13C metabolic flux analysis of recombinant Chinese hamster ovary (CHO) cell cultures

Abstract

We have applied 13C metabolic flux analysis (13C MFA) to develop a system-wide understanding of the central carbon metabolism of Chinese hamster ovary (CHO) cells, the most widely used host for industrial production of monoclonal antibodies (mAbs) and other therapeutic proteins. First, we assessed dynamic changes in CHO metabolism during four different stages of a fed-batch culture to discover metabolic phenotypes that were associated with peak mAb productivity. While glycolytic metabolism dominated during the initial growth phase, enhanced oxidative metabolism involving increased citric acid cycle (CAC) flux and oxidative pentose phosphate pathway (oxPPP) activity prevailed during later phases when mAb production was highest. Next, we applied 13C MFA to examine CHO lines that had been engineered to express Bcl-2Δ, an engineered anti-apoptotic protein, to elucidate its impact on cell growth and metabolism. Not only was integrated viable cell density (IVCD) and culture longevity increased by Bcl-2Δ, but the intracellular fate of pyruvate was significantly altered over the entire course of a batch culture. More pyruvate was oxidized by mitochondrial CAC metabolism during the initial lactate-producing phase, and more lactate was consumed relative to glucose during the later lactate-consuming phase of culture. Finally, 13C MFA was applied to compare the metabolic phenotypes of nine different mAb secreting CHO lines, as well as three non-producing host lines. Among four clones that did not express Bcl-2Δ, a linear correlation was found between specific antibody productivity and total NADH production (an indicator of oxidative metabolism). Expression of Bcl-2Δ led to a general increase in NADH production that was accompanied by even greater specific mAb productivity. Overall, an in-depth quantification of CHO intracellular metabolism was accomplished by this dissertation, with multiple findings that are applicable to industrial antibody production.