RU2809157C2 - Sterile chromatographic resin and its application in production methods - Google Patents

Abstract

FIELD: chromatography.

SUBSTANCE: method for reducing bioburden in a chromatography resin, comprising exposing a container with a composition of (i) a chromatography resin and (ii) a liquid containing benzyl alcohol and at least two antioxidants to a dose of gamma radiation sufficient to reducing bioburden in the container and chromatography resin, wherein at least two antioxidants are selected from the group consisting of: mannitol, sodium ascorbate, histidine and methionine; and benzyl alcohol and at least two antioxidants are present in amounts sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation. A composition for a container exposed to a dose of gamma irradiation, containing (i) a chromatography resin and (ii) a liquid containing benzyl alcohol and at least two antioxidants, where at least two antioxidants are selected from the group consisting of: mannitol, sodium ascorbate, histidine and methionine, and benzyl alcohol and at least two antioxidants are present in amounts sufficient to reduce the loss of binding capacity of the chromatography resin when treated with a dose of gamma radiation sufficient to reduce the bioburden in the composition.

EFFECT: preventing a decrease in the binding capacity of the chromatography resin after exposure to a dose of gamma irradiation.

29 cl, 2 dwg, 2 tbl, 2 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of US Provisional Patent Application Serial No. 62/726043, filed August 31, 2018; the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

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

BACKGROUND OF THE ART

Mammalian cells containing a nucleic acid that encodes a recombinant protein are often used to produce therapeutically or commercially important proteins. In the current landscape of product manufacturing processes, biotechnology companies are increasingly challenged to develop innovative solutions for highly flexible and cost-effective production of therapeutic protein-based drug substances. One strategy for efficiently isolating recombinant proteins is through methods that involve continuous chromatography (eg, using a closed system). One known limitation of continuous chromatography is the presence of contaminants in the system (eg, increased bioburden), which leads to product contamination, decreased product yield, and decreased flow (or increased pressure) in the system. For example, increased bioburden within a system may cause the system to shut down completely.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery that exposure of a chromatography resin to gamma radiation reduces the binding ability of the chromatography resin, and that irradiation in the presence of at least one alcohol can help prevent such a decrease in the binding ability of the chromatography resin. caused by gamma radiation. In view of this discovery, provided herein are methods for reducing bioburden in a chromatography resin that involve exposing a container comprising a composition comprising (i) a chromatography resin and (ii) a liquid including at least one alcohol to a dose of gamma radiation , sufficient to reduce the bioburden in the container and chromatography resin, wherein the at least one alcohol is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after/during exposure to a dose of gamma radiation. Also provided are bioburden reduced chromatography columns comprising a bioburden reduced chromatography resin prepared by any of the methods described herein; compositions comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol; methods of performing reduced bioburden column chromatography using at least one of such reduced bioburden chromatography columns; and integrated closed or substantially closed and continuous methods for producing purified recombinant protein with reduced bioburden, which involve the use of at least one of such chromatography columns with reduced bioburden. Any of the chromatography resins prepared by any of the methods described herein, any of the packed chromatography columns prepared by any of the methods described herein, any of the column chromatography methods, and any of the methods described herein document, can be sterile, completely sterile, aseptic or with reduced bioburden. Any of the chromatography resins produced by any of the methods described herein, any of the chromatography columns prepared by any of the methods described herein, and any of the methods described herein may be aseptic and sterile , absolutely sterile, aseptic or with reduced bioburden.

Provided herein are methods for reducing bioburden in a chromatography resin that comprise exposing a container containing a composition comprising (i) a chromatography resin and (i) a liquid containing at least one alcohol to a dose of gamma radiation sufficient to reduce the bioburden. in the container and the chromatography resin, wherein the at least one alcohol is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation.

In some embodiments, the method may further comprise placing the composition in a container prior to exposure.

In some embodiments, the container is a storage vessel.

In some embodiments, the container is a chromatography column.

In some embodiments, the container is a packed chromatography column.

In some embodiments, the composition is a slurry of precipitated chromatography resin.

In some embodiments, the composition is a wetted solid mixture.

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

In some embodiments, the at least one alcohol includes benzyl alcohol.

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

In some embodiments of any of the methods described herein, the liquid may further include at least one antioxidant and/or chelator.

In some embodiments, the liquid includes at least one antioxidant and/or chelator in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation.

In some embodiments of any of the methods described herein, the liquid includes at least one antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin , uric acid, lipoic acid, flavonoid, phenolpropanoic acid, lidocaine, naringin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl and dimethylmethoxychromanol.

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

In some embodiments, the liquid includes mannitol, sodium ascorbate, histidine, and methionine.

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

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

In some embodiments of any of the methods described herein, the liquid includes at least one chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium salt and 2,3-dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), metallothioneine and deferoxamine.

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

In some embodiments, the composition includes an affinity chromatography resin including a protein ligand.

In some embodiments, the protein ligand is Protein A.

In some embodiments, the composition includes an anion exchange chromatography resin.

In some embodiments, the anion exchange chromatography resin includes N-benzyl-N-methylethanolamine groups.

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

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

In some embodiments, the dose is from about 23 kGy to about 27 kGy.

In some embodiments of any of the methods described herein, exposure is carried out at a temperature ranging from about −25°C to about 0°C, inclusive.

In some embodiments of any of the methods described herein, exposure is carried out at a temperature ranging from about 0°C to about 25°C, inclusive.

Provided herein are reduced bioburden chromatography resins produced by any of the methods described herein.

In some embodiments, the resin has a sterility assurance level (SAL) of from about 1 x 10 ⁻⁸ to about 1 x 10 ⁻⁵ .

In some embodiments, the resin has a sterility assurance level (SAL) of from about 1 x 10 ⁻⁷ to about 1 x 10 ⁻⁶ .

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

In some embodiments, the chromatography resin includes an affinity chromatography resin including a protein ligand.

In some embodiments, the protein ligand is Protein A.

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

In some embodiments, the anion exchange chromatography resin includes N-benzyl-N-methylethanolamine groups.

Provided herein are methods for preparing a packed chromatography column with reduced bioburden, which comprise providing any of the reduced bioburden chromatography resins described herein; and packing the column with reduced bioburden chromatography resin under aseptic conditions.

Provided herein are packed chromatography columns with reduced bioburden produced by any of the methods described herein.

Provided herein are packed chromatography columns with reduced bioburden produced by any of the methods described herein.

In some embodiments, the resin in the packed column has a Sterility Assurance Level (SAL) of from about 1 x 10 ⁻⁸ to about 1 x 10 ⁻⁵ .

In some embodiments, the resin has a sterility assurance level (SAL) of from about 1 x 10 ⁻⁷ to about 1 x 10 ⁻⁶ .

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

In some embodiments, the resin includes an affinity or pseudoaffinity chromatography resin including a protein ligand.

In some embodiments, the ligand is protein A.

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

In some embodiments, the anion exchange chromatography resin includes N-benzyl-N-methylethanolamine groups.

Provided herein are compositions that include (i) a chromatography resin and (ii) a liquid comprising at least one alcohol, wherein the at least one alcohol is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin when treated with a dose of gamma radiation sufficient to reduce the bioburden in the composition.

In some embodiments, the composition is a slurry of precipitated chromatography resin.

In some embodiments, the composition is a wetted solid mixture.

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

In some embodiments, the at least one alcohol includes benzyl alcohol.

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

In some embodiments, any of the compositions described herein, the liquid further includes at least one antioxidant and/or chelator.

In some embodiments, the liquid further includes at least one antioxidant and/or chelator in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation.

In some embodiments, any of the compositions described herein, the liquid includes at least one antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin , uric acid, lipoic acid, flavonoid, phenolpropanoic acid, lidocaine, naringin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl and dimethylmethoxychromanol.

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

In some embodiments, the liquid includes mannitol, sodium ascorbate, histidine, and methionine.

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

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

In some embodiments, any of the compositions described herein, the composition includes at least one chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium salt and 2,3-dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), metallothioneine and deferoxamine.

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

In some embodiments, any of the compositions described herein, the resin includes an affinity chromatography resin including a protein ligand.

In some embodiments, the protein ligand is Protein A.

Provided herein are methods of performing reduced bioburden column chromatography that comprise (a) providing any of the packed reduced bioburden chromatography columns described herein; and (b) performing column chromatography using a packed bioburden-reduced chromatography column and a bioburden-reduced buffer in a closed system.

In some embodiments, bioburden reduced column chromatography using a packed bioburden reduced chromatography column is performed continuously over a period of time of at least 4 days.

In some embodiments, reduced bioburden column chromatography using a packed reduced bioburden chromatography column is performed continuously over a period of at least 5 days.

In some embodiments, reduced bioburden column chromatography using a packed reduced bioburden chromatography column is performed continuously over a period of time of at least 7 days.

In some embodiments, reduced bioburden column chromatography using a packed reduced bioburden chromatography column is performed continuously over a period of time of at least 14 days.

In some embodiments, bioburden reduced column chromatography using a packed bioburden reduced chromatography column is performed continuously over a period of time of at least 28 days.

In some embodiments, the resin in the reduced bioburden chromatography column pack in (a) has a percentage binding capacity of from about 75% to about 100% compared to the same resin not subjected to gamma irradiation treatment.

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

In some embodiments, the resin includes an affinity chromatography resin including a protein ligand.

In some embodiments, the protein ligand is Protein A.

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

Provided herein are integrated, closed and continuous methods for the production of purified recombinant protein with reduced bioburden, which include (a) providing a liquid culture medium containing the recombinant protein that is essentially cell-free; and (b) continuously feeding a liquid culture medium into a multi-column chromatography system (MCCS) containing at least one of any of the packed reduced bioburden chromatography columns described herein; In this case, the method uses a buffer with reduced bioburden, the method is integrated, and it is carried out continuously from the liquid culture medium to the eluate leaving the MCCS, which is the purified recombinant protein.

In some embodiments, at least two different separate operations are performed in the MCCS.

In some embodiments, the method involves switching columns.

In some embodiments, the MCCS performs separate recombinant protein capture and virus inactivation steps.

In some embodiments, the MCCS performs separate recombinant protein capture and purification steps.

In some embodiments, the MCCS contains at least two packed chromatography columns with reduced bioburden.

In some embodiments, the MCCS is a batch countercurrent chromatography system.

In some embodiments, the MCCS comprises a series of affinity or pseudoaffinity chromatography, cation exchange chromatography, anion exchange chromatography, or size exclusion chromatography columns, or any combination thereof.

In some embodiments, the MCCS comprises an affinity chromatography column, wherein the affinity chromatography is performed using a method comprising a capture mechanism selected from the group consisting of a protein A capture mechanism, a substrate capture mechanism, a binding capture mechanism with an antibody or antibody fragment, a capture mechanism by binding to an aptamer, and a capture mechanism by binding to a cofactor.

In some embodiments, affinity chromatography is performed using a protein A capture mechanism, wherein the recombinant protein is an antibody or antibody fragment.

Provided herein are integrated, closed and continuous methods for the production of purified recombinant protein with reduced bioburden, which include (a) providing a liquid culture medium containing the recombinant protein that is essentially cell-free; (b) continuously supplying a liquid culture medium to the first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein in liquid culture medium using MCCS1; (d) obtaining an eluate leaving MCCS1, which includes the recombinant protein, and continuously feeding the eluate to a second multicolumn chromatography system (MCCS2); (e) continuously feeding the recombinant protein from the eluate into the MCCS2 and subsequently eluting the recombinant protein to thereby obtain a purified recombinant protein, wherein the method uses a buffer with reduced bioburden, the method is integrated and is carried out continuously from the liquid culture medium to the purified recombinant protein and wherein at least one column in MCCS1 and/or MCCS2 comprises any of the reduced bioburden packed chromatography columns described herein.

In some embodiments, at least two different separate operations are performed on MCCS1 and/or MCCS2.

In some embodiments, the method involves switching columns.

In some embodiments, MCCS1 performs separate recombinant therapeutic protein capture and viral inactivation operations.

In some embodiments, MCCS2 performs separate purification and refinement operations on the recombinant protein.

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

In some embodiments, MCCS1 is a first periodic countercurrent chromatography system (PCCS1).

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

In some embodiments, affinity chromatography is performed using a capture mechanism selected from the group consisting of a protein A capture mechanism, a substrate capture mechanism, an antibody or antibody fragment capture mechanism, an aptamer capture mechanism, and uptake mechanism through binding to a cofactor.

In some embodiments, affinity chromatography is performed using a protein A capture mechanism, wherein the recombinant protein is an antibody or antibody fragment.

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

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

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

In some embodiments of any of the methods described herein, the method is carried out continuously over a period of time of at least 4 days.

In some embodiments, the method is carried out continuously over a period of at least 5 days.

In some embodiments, the method is carried out continuously over a period of time of at least 7 days.

In some embodiments, the method is carried out continuously over a period of time of at least 14 days.

In some embodiments, the method is carried out continuously over a period of time of at least 28 days.

When used herein, the singular form of a noun means one or more objects of a specific noun. For example, the expression “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 the article(s). For example, bioburden may refer to self-replicating biological contaminants present in a composition containing a chromatography resin or in a chromatography resin as a packing (e.g., self-replicating biological contaminants present in a chromatography resin as a packing 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 within the chromatography resin within the chromatography column (e.g., biological contaminants on the interior surface of the chromatography column and biological contaminants in the chromatography resin as a packing within the chromatography column). Bioburden can also refer to self-replicating biological contaminants present within a fluid (eg, a buffer used in any of the methods or processes described 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. Exemplary methods for determining bioburden are described herein. Additional methods for determining bioburden are known in the art.

The term "bioburden reduction" is known in the art and means a reduction (e.g., detectable reduction) in the level of self-replicating biological contaminants present in a composition (e.g., solid or liquid) and/or on a surface (e.g., external and/or internal surface) of the product(s). Non-limiting examples of methods for reducing bioburden in a chromatography resin (eg, a chromatography packing resin), buffer, and/or chromatography column (eg, a packed chromatography column) are described herein. Additional methods for reducing bioburden in any of the compositions described 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 the composition containing the chromatography resin, e.g. in suspension). For example, a reduced bioburden chromatography resin may be a resin exposed to a dose of gamma radiation sufficient to reduce the level of self-replicating biological contaminants in the chromatography resin (e.g., in a composition comprising a chromatography resin exposed to a dose of gamma radiation sufficient to reduce the level of self-replicating biological contaminants in the chromatography resin). For example, the reduced bioburden chromatography resin may be a resin that has been exposed to radiation ranging from about 1 kGy to about 15 kGy, from about 1 kGy to about 20 kGy of gamma radiation, from about 1 kGy to about 25 kGy of gamma radiation, from about 1 kGy to about 30 kGy of gamma radiation, or from about 1 kGy to about 35 kGy of gamma radiation. Exemplary methods for reducing bioburden in a chromatography resin are described herein. Additional methods for reducing bioburden in a chromatography resin are known in the art.

The term “reduced bioburden chromatography column” means a chromatography column (e.g., a packed chromatography column) that contains a treated chromatography resin (e.g., a gamma-irradiated chromatography resin) that has a level of self-replicating biological contaminants that is less than the level of self-replicating biological contaminants. biological contaminants present in an identical chromatography column that contains untreated chromatography resin. For example, a reduced bioburden chromatography column may contain a treated chromatography resin having a sterility assurance level of at least or about 1 x ¹⁰⁻⁶ , 1 x ¹⁰⁻⁷ , 1 x ¹⁰⁻⁸ , 1 x ¹⁰⁻⁹ , or 1 × 10 ⁻¹⁰ .

The term "reduced bioburden 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) that is characterized by a level of self-replicating contaminant(s). contaminant(s) that is less than the level of self-replicating contaminant(s) found in an identical, untreated liquid. For example, a reduced bioburden buffer may have a sterility assurance level of at least or about 1 x ¹⁰⁻⁶ , 1 x ¹⁰⁻⁷ , 1 x ¹⁰⁻⁸ , 1 x ^10−9, or 1 x 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 may apply to a gamma-irradiated chromatography resin, the inner surface and contents (eg, chromatography resin) of a chromatography column, and/or a buffer. A completely sterile composition or method may be pure (as this term is known in the art).

“Sterile” or “sterility” are terms used to describe a composition or method that has a sterility assurance level of about 1.0 x 10 ⁻⁶ or less (e.g., about 1.0 x 10 ⁻⁷ or less, about 1.0 × 10 ⁻⁸ or less, approximately 1.0 × 10 ⁻⁹ or less, or 1 × 10 ⁻¹⁰ ). Determining whether a composition or method is sterile can be accomplished using a number of validated processes known in the art. For example, a sterile composition or method may contain no viable self-replicating biological contaminants (eg, any of the self-replicating biological contaminants described herein) at all. A sterile composition or method may also be pure (as this term is known in the art).

The term "sterilization" means a validated process used to render a composition sterile (as defined herein). The degree of inactivation of indicative persistent 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 level of confidence that absolute sterility is achieved within a batch of treated units. The probability is usually calculated from the results of inactivation studies performed during validation and is expressed in the form 1 × 10 ⁻ⁿ .

The term “aseptic” is used to describe a composition or method that does not contain/do not contain disease-causing 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 this term is known in the art).

The term "individual step" is a term from 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, the individual operation may be filtration (e.g., removing contaminant bacteria, yeast, viruses and/or mycobacteria and/or particulate matter from a liquid containing the recombinant protein), capture, epitope tag removal, purification, retention or storage, fine purification, inactivation virus, adjusting the ion concentration and/or pH of the fluid comprising the recombinant protein, and removing unwanted salts.

The term "capture" means a step that is carried out to partially purify or isolate (e.g., providing 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% purity 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 diluted liquid culture medium (e.g., proteins of the culture medium or one or more other components (e.g., DNA, RNA or other proteins) present in or secreted by the mammalian cell cell). Typically, capture is performed using a chromatography resin that binds the recombinant protein (eg, using affinity chromatography). Non-limiting methods for capturing recombinant protein from liquid culture medium or dilute liquid culture medium are described herein and others are known in the art. The recombinant protein can be captured from the liquid culture medium using at least one chromatography column and/or chromatography membrane (eg, any of the chromatography columns and/or chromatography membranes described herein).

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

The term "fine purification" is a term in the art and means a step that is performed to remove remaining trace or small amounts of contaminants or impurities from a fluid comprising a recombinant protein (e.g., a recombinant therapeutic protein) that is of near final purity. cleanliness For example, fine purification can be accomplished by passing a fluid comprising the recombinant protein through chromatography column(s) or membrane absorber(s) that selectively bind either to the target recombinant protein or to small amounts of contaminants or impurities present in the fluid comprising the recombinant protein. In such an example, the eluate/filtrate from the chromatographic column(s) or membrane absorber(s) 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 particulate matter (eg, precipitated proteins) from a liquid (eg, liquid culture medium or fluid provided in any of the methods described herein).

The term "eluate/filtrate" is a term in the art and means a fluid that exits a chromatography column or chromatography membrane that includes a detectable amount of a recombinant protein (eg, a recombinant therapeutic protein).

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

The term "continuous method" means a method in which a fluid is continuously fed 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 is continuously supplied from a bioreactor through an MCCS. Another example of a continuous method is a method in which a liquid culture medium including a recombinant protein is continuously fed from a bioreactor through the first and second MCCS (MCCS1 and MCCS2). Additional examples include a method in which a liquid culture medium comprising a recombinant protein is continuously supplied through an MCCS, a method in which a liquid culture medium comprising a recombinant protein is continuously supplied through an MCCS1 and MCCS2, or a method in which a fluid comprising a recombinant protein is continuously supplied recombinant protein, via MCCS2.

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

The term “therapeutic protein drug substance” means a recombinant protein (e.g., immunoglobulin, protein fragment, engineered protein, or enzyme) that has been sufficiently purified or separated from contaminating proteins, lipids, and nucleic acids (e.g., contaminating proteins, lipids, and nucleic acids present in a liquid culture medium or derived from a host cell (e.g., a mammalian, yeast, or bacterial host cell)), as well as biological contaminants (e.g., viral and bacterial contaminants), and which can be formulated into a pharmaceutical product without any additional significant cleaning and/or decontamination steps.

The term "multi-column chromatography system" or "MCCS" means a system of a total of two or more interconnected or switched chromatography columns and/or chromatography membranes. A non-limiting example of a multicolumn chromatography system is a batch countercurrent chromatography (PCC) system comprising a total of two or more interconnected or switching chromatography columns and/or chromatography membranes. Additional examples of multicolumn chromatography systems are described 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% pure (e.g., at least or about 95%, 96%, 97%, 98%, or at least or about 99% purified, or about 100% purified) from said substance (eg, a mammalian cell or a contaminating protein, nucleic acid, carbohydrate or lipid forming a mammalian cell).

The term "mammalian cell" means any cell of any mammal or any cell 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 described herein. Additional examples of mammalian cells are known in the art.

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

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

The term "liquid culture medium" means a fluid medium that contains sufficient nutrients to support the growth and proliferation of a cell (eg, a mammalian cell) 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, nicotinamide, 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 product extracted from the mammal (formulated liquid culture medium). In some embodiments, the liquid culture medium may include trace metals, mammalian growth hormone, and/or mammalian growth factor. Another example of a liquid culture medium is minimal medium (eg, medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture medium are described herein. Additional examples of liquid culture medium are known in the art and are commercially available. The liquid culture medium may include mammalian cells at any density. For example, as used herein, the volume of liquid culture medium removed from the bioreactor may essentially be free of mammalian cells.

The term “liquid culture medium not containing animal components” 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 that includes serum from the blood of a mammal.

The term "chemically defined liquid culture medium" is a term from 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, since such preparations typically comprise 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., a variable domain, framework sequence and/or constant domain sequence). The immunoglobulin may include, for example, at least 15 amino acids from an immunoglobulin light chain, for example, at least 15 amino acids from 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 a trispecific antibody, or a dimeric, trimeric or multimeric antibody, or a diabody, an Affibody®, or a 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 described 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 of at least or about 4 amino acids in length, at least or about 5 amino acids in length, at least or about 6 amino acids in length, at least or about 7 amino acids in length, at least or about 8 amino acids, at least or about 9 amino acids, at least or about 10 amino acids, at least or about 11 amino acids, at least or about 12 amino acids, at least or about 13 amino acids, at least or about 14 amino acids, at least or about 15 amino acids, at least or about 16 amino acids, at least or about 17 amino acids, at least or about 18 amino acids, at least or about 19 amino acids, or at least or about 20 amino acids or longer than 20 amino acids. The recombinant protein fragment can be produced using any of the methods described herein.

