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					Journal of Automated Methods & Management in Chemistry
Vol. 24, No. 3 (May–June 2002) pp. 61–98




Abstracts of papers presented at the ISLAR
(International Symposium on Laboratory
Automation and Robotics) 2001
The 19th International Symposium on Laboratory Automation and Robotics provided presentations on state-of-the-ar t
developments in laboratory automation and robotics. The symposium programme included papers and posters on all
aspects of the technology. These comprised: managing laboratory automation (drug discovery); bioanalytical analysis;
managing laboratory automation in drug discovery development and QC laboratory; functional genomics strategies and
high throughput screening; advanced integration strategies; method development and global methods transfer;
compound handling and logistics; combinatorial chemistry and automated synthesis; high throughput LC-MS-MS;
increasing eæ ciency in dissolution testing; lead optimization; strategies for UHTS; increasing throughput for ADME
toxicology; data management/data handling and bioinformatics; using contract laboratories to increase productivity;
assay miniaturization; process optimization; compliance and automationÐ the regulatory perspective; novel high
throughput screening technologies; compliance and automationÐ the industry perspective. Several discussion sessions
were included and activated, and provided interactive communication on a wider range of subjects.
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 and each poster are included here. Full presentations of several of these papers will appear in
later editions of this journal.

Designing custom automation systems to                             t the            require precise control of sampling and temperature. A
needs of early development                                                          typical degradation study is usually conducted over a
                                                                                    period of weeks. To compare studies in a relative manner
Adam M. Fermier, The R. W. Johnson Pharmaceutical                                   for excipient compatibility studies, these times and
Research Institute, Raritan, NJ, USA                                                temperatures must be maintained. It is an ideal applica-
Co-authors: John Troisi, Ramon L. Rodriguez, and James V.                           tion for automation because of its repetitive nature. The
Weber, Michael P. Graham and Erin C. Herita                                         system allows for 16 reactions to be conducted in parallel.
The pharmaceutical industry is under extreme pressure                               The software keeps track of temperature and times of
to reduce the costs of bringing drugs to market. Thus, to                           sampling.
achieve this goal the entire drug development process
                                                                                    In this presentation we will review criteria we have found
requires continued ® ne tuning and the introduction of
                                                                                    to be successful in designing the instrumentation and
new technologies. Drug discovery has been moving
                                                                                    identifying processes that would be ideal to automate.
through this change over the past decade. Drug devel-
opment is now looking at that same daunting task of
increasing throughput while maintaining compliance                                  Bringing Caco-2 studies from moderate- to high-
and headcount restraints. Custom automation systems                                 throughput screenin g
provides one opportunity to attain this goal.
                                                                                    Dawn Alderman, Neurogen Corporation, Branford, CT, USA
We have been investigating and building custom auto-
mation into our drug development process. For example,                              High throughput screening (HTS) technologies have
we have modi® ed a Zymark Benchmate for weighing                                    been widely used in many areas of drug discovery.
small quantities (1± 50 mg) of dry powders. The addition                            Human Caco-2 cells provide an in vitro model of
of weighing dry powders from a stock tube enables many                              drug bioavailability. They are derived from a colorectal
advantages and increases the ¯ exibility of the Benchmate.                          adenocarcinoma cell line and exhibit functional
One application we have been using this robot for is to                             characteristics of the lower small intestinal tract.
weigh individual vials of drug substance into HPLC vials                            They diå erentiate as a monolayer with apical and
for subsequent accelerated solid state degradation studies.                         basal directionality that mimics the intestinal
This typically requires about 20± 40 weighings for a single                         barrier. Involvement of P-glycoprotein and other active
study. A typical degradation program would have a                                   transport systems expressed endogenousl y in¯ uence drug
dozen studies for a single compound. The weighing robot                             absorption.
not only saves time but also oå ers some intrinsic safety
                                                                                    Currently, most Caco-2 studies have low to moderate
because the analyst is not directly exposed to the
                                                                                    throughput due to assay complexity. Many diå erent
material.
                                                                                    assay formats exist and are commercially available.
We have also designed a custom automated system to                                  Becton Dickinson provides a 3-day assay system and a
conduct accelerated degradation studies. These studies                              10-day assay protocol. The 3-day system includes a

                         Journal of Automated Methods & Management in Chemistry ISSN 1463± 9246 print/ISSN 1464± 5068 online # 2002 ISLAR   61
                                                                    http://www.tandf.co.uk/journals
                                                                  DOI: 10.1080/14639240210141787
Abstracts of papers presented at the 2001 ISLAR


media kit, vital to the growth and diå erentiation of the      Currently, samples are evaluated for Papp (apparent
cells used within a 3-day period. The 10-day protocol          permeability) using this published equation:
suggests that the cells can be seeded and used after a 10-
                                                                         Papp …10¡6 cm s¡1 † ˆ …Vd =A†…dC=dT†:
day growth period without the requirement of the
enhanced media necessary for the 3-day system. The             This is a rate distance measurement, where Vd is the
10-day assay media that we currently use has an anti-          volume of the donor compartment. A is the surface area
biotic in it to protect against the potential bacterial        of the monolayer, dC is the change in concentration and
contamination that may occur with the cells seeded in          dT is the change in time.
the plate for 10 days. The antibiotic condition may aå ect
the data when comparing with the 3-day system. Neuro-          The Caco-2 cell model mimics the dynamics of the
gen is currently evaluating these assays for any potential     human lower small intestinal tract. The potential for this
diå erences.                                                   model to move towards a high-throughpu t format oå ers
                                                               the high-throughpu t compound screening in the future,
The Caco-2 model does not take into consideration ® rst-       which is essential to modern pharmaceutical develop-
pass metabolism. The detection method also creates a           ment. Optimizing the current cell density and volume
signi® cant bottleneck. Currently LC/MS/MS is being            speci® cations serve to strengthen this assay format, and is
used. By using HTS technologies, such as laboratory            currently a major hindrance in its development.
automation and miniaturization, we have shown that
the throughput of the Caco-2 assay can be signi® cantly        Rational approach for selecting extraction par-
increased. The introduction of the TECAN GENESIS               ameters for homogenizer-base d tablet assay
has allowed the automation of the drug addition to the         methods
Caco-2 assay plates. It has also allowed for automated
sampling. The TECAN GENESIS that is currently in               Alger Salt, GlaxoSmithKline, Research Triangle Park, NC,
our facility has 1 ml Te¯ on-coated syringes, and a 21-        USA
plate deck design. The programs being used have been           This poster will present a rational approach for choosing
written in-house using the GENESIS software.                   extraction parameters, speci® cally homogenizer times
We have evaluated the 3-day assay system and are               and speeds for tablet test methods that employ the
currently evaluating the 10-day protocol, in 24- and           TPW II workstation or similar homogenization-base d
96-well formats. We have found that our data are of            sample extraction procedures. This approach is based
good quality and are reproducible in-house. We are still       on the assumption that extractions can be modelled using
evaluating many aspects of the 96-well assay format.           a ® rst-order rate equation. The extraction pro® le, a curve
                                                               showing the amount of drug dissolved versus time, can be
Eå ective cell density and the volumes used in each assay
                                                               characterized by a single number k, the ® rst-order rate
format have been assessed. We began by investigating a
                                                               constant.
couple of cell densities. We have evaluated 12.5 K/well
(Becton Dickinson protocol) and 25 K/well. Well vol-           The amount of drug extracted at a given time can be
umes have also been assessed. Initially 100 ml in the apical   estimated or predicted if the rate constant is known.
ports and 250 ml in the basal ports was used. However,         Conversely, the amount of time required to reach a given
this condition is associated with a suspected wicking          amount of drug dissolved can be estimated. These
problem. The sample under the plate lid as well as the         assumptions allow one to model the process and predict
volume moving up the interior wall of the basal compart-       a combination of parameters to ensure that the tablet is
ments has been observed. This is a possible suspect for        completely dispersed and that the analyte is extracted
interwell contamination. We are further developing this        quantitatively. A formal protocol can then be designed
assay and will evaluate this phenomenon. We have               and performed to validate the extraction process. The
attempted to cut the volumes from 100 ml in the apical         poster will describe this approach and illustrate it with a
compartment to 80 ml. The basal well we have cut the           case study.
volume from 250 to 200 ml.
There are many assay conditions that have been pub-            Introduction of new technology into the pharma-
lished that vary between laboratories. Many publications       ceutical quality-contro l environment: ‘cutting
suggest that a pH gradient from 6.5 (apical compart-           costs without cutting corners’
ments) to 7.4 (basal compartments) is the best model,          Andy Boughey, AstraZeneca, UK
representing the conditions of the lower small intestine.
Another assay condition that varies between laboratories       Ever increasing demands at the drug-discovery level in
is what concentration of drug to use. High concentrations      recent years have lead to signi® cant advances in auto-
of drug create the risk of saturating the P-glycoprotei n      mation. This in turn has lead to increasing numbers of
transporte r as well as the other active transport mechan-     drug candidates and correspondingly more new products
isms. We are currently evaluating our Caco-2 studies in        being manufactured, with a subsequent impact on the
modi® ed HBSS buå er at 5 mm drug concentration using          Quality Assurance Department.
the pH gradient. Again, cell density is a factor that varies   Automation plays a key role in the future of quality
between laboratories. Becton Dickinson recommends a            control. It is recognized that signi® cant cost savings can
cell density in a 24-well plate to be 200 K/well and in a      be made with the introduction of automation. However,
96-well plate to be 12.5 K/well. We are currently using        there are equally important bene® ts to be gained with
this recommendation for both assay formats discussed.          respect to cGMP.

62
Abstracts of papers presented at the 2001 ISLAR


This presentation will discuss the pressures being placed      to track all compound and assay plates entering the
on quality control and the bene® ts and concerns of            system.
introducing new technologies.
                                                               Reformatting chemical libraries with the StaccatoTM
It is easy to support the introduction of new technology       system is very eæ cient. With its large storage capacity
when the bene® ts can be expressed in terms of dollars         and ¯ exible con® gurations, reformatting can be done
saved, but what is the true value of improved regulatory       through a variety of methods. Using the Rapid head
compliance?                                                    con® guration, 300 96-well plates can be reformatted to
                                                               75 384-well plates in one 8-h day. Using the small volume
                                                               ® xed-tip con® guration, one can convert 200 96-well
High-throughput analysis of free amino acids in                 plates to 50 384-well plates in one 8-h day. Although
biological matrices                                             ® xed tip con® gurations yield less throughput due to
Arthur Rugg, Cereon Genomics LLC, Cambridge, MA, USA             implementation of wash protocols, there are no consum-
Co-authors: Shaoxia Yu and Lily Li                               able costs. It also gives you the ability to reformat further
                                                                 from 384- to 1536-well plates, with an even smaller
Traditionally, chromogenic or ¯ uorescent derivatization         volume 384-cannula array designed for 1536 applica-
(FD) coupled with high-pressure chromatograph y                  tions.
(usually 20± 30 min/separation ) is used for amino acid
analysis due the amino acids’ lack of UV absorbance. To        The independent Z-axis probes provide the ability to
detect an alternated change in the levels of free amino        cherry pick select compounds, assemble serial dilution
acids through the screening of thousands of biological         plates, and manipulate compounds and reagents for
samples, the analysis of 20 amino acids using an HPLC/         ADMET studies. Performing parallel processing of plate
FD approach is tedious and time consuming. Therefore,          types using the ` on the ¯ y’ array swap capabilities, the
a high-speed 1.5 min per sample LC/MS/MS method has            Staccato TM is capable of reformatting from 96- to 384-
been developed, validated and implemented to analyse           well plates using the 96-cannul a array. After dropping oå
20 free amino acidsÐ in biological matrices.                   the 96 array, it can then pick up the 384 array and
                                                               further dilute or deliver compounds directly to assay
                                                               plates for execution with no human intervention.
Multitasking, scalability and exibility: the Stac-
                                                               The presented pipetting specs are based on ® ve trays run
cato TM automated workstation
                                                               with three transfers performed per tray. A 0.5 ml wet
Matt Boeckeler, Neurogen Corporation, Branford, CT, USA        transfer produces excellent precision with an average
                                                               %CV of 6.4, and an accuracy range of 1.06± 7.3%. A
With drug-discovery technology advancing and chang-
                                                               0.5-ml dry transfer yields an average precision %CV of
ing as rapidly as it is, there is a growing need for           8.6, and an accuracy range of 0.03± 10.1%. Dry transfers
automated equipment to have the capability to change           of 1 ml yield a precision average %CV of 5.8 and an
with these changes in technology. The use of single-
                                                               accuracy range of ± 1.5 to 4.4%. Volumes > 1 ml yield
workstation approaches to liquid handling is decreasing        similar results to the 1-ml numbers.
due to the increasing need for a system that can perform
various liquid-handling tasks on the same platform.            System use at Neurogen Corporation entails taking in
               TM                                              chemical entities from our existing archive or from our
The Staccato         automated workstation is an extremely
                                                               high-speed synthesis group and performing compound
versatile liquid handler with the capabilities of handling
                                                               dilution procedures, or reformatting processes on the
plate formats ranging from 96- to 1536-well plates. The
                                                               plates and transferring the compounds directly into assay
` Drag and Drop’ architecture of the software allows for
                                                               plates, all within the same method. On this platform, the
 ¯ exibility and ease in process development. With the use
                                                               system can also build dose± response plates, prepare
  of CLARATM 2001 software, method development and
                                                               compounds for interdepartmenta l support or execute a
  system recon® guration become less intensive. Its scal-
                                                               variety of assays.
  ability lends itself to supporting changes in platforms as
  emerging technologies arise.                                 After compounds are in the proper format and concen-
               TM                                              tration, with the use of the CavroTM dispensers, multiple
The Staccato     system can be equipped or con® gured
                                                               reagent addition to assay plates can be accomplished
with many components and options to ® t your liquid-
                                                               with the proper scheduling of processes. In essence, the
handling needs. Using the SciClone ALH as the base
                                                               system can take compounds from their starting format
liquid handler, the system can come equipped with a
                                                               and concentrations, manipulate the plates to accommo-
disposable tip Rapid head, or a small volume ® xed tip
                                                               date assay-speci® c plate formats and conditions, then
head with 96 or 384 cannula arrays. To augment the
                                                               proceed to add the appropriate reagents via the CavroTM
liquid-handling capabilities of the SciClone, up to four
                                                               dispensers, bringing the assay from compound delivery,
separate CavroTM bulk-dispensin g manifolds can be
                                                               through reagent addition. Dispensing by the CavroTM
mounted on the head for reagent additions. Liquid
                                                               syringes for volumes of 10± 2500 ml all yield similar results.
handling can be further enhanced by the addition of an
                                                               Across plate precision yields an average%CV of 3.8 with
eight tip, independent Z-axis probe used for various
                                                               an accuracy range of 1.1± 5.5%. Non-contact dispensing
liquid-handling applications. Integration of the Auto-
                                                               of reagents eliminates cross-well contamination and with
stack with the SciClone gives an exceptionally large
                                                               multiple manifolds, various reagents can be used.
storage capacity for unattended operation. The use of
on-board barcode readers in conjunction with the               Replication of compound data generated by the 384-
CLARATM Data Manager, gives the system the ability             cannula array at a dry transfer volume of 0.5 ml is quite

                                                                                                                           63
Abstracts of papers presented at the 2001 ISLAR


good. Dry dispensing of such a small volume is not easily      Update from PQRI’s Blend Uniformity Working
achieved, but obtainable with acceptable %CV using             Group: balancing workload with batch homogene-
particular pipetting methods. Using the same pipetting         ity assurance
methods and comparing data to proven control methods
                                                               Garth Boehm, Purepac Pharmaceutical Co., Elizabeth, NJ, USA
for drug delivery yields very acceptable %CVs and
correlation.                                                   The Product Quality Research Institute (PQRI) is a
                                                               consortium between industry, academia, and FDA,
CLARATM 2001 Software is used for scheduling methods           which aims to provide a scienti® c basis for the develop-
and integrating peripherals into the existing system. It       ment of regulatory policy. One of PQRI’ s ® rst initiatives
communicates with adapter modules that translate com-          was to form a Blend Uniformity Working Group
mands between CLARATM and the ICP of the periph-               (BUWG) charged with providing a scienti® c basis for
eral module, triggering program execution of the               continued development of FDA policy on BU testing.
instrument. This method of integration makes the scal-         The BUWG sought wider industry input through a
ability of the system much easier and more eæ cient than       survey of current BU testing practices, and a public
previous conventional methods. Upon process comple-            Workshop. Using this input together with the experience
tion, the ICP sends a message back to CLARATM                  of the BUWG members and colleagues, a draft proposal
through the adapter to con® rm completion, or send             has been developed and re® ned. The proposal provides a
notice of an error in execution. The system’ s open            guide for appropriate testing during scale-up and valida-
architecture, in conjunction with the CLARATM soft-            tion, and for subsequent routine manufacture. The
ware, allows for ¯ exibility and ease when modifying or        proposal is based around initially establishing the rela-
implementing new assay platforms and technologies.             tionship between BU testing and strati® ed testing of the
                                                               resulting dosage units. The type and amount of testing
The CLARATM Data Manager gives you the ability to              recommended during routine manufacture depends on
manipulate and ® lter incoming data with the use of the        the outcome and robustness determined from the valida-
Data Manager’ s ZyMap. Data can be con® gured into             tion testing. The BUWG is currently seeking data from
text ® les, Excel sheets or directly transported to a          industry to challenge the proposal with real data to
database via ODBC connections. However, the current            establish the validity of the suggested approach. The
version of the Data Manager is unable to catch critical        next step will be to seek public comment on the BUWG
eventsÐ events that would trigger the data to be sent to       proposal.
the database, such as an ` end of run’ event. This inability
does not yet allow the direct triggers to export data to a
database. A temporary remedy supplied by Zymark is a           Views from the past: HTS in the Molecular Bio-
developer’ s kit (a collection of APIs) that gives you the     chemistry Department, Glaxo Wellcome, Inc.
ability to build your own Data Manager using prede® ned        Brent Butler, GlaxoSmithKline, Research Triangle Park, NC,
templates. Using this method could require extensive           USA
time and programming on the user’ s end depending on           Co-authors: Cole Harris and Steve Blanchard
the process and data being captured. However, an
updated version of the Data Manager is anticipated to          The pharmaceutical industry saw a tremendous increase
be released at the end of October that would alleviate this    in screening throughput in the 1990s with the intro-
problem.                                                       duction of advanced robotics and liquid-handling
                                                               systems, as well as homogeneous assay formats conducive
Although the StaccatoTM system has many bene® ts and           to higher density assay plates. During this time, there
capabilities for liquid-handling needs, there were some        have been numerous debates on the advantages and
implementation issues along the way. The biggest setback       disadvantage s of centralized versus decentralized screen-
experienced was with limitations of the Data Manager.          ing sites. There have also been discussions about the use
One of the top reasons for the selection of the Zymark         of large, integrated robotic systems compared with small
system over other vendors’ was the ease of database            bench-top robotics.
connectivity and data export. Another issue was in the         Glaxo Wellcome Research Triangle Park has screened in
design of the ® xed tip arrays. Great care must be taken       a decentralized manner using both large automation
not to damage their Te¯ on coating. Any scarring or            platforms and small bench-top systems. In addition, the
scraping of the material will cause variation in pipetting     screens have migrated from 96- to 384-well plates. This
procedures especially at small volumes. Service for the        talk will focus on the lessons learned from the screening
most part is good; however, the redesign and complexity        eå ort in the Molecular Biochemistry Department of
of the system left the need for more time in training of       Glaxo Wellcome.
technicians to come up to speed in the overall workings of
the system.
                                                               Fully automated screening of intracellula r cal-
Overall, Neurogens experience with the StaccatoTM              cium using a novel detector
system has been a good one. All systems have their
bene® ts and drawbacks. The most important thing is            Brian Rasnow, Amgen, Inc., Thousand Oaks, CA, USA
the cooperation and speed of assistance received when          Co-authors: Jack Lile, Karen Kearns, James Treanor and Peter
problems do arise and eæ ciency in coming up with              Grandsard
solutions. In this aspect, Zymark has shown dedication         We have constructed a fully automated high-throughput ,
and commitment to their products and customers.                cell-based screening system that measures aequorin lumi-

