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A multiparameter optimization in middle-down analysis of monoclonal antibodies by LC–MS/MS

J. Mass Spectrom.
May 4, 2023

In this study, reaseachers from Sanofi describe aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. 


In antibody-based drug research, a complete characterization of antibody proteoforms covering both the amino acid sequence and all posttranslational modifications remains a major concern. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies but does not allow the diversity of proteoforms to be assessed. Middle-down and top-down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. 


MS spectra were deconvolved with Genedata Expressionist® software using a time-resolved deconvolution and the Maximum Entropy (MaxEnt) algorithm.


Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states, and the combination of replicates.


This works represents one of the most comprehensive studies performed on modern platforms offering experimental capabilities, such as multiplexing of charge states, previously not available.