RU2786078C2 - Methods for sterile chromatography and production - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to methods for chromatography, using gamma-irradiated chromatographic resin, for purification of recombinant protein. The method includes: (a) obtainment of a chromatographic column containing gamma-irradiated cation-exchanging and/or anion-exchanging chromatographic resin; (b) implementation of the first chromatography cycle through columns, where the first chromatography cycle includes reduction of a binding capacity of gamma-irradiated cation-exchanging and/or anion-exchanging chromatographic resin by subjecting gamma-irradiated cation-exchanging and/or anion-exchanging chromatographic resin to impact of a denaturing buffer containing one or more of urea, guanidine hydrochloride, and Triton™ X-100; and (c) implementation of at least one additional chromatography cycle through the column, where the specified column is subjected to impact of the specified denaturing buffer during each of at least one additional chromatography cycle, and where a flow rate, a volume, and a concentration of the denaturing buffer are selected such that there is essentially a reduction of a binding capacity lost because of gamma-irradiation of cation-exchanging and/or anion-exchanging resin. In addition, the invention relates to integrated, closed, or essentially closed, and continuous methods for the production of recombinant protein.

EFFECT: use of a denaturing buffer allows for reduction of a binding capacity of gamma-irradiated chromatographic resin lost during multiple chromatography cycles.

64 cl, 8 dwg, 2 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to US Provisional Application No. 61/928906, filed January 17, 2014, incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY

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

BACKGROUND OF THE INVENTION

Mammalian cells comprising a nucleic acid encoding a recombinant protein are often used to produce therapeutically or commercially important proteins. With today's diverse product pipelines, biotech companies are increasingly forced to develop innovative solutions for the highly flexible and cost-effective production of therapeutic protein drugs. One strategy for efficient isolation of recombinant proteins is the use of methods involving continuous chromatography (eg, using a closed system). One known limitation of continuous chromatography is the presence of contaminants in the system (eg, increased bioburden), resulting in a contaminated product, reduced product yield, and reduced flow rate (or increased pressure) in the system. For example, an increased bioburden in a system can cause the system to shut down completely.

SUMMARY OF THE INVENTION

The present invention is based at least in part on the discovery that gamma irradiation of a chromatography resin results in a reduction in the binding capacity of the resin (e.g., a steady decrease in the binding capacity of the resin over many chromatography cycles), and that the binding capacity of a gamma irradiated resin can be restored. exposing the resin to a denaturing buffer. In light of this discovery, the present invention relates to methods for performing chromatography using a gamma-irradiated chromatographic resin, including obtaining a chromatographic column, including a gamma-irradiated chromatographic resin; the implementation of the first cycle of chromatography using a column, where the cycle includes exposing the chromatographic resin to a denaturing buffer; and performing at least one additional round of column chromatography. In addition, the invention relates to integrated, closed, or essentially closed, and continuous methods for the production of a recombinant protein, including the use of at least one chromatographic column comprising a gamma-irradiated chromatographic resin, where the gamma-irradiated chromatographic resin is exposed to a denaturing buffer during each cycle of the method, and the method uses a reduced bioburden buffer. Any of the methods presented herein may be bioburden-reduced, sterile, aseptic, or completely sterile methods (as defined herein). Any of the methods presented herein may be a combination of aseptic and bioburden-reduced, sterile or completely sterile methods.

The present invention relates to methods for performing chromatography using a gamma-irradiated chromatographic resin, including: (a) obtaining a chromatographic column containing a gamma-irradiated chromatographic resin; (b) performing a first cycle of column chromatography, wherein the cycle includes exposing the chromatographic resin to a denaturing buffer; and (c) performing at least one additional round of column chromatography. In some embodiments of these methods, performing the cycles in (b) and/or (c) includes the steps of: (a) capturing the recombinant protein by exposing the chromatographic resin to a liquid containing the recombinant protein; (b) washing the chromatographic resin by exposing the chromatographic resin to a wash buffer; (c) eluting the recombinant protein by exposing the chromatographic resin to an elution buffer; and (d) regenerating the chromatographic resin by exposing the chromatographic resin to a denaturing buffer.

In some examples of any of the methods provided herein, the liquid containing the recombinant protein is a liquid culture medium. In some embodiments of any of the methods presented herein, cycles (b) and (c) are performed using a closed and integrated system. In some examples of any of the methods provided herein, the buffer is a reduced bioburden buffer (eg, a reduced bioburden buffer obtained by filtration).

In some examples of any of the methods provided herein, the denaturing buffer comprises one or more of urea, guanidine hydrochloride, and Triton ^TM X-100. In some embodiments of any of the methods provided herein, cycle (b) further comprises exposing the chromatographic resin to a wash buffer comprising about 0.5 M to about 1.5 M sodium hydroxide, after exposure to a denaturing buffer. In some embodiments that use a chromatographic affinity resin comprising a protein ligand, cycle (b) further comprises exposing the chromatographic resin to a wash buffer comprising about 1 mM to about 100 mM sodium hydroxide. In some examples of any of the methods presented herein, the column is part of a multi-column chromatography system (MCCS) (eg, a periodic countercurrent chromatography system (PCCS).

In some embodiments of any of the methods provided herein, the chromatography resin is an anion exchange chromatography resin, a cation exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography resin, an affinity chromatography resin, or any combination thereof. In some examples, the chromatographic resin is an anion exchange chromatographic resin.

In some examples of any of the methods provided herein, the chromatographic resin is treated with a dose of gamma radiation from about 10 kGy to about 40 kGy (for example, from about 15 kGy to about 35 kGy, or from about 20 kGy to about 30 kGy). In some embodiments of any of the methods provided herein, four or more (e.g., nine or more, fourteen or more, nineteen or more, twenty-four or more, twenty-nine or more, or thirty-nine or more) additional chromatography cycles are carried out. . In some examples of any of the methods provided herein, step (c) is performed over a period of at least 4 days (e.g., at least 5 days, at least 7 days, at least 14 days, or at least at least 28 days). In some examples of any of the methods provided herein, the recombinant protein is a recombinant therapeutic protein.

In addition, the invention relates to integrated, closed or substantially closed, and continuous methods for the production of purified recombinant protein, including: (a) obtaining a liquid culture medium comprising a recombinant protein, essentially free of cells; and (b) continuously feeding the liquid culture medium into a multi-column chromatography system (MCCS) comprising at least one chromatography column containing a gamma-irradiated chromatography resin, where the chromatography resin is exposed to a denaturing buffer during each cycle; where 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 from MCCS, which is a purified recombinant protein. In some embodiments of these methods, at least two different typical operations are performed by MCCS. In some examples of any of the methods presented herein, MCCS includes switching columns. In some embodiments of any of the methods provided herein, all columns in the MCCS contain a gamma irradiated chromatographic resin.

In some examples of any of the methods provided herein, MCCS performs routine recombinant protein capture and virus inactivation or routine recombinant protein capture and purification. In some embodiments of any of the methods presented herein, the MCCS is a batch countercurrent chromatography system. In some examples of any of the methods provided herein, the MCCS includes a plurality of columns for affinity chromatography, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, or hydrophobic interaction chromatography, or any combination thereof. In some embodiments of any of the methods provided herein, where the MCCS includes an affinity chromatography column, the affinity chromatography is performed using a capture mechanism selected from the group consisting of: protein A binding capture mechanism, substrate binding capture mechanism, an antibody or antibody fragment-binding capture mechanism, an aptamer-binding capture mechanism, and a cofactor-binding capture mechanism. In some examples of any of the methods provided herein, affinity chromatography is performed using a protein A binding capture mechanism and the recombinant protein is an antibody or antibody fragment. In some examples of any of the methods provided herein, the recombinant protein is a therapeutic recombinant protein. Some embodiments of any of the methods provided herein further include formulating the purified recombinant protein into a pharmaceutical composition. In some embodiments of any of the methods provided herein, the denaturing buffer comprises one or more of urea, guanidine hydrochloride, and Triton ^TM X-100. In some examples of any of the methods provided herein, the chromatographic resin is exposed to a wash buffer comprising from about 0.5 M to about 1.5 M sodium hydroxide after exposure to the denaturing buffer in each cycle.

In addition, the invention relates to integrated, closed, or essentially closed, and continuous methods for the production of a recombinant protein, including: (a) obtaining a liquid culture medium comprising a recombinant protein, essentially containing no cell; (b) continuously feeding the liquid culture medium into the first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein from the liquid culture medium using MCCS1; (d) obtaining an eluate from MCCS1 containing the recombinant protein and continuously feeding the eluate into a second multi-column chromatography system (MCCS2); (e) continuously feeding the recombinant protein from the eluate into MCCS2 and then eluting the recombinant protein to thereby obtain a purified recombinant protein, where: the method uses a buffer with reduced bioburden, the method is integrated, and it is carried out continuously from a liquid culture medium to a purified recombinant protein, and at least one column in MCCS1 and/or MCCS2 is a chromatographic column containing a gamma-irradiated chromatographic resin, and the chromatographic resin is exposed to a denaturing buffer during each cycle of the method. In some examples of any of the methods presented herein, MCCS1 and/or MCCS2 perform at least two different typical operations. In some examples of any of the methods presented herein, the use of MCCS1 or MCCS2, or both, involves switching columns.

In some embodiments of any of the methods provided herein, the MCCS1 additionally performs routine recombinant protein capture and virus inactivation operations. In some embodiments of any of the methods provided herein, MCCS2 performs routine purification and fine purification of the recombinant protein. In some examples of any of the methods presented in the present description, at least two chromatographic columns are used in MCCS1 and/or MCCS2. In some examples of any of the methods presented herein, all chromatographic columns in MCCS1 and MCCS2 are chromatographic columns containing gamma irradiated chromatographic resin. In some examples of any of the methods presented in the present description, MCCS1 is the first system of periodic countercurrent chromatography (PCCS1). In some embodiments of any of the methods provided herein, capture is performed using affinity chromatography, cation exchange chromatography, anion exchange chromatography or size exclusion chromatography, hydrophobic interaction chromatography, or any combination thereof. In some examples of any of the methods provided herein, capture is performed using affinity chromatography using a capture mechanism selected from the group: protein A binding capture mechanism, substrate binding capture mechanism, antibody or antibody fragment binding capture mechanism, aptamer-binding capture mechanism; and a cofactor-binding capture mechanism. In some embodiments of any of the methods provided herein, affinity chromatography is performed using a protein A binding capture mechanism and the recombinant protein is an antibody or antibody fragment.

In some embodiments of any of the methods presented herein, the MCCS2 is a second periodic countercurrent chromatography (PCCS2) system. In some examples of any of the methods provided herein, the recombinant protein is a therapeutic recombinant protein. Some embodiments of any of the methods provided herein further include formulating the purified recombinant protein into a pharmaceutical composition. In some examples of any of the methods provided herein, the method is carried out continuously for a period of at least 4 days (e.g., at least 5 days, at least 7 days, at least 14 days, or at least 28 days) .

In some embodiments of any of the methods provided herein, the chromatography resin is an anion exchange chromatography resin, a cation exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography resin, an affinity chromatography resin, or any combination thereof. In some examples of any of the methods provided herein, the chromatographic resin is an anion exchange chromatographic resin. In some examples of any of the methods provided herein, the chromatographic resin is treated with a dose of gamma radiation from about 10 kGy to about 40 kGy (for example, from about 15 kGy to about 35 kGy, or from about 20 kGy to about 30 kGy). In some embodiments of any of the methods provided herein, the denaturing buffer comprises one or more of urea, guanidine hydrochloride, and Triton ^TM X-100. In some embodiments of any of the methods provided herein, the chromatographic resin is exposed to a wash buffer comprising from about 0.5 M to about 1.5 M sodium hydroxide after exposure to the denaturing buffer. In some embodiments of any of the methods provided herein, where the chromatography resin is a protein ligand affinity resin (e.g., protein A ligand), the chromatography resin is exposed to a wash buffer comprising from about 1 mM to about 100 mM sodium hydroxide, after exposure to denaturing buffer.

As used herein, a singular noun is one or more specific nouns. For example, the phrase "chromatographic column" is "one or more chromatographic columns".

The term "gamma irradiated chromatographic resin" means a chromatographic resin subjected to gamma irradiation. For example, a gamma irradiated chromatographic resin may be a chromatographic resin subjected to a dose of gamma irradiation sufficient to reduce the bioburden of the chromatographic resin. In some examples, a gamma-irradiated chromatography resin is exposed to a dose of about 1 kGy to about 15 kGy, a dose of about 1 kGy to about 20 kGy of gamma radiation, a dose of about 1 kGy to about 25 kGy of gamma radiation, a dose of about 1 kGy to about 30 kGy of gamma radiation, or a dose of about 1 kGy to about 35 kGy of gamma radiation. The gamma-irradiated chromatographic resin may have a sterility assurance level of about or less than 1×10 ^-6 , 1×10 ^-7 , 1×10 ^-8 , 1×10 ^-9 or 1×10 ^-10 . Examples of methods for gamma irradiation of a chromatographic resin are presented in the present description. Additional methods for gamma irradiation of a chromatographic resin are known in the art.

