Rapid Peptide Mapping of Monoclonal Antibodies with Direct Infusion Mass Spectrometry

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. Competing Interest Statement J.J.C. is a consultant for Thermo Fisher Scientific and Seer. J.J.C and L.M.S. co-founded a company, CeleramAb Inc., to commercially distribute this methodology. A.M.G.T. and M.P.G. are employees of AbbVie Inc. J.A. and M.B. are employees of Lilly and Company B.R.P., G.L., and H.P.G. are employees of Johnson & Johnson Innovative Medicine W.P. and W.S. are employees of Genentech Inc. I.J.M., C.W., and A.S.H. are consultants of CeleramAb A.Z.S., A.S.H., C.W., M.M., K.L.M., and J.J.C. are inventors of intellectual property regarding this work. The remaining authors declare no competing interests.