RU2826038C2 - Methods of creating ultrahigh density cell banks - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to creation of blood banks. Method of obtaining a bank of frozen mammalian cells directly from a population of cultured cells involves culturing mammalian cells in a perfusion bioreactor at pH from 6.8 to 7.2, wherein the concentration of the population of cultured mammalian cells is from 2×10⁷ to 5×10⁷ cells/ml and supply of O₂ to maintain the value of dissolved oxygen (DO) ≥40% to obtain a cultured population of cells with ultrahigh density; adding a cryoprotectant to the cultured ultrahigh-density cell population; freezing at a controlled rate and obtaining a bank of frozen cells with ultrahigh density with a cell concentration of at least 1.0×10⁸ cells/ml.

EFFECT: proposed method of producing a bank of frozen mammalian cells enables to obtain a bank of frozen cells with a cell concentration of at least 1_0×10⁸ cells/ml without a high-quality cell concentration step and cell viability after defrosting of at least 95%.

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Description

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/052,257, filed September 18, 2014. Where permitted, the foregoing application is incorporated by reference for any and all purposes.

PREREQUISITES FOR THE CREATION OF THE INVENTION

Cell banking is widely used to store frozen, characterized cell starting materials that can be thawed for use in a variety of applications, including the production of therapeutically relevant proteins. Typically, cryopreserved starting materials are stored at lower densities (e.g., approximately 1 or 2 x 10 ⁷ cells/mL) or centrifuged into higher density aliquots for storage. Lower density starting materials do not allow efficient inoculation of large volume cultures, and concentration methods can damage the cells, thereby reducing the viability of the frozen starting materials. For these reasons, previous cell banking methods are relatively inefficient and ultimately do not allow rapid production of high density cell cultures from frozen starting materials. Accordingly, there is a need for improved cell banking methods.

BRIEF DESCRIPTION

The present disclosure provides improved methods for creating a cell bank with ultra-high density. In some embodiments, the methods of the present invention use improved cell culture methods under perfusion conditions that allow for the production of ultra-high density cell cultures that can be cryopreserved at unexpectedly high cell densities without the need for any cell concentration steps, while maintaining excellent cell viability for further use in cell culture production.

Accordingly, in one aspect, the present disclosure provides a method for producing a frozen ultra-high density cell bank directly from a population of cultured cells, the method comprising: culturing cells in a perfusion bioreactor to produce an ultra-high density cell population with a concentration of at least 1.0× ¹⁰⁸ cells/mL, wherein said perfusion bioreactor is connected to a cell retention system; and adding a cryoprotectant to the ultra-high density cell population to produce a frozen ultra-high density cell bank, wherein the frozen ultra-high density cell bank has a concentration of at least 1.0× ¹⁰⁸ cells/mL, and wherein there is no additional concentration step between culturing the cells and adding the cryoprotectant to the ultra-high density cell population.

In some embodiments, the cell retention system comprises an alternating tangential flow filtration system comprising a filter. In some embodiments, the filter has a surface area of at least about 0.08 ^m2 . In some embodiments, the filter has a surface area of from about 0.08 ^m2 to about 0.3 ^m2 , from about 0.3 ^m2 to about 0.5 ^m2 , from about 0.5 ^m2 to about 1.0 ^m2 , from about 0.7 ^m2 to about 0.8 ^m2 , from about 1.0 ^m2 to about 2.0 ^m2 , from about 2.0 ^m2 to about 3.0 ^m2 , from about 3.0 ^m2 to about 4.0 ^m2 , or from about 4.0 ^m2 to about 5.5 ^m2 . In some embodiments, the filter is characterized by a pore size selected from the group consisting of 0.2 μm, 0.4 μm, and 0.65 μm. In other embodiments, the filter is characterized by a pore size selected from the group consisting of 0.7 μm, 1.2 μm, and 7 μm.

In some embodiments, the ultra-high-density cell population is characterized by a viable cell density selected from the group consisting of about 1.0× ¹⁰⁸ cells/mL, about 1.1× ¹⁰⁸ cells/mL, about 1.2× ¹⁰⁸ cells/mL, about 1.3× ¹⁰⁸ cells/mL, about 1.4× ¹⁰⁸ cells/mL, about 1.5× ¹⁰⁸ cells/mL, about 1.6× ¹⁰⁸ cells/mL, about 1.7× ¹⁰⁸ cells/mL, about 1.8× ¹⁰⁸ cells/mL, about 1.9× ¹⁰⁸ cells/mL, and about 2.0× ¹⁰⁸ cells/mL.

In some embodiments, cryopreservation comprises adding dimethyl sulfoxide (DMSO) to the ultra-high-density cell population at a final concentration of about 5% to about 10% v/v. In some embodiments, cryopreservation comprises freezing at least a portion of the ultra-high-density cell population in a container suitable for storage under cryopreservation conditions.

In some embodiments, the container is a vial. In some embodiments, the ultra-high density frozen cell bank contains approximately 4.5×10 ⁸ cells/vial.

In some embodiments, the container is a cryopack. In some embodiments, the volume of the cryopack is from about 5 to about 150 ml. In some embodiments, the cell density of the ultra-high density frozen cell bank is at least about 1.0×10 ⁸ cells/ml.

In some embodiments, the perfusion rate in the perfusion bioreactor is from about 0.02 nL/cell/day to about 0.5 nL/cell/day. In some embodiments, the perfusion rate in the perfusion bioreactor is from 0 to 15 reactor volumes per day.

In some embodiments, the pH of the cell culture of the perfusion bioreactor is from about 6.8 to about 7.2.

In some embodiments, the dissolved oxygen (DO) concentration in the cell culture of the perfusion bioreactor is at least about 30%.

In some embodiments, the bioreactor is a bag-type bioreactor. In some embodiments, the bioreactor includes an integrated filter.

In some embodiments, the ultra-high density frozen cell bank has a cell viability after thawing of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%.

In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are selected from the group consisting of CHO, CHO-DBX11, CHO-DG44, CHO-S, CHO-K1, Vero, BHK, HeLa, COS, MDCK, HEK-293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0, CRL7030, HsS78Bst cells, PER.C6, SP2/0-Agl4, and hybridoma cells. In some embodiments, the cells are transfected cells. In some embodiments, the cells express a therapeutic protein.

In some embodiments, the perfusion bioreactor comprises a bioreactor in the form of an elastic bag; the filter has a surface area of at least 0.3 ^m2 and a molecular weight cutoff value (MWCO) of at least 50 kDa; the cryoprotectant added to the ultra-high-density cell population is DMSO; and the ultra-high-density frozen cell bank contains from about 5 to about 10% v/v DMSO.

In some embodiments, the pH and DO of the culture are controlled by automated methods. In some embodiments, the pH and DO of the culture are controlled by non-automated methods. In some embodiments, the pH and DO are controlled by any one or more of the following: adjusting the mixture of gases that are introduced into the culture, adjusting the shaking speed of the bioreactor, or adjusting the shaking angle of the bioreactor. In some embodiments, the bioreactor is shaken at 15 rpm with a shaking angle of 8°. In some embodiments, the bioreactor is shaken at 22 rpm with a shaking angle of 10°. In some embodiments, the bioreactor is shaken at 25 rpm with a shaking angle of 12°.

In some embodiments, the ultra-high-density cell population is cooled and maintained at a temperature of about 4°C prior to and during addition of the cryoprotectant and distribution. In some embodiments, the ultra-high-density cell population is maintained at a temperature of about 20°C to about 26°C during addition of the cryoprotectant and distribution. In some embodiments, the ultra-high-density cell population is maintained at an uncontrolled low temperature using an ice water bath during addition of the cryoprotectant and distribution.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 is a graphical representation of a method for creating a super-high density cell bank.

Figure 2 is a graph showing viable cell density (cells/mL), perfusion rate (RV(reactor volume)/day), and relative cell perfusion rate 10X (nL/cell/day) in an exemplary rCHO 1 cell line culture.

Figure 3 is a graph showing the autonomous pH profile in a culture of rCHO 1 cell line.

Figure 4A-C are a series of graphs showing the cell growth profile after thawing (A), the percentage of cells in the late apoptotic stage after thawing (B), and the specific productivity (units/E9 cells/day) for a 5 ml aliquot of an ultra-high density (HD) cell bank of the rCHO 1 cell line.

Figure 5 is a graph showing viable cell density (cells/mL), perfusion rate (RV/day), and specific cell perfusion rate 10X (nL/cell/day) in an exemplary rCHO 2 cell line culture.

Figure 6 is a graph showing the autonomous pH profile in a culture of the rCHO 2 cell line.

