expanding the compound management toolkit: leveraging technology to streamline sample qa/qc in high...
DESCRIPTION
The continual evolution of compound management labs in terms of sample capacity and capabilities provides opportunities to improve the associated QA and QC operations. As new technologies enable support of greater plate densities and library sizes, new approaches to QA and QC must follow suit. • Review of potential for error in compound management labs and impact of new technology on QA/QC efficiency • Leveraging sample color to quickly identify problems • Novel methods for monitoring the health of large sample libraries over time • Correlating visual properties of solubilized samples to experimental results and calculated propertiesTRANSCRIPT
Expanding the Compound Management toolkit
Pierre BaillargeonHTS/Lead Identification
About Scripps Florida
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I. Introduction to Lead Identification and Compound Management at Scripps Florida
II. Review of issues with DMSO solvated compounds in HTS libraries and methods for addressing these issues
III. An introduction to the Plate Auditor
IV. Impact of Plate Auditor results on our CM & HTS operations
Integrated Assay Development, uHTS and Compound Management
• >982K Compound Screening Collection• >622K Proprietary (largest in
academia, ~30k unique compounds, focused sub-libraries, professionally curated)
• >360K Public Domain (NIH)• Integrated Compound QC (LC-MS, Plate
Auditor)
The Lead Identification Laboratories at Scripps
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Where robotics, chemistry and biology join forces to help discover new drugs
• Located in Jupiter, FL• >175 targets screened in over 185 primary
campaigns to generate more than 60 million data points for academic & industrial collaborators
• S1P1 drug candidate entered phase 1 clinical trials in January 2011
Issues With Compound Libraries
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I. Introduction to Lead Identification and Compound Management at Scripps Florida
II. Review of issues with DMSO solvated compounds in HTS libraries and methods for addressing these issues
III. An introduction to the Plate Auditor
IV. Practical applications of Plate Auditor
Compound Management Challenges
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Job cuts
Budget cuts limiting technology investment
Building a library to meet new research requirements
Sample data management
Integration of automation technology
New automation technology
Sample quality
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
16.70%
16.70%
66.70%
66.70%
33.30%
33.30%
66.70%
What are your primary concerns in your current role or your department?
Graph reproduced from the 2012 IQPC ‘Setting up Compound Management Globally’ Compound Management report
http://www.compoundmanager.com/uploadedFiles/EventRedesign/UK/2012/May/11314006/Assets/IQPC---Compound-management-060112.pdf
% of respondents
Compound Management Responsibilities & Customers
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Compound Management
HTS
DMPK & Pharm
External
Collaborator
Therapeutic Areas
Compound
Vendors
Medicinal Chemistry
What role does CM play at Scripps?• HTS Library Procurement
• HTS Library Stewardship
• HTS Library QC (via LC-MS)
• Cherry pick for HTS Core
• Compound re-synthesis & restock
• Receive & reformat samples from external collaborators
• Ship samples to external collaborators
• Medicinal Chemistry support
Compound Management Responsibilities & Customers
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Compound Management
HTS
DMPK & Pharm
External
Collaborator
Therapeutic Areas
Compound
Vendors
Medicinal Chemistry
What operational challenges are encountered during these interactions?• Verifying quality of external
samples & sample data
• Degradation or precipitation of samples over time
• Enforcing internal QA/QC standards
• Tracking sample properties & genealogy in a way that can be easily audited in the future
• Managing large sample libraries which are constantly in flux (multiple copies, samples becoming depleted, new acquisitions, etc.)
Compound Management Responsibilities & Customers
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How do we currently address these issues and what are the limitations?
• LC-MS (time consuming)
• Acoustic auditing (time consuming, labware dependent)
• Manual visual inspection (time consuming, subjective)
• Suspension of solids or precipitated samples (sonicating, heating, solvent change, etc.)
What operational challenges are encountered during these interactions?• Verifying quality of external
samples & sample data
• Degradation or precipitation of samples over time
• Enforcing internal QA/QC standards
• Tracking sample properties & genealogy in a way that can be easily audited in the future
• Managing large sample libraries which are constantly in flux (multiple copies, samples becoming depleted, new acquisitions, etc.)
Opportunities For Error in Sample Distribution
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COMPOUND SUBMISSION• Has the correct sample been put into the labware?• Is the volume and concentration of the sample accurately recorded?
