In the spike protein of SARS-CoV-2, the receptor-binding domain (RBD) contains multiple dominant neutralizing epitopes and can be used as an antigen for developing COVID-19 vaccines and neutral antibodies. Affinity chromatography is one of the most extensively used methods for rapid one-step protein purification. However, there is a lack of commercially available affinity ligands for RBD purification. Here, we report the rapid isolation of a nanobody suitable for purifying RBD as an affinity ligand from immune phage display libraries. After bio-panning, the enriched clones were sequenced on next-generation sequencing (NGS) platforms and classified into four groups based on the CDRH3 amino acid sequence. The representative sequences with high nanomolar affinities to RBD were further categorized into two groups via epitope binning analysis. Finally, from the two epitope bins, we found that SS3 showed easy elution under a mild eluting condition and could be used as a functional affinity ligand to purify RBD. These results also indicate that categorizing the bio-panned sequences via high-throughput sequencing (HTS) techniques followed by epitope binning represents a fast workflow to select specific binders with desired properties.
Animal-derived anti-IgG secondary antibodies are currently employed to stain and screen of human monoclonal antibody(mAb)-producing cells, but using animal-derived antibodies may raise the concerns of high cost, complicated operations and biological safety issues in biopharmaceutical manufacturing. Nanobodies(VHHs) are attractive forms of antibodies for their straightforward engineering and expression in both eukaryotic and prokaryotic systems. Using phage-displayed immune llama VHH library, we identified new anti-Fc VHHs that could bind to human Fc with high affinity. In GFP fusion format, the anti-Fc VHH-GFP generated dramatically stronger FACS signals than AF488 conjugated anti-IgG antibodies when used for staining mAb-producing CHO cells. Furthermore, preparative sorting of CHO cells based on anti-Fc VHH-GFP staining resulted in the enrichment of cell lines capable of synthesizing mAb at high productivity. This safe and cost-efficient anti-Fc VHH-GFP may optimize the process of generating highly productive cell lines for therapeutic mAb production compared to conventional animal-derived fluorescent antibodies.
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