Speaker
Description
Monoclonal antibodies (mAbs) have revolutionized biotherapeutics, offering effective treatments for various diseases. Antibody-drug conjugates (ADCs) combine mAbs’ specificity with potent cytotoxic drugs linked via Lys- or Cys-conjugation, targeting malignant cells. However, heterogeneity in payload attachment challenges ADCs' safety and efficacy. We employed a middle-down (MD) mass spectrometry (MS) approach to investigate a SiLu ADC mimic with variable drug-to-antibody ratio (DAR) using the Orbitrap Ascend BioPharma Tribrid mass spectrometer. By integrating native MS of 100 kDa F(ab’)2 subunits with disulfide-reduced 25 kDa subunit analysis under denaturing conditions, we achieved thorough ADC characterization and precise localization of payload conjugation sites. Native MS analysis of F(ab’)2 subunits isolated specific DARs with high purity, determining major payload occupancy combinations for DARs 2-8. Our disulfide-reduced subunit analysis aimed to localize payloads on Fd’, which has up to three conjugation sites. While higher-energy collisional dissociation (HCD) and ultraviolet photodissociation (UVPD) provided limited diagnostic ions, Electron Transfer Dissociation (ETD) and ETD followed by supplemental HCD energy (EThcD) localized the conjugation site in the most abundant Fd’ species with a single payload at the MS2 level. Minor single payload species and double payload species required spectral decongestion via proton transfer charge reduction (PTCR) at the MS3 level for higher ion count and sequence coverage. EThcD MS2-PTCR MS3 uniquely localized payloads for all species combinations, achieving ~60% sequence coverage. This approach provided unambiguous localization of drug-payload attachment sites through PTCR following EThcD on the Orbitrap Ascend BioPharma MS.
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