Broad-spectrum affinity chromatography of SARS-CoV-2 and Omicron vaccines from ligand screening to purification

Emerging variants of SARS-CoV-2 pose great technological and regulatory challenges to vaccine manufacturing, especially in downstream processing. To address this dilemma, the development of broad-spectrum affinity chromatography for the purification of wild-type SARS-CoV-2 and its variants is crucial. We propose a comprehensive strategy to achieve this goal via the identification of high-affinity peptides by affinity selection of phage display and next-generation sequencing (NGS) and the evaluation of chromatographic performance. Two peptides targeting the angiotensin-converting enzyme 2 (ACE2)-binding motif on the receptor-binding domain (RBD), HFVKTPARWAWG (SP-HFV) and HYRTSHWHHLLG (SP-HYR), were obtained from the most abundant sequences of the enriched phage library. They exhibited nanomolar affinity for the RBD and trimeric spike protein (Trimer S), and had broad-spectrum affinity for all the RBDs from the variants. Molecular dynamics simulations revealed the different binding regions of SP-HFV and SP-HYR in the ACE2-binding motif and key residues contributing to binding. After SP-HYR was coupled onto agarose matrices, chromatographic results showed that the RBD and Trimer S from the wild-type and Omicron variant could be adsorbed at pH 6.0-6.5 and eluted by increasing the salt concentration, exhibiting broad-spectrum and mild-elution characteristics of affinity chromatography. Finally, the affinity chromatography was applied for the purification of inactivated SARS-CoV2 and Omicron vaccines, affording high yields (84.5-93.0 %) and purities (81.3-98.0 %), and great resistance to harsh cleaning-in-place in 20 cycles. This work clearly demonstrated the commercial potential of broad-spectrum affinity chromatography for vaccine purification to address the rapid variation of pathogenic viruses.