Automation of Anti-Drug Antibody Enrichment Using Streptavidin PhyTip® Columns for Sample Pretreatment in an Immunogenicity Assay.

Abstract

The administration of biotherapeutics has the potential to induce potent immune responses, including the induction of anti-drug antibodies (ADA) (1). Detection and characterization of ADA in clinical trials is an important component of a comprehensive immunogenicity program (2). ADA testing is mostly accomplished with ligand-binding assays (LBA). While LBA can be robust and specific, they are susceptible to various sources of interference (3). Of particular concern, interference from residual drug present in patient samples may lead to loss of ADA detection, producing false negative results. To mitigate drug interference, sample pretreatment procedures have been developed to enrich ADA from samples with high drug concentrations (4, 5). The pretreatment procedures can vary considerably but typically involve an initial step to liberate ADA from ADA-drug complexes and a subsequent step to affinity capture ADA and acid elute for detection. While these procedures can be highly effective, they have certain drawbacks when performed manually. First, the procedures are time-consuming and require near constant attention. Second, because of the manual workflow, the procedures can produce variable results between experiments and between different analysts with negative consequences for assay robustness. To address these drawbacks, we developed an automated procedure to enrich ADA by leveraging dual flow chromatography (DFC) and Streptavidin PhyTip® columns on a liquid-handling system (6). The enriched ADA were then tested in an LBA to evaluate the effectiveness of the automated enrichment procedure.