Decoupling Growth and Protein Production in CHO Cells: A Targeted Approach

Abstract

Fed-batch cultures of Chinese Hamster Ovary cells have been used to produce high quantities of biotherapeutics, particularly monoclonal antibodies. However, a growing number of next-generation biotherapeutics, such as bi-specific antibodies and fusion proteins, are difficult to express using standard fed-batch processes. Decoupling cell growth and biotherapeutic production is becoming an increasingly desired strategy for the biomanufacturing industry, especially for difficult-to-express products. Cells are grown to a high cell density in the absence of recombinant protein production (the growth phase), then expression of the recombinant protein is induced and cell proliferation halted (the production phase), usually by combining an inducible gene expression system with a proliferation control strategy. Separating the growth and production phases allows cell resources to be more efficiently directed toward either growth or production, improving growth characteristics and enhancing the production of difficult to express proteins. However, current mammalian cell proliferation control methods rely on temperature shifts and chemical agents, which interact with many non-proliferation pathways, leading to variable impacts on product quality and culture viability. Synthetic biology offers an alternative approach by strategically targeting proliferation pathways to arrest cell growth but have largely remained unused in industrial bioproduction. Due to recent developments in microbial decoupling systems and advances in available mammalian cell engineering tools, we propose that the synthetic biology approach to decoupling growth and production needs revisiting.

Acetate accumulation and regulation by process parameters control in Chinese hamster ovary cell culture

“…Temperature is a primary process parameter that globally regulates cell proliferation and metabolism 26 . Clones A and E, derived from two sister CHO‐K1 cell lines that share the same parental host, harbored the same monoclonal antibody molecule.…”

“…Temperature is a primary process parameter that globally regulates cell proliferation and metabolism. 26 profiles. Higher acetate accumulation (g) was achieved in the condition of Clone E-TS to 33 C, the corresponding cell specific glucose consumption rate (q Gluc. )…”

Resource-aware construct design in mammalian cells

“…Our work also highlights the importance of polyAs in defining the resource landscape of synthetic constructs and points to the need of a deeper understanding of the contribution of different expression resources in different genetic backgrounds. Other mitigation strategies have been proposed to address the shortcomings caused by resource competition in mammalian cells 15,16,[41][42][43][44] . One such example is the use of incoherent feed-forward loops (iFFLs).…”

An arrayed CRISPR screen reveals Myc depletion to increase productivity of difficult-to-express complex antibodies in CHO cells

“…Previous studies have shown that the productivity of DTE antibodies can be enhanced by decoupling growth and production phases ( 32–34 ). This results in the downregulation of cell proliferation and a redistribution of cellular resources toward increased recombinant protein production ( 35 ). Common strategies to halt proliferation and increase productivity included mild hypothermic cultivation ( 36 ), osmotic stress ( 37 ), addition of chemical agents ( 38 ) or genetic engineering ( 34 ).…”