Efficient Screening and Design of Variable Domain of Heavy Chain Antibody Ligands Through High Throughput Sequencing for Affinity Chromatography to Purify Fab Fragments

To design an affinity ligand for purification of antigen-binding fragment (Fab) antibody, variable domain of heavy chain antibody (VHH) phage libraries were constructed from Fab-immunized Alpaca and subjected to biopanning against Fabs. To find the specific binders, we directly applied high-throughput sequencing (HTS) analysis of the VHH sequences in the panned phages on next-generation sequencer. The efficiently enriched sequences were aligned for construction of the phylogenetic tree to be categorized into five groups. VHHs from three major groups were first selected to analyze their properties as an affinity ligand. However, those VHHs were not suitable as an affinity ligand because of lack of resistance against alkaline pH and/or difficulty in acidic elution from the affinity column. So, we further searched the candidates from minor group sequences. Among five, one VHH showed the binding ability but with low affinity against Fabs. Therefore, we improved its affinity-by-affinity maturation through error-prone PCR library techniques. The final designed VHH showed highly alkaline pH resistance and easy acidic elution together with high affinity to Fabs. These results indicate that HTS techniques combined with biopanning and followed by error-prone PCR library techniques is powerful in designing specific binders with desired properties.