Antibody–drug conjugates (ADCs) present unique analytical challenges due to their structural complexity and heterogeneity. This presentation highlights the development of a hydrophobic interaction chromatography (HIC) method for resolving and characterizing drug-load variants in site-specifically conjugated pyrrolobenzodiazepine dimer-based ADCs (PBD-ADCs). Method optimization strategies—including column selection, mobile phase composition, and gradient design—will be discussed to demonstrate how optimal separation of species with distinct drug-to-antibody ratios (DAR) was achieved. Fractionation of HIC-resolved DAR species under native conditions, followed by mass spectrometric characterization, provides an enhanced understanding of product heterogeneity during early-stage development, supporting critical decision-making in ADC process and product development.
