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Extractables and Leachables from Single Use Bioprocess Systems

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1980Founded in

P harmaceutical

Medical Devices

FoodPackaging

A Leaderin Plastics Analysis

IDENTIFICATIONQUANTIFICATION

&LeachablesExtractables

1,000Ove

r

Projects Annually

Chemists80%Of EmployeesAre Degreed

ANALYSISInvestigative

CracOff-Odor

P olymern& Additive ID

Deformulation

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Overview• E&L Background• E&L Study Overview

– Model Device– Extraction Conditions– Scouting Methods– Qual Methods

• Scouting Method Results• Qualitative Results

– Mass Spectroscopy Software Tools

– Method Sensitivity

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What are Leachables & Extractables?

Leachables are :organic and inorganic chemical entities that migrate from a packaging/delivery system, packaging component, or packaging material of construction into an associated drug product under normal conditions of storage and use or during accelerated drug product stability studies.

USP 1663 & 1664

Extractables are:Organic and inorganic chemical entities that can be released from a pharmaceutical packaging/delivery system, packaging component, or packaging material of construction under laboratory conditions..

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Introduction to Es and LsExamples of E&Ls

– Small molecules present in a polymer system including: Antioxidants Surfactants Slip agents Plasticizers Acid scavengers Cross linking agents Residual monomers and oligomers Polymerization side products Process Impurities

– Extractables may not be leachables depending on their solubility and the use conditions of the device.

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ExtractableCompounds

Observed Leachables

The Regulatory Landscape

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USP <661> USP <1663> USP <1664>

USP <661.3> BPOG BPSA

Pharma/Bio ManufacturingPharmaceutical Packaging

ToxicologyChemical Analysis

E&L Study

Protocol

Background Information

Study Design Protocol

Device Composition

Use Conditions

Analysis Techniques

Sample Selection

Extraction Conditions

Sample and Control

PreparationExtraction Method

Development

Toxicological Evaluation

Identification

Quantitation

Steps in an Extractables and Leachables Study

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Bioprocess System

Three Component System

• Single Use Bioprocess Bag (LDPE and EVA)

• Nylon Filter with Polypropylene Filter Housing

• Thermoplastic Elastomer Tubing

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ToxicologyChemical Analysis

E&L Study

Protocol

Background Information

Study Design Protocol

Device Composition

Use Conditions

Analysis Techniques

Sample Selection

Extraction Conditions

Sample and Control

PreparationExtraction Method

Development

Toxicological Evaluation

Identification

Quantitation

Steps in an Extractables and Leachables Study

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Single Use Bioprocess System

Extraction Conditions

Extractables•Cut and Cover•50°C for 72hr at 50 RPM

Leachables•Continuous•Recirculation at 37°C for 72hr

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Extraction Solvents and Samples

Water

Ethanol\Water

50/50

Ethanol

Saline

Bioprocess Bag

Pharma Tubing

Disk Filter

Complete System

X

X

X

X

X

X

X

X

X

X

X

X

X

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Saline = 137 mM NaCl, 2.7 mM KCl and 10 mM phosphate buffer solution (pH 7.4)

TOCUV-Vis

Grav

Scouting Methods

FTIR

Scouting Methods

Bulk Identification

Conjugation Double Bonds

Non-Volatile Residue

Organic Carbon

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DH

S-M

S

Very Volatile Compounds (VVOC) <100°C

Volatile Compounds (VOC) 100-260°C

Non-Volatile and Ionizable

Elemental Analysis

Analyte , Boiling PointTechnique

Instrumental Techniques

Semi Volatiles (SVOC) 260-400°C

ICP-

MS

QTO

F-G

CMS

QTO

F-LC

MS

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Analytical SensitivityAnalytical Evaluation Threshold

AET – 50 ng/ml

AET – 25 ng/ml

Must Identify

Must Identify

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Analytical Evaluation Threshold (AET)

Relative value based on:• The Safety Concern Threshold (SCT)• The drug product configuration• Analytical techniques/methods used

The threshold at or above which a particular leachable and/or extractable should be identified • Drug Products vs

Medical Devices• The Effects of Standard

Selection

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AET ValueAnalytical Evaluation Threshold

