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.

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. 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. 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. 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. 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. 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. 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. 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. 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. Washing by Centrifugation
10Reproducible/homogenous; Low %CV; No interference with cell layer.

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
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100
150
200
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350
0 50 100 150 200 250 300 350
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Run 2
0.1
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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. 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
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10000
0.1 1 10 100 1000 10000
Run1
Run 2
0.1
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0.1 1 10 100 1000 10000
Run1
Run 2
0.1
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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
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. 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. Biology/Pharmacology Team→Happy
Updated Project Status
Program Team→Comfortable
Automation Team→Not so Happy

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. 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. 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. 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. 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. 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
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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. 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. • 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. 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?