# Production of Clinical-Grade SARS-CoV-2 Spike Ferritin Nanoparticle Protein Immunogen by Transient Transfection

## TL;DR

Researchers developed a clinical-grade SARS-CoV-2 vaccine candidate using a nanoparticle-based immunogen that can be stored and transported under permissive conditions.

## Contribution

A scalable, CGMP-compliant method for producing a stable SARS-CoV-2 Spike Ferritin Nanoparticle vaccine with broad neutralizing activity.

## Key findings

- SpFN yields ~10 mg per liter and remains stable for two years at −20 °C and a month at room temperature.
- SpFN with ALFQ adjuvant induced potent neutralizing antibodies against multiple SARS-CoV-2 variants in mice.
- The method provides a framework for rapid recombinant protein vaccine production for emerging pathogens.

## Abstract

Background/Objectives: In response to the COVID-19 pandemic, we developed a vaccine candidate against SARS-CoV-2. Spike Ferritin Nanoparticle (SpFN) comprises 24 identical prefusion-stabilized spike proteins anchored to a self-assembled nanoparticle. Organized along the three-fold axis of the ferritin particle, eight SARS-CoV-2 spike trimers are presented per nanoparticle. Methods: Here, we describe the CGMP processes for manufacturing SpFN using transient transfection of Expi293F cells. Results: The final yield of SpFN was ~10 mg per liter of media supernatant. The resulting protein is stable in cold storage for two years at −20 °C, as well as for a month at room temperature, and can withstand multiple freeze/thaw cycles. SpFN material produced using the CGMP protocols adjuvanted with Army Liposomal Formulation-QS-21 (ALFQ) elicited potent neutralizing antibodies against WA-1, Alpha, Beta, and Delta variants in mice as measured by a pseudovirus neutralization assay. Conclusions: This work demonstrates rapid development and scaled-up production of clinical-grade SARS-CoV-2 vaccine protein material, allowing permissive storage and transport conditions, and serves as a framework for recombinant protein production for future emergent pathogens.