Highly Efficient Purification of Recombinant VSV-∆G-Spike Vaccine against SARS-CoV-2 by Flow-Through Chromatography.

Elad Lerer,Eyal Epstein,Arik Monash,Ziv Oren,Lilach Levin,Yaakov Adar,Arik Makovitzki,Ran Zichel,Edith Lupu,Einat Toister,Rona Levy,Eyal Dor,Meni Girshengorn,Niva Natan,Yaron Kafri,Lilach Cherry

doi:10.3390/biotech10040022

Abstract

This study reports a highly efficient, rapid one-step purification process for the production of the recombinant vesicular stomatitis virus-based vaccine, rVSV-∆G-spike (rVSV-S), recently developed by the Israel Institute for Biological Research (IIBR) for the prevention of COVID-19. Several purification strategies are evaluated using a variety of chromatography methods, including membrane adsorbers and packed-bed ion-exchange chromatography. Cell harvest is initially treated with endonuclease, clarified, and further concentrated by ultrafiltration before chromatography purification. The use of anion-exchange chromatography in all forms results in strong binding of the virus to the media, necessitating a high salt concentration for elution. The large virus and spike protein binds very strongly to the high surface area of the membrane adsorbents, resulting in poor virus recovery (<15%), while the use of packed-bed chromatography, where the surface area is smaller, achieves better recovery (up to 33%). Finally, a highly efficient chromatography purification process with CaptoTM Core 700 resin, which does not require binding and the elution of the virus, is described. rVSV-S cannot enter the inner pores of the resin and is collected in the flow-through eluent. Purification of the rVSV-S virus with CaptoTM Core 700 resulted in viral infectivity above 85% for this step, with the efficient removal of host cell proteins, consistent with regulatory requirements. Similar results were obtained without an initial ultrafiltration step.

Highlights

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 respiratory disease, which to date has infected over 220 million people and killed over 4.6 million [1,2], initiating a global economic and social crisis [3,4]
The SARS-CoV-2 virus is an enveloped single-stranded positive sense RNA virus that enters host cells by mediation of a transmembrane spike (S) glycoprotein binding to an angiotensin-converting enzyme (ACE2) [5]
Large aggregates, and insoluble contaminants were clarified using a filter train composed of 3 μm and 1.2 μm inert polypropylene depth filters and a 0.45/0.2 μm membrane filter

Summary

Introduction

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 respiratory disease, which to date has infected over 220 million people and killed over 4.6 million [1,2], initiating a global economic and social crisis [3,4]. The SARS-CoV-2 virus is an enveloped single-stranded positive sense RNA virus that enters host cells by mediation of a transmembrane spike (S) glycoprotein binding to an angiotensin-converting enzyme (ACE2) [5]. The S protein comprises two functional subunits, the S1 subunit responsible for binding to the host cell receptor and the S2 subunit responsible for the fusion of the viral and cellular membranes [6]. Entry of the coronavirus into the cells is a complex process that requires proteolytic cleavage of the S protein, promoting virus–cell fusion and entry [7].

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