The term "chromatographic column containing or including a gamma-irradiated chromatographic resin" means a chromatographic column comprising a gamma-irradiated chromatographic resin (as defined herein). For example, such a chromatography column may include a gamma-irradiated chromatography resin exposed to a dose of about 1 kGy to about 15 kGy, a dose of about 1 kGy to about 20 kGy of gamma radiation, a dose of about 1 kGy to about 25 kGy of gamma radiation, doses from about 1 kGy to about 30 kGy of gamma radiation; or doses from about 1 kGy to about 35 kGy of gamma radiation. For example, such a chromatographic column may contain a gamma-irradiated chromatographic resin having a sterility assurance level of up to or less than about 1×10 ^-6 , 1×10 ^-7 , 1×10 ^-8 , 1×10 ^-9 or 1×10 ^-10 . In some embodiments, the chromatography column containing or including gamma irradiated chromatography resin has reduced bioburden or is sterile, completely sterile, or aseptic (as defined herein) (i.e., the internal surfaces and contents of the chromatography column containing or including gamma irradiated chromatographic resin, has a reduced bioburden, is sterile, absolutely sterile, or aseptic). In some embodiments, the implementation of the chromatographic column containing or including gamma-irradiated chromatographic resin is aseptic and has a reduced bioburden, is sterile or completely sterile.

The term "denaturing buffer" means a liquid containing a sufficient amount of one or more chemicals (eg, detergents, reducing agents, acids, bases, chaotropic agents, organic solvents, or cross-linking agents, or any combination thereof) to cause protein denaturation. Non-limiting examples of denaturing buffers are presented in the present description. Additional examples of denaturing wash buffers are known in the art. Methods for determining protein denaturation are also well known in the art (eg, direct (eg, spectroscopy) or indirect (eg, by protein activity (eg, enzymatic activity or protein-binding activity)) assays to determine protein denaturation). Non-limiting examples of methods for determining protein denaturation are provided in the present description. In any of the methods provided herein, a denaturing buffer may be used to regenerate a chromatography column comprising a gamma-sterilized chromatography resin (eg, any chromatography resin or combination or chromatography resins provided herein).

The term "bioburden reduced buffer" means a treated (e.g., filtered, autoclaved, or gamma-irradiated) fluid (e.g., treated with a buffer solution) having a level of self-replicating biological contaminants that is less than the level of self-replicating biological contaminants found in an identical untreated fluid. Non-limiting examples of self-replicating biological contaminants can be bacteria (e.g., gram-positive or gram-negative bacteria or bacterial or fungal spores), mycobacteria, viruses (e.g., vesivirus, Cache Valley virus, parvovirus, herpes virus, and bunyavirus), parasites, fungi, yeast, and protozoa. For example, a reduced bioburden buffer may have a sterility assurance level of up to or less than about 1×10 ^-6 , 1×10 ^-7 , 1×10 ^-8 , 1×10 ^-9 , or 1×10 ^-10 .

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

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

The term "sterilization" means a validated method used to render a composition sterile (as defined herein). The degree of inactivation of resistant indicator self-replicating biological contaminants (eg, bacteria) during the treatment process 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 has been achieved in a batch of processed units. Probability is generally calculated from the results of inactivation studies carried out during the validation and expressed as 1×10 ^-n .

The term "aseptic" is used to describe a composition or method that is free of disease or symptom-causing self-replicating biological contaminants and/or proteins (e.g., any of the self-replicating biological contaminants described herein, toxins (e.g., endotoxins), or inflammatory proteins). The aseptic composition or method may also be pure (as the term is known in the art).

The term "chromatographic cycle" or "chromatographic cycle" is a term known in this field and means all the steps carried out in one round of chromatography using one chromatographic column. For example, the chromatography run may include the step of equilibrating the chromatography column with a buffer, passing a sample including the recombinant protein through the chromatography column, eluting the recombinant protein from the chromatography column, and washing the chromatography column by passing a denaturing buffer through the column. Additional examples of steps carried out in the chromatography cycle are presented in the present description. Additional examples of steps carried out in a chromatography run are also well known in the art.

The term "typical operation" is a term known in this field, and means a functional step that can be performed in a method for purifying a recombinant protein from a liquid culture medium. For example, a typical operation may be filtration (e.g., removal of bacterial, yeast, virus, or mycobacterial contaminants and/or particulate matter from a fluid, including recombinant protein), capture, epitope tagging, purification, storage, fine purification, virus inactivation, concentration adjustment ions and/or pH of the fluid containing the recombinant protein, and removal of unwanted salts.

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

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

The term "fine cleaning" is a term known in the art and means a step performed to remove remaining traces or small amounts of contaminants or impurities from a liquid containing a recombinant protein (e.g., a recombinant therapeutic protein) close to the final desired purity. For example, fine purification can be performed by passing the liquid containing the recombinant protein through chromatographic columns or membrane absorbents that selectively bind to the recombinant target protein or small amounts of contaminants or impurities present in the liquid containing the recombinant protein. In such an example, the eluate/filtrate of chromatographic columns or membrane absorbents includes a 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 liquid present in any of the methods presented herein).

The term "eluate/filtrate" is a term known in this field, and means a liquid obtained from a chromatographic column or chromatographic membrane, including a detectable amount of recombinant protein (eg, recombinant therapeutic protein).

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

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

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

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

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

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

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

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

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

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

The term "liquid culture medium containing no animal component" means a liquid culture medium that does not include any components (eg, proteins or serum) derived from a mammal.

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

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

The term "chemically defined liquid culture medium" is a term known in this field, 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 calf serum, bovine serum albumin, or human serum albumin, as these preparations typically include a complex mixture of albumins and lipids.

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

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

The term "protein fragment" or "polypeptide fragment" means a portion of a polypeptide sequence of at least or about 4 amino acids, at least or about 5 amino acids, at least or about 6 amino acids, at least or about 7 amino acids, at least at least or about 8 amino acids, at least or about 9 amino acids, at least or about 10 amino acids, at least or about 11 amino acids, at least or about 12 amino acids, at least or about 13 amino acids, at least or about 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 long, or more than 20 amino acids in length. A fragment of the recombinant protein can be obtained by any of the methods presented in the present description.

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

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

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

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

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

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

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

DESCRIPTION OF THE DRAWINGS

Figure 1 is a plot of binding isotherms at t0 of an untreated multimodal resin with anion exchange and hydrophobic groups (AE resins) and a 25 kGy gamma irradiated AE resin (no chromatography cycles).

Figure 2 is a plot of the percentage of normalized binding capacity of untreated AE resin gamma irradiated at 15 kGy AE resin and gamma irradiated at 25 kGy AE resin over multiple cycles of column chromatography when buffered with 700 mM arginine, 100 mM acetate (pH 3.0) and then a 1 N NaOH solution is used to wash each resin (after elution of the recombinant protein) in each cycle. The percentage binding capacity of each resin was normalized to the binding capacity of the untreated resin AE at time 0 (t0), which was also found to be very similar to the gamma irradiated resin at t0 (27±2 mg/ml).

Figure 3 is a graph showing the average rate of decline in binding capacity over multiple cycles of column chromatography for untreated AE resin, 15 kGy gamma-irradiated AE resin, and 25 kGy AE gamma-irradiated if buffer of 700 mM arginine, 100 mM acetate (pH 3.0), and then a 1 N NaOH solution are used to wash each resin (after elution of the recombinant protein) in each cycle.

Figure 4 is a graph of the percentage normalized binding capacity of raw AE resin over multiple cycles of column chromatography using a single column chromatography comprising raw AE resin washed with 700 mM arginine, 100 mM acetate (pH 3.0), and then with 1 N NaOH after elution of the recombinant protein in each cycle (low pH, arginine, raw resin), or using a single chromatographic column including 25 kGy gamma-irradiated AE Resin, washed after elution of the recombinant protein (in each cycle) with 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5 (low pH, urea); 6 M guanidine HCl (pH 2.5) (low pH, guanidine); 0.5% Triton-X 100 in 0.1 M acetic acid (pH 2.5) followed by 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) (low pH, Triton) ; 700 mM arginine, 100 mM acetate (pH 3.0) (low pH, arginine); or 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) (low pH, organic) followed by 1 N NaOH solution. The percentage binding capacity of each resin during each cycle was normalized to the binding capacity of the untreated resin AE at time 0 (t0), which was also found to be very similar to the gamma irradiated resin at t0 (27±2 mg/ml).

Figure 5 is a typical multi-column chromatography (MCC) profile showing eluate absorbance at 280 nm over multiple cycles of column chromatography performed using three chromatographic columns including AE resin irradiated at 25 kGy if each resin was washed (after elution of the recombinant protein) in each cycle using 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5, and then a solution of 1 N NaOH. Peaks representing bound protein released from AE resin upon exposure to 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5 denaturing buffer in three different cycles are indicated by an arrow. The chromatogram shows the UV traces for all three chromatographic columns used to run the MCC.

Figure 6 is a graph of the percentage of isolated recombinant protein bound to the chromatography resin per cycle over multiple cycles of column chromatography using a single column chromatography comprising crude AE resin washed with 700 mM arginine, 100 mM acetate (pH 3.0), and then with 1 N NaOH solution after elution of the recombinant protein in each cycle (low pH, arginine, raw resin), or using a single chromatographic column including 25 kGy gamma irradiated AE resin washed after elution of the recombinant protein (in each cycle a) 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5 (low pH, urea); 6 M guanidine HCl (pH 2.5) (low pH, guanidine); 0.5% Triton-X 100 in 0.1 M acetic acid (pH 2.5) followed by 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) (low pH, Triton) ; or 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) (low pH, organic); followed by a 1 N NaOH solution.

Figure 7 is a plot of host cell protein (ng/mg) present in the eluate over multiple cycles of column chromatography using a single column chromatography comprising raw AE resin washed with 700 mM arginine, 100 mM acetate (pH 3.0), and then with 1 N NaOH solution after elution of the recombinant protein in each cycle (low pH, arginine, raw resin), or using a single chromatographic column including 25 kGy gamma irradiated AE resin washed after elution of the recombinant protein (in each cycle a) 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5 (low pH, urea); 6 M guanidine HCl (pH 2.5) (low pH, guanidine); 0.5% Triton-X 100 in 0.1 M acetic acid (pH 2.5) followed by 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) (low pH, Triton) ; or 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) (low pH, organic); followed by a 1 N NaOH solution.

Figure 8 is a list of lysosomal storage diseases and recombinant therapeutic enzymes that can be used to treat each disease.

DETAILED DESCRIPTION

The present invention relates to methods for performing chromatography using a gamma-irradiated chromatographic resin, including obtaining a chromatographic column comprising a gamma-irradiated chromatographic resin and performing a first cycle of chromatography using a column, where the cycle includes exposing the chromatographic resin to a denaturing buffer. In addition, the invention relates to integrated, closed, or essentially closed, and continuous methods for the production of a recombinant protein, including the use of at least one chromatographic column comprising a gamma-irradiated chromatographic resin, where the gamma-irradiated chromatographic resin is exposed to a denaturing buffer during each cycle of the method, and the method uses a reduced bioburden buffer. Non-limiting aspects of these methods are described below. As is known in the art, the various aspects described below can be used in any combination without limitation.

Gamma irradiated chromatography resin

A wide range of different types of chromatographic resin known in this field (or combinations thereof) can be subjected to gamma irradiation by methods known in this field. For example, an isotope such as cobalt-60 or cesium-137 is used as the source of gamma rays. The chromatographic resin subjected to gamma irradiation may be in a packed chromatographic column. In other examples, the gamma-irradiated chromatographic resin is in a sealed container (eg, a suspension in a sealed container).

Chromatographic resin can be subjected to gamma irradiation at a temperature of from about -25°C to about 0°C, inclusive, or from about 0°C to about 25°C, inclusive. The chromatographic resin can be exposed to a dose of gamma radiation 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 , from about 10 kGy to about 45 kGy, from about 10 kGy to about 40 kGy, from about 10 kGy to about 35 kGy, from about 10 kGy to about 30 kGy, from about 15 kGy to about 50 kGy, from about 15 kGy up to about 45 kGy, from about 15 kGy to about 40 kGy, from about 15 kGy to about 35 kGy, or from between 20 kGy and approximately 30 kGy.

The gamma irradiated chromatography resin may be an anion exchange chromatography resin, a cation exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography resin, an affinity chromatography resin, or any combination thereof. Non-limiting examples of affinity chromatography resins include peptide ligand, protein ligand (eg, protein A or protein G), aptamer ligand, substrate ligand, product ligand, metal ligand, and cofactor ligand resins. The gamma irradiated chromatography resin may be a bimodal chromatography resin (eg, with features of an anion exchange resin and hydrophobic interaction chromatography resin).

