Have you ever wondered why some antibodies can combat viral mutations but have yet to be widely used in new drugs? The answer lies in the properties of “polyclonal antibodies (pAbs).” They can recognize multiple epitopes of an antigen, making them more resistant to mutations than monoclonal antibodies (mAbs). However, their complex composition has long posed a "sequencing dilemma.” Traditional methods either rely on scarce B cells germlines or a "guess" based on existing databases, resulting in slow and inaccurate outputs. Even direct sequencing via mass spectrometry generates overly complex data that is particularly challenging to assemble.
Thanks to the relentless efforts of our research team at Bioinformatics Solutions Inc. (BSI), this long-standing bottleneck has finally been overcome! On October 13th, the article titled "Sequencing of Polyclonal Antibodies by Integrating Intact Mass, Middle-Down and De Novo Bottom-Up Mass Spectrometry" was published in Molecular & Cellular Proteomics. By combining three mass spectrometry techniques—bottom-up, middle-down, and intact mass—the study achieves fully automated, database-independent sequencing of polyclonal antibodies with an accuracy of over 99%, ushering in a new era for pAb-based drug development.
Lei Xin, Wenting Li, and Shuyang Zhang from BSI are co-first authors of the article, with Baozhen Shan and Academician Ming Li serving as corresponding authors. This work represents a collaborative effort across teams specializing in algorithms, software development, liquid phase separation technology, and mass spectrometry.
Xin, L., Li, W., Zhang, S., Tran, N. H., Chen, Z., Ma, J., ... & Shan, B. (2025). Sequencing of Polyclonal Antibodies by Integrating Intact Mass, Middle-Down and De Novo Bottom-Up Mass Spectrometry. Molecular & Cellular Proteomics, 101088. doi:10.1016/j.mcpro.2025.101088
Abstract
Polyclonal antibodies represent nature's approach to robust immunity, targeting multiple sites on pathogens, but their complex mixtures have remained largely unsequenceable, limiting their therapeutic potential. While monoclonal antibodies dominate therapeutics due to their reproducibility, polyclonal antibodies offer superior resilience against viral mutations and broader target recognition. Current pAb sequencing attempts have shown limitations, requiring germline databases or B-cell sequencing. Due to the highly variable nature of antibodies, as well as the possibility of unavailable B-cells, there is a need for a purely mass spectrometry and de novo sequencing based solution. Here we present PolySeq.AI, an automated de novo workflow that combines bottom-up, middle-down, and intact mass analysis to accurately sequence pAb samples without relying on external databases. PolySeq.AI achieved >99% sequencing accuracy across all tested samples, including an mAb mixture from the HB-95 cell line and a four mAb mixture, with complete bottom-up coverage and strong middle-down fragment support. Importantly, recombinant antibodies produced from our de novo sequences of HB-95 antibodies retained full binding capabilities to HLA-I complexes, confirming the accuracy and efficacy of our pAb de novo sequencing workflow.