Abstract
Immunogenicity testing to detect and characterize anti-drug antibody (ADA) is required for almost all biotherapeutics. Monoclonal antibody biotherapeutics usually have long half-lives and for high-dose indications such as oncology, high level of drug will be present in the testing samples and interfere with ADA and/or neutralization antibody (NAb) measurement. To overcome this drug interference, acid-dissociation-based sample pre-treatment such as Bead-Extraction and Acid Dissociation (BEAD) has been successfully applied. The main concern for these acid-dissociation-based methods, however, is that harsh acid treatment could denature positive control Abs as well as NAb species in testing samples. In addition, high amount of biotinylated drug is needed in order to have effective competition with high level of drug in the samples, which in turn requires expensive magnetic beads. And the whole process of magnetic beads handling is tedious if doing manually and often causes trouble during assay transfer. Here, we describe a novel method which we named as Precipitation, Acid Dissociation and Biotin-drug as Assay Drug (PABAD). This novel method will need only one step of acid dissociation, with much milder and shorter acid treatment to maximally preserve NAb activity. In addition, only a fraction of biotinylated-drug is needed and there is no need to use additional streptavidin (SA)-plate or SA-magnetic beads for extraction. Compared to a BEAD-based assay, PABAD demonstrates significantly improved recovery of acid-sensitive NAb positive controls (PCs) and similar recovery of acid-resistant NAb PCs.
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Funding and all the materials that are not commercially available for this research were provided by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.
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Conception, design, or planning of the study: DW, WX, and RH. Acquisition of the data: DW, JW, and TW.
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DW, JW, TW, FF, TS, RH, and WX are current employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and may own stock and/or stock options in Merck & Co., Inc., Rahway, NJ, USA.
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Wickramarachchi, D., Wagner, J., Woo, T. et al. A Novel Neutralization Antibody Assay Method to Overcome Drug Interference with Better Compatibility with Acid-Sensitive Neutralizing Antibodies. AAPS J 25, 18 (2023). https://doi.org/10.1208/s12248-023-00783-9
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DOI: https://doi.org/10.1208/s12248-023-00783-9