Figure 7 is a graph showing cell growth based on viable cell density Xv (cells/mL, solid lines) and viability (dashed lines) for the rCHO 2 cell line when seeded at 0.5× ¹⁰⁶ viable cells/mL in 250 mL and 3 L shake flasks.

Figure 8 is a graph showing cell growth based on viable cell density Xv (cells/mL, solid lines) and viability (dashed lines) for the rCHO 2 cell line when seeded at 1.0× ¹⁰⁶ viable cells/mL in 250 mL and 3 L shake flasks.

Figure 9 is a graph showing the percentage of late apoptotic cells and dead cells in a culture of rCHO 2 cell line seeded at 0.5× ¹⁰⁶ viable cells/mL in 250 mL and 3 L shake flasks.

Figure 10 is a graph showing the percentage of late apoptotic cells and dead cells in a culture of rCHO 2 cell line seeded at 1.0× ¹⁰⁶ viable cells/mL in 250 mL and 3 L shake flasks.

Figure 11 is a graph showing the specific productivity (SPR) in shake flask cultures of the rCHO 2 cell line at 0.5×10 ⁶ viable cells/mL in 250 mL and 3 L shake flasks.

Figure 12 is a graph showing the specific productivity (SPR) in shake flask cultures of the rCHO 2 cell line at 1.0× ¹⁰⁶ viable cells/mL in 250 mL and 3 L shake flasks.

Figure 13 is a graph showing cell growth in WAVE bags based on viable cell density Xv (cells/mL, solid lines) and viability (dashed lines) for the rCHO 2 cell line.

Figure 14 is a graph showing the percentage of late apoptotic cells and dead cells in a culture of rCHO 2 cell line.

Figure 15 is a graph showing viable cell density (cells/mL), perfusion rate (RV/day), and specific cell perfusion rate 10X (nL/cell/day) in an exemplary rCHO 3 cell line culture.

Figure 16 is a graph showing the autonomous pH profile in a culture of the rCHO 3 cell line.

Figure 17A-C are a series of graphs showing cell growth (solid lines) and viability (dashed lines) (A); the percentage of late apoptotic and dead cells (B); and the specific productivity (SPR) (C) in shake flask cultures of the rCHO 3 cell line.

Figure 18 is a graph comparing viable cell density (cells/mL, solid lines) and viability (dashed lines) with internal medium (black) and CD CHO medium (gray) in an illustrative rCHO 3 cell line culture.

Figure 19 is a graph comparing the perfusion rate (circles, solid lines) and specific perfusion rate of cells (triangles, dotted lines) with internal medium (black) and CD CHO medium (gray) in an illustrative rCHO 3 cell line culture.

Figure 20 is a graph comparing viable cell density (cells/mL, solid lines) and viability (dashed lines) with CD CHO medium supplemented with Efficient Feed B (black) and CD CHO medium (gray) in an exemplary rCHO1 cell line culture.

Figure 21 is a graph comparing the perfusion rate (circles, solid lines) and specific perfusion rate of cells (triangles, dotted lines) with CD CHO medium supplemented with Efficient Feed B (black) and CD CHO medium (gray) in an illustrative rCHO 1 cell line culture.

Figure 22 is a graph showing the viable cell density (cells/mL, solid lines) and viability (dashed lines) using a 20 L WAVE bioreactor (10 L working volume, black) and a 10 L WAVE bioreactor (5 L working volume, gray) in an illustrative culture of the rCHO 3 cell line.

DETAILED DESCRIPTION

The present disclosure provides a method for creating a cryobank of ultra-high density cells, which comprises using a perfusion culture system associated with a cell holding device other than a centrifuge.

I. Definitions

As used herein, the term "batch culture" refers to a cell culture method in which a portion of fresh culture medium is inoculated with cells that rapidly enter the logarithmic growth phase and in which the culture medium is not continuously removed and replaced with fresh medium.

As used herein, the term "fed-batch culture" refers to a cell culture method in which a portion of fresh culture medium is first inoculated with cells and additional culture nutrients are supplied (continuously or in discrete increments) to the culture during the culture process, with or without periodic harvesting of the cells and/or product until the culture is terminated.

As used herein, the expression "perfusion culture" refers to a cell culture method in which a portion of fresh medium is inoculated with cells that rapidly enter the logarithmic growth phase (as defined above) and in which the culture medium is continuously removed from the culture and replaced with fresh medium.

As used in this document, the term "bioreactor" refers to a cell culture vessel.

In one embodiment, the bioreactor is a "bag bioreactor." The "bag bioreactor" is a sterile chamber capable of receiving a liquid medium and a cell inoculum, which further comprises connectors, ports, adapters, and flexible tubing. In one embodiment, the chamber is made of plastic. In a particular embodiment, the chamber is made of a multilayer transparent plastic sheet. In another particular embodiment, the chamber is made of a multilayer transparent plastic sheet and comprises a liquid contact layer made of ethylene vinyl acetate/low density polyethylene USP Class VI copolymer, while the outer layer is made of low density polyethylene.

In addition, connectors, ports, and adapters may be made of any type of plastic, including but not limited to polyethylene, polypropylene, and polycarbonate; while tubing may be made of any type of plastic, including but not limited to thermoplastic elastomer or silicone (e.g., platinum-based silicone).

Suitable flexible bag bioreactors are generally found in the art and include, without limitation, those described in U.S. Patent No. 6,544,788, which is incorporated herein by reference in its entirety.

The bioreactor in the form of an elastic bag can be partially filled with a culture medium and then filled with air to a rigid state. It can then be placed on a rocking platform (e.g., a BASE20/50EHT rocking unit from GE Life Sciences) that moves back and forth at a predetermined rocking angle and a predetermined rocking speed. This rocking motion causes wave-like movements in the culture medium, promoting mixing and oxygen transfer to improve cell culture performance. The predetermined rocking angle can be at least about 4 degrees, such as about 4 degrees, 5 degrees, 6 degrees, 7 degrees, 8 degrees, 9 degrees, 10 degrees, 11 degrees, or 12 degrees. In addition, the predetermined swing speed can be set as a revolution per minute (rpm) speed that is at least approximately 6 rpm, such as approximately 6 rpm, approximately 7 rpm, 8 rpm, 9 rpm, 10 rpm, 11 rpm, 12 rpm, 13 rpm, 14 rpm, 15 rpm, 16 rpm, 17 rpm, 18 rpm, 19 rpm, 20 rpm, 21 rpm, 22 rpm, 23 rpm, 24 rpm, 25 rpm, 26 rpm, 27 rpm, 28 rpm, 29 rpm, 30 rpm, 31 rpm, 32 rpm, 33 rpm, 34 rpm, 35 rpm, 36 rpm, 37 rpm, 38 rpm, 39 rpm, or 40 rpm. In a particular embodiment, the rpm is approximately 22 rpm.

As used in this document, the term "cell retention system" refers to any device capable of separating cells and waste products from a medium by means of a filter. Filters may include membrane, ceramic or metal filters of any shape, including spiral, tubular or plate filters. Filters may have different surface areas. For example, the surface area of the filter may be from about 0.08 ^m2 to about 5.5 ^m2 , such as about 0.08 ^m2 , 0.09 ^m2 , 0.1 ^m2 , 0.2 ^m2 , 0.3 ^m2 , 0.4 m2, ^{0.5 m2} , 0.6 ^m2 , 0.7 ^m2 , 0.77 ^m2 , 0.8 ^m2 , 0.9 ^m2 , 1.0 ^m2 , 1.1 ^m2 , 1.2 ^m2 , 1.3 ^m2 , 1.4 ^m2 , 1.5 ^m2 , 1.6 ^m2 , 1.7 ^m2 , 1.8 m ² , 1.9 ^m2 , 2.0 ^m2 , 2.1 ^m2 , 2.2 m2, 2.3 ^m2 , 2.4 ^m2 , 2.5 ^m2 , 2.6 ^m2 , 2.7 ^m2 , 2.8 ^m2 , 2.9 m2, 3.0 ^m2 , 3.1 ^m2 , 3.2 ^{m2 ,} 3.3 ^{m2 ,} 3.4 ^m2 , 3.5 ^m2 , 3.6 ^m2 , 3.7 ^m2 , 3.8 ^m2 , 3.9 ^m2 , 4.0 ^m2 , 4.1 ^m2 , 4.2 ^m2 , 4.3 ^m2 , 4.4 ^m2 , 4.5 m2 ^, 4.6 ^m2 , 4.7 ^m2 , 4.8 ^m2 , 4.9 ^m2 , 5.0 ^m2 , 5.1 ^m2 , 5.2 ^m2 , 5.3 ^m2 , 5.4 ^m2 or 5.5 m ² . In some embodiments, the nominal molecular weight cutoff value (MWCO) of the filter module is from about 10 kilodaltons (kDa) to about 100 kDa, for example, it is about 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or 100 kDa. In other embodiments, the mesh size of the filter module is from about 0.1 μm to about 7 μm, such as about 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, 3.0 μm, 3.1 μm, 3.2 μm, 3.3 μm, 3.4 μm, 3.5 μm, 3.6 µm, 3.7 µm, 3.8 µm, 3.9 µm, 4.0 µm, 4.1 µm, 4.2 µm, 4.3 µm, 4.4 µm, 4.5 µm, 4.6 µm, 4.7 µm, 4.8 µm, 4.9 µm, 5.0 µm, 5.1 µm, 5.2 µm, 5.3 µm, 5.4 µm, 5.5 µm, 5.6 µm, 5.7 µm, 5.8 µm, 5.9 µm, 6.0 µm, 6.1 µm, 6.2 µm, 6.3 µm, 6.4 µm, 6.5 µm, 6.6 µm, 6.7 µm, 6.8 µm, 6.9 µm or 7.0 µm.

