(International Symposium on Laboratory Automation and Robotics) 1998

Journal of Automated Methods & Management in Chemistry, Vol. 21, No. 3 (May-June 1999) pp. 65-105

Abstracts of papers presented at the ISLAR (International Symposium on Laboratory Automation and Robotics) 1998

The 16th International Symposium on Laboratory Automation and Robotics provided the largest number of technical presentations ever offered at this meeting. State-of-the-art development in laboratory automation and robotics were reflected in the symposium programme which included papers and posters on all aspects of the technology--drug discovery research, data handling and data management, chemical analysis, bio-analytical applications, managing laboratory automation, dissolution testing, pharmaceutical analysis, automation and combinatorial chemistry, validating automated methods and advanced topics. Several new workshops and discussion sessions were included and activated, and provided even more interactive communication. Although the programme was very comprehensive, the Symposium was designed to provide time for both formal and informal exchange of information. The technical presentations were organized into concurrent sessions with grouped papers on related topics. Abstracts for each paper (except two that were not available when going to press) and each poster are included here and the proceedings of the Symposium will be published as a CD-Rom early in 1999. Full presentations of several of these papers will appear in later editions of this journal. Integrating drug discovery technologies The analytical development laboratory provides multiple opportunities for increasing productivity through auto- ames A. Bristol, Parke-Davis Pharmaceutical Research, Ann mation and robotics. Today's generation of analytical Arbor, MI, USA scientists has arrived on the job accustomed to using and The pharmaceutical industry today is entering a new era developing automated approaches to their work. They wherein a number of new and enabling technologies are expect automation, and where it does not exist, will seek maturing simultaneously. These enabling technologies to add it. There are many approaches to automation of include combinatorial chemistry, ultra-high-throughput an analytical technique. The approach can vary depend- screening, genomics, pharamacogenomics, proteomics, ing on the scope and impact of the application, but also data management, high-throughput methodology for whether it is being developed by an analytical scientist or toxicology, and ADME parameters. In addition, new an engineer. A challenge exists in maintaining the and improved automation techniques will have a dra- automation creativity of individual scientists, while pro- matic impact on optimization of these new technologies. viding a mechanism for an integrated and coordinated At the same time, the unprecedented productivity and approach to development within a multi-site, multi- financial success of new pharmaceutical agents and the functional organization. From a diverse mix of analytical effect of mergers leading to vastly larger single companies chemists, biochemists, process control scientists, software have created unprecedented demands for continued developers and mechanical engineers, have come a wide improvements in productivity that will be translated range of automated systems. An overview of these systems directly to increased sales and profits. A further factor will be provided with emphasis on the successes and is the increasing trend to outsource key discovery and challenges of the various development approaches which development activities to biopharmaceutical companies were utilized. The ongoing evolution into the current and contract research organizations. team approach to automation development will be presented. These enabling technologies have the potential to trans- form pharmaceutical discovery into a highly efficient technological advanced process which will demand ap- Quality control: organization, cycle times, compli- propriate resourcing and integration for optimal effi- ance and efficiency ciency. These developments are the challenges for drug discovery scientists in the future who, in addition to j. M. Hawkins, Janssen Pharmaceutica, Titusville, NJ, USA having established expertise within their own scientific Understanding the contributions that the laboratory can disciplines, will necessarily have to work effectively at the make in product/process development, process improve- interface of these intersecting technologies and scientific ment, market surveillance and general business is key to disciplines. the pharmaceutical business today. Poor laboratory practice yields compliance issues, increased cost, in- Coordinated development of multi-site/multi- creased cycle time and delayed product introductions. functional analytical robotics applications: chal- In my presentation, I hope to prompt discussion and gain lenges and visions agreement and recognition of the laboratory's contribu- tion in meeting the expectations of our pharmaceutical E. F. McNiff, Bristol-Myers Squibb, Princeton, NJ, USA customers. I will cover key areas of customer satisfaction,

Journal of Automated Methods & Management in Chemistry ISSN 1463-9246 print/ISSN 1464-5068 online (C) 1999 ISLAR 65 http://www.tandf.co.uk/JNLS/auc.htm http://www, taylorandfrancis.com/JNLS/auc.htm Abstracts of papers presented at the 1998 ISLAR the role of the quality control and quality assurance trends towards assay miniaturization, industrial-strength laboratories, measures of accountability and progress, automation and new detection technologies further ex- and an example of how our robotics program is helping tend the view that more is better. However, finding more Janssen meet our goals. leads for more novel targets does not necessarily shorten the time to develop a chemical lead into a drug or increase the likelihood of success for an individual Automation in the lab: does the tail wag the dog? project. Simon Jones, Genetics Institute, Cambridge, MA, USA In the fiture, therefore, an emphasis needs to be placed Intensive automation of repetitive processes in the lab is on using HTS resources and expertise to improve the viewed as an attractive panacea for several reasons. First quality of chemical leads, not simply find more of them. is the assumption that removal of the human element in Concepts in automation, data management and stream- process leads to consistently accurate data sets in a lined assay design which have been developed within the shorter period of time. Second, and deriving from the HTS environment will be applied to other aspects of drug first, is that scientists are 'freed' to focus their talents on discovery, e.g. target validation and secondary screening. more creative pursuits. Third, head count can be stabil- Broader adoption of the tools of HTS may be able to ized and even reduced. Based on this rationale, a virtuous achieve improvements in drug discovery time lines and circle is established that pleases all parties in a biophar- cost savings which cannot occur through more and faster maceutical endeavour. HTS. However, in reality several issues were overlooked and unanticipated problems have arisen. First, and the most Libraries of molecules, screening and optimiza- serious, is the displacement of the bottleneck in sample tion of hits: a cost-effective approach handling to processes up- and downstream of the automated procedure, e.g. data analysis, compound inventory Nigel R. A. Beeley, Amylin Pharmaceuticals, San Diego, CA, and secondary assays in high-throughput screening. USA Second, the daily function of automated units requires As a we need repetitive and accurate operation. This frequently leads product-oriented biotechnology company solutions for libraries of molecules, to the 'burn out' phenomenon and turnover of personnel. inexpensive acquiring data and auto- Third, a substantial financial investment is required not introducing screening robotics, handling only in equipment and bulk materials, but in training mating chemistry when compared to the larger pharmaceutical and personnel. Fourth, a whole new breed of scientist has companies technology-oriented start-up We now have access to over 250000 com- developed to adapt existing bench processes or create companies. for from the new ones for automated systems. pounds screening purposes following: (i) pharmacophore-based selective purchasing from several These issues and their resolution in small molecule drug purveyors of compounds; (ii) risk-sharing arrangements discovery will be presented. giving access to relatively large numbers of compounds; and (iii) participation in a combinatorial chemistry consortium. We have put in place the necessary infor- The future of in high-throughput screening drug matics and screening robotics to identify hits and analyse discovery data from the above. In addition, we have re-tooled our John Babiak, Robotics and Automation, Wyeth-Ayerst Research, chemistry effort to tbcus on optimization of hits, making Princeton, NJ and Pearl River, NY, USA extensive use of parallel synthesis. Some recent examples from our anti-obesity and anti-diabetic programs will be High-throughput screening (HTS), in conjunction with presented. genomics, combinatorial chemistry and molecular modelling, provides pharmaceutical companies with therapeutic opportunities never before possible. HTS involves The HTS infrastructure---the right tools for the using automated equipment to test a large number of task samples against a novel molecular or cellular target to identify a reasonable number of active molecules in a Mark Beggs, Janssen Research Foundation, Belgium timely fashion. Under the best of circumstances, the High-throughput screening is a key component of active molecules identified HTS can be the chemical (HTS) by the pharmaceutical lead identification process leads which will be optimized into important new atJanssen Research Foundation. Over recent years, the pharma- marketed Because most discovery efforts fail drugs. drug ceutical industry has experienced significant increases in to result in a marketed the on drug, primary pressure the throughput capabilities of its HTS functions. In those HTS is to do more--more and more test targets companies where HTS has been effectively deployed, it is samples--to increase the likelihood of success. now possible to screen the entire corporate compound Many HTS groups within pharmaceutical companies collection against a pharmacological target within a have done more through the use of automation, im- timescale of several weeks to a few months. This cap- proved process design and hard work. It is now common ability has been realized, not as a result of the purchase of for HTS groups to test several hundred thousand samples any one particular piece of hardware, but rather through against 20 or more novel targets each year. These levels of the development of a truly effective HTS infrastructure productivity are roughly 10-fold greater than what was which matches the needs of the parent organization. typically achieved just 2 or 3 years ago. The popular Central to this development is the need to understand

66 Abstracts of papers presented at the 1998 ISLAR how to effectively combine the use of the different types bution is almost certainly small. One must begin to ask of hardware available to the HTS specialist. the question: If we in development are indeed gong to integrate robotics into our repertoire of analytical tools The use of both modular workstations and single-arm and techniques, what needs to be done to make this robotic has most HTS systems underpinned groups' happen? operations. Recent advances in the field of multiple- arm robotic systems and dedicated automation systems Recently joining a new organization helps one establish offer even further potential for increasing productivity. perspective and gives a new beginning and new ideas. This talk will describe our experience with the use of such One approach being tried is to firmly 'build automation technologies within Janssen Research Foundation's HTS in' when designing methods and sample preparation group. schemes. This approach accomplishes several things: (1) shifting the paradigm of conventional sample Implementation of analytical technologies in a preparation to that compatible with automation pharmaceutical development organizationmlook- (i.e. homogenization); ing into the next millennium (2) translating/transferring manual methods to an Nigel North, Pharmaceutical Technologies, SmithKline Beecham automated system becomes more facile; Pharmaceuticals, Harlow, Essex, UK (3) persons utilizing both see more equivalency between them, and hence usage is more readily Managing the implementation of new technology in a accepted. pharmaceutical development environment has provided challenges and opportunities to obtain the benefits from Using several examples, aspects of automation applied to automation. Successful technologies, e.g. laboratory ap- the challenges we all face as we move into the 21st plication of new a dedicated resource. techniques requires century will be highlighted. Within our department, this resource was initially a single person that has since evolved into a team dedicated to the investigation and development of robotics and Automated analytical equipment for routine test- non-invasive analytical techniques, including near-infra- ing used in the quality control labs red. Pharmaceutical development is an important interface between research and commercial manufacturing. H. Emmerich, S. Kurschat, W. Seifert, A. Siegle and M. In research, the success of genomics and combinatorial Fischer, Byk Gulden, Lomberg Chemische Fabrik GmbH, Singen, chemistry will result in a significant increase in the Germany number of development compounds. This increase com- In the past 10 years, laboratory automation (previously bined with the desire of commercial manutZacturing to used primarily in clinical-chemical analysis) became a move towards parametric release puts an emphasis on the more important tool for routine testing in pharmaceutical need for rapid analytical methods. Some ideas on the quality control. The use of automated or semi-automated techniques that will be required to meet these goals will laboratory can decrease the lab flow-through be described together with their impact on automation. equipment time of the samples for release testing of raw materials and finished products as well as for stability studies, thus Laboratory automation in pharmaceutical devel- saving manual capacity for other analytical work. There- opment: into the new millennium fore, to improve elticiency, the first automated equipment was introduced into the quality control labs of Byk Stephen Scypinski, Analytical Development, The R. W. Johnson Gulden in the early 1990s. These instruments were Pharmaceutical Research Institute, Raritan, NJ, USA prototype robotic systems which automated the release of an contrast medium and studies Incredibly, the use of laboratory automation and robotics testing X-ray stability of in pharmaceutical analysis and drug development is enteric coated tablets (including assay and purity After the and almost 20 years old. Those of us who have attended testing). implementation qualification for each the ISLAR over the past years have heard success stories, phase (approx. year system), equipment has been used over a of about 4 for routine trials and tribulations of automating many types of period years pharmaceutical dosage from assays. More recently, the testing and benchmarking studies. The results show that the lab flow time for the of one contrast automation wave has hit the drug discovery area quite through samples medium batch can be cut in half the automated hard, and it is amazing to see the rapid implementation using of automation and robotics in areas, e.g. high-throughput method compared to the corresponding manual procedure h instead of 16 The number of batches that screening and combinatorial chemistry. If one compares (8 h). can be or no the 'ramps of utilization' between discovery and devel- tested per day, week month is longer limited the hours of the but the opment in its utilization of automation, the question to be by working analysts, only by asked is... WHY? capacity of the equipment. Due to these positive experiences, a further automated instrument was ordered in I have asked myself the aforementioned question and January 1998 for the analysis of a new X-ray contrast have tried to answer it by factoring in the rigidities we in medium produced for the USA market. The first eco- development are confronted with, e.g. regulatory nomical data for this robotic system show the same methods, cGMP compliance, method system and com- significant drop in the lab flow-through time of the puters contributing to the non-usage of robotics in drug samples as m.a. tbr the other robotic systems (10 h instead development and pharmaceutical analysis, their contri- of 20 h).

67 Abstracts of papers presented at the 1998 ISLAR

Developing and managing the automated labora- used to perform all the vacuum steps, conditioning, tory washing and elution steps. A robotic sample processor is used to carry out sample loading. Muhammad Albrakeh, Iltifat Hasan and Richard Ashley, Barr Laboratories, Pomona, NY, USA Further developments of the system to allow automated sample concentration, reconstitution of sample extracts The of new development laboratory automated methods and protein precipitation in the 96-well format will be is at the forefront of R&D and QC. This technology presented. enables higher output, reduces waste and promotes safer laboratory practices, while cutting down operating costs. Working with the manufacturer, we have developed an Managing such a lab can be both challenging and on-line system linking the Zymark 96-well SPE system to rewarding. Barr Labs will discuss first hand the process a triple quandrapole mass spectrometer. A fully script- of managing the automated laboratory. Topics include able version of sample control coupled with the ability to IQ]OQ, preventive maintenance and calibration, necess- share files between the Zymark PC and the mass spectro- ary training for scientists and systems users, management meter Macintosh allow a dynamic link to be set up. suggestions, first-hand experiences, benefits and pitfalls, and tips which will carry us into the next millenium. Equipment discussed include: Zymark MultiDose Plus, Automation of the analysis of Loracarbef in Zymark Tablet Processing Workstations (TPW II), Zy- human plasma mark BenchMate II, and Zymark--fully automated-- T. A. Mihalski and A. Witkowski, BAS Analytics, 2701 Kent Batch Dissolution Systems with USP apparatus I & II for Avenue, West Lafaryette, IN, 47906, USA complete on-line dissolution testing, utilizing both UV and HPLC methods. A recent clinical study required the quantitation of the antibiotic Loracarbef in human plasma. An HPLC-UV method for this compound base on manual solid phase Succeeding with bioanalytical automation in a extraction had already been published [Kovach, P. M., matrix environment Lantz, R. J., and Brier, G., J. Chrom., 567 (1991), 129- but it was believed that automation of the pro- Glenn A. Smith and R. A. Biddlecombe, Glaxo 139], Wellcome, cedure would increase Therefore, a Research USA sample throughput. Triangle Park, NC, direct transfer of the manual method to an automated The International Division of Bioanalysis and Drug one was conducted utilizing the RapidTraceTM SPE Metabolism (BioMet) at Glaxo Wellcome is currently Workstation. The resulting assay was fully validated comprised of distinct departments, each serving a specific and found to be very robust. Use of the RapidTraceTM role in the drug development process. These departments proved to increase both assay precision and sample are organized into smaller project teams whose members throughput. collectively contain the necessary skill sets required to advance compounds through their portion of the development process and onto the next department. Expertise Comparison of automated liquid-liquid extraction with solid phase extraction using a Zymark Rapid in areas, e.g. bioanalysis, HPLFC, LCMS and automa- TM tion are no longer centralized but instead are spread Trace instrument throughout the divisional groups, creating a flat and Thierry D. Mann, Dianne Boland and Michael F. Burke, balanced organizational structure. Department of Chemistry, University of Arizona, Tucson, Az, The strengths, weaknesses, opportunities and threats of USA such a matrix structure will be cited, and the implications The use of LC/MS in a wide variety of analyses has they have on laboratory automation will be discussed. caused a rethinking of the need for highly selective Specific examples of successful communication, team- sample preparation techniques in some laboratories. It work and personnel development strategies, as well as is generally accepted that solid phase extraction (SPE) automation technologies and systems that enhance effi- provides greater potential selectivity than liquid-liquid ciency in a matrix environment, will be shared. extraction; however, the mass spectrum provides a great deal of selectivity in many cases. If selectivity is not the LLC can to be Fully automated SPE-LC-MS-MS: the continuing required, experiment be argued and less Another of the story simpler expensive. advantages that solid phase extraction (SPE) has over liquid-liquid R. A. Biddlecombe and S. Pleasance, Department of International extraction (LLE) has been its amenability to automation. Bioanalysis, Glaxo Wellcome, Ware, Hertfordshire, UK Recent advances in laboratory automation equipment though, have made it possible to perform simple liquid- We have previously reported on the application of a fully liquid extractions in parallel. The of this automated stand-alone system for SPE in the 96-well purpose presentation is to do a direct comparison of SPE and format. Based around a Zymark XP robot, these systems LLE using automation provided by the RapidTrace use a cooled storage carousel, which acts as both a system. warehouse for all the labware and a refrigerated storage unit for the sample extracts, and a custom-built SPE Two formats for performing liquid-liquid extractions station. The SPE station incorporates a vacuum mani- have been investigated. The first utilizes a thin mem- fold, a RAS\RAM station for rapid reagent addition and brane that is transparent to organic solvents. The control a nine- or 12-port solvent switching valve. This station is of the flow rate of the solvents employed in the experi-

68 Abstracts of papers presented at the 1998 ISLAR ment by the RapidTrace allows the issues of solvent all wetted materials are Teflon, PPS, glass or stain- densities, membrane wetting and contact time with the less steel; sample to be addressed. The second format for liquid- small footprint is accommodated in a standard fume liquid extraction involves the use of a mechanically hood. supported stationary liquid phase. In this case, a porous solid support, e.g. diatomaceous earth, is used to support A variety of chemistries have been performed to date. a thin layer of sample solution. The extracting solvent is These procedures will be described and compared with then applied to the cartridge and passes through, while manual methods. extracting the analytes. Alternatively, the extracting solvent is used to saturate the surface of the sorbent and the sample is then loaded onto the column. The analytes of Generation of a directed metalloprotease library interest are then eluted using an appropriate solvent. It by solid phase synthesis should be noted that this approach, like SPE, has effectively two separate extraction steps. The extraction of j. M. Salvino, New Lead Generation Department, Rhone acidic, neutral and basic analytes from serum and urine Poulenc Rorer, Collegeville, PA, USA will be presented with an emphasis on analysis time, Metalloproteases are an important class of enzymes extraction selectivity and other factors that affect recovery. involved in processing inactive precursor proteins to their biological active forms. Selective inhibitors of specific have led to Experience with a workstation-based multiple metalloproteases important therapeutic parallel solid phase synthesis system co-devel- agents. The design and synthesis of a library of novel, oped by Zymark Corporation and ZENECA Phar- low molecular weight hydroxamic acids has been under- maceuticals taken. This library was constructed on solid phase using a seven-step sequence, wherein the final hydroxamic acid Richard A. Wildonger and ames B. Campbell, Chemical formation was performed using a novel in-house devel- Technology Section, Lead Discovery Department, ZENECA oped linker. Biological testing against numerous metallo- Pharmaceuticals, Wilimington, DE, USA proteases has revealed potent, selective inhibitors. Details concerning the scope and limitations of the synthesis, to the In response the rapid advances in high-throughput challenges in automation and biological activity of the synthesis arena and in consideration of our requirements, inhibitors will be discussed. we entered into a collaboration with Zymark Corpora- tion aimed at co-developing a workstation-based solid phase multiple parallel synthesis system. Thus, we set out Automated synthesis at AgrEvo to automate selected processes associated with multiple parallel solid phase synthesis, e.g. diversity reagent pre- Brian A. Moloney, Automated Synthesis and Combinatorial paration, synthesis (reagent addition, washing, mixing and Chemistry, AgrEvo UK, Chesterford Park, Saffron Walden, short-term incubation), off-line incubation and product Essex, UK cleavage. From the start, we held to the tenet that it is the This paper will describe the different approaches taken to chemistry that should serve to define the shape of the automated at AgrEvo in both England and automation platform rather than the converse. synthesis Germany. The approach taken in the UK is to have a We will describe the modules completed to date which series of workstations, centred around a Zymark robotic include the diversity reagent preparation station, the platform. Separate evaporation and chromatography synthesis platform, incubation station and cleavage workstations are used to maximize the sample through- station. The synthesis platform is unique in that it put. In Frankfurt, an ISRA robot with on-line evapora- exemplifies three modes of liquid addition, i.e. tion and chromatography is used to minimize operator plumbed-serial, plumbed-parallel and robot-mediated involvement. Each of these approaches will be discussed plumbed-serial. Furthermore, spent reagents, impurities in detail in the presentation. and soluble by-products are removed by filtration using positive pressure differentials rather than vacuum, thus avoiding atmospheric contamination. Features of the Parallel organic synthesis and combinatorial drug synthesis platform include: discovery at Affymax ninety-six reaction positions (4 x 24 position reac- V. Antonenko, Affymax Research Institute, Santa Clara, CA, tion blocks); USA parallel addition of reaction solvents and common reagents; robot-mediated delivery of diversity reagents (100); gentle agitation by intermittent sparge with inert A multi-task consumer products robotics auto- gas; mation system heating to 150C or cooling to -75C with an additional reflux zone for vapour condensation at Jonathan J. Zieman, Paul L. Morabito, Ramasamy Tamilar- reflux; asan, The Dow Chemical Company, Midland, MI, USA; Paul resin bed washing by parallel means; D. Hazelwood, The Dow Chemical Company, Sarnia, Ontario, quick disconnects for rapid removal of reactor Canada, and Kevin Wagers, S. C. Johnson Wax, Racine, WI, blocks for remote processing; USA