Chromatography columns incorporating gamma irradiated chromatography resin

The methods provided herein include the use of a chromatography column comprising a gamma irradiated chromatography resin, and the methods presented herein include the use of one or two MCCSs comprising at least one chromatography column comprising a gamma irradiated chromatography resin. The gamma irradiated chromatographic resin may be any type of resin described herein (or any type of chromatographic resin known in the art). Gamma-irradiated chromatographic resin can be obtained by any of the methods presented in the present description or known in this field.

Such chromatographic columns can be obtained by filling the chromatographic column with untreated chromatographic resins and exposing the packed column to gamma irradiation (eg, using any of the treatments and conditions presented herein). In other examples, chromatography columns including gamma-irradiated resins can be prepared by exposing the chromatography resin to gamma irradiation (eg, the chromatography resin in a container) and filling the chromatography column with the gamma-irradiated chromatography resin. In such methods, the gamma irradiated chromatographic resin may be present as a suspension in a container and the chromatographic column is loaded in a bioburden reduced fume hood. In other methods, the chromatography resin may be gamma-irradiated as a solid mixture in a container and the gamma-irradiated chromatography resin may be slurried using a reduced bioburden buffer (e.g., prepared in a bioburden reduced fume hood) and the resulting slurry used to fill the chromatography chamber. columns in a fume hood with reduced bioburden. In some of these examples, the chromatography column may be treated to reduce bioburden (eg, autoclaved, gamma irradiated, or exposed to ethylene oxide) prior to filling.

A chromatographic column comprising a gamma-irradiated chromatographic resin may have a sterility assurance level (SAL) of about ^{1x10 -3} to about ^{1x10 -12} , about ^{1x10 -4} to about ^{1x10 -12} , from 1 ×10 ^-5 to about ^{1x10 -11} , from about ^{1x10 -5} to about ^{1x10 -10} , from about ^{1x10 -5} to about ^{1x10 -9} , from about ^{1x10 -6} to approximately 1×10 ^-9 or from approximately 1×10 ^-6 to approximately 1×10 ^-8 inclusive.

Buffers with reduced bioburden

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

Denaturing buffers

The methods presented in the present description include the use of a denaturing buffer. Denaturing buffers include a sufficient amount of one or more chemicals (eg, detergents, reducing agents, acids, chaotropic agents, organic solvents, or cross-linking agents, or any combination thereof) to denature the protein. Non-limiting examples of detergents that can be included in the denaturing buffer include Triton X-100, sodium dodecyl sulfate, and ethyltrimethylammonium bromide. Non-limiting examples of organic solvents that can be included in the denaturing buffer include ethanol, butanol, phenol, propanol, and methanol. Non-limiting examples of reducing agents that can be included in the denaturing buffer include 2-mercaptoethanol, dithiothreitol, and tris(2-carboxyethyl)phosphine. Non-limiting examples of acids that can be included in denaturing buffers include acetic acid, trichloroacetic acid, and sulfosalicylic acid. Non-limiting examples of chaotropic agents that can be included in the denaturing buffer include urea (e.g., 6 to 9 M urea), thiourea, guanidine chloride (e.g., 5 to 7 M guanidine chloride), lithium perchlorate (e.g., 4 to 7 M lithium perchlorate) or lithium acetate. Non-limiting examples of crosslinkers that can be included in the denaturing buffer include formaldehyde and glutaraldehyde.

Non-limiting examples of denaturing buffers include: 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5 (eg, then 1 N NaOH); 6 M guanidine HCl, pH 2.5 (eg followed by 1 N NaOH or 1 M NaOH and 1 M NaCl); and 0.5% Triton-X 100 in 0.1 M acetic acid, pH 2.5 (e.g., then 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol, pH 2.5, then 1 N NaOH) .

Recombinant Therapeutic Proteins

As presented in the present description, the recombinant protein may be a recombinant therapeutic protein. Non-limiting examples of recombinant therapeutic proteins that can be produced by the methods provided herein include immunoglobulins (including immunoglobulin light and heavy chains, antibodies or antibody fragments (e.g., any of the antibody fragments provided herein), enzymes (e.g., galactosidase ( for example, alpha-galactosidase), Myozyme® or Cerezyme®), proteins (eg, erythropoietin, tumor necrosis factor (TNF), or human interferon alpha or beta), or immunogenic or antigenic proteins or protein fragments (eg, proteins for use in a vaccine) Non-limiting examples of recombinant therapeutic enzymes that can be used to treat a variety of lysosomal storage diseases are shown in Figure 8. A recombinant therapeutic protein can be an engineered antigen-binding polypeptide that includes at least one multifunctional recombinant protein framework (see, for example, recombinant inant 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 their entirety)). Неограничивающие примеры рекомбинантных терапевтических белков, являющихся антителами, включают: панитумумаб, омализумаб, абаговомаб, абциксимаб, актоксумаб, адалимумаб, адекатумумаб, афелимомаб, афутузумаб, алацизумаб, алацизумаб, алемтузумаб, алирокумаб, алтумомаб, аматуксимаб, анатумомаб, аполизумаб, атинумаб, тоцилизумаб, базиликсимаб , bevacizumab, belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab, etaracizumab, golimumab, infliximab, natalizumab, palivizumab, panitumumab, pertuzumab, tocibubizumab, irastuzumab, Additional examples of recombinant therapeutic antibodies that can be obtained by the methods presented in the present description are known in this field. Additional non-limiting examples of recombinant therapeutic proteins that can be prepared/purified by the methods of the present invention include: alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, factor coagulation IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa, and alteplase.

The secreted soluble recombinant therapeutic protein can be isolated from the culture liquid (eg, the first and/or the second culture liquid) by removing or otherwise separating the culture liquid from the cells (eg, mammalian cells). Many different methods are known in the art for removing liquid culture medium from cells (eg, mammalian cells), including, for example, centrifugation, filtration, pipetting, and/or aspiration. The secreted recombinant therapeutic protein can then be isolated and further purified from the liquid culture medium 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 cut-off filtration).

Chromatography cycle

As is well known in the art, the steps in a chromatography run may differ depending on the chromatography resin, the buffers used to carry out each step in the run, and the biophysical characteristics of the recombinant target protein (eg, recombinant therapeutic protein). For example, an affinity chromatography column may include the steps of loading the affinity chromatography column with a liquid comprising the recombinant target protein, washing the column to remove unwanted biological material (e.g., contaminating proteins and/or small molecule compounds), eluting the recombinant target protein bound to the column, and rebalancing the column. A chromatography cycle using a cation exchange and/or anion exchange chromatography column, where the recombinant target protein is bound to the chromatographic resin in a loading step, may include the steps of loading the column with a liquid containing the target protein, washing the column to remove unwanted biological material, eluting the recombinant target protein bound to the column, and re-equilibrate the column. In other examples, a chromatography cycle using a cation exchange and/or anion exchange chromatography column, where the unwanted biological material binds to the chromatography resin during a loading step while the recombinant target protein does not bind, may include the steps of loading the column with a liquid comprising the target protein. , collecting the recombinant target protein in the eluate and re-equilibrating the column. As is well known in the art, any of the individual steps of the chromatography run may include a single buffer or multiple buffers (eg, two or more buffers), and one or more of any of the individual steps of the chromatography run may include a buffer gradient. Any combination of various well-known aspects of a single chromatography run can be used in these methods in any combination, e.g., different chromatographic resins, flow rates, buffers, column volumes excluded, column layer volumes, volumes of buffer used in each step, volumes of liquid including target protein, and the amount and types of buffers used in each step.

Methods for Performing Chromatography Using Gamma Sterilized Resin

The present invention relates to methods for performing chromatography using a gamma-irradiated chromatographic resin. These methods include obtaining a chromatography column comprising a gamma-irradiated chromatography resin, performing a first round of column chromatography, where the cycle includes exposing the chromatographic resin to a denaturing buffer, and performing at least one additional round of column chromatography. The gamma irradiated chromatographic resin may be any type of chromatographic resin and/or may be any of the gamma irradiated chromatographic resins described herein or known in the art. The chromatographic column may be any chromatographic column including a gamma irradiated chromatographic resin as described herein or known in the art. The recombinant protein may be a recombinant therapeutic protein (eg, any of the recombinant therapeutic proteins provided herein or known in the art).

In some examples, the column is part of a multi-column chromatography system (MCCS), for example, may be part of a periodic countercurrent chromatography system (PCCS).

The denaturing buffer may be any of the examples of denaturing buffers provided herein or known in the art. For example, the denaturing buffer may include one or more of urea, guanidine hydrochloride and Triton ^TM X-100. In some examples, the chromatography resin is exposed to a denaturing buffer for a period of at least 1 minute to about 2 hours (e.g., 1 minute to about 1.5 hours, about 1 minute to about 1.0 hour, from about 1 minute up to about 55 minutes, from about 1 minute to about 50 minutes, from about 1 minute to about 45 minutes, from about 1 minute to about 40 minutes, from about 1 minute to about 40 minutes, from about 1 minute to about 35 minutes, about 1 minute to about 30 minutes, about 1 minute to about 25 minutes, about 1 minute to about 20 minutes, about 1 minute to about 15 minutes, or about 1 minute to about 10 minutes). In some examples, exposing the chromatographic resin to a denaturing buffer comprises passing from about 0.5 times the bed volume to about 10 times the bed volume (e.g., from about 0.5 times the bed volume to about 9.0 times the bed volume, from about 0.5 times the layer volume to about 8.0 times the layer volume, from about 0.5 times the layer volume to about 7.0 times the layer volume, from about 0.5 times the layer volume to about 6, 0 times the layer volume, from about 0.5 times the layer volume to about 5.0 times the layer volume, from about 0.5 times the layer volume to about 4.0 times the layer volume, from about 0.5- from about 0.5 times the layer volume to about 3.0 times the layer volume, from about 0.5 times the layer volume to about 2.5 times the layer volume, from about 0.5 times the layer volume to about 2.5 times the layer volume , from app about 0.5 times bed volume to about 2.0 times bed volume, or from about 0.5 times bed volume to about 1.5 times bed volume) of denaturing buffer through a chromatographic column. In some examples, the first chromatography cycle may include exposing the chromatographic resin to a wash buffer containing about 0.5 M to about 1.5 M sodium hydroxide after exposure to a denaturing buffer.

The first round of chromatography carried out with the column may be any round of chromatography as described herein or known in the art, including exposing the chromatographic resin to a denaturing buffer. The at least one additional chromatography run carried out by the column may be any chromatography run described herein or known in the art. For example, the first chromatography cycle and at least one additional chromatography cycle may include the steps of: capturing the recombinant protein by exposing the chromatographic resin to a liquid containing the recombinant protein; washing the chromatography resin by exposing the chromatography resin to a wash buffer, eluting the recombinant protein by exposing the chromatography resin to an elution buffer, and regenerating the chromatography resin by exposing the chromatography resin to a denaturing buffer. In some examples, the liquid comprising the recombinant protein is a liquid culture medium (eg, a liquid culture medium harvested from a perfusion or batch culture).

The first cycle of chromatography and at least one additional cycle of chromatography can be performed using a closed and integrated system (for example, any of the examples of closed and integrated systems presented in the present description or known in this field). For example, the first chromatography run and at least one additional chromatography run may be performed using a closed and integrated system where the buffer is a bioburden reduced buffer (eg, all buffers used in the first and at least one additional run). As is well known in the art, the bioburden reduced buffer can be prepared in a variety of different ways (eg, by filtration, autoclaving, or heat treatment).

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

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

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

Some methods include continuously feeding a liquid culture medium into a multi-column chromatography system (MCCS) comprising at least one chromatography column comprising a gamma-sterilized resin, where the chromatography resin is exposed to a denaturing buffer during each cycle (e.g., exposed to any of the denaturing buffers presented in the present description or known in this field, for any period of time presented in the present description). These methods use a reduced bioburden buffer, are integrated, and run continuously from a liquid culture medium to an MCCS eluate that is a purified recombinant protein (eg, a therapeutic protein drug).

Some methods include continuously feeding the liquid culture medium into the first MCCS (MCCS1), capturing the recombinant protein from the liquid culture medium using the MCCS1, obtaining an eluate from the MCCS1 containing the recombinant protein, and continuously feeding the eluate into the second MCCS (MCCS2), and continuously feeding the recombinant protein from the eluate into MCCS2 and then eluting the recombinant protein to thereby obtain a purified recombinant protein, wherein at least one column in MCCS1 and/or MCCS2 is a chromatography column comprising a gamma-irradiated chromatography resin, and the chromatography resin is subjected to exposure to a denaturing buffer during each cycle of the process (eg, exposure to any of the denaturing buffers provided herein or known in the art for any period of time provided herein). These methods use a reduced bioburden buffer, are integrated, and run continuously from liquid culture medium to purified recombinant protein.

In some examples, each chromatography column used in MCCS, MCCS1 and/or MCCS2 includes a gamma irradiated chromatographic resin. Some embodiments further include the step of formulating the purified recombinant protein into a pharmaceutical composition.

