Antibody-Drug Conjugate Synthesis and Purification

Antibodies were dialyzed with BupH™ PBS Packs (Thermo Fisher Scientific) and conjugated with smcc-DM1 (MedChemExpress, Monmouth Junction, NJ, USA) at RT at a molar ratio of 1:20 for 1.5 h. The mixture was centrifuged at 21,000× g for 20 min at 20 °C, to remove aggregates. ADCs were purified using Sephadex G-25 PD-10 desalting column (GE Healthcare, Chicago, IL, USA) and stored in 10 mM sodium succinate, 6% (v/v) sucrose, and 0.05% (v/v) Tween-20 pH 5.0. The ADCs were further analyzed using SDS-PAGE and ultraviolet spectrometry. Analysis of the DAR ratio was performed as previously described [32 (link)].