A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations

Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity. We developed a simple RP-HPLC/MRM-MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10 nM with 370 pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step.