Development of a High-Throughput Size-Exclusion Chromatography-Mass Spectrometry Workflow for Real-Time Monitoring of...

The manufacturing of cysteine-based antibody-drug conjugates (ADCs) requires precise control of the drug-to-antibody ratio (DAR), a critical quality attribute influencing product safety and efficacy. Conventional analytical techniques used for DAR determination, such as hydrophobic interaction chromatography (HIC) and reversed-phase high-performance liquid chromatography (RP-HPLC), are low-throughput and unsuitable for real-time process monitoring, limiting process development efficiency. To address this challenge, we developed a high-throughput size-exclusion chromatography-mass spectrometry (SEC-MS) workflow with and without postcolumn denaturation (PCD) functionality, enabling real-time monitoring of both reduction and conjugation kinetics during cysteine-based ADC manufacturing. Using a short SEC column coupled to a mass spectrometer, analysis time can be performed under 2 min per sample. This workflow was applied to rapidly optimize critical process parameters, including reductant concentration and pH during reduction, and drug-linker ratio during conjugation. The approach provided deeper mechanistic insights into reaction kinetics and supports process analytical technology (PAT)-enabled strategies for advanced process control. Overall, this high-throughput SEC-MS workflow accelerates ADC development while improving manufacturing robustness.