As used in this document, the term "cryopreservation" refers to a process by which cells, tissues or any other substance susceptible to damage caused by time or enzymatic or chemical activity are preserved by cooling and storage at sub-zero temperatures.

As used herein, the term "creating a cryobank" refers to a process by which cells are mixed with a cryoprotectant (e.g., DMSO with or without hydroxyethyl starch (HES)) and placed in a container suitable for cryopreservation storage. These containers are then frozen using techniques well known in the art and stored at low temperatures, typically from about -130°C to about -196°C. The collection of cells obtained by this process constitutes a cell bank.

In one embodiment, the cell bank is an ultra-high density cell bank. As used herein, the term "ultra-high density cell bank" refers to aliquots of a generated cryobank of cells that have been frozen at an ultra-high density, wherein the density is at least about 1×10 ⁸ viable cells/mL, such as about 1×10 ⁸ viable cells/mL, about 1.1×10 ⁸ viable cells/mL, about 1.2×10 ⁸ viable cells/mL, about 1.3×10 ⁸ viable cells/mL, about 1.4×10 ⁸ viable cells/mL, about 1.5×10 ⁸ viable cells/mL, about 1.6×10 ⁸ viable cells/mL, about 1.7×10 ⁸ viable cells/mL, about 1.8×10 ⁸ viable cells/mL, about 1.9×10 ⁸ viable cells/ml, approximately 2×10 ⁸ viable cells/ml, approximately 3×10 ⁸ viable cells/ml, approximately 4×10 ⁸ viable cells/ml, or approximately 5×10 ⁸ viable cells/ml. The cells may be frozen according to any method available in the art and in any container suitable for storage under cryopreservation conditions.

In another embodiment, the cell bank is a master cell bank. As used herein, the term "master cell bank" refers to a cell culture (e.g., fully characterized cells) that has been grown from a single clone, distributed into storage containers (e.g., distributed into containers in a single operation), and stored under cryopreservation conditions as described above. In some embodiments, the cells are suitable for subsequent use in the production of a cell culture and further production of therapeutically significant proteins produced therefrom.

In another embodiment, the cell bank is a working cell bank. As used herein, the term "working cell bank" refers to a culture of cells (e.g., fully characterized cells) that has been grown from a single vial of a master cell bank or from two pooled vials of a master cell bank, distributed into storage containers (e.g., distributed into containers in a single operation), and stored under cryopreservation conditions as described above. In some embodiments, the cells are suitable for subsequent use in the production of a cell culture and further production of therapeutically significant proteins produced therefrom.

In another embodiment, the cell bank is a mini cell bank. As used herein, the term "mini bank" refers to aliquots of cells that have been cryopreserved according to the "cryobanking" methods (described above), but consist of a smaller number of samples than are typically used to create a cell bank. This type of bank can typically be used to optimize the conditions considered for cryopreserving a cell line prior to creating cell banks, such as a "master cell bank". As an example, a "mini bank" is used to determine the optimal cell density for the ultra-high-density cell banking method described in the present disclosure.

As used herein, the term "container suitable for storage under cryopreservation conditions" includes any container that can be used under conditions suitable for storage of cells, from about -130°C to about -196°C. These containers include, but are not limited to, vials that are made of materials suitable for cryopreservation. These materials include polymers (e.g., polytetrafluoroethylene, polystyrene, polyethylene, or polypropylene). In addition, surface treatments can be applied to the surface of the cryopreservation vial to improve cryopreservation conditions (e.g., hydrophilic coatings that reduce adsorption and denaturation). In exemplary embodiments, the volume of the vial may be greater than about 0.1 ml, such as the volume of the vial may be about 0.1 ml, about 0.5 ml, about 0.75 ml, about 1 ml, about 1.5 ml, about 2 ml, about 2.5 ml, about 4.5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, or about 50 ml. The container may also be a cryopack, which may have a volume of about 30 ml, about 50 ml, about 100 ml, about 150 ml, about 200 ml, about 300 ml, about 400 ml, or about 500 ml.

As used herein, the term "cryopack" refers to a sterile chamber that is capable of receiving a liquid medium, is suitable for storing cells from about -130°C to about -196°C, and may further comprise connectors, ports, adapters, and flexible tubing. The cryopack may be made of any suitable material, including, but not limited to, polymers such as polytetrafluoroethylene (PTFE), polystyrene, polyethylene, polypropylene, fluorinated ethylene propylene (FEP), polyolefin, and ethylene vinyl acetate (EVA). Illustrative cryopreservation bags include, but are not limited to, KryoSure® cryopreservation bags (Saint-Gobain), PermaLife™ bags (OriGen Biomedical), CryoStore freezing bags (OriGen Biomedical), Freeze-Pak™ biocontainers (Charter Medical), and the bags described in U.S. Provisional Patent Application No. 62/037,181 (Merial Ltd., a Sanofi Company), which is incorporated herein by reference in its entirety. In some embodiments, a cryopreservation bag may support a closed phase cell banking system (e.g., by using at least two sterilely sealable tubes to allow filling of a "closed system" of the bag).

As used in this document, the term "shake flask" refers to a vessel used as a culture flask in which the medium and cell culture are continuously stirred during incubation.

As used herein, the term "shake flask seeding system" refers to a method of cell propagation in which an aliquot of cells is first cultured (seeded) in a shake flask and grown therein. The cells are cultured according to their growth rate and are typically divided into larger and/or multiple vessels during their growth until the biomass reaches a level sufficient to inoculate the bioreactor.

As used in this document, the term "inoculation density" refers to the initial cell density at which the flask or bioreactor is inoculated.

As used herein, the term "therapeutically significant protein" refers to any protein that can be used to effect treatment of a disease or disorder or to treat a disease or disorder in an animal, including mammals such as mice, rats, monkeys, primates and humans. Such proteins may include, but are not limited to, binding polypeptides such as monoclonal antibodies, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, artificial protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins and thrombolytics.

As used herein, the term "binding polypeptide" or "binding polypeptide" refers to a polypeptide (e.g., an antibody) that comprises at least one binding site that is responsible for selectively binding to a target antigen of interest (e.g., a human antigen). Exemplary binding sites include a variable domain of an antibody, a ligand binding site of a receptor, or a ligand binding site of a receptor. In certain aspects, binding polypeptides comprise multiple (e.g., two, three, four, or more) binding sites.

As used herein, the term "antibody" refers to aggregates (e.g., intact antibody molecules, antibody fragments, or variants thereof) that have significant, known, specific immunoreactivity toward an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins contain light and heavy chains, with or without interchain covalent linkage between them. The basic structures of immunoglobulins in vertebrate organisms are relatively well understood.

The general term "antibody" includes five distinct classes of antibodies that can be distinguished by biochemical properties. All five classes of antibodies are clearly within the scope of the present disclosure; the following discussion will primarily focus on immunoglobulin molecules of the IgG class. With respect to IgG, immunoglobulins include two identical light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000-70,000 daltons. The four chains are joined by disulfide bonds in a "Y" configuration, where the connection of the light chains to the heavy chains begins at the mouth of the "Y" and continues through the variable region.

As used in this document, the term "dissolved oxygen" or "DO" is the percentage of dissolved oxygen gas present in a given liquid (such as cell culture medium) based on air saturation.

As used herein, the term "cell specific perfusion rate" (CSPR) refers to the rate at which cell culture medium is added to a cell culture, expressed as the volume of medium added per viable cell per day (Ozturk, SS. Engineering challenges in high density culture systems. Cytotechnology. 1996; 22:3-16).

