Methods for continuous cell culture

Abstract

This provides a continuous culture process for culturing populations of shear-sensitive cells. [Solution] A continuous culture process for culturing a population of cells consisting of shear-sensitive cells, comprising controlling the gas outflow rate of a bioreactor system so that the gas outflow rate does not exceed 20 m / s, wherein the bioreactor system includes at least 25 L of culture medium, and the population of cells is 20 × 10 6 From cells / mL to 15 × 10 7 The process is characterized by achieving a steady-state viable cell concentration within the range of cells / mL, and the gas efflux rate being controlled at least until the bioreactor system reaches steady-state conditions.

Description

[Technical Field]

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

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

[0003] This disclosure particularly relates to cell products, such as recombinant proteins, such as glycoproteins, e.g. For example, a continuous culture method for generating (e.g., manufacturing) antibody drugs or fusion proteins. The method is provided. In some cases, the method described herein is a continuous culture method (e.g., perfusion). This disclosure enables the large-scale production of recombinant proteins using a culture method. In large-scale cell cultures (e.g., at least 25L, e.g., at least 100L), Current continuous cell culture techniques with sufficient viable cell concentration and reduced cell viability (e.g., irrigation Identify and address challenges in fluid cell culture. This disclosure partially addresses shear-sensitive cells. For large-scale (e.g., at least 25L, e.g., at least 100L) continuous culture of cells We provide a method and system for this purpose.

[0004] This disclosure relates to the large-scale culture of shear-sensitive cells and the gas outflow from bioreactor systems. It provides insight that this can be achieved by controlling speed. For example, maximum Controlled and / or maintained within a specified amount and / or range so as to be 20 m / s. A bioreactor system having a gas outflow rate (e.g., perfusion bioreactor system Large-scale culture of shear-sensitive cells in (M) is approximately 20 × 10 6 Approximately 15 cells / mL ×10 7 To achieve a steady-state viable cell concentration of shear-sensitive cells within the range of cells / mL. It is possible.

[0005] In some cases, a bioreactor system (e.g., a perfusion bioreactor system) may be used. ) In this, a population of cells containing or consisting of shear-sensitive cells is cultured to determine A method comprising achieving a steady-state viable cell concentration, wherein the steady-state viable cell concentration is 20 × 10 6 From cells / mL to 15 × 10 7 The cells / mL range is within the bioreactor system The method 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 is provided with a speed of approximately 15 m / s. Below, 14m / s or less, 13m / s or less, 12m / s or less, 11m / s or less, 10m / s or less The gas outflow rate is controlled to a rate of 9 m / s or less, or 8 m / s or less. In one embodiment, the bioreactor system includes a sparger. Several implementations In terms of morphology, spargers are rated for speeds of approximately 15 m / s or less, 14 m / s or less, 13 m / s or less, and 12 m / s or less. Rates of 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. It has a controlled gas outflow rate.

[0006] In some cases, a bioreactor system containing at least 25L of culture medium (for example) This includes culturing shear-sensitive cells in a perfusion bioreactor system. The method involves a bioreactor system having a gas outflow rate of up to 20 m / s. Furthermore, shear-sensitive cells are approximately 20 x 10 6 Approximately 15 × 10 cells / mL 7 cells / mL A method is provided in which the cells are present at a steady-state viable cell concentration within a range. In some embodiments, Bioreactor systems (e.g., perfusion bioreactor systems) are suitable for applications with a flow rate of 15 m / s or less. Below, 14m / s or less, 13m / s or less, 12m / s or less, 11m / s or less, 10m / s or less The gas outflow rate is less than or equal to 9 m / s, or less than or equal to 8 m / s. Several implementations In this configuration, the gas outflow rate is controlled throughout the entire culture process. In some embodiments, The gas outflow rate is controlled at least until the bioreactor system reaches steady-state conditions. To be controlled.

[0007] In some cases, at least 25 L (e.g., at least 200 L) of culture medium and the A bioreactor with a gas outflow velocity of up to 20 m / s (for example, a maximum of 10 m / s) In the system, a population of cells consisting of shear-sensitive cells is cultured, 20 × 10 6 cell From / mL to 15 × 10 7 Achieve a steady-state viable cell concentration in the culture medium within the range of cells / mL. A method is provided. In some embodiments, the steady-state live cell concentration is set over a period of 5 days. The concentration of living cells fluctuates by up to 20% over time.

[0008] In some cases, the gas outflow rate of the bioreactor system is controlled so as not to exceed a rate of 20 m / s, in a continuous culture process for culturing a population of cells composed of shear-sensitive cells, wherein the bioreactor system contains at least 25 L of culture medium, and the population of cells achieves a steady-state viable cell concentration within the range of 20×10 cells / mL to 15×10 cells / mL. In some embodiments, the gas outflow rate is controlled at 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 6 embodiments, the sparger gas outflow rate is controlled at a rate of about 15 m / s or less, 14 m / s or less, 7 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 outflow rate is controlled for at least a portion of the period during which the shear-sensitive cells are cultured. In some embodiments, the control of the gas outflow rate begins when the population of cells (e.g., a population of shear-sensitive cells, e.g., inoculum) is added to the bioreactor system (e.g., when the population of cells is mixed with the culture medium of the bioreactor system). In some embodiments, the control of the gas outflow rate is after the population of cells (e.g., a population of shear-sensitive cells, e.g., inoculum) is added to the bioreactor system (e.g., when the population of cells is mixed with the culture medium of the bioreactor system).

[0009] ​​​​​​​​​​​​​It will start after approximately 2 hours, 6 hours, 12 hours, 24 hours, or 48 hours.

[0010] In some embodiments, the control of the gas outflow rate is performed on the cultured cells during the culture process. It starts when the cells reach a certain density. In some embodiments, the gas outflow rate is controlled. The bioreactor system has at least 1 × 10 6 cells / mL, 2×10 6 cell / mL, 5 x 10 6 cells / mL, or 10 × 10 6 Shear-sensitive cells at a rate of cells / mL It starts when it has a concentration of I

[0011] In some embodiments, the gas outflow rate is fixed by at least the bioreactor system. It is controlled until a steady state condition is reached. In some embodiments, the steady state condition is 5 days. This includes having a viable cell concentration that fluctuates by up to 20% over a period of time. Several embodiments So, the gas outflow rate is determined by the cell population (for example, a population of shear-sensitive cells, for example, inoculated ) when added to a bioreactor system (for example, when a population of cells is bio (When mixed with the culture medium of the reactor system) the culture is (for example, for a period of 5 days) (With a viable cell concentration that fluctuates by up to 20%), controlled until a steady state condition is reached. In some embodiments, the gas outflow rate is such that the bioreactor system is at least 1 ×10 6 When the cell concentration is such that it is shear-sensitive, the culture is ( For example, under steady-state conditions (with a viable cell concentration that fluctuates by up to 20% over a 5-day period) It will be controlled until it reaches its goal.

[0012] In some embodiments, the gas outflow rate is controlled throughout the entire culture process. In one embodiment, the gas outflow rate is at least about 10 days, at least about 15 days, At least approximately 20 days, at least approximately 25 days, at least approximately 30 days, at least approximately 4 Controlled for 0 days, at least approximately 50 days, or at least approximately 60 days of culture. In some embodiments, the gas outflow rate is determined by the cell population (for example, a cluster of shear-sensitive cells). When a group (for example, an inoculation) is added to a bioreactor system (for example, cells) (When the population is mixed with the culture medium of the bioreactor system) it is controlled and less 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 less Both are continued for approximately 60 days of incubation. In some embodiments, the gas efflux rate is bio The reactor system has at least 1 × 10 6 Shear-sensitive cell concentration is cells / mL When the culture has such characteristics, (for example, when the life changes by up to 20% over a period of 5 days) The cells are controlled until steady-state conditions (with sufficient cell concentration) are reached, for at least about 10 days, and less Approximately 15 days, at least approximately 20 days, at least approximately 25 days, at least approximately 30 days , culture for at least approximately 40 days, at least approximately 50 days, or at least approximately 60 days It continues for a while.

[0013] In some cases, the perfusion bioreactor system should not exceed a rate of 20 m / s. Perfusion culture for culturing shear-sensitive cells, including controlling the gas outflow rate. The perfusion bioreactor system comprises at least 25 L of culture medium. The culture medium contains shear-sensitive cells, and these shear-sensitive cells are approximately 20 × 10 6 cell From / mL to approximately 15 × 10 7 The process is present at steady-state viable cell concentrations within the range of cells / mL. A is provided. In some embodiments, the gas outflow rate of the perfusion bioreactor system The speed is controlled so that it does not exceed a rate of 10 m / s.

[0014] In some embodiments, the steady-state viable cell concentration of shear-sensitive cells is such that the upper limit is greater than the lower limit. It is a quantity that is also large, within a range limited by the lower and upper limits. In some embodiments The lower limit is approximately 20 × 10 6 cells / mL, approximately 25×10 6 cells / mL, approximately 30×10 6 Thin cells / mL, approximately 35×10 6 cells / mL, approximately 40×10 6 cells / mL, approximately 45×10 6 cell / mL, approx. 50×10 6 cells / mL, approximately 60×10 6 cells / mL, approximately 70×10 6 cell / mL, approx. 80×10 6 Cells / mL, or approximately 90 × 10⁻⁶ 6 It may be cells / mL. In some embodiments, the upper limit is approximately 40 × 10 6 cells / mL, approximately 45×10 6 cells / mL , about 50×10 6 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, approx. 11×10 7 cells / mL, approximately 12×107 cells / mL, approximately 13×10 7 cells / mL, approximately 1 4 x 10 7 Cells / mL or approximately 15 × 10⁴ 7 Cells / mL may also be acceptable.

