pharmaceutical impurity analysis • definitions - background • regulatory agencies and...

Pharmaceutical Impurity Analysis: The Importance of Selectivity, Sensitivity and Mass Accuracy in the Identification of Extractable and Leachable Compounds in API and Biologics

David A. Weil, Ph.D.

Center of Excellence

Agilent Technologies

Schaumburg, IL USA

Outline

• Definitions - Background

• Regulatory Agencies and Organizations

• Small Molecule versus Biologics

• Setting Detection Limits as function of Risk Factors

• Agilent Extractable/Leachable Workflow: Acc Mass, MS/MS

• Examples: • Reflux AL Foil Closure: MS/MS

• Polypropylene Bottle: Is APCI Best?

• Silicone tubing: Chromatography Matters

• Questions:

BioPharma Road Show July 2014

2

Definitions:

Extractable:

• Chemical compounds that can be removed from a primary container or component material into the drug or biologic by exertion of an artificial or exaggerated force, potential to migrate from polymeric construct into the drug product

= What can come out

Leachable:

• Chemical components that migrate from a container-closure system into the final packaged drug product under normal storage conditions. Direct contact with formulation under normal conditions

• Subset of Extractable

=What DOES come our


3


5

Common Sources of Extractable and Leachable

Plastic Components: Polymers Additives, Phthalates,

Lubricants, Fatty Acids, Nitrosamines

Elastomers/Rubber: Vulcanizing Agent, PAHs, Accelerators

Antioxidants, Carbon Black,

Inks/Labels: Azo Dyes, Aromatic Amines

Adhesives: Antioxidants (AO), Catalyst Residues,

Heavy Metals, Silicones, Surfactants

Pigments: Inorganic (TiO2, FeO’s), Organic Pigments

Cyclic Oligomers: Polybutylene terephthalate (PBT) polyester

Silicone Cyclic Oligomers


6

Container Closure System: Drug Packaging

• The sum of the packaging components that together contain and protect the dosage form. Includes both primary and secondary packing components

What Drug Packaging Should Do:

• Storage: adequately preserve the integrity of the drug

• Deliver the drug (Inhalers, prefilled syringes, ophthalmic, patches

• Protect the Drug during shelf life


7

Container Closure System: Drug Packaging

What Drug Packing Should Never Do

• Contribute harmful components to the drug

• Increase toxicity of the product

• Add Genotoxic or Carcinogenic Components

• Impact the stability and Efficacy of Drug

• Drug Manufacturing may also contribute impurities not from

packaging but from production and storage equipment such

as filters, gaskets, tubing, storage bags, cleaning process


8

Why Worry about Extractable/Leachable


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Johnson and Johnson recalls over

43 Over-the-counter (OTC) children’s

Medicines manufactured by McNeil

Consumers Healthcare

2014 to date 21 FDA Product Recalls > 60 in 2013


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Particulates, Mold, Moisture Stability

Regulatory Agencies

• Federal Food and Drug and Cosmetic Acid

• Good Manufacturing Practices – 21 CFR

• EU Directives, CFR 211.94tems and Drug Product Container and Closures, etc.

• Center for Drug Evaluation and Research (CDER)

• Health Canada

• International Committee on Harmonization ICH

• European Medicines Evaluation Agency (EMEA)

• Standards Compendia • United States Pharmacopeia New Standards for Heavy Metals 2015

• Chapters <661>, <661.1>, <1663>, <1664.1> under review new protocols


12

Contract Analytical Testing Labs: Global to Regional

August 20, 2014

Confidentiality Label

14

Biologics Typically Differ from Traditional Small Molecule Drugs for the following Reasons

• Large MW and complex structure ( multiple domains)

• Post-translational modifications:

- Glycosylation, Proteolysis, Acylation, Sulfation, others

• Intentional Modifications: PEGylation, Conjugation, etc.

