CX5-14 film validation guide - Thermo Fisher...

CX5-14 film validation guideFive-layer, 14 mil cast fi lm

DOC0017 • Revision C

November 2016

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™ CX5-14 film is

developed specifically for liquid handling, storage, and transportation

in the biopharmaceutical industry.

Thermo Scientific CX5-14 filmFive-layer, 14 mil cast film

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

CX5-14 film specifications 4

Biocompatibility 5

Physical properties 6

Mechanical properties 7

Extractables 8

Contents

4

CX5-14 film specifications

Property Test protocol Average values

Physical data (post–gamma irradiation, 25–40 kGy)

Tensile strength ASTM D882 2316 psi 16.0 MPa

Elongation ASTM D882 476%

Yield strength ASTM D882 1238 psi 8.5 MPa

2% Secant modulus ASTM D882 37898 psi 261.3 MPa

Tensile toughness ASTM D882 235 lbf-in 2.7 kN-cm

Puncture resistance ASTM F1306 26 lbf 116 N

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

O2 transmission rateASTM D3985, 0% relative humidity (RH) outside, 90% RH inside, 23°C

0.024 cc/100 in2/day

0.37 cc/m2/day

CO2 transmission rateMocon method, 0% RH outside, 100% RH inside, 23°C

0.089 cc/100 in2/day

1.38 cc/m2/day

Water vapor transmission rate

ASTM F1249, 0% RH outside, 100% RH inside, 23°C

0.023 g/100 in2/day

0.35 g/m2/day

HazeASTM D1003 (outside dry/inside dry)

70%

Glass transition temperature ASTM E1640 −19ºF −28ºC

Film gauge Internal study 0.014 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 1137:2006 2.5–4 Mrad 25–40 kGy

Biocompatibility data (post–gamma irradiation, >50 kGy)

USP Class VI USP <88> Pass

Cytotoxicity USP <87> Pass

Bacterial endotoxin USP <85> 0.006 EU/mL

Heavy metals USP <661> <1 ppm

Buffering capacity USP <661> <1 mL

Non-volatile residue USP <661> <1 mg

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

Thermo Scientific™ CX5-14 film is a five-layer, 14 mil cast film produced in a cGMP facility. The outer layer is a polyester elastomer coextruded with an ethyl vinyl alcohol (EVOH) barrier layer and a low-density polyethylene product contact layer. CX5-14 film is manufactured using non-animal origin components.

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

Polyester

Tie

EVOH

Tie

Polyethylene

Schematic cross section

Fluid contact surface

Schematic 3D view

0.8

0.9

1.0

0.9

10.4

www.thermofisher.com/sut 5

• 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.

ResultsA summary of biocompatibility testing is shown in Table 1.

OverviewBiocompatibility 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.

MethodsFollowing are the test methods used to help ensure the biocompatibility of the films. The test methods that were used include:

• USP <88> Class VI

– Acute systemic injection: An extract of the test article was prepared for 72 hours at 50°C and was injected intravenously into an animal model. Signs of toxicity were monitored.

– Intracutaneous reactivity: An extract of the test article was prepared for 72 hours at 50°C and injected under the skin of an animal model, and signs of irritation were monitored.

– Intramuscular implantation: The test article was implanted into the muscle tissue of an animal model, and the resulting tissue sections were examined grossly for signs of infection, necrosis, discoloration, and hemorrhage.

• USP <87> Cytotoxicity: An extract of the test article was prepared for 24 hours at 37°C in E-MEM cell culture medium and cultured with L-929 mouse fibroblast cells. 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.

Biocompatibility

Table 1. Summary of biocompatibility testing.

Test Results

USP Class VI USP <88> Pass

Cytotoxicity USP <87> Pass

Bacterial endotoxin USP <85> 0.006 EU/mL

Heavy metals USP <661> <1 ppm

Buffering capacity USP <661> <1 mL

Non-volatile residue USP <661> <1 mg

Residue on ignition USP <661> <1 mg

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

ConclusionSamples manufactured from the CX5-14 film were tested for biocompatibility per USP and EP protocols. There were no signs of toxicity, irritation, inflammation, or cytotoxicity.

6

OverviewPermeability 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.

• Water vapor transmission rate: Test articles were conditioned at 23°C and analyzed for water vapor transmission rate using a MOCON Permatran-W™ 700 test system per ASTM F1249. The film was oriented so that the inner layer of the film was exposed to 100% 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 transmission rate than ambient conditions (approximately 0% outside RH). Final water vapor transmission rates were recorded when the system equilibrated.

