Optimizing acid type and pH for dissociation of biotin-labeled drug from streptavidin-coated bead in BEAD ADA assays

BACKGROUND/AIM: The biotin-drug extraction with acid dissociation (BEAD) assay is used to enhance drug tolerance when detecting anti-drug antibodies (ADAs) during biotherapeutic development. A critical step of it is the acid-induced elution of ADA from the biotin-labeled drug immobilized on streptavidin-coated-beads (SA-beads). Although biotin binds streptavidin extremely tight, the dissociation dynamics between the biotin-labeled drug or the entire ADA-biotin-drug complex and SA-beads remain uncharacterized under acidic conditions. This study characterizes biotin-labeled drug dissociation and develops a systematic approach to optimize BEAD. METHOD/RESULT: Acid-induced dissociation was observed in a modified ADA bridging assay and the Biolayer Interferometry (BLI) analysis. Dissociation magnitude depended on the acid type and pH, with reduced sensitivity to acid concentration and exposure time. Acetic acid at pH 2.65, lactic acid at pH 2.23, and glycine at pH 3.00 produced the greatest dissociation, whereas acids at pH 4 produced the slowest rate. From these findings, we developed an optimization workflow focusing on concentrations of biotin- and ruthenium-labeled drugs in Master-Mix, SA-bead amount, and initial biotin-labeled drug. CONCLUSION: The manuscript proposes a fit-for-purpose optimization framework and provides guidance in acid selection and reagent tuning to improve drug tolerance and assay efficiency across ADA workflows.