case studies outlining analytical approaches, challenges and

Copyright © 2011 Eli Lilly and Company 1

Characterization of Extractables and LeachablesAssociated with Pharmaceutically Relevant Materials: Case Studies Outlining Analytical

Approaches, Challenges and Examples

Douglas KiehlEli Lilly & Company, Indianapolis, IN, USA

Some Key Definitions


Copyright © 2011 Eli Lilly and Company 3Copyright © 2011 Eli Lilly and Company 3

E&L evaluation is a Regulatory expectation for materials contacting or with potential to contact API or drug product

‐ container/closure systems‐ device components‐mfg. process materials‐ ICH Q3, Q6(A,B), Q7A, Q8, 21 CFR 211, EMEA/205/04

Why…

Polymer Supply Chain for Pharmaceutically Relevant Materials

Components for• Packaging• Devices• Container/closure• Processing

• Colorants• Processing aids• Lubricants

End products &Suppliers

N• Antioxidants• Processing aids• Light stabilizers• Antistatic agents

Masterbatcher

N‐1• Bulk process chemicals

• Storage stabilizers

Monomer

N‐3• Initiators/Catalysts• Storage stabilizers• Antioxidants• Processing aids

Polymer

N‐2

Natural Raw

Material

N‐4


Knowledge Space

QBD and Extractable/Leachable Design Space

AB

C

D

E

F G

H

I

JK

LMNP

Extractables

Copyright © 2011 Eli Lilly and Company 5Figure courtesy of ELSIE

Drug Product ALeachables Drug Product B

Leachables

A

C

E

I

Product BDesign Space

Product ADesign Space

B

C

DJK

M Each unique formulation will have a unique leachables design space

MNP

Control Space

J

K

M

Leachables

Control Space refers to the normalvariation range of the product within conditions of intended use

Extractable/Leachable Assessment Example

Evaluate design and define components

Stage II Extractables Study• potential leachables• safety/risk assessment of

extractables

Stage I Extractables Study• profile extractables• gain knowledge and understanding of material

Assess and select component raw

materials

Safety/risk assessment of

leachablesConclude and submit

data

Develop and validate analytical methods to

identify and quantify target leachables

Leachable Study• drug product stability storage• monitor target leachables

Product Life CycleMonitor internal and

supplier initiated changes




extractables



materials



data






Extractable/Leachable Assessment Example


Evaluate Design and Define Components

• The container/closure or device components are part of the drug product

• What are some desired performance characteristics of a C/C or device?‐ Accurate, reproducible and reliable delivery of drug product‐ Robust physical and mechanical operation and construction‐ Protection of drug product across range of intended use conditions

• Manufacturability—complexity, reproducibility and reliability

• Patient‐friendly characteristics‐ Convenience and ease of use‐ Appearance and dimensions‐ Ruggedness across diverse patient population




extractables



materials



data






Extractable/Leachable Assessment Approach Example


Assess and Select Component Raw Materials

• Obtain as much information as possible from component vendor about raw materials and production of components

• Complete list of chemicals and additives, under CDA if necessary

• As much history from “upstream” processors as available

• List of extractables if testing completed by vendor, including conditions under which extractables were determined‐ Extraction solvents, methods and conditions‐ Analytical conditions

• In‐use history of component lot in industry, if applicable




extractables



materials



data








Study Conditions PurposeAnalytical Techniques

Stage I Extractables

• Aggressive• Water, IPA, Hexane• Reflux

• Establish extractables profile• Gain understanding of material

HS GC‐MSGC‐MSLC‐MS, w/PDAICP‐MSIC

Stage II Extractables

• Milder conditions than reflux

• Water, IPA, buffers,“model solvents”

• May include placebo

• Identify extractables that may not be stable to reflux

• Simulate worst case drugproduct—packaging contact

• Impact of excipients

Same as above

Leachables

• Drug product on stabilitystorage

• Conditions of intended use

• Determine real‐time leachables

• Monitor leachables over shelf life of product

Determined with toxassessment of extractables



Extractable Compound Evaluation

• One of the most important and analytically challenging steps is the initial establishment of a comprehensive extractable compound profile‐ Unknown compound structural characterization is not trivial

• Establishing an extractable compound profile is important to enabling understanding of a material

