Automation Highlights from Literature

AUTOMATED ENZYME SCREENING METHODS FOR THE PREPARATION OF ENANTIOPURE PHARMACEUTICAL INTERMEDIATES

The last two decades, biocatalysts have been used increasingly in the preparation of drug intermediates and metabolites. In the synthesis of chiral compounds, enzymes have emerged as attractive alternatives to conventional chemical methods. The use of this technology, however, is still limited partly due to the lack of an efficient and practical high-throughput screening process. This process is required to identify the desired biocatalysts from hundreds of potential enzymes as well as a high number of reaction parameters (e.g. solvent, solvent content, pH, temperature, time, and cosubstrates), which have to be tested. A group from Pfizer describe their highly efficient approach to automated enzyme screening (D. R. Yazbeck, J. Tao, C. A. Martinez, B. J. Kline, S. Hu, Adv. Synth. Catal., 2003, 345, 524). By using a Tecan Genesis pipetting robot, they first produced enzyme screening kits with enzyme solutions dispensed into 96-well plates which then were stored at low temperatures.

Whenever a substrate has to be tested, a screening kit is thawed and the substrate, solvent, and cosubstrates are added and the plates are incubated. Finally the reaction outcome is determined by fast analytical methods (HPLC, CE, GC, and TLC). Using their procedure, only 10 mg of substrate are required to screen 100 reactions.

Two resolution processes of different racemic esters by selective enzymatic hydrolysis are described. Using this high-throughput approach, it was possible to drastically increase the yield and enantiomeric excess of the desired products. The authors state that both these processes could not have been identified. This technology has great potential for other enzymatic reactions and probably also in a similar way for other catalyzed reactions.

HIGH-THROUGHPUT AND COMBINATORIAL METHODS IN POLYMER SCIENCE

Over the last decades, combinatorial methods and high-throughput technologies, as well as parallel synthesis and characterization methods, have drastically changed the way research is done in the life sciences industries. Over the last few years, a similar trend is evolving in material science research. As a logical consequence of the successful use of these methods for parallel synthesis and screening of new drug candidates in the pharmaceutical industry, high-throughput methods are now also advancing into polymer research. However to be useful in this field, automated equipment has to fulfill totally different requirements. Systems became commercially available only recently. A special issue of Macromolecular Rapid Communication dedicated to “High-Throughput and Combinatorial Methods in Polymer Science” contains a collection of articles from the leading academic and industrial laboratories (Macromol. Rapid Commun., 2003, 24, 1–148). The current status of the technology and the commercially available systems are presented and critically discussed. It is highly probable that this interesting collection of articles will stimulate scientific discussion between experts in the field of combinatorial polymeric research.

HIGH-THROUGHPUT EXPERIMENTATION IN HOMOGENEOUS CATALYSIS RESEARCH FOR FINE CHEMICALS

The use of high-throughput experimentation (HTE) in homogeneous catalysis research for the production of fine chemicals is an important breakthrough. In the past, stoichiometric chemistry was often preferred because of time to market constraints. Now, HTE allows more economic catalytic solutions to be found within a very short time frame. J. G. de Vries and A. H. M. de Vries from DSM summarize their experience with high-throughput experimentation in catalyst research in a microreview (J. G. de Vries and A. H. M. de Vries, Eur. J. Org. Chem. 2003, 799). They give examples of catalyst lead generation and optimization based on their research program aimed at developing low-cost aromatic substitution reactions (C-H activation, Heck and Suzuki coupling reactions). An example of HTE in mechanistic research, which stems from their work in asymmetric hydrogenation with low-cost monodentate phosphoramidite ligands, is also discussed. By using automated equipment like the Chemspeed ASW 2000 and the Argonaut Endeavor (for high-pressure reactions), the authors have been able to reduce to three weeks the time needed to find a catalyst and suitable conditions. They also provide examples where it is doubtful that the catalyst lead and conditions would have been found if a classical single-reaction approach had been used. Both the quality and the output of their process research and development unit were increased by the introduction of high-throughput experimentation.

USE OF THE ULTRA LOW TEMPERATURE RC1E REACTION CALORIMETER

Reaction calorimetry is used to determine thermodynamic and kinetic data, which can be used in process design and optimization for any given chemical process. The Mettler RC1 Reaction Calorimeter is a computer controlled, electronically safeguarded automated laboratory reactor for the performance of isothermal and adiabatic reactions. Darren Green and Paul Fenwick from the Hazard Evaluation Group at Rhodia Chirex describe how they use the RC1e Reaction Calorimeter for ultra low temperatures down to −70 °C (D. Green, P. Fenwick, Organic Process Research & Development, 2003, 7, 214). Minimum operating temperature, temperature stability, baseline stability, cool down rates, and controllability of additions were tested and the issues and problems related to these low temperatures are discussed critically. It is possible to carry out real chemical processes at temperatures of −70 °C. However careful experiment planning is required to ensure the reactor temperature is controlled within the desired range. Detailed operational recommendations are given in order to perform successful experiments.

