Method for preparing mammalian cells for perfusion cell culture

Abstract

The present disclosure provides methods of preparing mammalian cells for use in a perfusion cell culture process that improve the growth and productivity of the cells. Also provided are cell culture methods involving subjecting mammalian cells to one or more perfusion procedures involving producing a solid phase and a liquid phase with a continuous flow centrifuge, and utilizing at least a portion of the solid phase to maintain, maintain and / or initiate a new cell culture. Perfusion bioreactor systems and components thereof are also provided.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 345,796, filed on May 25, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] The present disclosure generally relates to methods of preparing mammalian cells for use in perfusion cell culture processes that improve the growth and productivity of the cells. More specifically, the present disclosure relates to improved cell culture methods in which mammalian cells are subjected to one or more perfusion procedures that involve the use of a continuous flow centrifuge to produce a solid phase and a liquid phase, and the use of at least a portion of the solid phase to maintain, hold and / or initiate new cell cultures. The present disclosure also relates to perfusion bioreactor systems and components thereof. Background Art

[0003] Despite significant advances in the field over the past few decades, biomanufacturing processes involving industrial-scale culture of mammalian cells and production of products therein remain fraught with significant challenges. For example, typical bioreactor systems used for such processes must maintain a sterile, closed environment, which makes the addition of fresh cell culture media and the removal of spent cell culture media problematic. As a result, cells grown under batch or fed-batch process conditions typically face nutrient limitations when their cell culture media are depleted of important components. In addition, the accumulation of unwanted metabolic byproducts in the cell culture may further limit growth and may cause damage to the desired product. Therefore, there remains a strong need for improved cell culture systems and methods that can increase growth and improve productivity by addressing these and other limitations.

[0004] Compared with bacterial cell culture, mammalian cell culture generally has lower production rate and produces lower production yield. Therefore, a large number of studies have focused on mammalian cell culture conditions and methods that can optimize polypeptide output, i.e., conditions and methods that support high cell density and high protein titer. For example, it has been determined that limiting feeding glucose to mammalian cell culture in a batch feed process can control lactate production without the need for constant rate feeding glucose (see, e.g., U.S. Patent Application Publication No. US20050070013).

[0005] Two cell culture processes are mainly used for large-scale protein production: batch feed process and perfusion process. The main goal of these methods is to add nutrients (such as glucose) when they are consumed by cells, and to remove metabolic wastes (such as lactic acid and ammonia) when they are produced. In the batch feed process, cells are usually inoculated with glucose-containing medium at the beginning of culture and at one or more time points after the beginning of culture but before the termination of culture. Although this method can help control the lactic acid production of cultured cells at a relatively low level, it is impossible to reach the maximum cell density, growth rate and cell viability level due to glucose limiting conditions. Therefore, the number of cells and / or the quantity of products produced by cells are not maximized.

[0006] During the perfusion process, the cells also receive an inoculum basal medium, and when the cells reach the desired cell density, perfusion of the cells is initiated so that the spent medium is replaced with fresh medium. The perfusion process allows the culture to reach higher cell densities and, therefore, can produce large quantities of cells and / or products. However, at larger, industrially relevant production scales, the perfusion process requires extremely large quantities of fresh cell culture medium. In addition, when the spent medium is removed, any product secreted by the cells into the medium is lost. This requires a separate harvesting step to capture the product in the spent medium, which otherwise would result in an overall loss of efficiency if the spent medium is discarded.

[0007] Therefore, there is a need for improved large-scale cell culture methods that can maximize cell viability, cell concentration, and the amount of protein produced, and minimize product losses in spent culture medium. The present disclosure can address these and other needs. Summary of the invention

[0008] Provided herein is a cell culture method, comprising using a continuous flow centrifuge to perfuse a cell culture to produce a solid phase and a liquid phase, and returning at least a portion of the solid phase and a volume of cell culture medium to a culture vessel to maintain, hold and / or start a new cell culture. In certain embodiments, these methods can be used to reach a perfusion rate ranging from about 0.5 to about 6 or more vessel volumes / day (VVD). The continuous flow centrifuge is used as a part of cell culture, i.e., as a part of the "upstream" process of producing, for example, a therapeutic polypeptide or protein (such as an antibody or fusion protein), which is contrary to the use of a continuous flow centrifuge as a part of clarification (as a part of the "downstream" purification of such therapeutic polypeptides or proteins produced by cell culture).

[0009] In a first aspect, a method is provided herein of culturing mammalian cells that have been engineered to produce proteins, e.g., therapeutic proteins (such as antibodies), before clarification in preparation for protein purification; i.e., in a pre-production vessel, e.g., a bioreactor. In a specific embodiment, the method comprises placing a plurality of mammalian cells and a volume of culture medium in a culture vessel to produce a cell culture. In other specific embodiments, the method comprises culturing the cell culture to a cell density greater than 1% hematocrit (PCV). In a specific embodiment, the method comprises subjecting the cell culture to a perfusion procedure. In a more specific embodiment, the perfusion procedure comprises transferring at least a portion of the cell culture to a continuous flow centrifuge. In a more specific embodiment, the perfusion procedure comprises operating a continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV. In a more specific embodiment, the perfusion procedure comprises returning the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate ranging from 0.5 to 6 vessel volumes / day (VVD). In specific embodiments, a low perfusion rate of about 0.7 VVD is used, and / or a high perfusion rate of about 4 to about 5 VVD is used.

[0010] In specific embodiments, after the perfusion procedure is completed, the cell culture can have a cell density of 0.2% PCV or greater, such as 0.2% to about 30% PCV.

[0011] In some embodiments, the perfusion program includes increasing or decreasing the perfusion rate in a constant manner. In some embodiments, the perfusion program includes increasing or decreasing the perfusion rate in a variable manner.

[0012] In certain embodiments, the time period for performing a perfusion procedure or a cell culture method comprising such a perfusion procedure is between 0.5 hours and up to about 5 hours, such as about 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5 hours or longer. In certain embodiments, the time period for performing a perfusion procedure or a cell culture method comprising such a perfusion procedure is between about 5 hours and up to about 24 hours, such as about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours. In certain embodiments, the time period for performing a perfusion procedure or a cell culture method comprising such a perfusion procedure is between about 1 day and up to about 20 days, such as about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 days. In other embodiments, the perfusion procedure or the cell culture method comprising the perfusion procedure is performed for up to 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 days. In addition, the perfusion procedure according to the above embodiments can be performed in a semi-continuous, discontinuous or "intermittent" manner, wherein the perfusion procedure is performed, for example, once a day over a period of several days.

[0013] In certain embodiments, the continuous flow centrifuge comprises a disc stack bowl. In certain embodiments, the continuous flow centrifuge comprises a tubular bowl. In certain embodiments, the continuous flow centrifuge may have an operating speed ranging from 3,000 to 10,000 revolutions per minute (RPM). In certain embodiments, the continuous flow centrifuge comprises sterilizable components. In certain embodiments, the continuous flow centrifuge comprises disposable components. In certain embodiments, the continuous flow centrifuge may have an operating speed ranging from 1,000 to 10,000 revolutions per minute (RPM). 2 Up to 200,000m 2In certain embodiments, after completing the centrifugation procedure, the cell culture may have a viability percentage greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%. In certain embodiments, the cell culture maintains a viability percentage greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% over a period of 1 to 90 days (e.g., for, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 days). In certain embodiments, the mammalian cell comprises a recombinant mammalian cell. In certain embodiments, the recombinant mammalian cell includes a recombinant Chinese hamster ovary (CHO) cell. In certain embodiments, the cell culture may have a lactate concentration less than or equal to 4g / L. In certain embodiments, the recombinant mammalian cell may produce a secretion product. In certain embodiments, the secretion product includes a recombinant protein. In certain embodiments, the recombinant protein may be an antibody. In certain embodiments, the culture vessel may have a working volume greater than or equal to 80L. In certain embodiments, the culture vessel may have a total volume ranging from 100L to 3,000L. In certain embodiments, the culture vessel may have a total volume of 100L. In certain embodiments, the culture vessel may have a total volume of 3,000L. In certain embodiments, the culture vessel has a total volume ranging from 100L to 30,000L. In certain embodiments, the culture vessel has a total volume ranging from 5,000L to 30,000L. In certain embodiments, the culture vessel has a total volume ranging from 100L to 6,000L. In some embodiments, the method may include transferring at least a portion of the cell culture to a different culture vessel to start a second cell culture. In certain embodiments, the second cell culture has an initial cell density in the range of greater than or equal to 0.1% to 10% PCV. In some embodiments, the method may include transferring at least a portion of the cell culture to a production culture vessel to start a production culture with a starting cell density in the range of greater than or equal to 0.1% to 10% PCV. In some embodiments, the method may include culturing the production culture under batch or fed-batch process conditions. In some embodiments, the method may include separating a liquid phase from a continuous flow centrifuge and subjecting the secreted product therein to a purification procedure.In some embodiments, the method can include adding fresh culture fluid (eg, cell culture medium and / or cell culture media) to the culture vessel.

[0014] In certain embodiments, the cell culture methods provided herein include producing a mammalian cell culture having a cell density greater than or equal to 0.1% PCV. In certain embodiments, the method includes placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel to produce a cell culture. In certain embodiments, the method includes culturing the cell culture to a cell density greater than or equal to 10% PCV (e.g., about 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or about 30%). In certain embodiments, the method includes perfusing the cell culture. In certain embodiments, the perfusion procedure includes transferring at least a portion of the cell culture to a continuous flow centrifuge. In certain embodiments, the perfusion procedure includes operating a continuous flow centrifuge to produce a solid phase with a cell density ranging from about 1% to about 10%, about 20%, about 30%, about 40% or about 50% PCV. In a specific embodiment, the cell density ranges from about 20% to about 40%. In more specific embodiments, the cell density ranges from about 30% to about 35%. In certain embodiments, the perfusion procedure includes returning a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. In certain embodiments, after completing the perfusion procedure, the cell culture density increases or has increased by greater than 0.1% PCV, such as greater than or equal to 1% PCV or greater than or equal to 10% PCV. In specific embodiments, the solid phase cell density ranges from about 20% PCV to about 40% PCV.

[0015] In certain embodiments, the cell culture methods provided herein include producing cells comprising at least 4.8 x 10 12 In a specific embodiment, the method includes placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of at least, no greater than, or about 80 L to produce a cell culture having a starting cell density greater than or equal to 1 million cells / mL. In a specific embodiment, the method includes culturing the cell culture to a cell density greater than or equal to 1% PCV, or greater than or equal to 10% PCV. In a specific embodiment, the method includes subjecting the cell culture to a perfusion procedure. In a specific embodiment, the perfusion procedure includes transferring at least a portion of the cell culture to a continuous flow centrifuge comprising a disposable disc stack rotor and comprising a range of 1,000 to 200,000 m 2In a specific embodiment, the method comprises operating a continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV (e.g., about 10%, about 20%, about 30%, about 40%, or about 50% PCV). In a specific embodiment, the method comprises returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. In a specific embodiment, after completing the perfusion procedure, the cell culture has a cell density greater than or equal to 60 million cells / mL.

[0016] In certain embodiments, provided herein are methods for producing a total of at least 2.16 x 10 14 cells or about 1.5-2.5x10 6 In a specific embodiment, the method comprises placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of up to, at least, or about 3,000 L or 3,600 L to produce a culture vessel having a cell density greater than or equal to 1 million cells / mL (1 x 10 6 In a specific embodiment, the method comprises growing the cell culture to a cell density greater than or equal to 10% PCV. In a specific embodiment, the method comprises subjecting the cell culture to a perfusion procedure. In a specific embodiment, the perfusion procedure comprises transferring at least a portion of the cell culture to a continuous flow centrifuge comprising a disposable disc stack rotor and further comprising a range of 1,000 to 200,000 m 2 In specific embodiments of the methods provided herein, the perfusion procedure comprises operating a continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV (e.g., up to about 10%, about 20%, about 30%, about 40%, or about 50% PCV). In certain embodiments, the perfusion procedure comprises returning at least a portion of the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. In certain embodiments, after completing the perfusion procedure, the cell culture has a cell density greater than or equal to 60x10 6 The cell density was 10 cells / mL.

[0017] In certain embodiments, the cell culture produced by the methods described herein is a part of the seed culture in the pre-production cell culture. For example, the cell culture produced by the methods described herein can be used to inoculate another larger bioreactor, which constitutes a further step of the pre-production cell culture. In a specific embodiment, the cells in the cell culture are concentrated using a centrifuge, for example, a continuous flow centrifuge, and the resulting concentrated cells (i.e., solid phase or heavy phase) are used to start another cell culture vessel, for example, a bioreactor. In certain embodiments, the bioreactor is a pre-production bioreactor. In certain other embodiments, the bioreactor is a production bioreactor. In a specific embodiment, a solid phase or heavy phase is used to start more than one, for example, 2, 3 or more other cell cultures in 2, 3 or more cell culture vessels (e.g., bioreactors). In certain specific embodiments, a heavy phase or solid phase is used to start 2 or 3 production cell cultures.

[0018] These and further aspects are further explained in the remainder of this disclosure, including the examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram of a system for perfusion bioprocessing according to one or more embodiments.

[0020] Figure 2 is a flow chart of a process for culturing mammalian cells according to one or more embodiments.

[0021] Figure 3 is a flow chart of a process for culturing mammalian cells according to one or more embodiments.

[0022] Figure 4 Flow diagram of a process for producing a mammalian cell inoculum culture having a cell density greater than 0.1% PCV according to one or more embodiments.

[0023] Figure 5 To produce according to one or more embodiments at least 4.8x10 12 Flow chart of the process for inoculating a culture of mammalian cells with 100 cells.

[0024] Figure 6 To generate a system according to one or more embodiments comprising at least 2.16x10 14 Flow chart of the process for inoculating a culture of mammalian cells with 100 cells. DETAILED DESCRIPTION

[0025] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the following literature, such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel et al., ed., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0026] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range (based on one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise) and any other specified value or intervening value within the specified range are encompassed within the described embodiments. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also encompassed by the described embodiments, subject to any explicitly excluded limits within the specified ranges. Where the specified range includes one or both limits, ranges excluding one or both of those included limits are also included in the described embodiments.

[0027] In the following description, numerous specific details are set forth to provide a more thorough understanding of the technology described herein. However, it is apparent to one skilled in the art that the technology described herein can be practiced without one or more of these specific details. In other cases, well-known features and procedures well known to those skilled in the art are not described to avoid obscuring the current description of the various embodiments.