Erucamide – 25 ng/ml

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Irganox 1010 – 25 ng/ml

CompoundStandard Erucamide Irganox 1010Molecular Formula RRF Percent of

True Value RRF Percent of True Value

Erucamide C22H43NO 1.0000 100% 1.554 155%Oleamide C18H35NO 0.9742 97% 1.514 151%

Octadecanamide C18H37NO 0.7859 79% 1.221 122%Dioctyl phthalate C24H38O4 1.5950 160% 2.479 248%

mono-Butyl phthalate C12H14O4 0.0972 10% 0.151 15%Benzyl butyl phthalate C19H20O4 1.1127 111% 1.729 173%

Didecyl phthalate C28H46O4 1.8571 186% 2.886 289%Irganox 1010 C73H108O12 0.6434 64% 1.000 100%Irganox 1135 C25H42O3 0.5448 54% 0.847 85%Irganox 259 C40H62O6 2.1573 216% 3.353 335%

Irganox 3114 C48H69N3O6 0.5553 56% 0.863 86%Irganox 1141 C24H42O 0.0015 0.2% 0.002 0.2%Caprolactam C6H11NO 0.598 60% 0.929 93%

Triallyl-triazine-trione C12H15N3O3 0.006 1% 0.009 1%

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CompoundDetector MS UV-VIS CAD

Molecular Formula RRF % of True

Value RRF % of True Value RRF % of True

ValueFatty Amides

Erucamide C22H43NO 1.0000 100% 1.0000 100%Oleamide C18H35NO 0.9742 97% 0.7407 74%

Octadecanamide C18H37NO 0.7859 79% 0.9790 98%Phthalates

Dioctyl phthalate C24H38O4 1.0000 100% 1.000 100% 1.000 100%mono-Butyl phthalate C12H14O4 0.0610 6% 1.845 184% 0.186 19%Benzyl butyl phthalate C19H20O4 0.6976 70% 1.548 155% 0.150 15%

Didecyl phthalate C28H46O4 1.1643 116% 0.811 81% 3.160 316%Antioxidants

Irganox 1010 C73H108O12 1.0000 100% 0.256 100% 1.000 100%Irganox 1135 C25H42O3 0.8467 85% 0.522 52% 0.119 12%Irganox 259 C40H62O6 3.3527 335% 0.916 92% 0.861 86%

Irganox 3114 C48H69N3O6 0.8630 86% 1.921 192% 1.048 105%Irganox 1141 C24H42O 0.0023 0.2% 0.389 39% 0.448 45%

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Compound Class

MS UV-VIS CADMean RRF

STD. Dev. % RSD Mean

RRFSTD. Dev. % RSD Mean

RRFSTD. Dev. % RSD

Fatty Amides 0.9200 0.1168 13% 0.9065 0.1440 16%

Phthalates 0.7307 0.4866 67% 1.301 0.479 37% 1.1239 1.4127 126%

Antioxidants 1.2130 1.2596 104% 0.950 0.601 63% 0.6952 0.3994 57%

All Compounds 0.9790 0.8320 85% 1.106 0.548 50%* 0.891 0.802 90%

Relative Quantitation Results

* Value excludes Fatty Amides which have no chromophore.

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Optimizing Standard and Detector Type Significantly Reduces Response Factor Variation

ToxicologyChemical Analysis

E&L Study

Protocol

Background Information

Study Design Protocol

Device Composition

Use Conditions

Analysis Techniques

Sample Selection

Extraction Conditions

Sample and Control

PreparationExtraction Method

Development

Toxicological Evaluation

Identification

Quantitation

Steps in an Extractables and Leachables Study

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Gravimetric Analysis & TOCThe Foundational Methods for Extractables Quantitation

Sample Solvent Corrected Residue (mg/device)

TOC (mg/device)

Bag

Water 0.00550/50 Water/Ethanol 3.650

Ethanol 29.245Saline 2.607

Tubing

Water 2.01950/50 Water/Ethanol 2.126

Ethanol 2575.819Saline 2.078

Filter

Water 0.10650/50 Water/Ethanol 0.349

Ethanol 0.158Saline 0.243

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FTIRScouting Methods – Functional Group Identification

Blue = Ethanol Tubing ExtractRed = Mineral Oil

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UV-VISScouting Methods – Chromophore Containing Species

