Aegis5-14 film validation guide

Five-layer, 14 mil cast film

DOC0005 • Revision E

December 2017
Thermo Scientific Aegis5-14 film
Five-layer, 14 mil cast film
Thermo Scientific™ BioProcess Containers (BPCs) are built to meet
your single-use upstream and downstream bioprocessing needs.
Our films are engineered to meet the most demanding requirements
of your bioproduction processes. Thermo Scientific™ Aegis™5-14
film is developed specifically for liquid handling, storage, and
transportation in the biopharmaceutical industry.

Key benefits
• Good toughness and puncture resistance to
maximize security
• Highly flexible and stretchable material
• Non-animal origin formulation
• High barrier properties maximizing stability
of content
• One film for your entire workflow from 50 mL to 2000 L
• Standard and custom configurations available in 2D
pillow-style with seam or panel ports, and 3D square
tube–style configurations with top– and bottom–panel
porting options supplied gamma irradiated and ready
to use
Contents

Aegis5-14 film specifications 4

Biocompatibility 5

Physical properties 6

Mechanical properties 7

Extractables 8
Aegis5-14 film specifications
Thermo Scientific™ Aegis5-14 film is
Property Test protocol Average values
a five-layer, 14 mil cast film produced
in a cGMP facility. The outer layer is a Physical data (post–gamma irradiation, 25–40 kGy)
polyester elastomer coextruded with Tensile strength ASTM D882 2392 psi 16.5 MPa
an ethyl vinyl alcohol (EVOH) barrier Elongation ASTM D882 487%
layer and a low-density polyethylene
Yield strength ASTM D882 1362 psi 9.4 MPa
product contact layer. Aegis5-14
film is manufactured using non-animal 2% Secant modulus ASTM D882 43389 psi 299 MPa
origin components. Tensile toughness ASTM D882 243 lbf-in 2.7 kN-cm

Puncture resistance ASTM F1306 25 lbf 111 N

Seam strength ASTM F88 31 lbf/in 54 N/cm

Polyester 0.8
ASTM D3985, 0% relative 0.023 0.36
Tie 0.9 O2 transmission rate humidity (RH) outside, 90% RH
cc/100 in2/day cc/m2/day
inside, 23°C
EVOH 1.0
Mocon method, 0% RH 0.087 1.35
Tie 0.9 CO2 transmission rate
outside, 100% RH inside, 23°C cc/100 in2/day cc/m2/day

Water vapor ASTM F1249, 0% RH outside, 0.023 0.35

transmission rate 100% RH inside, 23°C g/100 in2/day g/m2/day

ASTM D1003
Haze 68%
(outside dry/inside dry)
Polyethylene 10.4 Glass transition temperature ASTM E1640 −24ºF −31ºC

Film gauge Internal study 0.14 in. 0.356 mm

Film contact material NA Polyethylene

Temperature range† Internal study −112ºF to 140ºF −80ºC to 60ºC

10–6 Sterility assurance level ANSI/AAMI/ISO 11137:2006 2.5–4 Mrad 25–40 kGy
Fluid contact
surface
Biocompatibility data (post–gamma irradiation, >50 kGy)

Schematic cross section USP Class VI USP <88> Pass

Cytotoxicity USP <87> Pass
Bacterial endotoxin USP <85> 0.005 EU/mL
Heavy metals USP <661> <1 ppm
Buffering capacity USP <661> <1 mL
Non-volatile residue USP <661> <1 mg
Schematic 3D view
Residue on ignition USP <661> <1 mg
Hemolysis ISO 10993-4 Nonhemolytic
Appearance EP <3.2.2.1> Pass
Acidity and alkalinity EP <3.2.2.1> Pass
Absorbance EP <3.2.2.1> Pass
Reducing substances EP <3.2.2.1> Pass
Transparency EP <3.2.2.1> Pass

All tests are run post–gamma irradiation unless otherwise noted. † Sub-zero conditions require proper support and handling.

