Journal of Laboratory Automation

Biochemical markers of bone turnover suffer from large analytical and natural fluctuations (20-30%), making small differences in bone resorption impossible to resolve. This limits the clinical utility of such markers for individuals with the skeletal complications associated with many disease states (e.g., metastatic cancer, renal failure, osteoporosis). We are developing the capability to measure small changes (5-10%) in bone turnover rate in vivo by tagging the living skeleton with ⁴¹Ca. Among the stable and radioactive calcium isotopes, only ⁴¹Ca is useful for direct quantitation of bone turnover because it is extremely rare in nature and radiologically benign (10⁵ years half-life, pure electron capture decay). The ratio of this tracer to total calcium remains quantifiable in body fluids and excreta via accelerator mass spectrometry (AMS) for many years following a single physiological-sized oral or intravenous dose. The highly automated AMS instrumentation and streamlined sample preparation allows a single operator to prepare or run more than 100 samples per day—significantly more than other ⁴¹Ca programs worldwide. We intend to exploit these measurements for earlier diagnosis of pathological processes and interactive intervention with therapeutic agents, allowing modulation of these agents to obtain the best individual result for a patient.

Automated parallel synthesis with multivariate analysis was employed in the optimization of a Pd-catalyzed three-component cascade reaction of 7-buta-2,3-dienyl-1,3-dimethyl-3,7-dihydropurine-2,6-dione with iodobenzene and morpholine (see Scheme 1). Initially a range of reaction factors were analyzed using experimental design to optimize for selectivity, conversion, and impurity levels. Solvents and bases were screened separately as the substantial number of discrete variables involved is unsuited to the fractional factorial design used. Instead, a solvent principal component analysis (PCA) model was used in selection of solvents for screening, so gaining maximum variation in solvent properties. Similarly, tertiary amines were chosen by pKa for screening as bases alongside inorganic salts. This investigation yielded twofold results, not only in enhancement of the reaction selectivity, but since significantly dierent findings were obtained on changing the solvent, the importance of the sequence of experimentation is also emphasized.

A fully automated chip-based nanoelectrospray (nanoESI) system, NanoMate® 100 (Advion Bio-Sciences, Inc., Ithaca, NY), was evaluated for its application on quantitative bioanalysis of small molecules in support of exploratory pharmacokinetic (PK) studies. The NanoMate® 100 was compared with the conventional autosampler coupled with liquid chromatography-electrospray (LC-ESI) interface. An API® 3000 triple quadrupole mass spectrometer (Applied Biosystems, Inc., Foster City, CA) was used for the evaluation. The results show that the NanoMate® 100 performs comparably to LC-ESI in terms of standard curve fitting, low limit of quantitation (LLOQ), dynamic range, accuracy, and precision. Parallel analyses of exploratory PK study samples show high correlation (R ² = 0.971) between the NanoMate® 100 and the LC-ESI. The NanoMate® 100 exhibits advantages in carryover, sample consumption, sample cycle time, and the ability to be full automated. Despite these advantages, the necessarily rigorous sample preparation process limits the application of the NanoMate® 100 for quantitative analysis in areas such as exploratory PK studies, which often involve multiple compounds in one study and require rapid turnaround. However, the NanoMate® 100 has great potential in qualitative work (e.g., metabolite identification) as well as in high-throughput quantitative analysis of compound in the development stage (i.e., a single analyte with a well-established sample extraction method). (JALA 2004;9:109-16)

Optimizing the buffer conditions of the selection of nucleic acid binding species (aptamers), increases the likelihood of producing a target aptamer. Aptamers, with high target affinity and specificity, are often compared to antibodies, as aptamers emerge in the industry as diagnostic and therapeutic tools. The increased demand for aptamers encourages high-throughput aptamer generation. The selection buffer conditions may vary as widely as the selection targets, and therefore buffer optimization is helpful if not required for effective aptamer selections. Such optimization work is time consuming and repetitious, which bodes well for high-throughput applications. To accommodate this, an automated buffer testing protocol has been developed to test target-to-unselected RNA pool binding in the presence of 96 different buffer conditions. The dynamic program may vary the monovalent salt(s) identity, monovalent salt(s) concentration, divalent salt(s) identity, divalent salt concentration, buffer identity, buffer concentration, and pH. The optimized buffer conditions likely increase the probability of a successful selection and therefore promote higher ratios of successful aptamer selections against a variety of targets.