As used herein, the term "approximately" refers to a tolerance range of 10% around a stated value. Therefore, when the term "approximately" is used to vary a stated value, the stated range will cover any number within ±0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the stated value.

II. Cell culturing under perfusion conditions

Traditional cell culture involves the "batch" method. In this type of culture, fresh medium is inoculated with cells, which rapidly enter a logarithmic growth phase. As these cells grow and divide, they consume available nutrients from the medium and excrete harmful waste products. Over time, the culture enters a stationary growth phase and finally a death phase. Although modifications to the batch method have made it more efficient over time, the modified batch protocols that have been created still result in cycles of rapid growth and death. In addition, the batch method has a limited ability to achieve the necessary cell densities to support ultra-high-density cell banking.

The "fed-batch" method refers to a further improvement in cell culture over traditional "batch" culture methods. Although this process allows for higher growth in cell density, it is still limited in its ability to efficiently grow high-density cell cultures and, therefore, efficiently generate cells for high-density cell banking.

In preferred embodiments, the present invention employs a perfusion culture method. Perfusion culture is a method for growing cells in which a fresh batch of medium is inoculated with cells that rapidly enter the logarithmic growth phase (as described above) and in which the nutrient medium is continuously removed from the culture and replaced with fresh medium. In this way, the culture is continuously supplied with fresh medium with a high nutrient content, while the medium containing waste products and lower nutrient levels is removed. This type of culture allows for logarithmic cell growth to be maintained, and in which at least half of the culture volume is changed daily, and cell density values can be much higher than in traditional or modified batch culture (an increase of 2 to more than 10 times). In one embodiment of the present invention, the cell specific perfusion rate (CSPR) can be from about 0.02 nL/cell^-1/day^-1up to approximately 0.5 nl/cell^-1/day^-1; for example, it can be approximately 0.02 nL/cell^-1/day^-1, 0.025 nl/cell^-1/day^-1, 0.05 nl/cell^-1/day^-1, 0.1 nl/cell^-1/day^-1, 0.2 nl/cell^-1/day^-1, 0.3 nl/cell^-1/day^-1, 0.4 nl/cell^-1/day^-1 or 0.5 nl/cell^-1/day^-1In another embodiment of the present invention, the perfusion rate may be measured in reactor volumes per day and may range from 0 to 15 reactor volumes per day, for example, can be about 0 reactor volumes per day, about 0.5 reactor volumes per day, about 1 reactor volume per day, about 2 reactor volumes per day, about 3 reactor volumes per day, about 4 reactor volumes per day, about 5 reactor volumes per day, about 6 reactor volumes per day, about 7 reactor volumes per day, about 8 reactor volumes per day, about 9 reactor volumes per day, about 10 reactor volumes per day, about 11 reactor volumes per day, about 12 reactor volumes per day, about 13 reactor volumes per day, about 14 reactor volumes per day, or about 15 reactor volumes per day. In a particular embodiment, the perfusion culturing can be carried out in a bioreactor with at least one dip tube.

In some embodiments, the pH, temperature, dissolved oxygen (DO) concentration, and osmolarity of the culture can be adjusted to maximize the viability and productivity of the culture. One method for controlling the DO and pH of the cultures is by means of an automatic feedback controller. This type of automatic controller operates using microprocessor computers to monitor and regulate the pH and DO of the culture and thereby maintain optimal conditions for cell growth. However, such automatic feedback control systems are expensive. Accordingly, in some embodiments, a non-automated method for controlling these parameters can be used. In one exemplary embodiment, any of the following can be used: adjusting the gas mixture supplied to the culture, adjusting the WAVE® rocking speed, or adjusting the rocking angle of the culture to control selected parameters (e.g., pH or DO). In some embodiments, both automatic feedback control and non-automated control can be used.

In one embodiment, the initial level of carbon dioxide gas is about 10% and the initial level of oxygen gas is about 20% at an air flow rate of about 0.2 liters per minute (L/min). If the pH does not exceed 6.9, the CO ₂ set point can be reduced from 10% to 5%. If after the time point the pH still does not exceed 6.9, the CO ₂ set point can be further reduced from 5% to 0%. If after the time point the pH still does not exceed 6.9, the perfusion rate can be increased. If the DO is not more than 45%, the O ₂ set point should be increased from 20% to 30%. If after the time point the DO is not more than 45%, the O ₂ level should be increased from 30% to 40%, the swing speed should be increased to 25 rpm, and the swing angle should be changed to about 12°.

i. Cell culture medium

Any type of culture medium suitable for culturing cells can be used in the methods of the present invention. Guidelines for selecting a suitable cell medium are well known in the art and are provided, for example, in Chapters 8 and 9 of Freshney, R. I. Culture of Animal Cells (basic techniques manual), 4th edition 2000, Wiley-Liss; and in Doyle, A., Griffiths, J. B., Newell, D. G. Cell & Tissue Culture: Laboratory Procedures 1993, John Wiley & Sons. Each of these references is hereby incorporated by reference in its entirety. Other methods for producing and maintaining cell cultures under animal-derived component- and protein-free conditions (including methods involving CHO cells) are known in the art, such as those described in International Patent Application Nos. WO 97/05240, WO 96/26266, and WO 00/11102, and U.S. Patent Nos. 6,100,061, 6,475,725, and 8,084,252. Each of the preceding documents is incorporated herein by reference in its entirety. In one embodiment of the present invention, an animal-derived component-free (ADC) medium may be used. A conventional synthetic medium with minimal requirements may contain inorganic salts, amino acids, vitamins, a carbohydrate source, and water. In a particular embodiment of the present invention, the medium that can be used is CD-CHO (GIBCO, Invitrogen Corp., a medium that does not contain components of animal origin, which is chemically defined and does not contain proteins, hydrolysates or components of unknown origin). In addition, the medium can contain additional components, including glutamine and/or methotrexate, or other factors that can promote growth or adhesion. In a particular embodiment, the additional component can be GLUTAMAX-1 or L-glutamine added in an amount of from about 2 mM to about 8 mM, such as about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM or about 8 mM.

ii. Host cells and expression vectors

In some embodiments, the cells used in the cell banking method of the present invention are host cells that carry an expression construct for expressing a therapeutically significant protein or other polypeptide of interest. Any cell that can be used to express a polypeptide of interest (e.g., a binding polypeptide) can be used in accordance with the methods described herein. The cells can optionally contain naturally occurring or recombinant nucleic acid sequences; for example, an expression vector that encodes the polypeptide of interest. The expression vector can optionally contain suitable transcriptional and translational regulators and can be constructed using recombinant DNA technology known in the art. The expression vectors can be transferred into any host cell using methods known in the art, and the transformed cells can then be cultured in accordance with the method of the present invention to create a high-density cell bank. In addition, the high density cell bank can then be thawed and cultured according to methods known in the art to obtain the encoded protein of interest and, if desired, such protein can then be purified.

In some embodiments, various host cell expression systems can be used to produce therapeutically significant proteins. In addition, the host cell expression system can be a mammalian cell system (e.g., CHO, CHO-DBX11, CHO-DG44, CHO-S, CHO-K1, Vero, BHK, HeLa, COS, MDCK, HEK-293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0, CRL7030, HsS78Bst, PER.C6, SP2/0-Agl4, and hybridoma cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells. Viral expression systems can also be used in conjunction with mammalian cells (see, e.g., Logan et al., 1984, Proc. Nat. Acad. Sci. USA 8: 355-359, incorporated herein by reference in its entirety). Expression efficiency can be enhanced by the inclusion of elements including (but not limited to) suitable transcriptional enhancer elements and transcription terminators (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544, incorporated herein by reference in its entirety).

In other embodiments, a host cell strain can be selected that modulates the expression of the inserted sequences or that modifies and processes the gene product in a certain desired manner. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable host cell lines or systems can be selected to ensure proper modification and processing of the expressed polypeptide (e.g., a binding polypeptide). Such cells include, for example, stable mammalian cell lines and animal cells, as well as insect cell lines, fungal cells, and yeast cells.

iii. Bioreactors

Any bioreactor suitable for culturing cells under perfusion culture conditions can be used in the methods of the present invention. The bioreactor can be inoculated with an aliquot of cells at an appropriate seed density (such as a flask of cells or cells from a stock culture, such as from a shake flask or shake flask seeding system, that have been cultured to a given density). The appropriate seed density for the culture depends on several factors, including the type of cells used and the bioreactor into which the inoculation is performed. The appropriate seed density can be determined using methods available in the art.