4
Biocompatibility
Overview
Biocompatibility testing was conducted in order to help
ensure that the film and other contact surfaces have no
adverse effects on any biological material that may be
contained within the sample. USP <88> Class VI tests for
in vivo reactions to the material, USP <87> tests for in vitro
reactions (cytotoxicity), USP <661> tests for assessment
of the physicochemical properties of the film, and
USP <85> tests for the presence of bacterial endotoxins.
• EP 3.2.2.1 Plastic containers for aqueous solutions
for parental infusion: Testing is done to characterize
the suitability and functionality of the materials
used in the construction of polyethylene-based
BioProcess Containers.
Results
A summary of biocompatibility testing is shown in Table 1.

Methods Table 1. Summary of biocompatibility testing.

Following are the test methods used to help ensure the
Test Results
biocompatibility of the films. The test methods that were
USP Class VI USP <88> Pass
used include:
Cytotoxicity USP <87> Pass
• USP <88> Class VI
Bacterial endotoxin USP <85> 0.005 EU/mL
––Acute systemic injection: An extract of the test article
Heavy metals USP <661> <1 ppm
was prepared for 72 hours at 50°C and was injected
intravenously into an animal model. Signs of toxicity Buffering capacity USP <661> <1 mL

were monitored. Non-volatile residue USP <661> <1 mg

––Intracutaneous reactivity: An extract of the test article Residue on ignition USP <661> <1 mg
was prepared for 72 hours at 50°C and injected under Appearance EP <3.2.2.1> Pass
the skin of an animal model, and signs of irritation were
Acidity and alkalinity EP <3.2.2.1> Pass
monitored.
Absorbance EP <3.2.2.1> Pass
––Intramuscular implantation: The test article was
Reducing substances EP <3.2.2.1> Pass
implanted into the muscle tissue of an animal model,
and the resulting tissue sections were examined grossly Transparency EP <3.2.2.1> Pass
for signs of infection, necrosis, discoloration, and
hemorrhage.
Conclusion
• USP <87> Cytotoxicity: An extract of the test article
Samples manufactured from the Aegis5-14 film were tested
was prepared for 24 hours at 37°C in E-MEM cell culture
for biocompatibility per USP and EP protocols. There were
medium and cultured with L-929 mouse fibroblast cells.
no signs of toxicity, irritation, inflammation, or cytotoxicity.
The resulting cell culture was monitored for morphological
changes and loss of viable cells.

• USP <661> Physiochemical test for plastics: An

extract of the test article was prepared for 24 hours at
70°C and analyzed for non-volatile residue, residue on
ignition, heavy metals, and buffering capacity.

• USP <85> Bacterial endotoxin testing: Limulus

amoebocyte lysate (LAL) testing quantifies the
presence of bacterial endotoxins on a sample after
gamma irradiation.

thermofisher.com/sut 5
Physical properties
Overview
Permeability gases and water vapor are important properties
of the film. Resistance to the transmission of oxygen,
carbon dioxide, and other gases is important in controlling
the pH and chemical properties of a single-use container’s
fluid content. Resistance to the transmission of water
vapor is also important to the control of the concentration
of a container’s fluid content. Factors that affect a film’s
permeability characteristics include film composition, film
thickness, temperature, and relative humidity (RH).
Methods
• Sample preparation: Test articles consisted of
10.8 x 10.8 cm (4.25 x 4.25 in.) swatches. The film
was always oriented so that the inner layer of the film
was exposed to relative humidity.
• O2 transmission rate: Test articles were conditioned
at 23°C and analyzed for O2 transmission rate using
a MOCON OX-TRAN™ 2/21 test system per ASTM
D3985. The film was oriented so that the inner layer
of the film was exposed to 100% RH O2 gas and the
outer layer of the film exposed to 0% RH N2 gas. These
conditions simulate storage conditions, with the inner
layer representing fluid contact (100% RH) and the outer
layer representing atmospheric contact (0% RH). Final
O2 transmission rates were recorded when the system
equilibrated.
Results
Results for the above transmission rate testing protocols are
shown in Table 2.

