Process Intensification for Recombinant Marburg Virus Glycoprotein Production Using Drosophila S2 Cells

Abstract

Marburg marburgvirus (MARV) is a highly virulent human pathogen with limited therapeutic options. Recombinant MARV glycoprotein (GP) produced in Drosophila Schneider 2 (S2) cells has been extensively investigated as potential vaccine antigen with promising efficacy demonstrated in nonhuman primate models. However, the existing production process for MARV-GP involving static batch cell cultures with limited scalability and process control show lower than desirable yields. Here, we assessed various process intensification strategies in single-use orbital shaken bioreactors (OSBs) or rocking bioreactors (WAVE) and report maximum viable cell concentrations (VCCs) of 31.6 × 10⁶ cells/mL in batch, 69.5 × 10⁶ cells/mL in fed-batch (FB), and up to 210.0 × 10⁶ cells/mL in perfusion mode. By changing from a glucose-only feed to a CellBoost5 feed, MARV-GP yields were increased by over two-fold. Implementation of perfusion cultures achieved a peak MARV-GP concentration of 57.4 mg/L and a 540% higher space-time yield compared to the FB process in the 50 L WAVE system. However, maximum cell-specific productivities were achieved at a VCC of 85 × 10⁶ cells/mL and decreased with increasing cell concentrations. Glycoanalysis revealed a uniform paucimannosidic N-glycan profile, predominantly α-1,6-core-fucosylated Man3F (F(6)M3) structures, across all production modes. Notably, transitioning pH control from CO₂ to phosphoric acid shifted glycan profiles toward higher mannose forms, highlighting the influence of culture conditions on glycosylation.