• Abundance of both hydrophilic and hydrophobic sites may

render biologic drugs more efficient extraction media

• Produced by complex manufacturing process employing

living organisms

• Susceptible to Microbial contamination Sensitive to Heat


16

Ref: Ingrid Markovic, CBER Presentation USP/PQRI E/L Workshop April 2014

Special Concerns about E/L Effect on Biologics

• Leachable may affect protein products by causing:

Oxidation

Unfolding

Aggregation

Increase in particulates

Formation of clipped variants

Formation of Protein Adducts

Post translational events during fermentation (glycosylation)

Altered protein translation

Ref: Wang, Ignatius, Thakkar, J of Pharma. Sci, 103, (2014) 1315-1330

Ref: Ingrid Markovic, CBER April 2014


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Impact Impurities from Container Closure System on Protein Stability

1. Silicone Oil – Syringe Barrels/Plungers Proteins absorb onto silicone oil droplet surface leading to loss of

selected proteins, aggregation, particle formation

Addition of surfactants or Adjust pH may prevent effect

2. Heavy Metals, Zn, Tungsten at ppm cause aggregation

3. pH Changes..

4. Thiuram disulfides: Thiol-Disulfide Exchange

5. IV-Bag Storage: Phthalates,Sterilization, Freezing..

6. Drug Delivery Systems: Peroxides, PLA, Free Radicals


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Risk Assessment Table by Dosage Forms


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Safety Thresholds from PQRI L/E Working Group

Safety Concern Threshold: (SCT) 0.15 ug per day, which is

defined as the threshold below which an individual leachable

would have a dose so low as to present negligible safety

concerns from carcinogenic and non-carcinogenic toxic effects.

Qualification Threshold: (QT) 5 ug per day: Threshold below

which a given leachable is not considered for safety

qualification (toxicological assessments) unless the leachable

presents structure-activity relationship (SAR) concerns

Analytical Evaluation Threshold: (AET) is determined by

consideration of the SCT and the specific drug product delivery

configuration (number of doses in a Drug Product vs. single

dose


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Special Case Compounds

• Highly toxic compounds known to be carcinogenic,

or have a structure that could cause damage to

DNA are considered special case compounds…

• Nitrosoamines

• PAH’s

• Mercaptobenzothiazoles

• Azo Dyes

• Safety Threshold for detection dependent on drug

formulation and delivery method/site


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Extractable and Leachable Workflow

Data

Analysis LC/MS

Analysis

Sample

Preparation

GC/MS

Analysis

Separations

Quant QQQ

Volatile

Residues

Non-Volatile

Residues TOF/QTOF

Impurity

Profiling

Targeted

Non-Targeted

Data

Analysis

Objective: Detect low-level Organic/Inorganic Impurities in Drugs: Tablets,

Capsules, Suspension, Transdermal, Meter Does Inhalers (MDI) and

Packaging Films (include food)

Page 25


ICP/MS

ICP/AA

Heavy Metals

Extraction Method Critical Step in Analysis


26

Besides Solvent Composition one must also optimize the following:

Extraction time, temperature, cycles, method, physical

Key not to be so far away from how API/Biologic interacts with CCS

Extractable Inorganic Impurities

Inorganic Impurities can result from the manufacturing

process and are generally known, identified and include:

Reagents, Ligands, Catalysts

Heavy Metals

Inorganic Salts

Manufacturing Aids (charcoal, filters)

Page 27


Elemental/Inorganic Impurity Analysis ICP-MS

• Reliable trace-level analysis of all 16 elements whose limits are defined in USP<232>.

• Low detection limits ensure that all regulated elements can easily be determined at or below regulated levels, and even when large sample dilutions are required.

• Can also be used in combination with a HPLC, GC, and CE, providing several options for separation (or speciation) of the different chemical forms of the elements.

