Continuous production of recombinant adeno-associated virus in the insect cell/baculovirus expression vector system

Abstract Continuous production processes may offer significant advantages for biotherapeutic manufacturing, including increased productivity, consistent product quality, reduced facility footprint, and decreased process turnaround time. Despite these benefits, the in-situ formation of defective recombinant baculovirus expression vectors (BEVs) hinders the continuous manufacturing of recombinant adeno-associated viruses (rAAV) in the baculovirus expression vector system. This study investigates an approach of reducing defective viruses through the infusion of standard recombinant baculovirus (rBV) and the compartmentalization of early- and late-stage infected cells, resulting in stable rAAV production. In this study, rAAVs were continuously produced in a series of cascading reactors, comprising a feeder reactor, an infection reactor, and a production reactor. Residence times and transfer rates across the three reactors were optimized based on the production kinetics of rBV and rAAV derived from our mechanistic model. The majority of rBV was produced within the production reactor, thereby reducing the impact of defective viruses in the infection reactor, enabling continuous rAAV production. This study demonstrates the successful implementation of a continuous rAAV production process, yielding over 5×10 10 vg/mL per day for 4 weeks. This work represents the first reported continuous rAAV production process utilizing the Sf9/BEVS platform and establishes engineering know-how for overcoming manufacturing challenges associated with rAAV-based gene therapies.