The methods provided herein provide continuous and rapid production of purified recombinant protein from a liquid culture medium containing the recombinant protein. For example, the time period between feeding the liquid culture medium comprising the therapeutic protein into MCCS or MCCS1 and eluting the recombinant protein from MCCS or MCCS2, respectively, may be, for example, from about 4 hours to about 48 hours, inclusive, for example, from about 4 hours to about 40 hours, from about 4 hours to about 35 hours, from about 4 hours to about 30 hours, from about 4 hours to about 28 hours, from about 4 hours to about 26 hours, from about 4 hours to about 24 hours , from about 4 hours to about 22 hours, from about 4 hours to about 20 hours, from about 4 hours to about 18 hours, from about 4 hours to about 16 hours, from about 4 hours to about 14 hours, from about 4 hours up to about 12 hours, from about 6 hours to about 12 hours, about t about 8 hours to about 12 hours, from about 6 hours to about 20 hours, from about 6 hours to about 18 hours, from about 6 hours to about 14 hours, from about 8 hours to about 16 hours, from about 8 hours to about 14 hours, from about 8 hours to about 12 hours, from about 10 hours to 20 hours, from about 10 hours to 18 hours, from about 10 hours to 16 hours, from about 10 hours to 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 owls up to about 40 hours, from about 20 hours to about 35 hours, or from about 20 hours to about 30 hours, inclusive. In other examples, the time period between the feeding of the liquid culture medium comprising the recombinant protein into MCCS or MCCS1 and the elution of the recombinant protein from MCCS or MCCS2, respectively, is, for example, more than about 4 hours to less than about 40 hours, inclusive, for example, more less than about 39 hours, approximately 38 hours, approximately 37 hours, approximately 36 hours, approximately 35 hours, approximately 34 hours, approximately 33 hours, approximately 32 hours, approximately 31 hours, approximately 30 hours, approximately 29 hours, approximately 28 hours, approximately 27 hours, approximately 26 hours, approximately 25 hours, approximately 24 hours, approximately 23 hours, approximately 22 hours, approximately 21 hours, approximately 20 hours, approximately 19 hours, approximately 18 hours, approximately 17 hours, approximately 16 hours , approximately 15 hours, approximately 14 hours, approx. approximately 13 hours, approximately 12 hours, approximately 11 hours, approximately 10 hours, approximately 9 hours, approximately 8 hours, approximately 7 hours, approximately 6 hours, approximately 5 hours or approximately 4.5 hours inclusive.

Non-limiting aspects of MCCS that can be used in any of these methods (MCCS, MCCS1 and/or MCCS2) are described in US Provisional Applications Nos. 61/775060 and 61/856390 (each of which is incorporated herein by reference).

Some exemplary methods do not use a storage step (eg, do not use a reservoir (eg, tank) in the entire method). Others may use a maximum of 1, 2, 3, 4 or 5 reservoirs (eg tanks) in the entire process. In any of the methods provided herein, a maximum of 1, 2, 3, 4, or 5 reservoirs (e.g., tanks) can be used in the entire process, where each tank stores the recombinant protein for a total of a total period of time, for example, from about 5 minutes to less than about 6 hours, inclusive, such as from about 5 minutes to about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes, inclusive.

Some methods use one, two, three, four, five, or six reservoirs (e.g., tanks) and may have a capacity ranging from, for example, 1 ml to about 300 ml, inclusive, for example, from 1 ml to about 280 ml. , approximately 260 ml, approximately 240 ml, approximately 220 ml, approximately 200 ml, approximately 180 ml, approximately 160 ml, approximately 140 ml, approximately 120 ml, approximately 100 ml, approximately 80 ml, approximately 60 ml, approximately 40 ml, approximately 20 ml or approximately 10 ml (inclusive). Any reservoirs (e.g., tanks) used (in any of the methods described herein) to store liquid prior to being fed into the MCCS or MCCS1 may have a capacity ranging from, for example, 1 ml to about 100%, inclusive, for example, 1 ml to about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%, inclusive, of the loading volume of the first column MCCS or MCCS1. Reservoirs (eg, tanks) may be used to store eluate from MCCS1 until it enters MCCS2 and may have a capacity ranging from, for example, 1 ml to about 100% inclusive, for example, from 1 ml to about 90%, about 80 %, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 10% or approximately 5% inclusive of the loading volume of the first MCCS2 column.

Various additional aspects of these methods are described in detail below, and they can be used in any combination in the methods presented in the present description, without limitation. Exemplary aspects of the presented methods are described below; however, one of skill in the art will appreciate that additional steps may be added to the methods provided herein, and other materials may be used to carry out any of the steps of the methods presented herein.

Liquid culture medium

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

The liquid culture medium obtained from the recombinant cell culture can be filtered or purified to obtain a liquid culture medium substantially free of cells and/or viruses. Methods for filtering or purifying a liquid culture medium to remove a cell are known in the art (eg, filtration at a pore size of 0.2 μm and filtration using an Alternating Tangential Flow (ATF ^TM ) system). Recombinant cells can also be removed from the liquid culture medium using centrifugation and removal of the supernatant, which is a liquid culture medium essentially free of cells, or by allowing the cells to settle under gravity to the bottom of a container (e.g., bioreactor) containing the liquid medium. for culturing, and removing the liquid culture medium (liquid culture medium containing essentially no cell) away from the settled recombinant cells.

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

The liquid culture medium may be any type of liquid culture medium described herein or known in the art. For example, the liquid culture medium can be selected from the group of: a liquid culture medium containing no animal component, a liquid culture medium containing no serum, a liquid culture medium containing serum, a chemically defined liquid culture medium, and a liquid culture medium for cultivation that does not contain protein. In any of the methods provided herein, the liquid culture medium obtained from the culture can be diluted by adding a second liquid (eg, buffer) prior to its feeding into MCCS or MCCS1.

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

Multicolumn Chromatography Systems

The methods presented herein include the use of MCCS or two or more (eg, two, three, four, five, or six) multi-column chromatography systems (MCCS) (eg, MCCS1 and MCCS2). The MCCS may include two or more chromatographic columns, two or more chromatographic membranes, or a combination of at least one chromatographic column and at least one chromatographic membrane. In non-limiting examples, MCCS (e.g., MCCS, MCCS1 and/or MCCS2 in any of the methods herein) may include four chromatographic columns, three chromatographic columns and a chromatographic membrane, three chromatographic columns, two chromatographic columns, two chromatographic membranes, and two chromatographic membranes. columns and one chromatographic membrane. Additional examples of combinations of chromatographic columns and/or chromatographic membranes for use by one of skill in the art in MCCS can be provided (eg, MCCS, MCCS1 and/or MCCS2 in any of the methods presented herein). The individual chromatographic columns and/or chromatographic membranes present in the MCCS may be identical (e.g., have the same shape, volume, resin, capture mechanism, and typical operation) or different (e.g., have one or more different shapes, volumes, resins, grippers and/or typical operations). Through the individual chromatographic columns and/or chromatographic membranes present in the MCCS (e.g., MCCS, MCCS1 and/or MCCS2 in any of the methods presented herein), a single operation can be performed (e.g., a capture, purification, or fine purification operation) or different routine operations (for example, different routine operations, for example, selected from the group of capture, purification, fine purification, virus inactivation, correction of ion concentration and/or pH of the fluid containing the recombinant protein, and filtration). For example, in the exemplary methods provided herein, at least one chromatographic column and/or chromatographic membrane in an MCCS or MCCS1 performs a typical recombinant protein capture operation.

One or more chromatography columns that may be present in MCCS (e.g., present in MCCS, MCCS1 and/or MCCS2) may have a resin volume, such as from about 1 ml to about 2 ml, about 5 ml, about 10 ml, about 15 ml, approximately 20 ml, approximately 25 ml, approximately 30 ml, approximately 35 ml, approximately 40 ml, approximately 45 ml, approximately 50 ml, approximately 55 ml, approximately 60 ml, approximately 65 ml, approximately 70 ml, approximately 75 ml , approximately 80 ml, approximately 85 ml, approximately 90 ml, approximately 95 ml or approximately 100 ml, inclusive. One or more chromatographic columns that may be present in 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, from about 2 ml to about 70 ml, from about 2 ml to about 60 ml, from about 2 ml to about 50 ml, from about 5 ml to about 50 ml, from about 2 ml to about 45 ml , from about 5 ml to about 45 ml, from about 2 ml to about 40 ml, from about 5 ml to about 40 ml, from about 2 ml to about 35 ml, from about 5 ml to about 35 ml, from about 2 ml to about 30 ml, from about 5 ml to about 30 ml, from about 2 ml to about 25 ml, from about 5 ml to about 25 ml, from about 15 m l 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 chromatographic columns in MCCS (eg, MCCS, MCCS1 and/or MCCS2) used in any of the methods presented herein may have essentially the same resin volume or different resin volumes. The flow rate used for one or more chromatographic columns in the MCCS (e.g., MCCS, MCCS1 and/or MCCS2) may be, for example, from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0. 2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute 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 about 15.0 ml/minute).

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

One or more chromatographic membranes that may be present in MCCS (e.g., present in MCCS, MCCS1 and/or MCCS2) may have a bed volume, e.g., from about 1 ml to about 500 ml (e.g., 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 approx. 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, about 2 ml to about 60 ml, about 5 ml to about 60 ml, about 1 ml to about 40 ml, about 2 ml to about 40 ml, about 5 ml to about 40 ml, 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 for about 10 ml or from about 2 ml to about 10 ml).

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

The MCCS, MCCS1 and/or MCCS2 may be a Periodic Counter Current Chromatography (PCCS) system. For example, PCCS may include two or more chromatographic columns (eg, three columns or four columns) switchable for continuous elution of the recombinant protein from two or more chromatographic columns. The PCCS may include two or more chromatographic columns, two or more chromatographic membranes, or at least one chromatographic column and at least one chromatographic membrane. The operation on the column (cycle) typically consists of loading, washing, elution and regeneration steps. In PCCS, a plurality of columns are used to carry out the same steps individually and continuously in a cyclic manner. Because columns are used in series, the eluate from one column is subjected to capture on another column. This unique feature of PCCS makes it possible to load the resin close to its static bonding capability instead of the dynamic bonding capability that is typical in batch chromatography. As a result of continuous cycles and elution, the liquid entering the PCCS is processed continuously and an eluate containing the recombinant protein is continuously obtained.

The column switching strategy is used to advance from one step to the next in the PCCS cycle. Examples of column switching that can be used in PCCS are described in US Provisional Patent Applications Nos. 61/775060 and 61/856390. For example, the column switching method can use two automated column switching operations: the first of which is associated with the appearance of the original product, while the second corresponds to the saturation of the column. Determining when to perform column switching operations can be done by monitoring the concentration of recombinant protein (eg, monitoring by UV monitoring) in the eluate from each chromatographic column located in the PCCS. For example, column switching can be determined using any PAT instrument capable of linear measurement of product concentration with feedback control. The PAT instrument is capable of real-time linear measurement of product concentration with feedback control. As is known in the art, column switching can also be done based on time or amount of liquid (eg buffer) passed through one or more chromatographic columns and/or chromatographic membranes in the MCCS, MCCS1 and/or MCCS2.

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

One or more exemplary operations that can be performed by MCCS or MCC1 and/or MCCS2 used in the methods of the present disclosure include, for example, capture of a recombinant protein, inactivation of viruses present in a fluid comprising the recombinant protein, purification of the recombinant protein, fine purification the recombinant protein, storing a fluid comprising the recombinant protein (for example, using any of the example tanks provided herein), filtering or removing particulate matter and/or cells from the fluid comprising the recombinant protein, and adjusting the ion concentration and/or pH of the fluid containing a recombinant protein.

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

A typical operation to inactivate viruses present in a liquid comprising a recombinant protein can be performed using MCCS, MCCS1 and/or MCCS2 (for example, including, for example, a chromatographic column, a chromatographic membrane, or a storage tank in which the liquid comprising the recombinant protein can be incubated , at a pH of about 3.0 to about 5.0 (e.g., about 3.5 to about 4.5, about 3.5 to about 4.25, about 3.5 to about 4.0, about 3.5 to about 3.8, or about 3.75) for a period of at least 30 minutes (e.g., a period of about 30 minutes to 1.5 hours, a period of about 30 minutes to 1.25 hours, a period of approximately 0.75 hours to 1.25 hours or a period of approximately 1 hour).

