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

Bárbara Fernandes; Marcos Sousa; Rute Castro; Anja Schäfer; Julia Hauser; Kai Schulze; Mario Amacker; Marco Tamborrini; Gerd Pluschke; Paula M Alves; Sylvain Fleury; António Roldão

doi:10.3389/fbioe.2022.879078

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

Front Bioeng Biotechnol. 2022 May 20:10:879078. doi: 10.3389/fbioe.2022.879078. eCollection 2022.

Authors

Bárbara Fernandes^{1

2}, Marcos Sousa^{1

2}, Rute Castro¹, Anja Schäfer^{3

4}, Julia Hauser^{3

4}, Kai Schulze⁵, Mario Amacker^{6

7}, Marco Tamborrini^{3

4}, Gerd Pluschke^{3

4}, Paula M Alves^{1

2}, Sylvain Fleury⁶, António Roldão^{1

2}

Affiliations

¹ iBET-Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
² ITQB NOVA-Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
³ Swiss Tropical and Public Health Institute, Basel, Switzerland.
⁴ University of Basel, Basel, Switzerland.
⁵ Helmhotz Center for Infecion Research, Braunschweig, Germany.
⁶ Mymetics SA, Épalinges, Switzerland.
⁷ Department of Pulmonary Medicine, Bern University Hospital, University of Bern, Bern, Switzerland.

Abstract

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) 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 PfCyRPA 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 PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA 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 PfCyRPA 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, PfCyRPA 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-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

Keywords: BEVS; PfCyRPA; bioprocess engineering; insect cells; malaria vaccine.