69 Abstracts of papers presented at the 1998 ISLAR

An automation system to prepare and test cleaning products by acidic hydrolytic extraction and quan- formulations according to large and complex experi- titative gas chromatography mental designs was implemented in the former Dow Brands Business group. Temperature-dependent phase Lee Pieta and Steven J. Salata, Nabisco, East Hanover, NJ, separation and continuous refractive index measurement USA as well as microemulsion room temperature turbidity In its Nutritional Labeling and Education Act of 1993 may be performed on the prepared formulations, or for (NLEA), the US Food and Drug Administration (USF- product or competitive analysis. Quaternary amine and DA) defined total fat as the sum of fatty acids expressed bleach titrations can be performed using a Brinkmann as triglycerides. Virtually all packaged foods regulated by Titrator integrated into the system. The system will the USFDA are required to carry specific nutritional handle 10 formulation solutions, including relatively information on total and saturated fat. The total fat viscous solutions, and prepare and/or test up to 240 method adopted by the Association of Official Analytical samples per run. All formulations are individually speci- Chemist (AOAC) is essentially a combination of two fied allowing maximum flexibility in experimental de- official methods. A total extract is obtained by first TM lipid sign. A Visual Basic Graphic User Interface (GUI) the with an acid. The released fats are TM digesting sample oversees the Zymark System V controller, and pro- extracted into ethyl and petroleum ethers. Alter gently vides data input and output with Excel Spreadsheets by evaporating off the ethers, the lipids are saponified and Object Linking and Embedding (OLE). Custom auto- The total fatty acid content is determined TM methylated. mation peripherals along with the required Easylab Nabisco, in partner- TM using capillary gas chromatography. code were produced including a SclLog Expert Pump ship with Zymark Corporation, has implemented a to provide one high viscosity solution delivery channel. laboratory automation system as the solution to a time- Further tests considered were viscosity, conductivity and consuming and technique-oriented procedure. competitive analysis for per cent solids. The system has significant positive economic impact and allows much more rapid formulation of cleaning products in addition A method for the determination of amprenavir in to reducing the tedium of repetitive manual operations. human plasma and serum using a Zymark/Tecan robotics system and LC,/MS/MS detection in a 96- well format Development of a fully automated analysis for the riboflavin vitamers in unfortified foods Cindy M. Rawls and Glenn A. Smith, International Bioanalysis, Glaxo Wellcome, Research Triangle Park, NC, USA L. F. Russell, L. Brooks and K. McRae, Agriculture & Agri- Food Canada, Kentville, Nova Scotia, Canada A sensitive, robust, high-throughput method for the determination of amprenavir, an investigational HIV Demands for more and better nutrient composition data protease inhibitor, was developed and validated for and the necessity to test large numbers of food samples in bioanalytical support and clinical trials. This protein order to generate representative nutrient values is fuelling precipitation method utilizes an integrated system con- the need for laboratory automation. A fully automated sisting of a Zymate II robot, a Tecan RSP 8051, a method has been developed for the simultaneous analysis reagent addition station (RAS) with a 25-port solvent of riboflavin and its coenzymes, flavin mononucleotide selection valve for all liquid additions, and a custom (FMN) and flavin adenine dinucleotide (FAD) in un- microplate vortex and centrifuge. fortified foods. It combined robotic extraction with HPLC quantitation. The robotic-HPLC method was Amprenavir and its internal standard (a stable isotope of compared with the HPLC quantitation preceded with a amprenavir) are detected on an API-300 triple-quadru- manual extraction. The manual extraction and HPLC pole mass spectrometer by atmospheric pressure chemical separation have been shown to provide total ribvoflavin ionization tandem mass spectrometry (APCI/MS/MS) in results that are comparable with the standard AOAC positive ion multiple reaction monitoring (MRM) mode. International fluorometric method. The two methods The assay has a dynamic range of 10-5000 ngml -l. were compared using foods that are significant dietary The robotics system processes two 96-well plates in less sources of riboflavin, e.g. liver, beef steak, eggs and milk. than 2 h including sample dilutions. Data presented from The results from the fully automated method compared two clinical trials in which more than 3500 samples were favourably with those obtained using manual extraction. analysed over 20 analytical runs demonstrated excellent The robotic extraction was faster than its manual accuracy and precision in both serum and plasma. counterpart and permitted determinations to be conducted under conditions that protected the riboflavin Validation test plan for the Packard Multi vitamers fi'om degradation and loss during analysis. As TM PROBE robotics system a result, the automated method is suitable for unattended analysis of the large numbers of samples that are needed Clark V. Williard, Jason D. Morse and Thomas D. Oglesby, to provide a representative estimate of the mean vitamer Taylor Technology, Princeton, NJ, USA content and its inherent variability in unfortified food products. A fully automated 96-well solid phase extraction method was developed for use in analytical support of GLP studies. A Packard MultiPROBETM robotics system Development and validation of an automated was used to automate the preparation and extraction of method for the determination of total fat in food standards, QCs and study samples. As part of the

70 Abstracts of papers presented at the 1998 ISLAR validation scheme for the method and in accordance with developing a robust, high-throughput assay. In particu- regulated guidelines for computer-controlled systems, a lar, these aspects of method optimization are discussed in validation plan was devised and executed. A validation detail: conditioning solvent volumes, vacuum considera- protocol was created which included a description of the tions, sample pretreatment options, sample dilution, system, a test plan, reference to related SOPs and wash solvent strategies to eliminate proteins, elution documentation of testing. It addressed requirements of solvent optimization and automation considerations. user verification of the vendor systems and source codes, The 96-well plate format is ideal for examining multiple change control procedure, application system documen- parameters within a single plate. A method can literally tation, and the system's physical analytical performance be optimized within a single plate, greatly reducing the parameters including position control and volumetric time required tbr method development. pipetting. The validation plan and results will be presented. Additionally, the plan will be discussed with Automated liquid/liquid extractions using Varian respect to fulfilment of recent regulatory mandates and TM guidelines. Chem Elut cartridges and a Zymark XP robotics system Christine M. Grosse and Glenn A. Direct injection of plasma for high-throughput John R. Alianti, Smith, drug metabolism HPLC analysis International Bioanalysis, Glaxo Wellcome, Research Triangle Park, NC, USA Art Sims, Joe Takarewski and Peter Duriga, Cohesive Tech- MA, USA Diatomaceous earth (DE) is a silica-based substance nologies, composed of the exoskeletons of fossilized unicellular A major time-limiting step in measuring drugs in plasma algae called diatoms. As the diatoms died, their skeletal is sample preparation. Steps necessary to remove the remains blanketed the floors of ancient sea bottoms, drugs of interest fi'om plasma prior to injection into a lakebeds and lagoons. Today, DE is excavated, processed HPLC typically require more time than the analysis. and purified for use in a variety of products including Recent advances in automation of solid phase extraction insecticides and filtration systems. have reduced the amount of hands-on time (SPE) greatly One important application for DE is in the pharmaceu- required for clean but even automated, this sample up, tical industry. Because DE is extremely hydrophilic, process is still prohibitive to high sample throughput. aqueous-based polar drug compounds and their metabo- This poster describes new, dynamic flow technology that lites readily adsorb to this matrix. The compounds can allows lbr direct injection of plasma samples and new then be extracted from the matrix an immiscible levels of sample throughput. using solvent, e.g. ethyl acetate, methylene chloride or chloro- Turbulent flow chromatography, and the resulting con- form vective mass has removed the transfer, time-consuming At Glaxo Wellcome, several automated and clean traditionally required in plasma drug LC/MS/MS sample up HPLC bioanalytical assays have been validated using analysis by allowing direct injection of plasma onto a this procedure because it offers numerous advantages specialized column. Relatively large la), phase-coated (50 over conventional solid phase and liquid/liquid extrac- particles retain the drugs of interest, while the plasma is tions. This poster describes the advantages and disad- eluted to waste. The drugs can then be quickly eluted vantages of this technique, the system layout, the and measured by or mass-specific detectors. UV/VIS extraction process, automated vial filling and future This will show data tiom direct plasma injection poster trends, e.g. 96-well plate technology. and sample analysis cycle times as short as 90 s.

Automated 96-well for high- Method optimization approaches using 96-well SPE-LC/MS/MS in a contract lab: prob- SPE disk throughput bioanalysis plates lems and solutions David Lawless and David A. Wells, 3M J. Ehresman, Greg Luca C. Matassa and Patricia E. Paterson, Maxxam Analytics, Filtration Products, St. Paul, MN, USA Mississauga, Ontario, Canada solid extraction in microtitre Ninety-six-well phase (SPE) Fast bioanalytical analysis techniques, e.g. LC/MS/MS, plate tbrmat offers a rapid sample preparation approach require equally fast, or faster, sample preparation tech- lbr the analysis of drugs and metabolites in biological niques to keep pace. Automated 96-well SPE with fluids. Beyond the gains achieved by the microtitre LC/ has been used successfully in our laboratory for 4 format and parallel processing of 96 samples, the 3M MS/MS years and has provided significant advantages over Empore disk technology particles: 10% PTFE, (90% manual techniques. There are, however, some specific provides an additional gain in throughput. The w/w) problems which must be solved in order to take full use of disks, instead of traditional beds, packed particle advantage of the technique. Both problems and solutions allows for smaller solvent volumes and the ability to of using automated 96-well SPE will be discussed in this eliminate the and reconstitution to dry-down step prior presentation. analysis. The conditions under which disks should opti- mally be used are slightly different than with traditional Major components include 96-well SPE plate (Waters, products. This presentation will discuss proper usage and 3M, Varian), a robotic liquid handling system (Packard disk plates for maximal efficiency. Also, optimization of Multiprobe 104 or 208), and an LC/MS/S and auto- disk-based SPE assays will be discussed with emphasis on sampler (PE-Sciex API III-plus and Hitachi 7250).

71 Abstracts of papers presented at the 1998 ISLAR

Automated 96-well SPE is best applied in high-volume extraction conditions. Our unique approach in using bioanalytical labs. It eliminates rate-limiting steps of automated extraction was PhASSETS (R) (Phoenix manual column preparation and liquid transfers by using Automated Simple Sample Extraction Treatment 96-well plates coupled to robotic handling of samples, System), an automated extraction system composed of liquids and extracts, thereby providing enhanced a liquid pipetting station and a special extraction throughput and accuracy. On the downside, the tech- cartridge. This system was designed and developed nique is semi-automated, requiring manual intervention at Phoenix International Life Sciences. This multi-pur- at some points. Process re-engineering/staff training and pose, automated extraction system has been responsible purchase of expensive hardware and consumables is for the validation of over 60 analytical methods, using required. The automation must also be adaptable to different means of detection, e.g. HPLC, HPLC]MS, various tubes sizes/configurations and sample matrices HPLC/MS/MS, GC, GC/MS and immunochemistry from multiple sources. assays. The PhASSETS (R) extraction system makes use of the liquid/liquid extraction theory (adsorption/deso- with a treated solid Fast on-line Prospekt (R) SPE/HPLC/MS/MS method rption) coupled specially support for the quantification of methylphenidate in hu- cartridge. man plasma (potassium oxalate) The extraction of these drugs from the biological Robert Masse, Christian Lemoyne and Claude Amestoy, Phoenix matrix (human plasma/EDTA) can be accomplished International Life Sciences, St-Laurent, Quebec, Canada, Daniel with a speed of 120 samples in 1.5 h, leaving the analysts Carazzato, Bristol-Myers Squibb (Canada), St-Laurent, Que- free to perform other duties. Statistical analysis on bec, Canada, Alain Carrier, Triangle (Canada), Montreal, between-batch quality control samples led to the follow- Quebec, Canada, and Denis Lessard, Bioanalytical Innovation, ing results. Levis, Quebec, Canada Felodipine Methylphenidate is a mild CNS stimulant with effects D A B C similar to that of amphetamine and is frequently pre- QC QC QC QC 40.44 3.033 6.066 scribed for children with attention-deficit 121.32 hyperactivity. pg/ml pg/ml pg/ml pg/ml A fast automated SPE/HPLC/MS/MS method was developed to quantify methylphenidate in human plasma Mean 43.311 124.485 3.0743 6.3292 at 0.100ng/ml. Human plasma (100ml) containing SD 4.3202 5.9277 48.6741 172.5209 methylphenidate was diluted with water, extracted with % CV 10.0 4.8 1.6 2.8 a fully automated on-line solid phase sample clean-up Nominal (%) 107.1 102.6 101.4 101.1 and injection system Prospekt (R) 9200 SPE Recovery (Varian (mean) (%) 96.95 system). Separation and quantification were performed N 10 10 9 9 with HPLC/MS/MS. The resultant total run time was 3min. A full validation was completed, linearity was observed in the range of 0.100-10ng/ml (r > 0.9997). Observed recoveries for methylphenidate at 0.300 ng/ml, 3.999ng/ml and 7.998ng/ml were 72.0%, 66.8% and Nifedipine 68.7%, respectively. The between-batch CV% for QC QC D QC A 1.52 Qc Qc c samples at 0.100ng/ml, 0.300ng/1, 3.999ng/ml and 0.51 pg/ml 50.54 70.75 7.998 ng/ml were 7.6%, 5.0%, 4.0% and 3.5%, respect- ng/ml ng/ml ng/ml Further validation and data will be ively. stability Mean 0.455 1.561 52.662 70.621 presented. SD 0.0724 0.1137 2.1257 2.2034 % CV 15.9 7.3 4.0 3.1 Nominal (%) 89.3 102.7 104.2 99.8 Automated analyses of three selected calcium Recovery channel blockers: fetodipine, nifedipine and nisol- 84.13 (R) (mean) (%) dipine by the Phoenix PhASSETS extraction N 6 6 6 6 robot coupled with gas chromatography Carl Dourambels, Reng Michaud, ohanne Bouchard, Isabelle Barbeau, Alain Arseneault and Robert Massg, Phoenix Life Sciences, Montrgal, Quebec, Canada Nisoldipine The Sample Preparation and Automation (SPA) and Qc D QC A 0.149 QC B QC c 0.050 ng/ml 2.980 5.961 Gas at Phoenix Chromatography (GC) departments ng/ml ng/ml ng/ml International Life Sciences have jointly developed a set of unique methods for the analysis of selected calcium Mean 0.494 124.485 3.1586 6.0734 channel blockers (CCBs), i.e. felodipine, nifedipine and SD 0.003 12 5.9277 0.149 56 0.185 79 nidoldipine, by gas chromatography either coupled to a % CV 6.3 4.8 4.7 3.1 mass-selective detector (MSD) or an electron capture Nominal (%) 98.8 102.6 106.0 101.9 detector The three drugs are structurally similar, Recovery (ECD). (mean) (%) 85.23 having close chemical and physical properties, and tend N 6 5 6 6 to react in similar ways when exposed to the same

72 Abstracts of papers presented at the 1998 ISLAR

Felodipine and nisoldipine were analysed by GC/MSD, Recovery of both compounds from (sodium EDTA) while nifedipine was analysed by GC/ECD. The maxi- human plasma was 91.5% for loratadine and 84.9% for mum run times for these analyses were 6 min per sample. descarboethoxy loratadine. Analytical batches typically numbered 95 samples. Chro- The use of Sciex API-III (R), coupled with matography systems were very stable, and no contamina- LC/MS/MS Waters Alliance (R) injector, led to 2min run time per tion or interference at analyte retention times was found sample, with no contamination or interference at the in all blank plasma samples tested. The use of the drug's retention times, determined on 10 different blank PhASSETS (R) extraction system coupled with GC was plasma samples. The use of a simple mobile phase and found to be ideal for these analyses. analytical HPLC column also tends to minimize all variations on the different systems, thus making this a and robust method for all hands involved in the The joint automated analysis of loratadine and rugged descarboethoxy loratadine (major metabolite) by process. Phoenix PhASSETS (R) extraction system and liquid chromatography coupled to tandem mass spectro- Evaluation of 96-well solid phase extraction (SPE) metry technology using methotrexate and aminopterin as drug probes both off-line and using the TOM- Reng Carl Dourambeis and Robert Massg, Phoenix Michaud, TEC Quadra 96 automated workstation International Life Sciences, Montrgal, Quebec, Canada Carl Dourambeis, Rigas Voyatzis, Trang Nguyen and Robert The Sample Preparation and Automation (SPA) depart- Massg, Phoenix International Life Sciences, Montreal, Quebec, ment at Phoenix International Life Sciences has devel- Canada oped a unique method for the joint determination of loratadine and its major metabolite, descarboethoxy Ninety-six-well technology has been investigated using loratadine, from human plasma by LC/MS/MS at a methotrexate and aminopterin as drug and internal quantitation limit of 50pg/ml. PhASSETS (Phoenix standard (IS) probes, respectively. The flow character- Automated Simple Sample Extraction Treatment istics and regeneration of the 96-well extraction blocks System), an internally developed extraction system has showed differences from manufacturer to manufacturer. been used to validate this method and over 60 other Extraction solvent effects at various levels during drug analytical methods for HPLC, HPLC/MS, GC, GC/MS extraction showed the dependence of the recovery on the and immunochemistry departments at Phoenix. This step in which different solvents were employed. Variable system makes use of liquid/liquid extraction theory volumes of sample loaded during the extraction showed (adsorption/desorption) on a solid support termed very little dependence on the sample volume used. 'thin-film liquid/liquid extraction'. The PhASSETS Mechanical and chemical weakness in the extraction robot uses multiple liquid/liquid additions to help selec- manifolds and extraction blocks are discussed. Results tively improve the recovery of the analyte(s) from the will show a comparison between various vendors' prod- biological matrix. The following results were obtained. ucts as well as optimization techniques leading to the development of a 96-well method for the isolation and purification of methotrexate and the internal standard, aminopterin. HPLC and LC/MS/MS are used as detec- Loratadine tion systems. Results of this work are presented. QC A QC B QC C ng/ml ng/ml ng/ml QC D High-throughput analysis of drugs from biological Actual matrices with a water-wettable sorbent in a 96- concentration 0.1506 6.0240 7.5300 well extraction plate Mean 0.14926 5.70400 7.20208 SD 0.010 128 0.235934 0.288902 Yung-Fong Cheng, Laura Bean, Robert Bonin, Ed Bouvier, (%) CV 6.8 4.1 4.0 9.4 Mark Capparella, Pare Iraneta, Uwe D. Neue, and Dorothy Nominal (%) 99.1 94.7 95.6 102.2 J. Phillips, Waters Corporation, Milford, MA, USA N 21 22 22 24 Sample preparation is required to maximize the quality of the analytical results and to minimize the downtime of expensive, high-overhead analytical tools e.g. LC/MS/ Descarboethoxy loratadine MS systems. Historically, liquid-liquid extraction and protein precipitation have been the bottleneck in labora- A B c QC QC QC as are not easy to automate. ng/ml ng/ml ng/nl QC D tory productivity they Presently, solid-phase extraction (SPE) has been proven Actual to be more readily automated for high sample through- concentration 0.158 3 6.330 3 7.912 9 0.0528 put. The most common high-throughput sample pre- Mean 0.16966 6.04556 7.561 63 0.05645 paration is solid-phase extraction in a 96-well format. SD 0.018 167 0.572 729 0.531 295 0.006 091 The enabling technology for 96-well plates is the single (%) CV 10.7 9.5 7.0 10.8 reversed-phase sorbent that can be used to extract all Nominal (%) 107.2 95.5 95.6 106.9 classes from fluids. A sorbent and N 21 22 22 22 drug biological genetic a simple SPE method are the keys to automating the

73 Abstracts of papers presented at the 1998 ISLAR analyses for pharmacokinetics studies in drug discovery of the initial sample transfer, ordinary steps in sample and the clinical trial samples in drug development. preparation, e.g. the addition of internal standard and sample treatment reagents, mixing, solid phase and This paper shows that using the generic OasisTM HLB liquid-liquid extraction, evaporation (under nitrogen or sorbent, in a 96-well format, can increase sample pre- by reconstitution, sample clarification (by paration productivity. With a simple and well thought vacuum), or and HPLC injection were all out SPE method development strategy using the poly- centrifuge filtration) TM in the 96-well format. Several of small meric Oasis sorbent, we were able to obtain excellent performed pieces were and custom built in-house to results for a wide range of compounds. Recoveries greater equipment designed achieve than 85% and variability less than 5% were obtained on the best efficiency. Typical sample preparation OasisTM HLB extraction plates for basic compounds (e.g. times for solid phase extraction were reduced to approxi- antidepressants and their metabolites) and polar drugs mately 2.5 h per 96 samples from approximately 5.5 h. (e.g. procainamide and ranitidine). More importantly, Typical sample preparation times for liquid/liquid ex- because the OasisTM HLB extraction sorbent is universal, traction were reduced to approximately 2.5h per 96 one general method was used for extracting many samples from approximately 6 h. different drugs covering a wide polarity range. Biological sample preparation by on-line solid High-throughput extraction with a novel poly- phase extraction meric solid-phase sorbent for screening drug candidates Min Chang, Tong Shen and Maury Chu, Abbott Laboratories, Abbott Park, IL, USA Judy L. Carmody, Pamela Iraneta, Yung-Fong Cheng, Dorothy Phillips and Uwe Neue, Waters Corporation, Milford, MA, Two types of unattended on-line solid phase extraction USA procedures were developed. The first procedure uses a restricted surface reverse phase cartridge to extract the Rapid and reproducible quantitation of basic and polar analytes from the plasma or urine. Proteins and other drug compounds from biological matrices has historically interfering components were washed off with an aqueous been a challenging, time-consuming endeavour. Necess- mobile phase. After the initial wash, analytes were eluted ary sample preparation techniques used to concentrate or into the analytical column by the analytical mobile phase clean matrices before HPLC of GC have up analysis which was also used for the chromatographic separation. proven to be either tedious and labour or intensive, The extraction cartridge was regenerated alter the irreproducible. Presently, the most commonly used tech- analytes eluted into the analytical column. The same nique is reversed-phase solid phase extraction using extraction cartridge was used for the entire analytical porous silica bonded with The major disadvantage C18. run. The other utilized an OSP-2A of employing this sorbent with basic compounds, e.g. procedure (JM Each was doxepin, elution of basic Science) sample preparation unit. sample prevents complete compounds be and results in low, variable results. extracted onto a separate extraction cartridge (may re-used) after activation and conditioning. The extrac- Highly reproducible and uncomplicated SPE methods tion procedure was essentially the same as the previously were developed for these types of basic drug compounds described procedure. However, as the sample extraction covering a wide polarity range using the novel polymeric and the elution and of the extraction c> regeneration Oasis HLB sorbent. Recoveries greater than 90% and cartridge may occur in parallel, extensive cartridge reproducibilities less than 5% RSD were realized even washing and drying can be performed without increasing with using the Oasis HLB sorbent in 96-well plate analysis time. A system with four sample preparation format for high throughput. Most importantly, because solvents and air will be Both ? drying presented. pro- Oasis HLB sorbent is a water-wettable polymer, these cedures were used to support clinical and toxicity studies. results were achieved without concern as to whether or not the sorbent ran dry. Straightforward method development techniques aimed at offering solutions to prob- Operational qualification of a BenchMate tablet lems associated with the sample preparation of drugs in processing workstation (TWP) biological matrices will be presented. John Leedes, Whitehall Robins, Richmond, VA, USA Integrated biological sample preparation in the 96- Recently, Whitehall Robins Healthcare Pharmaceutical well format Research and Development obtained a TPW. This workstation was the first fully automated sample preparation Min Chang, Jack Conrad, Enid Quinn, Matthew Rieser, Tong device brought into our laboratory for routine NDA Shen, Willium LaBeau, Bahaa Q,asawa and Maury Chu, Abbott product analysis. To satisfy GMP requirements, it was Abbott USA Laboratories, Park, IL, necessary to develop a qualification and calibration In order to gain the full benefit of parallel sample scheme to ensure that this unit was functioning properly. processing in the 96-well format, every step in a sample A protocol was written and approved which describes the preparation scheme must be compatible and preferable operational qualification (OQ) testing of the automated all in the same format. At Abbott Laboratories, inte- device. The resulting document, described in this poster, grated sample preparation in the 96-well format was used satisfies all GMP requirements and serves as the calibra- to prepare plasma and urine samples. With the exception tion method in our metrology program.

