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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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)