• ICP-MS achieves low detection limits for almost all elements, including those found in the more extensive analyte list proposed in the ICH Q3D, such as Au and Tl

Agilent 7700 Series ICP-MS

Page 28


Instruments for Extractable/Leachable Analysis

5975C

GC/MS

QQQ

6400 Series

TOF

6200 series

7000B

GC/QQQ

Hi-DEF Q-TOF

6500 series 7700

GC/QTOF

SFC/MSD/QTOF


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QTOF Ion Sources

Dual-Spray Electrospray Source (ESI)

Agilent Jet Stream Electrospray

Technology (AJS)

Atmospheric Pressure

Chemical Ionization (APCI)

Multimode: Operates in ESI-only,

APCI-only, or mixed mode (ESI+APCI)

Atmospheric Pressure Photoionization

(APPI)

DART (IonSense)

MassTech AP-MALDI

Dual ESI

AJT ESI

APPI

APCI

Multimode

ASTS Houston High Resolution Analysis of Oligomeric and

Polymeric Materials

Extractable Leachable Data Analysis Workflow • Combination of UHPLC separation and accurate mass TOF technology

• Effective data mining algorithms to easily FIND compounds in a sample

• COMPARE samples or sample sets to identify differences

• AMRT DBs and MS/MS Lib Search to simplify ID of targeted compounds

• Identification: Elemental Formula use Molecular Formula Generation (MFG)

• Identification: Structure ID by using MS/MS Libraries

• Identification: Structure ID using Molecular Structure Correlation Software

• Full AUTOMATION of data acquisition, processing and reporting

TOF/

Q-TOF

Analysis

ID via

AMRT

DBs

Molecular

Feature

Extraction

MFG of

Unknowns

w/ MSMS

MSMS

Structural

Correlation

ID via

MSMS

libraries

Differential

Analysis

Automation


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Identification: Accurate Mass Databases

TOF/

Q-TOF

Analysis

ID via

AMRT

DBs

Molecular

Feature

Finding

MFG of

Unknowns

w/ MSMS

MSMS

structural

correlation

ID via

MSMS

libraries

Automation

OPTION:

Differential

Analysis

Identification of targeted compounds via AM or AMRT databases

• Personal Compound Databases (PCD)

• PCD for extractable and leachable with over 1150 compounds

• The addition of retention time into the database search (PCD) enables

identification of isobaric compounds


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Personal Compound Databases (PCDL)

Accurate Mass and MS/MS Library Searchable Databases


Page 33

Polymer Additive Database contains

over 1250 Compounds Building MS/MS Searchable Library

User Generated Polymer Additive Database

Millipore

Page 34

Siloxanes Added

Irganox 1024 Accurate Mass Measurement

0.03 ppm

0.33 ppm

0.63 ppm

-0.53 ppm


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All Adducts Used!

Silicone Tubing C50H150Si25O25 [M+NH4]+ D25

Millipore

Page 36

Isotope Fidelity for Si25O25C50H150

Millipore

Page 37

Wide In-Scan Dynamic Range: Detection of Irgafos 168 Phosphate

Millipore

Page 38

Irgafos 168 Phosphate 0.57 ppm Error [M+H]+

Millipore

Page 39

Accurate Mass MS/MS Structural Information

Extractables and Leachables Examples

Degradation Product of Irganox 1010 [C56H84O10]

Identification using MS/MS by 10,20,40 eV CID of D10

Millipore

Page 41

10 eV

20 eV

40 eV

MassHunter MS/MS Structure Correlation (MSC) Confirm proposed structures and aid true unknown analysis

Provides highest confidence in confirmation of proposed

structures in minutes instead of hours

• Assigns fragment ions to substructures of the proposed parent structure

− Metabolite ID

− Metabolomics – compound identification

• Aids in the determination of true unknown compounds beyond Molecular Formula

Generation via parallel MSC of all structures retrieved for a unique Molecular

Formula


42

Analysis of IPA Reflux of Al Foil Closure


Page 43

IPA Extract from Foil – MFE APCI (+)


Page 44

Compounds Sorted by Abundance


Page 45

Major Compound C24H24O10 0.19 ppm Error


Page 46

Isotope Pattern Matching


Red Boxes are Theoretical

Isotope Pattern

Page 47

MS/MS of [M+H]+ C24H24O10 RT 6.9 minutes


Phthalate

C2H4O Losses

Page 48

MS/MS Data [M+NH4]+ peak for C24H24O10 at RT 7.0


Page 49

MS/MS of [M+H]+ C24H24O10 at RT 7.27 mins


Structural Isomer

MSMS Key

Page 50

Molecular Structure Correlator of [M+H]+ C24H24O10


Page 51


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Int J of Anal

Chem, 2009

Diethyleneglycol Terephthalate polyester polyols may have cyclic oligomers.


