Droplet Microfluidics Coupled to Capillary Electrophoresis and Mass Spectrometry for Pharmacological Applications

Abstract

Modern drug discovery relies on high-throughput screening for lead generation and protein target evaluation. There is a critical need to develop assays that not only have large-scale screening capabilities but provide insight of greater physiological relevance. It is in this space that biophysical methods are used to extract high information content datasets; however, throughput is often limited by sample introduction to the instrument. Droplet microfluidics represents an attractive approach for coupling high-throughput sampling with analytical and biophysical approaches. The aim of this thesis is to improve high-throughput workflows coupled to to capillary electrophoresis and ion mobility-mass spectrometry for the analysis of enzymatic activity and intact protein analysis. Optical assays are a fast and simple approach for screening small molecule activity against enzymatic reactions but can be prone to high false discovery rates. A capillary electrophoresis method was developed to screen against Sirtiuin-5 activity, where substrate and product peptides were separated and detected. Using a commercial capillary electrophoresis system, eight novel Sirtuin-5 inhibitors were confirmed through dose-response analysis. This assay was then coupled to a microchip capillary electrophoresis platform coupled to droplet sample introduction via an density-based oil drain. A sampling rate of 0.1 Hz, using only 4 nL sample volumes. This was applied to screen over 160 Sirtuin-5 samples. Future work is focused on developing a label-free mass spectrometry assay with on-line reagent addition. Native mass spectrometry coupled to ion mobility (IM-MS) has become an important tool for the investigation of protein structure and dynamics upon ligand binding. Additionally, collisional activation or collision induced unfolding (CIU) can further probe conformational changes induced by ligand binding. In this work we explore the high-throughput capabilities of CIU fingerprinting. Fingerprint collection times were reduced 10-fold over traditional data collections through the use of improved smoothing and interpolation algorithms. Fast-CIU was then coupled to a droplet sample introduction approach and applied to a 96-compound screen against Sirtuin-5. Over 20 novel Sirtuin-5 binders were identified, and it was found that Sirtuin-5 inhibitors will stabilize specific Sirtuin-5 gas-phase conformations. This work demonstrates that droplet-CIU can be implemented as a high-throughput biophysical characterization approach. Future work will focus on improving the throughput of this workflow and on automating data acquisition and analysis. Monoclonal antibodies (mAbs) are an important class of biotherapeutics, but the diversity and structural complexity of mAbs create an analytical challenge. Moreover, mAbs are produced through recombinant DNA technology and grown in mammalian cells, giving rise to complex background matrices and possible post-translational modification (PTM). A miniaturized MS-friendly mAb purification is developed and couped to droplet-MS. In-droplet calibrations were performed and an ultimate LOD of 0.15 mg/mL and LOQ of 2.6 mg/mL were achieved. This method was then applied to screen mAb production in 48 cell expression conditions with a rate of 0.04 Hz, identifying the most productive media conditions. This work was complemented by high resolution MS for PTM profiling, identifying a range of glycoforms on the intact mAb species. Future goals are aimed at coupling on-line sample preparation and droplet sample introduction to high resolution MS for high-throughput glycoform characterization.