Methods for continuous cell culture

Abstract

To provide continuous culture methods for producing a cell product such as a recombinant protein, a glycoprotein, an antibody agent or a fusion protein.SOLUTION: A method comprises: culturing a population of cells consisting of shear-sensitive cells in a bioreactor system with at least 25 L of culture media and a gas exit velocity that is at most 20 m / s to achieve a steady state viable cell concentration in the culture media within a range of 20×106 cells / mL to 15×107 cells / mL.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 62 / 727,976, filed on 6 September 2018, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Continuous cell culture methods and systems are becoming widespread in biomanufacturing. However, these methods and systems are associated with many precautions and troublesome problems. Therefore, there is still a need to improve continuous cell culture methods and systems. [Overview of the project] [Means for solving the problem]

[0003] This disclosure provides, in particular, a continuous culture method for producing (e.g., manufacturing) cell products, such as recombinant proteins, glycoproteins, antibody drugs, or fusion proteins. In some cases, the methods herein enable large-scale production of recombinant proteins using continuous culture methods (e.g., perfusion culture methods). This disclosure identifies and addresses the challenges of current continuous cell culture techniques (e.g., perfusion cell culture) that have insufficient viable cell concentrations and reduced cell viability in large-scale cultures (e.g., at least 25 L, e.g., at least 100 L) of certain cells. This disclosure provides, in part, a method and system for large-scale (e.g., at least 25 L, e.g., at least 100 L) continuous culture of shear-sensitive cells.

[0004] This disclosure provides insight that large-scale culture of shear-sensitive cells can be achieved by controlling the gas outflow rate of a bioreactor system. For example, large-scale culture of shear-sensitive cells in a bioreactor system (e.g., a perfusion bioreactor system) having a gas outflow rate controlled and / or maintained within a specified amount and / or range, such that it is up to 20 m / s, is approximately 20 × 10⁶ 6 Approximately 15 × 10 cells / mL 7 This can provide a steady-state viable cell concentration of shear-sensitive cells within the range of cells / mL.

[0005] In some cases, a method comprising culturing a population of shear-sensitive cells, or a population of cells consisting thereof, in a bioreactor system (e.g., a perfusion bioreactor system) to achieve a steady-state viable cell concentration, wherein the steady-state viable cell concentration is 20 × 10⁻⁶ 6 From cells / mL to 15 × 10 7 A method is provided in which the bioreactor system is within the range of cells / mL and includes at least 25 L of culture medium and a gas outflow rate of up to 20 m / s. In some embodiments, the bioreactor system has a gas outflow rate controlled to a rate of about 15 m / s or less, 14 m / s or less, 13 m / s or less, 12 m / s or less, 11 m / s or less, 10 m / s or less, 9 m / s or less, or 8 m / s or less. In some embodiments, the bioreactor system includes a sparger. In some embodiments, the sparger has a gas outflow rate controlled to a rate of about 15 m / s or less, 14 m / s or less, 13 m / s or less, 12 m / s or less, 11 m / s or less, 10 m / s or less, 9 m / s or less, or 8 m / s or less.

[0006] A method comprising, in some cases, culturing shear-sensitive cells in a bioreactor system (e.g., a perfusion bioreactor system) containing at least 25 L of culture medium, wherein the bioreactor system has a gas outflow rate of up to 20 m / s Furthermore, shear-sensitive cells are approximately 20 x 10 6from about 15×10 cells / mL 7 There is provided a method of being present at a steady-state viable cell concentration within the range of from about 15×10 cells / mL. In some embodiments, a bioreactor system (e.g., a perfusion bioreactor system) has a gas outlet velocity that is 15 m / s or less, 14 m / s or less, 13 m / s or less, 12 m / s or less, 11 m / s or less, 10 m / s or less, 9 m / s or less, or 8 m / s or less. In some embodiments, the gas outlet velocity is controlled throughout the culturing process. In some embodiments, the gas outlet velocity is controlled at least until the bioreactor system reaches steady-state conditions.

[0007] In some cases, culturing a population of cells comprising shear-sensitive cells in a bioreactor system having at least 25 L (e.g., at least 200 L) of culture medium and a gas outlet velocity that is at most 20 m / s (e.g., at most 10 m / s), to achieve a steady-state viable cell concentration in the culture medium within the range of from 20×10 6 cells / mL to 15×10 7 There is provided a method of achieving a steady-state viable cell concentration in a culture medium within the range of from 20×10 cells / mL to 15×10 cells / mL. In some embodiments, the steady-state viable cell concentration is a viable cell concentration that varies by at most 20% over a 5-day period.

[0008] In some cases, a continuous culturing process for culturing a population of cells comprising shear-sensitive cells, comprising controlling the gas outlet velocity of a bioreactor system so as not to exceed a rate of 20 m / s, wherein the bioreactor system comprises at least 25 L of culture medium, and the population of cells is from 20×10 6 cells / mL to 15×10 7A process is provided for achieving a steady-state viable cell concentration within the range of cells / mL. In some embodiments, the gas efflux rate is controlled to a rate of approximately 15 m / s or less, 14 m / s or less, 13 m / s or less, 12 m / s or less, 11 m / s or less, 10 m / s or less, 9 m / s or less, or 8 m / s or less. In some embodiments, the bioreactor system includes a sparger. In some embodiments, the sparger gas efflux rate is controlled to a rate of approximately 15 m / s or less, 14 m / s or less, 13 m / s or less, 12 m / s or less, 11 m / s or less, 10 m / s or less, 9 m / s or less, or 8 m / s or less.

[0009] In some embodiments, the gas efflux rate is controlled for at least a portion of the time the shear-sensitive cells are cultured. In some embodiments, control of the gas efflux rate begins when the cell population (e.g., a population of shear-sensitive cells, e.g., inoculated) is added to the bioreactor system (e.g., when the cell population is mixed with the culture medium of the bioreactor system). In some embodiments, control of the gas efflux rate begins approximately 2, 6, 12, 24, or 48 hours after the cell population (e.g., a population of shear-sensitive cells, e.g., inoculated) has been added to the bioreactor system (e.g., when the cell population is mixed with the culture medium of the bioreactor system).

[0010] In some embodiments, control of the gas outflow rate is initiated when the cultured cells reach a certain density during the culture process. In some embodiments, control of the gas outflow rate is initiated when the bioreactor system reaches at least 1 × 10⁻⁶ 6 cells / mL, 2×10 6 cells / mL, 5×10 6 cells / mL, or 10 × 10 6 Start when the concentration of shear-sensitive cells is cells / mL.

[0011] In some embodiments, the gas efflux rate is controlled at least until the bioreactor system reaches steady-state conditions. In some embodiments, steady-state conditions include having a viable cell concentration that fluctuates by up to 20% over a 5-day period. In some embodiments, the gas efflux rate is controlled from the time when a population of cells (e.g., a population of shear-sensitive cells, e.g., inoculated) is added to the bioreactor system (e.g., when the population of cells is mixed with the culture medium of the bioreactor system) until the culture reaches steady-state conditions (e.g., having a viable cell concentration that fluctuates by up to 20% over a 5-day period). In some embodiments, the gas outflow rate of the bioreactor system is at least 1 × 10⁻⁶ 6 The culture is controlled from a shear-sensitive cell concentration of cells / mL until it reaches a steady state condition (for example, with a viable cell concentration that fluctuates by up to 20% over a 5-day period).

[0012] In some embodiments, the gas efflux rate is controlled throughout the entire culture process. In some embodiments, the gas efflux rate is controlled for a culture period of at least about 10 days, at least about 15 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days. In some embodiments, the gas efflux rate is controlled from the time when a population of cells (e.g., a population of shear-sensitive cells, e.g., inoculation) is added to the bioreactor system (e.g., when the population of cells is mixed with the culture medium of the bioreactor system) and continues for at least about 10 days, at least about 15 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days. In some embodiments, the gas efflux rate is controlled when the bioreactor system is at least 1 × 10 6The culture is controlled from a point where it has a shear-sensitive cell concentration of cells / mL until it reaches a steady state condition (for example, with a viable cell concentration that fluctuates by up to 20% over a period of 5 days), and continues for at least approximately 10 days, at least approximately 15 days, at least approximately 20 days, at least approximately 25 days, at least approximately 30 days, at least approximately 40 days, at least approximately 50 days, or at least approximately 60 days.

[0013] A perfusion culture process for culturing shear-sensitive cells, which in some cases includes controlling the gas outflow rate of the perfusion bioreactor system so as not to exceed a rate of 20 m / s, wherein the perfusion bioreactor system comprises at least 25 L of culture medium, the culture medium comprises shear-sensitive cells, and the shear-sensitive cells are approximately 20 × 10⁶ 6 Approximately 15 × 10 cells / mL 7 A process is provided that exists at a steady-state viable cell concentration within the range of cells / mL. In some embodiments, the gas outflow rate of the perfusion bioreactor system is controlled so as not to exceed a rate of 10 m / s.

[0014] In some embodiments, the steady-state viable cell concentration of shear-sensitive cells is within a range limited by a lower and upper limit, where the upper limit is greater than the lower limit. In some embodiments, the lower limit is approximately 20 × 10⁻⁶ 6 cells / mL, approximately 25×10 6 cells / mL, approximately 30×10 6 cells / mL, approximately 35×10 6 cells / mL, approximately 40×10 6 cells / mL, approximately 45×10 6 cells / mL, approximately 50×10 6 cells / mL, approximately 60×10 6 cells / mL, approximately 70×10 6 cells / mL, approximately 80×10 6 Cells / mL, or approximately 90 × 10⁻⁶ 6 The cell count may be cells / mL. In some embodiments, the upper limit is about 40 × 10 6 cells / mL, approximately 45×10 6 cells / mL, approximately 50×106 cells / mL, approximately 60×10 6 cells / mL, approximately 70×10 6 cells / mL, approximately 80×10 6 cells / mL, approximately 90×10 6 cells / mL, approximately 10×10 7 cells / mL, approximately 11×10 7 cells / mL, approximately 12×10 7 cells / mL, approximately 13×10 7 cells / mL, approximately 14×10 7 Cells / mL or approximately 15 × 10⁴ 7 Cells / mL may also be acceptable.

[0015] In some embodiments, the perfusion bioreactor system has a controlled level of dissolved carbon dioxide. In some embodiments, the culture medium contains dissolved carbon dioxide at levels of 120 mmHg or less, 115 mmHg or less, 110 mmHg or less, 105 mmHg or less, 100 mmHg or less, 95 mmHg or less, 90 mmHg or less, 85 mmHg or less, or 80 mmHg or less.

