Validation of a Multiplex Assay for Simultaneous Quantification of Amyloid-β Peptide Species in Human Plasma with Utility for Measurements in Studies of Alzheimer's Disease Therapeutics

The aim of this study was to validate the INNO-BIA plasma amyloid-beta (A beta) forms assay for quantification of A beta(1-40) and A beta(1-42) according to regulatory guidance for bioanalysis and demonstrate its fitness for clinical trial applications. Validation parameters were evaluated by repeated testing of human EDTA-plasma pools. In 6 separate estimates, intra-assay coefficients of variation (CV) for repeated testing of 5 plasma pools were <= 9% and relative error (RE) varied between -35% and +22%. Inter-assay CV (n = 36) ranged from 5% to 17% and RE varied from -17% to +8%. Dilutional linearity was not demonstrated for either analyte using diluent buffer, but dilution with immuno-depleted plasma by 1.67-fold gave results within 20% of target. Analyte stability was demonstrated in plasma at 2-8 degrees C for up to 6 h. Stability during frozen storage up to 12 months and through 3 freeze-thaw cycles at <=-70 degrees C was also demonstrated in 5 of 6 individuals but deteriorated thereafter. Neither semagacestat nor LY2811376 interfered with the assay but solanezumab at 500 mg/L reduced recovery of A beta(1-42) by 53%.Specimens from a Phase I human volunteer study of the beta-secretase inhibitor LY2811376 were tested at baseline and at intervals up to 12 h after single oral doses, demonstrating a clear treatment effect. During 1,041 clinical assay runs from semagacestat studies over 10 months, the CV for plasma quality control pools at three levels were <= 15% and RE were <10%. In conclusion, the INNO-BIA plasma assay was successfully validated and qualified for use in clinical research.