Scalable Production of Recombinant Vesicular Stomatitis Virus Pseudoparticles Using HEK293 Suspension Cultures

ABSTRACT Recombinant vesicular stomatitis virus (rVSV) pseudoparticles displaying foreign glycoproteins are valuable for various applications, including vaccine vectors, oncolytic viruses, and research tools. Replication incompetent rVSV pseudoparticles, which do not encode their envelope proteins necessary for infection, offer increased versatility and rapid manufacturing. Traditionally, pseudotyping has been achieved using adherent host cells transiently expressing foreign glycoproteins, followed by infection of the host cells with a glycoprotein G (VSV‐G) presenting virus that is deficient in glycoprotein genes. This adherent production method is challenging to scale up, which limits many applications. We developed a high‐yield and scalable process using suspension adapted human embryonic kidney (HEK) 293F cells to produce replication incompetent rVSV. Using a multivariate approach, we optimized production duration to minimize the negative impact of residual transfection components and cellular waste products on viral infection and propagation. This eliminated the need for media exchange and enhanced process scalability. Key process parameters such as multiplicity of infection (MOI) and production duration were optimized to improve pseudoparticle productivity. The suspension process was scaled up to 2 L stirred tank bioreactors, yielding 7.4 × 10 9 fluorescent forming units (FFU)/mL for VSV‐G vector propagation and 2.4 × 10 6 FFU/mL for pseudotyping with the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein, achieving 8‐fold and 50‐fold higher productivity, respectively, than previous adherent processes. The pseudoparticles produced were fully neutralized by an anti‐SARS‐CoV‐2 antibody, further validating the quality of the pseudoparticles from this suspension manufacturing process.