RU2790876C2 - Method for production of recombinant therapeutic protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to a method for biological production of recombinant therapeutic protein. The method provides for a system that ensures the flow of the first volume of liquid containing recombinant therapeutic protein from the first vessel to the second vessel through the volume of sterilizing gas contained in the upper part of the second vessel, and purification of the recombinant therapeutic protein by ensuring the flow of the second volume of liquid containing recombinant therapeutic protein from the second vessel into at least one chromatographic column and/or at least one chromatographic membrane, producing the recombinant therapeutic protein. Moreover, the first vessel is a bioreactor, a chromatography system, a microfiltration system or an ultrafiltration/diafiltration system.

EFFECT: implementation of the invention provides fewer manual manipulations with the sterilized system and reduces the risk of contamination of the sterilized system.

17 cl, 2 dwg, 1 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No. 62/068181, filed Oct. 24, 2014, the entire contents of which are incorporated herein by reference.

FIELD OF TECHNOLOGY

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

BACKGROUND OF THE INVENTION

Mammalian cells containing a nucleic acid that encodes a recombinant protein are often used to produce proteins of therapeutic or commercial importance. In the current environment of product manufacturing processes, biotechnology companies are increasingly required to develop innovative solutions for the highly flexible and cost-effective production of therapeutics (eg, therapeutic protein-based drugs).

In a continuous biological manufacturing process, it is often necessary to remove fluids from a sterile process vessel. The removal of such fluid may be either a continuous flow or an intermittent flow based on some predetermined signal. Removal must be carried out in such a way as to protect the sterility of the vessel from which the fluid is removed. This can be of particular difficulty in biological production where the fluid flow has the ability to promote biological growth, which may eventually grow progressively in a sterile process vessel. The main method currently used is a batch transfer method in which the waste streams are collected in a second sterile vessel, and when this vessel is full, it is then disconnected from the sterile processing vessel, and then the waste is discharged. This process is not ideal because it is batchwise (not continuous) and takes a long time to process and sterilize the vessels. Such manipulations can also lead to risk factors in the process if any step is not performed. Alternatively, the same process can be carried out with pre-sterilized bags, however, the cost of the bags may be prohibitive for a continuous process, and the use of bags does not eliminate the risk factors in the process.

SHORT DESCRIPTION

The present invention is based in part on the development of a release process and associated hardware to allow intermittent or continuous release of fluid streams (eg, removal) from a sterilized system (eg, a sterile process vessel) that contains a sterile process stream. The isolation process uses a separating vessel to separate the sterile process stream from the environment and waste streams. The separating vessel is only partially filled and maintains a headspace in the vessel, with the headspace containing a sterilizing agent. A sterilizing agent (eg, a sterilizing gas (eg, a gas containing ozone, ethylene oxide, nitrogen dioxide, or evaporated hydrogen peroxide)) may be sparged into the vessel or introduced directly into the headspace of the vessel. The sterilizing agent maintains a sterilizing atmosphere within the headspace of the vessel that provides separation between the incoming sterile process stream and the outgoing fluid stream (eg, waste stream). The concentration of the sterilizing agent (eg sterilizing gas) is controlled within the headspace of the vessel to provide the necessary sterilizing atmosphere.

In one aspect, the invention provides a method for preventing contamination of a sterilized system, the method comprising providing a system comprising a first vessel, wherein the first vessel contains liquid, allowing a first volume of liquid to flow from the first vessel, through a volume of sterilizing gas, into the second vessel. Sterilized systems contemplated herein include, without limitation, biological manufacturing systems and pharmaceutical manufacturing systems. The first vessel is a sterilized vessel. In exemplary embodiments, the first vessel contains a component of a biological production system. For example, the first vessel may be (e.g., any of the exemplary bioreactors described herein or known in the art) one or more chromatography system components (e.g., a chromatographic column), one or more microfiltration system components, or one or more system components for ultrafiltration/diafiltration (UF/DF). For a biological production system, the liquid of the first vessel may be a liquid culture medium and/or a liquid containing the recombinant therapeutic protein. In some embodiments, the first vessel fluid contains a cell containing the recombinant therapeutic protein. The recombinant therapeutic protein may be a protein secreted from the cell or not secreted from the cell.

In some aspects, the sterilizing gas is selected from the group consisting of ozone, ethylene oxide, nitrogen dioxide, vaporized hydrogen peroxide (eg, ozone-containing gas, ethylene oxide-containing gas, nitrogen oxide-containing gas, and hydrogen peroxide-containing gas).

The first volume of liquid flowing from the first vessel to the second vessel may be a waste stream. In another aspect, the first volume of fluid flowing from the first vessel into the second vessel contains the recombinant therapeutic protein. Alternatively, the first volume of fluid flowing from the first vessel into the second vessel does not contain the recombinant therapeutic protein (i.e., the first volume of liquid is a waste stream or contains culture medium prior to cell culture initiation). The first liquid volume may contain fermentation by-products.

In one aspect, the methods disclosed herein further include allowing a second volume of liquid to flow from the second vessel to a recombinant protein purification and post-purification unit. For example, the method disclosed herein may further comprise allowing a second volume of liquid from a second vessel to flow into a first multi-column chromatography system (MCCS1), collecting said recombinant therapeutic protein from the liquid culture medium using the MCCS1, wherein the MCCS1 eluate containing the recombinant the therapeutic protein is continuously fed into a second multi-column chromatography system (MCCS2), and purification and post-purification of the recombinant therapeutic protein using MCCS2, wherein the eluate from MCCS2 is the recombinant therapeutic protein; and wherein the process is co-located and proceeds continuously from said liquid in the first vessel to the eluate from MCCS2, which is the recombinant therapeutic protein. In some embodiments, the implementation of the second volume of liquid contains the recombinant protein.

In one aspect, the methods disclosed herein further include allowing a second volume of fluid to flow from the second vessel into a reservoir to accommodate the biological waste stream. The reservoir may be, for example, a waste disposal bin, or a beaker, or other container for storing and/or disposing of liquid waste.

The disclosure also provides a system for separating sterile process streams from non-sterile environments. In one aspect, the system includes a first vessel containing a fluid outlet; and at least one second vessel comprising a fluid inlet in fluid communication with the fluid outlet of the first vessel and configured such that the fluid entering the second vessel passes through the free space filled with the sterilizing gas inside the second vessel, a fluid outlet configured such that the fluid exiting the second vessel is removed below the sterilizing gas-filled free space inside the second vessel, at least one gas inlet and at least one outlet for gas. In some examples, the first vessel is a sterilized vessel. In an exemplary embodiment, the first vessel is a component of a biological production system. For example, the first vessel is a fluid tube (e.g., any of the exemplary bioreactors described herein or known in the art), one or more chromatography system components (e.g., a chromatographic column), one or more microfiltration system components or one or more ultrafiltration/diafiltration system components. The bioreactor is, for example, a perfusion bioreactor, a fed-batch bioreactor, a production bioreactor or an inoculum growth bioreactor. In some embodiments, the fluid outlet of the second vessel is in fluid communication with the recombinant protein purification and purification unit. In an exemplary embodiment, the first vessel and the second vessel are placed on a stand.

In some aspects, the systems disclosed herein further comprise a fluid tube located between the first vessel and the second vessel, and optionally further comprise a filter disposed in the fluid tube between the first vessel and the second vessel and configured to be removed particulate matter from the fluid in the fluid tube. The systems disclosed herein may also include a pumping system (eg, a pump), the pumping system being housed in a fluid conduit. In some examples, the systems disclosed herein include a pump in fluid communication with the fluid outlet of the first vessel, a pump in fluid communication with the fluid outlet of the composite vessel, or both. In one embodiment, the pumping system is configured to remove a volume of fluid from the outlet of the vessel and allow the volume to flow into the fluid inlet of the second vessel.