[0015] In some embodiments, the perfusion bioreactor system controls the level of dissolved It contains carbon dioxide. In some embodiments, the culture medium has a humidity of 120 mmHg or less, 11 5mmHg or less, 110mmHg or less, 105mmHg or less, 100mmHg or less, 95 The values ​​are below mmHg, below 90 mmHg, below 85 mmHg, or below 80 mmHg. Contains dissolved carbon dioxide from Bell.

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

[0017] In some embodiments, the cell culture system has a solvent level of 120 mmHg or less. It contains carbon dioxide. In some embodiments, it is used in continuous cell culture systems (e.g., perfusion). The cell culture system maintains a dissolved oxygen level in the range of approximately 20 mmHg to 120 mmHg. It contains oxidized carbon. In some embodiments, the dissolved carbon dioxide is greater than the upper limit. It is present in continuous cell culture medium in amounts limited by the lower and upper limits. In one embodiment, the lower limits are approximately 20 mmHg, approximately 30 mmHg, approximately 40 mmHg, and approximately 5 It may be 0 mmHg, approximately 60 mmHg, or approximately 70 mmHg. Several implementations In this state, the upper limits are approximately 50 mmHg, 60 mmHg, 70 mmHg, and 80 mmHg. The blood pressure was approximately 90 mmHg, 100 mmHg, 110 mmHg, or 120 mmHg. That's fine.

[0018] In some embodiments, the perfusion bioreactor system has at least 50 L, and less At least 100L, at least 200L, at least 500L, at least 1,000L, Or it includes at least 2,000 L of culture medium.

[0019] In some embodiments, continuous culture of shear-sensitive cells is performed for at least 10 days. This is carried out over time. In some embodiments, continuous culture of shear-sensitive cells is performed for about 30 to 30 days. It will be implemented over a period of approximately 60 days.

[0020] In some embodiments, continuous culture of shear-sensitive cells is performed for 10 to 180 days. The process is carried out for a period within the range. In some embodiments, continuous culture of shear-sensitive cells is performed. , implemented for a period of time within the 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 10 days, about 15 days, about 20 days, and about 2 5 days, approximately 30 days, approximately 35 days, approximately 40 days, approximately 50 days, or approximately 60 days This is also acceptable. In some embodiments, the upper limit is about 30 days, about 35 days, about 40 days, about 5 0 days, approximately 60 days, approximately 70 days, approximately 80 days, approximately 90 days, approximately 100 days, approximately 120 days The interval could be approximately 140 days, 160 days, or 180 days.

[0021] In some embodiments, the serial culture method of the present disclosure includes the step of measuring the viable cell concentration. This includes. In some embodiments, the measured live cell concentration is at least 30 × 10 6 Thin Cells / mL, at least 40 × 106 cells / mL, or at least 50 × 10 6 cells / m It is L.

[0022] In some embodiments, shear-sensitive cells for culture according to the present disclosure are mammary glands It is a physical cell.

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

[0024] In some embodiments, the shear-sensitive cells are human cells. In this context, a human cell line sensitive to shear is HEK293: Human fetal kidney 293;HT- 1080: Derived from fibrosarcoma with an epithelial-like phenotype; PER.C6: Adenovirus E1 gene Derived from immortalized human embryonic retinal cells transfected by offspring; CAP: Ade Derived from human amniotic cells immortalized by the novirus type 5 E1 gene; HKB-11:HEK Prepared by polyethylene glycol fusion of 293-S and human B cell lines; and HuH-7: Selected from human hepatocellular carcinoma. In some specific embodiments, shear Cells sensitive to this include HEK293 cells, fibrosarcoma HT1080 cells, PER.C6 cells, and C The cells are selected from AP cells, HKB-11 cells, and HuH-7 cells.

[0025] In some embodiments, shear-sensitive cells for use according to this disclosure are cell production It contains or is manipulated to express a substance. In some embodiments, it is cultured Shear-sensitive cells contain or express cell products. Several implementations In this state, cell products are nucleic acids, lipids, peptides, and / or proteins, or These include. In some embodiments, the cell product is a recombinant protein. In some embodiments, the recombinant protein is a glycoprotein. Therefore, the glycoprotein is an Fc-containing glycoprotein. In some embodiments, the glycoprotein The protein is an antibody drug. In some embodiments, the antibody drug is a monoclonal antibody. In some specific embodiments, the monoclonal antibody is ustekinumab. .

[0026] In some embodiments, the shear-sensitive cells of this disclosure are used in human or animal therapeutic applications. Antibody drugs approved, for example, in the secondary approval process, for therapeutic or diagnostic use. Includes nucleic acids encoding. In some specific embodiments, the shear-sensitive cells of the Disclosure It contains the nucleic acid that codes for ustekinumab.

[0027] In some embodiments, the methods and / or processes of the present disclosure involve at least one cell To isolate cell products from a portion and / or from at least a portion of the culture medium This includes isolation. In some embodiments, the cell product is a recombinant protein, for example. For example, glycoproteins, such as Fc-containing glycoproteins, such as antibody drugs, such as monoc This is a ronal antibody.

[0028] In some embodiments, the cell culture medium is concentrated in the range of 1 ppm to 500 ppm. It also includes an antifoaming agent (e.g., Antifoam C). In some embodiments, it is shear sensitive. These cells are cultured in a cell culture medium containing a shear force protectant (e.g., Pluronic F-68). They are cultured in a certain environment. In some embodiments, shear-sensitive cells (for example, when sheared) are used. A population of sensitive cells was treated with Pluronic F at concentrations ranging from 1 g / L to 15 g / L. They are cultured in a cell culture medium containing -68. In some embodiments, they are shear-sensitive. A population of cells is exposed to an antifoaming agent (e.g., Antifoam C) in a range of 1 ppm to 500 ppm. The concentration within the range and the shear force protectant (e.g., Pluronic F-68) are 1 g / L or The cells are cultured in a culture medium containing the substance at a concentration within the range of 15 g / L.

[0029] In some embodiments, the perfusion bioreactor system includes a bioreactor tank. This includes. In some embodiments, the bioreactor tank is a stirred bioreactor It is a tank. In some embodiments, the bioreactor tank has a capacity of at least 50 L , 100L, 200L, 250L, 400L, 500L, 600L, 800L, 1,00 It has a capacity of 0L or 2,000L. In some specific embodiments, BioRia The tanks have capacities of approximately 200L, 250L or more.

[0030] In some embodiments, a bioreactor system (e.g., a perfusion bioreactor) is used. The system includes a cell retention device. In some embodiments, the cell retention device , continuous centrifuge, alternating tangential flow filter (ATF), tangential TFF (Transcatheter Flow Membrane) filters, dynamic filters, spin filters, ultrasonic filters Wave and dielectrophoretic separators, or gravity settlers, or including them. In some specific embodiments, the cell retention device is or includes an ATF.

[0031] In some embodiments, the bioreactor system is a stirred-tank type bioreactor - Includes cell retention devices, culture medium supply, and waste collection.

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

[0033] In some cases, large-scale (e.g., at least 25L, e.g.,) shear-sensitive cells may be required. A method for continuous culture (at least 100 L) including recombinant protein-coding nucleic acids To prepare or obtain cells sensitive to stun, and to have a controlled gas outflow rate Cells are cultured in an ioreactor system under conditions sufficient for recombinant protein expression. A method is provided which includes nourishing. In some embodiments, a bioreactor The system will take up to 20 m / s (for example, about 5 m / s) to reach steady-state conditions. It has a gas outflow rate that is controlled to a rate of approximately 10 m / s. In some embodiments, The bioreactor system maintains a maximum flow rate of 20 m / s (for example, from approximately 5 m / s to approximately) throughout the culture period. It has a gas outflow rate controlled to a rate of 10 m / s. In some embodiments, steady state The concentration of living cells under normal conditions is approximately 20 × 10⁻⁶. 6 Approximately 15 × 10 cells / mL 7 cells / mL It is within the range. In some embodiments, the concentration of living cells under steady-state conditions is less 40 x 10 6 The concentration is cells / mL.

[0034] In some cases, this method generates a continuous culture of protein preparations of shear-sensitive cells. In bioreactor systems with controlled gas outflow rates, shear sensitivity A method is provided which includes continuous culture of cells. In some embodiments, bio The reactor system has a capacity of at least 25L, at least 100L, or at least 20 Includes a bioreactor tank having a capacity of 0 L. In some embodiments, sys It takes up to 20 m / s (for example, from about 5 m / s to about 10 m / s) for the system to reach steady state conditions. The gas outflow rate is controlled to the rate of ). In some embodiments, a bioreactor system The rate is controlled to a maximum of 20 m / s (e.g., from about 5 m / s to about 10 m / s) throughout the culture period. It has a gas outflow rate. In some embodiments, the viable cell concentration under steady state conditions It is approximately 20 x 10 6 Approximately 15 × 10 cells / mL 7 It is within the range of cells / mL. In this embodiment, the viable cell concentration under steady-state conditions is at least 40 × 10 6 cells / m L is the method for producing the protein preparation is a cell and / or This includes isolating a protein or a mixture of proteins from a cell culture medium.

[0035] These and other aspects of the present invention are described in more detail below and in the claims. It will be listed.

[0036] The drawings included in this specification, which consist of the following figures, are for illustrative purposes only and are not intended to limit the scope of this specification. It is not. [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 viability 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 are, unless explicitly stated otherwise, This aligns with the meaning understood within the relevant technical field. Clear definitions of specific terms are provided below. The meanings of these and other terms will, in particular, be determined from the context throughout this specification by those skilled in the art. This should be obvious to them.

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

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

[0052] When used herein, "about" or "approximately" applies to one or more values ​​in question. The term "approximately" refers to a value similar to the reference value described. In certain embodiments, it means "approximately". The term "approximately" refers to 25%, 20%, 19%, 18%, and 1% of the stated baseline value. 7%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, This refers to a range of values ​​that fall within the following ranges: 6%, 5%, 4%, 3%, 2%, and 1% or less.

