Automation and Informatics to Profile

Degradation Products

John P. Walsh
February 28, 2007
PITTCON 2007: 1900-5

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 Background – New kalypsys approach to degradation

Queuing System – Run timing with CMG link

Chamber Integration – Proprietary dual-chamber design

stability
LC/MS Analysis – Proprietary MUX integration
chemical and photo
Analysis Data – ACD Labs IntelliXtract data reduction

Stability Assay Manager – Intranet-based data management

Wrap-up – Conclusion and future direction

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background
previous approach to degradation

• Every stability experiment treated as a “one-off”

• Prepared manually in scintillation vial requiring large sample amounts

• Incubation on a dry bath incubator for a period of time (variable)

• Historical tracking of degradation work was difficult

– no database log for which compounds were assessed
– all data captured in Scientist’s notebook

• Data acquisition performed on LC/UV system

– long gradients with manual data processing

• Chemical stability only (acid, base, oxidative)

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strategy
new approach to degradation
• Project based approach to stability
– workflow integration
– project management tools for information management
– data warehousing in the company database

• Integration with the Kalypsys “Profiler” system

– flexible automation workstation (liquid handler & robo fingers)
– central laboratory approach
– use existing automation “plug-ins”

• Automated sample prep

• Prove concept with classic assay prep

– large sample consumption vials
– dry bath incubators for temperature control
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strategy
new approach to degradation

• Data acquisition with LC/UV/MS

– shorter gradients
– exact mass for identification of degradants

• Data processing with ACD Labs intelliXtract

• Chemical stability (acid, base, neutral, oxidative)

• Photo stability
Proposed Workflow:

liquid handler
sample ACD Labs data
sample prep
queue w/ online LC/MS
processing manager

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 strategy Sample Liquid Handler ACD Labs
Data Manager
workflow Queue w/ online LC/MS Processing

• Proprietary Informatics resources at Kalypsys allow for well

connected data management solutions

– sample queuing system integrated with Compound Management

– assay manager to track each stability data set
– implementation of ACD Labs IntelliXtract

• Hardware innovations provides means for sample analysis

– stability-indicating & Discovery stability assay union

– in-house stability chamber design and construction
– patent pending MUX LC/MS to liquid handler integration for
online sample analysis
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queuing system sample
liquid handler
sample prep
ACD Labs
data manager
experiment initiation queue
w/ online LC/MS
processing

• Queuing system “plug-in” adapted for stability work

– channel all requests into central location
– allows pacing of work

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queuing system sample
liquid handler
sample prep
ACD Labs
data manager
plating management queue
w/ online LC/MS
processing

• Separate queues for each stability experiment (chem or photo)

– unique plating from Compound Management Group
– can insert logic into queue (i.e. chemical precedes photo
stability)

Compound
Management
Plating

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chamber design sample
liquid handler
sample prep
ACD Labs
data manager
concept to implementation queue
w/ online LC/MS
processing

Dual chamber design

– discreet chemical stab chamber
– discreet photo stab chamber

CAD designed from 80/20 toolbox

Constrained to automation platform

– size of entire unit
– access of robo-fingers

“Clean” air and exhaust chambers

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photo stability sample
liquid handler
sample prep
ACD Labs
data manager
chamber requirements queue
w/ online LC/MS
processing

Chamber designed on ICH Guidance

– Q1B Option 1
– dark controls

96-well format
– 48 sample capacity
– foil cover for “dark” samples

Incubator maintains temperature

– reduces effect of heat output from
metal halide bulb

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photo stability sample
liquid handler
sample prep
ACD Labs
data manager
chamber validation queue
w/ online LC/MS
processing

ICH Q1B guidance provides validation procedure

Actinometric standard
– 2% w/v Quinine Monohydrochloride Dihydrate

Guidance requires 10% loss of absorbance

Chamber provided endpoint at ~24hrs

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chemical stability sample
liquid handler
sample prep
ACD Labs
data manager
assay parameters queue
w/ online LC/MS
processing