[0028] All references cited throughout this disclosure, including patent applications and publications, are incorporated by reference in their entirety.

[0029] I. Definitions

[0030] "Comprising" means that the recited elements are required in the composition / method / kit, but other elements may be included to form a composition / method / kit, etc. within the scope of the claim.

[0031] "Consisting essentially of" is intended to limit the scope of the described compositions or methods to certain materials or steps that do not materially affect the basic and novel characteristics of the described embodiment.

[0032] "Consisting of" means excluding from the composition, method or kit any elements, steps or ingredients not specified in the claim.

[0033] The terms "culture" and "cell culture" as used interchangeably herein refer to suspension of a cell population in a cell culture medium under conditions suitable for survival and / or growth of the cell population. As used herein, these terms may refer to a combination of a cell population (e.g., an animal cell culture) and a culture medium in which the population is suspended.

[0034] The terms "medium," "cell culture medium," and "culture medium" are used interchangeably herein to refer to a solution containing nutrients that nourish cells (e.g., mammalian cells), and may also refer to the culture medium that is combined with cells. The term "seeding medium" refers to the culture medium used to produce an inoculum cell culture. The term "production medium" refers to the culture medium used to produce a production cell culture.

[0035] As used herein, the term "batch culture" refers to a method of culturing cells in which all components ultimately used to culture the cells, including the cell culture medium as well as the cells themselves, are provided at the beginning of the culture process. Batch culture is usually stopped at a certain specified time point, and the cells and / or components in the culture medium are harvested and optionally purified.

[0036] As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture for a period of time after the culture process begins. The components provided typically include nutrient supplements for cells that are depleted during the culture process. Fed-batch culture is typically stopped at a specified time point, and cells and / or components in the culture medium are harvested and optionally purified.

[0037] As used herein, the term "perfusion culture" refers to a method of culturing cells in which additional fresh medium is added to the culture (after the culture process begins) continuously or intermittently over a certain time period, and the spent medium is removed simultaneously. Fresh medium typically provides nutrient supplements for cells that have exhausted previously existing nutrient supplements during the culture process. Cell culture products, such as proteins (e.g., antibodies) that may be present in the spent culture medium are optionally purified. Perfusion also allows the removal of unwanted cell waste (e.g., excess metabolites, such as lactate) from cell cultures grown in bioreactors. Perfusion culture can be integrated into a continuous flow bioprocessing workflow to achieve constant product production and / or purification.

[0038] As used herein, the term "perfusion rate" refers to the rate at which cell culture medium is removed for perfusion culture and replaced with fresh cell culture medium (e.g., medium exchange rate). In some embodiments, the perfusion rate is measured in vessel volumes per day or VVD. In some embodiments, the perfusion rate is measured in units of volume per unit time, such as liters per minute (LPM), etc.

[0039] As used herein, the term "bioreactor" or "fermenter" or "vessel" or "culture vessel" refers to any vessel used for the growth of prokaryotic or eukaryotic cell cultures, such as animal cell cultures (e.g., mammalian cell cultures). When the term "vessel volume per day (VVD)" is used herein to describe the cell culture perfusion flow rate, for example, the "vessel" can be a bioreactor, a disposable bioreactor, a fermenter, or a culture vessel. A bioreactor can be of any size, provided that it is suitable for cell culture. Typically, the bioreactor will be at least 100 mL, and can be at least about 1, 10, 20, 80, 100, 250, 300, 350, 400, 450, 500, 1,000, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 7,500, 8,000, 10,000, 12,000, 15,000, 16,000, 18,000, 20,000, 22,000, 24,000, 26,000, 28,000, or 30,000 liters, or more, or any intermediate volume. Production volumes can range from 20 to 80 litres, 80 to 100 litres, 100 to 250 litres, 250 to 300 litres, 300 to 350 litres, 350 to 400 litres, 400 to 450 litres, 450 to 500 litres, 500 to 1,000 litres, 1,000 to 2,000, 2,000 to 2,500, 2,500 to 3,000 litres, 3,000 to 3,500, 3,500 to 4,000, 4,000 to 4,500, 4,500 to 5,000, 5,000 15,000 litres to 16,000 litres, 16,000 litres to 18,000 litres, 18,000 litres to 20,000 litres, 20,000 litres to 22,000 litres, 20,000 litres to 24,000 litres, 24,000 litres to 26,000 litres, 26,000 litres to 28,000 litres or 28,000 litres to 30,000 litres. The internal conditions of the bioreactor, which include but are not limited to dissolved oxygen (dO2), pH, as well as temperature, agitation, foaming, and pressure, are typically controlled during the culture cycle.

[0040] The terms "inoculated culture" and "inoculated cell culture" as used interchangeably herein refer to a cell culture that is primarily used to produce cell masses (i.e., to increase the number of viable cells in the culture) to reach a target cell density, which can be used to start a larger volume of cell culture (e.g., a larger inoculated cell culture, or a production cell culture). In some embodiments, the inoculated cell culture can be transferred as a whole to a production bioreactor and merged with a production cell culture medium to start a production cell culture. In some embodiments, the inoculated cell culture can be transferred as a whole to a larger inoculated cell culture bioreactor and merged with an additional inoculated cell culture medium to start an inoculated cell culture with a larger volume. In some embodiments, a first portion of the inoculated culture can be transferred to a production bioreactor and merged with a production cell culture medium to start a production cell culture, and a second portion of the inoculated culture can be merged with the inoculation culture medium to start a new inoculated cell culture. In general, one or more inoculated cell cultures grown before starting a production cell culture are referred to as "pre-production cell cultures."

[0041] As used herein, the term "inoculated bioreactor" refers to a bioreactor for containing inoculated cell cultures. Inoculated bioreactors are generally used for the production of cell blocks and generally have a smaller volume than production bioreactors. The volume of a large-scale cell culture inoculated bioreactor is generally greater than about 100 mL, usually at least about 10 liters, and can be 20, 50, 80, 100, 250, 300, 350, 400, 450, 500, 1,000, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000 liters or more, or any intermediate volume.

[0042] The terms "production culture" and "production cell culture," as used interchangeably herein, refer to a cell culture that is primarily used to produce a product and generally represents the last or final step in a cell culture process. In some embodiments, a production culture can be used to produce cells as a product. In some embodiments, a production culture can be used to produce a product within cells cultured in the production culture. In some embodiments, a production culture can be used to produce a product secreted by cells cultured in the production culture.

[0043] As used herein, the term "production bioreactor" refers to a bioreactor for accommodating production cell cultures. Production bioreactors are generally used to produce target cells or protein products. The volume of a large-scale cell culture production bioreactor is generally greater than about 100 mL, usually at least about 10 liters, and can be 20, 80, 100, 250, 300, 350, 400, 450, 500, 1,000, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 7,500, 8,000, 10,000, 12,000, 15,000, 16,000, 18,000, 20,000, 22,000, 24,000, 26,000, 28,000 or 30,000 liters or more, or any intermediate volume. The volume of the production bioreactor may be 20 liters to 80 liters, 80 liters to 100 liters, 100 liters to 250 liters, 250 liters to 300 liters, 300 liters to 350 liters, 350 liters to 400 liters, 400 liters to 450 liters, 450 liters to 500 liters, 500 liters to 1,000 liters, 1,000 liters to 2,000, 2,000 liters to 2,500 liters, 2,500 liters to 3,000 liters, 3,000 liters to 3,500 liters, 3,500 liters to 4,000 liters, 4,000 liters to 4,500 liters, 4,500 liters to 5,000 liters, 5 000 litres to 7,500 litres, 7,000 litres to 8,000 litres, 8,000 litres to 10,000 litres, 10,000 litres to 12,000 litres, 12,000 litres to 15,000 litres, 15,000 litres to 16,000, 16,000 litres to 18,000 litres, 18,000 litres to 20,000 litres, 20,000 litres to 22,000 litres, 20,000 litres to 24,000 litres, 24,000 litres to 26,000 litres, 26,000 litres to 28,000 litres or 28,000 litres to 30,000 litres.

[0044] Suitable bioreactors (e.g., inoculation bioreactors or production bioreactors) may include (i.e., be composed of) any material suitable for maintaining cells suspended in a culture medium under the desired culture conditions and facilitating cell growth, maintaining cell viability, and / or product production, including glass, plastic, or metal. Generally, the material should not interfere with the expression or stability of products (e.g., protein products) produced and / or secreted from the cultured cells. Generally, one of ordinary skill in the art will be able to readily select a suitable bioreactor and its operating conditions for use in implementing the methods described herein.

[0045] As used herein, the term "cell density" refers to the number of cells present in a given volume of culture medium. As used herein, the term "viable cell density" refers to the number of live (surviving) cells present in a given volume of culture medium. Various sensors or probes can be incorporated into a cell culture system to directly measure the conditions in a cell culture vessel. For example, optical probes can be used to monitor cell growth, but they may not be able to distinguish between live cells and dead cells. Radio frequency impedance (RFI) probes can be used to monitor cell growth, and live cells can also be detected by capacitance measurement to determine live cell density. Cell samples can be obtained from the culture once or multiple times during cell culture, and the percentage of viability is determined by using spectroscopy, for example, Raman spectroscopy, Alamar Blue dye staining, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Trypan Blue staining, electronic particle counting, EdU assay, XTT assay, WST-1 assay, luminescent ATP assay, etc. The number of viable cells per mL can then be determined.

[0046] As used herein, the term "hematocrit" or "PCV" refers to the ratio of the volume of a sample of cell culture fluid occupied by red blood cells to the volume of a sample of cell culture fluid after centrifugation of the cell culture fluid sample. As with cell density, optical and RFI probes can be used to determine PCV.

[0047] The term "cell viability" as used herein refers to the ability of cells in culture to survive under given culture conditions or experimental changes. As used herein, the term also refers to the living cell portion relative to the total number of live and dead cells in the culture at that time within a certain time. Radio frequency impedance (RFI) probes can be used to monitor cell growth, and live cells can also be detected by capacitance measurement to determine the live cell density. Cell samples can be obtained from the culture once or multiple times during cell culture, and the percentage of viability is determined by using alamar blue dye staining, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, electronic particle counting, EdU assay, XTT assay, WST-1 assay, luminescent ATP assay, etc.

[0048] As used herein, the terms "polypeptide" and "polypeptide product" are synonymous with the terms "protein" and "protein product", respectively, and as generally understood in the art, refer to at least one amino acid chain connected via continuous peptide bonds. In certain embodiments, "target protein" or "target polypeptide" etc. are proteins encoded by exogenous nucleic acid molecules that have been transformed into host cells. In certain embodiments, wherein an exogenous DNA molecule (host cell has been transformed) encodes a "target protein", the nucleic acid sequence of the exogenous DNA determines the amino acid sequence. In certain embodiments, "target protein" is a protein encoded by a nucleic acid molecule that is endogenous to the host cell. In certain embodiments, the expression of such endogenous target proteins is changed by transfecting host cells with exogenous nucleic acid molecules, which may, for example, contain one or more regulatory sequences and / or encode proteins that enhance the expression of target proteins.

[0049] As used herein, the term "titer" refers to the total amount of a target product (e.g., a polypeptide) produced by a cell culture (e.g., an animal cell culture) divided by a given volume of culture medium. Thus, "titer" refers to the concentration of a target polypeptide. Titers are usually expressed in units of grams or milligrams of polypeptide per milliliter or liter of culture medium.

[0050] An "isolated" product (e.g., an antibody) is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the product, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the product is (1) purified to greater than 95% by weight of the product as determined by the Lowry method, and preferably greater than 99% by weight; (2) purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer; or (3) purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver dye. An isolated product includes an in situ product within a recombinant cell, since at least one component of the product's natural environment will not be present. Typically, however, an isolated product will be prepared by at least one purification step.

[0051] The terms "σ value" and "σ factor" as used interchangeably herein with respect to a centrifuge refer to an operating constant that represents the geometry and speed of a centrifuge. The σ factor can be used to compare two or more centrifuges having different sizes, geometries, and / or operating speeds.

[0052] The term "solid phase" in the context of the cell culture methods described herein refers to the stream from a continuous centrifuge comprising the bulk of cells separated from the culture medium (hereinafter referred to as the "liquid phase").

[0053] The term "liquid phase" in the context of the cell culture methods described herein refers to the stream from a continuous centrifuge which comprises primarily liquid, such as cell culture medium, and has few or no cells.

[0054] The terms "solid to liquid phase ratio" and "split ratio" as used interchangeably herein refer to the ratio of the volume of the solid phase to the volume of the liquid phase combined to maintain, hold and / or start a new cell culture. For example, for a 50:50 solid to liquid split ratio, the centrifuge can be adjusted so that 50% of the centrifuge inlet flow flows volumetrically to the solids outlet and 50% of the centrifuge inlet flow flows volumetrically through the liquid outlet. In the same manner, for a 75:25 split ratio, 75% of the volume flow of the centrifuge inlet flow flows to the solids outlet and 25% of the volume flow of the centrifuge inlet flow flows through the liquid outlet, and for a 90:10 split ratio, 90% of the volume flow of the centrifuge inlet flow flows to the solids outlet and 10% of the volume flow of the centrifuge inlet flow flows through the liquid outlet.

[0055] As used herein, the term "residence time" means the time that cells are outside of a cell culture vessel, such as the time during perfusion centrifugation. The residence time includes the time that cells are within a centrifuge, as well as the time that cells are transported between the cell culture vessel and the centrifuge.

[0056] II. Specific implementation methods

[0057] Aspects of the present disclosure include methods involving perfusion of a cell culture using a continuous flow centrifuge to produce a solid phase and a liquid phase, and returning at least a portion of the solid phase and a volume of cell culture medium to a culture vessel to maintain, hold and / or initiate a new cell culture. In some embodiments, the methods optionally include returning a portion of the liquid phase of the original cell culture to the new cell culture.

[0058] Mammalian cell cultures typically consist of a growth phase, during which cells are grown to a target density (e.g., target cell concentration or hematocrit), and a production phase, during which the cells grow less but produce larger amounts of product (e.g., recombinant protein) than was produced by the cell culture during the growth phase.