64
Abstracts of papers presented at the 2001 ISLAR


nescence kinetics as an indicator of intracellular calcium   This presentation will discuss the coupling of ASE with
concentration. The system uses a modi® ed Zymark             liquid-handling apparatus to produce plant extracts free
Rapidplate96 pipetting station to add drug candidates        from interfering coextracted compounds. This combined
and known modulators to aequorin-expressin g cells           system produces natural product extracts that are ready
grown in microtitre plates. Simultaneous with compound       for biological or chromatographi c assay. The comparison
addition, custom optics and a high-performance chilled       of results from this procedure to those obtained without
CCD camera added to the Rapidplate96 take an image           automation will be discussed.
sequence of the plate bottom. Compounds that aå ect
calcium entry or release from intracellular stores can be
analysed using this system. Agonists and antagonists can     Determinatio n of cytotoxic e´ ects of di´ erent
be detected in the same screen by imaging both during        compounds using a multiparametri c cell-based
compound addition and during a subsequent addition of        cytotoxicity assay
known agonists.
                                                             Chandrasekaran Vasudevan, Cellomics, Inc., Pittsburgh, PA,
The 96-channel imaging ¯ ash luminometre is integrated       USA
with a Beckman CO2 incubator, Zymark Twister, tip            Co-authors: Oleg Lapets, Nancy Kerkvliet, Greg LaRocca,
store, ORCA robot, and other peripherals to perform          Rachael Krally, Lisa Lemmler, Patricia Petrosko, Megan Weiss,
fully automated screening of up to 72 microtitre plates      Joseph Zock and Richik Ghosh
with a cycle time of several minutes per plate (about
                                                             In vitro screening of active compounds for toxic eå ects
10 000 high-content, cell-based assays/system per day).
                                                             earlier in the drug-discovery process will help identify
The system is controlled with Amgen’ s automation soft-
                                                             problems and reduce the high failure rate of current
ware (` Synchronicity’ ). Analogue and digital signal pro-
                                                             clinical candidates. Cell-based toxicity assays are crucial
cessing are performed in real time to remove artefacts
                                                             in this eå ort, but are limited by the result generated,
from cosmic rays and bad pixels, and normalize any
                                                             usually cell death. Thus, we have developed a more
systematic eå ects from geometrical and biological hetero-
                                                             sensitive multiparameter cell-based cytotoxicity assay
geneity across the plates. This reduces the about 1.5 MB/
                                                             that quanti® es early changes to key aspects of cellular
plate of raw data to about 10 kB of time series, which can
                                                             physiology that can lead to cellular toxicity.
be further compressed and the compounds classi® ed using
principal component analysis and other methods.              This assay uses our ArrayScan1HCS System High
                                                             Content Screening (HCS) platform that simultaneousl y
We have validated the system for ion channel and GPCR
                                                             measures changes in nuclear morphology, cell perme-
HTS and dose± response characterization, and it has
                                                             ability and lysosomal physiology for individual cells, and
screened thousands of plates. We will discuss and contrast
                                                             also changes in cell density in microplate wells. Dose±
its design and performance with other devices that have
                                                             response and time-course data demonstrating the assay’ s
comparable capabilities, and explore some of the data
                                                             multiparametric nature will be presented for sample
and information processing challenges presented by high-
                                                             compounds that aå ect the cellular targets in various
throughput, high-content screening systems.
                                                             cultured cell lines and also rat primary hepatocytes.
                                                             Our data show the assay’ s ability to capture cellular
                                                             reactions to compounds and correlate multiple toxicolo-
Automated production of medicinal agents from                gical indicators at a single-cell level. This assay represents
plant matrices                                               a distinct advantage for HCS as a screening tool early in
                                                             the drug-discovery process.
Bruce E. Richter, Dionex Corporation, Salt Lake City, UT,
USA
Co-author: Richard E. Carlson
                                                             Development of a high-throughput, homogeneous,
Interest in the pharmacologically active compounds           cell-based assay for screening inhibitors of mul-
found in plant tissues is growing. The extraction tech-      tiple drug-resistanc e pumps on FLIPR or FLEX-
niques normally used to remove these compounds from          Station systems
plant tissues require long periods and copious amounts of
solvents. Accelerated solvent extraction (ASE) has been      Christopher Silva, Molecular Devices Corporation, Sunnyvale,
                                                             CA, USA
proven eå ective in removing target compounds from a
variety of plant tissues. Using ASE, the extraction of       Co-authors: Kelly J. Cassutt, Anne T. Ferguson and Jesse Twu
compounds from medicinal plants is completed in about        Multiple drug resistance (MDR) pumps expressed on the
15 min using only 20 ml solvent. Extraction of the target    surface of normal cells are involved in eliminating toxic
compounds may be only a part of the isolation process.       compounds generated by or exogenously introduced to
Interfering compounds such as waxes, pigments and            the cells. In cancer chemotherapy, these pumps may
tannins can be coextracted.                                  become over-expresse d by the tumour cells and render
                                                             the drug treatment ineå ective following relapse. MDR
These interferences must be removed before the extracts
                                                             pumps are also normally expressed at the blood-brain
can be subjected to screening techniques. Solid-phase
                                                             barrier (BBB) and as such give the brain ` sanctuary ’ from
extraction (SPE), liquid± liquid extraction (LLE) and
                                                             chemotherapeuti c drugs.
preparative liquid chromatograph y are the techniques
most often used for removing interferences from plant        Our goal at Molecular Devices is to provide a tool to
extracts. However, these steps are separate and not          screen libraries for compounds that inhibit the MDR
coupled with the extraction process.                         pumps. Such compound leads could be important for

                                                                                                                        65
Abstracts of papers presented at the 2001 ISLAR


inhibiting MDR, which might otherwise lead to failure of       Direct analysis of basic drugs in cell culture lysate
the therapeutic regimen. The compound leads identi® ed         using online extraction LC/MS/MS
might also help facilitate the absorption or retention of
other useful therapeutics for disease treatment.               Claude R. Mallet, Waters Corporation, Milford, MA, USA
                                                               Co-authors: Je¡ Mazzeo and Uwe D. Neue
The assay is designed primarily for screening inhibitors
against the two most common MDR pumps, P-glycopro-             During the last 10 years, pharmaceutical companies have
teins (P-gps) and multidrug resistance-associate d proteins    constantly pushed for shorter analysis time to breach the
(MRPs). Both classes of pumps are known to expel a             1000 analyses per day barrier. With this demand for
variety of mechanistically and structurally unrelated          high-speed analysis, new techniques such as 96-well
cytotoxic drugs such as anthracyclines, tacanes, vinca         plates, fast gradients or ultrahigh-¯ ow chromatograph y
alkaloids, and epipodophyllotoxin s as well as some ¯ uor-     are showing promising results.
escence substrates or indicators from the cells. For a given
assay the compound library is prepared in a 96- or 384-        We have focused on online extraction techniques for
well plate and allowed to incubate with cells at room          high-throughpu t analysis. In a previous study we inves-
temperature for 15 min or longer. The microplate is            tigated the potential of online extraction for the analysis
placed in the FLIPR or FLEXStation System followed             of basic and acidic drugs in rat plasma. Recently, we
by addition of the ¯ uorescence indicator by the instru-       have turned our attention to other types of matrices, e.g.
ment. The kinetic readout of the results can then be           the study of toxicity of drugs in cell cultures used in pre-
collected in a real-time mode. The results are expressed as    and post-discovery phases. Several chemical and physical
the change in relative ¯ uorescence units (RFU) over           lysate methods were evaluated. The extracts were in-
time, usually 5-8 min after the start of the experiment.       jected onto an extraction column at high ¯ ow rate (i.e.
Inhibition of the MDR pumps is indicated by the                4 ml min¡1 ) [1± 3] to remove macromolecular compounds
increase in RFU above the baseline control.                    such as proteins, but trap smaller analytes on the head of
                                                               the column. Several con® gurations for direct injection are
Using the cell line MCF-7/ADR* (resistant to adriamy-          possible. In the simplest con® guration, the extraction
cin), we showed that the baseline change in counts             column is connected directly to the MS/MS system.
without inhibitor is nearly zero while 30 mm cyclosporin       Other versions are con® gured with a single or a dual
A (a known inhibitor to P-gp) increased the RFU to             extraction column coupled to an analytical column. It is
> 5000. These results indicate that the P-gp expression        often necessary to split the ¯ ow. However, in cases where
level is high enough to keep dye completely out of the         sensitivity is low, this option is not recommended. For
cells and the MDR pumps were inhibited eå ectively by          eæ cient high-speed analysis, the use of a second pump
cyclosporin A. Various tumour and non-tumour cell lines        and a 10-port valve is also a good choice. One line (high
including the parental MCF-7, Caco-2, and T-cell line          ¯ ow rate) can be dedicated to the extraction column,
CEM were also tested and showed parallel results. Very          while the other (low ¯ ow rate) drives the analytical
little or no DMSO interference to the assay was observed.
                                                                column and the mass spectrometer.
We have developed a robust and reproducible homo-              A three-valve con® guration using two extraction columns
geneous cell-based assay for high-throughpu t screening of
                                                               was used for the analysis of basic drugs in cell culture
compound libraries for inhibitors of MDR pumps. The
                                                               lysate. The online analysis was performed on an Oasis
ease of use inherent in the format with no wash step
                                                               HLB extraction column (2:1 £ 30 mm, 25 mm) using a
involved along with the rich kinetic data previously
                                                               Waters Alliance 2790TM in the gradient mode and a 515
unavailable to the large-scale MDR studies indicate that
the FLIPR MDR Assay Kit will be a time-saving and              stand-alone pump in the isocratic mode. The extracted
cost-eå ective tool in HTS screening of inhibitors of MDR      analytes were forward ¯ ushed into an XTerra column
pumps.                                                         (2:1 £ 30 mm, 3.5 mm), which was added to provide
                                                               additional separation power. The drugs were quanti® ed
                                                               using a MicroMass UltimaTM triple-quadrupole mass
                                                               spectrometer equipped with an electrospray source and
                                                               set in multiple reaction-monitorin g mode (MRM).

Custom hit-picking robotic system

Claude Dufresne, Merck Research Laboratories, Rahway, NJ,      References
USA
Co-authors: Christopher Napolitano and Keith Silverman         1. Xia, Y. Q., Whigan, D. B., Powell, M. L. and Jemal, M., 2000,
                                                                  Rapid Commun. Mass Spectrom., 14, 105.
An in-house integrated automated ` cherry picking’             2. Ayrton, J., Dear, G. J., Leavens, W. J., Mallett, D. N. and
system will be described. It is designed to provide backup        Plumb, R. S., 2000, J. Chromatogr. A, 828, 199.
                                                               3. Eeckhout, N. V., Perez, J. C., Clearboudt, J., 2000, Vandeputte,
samples of natural products’ ` hits’ to biologists perform-
                                                                  R. and Van Peteghen, C. Rapid Commun. Mass Spectrom., 14, 280.
ing primary screening. The system uses a Tomtec
MegaStor for high-capacit y source plate storage. The
system is controlled by custom software, written in Visual
Basic 6.0, and makes use of ActiveX controls for each of
the system components.

66
Abstracts of papers presented at the 2001 ISLAR


A look to the future: screenin g in the Systems                   MS assays for these compounds. Challenges encountered
Biology Department, GlaxoSmithKline , Inc.                        during assay development and details of the method will
                                                                  be presented. Transfer of these automated o¥ine assays
Cole O. Harris, GlaxoSmithKline, Research Triangle Park, NC,      to online automated SPE and/or o¥ine automated 96-
USA
                                                                  well SPE will also be discussed.
Co-authors: Brent T. Butler, Steven G. Blanchard and David C.
Morris
Screening at the newly formed GlaxoSmithKline Re-
search Triangle Park site will be heavily dependent on
optimizing the successful processes incorporated over the
previous 5 years in the Molecular Biochemistry Depart-            Development and validation of an automated pool-
ment at Glaxo Wellcome. This will require expansion of            ing process for a complian t preparation of blood
the developing approaches to research aided by successful         plasma pools destined for molecular testing
utilization of existing automation equipment. GlaxoS-
mithKline will compartmentalize these processes to ex-            Danuta Kierek-Jaszczuk, Cangene Corporation, Winnipeg,
ploit the bene® ts of speci® c strategies, further de® ning the   Manitoba, Canada
process of moving chemistry towards hit identi® cation.           Co-authors: Mike Labossiere, Beverly Belanger, Patti Hosler and
This discussion will focus on accommodating a larger              LeeAnne Macaulay
infrastructure that provides greater target numbers and           Blood-borne viruses such as HCV, for example, pose a
larger libraries, as well as complete follow-up screening         danger of being transmitted from aå ected donor(s) onto
using the automation resources available.
                                                                  the recipients of the blood/plasma or thereof derived
                                                                  pharmaceuticals. The use of nucleic acid testing/nucleo-
Distributing mammalian cell lines for HTS using                   tide-ampli® cation testing (NAT) was shown to shorten
the Hydra-96 Microdispense r                                      the ` window period’ , i.e. a period when donors are in a
                                                                  state of active but serologically undetectable infection.
Courtney T. Ward, MDS Pharma Services, Bothell, WA, USA           When applied to the plasma pools assembled from the
                                                                  donations destined for the manufacture of the plasma-
Reliable and consistent distribution of cells into micro-         based therapeutics, HCV-speci® c NAT would decrease
plates is a key aspect of the performance of cell-based           the risks of HCV transmission onto the recipients of such
screening assays. Typically, this has been done using a           therapeutics if virus-contaminate d donation(s) are iden-
hand-held multichannel pipettor in a biosafety cabinet, a         ti® ed and removed from the fractionation pool.
task that is both labor-intensive and slow. We have found
that this step can be accelerated signi® cantly using a 96-       Accordingly, an automated plasma-pooling process and
channel pipettor such as the Hydra-96 Microdispenser              pooling-driven back-tracing algorithm have been devel-
whose physical pro® le permits the use of the instrument          oped and validated at Cangene to support the produc-
in a standard tissue-culture hood. This device consists of        tion of WinRho and VariZig therapeutics bearing lesser
an array of 96 glass syringes (290 ml) with ® xed needles         risk for HCV transmission. The validated Microlab
where the plungers move in unison under microprocessor            ATplus 2 automated liquid handler has been used for
control. Using four mammalian cell lines, we compared             the preparation of the plasma mega (master) pools in a
the pipetting performance of the Hydra-96, a hand-held            three-stage process where ® nal mega pools were as-
pipettor, and a disposable tip-based 96-channel pipetting         sembled from the intermediary secondary pools and
workstation. The results indicate that the well-to-well           these, in turn, from the subpools. The automated bar-
dispense precision and cell viability are equivalent for all      code-reading function of the pooling instrument, in a
three devices.                                                    combination with a suitable barcode-labelling system of
                                                                  the individual plasma donation- and pool-holding con-
                                                                  tainers, allowed for the positive identi® cation of both the
Automated o² ine solid-phase extraction for polar                 individual donations and assembled pools. The pipetting
new chemical entities using the Zymark Rapid                      reports generated by the instrument facilitated a full
TraceTM systems                                                   traceability of all pipetting events whereas gravimetric
                                                                  in-process veri® cation assured pipetting accuracy.
Daksha Desai-Krieger, R. W. Johnson PRI, Springhouse, PA,
USA                                                               A pooling scheme-driven back-tracing algorithm has also
                                                                  been developed and validated for singling out the indi-
A generic automated o¥ine SPE method using the                    vidual contaminate d donation(s). This algorithm in-
Zymark Rapid TraceTM systems was developed for three              volved four-stage back-testing and was based on the
very polar new chemical entities (NCEs) undergoing
                                                                  assumption that all NAT-positive test results are true-
development at RWJPRI, namely a neuroaminidase
                                                                  positives. It allowed for identi® cation of viral contamina-
inhibitor, a cephalosporin and a thrombin inhibitor.              tion of a single donation by testing 26 samples, i.e. one
The polar characteristics of these compounds made it
                                                                  ® nal pool, ® ve secondary pools, 12 subpools and eight
impossible to carry out liquid± liquid extraction as a
                                                                   individual donations. The developed and validated pro-
sample clean-up step for these compounds.
                                                                   cesses allow for cGMP-compliant preparation of the
Our goal was to obtain simple automated SPE methods                plasma pools and regulatory required back-tracing of
as sample clean-up for these polar compounds from                  the viral contamination to individual plasma dona-
biological matrices and develop quantitative LC-MS/                tion(s).

                                                                                                                              67
Abstracts of papers presented at the 2001 ISLAR


Extending the reach of miniaturizatio n technology              revert the gene pro® le of the diseased state back towards
across the screening laboratory                                 ` normal’ .
David A. Dunn, Pharmacopeia, Inc.                               This presentation will describe some of the approache s
Co-authors: James R. Beasley, Paul McCoy, Barbara Strohl,       being taken by Psychiatric Genomics, Inc., to automate
Robert Swanson, Ti¡any Walker and Jingchun Yang                 each of the steps in the drug-discovery process using the
                                                                new paradigm of automated drug discoveryÐ ` Multi-
Assay miniaturization and the implementation of high-           Parameter High Throughput Screening’ (MPHTSTM).
density 1536-microwell screening is vital to our need to
increase the eæ ciency of primary screening and early
phase lead discovery. To serve this need, we have               Comparison of ELISA between automation and
developed a 1536 uHTS platform that employs microwell           manual testing for measuring ARANESPTM in
plates, novel ¯ uid handling and optical detection tech-        rat serum
nologies. Full use of this platform requires the ability to
develop assays eæ ciently for a variety of biological targets   De Chen, Amgen, Inc., Thousand Oaks, CA, USA
in a straightforward and expeditious manner. Having a           Co-authors: Yan Wang, Monica Zordich, Han Gunn and Sharon
portfolio of miniaturizable assay technologies that can be      Baughman
easily developed into ultrahigh-throughpu t screens for         This presentation compares the results of measuring
any member of a class of biological targets would               ARANESPTM in rat serum, obtained manually or via
enhance and extend the utility of this technology. We           a fully automated immunoassay system. ARANESPTM
have evaluated a variety of assay technologies that are         is a new erythropoietic protein currently used in clinical
suitable for uHTS of kinase and GPCR targets. This              trials for the treatment of anaemia related to chronic
presentation will discuss the relative merits of these          renal failure.
technologies in assembling a portfolio of uHTS assays
for screening these target classes.                             The Tecan Genesis workstation 200 was developed to
                                                                perform the ARANESPTM quantitative ELISA. It was
                                                                equipped with eight pipetting ceramic needles capable of
Prequali cation of pharmaceutica l leads                        pipetting 5± 1000 ml, a shaking incubator, a Columbus
                                                                microplate washer, microplate hotels and a microplate
David Casebier, ArQule, Inc., Woburn, MA, USA
                                                                reader. A robotic arm transported microplates and lids
Reliable predictive models and increased throughput of          between the peripherals on the workstation deck.
ADME screens currently enable the design, construction
                                                                Seven independent studies were performed on various
and pro® ling of chemical libraries, providing con® dence
                                                                days by the TECAN and manually. Standards, quality
for series selection in hit-to-lead discovery. Predictive
                                                                controls and known samples were loaded in triplicate on
models for metabolism, absorption, BBB as well as
                                                                the plates in each assay. The data generated showed no
diversity measurements assist in library design and
                                                                signi® cant diå erence. ANOVA analysis con® rmed this
reagent selection. Robotic syntheses then generate spa-
                                                                conclusion.
tially addressed, high-quality libraries, which in turn are
pro® led for ADME characteristics. Integration of these         Fully automated systems can be introduced to quantita-
technologies allows for the prioritization of lead series       tive immunoassay for protein measurement in serum.
using data indicative of developmental survivability.           Technical advances in methodologies, robotics and com-
                                                                puterization will lead to signi® cant enhancements in the
                                                                capacity of immunoassay, resulting in signi® cant cost and
Automation of identi cation and screening of                    eæ ciency gains.
genomic targets related to psychiatric disease
David M. Evans, Psychiatric Genomics, Inc., Gaithersburg,       LC-MS/MS assay for the quantitation of RWJ-
MD, USA
                                                                270201 using the Zymark Rapid Trace SPE work-
Psychiatric Genomics, Inc., is interested in discovering        station
the underlying genetic causes of psychiatric diseases and
                                                                Denise Preston, R. W. Johnson PRI, Spring House, PA, USA
identifying therapeutics for them. Many of these diseases
                                                                Co-authors: Daksha Desai-Krieger, Virginia Scott and R. John
are multigeneic in nature and have not previously been
                                                                Stubbs
well characterized owing to the diæ culty in obtaining the
appropriate tissue samples and the lack of technologies         A simple, automated SPE extraction and LC-MS/MS
that could detect the changes in gene expression pro® les       assay was developed for the extraction and quantitation
within the brain regions.                                       of two NCEs, RWJ-270201 and RWJ-270204, being
                                                                evaluated as viral neuroaminidase inhibitors intended
In recent years, several tools and technologies have been
                                                                for use in the treatment of in¯ uenza A and B. A solid-
developed that allow multiple parameters to be tested in
                                                                phase extraction method was developed for these analo-
a single experiment. Micro-array technology allows the
                                                                gues using Zymark Rapid Trace SPE workstations.
discrimination of changes in many genes at one time, and
by comparing ` normal’ versus diseased tissue, it is poss-      Extraction of plasma samples was performed using IST
ible to identify gene expression patterns quickly that are      Phenyl SPE cartridges (200 mg 3 ml¡1 ) conditioned with
altered due to the disease. Having identi® ed the disease       methanol and water. Sample clean-up was achieved by
gene pro® le, it is important to be able to develop screens     rinsing cartridges with methyl-t-butyl ether. The com-
to examine whether small chemical compounds can                 pounds of interest were eluted using ethanol:water (90:10

68
Abstracts of papers presented at the 2001 ISLAR


v/v) and subsequently evaporated under nitrogen to           New technique for a high-throughpu t solubility
dryness. The residue obtained was reconstituted before       assay
injection on an LC-MS/MS in the positive-ion APCI
mode.                                                        Dima Voloboy, pION, Inc., Woburn, MA, USA
                                                             Co-authors: Chau M. Du, M. Stra¡ord, K. Tsinman and A.
The developed assay demonstrate d good precision and         Avdeef
accuracy and was linear over a broad curve range. The
method developed for these analogues was routinely used      We have successfully adapted the classical shake-¯ ask
to support preclinical pharmacokinetic studies and was       method to the 96-well microtitre plate format to obtain
later adapted for use with 96-well plate technology in       near shake-¯ ask-quality results at high-throughpu t
support of clinical studies.                                 speeds. The new instrument uses a robotic ¯ uidics system
                                                             and a parallel detection system employing a microtitre
                                                             plate UV (190-500 nm) spectrophotometer . Samples are
                                                             introduced as DMSO stock solutions.
                                                             The essence of the approach is based on making UV
Fully automated protein precipitatio n LC/MS/MS              concentration reference solutions from moleculesÐ espe-
assay using a 96-well plate technology and track             cially ionizable moleculesÐ whose UV spectroscopic
robotic system                                               properties are unknown at the start of the assay. This
Scott Paul Depee, GlaxoSmithKline, Research Triangle Park,   can be achieved in either of two ways, with or without
NC, USA                                                      cosolvents.
Co-author: Brent T. Butler                                   A unique computational method was developed to ex-
The need arose in our department to develop a faster and     tract the aqueous intrinsic solubilities of drug molecules
safer yet user-friendly means of sample preparation          from data distorted by DMSO drug binding or drug±
before sample analysis. With shorter mass spectrometer       drug aggregation reactions.
method run times, the rate-limiting factor was the           An improved method for determining concentration by
sample-processing procedure. To expedite this pro-           UV spectrophotometr y was derived. It uses a novel peak-
cedure, automation through robotics and 96-well plate        shape algorithm for adjusting weights in a whole spec-
technology was implemented. Not only is this robotic         trum-weighted regression analysis, matching spectra of
protein precipitation assay process faster than manual       reference solutions (of known concentration, under con-
preparation, but also the precision and accuracy are         ditions avoiding or suppressing precipitation) to solutions
comparable. In addition, our staå has less exposure to       containing an analyte of unknown concentration (due to
potential pathogens in the samples, therefore increasing     precipitation).
safety. This robotic system is operated by very user-
friendly software that enables the processing of samples     A quality-assessment scheme has been developed around
either in a fully or semi-automated manner. The system       the standard drugs diclofenac, indomethacin, ¯ urbipro-
can manage 96 samples in < 1:5 h.                            fen, chlorpromazine, phenazopyridine, verapamil, pirox-
                                                             icam and griseofulvin.
                                                             The new solubility method, which allows determination
                                                             at one pH or as many as 96 pHs, has a limit of detection
                                                             of about 0.1 mg ml¡1 , as demonstrated by measurements
Automated o² ine solid-phase extraction for polar
                                                             of the intrinsic solubilities of terfenadine and tamoxifen.
new chemical entities using the Zymark Rapid
Trace systems                                                About 200 assays may be performed per day on the
                                                             instrument.
Daksha Desai-Krieger, R. W. Johnson PRI, Springhouse, PA,
USA
A generic automated o¥ine SPE method using the
Zymark Rapid Trace( systems was developed for three
very polar new chemical entities (NCEs) undergoing
                                                             Automated solution-phas e synthesis work ow at
development at RWJPRI, namely a neuroaminidase
                                                             Schering AG
inhibitor, a cephalosporin and a thrombin inhibitor.
The polar characteristics of these compounds made it         Christoph M. Huwe, Schering AG, Berlin, Germany
impossible to carry out liquid± liquid extraction as a
sample clean-up step for these compounds. Our goal           At Schering we have established a ¯ exible, integrated
was to obtain simple automated SPE methods as sample         solution-phase synthesis, puri® cation and reformatting
clean-up for these polar compounds from biological           work¯ ow based on Chemspeed- and Zymark-automate d
matrices and develop quantitativ e LC-MS/MS assays           synthesizers, parallel normal-phase chromatograph y as
for these compounds. Challenges encountered during           well as reversed-phase HPLC-MS puri® cation equip-
assay development and details of the method will be          ment, and a highly automated Zymark reformatting
presented. Transfer of these automated o¥ine assays to       robot. In this presentation, the special features of the
online automated SPE and/or o¥ine automated 96-well          chosen equipment, the ideas behind the system design
SPE will also be discussed.                                  and the respective work¯ ows will be discussed.

