386102 13C Flux Analysis of Metabolic Phenotypes Associated with Apoptotic Resistance in CHO Cells

Monday, November 17, 2014: 4:09 PM
206 (Hilton Atlanta)
Allison McAtee1, Neil Templeton1, Abasha Lewis2, Kevin Smith3, Michael J. Betenbaugh2 and Jamey D. Young1, (1)Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, (2)Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, (3)Janssen Pharmaceuticals (J&J), Spring House, PA

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.

Extended Abstract: File Not Uploaded