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

The term “secreted protein” or “secreted recombinant protein” means a protein (e.g., a recombinant protein) that originally included at least one secretion signal sequence when translated within a mammalian cell, and as a result of at least partial enzymatic cleavage of the secretion signal sequence in in a mammalian cell it is secreted at least partially into the extracellular space (eg, liquid culture medium). Those skilled in the art will appreciate that a “secreted” protein does not have to be completely separated from the cell to be considered a secreted protein.

The term "perfusion bioreactor" means a bioreactor containing a number of cells (eg, mammalian cells) in a first liquid culture medium, wherein culturing the cells present in the bioreactor involves periodically or continuously removing the first liquid culture medium and, simultaneously or shortly thereafter, , adding almost the same volume of a second liquid culture medium to the bioreactor. In some examples, the volume of the first liquid culture medium removed and added is gradually varied (e.g., increased or decreased) over discrete periods of time (e.g., a period of time of about 24 hours, a period of time from about 1 minute to about 24 hours, or a period of time more than 24 hours) during the culture period (e.g. rate of refeeding the culture medium daily). The proportion of media removed and replaced each day may vary depending on the specific cells being cultured, the initial seed density, and the cell density at a particular time point. "RV" or "reactor volume" means the volume of culture medium present at the beginning of the culture process (eg, the total volume of culture medium present after inoculation).

The term "fed-batch bioreactor" is a term from the art and means a bioreactor containing a number of cells (eg, mammalian cells) in a first liquid culture medium, wherein culturing the cells present in the bioreactor involves periodically or continuously adding a second liquid culture medium. medium 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 fed-batch culture, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch culture, a second liquid culture medium is added as a dry powder.

The term "clarified liquid culture medium" means a liquid culture medium obtained by culturing bacterial or yeast cells that is substantially free (e.g., at least 80%, 85%, 90%, 92%, 94%, 96% pure) 98% or 99%) bacterial or yeast cells.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention relates. Methods and materials are described herein for use in the present invention; however, other suitable methods and materials known in the art can 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 by reference in their entirety. In the event of any conflict, this description, including definitions, will control.

Other features and advantages of the present invention will become apparent from the following detailed description and figures, as well as from the claims.

DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 is a graph showing the percentage binding capacity (compared to an unirradiated chromatography resin loaded with the same material) for MabSelect ^™ SuRe ^™ (Protein A Binding Chromatography Resin) over multiple chromatography runs after exposure to 40-49 kGy radiation, in the presence of one of the following buffers: (i) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine and 25 mM mannitol in 50 mM sodium phosphate buffer ("SMM'H"); (ii) 2% v/v benzyl alcohol ("2% BA") and (iii) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol and 2% v/v. benzyl alcohol in 50 mM sodium phosphate buffer ("SMM'H+2% BA").

Fig. 2 is a graph showing the percentage binding capacity (compared to an unirradiated chromatography resin loaded with the same material) for Capto Adhere chromatography resin over multiple chromatography cycles after exposure to 28-34 kGy or 40-49 kGy kGy of radiation (described in Example 2), in the presence of one of the following buffers: (i) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine and 25 mM mannitol in 50 mM sodium phosphate buffer ("SMM'H") or (ii) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol and 2% v/v. benzyl alcohol in 50 mM sodium phosphate buffer ("SMM'H+2% BA").

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods for reducing bioburden in a chromatography resin that comprise exposing a container comprising a composition comprising (i) a chromatography resin and (ii) a liquid comprising at least one (e.g., two, three, four, or five) alcohols , exposure to a dose of gamma radiation sufficient to reduce the bioburden in the container and chromatography resin, wherein at least one alcohol is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after/during exposure to a dose of gamma radiation. Also provided are bioburden reduced chromatography columns comprising a bioburden reduced chromatography resin prepared by any of the methods described herein; compositions comprising (i) a chromatography resin and (ii) a liquid comprising at least one (eg, two, three, four or five) alcohol; methods of performing bioburden-reduced column chromatography using at least one of such bioburden-reduced chromatography columns; and integrated closed or substantially closed and continuous methods for producing purified bioburden-reduced recombinant protein that involve the use of at least one of such chromatography columns with reduced bioburden. Non-limiting aspects of such methods and processes are described below. As can be understood from the prior art, the various aspects described below can be used in any combination without limitation.

Compositions containing a chromatography resin and at least one alcohol

Provided herein are compositions comprising (i) a chromatography resin (e.g., any of the chromatography resins described herein or known in the art) and (ii) a liquid comprising at least one (e.g., two, three, four or five) alcohol (for example, any of the illustrative alcohols described herein or known in the art), wherein at least one alcohol is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin when treated with a gamma dose radiation sufficient to reduce the bioburden in 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 (e.g., a protein or peptide ligand affinity chromatography resin, e.g., a protein A or protein G binding chromatography resin).

The composition may, for example, be a slurry of precipitated chromatography resin. In some examples, the composition may be a wetted or wet solid mixture. In some examples, the composition is a chromatography resin placed in a liquid.

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

In some examples of any of the compositions, the total combined concentration of one or more alcohols in the liquid or composition is from about 0.01% v/v. to about 20% v/v, from about 0.01% v/v. to about 19% v/v, from about 0.01% v/v. to about 18% v/v, from about 0.01% v/v. to about 17% v/v, from about 0.01% v/v. to about 16% v/v, from about 0.01% v/v. to about 15% v/v, from about 0.01% v/v. to about 14% v/v, from about 0.01% v/v. to about 13% v/v, from about 0.01% v/v. to about 12% v/v, from about 0.01% v/v. to about 11% v/v, from about 0.01% v/v. to about 10% v/v, from about 0.01% v/v. to about 9% v/v, from about 0.01% v/v. to about 8% v/v, from about 0.01% v/v. to about 7% v/v, from about 0.01% v/v. to about 6% v/v, from about 0.01% v/v. to about 5% v/v, from about 0.01% v/v. to about 4.5% v/v, from about 0.01% v/v. to about 4.0% v/v, from about 0.01% v/v. to about 3.5% v/v, from about 0.01% v/v. to about 3.0% v/v, from about 0.01% v/v. to about 2.5% v/v, from about 0.01% v/v. to about 2.2% v/v, from about 0.01% v/v. to about 2.0% v/v, from about 0.01% v/v. to about 1.8% v/v, from about 0.01% v/v. to about 1.6% v/v, from about 0.01% v/v. to about 1.4% v/v, from about 0.01% v/v. to about 1.2% v/v, from about 0.01% v/v. to about 1.0% v/v, from about 0.01% v/v. to about 0.8% v/v, from about 0.01% v/v. to about 0.6% v/v, from about 0.01% v/v. to about 0.4% v/v, from about 0.01% v/v. to about 0.2% v/v, from about 0.01% v/v. to about 0.1% v/v, from about 0.01% v/v. to about 0.05% v/v, from about 0.05% v/v. to about 20% v/v, from about 0.05% v/v. to about 19% v/v, from about 0.05% v/v. to about 18% v/v, from about 0.05% v/v. to about 17% v/v, from about 0.05% v/v. to about 16% v/v, from about 0.05% v/v. to about 15% v/v, from about 0.05% v/v. to about 14% v/v, from about 0.05% v/v. to about 13% v/v, from about 0.05% v/v. to about 12% v/v, from about 0.05% v/v. to about 11% v/v, from about 0.05% v/v. to about 10% v/v, from about 0.05% v/v. to about 9% v/v, from about 0.05% v/v. to about 8% v/v, from about 0.05% v/v. to about 7% v/v, from about 0.05% v/v. to about 6% v/v, from about 0.05% v/v. to about 5% v/v, from about 0.05% v/v. to about 4.5% v/v, from about 0.05% v/v. to about 4.0% v/v, from about 0.05% v/v. to about 3.5% v/v, from about 0.05% v/v. to about 3.0% v/v, from about 0.05% v/v. to about 2.5% v/v, from about 0.05% v/v. to about 2.2% v/v, from about 0.05% v/v. to about 2.0% v/v, from about 0.05% v/v. to about 1.8% v/v, from about 0.05% v/v. to about 1.6% v/v, from about 0.05% v/v. to about 1.4% v/v, from about 0.05% v/v. to about 1.2% v/v, from about 0.05% v/v. to about 1.0% v/v, from about 0.05% v/v. to about 0.8% v/v, from about 0.05% v/v. to about 0.6% v/v, from about 0.05% v/v. to about 0.4% v/v, from about 0.05% v/v. to about 0.2% v/v, from about 0.05% v/v. to about 0.1% v/v, from about 0.1% v/v. to about 20% v/v, from about 0.1% v/v. to about 19% v/v, from about 0.1% v/v. to about 18% v/v, from about 0.1% v/v. to about 17% v/v, from about 0.1% v/v. to about 16% v/v, from about 0.1% v/v. to about 15% v/v, from about 0.1% v/v. to about 14% v/v, from about 0.1% v/v. to about 13% v/v, from about 0.1% v/v. to about 12% v/v, from about 0.1% v/v. to about 11% v/v, from about 0.1% v/v. to about 10% v/v, from about 0.1% v/v. to about 9% v/v, from about 0.1% v/v. to about 8% v/v, from about 0.1% v/v. to about 7% v/v, from about 0.1% v/v. to about 6% v/v, from about 0.1% v/v. to about 5% v/v, from about 0.1% v/v. to about 4.5% v/v, from about 0.1% v/v. to about 4.0% v/v, from about 0.1% v/v. to about 3.5% v/v, from about 0.1% v/v. to about 3.0% v/v, from about 0.1% v/v. to about 2.5% v/v, from about 0.1% v/v. to about 2.2% v/v, from about 0.1% v/v. to about 2.0% v/v, from about 0.1% v/v. to about 1.8% v/v, from about 0.1% v/v. to about 1.6% v/v, from about 0.1% v/v. to about 1.4% v/v, from about 0.1% v/v. to about 1.2% v/v, from about 0.1% v/v. to about 1.0% v/v, from about 0.1% v/v. to about 0.8% v/v, from about 0.1% v/v. to about 0.6% v/v, from about 0.1% v/v. to about 0.4% v/v, from about 0.1% v/v. to about 0.2% v/v, from about 0.2% v/v. to about 20% v/v, from about 0.2% v/v. to about 19% v/v, from about 0.2% v/v. to about 18% v/v, from about 0.2% v/v. to about 17% v/v, from about 0.2% v/v. to about 16% v/v, from about 0.2% v/v. to about 15% v/v, from about 0.2% v/v. to about 14% v/v, from about 0.2% v/v. to about 13% v/v, from about 0.2% v/v. to about 12% v/v, from about 0.2% v/v. to about 11% v/v, from about 0.2% v/v. to about 10% v/v, from about 0.2% v/v. to about 9% v/v, from about 0.2% v/v. to about 8% v/v, from about 0.2% v/v. to about 7% v/v, from about 0.2% v/v. to about 6% v/v, from about 0.2% v/v. to about 5% v/v, from about 0.2% v/v. to about 4.5% v/v, from about 0.2% v/v. to about 4.0% v/v, from about 0.2% v/v. to about 3.5% v/v, from about 0.2% v/v. to about 3.0% v/v, from about 0.2% v/v. to about 2.5% v/v, from about 0.2% v/v. to about 2.2% v/v, from about 0.2% v/v. to about 2.0% v/v, from about 0.2% v/v. to about 1.8% v/v, from about 0.2% v/v. to about 1.6% v/v, from about 0.2% v/v. to about 1.4% v/v, from about 0.2% v/v. to about 1.2% v/v, from about 0.2% v/v. to about 1.0% v/v, from about 0.2% v/v. to about 0.8% v/v, from about 0.2% v/v. to about 0.6% v/v, from about 0.2% v/v. to about 0.4% v/v, from about 0.4% v/v. to about 20% v/v, from about 0.4% v/v. to about 19% v/v, from about 0.4% v/v. to about 18% v/v, from about 0.4% v/v. to about 17% v/v, from about 0.4% v/v. to about 16% v/v, from about 0.4% v/v. to about 15% v/v, from about 0.4% v/v. to about 14% v/v, from about 0.4% v/v. to about 13% v/v, from about 0.4% v/v. to about 12% v/v, from about 0.4% v/v. to about 11% v/v, from about 0.4% v/v. to about 10% v/v, from about 0.4% v/v. to about 9% v/v, from about 0.4% v/v. to about 8% v/v, from about 0.4% v/v. to about 7% v/v, from about 0.4% v/v. to about 6% v/v, from about 0.4% v/v. to about 5% v/v, from about 0.4% v/v. to about 4.5% v/v, from about 0.4% v/v. to about 4.0% v/v, from about 0.4% v/v. to about 3.5% v/v, from about 0.4% v/v. to about 3.0% v/v, from about 0.4% v/v. to about 2.5% v/v, from about 0.4% v/v. to about 2.2% v/v, from about 0.4% v/v. to about 2.0% v/v, from about 0.4% v/v. to about 1.8% v/v, from about 0.4% v/v. to about 1.6% v/v, from about 0.4% v/v. to about 1.4% v/v, from about 0.4% v/v. to about 1.2% v/v, from about 0.4% v/v. to about 1.0% v/v, from about 0.4% v/v. to about 0.8% v/v, from about 0.4% v/v. to about 0.6% v/v, from about 0.6% v/v. to about 20% v/v, from about 0.6% v/v. to about 19% v/v, from about 0.6% v/v. to about 18% v/v, from about 0.6% v/v. to about 17% v/v, from about 0.6% v/v. to about 16% v/v, from about 0.6% v/v. to about 15% v/v, from about 0.6% v/v. to about 14% v/v, from about 0.6% v/v. to about 13% v/v, from about 0.6% v/v. to about 12% v/v, from about 0.6% v/v. to about 11% v/v, from about 0.6% v/v. to about 10% v/v, from about 0.6% v/v. to about 9% v/v, from about 0.6% v/v. to about 8% v/v, from about 0.6% v/v. to about 7% v/v, from about 0.6% v/v. to about 6% v/v, from about 0.6% v/v. to about 5% v/v, from about 0.6% v/v. to about 4.5% v/v, from about 0.6% v/v. to about 4.0% v/v, from about 0.6% v/v. to about 3.5% v/v, from about 0.6% v/v. to about 3.0% v/v, from about 0.6% v/v. to about 2.5% v/v, from about 0.6% v/v. to about 2.2% v/v, from about 0.6% v/v. to about 2.0% v/v, from about 0.6% v/v. to about 1.8% v/v, from about 0.6% v/v. to about 1.6% v/v, from about 0.6% v/v. to about 1.4% v/v, from about 0.6% v/v. to about 1.2% v/v, from about 0.6% v/v. to about 1.0% v/v, from about 0.6% v/v. to about 0.8% v/v, from about 0.8% v/v. to about 20% v/v, from about 0.8% v/v. to about 19% v/v, from about 0.8% v/v. to about 18% v/v, from about 0.8% v/v. to about 17% v/v, from about 0.8% v/v. to about 16% v/v, from about 0.8% v/v. to about 15% v/v, from about 0.8% v/v. to about 14% v/v, from about 0.8% v/v. to about 13% v/v, from about 0.8% v/v. to about 12% v/v, from about 0.8% v/v. to about 11% v/v, from about 0.8% v/v. to about 10% v/v, from about 0.8% v/v. to about 9% v/v, from about 0.8% v/v. to about 8% v/v, from about 0.8% v/v. to about 7% v/v, from about 0.8% v/v. to about 6% v/v, from about 0.8% v/v. to about 5% v/v, from about 0.8% v/v. to about 4.5% v/v, from about 0.8% v/v. to about 4.0% v/v, from about 0.8% v/v. to about 3.5% v/v, from about 0.8% v/v. to about 3.0% v/v, from about 0.8% v/v. to about 2.5% v/v, from about 0.8% v/v. to about 2.2% v/v, from about 0.8% v/v. to about 2.0% v/v, from about 0.8% v/v. to about 1.8% v/v, from about 0.8% v/v. to about 1.6% v/v, from about 0.8% v/v. to about 1.4% v/v, from about 0.8% v/v. to about 1.2% v/v, from about 0.8% v/v. to about 1.0% v/v, from about 1.0% v/v. to about 20% v/v, from about 1.0% v/v. to about 19% v/v, from about 1.0% v/v. to about 18% v/v, from about 1.0% v/v. to about 17% v/v, from about 1.0% v/v. to about 16% v/v, from about 1.0% v/v. to about 15% v/v, from about 1.0% v/v. to about 14% v/v, from about 1.0% v/v. to about 13% v/v, from about 1.0% v/v. to about 12% v/v, from about 1.0% v/v. to about 11% v/v, from about 1.0% v/v. to about 10% v/v, from about 1.0% v/v. to about 9% v/v, from about 1.0% v/v. to about 8% v/v, from about 1.0% v/v. to about 7% v/v, from about 1.0% v/v. to about 6% v/v, from about 1.0% v/v. to about 5% v/v, from about 1.0% v/v. to about 4.5% v/v, from about 1.0% v/v. to about 4.0% v/v, from about 1.0% v/v. to about 3.5% v/v, from about 1.0% v/v. to about 3.0% v/v, from about 1.0% v/v. to about 2.5% v/v, from about 1.0% v/v. to about 2.2% v/v, from about 1.0% v/v. to about 2.0% v/v, from about 1.0% v/v. to about 1.8% v/v, from about 1.0% v/v. to about 1.6% v/v, from about 1.0% v/v. to about 1.4% v/v, from about 1.0% v/v. to about 1.2% v/v, from about 1.2% v/v. to about 20% v/v, from about 1.2% v/v. to about 19% v/v, from about 1.2% v/v. to about 18% v/v, from about 1.2% v/v. to about 17% v/v, from about 1.2% v/v. to about 16% v/v, from about 1.2% v/v. to about 15% v/v, from about 1.2% v/v. to about 14% v/v, from about 1.2% v/v. to about 13% v/v, from about 1.2% v/v. to about 12% v/v, from about 1.2% v/v. to about 11% v/v, from about 1.2% v/v. to about 10% v/v, from about 1.2% v/v. to about 9% v/v, from about 1.2% v/v. to about 8% v/v, from about 1.2% v/v. to about 7% v/v, from about 1.2% v/v. to about 6% v/v, from about 1.2% v/v. to about 5% v/v, from about 1.2% v/v. to about 4.5% v/v, from about 1.2% v/v. to about 4.0% v/v, from about 1.2% v/v. to about 3.5% v/v, from about 1.2% v/v. to about 3.0% v/v, from about 1.2% v/v. to about 2.5% v/v, from about 1.2% v/v. to about 2.2% v/v, from about 1.2% v/v. to about 2.0% v/v, from about 1.2% v/v. to about 1.8% v/v, from about 1.2% v/v. to about 1.6% v/v, from about 1.2% v/v. to about 1.4% v/v, from about 1.4% v/v. to about 20% v/v, from about 1.4% v/v. to about 19% v/v, from about 1.4% v/v. to about 18% v/v, from about 1.4% v/v. to about 17% v/v, from about 1.4% v/v. to about 16% v/v, from about 1.4% v/v. to about 15% v/v, from about 1.4% v/v. to about 14% v/v, from about 1.4% v/v. to about 13% v/v, from about 1.4% v/v. to about 12% v/v, from about 1.4% v/v. to about 11% v/v, from about 1.4% v/v. to about 10% v/v, from about 1.4% v/v. to about 9% v/v, from about 1.4% v/v. to about 8% v/v, from about 1.4% v/v. to about 7% v/v, from about 1.4% v/v. to about 6% v/v, from about 1.4% v/v. to about 5% v/v, from about 1.4% v/v. to about 4.5% v/v, from about 1.4% v/v. to about 4.0% v/v, from about 1.4% v/v. to about 3.5% v/v, from about 1.4% v/v. to about 3.0% v/v, from about 1.4% v/v. to about 2.5% v/v, from about 1.4% v/v. to about 2.2% v/v, from about 1.4% v/v. to about 2.0% v/v, from about 1.4% v/v. to about 1.8% v/v, from about 1.4% v/v. to about 1.6% v/v, from about 1.6% v/v. to about 20% v/v, from about 1.6% v/v. to about 19% v/v, from about 1.6% v/v. to about 18% v/v, from about 1.6% v/v. to about 17% v/v, from about 1.6% v/v. to about 16% v/v, from about 1.6% v/v. to about 15% v/v, from about 1.6% v/v. to about 14% v/v, from about 1.6% v/v. to about 13% v/v, from about 1.6% v/v. to about 12% v/v, from about 1.6% v/v. to about 11% v/v, from about 1.6% v/v. to about 10% v/v, from about 1.6% v/v. to about 9% v/v, from about 1.6% v/v. to about 8% v/v, from about 1.6% v/v. to about 7% v/v, from about 1.6% v/v. to about 6% v/v, from about 1.6% v/v. to about 5% v/v, from about 1.6% v/v. to about 4.5% v/v, from about 1.6% v/v. to about 4.0% v/v, from about 1.6% v/v. to about 3.5% v/v, from about 1.6% v/v. to about 3.0% v/v, from about 1.6% v/v. to about 2.5% v/v, from about 1.6% v/v. to about 2.2% v/v, from about 1.6% v/v. to about 2.0% v/v, from about 1.6% v/v. to about 1.8% v/v, from about 1.8% v/v. to about 20% v/v, from about 1.8% v/v. to about 19% v/v, from about 1.8% v/v. to about 18% v/v, from about 1.8% v/v. to about 17% v/v, from about 1.8% v/v. to about 16% v/v, from about 1.8% v/v. to about 15% v/v, from about 1.8% v/v. to about 14% v/v, from about 1.8% v/v. to about 13% v/v, from about 1.8% v/v. to about 12% v/v, from about 1.8% v/v. to about 11% v/v, from about 1.8% v/v. to about 10% v/v, from about 1.8% v/v. to about 9% v/v, from about 1.8% v/v. to about 8% v/v, from about 1.8% v/v. to about 7% v/v, from about 1.8% v/v. to about 6% v/v, from about 1.8% v/v. to about 5% v/v, from about 1.8% v/v. to about 4.5% v/v, from about 1.8% v/v. to about 4.0% v/v, from about 1.8% v/v. to about 3.5% v/v, from about 1.8% v/v. to about 3.0% v/v, from about 1.8% v/v. to about 2.5% v/v, from about 1.8% v/v. to about 2.2% v/v, from about 1.8% v/v. to about 2.0% v/v, from about 2.0% v/v. to about 20% v/v, from about 2.0% v/v. to about 19% v/v, from about 2.0% v/v. to about 18% v/v, from about 2.0% v/v. to about 17% v/v, from about 2.0% v/v. to about 16% v/v, from about 2.0% v/v. to about 15% v/v, from about 2.0% v/v. to about 14% v/v, from about 2.0% v/v. to about 13% v/v, from about 2.0% v/v. to about 12% v/v, from about 2.0% v/v. to about 11% v/v, from about 2.0% v/v. to about 10% v/v, from about 2.0% v/v. to about 9% v/v, from about 2.0% v/v. to about 8% v/v, from about 2.0% v/v. to about 7% v/v, from about 2.0% v/v. to about 6% v/v, from about 2.0% v/v. to about 5% v/v, from about 2.0% v/v. to about 4.5% v/v, from about 2.0% v/v. to about 4.0% v/v, from about 2.0% v/v. to about 3.5% v/v, from about 2.0% v/v. to about 3.0% v/v, from about 2.0% v/v. to about 2.5% v/v, from about 2.0% v/v. to about 2.2% v/v, from about 2.2% v/v. to about 20% v/v, from about 2.2% v/v. to about 19% v/v, from about 2.2% v/v. to about 18% v/v, from about 2.2% v/v. to about 17% v/v, from about 2.2% v/v. to about 16% v/v, from about 2.2% v/v. to about 15% v/v, from about 2.2% v/v. to about 14% v/v, from about 2.2% v/v. to about 13% v/v, from about 2.2% v/v. to about 12% v/v, from about 2.2% v/v. to about 11% v/v, from about 2.2% v/v. to about 10% v/v, from about 2.2% v/v. to about 9% v/v, from about 2.2% v/v. to about 8% v/v, from about 2.2% v/v. to about 7% v/v, from about 2.2% v/v. to about 6% v/v, from about 2.2% v/v. to about 5% v/v, from about 2.2% v/v. to about 4.5% v/v, from about 2.2% v/v. to about 4.0% v/v, from about 2.2% v/v. to about 3.5% v/v, from about 2.2% v/v. to about 3.0% v/v, from about 2.2% v/v. to about 2.5% v/v, from about 2.5% v/v. to about 20% v/v, from about 2.5% v/v. to about 19% v/v, from about 2.5% v/v. to about 18% v/v, from about 2.5% v/v. to about 17% v/v, from about 2.5% v/v. to about 16% v/v, from about 2.5% v/v. to about 15% v/v, from about 2.5% v/v. to about 14% v/v, from about 2.5% v/v. to about 13% v/v, from about 2.5% v/v. to about 12% v/v, from about 2.5% v/v. to about 11% v/v, from about 2.5% v/v. to about 10% v/v, from about 2.5% v/v. to about 9% v/v, from about 2.5% v/v. to about 8% v/v, from about 2.5% v/v. to about 7% v/v, from about 2.5% v/v. to about 6% v/v, from about 2.5% v/v. to about 5% v/v, from about 2.5% v/v. to about 4.5% v/v, from about 2.5% v/v. to about 4.0% v/v, from about 2.5% v/v. to about 3.5% v/v, from about 2.5% v/v. to about 3.0% v/v, from about 3.0% v/v. to about 20% v/v, from about 3.0% v/v. to about 19% v/v, from about 3.0% v/v. to about 18% v/v, from about 3.0% v/v. to about 17% v/v, from about 3.0% v/v. to about 16% v/v, from about 3.0% v/v. to about 15% v/v, from about 3.0% v/v. to about 14% v/v, from about 3.0% v/v. to about 13% v/v, from about 3.0% v/v. to about 12% v/v, from about 3.0% v/v. to about 11% v/v, from about 3.0% v/v. to about 10% v/v, from about 3.0% v/v. to about 9% v/v, from about 3.0% v/v. to about 8% v/v, from about 3.0% v/v. to about 7% v/v, from about 3.0% v/v. to about 6% v/v, from about 3.0% v/v. to about 5% v/v, from about 3.0% v/v. to about 4.5% v/v, from about 3.0% v/v. to about 4.0% v/v, from about 3.0% v/v. to about 3.5% v/v, from about 3.5% v/v. to about 20% v/v, from about 3.5% v/v. to about 19% v/v, from about 3.5% v/v. to about 18% v/v, from about 3.5% v/v. to about 17% v/v, from about 3.5% v/v. to about 16% v/v, from about 3.5% v/v. to about 15% v/v, from about 3.5% v/v. to about 14% v/v, from about 3.5% v/v. to about 13% v/v, from about 3.5% v/v. to about 12% v/v, from about 3.5% v/v. to about 11% v/v, from about 3.5% v/v. to about 10% v/v, from about 3.5% v/v. to about 9% v/v, from about 3.5% v/v. to about 8% v/v, from about 3.5% v/v. to about 7% v/v, from about 3.5% v/v. to about 6% v/v, from about 3.5% v/v. to about 5% v/v, from about 3.5% v/v. to about 4.5% v/v, from about 3.5% v/v. to about 4.0% v/v, from about 4.0% v/v. to about 20% v/v, from about 4.0% v/v. to about 19% v/v, from about 4.0% v/v. to about 18% v/v, from about 4.0% v/v. to about 17% v/v, from about 4.0% v/v. to about 16% v/v, from about 4.0% v/v. to about 15% v/v, from about 4.0% v/v. to about 14% v/v, from about 4.0% v/v. to about 13% v/v, from about 4.0% v/v. to about 12% v/v, from about 4.0% v/v. to about 11% v/v, from about 4.0% v/v. to about 10% v/v, from about 4.0% v/v. to about 9% v/v, from about 4.0% v/v. to about 8% v/v, from about 4.0% v/v. to about 7% v/v, from about 4.0% v/v. to about 6% v/v, from about 4.0% v/v. to about 5% v/v, from about 4.0% v/v. to about 4.5% v/v, from about 4.5% v/v. to about 20% v/v, from about 4.5% v/v. to about 19% v/v, from about 4.5% v/v. to about 18% v/v, from about 4.5% v/v. to about 17% v/v, from about 4.5% v/v. to about 16% v/v, from about 4.5% v/v. to about 15% v/v, from about 4.5% v/v. to about 14% v/v, from about 4.5% v/v. to about 13% v/v, from about 4.5% v/v. to about 12% v/v, from about 4.5% v/v. to about 11% v/v, from about 4.5% v/v. to about 10% v/v, from about 4.5% v/v. to about 9% v/v, from about 4.5% v/v. to about 8% v/v, from about 4.5% v/v. to about 7% v/v, from about 4.5% v/v. to about 6% v/v, from about 4.5% v/v. to about 5% v/v, from about 5% v/v. to about 20% v/v, from about 5% v/v. to about 19% v/v, from about 5% v/v. to about 18% v/v, from about 5% v/v. to about 17% v/v, from about 5% v/v. to about 16% v/v, from about 5% v/v. to about 15% v/v, from about 5% v/v. to about 14% v/v, from about 5% v/v. to about 13% v/v, from about 5% v/v. to about 12% v/v, from about 5% v/v. to about 11% v/v, from about 5% v/v. to about 10% v/v, from about 5% v/v. to about 9% v/v, from about 5% v/v. to about 8% v/v, from about 5% v/v. to about 7% v/v, from about 5% v/v. to about 6% v/v, from about 6% v/v. to about 20% v/v, from about 6% v/v. to about 19% v/v, from about 6% v/v. to about 18% v/v, from about 6% v/v. to about 17% v/v, from about 6% v/v. to about 16% v/v, from about 6% v/v. to about 15% v/v, from about 6% v/v. to about 14% v/v, from about 6% v/v. to about 13% v/v, from about 6% v/v. to about 12% v/v, from about 6% v/v. to about 11% v/v, from about 6% v/v. to about 10% v/v, from about 6% v/v. to about 9% v/v, from about 6% v/v. to about 8% v/v, from about 6% v/v. to about 7% v/v, from about 7% v/v. to about 20% v/v, from about 7% v/v. to about 19% v/v, from about 7% v/v. to about 18% v/v, from about 7% v/v. to about 17% v/v, from about 7% v/v. to about 16% v/v, from about 7% v/v. to about 15% v/v, from about 7% v/v. to about 14% v/v, from about 7% v/v. to about 13% v/v, from about 7% v/v. to about 12% v/v, from about 7% v/v. to about 11% v/v, from about 7% v/v. to about 10% v/v, from about 7% v/v. to about 9% v/v, from about 7% v/v. to about 8% v/v, from about 8% v/v. to about 20% v/v, from about 8% v/v. to about 19% v/v, from about 8% v/v. to about 18% v/v, from about 8% v/v. to about 17% v/v, from about 8% v/v. to about 16% v/v, from about 8% v/v. to about 15% v/v, from about 8% v/v. to about 14% v/v, from about 8% v/v. to about 13% v/v, from about 8% v/v. to about 12% v/v, from about 8% v/v. to about 11% v/v, from about 8% v/v. to about 10% v/v, from about 8% v/v. to about 9% v/v, from about 9% v/v. to about 20% v/v, from about 9% v/v. to about 19% v/v, from about 9% v/v. to about 18% v/v, from about 9% v/v. to about 17% v/v, from about 9% v/v. to about 16% v/v, from about 9% v/v. to about 15% v/v, from about 9% v/v. to about 14% v/v, from about 9% v/v. to about 13% v/v, from about 9% v/v. to about 12% v/v, from about 9% v/v. to about 11% v/v, from about 9% v/v. to about 10% v/v, from about 10% v/v. to about 20% v/v, from about 10% v/v. to about 19% v/v, from about 10% v/v. to about 18% v/v, from about 10% v/v. to about 17% v/v, from about 10% v/v. to about 16% v/v, from about 10% v/v. to about 15% v/v, from about 10% v/v. to about 14% v/v, from about 10% v/v. to about 13% v/v, from about 10% v/v. to about 12% v/v, from about 10% v/v. to about 11% v/v, from about 11% v/v. to about 20% v/v, from about 11% v/v. to about 19% v/v, from about 11% v/v. to about 18% v/v, from about 11% v/v. to about 17% v/v, from about 11% v/v. to about 16% v/v, from about 11% v/v. to about 15% v/v, from about 11% v/v. to about 14% v/v, from about 11% v/v. to about 13% v/v, from about 11% v/v. to about 12% v/v, from about 12% v/v. to about 20% v/v, from about 12% v/v. to about 19% v/v, from about 12% v/v. to about 18% v/v, from about 12% v/v. to about 17% v/v, from about 12% v/v. to about 16% v/v, from about 12% v/v. to about 15% v/v, from about 12% v/v. to about 14% v/v, from about 12% v/v. to about 13% v/v, from about 13% v/v. to about 20% v/v, from about 13% v/v. to about 19% v/v, from about 13% v/v. to about 18% v/v, from about 13% v/v. to about 17% v/v, from about 13% v/v. to about 16% v/v, from about 13% v/v. to about 15% v/v, from about 13% v/v. to about 14% v/v, from about 14% v/v. to about 20% v/v, from about 14% v/v. to about 19% v/v, from about 14% v/v. to about 18% v/v, from about 14% v/v. to about 17% v/v, from about 14% v/v. to about 16% v/v, from about 14% v/v. to about 15% v/v, from about 15% v/v. to about 20% v/v, from about 15% v/v. to about 19% v/v, from about 15% v/v. to about 18% v/v, from about 15% v/v. to about 17% v/v, from about 15% v/v. to about 16% v/v, from about 16% v/v. to about 20% v/v, from about 16% v/v. to about 19% v/v, from about 16% v/v. to about 18% v/v, from about 16% v/v. to about 17% v/v, from about 17% v/v. to about 20% v/v, from about 17% v/v. to about 19% v/v, from about 17% v/v. to about 18% v/v, from about 18% v/v. to about 20% v/v, from about 18% v/v. up to approximately 19% v/v or from about 19% v/v. up to approximately 20% v/v