                                                                                                                     69
Abstracts of papers presented at the 2001 ISLAR


Intelligent automation of HPLC method develop-                    ‘Cradle to grave’ tracking of synthetic combina-
ment                                                              torial libraries

Douglas R. Myers, Intelligent Laboratory Solutions, Naperville,   Phil Small, Tripos Receptor Research, Bude, UK
IL, USA                                                           A process-integratin g design, synthesis and analysis of
Co-author: Je¡rey D. DeCicco                                      combinatorial libraries has been implemented at Tripos
                                                                  Receptor Research. Crucial to the success of this process
Developing new HPLC methods is a time-consuming and               has been the development of a proprietary informatics
knowledge-intensive task. We have developed an online             system. This has so far been developed to manage reagent
software package called ChromSmart that automates                 inventory, track samples and record data from synthesis
HPLC method development for both normal and re-                   and analysis to provide a valuable database of compound
versed-phase chromatograph y for chiral and non-chiral            information.
sample mixtures. Unlike other software packages that
assist with o¥ine HPLC modelling, ChromSmart uses                 The combination of Tripos’ proprietary design software
online arti® cial intelligence technology to capture and          with automated synthesis and informatics leads to the
implement the method-developmen t knowledge of expert             production of drug-like libraries with well-de® ned purity
chromatographers . ChromSmart contains a real-time                and full synthetic history.
intelligent experiment planner and rule-based engine              This presentation will cover what were considered the
that execute the HPLC methods, monitor results and                important aspects involved in setting up and managing
dynamically update the development plan. A complete               an automated chemistry facility.
audit trail of the decision-making and results is gener-
ated.
                                                                  Imaging technology in ultrahigh-throughpu t
ChromSmart comes with a core set of chromatograph y               screening: overcoming the bottleneck of plate
knowledge that automates method development. Based                reader throughput
on user preferences, such as retention times and resolu-
tion, ChromSmart develops a method-developmen t                   E. Michael August, Boehringer Ingelheim Pharmaceuticals, Inc.,
strategy. This strategy is executed and can dynamically           Ridge¢eld, CT, USA
change based on information from online, real-time                Co-authors: Lori Patnaude and Carol A. Homon
experimental results. Features include automated                  Screening throughput is limited by its slowest step, and
equilibration, detection and cleaning of retained                 thus a major goal of uHTS must be to identify and
components, gradient and isocratic optimization, peak             overcome these bottlenecks. With the advent of high-
purity analysis, and peak tracking. When ChromSmart               density liquid-handling devices capable of delivering
® nds a method that meets or exceeds the user’ s criteria, it     liquid to 384 or more wells at rates approaching 1
 noti® es the user and continues to develop another               plate min¡1 , the bottleneck has shifted from reagent
 method for the next sample. ChromSmart operates                  delivery to plate reading. Conventional plate readers
 24 £ 7 £ 365 signi® cantly increasing productivity of            require several minutes to obtain data from an entire
 equipment and personnel.                                         plate.
                                                                  Imaging technology allows the detection of an entire
                                                                  plate simultaneously. Coupled with rapid, sensitive im-
                                                                  age-analysis software, such a system enables data capture
                                                                  to keep pace with reagent delivery. We present here
                                                                  screening results from a recent enzymatic Del® a uHTS
Laboratory automation using Zymark robots and                     campaign comparing the Wallac Viewlux Imaging
UV breoptics from Delphian Technology                             system with the LJL Analyst. Several hundred plates in
                                                                  both 96- and 384-well formats were read on both readers
Je¡rey B. Medwid, Wyeth-Ayerst Research, Pearl River, NY,
                                                                  and the data were analysed with a variety of statistical
USA
                                                                  parameters.
Co-authors: Jerry Edgar, Syed Rahman and Scott Keenan
                                                                  Similar comparisons of other assay formats will be
In the fast-paced pharmaceutical business, quicker turn-          discussed. The inherent ¯ exibility of imaging systems is
around times and larger sample loads face every analy-            highly compatible with assay miniaturization and the
tical laboratory manager. Unfortunately, this must be             trend to higher-density plate formats. However, we must
accomplished with no signi® cant increase in analyst              also realize that the bottleneck in the overall screening
resources. Laboratory automation is the only answer.              process has not been removed, but surely shifted to
Our research automation laboratory contains several               another step.
Zymark robots including the Multidose Dissolution
Workstation s and Tablet Processing Workstations . The
latest addition to our laboratory is a UV Fiber Optic             Finally the paperless and compliant laboratory
Dissolution Workstation manufactured by Delphian
                                                                  Ed Halpin, VelQuest Corporation, Hopkinton, MA, USA
Technology. This presentation will highlight some of
our recent work accomplished using these workstations             Compliance and cost have long been at odds in the
and some of our other initiatives in automation.                  modern regulated laboratory. Focusing on one meant
                                                                  sacri® cing the other. Regulatory compliance today is a

70
Abstracts of papers presented at the 2001 ISLAR


labour-intensive, paper-based operation. The ` paperless’      followed by adverse reactions and toxicity that add up
laboratory has been a goal of the pharmaceutical in-           to 21% of the causes for withdrawal from development.
dustry for over the past decade. The con¯ uence of
technology and innovation has reached a point where            In most of the cases the lack of a complete picture of the
the paperless laboratory is now economically and tech-         drug’ s properties is not available at the time the decision
nologically practical. Good manufacturin g practices and       to select a respective candidate is taken. In part this is
21CFR Part 11 de® ne the requirements to attain a              due to the current process of drug discovery where
paperless, compliant laboratory.                               information is accumulated stepwise rather than in a
                                                               de® ned parallel manner. The choice of the candidate
The solution would enable direct electronic data capture       taken forward is often dominated by emphasizing the
from human observations , instruments and networked            aspect of potency rather than addressing early on selec-
PCs replacing paper notebooks, ® les and control sheets.       tivity and drug property issues. On the other hand, the
The solution would enhance the collection of data and          companies have accumulated vast amounts of in-vitro and
metadata by dynamically linking it to the procedure by         in-vivo data that may be used to re® ne the decision matrix
which it was collected. Reviews, audits, investigations        either by retro- or prospective interpretation of existing
and approvals would be electronic processes, replacing         results. As a consequence and in order to take the
the current paper systems. Redundant checking would be         serendipity out of such fundamental decisions, many
replaced by ` review by exception’ and resources reallo-       pharmaceutical companies have started broad range
cated towards the real goal of bringing new human              pro® ling (i.e. testing the drugs in parallel against a panel
therapies to the market more rapidly. The solution must        of diå erent in-vitro model, both in pharmacology and
also complement and exchange data with other labora-           ADME/PK) in the preclinical phase and have put
tory applications such as Laboratory Information Man-          emphasis on the development and automation of second-
agement Systems (LIMS) and Chromatograph y Data                ary tests. High throughput pro® ling using robotics and
Systems (CDS).                                                 automated workstations allows to accumulate today a
                                                               broader knowledge on the hits identi® ed in the primary
                                                               screening. Both pharmacologicalÐ speci® city and selec-
                                                               tivity versus other targets, such as enzymes and recep-
Generating predictive models from high-through-                torsÐ and pharmaceutical propertiesÐ physicochemical
put pro ling                                                   parameters or metabolic and in-vitro safety aspectsÐ of
Dragos Horvath, 128 rue Danton, Rueil Malmaison, France        the candidates are evaluated in depth.8
Co-author: Michael Entzeroth
                                                               Through the progress in linking of high-throughpu t
Current drug development is characterized by a strong          pharmacological, physicochemical and ` in-silico’ methods
belief in high-throughpu t methods, both in combinatorial      with in-vitro approaches, progress is being made in
chemistry and screening. The trigger for constantly            predicting drug properties.8 The goal to predict in-vitro
increased performance is the need the industry to adjust       and, ® nally, in-vivo properties of drugs has become
to the race against time in order to secure shares on the      reasonable by linking the chemical structure via mol-
market place. Over many years the industry has tried to        ecular descriptors, area descriptors or ComPharm ® elds
keep pace by the constant introduction of new tech-            to certain in-vitro pro® les that again can be correlated to
nologies. The associated costs for drug development            the corresponding in-vivo eå ects (® gure 1).
increased over the last years. Aggregated R&D expendi-
tures for new chemical entities (NCE) peaked in 1996           An example for pharmacophor e ® ngerprints are the
close to US$500m.1 Recently, Lehman Brothers esti-             Fuzzy Bipolar Pharmacophore Autocorrelograms
mated the total costs for a launched product to account        (FBPA9 ) monitoring the numbers of atom pairs within
up to US$635m,2 while for drugs entering clinical trials       each of the 252 ˆ 21 £ 12 categories that can be de® ned
between 1972 and 1982 these costs were estimated to add        in terms of the 21 combinations of six pharmacophoric
up to US$312m.3 Research-based companies will invest           features a,b2{Hydrophobicity , Aromaticity, Hydrogen
$26.4 billion in R&D in 2000, a 10.1 percent increase          Bond Acceptor & Donor, Cation, Anion} times 12
over 1999. 4 The number New Chemical Entities ap-              considered distance ranges ¢2{(3..4), (4..5), . . . ,
                                                                             ®
                                                               (14..15)} [A]. For similarity searches and structural
proved by the FDA has not changed signi® cantly over
the last years (1997: 39, 1998: 30, 1999: 35), indicating      comparisons, these ® ngerprints are extremely powerful
that the success rate is not related to the initial eå orts.   in combination with ComPharm ® eld descriptors10 that
                                                               are taken as the intensities of the empirically de® ned
The portion of IND applications that fail has been             ` pharmacophor e ® elds’ generated by the atoms of the
estimated to approximatel y 20% in phase II, 60% in             considered compound at the space points occupied by a
phase II and most importantly 87% in phase I.5 83% of           reference structure, in a con® guration corresponding to
the drugs for which INDs were ® led between 1964 and            its optimal alignment with respect to this reference.
1989 were dropped before reaching NDA status.6;7 For
the pharmaceutical companies each dropout during the           The data mining approach described in this report is part
development is associated with huge costs that reduce the      of the BioPrintTM project11 that makes use of a broad
pro® t derived from NCEs that reached the market. The          spectrum pro® ling of marketed drugs and candidates in
failure of the candidates can be primarily attributed to       more than 90 diå erent in-vitro models. The marketed
eæ cacy, safety and economic concerns. The predominant         drugs, included in the test collection, comprise more than
reason for failure, however, are inappropriate pharma-         1,500 compounds that are available in the US pharma-
cokinetic properties of the drug candidate (39.4%),            cies today. Important are also the drug candidates

                                                                                                                         71
Abstracts of papers presented at the 2001 ISLAR



                                    Chemical                           Molecular                     In Vitro
                                    Structure                          Descriptors                    Profile               In Vivo Effects

                            F
                                                 HC
                                                 3        N                                                                 ADME/PK
                                                              S

                                                          N                                                                 Adverse effects
                                        N
                                                      O
                                                                                                                            Therapeutic effects
                                F
                                                                                                   In Vitro tests
                                                                        Pharmacophore          Receptors/Enzymes
                                                                         Fingerprints             Cellular Tests
                                                                       Area Descriptors         In Vitro ADME- T
                                                                                                         ADME-
                                                                       ComPharm Fields              properties

Figure 1. Predictive modelling of the properties of compounds correlates chemical structures via molecular descriptors with ADME/ëK and
therapeutic e¡ects.

included which have failed to make it all way through the                                          pounds. By contrast to logP models that mostly rely on
diå erent phases of preclinical and clinical drug develop-                                         incremental group contributions and/or molecular de-
ment. The models and predictive tools generated open a                                             scriptors of the average polarity of the solvent-accessible
new approach for candidate selection and early discovery                                           surface (an aspect which is in our model successfully
activities, such as target identi® cation or library design                                        captured in terms of PTA descriptors), a logD model
and will result in the long term in substantial cost savings                                       needs to account for proteolytic eå ects in the aqueous
due to reduced failure rates and time spent on the                                                 buå er of pH ˆ 7.4 used for experimental determinations.
research and development process. In-silico, linear models                                         In our models, proteolytic ionization of acids/bases is
to describe solubility and permeability as well as predic-                                         implicitly accounted for by the pharmacophor e descrip-
tions in pharmacology based on predictive neighborhood                                             tors relying on rule-based assignments of a positive/
behavior are presented.                                                                            negative charge status to ionizable groups. Furthermore,
                                                                                                   bipolar pharmacophor e elements or ® eld overlap terms
                                                                                                   successfully account for potential pKa shifts due to
Linear ADME models                                                                                 neighboring groups of the ionizable center.
The interest in an in-silico logD prediction model resides
both in its applicability in library design and its further
                                                                                                   Linear apparent permeabilit y models
use as a calculated molecular descriptor in other struc-
ture-activity relationships, if the activity under study is                                        The main challenge in predicting the apparent perme-
expected to relate to the overall lipophilicity of com-                                            ability of compounds through a Caco-2 cell monolayer is

                                                                                 6


                                                                                 5


                                                                                 4


                                                                                 3


                                                                                 2
              Pred. LogD




                                                                                 1


                                                                                 0
                           -4               -3                    -2     -1           0        1          2         3        4        5           6

                                                                                 -1


                                                                                 -2


                                                                                 -3


                                                                                 -4
                                                                                          E xpt. L ogD

Figure 2. Predicted versus experimental log D. The properties of compounds from a diverse library of combinatorial compounds (squares)
and medicinal chemistry (circles) were calculated from a model based on a training set of 320 entities (rhomboids).

72
Abstracts of papers presented at the 2001 ISLAR


the in-silico recognition of molecules which are trans-             number of descriptors that would be required to enter
ported by one of the active in- or e¥ux mechanisms.                 the model is much larger than the number of examples of
This will enable the introduction of speci® c correction            e¥uxed compounds currently included in the data setÐ
factors explaining the diå erence between the actual                note that multiple e¥ux/transportation mechanisms with
membrane crossing rate and the rate expected on the                 potentially diå erent pharmacophor e characteristics co-
basis of purely passive transmembrane diå usion. While              exist in Caco-2 cells, and that enough compounds
the latter aspect of predicting the passive diå usion rates         exemplifying each one of them would be required to
has been tackled in various publications, little progress           solve the problem.
has been made to understand the problem of transported/
                                                                    The introduction of ComPharm descriptors overriding
e¥uxed compounds, routinely discarded as ` expectable’
                                                                    the limitations of bipolar pharmacophor e terms is indeed
outliers in previous work. By contrast, the herein de-
                                                                    seen (® gure 3) to signi® cantly improve the permeability
scribed modeling eå ort focused on pharmacophor e de-
                                                                    prediction of the outliers from the previous model. These
scriptors in order to search for potential pharmacophor e
                                                                    ® eld descriptors were obtained on the basis of super-
motifs that may characterize e¥ux and/or transporta-
                                                                     imposition models of BioPrintTM compounds against
tion, while descriptors of the molecular surface properties
                                                                     some of the most heavily e¥uxed/transported substances
and the generic calculated logD descriptors were sup-
                                                                     encountered in this set.
posed to account for passive membrane crossing proper-
ties. The calculated logD (or, even better, the
experimental logD values) are relevant variables of the
linear log(P A± B) apical-to-basa l apparent permeability           Solubility model
model. A positive coeæ cient for the linear contribution,           A categorical solubility model based on a linear equation
together with a negative term in logD 2 characterize an             has been developed (® gure 4) in order to predict the
optimal logD range maximizing the passive membrane                  solubility class of the compounds: (1) Low: S < 10 mM;
crossing rate. This makes physical sense, since too hydro-          (2) Medium: 10 mM < S < 100 mM; (3) High:
philic compounds never leave the aqueous phase, while               S > 100 mM. This model (not shown here) included 12
too hydrophobic ones tend to accumulate in the mem-                 explaining variables, such as logD and logD2 and various
branes.                                                             Pharmacophore Type Areas (PTA) and bipolar pharma-
In spite of the important number of bipolar pharmaco-               cophore elements that predicts drug solubility from its
phore elements entering the model, a series of heavily              structural features. With encouraging results it has also
e¥uxed compounds are nevertheless mispredicted by the               been compared with other solubility prediction software
model and appear as ` false permeable’ . Most of the                packages and shown its advantages with 86% of the
outliers in the marked area are shown (® gure 3, circled            drugs predicted in the correct solubility class.
area) to be indeed e¥uxed molecules, since their meas-
ured basal-to-apica l apparent permeabilities were sig-
ni® cantly higher that the corresponding apical-to-basa l           Predictive neighborhood behaviour (PNB) models
values.
                                                                    By contrast with linear approache s that require a speci® c
It can be therefore concluded that either (a) e¥ux                  equation for each of the properties that are to be
cannot be understood in terms of bipolar pharmacophor e             predicted in terms of molecular descriptors, PNB models
descriptors only or (b) e¥ux might in principle be                  rely on the measured properties of related compounds
characterized in terms of such descriptors, but the                 (e.g. nearest neighbors in descriptors space) from the


                                                      2.5
                                                         2
                                                      1.5
           Pred. LogPa-b




                                                         1
                                                      0.5
                                                         0
                           -2            -1           -0.5 0                       1                     2
                                                        -1
                                                      -1.5
                                                        -2
                                                        Expt. LogPa-b
Figure 3. Improvement of the permeability prediction of e¥uxed compounds due to the use of ComPharm ¢eld descriptors in the model.
Training set (rhombs); test set (squares).

                                                                                                                               73
Abstracts of papers presented at the 2001 ISLAR



                  Expt. Class ->                      H              M               L               Tot.Pred
                  Pred. Class:H                      265             19               2                286
                  Pred. Class:M                      15              15              12                 42
                  Pred. Class:L                       0               0              12                 12
                  Tot.ExptClass                      280             34              26                340

            Predicted in correct class (green)=(265+15+12)/340=86%
            Predicted in neighboring class (yellow)=(15+19+12+0)/340=13.5%
            Predicted in WRONG class(red)=2/340=0.5%
Figure 4. Comparison of the predicted solubility class (vertical) of compounds versus the experimental class (horizontal) using the
BioPrintTM solubility model.




Figure 5. Receptor pro¢le of SCH23390, 10 ·m. (upper) Predicted pro¢le (each bar represents the percentage inhibition in a
pharmacological model ‡ RMS prediction error) based on the activity of its nearest neighbours; (lower) experimental data in the same
assay panel.


BioPrint set in order to provide an estimation of the                cluded in the original data set, however, tested later for
property value of a new molecule. The PNB algorithm                  comparison.
works in principle like a ` fuzzy data base querying tool’ in
the sense that if molecule X is not included in the
BioPrint database, the BioPrint compounds that are most
similar to X are retrieved and used for the evaluation of            Conclusion
the properties of X. The property of the unknown
molecule is computed as a weighted average of the                    During the last decade technologies such as ultrahigh-
experimentally determined properties PN(x) of its near-              throughput screening and combinatorial chemistry have
est neighbors N(X) from the BioPrint set, where the                  signi® cantly contributed to the advances in drug dis-
weighing factors are decreasing functions of the mol-                covery. The increase in R&D costs associated and the
ecular dissimilarity scores that de® ne how ` near’ a                bottlenecks further down the development as well as the
neighbor N(X) is with respect to X. These dissimilarity              high dropout rates have increased the demand for an
scores are Dice scores calculated on the basis of FBPA,              alternative approach. BioPrintTM combines molecular
PTA and EFO descriptors, the intervening empirical                   descriptors, high-throughpu t pro® ling with in-vivo drug
parameters being speci® cally ® ne-tuned in order to maxi-           pro® les. It takes advantage of data mining technologies
mize the predictive power with respect to a particular               to generate predictive models that link the three com-
property. Figure 5 shows the example in the case of                  ponents. These models help to build a eå ective strategy
SCH23390, a D1 receptor ligand, which was not in-                    that will foster drug discovery in the new millenium.