[0016] In some specific embodiments, the perfusion bioreactor system has a gas outflow rate of 10 m / s or less and contains dissolved carbon dioxide at a level of 80 mmHg or less.

[0017] In some embodiments, the cell culture system has a dissolved carbon dioxide level of 120 mmHg or less. In some embodiments, the continuous cell culture system (e.g., a perfusion cell culture system) has a dissolved carbon dioxide level in the range of about 20 mmHg to about 120 mmHg. In some embodiments, the dissolved carbon dioxide is present in the continuous cell culture medium in an amount limited by a lower limit and an upper limit, where the upper limit is greater than the lower limit. In some embodiments, the lower limit may be about 20 mmHg, about 30 mmHg, about 40 mmHg, about 50 mmHg, about 60 mmHg, or about 70 mmHg. In some embodiments, the upper limit may be about 50 mmHg, about 60 mmHg, about 70 mmHg, about 80 mmHg, about 90 mmHg, about 100 mmHg, about 110 mmHg, or about 120 mmHg.

[0018] In some embodiments, the perfusion bioreactor system includes at least 50 L, at least 100 L, at least 200 L, at least 500 L, at least 1,000 L, or at least 2,000 L of culture medium.

[0019] In some embodiments, the continuous culture of shear-sensitive cells is carried out for a period of at least 10 days. In some embodiments, the continuous culture of shear-sensitive cells is carried out for a period of about 30 to about 60 days.

[0020] In some embodiments, the continuous culture of shear-sensitive cells is carried out for a period ranging from 10 to 180 days. In some embodiments, the continuous culture of shear-sensitive cells is carried out for a period of time limited by a lower limit and an upper limit, where the upper limit is greater than the lower limit. In some embodiments, the lower limit may be about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 50 days, or about 60 days. In some embodiments, the upper limit may be about 30 days, about 35 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days, about 90 days, about 100 days, about 120 days, about 140 days, about 160 days, or about 180 days.

[0021] In some embodiments, the serial culture method of the present disclosure includes a step of measuring the viable cell concentration. In some embodiments, the measured viable cell concentration is at least 30 × 10 6 Cells / mL, at least 40 × 10 6 cells / mL, or at least 50 × 10 6 The concentration is cells / mL.

[0022] In some embodiments, the shear-sensitive cells for culturing according to this disclosure are mammalian cells.

[0023] In some embodiments, the shear-sensitive cells are mammalian cells. In some embodiments, the shear-sensitive cells are murine cells. In some embodiments, the shear-sensitive cells are derived from a mouse cell line. In some embodiments, the shear-sensitive cell line is a mouse myeloma cell line. In some specific embodiments, the shear-sensitive cells are selected from NS0 cells and SP 2 / 0 cells. In some specific embodiments, the shear-sensitive cells for use according to this disclosure are SP 2 / 0 cells.

[0024] In some embodiments, the shear-sensitive cells are human cells. In some embodiments, the shear-sensitive human cell lines are HEK293: human fetal kidney 293; HT-1080: fibrosarcoma-derived with an epithelial-like phenotype; PER.C6: human embryonic retinal cells immortalized by transfection with adenovirus E1 gene; CAP: human amniotic cells immortalized with adenovirus type 5 E1 gene; HKB-11: HEK 293-S and human B cell lines are prepared by polyethylene glycol fusion; as well as HuH-7, selected from human hepatocellular carcinoma. In some specific embodiments, shear-sensitive cells are selected from HEK293 cells, fibrosarcoma HT1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, and HuH-7 cells.

[0025] In some embodiments, shear-sensitive cells for use according to this disclosure contain or are manipulated to express cell products. In some embodiments, cultured shear-sensitive cells contain or express cell products. In some embodiments, cell products are nucleic acids, lipids, peptides, and / or proteins, or contain them. In some embodiments, cell products are recombinant proteins. In some embodiments, recombinant proteins are glycoproteins. In some embodiments, glycoproteins are Fc-containing glycoproteins. In some embodiments, glycoproteins are antibody drugs. In some embodiments, antibody drugs are monoclonal antibodies. In some specific embodiments, monoclonal antibodies are ustekinumab.

[0026] In some embodiments, the shear-sensitive cells of this disclosure include nucleic acids encoding antibody drugs approved, for example, in a secondary approval process, for therapeutic or diagnostic use in humans or animals. In some specific embodiments, the shear-sensitive cells of this disclosure include nucleic acids encoding ustekinumab.

[0027] In some embodiments, the methods and / or processes of the present disclosure include isolating a cell product from at least a portion of cells and / or isolating a cell product from at least a portion of a culture medium. In some embodiments, the cell product is a recombinant protein, e.g., a glycoprotein, e.g., an Fc-containing glycoprotein, e.g., an antibody drug, e.g., a monoclonal antibody.

[0028] In some embodiments, the cell culture medium also includes an antifoaming agent (e.g., Antifoam C) at a concentration ranging from 1 ppm to 500 ppm. In some embodiments, shear-sensitive cells are cultured in a cell culture medium containing a shear force protectant (e.g., Pluronic F-68). In some embodiments, shear-sensitive cells (e.g., a population of shear-sensitive cells) are cultured in a cell culture medium containing Pluronic F-68 at a concentration ranging from 1 g / L to 15 g / L. In some embodiments, a population of shear-sensitive cells is cultured in a culture medium containing an antifoaming agent (e.g., Antifoam C) at a concentration ranging from 1 ppm to 500 ppm and a shear force protectant (e.g., Pluronic F-68) at a concentration ranging from 1 g / L to 15 g / L.

[0029] In some embodiments, the perfusion bioreactor system includes a bioreactor tank. In some embodiments, the bioreactor tank is an agitated bioreactor tank. In some embodiments, the bioreactor tank has a capacity of at least 50 L, 100 L, 200 L, 250 L, 400 L, 500 L, 600 L, 800 L, 1,000 L, or 2,000 L. In some specific embodiments, the bioreactor tank has a capacity of about 200 L, about 250 L or more.

[0030] In some embodiments, the bioreactor system (e.g., a perfusion bioreactor system) includes a cell retention device. In some embodiments, the cell retention device is or includes a continuous centrifuge, an alternating tangential flow filter (ATF), a tangential flow membrane filter (TFF), a dynamic filter, a spin filter, an ultrasonic and dielectrophoretic separator, or a gravity sedimentation device. In some specific embodiments, the cell retention device is or includes an ATF.

[0031] In some embodiments, the bioreactor system includes a stirred-tank bioreactor, a cell retention device, a culture medium supply, and waste collection.

[0032] In some embodiments, the bioreactor system (e.g., a perfusion bioreactor system) includes a sparger. In some embodiments, the bioreactor system includes a drill-hole sparger. In some embodiments, the bioreactor system includes an open-pipe sparger. In some embodiments, the bioreactor system includes a sintered sparger.

[0033] In some cases, a method is provided for large-scale (e.g., at least 25 L, e.g., at least 100 L) continuous culture of shear-sensitive cells, comprising preparing or obtaining shear-sensitive cells containing recombinant protein-coding nucleic acids, and culturing the cells in a bioreactor system having a controlled gas outflow rate under conditions sufficient for recombinant protein expression. In some embodiments, the bioreactor system has a gas outflow rate controlled to a maximum of 20 m / s (e.g., about 5 m / s to about 10 m / s) until the system reaches steady-state conditions. In some embodiments, the bioreactor system has a gas outflow rate controlled to a maximum of 20 m / s (e.g., about 5 m / s to about 10 m / s) throughout the culture. In some embodiments, the viable cell concentration at steady-state conditions is about 20 × 10⁻¹⁶ 6 Approximately 15 × 10 cells / mL 7 It is within the range of cells / mL. In some embodiments, the live cell concentration under steady-state conditions is at least 40 × 10⁶ 6 The concentration is cells / mL.

[0034] In some cases, a method is provided for producing a continuous culture of protein preparations of shear-sensitive cells, comprising continuous culture of shear-sensitive cells in a bioreactor system having a controlled gas outflow rate. In some embodiments, the bioreactor system includes a bioreactor tank having a capacity of at least 25 L, at least 100 L, or at least 200 L. In some embodiments, the gas outflow rate is controlled to a maximum of 20 m / s (e.g., about 5 m / s to about 10 m / s) until the system reaches steady-state conditions. In some embodiments, the bioreactor system has a gas outflow rate controlled to a maximum of 20 m / s (e.g., about 5 m / s to about 10 m / s) throughout the culture. In some embodiments, the viable cell concentration at steady-state conditions is about 20 × 10⁻¹⁶ 6 Approximately 15 × 10 cells / mL 7 It is within the range of cells / mL. In some embodiments, the live cell concentration under steady-state conditions is at least 40 × 10⁶ 6 The concentration is cells / mL. In some embodiments, a method for producing a protein preparation includes isolating a protein or a mixture of proteins from cells and / or cell culture media.

[0035] These and other aspects of the present invention are described in further detail below and in the claims.

[0036] The drawings included in this specification, which consist of the following figures, are for illustrative purposes only and not to limit. [Brief explanation of the drawing]

[0037] [Figure 1] A schematic diagram of an example perfusion culture system with an alternating tangential flow as an example cell retention device is shown. As shown, the example perfusion culture system may include a production bioreactor (e.g., a stirred bioreactor tank), a cell retention device (e.g., alternating tangential flow), a supply medium, and a waste discharge system.

[0038] [Figure 2] This shows the viable cell concentrations (10⁶ cells / mL) of shear-sensitive cells (e.g., SP2 / 0 cells) cultured by perfusion culture on a scale of 3L to 100L. Cells grown in 3L (square), 5L (filled triangle), 15L (rhomboid), and 100L (outlined triangle) cultures showed comparable cell proliferation and steady-state viable cell concentrations.

[0039] [Figure 3] The viable cell concentration (10⁶ cells / mL) (upper panel) and viability percentage (lower panel) of shear-sensitive cells (e.g., SP2 / 0 cells) cultured by perfusion culture on a scale ranging from 3L to 200L are shown. Filled circles represent a 100L culture process, open squares represent conventional 3L control culture data, filled and open triangles represent a further 3L culture process, and open diamonds represent a 200L culture process. Initial cell proliferation was observed in all samples, but the 200L culture system showed both decreased cell proliferation and a decline in viable cell concentration, which began on day 4.

