High-Speed Automated Microdroplet Reactions with Ion-Mobility for Rapid Therapeutic Protein Characterization.

In the field of protein therapeutic discovery, it is crucial to characterize molecules quickly and precisely, while using minimal sample amounts. Traditional techniques often require significant amounts of samples (10-20 μg) and can introduce post-translational modification (PTM) artifacts that complicate characterization. This study presents an innovative approach using automated microdroplet-based reactions for ultrafast protein digestion and reduction in under one millisecond. The primary aim is to establish a rapid and reliable method for the characterization of protein therapeutics, essential for confirming the identity of protein molecules and assessing potential liabilities early in the discovery process. Furthermore, we have integrated ion mobility separation with microdroplet reactions to better understand the resulting mass species. Our findings demonstrate that this technique enhances the throughput of protein therapeutic characterization without compromising on accuracy. It is effective across a range of therapeutic protein formats including IgG1 antibodies, Fc-cytokine fusion proteins, and antibody-drug conjugates (ADCs). To the best of our knowledge, this is the first report to apply online microdroplet-based reactions across multiple therapeutic modalities while integrating ion mobility separation, establishing a high-throughput workflow for comprehensive protein analysis.