Journal of Laboratory Automation

Successful high-throughput screening requires methods for determining the precision of liquid dispensing. We compared absorbance and fluorescence methods. We found that plate-to-plate variability was large enough to mask the difference in optical methods. When a dual-dye method was used, we found that absorbance and fluorescence gave similar results in 96- and 384-well plates. In 1536-well plates, the two methods gave differing results, but centrifugation reduced the difference, suggesting that entrapped air bubbles or unsettled meniscus shapes artificially raised the coefficients of variation of fluorescence reads.

Multiparallel and combinatorial syntheses have enabled chemists to produce a vast number of chemical compounds for hit- and lead-finding campaigns. For high-throughput screening (HTS), these compound collections are typically dissolved in dimethylsulfoxid (DMSO) and stored at temperatures ranging from —80 °C to room temperature (RT). Having compounds readily available as DMSO solutions greatly facilitates HTS. However, there are a number of stability and solubility issues associated with compounds stored as DMSO solutions. ^{1 –8} To ensure compound integrity for a long period of time, we have developed a simple dry compound storage concept called DotFoil, from where compounds can be redissolved in a fast, reliable, and easy way and directly used in conventional 96- or 384-well plate based HTS (1536-well format is not supported at this time). Our results indicate that compounds are more stable if stored as dry film on DotFoils, compared to storage as DMSO solutions at +4 °C or RT. Redissolving the dry film of very apolar compounds like triphenylamine, even with a very small extraction volume of 2 μL DMSO, allows > 80% of the total amount of compound to be recovered in solution using the current prototype equipment. Cross contamination between individual wells during the process of redissolving compounds was negligible. Composition of our prototype equipment, procedure, and equipment for the extraction step to redissolve the dry film are described.

Protein stability is a standard metric to study the relationship between protein sequence and stability. Here we show that laboratory automation can be used to increase both the precision and the throughput of stability measurements. A laboratory automation workstation was used to purify his-tagged eglin c proteins in 96-well format and to prepare protein solutions for solvent denaturation on a semiautomated titrating fluorometer. Using this setup, we have attained a throughput of about 20 stability measurements per day. The robotics facilitate precise stability measurements in which the standard deviation of values from multiple protein preparations (±0.05 kcal/mol) differs little from multiple measurements from a single preparation (±0.04 kcal/mol), while the measurement errors in the literature ranges from 0.10-0.60 kcal/mol. Our approach can be applied to evaluate the consequences of mutation in any fast-folding protein. Large numbers of high-precision stability values derived from mutation are useful for parameterizing models about protein stability determinants and testing models for predicting protein stability and structure based on protein sequence information.

Despite advances in laboratory automation, many sample handling tasks remain a manual process. SHOW (Sample Handling Operation Wizard) is a computerized system developed to guide manual sample handling. It consists of an illumination station equipped with a flat panel computer monitor, a sample tray holding up to four microtiter plates and up to eight vials, and a computer installed with custom software. The sample tray, loaded with plates and vials, is placed on the illumination station, which displays images of wells and vials directly below. Plate format can be set by the user to fit 24-, 48-, 96-, or 384-well plates. By illuminating the wells or vials involved in a sample handling step of a predefined protocol, manual operations can be performed with precise guidance from the system, and sample properties or experimental data can be recorded by clicking on the corresponding well images. As a result, the risk of locating a wrong sample or placing a sample at a wrong location is minimized, and sample handling operations become more efficient and less stressful.

Fully automating laboratory processes is a nontrivial task made more difficult by a lack of standardization between components. Each device in the system requires custom interfaces and translators to permit sharing of data and remote control by a central protocol. The BioCube System from Protedyne features the LILY data management software that enables communication between external databases, the robot controller, and integrated third-party devices. Data is formatted and passed internally as XML files to provide a generic interface for all the components. The resulting system allows the process to begin with the input of samples, and finish with the automatic export of data to a variety of data management software.

Pharmaceutical profiling is the characterization of the physicochemical and metabolic properties of compounds during drug discovery. The data alerts project teams to compound liabilities, helps to diagnose complex in vivo processes such as bioavailability, helps to better plan and interpret discovery experiments, and provides feedback for synthetic chemical modifications to the structure that are designed to improve properties. Automation plays an important role in obtaining diverse sets of property data for thousands of compounds per year with short turnaround times that match discovery workflow. Key elements of automation in profiling include liquid handling, high capacity detection, and data processing and reporting. Key information is obtained from high throughput assays for integrity, permeability, solubility, stability, cytochrome P450 inhibition, lipophilicity, and pKa. Automated assays for these properties are discussed.

The merger between Astra AB (Sweden) and Zeneca Group PLC (U.K.) to form AstraZeneca in April 1999 placed considerable demands on the compound management capabilities within the merged company. A strategy was developed and implemented to ensure that the compound management infrastructure fully underpinned current and future drug discovery processes.

This overview of compound management within AstraZeneca includes the premerger capabilities, the postmerger activities, and key elements of the AstraZeneca strategy that led to the new fully operational and automated compound management facility at Alderley Park.

The tools in place will allow the technologies to change in the various downstream activities with sufficient flexibility to adapt to new demands. New screening strategies are now enabled, whether materials are chosen as selected sets, by rational theories, better use of the big screening capability, or to support difficult targets and assays; the choice is there for the discovery scientists to now exploit.