[0040] [Figure 4] The viable cell concentrations (10⁶ cells / mL) of shear-sensitive cells (e.g., SP2 / 0 cells) cultured by perfusion culture using varying gas outflow rates at different scales are shown. Filled circles represent a 100L culture process, open squares represent a 3L control culture, and open diamonds represent a 200L culture process. At time point (1), 2L of the culture medium from the 200L culture process is transferred to 3L of culture, represented by a filled triangle. At time point (2), the control gas outflow rate of the 200L culture is reduced to 10 m / s or less. At time point (3), the control gas outflow rate of the 200L culture is again increased to at least 20 m / s.

[0041] [Figure 5]This shows several serial cell culture runs using a 3L perfusion bioreactor at various gas efflux rates (GEV). The upper panel shows the viable cell concentration (VCC) over time, and the lower panel shows the viability percentage over time. FU27 (diamond) represents the control sample. FU28 (white square) represents a GEV of 10 m / s up to day 9, and then a GEV of 16 m / s thereafter. FU29 (white triangle) represents a GEV of 13 m / s, with culture rapidly declining on day 12. FU30 (X's) represents a GEV of 16 m / s up to day 9, and then a GEV that was reduced to 10 m / s thereafter.

[0042] [Figure 6] The viable cell concentration (10⁶ cells / mL) (upper panel) and survival percentage (lower panel) of shear-sensitive cells (e.g., SP2 / 0 cells) cultured at different dissolved CO₂ levels in a perfusion culture system are shown.

[0043] [Figure 7A] This figure compares the continuous culture performance of shear-sensitive cells at various scales while controlling GEV and dissolved CO2. The upper panel of Figure 7A shows the viable cell concentration (VCC) over time, and the lower panel of Figure 7A shows the viability percentage over time. [Figure 7B] This figure compares the continuous culture performance of shear-sensitive cells at various scales while controlling GEV and dissolved CO2. The upper panel of Figure 7B shows the gas efflux rate over time, and the lower panel of Figure 7B shows the dissolved CO2 over time.

[0044] [Figure 8] A table is shown outlining predictive models for various parameters for a 250L culture (for example, using a SUB250 bioreactor system).

[0045] [Figure 9]The graph shows the total glycan levels (top panel) and sialic acid content (bottom panel) for continuous cell cultures of shear-sensitive cells at various scales. For each, the leftmost bar represents a 3L culture process, the middle bar represents a 100L culture process, and the rightmost bar represents a 250L culture process.

[0046] [Figure 10] The predicted dissolved CO2 (upper panel) and gas efflux rate (lower panel) (black solid line) for a 1000L continuous culture of shear-sensitive cells are shown, compared to demonstrated culture processes in 250L (represented by triangles) and 100L (represented by circles).

[0047] [Figure 11] A table is shown outlining predictive models for various parameters for a 1000L culture (for example, using a SUB1000 bioreactor system).

[0048] [Figure 12] Here is an example equation for determining the gas outflow rate. [Modes for carrying out the invention]

[0049] Specific definition In general, the technical terms used herein have the same meaning as they are understood in the art unless explicitly indicated otherwise. Clear definitions of certain terms are given below. The meanings of these and other terms will, in particular, be evident to those skilled in the art from the context throughout this specification.

[0050] References or relevant portions thereof cited herein are incorporated herein by reference.

[0051] To facilitate understanding of the present invention, certain terms are first defined below. Further definitions of the following terms and other terms are provided throughout the specification.

[0052] As used herein, the terms “about” or “approximately” applied to one or more values ​​in question refer to values ​​similar to the reference value described. In certain embodiments, the terms “about” or “approximately” refer to a range of values ​​that are within or below 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% of the reference value described.

[0053] As used herein, the term “antibody” has the meaning understood in the art and refers to an immunoglobulin (Ig) that specifically binds to a particular antigen. As is known to those skilled in the art, naturally produced antibodies generally consist of four polypeptide chains: two heavy (H) chains and two light (L) chains. Each heavy and light chain has a variable region (hereinafter referred to as HCVR or V, respectively). H and LCVR or V L It consists of a heavy chain (abbreviated as C) and a steady region. The steady region of the heavy chain is C H 1. C H 2 and C H 3 domains (and optionally, C in the case of IgM and IgE) H It contains 4 domains. The constant region of the light chain is one domain, C L It consists of V. H and V L The region further includes a highly variable region called the Complementarity Determination Region (CDR), which is interspersed with a more conserved region called the Framework Region (FR). H and V L It consists of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The immunoglobulin molecule may be any type (e.g., IgM, IgD, IgG, IgA, and IgE), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass.

[0054] As used herein, the term “antibody drug” refers to a drug that specifically binds to a particular antigen. In some embodiments, this term encompasses any polypeptide having sufficient immunoglobulin structural elements to confer specific binding. In this context, suitable antibody drugs include, but are not limited to, monoclonal antibodies, polyclonal antibodies, humanized antibodies, primate-like antibodies, chimeric antibodies, human antibodies, bispecific or multispecific antibodies, single-domain antibodies (e.g., shark single-domain antibodies (e.g., IgNAR or its fragments)), conjugated antibodies (i.e., antibodies conjugated or fused with other proteins, radiolabeled substances, cytotoxins, etc.), Small Modular ImmunoPharmaceuticals ("SMIPs" trademark), single-chain antibodies, camelid antibodies, antibody fragments, etc. In some embodiments, this term may refer to staple peptides. In some embodiments, this term may refer to antibody-like conjugated peptide mimetics. In some embodiments, this term may refer to antibody-like conjugated scaffold proteins. In some embodiments, this term may refer to monobodies or adnectins. In many embodiments, the antibody drug is a polypeptide or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs). In some embodiments, the antibody drug is a polypeptide comprising, or comprising, at least one CDR (e.g., at least one heavy-chain CDR and / or at least one light-chain CDR) whose amino acid sequence is substantially identical to that found in the reference antibody. In some embodiments, the included CDR is substantially identical to the reference CDR, in which case it is either identical in sequence or contains between one and five amino acid substitutions when compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, in which case it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, in which case it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.In some embodiments, the included CDR is substantially identical to the reference CDR, in which case at least one amino acid in the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the included CDR is substantially identical to the reference CDR, in which case one to five amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the included CDR is substantially identical to the reference CDR, in which case at least one amino acid in the included CDR is substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the included CDR is substantially identical to the reference CDR, in which case one to five amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence otherwise identical to the reference CDR. In some embodiments, the antibody drug is a polypeptide or comprises the same, whose amino acid sequence contains structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody drug is a polypeptide protein having a binding domain that matches or largely matches the immunoglobulin binding domain. In some embodiments, the antibody drug is a polypeptide or comprises the same, which contains all the CDRs found in a particular reference antibody chain or multiple chains (e.g., heavy and / or light chains).

[0055] As used herein, the terms “biologics,” “biological therapies,” and “biologic products” are used synonymously and refer to polypeptides and protein products. For example, as used herein, biologics include, for example, naturally occurring or recombinant products expressed in cells, such as proteins, glycoproteins, fusion proteins, growth factors, vaccines, blood factors, thrombolytic agents, hormones, interferons, interleukin-based products, antibody drugs (e.g., monoclonal antibodies, bispecific antibodies, etc.), and therapeutic enzymes. Physical products are authorized under Section 351(a) of the Public Health (PHS) Act based on a “Biologics Product Authorization Application” or “BLA,” while biosimilars and alternative biologics that reference a BLA as a reference product are authorized under Section 351(k) of the PHS Act. Section 351 of the PHS Act is codified as 42 USC 262. Other biologics may also be authorized under Section 505(b)(1) of the Federal Food and Cosmetic Act, or as a simplified application under Sections 505(b)(2) and 505(j) of the Hatch Waxman Act, Section 505 is codified as 21 USC 355.

[0056] As used herein, “gas efflux rate” refers to the rate of gas efflux from a gas source in a bioreactor system, e.g., a gas source for aerating a culture medium (e.g., supplying air and / or oxygen). In some embodiments, the gas efflux rate refers to the rate at which gas flows out from one or more gas source openings (e.g., holes) and into the culture medium. In some embodiments, the gas efflux rate is calculated using the equation shown in Figure 12. In some embodiments, the bioreactor system includes a sparger, and the gas efflux rate refers to the rate of gas efflux from one or more sparger openings (e.g., holes). In some embodiments, the bioreactor system includes a sparger, and the gas efflux rate refers to the average rate of gas efflux from the openings of the sparger.

[0057] As used herein, “glycoprotein” refers to an amino acid sequence comprising one or more oligosaccharide chains (e.g., glycans) covalently bonded to the amino acid sequence. Examples of amino acid sequences include peptides, polypeptides, and proteins. Examples of glycoproteins include glycosylated antibodies, antibody drugs, and antibody-like molecules (e.g., Fc fusion proteins). Examples of antibodies include monoclonal antibodies and / or their fragments, polyclonal antibodies and / or their fragments, and Fc domain-containing fusion proteins (e.g., fusion proteins containing the Fc region of IgG1 or its glycosylated portion).

[0058] The term “isolated,” as used herein, means (1) separated from at least a portion of the components that were originally associated with the substance when it was first produced (whether in nature and / or in a laboratory setting), and / or (2) designed, produced, prepared, and / or manufactured by human hands. The isolated substance and / or component may be separated from more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% of the other components that were originally associated with the substance. In some embodiments, the isolated agent is ultrapure of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99%. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after being mixed with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the isolation percentage or purity of the substance is calculated without such carriers or excipients. In some embodiments, a biomolecule such as a naturally occurring polypeptide or polynucleotide is considered “isolated” if a) its origin or source is unrelated to some or all of the components that accompany it in its natural state in nature; b) it substantially does not contain other polypeptides or nucleic acids of the same species from which it is produced in nature; or c) it is expressed by a cell or other expression system that is not the species that produces it in nature, or is otherwise associated with components from there. Therefore, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system different from the one that produces it in nature is considered an “isolated” polypeptide. Or, or further, in some embodiments, a polypeptide subjected to one or more purification techniques may also be considered an “isolated” polypeptide to the extent that a) it is related in nature; and / or b) it is separated from other components related when it was first produced.

[0059] As used herein, “recovery” means a process, for example, using purification techniques known in the art, to render a drug or element substantially free of other pre-related components, such as by isolation. In some embodiments, the drug or element is recovered from natural sources and / or sources containing cells.

