Approaches to Resolve False Reporting in Neutralizing Antibody Assays Caused by Reagent Leaching from Affinity...

Area of Science:

• Biopharmaceutical Analysis
• Immunogenicity Testing
• Assay Development

Background:

• Drug tolerance is a critical challenge in developing neutralizing antibody (NAb) assays for biotherapeutics.
• Solid-phase extraction with acid dissociation (SPEAD) is a common pre-treatment method to enhance drug tolerance.
• Biotin-drug conjugate leaching during SPEAD can lead to inaccurate NAb assay results (false positives/negatives).

Purpose of the Study:

• To evaluate mitigation strategies for biotin-drug conjugate leaching in SPEAD-based NAb assays.
• To compare streptavidin capture and covalent drug capture methods for reducing leaching.
• To optimize assay conditions for improved drug tolerance and sensitivity.

Main Methods:

• Assessed biotin-drug conjugate leaching using streptavidin-coated plates with varying biotin molar challenge ratios (MCR).
• Evaluated an alternative covalent capture method using aldehyde-activated plates.
• Compared both methods under different elution pH, incubation times, and MCR conditions.

Main Results:

• Both streptavidin and covalent capture methods significantly reduced drug/biotin-drug conjugate leaching.
• Optimized streptavidin capture with a 50:1 MCR demonstrated high binding capacity.
• The optimized assay achieved a sensitivity of 153 ng/mL and drug tolerance up to 50 μg/mL.

Conclusions:

• Optimized streptavidin capture with a high MCR is an effective strategy to mitigate drug conjugate leaching.
• This approach yields a reliable NAb assay with improved drug tolerance and sensitivity.
• The findings support the development of more robust NAb assays for biotherapeutic characterization.