Exometabolome profiling reveals activation of the carnitine buffering pathway in fed‐batch cultures of CHO cells co‐fed with glucose and lactic acid

Abstract Adjustments to CHO cell physiology were recently observed during implementation of a Raman spectroscopy‐based glucose and lactate control strategy. To further understand how these cells, under monoclonal antibody (mAb) production conditions, utilized the extra lactic acid fed, we performed a comprehensive semi‐quantitative and time‐dependent analysis of the exometabolome. This study focused on the CHO cell's metabolic shift from the fifth day of culture. We compared relative levels of extracellular metabolites in the absence or presence of a 2 g/L lactic acid setpoint while glucose was kept at 4 g/L. Our hypothesis is that extra lactic acid would supply more pyruvate, favoring oxidative phosphorylation. We subsequentially uncovered several carnitine derivatives as biomarkers of the simultaneous activation of TCA anaplerotic pathways as well as a carbon‐buffering pathway. CHO cells exhibited a balance between intermediates from ( i ) amino acid catabolism, ( ii ) fatty acid β ‐oxidation, and ( iii ) pyruvate from glycolysis and lactic acid; and the secretion of their intermediate carnitine derivatives. In addition, 3‐hydroxy‐methyl‐glutaric acid (HMG) and mevalonate syntheses were found as biomarkers of alternative acyl group removal. Together, under a limited capacity to assimilate the surplus of acyl‐CoA groups as well as an ability to maintain the acyl‐CoA: free CoA ratio for proper and continuous functioning of the TCA cycle, CHO cells activate the carnitine‐buffering system, HMG, and mevalonate pathways.