A stable all-D peptide ligand inspires broad-spectrum affinity and high performance in the purification of COVID-19 vaccines.

Affinity peptides are an appealing class of ligands for affinity chromatography. However, the application of peptide ligands in biopharmaceutical manufacturing is impeded by their poor stability, susceptibility to degradation and short half-lives. Here, the affinity peptide identified previously, HWKAVNWLKPWT (L-HWK), was reformulated by substituting all the residues with their d-amino acid counterparts, and the d-enantiomer of l-HWK (D-HWK) was investigated to evaluate the binding affinity and molecular mechanism with the receptor binding domains (RBDs) on COVID-19 spike (S) proteins. The results showed that d-HWK had higher affinities than l-HWK for the wild-type (wt) RBD, trimer S protein (TrimerS), and RBDs from variants of concern. Molecular dynamics simulations further revealed that d- and l-HWK had the same binding pocket on the RBD and a more stable binding conformation was obtained in d-HWK binding, which was supported by enhanced intermolecular π-π stacking interactions and a more extensive hydrogen bond network. The chromatographic results demonstrated that Sep6FF-d-HWK chromatography had broader binding conditions for the wt-RBD and TrimerS than Sep6FF-l-HWK chromatography, and that the adsorbed targets could be efficiently eluted under mild conditions. Seng4FF-d-HWK chromatography was used to successfully purify wt-COVID-19 and Omicron vaccines. Most importantly, d-HWK exhibited great tolerance to enzymatic hydrolysis, and Seng4FF-d-HWK improved the purification performance and stability of inactivated Omicron variant vaccines across 20 purification cycles. The research is crucial for the application of broad-spectrum affinity chromatography in the purification of COVID-19 vaccines and provides a more effective strategy for accelerating the development of affinity chromatography for an unknown pandemic occurring in the future.