Nicotinamide Reverses the Warburg Effect in CHO Cell Culture

The Warburg effect, the preferential conversion of glucose-derived pyruvat to lactate despite available oxygen, is a key feature of Chinese hamster ovary (CHO) cell culture. Lactate accumulation in recombinant protein-producing cell culture is an inefficient usage of glucose, as well as being deleterious to cells. Lactate accumulation lowers culture pH, requiring base addition to maintain bioreactor pH setpoint, which subsequently leads to hyperosmolarity, adversely impacting cell growth, productivity and product quality. A key driver for the Warburg effect, and hence lactate accumulation, is the need to regenerate NAD⁺ consumed during glycolysis. Since oxidative phosphorylation has limited capacity to recycle NADH back to NAD⁺ at high glycolytic fluxes, cells rely on lactate dehydrogenase (LDH) to convert pyruvat to lactate, simultaneously regenerating NAD⁺ and sustaining glycolysis. Thus, providing the cells' capacity to generate more NAD⁺ would decrease the reliance on the Warburg effect. In this study, feeding the NAD⁺ precursor nicotinamide (NAM) leads to reversal of the Warburg effect, inducing the "lactate shift" 3 days earlier in cell culture and reducing peak lactate concentration by 40%. Transcriptomic analysis further confirms this metabolic shift, with an upregulation of key mitochondrial electron transport chain genes. NAM supplementation increased Fc galactosylation without affecting fucosylation, sialylation, or high-mannose species. These results identify NAD⁺/NADH balance as a key regulator of the Warburg effect and demonstrate NAM supplementation as a simple, cost-effective strategy to mitigate lactate accumulation and improve metabolic efficiency in CHO cell cultures.