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					        Conversion of manual access of 384 lidded
        plates to an automated storage and retrieval system


                                   Pavel Rychetsky
                                   Martha Ackerman
                                   Patti Willson

                                   Biology Automation

                                   Selectide, a subsidiary of Aventis Pharmaceuticals Inc.,
                                   Tucson, Arizona, USA

                                   pavel.rychetsky@aventis.com



                                                       MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   1
a subsidiary of Aventis Pharmaceuticals Inc.
        Presentation outline




          • Non-traditional compound delivery with pin tools using
          one library copy

          • Efficient HTS operation - need for simple storage solution

          • TekCel storage




                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   2
a subsidiary of Aventis Pharmaceuticals Inc.
         Factors for decision on automated storage

         • Storage tightly integrated with HTS operation
                   • Compounds produced in plates
                   • Limited amount of compound available
                   • HTS utilizing exclusively pin tools for compound transfer
                   • Centralized HTS; flexibility for future use of workstations

         • Unattended HTS operation
         • Cherry-picking from 384-well plates
         • Space limitations
         • Cost
         • Sealing

                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   3
a subsidiary of Aventis Pharmaceuticals Inc.
            Amount of compounds produced by
            CombiChem technologies at Selectide Aventis

        1. On-bead binding assay with colorimetric or fluorescent
        detection (1992-1994)
                             Individual bead (peptide) - 0.2 nmol

        2. Solution assays
           • Compounds released from single beads using double cleavable linker
           • Loose resin in DWP
              • Mixtures, 4 -12 cmpds/well, 0.5 umol/cmpd (100 ul 5 mM)
              • Singles, 2 - 10 umol
           • NanoKans (Irori), 5 umol
           • MicroKans (Irori), 30 umol
           • Lantern (Chiron), 12 umol

                                                  MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   4
a subsidiary of Aventis Pharmaceuticals Inc.
       Plate logistics for CombiChem libraries


      Dry cmpds                                     Dry cmpds                                     Archive
                                                     Dry cmpds                                    Dry, sealed, -20oC
       96 DWP                                         Dry cmpds
                                                     96 MTP
                                                      96 MTP
                                                        96 MTP

                                               Add 85% DMSO

                                                      DMSO 5 mM
                                                        96 MTP

                                         Retest                      Screen                         Screen
                                       384, 5 mM                    384, 5 mM                     384, 0.5 mM
                     Cherry-picking copy, 45 ul                           Pin-tooling copies, 50 ul

                                                       MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky              5
a subsidiary of Aventis Pharmaceuticals Inc.
     Compound transfer using pin tools -
         Hardware
          • Beckman HDRT (High-Density Replicating Tool) for Biomek2000
                               D. Brandt, J. Biomol. Screen., 2 (2), 111, 1997; Poster, SBS 2001, Vancouver, Canada
          • V&P Scientific
                 • pin tool adapter for CyBio CyBi-well pipettor
                 • pin tool adapters for Beckman Biomek2000




                                                                                            Beckman                V&P Scientific
                                                                                            - machined             - pressed in
                                                                                            steel                  aluminum base
                                                                                                                   - plain, slotted,
                                                              MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   hydrophobic         6
a subsidiary of Aventis Pharmaceuticals Inc.
             Compound transfer using pin tools -
                 Transferred Volumes
             • Dependent on volume in destination and source plates
             • 7 -150 nl transfer for different size pins
             • CV ranges:
                  • 6 - 11% for 384-well screening plates (40 ul volume)
                  • 10 - 17% for 1536-well screening plates (3-6 ul volume)
                                                   Dependence of transferred volume on volume in source and destination plates
                                                                         400

                                                                         350
                                               Volume transferred [nl]




                                                                         300

                                                                         250

                                                                         200

                                                                         150

                                                                         100                                   Destination 50 ul PBS

                                                                         50                                    Source 30 ul DMSO

                                                                          0
                                                                               15     20    25     30     35     40     45     50     55    60     65     70

                                                                           Volume in Source plates (Destin. 50 ul) or Destination plates (Source 30 ul) [ul]
                                                                                        MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky                     7
a subsidiary of Aventis Pharmaceuticals Inc.
          Compound transfer using pin tools -
              Washing

                                               Carryover
      No wash                                   10.80 %
      1x H20                                     0.18 %
      1x H20, 1x EtOH                            0.09 %
      1x H20, 2x EtOH                            0.02 %




                                                       MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   8
a subsidiary of Aventis Pharmaceuticals Inc.
          Compound transfer using pin tools -
              Comparison with pipetting

            Dose response curves for enzymatic assay, 60 nl transfer of
            inhibitor

                                                                              100

                                                                               80




                                                               % of Control
                       Pins - 384 Beckman pins,
                                                                               60
                       0.045’’ diameter
                       Pipettor - Beckman Multimek,                            40
                       96 channel
                                                                               20

                                                                                0
                                                                                    0.01               0.1              1
                                                                                                     [inhibitor](uM)

                                                                                           pipet transfer (IC50 = 0.19 +/- 0.02 uM)
                                                                                           hdr transfer (IC50 = 0.13 +/- 0.01 uM)


                                                      MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky                              9
a subsidiary of Aventis Pharmaceuticals Inc.
            Compound transfer using pin tools -
                Summary

        Advantages
        • Saving of compounds

        • Elimination of intermediate dilution plates
             • saves work
             • savings in consumables
             • eliminates potential compound precipitation and/or degradation during
             incubation in water-based buffers in dilution plates

        • Applicable to higher density formats (1536 format, 7 or 20 nl transfer using
        V&P Scientific pins, Beckman MicroPins)

        • Prevention of transfer of precipitated compounds

        • Excellent/acceptable CV values


                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   10
a subsidiary of Aventis Pharmaceuticals Inc.
                Compound transfer using pin tools -
                    Summary (Cont.)