In one aspect, the systems disclosed herein comprise a headspace filled with a sterilizing gas within a second vessel. The sterilizing gas may be, for example, a gas selected from the group consisting of ozone, ethylene oxide, nitrogen dioxide or vaporized hydrogen peroxide. In some embodiments, at least one gas inlet is connected to one or more gas bubbling elements that allow gas to be released into the second vessel and delivered to the headspace. The term "bubbling element" refers to a porous element (eg, filter, open tube, or frit) for bubbling gas through a liquid. In order to fill the free space, the second vessel comprises at least one gas inlet in gas phase communication with a system for generating or delivering a sterilizing gas or for generating and delivering a sterilizing gas. In some embodiments, the sterilant gas generation or delivery system is an ozone generation or delivery system or an ozone generation and delivery system. In some embodiments, the sterilizing gas delivery system is bottled gas. Further, in some embodiments, the second vessel includes at least one gas outlet configured to continuously or intermittently withdraw gas from the second vessel. For systems using ozone, the gas outlet is in gas phase communication with the ozone destruction unit. To monitor the concentration or amount of sterilant gas contained in the second vessel, the system may include, for example, a probe or sensor for dissolved gas to monitor the concentration of sterilant gas in the headspace of the second vessel.

In one aspect, the first vessel includes a fluid outlet in fluid communication with the fluid inlet of the second vessel. In an exemplary embodiment, the second vessel comprises a fluid inlet configured such that the volume of liquid entering the second vessel passes through the free space (e.g., a fluid inlet located on the second vessel at a position above the liquid level), a fluid outlet configured such that the liquid exiting the second vessel flows below the free space filled with the sterilizing gas (for example, a fluid outlet located on the second vessel at a position below the liquid level), at least one inlet a gas port and at least one gas outlet, the fluid inlet being in fluid communication with the first vessel. Preferably, the volume of sterilizing gas is located within the free space of the second vessel. In order to fill the headspace, the sterilizing gas may be bubbled into the second vessel or injected directly into the headspace of the second vessel. In some examples, the second vessel is at least partially filled with liquid.

In some embodiments, the liquid in the first vial contains the recombinant therapeutic protein.

In the context of this document, the singular form of a noun denotes one or more specific nouns. For example, the expression "recombinant mammalian cell" means "one or more recombinant mammalian cells".

The term "vessel" is well known in the art and means a device (eg container) of any shape or size with an internal volume suitable to hold a volume of liquid or gas. The vessel can be open (i.e., a device that interacts directly with its environment) or closed (i.e., an isolated device that does not interact with its environment in any way). The term "vessel" includes, for example, a device with an internal volume suitable for culturing a number of cells (eg, recombinant mammalian cells) in a liquid culture medium under a controlled set of physical conditions that allow the maintenance or proliferation of cells. Non-limiting examples of vessels are fluid tubes, bioreactors (e.g., any of the illustrative bioreactors described herein or known in the art), one or more chromatography system components (e.g., a chromatographic column), one or more system components for microfiltration, one or more UF/Diafiltration system components, beakers, receivers or tubes.

The term "sterilization" is known in the art and refers to any approved process used to obtain a sterile composition, for example, a process that eliminates (removes) or destroys all forms of life, including pathogens transmitted by various routes (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in a drug, or in a compound such as a biological culture medium. Sterilization can be achieved by the application of heat, chemicals (eg gas), radiation, high pressure or filtration, or combinations thereof.

The term "sterilizing gas" in the context of this document refers to a gas or gaseous composition that makes it possible to obtain a sterile composition, for example, in a process by which all forms of life are eliminated (removed) or destroyed, including causative agents of diseases transmitted by various routes (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in a drug, or in a compound such as a biological culture medium.

"Absolutely sterile" or "absolutely sterile" are terms used to describe a composition or process that/which is/are completely free/do not include self-replicating biological contaminants. For example, the term can be applied to a gamma irradiated vessel, internal surface and contents of the vessel and/or buffer. An absolutely sterile composition or process may be pure (as the term is known in the art).

"Sterile" or "sterility" are terms used to describe a composition or process that has a guaranteed level of sterility of about or less than 1.0 x 10 ^-6 (e.g., about or less than 1.0 x 10 ^-7 , about or less than 1 .0×10 ^-8 , about or less than 1.0×10 ^-9 or 1×10 ^-10 ). Determining whether a composition or process is sterile can be investigated using a number of validated manufacturing processes known in the art. For example, a sterile composition or process may be completely devoid of viable self-replicating biological contaminants (eg, any of the self-replicating biological contaminants described herein). A sterile composition or process may also be pure (as the term is known in the art). There is no contamination in sterile cell culture.

The term Assured Level of Sterility or "SAL" is known in the art and refers to the level of confidence that absolute sterility has been achieved within a batch of processed units. Probability is generally calculated from the results of inactivation studies performed during validation and expressed in the form 1×10 ^-n .

The terms "sterilized vessel" and "sterile process vessel" are used interchangeably and refer to a vessel that has been subjected to a sterilization process. As used herein, the terms "sterilized vessel" or "sterile processing vessel" include, for example, a vessel containing a controlled bioburden monoculture (eg, a bioburden controlled monoculture of recombinant mammalian cells). As used herein, the term "sterile system" refers to a system containing a set of one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) sterile process vessels that work together to achieve a specific result (eg, expression and purification of a recombinant protein from a liquid culture medium). "Sterile system" refers to a system of a total of two or more interconnected or switching vessels, wherein at least one or more vessels of the system is a sterilized vessel.

In the context of this document, the term "biological production system" or "bioproduction system" refers to a system for producing a biological drug. The term "pharmaceutical manufacturing system" refers to a system for producing a small molecule drug (eg, drug, prodrug, or drug product). The components of biological production systems and pharmaceutical systems contemplated herein include, for example, one or more bioreactors for culture initiation and growth, flasks, fluid tubes, vessels, one or more chromatography system components (e.g., chromatographic column, pumps, process vessels), one or more components of a filtration system (e.g. one or more components of a microfiltration system or one or more components of an ultrafiltration/diafiltration system) and other devices used to isolate and purify the drug. Systems may be open, closed, co-located, or continuous, as defined herein, or as otherwise generally understood by one skilled in the art.

The term "biological drug" in the context of this document refers to any therapeutic substance produced or obtained from a living organism or its products, which is used in the prevention, diagnosis or treatment of pathology. Thus, a biological drug or biological pharmaceutical product is a medicinal product obtained using biotechnology, for example, a protein (for example, a recombinant therapeutic protein), or a nucleic acid (DNA, RNA or antisense oligonucleotides) used for therapeutic or in vivo diagnostic purposes.

The term "small molecular weight drug" in the context of this document refers to a therapeutic agent with a small molecular weight, which is used in the prevention, diagnosis or treatment of pathology. The therapeutic agent is typically synthesized by organic chemistry, but may also be isolated from natural sources such as plants, fungi, and microorganisms.

In the context of this document, the first vessel is in "gas phase communication" with the second vessel by connecting the first and second vessels through a device or tube allowing flow or communication in the gas phase between the vessels. Similarly, the first vessel is in "fluid communication" with the second vessel by connecting the first and second vessels through a device or tube allowing flow or fluid communication between the vessels. In accordance with the teachings of the present invention, the terms "fluid communication" and "gas phase communication" are intended to be synonymous terms. In this regard, the fluid is intended to include a substance, either a liquid or a gas, configured to flow or adhere to the outer boundary of its container. In this regard, not only a liquid falls under the definition of a fluid, but also a gas, since the gas can flow and adhere to the outer boundary of the container within which it is present.

