Lean Screening

Lean Screening is a simplified system approach for Ultra High Throughput Screening. The term lean is plagiarized from the manufacturing sector. It minimizes handling and inventory to move to a just in time environment. Lean Screening utilizes only two pieces of equipment that can be replicated to fit any system throughput requirement. One unit is an automated plate retrieval system (Mega-Stor™ — Figure 1 ). Each unit can hold 1675 microplates in a dry temperature controlled environment. From a database work list it can deliver or put away microplates in a cassette (25 or 50 plate), format. The other element is a cassette stacker fed pipetting workstation (Quadra® — Figure 2 ). It can pipette from 96 well plates into either 96 or 384 well formats. An automated multiple station shuttle incorporates reagent additions and tip washing into the automated protocols. Both of these elements are tied together with a software system that tracks each microplate through bar codes to the final assay.

Figure 1

Mega-Stor™ Plate Retrival System

Figure 2

Quatra-Plus™ Plate Processing Workstation

Since only two basic devices are used in the system, it is easily expanded to meet the throughput required. As the library expands additional storage units are added. As the volume of assay plates increases additional pipetting workstations are added. Having multiple identical units provides simplicity and flexibility in operation. With only two devices to learn, operating personnel become very familiar with the system operation. This increases the flexibility of use, and above all reliability in operation.

In summary form, the 96 well compound plates are stored in the retrieval systems. They are selected and output in cassette formats according to the daily work list. The cassettes are moved manually to the next available pipetting workstation. The assay plate is prepared in the desired format, 96 or 384, with the required amount of compound delivered in the buffer of choice for the specific assay. The end user of the assay plates has only to add the common reagents, i.e., ligand, label etc., and the standards and controls, for the specific assay. This could be done with a simple dispenser or pipetting station.

The real challenge in moving to a Ultra High Throughput Screening (UHTS), System is not adding the common reagents. It is being able to deliver 100,000 discreet compound samples, of known value, from the compound library to the assay plate wells everyday. The logistics of the entire operation are detailed as follows.

The process starts with the originating library. Compounds, normally chemically derived, are weighed out and solvated with DMSO to create a liquid phase for pipetting. Weighing the initial compound is required to define validity of the assay results. It is not economical to weigh nanogram and picogram quantities. When milligram quantities of small molecular weight compounds are solvated, they normally end up with 5 to 10mL of solution, of a known molarity in 100% DMSO. The cost of each 5mL of compound solution is of significance, particularly since this cost may be replicated for 500,000 compounds in the library. Thus the need to preserve the compound solution efficacy for as long as possible.

Over the past 10 years of High Throughput Screening (HTS) experience, several guidelines have become accepted practices:

• The affinity that DMSO has for water is detrimental. Thus solvated compounds are stored at frozen temperatures ranging from −4°C down to −80°C, depending on how long they are to be stored.

• Frozen compound solutions must be thawed for use. Each freeze/thaw cycle presents the possibility of moisture in the compound, and loss of potency. It is desirable to minimize the freeze/thaw cycles of the compound mother plates.

• Some compounds, while soluble in 100% DMSO, fall out of solution in a lesser percentage. Many assays cannot tolerate more than 1% DMSO. Thus the final 100 fold dilution from the compound storage plate (100% DMSO), should be made directly to the assay plate (1% DMSO), without a predilution. Any dilution for the purposes of molarity should be made in 100% DMSO.

The accepted practice, in handling the 5mL of solution from the weighing operation, is to aliquot it into smaller units, either as sets of plates or individual tubes. Typically the 5mL is divided into 10 sets with 0.5mL per set. In this manner 9 sets of compound mother plates may be deep frozen at −20°C or −80°C, for extended storage. One set is then used to support the screening program for say a 6 month period. As this using set reaches its expiration date, it is discarded and another set is utilized. In this manner, the life of the weighing operation is prolonged to 5 years. Efficient handling of this using set of mother plates is the basis of this report.

