Exometabolome profiling reveals activation of the carnitine buffering pathway in fed‐batch cultures ofCHOcells 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.

Understanding the effect of temperature downshift onCHOcell growth, antibody titer and product quality by intracellular metabolite profiling and in vivo monitoring of redox state

“…It has been shown that the oxidation of lactate to pyruvate balances the intracellular redox state to maintain the rate of oxidative phosphorylation, so changes in the intracellular redox state are directly influenced by the competition between glycolysis and oxidative phosphorylation 37 . The NAD + /NADH ratio, and intracellular pyruvate level are the driving forces for the switch from aerobic glycolytic metabolism to hyperoxidative metabolism in CHO cells, and changes in lactate levels vary with these parameters 44,45 . Usually, in the rapid glycolysis stage, the reducing power NADH is continuously generated, and the reduction of pyruvate to lactate can quickly balance the intracellular redox state to maintain the rate of glycolysis; when the rate of oxidative phosphorylation is rapid, intracellular NADH continuously enters the mitochondria to provide reducing power, and redox balance is then maintained by the consumption of lactate to produce NADH.…”

“…37 The NAD + /NADH ratio, and intracellular pyruvate level are the driving forces for the switch from aerobic glycolytic metabolism to hyperoxidative metabolism in CHO cells, and changes in lactate levels vary with these parameters. 44,45 Usually, in the rapid glycolysis stage, the reducing power NADH is continuously generated, and the reduction of pyruvate to lactate can quickly balance the intracellular redox state to maintain the rate of glycolysis; when the rate of oxidative phosphorylation is rapid, intracellular NADH continuously enters the mitochondria to provide reducing power, and redox balance is then maintained by the consumption of lactate to produce NADH. Therefore, it can be considered that the redox balance drives the consumption of lactate, which is consistent with our findings.…”