• Water vapor transmission rate: Test articles were Table 2. Summary of data for transmission
conditioned at 23°C and analyzed for water vapor rate testing.
transmission rate using a MOCON Permatran-W™
Test Test conditions Results
700 test system per ASTM F1249. The film was oriented
Water vapor 0% RH outside,
so that the inner layer of the film was exposed to 100% 0.023 cc/100 in2/day
transmission rate 100% RH inside, 23°C
RH water vapor and the outer layer of the film exposed
to 0% RH N2 gas. These conditions simulate the worst-
case scenario and represents a higher water vapor 0% RH outside,
CO2 transmission rate 0.087 cc/100 in2/day
100% RH inside, 23°C
transmission rate than ambient conditions (approximately
0% outside RH). Final water vapor transmission rates
were recorded when the system equilibrated. 0% RH outside,
O2 transmission rate 0.023 cc/100 in2/day
90% RH inside, 23°C
• CO2 transmission rate: Test articles were conditioned
at 23°C and analyzed for CO2 transmission rate using
a MOCON Permatran-C™ 4/41 test system per ASTM Conclusion
D3985. The film was oriented so that the inner layer BPCs manufactured from the Aegis5-14 film were tested
of the film was exposed to 100% RH CO2 gas and the for gas and water vapor permeability. These results show
outer layer of the film was exposed to 0% RH N2 gas. the capability of the BPCs to resist loss of water vapor and
These conditions simulate the worst-case scenario transmission of CO2 and O2, thus showing their suitability for
and represent a higher CO2 transmission rate than use in storing solutions where pH, chemical stability, and
ambient conditions (approximately 0% outside RH). chemical concentration are a concern.
Final CO2 transmission rates were recorded when the
system equilibrated.

6
Mechanical properties
Overview
The mechanical properties of a film are important to the
integrity of a BPC and its suitability for use under varying
conditions. The mechanical properties evaluated were
tensile strength, elongation, yield strength, secant modulus,
tensile toughness, puncture resistance, and seam strength.
• Tensile strength is the maximum amount of stress
that a material can handle before breaking
• Elongation is a measure of the ability of a material to
resist changes of shape without crack formation
• Yield strength is the minimum amount of stress on
a material at which it begins to permanently deform
• Tensile toughness is a measure of the ability of a
material to absorb energy and deform up to the
point of failure (fracturing)
• Glass transition temperature: Testing was based on
ASTM E1640: Standard Test Method for Assignment of
the Glass Transition Temperature by Dynamic Mechanical
Analysis (DMA). Samples were analyzed using the DMA
method in tension mode: –145 to 50°C at 3°C per minute
using TA Instruments DMA Q800 equipment. The test
was terminated when the sample yielded.
• Haze: Testing was based on ASTM D1003: Standard
Test Method for Haze and Luminous Transmittance of
Transparent Plastics. Material samples were conditioned
for at least 24 hours at 23°C ± 2°C at 50% relative
humidity. Values were measured using the Hazemeter
method using Byk Haze-Gard I equipment.
Results
The results of mechanical testing are shown in Table 3.

• Secant modulus is the measure of a material’s Table 3. Summary of data for mechanical testing.
elasticity and stiffness
Test Results
• Puncture resistance is a measure of the amount
Tensile strength (psi) 2392 psi
of force required to pierce a material
Elongation (%) 487%
• Seam strength is a measure the force required
to rupture a seam Yield strength (psi) 1362 psi

2% Secant modulus (psi) 43389 psi

Methods
Tensile toughness 243 lbf-in
• Tensile properties: Tensile strength, elongation, yield
strength, tensile toughness (tear resistance), and secant Puncture resistance 25 lbf

modulus testing were based on ASTM D882: Standard Seam strength 31 lbf/in
Test Method for Tensile Properties of Thin Plastic
Haze 68%
Sheeting. Test articles were placed into the grips of the
Instron™ 5565 equipment’s mechanical test frame and Glass transition temperature –24oC
pulled at 50.8 cm (20 in.) per minute.
Conclusion
• Puncture resistance: Testing was based on ASTM
BPCs manufactured from the Aegis5-14 film were tested to
F1306: Standard Test Method for Slow Rate Penetration
verify mechanical properties. These BPCs proved capable of
Resistance of Flexible Barrier Films and Laminates. Test
resisting the stresses that are incurred during shipping and
articles were installed in the Instron 5565 equipment’s
regular use.
fixture. The probe speed was 2.54 cm (1 in.) per minute
and the test article was tested until puncture.

