A postulation of the acetate metabolic pathway in recombinant CHO cells with high-yield bispecific antibody expression

Chinese hamster ovary (CHO) cells are the primary host for biopharmaceutical production, yet metabolic imbalances and acidic byproduct accumulation frequently compromise efficiency. Although lactate metabolism is well characterized, acetate metabolism remains poorly understood. CHO-C (CHO-K1-derived cell line expressing PD-1/VEGF bispecific antibody) and CHO-B (CHO-K1-derived cell line expressing anti-EGFRvIII antibody C12) were used as research models in this study, aiming to investigate the origin of acetate in CHO cells and identify its genes associated with acetate accumulation, thereby alleviating pH acidification and restoring antibody expression impaired by acetate accumulation. Using 13 C-labeled pyruvate, pyruvate was shown to contribute to acetate formation in CHO cells. In high-acetate CHO-C cells, LDHA, LDHB and DLAT mRNA levels were upregulated nearly 2 times versus low-acetate CHO-B controls. SiRNA knockdown showed that LDHA and DLAT inhibition reduced acetate production by approximately 30%, whereas LDHB knockdown did not reduce acetate and even increased it. This study provides evidence that acetate accumulation in industrial CHO cells may derive from pyruvate conversion and be associated with LDHA and DLAT, while the role of LDHB remains unclear. LDHA and DLAT are identified as candidate contributors to acetate synthesis, and their knockdown reduces acetate accumulation in this model. However, because only one clone per antibody format was compared, the relative contribution of antibody format versus clonal background remains to be determined. These insights provide a basis for optimizing CHO cell culture in biomanufacturing.