[0060] Generally, as used herein, “protein” is a polypeptide (i.e., a chain of at least two amino acids linked to one another by peptide bonds). Proteins may also contain non-amino acid portions (e.g., glycoproteins) and / or may be otherwise processed or modified. Those skilled in the art will understand that “protein” may be a complete polypeptide chain produced by a cell (with or without a signal sequence) or a functional protein thereof. Those skilled in the art will further understand that proteins may include two or more polypeptide chains linked, for example, by one or more disulfide bonds or otherwise associated.

[0061] The term "protein preparation," as used herein, refers to a mixture of proteins obtained according to a particular method of production. The proteins in a protein preparation may be the same or different; that is, a protein preparation may contain multiple copies of the same protein and / or a mixture of different proteins. In some embodiments, a protein preparation includes a glycoprotein preparation. A glycoprotein preparation is a composition or mixture comprising at least one glycoprotein. In some cases, a glycoprotein preparation contains multiple copies of the same protein (i.e., having the same amino acid sequence) but has a mixture of glycans associated with the protein. In some cases, a glycoprotein preparation is prepared using methods and / or systems provided herein. The production method may include a recombinant preparation step using cultured cells engineered to express the protein in the protein preparation (or to express the protein at an appropriate level or under appropriate conditions). In some embodiments, the production method may include an isolation step in which the protein is isolated from a specific component of the engineered cell (e.g., by lysing the cell and pelleting the protein component by centrifugation). In some embodiments, the production method may also include a purification step in which the protein in the protein preparation is separated (e.g., by chromatography) from other cellular components, such as other proteins or organic components used in previous steps. These steps are not limiting, and it will be understood that any number of additional production steps may be included. Different protein preparations may be prepared by the same production method but on different occasions (e.g., different runs or preparations). Alternatively, different protein preparations may be prepared by different production methods. The two production methods may differ in any way (e.g., expression vector, engineered cell type, culture conditions, isolation procedure, purification conditions, etc.).

[0062] As used herein, “samples” refers to separately obtained samples. In some embodiments, the evaluation of isolated samples includes evaluation of samples from the same culture run (e.g., at different points in time during preparation) or from different culture runs (e.g., different cycles of culture).

[0063] As used herein, “sparging” or “gas sparging” refers to the aeration or addition of a gas (e.g., air and / or O2) to a cell culture medium. Generally, gas sparging refers to the process of bubbling a gas directly into a culture medium (e.g., by a sparger). In some embodiments, sparging is used to achieve a dissolved O2 concentration of at least about 20%, or between about 20% and about 100%.

[0064] As used herein, “steady state” or “steady state conditions,” when used in reference to a cell culture system or process, means that the cell culture has a viable cell concentration that fluctuates by up to 20% over a period of at least 5 days (e.g., from 5 days to about 60 days). In some embodiments, the cell culture system has a viable cell concentration that fluctuates by up to 20% over a period of 5 days. In some embodiments, the cell culture system has a viable cell concentration that fluctuates by up to 15%, up to 10%, or up to 5% over a period of at least 5 days (e.g., 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, 50 days, 60 days). In some embodiments, the cell culture system has a viable cell concentration that fluctuates by up to 15%, up to 10%, or up to 5% over a period of 5 days. In some embodiments, the cell culture system has a viable cell concentration of shear-sensitive cells that fluctuates by up to 10% (i.e., within a range of plus or minus 10%) over a period of at least 5 days (e.g., 5, 10, 15, 20, 25, 30, 40, 50, 60 days). In some embodiments, the cell culture system has a viable cell concentration of shear-sensitive cells that fluctuates by up to 10% (i.e., within a range of plus or minus 10%) over a period of 5 days. In some embodiments, a steady-state viable cell concentration is achieved and / or maintained by draining from the bioreactor system (e.g., removal of excess cells).

[0065] All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, professional texts, and web pages, are incorporated in their entirety by explicit reference, regardless of the form of such documents and similar materials. If one or more of the incorporated documents and similar materials, including but not limited to defined terms, usage of terms, and the technology described, differ from or contradict this application, this application shall prevail. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein in any way.

[0066] This disclosure partially identifies and addresses the challenges of current continuous cell culture techniques (e.g., perfusion cell culture) where, in large culture volumes (e.g., at least 100 L, e.g., at least 200 L), certain cells have insufficient viable cell concentrations and reduced cell viability. This disclosure encompasses the recognition that scaling up high cell density continuous cell culture processes can be difficult due to the high demands on oxygen supply and dissolved carbon dioxide (pCO2) removal. This disclosure analyzes many different variables associated with large-scale continuous cell culture.

[0067] This disclosure encompasses the recognition that the importance of aeration increases with bioreactor volume and cell concentration. In particular, this disclosure provides insight that controlling the gas outflow rate of a bioreactor system can improve the performance of large-scale continuous culture of specific cells (e.g., shear-sensitive cells).

[0068] In some embodiments, the present disclosure provides methods and processes for large-scale (e.g., at least 100 L, e.g., at least 200 L) continuous culture (e.g., perfusion culture) of shear-sensitive cells, wherein the gas outflow rate of the bioreactor system is controlled and / or maintained within a specified amount and / or range. For example, large-scale continuous culture (e.g., perfusion bioreactor system) of shear-sensitive cells in a bioreactor system (e.g., perfusion bioreactor system) having a gas outflow rate of up to 20 m / s (e.g., up to 10 m / s) A method for large-scale culture is provided. The provided method is (for example, 20 × 10 6 From cells / mL to 15 × 10 7 This enables the formation of high-density cell cultures (with steady-state live cell concentrations within the range of cells / mL).

[0069] Continuous culture method and process This disclosure provides, in part, methods, processes, and / or systems for large-scale (e.g., at least 25 L, e.g., at least 100 L, e.g., at least 200 L) continuous culture (e.g., perfusion cell culture) of shear-sensitive cells (e.g., a population of cells containing shear-sensitive cells, e.g., a population of cells consisting of shear-sensitive cells). In some embodiments, the large-scale culture is from 25 L to 3,000 L. In some embodiments, the large-scale culture is 25 L, 50 L, 100 L, 200 L, 250 L, 400 L, 500 L, 600 L, 800 L, 1000 L, 1200 L, 1500 L, 2000 L, 3000 L or more. In some embodiments, the large-scale culture is 100 L or more. In some specific embodiments, the large-scale culture is 200 L or more. In some specific embodiments, the large-scale culture is 250 L or more.

[0070] In some cases, continuous cell culture systems (e.g., perfusion cell culture systems) are suitable for cells sensitive to high-density shear (e.g., at least 20 × 10⁶ cells). 6 cells / mL, e.g., at least 30 × 10⁶ 6 cells / mL, e.g., at least 40 × 10⁶ 6 This refers to a large-scale culture (e.g., 200 L or more) capable of generating a steady-state viable cell concentration of cells / mL.

[0071] In some cases, the methods, processes, and / or systems of continuous cell culture systems are approximately 20 × 10 6 Approximately 15 × 10 cells / mL 7 The steady-state viable cell concentration of shear-sensitive cells is and / or achieved within the range of cells / mL. In some embodiments, the steady-state viable cell concentration of shear-sensitive cells is an amount within a range limited by a lower limit and an upper limit, where the upper limit is greater than the lower limit. In some embodiments, the lower limit is about 20 × 10⁻⁶ 6 cells / mL, approximately 25×10 6 cells / mL, approximately 30×10 6 cells / mL, approximately 35×10 6 cells / mL, approximately 40×10 6 cells / mL, approximately 45×106 cells / mL, about 50×10 6 cells / mL, about 60×10 6 cells / mL, about 70×10 6 cells / mL, about 80×10 6 cells / mL, or about 90×10 6 cells / mL may be. In some embodiments, the upper limit is about 40×10 6 cells / mL, about 45×10 6 cells / mL, about 50×10 6 cells / mL, about 60×10 6 cells / mL, about 70×10 6 cells / mL, about 80×10 6 cells / mL, about 90×10 6 cells / mL, about 10×10 7 cells / mL, about 11×10 7 cells / mL, about 12×10 7 cells / mL, about 13×10 7 cells / mL, about 14×10 7 cells / mL or about 15×10 7 cells / mL may be.

[0072] Generally, the cell culture method of the present disclosure includes culturing at a temperature within the range of 25°C to 40°C under the gravity encountered on Earth.

[0073] In some embodiments, a continuous cell culture system (e.g., a perfusion cell culture system) has a gas outflow rate within the range of about 1 m / s to about 20 m / s. In some embodiments, a continuous cell culture system (e.g., a perfusion cell culture system) has a gas outflow rate within the range of about 1 m / s to about 10 m / s. In some embodiments, a continuous cell culture system (e.g., a perfusion cell culture system) has a gas outflow rate not exceeding 20 m / s. In some embodiments, a continuous cell culture system (e.g., a perfusion cell culture system) has a gas outflow rate not exceeding 10 m / s.

[0074] In some embodiments, the continuous cell culture system (e.g., the perfusion cell culture system) has a gas outflow rate within a range limited by the lower and upper limits, where the upper limit is greater than the lower limit. In some embodiments, the lower limit is about 1 m / s, about 2 m / s, about 3 m / s, about The speed may be 4 m / s, approximately 5 m / s, approximately 6 m / s, approximately 7 m / s, approximately 8 m / s, approximately 9 m / s, or approximately 10 m / s. In some embodiments, the upper limit may be approximately 5 m / s, approximately 6 m / s, approximately 7 m / s, approximately 8 m / s, approximately 9 m / s, approximately 10 m / s, approximately 11 m / s, approximately 12 m / s, approximately 13 m / s, approximately 14 m / s, approximately 15 m / s, approximately 16 m / s, approximately 18 m / s, or approximately 20 m / s.

[0075] In some embodiments, the continuous cell culture system (e.g., perfusion cell culture system) has dissolved carbon dioxide at a level not exceeding 120 mmHg. In some embodiments, the continuous cell culture system (e.g., perfusion cell culture system) has dissolved carbon dioxide at a level in the range of about 20 mmHg to about 120 mmHg. In some embodiments, dissolved carbon dioxide is present in the continuous cell culture medium in an amount limited by a lower limit and an upper limit, where the upper limit is greater than the lower limit. In some embodiments, the lower limit may be about 20 mmHg, about 30 mmHg, about 40 mmHg, about 50 mmHg, about 60 mmHg, or about 70 mmHg. In some embodiments, the upper limit may be about 50 mmHg, about 60 mmHg, about 70 mmHg, about 80 mmHg, about 90 mmHg, about 100 mmHg, about 110 mmHg, or about 120 mmHg.

