Method for preparing mammalian cells for perfusion cell culture

Abstract

A method for preparing mammalian cells for a perfusion cell culture process that improves cell growth and productivity is provided. Also provided is a cell culture method that includes subjecting mammalian cells to one or more perfusion procedures, which includes generating a solid phase and a liquid phase with a continuous flow centrifuge and utilizing at least a portion of the solid phase to sustain, maintain, and / or initiate a new cell culture. Also provided are perfusion bioreactor systems and components thereof.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 345,796, filed May 25, 2022, the entire content of which is incorporated herein by reference.

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

Background Art

[0003] Despite significant advancements in the art over the past several decades, biomanufacturing processes, including the industrial-scale culture of mammalian cells and the production of products therein, still pose significant challenges. For example, typical bioreactor systems used in such processes must maintain a sterile closed environment, which poses problems with the addition of fresh cell culture medium and the removal of spent cell culture medium. Thus, cells grown under batch or fed-batch process conditions often face nutrient limitations when their cell culture medium becomes depleted of important components. Additionally, the accumulation of undesirable metabolic by-products in the cell culture can further limit growth and damage the desired product. Accordingly, there is still a strong need for improved cell culture systems and methods that can enhance growth and productivity by addressing these and other limitations.

[0004] Compared to bacterial cell cultures, mammalian cell cultures typically have lower production rates and result in lower production yields. Accordingly, a significant amount of research has 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 titer proteins. For example, it has been determined that restricted supply of glucose to mammalian cell cultures in a fed-batch process controls lactate production without requiring a constant rate supply of glucose (see, e.g., U.S. Patent Application Publication No. 20050070013).

[0005] Two cell culture processes, namely fed-batch processes and perfusion processes, are mainly used for large-scale protein production. The main objective of these methods is to add nutrients, such as glucose, when being consumed by the cells and to remove metabolic waste products, such as lactate and ammonia, when being produced. In a fed-batch process, the cells typically receive an inoculum medium containing glucose at the start of the culture and at one or more time points after the start but before the end of the culture. This approach can help control lactate production by the cultured cells at relatively low levels, but due to glucose-limiting conditions, maximum cell density, growth rate, and cell viability levels are not achieved. As a result, the number of cells and / or the amount of product produced by the cells is not maximized.

[0006] In a perfusion process, the cells also receive an inoculum basal medium, and when the cells reach the desired cell density, cell perfusion is initiated such that the spent medium is replaced with fresh medium. The perfusion process allows the culture to achieve higher cell density and thus enables the production of large amounts of cells and / or product. However, at larger industrially relevant production scales, the perfusion process requires very large amounts of fresh cell culture medium. Furthermore, any product secreted by the cells into the medium is lost when the spent medium is removed. This requires a separate recovery step to capture the product in the spent medium, or otherwise results in an overall efficiency loss if the spent medium is discarded.

[0007] Accordingly, there is a need for an improved method of large-scale cell culture that can maximize cell viability, cell concentration, and the amount of protein produced, and minimize product loss in the spent culture medium. The present disclosure addresses these and other needs. SUMMARY OF THE INVENTION

[0008] Disclosed herein is a cell culture method that includes performing a perfusion procedure on a cell culture using a continuous flow centrifuge to generate a solid phase and a liquid phase, and returning at least a portion of the solid phase and a fixed volume of cell culture medium to a culture vessel to sustain, maintain, and / or initiate a new cell culture. In some embodiments, the method can be used to achieve a perfusion rate in the range of from about 0.5 to up to about 6 vessel volumes per day (VVD) or more. The continuous flow centrifuge is used as part of the cell culture, i.e., as part of an "upstream" process for the production of a therapeutic polypeptide or protein such as, for example, an antibody or a fusion protein, as opposed to use of the continuous flow centrifuge as part of the clarification, as part of the "downstream" purification of such a therapeutic polypeptide or protein produced by the cell culture.

[0009] In a first aspect, provided herein is a method for culturing mammalian cells, such as mammalian cells engineered to produce a protein, e.g., a therapeutic protein such as an antibody, prior to clarification in the preparation of protein purification. That is, it is in a pre-production vessel, e.g., a bioreactor. In a specific embodiment, the method includes placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel to generate a cell culture. In another specific embodiment, the method includes culturing the cell culture to a cell density of greater than 1% packed cell volume (PCV). In a specific embodiment, the method includes performing a perfusion procedure on the cell culture. In a more specific embodiment, the perfusion procedure includes transferring at least a portion of the cell culture to a continuous flow centrifuge. In a more specific embodiment, the perfusion procedure includes operating the continuous flow centrifuge to generate a solid phase having a cell density of 1% PCV or greater. In a more specific embodiment, the perfusion procedure includes returning the solid phase and a fixed 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 a specific embodiment, 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 a specific embodiment, after completion of the perfusion procedure, the cell culture can have a cell density of 0.2% PCV or greater relative to, e.g., 0.2% to about 30% PCV.

[0011] In certain embodiments, the perfusion procedure includes increasing or decreasing the perfusion rate in a defined manner. In certain embodiments, the perfusion procedure includes increasing or decreasing the perfusion rate in a variable manner.

[0012] In certain embodiments, the perfusion procedure, or the period during which a cell culture method including such a perfusion procedure is carried out, ranges from 0.5 hours to a maximum of about 5 hours, such as about 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5 hours or more. In certain embodiments, the perfusion procedure, or the period during which a cell culture method including such a perfusion procedure is carried out, ranges from about 5 hours to a maximum of 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 perfusion procedure, or the period during which a cell culture method including such a perfusion procedure is carried out, ranges from about 1 day to a maximum of 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 including the perfusion procedure, is carried out for a maximum of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 days. Further, the perfusion procedure according to the above embodiments may be carried out in a semi - continuous, discontinuous or "interrupted" mode, and the perfusion procedure may be carried out, for example, once a day over several days.

[0013] In certain embodiments, the continuous - flow centrifuge comprises a disk 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 in the range of 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 is 1,000m 2 ~200,000m 2It may have a sigma value within a range. In certain embodiments, after completion of the centrifugation procedure, the cell culture may have a survival percentage of 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% or more. In certain embodiments, the cell culture may maintain a survival percentage of, for example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% over a period of, for example, 1 to 90 days, such as 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 cells include recombinant mammalian cells. In certain embodiments, the recombinant mammalian cells include recombinant Chinese hamster ovary (CHO) cells. In certain embodiments, the cell culture may have a lactate concentration of 4 g / L or less. In certain embodiments, the recombinant mammalian cells may produce a secreted product. In certain embodiments, the secreted 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 of 80 L or more. In certain embodiments, the culture vessel may have a total volume in the range of 100 L to 3000 L. In certain embodiments, the culture vessel may have a total volume of 100 L. In certain embodiments, the culture vessel may have a total volume of 3000 L. In certain embodiments, the culture vessel has a total volume in the range of 100 L to 30,000 L. In certain embodiments, the culture vessel has a total volume in the range of 5,000 L to 30,000 L. In certain embodiments, the culture vessel has a total volume in the range of 100 L to 6,000 L. In some embodiments, the method may include transferring at least a portion of the cell culture to a different culture vessel to initiate a second cell culture. In certain embodiments, the second cell culture has an initial cell density in the range of 0.1% or more to 10% PCV or less.In some embodiments, the method may include transferring at least a portion of the cell culture into a production culture vessel and initiating a production culture having an initial cell density in the range of 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 isolating the liquid phase from a continuous flow centrifuge and performing a purification procedure on the secreted product therein. In some embodiments, the method may include adding fresh culture medium (e.g., cell culture medium and / or cell culture medium) to the culture vessel.

[0014] In certain embodiments, the cell culture method provided herein includes generating a culture of mammalian cells having a cell density of 0.1% PCV or greater. In certain embodiments, the method includes placing a plurality of mammalian cells and a fixed volume of cell culture medium into a culture vessel to generate a cell culture. In certain embodiments, the method includes culturing the cell culture to a cell density of 10% PCV or greater, such as about 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or about 30%. In certain embodiments, the method includes performing a perfusion procedure on the cell culture. In certain embodiments, the perfusion procedure includes transferring at least a portion of the cell culture into a continuous flow centrifuge. In certain embodiments, the perfusion procedure includes operating the continuous flow centrifuge to generate 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. In a specific embodiment, the cell density is in the range of about 20% to about 40%. In a more specific embodiment, the cell density is in the range of about 30% to about 35%. In certain embodiments, the perfusion procedure includes returning a portion of the solid phase and a fixed 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 completion of the perfusion procedure, the cell culture density increases by more than 0.1% PCV, such as by 1% PCV or more or 10% PCV or more, or has increased. In a specific embodiment, the solid phase cell density is in the range of about 20% PCV to about 40% PCV.

[0015] In certain embodiments, the cell culture method provided herein includes generating a culture of mammalian cells comprising at least 4.8×10 12 cells. In a specific embodiment, the method includes placing a plurality of mammalian cells and a fixed volume of cell culture medium into a culture vessel having a working volume of at least 80 L, 80 L or less, or about 80 L to generate a cell culture having a starting cell density of 1 million cells / mL or greater. In a specific embodiment, the method includes culturing the cell culture to a cell density of 1% PCV or greater or 10% PCV or greater. In a specific embodiment, the method includes performing a perfusion procedure on the cell culture. 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 disk stack bowl and having a sigma factor in the range of 1,000 to 200,000 m 2 . In a specific embodiment, the method includes operating the continuous flow centrifuge to generate a solid phase having a cell density of 1% PCV or greater, such as about 10%, about 20%, about 30%, about 40% or about 50% PCV. In a specific embodiment, the method includes returning at least a portion of the solid phase and a fixed volume of 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 completion of the perfusion procedure, the cell culture has a cell density of 60 million cells / mL or greater.

[0016] In certain embodiments, provided herein is a method for generating an inoculum culture of mammalian cells comprising a total of at least 2.16×10 14 cells, or about 1.5 - 2.5×10 6 cells / mL. In a specific embodiment, the method includes inoculating a culture vessel with a cell density of 1 million cells / mL (1×10 6To produce a cell culture having a starting cell density of at least, at most, or about 3,000 L or 3,600 L working volume, a plurality of mammalian cells and a fixed volume of cell culture medium are placed in a culture vessel having a working volume of at least, at most, or about 3,000 L or 3,600 L. In a specific embodiment, the method includes culturing the cell culture to a cell density of 10% PCV or more. In a specific embodiment, the method includes performing a perfusion procedure on the cell culture. In a specific embodiment, the perfusion procedure transfers at least a portion of the cell culture to a continuous flow centrifuge further including a disposable disk stack bowl having a sigma factor in the range of 1,000 to 200,000 m 2 . In a specific embodiment of the method provided herein, the perfusion procedure includes operating the continuous flow centrifuge to produce a solid phase having a cell density of 1% PCV or more, such as up to about 10%, about 20%, about 30%, about 40% or about 50% PCV. In a particular embodiment, the perfusion procedure includes returning at least a portion of the solid phase and a fixed volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. In a particular embodiment, after completion of the perfusion procedure, the cell culture has a cell density of 60×10 6 cells / mL or more.

[0017] In certain embodiments, the cell culture produced by the methods described herein is part of a seed train in pre-production cell culture. For example, the cell culture produced by the methods described herein may be used to seed another larger bioreactor that constitutes a further step in pre-production cell culture. In a specific embodiment, the cells in the cell culture are concentrated using a centrifuge, such as a continuous flow centrifuge, and the resulting concentrated cells (i.e., the solid or heavy phase) are used to initiate another cell culture vessel, such as 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, the solid or heavy phase is used to initiate two, three, or more cell culture vessels, such as two or more, for example two, three, or more other cell cultures within a bioreactor. In certain specific embodiments, the heavy or solid phase is used to initiate two or three production cell cultures.

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

[0019]

Figure 1

[0020]

Figure 2

[0021]

Figure 3

[0022]

Figure 4

[0023]

Figure 5

[0024]

Figure 6

DETAILED DESCRIPTION OF THE INVENTION

[0025] The practice of the present disclosure, unless otherwise described, uses 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 described in documents such as ‘‘Molecular Cloning: A Laboratory Manual’’, second edition (Sambrook et al., 1989); ‘‘Oligonucleotide Synthesis’’ (M.J. Gait, ed., 1984); ‘‘Animal Cell Culture’’ (R.I. Freshney, ed., 1987); ‘‘Methods in Enzymology’’ (Academic Press, Inc.); ‘‘Current Protocols in Molecular Biology’’ (F.M. Ausubel et al., eds., 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] When a range of values is provided, unless otherwise indicated in context, each intervening value between the upper and lower limits of that range to one tenth of the unit of the lower limit, and any other stated value or intervening value within the stated range, is understood to be included in the described embodiments. It is also understood that the upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limits within the stated range. When the stated range includes one or both of the 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 in order to provide a more thorough understanding of the technologies described herein. However, it will be apparent to one of ordinary skill in the art that the technologies described herein may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the description of the various embodiments.

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

[0029] I. Definitions "Comprising" means that the listed elements are required in the composition / method / kit, but other elements may be included to form the composition / method / kit etc. within the scope of the claims.

