Expanding the Compound Management toolkit: leveraging technology to streamline sample QA/QC in high throughput environments

Expanding the Compound
Management toolkit

Pierre Baillargeon
HTS/Lead Identification

About Scripps Florida

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

The Scripps Research Institute © 2012 – All rights reserved

The Lead Identification Laboratories at Scripps

Where robotics, chemistry and biology join forces to help discover new drugs

Integrated Assay Development, uHTS and Compound Management
• Located in Jupiter, FL • >982K Compound Screening Collection
• >175 targets screened in over 185 primary campaigns to • >622K Proprietary (largest in
generate more than 60 million data points for academic & academia, ~30k unique compounds, focused
industrial collaborators sub-libraries, professionally curated)
• S1P1 drug candidate entered phase 1 clinical trials in • >360K Public Domain (NIH)
January 2011 • Integrated Compound QC (LC-MS, Plate Auditor)


Issues With Compound Libraries




IV. Practical applications of Plate Auditor


Compound Management Challenges
What are your primary concerns in your current role or your department?

Sample quality 66.70%

New automation technology 33.30%

Integration of automation technology 33.30%

Sample data management 66.70%

Building a library to meet new research requirements 66.70%

Budget cuts limiting technology investment 16.70%

Job cuts 16.70%

% of respondents
0% 20% 40% 60% 80% 100%

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


Compound Management Responsibilities & Customers

What role does CM play at
HTS
Scripps?
• HTS Library Procurement
• HTS Library Stewardship
Medicinal DMPK &
Chemistry Pharm
• HTS Library QC (via LC-MS)
• Cherry pick for HTS Core
• Compound re-synthesis & Compound
Management
restock
• Receive & reformat samples
from external collaborators Compound External
Vendors Collaborator
• Ship samples to external
collaborators
• Medicinal Chemistry support Therapeutic
Areas



What operational challenges are
encountered during these HTS

interactions?
• Verifying quality of external
samples & sample data
Medicinal DMPK &
Chemistry Pharm
• Degradation or precipitation of
samples over time
• Enforcing internal QA/QC
Compound
standards Management

• Tracking sample properties &
genealogy in a way that can be
easily audited in the future Compound External
Vendors Collaborator
• Managing large sample libraries
which are constantly in flux
(multiple copies, samples
Therapeutic
becoming depleted, new Areas
acquisitions, etc.)



What operational challenges are How do we currently address
encountered during these these issues and what are the
interactions? limitations?
• Verifying quality of external
samples & sample data • LC-MS (time consuming)
• Degradation or precipitation of
samples over time
• Acoustic auditing (time
• Enforcing internal QA/QC consuming, labware
standards dependent)

• Tracking sample properties &
genealogy in a way that can be • Manual visual inspection (time
easily audited in the future consuming, subjective)

• Managing large sample libraries
which are constantly in flux
(multiple copies, samples • Suspension of solids or
becoming depleted, new precipitated samples
acquisitions, etc.) (sonicating, heating, solvent
change, etc.)


Opportunities For Error in Sample Distribution

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




IV. Practical applications of Plate Auditor


Why Do We Need Plate Auditor?
Are empty/partially filled Do these wells
wells in Quadrant 1? contain precipitate?

My db says
this well is
empty. Does
it actually
have sample
in it?

My HTS detection format is sensitive to Are these wells partially
purple compounds. Where are they? filled?

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


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


Plate Auditor Features: Instrument Design


Plate Auditor Features: Color Recognition & Classification
Before Processing

Limit of
Color
Dye Used Detection
Detected
(uM)
Red Allura Red 35.1
Green Brilliant Green 9.0
Blue Euroglaucine 0.6
Yellow Tartrazine 12.7
Violet Gentian violet 1.3
After Processing


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


Plate Auditor Features: Low Volume Detection

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.)

40uL 20uL 10uL 8uL 4uL 2uL 1uL

Plate Auditor distinguishes normal wells (“full”) from empty/partially filled
wells (“low volume”)


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”

PLATE AUDITOR AUDITS CORPORATE DB RECORDS


Addressing Opportunities For Error in Sample Distribution

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
To date, Scripps has run over This includes Scripps Florida‟s
1,000 plates through Plate internal 622k Drug Discovery
Auditor resulting in over library and the NIH‟s Molecular
1,000,000 wells which have been Libraries-Small Molecule
analyzed for color, precipitate Repository (MLSMR) 362k library.
and low volume artifacts.

How can we leverage all of this new data to improve our workflow?


Practical Examples: HTS Library Analysis
To date, Scripps has run over This includes Scripps Florida‟s
1,000 plates through Plate internal 622k Drug Discovery
Auditor resulting in over library and the NIH‟s Molecular
1,000,000 wells which have been Libraries-Small Molecule
analyzed for color, precipitate Repository (MLSMR) 362k library.
and low volume artifacts.
How can we leverage all of this new data to improve our workflow?
1) Identify LIMS database errors in real time

2) Use sample color to track fidelity of CM processes & sample quality

3) Use sample color as a predictor of sample performance in HTS

4) Precipitate results measure effectiveness of solvation SOPs

5) Using precipitate detection to monitor library health

6) Other interesting observations: Labware QC


Plate Auditor data usage at Scripps Florida
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


Identifying LIMS database errors in real time
When checking reformatted plates, Plate Auditor finds high # of LIMS disagreement wells…

… examining the plate detail view reveals a pattern in quadrant 1…

… checking the LIMS record reveals samples in quadrant 1 are not registered …


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.

