Antibody sequence diversity identified through multiple in-parallel NGS-based phage display panning campaigns.

Monoclonal antibodies (mAbs) are crucial for developing assays to support vaccine development. Our in-house tool antibody generation involves phage panning using HuCAL Platinum human Fab library. To understand the antibody sequence diversity and reproducibility, we performed four parallel panning campaigns against a virus-like particle antigen (VLP, L1 protein from HPV18), and monitored the enrichment of antibody sequences from each round of panning. Long-read NGS platforms from both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) were used in deep analysis of antibody sequences. Results from both sequencing platforms were consistent, with comparable diversity and frequency rankings. Over 72% of top frequency ranked antibody sequences were in common between respective round three and round four. ELISA results showed no additional enrichment of antibodies with strong ELISA EC 50 values in round four compared to round three; therefore, further panning beyond round three is unnecessary. Surprisingly, top frequency ranked antibody sequences from the four parallel panning campaigns were vastly different. Across the four parallel panning campaigns, up to 68% of top ranked antibody sequences identified in round three were unique. Therefore, for large-diversity libraries, additional panning campaigns can yield more antibody candidates with unique sequences.