74 Abstracts of papers presented at the 1998 ISLAR

Integrating high-throughput technology into This approach could prove especially tedious for multi- separation science component products. The latest version (2.1) employs the MultiLink Excel-based software for data quantitation. Adam M. Fermier, Norberto A. Guzman and Stephen Scypinski, Additionally, version 2.1 has an interface with the Analytical Development Department, The R. W. Johnson Hewlett-Packard (HP) 8453 UV Spectrometer. The Pharmaceutical Research Institute Raritan, NJ, USA HP software contains programs for manipulating data The term 'high-throughput' has become synonymous from single and multiple measurements. This poster will with the streamlining of processes in the laboratory. Up discuss the process by which a multi-component analysis to now, the application of high-throughput technology (MCA) method is developed and validated using the has, for the most part, been restricted to drug discovery. MultiDose 2.1-HP8453-HP ChemStation UV software Indeed, high-throughput screening (HTS) has revolution- platform. A detailed description of the hardware and ized the manner in which new molecular entities (NMEs) software configuration is presented, along with analytical are derived from positive assay screens. In the area of validation results for the product tested. pharmaceutical development, whose mission is to trans- form an NME candidate into a marketable product, there A vision-based system for automatic determina- is a continual quest for increased productivity, in order to tion of antibiotic content minimize the total development time between candidate selection and the filling of the appropriate regulatory Margarida Machado, De_partamento de Electrdnica, INETI, documentation to apply for product registration. Azinhaga dos Lameiros, Estrada do Pa,co do Lumiar, 1699 Lisboa Codex, With this mindset, the analytical laboratory is expected Portugal not only to analyse an increased number of samples with Antibiotic content determination is essential not only for a high degree of accuracy and precision, but is also controlling the quality of pharmaceutical and other expected to develop and validate methodology, conduct products in which the antibiotics are used as additives, physiochemical characterization studies, etc. in a time- but also to ensure compliance with legislation. The constrained fashion. Pharmaceutical analysts rely heavily microbiological method for evaluation of antibiotic con- upon separation science to conduct their daily activities; tents is based on the observation of specific microorgan- however, separations are by definition one of the slowest isms' reaction to these chemical substances. This reaction of analytical techniques. In our laboratory, we have takes the form of inhibition halos that appear in the begun to apply the concepts of high-throughput tech- culture medium, around circular cavities where the nology to chemical separations. In this regard, we have antibiotic was introduced. As the halos' diameters are constructed an instrument capable of parallel processing directly proportional to the antibiotic concentration, it is up to eight simultaneous samples utilizing capillary array possible to evaluate sample contents by comparing their technology. responses with those from standards with known activity. The reasons for our choice of capillary technology were Traditionally, the reading of the halos' diameters is several. First, a capillary array instrument would be performed manually by human operators with the help capable of performing both chromatographic and elec- of a digital calliper. These readings involve repetitive trophoretic separations, thereby rendering it more versa- manipulations which, besides being time consuming and tile. Secondly, capillary separations are faster and do not tedious, demand great precision. utilize large volumes of reagents or sample. Finally, they The vision system presented evaluates antibiotic content conserve space. The capillary array instrument described from images of Petri dishes with inoculated culture and of here consists eight columns running in parallel. Detec- eight inhibition halos, corresponding to four different tion is accomplished by monitoring the absorbance of the dilutions of the standard and the sample. eight channels simultaneously with a fibre optic system. The system is controlled via a personal computer inter- The automatic determination method has four phases. In face. The design, testing and demonstrated usage of the the first one, an initial segmentation is performed trying system will be presented in this poster. to obtain the halo's contours. This segmentation is executed taking into account global information obtained from the histogram. Then a validation analysis is Development and validation of an automated carried out in order to identify the halos and eliminate method for dissolution testing of a multi-com- error situations, like, e.g. the consideration of a halo's ponent antiviral drug using the Zymark Multi- hole instead of the halo itself. In this analysis, factors, e.g. Dose Workstation the object's circularity, area and centre position are considered. Situations of non-detection are also analysed. Toni M. Terry, Glaxo Wellcome, Research Triangle Park NC, USA After this second phase, a third one is triggered in case of possible errors detected. It consists of a second segmenta- The Zymark MultiDose Dissolution Workstation has tion, performed in restricted areas and using local intensity been used as a standard platform tbr automated dissolu- information, followed by another error analysis. For the tion testing with USP Apparatus II conditions for solid halos identified, a diameter determination procedure is dosage forms of pharmaceutical products. Early versions executed. This procedure tries to minimize the fact that of the MultiDose system allowed for both off-line and on- halos are not 'perfect' circles, by averaging the distances line testing by chromatographic and spectroscopic meas- between opposite contour points situated at equally spaced urement methods. Subsequent nanipulation of the data angular intervals. Finally the content is evaluated using had to be performed manually or using spreadsheets. the diameter's values according to the standard norm.

75 Abstracts of papers presented at the 1998 ISLAR

Besides the automatic capability, the system also offers ing 200 puffs each. Each analyst actuated l0 canisters the possibility of diameter's correction, either automatic, each and recorded shot weights prior to actuation and semi-automatic or manual, so that the user can observe every 10 actuations hence. the effects of content value. An automatic transfer of data to a worksheet is also available. Using statistical tests, the data collected show that the MDI FD-10 Stations are statistically equivalent with The tests performed on two antibiotics, Zinc Bacitracin manual actuation and weighing of MDI canisters. A and Tylosin, for three different content values showed a summary of the data is shown in the table. mean difference inferior to 2.5% of the expected theoretical value, between content values obtained from manually determined diameter values and those given Means of actuation by the system in automatic mode without corrections. A standard deviation inferior to 2.5% was observed. The MDI MDI values with automatic correction are of the same order. Manual FD- 10#1 FD-10#2 Mean shot weight (% of target) 97.5 97.9 98.3 SD 3.352 1.451 1.307 A robotic system for skin permeation test in CV 3.4 1.5 1.3 pharmaceutical research development n 598 600 599 Min. shot weight (% of target) 80 90.9 95.4 P. Marty, C. Faure and F. Laroche, Institut de Recherche Pierre Max. shot weight (% of target 109 104 103 Fabre, Rue Jean Rostand, 31319 Labege Innopole, Cedex, France In vitro skin permeation tests using static diffusion cells are commonly used in pharmaceutical development to assess The results of automated actuation of MDI canisters the delivery of dermal or transdermal dosage forms using the MDI FD-10 Station reflect lower coefficient through biological (skin) or artificial membranes. Among of variances (CV) and standard deviations (SD) when the various models described, the Franz cells model is compared with manual actuation. In addition to the currently still the reference even though it was initially statistical equivalence of the results, the tremendous designed for manual sampling (using a syringe), at reduction in risk of repetitive strain injuries when using different time intervals, of the receptor medium. For this the MDI FD-10 Station when compared to manual type of implementation, a robotic system has been actuation of MDI canisters is important. Not only will developed to automate sampling for U5 Franz cells the MDI FD-10 Station significantly reduce the user's arranged on three thermostatically controlled trays. exposure to repetitive strain injury-related activity, but it The advantage of this system is that it provides increased will also provide the analyst's need to shake and fire capacity, productivity and reproducibility of the basic multiple MDI canisters needed for testing. permeation test, while still using the standard cell model. Improved reproducibility is obtained through better Automated sample preparation of Roxifiban temperature regulation, better control of the displaced tablets: transfer of a manual method to a robotic volumes (sampled quantities and replaced volumes) and, workstation more generally, by creating uniform manual operations for the methods currently being performed. William Shamrock and David K. Lloyd, Pharmaceutical R&D, The DuPont Merck Pharmaceutical Co., Experimental Station, Wilmington, DE, USA Validation of an automated waste shot workstation--determination of weight of actuation Automation offers obvious advantages for the preparation of tablets prior to analysis by HPLC, including Mark . Richards and Robin V. Platt, Rhdne-Poulenc Rorer, unattended operation, minimization of human interven- Holmes Chapel, Cheshire, UK tion and an electronic audit trail. However, significant effort has to be put in up front to develop and validate an The process of validating methods for use on an auto- automated if it is to mated workstation (MDI FD-10 Station, InnovaSys- method, particularly required closely follow an existing manual method. Here, method transfer tems, Pennsauken, NJ, USA) for the handling of waste for a be shots fiom metred dose inhalation products is best Roxifiban, fibrinogen receptor antagonist, will discussed. A Tablet Workstation II managed when broken into various tasks shot weight Zymark Processing (i.e. all robotic equivalency, medication delivery equivalency). This pos- (TPWII) was used for sample preparations. Manual methods for assay and degradation ter will present the activities related to determination of composite products testing of a tablet formulation were transferred average weight of actuation (as per cent of target to the TPWII. The method involved the weight); comparing shot weights of manually actuated dissolving form filtration of the homo- MDI canisters with canisters actuated on two different dosage by homogenization, dilution of the filtrate and transfer to MDI FD-10 Stations. Shot weight data are presented as genate, autosampler vials. Obstacles to a quick transfer included limitations in a per cent of target shot weight. the volume capabilities of the TPWII, poorly soluble Using two MDI FD-10 Stations, 60 canisters, containing analytes, and proper conditioning of the transfer lines 200 puffs each, were actuated. Weights were recorded and filter. After resolving these issues, a validated method prior to placement in the MDI FD-10 and at every 10 was achieved. Spiked recoveries were from 99.4% to actuations hence. Manual shot weights were collected 101.1% (RSDs <0.5%). A cross-validation between using three analysts actuating 30 MDI canisters contain- automated and manual assay methods was compared

76 Abstracts of papers presented at the 1998 ISLAR by Westlake analysis giving a 0.7% calculated interval of This poster will present current technology that can be the 95% confidence level. Carry-over was 0.07% after 20 applied to optimize automated dissolution testing. Op- sample preparations at the highest tablet strength. tions range from parallel bath sampling with multiple sampling probes and multiple flow injection valves for direct sample analysis to serial sampling with a single Automation of Andersen impaction testing of probe and multiple flow injection valves for automated pressurized metered dose inhalers analysis with multiple HPLC systems from a common Jonathan Faulkes, Rob Whyard and Astra Chamwood, Lough- sampling and analysis platform. The control options borough, UK; Malcolm Smith, Novi Systems, Maidstone, UK required to achieve these levels of automation will be presented. The Anderson impactor is an established device for characterizing the size distribution of particles in an air flow. This manual apparatus has become the standard An open BenchMateTM system for the analysis of technique for determining the particle size distribution of cleaning validation samples the delivered dose from pharmaceutical inhalation products. As a manual method it is time consuming, resource P. Deuringer and R. SchO'nneis, Bayer AG, Business Group intensive and repetitive. Automating Andersen testing Pharma, Quality Assurance, D-51368 Leverkusen, Germany has a been high priority within the pharmaceutical Cleaning validations are necessary to verify that the industry. pharmaceutical processing equipment is free of contam- A collaborative project between Astra Charnwood and ination from both active substances and cleaning agent the automation company Novi Systems has led to the after its use. The cleaning validation procedures for successful automation of Andersen testing. This was a complex equipment results in a multitude of samples. TM (R) very difficult automation project due to the design of the The BenchMate system of Zymark Corporation was manual device. To aid acceptance by regulatory auth- found to be a suitable unit to analyse the large sample orities, the internal construction of the Andersen im- load. pactor could not be significantly altered. In the QA laboratories, the swab samples which were The philosophy of the automated system is discussed taken from the equipment are directly transferred to the along with the operation and parameters that may be Zymark(R) BenchMateTM test tubes. altered to develop an automated test method. Validation Some were encountered when an 'off the data are also presented showing the equivalence between problems using shelf' available BenchMateTM auto and manual determinations. The potential resource commercially savings and reduction on the variability of results from the cannula hit the swab sample in the tube while going from a manual to an automated system are also filling the sample tubes with solvent, so that the discussed. balance was influenced; the cannula became clogged with swap materials; Dissolution bath sampling and analysis optimiza- sample solution was lost because the dipping speed tion of the cannula is very fast. Charles R. Frey, Gilson, Middleton, WI, USA Therefore, the critical filling and sampling procedures Dissolution testing guidelines require precise and accu- were reprogrammed using the open-mode operation. rate control of temperature, stirring configuration and Additionally, the time-consuming manual input for mul- sampling time. Due to the extended length of tests and tiple methods and the data transfer were optimized by time and TM sampling requirements, sampling analysis pro- connecting a PC. An Easyfill station was added to cedures are typically automated. Depending on the level back-up samples. of automation that can be achieved, actual sampling and analysis procedures may be less than ideal with significant sampling time variations and analysis inconsisten- Automating a 'simple' moisture method cies. Steven Salata, Nabisco, R&D, East Hanover, NJ, USA Optimization of sampling and analysis procedures can J. significantly improve dissolution test performance. Bath Nabisco's earliest venture into robotic laboratory auto- sampling can be performed serially (one bath at a time) mation started in 1986. The test chosen was moisture by or in parallel (all baths simultaneously) with the latter vacuum oven weight loss, one of the most common in the being the preferred option. Analysis is typically per- food industry. It was considered 'simple' in the hands of formed in a serial manner using either a direct sample any technicians, so improved productivity and precision analysis or a chromatographic separation for specific was the goal. That system was developed, refined and analyte detection. If multiple analytes are involved, used routinely over the course of the next 5 years. multiple separations may be required. In addition to Constant tweaking and tuning by the chemist who was sampling and analysis optimization, integration of these the 'owner' of the system allowed it to operate reliably processes into a single optimized sampling/analysis plat- enough to justify its space and maintenance cost, but form offers the advantage of accurate and reproducible disappointingly, it was not as 'good' as an experienced control of all sampling and analysis operations without technician, even if its repertoire was limited to sample analyst interaction and the associated potential for error. types that were homogeneous, easily pourable powders.

77 Abstracts of papers presented at the 1998 ISLAR

As our laboratory staff turned over, new users had no The XP robotics system enables the analyst to continu- original developers around to coach them. Inconsisten- ously prepare samples and proceed directly to the instru- cies appeared in routine validation check samples with- mental phase of the analysis. out obvious cause. Increased concern with GLP and In this presentation, the XP robotics system will be higher with fewer hands provided achieving productivity described with examples of programming which coordi- reason anew to redesign the system. This poster illustrates nates the extraction and derivatization procedures with how that led to subtle but implementation identifying the analytical processes. Photographs of different stages critical non-robotic factors and an extra unanticipated of the extraction procedures will be presented with phase of the project to assure data quality. accompanying descriptions of the advantages of this protocol. A robotic system designed for multiple applications Automation of residue analytical methods for determination of pesticides in soil Danh Nguygen and Martin Phillippi, The Clorox Company, Technical Center, Pleasanton, CA, USA Herbert Sommer, Volker Koch and Markus Schuld, Bayer AG, Agriculture Center Monheim, FRG Due to the relative high cost of robotics compared with other laboratory equipment and the high impetus in A new residue analytical method for determination of a industry to regulate staff costs while increasing produc- new plant protection compound and its metabolites in tivity, we have worked toward developing a robotic soil has been developed. Different analytical techniques system capable of readily performing multiple applica- were used to improve precision, repeatability and repro- tions. Although it is not unreasonable to utilize PyTech- ducibility of methods for the determination of pesticides nology capability for interchanging stations or other in soil and to reduce costs. For this, the most time- table modifications, we have found that the per cent consuming steps have been automated or eliminated. utilization drops dramatically when anything but minor Three main modules are used: set-up changes are required between applications. In addition, changing certain stations may present safety ASE 200 extractor concerns due to their weight, size and table location Zymate XP robotic system presenting a whole new set of issues. Component switch- API 365 or Quattro II--HPLCmMS/MS system ing between applications may also be a potential source of error if it is overlooked by the end user. After extraction of soil samples with an ASE 200 extractor, the crude extracts are transferred to a Zymate At our Research and Development Center, there is no XP laboratory robotics system that performs evapora- single, redundant application that would justify the tion, reconstitution, centrifugation and filling into HPLC purchase of a robotic system if the criteria were that vials. If necessary, sample preparation is completed by a the purchase price be recouped in 1-2 years of service. In solid phase extraction station on the robotic system. addition, changing and evolving product lines and Because of the high selectivity of HPLC-MS/MS, clean project priorities cannot ensure the long-term utilization up is not currently used. The active ingredient and three of an automated system based only on a single applica- metabolites are ionized using an electrospray interface. tion. The table layout and programming of our Zymark For quantification of the analytes, internal standards are XP robotic system were designed in such a way that added post-column utilizing a second injection port to multiple applications can be easily implemented on it by compensate possible matrix effects in the ion source. the end-user with minimal training. The types of applications, table modifications/adapters utilized, and peripheral equipment which enhance the system's versatility Comparison of commonly used robots for labora- and multiple application capabilities will be discussed. tory automation Rodney Stockton, SLR Systems, Vancouver, WA, USA Extraction and derivatization of acid and neutral The most commonly used commercial robots utilized in impurities in illicit heroin using the XP robotics laboratory automation systems will be presented. Im- system portant parameters, e.g. speed, reach, payload, communication and software descriptions will be compared Sam D. Cooper, Drug Enforcement Administration, Special between the various models. The presentation will in- Testing and Research Laboratory, McLean, VA, USA clude a purely physical description as well as more based upon the author's experi- The instrumental phase being used in this laboratory for subjective comparisons ence. identifying acidic and neutral impurities in heroin samples requires approximately 60 min for an acceptable Robots to be included (but certainly not limited to) in separation of all components. The manual method for this comparison are manufactured by Beckman, Cavro, extraction and derivatization of components required CRS Robotics, Fanuc, Hamilton, Hudson Robotics, approximately 30min. However, due to the instability Mitsubishi, Motoman, Panasonic, Seiko and Zymark. of the derivatized components, this manual method, even When multiple models from one manufacturer are avail- with an autosampler, did not provide a workable solution able, all models normally utilized in laboratory opera- to the problem of increasing productivity. tions will be covered.

78 Abstracts of papers presented at the 1998 ISLAR

Formulation tools for cosmetic and personal care PyTechnology system employs a Zymate II robot with a laboratories Zymark storage carousel and a print and apply labeller. The labeller has been modified to house a barcode reader Stephen D. Phelan, Formation Systems, Westborough, MA, for plate verification. It takes about min to get a plate, USA print and apply a barcode, and return the plate. The Over the past decade, there have been striking advances label contains both barcode and human-readable inlbr- in computer hardware and software. Surprisingly little of mation. The labelled target plates are subsequently this technology has trickled down to the R&D labora- loaded on a compound handling robot [Frezza, J., tories of household product manufacturers, which are Tomlinson, J. j., Klemp, M. and Butler, B. T., Fully distinguished by a much greater degree of variability automated compound distribution to multiple biochem- than laced by discrete manulacturers. Recently, however, ical targets. International Symposium on Laboratory Automation tools for both R&D and production chemists in process and Robotics, Boston, MA, October 1997] for processing. companies have become available. These tools address The system is controlled through a Visual Basic front end formula and across the en- development management that retrieves barcode information from an ORACLE companies not to the terprise, helping only improve database. The barcode information is downloaded to an productivity of their chemists but also to achieve faster ASCII text file as a feature of the database software. A time to market. Such tools encompass marketing claims Visual Basic interface processes this information and and specifications management; laboratory tests, transfers the data to EasyLab variables for methods and results; formula creation; guidelines and appropriate use by the barcode labeller. restrictions; formula approvals and distribution; production support; and manufacturing feedback. Use of these tools can drastically increase the value of the formula The new era in high-throughput screening: minia- base, which is the primary intellectual property of the turized assays with 1536 Wells consumer goods manufacturer. Giinther Knebel, Greiner GmbH, Frickenhausen, Germany Reaction screening and optimization by auto- The necessity of miniaturized low-volume assay plates is mated workstation-based systems: Part I mainly driven by limited availability of test material, savings in reagents, higher throughput and finally, less L. C. Hsu, E. Webb and L. Pin, SmithKline Beecham cost well. Today, 96-well and 384-well plates are in USA per Pharmaceuticals, King of Prussia, PA, use, but significant developments must evolve to meet The shortening of drug development timelines has caused further requirements to reduce cost per assay. This Chemical Development to rethink its strategies for prod- constraint leads directly toward even higher density uct development. One approach has been to incorporate plates. robotics and automation into new drug development In close and Greiner paradigms. This paper will discuss the hardware and cooperation, Bayer (Germany) developed a new high-density plate on a non-proprietary the custom chromatographic configuration required for basis that fulfills the in automated screening and optimization of the Suzuki coupling reac- requirements fully To utilize most efficiently, the focus tion. The reactions were performed in a STEM block and systems. space only was 1536 wells, a four-fold increase over a 384-well plate. the samples were transferred to a Zymark BenchMate The centre of a of 16 wells is still at workstation for further sample preparation, including group unchanged 9.0 mm, while the well-to-well is 2.25 mm. A well dilution and filtration. The treated samples were then pitch volume of 12 gl allows and cell-based assays to analysed by an HPLC system that was directly connected bioassays be carried out. A new optimized well geometry in a to the BenchMate workstation. Finally the chromato- has been nade to overcome wick- graphic system was configured so that data can be shared square-rounded shape between Synthetic Chemistry and Environmental Re- ing in the corners. search Laboratory via SmithKline Beecham network. Furthermore, there has been strong evidence that clear This automated approach has the potential for accelerat- bottom plates will be required. A new moulding and ing drug development and process optimization in process technology enables us to manufacture unique Chemical Development. The modification of BenchMate clear bottom plates with ultra-thin films (751am). The and the intranet data infrastructure will be discussed in film bottom offers excellent optical properties, reduces Part II of the series. light tunnelling and minimizes well-to-well crosstalk. White opaque and black clear bottom microplates are advantageous when microscopic monitoring is required A custom for and Zymate system printing apply- cell-based These clear bottom will ing bareode labels (e.g. assays). plates have a major impact on assay application and new Brent T. Butler and Joan Frezza, Department of Molecular instrumentation for the detection of luminescence or Biochemistry, Glaxo Wellcome, Research Triangle Park, NC, fluorescence assays. USA; Joseph D. Simpkins and Anne Shrago, Department of This talk will cover the of solid and clear Research Technologies, GlaxoWellcome, Research Triangle application Park, NC, USA bottom plates in automated assays. Also, we will report on how liquid handling and imaging can be combined, We have developed an automated plate labeller for and ongoing developments for updated 1536-well ver- labelling both 96- and 384-well assay target plates. This sions.

