A single homogeneous assay for simultaneous measurement of bispecific antibody target binding

Bispecific antibodies (BsAbs) are engineered to simultaneously bind two different antigens, and offer promising clinical outcomes for various diseases. The dual binding properties of BsAbs may enable superior efficacies and/or potencies compared to standard monoclonal antibodies (mAbs) or combination mAb therapies. Characterizing BsAb binding properties is critical during biotherapeutic development, where data is leveraged to predict efficacy and potency, assess critical quality attributes and improve antibody design. Traditional single-target, single-readout approaches (e.g., ELISA) have limited usefulness for interpreting complex bispecific binding, and double the benchwork. To address these deficiencies, we developed and implemented a new dual-target/readout binding assay that accurately dissects the affinities of both BsAb binding domains directly and simultaneously. This new assay uses AlphaPlex® technology, which eliminates traditional ELISA wash steps and can be miniaturized for automated workflows. The optimized BsAb AlphaPlex assay demonstrates 99-107% accuracy within a 50-150% linear range, and detected >50% binding degradation from photo- and thermal stress conditions. To the best of our knowledge, this is the first instance of a dual-target/readout BsAb AlphaPlex assay with GMP-suitable linear range, accuracy, specificity, and stability-indicating properties. As a highly customizable and efficient assay, BsAb AlphaPlex may be applicable to numerous bispecific formats and/or co-formulations against a variety of antigens beyond the clinical therapeutic space.