COMPOUND PROCESSING• Has the sample store delivered the correct samples?• Has the liquid handling automation pipetted the samples properly?• Has the plate been registered correctly?• Is the sample itself of high quality?
COMPOUND DELIVERY• Do we have a record of what we are delivering?
Room for improvement! How can we QC samples more efficiently?
Introduction to Plate Auditor
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I. Introduction to Lead Identification and Compound Management at Scripps Florida
II. Review of issues with DMSO solvated compounds in HTS libraries and methods for addressing these issues
III. An introduction to the Plate Auditor
IV. Practical applications of Plate Auditor
Are empty/partially filled wells in Quadrant 1?
Do these wells contain precipitate?
Are these wells partially filled?
My db says this well is
empty. Does it actually
have sample in it?
My HTS detection format is sensitive to purple compounds. Where are they?
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Why Do We Need Plate Auditor?
Plate Auditor Applications & Features
• Instant detection of errors from compound reformatting • Detect missed/partially filled wells, quadrant effects
• “Pre-screen” compound plates for HTS assay interferents• Detect colored compounds, suspended materials
• Identify database entry errors in corporate LIMS db• Determine which full/empty wells in plate differ from the full/empty well assignments in corporate db
• Periodic check-ups on the “health” of stored compounds• Detect ppt. formation, evaporation
• Detection of compound solubility• Identify HTS library samples that have “crashed out” of solution
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What is Plate Auditor?
• A custom HTS plate reader, incorporating recent advances in machine vision, image analysis, & the spectroscopy sciences
• It saves labor by automatically identifying & annotating issues specific to compound libraries:
• Colored compounds• Precipitate / Crystallization• Low volume / Full wells
• It can be used in “stand-alone” mode, or integrated with automation
• Works with microtiter plates
• Fast: reads a 384 well plate in <1 minute
• All measurements are non-contact
• User-friendly, intuitive analysis software
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Plate Auditor Features: Instrument Design
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Plate Auditor Features: Color Recognition & Classification
Color Detected Dye Used
Limit of Detection
(uM)
Red Allura Red 35.1
Green Brilliant Green 9.0
Blue Euroglaucine 0.6
Yellow Tartrazine 12.7
Violet Gentian violet 1.3Befo
re P
rocessin
gA
fter
Pro
cessin
g
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Plate Auditor Features: Precipitate Detection
Compound plates contain precipitates which are masked by color:
Plate Auditor precipitate analysis is:• Insensitive to color• Able to detect ppt. beyond “naked eye” inspection
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Plate Auditor Features: Low Volume Detection
Plate Auditor distinguishes normal wells (“full”) from empty/partially filled wells (“low volume”)
40uL 20uL 10uL 8uL 4uL 2uL 1uL
What can cause a well to have a low volume?• Incorrectly reported
volume in LIMS
• Oversampled by cherry picking operations
• Evaporation
• Liquid handling errors (clogged tips, samples transferred to incorrect wells/quadrants, etc.)
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Plate Auditor Features: LIMS/Corporate Database Auditing
RESULTS OF COMPOUND PLATE QUERY IN CORPORATE DATABASE:• Scripps database displays several wells as
empty (white rectangles in figure on left)
RESULTS OF THE SAME COMPOUND PLATE QUERY IN Plate Auditor DATABASE:• Plate Auditor compares instrument results
to records archived in corporate db
• Plate Auditor identifies discrepancies with corporate db records(red “x’s” in figure on left):
• Colored compounds (upper left)• Full wells that corporate db assigns as “empty”
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PLATE AUDITOR AUDITS CORPORATE DB RECORDS
Introduction to Plate Auditor
The Scripps Research Institute © 2012 – All rights reserved
I. Introduction to Lead Identification and Compound Management at Scripps Florida
II. Review of issues with DMSO solvated compounds in HTS libraries and methods for addressing these issues
III. An introduction to the Plate Auditor
IV. Practical applications of Plate Auditor
Addressing Opportunities For Error in Sample Distribution
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What new information do we obtain with Plate Auditor in the process?• We know which wells have been filled and which have low volume• We know which wells contain colored samples• We know which wells contain compounds that have precipitated
What do we do with this information?• Compare filled & low volume wells against LIMS to verify proper liquid handling & plate
registration• Compare ‘color fingerprint’ of samples against database of known samples to identify
issues with incorrect concentration & samples• Take measures to put precipitated samples back into solution (sonication, heating, etc.)• Keep a historical record of the health of the plate at the point when it was delivered (a
receipt of transaction)
Practical Examples: HTS Library Analysis
This includes Scripps Florida’s internal 622k Drug Discovery library and the NIH’s Molecular Libraries-Small Molecule Repository (MLSMR) 362k library.
