Real-Time Bioconjugation Reaction Monitoring of Antibody–Drug Conjugates with Multiattribute High-Throughput Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is a current gold standard for determining the drug-to-antibody ratio (DAR) and drug load distribution (DLD) in antibody-drug conjugates (ADCs). In this study, we developed several HIC methods using ammonium tartrate for comprehensive ADC characterization and bioconjugation reaction monitoring. We first demonstrated that ammonium tartrate delivers a separation performance comparable to conventional ammonium sulfate while offering superior mass spectrometry compatibility. We then established optimized platform HIC methods for analyzing both interchain and engineered cysteine-conjugated ADCs. Furthermore, we developed a rapid 10 min, multiattribute HIC method as a process analytical technology (PAT) for real-time bioconjugation reaction monitoring. This multiattribute HIC PAT method was used to simultaneously track DAR, DLD, and drug-linker (DL) concentration within complex reaction mixtures. Through advanced PAT tools, we gained critical mechanistic insights into the kinetics of ADC conjugation. For example, we discovered that the DL dissolution rate could limit reaction progression in slurry reactions, an insight not possible through conventional technologies such as ultraviolet spectroscopy. These findings enabled the identification of critical process parameters, informing targeted process development that ultimately reduced reaction times by 90% with a designed average DAR and lower undesired overconjugation.