Development of an electrochemiluminescence-based bridging assay to detect antibodies against a PTH inverse agonist in human plasma.

BACKGROUND: Therapeutic peptide-induced anti-drug antibodies (ADA) can have various effects, including adverse events and drug-neutralizing activity which can lead to loss of efficacy. This has led regulatory agencies to require nonclinical and clinical testing of biotherapeutics to assess immunogenicity risk. RESEARCH DESIGN AND METHODS: An electrochemiluminescence bridging assay was developed to detect antibodies against parathyroid hormone receptor inverse agonist (PTH-IA), a novel synthetic peptide that is being developed to treat Jansen's Metaphyseal Chondrodysplasia. Biotinylated PTH-IA and ruthenylated PTH-IA reagents were synthesized via amine-coupling chemistry, and rat polyclonal anti-PTH-IA positive control antibody was generated by immunizing rats with PTH-IA conjugated to keyhole limpet hemocyanin. The reagents were utilized to determine cut points for the screening and confirmatory assays and assay sensitivity in drug-naïve human plasma. RESULTS: Sensitivity of the screening assay was determined to be 3.61 ng/mL at cut point. The cut point for the confirmatory assay was determined to be 9.34% inhibition, and the cut point factor used to establish the floating plate cut point from the negative control signal was 1.055. The assay was found to be selective and reproducible. CONCLUSION: The assay's performance characteristics demonstrated its suitability for detecting anti-PTH-IA antibodies in human plasma.