End-To-End\nAutomated Intact Protein Mass Spectrometry\nfor High-Throughput Screening and Characterization of Bispecific and\nMultispecific Antibodies

Bispecific antibodies\n(bsAbs) and multispecific antibodies\n(msAbs)\nrepresent a promising frontier in therapeutic antibody development,\noffering unique capabilities not achievable with traditional monoclonal\nantibodies. Despite their potential, significant challenges remain\ndue to their increased molecular complexity. One prominent challenge\nis the correct assembly of light and heavy chains, as improper pairing\nleads to mispaired or incompletely assembled species that lack therapeutic\nefficacy and possess undesired properties, impairing the developability,\nmanufacturability, and safety. There is a critical need for rapid,\nsensitive analytical tools to monitor and control these undesired\nspecies and ensure the quality assessment of bsAbs and msAbs. To address\nthis need, we present a novel high-throughput, format-agnostic intact\nmass workflow that significantly enhances the efficiency of detecting\nand quantifying biotherapeutic products and related impurities. This\nworkflow integrates automated sample preparation, novel high-resolution\nrapid mass detection powered by SampleStream-MS, and an advanced data\nanalysis pipeline. It offers increased throughput and data quality\nwhile substantially reducing analysis turnover time and labor. This\nwas demonstrated in a pilot program where ∼800 multispecific\nantibodies were processed in 10 working days. The article details\nthe evaluation and validation of our method, demonstrating its repeatability\nand intermediate precision in terms of measurement accuracy and relative\nquantification of various product-related species. We underscore the\ntransformative potential of this end-to-end high-throughput workflow\nin expediting bispecific and multispecific antibody discovery, optimizing\nproduction processes, and ensuring high-quality development and manufacturing\nfor therapeutic antibodies.