74
Abstracts of papers presented at the 2001 ISLAR


References                                                                    a very sensitive and ultrahigh-throughpu t assay format.
                                                                              The collaboration between Sugen and Evotec OAI is
 1. Kettler, H., 1999, J. Comm. Biotech., 6, 60.                              focused on phosphatase s and has demonstrated successful
 2. Kettler, H., 1999, Updating the Costs of a New Chemical Entity            adaptation of 2D-FIDA anisotropy detection for phos-
    (London: OHE).
 3. DiMasi, J. D., Hansen, R., Grabowski H. G. and Lasagna, L.,               phatase assays at the 1 ml/well level. A discussion of this
    1991, J. Health Econ., 10, 107.                                           assay format and the uHTS screening results on the
 4. PhRMA., Pharmaceutical Industry Pro¢le 2000, 20.                          automated EVOscreen Mark II system for several phos-
 5. Kennedy, T, 1997, Drug Disc. Today, 2, 436± 444.                          phatases will be covered.
 6. DiMasi, J. A., 1995, Clin. Pharmacol. Ther., 58, 1.
 7. Prentis, R. A. and Walker, S. R., 1986, Br. J. Clin. Pharmacol.,
    21, 437.
 8. Smith, D. A. and van de Waterbeemd, H., 1999, Curr. Opin. Chem.           Considerations in dissolution automation
    Biol., 3, 373.
 9. Horvath, D., 2001, in A. K. Ghose and V. N. Viswanadhan
                                                                              G. Bryan Crist, VanKen Technology Group, Cary, NC, USA
    (eds), Combinatorial Library Design and EvaluationöPrinciples, Software   Among the future challenges for the pharmaceutical
    Tools and Applications in Drug Discovery (New York: Marcel
    Dekker), 429.
                                                                              laboratory exists the necessity to automate dissolution
10. Horvath, D., 2001, in M. Diudea (ed.), QSPR/QSAR Studies by               methods to allow maximum throughput with optimum
    Molecular Descriptors (New York: Nova), 395.                              compliance. This presentation will provide suggestions
11. Entzeroth, M., Chapelain, B., Guilbert, J. and Hamon, V.,                 for determining the level of automation needed to obtain
    2000, JALA, 5, 69.                                                        peak performance as well as providing insight into
                                                                              regulatory compliance issues. Associated topics to be
                                                                              discussed include: novel dosage forms today and provid-
Powder-dispensin g capabilitie s to the Zymark
                                                                              ing analytical challenges in the future, alternate methods
Benchmate system
                                                                              of analysis, the dissolution apparatus of the future and the
Erin C. Heritage, R. W. Johnson Pharmaceutical Research                       involvement of new analytical approaches. Instrumenta-
Institute, Raritan, NJ, USA                                                   tion ¯ exibility will be required in the dissolution labora-
                                                                              tory of the future to utilize fully pooled sampling
One of the most fundamental routines for a chemist is
                                                                              techniques, online UV, online HPLC, capillary electro-
the preparation of samples from dry powdered samples.
                                                                              phoresis and even atomic absorption for nutritional
There have been many expensive attempts to automate
                                                                              analysis.
this process. We demonstrat e powder dispensing using
an existing robot workstation and some common                                 Semi-automatio n to total automation concepts including
disposables.                                                                  in situ methods of analysis will also be discussed. As novel
                                                                              concepts evolve each day, engineers working with scien-
Powder dispensing was accomplished by building a
                                                                              tists will lead to more eæ cient, better-integrated dissolu-
dispensing station on an integrated weighing robotÐ
                                                                              tion instrumentation in the future.
the Zymark Benchmate. The operating principle of the
station is based on applying a vacuum to an airline
connected to a pipette tip. The system is designed to                         Update from PQRI’s Blend Uniformity Working
transfer dry powder from four-dram vials to 16 £ 100 test                     Group: balancing workload with batch homogene-
tubes. The user can specify target weight and replications                    ity assurance
for each compound through a custom-designed Visual
Basic program.                                                                Garth Boehm, Purepac Pharmaceutical Co., Elizabeth, NJ, USA
The sample vial is basically carried to the ® xed location                    The Product Quality Research Institute (PQRI) is a
of the pipette tip, placing the tip into the powder. The                      consortium between industry, academia, and FDA that
vacuum is applied to the airline and the vial removed.                        aims to provide a scienti® c basis for the development of
The vacuum holds a small amount of sample powder on                           regulatory policy. One of PQRI’ s ® rst initiatives was to
the pipette tip. A test tube is then moved into position                      form a Blend Uniformity Working Group (BUWG)
below the pipette tip, the vacuum is released and the                         charged with providing a scienti® c basis for continued
powder is dispensed into the 16 £ 100 test tube. When                         development of FDA policy on BU testing. The BUWG
the target weight is reached, sample preparation (e.g.                        sought wider industry input through a survey of current
dilution to concentration and mixing) can then be                             BU testing practices and a public workshop. Using this
accomplished on the same workstation.                                         input together with the experience of the BUWG mem-
                                                                              bers and colleagues, a draft proposal was developed and
                                                                              re® ned. It provides a guide for appropriate testing during
uHTS of phosphatases using 2D-FIDA anisotropy                                 scale-up and validation, and for subsequent routine
detection                                                                     manufacture. The proposal is based around initially
                                                                              establishing the relationship between BU testing and
Flora Tang, Sugen, Inc., South San Francisco, CA, USA
                                                                              strati® ed testing of the resulting dosage units. The type
Various parameters of ¯ uorescence measurement, such as                       and amount of testing recommended during routine
translational diå usion, brightness and rotation, have                        manufacture depends on the outcome and robustness
been widely recognized as readout fully amenable to                           determined from the validation testing. The BUWG is
homogenous, miniaturized uHTS. A novel detection                              currently seeking data from industry to challenge the
system using Evotec’ s FCSplus reader, which combines                         proposal with real data to establish the validity of the
the ¯ uorescence-intensity distribution analysis (FIDA)                       suggested approach. The next step will be to seek public
anisotropy detection with confocal microscopy, provides                       comment on the BUWG proposal.

                                                                                                                                       75
Abstracts of papers presented at the 2001 ISLAR


Potential impacts of implementing automated                   chemistries and separation steps. While some of the very
dissolution                                                   large complex robotics systems could automate the
                                                              complete sequence of operations comprising an assay, a
Gerard Schneider, LEC Consulting, Blairstown, NJ, USA         higher level of productivity is achieved by paying careful
The Sotax AT-70 Smart oå ers the ability to automate          attention to the work¯ ow. Determining the optimum
fully both USP Apparatus 1 and 2 dissolution methods.         assay conditions, standardizing some of the operations
This system, used in conjunction with other eæ ciency-        and selectively introducing automation bring in the best
driven technologies such as premanufactured medium            results and bene® ts.
and online spectrophotometri c analysis of samples, can
profoundly reduce the resources required for conven-
tional dissolution testing while enhancing the consistency    Improving the quality and speed of automated
of results. Most importantly, these eæ ciency gains can be    sample transfers
achieved within the parameters of a compliance-con-           Glenn Smith, GlaxoSmithKline, Research Triangle Park, NC,
scious industry.                                              USA
The eå ects of implementing such technology will be           Co-authors: Jimmy Bruner, Charles Buckner and Bob Biddle-
discussed. The discussion will address the eå ect that        combe
eæ cient, automated dissolution testing has on the prod-      The quantitative transfer of biological ¯ uids is the
uct development area, where companies are focussed on         most critical aspect of automated sample preparation.
delivering products to market in increasingly less time, as   The sampling of plasma, serum, blood or urine represents
well as marketed product stability and release labora-        the most diæ cult and time-consuming step in most
tories where QC managers are asked to reduce sample           bioanalytical extraction procedures. We will present
turnaround times without increasing resources. Examples       two practical solutions that address these separate
of eæ ciency gains will be given in both cases.               problems:
                                                                . automating ` diæ cult’ sample transfers with higher
Assay automation: let humans do what they are                     quality;
good at and take robotics to the limit                          . signi® cantly increasing the processing speed of most
                                                                  ` simple’ sample transfers.
Gladys Range, Human Genome Sciences, Rockville, MD, USA
In the automation of any process, there are basic steps       First, most laboratorie s automate sampling on a mul-
that when carefully suited and applied to a particular        tiple-tip liquid-handling workstation such as a Packard
laboratory assay or work¯ ow, serve as a tool for eå ective   MultiPROBE 1 or Tecan GenesisTM. Today, samples are
automation. Biological validation, time and high-             most commonly transferred from various size tubes to a
throughput screening assays are, by nature, very diå er-      deep-well plate or a 96-well solid-phase extraction (SPE)
ent, and are designed with diå erent criteria in mind. A      block. Despite their widespread use, these systems cannot
biological assay is a complex system designed to yield        perform sampling with suæ cient precision and accuracy
biological activity using qualitative measurements. Issues    for certain applications (e.g. samples containing clots or
of cost, amount and quality of reagents, although import-     other insoluble matter; samples with low or insuæ cient
ant, do not play a paramount role in development. The         volumes).
biological assay is usually a manual assay that requires      We will show how the integration of an analytical
signi® cant change before it is automation ready.             balance on an x-y-z liquid-handling deck can easily
                                                              improve the overall accuracy of assay data. The applica-
High-throughpu t assays in contrast are speci® c, fast,
                                                              tion also allows the workstation to process samples that
reproducible, low standard deviations, low cost and
                                                              would otherwise need to be transferred manually.
usually can be automated as much as mechanically and
                                                              Samples are aspirated in parallel then dispensed one at
humanly possible.
                                                              a time, allowing the balance to capture the exact weight
Key elements in assay automation are sample sourcing,         (ˆ> volume) of each sample that was added to a deep-
screening design, robotics’ hardware and data manage-         well or SPE plate. The data are used to correct auto-
ment. These elements need individual careful analysis, a      matically for any diå erences in sample weights, usually
master plan and follow-up. A challenge in automation is       by pasting the results directly in the ` dilution factor’
to enable individual laboratory personnel to contribute       column of an analytical system’ s worklist.
to the ® ne ongoing activity of building a database by
                                                              Second, for most other applications in which reliable
doing their work without stopping to write in a labora-
                                                              transfers are easily achieved without gravimetric con-
tory book or even to enter information in a computer.
                                                              ® rmation, the speed of sample processing remains a rate-
These in return will prevent redundancy and will im-
                                                               limiting step. Most systems require about 20± 45 min to
prove accuracy and completeness.
                                                               transfer 96 samples. This time is signi® cant and limiting
Second-generatio n laboratory robotics and liquid hand-        compared with the 1± 2 min required for other steps such
lers are now designed to handle very high-throughputs ,        as the addition of a reagent using a 96-channel pipetting
very dense formats and very small-volume applications.         station (e.g. Zymark RapidPlateTM-96, Tomtec Quadra
Likewise, the recent development of sophisticated ¯ uor-
                                                               96TM, Apricot PP550TM).
escence systems operating at longer wavelengths, using
time-resolve signals, and ® breoptics’ technology reduce      We will detail how custom sample racks can be used to
read time and facilitate moving away from less desired        speed up tube-to-plate sample transfers dramatically.

76
Abstracts of papers presented at the 2001 ISLAR


These novel racks have a standard microplate footprint          cell-speci® c actions on key endocrine mechanisms. Iden-
and can be used on any 96-channel pipetting station. The        ti® cation of genes induced or inhibited by TNF and other
racks, which consist of complimentary pairs, each hold 48       cytokines may lead to the discovery of novel targets for
sample tubes arranged in an alternating (staggered)             drug development.
sequence. With these racks, 48 samples are transferred
simultaneousl y and 96 are transferred in as little as 2 min.
The racks work with most brands of 13-mm outer                  CrystalScreen : a novel microplate for automated
diameter cryotubes and 11-mm outer diameter micro-              protein crystallizatio n
centrifuge tubes commonly used in bioanalytical labora-
                                                                Gunther Knebel, Greiner Bio-One GmbH, Frickenhausen,
tories.
                                                                Germany
With these two inexpensive workstation modi® cations we         Co-authors: Lajos Nyarsik1, Patrick Umbach, Martin Horn,
                                                                                    è
can now automate 100% of our applications and sig-              Thomas Przewieslik, Wolfram Saenger, Hans Lehrach, Peter
ni® cantly speed up the majority of these assays.               Opfermann and Holger Eickho¡
                                                                The three-dimensional structure of any protein plays a
Identi cation of cytokine-regulate d genes associ-              key role in the process of understanding the exact
ated with infertility and obesity by DNA gene-chip              function of these macromolecules. One proven approach
micro-arrays                                                    to structural information of proteins is based on X-ray
                                                                diå raction of single crystals. In the past, automated
Grace Wong, Serono Reproductive Biology Institute, Randolph,    crystallization was restricted because a reliable hardware
MA, USA                                                         platform and well-suited microplates for high-throughpu t
Co-authors: H. Yarovoi, Q. Chen, S. Nataraja, Q. Xu, C. Liu,    screening were missing.
W. Mesadie J. Lai, J. Straaubhaar, J. Strickler, M. Tepper,
M. Dreano, G. Garotta, S. Fumero, T. Wells, A. Eshkol and S.    The Max-Planck-Institut e for Molecular Genetics
Arkinstall                                                      (MPIMG), the Protein Structure Factory (PSF) and
                                                                Greiner Bio-One have collaborated to develop a unique
Although the sequence of the human genome has been              96-well protein crystallization microplate (Crystal-
determined, the regulation and function of many genes           ScreenTM) with a standardized microplate footprint for
remain largely unknown. Cytokines are tightly regulated         high-throughput applications. Each of the 96 mother
soluble proteins transiently produced by cells in response      liquor wells corresponds to three crystallization wells.
to immunological stimulation or disease manifestation.          This allows checkerboard screening with up to 288
The expression of these proteins has been linked to many        crystallization options per plate to investigate optimal
diseases including cancer, AIDS, obesity, autoimmunity,         crystal growth followed by 3D-structure analysis. In
immunode® ciency and infertility, suggesting that cyto-         combination with a preformed lid, this microplate en-
kines, their receptors, as well as cytokine-induced genes       ables high-throughpu t multichannel screening of proteins
are potential targets for new drug development. Micro-          in automated systems.
arrays such as gene chips and protein arrays are high-
throughput technologies for drug discovery in the post-         The system in use at MPIMG and PSF allows simul-
genomic era.                                                    taneous sitting-drop and hanging-dro p vapour diå usion
                                                                crystallization experiments at reduced costs. A huge
We employed DNA transcriptiona l pro® ling micro-ar-            storage system for 10 000 crystallization microplates and
rays to detect genes under control of cytokines in selected     a pipetting device based on solenoid inkjet technology
experimental models. Hence, in ovarian granulosa cells,         enables one to set up a complete plate with 96 crystal-
TNF stimulates DNA synthesis but inhibits FSH-induced           lization conditions in < 3 min. All crystallization wells
oestrogen production. These results suggest that identi® -      are inspected in regular intervals with an automated
cation of TNF inducible ` master control genes’ may
                                                                camera-based detection system to inspect the crystal
reveal new targets for infertility and possibly other           growth.
diseases associated with oestrogen. TNF induces more
than 70 genes in rat ovaries in vivo, including genes for
cytokines, kinases, receptors, transcription factors and        Seamless integration of information in a pharma-
enzymes. In contrast to its inhibitory eå ect on oestrogen      ceutical development environment: integrating
production in the ovary, TNF stimulates production of           technology from LabWare, NuGenesis, VelQuest
this sex steroid in human pre-adipocytes and adipocytes         and Waters
derived from more than 10 patients. Interestingly, hu-
man pre-adipocytes/adipocytes themselves produce TNF            Guy Talbot, Purdue Research Center, Ardsley, NY, USA
as well as other cytokines and our data suggest a positive
                                                                This presentation will describe Purdue Pharma’ s ap-
autocrine/paracrine mechanism for control of oestrogen
                                                                proach to an integrated solution between Laboratory
production by fat cells. In addition to TNF, 16 cytokines
                                                                Information Management Systems (LIMS), Chromato-
including LT-a, IL-1, IL-6, IL-11, LIF and oncostatin
                                                                graphy Data Systems (CDS), Process Management and
M all stimulate oestrogen production in human fat cells.
                                                                Compliance Systems (PMC) and Scienti® c Data Man-
Other cytokines such as TGF-­ 1, TGF-­ 2, VEGF,                 agement Systems (SDMS). It will describe the vendor-
Rantes, IL-4, IL-10, IL-12, IL-18, CD40 ligand and              selection process and how Purdue Pharma created an
TNF-related proteins such as LT-b, TWEAK, RANK                  environment to produce a best-of-breed information sol-
Ligand, APRIL and BAFF have no inducing eå ect.                 ution to creating the paperless laboratory. Working
These results indicate that TNF has diå erential and            towards the creation of a paperless laboratory has the

                                                                                                                       77
Abstracts of papers presented at the 2001 ISLAR


potential to result in signi® cant bene® ts including re-     now possible to fabricate high-density arrays of speci® ed
source liberation and an electronic compliance platform       DNA sequences that include every known gene of an
for process management and data review.                       organism on a single glass slide. Labelled RNA or DNA
                                                              targets (such as mRNAs obtained from cells, tissues or
This paper will describe the process of working with four
                                                              organisms under diå erent conditions) can be analysed by
vendors to create a complementary data-managemen t
                                                              hybridization on the array. Diå erences in the levels of
solution to Purdue Pharma’ s needs. The process of
                                                              expression for thousands of genes can thus be assessed all
logging in samples, creating worklists, executing analy-
                                                              at the same time in a single, simple experiment. Geno-
tical methods, capturing data electronically, storing
                                                              mics, informatics and automation are playing increas-
electronic records, and submitting reports will be re-
                                                              ingly important roles as discovery tools in the basic
viewed. After data creation, electronic data review will
                                                              biological sciences, and as diagnostic and rational ther-
be discussed enabling electronic ` instant replay’ of la-
                                                              apeutic aids in the clinical arena. We discuss the use of
boratory information.
                                                              automation to increase productivity in micro-array fab-
The primary bene® ts of this complementary solution           rication and describe how automated procedures increase
approach hold the following possibilities.                    the quality of results in micro-array experimentation.
     . Increased capacity by liberating valuable resources
       from rework loops and the manual data review pro-
       cess.                                                  Use of the Bio-Tek Precision 2000TM automated
     . Leveraging the approach to validation of the overall   pipetting system for micro-arra y sample prepara-
       solution saving time associated with multiple com-     tion
       puter validation plans and execution.
     . Promise of reduced time to market for new drugs by     Paul Held, Bio-Tek Instruments, Winooski, VT, USA
       using a paperless environment where possible.          Co-author: Gavin Picket

This presentation will describe the challenges, successes     The production of micro-arrays requires the spotting of
and experiences surrounding the task of integrating           large numbers of unique DNA fragments onto several
complementary information systems in a pharmaceutical         diå erent substrates. While several commercially available
development environment.                                      instruments have automated this spotting task, the
                                                              sample preparation, culture propagation and mainten-
                                                              ance of the DNA library are often performed manually
Using laboratory automation to prepare com-                   with multichannel pipettes in 8 £ 12, 96-well, or 16 £ 24,
pounds precisely for HTS                                      384-well formats. Manual multichannel pipettes, while
                                                              more eæ cient than single-channel pipettes, still represent
Haissam Abdelhamid, Purdue Pharma LP, Cranbury, NJ, USA       a large amount of pipetting with many opportunities for
Pharmaceutical companies are constantly expanding             pipetting errors. Here we describe the use of the Precision
their compound inventory and reorganizing their screen-       2000 TM automated pipetting system to carry out many of
ing plates in an eå ort to ensure new chemical entities can   the necessary pipetting tasks required for the preparation
be identi® ed as rapidly as possible. Whether these new       of samples for micro-array spotting. These steps include:
compounds come from libraries (focussed or diverse) or        the propagation of plasmid libraries, PCR reaction
medicinal chemistry eå orts, robotics has become an           preparation, treatment of PCR products for agarose gel
essential tool when preparing compounds for high-             electrophoresis and the reconstitution of lyophilized
throughput screening (HTS). Using robotics to automate        samples before micro-array spotting. The Precision
routine laboratory tasks not only increases the rate at       2000 has a completely con® gurable six-station platform
which these activities can be completed, but also reduces     to hold the required pipette tips, reagent troughs and
incidents of human error. In our laboratories, we have        microplates (96 and 384 well) for ¯ uid transfer. The
integrated several robotic systems to aid in compound         platform is removable, allowing for multi-user friendli-
dissolution and HTS plate preparation. These enhance-         ness, easy cleaning and set-up of the instrument. The
ments have saved time and increased the reliability of our    eight-channel pipette arm moves up and down as well as
screening inventory, particularly when coupled with our       side to side, while the platform moves front to back to
in-house data-managemen t and inventory tracking soft-        provide complete access to all locations on the work
ware. Examples of laboratory automation used at Purdue        platform and complete con® gurability. The pipette arm
over that past year will be presented.                        uses a proprietary technology to pick up and seal reliably
                                                              any standard tip with individual free-¯ oating barrels that
                                                              compensate for tips out of position. An optional rapid
DNA micro-arra y fabrication and processing :                 dispense eight-channel manifold, which uses a precise
automation in the laboratory                                  bidirectional syringe pump to dispense accurately and
                                                              rapidly ¯ uids from a large unpressurized reservoir, is also
Katrine Verdun, BIOGEM, Division of Biology, University of
                                                              available. The Precision 2000 has a built-in micropro-
California San Diego, La Jolla, CA, USA
                                                              cessor that controls all movements. The ¯ exible software,
Co-authors: Richard Rouse and Gary Hardiman
                                                              both onboard and PC-based, provides complete pro-
The con¯ uence of robotics, biotechnology, computer           gramming capabilities. For more complete automation,
sciences and the completion of genome sequencing eå orts      robotics interfaces can be developed using ActiveX1
for several organisms have resulted in revolutionary          software commands. The Precision 2000’ s small size, with
changes in how biomedical research is carried out. It is      a 15 £ 21-inch footprint and a height of 16 inches, allows

78
Abstracts of papers presented at the 2001 ISLAR


                                                                preparation have been determined and, second, the most
                                                                important ones have been pointed out in order that they
                                                                be validated. This has led to the establishment of a
                                                                transfer protocol, which is in two parts:
                                                                   . development of the automatic method (study and
                                                                     optimization of all parameters);
                                                                   . validation of the automatic method.


