Metabolic Footprint of Drosophila S2 Cells: Findings During the Production of a Recombinant Rabies Virus Glycoprotein

Over the past 50 years, the Drosophila melanogaster S2 cells have been valued for their ability to synthesize therapeutic molecules at high yield. To further increase protein expression, it is imperative to improve cellular performance, which is intrinsically linked to cell metabolism. Nevertheless, information on S2 metabolism, including pathways, components, and cellular compartments, remains limited, hindering advances in S2 cellular performance. Herein, using a genetically modified S2 cell line expressing the recombinant rabies virus glycoprotein (RVGP), we investigated the stress caused by RVGP production on S2 cells. Batch cultures using wild- and rec-types were performed, and 27 compounds were quantified over 192 h. The extracellular metabolome affected the rec-S2 growth kinetics after RVGP expression was activated. Although RVGP was produced in high amounts, we identified a substrate limitation for rec-S2 cell growth (glutamine), changes in amino acid routes due to RVGP biosynthesis (leucine, serine, glycine, and valine), and metabolites that might be affecting rec-S2 cell growth (acetate, pyruvate, citrate, and malate). Organic acid analysis indicated that malate and acetate production are correlated with RVGP production. This work revealed metabolic correlations in S2 cells that may have direct implications for media optimization and yield maximization, thereby improving S2 performance for scale-up.