Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

SLAS Screen Design and Assay Technology Special Interest Group SLAS2017 Presentation

666 views

Published on

Adam Weinglass and Mary Jo Wildey from Merck & Co. share their winning presentation from SLAS2017 in Washington, DC. Join the conversation in the SLAS Screen Design and Assay Technology Special Interest Group LinkedIn group at https://www.linkedin.com/groups/3867725.

Published in: Government & Nonprofit
  • Be the first to comment

SLAS Screen Design and Assay Technology Special Interest Group SLAS2017 Presentation

  1. 1. Screen Design and Assay Technology Special Interest Group (SIG) February 6th, 2017 Cell-Based In Vitro Assay Automation: Balancing Technology & Data Reproducibility/Predictability Adam Weinglass, Mary Jo Wildey Merck & Co., Inc., Kenilworth, NJ, USA
  2. 2. Challenge • Supporting a given GPCR program with structurally distinct classes of molecules classes at different stages of optimization. • Ensuring In vitro support for a target executed within Merck and at a CRO. • Across companies, scientists’ experience & access to instrumentation and automation varies. • Driving Chemistry SAR requires reproducible data between operators and sites. • Alignment of protocols & technologies critical-requires communication. • All data is uploaded to the same database where common tools are used for analysis. Mission High quality, reproducible & comparable data across sites every week!
  3. 3. Agonism of Gq-coupled GPCR monitored using IP1 accumulation assays in Tier 1 & 2 of ROP GPR40 Agonist • Homogeneous Time-Resolved Fluorescence (HTRF) based assay to measure the accumulation of D-myo-inositol monophosphate (IP1) – a metabolite of IP3. • LiCl in the assay buffer inhibits phosphatases which degrade IPs & leads to IP1 accumulation. • Competition – intracellular accumulation of IP1 inhibits the FRET signal. Assay Procedure Day 1: • Seed cells in 384 well plates in growth medium. • Incubate cells overnight at 37ºC/5% CO2 Day 2: • Remove growth media from cells. • Add Stimulation buffer containing LiCl. • Treat with compound (Echo transfer) for 1 hour @ 37ºC/5% CO2. • Lyse cells and add FRET reagents. • Read on the Envision.
  4. 4. GPCR1/HEK293 IP1 Accumulation assay performing acceptably at CRO using statistical criteria (MSR Analysis) Potency Mean Ratio; Minimum Significant Ratio • Classical Z’>0.4 does not guarantee reproducible EC50/IC50-need a ‘global reliability index’ of a dose response screen. • Minimum Significant Ratio (MSR) defines the ‘smallest potency ratio between any pair of compounds that is statistically significant. [SD is the standard deviation of the replicate EC50 results from an N=2]. SD MSR *22 10= [Eastwood et al. (2008), JBS] [Andy Lau (MRL Statistics)]
  5. 5. Issue: Chemistry concerned about EC50 variability observed in a new structural series of cpds in GPCR1/HEK293 Assay @ CRO: Variability not captured in GPCR1 Tracking Metrics • Performance formally evaluated periodically by MSR analysis. • Ref. EC50, Z’ & Signal/Baseline tracked in every assay. • Tracking metrics not ‘designed’ around distinct structural series- relevant to ‘well-behaved’ series. MSR Analysis Tracking Metrics
  6. 6. Investigation 1: Evaluate variability of structurally diverse series within MRL using GPCR1/HEK293 assay Dump/Pat N=1 vs. N=2 (separate days) 1 10 100 1000 10000 1 10 100 1000 10000 Run1 Run 2 0.1 1 10 100 1000 10000 10 100 1000 10000 Ratio Geometric Mean Potency Ratio vs Geometric Mean Potency Ratio MR RLs LsA 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 Run1 Run 2 0 50 100 150 200 250 300 0 50 100 150 200 250 300 Run1 Run 2 MR 0.5, MSR 19 MR 1.2, MSR 5.81 Potency Efficacy KW CRO Variability within Merck. Variability between sites. Dump/Pat @ KW, single operator Dump/Pat: KW vs CRO