                                                                Accelerating knowledge transfer for improved
                                                                lead-candidat e selection
                                                                John P. Helfrich, NuGenesis Technologies Corporation, West-
                                                                borough, MA, USA
                                                                Today’ s modern drug-discovery and development re-
                                                                search groups are expected to improve the eæ ciency of
                                                                delivering NCEs to the clinic. The new high-throughpu t
                                                                processes are dramatically increasing the raw number of
                                                                data sets that must be interpreted for decision support
                                                                across a global research team eå ort. The NuGenesis
it to be used almost anywhere including most biological         SDMS database serves as the centralized repository for
safety cabinets or chemical fume hoods.                         analytical reports, compound presentation or summary
                                                                documents, project reports, and instrumental raw data.
                                                                This database can then be extracted to work in conjunc-
Transposition and validation of a manual method                 tion with your LIMS, Enterprise Data Management
of sample preparation on the TPWII                              System and/or local specialized visualization and statis-
Heleé ne Brillard, Novartis Pharma, Orleans la Source, France   tical data software products. If your data sources save or
  è                                    è
Co-authors: Laurent Frances, Florence Dupas, Samir Haddouchi    print, the NuGenesis SDMS platform can get it, auto-
and Olivier Garinot                                             matically save it and allow fast eæ cient utilization across
                                                                the entire enterprise. After all, the data become informa-
The Analytical Development Laboratory NOVARTIS                  tion that when eå ectively communicated turns into
that works conjointly with the Pharmaceutical Develop-          critical-path knowledge for decision support in drug
ment Department had recently acquired the Zymark                discovery.
Tablet Processing Workstation , TPWII.
Within this laboratory, analytical methods are developed
to perform manual analysis. Consequently, we developed          Maximum return (strategic investments for high-
a procedure to transfer successfully a manual analytical        throughput screening)
method to this robotic system. First, the whole par-
ameters of the TPWII that have an in¯ uence on sample           James LaRocque, Wyeth-Ayerst Research, Pearl River, NY,
                                                                USA
                                                                Advanced technologies theoretically enable modern HTS
                                                                laboratories to screen expanding libraries quickly against
                                                                a drastically longer list of targets while reducing reagent
                                                                costs and maintaining more rigorous quality of results.
                                                                Even in large pharmaceutical corporations, however, the
                                                                collision of ® scal reality and the cost of state-of-the-ar t
                                                                HTS technologies make getting the most out of capital
                                                                investments a critical skill for success. Wyeth-Ayerst
                                                                Research has made cost-eå ective investments in Packard
                                                                CCS PlateTraks and Wallac CCD imagers, while also
                                                                continually upgrading our Zymark-integrate d systems
                                                                with new components and detectors.
                                                                This combination of equipment provides maximum
                                                                throughput for 384-well formats and supports the transi-
                                                                tion to 1536-well formats with relatively modular units
                                                                that can be programmed for multiple tasks. While the
                                                                management of individual screening projects is delegated
                                                                to individual scientists, the complexity of the automation
                                                                infrastructure oå ers ample opportunity for the develop-
                                                                ment of specialty skills, creating a well-rounded HTS
                                                                staå . By practising both individual initiative and co-
                                                                operativity, a relatively small group is empowered to

                                                                                                                          79
Abstracts of papers presented at the 2001 ISLAR


achieve eå ective sample plate replication, 384- and 1536-     our MetLab TM software suite for back-end chemometric-
well HTS, and rate-based hit characterization.                 based data processing techniques to con® rm automati-
                                                               cally expected and unknown metabolites.

Automated LC/MS analysis of biomolecule s using
ProMass                                                        Development of a high-throughpu t biochemical
                                                               pro ling platform
Je¡ Whitney, Novatia, Princeton, NJ, USA
Co-authors: Mark E. Hail and David J. Detlefsen                Je¡rey Murray, Paradigm Genetics, Inc., Research Triangle
                                                               Park, NC, USA
The recent increase in genomic and proteomic discovery         Co-authors: Ioana Popa-Burke and Chris Beecher
has increased the need for high-throughpu t automated
tools for biomolecule characterization. The ¯ ood of new       Paradigm Genetics, Inc., has industrialized the process of
protein targets will ultimately demand more eæ cient           gene-function discovery for human health, nutrition,
tools for the evaluation of expressed proteins for drug        crop production and industrial products. The company
discovery and structural biology studies. ProMass is an        has designed the GeneFunction FactoryTM, an indus-
automated biomolecule deconvolution and reporting              trial-scale laboratory that explores the function of genes
program used to process LC/ESI/MS data or single               in organisms by integrating state-of-the-ar t sequencing
ESI mass spectra to derive molecular mass information.         technology with phenotype, metabolite and gene-expres-
We recently integrated ProMass with the ThermoFinni-           sion pro® ling to collect hundreds of data points through a
gan Xcalibur data system to create a version of ProMass        single technology platform.
known as ProMassXcal iTM. ProMassXcal i processes en-          The Metabolic Pro® ling group is responsible for monitor-
tire Xcalibur sample sequences, deconvolutes mass spec-        ing changes in the biochemical pro® les of organisms that
tra from LC/MS data and produces web-based reports.            occur over the course of development in response to stress
ProMassXcali uses a novel cross-platform deconvolution         or induced genetic modi® cations. This is accomplished by
algorithm known as ZNovaTM. ZNova incorporates a               using LC-TOF-MS, GC-TOF-MS and ICP-MS instru-
unique charge-state scoring method that assigns the            ments. We describe here the innovative high-throughpu t
charge states of all signals in the ESI mass spectra and       processes developed for the cataloguing, storing in a dry
transforms the input ESI mass spectra to produce zero-         environment, grinding, dispensing and extraction/deri-
charge mass spectra (i.e. molecular mass information).         vatization of samples using custom robots, as well as
ZNova incorporates signal-processing techniques and            proprietary instruments such as the ` Mash-A-Matic and
unique logic that allow application to low charge state        ` Buster’ .
spectra and data of low signal-to-nois e ratio. As a result,
ZNova can be used to process data from a wide variety of
biomolecules including large proteins, oligonucleotides,       Role of automation and robotics in high-through-
peptides, etc. In this poster, an overview of the Pro-         put ADME pro ling of potential drug candidates
MassXcali/ZNova system will be presented along with            Kelly M. Jenkins, Bristol-Myers Squibb Co., San Diego, CA,
selected applications which highlight its utility in a high-   USA
throughput environment.                                        Co-authors: Robyn A. Rourick, Reginald Angeles, Marianne
                                                               Teopaco-Quintos and Daniel B. Kassel

Automated metabolite con rmation and identi -                  Early determinations of pharmaceutical properties can
cation using LC/MS and intelligent chemometric s               serve as predictors of a compound’ s likely developmental
                                                               success. Our laboratory has implemented high-through -
Je¡ Whitney, Novatia, Princeton, NJ, USA                       put ADME assays that address absorption, metabolism
                                                               and physicochemical properties in an eå ort to minimize
In recent years, drug-discovery researchers have placed
                                                               discovery to market attrition. While trying to meet the
greater emphasis on obtaining qualitative measures of
drug candidate ` quality’ by measuring ADME/Tox                throughput demands of parallel synthesis, we established
                                                               an integrated solution for ADME assays which incorpo-
tendencies earlier in the discovery process. This has led
                                                               rates a SAGIANTM core system for the determination of
to the development of in-vitro assay methodologies for
measuring metabolic stability, cell permeability, solubi-      both metabolic stability in human liver microsomes
                                                               (HLM) and cytochrome P450 (CYP) inhibition. This
lity, toxicity, etc. The challenge today is to automate
                                                               automated solution has allowed an increase in capacity,
fully all aspects of these assays by integrating intelligent
data analysis and interpretation tools into one simple-to-     throughput and reliability for both ADME assays.
use solution.                                                  The HLM assay uses a MultimekTM 96-channel pipettor
                                                               for liquid handling. The analysis plates are transferred
This talk will focus on the analytical methods and
                                                               o¥ine for ® nal analysis using high-throughput parallel
intelligent data analysis approaches we have employed
                                                               LC/MS. The CYP inhibition method uses a combination
to assess rapidly metabolic stability and metabolite
                                                               of liquid handlers and a ¯ uorescence plate reader to
identi® cation. With the use of SmartLCMSTM tech-
                                                               perform a single concentration pro® le assay for 88
nology, we will demonstrate the rapid assessment of
                                                               compounds. CYP inhibition is measured for both
metabolic stability of a compound followed by automatic
                                                               CYP3A4 and 2D6 isozymes.
detailed re-analysis using on-the-¯ y intelligent data-
analysis techniques. In addition to up-front intelligent       This system represents a fully integrated approach in
automation, we will illustrate the preliminary results of      support of high-throughpu t ADME evaluation in a drug-

80
Abstracts of papers presented at the 2001 ISLAR


discovery environment. The core system concept creates        anywhere around the world using Web technology. This
a plug-and-play approach that combines a series of            presentation will focus on the principles of good data
modular stations to build a robotic system, which is          management that allow for pharmaceutical and biotech
¯ exible, upgradable and easily recon® gurable when           discovery facilities to leverage data as an asset, protect
 assays change or are newly developed. The application        valuable intellectual property and ensure the accurate
 of these strategies as a means of assessing metabolic        and easy creation of complete compound documents
 stability and CYP inhibition of our combinatorial li-        throughout the drug-discovery and development process.
 braries is discussed.

                                                              Elutri-Zone MS: a new SPE system for rapid
Novel homogeneous FRET-based assay for endo-                  sample processing and LC/MS analysis
peptidase inhibitors: assay development , steady-
state kinetics, high-throughpu t screening and                John Janiszewski, P¢zer Global R&D, Groton, CT, USA
miniaturization                                               Co-authors: R. M. Olech, R. A. Pranis, J. R. Jacobson, C. A.
                                                              Perman, B. A. Boman, J. Soldo, R. Speziale, T. W. Astle,
Joe Bradley, P¢zer Global R&D, Sandwich, UK                   M. J. Cole, J. S. Janiszewski and K. W. Whalen
Co-authors: Chris Chambers, Helen Boyd, Emma Faure, Joe
Bradley, Simon Dales, Neil Benson, Wilma Keighley and         To increase sample throughput, many recent LC/MS
Andreas Sewing                                                methods have described techniques to shorten and/or
                                                              simplify the chromatograph y step(s) before MS analysis.
The growth in compound numbers and subsequent need            We describe a discrete solid-phase extraction step using
for increases in throughput and reduction in cost are key     Empore SPE membrane to capture the analyte, followed
drivers of developments in the ® eld of high-throughpu t      by an elution step that features the rapid quantitative
screening. Increasingly, there is a move towards ¯ uores-     transfer of the elutri zone (pure zone) into the MS. This
cence-based assay technologies, which are ideally suited      application was designed primarily for bioanalytical
for screening because they are versatile, homogeneous         support of high-throughpu t ADME screening.
and amenable to both automation and miniaturization.
                                                              An SPE Card having the outer dimensions of a microtitre
We developed a novel FRET-based assay to identify             plate was developed by molding a plastic frame around a
inhibitors of a metallo-endopepidase . This assay uses a      sandwich of 3M Empore sorbent (0.5 mm thickness,
novel substrate with a low Km , thus making it cost-          8 mm particles) and micro® bre support material.
eå ective for high-throughpu t screening. We have success-    Ninety-six discrete elution zones (7 mm diameter, 9 mm
fully transferred and validated this assay onto our           centres) were welded into the sheet. The SPE Card was
Robolab linear track-screening platform. A full HTS           designed to ® t in a modi® ed cell harvester (Tomtec) that
campaign was subsequently conducted in 384-well for-          facilitates SPE processing. The harvester performs sor-
mat, achieving a maximum throughput of 75 000 data            bent activation, load and wash steps. Once the analyte
points within 24 h.                                           has been retained, an interference wash is completed and
This poster will illustrate the HTS process from assay        the plate transferred to an elution device (Elutrix,
development to high-throughpu t screening using state-of-     Tomtec). Each well was eluted directly into the MS
the-art technology.                                           using ¯ ow rates ranging from 1.0 to 3.0 ml min ¡1 . A
                                                              single HPLC pump delivered eluent composed of 5 mm
                                                              Ammonium Acetate combined with 50± 90% methanol
Accelerating knowledge transfer for improved                  or acetonitrile.
lead-candidat e selection                                     The SPE Card and Harvester protocol allow rapid (2±
John Helfrich, NuGenesis Technologies Corporation, Westbor-   5 min) o¥ine sample clean-up followed by direct elution
ough, MA, USA                                                 into the MS detector. This protocol results in the
                                                              concentration of the sample and reduces liquid-handling
Managing and tracking data through the discovery pro-
                                                              steps as dry plates are transferred directly to the Elutrix
cess requires a compilation of many diå erent types of        for LC/MS processing. Owing to the small particle size of
analytical, biological and image output. This includes the    the Empore sorbent, the SPE Card can mimic the
collection, storage and management of relevant scienti® c
                                                              performance of discrete HPLC columns, similar to those
information about lead candidates, as well as immediate       used in high-throughpu t sample analysis or in online
access to this information for complete compound docu-        clean-up protocols. Relevant parameters and preliminary
ment creation. Data collaboration across the discovery
                                                              results will be described here.
arena, even across the entire enterprise, is critical to
making crucial go/no go decisions about subsequent lead
candidate development.                                        Combined gravimetri c and colorimetri c method
The NuGenesis1 Scienti® c Data Management System              for the calibration and performance testing of
(SDMS) allows discovery scientists electronically to view,    liquid-handlin g workstations in a GLP laboratory
share, reuse and access data within the laboratory, as well
                                                              John R. Alianti, GlaxoSmithKline, Research Triangle Park,
as throughout the enterprise. You can easily capture
                                                              NC, USA
laboratory and report data produced in any Windows-
                                                              Co-author: Glenn Smith
based software application. These data can be catalogued
automatically and accurately for easy retrieval. Immedi-      Good laboratory practices (GLPs) require that
ate access to this electronic information is possible from    appropriate calibration and performance testing be con-

                                                                                                                       81
Abstracts of papers presented at the 2001 ISLAR


ducted on all pipetting devices, including automated           Octave system: a new automated instrument for
liquid-handling systems. Routine evaluation of the             the analysis of molecular interaction s
equipment must be performed to assess both the accuracy
and reproducibility (precision) of liquid transfers.           Robert J. Kaiser, Prolinx, Inc., Bothell, WA, USA
                                                               Co-authors: Karin A. Hughes, Guisheng Li, Deborah D. Lucas,
Additionally, the testing methods and acceptance
                                                               Kevin P. Lund, Chris Pershing, Douglas A. Spicer, Mark L.
criteria must adhere to established company-wide
                                                               Stolowitz, Jean P. Wiley, Steve Bailey, Michael Baum, Erik
policies (SOPs). These methods must be sensitive as
                                                               Engstrom, Scott MacInnes, Rich Ward, Craig Yamamoto,
well as being robust and practical. Likewise, the data
                                                               George de la Torre, Lynn Hilt, Charles J. Hutchings, Arturo
generated must be easily analysed, interpreted and
                                                               Kozel, Terry A. Weeks, Dwight Bartholomew, Rick Carr, Keren
documented.                                                    Deng, Jerry Elkind, Tom Martin and John Quinn
Pipetting accuracy is determined by weighing 96-well           Surface plasmon resonance (SPR) technology is a power-
plates (in replicates of three) before and after dispensing    ful, label-free method for the analysis of molecular inter-
a speci® ed volume of liquid. System accuracy at various       actions. Prolinx1, Inc., has developed a new automated
volumes is determined and the results used to calibrate        instrument, the Octave Molecular Interaction Analysis
the workstation as appropriate (e.g. Zymark RapidPlate         System, that allows this powerful analytical platform to
Syringe Calibration Factors, Tecan GENESIS Liquid              become ubiquitous in life-science research and drug-
Classes, Packard MultiPROBE II Performance Files).             discovery laboratories. This technological breakthrough
The between-tip precision, individual-tip precision and        is the result of combining the miniaturized Texas Instru-
individual-tip accuracies are all ascertained by sub-          ments SpreetaTM 2000 SPR sensor with the Prolinx
sequent colorimetric testing using a photometric micro-        VersalinxTM Chemical Aæ nity Tools. These technologies
plate reader. A preformatted Microsoft Excel spreadsheet       have made possible the development of an instrument of
containing embedded formulas automatically analyses            moderate cost that incorporates eight independent sen-
the data and generates a report.                               sors operating in parallel. The sensor surfaces can be
                                                               readily and eæ ciently modi® ed with molecular targets,
The procedure developed combines the simplicity of             and exhibit low non-speci® c binding. Samples are intro-
gravimetric measurement with the speed of colorimetric         duced to the sensors from standard microwell plates using
microplate reading. The process is generic and can be          an integrated liquid-handling robot. This new instru-
used to assess or compare the accuracy and precision of        ment will signi® cantly increase the throughput of SPR-
any workstation. Automated data analysis and standar-          based molecular interaction analysis in basic biological
dized report generation facilitate the process and ensure      science and drug-discovery applications.
regulatory compliance.


                                                               Increased throughput e ciency with dual-column
                                                               technology in LC/MS/MS sample analysis
                                                               Kathryn B. O’Mara, GlaxoSmithKline, Research Triangle
Implementing automated sample preparation for                  Park, NC, USA
GLP and non-GLP bioanalysis to increase produc-                Co-authors: Lisa St John-Williams and John A. Dunn
tivity in a contract research organizatio n
                                                               Recent advances in automation have signi® cantly re-
John R. Kagel, Charles River Discovery and Development         duced the amount of time it takes to prepare clinical
Services, Worcester, MA, USA                                   samples for LC/MS/MS analysis. As a result, the eæ -
Co-authors: Larry E. Elvebak, Brian E. Lilley, Jakal M. Amin   ciency bottleneck has shifted from sample preparation
and James A. Jersey                                            time to analytical run time on the mass spectrometer. To
                                                               reduce the mass spectrometry analysis time, the use of
Client services for analysis and bioanalysis must: provide     dual-column technology was evaluated for three single-
reliable results, meet increasingly aggressive timelines,      column validated LC/MS/MS methods.
meet increasingly lower unit costs and be performed
under appropriate regulatory conditions. Automation            For each of the three methods, the HPLC system was
was used successfully to satisfy these criteria regarding      enhanced with switching valves and the appropriate
sample preparation for high-throughpu t LC/MS/MS               programming to allow the unit to function in the dual-
GLP and non-GLP bioanalysis in a CRO. The ® rst                column mode. A semi-automated liquid handler was used
phase in automating sample preparation involved pro-           to perform either protein precipitation or solid-phase
cessing samples in a 96-well format using 96-tip parallel      extraction using 96-well technology. A triple quadrupole
pipetting workstations (e.g. Tomtec) in an open-access         mass spectrometers was used to acquire data using mul-
environment. The impact of this implementation was to          tiple reaction monitoring.
increase productivity by at least twofold. The second          The three quantitative LC/MS/MS methods using dual-
phase in automating sample preparation used a liquid           column technology are currently being used in a high-
handler (e.g. Tecan Genesis) for the preparation of            throughput laboratory. Each method has been validated
calibration standards and automated reformatting and           to demonstrate acceptable accuracy and precision. Ro-
dilution of samples from tubes or vials into 96-well plates.   bustness has also been demonstrated by the analysis of
In addition, calibration and compliance issues related to      thousands of samples in a GLP environment. For each
automated sample preparation will be discussed.                method, the analytical run time has been reduced by

82
Abstracts of papers presented at the 2001 ISLAR


50% and instrument eæ ciency and sample throughput             ketoconazole > piroxicam > caå eine > metoprolol     (pro-
have been increased twofold.                                   posed BCS internal standard) > terbutaline.
                                                               Stirring during assay signi® cantly increased the observed
                                                               permeabilities for highly mobile molecules.
Rapid, e cient method for determining metabolic
stability                                                      In addition to permeability measurements, membrane
                                                               retention of compounds was determined. Yangonin,
Kelly Johnson, Waters Corporation, Milford, MA, USA
                                                               desmethoxyyangonin , ketoconazole and phenazopyridine
Co-authors: John Erve, Andre Dandeneau and Beverly Kenney
                                                               were > 60% retained by the arti® cial membranes con-
Modern drug discovery has been transformed by the              taining phospholipids.
automation of research. The resulting explosion of data
                                                               The in¯ uence of hydrogen bonding was explored by
in the discovery pipeline, combined with the pressure to
                                                               determining permeabilities using ® lters coated with do-
reduce costs and speed up drug-discovery cycles, provides
                                                               decane free of phospholipids. The membrane transport of
a strong demand for fast and selective analytical methods
                                                               phenazopyridine (strong hydrogen-bon d donor) is about
to produce quality data.
                                                               twice as fast and retention is about twice reduced in the
In vitro metabolic stability assays provide a rapid estima-    inert lipid membranes compared with phospholipid-
tion of new chemical entities in the discovery phase of        based membranes.
pharmaceutical development. These multistep assays
                                                               In the ® lter-IAM method, concentrations were deter-
routinely incorporate robotic liquid-handling systems to
                                                               mined by microtitre plate UV (190± 500 nm) spectro-
automate the incubation step of test compounds with
                                                               photometry and by LC/MS. Higher-throughpu t was
human liver microsomes in 96-well plates. Metabolic
                                                               achieved with direct UV by the use of 96-well microtitre
stability is subsequently measured by LC/MS as the
                                                               plate formats and with LC/MS by the use of cassette
amount of substrate metabolized (expressed as the per-
                                                               dosing (5-in-1).
centage remaining of the initial substrate). The resulting
information is crucial in the selection process in determin-
ing a compound’ s potential ` drugability’ .
                                                               Seamless integration of information in a pharma-
Given the large number of samples generated by the             ceutical development environment: integrating
liquid-handling systems in metabolic stability assays,         technology from LabWare, NuGenesis, VelQuest
employing automation and higher throughput in every            and Waters
step of the assay has become a necessity. This presenta-
tion will demonstrate the use of high-throughpu t LC/MS        Kurt Roinestad, Purdue Pharma LP, Ardsley, NY, USA
methods to analyse samples produced in a metabolic             This presentation will describe Purdue Pharma’ s ap-
stability assay. We will develop LC/MS methods that            proach to an integrated solution between Laboratory
signi® cantly increase overall throughpu t without sacri® -    Information Management Systems (LIMS), Chromato-
cing data quality. By incorporating such factors as            graphy Data Systems (CDS), Process Management and
parallel sample processing, high sample capacity, alter-       Compliance Systems (PMC), and Scienti® c Data Man-
nating column regeneration and smaller diameter col-           agement Systems (SDMS). It will describe the vendor-
umns to reduce cycle times, we can analyse the large           selection process and how Purdue Pharma created an
number of samples produced and ultimately help ex-             environment to produce a best-of-breed information sol-
pedite the drug-discovery process.                             ution to creating the paperless laboratory. Working
                                                               towards the creation of a paperless laboratory has the
                                                               potential to result in signi® cant bene® ts including re-
Filter-immobilize d arti cial membrane ( lter-                 source liberation and an electronic compliance platform
IAM) permeabilit y assay: a new high-throughpu t               for process management and data review.
in vitro drug-absorptio n model
                                                               This paper will describe the process of working with four
Konstantin Tsinman, pION, Inc., Woburn, MA, USA                vendors to create a complementary data management
Co-authors: A. Avdeef, D. Voloboy, M. Stra¡ord and B. Kenney   solution to Purdue Pharma’ s needs. The process of
The assessment of passive transport properties of over 20      logging in samples, creating work-lists, executing analy-
drug and natural product molecules was made using the          tical methods, capturing data electronically, storing
in vitro absorption model based on ® lter-immobilized          electronic records and submitting reports will be re-
arti® cial membranes (® lter-IAM), assembled from phos-        viewed. After data creation, electronic data review will
phatidylcholin e in dodecane, in buå er solutions at           be discussed enabling electronic ` instant replay’ of la-
pH 7.4.                                                        boratory information.
Several of the compounds were lactones extracted from          The primary bene® ts of this complementary solution
the roots of the kava-kav a plant. Experiments were            approach hold the possibilities to do the following.
designed to test the eå ects of stirring during assays and
                                                                 . Increased capacity by liberating valuable resources
the eå ects of varying the assay times.
                                                                   from rework loops and the manual data review
The highly mobile kava lactones permeated in the order             process.
dihydromethisticin > yangonin > kavain > methisticin >           . Leveraging the approach to validation of the overall
desmethoxyyangonin . Other molecules in the study                  solution saving time associated with multiple
ranked: phenazopyridine > testosterone > propranolol >             computer validation plans and execution.

