Contenu connexe Similaire à SLAS2014 Screen Design and Assay Technology SIG Presentation (20) Plus de SLAS (Society for Laboratory Automation and Screening) (11) SLAS2014 Screen Design and Assay Technology SIG Presentation1. DOE challenges &
opportunities
agenda
WAYNE J LEVIN
PREDICTUM INC.
LEVIN@PREDICTUM.COM
!
WWW.PREDICTUM.COM
About Predictum
Use the right methods (and there are more of them)
Build a system
Assume nothing
PREDICTUM INC. WWW.PREDICTUM.COM ©2014
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About Predictum
we increase productivity
more exploitable insights in less time, effort, cost
less frustration
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DOE purpose
look at numerous effects comprehensively yet isolate each
effect’s influence independently of the other effects
in less time, effort and cost
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2. use latest & greatest methods
Definitive Screening Designs
latest in computer-generated optimal designs
Split-plot designs
hard to change factors
definitive screening
more independence of effects
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Optimal Design -20 runs
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Definitive screening - 21 runs
Optimal Design
Definitive screening
Sacrifice some D-efficiency, but not much
Zero correlation among main
effects and 2-way interactions
Some correlation among main
effects and 2-way interactions
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3. split-plot designs
The concept of hard to
change is broader than you
might think.
!
Here multiple pipetting
constitutes hard to change
where materials are the same
across wells
it’s a multivariate universe
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Scenario
The tool is typical of HTS, in that there is limited
ways a chemical can be varied within a plate.
4. generating errors
Under REML
highlighted items
show type I
errors if analyzed
traditionally
generating errors
DOE can be a lot of work
take the time, make the effort
longer more complex experiment designed right often yields
more correct insights than a series of small experiments
if not done correctly, DOEs will generate
type I and type II errors
confusion and frustration
!
Under
Traditional
highlighted items
show type II
errors if analyzed
traditionally
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R&D and Improvement Initiatives are launched and completed in isolation.
build a system
time
Copyright © 2013 Predictum Inc.
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Confidential
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5. This is typically what happens.
Insufficient institutional memory.
Each project is isolated.
What if they were connected? What if
they started with all relevant
information previously acquired and
paid for?
!
Slippage on retaining acquired, prior insights. Must
pay to re-acquire what was previously known.
time
Copyright © 2013 Predictum Inc.
Confidential
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assume nothing
be methodical
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time
Copyright © 2013 Predictum Inc.
Confidential
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6. Utilizing Design of Experiment Statistical
Models to Improve Assay Development
in High Throughput Biology
Diana Ballard of Predictum Inc., Samuel Hasson of NIH, Wayne Levin of Predictum Inc.
Overview
Step 1
All 6 plates
Nozzle Buffer
Type
1
HEPES
2
Tris
3
Tris
4
HEPES
5
Tris
6
HEPES
7
Tris
8
HEPES
similar tests assume that the entire experiment
pH PK Enzyme
concentration
8
100 uM
7
10 uM
8
10 uM
7
10 uM
8
100 uM
8
10 uM
7
100 uM
7
100 uM
Nozzle
1
2
3
4
5
6
7
8
REML
ANOVA
Buffer Type
<.0001
<.0001
PK Enzyme concentration*Buffer Type*Nested Packaging
of assay
<.0001
<.0001
pH
Buffer Type*pH *PK Substrate [Nested Packaging of assay]
Buffer Type*PK Enzyme concentration
pH*PK Substrate[Nested Packaging of assay]
PK Enzyme concentration
PK Enzyme concentration*Timing*Nested Packaging of assay
Magnesium ion concentration*pH
pH*Buffer Type
pH*Timing*Nested Packaging of assay
Buffer Type*Magnesium ion concentration*PK Substrate
[Nested Packaging of assay
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
<.0001
0.0004
0.0006
<.0001
0.0186
0.0111
0.2577
0.0377
0.0432
0.0651
0.0669
0.1765
0.2379
Factor / Effect
PK Enzyme
concentration
Step 2
Error Term in the model
PK Enzyme concentration
* Buffer Type * pH *
Iteration
Source (Partial List of 85)
Buffer Type
Column within plate
Experimental unit (EU)
The wells on all plates that had unique bottle setups for PK Enzyme.