In some examples of any of the compositions described herein, the liquid may further include at least one (eg, two, three, four or five) antioxidant and/or chelator. In some examples of any of the compositions described herein, the liquid may further include at least one (e.g., two, three, four, or five) antioxidant and/or chelator in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after exposure gamma radiation doses.

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

In some examples of any of the compositions described herein, the liquid may contain at least one (e.g., one, two, three, or four) of methionine (or, alternatively, cysteine or glutathione), sodium ascorbate, histidine, and mannitol . In some examples of any of the compositions described herein, the liquid may contain methionine (or, alternatively, cysteine or glutathione), sodium ascorbate, histidine and mannitol. In some examples, any of the compositions described herein, the liquid may include (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 about 95 mM to about 105 mM) mannitol; (ii) 75 mM to about 125 mM (e.g., about 80 mM to about 120 mM, about 85 mM to about 115 mM, about 90 mM to about 110 mM, or about 95 mM to about 105 mM) 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 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 (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, or about 25 mM to about 35 mM) sodium ascorbate, or (vii) about 5 mM to about 45 mM (e.g., from about 10 mM to about 40 mM, about 15 mM to about 35 mM, or about 20 mM to about 30 mM) sodium ascorbate, about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, from 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., about 10 mM to about 40 mM, about 15 mM to about 35 mM or about 20 mM to about 30 mM) mannitol and about 5 mM to about 45 mM (e.g., about 10 mM to about 40 mM, about 15 mM to about 35 mM, or approximately 20 mM to approximately 30 mM) histidine. In some examples of any of the compositions described herein, the liquid may be a buffer solution (eg, phosphate buffer solution, e.g., sodium phosphate buffer solution, such as 50 mM sodium phosphate, pH 6.0).

In some embodiments, any of the compositions described herein, the liquid may further include at least one (e.g., two, three, four, or five) chelator (e.g., at least one chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium salts of 2,3-dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), metallothioneine and deferoxamine).

Also provided herein is a container (e.g., a storage vessel, e.g., a plastic container, or a chromatography column) comprising a composition (e.g., any of the exemplary compositions described herein) comprising (i) a chromatography resin (e.g., any of the chromatography resins described herein or known in the art) and (ii) a liquid comprising at least one alcohol (for example, any of the exemplary alcohols described herein or known in the art), wherein at least the alcohol alone is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin when treated with a dose of gamma radiation sufficient to reduce the bioburden in the composition. For example, a container (e.g., a storage container, such as a plastic container, or a chromatography column) may have an internal volume of, for example, at least about 1 ml, 5 ml, at least about 10 ml, at least about 20 ml, at least about 30 ml, at least about 40 ml, at least about 50 ml, at least about 60 ml, at least about 70 ml, at least about 80 ml, at least about 90 ml , at least about 100 ml, at least about 110 ml, at least about 120 ml, at least about 130 ml, at least about 140 ml, at least about 150 ml, at least about 160 ml, at least about 170 ml, at least about 180 ml, at least about 190 ml, at least about 200 ml, at least about 210 ml, at least about 220 ml, at least about 230 ml, according to 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 slurry of precipitated liquid chromatography resin. In some examples, the container includes a chromatography resin as a packing (eg, placed in a liquid).

In any of the compositions provided herein, the liquid may further contain at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) antioxidant and/or at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) chelator. Any of the antioxidants that may be included in any of the compositions provided herein may have the ability to quench one or more of the following reactive oxygen and/or nitrogen species: hydroxyl radical, carbonate radical, superoxide anion, peroxyl radical, peroxynitrite, dioxide nitrogen 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, phenolpropanoic acid, lidocaine, naringin, 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 that include an antioxidant enzyme as an antioxidant may further include one or more substrates for the enzyme. The antioxidant can be identified using a number of methods known in the art, including, for example, spin trap, redox-sensitive dyes and chemiluminescent assays.

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

The concentration of each of the chelators and/or antioxidants that may be included in any of the compositions provided herein may be from about 0.1 mM to about 150 mM (for example, from about 0.1 mM to about 150 mM, from about 0.1 mM to about 125 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 25 mM, about 0.1 mM to about 20 mM, 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, from about 1 mM to about 30 mM, from about 1 mM to about 25 mM, from about 5 mM to about 150 mM, from about 5 mM to about 125 mM, from about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, from about 5 mM to about 25 mM, from about 10 mM to about 150 mM, from about 10 mM to about 125 mM, from about 1 mM to about 100 mM, from about 10 mM to about 80 mM, from about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 20 mM to about 150 mM , from about 20 mM to about 125 mM, from about 20 mM to about 100 mM, from about 20 mM to about 80 mM, from about 20 mM to about 60 mM, from about 20 mM to about 50 mM, from 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, about 40 mM to about 90 mM, about 40 mM to about 80 mM, about 40 mM to about 70 mM, about 40 mM to about 60 mM, about 50 mM to about 150 mM, 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, 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 may contain one or more of mannitol in an amount of 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, 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 , from about 90 mM to about 110 mM, from about 95 mM to about 105 mM, from about 5 mM to about 50 mM, from about 5 mM to about 45 mM, from about 5 mM to about 40 mM, from about 5 mM to about 35 mM, from about 10 mM to about 35 mM, from about 15 mM to about 35 mM, or from about 20 mM to about 30 mM mannitol); methionine (or alternatively cysteine or glutathione) in an amount of 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 , 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, 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, 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, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 20 mM to about 25 mM); sodium ascorbate in an amount of from 5 mM to about 150 mM (for example, 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 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, about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM sodium ascorbate) and histidine in an amount of from 5 mM to about 150 mM (e.g., about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, about 60 mM to about 140 mM, about 70 mM to about 130 mM, about 80 mM to about 120 mM, about 90 mM to about 110 mM, about 95 mM to about 105 mM, from about 30 mM to about 70 mM, about 35 mM to about 65 mM, about 40 mM to about 60 mM, about 45 mM to about 55 mM, about 20 mM to about 50 mM, about 25 mM to about 45 mM, 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, from about 20 mM to about 30 mM or from about 20 mM to about 25 mM).

Non-limiting examples of any of the compositions may 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, in a 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), about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM or about 30 mM to about 35 mM) histidine and about 10 mM to about 50 mM (e.g., about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM, or about 30 mM to about 35 mM) sodium ascorbate (e.g., in a buffer solution, e.g., phosphate buffer such as 50 mM sodium phosphate, pH 6.0) or (vii) about 5 mM to about 45 mM (e.g. , about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 23 mM to about 27 mM) sodium ascorbate, about 5 mM to about 45 mM (e.g. , about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, or about 23 mM to about 27 mM) methionine (or, 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 (e.g. in a buffer solution, for example, a phosphate buffer such as 50 mM sodium phosphate, pH 6.0).

Non-limiting gamma radiation dose levels sufficient to reduce the bioburden in any of the compositions provided herein are described below. Additional gamma dose levels sufficient to reduce the bioburden in any of the compositions provided herein are known in the art. For example, any of the compositions described herein may be exposed to gamma radiation at any of the dose levels, at any of the gamma radiation intensities, and/or at any of the gamma radiation exposure temperatures described herein. document (in any combination). The bioburden in a composition can be determined, for example, by taking a sample from the composition, which may contain self-replicating biological contaminant(s) present in the composition, for example, by Stomacher homogenization, sonication, shaking, stirring vortex, wash, mix or collect a swab, and qualitatively or quantitatively determine the level of self-replicating biological contaminant(s) 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 alcohol, at least one antioxidant, and/or at least one chelator sufficient to reduce the loss of binding capacity of the chromatography resin when treated with a dose of gamma radiation sufficient to reduce the bioburden in the composition may be determined by for example, using the methods described in the examples. For example, the level of reduction in binding capacity of a chromatography resin subjected to gamma irradiation treatment in the presence of an amount of at least one alcohol (and optionally additionally in the presence of at least one antioxidant and/or chelator) can be compared to the level of reduction in binding capacity a chromatography resin treated with the same dose of gamma radiation in the absence of at least one alcohol (and optionally at least one antioxidant and/or chelator), wherein the reduction in the binding capacity of the chromatography resin exposed to gamma radiation is reduced in the presence of at least one alcohol (and optionally additionally in the presence of at least one antioxidant and/or chelator), compared to a chromatography resin exposed to gamma radiation in the absence of at least one alcohol (and optionally at least one antioxidant and/or chelator) indicates that at least one alcohol (and optionally an antioxidant and/or chelator) was present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin upon gamma irradiation treatment. Exemplary methods for determining the binding capacity of a chromatography 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 in chromatography resin

This document provides methods for reducing bioburden in a chromatography resin. Such methods include the step of exposing a container comprising a composition comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol (for example, any of the exemplary compositions comprising a chromatography resin and a liquid including at least one alcohol described herein), exposure to a dose of gamma radiation sufficient to reduce the bioburden in the container and chromatography resin, wherein at least one alcohol is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation ( or during it).

Methods of reducing bioburden in a chromatography resin are also provided which comprise exposing a container comprising a composition comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after /during exposure to gamma radiation (for example, any of the compositions comprising a chromatography resin and a liquid including at least one alcohol described herein), exposure to gamma radiation with an intensity of from about 0.1 kGy/hour to about 6 kGy/hour (for example, from about 0.1 kGy/hour to about 5.5 kGy/hour, from about 0.1 kGy/hour to about 5.0 kGy/hour, from about 0.1 kGy/hour hour to about 4.5 kGy/hour, from about 0.1 kGy/hour to about 4.0 kGy/hour, from about 0.1 kGy/hour to about 3.5 kGy/hour, from about 0.1 kGy /hour to about 3.0 kGy/hour, from about 0.1 kGy/hour to about 2.5 kGy/hour, from about 0.1 kGy/hour to about 2.0 kGy/hour, from about 0.1 kGy/hour to about 1.5 kGy/hour, from about 0.1 kGy/hour to about 1.0 kGy/hour, from about 0.5 kGy/hour to about 6 kGy/hour, from about 0.5 kGy /hour to about 5.5 kGy/hour, from about 0.5 kGy/hour to about 5.0 kGy/hour, from about 0.5 kGy/hour to about 4.5 kGy/hour, from about 0.5 kGy/hour to about 4.0 kGy/hour, from about 0.5 kGy/hour to about 3.5 kGy/hour, from about 0.5 kGy/hour to about 3.0 kGy/hour, from about 0. 5 kGy/hour to about 2.5 kGy/hour, from about 0.5 kGy/hour to about 2.0 kGy/hour), and/or at a temperature of from about 4°C to about 25°C (for example , 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), providing a dose of gamma radiation sufficient to reduce the bioburden in the container and chromatography resin.

