Application of camelid nanobody binders derived from a novel full-synthetic VHH library in immunoaffinity purification of recombinant coagulation factor VII

In our previous study, we proposed a novel bioinformatics approach for identifying complementarity determining region 3 (CDR-3) of Camelid antibodies and their randomization for natural recapitulation with similar amino acid residues. The current study aims to assess the efficacy of binders found in the library generated by the aforementioned bioinformatics method in developing an immunoaffinity column to purify recombinant coagulation factor VII (rFVII). As alternatives to whole antibodies, single-domain Camelid nanobodies (VHHs) have gained much popularity as they offer unique advantages, including a low production cost, small size, and high stability. In this study, two rFVII binders, namely VHH19 and VHH24, were expressed in a bacterial host and coupled to Sepharose CL-4B cross-linked beads following purification. The VHH binders showed a high specificity to rFVII as compared to other non-specific antigens found in human plasma. The coupled resin showed stable performance with a binding capacity of 2.3 ± 0.15 mg rFVII per mL of coupled Sepharose in optimal binding conditions. The resin was also compared with the commercial nanobody-based immunoaffinity resin VIISelect in terms of binding capacity. Our findings suggest that VHH binders obtained through a purely in silico randomization process (fully synthetic) could be effectively exploited in developing an immunoaffinity chromatography column for purifying rFVII.