• 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 D3985. The film was oriented so that the inner layer of the film was exposed to 100% RH CO2 gas and the outer layer of the film was exposed to 0% RH N2 gas. These conditions simulate the worst-case scenario and represent a higher CO2 transmission rate than ambient conditions (approximately 0% outside RH). Final CO2 transmission rates were recorded when the system equilibrated.

Physical properties

• 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.

ResultsResults for the above transmission rate testing protocols are shown in Table 2.

ConclusionBPCs manufactured from the CX5-14 film were tested for gas and water vapor permeability. These results show the capability of the BPCs to resist loss of water vapor and transmission of CO2 and O2, thus showing their suitability for use in storing solutions where pH, chemical stability, and chemical concentration are a concern.

Table 2. Summary of data for transmission rate testing.

Test Test conditions Results

Water vapor

transmission rate0% RH outside,100% RH inside, 23°C

0.023 cc/100 in2/day

CO2 transmission rate0% RH outside, 100% RH inside, 23°C

0.089 cc/100 in2/day

O2 transmission rate0% RH outside, 90% RH inside, 23°C

0.024 cc/100 in2/day


OverviewThe 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)

• Secant modulus is the measure of a material’s elasticity and stiffness

• Puncture resistance is a measure of the amount of force required to pierce a material

• Seam strength is a measure the force required to rupture a seam

Methods• Tensile properties: Tensile strength, elongation, yield

strength, tensile toughness (tear resistance), and secant modulus testing were based on ASTM D882: Standard Test Method for Tensile Properties of Thin Plastic Sheeting. Test articles were placed into the grips of the Instron™ 5565 equipment’s mechanical test frame and pulled at 50.8 cm (20 in.) per minute.

• Puncture resistance: Testing was based on ASTM F1306: Standard Test Method for Slow Rate Penetration Resistance of Flexible Barrier Films and Laminates. Test articles were installed in the Instron 5565 equipment’s fixture. The probe speed was 2.54 cm (1 in.) per minute and the test article was tested until puncture.

Mechanical properties

• 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.

ResultsThe results of mechanical testing are shown in Table 3.

Table 3. Summary of data for mechanical testing.

Test Results

Tensile strength (psi) 2316 psi

Elongation (%) 476%

Yield strength (psi) 1238 psi

2% Secant modulus (psi) 37989 psi

Tensile toughness 235 lbf-in

Puncture resistance 26 lbf

Seam strength 31 lbf/in

Haze 70%

Glass transition temperature –28oC

ConclusionBPCs manufactured from the CX5-14 film were tested to verify mechanical properties. These BPCs proved capable of resisting the stresses that are incurred during shipping and regular use.

8

Executive summaryBPCs are widely used in biopharmaceutical processes. Buffers, media, and other process liquids are stored in the polyethylene (PE) BPCs integrated with other components such as filters, tubing, and connectors. Since these BPCs are used in critical bioprocessing operations, their interactions with biopharmaceutical fluids and final drug 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 of extractables. Extractables are substances that can be forced into solution from the BPCs using common solvents and physical conditions that are expected to be more aggressive than normal conditions of use. The goal of this extractables study was to supply the worst-case extractable data to support end users in their validation studies.

During the study, high-performance liquid chromatography (HPLC), gas chromatography (GC) and inductively coupled plasma (ICP) separation techniques, and mass-spectrometry (MS) identification techniques were used.

ObjectiveThe 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.

MethodsExtraction solvents: The 5 extraction solvents are water-for-injection (WFI), 20% ethanol (EtOH), 4 M sodium chloride (NaCl), 3 M sodium hydroxide (NaOH), and 2 M hydrochloric acid (HCl).

Sample Preparation: 500 mL BPCs were filled with 216 mL of the common solvents listed above using a graduated cylinder and funnels that were rinsed with the common solvents before use.

Extractables

Control samples were prepared by filling multiple 20mL headspace vials with the appropriate extraction sample. Control samples for inorganic extractables testing were stored in chemically resistant Polytetrafluoroethylene (PTFE) vials.

Storage conditions

• Samples and controls were stored in a chamber qualified at 60°C ± 2°C.

• Chamber qualification, temperature monitoring and contingency planning were conducted by the test laboratory's Standard Operating Procedures (SOPs).

Time points

• At 24 hours, 30 days ± 8 hours, and 90 days ± 8 hours, one bag containing each of the common solvents was collected for sampling and analysis.

