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Evaluation of the Assembly Algorithm for De Novo Sequencing of Antibodies and Its Application in Analyzing Pan-Coronavirus Neutralizing Antibodies

ABSTRACT: Humanized monoclonal antibodies, generated by grafting complementarity-determining regions (CDRs), offer significant advantages in terms of safety and efficacy as therapeutic agents. The accurate determination of heavy- and light-chain CDR sequences is crucial for the development and production of recombinant antibodies. In this study, we performed de novo sequencing on three humanized pan-coronavirus neutralizing antibodies (3D11, C1520, 5A6) utilizing an integrated SP-MEGD pretreatment method combined with a bottom-up mass spectrometry approach. The antibody sequences were reconstructed employing three assembly algorithms: PEAKS AB, Stitch, and Fusion. A systematic comparison was performed to assess the performance, specifically the coverage and accuracy of assembly, of these three algorithms. The results indicate that the Fusion algorithm achieves 100% coverage and accuracy under the current experimental conditions, for both light and heavy chains across all three antibodies while maintaining stability in high-variability regions of CDRs without introducing additional sequence insertions. In contrast, PEAKS AB and Stitch exhibited fragment missing, insertions, or misassembled segments in certain areas. In conclusion, the robust performance demonstrated by the Fusion algorithm provides a reliable foundation for sequence analysis that supports rational design and development efforts concerning pan-coronavirus neutralizing antibodies.

ORGANISM(S): Homo Sapiens

SUBMITTER: Ningshao Xia

PROVIDER: PXD070714 | iProX | Fri Nov 14 00:00:00 GMT 2025

REPOSITORIES: iProX

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