  7. 7. Problem Statement Both Intra- & Inter-site metrics for assays struggling to meet criteria for SAR support with Dump/Pat • Structural series identified that perform inconsistently in GPCR1 (and GPCR2) IP1 assays; variability in EC50 observed when using the “Dump/Pat” method to remove growth media: • Variability between companies (MRK vs CRO), departments (Biology vs Assay Operations) & biologists (scientist 1 vs. scientist 2). • How can we modify these primary assays to generate reproducible results regardless of who or where the assay is run? – ‘Simplify’ the assay protocol to eliminate inconsistencies • Based on the high plasma protein binding of chemical matter, considered the following steps to improve consistency. – Run in serum, so residual amounts from Dump/Pat no longer an issue. – Find a means to consistently remove residual serum (i.e. move away from Dump/Pat) that can be applied to all orthogonal assays across sites.
  8. 8. Methods for Removing Growth Media • Dump/Pat – Widely used “traditional” technique for adherent cell based assays – Manually invert plate, shake/flick plate several times over a waste reservoir, blot the plate on paper towels several times – Highly inconsistent between operators as well as with the same operator within a day or across days • Cell Suspension – Eliminate the cell adherent process (no overnight incubation); same day assay – Complete removal of serum in the assay (Wash cells in assay buffer) – Aligns with the protocol used for cAMP assays • Centrifugal Washer (BlueWasher) – Uses centrifugation to remove buffer from assay plates, designed to be used as an automated “dump/pat’ paradigm – No contact with cell monolayer – Variety of programming options which can be used with or without a dispensing protocol • Plate Washer/Dispenser (BioTek, GNF, PlateMate etc.) – Automated method to aspirate from and dispense into an assay plate – Complicated programming, risk of losing cells, known residual volume remaining in the wells, variable between cell types, very finicky
  9. 9. 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 Run1 Run 2 0.1 1 10 100 0.1 10 1000 Ratio Geometric Mean Potency Ratio vs Geometric Mean Potency Ratio MR RLs LsA Ref Line 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Run1 Run 2 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 Run1 Run 2 MR 1.1, MSR 5.14 Potency Efficacy MR 7.23, MSR 45.9 KWDump/Pat KW Suspension Unacceptable variability. Cpds more potent in suspension mode & capture ‘false negatives’ Dump/Pat vs. Cell Suspension Suspension @ KW, same operator, separate days: Solid Correlation Methods for Removing Growth Media Suspension cells: Challenging assay performance using GPCR1/HEK293 cells and a wrinkle/opportunity with structurally distinct series
  10. 10. Washing by Centrifugation 10Reproducible/homogenous; Low %CV; No interference with cell layer.
  11. 11. 0.1 1 10 0.1 10 1000 Ratio Geometric Mean Potency Ratio vs Geometric Mean Potency Ratio MR RLs LsA Ref Line MR 0.84, MSR 2.7 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 Run1 Run 2 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Run1 Run 2 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 Run1 Run 2 Potency Efficacy KW Bluewasher KWDump/Pat MR 12.76, MSR 42.79 Dump/Pat vs. Bluewasher Bluewasher @ KW, same operator, separate days: Strong Correlation Acceptable variability. Cmpds are more potent in Bluewasher paradigm & capture ‘false negatives’ Methods for Removing Growth Media Centrifugal washing: Acceptable assay performance using GPCR1/HEK293 cells and the same wrinkle/opportunity
  12. 12. Comparison of Growth Media Methods for GPCR1/HEK293 Cells: Structurally distinct series is sensitive to media removal paradigm - centrifugal washer provides acceptable assay statistics Dump/Pat vs. Suspension Dump/Pat vs. Bluewasher Suspension vs. Bluewasher 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 Run1 Run 2 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 Run1 Run 2 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 Run1 Run 2 Dump/Pat Dump/Pat Suspension SuspensionBluewasher Bluewasher MR 12.76, MSR 42.79 MR 1.2, MSR 5.88MR 7.23, MSR 45.9 Across several assays/programs, centrifugal washing provides a reliable & consistent means to remove growth media within the context of a statistically robust assay GPCR1/HEK293 IP1 Assay GPCR1/HEK293 IP1 Assay
