US-12624336-B2 - Methods of producing recombinant proteins

Abstract

In certain embodiments, this disclosure provides novel methods of increasing the viable cell density of an N−1 large-scale bioreactor cell culture, comprising culturing a host cell expressing a recombinant poly peptide of interest in a non-perfusion-based culture system, and wherein the viable cell density is increased to at least 5×10 6 cells/mL. In certain embodiments, the disclosure provides novel methods for large-scale production of a recombinant polypeptide of interest, comprising: (1) culturing a host cell expressing a recombinant polypeptide of interest in an N−1 stage in a non-perfusion-based culture system, wherein the viable cell density is increased to at least 5×10 6 cells/mL; and (2) culturing N fed-batch production cells in an enriched media with high-seed density at least 1.5×10 6 cells/mL, wherein the N fed-batch production cells are inoculated from the N-1 stage in a non-perfusion-based culture system.

Inventors

• Jianlin Xu
• ANDREW YONGKY
• Jun Tian
• Michael C. BORYS
• Zhengjian Li

Assignees

• BRISTOL-MYERS SQUIBB COMPANY

Dates

Publication Date

20260512

Application Date

20190702

Claims (13)

1. 1 . A method for large-scale production of a recombinant polypeptide of interest comprising: (1) culturing a host cell expressing the recombinant polypeptide of interest in an N−1 stage, non-perfusion-based culture system, wherein the host cells are cultured in an enriched medium to obtain an N−1 stage viable cell density of at least 15×10 6 cells/mL, wherein the host cells are CHO cells; and (2) inoculating an N production culture system at high-seed density of at least 2×10 6 cells/mL with host cells from the N−1 stage, non-perfusion-based culture system, wherein the enriched medium comprises an increased amount of a carbon source relative to non-enriched medium, wherein the carbon source is glucose.
2. 2 . The method of claim 1 , wherein the N production culture system is a fed-batch bioreactor.
3. 3 . The method of claim 2 , wherein the fed-batch bioreactor is at least 1,000 L, at least 5,000 L, at least 10,000 L, at least 15,000 L, or at least 20,000 L.
4. 4 . The method of claim 1 , wherein the enriched medium is enriched by a feed media at least 5%, at least 10%, at least 15%, at least 20% relative to non-enriched medium.
5. 5 . The method of claim 1 , wherein the enriched medium further comprises an increased amount of nutrients relative to non-enriched medium, wherein the nutrients are selected from amino acids, lipids, vitamins, minerals, and polyamines.
6. 6 . The method of claim 1 , wherein the titer of the polypeptide of interest is at least 100 mg/L, at least 1 g/L, at least 3 g/L, at least 5 g/L or at least 10 g/L.
7. 7 . The method of claim 1 , wherein the host cell is cultured in a basal media or an enriched basal media to obtain an N production stage viable cell density of at least 5×10 6 , or at least 10×10 6 viable cells per mL.
8. 8 . The method of claim 1 , further comprising the step of isolating the polypeptide of interest, wherein the polypeptide of interest is an antibody or antigen-binding fragment.
9. 9 . The method of claim 8 , wherein the antibody binds an antigen, wherein the antigen is PD-1, PD-L1, LAG-3, TIGIT, GITR, CXCR4, CD73 HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, EGFR, HER3, or HER4.
10. 10 . The method of claim 1 , wherein the N production culture system is inoculated at high-seed density of at least 3×10 6 cells/mL with host cells from the N−1 stage, non-perfusion-based culture system.
11. 11 . The method of claim 1 , wherein the N production culture system is inoculated at high-seed density of at least 5×10 6 cells/mL with host cells from the N−1 stage, non-perfusion-based culture system.
12. 12 . The method of claim 1 , wherein the non-perfusion-based culture system is a batch bioreactor.
13. 13 . The method of claim 1 , wherein the non-perfusion-based culture system is a fed-batch bioreactor.