[0030] "Consisting essentially of" means a limitation of the scope of a composition or method to specific materials or steps that do not substantially affect the basic and novel features (s) of the described embodiments.

[0031] "Consisting of" means excluding from the composition, method or kit any element, step or component not specified in the claims.

[0032] As used interchangeably herein, the terms "culture" and "cell culture" refer to a population of cells suspended in a cell culture medium under conditions suitable for the survival and / or growth of the cell population. As used herein, these terms can refer to a combination comprising a cell population (e.g., an animal cell culture) and the medium in which the population is suspended.

[0033] The terms "medium", "cell culture medium", and "culture medium" are used interchangeably herein and refer to a solution containing nutrients that nourish cells, such as mammalian cells, and can also refer to the medium in combination with the cells. The term "inoculation medium" refers to the medium used to generate an inoculated cell culture. The term "production medium" refers to the medium used to generate a production cell culture.

[0034] As used herein, the term "batch culture" refers to a method of culturing cells in which all of the components ultimately used for culturing the cells, including the cell culture medium and the cells themselves, are provided at the start of the culture process. A batch culture is typically stopped at a designated time point, and the cells and / or components in the medium are recovered and optionally purified.

[0035] As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture at some point after the start of the culture process. The components provided typically include nutritional supplements for cells that have become depleted during the culture process. A fed-batch culture is typically stopped at a designated time point, and the cells and / or components in the medium are recovered and optionally purified.

[0036] As used herein, the term "perfusion culture" refers to a method of culturing cells in which additional fresh medium is added continuously or intermittently over a period of time to the culture (after the start of the culture process), while at the same time spent medium is removed. The fresh medium typically provides nutrient supplementation to cells that have exhausted the existing nutrient supplementation during the culture process. Cell culture products such as proteins (e.g., antibodies) that may be present in the spent medium are optionally purified. Perfusion also enables the removal of unwanted cell waste (e.g., excess metabolites such as lactate) from the cell culture growing in the bioreactor. Perfusion culture can be integrated into a continuous flow bioprocess workflow for the production and / or purification of a given product.

[0037] As used herein, the term "perfusion rate" refers to the rate at which cell culture medium is removed and replaced with fresh cell culture medium (e.g., at a moderate exchange rate) for perfusion culture. In some embodiments, the perfusion rate is measured in vessel volume per day, or VVD. In some embodiments, the perfusion rate is measured in units of volume per unit time, e.g., liters per minute (LPM), etc.

[0038] As used herein, the terms "bioreactor" or "fermenter" or "vessel" or "culture vessel" refer 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 Volumes per Day (VVD)" is used herein to describe the cell culture perfusion flow rate, for example, the "vessel" can be a bioreactor, a single-use bioreactor, a fermenter or a culture vessel. A bioreactor can be of any size as long as it is useful for culturing cells. Typically, a bioreactor is 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 can be in volumes of 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 liters to 7,500 liters, 7,000 liters to 8,000 liters, 8,000 liters to 10,000 liters, 10,000 liters to 12,000 liters, 12,000 liters to 15,000 liters, 15,000 liters to 16,000, 16,000 liters to 18,000 liters, 18,000 liters to 20,000 liters, 20,000 liters to 22,000 liters, 20,000 liters to 24,000 liters, 24,000 liters to 26,000 liters, 26,000 liters to 28,000 liters, or 28,000 liters to 30,000 liters. Without limitation, the dissolved oxygen (dO. 2 ), pH, and temperature, agitation, foaming, and pressure, the internal conditions of the bioreactor, are typically controlled during the cultivation period.

[0039] As used interchangeably herein, the terms "inoculum culture" and "inoculated cell culture" refer to a cell culture used primarily to generate cell mass (i.e., increase the number of live cells in the culture) to reach a target cell density that can be used to initiate 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 in its entirety to a production bioreactor and combined with a production cell culture medium to initiate a production cell culture. In some embodiments, the inoculated cell culture can be transferred in its entirety to a larger inoculated cell culture bioreactor and combined with additional inoculated cell culture medium to initiate an inoculated cell culture having a larger volume. In some embodiments, a first portion of the inoculum culture can be transferred to a production bioreactor and combined with a production cell culture medium to initiate a production cell culture, and a second portion of the inoculum culture can be combined with an inoculum culture medium to initiate a new inoculated cell culture. Generally, one or more inoculated cell cultures grown prior to the initiation of a production cell culture are referred to as "pre-production cell cultures".

[0040] As used herein, the term "inoculation bioreactor" refers to a bioreactor used to contain an inoculated cell culture. Inoculation bioreactors are generally used for the production of cell mass and generally have a smaller volume than production bioreactors. The volume of a large-scale cell culture inoculation bioreactor is generally greater than about 100 mL, typically 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.

[0041] As used interchangeably herein, the terms "production culture" and "production cell culture" refer to cell cultures that are primarily used to generate a product and generally represent the last or final step of a cell culture process. In some embodiments, a production culture can be used to generate cells as a product. In some embodiments, a production culture can be used to generate a product within cells cultured in the production culture. In some embodiments, a production culture can be used to generate a product secreted by cells cultured in the production culture.

[0042] As used herein, the term "production bioreactor" refers to a bioreactor used to contain a production cell culture. Production bioreactors are generally used for the production of a desired cell or protein product. The volume of a large-scale cell culture production bioreactor is generally greater than about 100 mL, typically 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 production bioreactor can be 20 to 80 liters, 80 to 100 liters, 100 to 250 liters, 250 to 300 liters, 300 to 350 liters, 350 to 400 liters, 400 to 450 liters, 450 to 500 liters, 500 to 1,000 liters, 1000 to 2,000, 2000 to 2,500 liters, 2500 to 3,000 liters, 3000 to 3,500 liters, 3500 to 4,000 liters, 4000 to 4,500 liters, 4500 to 5,000 liters, 5000 to 7,500 liters, 7000 to 8,000 liters, 8000 to 10,000 liters, 10,000 to 12,000 liters, 12,000 to 15,000 liters, 15,000 to 16,000, 16,000 to 18,000 liters, 18,000 to 20,000 liters, 20,000 to 22,000 liters, 20,000 to 24,000 liters, 24,000 to 26,000 liters, 26,000 to 28,000 liters, or 28,000 to 30,000 liters in volume.

[0043] A suitable bioreactor (e.g., an inoculation bioreactor or a production bioreactor) is suitable for retaining cells suspended in a medium under desired culture conditions and can include (i.e., be composed of) any material (including glass, plastic, or metal) that promotes cell growth, maintenance of cell viability, and / or production of a product. Generally, the material(s) should not interfere with the expression or stability of products produced and / or secreted within the cultured cells, such as protein products. One of ordinary skill in the art will be able to readily select a bioreactor suitable for use in practicing the methods described herein and its operating conditions.

[0044] As used herein, the term "cell density" refers to the number of cells present in a given volume of medium. As used herein, the term "viable cell density" refers to the number of live (viable) cells present in a given volume of medium. Various sensors or probes may be incorporated into the cell culture system to directly measure the conditions within the cell culture vessel. For example, optical probes may be used to monitor cell growth, although they may not be able to distinguish between live and dead cells. Radio frequency impedance (RFI) probes may be used to monitor cell growth, and viable cell density may be determined by detecting live cells through capacitance measurements. Samples of cells may be obtained from the culture one or more times during cell culture, and the percent viability may be determined by use of spectroscopy, such as 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, and the like. The number of viable cells per milliliter can then be determined.

[0045] As used herein, the term "packed cell volume" or "PCV" refers to the ratio of the volume of a sample of cell culture medium occupied by packed cells after centrifugation of the sample of cell culture medium to the volume of the sample of cell culture medium. Similar to cell density, PCV may be determined using optical and RFI probes.

[0046] As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term as used herein also refers to the portion of cells that are surviving at a particular time point relative to the total number of live and dead cells in the culture at that time point. Cell growth may be monitored using radio frequency impedance (RFI) probes, and viable cell density may be determined by detecting live cells through capacitance measurements. Samples of cells may be obtained from the culture one or more times during cell culture, and the percentage viability may be determined by use of 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, and the like.

[0047] As used herein, the terms "polypeptide" and "polypeptide product" are synonymous with the terms "protein" and "protein product", respectively, and refer to at least one chain of amino acids linked via contiguous peptide bonds, as generally understood in the art. In certain embodiments, a "protein of interest" or "polypeptide of interest", etc., is a protein encoded by an exogenous nucleic acid molecule transformed into a host cell. In certain embodiments where the exogenous DNA molecule into which the host cell is transformed encodes a "protein of interest", the nucleic acid sequence of the exogenous DNA determines the amino acid sequence. In certain embodiments, the "protein of interest" is a protein encoded by a nucleic acid molecule endogenous to the host cell. In certain embodiments, the expression of such an endogenous protein of interest is altered, for example, by transfecting the host cell with an exogenous nucleic acid molecule that may contain one or more regulatory sequences and / or may encode a protein that enhances the expression of the protein of interest.

[0048] As used herein, the term "titer" refers to the total amount of a product of interest (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 the polypeptide of interest. Titers are typically expressed in units of grams or milligrams of polypeptide per milliliter or liter of medium.

[0049] An "isolated" product (e.g., an antibody) is one that has been identified, separated, and / or recovered from the components of its natural environment. The contaminants of its natural environment are substances that can interfere with the diagnostic and therapeutic uses of the product, and these can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the product is purified to (1) greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. An isolated product includes a product in situ within a recombinant cell since at least one component of the product's natural environment is absent. However, usually, an isolated product is prepared by at least one purification step.

[0050] As used interchangeably herein with respect to centrifuges, the terms "sigma value" and "sigma factor" refer to operating constants that represent the shape and speed of a centrifuge. The sigma factor can be used to compare two or more centrifuges having different sizes, shapes, and / or operating speeds.

[0051] As used herein in connection with the cell culture methods described herein, the term "solid phase" refers to the stream from a continuous centrifuge that contains the majority of the cells separated from the culture medium (hereinafter "liquid phase").

[0052] As used herein in connection with the cell culture methods described herein, the term "liquid phase" refers to the stream from a continuous centrifuge that contains primarily liquid, e.g., cell culture medium, and little or no cells.

[0053] When the terms "solid-to-liquid ratio" and "split ratio" are used interchangeably herein, they refer to the ratio of the volume of the solid phase to the volume of the liquid phase that are combined to sustain, maintain, and / or initiate a new cell culture. For example, a centrifuge may be adjusted to flow 50% of the centrifuge inlet stream to the solid outlet and 50% of the centrifuge inlet stream to the liquid outlet at a volumetric flow rate for a 50:50 solid-to-liquid split ratio. Similarly, 75% of the volumetric flow rate at the centrifuge inlet flows to the solid outlet, 25% of the centrifuge inlet flows through the liquid outlet at a 75:25 split ratio, 90% of the volumetric flow rate at the centrifuge inlet flows to the solid outlet, and 10% of the centrifuge inlet flows through the liquid outlet at a 90:10 split ratio.

[0054] As used herein, the term "residence time" means the time that cells are outside of the cell culture vessel, e.g., during perfusion centrifugation. The residence time includes the time that cells are within the centrifuge and the time that cells are passing between the cell culture vessel and the centrifuge. II. Detailed Description

[0055] Aspects of the present disclosure include methods of performing a perfusion procedure on a cell culture using a continuous flow centrifuge to generate a solid phase and a liquid phase, and returning at least a portion of the solid phase and a fixed volume of cell culture medium to a culture vessel to sustain, maintain, and / or initiate a new cell culture. In some embodiments, the method optionally includes returning a portion of the liquid phase of the original cell culture to the new cell culture.

[0056] Mammalian cell cultures generally consist of a growth phase in which cells grow to a target density (e.g., a target cell concentration or packed cell volume), and a production phase in which cell growth is low, but more product (e.g., a recombinant protein) is produced than was produced by the cell culture during the growth phase.

[0057] During the growth phase of the cell culture, the cells are first mixed with a medium (i.e., the inoculation medium) to form a cell culture. Typically, the cell culture medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements that the cells require for at least minimal growth and / or survival, although not limited thereto. The culture medium may also contain components that enhance growth and / or survival above the minimum rate, including hormones and growth factors. The medium is preferably formulated to have a pH and salt concentration optimal for cell survival and growth. In at least one embodiment, the culture medium is a defined culture medium. A defined medium is a medium in which all components have known chemical structures. In other embodiments, the medium may contain amino acids (s) derived from any source or method known in the art, including but not limited to single amino acid addition (s), or addition of peptone or protein hydrolysate (s) (including animal or plant source (s)), which may include amino acids (s) from such sources. In yet other embodiments, the culture medium used during cell growth may contain a concentrated medium, i.e., a medium that contains nutrients at a higher concentration than is normally required and is normally provided to the growth culture. One of ordinary skill in the art will recognize which cell media, inoculation media, etc. are appropriate for use with a cell culture of a particular cell type, e.g., animal cells (e.g., CHO cells), as well as agents designed to control the amounts of glucose and other nutrients (e.g., glutamine, iron, trace elements), or other culture variables that the medium should contain (e.g., amount of foaming, weight osmolarity) (see, e.g., Mather, J.P., et al. (1999) ‘‘Culture media, animal cells, large scale production,’’ Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, Vol. 2:777-85; U.S. Patent Application Publication No. 2006 / 0121568, each of which is incorporated herein by reference in its entirety). The embodiments described herein contemplate variants of such known media, including, for example, nutrient-enriched variants of such media.