[0059] During the growth phase of the cell culture, the cells are first mixed with a culture medium (i.e., an inoculation medium) to form a cell culture. Typically, the cell culture medium provides but is not limited to the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for the minimum growth and / or survival of the cells. The culture medium may also contain components that enhance growth and / or survival beyond the minimum rate, including hormones and growth factors. The culture medium is preferably formulated into the pH and salt concentration that are most suitable for cell survival and proliferation. In at least one embodiment, the culture medium is a defined culture medium. The defined culture medium is a culture medium in which all components have a known chemical structure. In other embodiments, the culture medium may contain amino acids derived from any source or method known in the art, including but not limited to amino acids derived from a single amino acid addition or from peptone or protein hydrolyzate addition (including animal or plant sources). In other embodiments, the culture medium used during the cell growth phase may contain a concentrated culture medium, that is, a culture medium containing a higher concentration than the concentration of nutrients that are usually required and usually provided to the growth culture. Those skilled in the art will recognize what cell culture media, inoculation media, etc. are suitable for cell culture of a particular cell type, such as animal cells (e.g., CHO cells), as well as the amount of glucose and other nutrients (e.g., glutamine, iron, trace elements) or reagents intended to control other culture variables (e.g., foaming amount, osmotic pressure) that the culture medium should include (see, e.g., Mather, JP, et al. (1999) "Culture media, animal cells, large-scale production", Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bio separation, Vol. 2: 777-85; U.S. Patent Application Publication No. US2006 / 0121568; each of which is incorporated herein by reference in its entirety). The embodiments described herein contemplate variations of such known culture media, including, for example, nutrient-enriched variations of such culture media.

[0060] Those skilled in the art will also recognize at what temperature and / or concentration a particular cell line should be cultured. For example, most mammalian cells, such as CHO cells, grow well in the range of about 35°C to 39°C (preferably at 37°C), while insect cells are typically cultured at 27°C.

[0061] The methods according to embodiments of the present disclosure can utilize any suitable recombinant host cell, such as a prokaryotic or eukaryotic host cell, i.e., a cell transfected with an expression construct containing a nucleic acid encoding a polypeptide of interest (e.g., an antibody).Many mammalian cell lines are suitable host cells for recombinant expression of a polypeptide of interest. Mammalian host cell lines include, for example but not limited to, COS, PER.C6, TM4, VERO076, MDCK, BRL-3A, W138, Hep G2, MMT, MRC 5, FS4, CHO, 293T, A431, 3T3, CV-1, C3H10T1 / 2, Colo205, 293, HeLa, L cells, BHK, HL-60, FRhL-2, U937, HaK, Jurkat cells, Rat2, BaF3, 32D, FDCP-1, PC12, Mix, murine myeloma (e.g., SP2 / 0 and NSO) and C2C12 cells, as well as transformed primate cell lines, hybridomas, normal diploid cells, and cell lines derived from in vitro cultures of primary tissues and primary explants. Any eukaryotic cell capable of expressing the product of interest can be used in conjunction with the embodiments described herein. Many cell lines are available from commercial sources, such as the American Type Culture Collection (ATCC). In various embodiments, the cell culture employs hybridoma cells. In various embodiments, the cell culture employs CHO cells.

[0062] In specific embodiments, the cell culture methods provided herein can be used to culture cells that produce any type of therapeutic polypeptide. In certain embodiments, the therapeutic polypeptide is a fusion protein, such as a fusion protein including an antibody Fc portion or human serum albumin. In other specific embodiments, the therapeutic polypeptide is an antibody or antibody fragment, such as a monoclonal antibody, a monospecific antibody, a bispecific antibody (with or without a common light chain), a bispecific T cell engager (BiTE), a bispecific (mab) 2 antibody; a bispecific F (mab) 2 antibody; a single domain bispecific diabody (scBsDb), a single chain bispecific tandem variable domain (scBsTaFv), a trispecific NK cell engager therapy (TriNKET), a dual affinity retargeting protein (DART), a bispecific diabody, a tandem diabody (TandAb), a half antibody (e.g., an antibody comprising an Fc portion but only one CH-VH:CL-VL pair), a trifab contorsbody (as described in U.S. Patent Application Publication No. WO 2019 / 086395); a quadroma, a scFv, a docked and locked trivalent fab (DNL-(Fab) 3, a single domain antibody, a bispecific single domain antibody, etc.

[0063] In a specific embodiment, the therapeutic polypeptide is atezolizumab, abagovomab, abciximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, alacizumab, alemtuzumab, alirocumab, atumomab pentetate, or pentetate), amatuximab, amivantamab, anatumomab, andecaliximab, anetumab, anifrolumab, ansuvimab, anrukinzumab, apolizumab, aprutumab, ascrinvacumab, aselizumab, atidortoxumab, atinumab, atoltivimab, torolimumab, avelumab, atuximab zintuxizumab), bamlanivimab, bapineuzumab, basiliximab, bavituximab, bebtelovimab, bectumomab, begelomab, belantamab, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bermekimab, bersanlimab, bertilimumab, besilesomab, bevacizumab,bezlotoxumab, biciromab, bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab , briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camidanlumab, camrelizumab, canakinumab, cantuzumab, cantuzumab, caplacizumab, casirivimab, capromab, carlumab, carotuximab, catumaxomab, cedelizumab, cemiplimab, cergutuzumab, certolizumab, cetrelimab, cetuximab, cibisatamab, cilgavimab, cirmtuzumab, citatuzumab uzumab), cixutumumab, clazakizumab, clenoliximab, clivatuzumab, codrituzumab, cofetuzumab, coltuximab, conatumumab, concizumab, cosfroviximab, crenezumab, crizanlizumab,Crotedumab, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab, daratumumab, dectrekumab, demcizumab, denintuzumab, denosumab, depatuxizumab, derlotuximab, denosumab detumomab, dezamizumab, dinutuximab, denosumab, diridavumab, domagrozumab, dorlimomab, dostarlimab, drozitumab, duligotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab. Ecromeximab. Eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab umab), emapalumab, emibetuzumab, emicizumab, enapotamab, enavatuzumab, enfortumab, enlimomab, enoblituzumab, enokizumab, enoticumab. Ensituximab, epcoritamab, epitumomab, epratuzumab,Eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etesevimab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farisimab cimab), farletuzumab, fasinumab, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frovocimab, frunevetmab, fullanumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, golimumab avilimomab), gedivumab, gemtuzumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab, glofitamab, golimumab, gomiliximab, gosuranemab, guselkumab, ianalumab,ibalizumab, ibritumomab, icrucumab, ifabotuzumab, igovomab, iladatuzumab, imalumab, imaprelimab, imciromab, imdevimab, imgatuzumab, inclacumab, indatuximab, indutumab satumab), inebilizumab, infliximab, intetumumab, inolimomab, inotuzumab, ipilimumab, iomab-B, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab iximab), labetuzumab, lacnotuzumab, laditratuzumab, lampalizumab, lanadelumab, landogrozumab, laprituximab, larcaviximab, lebrikizumab, lecanemab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifastuzumab, ligelizumab, loncastuximab, losatuxizumab, lilotomab,Lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab, lucatumumab, lulizumab, lumiliximab, lumretuzumab, lupartumab, luspatercept, lutikizumab, maftivimab, mapatumumab mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab, mitumomab, zatuximab dotuximab), mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab, muromonab-CD3, nacolomab, namilumab, naptumomab, naratuximab, narnatumab, natalizumab alizumab), navicixizumab, navivumab, naxitamab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab,Obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odesivimab, odulimomab, ofatumumab, olaratumab, oleclumab, olendalizumab, olokizumab, omalizumab, omburtamab ), onartuzumab, ontuxizumab; onvatilimab, opicinumab, oportuzumab, oregovomab, orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pajama pagibaximab, palivizumab, pamrevlumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab, pintumomab, placulumab, prezalumab, plozalizumab, pogalizumab, polatuzumab, ponezumab, porgaviximab,prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab ), raxibacumab, ravagalimab, ravulizumab, refanezumab, regavirumab, regdanvimab, relatlimab, remtolumab, reslizumab, retifanlimab, rilotumumab, rinucumab, risankizumab, rituximab Rituximab, rivabazumab, robatumumab, rmab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalvalpituzumab, rovelizumab, rozanolixizumab, ruplizumab, sacituzumab uzumab), samalizumab, samrotamab, sarilumab, satralizumab, satumomab, secukinumab, selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, sifalimumab,siltuximab, simtuzumab, siplizumab, sirtratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, sotrovimab, spartalizumab, spesolimab, stamolumab Stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab, tadocizumab, tafasitamab, talacotuzumab, talizumab, talquetamab, tamtuv etmab), tanezumab, taplitumomab, tarextumab, tavolimab, teclistamab, tefibazumab, telimomab, telisotuzumab, terituzumab, tenatumomab, teneliximab, teplizumab, tepoditamab, tetulimab teprotumumab), tesidolumab, tetulomab, tezepelumab, TGN1412, tibulizumab, tildrakizumab, tigatuzumab, timigutuzumab, timolumab, tiragolumab, tiragotumab, tislelizumab, tisotumab,tixagevimab, TNX-650, tocilizumab, tomozuotuximab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, trevogruma b), tucotuzumab, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab, vanalimab, vandortuzumab, vantictumab, vanucizumab ), vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, vilobelimab, visilizumab, vobarilizumab, volociximab, vonlerol izumab, vopratelimab, vorsetuzumab, votumumab, vunakizumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenocutuzumab, ziralimumab, zolbetuximab, or zolimomab.

[0064] The construction of recombinant cells (e.g., hybridoma cells and CHO cells) that produce products is well known in the art. In some embodiments, the product may be a recombinant protein, such as a recombinant antibody, including, for example, a multispecific antibody. In some embodiments, the product may be a secreted product, which is secreted by the cell into a cell culture medium. In some embodiments, the product may be an intracellular product, which, once produced by the cell, remains contained within the cell, or remains contained within the cell wall, as in the case of certain production microorganisms (such as, for example, certain bacteria). In some embodiments, the product may be an organelle, or even a cell itself, as in the case of a cell therapy product.

[0065] The method according to an embodiment involves starting a system having a range of about 250,000 (x10 6 ) cells / mL up to about 25x10 6 5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, or 24.5 million cells / mL). Methods according to various embodiments involve initiating a cell culture having a starting cell density in the range of about 20 x 10 6 cells / mL up to about 25x10 6 The method according to some embodiments involves starting a cell culture with a starting cell density of about 22.5 x 10 6 In some embodiments, the methods involve initiating an inoculated cell culture with a starting cell density of about 0.5 to about 2 million cells / mL. In some embodiments, the methods involve initiating an inoculated cell culture with a starting cell density in the range of about 2 to about 5 million cells / mL. In some embodiments, the methods involve initiating an inoculated cell culture with a starting cell density in the range of about 5 to about 7.5 million cells / mL. In some embodiments, the methods involve initiating an inoculated cell culture with a starting cell density in the range of about 7.5 to about 10 million cells / mL.

[0066] In certain embodiments, the cell culture methods provided herein maintain a viability percentage of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% throughout the progress of the cell culture process.

[0067] In some embodiments, the methods involve initiating an inoculum cell culture having a starting cell density ranging from about 0.1% hematocrit (PCV) up to about 4% PCV, such as about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, or 3.9% PCV. In some embodiments, the inoculum cell culture has an initial % PCV ranging from 0.1 to 0.5% PCV. In some embodiments, the inoculum cell culture has an initial % PCV ranging from 0.5 to 1%. In some embodiments, the inoculated cell culture has an initial %PCV in the range of 1 to 1.5%. In some embodiments, the inoculated cell culture has an initial %PCV in the range of 1.5 to 2%. In some embodiments, the inoculated cell culture has an initial %PCV in the range of 2 to 2.5%. In some embodiments, the inoculated cell culture has an initial %PCV in the range of 2.5 to 3%. In some embodiments, the inoculated cell culture has an initial %PCV in the range of 3 to 3.5%. In some embodiments, the inoculated cell culture has an initial %PCV in the range of 3.5 to 4%. In some embodiments, the methods involve starting a culture with a PCV in the range of about 0.1% up to about 10%. PCV (such as about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6 or 9.8% PCV) of new cell cultures.

[0068] Methods according to embodiments described herein involve growing the inoculated cell culture to a first target cell density as described above prior to subjecting at least a portion of the inoculated cell culture to a perfusion procedure. In some embodiments, the first target cell density is in the range of about 50 x 10 6 cells / mL up to about 150x10 6 cells / mL, such as about 60, 70, 80, 90, 100, 110, 120, 130 or 140 x 10 6 In some embodiments, the methods involve growing the inoculum cell culture to a target cell density in the range of about 50 to about 100 million cells / mL. In some embodiments, the methods involve growing the inoculum cell culture to a target cell density in the range of about 60 to about 100 million cells / mL. In some embodiments, the methods involve growing the inoculum cell culture to a target cell density in the range of about 100 x 10 6 cells / mL to about 150x10 6 The target cell density is cells / mL.

[0069] In some embodiments, the methods involve growing a seeded cell culture, e.g., building a cell mass or reaching a target cell density ranging from about 10% hematocrit (PCV) up to about 30% PCV, such as about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29% PCV. In some embodiments, the methods involve growing a seeded cell culture to a target cell density ranging from about 10 to about 15% PCV. In some embodiments, the methods involve growing a seeded cell culture to a target cell density ranging from about 15 to about 20% PCV. In some embodiments, the methods involve growing a seeded cell culture to a target cell density ranging from about 20 to about 25% PCV. In some embodiments, the methods involve growing a seeded cell culture to a target cell density ranging from about 25 to about 30% PCV.

[0070] In certain embodiments, the cell culture (e.g., inoculated cell culture) produced by the methods described herein is part of a seed culture in a pre-production cell culture. For example, the cell culture produced by the methods described herein can be used to inoculate another larger bioreactor, which constitutes a further step of a pre-production cell culture. In a specific embodiment, the cells in the cell culture are concentrated using a centrifuge, e.g., a continuous flow centrifuge, and the resulting concentrated cells (i.e., solid phase or heavy phase) are used to start another cell culture vessel, e.g., a bioreactor. In certain embodiments, the bioreactor is a pre-production bioreactor. In certain other embodiments, the bioreactor is a production bioreactor.

[0071] The cell culture methods described herein can further be used to start multiple downstream cell cultures so that cell blocks can be constructed in parallel, or so that product production can be performed in parallel in multiple vessels. In specific embodiments, for example, a solid phase or a heavy phase is used to start more than one, for example, 2, 3 or more other cell cultures in 2, 3 or more cell culture vessels (e.g., bioreactors). Such other cell cultures can be, for example, additional (downstream) pre-production cell cultures, or can be production cell cultures. In certain specific embodiments, a heavy phase or a solid phase is used to start 2 or 3 or more pre-production cell cultures. In certain specific embodiments, a heavy phase or a solid phase is used to start 2 or 3 or more production cell cultures. Such a start-up can include collecting a heavy phase or a solid phase after centrifugation is complete, dividing the heavy phase or the solid phase into the number of pre-production or production cultures to be started, and starting these cultures. Such a start-up can also include collecting a heavy phase or a solid phase continuously or discontinuously during centrifugation, and during centrifugation, dividing the heavy phase or the solid phase into the number of pre-production or production cultures to be started, and starting these cultures.