HIGH-THROUGHPUT KINETIC INVESTIGATIONS OF ASYMMETRIC HYDROGENATIONS WITH MICRODEVICES

Today, asymmetric catalytic hydrogenation is a very important field due to the pioneering work of H. B. Kagan, W. S. Knowles, and R. J. Noyori. However the gap between the discovery of new chiral catalysts and the industrial use of this reaction is still large. This is due mainly to the molecular diversity of the chiral catalysts and ligands but also to the lack of knowledge for the prediction of enantiomeric excess under a range of process parameters such as pressure, temperature, concentrations, mixing etc. C. de Bellfon et al. evaluate a high-throughput test based on a micromachined mixer for molecular gas-liquid reactions for the kinetic investigation of the asymmetric hydrogenation of methyl Z-(α)-acetamidocinnamate with a chiral rhodium catalyst in aqueous phase (C. de Bellefon, N. Pestre, T. Lamouille, P. Grenouillet, V. Hessel, Adv. Synth. Catal., 2003, 345, 190). Up to 214 tests were performed in a short time with an average inventory of Rh/test as low as 14 μg. Comparisons with traditional batch experiments are provided.

HIGH-THROUGHPUT CATALYTIC SCIENCE: PARALLEL ANALYSIS OF TRANSIENTS IN CATALYTIC REACTIONS

In the field of heterogeneous catalysis, variations in temperature, pressure, or reactant concentration are commonly encountered under real operating conditions.

Historically, catalysis studies have been performed by testing each single catalyst formulation. However, as these catalysts are very complex systems, using a single reactor to study thousands of different catalyst formulations would entail a prohibitive amount of time. High-throughput experimentation has created new opportunities in this field of research in the last few years. J. A. Lauterbach and coworkers have developed a new parallel analytical method based on Fourier transform infrared (FTIR) to study the catalytic reduction of NO_x from automobile exhaust in a 16-channel microreactor (R. J. Hendershot, P. T. Fanson, C. M. Snively, J. A. Lauterbach, Angew. Chem., 2003, 115, 1184). During the transition, spectral images can be collected every three seconds from the effluents from all 16 reactors simultaneously. This approach enabled them not only to compare different catalysts but also get insight into the elementary reaction steps.

LABORATORY AUTOMATION AND ROBOTICS

A technology feature, “Automation on the move”, by Tim Chapman in Nature (T. Chapman, Nature, 6 February 2003, 421, 661) presents an interesting overview on current status of the technology and future trends with special focus on laboratory automation in the pharmaceutical industry. Applications in high-throughput screening and reagent delivery in the nanoliter range are discussed as well as protein structure determination by high-throughput crystallization approaches, systems for compound storage and retrieval, and the importance of efficient LIMS software to control the overall HTS process in an industrial production-like environment.

HIGH-THROUGHPUT MANUAL PARALLEL SYNTHESIS

S. W. Gerritz et al. from Glaxo SmithKline Inc. report a method for the high-throughput manual solid-phase parallel synthesis of libraries comprising thousands of discrete samples using pellicular supports (i.e. SynPhase crowns and lanterns) and a suite of novel tools and techniques (S. W. Gerritz et al., J. Comb. Chem., 2003, 5, 110). Crowns comprise an inert polyethylene core and a surface-grafted reactive polymer (i.e. polystyrene), which is further functionalized with linkers amenable for solid-phase synthesis. In parallel solid-phase synthesis, crowns have three distinct advantages over resin: (1) No resin segregation equipment is required because crowns are intrinsically segregated from one another, (2) the loading of a single crown is sufficient to produce multimilligram quantities of material, and (3) the 96-well compatible pin format allows direct cleavage of samples from crowns into 96-well plate.

Further key aspects of this approach include the combination of a split-split-split synthesis strategy with spatial encoding to differentiate thousands of crowns, the rapid washing and filtration of up to 48 reaction vessels in parallel, the application of an inexpensive and environmentally friendly technique to remove trifluoroacetic acid from sixteen 96-well plates in parallel, and a high-throughput method for removing cleaved crowns from reusable pin racks. Tens of thousands of discrete samples have been produced using this conceptually and operationally straightforward strategy.

