Chinese Hamster Ovary (CHO) cells are a major host for therapeutic recombinant protein production. Frequently, high substrate consumption rates lead to accumulation of toxic metabolites, resulting in lower cell viability and protein production. Thus, it is imperative to understand the metabolic fluxes in CHO cells as a function of culture conditions and the subsequent effect on the protein production. We have performed metabolic flux analysis of CHO cell culture in stationary phase by isotopomer balancing. The effects of process parameters such as dissolved oxygen and CO₂ on the metabolic fluxes and protein production were also investigated. Interestingly, analysis of the glycolytic and pentose phosphate pathway (PPP) fluxes indicates that almost all glucose flux is diverted towards NADPH production. Additionally, labeling analysis of the TCA cycle intermediates indicates low flux from pyruvate to citrate. We will present physiological insights gained from the metabolic flux analysis.

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