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JP-7856698-B2 - Methods for continuous cell culture

JP7856698B2JP 7856698 B2JP7856698 B2JP 7856698B2JP-7856698-B2

Inventors

  • イン， ジン
  • ザン， ルー

Assignees

  • モメンタ ファーマシューティカルズ インコーポレイテッド

Dates

Publication Date
20260511
Application Date
20240705
Priority Date
20180906

Claims (16)

  1. A method comprising culturing a population of shear-sensitive mammalian cells in a perfusion bioreactor system having a gas outflow rate of 7 to 10 m/s and a dissolved carbon dioxide level of up to 80 mmHg, to achieve a steady-state viable cell concentration in the culture medium ranging from 20 × 10⁶ cells/mL to 15 × 10⁷ cells/mL, wherein the shear-sensitive mammalian cells are HEK293 cells, fibrosarcoma HT1080 cells, PER. C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, or SP2/0 cells .
  2. The method according to claim 1, wherein the shear-sensitive mammalian cell is a human cell or a murine cell.
  3. The method according to claim 1, wherein the gas outflow rate is controlled at least until the bioreactor system reaches the steady-state conditions.
  4. The method according to claim 3, wherein the steady-state conditions include having a viable cell concentration that fluctuates by up to 20% over a period of 5 days.
  5. The method according to claim 1, wherein the gas outflow rate is controlled throughout the entire culture.
  6. The method according to claim 1, wherein the culturing further comprises draining or removing excess cells and/or non-living cells.
  7. The method according to claim 1, wherein the culturing is carried out for a period of 30 to 60 days.
  8. The method according to claim 1, further comprising measuring the concentration of living cells.
  9. The 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 method according to claim 8, wherein the amount is cells/mL.
  10. The method according to claim 1, wherein the bioreactor system includes a cell retention device, the cell retention device being or including a continuous centrifuge, an alternating tangential flow filter (ATF), a tangential flow membrane filter (TFF), a dynamic filter, a spin filter, an ultrasonic and dielectrophoretic separator, and/or a gravity sedimentation device.
  11. The bioreactor system according to claim 1, wherein the bioreactor system includes a sparger.
  12. The method according to claim 11, wherein the sparger is a drill hole sparger or an open pipe sparger.
  13. The method according to claim 1, wherein the culture of the population of cells is carried out under conditions that express a cell product, the cell product comprising one or more of nucleic acids, lipids, peptides, and proteins.
  14. The method according to claim 13, wherein the cell product is an antibody drug.
  15. The method according to claim 13, further comprising isolating the cell product from at least a portion of the shear-sensitive mammalian cells and/or isolating the cell product from at least a portion of the culture medium.
  16. Control the gas outflow rate of the perfusion bioreactor system so that it is between 7 and 10 m/s, and Control the dissolved carbon dioxide level in the culture medium so that the dissolved carbon dioxide level reaches a maximum of 80 mmHg. A continuous culture process for culturing a population of cells consisting of shear-sensitive mammalian cells, including, The aforementioned cell population is 20 × 10 6 Cells/mL to 15 x 10 7 The process achieves a steady-state viable cell concentration within the range of cells/mL, and the shear-sensitive mammalian cells are HEK293 cells, fibrosarcoma HT1080 cells, PER. C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, or SP2/0 cells.

Description

Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 62/727,976, filed on 6 September 2018, which is incorporated herein by reference in its entirety. Continuous cell culture methods and systems are becoming widespread in biomanufacturing. However, these methods and systems are associated with many precautions and troublesome problems. Therefore, there is still a need to improve continuous cell culture methods and systems. 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 generating bioreactor (e.g., a stirred bioreactor tank), a cell retention device (e.g., alternating tangential flow), a supply medium, and a waste discharge system. 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 are shown. 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. 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. The viable cell concentration ( 10⁶ cells/mL) of shear-sensitive cells (e.g., SP2/0 cells) cultured by perfusion culture using varying gas outflow rates at different scales is shown. Filled circles represent a 100 L culture process, open squares represent a 3 L control culture, and open diamonds represent a 200 L culture process. At time point (1), 2 L of the culture medium from the 200 L culture process is transferred to 3 L of culture, and this culture condition is represented by a filled triangle. At time point (2), the control gas outflow rate of the 200 L culture is reduced to 10 m/s or less. At time point (3), the control gas outflow rate of the 200 L culture is again increased to at least 20 m/s. This shows several serial cell culture runs using a 3L perfusion bioreactor at various gas outflow 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 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 reduced to 10 m/s thereafter. 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. This figure shows a comparison of 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.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. A table is shown outlining predictive models for various parameters for a 250L culture (for example, using a SUB250 bioreactor system). 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. 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 of 250L (represented by triangles) and 100L (represented by circles). A table is shown outlining predictive models for various parameters for a 1000L culture (for example, using the SUB1000 bioreactor system). Here is an example equation for determining the gas outflow rate. Specific Definitions Generally, technical terms used herein have the same meaning as they are understood in the art unless expli