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

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

A typical storage operation for a liquid comprising a recombinant protein can be performed using an MCCS (e.g. a MCCS, MCCS1 and/or MCCS2) comprising at least one reservoir (e.g. tank) or a maximum of 1, 2, 3, 4 or 5 reservoirs (e.g. tanks) in MCCS or in MCCS1 and MCCS2 in combination. For example, each of the reservoirs (eg, tanks) that can be used to perform this typical operation may have a volume of from about 1 ml to about 1 liter (for example, from about 1 ml to about 800 ml, from about 1 ml to about 600 ml, from about 1 ml to about 500 ml, from about 1 ml to about 400 ml, from about 1 ml to about 350 ml, from about 1 ml to about 300 ml, from about 10 ml to about 250 ml, from about 10 ml to about 200 ml, from about 10 ml to about 150 ml, or from about 10 ml to about 100 ml). Reservoirs (eg, tanks) used in the methods presented herein may have a capacity of, for example, from 1 ml to about 300 ml, inclusive, for example, from 1 ml to about 280 ml, about 260 ml, about 240 ml , approximately 220 ml, approximately 200 ml, approximately 180 ml, approximately 160 ml, approximately 140 ml, approximately 120 ml, approximately 100 ml, approximately 80 ml, approximately 60 ml, approximately 40 ml, approximately 20 ml, or approximately 10 ml, inclusive . Any of the reservoirs (e.g., tanks) used (in any of the methods described herein) to store liquid prior to entering the MCCS or MCCS1 may have a capacity ranging from, for example, 1 ml to about 100% inclusive, from about 1 ml to approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 10% or approximately 5% of the loading volume of the first MCCS or MCCS1 column . Any of the reservoirs (e.g., tanks) used to store the eluate from MCCS1 (including the recombinant protein) until it enters MCCS2 may have a capacity ranging from, for example, 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, of the loading volume of the first MCCS2 column.

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

Typical operations for filtering a liquid comprising a recombinant protein can be performed using an MCCS (e.g., MCCS, MCCS1 and/or MCCS2) including, for example, a filter, or a chromatographic column or chromatographic membrane comprising a molecular sieve resin. As is known in the art, a wide variety of submicron filters are available in the art (e.g., filters with pore sizes 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) that remove any deposited material and/or cells (e.g., precipitated unfolded protein; precipitated unwanted host cell proteins; precipitated lipids; bacteria; yeast cells; fungal cells; mycobacteria and/or mammalian cells). Filters having a pore size of about 0.2 µm or less than 0.2 µm are known to effectively remove bacteria from a liquid containing a recombinant protein. As is known in the art, a chromatographic column or a chromatographic membrane comprising a molecular sieve resin can also be used to perform a typical operation for filtering a liquid comprising a recombinant protein into an MCCS (e.g., MCCS, MCCS1 and/or MCCS2).

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

By means of MCCS, MCCS1 and/or MCCS2, two or more typical operations can be performed. For example, each of the MCCS, MCCS1 and/or MCCS2 can perform at least the following typical operations: the capture of the recombinant protein and the inactivation of viruses present in the fluid containing the recombinant protein; capturing the recombinant protein, inactivating viruses present in the fluid comprising the recombinant protein, and adjusting the ion concentrations and/or pH of the fluid comprising the recombinant protein; purification of the recombinant protein and fine purification of the recombinant protein; purifying the recombinant protein, finely purifying the recombinant protein, and filtering the liquid comprising the recombinant protein, or removing precipitates and/or particulates from the liquid comprising the recombinant protein; and purifying the recombinant protein, finely purifying the recombinant protein, filtering the liquid comprising the recombinant protein, or removing precipitates and/or solids from the liquid comprising the recombinant protein, and adjusting the ion concentration and/or pH of the liquid comprising the recombinant protein.

Recombinant protein capture

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

The liquid culture medium can be fed (loaded) into MCCS or MCCS1 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 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 liquid culture medium comprising the recombinant protein may be obtained from any of the exemplary sources provided herein or known in the art.

Some examples further include the optional step of filtering the liquid culture medium before it is fed into the MCCS or MCCS1. Any of the exemplary methods for filtering a liquid culture medium or liquid comprising a recombinant protein provided herein, or any of the filtration methods known in the art, can be used to filter a liquid culture medium comprising a recombinant protein before it is fed into MCCS or MCCS1. .

In the methods presented herein, capture of the recombinant protein from the liquid culture medium is performed using MCCS or MCCS1. As is known in the art, to achieve capture of a recombinant protein, at least one chromatographic column or at least one chromatographic membrane in an MCCS or MCCS1 must include a resin that uses a capture mechanism (e.g., any of the examples of capture mechanisms provided herein). ), or include a resin with which cation exchange, anion exchange, molecular sieve or hydrophobic interaction chromatography can be performed. For example, if the recombinant protein is an antibody or antibody fragment, the capture system may be a protein A-binding capture mechanism or an antigen-binding capture mechanism (where the antigen specifically recognized by the recombinant antibody or antibody fragment is captured). If the recombinant protein is an enzyme, the capture mechanism may use an antibody or antibody fragment that specifically binds to the enzyme to capture the recombinant enzyme, a substrate for the enzyme to capture the recombinant enzyme, an enzyme cofactor to capture the recombinant enzyme, or if the recombinant enzyme includes a label, a protein, a chelate a metal or an antibody (or antibody fragment) that specifically binds to a label present in the recombinant enzyme. Non-limiting examples of resins that can be used to capture a recombinant protein are provided herein, and additional resins that can be used to capture a recombinant protein are known in the art. One non-limiting example of a resin that uses a protein A-binding capture mechanism is MabSelect SuRe resin (GE Healthcare, Piscataway, NJ).

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

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

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

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

The wash buffer can be passed through at least one chromatographic column or chromatographic membrane at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml /minute to about 10 ml/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, from about 1.0 ml/minute to approximately 15.0 ml/minute). The volume of wash buffer used (e.g., the combined total volume of wash buffer used when multiple wash buffers are used) can be, for example, from about 1x column volume (CV) to about 15x CV (e.g., from about 1x CV to about 14 times CV, from about 1 times CV to about 13 times CV, from about 1 times CV to about 12 times CV, from about 1 times CV to about 11 times CV, from about 2- from about 3 times CV to about 11 times CV, from about 3 times CV to about 11 times CV, from about 4 times CV to about 11 times CV, from about 5 times CV to about 11 times CV, or from about 5x CV to approximately 10x CV). The total flush 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 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 at least one chromatographic column or chromatographic membrane in MCCS or MCCS1 containing a resin with which a typical capture operation can be performed, the recombinant protein is eluted from at least one chromatographic column or chromatographic membrane by passing the elution buffer through at least one chromatographic a column or chromatographic membrane in MCCS or MCCS1, including resin, through which you can perform a typical capture operation. The elution buffer can be passed through at least one chromatographic column or chromatographic membrane comprising a resin that can perform a typical capture operation at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5.0 mg/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, from about 1.0 ml/minute to about 15.0 ml /minute). The volume of elution buffer used to elute the recombinant protein from each of at least one chromatographic column or chromatographic membrane comprising a resin with which a typical purification operation can be carried out can be, for example, from about 1 times the column volume (CV) to about 15 times CV (e.g., about 1 times CV to about 14 times CV, about 1 times CV to about 13 times CV, about 1 times CV to about 12 times CV, about 1 times CV to about 11 times CV, about 2 times CV to about 11 times CV, about 3 times CV to about 11 times CV, about 4 times CV to about 11 times CV, about 5 times CV to about 11x CV, or approximately 5x CV to approximately 10x 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, about 20 minutes to about 40 minutes). Non-limiting examples of elution buffers that can be used in these methods will depend on the capture mechanism and/or recombinant protein. For example, the elution buffer may include different concentrations of salts (for example, increased salt concentration), different pH (for example, increased or decreased salt concentration), or a molecule that will compete with the recombinant protein for binding to the resin, through which a typical capture operation can be performed. Examples of such elution buffers for each example of the capture mechanism presented in the present description are well known in this field.

After the recombinant protein has been eluted from at least one chromatographic column or chromatographic membrane in MCCS or MCCS1, including a resin with which a typical capture operation can be performed, and up to the next volume, the liquid culture medium can be loaded onto at least one chromatographic column or chromatographic membrane , at least one chromatographic column or chromatographic membrane can be equilibrated using regeneration buffer. Regeneration buffer may be passed through at least one chromatography column or chromatography membrane comprising a resin that can perform a typical capture operation at a flow rate of, for example, from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml /minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5.0 mg/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, from about 5.0 ml/minute to about 15.0 ml/minute, or from about 1.0 ml/minute to approximately 15.0 ml/minute). In some examples, the regeneration buffer is a denaturing buffer (eg, any of the denaturing buffers or combinations of denaturing buffers provided herein). The volume of regeneration buffer used to equilibrate at least one chromatographic column or chromatographic membrane containing a resin, through which a typical capture operation can be performed, can be, for example, from about 1 times the column volume (CV) to about 15 times the CV (e.g., approximately 1x CV to approximately 14x CV, approximately 1x CV to approximately 13x CV, approximately 1x CV to approximately 12x CV, approximately 1x CV to approximately 11x CV, about 2 times CV to about 11 times CV, about 3 times CV to about 11 times CV, about 2 times CV to about 5 times CV, about 4 times CV to about 11 times CV, about 5 times CV to about 11 times CV, or about 5 times CV to about 10 times CV).

In some of the methods provided herein, the MCCS or MCCS1 includes a reservoir that stores a liquid containing the recombinant protein at a low pH (e.g., pH below 4.6, below 4.4, below 4.2, below 4, 0, below 3.8, below 3.6, below 3.4, below 3.2, or below 3.0) for, for example, from about 1 minute to 1.5 hours (for example, about 1 hour), and inactivate viruses present in the fluid containing the recombinant protein. An example of a reservoir that can be used to perform a typical virus inactivation operation is a shake flask (e.g., a 500 ml shake flask, e.g., a 500 ml shake flask with a programmable stirrer plate) that can store a liquid containing the recombinant protein , for, for example, from about 1 minute to 1.5 hours, for example, until the liquid containing the recombinant protein is fed into MCCS2. The reservoir used to perform a typical virus inactivation operation may be a 500 ml shake flask with a programmable stirrer plate (e.g., a stirrer plate programmed to agitate (e.g., intermittently stir) the liquid in the reservoir, e.g., every 4 hours). Another example of a reservoir that can be used to perform a typical virus inactivation operation is a plastic bag (e.g., a 500 ml plastic bag) that can store a liquid containing the recombinant protein for, for example, from about 1 minute to 1.5 minutes. hours, for example, before feeding the liquid containing the recombinant protein into MCCS2. In some examples, the fluid containing 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 fed into a tank used for a typical virus inactivation operation. As will be known to those skilled in the art, a variety of other methods can be used to perform a typical virus inactivation operation. For example, UV irradiation of a liquid containing a recombinant protein can also be used to perform a typical virus inactivation operation. Non-limiting examples of reservoirs that can be used to carry out a typical operation to inactivate viruses present in a fluid containing a recombinant protein are presented in the present description.

The MCCS or MCCS1 may include a PCCS comprising four chromatographic columns, where at least three of the four chromatographic columns perform a typical operation for capturing the recombinant protein from a liquid culture medium (for example, using an MCCS comprising any of at least one chromatographic column, including a resin, through which you can perform a typical capture operation (for example, any of those presented in the present description)). In these examples, the fourth column from the PCC can perform a typical operation to inactivate viruses in a liquid containing a recombinant protein (for example, any of the examples of columns provided herein that can be used to achieve inactivation of viruses in a liquid containing a recombinant protein).

In some examples, a fluid comprising a recombinant protein is continuously eluted from MCCS1 (eg PCCS1) and continuously fed into MCCS2 (eg PCCS2). The percentage of recombinant protein isolated in the eluate from MCCS or MCCS1 (e.g., PCCS or PCCS1) may be, for example, at least 70%, at least 72%, at least 74%, at least 76%, at least at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94 %, at least 96% or at least 98%. An eluate from MCCS1 (eg PCCS1) can be fed into MCCS2 (eg PCCS2) using a variety of means known in the art (eg tubing). The eluate from MCCS1 (e.g., PCCS1) can be fed into MCCS2 (e.g., PCCS2) at a flow rate of, for example, from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, about 0.5 ml/minute to about 20 ml/minute, about 0.2 ml/minute to about 15 ml/minute, about 0.5 ml/minute to about 15 ml/minute , from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5.0 mg/minute, from about 0.5 ml/minute 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 of the methods provided herein may further include the step of adjusting the ion concentration and/or pH of the eluate from MCCS1 (eg, PCCS1) before it is fed into MCCS2 (eg, PCCS2). As described herein, the ion concentration and/or pH of an eluate from MCCS1 (eg, PCCS1) can be adjusted (before it is fed into MCCS2) by adding a buffer to the eluate (eg, using a buffer ramp tank). Buffer can be added to the MCCS1 eluate at a flow rate of, for example, from about 0.1 ml/minute to about 15 ml/minute (for example, from about 0.1 ml/minute to about 12.5 ml/minute, from about 0 1 ml/minute to about 10.0 ml/minute, from about 0.1 ml/minute to about 8.0 ml/minute, from about 0.1 ml/minute to about 6 ml/minute, from about 0, 1 ml/minute to 4 ml/minute or from about 0.5 ml/minute to about 5 ml/minute).