• Often involves characterization of complex mixtures of diverse organic molecules in a variety of matrices

• Multiple analytical techniques are required


• Toxicologist screens characterized extractables for potential problematic compounds or alerting structures

• Qualitative SAR for compounds for which sufficient toxicity data is not available

• Provides indication of potential problematic compounds early enough to permit a decision involving changes to the design or materials for a component or container/closure system

• Early enough to work with supplier to mitigate changes

Safety/Risk Assessment of Extractables




extractables



materials



data








• Toxicologist evaluates qualitative and quantitative real‐time storage leachables data

• Ensure that confirmed leachables are consistent with observed extractables

• Confirmed leachables are correlated with available toxicity data

• If toxicity data is not available, SAR correlation or in‐vivo/in‐vitrostudies may be necessary

• Additional considerations may include:‐ Drug product dose‐ Administration route‐ Dose frequency‐ Treatment duration‐ Patient population

Safety/Risk Assessment of Leachables




extractables



materials



data








Product Life Cycle Management

• Strategy for gaining knowledge of, and addressing change should be established

• Supplier/vendor should alert customers of any changes in composition, production or construction

• Supplier/vendor should be aware of any changes initiated by “upstream” processors

• Routine extractables testing to monitor variation across lots


Extractable Compound EvaluationThe structural characterization of diverse, unknown organic compounds, particularly if present in complex mixtures and multiple matrices, represents the greatest challenge in establishing an initial extractable compound profile for a given material. This often requires extensive effort, expertise and sophisticated capabilities.


Workflow for Identifying Unknowns with LC‐MS

Extract material withappropriate solvent and conditions

Localand external databases

LC‐MS ElementalcompositionCcHhNnOoSsFf

m/z

Concentrate and/or filter

sample

Exact mass spectra

Set elemental limit space

Process data

MSn

experiments on unknowns

Individual component

ID

170 190 210 230 250

0

200

400

600

800

Inte

nsity

221.1900

composition(individual

components)

Search elemental

Propose Structure

and retention time match

Elemental composition

No match

Interpret fragmentation

Authentic compound available?

Confirm Identity

HO

Yes

NoAdditional experiments

and techniques required

HO

Confirmed



How complex is “complex” for pharmaceutically relevant materials?

Chromatographic complexity

16 18 20 22 24 26 28 30 32 34 36Time (min)

20

40

60

80

100

Rel

ativ

e Ab

unda

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K

J

EDA,BF

G1,G,H

I

LM

N

O PQ

RS T ZU V W X YC

5 10 15 20 25 30 35 40 45Time (min)

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A,B’C’

D’Total ion chromatogram (m/z 80‐1500) of IPA extract of multi‐layer polymer film

How Complex is “Complex?”


Composite mass spectrum of all components

Mass spectral complexity

5 10 15 20 25 30 35 40 45Time (min)

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Co‐add mass spectra of all components in TIC




12 Da




460

462

464

466

468

4700.0

0.51.0

1.52.0“Compressed” spectrum



7 T

Ion accumulation, fragmentation and isolation

Image Courtesy of Thermo Scientific

(< 1 ppm)(100‐500K)

FT‐ICR MS


Why High Resolution MS?

ESI FT‐ICR MS mass spectrum of n‐butyl benzenesulfonamide

Isotopes

7T FT‐ICR MSR = 200,000 (FWHM)external calibration

FTMS + p ESI Full ms [80.00-1500.00]

214.0 214.5 215.0 215.5 216.0m/z

05

101520253035404550556065707580859095

100

Rel

ativ

e A

bund

ance

214.0894

215.0928216.0852

[M+H]+

215.08 215.09 215.10 215.11m/z

0

100

Rel

ativ

e Ab

unda

nce

215.0928

215.0895215.0866

13C

33S15N

Detail of [M+H+1]+

isotope peak


F0906_01459_01 #108-821 RT: 5.49-45.05 AV: 714 NL: 4.28E3T: FTMS + c ESI Full ms [80.00-1500.00]

350 400 450 500 550 600 650m/z

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity

413.

2667

577.

1347

634.

8768

451.

1001

430.

9145

498.

9019

566.

8893

659.

2121

615.

5333

362.