[0076] In some embodiments, the continuous culture of shear-sensitive cells (e.g., a population of cells containing shear-sensitive cells, e.g., a population of cells consisting of shear-sensitive cells) is carried out for a period ranging from about 10 days to about 180 days. In some embodiments, the continuous culture of shear-sensitive cells (e.g., a population of cells containing shear-sensitive cells, e.g., a population of cells consisting of shear-sensitive cells) is carried out for a period of time limited by a lower limit and an upper limit, where the upper limit is greater than the lower limit. In some embodiments, the lower limit may be about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 50 days, or about 60 days. In some embodiments, the upper limit may be about 30 days, about 35 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days, about 90 days, about 100 days, about 120 days, about 140 days, about 160 days, or about 180 days.

[0077] In some embodiments, the continuous culture of shear-sensitive cells (e.g., a population of cells containing shear-sensitive cells, e.g., a population of cells consisting of shear-sensitive cells) is carried out for a period of at least 10 days. In some embodiments, the continuous culture of shear-sensitive cells (e.g., a population of cells containing shear-sensitive cells, e.g., a population of cells consisting of shear-sensitive cells) is carried out for a period of about 30 to about 60 days.

[0078] Bioreactor system This disclosure provides a bioreactor system for culturing, for example, shear-sensitive cells (e.g., a population of cells containing shear-sensitive cells, e.g., a population of cells consisting of shear-sensitive cells) for large-scale continuous cell culture. The bioreactor system of this disclosure is suitable for continuous culture methods. In some embodiments, the bioreactor system of this disclosure is suitable for perfusion culture. Figure 1 shows a schematic diagram of an example of a bioreactor system suitable for the culture method of this disclosure. In some embodiments, the bioreactor system includes a bioreactor tank and a cell holding device. In some embodiments, the bioreactor system includes a bioreactor tank, a cell holding device, a culture medium supply, and waste collection. In some embodiments, the bioreactor system further includes a population of cells (e.g., a population of cells consisting of shear-sensitive cells, e.g., an inoculation) and a cell culture medium.

[0079] In some embodiments, the bioreactor system includes a stirred-tank type bioreactor. In some embodiments, (for example, a stirred-tank type bioreactor) The bioreactor tank has a capacity ranging from 25 L to 3,000 L. In some embodiments, the bioreactor tank (for example, in a stirred-tank type bioreactor) has a capacity of at least 25 L, 50 L, 100 L, 200 L, 250 L, 300 L, 400 L, 500 L, 600 L, 800 L, 1000 L, 1200 L, 1400 L, 1500 L, 1600 L, 1800 L, 2000 L, 2400 L, 2500 L, 2600 L, 2800 L, or 3000 L. In some embodiments, the stirred-tank type bioreactor has a capacity of at least 100 L. In some embodiments, the stirred-tank type bioreactor has a capacity of at least 200 L. In some embodiments, the stirred-tank type bioreactor has a capacity of at least 250 L. In some embodiments, the stirred-tank type bioreactor has a capacity of at least 500 L. In some embodiments, the agitated tank type bioreactor has a capacity of at least 1000 L.

[0080] In some embodiments, the bioreactor system includes a sparger. Generally, a sparger can be used to introduce air and / or oxygen into the cell culture medium. This disclosure includes the recognition that the choice of sparger may also affect the rate and degree of aeration and minimizing foaming. In some embodiments, the bioreactor system includes an open-pipe sparger and / or a drill-hole sparger.

[0081] In some embodiments, the bioreactor system includes a drill-hole spagger. In some embodiments, the drill-hole spagger has a hole that is in the range of 0.05 mm to 5.0 mm in size. In some embodiments, the drill-hole spagger has a hole that is in the range of 0.1 mm to 1.0 mm in size. In some embodiments, the drill-hole spagger has a hole that is in the range of size limited by a lower limit and an upper limit, with the upper limit being greater than the lower limit. In some embodiments, the lower limit may be about 0.05 mm, about 0.06 mm, about 0.07 mm, about 0.08 mm, about 0.09 mm, about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, or about 1.0 mm. In some embodiments, the upper limit may be about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1.0 mm, about 1.25 mm, about 1.5 mm, about 1.75 mm, about 2.0 mm, about 2.25 mm, about 2.5 mm, about 2.75 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, or about 5.0 mm.

[0082] In some embodiments, the bioreactor system includes a sintered sparger. In some embodiments, the bioreactor system includes a sparger located at the bottom of the bioreactor tank. In some embodiments, the bioreactor system includes a sparger located approximately in the center of the bioreactor tank.

[0083] In some embodiments, the bioreactor system includes a cell retention device. Suitable cell retention devices for use in the bioreactor systems of this disclosure include a continuous centrifuge, an alternating tangential flow filter (ATF), a tangential flow membrane filter (TFF), a dynamic filter, a spin filter, an ultrasonic and dielectrophoretic separator, and a gravity sedimentation chamber. In some embodiments, the bioreactor system of this disclosure includes a cell alternating tangential flow (ATF) device. In some embodiments, the bioreactor system includes a cell retention device having one or more ATFs. In some embodiments, the bioreactor system includes a cell retention device having two ATFs. In some embodiments, the bioreactor system includes a cell retention device having three or more ATFs.

[0084] In some embodiments, the bioreactor system includes an impeller device. In some embodiments, the impeller is a bottom unit mounted inside the bioreactor tank. In some embodiments, the impeller is located approximately in the center of the bioreactor tank. In some embodiments, the impeller device is magnetically driven. The bioreactor system has a power output of approximately 5 W / m 3 From approximately 500W / m 3 It has an impeller stirring rate that exhibits a volume power input P / V.

[0085] Shear-sensitive cells In some cases, the culture methods, processes, and systems of this disclosure are for the continuous culture of shear-sensitive cells. This disclosure includes the recognition that animal cells without cell walls are sensitive to mechanical stress from the culture fluid in a stirred tank bioreactor, such as agitation caused by impellers in the culture medium in the stirred tank to supply oxygen to the cells, as well as by the generation and bursting of bubbles. In excess, these mechanical stresses from the fluid can result in damage / death of animal cells. Furthermore, this disclosure includes the recognition that certain animal cells are more sensitive to shear due to mechanical stress than others. For example, mouse myeloma cells may be more sensitive to shear than certain CHO cell lines. This disclosure provides methods, processes, and systems suitable for large-scale, high-density culture of shear-sensitive cells.

[0086] It is known in the art that certain cells, such as animal cells, are sensitive to certain mechanical and / or aeration conditions during culture (see, for example, Gooch et al., Curr. Opin. Biotech. 4:193-196 (1993)). As used herein, shear-sensitive cells are any animal cells that exhibit a certain level of sensitivity to shear (e.g., reduced cell viability) under conventional culture conditions (e.g., conventional large-scale culture conditions). In certain examples, as described herein, when such shear-sensitive cells are cultured under the conditions described herein, such cells exhibit an increased level of viability.

[0087] In some embodiments, the shear-sensitive cells are mammalian cells. In some embodiments, the shear-sensitive cells are murine cells. In some embodiments, the shear-sensitive cells are derived from mouse cell lines. Examples of shear-sensitive murine (e.g., mouse) cell lines include, for example, mouse myeloma cell lines. In some specific embodiments, the shear-sensitive cells are selected from NS0 cells and SP 2 / 0 cells.

[0088] In some embodiments, the shear-sensitive cells are human cells. Examples of shear-sensitive human cell lines include HEK293: human fetal kidney 293; HT-1080: derived from fibrosarcoma with an epithelial-like phenotype; PER.C6: derived from human embryonic retinal cells immortalized by transfection with the adenovirus E1 gene; CAP: derived from human amniotic cells immortalized by the adenovirus type 5 E1 gene; HKB-11: produced by polyethylene glycol fusion of HEK293-S and human B cell lines; and HuH-7: derived from human hepatocellular carcinoma. In some specific embodiments, the shear-sensitive cells are selected from HEK293 cells, fibrosarcoma HT1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, and HuH-7 cells.

[0089] In some specific embodiments, the shear-sensitive cells for use according to this disclosure are SP 2 / 0 cells. In some embodiments, the shear-sensitive cells for use according to this disclosure contain or are engineered to express a cell product.

[0090] According to this disclosure, conventional molecular biology, microbiology, and recombinant DNA technologies within the scope of the technology in the art may be utilized. Such technologies are described in the literature (e.g., Sambrook, Fritsch & Maniatis, Molecular Biology). Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985);Oligonucleotide Synthesis (MJ Gait ed. 1984);Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. (1985));Transcription And Translation (BD Hames & SJ Higgins, eds. (1984));Animal Cell Culture (RI Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)).

[0091] In some embodiments, shear-sensitive cells expressing the cell products described herein are prepared using recombinant methods. Recombinant expression of genes, such as genes encoding polypeptides, such as antibody drugs described herein, may include the construction of an expression vector containing polynucleotides encoding the polypeptide. Once the polynucleotides are available, the vector for the preparation of the polypeptide can be prepared by recombinant DNA techniques using techniques known in the art. Known methods can be used to construct an expression vector containing the polypeptide coding sequence and appropriate transcriptional and translational regulatory signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0092] The expression vector may be introduced into shear-sensitive cells by conventional techniques, and the transfected cells can then be cultured by the methods described herein to produce cell products (e.g., protein preparations, recombinant proteins, glycoproteins, fusion proteins, antibody drugs).

[0093] Cell culture medium This disclosure provides methods, processes, and systems for continuous cell culture in a culture medium sufficient for the expression of cell products (e.g., shear-sensitive cells, e.g., a population of cells containing shear-sensitive cells, e.g., a population of cells consisting of shear-sensitive cells). The cell culture medium generally contains a suitable energy source and compounds that regulate the cell cycle. Generally, the culture medium contains, for example, amino acids, vitamins, inorganic salts, and glucose known to those skilled in the art. In some embodiments, the cell culture medium has a pH of 6 to 8. Culture media for animal cells are well established in the art and are customarily optimized by those skilled in the art for specific purposes and / or cell types.

[0094] In some embodiments, the cell culture medium also includes an antifoaming agent (e.g., Antifoam C) in a concentration ranging from 1 ppm to 500 ppm. In some embodiments, shear-sensitive cells are cultured in a cell culture medium containing a shear force protectant (e.g., Pluronic F-68). They are cultured in a cell culture medium containing Pluronic F-68 at concentrations ranging from 1 g / L to 15 g / L. In some specific embodiments, a population of shear-sensitive cells may be cultured in a cell culture medium containing serum (e.g., fetal bovine serum).