Some embodiments of any such methods may include, before and/or after the exposure step, storing a container or composition comprising a chromatography resin and a liquid including at least one alcohol (e.g., any of the containers or any of the compositions comprising the resin for chromatography and a liquid comprising at least one alcohol described herein) for a period of time ranging from about 1 hour to about 1 year, from about 1 hour to about 11 months, from about 1 hour to about 10 months, from about 1 hour to about 9 months, from about 1 hour to about 8 months, from about 1 hour to about 7 months, from about 1 hour to about 6 months, from about 1 hour to about 5 months, from about 1 hour to about about 4 months, from about 1 hour to about 3 months, from about 1 hour to about 2 months, from about 1 hour to about 1 month, from about 1 hour to about 2 weeks, from about 1 hour to about 1 week, from about 1 hour to about 5 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 12 hours, about 1 hour to about 6 hours,

from about 6 hours to about 1 year, from about 6 hours to about 11 months, from about 6 hours to about 10 months, from about 6 hours to about 9 months, from about 6 hours to about 8 months, from about 6 hours to about 7 months, from about 6 hours to about 6 months, from about 6 hours to about 5 months, from about 6 hours to about 4 months, from about 6 hours to about 3 months, from about 6 hours to about 2 months, from approximately 6 hours to approximately 1 month, approximately 6 hours to approximately 2 weeks, approximately 6 hours to approximately 1 week, approximately 6 hours to approximately 5 days, approximately 6 hours to approximately 2 days, approximately 6 hours to approximately 1 day, from about 6 hours to about 12 hours, from about 12 hours to about 1 year, from about 12 hours to about 11 months, from about 12 hours to about 10 months, from about 12 hours to about 9 months, from about 12 hours to approximately 8 months, approximately 12 hours to approximately 7 months, approximately 12 hours to approximately 6 months, approximately 12 hours to approximately 5 months, approximately 12 hours to approximately 4 months, approximately 12 hours to approximately 3 months months, from about 12 hours to about 2 months, from about 12 hours to about 1 month, from about 12 hours to about 2 weeks, from about 12 hours to about 1 week, from about 12 hours to about 5 days, from about 12 hours to approximately 2 days, approximately 12 hours to approximately 1 day, approximately 1 day to approximately 1 year, approximately 1 day to approximately 11 months, approximately 1 day to approximately 10 months, approximately 1 day to approximately 9 months , from about 1 day to about 8 months, from about 1 day to about 7 months, from about 1 day to about 6 months, from about 1 day to about 5 months, from about 1 day to about 4 months, from about 1 day up to about 3 months, from about 1 day to about 2 months, from about 1 day to about 1 month, from about 1 day to about 2 weeks, from about 1 day to about 1 week, from about 1 day to about 5 days, from about 1 day to about 2 days, from about 2 days to about 1 year, from about 2 days to about 11 months, from about 2 days to about 10 months, from about 2 days to about 9 months, from about 2 days to about 8 months, from about 2 days to about 7 months, from about 2 days to about 6 months, from about 2 days to about 5 months, from about 2 days to about 4 months, from about 2 days to about 3 months, from approximately 2 days to approximately 2 months, approximately 2 days to approximately 1 month, approximately 2 days to approximately 2 weeks, approximately 2 days to approximately 1 week, approximately 2 days to approximately 5 days, approximately 5 days to approximately 1 year, from approximately 5 days to approximately 11 months, from approximately 5 days to approximately 10 months, from approximately 5 days to approximately 9 months, from approximately 5 days to approximately 8 months, from approximately 5 days to approximately 7 months, from approximately 5 days to approximately 6 months, approximately 5 days to approximately 5 months, approximately 5 days to approximately 4 months, approximately 5 days to approximately 3 months, approximately 5 days to approximately 2 months, approximately 5 days to approximately 1 month months, from about 5 days to about 2 weeks, from about 5 days to about 1 week, from about 1 week to about 1 year, from about 1 week to about 11 months, from about 1 week to about 10 months, from about 1 weeks to approximately 9 months, approximately 1 week to approximately 8 months, approximately 1 week to approximately 7 months, approximately 1 week to approximately 6 months, approximately 1 week to approximately 5 months, approximately 1 week to approximately 4 months , from about 1 week to about 3 months, from about 1 week to about 2 months, from about 1 week to about 1 month, from about 1 week to about 2 weeks, from about 2 weeks to about 1 year, from about 2 weeks to about 11 months, from about 2 weeks to about 10 months, from about 2 weeks to about 9 months, from about 2 weeks to about 8 months, from about 2 weeks to about 7 months, from about 2 weeks to about 6 months, from about 2 weeks to about 5 months, from about 2 weeks to about 4 months, from about 2 weeks to about 3 months, from about 2 weeks to about 2 months, from about 2 weeks to about 1 month, from about 1 month to about 1 year, from about 1 month to about 11 months, from about 1 month to about 10 months, from about 1 month to about 9 months, from about 1 month to about 8 months, from about 1 month to about 7 months, from about 1 month to about 6 months, about 1 month to about 5 months, about 1 month to about 4 months, about 1 month to about 3 months, about 1 month to about 2 months, about 2 months to about 1 year, approximately 2 months to approximately 11 months, approximately 2 months to approximately 10 months, approximately 2 months to approximately 9 months, approximately 2 months to approximately 8 months, approximately 2 months to approximately 7 months, approximately 2 months to approximately 6 months, approximately 2 months to approximately 5 months, approximately 2 months to approximately 4 months, approximately 2 months to approximately 3 months, approximately 3 months to approximately 1 year, approximately 3 months to approximately 11 months, from about 3 months to about 10 months, from about 3 months to about 9 months, from about 3 months to about 8 months, from about 3 months to about 7 months, from about 3 months to about 6 months, from about 3 months months to approximately 5 months, approximately 3 months to approximately 4 months, approximately 4 months to approximately 1 year, approximately 4 months to approximately 11 months, approximately 4 months to approximately 10 months, approximately 4 months to approximately 9 months , from about 4 months to about 8 months, from about 4 months to about 7 months, from about 4 months to about 6 months, from about 4 months to about 5 months, from about 5 months to about 1 year, from about 5 months to about 11 months, from about 5 months to about 10 months, from about 5 months to about 9 months, from about 5 months to about 8 months, from about 5 months to about 7 months, from about 5 months to about 6 months, from about 6 months to about 1 year, from about 6 months to about 11 months, from about 6 months to about 10 months, from about 6 months to about 9 months, from about 6 months to about 8 months, from about 6 months to about 7 months, from about 7 months to about 1 year, from about 7 months to about 11 months, from about 7 months to about 10 months, from about 7 months to about 9 months, from about 7 months to about 8 months, from approximately 8 months to approximately 1 year, approximately 8 months to approximately 11 months, approximately 8 months to approximately 10 months, approximately 8 months to approximately 9 months, approximately 9 months to approximately 1 year, approximately 9 months to approximately 11 months, from about 9 months to about 10 months, from about 10 months to about 1 year, from about 10 months to about 11 months, or from about 11 months to about 1 year, for example, at a temperature of from about 4°C to about 40°C, from about 4°C to about 35°C, from about 4°C to about 30°C, from about 4°C to about 28°C, from about 4°C to about 26°C, from about 4°C to about 24°C, about 4°C to about 22°C, about 4°C to about 20°C, about 4°C to about 18°C, about 4°C to about 16 °C, from about 4°C to about 14°C, from about 4°C to about 12°C, from about 4°C to about 10°C, from about 4°C to about 8°C, from about 4 °C to approximately 6°C, approximately 6°C to approximately 40°C, approximately 6°C to approximately 35°C, approximately 6°C to approximately 30°C, approximately 6°C to approximately 28° C, from about 6°C to about 26°C, from about 6°C to about 24°C, from about 6°C to about 22°C, from about 6°C to about 20°C, from about 6° C to about 18°C, about 6°C to about 16°C, about 6°C to about 14°C, about 6°C to about 12°C, about 6°C to about 10°C , from about 6°C to about 8°C, from about 8°C to about 40°C, from about 8°C to about 35°C, from about 8°C to about 30°C, from about 8°C to about 28°C, from about 8°C to about 26°C, from about 8°C to about 24°C, from about 8°C to about 22°C, from about 8°C to about 20°C, from about 8°C to about 18°C, from about 8°C to about 16°C, from about 8°C to about 14°C, from about 8°C to about 12°C, from about 8°C to about 10°C, from about 10°C to about 40°C, from about 10°C to about 35°C, from about 10°C to about 30°C, from about 10°C to about 28°C, from about 10°C to about 26°C, about 10°C to about 24°C, about 10°C to about 22°C, about 10°C to about 20°C, about 10°C to about 18°C, from about 10°C to about 16°C, from about 10°C to about 14°C, from about 10°C to about 12°C, from about 12°C to about 40°C, from about 12°C to about 35°C, about 12°C to about 30°C, about 12°C to about 28°C, about 12°C to about 26°C, about 12°C to about 24 °C, from about 12°C to about 22°C, from about 12°C to about 20°C, from about 12°C to about 18°C, from about 12°C to about 16°C, from about 12 °C to approximately 14°C, approximately 14°C to approximately 40°C, approximately 14°C to approximately 35°C, approximately 14°C to approximately 30°C, approximately 14°C to approximately 28° C, from about 14°C to about 26°C, from about 14°C to about 24°C, from about 14°C to about 22°C, from about 14°C to about 20°C, from about 14° C to about 18°C, from about 14°C to about 16°C, from about 16°C to about 40°C, from about 16°C to about 35°C, from about 16°C to about 30°C , from about 16°C to about 28°C, from about 16°C to about 26°C, from about 16°C to about 24°C, from about 16°C to about 22°C, from about 16°C to about 20°C, from about 16°C to about 18°C, from about 18°C to about 40°C, from about 18°C to about 35°C, from about 18°C to about 30°C, from about 18°C to about 28°C, from about 18°C to about 26°C, from about 18°C to about 24°C, from about 18°C to about 22°C, from about 18°C to about 20°C, from about 20°C to about 40°C, from about 20°C to about 35°C, from about 20°C to about 30°C, from about 20°C to about 28°C, from about 20°C to about 26°C, about 20°C to about 24°C, about 20°C to about 22°C, about 22°C to about 40°C, about 22°C to about 35°C, from about 22°C to about 30°C, from about 22°C to about 28°C, from about 22°C to about 26°C, from about 22°C to about 24°C, from about 24°C to about 40°C, about 24°C to about 35°C, about 24°C to about 30°C, about 24°C to about 28°C, about 24°C to about 26 °C, from about 26°C to about 40°C, from about 26°C to about 35°C, from about 26°C to about 30°C, from about 26°C to about 28°C, from about 28 °C to approximately 40°C, approximately 28°C to approximately 35°C, approximately 28°C to approximately 30°C, approximately 30°C to approximately 40°C, approximately 30°C to approximately 35° C or from about 35°C to about 40°C.

In the methods described in this paragraph, the binding capacity of the gamma irradiated chromatography resin produced by such methods is greater than the binding capacity of the gamma irradiated chromatography resin that has been exposed to gamma irradiation at an intensity greater than 6.1 kGy/hour, and/or at a temperature greater than 25°C, or both.

The chromatography resin can be exposed to gamma radiation using methods known in the art. For example, an isotope such as cobalt-60 or cesium-137 can be used as a source of gamma rays. The chromatography resin may be exposed to gamma radiation at a temperature of about −25°C to about 0°C inclusive, or from about 0°C to about 25°C inclusive. The chromatography resin may be exposed to gamma radiation at a dose ranging 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, from about 1 kGy to about 65 kGy, from about 5 kGy to about 65 kGy, from about 10 kGy to about 60 kGy, from about 10 kGy to about 55 kGy, from about 10 kGy to about 50 kGy, about 10 kGy to about 45 kGy, about 10 kGy to about 40 kGy, about 10 kGy to about 35 kGy, about 10 kGy to about 30 kGy, about 15 kGy to about 50 kGy , 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 exposed to gamma radiation at a dose sufficient to provide a sterility assurance level of the chromatography resin of about 1 x 10 ⁻⁶ or less, about 1 x 10 ⁻⁷ or less, about 10 x 10 ⁻⁸ or less , about 1 × 10 ⁻¹¹ or less or about 1 × 10 ⁻¹² or less or from about 1 × 10 ⁻⁶ to about 1 × 10 ⁻¹² , from about 1 × 10 ⁻⁶ to about 1 × 10 ⁻¹¹ , from approximately 1 × 10 ⁻⁶ to approximately 1 × 10 ⁻¹⁰ , approximately 1 × 10 ⁻⁶ to approximately 1 × 10 ⁻⁹ , approximately 1 × 10 ⁻⁶ to approximately 1 × 10 ⁻⁸ , 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 approximately 1 × 10 ⁻⁷ to approximately 1 × 10 ⁻⁹ , from approximately 1 × 10 ⁻⁷ to approximately 1 × 10 ⁻⁸ , from approximately 1 × 10 ⁻⁸ to approximately 1 × 10 ⁻¹² , from approximately 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 .

The dose of gamma radiation sufficient to reduce the bioburden in the chromatography resin can be determined using methods known in the art. For example, the bioburden level in a chromatography resin treated with a gamma radiation dose can be compared with the bioburden level in an untreated chromatography resin (e.g., a control not exposed to gamma radiation), and the reduction in bioburden level in a exposed gamma radiation in the chromatography resin compared to untreated chromatography resin indicates that the gamma radiation dose is sufficient to reduce the bioburden in the chromatography resin. Exemplary methods for determining the level of bioburden in a composition (eg, chromatography resin) are described herein. Additional methods for determining the level of bioburden in a composition (eg, chromatography resin) are known in the art.

The chromatography resin in any such method 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., from about 5 amino acids to about 100 amino acids, from about 5 amino acids to about 90 amino acids, from about 5 amino acids to about 80 amino acids, from about 5 amino acids to about 70 amino acids 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 amino acids up 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 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-methylethanolamine groups).

The container that includes the chromatography resin may be a plastic container (eg, a cylindrical tube, a sealed box or clip box, or a sealed bag). Non-limiting examples of containers used in such methods include a storage vessel or a chromatography column. For example, a composition comprising (i) a chromatography resin and (ii) a liquid including at least one alcohol (e.g., any of the exemplary compositions described herein) may be presented in a sealed container (e.g., a slurry in sealed container or chromatography resin as a packing in a sealed container (such as a chromatography column)). The container used in the methods described herein may be a disposable chromatography column. In some embodiments, the container used in the methods described herein is a disposable chromatography column housed in a blister pack. The container (e.g., 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, from about 1 ml to about 50 ml, from about 1 ml to about 40 ml, from about 1 ml to about 30 ml, or from about 1 ml to about 20 ml).

A composition comprising (i) a chromatography resin and (ii) a liquid including at least one alcohol (eg, any of the exemplary compositions described herein) included in a container may be presented as a wetted or wet solid mixture. For example, the container may include a slurry of precipitated liquid chromatography resin. In some embodiments, the container may include a chromatography resin as a packing. For example, a container including a composition comprising (i) a chromatography resin and (ii) a liquid including at least one alcohol (e.g., any of the compositions described herein) is a packed chromatography column (e.g., where the resin is placed into a liquid comprising at least one alcohol). Some embodiments include placing a composition comprising (i) a chromatography resin and (ii) a liquid comprising at least one alcohol (eg, any of the compositions described herein) in a container prior to exposure.

Any of the alcohols, antioxidants and/or chelators described herein can be used in any combination using any combination of exemplary concentrations described herein. For example, the liquid may include at least one alcohol selected from the group consisting of benzyl alcohol, cyclohexanol, isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol, propan-2-ol, propan-1-ol, butane -1-ol, pentan-1-ol, hexadecan-1-ol, 2-phenylethanol, sec-phenylethanol, 3-phenyl-1-propanol, 1-phenyl-1-propanol, 2-phenyl-1-propanol, 2 -phenyl-2-propanol, 1-phenyl-2-butanol, 2-phenyl-1-butanol, 3-phenyl-1-butanol, 4-phenyl-2-butanol, DL-1-phenyl-2-pentanol, 5 -phenyl-1-pentanol and 4-phenyl-1-butanol. In some examples, the liquid may further include at least one antioxidant (e.g., at least one antioxidant selected from the group consisting of reduced glutathione, reduced thioredoxin, reduced cysteine, carotenoid, melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin , uric acid, lipoic acid, flavonoid, phenolpropanoic acid, lidocaine, naringin, fullerene, glucose, mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl and dimethylmethoxychromanol), and/or at least one a chelator (eg, at least one chelator selected from the group consisting of EDTA, DMPS, DMSA, metallothionein and deferoxamine).

Exemplary methods for determining/identifying the amount(s) of at least one alcohol, at least one antioxidant, and/or at least one chelator sufficient to reduce the loss of binding capacity of a chromatography resin upon treatment with a dose of gamma radiation, sufficient to reduce the bioburden in the composition are described herein. Additional methods for determining/identifying the amount(s) of at least one antioxidant and/or chelator sufficient to reduce the loss of binding capacity of a chromatography resin when treated with a dose of gamma radiation sufficient to reduce the bioburden in the composition are known in the art technology.

Also provided herein is a reduced bioburden chromatography resin prepared by any of the methods described herein (eg, a reduced bioburden chromatography resin presented in a storage container, e.g., an airtight storage container). A reduced bioburden chromatography resin produced using any of the methods described herein may have a sterility assurance level of about 1 x 10 ^-6 or less, about 1 x 10 ^-7 or less, about 10 x 10 ^-8 or less, about 1 × 10 ⁻¹¹ or less, or about 1 × 10 ⁻¹² or less, or from about 1 × 10 ⁻⁶ to about 1 × 10 ⁻¹² , from about 1 × 10 ⁻⁶ to about 1 × 10 ⁻¹¹ , from approximately 1 × 10 ⁻⁶ to approximately 1 × 10 ⁻¹⁰ , from approximately 1 × 10 ⁻⁶ to approximately 1 × 10 ⁻⁹ , from approximately 1 × 10 ⁻⁶ to approximately 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 . The reduced bioburden chromatography resin produced by any of the methods described herein may have a binding capacity of at least 74% (e.g., at least 76%, at least 78%, at least 80% , at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, according 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%, from 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%, the binding capacity of the same chromatography resin that is not treated to reduce its bioburden (e.g., not exposed to gamma radiation), and the same protein is used to test the binding capacity of both the chromatography resin produced using the methods described herein and a control not processed resin for chromatography.

Methods for obtaining a packed chromatography column with reduced bioburden

Also provided herein are methods for preparing a reduced packed chromatography column which comprise providing a reduced bioburden chromatography resin prepared by any of the methods described herein and packing the chromatography resin into a reduced bioburden column under aseptic or septic conditions. reduced bioburden. In some embodiments, a packed chromatography column with reduced bioburden can be prepared by subjecting the column to include a chromatography resin as a packing and a liquid including at least one alcohol (e.g., any of the exemplary liquids described herein, e.g., optionally further comprising at least one antioxidant and/or chelator) to exposure to a dose of gamma radiation sufficient to reduce the bioburden of the column and the chromatography resin as a packing, wherein the at least one alcohol is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin as a packing nozzles after exposure to a dose of gamma radiation.

Also provided are packed bioburden reduced chromatography column(s) obtained using the methods described herein. Any of the packed reduced bioburden chromatography columns produced using the methods described herein may have a sterility assurance level of about 1 x 10 ⁻⁶ or less, about 1 x 10 ⁻⁷ or less, about 10 x 10 ⁻⁸ or less, about 1 × 10 ⁻¹¹ or less, or about 1 × 10 ⁻¹² or less, or from about 1 × 10 ⁻⁶ to about 1 × 10 ⁻¹² , from about 1 × 10 ⁻⁶ to about 1 × 10 ⁻¹¹ , from approximately 1 × 10 ⁻⁶ to approximately 1 × 10 ⁻¹⁰ , from approximately 1 × 10 ⁻⁶ to approximately 1 × 10 ⁻⁹ , from approximately 1 × 10 ⁻⁶ to approximately 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 packed reduced bioburden chromatography columns produced by the methods described herein may contain at least one chromatography resin selected from the group consisting of an anion exchange chromatography resin, a cation exchange chromatography resin, an affinity chromatography resin ( for example, any of the affinity chromatography resins described herein or known in the art), hydrophilic interaction chromatography resins, and size exclusion chromatography resins. For example, any of the reduced bioburden packed chromatography columns described herein may contain an affinity chromatography resin including a protein ligand (eg, protein A). The packed reduced bioburden chromatography column described herein may contain an anion exchange chromatography resin (eg, an anion exchange chromatography resin including N-benzyl-N-methylethanolamine groups).

Methods for performing chromatography with reduced bioburden

The methods described herein involve the use of a packed reduced bioburden chromatography column provided herein, and the methods described herein involve the use of one or two MCCSs that include at least one packed reduced bioburden chromatography column provided in this document. The gamma irradiated chromatography resin may be any type of resin described herein (or any type of chromatography resin known in the art).

A packed chromatography column with reduced bioburden can be prepared using any of the methods described herein. For example, a packed chromatography column with reduced bioburden can be prepared by packing the chromatography column with a composition comprising chromatography resin(s) and a liquid including at least one alcohol (e.g., any of such compositions described herein) and subjecting the packed column to exposure to gamma radiation (for example, using any of the exposure modes and conditions described in this document). In other examples, a packed chromatography column with reduced bioburden can be prepared by subjecting a container including a chromatography resin and a liquid including at least one alcohol (e.g., any of the exemplary liquids described herein, for example, optionally further including at least one an antioxidant and/or at least one chelator), exposure to a dose of gamma radiation and loading the chromatography column with the resulting reduced bioburden chromatography resin. In such methods, the chromatography resin (present in the container during exposure to gamma radiation) can be suspended in the container and the chromatography column is filled in a bioburden-reduced fume hood. In other methods, a chromatography resin present in a container with a liquid comprising at least an alcohol (e.g., any of the liquids described herein, e.g., optionally further comprising at least one antioxidant and/or at least one chelator ), can be exposed to gamma irradiation as a wetted or wet solid mixture in a container, and a slurry 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 for filling a chromatographic column in a fume hood with reduced bioburden. In some such examples, the chromatography column may be subjected to bioburden reduction treatments (eg, autoclaving, gamma irradiation, or ethylene oxide) prior to packing.

