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

Abstract

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.

Molecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and Antigen

“…The Mw for the latter complex could be determined by native MS to be 1,255.2 ± 0.1 kDa. These findings are consistent with complete occupancy of both Fab-Ag-binding sites in each individual IgG molecule (Dyachenko et al, 2015) ( Figure S4A). We observed little cooperativity in Ag recruitment, as the number of Ags incorporated into the IgG-Ag complexes increased evenly throughout the titration without any sharp transition.…”

Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

“…Therefore, sCD38 was incubated with a specific and high-affinity human IgG1 monoclonal antibody (mAb) against CD38. With excess sCD38, mAb/sCD38 complexes were assembled at a predominant stoichiometry of 1:2 as assessed by native MS, 34 showing visibly better resolved charge states than for the uncomplexed sCD38 (Figure 2B). In Step 2, we used TEAA as an additive in the spray solution to reduce charging of the ion, further increasing the spectral spacing between adjacent charge states (Figure 2B).…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…A number of recent instrument modifications have also been made to Orbitrap mass analyzers to facilitate improved analysis of higher mass species under native conditions, such as monoclonal antibodies [133] and non-covalent protein assemblies approaching 1 MDa [134,135]. In particular, instrumentation modifications to a commercial Q Exactive system have enabled multi-stage fragmentation of non-covalent protein complexes to individual subunits, and then down to the peptide level, thereby enabling the characterization of protein complex stoichiometry and primary structure of individual subunits [135].…”

“…Middle-down analysis of mAbs, characterized by lower molecular weight precursors, has the potential to resolve subtle differences in the m/z domain, as well as to detect and identify low-abundance PTMs. Moreover, precursor ion selection with an analytical quadrupole on a commercial Q Exactive™ Orbitrap mass spectrometer is confined to an upper m/z limit of 2,500 Th [270], thereby precluding the ability to select the most abundant charge states of an intact mAb for MS 2 analysis. Conversely, limited mAb digestion (e.g., using IdeS enzyme) and ionization under denaturing conditions yield both the precursor and fragment ion species, which are amenable to analysis on a commercial Orbitrap™ MS [267].…”