Collaborative robotics to enable ultra-high-throughput IR-MALDESI.

Over the last 5 years, IR-MALDESI-MS (Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry) has been demonstrated for use in a range of high-throughput biochemical and cellular assays with remarkable sample acquisition rates up to 22 Hz for a single 384-well assay plate. With such high single plate acquisition rates, the rate limiting step becomes how fast subsequent plates can be presented to the MS for analysis. To make this transfer as fast as possible while maintaining safe operation in a laboratory environment, we developed a collaborative robotic plate transfer system (CRPTS) that combines a 6-axis robot with dual plate grippers, a 7th axis conveyor stage, and a 420-plate capacity sample loading window. As a demonstration of the throughput and flexibility of CRPTS, we performed a biochemical assay that monitored the oxidation of tris(2-carboxyethyl)phosphine (TCEP) to screen for nuisance compounds. Using continuous and step motion scan profiles, we analyzed 158,799 compounds contained in 448 assay plates over the course of 12.5 h (Z-Factor=0.87) and 17.5 h (Z-factor=0.99), respectively. Extrapolating these results enables the screening of a million compounds within 6-7 working days.

In-vitro modeling of intravenous drug precipitation by the optical spatial precipitation analyzer (OSPREY).

Intravenous (IV) administration of poorly water-soluble small molecule therapeutics can lead to precipitation during mixing with blood. This can limit characterization of pharmacological and safety endpoints in preclinical models. Most often, tests of kinetic and thermodynamic solubility are used to optimize the formulation for solubility prior to infusion in animals, but these do not capture the dynamic precipitation processes that take place during in-vivo administration. To better capture the fluid dynamic processes that occur during IV administration, we developed the Optical Spatial PREcipitation analYzer (OSPREY) as a method to quantify the amount and size of compound precipitates in whole blood using a flow-through system that mimics IV administration. Here, we describe the OSPREY device and its underlying imaging processing methods. We then validate the ability to accurately segment particles according to their size using monodisperse suspensions of microspheres (diameter 50 to 425 µm). Next, we use a tool compound, ABT-737, to study the effects of compound concentration, vessel flow rate, compound infusion rate and vessel diameter on precipitation. Finally, we use the physiological diameter and flow rate of rat femoral vein and dog saphenous vein to demonstrate the potential of OSPREY to model in-vivo precipitation in a controlled, dynamic in-vitro assay.

Ultra-High-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method that is being developed for high-throughput, label-free, biochemical screening of large-scale compound libraries. Here, we report the development of an ultra-high-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well, and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 µM 1-hydroxymidazolam and 12 µM dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with a ∼30 s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115200 samples from 300 microtiter plate reads consecutively over 5.54 h with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high-throughput compound screening.

An Automated Tube Labeler for High-Throughput Purification Laboratories.

Centralized high-throughput purification laboratories routinely produce large numbers of test tubes with fractions containing the purified compounds of interest interspersed with test tubes containing fractions collected from undesired peaks. Because the next step after purification entails the removal of the solvent in a centrifugal evaporator with multiple sample positions per rotor, select test tubes must be labeled prior to dry-down to track the identity of each compound. The diversity of test tube sizes and tray configurations from different chromatography system vendors complicates this labeling task. Therefore, the development of an automated tube labeler that can accommodate a multitude of test tube and tray sizes can reduce the chances of error as well as reduce the hands-on labor required to complete this tedious but essential task. Custom hardware and software have been implemented to inform and to enable the Pick-n-Place arm of a commercially available Tecan EVO robotic system to pick up and present select tubes, filled with purified chromatography fractions from a multitude of vendor trays, to a custom label application station integrated with a commercially available Zebra label printer. Particular challenges existed with accurately positioning tubes in Agilent G1364-84544 trays onto the deck of the instrument. The resulting instrument reduces hands-on time for labeling fractions by approximately 60%.

Engineering Chemistry Innovation.

Automation of chemistry at a pharmaceutical company commonly entails bringing commercial solutions in-house, reproducing manual processes with a robot, or integrating multiple instruments to eliminate human intervention. A strategy of industrializing proven approaches, while financially justifiable, however, does not encourage innovation. On the other hand, trying to automate unproven or difficult processes may seem to be risky but can actually accelerate the adoption, modification, or rejection of novel technologies. Having chemists and engineers work together to develop automation that accelerates the development and evaluation of innovative concepts is one blueprint for delivering a competitive advantage to an organization.

Automated library synthesis of cyclopropyl boronic esters employing diazomethane in a tube-in-tube flow reactor.