The term "perfusion bioreactor" is known in the art and means a bioreactor with an internal volume for culturing a number of cells (for example, recombinant mammalian cells) in a liquid culture medium, and having a means (for example, an outlet, an inlet, a pump, or other such device) for periodically or continuously withdrawing liquid culture medium from the bioreactor, and having a means (for example, an outlet, inlet, pump, or other such device) for adding essentially the same volume of replacement liquid culture medium to the bioreactor. The addition of the replacement liquid culture medium can be carried out substantially simultaneously or shortly after removal of the liquid culture medium from the bioreactor. The means for removing the liquid culture medium from the bioreactor and the means for adding the replacement liquid culture medium may be one device or system.

The term "production bioreactor" is a technical term and means a large scale bioreactor (for example, with an internal volume of more than 500 L, 1000 L, 5000 L, 10000 L, 20000 L, 50000 L or 100000 L). For example, the production bioreactor may be a perfusion bioreactor.

The term "fed-batch bioreactor" is a term in the art and means a bioreactor containing a number of cells (e.g., recombinant mammalian cells) in a first liquid culture medium, where culturing the cells present in the bioreactor comprises the periodic or continuous addition of a second liquid culture medium into 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 fed-batch culture, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch cultures, the second liquid culture medium is added as a dry powder.

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

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

The term "culturing" or "culturing a cell" means the maintenance or proliferation of a mammalian cell (eg, a recombinant mammalian cell) under a controlled set of physical conditions.

The term "mammalian cell culture" or "cell culture" means a liquid culture medium containing a number of mammalian cells that maintain or proliferate under a controlled set of physical conditions.

The term "liquid culture medium" or "culture medium" means a fluid medium that contains sufficient nutrients to allow a cell (eg, mammalian cell) to grow or proliferate in vitro . For example, the liquid culture medium may contain one or more of amino acids (eg, 20 amino acids), purine (eg, hypoxanthine), pyrimidine (eg, thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, nicotinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc and sodium bicarbonate. In some embodiments, the implementation of the liquid culture medium may contain serum from a mammal. In some embodiments, the implementation of the liquid culture medium does not contain serum or other extract from a mammal (liquid culture medium with a certain composition). In some embodiments, the liquid culture medium may contain trace metals, mammalian growth hormone, and/or mammalian growth factor. Another example of a liquid culture medium is a minimal medium (eg, a medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture medium are described in this document. Additional examples of liquid culture media are known in the art and are commercially available. The liquid culture medium may contain mammalian cells at any density. For example, within the context of this document, a volume of liquid culture medium removed from a production bioreactor may be substantially free of mammalian cells.

The terms "recombinant therapeutic protein" or "recombinant protein" is known in the art and means any therapeutic protein produced by recombinant DNA technology. As used herein, a "recombinant therapeutic protein" includes, for example, an antibody or antibody fragment, an enzyme, an engineered protein, or an immunogenic protein or protein fragment.

The term "protein fragment" or "polypeptide fragment" means a portion of a polypeptide sequence that is at least or about 4 amino acids long, at least or about 5 amino acids long, at least or about 6 amino acids long, at least or about 7 amino acids long, at least or about 8 amino acids, at least or about 9 amino acids, at least or about 10 amino acids, at least or about 11 amino acids, at least or about 12 amino acids, at least or about 13 amino acids, at least or about 14 amino acids, at least or about 15 amino acids, at least or about 16 amino acids, at least or about 17 amino acids, at least or about 18 amino acids, at least or about 19 amino acids, or at least or about 20 amino acids or longer than 20 amino acids T. A fragment of a recombinant protein can be obtained using any of the processes described in this document.

The term "engineered protein" means a polypeptide that is 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 result in increased stability and/or catalytic activity of the engineered enzyme), fusion proteins, antibodies (e.g., bivalent antibodies, trivalent antibodies, or diabodies) and antigen-binding proteins that contain at least one recombinant backbone sequence.

The term "combined process" means a process that is carried out using structural elements that function together to provide a certain result (for example, obtaining an isolated recombinant protein from a liquid culture medium).

The term "continuous process" means a process by which a fluid is continuously transported through at least part of the system.

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

The term "perfusion culture" is a term in the art and means culturing a cell culture in a vessel (e.g., a bioreactor), where culturing the cell culture in the vessel involves intermittent or continuous removal of a liquid culture medium present in the vessel (e.g., a liquid culture medium that is essentially does not contain a cell), and the simultaneous or shortly thereafter adding essentially the same volume of replacement liquid culture medium to the vessel. In some examples, gradually change (e.g., increase or decrease) the volume of liquid culture medium that is removed and the volume of replacement culture medium that is added over discrete periods (e.g., about a 24-hour period, a period ranging from about 1 minute up to about 24 hours, or a period greater than 24 hours) during the culture period (eg, culture medium refeed rate daily). The fraction of medium that is removed and replaced each day may vary depending on the specific cells to be cultured, initial seeding density and cell density at a particular point in time. "RV" or "reactor volume" means the volume of culture medium available at the start of the culture process (eg, the total volume of culture medium available after inoculation).

The term "fed-batch culture" is a term in the art and means a vessel (for example, a production bioreactor) containing a number of cells (for example, mammalian cells) in a liquid culture medium, where the cultivation of cells present in the vessel (for example, a production bioreactor), involves intermittent or continuous addition of fresh liquid culture medium to the vessel without substantial or significant removal of the liquid culture medium from the vessel during culture. The fresh liquid culture medium may be the same as the liquid culture medium present in the vessel at the time the culture was started. In some examples of fed-batch culture, the fresh liquid culture medium is a concentrated form of the liquid culture medium present in the vessel at the start of the culture. In some examples of fed-batch culture, fresh culture medium is added as a dry powder.

"Stand" is a term from the field of technology and in the context of this document refers to a three-dimensional solid structure that can function as a platform or support for the system described in this document. If the stand contains one or more structures that provide movement (for example, wheels, rollers, etc.), it can give mobility to the system or part of it. Non-limiting examples of stands are described in this document. Additional examples of stands are known in the art.

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

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

DESCRIPTION OF THE DRAWINGS

In FIG. 1 is a schematic diagram illustrating a typical system for separating sterile process streams from non-sterile media according to the present invention.

In FIG. 2 is a schematic diagram illustrating a typical system for separating sterile process streams from non-sterile media according to the present invention.

DETAILED DESCRIPTION

Disposal processes and associated hardware are provided herein for allowing fluid streams to be separated from a sterilized system (eg, a sterile process vessel) that contains a sterile process stream. The extraction processes described herein provide many advantages. For example, the release processes allow intermittent or continuous removal of fluid streams from the sterilized system, which allows for less manual manipulation of the sterilized system and a reduced risk of contamination of the sterilized system. For example, the isolation processes described herein provide for intermittent or continuous removal of liquid (e.g., waste streams, liquid containing recombinant therapeutic proteins) from the bioreactor, which in turn allows for less manual cell culture handling and reduced risk of contamination. cell cultures. Non-limiting aspects of these selection processes are described herein and may be used in any combination.

The methods described herein include causing volumes of fluid to flow from a first vessel to a second vessel, allowing volumes of fluid to flow from a third vessel to a fourth vessel, or allowing volumes of fluid to flow from a fifth vessel to a sixth vessel. As can be understood from the prior art, there are many ways in which a volume of fluid can flow from the first vessel to the second vessel, such as gravity flow or pump flow. Thus, in some aspects, the systems described herein may also comprise one or more (eg, two, three, four, or five) pumps (eg, automatic, eg, automatic peristaltic pumps). One or more pumps may be placed in a fluid tube placed between the first vessel and the second vessel. For example, the systems described herein may also include one or more pumps configured to remove a volume of fluid from the outlet of the first vessel and allow the volume to flow into the second vessel. In some examples, one or more pumps are configured to remove a volume of fluid from a sterile process vessel outlet and allow the volume to flow into a separating vessel fluid inlet, as described herein. In some examples, one or more pumps are in fluid communication with at least one fluid outlet of the separating vessel. The fluid may be removed from the sterile process vessel by a pumping system (eg, variable tangential flow filtration (ATF) or tangential flow fluid filtration (TFF) system).