In certain embodiments, the bioreactor can be disposable, for example, the bioreactor can be an elastic bag or a plastic flask that are connected to a cell holding device via flexible tubes. The structure can optionally include a feed tube and an output tube or an inoculation tube that can be sterilely welded or connected to a source of eukaryotic cells. In some embodiments, the volume of the bioreactor is at least about 1 L, such as about 1 L, 2 L, 5 L, 10 L, 20 L, 50 L, 75 L, 85 L, 100 L, 150 L or 400 L. In a particular embodiment of the present invention, the bioreactor is a flexible bag with a volume of 10 L, which has been equipped with one or two dip tubes that are used to remove the medium or product. Examples of disposable bioreactors include the bag-type WAVE® cell bioreactors (GE Healthcare, Pittsburgh, PA), such as the 20 L WAVE® bioreactor. These perfusion bioreactor systems are described, among other papers, in Singh, 1999, Disposable bioreactor for cell culture using wave-induced agitation, Cytotechnology, p.149-158, incorporated herein by reference in its entirety.

The working volume of the reactor is the volume occupied by the culture. The working volume may be, for example, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the culture, but preferably not more than approximately 75%.

In addition, the bioreactor may not be disposable. For example, the bioreactor may be made of stainless steel or glass. Other bioreactors suitable for use in the present invention include, but are not limited to, shake flasks, stirred vessels, bubble vessels, and disposable bags that can be stirred by rocking, shaking, or stirring.

In one embodiment, the bioreactor may be coupled to a cell retention system, including, but not limited to, in-line filters, cartridge screens, tangential flow filtration (TFF) systems, and alternating tangential flow filtration (ATF) systems.

III. Cell retention

Perfusion culturing is driven by the ability to remove nutrient-depleted and waste-containing medium from a culture while minimizing cell damage. Previous cell retention methods that separated depleted medium from cultured cells often damaged the cells, such as by creating shear forces. Such cell damage resulted in filter plugging and failure of perfusion devices, many of which were within the culture system. Accordingly, in one aspect, the present disclosure provides a method that utilizes a "cell retention system" that allows for medium exchange.

In one embodiment, the type of cell retention system used is an "in-line" type filter, in which the filter is located in the bioreactor chamber and is free to move within the chamber. The filter may be connected to one of the tubes exiting the chamber, allowing filtered medium to be collected from the culture. One example of an "in-line" type filter can be found in U.S. Patent No. 6,544,788, incorporated herein by reference in its entirety.

In another embodiment, the cell retention system used is a tangential flow filtration (TFF) system. In a TFF system, the culture medium is circulated from the culture vessel through a filter module and then back into the culture vessel by a pump attached to the tubing between the filter module and the culture vessel, creating a tangential flow through the filter module. A second pump is placed on the filtrate side of the filter module and is used to control the rate at which the filtrate is removed. The use of hollow fiber membrane filters is preferred in such a system because they are easier to sterilize and ensure that a uniform flow is maintained through the filter module. However, when hollow fiber filters are used in such a system, they are prone to clogging because the unidirectional flow causes solid particles to aggregate at the entrance to the lumen.

In a particular embodiment, the type of cell retention system used is an ATF type of cell retention system. In an ATF type cell retention system, the filtration compartment is connected to a storage vessel at one end and a diaphragm pump at the other. The pump first moves the medium from the vessel through the filter element into the pump and then reverses it to send the medium from the pump through the filter and back into the vessel, creating a bidirectional or alternating flow. This is called ATF because there is an alternating tangential flow across the filter module, i.e., there is one flow in the same direction to the membrane surfaces of the filter module (tangential to that surface), and there is another flow that is substantially perpendicular to those surfaces. This type of filtration has been reported in the literature since 2000 and is characterized by a fast, low shear, uniform flow. ATF filtration can be achieved by methods known in the art, such as those described in U.S. Patent No. 6,544,424, which is incorporated herein by reference in its entirety. In addition, variable tangential flow systems are commercially available from manufacturers such as Refine Technology and include various models such as the ATF2, ATF4, ATF6, ATF8, and ATF10 systems.

In another specific embodiment of the invention, the filter is a tubular membrane filter and may further be a hollow fiber filter.

As noted above, the methods of the present invention provide for efficient growth of cells to ultra-high densities. In a particular embodiment of the present invention, the culture is grown to a density of at least about 1×10 ⁸ cells/mL, such as about 1×10 ⁸ cells/mL, about 1.1×10 ⁸ cells/mL, about 1.2×10 ⁸ cells/mL, about 1.3×10 ⁸ cells/mL, about 1.4×10 ⁸ cells/mL, about 1.5×10 ⁸ cells/mL, about 1.6×10 ⁸ cells/mL, about 1.7×10 ⁸ cells/mL, about 1.8×10 ⁸ cells/mL, about 1.9×10 ⁸ cells/mL, or about 2×10 ⁸ cells/mL. Growing the culture to such high concentrations allows immediate cryopreservation of high-density starting materials without further concentration of the cell population by centrifugal or non-centrifugal methods.

IV. Cryopreservation and creation of a cell bank

Cryopreservation refers to the process by which cells, tissues or any other substance susceptible to damage caused by time or enzymatic or chemical activity are preserved by cooling and storing at sub-zero temperatures. The importance of cryopreservation of important cell lines cannot be underestimated, as (among other important advantages) it allows such lines to be maintained without maintaining them in permanent culture, reduces the risk of contamination and reduces the risk of genetic drift.

When using cryopreservation methods, it is vital to achieve and maintain these low temperatures without causing additional damage due to ice formation during the freezing process. Methods in the art traditionally use substances that reduce freezing damage to cells, called cryoprotectants. Cryoprotectants can be added to the cell culture medium prior to the freezing process. In a particular embodiment, the cryoprotectant used can be one or more of glycerol or dimethyl sulfoxide (DMSO). In addition, the cryoprotectant can be added with or without hydroxyethyl starch (HES). In another specific embodiment, DMSO can be added at a concentration of at least about 5%, such as it can be added at a concentration of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%.

The cryoprotectant-added culture can then be distributed into containers suitable for storage under cryopreservation conditions. In one embodiment, the container can be a vial made of a material that can include, but is not limited to, polymers such as polytetrafluoroethylene, polystyrene, polyethylene, or polypropylene. In a particular embodiment, the vial can have additional surface treatments to improve cryopreservation conditions (e.g., hydrophilic coatings that reduce adsorption and denaturation). In exemplary embodiments, the volume of the vial can be greater than about 0.1 ml, such as the volume of the vial can be about 0.1 ml, about 0.5 ml, about 0.75 ml, about 1 ml, about 1.5 ml, about 2 ml, about 2.5 ml, about 4.5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, or about 50 ml. In another embodiment, the container may also be a cryopack, the volume of which is greater than about 30 ml, for example, the volume of the cryopack may be about 30 ml, about 50 ml, about 100 ml, about 150 ml, about 200 ml, about 300 ml, about 400 ml, or about 500 ml. The cryopack may be made of any suitable material, including, but not limited to, polymers such as polytetrafluoroethylene, polystyrene, polyethylene, polypropylene, fluorinated ethylene propylene (FEP), polyolefin, and ethylene vinyl acetate (EVA). Illustrative single-use bioreactors include, but are not limited to, KryoSure® cryopreservation bags, PermaLife™ bags (OriGen Biomedical), CryoStore freezing bags (OriGen Biomedical), Freeze-Pak™ biocontainers (Charter Medical), and the bags described in U.S. Provisional Patent Application No. 62/037,181 (Merial Ltd., a Sanofi Company), which is incorporated herein by reference in its entirety.

These containers are then frozen using methods and devices well known in the art before being stored at low temperatures, typically from about -130°C to about -196°C. In one embodiment, the freezing technique used may be rate controlled and slow freezing (also called slow programmed freezing or SPF). The cell collection obtained in this manner is a cell bank.

In one embodiment, the cell bank may be, but is not limited to, an ultra-high density cell bank, a master cell bank, a working cell bank, or a mini bank.

V. Determination of cell viability

As indicated above, the methods of the present invention provide for the creation of a high density cell bank while maintaining excellent cell viability for subsequent use. As used herein, the term "cell viability" may be defined as the number or percentage of viable cells in a given sample. Cell viability may be determined using any method available in the art at any point in the described method for creating an ultra-high density cell bank. Commonly used methods for determining cell viability are largely based on the principle that living cells have intact cell membranes that are impermeable to certain dyes, such as trypan blue (diazo dye), eosin or propidium, whereas dead cells do not.

