chromatography resin including a protein ligand (eg, protein A or protein G). In some examples of any of the packed reduced bioburden chromatography columns provided herein, the resin includes an anion exchange chromatography resin (eg, the anion exchange chromatography resin includes N-benzyl-N-methyl-ethanolamine groups).

The present invention also relates to compositions comprising a chromatography resin and at least one antioxidant and/or chelating agent, wherein the at least one antioxidant and/or chelating agent is present in an amount sufficient to improve the loss of binding capacity of the chromatography resin after treatment with a gamma dose. radiation sufficient to reduce the bioburden of the composition. In some examples of any of the compositions provided herein, the composition is a suspension of a sedimented chromatography resin in a liquid comprising at least one antioxidant and/or chelating agent. In some examples, the liquid contains: (i) from 75 mM to about 125 mM (e.g., from 80 mM to about 120 mM, from about 85 mM to about 115 mM, from about 90 mM to about 110 mM, or from about 95 mM to approximately 105 mM) mannitol; (ii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) methionine (or alternatively cysteine or glutathione); (iii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) ascorbate sodium; (iv) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) histidine ; (v) 30 mM to about 70 mM (e.g., about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) methionine (or, alternatively, cysteine or glutathione) and about 30 mM to about 70 mM (eg, about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) histidine; (vi) about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) methionine (or, alternatively, cysteine or glutathione ), about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) histidine, and about 10 mM to about 50 mM mM (eg, about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) sodium ascorbate; or (vii) about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) sodium ascorbate, from about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) methionine (or, alternatively, cysteine or glutathione), about 5 mM to about 45 mM (e.g., from about 10 mM to about 40 mM, from about 15 mM to about 35 mM, or from about 20 mM to about 30 mM) mannitol; and from about 5 mM to about 45 mM (for example, from about 10 mM o about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) histidine. In some examples, the liquid is a buffer solution (eg, a phosphate buffer solution, such as a sodium phosphate buffer solution, such as 50 mM sodium phosphate, pH 6.0).

In some examples of any of the compositions presented herein, the composition is a solid mixture.

Some embodiments of any of the compositions provided herein may include at least one antioxidant selected from the group of: reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid , lipoic acid, flavonoid, phenylpropionic acid, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl and dimethylmethoxychromanol. Some embodiments of any of the compositions provided herein may include at least one (one, two, three or four) antioxidant selected from mannitol, sodium ascorbate, methionine (or alternatively cysteine or glutathione) and histidine. Some examples of any of the compositions provided herein include mannitol, sodium ascorbate, methionine (or alternatively cysteine or glutathione) and histidine. Some examples of any of the compositions presented herein may include at least one chelating agent selected from the group: ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanesulfonic acid sodium salt (DMPS), dimercaptosuccinic acid (DMSA ), metallothionein and deferoxamine. In some examples of any of the compositions provided herein, the chromatography resin may include at least one resin selected from the group of anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin. In some examples of any of the compositions provided herein, the resin includes an affinity chromatography resin including a protein ligand (eg, protein A or protein G).

The present invention also provides methods for performing bioburden-reduced column chromatography, comprising: (a) preparing any of the bioburden-reduced packed chromatography columns produced by any of the methods presented herein; and (b) performing column chromatography using a packed bioburden-reduced chromatography column and a bioburden-reduced buffer in a closed system. In some examples of any of the methods presented herein, bioburden reduced column chromatography using a packed bioburden reduced chromatography column is performed continuously over a period of at least 4 days (e.g., at least 5 days, at least 7 days , at least 14 days, at least 28 days or at least 60 days). In some embodiments of any of the methods presented herein, the resin in the reduced bioburden chromatography column pack (a) has a binding capacity percentage of from about 65% to about 100% (e.g., from about 75% to about 100%) of compared to the same resin not subjected to gamma irradiation. In some embodiments of any of the methods presented herein, the resin in the packed reduced bioburden chromatography column comprises at least one resin selected from the group of: anion exchange chromatography resin, cation exchange chromatography resin, affinity or pseudoaffinity chromatography resin, hydrophobic interactions and size exclusion chromatography resin. In some examples of any of the methods presented herein, the resin includes an affinity chromatography resin including a protein ligand (eg, protein A or protein G). In some embodiments of any of the methods presented herein, the resin includes an anion exchange chromatography resin.

The present invention also provides integrated, closed, or substantially closed, and continuous methods for producing purified recombinant protein with reduced bioburden, comprising: (a) providing a liquid culture medium comprising the recombinant protein substantially free of cells; and (b) continuously feeding the liquid culture medium into a multi-column chromatography system (MCCS) comprising at least one of any of the packed reduced bioburden chromatography columns produced by any of the methods presented herein; where the method uses a buffer with reduced bioburden, the method is integrated and is carried out continuously from the liquid culture medium to the MCCS eluate, which is the purified recombinant protein. In some embodiments of any of the methods presented herein, at least two different typical operations are performed by the MCCS. In some embodiments of any of the methods presented herein, the method includes switching columns. In some examples of any of the methods presented herein, the MCCS performs typical recombinant protein capture and virus inactivation operations or performs typical recombinant protein capture and purification operations.

In some embodiments of any of the methods presented herein, the MCCS includes at least two packed chromatography columns with reduced bioburden. In some examples of any of the methods presented herein, the MCCS is a batch countercurrent chromatography system. In some examples of any of the methods presented herein, the MCCS includes a plurality of affinity chromatography, cation exchange chromatography, anion exchange chromatography, or size exclusion chromatography columns, or any combination thereof. In some embodiments of any of the methods presented herein, the MCCS includes an affinity chromatography column and the affinity chromatography is performed in a method with a capture mechanism selected from the group of: protein A binding capture mechanism, substrate binding capture mechanism, binding capture mechanism an antibody or antibody fragment, an aptamer-binding capture mechanism, and a cofactor-binding capture mechanism. In some examples of any of the methods presented herein, the affinity chromatography is performed in a protein A capture capture mechanism and the recombinant protein is an antibody or antibody fragment.

The present invention also provides integrated, closed, or substantially closed, and continuous methods for producing purified recombinant protein with reduced bioburden, comprising: (a) providing a liquid culture medium comprising the recombinant protein substantially free of cells; (b) continuously feeding liquid culture medium into the first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein in liquid culture medium using MCCS1; (d) obtaining an eluate from MCCS1 including the recombinant protein, and continuously feeding the eluate into a second multicolumn chromatography system (MCCS2); (e) continuously feeding the recombinant protein from the eluate into MCCS2 and subsequent elution of the recombinant protein, thereby obtaining a purified recombinant protein, wherein: the method uses a buffer with reduced bioburden, the method is integrated and is carried out continuously from a liquid culture medium to the purified recombinant protein, and at least one column in MCCS1 and/or MCCS2 includes at least one of any of the reduced bioburden chromatography columns prepared by any of the methods presented herein. In some embodiments of any of the methods presented herein, at least two different types of operations are performed by MCCS1 and/or MCCS2. Some examples of any of the methods presented herein include column switching. In some examples of any of the methods presented herein, MCCS1 performs typical recombinant therapeutic protein capture and viral inactivation operations. In some embodiments of any of the methods presented herein, MCCS2 performs typical recombinant protein purification and refinement operations. In some embodiments of any of the methods presented herein, MCCS1 and/or MCCS2 includes at least two chromatography columns. In some examples of any of the methods presented herein, MCCS1 is a first periodic countercurrent chromatography system (PCCS1). In some examples of any of the methods presented herein, capture is accomplished using affinity chromatography, cation exchange chromatography, anion exchange chromatography, or size exclusion chromatography, or any combination thereof. In some examples of any of the methods presented herein, affinity chromatography is performed using a capture mechanism selected from the group: protein A binding capture mechanism, substrate binding capture mechanism, antibody or antibody fragment capture capture mechanism, binding capture mechanism aptamer and cofactor binding uptake mechanism. In some embodiments of any of the methods presented herein, the affinity chromatography is performed using a protein A binding capture mechanism, and the recombinant protein is an antibody or antibody fragment.

In some embodiments of any of the methods presented herein, the MCCS2 is a second periodic countercurrent chromatography system (PCCS2). In any of the methods presented herein, the recombinant protein is a therapeutic recombinant protein. Some embodiments of any of the methods presented herein further include formulation of the purified therapeutic recombinant protein into a pharmaceutical composition. Some embodiments of any of the methods presented herein can be performed continuously over a period of at least 4 days (e.g., at least 5 days, at least 7 days, at least 14 days, or at least 28 days). days).

The present invention also provides methods for reducing bioburden in chromatography, comprising (a) exposing a container containing a substantially dry chromatography resin to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin. Some embodiments of these methods further include drying the chromatography resin prior to step (a) to remove liquid from the chromatography resin. In some embodiments, the substantially dry chromatography resin does not contain a significant amount of antioxidant or a significant amount of chelating agent. In some embodiments, the container is a storage vessel. In some embodiments, the chromatography resin is covalently attached to the surface of an object (eg, a chip, membrane, or cassette). In some embodiments, the chromatography resin contains a protein ligand (eg, protein A or protein G). In some embodiments, the chromatography resin is an anion exchange chromatography resin (eg, a chromatography resin containing N-benzyl-N-methyl-ethanolamine groups). In some embodiments, the dose is from about 15 kGy to about 45 kGy (eg, from about 20 kGy to about 30 kGy). The present invention also relates to chromatographic resins with reduced bioburden, obtained by any of the methods presented in the present description. In some embodiments, the resulting reduced bioburden chromatography resin has a Sterility Assurance Level (SAL) of about 1x10 ^-8 to about 1x10 ^-5 (e.g., a SAL of about 1x10 ^-7 to about 1x10 ^-6 ) . In some embodiments, the reduced bioburden chromatography resin comprises at least one resin selected from the group of anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin. In some embodiments, the reduced bioburden chromatography resin comprises an affinity chromatography resin containing a protein ligand (eg, protein A). In some embodiments, the reduced bioburden chromatography resin comprises an anion exchange chromatography resin (eg, an anion exchange chromatography resin containing N-benzyl-N-methyl-ethanolamine groups). The present invention also relates to methods for producing a packed chromatography column with reduced bioburden, including producing a chromatography resin with reduced bioburden obtained by any of the methods presented in the present description; and loading the chromatography resin into a column with reduced bioburden under aseptic conditions. The present invention also relates to packed chromatography columns with reduced bioburden, obtained by any of the methods presented in the present description.

The present invention also provides integrated, closed-loop, and continuous methods for producing purified recombinant protein with reduced bioburden, comprising: (a) producing a liquid culture medium containing the recombinant protein that is substantially free of cells; and (b) continuously feeding the liquid culture medium into a multi-column chromatography system (MCCS) containing at least one packed chromatography column with reduced bioburden obtained by any of the methods presented herein; where the method uses a buffer with reduced bioburden, the method is integrated and is carried out continuously from the liquid culture medium to the MCCS eluate, which is the purified recombinant protein. The present invention also provides integrated, closed-loop and continuous methods for producing purified recombinant protein with reduced bioburden, comprising: (a) producing a liquid culture medium containing a substantially cell-free recombinant protein; (b) continuously feeding liquid culture medium into the first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein in liquid culture medium using MCCS1; (d) obtaining an eluate from MCCS1 containing the recombinant protein and continuously feeding the eluate into a second multi-column chromatography system (MCCS2); (e) continuously feeding the recombinant protein from the eluate into MCCS2 and subsequent elution of the recombinant protein, thereby obtaining a purified recombinant protein, wherein: the method uses a buffer with reduced bioburden, the method is integrated and is carried out continuously from a liquid culture medium to the purified recombinant protein, and at least one column in MCCS1 and/or MCCS2 includes a packed reduced bioburden chromatography column obtained by any of the methods presented herein.

As used herein, a singular noun is one or more concrete nouns. For example, the phrase “reduced bioburden chromatography column” is “one or more reduced bioburden chromatography columns.”

The term "bioburden" is known in the art and refers to the level of self-replicating biological contaminants present in the composition (eg, solid or liquid) and/or on the surface (eg, external and/or internal surface) of objects. For example, bioburden may refer to self-replicating biological contaminants present in a composition containing a chromatography resin or a packed column chromatography resin (e.g., self-replicating biological contaminants are present in a packed column chromatography resin in a packed chromatography column). In other examples, bioburden may refer to self-replicating biological contaminants on the interior surface of a chromatography column and/or in the chromatography resin in a chromatography column (e.g., biological contaminants on the interior surface of a chromatography column and biological contaminants in a chromatography resin for a packed column in a chromatography column). Bioburden can also refer to self-replicating biological contaminants present in a fluid (eg, a buffer used in any of the methods presented herein). Non-limiting examples of self-replicating biological contaminants include bacteria (eg, gram-positive or gram-negative bacteria or bacterial spores), mycobacteria, viruses (eg, vesivirus, Cache Valley virus, parvovirus, herpes virus, and bunyavirus), parasites, fungi, yeast, and protozoa. Examples of methods for determining bioburden are presented in the present description. Additional methods for determining bioburden are known in the art.

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

The term “bioburden-reduced chromatography resin” 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, e.g., a suspension). For example, a reduced bioburden chromatography resin may be a resin that has been gamma-irradiated at a dose sufficient to reduce the level of self-replicating biological contaminants in the chromatography resin (e.g., a composition comprising a chromatography resin that has been gamma-irradiated at a dose sufficient to reduce the level of self-replicating biological contaminants in the chromatography resin). contaminants in the chromatography resin). For example, a reduced bioburden chromatography resin may be a resin exposed to a dose of from about 1 kGy to about 15 kGy, from about 1 kGy to about 20 kGy of gamma irradiation, from about 1 kGy to about 25 kGy of gamma irradiation, from about 1 kGy to approximately 30 kGy of gamma irradiation or from approximately 1 kGy to approximately 35 kGy of gamma irradiation. Examples of methods for reducing the bioburden of a chromatography resin are presented herein. Additional methods for reducing the bioburden of a chromatography resin are known in the art.

The term "reduced bioburden chromatography column" means a chromatography column (e.g., a packed chromatography column) comprising a treated chromatography resin (e.g., a gamma-irradiated chromatography resin) having a level of self-replicating biological contaminants that is less than the level of self-replicating biological contaminants present in identical to the chromatography column comprising the untreated chromatography resin. For example, a reduced bioburden chromatography column may include a treated chromatography resin having a sterility assurance level of at least or about 1x10 ^-6 , 1x10 ^-7 , 1x10 ^-8 , 1x10 ^-9 or 1x10 ^{- 10} .

The term "bioburden reduced buffer" is known in the art and means a treated (e.g., filtered, autoclaved, and/or gamma-irradiated) liquid (e.g., a treated buffer solution) having a level of self-replicating biological contaminants that is less than the level of self-replicating biological contaminants , found in identical untreated liquid. For example, a reduced bioburden buffer may have 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 .

"Absolutely sterile" or "absolutely sterile" are terms used to describe a composition or method that is completely free of self-replicating biological contaminants. For example, the term can be used to refer to a gamma-irradiated chromatography resin, the inner surface and contents (eg, chromatography resin) of a chromatography column and/or buffer. A completely sterile composition or method may be pure (as that term is known in the art).

"Sterile" or "sterility" are terms used to describe a composition or method having a sterility assurance level of about or less than 1.0 x 10 ^-6 (e.g., about or less than 1.0 x 10 ^-7 , about or less than 1. 0×10 ^-8 , approximately or less than 1.0×10 ^-9 or 1×10 ^-10 ). Determining whether a composition or method is sterile can be accomplished using a number of validated preparation methods known in the art. For example, the sterile composition or method may be completely free of viable self-replicating biological contaminants (eg, any of the self-replicating biological contaminants described herein). A sterile composition or method may also be pure (as that term is known in the art).

The term "sterilization" means a validated method used to render a composition sterile (as defined herein). The degree of inactivation of resistant indicator self-replicating biological contaminants (eg, bacteria) during a processing method can be measured to determine whether sterility (as defined herein) of the composition has been achieved.

The term "sterility assurance level" or "SAL" is known in the art and refers to the confidence level of achieving absolute sterility in a batch of processed units. The probability is typically calculated taking into account the results of inactivation studies carried out during validation and expressed as 1×10 ^-n .

The term “aseptic” is used to describe a composition or method that does not contain disease- or symptom-causing self-replicating biological contaminants (eg, any of the self-replicating biological contaminants described herein). The aseptic composition or method may also be pure (as that term is known in the art).

The term "type operation" is a term known in the art and refers to a functional step that can be performed in a method for purifying a recombinant protein from a liquid culture medium. For example, a typical operation may be filtration (e.g., removing bacteria, yeast, viruses and/or mycobacteria contaminants and/or particulate matter from a liquid, including recombinant protein), capture, epitope tag removal, purification, storage, fine purification, virus inactivation, adjusting the ion concentration and/or pH of the liquid containing the recombinant protein, and removing unwanted salts.

The term "capture" means a step performed to partially purify or isolate (e.g., to obtain at least or about 5%, e.g., at least or about 10%, 15%, 20%, 25%, 30%, 40% , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least or about 95% pure by weight) and concentrating the recombinant protein (e.g., recombinant therapeutic protein) from one or more other components present in the liquid culture medium or dilute liquid culture medium (for example, proteins of the culture medium or one or more other components (for example, DNA, RNA or other proteins) present or secreted by the cell mammal). Typically, capture is accomplished using a chromatography resin that binds the recombinant protein (eg, affinity chromatography). Non-limiting methods for capturing recombinant protein from a liquid culture medium or dilute liquid culture medium are presented herein, and others are known in the art. The recombinant protein can be captured from a liquid culture medium using at least one chromatography column and/or chromatography membrane (eg, any of the chromatography columns and/or chromatography membranes presented herein).

The term “purification” means a step performed to isolate a recombinant protein (e.g., a recombinant therapeutic protein) from one or more other impurities (e.g., bulk impurities) or components present in a liquid comprising the recombinant protein (e.g., proteins from a liquid culture medium or one or more other components (eg, DNA, RNA, other proteins, endotoxins, viruses, etc.) present or secreted by the mammalian cell). For example, purification can be carried out during or after the initial capture step. Purification can be accomplished using a chromatography resin, membrane, or any other solid support that binds the recombinant protein or contaminants (eg, affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography, or molecular sieve chromatography). The recombinant protein can be purified from a liquid comprising the recombinant protein using at least one chromatography column and/or chromatography membrane (eg, any of the chromatography columns or chromatography membranes provided herein).

The term "fine purification" is a term known in the art and means a step performed to remove remaining traces or small amounts of contaminants or impurities from a liquid comprising a recombinant protein (eg, a recombinant therapeutic protein) to near the final desired purity. For example, fine purification can be accomplished by passing a liquid comprising the recombinant protein through chromatography columns or membrane absorbents that selectively bind to the target recombinant protein or small amounts of contaminants or impurities present in the liquid comprising the recombinant protein. In such an example, the eluate/filtrate of chromatography columns or membrane absorbents includes the recombinant protein.

The term "filtration" means the removal of at least a portion (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%) of unwanted biological contaminants (e.g., mammalian cells, bacteria, yeast cells, viruses or mycobacteria) and/or solid particles (eg, precipitated proteins) from a liquid (eg, liquid culture medium or liquid present in any of the methods presented herein).

The term "eluate/filtrate" is a term known in the art and means a liquid obtained from a chromatography column or chromatography membrane containing a detectable amount of a recombinant protein (eg, a recombinant therapeutic protein).

The term "integrated method" means a method carried out using structural elements that function together to produce a specific result (eg, purification of a recombinant protein from a liquid culture medium).

The term "continuous process" means a process in which liquid is continuously passed through at least a portion of the system. For example, a continuous method is a method in which a liquid culture medium including a recombinant protein from a bioreactor is continuously passed through an MCCS. Another example of a continuous method is a method in which a liquid culture medium including a recombinant protein from a bioreactor is continuously passed through the first and second MCCS (MCCS1 and MCCS2). Additional examples include a method in which a liquid culture medium including a recombinant protein is continuously passed through an MCCS, a method in which a liquid culture medium including a recombinant protein is continuously passed through an MCCS1 and MCCS2, or a method in which a liquid including a recombinant protein is continuously passed through MCCS2.

The term "closed-loop process" is a term known in the art and means a process that is carried out such that process components (e.g., chromatography resins and/or buffers) brought into contact with the recombinant protein, or liquids comprising the recombinant protein, not intentionally exposed to contaminants for a significant period of time (eg, not intentionally exposed to air for a significant period of time).

The term "therapeutic protein drug" means a recombinant protein (e.g., immunoglobulin, protein fragment, engineered protein, or enzyme) sufficiently purified or isolated from contaminant proteins, lipids, and nucleic acids (e.g., contaminant proteins, lipids, and nucleic acids present in a liquid culture medium, or from a host cell (eg, a mammalian host cell, yeast or bacteria)) and biological contaminants (eg, viral and bacterial contaminants), and can be formulated into a pharmaceutical without any additional significant purification steps and/or decontamination.

The term "multi-column chromatography system" or "MCCS" means a system of just two or more interconnected or switchable chromatography columns and/or chromatography membranes. A non-limiting example of a multi-column chromatography system is a batch countercurrent chromatography (PCC) system comprising just two or more interconnected or switchable chromatography columns and/or chromatography membranes. Additional examples of multi-column chromatography systems are presented herein and are known in the art.

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

The term "mammalian cell" means any cell from or derived from any mammal (eg, human, hamster, mouse, vervet monkey, rat, pig, cow or rabbit). For example, a mammalian cell may be an immortalized cell. In some embodiments, the mammalian cell is a differentiated cell. In some embodiments, the mammalian cell is an undifferentiated cell. Non-limiting examples of mammalian cells are provided herein. Additional examples of mammalian cells are known in the art.

The term "culture" or "cell culture" means the maintenance or proliferation of mammalian cells under a set of controlled physical conditions.

The term "mammalian cell culture" means a liquid culture medium comprising a plurality of mammalian cells maintained or proliferated under a set of controlled physical conditions.

The term "liquid culture medium" means a liquid containing sufficient nutrients to allow a cell (eg, a mammalian cell) to grow or proliferate in vitro . For example, the liquid culture medium may include one or more of: amino acids (e.g., 20 amino acids), purine (e.g., hypoxanthine), pyrimidine (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc and sodium bicarbonate. In some embodiments, the liquid culture medium may include mammalian serum. In some embodiments, the liquid culture medium does not include serum or other extract from the mammal (the specific liquid culture medium). In some embodiments, the liquid culture medium may include metal media, mammalian growth hormone, and/or mammalian growth factor. Another example of a liquid culture medium is a minimal medium (eg, a medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture media are provided herein. Additional examples of liquid culture media are known in the art and are commercially available. The liquid culture medium may include any density of mammalian cells. For example, as used herein, the volume of liquid culture medium removed from the bioreactor may be substantially free of mammalian cells.

The term “liquid culture medium not containing a component of animal origin” means a liquid culture medium that does not include any components (eg, proteins or serum) derived from a mammal.

The term “serum-free liquid culture medium” means a liquid culture medium that does not include mammalian serum.

The term "liquid culture medium containing serum" means a liquid culture medium comprising serum of a mammal.

The term "chemically defined liquid culture medium" is a term known in the art and means a liquid culture medium in which all chemical components are known. For example, the chemically defined liquid culture medium does not include fetal bovine serum, bovine serum albumin, or human serum albumin because these drugs typically include a complex mixture of albumins and lipids.

The term “protein-free liquid culture medium” means a liquid culture medium that does not include any protein (eg, any detectable protein).