CHEMICAL MICROARRAYS: A NOVEL APPROACH TO DRUG DISCOVERY

Since proteomics will establish the link between protein content of a cell and physiological changes which may be associated with malfunction and disease, it will give rise to numerous novel drug targets. Thus innovative technologies for the quick identification of chemical compounds are required.

S. Freundlib et al. reports the use of chemical microarrays as a new type of chip technology. (New Drugs, 2002, 54). It is based on function blind, label free imaging of the interactions between target proteins and small, information-rich lead-like molecules. No assay development is necessary. The platform rapidly delivers high quality compounds with a known pedigree allowing genome-scale drug discovery and chemical validation. For the production of chemical arrays, glass plates are coated with a thin gold layer. The immobilization of the compounds is mediated by an organic film containing a self-assembling monolayer (SAM). The SAM is optimized to prevent unspecific binding of proteins to the array surface and presents the ligands for protein binding. The analysis is done using a plasmon imager, which enables label free binding detection. A two-dimensional sensor array is imaged onto a spatially resolving detector and the wavelength of the light illuminating the sensor array is scanned. Parallel measurements are performed with 1,536 compounds on the senosor.

Chemical microarray technology brings chemistry into the drug discovery process at an earlier stage than traditional HTS approaches. Therefore it will considerably shorten the time needed to transfer genetic information derived from the genome project into pharmacologically active substances.

SECONDARY SCREENING WORKSTATIONS – THE EMERGENCE OF COMPACT TURNKEY SOLUTIONS

In a laboratory automation review, John Comley gives an overview on currently available and newly designed workstations focused on secondary screening application (J. Comley, Drug Discovery World, Winter 2002/2003, 31). With the high sample numbers processed in HTS, a shift in the bottleneck to obtain high quality data in secondary screening and assay development becomes obvious. The author compares the different approaches to assay automation and critically discusses the commercially available turnkey solutions from Tecan, Gyros, Beckman Coulter, Perkin Elmer Life Sciences, CyBio, Amersham Biosciences, Thermo Labsystems, and Zymark. The key benefits of turnkey workstations (i.e. standardization of assay technology, optimized streamlined process and application, very compact footprint, etc.), as well as modern application solutions like the LabCD-ADMET technology, are discussed. According to the author, these new workstations could be used to enhance the process by which selected screening sets or focused libraries based around leads are screened and facilitate decentralization and distribution of that screening capability across drug discovery departments and sites. Judging by the supplier focus on this market segment, it can be assumed that there is an unmet need in this area and systems of this type will impact on secondary screening over the coming years.

APPLICATION OF FLOW CYTOMETRY FOR IN VITRO TESTING - A TOOL TO MINIMIZE ANIMAL TESTING

The testing of pharmaceuticals in cell culture is a well-established method as an alternative to animal testing. Due to the high number of new substances from the drug discovery process, the development of new test methods is one major goal in biological screening. E. Kasper et al. (BIOforum international, 2002, 6) describe the use flow cytometry as a modern drug screening method. In flow cytometry, different characteristics of cells are examined at high speed. The single cells pass a laser beam at the interrogation point in the flow chamber. The laser light is scattered and can be detected. In addition, cells can be labeled with fluorescent dyes, which can be excited by the laser beam, and the emitted fluorescence can be detected. The dyes can be used to determine the actual status of the cells, such as vitality or metabolic state. Additional information on cell size and morphology, as well as intramolecular compounds like proteins, receptors and antigens, lipids, enzymes, RNS, and DNA, can be gained from the scattered light.

The application of flow cytometry proved quite suitable for the in vitro testing of substances with potential anti-tumor activity. The method could be a valuable tool in the future in the field of in vitro testing as an alternative to animal testing. Application fields can be found in various fields of medicine such as oncology, hematology, and immunology but also in bacteriology, food industry, or biotechnology. The automation of the flow cytometry process and the integration into complex HTS systems will make this new method even more interesting and available in automated biological screening processes.

SIMPLE AND ECONOMIC HIGH-THROUGHPUT PURIFICATION OF TRITYL-ON OLIGONUCLEOTIDES

Synthetic oligonucleotides are used for mutation detection and analysis in disease diagnosis. The demand for pure oligonucleotides in larger quantities is expected to increase even more with the development of DNA microarrays, which require thousands of oligos per chip. Whereas high-throughput synthesizers can generate more than 300 oligos per day, oligo purification is the biggest bottleneck in today's oligo production.

