The advent of combinatorial chemistry and high-throughput screening technologies has resulted in a large increase in the number of potential drug candidates being identified for development. Selecting the best compound to take forward into the drug development process is further complicated by development time frames that continue to shrink. Together, these factors have increased the need for an intelligent lead compound selection process that is based not only on biological activities but also on the pharmacological and chemical characteristics of the potential lead compounds. Failure to consider any of these characteristics could lead to significant and costly development problems, delays in getting the product to market, or the failure of the development project altogether.
Cellular permeability is one drug characteristic that is now being considered much earlier in the drug discovery process. Permeability assays using the Caco-2 colon carcinoma cell line are being used throughout the pharmaceutical industry to estimate the ability of potential drug compounds to cross the intestinal epithelium. Traditional permeability assays are very labor-intensive and have a throughput of fewer than 10 compounds per week. Fortunately, however, automation of permeability assays is possible. We have applied a Tecan Genesis 150 workstation configured specifically for cell permeability assays to improve the throughput of potential drug compounds. Using this system, the throughput of permeability assays has been improved 10-fold relative to traditional manual permeability assays and 4-fold relative to streamlined manual permeability screens.
Manual permeability assays
Traditionally, all aspects of permeability assays, including monolayer washing, addition of test compounds, and receiver compartment sampling at multiple time points, have been done manually. The numerous sampling intervals and replicate monolayers used for each transport direction of traditional assays add an even larger burden to performing the assay. As a result, the practical throughput of manual assays has been limited to just a few compounds per week. Throughput can be improved, however, by decreasing the number of replicate monolayers used and the number of sampling intervals examined for each compound. Still, even with such streamlining modifications, the assay remains labor-intensive, with a throughput of only about 10 compounds per day.
The optimum platform for automating the Caco-2 permeability assay can automate the required pipetting operations as well as perform nonpipetting operations. For example, the instruments ability to transport assay plates between the work surface and an enclosed incubator is preferable to performing the entire assay on a warmed surface. Likewise, instruments that can remove and replace plate lids allow the assay plates to be covered for extended incubations, thereby minimizing evaporation. The Tecan Genesis workstation is ideal for automating permeability assays because it can perform all of these tasks.
When configured for cell-permeability assays, the Tecan Genesis workstation combines an 8-pipette liquid-handling system, one or more shaking incubators capable of holding four 24-well permeability assay plates, various plate/lid racks, and temperature-controlled carriers and reagent reservoirs.
Unique to this workstation is a robotic manipulator arm (ROMA) that can pick up and move plates, lids, or permeability inserts to and from various locations within its work space. The workstation also includes software that dynamically schedules multiwell plate processing and automatically adjusts the schedule to account for process time variation. Together, the software, liquid handler, and robotic manipulator of the system allow the workstation to perform the entire Caco-2 permeability assay without manual intervention.
Another advantage of this workstation is its flexible pipetting software. With most traditional automation workstations, all pipette tips must perform the same operation simultaneously, and whatever solution is aspirated must be dispensed in the next programming step followed by tip washing. The Genesis workstation aspirates and dispenses using separately controlled tips, reducing cycle times on the platform, and therefore, assay times.
Automated permeability screening
Researchers perform an automated permeability assay by configuring the workstation with appropriate physiological buffers, lab ware, and test compounds. Multiwell insert systems containing Caco-2 cells are placed on the workstation and loaded into the temperature-controlled shaking incubator using the ROMA. Individually, the plates containing the inserts are brought to a prewarmed carrier on the workstation, where the plate lid is removed and the insert containing the Caco-2 cell monolayers is transferred to a new 24-well base plate. The growth medium is then removed from the insert chambers, and the cell monolayers are washed with assay buffer. Test compounds and assay buffers are then added to the donor and receiver compartments of the transport inserts, respectively, and a small sample is taken from the donor chamber and automatically diluted for analysis to verify the concentration of the test compound. The ROMA then retrieves the assay plate lid and returns the plate to the incubator for two hours. Following incubation, samples are taken from each chamber. The conical shape of the permeability inserts used at Pharmacia allows the liquid-handling arm to sample from either of the two chambers created by the placement of the insert. The final step involves adding a fluorescent marker compound to every well, performing another incubation step, and removing a sample to determine if the cell monolayer has been perturbed. This procedure, which was not part of the manual assay due to the increase in assay time, provides an important validation step in every assay that is performed.
The new automated Caco-2 cell permeability assay provides a throughput of 48 compounds per assay, four times the amount that could be produced by manual screening. The assay runs in about 6.5 h without manual intervention, saving approximately 2 h of manual labor per assay. The assay can now be performed much earlier in the drug discovery process (immediately after confirmation of activity through high-throughput and secondary screening) due to the increased throughput from automation. As a result, researchers can now determine permeability coefficients and flag poorly permeable compounds before preparing analogues and developing cell and animal models. The primary payback will be an acceleration of the drug discovery process, part of Pharmacias multimillion-dollar effort to move critical assays further up the drug discovery pipeline.