[0072] Methods according to embodiments described herein involve initiating a pulse having a range of about 0.25x10 6 cells / mL up to about 25x10 6 cells / mL (such as about 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, or 24.5 x 10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 0.5 x 10 6 cells / mL to about 2x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 2 x 10 6 cells / mL to about 5x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 5 x 10 6 cells / mL to about 7.5x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 7.5 x 106 cells / mL to about 10x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 10 x 10 6 cells / mL to about 12.5x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 12.5 x 10 6 cells / mL to about 15x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 15 x 10 6 cells / mL to about 17.5x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 17.5 x 10 6 cells / mL to about 20x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 20 x 10 6 cells / mL to about 22.5x10 6 In some embodiments, the methods involve initiating a production cell culture having a starting cell density in the range of about 22.5 x 10 6 cells / mL to about 25x10 6 The production cell culture was started at a starting cell density of 10 cells / mL.

[0073] Methods according to embodiments described herein involve growing the production cell culture to a first target cell density as described above prior to subjecting at least a portion of the production cell culture to a perfusion procedure. In some embodiments, the first target cell density is in the range of about 50 x 10 6 cells / mL up to about 150x10 6 cells / mL, such as about 60, 70, 80, 90, 100, 110, 120, 130 or 140 x 10 6 In some embodiments, the methods involve growing the production cell culture to a range of about 50 x 10 6 cells / mL to about 100x10 6 In some embodiments, the methods involve growing the production cell culture to a target cell density in the range of about 60 x 10 6 cells / mL to about 100x10 6In some embodiments, the methods involve growing the production cell culture to a target cell density in the range of about 100 x 10 6 cells / mL to about 150x10 6 The target cell density is cells / mL.

[0074] In some embodiments, the methods involve constructing a cell mass or reaching a first target cell density ranging from about 10% hematocrit (PCV) to about 30% PCV, such as about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29% PCV. In some embodiments, the methods involve growing a production cell culture to a target cell density ranging from about 10 to about 15% PCV. In some embodiments, the methods involve growing a production cell culture to a target cell density ranging from about 15 to about 20% PCV. In some embodiments, the methods involve growing a production cell culture to a target cell density ranging from about 20 to about 25% PCV. In some embodiments, the methods involve growing a production cell culture to a target cell density ranging from about 25 to about 30% PCV.

[0075] Aspects of the technology described herein relate to using a continuous flow centrifuge to perform a perfusion procedure on at least a portion of a cell culture (e.g., an inoculated cell culture or a production cell culture). Continuous flow centrifuges are known in the art and are described, for example, in PCT Publication No. WO2008 / 138345 and PCT Publication No. WO2020 / 229184, which are incorporated herein by reference in their entirety. A continuous flow centrifuge that can be used according to the embodiments described herein, but is not limited to a disc stack centrifuge, a tubular centrifuge, etc. In certain embodiments, a continuous flow centrifuge is constructed for sterilization, which means that one or more components (e.g., a drum) of the centrifuge can be subjected to a sterilization procedure to make it suitable for use under biomanufacturing conditions (e.g., according to cGMP regulations). In certain embodiments, a continuous flow centrifuge includes a disposable component, such as a disposable drum component. In such embodiments, the disposable component is configured to be easily separated from the remaining components of the device and can be easily discarded and / or replaced. New disposable components can then be attached to the centrifuge. The new disposable components may be pre-sterilized, or may be configured for sterilization, either separately from the remaining components of the centrifuge, or together with the remaining components of the centrifuge.

[0076] The continuous flow centrifuge according to the embodiments described herein is generally operated and the flow rate is suitable for separating cells in the liquid phase from the solid phase in the cell culture medium. In addition to the effect of the flow rate on the separation, it also has an effect on the residence time of the cells outside the production bioreactor. Increasing the flow rate can minimize the residence time while still achieving the desired cell separation in an optimal manner.

[0077] The continuous flow centrifuge according to the embodiments described herein generally also includes suitable inlet and outlet components to allow them to be operably connected to one or more bioreactors, one or more cell culture medium reservoirs, etc. Further, the continuous flow centrifuge according to the embodiments described herein can generally be configured to interoperate with standard cell culture manufacturing equipment (such as pumps, controllers, flow meters, etc.). The continuous flow centrifuge described herein is generally operated at a flow rate suitable for cell separation, wherein separation is achieved between the solid phase (cells) and the liquid phase (cell culture medium). In certain embodiments, suitable inlet and outlet components are designed to minimize the volume of the system and thus minimize the residence time of cells outside the cell culture vessel bioreactor (e.g., production bioreactor). In certain embodiments, the volume of the centrifugal system (including inlet and outlet components) can be achieved by minimizing the length and / or diameter of the pipeline, cannula or cannula flow test kit, while still being able to achieve the expected flow rate of the system.

[0078] Continuous flow centrifuges according to embodiments described herein may also typically include temperature controlled elements to provide suitable conditions for materials leaving the production bioreactor and entering the centrifugal system, during centrifugation within the system, the resulting solid phase leaves the system and is subsequently returned to the production bioreactor, and the resulting liquid phase leaves the system and is subsequently further processed downstream. The temperature control element may be passive or actively controlled. In one embodiment, the temperature control is passive. Here, the pipe, sleeve or sleeve flow kit may be insulated or non-insulated (as required), allowing passive temperature control to ambient temperature, or to the reduced temperature present at the operating point. For example, cold water (about 4°C to 10°C) can be used to cool the water, which in turn cools the centrifuge seals and minimizes the temperature increase caused by the centrifuge rotation. In a specific embodiment, the ambient temperature is reduced to minimize the temperature increase caused by friction at high drum speeds for cell separation. The pipe, sleeve or sleeve flow kit may be insulated or non-insulated, allowing passive temperature control via the ambient temperature at the operating point.

[0079] In other embodiments, temperature control is active. For example, a pipe, sleeve or sleeve flow test kit may be subjected to a heat exchanger, thereby allowing active temperature control to a specific temperature set point. The centrifuge may have a temperature control of the centrifuge drum to minimize the temperature increase caused by friction under the high drum speed required for cell separation. The centrifuge may have a temperature control of the mechanical seal to minimize the temperature increase caused by friction under the high drum speed required for cell separation. Further, the temperature control of the centrifuge drum and the mechanical seal may be set to ambient temperature or a reduced temperature to minimize the temperature rise during processing.

[0080] In certain embodiments of any method disclosed herein, the residence time of cells and cell culture medium is minimized so that the viability of cells in cell culture is maintained, or substantially not reduced. In certain embodiments, the residence time is less than 3 minutes, 2 minutes or 1 minute. In certain embodiments, the residence time is reduced by increasing culture medium and cells from culture vessels to centrifuges and returning to culture vessels and flow rates. In other embodiments, by using a larger conduit (e.g., sleeve) between culture vessels and centrifuges, a larger centrifuge etc. is used to reduce the residence time. In certain embodiments, a specific residence time causes a cell viability loss of no more than 3%, 2%, 1% or 0.5% during the culture period.

[0081] In certain embodiments of any of the methods disclosed herein, the temperature of the cell culture medium is adjusted or maintained during the residence time. In specific embodiments, the temperature is maintained during the residence time such that any temperature instant during the residence time is 4°C or lower, 3°C or lower, 2°C or lower, or 1°C or lower compared to the temperature of the medium in the cell culture vessel. In various embodiments, the temperature of the cell culture medium is maintained during the residence time using a water bath or heat exchanger, for example, during transport of the cell culture medium between a culture vessel and a centrifuge, between a centrifuge and a cell culture vessel, or between the two. For example, during Figure 1 In the embodiment, the temperature of the cell culture medium can be maintained such that any temperature is 4°C or less, 3°C or less, 2°C or less, or 1°C or less during transport from the cell culture vessel 120 through the sterile connection 103, the cell separation device 130, the solid phase outlet 106, and back to the cell culture vessel 120 through the sterile connection 105. In a specific embodiment, the cell culture medium is maintained at a temperature between about 30°C and about 39°C (e.g., between 31°C and 38°C, between 32°C and 38°C, between 33°C and 38°C, between 34°C and 38°C, between 35°C and 38°C, or between 36°C and 38°C) during the residence time. In certain embodiments, the centrifuge, such as a continuous centrifuge, such as a disposable centrifuge, includes a water cooling system that reduces heat addition to the cell culture medium within the centrifuge, such as heat addition at the seal.

[0082] In some embodiments, a continuous flow centrifuge may be characterized by a σ factor or σ value, which refers to an operating constant that represents the geometry and speed of the centrifuge. The σ factor can be used to compare two or more centrifuges having different sizes, geometries, and / or operating speeds, so that two different centrifuges having different operating parameters can be compared to each other to achieve similar operating performance. A continuous flow centrifuge according to embodiments of these techniques typically has a σ factor in the range of about 1,000 m / s. 2 to about 200,000m 2 (e.g., about 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000, 56,000, 57,000, 58,000, 59,000, 61,000, 62,000, 63,000 0, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 120,000, 140,000, 160,000, or 180,000m 2 ) . Continuous flow centrifuges according to embodiments of the technology described herein are typically configured to achieve operating speeds ranging from about 3,000 to about 10,000 RPM, such as about 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, or 9,500 RPM. Some disposable continuous flow centrifuges can operate at speeds ranging from about 3,000 RPM to about 6,000 RPM.

[0083] In use, a continuous flow centrifuge according to embodiments of the technology described herein can be configured to receive at least a portion of a cell culture (e.g., an inoculum cell culture or a production cell culture) and separate the received cell culture portion into a solid phase and a liquid phase, wherein the solid phase contains all or most of the cells in the received portion and the liquid phase contains primarily cell culture medium. In certain embodiments, the solid phase can have a final cell density ranging from about 1% PCV to about 10%, about 20%, about 30%, about 40%, or about 50% PCV.

[0084] After separation into a solid phase and a liquid phase, the centrifuge and / or its auxiliary components are configured to return the solid phase or a portion thereof and optionally a portion of the liquid phase to a new bioreactor (or original bioreactor) to maintain, hold and / or start a new cell culture. In addition to the solid phase and optionally a portion of the liquid phase, an appropriate amount of fresh cell culture medium may be combined with the solid phase (and optionally a portion of the liquid phase) to achieve the desired initial cell density of the new cell culture. In some embodiments, the initial cell density is in the range of about 0.25 x 10 6 cells / mL up to about 25x10 6 cells / mL, such as about 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, or 24.5 x 10 6 In some embodiments, the methods involve initiating a method having a range of about 0.5 x 10 6 cells / mL to about 2x10 6 In some embodiments, the methods involve initiating a new cell culture having an initial cell density in the range of about 2x10 6 cells / mL to about 5x10 6 In some embodiments, the methods involve initiating a new cell culture having an initial cell density in the range of about 5x10 6 cells / mL to about 7.5x10 6 In some embodiments, the methods involve initiating a new cell culture having an initial cell density in the range of about 7.5 x 10 6 cells / mL to about 10x10 6 In some embodiments, the methods involve initiating a new cell culture having an initial cell density in the range of about 10 x 10 6 cells / mL to about 12.5x10 6 In some embodiments, the methods involve initiating a production cell culture having an initial cell density in the range of about 12.5 x 10 6 cells / mL to about 15x10 6 In some embodiments, the methods involve initiating a production cell culture having an initial cell density ranging from about 15 to about 17.5 x 106 In some embodiments, the methods involve initiating a production cell culture having an initial cell density in the range of about 17.5 x 10 6 cells / mL to about 20x10 6 In some embodiments, the methods involve initiating a production cell culture having an initial cell density in the range of about 20 x 10 6 cells / mL to about 22.5x10 6 In some embodiments, the methods involve initiating a production cell culture having an initial cell density in the range of about 22.5 x 10 6 cells / mL to about 25x10 6 The production cell culture was grown at an initial cell density of 10 cells / mL.

[0085] In some embodiments, the methods involve initiating a method having a range of about 0.1% hematocrit (PCV) up to about 10% PCV (such as about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3. In some embodiments, the new cell culture has an initial cell density of 0.1 to 1%. In some embodiments, the new cell culture has an initial % PCV in the range of 1.1 to 2%. In some embodiments, the new cell culture has an initial % PCV in the range of 2.1 to 3%. In some embodiments, the new cell culture has an initial % PCV in the range of 3.1 to 4%. In some embodiments, the new cell culture has an initial % PCV in the range of 4.1 to 5%. In some embodiments, the new cell culture has an initial % PCV in the range of 5.1 to 6%. In some embodiments, the new cell culture has an initial % PCV in the range of 6.1 to 7%. In some embodiments, the new cell culture has an initial % PCV in the range of 7.1 to 8%. In some embodiments, the new cell culture has an initial % PCV in the range of 8.1 to 9%. In some embodiments, the new cell culture has an initial % PCV in the range of 9.1 to 10%.

[0086] Methods according to embodiments of the technology described herein can involve growing a new cell culture to a target cell density before subjecting at least a portion of the cell culture to a perfusion procedure. In some embodiments, the target cell density is in the range of about 50 million (x10 6 ) cells / mL up to about 150 million cells / mL, such as about 60, 70, 80, 90, 100, 110, 120, 130, or 140 million cells / mL. In some embodiments, the methods involve growing the new cell culture to a target cell density ranging from about 50 to about 100 million cells / mL. In some embodiments, the methods involve growing the new cell culture to a target cell density ranging from about 60 to about 100 million cells / mL. In some embodiments, the methods involve growing the new cell culture to a target cell density ranging from about 100 to about 150 million cells / mL.

[0087] In some embodiments, the methods involve growing new cell cultures, building cell clumps, or reaching a target cell density ranging from about 10% hematocrit (PCV) up to about 30% PCV, such as about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29% PCV. In some embodiments, the methods involve growing new cell cultures to a target cell density ranging from about 10 to about 15% PCV. In some embodiments, the methods involve growing new cell cultures to a target cell density ranging from about 15 to about 20% PCV. In some embodiments, the methods involve growing new cell cultures to a target cell density ranging from about 20 to about 25% PCV. In some embodiments, the methods involve growing new cell cultures to a target cell density ranging from about 25 to about 30% PCV.