[0058] One skilled in the art will also recognize at which temperature and / or concentration a particular cell line should be cultured. For example, most mammalian cells, such as CHO cells, grow well within the range of about 35°C to 39°C, preferably at 37°C, while insect cells are typically cultured at 27°C.

[0059] The methods according to embodiments of the present disclosure can use any suitable recombinant host cell, such as a prokaryotic host cell or a 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). Some mammalian cell lines are host cells suitable for the recombinant expression of polypeptides of interest. Examples of mammalian host cell lines include, but are not limited to, COS, PER.C6, TM4, VERO076, MDCK, BRL-3A, W138, Hep G2, MMT, MRC 5, FS4, CHO, 293T, A431, 3T3, CV-I, C3H10T1 / 2, Colo205, 293, HeLa, L cells, BHK, HL-60, FRhL-2, U937, HaK, Jurkat cells, Rat2, BaF3, 32D, FDCP-I, PC12, Mix, mouse myelomas (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 culture of primary tissues and primary explants. Any eukaryotic cell capable of expressing the product of interest may be used in connection with the embodiments described herein. A number of cell lines are available from commercial sources such as the American Type Culture Collection (ATCC). In various embodiments, the cell culture uses hybridoma cells. In various embodiments, the cell culture uses CHO cells.

[0060] In a specific embodiment, the cell culture method provided herein can be used to culture cells that produce any type of therapeutic polypeptide. In a particular embodiment, the therapeutic polypeptide is a fusion protein, such as a fusion protein comprising 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; bispecific F(mab) 2 antibody; single-domain bispecific diabody (scBsDb), single-chain bispecific tandem variable domain (scBsTaFv), trispecific NK cell engager (TriNKET), dual-affinity retargeting protein (DART), bispecific diabody, tandem diabody (TandAb), half-antibody (e.g., an antibody that includes an Fc portion but only one CH-VH:CL-VL pair), trifab contorsbody (described in International Publication No. WO 2019 / 086395); quadroma, scFv, dock-and-lock trivalent fab (DNL-(Fab) 3 , single-domain antibody, bispecific single-domain antibody, etc.

[0061] In a specific embodiment, the therapeutic polypeptide is atezolizumab, abagovomab, abciximab, abciximab, abciximab, abciximab, abciximab, abciximab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, alacizumab, alemtuzumab, alirocumab, altumomab pentate, amaciximab, amivantamab, anatumomab, andecaliximab, anetumab, anifrolumab, ansuvimab, anrukinzumab, apolizumab, aptumumab, ascrinvacumab, acelizumab, atezolizumab, atidortoxumab, atinumab, atorlivumab, atorlimumab, avabelumab, azintuxizumab, bamlanivimab, bapineuzumab, basiliximab, babiximab, beboterovimab, bectumomab, begeromab, belantamab, belimumab, bemarituzumab, benralizumab, bellirimatoxumab, belmekizumab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, bisirilumab, bimagrumab, bimekizumab, biltamab, bibatuzumab, breselumab, blinatumomab, blontuvetmab, brodalumab, brodalumab, brodalumab, bronchiquitumumab, brosimumab, cabiralizumab, camidanlumab, camrelizumab, canakinumab, canakinumab, canakinumab, caplacizumab, casirivimab, capromab, carlumab, carotuximab, catumaxomab, cedelizumab, semiprimab, selgantuzumab, certolizumab, cetrorelixumab, cetuximab, sibisatamab, silgabimab, siltuximab, sitaxizumab, sixatumumab, clazakizumab, clenoliximab, crizanlizumab, codrituzumab, cophixizumab, cortuximab, conatumumab, concizumab, cosfrovibiximab, crenezumab, crisantaspase, crotedumab, cusatuzumab, dacetuzumab, daclizumab, darolutamide, daratumumab, dectrekumab, demcizumab, denintuzumab, denosumab, depatuxizumab, deruxtecan, detumomab, desazumab, dinutuximab,Dinutuximab, Giridumab, Dmagrozumab, Dorlimomab, Dostarlimab, Dorzigotuzumab, Zolgotuzumab, Dupilumab, Dulvalumab, Ducesigumab, Dubortuxizumab, Ecromeximab, Eclizumab, Edobacumab, Edrecolomab, Efarizumab, Efungumab, Erdeltumab, Elezanumab, Elgemtuzumab, Erotuzumab, Elsirimumab, Emactuzumab, Emapalumab, Emibetuzumab, Emicizumab, Enapotamab, Enabatuzumab, Enfortumab, Enlimomab, Enoblituzumab, Enokizumab, Enotimab, Ensituximab, Epcoritamab, Epitumomab, Epratuzumab, Eptinezumab, Elenumab, Elirizumab, Eltuxomab, Etalizumab, Eteseveumab, Ethigilimab, Etorilizumab, Ebinaumab, Everolumab, Ekibivirmab, Fanolesomab, Faralimomab, Faricimab, Falretuzumab, Facinumab, Felvizumab, Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Filitumab, Flanbotuzumab, Fretikumab, Flotetuzumab, Fontrizumab, Foralumab, Forabilumab, Fleremab, Fresolimumab, Frovocimab, Flunevetumab, Fluranumab, Futsuximab, Galcanezumab, Galiximab, Gancotamab, Ganitumab, Gantenerumab, Gatipotuzumab, Gabirimomab, Gezibumab, Gemtuzumab, Geboxizumab, Gilvetumab, Gimcilumab, Girentuximab, Glembatumumab, Grofitamab, Gorimomab, Gomiriliximab, Goslanemab, Guselcumab, Ianalumab, Ibalizumab, Ibritumomab, Ikurkumab, Ifabotuzumab, Igobomab, Iladatuzumab, Imalumab, Imaprelimumab, Imisilomab, Imdevimab, Imgatuzumab, Inclacumab, Indatuximab, Indusatumab, Inebilizumab, Infliximab, Intertumumab, Inorlimomab, Inotuzumab, Ipilimumab, Iomab-B, Iratumumab, Isatuximab, Iskarimab, Istilatumab, Itrizumab, Ikezekizumab, Keligximab, Labetuzumab, Lacnotuzumab, Radilatumab, Lampalizumab, Ranadelumab, Landogrozumab, Laprituximab,Ralcabiximab, Lebrikizumab, Recanemab, Remaresomab, Rendilizumab, Renvelumab, Renzilumab, Redelimumab, Relonlimab, Resofabumab, Retrizumab, Rexatumumab, Ribivizumab, Riflazumab, Rigelizumab, Loncastuximab, Rosatuxizumab, Ritortumab, Linzumab, Lilimumab, Rodessizumab, Rokibizumab, Rolobizumab, Lucatumumab, Rulizumab, Lumiliximab, Rurezumab, Rupalizumab, Ruspatelcept, Rutiqizumab, Maftivimab, Mapatumumab, Margetuximab, Marstacimab, Maslimomab, Mabrilimumab, Matsuzumab, Mepolizumab, Metelimumab, Miratuximab, Minrezzumab, Militizumab, Milveximumab, Mitsumomab, Modotuximab, Mogamulizumab, Monalizumab, Morolimumab, Mosunizumab, Motavizumab, Moxatumumab, Muromonab-CD3, Nakoromab, Namilumab, Napumomab, Natalizumab, Navicixizumab, Navibumab, Nacitumab, Nebacumab, Necitumumab, Nemolizumab, Nerelemumab, Nesvacumab, Netakimab, Nimotuzumab, Nirsevimab, Nivolumab, Nofetumomab, Obinutuzumab, Okaratuzumab, Ocrelizumab, Odesivimab, Odrimomab, Ofatumumab, Olaratumab, Orelumab, Orendilizumab, Orokizumab, Omalizumab, Ombulatumab, Onartuzumab, Ontuxizumab, Onbatilimab, Opisimumab, Opurtuzumab, Oregovomab, Ortikumab, Oterixizumab, Otilimab, Otrexup, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Parvizumab, Pamrevlumab, Panitumumab, Pancumab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pacilizumab, Pidilizumab, Pinatuzumab, Pinimumab, Pralukast, Prezalumab, Prozartuzumab, Pogalizumab, Polatuzumab, Ponezumab, Porgabiximab, Prasinezumab,Presarizumab, Priliximab, Pritoxaximab, Pritumumab, Kilizumab, Racotumomab, Radretumab, Rafivirumab, Larapintuzumab, Ramucirumab, Ranevetmab, Ranibizumab, Laxibacumab, Rabagalimab, Rubrilizumab, Refanezumab, Legavilumab, Regdanvimab, Relatlimab, Remtulizumab, Reslizumab, Retifanlimab, Lirilumab, Linukumab, Risankizumab, Rituximab, Ribavizumab, Robatumumab, rmab, Lorelumab, Romilkimab, Romosozumab, Rontalizumab, Rossmantuzumab, Rovalpituzumab, Roberizumab, Rozanolixizumab, Rupilizumab, Sacituzumab, Samalizumab, Samlotamab, Sarilumab, Satralizumab, Satumomab, Secukinumab, Serclulimab, Serbanizumab, Setoxaximab, Setolusumab, Sevilumab, Sibrotuzumab, Sifalimumab, Silukizumab, Simtuzumab, Cypilizumab, Siltuximab, Silkumab, Sofituzumab, Soranebizumab, Solitomab, Sonepcizumab, Sonotuzumab, Sotrovimab, Spartalizumab, Specolimumab, Stumulumab, Suresomab, Sputabumab, Stimlimab, Subizumab, Subratoxumab, Tabalumab, Takatsuzumab, Tadocizumab, Tafasitamab, Taracotuzumab, Talisizumab, Talquetamab, Tamubetamab, Tanezumab, Tapritumumab, Tarextumab, Tabolumab, Teclistamab, Tefivazumab, Telimomab, Telisoztumab, Telisoztumab, Tenaatumomab, Teneliximab, Teprilizumab, Tepoditamab, Teprotumumab, Tescidolumab, Tetumomab, Tezepelumab, TGN1412, Chibulizumab, Childrakizumab, Chigatsuzumab, Chimiguzumab, Timolumab, Tiracolumab, Tirakozumab, Chisrelizumab, Chisotuzumab, Tixagevimab, TNX-650, Tocilizumab, Tomozotuximab, Tralizumab, Tosatoxumab, Tositumomab, Tobetumab, Tralokinumab, TrastuzumabTRBS07, trechalizumab, tremelimumab, treboglumab, tucotuzumab, tubirumab, ublituximab, urocuplumab, urelumab, ultuxizumab, ustekinumab, utomilumab, vadastuximab, banalizumab, bandrutuximab, banchikumab, banesimab, bapalizumab, barisacumab, baririlumab, baterilizumab, bedrizumab, bertuzumab, bepalimumab, besemumab, birovirumab, bisirilumab, bobarilizumab, borosikizumab, bonelirizumab, boplatelimab, borsetuzumab, botsumumab, bunakizumab, xantuzumab, XMAB-5574, zalutumumab, zanolimumab, zatsuximab, zenocutuzumab, zilalimumab, zolbetuximab, or zolimumab.

[0062] The construction of product-producing recombinant cells (e.g., hybridoma cells and CHO cells) is well known in the art. In some embodiments, the product can be a recombinant protein, such as a recombinant antibody including a multispecific antibody. In some embodiments, the product can be a secreted product secreted by the cells into the cell culture medium. In some embodiments, the product can be an intracellular product, which remains contained within the cells when produced by the cells, or remains contained within the cell wall, as in the case of certain production microorganisms such as certain bacteria. In some embodiments, the product can be a cell organelle or even the cell itself, as in the case of a cell therapy product.

[0063] The method according to the embodiment is from about 250,000 (×10 6 ) cells / mL to a maximum of about 25×10 6Starting an inoculated cell culture having an initial cell density in the range of cells / mL, for example, about 500,000, 750,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 10,500,000, 11,000,000, 11,500,000, 12,000,000, 12,500,000, 13,000,000, 13,500,000, 14,000,000, 14,500,000, 15,000,000, 15,500,000, 16,000,000, 16,500,000, 17,000,000, 17,500,000, 18,000,000, 18,500,000, 19,000,000, 19,500,000, 20,000,000, 20,500,000, 21,000,000, 21,500,000, 22,000,000, 22,500,000, 23,000,000, 23,500,000, 24 or 24,500,000 cells / mL. The methods according to various embodiments include starting an inoculated cell culture having an initial cell density in the range of from about 20×10 6 cells / mL to a maximum of about 25×10 6 cells / mL. The methods according to some embodiments include starting an inoculated cell culture having an initial density of about 22.5×10 6 cells / mL. In some embodiments, the method includes starting an inoculated cell culture having an initial cell density in the range of from about 500,000 to about 2,000,000 cells / mL. In some embodiments, the method includes starting an inoculated cell culture having an initial cell density in the range of from about 2,000,000 to about 5,000,000 cells / mL. In some embodiments, the method includes starting an inoculated cell culture having an initial cell density in the range of from about 5,000,000 to about 7,500,000 cells / mL. In some embodiments, the method includes starting an inoculated cell culture having an initial cell density in the range of from about 7,500,000 to about 10,000,000 cells / mL.

