Using MVDA with Stoichiometric Balances to Optimize Amino Acid Concentrations in Chemically Defined CHO Cell Culture Medium for Improved Culture Performance

Chemically defined (CD) media are routinely used in the production of biologics in Chinese Hamster Ovary (CHO) cell culture and provide enhanced raw material control. Nutrient optimized CD media is an important path to increase cell growth and monoclonal antibody (mAb) productivity in recombinant CHO cell lines. However, nutrient optimization efforts for CD media typically rely on multi-factorial and experimental design of experiment (DoE) approaches or complex mathematical models of cellular metabolism or gene expression systems. Moreover, the majority of these efforts are aimed at amino acids since they constitute essential nutrients in CD media as they directly contribute to biomass and protein production. In this study, we demonstrate the utilization of multi-variate data analytics (MVDA) coupled with amino acid stoichiometric balances (SBs) to increased cell growth and mAb productivity in efforts to reduce CD media development efforts. SBs measure the difference between theoretical demand of amino acids and the empirically measured fluxes to identify metabolic states of the cell. When coupled with MVDA, the statistical models were not only able to highlight key amino acids towards cell growth or productivity, but also provided direction on metabolic favorability of the amino acid. Experimental validation of our approach resulted in a 55% increase in total cell growth and about an 80% increase in total mAb productivity. Increased specific consumption of stoichiometrically balanced amino acids and decreased specific consumption of glucose was also observed in optimized CD media suggesting favorable consumption of desired nutrients and a potential for energy redistribution towards increased cellular growth or mAb productivity.

Osmolality Effects on CHO Cell Growth, Cell Volume, Antibody Productivity and Glycosylation

The addition of nutrients and accumulation of metabolites in a fed-batch culture of Chinese hamster ovary (CHO) cells leads to an increase in extracellular osmolality in late stage culture. Herein, we explore the effect of osmolality on CHO cell growth, specific monoclonal antibody (mAb) productivity and glycosylation achieved with the addition of NaCl or the supplementation of a commercial feed. Although both methods lead to an increase in specific antibody productivity, they have different effects on cell growth and antibody production. Osmolality modulation using NaCl up to 470 mOsm kg−1 had a consistently positive effect on specific antibody productivity and titre. The addition of the commercial feed achieved variable results: specific mAb productivity was increased, yet cell growth rate was significantly compromised at high osmolality values. As a result, Feed C addition to 410 mOsm kg−1 was the only condition that achieved a significantly higher mAb titre compared to the control. Additionally, Feed C supplementation resulted in a significant reduction in galactosylated antibody structures. Cell volume was found to be positively correlated to osmolality; however, osmolality alone could not account for observed changes in average cell diameter without considering cell cycle variations. These results help delineate the overall effect of osmolality on titre and highlight the potentially negative effect of overfeeding on cell growth.