Hydrophobic interaction chromatography (HIC) is commonly used to determine the drug-to-antibody ratio (DAR) and drug load distribution of antibody-drug conjugates (ADCs). However, identifying various DAR species separated by HIC is challenging due to the traditional use of mobile phases that are incompatible with mass spectrometry (MS). Existing approaches used to couple HIC with MS often encounter issues, such as complex instrumentation, compromised separation efficiency, and reduced MS sensitivity. In this study, we introduce a 22-min online native HIC-MS method for the separation and characterization of different DAR species in ADCs, addressing these challenges. The key novelty of this method is the use of ammonium tartrate, a kosmotropic and thermally decomposable salt, as the salt of HIC mobile phase, ensuring both excellent HIC separation and MS compatibility. Additionally, an ultrashort size exclusion chromatography step is integrated for online sample cleaning, enhancing MS sensitivity. This platform native HIC-MS method offers a rapid, sensitive, and robust solution for comprehensive profiling of DAR species in ADCs with a simple and cost-effective instrumental setup.
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