In some examples, the systems described herein may also include one or more (eg, two, three, four, or five) filters to remove unwanted biological contaminants (eg, mammalian cells, bacteria, yeast cells, viruses, or mycobacteria) and/ or particulate matter (eg, precipitated proteins) from a liquid (eg, liquid culture medium or fluid present in any of the systems or processes described herein).

In some aspects, the invention provides methods for preventing contamination of a sterilized system, comprising providing a system comprising a first vessel, wherein the first vessel contains a liquid, allowing a first volume of liquid to flow from the first vessel, through a volume of sterilizing gas, into the second vessel.

In some aspects, the invention provides methods for preventing contamination of a sterilized system, comprising providing a system comprising a first vessel, wherein the first vessel contains a liquid, allowing a first volume of liquid to flow from the first vessel, through a volume of sterilizing gas, into the second vessel. In some examples, the first vessel is a sterile process vessel, wherein the sterile process vessel includes a fluid outlet in fluid communication with the second vessel's fluid inlet. In some examples, the second vessel is a separating vessel, as described herein, and the volume of sterilizing gas is placed within the headspace of the separating vessel.

In some aspects, the invention provides systems for separating sterile process streams from non-sterile media. For some examples, the system comprises a sterile process vessel (eg, a first vessel) containing a fluid outlet and at least one separating vessel (eg, a second vessel), wherein at least one separating vessel comprises (i) an inlet for fluid, in fluid communication with the fluid outlet of the first vessel and configured in such a way that the fluid entering the second vessel passes through the free space filled with sterilizing gas inside the second vessel, (ii) the fluid outlet environment, configured so that the fluid leaving the second vessel is removed below the sterilizing gas-filled free space inside the second vessel, (iii) at least one gas inlet, and (iv) at least one gas outlet . In some examples, the systems disclosed herein further comprise a fluid tube placed between the first vessel and the second vessel.

The isolation processes disclosed herein use a vessel (i.e., a "separation vessel") to separate a sterile process stream from the environment and waste streams. In some examples, the separating vessel comprises (i) a fluid inlet in fluid communication with the fluid outlet of the sterile process vessel and configured such that fluid entering the separating vessel passes through the sterilizing gas filled void. a space inside the separating vessel, (ii) a fluid outlet configured such that the fluid exiting the separating vessel is removed below the sterilizing gas-filled free space inside the separating vessel, (iii) at least one gas inlet, and (iv) at least one gas outlet.

As can be understood from the prior art, the separating vessel may have a number of different volumes. For example, a separating vessel may have an internal volume

from about 0.20 L to about 20 L (for example, from about 0.20 L to about 18 L, from about 0.20 L to about 16 L, from about 0.20 L to about 14 L, from about 0, 20 L to about 12 L, from about 0.20 L to about 10 L, from about 0.20 L to about 9.0 L, from about 0.20 L to about 8.0 L, from about 0.20 L to about 7.0 L, from about 0.20 L to about 6.0 L, from about 0.20 L to about 5.0 L, from about 0.20 L to about 4.0 L, from about 0, 20 L to about 3.0 L, from about 0.20 L to about 2.0 L, from about 0.20 L to about 1.0 L, from about 0.50 L to about 18 L, from about 0, 50 L to about 16 L, from about 0.50 L to about 14 L, from about 0.50 L to about 12 L, from about 0.50 L to about 10 L, o t about 0.50 L to about 9.0 L, from about 0.50 L to about 8.0 L, from about 0.50 L to about 7.0 L, from about 0.50 L to about 6.0 L, from about 0.50 L to about 5.0 L, from about 0.50 L to about 4.0 L, from about 0.50 L to about 3.0 L, from about 0.50 L to about 2 .0 L, from about 0.50 L to about 1.0 L, from about 1.0 L to about 20 L, from about 1.0 L to about 18 L, from about 1.0 L to about 16 L, from about 1.0 L to about 14 L, from about 1.0 L to about 12 L, from about 1.0 L to about 10 L, from about 1.0 L to about 9.0 L, from about 1, 0 L to about 8.0 L, from about 1.0 L to about 7.0 L, from about 1.0 L to about 6.0 L, from about 1.0 L to about 5.0 L, from about1.0 L to about 4.0 L, about 1.0 L to about 3.0 L, about 1.0 L to about 2.0 L, about 1.0 L to about 1.0 L) , or about 0.20 L, about 0.50 L, about 1.0 L, about 2.0 L, about 3.0 L, about 4.0 L, about 5.0 L, about 6.0 L, approximately 7.0 L, approximately 8.0 L, approximately 9.0 L, approximately 10.0 L, approximately 12.0 L, approximately 14.0 L, approximately 16.0 L, approximately 18.0 L, or approximately 20 .0 l.

The separating vessel is only partially filled and maintains free space in the vessel. The headspace may contain a sterilizing agent (eg a sterilizing gas). In some examples, the sterilizing gas-filled headspace contained within the separating vessel spans from about 3% to about 97% of the total internal volume of the separating vessel; from about 5% to about 95% of the total internal volume of the separating vessel, for example, from about 10% to about 90% of the total internal volume of the separating vessel; from about 15% to about 85% of the total internal volume of the separating vessel; from about 20% to about 80% of the total internal volume of the separating vessel; from about 25% to about 75% of the total internal volume of the separating vessel; from about 30% to about 70% of the total internal volume of the separating vessel; from about 35% to about 65% of the total internal volume of the separating vessel; from about 40% to about 60% of the total internal volume of the separating vessel; from about 45% to about 55% of the total internal volume of the separating vessel; or approximately 5% of the total internal volume of the separating vessel; approximately 10% of the total internal volume of the separating vessel; approximately 15% of the total internal volume of the separating vessel, approximately 20% of the total internal volume of the separating vessel; approximately 25% of the total internal volume of the separating vessel; approximately 30% of the total internal volume of the separating vessel; approximately 35% of the total internal volume of the separating vessel; about 40% of the total internal volume of the separating vessel; approximately 45% of the total internal volume of the separating vessel; approximately 50% of the total internal volume of the separating vessel; approximately 55% of the total internal volume of the separating vessel; approximately 60% of the total internal volume of the separating vessel; approximately 65% of the total internal volume of the separating vessel; approximately 75% of the total internal volume of the separating vessel; approximately 80% of the total internal volume of the separating vessel; approximately 85% of the total internal volume of the separating vessel; approximately 90% of the total internal volume of the separating vessel; or approximately 95% of the total internal volume of the separating vessel.

Exemplary sterilizing gases for use in the systems and methods disclosed herein include, for example, ozone gas, ethylene oxide gas, nitrogen dioxide gas, and vaporized hydrogen peroxide (e.g., ozone-containing gas, ethylene oxide-containing gas, nitric oxide-containing gas , and a gas containing hydrogen peroxide), or any suitable mixture of such gases. In some examples, the sterilizing gas contained within the headspace of the separating vessel may be maintained, for example, at a temperature of from about 15°C to about 70°C, from about 20°C to about 65°C, from about 25°C to about 60 °C, from about 30°C to about 55°C, from about 35°C to about 50°C, or from about 40°C to about 45°C.

