Dissolved oxygen tension as a key driver for tailoring the size and expression of rabies virus-like particles

Rabies remains a major global health threat, motivating cost-effective vaccine platforms such as virus-like particles (VLPs) produced in the baculovirus–insect cell system. Because co-expression of rabies virus glycoprotein (RVGP) and matrix protein (RVM) is sensitive to the cellular microenvironment, we evaluated how dissolved oxygen (DO) setpoints (10%, 20%, and 30% air saturation) affect Sf9 cell kinetics and rabies VLP structural attributes. Batch cultures were performed in stirred-tank bioreactors with chemically defined medium, monitoring growth, specific oxygen consumption rate ( q O 2 ), death kinetics, viral titer, and protein expression by Western blot and transmission electron microscopy. Exponential-phase growth was similar at all DO levels (the maximum specific growth rate, μ max = 0.026 h −1 ), and q O 2 followed comparable trends: it decreased during growth, increased early after infection, and ended at 2 × 10 −11 mg O 2 /cell·min. In contrast, DO strongly affected VLP yield and size. At 10% DO, RVGP and RVM reached 8.49 and 186.43 mg/L, respectively, producing ~81.67 nm particles resembling native rabies virions and achieving 1.2 × 10 7 PFU/mL. At 30% DO, RVGP and RVM decreased to 0.72 and 84.46 mg/L, and particle size increased to ~108 nm. These results indicate that 10% air-saturation DO is optimal for producing homogeneous rabies VLPs with higher protein expression and viral titer.