[0053] As used herein, the term “antibody” has the meaning understood in the art. This refers to immunoglobulins (Ig) that possess and specifically bind to specific antigens. As such, antibodies produced in nature generally consist of two heavy (H) chains and two light (L) chains. It is composed of four polypeptide chains, each of which is a heavy chain and a light chain. In the book, they are referred to as HCVR or V respectively. H and LCVR or V L (Abbreviated as) and steady state It is composed of regions. The steady region of the heavy chain is C H 1, C H 2 and C H 3 domains (and Optionally, in the case of IgM and IgE, C 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 domain is called the Framework Domain (FR). Rather than being exposed, a preserved region is inserted between them, creating a super-convertible region called the Complementary Determination Region (CDR). Further includes the degenerated region. Each V H and V L It consists of three CDRs and four FRs. These are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR 1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules of any type. (e.g., IgM, IgD, IgG, IgA and IgE), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or their subclasses may also be included. stomach.

[0054] As used herein, the term “antibody drug” means a drug that specifically binds to a particular antigen. This refers to a drug that confers specific binding. In some embodiments, this term is sufficient to confer specific binding. It includes any polypeptide having immunoglobulin structural elements. Various implementations In this context, suitable antibody drugs include monoclonal antibodies, polyclonal antibodies, and human antibodies. Chimeric antibodies, primate-derived antibodies, chimeric antibodies, human antibodies, bispecific or multispecific antibodies, 1 Domain antibodies (e.g., shark single-domain antibodies (e.g., IgNAR or its fragmentation) (i.e., binds to other proteins, radiolabeled substances, cytotoxins, etc.) (or fused antibodies), Small Modular ImmunoPharmaceut icals ("SMIPs" trademark), single-chain antibodies, camelid antibodies, antibody fragments Examples include, but are not limited to, this term. In some embodiments, this term This can refer to a staple peptide. In some embodiments, this term can refer to an antibody. This term may refer to mimetics of linked peptides. In some embodiments, this term may mean: This term may refer to an antibody-like binding scaffold protein. In some embodiments, this term may refer to a mo This may refer to a nobody or adnectin. In many embodiments, the antibody drug is ami The no-acid sequence is one or more structural elements that are recognized by those skilled in the art as complementarity-determining regions (CDRs). A polypeptide containing an element, or containing the same. In some embodiments, an antibody drug The agent has at least one amino acid sequence that is substantially identical to that found in the reference antibody. CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) A polypeptide containing, or including, ) in some embodiments. The CDR is substantially identical to the reference CDR, and in this case, it is because the sequences are identical. Either contains between 1 and 5 amino acid substitutions when compared to a reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR. In this case, it refers to the CDR and at least 85%, 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , or exhibiting 100% sequence identity. In some embodiments, the included CDR is reference It is substantially identical to the reference CDR, and in this case, it is at least 96% and 9% identical to the reference CDR. It exhibits sequence identity of 6%, 97%, 98%, 99%, or 100%. Several implementations In this case, the included CD-R is substantially identical to the reference CD-R, and in this case, the included CD At least one amino acid in R is deleted, added, or replaced when compared to the reference CDR. Although it has been replaced, the included CDR contains the same amino acids as the reference CDR in all other respects. It has an array. In some embodiments, the included CDR is substantially identical to the reference CDR. In this case, 1 to 5 amino acids in the included CDR are compared to the reference CDR. In some cases, CDRs are missing, added, or substituted, but the included CDRs are otherwise reference C. It has the same amino acid sequence as DR. In some embodiments, the included CDR is a reference It is substantially identical to a CDR, and in this case, at least one amino acid in the CDR that is included. However, it has been replaced when compared to the reference CDR, but the included CDR is otherwise It has the same amino acid sequence as the reference CDR. In some embodiments, the included C The DR is substantially identical to the reference CDR, and in this case, it contains 1 to 5 of the CDRs included. Amino acids are deleted, added, or substituted compared to the reference CDR, but are not included. The CDR has the same amino acid sequence as the reference CDR in all other respects. Morphologically, antibody drugs have an amino acid sequence that, according to those skilled in the art, has an immunoglobulin variable domain. A polypeptide that contains, or includes, a recognized structural element. Several implementations Morphologically, antibody drugs either match or largely match the immunoglobulin-binding domain. It is a polypeptide protein having a binding domain. In some embodiments, Antibody drugs are found in a specific reference antibody chain or multiple chains (e.g., heavy chain and / or light chain). A polypeptide containing all CDRs, or containing all CDRs.

[0055] As used herein, "biologics," "biological therapies," and "biologic products" are used in this specification. The terms are used synonymously and refer to polypeptides and protein products. For example, this specification In this book, biological products include, for example, proteins, glycoproteins, fusion proteins, and growth factors. Vaccines, blood factors, thrombolytic agents, hormones, interferon, interleukinbe Products, antibody drugs (e.g., monoclonal antibodies, bispecific antibodies, etc.), and therapeutic drugs Contains naturally occurring or recombinant products expressed in cells, such as therapeutic enzymes. Biologics are subject to a "biological product authorization application" under Section 351(a) of the Public Health (PHS) Act. Or, it is approved based on "BLA," but the biosimilar references BLA as the reference product. And alternative biological products are authorized under Section 351(k) of the PHS Act. Section 351 is codified as 42 USC 262. Other biological products are F Section 505(b)(1) of the Ederal Food and Cosmetic Act Under the or Articles 505(b)(2) and 505 of the Hatch Waxman Act (j) In some cases, it may be approved as a simplified application under Section (j), Section 505 is 21 USC It is codified as 355.

[0056] As used herein, “gas outflow rate” refers to the gas flow rate of a bioreactor system. A supply source, for example, to aerate the culture medium (e.g., supply air and / or oxygen). This refers to the rate of gas leakage from the gas supply source. In some embodiments, the gas leakage rate is the rate of gas leakage. The body exits through one or more gas supply source openings (e.g., holes) and enters the culture medium. This refers to the rate. In some embodiments, the gas outflow rate is calculated using the equation shown in Figure 12. It is calculated by using a sparger. In some embodiments, the bioreactor system uses a sparger. Including the gas outflow rate from one or more sparger openings (e.g., holes) This refers to the rate of gas leakage. In some embodiments, the bioreactor system uses sparge Including the sparger, the gas outflow rate refers to the average rate of gas outflow from the opening of the sparger. vinegar.

[0057] As used herein, "glycoprotein" refers to one amino acid sequence covalently bonded to a single amino acid. This refers to an amino acid sequence containing the above-mentioned oligosaccharide chains (e.g., glycans). The categories include peptides, polypeptides, and proteins. An example is glycoprotein. In terms of quality, these include glycosylated antibodies, antibody drugs, and antibody-like molecules (e.g., Fc fusion proteins) Examples of antibodies include monoclonal antibodies and / or their flags. Menthol, polyclonal antibody and / or its fragment, and Fc domain-containing Fusion protein (for example, a fusion containing the Fc region of IgG1 or its glycosylated portion) Proteins are one example.

[0058] The term “isolated” as used herein means (1) (in nature) (and / or regardless of the experimental environment) when first produced, a small amount of the associated components (2) Designed, produced, prepared, and / or (3) separated from, and / or (4) designed, produced, prepared by hand. Or refers to manufactured substances and / or elements. Isolated substances and / or elements are about 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately Even if separated from other initially related components by 98%, approximately 99%, or more than approximately 99%, i. In some embodiments, the isolated drug is about 80%, about 85%, about 90%, about 9% 1%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 9 It is 9%, or about 99%, ultrapure. When used herein, the substance is substantially other If it contains no components, it is "pure". In some embodiments, by those skilled in the art As will be understood, a substance is, for example, one or more carriers or excipients (e.g., b Even after being mixed with certain other components (such as ferrous solvents, water, etc.), it still remains " Such embodiments may be considered "isolated" or even "pure." In this case, the isolation percentage or purity of the substance is, without such carriers or excipients. It is calculated. In some embodiments, as an example, naturally occurring polypeptides are used. Biomacromolecules such as polynucleotides a) have an origin or source in nature b) It is not related to some or all of the components that are present in its natural state; It substantially contains other polypeptides or nucleic acids of the same species from which it is produced in nature. c) Not expressed by cells or other expression systems that are not species that produce it in nature. A substance is considered "isolated" if it is associated with its components in any other way. Therefore, for example, in some embodiments, it is chemically synthesized or obtained from nature. Polypeptides synthesized in a different cell line than those produced by the same cell line are called "isolated" polypeptides. It is considered to be a cydo. Or, or further, in some embodiments, one or more The polypeptides subjected to the purification technology are a) related in nature; and / or b) "Isolation" refers to the degree to which it was separated from other related components when it was first produced. It may also be considered a polypeptide that has been "modified".

[0059] As used herein, “recover” means, for example, in the art. Using known purification techniques, for example, by isolation, a drug or element can be extracted from other pre-related substances. This refers to a process that substantially eliminates the active ingredient. In some embodiments, the drug Alternatively, the elements may be recovered from natural sources and / or sources containing cells.

[0060] Generally, when used herein, "protein" refers to polypeptides. A protein is a chain of at least two amino acids linked together by peptide bonds. The protein may also contain parts other than amino acids (for example, glycoproteins). And / or may be processed or modified in other ways. Those skilled in the art will know that "protein" is Complete polypeptides produced by cells (with or without signal sequences) You will understand that it may be a chain, or it may be a functional protein. Those skilled in the art will know that proteins are linked, for example, by one or more disulfide bonds, It may include two or more polypeptide chains linked together by other means. To understand this further.

