Abstract
INTRODUCTION
THE PROJECT
Discovery goals had set a challenging target for growth in candidate generation, and in order to achieve this a co-ordinated strategy to increase our lead-seeking capabilities was developed. This included significant investments in combinatorial chemistry, both internally and externally, in order to enrich our files with thousands of drug-like molecules. Our current compound file was expected to exceed 1.0 million by end 2000 and it was felt essential that these compounds be exposed to as many of our lead seeking targets as possible. In addition, parallel initiatives will increase the annual number of ultra and high throughput screens run.
This plan implied an enormous increase in sample handling and throughput in order to expose a growing number of biological targets, against an expanding file of screenable entities. Our ability to dispense samples for test on the scale required to support this effort was far beyond our existing manual capabilities, being a mixture of manual and semi-automated effort — labour intensive and prone to human error.
Following an intensive tendering and selection period, a Manchester (UK) based engineering company, RTS Thurnall, was selected to design and build Pfizer's Automated Liquid Sample Bank. On January 20th, 2000, almost 5 years to the day the original concept document had been presented to senior management, Pfizer formally accepted delivery of the ALSB.
As a stand-alone system, ALSB worked but it was now necessary to complete the integration of ALSB with Pfizer software packages DISCUS, ECADA and IMSO without which ALSB could not be used. Integration testing was conducted using test instances of the Pfizer databases. DISCUS (which holds the compound inventory), ECADA (which manages High Throughput Screening data) and the compound ordering application IMSO, which provides both a direct user interface for ALSB plus an interface to connect HTS experiment creation with ordering from ALSB. For the purposes of testing, these databases were populated with dummy data to ensure that incompatibilities could be identified and resolved before interfacing ALSB with the live applications and databases.
The ALSB liquid handling cells and transport system.
THE SYSTEM
For reasons of compound stability, we elected that samples should be stored at −20°C. In addition the system should prepare daughter plates for screening, and that these daughter plates should be prepared in as timely a manner as possible. Therefore, at the highest level, the ALSB was to consist of a frozen store, a station in which to thaw the samples, and liquid handling robotics, all connected by a transport system.
COLD STORE
The cold store is a 500 m2 room maintained at −20°C. This room houses the samples, held in racks of 90 tubes, and has a capacity in excess of 2.7 million samples. Following an order, the carousel level containing the sample rotates to a pre-set position where one of 12 tray pulling robots pulls the entire tray of 43 racks out of the carousel. The picking hand of one of the twin overhead gantry robots then selects either an individual tube from a rack, or the entire rack of samples, depending upon the order make-up. Rack selection and tube cherry picking occurs within the −20°C store thus removing the necessity to freeze/thaw any samples other than those required for the order. The gantry robots weigh approximately one ton, are around 8 feet long, and, over an operating envelope of 120 sq. ft., can position with an accuracy of 4 thousandths of an inch. Racks of tubes, identified by bar-code, are placed on the cold store out-feed conveyor and transported to the defrost station.
DEFROST STATION
Since samples in 100% DMSO at −20°C will almost certainly be solid, prior to aspiration the samples are gently thawed into the liquid state. Racks of samples are transferred from a port connecting the defrost station with the cold store, to the next available shelf of a paternoster oven contained in the defrost station. After a pre-set time, currently around 2 hours, the rack of samples, now thawed, is picked and placed on the conveyor leading into the laboratory environment of the ALSB. The air intake to the defrost station is controlled with respect to humidity to minimise exposure of the samples to water vapour in the environment.
LIQUID HANDLING CELLS
The ALSB system has twin liquid handling cells in which the samples are aspirated, diluted and dispensed into the format specified in the originating order. The hardware configuration of each cell is identical, and consists of an anthropomorphic arm, which can access a modified Tecan Genesis and stores of microplates, together with other support hardware. Selection of the liquid handling cell in which the order is to be processed is made by the system software with a priority of keeping samples from the same order together. If required, it is possible to manually override the system's LHC selection.
Incoming racks of tubes are picked by a 6-axis Staubli robotic arm, which traverses the length of the cell. The rack is placed on one of nine positions on twin platens designed to locate in the operating envelope of a Tecan Genesis robotic sample processor. This twin platen arrangement allows the Genesis to work on the contents of one platen while the Staubli robot unloads or loads the second platen. Dilution algorithm software calculates the most efficient and compound sparing aspirate-dispense protocol to achieve the delivered volume, compound concentration and DMSO concentration specified in the original order.
The Staubli robot collects the appropriate labware from in-feed stack locations, checking each selection by scanning its type identifying bar code. Prior to liquid handling, the system sprays the order information on a blank label on the final destination (daughter) plate. Once all labware is assembled the platen retracts and the Genesis probes pierce the tube septa to aspirate the sample. The probes are designed with an outer jacket open to the atmosphere to equalise pressure on each side of the sample tube. The silicon rubber septa were designed in-house, the exact formulation and curing process being arrived at after extensive testing to find a product which did not core but sealed well after repeated piercing. Each septa is able to withstand in excess of 160 piercing events, which is greater than the expected number of sampling events.
ALSB Cold Store Carousels and Pickiing Robotics
Once all liquid handling is complete, the transport rack is placed on an out-feed conveyor loop for return to the cold store. The order processor will have decremented the volume field in the database entry of each tube by the amount aspirated. Labware used in the order is sealed, daughter plates are delivered to the out-feed stacks, and any labware used for intermediate dilutions is dropped into a disposal bin. The capacity of each liquid handling cell allows up to 1200 microplates to be available at one time, together with an out-feed capacity of 700 plates.
TRANSPORT SYSTEM
The three major components of the ALSB, cold store, defrost station and liquid handling cells, are connected by a series of ten independent Flexlink conveyor systems. In order to optimise the use of space, the conveyors are on two levels, the upper dedicated to out-feed and the lower for in-feed, relative to the cold store.
DELIVERABLES
A conservative estimate of ALSB output rate for rack based orders is 500 unique racks per day with multiple copies of each rack possible (limited generally by liquid handling rate) and for tube based orders is 6000 tubes in a 24–hour period (limited by tube picking robotics). Most days a mix of tube and rack based orders results, thus maximising the usage of both liquid handling and robotic tube picking.
The customer of the ALSB output is not always a screening scientist, as many of our HTS screens are now run on fully automated systems. In some cases, the output from ALSB requires reformatting before delivery to the scientist and, here too, the recipient of the ALSB output plate is a robotic system. All output plates from ALSB bear a human readable label printed directly onto the plates at time of liquid handling in ALSB. This label gives details of the number of plates in the order and sequence of the individual plate, plus recipient's name, etc. This same information is also held in a barcode printed on the label, again at liquid handling, which can be read by the other automated devices. The barcode gives information about what should happen to this plate and which compounds it contains, so, for example, should plates get out of sequence during running of the assay, the data will still be associated with the correct compound via the barcode check.
CONCLUSION
In the few months the ALSB has been up and running, it has supplied samples to a wide variety of screens at all stages of the discovery process. The store is being used to supply our partners around the globe, and is a key component in Pfizer's global compound management strategy. We are however aware that the system is very new, and we are still on the steep part of the learning curve of how to optimise the tremendous capability of our ALSB.