The term “immunoglobulin” means a polypeptide comprising an amino acid sequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., the variable domain sequence framework sequence and/or constant domain sequence). The immunoglobulin, for example, may comprise at least 15 amino acids of an immunoglobulin light chain, for example, at least 15 amino acids of an immunoglobulin heavy chain. The immunoglobulin may be an isolated antibody (eg, IgG, IgE, IgD, IgA, or IgM), such as a subclass of IgG (eg, IgG1, IgG2, IgG3, or IgG4). The immunoglobulin may be an antibody fragment, for example, a Fab fragment, an F(ab′) ₂ fragment, or a scFv fragment. The immunoglobulin may also be a bispecific antibody or trispecific antibody, or a dimeric, trimeric or multimeric antibody, or a diabody, Affibody®, or Nanobody®. The immunoglobulin may also be an engineered protein comprising at least one immunoglobulin domain (eg, a fusion protein). Non-limiting examples of immunoglobulins are provided herein, and additional examples of immunoglobulins are known in the art.

The term “protein fragment” or “polypeptide fragment” means a portion of a polypeptide sequence comprising at least or about 4 amino acids, at least or about 5 amino acids, at least or about 6 amino acids, at least or about 7 amino acids, at least at least or about 8 amino acids, at least or about 9 amino acids, at least or about 10 amino acids, at least or about 11 amino acids, at least or about 12 amino acids, at least or about 13 amino acids, at least or about 13 amino acids about 14 amino acids, at least or about 15 amino acids, at least or about 16 amino acids, at least or about 17 amino acids, at least or about 18 amino acids, at least or about 19 amino acids, or at least or about 17 amino acids 20 amino acids in length, or more than 20 amino acids in length. The recombinant protein fragment can be obtained by any of the methods presented in the present description.

The term “engineered protein” means a polypeptide not naturally encoded by an endogenous nucleic acid present in an organism (eg, a mammal). Examples of engineered proteins include enzymes (eg, with one or more amino acid substitutions, deletions, insertions, or additions resulting in increased stability and/or catalytic activity of the engineered enzyme), fusion proteins, antibodies (eg, bivalent antibodies, trivalent antibodies, or diabodies), and antigen binding proteins comprising at least one recombinant framework sequence.

The term "secreted protein" or "secreted recombinant protein" means a protein (e.g., a recombinant protein) initially comprising at least one secretion signal sequence when it undergoes translation in a mammalian cell and through, at least in part, enzymatic cleavage of the signal sequence of secretion in a mammalian cell, secreted at least partially into the extracellular space (eg, liquid culture medium). Those skilled in the art will appreciate that a “secreted” protein may not completely dissociate from the cell to be considered a secreted protein.

The term "perfusion bioreactor" means a bioreactor comprising a plurality of cells (eg, mammalian cells) in a first liquid culture medium, wherein culturing the cells present in the bioreactor includes periodically or continuously removing the first liquid culture medium and simultaneously or shortly thereafter adding a bioreactor with substantially the same volume of a second liquid culture medium. In some examples, a stepwise change (e.g., increase or decrease) in the volume of the first liquid culture medium is observed being removed and added at incremental time intervals (e.g., about a 24 hour interval, a period from about 1 minute to about 24 hours, or a period greater than 24 hours ) during the culture period (e.g. by daily refeeding of the culture medium). The fraction of media removed and replaced each day may vary depending on the specific cells being cultured, the initial seeding density, and the cell density at a particular time point. "RV" or "reactor volume" means the volume of culture medium present at the start of culture (eg, the total volume of culture medium present after inoculation).

The term "batch bioreactor" is a term known in the art and means a bioreactor comprising a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, wherein culturing the cells present in the bioreactor involves batchwise or continuously adding a second liquid culture medium for culture to the first liquid culture medium without substantially or substantially removing the first liquid culture medium or the second liquid culture medium from the cell culture. The second liquid culture medium may be the same as the first liquid culture medium. In some examples of batch culture, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of batch culture, a second liquid culture medium is added in the form of a dry powder.

The term "purified liquid culture medium" means a liquid culture medium obtained from a culture of bacterial or yeast cells substantially free (e.g., at least 80%, 85%, 90%, 92%, 94%, 96 %, 98% or 99% free of bacteria or yeast cells.

Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one skilled in the art to which the present invention relates. Methods and materials are presented herein for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this description, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description, figures and claims.

DESCRIPTION OF DRAWINGS

Figure 1 is a graph showing the static resin binding curve (mg/mL protein) for untreated GE Mab Select SuRe ^™ resin, 25 kGy gamma-irradiated GE Mab Select SuRe ^™ resin, 15 kGy gamma-irradiated JSR resin LifeSciences Amsphere ProA JWT203 and 25 kGy gamma-irradiated Kaneka KanCap A resin.

Figure 2 is a table showing the absorbance at 280 nm (protein A absorbance) of supernatant from GE Mab Select SuRe ^™ resin after 30 kGy gamma irradiation and from JSR LifeSciences Amsphere ProA JWT203 resin after 30 kGy gamma irradiation.

Figure 3 is a graph showing the amount of protein in the eluate over multiple chromatography runs using untreated JSR LifeSciences Amsphere ProA JWT203 resin (first eluate) or 15 kGy gamma irradiated JSR LifeSciences Amsphere ProA JWT203 resin (gamma eluate). -irradiated resin).

Figure 4 is a chromatogram showing the elution profile of run 7 and run 11 by continuous chromatography performed using JSR LifeSciences Amsphere ProA JWT203 raw resin.

Figure 5 is a chromatogram showing the elution profile of run 7 and run 11 from continuous chromatography performed using 15 kGy gamma irradiated JSR LifeSciences Amsphere ProA JWT203 resin.

Figure 6 is a graph showing the amount of protein in the eluate over multiple cycles of continuous chromatography performed using untreated JSR LifeSciences Amsphere ProA JWT203 resin (first) or 15 kGy gamma irradiated JSR LifeSciences Amsphere ProA JWT203 resin (gamma).

Figure 7 is a plot of protein bound over multiple chromatography runs performed using untreated JSR LifeSciences Amsphere ProA JWT203 resin (first) or 15 kGy gamma irradiated JSR LifeSciences Amsphere ProA JWT203 resin (gamma).

Figure 8 is a graph showing the amount of protein in the eluate over multiple chromatography cycles performed using untreated JSR LifeSciences Amsphere ProA JWT203 resin (first) or 29 kGy gamma irradiated JSR LifeSciences Amsphere ProA JWT203 resin (gamma).

Figure 9 is a graph showing the amount of protein (anti-αβTCR antibody (IgG1) or anti-TGFβ antibody (IgG4)) in the eluate over multiple chromatography runs performed using untreated JSR LifeSciences Amsphere ProA JWT203 resin (first), gamma irradiated at a dose of 15 kGy of JSR LifeSciences Amsphere ProA JWT203 resin (15 kGy) or gamma-irradiated JSR LifeSciences Amsphere ProA JWT203 resin (29 kGy) at a dose of 29 kGy.

Figure 10 is a list of examples of recombinant enzyme proteins that can be used to treat specific disorders in humans.

Figure 11 is a plot of the protein eluate of untreated (first) and 29 kGy gamma-irradiated GE Mab Select SuRe ^™ resin over multiple chromatography runs when culture medium containing monoclonal IgG1 was used to load each resin.

Figure 12 is a graph of the amount of protein bound to untreated (first) and 29 kGy gamma-irradiated GE Mab Select SuRe ^™ resin over multiple chromatography runs when culture medium containing monoclonal IgG1 was used to load each resin.

Figure 13 is a graph of the amount of protein bound (mg bound protein/ml resin) to untreated (prime) and 29 kGy gamma-irradiated GE Mab Select SuRe ^™ LX resin after loading with various steady-state concentrations of IgG4 monoclonal antibody.

Figure 14 is a graph of static binding capacity (mg protein/mL resin) at steady state concentration of 2 mg/mL IgG4 antibody of untreated GE Mab Select SuRe ^™ LX resin (first LX) gamma irradiated to 29 kGy of GE Mab Select SuRe ^™ resin. LX (29 kGy LX), dry untreated GE Mab Select SuRe ^TM LX resin, and dry 29 kGy gamma-irradiated GE Mab Select SuRe ^TM LX resin, and 29 kGy gamma-irradiated GE Mab Select SuRe ^TM LX resin the presence of one of seven different test buffers containing at least one antioxidant.

Figure 15 is a graph of the amount of protein in the eluate over multiple chromatography runs performed using untreated GE Mab Select SuRe ^™ LX resin (first) or GE Mab Select SuRe ^™ LX resin gamma-irradiated at 29 kGy in the presence of 25 mM mannitol. , 25 mM histidine, 25 mM sodium ascorbate, 25 mM methionine, 50 mM sodium phosphate, pH 6.0.

Figure 16 is a graph of the amount of protein bound over multiple chromatography runs using untreated GE Mab Select SuRe ^™ LX resin (first) or GE Mab Select SuRe ^™ LX resin gamma-irradiated at 29 kGy in the presence of 25 mM mannitol. , 25 mM histidine, 25 mM sodium ascorbate, 25 mM methionine, 50 mM sodium phosphate, pH 6.0.

Figure 17 is an unreduced sodium dodecyl sulfate polyacrylamide gel showing IgG1 antibody eluate through multiple chromatography runs using untreated GE Mab Select SuRe ^TM LX resin (the first LX) and GE Mab Select SuRe ^TM LX resin dosed with gamma irradiation 29 kGy, in the presence of 25 mM mannitol, 25 mM histidine, 25 mM sodium ascorbate, 25 mM methionine, 50 mM sodium phosphate, pH 6.0.

DETAILED DESCRIPTION

The present invention relates to methods for reducing the bioburden of a chromatography resin, comprising exposing a container comprising a composition comprising a chromatography resin and at least one antioxidant and/or chelating agent to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein at least At least one antioxidant and/or chelating agent is present in an amount sufficient to improve the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation. The present invention also relates to chromatography columns with reduced bioburden containing a chromatography resin with reduced bioburden obtained by any of the methods presented in the present description, compositions including a chromatography resin and at least one chelating agent and/or antioxidant, methods of performing chromatography on columns with reduced bioburden using at least one of these chromatography columns with reduced bioburden, and integrated, closed, or substantially closed, and continuous methods for the production of purified recombinant protein with reduced bioburden, including the use of at least one of these chromatography columns with reduced bioburden. Non-limiting aspects of these methods are described below. As is known in the art, the various aspects described below can be used in any combination without limitation.

Compositions containing a chromatography resin and an antioxidant and/or chelating agent

The present invention relates to compositions comprising a chromatography resin (for example, any of the chromatography resins presented herein or known in the art) and at least one antioxidant and/or chelating agent (for example, any of the antioxidants and/or chelating agents presented herein or known in the art), wherein the at least one antioxidant and/or chelating agent is present in an amount sufficient to improve the loss of binding capacity of the chromatography resin after treatment with a dose of gamma radiation sufficient to reduce the bioburden of the composition. For example, the chromatography resin may be at least one of an anion exchange chromatography resin, a cation exchange chromatography resin, an affinity or pseudoaffinity chromatography resin, a hydrophobic interaction chromatography resin, and a size exclusion chromatography resin, or any combination thereof. In some examples, the chromatography resin is a resin including a protein or peptide ligand (eg, a protein or peptide ligand affinity chromatography resin, eg, a protein A or protein G chromatography resin).

The composition, for example, may be a suspension of a sedimented chromatography resin in a liquid comprising at least one antioxidant and/or chelating agent. For example, the liquid may contain at least one (eg, one, two, three or four) of methionine (or alternatively cysteine or glutathione), sodium ascorbate, histidine and mannitol. In some examples, the liquid contains methionine (or alternatively cysteine or glutathione), sodium ascorbate, histidine and mannitol. In some examples, the liquid contains: (i) from 75 mM to about 125 mM (e.g., from 80 mM to about 120 mM, from about 85 mM to about 115 mM, from about 90 mM to about 110 mM, or from about 95 mM to approximately 105 mM) mannitol; (ii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) methionine (or alternatively cysteine or glutathione); (iii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) ascorbate sodium; (iv) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) histidine ; (v) about 30 mM to about 70 mM (e.g., about 35 mM to about 65 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM) methionine (or, alternatively, cysteine or glutathione ) and from about 30 mM to about 70 mM (for example, from about 35 mM to about 65 mM, from about 40 mM to about 60 mM, or from about 45 mM to about 55 mM) histidine; (vi) about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) methionine (or, alternatively, cysteine or glutathione ), about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) histidine, and about 10 mM to about 50 mM mM (eg, about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) sodium ascorbate; or (vii) about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) sodium ascorbate, from about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) methionine (or, alternatively, cysteine or glutathione), about 5 mM to about 45 mM (e.g., from about 10 mM to about 40 mM, from about 15 mM to about 35 mM, or from about 20 mM to about 30 mM) mannitol, and from about 5 mM to about 45 mM (for example, from about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) histidine. In some examples, the liquid is a buffer solution (eg, a phosphate buffer solution, such as a sodium phosphate buffer solution, such as 50 mM sodium phosphate, pH 6.0). In some examples, the liquid is a buffer solution (eg, a phosphate buffer solution, such as sodium phosphate buffer solution (eg, 50 mM sodium phosphate, pH 6.0).

In some examples, the composition may be a solid mixture (eg, a dry solid mixture or a wet or wet solid mixture). The solid mixture may include at least one (eg, one, two, three or four) of methionine (or alternatively cysteine or glutathione), sodium ascorbate, histidine and mannitol. In some examples, the solid mixture contains methionine (or alternatively cysteine or glutathione), sodium ascorbate, histidine and mannitol. In some examples, the composition is a liquid-filled chromatography resin including at least one antioxidant and/or chelating agent. Non-limiting examples of such liquids are presented herein.

The present invention also provides a container (eg, a plastic container or chromatography column) comprising a chromatography resin (eg, any of the chromatography resins described herein or known in the art) and at least one antioxidant and/or chelating agent (eg , any of the antioxidants and/or chelating agents described herein or known in the art), wherein the at least one antioxidant and/or chelating agent is present in an amount sufficient to improve the loss of binding capacity of the chromatography resin after treatment with a dose of gamma radiation , sufficient to reduce the bioburden of the composition. For example, a container (e.g., a plastic container or chromatography column) may have an internal volume, such as 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, according to 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 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, for example, from about 1 ml to about 500 ml, from about 1 ml to about 50 ml, from about 5 ml to about 500 ml, from about 5 ml to about 400 ml, from about 5 ml to about 350 ml, from about 5 ml to about 300 ml, from about 5 ml to about 250 ml, from about 5 ml to about 200 ml, from about 5 ml to about 150 ml, from about 5 ml to about 100 ml, or from about 5 ml to about 50 ml. In some examples, the chromatography resin in the container is a suspension of sedimented chromatography resin in a liquid including at least one antioxidant and/or chelating agent. In some examples, the container includes a chromatography resin for a packed column (eg, packed in a liquid including at least one antioxidant and/or chelating agent).

Any of the compositions provided herein may contain at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) antioxidant and/or at least one (e.g., 2, 3 , 4, 5, 6, 7, 8, 9 or 10) chelating agent. Any of the antioxidants included in any of the compositions presented herein may have the ability to quench one or more of the following reactive oxygen and/or nitrogen species: hydroxyl radical, carbonate radical, superoxide anion, peroxide radical, peroxynitrite, nitrogen dioxide and nitric oxide. Non-limiting examples of antioxidants that may be included in any of the compositions provided herein include: reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoid , phenylpropionic acid, lidocaine, naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl and dimethylmethoxychromanol. Additional non-limiting examples of antioxidants include antioxidant enzymes (eg, 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. Additional examples of antioxidants include cysteine, taurine, mercaptopropionylglycine, N-acetylcysteine, garlic oil, diallyl sulfide, dihydrolipoic acid, and diallyl trisulfide. Some embodiments comprising an antioxidant enzyme as an antioxidant may further include one or more substrates for the enzyme. The antioxidant can be identified by a number of methods known in the art, including, for example, spin trap, redox sensitive dyes and chemiluminescence assays.

Any of the chelating agents included in any of the compositions provided herein may have the ability to bind a redox-active metal (e.g., Cu ²⁺ and Fe ²⁺ ) with high affinity (e.g., about or less than 1 TM, about or less 800 nM, about or less than 700 nM, about or less than 600 nM, about or less than 500 nM, about or less than 400 nM, about or less than 300 nM, about or less than 250 nM, about or less than 200 nM, about or less than 150 nM , about or less than 100 nM, about or less than 80 nM, about or less than 60 nM, about or less than 40 nM, about or less than 20 nM, or about or less than 1 nm). Non-limiting examples of chelating agents that may be included in any of the compositions provided herein include ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanesulfonic acid sodium salt (DMPS), dimercaptosuccinic acid (DMSA), metallothionein and deferoxamine .

The concentration of each of the chelating agents and/or antioxidants in any of the compositions provided herein can range from about 0.1 mM to about 150 mM (e.g., from about 0.1 mM to about 150 mM, from about 0.1 mM to about 125 mM, from about 0.1 mM to about 100 mM, from about 0.1 mM to about 80 mM, from about 0.1 mM to about 60 mM, from about 0.1 mM to about 50 mM, from about 0.1 mM to about 40 mM, from about 0.1 mM to about 30 mM, from about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, from about 0.1 mM to about 5.0 mM, from about 0.5 mM to about 150 mM, from about 0.5 mM to about 100 mM, from about 0.5 mM to about 50 mM , from about 0.5 mM to about 25 mM, from about 0.5 mM to about 15 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 125 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, from about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 5 mM to about 150 mM, about 5 mM to about 125 mM, about 5 mM to about 100 mM, from about 5 mM to about 80 mM, from about 5 mM to about 60 mM, from about 5 mM to about 50 mM, from about 5 mM to about 40 mM, from about 5 mM to about 30 mM, from about 5 mM to about 25 mM, about 10 mM to about 150 mM, about 10 mM to about 125 mM, about 1 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 10 mM to about 30 mM, from about 10 mM to about 25 mM, from about 20 mM to about 150 mM, from about 20 mM to about 125 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM , from about 20 mM to about 30 mM, from about 30 mM to about 150 mM, from about 30 mM to about 125 mM, from about 30 mM to about 100 mM, from about 30 mM to about 80 mM, from about 30 mM to about 60 mM, from about 30 mM to about 50 mM, from about 30 mM to about 40 mM, from about 40 mM to about 150 mM, from about 40 mM to about 125 mM, from about 40 mM to about 100 mM, from about 40 mM to about 90 mM, from about 40 mM to about 80 mM, from about 40 mM to about 70 mM, from about 40 mM to about 60 mM, from about 50 mM to about 150 mM, from about 50 mM to about 125 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 50 mM to about 60 mM, about 80 mM to about 150 mM, about 80 mM to about 125 mM, from about 80 mM to about 100 mM, about 100 mM to about 150 mM, or about 100 mM to about 125 mM).

In some examples, the compositions provided herein contain one or more of 5 mM to about 150 mM mannitol (e.g., about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM, from about 40 mM to about 150 mM, from about 50 mM to about 150 mM, from about 60 mM to about 140 mM, from about 70 mM to about 130 mM, from about 80 mM to about 120 mM, from about 90 mM to about 110 mM, about 95 mM to about 105 mM, about 5 mM to about 50 mM, about 5 mM to about 45 mM, about 5 mM to about 40 mM, about 5 mM to about 35 mM, about 10 mM to about 35 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM mannitol); 5 mM to about 150 mM (e.g., about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, from about 60 mM to about 140 mM, from about 70 mM to about 130 mM, from about 80 mM to about 120 mM, from about 90 mM to about 110 mM, from about 95 mM to about 105 mM, from about 30 mM to about 70 mM, from about 35 mM to about 65 mM, from about 40 mM to about 60 mM, from about 45 mM to about 55 mM, from about 20 mM to about 50 mM, from about 25 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 5 mM to about 45 mM, about 10 mM to about 40 mM, about 15 mM to about 35 mM, from about 20 mM to about 30 mM or about 20 mM to about 25 mM) methionine (or alternatively cysteine or glutathione); 5 mM to 150 mM sodium ascorbate (e.g., about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, about 60 mM to about 140 mM, about 70 mM to about 130 mM, about 80 mM to about 120 mM, about 90 mM to about 110 mM, about 95 mM to about 105 mM , from about 10 mM to about 50 mM, from about 15 mM to about 45 mM, from about 20 mM to about 40 mM, from about 25 mM to about 35 mM, from about 30 mM to about 35 mM, from about 5 mM to about 45 mM, from about 10 mM to about 40 mM, from about 15 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM sodium ascorbate); and from 5 mM to about 150 mM (e.g., from about 10 mM to about 150 mM, from about 20 mM to about 150 mM, from about 30 mM to about 150 mM, from about 40 mM to about 150 mM, from about 50 mM to about 150 mM, about 60 mM to about 140 mM, about 70 mM to about 130 mM, about 80 mM to about 120 mM, about 90 mM to about 110 mM, about 95 mM to about 105 mM , from about 30 mM to about 70 mM, from about 35 mM to about 65 mM, from about 40 mM to about 60 mM, from about 45 mM to about 55 mM, from about 20 mM to about 50 mM, from about 25 mM to about 45 mM, from about 30 mM to about 40 mM, from about 30 mM to about 35 mM, from about 5 mM to about 45 mM, from about 10 mM to about 40 mM, from about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 20 mM to about 25 mM) histidine.

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

Non-limiting doses of gamma radiation sufficient to reduce the bioburden of any of the compositions presented herein are described below. Additional doses of gamma radiation sufficient to reduce the bioburden of any of the compositions presented herein are known in the art. For example, any of the compositions presented herein may be subjected to gamma irradiation at any of the doses, at any of the gamma irradiation powers, and/or at any of the temperatures to produce the gamma irradiation presented herein (in any combination) . The bioburden of a composition can be determined, for example, by obtaining a sample of the composition that will contain self-replicating biological contaminants present in the composition, for example, by digestion, sonication, shaking, vortexing, washing, mixing or swabbing and qualitative or quantitative analysis the level of self-replicating biological contaminants present in the sample (for example, by placing the sample in a growth medium that will allow the biological contaminant to self-replicate, such as plating the sample on a Petri dish or passing the sample through a membrane).

An amount of at least one antioxidant and/or chelating agent sufficient to improve the loss of binding capacity of the chromatography resin after treatment with a dose of gamma radiation sufficient to reduce the bioburden of the composition can be determined, for example, by the methods described in the examples. For example, the level of reduction in binding capacity of a chromatography resin exposed to gamma irradiation in the presence of an amount of at least one antioxidant and/or chelating agent can be compared with the level of reduction in binding capacity of a chromatography resin treated with the same dose of gamma irradiation in the absence of at least one antioxidant and/or chelating agent, wherein reducing the level of decrease in binding capacity of a chromatography resin gamma-irradiated in the presence of at least one antioxidant and/or chelating agent, compared to a chromatography resin gamma-irradiated in the absence of at least one antioxidant and/or or chelating agent, indicates that at least one antioxidant and/or chelating agent was present in an amount sufficient to improve the loss of binding capacity of the chromatography resin after gamma irradiation. Examples of 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 a chromatography resin are known in the art.

Methods for reducing bioburden of chromatography resin

The present invention relates to methods for reducing the bioburden of a chromatography resin. These methods include the step of exposing a container comprising a composition comprising a chromatography resin and at least one antioxidant and/or chelating agent to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin, wherein the at least one antioxidant and/or chelating agent the agent is present in an amount sufficient to improve the loss of binding capacity of the chromatographic resin after exposure to a dose of gamma radiation.