                                                                                                                      83
Abstracts of papers presented at the 2001 ISLAR


     . Promise of reduced time to market for new drugs by        and reproducible method for measuring mRNA levels of
       using a paperless environment where possible.             a small number of genes. The assay’ s 96-well plate format
                                                                 and simple handling procedures makes it highly
This presentation will describe the challenges, successes        amenable to high-throughpu t robotics. This talk will
and experiences surrounding the task of integrating              describe a Zymark StaccatoTM Workstation designed to
complementary information systems in a pharmaceutical            perform the hybridization, ampli® cation and signal-
development environment.                                         detection steps of the Quantigene assay.


High-throughpu t robotic workstation for per-                    Multivariate calibration of a UV-VIS breoptic
forming gene-expressio n assays                                  probe used for direct measurement s in a Zymark
                                                                 XP-based automated dissolutio n system
Michael A. Kuziora, Gene Logic, Inc., Gaithersburg, MD, USA
                                                                 Lars A. Svensson, AstraZeneca, Molnda, Sweden
Metabolism and growth are dependent on a highly
                                                                 Co-authors: Rimstedt Eva and Svensk-Ankarberg Anna
orchestrated interplay of a variety of proteins within
cells. These proteins function as enzymes in metabolic           Correlation between drug dissolution results obtained by
pathways, signalling molecules for communication be-             both liquid chromatograph y (LC) and ultraviolet-visua l
tween and within cells, and as various cellular structural       (UV-VIS) spectrophotometry has been performed by
components. In a disease state, cells often modulate the         multivariate calibration.
amounts of speci® c proteins and may produce new                 The dissolution of tablets or capsules, stored and exposed
proteins not normally found in a particular type of cell.        to harsh conditions (high humidity/elevated tempera-
The alterations in the proteome observed in the diseased         tures) at diå erent degrees, was carried out by an auto-
state most often result from changes in gene expression          mated system based on the Zymark XP robot. The
levels within a cell. Gene Logic believes that pharmaceu-        system had the capability of withdrawing samples to be
tical companies can reduce the time, risk and cost associ-       subsequently prepared into LC vials at the same instance
ated with drug discovery if they know the expression             as an UV-VIS spectrum was recorded via a ® breoptic
levels of genes that play roles in the disease-associate d       probe. The same probe was used in all vessels and could
pathways. Such knowledge may help them discover drug             be attached directly adjacent to the ® lter tip for the
targets, screen drug leads, and predict toxic and phar-          sample withdrawal on one of the robotic hands.
macological responses to drug leads.                             The subsequent multivariate calibration was performed
Using the Aå ymetrix, Inc. GeneChip1 micro-array plat-           with Simca-P 8.0 (Umetrics AB, Sweden). Most of the
form, Gene Logic has measured the expression levels of           calibration is explained in two components, with the ® rst
thousands of genes from a diverse range of normal and            mainly corresponding to the UV spectrum and the
diseased tissues to create a reference gene expression           second taking into consideration the baseline oå set.
database called the Gene Express1 Suite. The bioinfor-
matic analysis tools incorporate d in the Gene Express Suite
facilitate the identi® cation of distinct sets of genes whose    High-throughpu t chemistry: an integration of
expression is consistently altered in a particular disease       chemistry, automation and informatics
state. The expression patterns of these gene sets become a
                                                                 Li Chen, Ho¡mann-La Roche, Inc., Nutley, NJ, USA
molecular ® ngerprint of the disease and thus not only
re¯ ect the disease status of metabolic pathways, but also       One of many impacts that combinatorial chemistry has
could also serve to indicate the eå ect of potential treat-      been made in the last decade is to stimulate intellectual
ments when aå ected cells are exposed to a potential drug.       creativity to invent new and more eæ cient ways of
                                                                 making new compounds. Although much eå ort has been
A primary goal of a pharmaceutical company is to
                                                                 investigated into developing synthetic methods, auto-
discover drugs that restore the normal functioning of
                                                                 mated synthesizers and analytical tools, the high produc-
the disease-aå ected pathways. In one scenario, a phar-
                                                                 tivity of compound library synthesis cannot be achieved
maceutical company could use the information in the
                                                                 without the integration of chemistry, automation and
Gene Express database to identify a small set of genes
                                                                 informatics into an eæ cient process. I will emphasize
that indicate a disease state. The expression of this gene
                                                                 what we have learned in how to establish an infrastruc-
set is then monitored when an appropriate cultured cell
                                                                 ture that integrates functional modules containing a
line that represents a disease state of interest is exposed to
                                                                 diverse set of tools for high-throughpu t organic synthesis
chemicals from a compound library. A potential drug
                                                                 applications. This modular approach provides the maxi-
can be identi® ed for further characterization if, for ex-
                                                                 mum eæ ciency of combinatorial tool functions to support
ample, it is found to restore expression levels of the gene
                                                                 multiple project teams for drug lead generation, explora-
set to normal levels.
                                                                 tion and optimization.
Unfortunately, the use of large micro-arrays to measure
                                                                 The agenda includes the following.
gene expression of a few genes in a high-throughpu t
screen is not economically feasible at this time. We               .   HTOS process design.
therefore investigated the use of alternative method-              .   Pro- and post-synthesi s automation.
ologies to measure gene expression. Bayer Corporation’ s           .   Synthetic chemistry method.
Quantigene TM assay, which uses branched DNA (b-                   .   HT analysis and puri® cation.
DNA) for signal ampli® cation, and provides a sensitive            .   Data management and chemo-informatics.

84
Abstracts of papers presented at the 2001 ISLAR


Living Chip TM: a nano uidic platform for ultra-                      acquisition of biochemical information, on the order of
high-throughput, massively parallel synthesis,                        107 measurements per day. Combining nanolitre reaction
storage and screening (MPS3 )                                         volumes and a simple interface to microtitre plates, the
                                                                      Living ChipTM format conserves compound libraries,
John R. Linton, BioTrove, Inc., Cambridge MA, USA                     increases analytical capabilities and decreases costs. The
The Living ChipTM is a nanotitre plate consisting of a                platform has applications in all aspects of drug discovery
uniform and addressable through-hole array. The                       from materials’ handling and storage, target ID and
though-hole s are nominally 300 mm square and 500 mm                  validation, to lead discovery and optimization.
deep, giving each well a 50-nl volume. Proprietary coat-              Cell culture in the Living ChipTM using yeast (S.
ings make the surfaces of the plates hydrophobic and the              cerevisiae) and bacteria (E. coli) shows similar growth
interior of the wells hydrophilic. This allows the samples            characteristics to cells grown in bulk. Additionally, E.
to be held in the wells by surface forces and prevents                coli cells expressing GFP inoculated into isolated channels
sample contamination from adjacent wells. Ten thou-                   show no crosstalk.
sand-well chips are currently in use and 100 000-well
chips are in production.                                              Diverse biological libraries can be introduced to the chip
                                                                      using a simple dip loading procedure. This technique was
Massively parallel mixing of assay components takes                   used by BioTrove collaborator Genofocus to dip load
place when chips containing diå erent components are                  cells expressing a lipase enzyme library into a Living
stacked such that the through-holes align. The chips are              ChipTM. The cells were grown up overnight and then
imaged using a CCD array for readouts such as absor-                  stacked with a chip containing a tagged substrate so that
bance, ¯ uorescence and luminescence. Detection is per-               wells containing clones with increased lipase activity
formed in a transmission geometry, taking advantage of                yielded a greater ¯ uorescence signal. The ` hits’ were
the bottomless wells. These key features and the sophis-              harvested by a puå of air from a microsolenoid valve
ticated robotics built by BioTrove allow for the rapid                positioned above the well, into a microtitre plate waiting


                  hydrophobic               hydrophilic


                                                                             125-300um


                                                                                                     >500um




Figure 1. The Living ChipTM: a nanotiter plate. The surfaces are hydrophobically coated and the wells are hydrophilic to contain and
isolate samples.




                                                                                                 5 cm square




Figure 2. 10 000 well chips (above) are currently in use and 100 000 well chips are in production.

                                                                                                                                 85
Abstracts of papers presented at the 2001 ISLAR


                                                                Automated compound dilution and presentatio n
                                                                for determinatio n of IC50 data
                                                                Malcolm Willson, Systems Research, GlaxoSmithKline, Steve-
                                                                nage, UK
                                                                Co-authors: David Brown and David Hayes
                                                                Programme-targete d SAR screening within Systems Re-
                                                                search at Stevenage calls for a process able to progress
                                                                compound activity determination (80 to > 320 com-
                                                                pounds/week) in real time, enabling ongoing chemistry
                                                                to continue based on known biological activity.
                                                                Eæ cient compound dilution and generation of assay
                                                                plates is required to meet weekly turn around times for
                                                                IC50 and cross-screening data generation.
                                                                Comparison of manual across-plate dilution, vertical
                                                                dilution down a series of plates and Z-dilutions within a
                                                                series of 384-well plates will be discussed.
Figure 3. Isolated wells: all wells loaded with media. Cell     Use of the twin head Biomek FX uses the ¯ exibility of
expressing GFP innoculated into wells show no crosstalk after   both the 96- and 384-tip heads, allowing Z-dilutions, a
culturing.                                                      very eæ cient method for automated generation of IC50 s.
                                                                By having space on the stock plates for several extra
                                                                compounds, a selected range of ` standards’ covering
below. Kinetic analysis con® rmed the discovery of a            several assays can be included to enable assay perform-
lipase mutant with a 20-fold higher activity relative to        ance and automation QC to be monitored for each set of
the wild-type. This screening consisted of 30 000 assays        compounds, along with suæ cient control/blanks for
performed by one person in less than 2 days, and would          statistical analysis of assay performance.
have taken several scientists weeks using conventional
means.                                                          Cloning of assay templates reduces IT resource require-
                                                                ments for data analysis as templates can be cloned rather
                                                                than written from scratch.
                                                                If compound numbers increase, the system can be
                                                                converted to run vertical dilutions down a series of plates
Next step in miniaturization : submicrolitre assays             (diå erent dilution per plate). However, this only works if
in 96-well formats                                              the assays are stable enough for the increased length of
                                                                assay due to the higher plate numbers.
M. J. Wildey, R. W. Johnson Pharmaceutical Research             Conversion to 1536 plates doing Z-dilutions from 384-
Institute, Raritan, NJ, USA                                     well stock plates is also an option that would be possible
Co-author: C. Fleming                                           when liquid-handling devices can cope with smaller
Some of the challenges and goals in many of today’ s            volumes for assays.
screening laboratories are to investigate, evaluate and
validate new techniques that will enable the reduction in
cost, provide an increase in eæ ciency and strengthen the
quality of screening data. Towards the end of reducing
cost, we have been a Beta test site for a novel 96-well         Lead generation and optimization : integrated data
submicrolitre assay system called Arteas. The architec-         mining and informatics in drug discovery
ture of Arteas oå ers a solution to the problem of              Charles J. Manly, Discovery Technologies, Neurogen Corpora-
evaporation in small-volume assays.                             tion, Branford, CT, USA
We have successfully performed a ¯ uorescent-base d en-         Drug discovery today includes considerable focus of
zyme assay in Arteas with a reaction volume of 400 nl.          laboratory automation and other resources on high-
We successfully reproduced published control IC50 s and         throughput technologies, but lead generation and opti-
routinely generate Z’ -factors in the range 0.6± 0.7. We        mization to clinical candidates continues to be a lengthy
will show these data and variability around some tests          and costly process. The real bene® t of today’ s tech-
performed with actual library compounds. Translation of         nologies is beyond the exploitation of each individually.
this device to production screening will be dependent           Only recently have signi® cant eå orts focused on eå ec-
upon the successful integration of nanoliquid handling          tively integrating these complex discovery disciplines to
onto our robotic platforms. Initially, we will integrate        realize their larger potential. Informatics, computationa l
this new liquid-handling option onto our Fast Track             chemistry, virtual screening and data mining play a large
robotic platform. Progress on this translation will be          role in this integration and in increasing the eæ ciency of
addressed.                                                      the drug-discover y process.

86
Abstracts of papers presented at the 2001 ISLAR


Survival skills for managing robotics and automa-               Unfortunately, as the project continued, con¯ icts
tion: how to outwit, outplay, outlast                           and diæ culties arose. Each group had its own expecta-
                                                                tions for the project that were not necessarily commu-
Maria DeGuzman, A¡ymetrix, Inc., Santa Clara, CA, USA           nicated with the others eå ectively. This led to many
Our experience in automating a genomics’ laboratory for         problems including delays, miscommunications, unrealis-
single nucleotide polymorphism (SNP) detection has              tic expectations, frustration and blame being placed on
shown us that automation projects are not often com-            one another. In addition, another facet compounded the
pleted in the planned or expected manner. With auto-            already diæ cult process: reality. We found several in-
mation playing an ever-increasing role in the research          stances where reality interfered in the development of the
and development laboratory, we will discuss why some            project including budget, time, resources and process
projects succeed while others fail. For instance, what are      bottlenecks. We also saw evidence of con¯ icts such as
the real bottlenecks to address? Does experience make a         when we needed to decide between 96- or 384-well plates,
diå erence? Is management expectation realistic? What           tubes or plates, custom or commercial or semi-automa-
are the advantages and disadvantage s of custom or              tion, or even con¯ icts between the scientists and the
commercial automation? What are the roles of the                engineers.
scientists, engineers, and laboratory managers in such
projects?                                                       Fortunately, we realized many of these issues and
                                                                managed them eå ectively. We also proceeded to perfect
The recent completion of our high-throughput screening          the process in other ways using protocol enhancements
project has enabled us to look back objectively and             and microscaling improvements. This increased our
answer these questions for our genomics’ application.
                                                                throughput to 1.4 Mb per day for every two people while
The high-throughpu t screening project at Aå ymetrix            reducing reagent costs.
began in 1999. The initial goals of the project were to
screen for SNPs across 40 unrelated individuals in an           At the conclusion of this project, approximately 25 000
automated fashion. In the initial stages of this project, the   genes were screened covering 8.2 Mb of the genome
process), which encompassed sample preparation to               (including genes, regulatory regions and STSs). Since
scanning, was done manually. This provided an output            most of the projects were tested against 40 individuals, we
of about 2.3 Mb of sequence a week for every 4.5 people.        screened 24 million bases of dsDNA and identi® ed 15 682
With the collaborative eå ort of the executive manage-          SNPs that have been deposited to the SNP public
ment group, the high-throughpu t screening laboratory,          database. In addition, much of the software, automation
the engineering group and the bioinformatics group, we          and protocols developed during this project have been
set out to accomplish this task.                                incorporated into other internal research laboratories.




                                                                                                                        87
Abstracts of papers presented at the 2001 ISLAR


Stand alone extractor for semi-automate d method              The Tissue Culture Infectious Dose 50% (TCID50)
development and validation                                    assay was used for the quantitation of viral titres used
                                                              in vaccine products.
Maria Styslo-Zalasik, R. W. Johnson Pharmaceutical Research
Institute, Raritan, NJ, USA                                   These assays required sterility, therefore requiring all
Co-author: Kathleen Cirillo-Penn                              equipment to be housed in Class II microbiological safety
                                                              cabinets. The TCID50 assay was programmed on a
In the past 10 years, laboratory automation has become        Tecan RSP 200 using eight disposable-tip liquid-hand-
increasingly important for routine chemical analysis          ling probes in conjunction with a robotic manipulator
testing in the pharmaceutical industry. The use of            arm. Culturing of plates containing tissue culture, to
automatization or semi-automated laboratory equipment         support the TCID50 assay, was automated using a
can reduce the laboratory ¯ ow-through time of the            Zymark Twister coupled to a Multidrop dispenser.
samples in the QC area. Likewise, automated and semi-
automated equipment can be used as a tool for research        Scheduling and liquid-handling software were combined
during method development and validation.                     to execute this automated assay. The use of the ` joblist’
                                                              function allowed the operator to select from a databas e of
This presentation will illustrate how semi-automate d         prede® ned dilutions within the Logic pipetting software.
sample preparation can aå ord rapid optimization of
method parameters during early-stage method develop-          A formal comparison of the automated TCID50 assay
ment and validation. The potential dissolution rate and       against the manual TCID50 was performed and proved
solubility issues with high sample concentrations can be      comparability.
avoided through the evaluation of data from parameter
optimization of the sample preparation using semi-auto-
mated instrumentation . Examples for the use of a stand       Integrated approach to high-throughpu t sample
alone extractor in an assay/purity method validation will     processing , characterizatio n and puri cation
be presented.
                                                              Michael L. Moore, GlaxoSmithKline, King of Prussia, PA,
                                                              USA
New automated instrument for the characteriza -               The extension of high-throughpu t synthesis in support of
tion of biomolecula r interaction s                           lead optimization has imposed increasingly stringent
                                                              requirements on compound characterization, purity
Mark L. Stolowitz, Prolinx, Inc., Bothell, WA, USA            and accurate concentration determination. We devel-
Surface plasmon-resonanc e technology is a powerful,          oped an integrated and highly eæ cient process for
label-free method for the analysis of biomolecular inter-     compound analysis, puri® cation and sample processing
actions. However, the use of this technology is limited by    with a capacity of 100 000 compounds/year at a 20-mg
the cost, throughput and complexity of existing instru-       scale. Puri® cation is driven by ultrahigh-throughpu t
mentation and chemistries. Prolinx has developed a new        analytical LC/MS, which minimizes the number of
instrument that will address these limitations and allow      fractions collected and analysed. Custom software with
this powerful analytical platform to become ubiquitous in     a browser-based front end is employed to track samples
life science research and drug-discovery laboratories.        and provide the required data to robotic workstations.

This technological breakthroug h is the result of combin-
ing the Texas Instruments Spreeta1 2000 chip with             Managing laboratory automation in the postge-
Prolinx’ s VersalinxTM Chemical Aæ nity Tools. These          nomic era
technologies enabled the development of an instrument
of moderate cost that incorporates eight parallel sensor      Michael R. Kozlowski, Axiom Biosciences, San Diego, CA,
surfaces that can be eæ ciently modi® ed with molecular       USA
targets and exhibit low non-speci® c binding. The pre-        The completion of the cloning of the human genome has
sentation will encompass technology, design and applica-      provided the drug-discovery community with a wealth of
tions of this new instrument.                                 potential drug targets. At the same time, it has produced
                                                              challenges to the way in which drug discovery is done.
                                                              Formerly, the mandate of drug discovery was to screen a
Automation of an infectivity assay for the quanti-            relatively small number of well-validated targets against
tative analysis of virus in biopharmaceutica l                an immense number of compounds. Now, drug-discovery
products                                                      scientists are faced with processing a very large number of
                                                              minimally validated targets. Most targets were formerly
Mervyn Cadette, GlaxoSmithKline, Beckenham, UK
                                                              single proteins, which are amenable to screening in
Relative to chemical assays in a pharmaceutical devel-        highly reductionist systems. Now it is clear that most
opment environment, biological assays are relatively ` low    biological processes, including those contributing to
throughput’ in comparison. However, the complexity of         pathological states, must be the result of complex inter-
assays associated with biological products is often of a      actions between proteins. A way must be found to address
diå erent magnitude. Biological assays become attractive      this new level of complexity. In addition to these
automation candidates based upon their complexity             challenges, the availability of the entire sequence of the
coupled with high demand for their use in biopharma-          human genome raises expectations for the rapid intro-
ceutical analysis.                                            duction of more, and better, drugs.

88
Abstracts of papers presented at the 2001 ISLAR


These challenges demand new ways of thinking about            protein therapeutics and other bioactive molecules will
how we carry out drug discovery, and about drug-              be presented.
discovery automation, from assay development to AD-
MET pro® ling. This talk will discuss some of these new
approaches.


High-throughput screening inhibition assays to                Building an electronic data-handling, compliant,
evaluate the interaction of P zer proprietary com-            foundation for the QA analytical laboratorie s at
pounds with cytochrome s P450                                 Westborough

Michael West, P¢zer Global R&D, Groton, CT, USA               Mike Stroz, AstraZeneca, Westboro, MA, USA
Co-authors: Larry Cohen, Al¢n Vaz and Shawn Harriman
                                                              A signi® cant portion of QA laboratory activities are
A ¯ uorescence-base d drug interaction assay using recom-     dedicated to compliance and data handling in a paper-
binant CYPs and a cocktail of CYP-speci® c probes in          based system. AZ Westborough is implementing an
human liver microsomes was assessed as higher through-        electronic environment in the laboratories to ensure 21
put methods for evaluating the potential for inhibition of    CFR Part 11 compliance, improve eæ ciency and reduce
CYP1A2-, CYP2C9-, CYP2C19-, CYP2D6- and                       costs. The presentation will discuss the applications
CYP3A4-mediated metabolism. Comparisons of IC50               selected, and the validation and system architecture
obtained with the ¯ uorogenic and conventional drug           being installed to achieve these goals.
probes in recombinant CYPs were similar for CYP1A2,
2C9, 2C19 and 2D6, but not for CYP3A4.
Additionally, using a single-point estimated IC50 ap-
proach, compounds that were shown to be inhibitors
using conventional drug probes with human liver micro-
                                                              Online SPE by column switching: another form of
somes were also classi® ed as inhibitors in recombinant
                                                              bioanalytical laboratory automation
CYPs using the ¯ uorescent probes. For the cocktail
approach, it was shown that the CYP-speci® c reactions        Min Chang, Abbott Labs, Abbott Park, IL, USA
were not altered in the presence of multiple probes as        Co-authors: Huong Mai, Anita Shen, Brendan Swaine, Qin Ji
indicated by no distinguishable eå ect on Km or Vmax . As     and Tawakol El-Shourbagy
expected from this result, the IC50 s generated in the
cocktail incubations were in good agreement to those          Time event-controlled column switching valves have
obtained from individual incubations.                         been available to analytical scientists since the mid-
                                                              1970s. Earlier uses of the column-switching valve in-
                                                              cluded removing late elution peaks from the HPLC
GigaMatrix TM         ultrahigh-throughpu t       screening   run, autosampler sharing, two-dimensional HPLC se-
platform                                                      paration (heart cut), inline ® lter/guard column regenera-
                                                              tion, online concentration, unattended column selection
Mike La¡erty, Diversa, San Diego, CA, USA                     and fractions’ collection. Although there is at least one
The myriad of microbes inhabiting this planet represent       column-switching valve in an HPLC system (the injector
a tremendous repository of biomolecules for pharmaceu-        valve), the idea of the addition of another valve has not
tical, agricultural, industrial and chemical applications.    been widely accepted. Analytical scientists have turned
Diversa Corporation has the unique capability of              away from the technology possibly due to the lack of
accessing this microbial diversity by taking a culture-       ruggedness of the ® rst-generation air-actuated valve and
independent, recombinant approach to the discovery            other additional pieces of HPLC equipment including
of novel proteins and small molecules. Diversa’ s discovery   columns.
programme uses genes and gene pathways captured from          Recently, with the availability of commercial online
nucleic acids extracted directly from the environment,        solid-phase extraction system(s), the column-switching
which are then constructed into complex, 109-member           technique has become an acceptable option for bioana-
environmental libraries. These libraries often contain        lytical sample preparation. At Abbott Laboratory, we
up to 5000 diå erent microbial genomes and, thus,             have developed several online solid-phase extraction
require high-throughpu t screening methods to cover their     HPLC methods using automated valves, an internal
diversity eå ectively.                                        reversed-phase guard cartridge and HPLC equipment
Diversa has developed GigaMatrixTM, a new ultrahigh-          by Shimadzu and Agilent. Internal standard forti® ed
throughput screening platform. GigaMatrix plates have         plasma were clari® ed by either centrifugation or ® ltered
> 100 000 bottomless wells in the same footprint as a         before the HPLC injection. These methods have been
microtitre plate. The platform is automated and capable       used successfully to analyse two Abbott compounds and
of screening 1 billion clones per day. Less equipment time    their metabolites. This application of automated column-
and manpower are required and assay costs are drama-          switching valves has provided an alternative to o¥ine
tically reduced as compared with traditional microtitre       solid-phase extraction and liquid± liquid extraction and
plate-based screening. The power of the GigaMatrix            made it possible to select the best technology to increase
platform to discover novel enzymes, small molecules,          assay throughput and sensitivity.