[0064] In certain embodiments, the methods of cell culture provided herein maintain at least 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% survival percentage throughout the execution of the cell culture process.

[0065] In some embodiments, the method includes initiating an inoculated cell culture having an initial cell density in the range of from about 0.1% packed cell volume (PCV) to 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 inoculated cell culture has an initial % PCV in the range of 0.1 - 0.5% PCV. In some embodiments, the inoculated cell culture has an initial % PCV in the range of 0.5 - 1%. In some embodiments, the inoculated cell culture has an initial % PCV in the range of 1 - 1.5%. In some embodiments, the inoculated cell culture has an initial % PCV in the range of 1.5 - 2%. In some embodiments, the inoculated cell culture has an initial % PCV in the range of 2 - 2.5%. In some embodiments, the inoculated cell culture has an initial % PCV in the range of 2.5 - 3%. In some embodiments, the inoculated cell culture has an initial % PCV in the range of 3 - 3.5%. In some embodiments, the inoculated cell culture has an initial % PCV in the range of 3.5 - 4%. In some embodiments, the method includes initiating a new cell culture having an initial cell density in the range of from about 0.1% packed cell volume (PCV) to 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.

[0066] The method according to the embodiments described herein includes growing the inoculated cell culture to a first target cell density as described above before performing a perfusion procedure on at least a portion of the inoculated cell culture. In some embodiments, the first target cell density is from about 50×10 6 cells / mL to a maximum of about 150×10 6 cells / mL, such as about 60, 70, 80, 90, 100, 110, 120, 130, or 140×10 6 cells / mL. In some embodiments, the method includes growing the inoculated cell culture to a target cell density in the range of from about 50 million to about 100 million cells / mL. In some embodiments, the method includes growing the inoculated cell culture to a target cell density in the range of from about 60 million to about 100 million cells / mL. In some embodiments, the method includes growing the inoculated cell culture to a target cell density in the range of from about 100×10 6 cells / mL to about 150×10 6 cells / mL.

[0067] In some embodiments, the method includes growing the inoculated cell culture, such as building a cell mass, or achieving a target cell density in the range of from about 10% packed cell volume (PCV) to a maximum of 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 method includes growing the inoculated cell culture to a target cell density in the range of from about 10 to about 15% PCV. In some embodiments, the method includes growing the inoculated cell culture to a target cell density in the range of from about 15 to about 20% PCV. In some embodiments, the method includes growing the inoculated cell culture to a target cell density in the range of from about 20 to about 25% PCV. In some embodiments, the method includes growing the inoculated cell culture to a target cell density in the range of from about 25 to about 30% PCV.

[0068] In certain embodiments, a cell culture produced by the methods described herein (e.g., an inoculated cell culture) is part of a seed train in pre-production cell culture. For example, a cell culture produced by the methods described herein may be used to seed another, larger bioreactor that constitutes a further step in 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., the solid or heavy phase) are used to initiate 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.

[0069] The cell culture methods described herein can be further used to initiate multiple downstream cell cultures such that cell masses can be constructed in parallel or such that product production can be carried out in parallel in multiple vessels. In a specific embodiment, for example, the solid or heavy phase is used to initiate two, three or more cell culture vessels, e.g., two or more, such as two, three or more other cell cultures within a bioreactor. Such other cell cultures can be, for example, additional (downstream) pre-production cell cultures or production cell cultures. In certain specific embodiments, the heavy or solid phase is used to initiate two or more pre-production cell cultures. In certain specific embodiments, the heavy or solid phase is used to initiate two or more production cell cultures. Such initiation can include collecting the heavy or solid phase after centrifugation is complete, dividing it among the number of pre-production or production cultures to be initiated, and initiating the cultures. Such initiation can also include collecting the heavy or solid phase continuously or discontinuously during centrifugation, dividing it among the number of pre-production or production cultures to be initiated during centrifugation, and initiating the cultures.

[0070] The methods according to the embodiments described herein range from about 0.25 (×10 6 ) cells / mL to a maximum of about 25×10 6cells / 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 24.5×10 6 starting a production cell culture having an initial cell density in the range of about 0.5×10 6 cells / mL to about 2×10 6 starting a production cell culture having an initial cell density in the range of about 2×10 6 cells / mL to about 5×10 6 starting a production cell culture having an initial cell density in the range of about 5×10 6 cells / mL to about 7.5×10 6 starting a production cell culture having an initial cell density in the range of about 7.5×10 6 cells / mL to about 10×10 6 starting a production cell culture having an initial cell density in the range of about 10×10 6 cells / mL to about 12.5×10 6 starting a production cell culture having an initial cell density in the range of about 12.5×10 6 cells / mL to about 15×10 6 starting a production cell culture having an initial cell density in the range of about 15×10 6 cells / mL to about 17.5×10 6 starting a production cell culture having an initial cell density in the range of about 17.5×10 6 cells / mL to about 20×10 6including initiating a production cell culture having an initial cell density in the range of cells / mL. In some embodiments, the method is about 20×10 6 cells / mL to about 22.5×10 6 including initiating a production cell culture having an initial cell density in the range of cells / mL. In some embodiments, the method is about 22.5×10 6 cells / mL to about 25×10 6 including initiating a production cell culture having an initial cell density in the range of cells / mL.

[0071] The method according to the embodiments described herein includes growing the production cell culture to a first target cell density as described above before performing a perfusion procedure on at least a portion of the production cell culture. In some embodiments, the first target cell density is about 50×10 6 cells / mL to up to about 150×10 6 cells / mL, such as in the range of about 60, 70, 80, 90, 100, 110, 120, 130, or 140×10 6 cells / mL. In some embodiments, the method includes growing the production cell culture to a target cell density in the range of about 50×10 6 cells / mL to about 100×10 6 cells / mL. In some embodiments, the method includes growing the production cell culture to a target cell density in the range of about 60×10 6 cells / mL to about 100×10 6 cells / mL. In some embodiments, the method includes growing the production cell culture to a target cell density in the range of about 100×10 6 cells / mL to about 150×10 6 cells / mL.

[0072] In some embodiments, the method includes constructing a cell mass or achieving a first target cell density in the range of about 10% packed cell volume (PCV) to 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 method includes growing a production cell culture to a target cell density in the range of about 10 to about 15% PCV. In some embodiments, the method includes growing a production cell culture to a target cell density in the range of about 15 to about 20% PCV. In some embodiments, the method includes growing a production cell culture to a target cell density in the range of about 20 to about 25% PCV. In some embodiments, the method includes growing a production cell culture to a target cell density in the range of about 25 to about 30% PCV.

[0073] Aspects of the technology described herein include performing a perfusion procedure on at least a portion of a cell culture (e.g., an inoculated cell culture or a production cell culture) using a continuous flow centrifuge. Continuous flow centrifuges are known in the art and are described, for example, in International Publication No. WO 2008 / 138345 of PCT Publication Number and International Publication No. WO 2020 / 229184 of PCT Publication Number, the disclosures of which are incorporated herein by reference in their entirety. Continuous flow centrifuges that can be used according to the embodiments described herein include, but are not limited to, disk stack centrifuges, tubular centrifuges, etc. In certain embodiments, the continuous flow centrifuge is configured for sterilization, which means that one or more components of the centrifuge (e.g., the bowl) can be subjected to a sterilization procedure to make it suitable for use under biomanufacturing conditions (e.g., in accordance with cGMP regulations). In certain embodiments, the continuous flow centrifuge includes disposable components such as a disposable bowl component. In such embodiments, the disposable component is configured to be easily separable from the remaining components of the device and can be easily discarded and / or replaced. A new disposable component can then be attached to the centrifuge. The new disposable component can be pre-sterilized or configured to be sterilized separately from or together with the remaining components of the centrifuge.

[0074] The continuous flow centrifuge according to the embodiments described herein is generally operated at a flow rate suitable for separating cells in the solid phase from the cell culture medium in the liquid phase. The flow rate affects not only the separation but also the residence time of the cells outside the production bioreactor. By increasing the flow rate, the residence time can be minimized while optimally enabling the intended separation of the cells.

[0075] The continuous flow centrifuge according to the embodiments described herein also generally comprises suitable inlet and outlet components to enable operable connection to one or more bioreactors, one or more cell culture media reservoirs, etc. Further, the continuous flow centrifuge according to the embodiments described herein can generally be configured for interoperability with standard cell culture manufacturing equipment such as pumps, controllers, flow meters, etc. The continuous flow centrifuge described herein generally operates at a flow rate suitable for cell separation in which separation is achieved between a solid phase (cells) and a liquid phase (cell culture medium). In certain embodiments, the suitable inlet and outlet components are designed to minimize the residence time of cells outside of the system volume, and thus the cell culture vessel bioreactor, e.g., the production bioreactor. In certain embodiments, the volume of the centrifuge system (including inlet and outlet components) can be achieved by minimizing the length and / or diameter of the piping, tubing or tubing flow kit while still enabling the intended flow rate of the system.

[0076] The continuous flow centrifuge according to the embodiments described herein may also generally include temperature control elements for providing suitable conditions for the material exiting the production bioreactor and entering the centrifuge system, the solid phase exiting the system prior to returning to the production bioreactor, and the liquid phase exiting the system prior to further downstream processing during centrifugation within the system. The temperature control elements may be controlled passively or actively. In one embodiment, the temperature control is passive. Here, the piping, tubing, or tubing flow kit may be (optionally) insulated or non-insulated to allow for passive temperature control to ambient temperature or to a reduced temperature present at the operating point. For example, cooling water (about 4°C to 10°C) may be used to cool the water, thereby cooling the centrifuge seal and minimizing the temperature rise due to the rotation of the centrifuge. In a specific embodiment, the ambient temperature is reduced to minimize the temperature rise from frictional forces at high bowl speeds for cell separation. The piping, tubing, or tubing flow kit may be insulated or non-insulated and allow for passive temperature control via the ambient temperature at the operating point.

[0077] In other embodiments, temperature control is active. For example, the piping, tubing or tubing flow kit can be provided to a heat exchanger that enables active temperature control to a specific temperature set point. The centrifuge can have temperature control of the centrifuge bowl to minimize the temperature rise from the frictional force at the high bowl speeds required for cell separation. The centrifuge can have temperature control of the mechanical seal to minimize the temperature rise from the frictional force at the high bowl speeds required for cell separation. Further, the temperature control of the centrifuge bowl and the mechanical seal may be provided as ambient temperature or temperature reduction to minimize the temperature rise during processing.

[0078] In certain embodiments of any of the methods disclosed herein, the residence time of the cells and cell culture medium is minimized such that the viability of the cells in the cell culture is maintained or does not substantially decrease. In certain embodiments, the residence time is less than 3 minutes, 2 minutes or 1 minute. In certain embodiments, the residence time is shortened by increasing the flow rate of the culture medium and cells from the culture vessel to centrifugation and back to the culture vessel. In other embodiments, the residence time is shortened by use of a larger conduit, such as tubing, between the culture vessel and the centrifuge, use of a larger centrifuge, etc. In certain embodiments, a particular residence time results in a loss of 3%, 2%, 1% or 0.5% or less of the cell viability during culture.

[0079] 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, during the residence time, the temperature is maintained such that any temperature transient is 4°C or less, 3°C or less, 2°C or less, or 1°C or less during the residence time as compared to the temperature of the medium within 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 a heat exchanger, for example, while the cell culture medium passes between the culture vessel and the centrifuge, between the centrifuge and the cell culture vessel, or both. In FIG. 1, for example, the temperature of the cell culture medium can be maintained such that any temperature transient while returning from the cell culture vessel 120 through the sterile connection 103, the cell separation device 130, the solid phase outlet 106, and the sterile connection 105 back to the cell culture vessel 120 is 4°C or less, 3°C or less, 2°C or less, or 1°C or less. In specific embodiments, the cell culture medium is maintained at a temperature of about 30°C to about 39°C, such as 31°C to 38°C, 32°C to 38°C, 33°C to 38°C, 34°C to 38°C, 35°C to 38°C, or 36°C to 38°C during the residence time. In certain embodiments, a centrifuge, such as a continuous centrifuge, such as a single-use centrifuge, includes a water cooling system that reduces heat addition to the cell culture medium within the centrifuge, such as heat addition during sealing.

[0080] In some embodiments, a continuous flow centrifuge can be characterized by a sigma factor or sigma value that refers to an operating constant representing the shape and speed of the centrifuge. The sigma factor can be used to compare two or more centrifuges having different sizes, geometries, and / or operating speeds, such 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 the technology generally has a sigma value of about 1,000 m 2 ~ about 200,000 m 2, for example, 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, 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 180,000 m 2 has a sigma factor in the range of. The continuous flow centrifuge according to embodiments of the technology described herein is generally configured to achieve an operating speed in the range of about 3,000 to about 10,000 RPM, for example, 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 single-use continuous flow centrifuges can operate at speeds in the range of about 3,000 RPM to about 6,000 RPM.

[0081] In use, the 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 inoculated cell culture or a production cell culture) and separate the received portion of the cell culture into a solid phase that includes all or a substantial portion of the cells of the received portion and a liquid phase that mostly includes the cell culture medium. In certain embodiments, the solid phase can have a final cell density in the range of about 1% PCV to about 10%, about 20%, about 30%, about 40% or about 50% PCV.

