Assay development considerations to improve drug tolerance in direct competitive ligand binding neutralizing antibody assays, pretreatment strategies

• Published in: Journal of immunological methods Vol. 517; p. 113484
• Main Authors: Rathi, Alok, Rinker, Sherri, Niu, Hongmei, Carter, Carina, Kumar, Seema, Cowan, Kyra
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-06-2023
• Subjects: Acid dissociation; Affinity capture elution; Antibodies, Neutralizing; Biological Assay; Competitive ligand binding assay; Drug Tolerance; Dynabeads extraction; Ligands; Neutralizing anti-drug antibodies; Competitive ligand binding assay; Dynabeads extraction; Neutralizing anti-drug antibodies; Drug tolerance; Affinity capture elution; Acid dissociation
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2023.113484

Neutralizing anti-drug antibodies (ADAs) may affect safety, efficacy, and pharmacokinetic profile of a biotherapeutic drug and thus their assessment is of particular importance during immunogenicity testing. Neutralizing antibody (NAb) assays typically rely on NAbs ability to block the drug-target interaction. Higher NAb concentration and/or higher binding affinity of NAb to the drug, lowers the drug-target binding interaction. However, in the presence of high concentrations of residual circulating drug, as often seen for drugs with longer half-lives or in repeat-dose studies, NAbs may exist as drug bound complexes. In direct NAb assay formats, the NAb-drug complexes present in the sample could result in the NAb being unable to block the drug-target interaction eventually leading to a false negative response. The residual free circulating drug present in the sample may bind to the target in the NAb assay thereby competing with the drug used in the assay and inhibiting the assay signal, leading to a false positive response. For traditional ADA assays, multiple approaches involving acid treatment have been described to mitigate circulating drug interference issue. Here, we report two acid-treatment approaches that utilize the Dynabeads extraction with acid dissociation and Affinity Capture Elution (ACE) principle to improve drug tolerance in NAb assays.
•Competitive ligand binding neutralizing antibody assays suffers from poor drug tolerance.•Neutralizing antibodies (NAb) are detected by their ability to block the drug-target binding interaction.•Circulating drug in patient samples could result in false positive or false negative assay responses.•Acid treatment methods like Dynabeads extraction and affinity capture elution can help improve drug tolerance in NAb assays.