To put specific values on the task, we assume a UHTS operation that is screening 100,000 compounds per day through 5 different assays, using both 384 and 96 well formats. The total using library is 500,000 compounds stored frozen at −20°C in 5682 deepwell microplates (88 compounds/plate), with at starting volume of 0.5mL per well. In general terms, the procedure is to replicate the mother plates into an active set of 5682 microplates in the 96 well format with 80 microliters per well. This active set of daughter plates is to be stored in 4 automated storage retrieval units (Mega-Stor™). They will be stored non-frozen (8 to 10°C), in a dry environment.

Daily, 1136 plates (100,000 compounds), will be retrieved, according to a database generated work list. They will be output in a 25 or 50 plate cassette format for direct transfer to the pipetting workstations. After the expiration date of this active set of 5682 daughter plates, they will be discarded and replaced with a new set replicated from the mother plate set in use for this period.

By storing the active daughter plates non-frozen, they are available each day for immediate use in making assay plates. Assume the using life is limited to 4 weeks. There is increasing information from several sources to indicate very acceptable performance over this time period. Periodic quality control tests with known assay results can be incorporated into the assay protocols for verification.

The cassettes of daughter plates are stacked in order of use for the specific assays. This order is established by the library database. The plate retrieval system picks according to the work list. The pipetting workstations are 96 well small volume pipettors (Quadra — Figure 2 ). They can produce 96 well plates or they can reformat four 96 well plates into one 384 well plate. They have the precision to dispense 0.5μL with a Cv of 2–3%. This allows the direct 100 fold dilution of DMSO to a 50μL or greater assay. Each pipetting workstation has two sets of cassette type plate stackers. One set feeds in the compound daughter plates while the other feeds in the outbound assay plate. Each has a multi-station automated shuttle. Two stations are dedicated to plate handling. A third station has an ultrasonic tip washing station. Other stations are available for the buffers to be used. The pipetting workstation is also connected to the database, which can control its programming. Barcodes on all of the plates, track the audit trail.

The assay plates, with compound, are delivered in the buffer of choice for that specific assay. This has several advantages. In the pipetting workstation there is essentially no time lost in adding the buffer. There is a distinct advantage in improved precision. The pipettors are designed to transfer 500 nanoliter aliquots. This allows a 100:1 dilution of the compound into a 50μL assay volume. If the 0.5μL of compound is washed out with 25μL of the assay buffer, a Cv of 2–3% is obtained. If 0.5μL is dispensed dry, and then resuspended later with the assay buffer, the Cv will be 6–9% using the same conditions. However, to dispense dry into a plate, requires special attention to the pipetting conditions. Dispensing 0.5μL to a dry well requires the tip to touch off on the bottom of the well. Variations in the molded plate and how it is handled from the stacker, as well as how it sits on the plate nest, are critical factors. This adds uncontrolled factors, which lead to reliability problems to a fully automated, unobserved system.

There is some concern as to whether a 0.5μL spot of compound in 100% DMSO can be consistently resuspended with an aqueous buffer, if it is allowed to dry. Another advantage of supplying the compound in the buffer of choice is that the final user only adds the common reagents for the assay. Ligand, label, etc., can be added with a multiple dispenser, or pipettor along with the standards and controls for that assay.

The above are the advantages of dispensing compounds in the assay buffer of choice. A disadvantage is that the life of the assay plate is shortened. The diluted compound is now in an aqueous solution. The time between compound addition and completion of the assay components may be shortened. Another requirement for the just in time lean screening approach is that more planning is required at the database level. Compound plates must be selected in order for the specific assays. What buffer is to be used? Will the assay be in 96 or 384 well format? The reward is a streamlined semi-automated system that only requires the manual intervention of moving cassettes of 25 or 50 plates from one station to another. Bar codes maintain the audit trial.

System Flow Schematic

Another area to be addressed at the time of making the assay plates is compound recall or “Cherry Picking”. The automated storage system, if loaded with 384 well plates, can hold in excess of 500,000 discrete compounds. A pipettor head is available for this unit to perform the recall function, or cherry picking. The desired compound plate is withdrawn. An aliquot of the desired compound is aspirated and dispensed into an output plate. The output plates are fed in and out of the picker via the cassette stackers.

