Utilizing Biozen Native RP-5 Column for Rapid RPLC HRMS Separation and Identification of Native, Intact Antibody Drug Conjugate Species

Antibody-Drug Conjugates (ADCs) are a powerful class of targeted cancer therapeutics that deliver cytotoxic agents directly to cancer cells while minimizing systemic toxicity. A critical quality attribute in ADC development is the drug-to-antibody ratio (DAR), which impacts efficacy and safety. Conventional methods for DAR analysis, such as Hydrophobic Interaction Chromatography (HIC) coupled with offline mass spectrometry (MS), can be time-consuming and introduce variability due to required buffer exchanges. This study demonstrates the application of the Biozen Native RP-5 column, a non-porous, hydrophilic reversed-phase sorbent, for the chromatographic separation and online MS identification of intact ADCs in their native state. Using three commercially available cysteine-linked ADCs Padcev, Polivy, and Tivdak the method effectively resolved all DAR species, including positional isomers. High-resolution mass spectrometry (HRMS) provided positive identification of DAR species with mass errors below 50 ppm. The study highlights the robustness and efficiency of this approach in ADC characterization, offering a streamlined workflow that improves reliability and reproducibility for drug development.