What and How Much Information Can We

Really Extract in an Automated Manner

from LC/MS Data
John P. Walsh1, Robyn A. Rourick1, Mark Bayliss2 and Vitaly Lashin3

1Kalypsys,
Inc., 10420 Wateridge Circle, San Diego, CA 92121;
2Advanced Chemistry Development, Inc., 110 Young Street,

Toronto, ON, Canada, M5C 1T4;

3Advanced Chemistry Development, Bakuleva 6, Str. 1,

Moscow, Russia, 117513

ASMS 2006 Conference. Seattle, WA

May 30, 2006
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Presentation Outline

• The Kalypsys Model

• The Analysis Challenge

• The Informatics Challenge

• Metabolism Case Study

• The Road Forward

2
 Kalypsys has technology resources that are
unmatched in the industry

uHigh-throughput automation
Gene silencing & Protein tech
Chemistry-intensive technology

< we continue to evolve our technology to ensure

dominance in the field >
3
Proprietary Lead Discovery Technologies
Fully-Automated Hit Picker
Assay Development Workstations

Assay
Development Pharma
Hit IC50/EC50 Lead
Screening Preclinical
Compound Selection Confirmation Profiling
Studies
Synthesis

- Primary Screening System

- Follow-up Screening System
- Integrated Compound Storage

Fully-Automated uHTS System

4
 …parallel compound profiling increases our
knowledge of each compound studied

ultra
high throughput screen medicinal
uHTS chemistry

reduces time to clinic

target
selectivity
identify compounds with novel mechanisms
potency

ADMET earlier understanding of toxicology

5
The Analytical Challenge - Objectives
– On-the-fly LC/MS profiles of compound library
– Purity
– ADME
– Stream-lined interface for acquiring samples
– Speed
– System monitoring

– Automated processing of raw data

– Little user intervention
– Data archival

6
The LC/MS Analysis Constraints
• Throughput requirements to handle the volume of hits
(>1200 per day)
– fast run-times
– minimal cycle time
– more than one sample at a time
– fast acquisition times on detectors

• Minimal sample consumption (<10uL)

• Minimal to no dilution necessary for injection
• Minimize downtime
• Easy and flexible integration into the workflow
• Maximize information output
7
The Analysis Solution
Bring the Analysis to the Samples!

• Extend Hit-picker system to autosampler

– Direct access to compound collection
– Tecan becomes assay station
– No sample waste

• Parallel LC/MS System

– 4 x 1525u HPLC pumps
– 4 x HPLC columns
– 4-channel dual-λ UV detector
– 5-channel MUX LCT Time-of-Flight Mass Spec (Lock-mass)

8
System Design

9
System Integration

• Mounted Valco valves on

Tecan
• Top Loading Valves for
minimal injector void
volume
• Custom tips
manufactured for
interface to valves
• Defined locations for
injection one at a time or
all at once

10
Workflow Enhancements

List of
Compounds

Transfer Data Manual

Compounds LC/MS Processing Upload
to Analysis Analysis at
Plate Transfer Plate Workstation
across Depts.

Company
On-the-fly Data Database
4x LC/MS Processing and
Upload
Purity
Analysis

11
The New Information Challenge

Sample Data Data Reporting &

Submission Acquisition Processing Searching

12
The New Information Challenge

Sample Data Data Reporting &

Submission Acquisition Processing Searching

13
Informatics Challenges – Front-End
Sample Data Data Reporting &
Submission Acquisition Processing Searching

• Can handle compound or plate identifiers

• Database query for relevant information

– sufficient quantity on the hitpicker?
– analyzed previously?

• Intelligent arrangement of queue order

• Generation of relevant instrument files

– “hit list” generated for Hitpicker work
– analysis sequence created for MUX acquisition

14
The Informatics Challenges – Back-End
Sample Data Data Reporting &
Submission Acquisition Processing Searching

ACD Automation Server

• An application for automated processing
– sequentially performs processing tasks

• Provides rigorous assessment of results

– CODA detection algorithm for finding peaks
– accurate mass confirmation

• Integrates with detailed ACD tools

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Data Storage & Searching - Value Added
Sample Data Data Reporting &
Submission Acquisition Processing Searching

ACD SpecDB
• All processed results are stored in SpecDB files
• “Power user” functionality
• Ability to change processed results before finalization

Company database

• Final results are uploaded to database

• Textual peak tables are stored for searching
• Links to data are passed for traceability
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Profiler Expansion
• Expand Integration into a Property Profiler System
• Rapidly gather SAR relevant information rapidly after screen