• A matching control sample was also removed for each of 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 methodVolatileextractables

A gradient GC-MS method was used.

Table 4. Volatile extractable reference standards

Reference standard number

Name Manufacturer Purity

RS690 Octamethyltetrasiloxane (D4) Aldrich 99.6%


RS655 Dodecane (C12) Fluka 100.0%

RS863 Dodecane (C12) Fluka 99.9%

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

RS893 2,4-ditert-butyl phenol Aldrich 99.5%


Semi-volatile extractables A gradient GC-MS method was used.

Non-volatile extractables A gradient UPLC-PDA-MS method was used.

Inorganic extractables

• The inorganic extractables analysis was performed by NSF International.

• Analysis of the below listed metals was conducted.

Extractables

Table 5. Semi-volatile extractable reference standards





RS572 Butylated hydroxytoluene Chem Service 99.5%

RS813 2,4-ditert-butyl phenol Aldrich 99.5%

RS896 4-cumylphenol Aldrich 98.9%

Table 6. Non-volatile extractable reference standards



RS800 Cyanox 1790 Aldrich 98.8%

RS622 Stearic Acid Fluka 99.7%

RS880 Stearic Acid Fluka 99.7%

RS805 Irgafos 168 Aldrich 98%

RS567 Irganox 1010 BASF 99.2%

RS859 Oleamide Sigma 100%

RS881a Oleamide Sigma 100%

Aluminum Cobalt Iridium Niobium Selenium Tungsten

Antimony Copper Iron Palladium Silver Uranium

Arsenic Dysprosium Lanthanum Phosphorus Sodium Vanadium

Barium Erbium Lead Platinum Strontium Ytterbium

Beryllium Europium Lithium Potassium Tantalum Yttrium

Bismuth Gadolinium Lutetium Preseodymium Tellurium Zinc

Boron Gallium Magnesium Rhenium Terbium Zirconium

Cadmium Germanium Manganese Rhodium Thallium

Calcium Gold Mercury Rubidium Thorium

Cerium Hafnium Molybdenum Ruthenium Thulium

Cesium Holmium Neodymium Samarium Tin

Chromium Indium Nickel Scandium Titanium

Total organic carbon (TOC)The TOC conditions listed in the current version of the test laboratory's internal protocol used.

pH testingThe pH conditions listed in the current version of USP General Chapter <791> were used.

10

Results Tables 7-16 report the results of extractables testing

Extractables

Table 7. Estimated limits of detection (LODs) for volatile by headspace GC-MS and semivolatile by direct inject GC-MS extractables

Quantitation standardLOD (μg/mL)

24 hour 30 day 90 day

Volatiles

Analysis

Octamethylcyclotetrasiloxane 0.010 0.007 0.004

Dodecane 0.032 0.017 0.008

Semi-

Volatiles

Analysis

Octamethylcyclotetrasiloxane 0.272 0.108 0.125

Butylated Hydroxytoluene 0.509 0.202 0.252

2,4 di-tert-butylphenol 0.604 0.240 0.278

Table 8. Estimated limits of LODs for nonvolatile extractables by UPLC-PDA-MS

Quantitation

standard

LOD (μg/mL)

24 hour30 day

(WFI, EtOH)

