Automated Real Time High-Speed Microdroplet Reactions to Increase the Throughput of Therapeutic Protein Characterization

Background/Motivation: Microdroplet reactions, known for their ability to accelerate reactions significantly (e.g., achieving ultrafast protein digestion in microdroplets in less than 1 ms), have garnered considerable interest. This study integrates a microdroplet protein reduction/digestion technique with automated sample flow injection and online mass spectrometry (MS) analysis. The goal is to establish a rapid and robust method for the structural characterization of protein therapeutics, crucial for confirming identity and assessing the liabilities of a protein therapeutic candidate. Problem Statement: In the early discovery stage of protein therapeutics, there is a pressing need for swift and robust characterization of molecules with minimal sample amounts. Conventional sample processing methods require higher amount (10-20 µg) of sample and have been shown to induce post-translational modification (PTM) artifacts. Automated microdroplet-based reactions present a potential solution for the rapid screening of these therapeutic candidates. Conclusions/implications: This work presents the first instance of combining microdroplet-based reactions with protein therapeutics beyond monoclonal antibodies. The entire analysis workflow, encompassing the reaction and MS analysis, takes only 3 minutes. Given the technique's applicability to various protein therapeutic modalities, it emerges as a valuable tool for the rapid and robust characterization of these molecules.