                                                                                                                     89
Abstracts of papers presented at the 2001 ISLAR


Automation and compliance in quality control                 Applications of the Zymark Staccato for in vitro
laboratories                                                 drug metabolism studies
Muhammad Albarakeh, Barr Laboratories, Inc., Pomona, NY,     Heather Sulkowski, Boehringer Ingelheim Pharmaceuticals, Inc.,
USA                                                          Ridge¢eld, CT, USA
Co-authors: Richard Ashley and Timothy Breuninger            Co-authors: J. Richard Mount¢eld, Donald Tweedie and Drane   è
                                                             O’Brien
Current laboratory demands related to the high through-
put of samples, the need to keep costs and expenses          Advances in genomics, combinatorial chemistry and
minimal, yet keep the release function ¯ owing to prevent    pharmacology screening have led to new challenges
product backorder, have necessitated the use of labora-      within drug metabolism due to large numbers of com-
tory automation and robotics systems. As a result of this    pounds requiring in vitro evaluation. To meet these
automation, huge amounts of samples and related data         demands within Boehringer Ingelheim Pharmaceuticals,
are processed and generated.                                 Inc., the Zymark Staccato was evaluated as a robotic tool
                                                             for conducting multiple in vitro assays. The criteria for an
Without a properly controlled and validated system, the      automated system included the following.
very tools implemented to process high amounts of
samples can bring your release function to a halt. Over-        . A 96-well head allowing for maximum sample
views on how to prevent cGMP logs jam and reach a                 throughput.
` zero backorder’ release of product will be presented.         . Thermal block to maintain constant temperature.
                                                                . Flexible deck layout.
                                                                . Open-access capability

Methods’ development for monoclonal antibody                 As part of the evaluation process, assays were compared
screening: how to think like a robot                         using both manual and automated methods. The results
                                                             are described in this presentation
Nanci E. Donacki, MedImmune, Inc., Gaithersburg, MD, USA
                                                             Automation of several key in vitro metabolism assays has
A single fusion for the development of monoclonal            been achieved. The results indicate that the automated
antibodies will often generated 20 or more microplates       assays allow for a higher throughput while maintaining a
that need to be screened. ELISA (enzyme-linked im-           high degree of precision. The work will be extended to
munosorbent assay) is the most common method for             additional in vitro assays, and the strategy of comparing
screening newly developed monoclonal antibodies for          manual versus automated processes will be continued as
antibody production and speci® city. The method,             part of the validation procedures.
although speci® c for the antibodies, is highly repetitive
for each step and can easily be automated. However, the
way the assay is performed at the benchtop is not always
the best way for an automated system to run the assay.       Analytical method validation: an automated soft-
Tips and techniques for transferring an assay from bench-    ware approach
top to automation will be presented.
                                                             Patricia A. Fowler, Waters Corporation, Milford, MA, USA
                                                             Co-author: Michael Swartz
                                                             Method validation is a tedious process performed to
Lean manufacturing and six sigma: an evaluation              determine if an analytical method meets the require-
of the impact of these concepts on laboratory                ments for its intended purpose. In the regulated labora-
automation                                                   tory, method validation may take many days to perform
                                                             the necessary analytical tests. Data reduction and the
Nigel North, Pharmaceutical Development, GlaxoSmithKline,    statistical analysis of results performed can be a very
Ware, UK                                                     time-consuming process. There is also a greater poss-
                                                             ibility of introducing error when calculations are per-
The concepts of lean manufacturing and six sigma are
                                                             formed manually. With the use of automated software to
now beginning to be applied to pharmaceutical manu-          perform these calculations, method validation is much
facturing processes. Lean manufacturing has been suc-
                                                             faster and easier, with less chance for error.
cessfully applied for many years in the automotive and
aerospace industries with the key goal of reducing waste.    In this poster, we will show that an analytical method
Six sigma is a more recent concept involving reducing        was validated using automated software. Chromato-
variation in manufacturing processes which has been          graphic results were directly accessed from a relational
implemented in the semiconductor industry resulting in       database bypassing manual intervention. Statistical cal-
signi® cant cost savings. The combination of lean manu-      culations were performed automaticall y and a report
facturing and six sigma provides a powerful combination      generated showing the results of the analyses from the
of principles to deliver robust manufacturing processes      Student, Cochran, Dixon and Fisher tests. Graphs were
with the elimination of non-value-adde d activities. The     generated representing the results of the statistical analy-
eå ect of these concepts on how we approach automation       sis. In addition, we will show that the data reduction and
in the laboratory will be examined together with provid-     statistical calculations necessary to validate the method,
ing some perspectives on new technology that will be         complete with the necessary documentation and report
required to meet these challenges.                           generation, are completed in signi® cantly less time.

90
Abstracts of papers presented at the 2001 ISLAR


Streamlining the dissolution test: automated                   Development of a lead identi cation platform for
HPLC techniques                                                kinase drug discovery
Patricia A. Fowler, Waters Corporation, Milford, MA, USA       Paul Gallant, Millennium Pharmaceuticals, Inc., Cambridge,
Co-authors: Kelly A. Johnson, Michael E. Swartz and Charles    MA, USA
H. Frasier                                                     The goal of any drug-discovery process is to eliminate
                                                               quickly ` poor’ leads while rapidly advancing those with
The fast pace of the pharmaceutical industry requires          the highest potential. Millennium has used a combina-
laboratories to reduce the analytical burden of their          tion of novel and mature technologies to establish a
test procedures and increase productivity while still          system to progress novel kinase targets rapidly through
satisfying regulatory compliance. There are several ways -     assay development, high-throughpu t screening, hit vali-
to meet these challenges in the dissolution laboratory.        dation and hit characterization. By selecting focused
By automating the dissolution process, pharmaceutical          technologies, automation, secondary screens and IT, an
laboratories eliminate the slight variations that may          integrated process has been constructed which is capable
occur in manual methods, insuring reproducible                 of handling multiple targets quickly while identifying the
data, higher throughput and cost reduction. Vali-              compounds with the highest potential for lead optimiza-
dated single-source software control of the entire             tion. This presentation will discuss the platforms chosen
system, as well as dissolution data acquisition, calcu-        for assay development, high-throughpu t screening, hit
lations and reporting, can further streamline the work         con® rmation and potency/selectivity determinations. A
¯ ow while maintaining FDA compliance with 21 CFR              process will be described to show how using a standard
 Part 11.                                                      well-de® ned system can eå ectively move multiple kinase
Automated online HPLC dissolution systems can                  targets rapidly into the ` Lead Optimization’ stage.
pool dissolution samples signi® cantly to save time.
Similarly, automated sampling at shorter intervals             Optimization and automation of bioanalytical
and analysis of a large number of samples by online            methods
HPLC may provide a more complete solution for
the decision-making process in the early stages of             Peter D. Bryan, Forest Laboratories, Abbott Park, IL, USA
drug development. In addition, these systems must be           Co-authors: Min S. Chang and Anita Shen
capable of handling increasingly complex formulations          Now that LC-MS has become the technique of choice in
such as multiple actives and widely varying dosage levels,     the GLP bioanalytical laboratory, the work¯ ow bottle-
as well as diå erent media types such as buå ers and           neck has shifted from the separation± detection to the
surfactants.                                                   sample-processing portion of the bioanalytical method.
                                                               To address the sample-processing bottleneck, eå orts have
Our poster shows applications used to help reduce the
                                                               been concentrated on the conversion and validation of
analytical burden and increase the sample throughput
                                                               existing liquid± liquid extraction (LLE) to solid-phase
and productivity in the laboratory.
                                                               extraction (SPE) methods. Profound increases in sample
                                                               throughput have been achieved using LC-MS with
                                                               automated SPE in the 96-well format over manual
                                                               LLE extraction HPLC methods. Validation of auto-
                                                               mated 96-well format SPE methods have shown their
                                                               equivalence to manual LLE extraction. Automated 96-
Homogeneous high-throughput SNP assay direct
                                                               well format SPE is also much less tedious and allows the
from genomic DNA
                                                               analyst to concentrate more on data analysis and com-
Patrick B. Cahill, Genome Therapeutics Corporation, Waltham,   pliance issues. For routine analysis, transfer from clinical
MA, USA                                                        tubes to the 96-well format has been accomplished using
Co-authors: Dina Weymouth, Cori Gustafson, James Hurley,       either the Biomek 2000 or the Hamilton MicroLab AT.
Michele Bakis, Veena Kamath, Doug Smith and Lynn Doucette-     Automated SPE is then performed on the Biomek 2000.
Stamm                                                          Additionally, semi-automated method development for
                                                               96-well format SPE has been automated using the
Genome Therapeutics Corp has developed a                       Biomek 2000.
high-throughpu t SNP assay based on the ability of
some DNA polymerases to proofread the sequence
as they extend. This Exo-Proofreadin g SNP assay               How small is enough?
can be accomplished, from sample addition to detection,
in one tube. In addition, this SNP assay has the               Peter Grandsard, Amgen, Inc., Thousand Oaks, CA, USA
capability to be performed directly on genomic                 Co-authors: Jim Petersen, Brian Rasnow, Mike Johnson, Chuck
DNA. Results will be presented for this assay on multiple      Li, Les Walling and Doug Overland
SNPs screened on large populations. The results will           A signi® cant technical and organizational challenge
be compared with ASO and RFLP data with dis-                   facing the biopharmaceutica l industry is the incorpora-
crepancies resolved by sequencing. This assay provides         tion of miniaturization technologies into their business.
a new, highly robust and fast method for large-scale SNP       The successful application of these technologies requires
screening required for human disease and pharmacoge-           answers to the following questions. What is this new
netic studies.                                                 miniaturization technology? Which technologies should

                                                                                                                           91
Abstracts of papers presented at the 2001 ISLAR


be applied to which work¯ ows? Why? When should they               plied, thus increasing sample throughput and improving
be implemented? Should a technology be implemented                 the quality of decisions impacting compound selection
early, through some sort of technology access programme            and evaluation.
(TAP), or should one wait until it becomes commercially
                                                                   This presentation will describe sample-handling method-
available? Our views on these matters will be presented.
                                                                   ologies and automated instrumentation developed to
In collaboration with other Amgen R&D groups, the                  conduct solubility measurements as a function of pH.
Research & Automation Technologies department has                  The use of these methodologies and instrumentation has
been actively cultivating critical collaborators with              improved both throughput and eæ ciency while main-
knowledge of submicrolitre liquid-handling, microma-               taining the accuracy that can be achieved with manual
chining and detection technologies. For example, our               methods. The accuracy and precision of the system will
TAP with Caliper, Inc., has increased our institutional            be illustrated using data on model compounds as well as
understanding of miniaturization applications in the               drug candidates. Finally, we will describe the strategy
drug-discovery process ¯ ow, most particularly in small            and process used to prepare samples for stability deter-
molecule screening and DNA analysis.                               minations at user-de® ned pHs, and how this process is
                                                                   done in concert with the solubility determinations.
Miniaturization at Amgen has invoked a cascade of new
projects or at least studies. The goals of these projects are
to ® nd solutions for interfacing any miniaturized plat-
                                                                   Development of novel micro-arra y technology for
form to the ` macro-world’ , such that the advantages of
                                                                   cell-based assays
miniaturization are not undone by integration or certain
interfaces. Operations that need to undergo changes to             Quiyang Zhang, Cytoplex Biosciences, Plano, TX, USA
bene® t fully from miniaturization include the storage and         Co-author: Alex Freeman
retrieval of small molecules, detection and compound
                                                                   Cell-based assays are increasingly used for high-through -
handling during screening, and information manage-
                                                                   put screening in drug-discovery programmes because
ment. We will discuss some of our ® ndings and solutions.
                                                                   of their high information content. For the implementa-
                                                                   tion of miniaturized cell assay, micro-arraying is an
                                                                   essential part of the technology. Initially, we had per-
Automation         and     tracking       of      combinatoria l
                                                                   formed cell-adhesion studies and evaluated ® ve diå erent
libraries
                                                                   substratesÐ polystyrene, polycarbonate , silicon, glass and
Phil Small, Tripos Receptor Research, Bude, UK                     PDMSÐ and found that with polylysine coating, the
                                                                   glass, silicon and PDMS substrates served well for cell
A process-integratin g design, synthesis and analysis of
                                                                   attachment. However, the conventional well-less spotting
combinatorial libraries has been implemented at Tripos
                                                                   and assaying method was diæ cult to implement in
Receptor Research. Crucial to the success of this process
                                                                   producing a predetermined array pattern of cells.
has been the development of a proprietary informatics
system. This has so far been developed to manage reagent           To overcome the above diæ culty, we further developed
inventory, track samples, and record data from synthesis           both bottomed and bottomless well arrays to address the
and analysis to provide a valuable database of compound            need for cell culturing and assays. PDMS and glass were
information.                                                       used as the bottom surfaces for both bottomless well
                                                                   silicon substrates and bottomed wells. Novel liquid distri-
The combination of Tripos’ s proprietary design software
                                                                   bution arrays were fabricated in silicon to facilitate initial
with automated synthesis and informatics leads to the
                                                                   coating and liquid exchange from the entire array. The
production of drug-like libraries with well-de® ned purity
                                                                   arrays were implemented for culture of adherent and
and full synthetic history.
                                                                   non-adherent cells, immunochemical assays, and auto-
This presentation will cover what were considered the              matic liquid transfer all in high-density format. The
important aspects involved in setting up and managing              current array platform will be also useful in cell library
an automated chemistry facility.                                   construction, combinatorial library synthesis, and con-
                                                                   tinuous homogeneous and heterogeneous assays.

Automating pH solubility and stability determina-
tions conducted in support of early development                    Adaptive powder-dispensin g system
Phil Waters, GlaxoSmithKline, Research Triangle Park, NC,          Rajesh K. Maheshwari, Schering-Plough Research Institute,
USA                                                                Union, NJ, USA
Co-authors: James Ormand, David Igo and Pingyun Chen               Co-authors: Annaniy Berenshteyn, Gary Kowalski and Joseph
                                                                   Norgard
Evaluation of the equilibrium solubility and chemical
stability of compounds as a function of pH aids our                This poster is about a robotic powder dispensing system
understanding of the bioavailability of a drug candidate.          we have developed. It can dispense on average
Additionally, these data lead to rapid development of              10 mg § 5% from any of the 288 dispensers on its
formulations for use in preclinical and clinical studies.          carousel, into a sealed 24/96-well deep-well microtitre
Conducting these measurements often requires a signi® -            plate. The powders are kept in a sealed environment and
cant amount of human labour and time. Reducing the                 are diå erentiated for dispensing purposes by their
labour and time burden is expected to increase the                 dispensing parameters, which are stored by in a
frequency with which these measurements can be ap-                 Microsoft Access database . The databas e entry is pointed

92
Abstracts of papers presented at the 2001 ISLAR


to by a barcode label on the dispenserÐ           hence, adaptive   Comparison and contrast of di´ erent dissolution -
dispensing.                                                         sampling systems using the Agilent 8453 UV-VIS
                                                                    in a QC laboratory
The user interface for this multiprocessor system consists
of 250 000 lines of Multithreaded/Multitasking Visual               Reem Malki, Andrx Pharmaceutical, Fort Lauderdale, FL, USA
C‡‡ code. An Access database holds dispensing                       Co-author: Michael K. Michaels
parameters’ information for each powder. A Dynamic                  An integral part of an eæ cient QC laboratory is thorough
Query Screen has been designed to make it easy for a                planning. With a new facility about to open and many
chemist to view the database selectively.
                                                                    diverse projects in the pipeline, the proper approach to a
Such a system can ® nd other uses such as distributing              successful implementation is to think, research, plan,
compounds in the compound distribution centre, dispen-              budget, set up, test, comply and train.
sing lyophalized microorganisms, dispensing resins for              We knew as a laboratory that we could not function with
combinatorial chemistry, and any other application                  only one type of dissolution system. Our research led us to
when powder is to be delivered precisely in a controlled            two automated options. The ® rst, a peristaltic pump
environment.                                                        sampling system using a single-¯ ow cell approach, oå ers
                                                                    the detection of the sample. The second, a syringe pump
                                                                    sampling system using a multi¯ ow cell approach, pro-
                                                                    vided us the options to detect, detect and collect, and
Automated system for compound library screen-                       collect and dilute the sample. Each of these dissolution
ing by MTS cell-viabilit y assays                                   set-ups is paired with an HP8453 UV-VIS while remain-
                                                                    ing 21 CFR Part 11 compliant.
Randall Engler, Kendro Lab Products, Newtown, CT, USA
Co-authors: C. Elliot and R. Moody
                                                                    Cross-functiona l team approach to implementin g
Cell proliferation, cytotoxicity and other viability assays
                                                                    and managing laboratory automation in the qual-
play an important role the drug-discovery process. Many
                                                                    ity control laboratory
screening programmes target anticancer compounds,
requiring in-vitro characterizatio n of eæ cacy. Addition-          Timothy Reilly, Novartis Pharmaceutical Corporation, Su¡ern,
ally, all potential therapeutic compounds must be char-             NY, USA
acterized for their cytotoxicity regardless of the target
                                                                    Poorly de® ned project goals and an inadequate infra-
(ADME/Tox).
                                                                    structure have hindered previous attempts to initiate
Cytokinetics has developed a system to automate the                 automation projects in the Novartis Quality Control
process of reagent addition, incubation and reading                 laboratory. This haphazard approach has been replaced
of cell viability assays using the MTS method of                    with well-de® ned project expectations and agreement
Promega, Inc. The integrated system includes hardware               among the functional groups that make up the project
from a variety of sources and software developed by                 infrastructure to support aggressively the automation
Cytokinetics.                                                       project goals.
Control of environmental conditions including CO2 ,
temperature and humidity are important factors in                   New concept for automation of dissolution tests
accurate and reproducible cell-based assays. The system             for biowaiver studies
described here includes the Heraeus1 Cytomat 1 6000
series incubator from Kendro for the incubation of cells.           Rolf Rolli, Sotax Ltd, Allschwil, Switzerland
                                                                    New FDA regulations allow biowaivers for Class I drugs
                                                                    based on solubility, permeability and dissolution testing.
                                                                    BCS-based biowaivers can be requested for signi® cant
Automation development for high-throughpu t                         post-approva l changes (e.g. Level 3 changes in com-
phage library screening                                             ponents and compositions) to a rapidly dissolving im-
                                                                    mediate release product containing a highly soluble,
Randy Yen, Genentech, Inc., San Francisco, CA, USA                  highly permeable drug substance, provided that dissolu-
Co-authors: Sherry Yeh and Suki Hyare                               tion remains rapid for the post-change product and both
                                                                    pre-/post-change products exhibit similar dissolution
Phage display technology is a powerful tool in genomic
                                                                    pro® les. Dissolution tests have to be performed at pHs
and drug development. It allows scientists to screen
                                                                    1, 4.5 and 6.8. With this regulation, automation of
quickly billions of peptides, antibodies and cellular
                                                                    dissolution testing gains further importance with respect
proteins for binding to a target. It is a vital tool in studies
                                                                    to pH changes.
aimed at identifying molecules that bind to a speci® c
target and at improving particular features of pre-exist-           A fully automated test system with the necessary software
ing molecules. At Genentech, we automated the binding               is described. This solution oå ers one the capability to run
assays for the speci® c target to screening the phage               up to 15 USP 2 tests in series. With such a system, all
library. By running fully automated 384-well assay on               steps are fully automated, from medium preparation, to
the robotic system, we screened thousands of clones in a            tablet input and up to the printout of the reports. The
multiple target in 1 day and ® nished each library in a             system includes a very eæ cient cleaning device that
short time.                                                         prevents any carry-over between tests. Tests requiring

                                                                                                                             93
Abstracts of papers presented at the 2001 ISLAR


baskets are handled with the Basket-Station . This system      The set-up of the DNA sequencing reaction is the next
allows up to 10 USP 1 tests to be analysed. With this          process that has been selected for improvement. Cur-
automated dissolution concept, biowaiver studies are           rently, requests for sequencing from the Therapeutic
executed rapidly allowing for accelerated drug develop-        Area laboratories are (1) submitted online, (2) the
ment and SUPAC changes.                                        sample information is transferred to the 3700 plate record
                                                               and (3) the addition of DNA and primers to the thermal
                                                               cycle plate is performed manually.
Integration of reagent management and computa-
tionally biased combinatoria l synthesis                       Manual chemistry requires up to 4 h of hands-on time per
                                                               day depending on the number of samples. While a liquid
Scott M. Harris, DuPont Pharmaceuticals Research Labora-       handler working oå a ® le can perform the hit-picking
tories, San Diego, CA, USA                                     required for core terminator chemistry, the set-up of
One of the key aspects to successful high-throughpu t drug     the source racks is time-consuming and a possible source
discovery is the ability to integrate several functions.       of errors. The poster will present the use of Web
These include medicinal chemistry, computationa l de-          submission, individually bar-coded sample tubes and a
sign, high-throughpu t synthesis and puri® cation. A po-       liquid handler for automating chemistry set-up in a core
tential bottleneck in this process is a lack of established    DNA sequencing facility.
chemistries linked with readily available commercial and
proprietary building blocks. Early in the process, it is
critical that a protocol exists that allows for planning and   Micro-arra y process industrializatio n
synthesizing libraries, developing initial structure± activ-
ity relationships and validating screening leads.              Shane Weber, Millennium Pharmaceuticals, Cambridge, MA,
                                                               USA
It is also important to have access to novel building
blocks and reagents for preparing targeted or focused          Millennium’ s nylon cDNA micro-array platform is suc-
libraries. DuPont Pharmaceuticals has developed a              cessfully ® lling our drug- and marker-discovery plat-
simple custom Web-based monomer request system that            forms. This platform is eå ective because of its
uses a JAVA GUI that allows for tracking of orders and         sensitivity, gene content, throughput eæ ciency and cost.
report generation. The data are housed in a Reagent            The platform’ s eå ectiveness results from the industria-
Inventory Tracking System using Oracle and the physi-          lized process ¯ ow of liquid handling, printing and
cal reagents are stored in a controlled ventilated cabinet     information tracking. Eå ectiveness is constantly assessed
system. The typical throughput and average turn around         by measurements of quality and eæ ciency. The process
time will be discussed. The overall bene® ts of this           ¯ ow and quality assessment will be reviewed.
programme are lower costs, better inventory control
and an increase in eæ ciency in library production and
chemistry.                                                     Mass-directe d      puri cation       of    combichem
                                                               libraries
Automation of chemistry in a core DNA-sequen-                  William R. Hall, GlaxoSmithKline, Research Triangle Park,
cing facility                                                  NC, USA
Shawn Hallowell, P¢zer Global R&D, Groton, CT, USA             Co-authors: Melissa Lindsay and Dean Phelps
Co-authors: Suzanne P. Williams, Yevette C. Clancy, Melissa    The Library Puri® cation Group at Research Triangle
T. Cronan, Shawn E. Hallowell, Michael Polchanino¡, Lance      Park is responsible for puri® cation of both large and
Ryley and Janice L. Palmer                                     small libraries from discovery and targeted research.
Conversion of our core DNA sequencing laboratory to            We use mass-directed preparative HPLC to purify
the 3700 DNA analyser (AB) and sequencer for BioLIMS           library samples with masses of 5± 100 mg per well. Our
sequence analysis and databas e software (AB, genecodes)       system incorporates at-column dilution, separate waste
has greatly reduced the amount of manual intervention          collection for each sample and UV veri® cation of peak
required for generating, analysing and maintaining DNA         collection. Eæ cient evaporation of solvents, robotic li-
sequence data.                                                 quid handling and reformatting, and fast QC on puri® ed
                                                               samples allows for high-throughpu t of puri® ed libraries.




