Rapid Peptide Mapping of Monoclonal Antibodies with Direct Infusion Mass Spectrometry

Salome, A. Z.; Morgenstern, M.; Hebert, A. S.; Wenger, C. D.; Sinitcyn, P.; Anderson, B. J.; Chlystek, J. S.; Serrano, L. R.; Mertz, K. L.; Miller, I. J.; Miller-Galow, E.; Godamudunage, M. P.; Batt, M.; Patel, B. R.; Lee, G.; Smith, L. M.; Quarmby, S. T.; George Thompson, A. M.; Ahn, J.; Gunawardena, H. P.; Coon, J. J.

doi:10.64898/2026.05.14.725248

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bioRxiv · 10.64898/2026.05.14.725248

Rapid Peptide Mapping of Monoclonal Antibodies with Direct Infusion Mass Spectrometry

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Abstract

Peptide mapping is a critical tool for characterizing biotherapeutic proteins and is essential for the development of monoclonal antibody drugs. Here we describe a new direct infusion technology that streamlines peptide mapping data collection and analysis, accelerating the method by up to 100-fold. This method, which we term RaPiD-mAb-MS, combines high-throughput plate-based sample preparation with direct infusion mass spectrometry analysis. RaPiD-mAb-MS allows analysis of 96 samples within [~] 1.5 to 2 hours, routinely achieves >95% sequence coverage, and has been successfully applied to 28 unique antibodies and over 2,000 samples. Here we demonstrate that RaPiD-mAb-MS detects and quantifies oxidation, deamidation, isomerization, glycosylation, and sequence variants with results comparable to conventional LC-MS based methods in a fraction of the time. Further, by eliminating chromatography, data analysis is greatly streamlined and simplified. By allowing for the collection of [~] 1,000 peptide maps per day, RaPiD-mAb-MS is positioned to accelerate all phases of antibody-based drug discovery & development and sets the stage for collection of massive datasets that would allow artificial intelligent prediction of optimal antibody variants and formulations.

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Salome, A. Z., Morgenstern, M., Hebert, A. S., Wenger, C. D., Sinitcyn, P., Anderson, B. J., Chlystek, J. S., Serrano, L. R., Mertz, K. L., Miller, I. J., Miller-Galow, E., Godamudunage, M. P., Batt, M., Patel, B. R., Lee, G., Smith, L. M., Quarmby, S. T., George Thompson, A. M., Ahn, J., Gunawardena, H. P., Coon, J. J.. 2026-05-16. Rapid Peptide Mapping of Monoclonal Antibodies with Direct Infusion Mass Spectrometry. https://doi.org/10.64898/2026.05.14.725248

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