9270

643.

5647

631.

5647

429.

1183

414.

2700

335.

1258

594.

1615

681.

1946

578.

1383

660.

2158

645.

5803

599.

1169

469.

1105

452.

1036

391.

2846

533.

1783

492.

8841

529.

2069

551.

1887

447.

2930

511.

1964

696.

8456

682.

1982

560.

8713

356.

9097

389.

2513

487.

4881

331.

2097

447.

1287

469.

3142

515.

8922

609.

1599

499.

4882

699.

2046

336.

1293

569.

5277

< 1 ppm mass accuracy narrows possible choices for elemental composition…

To Extol the Virtues of Accurate Mass Measurement…


…but many structures are still possible…

Correct structure

(All C22H20O9!!)

Correlating Structure with Formula



1. Identification of a packaging‐related impurity detected in API material

2. Characterization of unknown extractables from PETG bottles with HDPE caps

3. Characterization of unknown extractables from an elastomer material under evaluation for device components

4. Qualitative comparison of 60 mL polypropylene syringes from two sources

5. Characterization of unknown impurities in bioprocessing equipment high volume filters

Five Case Studies will be examined:

• API powder on accelerated stability (40 °C, 75% RH) exhibited a previously unidentified impurity peak at 0.3% when assayed by HPLC/UV with related substances method

• Accurate mass data indicated a proposed formula of C16H33NO3 and MS/MS data were indicative of an long‐chain hydroxylated amide

• Materials composition list for the packaging material was obtained from the vendor, revealed the presence of a commercial dodecanamide anti‐static agent incorporated into the film at 0.5%

• A sample of the anti‐static agent was acquired and confirmed identity

Case Study #1


T W C o f D A D S p e c t ra l D a ta : f r o m Q S 0 6 1 0 _ 0 9 1 6 7 _ 0 1 ( r e c a lib ra te d ) .w if f M a x . 7 .3 e 4 m A U .

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0T im e , m in

0 .0

5 0 0 0 .0

1 .0 e 4

1 .5 e 4

2 .0 e 4

2 .5 e 4

3 .0 e 4

3 .5 e 4

4 .0 e 4

4 .5 e 4

5 .0 e 4

5 .5 e 4

6 .0 e 4

6 .5 e 4

7 .0 e 47 .3 e 4 1 1 .0 7

1 9 .3 3

T IC o f + T O F M S : f r o m Q S 0 6 1 0 _ 0 9 1 6 7 _ 0 1 ( r e c a lib ra te d ) .w if f M a x . 1 .3 e 5 c p s .

2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0T im e , m in

0 .0 0

1 .0 0 e 4

2 .0 0 e 4

3 .0 0 e 4

4 .0 0 e 4

5 .0 0 e 4

6 .0 0 e 4

7 .0 0 e 4

8 .0 0 e 4

9 .0 0 e 4

1 .0 0 e 5

1 .1 0 e 5

1 .2 0 e 5

1 .2 6 e 5 1 1 .1 1

1 9 .3 7

Case Study #1

Unknown

API

API

O

HN

OH

OH

N,N-bis(2-hydroxyethyl)dodecanamide

Chemical Formula: C16H34NO3+

Exact Mass: 288.2533


Unknown[M+H]+ = m/z 288.2539

Total ion chromatogram(m/z 80‐1500)

PDA chromatogram(200‐400 nm)

• Anti‐static compound N,N‐bis(2‐hydroxyethyl)dodecanamideapparently migrating from packaging film into the API at the contact interface

• An alternate packaging film was available without incorporated anti‐static compounds

• Utilization of the alternate packaging material resulted in no additional impurity peaks

Case Study #1


• Polyethylene terephthalate glycol (PETG) bottles and corresponding HDPE caps (no liner) were exposed to IPA at 50 °C for 14 days

• GC‐MS and HPLC/PDA analysis indicated a number of unknown compounds in the IPA extracts

• IPA solution was submitted to the Characterization MS lab for evaluation of unknown compounds by Orbitrap LC‐MS

• PETG bottles: accurate mass data confirmed the presence of a commercial antioxidant. The majority of compounds observed represent cyclic and acyclic PETG oligomers.