[0082] 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 part thereof, and optionally a part of the liquid phase, to a new bioreactor (or the original bioreactor) in order to sustain, maintain and / or initiate a new cell culture. In addition to the solid phase, and optionally a part of the liquid phase, an appropriate amount of fresh cell culture medium can be combined with the solid phase (and optionally a part of the liquid phase) to achieve the desired initial cell density for the new cell culture. In some embodiments, the initial cell density is about 0.25×10 6 cells / mL to a maximum of about 25×10 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 24.5×10 6 cells / mL. In some embodiments, the method includes initiating a new cell culture having an initial cell density in the range of about 0.5×10 6 cells / mL to about 2×10 6 cells / mL. In some embodiments, the method includes initiating a new cell culture having an initial cell density in the range of about 2×10 6 cells / mL to about 5×10 6 cells / mL. In some embodiments, the method includes initiating a new cell culture having an initial cell density in the range of about 5×10 6 cells / mL to about 7.5×10 6 cells / mL. In some embodiments, the method includes initiating a new cell culture having an initial cell density in the range of about 7.5×10 6 cells / mL to about 10×10 6 cells / mL. In some embodiments, the method includes initiating a new cell culture having an initial cell density in the range of about 10×10 6 cells / mL to about 12.5×10 6including starting a production cell culture having an initial cell density in the range of cells / mL. In some embodiments, the method is about 12.5×10 6 cells / mL to about 15×10 6 including starting a production cell culture having an initial cell density in the range of cells / mL. In some embodiments, the method is about 15 to about 17.5×10 6 including starting a production cell culture having an initial cell density in the range of cells / mL. In some embodiments, the method is about 17.5×10 6 cells / mL to about 20×10 6 including starting a production cell culture having an initial cell density in the range of cells / mL. In some embodiments, the method is about 20×10 6 cells / mL to about 22.5×10 6 including starting a production cell culture having an initial cell density in the range of cells / mL. In some embodiments, the method is about 22.5×10 6 cells / mL to about 25×10 6 including starting a production cell culture having an initial cell density in the range of cells / mL.

[0083] In some embodiments, the method includes initiating a new cell culture having an initial cell density in the range of from about 0.1% packed cell volume (PCV) to 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. In some embodiments, the new cell culture has an initial % PCV in the range of 0.1 - 1%. In some embodiments, the new cell culture has an initial % PCV in the range of 1.1 - 2%. In some embodiments, the new cell culture has an initial % PCV in the range of 2.1 - 3%. In some embodiments, the new cell culture has an initial % PCV in the range of 3.1 - 4%. In some embodiments, the new cell culture has an initial % PCV in the range of 4.1 - 5%. In some embodiments, the new cell culture has an initial % PCV in the range of 5.1 - 6%. In some embodiments, the new cell culture has an initial % PCV in the range of 6.1 - 7%. In some embodiments, the new cell culture has an initial % PCV in the range of 7.1 - 8%. In some embodiments, the new cell culture has an initial % PCV in the range of 8.1 - 9%. In some embodiments, the new cell culture has an initial % PCV in the range of 9.1 - 10%.

[0084] The method according to embodiments of the technology described herein may include growing a new cell culture to a target cell density before performing a perfusion procedure on at least a portion of the cell culture. In some embodiments, the target cell density is about 50 million (×10 6) It ranges from about 100,000 cells / mL to a maximum of about 150 million cells / mL, such as about 6 million, 70 million, 80 million, 9 million, 100 million, 110 million, 120 million, 130 million or 140 million cells / mL. In some embodiments, the method includes growing a new cell culture to a target cell density in the range of about 5 million to about 100 million cells / mL. In some embodiments, the method includes growing a new cell culture to a target cell density in the range of about 60 million to about 100 million cells / mL. In some embodiments, the method includes growing a new cell culture to a target cell density in the range of about 100 million to about 150 million cells / mL.

[0085] In some embodiments, the method includes growing a new cell culture, constructing a cell mass, or achieving a target cell density in the range of about 10% packed cell volume (PCV) to a maximum of 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 method includes growing a new cell culture to a target cell density in the range of about 10 to about 15% PCV. In some embodiments, the method includes growing a new cell culture to a target cell density in the range of about 15 to about 20% PCV. In some embodiments, the method includes growing a new cell culture to a target cell density in the range of about 20 to about 25% PCV. In some embodiments, the method includes growing a new cell culture to a target cell density in the range of about 25 to about 30% PCV.

[0086] Using the various methods described herein, a desired perfusion rate for cell culture can be achieved. The rate of perfusion can be any rate appropriate for the cell culture. For example, the rate of perfusion can range from about 0.5 vessel volume per 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 from 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.

[0087] 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 rate of perfusion can remain constant over a period of time, or can vary (i.e., increase or decrease) over the period of perfusion, or can be any combination thereof. Further, an increase or decrease in the perfusion rate can be applied in any manner known in the art, including a stable change over time, such as a stable increase during the perfusion period, or a series of changes over time, such as a series of stable changes, a series of stepwise changes (e.g., the perfusion rate can be increased or decreased stepwise), or any combination thereof. Perfusion can be applied continuously or intermittently, as described above. The timing of the start and stop of the perfusion period(s), and the timing of any change in perfusion, can be predetermined, for example, at set times or intervals, or based on monitoring of some parameters or criteria of the cell culture.

[0088] The perfusion procedure according to the embodiments of the present disclosure can be carried out over a period that can range from several hours to several days. For example, in some embodiments, the period during which the perfusion procedure, or a cell culture method including such a perfusion procedure, is carried out is in the range from 0.5 hour to a maximum of about 5 hours, such as about 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5 hours or more. In some embodiments, the period during which the perfusion procedure, or a cell culture method including such a perfusion procedure, is carried out is in the range from about 5 hours to a maximum of 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 period during which the perfusion procedure, or a cell culture method including such a perfusion procedure, is carried out is in the range from about 1 day to a maximum of 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 a cell culture method including the perfusion procedure, is carried out for a maximum of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 days. Further, the perfusion procedure according to the above embodiments may be performed in a semi - continuous, discontinuous or "interrupted" mode, and the perfusion procedure is, for example, carried out once a day over several days.

[0089] The methods and systems according to the embodiments described herein can be designed to maintain a target cell viability throughout the perfusion procedure such that the cell culture can maintain a cell viability above a desired target value. In some embodiments, the method results in a cell culture viability of 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% or more throughout the period of the perfusion procedure.

[0090] Methods and systems according to embodiments of the technology described herein can be designed to maintain a target cell viability over the period of a cell culture such that the cell culture can maintain a cell viability above a desired target value over the entire period of the culture. In some embodiments, the method results in a cell culture viability of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more over the duration of the entire cell culture.

[0091] Methods and systems according to embodiments of the technology described herein can be designed to maintain a target concentration of cell culture waste or by-products below a target level. For example, in some embodiments, the method results in a cell culture lactate (lactate) concentration of 4 g / L or less, such as 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 or less over the period of a perfusion procedure. In some embodiments, the method results in a cell culture ammonium concentration of 4 mM or less, such as 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 or less over the period of a perfusion treatment. In some embodiments, the method results in a cell culture pCO 2 concentration of 150 mmHg or less, such as 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 mmHg or less over the period of a perfusion procedure.

[0092] In some embodiments, the methods and systems described herein are configured for industrial-scale use at cell culture bioreactor volumes of 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 of 80 L or more. In some embodiments, the cell culture bioreactor has a total volume in the range of 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.

[0093] Aspects of the technology described may include methods that involve transferring at least a portion of a first cell culture from one bioreactor to another bioreactor to initiate a second cell culture. In certain embodiments, the method includes combining a desired volume of solid phase discharged from a continuous flow centrifuge with a desired volume of fresh cell culture medium to sustain, maintain and / or initiate 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.

[0094] In some embodiments, where the new cell culture is an inoculated cell culture, the method further comprises culturing the new inoculated cell culture for a period of time to generate a target cell density. In some embodiments, the new inoculated cell culture is cultured for 1 to 7 days, such as 2, 3, 4, 5, or 6 days.

[0095] In some embodiments, where the new cell culture is a production cell culture, the method further comprises culturing the production cell culture under batch or fed-batch process conditions. In some embodiments, the method further comprises performing a recovery procedure on the cell culture to separate the cells from the cell culture medium at a given time point, and performing a purification procedure on the recovered harvested cell culture fluid (HCCF). In some embodiments, the method comprises culturing the production culture for 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 performing the recovery procedure.

[0096] In a preferred embodiment, a method for culturing mammalian cells comprises placing a plurality of mammalian cells and a certain amount of cell culture medium into a culture vessel to generate a cell culture; culturing the cell culture to a cell density of 1% packed cell volume (PCV) or more; performing a perfusion procedure on the cell culture, the perfusion procedure including transferring at least a portion of the cell culture to a continuous flow centrifuge; operating the continuous flow centrifuge to generate a solid phase having a final cell density of 1% PCV or more, such as about 10%, about 20%, about 30%, about 40% or about 50% PCV; returning the solid phase and a certain 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. After completion of the perfusion procedure, the cell culture has a cell density of 0.2% PCV or more. In some embodiments, the perfusion rate is in the range of 2 to 4 VVD. In some embodiments, the perfusion rate can be in the range of 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.

[0097] In a preferred embodiment, the method comprises generating a culture of mammalian cells having a cell density of 1% PCV or greater, the method comprising placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel to generate a cell culture, culturing the cell culture to a cell density in the range of 10% - 30% PCV, such as 10% - 15% PCV, 15% - 20% PCV, 20% - 25% PCV, or 25% - 30% PCV, performing a perfusion procedure on the cell culture, the perfusion procedure comprising transferring at least a portion of the cell culture to a continuous flow centrifuge, operating the continuous flow centrifuge to generate a solid phase having a cell density greater than the cell density % PCV of the cell culture, for example, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% greater than the cell density of the cell culture, for example, 15% - 20% PCV, 20% - 25% PCV, or 25% - 30% PCV, 30% - 35% PCV, 35% - 40% PCV or greater, or greater than 40% PCV, returning at least a portion of the solid phase and a fixed volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 - 6 vessel volumes per day (VVD), such as 1, 2, 3, 4, or 5 VVD. After completion of the perfusion procedure, the cell culture has a cell density of 0.2% PCV or greater, such as 0.2% - about 30% PCV. Preferably, the perfusion rate is 3.5 - 6 VVD. More preferably, the perfusion rate is 5 - 6 VVD.

[0098] In one embodiment, the method comprises generating a culture of mammalian cells comprising at least or about 4.8×10 12 cells, the method comprising placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel having a working volume of 80 L to generate a cell culture having a starting cell density of 1 million cells / mL or greater, culturing the cell culture to a cell density of 10% PCV or greater, performing a perfusion procedure on the cell culture, the perfusion procedure comprising transferring at least a portion of the cell culture to a 10,000 - 200,000 m 2Transfer the cell culture to a continuous flow centrifuge having a sigma factor in the range of, perform a perfusion procedure on the cell culture, operate the continuous flow centrifuge to generate 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 return at least a portion of the solid phase and a fixed volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. After completion of the perfusion procedure, the cell culture has 60×10 6 cells / mL or higher cell density. In a specific embodiment, the solid phase cell density is in the range of about 20% to about 40%. In a more specific embodiment, the cell density is in the range of about 30% to about 35%.

[0099] In another embodiment, a method comprising generating a culture of mammalian cells containing at least 1.80×10 14 cells, comprising placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel with a working volume of 3000 L to generate a cell culture having a starting cell density of 1×10 6 cells / mL or higher, culturing the cell culture to a cell density of 10% PCV or higher, and performing a perfusion procedure on the cell culture, the perfusion procedure comprising transferring at least a portion of the cell culture to a continuous flow centrifuge having a sigma factor in the range of 10,000 to 200,000 m 2 performing a perfusion procedure on the cell culture, operating the continuous flow centrifuge to generate 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 fixed volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. After completion of the perfusion procedure, the cell culture has 60×10 6 cells / mL or higher cell density. In a specific embodiment, the solid phase cell density is in the range of about 20% to about 40%. In a more specific embodiment, the cell density is in the range of about 30% to about 35%.

[0100] In a preferred embodiment, the method is at least 2.16×10 14generating a culture of mammalian cells comprising individual cells, the method comprising placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel with a working volume of 3600 L to generate a cell culture having a starting cell density of 1 million cells / mL or more; culturing the cell culture to a cell density of 10% PCV or more; and performing a perfusion procedure on the cell culture, the perfusion procedure comprising transferring at least a portion of the cell culture to a continuous flow centrifuge having a sigma factor in the range of 10,000 to 200,000 m 2 transferring at least a portion of the cell culture to a continuous flow centrifuge having a sigma factor in the range of 10,000 to 200,000 m; operating the continuous flow centrifuge to generate 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 fixed volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. After completion of the perfusion procedure, the cell culture has a cell density of 60 million cells / mL or more. In a specific embodiment, the solid phase cell density is in the range of about 20% to about 40%. In a more specific embodiment, the cell density is in the range of about 30% to about 35%.