[0088] The various methods described herein can be used to achieve the perfusion rate required for cell culture. The perfusion rate can be any rate suitable for cell culture. For example, the perfusion rate can range from about 0.5 vessel volume / day (VVD) to about 10 VVD, such as about 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5 or about 9.75 VVD. In some embodiments, the perfusion rate ranges from about 0.5 to about 6 VVD, such as 0.5 to about 4 VVD, or about 0.7 VVD to about 6 VVD. In some embodiments, the perfusion rate ranges from about 4 to about 6 VVD. In some embodiments, the perfusion rate ranges from about 6 to about 8 VVD. In some embodiments, the perfusion rate ranges from about 8 to about 10 VVD.

[0089] Aspects of the present disclosure include variations of perfusion procedures that can be used to achieve any of a variety of perfusion rates for cell culture. For example, the perfusion rate can remain constant over a period of time, or can change (i.e., increase or decrease) during a perfusion cycle, or any combination thereof. Further, the increase or decrease in perfusion rate can be applied in any manner known in the art, including but not limited to a steady change over time, e.g., a steady increase during a perfusion cycle; or a series of changes over time, e.g., a series of steady changes, a series of stepwise changes (e.g., the perfusion rate can be gradually increased or decreased), or any combination thereof. As noted above, perfusion can be applied in a continuous manner or in an intermittent manner. The timing of starting and stopping a perfusion cycle and any changes in perfusion can be predetermined, e.g., predetermined at set times or time intervals, or predetermined based on monitoring of certain parameters or criteria for cell culture.

[0090] The perfusion procedure according to the embodiments of the present disclosure can be performed over a time period that can range from several hours to several days. For example, in some embodiments, the time period for performing the perfusion procedure or the cell culture method including such a perfusion procedure is between 0.5 hours and about 5 hours, such as about 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5 hours or longer. In some embodiments, the time period for performing the perfusion procedure or the cell culture method including such a perfusion procedure is between about 5 hours and about 24 hours, such as about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours. In some embodiments, the time period for performing the perfusion procedure or the cell culture method including such a perfusion procedure is between about 1 day and about 20 days, such as about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 days. In other embodiments, the perfusion procedure or the cell culture method comprising the perfusion procedure is performed for up to 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 days. In addition, the perfusion procedure according to the above embodiments can be performed in a semi-continuous, discontinuous or "intermittent" manner, wherein the perfusion procedure is performed, for example, once a day over a period of several days.

[0091] The methods and systems according to the embodiments described herein can be designed to maintain target cell viability throughout the perfusion procedure, such that the cell culture can maintain cell viability at or above a desired target value. In some embodiments, the methods produce a cell culture viability greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% throughout the duration of the perfusion procedure.

[0092] Methods and systems according to embodiments of the technology described herein can be designed to maintain target cell viability throughout the cell culture duration, such that the cell culture can maintain cell viability at or above the target value required for the entire culture duration. In some embodiments, these methods produce greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% cell culture viability throughout the cell culture duration.

[0093] Methods and systems according to embodiments of the technology described herein can be designed to maintain a target concentration of cell culture waste or byproducts at or below a target level. For example, in some embodiments, the methods produce a cell culture lactate (lactate) concentration of less than or equal to 4 g / L, such as less than or equal to 3.75, 3.5, 3.25, 3.0, 2.75, 2.5, 2.25, 2.0, 1.75, 1.5, 1.25, 1.0, 0.75, 0.5, 0.25, 0.2, or 0.1 g / L throughout the duration of the perfusion procedure. In some embodiments, the methods produce a cell culture ammonia concentration of less than or equal to 4 mM, such as less than or equal to 3.75, 3.5, 3.25, 3.0, 2.75, 2.5, 2.25, 2.0, 1.75, 1.5, 1.25, 1.0, 0.75, 0.5, 0.25, 0.2, or 0.1 mM, throughout the duration of the perfusion procedure. In some embodiments, the methods produce a cell culture pCO concentration of less than or equal to 150 mmHg, such as less than or equal to 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 mmHg, throughout the duration of the perfusion procedure.

[0094] In some embodiments, the methods and systems described herein are configured for industrial scale use in a cell culture bioreactor volume of about or greater than about 20 L, such as 50, 80, 100, 250, 300, 350, 400, 450, 500, 1,000, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 7,500, 8,000, 10,000, 12,000, 15,000, 16,000, 18,000, 20,000, 22,000, 24,000, 26,000, 28,000, or 30,000 liters or more. In some embodiments, the cell culture bioreactor has a working volume greater than or equal to 80 L. In some embodiments, the cell culture bioreactor has a total volume ranging from 100 L to 3,000 L. In some embodiments, the cell culture bioreactor has a total volume of 100 L. In some embodiments, the cell culture bioreactor has a total volume of 3,000 L. In some embodiments, the cell culture bioreactor has a total volume in the range of 100 L to 30,000 L. In some embodiments, the cell culture bioreactor has a total volume in the range of 5,000 L to 30,000 L. In some embodiments, the cell culture bioreactor has a total volume in the range of 100 L to 6,000 L.

[0095] Aspects of the described technology may include methods involving transferring at least a portion of a first cell culture from one bioreactor to another bioreactor to start a second cell culture. In certain embodiments, these methods involve combining a desired volume of solid phase discharged from a continuous flow centrifuge with a desired volume of fresh cell culture medium to maintain, maintain and / or start a new cell culture. In certain embodiments, the new cell culture is an inoculum cell culture. In certain embodiments, the new cell culture is a production cell culture.

[0096] In some embodiments, when the new cell culture is a seeded cell culture, these methods involve further culturing the new seeded cell culture for a period of time to produce a target cell density. In some embodiments, the new seeded cell culture is cultured for a period of time ranging from 1 to 7 days (such as 2, 3, 4, 5 or 6 days).

[0097] In some embodiments, when the new cell culture is a production cell culture, the methods further involve culturing the production cell culture under batch or fed-batch process conditions. In some embodiments, the methods further involve subjecting the cell culture to a harvesting procedure to separate the cells from the cell culture medium at a given time point, and subjecting the harvested cell culture fluid (HCCF) to a purification procedure. In some embodiments, the methods involve culturing the production culture for a period of time ranging from 1 to 20 days, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 days prior to subjecting the production culture to a harvesting procedure.

[0098] In a preferred embodiment, the method includes placing a plurality of mammalian cells and a volume of culture medium in a culture vessel to produce a cell culture; culturing the cell culture to a cell density greater than or equal to 1% hematocrit (PCV); subjecting the cell culture to a perfusion procedure, wherein the perfusion procedure includes: transferring at least a portion of the cell culture to a continuous flow centrifuge; operating the continuous flow centrifuge to produce a solid phase having a final cell density greater than or equal to 1% PCV, such as about 10%, about 20%, about 30%, about 40% or about 50% PCV; and returning the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 vessel volumes per day (VVD) (such as at least 1, 2, 3, 4 or 5 VVD), wherein after completing the perfusion procedure, the cell culture has a cell density greater than or equal to 0.2% PCV. In some embodiments, the perfusion rate ranges from 2 to 4 VVD. In some embodiments, the perfusion rate can range from about 0.5 vessel volumes per day (VVD) to about 10 VVD, such as at least or about 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75 VVD, or about 10.0 VVD.

[0099] In a preferred embodiment, a method includes producing a mammalian cell culture having a cell density greater than or equal to 1% PCV, the method comprising: placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel to produce the cell culture; culturing the cell culture to a cell density in a range of 10% to 30% PCV, such as 10% to 15% PCV, 15% to 20% PCV, 20% to 25% PCV, or 25% to 30% PCV; subjecting the cell culture to a perfusion procedure, wherein the perfusion procedure comprises: transferring at least a portion of the cell culture to a continuous flow centrifuge; operating the continuous flow centrifuge to produce a solid phase having a cell density greater than the % PCV of the cell culture, such as greater than at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the % PCV of the cell culture, such as greater than or equal to 15% to 20% PCV, 20% to 25% PCV, or 25% to 30% PCV, 30% to 35% PCV, PCV, 35% to 40% PCV or greater than 40% PCV; and returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 to 6 vessel volumes per day (VVD), such as 1, 2, 3, 4 or 5 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 0.2% PCV (e.g., 0.2% to about 30% PCV). Preferably, the perfusion rate is 3.5 to 6 VVD. Preferably, the perfusion rate is 5 to 6 VVD.

[0100] In one embodiment, a method includes producing a nanostructured carbon nanotube comprising at least or about 4.8 x 10 12 The method comprises: placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of 80 L to produce a cell culture having a starting cell density greater than or equal to 1 million cells / mL; culturing the cell culture to a cell density greater than or equal to 10% PCV; subjecting the cell culture to a perfusion procedure, wherein the perfusion procedure comprises: transferring at least a portion of the cell culture to a medium having a range of 10,000 to 200,000 m 2 The method comprises the steps of: operating the continuous flow centrifuge to produce a solid phase having a cell density in the range of about 1% to about 10%, about 20%, about 30%, about 40%, or about 50% PCV; and returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a PCV greater than or equal to 60×10 6In a specific embodiment, the solid phase cell density ranges from about 20% to about 40%. In a more specific embodiment, the cell density ranges from about 30% to about 35%.

[0101] In another embodiment, a method is provided herein comprising producing a 14 The method comprises: placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of 3000 L to produce a culture vessel having a volume greater than or equal to 1×10 6 cells / mL; culturing the cell culture to a cell density greater than or equal to 10% PCV; subjecting the cell culture to a perfusion procedure, wherein the perfusion procedure comprises: transferring at least a portion of the cell culture to a medium having a range of 10,000 to 200,000 m 2 The method comprises the steps of: operating the continuous flow centrifuge to produce a solid phase having a cell density in the range of about 1% to about 10%, about 20%, about 30%, about 40%, or about 50% PCV; and returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a PCV greater than or equal to 60×10 6 In a specific embodiment, the solid phase cell density ranges from about 20% to about 40%. In a more specific embodiment, the cell density ranges from about 30% to about 35%.

[0102] In a preferred embodiment, a method comprises generating a 14 The method comprises: placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of 3600 L to produce a cell culture having a starting cell density greater than or equal to 1 million cells / mL; culturing the cell culture to a cell density greater than or equal to 10% PCV; subjecting the cell culture to a perfusion procedure, wherein the perfusion procedure comprises: transferring at least a portion of the cell culture to a medium having a range of 10,000 to 200,000 m 2A continuous flow centrifuge having a σ factor of 100%, operating the continuous flow centrifuge to produce a solid phase having a cell density ranging from about 1% to about 10%, about 20%, about 30%, about 40%, or about 50% PCV; and returning at least a portion of the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate ranging from 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 60 million cells / mL. In a specific embodiment, the solid phase cell density ranges from about 20% to about 40%. In a more specific embodiment, the cell density ranges from about 30% to about 35%.

[0103] Thus, provided herein are exemplary embodiments of methods of culturing mammalian cells:

[0104] Embodiment 1: A method for culturing mammalian cells, comprising: (a) placing a plurality of mammalian cells and a volume of culture medium in a culture vessel to produce a cell culture; (b) culturing the cell culture to a cell density greater than or equal to 1% hematocrit (PCV); (c) subjecting the cell culture to a perfusion procedure during step (b), wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV (e.g., about 10%, about 20%, about 30%, about 40% or about 50% PCV); and (iii) returning the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 vessel volumes per day (VVD), wherein after completion of the perfusion procedure (e.g., after 7 to 8 days), the cell culture has a cell density of 0.2% PCV. PCV or greater (e.g., 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, or 9.8%) PCV, or 10%, 15%, 20%, 25% or 30% PCV) of cell density. Example 2: The method according to Example 1, wherein the perfusion rate ranges from 2 to 4 VVD. Example 3: The method according to Example 1 or 2, wherein the perfusion program includes increasing or decreasing the perfusion rate in a constant manner. Example 4: The method according to Example 1 or 2, wherein the perfusion program includes increasing or decreasing the perfusion rate in a variable manner. Example 5: The method according to any one of Examples 1 to 4, wherein the perfusion program is performed continuously over a time period ranging from 1 to 7 days. Example 6: The method according to any one of Examples 1 to 4, wherein the perfusion program is performed in a semi-continuous manner over a time period ranging from 1 to 7 days. Example 7: The method according to any one of Examples 1 to 6, wherein the continuous flow centrifuge comprises a disc stack rotor. Example 8: The method according to any one of Examples 1 to 6, wherein the continuous flow centrifuge comprises a tubular rotor. Embodiment 9: The method of any one of embodiments 1 to 8, wherein the continuous flow centrifuge has an operating speed ranging from 3,000 to 10,000 RPM. Embodiment 10: The method of any one of embodiments 1 to 9, wherein the continuous flow centrifuge comprises sterilizable components.Embodiment 11: The method of any one of embodiments 1 to 10, wherein the continuous flow centrifuge comprises disposable components. Embodiment 12: The method of any one of embodiments 1 to 11, wherein the continuous flow centrifuge has a range of 1,000 m. 2 Up to 200,000m 2σ value. Embodiment 13: The method according to any one of embodiments 1 to 12, wherein after completing the centrifugation procedure, the cell culture has a viability percentage greater than or equal to 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%). Embodiment 14: The method according to any one of embodiments 5 to 13, wherein the cell culture maintains a viability percentage greater than or equal to 85% over a period of 1 to 7 days. Embodiment 15: The method of any one of embodiments 1 to 14, wherein the cell culture medium is maintained at a temperature between about 30°C and about 39°C (e.g., between 31°C and 38°C, between 32°C and 38°C, between 33°C and 38°C, between 34°C and 38°C, between 35°C and 38°C, or between 36°C and 38°C) during the residence time. Embodiment 16: The method of any one of embodiments 1 to 15, wherein the mammalian cells comprise recombinant mammalian cells. Embodiment 17: The method of embodiment 16, wherein the recombinant mammalian cells comprise recombinant Chinese hamster ovary (CHO) cells. Embodiment 18: The method of embodiment 17, wherein after completion of the centrifugation procedure, the cell culture has a lactate concentration of less than or equal to 4 g / L. Embodiment 19: The method of any one of claims 16 to 18, wherein the recombinant mammalian cells produce a secreted product. Embodiment 20: The method of embodiment 16, wherein the secreted product comprises a recombinant protein. Embodiment 22: The method of embodiment 21, wherein the recombinant protein is an antibody. Embodiment 23: The method according to any one of Embodiments 1 to 22, wherein the culture vessel has a working volume greater than or equal to 80 L. Embodiment 24: The method according to any one of Embodiments 1 to 23, wherein the culture vessel has a total volume in the range of 100 L to 30,000 L. Embodiment 25: The method according to any one of Embodiments 1 to 23, wherein the culture vessel has a total volume in the range of 5,000 L to 30,000 L. Embodiment 26: The method according to any one of Embodiments 1 to 23, wherein the culture vessel has a total volume in the range of 100 L to 6,000 L. Embodiment 27: The method according to any one of Embodiments 1 to 26, further comprising transferring at least a portion of the cell culture to a different culture vessel to start a second cell culture. Embodiment 28: The method according to Embodiment 27, wherein the second cell culture has an initial cell density in the range of 0.1% to 10% PCV. Embodiment 29: The method of any one of embodiments 1 to 28, further comprising transferring at least a portion of the cell culture to a production culture vessel to initiate a production culture having a starting cell density in the range of 0.1% to 10% PCV.Embodiment 30: The method according to embodiment 29, further comprising culturing the production culture under batch or fed-batch process conditions. Embodiment 31: The method according to any one of embodiments 1 to 30, further comprising adding fresh culture fluid to the culture vessel.