[0061] When used herein, the term "protein preparation" refers to a specific method of production. Therefore, it refers to the resulting mixture of proteins. The proteins in the protein preparation are the same. It may or may not be the same protein; that is, the protein preparation may contain the same protein It may include multiple copies and / or mixtures of different proteins. Several embodiments So, protein preparations include glycoprotein preparations. Glycoprotein preparations are few Each is a composition or mixture containing one glycoprotein. In some cases, glycoproteins Protein preparations are made from multiple copies of the same protein (i.e., having the same amino acid sequence). It contains, but has a mixture of proteins and related glycans. In some cases, glycoproteins The preparations are prepared using the methods and / or systems provided herein. The production method involves expressing the protein in the protein preparation (or at an appropriate level). Using cultured cells that have been manipulated (to express proteins under appropriate conditions) The method may include a recombinant preparation step. In some embodiments, the production method is such that the protein , from specific components of manipulated cells (for example, by lysing the cells and centrifuging the proteins) The process may include an isolation step in which the qualitative components are isolated (by forming pellets). In this embodiment, the production method also involves the protein in the protein preparation being used in other cells. minutes, for example, from other proteins or organic components used in the previous step (for example, The process may include purification steps (separated by chromatography). These steps are not limited to It will be understood that this is the target and may include any number of additional generation steps. The protein preparations are produced using the same method but under different circumstances (e.g., different circumstances). It may be prepared in a row or in a preparation. Alternatively, different protein preparations may produce different results. It may be prepared by method. The two production methods may differ in any way (for example, (e.g., expression vector, manipulated cell type, culture conditions, isolation procedure, purification conditions).

[0062] As used herein, "sample(s)" refers to a sample(s) obtained separately. It refers to. In some embodiments, the evaluation of isolated samples is performed using the same culture run (e.g., From different points in time during preparation, or from different culture runs (e.g., different culture cycles) This includes evaluation of samples from ).

[0063] As used herein, “sparging” or “gas sparging” refers to cell This refers to the aeration or addition of gas (e.g., air and / or O2) to the culture medium. Generally, Gas sparging is the process of directly adding gas to a culture medium (for example, using a sparger). This refers to the process of sparging. In some embodiments, sparging is performed for at least about 20 minutes. It is used to achieve a dissolved O2 concentration of %, or between approximately 20% and 100%.

[0064] As used herein, “steady state” or “steady state conditions” means a cell culture system When used in reference to a stem or process, the cell culture is performed for at least 5 days. For example, a viable cell concentration that fluctuates by up to 20% over a period of 5 to approximately 60 days. This means that, in some embodiments, the cell culture system is used over a period of 5 days. The live cell concentration has a variation of up to 20%. In some embodiments, the cell culture system , at least 5 days (for example, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days) Up to 15%, up to 10%, or over a period of 40, 50, or 60 days. It has a viable cell concentration that fluctuates by up to 5%. In some embodiments, the cell culture system is The viable cell concentration fluctuates by up to 15%, up to 10%, or up to 5% over a 5-day period. In some embodiments, the cell culture system is used for at least 5 days (for example, 5 days). Intervals: 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, 50 days, 60 days Changes of up to 10% (i.e., within a range of plus or minus 10%) over the period of time. It has a viable cell concentration of cells sensitive to moving shear. In some embodiments, the cell culture system The stem can change by up to 10% (i.e., plus or minus 10%) over a 5-day period. It has a viable cell concentration of shear-sensitive cells that fluctuates within a range. In some embodiments, The system is brought to a steady state by the discharge of waste from the bioreactor system (e.g., removal of excess cells). The concentration of living cells is achieved and / or maintained.

[0065] This includes, but is not limited to, patents, patent applications, papers, books, professional texts, and web pages. Not applicable to all documents and similar materials cited in this application, such documents and similar materials. Regardless of the format of the similar materials, the whole is clearly incorporated by reference. This includes, but is not limited to, words, terminology usage, and the technologies described, but also incorporates other elements. If one or more documents or similar materials differ from or contradict this application, this application shall take precedence. The section headings used herein are for organizational purposes only and are not included in the description. It should not be interpreted as something that limits the subject matter in any way.

[0066] This disclosure partially relates to large culture volumes (e.g., at least 100 L, e.g., less In both 200L tanks, certain cells exhibit insufficient viable cell concentrations and reduced cell viability. This identifies the challenges of current continuous cell culture technologies (e.g., perfusion cell culture) and addresses them. This disclosure relates to the scaling up of high cell density continuous cell culture processes, including oxygen supply and solubility. We acknowledge that this can be difficult due to the high demands for removal of residual carbon dioxide (pCO2). This disclosure includes an analysis of many different variable products associated with large-scale continuous cell culture.

[0067] This disclosure acknowledges the recognition that the importance of aeration increases with bioreactor volume and cell concentration. This disclosure includes, in particular, the ability to control the gas outflow rate of a bioreactor system. This improves the performance of large-scale continuous cultures of specific cells (for example, shear-sensitive cells). It provides insight that this is possible.

[0068] In some embodiments, the disclosure relates to large-scale (e.g., at least) shear-sensitive cells. Method for continuous culture (e.g., perfusion culture) in 100L, for example, at least 200L and process, wherein the gas outflow rate of the bioreactor system is a specified amount and To provide methods and processes that are controlled and / or maintained within a range. For example, A bioreactor with a gas outflow rate of up to 20 m / s (for example, up to 10 m / s) - Large number of shear-sensitive cells in a system (e.g., perfusion bioreactor system) A method for large-scale culture is provided. The provided method is (for example, 20 × 10 6 cells / mL 15 x 10 7 (A form of high-density cell culture having a steady-state viable cell concentration within the range of cells / mL) It makes success possible.

[0069] Continuous culture method and process This disclosure partially relates to shear-sensitive cells (e.g., cells containing shear-sensitive cells). Large-scale populations (e.g., populations of cells consisting of shear-sensitive cells) 25L, for example, at least 100L, for example, at least 200L) Continuous culture (for example The present invention provides methods, processes, and / or systems for perfusion cell culture. In some embodiments, large-scale cultures range from 25 L to 3,000 L. Large-scale cultivation is available in 25L, 50L, 100L, 200L, 250L, 400L, and 500L sizes. L, 600L, 800L, 1000L, 1200L, 1500L, 2000L, 3000 It is L or more. In some embodiments, large-scale culture is 100 L or more. In certain embodiments, large-scale cultivation is 200 L or more. Therefore, large-scale cultivation is defined as 250L or more.

[0070] In some cases, continuous cell culture systems (e.g., perfusion cell culture systems) are high density Shear-sensitive cells (e.g., at least 20 × 10) 6cells / mL, for example, at least 30 x 10 6 cells / mL, e.g., at least 40 × 10⁶ 6 The steady state is cells / mL. This refers to a large-scale culture (e.g., 200L or more) capable of generating a viable cell concentration.

[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 Shear-sensitive nanoparticles within the range of cells / mL The cells have and / or achieve a steady-state viable cell concentration. In some embodiments, The steady-state viable cell concentration of signal-sensitive cells is such that the upper limit is greater than the lower limit, and the lower and upper limits are... This is an amount within a limited range. In some embodiments, the lower limit is about 20 × 10 6 Thin cells / mL, approximately 25×10 6 cells / mL, approximately 30×10 6 cells / mL, approximately 35×10 6 cell / mL, approx. 40×10 6 cells / mL, approximately 45×10 6 cells / mL, approximately 50×10 6 cell / mL, approximately 60×10 6 cells / mL, approximately 70×10 6 cells / mL, approximately 80×10 6 cells / m L, or approximately 90 x 10 6 The cell / mL may be. In some embodiments, the upper limit is , about 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, approx. 90×106 cells / mL, about 10×10 7 cells / mL, about 11×10 7 cells / mL, about 1 2×10 7 cells / mL, about 13×10 7 cells / mL, about 14×10 7 cells / mL or about 15×10 7 cells / mL may be sufficient.

[0072] Generally, the cell culture method of the present disclosure involves culturing at a temperature within the range of 25°C to 40°C under the gravity encountered on Earth. It includes culturing 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. In some embodiments, a continuous cell culture system (e.g., a perfusion cell culture system) has a gas outflow rate within a range bounded 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 1 m / s, about 2 m / s, about 3 m / s, about

[0074] In some embodiments, a continuous cell culture system (e.g., a perfusion cell culture system) has a gas outflow rate within a range bounded 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 1 m / s, about 2 m / s, about 3 m / s, about 4 m / s, about 5 m / s, about 6 m / s, about 7 m / s, about 8 m / s, about 9 m / s or about 10 m / s. In some embodiments, the upper limit may be about 5 m / s, about 6 m / s, about In some embodiments, the upper limit may be about 5 m / s, about 6 m / s, about 7m / s, approx. 8m / s, approx. 9m / s, approx. 10m / s, approx. 11m / s, approx. 12m / s, approx. 1 3 m / s, approximately 14 m / s, approximately 15 m / s, approximately 16 m / s, approximately 18 m / s, or approximately 20 m / s is also acceptable.

[0075] In some embodiments, a continuous cell culture system (e.g., a perfusion cell culture system) , it has dissolved carbon dioxide at a level not exceeding 120 mmHg. In some embodiments For continuous cell culture systems (e.g., perfusion cell culture systems), the temperature range is approximately 20 mmHg to approximately It has dissolved carbon dioxide levels in the range of 120 mmHg. In some embodiments, Dissolved carbon dioxide is within a range limited by the lower and upper limits, where the upper limit is greater than the lower limit. It is present in the continuous cell culture medium in a certain quantity. In some embodiments, the lower limit is about 20 mmHg Approximately 30mmHg, 40mmHg, 50mmHg, 60mmHg, or 70mm It may also be mHg. In some embodiments, the upper limit is about 50 mmHg, about 60 mmHg. Hg, approx. 70mmHg, approx. 80mmHg, approx. 90mmHg, approx. 100mmHg, approx. 110 mmHg, or approximately 120 mmHg.