94
Abstracts of papers presented at the 2001 ISLAR


Establishment and management of the Bristol-                   HCMV exonuclease in 50 mm HEPES, pH 7.6, which
Myers Squibb Automated Screening Core                          produced a 50% reduction in mP in 8.3 min. We
                                                               screened 117 843 compounds at 10 mm (2.5% DMSO).
Steven F. Innaimo, Bristol Myers Squibb PRI, Wallingford,      The screen identi® ed 647 compounds that inhibited the
CT, USA                                                        HCMV exonuclease3 30%.
As part of the evolution and industrialization of the BMS
Lead Discovery organization, a core group of specialized
scientists focused solely on automated screening was           High-throughpu t protein function screening for
assembled. The mission of the Automated Screening Core         immune system target validation
is to adapt and prosecute high-throughpu t screen cam-         Stewart D. Chipman, Immunex, Corporation, Seattle, WA, USA
paigns on fully automated robotic screening systems.
                                                               Validation of the biochemical function of a novel gene/
This talk will cover the implementation of the BMS             protein in the pathophysiolog y of a disease is the rate-
automated screening infrastructure, the rationale for          limiting step in novel target validation for drug discov-
the establishment of the Automated Screening Core              ery. Many groups have applied the technology and
and the organizationa l impact of such a group.                methodology developed for small molecule high-through-
                                                               put screening and clinical testing to assays that test for
                                                               novel protein function. This presentation will address
Determining feasibility and parameter values for               how we have integrated gene discovery and expression
compound orders based on transfer amounts,                     analysis, protein production and inventory capability,
liquid-handler capabilitie s and container-volum e             cell-based protein function assays, laboratory automation
capacities                                                     tools and data management systems into an integrated
Steven Ho¡man, Bristol-Myers Squibb PRI, Princeton, NJ,        target discovery and validation platform for immune
USA                                                            system targets. Topics to be discussed are automated
Co-author: Mark F. Russo                                       and multiplexed cytokine detection, cognate ligand
                                                               detection, calcium mobilization, high-throughpu t optical
An algorithm is described for determining the feasibility      imaging, large-scale cell preparation, large-scale protein
and calculating parameter values for one or more               expression and puri® cation, workstation style laboratory
compound processing orders. Feasibility is determined          automation and automated data handling.
primarily by applying a series of inequality constraints
that result in a valid target concentration range within
which orders can be processed. These inequality con-           Laboratory automation: a cost-e´ ective solution
straints are obtained from initial compound amounts,           to increasing laboratory workloads
source and destination container-volum e capacities, the
liquid-handler transfer volume range and the amount of         Stephen Green, Forest Laboratories Ireland Ltd, Dublin, Ireland
compound to be processed. If feasible, calculated par-         Forest Laboratories, Inc., markets Celexa, an SSRI
ameter values include the dilution, transfer and top-oå        antidepressant, in the USA. The bulk tablets are pro-
volumes necessary to ful® l the order. Sample code             duced at the manufacturing site in Dublin, Ireland.
implementing the algorithm is given in Microsoft Visual        Market projections in 1999 indicated a 250% increase
Basic 1.                                                       in volumes to be produced at the Dublin site. Several
                                                               laboratory strategies to cope with this increased produc-
                                                               tion were assessed and the use of automated robotic
High-throughput screen to detect inhibitors of the             systems was shown to be the most cost-eå ective solution.
3 0 -5 0 -HCMV exonuclease
                                                               The introduction of the automation into the laboratory
Steven J. Conrad, Pharmacia Corporation, Kalamazoo, MI,        had several phases. Initially, there were physical changes
USA                                                            required to the laboratory layout to accommodate the
Co-authors: Thomas W. Pitts, Nancee Oien, Robert A. Anstadt,   robots. Second, there was the quali® cation of the robotic
Roger A. Poorman, Peter A. Wells, Michael W. Wathen and        systems and the training of the initial users. Finally, there
Yoshihiko Yagi                                                 was the development and validation of several analytical
We carried out a ¯ uorescence polarization-base d (FP)         methods, which was performed in conjunction with the
high-throughpu t screen (HTS) to detect inhibitors of the      Zymark MTOV group.
3 0 -5 0 -exonuclease activity of the human cytomegalovirus
DNA polymerase (the HCMV exonuclease). We used a
                                                               Characterizatio n and e´ ective use of the molecu-
23-mer ssDNA substrate labelled with TAMRA at the 3 0
                                                               lar properties of reaction vessel surfaces in
end. The emitted light from TAMRA in an intact
                                                               high-throughpu t assay development
substrate molecule was highly polarized (about
240 mP), but, upon substrate digestion with the HCMV           Sven Erik Rasmussen, Nalge Nunc International, Naperville, IL,
exonuclease, the emission from TAMRA free in solution          USA
exhibited a lower polarization (about 40 mP).                  Co-author: Nalge Nunc
The assay was designed to run in a 384-well format with        An understanding of the molecular surface of a reaction
a ® nal volume of 50 ml. The Km of the HCMV exonu-             vessel is critical to achieving the sensitivity and speci® city
clease for the substrate was determined to be 20 nm. In        for high-throughpu t or high-content molecular and cell-
the assay, 50 nm substrate was digested by 0:01 £ stock        based assays. We developed and characterized several

                                                                                                                           95
Abstracts of papers presented at the 2001 ISLAR


molecular surfaces for reaction vessels that can be eå ec-       strates. Data from oligonucleotide arrays as well as cDNA
tively used in genomic and proteomic assays. These               arrays will be presented.
surfaces, in conjunction with the physical properties of
the substrate material (optical or thermal properties, for
example) along with format (MicroWell, slide, or chip),          Flexibility paradigm in laboratory automation
are compared with regard to assay performance.                   management
Through XPS analysis (also known as ESCA), the mol-              T. C. Ramaraj, Roche Discovery Technologies, Ho¡mann-La
ecular and atomic compositions of various MicroWell              Roche, Nutley, NJ, USA
surfaces have been de® ned. These data will be used to           Considerable developments and rapid changes have
demonstrate the speci® c utilization of the surface in           taken place in high-throughpu t screening and compound
molecular and cell-based assays. For example, a hydro-           management in the last 5 years. This is a result of a
philic, highly charged surface, MultiSorp, preferentially        concerted eå ort between the pharmaceutical industry,
binds complex phospholipid molecules while excluding             laboratory automation companies, instrument companies
other molecules, including glycoproteins such as IgG.            and manufacturer s of consumable laboratory ware. The
Another ` high-binding’ hydrophilic surface, MaxiSorp,           evolution of new automation technologies has fuelled the
binds a signi® cant amount of glycoproteins such as              growth and prospects for various modes of automation in
immunoglobins. These surfaces and others such as                 virtually all phases of drug-discovery research. Here, we
the Nunclon Delta cell culture surface and non-treated           will try to highlight our experiences in managing a
sterile polystyrene surfaces are molecularly de® ned by          complex HTS and compound management infrastruc-
ESCA techniques accompanied by speci® c procedural               ture at Hoå mann-La Roche.
examples.
                                                                 The infrastructure consists of automation platforms that
Well geometry, round, square, columnar or ` shallow’ ,           have evolved over several years. All require proper care
in¯ uences assay performance, particularly when an ac-           and maintenance for sustained performance. Some re-
tive surface is used. Additional parameters come into            cently installed con® gurations require continued valida-
eå ect when well volumes are miniaturized as in a 1536-          tion to achieve expected levels of performance and
well plate. The eå ect of a molecular surface changes            reliability to match ever-increasing throughput require-
again as the assay is further miniaturized to a two-             ments. Promising new automated screening technologies
dimensional format as in a slide or chip and the in¯ uence       require careful evaluation. Validation of these new tech-
of the substrate material may increase.                          nologies also require allocation of signi® cant resources for
Also important to the development of a speci® c, sensitive       meeting demanding requirements of miniaturization,
and eæ cient assay are the physical characteristics of           speed, ¯ exibility, cost eæ ciency and increased perform-
various substrate materials. With regard to optical prop-        ance levels. It is no easy task to steer winning method-
erties, the ¯ uorescent and re¯ ective properties of solid       ologies to their fullest potential and at the same time take
black and solid white as well as black or white optical          a risk in exploring unproven avenues and technologies.
bottom plates (OBP) were compared by measuring                   To maintain a balanced perspective among available
signal-to-noise ratios, light cross talk and sensitivity. Data   choices requires ¯ exibility in approach, quick implemen-
and practical applications will be discussed.                    tation strategies, a diverse pool of expertise and talent
                                                                 that can work as a team, contribute as individuals, share
                                                                 the successes as well as failures, learn from the mistakes,
                                                                 revise the expectations and still meet corporate goals and
New polymer substrate for DNA micro-array s                      objectives every year. The presentation will also include
Svend Erik Rasmussen, Nalge Nunc International, Naperville,      examples from some of our most recent integration
IL, USA                                                          experiences, the upgrade of existing systems, the imple-
Co-authors: T. Kristensen, K. HolmstrÖm and L. Pedersen          mentation of new detection modes in existing instruments
                                                                 and our experience with latest 384-well pipetting systems.
The quality and surface properties of the micro-array
substrates are fundamental factors for a successful DNA
micro-array system. At present, coated glass-slides              Variety of automation tools for drug metabolism
(amine-, alehyde- or epoxy-coated) are the dominating
substrates on the market. However, an often-encountered          Thomas Lloyd, DuPont Pharmaceutical Co., Newark, DE, USA
problem using these substrates is a non-homogenous               Automation continues to be developed for new diversi® ed
surface, which can lead to problems with spot uniformity         applications within drug metabolism. The choice of an
and morphology. Nunc A/S has developed a pre-acti-               automated tool can vary considerably depending on the
vated, but uncoated, NucleoLinkTM polymer MicroAr-               speci® c requirements of the application. An overview of
ray Slide that can be used, without any further                  the automated tools that have been introduced at
modi® cation, for creating DNA micro-arrays. The Nu-             DuPont over the last 5 years will be presented along
cleoLinkTM polymer MicroArray Slide can be used for              with speci® c application examples. An evaluation is
both covalent attachment of DNA as well as immobiliza-           oå ered for how the diå erent tools have proliferated
tion of DNA by electrostatic forces and/or ionic bonds.          throughout this function.
Studies of auto¯ uorescence properties, DNA-binding ca-          Automation application areas include bioanalytical
pacity and DNA-hybridization eæ ciency have been                 sample preparation techniques including solid-phase
performed and compared with conventional glass sub-              extraction, liquid± liquid extraction, protein precipitation

96
Abstracts of papers presented at the 2001 ISLAR


and online approaches for in vivo samples, in vitro sample        data points to the desirability of more frequent sampling
preparation processes, sample handling, data handling             points to validate a dissolution pro® le. As a result, these
and training. Application areas include many functions            developments have signi® cantly increased the require-
within drug metabolism ranging from discovery through             ments of equipment, personnel, data processing and
clinical trials.                                                  validation, which translates into increased production
                                                                  time for oral dosage forms. Thus, automation in dissolu-
                                                                  tion testing has become a necessity in order to handle
Characterizatio n and e´ ective use of the molecu-                these demands.
lar properties of reaction vessel surfaces to
achieve optimal assay performanc e                                Automation in dissolution testing is not new to the
                                                                  pharmaceutical industry. Simultaneous to the develop-
Tom Cummins, Nalge Nunc International, Rochester, NY, USA         ment in pharmaceutical technology over the past decade,
Co-authors: Janne N. Knudsen, Lena B. Larsen, Svend Erik          there have been advances in automated dissolution
Rasmussen, Tom Cummins and Joseph Granchelli                      testing, although at a slower pace. The virtual explosion
An understanding of the molecular surface of a reaction           in the diverse and yet speci® c requirements associated
vessel is critical to achieving the sensitivity and speci® city   with a plethora of dosage form types and their respective
for high-throughpu t or high-content molecular- and cell-         functions have curtailed the outright all-encompassing
based assays. We developed and characterized several              development in automated dissolution testing. While the
molecular surfaces for reaction vessels that can be used          advantages of automated dissolution testing are unequi-
eå ectively in genomic and proteomic assays. These                vocal, the limitations of automated dissolution testing
surfaces, in conjunction with the physical properties of          systems cannot be refuted. There is a constant quest to
the substrate material (optical or thermal properties, for        improve an existing automated system that meets speci® c
example) along with the format, are compared with                 requirements, either a unit function of the test or to
regard to assay performance.                                      accomplish a speci® c expectation of a dosage form, thus
                                                                  limiting the universality of the automated system.
Through ESCA analysis, the molecular and atomic
compositions of various MicroWell1 surfaces have been             Robert Kennedy once said, ` Some people watch things
de® ned. These data will be used to demonstrate the               happen and ask the question, why; and there are those
speci® c use of the surface in molecular and cell-based           that dream of things and ask the question, why not!’ It is
assays. For example, a hydrophilic, highly charged sur-           beyond doubt that an automated dissolution testing
face preferentially binds complex phospholipid molecules          system that addresses the current limitations of the
while excluding other molecules, including glycoproteins          existing ones and yet is ¯ exible and universal enough is
such as IgG. Another ` high-binding’ hydrophilic surface,         the need of the day. Going beyond the minimum
MaxiSorp TM, binds a signi® cant amount of glycoproteins          expectations of such an automated dissolution test into
such as immunoglobins.                                            its utility during early drug development and above all in
Well geometry, round, square, columnar or ` shallow’ ,            simultaneous prediction of bioavailability, can be a
in¯ uences assay performance, particularly when an ac-            dream worth striving for. A vision for such an automated
tive surface is used. Additional parameters come into             dissolution test system will be presented.
eå ect when well volumes are miniaturized as in a 1536-
well plate.
Also important to the development of a speci® c, sensitive
and eæ cient assay are the physical characteristics of
various substrate materials. With regard to optical prop-         Development of a radiofrequency identi cation
erties, the ¯ uorescent and re¯ ective properties of solid        (RFID) data carrier interface with an analytical
black and solid white as well as black or white optical           balance
bottom plates (OBP) were compared by measuring                    Je¡rey Veitch, GlaxoSmithKline, Ware, UK
signal-to-nois e ratios, light cross-talk and sensitivity.        Co-authors: Tony Allcock and James Ormand
Data and practical applications will be discussed.
                                                                  The use of process instrumentation such as analytical
                                                                  balances within the laboratory is commonplace and the
Intelligent automated dissolution                 system!    A    need for capturing related process measurement data are
challenge worth striving for                                      often a necessity. Quite often these data need to be
Umesh V. Banakar, Pharm-Assist International, Carmel, IN,         exported to databases and invariably to laboratory
USA                                                               information management systems (LIMS). Laboratories
Co-author: Manoj Bagool                                           regularly use process equipment such as balances, pH
                                                                  meters, etc. that are not attached to a dedicated PC. At
Dissolution testing requirements have been extended to            GlaxoSmithKline, we have developed a way of capturing
all oral dosage forms over the past decade. Additionally,         process data (e.g. weight values) directly to radiofre-
the dissolution regulations for pharmaceutical formula-           quency identi® cation (RFID) smart labels, where these
tions have been tightened world-wide. Furthermore, the            labels are used as data carriers.
past decade has also witnessed an increase in popularity
of modi® ed release pharmaceutical s that inherently re-          This evaluation has proven to be a success and is
quire extended dissolution protocols. The increased               potentially the stepping stone towards a more speci® c
emphasis on correlating dissolution and bioavailability           use of RFID within GlaxoSmithKline.

                                                                                                                           97
Abstracts of papers presented at the 2001 ISLAR


System integration for genomics using biorobot                 Dispensing precision for the SciCloneTM auto-
workstations                                                   mated liquid-handlin g workstation for 96-channel
                                                               pipetting
Achim Wehren, QIAGEN Hilden, Germany, and Valencia, CA,
USA                                                            Rudy Willebrords, Janssen Research Foundation, Beerse,
Co-authors: Fred Siegmanand Carola Schade                      Belgium
                                                               One of the ® rst steps in drug discovery involves identi® ca-
Automated robotic workstations are frequently used as          tion of novel compounds that interfere with therapeuti-
stand-alone systems for sample puri® cation, reaction set-     cally relevant biological processes. Identi® cation of ` lead’
up or sample re-array in the ® eld of genomics. However,       compounds in all therapeutic areas included in a drug-
a variety of external instruments often must be integrated     discovery programme requires labour-intensive evalua-
to provide full automation of several sequential tasks or to   tion of numerous samples in a battery of therapy-targete d
increase the range of applications that can be performed.      biological assays. To accelerate the identi® cation of lead
QIAGEN1 Instruments provides complete solutions                compounds, JRF has developed an automated high-
to automate molecular biology and liquid-handling              throughput screening (HTS) based on the unattended
applications by integrating BioRobot workstations              operation of a custom Zymark-tracked robot system.
with a wide range of accessory instruments, including          Automation of enzymatic and cellular assays was realized
                                                               with this system adapted to the handling of microtitre
thermal cyclers, spectrophotometer s and the BioRobot
                                                               plates. The microtitre-plate technology is the basis of our
RapidPlate 1, a 96-channel pipetting system with cap-
                                                               screening. All compounds within our chemical library are
abilities for both 96- and 384-well pipetting. These
                                                               stored and distributed in micronic tube racks or micro-
integrated systems provide rapid and fully automated
                                                               titre plates for screening. An eæ cient in-house-develope d
processing for applications such as DNA template nor-
                                                               mainframe-base d laboratory information management
malization, PCR, sequencing reaction set-up and sample
                                                               system supported all screening activities. Our experience
transfer tasks.                                                at JRF has shown that the preparation of test compounds
BioRobot 1 3000 extended-arm systems are designed              and making serial dilutions have been rate-limiting steps
to accommodate complementary instruments on both               in the overall screening process. To increase compound
the left and right sides. Additional integration of            throughput, it was necessary both to optimize the
the BioRobot TwisterTM increases system storage ca-            robotized assays and automate the compound supply
pacity for microplates, blocks and disposable tips, and        processes. In HTS applications, one of the primary
allows the integration of accessory instruments with both      requirements is highly accurate and precise pipetting
extended-arm and standard BioRobot workstation con-            of microlitre volumes of samples into microplates.
® gurations.                                                   The SciCloneTM is an automated liquid-handling work-
                                                               station capable of both 96- and 384-channel high-preci-
BioRobot 1 8000 robotic workstations can be integrated         sion pipetting. For high-throughpu t applications, the
with accessory instruments using the BioRobot Twister II       SciClone TM instrumentation can pipette a variety of
external arm for walkaway front-end processing, e.g.           liquid solutions with a high degree of accuracy and
from plasmid puri® cation to ® nal reaction set-up. The        precision between microplates (interplate variability)
large size of the BioRobot 8000 worktable and the high         and tip-to-tip (intraplate variability) within a single
storage capacity of the Twister II robotic arm allow           plate. The focus of this presentation is to review the
unattended operation over extended periods.                    liquid-handling performance of the SciCloneTM system
                                                               as a multipurpose instrument for pipetting aqueous or
QIAsoftTM, the BioRobot Operating System, commu-               organic solutions, and virus suspensions into 96- and 384-
nicates with external instrument software, controls pro-       well microplates. The capabilities of the system and the
cesses performed on the BioRobot and coordinates the           resulting bene® ts for our screening activities will be
actions of the BioRobot and external instruments.              described.




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