UV Absorbance

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ToxicologyChemical Analysis

Protocol

Background Information

Study Design Protocol

Device Composition

Use Conditions

Analysis Techniques

Sample Selection

Extraction Conditions

Sample and Control

PreparationExtraction Method

DevelopmentToxicological Evaluation

Identification

Quantitation

Steps in an Extractables and Leachables Study

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Extraction

Identification

Scouting

Qualitative Analysis

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QTOF-GCMSICP-MS

QTOF-LCMSDHGCMS

Dynamic Headspace Mass Spectrometry

Very Volatile Compounds (VVOC) <100 °C

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Elemental Analysis

ICP-MS

As Cd Hg Pb Co Ni Cr Mo Sb Ba Class (ICH

Guidelines) 1 1 1 1 2A 2A 3 3 3 3

Salin

e

Filters 0.102 <DL 0.068 <DL <DL <DL 0.1258 0.1428 356.7 0.017Tube <DL 1.206 1.742 1.072 25.46 <DL 1.34 <DL 4.02 1.206Bag 0.312 0.624 1.404 1.56 <DL <DL 0.624 <DL 158.7 44.6

Flow Through 4.5 0.6 5.7 21 24.6 <DL 11.7 7.8 77.4 <DL

Wat

er Filters 0.238 <DL 0.017 <DL <DL <DL 0.0986 <DL 68.1 0.6766Bag <DL 1.34 0.67 3.618 <DL <DL <DL <DL <DL 13.67

Tube <DL 2.652 0.936 2.028 50.4 26. 4 <DL 0.624 <DL 8.892

Results in ng per Device

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ICP-MS

As Cd Hg Pb Co Ni Cr Mo Sb Ba Class (ICH

Guidelines) 1 1 1 1 2A 2A 3 3 3 3

Sal

ine

Filters 0.102 <DL 0.068 <DL <DL <DL 0.1258 0.1428 356.7 0.017Tube <DL 1.206 1.742 1.072 25.46 <DL 1.34 <DL 4.02 1.206Bag 0.312 0.624 1.404 1.56 <DL <DL 0.624 <DL 158.7 44.6

Flow Through 4.5 0.6 5.7 21 24.6 <DL 11.7 7.8 77.4 <DL

Wat

er Filters 0.238 <DL 0.017 <DL <DL <DL 0.0986 <DL 68.1 0.6766Bag <DL 1.34 0.67 3.618 <DL <DL <DL <DL <DL 13.67

Tube <DL 2.652 0.936 2.028 50.4 26. 4 <DL 0.624 <DL 8.892

Results in ng per Device

Saline ExtractsNa – 350,000 ng/mL

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QTOF-GCMS: Bioprocess Bag Extractables

Ethanol

50% Ethanol

Water

Agilent 7200 QTOF GCMSEI; DB-5MS

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Ethanol

50%Ethanol

Water

Agilent 6545 QTOF LCMSESI positive ionization

QTOF-LCMS: Bioprocess Bag Extractables

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107 Counts

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI, Positive Ionization

QTOF LCMS Ethanol Extract of Bioprocess Bag

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106 Counts

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI ,Positive Ionization

QTOF LCMS Ethanol Extract of Bioprocess Bag

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105 Counts

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI ,Positive Ionization

QTOF LCMS Ethanol Extract of Bioprocess Bag

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Feature (Compound) FindingUsing Recursive Analysis

AgilentMass Profiler Professional

AgilentProfinder

FilteringStatistical Analysis

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MS Advanced Qualitative Workflow

Recursive Feature Finding

Find Compounds

Formula GenerationDatabases

Compare ID Compounds

Statistical Analysis

Fold Change Filtering

Abundance Filtering

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Advanced Qualitative Workflow

Recursive Feature Finding

Find Compounds

Formula GenerationDatabases

Compare ID Compounds

Statistical Analysis

Fold Change Filtering

Abundance Filtering

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105 Counts

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI ,Positive Ionization

QTOF LCMS Ethanol Extract of Bioprocess Bag

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Total Compounds Detected – QTOF LCMS

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI + Ionization

Tubing

Filter

Bag

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Advanced Qualitative Workflow

Recursive Feature Finding

Find Compounds

Formula GenerationDatabases

Compare ID Compounds

Statistical Analysis

Fold Change Filtering

Abundance Filtering

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726 Compounds

288 Compounds from Control

Significance and Fold Change

Filtering

575 Extractable Compounds

Recursive Feature FindingAgilent Profinder

Agilent Mass Profiler

Professional

Bioprocess Bag Ethanol Extract

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI, + Ionization

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Advanced Qualitative Workflow

Recursive Feature Finding

Find Compounds

Formula GenerationDatabases

Compare ID Compounds

Statistical Analysis

Fold Change Filtering

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Abundance Filtering

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With this many Compoundswhich areImportant?

