Monitoring mAb proteoforms in mouse plasma using an automated immunocapture combined with top-down and middle-down mass spectrometry

17 March 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Monoclonal antibodies (mAbs) have established themselves as the leading biopharmaceutical therapeutic modality. Once the developability of a mAb drug candidate has been assessed, an important step is to check its in vivo stability through pharmacokinetics (PK) studies. The gold standard is ligand-binding assay (LBA) and LC–MS (Liquid Chromatography- Mass Spectrometry) performed at the peptide level (bottom-up approach). However, these analytical techniques do not allow to address the different mAb proteoforms that can arise from biotransformation. In recent years, top-down and middle-down mass spectrometry approaches have gained popularity to characterize proteins at the proteoform level but are not yet widely used for PK studies. We propose here a workflow based on an automated immunocapture followed by top-down and middle-down LC-MS/MS approaches to characterize mAb proteoforms spiked in mouse plasma. We demonstrate the applicability of our workflow on a large concentration range using pembrolizumab as a model. We also compare the performance of two state-of-the-art Orbitrap platforms (Tribrid Eclipse and Exploris 480) for these studies. The added value of our workflow for an accurate and sensitive characterization of mAb proteoforms in mouse plasma is highlighted.


middle-down mass spectrometry
monoclonal antibody
top-down mass spectrometry

Supplementary materials

Supplementary Information
Supplementary information combining details on targeted LC-MS/MS acquisition methods for middle-down analysis and complementary results (tables & figures).


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