• Initial Implementation Assays

− Solubility
− PAMPA
− Log P
− Log D
− Metabolic Stability
− Chemical Stability
• Secondary Assays
− Protein Binding
− Caco-2
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Metabolite ID Approach
• Provide processing to support detailed elucidation
– Adaptable to high-throughput data

• Equipment is not altered, methods are not changed

– Effective algorithms and processing will extract data

• Reduce false positive reporting

• Expansion capable to allow for multiple applications and

future development

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IntelliXtract Objectives
• BOTTLENECK = Data Reduction and Analysis

• Focus solely on the data analysis part of the problem

• Software requirements

– Auto componentization/deconvolution
– Minimization of software based settings on a sample
by sample basis

– Critical analysis of ion clusters

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Data Reduction Strategy
• Perform chromatographic peak extraction on the dataset

• Componentize the extracted peaks

• Determine the 12C, 13C, (14C), 12C+2, 12C+3, 12C+n… ions for each
ion cluster series

• Determine the most probable molecular ion 12C based on the

presence/absence of supporting adduct ions

• Determine the most probable metabolites based on a mass delta

lookup list

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Metabolism Case Study

IntelliXtract In Vitro Metabolite Analysis

of KLYP A

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Instrument Traces 09:26:52
052306_30minCID_909028_01 3: Diode Array
0.39 0.80 Range: 1.223e-3

2.5e-4

2.25e-4

2.0e-4

1.75e-4
UV
1.5e-4
AU

1.25e-4 1.25 8.47

2.70
1.0e-4
2.94
7.5e-5 7.10
7.85
5.0e-5
2.27
2.5e-5 6.24

0.0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
052306_uSomes_None_06 1: TOF MS ES+
0.65 0.71 TIC
8.87e3

MS
8.78
%

1.10
7.43 8.16

6.58 6.82 7.63

8.41
5.72 5.92 6.49 9.28 9.71 10.81 11.24
3.26 3.32 3.83 4.64 4.95 10.24
4.78
1.55
2.28

11 Time
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

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IntelliXtract: Peak 1 Post-Selection

Diagnostic Fragments

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IntelliXtract: Peak 2 Post-Selection

Diagnostic Fragments
NL 269 maps to parent
NL 300 maps to 16+parent

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IntelliXtract: Peak 3 Post-Selection

Diagnostic Fragments
NL 269 maps to parent
NL 300 maps to 16+parent

25
Data Reduction Statistics
Following data processing

• 4294 chromatographic like peaks remaining

• 2075 peaks after +/-100mDa mass filtering (952 w/ 50mDa)

• 221 12C identified ions (+/- 100mDa mass filtering)

• 66 [M+H]+ Ions (12C, 12C/13C = Pass)

• 6 potential ions consistent with metabolite knowledge based

modifications

• 3 components containing structurally relevant fragments

[parent + 2 mono-oxidated]

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 IntelliXtract: Component Table
• Lists all the components in table format

• Controls which masses appear in the spectra and chromatogram

• Provides additional insight into potential metabolites

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IntelliXtract: Additional Metabolites

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Data Reduction Summary

Molecule m/z Fragments Neutral Loss Notes

159 NL = 269
KLYP A 428 Parent
144 NL = 284

175 NL = 269
M1 (+16) 444 +16 on Portion B of Parent
144 NL = 300

175 NL = 269
M2 (+16) 444 +16 on Portion B of Parent
144 NL = 300

M3 (+16) 444

M4 (+16) 444

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Conclusions
• Validation of IntelliXtract method with previous results

• IntelliXtract confirmed metabolites that were not detected by

manual inspection
– Indicates powerful data extraction capabilities that exceed
traditional potential

• Precision of the software, coupled with the integrated MUX

Profiler, will lead to many applications.
• Metabolite ID, degradant studies, CMC

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Implementation Strategy
• Build upon current ACD package for high-throughput Purity Analysis
using the MUX
– Re-mine existing data sets processed by Automation Server
– Identify and characterize impurities/degradants in libraries

• Evaluate stability samples, utilizing the power to analyze pathways

– Metabolic Stability, Chemical Stability

• Take to production on the Profiler platform

– Complete assay and analysis automation

• Processing and data management on the Automation Server

– Reduce the data reduction/analysis bottleneck
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Acknowledgements
Chemistry Informatics
Charles Taylor Qiner Yang
Xing Cheng
Peter Simms

Screening ACD
Paul Queeney Andrew Anderson
Gavin Shear
Eric Milgram
IT
Shawn Constable

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