Analytical Chemistry

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The Analytical Chemistry team has continued to perform the core responsibility of purifying samples with material in the range of milligrams to grams. Major and minor components (< 0.1 percent) have been isolated for additional testing and characterization. The teams automated sample processing protocol allows for dispensing into 1D barcoded vials, Matrix 2D barcoded tubes and/or 96-well plates for efficient tracking, storing, and testing. The entire process from receiving the sample to final plating is completed within one week. While the group uses a variety of liquid chromatographs to determine identity and purity, single quadrupole liquid chromatography/mass spectrometry instrumentation is utilized for high-throughput automated analysis. Due to the wide variety of analytes tested, the teams range of analytical detectors includes ultraviolet (UV), mass spectrometry (MS; positive and negative mode), and evaporative light scattering detector and fluorescence (ELSD). Time-of-flight mass spectrometry (TOF-MS) is employed to achieve formula confirmation and identity determination of unknowns. The chemical and enantiomeric purity of chiral compounds is routinely determined within the groups full-scale chiral laboratory. Methods development with the chiral chromatography screening protocol involves the utilization of various chiral stationary phases in conjunction with multiple mobile phase conditions. The use of an inline chiral detector allows for the determination of relative optical rotation. Sample purification on a scale of up to hundreds of milligrams is possible. We have expanded our chiral purification capabilities through the addition of two new semi-preparative chiral columns. These columns contain chiral stationary phases unique from our other columns enabling a wider range of separation methods and conditions. Our vibrational circular dichroism (VCD) spectrometry platform continues to be developed to determine the absolute stereochemistry for the ever increasing number of chiral compounds being synthesized at NCATS. Progress is being made in the deployment of a conformational analysis software onto the NCATS high-performance computing (HPC) cluster. This will greatly reduce the computing time needed to generate predicted VCD spectra for comparison with experimental results. Additionally, this software can be applied to the computer-assisted 3D structure elucidation (CASE-3D) platform we are developing, which will utilize isotropic and anisotropic NMR data. The expansion of DPIs focus beyond small molecules to alternate therapeutic modalities necessitates constant advancement in our NMR and MS analytical capabilities through the acquisition and incorporation of more advanced instrumentation and techniques in order to detect, identify, quantify, and validate these new chemical entities. For NMR spectroscopy, we continued the implementation of routine methods for the analysis of peptides. Ligand-protein binding experiments were utilized to investigate protein-protein interactions (PPIs) for various disease states to aid in therapeutic development. Explorations into the use of Mestrelabs Stereofitter software were initiated to determine the 3D conformation of chiral small molecules. For mass spectrometry, we acquired a field asymmetric ion mobility spectrometry (FAIMS) Pro module for installation on our Thermo Fisher Orbitrap Fusion Lumos MS system, which will be extremely beneficial to proteomics analysis. The FAIMS Pro interface is an ion separation technique based on differences in an ions mobility between two electrodes at varying electric fields. This technique lets selected ions pass from the ion source to the mass spectrometer improved selectivity (greater signal-to-noise ratio), improved detection limits, and increased throughput. These improvements not only minimize the cell counts require per sample but will enable the discovery of low quantity proteins within the cells. Another advancement to our proteomics program was the development of a universal 384-well proteomic sample preparation platform for high-throughput screening and biomarker discovery. The workflow utilizes an Agilent Bravo liquid handler to automate the majority of process steps leading to greater efficiency, higher throughput, and reduced costs. The proteomics sample preparation platform was so successful that we are in the process of acquiring another Agilent Bravo system to meet research demands. Our work on antibody-drug conjugate (ADC) screening project continues to expand and we are currently purchasing an high performance mass spectrometer that is designed for intact protein analysis in the native state to accommodate these growing needs. We completed the installation of a second Agilent RapidFire-QQQ high-throughput mass spectrometry system. This allows us to utilize one RapidFire system to methods development and small-scale screens (<10,000 samples) while the other system is a full production unit dedicated to large-scale biological assays. The Sample Management and Resource Tracking (SMART) laboratory information management system (LIMS), in conjunction with our centralized purification platform is utilized at DPI for sample submission, compound purification, compound processing, sample registration, compound inventory, sample tracking, data retrieval, and data management. This semi-automated workflow is continuously being modified and expanded based on changing scientific needs. We are in the process of replacing our aging Sirius MultiTasker liquid handling system with a more advanced Sirius OmniTasker platform. In addition to greater automated capabilities, the OmniTasker was designed to hold two process batches of samples compared to the current single batch improving efficiency and productivity. Furthermore, we have acquired a Sirius MicroTasker customized for preparing samples within NMR tubes that will provide a time savings to the medicinal chemists. In association with the A Specialized Platform for Innovative Research Exploration (ASPIRE) program, Analytical Chemistry has continued development toward a fully automated purification system. We have been focused on evaluating various evaporative technologies for the removal of solvent in less time than the 20 hours currently required for lyophilization. Additionally, we have acquired a Biosero automated mobile robot to transfer sample racks and tubes between physically separated instruments and equipment within the lab that are part of an automated workflow. The DPI initiative of supplanting our current outdated registration system with GSRS, a chemical registration and management software developed at NCATS and currently utilized by the FDA, spearheaded by Informatics, Analytical Chemistry, and Compound Management has made significant progress. We have also implemented the processes and protocols we developed for the purification and management of DEA controlled substances as part of NCATSs involvement in the NIH HEAL Initiative The NCATS Scientific Ordering System (SOS), the primary system for the requisition, organization, and management of all orders at DPI, has seen some significant upgrades and increased functionality. Continued expansion of functionality, as well as development of direct interfacing with POTS, will allow SOS to expand its usage to all NCATS to better manage spending on a project-by-project basis Direct connection of SOS with POTS through an application programming interface (API) improves efficiency and expediency of order submission for both order managers and purchasing agents. Development and implementation of a controlled substance database and resolver allows for tracking of DEA controlled substances through the SOS ordering and delivery process. Many of the added features were to improve usability and facilitate greater ease of ordering, which include bu