HPPA as assay substrate for improved capturing of sample variability during cut point analyses in immunogenicity testing

AIMS: Immunogenicity testing is a key component of protein drug development, with ADA bridging assays recognized as the gold standard method. These assays employ labeled therapeutic drugs for capture and detection, with the substrate playing a critical role in generating a detectable signal to differentiate the presence of anti-drug antibodies (ADAs) from nonspecific binding. This study investigates the impact of substrate choice on the assay’s ability to capture individual sample variability, focusing on screening and confirmatory cut points (SCP and CCP). METHODS: We compared the colorimetric substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with the fluorescent substrate 3-(4-hydroxyphenyl)propionic acid (HPPA) in ELISA-based ADA bridging assays for two monoclonal antibodies. RESULTS: Switching the substrate from ABTS to HPPA improved the differentiation of individual signals from baseline noise, enabling a more robust ADA identification. Additionally, the use of HPPA resulted in higher SCP and CCP values. Further investigation revealed that technical reader noise affected a normally distributed sample set when using ABTS. CONCLUSION: The substrate switch, while maintaining all other assay parameters, proved to be a convenient approach to increase SCP and CCP while improving the ability to capture individual sample variability.

Jordan G, Pohler A, Jany C, Bach T, and Staack R F. (2025) HPPA as assay substrate for improved capturing of sample variability during cut point analyses in immunogenicity testing. Bioanalysis 17(15):951-958 . [article]