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P1002
Validation of Automated High-Throughput Plasma Protein Binding Assays
Presenter Michael Shanler, BD Biosciences, USA
Additional Authors: Andrew Mason, Ronell Crocker, Elke Perloff, David Stresser
The degree of plasma protein binding (PPB) of a test article is a parameter that determines which fraction of the total plasma concentration of a compound is bound to plasma proteins and thus generally considered not available for interaction with its biological target (receptors, enzymes, transporters, etc.). Determination of the free (%Fu) and bound (%Bound) fractions of a test article in plasma is therefore a critical parameter, which is routinely determined in the process of drug discovery and development. PPB assays by HT equilibrium dialysis using the RED (“Rapid Equilibrium Dialysis”) device with LCMS quantitation allow for the screening of large numbers of compounds with fast turnaround times.
Results from HT Plasma Protein Binding (PPB) Assays performed at BD Gentest™ Contract Research Services, are compared using both manual and automated methods. A panel of compound stocks (1 & 10mM), including common standards such as Warfarin, Propranalol, Verapamil, Citalopram, and Linezolid are compared. Data showing tight correlation to published values is presented on multiple species including human, rat, dog, and mouse. The plasma protein binding assay was validated at two donor concentrations, and the Intra-plate, plate-to-plate, assay-to-assay and operator-to-operator variability was assessed.
The procedure used for automated assays performed on a Tecan Freedom EVO200 coinfigured with a 96-Multichannel Arm (MCA), span 8 tips, and automated tip-loading system is reviewed. The instrument’s deck layout for performing this assay in both singlicate and duplicate is reviewed.