thermofisher.com/sut 7
Extractables
Executive summary funnels that were rinsed with the common solvents
BPCs are widely used in biopharmaceutical processes. before use.
Buffers, media, and other process liquids are stored in the
• Control samples were prepared by filling multiple 20 mL
polyethylene (PE) BPCs integrated with other components
headspace vials with the appropriate extraction sample.
such as filters, tubing, and connectors. Since these
Control samples for inorganic extractables testing were
BPCs are used in critical bioprocessing operations, their
stored in chemically resistant Polytetrafluoroethylene
interactions with biopharmaceutical fluids and final drug
(PTFE) vials.
formulations must be well understood and validated by
end users. To ensure the quality of the BPCs, analytical
testing is conducted for the identification and quantification Storage conditions
of extractables. Extractables are substances that can be
• Samples and controls were stored in a chamber qualified
forced into solution from the BPCs using common solvents
at 60°C ± 2°C.
and physical conditions that are expected to be more
aggressive than normal conditions of use. The goal of this • Chamber qualification, temperature monitoring and
extractables study was to supply the worst-case extractable contingency planning were conducted by the test
data to support end users in their validation studies. laboratory's Standard Operating Procedures (SOPs).

During the study, high-performance liquid chromatography Time points

(HPLC), gas chromatography (GC) and inductively coupled
• At 24 hours, 30 days ± 8 hours, and 90 days ± 8 hours,
plasma (ICP) separation techniques, and mass-spectrometry
one bag containing each of the common solvents was
(MS) identification techniques were used.
collected for sampling and analysis.

• A matching control sample was also removed for each of

Overview
The objective of this study was to provide a comprehensive
assessment of metals, volatile, semi-volatile, and non-volatile
organic compounds that can potentially be extracted from
fluid contact surface of gamma-irradiated BPCs.
The selection of the tested solutions has been done to
cover a range of worst-case conditions for the extractable
analyses. The intent of this extractable analysis is to
provide qualitative and quantitative information to assess
toxicological risks, and to evaluate the overall safety of
BPCs for long-term storage.
the representative solvents at the same time points.
• All analyses for organic extractables were completed
within 7 days of the sample date.
• All analyses for inorganic extractables were completed
within 5 days of the sample date.
Analytical test method
Volatile extractables
A gradient GC-MS method was used.

Methods Table 4. Volatile extractable reference standards

Extraction solvents Reference
standard Name Manufacturer Purity
• The 5 extraction solvents are water-for-injection (WFI), number
20% ethanol (EtOH), 4 M sodium chloride (NaCl),
RS690 Octamethyltetrasiloxane (D4) Aldrich 99.6%
3 M sodium hydroxide (NaOH), and 2 M hydrochloric
acid (HCl). RS864 Octamethyltetrasiloxane (D4) Aldrich 98.9%

RS655 Dodecane (C12) Fluka 100.0%

Sample Preparation RS863 Dodecane (C12) Fluka 99.9%

RS897 1,3-ditert-butyl benzene Aldrich 97.4%

• 500mL BPCs were filled with 216mL of the common
solvents listed above using a graduated cylinder and RS893 2,4-ditert-butyl phenol Aldrich 99.5%

8
Extractables
Semi-volatile extractables Inorganic extractables
A gradient GC-MS method was used.
• The inorganic extractables analysis was performed by
Table 5. Semi-volatile extractable reference standards NSF International.

Reference • Analysis of the below listed metals was conducted.

standard Name Manufacturer Purity
number Aluminum Cobalt Iridium Niobium Selenium Tungsten
RS690 Octamethyltetrasiloxane (D4) Aldrich 99.6% Antimony Copper Iron Palladium Silver Uranium

RS864 Octamethyltetrasiloxane (D4) Aldrich 98.9% Arsenic Dysprosium Lanthanum Phosphorus Sodium Vanadium

RS572 Butylated hydroxytoluene Chem Service 99.5% Barium Erbium Lead Platinum Strontium Ytterbium

RS813 2,4-ditert-butyl phenol Aldrich 99.5% Beryllium Europium Lithium Potassium Tantalum Yttrium

RS896 4-cumylphenol Aldrich 98.9% Bismuth Gadolinium Lutetium Preseodymium Tellurium Zinc