• HDPE caps: accurate mass data confirmed the presence of a commercial antioxidant and a related degradation product, as well as erucamide (a slip agent)

Case Study #2


Case Study #2


RT: 0.00 - 47.38

0 5 10 15 20 25 30 35 40 45Time (min)

0

50000

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150000

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250000

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uAU

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O

O O

O

O

OO

O

O OO

O

O

OOO

O

OO

O

Irgafos 168 phosphate

Polyethylene terephthalate oligomers(acyclic and cyclic)

O

OO O

OO

O

O

O

OO

OO

OO

HO

O

OO

O

O

OO

O

O

O

O

O

HO

OP

OO

O

PETG bottles, IPA extract

Case Study #2




RT: 0.29 - 47.46

5 10 15 20 25 30 35 40 45Time (min)

0

20000

40000

60000

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uAU

0

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6.49E6TIC MS f0906_01461_01

NL:8.18E4Total Scan PDA f0906_01461_01

*

*

*

* = common system background peaks

*

ErucamideDeg Productof Irgafos 168

Irgafos 168 phosphate

OP

O

OHO

O NH2

OP

OO

O

HDPE caps, IPA extract

• Elastomer material under evaluation for fabrication of drug delivery device components

• Coupons of elastomer were refluxed for 60 min in IPA and H2O

• The IPA and H2O extracts were evaluated using FT‐ICR LC‐MS

• Compounds characterized in the IPA extracts included several octylsulfinylmethylphenol and other antioxidans and stabilizers, aminocrotonic acid esters, and a series of alkyl‐substituted PEG homologs

• The H2O extracts were did not reveal the presence of any detectable extractable compounds, and were consistent with blank H2O injections

Case Study #3


RT: 19.89 - 46.12

20 22 24 26 28 30 32 34 36 38 40 42 44 46Time (min)

0

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NL:3.28E6TIC MS FT1007_04181_FM480_04

NL:1.96E5Total Scan PDA FT1007_04181_FM480_04

Case Study #3



Expanded region


Elastomer, IPA extract

RT: 19.89 - 46.12

20 22 24 26 28 30 32 34 36 38 40 42 44 46

Time (min)

0

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Case Study #3




(Alkyl PEG ethers)

Irganox 1010

Irganox 1010Likely deg. product

HO

SO

SO

HO

SO

SO O

O

O

OH

HO

O O

HO

OH

Palmiticacid

HO

S

SO

OO

OH

O

O

HO

OO

HO

O

O

OH

IsomersOctadecylamine

Didecylphthalate

HO

O

O

O

O

NH2

Aminocrotonic acid esters

Elastomer, IPA extract

Case Study #3




RT: 0.00 - 49.95

0 5 10 15 20 25 30 35 40 45Time (min)

0

20000

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Elastomer, H2O extract

• Qualitative evaluation of 60 mL polypropylene syringes from two sources for any apparent/overt differences in plunger lubricant and barrels

• Syringes were extracted with CH2Cl2, extracts evaluated using FT‐ICR LC‐MS

• Both syringes revealed presence of PDMS oligomers indicative of silicone oil

• One syringe indicated presence of large amount of erucamide and various fatty acids and amides

• The other syringe revealed presence of two commercial antioxidant additives, as well as related degradation products

• Information permitted a quick, high‐level decision regarding syringe choice before commencing a formal extractables evaluation

Case Study #4


Case Study #4


FT‐ICR LC‐MS total ion chromatograms (m/z 80‐1500) of combined CH2Cl2 extracts of polypropylene syringes from two different sources

l ll

Polydimethylsiloxane oligomers

l ll l l l l l l l

l l ll l l l l

ll

Various fatty acids and amides

Erucamide

Irganox 565

Aminotriazine Deg. Product of Irganox 565

Irgafos 168phosphate

Polydimethylsiloxane oligomers

Iminoquinone Deg. Product of Irganox 565

Case Study #4


Irganox 565 and observed degradation products

Allen, David W.; Leathard, David A.; Smith, Christine, Chemistry & Industry (1989), 2, 38‐9

HNN

N

HO

N

S

S

NN

N

O

N

S

S

H2NN

NN

S

S

Irganox 565

Iminoquinone deg. product Aminotriazine deg. product

• Inspection of a high flow filtration unit for bioproduct processing revealed the presence of an unidentified precipitate