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

Plate Auditor data can be used to monitor consistency of sample color over time…

We now can query and identify color inconsistencies across entire compound collections or
the same sample distributed across multiple working copies of the library.



Why should we care about sample color?


Running color comparison reveals
color inconsistencies…

Sample 1

Sample 2

Two copies of plate prepared by external vendor
reveal inconsistencies
Sample 3

Sample 4

Sample 5

Color data identifies problematic samples earlier in CM processes


Sample color is indicative of compound quality (MLSMR)
2011 vs. 2009

Degraded sample (left) vs.
expected sample (right), both
verified via LC-MS

2011 vs. 2011

Expected sample (left) copy 2 vs.
failed sample (right, precipitated)
from copy 1, both verified via LC-MS

Cherrypicked samples vs. screening deck
Expected cherrypicked samples
(left, middle) vs. failed screening
deck sample (right), all verified via
LC-MS

Plate Auditor results predict LC-MS results


Sample color is predictive of its performance in HTS

Why else should we care about sample color?

Typical primary screen plate Typical confirmation screen
plate

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

Is there correlation between sample color and various
assay properties?

% Colored samples % of hits containing color vs. HTS readout technology
12/2007-12/2011 N=90 assays
18% 80%
16% 70%
14% 60%
12%
50%
10%
40% Readout Technology
8%
30% Average % hits
6%
20% Overall average % hits
4%
2% 10%
0% 0%
MLPCN HTS compound HTS hits
collection

Sample color data has impacted HTS assay development (biologists) &
follow up of HTS hits by Project Chemists


Precipitate results measure effectiveness of solvation SOPs
How can we monitor changes in precipitate? What impact does it have on CM operations?

Heat, mixing
solvent
change, sonicat
ion, etc…

Tubes containing DMSO Tubes containing DMSO
solvated samples (pre- solvated samples (post-
sonication) sonication)

Quantitative precipitate monitoring improved compound solvation SOPs


Using precipitate detection to monitor library health

“Normal” screening plate Precipitate filled screening plate

Plate Auditor automatically identifies plates with high levels of precipitate


Using precipitate detection to monitor library health

Plate Auditor helps to identify solubility issues when
analyzing samples from sublibraries … A

% of library
Library containing
precipitate

Sublibrary A 0.1% B
Sublibrary B 2.5%
Sublibrary C 0.8%

… providing a useful dataset when
communicating problems with
suppliers, collaborators and CM staff C
internally.


Using precipitate detection to monitor library health freezer)
BioFocus comparison: HTS copy 3 (in use) vs. HTS copy 4 (from

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

Plate # Copy 3 Copy 4
2 10 34
3 17 60
4 34 97
5 65 128
6 24 39
TOTAL 150 358
% increase 139%

Helps identify existing samples that need re-solvation...or remediation


Interesting Observations: Labware QC

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.



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.



Plate Auditor images can be used to document plate damage and
deformities



Are all wells created equally? .28mm offset

2.25mm

2.25mm

.28mm offset



What impact do offset wells have on experiments?

Plate with offset wells Plate with no offset wells

Plate from
different
vendor

Plate Auditor provides data to communicate problems with third parties


Impact Review
Review of Plate Auditor impact at Scripps Florida
1) Detect LIMS database errors in real time:
• LIMS DB errors are detected & corrected immediately upon plate creation
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 results
3) 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 rate
4) Use precipitate as a measure of solvation effectiveness:
• Quantitative precipitate monitoring improved compound solvation SOPs
5) Use precipitate as an indicator of library health:
• Helps identify existing samples that need re-solvation...or remediation
6) Other interesting observations:
• Images can be used to document plate damage and deformations
• Images can be used to communicate problems with third parties


Impact of Plate Auditor at Scripps

What operational challenges are
encountered during these
interactions?
• Verifying quality of external • Received samples are quickly QC‟d
samples & sample data upon arrival; Plate Auditor results are
then compared to the vendor‟s plate
• Degradation of samples over map.
time • Plate Auditor periodically audits
• Enforcing internal QA/QC compound library over its lifecycle
standards • Automated QA/QC of samples removes
subjective analysis and prevents CM
• Tracking sample properties & staff burnout
genealogy in a way that can be
easily audited in the future • Plate Auditor plate “snapshots” allow
us to easily & visually track sample
• Managing large sample libraries genealogy
which are constantly in flux • Routine QC of large libraries now
(multiple copies, samples possible by automating labor
becoming depleted, new intensive visual inspections
acquisitions, etc.)


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


Acknowledgements
Scripps Florida
Funding Corporation

http://hts.florida.scripps.edu bpierre@scripps.edu

Lead Identification:
Peter Hodder
Pierre Baillargeon
Peter Chase
Joseph Datsko
Christina Eberhart
Imarhia Enogieru
Ross Einsteder
Katharine Emery
Virneliz Fernandez-Vega
Lina DeLuca
Franck Madoux
Becky Mercer
Laura Pedro-Rosa
Christine Pinello
Louis Scampavia
Timothy Spicer


Expanding the Compound Management toolkit: leveraging technology to streamline sample QA/QC in high throughput environments

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