53

Analysis of Rubber Extract


Page 57

Overlay TIC with Major Components (APCI-)


Irganox 1010

Stearic Acid

Palmitic Acid

Page 58

MS and MS/MS of [M-H]- of Irganox 1010


Page 59

Mass Profiler Foil versus Rubber Differential Analysis Impurity Profiling


Page 60

Erucamide C22H43NO MW 337.3344 1.5 fold change between Foil and Rubber


Page 61

17 only in the Foil Extracts top formula hits


Page 62

Mass Profiler Unique in Rubber Extract


Page 63

Easy to Identify Fragment Compounds Produced by APCI Source

Identification of Extractable Compounds from

Polypropylene Bottle Comparing APCI, Multimode, Jet Stream and GP-APCI

Polypropylene IPA Extract Positive Ion

APCI

Multimode APCI only

Multimode APCI/ESI

Jet Stream


65

Detection of Irgafos 168, Phosphate [M+H]+

APCI

APCI

Jet Stream

Jet Stream

MM: Mixed

MM: APCI MM: Mixed

Irgafos 168

Irgafos 168

Phosphate


66

Detection of Irgafos 168 and Phosphate All Adducts

Jet Stream

MM: APCI APCI

APCI

Jet Stream

MM: APCI MM: Mixed

MM: Mixed

MM: Mixed

AJS

MM: APCI

APCI

Ionic species poorer

Ionization with APCI

Irgafos 168

Phosphate

Irgafos 168


67

Compare TIC’s: Multi-Mode Mixed versus APCI

APCI Only

ESI And APCI

Intensity Compound Dependent

2X 4X

0.8X


68

Components using Data Mining (MFE) APCI+


70

MS/MS m/z 537.5350 [M+H]+ C34H68N2O2


71

MS/MS m/z 565.5669 [M+H]+ C36H72N2O2


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MS/MS m/z 593.5977 [M+H]+ C38H76N2O2


73

Molecular Structure Correlator C34H68N2O2


74

Possible Structures for C34H68N2O2

N,N'-ethane-1,2-diyldihexadecanamide

N,N'-1,2-Ethanediylbis(2-pentylundecanamide)

N,N'-1,6-Hexanediylditetradecanamide

N,N'-Dihexadecyloxamide


75

Molecular Structure Correlator C38H76N2O2


76

Total of Six Possible Structures

N,N'-Ethylenebis(stearamide)

N,N'-1,2-Ethanediylbis(16-methylheptadecanamide)

N,N'-Dihexadecylhexanediamide


77

Internet Search Confirms Database Hit


78

• Increases MS flexibility by

enabling both GC and LC

compatibility

• Analysis of wider range of

compounds

• Proven QTOF LC/MS platform

• Proven 7890 GC performance

• Excellent sensitivity and

mass accuracy performance

• Dedicated GC QTOF offers

electron impact (EI) ionization

G3212A - GC APCI Interface

ASTS Houston High Resolution

Analysis of Oligomeric and Polymeric

Materials

MS/MS of Irgafos Phosphate m/z 663 (PP Extract)

MS/MS of Irgafos 168 m/z 647

Top Components in PP IPA Extract GC/APCI Exclude Component also in Blank

GC/APCI Accurate Mass of low molecular

weight additives and also residual solvents.

Top Components in PP IPA Extract GC/APCI Exclude Component also in Blank

Summary

• High Mass Accuracy in both MS and MSMS modes enables

one to proposed elemental composition information for

impurity from all Adducts formed.

• Accurate mass polymer additive database and MS/MS

library searching can be used to identify targeted

compounds.

• Molecular structure correlation software provided means to

assist in the interpretation of accurate mass MS/MS data

linked to chemspider, SDF files or individual databases.


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What to learn More . Books


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ISBN: 978-0-470-28176-5

June 2009

ISBN: 978-0-470-17365-7

January 2012

Meetings and Conferences:


87

USP

Questions and Answers


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pharmaceutical impurity analysis • definitions - background • regulatory agencies and...

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