Scalable Process for High-Yield Production of CyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

cysteine-rich protective antigen (CyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield CyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality CyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved CyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest CyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, CyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-CyRPA antibodies that impeded the multiplication of . This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant CyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.