Measurement of Free Versus Total Therapeutic Monoclonal Antibody in Pharmacokinetic Assessment is Modulated by Affinity, Incubation Time, and Bioanalytical Platform

Talbot JJ, et al., 17(6):1446-54, AAPS J, 2015

Ligand binding assays (LBAs) play a critical role in quantifying serum therapeutic protein (TP) levels in pharmacokinetic and pharmacodynamic assessments. Described herein is the impact of the bioanalytical platform, reagent-antibody affinity, and incubation time on obtaining free or total TP levels. An ELISA-based LBA that quantifies humanized anti-sclerostin monoclonal antibody (TPx) was used as the model system. All kinetic experiments were performed by Bio-Layer Interferometry (BLI) on a Pall ForteBio Octet RED384 system equipped with Streptavidin (SA) Biosensor probes. The sensor tips were loaded with biotinylated-Anti-Fc mAb, which were subsequently captured with TPx. Sensor probes were then dipped in wells containing Sclerostin (Scl) protein. Kinetic parameters (kon, koff, and KD values) were determined. The importance of having a complete picture of the binding interaction from a BLI kinetic assay as compared to the use of end-point data from ELISA is higlighted. Overall results of this investigation provide a better understanding of how the choice of reagents and the platform for LBAs can affect accurate quantitation of either the free or total TP concentrations.

Read More