The planning and establishment of asample preparation laboratory for drugdiscovery

Claude DufresneNatural Products Drug Discovery, Automation & Informatics Group, MerckResearch Laboratories, R80Y-340, 126 E. Lincoln Ave, Rahway, NJ 07065,USA

Nature has always been a productive source of new drugs. Withthe advent of high-throughput screening, it has now becomepossible to rapidly screen large sample collections. In addition toseeking greater diversity from natural product sources (micro-organisms, plants, etc.), fractionation of the crude extracts prior toscreening is becoming a more important part of our e¶ orts. Assample preparation protocols become more involved, automation canhelp to achieve and maintain a desired sample throughput. Toaddress the needs of our screening program, two robotic systemswere designed. The � rst system processes crude extracts all the wayto 96-well plates, containing solutions suitable for screening inbiological and biochemical assays. The system can dissolve crudeextracts, fractionate them on solid-phase extraction cartridges, dryand weigh each fraction, re-dissolve them to a known concentra-tion, and prepare mother plates. The second system replicatesmother plates into a number of daughter plates.

Introduction

Nature is unmatched in its ability to create small organicmolecules enriched with biological potency and structur-al diversity. Chemical diversity is currently soughtthrough biodiversity of natural product-producingsources: microorganisms (prokaryotes and eukaryotes),plants, marine macroorganisms , insects. From 1983 to1994, of the 520 new Food and Drug Administrationapproved drugs, nearly 40% were derived from naturalproducts (30 are unmodi® ed natural products, 127 aresemi-synthetic derivatives, and 46 are synthetic com-pounds modelled after a natural product parent) [1].

In screening natural sources, at the minimum, a crudeextract is generated. This is usually accomplished bytreating the source with an organic solvent (EtOAc,MEK, etc). Crude extracts can then be partitionedbetween various solvents to minimize interferences(fatty acids, salts, etc). In addition to seeking greaterdiversity of samples from the intrinsic diversity of naturalproducts sources, fractionation of the crude extracts priorto screening is becoming a more important part of oure ¶ orts. Fractionated samples contain a smaller number ofindividual compounds and are less likely to give erro-neous assay results (false positives and/or masked activ-ities). Fractionation thus improves the quality ofscreening samples. However, increased sample processingincreases the possibilities of degrading unstable com-pounds, as well as ` losing’ others owing to irreversiblebinding to chromatography supports. Furthermore, pro-

cessing time, whether using manpower or automatedequipment is costly. The most important downside offractionation as part of a sample preparation process, isthe increase in costs resulting from the increased numberof samples. However, with the advent of high-throughputscreening (HTS) and higher density formats, screeningincreasingly larger sample collections is becoming less of aproblem.

To partly address the needs of our screening program,automated systems were designed. Processes were dividedbetween two robotic systems. The ® rst system, the samplepreparation system, processes samples from crude ex-tracts all the way to 96-well mother plates, containingsolutions suitable for screening assays. The second systemreplicates mother plates into a number of daughterplates. The process of fractionating samples using solid-phase extraction (SPE) will not be covered in thispresentation.

General considerations

Following the initial extraction, samples are usuallyhandled as solutions in tubes or vials. When drying isperformed, tubes are preferred for their round shapedbottom. Volumes vary from a few millilitres to severalhundred millilitres. The end product of the ® rst systemhad to be deep well mother plates. For convenience and¯ exibility, we decided to use racks of tubes that wouldhave the same outside dimensions as that of a 96-wellplate. In that way, the same robot gripper could be usedfor both plates and racks of tubes. Furthermore, the samestorage devices, such as carousels, could be used for bothtypes of container. After looking at various possibilities,we chose 16 mm ID tubes. With that diameter, racks of24 tubes …4 £ 6† can have the required footprint. Inaddition, these racks would map nicely to the fourquadrants of a 96-well plate. Even though we currentlyuse only 80 of the 96 wells, as a company standard forsample distribution, designing the racks to map to fullplates would allow us to avoid obsolescence as long as the96-well format remained in existence. We would there-fore use only 20 of the 24 tube locations, but would designthe equipment to be able to use all positions. Tube heightwas selected as 100 mm. Even though higher tubes(150 mm) provided for additional volume capacity(15 ml vs 10 ml), they had several problems: drying wasmore problematic in the longer tubes, storage density wasdecreased, and access with liquid handlers was moredi µ cult (clearance, reach). Our processes were thusadapted to a 10 ml working volume.