The present invention also provides methods for reducing the bioburden of a chromatography resin, comprising exposing a container comprising a composition comprising the chromatography resin (and optionally at least one antioxidant and/or chelating agent in an amount sufficient to improve the loss of binding capacity of the chromatography resin after exposure to gamma irradiation ) exposure to gamma radiation at powers from about 0.1 kGy/h to about 6 kGy/h (e.g., from about 0.1 kGy/h to about 5.5 kGy/h, from about 0.1 kGy/h to approximately 5.0 kGy/h, approximately 0.1 kGy/h to approximately 4.5 kGy/h, approximately 0.1 kGy/h to approximately 4.0 kGy/h, approximately 0.1 kGy/h to approximately 3.5 kGy/h, from approximately 0.1 kGy/h to approximately 3.0 kGy/h, from approximately 0.1 kGy/h to approximately 2.5 kGy/h, from approximately 0.1 kGy/ h to about 2.0 kGy/h, from about 0.1 kGy/h to about 1.5 kGy/h, from about 0.1 kGy/h to about 1.0 kGy/h, from about 0.5 kGy /h to approximately 6 kGy/h, from approximately 0.5 kGy/h to approximately 5.5 kGy/h, from approximately 0.5 kGy/h to approximately 5.0 kGy/h, from approximately 0.5 kGy/ h to about 4.5 kGy/h, from about 0.5 kGy/h to about 4.0 kGy/h, from about 0.5 kGy/h to about 3.5 kGy/h, from about 0.5 kGy /h to about 3.0 kGy/h, from about 0.5 kGy/h to about 2.5 kGy/h, from about 0.5 kGy/h to about 2.0 kGy/h) and/or at temperature from about 4°C to about 25°C (e.g., from about 4°C to about 20°C, from about 4°C to about 15°C, from about 4°C to about 10°C, from about 10° C to about 25°C, from about 10°C to about 20°C, from about 10°C to about 15°C, or from about 15°C to about 25°C) in the case of a gamma radiation dose sufficient to reducing the bioburden of the container and chromatography resin. In the methods described in this paragraph, the level of binding capacity of the gamma-irradiated chromatography resin produced by these methods is higher than the level of binding capacity of the gamma-irradiated chromatography resin gamma-irradiated at powers above 6.1 kGy/h and/or at temperatures above 25 °C, or both.

The chromatography resin can be subjected to gamma irradiation by methods known in the art. For example, an isotope such as cobalt-60 or cesium-137 can be used as a gamma ray source. The chromatography resin can be subjected to gamma irradiation at a temperature of from about -25°C to about 0°C inclusive, or from about 0°C to about 25°C inclusive. The chromatography resin can be exposed to a gamma radiation dose of from about 0.1 kGy to about 100 kGy, from about 1 kGy to about 100 kGy, from about 1 kGy to about 90 kGy, from about 1 kGy to about 80 kGy, from about 1 kGy to about 70 kGy, about 1 kGy to about 65 kGy, about 5 kGy to about 65 kGy, about 10 kGy to about 60 kGy, about 10 kGy to about 55 kGy, about 10 kGy to about 50 kGy , from about 10 kGy to about 45 kGy, from about 10 kGy to about 40 kGy, from about 10 kGy to about 35 kGy, from about 10 kGy to about 30 kGy, from about 15 kGy to about 50 kGy, from about 15 kGy to about 45 kGy, from about 15 kGy to about 40 kGy, from about 15 kGy to about 35 kGy, from about 20 kGy to about 30 kGy, or from about 23 kGy to about 27 kGy. The chromatography resin may be subjected to gamma irradiation at a dose sufficient to obtain a sterility assurance level of the chromatography resin of about or less than 1×10 ^-6 , about or less than 1×10 ^-7 , about or less than 10×10 ^-8 , about or less than 1× 10 ^-11 , or approximately or less than 1×10 ^-12 , or between approximately 1×10 ^-6 and approximately 1×10 ^-12 , between approximately 1×10 ^-6 and approximately 1×10 ^-11 , between approximately 1×10 ^-6 and about 1×10 ^-10 , from about 1×10 ^-6 and about 1×10 ^-9 , from about 1×10 ^-6 and about 1×10 ^-8 , from 1×10 ^-6 to about 1× 10 ^-7 , from approximately 1×10 ^-7 and approximately 1×10 ^-12 , from approximately 1×10 ^-7 and approximately 1×10 ^-11 , from approximately 1×10 ^-7 and approximately 1×10 ^-10 , from from approximately 1×10 ^-7 and approximately 1×10 ^-9 , from approximately 1×10 ^-7 and approximately 1×10 ^-8 , from approximately 1×10 ^-8 and approximately 1×10 ^-12 , from approximately 1×10 ^{- 8} and about 1×10 ^-11 , from about 1×10 ^-8 and about 1×10 ^-10 , or from about 1×10 ^-8 and about 1×10 ^-9 .

The dose of gamma irradiation sufficient to reduce the bioburden of the chromatography resin can be determined by methods known in the art. For example, the bioburden level of a gamma-irradiated chromatography resin can be compared with the bioburden level of an untreated (eg, control, non-gamma-irradiated) chromatography resin, and the reduction in the bioburden level of a gamma-irradiated chromatography resin compared to an untreated chromatography resin indicates that the dose of gamma radiation is sufficient to reduce the bioburden of the chromatography resin. Examples of methods for determining the bioburden level of a composition (eg, chromatography resin) are presented herein. Additional methods for determining the bioburden level of a composition (eg, chromatography resin) are known in the art.

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

The container comprising the chromatography resin may be a plastic container (eg, a cylindrical tube, a sealed or secured box, or a sealed bag). Non-limiting examples of containers used in these methods include a storage vessel or a chromatography column. For example, a composition comprising a chromatography resin and at least one antioxidant and/or chelating agent may be contained in a sealed container (eg, a suspension in a sealed container or a chromatography resin for a packed column in a sealed container (eg, a chromatography column)). The container used in the methods presented herein may be a disposable chromatography column. In some embodiments, the container used in the methods presented herein is a disposable chromatography column housed in a blister pack. The container (e.g., a storage vessel or chromatography column) may have a total internal volume of from about 1 ml to about 1 L (e.g., from about 1 ml to about 900 ml, from about 1 ml to about 800 ml, from about 1 ml to about 700 ml, from about 1 ml to about 600 ml, from about 1 ml to about 500 ml, from about 1 ml to about 450 ml, from about 1 ml to about 400 ml, from about 1 ml to about 350 ml, from about 1 ml to about 300 ml, from about 1 ml to about 250 ml, from about 1 ml to about 200 ml, from about 1 ml to about 150 ml, from about 1 ml to about 100 ml, from about 1 ml to about 75 ml, about 1 ml to about 50 ml, about 1 ml to about 40 ml, about 1 ml to about 30 ml, or about 1 ml to about 20 ml).

The composition containing the chromatography resin and at least one antioxidant and/or chelating agent included in the container may be in a solid mixture (eg, a dry or wetted solid mixture). For example, the container may include a suspension of the sedimented chromatography resin in a liquid (eg, a buffer solution) containing at least one antioxidant and/or chelating agent. In some embodiments, the container may include a chromatography resin for a packed column. For example, a container including a composition comprising a chromatography resin and at least one antioxidant and/or chromatography resin is a packed chromatography column (e.g., in which the resin is filled with a liquid (e.g., a buffer solution) containing at least one antioxidant and/or chromatographic resin). Some embodiments include placing a composition comprising a chromatography resin and at least one antioxidant and/or chelating agent in a container prior to exposure.

Any of the antioxidants and/or chelating agents presented herein can be used in any combination, using any combination of the exemplary concentrations presented herein. For example, the composition may include at least one antioxidant selected from: reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, flavonoid, phenylpropionic acid, lidocaine , naringenin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl and dimethylmethoxychromanol, and/or at least one chelating agent selected from: EDTA, DMPS, DMSA, metallothionein and deferoxamine.

Provided herein are examples of methods for determining/identifying amounts of at least one antioxidant and/or chelating agent sufficient to improve the loss of binding capacity of a chromatography resin after treatment with a dose of gamma radiation sufficient to reduce the bioburden of the composition. Additional methods for determining/identifying amounts of at least one antioxidant and/or chelating agent sufficient to improve the loss of binding capacity of a chromatography resin after treatment with a dose of gamma radiation sufficient to reduce the bioburden of the composition are known in the art.

The present invention also relates to a reduced bioburden chromatography resin prepared by any of the methods presented herein (eg, a reduced bioburden chromatography resin provided in a storage container, eg, a sealed storage container). The reduced bioburden chromatography resin produced by any of the methods presented herein may have a sterility assurance level of about or less than 1x10 ^-6 , about or less than 1x10 ^-7 , about or less than 10x10 ^-8 , about or less than 1×10 ^-11 , or about or less than 1×10 ^-12 , or from about 1×10 ^-6 to about 1×10 ^-12 , from about 1×10 ^-6 to about 1×10 ^-11 , from about 1x10 ^-6 to about 1x10 ^-10 , from about 1x10 ^-6 to about 1x10 ^-9 , from about 1x10 ^-6 to about 1x10 ^-8 , from 1x10 ^-6 to about 1×10 ^-7 , from about 1×10 ^-7 to about 1×10 ^-12 , from about 1×10 ^-7 to about 1×10 ^-11 , from about 1×10 ^-7 to about 1×10 ^{- 10} , from about 1x10 ^-7 to about 1x10 ^-9 , from about 1x10 ^-7 to about 1x10 ^-8 , from about 1x10 ^-8 to about 1x10 ^-12 , from about 1 ×10 ^-8 to about 1x10 ^-11 , from about 1x10 ^-8 to about 1x10 ^-10 or from about 1x10 ^-8 to about 1x10 ^-9 . The reduced bioburden chromatography resin produced by any of the methods presented herein may have a binding capacity of at least 74% (e.g., at least 76%, at least 78%, at least 80%, at least 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 from about 74% to about 95%, from about 74% to about 95%, from about 76% to about 95%, at least about 78% to about 95%, from about 80% to about 95%, or about 74% to about 90%, about 76% to about 90%, about 78% to about 90%, or about 80% to about 90% of the binding capacity of the same chromatography resin not treated to reduce its bioburden (eg, not subjected to gamma irradiation) if the same protein is used to test the binding ability of a chromatography resin produced by the methods presented herein and a control, untreated chromatography resin.

Methods for obtaining a packed chromatography column with reduced bioburden

The present invention also provides methods for preparing a packed chromatography column with reduced bioburden, comprising preparing a chromatography resin with reduced bioburden obtained by any of the methods presented herein, and packing the chromatography resin into a reduced bioburden column under aseptic or reduced bioburden conditions. In some embodiments, a packed chromatography column with reduced bioburden can be obtained by exposing the column, including a loaded chromatography resin and at least one antioxidant and/or chelating agent, to a dose of gamma radiation sufficient to reduce the bioburden of the column and packed chromatography resin. columns wherein the at least one antioxidant and/or chelating agent is present in an amount sufficient to improve the loss of binding capacity of the packed column chromatography resin after exposure to a dose of gamma radiation.

The present invention also relates to packed chromatography columns with reduced bioburden, obtained by the methods presented in the present description. Any of the packed reduced bioburden chromatography columns produced by the methods presented herein may have a sterility assurance level of about or less than 1×10 ^-6 , about or less than 1×10 ^-7 , about or less than 10×10 ^-8 , approximately or less than 1×10 ^-11 , or approximately or less than 1×10 ^-12 , or from approximately 1×10 ^-6 to approximately 1×10 ^-12 , from approximately 1×10 ^-6 to approximately 1×10 ^-11 , from about 1×10 ^-6 to about 1×10 ^-10 , from about 1×10 ^-6 to about 1×10 ^-9 , from about 1×10 ^-6 to about 1×10 ^-8 , from 1×10 ^-6 to about 1×10 ^-7 , from about 1×10 ^-7 to about 1×10 ^-12 , from about 1×10 ^-7 to about 1×10 ^-11 , from about 1×10 ^-7 to about 1×10 ^-10 , from about 1×10 ^-7 to about 1×10 ^-9 , from about 1×10 ^-7 to about 1×10 ^-8 , from about 1×10 ^-8 to about 1×10 ^-12 , from about 1×10 ^-8 to about 1×10 ^-11 , from about 1×10 ^-8 to about 1×10 ^-10 , or from about 1×10 ^-8 to about 1×10 ^-9 . Any of the reduced bioburden packed chromatography columns produced by the methods presented herein may contain at least one chromatography resin selected from the group: anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin (e.g., any of the affinity chromatography resins presented herein or known in the art), hydrophobic interaction chromatography resin and size exclusion chromatography resin. For example, any of the packed reduced bioburden chromatography columns provided herein may include an affinity chromatography resin including a protein ligand (eg, protein A). The packed reduced bioburden chromatography column provided herein may include an anion exchange chromatography resin (eg, an anion exchange chromatography resin including N-benzyl-N-methyl-ethanolamine groups).

Methods for performing chromatography with reduced bioburden

The methods provided herein include the use of a packed reduced bioburden chromatography column as set forth herein, and the methods provided herein include the use of one or two MCCSs comprising at least one packed reduced bioburden chromatography column as set forth in this description. The gamma irradiated chromatography resin can be any type of resin presented herein (or any type of chromatography resin known in the art).

A packed chromatography column with reduced bioburden can be prepared by any of the methods presented herein. For example, a packed chromatography column with reduced bioburden can be obtained by packing the chromatography column with a composition comprising chromatography resins and at least one antioxidant and/or chelating agent (for example, any of such compositions presented herein) and exposing the packed column to gamma exposure (eg, using any of the exposures and conditions presented herein). In other examples, a packed chromatography column with reduced bioburden can be prepared by exposing a container including a chromatography resin and at least one antioxidant and/or chelating agent to a dose of gamma radiation and packing the chromatography column with the resulting reduced bioburden chromatography resin. In such methods, the chromatography resin (present in the container when subjected to gamma irradiation) may be suspended in the container and the chromatography column is packed in a bioburden-reduced fume hood. In other methods, a chromatography resin present in a container with at least one antioxidant and/or chelating agent can be gamma-irradiated as a solid mixture in the container, and a suspension of the resulting bioburden-reduced chromatography resin can be prepared using a bioburden-reduced buffer ( e.g., produced in a bioburden-reduced fume hood), and the resulting suspension is used to fill a chromatography column in a bioburden-reduced fume hood. In some of these examples, the chromatography column may be treated to reduce bioburden (eg, autoclave, gamma irradiation, or ethylene oxide) prior to packing.

A packed, reduced bioburden chromatography column used in any of the methods presented herein may have a Sterility Assurance Level (SAL) of about 1x10 ^-3 to about 1x10 ^-12 , about 1x10 ^-4 to about 1×10 ^-12 , from 1×10 ^-5 to about 1×10 ^-11 , from about 1×10 ^-5 to about 1×10 ^-10 , from about 1×10 ^-5 to about 1×10 ^-9 , from about 1×10 ^-6 to about 1×10 ^-9 or from about 1×10 ^-6 to about 1×10 ^-8 , inclusive.

Buffers with reduced bioburden

The methods presented herein can be carried out using one or more buffers with reduced bioburden. As is known in the art, the reduced bioburden buffer can be any type of buffer used in a chromatography run (eg, a buffer used in any of the steps of a chromatography run or in any of the typical operations presented herein). Examples of methods to reduce buffer bioburden include filtration (0.2 μm pore size filtration), autoclaving, and gamma irradiation. Additional methods for reducing buffer bioburden are known in the art. The reduced bioburden buffer may have a sterility assurance level of about 1x10 ^-3 to about 1x10 ^-12 , about 1x10 ^-4 to about 1x10 ^-12 , 1x10 ^-5 to about 1x10 ^-11 , from about 1×10 ^-5 to about 1×10 ^-10 , from about 1×10 ^-5 to about 1×10 ^-9 , from about 1×10 ^-6 to about 1×10 ^-9 or from about 1×10 ^-6 to approximately 1×10 ^-8 , inclusive.

Recombinant therapeutic proteins

As provided herein, the recombinant protein may be a recombinant therapeutic protein. Non-limiting examples of recombinant therapeutic proteins that can be produced by the methods presented herein include immunoglobulins (including immunoglobulin light and heavy chain, antibodies or antibody fragments (e.g., any of the antibody fragments provided herein), enzymes (e.g., galactosidase ( e.g., alpha-galactosidase), Myozyme® or Cerezyme®), proteins (e.g., erythropoietin, tumor necrosis factor (TNF), or human interferon alpha or beta) or immunogenic or antigenic proteins or protein fragments (e.g., proteins for use in a vaccine) Non-limiting examples of recombinant therapeutic enzymes that can be used to treat a variety of lysosomal storage diseases are presented in Figure 10. The recombinant therapeutic protein may be an engineered antigen binding polypeptide comprising at least one multifunctional recombinant protein scaffold (see, for example, recombinant antigen binding proteins, described in Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009; and US Patent Application Publication No. 2012/0164066 (incorporated herein by reference in its entirety)). Non-limiting examples of recombinant therapeutic antibody proteins include: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatum omab, apolizumab, atinumab, tocilizumab, basiliximab , bektumomab, belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab, etaracizumab, golimumab, infliximab, natalizumab, palivizumab, panitumumab, pertuzuma b, ranibizumab, rituximab, tocilizumab and trastuzumab. Additional examples of recombinant therapeutic antibodies that can be produced by the methods presented herein are known in the art. Additional non-limiting examples of recombinant therapeutic proteins that can be produced/purified by the methods of the present invention include: alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, factor coagulation IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa and alteplase.

The secreted, soluble recombinant therapeutic protein can be isolated from the liquid culture medium (eg, the first and/or second liquid culture medium) by removing or otherwise physically separating the liquid culture medium from the cells (eg, mammalian cells). Many different methods are known in the art to remove liquid culture medium from cells (eg, mammalian cells), including, for example, centrifugation, filtration, pipetting and/or aspiration. The secreted recombinant therapeutic protein can then be isolated and further purified from the liquid culture medium by a variety of biochemical methods, 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 (eg, molecular weight cutoff filtration).

Chromatography cycle

As is well known in the art, the steps of a chromatography cycle may differ depending on the chromatography resin, the buffers used to carry out each step of the cycle, and the biophysical characteristics of the target recombinant protein (eg, the recombinant therapeutic protein). For example, affinity chromatography on a column may include the steps of loading an affinity chromatography column with a liquid including a recombinant target protein, washing the column to remove unwanted biological material (e.g., contaminating proteins and/or small molecules), eluting the recombinant target protein bound to the column, and re-equilibration of the column. A chromatography cycle using a cation exchange and/or anion exchange chromatography column, where the recombinant target protein is bound to the chromatography resin in a loading step, may include the steps of loading the column with a liquid including the target protein, washing the column to remove unwanted biological material, eluting the recombinant target protein , contacting the column, and re-equilibrating the column. In other examples, a chromatography run using a cation exchange and/or anion exchange chromatography column, where the unwanted biological material is bound to the chromatography resin in a loading step while the recombinant target protein is not bound, may include the steps of loading the column with a liquid including the target protein, collecting the recombinant target protein in the eluate and re-equilibrating the column. As is well known in the art, any of the individual steps of the chromatography run may include a single buffer or multiple buffers (eg, two or more buffers), and one or more of any of the individual steps of the chromatography run may include a buffer gradient. Any of combinations of various well-known aspects of a single chromatography run can be used in these methods in any combination, for example, various chromatography resins, eluates, buffers, column volumes excluded, column bed volumes, volumes of buffer used in each step, volumes of liquid including protein -target, and the range and types of buffers used at each stage.

Methods for performing chromatography on columns with reduced bioburden

The present invention relates to methods for performing chromatography with reduced bioburden. These methods include preparing a packed chromatography column with reduced bioburden obtained by any of the methods presented herein, and performing column chromatography using a packed chromatography column with reduced bioburden. The packed reduced bioburden chromatography column may include at least one of any of the chromatography resins provided herein in any combination. For example, the chromatography resin present in a packed reduced bioburden chromatography column may be an affinity resin including a protein ligand (eg, protein A) or may include an anion exchange chromatography resin. The packed reduced bioburden chromatography column may have any of the example internal volumes presented herein. The packed reduced bioburden chromatography column can have any shape (eg, cylinder, sub-cylindrical shape, or elliptical shape) described herein or known in the art. Column chromatography, performed by any method, can be used to purify or isolate a recombinant protein (eg, any of the recombinant therapeutic proteins described herein or known in the art). In some examples, the packed, reduced bioburden chromatography column is part of a multicolumn chromatography system (MCCS), for example, may be part of a batch countercurrent chromatography system (PCCS).

The column chromatography performed may include at least one cycle of chromatography as described herein or known in the art. For example, at least one chromatography cycle may include the steps of: capturing the recombinant protein by exposing the chromatography resin to a liquid including the recombinant protein; washing the chromatography resin by exposing the chromatography resin to a wash buffer, eluting the recombinant protein by exposing the chromatography resin to an elution buffer; and regenerating the chromatography resin by exposing the chromatography resin to a regeneration buffer. In some examples, the liquid comprising the recombinant protein is a liquid culture medium (eg, a liquid culture medium collected from a perfusion or batch culture) or a diluted liquid culture medium (eg, a culture medium diluted with a buffer).

Column chromatography can be performed using a closed-loop and integrated system (eg, any of the examples of closed-loop and integrated systems presented herein or known in the art). For example, column chromatography can be performed using a closed and integrated system where the buffer is a reduced bioburden buffer. As is well known in this field, a buffer with reduced bioburden can be obtained using a variety of different methods (eg, obtained by filtration, autoclaving or thermal processing).

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

In some embodiments, the chromatography resin in a packed bioburden chromatography column has a binding capacity percentage of from about 75% to about 100% (e.g., from about 76% to about 98%, from about 76% to about 96%, from about 76 % to about 94%, from about 76% to about 92%, from about 76% to about 90%, from about 78% to about 100%, from about 78% to about 98%, from about 78% to about 96% , from about 78% to about 94%, from about 78% to about 92%, from about 78% to about 90%, from about 80% to about 100%, from about 80% to about 98%, from about 80% to about 96%, from about 80% to about 94%, from about 80% to about 92%, from about 80% to about 90%, from about 82% to about 100%, from about 82% to about 98%, from about 82% to about 96%, from about 82% to about 94%, from about 82% to about 92%, from about 82% to about 90%, from about 84% to about 100%, from about 84% to about 98%, from about 84% to about 96%, from about 84% to about 94%, from about 84% to about 92%, from about 84% to about 90%, from about 86% to about 100%, from about 86% to about 98%, about 86% to about 96%, about 86% to about 94%, about 86% to about 92%, about 88% to about 100%, about 88% to about 98%, from about 88% to about 96%, from about 88% to about 94%, from about 90% to about 100%, from about 90% to about 98%, from about 90% to about 96%, from about 92% to about 100% or about 92% to about 98%) compared to the same resin not subjected to gamma irradiation (if the same protein is used to test the binding ability of both resins) (e.g., assessed directly after gamma -irradiation).

Integrated, closed-loop, or substantially closed-loop, and continuous methods for producing recombinant protein

The present invention relates to integrated, closed, or substantially closed, and continuous methods for the production of purified recombinant protein (eg, recombinant therapeutic protein). These methods involve providing a liquid culture medium comprising a recombinant protein (eg, a recombinant therapeutic protein) substantially free of cells.