Boron Gallium Magnesium Rhenium Terbium Zirconium

Cadmium Germanium Manganese Rhodium Thallium

Non-volatile extractables Calcium Gold Mercury Rubidium Thorium
A gradient UPLC-PDA-MS method was used.
Cerium Hafnium Molybdenum Ruthenium Thulium

Cesium Holmium Neodymium Samarium Tin

Table 6. Non-volatile extractable reference standards
Chromium Indium Nickel Scandium Titanium
Reference
standard Name Manufacturer Purity
number
Total organic carbon (TOC)
RS800 Cyanox 1790 Aldrich 98.8% The TOC conditions listed in the current version of the test
RS622 Stearic Acid Fluka 99.7% laboratory's internal protocol used.

RS880 Stearic Acid Fluka 99.7%

pH testing
RS805 Irgafos 168 Aldrich 98% The pH conditions listed in the current version of USP
RS567 Irganox 1010 BASF 99.2% General Chapter <791> were used.

RS859 Oleamide Sigma 100%

RS881a Oleamide Sigma 100%

thermofisher.com/sut 9
Extractables

Results
Tables 7-16 report the results of extractables testing

Table 7. Estimated limits of detection (LODs) for Table 8. Estimated limits of LODs for nonvolatile
volatile by headspace GC-MS and semivolatile by extractables by UPLC-PDA-MS
direct inject GC-MS extractables
LOD (μg/mL)
LOD (μg/mL)
Quantitation
Quantitation standard
standard 30 day 30 day
24 hour 30 day 90 day 24 hour 90 day
(WFI, EtOH) (HCI, NaOH, NaCI)
Volatiles Octamethylcyclotetrasiloxane 0.010 0.007 0.004
Irganox 1010 (MS) 0.95 1.05 1.60 0.46
Analysis
Dodecane 0.032 0.017 0.008
Stearic Acid 79.72 51.52 38.33 8.48
Octamethylcyclotetrasiloxane 0.272 0.108 0.125
Semi- Oleamide 0.24 0.14 1.18 0.49
Volatiles Butylated Hydroxytoluene 0.509 0.202 0.252 Irgafos 168 0.20 0.55 0.56 0.40
Analysis
2,4 di-tert-butylphenol 0.604 0.240 0.278 Irganox 1010 (PDA) 0.14 0.10 0.11 0.04

Table 9. Observed volatile, semivolatile, nonvolatile, inorganic extractables,

pH and TOC for WFI

Solvent and Incubation condition Target compounds Extracted compound ppm

VOC Non Detected (ND) ND

SVOC ND ND

WFI NVOC ND ND
24 hours, 60°C Metals ND ND
pH - 5.51
TOC - 7.61
VOC ND ND
SVOC ND ND

WFI NVOC ND ND
30 days, 60°C Metals ND ND
pH - 5.05
TOC - 11.6
VOC ND ND
SVOC ND ND

WFI NVOC ND ND
90 days, 60°C Metals Boron 0.48
pH - 4.39
TOC - 12.3

10
Extractables

Table 10. Observed volatile, semivolatile, nonvolatile and inorganic extractables for 20% EtOH

Solvent and Incubation condition Target compounds Extracted compound ppm

VOC ND ND
SVOC ND ND
20% EtOH
NVOC ND ND
24 hours, 60°C
Metals Sodium 22
pH - 5.41
VOC ND ND
VOC ND ND

20% EtOH SVOC ND ND

30 days, 60°C NVOC ND ND
Metals ND ND
pH - 5.80
VOC ND ND
VOC ND ND

20% EtOH SVOC ND ND

90 days, 60°C NVOC ND ND
Metals Boron 0.77
pH - 4.46

thermofisher.com/sut 11
Extractables
Table 11. Observed volatile, semivolatile, nonvolatile and inorganic extractables for 4 M NaCl

Solvent and Incubation condition Target compounds Extracted compound ppm

VOC ND ND
SVOC ND ND
4 M NaCI
NVOC ND ND
24 hours, 60°C
Metals Potassium 54
pH - 5.73
VOC ND ND
SVOC ND ND
4 M NaCI
NVOC ND ND
30 days, 60°C
Metals Potassium 29
pH - 4.56
VOC ND ND
SVOC ND ND
4 M NaCI
NVOC ND ND
90 days, 60°C
Metals Potassium 130
pH - 4.51

