Target engagement has become increasingly important for proving drug MOA and understanding dose-dependent PD effects. However, reliable measurements of free/total targets in the presence of high levels of drug can be challenging due to the dynamic nature of drug-target binding. I’ll describe an LC–MS approach to measuring free/total complement C5 in monkey serum as a TE biomarker for anti-C5 therapy. The total C5 was measured by direct quantitation of C5-specific peptide from serum digest, whereas free C5 was measured by immunoaffinity removal of drug-bound C5 via protein A followed by analysis of the C5 peptide. This method circumvents free C5 immunocapture and thus minimizes the shift of the drug-target binding equilibrium. Mathematical simulation was developed to correct the impact of sample dilution on drug-target dissociation and thus free C5 measurement. The fit-for-purpose assays were fully validated and have been utilized to aid in PK/PD correlation analysis and human dose projection.
learn an LC-MS approach to addressing issues with current techniques for assessment of target engagement.
learn how to measure total and free protein target as another dimension to assess PD/PK correlation analysis