The efficient synthesis of cyclopropyl boronic esters in library format using a diazomethane flow reactor has been achieved. A pivotal component of the system is a fully automated tube-in-tube reactor allowing for safe handling of hazardous diazomethane on repeated small scale and for the generation of larger quantities of product. The setup enables the repeated execution of Pd-catalyzed cyclopropanation reactions without compromising its operation over time.

An automated synthesis-purification-sample-management platform for the accelerated generation of pharmaceutical candidates.

A flexible and integrated flow-chemistry-synthesis-purification compound-generation and sample-management platform has been developed to accelerate the production of small-molecule organic-compound drug candidates in pharmaceutical research. Central to the integrated system is a Mitsubishi robot, which hands off samples throughout the process to the next station, including synthesis and purification, sample dispensing for purity and quantification analysis, dry-down, and aliquot generation.

Purification of drugs from biological fluids by counter-current chromatography.

Experiments were performed to demonstrate the potential of counter-current chromatography (CCC) for the isolation of drugs and their metabolites from biological matrices relevant to the metabolism studies of pharmaceutical research. Examples of typical drugs are spiked into biological media ex vivo to provide test samples for analysis. A mass spectrometer hyphenated to a CCC allows for the detection of small molecule drugs within the matrix through selected ion monitoring, and fraction collection can provide material for further structural elucidation by NMR.

Purification of high-throughput organic synthesis libraries by counter-current chromatography.

Experiments were performed to evaluate whether counter-current chromatography (CCC) could function as an alternative purification method to reversed-phase high-performance liquid chromatography (RP-HPLC) and normal-phase supercritical fluid chromatography (SFC). RP-HPLC and SFC are the routine methods currently used in our high-throughput purification (HTP) facility for the purification of high-throughput organic synthesis (HTOS) libraries and medicinal chemistry reaction mixtures. Pre-equilibration of the solvent mixture layers was not mandatory for effective chromatography when hexanes-ethyl acetate-methanol-water (HEMW) solvent mixtures were used. Key to the use of CCC for high-throughput applications is the ability to effectively select a solvent system appropriate to each library member. Pilot-scale CCC elution time was used to estimate a starting solvent ratio and RP-HPLC retention time was then used to adjust solvent ratios within a particular library. It was also found that dimethyl sulfoxide (DMSO) and DMSO-methanol were suitable as sample injection solvents when using the HEMW solvent systems.

Construction and validation of an automated flow hydrogenation instrument for application in high-throughput organic chemistry.

This manuscript details the construction of a fully automated flow hydrogenation apparatus for use in high-throughput organic synthesis. The instrument comprises of a Bohdan robot platform coupled with a ThalesNano H-cube hydrogenator and a series of solvent valves and pumping mechanisms. Using this instrument, we have been able to fully automate a number of key transformations that could not otherwise be conveniently undertaken in a high-throughput manner.

A self-contained 48-well fatty acid oxidation assay.

The modulation of fatty acid metabolism and especially the stimulation of fatty acid oxidation in liver or skeletal muscle are attractive therapeutic approaches for the treatment of obesity and the associated insulin resistance. However, current beta-oxidation assays are run in very low throughput, which represents an obstacle for drug discovery in this area. Here we describe results for a 48-well beta-oxidation assay using a new instrument design. A connecting chamber links two adjacent wells to form an experimental unit, in which one well contains the beta-oxidation reaction and the other captures CO(2). The experimental units are sealed from each other and from the outside to prevent release of radioactivity from the labeled substrate. CO(2) capture in this instrument is linear with time and over the relevant experimental range of substrate concentration. Cellular viability is maintained in the sealed environment, and cells show the expected responses to modulators of beta-oxidation, such as the AMP kinase activator 5-aminoimidazole carboxamide riboside. Data are presented for different lipid substrates and cell lines. The increased throughput of this procedure compared with previously described methods should facilitate the evaluation of compounds that modulate fatty acid metabolism.

High throughput electrophysiology using a fully automated, multiplexed recording system.

The drug discovery process centers around finding and optimizing novel compounds active at therapeutic targets. This process involves direct and indirect measures of how compounds affect the behavior of the target in question. The sheer number of compounds that must be tested poses problems for classes of ion channel targets for which direct functional measurements (e.g., traditional patch-clamping) are too cumbersome and indirect measurements (e.g., Ca(2+)-sensitive dyes) lack sufficient sensitivity or require unacceptable compromises. We present an optimized process for obtaining large numbers of direct electrophysiological measurements (two-electrode voltage-clamp) from Xenopus oocytes using a combination of automated oocyte handling, efficient and flexible liquid delivery, parallel operation, and powerful integrated data analysis. These improvements have had a marked impact, increasing the contribution electrophysiology makes in optimizing lead compound series and the discovery of new ones. The design of the system is detailed along with examples of data generated in support of lead optimization and discovery.