Amino acids, including asparagine, aspartate, glutamine, and glutamate, play important roles in the purine and pyrimidine biosynthesis as well as serve as anaplerotic sources fueling the tricarboxylic acid (TCA) cycle for mitochondrial energy generation in mammalian cells. Despite extensive studies on glutamine and glutamate in CHO cell cultures, the roles of asparagine and aspartate, especially in feed media, remain underexplored. In this study, we utilized the CHO genome scale model to first deeply characterize the intracellular metabolic states of CHO cells cultured in different combinations of basal and feed media to understand the traits of asparagine/aspartate-dependent and glutamate-dependent feeds. Subsequently, we identified the critical role of asparagine and aspartate in the feed media as anaplerotic sources and conduct in silico simulations to ascertain their optimal ratios to improve cell culture performance. Finally, based on the model simulations, we reformulated the feed media by tailoring the concentrations of asparagine and aspartate. Our experimental data reveal a CHO cell preference for asparagine compared with aspartate, and thus maintaining an optimal ratio of these amino acids is a key factor for achieving optimal CHO cell culture performance in biopharmaceutical production.

Designing and selecting cell culture media and their feeding are a key strategy to maximize culture performance in industrial biopharmaceutical processes. However, mammalian cells are very sensitive to their culture environment, requiring specific nutritional needs to grow and produce high-quality proteins such as antibodies, depending on cell lines and operational conditions. In this regard, previously we developed data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on Chinese hamster ovary (CHO) cell culture performance, allowing us to design and reformulate basal media. To expand our exploration for media development research further, we evaluated two chemically defined feed media, A and B, in ambr15 bioreactor runs using a monoclonal antibody-producing CHO K1 cell line. We observed a significant impact of feed media on cell growth, longevity, viability, productivity and toxic metabolites production. Specifically, concentrated feed A was not sufficient to support prolonged cell culture and high titer compared to feed B. The framework systematically characterized the major metabolic bottlenecks in the TCA cycle and its related amino acid transferase reactions, thereby identifying key design components, such as asparagine, aspartate, and glutamate, which are needed for highly productive cell cultures. Based on our results, we subsequently reformulated the feeds by adjusting the amounts of those amino acids and successfully validated their effectiveness in promoting cell growth and/or titer.