Ozone provides many benefits as a sterilizing gas. Ozone is a very effective sterilizing agent due to its strong oxidizing properties, which are capable of destroying a wide range of pathogens, including prions. The high reactivity of ozone-based products that consume ozone can be destroyed by passing the ozone over a simple catalyst that converts ozone to oxygen. This also means that the cycle time is relatively short. In some examples, the headspace contains ozone, such as a gas containing ozone, with an ozone concentration of at least about 3000 ppm, such as at least about 4000 ppm, at least about 5000 ppm, at least about 6000 ppm, at least at least about 7,000 ppm, at least about 8,000 ppm, at least about 9,000 ppm, at least about 10,000 ppm, at least about 15,000 ppm, at least about 20,000 ppm, at least about 50,000 ppm, at least about 100,000 ppm, at least about 500,000 ppm, or at least about 1,000,000 ppm.

Ethylene oxide has microbiocidal properties and can kill all known viruses, bacteria, and fungi, including bacterial spores, and is compatible with most materials (eg, sterile process vessels used in biological manufacturing processes). In some examples, the headspace contains ethylene oxide, such as a gas containing ethylene oxide, with an ethylene oxide concentration of at least about 500 ppm, such as at least about 850 ppm, at least about 1000 ppm, at least about 2000 ppm, at least at least about 3000 ppm, at least about 4000 ppm, at least about 5000 ppm, at least about 6000 ppm, at least about 7000 ppm, at least about 8000 ppm, at least about 9000 ppm, at least about 10,000 ppm, at least about 15,000 ppm, at least about 20,000 ppm, at least about 50,000 ppm, at least about 100,000 ppm, at least about 500,000 ppm, or at least about 1,000,000 ppm.

Nitrogen dioxide gas (NO ₂ ) is effective as a sterilant against a wide range of microorganisms including known bacteria, viruses and spores. In some examples, the headspace contains nitrogen dioxide, such as a gas containing nitrogen dioxide, with an ethylene oxide concentration of at least about 500 ppm, at least about 850 ppm, at least about 1000 ppm, at least about 2000 ppm, at least at least about 3000 ppm, at least about 4000 ppm, at least about 5000 ppm, at least about 6000 ppm, at least about 7000 ppm, at least about 8000 ppm, at least about 9000 ppm, at least about 10,000 ppm, at least about 15,000 ppm, at least about 20,000 ppm, at least about 50,000 ppm, at least about 100,000 ppm, at least about 500,000 ppm, or at least about 1,000,000 ppm.

Hydrogen peroxide (H ₂ O ₂ ) has good sterilizing properties and can be split into water and oxygen. In some examples, the headspace contains hydrogen peroxide, such as a gas containing hydrogen peroxide, with an ethylene oxide concentration of at least about 5 ppm, at least about 5 ppm, at least about 10 ppm, at least about 50 ppm, at least at least about 100 ppm, at least about 250 ppm, at least about 500 ppm, at least about 850 ppm, at least about 1000 ppm, at least about 2000 ppm, at least about 3000 ppm, at least about 4000 ppm, at least about 5000 ppm, at least about 6000 ppm, at least about 7000 ppm, at least about 8000 ppm, at least about 9000 ppm, at least about 10000 ppm, at least about 15,000 ppm, at least about 20,000 ppm, at least about 50,000 ppm, according to at least about 100,000 ppm, at least about 500,000 ppm, or at least about 1,000,000 ppm.

The separating vessel may further comprise a component for monitoring the concentration of the sterilant (eg, sterilizing gas) within the headspace of the chamber in order to monitor the sterilizing atmosphere. For example, the separating vessel may include a sensor to monitor the sterilant gas concentration within the headspace, or a sensor (eg, a dissolved gas probe) to monitor the dissolved gas concentration of the liquid contained in the separating vessel.

In some examples, the liquid-filled space within the separating vessel represents from about 3% to about 97% of the total volume of the separating vessel; from about 5% to about 95% of the total volume of the separating vessel; from about 10% to about 90% of the total volume of the separating vessel; from about 15% to about 85% of the total volume of the separating vessel; from about 20% to about 80% of the total volume of the separating vessel; from about 25% to about 75% of the total volume of the separating vessel; from about 30% to about 70% of the total volume of the separating vessel; from about 35% to about 65% of the total volume of the separating vessel; from about 40% to about 60% of the total volume of the separating vessel; from about 45% to about 55% of the total volume of the separating vessel; or approximately 5% of the total volume of the separating vessel; approximately 10% of the total volume of the separating vessel; approximately 15% of the total volume of the separating vessel; approximately 20% of the total volume of the separating vessel; approximately 25% of the total volume of the separating vessel; approximately 30% of the total volume of the separating vessel; approximately 35% of the total volume of the separating vessel; approximately 40% of the total volume of the separating vessel; approximately 45% of the total volume of the separating vessel; approximately 50% of the total volume of the separating vessel; approximately 55% of the total volume of the separating vessel; approximately 60% of the total volume of the separating vessel; approximately 65% of the total volume of the separating vessel; approximately 75% of the total volume of the separating vessel; approximately 80% of the total volume of the separating vessel; approximately 85% of the total volume of the separating vessel; approximately 90% of the total volume of the separating vessel; or approximately 95% of the total volume of the separating vessel.

The separating vessel may include at least one gas inlet for the purpose of introducing a sterilizing gas into the head space of the separating vessel. As can be understood from the prior art, there are many ways in which gas can be introduced into the headspace of a vessel. For example, the gas may be bubbled into the vessel or injected directly into the headspace of the vessel. Thus, at least one gas inlet can be connected to one or more gas bubbling elements which allow gas to be released into the separating vessel. The gas inlet may be in gas phase communication via tubing with a system for generating or delivering a sterilizing gas, or for generating and delivering a sterilizing gas (eg, ozone, ethylene oxide, nitrogen dioxide, or vaporized hydrogen peroxide). For example, the gas inlet may be in gas phase communication with the ozone generating system, as is well known in the art.

The separating vessel may comprise at least one gas outlet configured to continuously or intermittently remove gas from the free space of the separating vessel. As can be understood from the prior art, the gas outlet can be configured to automatically discharge the gas, the pressure of the gas in the headspace must be excessive. The gas outlet may be in gas phase communication with the unit configured to contain, destroy, or reduce the amount of sterilizing gas. For example, the gas outlet may be in gas phase communication with the ozone destruction unit. Ozone destroyers are known in the art and can be catalytic, thermal, thermocatalytic or activated carbon. The catalytic blocks can either use magnesium dioxide or be coated with aluminum and palladium and destroy ozone at a temperature of approximately 50°C. Thermal breakers typically operate at approximately 120°C.

In some examples, the separation vessel described herein comprises at least one fluid inlet in fluid communication with at least one fluid outlet of the sterile process vessel and configured such that the fluid entering the separating vessel passes through the free space filled with sterilizing gas inside the separating vessel. In some aspects, at least one fluid inlet of the separating vessel is in fluid communication with at least one fluid outlet of the sterile process vessel via a fluid conduit.

In some examples, the separating vessel described herein includes at least one fluid outlet. For some exemplary system configurations, at least one separating vessel fluid outlet is in fluid communication with the recombinant protein purification and purification unit. Thus, in some aspects, the methods disclosed herein include allowing a volume of liquid to flow from a separating vessel (eg, a second vessel) to a recombinant protein purification and post-purification unit.

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 liquid culture medium or one or more other components (eg, DNA, RNA, other proteins, endotoxins, viruses, etc.) present in or secreted by the mammalian cell). For example, cleaning can be done during or after the primary collection step. Purification can be carried out using any method known in the art, for example, using a resin, membrane, or any other solid support that binds either the recombinant protein or contaminants (for example, by using affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography or molecular sieve chromatography). The recombinant protein can be purified from a fluid containing the recombinant protein using at least one chromatographic column and/or chromatographic membrane (eg, any of the chromatographic columns or chromatographic membranes described herein).