To date, Scripps has run over 1,000 plates through Plate Auditor resulting in over 1,000,000 wells which have been analyzed for color, precipitate and low volume artifacts.
How can we leverage all of this new data to improve our workflow?
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Practical Examples: HTS Library Analysis
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This includes Scripps Florida’s internal 622k Drug Discovery library and the NIH’s Molecular Libraries-Small Molecule Repository (MLSMR) 362k library.
To date, Scripps has run over 1,000 plates through Plate Auditor resulting in over 1,000,000 wells which have been analyzed for color, precipitate and low volume artifacts.How can we leverage all of this new data to improve our
workflow?
2) Use sample color to track fidelity of CM processes & sample quality3) Use sample color as a predictor of sample performance in HTS4) Precipitate results measure effectiveness of solvation SOPs
5) Using precipitate detection to monitor library health
1) Identify LIMS database errors in real time
6) Other interesting observations: Labware QC
Plate Auditor data usage at Scripps Florida
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1) Well data classified via Plate Auditor GUI
2) Well data merged with sample information from Corporate Plate DB and stored in Plate Auditor DB
3) Resulting data set exported from Plate Auditor DB and merged with HTS, Cheminformatics and LC-MS results
By making Plate Auditor data available in the same web-based informatics portal as other HTS data, project biologists & chemists can easily examine HTS library samples for volume, color and precipitate issues
… examining the plate detail view reveals a pattern in quadrant 1…
Identifying LIMS database errors in real time
When checking reformatted plates, Plate Auditor finds high # of LIMS disagreement wells…
… checking the LIMS record reveals samples in quadrant 1 are not registered …
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Identifying LIMS database errors in real time
… CM team updates LIMS record and runs plate through Plate Auditor to confirm the corrected LIMS record matches the physical locations of samples within the plate.
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Real time detection of registration errors saves CM time and resources
LIMS Comparison feature also used to detect formatting errors
Using Sample color to track CM processes & sample quality
We now can query and identify color inconsistencies across entire compound collections or the same sample distributed across multiple working copies of
the library.
Plate Auditor data can be used to monitor consistency of sample color over time…
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Using Sample color to track CM processes & sample quality
Why should we care about sample color?
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Two copies of plate prepared by external vendor reveal inconsistencies
Using Sample color to track CM processes & sample quality
Running color comparison reveals color inconsistencies…
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
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Color data identifies problematic samples earlier in CM processes
Sample color is indicative of compound quality (MLSMR)
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Degraded sample (left) vs. expected sample (right), both verified via LC-MS
Expected sample (left) copy 2 vs. failed sample (right, precipitated) from copy 1, both verified via LC-MS
Expected cherrypicked samples (left, middle) vs. failed screening deck sample (right), all verified via LC-MS
Plate Auditor results predict LC-MS results
2011 vs. 2009
2011 vs. 2011
Cherrypicked samples vs. screening deck
Sample color is predictive of its performance in HTS
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Typical primary screen plate
Typical confirmation screen plate
Why else should we care about sample color?
With the Plate Auditor, can we show a true correlation between sample color and its hit activity?
Sample color is predictive of its performance in HTS
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Is there correlation between sample color and various assay properties?
ABSBLA
CALCIU
MCNGC
EPIA
BS
FLIN
T
FLUO8 FP
HTRFLU
MI
QFRET
TRFR
ET0%
10%
20%
30%
40%
50%
60%
70%
80%
% of hits containing color vs. HTS readout technology
N=90 assays
Readout Technology Average % hitsOverall average % hits
Sample color data has impacted HTS assay development (biologists) & follow up of HTS hits by Project Chemists
MLPCN HTS compound collection
HTS hits0%
2%
4%
6%
8%
10%
12%
14%
16%
18%
% Colored samples 12/2007-12/2011
Precipitate results measure effectiveness of solvation SOPs
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Tubes containing DMSO solvated samples (pre-sonication)
Tubes containing DMSO solvated samples (post-sonication)
How can we monitor changes in precipitate? What impact does it have on CM operations?
