High-titer manufacturing of SARS-CoV-2 Spike-pseudotyped VSV in stirred-tank bioreactors

Hayley M Todesco; Chris Gafuik; Cini M John; Erin L Roberts; Breanna S Borys; Alexis Pawluk; Michael S Kallos; Kyle G Potts; Douglas J Mahoney

doi:10.1016/j.omtm.2024.101189

High-titer manufacturing of SARS-CoV-2 Spike-pseudotyped VSV in stirred-tank bioreactors

Mol Ther Methods Clin Dev. 2024 Jan 17;32(1):101189. doi: 10.1016/j.omtm.2024.101189. eCollection 2024 Mar 14.

Authors

Hayley M Todesco^{1

2

3}, Chris Gafuik^{1

2

3}, Cini M John^{1

2

3}, Erin L Roberts⁴, Breanna S Borys⁵, Alexis Pawluk⁵, Michael S Kallos^{4

5}, Kyle G Potts^{1

2

3}, Douglas J Mahoney^{1

2

3

6

7}

Affiliations

¹ Arnie Charbonneau Cancer Institute, Faculty of Medicine, University of Calgary, Calgary, AB, Canada.
² Alberta Children's Hospital Research Institute, Faculty of Medicine, University of Calgary, Calgary, AB, Canada.
³ Snyder Institute for Chronic Disease, Faculty of Medicine, University of Calgary, Calgary, AB, Canada.
⁴ Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada.
⁵ Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada.
⁶ Department of Microbiology, Immunology and Infectious Disease, Faculty of Medicine, University of Calgary, Calgary, AB, Canada.
⁷ Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB, Canada.

Abstract

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×10⁶ cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×10⁸ plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 10⁶ PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.

Keywords: COVID-19; Cytodex 1; SARS-CoV-2; VSV; Vero; microcarrier; omicron; spinner flask; vaccine.