Preliminary results show trends with the buffer matrix solutions and lysozyme:unselected pool binding. In general, an inverse relationship between lysozyme binding and monovalent salt concentration is observed. (JALA 2004;9:117-22)

Many bottlenecks in drug discovery have been addressed with the advent of new assay and instrument technologies. However, storing and processing chemical compounds for screening remains a challenge for many drug discovery laboratories. Although automated storage and retrieval systems are commercially available for medium to large collections of chemical samples, these samples are usually stored at a central site and are not readily accessible to satellite research labs.

Drug discovery relies on the rapid testing of new chemical compounds in relevant biological assays. Therefore, newly synthesized compounds must be readily available in various formats to biologists performing screening assays. Until recently, our compounds were distributed in screw cap vials to assayists who would then manually transfer and dilute each sample in an “assay-ready” compound plate for screening. The vials would then be managed by the individuals in an ad hoc manner.

To relieve the assayist from searching for compounds and preparing their own assay-ready compound plates, a newly customized compound storage system with an ordering software application was implemented at our research facility that eliminates these bottlenecks. The system stores and retrieves compounds in 1 mL-mini-tubes or microtiter plates, facilitates compound searching by identifier or structure, orders compounds at varying concentrations in specified wells on 96- or 384-well plates, requests the addition of controls (vehicle or reference compounds), etc. The orders are automatically processed and delivered to the assayist the following day for screening. An overview of our system will demonstrate that we minimize compound waste and ensure compound integrity and availability. (JALA 2004;9:123-7)

PCR-based fluorescent detection assays for the relative and quantitative measurement of gene expression, such as Taq Man™, LUX™, and SYBR Green™, are currently in wide spread use due to their general applicability, low cost, reproducibility, accuracy, and ease of use. One current limitation of quantitative PCR (Q-PCR) is the lack of a fully integrated and high-throughput method for general genomic and diagnostic applications. Here we report a reliable and high-throughput system for the automated extraction of RNA, first-strand cDNA synthesis, quality control measures, consecutive real-time PCR amplification, and primary data analysis. As described, this procedure utilizes commonly available reagents and pre-packaged “kits” for RNA extraction, first strand cDNA synthesis, Q-PCR, liquid handling, and capillary electrophoresis that are generally applicable to a wide variety of robotic platforms. (JALA 2004;9:128-34)

Advances in automation have enabled screening methods, such as enzymatic assays and ELISAs, to be performed in a high-throughput fashion. Instruments can perform each of the steps more rapidly and with fewer errors than humans, however, in many cases, humans still perform the key steps of integrating the instruments and Laboratory Information Management Systems (LIMS). Typically, the screening process involves multiple steps for sample testing, manual data entry and design of protocols to handle the positive samples, and final processing of positives. Fully integrated systems such as Protedyne's BioCube™ System combine liquid handling, LIMS, plate washing, and plate reading in a device that is controlled via a single user interface. As an example, we present the results of automating plate reading and retesting of positive samples in a model protocol based on ELISA testing. By performing an automated initial screening of results, our system enables positive samples to be retested at higher stringency or sent to a technician for corroboration of results. The screening process then becomes a single protocol that starts with a sample set and results in a data set of tested and retested/reflux tested positives as well as re-arrayed plates of positive samples that are ready for additional downstream processing. (JALA 2004;9:135-9)