In one embodiment, trypan blue can be used to stain a portion of the cells and thus determine the presence of cells with intact membranes (not stained) and the presence of cells with disrupted membranes (blue). These cells can then be counted to determine the number of both live and dead cells in the culture and presented as a percentage to indicate the relative viability of the culture.

In a particular embodiment, cell viability may be determined using a culture that has been grown to an ultra-high density but has not yet been frozen (i.e., before freezing or before thawing the culture). In a particular embodiment, cell viability may be determined using a culture that has been frozen and then thawed (i.e., a post-freeze or post-thaw culture).

In a particular embodiment, the cell viability is at least about 60%, such as about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the cell viability after thawing is at least about 80%, such as at least about 85%, 90%, or 95%, including up to 100%.

VI. Therapeutic proteins

The cells obtained by the method for creating a cryobank of cells with ultra-high density according to the present invention can be used in a later stage of protein production. For example, cells expanded in a bioreactor and frozen in aliquots of a high-density bank according to the methods of the present invention can be used in the production of biological substances, including therapeutically important proteins. Such biological substances can include, but are not limited to: viruses (see, for example, WO 200138362), binding polypeptides such as antibodies, for example, monoclonal antibodies and fragments thereof, for example, Fab fragments; Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, artificial protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins and thrombolytics. These biological substances can be obtained by any method available in the art. In one embodiment, the present invention relates to a method for producing a biological substance, wherein the method comprises culturing cells capable of expressing the biological substance under conditions suitable for producing the biological substance, wherein the cells were obtained from a bank of frozen ultra-high-density cells obtained using perfusion culturing. The biological substance may be (without limitation) a protein (e.g., any therapeutically significant protein).

Examples

The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents and published patent applications cited in this application are herein incorporated by reference in their entirety.

Example 1. General methods

The following conditions were common to the experiments outlined in Examples 3-6 below.

The pH was set at 7.0 ± 0.1. The pH of the culture was maintained at the setpoint by an automatic feedback control system using the WAVE POD controller. The automatic controller varied the _CO2 concentration in the headspace gas and additionally controlled the addition of base ( _CO2 /base control circuit). When the controller registered a pH below the selected pH setpoint, an attempt was first made to achieve the pH setpoint by decreasing the _CO2 level. If the pH setpoint was not achieved within the specified time, base was added. At pH above the pH setpoint, the _CO2 concentration was increased.

The DO (dissolved oxygen) value was set to ≥ 40%. The DO of the culture was maintained at the set value by an automatic feedback control system using the WAVE POD controller. The oxygen concentration was automatically varied from 21% to 50% ( _O2 concentration control circuit). Additional pure oxygen gas was manually supplied to the headspace in combination with manual adjustment of the rocking conditions to maintain DO ≥ 40%. Specifically, when the current DO was approximately 40% and the _O2 concentration via the POD controller was increased from 21% to >30%, the rocking speed and angle were increased from 22 rpm/10° to 25 rpm/12°. If the POD _O2 again became > 30%, the oscillation speed and angle were increased from 25 rpm/12° to 30 rpm/12°, or pure _O2 was added to the headspace (the latter was preferred). Finally, the volume percent pure _O2 in the gas was gradually increased to increase the total headspace _O2 . The manual addition strategy resulted in an increase of approximately 10% per day by adjusting the ratio of monitored volumes between POD-read gaseous oxygen (21%-50% provided by the POD regulator) and additional pure gaseous oxygen (100% monitored by the rotameter). The total gas flow rate was increased slightly to facilitate gas transfer.

The CSPR (cell specific perfusion rate) of the cultures was kept nearly constant, with the perfusion rate increased daily based on the measured cell density.

Example 2. Development and implementation of a protocol for creating a cell bank with ultra-high density

The protocol for establishing an ultra-high-density cell cryobank was started with a 2 mL vial of master cell bank cells (working volume 1.5 mL) at an approximate density of 2.0-2.4× ¹⁰⁷ viable cells/mL (normal cell cryopreservation condition). The cells were then grown by culturing under perfusion conditions. After adding DMSO to the cell culture, this ultra-high-density cell culture was distributed into individual 5 mL vials (working volume approximately 4.5 mL) or cryopreservation bags (working volume approximately 100 mL) suitable for cryopreservation and storage as an ultra-high-density cell bank (at least approximately 10× ¹⁰⁷ cells/mL). A schematic representation of this protocol is shown in Figure 1.

Example 3. Ultra-high-density cell culture and cell banking using rCHO 1.

The parameters for the next experiment were as follows:

1) _CO2 was reduced to 0% when Xv ≥ approximately 3× ¹⁰⁷ viable cells/mL (on day 9), then base was added if necessary;

2) WAVE speed and angle: initially 22 rpm/10° (from day 0 to day 8); increased to 25 rpm/12° when O ₂ as measured by POD increased from 21% to > 30% (on day 8), and then to 30 rpm/12° (on day 9) to facilitate gas transfer (delivery of O ₂ and removal of CO ₂ );

3) Supplemental pure _O2 (100%): manually delivered into the headspace when _O2 as indicated by POD > 30% and Xv ≥ approximately 6× ¹⁰⁷ viable cells/mL (on day 11);

4) Total gas flow: 0.2 L/min (liters per minute) from day 0 to day 10, increased to 0.4 L/min on day 10 when Xv > 4×10 ⁷ viable cells/mL, and then to approximately 0.5 L/min on day 11 (POD O ₂ : net O ₂ = 3:1, total O ₂ : approximately 62%); and

5) Cell specific perfusion rate: target ≥ approximately 0.2 nL/cell/day initially; perfusion rate was increased gradually to 12 RV/day (note: RV is defined as reactor volume). CSPR on the last day was 0.1 nL/cell/day to 0.2 nL/cell/day.

6) Inoculum system and bioreactor media: CD CHO with 4 mM L-glutamine

7) Bioreactor: GE custom Cellbag 10L perfusion bioreactor with two dip tubes; working volume: 5L;

8) Bioreactor inoculum: shake flask inoculation system; inoculum density: approximately 5×105 viable cells/mL

9) Cell retention methods: ATF4 (pore size 0.2 µm)

10) Cell culture temperature: 37°C

11) Freezing: CryoMed™ Variable Speed Freezing Chamber

Ultra-high density (UHD) cell culture and cell banking (after freezing and thawing) were tested on the rCHO 1 cell line. The Xv value of the perfusion culture reached approximately 1.11× ¹⁰⁸ viable cells/mL at day 12 with high viability (>97% throughout the process). Figure 2 shows the viable cell density (cells/mL), perfusion rate in reactor volumes/day, and CSPR 10X (10X cell specific perfusion rate in nL/cell/day) of the ultra-high density perfusion culture. Figure 3 shows the autonomous pH profile of the culture. The culture was harvested directly without concentration to create two 5 mL UHD vials at two DMSO storage temperatures: a well-controlled cold temperature using a 4 °C jacketed stirrer and an uncontrolled cold temperature using a stirrer cooled in an ice water bath. Figure 4A-C show the post-freezing parameters of 5 mL UHD vials obtained under the above cell culture conditions. Both UHD vials showed rapid cell growth after freezing, very high viability and low apoptosis. However, the more efficient jacketed stirrer temperature control resulted in slightly improved subsequent bank recovery and growth compared to the more passive ice water bath temperature control.

Example 4. Ultra-high-density cell culture and cell banking using rCHO 2.

The parameters for the next experiment were as follows:

1) CO2 was reduced to 0% when Xv≥ approximately 5× ¹⁰⁷ viable cells/mL (on day 9), then base was added if necessary.

2) WAVE speed and angle: initially 22 rpm/10° (day 0 to day 7); increased to 25 rpm/12° when O ₂ as measured by POD increased from 21% to > 30% (day 7) to facilitate gas transfer (to supply O ₂ and remove CO ₂ ).

3) Supplemental pure _O2 (100%): manually delivered into the headspace when _O2 as indicated by POD > 30% and Xv ≥ approximately 3× ¹⁰⁷ viable cells/mL (on day 8).

4) The total gas flow was initially 0.2 L/min (from day 0 to day 8) and increased to 0.4 L/min on day 8 when Xv ≥ approximately 3× ¹⁰⁷ viable cells/mL (POD _O2 : net _O2 =3:1, total _O2 : approximately 62%), and then to approximately 0.5 L/min on day 9 (POD _O2 : net _O2 =1:1, total _O2 : approximately 75%).

5) Specific cell perfusion rate: the target was ≥ 0.2 nL/cell/day initially, but the perfusion rate was increased gradually to 12 RV/day. On the last day, 0.1 nL/cell-day < CSPR < 0.2 nL/cell/day.