[0105] Example 33: A method for producing a culture of mammalian cells having a cell density greater than or equal to 0.1% PCV, the method comprising: (a) placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel to produce a cell culture; (b) culturing the cell culture to a cell density greater than or equal to 10% PCV; (c) subjecting the cell culture to a perfusion procedure during step (b), wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to produce a solid phase having a cell density ranging from about 1% to about 10%, about 20%, about 30%, about 40% or about 50% PCV; and (iii) returning a portion of the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate ranging from 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 0.1% PCV. In certain embodiments, after the perfusion procedure is completed, the cell density of the cell culture increases or has increased by greater than or equal to 0.1% PCV, such as greater than or equal to 0.1% to about 30% PCV. In specific embodiments, the solid phase cell density ranges from about 20% to about 40%. In more specific embodiments, the cell density ranges from about 30% to about 35%.

[0106] Example 34: A method for producing a 12 A method for culturing a mammalian cell having a volume of greater than or equal to 1×10 cells, the method comprising: (a) placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of 80 L to produce a culture vessel having a volume of greater than or equal to 1×10 6 (a) preparing a cell culture having a starting cell density of 1,000 cells / mL; (b) growing the cell culture to a cell density greater than or equal to 10% PCV; and (c) subjecting the cell culture to a perfusion procedure during step (b), wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge comprising a disposable disc stack rotor and comprising a flow rate ranging from 1,000 to 200,000 m / s. 2(ii) operating the continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV; and (iii) returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a PCV greater than or equal to 60×10 6 The cell density was 10 cells / mL.

[0107] Example 35: A method for producing a 14 A method for culturing a plurality of mammalian cells, the method comprising: (a) placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of 3,000 L to produce a culture vessel having a working volume of greater than or equal to 110 L; 6 (a) preparing a cell culture having a starting cell density of 1,000 cells / mL; (b) growing the cell culture to a cell density greater than or equal to 10% PCV; (c) subjecting the cell culture to a perfusion procedure, wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge comprising a disposable disc stack rotor and comprising a flow rate ranging from 1,000 to 200,000 m / s; 2 (ii) operating the continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV; and (iii) returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 60×10 6 The cell density was 10 cells / mL.

[0108] Cell banking and / or seed culture or pre-production cell block increase: In certain embodiments, the cell culture methods disclosed herein may include or may be used for cell bank establishment. For example, in one aspect, a method for cell banking is provided herein, comprising: (a) placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel to produce a cell culture; (b) culturing the cell culture to a cell density greater than or equal to 1% hematocrit (PCV); (c) subjecting the cell culture during step (b) to a perfusion procedure, wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to produce a solid phase (heavy phase); (iii) dividing the solid phase into a first portion and a second portion returned to the cell culture vessel, and (iv) returning the first portion to the cell culture vessel (bioreactor); and (d) combining the second portion with a cryopreservative. In a specific embodiment, the second portion is frozen after step (d). In a specific embodiment, the first portion of the solid phase and a volume of cell culture medium are returned to the cell culture vessel. In a more specific embodiment, the first portion of the solid phase and the cell culture medium are returned to the cell culture vessel to achieve a perfusion rate in the range of 0.7 to 6 vessel volumes per day (VVD). In a more specific embodiment, after the perfusion procedure is completed, the cell culture has a cell density of 0.2% PCV or greater (e.g., 0.2% to about 30% PCV or greater). In certain embodiments, the cryopreservative is dimethyl sulfoxide (DMSO) or a growth medium supplemented with DMSO. In a specific embodiment, DMSO has a final concentration of 5% to 10% by volume (vol. / vol.). In certain embodiments, the second portion of the heavy phase has a cell density of or is adjusted to a cell density of at least 1x10 8 In more specific embodiments, the second portion of the heavy phase has a cell density of or is adjusted to a cell density of at least, about, or no more than 1.0 x 10 8 cells / mL, 1.1x10 8 cells / mL, 1.2x10 8 cells / mL, 1.3x10 8 cells / mL, 1.4x10 8 cells / mL, 1.5x10 8 cells / mL, 1.6x10 8 cells / mL, 1.7x10 8 cells / mL, 1.8x10 8 cells / mL, 1.9x10 8 cells / mL, 2.0x10 8Cells / mL. In certain embodiments, the first portion of the solid phase includes up to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or up to 50% of the total volume of the solid phase. In certain embodiments, wherein the first portion of the solid phase includes up to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or up to 50% of the total volume of the solid phase, the second portion of the solid phase includes or substantially includes the remainder of the solid phase. In certain embodiments, during cell culture, the solid phase is discretely divided into the first and second portions multiple times. In certain embodiments, during cell culture, the solid phase is divided into the first and second portions once. In certain embodiments, during cell culture, the solid phase is continuously divided into the first and second portions. In certain embodiments, the solid phase or the second portion of the solid phase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% or 95% cell viability. %. In the above embodiments, the centrifuge can be a continuous flow centrifuge, such as a single continuous flow centrifuge. In some embodiments, two or more continuous flow centrifuges are used (e.g., in parallel). In additional embodiments, cells or cell masses produced from one or more continuous flow centrifuges are used to start or inoculate a culture in one of a plurality of pre-production bioreactors, or to inoculate or transfer a culture to one or more production bioreactors.

[0109] In certain embodiments, the cell bank is part of a pre-production cell culture of cells genetically modified to produce a protein of interest, such as a therapeutic protein or polypeptide. In certain embodiments, the therapeutic protein or polypeptide is a fusion protein, such as a fusion protein including an antibody Fc portion or human serum albumin. In other specific embodiments, the therapeutic protein or polypeptide is an antibody or antibody fragment, such as a monoclonal antibody, a monospecific antibody, a bispecific antibody (with or without a common light chain), a bispecific T cell engager (BiTE), a bispecific (mab) 2 antibody; a bispecific F (mab) 2 antibody; a single domain bispecific diabody (scBsDb), a single chain bispecific tandem variable domain (scBsTaFv), a trispecific NK cell engager therapy (TriNKET), a dual affinity retargeting protein (DART), a bispecific diabody, a tandem diabody (TandAb), a half antibody (e.g., an antibody comprising an Fc portion but only one CH-VH:CL-VL pair), a trifab contorsbody (as described in PCT application publication No. WO 2019 / 086395; a quadroma, a scFv, a docking and locking trivalent fab (DNL-(Fab) 3, a single domain antibody, a bispecific single domain antibody, etc.

[0110] III. System

[0111] Various systems described herein and commercially available may be suitable for performing the presently disclosed methods. Systems may be based on batch feeding and / or continuous flow. Systems may include stirring systems based on stirred tanks or shaking. Such systems may be designed for single use or repeated use. For example, a centrifuge (e.g., a device suitable for inclusion in a perfusion method) may include disposable components (e.g., a disposable drum). Examples of batch feeding systems that can also support continuous flow or perfusion techniques for performing the methods described herein include, but are not limited to, any of the HyPerforma TM Systems (e.g., HyPerforma TM 5:1 disposable bioreactor, Jacketed, AC motor, Load Cells, catalog number: SUB00508100), which is manufactured by ThermoFisher Scientific TM Manufacturing; any kind or STR systems (e.g., Biostat Generation 3featuring Automation) by Sartorious TM Manufacturing; Any Allegro TM system, which consists of These systems can be modified or adapted to include perfusion-based devices and systems. A non-limiting example of a perfusion system can include alternating tangential flow (ATF) filtration using hollow filter fibers (e.g., from Repligen TM Another non-limiting example of a perfusion system may include tangential flow filtration (e.g., from Flow Sciences, Inc. TM products). Additional examples of tangential flow filtration (TFF) systems are provided by Sartorious TM Production (e.g. 150Auto Disposable Tangential Flow Filtration System). Depth filtration can also be used in the perfusion methods described herein. Additional non-limiting examples of perfusion system components may include acoustic sedimentation based perfusion technology (e.g., from SonoSep Technologies TMProducts). Continuous flow systems can be combined with one or more systems described herein and other commercially available systems. A variety of centrifugal systems can be included with the batch feeding and perfusion systems described herein to perform the methods described herein. Preferably, the centrifuge used in the cell culture method of the present invention includes disposable components that contact cells and / or cell culture medium, such as a disposable drum or a drum embedded component. In certain embodiments, the continuous centrifuge includes a disposable centrifugal pump. Non-limiting examples of such systems include those available from Alfa Laval TM CultureOne TM (For example, Alfa Laval CultureOne Primo TM Other suitable commercial systems include the Culturefuge 400B available from Alfa Laval, the GEA Available from Sartorius TM of DynaSpin from Thermo Fisher TM and can be purchased from of In certain embodiments, a single continuous flow centrifuge is used. In other embodiments, two or more continuous flow centrifuges are used (e.g., in parallel). In additional embodiments, cells or cell masses produced from one or more continuous flow centrifuges are used to start or inoculate a culture in one of a plurality of pre-production bioreactors, or to inoculate or transfer a culture to one or more production bioreactors.

[0112] Figure 1 Schematic diagram of a system for culturing cells according to one or more embodiments of the cell culture methods provided herein, the system comprising a perfusion and cell separation device (in some embodiments, a centrifuge; in more specific embodiments, a continuous flow centrifuge) 100. Generally, a cell culture system for performing the methods described herein involves inoculating a cell culture medium (e.g., Gibco) in a cell culture vessel 120 with suitable cells (e.g., cell types described herein, recombinant mammalian cells, recombinant CHO cells, and any other commercially available cell types) from an inoculation vessel 110. TMCHO perfusion medium) to grow a cell culture in a cell culture vessel 120. The culture medium including the cells can be transferred (e.g., pumped) from the inoculation vessel 110 to the cell culture vessel 120 via a sterile connection 101. The inoculated cell culture (e.g., an inoculated culture of mammalian cells) can have a cell density greater than or equal to 0.1% PCV. The inoculated cell culture (e.g., an inoculated culture of mammalian cells) can have a cell density greater than or equal to 1% PCV.

[0113] Inoculation is preferably performed under sterile conditions by depositing (e.g., pumping through a sterile connection) a plurality of mammalian cells included in a volume of cell culture medium into a culture vessel to produce a cell culture. In a specific embodiment, inoculation can occur under sterile conditions by depositing (e.g., pumping through a sterile connection) a plurality of mammalian cells included in a volume of cell culture medium into a culture vessel having a working volume of, for example, 80 L to produce a cell culture having a starting cell density greater than or equal to 1 million cells / mL. In another specific embodiment, inoculation can occur under sterile conditions by depositing (e.g., pumping through a sterile connection) a plurality of mammalian cells and a volume of cell culture medium into a culture vessel having a working volume of 3,000 L or 3,600 L to produce a cell culture having a starting cell density greater than or equal to 1 million cells / mL.

[0114] Once the culture has expanded to a cell density greater than or equal to 10% PCV (or alternatively 1%), at least a portion of the culture can be transferred from the cell culture vessel 120 to the cell separation device 130 via sterile connection 103 (eg, using a peristaltic pump).

[0115] In a specific embodiment of the cell culture system provided herein, the cell separation device 130 preferably removes a portion of the spent or exhausted culture medium and returns a portion, preferably substantially all, of the cells to the cell culture vessel 120. In addition to removing the exhausted culture medium from the cell culture vessel 120, a fresh culture medium and / or a plurality of culture mediums are preferably transferred to the cell culture vessel from the fresh fluid vessel 170 via a sterile connection 171. In such a system, fresh culture medium can be added back to the cell culture when the exhausted culture medium is removed. In a specific embodiment, the cell culture vessel 120 has a working volume or total volume greater than or equal to 80L. In other specific embodiments, the cell culture vessel 120 has a working volume or total volume ranging from 100L to 3,000L. In a specific embodiment, the cell culture vessel 120 has a working volume or total volume of 100L. In another specific embodiment, the cell culture vessel 120 has a working volume or total volume of 3,000L. Preferably, the process of spent culture medium removal, cell separation, and cell return to the cell culture vessel 120 is continuously performed during at least part of the time of culturing cells.

[0116] In some systems, the cell separation device 130 may include a filtration system (e.g., depth filtration, ATF and / or TFF). In a specific embodiment of the cell culture method provided herein, the cell separation device 130 includes a centrifuge or is a centrifuge. In certain embodiments, the cell separation device 130 combines filtration and centrifugation. Before or during use, a flushing liquid can be used to prime one or more components of the system, such as the cell separation device 130. In the case where the cell separation device 130 includes a centrifuge that can be used for continuous or semi-continuous applications, the flushing fluid can be used to drive away or flush away a portion of the cell culture transferred from the cell culture vessel 120 to the cell separation device 130. In certain embodiments, the centrifuge is a single continuous flow centrifuge. In aspects of such embodiments, two or more continuous flow centrifuges are used (e.g., in parallel). In additional embodiments, cells or cell blocks produced from one or more continuous flow centrifuges are used to start or inoculate a culture in one of a plurality of pre-production bioreactors, or a culture is inoculated or transferred to one or more production bioreactors.

[0117] The flushing fluid may include a buffer and / or purified water. The buffer may be suitable for maintaining healthy cells (e.g., a phosphate buffered saline (PBS) solution at an appropriate pH so that the cells are less likely to dissolve). The flushing fluid may be pumped from the flushing liquid vessel 160 to the cell separation device 130 via a sterile connection 111. In many embodiments, the flushing fluid may be used to empty the cell separation device 130 to preserve cells and / or products. In some systems, the flushing fluid may be used to remove solids from the walls of the cell separation device 130 (e.g., a centrifuge).