79 Abstracts of papers presented at the 1998 ISLAR

Intranet control over several liquid handling work POLARA: a versatile architecture for scheduling cells via active XTM and running microplate assays Jeffrey A. Guss, Bristol-Myers Squibb, Wallingford, CT, USA Daniel Sipes, Joyce Kuoha and Jonathan Rosen, Ligand Pharmaceuticals, San Diego, CA, USA In an effort to streamline deployment and maintenance POLARA (Programming Architecture for Laboratory of liquid handling applications over several work cells, a is a Windows NT-based software novel approach using Active XTM was used to program a Automation) package for laboratory automation CRS Robotics user interface coupled to vender-specific software. The produced by Ontario, An overview of the benefits of undertaking this project are that it provides (Burlington, Canada). installation and of POLARA on an single-point application development, increased security operation existing custom CRS Robotics for cell-based in and a thin operating environment. In addition, the system assays will be application can be run 'off-line' from the work cells. This microplates presented. capability allows programming and debugging from TM remote locations. The Active X Intranet application NMR and automated reaction monitoring contains an Access 97 database, interface tools for the liquid handling work cells, Hamilton Eclipse 4.1 soft- M. Armitage, J. Richards, K. Sutcliffe and S. Moore, Smith- ware, liquid handling method selection, and a link to a Kline Beecham Pharmaceuticals, Tonbridge, Kent, UK TM paging system. The combination of the Active X Chemical Development within SmithKline Beecham is Intranet application components and the consolidation charged with developing an efficient process for the of deployment to a distinct location makes tracking work manufacture of drug substance. Such a process must cell activity and any errors a more manageable task. be demonstrated to be safe, robust, economically viable and environment friendly at a manufacturing scale. A thorough understanding of the chemistry using reaction Fluorescence-based assays in microwell plate for- monitoring can help achieve these goals. mat for high-throughput drug screening Monitoring has traditionally been performed off-line Steven F. Gessert, Suzanne W. Jones, A. Lee Lang, Toddy using techniques, e.g. TLC or HPLC, but NMR poten- Sewell and Sydney B. Shaw, Chromagen, San Diego, CA, USA tially gives much more information about the pathway of a given reaction. An on-line NMR reaction monitoring Rapid advances in the generation of potential drug system therefore would be beneficial to the scale-up targets through genomics and concurrent strides in the process. Whilst proprietary systems specifically designed creation of drug candidates through combinatorial chem- to perform these tasks are becoming available, we tried to istry have dramatically increased the demand for sensi- implement such a system with what was available to us. tive, rapid, automatable drug screening assay formats. This poster describes our experience with a reaction Chromagen has developed fluorescence-based gene monitoring set-up using an NMR spectrometer fitted transcription and protease assays in a high-throughput- with an LC-NMR probe and a Gilson 231 autosampler. compatible microwell plate format. These assays incorporate three of Chromagen's proprietary core The Gilson 231 autosampler consists of an XYZ robotic technologies: arm and a diluter unit. The instrument is programmable and capable of sampling, dilution and injection of (1) novel fluorophores and fluorogenic enzyme sub- aliquots of sample, and should therefore provide a suit- strates specifically developed for simultaneous able system for introduction of a sample into the NMR multianalyte detection; spectrometer. Our goal was to automate the system as far as such that unattended could be microwell-based, extraction chemis- possible operation (2) solid-phase achieved. tries for the isolation and purification of nucleic acids and peptides; and a scanning photon-counting spectrofluorometer Automated liquid handling confirmation of a capable of providing simultaneous multi-target Tecan Genesis detection. Jimmy Bruner, Rusty Czerwinski and James Ormand, Glaxo Welcome, Research Triangle Park, NC, USA The gene transcription assays [developed for the human An application has been developed to perform auto- cytokines tumour necrosis factor-alpha and (TNF- mated gravimetric confirmation of the Tecan Genesis's interleukin-6 (IL-6)] utilize DNA probes designed to liquid handling system. The application was developed in recognize specific sequences in target mRNA, and re- response to the time-consuming manual liquid handling quire neither nucleotide amplification nor reporter gene confirmation required for Good Laboratory Practice constructs. Detection levels in the attomole range (10E- compliance. The system consists of a Tecan 8 of specific mRNA are routinely achieved using (GLP) mole) Genesis 100, a Sartorius RC250S balance, lysates from 10E5 cells per assay well. The mRNA assay analytical and a custom ActiveX DLL for communicating with platform is applicable to numerous genes and can be Tecan Logic version 2.11. The system components were optimized for cell lines for a variety of organisms. The integrated using Microsoft Excel 97. protease assays developed for the coagulation cascade are homogenous assays and measure kinetics of substrate First, an ActiveX DLL was created to allow communica- metabolism. tions between external applications and Tecan Logic

80 Abstracts of papers presented at the 1998 ISLAR version 2.11. The LogicPipeComm.DLL consist of two high-throughput screening (UHTS) on post-assay data components, connection and communications. The con- processing. Data collection, storage, back-up and protec- nection component was created in Microsoft Visual tion are only a small part of the overall problem. The C + + and connects to the Logic pipe. The communica- more challenging task involves presenting this large tions component was created in Microsoft Visual Basic volume of data in such a way as to allow scientists to version 5.0 to access the connection component for a pipe make correct, timely decisions about potential errors in handle, and to write data to and from the Logic pipe. plate processing or treatment during the assay; to mark specific compounds, rows or entire plates for retesting in a Second, an Excel 97 VBA Basic application) was (Visual particular assay; to quickly find statistically significant created. The Excel application integrates the LogicPipe- 'hits'; and to assign a compound for further testing in Comm.DLL, Sartorius RC250S balance, and analytical This one approach at Tecan version 2.11. The controls all subsequent assays. poster presents Logic application addressing these issues, and explores future possibilities balance operations, starts the Tecan calibration method, with regards to data presentation and 'pre-analysis' of the receives data from the balance, and calculates the mean, data generated during UHTS. standard deviation and per cent bias of replicate aliquots. The LogicPipeComm.DLL gives us the capability to control Tecan Logic via external programming plat- Strategies for high-throughput HPLG purification forms, e.g. Visual Basic and Excel. Integration of the for combinatorial chemistry Tecan and a balance reduces the time the scientist must j. Carmody, U. Neue, R. Crowley and C. Andres, Waters spend recording weight data needed for GLP compli- Corporation, Milford, MA, USA ance. Traditionally, the search for biologically active molecules has involved the synthesis of one-molecule-at-a-time Versatile object weighing on a Zymark BenchMate discovery strategies. This has proven to be a very time- consuming and labour-intensive process for the discovery ,immy Bruner, David Igo and Sonia Shamdasani, Glaxo Well- of new drugs, catalysts and materials. New combinatorial come, Research Park, NC, USA Triangle chemistry techniques have reduced synthesis times by An application has been developed to perform fully allowing simultaneous generation of a large number of automated weighing of various objects on the Zymark chemical variants, several of which may be active leads. BenchMate. The application, known as 'ZyWeighing', Once this lead generation process is complete, the was developed in response to the time-consuming manual resultant combinatorial library is subjected to high- method of weighing containers and contents used in throughput screening techniques, one of which is HPLC. chemical investigations. The ZyWeighing system consists After biological activity testing, there is still the need to of a standard Zymark BenchMate, Zymark EasyServ isolate the active molecules without creating a bottleneck OLE Server, custom object carriers, and custom pro- in the laboratory. grams. Lead optimization, for many compounds, requires that The software consists of three elements. First, a Visual only a few micrograms or milligrams of a compound be Basic program was developed using OLE technology to purified to confirm the preliminary results. In this paper, access the EasyServ OLE and Microsoft Excel. Second, we will show straightforward HPLC strategies to devel- an Excel workbook was created for capturing the experi- oping fast gradient methods to quickly scale-up and ment and weight data. Finally, EasyLab programs were purify target compounds from other inactive combina- created for running on the BenchMate Controller. tions Customer carries were developed for objects of various size. Each carrier was designed to fit the standard Strategies for high-throughput HPL(] analysis for BenchMate rack. combinatorial chemistry The system has been shown to reduce the amount of time j. Carmody, U. Neue, R. Crowley and C. Andrews, Waters a scientist spends performing weighing operations for 4 h Corporation, Milford, MA, USA to 30 min for a standard set of 100 samples. Precision was assessed by weighing 50 individual samples 100 times Traditionally, the search for biologically active molecules using the resident Sartorius Model XH110 balance. The has involved the synthesis of one-molecule-at-a-time standard deviation was less than or equal to 0.1 mg. discovery strategies. This has proven to be a very time- Accuracy was examined using standards having masses consuming and labour-intensive process for the discovery ranging from 5mg to 2g. The weights ranged from of new drugs, catalysts and materials. New combinatorial 100.00 to 101.00% of their theoretical values. chemistry techniques have reduced synthesis times by allowing simultaneous generation of a large number of chemical variants, several of which may be active leads. Ultra-high-throughput data analysis Once this lead generation process is complete, the resultant combinatorial library is subjected to high- Neil Carlson, Nick Ware and Michelle Palmer, Assay Develop- throughput screening techniques, one of which is HPLC. ment Services, Tropix, MA, USA Bedford, Due to the plethora of compounds generated by the Now that it is possible to run 100000 assays in a 24-h combinatorial chemistry technique, minimizing analysis period, it is critical to look beyond the physical issues of time is paramount to meeting the major challenge of running these assays, and to address the impact of ultra- isolating the desired compound from other indigenous

81 Abstracts of papers presented at the 1998 ISLAR material as quickly as possible. Optimization of the Does your liquid handler pipette accurately? HPLC screening process to achieve shorter analysis times Ormand, Jimmy Brunet, Sarva Tadepalli and Cole is not always intrinsically straightforward. In order to James Harris, Glaxo Wellcome, Research Triangle Park, NC, USA achieve compressed analysis times, there is a need to more completely understand the effect that the column Many bioassays require organic solvents to solubilize characteristics (i.e. particle size and column length) and high concentrations of compounds, precipitate proteins, the operational parameters (i.e. flow rate and gradient quench enzymatic reaction, etc. Manufacturers, e.g. time) have on the selectivity and resolution of the Tecan, Packard and Beckman have designed a variety separation; the resolution and the selectively being the of liquid handlers to automate these bioassays. Water is two characteristics of the separation most affected by the benchmark liquid used when evaluating the accuracy changes in the column and operational parameters. and precision of liquid handling equipment. But, how accurately does this equipment handle non-aqueous In this paper, we will show straightforward HPLC liquids? strategies to developing fast gradient methods to quickly resolve target compounds from other inactive combina- In this example, preparation of plasma standards from tions. acetonitrile stock solutions using a Tecan Genesis 150 with Logic version 1.85 revealed a problem with the accuracy of delivering organic solvents within the normal Sample dissolution and replication station for volume range of the instrument. The observed concen- plates from combinatorial chemistry trations of standards prepared using the automated tech- were fbund to be 15% lower across the range Gerhard Mihm, Department Chemical Research, Screening nique of when to manually standards for two Support, Boehringer Ingelheim Pharma KG, Biberach, Germany compared prepared separate runs. Gravimetric measurements revealed that As a result of the rapidly increasing number of com- he system was only delivering 84% of the requested pounds prepared in the combinatorial chemistry groups, volume of acetonitrile, and similar results were observed there is an urgent need in the chemical repositories to with methanol. The solution to this problem was found in optimize the processing of the samples for high-through- the recently released Tecan Logic version 2.11 through put screening (HTS). Samples have to be dissolved and the use of liquid classes. into several and tubes in order to transfer aliquoted plates This poster will discuss the challenges associated with them to the HTS groups. In addition, an efficient and handling non-aqueous liquids and how manulacturers, reliable tracking of the platemaps is mandatory. We have e.g. Tecan and Packard handle these issues. designed and implemented, together with Zymark UK and Zymark Germany, a workstation-type robotic system based on the Zymark RapidPlate, including a robotic A flexible, generic robotic system that performs arm to handle the plates. Plates are tracked by barcode protein precipitation assay for research support labels. Data management is via file transfer between the L. DuPont Pharma- workstation and the dispensary ORACLE database. j. A. Short, E. D. Lynch and T. Lloyd, ceuticals Company, Newark, DE, USA As a drug discovery program begins to narrow the search Efficient handling of compound solutions for HTS for a specific compound to advance into drug develop- and follow-up assays using a new robotic system ment, the final candidate compounds within that re- Gerhard Mihm, Department of Chemical Research, Screening search class are studied in vivo in different animal Support Unit, and Rudolf Haider, IS Department, Research populations. Research support bioanalysis involves these Support Team, Boehringer Ingelheim Pharma KG Biberach, first in vivo sample analyses of a variety of closely related Birkendorferstrasse 65, Germany compounds. These studies are characterized by small sample batches typically 20-80 samples per compound, The demands for rapid and reliable handling of com- where analysis of multiple compounds is often required pound solutions for high-throughput screening activities within a single analytical run. There are lots of studies for and follow-up assays are increasing steadily. In addition, compounds closely related in structure, therefore leading the numbers to be stored and manipulated in the to the generation of a large number of different standard compound dispensaries are also increasing due to the curves for quantitation. Sample matrices vary consider- activities in combinatorial chemistry. ably and sample volumes are often small (200tl). Because these are the first in vivo studies with these In order to meet those needs, we have implemented an are forced to rather broadly automated storage and retrieval system lbr compound compounds, analysts guess solutions in microtubes, microtubes in the corresponding at sample concentrations. racks and microtitre plates (REMPASS). The samples The investment in method development at this stage is are stored in special storage racks at 4C and manipu- typically measured in hours, with almost all of the lated with a 14axes robotic system. The coordinates of the research support work involving protein precipitation samples are kept in a dedicated database. Requests for for sample preparation. The scientists performing these compounds are processed via the database used in the studies typically have broad responsibilities, with the compound repository for the administration of all bioanalytical component only comprising a small portion samples. The robotic system used for the manipulation of their duties. As such, an automated platform for these of the samples is presented here as well as the interface to applications must have a friendly user interface such that the dispensary database. users can walk up and quickly run the system with

82 Abstracts of papers presented at the 1998 ISLAR minimal training or understanding of the automation. Rich Garretson and Donald G. Sheer, Millipore Corporation, The system must have a high degree of flexibility to Bedford, MA, USA; Melanie Lin, Ken Parker and Martin accommodate the method changes associated with each Hornshaw, PerSeptive Biosystems, Framingham, MA, USA set of samples between or within analytical runs. The ability to remove salt and detergent from protein or Such a system has been created and implemented. peptide samples at the nanogram level has become rate- Features of this system will be presented. Some of the limiting in structural characterization studies. In many more formidable challenges to this approach, encoun- cases, the high sensitivity afforded by mass spectrometry tered in working with the Factor Xa class of compounds, is compromised by interfering contaminants that suppress will be explored and discussed. fragment ionization and obscure mass peaks. This com- plication along with the difficulties associated with microlitre volume and nanogram mass handling makes Automated HTS monitoring technology: laser sample preparation a significant challenge. To address fluorescence methodology for fingerprinting food, these demands, an adsorptive pipette tip containing beverage and pharmaceutical products previously evaluated reverse phase and ion exchange resins [J. Biomolecular Techniques (1997), 1, 0001, 0009] R. P. Gill, W. Smith and R. H. Selinfreund, Lion Laboratories, is presented for direct mass spectrometry. Sample recov- Saybrook, CT, USA ery, fidelity and reproducibility were demonstrated using HPLC and MALDI. The ability of these devices to The absorption of light by matter is related to molecular remove salt and detergents enabled the acquisition of structure. Some molecules can momentarily store excellent MALDI spectra for a variety of controlled energy by the excitation of electrons from the ground digests. state to excited singlet states. Relaxation fi'om an excited singlet state results in fluorescence via the emission of light at a different wavelength. The interaction of Use of the (lytofluor 4000 microplate fluorimeter fluorescent dyes with complex mixtures, e.g. foods and and twister plate loading robot for high-through- beverages, is highly reproducible, quantifiable, and can put yeast viability screens be used as a particularly valuable method to characterize Christopher L. Haber and James C. McGuire, Discovery Tech- mixtures. nologies, Pharmacia & Upjohn, Kalamazoo, MI, USA Using a proprietary library of dyes and the resulting Fluorescent dyes have been used extensively as indicators interaction(s) with single or multiple analytes, Lion of cell viability. Because of their robustness and ease of Laboratories has developed a method to numerically use, they are easily incorporated into high-throughput, describe fluorescent fields for foods and beverages. This whole cell screens with cell viability end-points. We have process in the most simple terms can be thought of as routinely used the alarmarBlue redox indicator in such mapping the fluorescent field of an analyte or mixture of screens and the Cytofluor 4000 microplate fluorimeter for analytes via interaction with specific dyes or dye com- determining the fluorescence intensity of the samples. binations. The dye analyte interactions may involve Recently, a robotic plate handling arm (the Twister) complexation and]or the formation of ionic or covalent has become available for the Cytofluor 4000. Here we bonds. The fluorescence utilized can be in the ultraviolet, describe the performance of the Twister-outfitted Cyto- infrared or visible regions of the electromagnetic spec- fluo in screening for least viability using 96-well and trum, and is expressed in a digital ttshion to develop a 384-well plates. The Twister currently holds up to 20 multiple fluorescent profile of a mixture designated as a plates. The software allows multiple plate data in a single fingerprint. In certain cases, this can be used as a specific file, and can accommodate > 20 plates per file (sequen- assay for a single analyte that may be either a key tial runs of up to 20 plates each). Raw data are exported ingredient or an undesirable contaminant; or more in a variety of file formats lbr use in data processing generally, the fingerprint can be used to differentiate programs. Throughput with 96-well plates with the authentic from non-authentic samples. Twister was 45 plates/h, 15 plates/h with 384-well plates. This system was used to screen at 116000 compound Lion has developed this process utilizing 96-well plates, library against a Saccharomyces cerevisiae permeability robotics and automated dilution/sampling techniques to mutant. provide a technology (patented) to assess authenticity of complex mixtures. Applications include spirits, beverages, fruit juices, flavours, natural extracts, e.g. vanilla, High-performance automated chemistry: syn- as well as individual ingredients, e.g. caffeine. The tech- thesis and beyond is also of value to characterize or nology fingerprint Richard Gray and Clare Ruddick, The Technology Partnership, chexnical processes, and it allows a simple mechanism Melbourn, UK for protection of intellectual property. The poster includes an overview of test design, robotic HTS infra- This poster illustrates various key features of the Myriad structure, sample handling and data management/ Core System (MCS) and Personal Synthesizer (PS) presentation. which were developed at The Technology Partnership (TTP) in collaboration with seven leading pharmaceutical companies. In particular, it will emphasize the most Microadsorptive tips for sample preparation recent developments which expand the range of chem- prior to mass spectrometry istry possible, including reactions with reactive gases at

83 Abstracts of papers presented at the 1998 ISLAR up to 4 bar pressure and reactions involving distillation A powerful new screening technology utilizing steps. The new automated liquid-liquid extraction capillary electrophoresis module is also described. This poster addresses the most common solution phase work-up procedure in a con- Y. M. Dunayevsky, L. G. Rashkovetsky and D. E. Hughes, venient and efficient instrument. Cetek Corporation, Marlborough, MA, USA A new screening technology based on laser-induced fluorescence with high-resolution capillary electrophor- The automated synthesis of a solid phase Ugi esis (CE) has recently been developed. This technology library greatly enhanced the ability to discover valuable 'hits' in George C. Morton, Timothy F. Herpin, Joseph M. Salvino, complex mixtures, e.g. natural samples (crude extracts, Jonathan S. Mason, Sheng-Yuh Tang, Gerald McGeehan and broth), combinatorial chemistry libraries and combina- Richard Labaudienier, Lead Dis.covery, Rhone-Poulenc Rorer torial biology mixtures. Examples using several targets Central Research, Collegeville, PA, USA and discriminating between strong, moderate and weak binding hits will be shown. With natural samples, these A novel resin-bound isonitrile was used in the Ugi multi- very sensitive CE assays can follow active fractions component reaction to generate a library of dipeptoid through multiple purification steps, and typically find carboxylic acids. One thousand, one hundred and thirty- numerous hits per active sample. six compounds were synthesized on two ACT 496 MOS robotic synthesizers. A Tecan RSP 5051 liquid handling robot was used to transfer a slurry of the resin into the A high-throughput luciferase gene reporter assay system: LucLiteTM, LumiGount(R) microplate lu- ACT reaction blocks and also to transfer the final prod- TM ucts. The automation protocol will be presented along minometer and Twister universal microplate with the general reaction scheme. handler Li Li and Steven Lane, Packard Instrument Company, Meriden, CT, SA Managing combinatorial chemistry analytical data using an electronic notebook interface and Reporter gene assays have been widely used in studying open database technology regulation of gene expression. The bioluminescent luciferase reporter assay has become the method of choice in Tom Clemow, Howard L Cannon and Curtis Marx, Groton reporter gene studies, especially in many drug-screening NeoChem, Acton, MA, USA applications, because it is the most sensitive, simple and rapid assay for quantitating transcriptional activity. In Improvements in combinatorial organic synthesis have this paper, we present a simple procedure or automating made producing thousands or tens of thousands of a luciferase reporter gene assay using the LucLiteTM compounds per month practical. Today's HPLC, MS luciferase reporter system, the LumiCount(R) microplate and NMR instruments accommodate or quantitative luminometer and the TwisterTM universal microplate qualitative analysis of thousands of samples per month handler. The LucLite reagent kit provides true long-lived with a limited number of instruments. glow luminescence signal for detection and a simple Existing data management tools, however, cannot easily homogeneous assay procedure. Tle LumiCount micro- or automatically combine data from multiple analytical plate luminometer enhances these advantages with a techniques, or with structural, activity and compound high-performance detection design, allowing detection location data. Without suitable management software, of firefly luciferase at the attomole concentration level analytical data are disconnected from other drug disin both 96- and 384-well microplate formats. The signal covery data. Future improvements in synthetic, analyti- is linear over an extended enzyme concentration range of cal and screening automation will exacerbate this five orders of magnitude using one instrument setting. bottleneck in drug discovery. The same attomole level sensitivity and extended linear detection range are achieved using the Twister universal The NeoLink/cc software automatically imports chroma- microplate handler, allowing for an increased sample tographic, MS and NMR results into ORACLE or any throughput and walk-away convenience. The resulting ODBC compliant database. This database also contains performance in sensitivity, throughput, accuracy and cost links to other drug discovery data including chemical advantages makes this combination of reagents, structure, compound location and activity. The software instrument and plate stacker an ideal system for micro- interface includes powerful data query and data display plate-based gene regulation studies and high-throughput functionality. The notebook interface provides a common drug-screening applications. viewer for raw and procesed data from different analytical techniques. Once data are associated in a notebook format, they can be saved and forwarded to other New techniques in solid-phase synthesis: gaseous researchers to support rehearsal or production syntheses. chemistry for parallel array synthesis Data extraction and association can be automated using Thomas J. Baiga, Charybdis Technologies, Carlsbad, CA, data templates for routine data analysis and reporting. P. This software tool USA; Gregory Roth, Boehringer Ingelheim Pharmaceuticals, effectively links analytical QC data to Ridgefield, CT, USA chemical library and HTS databases for more comprehensive and timely access to chemical synthesis intbrma- As part of an on-going program to further develop tion. the versatility of the Calypso Reaction Block System,

84 Abstracts of papers presented at the 1998 ISLAR

Charybdis teamed up with Boehringer Ingelheim to dispense in a non-contact mode for easier mechan- advance the state-of-the-art solid-phase synthesis para- ical alignment; digm. These new techniques involved gaseous chemis- rapid dispensing speed; and tries, where active synthesis reagents were delivered to wide dynamic dispense volume range (low nanoli- the Reaction Block as gases using the Calypso Gas tres to mid microlitres). Manifold. Three new chemical methodologies were developed: Pd-catalysed aromatic nitro group reaction, Pd- Increasingly, there is interest in using high-density micro- catalysed hydrogenolysis as a Wang resin linker cleavage plates (384 wells and greater) for screening applications protocol, and Pd-catalysed Still-type carbonyl insertion. to achieve higher throughput and reduce plastic and costs. Because these have a well These chemistries were originally developed manually in reagent microplates volume as small as 2 the typical dispense volume is the Calypso Reaction Blocks. But to further enhance and lal, in the sub-microlitre or nanolitre With its explore this methods development program, several range. ability to this volume the automation platforms have been integrated, notably the quantitatively dispense in range, Iliad PS2 Personal Synthesis System, the Calypso 4X nQUAD technology has found recent application as a useful This Shaker Workstation and the RapidPlate (R) multichannel technology in microplate applications. poster will show the characteristics and practical pipetter. The RapidPlate represents the newest product mechanism, issues in the to in an extremely versatile line of workstations to be applying nQUAD technology microplate integrated with the Calypso System. screening applications.

