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Diagnostic Technologies and Molecular and Cellular Profiling: Biomarker Assay Development, Validation, and Qualification

Qualification of two multi-plexed immunoassays utilizing meso-scale discovery (MSD) technology for quantitation of angiogenic cytokines used as exploratory biomarkers

Amgen, Thousand Oaks, CA

Abstract

A15

Biomarkers used in clinical trials as pharmacodynamic endpoints have been an emerging focus. Attention has been given to the evaluation of biomarkers for utility to inform and guide clinical trials. Biomarker assay results can be used to correlate markers of interest to dose or efficacy of the drug. Biomarker assay qualification is necessary to ensure that the data produced is reliable. To measure levels of angiogenic biomarkers in clinical trials of anti-angiogenic therapeutics, we used two multi-plexed MSD assays. .We selected this assay format because multi-plexing conserves sample, allowing multiple analytes to be tested in the same assay. Another beneficial feature of MSD assays is that they have large dynamic ranges, which reduces the need to re-analyze samples. One of the MSD multi-plexed assays is a 4-plex that includes bFGF (basic fibroblast growth factor), PlGF (Placental Growth Factor), VEGF (Vascular Endothelial Growth Factor) and soluble Flt-1 (VEGFR1). The second MSD multi-plexed assay includes soluble KDR (VEGFR2) and soluble cKit. Assay parameters evaluated during qualification include sample collection, sample stability, assay reagent stability, inter- and intra- assay precision, accuracy, sample parallelism, linearity, specificity, interference and assay range. For the anti-angiogenic panel of assays, sample stability, inter-assay precision and interference had the greatest impact on the accuracy of results. During assay qualification, VEGF and bFGF were shown to be unstable over multiple freeze/thaw cycles. Changes in sample preparation reduced this instability, and increased consistency of results on multiple sample preparations from single individuals. Inter-assay variability of Flt-1 and VEGF was high, and interference studies revealed the impact of receptor-ligand binding on the ability to accurately quantitate some analytes. In summary, assay qualification is used to develop the appropriate sample collection and assay conditions necessary to obtain reliable biomarker results.