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.

双特异性抗体 (BsAb) 被设计为同时结合两种不同的抗原，并为各种疾病提供有希望的临床结果。与标准单克隆抗体 (mAb) 或联合 mAb 疗法相比，BsAb 的双重结合特性可以实现卓越的疗效和/或效力。在生物治疗药物开发过程中，表征 BsAb 结合特性至关重要，其中数据可用于预测疗效和效力、评估关键质量属性并改进抗体设计。传统的单一目标、单一读数方法（例如 ELISA）在解释复杂的双特异性结合方面的用处有限，而且工作量增加了一倍。为了弥补这些不足，我们开发并实施了一种新的双靶点/读出结合测定法，可以直接和同时准确地剖析两个 BsAb 结合域的亲和力。这种新的检测使用 AlphaPlex® 技术，该技术消除了传统的 ELISA 洗涤步骤，并且可以针对自动化工作流程进行小型化。

优化的 BsAb AlphaPlex 检测在 50-150% 的线性范围内显示出 99-107% 的准确度，并检测到光和热应力条件下>50% 的结合降解。据我们所知，这是具有 GMP 适合的线性范围、准确性、特异性和稳定性指示特性的双靶点/读出 BsAb AlphaPlex 检测的第一个实例。作为一种高度可定制且高效的检测方法，BsAb AlphaPlex 可适用于针对临床治疗领域之外的各种抗原的多种双特异性形式和/或复合制剂。