Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development.

Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for labeling antibodies was evaluated and is presented in this manuscript. In addition, simple methods for evaluation and qualification of streptavidin-coated plates and overcoming soluble target interference in solution ELISA have also been investigated and highlights of these investigations are discussed. The selection of the solution ELISA format was based on availability of generic reagents, achievement of optimal drug tolerance and robust assay performance on a platform that is readily available in many laboratories. This approach removed the heavy reliance on specialized equipment sourced from a single vendor and assay conditions described here are broadly applicable to other immunogenicity assays across many biologics both during clinical development setting and in the post-marketing arena.