• Accurate mass data (FT‐ICR LC‐MS) and MS/MS fragmentation for the precipitate were consistent with a homologous series of benzalkoniumsurfactant compounds, common in many commercial disinfectant solutions

• Materials composition list and MSDS for all components in the system were requested from vendors. A series of high‐flow cartridge filters were found to be shipped in a solution containing approx. 0.1% benzalkoniumchloride (C12‐C16 chain length)

• Prescribed pre‐wash procedure from vendor not effective

• Altering pre‐wash procedure to ensure complete removal of the benzalkonium compounds resolved the issue

Case Study #5 (Residual Impurities)


Case Study #5


FT‐ICR LC‐MS total ion chromatogram (m/z 80‐1500) of unknown precipitate in ACN

RT: 0.00 - 45.44

0 5 10 15 20 25 30 35 40 45Time (min)

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:

N N

N

T: FTMS + c ESI Full ms [80.00-1500.00]

280 290 300 310 320 330 340 350 360 370 380 390 400 410 420m/z

0

Inte

nsity

332.3313C23 H42 N = 332.3312

3.5 RDBE0.4623 ppm

304.3000C21 H38 N = 304.2999

3.5 RDBE0.3218 ppm

360.3628C25 H46 N = 360.3625

3.5 RDBE0.8424 ppm

A

B

C

Case Study #5


N

N

N

ESI FT‐ICR full scan mass spectrum of unknown precipitate in ACN

Case Study #5


FTMS + c ESI d Full ms2 [email protected] [70.00-315.00]

210 211 212 213 214 215m/z

0

Inte

nsity

212.2372C14H30N = 212.2373

0.5 RDBE-0.4241 ppm


239 240 241 242m/z

0

Inte

nsity

240.2684C16H34N = 240.2686

0.5 RDBE-0.5442 ppm


266 268 270m/z

0

Inte

nsity

268.2998C18H38N = 268.2999

0.5 RDBE-0.2447 ppm

N

10N

12

N

14

-H -H -Hm/z 212 m/z 240 m/z 268

m/z 304.3 m/z 332.3 m/z 360.3

ESI FT‐ICR MS2 mass spectra of unknown precipitate in ACN

A key step in an extractables/leachables assessment is the establishment of a comprehensive extractable compound profile for materials and components

Characterization of unknown extractable compounds, particularly if present in complex mixtures of organic molecules, is not a trivial exercise and often requires sophisticated capabilities, deep expertise, and knowledge of material and component composition and history

Extractable and leachable assessment of given materials represents a dynamic, rather than static target since development and use of new polymers, elastomers, processing agents and additives, as well as upstream changes, affect lot‐to‐lot material composition and properties

Establishment of comprehensive, accessible exact mass spectral libraries will aid greatly with screening polymer and elastomer extracts for common additives and extractables (creation of a comprehensive library via a Lilly‐Thermo Scientific collaboration is currently in progress using Q‐Exactive technology)

Conclusions


Acknowledgments


Gene InmanBryan CastleSteve BaertschiBrian FahieLeslie KingSusan JanesRandy ThackreyEvgenia PindelGuangliang (Greg) PanMark Copeland

Michael HarrisonMihaela SimianuSteve BandyMatt ClemensMatt EarleyK. Wayne TaylorDavid RobbinsSteve ThomasCourtney CallisWolfgang Mueller

Thermo ScientificEsther LewisIain MylchreestJohn ButlerYinying HuangDavid PeakePatrick BennettKate Comstock

Thank you!


Back‐up slides


Definitions ‐ Thresholds


• Safety Concern Threshold (SCT): Total Daily Intake (TDI) threshold below which a leachable present negligible safety concerns from carcinogenic and noncarcinogenic toxic effects

• Analytical Evaluation Threshold (AET): Threshold at or above which extractables or leachables need to be identified, quantitated, and reported for a toxicological assessment

• Qualification Threshold (QT): Threshold below which a given non‐carcinogenic leachable would present negligible safety concerns, unless the leachable presents structure‐activity relationship (SAR) concerns

AET μg/ml = SCT μg/dayDose ml/day

case studies outlining analytical approaches, challenges and

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