Limiting degradation of reactive antibody drug conjugate intermediates in HPLC method development

The development of an accurate HPLC method for a reactive linker drug intermediate for antibody drug conjugates (ADCs) is challenging as the linker drug is designed to be reactive. This reactivity can lead to the generation of artifact peaks in the chromatograms and deliver inaccurate impurity content results. In this study, the linker drug contains an amine reactive tetrafluorophenyl (TFP) ester that readily undergoes hydrolysis and internal succinimide cyclization in aqueous solution. While complete elimination of the on-column degradation was nearly impossible, optimization of the critical chromatography conditions guided by chemical kinetics minimizes the degree of on-column degradation enabling an accurate assay and purity method for this reactive linker drug intermediate. Kinetics of the linker drug reactions were studied in different solution pHs and temperatures while the reaction rates were used to guide the selection of the optimum diluent, mobile phase pH, and column temperature to minimize the on-column degradation. An UHPLC column was used to achieve fast analysis to further reduce the degree of on-column degradation. The actual amount of on-column degradation of the final HPLC method was determined to be <0.1%, lower than the ICH reporting limit of, therefore demonstrate the effectiveness of the strategy.