Elucidating lactate metabolism in industrial CHO cultures through the combined use of flux balance and principal component analyses

Overflow metabolism in the form of lactate accumulation in proliferating mammalian cell cultures results in significant process design challenges for industrial bioprocesses. While lactate metabolism in CHO cell cultures naturally switches from lactate production (LP) to lactate consumption (LC) both in batch and fed-batch cultures, neither the exact mechanism nor what triggers the metabolic switch are well understood. Herein, a computational methodology based on flux balance analysis to analyse experimental data from multiple industrial CHO cell lines in order to identify key differences between the two metabolic states is presented. Experimentally determined uptake and secretion rates from the LP and LC states of four industrial cell lines were used to constrain a CHO genome-scale model. Subsequently, a large number of sampled flux distributions were retrieved from the space of feasible solutions for each state (LP, LC) and cell line using an Artificial Centering Hit-and-Run algorithm. The sampled flux distributions were labelled and randomised before being analysed by principal component analysis (PCA). PCA was able to identify and completely separate samples from the two metabolic states. Based on a detailed analysis of PCA loadings a mechanism detailing the function and switch of lactate metabolism is proposed. Briefly, we hypothesize that (I) the production of lactate is linked to the regeneration of the NAD+ pool in the cytosol as a result of large passive glucose intake, (II) the switch in lactate metabolism is regulated by (i) the concentration difference between extracellular and intracellular lactate and (ii) the transmembrane proton gradient.