Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods

Nicole Cibelli,Farah Vejzagic,Tina Khin,Xin Wang,Kristin Leach,Gabriel Arias,Q Paula Lei,Renata Skubutyte,Aakash Patel,Niutish Bastani,Mckenzie Figur,Karen Vickery,Krishana Gulla,Alison Vinitsky,Shireen Khayat ,W Chuenchor ,Yaroslav Tsybovsky,Ivan Loukinov,Misa Mai,William R Shadrick ,Daniel B Gowetski

doi:10.1038/s41598-022-07485-w

Nicole Cibelli, Farah Vejzagic + Show 19 more

Open Access

https://doi.org/10.1038/s41598-022-07485-w

Copy DOI

Abstract

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.

Highlights

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development
Glycosylation around the receptor binding domain (RBD) of the spike protein is of specific interest, as it may play an important role in antibody r ecognition[21]
To rapidly respond to the SARS-CoV-2 pandemic, large quantities of soluble, stabilized spike ectodomain protein are needed as a vaccine candidate and as a reagent for therapeutic and diagnostic development

Summary

Introduction

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. Population-wide serological detection of SARS-CoV-2-specific antibodies with a spike protein ELISA is a useful tool for surveillance and containment, with throughput and cost benefits over PCR-based virus assays[7]. To supply these significant endeavors, a scalable, economical, rapid spike protein production protocol is of critical importance. Except for lentil lectin, these methods require the inclusion of a tag in the sequence of the molecule and, generally, a protease-mediated cleavage step following purification While these affinity methods yield a highly pure product and require little optimization or development work, they are difficult to scale to large manufacturing campaigns. When size-exclusion chromatography (SEC) is applied as a polish step after affinity c hromatography[9,13,18], facility fit challenges arise; the required large column volumes and small load volumes necessitate an extra concentration step prior to chromatography or many cycles when manufactured at large scale

Methods

Results

Conclusion