     Disadvantages

     • Limited flexibility in adjusting volumes of transferred compound
     • Dependence of transferred volume on volume in both source and destination
     plates (equal volume of compound stocks in plates is necessary; edge effects)
     • Fragility of lower volume pins (expensive consumable)
     • Custom hardware modifications necessary for lower volume pin sets
     • Calibration curves needed for new sets of pins
     • Transferred volume can be dependent on compound characteristics (viscosity,
     surface tension)
     • Lower concentration compound stock copy was necessary before the low
     volume pins were available
     • Problems with certain cell-lines (temporary high local DMSO concentration ?)

                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   11
a subsidiary of Aventis Pharmaceuticals Inc.
       Unattended HTS operation

     • Tip washing
               • DMSO (recirculating reservoir)
               • water (fresh or recirculated)
               • elimination of potential problems with automated tip delivery
               • consumables savings
               • higher throughput

     • Pin tool washing

     • Self - refilling cooled reservoirs (recirculating, optical liquid sensing)

     • Automated paging for error reporting

     • Reliable hardware (excellent service support by Beckman)

     • Manual delidding (most error-prone operation caused usually by
     condensation water)

                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   12
a subsidiary of Aventis Pharmaceuticals Inc.
       Integrated HTS/plate logistics operation

      Staff: 3 biologists, 1/3 engineer (1999)

      Hardware: 1 robotic system Beckman Core
                 •         ORCA arm
                 •         Multimek pipettor
                 •         Biomek 2000 for pin tool transfers
                 •
                 •
                 •
                           Wallac MicroBeta
                           Wallac Victor
                           CO2 incubator
                                                                                                    $
                 •         sealer                                                          $500,000
                                                                                       including readers
                 •         delidder
                 •         shaker
                 •         Skatron washer
                 •         Carousel; 180 plates maximum capacity

                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky          13
a subsidiary of Aventis Pharmaceuticals Inc.
   Integrated HTS/plate logistics operation (Cont.)
   Functions (1999):
   1. Plate logistics (1,200 96-well plates -> 900 384-well plates)
                        Bar coding plates
                        Dissolution & reformatting libraries from 96 to 384-well plates
                        Plate storage (racks, -20oC freezers, 384-well plates in DMSO,
                                96-well plates with dry archive cmpds, sealing archive plates)
                        Inventory management/Plate registration
                        Support for 1536 screening on workstations with 384-well cmpd plates
                        Cherry-picking from 384-well plates (added later)
   2. Assay adaptation to HTS (17 assays)
   3. HTS
                        All HTS in 384-well format
                        7,000 384 well plates/year
                        1536 screening on workstations
   4. Import HTS data into database (idbs ActivityBase; protocol design)
   5. Projects (assay transfer, assay development, HTS, follow-up, etc.)
                                                         MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   14
a subsidiary of Aventis Pharmaceuticals Inc.
            Estimated average distribution of tasks to
            prepare for HTS run - 2 hrs, 100 plates

        Unload compound plates from robot carousels                                     - 20 min
        Load compound plates (manual delidding)                                         - 40 min
        Unload assay plates                                                             - 5 min
        Load assay plates                                                               - 10 min
        Clean & fill reagent reservoirs                                                 - 20 min
        Change tip wash solution                                                        - 20 min
        Schedule assay                                                                  - 1 min


        50% time spent on loading/unloading compound plates




                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   15
a subsidiary of Aventis Pharmaceuticals Inc.
            Problems with storing compound plates in
            racks in freezers

        • Handling heavy frozen racks from difficult to access shelves
        • Small injuries
        • Vacuum-closed freezer door
        • Manual lidding/delidding plates; contact with DMSO & cmpds
        • Stress from fear of dropping racks (hasn’t happened yet)
        • Dropped plates (very rare)

        Other disadvantages of storage of lidded plates in freezer racks
        • Prolonged exposure of compounds to lab environment during HTS runs
        • Evaporation in outer wells due to the use of loose lids
        • Condensation water causing problems with delidding and cross-
                contamination
        • Cherry-picking: loading carousels with 384-well plates is slow

                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   16
a subsidiary of Aventis Pharmaceuticals Inc.
        Integrated storage system by TekCel

     1. Modular automated storage system
        - Server
             Interface between processing stations and storage units
        - Storage units
             1,000 plates/units

