Chinese Hamster Ovary (CHO) cells are the predominant hosts for recombinant protein therapeutics production. CHO cells in high cell density fed-batch cultures exhibit enhanced consumption of nutrients including glucose and amino acids. These substrates, primarily used for biomass production and protein synthesis, may be diverted towards metabolic pathways generating by-products. Very few studies have examined the metabolism of amino acids in CHO cell cultures. Thus, we applied comprehensive ¹³C labeling assisted metabolomics and advanced flux analysis tools to elucidate the metabolic flow of all 20 amino acids and glucose in two different IgG producing CHO cell lines (CHO-K1 and CHO-GS). Custom media formulations each depleted with a single amino acid (e.g., valine) were supplemented with a fully labeled ¹³C amino acid (here, [U-¹³C] valine). Carbon enrichment flows were tracked and changes in amino acids metabolism were quantified when cells transitioned from growth phase to stationary phase using GCMS based analytical methods. As cells metabolized labeled amino acids, this resulted in a redistribution of ¹³C-atoms which we quantified for extracellular metabolites IgG product, free intracellular metabolites, biomass and lipids. This allowed us to analyze relative pathway activities, assess nutrient contributions to metabolite production and calculate fraction of each amino acid that was used for cell growth, protein production, and energy generation. Mass isotopomer distributions and labeling dynamics yielded information about metabolic pathways and changes in metabolism respectively. Some differences in amino acids utilization were also observed between the two different CHO cell lines. Prior studies have employed LCMS based metabolomics to identify cytotoxic metabolites generated by cells via amino acid catabolism. Using ¹³C labeling assisted GCMS based metabolomics and commercially available standards, we were able to identify and evaluate some of these previously identified cytotoxic metabolites. Integrating all of this data together, we are building stoichiometric and kinetic models of CHO cell metabolism. This knowledge will help elucidate strategies including media design and feeding to modulate amino acids levels in CHO cell cultures sufficient for protein synthesis while limiting unwanted byproduct synthesis.

Michael Betenbaugh

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