Enhanced middle-down MS characterization of antibody-based therapeutics using ion-ion and ion-photon activation

The antibody scaffold is a powerful tool for the development of advanced biotherapeutics, including antibody-drug conjugates (ADCs). However, the inherent microheterogeneity of monoclonal antibodies (e.g., terminal modifications/amino acid clipping, amino acid side chain modifications such as oxidation, glycosylation) coupled with the hard-to-predict results of bioconjugation, require improvements in analytical tools to achieve a thorough characterization of their critical quality attributes. Mass spectrometry is used throughout all developmental stages of therapeutic mAbs and ADCs, primarily by applying a peptide mapping (or “bottom-up”) approach. Peptide-centric strategies, while useful, suffers of several limitations, which in the case of ADCs can complicate the identification of payload conjugation sites. Here, we demonstrate that a “middle-down” workflow can generate information-rich and easy-to-interpret data through the use of ion-ion and ion-photon activation. The combination of advanced fragmentation techniques (e.g., electron transfer dissociation with various supplemental activations) and proton transfer reactions enables the unambiguos mapping of payload conjugation sites of an ADC mimic in a single liquid chromatography – tandem mass spectrometry (LC-MS/MS) run. Additionally, new software solutions facilitate the interpretation of middle-down mass spectra and enabled the seamless integration of amino acid sequence information derived from distinct LC-MS/MS runs.