Impact of Mobile Phase Conditions and Temperature on Antibody Drug Conjugates Separation using Biozen Native RP-5 Column

Antibody-drug conjugates (ADCs) require precise analytical techniques for characterization due to their complex hydrophobic properties. This study evaluates the Biozen Native RP-5 column for LC-HRMS analysis of intact cysteine-linked ADCs, examining the impact of mobile phase ionic strength, pH, and temperature on retention, peak shape, selectivity, and MS sensitivity. Results show that lower ionic strength enhances retention and MS signal, while lower pH affects retention and resolution of additional peaks, potentially revealing positional isomers. Elevated temperature reduces retention but improves peak resolution. This method enables rapid, MS-compatible ADC characterization, optimizing analytical workflows for biotherapeutic development.