     2. Unique plate sealing lids
                                                                             TekCel
                                                                             Server
                                               TekCel         TekCel
                                                                                               HTS robot/
                                               Store          Store                            Cherry-picking station



                                                        MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky              17
a subsidiary of Aventis Pharmaceuticals Inc.
                Modular storage by TekCel -
                    Cherry-picking


                                                                        PlateServer A
                                                                              •      Thawing
              PlateStore (1,000 sealed plates)                                •      Unsealing
                                                                              •      Presenting plates




                                                                         A
                       1                       2   3        4                      Cherry-picking station




                                                       MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   18
a subsidiary of Aventis Pharmaceuticals Inc.
                Modular storage by TekCel -
                    HTS support
                PlateStore (independent mobile units)

                                                  1        2

                                                  3        4


                                                      PlateServer B                                          PlateServer C
                                   B                                                          C
                 1                                                                 4
                                               Screening Robot 1                                     Screening Robot 2




                                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky            19
a subsidiary of Aventis Pharmaceuticals Inc.
          Modular storage by TekCel


        Pro’s                                                          Con’s
        Supports multiple systems                                      Lower storage density
         • Fits into existing facilities
                                                                       Thawing whole plates
        Expandable
         • Add modules as library grows

        Redundancy
        Fits in current facilities
        Transportable
        Less cost/square foot

                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   20
a subsidiary of Aventis Pharmaceuticals Inc.
                                                                 TekCel
                                                                 PlateServerTM

                                                             • Anhydrous inert atmosphere
                                                             • Hand-off arm with position
                                                             adjustment
                                                             • Front access for manual
                                                             loading/unloading 8 cassettes with 34
                                                             plates, 272 total
                                                             • Supports multiple PlateStoresTM
                                                             • Exchange track
                                                             • Thawing capability
                                                             • Bar code scanner
                                                             • 0.9 m x 1.5 m footprint
                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   21
a subsidiary of Aventis Pharmaceuticals Inc.
              TekCel PlateServerTM




                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   22
a subsidiary of Aventis Pharmaceuticals Inc.
            TekCel PlateStoreTM


       • Anhydrous inert atmosphere
       • -20 °C to RT
       • Exchange track
       • Random access
       • Tracking
       • Mobile (AirSled technology)
       • 1,000 sealed microplates
       • 0.9 m x 1.2 m footprint




                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   23
a subsidiary of Aventis Pharmaceuticals Inc.
               TekCel PlateStoreTM




     Exchange track links modules
     together




                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   24
a subsidiary of Aventis Pharmaceuticals Inc.
            TekCel SealTiteTM


      • Automatic or manual sealing and unsealing
      • Personal exposure eliminated
      • Limited cross contamination due to vertical removal of cover
      • Shorter exposure of compound stocks to lab environment (few
      minutes)
      • Covers
         • Reusable
         • Stackable
         • Inert
         • Low temperatures compatible
         • Sealing by spring mechanism

                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   25
a subsidiary of Aventis Pharmaceuticals Inc.
                TekCel PlateSealTM




                                               MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   26
a subsidiary of Aventis Pharmaceuticals Inc.
              Database structure

                                                                                Cluster1 Database




                                                                                   Plate Server          Plate Store 1     Plate Store 2



                                                                                 Device Database        Device Database   Device Database

                                                       Tekcel World Database



                                                                                Cluster 2 Database




                                                                                   Plate Server          Plate Store 1     Plate Store 2



                                                                                 Device Database        Device Database   Device Database

                                          Customer’s
                                          Backbone




                                                                  MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky                        27
a subsidiary of Aventis Pharmaceuticals Inc.
         Software Communication

                                                                                 All devices within a cluster communicate
               Data Objects
                                                             Error               through a Message Center
               Logs all data                                Handling
                                                            Logs all data
                                                                                 Each device logon to a Message Center

                                                                                 Devices pass to the message center the
                                               Messaging                         types of messages they wish to receive
                                                Center
                                                                                 All Messages are formatted in XML


                Main                                         Hardware
              Application                                     Objects
                 Update GUI                                 Robots, Barcode
                                                           Readers, Track etc…




                                                                 MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky    28
a subsidiary of Aventis Pharmaceuticals Inc.
       TekCel storage - Summary


                           • Complete automated HTS process
                           • Automated cherry-picking
                           • Mobile storage
                                   Minimal space requirements
                                   Flexible, expandable, enclosed
                           • Combination robots, stackers, conveyors
                           • Cost effective
                           • Modular; grows with collection
                           • Easy integration with robotics systems
                           • Fast, 60 - 160 plates/hr
                           • Efficient sealing
                                    Automated
                                    Inert atmosphere
                                                   MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   29
a subsidiary of Aventis Pharmaceuticals Inc.
       Acknowledgements




                           • Martha Ackerman
                           • Christine Mortel
                           • Patti Willson
                           • Todd Smith


                           • Ernie Woods
                           • Julian Warhurst, Steve Fillers, Marsha Paul


                                                MipTec ICAR, Basel, June 20, 2001, Pavel Rychetsky   30
a subsidiary of Aventis Pharmaceuticals Inc.

				
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