Screening Clones for Monoclonal Antibody Production Using Droplet Microfluidics Interfaced to Electrospray Ionization Mass Spectrometry.

As one of the most critical steps in process development for protein therapeutics, clone selection and cell culture optimization require a large number of samples to be screened for high titer and desirable molecular profiles. Typical analytical techniques, such as chromatographic approaches, often take minutes per sample which are inefficient for large-scale screenings. Droplet microfluidics coupled to mass spectrometry (MS) represents an attractive approach due to its low volume requirements, high-throughput capabilities, label-free nature, and ability to handle complex mixtures. In this work, we coupled a modified protein cleanup protocol with a droplet-MS workflow for mAb titer screening to guide clone selection. With this droplet approach we achieved a throughput of 0.04 samples/s with an LoD of 0.15 mg/mL and an LoQ of 0.45 mg/mL. To test its performance in a real-world setting, this workflow was applied to a 35-clone screen, where the top 20% producing clones were identified. In addition, we coupled our sample cleanup protocol to a high-resolution MS and compared the glycan profiles of the high titer clones. This work demonstrates that droplet-MS provides a rapid way of clone screening and cell culture optimization based on titer and molecular structure of the expressed proteins. Future work is aimed at increasing the throughput and automation of this droplet-MS technique.

Ion Mobility-Mass Spectrometry Coupled to Droplet Microfluidics for Rapid Protein Structure Analysis and Drug Discovery.

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; however, larger scale screens have not been implemented due to limitations associated with throughput and sample introduction. 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 using 40 nL droplet sample volumes and 2 s dwell times at each collision voltage. This workflow, which increased throughput by ∼16-fold over conventional nanospray CIU methods, was applied to a 96-compound screen against Sirtuin-5, a protein target of clinical interest. 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.

A microfluidic chip for on-line derivatization and application to in vivo neurochemical monitoring.

Microfluidic chips can perform a broad range of automated fluid manipulation operations for chemical analysis including on-line reactions. Derivatization reactions carried out on-chip reduce manual sample preparation and improve experimental throughput. In this work we develop a chip for on-line benzoyl chloride derivatization coupled to microdialysis, an in vivo sampling technique. Benzoyl chloride derivatization is useful for the analysis of small molecule neurochemicals in complex biological matrices using HPLC-MS/MS. The addition of one or more benzoyl groups to small, polar compounds containing amines, phenols, thiols, and certain alcohols improves reversed phase chromatographic retention, electrospray ionization efficiency, and analyte stability. The current derivatization protocol requires a three-step manual sample preparation, which ultimately limits the utility of this method for rapid sample collection and large sample sets. A glass microfluidic chip was developed for derivatizing microdialysis fractions on-line as they exit the probe for collection and off-line analysis with HPLC-MS/MS. Calibration curves for 21 neurochemicals prepared using the on-chip method showed linearity (R2 > 0.99), limits of detection (0.1-500 nM), and peak area RSDs (4-14%) comparable to manual derivatization. Method temporal resolution was investigated both in vitro and in vivo showing rapid rise times for all analytes, which was limited by fraction length (3 min) rather than the device. The platform was applied to basal measurements in the striatum of awake rats where 19 of 21 neurochemicals were above the limit of detection. For a typical 2 h study, a minimum of 120 pipetting steps are eliminated per animal. Such a device provides a useful tool for the analysis of small molecules in biological matrices which may extend beyond microdialysis to other sampling techniques.

Droplet sample introduction to microchip gel and zone electrophoresis for rapid analysis of protein-protein complexes and enzymatic reactions.

Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by microchip electrophoresis. Here, microchip gel electrophoresis separations of proteins are achieved in 2.6 s with 1200 V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to microchip electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for microchip gel electrophoresis and free-solution electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10 s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device. Graphical abstract.

Advances in capillary electrophoresis and the implications for drug discovery.

INTRODUCTION: Many screening platforms are prone to assay interferences that can be avoided by directly measuring the target or enzymatic product. Capillary electrophoresis (CE) and microchip electrophoresis (MCE) have been applied in a variety of formats to drug discovery. CE provides direct detection of the product allowing for the identification of some forms of assay interference. The high efficiency, rapid separations, and low volume requirements make CE amenable to drug discovery. Areas covered: This article describes advances in capillary electrophoresis throughput, sample introduction, and target assays as they pertain to drug discovery and screening. Instrumental advances discussed include integrated droplet microfluidics platforms and multiplexed arrays. Applications of CE to assays of diverse drug discovery targets, including enzymes and affinity interactions are also described. Expert opinion: Current screening with CE does not fully take advantage of the throughputs or low sample volumes possible with CE and is most suitable as a secondary screening method or for screens that are inaccessible with more common platforms. With further development, droplet microfluidics coupled to MCE could take advantage of the low sample requirements by performing assays on the nanoliter scale at high throughput.