"Fit-for-purpose" method validation and application of a biomarker (C-terminal telopeptides of type 1 collagen) in denosumab clinical studies.

Biomarkers are used to study drug effects, exposure-response relationships, and facilitate early decision making during development. Denosumab, a fully human monoclonal antibody against receptor activator of nuclear factor-kappaB ligand, profoundly inhibits bone resorption. C-terminal telopeptides of type I collagen (CTx), a bone resorption biomarker, provides early indications of denosumab effectiveness and informs protracted clinical outcomes (e.g., bone mineral density). Because of the dynamic relationship between denosumab and CTx, a precise and robust assay was desired. Thus, we adopted a fit-for-purpose approach to modify and validate a commercial CTx diagnostic kit to meet the intended applications of a quantitative pharmacodynamic biomarker for denosumab development. Seven standards were prepared to replace five calibrators provided in the kit. Three quality controls (QC) and two sample controls were used to characterize and monitor assay performance. Robotic workstations were used for standard and QC preparation and assay execution. Method validation experiments were conducted with rigor and procedures similar to those used for drug bioanalysis. The method demonstrated a linear range of 0.0490-2.34 ng/mL with four-parameter logistic regression. Inter-assay total error of validation samples in serum was < or = 26.7%. Extensive tests were conducted on selectivity in sera from target populations, specificity, stability, parallelism, and dilutional linearity. Applications to samples from numerous clinical studies confirmed that the CTx method was reliable, robust, and fit for use as an early indicator of denosumab effectiveness. Refinement supported the confidence for use in pharmacokinetic/pharmacodynamic modeling, dose selections, correlation to clinical effects, and formulation bioequivalence work.