Advances in technology have led to a shift for peptide quantification from traditional ligand-binding assays to LC-MS/MS-based analysis, which presents challenges, in other assay sensitivity, specificity and ruggedness, in addition to lacking of regulatory guidance, especially for the hybrid assay format. Methodology & results: This report communicates a strategy that has been employed in our laboratories for method development and assay validation, and exemplified in a case study of MK-2640, a glucose-responsive insulin, in multiple matrices. Intact MK-2640 was monitored, while immunoaffinity purification and SPE were used to support the rat/dog GLP and clinical studies, respectively. The rationale and considerations behind our approach, as well as the acceptance criteria applied to the assay validation are discussed.
Keywords: GRI; glucose-responsive insulin; hybrid assay; peptide quantification.