The term "finishing" is a term in the art and means a step performed to remove remaining trace or small amounts of contaminants or impurities from a fluid containing a recombinant therapeutic protein, bringing it closer to the final required purity. For example, post-purification can be accomplished by passing a fluid containing the recombinant therapeutic protein through chromatographic column(s) or membrane absorber(s) that selectively binds either to the target recombinant therapeutic protein or to small the amounts of contaminants or impurities present in the fluid containing the recombinant therapeutic protein. In such an example, the eluate/filtrate of the chromatographic column(s) or membrane absorber(s) contains the recombinant therapeutic protein.

For example, the invention provides methods comprising: allowing a volume of liquid containing a recombinant protein to flow from a separating vessel (e.g., a second vessel) into a first multi-column chromatography system (MCCS1), collecting said recombinant therapeutic protein from a liquid culture medium using MCCS1, this MCCS1 eluate containing the recombinant therapeutic protein is continuously fed into the second system for multi-column chromatography (MCCS2); and purification and post-purification of the recombinant therapeutic protein using MCCS2, wherein the eluate from MCCS2 is the recombinant therapeutic protein; and while the technological process is combined and is carried out continuously from the specified first vessel to the eluate from MCCS2, which is a recombinant therapeutic protein.

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

The term "collection" means a step performed to partially purify or isolate (e.g., provide 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), concentrating and stabilizing 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., culture medium proteins or one or more other components (e.g., DNA, RNA, or other proteins) present in the mammalian cell or secreted her). Typically, the collection is carried out using a resin that binds the recombinant protein (for example, by using affinity chromatography). Non-limiting methods for harvesting recombinant protein from liquid culture medium or dilute liquid culture medium are described herein and others are known in the art. The recombinant protein can be harvested 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 described herein).

The term "eluate/filtrate" is an art term and refers to the fluid that exits a chromatographic column or chromatographic membrane that contains a detectable amount of a recombinant protein (eg, a recombinant therapeutic protein).

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

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

For some exemplary system configurations, at least one separating vessel fluid outlet is in fluid communication with a waste material receiving and/or accommodating reservoir (e.g., a biological process fluid accommodating vessel, receptacle, or unit known to those skilled in the art). this technical field).

Recovery processes and systems allow fluid streams to be recovered from a sterilized system vessel (eg, a sterile process vessel) that contains a sterile process stream. In some examples, a sterile process vessel contains a sterile process stream and includes at least one fluid outlet to remove fluid from the vessel. For the processes and systems described herein, at least one fluid outlet is in fluid communication with at least one fluid inlet of the separating vessel, wherein the fluid inlet of the separating vessel is configured in such a manner that the fluid entering the separating vessel passes through the free space filled with sterilizing gas inside the separating vessel.

As can be understood from the prior art, a sterile process vessel may have a number of different volumes. For example, a sterile stage process vessel may have an internal volume of from about 0.50 L to about 200 L (e.g., from about 0.50 L to about 180 L, from about 0.50 L to about 160 L, from about 0. 50 L to about 140 L, from about 0.50 L to about 120 L, from about 0.50 L to about 100 L, from about 0.50 L to about 90 L, from about 0.50 L to about 80 L , from about 0.50 L to about 70 L, from about 0.50 L to about 60 L, from about 0.50 L to about 50 L, from about 0.50 L to about 40 L, from about 0.50 L to about 30 L, from about 0.50 L to about 20 L, from about 0.50 L to about 10 L, from about 0.50 L to about 5.0 L, from about 1.0 L to about 200 l, from about 1.0 l to about 180 l, from about 1 0 L to about 160 L, from about 1.0 L to about 140 L, from about 1.0 L to about 120 L, from about 1.0 L to about 100 L, from about 1.0 L to about 90 l, from about 1.0 l to about 80 l, from about 1.0 l to about 70 l, from about 1.0 l to about 60 l, from about 1.0 l to about 50 l, from about 1, 0 L to about 40 L, from about 1.0 L to about 30 L, from about 1.0 L to about 20 L, from about 1.0 L to about 10 L, from about 1.0 L to about 5, 0 L, from about 1.5 L to about 200 L, from about 1.5 L to about 180 L, from about 1.5 L to about 160 L, from about 1.5 L to about 140 L, from about 1 .5 L to about 120 L, from about 1.5 L to about 100 L, from about 1.5 L to about 90 L, from about 1.5 L to about 80 L, from about 1.5 L to about 70 L, from about 1.5 L to about 60 L, from about 1.5 L to about 50 L, from about 1.5 L to about 40 L, from about 1.5 L to about 30 L, from about 1.5 L to about 20 L, from about 1.5 L to about 10 L, from about 1.5 L to about 5.0 L , from about 2.0 L to about 200 L, from about 2.0 L to about 180 L, from about 2.0 L to about 160 L, from about 2.0 L to about 140 L, from about 2.0 L to about 120 L, from about 2.0 L to about 100 L, from about 2.0 L to about 90 L, from about 2.0 L to about 80 L, from about 2.0 L to about 70 L, from about 2.0 L to about 60 L, from about 2.0 L to about 50 L, from about 2.0 L to approx. significantly 40 L, from about 2.0 L to about 30 L, from about 2.0 L to about 20 L, from about 2.0 L to about 10 L, from about 2.0 L to about 5.0 L, from about 2.5 liters to about 200 liters, from about 2.5 liters to about 180 liters, from about 2.5 liters to about 160 liters, from about 2.5 liters to about 140 liters, from about 2.5 liters to about 120 liters, from about 2.5 liters to about 100 liters, from about 2.5 liters to about 90 liters, from about 2.5 liters to about 80 liters, from about 2.5 liters to about 70 liters, from about 2.5 L to about 60 L, from about 2.5 L to about 50 L, from about 2.5 L to about 50 L, from about 2.5 L to about 40 L, from about 2.5 L to about 30 L, from about 2.5 L to about 20 L, from about 2.5 L to about 10 L, from about 2.5 L to about 5.0 L, from about 5.0 L to about 200 L, from about 5.0 L to about 180 L, from about 5.0 L to about 160 L, from about 5.0 L to about 140 L, from about 5.0 L to about 120 L, from about 5.0 L to about 100 L, from about 5.0 L to about 90 L, from about 5.0 L to about 80 L, from about 5.0 L to about 70 L, from about 5.0 L to about 60 L, from about 5.0 L to about 50 L, from about 5.0 L to about 40 L, from about 5.0 L to about 30 L, from about 5.0 L to about 20 L, or from about 5.0 L to about 10 L).

In some examples, the vessel that contains the sterile process stream is a component of a biological manufacturing system. Components of biological production systems contemplated herein include, for example, a flask, a fluid tube, a bioreactor, one or more chromatography system components (e.g., a chromatographic column), one or more microfiltration system components, or one or more system components. for ultrafiltration/diafiltration.

In some embodiments, the bioreactor is a perfusion bioreactor, a fed-batch bioreactor, or a production bioreactor. The perfusion bioreactor can be any of the exemplary perfusion bioreactors described herein or known in the art. For example, the perfusion bioreactor may be made of stainless steel or plastic (eg, a plastic sterile bag). The inner surface of the perfusion bioreactor may have at least one coating (eg, at least one coating of gelatin, collagen, poly-L-ornithine, polystyrene, and laminin) and, as is known in the art, one or more O ₂ bubbling holes. , CO ₂ and N ₂ into the liquid culture medium and a device for stirring the liquid culture medium. The perfusion bioreactor may also be equipped with a mechanical device that allows a volume of fluid (e.g., liquid culture medium) to be removed from the bioreactor, and optionally a filter within the mechanical device that removes cells from the fluid during the process of removing the fluid from the bioreactor ( for example, a variable tangential flow (ATF) system, a tangential flow filtration (TFF) system, or a filtration system as described in US Provisional Application No. 61/878502). The bioreactor may be equipped with one or more pumps and one or more reservoirs for holding the removed fluid.