Table 12. Observed volatile, semivolatile, nonvolatile and inorganic extractables for 3 M NaOH

Solvent and Incubation condition Target compounds Extracted compound ppm

VOC ND ND
SVOC ND ND
3 M NaOH
NVOC ND ND
24 hours, 60°C
Metals Potassium 40
pH - 14
VOC ND ND
SVOC ND ND
3 M NaOH
NVOC ND ND
30 days, 60°C
Metals Potassium 19
pH - 14
VOC ND ND
SVOC ND ND
3 M NaOH
NVOC ND ND
90 days, 60°C
Metals Potassium 52
pH - 14

12
Extractables
Table 13. Observed volatile, semivolatile, nonvolatile and inorganic extractables for 2 M HCl

Solvent and Incubation condition Target compounds Extracted compound ppm

VOC ND ND
SVOC ND ND
2 M HCl
NVOC ND ND
24 hours, 60°C
Metals ND ND
pH - 0
VOC ND ND
SVOC ND ND
2 M HCl
NVOC ND ND
30 days, 60°C
Metals Sodium 6
pH - 0
VOC ND ND
SVOC ND ND
2 M HCl
NVOC ND ND
90 days, 60°C
Metals Boron 0.65
pH - 0

Table 14. Control sample pH values

pH

Solvent Control

24 hour 30 day 90 day

WFI 8.19 7.09 7.15

20% EtOH 6.53 6.06 6.97
4 M NaCl 5.89 6.27 6.40
3 M NaOH 14 14 14
2 M HCl 0 0 0

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Extractables
Table 15. Control sample TOC values

Total organic carbon (ppm)

Solvent
24 hour 30 day 90 day

WFI 4.32 4.88 4.57

Table 16. Control sample inorganic extractable values

Incubation Target Extracted

Solvent ppm
Conditions Compounds Compound

24 Hours, 60°C Metals ND ND

WFI 30 Days, 60°C Metals ND ND
90 Days, 60°C Metals Boron 0.790
24 Hours, 60°C Metals ND ND
20% EtOH 30 Days, 60°C Metals ND ND
90 Days, 60°C Metals Boron 0.590
24 Hours, 60°C Metals Potassium 51
30 Days, 60°C Metals Potassium 25
4 M NaCl
Boron 1.200
90 Days, 60°C Metals
Potassium 79
Boron 1.300
24 Hours, 60°C Metals
Potassium 24
3 M NaCl
30 Days, 60°C Metals Potassium 14
90 Days, 60°C Metals Potassium 43
24 Hours, 60°C Metals ND ND
2 M HCI 30 Days, 60°C Metals Boron 1.500
90 Days, 60°C Metals Boron 2.8

14
Bioprocessing films for all
your bioproduction needs

BPCs are built to meet your single-use bioprocessing One partner

needs, whether upstream, for cell culture and fermentation, We offer comprehensive documentation for all our films,
or downstream for sophisticated applications, or simply as well as the following benefits:
for holding and transferring systems in your cGMP
• Outstanding physical characteristics for superior
bioprocessing facilities.
puncture resistance and content integrity

Four films, engineered for the most • Coextruded structure to minimize delamination potential
demanding applications
• Greater cleanliness and low leachable/extractable profiles
Our films are engineered to meet the most demanding
to support end-product purity and quality
requirements of your bioproduction processes.
Choose between: • Non–animal origin formulations for safer contact surfaces

• Thermo Scientific™ CX5-14 polyethylene film—one of the • High gas- and liquid-barrier properties for better
most widely used films in the industry, proven over 10 years content stability

• Thermo Scientific™ ASI™ 26/77 polyethylene film—
a two-layered film with extremely low extractables, for
high-value application
• Thermo Scientific Aegis 5-14 polyethylene film—
™ ™
• Films to meet your bioproduction needs across the
entire bioprocess stream
• Validation packages for assurance of product performance

for high-value applications

• Thermo Scientific™ ASI™ 28 ethyl vinyl acetate film—

a robust, four-layered film with excellent oxygen- and
moisture-barrier properties

Find out more at thermofisher.com/sut

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unless otherwise specified. DOC0005 REV E 1217