Metabolic Flux Analysis Reveals Cell Line‐Specific Rewiring in CHO Cells Following TCA Cycle Intermediate Feeding for Bioprocess pH Control

ABSTRACT Chinese hamster ovary (CHO) cells are the leading host for recombinant therapeutic protein production in the biopharma industry. In this study, we investigated how feeding acidic forms of tricarboxylic acid (TCA) cycle intermediates—malic acid, succinic acid, and α‐ketoglutaric acid—affects cell culture performance and metabolism in two industrial IgG‐producing CHO cell lines. These intermediates were used as pH control agents to replace conventional CO 2 sparging, enabling simultaneous modulation of the bioreactor environment and cell metabolism. Carbon‐13 metabolic flux analysis ( 13 C‐MFA) revealed substantial rewiring of central carbon and nitrogen metabolism under all fed conditions, with distinct responses between cell lines. Intermediate‐fed cultures exhibited enhanced TCA cycle fluxes, reduced glucose dependency, decreased lactate accumulation, and altered routing of pyruvate. Notably, α‐ketoglutaric (α‐KG) acid feeding triggered divergent nitrogen assimilation phenotypes: one cell line enhanced glutamine biosynthesis and ammonium clearance, while the other accumulated glutamate with minimal glutamine production. These metabolic adaptations were accompanied by shifts in redox balance and delayed but measurable increases in cell‐specific IgG productivity. Our findings highlight the compound‐ and cell line‐specific nature of metabolic responses to TCA cycle intermediate feeding and support its use for pH control and bioprocess optimization.