Application of metabolomics to improve recombinant AAV production in the Sf9/baculovirus system

The Sf9-baculovirus expression system is widely used to produce recombinant adeno associated virus (rAAV) vectors for gene therapy applications. The manufacturing process generally involves baculovirus infection of Sf9 cells at low cell density, as infection at higher cell densities results in lower specific productivity. This “cell-density effect” is thought be a result of nutrient or metabolic deficiency; however, the exact mechanism and its resolution is not well defined yet.
In this study, Sf9 cells cultivated in an in-house medium, co-infected with baculovirus at different cell densities were examined to understand the metabolic phenomena driving the cell-density-effect. Using steady state metabolomics, we tracked metabolite levels across glycolysis, the TCA cyle, amino acid catabolism as well as nucleotide synthesis and degradation pathways, enabling the identification of potentially limiting metabolites. These novel results were matched with a detailed carbon balance flux analysis to investigate major carbon utilization pathways. Sf9 cells cultivated with C-13 labelled Glucose were infected at different cell densities and C-13 flux analysis was performed across major metabolic pathways. These studies show that Sf9 cells experience major decrease in glycolysis and TCA cycle fluxes when infected with Baculovirus at high cell densities. We were also able to identify the major amino acids catabolized after baculovirus infection. Additionally, certain metabolites had different utilization rates between low and high cell density infections.
These in-depth studies to understand Sf9 metabolism pre and post baculovirus infection have been instrumental in designing an in-house feed supplement that boosts AAV productivity by enabling high cell density infections.