[0076] In some embodiments, shear-sensitive cells (e.g., cells containing shear-sensitive cells) are used. Continuous culture of a cell population (for example, a population of cells consisting of shear-sensitive cells) takes approximately 10 days. The process is carried out over a period of approximately 180 days. In some embodiments, shear-sensitive Cells (for example, a population of cells including shear-sensitive cells, for example, from shear-sensitive cells) The continuous culture of a population of cells is limited by the lower and upper limits, where the upper limit is greater than the lower limit. It is carried out for a specified amount of time within a given range. In some embodiments, the lower limit is about 10 days. Approximately 15 days, approximately 20 days, approximately 25 days, approximately 30 days, approximately 35 days, approximately 40 days, approximately 50 days It may be between, or about 60 days. In some embodiments, the upper limit is about 30 days. Approximately 35 days, approximately 40 days, approximately 50 days, approximately 60 days, approximately 70 days, approximately 80 days, approximately 90 days Between approximately 100 days, 120 days, 140 days, 160 days, or 180 days It's okay to have it.

[0077] In some embodiments, shear-sensitive cells (e.g., cells containing shear-sensitive cells) are used. Continuous culture of a population of cells (for example, a population of cells consisting of shear-sensitive cells) is at least The procedure is carried out over a period of 10 days. In some embodiments, shear-sensitive cells (e.g., A group of cells that includes cells sensitive to shear, for example, a group of cells consisting of cells sensitive to shear. Continuous culture is carried out for a period of approximately 30 to 60 days.

[0078] Bioreactor system This disclosure relates to large-scale continuous cell culture, for example, shear-sensitive cells (e.g., shear A population of cells containing cells sensitive to shear (for example, a population of cells consisting of cells sensitive to shear) The present disclosure provides a bioreactor system for nourishing organisms. This is suitable for continuous culture methods. In some embodiments, the bioreactor of the present disclosure The stem is suitable for perfusion culture. Bioreactor system suitable for the culture method of this disclosure A schematic diagram illustrating an example is shown in Figure 1. In some embodiments, the bioreactor system , including bioreactor tanks and cell retention devices. In some embodiments, The bioreactor system includes a bioreactor tank, a cell retention device, a culture medium supply, and Includes waste collection. In some embodiments, the bioreactor system is used for cells. Population (e.g., a population of cells consisting of shear-sensitive cells, e.g., inoculation) and cell culture It also includes the earth.

[0079] In some embodiments, the bioreactor system is a stirred-tank type bioreactor -Includes. In some embodiments, (for example, of a stirred tank type bioreactor) Oreactor tanks have capacities ranging from 25L to 3,000L. In terms of application configuration, the bioreactor tank (for example, in a mixed-tank type bioreactor) is, At least 25L, 50L, 100L, 200L, 250L, 300L, 400L, 50 0L, 600L, 800L, 1000L, 1200L, 1400L, 1500L, 160 0L, 1800L, 2000L, 2400L, 2500L, 2600L, 2800L, Or it has a capacity of 3000L. In some embodiments, a stirring tank type bioreactor - has a capacity of at least 100L. In some embodiments, a stirring tank type The bioreactor has a capacity of at least 200 L. In some embodiments, The agitated tank type bioreactor has a capacity of at least 250 L. In this embodiment, the agitated tank type bioreactor has a capacity of at least 500 L. In some embodiments, the agitated tank type bioreactor has at least 1000 It has a capacity of L.

[0080] In some embodiments, the bioreactor system includes a sparger. Generally A sparger is used to introduce air and / or oxygen into the cell culture medium. can be achieved. The present disclosure includes the recognition that the selection of the sparger can also affect the rate and degree of aeration and minimize 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 sparger. In some embodiments, the drill hole sparger has holes with sizes in the range of 0.05 mm to 5.0 mm. In some embodiments, the drill hole sparger has holes with sizes in the range of 0.1 mm to 1.0 mm. In some embodiments, the drill hole sparger has holes with sizes 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 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 is disposed at the bottom of the bioreactor tank and includes a sparger. In some embodiments, the bioreactor system includes a sparger disposed substantially at the center within the bioreactor tank.

[0083] In some embodiments, the bioreactor system includes a cell retention device. Suitable cell retention devices for use in the disclosed bioreactor system include continuous centrifuges, alternating tangential flow filters (ATF), tangential flow membrane filters (TFF), dynamic filters, spin filters, ultrasonic and dielectrophoretic separators, and gravity settlers. In some embodiments, the bioreactor system of the present disclosure includes a cell alternating tangential flow (ATF) device. In some embodiments, the bioreactor system includes a cell retention device with one or more ATFs. In some embodiments, the bioreactor system includes a cell retention device with two ATFs. In some embodiments, the bioreactor system includes a cell retention device with three or more ATFs.

[0084] In some embodiments, the bioreactor system includes an impeller device. In some embodiments, the impeller is at the bottom mounted within the bioreactor tank. In some embodiments, the impeller is disposed substantially at the center within the bioreactor tank. In some embodiments, the impeller device is magnetically driven. The bioreactor system has a volumetric power input P / from about 5 W / m 3 to about 500 W / m 3 ​ It has an impeller stirring rate that exhibits V.

[0085] Shear-sensitive cells In some cases, the culture methods, processes, and systems of this disclosure may be used for shear-sensitive cells. This disclosure relates to the continuous culture of animal cells without cell walls in a stirred tank type bio Mechanical stress caused by the culture fluid in an oxygenator, for example, to supply oxygen to cells. In the culture medium in the stirring tank, the impeller generates and bursts bubbles. This includes the recognition that they are sensitive to stirring and other agitation. In excessive cases, these fluids can cause mechanical problems. Stress can lead to damage / death of animal cells. Furthermore, this disclosure applies to certain animal cells. This includes the recognition that cells are more sensitive to shear caused by mechanical stress than other materials. Mouse myeloma cells may be more sensitive to shear than certain CHO cell lines. This paper presents methods, processes, and systems suitable for large-scale, high-density culture of shear-sensitive cells. To provide.

[0086] Certain cells, such as animal cells, respond to specific mechanical and / or aeration conditions during culture. It is known in the art that it is sensitive (for example, Gooch et a L., Curr. Opin. Biotech. 4:193-196 (1993 (See reference). When used herein, shear-sensitive cells are cultured under conventional conditions (e.g., For example, it exhibits a certain level of sensitivity to shear under conventional large-scale culture conditions. For example, any animal cell (showing reduced cell viability). As described herein, in certain cases, cells that are sensitive to such shear are When cultured under the conditions described, such cells exhibit an increased level of viability. Yes.

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

[0088] In some embodiments, shear-sensitive cells are human cells. Examples of shear-sensitive human cell lines include, for example, HEK293: human embryonic 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 a human B cell line; and HuH-7 : derived from human hepatocellular carcinoma. In some particular embodiments, the shear-sensitive cells are selected from HEK293 cells, fibrosarcoma HT1080 cells, PER.C6 cells, CAP cells, H KB-11 cells and HuH-7 cells.

[0089] In some particular embodiments, the shear-sensitive cells for use according to the present disclosure are SP 2 / 0 cells. In some embodiments, the shear Cells sensitive to this are either equipped with or manipulated to express cell products.

[0090] According to this disclosure, conventional molecular biology, microbiology, and compounding within the scope of the art in the said art field DNA replacement technology may be used. Such technology is described in the literature (for example). , Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Ed ition (1989) Cold Spring Harbor Laborato ry Press, Cold Spring Harbor, NY;DNA C loning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); eotide synthesis (MJ Gait ed. 1984);N ucleic Acid Hybridization (BD Hames & SJ Higgins eds. (1985));Transcriptio n And Translation (BD Hames & SJ H iggins, eds. (1984));Animal Cell Culture (RI Freshney, ed. (1986));Immobilize d Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecule r Cloning (1984);FM Ausubel et al. ds.), Current Protocols in Molecular Bio (See logy, John Wiley & Sons, Inc. (1994)) .

[0091] In some embodiments, the cell products described herein are shear-sensitive to expressing the cell products described herein. These cells are produced using recombinant methods. Antibody drugs, etc., as described herein. Recombinant expression of genes such as polypeptide-coding genes is a way of expressing polypeptides. The construction of an expression vector containing polynucleotides may also be included. If obtained, the vector for polypeptide production is known in the art. It can be produced using recombinant DNA technology that employs the following techniques. Polypeptide coding In order to construct an expression vector containing the sequence and appropriate transcription and translation regulatory signals, Knowledge methods can be used. These methods include, for example, in vitro recombination. Examples include DNA technology, synthesis technology, and in vivo genetic engineering.

[0092] The expression vector may be introduced into shear-sensitive cells using conventional techniques, Sfection cells are then cultured by the method described herein to produce fine cells. Cellular products (e.g., protein preparations, e.g., recombinant proteins, e.g., glycoproteins) It can produce various substances, such as fusion proteins or antibody drugs.

[0093] Cell culture medium This disclosure relates to the expression of cell products in a culture medium sufficient for the expression of cell products (for example, shear-sensitive cells, For example, a population of cells that includes shear-sensitive cells, for example, a cell population consisting of shear-sensitive cells The present invention provides methods, processes, and systems for continuous cell culture (of a population of cells). Culture media generally contain appropriate energy sources and compounds that regulate the cell cycle. The culture medium comprises, for example, amino acids, vitamins, inorganic salts, and glucose, which are known to those skilled in the art. In some embodiments, the cell culture medium has a pH of 6 to 8. For animal cell culture The culture medium is well established in the art and is suitable for a specific purpose and / or cell type Therefore, it is conventionally optimized by those skilled in the art.

[0094] In some embodiments, the cell culture medium is defoamed in the range of 1 ppm to 500 ppm. The agent (e.g., antiform C) is also included. In some embodiments, shear-sensitive cells This is a cell culture medium containing a shear force protectant (e.g., Pluronic F-68). They are cultured. In some embodiments, shear-sensitive cells are cultured at 1 g / L to 15 g / Cells are cultured in a cell culture medium containing Pluronic F-68 at concentrations within the range of L. In some specific embodiments, a population of shear-sensitive cells is found in serum (e.g., bovine fetus). They may be cultured in a cell culture medium containing (fetal serum).

