2025 AIChE Annual Meeting

(70h) Metabolic Rewiring of CHO Cell Growth By Supplementing the Cell Culture Medium with Citrate and ?-Ketoglutarate As the TCA Cycle Intermediates

Authors

Vaughn Perveiler, GlaxoSmithKline
Blake Askenas, GlaxoSmithKline
Kyle Kleinberg, GlaxoSmithKline
The biochemical pathways related to Chinese Hamster Ovary (CHO) cell metabolism are complex, and perturbations to this network impact the cell culture growth and cellular functions supporting recombinant protein production. There is an opportunity to rewire CHO’s metabolism by modulating the cell culture medium they are exposed to. We characterize the impact of two tricarboxylic-acid (TCA) cycle intermediates, citrate and α-ketoglutarate (aKG), on cell growth. Our proprietary host cell lines and in-house media-platform were used. The rationale was to test the TCA cycle activity exhibited by CHO cells during growth. Spent media analysis was performed based on liquid-chromatography/mass-spectrometry (LC/MS).

To characterize the effect of citrate addition to basal media, we selected a mAb-production process with low growth rate at 2.0mM. A 0-2mM citrate-concentration range was tested to show that above 1 mM citrate became inhibitory of growth compared to control. In the same project, we evaluated the 0-4mM aKG concentration range to discover no impact on growth. aKG was completely consumed and production of glutamate as byproduct. Glutamate was secreted with an average conversion of 64% for every aKG consumed at concentrations above 0.5mM.

In conclusion, low incorporation of aKG and citrate into TCA cycle indicates that CHO cells rely more on glycolysis than TCA cycle during exponential growth phase. However, we found the presence of transamination activities based on the active co-assimilation of aKG and aspartate. These results elucidate the metabolic rewiring of CHO cells in the transition from exponential to stationary phase of growth.