[0101] Accordingly, exemplary embodiments of a method for culturing mammalian cells are provided herein.

[0102] Embodiment 1: A method for culturing mammalian cells, comprising: (a) placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel to generate a cell culture; (b) culturing the cell culture to a cell density of 1% packed cell volume (PCV) or more; (c) during step (b), performing a perfusion procedure on the cell culture, the perfusion procedure comprising: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to generate a solid phase having a cell density of 1% PCV or more, such as about 10%, about 20%, about 30%, about 40% or about 50% PCV; (iii) returning the solid phase and a fixed 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), and after completion of the perfusion procedure (e.g., after 7 to 8 days), the cell culture having a cell density of 0.2% PCV or more, such as 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, and performing a perfusion procedure on the cell culture during step (b). Embodiment 2: The method according to Embodiment 1, wherein the perfusion rate is in the range of 2 to 4 VVD. Embodiment 3: The method according to Embodiment 1 or 2, wherein the perfusion procedure includes increasing or decreasing the perfusion rate in a constant pattern. Embodiment 4: The method according to Embodiment 1 or 2, wherein the perfusion procedure includes increasing or decreasing the perfusion rate in a variable pattern. Embodiment 5: The method according to any one of Embodiments 1 to 4, wherein the perfusion procedure is continuously performed over a period in the range of 1 to 7 days. Embodiment 6: The method according to any one of Embodiments 1 to 4, wherein the perfusion procedure is semi - continuously performed over a period in the range of 1 to 7 days.Embodiment 7: The method according to any one of Embodiments 1 to 6, wherein the continuous flow centrifuge comprises a disc stack bowl. Embodiment 8: The method according to any one of Embodiments 1 to 6, wherein the continuous flow centrifuge comprises a tubular bowl. Embodiment 9: The method according to any one of Embodiments 1 to 8, wherein the continuous flow centrifuge has an operating speed in the range of 3,000 to 10,000 RPM. Embodiment 10: The method according to any one of Embodiments 1 to 9, wherein the continuous flow centrifuge comprises sterilizable components. Embodiment 11: The method according to any one of Embodiments 1 to 10, wherein the continuous flow centrifuge comprises disposable components. Embodiment 12: The continuous flow centrifuge is 1,000 m. 2 ~200,000 m 2The method according to any one of Embodiments 1 to 11, having a sigma value within the range. Embodiment 13: After completion of the centrifugation procedure, the cell culture has a survival percentage of 80% or more, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, the method according to any one of Embodiments 1 to 12. Embodiment 14: The method according to any one of Embodiments 5 to 13, wherein the cell culture maintains a survival percentage of 85% or more over a period of 1 to 7 days. Embodiment 15: The method according to any one of Embodiments 1 to 14, wherein the cell culture medium is maintained at a temperature of about 30°C to about 39°C, such as 31°C to 38°C, 32°C to 38°C, 33°C to 38°C, 34°C to 38°C, 35°C to 38°C or 36°C to 38°C during the residence time. Embodiment 16: The method according to any one of Embodiments 1 to 15, wherein the mammalian cells include recombinant mammalian cells. Embodiment 17: The method according to Embodiment 16, wherein the recombinant mammalian cells include recombinant Chinese hamster ovary (CHO) cells. Embodiment 18: The method according to Embodiment 17, wherein after completion of the centrifugation procedure, the cell culture has a lactate concentration of 4 g / L or less. Embodiment 19: The method according to any one of Embodiments 16 to 18, wherein the recombinant mammalian cells produce a secreted product. Embodiment 20: The method according to Embodiment 16, wherein the secreted product includes a recombinant protein. Embodiment 22: The method according to 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 of 80 L or more. 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 initiate 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 according to any one of embodiments 1 to 28, further comprising transferring at least a part of the cell culture to a production culture vessel and starting 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 medium to the culture vessel.

[0103] Embodiment 33: A method for generating a culture of mammalian cells having a cell density of 0.1% PCV or more, comprising: (a) placing a plurality of mammalian cells and a fixed volume of cell culture medium in a culture vessel to generate a cell culture; (b) culturing the cell culture to a cell density of 10% PCV or more; and (c) performing a perfusion procedure on the cell culture during step (b), the perfusion procedure comprising: (i) transferring at least a part of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to generate 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 (iii) returning a part of the solid phase and a fixed volume of the cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD, and performing a perfusion procedure on the cell culture during step (b), after completion of the perfusion procedure, the cell culture having a cell density of 0.1% PCV or more. In a particular embodiment, after completion of the perfusion procedure, the cell culture has a cell density that increases by 0.1% PCV or more, for example 0.1% to about 30% PCV, or is increasing. In a specific embodiment, the solid phase cell density is in the range of about 20% to about 40%. In a more specific embodiment, the cell density is in the range of about 30% to about 35%.

[0104] Embodiment 34: At least 4.8×10 12A method for generating a culture of mammalian cells containing a number of cells, comprising: (a) placing a plurality of mammalian cells and a fixed volume of cell culture medium into a culture vessel having a working volume of 80 L to generate a cell culture having a starting cell density of 1×10 6 cells / mL or higher; (b) culturing the cell culture to a cell density of 10% PCV or higher; and (c) performing a perfusion procedure on the cell culture during step (b), the perfusion procedure comprising: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge equipped with a disposable disc stack bowl and having a sigma factor in the range of 1,000 to 200,000 m 2 ; (ii) operating the continuous flow centrifuge to generate a solid phase having a cell density of 1% PCV or higher; and (iii) returning at least a portion of the solid phase and a fixed volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD, and after completion of the perfusion procedure, the cell culture having a cell density of 60×10 6 cells / mL or higher, and performing a perfusion procedure on the cell culture during step (b).

[0105] Embodiment 35: A method for generating a culture of mammalian cells containing at least 2.16×10 14 cells, comprising: (a) placing a plurality of mammalian cells and a fixed volume of cell culture medium into a culture vessel having a working volume of 3,000 L to generate a cell culture having a starting cell density of 1 10 6 cells / mL or higher; (b) culturing the cell culture to a cell density of 10% PCV or higher; and (c) performing a perfusion procedure on the cell culture, the perfusion procedure comprising: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge equipped with a disposable disc stack bowl and having a sigma factor in the range of 1,000 to 200,000 m 2transferring to a continuous flow centrifuge containing a sigma coefficient in the range of, (ii) operating the continuous flow centrifuge to produce a solid phase having a cell density of 1% PCV or more, and (iii) returning at least a portion of the solid phase and a fixed volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. After completion of the perfusion procedure, the cell culture has 60×10 6 performing a perfusion procedure on the cell culture having a cell density of cells / mL or more.

[0106] Cell banking and / or expansion of seed train or pre-production cell volume: In certain embodiments, the cell culture methods disclosed herein may include or be used for cell banking. In one aspect, for example, a method of cell banking comprising: (a) placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel to generate a cell culture; (b) culturing the cell culture to a cell density of 1% packed cell volume (PCV) or more; (c) performing a perfusion procedure on the cell culture of step (b), the perfusion procedure comprising: (i) transferring at least a portion of the cell culture to a continuous flow centrifuge; (ii) operating the continuous flow centrifuge to generate a solid phase (heavy phase); (iii) dividing the solid phase into a first portion and a second portion that are returned to the cell culture vessel; (iv) returning the first portion to the cell culture vessel (bioreactor); and (d) combining the second portion with a cryopreservative. Also provided herein is a method comprising performing the perfusion procedure on the cell culture of step (b). In a specific embodiment, the second portion is frozen after step (d). In a specific embodiment, the first portion of the solid phase is returned to the cell culture vessel together with a fixed volume of cell culture medium. In a more specific embodiment, returning the solid phase and the first portion of the cell culture medium to the cell culture vessel achieves a perfusion rate in the range of 0.7 to 6 vessel volumes per day (VVD). In a more specific embodiment, after completion of the perfusion procedure, the cell culture has a cell density of 0.2% PCV or more, for example 0.2% to about 30% PCV or more. 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% (volume / volume). In certain embodiments, the second portion of the heavy phase has a cell density of at least 1×10 8 cells / milliliter (cells / mL) or is adjusted to this cell density. In a more specific embodiment, the second portion of the heavy phase has at least 1.0×10 8 cells / mL, 1.1×10 8 cells / mL, 1.2×10 8 cells / mL, 1.3×10 8 cells / mL, 1.4×108 cells / mL, 1.5×10 8 cells / mL, 1.6×10 8 cells / mL, 1.7×10 8 cells / mL, 1.8×10 8 cells / mL, 1.9×10 8 cells / mL, 2.0×10 8 The cell density of cells / mL, approximately this cell density, or has a cell density equal to or lower than this, or is adjusted to this cell density. In certain embodiments, the first portion of the solid phase comprises 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, the first portion of the solid phase comprises up to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or up to 50% of the total volume of the solid phase, and the second portion of the solid phase comprises or substantially comprises the remainder of the solid phase. In certain embodiments, the solid phase is divided into the first portion and the second portion at a plurality of discrete times during cell culture. In certain embodiments, the solid phase is divided into the first portion and the second portion at one time during cell culture. In certain embodiments, the solid phase is continuously divided into the first portion and the second portion during cell culture. In certain embodiments, 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%. %. In the above embodiments, the centrifuge may be a continuous flow centrifuge, for example a single continuous flow centrifuge. In some embodiments, two or more continuous flow centrifuges are used, for example in parallel. In a further embodiment, cells or cell aggregates generated from one or more continuous flow centrifuges are used to initiate or inoculate a culture in one or more pre-production bioreactors, or to inoculate or transfer the culture to one or more production bioreactors.

[0107] In certain embodiments, the above cell banking 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 comprising 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; bispecific F(mab) 2 antibody; single domain bispecific diabody (scBsDb), single chain bispecific tandem variable domain (scBsTaFv), trispecific NK cell engager (TriNKET), dual affinity retargeting protein (DART), bispecific diabody, tandem diabody (TandAb), half antibody (e.g., an antibody that includes an Fc portion but only one CH-VH:CL-VL pair), trifab contorsbody (described in PCT Application Publication No. WO 2019 / 086395); quadroma, scFv, dock and lock trivalent fab (DNL-(Fab) 3 , single domain antibody, bispecific single domain antibody, etc. III. SYSTEM

[0108] The various systems described herein and commercially available may be suitable for implementing the methods of the present disclosure. The systems may be fed-batch or continuous flow. The systems may include a stirred tank or an agitation system based on rocking. Such systems may be designed for single use or reuse. For example, a centrifuge (e.g., an apparatus suitable for inclusion in a perfusion method) may include disposable components (e.g., a single-use bowl). Examples of fed-batch systems that may support continuous flow or perfusion techniques for implementing the methods described herein include any one of the HyPerforma™ systems (e.g., HyPerforma™ 5:1 single-use bioreactor, jacketed, AC motor, load cell, catalog number SUB00508100) manufactured by Thermo Fisher Scientific™, any one of the Ambr® or Biostat® STR systems (e.g., Biostat STR® Generation 3 featuring Biobrain® Automation) manufactured by Sartorious™, and any one of the Allegro systems manufactured by Pall®; however, the systems are not limited thereto. These systems may be modified or adapted to include perfusion-based equipment and systems. One non-limiting example of a perfusion system may include alternating tangential flow (ATF) filtration utilizing hollow filter fibers (e.g., a product supplied by Repligen™). Another non-limiting example of a perfusion system may include tangential flow filtration (e.g., a product supplied by Flow Sciences, Inc™). Further examples of tangential flow filtration (TFF) systems are manufactured by Sartorious™ (e.g., Sartoflow® 150 automatic single-use tangential flow filtration system). Depth filtration may also be used in the perfusion methods described herein.Further non-limiting examples of perfusion system components can include acoustic settler-based perfusion technology (e.g., products supplied by SonoSep Technologies™). Continuous flow systems may incorporate one or more of the systems described herein, as well as other commercially available systems. To perform the methods described herein, various centrifugation systems may be included in the fedbatch and perfusion systems described herein. Preferably, the centrifuge used in this cell culture method includes disposable components that contact the cells and / or cell culture medium, such as a disposable bowl or bowl insert assembly. In certain embodiments, a continuous centrifuge includes a single-use centrifugal pump. Non-limiting examples of such systems include CultureOne™ available from Alfa Laval™ (e.g., the Alfa Laval CultureOne Primo™ single-use cell separator). Other suitable commercially available systems include the Culturefuge 400 B available from Alfa Laval, the GEA kytero® available from GEA Group Aktiengesellschaft, the Ksep® available from Sartorius™, the DynaSpin™ available from Thermo Fisher, and the Unifuge® available from CARR®. In certain embodiments, a single continuous flow centrifuge is used. In other embodiments, two or more continuous flow centrifuges are used, for example, in parallel. In further embodiments, cells or cell aggregates generated from one or more continuous flow centrifuges are used to initiate or inoculate culture in one or more pre-production bioreactors, or to inoculate or transfer the culture to one or more production bioreactors.