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

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

Fine purification and recombinant protein purification

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

At least one chromatographic column or chromatographic membrane that can be used to perform a typical recombinant protein purification operation may include a resin that uses a capture mechanism (for example, any of the capture mechanisms presented in the present description or known in this field), or a resin that can be used to perform anion exchange, cation exchange, molecular sieve or hydrophobic interaction chromatography. The at least one chromatographic column or chromatographic membrane that can be used to perform a typical fine purification operation of a recombinant protein may include a resin that can be used to perform anion exchange, cation exchange, molecular sieve, or hydrophobic interaction chromatography (e.g., any of examples of resins for carrying out anion exchange, cation exchange chromatography, molecular sieve chromatography or hydrophobic interaction chromatography, presented in the present description or known in this field).

Size, shape, and volume of at least one chromatographic column or chromatographic membrane that can be used to perform a typical recombinant protein purification operation, and/or size and shape of at least one chromatographic membrane that can be used to perform a typical recombinant protein fine purification operation , may be any of a combination of examples of sizes, shapes and volumes of chromatographic columns or chromatographic membranes presented in the present description. As known to one skilled in the art, the step of purifying or fine-purifying a recombinant protein, for example, may include the steps of loading, washing, eluting, and equilibrating at least one chromatographic column or chromatographic membrane used to perform a typical recombinant protein purification or fine-purification operation. Typically, the elution buffer leaving the chromatographic column or chromatographic membrane used to perform a typical purification operation includes a recombinant protein. Typically, the addition buffer and/or wash buffer exiting the chromatographic column or chromatographic membrane used in a typical fine purification operation includes a recombinant protein.

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

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

The wash buffer can be passed through at least one chromatographic column or chromatographic membrane at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml /minute to about 10 ml/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, or from about 1.0 ml/minute to approximately 15.0 ml/minute). The volume of wash buffer used (e.g., the combined total volume of wash buffer used when multiple wash buffers are used) can be, for example, from about 1x column volume (CV) to about 15x CV (e.g., from about 1x CV to about 14 times CV, about 1 times CV to about 13 times CV, about 1 times CV to about 12 times CV, about 1 times CV to about 11 times CV, about 2 times CV to about 11 -x CV, approximately 3x CV to approximately 11x CV, approximately 4x CV to approximately 11x CV, approximately 2.5x CV to approximately 5.0x CV, approximately 5x CV to about 11 times CV, or about 5 times CV to about 10 times CV). The total flush 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 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 at least one chromatographic column or chromatographic membrane used to perform a typical recombinant protein purification operation, the recombinant protein is eluted from at least one chromatographic column or chromatographic membrane by passing the elution buffer through at least one chromatographic column or chromatographic membrane used to perform a typical recombinant protein purification operation. The elution buffer can be passed through at least one chromatographic column or chromatographic membrane that can be used to perform a typical recombinant protein purification operation at a flow rate of from about 0.2 ml/minute to about 25 ml/minute (for example, from about 0. 2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute to about 6.0 ml/minute, from about 1.0 ml/minute to about 5, 0 mg/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, or from about 1.0 ml/minute to about 15.0 ml/minute). The volume of elution buffer used to elute the recombinant protein from each of at least one chromatographic column or chromatographic membrane that can be used to perform a typical recombinant protein purification operation can be, for example, from about 1 times the column volume (CV) to about 25 -fold CV (e.g., approximately 1-fold CV to approximately 20-fold CV, approximately 15-fold CV and approximately 25-fold CV, approximately 1-fold CV to approximately 14-fold CV, approximately 1-fold CV to about 13 times CV, about 1 times CV to about 12 times CV, about 1 times CV to about 11 times CV, about 2 times CV to about 11 times CV, about 3 times CV to about 11x CV, approximately 4x CV to approximately 11x CV, approximately 5x CV to approx. about 11 times CV or about 5 times CV to about 10 times 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, about 20 minutes to about 40 minutes, or about 30 minutes to 1.0 hour). Non-limiting examples of elution buffers that can be used in these methods will depend on the resin and/or the biophysical properties of the recombinant protein. For example, the elution buffer may include a different salt concentration (eg, increased salt concentration), a 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 eluting buffers for each of the examples of capture mechanisms presented herein are well known in the art.

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

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

The size, shape, and volume of at least one chromatographic column or chromatographic membrane that can be used to perform a typical recombinant protein fine purification operation, and/or the size and shape of at least one chromatographic membrane that can be used to perform a typical recombinant protein fine purification operation protein, may be any of a combination of examples of sizes, shapes and volumes of chromatographic columns or chromatographic membranes presented in the present description. For example, the size of at least one chromatographic column or chromatographic membrane that can be used to perform a typical recombinant protein fine purification operation may have 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, about 5.0 ml to about 30 ml, about 0.5 ml to about 25 ml, about 0.2 ml to about 10 ml, or about 0.2 ml to about 5 ml). The flow rate of a liquid comprising a recombinant protein loaded onto at least one chromatographic column or chromatographic membrane that can be used to perform a typical recombinant protein fine purification operation can 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 liquid containing the recombinant protein loaded onto at least one chromatographic column or chromatographic membrane that can be used to perform a typical recombinant protein fine purification operation can be, for example, from about 1.0 ml to about 250 ml (for example, 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 chromatographic column or chromatographic membrane used for fine purification may be an anion exchange resin or a cation exchange resin. The resin in at least one chromatographic column or chromatographic membrane used for a typical fine purification operation may be a cation exchange resin (eg, Sartobind® Q resin, Sartorius, Goettingen, Germany).

After the loading step, a displacement step is performed (eg, a displacement buffer is passed through at least one chromatographic membrane or chromatographic membrane to collect recombinant protein substantially non-binding to at least one chromatographic column or chromatographic membrane). In these examples, the displacement buffer may be passed through at least one chromatographic column or chromatographic membrane at a flow rate of from about 0.2 ml/minute to about 50 ml/minute (e.g., from about 1 ml/minute to about 40 ml/minute , from about 1 ml/minute to about 30 ml/minute, from about 5 ml/minute to about 45 ml/minute, from about 10 ml/minute to about 40 ml/minute, from about 0.2 ml/minute to about 20 ml/minute, from about 0.5 ml/minute to about 20 ml/minute, from about 0.2 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 15 ml/minute, from about 0.5 ml/minute to about 10 ml/minute, from about 0.5 ml/minute and about 14 ml/minute, from about 1.0 ml/minute to about 25.0 ml/minute, or from about 1 .0 ml/minute to approximately 15.0 ml/minute). The volume of displacement buffer used can be, for example, from about 1 times the column volume (CV) to about 100 times the CV (e.g., from about 1 times the CV to about 90 times the CV, from about 1 times the CV to about 80 -fold CV, from about 1-fold CV to about 70-fold CV, from about 1-fold CV to about 60-fold CV, from about 1-fold CV to about 50-fold CV, from about 1-fold CV to about 40 times CV, from about 1 times CV to about 30 times CV, from about 1 times CV to about 20 times CV, from about 1 times CV to about 15 times CV, from about 5 times CV to about 20 times CV, about 5 times CV to about 30 times CV, about 1 times CV to about 14 times CV, about 1 times CV to about 13 times CV, about 1 times CV to about 12 times CV, about 1 times CV to about 11 times CV, about 2 times CV to about 11 times CV, about 3 times CV to about 11 times times CV, approximately 4 times CV to approximately 11 times CV, approximately 2.5 times CV to approximately 5.0 times CV, approximately 5 times CV to approximately 11 times CV, or approximately 5 times CV to approximately 10 times CV). The total displacement time may be, for example, from about 1 minute to about 3 hours (for example, 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 10 minutes, about 2 minutes to 15 minutes, or about 2 minutes to 30 minutes). The combined concentration of recombinant protein present in the eluate passed through the column during the loading step and the displacement step may be, for example, from about 0.1 mg/ml to about 100 mg/ml of recombinant protein (for example, from about 0.1 mg/ml 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/m l to about 10 mg/ml, or from about 1 mg/ml to about 5 mg/ml of recombinant protein).

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

In other examples, one or more chromatographic columns and/or chromatographic membranes used to perform a typical fine purification operation includes a resin that selectively binds or retains impurities present in a liquid containing a recombinant protein, and instead of regenerating one or more columns and/or membranes one or more columns and/or membranes are replaced (eg, replaced with essentially similar columns and/or membranes) after reaching or essentially reaching the binding capacity of the resin in one or more columns and/or membranes.

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

Some embodiments of this example may further include the step of correcting the ion concentration and/or pH of the eluate from the three chromatographic columns in the PCCS prior to feeding the eluate into the chromatographic membrane in the PCCS. As described herein, the ion concentration and/or pH of the PCCS three column eluate can be adjusted (before it is fed into the PCCS chromatographic membrane in this example) by adding a buffer to the PCCS three column eluate (e.g., with using a tank for linear buffer correction). Buffer can be added to the eluate at a flow rate of, for example, from about 0.1 ml/minute to about 15 ml/minute (for example, from about 0.1 ml/minute to about 12.5 ml/minute, from about 0.1 ml/minute to about 10.0 ml/minute, from about 0.1 ml/minute to about 8.0 ml/minute, from about 0.1 ml/minute to about 6 ml/minute, from about 0.1 ml /minute to 4 ml/minute or from about 0.5 ml/minute to about 5 ml/minute).

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

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

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

Cultivation methods

Some of the methods provided herein further include the step of culturing cells (e.g., recombinant mammalian cells) secreting the recombinant protein in a bioreactor (e.g., perfusion bioreactor or batch bioreactor) comprising a liquid culture medium, wherein the volume of the liquid culture medium is , essentially free of cells (eg, mammalian cells), is continuously or intermittently removed from the bioreactor (eg, perfusion bioreactor) and fed into MCCS or MCCS1. The bioreactor may have a volume of, for example, from about 1 L to about 10,000 L (e.g., from about 1 L to about 50 L, from about 50 L to about 500 L, from about 500 L to about 1000 L, from 500 L to about 5000L, from 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 about 2000 L or from about 10 L to about 1000 L). The amount of liquid culture medium present in the bioreactor may be, for example, from about 0.5 L to about 5000 L (e.g., from about 0.5 L to about 25 L, from about 25 L to about 250 L, from about 250 liters to about 500 liters, from 250 liters to about 2500 liters, from about 250 liters to about 5000 liters, from about 2500 liters to about 5000 liters, from about 0.5 liters to about 5000 liters, from about 0, 5 L to about 4000 L, from about 0.5 L to about 3000 L, from about 0.5 L to about 2.500 L, from about 50 L to about 2.500 L, from about 5 L to about 50 L, from about 5 L to about 2000 L, from about 5 L to about 1,500 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 batch bioreactor or a perfusion bioreactor. Non-limiting examples and various aspects of cell culture (eg mammalian cell culture) are described below and can be used in any combination.

Cells

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

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

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

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

Culture media

Liquid culture media are known in the art. Liquid culture media (e.g., first and/or second tissue culture media) can be supplemented with mammalian serum (e.g., fetal bovine serum and bovine serum) and/or growth hormone or growth factor (e.g., insulin, transferrin, and epidermal growth factor) . Alternatively or additionally, the liquid culture media (e.g., the first and/or the second liquid culture medium) may be a chemically defined liquid culture medium, a liquid culture medium containing no animal component, a liquid culture medium containing no serum, or liquid culture medium containing serum. Non-limiting examples of chemically defined liquid culture media, liquid culture media containing no animal component, liquid culture media containing no serum, and liquid culture media containing serum are commercially available.

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

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

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

Additional features of exemplary bioreactors

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

Temperature

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

CO ₂

The culture step provided herein may further include exposing the liquid bioreactor culture medium to an atmosphere including most or about 15% CO ₂ (e.g., most 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 mostly or about 1% CO ₂ ).

Perfusion bioreactor

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

Alternatively, the volume removed and added can be varied (eg, incrementally increased) every 24 hour period (or alternatively, an incremental period of time from 1 hour to about 24 hours, or an incremental period of time greater than 24 hours) during the culture period. For example, the volume of the first liquid culture medium removed and the volume of the second liquid culture medium added during each period of 24 hours (or alternatively, an incremental period of time from about 1 hour to more than 24 hours, or an incremental period of time more than 24 hours) during the period cultures can be increased (e.g., gradually or in non-uniform increments) over the culture period from a volume of 0.5% to about 20% of the volume of the bioreactor, or the volume of the first liquid culture medium, up to 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 can be removed, for example, using a mechanical system that can remove the first volume of the first liquid culture medium from the bioreactor (for example, the first volume of the first liquid culture medium, essentially containing no cells from the bioreactor) . Alternatively or additionally, the first volume of the first liquid culture medium can be removed by filtration or gravity flow of the first volume of the first liquid culture medium through a sterile molecular weight cut-off membrane excluding cells (e.g., mammalian cells).