Some methods involve continuously feeding a liquid culture medium into a multicolumn chromatography system (MCCS) comprising at least one packed bioburden-reduced chromatography column as described herein, wherein the methods use a bioburden-reduced buffer, the methods are integrated, and are carried out continuously from the liquid culture medium to the eluate from the MCCS, which is a purified recombinant protein (eg, a therapeutic protein drug).

Some methods include continuously feeding a liquid culture medium into a first MCCS (MCCS1), capturing a recombinant protein from a liquid culture medium using MCCS1, obtaining an eluate from MCCS1 including the recombinant protein, and continuously feeding the eluate into a second MCCS (MCCS2), and continuously feeding the recombinant protein from the eluate into MCCS2 and subsequent elution of the recombinant protein, thereby obtaining a purified recombinant protein, wherein at least one column in MCCS1 and/or MCCS2 is a packed reduced bioburden chromatography column as provided herein, in methods use a buffer with reduced bioburden, the methods are integrated and are carried out continuously from the liquid culture medium to the purified recombinant protein.

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

The methods presented herein provide the continuous and rapid production of purified recombinant protein from a liquid culture medium containing the recombinant protein. For example, the period of time between feeding a liquid culture medium comprising a therapeutic protein into the MCCS or MCCS1 and elution of the recombinant protein from the MCCS or MCCS2, respectively, can be, for example, from about 4 hours to about 48 hours inclusive, for example, from about 4 hours to about 40 hours, from about 4 hours to about 35 hours, from about 4 hours to about 30 hours, from about 4 hours to about 28 hours, from about 4 hours to about 26 hours, from about 4 hours to about 24 hours , from about 4 hours to about 22 hours, from about 4 hours to about 20 hours, from about 4 hours to about 18 hours, from about 4 hours to about 16 hours, from about 4 hours to about 14 hours, from about 4 hours to about 12 hours, from about 6 hours to about 12 hours, from about 8 hours to about 12 hours, from about 6 hours to about 20 hours, from about 6 hours to about 18 hours, from about 6 hours to about 14 hours, from about 8 hours to about 16 hours, from about 8 hours to about 14 hours, from about 8 hours to about 12 hours, from about 10 hours to 20 hours, from about 10 hours to 18 hours, from about 10 hours to 16 hours , from about 10 hours to about 14 hours, from about 12 hours to about 14 hours, from about 10 hours to about 40 hours, from about 10 hours to about 35 hours, from about 10 hours to about 30 hours, from about 10 hours to about 25 hours, from about 15 hours to about 40 hours, from about 15 hours to about 35 hours, from about 15 hours to about 30 hours, from about 20 hours to about 40 hours, from about 20 hours to about 35 hours, or from approximately 20 hours to approximately 30 hours, inclusive. In other examples, the period of time between feeding the liquid culture medium comprising the recombinant protein into the MCCS or MCCS1 and elution of the recombinant protein from the MCCS or MCCS2, respectively, is, for example, more than about 4 hours and less than about 40 hours, inclusive, for example, more than approximately 4 hours or less 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.

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

Some example methods do not use a storage step (eg, do not use a reservoir (eg, tank) in the entire method). Others may use a maximum of 1, 2, 3, 4 or 5 reservoirs (eg tanks) in the entire method. In any of the methods presented herein, a maximum of 1, 2, 3, 4 or 5 reservoirs (e.g., tanks) may be used throughout the process, with each tank only holding the recombinant protein for a total time period of, for example, about 5 minutes to less than about 6 hours, inclusive, 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, inclusive.

Some methods use one, two, three, four, five or six reservoirs (e.g., tanks) and may have a capacity of, for example, from 1 ml to about 300 ml, inclusive, for example, from 1 ml to about 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 (inclusive). Any reservoirs (eg, tanks) used (in any of the methods presented herein) to store liquid prior to feeding into the MCCS or MCCS1 may have a capacity of, for example, 1 ml up to and including about 100%, such as 1 ml to about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% inclusive of the loading volume of the first MCCS or MCCS1 column. Reservoirs (eg, tanks) may be used to store the eluate from MCCS1 prior to entering MCCS2, and may have a capacity of, for example, from 1 ml to about 100% inclusive, for example from 1 ml to about 90%, about 80 %, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 10%, or approximately 5% inclusive of the loading volume of the first MCCS2 column.

Various additional aspects of these methods are described in detail below, and they can be used in any combination in the methods presented herein, without limitation. Examples of aspects of the presented methods are described below; however, one skilled in the art will appreciate that additional steps can be added to the methods presented herein, and other materials can be used to implement any of the steps of the methods presented herein.

Liquid culture medium

Liquid culture medium comprising a recombinant protein (eg, a recombinant therapeutic protein) substantially free of cells can be obtained from any source. For example, the liquid culture medium can be obtained from a culture of recombinant cells (eg, a culture of recombinant bacterial, yeast or mammalian cells). The liquid culture medium can be obtained from a batch culture of cells (e.g., mammalian cells) (e.g., a batch bioreactor comprising a culture of mammalian cells secreting a recombinant protein) or a perfusion culture of cells (e.g., mammalian cells) (e.g., a perfusion bioreactor comprising a culture mammalian cells secreting recombinant protein). The liquid culture medium may also be a purified liquid culture medium from a culture of bacterial or yeast cells secreting the recombinant protein.

The liquid culture medium obtained from the recombinant cell culture can be filtered or purified to obtain a liquid culture medium substantially free of cells and/or viruses. Methods for filtering or purifying a liquid culture medium to remove cells are known in the art (eg, 0.2 μm filtration and Alternating Tangential Flow (ATF ^™ ) system filtration). Recombinant cells can also be removed from the liquid culture medium using centrifugation and removal of the supernatant, which is a liquid culture medium substantially free of cells, or by allowing the cells to settle by gravity to the bottom of a container (eg, a bioreactor) containing the liquid medium for cultivation, and removing the liquid culture medium (liquid culture medium substantially free of cells) away from the settled recombinant cells.

The liquid culture medium can be prepared from a culture of recombinant cells (eg, recombinant bacteria, yeast, or mammalian cells) producing any of the recombinant proteins (eg, recombinant therapeutic proteins) described herein or known in the art. Some examples of any of the methods presented herein may further include the step of culturing recombinant cells (eg, recombinant bacteria, yeast or mammalian cells) producing a recombinant protein (eg, a recombinant therapeutic protein).

The liquid culture medium may be any of the types of liquid culture medium described herein or known in the art. For example, the liquid culture medium may be selected from the group of: a liquid culture medium not containing an animal component, a liquid culture medium not containing serum, a liquid culture medium containing serum, a chemically defined liquid culture medium, and a liquid culture medium for cultivation, does not contain protein. In any of the methods presented herein, the liquid culture medium obtained from the culture can be diluted by adding a second liquid (eg, a buffer) prior to feeding it into the MCCS or MCCS1.

Liquid culture medium comprising the recombinant protein, substantially free of cells, can be stored (e.g., below about 15°C (e.g., below about 10°C, below about 4°C, below about 0°C, below about -20°C, below about -50°C, below about -70°C, or below about -80°C) for at least 1 day (e.g., at least about 2 days, at least about 5 days , at least about 10 days, at least about 15 days, at least about 20 days, or at least about 30 days) before feeding the liquid culture medium into the MCCS or MCCS 1. Alternatively, in some examples, the liquid culture medium is subjected to feeding into the MCCS or MCCS1 directly from the bioreactor (eg, feeding into the MCCS or MCCS1 directly from the bioreactor after a filtration or purification step).

Multi-column chromatography systems

The methods presented herein include the use of an MCCS or two or more (eg, two, three, four, five or six) multi-column chromatography systems (MCCS) (eg, MCCS1 and MCCS2). The 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 non-limiting examples, the MCCS (e.g., MCCS, MCCS1, and/or MCCS2 in any of the methods herein) may include four chromatography columns, three chromatography columns and a chromatography membrane, three chromatography columns, two chromatography columns, two chromatography membranes, and two chromatography columns and one chromatographic membrane. Additional examples of combinations of chromatography columns and/or chromatography membranes for use by one skilled in the art in MCCS (eg, MCCS, MCCS1 and/or MCCS2 in any of the methods presented herein) may be provided without limitation. The individual chromatography columns and/or chromatography membranes present in the MCCS may be identical (e.g., have the same shape, volume, resin, capture mechanism, and operation type) or different (e.g., have one or more different shapes, volumes, resins, gripping mechanisms and/or typical operations). The individual chromatography columns and/or chromatography membranes present in the MCCS (e.g., MCCS, MCCS1, and/or MCCS2 in any of the methods presented herein) can perform the same generic operation (e.g., a generic capture, purification, or fine purification) or various typical operations (for example, various typical operations selected, for example, from the group of capture, purification, fine purification, virus inactivation, correction of ion concentration and/or pH of the liquid containing the recombinant protein, and filtration). For example, in the exemplary methods presented herein, a typical recombinant protein capture operation is performed by at least one chromatography column and/or chromatography membrane in the MCCS or MCCS1.

One or more chromatography columns that may be contained in the MCCS (e.g., contained MCCS, MCCS1 and/or MCCS2) may have a resin volume of, for example, from about 1 ml to about 2 ml, about 5 ml, about 10 ml, about 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, inclusive. The one or more chromatography columns that may be contained in the MCCS (e.g., contained in the MCCS, MCCS1 and/or MCCS2) may have a resin volume of from about 2 ml to about 100 ml, from about 2 ml to about 90 ml, from about 2 ml to about 80 ml, from about 2 ml to about 70 ml, from about 2 ml to about 60 ml, from about 2 ml to about 50 ml, from about 5 ml to about 50 ml, from about 2 ml to about 45 ml , from about 5 ml to about 45 ml, from about 2 ml to about 40 ml, from about 5 ml to about 40 ml, from about 2 ml to about 35 ml, from about 5 ml to about 35 ml, from about 2 ml to about 30 ml, from about 5 ml to about 30 ml, from about 2 ml to about 25 ml, from about 5 ml to about 25 ml, from about 15 ml to about 60 ml, from about 10 ml to about 60 ml, from about 10 ml to about 50 ml and from about 15 ml to about 50 ml. One or more chromatography columns in MCCS (eg, MCCS, MCCS1 and/or MCCS2) used in any of the methods presented herein may have substantially the same volume of resin or may have different volumes of resin. The flow rate used for one or more chromatography columns in an MCCS (e.g., MCCS, MCCS1, and/or MCCS2) may be, for example, from about 0.2 mL/minute to about 25 mL/minute (e.g., from about 0. 2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, about 0.5 ml/minute to about 10 ml/minute, about 0.5 ml/minute to about 14 ml/minute, about 1.0 ml/minute to about 25.0 ml /minute or from about 1.0 ml/minute to about 15.0 ml/minute).

One or more chromatography columns in an MCCS (eg, MCCS, MCCS1 and/or MCCS2) may have substantially the same shape or may have substantially different shapes. For example, one or more chromatography columns in an MCCS (eg, MCCS, MCCS1 and/or MCCS2) may be substantially circular in shape or may be substantially oval in shape.

One or more chromatography membranes that may be present in the MCCS (e.g., located in the MCCS, MCCS1, and/or MCCS2) may have a bed volume, for example, from about 1 ml to about 500 ml (for example, from about 1 ml to about 475 ml, from about 1 ml to about 450 ml, from about 1 ml to about 425 ml, from about 1 ml to about 400 ml, from about 1 ml to about 375 ml, from about 1 ml to about 350 ml, from about 1 ml to about 325 ml, from about 1 ml to about 300 ml, from about 1 ml to about 275 ml, from about 1 ml to about 250 ml, from about 1 ml to about 225 ml, from about 1 ml to about 200 ml , from about 1 ml to about 175 ml, from about 1 ml to about 150 ml, from about 1 ml to about 125 ml, from about 1 ml to about 100 ml, from about 2 ml to about 100 ml, from about 5 ml to about 100 ml, from about 1 ml to about 80 ml, from about 2 ml to about 80 ml, from about 5 ml to about 80 ml, from about 1 ml to about 60 ml, from about 2 ml to about 60 ml, from about 5 ml to about 60 ml, from about 1 ml to about 40 ml, from about 2 ml to about 40 ml, from about 5 ml to about 40 ml, from about 1 ml to about 30 ml, from about 2 ml to about 30 ml, from about 5 ml to about 30 ml, from about 1 ml to about 25 ml, from about 2 ml to about 25 ml, from about 1 ml to about 20 ml, from about 2 ml to about 20 ml, from about 1 ml to about 15 ml, about 2 ml to about 15 ml, about 1 ml to about 10 ml, or about 2 ml to about 10 ml).

In any of the methods presented herein, when using MCCS, MCCS1 and/or MCCS2, one or more (for example, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen , fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, or twenty-four) different types of buffer with reduced bioburden. As is known in the art, one or more types of reduced bioburden buffer used in MCCS, MCCS1 and/or MCCS2 in the methods presented herein will depend on the resin present in the chromatography columns and/or chromatography membranes of MCCS, MCCS1 and /or MCCS2, the biophysical properties of the recombinant protein and the typical operation (eg, any of the examples of typical operations presented herein) carried out through specific chromatography columns and/or chromatography membranes MCCS, MCCS1 and/or MCCS2. The amount and type of buffer used when using MCCS, MCCS1 and/or MCCS2 in any of the methods presented herein can also be determined by one skilled in the art (eg, as discussed in more detail below). For example, the volume and types of buffer used when using MCCS, MCCS1 and/or MCCS2 in any of the methods presented herein can be selected to optimize one or more of the following in the purified recombinant protein (e.g., recombinant protein drug product): total yield of the recombinant protein, activity of the recombinant protein, level of purity of the recombinant protein, and removal of biological contaminants from the liquid containing the recombinant protein (eg, liquid culture medium) (eg, absence of active viruses, mycobacteria, yeast, bacteria or mammalian cells).

MCCS, MCCS1 and/or MCCS2 may be a periodic countercurrent chromatography system (PCCS). The PCCS, for example, may include two or more chromatography columns (eg, three columns or four columns) switched to continuously elute recombinant protein from two or more chromatography columns. The 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) typically consists of loading, washing, elution, and regeneration steps. PCCS uses multiple columns to perform the same steps individually and continuously in a cyclic manner. Because columns are used in series, and the eluate from one column is captured on another column. This unique feature of PCCS allows the resin to be loaded close to its static binding capacity instead of the dynamic binding capacity that is typical in batch chromatography. Through continuous cycling and elution, the liquid entering the PCCS is continuously processed and an eluate containing the recombinant protein is continuously produced.

The column switching strategy is used to advance from one stage to another in the PCCS cycle. Examples of column switching that can be used in PCCS are described in US Provisional Patent Application Nos. 61/775,060 and 61/856,390. For example, a column switching method may employ two automated switching operations per column: the first associated with the appearance of the original product, while the second corresponds to saturation of the column. Determining when column switching operations are necessary can be accomplished by monitoring the recombinant protein concentration (eg, monitoring via UV monitoring) in the eluate from each chromatography column contained in the PCCS. For example, column switching can be determined using any PAT instrument capable of linearly measuring recombinant protein concentration with feedback control. The PAT instrument is capable of linear, real-time measurement of recombinant protein concentration with closed-loop control. As is known in the art, column switching can also be performed based on the time or amount of liquid (eg, buffer) passed through one or more chromatography columns and/or chromatography membranes in MCCS, MCCS1 and/or MCCS2.

In PCCS, the treatment time (RT) of the recombinant protein on each chromatography column and/or chromatography membrane located in the PCCS can be reduced without increasing the column/membrane size because the emerging product from the first column/membrane can be captured on another column/membrane in the PCCS. A continuous process system can be designed for a liquid culture process at any perfusion rate (D) by varying the column/membrane volume (V) and RT using the equation: V=D*RT.

One or more typical operations that can be performed by MCCS or MCC1 and/or MCCS2 used in the methods presented herein include, for example, capturing the recombinant protein, inactivating viruses present in the liquid comprising the recombinant protein, purifying the recombinant protein, fine purifying the recombinant protein, retaining the liquid comprising the recombinant protein (for example, using any of the example tanks provided herein), filtering or removing particulate matter and/or cells from the liquid comprising the recombinant protein, and adjusting the ion concentration and/or pH liquid containing the recombinant protein.

In some embodiments, the MCCS or MCCS1 includes at least one chromatography column and/or chromatography membrane through which a typical recombinant protein capture operation is performed. A typical capture operation can be performed using at least one chromatography column and/or chromatography resin, for example, which uses a capture mechanism. Non-limiting examples of capture mechanisms include a protein A-binding capture mechanism, an antibody or antibody fragment-binding capture mechanism, a substrate-binding capture mechanism, an aptamer-binding capture mechanism, a tag-binding capture mechanism (e.g., a polyhistidine tag-based capture mechanism), and a uptake with cofactor binding. Capture can also be accomplished using a resin that can be used to perform cation exchange or anion exchange chromatography, molecular sieve chromatography, or hydrophobic interaction chromatography. Non-limiting examples of resins that can be used to capture recombinant protein are provided herein. Additional examples of resins that can be used to capture recombinant protein are known in the art.

A typical operation for inactivating viruses present in a liquid comprising a recombinant protein may be performed using an MCCS, MCCS1 and/or MCCS2 (e.g., comprising, for example, a chromatography column, a chromatography membrane, or a storage tank in which the liquid comprising the recombinant protein may be incubated at a pH of from about 3.0 to about 5.0 (e.g., from about 3.5 to about 4.5, from about 3.5 to about 4.25, from about 3.5 to about 4.0, from about 3 .5 to about 3.8 or about 3.75) for a period of at least 30 minutes (e.g., a period from about 30 minutes to 1.5 hours, a period from about 30 minutes to 1.25 hours, a period from about 0 .75 hours to 1.25 hours or a period of approximately 1 hour)).

A typical recombinant protein purification operation can be performed using one or more MCCS (eg, MCCS, MCCS1 and/or MCCS2), including, for example, a chromatography column or a chromatography membrane including a resin, for example, that uses a capture system. Non-limiting examples of capture mechanisms include a protein A-binding capture mechanism, an antibody or antibody fragment-binding capture mechanism, a substrate-binding capture mechanism, an aptamer-binding capture mechanism, a tag-binding capture mechanism (e.g., a polyhistidine tag-based capture mechanism), and a uptake with cofactor binding. Purification can also be accomplished using a resin that can be used to perform cation exchange or anion exchange chromatography, molecular sieve chromatography, or hydrophobic interaction chromatography. Non-limiting examples of resins that can be used to purify recombinant protein are provided herein. Additional examples of resins that can be used to purify recombinant protein are known in the art.

A typical recombinant protein purification operation can be performed using one or more MCCS (e.g., MCCS, MCCS1 and/or MCCS), including, for example, a chromatography column or chromatography membrane including a resin, for example, which can be used to perform cation exchange, anion exchange chromatography , molecular sieve chromatography or hydrophobic interaction chromatography. Non-limiting examples of resins that can be used for fine purification of recombinant protein are presented herein. Additional examples of resins that can be used for fine purification of recombinant protein are known in the art.

A typical storage operation for a liquid comprising a recombinant protein may be performed using an MCCS (eg, MCCS, MCCS1, and/or MCCS2) comprising at least one reservoir (eg, a tank) or a maximum of 1, 2, 3, 4, or 5 reservoirs ( e.g. tanks) in MCCS or a combination of MCCS1 and MCCS2. For example, each of the reservoirs (e.g., tanks) that may be used to perform this exemplary operation may have a volume of from about 1 ml to about 1 L (e.g., from about 1 ml to about 800 ml, from about 1 ml to about 600 ml, from about 1 ml to about 500 ml, from about 1 ml to about 400 ml, from about 1 ml to about 350 ml, from about 1 ml to about 300 ml, from about 10 ml to about 250 ml, from about 10 ml to about 200 ml, from about 10 ml to about 150 ml, or from about 10 ml to about 100 ml). Reservoirs (e.g., tanks) used in the methods presented herein may have a capacity of, for example, from 1 ml to about 300 ml inclusive, for example, from 1 ml to about 280 ml, about 260 ml, about 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, inclusive . Any of the reservoirs (e.g., tanks) used (in any of the methods presented herein) to hold liquid before it enters the MCCS or MCCS1 may have a capacity of, for example, from 1 ml to and including about 100% of about 1 ml to approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 10%, or approximately 5% inclusive of the loading volume of the first MCCS column or MCCS1. Any of the reservoirs (e.g., tanks) used to hold the eluate from MCCS1 (including the recombinant protein) before it enters MCCS2 may have a capacity of, for example, from 1 ml up to and including about 100%, for example, from about 1 ml to about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% inclusive of the loading volume of the first MCCS2 column.

Each of the reservoirs (e.g., tanks) can hold liquid comprising the recombinant protein for at least 10 minutes (e.g., at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours or at least 6 hours). In other examples, reservoirs (e.g., tanks) hold the recombinant protein for only a total period of time, e.g., from about 5 minutes to less than about 6 hours, inclusive, e.g., from about 5 minutes to about 5 hours, about 4 hours, about 3 hours, approximately 2 hours, approximately 1 hour, or approximately 30 minutes, inclusive. Reservoirs (eg, tanks) can be used to store and cool (eg, less than 25°C, less than 15°C, or less than 10°C) the liquid comprising the recombinant protein. The reservoir may be of any shape, including a circular cylinder, an oval cylinder, or an approximately rectangular sealed and leak-proof bag.

Typical filtration operations for a liquid comprising a recombinant protein can be performed using an MCCS (eg, MCCS, MCCS1 and/or MCCS2) comprising, for example, a filter, or a chromatography column or chromatography membrane including a molecular sieve resin. As is known in the art, a wide variety of submicron filters are available in the field (e.g., filters with pore size less than 1 μm, less than 0.5 μm, less than 0.3 μm, approximately 0.2 μm, less than 0.2 μm, less 100 nm, less than 80 nm, less than 60 nm, less than 40 nm, less than 20 nm, or less than 10 nm) with which to remove any precipitated material and/or cells (e.g., precipitated unfolded protein; precipitated unwanted host cell proteins; precipitated lipids; bacteria; yeast cells; fungal cells; mycobacteria and/or mammalian cells). Filters having a pore size of approximately 0.2 μm or less than 0.2 μm are known to effectively remove bacteria from a liquid containing a recombinant protein. As is known in the art, a chromatography column or chromatography membrane comprising a molecular sieve resin can also be used to perform a typical filtration operation of a liquid comprising a recombinant protein into an MCCS (eg, MCCS, MCCS1 and/or MCCS2).

Typical operations for adjusting the ion concentration and/or pH of a liquid comprising a recombinant protein can be accomplished using an MCCS (e.g., MCCS, MCCS1, and/or MCCS2) including and utilizing a buffer adjustment reservoir (e.g., a linear buffer adjustment reservoir), with by which a new buffer solution is added to the liquid containing the recombinant protein (for example, between the columns in MCCS, MCCS1 and/or MCCS2, or after the last column in the penultimate MCCS (for example, MCCS1) and before feeding the liquid containing the recombinant protein into the first column next MCCS (eg MCCS2)). As is known in the art, a linear buffer correction reservoir can be of any size (e.g., greater than 100 mL) and can include any buffer solution (e.g., a buffer solution having one or more of: an increased or decreased pH relative to the liquid comprising recombinant protein, an increased or decreased concentration of ions (e.g., salts) relative to the liquid comprising the recombinant protein, and/or an increased or decreased concentration of an agent that competes with the recombinant protein for binding to the resin present in at least one chromatography column or at least one chromatography membrane in the MCCS (eg MCCS, MCCS1 and/or MCCS2)).