  13. 13. 13 Which assay reality is the right one? GPCR1/HEK293 Dump/Pat • Benchmark to in vivo efficacy. • Reliable for certain classes. GPCR1/HEK293 Centrifugal Washer • Closer to serum-free ‘intrinsic’ potency. • Improved consistency in the data. • Decreases the rate of “false” negatives. MSR=42.8 on test set with distinct structural class
  14. 14. GPCR1 assay format changed/clarified SAR Relatively inactive historic cpds suddenly aligned with modified media removal paradigm In vitro profile aligns better with translatable assays O O N OH O L-001842278 hGPR40 IP1 EC50 224 nM hGPR120 IP1 EC50 424 nM L-002485812 (Racemic) hGPR40 EC50 103 nM hGPR120 EC50 318 nM O O N OH O L-002190770 GPR120 LID Compound h GPR40 IP1 EC50 3440 nM Really?? hGPR120 IP1 EC50 105 nM O O N OH O L-002312918 hGPR40 IP1 EC50 324 nM hGPR120 IP1 EC50 121 nM O O N OH O F O O N OH O L-002190770 GPR120 LID Compound hGPR40 IP1 EC50 154 nM Bluewasher; Cpd A (Racemic) hGPCR1 EC50 103 nM Bluewasher; Cpd C hGPCR1 EC50 324 nM Bluewasher; Cpd B hGPCR1 EC50 224 nM Dump/Pat Historic Data; Cpd X hGPCR1 EC50 3440 nM-?? Bluewasher; Cpd X hGPCR1 EC50 154 nM
  15. 15. Biology/Pharmacology Team→Happy Updated Project Status Program Team→Comfortable Automation Team→Not so Happy
  16. 16. Methods for Removing Growth Media • Dump/Pat – The widely used “traditional” technique for adherent cell based assays – Manually invert plate, shake/flick plate several times over a waste reservoir, blot the plate on paper towels several times – Highly inconsistent between operators as well as with the same operator within a day or across days • Cell Suspension – Eliminate the cell adherent process (no overnight incubation); same day assay – Complete removal of serum in the assay (Wash cells in assay buffer) – Aligns with the protocol used for cAMP assays • Centrifugal Washer (BlueWasher) – Uses centrifugation to remove buffer from assay plates, designed to be used as an automated “dump/pat’ paradigm – No contact with cell monolayer – Variety of programming options which can be used with or without a dispensing protocol • Plate Washer/Dispenser (BioTek, GNF, PlateMate etc.) – Automated method to aspirate from and dispense into an assay plate – Aspirate/dispense heights and positions must be programmed and periodically checked to minimize risk of losing cells – Residual volume remaining in the wells – Variable between cell types
  17. 17. Plate Washers • Several vendors and models of plate washers available for walk-up use or robotic integration – Automated method to aspirate from and dispense into an assay plate – Aspirate/dispense heights and positions must be programmed and periodically checked to minimize risk of losing cells – Biotek EL406 is currently the favored online and off-line plate washer • In an effort to standardize, minimize error, and improve efficiency of the GPCR1 and GPCR2 IPOne assays, the manual method was successfully transferred to an integrated robotic system
  18. 18. Walk-Up vs. Integrated Automation Walk-Up Protocol Integrated Automation Protocol Manual movement of assay plates between instruments Hands free automation of assay steps Imprecise plate processing times Considerable reduction of biologist’s time in assay execution Less accurate incubation times of assay plates with compound/agonist Software-controlled plate pace timings and plate order processing Increased chance of human error such as plates being processed out of order or being dropped Accurate incubation timings throughout assay execution Limited number of plates possible per assay run with numerous compound plates being screened against multiple cell lines Capacity for increased number of compounds across multiple cell lines which can be processed in parallel
