Continuous Production of Influenza VLPs Using IC-BEVS and Multi-Stage Bioreactors

The insect cell-baculovirus expression vector system (IC-BEVS) has been an asset to produce biologics for over 30 years. With the current trend in biotechnology shifting toward process intensification and integration, developing intensified processes such as continuous production is crucial to hold this platform as a suitable alternative to others. However, the implementation of continuous production has been hindered by the lytic nature of this expression system and the process-detrimental virus passage effect. In this study, we implemented a multi-stage bioreactor setup for continuous production of influenza hemagglutinin-displaying virus-like particles (HA-VLPs) using IC-BEVS. A setup consisting of one Cell Growth Bioreactor simultaneously feeding non-infected insect cells to three parallel Production Bioreactors operated at different residence times (RT) (18, 36, and 54 h) was implemented; Production Bioreactors were continuously harvested. Two insect cell lines (neutral pH-adapted High Five and Sf9) and two recombinant baculovirus (rBAC) constructs (one that originates from a bacmid, rBAC_bacmid, and another of non-bacteria origin, rBAC_flashbac) were tested. Combining rBAC_flashbac with Sf9 cells was the most efficient approach, allowing consistent HA-VLPs titers (34 ± 14 HA titer/mL) and rBAC titers (10⁸-10⁹ pfu/mL) throughout the period of continuous operation (20 days). Cell growth kinetics and viability varied across RT, and higher RT was associated with increased expression of HA-VLPs, independent of the cell line and rBAC used; RT of 54 h allowed to maximize titers. The presence of particles resembling HA-VLPs was confirmed by transmission electron microscopy throughout the continuous operation. This work showcases the implementation of a process for continuous production of a promising class of biotherapeutics (i.e., VLPs), and paves the way for establishing continuous, integrated setups using the IC-BEVS expression system.