[0095] Cell products In some cases, shear-sensitive cells or populations of cells consisting of shear-sensitive cells are continuously cultured using the methods, processes, and / or of the present disclosure to express cell products. In some embodiments, the cell products are or include nucleic acids, lipids, peptides, and / or proteins.

[0096] In some embodiments, the cell product is a recombinant protein. In some embodiments, the shear-sensitive cells of this disclosure contain nucleic acids encoding the recombinant protein. In some embodiments, the cell product is a glycoprotein. In some embodiments, the recombinant protein is a fusion protein. In some embodiments, the shear-sensitive cells of this disclosure contain nucleic acids encoding the fusion protein. In some embodiments, the recombinant protein is an Fc fusion protein. In some embodiments, the shear-sensitive cells of this disclosure contain nucleic acids encoding the Fc fusion protein.

[0097] In some embodiments, the cell product is an antibody drug. In some embodiments, the shear-sensitive cells of this disclosure contain nucleic acids encoding an antibody drug. In some embodiments, the cell product is a monoclonal antibody.

[0098] In some embodiments, the shear-sensitive cells of this disclosure include nucleic acids encoding glycoprotein complexes (e.g., Fc regions or Fc fragments comprising one or more N-glycosylation sites bound to or fused with one or more heterologous regions). Heterologous regions include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic drugs, synthetic drugs, inorganic molecules, and organic molecules. In some cases, the glycoprotein complex is a fusion protein comprising a peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or antibody mimetic fused with an Fc region such as a glycosylated Fc region. The fusion protein may include a linker region that connects the Fc region to the heterologous region (see, for example, Hallewell et al. (1989), J. Biol. Chem. 264, 5260-5268; Alfthan et al. (1995), Protein Eng. 8, 725-731; Robinson & Sauer (1996)).

[0099] In some embodiments, the shear-sensitive cells of this disclosure include nucleic acids encoding proteins approved, for example, in a secondary approval process, for therapeutic or diagnostic use in humans or animals.

[0100] In some embodiments, the shear-sensitive cells of this disclosure include nucleic acids encoding a protein having the same primary amino acid sequence as a protein approved, for example, in a secondary approval process, for therapeutic or diagnostic use in humans or animals. In some embodiments, the shear-sensitive cells of this disclosure include nucleic acids encoding a protein that differs from the approved therapeutic or diagnostic protein by no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 residues. In some embodiments, the shear-sensitive cells of this disclosure include nucleic acids encoding a protein having at least 90, 95, 98, 99%, or 100% sequence identity with that of the approved therapeutic or diagnostic protein. "Same primary amino acid sequence," "Primary amino acid sequence differing by no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 residues," "At least 98% or more sequence identity." The term "sequence having identity," or similar terms, refers to the level of identity between primary amino acid sequences. In some embodiments, the protein preparation or product contains amino acid variants, e.g., species with different terminal residues, e.g., one or two terminal residues. In some embodiments of such cases, the sequence identity being compared is the identity between the most abundant (e.g., most abundant active) species of primary amino acid sequences in each product being compared. In some embodiments, sequence identity refers to the amino acid sequence encoded by the nucleic acid that can be used to produce the product.

[0101] In some embodiments, the shear-sensitive cells of this disclosure include nucleic acids encoding proteins not authorized for therapeutic or diagnostic use in humans or animals.

[0102] Examples of recombinant proteins that are not restricted include abatacept (Orencia®, Bristol-Myers Squibb), absiximab (ReoPro®, Roche), adalimumab (Humira®, Bristol-Myers Squibb), aflibercept (Eylea®, Regeneron Pharmaceuticals), alefacept (Amevive®, Astellas Pharma), alemtuzumab (Campath®, Genzyme / Bayer), basiliximab (Simulect®, Novartis), and belatacept (Nulojix®, Bristol-Myers Squibb), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Roche), canakinumab (Ilaris®, Novartis), brentuximab vedotin (Adcetris®, Seattle Genetics), certolizumab (CIMZIA®, UCB, Brussels, Belgium), cetuximab (Erbitux®, Merck-Serono), daclizumab (Zenapax®, Hoffmann-La Roche), deniroikin difutitox (Ontak®, Eisai), denosumab (Prolia®, Amgen;Xgeva (registered trademark, Amgen), eculizumab (Soliris (registered trademark, Alexion Pharmaceuticals)), efalizumab (Raptiva (registered trademark, Genentech)), etanercept (Enbrel (registered trademark, Amgen-Pfizer)), gemtuzumab (Mylotarg (registered trademark, Pfizer)), golimumab (Simponi (registered trademark, Janssen)), ibritumomab (Zevalin (registered trademark, Spectrum Pharmaceuticals)), infliximab (Remicade (registered trademark, Centocor)), ipilimumab (Yervoy (trademark, Bristol-Myers Squibb)), muromonab (Orthoclone OKT3 (registered trademark, Janssen-Cilag)), natalizumab (Tysabri (registered trademark, Biogen)) Idec, Elan), ofatumumab (Arzerra®, GlaxoSmithKline), omalizumab (Xolair®, Novartis), palivizumab (Synagis®, MedImmune), panitumumab (Vectibix®, Amgen), ranibizumab (Lucentis®, Genentech), rilonacept (Arcalyst®, Regeneron); Examples include rituximab (MabThera®, Roche), tocilizumab (Actemra®, Genentech; RoActemra, Hoffman-La Roche), tositumomab (Bexxar®, GlaxoSmithKline), trastuzumab (Herceptin®, Roche), and ustekinumab (Stelara®, Janssen).

[0103] In some specific embodiments, the cell product is ustekinumab. Morphologically, shear-sensitive cells contain nucleic acids that encode ustekinumab.

[0104] In some embodiments, the shear-sensitive cells contain nucleic acids encoding an antibody drug comprising a heavy chain variable domain as described in SEQ ID NO: 1 and a light chain variable domain as described in SEQ ID NO: 2. In some embodiments, the shear-sensitive cells contain nucleic acids encoding an antibody drug comprising a heavy chain comprising the sequence of SEQ ID NO: 1 and a light chain comprising the sequence of SEQ ID NO: 2. In some embodiments, the shear-sensitive cells contain nucleic acids encoding an antibody drug comprising the HCDR1, HCDR2, and HCDR3 sequences as described in SEQ ID NO: 1 and the LCDR1, LCDR2, and LCDR3 sequences as described in SEQ ID NO: 2.

[0105] Sequence ID 1 - Ustekinumab heavy chain sequence (bold indicates the variable domain sequence including the underlined CDR sequence) [ka]

[0106] Sequence ID 2 - Ustekinumab light chain sequence (bold indicates the variable domain sequence including the underlined CDR sequence) [ka]

[0107] Pharmaceutical composition Cell products (e.g., recombinant proteins, e.g., glycoproteins, e.g., antibody drugs) produced or manufactured using any method, system and / or process described herein can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions may be useful for the prevention and / or treatment of diseases. Pharmaceutical compositions containing recombinant proteins (e.g., glycoproteins, e.g., antibody drugs) can be formulated by methods known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins, 2000). Pharmaceutical compositions may be administered parenterally in the form of injectable formulations comprising sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, a pharmaceutical composition may contain cell products (e.g., recombinant proteins, e.g., glycoproteins, e.g., antibody drugs) in a pharmaceutically acceptable vehicle or medium, e.g., sterile water and physiological fluids. It can be formulated by appropriately combining it with saline solution, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, diluents, vehicles, preservatives, and binders, and then mixing it into unit dosage forms required in generally accepted pharmaceutical practices. The amount of active ingredient contained in the pharmaceutical preparation is such that a suitable dose within the specified range is provided.

[0108] The administration route can be, for example, parenteral administration by injection, nasal administration, pulmonary administration, or transdermal administration. Administration may be systemic or local by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0109] The appropriate means of administration can be selected based on the patient's age and condition. A single dose of a pharmaceutical composition containing cell products (e.g., recombinant proteins, glycoproteins, antibody drugs) may be selected from a range of 0.001 mg / kg body weight to 1000 mg / kg body weight. In some embodiments, the dose may be selected from a range of 0.001 mg to 100,000 mg, but this disclosure is not limited to such a range. The dose and method of administration will vary depending on the patient's weight, age, condition, etc., and can be appropriately selected as needed by those skilled in the art.

[0110] This disclosure is further illustrated by the following embodiments. The embodiments are provided solely for illustrative purposes and should not be construed in any way as limiting the scope or content of this disclosure. [Examples]

[0111] Example 1: Conventional conditions for continuous culture of shear-sensitive cells are unsuitable for scaled-up processes. This example describes the identification of scale-up challenges in the continuous culture of shear-sensitive cells. In particular, this example shows the finding that large-scale (e.g., 200 L) perfusion culture of shear-sensitive cells without control of gas efflux rate resulted in reduced cell viability and insufficient viable cell concentration for biological production. Specifically, this example demonstrates the scale-up of a perfusion culture method for an example shear-sensitive cell line, SP2 / 0, which has been engineered to contain nucleic acids encoding example cell products (e.g., antibody drugs, e.g., monoclonal antibodies).

[0112] Conventional conditions for continuous cell culture (e.g., perfusion cell culture) of example shear-sensitive cells (e.g., SP2 / 0) expressing example antibody drugs were evaluated using a small-scale continuous culture system (e.g., up to 100 L), and the results are shown in Figure 2. Specifically, shear-sensitive cells grown in 3 L, 5 L, 15 L, and 100 L SUB100 disposable reactor systems showed comparable cell proliferation and steady-state viable cell concentrations. Shear-sensitive cells cultured in 3 L, 5 L, 15 L, and 100 L also had comparable productivity and product quality (data not shown).

[0113] We attempted to scale up a continuous cell culture (e.g., perfusion cell culture) of shear-sensitive cells, SP2 / 0, to 200L, and the results are shown in Figure 3. Specifically, we used a stirred tank type bioreactor system, as described below. [Table 1-1] [Table 1-2]

[0114] Initial growth was observed in all samples, but as shown in Figure 3, the 200L culture process using the GE / Xcellerex XDR200 disposable reactor system showed decreased cell growth (viable cell concentration of shear-sensitive cells (10)) starting from day 4. 6 Both a decrease in cells per mL (top) and a decrease in viability (percentage of viability (bottom)) were observed. Therefore, this example indicates that an improved method is needed for successful continuous culture of shear-sensitive cells on a large scale (e.g., at least 200 L).