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

Batch bioreactor

The culture step described herein can be carried out using a batch bioreactor. Culture of the cells in a batch bioreactor includes, during most of the culture period, adding (eg, batch or continuous addition) to the first culture liquid a second volume of the second culture liquid. The second liquid culture medium can be continuously added (for example, at a volume addition rate of 0.1% to 300% (for example, 1% to 250%, 1% to 100%, 100% to 200%, 5 % to 150%, 10% to 50%, 15% to 40%, 8% to 80%, or 4% to 30%) of the volume of the bioreactor or the volume of the first liquid culture medium for any specified period of time (e.g. , over a period of 24 hours, over an incremental period of time from about 1 hour to about 24 hours, or over an incremental period of time over 24 hours)) or periodically (for example, every three days, every other day, daily, twice a day, three times a day, four times a day, or five times a day), or any combination thereof. When performed intermittently, the volume added (for example, over a period of about 24 hours, over an incremental period of time from about 1 hour to about 24 hours, or over an incremental period of time over 24 hours) can be, for example, from 0.1% to 300 % (e.g. 1% to 200%, 1% to 100%, 100% to 200%, 5% to 150%, 10% to 50%, 15% to 40%, 8% to 80% or 4% to 30%) of the volume of the bioreactor or the volume of the first liquid culture medium. The added second volume of the second liquid culture medium can, in some cases, be kept approximately the same for each period of 24 hours (or alternatively, an incremental period of time from about 1 hour to about 24 hours, or an incremental period of time greater than 24 hours) for all or part of cultivation period. As is known in the art, the rate at which the second volume of the second liquid culture medium is added (volume/time unit) can be varied during all or part of the culture period. For example, the added volume of the second liquid culture medium can be changed (e.g., gradually increased) during each period of 24 hours (or alternatively, an incremental period of time from 1 hour to about 24 hours, or an incremental period of time greater than 24 hours) during the culture period. For example, the added volume of the second liquid culture medium for each period of 24 hours (or alternatively, an incremental period of time from about 1 hour to more than 24 hours, or an incremental period of time more than 24 hours) during the culture period can be increased (for example, gradually or with in unequal increments) during the culture period from 0.5% to about 20% of the volume of the bioreactor or the volume of the first liquid culture medium to about 25% to about 150% of the volume of the bioreactor or the volume of the first liquid culture medium. The rate at which the second volume of the second liquid culture medium is added (volume/time unit) may be approximately the same for all or part of the culture period.

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

In some cases, the addition to the first liquid culture medium of the second volume of the second liquid culture medium is not carried out for at least 1 hour (for example, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 14 hours, within 16 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours, within 96 hours, or 96 hours later) after inoculation of mammalian cells in the bioreactor. Batch cell culture medium is typically collected at the end of the culture period and used in any of the methods described herein, however, cell batch culture medium can also be collected at one or more times during the culture period and use in any of the ways presented in the present description.

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

Examples of biological production systems

Examples of biological production systems applicable to the implementation of the methods presented in the present description and including MCCS or MCCS1 and MCCS2, described in provisional patent applications US No. 61/775060 and 61/856390 (incorporated by reference). In these exemplary systems, at least one (eg, at least two, three, four, five, or six) chromatography column is present in the MCCS or MCCS1 and/or MCCS2, comprising a gamma irradiated chromatographic resin. For example, the entire system may include a total of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty chromatographic columns including gamma irradiated chromatographic resin. For example, MCCS, MCCS1, and/or MCCS2 may include (or each may include) one, two, three, four, five, six, seven, eight, nine, or ten chromatography columns comprising a gamma irradiated chromatographic resin.

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

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

Additional examples of system structures and features

The MCCS or MCCS1 may include an inlet through which a liquid (eg, liquid culture medium containing essentially no cells) may enter the MCCS or MCCS1, respectively. The inlet may be any structure known in the art for such purposes. It may include, for example, threads, stiffeners, or a seal collar to allow the insertion of the liquid conduit such that once the liquid conduit is inserted into the inlet, liquid will flow into the MCCS or MCCS1 through the inlet without significant fluid leakage from the inlet. Non-limiting examples of inlets that can be used in the systems of the present invention are known and understood by those skilled in the art.

The MCCS or MCCS1 may include at least two chromatographic columns, at least two chromatographic membranes, or at least one chromatographic column and at least one chromatographic membrane and an inlet. MCCS or MCCS1 may be any of the examples of MCCS provided herein or have one or more of any of the examples of MCCS features (in any combination) provided herein. Chromatographic columns and/or chromatographic membranes present in MCCS or MCCS1 may have one or more of any of the examples of shapes, sizes, volumes (layer volumes) and/or typical operations presented in the present description.

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

Through the two or more chromatographic columns and/or chromatographic resins present in the MCCS or MCCS1, one or more routine operations (e.g., recombinant protein capture, recombinant protein purification, recombinant protein fine purification, virus inactivation, ion concentration adjustment, and/or pH of a liquid containing a recombinant protein, or filtration of a liquid containing a recombinant protein). In non-limiting examples, typical operations can be performed by MCCS or MCCS1 to capture a recombinant protein from a liquid (eg, a liquid culture medium) and inactivate viruses present in a liquid containing the recombinant protein. By means of MCCS or MCCS1, any combination of two or more typical operations presented in the present description or known in this field can be performed.

Chromatographic columns and/or chromatographic membranes present in MCCS or MCCS1 can be connected or moved relative to each other using a switching mechanism (eg, a column switching mechanism). The MCCS or MCCS1 may also include one or more (eg, two, three, four, or five) pumps (eg, automated, eg, automated peristaltic pumps). Column switching may trigger a determination of the level of recombinant protein, as determined by UV absorption, corresponding to a particular level of recombinant protein in the fluid passing through the MCCS or MCCS1 (e.g., inlet fluid and/or eluate from one or more chromatographic columns and/or chromatographic membranes in the MCCS or MCCS1), a specific volume of liquid (eg buffer) or a specific period of time. Column switching generally means the mechanism by which at least two different chromatographic columns and/or chromatographic membranes in MCCS or MCCS1 (e.g., two or more different chromatographic columns and/or chromatographic membranes present in MCCS1 or MCCS2) are allowed to pass through different steps (eg, equilibration, loading, elution or washing) for essentially the same time during the implementation of at least part of the method.

The MCCS or MCCS1 may be a Periodic Counter Current Chromatography (PCCS) system. For example, a PCCS that is MCCS or MCCS1 (i.e., PCCS or PCCS1, respectively) may include four chromatographic columns, where the first three columns perform a typical operation to capture the recombinant protein from a liquid (for example, liquid culture medium) and through the fourth PCCS columns perform a typical virus inactivation operation in a liquid containing a recombinant protein. In a PCCS that is MCCS or MCCS1, the column switching mechanism can be used. The PCC system can use a modified ÄKTA system (GE Healthcare, Piscataway, NJ) that can use up to, for example, four, five, six, seven, or eight columns or more.

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

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

Second MCCS

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

Through the chromatographic columns and/or chromatographic membranes present in MCCS2, you can perform one or more typical operations (for example, any of the typical operations presented in the present description, or any combination of the typical operations presented in the present description). In non-limiting examples, typical operations for purifying recombinant protein from a liquid and finely purifying a recombinant protein present in a liquid containing the recombinant protein can be performed by MCCS2. In other non-limiting examples, the MCCS2 can perform typical operations of purifying a recombinant protein present in a liquid, finely purifying a recombinant protein present in a liquid, and filtering a liquid containing the recombinant protein. In another example, the MCCS2 can perform typical operations of purifying a recombinant protein present in a fluid, finely purifying a recombinant protein present in a fluid, filtering a fluid comprising a recombinant protein, and adjusting the ion concentration and/or pH of a fluid comprising a recombinant protein. By means of MCCS2, any combination of two or more typical operations presented in the present description or known in this field can be performed.

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

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

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

MCCS2 may optionally include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one , twenty-two, twenty-three, or twenty-four) buffer linear correction tanks and/or buffer tanks. In other examples, the MCCS2 may include one or more (e.g., two, three, four, five, or six) tanks (e.g., any of the tanks provided herein) that can store liquid that cannot be rapidly delivered to one or several of the chromatographic columns and/or chromatographic membranes in MCCS2.

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

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

The systems described herein may optionally include a liquid conduit located between the final chromatographic column or chromatographic membrane in the MCCS2 and the outlet port. The systems described herein may further include one or more filters in fluid communication with a liquid conduit located between the final chromatographic column or chromatographic membrane in the MCCS2 and the outlet, such that the filter can remove, for example, precipitated material, solid particles or bacteria from the liquid present in the liquid conduit located between the final chromatographic column or chromatographic membrane in the MCCS2 and the outlet port.

Some examples of systems presented herein also include a bioreactor in fluid communication with an MCCS or MCCS1 inlet. Any of the examples of bioreactors described herein or known in the art can be used in the systems of the present invention.

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

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

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

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

EXAMPLES

Example 1 Effect of Gamma Irradiation on the Binding Capacity of a Chromatographic Resin at t0

A series of experiments were carried out to investigate the effect of gamma irradiation on the binding capacity of a bimodal chromatographic resin having anion exchange and hydrophobic properties (AE resin). The AE resin used in these experiments was Capto Adhere (GE Healthcare Life Sciences) having an N-benzyl-N-methyl-ethanolamine ligand, an average particle size of 75 µm, and an ion exchange capacity of 0.09-0.12 mmol Cl ^-1 / ml of medium. Resin AE was left untreated or treated to reduce the bioburden of the resin (gamma irradiated at a dose of 25 kGy). Irradiation was carried out using 0.2 ml of resin suspended in 50 mm sodium phosphate, pH 7.0.

The amount of protein (Fabrazyme®) bound to untreated AE Resin (in liquid culture medium) and 25 kGy gamma-irradiated AE Resin at t0 (no chromatography performed) was determined at different concentrations of the target protein (Fabrazyme®). 1 shows binding isotherms at t0 for untreated and 25 kGy gamma irradiated AE resin. These data indicate that the binding of the target protein does not change after gamma irradiation of the resin at t0.

Example 2 Effect of Gamma Irradiation on the Binding Capacity of a Chromatography Resin over Multiple Cycles and the Use of Denaturing Buffers to Reduce Loss of Binding Capacity

Experiments were carried out to test the effect of gamma irradiation on the binding capacity of the chromatographic resin over multiple cycles. In multi-column chromatography, each of the columns used is loaded with protein (Fabrazyme® in liquid culture medium) to its binding capacity. The column was equilibrated and washed with 20 mM MES (2-( N -morpholino)ethanesulfonic acid), pH 7.0. Elution buffer, 200 mM arginine, 270 mM MES, 20% ethylene glycol, pH 7.5, was used to elute the bound protein from the columns. To test the lifetime properties of the gamma irradiated chromatography resin in this example, columns containing untreated or 25 kGy gamma irradiated AE chromatography resin (prepared as described in Example 1) were run using a Multi Column Chromatography System (MCCS ) or individual columns were used to recycle chromatography under conditions simulating MCCS conditions (each column loaded to its static binding capacity prior to washing and elution). Each of the washing and elution steps in the experiments described in this example was carried out in stages.

Multiple column chromatography runs were performed using a separate chromatography column comprising AE resin (raw, 15 kGy gamma irradiated or 25 kGy gamma irradiated resin) and wash buffer 700 mM arginine, 100 mM acetate (pH 3.0 ), followed by a 1 N NaOH solution (after elution of the recombinant protein) in each cycle. The percentage of normalized binding capacity (compared to untreated resin at t0) was determined over multiple cycles. The data obtained from these experiments indicate that although the binding capacity of the untreated and gamma irradiated resin was similar at t0, the binding capacity of the untreated and gamma irradiated resin showed different rates of decline in binding capacity over multiple chromatography cycles (Figure 2) . Although the untreated resin showed a decrease in binding capacity over multiple chromatography cycles, the gamma-irradiated resin showed a greater decrease in binding capacity over multiple chromatography cycles (Figure 2). The calculated rate of decline in binding capacity for each resin in this set of experiments is also shown in Figure 3. These data indicate that the use of gamma irradiation to reduce chromatography bioburden results in a reduction in resin binding capacity, progressively deteriorating over multiple chromatography cycles. Carried out a number of experiments to determine which of the many denaturing buffers can be used to restore the loss of binding capacity of the gamma-irradiated chromatographic resin. In these experiments, chromatography runs using raw AE Resin were run using 700 mM arginine, 100 mM acetate (pH 3.0) wash buffer followed by 1 N NaOH solution (low pH, arginine). Chromatography runs were also performed using a 25 kGy gamma-irradiated resin using one of the following combinations of buffers to wash the resin after elution of the recombinant protein in each run: 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2, 5 followed by 1 N NaOH solution (low pH, urea); 6 M guanidine HCl (pH 2.5) followed by 1 N NaOH (or 1 M NaOH + 1 M NaCl) solution (low pH, guanidine); 0.5% Triton-X 100 in 0.1 M acetic acid (pH 2.5) followed by a solution of 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) followed by solution 1 H NaOH (low pH, Triton); 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) followed by 1 N NaOH solution (low pH, organic); or 700 mM arginine, 100 mM acetate (pH 3.0) followed by 1 N NaOH solution (low pH, arginine). Normalized percent binding capacity (normalized by the binding capacity of untreated resin at t=0), percent recombinant protein recovery (relative to the amount of recombinant protein present in the fluid loaded onto the resin) per cycle, and host cell protein levels (ng/mg) were determined. ) present in the eluate per cycle. The data presented in Figure 4 indicates that all denaturing buffers tested were able to reduce the loss of binding capacity of the gamma irradiated chromatography resin, with the exception of 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) and 700 mM arginine, 100 mM acetate (pH 3.0). The data presented in Figure 4 shows that with all denaturing buffers except 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) and 700 mM arginine, 100 mM acetate (pH 3.0), it is possible to effectively purify the gamma-irradiated chromatography resin and maintain the binding capacity of the gamma-irradiated chromatographic resin for multiple chromatography cycles.