Step 1 had 8 unique bottles, repeating step 1 in iteration 2. Across
both iterations, there are 16 EU.
The wells on all plates that were treated by one of the 16 bottles
made for step 1.
PK Enzyme concentration
* Buffer Type * pH *
Iteration
Magnesium concentration
* Iteration
Magnesium ion
concentration
Nested packaging
of assay
Magnesium
solution
5 uM
water
1 2 3 4 5 6 7 8
Introduction
Step 3
Changed on a per plate basis. Any plate could have gotten either
setting. Across both iterations, there are 12 EU.
Plate * Iteration
PK Enzyme
concentration *
Magnesium ion
concentration
Buffer Type *
Magnesium ion
concentration
The intersection of the EU for PK Enzyme and the EU for
Magnesium ion concentration. 16 x 4 = 64 EU across
both iterations.
Buffer Type *
Nested packaging
of assay
All 6 plates
The wells on all plates that had unique bottle setups for Step 2 and
Magnesium concentration. Across both iterations, there are 4 EU.
The intersection of the EU for Buffer Type and the EU for
Nested packaging of assay. 16 EU x 12 EU = 192 EU
across both iterations.
PK Enzyme concentration
* Buffer Type * pH *
Magnesium concentration
* Iteration
PK Enzyme concentration
* Buffer Type * pH *
Magnesium concentration
* Iteration
PK Enzyme concentration
* Buffer Type * pH * plate *
Iteration
The intersection of the EU for Buffer Type and the EU for
Magnesium ion concentration. 16 x 4 = 64 EU across
both iterations
The inclusion of two iterations is critical
Prob > F
FIG 1
Visualize
Buffer Type *
Magnesium *
PK Substrate
[Nested
Packaging
of Assay]
All 6 plates
handled in classical full and fractional factorial designs leading them to provide
BSA
0.2%
concentration water
solution
1 2 3 4 5 6 7 8
Prob > F
instead of the experimental unit to
Plate
Methods
OBJECTIVES:
Step 4
1
2
3
the experimental unit for this model
FIG 2
2800
PK Substrate
400nM
40nM
PK Substrate 400nM
combined with 40nM
Kinase Glo
4
5
6
2750
2700
PK Enzyme
2650
10
100
2600
2550
FACTORS: All eight of the following factors are difficult to vary on the Thermo Multi Drop
Plate
Step 5
1
2800
2
3
Tris
2750
2700
Buffer Type
2650
Timing
5 min
10 min
4
5
6
Conclusions
2600
HEPES
2550
10
20
30
40
50
60
70
80
90
100 110
Tris
HEPES
Plate
Step 6
RESPONSE:
Kinase Glo
4 uL
nothing
1
2
3
4
5
6
DESIGN NOTES:
Step 7
Read all 6 plates
Step 8
Wait Several Days
Do steps 1-7 again
All 6 plates
Copyright © 2013 Predictum Inc.
experimental design and we found a straightforward method to implement its use in DOE
8. ACT
•what was learned,
that if proven valid,
can be implemented?
•new questions/
insights sought?
•what next?
STUDY
•what happened that
was expected?
•what did not happen
that was expected?
•what happened that
was not expected?
PLAN
•insights sought
(specify model: main
effects, interactions,
quadratics)
•responses & goals
(maximize, minimize,
match target)
•factors & levels
•identify difficult to
change factors
•constraints on factors
& levels
•design experiment
(evaluate properties)
•detail expectations
•operational
definitions of run
changes and response
measurement
•list all assumptions