The push toward higher cell densities and product titers in mammalian cell bioprocesses is often accompanied by accumulation of inhibitory metabolites, specifically lactate and ammonia. Accumulation of these toxic by-products is a potent trigger of apoptosis, which is responsible for roughly 80% of cell death in a typical bioreactor. Additionally, lactate accumulation is an indication that carbon sources are being used inefficiently, thus reducing yields of product protein and biomass. Therefore, we are investigating the causes of lactate accumulation and how it can be reversed by metabolic engineering and/or medium optimization strategies.
We and others have observed that CHO cells are capable of not only producing but also consuming lactate under certain conditions. The switch to lactate consumption often accompanies the depletion of a preferred carbon source (e.g., glucose or glutamine) from the growth medium. In shake flask experiments, we observed that the specific lactate production rate was halved when the medium glutamine concentration was reduced from 8 to 4 mM. This corresponded with more efficient growth (30% higher specific growth rate and 40% higher peak viable cell density) as well as a 70% decrease in specific glucose consumption rate. Therefore, by reducing glutamine availability we were able to shift CHO cells to a more efficient metabolic state involving both increased growth rate and decreased by-product accumulation.
We are conducting 13C metabolic flux analysis and metabolomic profiling experiments to further elucidate the mechanism by which CHO cells switch from a high to a low lactate-accumulating state. We are also investigating the effects of anti-apoptotic proteins to modulate these metabolic shifts, which is a novel aspect of our work that builds upon recent findings that these proteins can exert a strong influence on lactate metabolism in CHO cells (Dorai et al., Biotechnol. Bioeng. 103:592-608, 2009). We will present results on several wild-type and engineered CHO cell lines, including the commercially available CHO-S line and three adapted CHO-K1 lines. Two of the CHO-K1 lines have been transfected with either the Bcl-2Δ or Bcl-xL anti-apoptotic genes. We will discuss how these genetic manipulations can be combined with improved medium design to redirect metabolic fluxes in CHO cells and thereby reduce the accumulation of inhibitory by-products.
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