Two or more typical operations can be performed by MCCS, MCCS1 and/or MCCS2. For example, each of MCCS, MCCS1 and/or MCCS2 can perform at least the following exemplary operations: capturing the recombinant protein and inactivating viruses present in the liquid including the recombinant protein; capturing the recombinant protein, inactivating viruses present in the liquid comprising the recombinant protein, and adjusting the ion concentrations and/or pH of the liquid comprising the recombinant protein; recombinant protein purification and recombinant protein fine purification; purifying the recombinant protein, finely purifying the recombinant protein, and filtering the liquid including the recombinant protein, or removing sediments and/or solids from the liquid including the recombinant protein; and purifying the recombinant protein, finely purifying the recombinant protein, filtering the liquid including the recombinant protein, or removing sediments and/or solids from the liquid including the recombinant protein, and adjusting the ion concentration and/or pH of the liquid including the recombinant protein.

Recombinant protein capture

The methods of the present invention include the step of capturing a recombinant protein using MCCS or MCCS1. As is known in the art, a liquid culture medium comprising a recombinant protein can be continuously fed into MCCS or MCCS1 using a variety of different methods. For example, the liquid culture medium may be actively pumped into the MCCS or MCCS1, or the liquid culture medium may be fed into the MCCS or MCCS1 using gravity. The liquid culture medium may be stored in a reservoir (e.g., a storage tank) prior to feeding it into the MCCS or MCCS1, or the liquid culture medium may be actively pumped from a bioreactor containing a cell culture (e.g., mammalian cells secreting recombinant protein into the culture medium) in MCCS or MCCS1.

The liquid culture medium can be fed into the MCCS or MCCS1 at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml /minute to about 10 ml/minute, from about 0.5 ml/minute to about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, from about 1.0 ml/minute to approximately 15.0 ml/minute). Liquid culture medium comprising the recombinant protein can be obtained from any of the exemplary sources presented herein or known in the art.

Some examples further include the optional step of filtering the liquid culture medium prior to feeding it into the MCCS or MCCS1. To filter the liquid culture medium including the recombinant protein before feeding it into the MCCS or MCCS1, any of the example methods for filtering the liquid culture medium or liquid including the recombinant protein presented herein, or any filtration methods known in the art, can be used. .

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

Non-limiting examples of sizes and shapes of chromatography columns or chromatography membranes found in MCCS or MCCS1 that can be used to capture recombinant protein are provided herein. The liquid culture medium fed into MCCS or MCCS1 may include, for example, from about 0.05 mg/ml to about 100 mg/ml recombinant protein (for example, from about 0.1 mg/ml to about 90 mg/ml, from about 0.1 mg/ml to about 80 mg/ml, from about 0.1 mg/ml to about 70 mg/ml, from about 0.1 mg/ml to about 60 mg/ml, from about 0.1 mg/ml to about 50 mg/ml, from about 0.1 mg/ml to about 40 mg/ml, from about 0.1 mg/ml to about 30 mg/ml, from about 0.1 mg /ml to about 20 mg/ml, from 0.5 mg/ml to about 20 mg/ml, from about 0.1 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 15 mg /ml, from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml recombinant protein). The average time required for the recombinant protein to bind to the resin used to perform a typical capture operation may be, for example, from about 5 seconds to about 10 minutes (e.g., from about 10 seconds to about 8 minutes, from about 10 seconds to about 7 minutes, from about 10 seconds to about 6 minutes, from about 10 seconds to about 5 minutes, from about 30 seconds to about 5 minutes, from about 1 minute to about 5 minutes, from about 10 seconds to about 4 minutes, from about 30 seconds to about 4 minutes, or from about 1 minute to about 4 minutes).

As is known in the art, to capture recombinant protein using the chromatography columns or chromatography membranes contained in the MCCS or MCCS1, it is necessary to perform sequential chromatographic steps of loading, washing, elution and regeneration of the chromatography columns or chromatography membranes contained in the MCCS or MCCS1. Any of the example flow rates, buffer volumes, and/or lengths of time provided for each sequential chromatographic step presented herein can be used in one or more of these different sequential chromatographic steps (e.g., one or more of the sequential loading chromatographic steps, washing, elution and regeneration of chromatography columns or chromatography membranes contained in MCCS or MCCS1 used to capture the recombinant protein). Non-limiting examples of flow rates, buffer volumes, and/or lengths of time allocated for each sequential chromatographic step that can be used to capture chromatography columns and/or chromatography membranes in MCCS or MCCS1 (eg, PCCS or PCCS1) are provided below. In addition, examples of buffers that can be used in MCCS and/or MCCS1 are described below.

An MCCS or MCCS1 comprising at least one chromatography column and/or a chromatography membrane including a resin through which a typical capture operation can be performed (e.g., any of the example resins that can be used for capture provided herein) can be loaded with a liquid medium for cultivation comprising the recombinant protein using any of the loading flow rates (feeding rates) described above. In some examples, a single chromatography column or a single chromatography membrane including a resin through which a typical capture operation can be performed is loaded for, for example, about 10 minutes to about 90 minutes (for example, about 15 minutes to about 90 minutes, from about 20 minutes and 80 minutes, from about 30 minutes and 80 minutes, from about 40 minutes to about 80 minutes, from about 50 minutes to about 80 minutes, and from about 60 minutes and 80 minutes). In some examples, where the MCCS or MCCS1 includes at least two chromatography columns comprising a resin through which a typical capture operation in the series can be performed, the time required to load two of the chromatography columns in the series is, for example, from about 50 minutes to about 180 minutes (e.g., from about 60 minutes to about 180 minutes, from about 70 minutes to about 180 minutes, from about 80 minutes to about 180 minutes, from about 90 minutes to about 180 minutes, from about 100 minutes to about 180 minutes, from about 110 minutes to about 150 minutes and from about 125 minutes to about 145 minutes).

After loading the recombinant protein onto at least one chromatography column or chromatography membrane in an MCCS or MCCS1 comprising a resin through which a typical capture operation can be performed, the at least one chromatography column or chromatography membrane is washed with at least one wash buffer. As is known in the art, at least one (e.g., two, three, or four) wash buffer is designed to elute all or most of the non-recombinant protein proteins from at least one chromatography column or chromatography membrane, while not interfering with the interaction of the recombinant protein with the resin.

The wash buffer can be passed through at least one chromatography column or chromatography membrane at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml /minute to about 10 ml/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, from about 1.0 ml/minute to approximately 15.0 ml/minute). The volume of wash buffer used (e.g., the combined total volume of wash buffer used when multiple wash buffers are used) can be, for example, about 1 times the column volume (CV) to about 15 times the CV (e.g., about 1 times the CV to about 14 times CV, from about 1 times CV to about 13 times CV, from about 1 times CV to about 12 times CV, from about 1 times CV to about 11 times CV, from about 2- multiple of CV to approximately 11 times CV, approximately 3 times CV to approximately 11 times CV, approximately 4 times CV to approximately 11 times CV, approximately 5 times CV to approximately 11 times CV, or approximately 5x CV to approximately 10x CV). The total wash time may be, for example, from about 2 minutes to about 3 hours (e.g., from about 2 minutes to about 2.5 hours, from about 2 minutes to about 2.0 hours, from about 5 minutes to about 1.5 hours, from about 10 minutes to about 1.5 hours, from about 10 minutes to about 1.25 hours, from about 20 minutes to about 1.25 hours, or from about 30 minutes to about 1 hour).

After washing the at least one chromatography column or chromatography membrane in MCCS or MCCS1, including a resin through which a typical capture operation can be performed, the recombinant protein is eluted from the at least one chromatography column or chromatography membrane by passing the elution buffer through at least one chromatography a column or chromatography membrane in MCCS or MCCS1 including a resin through which a typical capture operation can be performed. The elution buffer may be passed through at least one chromatography column or chromatography membrane comprising a resin through which a typical capture operation may be performed at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5.0 mg/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, or from about 1.0 ml/minute to about 15.0 ml /minute). The volume of elution buffer used to elute the recombinant protein from each of the at least one chromatography column or chromatography membrane including a resin through which a typical purification operation can be performed can be, for example, from about 1 times the column volume (CV) to approximately 15 times CV (e.g., approximately 1 times CV to approximately 14 times CV, approximately 1 times CV to approximately 13 times CV, approximately 1 times CV to approximately 12 times CV, approximately 1 times CV to approximately 11 times CV, approximately 2 times CV to approximately 11 times CV, approximately 3 times CV to approximately 11 times CV, approximately 4 times CV to approximately 11 times CV, approximately 5 times CV to approximately 11 times CV, or approximately 5 times CV to approximately 10 times CV). The total elution time may be, for example, from about 2 minutes to about 3 hours (e.g., from about 2 minutes to about 2.5 hours, from about 2 minutes to about 2.0 hours, from about 2 minutes to about 1.5 hours, from about 2 minutes to about 1.5 hours, from about 2 minutes to about 1.25 hours, from about 2 minutes to about 1.25 hours, from about 2 minutes to about 1 hour, from about 2 minutes to about 40 minutes, about 10 minutes to about 40 minutes, or about 20 minutes to about 40 minutes). Non-limiting examples of elution buffers that can be used in these methods will depend on the capture mechanism and/or recombinant protein. For example, the elution buffer may include different salt concentrations (eg, increased salt concentration), different pH (eg, increased or decreased salt concentration), or a molecule that will compete with the recombinant protein for binding to the resin, through which a typical capture operation can be performed. Examples of such elution buffers for each example of the capture mechanism presented herein are well known in the art.

After the recombinant protein has been eluted from at least one chromatography column or chromatography membrane into an MCCS or MCCS1 comprising a resin through which a typical capture operation can be performed, and up to the next volume, the liquid culture medium can be loaded onto the at least one chromatography column or chromatography membrane , at least one chromatography column or chromatography membrane can be equilibrated using a regeneration buffer. The regeneration buffer may be passed through at least one chromatography column or chromatography membrane comprising a resin through which a typical capture operation may be performed at a flow rate of, for example, about 0.2 mL/minute to about 25 mL/minute (e.g., about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml /minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5.0 mg/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, from about 5.0 ml/minute to about 15.0 ml/minute, or from about 1.0 ml/minute to about 15.0 ml/minute). The volume of regeneration buffer used to equilibrate the at least one chromatography column or chromatography membrane including a resin through which a typical capture operation can be performed may be, for example, from about 1 times the column volume (CV) to about 15 times the CV (e.g., about 1 times CV to about 14 times CV, about 1 times CV to about 13 times CV, about 1 times CV to about 12 times CV, about 1 times CV to about 11 times CV, approximately 2 times CV to approximately 11 times CV, approximately 3 times CV to approximately 11 times CV, approximately 2 times CV to approximately 5 times CV, approximately 4 times CV to approximately 11 times CV, approximately 5 times CV to approximately 11 times CV, or approximately 5 times CV to approximately 10 times CV).

In some of the methods presented herein, the MCCS or MCCS1 includes a reservoir in which a liquid comprising the recombinant protein is stored at a low pH (e.g., pH below 4.6, below 4.4, below 4.2, below 4, 0, below 3.8, below 3.6, below 3.4, below 3.2, or below 3.0) for, for example, about 1 minute to 1.5 hours (for example, about 1 hour), and inactivate viruses present in the liquid containing the recombinant protein. An example of a container that can be used to perform a typical virus inactivation operation is a shake flask (e.g., a 500 mL shake flask, e.g., a 500 mL shake flask with a programmable stir plate) in which a liquid including the recombinant protein can be stored , for example, from about 1 minute to 1.5 hours, for example, before feeding the liquid including the recombinant protein into MCCS2. The reservoir used to perform a typical virus inactivation operation may be a 500 mL shake flask with a programmable stir plate (eg, a stir plate programmable to agitate (eg, periodically stir) the liquid in the reservoir, eg, every 4 hours). Another example of a reservoir that can be used to perform a typical virus inactivation operation is a plastic bag (for example, a 500 ml plastic bag) in which a liquid comprising the recombinant protein can be stored for, for example, about 1 minute to 1.5 hours, for example, before feeding the liquid including the recombinant protein into MCCS2. In some examples, the liquid comprising the recombinant protein may already have a low pH (e.g., pH below 4.6, below 4.4, below 4.2, below 4.0, below 3.8, below 3.6, below 3 ,4, below 3.2 or below 3.0) when feeding into a reservoir used to carry out a typical virus inactivation operation. As is known to those skilled in the art, many other methods can be used to carry out a typical virus inactivation operation. For example, to carry out a typical virus inactivation operation, UV irradiation of a liquid containing a recombinant protein can also be used. Non-limiting examples of reservoirs that can be used to perform a typical operation of inactivating viruses present in a liquid comprising a recombinant protein are presented herein.

MCCS or MCCS1 may include a PCCS including four chromatography columns, wherein at least three of the four chromatography columns perform a typical capture operation of the recombinant protein from a liquid culture medium (e.g., using an MCCS including any of at least one chromatography column, comprising a resin through which a typical gripping operation (eg, any of those presented herein) can be performed). In these examples, the fourth PCC column can perform a typical operation of inactivating viruses in a liquid including a recombinant protein (eg, any of the example columns presented herein that can be used to achieve inactivation of viruses in a liquid including a recombinant protein).

In some examples, the liquid comprising the recombinant protein is continuously eluted from MCCS1 (eg, PCCS1) and continuously fed into MCCS2 (eg, PCCS2). The percentage of recombinant protein recovered in the eluate from MCCS or MCCS1 (e.g., PCCS or PCCS1) may be, for example, at least 70%, at least 72%, at least 74%, at least 76%, at least 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 (eg, PCCS1) can be fed into MCCS2 (eg, PCCS2) using a variety of means known in the art (eg, tubing). The eluate from MCCS1 (e.g., PCCS1) can be fed into MCCS2 (e.g., PCCS2) at a flow rate, for example, from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, about 0.5 ml/minute to about 20 ml/minute, about 0.2 ml/minute to about 15 ml/minute, about 0.5 ml/minute to about 15 ml/minute , from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5.0 mg/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, from about 5.0 ml/minute to about 15.0 ml/minute, from about 15 ml/minute to about 25 ml/minute or about 1.0 ml/minute to about 15.0 ml/minute).

Some methods presented herein may further include the step of adjusting the ion concentration and/or pH of the eluate from MCCS1 (eg, PCCS1) prior to feeding it into MCCS2 (eg, PCCS2). As presented herein, the ion concentration and/or pH of the eluate from MCCS1 (eg, PCCS1) can be adjusted (prior to feeding it into MCCS2) by adding a buffer to the eluate (eg, using a buffer linear correction reservoir). The buffer can be added to the MCCS1 eluate at a flow rate of, for example, about 0.1 ml/minute to about 15 ml/minute (for example, about 0.1 ml/minute to about 12.5 ml/minute, from about 0 .1 ml/minute to about 10.0 ml/minute, from about 0.1 ml/minute to about 8.0 ml/minute, from about 0.1 ml/minute to about 6 ml/minute, from about 0. 1 ml/minute to 4 ml/minute or from about 0.5 ml/minute to about 5 ml/minute).

The methods presented herein may further include the step of storing (and optionally also cooling) the eluate from MCCS1 prior to feeding the eluate from MCCS1 to MCCS2. As presented herein, this storage step can be accomplished using any of the tanks (eg, reserve tanks) presented herein.

The methods presented herein may also include the step of filtering the eluate from MCCS1 prior to feeding the eluate to MCCS2. Any of the example filters or filtration methods presented herein can be used to filter the eluate from MCCS1 prior to feeding the eluate into MCCS2.

Fine purification and purification of recombinant protein

MCCS, MCCS1 and/or MCCS2 can be used to perform a typical purification and fine-purification operation of a recombinant protein. For example, MCCS2 can be used to carry out the purification and purification operation of a recombinant protein, and the eluate from MCCS2 is a protein drug. MCCS, MCCS1 and/or MCCS2 may include at least one (e.g., two, three, or four) chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation, and at least one (e.g., two, three or four) a chromatography column or chromatography membrane that can be used to perform a typical fine purification operation of a recombinant protein.

At least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation 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 chromatography, cation exchange chromatography, molecular sieve chromatography or hydrophobic interaction chromatography. At least one chromatography column or chromatography membrane that can be used to perform a typical purification operation of a recombinant protein may include a resin that can be used to perform anion exchange chromatography, cation exchange chromatography, molecular sieve chromatography, or hydrophobic interaction chromatography (e.g., any of examples of resins for performing anion exchange, cation exchange chromatography, molecular sieve chromatography or hydrophobic interaction chromatography presented herein or known in this field).

The size, shape and volume of at least one chromatography column or chromatography membrane that can be used to carry out a typical purification operation of a recombinant protein, and/or the size and shape of at least one chromatography membrane that can be used to carry out a typical purification step of a recombinant protein , may be any combination of exemplary sizes, shapes, and volumes of chromatography columns or chromatography membranes presented herein. As is known to one skilled in the art, the purification or purification step of a recombinant protein, for example, may include the steps of loading, washing, eluting, and equilibrating at least one chromatography column or chromatography membrane used to perform a typical recombinant protein purification or purification operation. Typically, the elution buffer exiting the chromatography column or chromatography membrane used to perform a typical purification operation includes the recombinant protein. Typically, the loading buffer and/or wash buffer exiting the chromatography column or chromatography membrane used for a typical purification operation includes the recombinant protein.

For example, the size of at least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation may have a volume, for example, from about 2.0 ml to about 200 ml (for example, from about 2.0 ml to about 180 ml, from about 2.0 ml to about 160 ml, from about 2.0 ml to about 140 ml, from about 2.0 ml to about 120 ml, from about 2.0 ml to about 100 ml, from 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 40 ml 30 ml, about 5.0 ml to about 30 ml, or about 2.0 ml to about 25 ml). The flow rate of a liquid comprising a recombinant protein loaded onto at least one chromatography column or at least one chromatography membrane that can be used to perform a typical recombinant protein purification operation may be, for example, from about 0.1 ml/minute to about 25 ml/minute (e.g., about 0.1 ml/minute to about 12.5 ml/minute, about 0.1 ml/minute to about 10.0 ml/minute, about 0.1 ml/minute to approximately 8.0 ml/minute, approximately 0.1 ml/minute to approximately 6 ml/minute, approximately 0.1 ml/minute to 4 ml/minute, approximately 0.1 ml/minute to approximately 3 ml/ minute, from about 0.1 ml/minute to about 2 ml/minute or about 0.2 ml/minute to about 4 ml/minute). The concentration of the recombinant protein in the liquid loaded onto at least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation may be, for example, from about 0.05 mg/ml to about 100 mg/ml recombinant protein (e.g., from about 0.1 mg/ml to about 90 mg/ml, from about 0.1 mg/ml to about 80 mg/ml, from about 0.1 mg/ml to about 70 mg/ml, from about 0.1 mg/ml to about 60 mg/ml, from about 0.1 mg/ml to about 50 mg/ml, from about 0.1 mg/ml to about 40 mg/ml, from about 0.1 mg/ml ml to about 30 mg/ml, from about 0.1 mg/ml to about 20 mg/ml, from 0.5 mg/ml to about 20 mg/ml, from about 0.1 mg/ml to about 15 mg/ ml, about 0.5 mg/ml to about 15 mg/ml, about 0.1 mg/ml to about 10 mg/ml, or about 0.5 mg/ml to about 10 mg/ml recombinant protein). The resin in at least one chromatography column or chromatography membrane used to perform a typical purification operation may be a resin that can be used to perform anion exchange or cation exchange chromatography. The resin in at least one chromatography column or chromatography membrane used to perform a typical purification operation may be a cation exchange resin (eg, Capto-S resin, GE Healthcare Life Sciences, Piscataway, NJ).

After loading the recombinant protein onto at least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation, the at least one chromatography column or chromatography membrane is washed with at least one wash buffer. As is known in the art, at least one (e.g., two, three, or four) wash buffer is designed to elute all proteins other than the recombinant protein from at least one chromatography column or chromatography membrane without interfering with the interaction of the recombinant protein with the resin or other elution of the recombinant protein.

The wash buffer can be passed through at least one chromatography column or chromatography membrane at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml /minute to about 10 ml/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, or from about 1.0 ml/minute to approximately 15.0 ml/minute). The volume of wash buffer used (e.g., the combined total volume of wash buffer used when multiple wash buffers are used) can be, for example, from about 1 times the column volume (CV) to about 15 times the CV (e.g., from about 1 times the CV to approximately 14 times CV, approximately 1 times CV to approximately 13 times CV, approximately 1 times CV to approximately 12 times CV, approximately 1 times CV to approximately 11 times CV, approximately 2 times CV to approximately 11 - times CV, approximately 3 times CV to approximately 11 times CV, approximately 4 times CV to approximately 11 times CV, approximately 2.5 times CV to approximately 5.0 times CV, approximately 5 times CV to approximately 11 times CV or approximately 5 times CV to approximately 10 times CV). The total wash time may be, for example, from about 2 minutes to about 3 hours (e.g., from about 2 minutes to about 2.5 hours, from about 2 minutes to about 2.0 hours, from about 5 minutes to about 1.5 hours, from about 10 minutes to about 1.5 hours, from about 10 minutes to about 1.25 hours, from about 20 minutes to about 1.25 hours, from about 30 minutes to about 1 hour, from about 2 minutes to 10 minutes, from about 2 minutes to 15 minutes, or from about 2 minutes to 30 minutes).

After washing the at least one chromatography column or chromatography membrane used to perform a typical recombinant protein purification step, the recombinant protein is eluted from the at least one chromatography column or chromatography membrane by passing the elution buffer through the at least one chromatography column or chromatography membrane used to carry out a typical recombinant protein purification operation. The elution buffer can be passed through at least one chromatography column or chromatography membrane, which can be used to perform a typical recombinant protein purification operation, at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0. 2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5, 0 mg/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, or from about 1.0 ml/minute to about 15.0 ml/minute). The volume of elution buffer used to elute the recombinant protein from each of the at least one chromatography column or chromatography membrane that may be used to perform a typical recombinant protein purification operation may be, for example, from about 1 times the column volume (CV) to about 25 -fold CV (e.g., from about 1 times CV to about 20 times CV, from about 15 times CV and about 25 times CV, from about 1 times CV to about 14 times CV, about 1 times CV to approximately 13 times CV, approximately 1 times CV to approximately 12 times CV, approximately 1 times CV to approximately 11 times CV, approximately 2 times CV to approximately 11 times CV, approximately 3 times CV to approximately 11 times CV, approximately 4 times CV to approximately 11 times CV, approximately 5 times CV to approximately 11 times CV, or approximately 5 times CV to approximately 10 times CV). The total elution time may be, for example, from about 2 minutes to about 3 hours (e.g., from about 2 minutes to about 2.5 hours, from about 2 minutes to about 2.0 hours, from about 2 minutes to about 1.5 hours, from about 2 minutes to about 1.5 hours, from about 2 minutes to about 1.25 hours, from about 2 minutes to about 1.25 hours, from about 2 minutes to about 1 hour, from about 2 minutes to about 40 minutes, about 10 minutes to about 40 minutes, about 20 minutes to about 40 minutes, or about 30 minutes to 1.0 hour). Non-limiting examples of elution buffers that can be used in these methods will depend on the resin and/or the biophysical properties of the recombinant protein. For example, the elution buffer may include a different salt concentration (eg, increased salt concentration), different pH (eg, increased or decreased salt concentration), or a molecule that will compete with the recombinant protein for binding to the resin. Examples of such elution buffers for each of the examples of capture mechanisms presented herein are well known in the art.