The volume of liquid can be removed, for example, using a mechanical system and/or by passing or gravity flow of the volume through a sterile molecular weight cut-off membrane that excludes mammalian cells present in the volume.

As can be understood in the art, the vessel that contains the sterile process stream can be any apparatus used in the art for the purpose of culturing mammalian cells (eg, a flask (eg, a stirred flask), a spin tube, or a bioreactor). For example, the vessel that contains the sterile process stream may be any apparatus used in the art for culturing recombinant mammalian cells. The vessel may contain internal stirring means (eg a stirrer), or the vessel may be shaken externally (eg by using a rotating and/or rocking platform). The vessel can be made of stainless steel or plastic (eg plastic sterile bag). In some embodiments, the vessel may be a disposable bioreactor (e.g., 3 L MilliporeTM Mobius® Cellready Disposable Bioreactor, 20 L Pierre Guerin ATM1 NucleoTM Disposable Bioreactor, 50 L Sartorius Cultibag STRTM Disposable Bioreactor, 20 L Sartorius Cultibag RMTM Bioreactor , 50 L Sartorius Cultibag OrbitalTM, 5 L GE Wave Bioreactor 2/10 System, 25 L GE Wave Bioreactor 20/50 System, 200 L GE Wave Bioreactor 200 System, or GE Wave Bioreactor 500/1000 System with a volume of 500 l). The inner surface of the vessel may have at least one coating (for example, at least one coating of gelatin, collagen, poly-L-ornithine, polystyrene and laminin) and, as is known in the art, one or more holes for sparging O ₂ . CO ₂ and N ₂ in the first liquid culture medium. The vessel may be equipped with one or more sensors. If the vessel is made of a non-rigid plastic material (eg, a plastic sterile bag), the vessel may be associated with an external support that surrounds and supports the vessel.

The recombinant mammalian cell may be a human, mouse, hamster or monkey cell. For example, a recombinant mammalian cell may refer to a cell line, e.g., Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells, CHO-K1 cells, C02.31 clonal cells, A14.13 clonal cells, C02.57 clonal cells, and F05.43 clonal 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 (Vero) renal epithelium or Madin-Darby dog (Cocker Spaniel) (MDCK) renal epithelial cells.

A nucleic acid encoding a recombinant protein can be introduced into a mammalian cell to produce a recombinant mammalian cell using a variety of methods known in the art of molecular biology and molecular genetics. Non-limiting examples include transfection (eg, lipofection), transduction (eg, infection with a lentivirus, adenovirus, or retrovirus), and electroporation. In some cases, the nucleic acid that encodes the recombinant protein is not stably integrated into the chromosome of the recombinant mammalian cell (transient transfection), while in other recombinant mammalian cells the nucleic acid is integrated into the chromosome. Alternatively or in addition, the nucleic acid encoding the recombinant protein may be present in the plasmid and/or in a mammalian artificial chromosome (eg, human artificial chromosome). Alternatively or in addition, the nucleic acid can be introduced into a mammalian 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 containing the nucleic acid may optionally also contain a selectable marker (eg a gene that confers resistance to hygromycin, puromycin or neomycin) in a mammalian cell.

Liquid culture media (culture media) are known in the art. Liquid culture media can be supplemented with mammalian serum (eg, fetal bovine serum and bovine serum) and/or growth hormone or growth factor (eg, insulin, transferrin, and epidermal growth factor). Any of the liquid culture media described herein may be selected from the group consisting of a liquid culture medium containing no animal components, a liquid culture medium containing no serum, a liquid culture medium containing serum, a liquid culture medium with a specific chemical composition and liquid culture medium containing no proteins. Non-limiting examples of chemically defined liquid culture media, animal-free liquid culture media, serum-free liquid culture media, and serum-containing liquid culture media are commercially available.

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

Many different liquid culture media are known in the art that can be used to culture cells (eg, mammalian cells) at any stage of any of the methods described herein. Media components that may also be used in the processes of the present invention include, but are not limited to chemically known hydrolysates (CDs), 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.

A liquid culture medium derived from a culture of recombinant mammalian cells can be filtered or purified to provide a liquid culture medium that is substantially free of cells and/or viruses. Methods for filtering or purifying a liquid culture medium to remove cells are known in the art (e.g., 0.2 µm filtration, variable tangential flow filtration (ATF ^™ ), tangential flow filtration (TFF) system, or any of the systems described in U.S. Provisional Application No. 61/878502). Recombinant cells can also be removed from the liquid culture medium using centrifugation and removal of the settled liquid, which is a liquid culture medium that is substantially cell-free, or by allowing the cells to settle to the gravity bottom of a container (e.g., vessel) containing the liquid culture medium. medium, and removing the liquid culture medium (liquid culture medium, which essentially does not contain cells), which is located at some distance from the settled recombinant mammalian cells. In some embodiments, one or more (eg, two, three, or all) of the first culture medium, the second culture medium, the third culture medium, and the fourth culture medium are identical.

The liquid culture medium used in any of the steps in any of the methods described herein may be any of the types of liquid culture medium described herein or known in the art. In any of the exemplary methods for isolating a recombinant protein described herein, a liquid culture medium obtained from a culture of producer cells can be diluted by adding a second fluid (eg, buffer).

A liquid culture medium containing a recombinant protein (e.g., a recombinant therapeutic protein) that is substantially cell-free can be stored (e.g., below about 15°C (e.g., below about 10°C, below about 4°C, below about 0°C, below about -20°C, below about -50°C, below about -70°C, or below about -80°C) for at least 1 day (e.g., at least about 2 days, at least about 5 days, at least about 10 days, at least about 15 days, at least about 20 days, or at least about 30 days) before isolation of the recombinant protein (e.g., before loading the liquid culture medium into the first MCCS ( for example, the first PCCS)). used to isolate the recombinant protein.

The recombinant protein may be a recombinant therapeutic protein. Non-limiting examples of recombinant therapeutic proteins that can be obtained using the methods presented herein include immunoglobulins (including immunoglobulin light and heavy chains, antibodies or antibody fragments (for example, any of the antibody fragments described herein), enzymes ( for example, galactosidase (eg, alpha-galactosidase), Myozyme® or Cerezyme®), proteins (eg, human erythropoietin, tumor necrosis factor (TNF), or interferon alfa or beta), or immunogenic or antigenic proteins or protein fragments (eg, proteins for use in a vaccine.) The recombinant therapeutic protein can be an engineered antigen-binding polypeptide that contains the backbone of at least one multifunctional recombinant protein (see, for example, recombinant antigen-binding proteins described in Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009 and U.S. Patent Application Publication No. 2012/0164066 (BK incorporated herein by reference in its entirety)). Неограничивающие примеры рекомбинантных терапевтических белков, которые представляют собой антитела, включают: панитумумаб, омализумаб, абаговомаб, абциксимаб, актоксумаб, адалимумаб, адекатумумаб, афелимомаб, афутузумаб, алацизумаб, алацизумаб, алемтузумаб, алирокумаб, альтумомаб, аматуксимаб, аматуксимаб, анатумомаб, анрукинзумаб, аполизумаб , арцитумомаб, атинумаб, тоцилизумаб, базилизимаб, бектумомаб, белимумаб, бевацизумаб, безилезомаб, безлотоксумаб, бициромаб, канакинумаб, цертолизумаб, цетукцимаб, циксутумумаб, даклизумаб, деносумаб, денсумаб, экулизумаб, эдреколомаб, эфализумаб, эфунгумаб, эпратузумаб, эртумаксомаб, этарацизумаб, фигитумумаб , голимумаб, ибритутомаб тиуксетан, иговомаб, имгатузумаб, инфликсимаб, инолимомаб, инотузумаб, лабетузумаб, лебрикизумаб, моксетумомаб, натализумаб, обинутузумаб, ореговомаб, паливизумаб, панитумумаб, пертузумаб, ранибизумаб, ритуксимаб, тоцилизумаб, тозитумомаб, тралокинумаб, тукотузумаб, трастузумаб, велтузумаб, zalutumumab and zatuximab. Additional examples of recombinant therapeutic antibodies that can be obtained using the methods described in this document are known in the art. Additional non-limiting examples of recombinant therapeutic proteins that can be produced using the methods of the present invention include: alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, factor coagulation IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa, insulin or insulin analogues, mecasermin, factor VIII, factor VIIa, antithrombin III, protein C, human albumin, erythropoietin, granulocyte colony stimulating factor, granulocyte- macrophage colony stimulating factor, interleukin-11, laronidase, idursulfase, galsulfase, α-1-proteinase inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alfa (eg, Thyrogen®), and alteplase. Additional examples of recombinant proteins that can be produced using the methods of the present invention include acid α-glucosidase, zalglucosidase alfa (eg, Myozyme® and Lumizyme®), α-L-iduronidase (eg, Aldurazyme®), iduronate sulfatase, heparan -N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase (for example, Cerezyme® and Ceredase®), galactosylceramidase, α-galactosidase-A (e.g. Fabrazyme®), acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A and hexosaminidase B.

