Automation of Pre-coated PAMPA Plates Improves Predictability ...

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(A) Traditional PAMPA Process Flowchart96 well Filter Plate, uncoated 96 well Filter Plate, coated with lipidsCoat filterAdd donorplate with& acceptorlipidssolutions96 well Receiver PlateAcceptor solutionDonor solutionCoupleplatestogether forpermeationIncubateplates forseveralhoursAnalyze concentrations of donorand acceptor solutions using UVplate reader or using LC-MS.Calculate permeability from thedonor and acceptor concentrations.(B) New PAMPA Process Flowchart using Corning® Gentest Pre-coated PAMPA Plate System96 well Filter Plate, pre-coated with lipidsAcceptor solutionAdd donor& acceptorsolutions96 well Receiver PlateDonor solutionCoupleplatestogether forpermeationIncubateplates forseveralhoursAnalyze concentrations of donorand acceptor solutions using UVplate reader or using LC-MS.Calculate permeability from thedonor and acceptor concentrations.Figure 1. Experimental Setup of PAMPA• In the traditional PAMPA method, the user needs to prepare the artificial membrane by coatingthe filter plate with a lipids solution• In the new PAMPA method, the Corning Gentest Pre-coated PAMPA Plate System is ready to use,allowing the user to skip the step of preparing the artificial membraneTraditional PAMPA membrane constructionPorous filterArtificialmembraneCorning Gentest PAMPA membrane constructionPorous filterArtificialmembraneFigure 2. Comparison of the Traditional PAMPA Membrane and the Corning Gentest PAMPAMembraneTraditional PAMPA membrane construction:• Coated by user (by adding a solution of lipids in each well)• The porous filter is soaked with a solution of lipids• Excessive solvents• Long permeation pathwayCorning Gentest PAMPA membrane construction:• Pre-coated by manufacturer• A lipid-oil-lipid tri-layer structure is constructed in the pores of the porous filter• No excessive solvents• Short permeation pathway (closer to the biological membrane)Figure 3. Automated PAMPA Deck Layout on a Tecan® Genesis 150 Configured with an IntegratedRobotic Arm, UV Plate Reader, and IncubatorA standard Tecan Genesis 150 robot deck had the capacity to fit all the necessary consumables torun the assay: one (1) Corning Gentest Pre-coated PAMPA Plate System (Cat. No. 353015); three(3) Falcon® 96 Well UV Transparent Microplates (Cat. No. 353261), one (1) Falcon 96 well 2 mLPolypropylene Storage Plate (Cat. No. 353966); three (3) 200 µL and two (2) 1000 µL disposablepipet tip racks; and one (1) “dummy” receiver plate was used to rest the PAMPA insert during accessto the donor wells. Integrated to the robot are a UV Plate reader, temperature controlled incubator,and multiple hotel shelves.2
Figure 4. The Integrated Robot Gripper and Pipette Tips are Fully Compatible with the Corning®Gentest Pre-coated PAMPA Plate SystemThe Tecan® RoMa (robotic arm) was programmed with vectors to retrieve and manipulate individualcomponents of the assembly (lids, receiver, and donor components). The RoMa also transferredthe UV assay plates to the plate reader. Both 200 µL and 1000 µL conductive Tecan DiTi (disposabletips) were programmed to reproducibly aspirate and dispense with liquid level sensing and a1.0 mm offset to minimize the risk of a membrane puncture or tear.Permeability Pe (10 -6 cm/s)12108642Plate 1 (1-month old)Plate 2 (6-month old)Plate 3 (12-month old)Permeability Pe (10 -6 cm/s)12108642Manual AssayAutomated Assay0famotidineamiloridefurosemidehydrochlorothiazideantipyrinecaffeinemetoprololnaproxen0famotidineamiloridefurosemidehydrochlorothiazideantipyrinecaffeinemetoprololnaproxenFigure 5. Reproducibility and Stability ofthe Corning Gentest Pre-Coated PAMPAPlate SystemComparison of PAMPA permeability valuesof 8 compounds obtained using 3 pre-coatedplates prepared at different times: Plate 1 wasprepared one month before the day of assay,Plate 2 was prepared 6 months before the dayof assay, Plate 3 was prepared one year beforethe day of assay.Figure 6. Automated Assay Improved CVCompared to Manual AssayComparison of PAMPA permeability values of 8compounds obtained using manual assay andautomated assay methods. The automatedassay method produced better CVs.A. Traditional PAMPA Membrane B. Corning Gentest PAMPA MembraneHuman Absorption (%FA)False negativeLow PermeabilityHigh PermeabilityFalse positiveHuman Absorption (%FA)False negativeLow PermeabilityHigh PermeabilityFalse positiveLog P e (cm/s)Log P e (cm/s)Figure 7. Corning Gentest PAMPA Membrane Improves Correlation with Human Absorption DataComparison of the performance of traditional PAMPA membrane and the Corning Gentest PAMPAmembrane by analyzing the correlation of the permeability data with the human absorption datafor a set of 35 compounds. The permeability data of the traditional PAMPA membrane and thehuman absorption data were cited from Reference 3. The permeability data of the Corning GentestPre-Coated PAMPA Plate System membrane were obtained using UV VIS measurements; bothdonor and acceptor buffers were PBS, pH 7.4; and the PAMPA plate system was incubated at roomtemperature for 5 hours without agitation.3
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Automation of Pre-coated PAMPA Plates Improves Predictability ...

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