Method development of purity and assay HPLC method for unstable antibody-drug conjugate linker regulated starting material BCN-OH

BCN-OH is a critical raw material in an innovative payload-linker platform utilizing azido-sugar site-specific conjugation technology, which offers superior conjugation efficiency and therapeutic index. However, BCN-OH exhibits extreme instability, with solution stability of less than one hour and high susceptibility to oxidation upon air exposure. These characteristics pose significant challenges for impurity profiling and specification setting during manufacturing. To address this, we aimed to develop a robust and reproducible HPLC method capable of accurately identifying and characterizing related substances in BCN-OH, ensuring reliable quality control and regulatory compliance. Through systematic optimization of chromatographic conditions-including column selection, temperature, mobile phase composition, sample diluents, and gradient profiles, the method achieved baseline separation of five specified impurities and resolved the main peak from its isomer. Incorporation of BHT as an antioxidant extended BCN-OH solution stability from under one hour to 24 hours, greatly improving operational feasibility. Additional measures, such as nitrogen storage and glove box handling, further minimized oxidation risk. The validated method demonstrated robustness, intermediate precision, and suitability for PPQ and routine QC, providing essential data for impurity specification setting and ensuring consistent quality of this highly unstable payload-linker intermediate.