After elution of the recombinant protein from at least one chromatography column or chromatography membrane used to perform a typical recombinant protein purification operation, and before loading the next volume of liquid containing the recombinant protein onto the at least one chromatography column or chromatography membrane, at least one The chromatography column or chromatography membrane must be equilibrated using a regeneration buffer. The regeneration buffer may be passed through at least one chromatography column or chromatography membrane used to perform a typical recombinant protein purification operation at a flow rate of, for example, from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5.0 mg/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, from about 5.0 ml/minute to about 15 .0 ml/minute or from about 1.0 ml/minute to about 15.0 ml/minute). The volume of regeneration buffer used to equilibrate the at least one chromatography column or chromatography membrane including a resin that can be used to perform a typical recombinant protein purification operation can be, for example, from about 1 times the column volume (CV) to about 15 -fold CV (e.g., from about 1 times CV to about 14 times CV, from about 1 times CV to about 13 times CV, from about 1 times CV to about 12 times CV, from about 1 times CV to approximately 11 times CV, approximately 2 times CV to approximately 11 times CV, approximately 3 times CV to approximately 11 times CV, approximately 2 times CV to approximately 5 times CV, approximately 2 times .5 times CV to approximately 7.5 times CV, approximately 4 times CV to approximately 11 times CV, approximately 5 times CV to approximately 11 times CV, or approximately 5 times CV to approximately 10 times multiple of CV). The concentration of the recombinant protein in the eluate of at least one chromatography column or chromatography membrane used to perform a typical recombinant protein purification operation may be, for example, from about 0.05 mg/ml to about 100 mg/ml recombinant protein (for example, from about 0.1 mg/ml to about 90 mg/ml, from about 0.1 mg/ml to about 80 mg/ml, from about 0.1 mg/ml to about 70 mg/ml, from about 0.1 mg/ml ml to about 60 mg/ml, from about 0.1 mg/ml to about 50 mg/ml, from about 0.1 mg/ml to about 40 mg/ml, from about 2.5 mg/ml to about 7, 5 mg/ml, from about 0.1 mg/ml to about 30 mg/ml, from about 0.1 mg/ml to about 20 mg/ml, from 0.5 mg/ml to about 20 mg/ml, from about 0.1 mg/ml to about 15 mg/ml, about 0.5 mg/ml to about 15 mg/ml, about 0.1 mg/ml to about 10 mg/ml, or about 0.5 mg /ml to approximately 10 mg/ml recombinant protein).

At least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation may include a resin that can be used to perform cation exchange chromatography, anion exchange chromatography, or molecular sieve chromatography. As is known in the art, fine purification of a recombinant protein using at least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein fine purification operation may include, for example, the steps of loading, displacement, and regeneration of at least one chromatography column or chromatographic membrane that can be used to perform a typical fine purification operation of a recombinant protein. For example, if loading, displacement, and regeneration steps are used to perform purification, the recombinant protein is not bound to the resin in at least one chromatography column or chromatography membrane used to perform a typical recombinant protein purification operation, and the recombinant protein is eluted from the at least one one chromatography column or chromatography membrane in the loading and displacement steps, and a regeneration step is used to remove any impurities from the at least one chromatography column or chromatography membrane before loading additional liquid including the recombinant protein onto the at least one chromatography column or chromatography membrane. Examples of flow rates and buffer volumes to be used in each of the loading, displacement and regeneration stages are described below.

The size, shape and volume of at least one chromatography column or chromatographic membrane that can be used to carry out a typical purification operation of a recombinant protein, and/or the size and shape of at least one chromatography membrane that can be used to carry out a typical purification step of a recombinant protein may be any combination of exemplary sizes, shapes and volumes of chromatography columns or chromatography membranes presented herein. For example, the size of at least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation may have a volume, for example, from about 0.5 ml to about 200 ml (for example, from about 0.5 ml to about 180 ml, from about 0.5 ml to about 160 ml, from about 0.5 ml to about 140 ml, from about 0.5 ml to about 120 ml, from about 0.5 ml to about 100 ml, from about 0.5 ml to about 80 ml, from about 0.5 ml to about 60 ml, from about 0.5 ml to about 40 ml, from about 5.0 ml to about 40 ml, from 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 liquid comprising the recombinant protein loaded onto at least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation may be, for example, from about 0.1 ml/minute to about 25 ml/ minute (e.g., from about 0.1 ml/minute to about 12.5 ml/minute, from about 0.1 ml/minute to about 10.0 ml/minute, from about 0.1 ml/minute to about 8, 0 ml/minute, from about 0.1 ml/minute to about 6 ml/minute, from about 0.1 ml/minute to 4 ml/minute, from about 0.1 ml/minute to about 3 ml/minute, from about 2 ml/minute to about 6 ml/minute, about 0.1 ml/minute to about 2 ml/minute, or about 0.2 ml/minute to about 4 ml/minute). The total volume of liquid comprising the recombinant protein loaded onto at least one chromatography column or chromatography membrane that can be used to perform a typical recombinant protein purification operation may be, for example, from about 1.0 ml to about 250 ml (e.g. from about 1.0 ml to about 225 ml, from about 1.0 ml to about 200 ml, from about 1.0 ml to about 175 ml, from about 1.0 ml to about 150 ml, from about 100 ml to about 125 ml, from about 100 ml to about 150 ml, from about 1.0 ml to about 150 ml, from about 1.0 ml to about 125 ml, from about 1.0 ml to about 100 ml, from about 1.0 ml to about 75 ml, from about 1.0 ml to about 50 ml, or from about 1.0 ml to about 25 ml). The resin in at least one chromatography column or chromatography membrane used to perform fine purification may be an anion exchange resin or a cation exchange resin. The resin in at least one chromatography column or chromatography membrane used to perform a typical fine purification operation may be a cation exchange resin (eg, Sartobind® Q resin, Sartorius, Goettingen, Germany).

Following the loading step, a displacement step is performed (eg, a displacement buffer is passed through at least one chromatography column or chromatography membrane to collect recombinant protein that does not substantially bind to the at least one chromatography column or chromatography membrane). In these examples, the displacement buffer can be passed through at least one chromatography column or chromatography membrane at a flow rate of from about 0.2 ml/minute to about 50 ml/minute (for example, from about 1 ml/minute to about 40 ml/minute , from about 1 ml/minute to about 30 ml/minute, from about 5 ml/minute to about 45 ml/minute, from about 10 ml/minute to about 40 ml/minute, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, or from about 1 .0 ml/minute to approximately 15.0 ml/minute). The volume of displacement buffer used may be, for example, from about 1 times the column volume (CV) to about 100 times the CV (e.g., from about 1 times the CV to about 90 times the CV, from about 1 times the CV to about 80 - times the CV, from about 1 times the CV to about 70 times the CV, from about 1 times the CV to about 60 times the CV, from about 1 times the CV to about 50 times the CV, from about 1 times the CV to approximately 40 times CV, approximately 1 times CV to approximately 30 times CV, approximately 1 times CV to approximately 20 times CV, approximately 1 times CV to approximately 15 times CV, approximately 5 times CV CV to approximately 20 times CV, approximately 5 times CV to approximately 30 times CV, approximately 1 times CV to approximately 14 times CV, approximately 1 times CV to approximately 13 times CV, approximately 1 times CV to approximately 12 times CV, approximately 1 times CV to approximately 11 times CV, approximately 2 times CV to approximately 11 times CV, approximately 3 times CV to approximately 11 times CV, approximately 4 times CV to approximately 11 times CV, approximately 2.5 times CV to approximately 5.0 times CV, approximately 5 times CV to approximately 11 times CV, or approximately 5 times CV to approximately 10 times CV). The total displacement time may be, for example, from about 1 minute to about 3 hours (e.g., from about 1 minute to about 2.5 hours, from about 1 minute to about 2.0 hours, from about 1 minute to about 1.5 hours, from about 2 minutes to about 1.5 hours, from about 1 minute to about 1.25 hours, from about 2 minutes to about 1.25 hours, from about 1 minute to about 5 minutes, from about 1 minute to about 10 minutes, about 2 minutes to about 4 minutes, about 30 minutes to about 1 hour, about 2 minutes to about 10 minutes, about 2 minutes to about 15 minutes, or about 2 minutes to about 30 minutes). The combined concentration of recombinant protein present in the eluate passed through the column during the loading step and the displacement step may be, for example, from about 0.1 mg/mL to about 100 mg/mL recombinant protein (e.g., from about 0.1 mg/mL recombinant protein). ml to about 90 mg/ml, from about 0.1 mg/ml to about 80 mg/ml, from about 0.1 mg/ml to about 70 mg/ml, from about 0.1 mg/ml to about 60 mg /ml, from about 0.1 mg/ml to about 50 mg/ml, from about 0.1 mg/ml to about 40 mg/ml, from about 2.5 mg/ml to about 7.5 mg/ml, from about 0.1 mg/ml to about 30 mg/ml, from about 0.1 mg/ml to about 20 mg/ml, from 0.5 mg/ml to about 20 mg/ml, from about 0.1 mg /ml to about 15 mg/ml, from about 0.5 mg/ml to about 15 mg/ml, from about 0.1 mg/ml to about 10 mg/ml, from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 5 mg/ml recombinant protein).

After the displacement step and before loading the next volume of liquid containing the recombinant protein onto at least one chromatography column or chromatography membrane that can be used for a typical fine purification operation, the at least one chromatography column or chromatography membrane must be regenerated using a buffer for regeneration. The regeneration buffer can be passed through at least one chromatography column or chromatography membrane, which can be used for a typical recombinant protein purification operation, at a flow rate of, for example, from about 0.2 ml/minute to about 50 ml/minute (for example , from about 1 ml/minute to about 40 ml/minute, from about 1 ml/minute to about 30 ml/minute, from about 5 ml/minute to about 45 ml/minute, from about 10 ml/minute to about 40 ml /minute, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute minute to about 25.0 ml/minute or from about 1.0 ml/minute to about 15.0 ml/minute). The volume of regeneration buffer used to regenerate at least one chromatography column or chromatography membrane that can be used for a typical fine purification operation can be, for example, from about 1 times the column volume (CV) to about 500 times the CV ( for example, from about 1 times CV to about 450 times CV, from about 1 times CV to about 400 times CV, from about 1 times CV to about 350 times CV, from about 1 times CV to about 300 - times the CV, from about 1 times the CV to about 250 times the CV, from about 1 times the CV to about 200 times the CV, from about 1 times the CV to about 150 times the CV, from about 1 times the CV to approximately 100 times CV, approximately 1 times CV to approximately 90 times CV, approximately 1 times CV to approximately 80 times CV, approximately 1 times CV to approximately 70 times CV, approximately 1 times CV to approximately 60 times CV, approximately 1X to approximately 50 times CV, approximately 1 times CV to approximately 40 times CV, approximately 1 times CV to approximately 30 times CV, approximately 1 times CV to about 20 times CV, from about 1 times CV to about 15 times CV, from about 5 times CV to about 20 times CV, from about 5 times CV to about 30 times CV, from about 1- multiple CV to approximately 14 times CV, approximately 1 times CV to approximately 13 times CV, approximately 1 times CV to approximately 12 times CV, approximately 1 times CV to approximately 11 times CV, approximately 2 times CV to approximately 11 times CV, approximately 3 times CV to approximately 11 times CV, approximately 4 times CV to approximately 11 times CV, approximately 2.5 times CV to approximately 5.0 times CV, approximately 5- multiple CV to approximately 11 times CV or approximately 5 times CV to approximately 10 times CV).

In other examples, one or more chromatography columns and/or chromatography membranes used to perform a typical fine purification operation include a resin that selectively binds or retains impurities present in the liquid comprising the recombinant protein, and instead of regenerating one or more columns and/or membranes one or more columns and/or membranes are replaced (eg, replaced with substantially similar columns and/or membranes) after resin binding capacity in one or more columns and/or membranes has been achieved or substantially achieved.

In some examples of these methods presented herein, MCCS2 includes a PCCS including three chromatography columns and one chromatography membrane, for example, where three chromatography columns in the PCCS perform a typical recombinant protein purification operation (e.g., using at least one chromatography column , which can be used to carry out a typical protein purification operation), and through a chromatographic membrane in the PCCS, a typical fine purification operation of a recombinant protein is carried out. In these examples, the chromatography membrane in the PCCS that can be used to perform a typical purification step for a therapeutic protein may be any of the examples of chromatography membranes provided herein that can be used to perform a typical purification step for a recombinant protein. Any of the column switching methods presented herein can be used to determine when to switch the first three chromatography columns and the chromatography membrane in the PCCS in this example.

Some embodiments of this example may further include the step of adjusting the ion concentration and/or pH of the eluate from the three chromatography columns in the PCCS prior to feeding the eluate into the chromatography membrane in the PCCS. As presented herein, the ion concentration and/or pH of the eluate from the three chromatography columns in the PCCS can be adjusted (prior to its feeding into the chromatography membrane in the PCCS in this example) by adding a buffer to the eluate from the three chromatography columns in the PCCS (for example, with using a reservoir for linear buffer correction). The buffer can be added to the eluate at a flow rate, for example, from about 0.1 ml/minute to about 15 ml/minute (for example, from about 0.1 ml/minute to about 12.5 ml/minute, from about 0.1 ml/minute to about 10.0 ml/minute, from about 0.1 ml/minute to about 8.0 ml/minute, from about 0.1 ml/minute to about 6 ml/minute, from about 0.1 ml /minute to 4 ml/minute or from about 0.5 ml/minute to about 5 ml/minute).

These examples may further include the step of storing the eluate from three chromatography columns in a PCCS in this example prior to feeding the eluate into a chromatography membrane (a chromatography membrane that can be used to perform a typical recombinant protein purification operation). As presented herein, this storage step can be accomplished using any of the tanks (eg, reserve tanks) presented herein.

These examples may also include the step of filtering the chromatography membrane eluate in an example PCCS system (a chromatography membrane eluate that can be used to perform a typical recombinant protein purification operation). Any of the example filters or filtration methods presented herein can be used to filter a chromatography membrane eluate in the example PCCS (a chromatography membrane eluate that can be used to perform a typical recombinant protein purification operation).

As is known to those skilled in the art, purified recombinant protein can be periodically eluted from MCCS or MCCS2 by any of the methods presented herein. For example, the purified recombinant protein can be eluted by any of the methods presented herein within, for example, about 30 seconds to about 5 hours (for example, about 1 minute to about 4 hours, about 1 minute to about 3 hours, from about 1 minute to about 2 hours, from about 1 minute to about 1.5 hours, from about 1 minute to about 1 hour, or from about 1 minute to about 30 minutes) with a frequency of, for example, from about 1 minute to about 6 hours (e.g., from about 1 minute to about 5 hours, from about 1 minute to about 4 hours, from about 1 minute to about 3 hours, from about 1 minute to 2 hours, from about 1 minute to 1 hour, or from about 1 minutes to 30 minutes), depending on, for example, the chromatography columns and/or chromatography membranes used in MCCS or MCCS1 and MCCS2.

Cultivation methods

Some of the methods provided herein further include the step of culturing cells (e.g., recombinant mammalian cells) secreting the recombinant protein in a bioreactor (e.g., a perfusion bioreactor or a batch bioreactor) including a liquid culture medium, wherein the volume of the liquid culture medium is , substantially free of cells (eg, mammalian cells), is continuously or periodically removed from a bioreactor (eg, a perfusion bioreactor) and fed into MCCS or MCCS1. The bioreactor may have a volume, for example, from about 1 L to about 10,000 L (for example, from about 1 L to about 50 L, from about 50 L to about 500 L, from about 500 L to about 1000 L, from 500 L to about 5000L, from approximately 500 L to approximately 10,000 L, from approximately 5,000 L to approximately 10,000 L, from approximately 1 L to approximately 10,000 L, from approximately 1 L to approximately 8,000 L, from approximately 1 L to approximately 6,000 L, from approximately 1 L to approximately 5000 L, approximately 100 L to approximately 5000 L, approximately 10 L to approximately 100 L, approximately 10 L to approximately 4000 L, approximately 10 L to approximately 3000 L, approximately 10 L to approximately 2000 L or from about 10 L to about 1000 L). The amount of liquid culture medium present in the bioreactor may be, for example, from about 0.5 L to about 5000 L (e.g., from about 0.5 L to about 25 L, from about 25 L to about 250 L, from about 250 L to about 500 L, from about 250 L to about 2500 L, from about 250 L to about 5000 L, from about 2500 L to about 5000 L, from about 0.5 L to about 5000 L, from about 0, 5 L to about 4000 L, from about 0.5 L to about 3000 L, from about 0.5 L to about 2,500 L, from about 50 L to about 2,500 L, from about 5 L to about 50 L, from about 5 L to about 2000 L, about 5 L to about 1,500 L, about 5 L to about 1000 L, or about 5 L to about 500 L). Cell culture can be carried out, for example, using a batch bioreactor or a perfusion bioreactor. Non-limiting examples and various aspects of cell culture (eg, mammalian cell culture) are described below and can be used in any combination.

Cells

Cells cultured in some of the methods presented herein may be bacteria (eg, gram-negative bacteria), yeast (eg, Saccharomyces cerevisiae , Pichia pastoris , Hansenula polymorpha , Kluyveromyces lactis , Schizosaccharomyces pombe , Yarrowia lipolytica or Arxula adeninivorans ) or cells mammals. The mammalian cell may be a suspension-growing cell or an adherent cell. Non-limiting examples of mammalian cells that can be cultured in any of the methods presented herein include: Chinese hamster ovary (CHO) cells (eg, CHO DG44 cells or CHO-K1s cells), Sp2.0, myeloma cells (eg, NS/ 0), B cells, hybridoma cells, T cells, human embryonic kidney (HEK) cells (eg, HEK 293E and HEK 293F), African green monkey kidney epithelial cells (Vero), and canine kidney Madin-Darby cells (Cocker- spaniel) (MDCK). In some examples in which adherent cells are cultured, the culture may also include a plurality of microcarriers (eg, microcarriers including one or more pores). Additional mammalian cells that can be cultured in any of the methods presented herein are known in the art.

A mammalian cell may include a recombinant nucleic acid (eg, a nucleic acid stably integrated into the genome of a mammalian cell) encoding a recombinant protein (eg, a recombinant protein). Non-limiting examples of recombinant nucleic acids encoding examples of recombinant proteins are described below, i.e. recombinant proteins that can be produced by the methods presented in the present description. In some cases, a mammalian cell cultured in a bioreactor (eg, any of the bioreactors presented herein) was obtained from a larger culture.

The nucleic acid encoding the recombinant protein can be introduced into a mammalian cell using a wide variety of techniques known in molecular biology and molecular genetics. Non-limiting examples include transfection (eg, lipofection), transduction (eg, lentiviral, adenoviral, or retroviral infection), and electroporation. In some cases, the nucleic acid encoding the recombinant protein is not stably inserted into the chromosome of a mammalian cell (transient transfection), while in others the nucleic acid is inserted. Alternatively or additionally, the nucleic acid encoding the recombinant protein may be present on a plasmid and/or on a mammalian artificial chromosome (eg, a human artificial chromosome). Alternatively or additionally, the nucleic acid can be introduced into a cell using a viral vector (eg, a lentiviral, retroviral, or adenoviral vector). The nucleic acid may be operably linked to a promoter sequence (eg, a strong promoter such as the β-actin promoter and the CMV promoter, or an inducible promoter). The vector comprising the nucleic acid may also optionally include a selectable marker (eg, a gene that confers resistance to hygromycin, puromycin, or neomycin to a mammalian cell).

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

Culture media

Liquid culture media are known in the art. Liquid culture media (e.g., first and/or second tissue culture medium) can be supplemented with mammalian serum (e.g., fetal bovine serum and bovine serum) and/or growth hormone or growth factor (e.g., insulin, transferrin, and epidermal growth factor) . Alternatively or additionally, the liquid culture media (e.g., the first and/or second liquid culture medium) may be a chemically defined liquid culture medium, a liquid culture medium that does not contain an animal component, a liquid culture medium that does not contain serum, or liquid culture medium containing serum. Non-limiting examples of chemically defined liquid culture media, animal-free culture media, serum-free liquid culture media, and serum-containing liquid culture media are commercially available.

The liquid culture medium typically includes an energy source (e.g., a carbohydrate such as glucose), essential amino acids (e.g., the core set of twenty amino acids and cysteine), vitamins and/or other organic compounds required in low concentrations, free fatty acids acids and/or trace elements. Liquid culture media (e.g., the first and/or second liquid culture medium), if desired, can be supplemented with, for example, a hormone or mammalian growth factor (e.g., insulin, transferrin, or epidermal growth factor), salts, and buffers (e.g., salts calcium, magnesium and phosphate salts), nucleosides and bases (eg adenosine, thymidine and hypoxanthine), protein and tissue hydrolysates and/or any combination of these additives.

A wide variety of different liquid culture media are known in the art that can be used to culture cells (eg, mammalian cells) in any of the methods presented herein. Media components that may also be useful in the methods of the present invention include, but are not limited to, chemically defined (CD) hydrolysates, for example, CD peptone, CD polypeptides (two or more amino acids), and CD growth factors. Additional examples of liquid tissue culture media and media components are known in the art.

Those skilled in the art will appreciate that the first liquid culture medium and the second liquid culture medium provided herein may be the same type or different media.

Additional features of example bioreactors

The interior surface of any of the bioreactors disclosed herein may have at least one coating (e.g., at least one coating of gelatin, collagen, poly-L-ornithine, polystyrene, and laminin), and is known in the art to one or more holes for bubbling O ₂ , CO ₂ and N ₂ into the liquid culture medium, and a stirring mechanism for mixing the liquid culture medium. A bioreactor can incubate cell culture in a controlled humidified atmosphere (e.g., above 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% humidity or 100%). The bioreactor may also be equipped with a mechanical device that can remove a volume of liquid culture medium from the bioreactor, and optionally a filter in the mechanical device that removes cells from the liquid culture medium when transferring the liquid culture medium from the bioreactor (e.g. ATF systems or cell filtration systems described in US Provisional Patent Application No. 61/878502).

Temperature

The mammalian cell culture step can be performed at a temperature of from about 31°C to about 40°C. Those skilled in the art will appreciate that the temperature can be changed at specific times during the culture phase, for example, hourly or daily. For example, the temperature may be changed (e.g., increased or decreased) after approximately one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, or approximately twenty days or more after initial seeding of cells (eg, mammalian cells) into the bioreactor. For example, the temperature may be increased (e.g., changed by up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 ,0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 , 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or up to or about 20°C). For example, the temperature may be reduced (e.g., changed by up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 ,0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 , 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or up to or about 20°C).

CO ₂

The culture step provided herein may further comprise exposing the liquid bioreactor culture medium to an atmosphere including substantially or about 15% CO ₂ (e.g., substantially or approximately 14% CO ₂ , 12% CO ₂ , 10 % CO ₂ , 8% CO ₂ , 6% CO ₂ , 5% CO ₂ , 4% CO ₂ , 3% CO ₂ , 2% CO ₂ or mostly or approximately 1% CO ₂ ).

