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.
Highlights
Today the evaluation of unwanted immunogenicity is a key component in the clinical safety evaluation of new biotherapeutic drugs and macromolecular delivery strategies
The evaluation of unwanted immunogenicity is an integral component in the overall clinical safety assessment of new candidate biotherapeutic drugs and macromolecular delivery s trategies[1,2,3,4]
For multiple reasons, including appreciable homology with unrelated serum immunoglobulins, anti-drug antibodies (ADA) testing of monoclonal antibodies (mAbs) relies heavily on the ‘bridging’ assay d esign[5,8,9,22]. This assay design takes advantage of paratope-specific interactions between a bivalent serum ADA and labeled versions of the monoclonal antibody therapeutic to form a bridging complex in which the resultant signal response is proportional to the concentration of ADA in the serum test sample
Summary
Today the evaluation of unwanted immunogenicity is a key component in the clinical safety evaluation of new biotherapeutic drugs and macromolecular delivery strategies. For multiple reasons, including appreciable homology with unrelated serum immunoglobulins, ADA testing of mAbs relies heavily on the ‘bridging’ assay d esign[5,8,9,22] This assay design takes advantage of paratope-specific interactions between a bivalent serum ADA and labeled versions of the monoclonal antibody therapeutic (i.e., often a biotinylated version for solid-phase capture reagent and a different labeled form for detection) to form a bridging complex in which the resultant signal response is proportional to the concentration of ADA in the serum test sample. Notwithstanding the commonly encountered technical challenges of drug tolerance[22,23] and/or target interference[24], bridging assays possess numerous attributes that make them appealing for use in ADA detection These include low nonspecific background, good assay sensitivity, indifference to detection of surrogate antibodies from different mammalian species, efficient detection of immunoglobulin isotypes and operational ease for high throughput[13,14,15]. Since both assay designs are highly drug tolerant, they have gained popularity for detecting ADA to mAb therapeutics that have a long terminal elimination half-life
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