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

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.

Neutralization of SARS-CoV-2 Variants by rVSV-ΔG-Spike-Elicited Human Sera

“…The process of BriLife ® generation was performed by serial passaging in Vero E6, followed by passaging in Vero cells, in a serum-free medium [5]. This process was accompanied by sequencing at key passages.…”

“…BriLife ® was developed and generated as previously described [2], followed by additional passaging in Vero cells (WHO Vero RCB 10-87) widely accepted by the WHO and regulatory agencies for the manufacturing of human viral vaccines, and further purified, formulated, and titered as previously described [5]. The BriLife ® sequence was analyzed by whole-genome sequencing (WGS, described below).…”

SARS-CoV-2 spike antigen quantification by targeted mass spectrometry of a virus-based vaccine

“…The VSV-ΔG-spike vaccine was produced in BioBLU® 5p Eppendorf single-use bioreactors in Vero cell that were absorbed to Fibra-Cels. The medium from the bioreactors, containing the released vaccine virus, was harvested and transferred to downstream process (DSP) that comprises several steps (as described earlier ( Lerer et al, 2021 ; Makovitzki et al, 2021 ). The purification steps include: endonuclease digestion of host cell DNA, followed by clarification by a train of decreasing cut off filters, chromatographic purification, and tangential flow filtration (TFF).…”

In-Line Monitoring of Downstream Purification Processes for VSV Based SARS-CoV-2 Vaccine Using a Novel Technique

“…The DSP of rVSV-∆G-spike comprises the several steps described in [ 9 , 10 ]. Briefly, MT or bioreactor harvested supernatants were subjected to endonuclease digestion of host cell DNA using 60 U/mL Denerase endonuclease (20804-5M, GMP grade, c-LEcta, Leipzig, Germany) in the presence of 2 mM MgCl 2 (Spectrum chemical, New Brunswick, NJ, USA) for 3 h in a shaker incubator (Innova 43R, Eppendorf, Hamburg, Germany) at 37 °C under constant and mild agitation.…”

“…The DSP must eliminate impurities, either process-related (e.g., DNase, nutrients, extractables and leachables) or product-related (e.g., host cell proteins (HCP), metabolites and host cell DNA (hc-DNA)). The DSP that was developed by the IIBR for the rVSV-∆G-spike vaccine is a multistep process that comprised endonuclease digestion, depth and membrane filter clarification, chromatographic purification and ultrafiltration [ 9 , 10 ].…”