of a 384-well plate washer with a Throughput organic for Integration synthesis discovery robotic plate handler library production: the automation of the Ugi multi-component condensation reaction Jim Breunig, Mitotix, Cambridge, MA, USA Traci Trotman, Charybdis Technologies, Carlsbad, CA, USA; The advantages of the 384-well plate format for high- Chris Hulme, Rhone-Poulenc Rorer, Collegeville, PA, USA throughput screening can be greatly enhanced with the capability to automate the delivery and removal of the Faced with the task of daunting automating the synthesis plates to the instrument. A recent integration of a of more than 3000 compounds per week, scientists of Skatron Instruments, EMBIA 384-well plate washer with Rhone-Poulenc have several tech- TM Rorer integrated a Zymark Corporation Twister universal microplate nologies into a unique combinatorial chemistry produc- handler was tested and evaluated. Results of ease of set- tion the facility. Utilizing Ugi multi-component up, ease of use, throughput, reliability and overall condensation reaction as a probe for molecular diversity suitability for the high-throughput laboratory are pre- in their discovery libraries, the RPR Team has success- sented and discussed. fully automated most solid-phase synthesis operations. Charybdis Technologies Calypso Reaction Blocks (96 Multifunctional robotics compatible washer for well, filtration were initially prepared with variant) high-throughput screening synthesis resin. This task was automated by integrating the Calypso RM onto a RapidPlate (R) 96 locator plate, Paul Held and Gary Barush, Bio-Tek Instruments, Winooski, and simultaneously dispensing resin materials as an VT, CrSA isopycnic slurry. The Calypso RBs were drained, washed and prepared for synthesis on the Iliad PS2 personal Today's research requires that instrumentation be cost synthesis system. The Iliad delivered reaction solvents effective, reliable and easy to use. Bio-Tek Instruments and diversity reagents to the 96-well reaction blocks. would like to introduce the Elx405 multifunctional Reaction incubation was performed off-line to maximize washer. Because today's assays require multiple washing the compound throughput for the task-specific work- formats, the Elx405 can be configured in three different station network. modes of operation: (i) a combination of 96-well or 384- well washer manitbld; (ii) a 96-well only manifold; and (iii) a 96-well version with magnetic bead capabilities. Use of the nQUAD technology for nanolitre dis- The 96/384 manifold has a unique design (patent pend- pensing in microplate applications ing) which provides for independent control of aspirate and dispense tube location and height, enabling bubble- Dennis Stachelek, Don Rose, Tony Lemmo and Tom Tisone, free fluid-dispense and overflow protection in 384-well Cartesian Technologies, Irvine, CA, USA plates. The 96-well manifold allows for overflow protec- Cartesian Technologies has developed a nanolitre, quan- tion, as well as 'bottom washing', and standard features titative aspirate and dispense (nQUAD) technology for have been included to make the Elx405 washer robotics use in microplate applications. The technology couples friendly and ideal for automation. Features, e.g. vacuum the precision displacement resolution of a stepper motor- detection, are provided to ensure that the vacuum pump driven syringe pump with the rapid actuation capability is switched on and vacuum vessel are connected. Flow of a micro-solenoid valve. The result is a dispenser protection alerts the user to an interruption of flow to the technology with the following features: dispensing manifold. The washer has a waste detection device, which through software prevents overflow of quantitative dispensing of nanolitre volumes (down waste into the vacuum pump. Because buffer is drawn to 4 nanolitres); from reservoirs by suction from a syringe pump, pressur-

85 Abstracts of papers presented at the 1998 ISLAR ized buffer containers are not required for operation. The determined by reference to a pathlength/volume stan- optional buffer switching module allows for the automatic dard curve. Dispense volumes between 30 gl and 325 lal switching from up to four different buffers. The washer can be tested directly with typical standard deviations of also has a separate priming trough to allow for automatic 0.003cm (or 0.5gl in a half-area well microplate). A priming ('autoprime') and automatic rinsing ('autorinse') difference method enables volumes from 4 lal to 30 gl to during wash procedures. The Elx405 is robotics friendly be accurately tested with a standard deviation of with RS232 connections that allow communication with 0.015 mm. The method is fast, accurate and ideally suited several robotic devices. Assay protocols are capable of to test automated liquid handling stations, especially 96- being downloaded from a separate PC or programmed channel pipettors. through a washer keypad. Besides being versatile, the washer is lightweight (< 13.5kg), compact (41 x 41 x 26 cm) and capable of 110 and 220 V operation. A novel fluorescent HTS platform using homogeneous miniaturized cell- and bead-baseds assays for drug discovery and development An automation-friendly high-performance and high-throughput (HTS) assay detection system Elana Swartzman, Sheri Miraglia, Lolita Evangelista, Kenton integrated with a high-capacity microplate hand- L. Lohman and Pau Yuan, PE Biosystems, Foster City, CA, Ier USA, David M. Heffelfinger, Ed Goldbery and Bala S. Manian, Biometric Imaging, Mountain View, CA, USA Amer E1-Hage, Carl Wright, Mike Biros and Doug Modlin, LJL BioSystems, Sunnyvale, CA, USA Detecting cellular biochemical or molecular events in reaction to exogenous drug stimuli in live cells has always An overview of the design, performance and features for TM been a challenge to drug development groups. Homo- laboratory integration of the Analyst will be pre- or 'no wash' on live cells have been TM geneous assays sented. Analyst is LJL BioSystems' new multi-mode especially difficult and have lacked the sensitivity needed detection platform for high-throughput screening (HTS) for effective evaluation of biological events. A number of for accelerated drug discovery. The instrument was instrument and biochemical technologies have been designed from conception to operate in an automated developed to address this issue. Most have concentrated laboratory environment as p_art of a workstation or on elucidating drug effects on live cells earlier in the drug robotics line set-up. Analyst has already been success- screening process. In most cases, however, high-through- fully installed and integrated into several automated put formats have been sacrificed for the increase in laboratories with robotics systems and workstations with information. minimal effort. An integrated microplate stacker option to the instrument improves throughput and efficiency, In a co-development effort, PE Biosystems and Biometric and a robot-ti'iendly microplate carrier reduces both set- Imaging have developed a new, two-colour, fluorescence- up time and process errors. In addition, a graphical user based instrumentation which enables the user to rapidly interface that runs on a local host in Windows 95 or NT screen large numbers of drug candidates on live cells facilitates system set-up and method development. We using homogeneous assay formats in standard microplate will highlight in this poster the integration of the Analyst formats up to 1536 wells. The instrument is based on the with the Zymark TwisterTM microplate handler as a laser scanning microvolume fluorometry technology de- specific example. veloped by Biometric Imaging tbr cytometric analyses. Homogeneous receptor/ligand studies, signal transduc- mechanisms and cell death determinations can all be Test liquid handling systems from 4tl to 3251 tion quickly and easily with Path(heckTM sensor made rapidly on live cells. Data are collected for each individual cell in the scan area, which is unique for high- Evelyn McGown, Kirk Schroeder and Dean Hafemen, Molecular throughput instrumentation. The two-colour format Devices Corporation, Sunnyvale, CA, USA provides the user with the ability to evaluate two cellular events in a cell in a As part of a laboratory quality system, pipettors and biological simultaneously single high- mode. dispensers should be checked regularly to verify perform- throughput ance. Gravimetric methods for pipettor calibration are In addition, two-colour homogeneous, bead-based ver- adequate for single-channel devices but are impractical sions of standard biochemical assays previously devel- for multichannel pipettors. Spectophotometric methods oped for solid phase technologies, e.g. immunoassays, are typically require an added dye, which is convenient and easily accomplished on this platform. potentially erroneous because the dispense volume of the liquid may be affected by the dye. The Molecular We will describe the specific, homogeneous, biochemical Devices SPECTRAmax(R) Microplate Spectrophot- systems and instrumentation that we have developed to ometers employ a patented PathCheck sensor which investigate the effect of candidate drugs on live cells in offers a fast, reliable method lbr checking performance high-throughput formats. of multichannel pipettors and dispensing stations using without added dispense reagent, dye. Detecting the binding of fluorescent neuropeptide The performance test is performed by dispensing the Y to GHO-KI cells expressing NPY receptor sub- reagent into a 96-well microplate and measuring the types using fluorometric microvolume assay tech- optical pathlength in each well. Precision is calculated nology (FMAT) in a high-throughput, cell-based directly from the pathlength data and accuracy is format

86 Abstracts of papers presented at the 1998 ISLAR

Sheri Miraglia, Elana Swartzman, Lolita Evangelista, Kenton Work-up in chemical organic synthesis consists of a series Lohman and Pau Yuan, Perkin Elmer Biosystems, Foster City, of post-reaction operations designed using differential CA, USA; Bala Manian, Biometric Imaging, Mountain View, chemical properties to remove excess reagent or starting CA, USA material, reagent products and, where possible, reaction by-products. Careful consideration of post-reaction op- The development of molecules that mimic, enhance and/ erations as a clean-up step can obviate the requirement or block the interaction of bioactive peptides with cell for purification. This has been an attractive feature of surface receptors, in particular the subset represented by some MPS at Alderley Park, which we have attempted to the 7-TM (transmembrane) G-protein-coupled recep- address with automated procedures. tors, is a major focus of pharmaceutical development. The rapid identification of potential leads is a crucial first Generally, work-up can be resolved into four operations: step in this process and would be greatly facilitated by filtration, solvent addition (dilution, trituration), wash- simple, homogeneous, high-throughput screening assays. ing and separation (partition). The ordering and selec- Currently, the scintillation proximity assay provides a tion of these four basic operations through an operator- homogeneous format for monitoring receptor-ligand controlled interface will, therefore, create a generic work- interactions; however, the assays are all bead-based and up program. This presentation will describe the flexibility require the use of radioactivity. We have developed a and power of a Zymate XP-based generic work-up technology, fluorometric microvolume assay technology protocol as well as the safeguards necessary for its smooth (FMAT), which enables detection in the red region and operation as an open-access facility. Thought is also given is compatible with various formats, including 96-, 384- to the development of a condensed, bench-top system for and 1536-well plates. Using this technology, we are able use in a standard chemistry laboratory fume cupboard. to detect the binding of fluorescent neuropeptide Y to transfected cells expressing either the neuropeptide Y Solid phase extraction as an efficient method of type receptor or type 2 receptor, and identify the IC- purification for automated synthesis 50 values of competing unlabelled peptide. Ligand binding assays were pertbrmed in high-density, low-vol- Marian Young, Harold Weller, Jacques Roberge and Walter ume 384-well plates. This technology can be used in a Ruediger, Bristol-Myers Squibb Pharmaceutical Research Insti- cell- or bead-based format, and is unique in that it is tute, Lawrenceville, NJ, USA simple, homogeneous, high throughput and non-radio- As pharmaceutical companies continue to search for new active. drugs, automated synthesis is becoming more widely used. These synthetic procedures often produce compounds that need purification before they are submitted Performance of Labsystems' microplate instru- tbr biological screening. ment interfaced with the Assist microplate handling device Solid phase extraction (SPE) is an automated high- throughput purification method that was originally used Ron Gulka, Labsystems, Franklin, MA, USA for concentration of biological and environmental samples. We have modified these methods to be useful The Labsystems' Assist microplate handling device easily for purification of synthetic compounds. Most commer- and economically automates the manipulation of 96- and cial instruments using SPE cartridges process one sample 384-well plates. The Assist can be interfaced to a wide at a time. Because each sample can take 20-60 min for range of Labsystems' microplate instrumentation, includ- purification, a modest automated run of 96 compounds ing dispensers, washers and readers. Highlighted will be can take several days to purify. the performance specifications of the Assist and the Multidrop 384 dispenser, Wellwash Ascent plate washer, We have developed with Bohdan an instrument that is Multiskan Ascent photometric reader, Fluoroskan Ascent capable of processing a set of 96 compounds purifying fluorometric reader, and Fluoroskan FL combination eight samples at a time. This instrument has the versa- fluorometric and luminometric reader. tility of collecting up to three fractions for each sample, and using either 3 or 6 ml cartridges. This capability has allowed the purification of 288 compounds in a single A versatile, fully automated open-access work-up day. We designed the graphical interlSce to be very user station designed to serve the needs of the rapidly friendly, as we encourage walk-up use of our automated expanding field of multiple parallel synthesis equipment. The development of instrumentation, mod- (MPS) ification of SPE resins and purification methods used to make SPE a useful and efficient method of puriting Gordon A. Hamlin, Zeneca Pharmaceuticals, Alderley Park, libraries of compounds will be discussed. Macclesfield, Cheshire, UK The rapid growth of multiple parallel synthesis (MPS) in Automation and library production at Selectide our laboratories has created a demand tbr a robust, easily Eric Kirsten Bjergarten, Marry Vanderstelt and accessed automated work-up procedure, as the manual Wegrzyniak, Daniel Schirlin, Selectide, a Hoechst Marion work-up of large numbers of small-scale reactions is both subsidiary of Roussel, Tucson, A USA time consuming and tedious. Manual work-up is also a z, rate-limiting step in the generation of large numbers of In order to complement the well-known 'one bead one compounds for test. compound' or 'split and mix' technology originally

87 Abstracts of papers presented at the 1998 ISLAR developed by Selectide, we have designed a system for standard PC and controlling LaMOSS through the high-speed synthesis (HSS) of combinatorial libraries in normal EasyLab high-speed connection. commercially available 96-well plates. Libraries are produced either as large collections of individual This connection became available to us with the use of Zymark Tools for WindowsTM, and relatively straight- compounds, or as low-complexity mixtures, thus drama- TM tically decreasing the need of deconvolution for hit forward user-interface was developed in Visual Basic structure determination while maintaining a high-syn- for writing and controlling a synthesis. Because this thetic throughput. approach demands a direct connection between PC and controller, the tube-transfer to the XP-system is now The system uses a workstation approach. A Zymate XP organized with the use of switches and inputs in both robotics arm distributes the plates among local robotics systems. units performing various organic synthesis tasks, where multiple microtitre plates are processed simultaneously. The top level software consists of a Synthesis Editor for The tasks include resin dispensing, reagent addition, writing and running the recipe and an Event Viewer for incubation, washing and cleavage. A description of the showing the controller status. The chemist chooses the different workstations, along with the strategies followed desired events via buttons and pop-up windows, and the for libraries synthesis on solid phase and/or solution phase program shows the recipe in 'chemical' language. Sub- will be discussed. sequently, the program adds the appropriate unit operations and parameters to be sent to the controller. In this way, the controller is handling a complete unit operation User-friendly top level software for controlling an by itself, so there still is a minimum of communication automated combinatorial chemistry system time involved. Ton Jenneboer and Will Kuijpers, NV Organon, Oss, The Netherlands Automated method transfer: 'a tale of two laboratories' At the end of 1996, a fully automated robot system was installed in our research laboratories to support combi- Timothy A. Diehl, Janssen Pharmaceutica, Titusville, NJ, natorial chemistry activities. The heart of the system is USA formed by LaMOSS (Labotec Modular Organic Syn- thesis System), a synthesizer capable of performing liquid 'It was the best of times, it was the worst of times', as phase as well as solid phase chemistry. LaMOSS is based Dickens wrote, captures the implementation process of on Zymark technology. Its features include nozzle-based automation systems in non-R&D laboratories. Imple- handling of reagents, building blocks, solvents and resin- menting automation in a quality assurance laboratory slurry (including split/pool procedures), temperature has always been a difficult task due in part to the control of the 48 reaction vessels and collection of the technical and managerial roadblocks. However, as auto- products in standard 16 x 100mm tubes. These tubes mation becomes an integral part of many corporations' can be transported with a shuttle to a Zymark XP robot strategies to increase efficiencies, the inertia to overcome where evaporation and liquid-liquid extraction can be these obstacles is being gained. The use of technology performed. transfer process helps to determine if any analytical method developed on a research instrument will work LaMOSS is provided with pre-programmed basic func- on a production instrument. However, these require- tions which perform the most simple tasks (e.g. nozzle- ments may also complicate the implementation process. movement, set switches). We have added so-called unit Factors both positive and negative that influence the operations, each of them performing a specific task using implementation of a global automation plan will be the basic functions (e.g. add liquids, transfer tubes). Until discussed. Experiences encompassing logistics, manage- recently, the actual synthesis was programmed in the ment, regulatory, compliance, communications and tech- recipe which contains a list of unit operations accom- nology transfer will be discussed in this presentation. panied by the selected parameters. This recipe had to be written prior to every synthesis. The disadvantage of this set-up was the complex and time-consuming program- Development and transfer of an analytical chem- ming of the syntheses. istry workstation to a manufacturing site Initially, LaMOSS was connected as local controller to Craig Bender, David Otto, Alan Wickman and Mark Schweit- the XP-controller to enable communication between zer, Searle Research and Development, Skokie, IL, USA both controllers in case of tube transport. In this set-up, syntheses were programmed entirely in the LaMOSS Automated analytical chemistry workstations can be very controller using EasyLab for WindowsTM and could be useful for a quality assurance lab at a pharmaceutical started either from there or by the XP-system. As a manufacturing site. Workstations can provide increased consequence of this set-up, manual interference in one sample throughput, increased turnaround times, and system (stop and abort) resulted in a stop of the other more accurate and precise results. Methods can be system as well. developed by Research and Development labs and transferred to the manufacturing site on the automated These experiences encouraged us to find a better way of equipment. Automating the analysis using the same controlling LaMOSS. This paper describes the develop- sample preparation parameters can ensure results that ment of user-friendly top level software, running on a agree between sites.

88 Abstracts of papers presented at the 1998 ISLAR

An automation project was undertaken by Searle Re- precision (interday and intraday precision of 7.0% and search and Development and Bohdan Automation 9.8%, respectively) and accuracy (interday and intraday (Mundelein, IL) to automate the analysis of dosage form relative errors of-t-2.7% and :t=6.7%, respectively), and assay and content uniformity testing. Two identical recovery (greater than 90%) over a calibration range of systems have been developed, one for Research and 5-1000ng/ml. The method was used to assay over 2500 Development, and one for our manufacturing site in samples, standards and controls in support of toxicology Puerto Rico. Each automated system can process up to studies in mouse, rat, dog and monkey. Benefits of the 30 samples by serial processing using the following steps: automation included saving of analyst time, and decreasing exposure of analyst to biological and chemical media into (1) dispense sample tube; hazards. Evaluation of automation performance (includ- vortex to (2) dissolve; ing failure rate, carryover and throughput filter benchmarks) (3) sample aliquot; will be discussed. Adaptation of the method for use with (4) dilute samples; and 96-well technology and insights gained during the study (5) inject sample onto HPLC. will be presented. System validation and transfer activities to Puerto Rico are currently underway. Protocols and methodologies Determination of 264W94 and 2169W94 by LC/MS/ have been developed by Searle R&D. This presentation MS using a 96-well SPE: a comparison of semi- will describe the activities that have taken place during automated and fully automated approaches the transfer of the automated system to Puerto Rico. Lisa St. ohn-Williams, Glenn A Smith, Ifathryn B. Ifenney and John A. Dunn, Glaxo Wellcome, Research Triangle Park, NC, High-throughput bioanalytical mass spectrometry USA using 96-well solid phase extraction The compound 264W94 is currently under investigation ohn aniszewski, Monica Swyden and Hassan Fouda, Drug as a hypercholesterolemic agent. An automated method Metabolism Department, Pfizer Central Research, Groton, CT, for the quantitative determination of the drug and its USA demethylated metabolite, 2169W94, in human serum Coupled with HPLC/atmospheric pressure ionization-- was developed and validated using solid phase extraction mass spectrometry, microtitre 96-well plate technologies in the 96-well plate format. The extracts were analysed for sample and liquid handling have dramatically in- using electrospray ionization LC/MS/MS. The com- creased our drug metabolism bioanalytical throughput. pounds are isolated from human serum using Waters' TM extraction The calibration Over the past several years, we have focused our efforts Oasis HLB plates. range of on developing and integrating several bioanalytical the assay was 0.1-100 ng/ml. throughput-enhancing approaches for use in our depart- Two different automation approaches were validated ment. These efforts have led to the development of and compared. The original method was developed using standardized protocols for 96-well solid phase extraction a Packard Multiprobe 104DT and vacuum manifold. methods development, assay validation and sample The method was later transferred to a fully automated analysis for the quantitation of drugs and metabolites Zymark XP robotics system interfaced with a Tecan RSP in biological fluids. The protocols feature the routine use 8051 liquid handling workstation. The latter system uses of X-Y liquid handlers from the outset of methods centrifugation to carry out the solid phase extraction, and development through assay validation and sample analy- is the first example demonstrating this technique using sis. The liquid handlers are used for standard curve these new robotics systems. preparation and sample transfer to the 96-well format. Standardized dilution templates and plate layouts are The validation data from both automated systems con- used, reducing the need for customized software pro- firm that the specificity, precision and accuracy of the gramming. The SPE plates are processed using a pro- assay are sufficient to support the quantitation of grammable 96-well pipettor, the Quadra 96. Once an 264W94 and 2169W94 in clinical trial samples. The analytical method has been developed, a single analyst problems encountered during the development of the can routinely process 400-600 samples in about 4 h. Most automated methods and the validation results will be of the analyst's time is taken in set-up and sorting of the presented. The two approaches will be compared, and samples prior to and while using the workstations. their unique advantages and disadvantages will be discussed.

Evaluations of validated assays using automated solid-phase extraction with LC.,/MS/MS for ana- A comparison of pressure- and vacuum-based lytes in plasma automated 96-well SPE systems for the LC-MS/ MS bioanalysis of haloperidol in plasma Naijia H. Huang, Lloyd R. Whitfield, John R. Kagel and David T. Rossi, Parke-Davis Pharmaceutical Research, Division of Jaap Wieling, Jelle Hempenius, Ruud J. J. M. Steenvoorden Warner-Lambert Company, Ann Arbor, MI, USA and Jan H. G. Jonkman, Pharma Bio-Research International B. V., 9470, AE Zuidlaren, The Netherlands Automated solid-phase extraction (SPE) using a Rapid- Trace workstation was developed with LC]MS/MS to Since the introduction of liquid chromatography with qualify a drug candidate in plasma for blood lipid tandem mass spectrometric detection (LC-MS/MS) as a regulation. The validated method was characterized for quantitative technique in the field of routine bioanalysis,

89 Abstracts of papers presented at the 1998 ISLAR sample preparation became the new throughput-limiting ever, it soon became evident that even conversion from step in the entire analytical process. Manual solid-phase 96-well formats to 384 was not going to be as simple as extraction (SPE) is a particular example of this situation. expected. New designs tbr 384-well microtitre plates are evolving to address the problems identified with the We have implemented various instrumentation for auto- earlier designs, e.g. wicking and pipetting with multiple mated 96-well SPE (SPE96), either based on positive tips. It was appearing that UHTS would have to wait for pressure (Hamilton Microlab) or based on negative for better plate designs, remodelled pipettors to specifically pressure (vacuum) (Packard MultiProbe) pipetting address 384-well formats and lower volumes, clinical samples, and the application of conditioning, pipetting and changes in quantitation techniques to imaging. In washing and elution solvents on the solid-phase bed the the issue of how to and move the (3M Empore extraction plates) and subsequent flow meantime, manage plates required for 100000 compounds needed to be through. To compare and validate both principles, we addressed. In the fall of 1996, an idea emerged around have implemented an SPE procedure tbr an LC-MS/MS the simple of TM instead of robots, method for haloperidol in human plasma on the Hamil- concept platemovers and reducing the HTS process to a series of individual ton and Packard systems, keeping the experimental con- steps separated by time. A robotic concept now called ditions for the extraction on the two systems as similar as TM a of possible. Besides validation results on precision and Allegro by Zymark would be series fully indepen- accuracy, some other per|brmance aspects were assessed, dent modules working together in a process-like manner very similar to an assembly line. The simple motions e.g. throughput, number of assays to be repeated due to TM blockings, human intervention, detection of levels and required of the platemovers would allow them to carry-over. move faster, make them far more dependable and much easier to program. These modules could be easily re- The validated calibration range was from 0.100 to ordered, passed or linked to create new procedures. The 50.0 ng/ml. The inaccuracy and imprecision were below system would be fully enclosed to allow control of the 15% at all concentration levels and for both systems, assay environment in terms of gas, temperature and although the coefficients of variation for the vacuum humidity. This new modular robotic system completely system were significantly better (two-sided F-test). This processes 1200 plates of a 96-well design in 24 h. Alle- was most likely caused by the Hamilton's flow detection gro'" will also allow for easy incorporation of new system using a pressure-drop monitor, leading to drying technology-based modules. As this new technology is out of the wells and subsequent channelling and irregular incorporated, the future throughput in terms of total wetting of disks, and thus less reproducibility within an number of wells could be even higher. Once AllegroTM SPE96 plate. Secondly, the larger carry-over of the has finished its initial test programs, assays that are pipetting equipment of the Hamilton system caused less compatible with a 384 format will be started. This precision. presentation will present the AllegroTM system applica- Although the sample throughput of the Packard systems tions as well as an evaluation of this new concept as it may be higher as a result of a four-needle configuration, pertains to performing UHTS and its integration in the current commercial systems cannot operate fully unat- research environment. tended; technicians must interfere by changing 96-well collectors underneath the plates, positioning sample (fell-based assay miniaturization for ultra-high- to HPLC plates, and transfer of the sample collectors the throughput screening systems. The Hamilton operates slower (current system applies only one needle), but operates fully unattended. Ilona Kariv, Anthony M. Mafia and Kevin R. Oldenburg, DuPont Merck Pharmaceuticals, Wilmington, DE, USA AllegroTM: moving the bar upwards Recent advancements in the fields of combinatorial chemistry and genomics greatly increased the size of R. W. Pharmaceutical Research Mary Jo Wildey, Johnson Inst., chemical libraries and the number of targets against Raritan, USA, Carol Ann Homon, Ingelheim NJ, Boehringer which they are screened. This substantial growth in Pharmaceuticals, CT, USA, and Hutchins, Ridgefield, Burleigh library size and target number necessitates changes in Zymark Corp., Hopkinton, MA, USA high-throughput (HTS) screening. One solution is to At the 1995 Society for Biomolecular Screening Con- miniaturize the HTS format, thus providing a possibility ference, the term ultra-high-throughput screening to increase throughput and save reagent cost. Miniatur- (UHTS) was introduced. UHTS was to be the next ization involves designing and adapting current HTS generation of screening and was defined as the screening assays to high-density lbrmats, either 1536-well (5gl) of 100 000 samples in an assay in a day. This new rate of plate for cell-based assays or 6144-well (0.7 gl) plate for screening would be required to test the million-com- soluble enzyme assays. Although some progress has been pound libraries enhanced through combinatorial chem- achieved in the last few years in adapting bacterial and istry of the future. At that time, most highly automated protein-based screens to high-density formats, mam- systems had a throughput of 100 plates a day. The major malian cell-based assay modification still remains a major limitations tbr these systems were the robotic designs and challenge. One of the primary obstacles is the ability to the time of analysis. Cost was to be a major factor for the dispense cells in a small volume, while maintaining cell screening of these new million-compound libraries. To integrity. This is especially true for functional assays maintain costs, new technologies would need to be relying on transcriptional machinery to express genes of incorporated into the screening solution. Miniaturization interest. Extensive assay run time creates the additional and nanotechnology became the new buzzwords. How- problem of liquid evaporation. In this study, we selected