The data presented in Figure 6 indicates that all denaturing buffers tested result in a stable percentage recovery of recombinant protein bound to the gamma irradiated chromatography resin per cycle over multiple cycles. The data presented in Figure 6 indicates that with each of the denaturing buffers tested, any amount of protein bound to the gamma irradiated chromatographic resin can be recovered per cycle to a similar extent. The data presented in Figure 7 indicates that the use of the various denaturing buffers tested results in acceptable levels of host cell protein in the recombinant protein eluate in each cycle.

The denaturing buffer used in each run is believed to restore the binding capacity of the gamma irradiated chromatographic resin by releasing the highly bound proteins from the resin. Representative chromatograms of multiple chromatography runs were recorded using three chromatographic columns comprising 25 kGy gamma-irradiated AE Resin, washed with 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5, and a solution of 1 N NaOH after elution of the recombinant protein (after each cycle) (figure 5). The chromatograms show that treatment of the gamma irradiated resin with denaturing buffer resulted in the release of essentially the same amount of protein from the gamma irradiated resin in each of the three chromatographic columns over multiple cycles (see arrows in Figure 5).

Overall, these data indicate that various denaturing buffers can be used to restore the binding capacity of the gamma irradiated chromatography resin to achieve the expected properties.

OTHER IMPLEMENTATION OPTIONS

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

Claims (89)

1. A method for performing chromatography using a gamma-irradiated cation-exchange and/or anion-exchange chromatographic resin to purify a recombinant protein, comprising: (a) obtaining a chromatographic column containing gamma-irradiated cation exchange and/or anion exchange chromatographic resin; (b) performing a first cycle of chromatography through columns, wherein the first cycle of chromatography comprises restoring the binding capacity of the gamma-irradiated cation exchange and/or anion exchange chromatography resin by exposing the gamma-irradiated cation exchange and/or anion exchange chromatography resin to a denaturing buffer containing one or more of urea , guanidine hydrochloride and Triton™ X-100; and (c) performing at least one additional chromatography cycle through a column, wherein said column is subjected to said denaturing buffer during each of at least one additional chromatography cycle, and wherein the flow rate, volume, and concentration of denaturing buffer are selected such that substantially restoring the binding capacity lost due to gamma irradiation of the cation exchange and/or anion exchange chromatography resin. 2. The method of claim 1, wherein performing the first chromatography run in step (b) and/or said at least one additional chromatography run in step (c) comprises the steps of: (a) capturing said recombinant protein by exposing a gamma-irradiated cation exchange and/or anion exchange chromatographic resin to a liquid containing said recombinant protein; (b) washing the gamma irradiated cation exchange and/or anion exchange chromatography resin by exposing the gamma irradiated cation exchange and/or anion exchange chromatography resin to a wash buffer; (c) eluting the recombinant protein by exposing the gamma-irradiated cation exchange and/or anion exchange chromatography resin to an elution buffer; and (d) restoring the binding capacity of the gamma-irradiated cation exchange and/or anion exchange chromatography resin by exposing the gamma-irradiated cation exchange and/or anion exchange chromatography resin to a denaturing buffer. 3. The method of claim 2, wherein the liquid containing said recombinant protein is a liquid culture medium. 4. The method of claim 1, wherein the first chromatography run in step (b) and said at least one additional chromatography run in step (c) are performed using a closed and integrated system. 5. The method of claim 4, wherein said denaturing buffer has a sterility assurance level of approximately 1 x 10 ^-6 or less. 6. The method of claim 1 wherein the denaturing buffer contains 6 to 9 M urea. 7. The method of claim 1 wherein the denaturing buffer contains 5 to 7 M guanidine hydrochloride. 8. The method of claim 1 wherein the denaturing buffer contains Triton™ X-100. 9. The method of claim 1, wherein the denaturing buffer is selected from the group consisting of: 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5; 6 M guanidine hydrochloride, pH 2.5; and 0.5% Triton™ X-100 in 0.1 M acetic acid, pH 2.5. 10. The method of claim 1, wherein the first chromatography cycle of step (b) further comprises, after exposing the gamma-irradiated cation exchange and/or anion exchange chromatography resin to a denaturing buffer, exposing the gamma-irradiated cation and/or anion exchange chromatography resin to a wash buffer, containing from about 0.5 M to about 1.5 M sodium hydroxide. 11. The method of claim 1 wherein the column is part of a multi-column chromatography system (MCCS). 12. The method of claim 11 wherein the MCCS is a Periodic Counter Current Chromatography System (PCCS). 13. The method of claim 1, wherein the gamma-irradiated cation exchange and/or anion exchange chromatographic resin is treated with a dose of gamma radiation from about 10 kGy to about 40 kGy. 14. The method of claim 13, wherein the gamma-irradiated cation exchange and/or anion exchange chromatographic resin is treated with a dose of gamma radiation from about 15 kGy to about 35 kGy. 15. The method of claim 14, wherein the gamma-irradiated cation exchange and/or anion exchange chromatographic resin is treated with a dose of gamma radiation from about 20 kGy to about 30 kGy. 16. The method of claim. 1, where step (c) includes the implementation of four or more additional cycles of chromatography. 17. The method of claim 16 wherein step (c) comprises performing nine or more additional chromatography runs. 18. The method of claim 17 wherein step (c) comprises performing fourteen or more additional chromatography runs. 19. The method of claim 18, wherein step (c) comprises performing nineteen or more additional chromatography cycles. 20. The method of claim 21 wherein step (c) comprises performing twenty-four or more additional chromatography runs. 21. The method of claim. 20, where step (c) includes the implementation of twenty-nine or more additional cycles of chromatography. 22. The method of claim 21 wherein step (c) comprises performing thirty nine or more additional chromatography runs. 23. The method of claim 1, wherein step (c) is carried out continuously for a period of at least 4 days. 24. The method of claim 23, wherein step (c) is carried out continuously for a period of at least 7 days. 25. The method of claim 24, wherein step (c) is carried out continuously for a period of at least 14 days. 26. The method of claim 25, wherein step (c) is carried out continuously for a period of at least 28 days. 27. The method of claim 1 wherein the recombinant protein is a recombinant therapeutic protein. 28. Integrated, closed and continuous method for the production of purified recombinant protein, including: (a) obtaining a liquid culture medium containing the recombinant protein, essentially free of cells; and (b) continuously feeding the liquid culture medium into a multi-column chromatography system (MCCS) comprising at least one chromatography column containing a gamma-irradiated cation exchange and/or anion exchange chromatography resin, and performing at least two chromatography cycles with at least one chromatographic column, where the specified at least one chromatographic column during each of the specified at least two cycles of chromatography is exposed to a denaturing buffer containing one or more of urea, guanidine hydrochloride and Triton™ X-100, and where the flow rate, volume and the concentration of the denaturing buffer is chosen such that there is essentially a recovery of the binding capacity lost due to gamma irradiation of the cation exchange and/or anion exchange chromatographic resin; wherein said denaturing buffer has a sterility assurance level of approximately 1×10 ^-6 or less, and said method is integrated, and is carried out continuously from the liquid culture medium to the MCCS eluate, which is a purified recombinant protein. 29. The method of claim 28, wherein at least two different routine operations are performed by the MCCS. 30. The method of claim 29, wherein the application of the MCCS includes switching columns. 31. The method of claim 28 wherein all columns in the MCCS contain a gamma irradiated cation exchange and/or anion exchange chromatography resin. 32. The method of claim 28, wherein the MCCS performs typical recombinant protein capture and virus inactivation operations. 33. The method of claim 28, wherein the MCCS performs typical recombinant protein capture and purification operations. 34. The method of claim 28, wherein the MCCS is a batch countercurrent chromatography system. 35. The method of claim 28, wherein the recombinant protein is a therapeutic recombinant protein. 36. The method of claim 35, further comprising formulating the purified recombinant protein into a pharmaceutical composition. 37. The method of claim 28 wherein the denaturing buffer contains 6 to 9 M urea. 38. The method of claim 28 wherein the denaturing buffer contains 5 to 7 M guanidine hydrochloride. 39. The method of claim 28 wherein the denaturing buffer contains Triton™ X-100. 40. The method of claim 28, wherein the denaturing buffer is selected from the group consisting of: 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5; 6 M guanidine hydrochloride, pH 2.5; and 0.5% Triton™ X-100 in 0.1 M acetic acid, pH 2.5. 41. The method of claim 28, wherein, in each of said at least two chromatography cycles, after exposing said at least one chromatographic column to a denaturing buffer, said at least one chromatographic column is exposed to a wash buffer containing from about 0 .5 M to about 1.5 M sodium hydroxide. 42. Integrated, closed and continuous method for the production of recombinant protein, including: (a) obtaining a liquid culture medium containing the recombinant protein, essentially free of cells; (b) continuously feeding the liquid culture medium into the first multi-column chromatography system (MCCS1); (c) capturing the recombinant protein from the liquid culture medium using MCCS1; (d) obtaining an eluate from MCCS1 containing the recombinant protein and continuously feeding the eluate into a second multi-column chromatography system (MCCS2); (e) continuously feeding the recombinant protein from the eluate into MCCS2 and then eluting the recombinant protein to thereby obtain a purified recombinant protein, where: the method is integrated, and it is carried out continuously from the liquid culture medium to the purified recombinant protein, and at least one column in MCCS1 and/or MCCS2 is a chromatographic column containing a gamma-irradiated cation exchange and/or anion exchange chromatographic resin, and this chromatographic resin is exposed to a denaturing buffer containing one or more of urea, guanidine hydrochloride, and Triton™ X-100 during each cycle of the process, and the flow rate, volume, and concentration of the denaturing buffer are chosen such that there is substantially recovery of the binding capacity lost by due to gamma irradiation of the cation exchange and/or anion exchange chromatographic resin. 43. The method of claim 42, wherein the MCCS1 and/or MCCS2 perform at least two different routine operations. 44. The method of claim 42, wherein the use of MCCS1 or MCCS2 or both includes switching columns. 45. The method of claim 42, wherein the MCCS1 additionally performs routine recombinant protein capture and virus inactivation operations. 46. The method of claim 42 wherein the MCCS2 performs routine purification and fine purification of the recombinant protein. 47. The method of claim 42, wherein at least two chromatographic columns are used in MCCS1 and/or MCCS2. 48. The method of claim 42, wherein all of the chromatographic columns in MCCS1 and MCCS2 are chromatographic columns containing a gamma-irradiated cation exchange and/or anion exchange chromatographic resin. 49. The method of claim 42, wherein the MCCS1 is a first periodic countercurrent chromatography system (PCCS1). 50. The method of claim 42 wherein the MCCS2 is the second Periodic Counter Current Chromatography System (PCCS2). 51. The method of claim 42, wherein the recombinant protein is a therapeutic recombinant protein. 52. The method of claim 51, further comprising formulating the purified recombinant protein into a pharmaceutical composition. 53. The method according to p. 28 or 42, where the method is carried out continuously for a period of at least 4 days. 54. The method of claim 53, wherein the method is carried out continuously for a period of at least 7 days. 55. The method of claim 54, wherein the method is carried out continuously for a period of at least 14 days. 56. The method of claim 55, wherein the method is carried out continuously for a period of at least 28 days. 57. The method according to claim 28 or 42, wherein the gamma-irradiated cation exchange and/or anion exchange chromatographic resin is treated with a dose of gamma radiation from about 10 kGy to about 40 kGy. 58. The method of claim 57, wherein the gamma-irradiated cation exchange and/or anion exchange chromatographic resin is treated with a dose of gamma radiation from about 15 kGy to about 35 kGy. 59. The method of claim 58, wherein the gamma-irradiated cation exchange and/or anion exchange chromatographic resin is treated with a dose of gamma radiation from about 20 kGy to about 30 kGy. 60. The method of claim 42, wherein the denaturing buffer contains 6 to 9 M urea. 61. The method of claim 42, wherein the denaturing buffer contains 5 to 7 M guanidine hydrochloride. 62. The method of claim 42 wherein the denaturing buffer contains Triton™ X-100. 63. The method of claim 42, wherein the denaturing buffer is selected from the group consisting of: 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5; 6 M guanidine hydrochloride, pH 2.5; and 0.5% Triton™ X-100 in 0.1 M acetic acid, pH 2.5. 64. The method of claim 42, wherein the gamma-irradiated cation exchange and/or anion exchange chromatography resin is exposed to a wash buffer containing about 0.5 M to about 1.5 M sodium hydroxide after exposure to the denaturing buffer.

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