A packed, reduced bioburden chromatography column used in any of the methods described herein may have a Sterility Assurance Level (SAL) of about 1 x 10 ⁻³ to about 1 x 10 ⁻¹² to about 1 x 10 ⁻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 and processes described herein can be carried out using one or more reduced bioburden buffers. As can be understood from the prior art, the reduced bioburden buffer can be any type of buffer used in a chromatography cycle (eg, a buffer used at any of the stages in the chromatography cycle or in any of the individual operations described herein). Exemplary methods for reducing bioburden in a buffer include filtration (0.2 μm pore size filtration), autoclaving, and exposure to gamma radiation. Additional methods for reducing the bioburden of a buffer are known in the art. The reduced bioburden buffer may have a sterility assurance level of about 1 x 10 ^-3 to about 1 x 10 ^-12 , about 1 x 10 ^-4 to about 1 x 10 ^-12 , 1 x 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 approximately 1 × 10 ⁻⁶ to approximately 1 × 10 ⁻⁸ , inclusive.

Recombinant therapeutic proteins

The recombinant protein described herein may be a recombinant therapeutic protein. Non-limiting examples of recombinant therapeutic proteins that can be produced using the methods provided herein include immunoglobulins (including immunoglobulins containing light and heavy chains, antibodies or antibody fragments (e.g., any of the antibody fragments described herein), enzymes (for example, galactosidase (for example, alpha-galactosidase), Myozyme® or Cerezyme®), proteins (for example, human erythropoietin, tumor necrosis factor (TNF) or interferon alpha or beta) or immunogenic or antigenic proteins or protein fragments (for example, proteins for use in a vaccine).The recombinant therapeutic protein may be an engineered antigen binding polypeptide that includes the backbone of at least one multifunctional recombinant protein (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 proteins that are antibodies include panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab , apolizumab, atinumab, tocilizumab, basiliximab , 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 using the methods described herein are known in the art. Additional non-limiting examples of recombinant therapeutic proteins that can be produced/purified using 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, coagulation factor IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa and alteplase.

The secreted soluble recombinant therapeutic protein can be obtained from a liquid culture medium (eg, a first and/or second liquid culture medium) by removing or otherwise physically separating the liquid culture medium from cells (eg, mammalian cells). There are many different methods known in the art for separating 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 recovered and further purified from the liquid culture medium using a variety of biochemical techniques, including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography, anion exchange chromatography or hydrophobic interaction chromatography, or any combinations thereof) and/or filtration (eg, molecular weight cutoff filtration).

Chromatography cycle

As is generally known in the art, the steps of a chromatography cycle may vary depending on the chromatography resin, the buffers used to conduct each step in the cycle, and the biophysical characteristics of the target recombinant protein (eg, recombinant therapeutic protein). For example, an affinity chromatography column may include the steps of loading the affinity chromatography column with a fluid including the target recombinant protein, washing the column to remove unwanted biological material (e.g., contaminating proteins and/or small molecules), eluting the target recombinant protein bound to the column, and re-equilibrating the column. A chromatography cycle using a cation and/or anion exchange chromatography column, where the target recombinant protein is bound to the chromatography resin in a loading step, may include the steps of loading the column with a fluid including the target protein, washing the column to remove unwanted biological material, eluting the target recombinant protein bound to the column and re-equilibrating the column. In other examples, a chromatography run using a cation and/or anion exchange chromatography column, where the unwanted biological material is bound to the chromatography resin during a loading step while the target recombinant protein is not bound, may include the steps of loading the column with a fluid comprising the target protein , collecting the target recombinant protein in a continuous flow and re-equilibrating the column. As is generally known in the art, any of the individual steps in a chromatography cycle may include one buffer or more buffers (eg, two or more buffers), and one or more of any of the individual steps in the chromatography cycle may include a buffer gradient. These methods may adopt any combination of various well-known aspects of a particular chromatography run in any combination, for example, different chromatography resin(s), flow rate(s), buffer(s), column dead volume value(s), column bed volume value(s), buffer volume value(s) used in each step, volume value(s) of fluid comprising the target protein, and the amount and type of buffer(s) used in each step.

Methods for Performing Column Chromatography with Reduced Bioburden Provided herein are methods for performing chromatography with reduced bioburden. Such methods involve providing a packed reduced bioburden chromatography column obtained using any of the methods described herein and performing column chromatography using the packed reduced bioburden chromatography column. The packed reduced bioburden chromatography column may contain at least one of any of the chromatography resins described herein in any combination. For example, the chromatography resin present in a packed reduced bioburden chromatography column may be an affinity chromatography 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 exemplary internal volume values described herein. The packed reduced bioburden chromatography column may have any shape (eg, cylindrical, near-cylindrical, or elliptical) described herein or known in the art. Column chromatography performed using such methods 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, bioburden-reduced 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 chromatography cycle described herein or known in the art. For example, the at least one chromatography cycle may comprise the steps of capturing the recombinant protein by contacting a chromatography resin with a liquid comprising the recombinant protein; washing the chromatography resin by contacting the chromatography resin with a wash buffer, eluting the recombinant protein by contacting the chromatography resin with the elution buffer, and reconstituting the chromatography resin by contacting the chromatography resin with the 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 fed-batch culture) or a diluted liquid culture medium (eg, a culture medium diluted in a buffer).

Column chromatography can be performed using a closed and integrated system (eg, any of the exemplary closed and integrated systems described 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 generally known in the art, a buffer with reduced bioburden can be obtained using a number of different methods (eg, obtained by filtration, autoclaving or heat treatment).

Column chromatography may include two or more (for example, 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 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 for a period of time 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, according to 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 reduced bioburden packed column chromatography resin has a binding capacity of about 75% to about 100% (e.g., about 76% to about 98%, about 76% to about 96%, about 76% to about 94%, about 76% to about 92%, about 76% to about 90%, about 78% to about 100%, about 78% to about 98%, about 78% to about 96 %, 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%, from about 86% to about 96%, from about 86% to about 94%, from about 86% to about 92%, from about 88% to about 100%, from 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 treatment (using the same protein to test the binding ability of both resins) (eg , when assessed immediately after exposure to gamma radiation).

Integrated closed or essentially closed and continuous processes for the production of recombinant protein

Provided herein are integrated closed or essentially closed and continuous methods for producing purified recombinant protein (eg, a recombinant therapeutic protein). Such methods involve providing a liquid culture medium comprising a recombinant protein (eg, a recombinant therapeutic protein) that is substantially free of cells.

Some methods involve the continuous supply of a liquid culture medium to a multicolumn chromatography system (MCCS) that includes at least one packed bioburden-reduced chromatography column provided herein, such methods using a bioburden-reducing buffer, being integrated, and they are carried out continuously from the liquid culture medium to the eluate leaving the MCCS, which is a purified recombinant protein (eg, a therapeutic protein drug).

Some methods include continuously feeding a liquid culture medium to a first MCCS (MCCS1), capturing the recombinant protein from the liquid culture medium using the MCCS1, obtaining an eluate leaving the MCCS1 that includes the recombinant protein, and continuously feeding the eluate to a second MCCS (MCCS2), and continuously feeding the recombinant protein from the eluate into MCCS2, and subsequently eluting the recombinant protein to thereby obtain a purified recombinant protein, wherein at least one column in MCCS1 and/or MCCS2 is a packed reduced bioburden chromatography column as provided herein, wherein The methods use a buffer with reduced bioburden, are integrated, and are carried out continuously from liquid culture medium to 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 provided herein. Some embodiments further include the step of forming the purified recombinant protein into a pharmaceutical composition.

The methods described herein provide continuous and rapid production of purified recombinant protein from a liquid culture medium containing the recombinant protein. For example, the time that elapses from supplying a liquid culture medium comprising a therapeutic protein to the MCCS or MCCS1 until the recombinant protein is eluted 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 approximately 40 hours, approximately 4 hours to approximately 35 hours, approximately 4 hours to approximately 30 hours, approximately 4 hours to approximately 28 hours, approximately 4 hours to approximately 26 hours, approximately 4 hours to approximately 24 hours 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 approximately 12 hours, approximately 6 hours to approximately 12 hours, approximately 8 hours to approximately 12 hours, approximately 6 hours to approximately 20 hours, approximately 6 hours to approximately 18 hours, approximately 6 hours to approximately 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 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 time that elapses from supplying a liquid culture medium comprising the recombinant protein to the MCCS or MCCS1 until the recombinant protein is eluted from the MCCS or MCCS2, respectively, is, for example, greater than about 4 hours and less than about 40 hours, inclusive. for example, more than about 4 hours and less than about 39 hours, about 38 hours, about 37 hours, about 36 hours, about 35 hours, about 34 hours, about 33 hours, about 32 hours, about 31 hours, about 30 hours, 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 such 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 exemplary methods do not use a retention step (eg, do not use a reservoir (eg, a buffer tank) in the entire process). In other cases, a maximum of 1, 2, 3, 4 or 5 tanks (eg buffer tanks) may be used in the entire process. In any of the methods described herein, a maximum of 1, 2, 3, 4 or 5 tanks (e.g., buffer tanks) may be used throughout the process, wherein each buffer tank holds the recombinant protein for only a total time period of for example, from 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 (eg, buffer 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 reservoir(s) (e.g., buffer container(s)) used (in any of the methods described herein) to contain the fluid prior to its delivery to the MCCS or MCCS1 may( may) be characterized by a capacity of, for example, from 1 ml to about 100% inclusive, for example, from 1 ml to about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30 %, about 20%, about 10%, or about 5%, inclusive, relative to the loading volume of the first MCCS or MCCS1 column. Reservoir(s) (e.g., buffer tank(s)) may be used to hold the eluate leaving MCCS1 before it enters MCCS2, and it(hey) has a capacity of, for example, from 1 ml to about 100% inclusive, for example from 1 ml to about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or 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, without limitation, in any combination in the methods provided herein. Illustrative aspects of the provided methods are described below; however, one skilled in the art will appreciate that additional steps can be added to the methods described herein, and other materials can be used to carry out any of the steps of the methods described herein.

Liquid culture medium

Liquid culture medium that includes a recombinant protein (eg, a recombinant therapeutic protein), essentially free of cells, can be obtained from any source. For example, the liquid culture medium may be obtained from a recombinant cell culture (eg, a recombinant bacterial, yeast, or mammalian cell culture). The liquid culture medium can be obtained from a fed-batch cell culture (e.g., mammalian cells) (e.g., from a fed-batch bioreactor comprising a culture of mammalian cells that secretes the recombinant protein) or a perfusion cell culture (e.g., mammalian cells) (e.g. , from a perfusion bioreactor containing a culture of mammalian cells that secrete the recombinant protein). The liquid culture medium may also be a clarified liquid culture medium from a culture of bacterial or yeast cells that secrete the recombinant protein.

The liquid culture medium obtained from the recombinant cell culture can be filtered or clarified to provide a liquid culture medium substantially free of cells and/or viruses. Methods for filtering or clarifying a liquid culture medium to remove cells are known in the art (eg, 0.2 μm pore size filtration or variable tangential flow (ATF ^™ ) filtration). Recombinant cells can also be removed from the liquid culture medium by using centrifugation and removal of the supernatant, which is a liquid culture medium essentially free of cells, or by allowing the cells to settle to the gravity bottom of a container (e.g., a bioreactor) containing the liquid culture medium, and removing the liquid culture medium (liquid culture medium essentially free of cells) that is located away from the pelleted recombinant cells.

The liquid culture medium can be obtained from a culture of recombinant cells (eg, recombinant bacterial, 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 described herein may further include the step of culturing recombinant cells (eg, recombinant bacterial, yeast, or mammalian cells) producing the recombinant protein (eg, 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 consisting of a liquid culture medium not containing animal components, a liquid culture medium not containing blood serum, a liquid culture medium containing blood serum, a liquid culture medium having a defined chemical composition, and a liquid culture medium protein-free environment. In any of the methods described herein, the liquid culture medium obtained from the culture can be diluted by adding a second fluid (eg, buffer) before it is supplied to the MCCS or MCCS1.

Liquid culture medium comprising the recombinant protein, substantially free of cells, can be stored (e.g., at a temperature lower than about 15°C (e.g., lower than about 10°C, lower than about 4°C, lower than about 0°C , lower than about −20°C, lower than about −50°C, lower than about −70°C, or lower than 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 introducing the liquid culture medium into the MCCS or MCCS1. Alternatively, in In some examples, the liquid culture medium is supplied to the MCCS or MCCS1 directly from the bioreactor (eg, supplied to the MCCS or MCCS1 directly from the bioreactor after a filtration or clarification step).

Multi-column chromatography systems

The methods described herein involve 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 comprise two or more chromatography columns, two or more chromatography membranes, or a combination of at least one chromatography column and at least one chromatography membrane. In non-limiting examples, an MCCS (e.g., MCCS, MCCS1, and/or MCCS2 in any of the methods herein) may comprise 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 chromatography membrane. Without limitation, one skilled in the art will be able to envision additional examples of combinations of chromatography columns and/or chromatography membranes for use in MCCS (eg, MCCS, MCCS1 and/or MCCS2 in any of the methods described herein). The individual chromatography columns and/or chromatography membranes present in an MCCS may be identical (e.g., have the same shape, volume, resin, capture mechanism, and distinct operation) or may be different (e.g., have one or more of different shape, volume, resin, gripping mechanism and/or separate operation). In the individual chromatography column(s) and/or chromatography membrane(s) present in the MCCS (e.g., MCCS, MCCS1, and/or MCCS2 in any of the methods described herein), you can carry out the same separate operation (for example, a separate capture, cleaning or fine cleaning operation) or different separate operations (for example, different separate operations selected, for example, from the group including capture, cleaning, fine cleaning, virus inactivation, concentration control ions and/or pH of the fluid comprising the recombinant protein, and filtration). For example, in the example methods described herein, at least one chromatography column and/or chromatography membrane in the MCCS or MCCS1 performs a separate capture step for the recombinant protein.

One or more chromatography columns that may be present in the MCCS (e.g., present in MCCS, MCCS1 and/or MCCS2) may have a resin volume of, for example, about 1 ml to about 2 ml, about 5 ml, about 10 ml, approximately 15 ml, approximately 20 ml, approximately 25 ml, approximately 30 ml, approximately 35 ml, approximately 40 ml, approximately 45 ml, approximately 50 ml, approximately 55 ml, approximately 60 ml, approximately 65 ml, approximately 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml, or about 100 ml inclusive. The one or more chromatography columns that may be present in the MCCS (e.g., present in 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, about 2 ml to about 70 ml, about 2 ml to about 60 ml, about 2 ml to about 50 ml, about 5 ml to about 50 ml, about 2 ml to about 50 ml 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 an MCCS (eg, MCCS, MCCS1 and/or MCCS2) used in any of the methods described 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 approximately 15 ml/minute, approximately 0.5 ml/minute to approximately 10 ml/minute, approximately 0.5 ml/minute to approximately 14 ml/minute, approximately 1.0 ml/minute to approximately 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 the same oval cylinder in shape.

One or more chromatographic membranes that may be present in the MCCS (e.g., present in MCCS, MCCS1 and/or MCCS2) may have a bed volume of, for example, 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, from about 2 ml to about 15 ml, from about 1 ml to about 10 ml, or from about 2 ml to about 10 ml).

One or more may be used (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 during use of MCCS, MCCS1 and/or MCCS2 in any of the methods described herein. As is known in the art, one or more types of reduced bioburden buffers used in MCCS, MCCS1 and/or MCCS2 in the methods described herein will depend on the resin present in the chromatography column(s) and/or in the chromatographic membrane(s) in MCCS, MCCS1 and/or MCCS2, on the biophysical properties of the recombinant protein and the individual operation (e.g., from any of the illustrative individual operations described herein) that is carried out using the specific(s) chromatographic column(s) and/or chromatographic membranes in MCCS, MCCS1 and/or MCCS2. One skilled in the art will also be able to determine the amount and type of buffer used during application of MCCS, MCCS1 and/or MCCS2 in any of the methods described herein (eg, discussed in more detail below). For example, the volume and type(s) of buffer used during use of MCCS, MCCS1, and/or MCCS2 in any of the methods described herein can be selected to optimize one or more of the following in the purified recombinant protein (e.g., drug recombinant protein formulation): overall yield of recombinant protein, activity of the recombinant protein, level of purity of the recombinant protein, and removal of biological contaminants from the fluid containing the recombinant protein (e.g., liquid culture medium) (e.g., ensuring the 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 may, for example, comprise two or more chromatography columns (eg, three columns or four columns) that can be switched to provide continuous elution of the recombinant protein from two or more chromatography columns. The PCCS may comprise two or more chromatography columns, two or more chromatography membranes, or at least one chromatography column and at least one chromatography membrane. The column operation (cycle) typically consists of loading, washing, elution, and regeneration steps. In PCCS, multiple columns are used to perform the same steps separately and continuously in a cyclic manner. Since the columns operate in series, the effluent and wash liquid from one column is captured by the other column. This unique feature of PCCS allows the resin to be loaded to values close to its static binding capacity, rather than the dynamic binding capacity values typical in batch chromatography. As a result of continuous cycling and elution, the fluid 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 Applications 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 feed breakthrough and the second associated with column saturation. Determining when column switching operations should occur can occur by monitoring the concentration of the recombinant protein (eg, tracking via UV tracking) in the eluate from each chromatography column present in the PCCS. For example, column switching can be detected using any PAT tool that provides on-line measurement of recombinant protein concentration with feedback control. The PAT tool provides real-time, in-line measurement of recombinant protein concentration with closed-loop control. As is known in the art, column switches can also be designed depending on the time or amount of fluid (eg, buffer) passing through one or more chromatography columns and/or chromatography membranes in MCCS, MCCS1 and/or MCCS2.

In PCCS, the retention time (RT) of the recombinant protein in each chromatography column and/or chromatography membrane present in the PCCS can be reduced without increasing the size of the column/membrane, since leakage from the first column/membrane can be captured on another column/membrane in the PCCS. A continuous process system can be designed to process the liquid culture medium at any perfusion rate (D) by varying the column/membrane volume (V) and RT using the equation: V=D × RT.

One or more separate operations that can be performed by MCCS or MCC1 and/or MCCS2 used in the methods described herein include, for example, capture of the recombinant protein, inactivation of viruses present in the fluid comprising the recombinant protein, purification of the recombinant protein , finely purifying the recombinant protein, retaining the fluid comprising the recombinant protein (e.g., using any of the exemplary buffer vessels described herein), filtering or removing particulate matter and/or cells from the fluid including the recombinant protein, and adjusting the concentration ions and/or pH of the fluid comprising the recombinant protein.

In some embodiments, the MCCS or MCCS1 includes at least one chromatography column and/or chromatography membrane that has a separate recombinant protein capture step. The separate 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 protein A capture mechanism, antibody or antibody fragment capture mechanism, substrate binding capture mechanism, aptamer-binding capture mechanism, tag-binding capture mechanism (e.g., tag-based capture mechanism). poly-His tags) and the mechanism of uptake via cofactor binding. Capture can also be carried out using a resin that can be used for cation exchange or anion exchange chromatography, molecular sieve chromatography or hydrophobic interaction chromatography. Non-limiting resins that can be used to capture recombinant protein are described herein. Additional examples of resins that can be used to capture recombinant protein are known in the art.

A separate step for inactivating viruses present in a fluid comprising a recombinant protein can be performed using MCCS, MCCS1 and/or MCCS2 (for example, which includes, for example, a chromatography column, a chromatography membrane or a retention vessel suitable for incubating the fluid comprising the recombinant protein at a pH of from about 3.0 to 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, about 3.5 to about 3.8, or about 3.75) over a period of time of at least 30 minutes (e.g., a period of time of from about 30 minutes to 1.5 hour, a period of time ranging from about 30 minutes to 1.25 hour, a period of time ranging from approximately 0.75 hour to 1.25 hour, or a period of time ranging from approximately 1 hour)).

A separate recombinant protein purification step can be performed using one or more MCCS (eg, MCCS, MCCS1 and/or MCCS2), which include, for example, a chromatography column or chromatography membrane that contains a resin, such as one that uses a capture system. Non-limiting examples of capture mechanisms include protein A capture mechanism, antibody or antibody fragment capture mechanism, substrate binding capture mechanism, aptamer-binding capture mechanism, tag-binding capture mechanism (e.g., tag-based capture mechanism). poly-His tags) and the mechanism of uptake via cofactor binding. Purification can also be carried out using a resin that can be used for cation exchange or anion exchange chromatography, molecular sieve chromatography or hydrophobic interaction chromatography. Non-limiting resins that can be used to purify recombinant protein are described herein. Additional examples of resins that can be used to purify recombinant protein are known in the art.

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

The separate operation of holding a fluid comprising a recombinant protein can be performed using an MCCS (eg, MCCS, MCCS1 and/or MCCS2) that includes at least one reservoir (eg, a buffer tank) or at most 1, 2, 3, 4 or 5 reservoirs (eg buffer tanks) in MCCS or in MCCS1 and MCCS2 combined. For example, each of the reservoirs (e.g., buffer tanks) that may be used to perform a given individual 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). The reservoir(s) (e.g., buffer container(s)) used in the methods described herein may have a capacity of, for example, from 1 ml to about 300 ml inclusive, for example , from 1 ml to approximately 280 ml, approximately 260 ml, approximately 240 ml, approximately 220 ml, approximately 200 ml, approximately 180 ml, approximately 160 ml, approximately 140 ml, approximately 120 ml, approximately 100 ml, approximately 80 ml, approximately 60 ml, approximately 40 ml, approximately 20 ml or approximately 10 ml inclusive. Any of the reservoirs (e.g., buffer tanks) used (in any of the methods described herein) to contain the fluid before it enters the MCCS or MCCS1 may have a capacity of, for example, 1 ml to about 100% inclusive, 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, relative loading volume of the first MCCS or MCCS1 column. Any of the reservoirs (e.g., buffer vessels) used to hold the eluate leaving MCCS1 (including the recombinant protein) before it enters MCCS2 may have a capacity of, for example, from 1 ml to about 100%, inclusive. 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, based on the loading volume first column of MCCS2.

Each of the reservoirs (e.g., buffer tanks) can hold the fluid 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, the reservoir(s) (e.g., buffer tank(s)) hold(s) the recombinant protein only for 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, about 2 hours, about 1 hour, or about 30 minutes inclusive. The reservoir(s) (e.g., buffer tank(s)) may be used to both contain and cool (e.g., less than 25°C, less than 15°C, or less than 10°C) the fluid , including recombinant protein. The reservoir may be of any shape, including a circular cylinder, an oval cylinder, or a roughly rectangular sealed and impermeable chamber.