90 Abstracts of papers presented at the 1998 ISLAR a cell-based assay utilizing luciferase reporter gene under cant challenges in relation to assay technology, screening control of the gene of interest 5 binding site in a stably logistics, automation and management of the processes. transfected Jurkat T cell line, as a model. The objective The challenges and the opportunities they present will be was to identify specific inhibitors of the expression of the discussed. gene of interest. Limitations and advantages in adapting this assay to high-density format, 1536-well plate, and technical considerations and challenges are further dis- Leveraging a discovery organization: the Sepracor cussed in this report. experience ames R. Hauske, Sepracor, Marlborough, MA, USA TM ExtremeScreen meeting the challenges of ultra- Discovery at Sepracor is shaped by the strategic impera- high-throughput screening tives of the ICE portfolio, i.e. our drug discovery efforts Michelle Palmer, Mark Roskey, Bonnie Tillotson, Neil Carlson, are directed toward the therapy areas of inflammation, Mike Nemzek and Irena Bronstein, PE Tropix, Bedford, MA, pain and urological diseases. We have attempted to USA enrich the highly successful ICE( approach by broadly defining each of the therapeutic areas to include a wide Extreme high-throughput screening (EHTS) at rates range of molecular targets with potential therapeutic beyond 100000 compounds/day is an emerging tech- advantage over existing approaches. These approaches nology. It is diflicult for a screening laboratory to are ultimately directed at disease with unmet, undermet, dedicate resources to all the new screening technologies medical need and significantly large patient populations. and incorporate them into their working environment. By focusing our efforts on the core competencies of The challenges of EHTS include selection of technologies synthetic chemistry, medicinal chemistry and combina- and formats, the associated learning curve, adaptation of torial chemistry, we have developed a proprietary and existing assay methodologies to new formats, and re- highly diverse corporate file of small, drug-like molecules. quired infrastructure and logistic issues. Our strategy of developing collaborative relationship Tropix ExtremeScreenTM is a new concept that provides with selected biotech partners with proprietary molecular a three-tiered approach to delivery advanced chemilu- targets, or proprietary platform technology, in the thera- minescent screening technologies. First, ExtremeScreen peutic areas of strategic interest to Sepracor provides a provides proprietary chemiluminescent dioxetane screen- means to most effectively leverage our ever-expanding ing reagents to meet the stringent demands of yielding corporate file of drug-like molecules. Combining our high-quality data across a wide range of assay types in a unique skills in combinatorial chemistry and medicinal single platform at very high-throughput levels in a cost- chemistry with the highly evolved HTS competency and effective manner. Second, ExtremeScreen rapidly devel- proprietary molecular targets of each of our biology-rich ops custom screening assays tbr deployment on screening collaborators has driven the rapid implementation of our systems at the user's site. Third, ExtremeScreen is a drug discovery strategy. We will describe the approach complete service to perform advanced screens at rates that we utilized to define the portfolio of Sepracor above 100000 samples per day with in-house or custo- Discovery, as well as the implementation of the discovery mer-supplied libraries, bringing extremely rapid turn- programs. around on large or small screening projects. The combination of the three elements significantly contri- Leveraging your technology investment: maintain- butes to accelerating the drug discovery timeline. ing the competitive edge in drug discovery Ralph Infantino, Schwedes, Margaret Reynolds, Mimi logistics and common sense: chal- anine Technology, Rivera and Mel Reichman, OSI Pharmaceuticals, Uniondale, lenges and opportunities in high-throughput NY, USA screening Recent advances in drug discovery technologies a Major, Lead Department, Pharmaceu- present John Discovery Zeneca wider range of opportunities that require careful evalua- ticals, Alderley Park, Cheshire, UK Macclesfield, tion for compatibility with existing systems. This can be High-throughput screening (HTS) is a key process in an arduous task for an established facility with much pharmaceutical lead identification. Screening through- invested in the current infrastructure. Because HTS is no puts of tens of thousands of compounds per assay run are longer the rate-limiting factor in discovery, established typical. Advances in chemistry are providing opportu- R&D sites specializing in it must carefully weigh its nities to access increasingly large compound sets. This options and aim for rapid return on investment. Issues development is fielling a trend towards greatly increased that must factor into decisions on increasing throughput screening capability. Effective systems need to be devised and capacity must address questions involving how for storage, retrieval, formulation and supply of com- quickly a screen must be completed versus the pace at pounds to screens. HTS operations need to be fuelled which assays are formatted and validated. Moreover, with novel drug targets, and screening systems capable of solutions should address how to leverage the resources efficiently delivering leads rather than hits are urgently required for lead follow-up, medicinal chemistry, com- required. There is a need to deploy the principles of high- pound acquisition and pharmacology. We will describe throughput screening against downstream functional our philosophy and operational approaches that allow us biology and the drug adsorption, metabolism and tox- to derive maximum value from our formidable existing icology bottlenecks. These requirexnents present signifi- infrastructure dedicated to HTS, while keeping pace with

91 Abstracts of papers presented at the 1998 ISLAR emerging technologies ad the ever-increasing need to Automated method validation of an oral product's increase throughput and capacity. assay and content uniformity Samuel Barrera, Rafael Cruz, David Ledn and Carlos Rivera, An overview of the methods validation process Bristol-Myers Squibb Company Bristol Laboratories Corporation, Quality Control, Mayaguez, PR, USA Patrick J. Faustino, Center for Drug Evaluation and Research, QC challenges for improving productivity, reducing Office of Pharmaceutical Science, Division of Product Quality cycle time, improving efficiency and maintaining a high Research, Laurel, MD, USA level of regulatory compliance require a high application of con- The methods validation process involves three areas: advanced technology. On the other hand, the centrated efforts of AR&D in a fast validation, evaluation and verification. These terms are drug discovery, pace defined by the Analytical Methodology Technical Com- of newly launched products and the constraints of cost reduction have led to more of labs in mittee (AMTC) of the FDA within the current revision independence QC under 'good guidance practices' (GGPs) of the 1987 method validations. This situation triggered the QC guidance: 'Guideline for submitting samples and analy- initiative undertaken at Bristol Laboratories Corporation in PR to validate the TPWI workstation for tical data for methods validation'. The three areas out- Mayaguez, the automated of and line the regulatory responsibility of the drug sponsor and sample preparation BuSpar assay content based on of the the agency. Validation is the responsibility of the drug uniformity previous applications lab in Evansville of this same company. This project sponsor to prove the analytical method will work accord- QC has opened the door for other applications that will result ing to its intended Evaluation is the purpose. responsi- in and other benefits of bility of the review chemist. Evaluation is the analysis of higher throughput, efficiency technology for success. the drug sponsor's validation of the analytical method. Evaluation also defines what analytical procedures and performance parameters are required and specified in the Keys to successful method validation and method 1987 guidance and the USP chapter 1225. Verification is transfer in the R&D environment the responsibility of two FDA field laboratories to establish the suitability of the method for regulatory purposes. dTon P. Sadowitz and Sam Li, Barr Laboratories, Pomona, NI, Further discussion of the methods validation process will USA also include I CH guidelines and FDA's Chemistry Automated techniques for sample testing are readily Manufacturing and Controls committee's review practice increasing in the R&D environment. With this increase and guidance development. comes the need for pertinent validation that streamlines the path for use of these techniques, and addresses the means to transfer these methods to other areas or Development and validation guidelines for auto- departments, either locally or remotely. This presenta- mated methods automation subgroup of the phar- tion will address the prevailing philosophies, techniques maceutical and analytical sciences group and protocols of the R&D automated method development team, and how they affect the automated method SmithKline Beecham John Stanley, Pharmaceuticals, Harlow, validation of solid dosage forms of assay, content uni- Essex, UK formity and impurities testing along with the transfer of The Automation subgroup of the PASG in the UK was these methods to the QC environment. set up at the beginning of 1996 and has since grown to include representatives from pharmaceutical companies, Automation and robotics--the key elements in vendors and contract laboratories. The purpose of the global standardization of physical test methods group was to exchange experiences on robotics and to influence vendor developments. However, it soon became Ramasamy Tamilarasan, Paul L. Morabito and Jonathan ,. clear that the most immediate need, and probably Zieman, The Dow Chemical Company, Midland, MI, USA; the greatest challenge, was to construct guidelines for Paul D. Hazelwood, The Dow Chemical Company, Sarnia, the development and validation of automated methods. Ontario, Canada Individual companies had gone through parallel Companies have realized that they have to embrace the development processes that relied on hard won world as their workplace and marketplace in order to experiences and considerable expertise. The novice robot grow and compete in the global market economy. They user is faced with a very steep learning curve, so are sprinting into the global marketplace at record clip, considerable effort could be saved by the pooling propelled by technology. The next-generation companies together of the collective expertise already available in will be so integrated with the world that they will be non- the group. national rather than multinational. The resulting document contains detailed recommenda- Globalization of products is forcing the companies to tions for single and composite dosage form assays, globally standardize the production and quality control degradation/impurity profiles and dissolution testing, test methods. This paper describes the development and and is suitable for any instrument that is capable of implementation of the robotic automated systems for performing these tests in a laboratory environment. The tensile, FLEXuRAL, hottack and heat seal tests of presentation highlights the important issues arising from polymeric materials. These systems handle rigid polymer the work of the PASG automation subgroup. samples of 6-8 inches long, polymer film samples of 6-13

92 Abstracts of papers presented at the 1998 ISLAR inches long, as thin as 0.001 inch. The other capabilities Two approaches used by Helene Curtis for printing bar of the systems include reading barcode labels, measuring code labels will be presented. The software presented are sample thickness and communicating to the testing two custom bar code label printing applications devel- instrument. These automated systems enabled the global oped in-house; one written in LabVIEWTM and the standardization of the test methods, improve the produc- other in Visual BasicTM. A comparison of LabVIEWTM tivity by nearly 150%, and provide consistent and and Visual BasicTM software for application development quality results in a cost-effective manner. will be discussed, along with techniques on interlacing custom software to the ZebraTM bar code printer. Interfacing databases with the bar code software will A new standard for polyurethane physical prop- also be covered. erty testing

Steven E. Robbins and Michael E. Rusak, Air Products and An automated sample preparation for determina- Chemicals, Allentown, PA, USA tion of total siloxanes in shampoo products using Polyurethane foam is found in hundreds of applications, inductively coupled plasma (ICP) spectroscopy furniture including cushions, automotive seating, appli- Kyoko Ida and M. Karita, Procter & Gamble Far East, ance and construction insulation, and carpet underlay. Higashinada-ku, Kobe, Japan Polyurethane foams are created by reaching isocyanate, water and polyol in the presence of catalysts, surfactants This paper describes an automated sample preparation and potentially other additives. The resultant polymer for the determination of total siloxanes in shampoo morphology resembles a sponge, a non-homogeneous products using inductively coupled plasma (ICP) spec- material with a varying cell size distribution. While the troscopy. A fully automated robotics system requires only cellular nature of the foams is exploited to meet certain setting blank sample tubes on the racks and putting performance characteristics, physical property measure- products into these tubes manually. Robot preparation ments must be based on samples large enough to gives us higher efficiency in extraction and lower RSD represent average behaviour. than current manual preparation. Industry standards, e.g. ASTM, define sample handling, Currently, we can analyse 30 samples per day by the conditioning, shape and size, as well as manual testing manual method at KTC. However, our new automated methods, for each physical property desired. Our experi- robotics method is expected to have the capability of ence has shown that these manual methods are subject to analysing 50 samples per day. Therefore, this automated operator and device variability. robotics method will supply us with much higher speed for determination of the siloxane percentage in shampoo During the past decade, we have developed standard products. automation approaches to improve sample handling, instrument control and data acquisition, while develop- The analytical data sets demonstrate accuracy, precision ing a collection of reusable hardware and software com- and reproducibility of the sample preparation. Linearity, ponents. Data reliability and reproducibility have accuracy and precision ensure the validity of the robotics improved greatly for each of the methods we have method. The method has a linearity working range from automated. These methods include density, airflow, com- 0 to 500ppm (correlation coefficient, R 0.9999). The pression set, Japanese wet set, tensile-tear, and dimen- method gives sufficient recoveries, R 101 + 1%. Rela- sional stability. This presentation will illustrate test tive standard deviation (RSD) is 0.42%. improvements and some of the automated techniques developed to achieve them. Database mining and knowledge discovery in modern drug research Custom bar code label printing software for laboratory automation G. M. Maggiora, M. S. Lajiness, D. W. Elrod, M. A. Johnson and T. R. Hagadone, Pharmacia & Upjohn, 301 Henrietta Saul Llamas and Stephen Dokoupil, Automation Laboratory, Street, Kalamazoo, MI, USA Helene Curtis Innovation Center, Unilever Home and Personal Care, Rolling Meadows, IL, USA A growing trend towards automation within the pharmaceutical industry has led to an explosive growth in the Barcodes are prominently used in the retail marketplace rate, amount and types of data that must be dealt with in and have found many uses in other fields, including the modern drug research. Within the space of only a few area of laboratory automation. Jars, prototype contain- years, highly sophisticated technologies have transformed ers, test tubes, sample vials and microtitre plates are just drug screening; DNA, RNA and protein sequencing; and a few of the items that are now bar coded. Bar codes are chemical synthesis increasing throughput in these key key to a successful robotics automation system. Bar codes areas by a thousand-fold or more. Computers provide the allow a robotics system to identify and track samples. Bar only effective means for managing this growing flood of codes also enable the use of embedded protocols within data. But managing data is not enough if the knowledge the bar code number that identify the tests to perform on embedded within it is to be obtained. It is here that a sample. In addition, bar code labels allow placement of newly emerging techniques of database mining and human-readable data, providing users in the laboratory knowledge discovery, already having considerable im- with quick descriptive information on a particular test pact in many areas of business and industry, may also sample. play an important role in pharmaceutical research. In

93 Abstracts of papers presented at the 1998 ISLAR high-throughput screening, e.g. numerous biological Advances in genomics continue to discover new explora- assays are run against corporate compound collections tory targets for drug development in large numbers. At 3- typically of several hundred-thousand compounds or Dimensional Pharmaceuticals, we have developed a more. Such a database represents a watershed of un- powerful drug discovery system that integrates our discovered biological knowledge, but the database must proprietary technology components of Probe Libraries, be properly 'mined' if the knowledge within is to be ThermoFluor(R)=AE High-Throughput Screening, Di- realized. The discovery of relationships, rules and pat- rected Diversity@=AE and Informatics. The strategy terns in databases of this size is, however, a daunting task for finding small molecule prototype drugs for functional at best and cannot be addressed with many of the tools denomics targets using our integrated drug discovery used successfully on much smaller datasets. The talk will systems will be described. present a brief overview of the salient issues in database mining and knowledge discovery that are applicable to pharmaceutical research. Several examples will be pro- Inhalation product testing: automated sample re- vided that illustrate how these methodologies can be used covery from Cascade Impactors to extract new knowledge that can potentially expedite Phil Waters, Glaxo Wellcome, Research Triangle Park, NC, the drug discovery process. USA The Andersen eight-stage Cascade Impactor is a stan- Quality control for high-throughput screening dard device used to characterize the aerodynamic particle size distribution of metered dose inhaler (MDI) and Michael W. Lutz and Jay Gill, Glaxo Wellcome, Research multiple dose powder inhaler (MDPI) products. The Triangle Park, NC, USA manual test typically requires assembly, dosing and separation of the Impactor unit; rinsing each stage/plate Robotic systems for biochemical assay introduce a new component into separate collection containers; adding set of requirements for analysis of quality control data. sufficient volume of solvent; and sample assay (typically The precision and accuracy of liquid dispensing robots by HPLC). This paper discusses a device developed by and complete robotic assay systems must be determined Glaxo Wellcome that automates the time-consuming and monitored. To properly interpret data, assay varia- rinse steps associated with the recovery of drug from bility must be quantified. Automatic detection of gross Cascade Impactor stages. A detailed description of the errors, systematic errors and random errors is necessary hardware and software configuration is presented, along for systems that run continuously. with analytical validation results for products currently This presentation addresses statistical methods and prac- tested using this system. tical concerns involved in quality control analysis for high-throughput screening. After a review of methods, Enhanced decision-making from analytical auto- examples are given that demonstrate how quality control mation statistics are applied in the area of high-throughput screening. Finally, the recommendations for best prac- Harnath Doddapaneni and John Jushchyshyn, SmithKline tices are discussed. Beecham Pharmaceuticals Research & Development, Upper Merion, PA, USA Automation initiatives have been traditionally viewed as Maximizing information content in combinatorial a resource-reducing tool. The ability of automation to libraries generate high-quality, reproducible and correlative data Steven L. Gallion, ArQule, Medford, MA, USA has been largely overlooked. This paper will present long-term dissolution data generated on development The process of library design and interpretation of SAR batches of drug product which compare manual ap- data should be guided by the ability to extract substan- proaches versus a segmented automated approach. The tive information during each design cycle. The successful data produced using automated methods reduce or elim- management of data during the entire drug discovery inate variation due to slight changes in manual tech- process is a function of the ability to not only track and niques which occur from analyst to analyst over a period store data, but to formulate and refine hypotheses that of years in a typical stability study. While the outcome of result in at least a semi-quantitative rationale for lead a stability exercise is equivalent, the data produced using optimization. This presentation will discuss the issues and automated procedures allow management decision-mak- strategies involved in the design of combinatorial libraries ing at a much earlier stage in the stability cycle when to deliver maximal information content. Examples in- compared to other approaches which are inherently more clude the rational selection of building block components variable. using a combination of medicinal chemistry expertise, computational analyses, crystallographic structures and diversity assessment tools. Development of an automated assay for sustained release tablets using the TPW-II Workstation John C. Lynch, Zeneca Pharmaceuticals, 1800 Concord Pike, From functional genomics to prototype drugs Wilmington, DE, USA Dale S. Dhanoa, 3-Dimensional Pharmaceuticals, Exton, PA, Automated composite and content uniformity assays for USA sustained release tablets have been developed using the

94 Abstracts of papers presented at the 1998 ISLAR

TPW-II Workstation. Use of the TPW-II has reduced A robotic compound dissolution system for the sample prepartation and analysis time from over 4 h per Pearl River Research Compound Bank sample to under 30 min per sample, significantly increasing sample throughput. Because the TPW-II homo- Kevin Olsen, Robotocist, Research Compound Bank, Wyeth genizes, filter and dilutes each sample, analysts spend Ayerst Research, Pearl River, Y, USA less time preparing samples and can focus on other In order to make more compounds available to high- responsibilities. throughput screening researchers, the Pearl River In order to successfully and efficiently automate methods Research Compound Bank and the Wyeth Ayerst using the TPW-II, analysts need to evaluate the cap- Biomedical Engineering Department collaborated on abilities and limitations of the workstation as well as the the construction of compound dissolution and distri- method development costs versus the advantages bution robots. (throughput, labour, safety) achieved by automating a The machines are modified industrial robots and specific assay. gantry are controlled with a Visual Basic interlhce running on a Our experience developing automated sustained release PC. Other than the basic robot, all hardware and soft- assays has enabled us to develop a method development ware development was developed by Biomedical Engin- template for the TPW-II. A simple, step-wise approach eering. By standardizing upon and then lnodifying a has been defined to guide automated method develop- basic platform, the engineers used this project to develop ment and provide the analyst with a reasonable estimate the expertise that was applied to subsequent projects. of the work required at any given stage of product development. This presentation will discuss this method The machines were designed to process 92 compounds specific from the per hour. Data entry is completely automated through development approach using examples and automated sustained release tablet assay. the use of bar codes on both the compound samples microplates. Liquid handling was engineered so that dissolved compounds would be completely utilized, and Development of an automated liquid store to there would be no residual aliquots at the end of the run. improve sample management in the lead discov- The units have been extremely reliable. During the fall of ery process 1996, they compiled a record of only three mechanical failures in 75 000 samples. Sue Holland, Peter Aperghis, Melanie O'Neill, Clive Battle, Tim Linsdell and Martin Sykes, Glaxo Wellcome Research & Development, Medicines Research Centre, Stevenage, Hefts, UK Validation of 1 1 compound delivery to dry plates using the Zymark RapidPlate The potential for identifying new lead compounds in the drug discovery process is increasing through the use of Brent T. Butler, Joan Frezza and Julie J. Tomlinson, faster and more efficient approaches to high-throughput Department of Molecular Biochemistry, Glaxo Wellcome, Re- screening (HTS) and larger, more diverse compound search Triangle Park, NC, USA collections. The rapid developments in robotic HTS systems and the expansion of sample libraries through The quality of data generated in screens may be com- combinatorial chemistry place demands on traditional promised by the instability of compounds delivered to approaches to sample supply which have made this those screens. 'Just in time' dilution of compounds will element of the lead identification process rate limiting. help to maintain the stability of these compounds. The ability to delivery gl of compound in neat DMSO to a In order to maintain pace with HTS, a sample supply variety of target plates is the basis for just in time strategy is required which will respond rapidly to screen- compound delivery and paramount to increasing the ing requests, provide the flexibility screeners want, and quality of screening data generated in those targets. This maintain control and accountability for the quality of also eliminates a dilution step in the compound handling samples produced. To achieve these goals, Glaxo Well- system, thus decreasing the time spent preparing com- come has developed an automated sample storage, pound plates for the numerous targets being screened. retrieval and processing system with the capacity of 3 million samples and an output rate of 15 million samples Results of experiments on various plate types indicate per year. The automated liquid store (ALS) comprises an that Zymark RapidPlates located on the compound environmentally conditioned sample storage facility and handling robot in the Molecular Biochemistry Depart- an input/output buffer integrated with three liquid ment, RTP are able to dispense gl of DMSO reliably handling robots (LHRs). The system will perform sample and accurately. The c.v. for all plates tested were less replication and discrete sample picking from 384-well then 10%, with accuracy between 95 and 110%. This source blocks into either 96- or 384-well microtitre plates. capability will allow compounds to be delivered to targets just in time, which will help to maintain the The scale of this facility has called for an industrial design stability of these compounds. The compound handling concept which is modular to allow reconfiguration of the robot will run control plates at regular intervals to LHRs to meet future business needs whilst maintaining monitor RapidPlate function. the core storage and retrieval infrastructure. This presentation will review the role of the ALS in the GW sample supply strategy, its principle elements and func- Use of a Zymark XP system for multiple com- tions, and the approach taken to managing the project. pound formatting tasks