[0109] FIG. 1 is a schematic diagram of a system for cell culture, including a perfusion and cell separation device (in certain embodiments, a centrifuge, and in more specific embodiments, a continuous flow centrifuge) 100, according to one or more embodiments of the cell culture methods provided herein. Generally, a cell culture system for practicing the methods described herein includes growing a cell culture in a cell culture vessel 120 by first inoculating an appropriate cell (e.g., a cell type described herein, a recombinant mammalian cell, a recombinant CHO cell, and any other commercially available cell type) from an inoculation vessel 110 into the cell culture medium (e.g., Gibco™ CHO perfusion medium) of the cell culture vessel 120. The medium containing 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 of 0.1% PCV or greater. The inoculated cell culture (e.g., an inoculated culture of mammalian cells) can have a cell density of 0.1% PCV or greater.

[0110] Inoculation is preferably performed under sterile conditions by placing a plurality of mammalian cells contained in a fixed volume of cell culture medium into the culture vessel (e.g., pumped via a sterile connection) to produce a cell culture. In a specific embodiment, inoculation can be performed under sterile conditions by depositing (e.g., pumping via a sterile connection) a plurality of mammalian cells contained in a fixed volume of cell culture medium in a culture vessel having a working volume of, for example, 80 L, to produce a cell culture having an initial cell density of 1 million cells / mL or greater. In another specific embodiment, inoculation can be performed under sterile conditions by placing a plurality of mammalian cells and a fixed volume of cell culture medium into a culture vessel having a working volume of 3,000 L or 3,600 L (e.g., pumped via a sterile connection) to produce a cell culture having a starting cell density of 1 million cells / mL or greater.

[0111] When the culture has expanded to a cell density of 10% PCV or greater (alternatively 1%), at least a portion of the culture can be transferred (e.g., pumped using a peristaltic pump) from the cell culture vessel 120 to the cell separation device 130 via a sterile connection 103.

[0112] In a specific embodiment of the cell culture system provided herein, the cell separator 130 preferably removes a portion of the used or depleted medium and returns a portion, preferably substantially all, of the cells to the cell culture vessel 120. In addition to removing the depleted medium from the cell culture vessel 120, fresh medium and / or culture medium is preferably transferred from the fresh fluid container 170 to the cell culture vessel via the sterile connection 171. In such a system, fresh medium can be returned to the cell culture while the depleted medium is being removed. In a specific embodiment, the cell culture vessel 120 has a working volume or total volume of 80 L or more. In other specific embodiments, the cell culture vessel 120 has a working volume or total volume in the range of 100 L to 3000 L. In a specific embodiment, the cell culture vessel 120 has a working volume or total volume of 100 L. In another specific embodiment, the cell culture vessel 120 has a working volume or total volume of 3000 L. Preferably, the process of removing the used medium, separating the cells, and returning the cells to the cell culture vessel 120 proceeds continuously over at least a portion of the time during which the cells are cultured.

[0113] In some systems, the cell separation device 130 may include a filtration system (e.g., depth, ATF and / or TFF). In specific embodiments of the cell culture methods 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. Prior to or during use, a flush fluid may be used to prime one or more components of the system, such as the cell separation device 130. If the cell separation device 130 includes a centrifuge that can be used in continuous or semi - continuous applications, a flush fluid may be used to expel or wash 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 one aspect of such embodiments, two or more continuous - flow centrifuges are used in parallel, for example. In further embodiments, cells or cell aggregates generated from one or more continuous - flow centrifuges are used to initiate or inoculate a culture in one or more pre - production bioreactors or to inoculate or transfer the culture to one or more production bioreactors.

[0114] The flush fluid may include a buffer solution and / or purified water. The buffer solution may be suitable for maintaining healthy cells (e.g., a phosphate - buffered saline (PBS) solution at an appropriate pH such that the cells are less likely to lyse). The flush fluid may be pumped from the wash solution container 160 to the cell separation device 130 via the sterile connection 111. In many embodiments, the flush fluid can be used to empty the cell separation device 130 in order to preserve cells and / or products. In some systems, the flush fluid may be used to remove solids from the walls of the cell separation device 130, such as from a centrifuge.

[0115] Some conventional centrifugal systems include outlets for the light and heavy phases. In such conventional systems, solids (e.g., cultured cells) accumulate within the centrifuge bowl and must be discharged (either intermittently or deliberately) or periodically scraped out. Such procedures interrupt the continuous flow. Such systems may be incorporated into the cell culture methods provided herein (e.g., as the cell separation device 130), but the cell culture methods preferably incorporate a continuous flow cell separator, e.g., a continuous flow centrifuge.

[0116] In the various systems described herein, the heavy phase may contain such solids (e.g., the solid phase containing cultured cells). The light phase (e.g., the liquid phase) may contain a product (e.g., a secreted product from the cells, a recombinant protein, and / or an antibody). The centrifugal systems (e.g., disk stack centrifuges, tubular centrifuges) used to practice the methods described herein may be used to separate the liquid phase from the solid phase. The solid phase may contain cells. In many systems, unlike in conventional systems, the solids may not be discharged from the centrifugation system but may be retained in the solid phase.

[0117] 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 that leads to a liquid phase collection container 150. The liquid phase transferred to the liquid phase collection container 150 may contain one or more products from the cell culture. Once isolated, purification procedures may be performed on the product (e.g., a product secreted from the cells).

[0118] Optionally, a portion of the liquid phase may be returned to the cell culture container 120 via the sterile connection 113.

[0119] 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 return cells to the cell culture container 120 via the sterile connection 113, and the light liquid phase outlet may contain spent medium.

[0120] In various systems, the cell separator 130 may include a solid phase outlet 106. In many systems, the solid phase outlet 106 may return all or a portion of the solid phase to the cell culture vessel 120 via a sterile connection 105. The solid phase may include cells packed at a high density. In some cases, the solid phase may include a highly viscous solution. The centrifuges described herein may operate continuously to produce a solid phase having a cell density in the range of 1% to 50% PCV, such as about 10%, about 10%, about 30%, about 40% or about 50% PCV. The centrifuges described herein may operate continuously to produce a solid phase having a cell density of 30% PCV or greater.

[0121] One or more pumps (e.g., a centrifugal pump or a peristaltic pump) may return at least a portion of the solid phase and a fixed volume of 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). After completion of the perfusion procedure, the cell culture has a cell density of 0.2% PCV or greater. Further, to replace depleted medium, fresh cell culture fluid may be pumped from the fresh cell culture medium container 170 through the sterile connection 171 into the cell culture vessel 120.

[0122] To achieve a perfusion rate in the range of 0.5 to 6 VVD, one or more pumps (e.g., a centrifugal pump or a peristaltic pump) may return at least a portion of the solid phase and a fixed volume of cell culture medium to the culture vessel 120. After completion of the perfusion procedure, the cell culture has a cell density of 0.1% PCV or greater. Further, to replace depleted medium, fresh cell culture fluid may be pumped from the fresh cell culture medium container 170 through the sterile connection 171 into the cell culture vessel 120.

[0123] To achieve a perfusion rate in the range of 0.5 to 6 VVD, one or more pumps (e.g., a centrifugal pump or a peristaltic pump) may return at least a portion of the solid phase and a fixed volume of cell culture medium to the culture vessel 120. After completion of the perfusion procedure, the cell culture is 60×10 6It has a cell density of cells / ml or higher. Further, in order to replace the depleted medium, fresh cell culture solution can be pumped into the cell culture vessel 120 from the fresh cell culture medium container 170 through the sterile connection part 171.

[0124] One or more pumps (e.g., a centrifugal pump or a peristaltic pump) may return at least a part of the solid phase and a fixed volume of cell culture medium to the cell culture vessel 120 in order to achieve a perfusion rate in the range of 2 - 6 VVD. Further, in order to replace the depleted medium, fresh cell culture solution can be pumped into the cell culture vessel 120 from the fresh cell culture medium container 170 through the sterile connection part 171.

[0125] One or more pumps may operate to increase or decrease the perfusion rate in a fixed pattern. One or more pumps may operate to increase or decrease the perfusion rate in a variable pattern. One or more pumps may operate continuously over a period in the range of 1 - 7 days. One or more pumps may operate semi - continuously over a period in the range of 1 - 7 days.

[0126] The centrifugation process can result in a cell culture having a survival percentage of 85% or higher. The centrifugation process can result in a cell culture that maintains a survival percentage of 85% or higher over a period of 1 - 7 days. The centrifugation process can result in a cell culture having a lactate concentration of 4 g / L or less.

[0127] The centrifuge can operate at a speed in the range of 3,000 - 10,000 RPM. The continuous - flow centrifuge can have a sigma value in the range of 1,000m 2 ~200,000m 2 and can have a sigma value in the range of.

[0128] The cell separation device 130 may include sterilizable components. For example, the cell separation device may be sealed. When the cell separation device 130 includes a centrifuge bowl (e.g., a disk stack or a tube), the rotating components (e.g., a drive shaft) may include one or more seals. In some cases, the sterilization components may include a heating component. Further, one or more sterile connectors may connect the continuous flow centrifuge to a pipe or line directly or indirectly connected to the cell culture vessel 120, the flush fluid vessel 160, one or more solid phase collection vessels 140, and / or the liquid phase collection vessel 150.

[0129] In some systems, the solid phase outlet 106 may transfer all or part of the solid phase to the solid phase collection vessel 140 via the sterile connection 107. In some systems, the solid phase collection vessel 140 may be a cell culture vessel. In some systems, the solid phase collection vessel 140 may be a plurality of cell culture vessels. The solid phase may be used to inoculate the medium in one or more solid phase collection vessels 140. For example, the system may transfer at least a portion of the cell culture to a different culture vessel to initiate a second cell culture. The second cell culture may have an initial cell density in the range of 0.1% to 10% PCV.

[0130] In some systems, the cell culture vessel 120 may include a production culture vessel. The system may include transferring at least a portion of the cell culture to the production culture vessel to initiate a production culture having an initial cell density in the range of 0.1% to 10% PCV. The production culture undergoes continuous processing conditions. The production culture undergoes batch processing conditions or fed-batch processing conditions.

[0131] The sterile connection parts 101, 103, 105, 107, 109, 111, 113 described in this specification may include pipes connected to containers and devices 110, 120, 130, 140, 150, 160 via one or more sterile connectors, and may be pumped using one or more pumps (e.g., peristaltic pumps). Pipes, pumps, and sterile connectors are commercially available from various manufacturers including Thermo Fisher Scientific (trademark), Sartorious (trademark), and Pall (registered trademark).

[0132] Without limitation, environmental conditions including cell density, cell viability, pH, dO 2 , 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 signals to a control system that reports the environmental conditions. The control system can then act 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. IV. Method of Use

[0133] Figure 2 is a flowchart of a process for culturing mammalian cells 200 according to one or more embodiments.

[0134] Step 1 includes placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel to generate a cell culture 202.

[0135] Step 2 includes culturing the cell culture to a desired cell density 204.

[0136] Step 3 includes performing a perfusion procedure on the cell culture 206.

[0137] 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 generate a solid phase. The perfusion procedure may include returning the solid phase and a fixed volume of cell culture medium to the culture vessel at a desired perfusion rate.

[0138] Figure 3 is a flowchart of a process for culturing mammalian cells 300 according to one or more embodiments.

[0139] Step 1 includes placing a plurality of mammalian cells and a fixed volume of culture medium into a culture vessel to generate 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 a secreted product. In various processes, the secreted product may include a recombinant protein. In various processes, the recombinant protein may include an antibody.

[0140] Various cultures may use a culture vessel having a working volume that can be 80 L or more. Various cultures may use a culture vessel having a total volume that can range from 100 L to 3000 L. Some cultures may use a vessel having a total volume of 100 L. Some cultures may use a vessel having a total volume of 3,000 L.

[0141] Step 2 includes culturing the cell culture to a cell density of 1% packed cell volume (PCV) 304 or more.

[0142] Step 3 includes performing a perfusion procedure on the cell culture 306.

[0143] Performing a perfusion procedure can involve returning a solid phase and a fixed volume of cell culture medium to a 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 is in the range of 2 to 4 VVD. Some perfusion procedures can involve increasing or decreasing the perfusion rate in a fixed pattern. Alternative perfusion procedures can involve increasing or decreasing the perfusion rate in a variable pattern.

[0144] The perfusion procedure can be carried out continuously over a period ranging from 1 to 7 days. Alternative perfusion procedures can be carried out semi - continuously over a period ranging from 1 to 7 days. The cell culture can maintain a survival percentage of 85% or more over a period of 1 to 7 days.

[0145] Performing a perfusion procedure can involve transferring at least a portion of the cell culture to a continuous - flow centrifuge. Some perfusion procedures can use a continuous - flow centrifuge equipped with a disk stack bowl. The continuous - flow centrifuge can have a sigma value in the range of 1,000m 2 ~200,000m 2 . The continuous - flow centrifuge can maintain a residence - time temperature in the range of about 30°C to about 39°C, such as 31°C to 38°C, 32°C to 38°C, 33°C to 38°C, 34°C to 38°C, 35°C to 38°C, or 36°C to 38°C.

[0146] Alternatively, the perfusion procedure can involve the use of a continuous - flow centrifuge equipped with a tubular bowl. Various suitable centrifuge systems are commercially available.

[0147] Performing a perfusion procedure can involve operating a continuous - flow centrifuge to produce a solid phase having a final cell density of 1% PCV or more, such as about 10%, about 20%, about 30%, about 40%, or about 50% PCV.

