Characterization of drug load variants in a thiol linked antibody-drug conjugate using multidimensional chromatography

The biological complexity associated with biotherapeutics such as antibody-drug conjugates (ADCs) requires extensive characterization to ensure product quality consistency, safety, and efficacy. ADCs generated via partial reduction of antibody interchain disulfide bonds result in a distribution of variants containing 0-8 conjugated drugs. Hydrophobic interaction chromatography (HIC) is a key analytical technique used to separate drug load variants of thiol linked ADCs and to calculate the average drug-to-antibody molar ratio (DAR). Salts present in HIC separations are not amenable to mass spectrometry (MS) detection, therefore confirmation of HIC peak identities have historically required offline fraction collection and subsequent MS analysis. We present a workflow comprised of three MS compatible two-dimensional liquid chromatography methods for higher throughput characterization of HIC peaks without manual fractionation. These methods are complementary and together provide DAR confirmation, identification of drug-load isomers, and localization of post-translational modifications to specific subunits. The results demonstrate an efficient mechanism for characterization of ADC HIC peaks and provided unique insight into differential HIC retention times based on conjugation sites and glycan structure.