Characterization Of Antibody‑Drug Conjugates Using 2D-LC And Native MS

By David L. Wong and Sarah M. Stow

Antibody drug conjugates (ADCs) continue to gain traction as targeted biotherapeutics, but maintaining control over drug-to-antibody ratio (DAR) remains essential for balancing efficacy and safety. Traditional 2D-LC/MS approaches—relying on hydrophobic interaction and reversed-phase separations—can introduce degradation due to harsh organic and acidic conditions, limiting analytical confidence. A refined workflow combines hydrophobic interaction chromatography with multiple heart-cutting and online desalting via size exclusion chromatography, enabling analysis under native conditions. Coupled with high-resolution mass spectrometry and advanced data processing, this approach preserves molecular integrity while delivering accurate DAR characterization.

For teams working across cell and gene or drug discovery pipelines, gain insight into a more reliable path to understanding ADC heterogeneity and stability.