The demand for high-throughput RNA isolation has been dramatically increasing with wide applications of RNAi, expression profiling, and molecular diagnosis. A comparison of various RNA isolation methods that have been adapted to high-throughput platforms, focusing on consistently high yield and quality of isolated RNA, reduction of cross-contamination, and simplicity and robustness of the protocol is presented. The streamlining of RNA isolation with RNA quantification by qRT-PCR and amplification for microarray analysis is also discussed. In general, a microspheric bead-based approach results in more consistent RNA recovery than glass fiber filter-based RNA isolation method, and RNA can be eluted in a smaller volume. This is because beads can be fully re-suspended in solution to enable more thorough mixing, washing, and elution, whereas the glass fiber matrix is fixed in a filter plate. (JALA 2004;9:140-5)

The High Throughput Biology (HTB) department at GlaxoSmithKline is developing in vitro models to better predict the efficacy of compounds in the clinic. The development and progression of disease is often associated with characteristic changes in gene expression. These “transcriptional profiles” (mRNA gene expression patterns associated with a given disease) can be used as biomarkers to monitor disease states. Therefore, we are utilizing transcriptional profiling as a way to understand diseases and the effects of pharmaceuticals on those diseases. Transcriptional profiling has numerous advantages over other techniques including highly sensitive and highly quantitative assays, simple assay development, and accessibility of nearly the entire genome to transcriptional readouts. Our transcriptional profiling strategy involves isolation of total RNA from samples followed by real-time quantitative PCR assays (Taqman® assays). (JALA 2004;9:146-9)

The in vitro selection of aptamers that bind to low molecular weight targets is commonly a tedious, time-consuming project. We have expanded current automated selection protocols to include aptamer selections against small molecules including the aminoglycosides neomycin, kanamycin, and tobramycin. This modified procedure decreases both the frequency of manual handling of the selection reagents and the time required to perform the experiment, generating aptamers against the chosen target at a much greater rate. Using this process, we have selected aptamers of good affinity against all three aminoglycosides chosen. The method is suitable for integration with high-throughput technologies, greatly expanding the possibility of discovering useful aptamers against other low weight targets. (JALA 2004;9:150-4)

A high-throughput cell-based fluorescence screening assay was used to determine the cytotoxic activity of various cytostatic reference agents, unknown compounds of natural sources and pharmaceutic standard excipients in different cell lines such as HEK-293 and DLD-1 cells. In this paper we exemplarily show results for a selection of compounds and excipients. The cytostatic agent piposulfan has a much higher cytotoxic activity in DLD-1 cells (EC₅₀=0.001 mg/mL) than in HEK-293 cells (EC₅₀=0.3 mg/mL) in contrast to busulfan and vinblastine where the EC₅₀ values are close together in both cell lines.

From 113 tested compounds from natural sources the cytotoxic activity of 75 compounds showed no difference in both cell lines, 34 of them had a higher activity in HEK-293 cells than in DLD-1 cells and 4 compounds showed less cytotoxic activity in HEK-293 cells than in DLD-1 cells. We demonstrate the rather potent cytotoxic action of Cremophor EL; this finding is in accordance with previously published observations. The presented results demonstrate that the used assay system is reliable, enables the ranking of the cytotoxic potential of compounds of various chemical classes, and allows the determination of cell type-specific cytotoxicity. (JALA 2004;9:159-62)

The obvious utility of microarray technology is its incredibly high feature density. An entire genome can be assayed in a single experiment. However, this same strength creates difficulties. First, what does one do with the transcription data of a gene whose function(s) are unknown? Second, in most applications, hundreds—not hundreds of thousands—of genes are regulated. Why collect replicate data on thousands of genes not involved in the problem at hand? In one sense, a high-density microarray is a fishing expedition that identifies the important players. Subsequently, efforts may be focused on the complete, albeit smaller set of regulated elements.