The secreted soluble recombinant protein can be recovered from the liquid culture medium by removing or otherwise physically isolating the liquid culture medium from the cells (eg, mammalian cells). Many different methods for removing liquid culture medium from cells (eg, mammalian cells) are known in the art, including, for example, centrifugation, filtration, pipetting, and/or aspiration. The secreted recombinant therapeutic protein can then be recovered and isolated from the liquid culture medium using various biochemical techniques, including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography, hydrophobic interaction chromatography, or anion exchange chromatography) and/or filtration (e.g., , molecular weight cut-off filtration).

The fluid may be removed from the sterile process vessel by continuous or intermittent removal. In some examples, the fluid removed from the sterile process vessel contains the recombinant protein. In some examples, the fluid removed from the sterile process vessel contains the culture medium. In some examples, the fluid removed from the sterile process vessel does not contain the recombinant protein.

EXAMPLES

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

Example 1

In FIG. 2 describes a typical system for separating sterile process streams from non-sterile media according to the present invention. The system may be any sterile process including, for example, a component of a biological manufacturing process stream. As shown in FIG. 2, the system includes a sterile process vessel (110) (eg, the first vessel) containing a fluid outlet (130). For a biological production process stream, the first vessel may be, for example, a fluid tube to allow fluid flow, a bioreactor (for example, any of the exemplary bioreactors described herein or known in the art), one or more chromatography system components. (eg, a chromatographic column), one or more components of a microfiltration system, or one or more components of an ultrafiltration/diafiltration system. The system described in Fig. 2 further comprises a separating vessel (120) (e.g., a second vessel) containing a fluid inlet (140) in fluid communication via a fluid tube (210) with a fluid outlet (130) of the first vessel (110) and made in such a way that the fluid entering the second vessel passes through the free space (150) filled with sterilizing gas inside the second vessel (120), the outlet (170) for the fluid, made in such a way that the fluid the medium leaving the second vessel is removed below the free space (150) filled with the sterilizing gas inside the second vessel (120). The second vessel contains at least one gas inlet (180) for supplying sterilizing gas through the gas tube (220) to fill the free space of the second vessel. The second vessel also contains at least one outlet (160) for gas, made with the possibility of continuous or periodic removal of gas from the release.

In FIG. 1 provides an exemplary embodiment for separating sterile process streams from non-sterile environments according to the present invention. For the system described in Fig. 1, waste streams from a sterile process vessel (e.g., first vessel, not shown) are in fluid communication with a separating vessel (e.g., second vessel), with the separating vessel configured such that fluid enters the top of the second vessel. and passes through the free space filled with sterilizing gas inside the second vessel. The second vessel further comprises a fluid outlet configured such that the fluid leaving the second vessel is removed below the sterilizing gas-filled headspace (i.e., below the fluid-filled portion of the second vessel) within the second vessel. In FIG. 2 further illustrates a pumping system comprising a pump configured to remove a volume of fluid through a second vessel outlet and cause the volume to flow through a reservoir to accommodate a biological waste stream. The second vessel contains at least one gas inlet in gas phase communication with a system for generating or delivering a sterilizing gas or for generating and delivering a sterilizing gas (for example, an ozone generating system) to fill the free space of the second vessel. In this embodiment, the sterilizing gas is bubbled into the second vessel. The second vessel also contains at least one gas outlet, configured to continuously or intermittently remove gas from the release.

OTHER IMPLEMENTATION OPTIONS

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

Claims (29)

1. A method for the production of a recombinant therapeutic protein, comprising: (a) providing a system comprising a first vessel and a second vessel, wherein the first vessel contains a liquid containing the recombinant therapeutic protein and the second vessel contains a volume of sterilizing gas; (b) allowing the first volume of liquid to flow from the first vessel and into the second vessel through the volume of sterilizing gas contained at the top of the second vessel, wherein the first vessel is a bioreactor, a chromatography system, a microfiltration system, or an ultrafiltration/diafiltration system, and (c) purifying the recombinant therapeutic protein by allowing a second volume of liquid containing the recombinant therapeutic protein to flow from the second vessel into at least one chromatographic column and/or at least one chromatographic membrane, thereby obtaining a recombinant therapeutic protein. 2. The method of claim 1 wherein the first vessel is a bioreactor. 3. The method of claim 1 wherein the first vessel is a chromatography system. 4. The method of claim 1 wherein the first vessel is a microfiltration system. 5. The method of claim 1 wherein the first vessel is an ultrafiltration/diafiltration system. 6. The method of claim 1, wherein the second vessel further comprises (i) a fluid inlet configured such that a volume of fluid entering the second vessel passes through the top; (ii) a fluid outlet configured such that liquid is removed below the sterilizing gas-filled free space within the second vessel; (iii) at least one gas inlet and (iv) at least one gas outlet, wherein the fluid inlet is in fluid communication with the first vessel. 7. The method of claim 1, wherein the first vessel includes a fluid outlet in fluid communication with the fluid inlet of the second vessel. 8. The method of claim 1, wherein step (c) comprises: (i) allowing a second volume of liquid containing the recombinant therapeutic protein to flow from the second vessel to the first multi-column chromatography system (MCCS1); (ii) collecting the recombinant therapeutic protein from the liquid using MCCS1, wherein the MCCS1 eluate containing the recombinant therapeutic protein is continuously fed into a second multi-column chromatography system (MCCS2); And (iii) purification and post-purification of the recombinant therapeutic protein using MCCS2, wherein the eluate from MCCS2 contains the recombinant therapeutic protein; And while the technological process is combined and is carried out continuously from the first vessel to the eluate from MCCS2, which contains the recombinant therapeutic protein. 9. The method according to any one of paragraphs. 1-8, wherein the recombinant therapeutic protein is an antibody or antibody fragment, an enzyme, an engineered protein, or an immunogenic protein or protein fragment. 10. The method of claim 9 wherein the recombinant therapeutic protein is an enzyme. 11. The method of claim 10 wherein the enzyme is α-galactosidase. 12. The method of claim 13 wherein the α-galactosidase is α-galactosidase-A. 13. The method of claim 9, wherein the recombinant protein is secreted from the cell. 14. The method of claim 1, wherein the sterilizing gas is selected from the group consisting of ozone, ethylene oxide, nitrogen dioxide, or vaporized hydrogen peroxide. 15. The method of claim 14 wherein the sterilizing gas is ozone. 16. The method of claim 14 wherein the sterilizing gas is ethylene oxide. 17. The method of claim 1 wherein the first vessel is sterilized.

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