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EP-4735575-A1 - SYSTEMS AND METHODS OF REAL TIME PREDICTION OF BIOMASS FOR CELLS CULTURED IN FIXED BED BIOREACTORS

EP4735575A1EP 4735575 A1EP4735575 A1EP 4735575A1EP-4735575-A1

Abstract

A method of monitoring biomass during a cell culture of cells in a bioreactor is provided. The method includes culturing the cells in the bioreactor using a cell culture medium perfused through the bioreactor; measuring at least one of a cell nutrient and a cell byproduct in the cell culture medium; determining at least one of a consumption rate of the cell nutrient and an accumulation rate of the cell byproduct; and predicting a cell number within the bioreactor at a specified culture time based on at least one of the consumption rate and the accumulation rate.

Inventors

  • FANG, YE
  • GORAL, VASILIY NIKOLAEVICH
  • HONG, YULONG
  • JEROME, Snold Junior
  • KREBS, Kathleen Anne
  • PIKULA, DRAGAN
  • SUN, Yujian

Assignees

  • Corning Incorporated

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A method of monitoring biomass during a cell culture of cells in a bioreactor, the method comprising: culturing the cells in the bioreactor using a cell culture medium perfused through the bioreactor; measuring at least one of a cell nutrient and a cell byproduct in the cell culture medium; determining at least one of a consumption rate of the cell nutrient and an accumulation rate of the cell byproduct; predicting a cell number within the bioreactor at a specified culture time based on at least one of the consumption rate and the accumulation rate.
  2. 2. The method of claim 1, wherein the bioreactor is a fixed bed bioreactor comprising a substrate configured for culturing cells attached to a surface of the substrate.
  3. 3. The method of claim 1 or claim 2, wherein the at least one cell nutrient is glucose or glutamine.
  4. 4. The method of any of claims 1-3, wherein the at least one cell byproduct is lactate or ammonia.
  5. 5. The method of any of claims 1-4, wherein the cell culture medium is a glucose- or a glutamine-rich cell culture medium.
  6. 6. The method of any of claims 1-5, wherein the measuring the at least one of the cell nutrient and the cell byproduct in the cell culture medium comprises taking multiple measurements of the cell nutrient or the cell byproduct, the multiple measurements being separated by a measurement interval that is less than a doubling time of the cells in the cell culture.
  7. 7. The method of claim 6, wherein the measurement interval is greater than or equal to a minimum interval time, the minimum interval time being a time at which the change in the level of the cell nutrient or the cell byproduct is larger than a measurement tolerance of measuring the cell nutrient or cell byproduct.
  8. 8. The method of claim 7, wherein the minimum interval time is greater than or equal to about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours.
  9. 9. The method of any of claims 1-8, wherein the measuring of the cell nutrient in the cell culture medium comprises measuring the cell nutrient multiple times per day of the cell culture.
  10. 10. The method of any of claims 1-9, wherein the measuring of the cell byproduct in the cell culture medium comprises measuring the cell byproduct multiple times per day of the cell culture.
  11. 11. The method of any of claims 1-10, wherein the predicting the cell number comprises using a mathematical model to calculate biomass at the specified culture time.
  12. 12. The method of any of claims 1-11, wherein the measuring comprises using an inline sensor in a perfusion line of the cell culture medium.
  13. 13. The method of any of claims 1-11, wherein the measuring comprises using an offline measurement of samples of the cell culture medium.
  14. 14. The method of any of claims 1-13, further comprising, after the determining of the consumption rate and the accumulation rate, comparing at least one of the consumption rate of a first cell nutrient and the accumulation rate of a first cell byproduct to at least one of the consumption rate of a second cell nutrient and the accumulation rate of a second cell byproduct.
  15. 15. The method of claim 14, wherein the comparing comprises comparing the consumption rate of glucose to the consumption rate of glutamine.
  16. 16. The method of claim 14 or claim 15, wherein the comparing comprises comparing the consumption rate of glucose to the accumulation rate of ammonia.
  17. 17. The method of any of claims 14-16, wherein the comparing comprises comparing the accumulation rate of lactate to the consumption rate of gutamine.
  18. 18. The method of any of claims 14-17, wherein the comparing comprises comparing the accumulation rate of lactate to the accumulation rate of ammonia.
  19. 19. The method of any of claims 14-18, further comprising determining an abnormality in the cell culture based on the comparing.
  20. 20. The method of any of claims 1-19, further comprising seeding cells in the bioreactor at a seeding density.