The individual filtration steps of the fluid comprising the recombinant protein can be performed using an MCCS (e.g., MCCS, MCCS1 and/or MCCS2) that includes, for example, a filter or chromatography column or chromatography membrane that contains a molecular sieve chromatography resin . As is known in the art, a wide variety of submicron filters are available in the art (e.g., a filter with a pore size of less than 1 μm, less than 0.5 μm, less than 0.3 μm, approximately 0.2 μm, less than 0.2 µm, less than 100 nm, less than 80 nm, less than 60 nm, less than 40 nm, less than 20 nm or less than 10 nm) which are suitable for removing any deposited material and/or cells (e.g. deposited unfolded protein; deposited unwanted cell proteins - host; precipitated lipids; bacteria; yeast cells; fungal cells; mycobacteria and/or mammalian cells). Filters with a pore size of approximately 0.2 μm or less than 0.2 μm are known to effectively remove bacteria from a fluid containing a recombinant protein. As is known in the art, it is also possible to employ a chromatography column or chromatography membrane comprising a molecular sieve chromatography resin in an MCCS (eg, MCCS, MCCS1 and/or MCCS2) to perform a separate filtration step of the fluid comprising the recombinant protein.

The individual steps of adjusting the ion concentration and/or pH of the fluid comprising the recombinant protein may be performed using an MCCS (e.g., MCCS, MCCS1, and/or MCCS2) that includes and utilizes a buffer adjustment reservoir (e.g., an adjustment reservoir in-line buffer) that ensures that new buffer solution is added to the fluid that includes the recombinant protein (e.g., between columns within an MCCS, MCCS1 and/or MCCS2, or after the last column in the penultimate MCCS (e.g., MCCS1) and before the fluid is introduced , including the recombinant protein, in the first column of the next MCCS (for example, MCCS2)). As can be understood from the prior art, the in-line buffer control reservoir can be of any size (e.g., greater than 100 mL) and can contain any buffer solution (e.g., a buffer solution that has one or more of an increased or decreased pH relative to the flow medium comprising the recombinant protein, an increased or decreased concentration of ions (e.g., salt) relative to the fluid including 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)).

In MCCS, MCCS1 and/or MCCS2, two or more separate operations can be performed. For example, each of MCCS, MCCS1 and/or MCCS2 can perform at least the following separate operations: capturing the recombinant protein and inactivating viruses present in the fluid including the recombinant protein; capturing the recombinant protein, inactivating viruses present in the fluid comprising the recombinant protein, and adjusting the ion concentration and/or pH of the fluid comprising the recombinant protein; recombinant protein purification and recombinant protein fine purification; purifying the recombinant protein, finely purifying the recombinant protein, and filtering the fluid including the recombinant protein, or removing sediments and/or solids from the fluid including the recombinant protein; and purifying the recombinant protein, finely purifying the recombinant protein, filtering the fluid including the recombinant protein, or removing sediments and/or solids from the fluid including the recombinant protein, and adjusting the ion concentration and/or pH of the fluid 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 can be understood from the prior art, a liquid culture medium comprising a recombinant protein can be continuously supplied to the MCCS or MCCS1 using a number of different methods. For example, the liquid culture medium can be actively pumped into the MCCS or MCCS1, or the liquid culture medium can be fed into the MCCS or MCCS1 using gravity. The liquid culture medium may be stored in a reservoir (e.g., a holding vessel) before being supplied to the MCCS or MCCS1, or the liquid culture medium may be actively pumped from a bioreactor comprising a cell culture (e.g., mammalian cells that secrete the 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 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 about 15 .0 ml/minute). Liquid culture medium comprising the recombinant protein can be obtained from any of the illustrative sources described herein or known in the art.

Some examples further provide the optional step of filtering the liquid culture medium before it is supplied to the MCCS or MCCS1. Any of the exemplary methods for filtering a liquid culture medium or fluid including a recombinant protein described herein, or any filtration methods known in the art, can be used to filter a liquid culture medium including a recombinant protein before it is submitted to the MCCS or MCCS1 .

In the methods described herein, capture of recombinant protein from liquid culture medium is carried out using MCCS or MCCS1. As can be understood from the prior art, in order to achieve capture of a recombinant protein, at least one chromatography column or at least one chromatography membrane in MCCS or MCCS1 must contain a resin that utilizes a capture mechanism (e.g., any of the exemplary capture mechanisms described in herein), or contain a resin suitable for performing 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 capture mechanism or an antigen capture mechanism (wherein the capture antigen is specifically recognized by the recombinant antibody or antibody fragment). If the recombinant protein is an enzyme, the capture mechanism 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, then a protein, metal chelate, or antibody (or antibody fragment) that specifically binds to a tag present in a recombinant enzyme. Non-limiting resins that can be used to capture recombinant protein are described 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 mechanism is Mab Select SuRe ^™ resin (GE Healthcare, Piscataway, NJ), JSR LifeSciences Amsphere ProA JWT203 (Sunnyvale, CA), and Kaneka KanCap A (Osaka , Japan).

Exemplary non-limiting sizes and shapes of chromatography column(s) or chromatography membrane(s) present in MCCS or MCCS1 that can be used to capture recombinant protein are described herein. The liquid culture medium that is supplied to the 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 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 for a particular 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 approximately 4 minutes or from approximately 1 minute to approximately 4 minutes).

As can be understood from the prior art, in order to capture a recombinant protein using the chromatographic column(s) or chromatographic membrane(s) present in MCCS or MCCS1, it is necessary to carry out successive chromatographic steps consisting of loading, washing, elution and regeneration of the chromatography column(s) or chromatography membrane(s) present in the MCCS or MCCS1. Any of the exemplary flow rates, buffer volume, and/or length of time allocated to each sequential chromatography step described herein may be applied to one or more of such various sequential chromatographic steps (e.g., one or more sequential chromatographic loading, washing, , elution and regeneration of the chromatography column(s) or chromatography membrane(s) present in the MCCS or MCCS1, which is used to capture the recombinant protein). Non-limiting values for the flow rate, buffer volume and/or length of time allocated for each successive chromatographic step that can be used for capture in the chromatography column(s) and/or chromatography membrane(s) in MCCS or MCCS1 ( for example in PCCS or PCCS1) are provided below. In addition, exemplary 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 chromatography membrane including a resin with which a separate capture operation can be performed (e.g., any of the exemplary capture resins described herein) can load the liquid culture medium comprising the recombinant protein using any of the loading rates (feed rates) described above. In some examples, one chromatography column or one chromatography membrane including a resin suitable for performing a separate capture operation is loaded in a time of, for example, from about 10 minutes to about 90 minutes (for example, from about 15 minutes to about 90 minutes, from about 20 minutes to 80 minutes, about 30 minutes to 80 minutes, about 40 minutes to about 80 minutes, about 50 minutes to about 80 minutes, and about 60 minutes to 80 minutes). In some examples, where MCCS or MCCS1 includes at least two chromatography columns connected in series that contain a resin suitable for performing a separate capture operation, the time required to load two chromatography columns connected in 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 that contains a resin suitable for performing a separate capture step, the at least one chromatography column or chromatography membrane is washed with at least one wash buffer. As can be understood from the prior art, at least one (e.g., two, three, or four) wash buffer is designed to elute all or most of the proteins other than the recombinant protein from the at least one chromatography column or chromatography membrane without disturbing the this 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 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). The volume of wash buffer used (e.g., the cumulative total volume of wash buffers used if more than one wash buffer is used) can be, for example, from about 1×column volume (CV) to about 15×CV (e.g., from about 1× CV to approximately 14×CV, approximately 1×CV to approximately 13×CV, approximately 1×CV to approximately 12×CV, approximately 1×CV to approximately 11×CV, approximately 2×CV to approximately 11×CV , from about 3xCV to about 11xCV, from about 4xCV to about 11xCV, from about 5xCV to about 11xCV, or from about 5xCV to about 10xCV). The total 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, which contains a resin suitable for performing a separate capture step, the recombinant protein is eluted from the at least one chromatography column or chromatography membrane by passing elution buffer through the at least one a chromatography column or chromatography membrane in MCCS or MCCS1, which contains a resin suitable for performing a separate capture operation. The elution buffer can be passed through at least one chromatography column or chromatography membrane that contains a resin suitable for performing a separate capture step 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 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 mL/minute, about 0.5 mL/minute to about 14 mL/minute, about 1.0 mL/minute to about 25.0 mL/minute, or 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 at least one chromatography column or chromatography membrane comprising a resin suitable for the individual purification step may be, for example, from about 1×column volume (CV) to about 15×CV (e.g., from about 1×CV to about 14×CV, from about 1×CV to about 13×CV, from about 1×CV to about 12×CV, from about 1×CV to about 11×CV, from about 2×CV to about 11×CV, from about 3×CV to about 11×CV, from about 4×CV to about 11×CV, from about 5×CV to about 11×CV, or from about 5×CV to approximately 10×CV). The total elution time may be, for example, from about 2 minutes to about 3 hours (for example, 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 such methods will depend on the capture mechanism and/or recombinant protein. For example, the elution buffer may provide 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 suitable for the separate capture operation. Examples of such elution buffers for each exemplary capture mechanism described herein are well known in the art.

After the recombinant protein has been eluted from the at least one chromatography column or chromatography membrane into an MCCS or MCCS1 that contains a resin suitable for performing a separate capture operation, and before a further volume of liquid culture medium can be loaded onto the at least one chromatography column or chromatography membrane, at least one chromatography column or chromatography membrane must be equilibrated using a regeneration buffer. The regeneration buffer can be passed through at least one chromatography column or chromatography membrane that contains a resin suitable for performing a separate capture step at a flow rate of, for example, 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 minute to about 5.0 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 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 at least one chromatography column or chromatography membrane that contains a resin suitable for performing a separate capture operation may be, for example, from about 1×column volume (CV) to about 15×CV (e.g. , from about 1xCV to about 14xCV, from about 1xCV to about 13xCV, from about 1xCV to about 12xCV, from about 1xCV to about 11xCV, from about 2xCV to about 11xCV, from about 3xCV to about 11xCV, from about 2xCV to about 5xCV, from about 4xCV to about 11xCV, from about 5xCV to about 11xCV, or from approximately 5×CV to approximately 10×CV).

In some methods described herein, the MCCS or MCCS1 includes a reservoir that allows the fluid comprising the recombinant protein to be retained 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 a period of time, for example, from about 1 minute to 1.5 hours (for example, approximately 1 hour) and inactivation of viruses present in the fluid comprising the recombinant protein. An example of a reservoir that can be used for a separate virus inactivation operation is a stirrer flask (e.g., a 500 mL stirrer flask, e.g., a 500 mL stirrer flask with a programmable stirrer plate) suitable for containing a fluid comprising a recombinant protein , for a period of time of, for example, from about 1 minute to 1.5 hours, for example, before supplying the fluid comprising the recombinant protein to the MCCS2. The reservoir used for a separate virus inactivation operation may be a 500 mL stirrer flask with a programmable stirrer plate (e.g., a stirrer plate with a set program for stirring (e.g., periodically stirring) the fluid in the reservoir, e.g., every 4 hours ). Another example of a reservoir that can be used for a separate virus inactivation step is a plastic bag (for example, a 500 ml plastic bag) suitable for holding a fluid comprising a recombinant protein for a period of time, for example, from about 1 minute up to 1.5 hours, for example, before introducing the fluid comprising the recombinant protein into the MCCS2. In some examples, the fluid 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 it is fed into a reservoir, which is used to carry out a separate virus inactivation operation. As will be appreciated by one skilled in the art, a number of other means can be used to perform a separate viral inactivation step. For example, UV irradiation of a fluid containing a recombinant protein can also be used to perform a separate viral inactivation step. Non-limiting examples of reservoirs that can be used to perform a separate step to inactivate viruses present in a fluid comprising a recombinant protein are described herein.

The MCCS or MCCS1 may include a PCCS comprising four chromatography columns, wherein at least three of the four chromatography columns have a separate step for capturing the recombinant protein from the liquid culture medium (e.g., using an MCCS that contains any of the at least one chromatography column which contains a resin suitable for performing a particular gripping operation (eg, any of those described herein)). In these examples, the fourth PCC column can perform a separate step to inactivate viruses in a fluid including a recombinant protein (e.g., any of the exemplary columns described herein that can be used to provide inactivation of viruses in a fluid including a recombinant protein).

In some examples, a fluid comprising the recombinant protein is continuously eluted from MCCS1 (eg, PCCS1) and continuously fed to MCCS2 (eg, PCCS2). The percentage of resulting recombinant protein in the eluate emerging 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 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least at least 94%, at least 96% or at least 98%). The eluate leaving MCCS1 (eg, PCCS1) can be supplied to MCCS2 (eg, PCCS2) using a variety of means known in the art (eg, tubing). The eluate leaving MCCS1 (e.g., PCCS1) can be supplied to MCCS2 (e.g., PCCS2) at a flow rate of, for example, about 0.2 ml/minute to about 25 ml/minute (for example, 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 to 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 from about 1.0 ml/minute to about 15.0 ml/minute).

Some methods described herein may further include the step of adjusting the ion concentration and/or pH of the eluate leaving MCCS1 (eg, PCCS1) before it is supplied to MCCS2 (eg, PCCS2). As described herein, the ion concentration and/or pH of the eluate leaving MCCS1 (eg, PCCS1) can be adjusted (before it is supplied to MCCS2) by adding a buffer to the eluate (eg, by using an in-line buffer control reservoir). The buffer can be added to the eluate leaving MCCS1 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 described herein may further include the step of holding or storing (and optionally also cooling) the eluate leaving MCCS1 before supplying the eluate leaving MCCS1 to MCCS2. As described herein, this retention or storage step can be carried out using any of the containers (eg, storage tanks) described herein.

The methods described herein may also include the step of filtering the eluate leaving MCCS1 before feeding the eluate to MCCS2. Any of the exemplary filters or filtration methods described herein can be used to filter the eluate leaving MCCS1 before feeding the eluate to MCCS2.

Recombinant protein fine purification and purification

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

At least one chromatography column or chromatography membrane that can be used to perform a separate recombinant protein purification step may contain 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 separate purification step of the recombinant protein may contain a resin that can be used to perform anion exchange chromatography, cation exchange chromatography, molecular sieve chromatography or hydrophobic interaction chromatography (for example, any of the exemplary resins for performing anion exchange chromatography, cation exchange chromatography, molecular sieve chromatography, or hydrophobic interaction chromatography described herein or known in the art).

The size, shape and volume of at least one chromatography column or chromatography membrane that can be used to carry out a separate purification step of a recombinant protein, and/or the size and shape of at least one chromatography membrane that can be used to carry out a separate purification step of a recombinant protein , may be any combination of exemplary sizes, shapes, and volumes of chromatography columns or chromatography membranes described herein. One skilled in the art will appreciate that the step of purifying or purifying a recombinant protein may, for example, include the steps of loading, washing, eluting, and equilibrating at least one chromatography column or chromatography membrane used to perform a separate purification or purification step of the recombinant protein. . Typically, the elution buffer exiting the chromatography column or chromatography membrane used for a separate purification step includes the recombinant protein. Typically, the loading and/or wash buffer exiting the chromatography column or chromatography membrane used for a separate 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 separate recombinant protein purification step may include a volume of, 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, from about 2.0 ml to about 60 ml, from about 2.0 ml to about 40 ml, from about 5.0 ml to about 40 ml, from about 2.0 ml to about 30 ml, from about 5.0 ml to about 30 ml, or from about 2.0 ml to about 25 ml). The flow rate of the fluid comprising the recombinant protein when loaded onto at least one chromatography column or at least one chromatography column that can be used to perform a separate purification step of the recombinant protein can 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 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 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 fluid loaded onto at least one chromatography column or chromatography membrane that can be used to perform a separate purification step of the recombinant protein 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 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 separate purification step 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 that is used to perform a separate purification step 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 for a separate purification step of the recombinant protein, the at least one chromatography column or chromatography membrane is washed with at least one wash buffer. As can be understood from the prior art, at least one (for example, 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 disturbing the interaction of the recombinant protein with resin, or otherwise eluting 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 to 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 cumulative total volume of wash buffers used if more than one wash buffer is used) can be, for example, from about 1×column volume (CV) to about 15×CV (e.g., from about 1× CV to approximately 14×CV, approximately 1×CV to approximately 13×CV, approximately 1×CV to approximately 12×CV, approximately 1×CV to approximately 11×CV, approximately 2×CV to approximately 11×CV, from about 3xCV to about 11xCV, from about 4xCV to about 11xCV, from about 2.5xCV to about 5.0xCV, from about 5xCV to about 11xCV, or from about 5×CV to approximately 10×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 separate purification step of the recombinant protein, 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 separate operation of recombinant protein purification. The elution buffer may be passed through at least one chromatography column or chromatography membrane, which may be used to perform a separate recombinant protein purification step, at a flow rate of 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 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, about 0.5 ml/minute to about 14 ml/minute, about 1.0 ml/minute to about 25.0 ml/minute, or 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 separate recombinant protein purification step may be, for example, from about 1×column volume (CV) to about 25xCV (e.g., about 1xCV to about 20xCV, about 15xCV to about 25xCV, about 1xCV to about 14xCV, about 1xCV to about 13xCV, from about 1xCV to about 12xCV, from about 1xCV to about 11xCV, from about 2xCV to about 11xCV, from about 3xCV to about 11xCV, from about 4xCV to about 11xCV, about 5xCV to about 11xCV, or about 5xCV to about 10xCV). The total elution time may be, for example, from about 2 minutes to about 3 hours (for example, 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 such methods will depend on the resin and/or the biophysical properties of the recombinant protein. For example, the elution buffer may provide 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 exemplary capture mechanisms described herein are well known in the art.

After the recombinant protein has been eluted from the at least one chromatography column or chromatography membrane used to perform a separate purification step of the recombinant protein, and before a further volume of fluid comprising the recombinant protein can be loaded onto the at least one chromatography column or chromatography membrane. membrane, at least one 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 for a separate recombinant protein purification step 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, about 0.5 ml/minute to about 14 ml/minute, about 1.0 ml/minute to about 25.0 ml/minute, 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 at least one chromatography column or chromatography membrane that contains a resin that can be used for a separate recombinant protein purification step can be, for example, from about 1×column volume (CV) to about 15 ×CV (e.g., from about 1×CV to about 14×CV, from about 1×CV to about 13×CV, from about 1×CV to about 12×CV, from about 1×CV to about 11×CV, from approximately 2×CV to approximately 11×CV, approximately 3×CV to approximately 11×CV, approximately 2×CV to approximately 5×CV, approximately 2.5×CV to approximately 7.5×CV, approximately 4 ×CV to about 11×CV, from about 5×CV to about 11×CV, or from about 5×CV to about 10×CV). The concentration of recombinant protein in the eluate emerging from at least one chromatography column or chromatography membrane used to perform a separate recombinant protein purification step 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, about 0.1 mg/mL to about 50 mg/mL, about 0.1 mg/mL to about 40 mg/mL, about 2.5 mg/mL to about 7.5 mg/ml, from about 0.1 mg/ml to about 30 mg/ml, from about 0.1 mg/ml to about 20 mg/ml, between 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 approximately 10 mg/ml recombinant protein).

At least one chromatography column or chromatography membrane that can be used to perform a separate purification step of the recombinant protein may contain a resin that can be used to perform cation exchange, anion exchange, or molecular sieve chromatography. As can be understood from the prior art, fine purification of a recombinant protein using at least one chromatography column or chromatography membrane, which can be used to carry out a separate fine purification operation of the recombinant protein, may include, for example, the steps of loading, running and regenerating at least one chromatographic column or chromatographic membrane, which can be used to carry out a separate operation for fine purification of the recombinant protein. For example, if loading, running, 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 that is used to perform a separate purification step of the recombinant protein, and the recombinant protein is eluted from at least one at least one chromatography column or chromatography membrane in the loading and running steps, and a regeneration step is used to remove any impurities from the at least one chromatography column or chromatography membrane before additional fluid comprising the recombinant protein can be loaded into the at least one one chromatography column or chromatography membrane. Exemplary buffer flow rates and volumes to be used in each of the loading, run and regeneration stages are described below.

The size, shape and volume of at least one chromatographic column or chromatographic membrane that can be used to carry out a separate fine purification step of a recombinant protein, and/or the size and shape of at least one chromatographic membrane that can be used to carry out a separate fine purification step of a recombinant protein protein may be any combination of exemplary sizes, shapes, and volumes of chromatography columns or chromatography membranes described herein. For example, the size of at least one chromatography column or chromatography membrane that can be used to perform a separate purification step of a recombinant protein may include a volume of, 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, from about 5.0 ml to about 30 ml, from about 0.5 ml to about 25 ml, from about 0.2 ml to about 10 ml, or from about 0.2 ml to about 5 ml) . The flow rate of the fluid comprising the recombinant protein when loaded onto at least one chromatography column or chromatography membrane that can be used for a separate fine purification step of the recombinant protein 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, from 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 the fluid comprising the recombinant protein loaded onto at least one chromatography column or chromatography membrane that can be used for a separate purification step of the recombinant protein 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 that is used to perform a separate fine operation may be a cation exchange resin (eg, Sartobind® Q resin, Sartorius, Göttingen, Germany).

Following the loading step, a running step is carried out (eg, the running buffer is passed through at least one chromatography membrane 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 running 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). 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, 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 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 running buffer used may be, for example, from about 1 x column volume (CV) to about 100 x CV (e.g., from about 1 x CV to about 90 x CV, from about 1 x CV to about 80 x CV, from about 1xCV to about 70xCV, about 1xCV to about 60xCV, about 1xCV to about 50xCV, about 1xCV to about 40xCV, about 1xCV to about 30 ×CV, from about 1×CV to about 20×CV, from about 1×CV to about 15×CV, from about 5×CV to about 20×CV, from about 5×CV to about 30×CV, from about 1 ×CV to approximately 14×CV, approximately 1×CV to approximately 13×CV, approximately 1×CV to approximately 12×CV, approximately 1×CV to approximately 11×CV, approximately 2×CV to approximately 11× CV, from about 3xCV to about 11xCV, from about 4xCV to about 11xCV, from about 2.5xCV to about 5.0xCV, from about 5xCV to about 11xCV, or from approximately 5×CV to approximately 10×CV). The total run 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 column effluent from the loading step and the run 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 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 distillation step and before the next volume of fluid comprising the recombinant protein can be loaded onto at least one chromatography column or chromatography membrane that can be used to perform a separate fine purification operation, at least one chromatography column or chromatography membrane must be regenerated using a regeneration buffer. The regeneration buffer can be passed through at least one chromatography column or chromatography membrane, which can be used for a separate purification step of the recombinant protein, at a flow rate of, for example, 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 to 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 may be used for a separate fine purification operation may be, for example, from about 1×column volume (CV) to about 500×CV (e.g. from about 1xCV to about 450xCV, from about 1xCV to about 400xCV, from about 1xCV to about 350xCV, from about 1xCV to about 300xCV, from about 1xCV to approximately 250×CV, approximately 1×CV to approximately 200×CV, approximately 1×CV to approximately 150×CV, approximately 1×CV to approximately 100×CV, approximately 1×CV to approximately 90×CV, approximately about 1xCV to about 80xCV, about 1xCV to about 70xCV, about 1xCV to about 60xCV, about 1xCV to about 50xCV, about 1xCV to about 40 ×CV, from about 1×CV to about 30×CV, from about 1×CV to about 20×CV, from about 1×CV to about 15×CV, from about 5×CV to about 20×CV, from about 5 ×CV to approximately 30×CV, approximately 1×CV to approximately 14×CV, approximately 1×CV to approximately 13×CV, approximately 1×CV to approximately 12×CV, approximately 1×CV to approximately 11× CV, from about 2xCV to about 11xCV, from about 3xCV to about 11xCV, from about 4xCV to about 11xCV, from about 2.5xCV to about 5.0xCV, from about 5xCV to about 11xCV or from about 5xCV to about 10xCV).