IQ® Technology, a homogeneous, universal-detection platform, originally designed for high-throughput screening (HTS) of kinases and phosphatases, has now been applied to protease screening. Representative enzymes from the major classes of proteases have been assayed in the IQ® format. Enzyme activity and compound inhibition data are presented for such enzymes as Trypsin, Matrix Metalloproteinase 3 (MMP-3) and Calpain 1. The technology has been tested in 96- to 384- to 1536-well microplate formats and is universally suited for automated screening. IQ® Technology is a direct, noncompetitive assay that does not require antibodies or radioisotopes. Fluorophore-labeled peptides are used as enzyme substrates. Kinase or phosphatase activity is quantified by direct measurement of the phosphorylation state of the substrate. For protease activity, cleavage is quantified with a peptide substrate containing a phospho-residue distal to the fluorphore. Cleavage of the substrate liberates the fluorphore-labeled terminus from the terminus containing the phospho-residue. Protease activity is measured by the change in fluorescence intensity that occurs when a proprietary compound binds specifically to phosphoryl groups on peptides and quenches the fluorescence. Iq® Technology can be used with any peptide sequence and is insensitive to high concentrations of ATP and substrate. The IQ® Technology has been validated against a large number of detergents, organics, and other reagents found in reaction mixtures and has been optimized for HTS applications exhibiting representative Z' values of 0.7. (JALA 2004;9:171-6)

This technical paper describes the utilization of a new automated liquid handler from Beckman Coulter, Inc., the Biomek® NX Laboratory Automation Workstation, for genomic and proteomic applications. For genomic applications, methodology for plasmid DNA purification using Promega Wizard® SV 96 reagents was developed for the Biomek NX. A single plate of bacterial pellets can be processed to purified plasmid DNA without user interaction after initial setup. DNA quantity and quality were assessed by spectrophotometric analysis, restriction digestion, PCR (The PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Homan La Roche, Ltd.), and capillary sequencing. Additionally, the plasmid preparation method was used to purify plasmid DNA from bacterial clones isolated in a bacterial two-hybrid screening procedure. In this case, the system quickly and efficiently prepared clones for rapid identification of target sequences. For proteomic applications, His-tag proteins were purified from bacterial cultures in a 96-well plate format. Following purification, a Bradford assay was used to determine the quantitative yields of the His-tag protein products in each of the aliquots from the purified samples. The AD 340 Automated Labware Positioner (ALP), an integrated absorbance reader, was used for absorbance measurements in the Bradford assay. Given the placement of this ALP on the deck of the Biomek NX, the entire process of protein purification and quantitation was performed in a complete walk-away automated format. Results obtained when purifying proteins, from both uninduced and induced bacterial cultures, on the worksurface of the Biomek NX will be described. (JALA 2004;9:177-84)

The Nanostream (Pasadena, CA) Veloce system, together with 24-column Brio cartridges, offers a novel approach to micro parallel liquid chromatography (μPLC). This system allows users to achieve unprecedented throughput for standard assays while matching the performance of conventional LC instrumentation, thus enabling routine compound purity assessment and physiochemical property profiling early in the drug discovery and development process.

The Veloce system—which includes instrumentation, software, and replaceable microfluidic cartridges—incorporates pressure-driven flow to achieve chromatograms comparable to conventional high performance liquid chromatography (HPLC) instrumentation for a broad class of analytical applications while offering a dramatic increase in sample analysis capacity. The system enables parallel chromatographic separations and simultaneous, real-time UV detection. Each Nanostream Brio cartridge, made of polymeric materials, incorporates 24 columns packed with standard (C-18) stationary phase material to achieve reverse phase separations. Mixing and distribution of the mobile phase to each of the 24 columns is precisely controlled in each cartridge. The system provides an ideal platform to accelerate assessment of compound purity and physicochemical properties (i.e., log P, CHI, etc.) for a large number of compounds. In addition, the 24-fold increase in sample analysis capacity allows standard curve generation and simultaneous analysis of multiple replicates of samples in a single run. (JALA 2004;9:185-91)