Scalable, continuous biomanufacturing processes have grown in importance to meet demand for smaller bioreactor sizes, lowered production costs, and improved quality attribute consistency. The Sf9/recombinant baculovirus (rBV) expression system demonstrates promise for virus-like particle (VLP) vaccine and gene therapy production. Here, we present a continuous rBV platform integrating an infection plug flow reactor (PFR) between stirred tank growth (gCSTR) and production (pCSTR) bioreactors. Cell expansion in the gCSTR included a ramp-up stage followed by continuous growth, reaching a steady state of 5106 cells/mL and >90% viability. Péclet number-fit tracer studies confirmed near-ideal plug flow in the PFR, yielding a 10 h residence time and progressive infection as measured by gp64 signaling. Finally, a pCSTR with a residence time of 48 h exhibited sustained recombinant protein production. An integrated pilot cascade incorporating all reactors ran continuously for 5 days, maintaining stable CSTR cell densities and a measurable increase in infected cell diameter from 14. 5 μm to 16. 1 μm. Western blotting and EM of ~ 100 nm VLPs in pCSTR effluent demonstrated platform success. Digital twin mechanistic models across four distinct stages of bioreactor operation and Hill-type relationships for rBV infection kinetics predicted cell growth and death for a 7-day run, demonstrating promise for designing continuous systems in silico and building a quantitative framework for scale-up and optimization. Our multi-stage reactor configuration represents a cell host- and product-agnostic production scheme, particularly for processes prone to product heterogeneity, and paves the way towards a true end-to-end continuous platform for myriad modalities in the future.
Sargunas et al. (2026) studied this question.