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

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 liquid chromatography-mass spectrometry (LC-MS) 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 liquid chromatography-tandem mass spectrometry (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.

Automated summary

Monoclonal antibodies (mAbs) are widely used in biopharmaceutical therapeutics, and assessing their in vivo stability is crucial for drug development. Current analytical techniques for pharmacokinetics (PK) studies, such as ligand-binding assay (LBA) and liquid chromatography-mass spectrometry (LC-MS), analyze mAbs at the peptide level, which limits the characterization of different mAb proteoforms. In this study, we propose a workflow using automated immunocapture followed by top-down and middle-down LC-MS/MS approaches to characterize mAb proteoforms in mouse plasma. The workflow was demonstrated using pembrolizumab as a model and the performance of two Orbitrap platforms was compared.