6) Inoculum system and bioreactor media: CD CHO with 4 mM L-glutamine

7) Bioreactor: GE custom Cellbag 10L perfusion bioreactor with one dip tube; working volume: 5L;

8) Bioreactor inoculum: shake flask inoculation system; inoculum density: approximately 5×105 viable cells/mL

9) Cell retention methods: ATF4 (pore size 0.2 µm)

10) Cell culture temperature: 37°C

11) Freezing: CryoMed™ Variable Speed Freezing Chamber

Ultra-high-density cell culture and cell banking (after freezing and thawing) were tested on the rCHO 2 cell line. Figure 5 shows the viable cell density (cells/mL), perfusion rate in reactor volumes/day, and CSPR 10X (10X cell specific perfusion rate in nL/cell/day) of the ultra-high-density perfusion culture. Figure 6 shows the offline pH profile of the culture. The culture was harvested to produce 5 mL UHD flasks and 100 mL UHD cryopreservation bags under two different DMSO storage conditions: 1) room temperature (RT, approximately 22-25°C); and 2) controlled cold temperature (CT, approximately 5°C using a 4°C lined stirrer).

Figures 7 and 8 present graphs showing the cell growth (Xv, solid lines) and viability (dashed lines) profiles in 250 mL and 3 L shake flasks for UHD flasks and packs obtained under the above-mentioned cell culture conditions at room temperature and under well-controlled cold storage conditions (RT and CT, respectively). These UHD flasks recovered well with rapid cell growth after freezing, high viability, low apoptosis, and comparable productivity when seeded at a concentration of 0.5×10⁶viable cells/ml and 1.0×10⁶viable cells/ml in 250 ml and 3 l shake flasks. No difference was observed between UHD cryopacks and cryovials under any of the storage conditions in terms of growth, viability, apoptosis, and productivity after thawing. However, vials and bags produced at a well-controlled low temperature using a 4°C lined stirrer exhibited faster growth, higher viability, and lower apoptosis compared to those produced at room temperature. Figures 9 and 10 show the apoptosis profiles after thawing of UHD banks seeded at a concentration of 0.5×10⁶viable cells/ml and 1.0×10⁶viable cells/ml, respectively, and Figures 11 and 12 show the specific productivity (SPR, RT- room temperature, CT- well-controlled cold temperature)

Example 5: Creation of an ultra-high-density cell bank in a 20 L WAVE bioreactor using rCHO 2.

The parameters for the next experiment were as follows:

1) Cell bank: 100 ml UHD cryopacket obtained in Example 2

2) Bioreactor medium: CD CHO with 4 mM L-glutamine.

3) Post-freezing bioreactor: GE Cellbag, 20L; working volume: 10L.

4) Bioreactor inoculum: thawed culture from a thawed cryopackage; inoculum density: approximately 5×105 viable cells/ml.

5) Bioreactor temperature: 37°C, O2: 20%, CO2: 5%, speed and swing angle: 22 rpm/8°

One 100 ml UHD cryopreservoir was thawed. One 20 L WAVE bioreactor (A) with a total working volume of 10 L was directly inoculated with 50 ml of the thawed culture. Another 50 ml was slowly diluted with cold medium first to reduce potential cell damage induced by the large osmotic gradient, which was then inoculated into a second 20 L WAVE bioreactor (B) with a total working volume of 10 L. Both bioreactors were operated under identical conditions. Aliquots from both bioreactors were transferred to control shake flasks (60 ml working volume) for growth comparison. Bioreactor cultures were also compared with shake flask cultures (60 mL/250 mL shake flasks and 1.5 L/3 L shake flasks) with inoculum from another thawed 100 mL cryopreservoir.

The cultures from the two WAVEs showed very comparable performance in terms of growth, viability, and apoptosis after cell thawing. And the cultures in both bioreactors were very comparable not only to those in the controlled shake flasks, but also to those thawed and grown in shake flasks. These data suggest that a single UHD cryopacket can be directly thawed in two 20L WAVE bioreactors without slow dilution with cold media, with comparable performance to shake flasks, making the seeding system a completely closed method, from bag thawing to growth in the WAVE bioreactor.

Figure 13 is a graph showing the cell growth profile (Xv solid lines) and viability (dashed lines) in 20 L WAVE bags for the UHD cryopack of the rCHO 2 cell line obtained according to the above-mentioned cell culture conditions at room temperature shown in Example 2.

Example 6. Ultra-high-density cell culture and cell bank establishment using rCHO 3.

The parameters for the next experiment were as follows:

1) _CO2 was reduced to 0% when Xv≥ approximately 2× ¹⁰⁷ viable cells/mL (on day 8), then base was added if necessary.

2) WAVE speed and angle: initially 22 rpm/10° (day 0 to day 7); increased to 25 rpm/12° when O ₂ as measured by POD increased from 21% to > 30% (day 7) to facilitate gas transfer (to supply O ₂ and remove CO ₂ ).

3) Supplemental pure _O2 (100%): manually delivered into the headspace when _O2 as indicated by POD > 30% and Xv ≥ approximately 3× ¹⁰⁷ viable cells/mL (on day 9).

4) Total gas flow: 0.2 L/min initially (from day 0 to day 9) and increased to 0.4 L/min on day 9 when Xv ≥ approximately 3× ¹⁰⁷ viable cells/mL (POD _O2 : net _O2 = 3:1, total _O2 : approximately 62%), and then to approximately 0.5 L/min on day 10 (POD _O2 : net _O2 = 1:1, total _O2 : approximately 75%), to approximately 0.6 L/min on day 12 (POD _O2 : net _O2 = 1:2, total _O2 : approximately 83%).

5) Cell specific perfusion rate: perfusion was started at 0.5 RV/day on day 2 and increased gradually to maintain a low CSPR ≥ 0.05 nL/cell/day

6) Inoculum system and bioreactor media: CD CHO with 4 mM L-glutamine

7) Bioreactor: GE custom Cellbag 10L perfusion bioreactor with one dip tube; working volume: 5L;

8) Bioreactor inoculum: shake flask inoculation system; inoculum density: approximately 5×105 viable cells/mL

9) Cell retention methods: ATF4 (pore size 0.2 µm)

10) Cell culture temperature: 37°C

11) Freezing: CryoMed™ Variable Speed Freezing Chamber

12) Evaluation of the established cell bank: shake flask (two passages) and 20L WAVE (one passage), Xv, viability, apoptosis, productivity were evaluated.

13) Medium for evaluation of the established cell bank: CD CHO with 4 mM L-glutamine

Ultra-high density cell culture and cell banking (after freezing and thawing) were tested on the rCHO 3 cell line. Figure 15 shows the viable cell density (cells/mL), perfusion rate in reactor volumes/day, and CSPR 10X (10X cell specific perfusion rate in nL/cell/day) of the ultra-high density perfusion culture. Figure 16 shows the offline pH profile of the culture. 5 mL UHD flasks and 100 mL UHD bags were prepared using ultra-high density perfusion culture under two different DMSO storage conditions: 1) room temperature (RT, approximately 22-25°C); and 2) controlled cold temperature (CT, approximately 5°C using a 4°C lined stirrer).

All thawed cultures from UHD flasks and UHD bags recovered well, showing rapid growth, low apoptosis and comparable productivity. UHD bags and flasks obtained under well-controlled cold storage conditions were comparable to those obtained at room temperature, showing faster growth and higher viability (>95%) at first passage. The thawed UHD bag had comparable performance in shake flasks and WAVE bioreactors. This also suggests that a single UHD cryopack could be directly thawed in two 20L WAVE bioreactors, making the plating system a fully closed process, from bag thawing to growth in the WAVE bioreactor. Figure 17A is a graph showing the cell growth (Xv, solid lines) and viability (dashed lines) profiles in 250 mL and 3 L shake flasks and 20 L WAVE bioreactors for UHD flasks and bags obtained under the above-mentioned cell culture conditions at room temperature and under well-controlled cold storage conditions (RT and CT, respectively). Figure 17B and C show the post-thawing apoptosis profiles and the specific productivity (SPR).

Example 7. Reduction in the use of medium in the ultra-high density culture method under perfusion conditions using rCHO 3.

