Measuring Antibody Drug Concentrations in Presence of a Reversal Drug

REGN-A is a monoclonal antibody that binds and activates its target and is currently under clinical development for cardiovascular indications. REGN-B, a one-arm, monovalent antibody, is being developed as a reversal agent for REGN-A to reverse its pharmacodynamic effects. In this talk, the specific bioanalytical challenges encountered during the phase I clinical study for REGN-A and REGN-B will be discussed.

Four ligand-binding assays have been developed and validated to measure free and total REGN-A, as well as free and total REGN-B, in human serum. During the clinical sample analysis in this phase I study, it was observed that the anticipated reduction of free REGN-A after administration of excess REGN-B was less pronounced at higher dose levels compared to lower dose levels. This resulted in quantitation of levels of free REGN-A at higher doses that were physiologically significant, even after addition of the reversal agent.

To support the pharmacokinetic evaluation of REGN-A and evaluate the reversibility of REGN-B in the clinical study, it was essential to understand whether this higher-than-expected quantitation of free drug was due to a bioanalytical issue. For this purpose, a comprehensive investigation was performed, and incurred study samples were analyzed at different dilutions beyond the 1:20 minimum-required-dilution (MRD). The investigation data demonstrated that excessive dilution beyond the MRD could lead to over-quantitation of free REGN-A in the presence of the reversal agent.

Additional efforts were made to shed light on the mechanism of this free drug over-quantitation related to over-dilution of samples. Characterization assays based on Asymmetrical Flow Field-Flow Fractionation coupled to Multi-Angle Light Scattering (A4F-MALS) and mass photometry were utilized to understand the dissociation of the REGN-A:REGN-B complex and the competition of the assay capture reagent with REGN-B molecules from the complex. The results showed that the REGN-A:REGN-B complex mainly exists as a stable 1:2 complex following up to approximately 100-fold dilution. However, moderate dissociation of the complex can occur at higher dilution level (1000-fold). In addition, excess assay capture reagent can displace at least one REGN-B molecule from the complex. These characterization results collectively demonstrated that the dissociation of the REGN-A:REGN-B complex upon dilution beyond MRD and the competition of the assay capture reagent with REGN-B in the complex could contribute to the over-quantitation of free REGN-A in the presence of REGN-B.

Based on the above bioanalytical investigation and characterization using orthogonal assay platforms, clinical study samples collected from subjects co-administrated with REGN-A and REGN-B were reanalyzed at MRD for the quantitation of free REGN-A. The reanalysis results demonstrated that, upon dosing of the reversal agent, free REGN-A levels dropped rapidly and significantly to below physiologically significant levels. The overall bioanalytical strategy successfully supported the understanding of the pharmacokinetics of REGN-A in the presence of the reversal agent as well as the pharmacodynamic interpretation, including the reversibility of REGN-B.