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Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

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High ionic strength dissociation assay reduces dimeric target interference in immunogenicity testing.

Alexander Pöhler¹, Cordula Jany¹, Joachim Butzer²

¹Roche Pharma Research & Early Development (pRED), Pharmaceutical Sciences, Bioanalysis & Biomarkers, Roche Innovation Center Munich, Roche Diagnostics GmbH, Nonnenwald 2, Penzberg, 82377, Germany.

|June 16, 2023

Summary

This summary is machine-generated.

High ionic strength dissociation assay (HISDA) effectively reduces target interference in anti-drug antibody (ADA) assays. This method eliminates background noise from dimeric targets, ensuring accurate results in routine drug development.

Keywords:

ELISA HISDA anti-drug antibody drug tolerance immunogenicity ligand-binding assay magnesium chloride target interference

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Area of Science:

Biopharmaceutical analysis
Immunogenicity testing
Assay development

Background:

Soluble target dimers in biological samples can cause interference in anti-drug antibody (ADA) assays.
This interference leads to elevated background values and potential false-positive results.
Accurate ADA assay development requires mitigation of such target-related interference.

Purpose of the Study:

To evaluate the efficacy of the high ionic strength dissociation assay (HISDA) in reducing target interference.
To assess HISDA's impact on two distinct ADA assay formats.
To determine if HISDA can improve the reliability of ADA assays in the presence of dimeric targets.

Main Methods:

Application of the high ionic strength dissociation assay (HISDA) to ADA assays.
Investigation of interference caused by homodimeric fibroblast activation protein (FAP).
Biochemical confirmation of homodimeric FAP dissociation under high ionic strength conditions.

Main Results:

HISDA successfully eliminated interference from homodimeric FAP.
Cut point determination was achievable after implementing HISDA.
Biochemical assays confirmed that high ionic strength dissociates homodimeric FAP.

Conclusions:

HISDA is a valuable tool for reducing interference from noncovalently bound dimeric targets in ADA assays.
The assay offers high drug tolerance while minimizing target interference.
HISDA requires minimal optimization, making it suitable for routine use in biopharmaceutical development.