B-174 Turning Polyclonal Antibodies into Monoclonals: A Proteomics-Driven Approach

Abstract Background Polyclonal antibodies (pAb), derived from immunized animals, are essential reagents used in antigen-specific experiments across diagnostics and research. However, pAb production faces challenges such as batch-to-batch variability, hindering research reproducibility and requires continued animal use and culling. One potential solution is determining the sequences of dominant antibody forms within a polyclonal mixture, enabling the development of recombinant monoclonal antibody mixtures. This approach allows for recombinant expression, replicating the binding characteristics of the original pAbs and promoting reproducibility in clinical research and laboratory medicine. Methods In this study, we employed a novel mass spectrometry-based method using the REpAb® polyclonal sequencing platform to sequence polyclonal antibodies directly from a purified protein mixture, completely de novo, without relying on prior sequence knowledge or databases. First, a purified polyclonal mixture was subjected to multi-enzyme digestion to generate shorter, overlapping peptides. Both middle-down and top-down mass spectrometry methods were then applied to analyze these peptides. The resulting data was de novo sequenced by tandem mass spectrometry (MS/MS), and the peptides were subsequently assembled into full-length antibody chains with correct pairing of the heavy and light antibody chains using machine learning-based bioinformatics. Protein lysates underwent analysis using an Orbitrap EclipseTM Series instrument (ThermoFisher Scientific, CA, US) coupled with the LC Evosep One (Evosep, Denmark). Results Following de novo sequencing of the polyclonal antibodies, chain assembly, and chain pairing, the dominant antibody forms within the pAb mixture were recombinantly expressed as monoclonal antibodies (mAbs) based on the derived sequences. The recombinant mAbs exhibited affinities in the mid-picomolar to low-nanomolar range and demonstrated comparable activity to the original polyclonal sample. Conclusion This study demonstrates the application of mass spectrometry and de novo protein sequencing technology to generate recombinant polyclonal antibody mixtures directly from a purified protein mixture. This approach enhances the application of polyclonal antibodies in research, laboratory, and diagnostic settings where reproducibility is essential. High affinity, antigen-specific antibodies were successfully captured and derived without the need for cell lines or nucleotide sequence data. By converting a polyclonal mixture into recombinant monoclonal forms, good antibody products can be immortalized and produced at a large scale, eliminating the irreproducibility issue inherent with pAbs.