[0095] Cell products In some cases, shear-sensitive cells or populations of cells consisting of shear-sensitive cells Cells are continuously cultured using the methods, processes, and / or of the present disclosure and express cell products. In some embodiments, the cell product is nucleic acid, lipid, peptide, and / or protein. It is of quality, or contains such quality.

[0096] In some embodiments, the cell product is a recombinant protein. In this state, the shear-sensitive cells of this disclosure contain nucleic acids that encode recombinant proteins. In some embodiments, the cell product is a glycoprotein. In some embodiments, Recombinant proteins are fusion proteins. In some embodiments, the following is a description of the present disclosure. Cells sensitive to the disruption contain nucleic acids that encode fusion proteins. In some embodiments, The recombinant protein is an Fc fusion protein. In some embodiments of this disclosure Shear-sensitive cells contain nucleic acids that encode Fc fusion proteins.

[0097] In some embodiments, the cell product is an antibody drug. Cells sensitive to shear in the disclosure contain nucleic acids encoding antibody drugs. In some embodiments, The cell product is a monoclonal antibody.

[0098] In some embodiments, the shear-sensitive cells of this disclosure are glycoprotein complexes (e.g.) If it contains one or more N-glycosylation sites that are bound or fused with one or more heterogeneous parts, It contains nucleic acids that encode an Fc region or Fc fragment. Heterogeneous parts include peptides. Tides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic drugs, synthesis Examples include, but are not limited to, drugs, inorganic molecules, and organic molecules. In some cases... Glycoprotein complexes are peptides fused with Fc regions such as glycosylated Fc regions, and polyproteins. Lipeptides, protein scaffolds, scFv, dsFv, diabodies, Tandab, also It is a fusion protein containing an antibody mimetic. The fusion protein connects the Fc region with the heterologous region. It may include a linker region (for example, Hallewell et al. (1 989), J. Biol. Chem. 264, 5260-5268;Alft han et al. (1995), Protein Eng. 8, 725-7 31; See Robinson & Sauer (1996).

[0099] In some embodiments, the shear-sensitive cells of this disclosure are used in human or animal therapeutic applications. For therapeutic or diagnostic use, for example, proteins approved in the secondary approval process It contains nucleic acids that code for quality.

[0100] In some embodiments, the shear-sensitive cells of this disclosure are used in human or animal therapeutic applications. For therapeutic or diagnostic use, for example, proteins approved in the secondary approval process It contains nucleic acids that encode proteins having the same primary amino acid sequence as the quality. Several implementations Morphologically, the shear-sensitive cells of this disclosure are compatible with approved therapeutic or diagnostic proteins. Differentiated by no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 residues It contains nucleic acids that encode proteins. In some embodiments, the shear-sensitive of the Disclosure The cells contain at least 90, 95, and 90 of the approved therapeutic or diagnostic proteins. 8. Contains nucleic acids encoding proteins with 99% or 100% sequence identity. "Same primary amino acid sequence", "1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 "Primary amino acid sequences that differ by no more than one residue," "Sequences identical by at least 98%." The term "sequence having identity," or similar terms, refers to the level of identity between primary amino acid sequences. Regarding the protein preparation or product, in some embodiments, the amino acid variant is used. Ants, for example, include species where one or two terminal residues differ. In some embodiments of such cases, the sequence identity being compared is in each product being compared. This refers to the identity between the primary amino acid sequences of the most abundant (e.g., the most abundant active) species. In that embodiment, sequence identity is used to determine which nucleic acids can be used to produce the product. Therefore, it refers to the coded amino acid sequence.

[0101] In some embodiments, the shear-sensitive cells of this disclosure are used in human or animal therapeutic applications. Contains nucleic acids that encode proteins not approved for therapeutic or diagnostic use.

[0102] Examples of recombinant proteins that are not limited include abatacept (Orencia®). Bristol-Myers Squibb, Absiximab (ReoPro (Registered) (Trademark), Roche), Adalimumab (Humira (Registered Trademark), Bristol-M Yers Squibb), Aflibercept (Eylea®, Regene (ron Pharmaceuticals), Alefacept (Amevive (Registered Trademark) (Registered Trademark), Astellas Pharma, Alemtuzumab (Campath (Registered Trademark) ), Genzyme / Bayer), basiliximab (Simulect(registered trademark), Novartis), Veratacept (Nulojix®, Bristol-M (Yers Squibb), belimumab (Benlysta®, GlaxoS) mithKline), bevacizumab (Avastin®, Roche), Nakinumab (Ilaris®, Novartis), Brentuximab Vedoti Adcetris (registered trademark, Seattle Genetics), Celltris Mab (CIMZIA®, UCB, Brussels, Belgium), Setsu Ximab (Erbitux®, Merck-Serono), daclizumab ( Zenapax (registered trademark), Hoffmann-La Roche, Deniroy King Fuchitox (Ontak®, Eisai), Denosumab (Prolia®) (Trademark), Amgen;Xgeva (Registered Trademark), Amgen), Eculizumab (Soli ris (registered trademark, Alexion Pharmaceuticals), Ephaliz Mab (Raptiva®, Genentech), etanercept (Enbr el (registered trademark), Amgen-Pfizer), gemtuzumab (Mylotarg (registered trademark) (Registered trademark), Pfizer, Golimumab (Simponi (Registered Trademark), Janssen) ), ibritumomab (Zevalin®, Spectrum Pharmac euticals), infliximab (Remicade®, Centoc or), ipilimumab (Yervoy (trademark), Bristol-Myers Squi bb), Muromonab (Orthoclone OKT3 (registered trademark), Janssen- Cilag), Natalizumab (Tysabri®, Biogen Idec, Elan), ofatumumab (Arzerra®, GlaxoSmithKl) ine), omalizumab (Xolair®, Novartis), palivizumab (Synagis®, MedImmune), Panitumumab (Vectib) ix (registered trademark), Amgen), ranibizumab (Lucentis (registered trademark), Ge nentech), Rilonacept (Arcalyst®, Regeneron Pharmaceuticals), Rituximab (MabThera® registered trademark), Roche), Tocilizumab (Actemra®, Genentech; Ro Actemra, Hoffman-La Roche) Toshitsumomab (Bexxar ( (Registered trademark), GlaxoSmithKline), Trastuzumab (Herceptin) (Registered Trademark), Roche), and ustekinumab (Stelara (Registered Trademark), Jan One example is ssen.

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

[0104] In some embodiments, shear-sensitive cells are as described in Sequence ID No. 1. The heavy chain variable domain and the light chain variable domain as described in Sequence ID No. 2 are included in the anti It contains nucleic acids that encode a somatic drug. In some embodiments, shear-sensitive cells are sequenced A nucleus encoding an antibody drug, comprising a heavy chain containing sequence number 1 and a light chain containing sequence number 2. Contains acid. In some embodiments, shear-sensitive cells are described in SEQ ID NO: 1. As described in HCDR1, HCDR2, and HCDR3 sequences and Sequence ID No. 2 The antibody drug codes for the LCDR1, LCDR2, and LCDR3 sequences as shown. It contains nucleic acids.

[0105] Sequence ID 1 - Ustekinumab heavy chain sequence (bold indicates the inclusion of the underlined CDR sequence) (Shows the variant domain sequence) [ka]

[0106] Sequence ID 2 - Ustekinumab light chain sequence (bold indicates the inclusion of the underlined CDR sequence) (Shows the variant domain sequence) [ka]

[0107] Pharmaceutical composition Generated using any method, system and / or process described herein or cell products produced (e.g., recombinant proteins, e.g., glycoproteins, e.g.) Antibody drugs can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions can treat diseases. Recombinant proteins (e.g., glycoproteins) may be useful in the prevention and / or treatment of [conditions]. Pharmaceutical compositions containing substances (for example, antibody drugs) can be formulated by methods known to those skilled in the art. (For example, Remington's Pharmaceutical Sciences) ences, 20th Ed., Lippincott Williams & W. (See Ilkins, 2000). The pharmaceutical composition is dissolved in water or other pharmaceutically acceptable liquid. It may be administered parenterally in the form of an injectable formulation containing a sterile solution or suspension for the body. Example For example, a pharmaceutical composition contains cell products (e.g., recombinant proteins, e.g., glycoproteins, For example, antibody drugs) in a pharmaceutically acceptable vehicle or medium, such as sterile water and physiological Saline solution, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, diluent, Hicle, preservatives, and binders are appropriately combined and subsequently required for generally accepted pharmaceutical practices. It can be formulated by mixing it with the required unit dosage form. The amount of active ingredient present is such that a suitable dose is provided within the specified range.

[0108] The route of administration is, for example, parenteral administration by injection, nasal administration, pulmonary administration, or transdermal administration. This is possible. Administration is systemic or by intravenous, intramuscular, intraperitoneal, or subcutaneous injection. It may be localized.

[0109] The appropriate means of administration can be selected based on the patient's age and condition. Cell products Pharmaceuticals containing (e.g., recombinant proteins, glycoproteins, antibody drugs) The single dose of the composition ranges from 0.001 mg / kg body weight to 1000 mg / kg body weight. It may be selected. In some embodiments, the dose is from 0.001 mg to 100.00 The dosage may be selected within the range of 0 mg, but is not limited to such a range. The dosage and method of administration will vary depending on the patient's weight, age, condition, etc. The person can make an appropriate choice as needed.