Varian introduced a new TOP (Trityl-on oligonucleotides purification) system for simple and economical high-throughput purification up to 200 nmole in scale and 50-mer in length. TOP includes a 96-well plate with removable cartridges containing a pH-stable, high affinity polymeric resin (functionalized styrene divinyl-benzene) as stationary phase. Several washing steps and subsequent on-column cleavage of the trityl groups provide more than 90% pure product. All extraction steps are performed by gravity flow. TOP achieves high yields over a wide range of oligo lengths (5–15 mer) and will purify up to 50 nmol of trityl-on oligos with 25 mg of polymeric resin.

96 oligo samples can be simultaneously purified within 45 min. by using multichannel pipettes. Automating the TOP process can increase the capacity to more than 1,000 oligonucleotides per day. (New Drugs, 2002, 4, 42)

PHYSICOCHEMICAL HIGH-THROUGHPUT SCREENING OF ADME PARAMETERS IN DRUG DISCOVERY

The use of genomics, bioinformatics, and combinatorial chemistry in modern drug discovery leads to an increasing number of drug candidates. Thus we see a rising demand of new powerful technologies for the determination of early ADME parameters. The solid supported liquid membrane Transil represents a perfect system for fast and efficient determination of lipid binding constants and lipid-water partition coefficients. Transil-HSA consists of covalently bound HSA immobilized on an inert surface. The surface was designed to have minimum interactions with drug molecules to prevent non-specific interactions between drug and immobilization layers. Transil-HSA is an appropriate tool for the determination of serum binding in vitro.

The compound of interest is added to the Transil beads. After the short incubation step, the supernatant is centrifuged and analyzed by UV spectroscopy or HPLC/MS. Transil and Transil-HSA are also available as “ready to go” 96-well microtiter plates for high-throughput screening of membrane affinities and serum binding. These plates consist of a filter plate prepipetted with buffer and Transil or Transil-HSA. The total assay time for a 96-well plate is less than 20 minutes using UV detection.

Nimbus Biotechnology offers high-throughput assays for the functional screening of early ADME parameters. These assays are designed to overcome crucial bottleneck situations in the drug discovery process. (New Drugs, 2002, 4, 26)

ULTRA-HIGH-THROUGHPUT SNP GENOTYPING FOR PHARMACOGENOMICS AND DRUG DISCOVERY

The study of SNPs is of great interest in the pharmaceutical industry because understanding their role in genetic variation will have a revolutionary impact on drug development. To meet the current and future needs for genotyping in the pharmaceutical industry, the SNPstream UHT platform was developed by Orchid BioSciences (New Drugs, 2002, 7, 26). The company's proprietary technology, SNP-IT, involves annealing an oligonucleotide primer immediately adjacent to a known SNP site. This step is followed by a single base extension reaction using DNA polymerase in the presence of labeled terminating nucleotides. The UHT technology combines multiplexed assays with high-density oligonucleotide arrays capable of screening up to 4608 SNPs simultaneously on a single plate. The SNPs are detected by the specific incorporation of a fluorescent dye using a multiplex thermocycled single-base primer extension reaction. This reaction is followed by a solid phase sorting on the microarray glass surface using a Universal TagArray or Zipcode format prior to readout. The system can handle SNP studies from a few hundred up to a ten of thousands of SNPs.

The system allows identification and documentation of all the SNPs that exist in the complex pathway that determine an individual pharmacogenetic profile. It offers the possibility of a systematic, predictive, and genetically based approach to medicine and drug development.

MINIATURIZATION AND AUTOMATION OF SOLID-PHASE ASSAYS

High-throughput screening requires laboratory instrumentation, which supports the use of 384-well microplates for reaching higher throughput and saving sample volumes and reagents. B. Andersson describes the conversion of a solid-phase assay from 96-well format to 384-well format using especially designed instrumentation (New Drugs, 2002, 1, 24). For performing a solid-phase immunoassay, coated microwells are required. Andersson used the HBsAg immunoassay (detection of hepatitis B surface antigen in human serum or plasma) for the assay miniaturization. In this assay sample and HRP, conjugated anti HBs are added to the antibody coated wells and incubated for two hours. After washing and adding a H₂O₂/TMB substrate/chromogen and a second incubation, the reaction is stopped and the plate is measured at a wavelength of 450 nm. The microplate is heated evenly from all sides to eliminate temperature gradients and edge effects.

The 96- and 384-well assay formats were compared by creating calibration curves with both types of plates. The observed curves were similar indicating that the assay may be performed in 384-well plates without losing sensitivity. The miniaturization of the assay leads to remarkable savings in samples and reagents. The assay performance with 384-well plates was similar to that obtained with 96-well plates. The Relay System provides automation for solid-phase assays as well as other types of assays.