(T1130-05-33) Drug Tolerance Enhancement in a Ligand Binding Neutralizing Antibody (NAb) Assay using Affinity Extraction

Purpose: In immunogenicity assays, the importance of drug tolerance in neutralizing antibody assay is crucial to ensure that positive samples can be correctly identified.  The ability to detect clinically relevant antibodies in study samples without interference of the drug is key to ensuring the safety, efficacy, and accurate pharmacokinetic profiles of biotherapeutic drugs.

Methods: Serial-affinity extraction steps were implemented to remove excess drug at 37 °C after initial acid treatment.   Samples were then incubated overnight with biotinylated drug coated streptavidin magnetic beads, forming biotin-drug-neutralizing antibody complex on the beads. After an overnight incubation, the streptavidin magnetic beads were washed to rid excess drug and any unbound complexes. A second acid treatment was applied to dissociate neutralizing antibodies from complexes bound on the streptavidin magnetic beads. Subsequently, samples were neutralized with sulfo-tag-labeled drug prior to immobilization via a biotinylated target coated on a streptavidin plate. The presence of neutralizing antibodies bound to sulfo-tag-labeled drug inhibited the interaction with the biotinylated target coated on streptavidin plate, resulting in reduced electrochemiluminescence signals.

Results: Using a preliminary cut point of 0.800 signal-to-noise ratio (SNR), assay sensitivity was demonstrated to be 500 ng/mL (SNR of 0.756) with serial drug extraction steps, and 1000 ng/mL (SNR 0.688) without an extraction step. For drug tolerance assessment without affinity extraction, positive control (PC) at 8000 ng/mL and 16,000 ng/mL were able to tolerate up to 0.500 mg/mL and 1.00 mg/mL of drug, with corresponding SNRs of 0.727 and 0.755, respectively. In comparison, drug tolerance assessment with serial extraction steps, PC at 4000 ng/mL was able to tolerate up to 1.00 mg/mL of drug with an SNR of 0.771.

Conclusion: Implementation of serial affinity extraction improved drug tolerance in the neutralizing antibody ligand binding assay.  In the presence of 1.00 mg/mL of drug, sample processed with serial extraction steps maintained signal-to-noise ratio below the preliminary cut point at positive control concentrations as low as 4000 ng/mL.  In contrast, without an extraction step, the same drug concentration resulted in a signal-to-noise ratio below the preliminary cut point at higher positive control level of 16,000 ng/mL.

References: FDA. (2019). “Immunogenicity Testing Therapeutic Protein Products – Developing and Validating Assays for Anti-Drug Antibody Detection.” (January): 3-27

Acknowledgements: Copyright, 2025 by Pharmaceutical Product Development Laboratories, part of Thermo Fisher Scientific. This poster, including the information contained herein and any associated commentary is provided as a service of PPD®.