Perfusion bioreactor

The cultivation step presented herein can be carried out using a perfusion bioreactor. Cultivating cells (e.g., mammalian cells) in a perfusion bioreactor involves removing from the bioreactor a first volume of a first liquid culture medium (e.g., including any concentration of mammalian cells, e.g., a first volume of a first liquid culture medium substantially free of cells) and adding into a first liquid culture medium and a second volume of a second liquid culture medium. Removal and addition can be done simultaneously or sequentially, or a combination of them. Additionally, removal and addition can be carried out continuously (e.g., at a rate at which a volume of 0.1% to 800% is removed and replaced (e.g., 1% to 700%, 1% to 600%, 1% to 500%, from 1% to 400%, from 1% to 350%, from 1% to 300%, from 1% to 250%, from 1% to 100%, from 100% to 200%, from 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 for any specified period of time (e.g., for period of 24 hours, over an incremental period of time from about 1 hour to about 24 hours, or over an incremental period of time greater than 24 hours)) or periodically (for example, every three days, every other day, daily, twice a day, three times a day , four times a day or five times a day), or any combination thereof. When performed periodically, the volume removed or replaced (e.g., over a period of about 24 hours, over an incremental period of time from about 1 hour to about 24 hours, or over an incremental period of time greater than 24 hours) can be, for example, from 0.1% up to 800% (for example, from 1% to 700%, from 1% to 600%, from 1% to 500%, from 1% to 400%, from 1% to 300%, from 1% to 200%, from 1 % to 100%, from 100% to 200%, from 5% to 150%, from 10% to 50%, from 15% to 40%, from 8% to 80% or from 4% to 30%) of the bioreactor volume or volume of the first liquid culture medium. The first volume of first liquid culture medium removed and the second volume of second liquid culture medium added may in some cases be maintained approximately the same during each 24 hour period (or, alternatively, an incremental time period of about 1 hour to about 24 hours or incremental time period more than 24 hours) during all or part of the culture period. As is known in the art, it is possible to vary the rate at which the first volume of the first liquid culture medium is removed (volume/time unit) and the rate at which the second volume of the second liquid culture medium is added (volume/time unit). The rate at which the first volume of the first liquid culture medium is removed (volume/time unit) and the rate at which the second volume of the second liquid culture medium is added (volume/time unit) may be approximately the same or may be different.

Alternatively, the volume removed and added may be varied (eg, gradually increased) during each 24 hour period (or, alternatively, an incremental time period of 1 hour to about 24 hours or an incremental time period greater than 24 hours) during the culture period. For example, the volume of the first liquid culture medium removed and the volume of the second liquid culture medium added during each period of 24 hours (or, alternatively, an incremental period of time from about 1 hour to more than 24 hours or an incremental period of time greater than 24 hours) during the period The culture may be increased (e.g., gradually or in uneven increments) during the culture period from a volume of from 0.5% to about 20% of the bioreactor volume, or the volume of the first liquid culture medium to from about 25% to about 150% of the bioreactor volume or the volume of the first liquid culture medium.

Those skilled in the art will appreciate that the first liquid culture medium and the second liquid culture medium may be the same type of media. In other cases, the first liquid culture medium and the second liquid culture medium may be different.

The first volume of the first liquid culture medium can be removed, for example, by a mechanical system that can be used to remove the first volume of the first liquid culture medium from the bioreactor (e.g., the first volume of the first liquid culture medium substantially free of cells from the bioreactor) . Alternatively or additionally, the first volume of the first liquid culture medium can be removed by filtering or gravity flowing the first volume of the first liquid culture medium through a sterile membrane with a molecular weight cutoff excluding cells (eg, mammalian cells).

The second volume of the second liquid culture medium can be added to the first liquid culture medium in an automated manner, for example, using a perfusion pump.

In some cases, removing a first volume of a first liquid culture medium (e.g., a first volume of a first liquid culture medium substantially free of mammalian cells) and adding a second volume of a second liquid culture medium to the first liquid culture medium does not occur within at least 1 hour (e.g. within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 14 hours, within 16 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours, within 96 hours or after 96 hours) after seeding mammalian cells in a bioreactor.

Batch bioreactor

The cultivation step presented herein can be carried out using a batch bioreactor. Cultivating cells in a batch bioreactor involves, during a major portion of the culture period, adding (eg, batch or continuous addition) to the first liquid culture medium a second volume of a second liquid culture medium. A second liquid culture medium may be added continuously (e.g., at a volume addition rate 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 for any specified period of time (e.g. , over a period of 24 hours, over an incremental period of time from about 1 hour to about 24 hours, or over an incremental period of time greater than 24 hours)) or periodically (e.g., every three days, every other day, daily, twice daily, three times a day, four times a day or five times a day), or any combination thereof. When carried out periodically, the volume added (e.g., over a period of about 24 hours, over an incremental period of time from about 1 hour to about 24 hours, or over an incremental period of time greater than 24 hours) can be, for example, from 0.1% to 300 % (for example, from 1% to 200%, from 1% to 100%, from 100% to 200%, from 5% to 150%, from 10% to 50%, from 15% to 40%, from 8% to 80% or from 4% to 30%) of the volume of the bioreactor or the volume of the first liquid culture medium. The added second volume of second liquid culture medium may in some cases be maintained approximately the same during each 24 hour period (or, alternatively, an incremental time period of about 1 hour to about 24 hours or an incremental time period greater than 24 hours) for all or part of cultivation period. As is known in the art, the rate at which the second volume of the second liquid culture medium is added (volume/unit time) can be varied during all or part of the culture period. For example, the added volume of the second liquid culture medium can be varied (eg, gradually increased) during each 24 hour period (or alternatively, an incremental time period of 1 hour to about 24 hours or an incremental time period greater than 24 hours) during the culture period. For example, the volume of second liquid culture medium added during each 24 hour period (or alternatively, an incremental time period of about 1 hour to more than 24 hours or an incremental time period of more than 24 hours) during the culture period can be increased (e.g., gradually or incrementally). in unequal increments) during the culture period from a volume of from 0.5% to about 20% of the volume of the bioreactor or the volume of the first liquid culture medium, to from about 25% to about 150% of the volume of the bioreactor or the volume of the first liquid culture medium. The rate at which the second volume of the second liquid culture medium is added (volume/unit time) may be approximately the same throughout all or part of the culture period.

Those skilled in the art will appreciate that the first liquid culture medium and the second liquid culture medium may be the same type of media. In other cases, the first liquid culture medium and the second liquid culture medium may be different. The volume of the second liquid culture medium can be added to the first liquid culture medium in an automated manner, for example, using a perfusion pump.

In some cases, the second volume of the second liquid culture medium is not added to the first liquid culture medium for at least 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 14 hours, within 16 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours, within 96 hours, or after 96 hours) after seeding the mammalian cells into the bioreactor. Batch cell culture media is typically collected at the end of the culture period and used in any of the methods presented herein, however, batch culture media can also be collected at one or more time points during the culture period and use in any of the methods presented in this description.

Those skilled in the art will appreciate that any of various culture parameters (eg, containers, volumes, rates or frequencies of culture changeovers, agitation frequency, temperature, media, and CO ₂ concentration) in any combination can be used to implement these methods. In addition, any of the mammalian cells described herein or known in the art can be used to produce the recombinant protein.

Examples of biological production systems

Examples of biological production systems useful for carrying out the methods presented herein and including MCCS or MCCS1 and MCCS2 are described in US Provisional Patent Application Nos. 61/775,060 and 61/856,390 (incorporated by reference). In these example systems, MCCS or MCCS1 and/or MCCS2 contains at least one (eg, at least two, three, four, five, or six) packed reduced bioburden chromatography columns as described herein. For example, an entire system may include as few as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty packed bioburden-reduced chromatography columns presented in this description. For example, MCCS, MCCS1 and/or MCCS2 may include (or each may include) one, two, three, four, five, six, seven, eight, nine or ten packed reduced bioburden chromatography columns as described herein.

For example, applicable systems may include an MCCS1 including an inlet and an MCCS2 including an outlet, or an MCCS including an inlet and an outlet. In some embodiments, MCCS1 and MCCS2 are fluidly coupled to each other. These systems can also be configured so that liquid can enter the inlet, through MCCS1 and MCCS2, and exit the production system through the outlet. These systems provide continuous and rapid production of therapeutic drug substances from liquid culture media. For example, the period of time between feeding a liquid (e.g., a liquid culture medium) comprising a therapeutic protein into MCCS1 and elution of the purified recombinant protein (e.g., a therapeutic protein drug) from the outlet of MCCS2 may be, for example, from about 4 hours to about 48 hours, inclusive.

Some example systems do not include a tank. In others, the system may include a maximum of 1, 2, 3, 4, or 5 tanks in the entire system (eg, where each tank stores the therapeutic protein for a total period of time, e.g., from about 5 minutes to about 6 hours, inclusive). The tanks may have a capacity ranging from 1 ml to approximately 300 ml, inclusive. Any tanks arranged in the system such that liquid enters the tanks before entering the MCCS1 or MCCS may have a capacity ranging from 1 ml up to and including approximately 100% of the loading volume of the first MCCS1 or MCCS column, respectively. Any tanks arranged in the system such that liquid enters the tanks before entering MCCS2 (and after leaving MCCS1) may have a capacity ranging from, for example, 1 ml up to and including approximately 100% of the loading volume of the first column of MCCS2.

Additional examples of system structures and features

The MCCS or MCCS1 may include an inlet through which a liquid (eg, a liquid culture medium substantially free of cells) can flow into the MCCS or MCCS1, respectively. The inlet port may be any structure known in the art for such purposes. It may include, for example, threads, ribs, or a sealing collar to allow the liquid conduit to be inserted such that upon insertion of the liquid conduit into the inlet port, liquid will flow into the MCCS or MCCS1 through the inlet port without significant leakage of liquid from the inlet port. Non-limiting examples of inlets that can be used in systems of the present invention are known and understood by those skilled in the art.

The MCCS or MCCS1 may include at least two chromatography columns, at least two chromatography membranes, or at least one chromatography column and at least one chromatography membrane and an inlet. MCCS or MCCS1 may be any of the examples of MCCS presented herein, or have one or more of any example of MCCS features (in any combination) presented herein. The chromatography columns and/or chromatography membranes present in the MCCS or MCCS1 may have one or more of any of the exemplary shapes, sizes, volumes (bed volumes), and/or routine operations presented herein.

The chromatography columns and/or chromatography membranes present in MCCS or MCCS1 may include one or more of any of the examples of resins presented herein or known in the art. For example, the resin included in one or more chromatography columns and/or chromatography membranes present in MCCS or MCCS1 may be a resin that utilizes a capture mechanism (e.g., protein A-binding capture mechanism, protein G-binding capture mechanism, antibody or antibody fragment-binding capture mechanism, substrate-binding capture mechanism, cofactor-binding capture mechanism, aptamer-binding capture mechanism, and/or tag-binding capture mechanism). The resin included in one or more of the MCCS or MCCS1 chromatography columns and/or chromatography membranes may be a cation exchange resin, an anion exchange resin, a molecular sieve resin, or a hydrophobic interaction chromatography resin, or any combination thereof. Additional examples of resins that can be used to purify recombinant protein are known in the art and can be included in one or more of the chromatography columns and/or chromatography membranes present in MCCS or MCCS1. The chromatography columns and/or chromatography membranes present in MCCS or MCCS1 may include the same and/or different resins (eg, any of the resins presented herein or known in the art for use in recombinant protein purification).

One or more typical operations (e.g., recombinant protein capture, recombinant protein purification, recombinant protein fine purification, virus inactivation, ion concentration correction, and/or pH of the liquid comprising the recombinant protein, or filtration of the liquid comprising the recombinant protein). In non-limiting examples, the MCCS or MCCS1 can perform typical operations of capturing a recombinant protein from a liquid (eg, a liquid culture medium) and inactivating viruses present in the liquid including the recombinant protein. Any combination of two or more typical operations described herein or known in the art can be performed by MCCS or MCCS1.

The chromatography columns and/or chromatography membranes present in the MCCS or MCCS1 can be connected or moved relative to each other using a switching mechanism (eg, a column switching mechanism). The MCCS or MCCS1 may also include one or more (eg, two, three, four, or five) pumps (eg, automated, eg, automated peristaltic pumps). Column switching may trigger detection of the level of recombinant protein, determined by UV absorbance, corresponding to the specific level of recombinant protein in the fluid passed through the MCCS or MCCS1 (e.g., the influent and/or eluate from one or more chromatography columns and/or chromatography membranes in the MCCS or MCCS1), a specific volume of liquid (eg buffer) or a specific period of time. Column switching generally refers to a mechanism by which at least two different chromatography columns and/or chromatography membranes in MCCS or MCCS1 (e.g., two or more different chromatography columns and/or chromatography membranes present in MCCS1 or MCCS2) are allowed to pass through through different steps (eg, equilibration, loading, elution or washing) in substantially the same amount of time during at least a portion of the process.

MCCS or MCCS1 may be a periodic countercurrent chromatography system (PCCS). For example, a PCCS that is MCCS or MCCS1 (i.e., PCCS or PCCS1, respectively) may include four chromatography columns, where the first three columns perform a typical capture operation of the recombinant protein from a liquid (e.g., a liquid culture medium) and the fourth PCCS columns perform a typical operation of inactivating viruses in a liquid containing recombinant protein. A PCCS that is MCCS or MCCS1 can use a column switching mechanism. The PCC system can use a modified ÄKTA system (GE Healthcare, Piscataway, NJ), which can use up to, for example, four, five, six, seven or eight columns or more.

MCCS or MCCS1 can be equipped with: one or more (e.g. two, three, four, five, six, seven, eight, nine or ten) UV monitors, one or more (e.g. two, three, four, five, six, seven, eight, nine or ten) valves, one or more (e.g. two, three, four, five, six, seven, eight, nine or ten) pH meters, and/or one or more (e.g. two, three , four, five, six, seven, eight, nine or ten) conductivity meters. The MCCS or MCCS1 can also be equipped with an operating system that uses software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ) to determine when column switching should occur (e.g., UV absorbance, liquid volume or time period), and influence on switching columns (triggering).

MCCS or MCCS1 may further include one or more (for example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three or twenty-four) linear buffer correction reservoirs and/or buffer reservoirs. In other examples, the MCCS or MCCS1 may include one or more (e.g., two, three, four, five, or six) tanks in which to store liquid that cannot readily flow to one or more chromatography columns and/or chromatography membranes in the MCCS or MCCS1. The systems presented herein may include one or more tanks (eg, the tank presented herein) in MCCS, MCCS1 and/or MCCS2. Other examples of systems presented herein do not include a tank in the MCCS, MCCS1 or MCCS2 or do not include a tank in the entire system. Other examples of systems presented herein include a maximum of one, two, three, four or five tanks (eg, any of the tanks presented herein) in an entire system.

Second MCCS

The second MCCS (MCCS2) in example systems includes at least two chromatography columns, at least two chromatography membranes, or at least one of the chromatography columns, and at least one of the chromatography membranes and an outlet. MCCS2 may be any of the examples of MCCS presented herein, or may have one or more of any example MCCS features (in any combination) presented herein. The chromatography columns and/or chromatography membranes present in MCCS2 may have one or more of: any of the shapes, sizes, volumes (bed volumes) and/or typical operations presented herein. Chromatography columns and/or chromatography membranes may include any of the examples of resins presented herein or known in the art. For example, the resin included in one or more of the chromatography columns and/or chromatography membranes present in MCCS2 may be a resin that utilizes a capture mechanism (e.g., protein A-binding capture mechanism, protein G-binding capture mechanism, with binding of an antibody or antibody fragment, a substrate-binding capture mechanism, a cofactor-binding capture mechanism, a tag-binding capture mechanism, and/or an aptamer-binding capture mechanism). Useful resins include, for example, a cation exchange resin, an anion exchange resin, a molecular sieve resin and a hydrophobic interaction chromatography resin. Additional examples of resins are known in the art. The chromatography columns and/or chromatography membranes present in MCCS2 may include the same and/or different resins (eg, any of the resins presented herein or known in the art for use in recombinant protein purification).

The chromatography columns and/or chromatography membranes present in MCCS2 can perform one or more generic operations (eg, any of the generic operations presented herein, or any combination of the generic operations presented herein). In non-limiting examples, MCCS2 can perform typical operations of purifying a recombinant protein from a liquid and finely purifying a recombinant protein present in a liquid including the recombinant protein. In other non-limiting examples, the MCCS2 can perform typical operations of purifying a recombinant protein present in a liquid, finely purifying a recombinant protein present in a liquid, and filtering a liquid including a recombinant protein. In another example, the MCCS2 can perform the typical operations of purifying a recombinant protein present in a liquid, finely purifying a recombinant protein present in a liquid, filtering a liquid including the recombinant protein, and adjusting the ion concentration and/or pH of the liquid including the recombinant protein. Any combination of two or more typical operations described herein or known in the art can be performed by MCCS2.

Chromatography columns and/or chromatography membranes present in MCCS2 can be connected or moved relative to each other through a switching mechanism (eg, a column switching mechanism). MCCS2 may also include one or more (eg, two, three, four or five) pumps (eg, automated, eg, automated peristaltic pumps). Column switching may trigger a determination of the level of recombinant protein, determined by the UV absorbance corresponding to a particular level of recombinant protein in the fluid passed through the MCCS2 (e.g., the influent and/or eluate from one or more chromatography columns and/or chromatography membranes in the MCCS2), a specific volume of liquid (eg buffer) or a specific period of time.

MCCS2 may be a batch countercurrent chromatography system (ie PCCS2). For example, PCCS2 may include three columns that perform a typical purification operation of a recombinant protein from a liquid, and a chromatographic membrane that performs a typical fine purification step of a recombinant protein present in a liquid. For example, the three columns that carry out the typical purification operation of a recombinant protein from a liquid may include, for example, a cation exchange resin, and the chromatographic membrane that carries out the typical fine purification step may include a cation exchange resin. In PCCS2 you can use a speaker switching mechanism. PCCS2 can use a modified ÄKTA system (GE Healthcare, Piscataway, NJ), which can use up to, for example, four, five, six, seven or eight columns, or more.

The MCCS2 can be equipped with: one or more (e.g. two, three, four, five, six, seven, eight, nine or ten) UV monitors, one or more (e.g. two, three, four, five, six, seven, eight, nine or ten) valves, one or more (e.g. two, three, four, five, six, seven, eight, nine or ten) pH meters and/or one or more (e.g. two, three, four, five, six, seven, eight, nine or ten) conductivity meters. The MCCS2 can also be equipped with an operating system that uses software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ) to determine when column switching should occur (e.g., based on UV absorbance, liquid volume, or period time), and influence on speaker switching.

MCCS2 may further include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one , twenty-two, twenty-three or twenty-four) linear buffer correction reservoirs and/or buffer reservoirs. In other examples, MCCS2 may include one or more (e.g., two, three, four, five, or six) tanks (e.g., any of the tanks described herein) that can store liquid that cannot readily flow into one or several of the chromatography columns and/or chromatography membranes in MCCS2.

MCCS2 includes an outlet through which the therapeutic protein drug can exit the system. The outlet may include, for example, threads, ribs, or a seal to allow insertion of a fluid conduit or vessel for storing purified recombinant protein (eg, a therapeutic protein drug). The outlet may include a surface that can be used to close a bioburden-reduced storage vessel or other such storage container onto the outlet to allow purified recombinant protein (e.g., a therapeutic protein drug) to flow directly into the bioburden-reduced storage vessel or container. Non-limiting examples of outlets that can be used in the systems of the present invention are known and understood by those skilled in the art.

The systems presented herein may also include a liquid pipeline located between MCCS1 and MCCS2. Any of the liquid conduits presented herein may be, for example, a tube made of, for example, polyethylene, polycarbonate or plastic. The liquid conduit located between MCCS1 and MCCS2 may further include one or more of the following in any combination: one or more linear buffer correction reservoirs in fluid communication with the liquid conduit and positioned such that the buffer stored in the linear buffer correction reservoirs correction buffer is added to the liquid present in the liquid pipeline; a tank (eg, any of the tanks described herein) in hydraulic connection with the liquid conduit and positioned such that it can store any excess liquid present in the liquid conduit that is not readily available to the MCCS2; and one or more filters disposed in the liquid conduit such that they can filter (eg, remove bacteria) liquid present in the liquid conduit. Any of the linear buffer correction reservoirs may include, for example, a volume of from about 0.5 L to 50 L of buffer (eg, at a temperature of 25°C, 15°C, or 10°C or lower).

The systems presented herein may optionally include a liquid conduit located between the final chromatography column or chromatography membrane in the MCCS2 and the outlet. The systems presented herein may further include one or more filters in hydraulic connection with a liquid conduit located between the final chromatography column or chromatography membrane in the MCCS2 and the outlet such that the filter can remove, for example, deposited material, solids particles or bacteria from the liquid present in the liquid tubing located between the final chromatography column or chromatography membrane in the MCCS2 and the outlet.

Some examples of systems presented herein also include a bioreactor in hydraulic connection to the MCCS or MCCS1 inlet. Any of the examples of bioreactors presented herein or known in the art can be used in the systems of the present invention.

Some examples of systems presented herein also include a pumping system. The pumping system may include one or more of the following: one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) pumps (e.g., any of the pumps described herein or known herein) area), one or more (e.g., two, three, four, or five) filters (e.g., any of the filters presented herein or known in the art), one or more (e.g., two, three, four, five, six, seven, eight, nine or ten) UV detectors and one or more (eg, two, three, four or five) tanks (eg, any of the tanks described herein). Some examples of systems presented herein further include a liquid piping located between the pump and the inlet of the MCCS or MCCS1 (eg, any of the examples of liquid piping presented herein or known in the art). In some examples, this particular fluid conduit 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) tanks (for example, any of the example tanks presented herein), where these pumps and/or tanks are in hydraulic connection with the liquid present in the liquid line.

Some examples of systems provided herein further include an additional liquid conduit connected to a liquid conduit between a pump and an inlet, wherein one end of the additional liquid conduit is in hydraulic connection with the bioreactor, and the other end is in hydraulic connection with a liquid conduit between the pump and inlet. This additional liquid conduit may include a filter through which cells can be removed from the liquid culture medium removed from the bioreactor (eg, an ATF cell retention system).

The systems presented herein enable the continuous production of purified recombinant protein (eg, a therapeutic protein drug). As is known in the art, systems can provide periodic elution of purified recombinant protein (eg, a therapeutic protein drug). The systems provided herein may also result in a net yield of purified recombinant protein (e.g., therapeutic protein drug) of at least about 5 g/day, at least about 10 g/day, at least about 15 g/day , at least about 20 g/day, at least about 30 g/day, or at least about 40 g/day over a continuous period of at least about 5 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days , at least about 80 days, at least about 90 days, or at least about 100 days.

Methods for reducing the bioburden of a chromatography resin comprising the use of a substantially dry chromatography resin

The present invention also provides methods for reducing the bioburden of a chromatography resin, comprising (a) exposing a container containing a substantially dry chromatography resin to a dose of gamma radiation sufficient to reduce the bioburden of the container and the chromatography resin. Some embodiments of these methods further include, prior to step (a), drying the chromatography resin to remove liquid from the chromatography resin. Drying of the chromatography resin can be accomplished using a heat treatment (eg, oven) or a desiccator. Additional methods for drying a chromatography resin are known in the art.

These methods can use any of the gamma irradiation conditions and doses presented herein. For example, the gamma irradiation dose may range from about 15 kGy to about 45 kGy (eg, from about 20 kGy to about 30 kGy). These methods can use any of the containers and chromatography resins presented herein. For example, the container may be a storage vessel or a chromatography column. The chromatographic resin in these methods may include a protein ligand (eg, protein A or protein G). In some examples, the chromatography resin may include an anion exchange chromatography resin (eg, a chromatography resin including N-benzyl-N-methyl-ethanolamine groups). In some examples, the chromatography resin is covalently attached to the surface of an object (eg, a chip, membrane, or cassette). In some embodiments, the substantially dry chromatography resin does not contain a significant amount of antioxidant or a significant amount of chelating agent. The present invention also relates to chromatographic resins with reduced bioburden, obtained by any of the methods presented in the present description.