  19. 19. GPCR1 and GPCR2 IP1 HighRes Automation Flow Read EnVision 615/665nM Media Exchange/Stimulation Buffer Addition Biotek Washer/peripump dispenser 10ul change tips Compound and controls addition Echo 555 50nl IP One Standard Curve Addition Agilent Bravo 10ul, columns 1&24 Detection Addition Multidrop Combi 10ul Incubation Cytomat C-24 Incubator 1 hour @37deg. C Incubation Cytomat C-24 Incubator 1 hour @RT Prep time: Automated/Unattended time: ~5 hours
  20. 20. Walk-up vs. Integrated Automation Assay Validation GPR120 IPONE Human cells: manual vs HiRes(2) Run 1 & Run 2 0.1 1 10 10 100 1000 10000 Ratio Geometric Mean Potency Ratio vs Geometric Mean Potency Ratio MR RLs LsA Ref Line Statistical Analysisof "Bland-Altman" Plot Equivalence Test Results Reproducibility Test Results N 18 MR 1.30 MSR (Within-Run) 1.81 RLs 1.12 LsA 0.72 1.50 2.34 Sig Diff Between SD 0.0908 Runs Test, p = 0.0017 GPR120 IPONE MOUSE cells: manual vs HiRes(2) Manual & HiRes 0.1 1 10 10 100 1000 10000 Ratio Geometric Mean Potency Ratio vs Geometric Mean Potency Ratio MR RLs LsA Ref Line Statistical Analysisof "Bland-Altman" Plot Equivalence Test Results Reproducibility Test Results N 18 MR 0.98 MSR (Within-Run) 1.87 RLs 0.84 LsA 0.53 1.15 1.83 Sig Diff Between SD 0.0957 Runs Test, p = 0.8205 •MR represents the average potency ratio across the 2 runs •MSR represents the largest potency ratio that can be considered a random change within a run of the assay MR acceptance criteria 1+/- 0.5 MSR acceptance criteria 3+/- 1.0 GPCR2 GPCR2
  21. 21. Correlation Across Multiple Cell Lines with Integrated Automation and the Biotek Washer Mouse Cell Line R2= 0.977 Manual vs. HiRes with 45-degree Line 10 100 1000 10000 10 100 1000 10000 Run1 Run 2 R2=0.976 Manual vs. HiRes with 45-degree Line 10 100 1000 10000 10 100 1000 10000 Run1 Run 2 Human Cell Line Walk-Up and Automated Assay show good correlation across both cell lines
  22. 22. Root Cause Discussion Observations • In-house biology team – Expert in vitro biologists – Most will prefer Dump/Pat – Competent with user-friendly (robust) walk-up automation – Relies on automation staff for fine tuning and QC on systems like plate washers • External biology team – Expert in vitro biologists – Prefers Dump/Pat – Competent with user-friendly (robust) walk-up automation – Do not have access to equivalent automation staff for fine tuning and QC on systems like plate washers • Automation team – Experts in walk-up and integrated automation – Understand the need to monitor and adjust devices such as plate washers for plate type, lot changes, cell line types
  23. 23. • Assumption that all methods were equivalent all the time so it was OK to substitute dump/pat for the plate washer • Underestimated the shortcomings of dump/pat and the impact of residual media • Couldn’t predict the chemistry sensitivity to the residual serum • We had the perfect storm…… ….and a new solution…. . . . that blurs the skillset lines of assayists and automation Root Cause Discussion Observations
  24. 24. Building Fit-for-Purpose In vitro Assays: Points for Discussion & Lessons Learned • How do we design in vitro cell-based functional assays to improve accuracy/translatability within the context of infrastructure? • Understand how broader team are using the data (e.g. ranking cpds vs. interpreting in vivo efficacy data/making human dose projections). • Ensure ‘identical’ conditions are used for orthogonal assays that may ultimately be compared (e.g. cross-species evaluation). • Be appreciative of the physiochemical properties of chemical matter being evaluated (e.g. PPB, sticking to plastics). • Try to project! Evaluate the skillset of all the different groups that may be involved in developing/running assays during program lifetime. • If assays are likely to be externalized to CRO’s or transfer between Departments, assess whether all groups have the same equipment infrastructure-do they need to?

×