Modification of a SARS-CoV-2 spike-RBD targeting nanobody for pH-dependent binding and its chromatographic engineering for purification of WT and variant S-RBDs with mild elution conditions.

For vaccine development against SARS-CoV-2, the spike (S) or spike receptor-binding domain (S-RBD) serves as the major antigen. Enhancing the efficiency and streamlining the process for S/S-RBD purification thus hold considerable practical significance. Here, we identify a basic and evolutionarily conserved region within S-RBD and select a nanobody targeting the region for structural-guided modifications. Histidine substitutions are introduced in the nanobody to enable pH-dependent binding to S-RBD. Two candidates, MNb-11 and MNb-14, are found to readily bind to S-RBD at pH 7.5 but lose the binding capacity below pH 5.0. During the chromatographic-purification trials, resins immobilized with MNb-11 or MNb-14 could purify both wild-type and variant S/S-RBD proteins in one step to a high level of purity and homogeneity. In addition, both modified nanobodies show superior stability across multiple binding-elution cycles. Taken together, our study presents a one-step affinity-chromatographic method for purifying the S/S-RBD protein, which should aid in vaccine development and production against SARS-CoV-2.