30 day

(HCI, NaOH, NaCI)90 day

Irganox 1010 (MS) 0.95 1.05 1.60 0.46

Stearic Acid 79.72 51.52 38.33 8.48

Oleamide 0.24 0.14 1.18 0.49

Irgafos 168 0.20 0.55 0.56 0.40

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

WFI 24 hours, 60°C

VOC Non Detected (ND) ND

SVOC ND ND

NVOC ND ND

Metals Potassium 7

pH - 5.08

TOC - 9.75

WFI 30 days, 60°C

VOC 2,4-Di-tert-butyl phenol 0.071, 0.027, 0.030

SVOC ND ND

NVOC ND ND

Metals ND ND

pH - 5.50

TOC - 11.3

WFI 90 days, 60°C

VOC 2,4-Di-tert-butyl phenol <0.008, 0.008, <0.008

SVOC ND ND

NVOC ND ND

Metals Boron 0.49

pH - 4.24

TOC - 12.8


Extractables

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


20% EtOH 24 hours, 60°C

VOC 1,3 – Di-tert-butyl benzene <0.032, <0.032, <0.032

SVOC 2,4-Di-tert-butyl phenol 0.625, 0.576, 0.438

NVOC Irgafos 168 Degradanta 1.084, 1.099, 1.083

Metals ND ND

pH - 4.87

20% EtOH 30 days, 60°C

VOC 1,3 – Di-tert-butyl benzene 0.024. 0.023, 0.023

VOC 2,4-Di-tert-butyl phenol 0.034, <0.017, <0.017


NVOC Irgafos 168 Degradanta 1.702, 1.745, 2.162

Metals ND ND

pH - 5.58

20% EtOH 90 days, 60°C

VOC 1,3 – Di-tert-butyl benzene 0.009, 0.010, 0.009

VOC 2,4-Di-tert-butyl phenol 0.014, 0.008, 0.009


NVOC Irgafos 168 Degradanta <0.400, <0.400, <0.400

Metals Boron 0.74

pH - 4.21

a Bis(2,4-Di-tert-butylphenyl) hydrogen phosphate

12

ExtractablesTable 11. Observed volatile, semivolatile, nonvolatile and inorganic extractables for 4 M NaCl


4 M NaCI 24 hours, 60°C

VOC ND ND

SVOC ND ND

NVOC ND ND

Metals Potassium 47

pH - 5.51

4 M NaCI 30 days, 60°C

VOC ND ND

SVOC ND ND

NVOC ND ND

Metals Potassium 30

pH - 4.84

4 M NaCI 90 days, 60°C

VOC ND ND

SVOC ND ND

NVOC ND ND

Metals Potassium 130

pH - 4.92

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


3 M NaOH 24 hours, 60°C

VOC ND ND


NVOC ND ND

Metals Potassium 40

pH - 14

3 M NaOH 30 days, 60°C

VOC ND ND

SVOC ND ND

NVOC ND ND

Metals Potassium 19

pH - 14

3 M NaOH

VOC ND ND

SVOC ND ND

NVOC ND ND

Metals Potassium 52

pH - 14


ExtractablesTable 13. Observed volatile, semivolatile, nonvolatile and inorganic extractables for 2 M HCl


2 M HCl 24 hours, 60°C

VOC ND ND

SVOC ND ND

NVOC ND ND

Metals ND ND

pH - 0

2 M HCl 30 days, 60°C

VOC ND ND

SVOC ND ND

NVOC ND ND

Metals Sodium 6

pH - 0

2 M HCl 90 days, 60°C

VOC ND ND

SVOC ND ND

NVOC ND ND

Metals Boron 0.74

pH - 0

Table 14. Control sample pH values

Solvent

pH

Control


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

14

Extractables

Table 16. Control sample inorganic extractable values

SolventIncubation

Conditions

Target

Compounds

Extracted

Compoundppm

WFI

24 Hours, 60°C Metals ND ND

30 Days, 60°C Metals ND ND

90 Days, 60°C Metals Boron 0.790

20% EtOH


30 Days, 60°C Metals ND ND


4 M NaCl

24 Hours, 60°C Metals Potassium 51

30 Days, 60°C Metals Potassium 25

90 Days, 60°C MetalsBoron Potassium

1.200 79

3 M NaCl

24 Hours, 60°C MetalsBoron Potassium

1.300 24



2 M HCI




Table 15. Control sample TOC values

SolventTotal organic carbon (ppm)


WFI 4.32 4.88 4.57

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One partnerWe offer comprehensive documentation for all our films, as well as the following benefits:

• Outstanding physical characteristics for superior puncture resistance and content integrity

• Coextruded structure to minimize delamination potential

• Greater cleanliness and low leachable/extractable profiles to support end-product purity and quality

• Non–animal origin formulations for safer contact surfaces

• High gas- and liquid-barrier properties for better content stability

• Films to meet your bioproduction needs across the entire bioprocess stream

• Validation packages for assurance of product performance

Bioprocessing films for all your bioproduction needs

BPCs are built to meet your single-use bioprocessing needs, whether upstream, for cell culture and fermentation, or downstream for sophisticated applications, or simply for holding and transferring systems in your cGMP bioprocessing facilities.

Four films, engineered for the mostdemanding applicationsOur films are engineered to meet the most demanding requirements of your bioproduction processes. Choose between:

• Thermo Scientific™ CX5-14 polyethylene film—one of the most widely used films in the industry, proven over 10 years

• 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— for high-value applications

• Thermo Scientific™ ASI™ 28 ethyl vinyl acetate film— a robust, four-layered film with excellent oxygen- and moisture-barrier properties

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