Description

SYSTEMS AND METHODS OF REAL TIME PREDICTION OF BIOMASS FOR CELLS CULTURED IN FIXED BED BIOREACTORS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application Serial No. 63/546,657 filed on October 31, 2023 and U.S. Provisional Application Serial No. 63/524,279 filed on June 30, 2023, the content of which are relied upon and incorporated herein by reference in their entirety. FIELD OF THE DISCLOSURE [0002] This disclosure general relates to systems and methods of monitoring and predicting cell cultures in bioreactor systems. In particular, the present disclosure relates to methods, protocols, systems, and models for biomass monitoring of a cell culture within a bioreactor system, and using oxygen uptake rate (OUR) to model cell attachment and growth. BACKGROUND [0003] In the bioprocessing industry, large-scale cultivation of cells is performed for purposes of the production of hormones, enzymes, antibodies, vaccines, therapeutic proteins, and cell therapies. Cell and gene therapy markets are growing rapidly, with promising treatments moving into clinical trials and quickly toward commercialization. However, one cell therapy dose can require billions of cells or trillions of viruses. As such, being able to provide a large quantity of cell products in a short amount of time is critical for clinical success. [0004] A significant portion of the cells used in bioprocessing are anchorage dependent, meaning the cells need a surface to adhere to for growth and functioning. Traditionally, the culturing of adherent cells is performed on two-dimensional (2D) cell-adherent surfaces incorporated in one of a number of vessel formats, such as T-flasks, petri dishes, cell factories, cell stack vessels, roller bottles, and other multilayered vessels (e.g., the HYPERStack® from Coming Inc.). These approaches can have significant drawbacks, including the difficulty in achieving cellular density high enough to make it feasible for large scale production of therapies or cells. [0005] Alternative methods have been suggested to increase volumetric density of cultured cells. These include microcarrier cultures performed in stir tanks; hollow fiber bioreactors, in which cells may form large three-dimensional aggregates as they proliferate in the interspatial fiber space; and packed-bed bioreactors. In packed-bed or fixed-bed bioreactors, a packed or fixed cell substrate is used to provide a surface for the attachment of adherent cells. Medium is perfused along the surface or through the semi -porous substrate to provide nutrients and oxygen needed for the cell growth. For example, packed bed bioreactor systems that contain a packed bed of support or matrix systems to entrap the cells have been previously disclosed U.S. Patent Nos. 4,833,083; 5,501,971; and 5,510,262. Packed bed matrices usually are made of porous particles as substrates or non-woven microfibers of polymer. [0006] One of the significant issues with traditional fixed bed bioreactors is the nonuniformity of cell distribution inside the bed. For example, the packed bed can function as a depth filter with cells predominantly trapped at the inlet regions or other regions of relatively low flow and/or high substrate density, resulting in a gradient of cell distribution during the inoculation step. In addition, due to random fiber packaging, flow resistance and cell trapping efficiency of cross sections of the packed bed are not uniform. For example, medium flows fast though the regions with low cell packing density and flows slowly through the regions where resistance is higher due to higher number of entrapped cells. This creates a channeling effect where nutrients and oxygen are delivered more efficiently to regions with lower volumetric cells densities and regions with higher cell densities are being maintained in suboptimal culture conditions. [0007] Another significant drawback of traditional packed bed systems disclosed in a prior art is the inability to efficiently harvest intact viable cells at the end of culture process. Harvesting of cells is important if the end product is cells, or if the bioreactor is being used as part of a “seed train,” where a cell population is grown in one vessel and then transferred to another vessel for further population growth. U.S. Patent No. 9,273,278 discloses a bioreactor design to improve the efficiency of cell recovery from the packed bed during cells harvesting step. It is based on loosening the packed bed matrix and agitation or stirring of packed bed particles to allow porous matrices to collide and thus detach the cells. However, this approach is laborious and may cause significant cells damage, thus reducing overall cell viability. [0008] In addition, because of the random arrangement of fibers in the traditional packed or fixed bed substrates, it can be difficult for bioreactor users to predict cell culture performance, since the su