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Sample preparation system

It was decided to structure our automated system aroundthe concept of using workstations where samples aretransported in groups as opposed to individually.Furthermore, for maximum ¯ exibility in end-user inter-actions, each workstation should be usable in both amanual and an automated mode. In manual mode, theuser would place racks of samples on the workstationdeck, while in automated mode, the robot would bringthe samples. This dual mode can be readily achieved byusing workstations that can be accessed from the rear bythe robot. The workstations then face the user as theynormally would in stand-alone con® gurations. Rearaccess can be accommodated through an opening in theback of the workstation or using a shuttle that extendsout of the side of the station (® gure 1). It was felt thisarrangement would ensure that our work could proceedin the event the full system itself was down due to any oneof several reasons.

Workstations

We selected Bohdan Automation, Inc. to build the work-stations. The ® rst workstation is a weigher/labeller(® gure 1). The workstation can label tubes (Zebraprinter) and weigh them using a four-place Mettlerbalance. A barcode reader is used to positively identifyevery tube. The manual area can accommodate six racksof tubes (144 tubes), while the side-mounted shuttle, forrear robot access, can hold one rack of 24 tubes.

Label application (® gure 2) involves several steps. As thelabel is printed, it is held on a vacuum transfer plate thatpivots to contact a revolving tube. As the tube revolves,the label is applied.

The second workstation (® gure 3), for dissolution, incor-porates a shower head canula (choice of six solvents), asonicator bath, a homogenizer probe, and a barcodereader.

The third workstation, for SPE, will be the subject of aseparate presentation. The dryer was built in house.Details of a slightly di ¶ erent model, designed for 96-well plates, have been published [2].

A Packard MultiProbe 104 was selected for its ease ofintegration and macro handling capabilities. Everystation (except for the dryer) uses its own control PC.

This is necessary to ensure full manual mode operationsat any time.

Robot

The A465 robot arm (CRS Automation Solutions, Inc.)was chosen for its payload capability. A metal rack of 24tubes, ® lled with solvent, weighs approximately 1 kgwhich is beyond the safe payload of most small laboratoryscale robots. In addition the A465 has a long reach whichallows the use of a storage carousel that is 32 inches high.With three out of every four shelves removed from theCRS carousel, we can store any combination of 48 racksof tubes or deep-well plates. The ® ngers of a standardmicrotitre plate gripper were modi® ed (® gure 4) to beable to grab either a deep-well plate or a rack of tubefrom the same taught position.

Room facility

With good estimates of system sizes, a laboratory wasselected to house the facility. As is almost always the case,there never is a vacant wide open room that is available.We selected a generic laboratory …16 0 £ 36 0†, moved outthe equipment, and renovated it. New air exhausts wereinstalled, as well as new services (high pressure air,

Figure 1. Bohdan weigher workstation.

Figure 2. Label application.

Figure 3. Bohdan dissolution workstation.

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nitrogen, vacuum, water, power, ethernet, phone). Acustom table, built with extruded aluminium sectionsfrom Item Products and designed to house the robottrack below the table surface, was installed. All utilitieswere built onto the table framework.

System integration

The system was integrated around CRS POLARA soft-ware (® gure 5). POLARA integrated systems currentlymake use of two integrator provided components: anActiveX component for user interface (during system

con® guration and method building) and a RAPL-3component (for instrument communications and interfa-cing with the system controller). Since RAPL-3 is de-signed to work via serial lines, every PC is connected tothe robot controller PC using serial lines (we use aCyclades Z-card). POLARA( also provides built-in sup-port for a PLC (we use Direct PLC) to monitor varioussensors.

Custom software was written to integrate the operation ofthe robot with our corporate registration database(NEXUS). As sets of 20 samples are formed early on inthe process, they are grouped in sets of four, assigned aquadrant in ultimate mother plates, registered and as-signed new set IDs. As tubes are weighed, the volume ofsolvent for re-dissolution is calculated, as well as theamount of diluent needed for ` heavy’ samples. Theresulting information (sample amount and concentra-tion) is automatically uploaded to NEXUS.

Experiences

After over a year of full operation, we have experiencedrelatively few failure types. In the beginning, the single-most occurring source of errors was label mis-application. When numerous attempts at adjusting theapplicator did not improve outcomes, we elected topurchase pre-labelled tubes. Subsequently, Bohdanmodi® ed the applicator mechanism with improvedresults. The time saving with the purchase of pre-labelledtubes is signi® cant.

