Acoustic Droplet Ejection High-Resolution Mass Spectrometry Enables High-Throughput Analysis of Proteins in Complex Biologic and Vaccine Matrices

Abstract Accelerating the discovery and development of life-saving therapeutics and vaccines requires a commensurate level of analytical innovations, especially high-throughput (HT) methodologies for simultaneous multicomponent analyses traditionally used in fast-paced laboratory settings. HT screening is a critical step to enable lead candidate design, selection, and formulation at aggressive timelines. Current methods used for this purpose, such as liquid chromatography-mass spectrometry (LC-MS)-based approaches, are intrinsically low-throughput. Such assays are less meaningful in settings where processing thousands of samples is required to guide critical decisions quickly. Acoustic droplet ejection-open-port interface-mass spectrometry (ADE-OPI-MS) is a cutting-edge technology that eliminates the need for LC by introducing nanoliter-scale samples to an MS via acoustic ejection at speeds of <1 s per sample while minimizing matrix effects and data variability compared to more established HT sample introduction methods. Previously, ADE-OPI-MS has been demonstrated for purified protein standards in simple matrices, but its use on proteins within complex matrices remains underexplored. Biopharmaceutical samples often contain salts, buffers, and other components that make sample detection challenging without some form of sample cleanup. Herein, an integrated workflow combining sample preparation, detection, and quantification for ADE-OPI-MS is introduced, streamlining relative quantification of products spiked into complex matrices. Using this analytical framework, effective quantification of myoglobin, virus-like particle L1 protein, and monoclonal antibodies in complex cell lysate and drug process matrices is straightforward while also minimizing manual labor with automatable sample preparation capable of analyzing a full 384-well plate in under 12.5 min.