In other examples, one or more chromatography columns and/or chromatography membranes used to perform a separate fine purification operation contain a resin that selectively binds or retains impurities present in the fluid comprising the recombinant protein, and instead of regenerating one or more columns and/or or membranes, one or more columns and/or membranes are replaced (e.g., replaced with substantially similar column(s) and/or membrane(s)) once the binding capacity of the resin in one or more columns and/or membranes reaches its limit or is essentially approaching the limit.

In some examples of such methods described herein, MCCS2 provides a PCCS containing three chromatography columns and one chromatography membrane, for example, wherein the three chromatography columns in the PCCS carry out a separate purification step for the recombinant protein (e.g., using at least one chromatography (chromatographic) column(s), which can be used to perform a separate protein purification step), and in a chromatographic membrane in the PCCS, a separate fine purification step of the recombinant protein is carried out. In these examples, the chromatography membrane in the PCCS that can be used to perform a separate purification step of the recombinant protein can be any of the exemplary chromatography membranes described herein that can be used to perform a separate purification step of the recombinant protein. Any of the column switching methods described 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 exiting the three chromatography columns in the PCCS before applying the eluate to the chromatography membrane in the PCCS. As described herein, the ion concentration and/or pH of the eluate effluent from the three chromatography columns in the PCCS can be adjusted (before it is applied to the chromatography membrane in the PCCS in this example) by adding a buffer to the eluate effluent from the three chromatography columns in the PCCS (eg by using a reservoir to control the buffer in the flow). The buffer may be added to the eluate at a 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).

Such examples may further include the step of retaining or storing the eluate exiting the three chromatography columns in a PCCS, in this example, before applying the eluate to a chromatography membrane (a chromatography membrane that can be used for a separate purification step of the recombinant protein). As described herein, this retention or storage step can be carried out using any of the containers (eg, storage tanks) described herein.

Such examples may also include the step of filtering the chromatography membrane effluent in an exemplary PCCS system (a chromatography membrane effluent that can be used to perform a separate recombinant protein purification step). Any of the exemplary filters or filtration methods described herein can be used to filter the eluate effluent from a chromatography membrane in this exemplary PCCS (eluate effluent from a chromatography membrane that can be used to perform a separate purification step of a recombinant protein).

One skilled in the art will appreciate that purified recombinant protein can be periodically eluted from MCCS or MCCS2 using any of the methods described herein. For example, any of the methods described herein can elute the purified recombinant protein within a period of, 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, about 1 minute to about 2 hours, about 1 minute or about 1.5 hours, about 1 minute to about 1 hour, or about 1 minute to about 30 minutes) at a frequency of, for example, about 1 minute to about 6 hours (e.g., about 1 minute to about 5 hours, about 1 minute to about 4 hours, about 1 minute to about 3 hours, about 1 minute to 2 hours, about 1 minute to about 1 hour or from about 1 minute to 30 minutes), depending on, for example, the chromatographic column(s) and/or chromatographic membrane(s) used in the MCCS or MCCS1 and MCCS2.

Cultivation methods

Some of the methods described herein further include the step of culturing cells (e.g., recombinant mammalian cells) that secrete the recombinant protein in a bioreactor (e.g., a perfusion bioreactor or a fed-batch bioreactor) that contains a liquid culture medium, wherein a certain volume A liquid culture medium that is substantially free of cells (eg, mammalian cells) is continuously or periodically removed from a bioreactor (eg, a perfusion bioreactor) and supplied to the MCCS or MCCS1. The bioreactor may have a volume of, 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 up to about 5000 L, from about 500 L to about 10000 L, from about 5000 L to about 10000 L, from about 1 L to about 10000 L, from about 1 L to about 8000 L, from about 1 L to about 6000 L, from about 1 L to about 5000 L, from about 100 L to about 5000 L, from about 10 L to about 100 L, from about 10 L to about 4000 L, from about 10 L to about 3000 L, from about 10 L to approximately 2000 L or approximately 10 L to approximately 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 (for example, 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 approximately 4000 L, approximately 0.5 L to approximately 3000 L, approximately 0.5 L to approximately 2500 L, approximately 50 L to approximately 2500 L, approximately 5 L to approximately 50 L, approximately 5 L to approximately 2000 L, from about 5 L to about 1500 L, from about 5 L to about 1000 L, or from about 5 L to about 500 L). Cell culture can be carried out, for example, using a fed-batch bioreactor or a perfusion bioreactor. Non-limiting examples and various aspects of cell culture (eg, mammalian cell culture) are described below and can be used in any combination.

Cells

The cells that are cultured in some of the methods described herein may be bacterial (eg, gram-negative bacteria), yeast (eg, Saccharomyces cerevisiae , Pichia pastoris , Hansenula polymorpha , Kluyveromyces lactis , Schizosaccharomyces pombe , Yarrowia lipolytica , or Arxula adeninivorans ) cells. ) or mammalian cells. The mammalian cell may be a cell that grows in suspension or an adherent cell. Non-limiting examples of mammalian cells that can be cultured in any of the methods described 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 cells), and canine kidney epithelial cells (Cocker-cells). spaniel) Madin-Darby (MDCK cells). In some examples where an adherent cell is cultured, the culture may also include a number of microcarriers (eg, microcarriers that contain one or more pores). Additional mammalian cells that can be cultured in any of the methods described 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) that encodes a recombinant protein (eg, a recombinant protein). Non-limiting examples of recombinant nucleic acids that encode exemplary recombinant proteins are described below, as are recombinant proteins that can be produced using the methods described herein. In some cases, the mammalian cell that is cultured in a bioreactor (eg, in any of the bioreactors described herein) is derived from a larger culture.

The nucleic acid encoding the recombinant protein can be introduced into a mammalian cell using a variety of methods known in the fields of molecular biology and molecular genetics. Non-limiting examples include transfection (eg, lipofection), transduction (eg, infection with a lentivirus, adenovirus, or retrovirus), and electroporation. In some cases, the nucleic acid that encodes the recombinant protein is not stably integrated into the chromosome of the mammalian cell (transient transfection), while in other cases the nucleic acid is integrated into the chromosome. Alternatively or in addition, the nucleic acid encoding the recombinant protein may be located on a plasmid and/or on a mammalian artificial chromosome (eg, a human artificial chromosome). Alternatively or in addition, the nucleic acid can be introduced into a cell using a viral vector (eg, lentiviral, retroviral or adenoviral vectors). 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 optionally also 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 that encodes a signal peptide for secretion at the N- or C-terminus of the recombinant protein, which is cleaved by an enzyme present in the mammalian cell and then released into the extracellular environment (for example, the first and/or a second liquid culture medium).

Culture media

Liquid culture media are known in the art. Liquid culture media (eg, first and/or second tissue culture media) can be supplemented with mammalian serum (eg, fetal bovine serum and bovine serum) and/or growth hormone or growth factor (eg, insulin, transferrin and epidermal growth factor) . Alternatively or in addition, the liquid culture medium (e.g., the first and/or second liquid culture medium) may be a liquid culture medium with a defined chemical composition, a liquid culture medium that does not contain animal components, a liquid culture medium that does not contain blood serum , or liquid culture medium containing blood serum. Non-limiting examples of chemically defined liquid culture media, animal-free liquid 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 (eg, a carbohydrate such as glucose), essential amino acids (eg, a set of twenty essential amino acids plus cysteine), vitamins and/or other organic compounds required in low concentrations, free fatty acids and /or microelements. Liquid culture media (for example, the first and/or second liquid culture medium) can be supplemented as necessary, for example, with a hormone or mammalian growth factor (for example, insulin, transferrin or epidermal growth factor), salts and buffers (for example, calcium, magnesium and phosphates), nucleosides and bases (eg, adenosine, thymidine and hypoxanthine), protein and tissue hydrolysates, and/or any combination of such additives.

A wide variety of different liquid culture media that can be used to culture cells (eg, mammalian cells) in any of the methods described herein are known in the art. Media components that can also be used in the methods of the present invention include, but are not limited to, chemically defined hydrolysates (CDs), such as CD peptone, CD polypeptides (of 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 described herein may be the same type of media or different media.

Additional Properties of Illustrative Bioreactors

The interior surface of any of the bioreactors described herein may have at least one coating (for example, at least one coating of gelatin, collagen, poly-L-ornithine, polystyrene and laminin) and, as is known in the art, one or more holes for bubbling O ₂ , CO ₂ and N ₂ into the liquid culture medium, as well as a device for mixing the liquid culture medium. The bioreactor can incubate the cell culture in a controlled humidified atmosphere (e.g., greater than 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% humidity, or humidity 100%). The bioreactor may also be equipped with a mechanical device that is configured to remove a volume of liquid culture medium from the bioreactor, and optionally a filter within the mechanical device that allows cells to be removed from the liquid culture medium during the process of transferring the liquid culture medium from the bioreactor (e.g. ATF system or cell filtration system described in US Provisional Patent Application No. 61/878,502).

Temperature

The mammalian cell culture step can be performed at a temperature of from about 31ºC to about 40ºC. Those skilled in the art will appreciate that during the culture step, the temperature may be changed at specific time(s), such as every hour or every 24 hours. For example, the temperature may be changed or shifted (e.g., increased or decreased) for 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 the initial seeding of the cell into the bioreactor (eg, mammalian cells). For example, the temperature may be shifted upward (e.g., changed to a value no greater than or equal to approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0, 8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or value not greater than or equal to approximately 20°C). For example, the temperature may be adjusted downward (e.g., changed to a value no greater than or equal to approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0, 8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or value not greater than or equal to approximately 20ºC).

_CO2

The culture step described herein may further comprise exposing the liquid culture medium in the bioreactor to an atmosphere comprising no more than or about 15% CO ₂ (e.g., no more than or about 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 not more than or approximately 1% CO ₂ ).

Perfusion bioreactor

The culture step described herein can be carried out using a perfusion bioreactor. Cultivating a cell (e.g., a mammalian cell) in a perfusion bioreactor involves removing from the bioreactor a first volume of a first liquid culture medium (e.g., characterized by any concentration of mammalian cells, e.g., a first volume of a first liquid culture medium that is substantially free of cells) and adding to the first liquid a culture medium of a second volume of a second liquid culture medium. Removal and addition can be done simultaneously or sequentially, or by combining them. Additionally, removal and addition may be performed continuously (e.g., at a rate at which a volume of 0.1% to 800% is removed or 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% up 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 given period of time (e.g. over a period of time of 24 hours, over a discrete period of time from about 1 hour to about 24 hours, or over a discrete period of time greater than 24 hours)) or periodically (for example, once every three days, once every two days , once a day, twice a day, three times a day, four times a day, or five times a day), or any combination thereof. If performed intermittently, the volume that is removed or replaced (e.g., over a period of time of approximately 24 hours, over a discrete time period of from about 1 hour to about 24 hours, or over a discrete time period of more than 24 hours ), may be, for example, from 0.1% 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 volume of the bioreactor or the 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 for each 24 hour period of time (or, alternatively, a discrete time period of from about 1 hour to about 24 hours). hours, or a discrete period of time greater than 24 hours) during all or part of the cultivation period. As is known in the art, 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) can 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) may be approximately the same or may be different.

Alternatively, the volumes removed and added may be varied (eg, incrementally increased) over each 24 hour period of time (or, alternatively, a discrete time period of 1 hour to about 24 hours, or a discrete time period, more 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 time period of 24 hours (or, alternatively, a discrete time period of from about 1 hour to more than 24 hours, or a discrete time period, greater than 24 hours) may be increased (e.g., gradually or in steps) during the culture period from a volume that is from 0.5% to about 20% of the volume of the bioreactor or the volume of the first liquid culture medium to a value of from about 25% to about 150% of the volume of the bioreactor 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 may be removed, for example, by a mechanical system that may cause the first volume of the first liquid culture medium to be removed from the bioreactor (e.g., the first volume of the first liquid culture medium that is substantially free of cells from the bioreactor). Alternatively or in addition, the first volume of the first liquid culture medium can be removed by allowing the first volume of the first liquid culture medium to drain or gravity flow through a sterile membrane with a molecular weight cutoff that excludes the passage of a cell (eg, a mammalian cell).

A second volume of the second liquid culture medium can be added to the first liquid culture medium automatically, for example, using a perfusion pump.

In some cases, the removal of the first volume of the first liquid culture medium (e.g., the first volume of the first liquid culture medium that is substantially free of mammalian cells) and the addition of the second volume of the second liquid culture medium to the first liquid culture medium are not performed for at least 1 hour (for example, for 2 hours, for 3 hours, for 4 hours, for 5 hours, for 6 hours, for 7 hours, for 8 hours, for 9 hours, for 10 hours, for 12 hours, for 14 hours, for 16 hours, for 18 hours, for 24 hours, for 36 hours, for 48 hours, for 72 hours, for 96 hours or 96 hours later) after sowing in mammalian cell bioreactor.

Fed-batch bioreactor

The cultivation step described herein can be carried out using a fed-batch bioreactor. Cultivating a cell in a fed-batch bioreactor involves, during most of the culture period, adding (eg, batchwise or continuously adding) a second volume of a second liquid culture medium to the first liquid culture medium. The addition of the second liquid culture medium can be done continuously (e.g., at a rate at which the volume increases by an amount of 0.1% to 300% (e.g., 1% to 250%, 1% to 100%, 100% up 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 the volume of the first liquid culture medium during any a specified period of time (e.g., over a period of time of 24 hours, over a discrete time period of from about 1 hour to about 24 hours, or over a discrete time period of more than 24 hours)), or periodically (for example, once every three days, once every two days, once a day, twice a day, three times a day, four times a day or five times a day), or any combination thereof. If carried out periodically, the volume that is added (for example, over a period of time of approximately 24 hours, over a discrete period of time of from about 1 hour to about 24 hours, or over a discrete period of time of more than 24 hours), may 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. In some cases, the second volume of added second liquid culture medium may be maintained at approximately the same level for each 24 hour period of time (or, alternatively, a discrete time period of from about 1 hour to about 24 hours, or a discrete time period more than 24 hours) during the entire cultivation period or part thereof. As is known in the art, the rate at which the second volume of the second liquid culture medium is added (volume/unit time) may vary during all or part of the culture period. For example, the volume of the second liquid culture medium added may be varied (e.g., gradually increased) over each 24 hour period of time (or, alternatively, a discrete time period of 1 hour to about 24 hours, or a discrete time period, more than 24 hours) during the culture period. For example, the volume of second liquid culture medium added for each time period of 24 hours (or, alternatively, a discrete time period of from about 1 hour to more than 24 hours, or a discrete time period of more than 24 hours) during the culture period, can be increased (e.g., gradually or by step increases) during the culture period from a volume that is from 0.5% to about 20% of the volume of the bioreactor or the volume of the first liquid culture medium to a value that is from about 25% to approximately 150% of the bioreactor volume 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 automatically, 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., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours). hours, for 7 hours, for 8 hours, for 9 hours, for 10 hours, for 12 hours, for 14 hours, for 16 hours, for 18 hours, for 24 hours, for 36 hours, for 48 hours, for 72 hours, for 96 hours or more than 96 hours) after seeding a mammalian cell into the bioreactor. Fed-batch cell culture medium is typically collected at the end of the culture period and used in any of the methods described herein, however, fed-batch cell culture medium can also be collected in one or multiple time points during the culture period and apply in any of the methods described herein.

It will be appreciated by those skilled in the art that any of a number of culture parameters (e.g., containers, volume values, rate or frequency of changing culture volumes, stirring frequency values, temperature values, media values, and CO ₂ concentration values) can be used to implement these methods. any combination. In addition, any mammalian cells described herein or known in the art can be used to produce the recombinant protein.

Illustrative Biological Production Systems

Examples of biological production systems used to perform the methods described herein, which include MCCS or MCCS1 and MCCS2, are described in US Provisional Patent Application Nos. 61/775,060 and 61/856,390 (incorporated by reference). In such exemplary systems, at least one (eg, at least two, three, four, five or six) packed reduced bioburden chromatography column provided herein is present in the MCCS or in MCCS1 and/or MCCS2. For example, the entire system may contain a total of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty packed chromatography columns with reduced bioburden provided for in this document. For example, MCCS, MCCS1 and/or MCCS2 may contain (or each may contain) one, two, three, four, five, six, seven, eight, nine or ten packed reduced bioburden chromatography columns provided herein.

For example, applicable systems may comprise an MCCS1 that includes an inlet and an MCCS2 that includes an outlet, or an MCCS that includes an inlet and an outlet. In some embodiments, MCCS1 and MCCS2 are in fluid communication with each other. Such systems can also be configured such that fluid can enter an inlet, pass through MCCS1 and MCCS2, and exit the production system through an outlet. Such systems provide continuous and time-efficient production of a therapeutic drug from a liquid culture medium. For example, the time elapsed between supply of a fluid (eg, liquid culture medium) comprising a therapeutic protein to MCCS1 and elution of the purified recombinant protein (eg, therapeutic protein drug) from the outlet of MCCS2 may be, for example, from about 4 hours up to and including approximately 48 hours.

Some exemplary systems do not contain buffer capacity. In other cases, the system may contain a maximum of 1, 2, 3, 4, or 5 buffer reservoirs throughout the system (e.g., where each buffer reservoir provides retention of the therapeutic protein for a total period of time, e.g., from about 5 minutes to about 6 hours inclusive). The buffer container(s) may have a capacity ranging from 1 ml to approximately 300 ml inclusive. Any buffer tanks located in the system such that fluid enters the buffer 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 buffer tanks located in the system such that fluid enters the buffer tanks before entering MCCS2 (and after exiting MCCS1) may have a capacity of, for example, 1 ml up to and including approximately 100% of the loading volume of the first column of MCCS2 .

Structures and Properties of the Supplementary Illustrative System

The MCCS or MCCS1 may include an inlet through which a liquid (eg, a liquid culture medium that is substantially free of cells) may flow into the MCCS or MCCS1, respectively. The inlet opening may have any structure known in the art for such purposes. It may include, for example, a screw thread, ribbing, or seal that allows a fluid conduit to be inserted such that upon insertion of the fluid conduit into the inlet port, fluid will enter the MCCS or MCCS1 through the inlet port without significant accidental leakage of fluid from the inlet port. inlet. Non-limiting inlets that can be used in systems of the present invention are known and will be understood by those skilled in the art.

The MCCS or MCCS1 may comprise 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 exemplary MCCSs described herein, or have one or more of any of the exemplary MCCS properties (in any combination) described herein. The chromatographic column(s) and/or chromatographic membrane(s) present in the MCCS or MCCS1 may be characterized by one or more of any illustrative shapes, sizes, volumes (layer volumes), and/or individual(s) operation(s) described in this document.

The chromatography column(s) and/or chromatography membrane(s) present in the MCCS or MCCS1 may contain one or more of any of the illustrative resins described herein or known in the art. For example, the resin contained in one or more chromatography columns and/or chromatography membranes present in MCCS or MCCS1 may be a resin that utilizes a capture mechanism (e.g., protein A capture mechanism, protein A capture mechanism G, capture mechanism by binding to an antibody or antibody fragment, capture mechanism by binding to a substrate, capture mechanism by binding to a cofactor, capture mechanism by binding to an aptamer and/or capture mechanism by binding to a tag). The resin contained in one or more MCCS or MCCS1 chromatography columns and/or chromatography membranes may be a cation exchange resin, an anion exchange resin, a molecular sieve chromatography 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 may be contained in one or more chromatography columns and/or chromatography membranes present in MCCS or MCCS1. The chromatography column(s) and/or chromatography membrane(s) present in the MCCS or MCCS1 may contain the same and/or different resins (e.g., any of the resins described herein or known in the art for use in recombinant protein purification).

One or more separate operations (e.g., recombinant protein capture, recombinant protein purification, recombinant protein fine purification, virus inactivation, ion concentration control, and/or pH of the fluid comprising the recombinant protein, or filtering the fluid comprising the recombinant protein). In non-limiting examples, the MCCS or MCCS1 may perform the separate steps of capturing a recombinant protein from a fluid (eg, a liquid culture medium) and inactivating viruses present in the fluid including the recombinant protein. The MCCS or MCCS1 may perform any combination of two or more separate operations described herein or known in the art.

The chromatography column(s) and/or chromatography membrane(s) 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 comprise one or more (eg, two, three, four or five) pumps (eg, automatic, eg, automatic peristaltic pumps). Column switching events can be triggered upon detection of a level of recombinant protein detected by UV absorption corresponding to a determined level of recombinant protein in the fluid passing through the MCCS or MCCS1 (e.g., the influent and/or eluate exiting one or more chromatography columns and/or chromatographic membranes in MCCS or MCCS1), a specific volume of liquid (eg buffer) or a specific elapsed time. Column switching generally means a mechanism by which at least two different chromatography columns and/or chromatography membranes in MCCS or MCCS1 (for example, two or more different chromatography columns and/or chromatography membranes present in MCCS1 or MCCS2) are allowed to pass through another step (eg, equilibration, loading, elution or washing) at substantially the same 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 comprise four chromatography columns, with the first three columns having a separate capture step for recombinant protein from a fluid (e.g., liquid culture medium ), and in the fourth column of PCCS, a separate operation of inactivating viruses in a fluid containing the recombinant protein is carried out. In PCCS, which is MCCS or MCCS1, a column switching mechanism can be used. The PCC system can use a modified ÄKTA system (GE Healthcare, Piscataway, NJ), which can operate, for example, up to four, five, six, seven or eight columns or more.

