Advances in early stage compound screening and the impact of high throughput, microfluidic mass spectrometry
More predictive, biologically-relevant high throughput screening techniques are necessary to improve early stage compound screening. Rejecting compounds early in development based on actionable, high quality safety and efficacy data from assays relevant to the target disease and human physiology will bring substantial benefits by avoiding the much higher expense of failure down the line in human clinical trials.
Mass spectrometry may be used to detect given analytes in a complex mixture based on their mass-to-charge ratio (m/z) with excellent selectivity and sensitivity, making mass spectrometry particularly relevant for trace-level quantitation of unmodified biochemical entities. A wide variety of chemistries, including those targets previously deemed intractable with conventional HTS technologies, may be detected using MS. Rapid assay development can be accomplished with MS, for neither surrogate substrates nor indirect detection approaches (optical, isotopic or antibody-based) are needed. Mass spectrometry may be used to quantify substrate and product directly, providing a percent conversion.
Mass spectrometry is also insensitive to many of the test-compound-specific interferences or artifacts that influence other assays (eg, auto-fluorescence or amended reactivity in the case of optical detection of fluorescent reporter groups, or crossreactivity in a coupled or antibody- based assay format). Nonetheless, mass spectrometry also presents several shortcomings. Mass spectrometers have a high capital and operational cost limiting the scalability of the platform.
Additionally, MS requires extensive upstream sample preparation and purification, usually through some form of chromatography, to isolate the analytes of interest from the confounding components in the assay, ultimately limiting the throughput of this platform.
The inherent advantages provided by MS-based analysis have led to a search for solutions to the problem of limited throughput. Ultra performance liquid chromatography (UPLC) systems such as the Waters Acquity® system use small micron-size beads to attain better separation with faster run times and lower system volumes.
Turbulent flow® chromatography from ThermoFisher Scientific permits higher flow velocities. Another approach is to use multiple liquid chromatography systems interfaced to a single MS. This amortises the cost of the latter while compensating for the relative slowness of the former. The commercially available Gilson-Waters multiplexed parallel sample acquisition interface (MUX) further epitomises these efforts. In this approach, a single MS is equipped with up to eight indexed electrospray ionisation sprayers.
The FlashQuant™ system from Life Technologies couples a MALDI ionisation source with a triple quadrupole mass spectrometer for the MS analysis of large numbers of samples that have been prepared offline by parallel solid phase extraction techniques.
Miniaturized, quantitative assays implemented in high throughput hold the promise to impact these important areas of need. Nanofluidic quantitative PCR moves the superior analytical properties of the PCR assay into high throughput screening by enabling the capability of quantitatively measuring gene expression in multiple genes in multiple specimens simultaneously with the potential of providing better quality information by biologically validating a target’s role in disease pathophysiology.
The dual concepts of miniaturization and quantification is further extended to screening functions in the discovery and development process where the goal is to generate safe and efficacious compounds for subsequent testing in humans. Introduction of high throughput mass spectrometry methods in the initial screening phase and following in vitro ADME/Tox testing phase allows testing of putative drug compounds with assays of greater biological relevance to the disease pathology combined with information on compound safety earlier in development.
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Journal of Chromatography & Separation Techniques