[0115] Example 2: Root cause analysis of scaled serial cell culture of shear-sensitive cells This example identifies the causes of the reduced cell growth and viability of shear-sensitive cells, SP2 / 0 cells, in large scale (e.g., 200 L or more) shear, and provides a method for large scale continuous cell culture (e.g., perfusion cell culture) that results in high density shear-sensitive cells (e.g., having a steady state viable cell concentration of at least 20×10 6 cells / mL, e.g., at least 30×10 6 cells / mL, etc.).

[0116] In a 200 L continuous culture system, various parameters were analyzed to understand why the cell growth and viability of shear-sensitive cells decreased. The protocols and conditions were reviewed to ensure there were no issues with the raw materials (e.g., media, media prep filters, antifoam agents, inoculum, etc.). Parameters such as dissolved oxygen (DO), pH, and temperature were kept constant between small scale and large scale cultures (data not shown).

[0117] It was identified that a gas efflux velocity (GEV) rate > 20 m / s may be a contributing factor to the reduced growth and viability. To evaluate this hypothesis, a portion of the culture fluid (specifically, 2 L of cell culture) was transferred from a 200 L bioreactor (XDR200 bioreactor) to a 3 L bench-top bioreactor with a maximum GEV of 3 m / s on day 8 of the culture. The viable cell concentrations of the remaining 200 L culture system and the 3 L bench-top bioreactor were monitored. As shown in Figure 4, the viable cell concentration of the 3 L bench-top bioreactor recovered rapidly. On the other hand, the sparger gas efflux velocity in the 200 L bioreactor was then controlled (reduced) to a rate of 10 m / s or less from day 9 to day 13 of the culture. During this time, the viable cell concentration recovered. Further, when the GEV was increased to at least 20 m / s on day 13, the viable cell concentration decreased rapidly. These results suggest that controlling the GEV to a level of 10 m / s or less promotes cell viability for a successful scale-up (e.g., to pilot scale).

[0118] To determine the GEV threshold, continuous cell culture was performed using a 3L perfusion bioreactor at various gas outflow rates (GEV), as shown in Figure 5. Specifically, an increase in GEV from 10 m / s to 16 m / s resulted in a rapid decrease in viable cell concentration. Similarly, maintaining a GEV of 13 m / s also resulted in a sharp final decrease in viable cell concentration after 12 days of culture. On the other hand, decreasing the GEV from 16 m / s to 10 m / s increased the viable cell concentration.

[0119] Therefore, this embodiment successfully scales up continuous culture of shear-sensitive cells to large-scale culture (e.g., 200 L or more, e.g., 250 L or more) by controlling the sparger gas outflow rate to less than 20 m / s (e.g., less than 10 m / s), and allows for the cultivation of high-density shear-sensitive cells (e.g., at least 20 × 10⁶). 6 cells / mL, e.g., at least 30 × 10⁶ 6 This demonstrates that it can be produced at a steady-state concentration of cells / mL.

[0120] The effect of dissolved CO2 on the cell culture viability of shear-sensitive cells was also evaluated, and the results are shown in Figure 6. Specifically, it was found that an example of shear-sensitive cells (SP2 / 0 cells) is sensitive to dissolved carbon dioxide (pCO2). It was found that cell culture performance is adversely affected when pCO2 is ≥ 90 mmHg.

[0121] This example demonstrates that cell proliferation and viability of shear-sensitive cells in large-scale continuous culture can be restored by controlling the gas outflow rate (e.g., to less than 10 m / s). Furthermore, this example also shows that controlling the pCO2 level is beneficial for large-scale, high-density culture of shear-sensitive cells.

[0122] Example 3: Large-scale continuous cell culture of shear-sensitive cells This example demonstrates the successful large-scale continuous culture of shear-sensitive cells applying the understandings of Examples 1 and 2. Specifically, continuous cell culture was performed in a 250 L perfusion reactor (SUB250) with both GEV and pCO2 controlled. Figures 7A and 7B provide comparisons of cell density and cell viability between 3 L, 5 L, 15 L, 100 L (SUB100) and 250 L (SUB250). Specifically, Figures 7A and 7B provide further analysis of cell viability using GEV and pCO2 controlled at various levels at different cell culture scales (e.g., 100 L, e.g., 250 L). The SUB100, 100 L bioreactor contains 570 × 0.18 mm pores, and the SUB250, 250 L bioreactor contains 760 × 0.233 mm pores. As shown in Figure 7A, continuous cell culture at a scale of 250 L (SUB250) with a controlled GEV of approximately 7 m / s and a maximum dissolved carbon dioxide of 80 mmHg showed cell viability similar to that of smaller cultures. Figure 8 provides an overview of predictive models for various parameters for 250 L cultures (e.g., using a SUB250 bioreactor system). In particular, the CO2 levels predicted using these models for dissolved CO2 at 100 L and 250 L were remarkably similar to the actual data obtained (not shown).

[0123] Figure 9 shows that these sequential cultures of shear-sensitive cells at 3 L, 100 L, and 250 L all exhibit comparable total glycan levels (upper panel) and comparable sialic acid content (lower panel). Thus, this example demonstrates the successful scale-up of high-cell-density shear-sensitive cells (SP2 / 0) to a 250 L scale (e.g., using a Thermo Fisher / HyClone SUB250 (250 L) agitated tank bioreactor) via a perfusion culture process (Figures 7A, 7B, and 9).

[0124] Therefore, this embodiment can also control pCO2 levels in shear-sensitive cells (e.g., at least 20 × 10⁻¹⁰). 6 cells / mL, e.g., at least 30 × 10⁶ 6cell It also demonstrates that it is beneficial for large-scale cultivation at high densities (e.g., at least 100 L, e.g., 250 L) with a steady-state concentration of / mL. By controlling pCO2 to less than 80 mmHg and the sparger gas outflow rate to less than 10 m / s, the inventors were able to successfully scale up their process to a 3 L to 250 L stirred-tank bioreactor.

[0125] Therefore, this embodiment supports the idea of ​​controlling the GEV from the sparger to a rate of <10 m / s while simultaneously maintaining pCO2 ≤80 mmHg for successful scale-up of perfusion culture of shear-sensitive cells.

[0126] Example 4: Scaling continuous cell culture to production scale This example demonstrates the modeling of various parameters for large-scale continuous culture of shear-sensitive cells at production scale (e.g., 1000L or more). As shown in Figure 10, the predicted parameters for dissolved CO2 (upper panel) and gas efflux rate (lower panel) for 1000L continuous culture of shear-sensitive cells are comparable to those demonstrated in 250L and 100L culture processes. Furthermore, Figure 11 shows a table outlining the predictive model for various parameters for 1000L culture (e.g., using a SUB1000 bioreactor system). Thus, it is expected that the parameters measured for large scale (e.g., pilot scale) will also apply to even larger cultures, such as those using a 1000L bioreactor system.