The MCCS or MCCS1 may be equipped with one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) UV tracking devices, 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 may 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 absorption, fluid volume or elapsed time) and which influences (initiates) column switching events.

MCCS or MCCS1 may further comprise 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) in-stream buffer control tanks and/or buffer tanks. In another example, the MCCS or MCCS1 may contain one or more (e.g., two, three, four, five, or six) buffer reservoirs that can hold fluid that cannot readily transfer to one or more chromatography columns and/or chromatography membranes in MCCS or MCCS1. The systems described herein may include one or more buffer tanks (eg, the buffer tank described herein) in MCCS, MCCS1 and/or MCCS2. Other examples of systems described in this document contain no buffer capacity in the MCCS, MCCS1 or MCCS2, or no buffer capacity in the entire system. Other examples of systems described herein contain a maximum of one, two, three, four, or five buffer tanks (eg, any of the buffer tanks described herein) throughout the system.

Second MCCS

The second MCCS (MCCS2) in exemplary systems comprises at least two chromatography columns, at least two chromatography membranes, or at least one chromatography column and at least one chromatography membrane and an outlet. MCCS2 may be any of the exemplary MCCSs described herein, or may be characterized by one or more of any of the exemplary MCCS properties (in any combination) described herein. The chromatographic column(s) and/or chromatographic membrane(s) present in MCCS2 may be characterized by one or more of any of the exemplary shapes, sizes, volumes (bed volumes), and/or distinct operations described herein document. The chromatographic column(s) and/or chromatographic membrane(s) may contain any of the exemplary resins described herein or known in the art. For example, the resin contained in one or more chromatography columns and/or chromatography membranes present in MCCS2 may be a resin that utilizes a capture mechanism (e.g., protein A capture mechanism, protein G capture mechanism, capture mechanism by binding to an antibody or antibody fragment, capture mechanism by binding to a substrate, capture mechanism by binding to a cofactor, capture mechanism by binding to a tag, and/or capture mechanism by binding to an aptamer). Useful resins include, for example, a cation exchange resin, an anion exchange resin, a molecular sieve chromatography resin, a hydrophobic interaction chromatography resin. Additional examples of resins are known in the art. The chromatography column(s) and/or chromatography membrane(s) present in MCCS2 may contain the same and/or different resins (e.g., any of the resins described herein or known in the art for use in recombinant protein purification).

The chromatography column(s) and/or chromatography membrane(s) present in MCCS2 may carry out one or more separate operations (e.g., any of the individual operations described herein, or any combination of individual operations described in this document). In non-limiting examples, MCCS2 can perform the separate steps of purifying a recombinant protein from a fluid and purifying a recombinant protein present in a fluid including the recombinant protein. In other non-limiting examples, MCCS2 may perform the separate operations of purifying a recombinant protein present in a fluid, finely purifying a recombinant protein present in a fluid, and filtering a fluid including the recombinant protein. In another example, MCCS2 may perform the separate operations of purifying the recombinant protein present in the fluid, finely purifying the recombinant protein present in the fluid, filtering the fluid including the recombinant protein, and adjusting the ion concentration and/or pH of the fluid including the recombinant protein . MCCS2 can perform any combination of two or more separate operations described herein or known in the art.

The chromatography column(s) and/or chromatography membrane(s) present in MCCS2 can be coupled or moved relative to each other using a switching mechanism (eg, a column switching mechanism). MCCS2 may also include one or more (eg, two, three, four or five) pumps (eg, automatic, eg, automatic peristaltic pumps). Column switching events can be triggered by detecting a level of recombinant protein, as detected by UV absorption, corresponding to a determined level of recombinant protein in the fluid passing through the MCCS2 (e.g., in the influent and/or eluate leaving one or more chromatography unit(s). ) column(s) and/or chromatographic membranes in MCCS2), a specific volume of liquid medium (eg buffer) or a specific elapsed time.

The MCCS2 may be a batch countercurrent chromatography system (ie PCCS2). For example, PCCS2 may comprise three columns that have a separate purification step for the recombinant protein from the fluid, and a chromatography membrane that has a separate purification step for the recombinant protein present in the fluid. For example, three columns that carry out a separate purification step for recombinant protein from a fluid may contain, for example, a cation exchange resin, and a chromatographic membrane that carries out a separate fine purification step can contain 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 operate, for example, up to 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 tracking devices, 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 may also be equipped with an operating system that uses software (eg, Unicorn-based software, GE Healthcare, Piscataway, NJ) to determine when a column switching event should occur (eg, based on UV absorbance, volume liquid or elapsed time) and which influences column switching events.

MCCS2 may further comprise 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) in-stream buffer control tanks and/or buffer tanks. In other examples, MCCS2 may contain one or more (e.g., two, three, four, five, or six) buffer tanks (e.g., any of the buffer tanks described herein) that can hold fluid that cannot readily move into one or more chromatography columns and/or chromatography membranes in MCCS2.

MCCS2 contains an outlet through which the therapeutic protein drug can exit the system. The outlet may include, for example, a screw thread, ribbing, or seal that allows the insertion of a fluid tube or vial configured to hold or store a purified recombinant protein (eg, a therapeutic protein drug). The outlet may include a surface that can be used to seal a reduced bioburden container or other such storage container at the outlet to allow a stream of purified recombinant protein (e.g., a therapeutic protein drug) to flow directly into the reduced bioburden container or storage container. Non-limiting outlets that can be used in the systems of the present invention are known and will be understood by those skilled in the art.

The systems described herein may also include a fluid conduit that is located between MCCS1 and MCCS2. Any of the fluid conduits described herein may be, for example, a pipe that is made of, for example, polyethylene, polycarbonate, or plastic. The fluid conduit located between MCCS1 and MCCS2 may further comprise one or more of the following in any combination: one or more in-stream buffer control reservoirs that are in fluid communication with the fluid conduit and are positioned such that the buffer , stored within the reservoir(s) to regulate the buffer in the flow, is added to the fluid present in the fluid tube; a buffer vessel (e.g., any of the buffer vessels described herein) that is in fluid communication with the fluid conduit and is positioned such that it can contain any excess fluid present in the fluid conduit that cannot apply to MCCS2 without delay; and one or more filters that are disposed in the fluid conduit such that they are capable of filtering fluid (eg, removing bacteria) present in the fluid conduit. Any of the in-stream buffer control 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 described herein may optionally include a fluid conduit located between the final chromatography column or chromatography membrane in the MCCS2 and the outlet. The systems described herein may further comprise one or more filters in fluid communication with a fluid conduit located between the final chromatography column or chromatography membrane in the MCCS2 and the outlet, whereby the filter can remove, for example, precipitated material, particulate matter or bacteria from the fluid present in the fluid tube located between the last chromatography column or chromatography membrane in the MCCS2 and the outlet.

Some example systems presented herein also provide a bioreactor that is in fluid communication with the MCCS or MCCS1 inlet. Any of the exemplary bioreactors described herein or known in the art can be used in the systems of the present invention.

Some example systems presented in this document also include a pumping system. The pumping system may comprise 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 pumps described herein or known in the art technique), one or more (e.g., two, three, four, or five) filters (e.g., any filters described 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 (e.g., two, three, four, or five) buffer tanks (e.g., any of the buffer tanks described herein). Some example systems provided herein further include a fluid conduit located between the pump and the inlet port of the MCCS or MCCS1 (eg, any of the exemplary fluid conduits described herein or known in the art). In some examples, such a particular fluid conduit may include one or more (e.g., two, three, or four) pumps (e.g., any pumps described herein or known in the art) and/or one or more (e.g., two, three or four) buffer tanks (e.g., any of the exemplary buffer tanks described herein), wherein such pump(s) and/or buffer tank(s) are in fluid communication with the fluid environment present in the fluid tube.

Some examples of systems described herein further include an additional fluid conduit connected to a fluid conduit located between the pump and the inlet, where one end of the additional fluid conduit is in fluid connection with the bioreactor and the other the end is in fluid connection with a fluid tube located between the pump and the inlet. Such additional fluid conduit may include a filter that is capable of removing cells from the liquid culture medium removed from the bioreactor (eg, an ATF cell retention system).

The systems provided herein provide 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 described herein may also result in an overall 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 bioburden in a chromatography resin that involve the use of a substantially dry chromatography resin

Also provided herein are methods of reducing bioburden in 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 in the container and the chromatography resin, wherein the substantially dry resin for chromatography provides a liquid comprising at least one alcohol, wherein the at least one alcohol is present in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation. Some embodiments of such methods further include, prior to step (a), drying the chromatography resin to substantially remove liquid (but not all liquid) from the chromatography resin. Drying of the chromatography resin can be accomplished using heat treatment (eg, an oven) or a desiccant. Additional methods for drying chromatography resin are known in the art.

Such methods may use any of the gamma radiation conditions and dose values described herein. For example, the gamma radiation dose may range from about 15 kGy to about 45 kGy (eg, from about 20 kGy to about 30 kGy). Such methods may use any of the containers, chromatography resins, and liquids comprising at least one alcohol described herein. For example, the container may be a storage vessel or a chromatography column. The chromatography resin in such 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 containing N-benzyl-N-methylethanolamine groups). In some examples, the chromatography resin is covalently attached to the surface of an article (eg, 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 chelator. Reduced bioburden chromatography resins produced by any of the methods described herein are also provided.

In some examples, the resulting reduced bioburden chromatography resin has a sterility assurance level (SAL) of about 1 x 10 ⁻⁸ to about 1 x 10 ⁻⁵ (e.g., a SAL of about 1 x 10 ⁻⁷ to about 1 x 10 ⁻⁶ ). The resulting reduced chromatography resin may include at least one resin selected from the group consisting of an anion exchange chromatography resin, a cation exchange chromatography resin, an affinity chromatography resin, a hydrophobic interaction chromatography resin, and a size exclusion chromatography resin. In some examples, the resulting reduced chromatography resin includes an affinity chromatography resin containing a protein ligand (eg, protein A). In some examples, the resulting reduced chromatography resin includes an anion exchange chromatography resin (eg, an anion exchange chromatography resin containing N-benzyl-N-methylethanolamine groups). Also provided are methods for preparing a packed bioburden chromatography column that comprise providing a bioburden reduced chromatography resin prepared by any of the methods described herein and packing the bioburden chromatography resin into the reduced bioburden column under aseptic conditions. Also provided are packed chromatography columns with reduced bioburden produced by any of the methods described herein.

Also provided are integrated, closed and continuous methods for producing purified recombinant protein with reduced bioburden, which include (a) providing a liquid culture medium comprising the recombinant protein that is substantially cell-free; and (b) continuously feeding a liquid culture medium into a multi-column chromatography system (MCCS) containing at least one packed reduced bioburden chromatography column obtained using any of the methods provided herein; where the method uses a reduced bioburden buffer, the method is integrated and is carried out continuously from the liquid culture medium to the eluate leaving the MCCS, which is the purified recombinant protein. Also provided are integrated, closed and continuous methods for producing purified recombinant protein with reduced bioburden, which include (a) providing a liquid culture medium comprising the recombinant protein that is substantially cell-free; (b) continuously supplying a liquid culture medium to the first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein in liquid culture medium using MCCS1; (d) obtaining an eluate leaving MCCS1, which includes the recombinant protein, and continuously feeding the eluate to a second multicolumn chromatography system (MCCS2); (e) continuously feeding the recombinant protein from the eluate into the MCCS2 and subsequently eluting the recombinant protein to thereby obtain a purified recombinant protein, wherein the method uses a buffer with reduced bioburden, the method is integrated and is carried out continuously from the liquid culture medium to the purified recombinant protein and wherein at least one column in MCCS1 and/or MCCS2 comprises a packed reduced bioburden chromatography column obtained using any of the methods provided herein. Such methods may utilize any of the exemplary aspects of the integrated, closed and continuous processes for producing purified recombinant protein with reduced bioburden described herein.

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

Also provided herein are methods for preparing a membrane, resin, coated material, chip, or cassette with reduced bioburden, which comprise subjecting a container comprising a composition comprising (i) a membrane, resin, coated material, chip, or cassette (e.g., a membrane to cellulose, agarose or sugar based, resin, coated material, chip or cassette) and (ii) a liquid comprising at least one alcohol (for example, and optionally at least one antioxidant and/or chelator), exposed to a dose of gamma radiation , sufficient to reduce the bioburden in the container and in the membrane, resin, coated material, chip or cassette, wherein the at least one alcohol is present in an amount sufficient to reduce damage to the membrane, resin, coated material, chip and cassette after exposure to a gamma dose - radiation. In some examples, the cartridge is a cartridge containing a resin (eg, any of the illustrative resins described 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 of its surfaces or a portion thereof. In some embodiments, the composition includes a membrane, a resin, a coated material, a chip or cartridge, and a liquid comprising at least an alcohol (eg, and optionally at least one antioxidant and/or at least one chelator). Any of the exemplary combinations and concentrations of alcohols, antioxidant(s) and/or chelator(s) described herein can be used in any of such methods. Any of the illustrative liquids described herein can be used in any of such methods. In some embodiments, the composition is a wetted or moistened dry material. Also provided herein is a reduced bioburden membrane, resin, coated material, chip, or cassette made using any of the methods described herein. In some examples, the container is a sealed storage container or vessel.

Also provided herein are methods for producing a membrane, resin, coated material, chip, and cassette with reduced bioburden, which comprise exposing a container comprising a substantially dry membrane, resin, coated material, chip, or cassette (e.g., a cellulose-based membrane, agarose or sugar, resin, coated material, chip or cassette) to a dose of gamma radiation sufficient to reduce the bioburden in the container and in the membrane, resin, coated material, chip or cassette. Some examples further include the step of drying the membrane, resin, coated material, chip, or cassette before 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 cartridge containing a resin (eg, any of the illustrative resins described herein or known in the art). Also provided herein is a reduced bioburden membrane, resin, coated material, chip, or cassette made using any of the methods described herein.

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

EXAMPLES

Example 1 Protective effect of alcohol on gamma-irradiated affinity chromatography resin

In the first set of experiments, MabSelect ^™ SuRe ^™ (Protein A binding chromatography resin) was irradiated in three different buffers at a dose of 40-49 kGy at a standard dose rate:

(1) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol in 50 mM sodium phosphate buffer (radiation 40-49 kGy at dose intensity >7.5 kGy/hour);

(2) 2% v/v benzyl alcohol (40-49 kGy at dose intensity >7.5 kGy/hour) and

(3) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, 2% benzyl alcohol in 50 mM sodium phosphate buffer - at higher radiation dose (40-49 kGy and higher dose intensity >7. 5 kGy/hour)

After irradiation with chromatography resins, separate chromatography columns were filled and several runs were performed using cell culture harvest material and a retention time of six minutes. Binding capacity values for breakthrough were recorded for irradiated resins and compared with those for unexposed (non-irradiated) MabSelect ^™ SuRe ^™ (protein A binding chromatography) resin.

The data obtained in this experiment are shown in Figure 1.

The data in Figure 1 demonstrates that buffer (3) provides an additive effect of the combination of both buffers (1) and (2) in maintaining binding capacity. In this case, 2% v/v. The benzyl alcohol in the buffer acts as a preservative and when combined with buffer (1) also provides some protective properties.

Table 1 shows that all measured product quality data did not differ significantly depending on the application of buffers (1)-(3), and are similar to the expected values observed for unexposed (non-irradiated) MabSelect ^™ SuRe ^™ (Protein A binding chromatography resins).

Table 1.

Buffer Number of cycles Quality data Receipt (%) HCP (ppm) Residual ProA (ppm) Unexposed to MSS 1 92 233 17 5 89 304 16 10 90 174 34 Buffer 3
SMM'H+2% BA 1 89 245 27 5 87 233 25 10 88 225 26 Buffer 1
SMM'H 1 89 274 34 5 93 273 37 10 94 245 25 Buffer 2
2% BA 1 89 310 26 5 87 294 27 10 88 287 25

These data demonstrate that irradiation of a chromatography resin in a liquid containing an alcohol (eg, benzyl alcohol) protects the resin from subsequent loss of binding capacity over multiple chromatography cycles.

Example 2: Protective effect of alcohol on gamma-irradiated Capto ^TM Adhere chromatography resin

Gamma irradiation GE Capto ^TM adhere (anion exchange chromatography resin with functional groups providing multimodality) involved irradiation in three different buffers at a dose of 28-49 kGy at standard dose rates:

(A) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, and 25 mM mannitol in 50 mM sodium phosphate buffer (28-34 kGy, at a dose rate of 2-3 kGy/hour);

(B) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol in 50 mM sodium phosphate buffer (40-49 kGy, at dose intensity >7.5 kGy/hour) and

(C) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, 2% v/v. benzyl alcohol in 50 mM sodium phosphate buffer (40-49 kGy and higher dose intensity >7.5 kGy/hour).

After irradiation with chromatography resins, separate chromatography columns were filled and several runs were performed using cell culture harvest material and a retention time of six minutes. Breakthrough binding capacity values were recorded for irradiated resins and compared to those for unexposed (non-irradiated) GE Capto ^TM adhere resin (anion exchange chromatography resin with functional groups providing multimodality).

Data from this experiment are shown in Figure 2. The data in Figure 2 demonstrates that there was no significant change in binding capacity when the resin was irradiated in the presence of benzyl alcohol, and the presence of benzyl alcohol provided an advantageous effect of increased storage time before exposure to gamma radiation.

Table 2 below shows that irradiation of the resin in the presence of benzyl alcohol does not result in any significant impact on other quality data regarding the effectiveness of the resin in protein purification.

Table 2.

Buffer Number of cycles Quality data HCP (mcg/mg) Purity (%) Test condition A
SMM'H with low dose (28-34 kGy) 1 229 92.32 10 279 90.64 18 218 91.31 Test condition B
SMM'H with high dose (40-49 kGy) 1 285 92.01 10 162 90.58 18 207 90.82 Test condition C
SMM'H+2% BA (40-49 kGy) 2 165 91.69 eleven 166 91.8 16 221 92.28

OTHER IMPLEMENTATION OPTIONS

It should be understood that while the present invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to be illustrative and does not limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims (39)

1. A method for reducing bioburden in a chromatography resin, comprising: exposing a container containing a composition comprising (i) a chromatography resin and (ii) a liquid containing benzyl alcohol and at least two antioxidants to a dose of gamma radiation sufficient to reduce the bioburden in the container and the chromatography resin, Where at least two antioxidants are selected from the group consisting of: mannitol, sodium ascorbate, histidine and methionine; And benzyl alcohol and at least two antioxidants are present in quantities sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation. 2. The method according to claim 1, where the container is a storage vessel. 3. The method according to claim 1, where the container is a chromatographic column. 4. The method according to claim 1, where the container is a filled chromatography column. 5. The method according to claim 1, where the composition is a suspension of precipitated chromatography resin. 6. Method according to any one of paragraphs. 1-5, where the concentration of benzyl alcohol in the liquid is from 0.01% v/v. up to 10% v/v 7. Method according to any one of paragraphs. 1-6, wherein the liquid further contains at least one chelator. 8. The method of claim 7, wherein the liquid contains at least one chelator in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation. 9. The method according to claim 1, where the liquid contains mannitol, sodium ascorbate, histidine and methionine. 10. The method according to claim 1, where the liquid contains (i) 30 mM to 70 mM methionine and 30 mM to 70 mM histidine; (ii) 10 mM to 50 mM methionine, 10 mM to 50 mM histidine, and 10 mM to 50 mM sodium ascorbate; or (iii) 5 mM to 45 mM sodium ascorbate, 5 mM to 45 mM methionine, 5 mM to 45 mM mannitol, and 5 mM to 45 mM histidine. 11. The method according to claim 7, where the liquid contains at least one chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium salt and 2,3-dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA ), metallothioneine and deferoxamine. 12. The method of claim 1, wherein the chromatography resin is selected from the group consisting of an anion exchange chromatography resin, a cation exchange chromatography resin, an affinity chromatography resin, a hydrophobic interaction chromatography resin, and a size exclusion chromatography resin. 13. The method of claim 12, wherein the composition comprises an affinity chromatography resin containing a protein ligand. 14. The method of claim 13, wherein the protein ligand is protein A. 15. The method according to claim 12, where the composition contains an anion exchange chromatography resin. 16. The method according to claim 15, wherein the anion exchange chromatography resin contains N-benzyl-N-methylethanolamine groups. 17. Method according to any one of paragraphs. 1-16, where the dose ranges from 15 kGy to 45 kGy. 18. The method according to claim 17, where the dose is from 20 kGy to 30 kGy. 19. The method according to claim 18, where the dose is from 23 kGy to 27 kGy. 20. Method according to any one of paragraphs. 1-19, where exposure is carried out at a temperature ranging from -25°C to 0°C inclusive. 21. Method according to any one of paragraphs. 1-19, where exposure is carried out at a temperature ranging from 0°C to 25°C inclusive. 22. A composition for a container exposed to a dose of gamma irradiation, containing (i) a chromatography resin and (ii) a liquid containing benzyl alcohol and at least two antioxidants, where at least two antioxidants are selected from the group consisting of: mannitol, sodium ascorbate, histidine and methionine, and benzyl alcohol and at least two antioxidants are present in amounts sufficient to reduce the loss of binding capacity of the chromatography resin when treated with a dose of gamma radiation sufficient to reduce the bioburden in the composition. 23. The composition according to claim 22, where the composition is a slurry of precipitated chromatography resin. 24. The composition according to claim 22 or 23, where the concentration of benzyl alcohol in the liquid is from 0.01% v/v. up to 10% v/v 25. Composition according to any one of paragraphs. 22-24, where the liquid further contains at least one chelator. 26. The composition of claim 25, wherein the liquid further contains at least one chelator in an amount sufficient to reduce the loss of binding capacity of the chromatography resin after exposure to a dose of gamma radiation. 27. The composition according to claim 22, where the liquid contains mannitol, sodium ascorbate, histidine and methionine. 28. The composition according to claim 25 or 26, where the liquid contains (i) 30 mM to 70 mM methionine and 30 mM to 70 mM histidine; (ii) 10 mM to 50 mM methionine, 10 mM to 50 mM histidine, and 10 mM to 50 mM sodium ascorbate; or (iii) 5 mM to 45 mM sodium ascorbate, 5 mM to 45 mM methionine, 5 mM to 45 mM mannitol, and 5 mM to 45 mM histidine. 29. The composition according to claim 25, where the composition contains at least one chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium salt and 2,3-dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA ), metallothioneine and deferoxamine.