[0118] Some traditional centrifugal systems include outlets for light phase and heavy phase. In such traditional systems, solids (e.g., cells being cultivated) may gather in the drum of the centrifuge and need to be (intermittently or intentionally) discharged or regularly scraped off. Such programs can interrupt continuous flow. Although such systems can be incorporated into the cell culture method provided herein (e.g., as cell separation device 130), the cell culture method is preferably combined with a continuous flow cell separator, such as a continuous flow centrifuge.

[0119] In various systems described herein, the heavy phase may include such solids (e.g., a solid phase including cultured cells). The light phase (e.g., liquid phase) may include products (e.g., secretory products from cells, recombinant proteins and / or antibodies). Centrifugal systems (e.g., disc stack centrifuges, tubular centrifuges) used to perform the methods described herein may be used to separate the liquid phase from the solid phase. The solid phase may include cells. In many systems, the solid may not be ejected from the centrifugal system as is the case in conventional systems, but may remain in the solid phase.

[0120] In various systems, the cell separation device 130 may include a liquid phase outlet 112. In various embodiments, the liquid phase outlet 112 may include a sterile connection 109 to a liquid phase collection vessel 150. The liquid phase transferred to the liquid phase collection vessel 150 may include one or more products from the cell culture. Once separated, the products (e.g., products secreted from the cells) may be subjected to a purification procedure.

[0121] Optionally, a portion of the liquid phase can be transferred back to the cell culture vessel 120 via the sterile connection 113 .

[0122] In some systems, the liquid phase outlet 112 may include a heavy liquid phase outlet and a light liquid phase outlet. In such systems, the heavy liquid phase outlet may transfer cells back to the cell culture vessel 120 via a sterile connection 113 and the light liquid phase outlet may include spent culture medium.

[0123] In various systems, the cell separation device 130 may include a solid phase outlet 106. In many systems, the solid phase outlet 106 can transfer all or part of the solid phase back to the cell culture vessel 120 via a sterile connection 105. The solid phase may include dense red blood cells. In some cases, the solid phase may include a highly viscous solution. The centrifuge described herein can be operated continuously to produce a solid phase with a cell density ranging from 1% to 50% PCV, for example, about 10%, about 10%, about 30%, about 40% or about 50% PCV. The centrifuge described herein can be operated continuously to produce a solid phase with a cell density greater than or equal to 30% PCV.

[0124] One or more pumps (e.g., centrifugal pumps or peristaltic pumps) can return at least a portion of the solid phase and a volume of the cell culture medium to the cell culture vessel 120 to achieve a perfusion rate in the range of 0.5 to 6 vessel volumes per day (VVD), wherein after completion of the perfusion procedure, the cell culture has a cell density of 0.2% PCV or greater. In addition, fresh cell culture fluid can be pumped from the fresh cell culture medium vessel 170 to the cell culture vessel 120 through the sterile connection 171 to replace the spent culture medium.

[0125] One or more pumps (e.g., centrifugal pumps or peristaltic pumps) can return at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel 120 to achieve a perfusion rate in the range of 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 0.1% PCV. In addition, fresh cell culture fluid can be pumped from the fresh cell culture medium vessel 170 to the cell culture vessel 120 through the sterile connection 171 to replace the spent culture medium.

[0126] One or more pumps (e.g., centrifugal pumps or peristaltic pumps) can return at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel 120 to achieve a perfusion rate in the range of 0.5 to 6 VVD, wherein after the perfusion procedure is completed, the cell culture has a volume greater than or equal to 60×10 6 In addition, fresh cell culture fluid can be pumped from fresh cell culture medium vessel 170 to cell culture vessel 120 via sterile connection 171 to replace the spent culture medium.

[0127] One or more pumps (e.g., centrifugal pumps or peristaltic pumps) can return at least a portion of the solid phase and a volume of the cell culture medium to the cell culture vessel 120 to achieve a perfusion rate in the range of 2 to 6 VVD. In addition, fresh cell culture fluid can be pumped from the fresh cell culture medium vessel 170 to the cell culture vessel 120 through the sterile connection 171 to replace the spent culture medium.

[0128] The one or more pumps may be operated to increase or decrease the perfusion rate in a constant manner. The one or more pumps may be operated to increase or decrease the perfusion rate in a variable manner. The one or more pumps may be operated continuously over a time period ranging from 1 to 7 days. The one or more pumps may be operated in a semi-continuous manner over a time period ranging from 1 to 7 days.

[0129] The centrifugation process can produce a cell culture having a viability percentage greater than or equal to 85%. The centrifugation process can produce a cell culture that maintains a viability percentage greater than or equal to 85% over a period of 1 to 7 days. The centrifugation process can produce a cell culture having a lactate concentration less than or equal to 4 g / L.

[0130] Centrifuges can operate at speeds ranging from 3,000 to 10,000 RPM. Continuous flow centrifuges can have speeds ranging from 1,000 to 2,000 RPM. 2 Up to 200,000m 2 The σ value of .

[0131] The cell separation device 130 may include sterilizable components. For example, the cell separation device may be airtight. When the cell separation device 130 includes a centrifuge drum (e.g., a disc-type stack or tubular), the rotating component (e.g., a drive shaft) may include one or more seals. In some cases, the sterilization component may include a heating component. In addition, one or more sterile connectors may couple the continuous flow centrifuge to a sleeve or pipeline that is directly or indirectly coupled to the cell culture vessel 120, the flushing liquid vessel 160, one or more solid phase collection vessels 140, and / or the liquid phase collection vessel 150.

[0132] In some systems, the solid phase outlet 106 can transfer all or part of the solid phase to a solid phase collection vessel 140 via a sterile connection 107. In some systems, the solid phase collection vessel 140 can be a cell culture vessel. In some systems, the solid phase collection vessel 140 can be a plurality of cell culture vessels. The solid phase can be used to inoculate the culture medium of one or more solid phase collection vessels 140. For example, the system can transfer at least a portion of the cell culture to a different culture vessel to start a second cell culture. The second cell culture can have an initial cell density in the range of 0.1% to 10% PCV.

[0133] In some systems, the cell culture vessel 120 may include a production culture vessel. The system may transfer at least a portion of the cell culture to the production culture vessel to initiate a production culture having a starting cell density in the range of 0.1% to 10% PCV. The production culture undergoes continuous process conditions. The production culture undergoes batch or fed-batch process conditions.

[0134] The sterile connections 101, 103, 105, 107, 109, 111, 113 described herein may include cannulas connected to vessels and devices 110, 120, 130, 140, 150, 160 through one or more sterile connectors and may be pumped using one or more pumps (e.g., peristaltic pumps). Cannulas, pumps, and sterile connectors are commercially available from various manufacturers, including Thermo Fisher Scientific TM , Sartorious TM and

[0135] Environmental conditions, including but not limited to cell density, cell viability, pH, dO2, pressure, and temperature, can be monitored by one or more sensors or probes within the cell culture vessel 120 or any other component of the system. In an automated cell culture process, one or more sensors or probes can send a signal to the control system to report the environmental conditions. The control system can then take action to adjust one or more of the environmental conditions by activating a pump, a heating element, or any auxiliary device capable of changing the environmental conditions.

[0136] IV. How to use

[0137] Figure 2 is a flow chart of a process 200 for culturing mammalian cells according to one or more embodiments.

[0138] Step 1 includes placing a plurality of mammalian cells and a volume of culture medium in a culture vessel to produce a cell culture 202 .

[0139] Step 2 includes growing the cell culture to a desired cell density 204 .

[0140] Step 3 includes subjecting the cell culture to a perfusion procedure 206 .

[0141] The perfusion procedure may include transferring at least a portion of the cell culture to a continuous flow centrifuge. The perfusion procedure may include operating the continuous flow centrifuge to produce a solid phase. The perfusion procedure may include returning the solid phase and a volume of cell culture medium to the culture vessel at a desired perfusion rate.

[0142] Figure 3 is a flow chart of a process 300 for culturing mammalian cells according to one or more embodiments.

[0143] Step 1 includes placing a plurality of mammalian cells and a volume of culture medium in a culture vessel to produce a cell culture 302. In some cultures, the mammalian cells may include recombinant mammalian cells. The recombinant mammalian cells may include recombinant Chinese hamster ovary (CHO) cells. The recombinant mammalian cells may produce secreted products. In various processes, the secreted products may include recombinant proteins. In various processes, the recombinant proteins may include antibodies.

[0144] Various cultures may use culture vessels with a working volume that may be greater than or equal to 80 L. Various cultures may use culture vessels with a total volume that may range from 100 L to 3,000 L. Some cultures may use vessels with a total volume of 100 L. Some cultures may use vessels with a total volume of 3,000 L.

[0145] Step 2 includes growing the cell culture to a cell density 304 of greater than or equal to 1% packed cell volume (PCV).

[0146] Step 3 includes subjecting the cell culture to a perfusion procedure 306 .

[0147] Performing a perfusion procedure may include returning a solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 vessel volumes per day (VVD). In some perfusion procedures, the perfusion rate ranges from 2 to 4 VVD. Some perfusion procedures may include increasing or decreasing the perfusion rate in a constant manner. Alternative perfusion procedures may include increasing or decreasing the perfusion rate in a variable manner.

[0148] The perfusion procedure can be performed continuously over a time period ranging from 1 to 7 days. An alternative perfusion procedure can be performed in a semi-continuous manner over a time period ranging from 1 to 7 days. The cell culture can maintain a viability percentage greater than or equal to 85% over a period of 1 to 7 days.

[0149] Performing a perfusion procedure may include transferring at least a portion of the cell culture to a continuous flow centrifuge. Some perfusion procedures may use a continuous flow centrifuge that includes a disc stack rotor. The continuous flow centrifuge may have a range of 1,000 m 2 Up to 200,000m 2 The continuous flow centrifuge can be maintained at a residence time temperature between about 30°C and about 39°C, for example, between 31°C and 38°C, between 32°C and 38°C, between 33°C and 38°C, between 34°C and 38°C, between 35°C and 38°C, or between 36°C and 38°C.

[0150] Alternatively, the perfusion procedure may include the use of a continuous flow centrifuge comprising a tubular rotor.A variety of suitable centrifugation systems are commercially available.

[0151] Performing a perfusion procedure can include operating a continuous flow centrifuge to produce a solid phase having a final cell density greater than or equal to 1% PCV, such as about 10%, about 20%, about 30%, about 40%, or about 50% PCV.

[0152] The perfusion procedure may include separating the liquid phase from the continuous flow centrifuge and performing a purification procedure on the secreted product therein.

[0153] The continuous flow centrifuge used in the perfusion procedures described herein may include sterilizable components. The sterilizable components may include one or more devices or processes described herein and elsewhere.

[0154] The continuous flow centrifuge used in the perfusion procedures described herein may include disposable components.

[0155] Continuous flow centrifuges used in perfusion procedures may include operating speeds ranging from 3,000 to 10,000 RPM.

[0156] In some processes for culturing mammalian cells, cultivation of the production culture is carried out under batch or fed-batch process conditions.

[0157] After completing the centrifugation procedure of the perfusion procedure, the cell culture may have a viability percentage greater than or equal to 85%. After completing the centrifugation procedure of the perfusion procedure, the cell culture may have a lactate concentration less than or equal to 4 g / L. After completing the perfusion procedure, the cell culture may have a cell density of 0.2% PCV or greater, such as 0.2% PCV to about 30% PCV.

[0158] The process of culturing mammalian cells can include transferring at least a portion of the cell culture to a different culture vessel to initiate a second cell culture. In some processes, the second cell culture can have an initial cell density ranging from 0.1% to 10% PCV.

[0159] The process of culturing mammalian cells can include transferring at least a portion of the cell culture to a production culture vessel to initiate a production culture having a starting cell density in the range of 0.1% to 10% PCV.

[0160] Figure 4 Flow diagram of a process 400 for producing a mammalian cell inoculum culture having a cell density greater than or equal to 0.1% PCV (eg, 0.1% to about 30% PCV) according to one or more embodiments.

[0161] Step 1 includes placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel to produce a cell culture 402 .

[0162] Step 2 includes growing the cell culture to a cell density 404 greater than or equal to 10% PCV.

[0163] Step 3 includes subjecting the cell culture to a perfusion procedure 406 .

[0164] The perfusion procedure may include transferring at least a portion of the cell culture to a continuous flow centrifuge. The perfusion procedure may include operating the continuous flow centrifuge to produce a solid phase having a cell density in a range of 1% to about 50% PCV. The perfusion procedure may include returning a portion of the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate in a range of 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 0.1% PCV to 30% PCV.

[0165] Figure 5To produce according to one or more embodiments at least 4.8x10 12 Flow diagram of process 500 for inoculating a culture of mammalian cells of 10 cells.

[0166] Step 1 includes placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having an 80 L working volume to produce a cell culture 502 having a starting cell density greater than or equal to 1 million cells / mL.

[0167] Step 2 includes growing the cell culture to a cell density 504 greater than or equal to 10% PCV.

[0168] Step 3 includes subjecting the cell culture to a perfusion procedure 506 .

[0169] The perfusion reaction may include transferring at least a portion of the cell culture to a continuous flow centrifuge comprising a disposable disc stack rotor and comprising a range of 1,000 to 200,000 m 2 The perfusion procedure can include operating the continuous flow centrifuge to produce a solid phase having a final cell density greater than or equal to 1% PCV, such as about 10%, about 20%, about 30%, about 40%, or about 50% PCV. The perfusion procedure can include returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 60 million cells / mL.

[0170] Figure 6 To generate a system according to one or more embodiments comprising at least 2.16x10 14 Flow diagram of process 600 for inoculating a culture of mammalian cells of 10 cells.

[0171] Step 1 includes placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of 3,000 L or 3,600 L to produce a cell culture 602 having a starting cell density greater than or equal to 1 million cells / mL.

[0172] Step 2 includes growing the cell culture to a cell density 604 of greater than or equal to 10% PCV.

[0173] Step 3 includes subjecting the cell culture to a perfusion procedure 606 .

[0174] The perfusion procedure may include transferring at least a portion of the cell culture to a continuous flow centrifuge comprising a disposable disc stack rotor and comprising a sigma factor in the range of 1,000 to 200,000 m2. The perfusion procedure may include operating the continuous flow centrifuge to produce a solid phase having a final cell density greater than or equal to 1% PCV, such as about 10%, about 20%, about 30%, about 40%, or about 50% PCV. The perfusion procedure may include returning at least a portion of the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 60 million cells / mL.

[0175] The processes in the methods described herein may be performed using the systems and apparatus described herein and elsewhere.