Analytical Evaluation Threshold

SCT = 0.15 µg/daySafety Concern ThresholdEach system provides 500 dosesEach patient takes 2 doses/day

AET =37.5 µg/DeviceEstimated Analytical Evaluation Threshold

0.15 µ𝑔𝑔𝑑𝑑𝑑𝑑𝑑𝑑

× 1 𝑑𝑑𝑑𝑑𝑑𝑑2 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑

× 500 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑆𝑆𝑑𝑑𝑑𝑑𝑆𝑆𝑑𝑑𝑆𝑆

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Estimated AET =37.5 µg/Device

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Adjusting AET

Mean Relative Response Factor = 0.78Standard Deviation = 0.646%RSD = 83%

AET =37.5 µg/DeviceEstimated Analytical Evaluation Threshold

AET = 6.4 µg/DeviceFinal Analytical Evaluation Threshold

37.5 µ𝑔𝑔𝐷𝐷𝑑𝑑𝐷𝐷𝐷𝐷𝐷𝐷𝑑𝑑

- 37.5 µ𝑔𝑔𝐷𝐷𝑑𝑑𝐷𝐷𝐷𝐷𝐷𝐷𝑑𝑑

× 0.83

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Estimated AET =37.5 µg/Device

Final AET =6.4 µg/Device

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Abundance Filtering at AET

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Advanced Qualitative Workflow

Recursive Feature Finding

Find Compounds

Formula GenerationDatabases

Compare ID Compounds

Statistical Analysis

Fold Change Filtering

Abundance Filtering

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Jordi Proprietary Additives and Polymer Database

Confirmation

Identification

Agilent E&L Database

High Resolution MSMS Fragmentation SpectraAnalysis of Reference Standards

Molecular Formula Generation

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Iden

tific

atio

n

Erucylamide C22H43NO ●Trioctyl Trimellitate C33H54O6 ● ●Oxidized Irgafos 168 C42H63O4P ●Caprolactam C6H11NO ● ● ●Oxidized Irgafos 168 Degradants Var. ●Hexanamide C6H13NO ● ● ●Caprolactam Dimer C12H22N2O2 ●Tetradecamethylcyclohexasiloxane C14H42O7Si7 ●Docosanamide C22H45NO ●Oleylmorpholine C22H43NO ●Eicosenamide C20H39NO ●Pentamethylpiperidinol C10H21NO ● ● ●Pentanamide C5H11NO ● ● ●Azacyclododecan-2-one C11H19NO ● ● ●

● - 50% Ethanol ● - Water ● - Saline

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Management of large datasets through

Statistical FilteringBackground and Trace Compounds

RemovedIdentificationsmade using Database Searching &Molecular Formula Generation

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Tubing Extractables by Solvent

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI, Positive Ionization

Ethanol50% Ethanol

Water

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Tubing Extractables by Solvent

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI, Positive Ionization

Ethanol50% Ethanol

Water

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Tubing Extractables vs. Saline

Extractables

Saline Extract

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI, Positive Ionization

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Tubing Extractables vs. Saline

Extractables

Saline Extract

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI, Positive Ionization

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Individual Parts vs Flow Extraction

Individual Parts

Flow ThroughExtraction

Agilent 1290 UHPLCAgilent 6545 QTOF LCMSDual ESI, Positive Ionization

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https://pixabay.com/en/autumn-leaves-colorful-fall-foliage-1649362/

Extract Preparation

Unknown Identification

Bioprocess System

Scouting Analyses by FTIR, TOC, Gravimetric and UV-VIS

Feature Finding, Statistical Analysis and Abundance Filtering using Agilent Profinder and MPP

Unknown Identification by Database and MFG

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Qualitative Screening (DHS, ICP-MS, QTOF-MS)