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

Abstract

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.

Fig. 1

Composite CTx standard curve. Data from eight validation runs performed over 3 days. The standards were 2.46 (anchor point), 2.34, 1.85, 0.820, 0.406, 0.205, 0.0800, 0.0490, and 0.0260 (anchor point) ng/mL. Mean OD were plotted against concentrations with four-parameter logistic regression of 1/Y ² weighting. The error bars were standard deviation of the mean OD, which typically increase with increase responses in an immunoassay

Fig. 2

Accuracy and precision of validation samples in buffer and human serum. The VSs were 2.34 ng/mL for ULOQ, 1.70 ng/mL for high QC, 0.500 ng/mL for mid QC, 0.150 ng/mL for low QC, 0.800 ng/mL for LLOQ1, and 0.0490 ng/mL for LLOQ2. The VSs were prepared in buffer (green- and blue-colored bars) or in human serum (red- and purple - colored bars). The total error bars were composed of the inter-assay accuracy (%RE, dotted areas) and inter-assay precision (%CV, downward lined areas)

Fig. 3

Spike recovery of 0.145 ng/mL of CTx from normal and patient sera. CTx at 0.145 ng/mL was spiked into individual serum of various populations: breast cancer patients (BC, N = 20), multiple myeloma patients (MM, N = 10), normal female (F, N = 20), normal male (M, N = 20), osteoporosis patients (Osteo, N = 18), and rheumatoid arthritis patients (RA, N = 10). The solid line is the mean spike recovery of the normal female sera as a control comparison to other groups. The dotted lines represent the ±20% from the nominal spiked value. The box included data within 25 to 7th quantiles with the median line in the middle and extended values in the whiskers

Fig. 4

Parallelism test. Three authentic samples of relatively high concentrations (filled square, triangle, and circle symbols) and the HSC (open circles) were each diluted at 1.5-fold, twofold, fivefold, and tenfold with assay buffer. The observed concentrations times the dilution was plotted against 1/dilution factor

Fig. 5

In-study monitoring CTx sample control charts. a, HSC; b, LSC. Centerlines are the overall mean concentrations of LSC and HSC at 0.137 and 0.633 ng/mL, respectively. The upper and lower lines are the mean ± 2 SD values. Two lots of reference materials were used for standards and QC preparations during the time span of 3 years. Cross symbols are values from lot 1 (up to day 316) and square symbols are values from lot 2. Different shades (colors) represent different kit lots

Fig. 6

a Pharmacodynamic profiles of patients dosed with denosumab in a phase 2 study. Doses were placebo (filled triangles), alendronate (open triangles), and denosumab Q6months, 14 and 60 mg (14 mg: filled circles, 60 mg: open circles). The Y-axis is mean CTx % change, with error bars of +standard error of the mean; X-axis is time in months. b Pharmacokinetic profiles of patients dosed with denosumab in a phase 2 study. Doses were denosumab Q6months, 14 and 60 mg (14 mg: open triangles, 60 mg: filled triangles). The Y-axis is log mean denosumab serum concentration, with error bars of +standard deviation; X-axis is time in months