The pipettors can create the 384 well compound plates at the same time they are making assay plates. Since the plates for cherry picking must remain non-frozen, their useful life is limited. However, they can be replenished on demand, since the system merely treats them as another assay requirement.

To maintain a smooth flow through the pipettors, all plates required from each compound plate are made sequentially. This is facilitated by the pipettors, since they can produce either 96 or 384 well plates and select from three or more buffers. This means that each output cassette may have plates for all of the required assays. The bar code on each assay plate tracks its production and identifies it.

The output cassette is then sorted for the specific assays. This may be done manually by wanding the bar code and manually restacking. A simple automated distribution system is also available ( Figure 3 ). A cassette to be sorted is placed at one end along with five or more receiving cassettes. The bar code on the plate automatically defines its destination cassette.

To review the logistics of the system, start with replicating the active set of 96 well compound daughter plates from the origin library plates. Only 80μL of compound is transferred. This 80μL will support the assays for four weeks. Using the baseline of 100,000 compounds per day from a 500,000 compound library the average plate will only be used four times (20μL available). 96 well pipetting workstations, with stackers, would replicate 5682 mother plates into the same number of active daughter plates. Using four workstations would require approximately 36 hours. This task would need to be repeated every four weeks. The mother plates would be returned to cold storage. The cassettes of active daughter plates would be transferred to the plate storage and retrieval units. The inventory would be established in the database as the plates are put away.

Each working day the database would generate a work list of compound plates to be retrieved. The storage retrieval units would select and stack the required plates in order in the prescribed cassettes for manual transfer to the pipetting workstations. To store 5682 plates would require four storage units. Each day they must retrieve 1136 plates (100,000 compounds). This would require something less than 8 hours of running time.

In the example stated earlier the workload is 100,000 compounds per day, through five assays. The 100,000 compounds equates to 1136 plates. This would be 5680 assay plates if all were 96 well and less by how many were 384. The pipetting station can process a 96 well plate in approximately 1.5 minutes or 6 minutes to reformat four 96 well plates into a 384 well plate. To accomplish this example workload on a daily basis (8 hours) would require about 20 pipetting workstations. They would supply considerable flexibility, since they are all replicates. Any one unit could handle any and all tasks.

Figure 3

Plate Sorter

In some HTS environments, the physical distance between the compound dispensary and the assay location inhibits the just in time approach described above. Another option entails the use of MicroTape ( Figure 4 ). This is a sprocket driven carrier tape with continuous 384 well patterns. Each well has a brim volume of 10μL. The carrier tape is polypropylene, as are the current 96 well plates for compound storage. Polypropylene is inert to DMSO and will withstand −80°C.

Figure 4

MicroTape™ Pattern

MicroTape is designed for automation (1). The wells may be filled with current 96 or 384 well pipettors. 100,000 discrete samples may be stored sealed and frozen in a roll 16 inches in diameter × 4 inches wide. It provides very compact storage. Multiple replicates of the compound library can be created from one freeze/thaw cycle of the Mother plates. Ten replicate rolls of compounds may be created at one time. Each roll contains 100,000 compounds in 5μL aliquots.

There is very little thermal mass in a roll of MicroTape permitting quick thawing. Since the compound is sealed until time of use, degradation due to moisture is not a concern. Each 384 well pattern has an identifying binary code punched into the MicroTape. It can be cut into individual 384 well patterns or used as a continuous roll. As a continuous roll, it serves as the compound infeed to an automated pipetting workstation.

The benefits of Lean Screening are realized throughout the laboratory. Non value added steps are minimized. Inventory of work in progress is eliminated. Compounds move directly to the assay plates in a just in time environment. The need for capital investment is tied directly to the throughput required. Each pipetting workcell is identical, and has the flexibility to meet any assay requirement 96 or 384. This multiplicity of common devices eliminates bottlenecks in that any instrument failure does not stop the process. The improved precision in pipetting provides better quality leads with less hit requalification.