[0127] Equal portions While this disclosure has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate, and not to limit, the scope of the invention as defined by the attached claims. Other aspects, advantages, and modifications are within the following claims. The inventions described in the original claims of this application are listed below. [Invention 1] In a bioreactor system with at least 25 L of culture medium and a gas outflow rate of up to 20 m / s, a population of shear-sensitive cells is cultured, resulting in 20 × 10⁻¹⁶ cells. 6 From cells / mL to 15 × 10 7 A method comprising achieving a steady-state viable cell concentration in the culture medium within the range of cells / mL. [Invention 2] The method according to Invention 1, wherein the gas outflow velocity is 15 m / s, 14 m / s, 13 m / s, 12 m / s, 11 m / s, 10 m / s, 9 m / s, 8 m / s or less. [Invention 3] The method according to invention 1 or 2, wherein the gas outflow rate is controlled at least until the bioreactor system reaches a steady state condition. [Invention 4] The method according to Invention 3, wherein the steady-state conditions include having a viable cell concentration that fluctuates by up to 20% over a period of 5 days. [Invention 5] The method according to any one of Inventions 1 to 4, wherein the gas outflow rate is controlled throughout the entire culture process. [Invention 6] The method according to any one of Inventions 1 to 5, wherein the bioreactor system is a perfusion bioreactor system. [Invention 7] The method according to Invention 6, wherein achieving the steady-state viable cell concentration by culturing a population of cells consisting of shear-sensitive cells in the perfusion bioreactor system includes draining or removing excess cells and / or non-viable cells. [Invention 8] The method according to any one of Inventions 1 to 7, wherein the culture medium has dissolved carbon dioxide at a level of up to 120 mmHg, up to 115 mmHg, up to 110 mmHg, up to 105 mmHg, up to 100 mmHg, up to 95 mmHg, up to 90 mmHg, up to 85 mmHg, or up to 80 mmHg. [Invention 9] (i) The gas outflow rate is 10 m / s or less, (ii) The culture medium has dissolved carbon dioxide at a maximum level of 80 mmHg. The method according to any one of inventions 1 to 8. [Invention 10] The method according to any one of Inventions 1 to 9, wherein the bioreactor system comprises at least 50 L, at least 100 L, at least 200 L, at least 500 L, at least 1,000 L, or at least 2,000 L of culture medium. [Invention 11] The method according to any one of inventions 1 to 10, wherein the culturing is carried out for a period of at least 10 days. [Invention 12] The method according to any one of inventions 1 to 11, wherein the culturing is carried out for a period of 30 to 60 days. [Invention 13] The method according to any one of inventions 1 to 12, further comprising measuring the concentration of living cells. [Invention 14] The measured viable cell concentration was at least 30 × 10 6 Cells / mL, at least 40 × 10 6 cells / mL, or at least 50 × 10 6 The method according to Invention 13, wherein the concentration is cells / mL. [Invention 15] The method according to any one of Inventions 1 to 14, wherein the shear-sensitive cells are mammalian cells. [Invention 16] The method according to any one of Inventions 1 to 15, wherein the shear-sensitive cells are human cells. [Invention 17] The method according to any one of Inventions 1 to 16, wherein the shear-sensitive cells are HEK293 cells, fibrosarcoma HT1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, or HuH-7 cells. [Invention 18] The method according to any one of Inventions 1 to 17, wherein the shear-sensitive cells are murine cells. [Invention 19] The method according to any one of inventions 1 to 15 and 18, wherein the shear-sensitive cells are a mouse myeloma cell line. [Invention 20] The method according to any one of inventions 1 to 15, 18, and 19, wherein the shear-sensitive cells are NS0 cells or SP 2 / 0 cells. [Invention 21] The bioreactor system is a method according to any one of inventions 1 to 20, comprising a cell retention device. [Invention 22] The method according to Invention 21, wherein the cell retention device is a continuous centrifuge, an alternating tangential flow filter (ATF), a tangential flow membrane filter (TFF), a dynamic filter, a spin filter, an ultrasonic and dielectrophoretic separator, and / or a gravity sedimentation device, or includes them. [Invention 23] The method according to invention 21 or 22, wherein the cell retention device is one or more ATFs, or includes one or more ATFs. [Invention 24] The bioreactor system comprises a bioreactor tank, the bioreactor tank having a capacity of at least 50L, 100L, 200L, 500L, 1,000L, or 2,000L, according to any one of inventions 1 to 23. [Invention 25] The method according to invention 24, wherein the bioreactor tank is a stirred tank type bioreactor. [Invention 26] The bioreactor system is the method according to any one of inventions 1 to 26, including a sparger. [Discussion 27] The method according to invention 26, wherein the sparger is a drill hole sparger or an open pipe sparger. [Invention 28] The method according to any one of Inventions 1 to 27, wherein the culture of the aforementioned population of cells is carried out under conditions that express a cell product. [Invention 29] The method according to Invention 28, wherein the cell product is a nucleic acid, a lipid, a peptide, and / or a protein, or comprises the same. [Invention 30] The method according to Invention 29, wherein the cell product is a recombinant protein. [Invention 31] The method according to invention 30, wherein the recombinant protein is a glycoprotein. [Invention 32] The method according to Invention 31, wherein the glycoprotein is an Fc-containing glycoprotein. [Invention 33] The method according to invention 31 or 32, wherein the glycoprotein is an antibody drug. [Invention 34] The method according to Invention 33, wherein the antibody drug is a monoclonal antibody. [Invention 35] The method according to invention 34, wherein the monoclonal antibody is ustekinumab. [Invention 36] The method according to any one of inventions 28 to 35, further comprising isolating the cell product from at least a portion of the shear-sensitive cells and / or isolating the cell product from at least a portion of the culture medium. [Invention 37] A continuous culture process for culturing a population of shear-sensitive cells, comprising controlling the gas outflow rate of a bioreactor system so as not to exceed a rate of 20 m / s, The bioreactor system includes at least 25 L of culture medium, and the cell population is 20 × 10 6 From cells / mL to 15 × 10 7 The process for achieving a steady-state live cell concentration within the range of cells / mL. [Invention 38] The bioreactor system has a gas outflow rate of 15 m / s, 14 m / s, 13 m / s, 12 m / s, 11 m / s, 10 m / s, 9 m / s, and 8 m / s or less, according to the process of invention 37. [Invention 39] The process according to invention 37 or 38, wherein the gas outflow rate is controlled at least until the bioreactor system reaches a steady state condition. [Invention 40] The process according to Invention 39, wherein the steady-state conditions include having a viable cell concentration that fluctuates by up to 20% over a period of 5 days. [Invention 41] The process according to any one of inventions 37 to 40, wherein the gas outflow rate is controlled throughout the entire culture process. [Invention 42] The process according to any one of inventions 37 to 41, wherein the bioreactor system is a perfusion bioreactor system. [Invention 43] The process according to Invention 42, wherein in the perfusion bioreactor system, culturing a population of cells consisting of shear-sensitive cells to achieve the steady-state viable cell concentration includes draining or removing excess cells and / or non-viable cells. [Invention 44] The process according to any one of inventions 37 to 43, further comprising controlling the dissolved carbon dioxide level of the cell culture medium. [Invention 45] The process according to Invention 44, wherein the culture medium has dissolved carbon dioxide at a level of 120 mmHg, 115 mmHg, 110 mmHg, 105 mmHg, 100 mmHg, 95 mmHg, 90 mmHg, 85 mmHg, or 80 mmHg or less. [Invention 46] (i) The gas outflow rate is 10 m / s or less, (ii) The dissolved carbon dioxide level is 80 mmHg or less. The process described in either invention 44 or 45. [Invention 47] The process according to any one of Inventions 37 to 46, wherein the perfusion bioreactor system comprises at least 50 L, at least 100 L, at least 200 L, at least 500 L, at least 1,000 L, or at least 2,000 L of culture medium. [Invention 48] The continuous culture process is carried out for a period of at least 10 days, as described in any one of inventions 37 to 47. [Invention 49] The continuous culture process is the process described in any one of inventions 37 to 48, which is carried out for a period of 30 to 60 days. [Invention 50] The process according to any one of inventions 37 to 49, wherein the process includes measuring the concentration of living cells. [Invention 51] The measured viable cell concentration was at least 30 × 10 6 Cells / mL, at least 40 × 10 6 cells / mL, or at least 50 × 10 6 The process described in Invention 50, wherein the result is cells / mL. [Invention 52] The process according to any one of inventions 37 to 51, wherein the shear-sensitive cells are mammalian cells. [Invention 53] The process according to any one of inventions 37 to 52, wherein the shear-sensitive cells are human cells. [Invention 54] The shear-sensitive cells are HEK293 cells, fibrosarcoma HT1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, or HuH-7 cells, according to any one of Inventions 37-53. [Invention 55] The process according to any one of inventions 37 to 52, wherein the shear-sensitive cells are murine cells. [Invention 56] The process according to any one of inventions 37-52 and 55, wherein the shear-sensitive cells are a mouse myeloma cell line. [Invention 57] The process according to any one of inventions 37-52, 52, and 56, wherein the shear-sensitive cells are NS0 cells or SP 2 / 0 cells. [Invention 58] The bioreactor system comprises a cell retention device, the process according to any one of inventions 37 to 57. [Invention 59] The process according to Invention 58, wherein the cell retention device is a continuous centrifuge, an alternating tangential flow filter (ATF), a tangential flow membrane filter (TFF), a dynamic filter, a spin filter, an ultrasonic and dielectrophoretic separator, or a gravity sedimentation chamber, or includes these. [Invention 60] The process according to invention 58 or 59, wherein the cell retention device is one or more ATFs, or comprises them. [Invention 61] The process according to any one of Inventions 37 to 60, wherein the bioreactor system includes a bioreactor tank having a capacity of at least 50 L, 100 L, 200 L, 500 L, 1,000 L, or 2,000 L. [Invention 62] The process according to invention 61, wherein the bioreactor tank is a stirred tank type bioreactor. [Invention 63] The bioreactor system comprises a sparger, the process according to any one of inventions 37 to 62. [Invention 64] The process according to invention 63, wherein the sparger is a drill hole sparger or an open pipe sparger. [Invention 65] The continuous culture process for culturing the population of cells is carried out under conditions that express a cell product, according to any one of inventions 37 to 64. [Invention 66] The process according to Invention 65, wherein the cell product is a nucleic acid, a lipid, a peptide, and / or a protein, or comprises them. [Invention 67] The process according to invention 66, wherein the cell product is a recombinant protein. [Invention 68] The process according to Invention 67, wherein the recombinant protein is a glycoprotein. [Invention 69] The process according to Invention 68, wherein the glycoprotein is an Fc-containing glycoprotein. [Invention 70] The process according to invention 68 or 69, wherein the glycoprotein is an antibody drug. [Invention 71] The process according to Invention 70, wherein the antibody drug is a monoclonal antibody. [Discussion 72] The process according to Invention 71, wherein the monoclonal antibody is ustekinumab. [Invention 73] The process according to any one of inventions 65 to 72, further comprising isolating the cell product from at least a portion of the shear-sensitive cells and / or isolating the cell product from at least a portion of the culture medium.

Claims

1. In a perfusion bioreactor system having dissolved carbon dioxide at a maximum level of 80 mmHg and a gas outflow rate of 7 to 10 m / s, a population of cells consisting of shear-sensitive mammalian cells is cultured, 20 × 10 ６ Cells / mL to 15 x 10 ７ A method comprising achieving a steady-state viable cell concentration in culture medium within the range of cells / mL, wherein the shear-sensitive mammalian cells are HEK293 cells, fibrosarcoma HT1080 cells, PER. C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, or SP2 / 0 cells.

2. The method according to claim 1, wherein the shear-sensitive mammalian cell is a human cell or a murine cell.

3. The method according to claim 1, wherein the gas outflow rate is controlled at least until the bioreactor system reaches the steady-state conditions.

4. The method according to claim 3, wherein the steady-state conditions include having a viable cell concentration that fluctuates by up to 20% over a period of 5 days.

5. The method according to claim 1, wherein the gas outflow rate is controlled throughout the entire culture.

6. The method according to claim 1, wherein the culturing further comprises draining or removing excess cells and / or non-living cells.

7. The method according to claim 1, wherein the culturing is carried out for a period of 30 to 60 days.

8. The method according to claim 1, further comprising measuring the concentration of living cells.

9. The method according to claim 8, wherein the measured live cell concentration is at least 30 × 10⁶ cells / mL, at least 40 × 10⁶ cells / mL, or at least 50 × 10⁶ cells / mL.

10. The method according to claim 1, wherein the bioreactor system includes a cell retention device, the cell retention device being or including a continuous centrifuge, an alternating tangential flow filter (ATF), a tangential flow membrane filter (TFF), a dynamic filter, a spin filter, an ultrasonic and dielectrophoretic separator, and / or a gravity sedimentation device.

11. The method according to claim 1, wherein the bioreactor system includes a sparger.

12. The method according to claim 11, wherein the sparger is a drill hole sparger or an open pipe sparger.

13. The method according to claim 1, wherein the culture of the population of cells is carried out under conditions that express a cell product, the cell product comprising one or more of nucleic acids, lipids, peptides, and proteins.

14. The method according to claim 13, wherein the cell product is an antibody drug.

15. The method according to claim 13, further comprising isolating the cell product from at least a portion of the shear-sensitive mammalian cells and / or isolating the cell product from at least a portion of the culture medium.

16. Controlling the gas outflow rate of the perfusion bioreactor system to be between 7 and 10 m / s, and Control the dissolved carbon dioxide level in the culture medium so that the dissolved carbon dioxide level reaches a maximum of 80 mmHg. A continuous culture process for culturing a population of cells consisting of shear-sensitive mammalian cells, including, The cell population achieves a steady-state viable cell concentration in the range of 20 × 10⁶ cells / mL to 15 × 10⁷ cells / mL, and the shear-sensitive mammalian cells are HEK293 cells, fibrosarcoma HT1080 cells, PER. C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, or SP2 / 0 cells in the process.

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