[0176] It will be apparent to those skilled in the art that various changes and modifications can be made in light of the techniques described herein without departing from the spirit or scope of the embodiments of the invention.

[0177] Examples

[0178] Example 1: 50% split ratio

[0179] A perfusion cell culture run was completed with a 50% ratio of solid to liquid phase returned to the production cell culture. For a 50:50 solid to liquid split ratio, the centrifuge was adjusted so that 50% of the centrifuge inlet flow volumetrically flowed to the solids outlet and 50% of the centrifuge inlet flow volumetrically flowed through the liquid outlet.

[0180] Example 2: 75% split ratio

[0181] A perfusion cell culture run was completed with a 75% ratio of solid to liquid phase returned to the production cell culture. For a 75:25 solid to liquid split ratio, the centrifuge was adjusted so that 75% of the centrifuge inlet flow volumetrically flowed to the solids outlet and 25% of the centrifuge inlet flow volumetrically flowed through the liquid outlet.

[0182] Example 3: 90% split ratio

[0183] A perfusion cell culture run was completed with a 90% ratio of solid to liquid phase returned to the production cell culture. For a 90:10 solid to liquid split ratio, the centrifuge was adjusted so that 90% of the centrifuge inlet flow volumetrically flowed to the solids outlet and 10% of the centrifuge inlet flow volumetrically flowed through the liquid outlet.

[0184] Example 4: Seven-day cell culture

[0185] This example demonstrates seven days of cell culture incorporating the present continuous centrifugation cell culture method without a clarification step in preparation for protein harvest.

[0186] Mammalian cells (Chinese hamster ovary (CHO) cells) expressing therapeutic antibodies were grown in a bioreactor with a working volume of 500 liters using a target actual volume of approximately 300 L. The hematocrit (PCV) at the start of the cell culture was 0.30% with a viable cell count (VCC) of 2.087 x 10 6 cells / mL, with a viability of 97.7%. After about 19 hours of culture, PCV rose to 0.40%, with VCC of 3.472x10 6 cells / mL, with a viability of 97.9%. At about 67 hours of culture, using Alfa Laval CultureOne Primo TM The continuous centrifuge was started with a perfusion program of 2.5 vessel volumes / 24 hour period (VVD), which resulted in a perfusion flow rate of 521 mL / min. The centrifuge was operated with a target split ratio of 50% (i.e., 50:50 solid and light phase) and an actual centrifuge inlet flow rate of 1040 mL / min. The solid phase was returned to the cell culture vessel and the light phase of the run was discarded. The advantages of this continuous centrifuge are the spin insert assembly (Spinsert TM ) is designed as a disposable, single-use component that simplifies sterility maintenance and provides gentle handling of cells (promoting higher cell viability). The centrifuge is supplemented with a peristaltic pump connected to the inlet and outlet ports. Cold water (approximately 4°C to 10°C) is used to cool the centrifuge seals and minimize the temperature increase caused by centrifuge rotation. The addition of culture medium is added at a flow rate equal to the liquid phase flow rate throughout the perfusion process to maintain the actual volume of the bioreactor. The addition is controlled by the bioreactor load cell, increasing the rotor speed from 3000RPM to 3200RPM and then to 3400RPM to optimize cell retention. At this point, the bioreactor PCV is about 2.2%, with a VCC of 1.24x10 7 cells / mL, and the viability was 97.1%. The cell culture perfusion procedure continued for the next approximately 46.5 hours, so the bioreactor PCV increased to 4.33% and the VCC increased to approximately 2.5x10 7 cells / mL, with a viability of 91.7%.

[0187] At about 113.5 hours, the perfusion flow rate was increased from 2.5 VVD to 3.7 VVD (equivalent to a target flow rate of 771 mL / min at a 300 L working volume), with an actual centrifuge inlet flow rate of 1540 mL / min. Five hours later, the bioreactor PCV was about 4.8%, the cell viability was 92.8%, and the VCC was 3.069x10 7 Over the next 22.5 hours, PCV increased to approximately 7.40%, and VCC increased to approximately 4.038x10 7 cells / mL, with a viability of 92.6%.

[0188] At approximately 137.5 hours after startup, the perfusion flow rate was increased from 3.7 VVD to 3.96 VVD (for reference, 3.96 VVD is equivalent to a target flow rate of 825 mL / min at a 300 L working volume), with an actual centrifuge inlet flow rate of 1650 mL / min. The centrifuge continued to operate at a target split ratio of 50%. After the change, the cell viability was 93.8%. Over the next 27 hours, the PCV rose to approximately 12.0%, the cell viability was 95.1%, and the VCC rose to approximately 5.66 x 10 7 cells / mL.

[0189] At approximately 164.5 hours after start-up, the perfusion flow rate was increased from 3.96 VVD to 5 VVD (for reference, 5 VVD is equivalent to a target flow rate of 1042.0 mL / min at a working volume of 300 L). The centrifuge continued to operate at a target split ratio of 50%, with an actual centrifuge inlet flow rate of 2080 mL / min. The solid phase flow rate was 1040 mL / min, and a manual peristaltic pump needed to be installed at this time. After this change, the bioreactor cell viability was 95.9%. Cell culture and centrifugation continued until approximately 181.5 hours after the start of the culture. Starting at 3.96 VVD, the solid phase was 14% PCV; at the end of the run at 5 VVD, the solid phase reached 32% PCV. At this time, the cell viability in the culture vessel was approximately 93.2%, with a PCV of approximately 16%, and a VCC of approximately 8.435x10 7 cells / mL while running at 5 VVD. Starting at about 3.96 VVD and about 14% PCV when the solid phase was first sampled, the hematocrit increased to about 32% PCV at about 5 VVD by the end of the run.

[0190] At each time point during this culture-centrifugation process, bioreactor cell viability was at least 91.4%, and at higher perfusion rates (above 3.7 VVD), bioreactor cell viability was routinely above 92%.

[0191] Although preferred embodiments of the present technology have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art may devise numerous variations, changes, and substitutions without departing from the described technology. It should be understood that various alternatives to the embodiments described herein may be used to practice the systems and methods. The following claim scope is intended to cover not only the methods and structures described, but also their equivalents.

Claims

1. A method for culturing mammalian cells, the method comprising: (a) placing a plurality of mammalian cells and a volume of culture medium in a culture vessel to produce a cell culture; (b) growing the cell culture to a cell density greater than or equal to 1% packed cell volume (PCV); (c) subjecting the cell culture to a perfusion procedure during step (b), wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV; and (iii) returning the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 to 6 vessel volumes per day (VVD), wherein after completion of the perfusion procedure, the cell culture has a cell density of 0.2% PCV or greater.

2. The method of claim 1, wherein the perfusion rate ranges from 2 to 6 VVD.

3. The method according to claim 1 or 2, wherein the perfusion program comprises increasing or decreasing the perfusion rate in a constant manner.

4. The method according to claim 1 or 2, wherein the perfusion program comprises increasing or decreasing the perfusion rate in a variably manner.

5. The method according to any one of claims 1 to 4, wherein the perfusion procedure is performed continuously over a period ranging from 1 to 7 days.

6. The method according to any one of claims 1 to 4, wherein the perfusion procedure is performed in a semi-continuous manner over a period of time ranging from 1 to 7 days.

7. The method of any one of claims 1 to 6, wherein the continuous flow centrifuge comprises a disc stack rotor.

8. The method of any one of claims 1 to 6, wherein the continuous flow centrifuge comprises a tubular bowl.

9. The method of any one of claims 1 to 8, wherein the continuous flow centrifuge has an operating speed ranging from 3,000 to 10,000 RPM.

10. The method of any one of claims 1 to 9, wherein the continuous flow centrifuge comprises sterilizable components.

11. The method of any one of claims 1 to 10, wherein the continuous flow centrifuge comprises disposable components.

12. The method according to any one of claims 1 to 11, wherein the continuous flow centrifuge has a range of 1,000 m 2 Up to 200,000m 2 The σ value of .

13. The method according to any one of claims 1 to 12, wherein after completion of the centrifugation procedure, the cell culture has a viability percentage greater than or equal to 85%.

14. The method of any one of claims 5 to 13, wherein the cell culture maintains a percent viability greater than or equal to 85% over a period of 1 to 7 days.

15. The method of any one of claims 1 to 14, wherein the mammalian cell comprises a recombinant mammalian cell.

16. The method of claim 15, wherein the recombinant mammalian cell comprises a recombinant Chinese Hamster Ovary (CHO) cell.

17. The method of claim 16, wherein after completing the centrifugation procedure, the cell culture has a lactate concentration less than or equal to 4 g / L.

18. The method of any one of claims 15 to 17, wherein the recombinant mammalian cell produces a secreted product.

19. The method of claim 15, wherein the secreted product comprises a recombinant protein.

20. The method of claim 19, wherein the recombinant protein is an antibody.

21. The method according to any one of claims 1 to 20, wherein the culture vessel has a working volume greater than or equal to 80 L.

22. The method according to any one of claims 1 to 21, wherein the culture vessel has a total volume in the range of 100L to 30,000L.

23. The method of any one of claims 1 to 21, wherein the culture vessel has a total volume in the range of 5,000L to 30,000L.

24. The method of any one of claims 1 to 21, wherein the culture vessel has a total volume ranging from 100 L to 6,000 L.

25. The method of any one of claims 1 to 24, further comprising transferring at least a portion of the cell culture to a different culture vessel to initiate a second cell culture.

26. The method of claim 25, wherein the second cell culture has an initial cell density in the range of 0.1% to 10% PCV.

27. The method of any one of claims 1 to 26, further comprising transferring at least a portion of the cell culture to a production culture vessel to initiate a production culture having a starting cell density in the range of 0.1% to 10% PCV.

28. The method of claim 27, further comprising culturing the production culture under batch or fed-batch process conditions.

29. The method according to any one of claims 1 to 28, further comprising separating the liquid phase from the continuous flow centrifuge and subjecting the secreted product therein to a purification procedure.

30. The method according to any one of claims 1 to 28, further comprising adding fresh culture medium to the culture vessel.

31. A method of producing a culture of mammalian cells having a cell density greater than or equal to 0.1% PCV, the method comprising: (a) placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel to produce a cell culture; (b) growing the cell culture to a cell density greater than or equal to 10% PCV; (c) subjecting the cell culture to a perfusion procedure during step (b), wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to produce a solid phase having a cell density ranging from about 1% to about 50% PCV; and (iii) returning a portion of the solid phase and a volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 0.1% PCV.

32. A method of producing a 12 A method for culturing a mammalian cell comprising: (a) placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of 80 L to produce a cell culture having a starting cell density greater than or equal to 1 million cells / mL; (b) growing the cell culture to a cell density greater than or equal to 10% PCV; (c) subjecting the cell culture to a perfusion procedure during step (b), wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge comprising a disposable disc stack rotor and comprising a centrifuge having a speed ranging from 1,000 to 200,000 m 2 σ factor; (ii) operating the continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV; and (iii) returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 60 million cells / mL.

33. A method of generating a 14 A method for culturing a mammalian cell comprising: (a) placing a plurality of mammalian cells and a volume of cell culture medium in a culture vessel having a working volume of 3,000 L to produce a cell culture having a starting cell density greater than or equal to 1 million cells / mL; (b) growing the cell culture to a cell density greater than or equal to 10% PCV; (c) subjecting the cell culture of step (b) to a perfusion procedure, wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge comprising a disposable disc stack rotor and comprising a centrifuge having a speed ranging from 1,000 to 200,000 m 2 σ factor; (ii) operating the continuous flow centrifuge to produce a solid phase having a cell density greater than or equal to 1% PCV; and (iii) returning at least a portion of the solid phase and a volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 to 6 VVD, wherein after completion of the perfusion procedure, the cell culture has a cell density greater than or equal to 60 million cells / mL.

34. A method for constructing a cell bank, comprising: (a) placing a plurality of mammalian cells and a volume of culture medium in a culture vessel to produce a cell culture; (b) growing the cell culture to a cell density greater than or equal to 1% packed cell volume (PCV); (c) subjecting the cell culture of step (b) to a perfusion procedure, wherein the perfusion procedure comprises: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to produce a solid phase (heavy phase); (iii) dividing the solid phase into a first portion which is returned to the cell culture vessel and a second portion, and (iv) returning the first portion to the cell culture chamber; (d) combining the second part with a cryopreservative.

35. The method of claim 34, wherein the second portion is frozen after step (d).

36. The method of claim 34, wherein the first portion of the solid phase is returned to the cell culture vessel to achieve a perfusion rate in the range of 0.7 to 6 vessel volumes per day (VVD).

37. The method of claim 34, wherein after completing the perfusion procedure, the cell culture has a cell density of 0.2% PCV or greater.

38. The method of claim 34, wherein the cryopreservative is dimethyl sulfoxide (DMSO) or growth medium supplemented with DMSO.

39. The method of claim 34, wherein the DMSO has a final concentration of 5% to 10% by volume (vol. / vol.).

40. The method of claim 34, wherein the second portion of the heavy phase has a cell density of or is adjusted to a cell density of at least 1 x 10 8 cells / ml (cells / mL).

41. The method of claim 34, wherein prior to adding the cryopreservative, the second portion of the heavy phase has or is adjusted to a cell density of at least, about, or no more than 1.0 x 10 8 cells / mL, 1.1x 10 8 cells / mL, 1.2x 10 8 cells / mL, 1.3x 10 8 cells / mL, 1.4x 10 8 cells / mL, 1.5x 10 8 cells / mL, 1.6x 10 8 cells / mL, 1.7x 10 8 cells / mL, 1.8x 10 8 cells / mL, 1.9x10 8 cells / mL or 2.0 x 10 8 cells / mL.

42. The method of claim 34, wherein the first portion of the solid phase comprises at most 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the total volume of the solid phase.

43. A method according to claim 42, wherein the second portion of the solid phase includes or substantially includes the remainder of the solid phase.

44. The method of claim 34, wherein during cell culture, the solid phase is discretely divided into the first part and the second part a plurality of times.

45. The method of claim 34, wherein the solid phase is separated into the first part and the second part once during cell culture.

46. ​​The method of claim 34, wherein during cell culture, the solid phase is continuously separated into the first part and the second part.

47. The method of claim 34, wherein the solid phase or the second portion of the solid phase has a cell viability of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% or 95%.

48. The method of any one of claims 1 to 47, wherein the perfusion program includes a temperature control element.

49. The method of claim 48, wherein the temperature control element is a passive temperature control.

50. The method of claim 48, wherein the temperature control element is an active temperature control.

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