(173d) 13C Flux Analysis of Metabolic Phenotypes Associated with Apoptotic Resistance in CHO Cells

Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins.  However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We have transfected Chinese hamster ovary (CHO) cells to express Bcl-2Δ, an engineered anti-apoptotic gene.  Another line was transfected to simultaneously express three other anti-apoptotic genes: E1B-19K, AVEN, and XIAPΔ. ¹³C metabolic flux analysis (MFA) was then applied to elucidate the metabolic alterations induced by these anti-apoptotic gene expressions.  Expression of Bcl-2Δ reduced lactate accumulation by redirecting the fate of intracellular pyruvate toward mitochondrial oxidation during the lactate-producing phase, and it significantly increased lactate re-uptake during the lactate-consuming phase.  This flux redistribution was associated with significant increases in biomass yield, peak viable cell density (VCD), and integrated VCD.  Additionally, Bcl-2Δ expression was associated with significant increases in isocitrate dehydrogenase and NADH oxidase activities, both rate-controlling mitochondrial enzymes. Our presentation will compare the metabolic impact of Bcl-2Δ expression with that of combinatorial expression of E1B-19K, AVEN, and XIAPΔ. This is the first comprehensive ¹³C MFA study to demonstrate that expression of anti-apoptotic genes has a significant impact on intracellular metabolic fluxes, especially in controlling the fate of pyruvate carbon, which has important biotechnology applications for reducing lactate accumulation and enhancing productivity in mammalian cell cultures.