Determining Antibody-Drug Conjugate Positional Isomer Distribution With Nonreduced CE-SDS And HIC

By Kyle Luttgeharm, Whitney Pike, Lisa Engelbart, and Sonja Schneider

Antibody–drug conjugates demand a clear view of both drug-to-antibody ratio (DAR) and where payloads attach, as each shapes efficacy. Hydrophobic interaction chromatography (HIC) excels at resolving DAR distributions, while nonreduced CE-SDS reveals fragmentation patterns linked to conjugation at interchain disulfides. By combining these orthogonal datasets with a system-of-equations approach, it becomes possible to estimate positional isomer abundance without specialized assays. Analysis of a SigmaMAb ADC mimic showed a dominant DAR4 population (41.5%) with drugs at both Fab disulfides, alongside notable DAR2f (19.3%) and DAR6fhh (22.9%) species, and no detectable DAR2h or DAR6ffh. This integrated workflow using Agilent CE-SDS and HIC platforms delivers rapid, actionable insight into ADC structure.

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