Multispecific antibodies stoichiometry at the single-molecule level

Multispecific antibodies (MAbs) are innovative biotherapeutic agents designed to target multiple antigens simultaneously, thus leveraging a specific immune response with minimised immune-related side effects. The development of MAbs is currently limited by the complex manufacturing process which leads to numerous mispaired variants that significantly reduce the production yield and increase the manufacturing cost. The traditional biophysical methods, such as mass spectrometry and chromatography, are hampered by the low throughput and insufficient resolution to effectively characterise the structural diversity of mispaired species. This project aims to address these limitations by developing and validating a high-throughput single-molecule fluorescence imaging sensor tool. The proposed method leverages single molecule fluorescence microscopy to precisely identify and quantify the mispaired species produced, aiming to optimise and accelerate the manufacturing process. The main objectives of this project include developing a single-molecule imaging workflow, devising an optimal fluorescent labelling strategy, automating the sample preparation by integrating microfluidic platforms, benchmarking the method against standard analytical techniques, and implementing the proposed method as a process analytical technology (PAT) in MAbs manufacturing. This interdisciplinary approach bridging engineering, biology and optics is expected to significantly impact both industrial and academic fields by developing a cost-effective, automated and quality-controlled system for the analysis and characterisation of mispaired species in MAbs
manufacturing. This project sets the stage for substantial advancements in the pharmaceutical industry and clinical research community by accelerating the development of these therapeutic agents and leading to societal benefits in terms of access to novel and improved therapeutics.