Footprint metabolomic profile shift of Sf9 insect cell line throughout growth and rabies VLP production.

The baculovirus/insect cell system is widely used for recombinant protein and virus-like particles (VLP) production, but its performance at high cell density is limited by infection-associated metabolic stress. In this study, a targeted metabolic footprinting approach was applied to characterize the extracellular metabolome of Spodoptera frugiperda Sf9 cells during baculovirus-mediated production of rabies VLP in chemically defined medium. Infected and uninfected cultures were cultivated in controlled bioreactor runs, and extracellular carbohydrates, amino acids, organic acids, and nucleosides were quantified by HPLC and LC-MS/MS. Metabolite data were converted into specific uptake and release rates, then also analyzed using Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Variable Importance in Projection scoring (VIP). Infection induced marked metabolic reprogramming, with increased glucose demand, dynamic shifts in amino acid consumption and release, and altered extracellular profiles over time. Although infected and control samples partially overlapped in PCA, supervised analysis improved group discrimination and identified sucrose, leucine, lysine, glycine, and pyruvate as the main variables associated with infection. Sequence-based compositional analysis of the recombinant proteins supported the relevance of amino acid-related metabolic changes, while transmission electron microscopy confirmed successful formation of rabies VLP with expected morphology. Together, these results provide a quantitative description of the metabolic burden imposed by rabies VLP production in Sf9 cells and highlight candidate metabolites for medium redesign and targeted feeding strategies to reduce the cell density effect and improve process productivity in baculovirus-based VLP manufacturing.