Tandem Native Mass-Spectrometry on Antibody-Drug Conjugates and Submillion Da Antibody-Antigen Protein Assemblies on an Orbitrap EMR Equipped with a High-Mass Quadrupole Mass Selector.

Native mass spectrometry is emerging as a powerful tool for the characterization of intact antibodies and antibody-based therapeutics. Here, we demonstrate new possibilities provided by the implementation of a high mass quadrupole mass selector on the recently introduced Orbitrap Exactive EMR mass spectrometer. This configuration allows precursor ion selection, and thus tandem mass spectrometry experiments, even on analytes with masses in the hundreds of kilodaltons. We apply tandem mass spectrometry to localize the drug molecules in the therapeutic antibody-drug conjugate brentuximab vedotin, which displays a heterogeneous drug load. Our tandem MS data reveal that drug conjugation takes place nonhomogeneously to cysteine residues both on the light and heavy chains. Next, we analyzed how many antigens bind to IgG hexamers, based on a recently described antibody mutant IgG1-RGY that forms hexamers and activates complement in solution. The fully saturated IgG1-RGY-antigen complexes displayed a stoichiometry of IgG:CD38 of 6:12, possessing a molecular weight of about 1.26 MDa and demonstrating that IgG assembly does not hamper antigen binding. Through tandem MS experiments, we retrieve information about the spatial arrangement and stoichiometry of the subunits within this complex. These examples underscore the potential of this further modified Orbitrap-EMR instrument especially for the in-depth characterization by native tandem mass spectrometry of antibodies and antibody-based constructs.