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

[0111] Example 1: Conventional conditions for continuous culture of shear-sensitive cells were scaled up. It is inappropriate for the process. This example describes the identification of scale-up challenges in the continuous culture of shear-sensitive cells. In particular, this embodiment is a large-scale (e.g.,) study of shear-sensitive cells without controlling the gas outflow rate. For example, perfusion culture (200L) resulted in reduced cell viability and insufficient biomass for biological production. This demonstrates the discovery of the presence of cell concentration. Specifically, this example shows an example of cell product Processed to contain nucleic acids encoding (for example, antibody drugs, e.g., monoclonal antibodies) This example demonstrates the scale-up of perfusion culture of SP2 / 0, a shear-sensitive cell line. .

[0112] A sequence of shear-sensitive cells (e.g., SP2 / 0) expressing an example antibody drug. Conventional conditions for continuous cell culture (e.g., perfusion cell culture) are applied to small-scale continuous culture systems. For example, the evaluation was performed using a maximum of 100L, and the results are shown in Figure 2. Specifically, a 3L glass was used. Reactor, 5L glass reactor, 15L glass reactor and 100L SUB Shear-sensitive cells grown in a 100-unit disposable reactor system are comparable to those of equivalent cells. Cellular growth and steady-state viable cell concentrations were shown. Cells were cultured in 3L, 5L, 15L, and 100L tanks. Shear-sensitive cells also possessed comparable productivity and product quality (data not shown). stomach).

[0113] As an example, continuous cell culture (e.g., perfusion cell culture) of shear-sensitive cells, SP2 / 0 We attempted to scale up to 200L, and the results are shown in Figure 3. Specifically, the following is an example of stirring A mixing tank type bioreactor system was used. [Table 1-1] [Table 1-2]

[0114] Initial growth was observed in all samples, but as shown in Figure 3, the GE / Xce llerex XDR200 disposable reactor system was used for a 200 L culture pro cess that began on day 4 with a decline in cell growth (viable cell concentration of shear-sensitive cells (10 6 cells / mL unit) (above)) and a decline in viability (percent viability (below)). Thus, this example shows that an improved method is needed to successfully continuously culture shear-sensitive cells at large scale (e.g., at least 200 L).

[0115] Example 2: Root Cause Analysis of Scale-Up Continuous Cell Culture of Shear-Sensitive Cells Analysis This example identifies the causes of the decreased cell growth and viability of shear-sensitive cells, SP2 / 0 cells, at large scale (e.g., 200 L or more) as described in Example 1, and provides a method for large-scale continuous cell culture (e.g., perfusion cell culture) that yields high density shear-sensitive cells (e.g., at least 20×10 6 cells / mL, e.g., at least 30×10 6 cells / mL or the like, e.g., having a steady-state viable cell concentration). To understand why cell growth and viability of shear-sensitive cells are low in a 200 L continuous culture system,

[0116] various parameters were analyzed. Protocols and conditions were reviewed to ensure that there were no issues with the raw materials (e.g., media, media prep filters, antifoam agents, inoculum materials, etc.). Parameters such as dissolved oxygen (DO), pH, and temperature were kept constant between small-scale and large-scale cultures (data not shown). ​​

[0117] A gas efflux rate (GEV) of >20 m / s is a contributing factor to the reduced growth and survival rates. It was identified as a possibility. To evaluate this hypothesis, a portion of the culture fluid (specifically, 2 After 8 days of culturing, transfer the L cell culture medium to a 200L bioreactor (XDR200 Bio). A 3L benchtop bioreactor with a maximum GEV of 3 m / s from the reactor. It was moved to the remaining 200L culture system and 3L benchtop bioreactor. The concentration of viable cells was monitored. As shown in Figure 4, a 3L benchtop bioreactor was used. The viable cell concentration recovered rapidly. Meanwhile, in a sparger in a 200L bioreactor... The gas outflow rate was then controlled to be less than 10 m / s from day 9 to day 13 of incubation. (It was reduced). During this time, the concentration of viable cells recovered. Furthermore, on the 13th day thereafter, GEV When the level was increased to at least 20 m / s, the concentration of living cells decreased rapidly. Our results show that controlling the GEV to a level of 10 m / s or less (for example, pyro This suggests that it promotes cell viability for successful scaling up (to a large scale).

[0118] To determine the GEV threshold, perform continuous cell culture using a 3L perfusion bioreactor. This was performed using various gas efflux rates (GEV), and the results are shown in Figure 5. Specifically, The increase in GEV from 10 m / s to 16 m / s resulted in a rapid decrease in the concentration of viable cells. Similarly, even when GEV is maintained at 13 m / s, the viable cell concentration drops sharply after 12 days of culture. It decreased to [value]. On the other hand, when GEV was reduced from 16 m / s to 10 m / s, the concentration of living cells increased. Added.

[0119] Therefore, in this embodiment, the sparger gas outflow rate is less than 20 m / s (for example, 10 By controlling the shear speed to less than m / s, continuous culture of shear-sensitive cells can be performed on a large scale. For example, successfully scale up to over 200L (for example, over 250L), and high density Shear-sensitive cells (for example, at least 20 × 10 6 cells / mL, for example, at least 30 x 10 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 were... This is shown in Figure 6. Specifically, an example of a shear-sensitive cell (SP2 / 0 cell) is dissolved diacid. It was found to be sensitive to pCO2. When pCO2 is ≥ 90 mmHg It was found that this process negatively impacts cell culture performance.

[0121] This example demonstrates that the cell proliferation and viability of shear-sensitive cells in large-scale continuous culture are related to gas flow. It can be restored to its original state by controlling the departure speed (for example, to less than 10 m / s). This demonstrates that the pCO2 level can also be controlled, and that the shear-sensitive system is also... This demonstrates its usefulness in large-scale culture of cells at high density.

[0122] Example 3: Large-scale continuous cell culture of shear-sensitive cells This example demonstrates the application of the understanding of Examples 1 and 2 to the large-scale reticular development of shear-sensitive cells. This shows the process of continuous culture. Specifically, it involves performing continuous cell culture in a 250L perfusion reactor (SUB25). 0) was performed while controlling both GEV and pCO2. Figures 7A and 7B show 3L and 5L , cell density between 15L, 100L (SUB100) and 250L (SUB250) and This provides a comparison of cell viability. Specifically, Figures 7A and 7B show the results of various cell cultures. GEV and p controlled at various levels in models (e.g., 100L, e.g., 250L) This provides further analysis of cell viability using CO2. SUB100, 100L bio The reactor includes a 570 x 0.18 mm hole and is a SUB250, 250L bioreater. The vent includes a hole measuring 760 x 0.233 mm. As shown in Figure 7A, the standard is 250L. GEV controlled to approximately 7 m / s and dissolved oxide up to 80 mmHg using a SUB250 model. Continuous cell culture using carbon showed cell viability similar to that of small-scale cultures. Figure 8 shows (example). For example, for a 250L culture using the SUB250 bioreactor system... This provides an overview of predictive models for various parameters, particularly for 100L and 250L. The CO2 levels predicted using these models for dissolved CO2 at L are: The data obtained was extremely similar to the actual data (not shown).

[0123] Figure 9 shows the results of these continuous cultures of shear-sensitive cells in 3L, 100L, and 250L. All have equivalent total glycan levels (top panel) and equivalent sialic acid content (bottom panel) This demonstrates that (L) is achieved. Therefore, this example shows high cell count by perfusion culture process. Density shear-sensitive cells (SP2 / 0) (e.g., Thermo Fisher / Hy Clone SUB250 (250L) Agitated Tank Type Bioreactor (250) The results demonstrate a successful scaling up to the scale of L (Figures 7A, 7B, and 9).

[0124] Therefore, this embodiment can also control pCO2 levels in shear-sensitive cells. (For example, at least 20 × 10 6 cells / mL, e.g., at least 30 × 10⁶ 6 cell Large-scale culture at high density (e.g., having a steady-state concentration of / mL) It is also shown to be beneficial for L (for example, 250 L). The inventors have shown that pCO2 is beneficial at 80 mmH By controlling the sparger gas outflow rate to less than g and further to less than 10 m / s, The inventors successfully transferred their process to a 3L to 250L agitated tank-type bioreactor. We were able to scale up.

[0125] Therefore, this example demonstrates the successful scale-up of perfusion culture of shear-sensitive cells. Therefore, the GEV from the sparger is controlled to a rate of <10 m / s while simultaneously setting pCO2 to ≤8 This supports maintaining a blood pressure of 0 mmHg.

[0126] Example 4: Scaling continuous cell culture to production scale This example demonstrates large-scale processing of shear-sensitive cells at production scale (e.g., 1000L or more). This shows a model of various parameters for continuous culture, as shown in Figure 10. Dissolved CO2 (upper panel) and gas for continuous culture of shear-sensitive cells in 1000L Predictive parameters for cell outflow rate (bottom panel) are for 250L and 100L cultures. It is equivalent to the level demonstrated in the process. Furthermore, Figure 11 shows (for example, SUB100) Various parameters for 1000L culture (using a bioreactor system) A table outlining the predictive model for the ter is shown. Therefore, for large scale (e.g., pyro The parameters measured for a 1000L bioreactor system (in terms of scale) This is expected to apply to even larger cultures, such as those conducted by [unspecified method].

[0127] Equal parts This disclosure has been described in conjunction with its detailed explanation, but the above explanation is within the scope of the attached claims. This is intended to describe, and not limit, the scope of the present invention as defined by the box. It should be understood that this is not intended. Other aspects, advantages, and modifications are as follows: It is within the scope of the patent claims.

Claims

1. A culture medium of at least 25 L and a bioreceptor with a gas outflow rate of up to 20 m / s In the actor system, a population of cells consisting of shear-sensitive cells is cultured for 20 × 10 ６ Cells / mL to 15 x 10 ７ Steady state cells in the culture medium within the range of cells / mL A method including achieving cellular concentration.

2. The gas outflow velocities were 15 m / s, 14 m / s, 13 m / s, 12 m / s, and 11 m / s. The method according to claim 1, wherein the speed is 10 m / s, 9 m / s, or 8 m / s or less.