[0148] Performing a perfusion procedure can involve isolating the liquid phase from the continuous - flow centrifuge and performing a purification procedure on the secreted products therein.

[0149] The continuous flow centrifuge used in the perfusion procedure described herein may comprise sterilizable components. The sterilization components may include one or more devices or processes described herein and elsewhere.

[0150] The continuous flow centrifuge used in the perfusion procedure described herein may comprise disposable components.

[0151] The continuous flow centrifuge used in the perfusion procedure may include an operating speed in the range of 3,000 to 10,000 RPM.

[0152] In some processes for culturing mammalian cells, the production culture is cultured under batch or fed-batch process conditions.

[0153] After completion of the centrifugation procedure of the perfusion procedure, the cell culture may have a viability percentage of 85% or more. After completion of the centrifugation procedure of the perfusion procedure, the cell culture may have a lactate concentration of 4 g / L or less. After completion of the perfusion procedure, the cell culture may have a cell density of 0.2% PCV or more, for example, from 0.2% PCV to about 30% PCV.

[0154] The process for culturing mammalian cells may 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 may have an initial cell density in the range of 0.1% to 10% PCV.

[0155] The process for culturing mammalian cells may 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.

[0156] Figure 4 is a flowchart of a process for generating a seeded culture of mammalian cells having a cell density of 400 or more of 0.1% PCV, according to one or more embodiments, for example, from 0.1% to about 30% PCV.

[0157] Step 1 includes placing a plurality of mammalian cells and a fixed volume of cell culture medium into a culture vessel to generate a cell culture 402.

[0158] Step 2 includes culturing the cell culture to a cell density 404 of 10% PCV or higher.

[0159] Step 3 includes performing a perfusion procedure on the cell culture 406.

[0160] 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 generate a solid phase having a cell density in the range of 1% to about 50% PCV. The perfusion procedure may include returning a portion of the solid phase and a fixed volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. After completion of the perfusion procedure, the cell culture has a cell density of 0.1% PCV to 30% PCV or higher.

[0161] FIG. 5 is a flowchart of a process for generating an inoculum culture of mammalian cells containing at least 4.8×10 12 cells 500 according to one or more embodiments.

[0162] Step 1 includes placing a plurality of mammalian cells and a fixed volume of cell culture medium into a culture vessel having a working volume of 80 L to generate a cell culture having a starting cell density of 1 million cells / mL 502 or higher.

[0163] Step 2 includes culturing the cell culture to a cell density of 10% PCV 504 or higher.

[0164] Step 3 includes performing a perfusion procedure on the cell culture 506.

[0165] The perfusion reaction includes transferring at least a portion of the cell culture to a disposable disk stack bowl including 1,000 to 200,000 m 2It may include transferring to a continuous flow centrifuge including a sigma coefficient in the range of. The perfusion procedure may include operating the continuous flow centrifuge to produce a solid phase having a final cell density of 1% PCV or more, 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 certain volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.5 to 6 VVD. After completion of the perfusion procedure, the cell culture has a cell density of 60 million cells / mL or more.

[0166] Figure 6 is a flowchart of a process for generating an inoculum culture of mammalian cells comprising at least 2.16×10 14 cells 600 according to one or more embodiments.

[0167] Step 1 includes placing a plurality of mammalian cells and a certain 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 of 1 million cells / mL 602 or more.

[0168] Step 2 includes culturing the cell culture to a cell density of 10% PCV 604 or more.

[0169] Step 3 includes performing a perfusion procedure on the cell culture 606.

[0170] The perfusion procedure may include transferring at least a portion of the cell culture to a continuous flow centrifuge including a disposable disk stack bowl and including a sigma coefficient 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 of 1% PCV or more, 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 certain volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 to 6 VVD. After completion of the perfusion procedure, the cell culture has a cell density of 60 million cells / mL or more.

[0171] The processes in the methods described in this specification may be performed using the systems and devices described in this specification and elsewhere.

[0172] It will be apparent to those skilled in the art that various changes and modifications can be made to the technology described in this specification without departing from the spirit or scope of the present embodiment.

Examples

[0173] Example 1: 50% Split Ratio A perfusion cell culture run was completed that returned a 50% ratio of solid to liquid phase to the production cell culture. The centrifuge was adjusted to flow 50% of the centrifuge inlet stream to the solid outlet and 50% of the centrifuge inlet stream to the liquid outlet at volumetric flow rates for a 50:50 solid-to-liquid split ratio.

[0174] Example 2: 75% Split Ratio Complete a perfusion cell culture run that returns a 75% ratio of solid to liquid phase to the production cell culture. Adjust the centrifuge to flow 75% of the centrifuge inlet stream to the solid outlet and 25% of the centrifuge inlet stream to the liquid outlet at volumetric flow rates for a 75:25 solid-to-liquid split ratio.

[0175] Example 3: 90% Split Ratio Complete a perfusion cell culture run that returns a 90% ratio of solid to liquid phase to the production cell culture. Adjust the centrifuge to flow 90% of the centrifuge inlet stream to the solid outlet and 10% of the centrifuge inlet stream to the liquid outlet at volumetric flow rates for a 90:10 solid-to-liquid split ratio.

[0176] Example 4: Cell Culture on Day 7 This example demonstrates a 7-day cell culture incorporating this continuous centrifuge cell culture method without a clarification step in the preparation for protein recovery.

[0177] Mammalian cells (Chinese hamster ovary (CHO) cells) capable of expressing a therapeutic antibody were grown in a bioreactor with a working volume of 500 liters using a target actual volume of approximately 300 L. The packed cell volume (PCV) at the start of cell culture was 0.30%, the viable cell count (VCC) was 2.087×10 6 cells / mL, and the viability was 97.7%. After culturing for approximately 19 hours, the PCV increased to 0.40%, the VCC was 3.472×10 6 cells / mL, and the viability was 97.9%. In a culture of approximately 67 hours, a perfusion procedure was initiated at 2.5 vessel volumes per day (VVD) using an Alfa Laval CultureOne Primo (trademark) continuous centrifuge to obtain a perfusion flow rate of 521 mL / min. The centrifuge was operated at a target split ratio of 50% (i.e., the solid phase and the light phase were 50:50) 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 was discarded for this run. The advantage of this continuous centrifuge is that the bowl insert assembly (Spinsert (trademark)) is designed as a disposable single-use assembly that simplifies the maintenance of sterility and provides gentle handling of the cells (promoting higher cell viability). Peristaltic pumps connected to the inlet and outlet ports were added to the centrifuge. Cold water (approx. 4°C - 10°C) was used to cool the centrifuge seal and minimize the temperature rise from the rotation of centrifugation. Medium addition throughout the perfusion process was added at a flow rate equal to the liquid phase flow rate to maintain the actual volume of the bioreactor, which was controlled by the load cell of the bioreactor. The bowl speed was increased from 3000 RPM to 3200 RPM to 3400 RPM to optimize cell retention. At this point, the bioreactor PCV was approximately 2.2%, the VCC was 1.24×10 7 cells / mL, and the viability was 97.1%. The cell culture perfusion procedure was continued for the next approximately 46.5 hours, as a result, the bioreactor PCV increased to 4.33%, the VCC increased to approximately 2.5×10 7 cells / mL, and showed a viability of 91.7%.

[0178] In 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 for a working volume of 300 L), and the actual centrifuge inlet flow rate was 1540 mL / min. After 5 hours, the bioreactor PCV was about 4.8%, the cell viability was 92.8%, and the VCC was 3.069×10 7 cells / mL. Over the next 22.5 hours, the PCV increased to about 7.40%, the VCC increased to about 4.038×10 7 cells / mL, and the viability was 92.6%.

[0179] At about 137.5 hours after the start, 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 for a working volume of 300 L), and the actual centrifuge inlet flow rate was 1650 mL / min. The centrifuge continued to operate at a target split ratio of 50%. The cell viability after the change was 93.8%. Over the next 27 hours, the PCV increased to about 12.0%, the cell viability was 95.1%, and the VCC was about 5.66×10 7 cells / mL increased.

[0180] At about 164.5 hours after the start, the perfusion flow rate was increased from 3.96 VVD to 5 VVD (for reference, 5 VVD corresponded to a target flow rate of 1042.0 mL / min for a working volume of 300 L). The centrifuge continued to operate at a target split ratio of 50%, and the actual centrifuge inlet flow rate was 2080 mL / min. The solid phase flow rate was 1040 mL / min, and at this point, it was necessary to install a manual peristaltic pump. After this change, the bioreactor cell viability was 95.9%. The cell culture with centrifugation continued until about 181.5 hours after the start of the culture. Starting from 3.96 VVD, the solid phase was 14% PCV, and at the end of the run at 5 VVD, the solid phase achieved 32% PCV. While running at 5 VVD, the cell viability in the culture vessel was about 93.2% at this point, the PCV was about 16%, and the VCC was about 8.435×10 7It was cells / mL. Starting from when the solid phase was first sampled, it began at approximately 3.96 VVD and approximately 14% PCV, and by the end of the run, at approximately 5 VVD, the packed cell volume increased to approximately 32% PCV.

[0181] At each time point during this culture - centrifugation process, the bioreactor cell viability was at least 91.4%, and at higher perfusion rates (above 3.7 VVD), the bioreactor cell viability was generally above 92%.

[0182] Preferred embodiments of the present technology have been shown and described herein, but 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 envision numerous variations, modifications, and substitutions without departing from the described technology. It should be understood that various alternative forms to the embodiments described herein may be used in implementing the system and method. The following claims are intended to cover not only the described methods and structures but also their equivalents.

Claims

1. A method for culturing recombinant CHO cells, (a) To produce a cell culture by placing multiple recombinant CHO cells and a certain volume of culture medium in a culture vessel, (b) Culturing the cell culture to a cell density of 1% filled cell volume (PCV) or more, (c) Perform a perfusion procedure on the cell culture during step (b) The perfusion procedure includes, (i) Transferring at least a portion of the cell culture to a continuous flow centrifuge, (ii) Operating the continuous flow centrifuge to generate a solid phase having a cell density of 1% PCV or more, (iii) Returning the solid phase and a certain volume of cell culture medium to the culture vessel to achieve a perfusion rate in the range of 0.7 to 6 vessel volumes / day (VVD), wherein after the completion of the perfusion procedure, the perfusion rate is achieved such that the cell culture has a cell density of 10% PCV or more. The culture vessel (a) has a working volume of 80 L or more, and / or (b) has a total volume in the range of 100 L to 30,000 L, A method wherein the perfusion procedure is carried out continuously over a period of time ranging from one to seven days.

2. The method according to claim 1, wherein the perfusion rate is in the range of 2 to 6 CVD.

3. The method according to claim 1, wherein the perfusion procedure includes increasing or decreasing the perfusion rate in a fixed or variable manner.

4. The method according to claim 1, wherein the continuous flow centrifuge comprises a disc stack bowl or a tubular bowl.

5. The method according to claim 1, wherein the continuous flow centrifuge has an operating speed in the range of 3,000 to 10,000 RPM.

6. The method according to claim 1, wherein the continuous flow centrifuge comprises sterilizable components.

7. The continuous flow centrifuge described above operates at 1,000 m ２ ~200,000m ２ The method according to claim 1, wherein the sigma value is in the range of .

8. The method according to claim 1, wherein, after completion of the centrifugation procedure, the cell culture has a viability percentage of 85% or more.

9. The method according to any one of claims 5 to 8, wherein the cell culture maintains a survival percentage of 85% or more over a period of 1 to 7 days.

10. The method according to claim 9, wherein, after the completion of the centrifugation procedure, the cell culture has a lactic acid concentration of 4 g / L or less.

11. The method according to claim 1, wherein the recombinant CHO cells produce secretory products, particularly recombinant proteins.

12. The method according to claim 11, wherein the recombinant protein is an antibody.

13. The method according to claim 1, wherein the culture vessel has a total volume in the range of 5,000 L to 30,000 L.

14. The method according to claim 1, wherein the culture vessel has a total volume in the range of 100 L to 6,000 L.

15. The method according to claim 1, 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.

16. (a) Isolating the liquid phase from the continuous flow centrifuge and performing a purification procedure on the secreted product therein, and / or (b) The method according to claim 1, further comprising adding fresh culture medium to the culture vessel.

17. A method for producing a culture of recombinant CHO cells having a cell density of 10% PCV or more, (a) To produce a cell culture by placing multiple recombinant CHO cells and a certain volume of cell culture medium in a culture vessel, (b) Culturing the cell culture to a cell density of 10% PCV or more, (c) Perform a perfusion procedure on the cell culture during step (b) The perfusion procedure includes, (i) Transferring at least a portion of the cell culture to a continuous flow centrifuge, (ii) Operating the continuous flow centrifuge to generate a solid phase having a cell density in the range of approximately 1% to approximately 50% PCV, (iii) Returning a portion of the solid phase and a certain 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 the completion of the perfusion procedure, the cell culture has a cell density of 10% PCV or more, and the perfusion rate is achieved. The culture vessel (a) has a working volume of 80 L or more, and / or (b) has a total volume in the range of 100 L to 30,000 L, The perfusion procedure is carried out continuously over a period of 1 to 7 days. Methods that include...

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