Rapid High-Yield Production of Functional SARS-CoV-2 Receptor Binding Domain by Viral and Non-Viral Transient Expression for Pre-Clinical Evaluation

Abstract

Vaccine design strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the Spike protein or its subunits as the main antigen target of neutralizing antibodies. In this work, we propose rapid production methods of an extended segment of the Spike Receptor Binding Domain (RBD) in HEK293SF cells cultured in suspension, in serum-free media, as a major component of a COVID-19 subunit vaccine under development. The expression of RBD, engineered with a sortase-recognition motif for protein-based carrier coupling, was achieved at high yields by plasmid transient transfection or human type-5-adenoviral infection of the cells, in a period of only two and three weeks, respectively. Both production methods were evaluated in 3L-controlled bioreactors with upstream and downstream bioprocess improvements, resulting in a product recovery with over 95% purity. Adenoviral infection led to over 100 µg/mL of RBD in culture supernatants, which was around 7-fold higher than levels obtained in transfected cultures. The monosaccharide and sialic acid content was similar in the RBD protein from the two production approaches. It also exhibited a proper conformational structure as recognized by monoclonal antibodies directed against key native Spike epitopes. Efficient direct binding to ACE2 was also demonstrated at similar levels in RBD obtained from both methods and from different production lots. Overall, we provide bioprocess-related data for the rapid, scalable manufacturing of low cost RBD based vaccines against SARS-CoV-2, with the added value of making a functional antigen available to support further research on uncovering mechanisms of virus binding and entry as well as screening for potential COVID-19 therapeutics.

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals

“…Hexa-His-tagged RBD expressed in HEK or Pichia cells. RBD was produced by Kamen and coworkers in HEK293SF (clone 293SF-3F6, National Research Council of Canada) suspension cells cultured in serum-free medium, in 3L-controlled bioreactors, according to their previously published protocol 45 (see ESI † for more information). The resulting N-terminally-His-tagged, glycosylated recombinant protein of approximately 38 kDa, designated as pTPA_SP-RBD-His, was purified by IMAC and characterized by SDS-PAGE and Western blot (Fig.…”

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

“…RBD contains 220 amino acid residues with nine cysteine residues and two N ‐ glycosylation sites [17]. The apparent molecular mass of RBD was determined to be ∼34 kDa, whereas that of the RBD amino acid sequence alone was ∼27 kDa [17]. N‐glycosylation and O‐glycosylation were both observed by analysis of RBD [18].…”

Expression of SARS-CoV-2 surface glycoprotein fragment 319–640 in E. coli, and its refolding and purification

“…We aimed to examine the antigenic properties of the surface glycoprotein fragment S319-640. S319-591 fragment was efficiently expressed in transiently transfected HEK293 cells, similar to RBD-containing fragments used in other serology tests 11 , 12 , 13 . However, S319-640 fragment was not detected in the conditioned media of HEK293 cells transiently transfected with the corresponding expression construct.…”