3. The gas outflow rate is such that the bioreactor system reaches steady state conditions at least The method according to claim 1 or 2, which is controlled until it is reached.

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

5. The gas outflow rate is controlled throughout the entire culture process, any one of claims 1 to 4. The method described in section [section number].

6. The bioreactor system is a perfusion bioreactor system, according to claims 1 to The method described in any one of item 5.

7. In the perfusion bioreactor system, the cells consisting of the shear-sensitive cells Culturing a population to achieve the aforementioned steady-state viable cell concentration is necessary to remove excess cells and / or non- The method according to claim 6, comprising the discharge or removal of living cells.

8. The culture medium has a maximum of 120 mmHg, a maximum of 115 mmHg, a maximum of 110 mmHg, and a maximum of 120 mmHg. Large 105mmHg, maximum 100mmHg, maximum 95mmHg, maximum 90mmHg, maximum 8 Claim 1, having dissolved carbon dioxide at a level of 5 mmHg or a maximum of 80 mmHg The method described in any one of items (7) to (7).

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 claims 1 to 8.

10. The bioreactor system has a capacity of at least 50 L, at least 100 L, and at least 200L, at least 500L, at least 1,000L, or at least 2,000 The method according to any one of claims 1 to 9, comprising 0 L of culture medium.

11. The culturing is carried out for a period of at least 10 days, any one of claims 1 to 10. The method described in section [section number].

12. The culturing is carried out for a period of 30 to 60 days, according to any one of claims 1 to 11. Methods used.

13. The method further comprises measuring the concentration of living cells, any one of claims 1 to 12. The method described in paragraph 1.

14. The measured live cell concentration was at least 30 × 10 ６ cells / mL, at least 40× 10 ６ cells / mL, or at least 50 × 10 ６ The cell / mL as described in claim 13. The method.

15. The shear-sensitive cells are mammalian cells, as described in any one of claims 1 to 14. Method of loading.

16. The shear-sensitive cells are human cells, as described in any one of claims 1 to 15. method.

17. The shear-sensitive cells mentioned above are HEK293 cells, fibrosarcoma HT1080 cells, and PER. Claims 1 to 16, which are C6 cells, CAP cells, HKB-11 cells, or HuH-7 cells. The method described in any one of the items.

18. The shear-sensitive cells are murine cells, according to any one of claims 1 to 17. Methods used.

19. The shear-sensitive cells are a mouse myeloma cell line, according to claims 1 to 15 and 18. The method described in item 1.

20. The shear-sensitive cells are NS0 cells or SP2 / 0 cells, claims 1-1 The method described in any one of paragraphs 5, 18, and 19.

21. The bioreactor system includes a cell retention device, any one of claims 1 to 20. The method described in item 1.

22. The cell retention device includes a continuous centrifuge and an alternating tangential flow filter. (ATF), Tangential Flow Membrane Filter (TFF), Dynamic Filter , spin filters, ultrasonic and dielectrophoretic separators, and / or gravity sediments. or the method according to claim 21, including the same.

23. Claim 21, the cell retention device is one or more ATFs, or includes one or more ATFs. Or the method described in 22.

24. The bioreactor system includes a bioreactor tank, and the bioreactor The tar tanks have capacities of at least 50L, 100L, 200L, 500L, 1,000L, and The method according to any one of claims 1 to 23, wherein the capacity is 2,000 L.

25. The bioreactor tank is a stirring tank type bioreactor, according to claim 24. Method of description.

26. The bioreactor system includes a sparger, any one of claims 1 to 26. The method described in section [section number].

27. The sparger is either a drill hole sparger or an open pipe sparger. A method according to claim 26.

28. The cultivation of the aforementioned cell population is carried out under conditions that express the cell product, claim The method described in any one of items 1 to 27.

29. The cell product is a nucleic acid, lipid, peptide, and / or protein, or The method according to claim 28, including the following.

30. The method according to claim 29, wherein the cell product is a recombinant protein.

31. The method according to claim 30, wherein the recombinant protein is a glycoprotein.

32. The method according to claim 31, wherein the glycoprotein is an Fc-containing glycoprotein.

33. The method according to claim 31 or 32, wherein the glycoprotein is an antibody drug.

34. The method according to claim 33, wherein the antibody drug is a monoclonal antibody.

35. The method according to claim 34, wherein the monoclonal antibody is ustekinumab.

36. The above method isolates the cell product from at least a portion of the shear-sensitive cells. and / or further isolation of the cell product from at least a portion of the culture medium. The method according to any one of claims 28 to 35, including the method described herein.

37. The gas outflow rate of the bioreactor system is controlled so that it does not exceed a rate of 20 m / s. A continuous culture process for culturing a population of cells consisting of shear-sensitive cells, including the following: It is S, The bioreactor system includes at least 25 L of culture medium and the collection of the cells The group is 20 x 10 ６ Cells / mL to 15 x 10 ７ Steady-state live cell concentration within the range of cells / mL The process for achieving the degree.

38. The bioreactor system has speeds of 15 m / s, 14 m / s, 13 m / s, and 12 m / s. The claim has a gas outflow rate of 11 m / s, 10 m / s, 9 m / s, 8 m / s or less. The process described in item 37.

39. The gas outflow rate is such that the bioreactor system reaches steady state conditions at least The process according to claim 37 or 38, which is controlled until it is completed.

40. The aforementioned steady-state conditions have a viable cell concentration that fluctuates by up to 20% over a period of 5 days. The process according to claim 39, including the following:

41. The gas outflow rate is controlled throughout the entire culture process, claims 37-40. The process described in any one of the following items.

42. Claim 37: The bioreactor system is a perfusion bioreactor system. The process described in any one of items 41-41.

43. In the perfusion bioreactor system, the cells consisting of the shear-sensitive cells Culturing a population to achieve the aforementioned steady-state viable cell concentration is necessary to remove excess cells and / or non- The process according to claim 42, comprising draining or removing living cells.

44. Claims 37 to further include controlling the dissolved carbon dioxide level of the cell culture medium. The process described in any one of paragraphs 43.

45. The culture media are 120 mmHg, 115 mmHg, 110 mmHg, and 105 mmHg. 100 mmHg, 95 mmHg, 90 mmHg, 85 mmHg, or 80 mmHg or less The process according to claim 44, wherein the dissolved carbon dioxide is at a level below a certain level.

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 according to any one of claims 44 or 45.

47. The perfusion bioreactor system has a capacity of at least 50 L, at least 100 L, and less at least 200L, at least 500L, at least 1,000L, or at least 2, The process according to any one of claims 37 to 46, comprising 000 L of culture medium.

48. The continuous culture process is carried out for a period of at least 10 days, according to claims 37 to 47. The process described in any one of the items.

49. The continuous culture process is carried out for a period of 30 to 60 days, as in any of claims 37 to 48. The process described in item 1.

50. The process includes measuring the concentration of living cells, any one of claims 37 to 49. The process described in item 1.

51. The measured live cell concentration was at least 30 × 10 ６ cells / mL, at least 40× 10 ６ cells / mL, or at least 50×10 ６ cells / mL, as described in claim 50 The process.

52. The shear-sensitive cells are mammalian cells, according to any one of claims 37 to 51. The process described.

53. The shear-sensitive cells are human cells, as described in any one of claims 37 to 52. The process.

54. The shear-sensitive cells mentioned above are HEK293 cells, fibrosarcoma HT1080 cells, and PER. Claims 37-5, which are C6 cells, CAP cells, HKB-11 cells, or HuH-7 cells. The process described in any one of item 3.

55. The shear-sensitive cells are murine cells, as per any one of claims 37 to 52. The process described in the section.

56. The shear-sensitive cells are a mouse myeloma cell line, according to claims 37-52 and 55. The process described in any one of the items.

57. The shear-sensitive cells are NS0 cells or SP2 / 0 cells, claim 37- The process described in any one of paragraphs 52, 52, and 56.

58. The bioreactor system includes a cell retention device, as of any of claims 37 to 57. The process described in item 1.

59. The cell retention device includes a continuous centrifuge and an alternating tangential flow filter. (ATF), Tangential Flow Membrane Filter (TFF), Dynamic Filter , spin filters, ultrasonic and dielectrophoretic separators, or gravity sediments, or the process according to claim 58, including those.

60. Claim 5, the cell retention device is one or more ATFs, or comprises them. The process described in 8 or 59.

61. The bioreactor system includes a bioreactor tank, and the bioreactor The tar tanks have capacities of at least 50L, 100L, 200L, 500L, 1,000L, and The process according to any one of claims 37 to 60, wherein the vessel has a capacity of 2,000 L.

62. The bioreactor tank is a stirring tank type bioreactor, according to claim 61. The process described.

63. The bioreactor system includes a sparger, as per any of claims 37 to 62. The process described in item 1.

64. The sparger is either a drill hole sparger or an open pipe sparger. A process according to claim 63.

65. The continuous culture process for culturing the aforementioned population of cells is performed under conditions that express cell products. The process according to any one of claims 37 to 64, carried out in [location].

66. The cell product is a nucleic acid, lipid, peptide, and / or protein, or The process according to claim 65, including these.

67. The process according to claim 66, wherein the cell product is a recombinant protein.

68. The process according to claim 67, wherein the recombinant protein is a glycoprotein.

69. The process according to claim 68, wherein the glycoprotein is an Fc-containing glycoprotein.

70. The process according to claim 68 or 69, wherein the glycoprotein is an antibody drug.

71. The process according to claim 70, wherein the antibody agent is a monoclonal antibody.

72. The process according to claim 71, wherein the monoclonal antibody is ustekinumab.

73. The process involves isolating the cell product from at least a portion of the shear-sensitive cells. to isolate the cell product from at least a portion of the culture medium. The process further includes the process according to any one of claims 65 to 72.

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