95 Abstracts of papers presented at the 1998 ISLAR

Richard Spann, Molecular Pharmacology, Berlex Bioscience capture can be fully addressed. This approach to data Richmond, CA, USA integrity can provide management with a high degree of confidence in their business data, and the ability to The Zymark XP laboratory automation system provides respond in a timely manner to external regulatory a versatile technology that can be fully upgraded with the latest enhancements at desired intervals. The modules agencies. include a Zymark XP arm, a Master Lab Station II, a At the third (top) level, the global needs of the business Power and Event Controller, a Fully Automated Cap- can be addressed. Today's business climate is fraught ping Station, a RapidPlate-96 and two Storage Carou- with corporate mergers, increasing process regulation, sels. new reporting constraints, and software that simply does The architecture is standard System V with operating not cut it anymore. All of these circumstances are system version 2.5. The interface PC is running Windows compelling from the standpoint of initiating business and the Zymark Utilities package. Previous versions of process re-engineering projects. Projects directed at the the Zymark OS only allowed I/O interactions with the data collection level preserve the viability of the business System V controller disk drive or selected files on the on the day-to-day operational level and serve to lay the hard drive via Easylink. Using functions supported under groundwork for utilizing the data in more creative ways. the Zymark Utilities, data I/O is now possible directly Projected directed at cross-department sharing of data with any available drive. (e.g. QA laboratory data with that of manufacturing), or data sharing globally within a department serve to create The system is being used to support compound format- new business efficiencies. Such large-scale projects, e.g. and distribution for screening ting high-throughput those of data warehousing, can be viewed as both The can four different activities: (HTS). setup perform strategic and defensive when consideration is given to dissolution of vials of then transfer to 96-well compound the preservation of historical data. plates; transfer from vials of solubilized compounds to 96- well plates; dissolution of compounds in vials; and consolidation of plates containing single compounds per Utilizing ACCESS to efficiently manage data in a well into plates containing 10 compounds per well. The quality control microbiology laboratory plates with 10 compounds/well can then be used in HTS assays. The matrix strategy employed allows for an C. R. Bierman and T. M. Kajs, The Procter & Gamble efficient means to identify HTS hits. Company, Mason, OH, USA The quality control constituent of the Health Care Laboratory data management in the QA environ- Microbiology Department at Procter & Gamble is ment responsible for microbial content testing (MCT) and Tricia Chen, Novartis Pharmaceuticals, E. Hanover, NJ, USA preservative challenge testing (MST) of all health care products developed at the facility. MCT and MST are Data management is a significant challenge for the required testing for product release and expiration data Pharmaceutical Quality Assurance organization. The determination. Put simply, data accumulated from these and myriad of regulations imposed by the FDA DEA two analyses, over time, determine the microbial stability and their subsequent translation into business practice profile for each health care product. These data are affords the industry many technical challenges with stored in the data documentation. respect to managing the data. From the point of data generation in its completely raw form to its fully pro- DDR is a system built on ACCESS software, which cessed and final resting places, the data undergo several manages MST and MCT data in an electronic, fully transformations and multiple moves. searchable format. It was developed using the collabora- At the lowest level, the commonplace business response is tive efforts of the Microbiology Department and Com- to utilize a data acquisition system to control the puter Management Systems. instruments and to ensure that the collection analytical Prior to the inception of DDR, the department tracked of follows defined laboratory data strictly (in-house) all data in hard copy. As tests were completed and The twin concerns of data and guidelines. integrity verified, data for each sample were entered into a control can be a collection auditing then addressed by relegating laboratory information management system. Data entry of routine low-level tasks data entry, checking, (e.g. proved to be time consuming and cumbersome. This and to the by com- storage retrieval) discipline imposed system did not offer a means of searching MST and For better control and efficient main- puter processes. MCT data to the microbiologists, the primary owners of a networked data is tenance, acquisition system the information. Microbiology's need for capturing total recommended strongly instead of stand-alone integrators. products stability was critical; therefore, the DDR system At the next level, we find the LIMS software with its two was developed to help streamline work processes, increase primary roles, i.e. to coordinate/orchestrate the testing the department's data management capability, and and to centralize the generated data. When all three provide electronic retrieval capability. stages of the process are properly structured, i.e. (i) the automation of data captures; (ii) the transfer of the data via networked (interfaced) laboratory instruments; and Using a custom-designed compound tracking (iii) the centralized storage of the test data, the audit- system to facilitate the automation of the produc- ability and trace-ability issues surrounding the data tion of the OptiverseTM lead generation library

96 Abstracts of papers presented at the 1998 ISLAR

L. Cameron, C. Garr and L. Schultz, MDS-Panlabs, 11804 continuous cycle of applications development follows. North Creek Parkway South, Bothwell, WA, USA The information management system that complements drug discovery operations must keep pace with the The desire for faster drug discovery led to the implemen- dynamic nature of R&D. We will provide an overview tation of combinatorial methodologies for small-molecule of our drug discovery informatics system. The modules synthesis. In 1995, MDS-Panlabs and began a Trpos are developed utilizing a combination of industry-stan- collaboration to the a produce Optiverse Library, dard tools, Oracle parallel synthesis library composed of 140 000 diversity development third-party components, > and ISIS as a database back-end, and a novel interface designed compounds. We have implemented a purifica- This is our current needs to tion such that purified will concept. system meeting process highly compounds track HTS and associated downstream data flow. It is join the OptiverseTM suite of compounds. In doing so, the also to meet future for closer track- information associated with the re- poised requirements explosion process of lead quires special attention. The challenge becomes tracking ing progression. this information and maintaining the compound-data link which is accomplished using a custom-designed The use of TRAP for lead structure identification compound tracking system (CTS) database. The focus of this presentation will be on the CTS and how it M. Kozlowski, Telik, S. San Francisco, CA, USA facilitates the modular high-throughput processes. TRAP is a proprietary technology that is based on understanding the biochemical description of a molecule and using that information in concert with structural Integrating good statistical practice in HTS (GSP) to minimize the number of to information management descriptors assays required find lead compounds for pharmaceutical targets. TRAP Frances P. Stewart, SmithKline Beecham Pharmaceuticals complements the drug discovery process by providing an R&D, King of Prussia, PA, USA efficient and inexpensive way to identify lead compounds for targets that are not amenable to a high-throughput The integrity of data is a quintessential construct in assay screening (HTS) format. TRAP is also an efficient way to the success of the HTS in As paradigm drug discovery. identify lead compounds and validate targets that have increases in both combina- technology high-throughput little pharmacological data. Terrapin employs TRAP, as torial and synthesis ultra-high-throughput screening, well as computational, medicinal and combinatorial assay information must evolve to address management chemistry, to find families of high-affinity compounds the amounts of data. The software increasing assay for pharmaceutical targets. The TRAP biochemical with robotics to and store integrated laboratory capture description of a molecule is derived from experimental the data must include the functionality to alert the user data. Competitive binding fluorescence polarization (FP) when statistical tests detect erratic results or shifts due to assays are used to determine the affinities of compound control or of across time. poor process instability reagents for a diverse panel of proteins. A high-throughput screen- At SmithKline Beecham the IT infrastructure Pharm., ing system has been designed around our FP assays to that supports high-throughput screening implements ensure the rapid addition of affinity data to our TRAP within our data components screening management database. This presentation describes the unique aspects that monitor the of the data and application quality of Terrapin's process of lead structure identification. alerts users to intervene when poor data quality is suspected. Baseline control limits are established for all HTS assays as part of the development criteria before Discovery and optimization with large encoded these assays progress to the 'live' status of production combinatorial libraries runs. These limits are used to flag bad data, which enables users to cancel the data and continue screening Daniel Chelsky, Pharmacopeia, Cranbury, NJ, USA or to suspend screening until the source of the problems Pharmacopeia has produced over four million com- are identified and corrected. This ensures that false pounds using its ECLIPS encoding technology. These positive/negative rates are minimized and that data compounds have been synthesized as approximately 85 posted to the corporate DB is valid. Our philosophy discrete sets of 'libraries' which can provide substantial towards data regulation and an overview of our screening SAR information at both the discovery and optimization data management software are discussed. phases of a program. Information obtained is not limited to the primary target and can be extended to selectivity as well as preliminary toxicity and metabolism, allowing The of a user interface for development dynamic early choices among related compounds in a series. Case drug discovery operations studies as well as the integration of 1536-microwell Ralph Infantino, Ildefonso Belliard, Chris Waller and Mel technologies will be described. Reichman, OSI Pharmaceuticals, Uniondale, NY, USA Data management for drug discovery poses a variety of Making the move to 384-well high-throughput challenges to an informatics group. Unlike certain screening 'mature' classic business operations, or e.g. accounting B. Orlova and purchasing, the operations model for drug discovery is in j. LaRocque, Czermik, N. J. Babiak, Wyeth- Ayerst Research, Pearl River, NY, USA its infancy and varies greatly from one company to the next. Commercial packages for HTS data management Recent advances in automation and assay detection require substantial customization. As needs change, a make the use of 384-well plates a viable solution to

97 Abstracts of papers presented at the 1998 ISLAR increase efficiency in high-throughput screening. We The myriad core system (MCS) is a new automated have evaluated the use of 384-well plates to run a cell- synthesizer developed by a consortia of pharmaceutical based receptor antagonist assay and an in vitro enzyme companies and The Technology Partnership (TTP). It is inhibition assay. A comparison of each assay run in 96- based on a flexible modular design and has capability for well and 384-well plates with regard to assay perform- both high-performance and high-throughput chemical ance, automation, reagent usage, efficiency and through- synthesis. The system incorporates several novel tech- put will be presented. nologies in reaction vessel design, reagent delivery and inert gas blanketing and is controlled by user-friendly software that enables multi-job scheduling. A description A second-generation approach to molecular diver- of the MCS and its use in the synthesis of solid and sity solution phase libraries will be presented. j. Christopher Phelan, Axys, South San Francisco, CA, USA The increasing sophistication and diversity of chemical A chemist's simple approach to robot program- structures available through combinatorial chemistry ming for reaction optimization: scheduling non- commensurately increases the challenge of how to choose identical procedures to make limited resources. We which compounds given Robert Osborne, Pharmaceuticals, Hallen, Bristol, UK will discuss the problem of molecular diversity in large Zeneca screening libraries for lead discovery. We will present a The use of statistical experimental design in the optimiza- new 'hybrid' approach for simultaneous optimization of tion of chemical processes requires the performance of molecular dissimilarity (a 'negative' property) and de- many pre-planned repetitive experiments and hence is an scribe drug-like parameters ('positive' properties) obvious candidate for automation. The programming of such automated sets of experiments is complicated by the need to vary conditions (temperature, time, addition Universal solvent exchanging device and reaction rate, etc.) between individual experiments as well as optimization tool in organic synthesis variation in the reagents used. This paper will present a llya Feygin and Leslie Walling, Pharmacopeia, Monmouth view of the particular requirements for the automation of Junction, NJ, USA process development compared to lead optimization and will present one simple approach, using standard soft- Reliable and fully programmable delivery and evacua- ware, to providing the flexibility required for successful tion of solvents and reagents into and out of the reaction automation in this area. vessels remain to be two of the biggest challenges in automated chemical research. Another major challenge Automated dose measurement stations---valuable is providing controllable temperature and level of agita- c tion independently in each of the vessels. players in the development of Turbuhaler inhalers Results of a joint effort between chemists and engineers at Pharmacopeia that successfully resolved these issues will Roger Appelqvist, Peter Andersson, Hans Lundba(k, Thomas be discussed. LO'gf, and Bo Olsson, Astra Draco AB, Lund, Sweden A novel device with an open frame architecture, and Turbuhaler is a multi-dose reservoir dry powder inhaler individually observable and accessible vessels allows for containing up to 200 doses. Within the product develop- either separate or batch processing of reactions. The ment work as well as for release control at the manu- device provides controlled temperature, programmable facturing site, the entire dose interval needs to be selection of delivered solvents and reagents, and three accessed. Automated systems are therefore required in different modes of agitation. the accomplishment of high-quality work at a high- throughput rate. At Astra Draco, the automation of dose A novel side-stirring mechanism placed on individual measurements started in 1985 and the development of vessels creates a controllable, two-dimensional vortex automated methods has since been an important part of capable ot" effectively agitating ditlirent resins. our R&D work. The number of robot stations has The rotating vessel-holding carousel provides a reliable increased from four stations in 1992 to over 40 identical circular alignment and engagement with the stationary stations today, located at different sites in Sweden, top and bottom manifolds. This new technique of filling Germany and USA. and draining vessels which was enhanced by special The robot stations are instruments which per- mating fittings has allowed for elimination of liquid complex form a number of tasks dose reduction of and the increase in including sample handling, handling valves, tubing and evaluation of the results. Different overall reliability of the system. This device is currently quantification configurations may be used with tst transformation going through laboratory testing, and it will most likely in a versatile Experi- be commercialized later this year. procedures resulting highly system. ences from the development of the automated systems will be discussed as well as different aspects of quality High-throughput automated organic synthesis assurance. using the myriad core system Nick Hird and Bill MacLachlan, SmithKline Beecham Pharma- Automated moisture testing by Karl Fischer titra- ceuticals, Harlow, Essex, UK tion and other methods

98 Abstracts of papers presented at the 1998 ISLAR

Colleen S. Nichol, Searle, Stokie, IL, USA Patrick J. Faustino, Ajaz S. Hussain and Karl P. Flora, Food and Drug Administration Center Drug Evaluation and Moisture is a the for testing major, high-volume analysis in Research Division of Product Quality Research, Laurel, MD, pharmaceutical laboratory. Moisture analysis is used in USA support of stability and formulation development. Moist- ure content is a very important parameter in the drug Automation has evolved rapidly since its introduction development process, because water in the drug product into the laboratory. Automation or laboratory robotics or drug substance can lead to instability, as demonstrated were used primarily in quality assurance and quality by product degradation, increases or decreases in hard- control laboratories, and gradually entered pharmaceu- ness, tiiability, etc. Therefore, the product should contain tical research laboratories where they currently play an minimal water and be stored in containers so as to reduce important role in the drug development and drug or prevent water absorption into the product. The water evaluation functions. In many laboratories, the rate of content is generally tested using a Karl Fischer titration. change is increasing as automation and research pri- At Searle, we have successfully automated a Karl Fischer orities are being challenged by advances in technologies titration workstation. We have investigated several that include combinatorial chemistry, novel in vitro and methods that will increase throughput, while at the same in vivo assays, and increasingly sophisticated analytical time decrease analysts' preparation time. Several differ- techniques. These challenges may require automation to ent approaches to automating moisture testing of phar- efficiently evaluate new molecular entities, new pre- maceutically relevant samples will be discussed. Some of clinical models or novel research endpoints. Changes in these approaches are manual Karl Fischer titration in a research priorities and requirements may require auto- humidity-controlled glove box, the automated moisture mation to more rapidly evolve to effectively utilize high- system from Bohdan, near-infrared spectroscopic throughput techniques to evaluate a complete range of methods, and the Brinkmann moisture system. Data will complex issues, e.g. chemistry, pharmaceutics, toxicity, be presented to show the successes and failures of the drug metabolism and drug-drug interactions. various moisture analysis systems examined at Searle. The impact of automation and the changes in regulatory research are equally striking. The regulatory research A simple customized automated procedure for laboratory has evolved from the initial use of laboratory sample dilution robotics as a tool to address safety issues, e.g. minimizing Sharon Chang, Ian Harry and Maria Guazzaroni, Pfizer, human exposure to pathological agents, to efficiency Control Laboratories, Quality Operations, Brooklyn, NY, USA issues involving rapid methods development and assay validation as research resources and priorities have A high-performance liquid chromatography (HPLC) changed. Regulatory science has continued its evolution, system with electrochemical detections (EC) was utilized focusing research efforts to efficiently provide scientific to assay a low UV-absorbed drug substance and its data that can facilitate regulatory decisions. Automation dosage forms. Two manual dilutions have to be made is playing a key role in our laboratory's evaluation of two for each sample. A Zymark BenchMate II Workstation pre-clinical models, an in vitro cell culture model (Caco-2) was evaluated to assist the Quality Operation Laboratory to determine intestinal permeability of drug substance in performing high volumes of sample preparation. and drug product, and an in vivo transgenic mouse model to determine the carcinogenicity of drugs. Several factors were considered in developing the pro- (TG.AC) These research efforts intramural automa- cedures for automation. supported by tion resources may help in the development of regulatory (i) The HPLC analysis method has a long run time. guidelines that will aid FDA and industry in the NDA (ii) The compound is susceptible to air. process. (iii) The dissolving solvent and the diluent contain different organic compositions. report documenting Bench- The final volume after Five-year summary (iv) sample changes slightly Mate instrument performance preparing 650 mixing. urine samples for 2,4-D analysis (v) There is an internal standard in the diluent. (vi) The sample solution contains sugar and has a K. K. Brown and J. L. McLaurin, US National Institute for different density from the standard solution. Occupational Safety and Health, Division of Biomedical and Behavioral Science, Cincinnati, OH, USA A simple dilution procedure using the BenchMate with a volumetric mode, without connecting it to the HPLC NIOSH conducted an exposure assessment study that system, was designed and validated to replace the required the development and application of a complex manual preparation. Instrument calibration was sched- clinical chemistry method for the analysis of 2,4-D in uled to ensure accuracy and precision of the performance. urine. One assumption that this methods was developed The automated dilution procedure is not only reliable upon was that robotic automation would free up personal and convenient, but also cost effective and environmen- work time in preparing samples, and thus enhance tally friendly. sample throughput and reporting response time. Another assumption was that robotics would improve QC and QC of the analytical data. A BenchMate workstation was The evolution of automation and research in used to automate the sample preparation. The robot regulatory science utilizes an analytical balance accurate to a tenth of a

99 Abstracts of papers presented at the 1998 ISLAR milligram which can be used to track the sample mass mark Rapid Trace SPE workstations. The samples are before and after each preparation step. eluted directly into appropriately labelled HPLC autosampler vials which are then capped, mixed and ana- For each batch during the study, the 30-sample analysed lysed, using a C18 column and an HPLC system mass data file was transferred from the robot to a output equipped with diode array and fluorescence detectors. spreadsheet and converted into a QC chart. In this Qc chart, the volume of liquid used in each sample prepara- This method of analysis eliminates most of the tedious tion step was calculated and plotted from the mass and time-consuming steps associated with liquid/liquid record. In this way, sample preparation variables were extraction and manual SPE procedures, and circumvents monitored, including the solid phase extraction cartridge the problems associated with the instability of the named load rinse and elute volumes. Along with charting vol- drugs when they are being handled at low concentra- ume, other parameters were plotted, e.g. reagent transfer tions. A highly reproducible procedure that requires only efficiency, per cent water added to the organic phase for the accurate allocation of the initial serum sample will be reversed-phase SPE, and evaporative losses. Thus, every presented. critical parameter of the sample preparation was recorded and monitored. This chart was especially helpful a SPE in characterizing the ruggedness of the new method. Confirmation of the NIDA five using single sorbent and an eight-solvent RapidTraceTM By accumulating these batch charts, a BenchMate per- system formance history was charted with each new batch of samples analysed. This performance history documented Thierry D. Mann and Michael F. Burke, Department of changes in method parameters, and established a per- Chemistry, University of Arizona, Tucson, AZ, USA formance baseline on which decisions were made about The NIDA five is a series of drugs that are most instrument maintenance and repair. Also, these records commonly analysed in forensic cases. These drugs include provided information for justifying the rejection of'out of the opiates morphine and codeine, THC, cocaine, PCP, control' data. This information was stored on CD for amphetamines, and the barbiturates. It would be ideal if CLIA documentation. there was one universal method for extracting all drugs. The BenchMate alone did not improve the response time This is not a realistic scenario, however, because many and for delivering exposure data to workers, but it did shift drugs have drastically different properties, in order must be the bottleneck from sample preparation to data reduc- for selectivity to prevail, extraction methods tion. Using the BenchMate did improve QA and Qc of tailored to certain functionalities of molecules. It is sample analysis by documenting the preparation of each possible, however, to use the single mixed-mode SPE sample. Some of the unprecedented advantages of using sorbent in combination with eight solvents to achieve 5. the BenchMate over manual sample preparation during high selectivity and efficiency extraction of the NIDA this study were as follows. (i) Automatic weighing of test Although liquid-liquid extraction has been the most tubes allowed for enhanced mathematical analysis of common method for sample preparation for forensic effect variables with respect of their desired response. analysis, solid phase extraction (SPE) has some distinct For example, the effect of SPE loading elution volumes advantages. Studies have shown that SPE is more rapid, on analyte recovery. (ii) After method development, that economical and efficient than LLE. Surveys of NIDA- same feature allowed the method to be characterized certified drug-testing labs have shown that approxi- more accurately. It would have been too labour intensive mately 50% of analyses utilize SPE techniques. Cocaine for a chemist to manually weigh and record the mass of a and THC analyses are by far the highest volume assays, tube before and after every manipulation in the method. and although SPE procedures for these drugs are well (iii) The analytical balance confirmed that the robot can accepted, it is not clear that SPE methods for other dispense reagents more accurately and precisely than the NIDA 5 are compatible with the solvents used in the reported precision for disposable pipettes. (iv) Moreover, previous analyses. the mass record allowed for increased electronic docu- of SPE mentation which fulfils the spirit of CLIA'88 that re- This presentation will address the simplification a TM automated so quests high levels of QA and QC for human samples with procedures using RapidTrace system is when potential medical diagnostic outcomes. that no solvent replacement necessary changing assays. The use of a single sorbent also simplifies the extraction procedures. The detection and quantitation of drugs in equine serum using Zymark RapidTrace SPE modules Steps to automation in a contract research organ- Robert McKenzie and Gina Dew#t, Michigan Department of ization Agriculture, East Lansing, MI, USA Robert Massg and Carl Dourambeis, Phoenix International Life A method was needed to reduce the time involved in the Sciences, Montreal, Quebec, Canada quantitation and confirmation of serum fluorsemide and Phoenix International is one of the fifth CROs and other of interest in largest phenylbutazone drugs equine research in the world, featuring serum. (contract organization) many services for the pharmaceutical industry. Part of Serum samples are placed in test tubes. Urea and buffer Phoenix's portfolio of services, outside clinical research, is spiked with an internal standard are added. The samples a bioanalytical laboratory service consisting of HPLC, are then submitted to solid phase extraction using Zy- GC, GC/MS, HPLC/MS, HPLC/MS/MS and immuno-

