09/04/2026

ADA for a small recombinant protein: development and troubleshooting

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The immunogenicity of biological therapeutics is one of the crucial criteria taken into account when selecting candidates to undergo clinical trials, as biotherapeutics may induce an unwanted immune response, increasing safety risks for the patients and reducing efficacy. As a result, robust and reliable methods to detect anti-drug antibodies (ADAs) are essential for an accurate immunogenicity assessment.

ADA assay development is technically complex and often requires iterative optimisation to overcome challenges such as drug interference, matrix effects and non-specific binding. The aim of this poster, presented at the 12th EBF Young Scientist Symposium 2026 (please see below for reference), is to describe the troubleshooting process followed during the development of an assay for the detection of ADA against a recombinant protein of 24kDa. During method development and due to the nature of the drug, several challenges were identified, including high background signal, insufficient drug tolerance, low sensitivity and low drug stability. High interference was caused by non-specific binding as well as interaction between labelled reagents. Additionally, interference was increased, probably due to the low structural stability of the drug, which would make epitope recognition challenging and result in high background and low sensitivity.

Optimisation strategies included the adjustment of capture and detection steps, reagent and washer protocols assessment, optimisation of acid dissociation and neutralisation conditions and implementation of alternative procedures (Standard Bridging Assay vs Affinity Capture Elution-AGL immunoassay).

Overall, the implementation of optimised assay conditions significantly reduced background signal and improved assay sensitivity, as well as achieved an optimal drug tolerance.