Quantitative precipitate monitoring improved compound solvation SOPs
Heat, mixing solvent change, sonication, etc…
Using precipitate detection to monitor library health
Precipitate filled screening plate“Normal” screening plate
Plate Auditor automatically identifies plates with high levels of precipitate
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Using precipitate detection to monitor library health
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Plate Auditor helps to identify solubility issues when analyzing samples from sublibraries …
Library% of library containing precipitate
Sublibrary A 0.1%
Sublibrary B 2.5%Sublibrary C 0.8%
A
B
C
… providing a useful dataset when communicating
problems with suppliers, collaborators and CM staff
internally.
BioFocus comparison: HTS copy 3 (in use) vs. HTS copy 4 (from freezer)
Plate # Copy 3 Copy 42 10 343 17 604 34 975 65 1286 24 39
TOTAL 150 358% increase 139%
Using precipitate detection to monitor library health
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Helps identify existing samples that need re-solvation...or remediation
Baseline precipitate levels can be established with Plate Auditor data
Representative plate from copy 3 of sublibrary B
Representative plate from copy 4 of sublibrary B
Interesting Observations: Labware QC
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Why do we care if there are air bubbles in the polypropylene?Air bubbles cause plates to be warped & unusable with automation.
When creating new compound plates, Scripps discards plates with air bubbles as precautionary measure.
Interesting Observations: Labware QC
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Air bubbles can also exist in 1536 well polypropylene plates.
In addition to plate warping, bubble artifact in 1536 well poses additional problems for machine vision QA/QC.
Interesting Observations: Labware QC
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Plate Auditor images can be used to document plate damage and deformities
Interesting Observations: Labware QC
Are all wells created equally?
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2.25mm
2.25mm
.28mm offset
.28mm offset
Plate with offset wells Plate with no offset wells
Plate from different vendor
What impact do offset wells have on experiments?
Interesting Observations: Labware QC
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Plate Auditor provides data to communicate problems with third parties
Impact Review
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Review of Plate Auditor impact at Scripps Florida
2) Using Sample color to track CM processes and sample quality:• Color data identifies problematic samples earlier in CM
processes• Plate Auditor results predict LC-MS results3) Use sample color as a predictor of sample performance in HTS:• Susceptibility of a HTS assay to color interference is
considered during assay development (biologists) and hit-follow up (chemists)
• Correlation between color and hit performance helps determine HTS false negative/positive rate4) Use precipitate as a measure of solvation effectiveness:
• Quantitative precipitate monitoring improved compound solvation SOPs
1) Detect LIMS database errors in real time:• LIMS DB errors are detected & corrected immediately upon
plate creation
5) Use precipitate as an indicator of library health:• Helps identify existing samples that need re-solvation...or
remediation6) Other interesting observations:• Images can be used to document plate damage and
deformations• Images can be used to communicate problems with third
parties
What operational challenges are encountered during these interactions?• Verifying quality of external
samples & sample data
• Degradation of samples over time
• Enforcing internal QA/QC standards
• Tracking sample properties & genealogy in a way that can be easily audited in the future
• Managing large sample libraries which are constantly in flux (multiple copies, samples becoming depleted, new acquisitions, etc.)
Impact of Plate Auditor at Scripps
• Received samples are quickly QC’d upon arrival; Plate Auditor results are then compared to the vendor’s plate map.
• Plate Auditor periodically audits compound library over its lifecycle
• Automated QA/QC of samples removes subjective analysis and prevents CM staff burnout
• Plate Auditor plate “snapshots” allow us to easily & visually track sample genealogy
• Routine QC of large libraries now possible by automating labor intensive visual inspections
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Plate Auditor Summary & More Information
Scripps has licensed the Plate Auditor technology to Brooks Life Science Systems for commercialization & sale. The Brooks Plate Auditor will be part of the Brooks sample analysis line of instrumentation.
Additional information, including Plate Auditor Pilot Screen results, can be found in “Monitoring of HTS compound library quality via a high-resolution image acquisition and processing instrument”. Baillargeon P, Scampavia L, Einsteder R, Hodder P. ; J. Lab Automation, June 2011
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Lead Identification:Peter HodderPierre BaillargeonPeter ChaseJoseph DatskoChristina EberhartImarhia EnogieruRoss EinstederKatharine EmeryVirneliz Fernandez-VegaLina DeLucaFranck MadouxBecky MercerLaura Pedro-RosaChristine PinelloLouis ScampaviaTimothy Spicer
http://hts.florida.scripps.edu [email protected]
Acknowledgements
Scripps Florida Funding
Corporation
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