In some examples, the resulting reduced bioburden chromatography resin has a Sterility Assurance Level (SAL) of about 1x10 ^-8 to about 1x10 ^-5 (eg, a SAL of about 1x10 ^-7 to about 1x10 ^-6 ). The resulting reduced bioburden chromatography resin may include at least one resin selected from the group consisting of: anion exchange chromatography resin, cation exchange chromatography resin, affinity chromatography resin, hydrophobic interaction chromatography resin, and size exclusion chromatography resin. In some examples, the resulting reduced bioburden chromatography resin includes an affinity chromatography resin containing a protein ligand (eg, protein A). In some examples, the resulting reduced bioburden chromatography resin includes an anion exchange chromatography resin (eg, an anion exchange chromatography resin including N-benzyl-N-methyl-ethanolamine groups). The present invention also relates to methods for producing a packed chromatography column with reduced bioburden, including producing a chromatography resin with reduced bioburden obtained by any of the methods presented in the present description; and loading the chromatography resin into a column with reduced bioburden under aseptic conditions. The present invention also relates to packed chromatography columns with reduced bioburden, obtained by any of the methods presented in the present description.

The present invention also provides integrated, closed and continuous methods for producing purified recombinant protein with reduced bioburden, comprising: (a) producing a liquid culture medium comprising the recombinant protein that is substantially free of cells; and (b) continuously feeding the liquid culture medium into a multi-column chromatography system (MCCS) comprising at least one packed reduced bioburden chromatography column obtained by any of the methods presented herein; where the method uses a buffer with reduced bioburden, the method is integrated and is carried out continuously from the liquid culture medium to the MCCS eluate, which is the purified recombinant protein. The present invention also provides integrated, closed-loop and continuous methods for producing purified recombinant protein with reduced bioburden, comprising: (a) producing a liquid culture medium comprising the recombinant protein that is substantially free of cells; (b) continuously feeding liquid culture medium into the first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein in liquid culture medium using MCCS1; (d) obtaining an eluate from MCCS1 including the recombinant protein, and continuously feeding the eluate into a second multicolumn chromatography system (MCCS2); (e) continuously feeding the recombinant protein from the eluate into MCCS2 and subsequent elution of the recombinant protein, thereby obtaining a purified recombinant protein, wherein: the method uses a buffer with reduced bioburden, the method is integrated and is carried out continuously from a liquid culture medium to the purified recombinant protein, and at least one column in MCCS1 and/or MCCS2 includes a packed reduced bioburden chromatography column obtained by any of the methods presented herein. These methods can use any of the example aspects of the integrated, closed-loop and continuous methods for producing purified recombinant protein with reduced bioburden presented herein.

Methods for producing membranes, resins, coated materials, chips and cassettes with reduced bioburden

The present invention also provides methods for preparing a membrane, resin, coated material, chip or cassette with reduced bioburden, comprising: exposing a container comprising a composition comprising the membrane, resin, coated material, chip or cassette (e.g., membrane, resin, material coated, cellulose, agarose or sugar-based chip or cassette) and at least one antioxidant and/or chelating agent, exposed to a dose of gamma radiation sufficient to reduce the bioburden of the container and membrane, resin, coated material, chip or cassette, wherein the at least one antioxidant and/or chelating agent is present in an amount sufficient to improve damage to the membrane, resin, coated material, chip and cassette following exposure to a dose of gamma radiation. In some examples, the cartridge is a resin-containing cartridge (eg, any of the examples of resins presented herein or known in the art). In some embodiments, the membrane, resin, coated material, chip, or cassette includes a protein A or protein G ligand covalently attached to at least one or a portion of its surface. In some embodiments, the composition includes a membrane, a resin, a coated material, a chip or cassette, and a liquid including at least one antioxidant and/or at least one chelating agent. Any of the example combinations and concentrations of antioxidants and/or chelating agents provided herein can be used in any of these methods. Any of the example liquids presented herein can be used in any of these methods. In some embodiments, the composition is a dry material. The present invention also relates to a membrane, resin, coated material, chip or cassette with reduced bioburden produced by any of the methods presented herein. In some examples, the container is a sealed container or storage vessel.

The present invention also provides methods for producing a membrane, resin, coated material, chip, and cassette with reduced bioburden, comprising: exposing a container comprising a substantially dry membrane, resin, coated material, chip, or cassette (e.g., membrane, resin , coated material, cellulose, agarose or sugar-based chip or cassette) to a dose of gamma radiation sufficient to reduce the bioburden of the container and membrane, resin, coated material, chip or cassette. Some examples further include the step of drying the membrane, resin, coated material, chip or cassette prior to the exposure step. In some embodiments, the membrane, resin, coated material, chip, or cassette includes a protein A or protein G ligand covalently attached to its surface. In some examples, the cartridge is a resin-containing cartridge (eg, any of the example resins presented herein or known in the art). The present invention also relates to a membrane, resin, coated material, chip or cassette with reduced bioburden produced by any of the methods presented herein.

The invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

EXAMPLES

Example 1 Effect of Gamma Irradiation on Static Binding Ability of Various Chromatography Resins

A series of experiments were carried out to test the effect of gamma irradiation on the static binding ability of three different Protein A affinity chromatography resins: GE Mab Select SuRe ^™ (a highly cross-linked agarose resin having an 85 µm particle size and functional epoxy groups linking Protein A with agarose), JSR LifeSciences Amsphere ProA JWT203 (a porous polymethacrylate resin having a particle size of ~50 µm and functional epoxy groups connecting protein A to the polymethacrylate) and Kaneka KanCap A (a highly cross-linked cellulose having a particle size of 65-85 µm with protein A, combined with cellulose through reductive amination). Each of the three resins was irradiated with 15 kGy gamma irradiation, with the exception of Mab Select SuRe ^™ , which was treated with 25 kGy radiation, and a control sample of GE Mab Select SuRe ^™ resin was left untreated, loaded with anti-TGFβ (IgG4) antibody, and antibody levels were determined against TGFβ in the eluate. Gamma irradiation of each resin in this example was carried out in 50 mM phosphate, pH 7.0. The static binding curves of each of the gamma irradiated resins and the untreated control resin are presented in Figure 1. These data indicate that gamma irradiation reduced the binding ability of each of the three Protein A chromatography resins tested, with JSR LifeSciences Amsphere ProA JWT203 resin exhibiting the smallest decrease in binding capacity in response to gamma irradiation at a dose of 15 kGy.

The following experiment was performed to determine whether gamma irradiation of JSR LifeSciences Amsphere ProA JWT203 resin or GE Mab Select SuRe ^™ resin resulted in the cleavage (and release) of protein A from the chromatography resin. In these experiments, JSR LifeSciences Amsphere ProA JWT203 resin or GE Mab Select SuRe ^TM resin was exposed to 30 kGy gamma irradiation and then the level of protein A in the supernatant was determined by measuring absorbance at 280 nm. Data from this experiment indicate that little protein A is released from either of the two test resins after gamma irradiation (Figure 2).

These data indicate that gamma irradiation results in an immediate decrease in the binding capacity of the affinity chromatography resin and that this loss of binding capacity is not the result of cleavage of the protein ligand from the resin.

Example 2 Multiple Effects of Gamma Irradiation on Resin Binding Capacity over Multiple Chromatography Cycles

A series of subsequent experiments were carried out to test the effect of gamma irradiation on the binding capacity of the chromatography resin over multiple chromatography cycles. These experiments were performed using JSR LifeSciences Amsphere ProA JWT203 resin because this resin exhibited the least amount of loss of binding capacity in response to gamma irradiation in Example 1. Gamma irradiation of each of the resins in this example was carried out in 50 mM phosphate, pH 7.0.

The first experiment was carried out to test the effect of gamma irradiation at a dose of 15 kGy on the ability of JSR LifeSciences Amsphere ProA JWT203 resin to bind to anti-TGFβ (IgG4) antibody over multiple chromatography cycles. Untreated JSR LifeSciences Amsphere ProA JWT203 resin was also used to perform multiple chromatography runs as a positive control. At the end of each cycle, the resins were washed with 0.1 N NaOH. The amount of anti-TGFβ antibody in the eluate from each run was determined. Data indicate that gamma irradiation at a dose of 15 kGy causes an immediate decrease in the binding capacity of JSR LifeSciences Amsphere ProA JWT203 resin by approximately 20%-25% (i.e. in the first cycle), but over many additional chromatography cycles the binding the capacity decreases only at a very slow rate (ie at a rate comparable to the rate of decrease in the binding capacity of untreated resin) (Figures 3, 6 and 7).

The minimal decrease in binding capacity of the 15 kGy gamma-irradiated JSR LifeSciences Amsphere ProA JWT203 resin after the initial chromatography run is also evident from a comparison of the chromatograms in run 7 and run 11 in these experiments (Figures 4 and 5). These figures show that both untreated and 15 kGy gamma-irradiated JSR LifeSciences Amsphere ProA JWT203 resins showed only modest, if any, change in binding capacity of these resins over multiple chromatography runs.

Additional experiments were performed to test the change in binding capacity of JSR LifeSciences Amsphere ProA JWT203 resin untreated or exposed to 29 kGy gamma irradiation over multiple chromatography cycles. In these experiments, the resins were washed with 0.1 N NaOH at the end of each cycle and at the beginning of each cycle, the resins were loaded with anti-αβTCR monoclonal antibody (IgG1). The data obtained again demonstrated an initial decrease in binding capacity of the 29 kGy gamma-irradiated resin of approximately 20% to 25% compared to the untreated resin (Figure 8). The binding capacity of the untreated and 29 kGy gamma-irradiated resin decreased only moderately over multiple additional chromatography cycles (at approximately the same rate for the untreated and 29 kGy gamma-irradiated resin) (Figure 8).

A series of endpoint experiments were performed to test the binding capacity of untreated, 15 kGy gamma-irradiated and 29 kGy gamma-irradiated JSR LifeSciences Amsphere ProA JWT203 resin over multiple chromatography cycles. At the end of each cycle, the resins were washed with 0.1 N NaOH and the resin was loaded with anti-TGFβ monoclonal antibody (IgG4) or anti-αβTCR (IgG1) monoclonal antibody (abTCR). Data indicate that 15 kGy and 29 kGy gamma irradiation results in substantially the same level of reduction in binding capacity of JSR LifeSciences Amsphere ProA JWT203 resin, and that the level of binding capacity of 15 kGy and 29 kGy gamma irradiated resin JSR LifeSciences Amsphere ProA JWT203 decreased moderately over multiple chromatography runs at the same rate as the untreated resin (Figure 9).

These data suggest that gamma irradiation of a protein ligand chromatography resin results in an immediate decrease in binding capacity, and that the loss of binding capacity remains relatively stable over multiple rounds of chromatography. These data suggest that gamma irradiation results in oxidative damage to the chromatography resin (e.g., a chromatography resin containing a protein or peptide ligand, such as an affinity chromatography resin) upon gamma irradiation, and that loss of binding capacity of the chromatography resin (e.g., chromatography resin containing a protein or peptide ligand, such as an affinity chromatography resin) gamma irradiation can be prevented, improved or reduced by gamma irradiation of the chromatography resin in the presence of at least one chelating agent and/or antioxidant.

Example 3: Effect of Gamma Irradiation on the Binding Capacity of GE Mab Select SuRe Resin ^TM

A series of experiments were performed to determine the effect of gamma irradiation on the binding ability of another protein ligand chromatography resin (GE Mab Select SuRe ^™ resin) over multiple chromatography cycles. The resin was buffer exchanged to 50 mM sodium phosphate, pH 7.0, and then subjected to 29 kGy gamma irradiation in a 30 mL Nalgene bottle. The resin was then filled into a 0.66 cm diameter column with a bed height of 3 cm (1 ml column). This column was then used to capture the IgG1 monoclonal antibody in cell culture media over multiple chromatography cycles. Untreated GE Mab Select SuRe ^™ resin was also used to perform multiple chromatography runs as a positive control. At the end of each cycle, the resins were washed with 0.1 N NaOH. The amount of IgG1 in the eluate from each cycle was determined. Data indicate that gamma irradiation at a dose of 29 kGy results in an immediate decrease in the binding capacity of GE Mab Select SuRe ^TM resin by approximately 25%-30% (ie, in the first cycle) (Figures 11 and 12). After the first chromatography cycle, 29 kGy gamma-irradiated GE Mab Select SuRe ^™ resin showed only a very low rate of decline in binding capacity over many additional chromatography cycles (i.e., a rate comparable to the rate of decline in binding capacity of untreated resin over many cycles chromatography) (Figures 11 and 12). These data are similar to those obtained for the JSR Protein A resin described in Example 2, and the mechanism for the loss of resin binding capacity as a result of gamma irradiation appears to be similar.

Example 4 Effect of Gamma Irradiation on the Capacity of MAb Select SuRe Resin ^TM LX to static linking

The data described in Example 1 indicate that gamma irradiation reduces the static binding ability of three different protein A chromatography resins (GE Mab Select SuRe ^™ , JSR LifeSciences Amsphere ^™ ProA JWT203 and Kaneka KanCapA ^™ ). Gamma irradiation was tested with another Protein A chromatography resin: GE Mab Select SuRe ^TM LX. GE Mab Select SuRe ^™ LX Resin has an agarose backbone and the same protein A ligand as Mab Select SuRe ^™ Resin, but has a higher ligand density than Mab Select SuRe ^™ Resin. The resin buffer was exchanged from 20% ethanol to 50 mM sodium phosphate, pH 6.0, and then subjected to gamma irradiation at a dose of 29 kGy. Static resin binding was determined as described in Example 1 (with the static resin binding ability being determined before and after gamma irradiation). The data indicate a similar decrease in the static binding ability of the resin after gamma irradiation (Figure 13). This gamma irradiation-induced decrease in GE Mab Select SuRe ^™ LX resin static binding ability is similar to the decrease in static binding ability observed with the three Protein A chromatography resins tested in Example 1 after gamma irradiation (Figure 1). The data presented in Figure 13 indicates that gamma irradiation of Mab Select SuRe ^™ LX results in an immediate decrease in the static binding ability of the resin.

Example 5: Reducing Gamma Irradiation-Induced Decrease in Chromatography Resin Binding Capacity

The data presented in the previous examples indicate that gamma irradiation of the chromatography resin at a dose that reduces the bioburden of the resin also leads to a decrease in the binding capacity of the resin: a decrease in the binding capacity of the resin by approximately 20%-40% compared to the binding capacity of the untreated control resin. The degree of reduction in binding capacity caused by gamma irradiation appears to be independent of the protein A ligand attached to the resin and/or the chemical properties of the resin particles. This additional set of experiments was carried out to identify buffer solutions that would prevent the loss of chromatography resin binding capacity that occurs during gamma irradiation. GE Mab Select SuRe ^™ LX was used as the chromatography resin for these experiments. GE Mab Select SuRe ^TM LX contains Protein A covalently immobilized on the porous matrix of each particle. The resin was first buffer exchanged from 20% ethanol to another buffer containing one or more antioxidants or a control buffer not including any antioxidants. The buffer solutions tested are presented in Table 1 below. In a control experiment, the resin was subjected to gamma irradiation as a dry composition in the absence of antioxidants. These resin samples were oven dried at 23°C for 2 days in such a way that gamma irradiation minimized water content. Each resin sample shown in Table 1 was irradiated with 29 kGy of gamma irradiation in a 30 mL Nalgene bottle. Static binding capacity was determined for gamma-irradiated resin samples and for untreated (unirradiated) resin samples using the methods described in Example 1.

Table 1. GE Mab Select SuRe Resin Samples Tested ^TM LX and buffer solutions

Resin Main buffer Dampeners Non-irradiated Mab Select SuRe LX 50 mM sodium phosphate, pH 6.0 No Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 No Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 100 mM mannitol Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 100 mM methionine Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 100 mM sodium ascorbate Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 100 mM histidine Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 50 mM methionine + 50 mM histidine Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 33 mM methionine + 33 mM histidine + 33 mM sodium ascorbate Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 25 mM sodium ascorbate + 25 mM methionine + 25 mM mannitol + 25 mM histidine Dried, non-irradiated Mab Select SuRe LX No No Dried Mab Select SuRe LX irradiated at 29 kGy No No

Data indicate that gamma irradiation of the chromatography resin in the presence of one or more antioxidants reduced the loss of binding capacity caused by gamma irradiation (Figure 14 and Table 2). A buffer of 25 mM sodium ascorbate, 25 mM methionine, 25 mM mannitol, 25 mM histidine, 50 mM sodium phosphate, pH 6.0 (hereinafter referred to as “SMMH” buffer) completely reduced the gamma-irradiation-induced loss of resin binding capacity (i.e., e. did not observe the loss of binding capacity observed when comparing the untreated control resin and the resin subjected to gamma irradiation at a dose of 29 kGy in the presence of SMMH buffer) (Figure 14 and Table 2). The dried resin sample gamma-irradiated at 29 kGy had the same binding capacity as the untreated resin sample. These data suggest that gamma irradiation of dry chromatography resin is another way to reduce the extent of gamma irradiation-induced loss of resin binding capacity.

Table 2. Specific binding properties of GE Mab Select SuRe resin samples tested ^TM LX

Resin Main buffer Dampeners SBC (mg/ml) Non-irradiated Mab Select SuRe 50 mM sodium phosphate, pH 7.0 No 50 Mab Select SuRe irradiated at 29 kGy 50 mM sodium phosphate, pH 7.0 No 32.8 Non-irradiated Mab Select SuRe LX 50 mM sodium phosphate, pH 6.0 No 70 Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 No 53 Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 100 mM mannitol 57 Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 100 mM methionine 64.5 Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 100 mM sodium ascorbate 66 Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 100 mM histidine 69 Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 50 mM methionine + 50 mM histidine 63.5 Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 33 mM methionine + 33 mM histidine + 33 mM sodium ascorbate 69 Mab Select SuRe LX irradiated at 29 kGy 50 mM sodium phosphate, pH 6.0 25 mM sodium ascorbate + 25 mM methionine + 25 mM mannitol + 25 mM histidine 74 Dried, non-irradiated Mab Select SuRe LX No No 75 Dried Mab Select SuRe LX irradiated at 29 kGy No No 74

An additional experiment was carried out to test the properties of the resin gamma-irradiated at a dose of 29 kGy in the presence of SMMH buffer and the untreated resin over multiple chromatography cycles. Each test resin was packed into a column with a diameter of 0.66 cm and a height of 3 cm. The columns were loaded with cell culture medium containing an IgG1 monoclonal antibody. As observed in the static binding capacity experiments (Figure 14), dynamic binding capacity was comparable for untreated resin and resin gamma irradiated at 29 kGy in the presence of SMMH buffer in all cycles (Figures 15 and 16). In addition, other critical quality and process parameters were also comparable: the percentage of high molecular weight substances measured by size exclusion high-performance liquid chromatography (SEC-HPLC) and the concentration of host cell protein (HCP) in the eluate (Table 3). Figure 17 is a polyacrylamide gel after non-denaturing sodium dodecyl sulfate electrophoresis, showing comparable eluate purity over multiple runs using untreated resin and resin gamma-irradiated at 29 kGy in the presence of SMMH buffer.

Table 3. Percentage of High Molecular Weight and Host Cell Protein in Untreated GE Mab Select SuRe Resin Eluate ^TM LX and GE Mab Select SuRe resins ^TM LX, gamma-irradiated in the presence of SMMH buffer

Protein A resin eluate %HMWS HCP (ng/mg) Protein A resin eluate %HMWS HCP (ng/mg) Raw resin eluate, cycle 1 1.55 173 Eluate with SMMH, cycle 1 1.82 107.6 Raw resin eluate, cycle 2 1.73 247.2 Eluate with SMMH, cycle 2 1.75 But Raw resin eluate, cycle 3 1.89 289.5 Eluate with SMMH, cycle 3 1.59 146.2

The percentage of high molecular weight substances was measured by size exclusion high performance liquid chromatography (SEC-HPLC) using a Tosoh G3000 SWxl column and the host cell protein concentration was measured using an ELISA assay.

Additional buffer solutions were tested for their ability to reduce gamma irradiation-induced loss of chromatography resin binding capacity. In these experiments, a single chromatography run was performed to determine binding capacity in dynamic mode. For each buffer tested, a column similar to that described above (diameter 0.66 cm, height 3 cm) was filled and loaded with cell culture medium containing monoclonal IgG1. As observed in the static binding capacity experiments (Figure 14), each buffer containing at least one antioxidant reduced the loss of resin binding capacity caused by gamma irradiation (Table 4). These data suggest that the presence of at least one antioxidant may reduce the loss of resin binding capacity caused by gamma irradiation. In addition, these data also suggest that chelating agents (which bind and prevent redox metals from forming reactive oxygen and nitrogen species) will also be able to reduce the loss of resin binding capacity caused by gamma irradiation.

Table 4. Data from one run of chromatography on GE MabSelect SuRe LX resin (untreated and irradiated at 29 kGy with or without quenchers) with cell culture containing IgG1

Resin properties Protein in eluate (mg) Bound protein (mg) Non-irradiated Mab Select SuRe LX 70 68 Mab Select SuRe LX irradiated at 29 kGy with SMMH 69 67 Mab Select SuRe LX irradiated at 29 kGy with 100 mM histidine 68 67 Mab Select SuRe LX irradiated at 29 kGy with 100 mM methionine 64 63 Mab Select SuRe LX irradiated at 29 kGy with 100 mM SA 62 61 Mab Select SuRe LX irradiated at 29 kGy 51 51

Overall, these data suggest that gamma irradiation of a chromatography resin in the presence of at least one antioxidant and/or at least one chelating agent can reduce the loss of resin binding capacity caused by gamma irradiation. These data also suggest that the presence of at least one antioxidant and/or at least one chelating agent protects the immobilized protein A ligand as well as the underlying resin matrix from damage caused by free radicals generated by gamma irradiation. The presence of at least one antioxidant and/or at least one chelating agent can also be used in other biotechnological applications involving gamma irradiation of the material at a dose that can cause damage to the matrix and/or functional groups. For example, compositions containing at least one antioxidant and/or at least one chelating agent can be used to prevent gamma irradiation-induced damage to cellulose membranes and other cellulose, agarose, or sugar-based resins, membranes, or materials.

OTHER IMPLEMENTATION OPTIONS

It should be understood that while the invention has been described in combination with the detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention as defined by the scope of the claims. Other aspects, advantages and modifications are included within the scope of the claims below.

Claims (8)

1. A composition for reducing the bioburden of a protein ligand-containing affinity chromatography resin after exposure to a dose of gamma radiation, comprising (i) an affinity chromatography resin containing a protein ligand, and (ii) a liquid containing from about 15 mM to about 35 mM ascorbate sodium, about 15 mM to about 35 mM methionine, about 15 mM to about 35 mM mannitol, and about 15 mM to about 35 mM histidine. 2. The composition according to claim 1, where the protein ligand is protein A. 3. The composition of claim 1 or 2, wherein said liquid contains from about 20 mM to about 30 mM sodium ascorbate, from about 20 mM to about 30 mM methionine, from about 20 mM to about 30 mM mannitol, and from about 20 mM to approximately 30 mM histidine. 4. Composition according to any one of paragraphs. 1-3, where the liquid is a buffer solution. 5. A container for reducing the bioburden of an affinity chromatography resin containing a protein ligand after exposure to a dose of gamma radiation, containing a composition according to any one of claims. 1-4. 6. The container according to claim 5, where the container is a storage vessel. 7. The container according to claim 5, where the container is a chromatography column. 8. The container according to claim 7, wherein the chromatography column is a packaged chromatography column.