Targeting Warburg effect in Chinese hamster ovary cell culture with pyruvate dehydrogenase kinase inhibitors

Production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is affected by the "Warburg effect" which is characterized by high lactate accumulation. Therefore, the present study explores the use of pyruvate dehydrogenase kinase (PDK) inhibitors dichloroacetate (DCA), metformin (M), (S)-3,3,3-trifluoro-2-hydroxy-2-methyl propionic acid (TPP), and 4-phenylbutyrate (PB) to counteract this metabolic bottleneck. To achieve optimal mAb yields, individual inhibitors (0.5-10 mM) were screened, followed by evaluation of their combinatorial effects. A combination of 5 mM DCA and 1 mM TPP (DCA + TPP) revealed ~2-fold enhancement in mAb yield (1.2 g/L) under fed-batch conditions, accompanied by a marked reduction in lactate accumulation as compared to control. The optimized DCA + TPP combination also exhibited 15%-20% enhancement in the mAb yield as compared to a single PDK inhibitor that is, DCA (5 mM) and TPP (1.0 mM) at the same concentration. Metabolic analysis revealed a 19.5-fold increase in pyruvate flux and a 2-fold increase in intracellular acetyl-CoA. The critical product quality attributes exhibited insignificant impact on charge variant (<3%), aggregation, and glycosylation of mAb. The amino acid consumption profiles predominantly showed depletion of glycine and tryptophan, whereas threonine and aspartic acid were primarily consumed in the control. Proteomic analysis exhibited upregulation and downregulation of various proteins, which significantly contribute to mAb's yield. Validation in Trastuzumab-producing CHO-S cells confirmed a 30% increase in mAb yield and a 10% improvement in cell density. These outcomes demonstrate that the combination of DCA + TPP is a promising metabolic intervention to mitigate the Warburg effect and enhance recombinant protein production in mammalian systems.