Abstract
Today the evaluation of unwanted immunogenicity is a key component in the clinical safety evaluation of new biotherapeutic drugs and macromolecular delivery strategies. However, the evolving structural complexity in contemporary biotherapeutics creates a need for on-going innovation in assay designs for reliable detection of anti-drug antibodies, especially for biotherapeutics that may not be well-suited for testing by a bridging assay. We, therefore, initiated systematic optimization of the direct binding assay to adapt it for routine use in regulatory-compliant assays of serum anti-drug antibodies. Accordingly, we first prepared a SULFO-TAG labeled conjugate of recombinant Protein-A/G to create a sensitive electrochemiluminescent secondary detection reagent with broad reactivity to antibodies across many species. Secondly, we evaluated candidate blocker-diluents to identify ones producing the highest signal-to-noise response ratios. Lastly, we introduced use of the ratio of signal responses in biotherapeutic-coated and uncoated wells as a data transformation strategy to identify biological outliers. This alternative data normalization approach improved normality, reduced skewness, and facilitated application of a parametric screening cut point. We believe the optimized direct binding assay design employing SULFO-TAG labeled Protein-A/G represents a useful analytical design for detecting serum ADA to biotherapeutics that lack an immunoglobulin Fc domain.