CMG supplied 10mg/mL DMSO stocks

2mg/mL stability solutions

– 0.02N HCl
– 0.06N NaOH
– 3% H2O2 (OTC)
– pH 7.4 phosphate buffer
– DMSO

Incubation at 40ºC

20 x 4mL vials in Al block

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chemical stability sample
liquid handler
sample prep
ACD Labs
data manager
sample analysis queue
w/ online LC/MS
processing

• Chemical stability sample analysis

online

• No reaction quenching required

• Accurate mass data collection on

MUX LCT Premier

• 8min cycle time

– 1x50mm 3um C18 columns (4)
– 0.200mL/min per column

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chemical stability sample
liquid handler
sample prep
ACD Labs
data manager
LC/MS integration queue
w/ online LC/MS
processing

Required connectivity between multiple instruments

– liquid handler for dispensing in valves
– valve control to start injections
– Masslynx for sample lists and LC/MS state

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analysis data sample
liquid handler
sample prep
ACD Labs
data manager
ACD Labs IntelliXtract queue
w/ online LC/MS
processing

• ACD Labs IntelliXtract Version 10.05

• Control (t0h) samples were compared with degraded samples (t2h, t4h)

• Control spectra was auto-annotated with diagnostic fragments

• Degraded samples were compared to the “reference” spectra

– differences in intensity (defined levels)
– mass shifting from parent and/or fragments

• Common modification list used to assess degradation pathway

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analysis data sample
liquid handler
sample prep
ACD Labs
data manager
ACD Labs IntelliXtract queue
w/ online LC/MS
processing

Bisacodyl Reference Spectra

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analysis data sample
liquid handler
sample prep
ACD Labs
data manager
ACD Labs IntelliXtract queue
w/ online LC/MS
processing

Bisacodyl in pH 7.4 phosphate buffer

degraded - t4h

control - t6m

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analysis data sample
liquid handler
sample prep
ACD Labs
data manager
ACD Labs IntelliXtract queue
w/ online LC/MS
processing

Bisacodyl in 3% hydrogen peroxide

degraded - t4h

control - t6m

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degradation profile sample
liquid handler
sample prep
ACD Labs
data manager
ACD Labs IntelliXtract queue
w/ online LC/MS
processing

Degradation Conditions Degradation Observations

DMSO No Degradants detected

De-acetylation 2X greater than

Buffer pH 7.4
control

Acid – 0.02M HCl No Degradants detected

Base – 0.06M NaOH No Degradants detected

Deacetylation, Di-deactylation
Oxidation – 3% H2O2
products

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data manager sample
liquid handler
sample prep
ACD Labs
data manager
stability archival queue
w/ online LC/MS
processing

• Study management “plug-in” adapted for stability work

– easy data mining
– referencing to experiments/reports

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data manager sample
liquid handler
sample prep
ACD Labs
data manager
experiment detail queue
w/ online LC/MS
processing

Each experiment item references a

details page

– experiment variables
– conditions
– notebook references
– structure
– data files

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wrap-up
conclusions

• Designed a stability program from a top-down perspective

– used project management tools
– derived a solution that integrates into a workflow

• Used existing informatics “plug-ins” for a rapid deployment

• Custom designed and built a stability chamber accessible to
automation
– discreet photo and chemical stability chambers
– integrated dry bath incubators for temp control

• Demonstrated photo degradation of an FDA standard

• Integrated LC/MS analysis online for real-time monitoring of rxns
• Deployed powerful and evolving ACD tools for data interrogation
• Created a warehouse for data archival and retrieval
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wrap-up
future direction

• Standardize the assay format (vials vs. plates)

• Evolve the informatics tools to auto-populate fields

• Optimize LC/MS system for robust and ideal performance

• Fiber optics for remote placement of lamps?

• Multiple column chemistry selection?

• Finalize all custom code for routine deployment

• Integrate Combine function of IntelliXtract into Automation Server

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acknowledgements

Kalypsys, Inc.
Charles Taylor
Robyn Rourick
Stewart Smith
Qiner Yang
Chris Petersen

ACD Labs
Mark Bayliss

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