With this source of error removed, the next level offailures that became apparent was human errors: mis-

Figure 4. Dual-mode custom gripper.

Figure 5. POLARA schedule view.

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placed racks or plates, programming errors. However, aswe got better at setting up runs and the most obvioussoftware bugs were corrected, less frequent but moredi µ cult to solve system problems began to surface. The® rst one was a design problem with our grippers. We hadelected to use a standard microtitre plate gripper fromCRS. That gripper has a maximum opening distance of5.25 00 and a minimum closure distance of 3.12 00. This isperfectly ® ne to handle plates in both portrait or land-scape orientation. However, our original dual modegripper used pins on the ® ngers to mate with holes onthe rack for a solid pickup. Clearances and range limita-tions of the gripper limited the size of the pins andmatching holes to about 0.075 00. As is now obvious, thiswas too small to ensure a 0% failure rate in matching upthe pins and the holes. Variations in human placement ofthe racks within the holding plates of the carousel were ofthe same magnitude as the hole diameter. There weretwo instances where racks were gripped incorrectly re-sulting in crashes. A new design using a shelf-like platehas not seen a failure yet. Since clearances in landscapemode are tight, we are replacing the gripper with a newmodel from CRS that o ¶ ers a wider maximum opening.After experimenting with various materials for ` pads’ forplate pickup (sandpaper, rubbers, etc.), we concludedthat sharp pins were the most reliable.

In parallel with system ® xes described above, instrumentcontrol software for the three Bohdan workstations wasrewritten in Visual Basic 6. This allowed for better errorhandling, faster workstation executions, and tight inte-gration with our databases.

Plate replication system

The plate replication system (® gure 6) had a simple goal:to replicate mother plates into a number of labelled andsealed daughter plates. Because of our experience withthe CRS arm and POLARA, we chose to use the same forthis system. Even though the payload of the A465 was notrequired, its long reach (about 34 00) made it possible to

design a system without a track, in a fairly small footprintof 4 0 £ 6 0.

The Beckman Multimek 96 was selected as the 96-wellpipetter because several were already installed in units inthe Company and for its 384 well indexing capability.The CRS carousel allowed for the storage of 96 daughterplates (0.7 ml) with every other shelf removed. The platelabeler selected is the Zymark Presto Labeler. It waschosen for its small footprint and for the availability of anOCX for easy integration. A portion of the cover had tobe cut o¶ to allow for gripper clearance during landscapeplate loading from the side. The plate sealer is the ALPS1000 heat sealer. For plate shipments adhesive sealers arenot appropriate and cap mats have a tendency to pop o ¶ .Heat sealers, however, require plates with chimneysaround the wells.

Since this system is fairly self-contained, we integrated itusing a single PC. The sealer is controlled directly fromthe RAPL-3 component of the Sealer ` Interface’ via asimple contact closure on a GPIO block. The Multimekis controlled from its own control application which inturn is launched from a small Visual Basic applicationthat communicates with the RAPL-3 component of thePOLARA ` Interface’ via a named pipe. The PrestoLabeler has a small custom Visual Basic applicationwhich incorporates the Zymark OCX. This also uses anamed pipe to communicate with POLARA.

Mother plates are housed in a simple stand alone hotel(® gure 7). A barcode scanner is used to manually scan inthe mother plates on the hotel. A Visual Basic applicationthen registers all of the daughter plates and generates thelist of barcodes to be applied on the daughter plates. Thewhole replication process is now taking about 1 minuteper plate.

Use of the integrated system has just begun. So far theonly source of failure has been with label application.The problem is erratic and di µ cult to reproduce andhence debug. We are working with Zymark to resolvethis problem.

Figure 6. Plate replication system. Figure 7. Plate replication system (side view).

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Acknowledgements

I wish to acknowledge the very special contribution ofMiguel Maccio to the implementation of the ® rst system.Thanks also to Kevin Blake, Gary Kath, Brian Uhrig,from our Bioelectronics Group, Gary Mallow, and ChrisNapolitano.

References

1. CRAGG, G. et al., 1997, Natural products in drug discovery anddevelopment. Journal of Natural Productions, 60, 52± 60.

2. MACCIO, M. A., BLAKE, K., HOLT, T. and DOHERTY, P. J ., 1999,Drying Saucer: a 96-well format microplate evaporator. Journal ofthe Association for Laboratory Automation, 4, 31± 35.

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