The rCHO 3 cell line was grown at a low cell specific perfusion rate (CSPR) of ≥ 0.025 nL/cell/day using an in-house developed medium. A conventional 10 L WAVE Cellbag coupled with ATF4 was used for perfusion culture. The culture reached a viable cell density of approximately 1.25 x 10 ⁸ viable cells/mL on day 11 with a perfusion rate of up to 3 reactor volumes/day. The culture viability was > 97% during bioreactor culture. The medium usage was reduced to approximately 50% compared to the same culture conditions using commercial medium. However, the observed cell growth was slightly faster than with the commercial CD CHO medium. The culture was directly harvested (and not concentrated) to create UHD banks in 5 mL vials and 100 mL cryopacks under well-controlled cold storage temperature in DMSO using a lined stirrer set at 4°C. Post-banking studies demonstrated that both vial and cryopack UHD banks recovered well after thawing in shake flasks with rapid growth, high viability (>95%) and comparable productivity. Additionally, one cryopack was thawed in two 20 L WAVE bioreactors and demonstrated comparable cell growth and viability to shake flask results. Figure 18 is a graph comparing viable cell density (cells/mL, solid lines) and viability (dashed lines) with the internal medium (black) and CD CHO medium (gray) in an exemplary rCHO 3 cell line culture. Figure 19 is a graph comparing perfusion rate (circles, solid lines) and specific cell perfusion rate (triangles, dashed lines) with the internal medium (black) and CD CHO medium (gray) in an exemplary rCHO 3 cell line culture.

Example 8. Reduction in the use of medium in the ultra-high density culture method under perfusion conditions using rCHO 1.

The rCHO 1 cell line was grown using commercial CD CHO medium supplemented with Efficient Feed B (EFB) and maintained at a low cell specific perfusion rate (CSPR) of ≥ 0.04 nL/cell/day. A 10 L GE WAVE Cellbag perfusion bioreactor with an integrated filter was used for perfusion culture. Commercial CD CHO medium without any nutrient supplement was also tested for comparison. The culture without EFB achieved a viable cell density of approximately 1.0 x 10 ⁸ viable cells/mL on day 13 with a perfusion rate of up to 8 reactor volumes/day. With the addition of 10% EFB to CD CHO medium from day 5 to day 10 and 20% EFB from day 10 to day 14, the culture achieved a viable cell density of approximately 1.03× ¹⁰⁸ viable cells/mL on day 14 at a perfusion rate of up to 4 reactor volumes/day. Therefore, the addition of EFB achieved comparable UHD perfusion cell culture with approximately 50% reduction in medium usage. Figure 20 is a graph comparing viable cell density (cells/mL, solid lines) and viability (dashed lines) between CD CHO medium supplemented with Efficient Feed B (black) and CD CHO medium (gray) in an exemplary rCHO1 cell line culture. Figure 21 is a graph comparing the perfusion rate (circles, solid lines) and specific cell perfusion rate (triangles, dotted lines) of CD CHO medium supplemented with Efficient Feed B (black) and CD CHO medium (gray) in an illustrative rCHO 1 cell line culture.

Example 9. Scaling up the process to obtain ultra-high density under perfusion conditions using rCHO 3.A

A 20L WAVE Cellbag was configured for connection to ATF4 to provide a 10L UHD perfusion culture. The rCHO 3 cell line was tested for growth in commercial CD CHO medium. The culture reached a viable cell density of approximately 1.1 x ¹⁰⁸ viable cells/mL at day 14 with a perfusion rate of up to 5 reactor volumes/day. The cell specific perfusion rate (CSPR) was maintained at ≥ 0.04 nL/cell/day and viability was >97%. Cell growth was comparable between the 10L WAVE Cellbag and a 5L working volume. Figure 22 shows the viable cell density (cells/mL, solid lines) and viability (dashed lines) using a 20 L WAVE Bioreactor (10 L working volume, black) and a 10 L WAVE Bioreactor (5 L working volume, gray) in an illustrative culture of the rCHO 3 cell line. The culture was directly harvested to yield approximately 90 x 100 mL cryopacks.

Example 10. Evaluation of ultra-high density cryopacks after one year of storage in freezing chambers under liquid nitrogen vapour storage conditions using rCHO 2 and rCHO 3.

100 mL UHD cryopreservoir jars (approximately 1.0 x 10 ⁸ viable cells/mL, 100 mL per cryopreservoir) of rCHO 2 and 3 cell lines were tested for post-thaw performance and compared to time 0 (when jars were frozen and transferred to liquid N ₂ vapor overnight for storage). Comparable cell growth, viability, and productivity were observed post-thaw at time 0 and over one year.

Claims (25)

1. A method for producing a bank of frozen mammalian cells at ultra-high density directly from a population of cultured cells, comprising: a) culturing mammalian cells in a perfusion bioreactor at a pH of 6.8 to 7.2, wherein the concentration of the cultured mammalian cell population is from 2×10 ⁷ to 5×10 ⁷ cells/mL and supplying O ₂ to maintain a dissolved oxygen (DO) value ≥ 40%, to obtain an ultra-high-density cultured cell population with a cell concentration of at least 1.0×10 ⁸ cells/mL, wherein said perfusion bioreactor is connected to a cell retention system; and b) adding a cryoprotectant to the ultra-high density cultured cell population; c) controlled rate freezing; and d) obtaining a bank of frozen cells with ultra-high density, wherein the ultra-high density frozen cell bank is characterized by a cell concentration of at least 1.0×10 ⁸ cells/mL and at least 95% cell viability after thawing and where there is no concentration step between culturing the cell population and adding the cryoprotectant to the ultra-high density cultured cell population. 2. The method according to claim 1, wherein the cultured ultra-high density cell population has a cell density of 1.1×10 ⁸ cells/ml, or 1.2×10 ⁸ cells/ml, or 1.3×10 ⁸ cells/ml, or 1.4×10 ⁸ cells/ml, or 1.5×10 ⁸ cells/ml, or 1.6×10 ⁸ cells/ml, or 1.7×10 ⁸ cells/ml, or 1.8×10 ⁸ cells/ml, or 1.9×10 ⁸ cells/ml, or 2.0×10 ⁸ cells/ml. 3. The method of claim 1 or 2, wherein adding the cryoprotectant comprises adding dimethyl sulfoxide (DMSO) to the ultra-high density cell population at a final concentration of 5 to 10% v/v. 4. The method according to any one of claims 1-3, wherein the ultra-high density frozen cell bank contains 4.5×10 ⁸ cells/vial or 100×10 ⁸ cells/cryopacket. 5. The method according to any one of paragraphs. 1-4, wherein the perfusion rate in the perfusion bioreactor is from 0.02 to 0.5 nl/cell/day. 6. The method according to any one of paragraphs. 1-5, wherein the perfusion rate in the perfusion bioreactor is up to 15 reactor volumes per day. 7. The method according to any one of claims 1 to 6, wherein the mammalian cells are selected from the group consisting of: CHO, CHO-DBX11, CHO-DG44, CHO-S, CHO-K1, Vero, BHK, HeLa, COS, MDCK, HEK-293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0, CRL7030, HsS78Bst cells, PER.C6, SP2/0-Agl4 and hybridoma cells. 8. The method according to any one of claims 1-7, wherein the mammalian cells are transfected cells or the mammalian cells express a therapeutic protein. 9. The method according to any one of claims 1-8, wherein the bioreactor is rocked at 25 rpm with a rocking angle of 12° or at 22 rpm with a rocking angle of 10°. 10. The method according to any one of claims 1-9, wherein the cultured ultra-high density cell population is maintained prior to and during the addition of the cryoprotectant by any of the following methods: (i) cooled and maintained at 4°C; or (ii) maintained at a temperature between 20 and 26 °C; or (iii) maintained at uncontrolled low temperature using an ice bath. 11. The method according to any one of claims 1 to 10, wherein the total gas flow rate is initially set at 0.2 liters per minute (L/min), with a subsequent increase in the total gas flow rate to 0.4 L/min when the cell concentration of the cultured mammalian cells is from 3×10 ⁷ to 4×10 ⁷ cells/mL. 12. A method for producing a bank of frozen, ultra-high-density mammalian cells, wherein the bank contains a cell concentration of at least 1.0×10 ⁸ cells/ml, the method comprising: a) adding a cryoprotectant directly to the ultra-high density cell culture in a bioreactor, wherein the bioreactor is connected to a non-centrifugal cell holding device; wherein the ultra-high density cell culture is obtained by culturing mammalian cells at a concentration of 2×10 ⁷ to 5×10 ⁷ cells/mL in a perfusion bioreactor at a pH of 6.8 to 7.2 and supplying O ₂ to maintain a dissolved oxygen (DO) value ≥ 40%; b) harvesting ultra-high density cell culture; and c) controlled rate freezing to produce a bank of frozen cells with ultra-high density; wherein the ultra-high density frozen cell bank is characterized by at least 95% viability after thawing in perfusion culture.