100 Abstracts of papers presented at the 1998 ISLAR chemistry laboratories. Phoenix also has a new drug Re-engineering an automated antiviral drug discovery group consisting of in vivo, in vitro and analytical screening: a novel approach of integrating a drug metabolism areas. Phoenix's service laboratory high-speed pipetting robot group typically analyses large volumes of samples requiring routine repetitive manual work. Rudy Willebrords and Ifoen Andries, anssen Research Founda- tion, Beerse, Belgium, Eugy Van Gool, Labotec VV, Teralfene, In today:s highly competitive contract research market- Belgium place, there is a strong need for cost reduction and In a continuous effort to optimize our drug screening, we accurate analytical data reporting which reflects on the set out new goals to improve reproducibility and increase pricing for the customer. One of the key factors to cost throughput. After 10 years of faithful service, our robot reduction is automation. Although, initially, automation system had to be replaced and re-engineered. requires an upfront capital expenditure, there will be longer term benefits ariving from this type of investment, A central XP-robot installed on a custom-designed track, including reproducible results, marketing ability of our in a conditioned room (temperature and humidity), up-to-date technology, and larger sample throughput remains the heart of the system. A multi-channel high- with the same staff availability. speed robotic pipettor (Zypettor) was integrated with the central robot to increase the sample throughput. Insuffi- Phoenix has established a laboratory group called cient number of microplate and tip box positions on the 'Sample Preparation and Automation (SPA)' to fulfil original Zypettor, with its relative slowness, forced us to the necessary tasks of evaluation, implementation and rebuild this station. We designed a new six-position technology transfer of suitable automation workstation turntable for microplates and tip boxes to interact with platforms and robotic systems. Development of a new the Zypettor in order to increase the number of micro- type of trust in this technology appears to be one of the plates as well as the speed of pipetting of the Zypettor. larger hurdles to overcome in terms of employee accep- Plates and disposables can be positioned in a wide or tance. Another hurdle comes from the large pharmaceu- narrow orientation to allow rapid pipettng in rows of tical companies in terms of method adaptation. Phoenix's eight or 12 by the Zypettor. This new dimension and challenge is to adapt their developed automated method- approach may create new opportunities in respect of ology into its internal laboratories. Phoenix needs to meet future configuration developments. the clients' requirements for automation technology and Efforts were also undertaken to the capabilities of still maintain a profitable edge with its capital spending. expand automated microplate measurements. Spectrophoto- Phoenix's initiative in SPA as both a multi-instrument metric as well as fluorometric assessments were developed automation development group and a virtual technology to measure the viability of virus- and mock-infected cells. transfer group are enabling Phoenix to maintain an edge The integration of a multi-reagent addition module over the competition by answering the clients' demands (Ras/Ram) and a Biotek 96-well washer were crucial for faster, accurate data generation and reporting. for the realization of those procedures. These experi- This talk will represent the current approach to auto- mental implementations result in a flexible robot system mation via SPA as well as interdepartmental coopera- designed to run cell-based and enzymatic assays. tion, and the challenges encountered. Also presented will be ideas learned the to key along way implement Evaluation of a transplanted Zymate microplate automation as well as how we are technology winning system for 384-well automation over staff support towards the necessary evolution of automation. T. C. Ramaraj, Robotics & Automation--High-Throughput Screening, Wyeth-Ayerst Research, Pearl River, NY, USA

A comparison of two methods for measuring We will summarize our efforts to implement automated format a kinase activity in a high-throughput format 384-well assays using Zymate microplate system. This Zymate microplate system was originally Maureen Laney, David Lesikar and David Liu, SCIOS, Dept. of designed and delivered to one of our sister companies for Research, Sunnyvale, CA, USA automation of an ELISA format assay in 96-well format. The system was also used tbr generation of diluted assay Two methods will be compared for measuring kinase plates from predilution plates. The original configuration activity in vitro in a high-throughput format. A primary included a high-speed 12-channel Zypettor module for consideration in choosing a method was for an assay that dilution. In addition, the Zypettor was used for adding would be widely applicable to a variety of kinase targets, substrate to assays and for serial dilution of compounds. with minimal changes required when the specific enzyme The system, with addition of pipetting stations, reagent target changed. One method is an indirect assay which addition dispensers and an online reader for fluorescent/ links kinase activity through two auxiliary enzymes to a spectrophotometric assays. An interesting aspect of this colorimetric readout. The other method is a radioactive presentation will be evaluation of the Zypettor module assay with a biotinylated substrate. The product is for sample addition and small-volume delivery of re- captured on the SAM'2"I M streptavidin membrane (Pro- agents to 384-well plates. Our attempt to automate a mega). Reactions can be applied to the filter in a high- fluorescent assay in the fully integrated system will be density array by the use of the Biomek high-density supplemented with data collected on throughput, effi- replicating tool (HDRT). Both methods are easily auto- ciency of plate handling and overall sensitivity of the mated for high throughput. assay as tailored to the Zymate system.

101 Abstracts of papers presented at the 1998 ISLAR

Automated cell assays using image analysis Brunswick, NJ, USA; Mike S. Lee, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, USA Thomas Hartmann, Ribozyme Pharmaceuticals, Boulder, CO, USA Considerable emphasis within the pharmaceutical industry has been directed toward acceleration of the drug We have developed a number of automated quantitative discovery-pharmaceutical development continuum. In procedures which allow us to perform cell-based assays, support of these initiatives, greater amounts of informa- e.g. apoptosis, cytotoxicity, drug delivery, gene expres- tion relevant to lead compound selection criteria will sion and proliferation assays. These assays can be used in serve to accelerate these stages. Combinatorial chemistry high-density plates and are amenable to plate readers is a widely applied approach for the generation of and robotics. Signal detection using an epifluorescence structural diversity and increased numbers of novel microscope provides single-cell sensitivity and a dynamic compounds with the goal of increasing the rate of novel range of at least three orders of magnitude. Automation lead compound discovery. Typically, mixtures of com- of the image acquisition and inaage analysis procedure pounds, or 'libraries', having predictable structural vari- allows unsupervised scanning of high-density plates. ation are tested for activity in a parallel format, thus Applications, previously pertbrmed with conventional increasing the probability of discovering active lead plate readers or fluorescence-activated cell sorters, can compounds. However, reliance on activity alone may take advantage of the instrument's specifications. In not be sufficient for selection of the best pharmaceutical particular, counting procedures and colocalization product. Pharmaceutical development issues focused on studies should benefit from the increased sensitivity, excipient interactions, chemical stability, metabolic sta- spatial resolution and multi-parameter capabilities of this bility, pharmacokinetics, toxicology and chemical process instrument. research heavily affect the commercial success of a drug. The combinatorial approach may be extended for pro- Automated analytical characterization of com- spective parallel analytical research in pharmaceutical binatorial libraries by LC]MS development. Combinatorial predictive development experiments may be conducted in order to rapidly obtain Olga Issakova, Victor Nikolaev, Shelly Wade, Stefan Walle and information relevant to early go/no go decisions based on Nikolai Sepetov, Selectide, a subsidiary of Hoechst Marion a wide matrix of development information. We have Rousel, Tucson, Arizona, USA utilized a predictive degradation strategy, aimed at the Combinatorial chemistry was developed several years identification of degradants in advance of their appear- ago as a tool used by a few companies for rapid ance during the drug development process. We employ a identification of new drug candidates. Today it is one strategy using standard LC/MS profiling and LC/MS/ of the fastest growing and fastest changing research fields. MS substructural analysis conditions to rapidly propose One of the biggest changes that has occurred in the field the structures of the degradants. Based on these studies, a within the last few years is the expansion of automation in degradant library is developed which includes infor- both synthesis and screening technologies. This change mation on molecular structures, chromatographic shifted the bottleneck fi'om production of new compounds behaviour, molecular weight, UV spectrum and sub- to their analytical characterization. structure-specific MS/MS product ions of the individual components. This library can then be used as a spring- Here we present a strategy developed in our lab for board for the structure dereplication and elucidation of automated analysis of libraries synthesized in different degradation products, impurities and process-related by- formats (low complexity mixtures, arrays of individual products encountered in samples throughput the drug compounds, etc.). The goal of analysis is to provide discovery-development-manufacturing continuum, as biologists with information about whether or not the well as candidate selection. As a result, in an effort to desired diversity was achieved, and give information to increase the capacity and throughput of the predictive chemists about the coupling success of the building blocks degradation profiling process, sample preparation was in each randomization. Approximately 10-20% of the critically evaluated. It was found that sample prepara- compounds (wells) in a library designed for lead generation is often very resource and time consuming, and tion, and 100% of compounds synthesized in arrays at ultimately a rate-limiting factor. Therefore, as an exten- the stage of library development or for SAR, are analysed sion of predictive models, we have developed a procedure by LC/MS in high-throughput fashion. Examples of the for automated sample preparation which involves a formats of data output used for the analysis will be Zymate robot. The utilization of these strategies as a presented. Aspects of quantitative analysis of combina- means of fulfilling and comparing selection criteria for a torial libraries will be discussed and illustrated utilizing number of potential drug candidates will be demon- the most recent examples. strated.

The impact of structure profile libraries on the Automated predictive profiling of drug candidates drug discovery-development continuum using LC/MS and robotics Robyn A. Rourick and Jeffrey L. Whitney, Bristol-Myers, Jeffrey L. Whitney, Robyn A. Rourick and Juan Cadavid, Squibb Pharmaceutical Research Institute, Wallingford, CT, Bristol-Myers Squibb Pharmaceutical Research Institute, USA; Kevin J. Volk, Saul W. Fink and Edward H. Kerns, Wallingford, CT, USA; Saul W. Fink and Kevin J. Volk, Bristol-Myers Squibb Pharmaceutical Research Institute, New Bristol-Myers Squibb Pharmaceutical Research Institute, New

102 Abstracts of papers presented at the 1998 ISLAR

Brunswick, NJ, USA; Mark E. Hail and Mike S. Lee, Bristol- must be versatile and represent good value for the Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, investment made. At BioFocus, we are essentially a USA service-based, problem-solving organization, which means that we are presented with a wide range of other Recent analytical initiatives aimed at accelerating the In order to meet drug discovery and early development continuum have people's starting points. tight deadlines, we cannot afford to be locked to one technology, but focused on obtaining greater amounts of structural must quickly make an assessment of the correct technique information on potential drug candidates earlier in the for the problem to hand. We use computation experi- process. In our pharmaceutical research and develop- mental design to constrain the number of chemicals we ment laboratories, analytical strategies based on standard need to in order to achieve maximal informa- and automated methods R. A. et al., synthesize LC/MS [Rourick, We have a of lOth AAPS Annual Meeting Proceedings Kerns, E. K. tion (predictive array designTM). variety (1994), automated and semi-automated synthetic methodologies et al., Nat. Prod., 57, 1391 (1994) are highly utilized to J. to both solid and solution syntheses, and rapidly provide this information. provide phase have constrained ourselves to high-quality end products. Critical factors affecting the early development of a drug include chemical stability, metabolic stability, formula- Understanding the physical and chemical limitations of tion development, process optimization and satty. Tra- the synthetic and analytical techniques allows integration ditionally, these factors are only addressed in a 'just in when appropriate. These issues will be addressed in the time' capacity during development, quite often nega- talk, illustrated with practical examples. tively affecting critical timelines. One proactive use of our methods involves LC/MS performing predictive Systems development strategy: a component- development profiles [Thompson, W. T. et al., Proceedings based approach, the overview of the 43rd ASMS Conference, 174 (1995)] on selected drug candidates during the discovery process. This approach Dinesh S. Thakur, Vincent W. Chen, Robi L. Banerjee, involves intentional chemical transformation of a poten- Matthew G. Holt and Kirk J. Leister, Pharmaceutical Research tial drug with a variety of sample conditions followed by Institute, Bristol-Myers Squibb Company, Syracuse, NY, USA LC/MS and LC/MS/MS analysis to elucidate any new components. Structures resulting from the transformation A component-based framework has been developed faster of a potential drug are assembled into a structure profile which facilitates software re-use and provides library (SPL) along with relevant source and reaction integration of laboratory automation systems. Implemen- parameters. Subsequently, the SPL is made available to tation of laboratory automation systems by the tradi- all downstream development areas for reference through- tional approach usually takes significant development out the lifetime of the drug candidate. In this way, time as both module-level and application-level software components in samples generated throughout preclinical are written custom to a particular application. In addi- development and Phase I-IV studies can be rapidly tion, modification and adaptation of an existing system identified by referencing the SPL. developed by the traditional approach, to new and enhanced applications, usually requires significant recon- Current initiatives aimed at accelerating the predictive figuration to system software. This is because modifica- profiling process involve automated analysis methods, tion and reconfiguration affect the way in which the intbrmation management systems, combinatorial incuba- module-level software interacts with each other. In order tion experiments [Volk, K. J. et al., Proceedings of the 44th to reduce the development time, and build flexible and ASMS Conference, 176 (1996)] and integrated robotic easily re-configurable systems, a component-based frame- methods [Whitney, J. L. et al., 15th ISLAR Conference work has been developed that addresses the issue of Proceedings (1997)]. These initiatives address the current software re-use and run-time configuration. This frame- rate-limiting steps, which include sample production, work is based on a services-based architecture and sample introduction, structure analysis and data man- supports stateless components that can be configured at agement. Examples will be shown illustrating our LC/ run-time. In addition, this framework supports poly- MS-based predictive profiling methods and integrated morphic behaviour for all its components, which allows robotic method development station. for integrated substitution of hardware offering similar functions into an existing system to enhance its function- Run-time has an additional benefit of Automation and computer design in chemical syn- ality. configuration laboratory automation systems fault tolerant. thesis: a small company perspective making This presentation outlines the basic overview of this Paul M. Doyle, Stephen R. Hopkins and Emma Barker, framework in the context of laboratory automation BioFocus, Sittingbourne Research Centre, Sittingbourne, Kent, systems, introduces the concepts involved and provides UK a high-level description of the architecture used to this framework. Currently, there are a bewildering array of technological develop solutions to the general problem of enhancing the throughput of chemical synthesis in the pharmaceutical Systems development strategy: component- and related industries. The degree of sophistication in based approach, the architecture chemical manipulation that can be achieved is impress- ive, although this complexity carries the penalty of cost. Vincent W. Chen, Matthew G. Holt, Robi L. Banerjee, Dinesh For the smaller laboratory, not only must there be a wide S. Thakur and Kirk J. Leister, Pharmaceutical Research variety of techniques available to the chemist, but they Institute, Bristol-Myers Squibb Company, Syracuse, NY, USA

103 Abstracts of papers presented at the 1998 ISLAR

The technical detail of the architecture for laboratory The peptidyl -ketoamide class of compounds are known automation systems built using the component-based to be both cysteine and serine protease inhibitors. Using framework will be described in this presentation. The solution phase, combinatorial unit synthesis was created interactions between the system coordinator, the system 45 000 discrete -kitoamide compounds (via the selec- repository, system scheduler, system logger and other tive monoacylation of diamines) that were arrayed in a peripheral components will be presented in the context spatially addressable, 96-well microtitre plate format. To of the underlying services-based architecture. Description address the effectiveness of this class of compounds of the communication interfaces between various com- against cysteine proteases, we tested their ability to ponents of an automated system will be explained. inhibit cruzain. Initial results yielded a series of three Several design patterns were used to develop this archi- lead compounds with sub-gM I Cs0 activity and up to tecture. The influence of these patterns on the design of > 10-fold selectivity versus cathepsin B and a panel of laboratory automation systems will be addressed. Also, serine proteases. The structure-activity relationships ob- the influence of unified modelling language on interface served from the initial screening of the spatially addres- design will be discussed. Integration of the system com- sable -ketoamide array facilitated the design and ponents with the peripheral components to build an synthesis of additional compounds, that yielded further automated system based on this framework will be increases in potency and specificity. The integration of presented with emphasis on run-time configuration and spatially addressable chemical synthesis and high- component reuse. In addition, the integration of this throughput screening accelerated the process of inhibitor architecture with enterprise applications, e.g. MS Ex- discovery and optimization for the protease cruzain. change and MS IIS will be addressed. The development of a laboratory component based on this framework will also be described. High-throughput screening--a multidisciplinary team effort

Systems development strategy: a component- A. E. Fletcher, M. S. Briggs, G. Foster, J. Kerby and N. Allen, based approach, the prototype Merck Sharp and Dohme, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, UK Robi L. Banerjee, Matthew G. Holt, W. Chen, Dinesh S. Thakur and Kirk Leister, Pharmaceutical Research Institute, The increasing demands on the pharmaceutical industry J. has led to the Bristol-Myers Squibb Company, Syracuse, NY, USA to enhance the drug discovery process introduction of automated high-throughput screening as This presentation provides a concrete example of an an 'industry standard'. However, the widespread implica- application developed using the component-based frame- tions of establishing such systems is frequently overlooked work. A sample inventory and control system was by many companies and can be in some cases the limiting developed for the purpose of aliquoting, distributing factor to efficient HTS. Any successful HTS operation and tracking biologic molecules in pharmaceutical drug requires a dedicated and committed multidisciplinary development. This presentation will provide an overview team to ensure efficient maximum productivity. At the of the workflow involved, a detailed description of the Neuroscience Research Centre we have established such a functional implementation of the robotic aliquoting team which encompasses representatives from scientific system, and a demonstration of the application with the (Sample Repository, HTS and Tissue Culture) and sup- real world implementation of the component-based port (Research Engineering, Purchasing and IT) staff. framework. The close interaction between the departments and the benefits of such a team will be reviewed together with an overview of the strategy employed for our HTS program. Accelerated discovery of cysteine protease inhibi- tots by the high-throughput bioassay of cz-ketoamide-based compounds created by automated Maximizing assay capability of older robotic parallel synthesis systems by incremental introduction of new technology to the screening laboratory H. Cheng, M. Cullinane, K. Brinner, C. Baldino and S. Chipman, Departments of Biology and Process Chemistry, M. Elizabeth Miller, Rohm and Haas Company, Spring House, ArQule, Medford, MA, USA; M. Laboissiere and C. Craik, PA, USA Department Pharmaceutical Chemistry, University Cali- of of The at Rohm and at San Francisco, San Francisco, CA, USA Agricultural Discovery Department fornia Haas Company has used a Zymark robotic workcircle to Cysteine proteases have been implicated in a wide variety perform fungitoxicity screening in microtitre plates for of human pathophysiological conditions. This class of about 10 years. At the time of delivery in 1988, the enzymes can be subdivided into > 14 different families system was considered state-of-the-art for handling mi- on the basis of sequence homology, the calpain and croplate assays, and was comprised completely of custom- papain families being the most significant. We chose a designed stations, as microplate-handling devices were papain-like cysteine protease, cruzain, to demonstrate almost unknown in those days. Since then, microplate how the integration of automated, parallel chemical handling on integrated robotic workcircles has become synthesis and high-throughput screening has accelerated routine, and a variety of 'off-the-shelf' plate handling the discovery and optimization of small organic com- stations are now available from different vendors, obviat- pound inhibitors. Cruzain is a protease isolated from ing the need for custom stations in many cases. In light of Trypanosoma cruzi, the causative agent in Chagas' disease. new developments in screening technology, do older

104 Abstracts of papers presented at the 1998 ISLAR integrated robotic systems still have a place in the low-throughput manual assay, to semi-automated, and laboratory? Depending upon the demands on the screen- finally to the development of the UHTS used for several ing organization, the answer can certainly be yes. We are nuclear receptors will be discussed. still using our original workcircle with custom stations, with only some minor upgrades made to the system. In order to accommodate newer assay procedures, however, Generic testing procedure for automated analyti- we have added several stand-alone workstations to the cal chemistry workstations laboratory, which are used for a variety of work processes Craig Bender, Searle R&D, Skokie, IL, USA not manageable on our Zymark system. This combined approach of using an integrated workcircle in concert with stand-alone units has been a very cost-effective way of enhancing our screening capabilities while continuing Automation of for to make full use of our investment in our original robotic liquid handling high-through- in 1536-well with the system. put screening microplates Robbins Hydra and automated plate positioning system Information--not just data: use of a robotic dis- David W. and Robbins solution system to provide rapid graphical feed- Batey Jim Stanchfield, Scientific, back to formulators Sunnyvale, CA, USA Distribution of and of Mark J. Dryfoos, Stability, Robotics & Automation Group, compounds dispensing reagents PHAD/ARD Novartis Pharmaceuticals Corporation, East used in high-throughput screening assays are readily Hanover, NJ, USA performed with Hydra microdispensers. The 96 (or 384) glass syringes of the Hydra are held in a fixed Early in the development stage of a solid dosage form, array positioned at the centre of each well in a standard many variants are produced. The sheer number of microplate. Precision mechanical components allow an samples to be tested can be overwhelming as formulators accuracy of +1% for dispensing I.tl with a coefficient of try to achieve an optimal balance of lubricants, disin- variation of less than or equal to 3% across the syringe tegrates, coating thickness, tablet hardness, etc. Because array. With syringes available in sizes ranging from these samples will neither be used in clinical trials nor in 1001al to ml, the Hydra is well suited for transfer of primary stability studies, a less formalized approach (i.e. bulk reagents as well as distribution of compounds in non-GMP) to their dissolution testing may be utilized. submicrolitre volumes down to 100nl. When equipped We have adopted just such a 'second-track' strategy in with the newly developed automated plate positioner, the the Stability, Robotics, & Automation Group, both to Hydra-96 and Hydra-384 are ideally suited for high- ease the burden on the GMP Dissolution Testing Lab, as throughput, repetitive dispensing procedures. All plate well as to provide rapid turnaround, high value-added and liquid handling functions can be controlled through results to the formulators. This capability is provided by user-defined parameters within the included software or an automated Apparatus 2 dissolution system featuring a through simple ASCII commands when used in an Zymate XP robot with online fast HPLC. Customized integrated system. The stage can accommodate a combi- spreadsheets, macros and presentation graphics templates nation of two plates of various well densities with a third allow for rapid data reduction and plotting. Several location dedicated as a wash reservoir that is filled and unique aspects of the system as well as the advantages emptied by peristaltic pumps. Settings for the positioning of this approach will be discussed and illustrated. of the plates during dispense/aspirate operations can be adjusted in increments of 5 tm. This level of positional accuracy allows consistent and reproducible access into An automated assay for ligands of nuclear recep- current 1536-well microplates as well as future high- tors density formats. For reformatting operations, any combination of 96- (Hydra-96 only), 384- and 1536-well Richard If. Harrison, Litao ,hang, Ien Locke and Geoff microplates can be in the two positions Rhone-Poulenc USA placed plate Schwartz, Rorer, Collegeville, PA, and re-arrayed through software protocols defined by Nuclear receptors are ligand-activated transcription fac- the user. Fluorescence signal was obtained for reactions tors. Their function in regulation of gene expression assembled and distributed into all wells of 1536-well makes them attractive targets for therapeutic interven- plates. By following recommended wash protocols for tion in various metabolic diseases. As part of an ongoing the glass syringes, the results indicated no carryover program aimed at regulation of these receptors, we have contamination between samples or reagents. Reaction developed a high-throughput assay for determination of components included compounds stored in 100% DMSO compounds than can bind to nuclear receptor targets of as well as aqueous reagents. Process time for reaction interest. The assay is a simple homogeneous system based assembly in 1536-well plates will be presented. The on scintillation proximity protocols, different from other Hydra-96 and Hydra-384 microdispensers, equipped with SPA assays for nuclear receptors in the use of unde- the automated plate positioner stage and software make rivitized ligand binding domain, and is capable of screen- up a complete high-throughput system for accurate, ing over 50000 compounds/week. The evolution, from a precise dispensing into today's high-density microplates.

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