Description

CROSS REFERENCE TO RELATED APPLICATIONS The present application is a U.S. National Stage of International Patent Application No. PCT/US2019/040298, filed Jul. 2, 2019, which claims the priority benefit of U.S. Provisional Application No. 62/693,606, filed Jul. 3, 2018, each of which is hereby incorporated by reference in their entirety. FIELD OF INVENTION The present invention generally relates to methods of increasing viable cell density during the N−1 culture stage with non-perfusion strategy for inoculation of N production bioreactors at high-seed density for cell culture manufacturing. BACKGROUND Proteins and polypeptides have become increasingly important as therapeutic agents. In most cases, therapeutic proteins and polypeptides are produced in cell culture, from cells that have been engineered and/or selected to produce unusually high levels of the polypeptide of interest. Control and optimization of cell culture conditions is critically important for successful commercial production of proteins and polypeptides. Many proteins and polypeptides produced in cell culture are made in a fed-batch process, in which cells are cultured for a period of time, and then the culture is terminated and the produced protein or polypeptide is isolated. The ultimate amount and quality of protein or polypeptide produced can be dramatically affected by the N−1 seed culture and the seed-density at N production. While efforts have been made to improve production of proteins and polypeptides in fed-batch culture processes, there remains a need for additional improvements. Perfusion cell culture can achieve much higher viable cell densities than conventional fed-batch cell culture systems. Perfusion cell culture provides a continuous supply of fresh media in the culture system, while removing waste products, which provides a rich environment for the cells to grow. In comparison to the conventional fed-batch production culture with low-seed density, the high-seed density fed-batch production culture inoculated with N−1 perfusion seed can achieve higher final titer within a short duration. However, perfusion cell culture becomes expensive when used in large-scale culture systems (e.g., greater than 200 L bioreactor) because of large quantities of cell culture media consumed. Also, perfusion cell culture can have complications from the cell retention system which prevents the cells from being removed from the cell culture system, especially for a large scale manufacturing. There is a particular need for the development of improved systems for producing proteins and polypeptides by large-scale cell culture at high-seed cell density with non-perfusion systems. BRIEF SUMMARY OF THE DISCLOSURE The present disclosure is directed to a method of increasing the viable cell density of a N−1 large-scale bioreactor cell culture, comprising culturing a host cell expressing a recombinant polypeptide of interest in a non-perfusion-based culture system, and wherein the viable cell density is increased to at least 5×106 cells/mL. In some embodiments, the non-perfusion-based culture system is a batch or fed-batch bioreactor. In some embodiments, the viable cell density at an N−1 stage is at least 5×106, at least 10×106, at least 15×106, at least 20×106, at least 25×106, or at least 30×106 viable cells per mL. In some embodiments, the cell viability is at least 80% on the last day of the N−1 stage, at least 85% on the last day of the N−1 stage, or at least 90% on the last day of the N−1 stage. In some embodiments of the invention, the host cell is cultured in an enriched media for an N−1 batch culture. In some embodiments, the host cell is cultured in a seed media with addition of a feed media for an N−1 fed-batch culture. In some embodiments of the invention, the media is enriched by a feed media at least 5% relative to non-enriched media, at least 10% relative to non-enriched media, at least 15% relative to non-enriched media, or at least 20% relative to non-enriched media. In some embodiments, the enriched media or feed media comprises an increased amount of a carbon source. In some embodiments, the carbon source is glucose. In some embodiments, the enriched media or feed media comprises an increased amount of nutrients. In some embodiments, the nutrients are selected from amino acids, lipids, vitamins, minerals, and polyamines. In some embodiments, the enriched media comprises an increased amount of a carbon source and nutrients. In some embodiments, the carbon source is glucose and the nutrients are selected from amino acids, lipids, vitamins, minerals, and polyamines. In some embodiments of the invention, the host cell is a mammalian cell. In some embodiments, the mammalian cell is selected from the group consisting of CHO, VERO, BHK, HEK, HeLa, COS, MDCK and hybridoma cells. In some embodiments, the host cell is a CHO cell. In some embodiments of the invention, the polypeptide of interest is a therapeutic polypeptide. In some embodiments