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

Area of Science:

• Biochemistry
• Immunology
• Pharmaceutical Science

Background:

• Immunogenicity testing is crucial for protein drug development.
• Anti-drug antibody (ADA) bridging assays are the gold standard for this testing.
• Assay substrates are critical for generating detectable signals and differentiating specific antibody binding from noise.

Purpose of the Study:

• To investigate the impact of substrate choice on ADA bridging assay performance.
• To evaluate how different substrates affect the capture of individual sample variability.
• To determine the influence of substrate on screening and confirmatory cut points (SCP and CCP).

Main Methods:

• Comparison of colorimetric (ABTS) and fluorescent (HPPA) substrates in ELISA-based ADA bridging assays.
• Assays were performed for two monoclonal antibodies.
• Analysis focused on signal differentiation, sample variability, and cut point values.

Main Results:

• The fluorescent HPPA substrate improved the differentiation of individual signals from baseline noise compared to the colorimetric ABTS substrate.
• Using HPPA led to higher SCP and CCP values, indicating a more robust assay.
• Technical reader noise impacted normally distributed samples when using the ABTS substrate.

Conclusions:

• Switching to the HPPA substrate is a convenient method to increase SCP and CCP in ADA bridging assays.
• The HPPA substrate enhances the assay's ability to capture individual sample variability.
• This substrate optimization improves the reliability of ADA identification in protein drug development.