Characterization of Single-Use BioProcess Container Systems Based on Thermo Scientific Aegis5-14 Film. Aegis5-14 Film Technical Paper

Transcription

1 Characterization of Single-Use BioProcess Container Systems Based on Thermo Scientific Aegis5-14 Film Aegis5-14 Film Technical Paper

2 Table of Contents Table of Contents Introduction... 3 Two-Dimensional Geometry... 4 PVC Mono-Layer... 4 EVA Mono-Layer... 4 EVA-Based Single-Web Multi-Layer Films... 4 Multi-Web Containers with Polyethylene Films for Larger Volumes... 5 Three-Dimensional Geometry... 5 Single-Web Multi-Layer Polyethylene Films... 5 Performance Requirements... 6 Ten Important Characteristics of Container Systems... 7 Discussion and Results 1. Biological Compatibility Tensile Properties Puncture Resistance and Puncture Strength Glass Transition Temperature Transportability Clarity Permeability ph Stability Extractable Compounds Cell Culture Growth Performance Overall Conclusions Aegis5-14 Film Characterization Thermo Scientific

3 Introduction The trend towards single-use containers and systems in the biopharmaceutical industry has led to a shift of concerns regarding cleaning and sterilization of conventional multi-use containers and systems to those regarding material characteristics and container/content interactions. The purpose of this document is to cover these concerns and explain our approach to them. Flexible container systems consist of plastic films, ports, tubing and fittings. Performance of a specific container system in a particular application depends on the materials and quality of construction as well as the conditions and constraints imposed by the application. The integrity of stored contents depends primarily on the characteristics of the film, the largest component of any flexible container system. Figure 1. Flexible container being used for cell culture medium hydration The volume capacity of flexible containers available on the market can range from 50mL to 10,000L. Large volume flexible container systems can be used as alternatives to traditional stainless steel bioprocessing systems. Small volume flexible containers may replace glass or rigid plastic containers used for applications such as sampling and storage. 3 Aegis5-14 Film Characterization Thermo Scientific

4 Two-Dimensional Geometry Historically, small volume flexible containers first appeared as two-dimensional (2D) pillow-style (flat until filled) containers for storage of blood and other medical solutions. The containers utilized materials with a special combination of properties: Optical transparency: Allows visual inspection of contents Flexibility: Allows addition or removal without introducing air Wide operating temperature range of -80 C to +60 C: Enables freezing or heating stored contents Chemical compatibility with many commonly used medical solutions: Enables use in many medical applications Resistance to degradation from sterilization Ease of manufacture PVC Mono-Layer The first flexible containers typically used poly vinyl chloride (PVC) films. PVC, by itself is a brittle material not suitable for flexible containers but becomes a soft and pliant material through the addition of plasticizers such as di-octyl phthalate (DOP). Flexible PVC was used to make a variety of small volume flexible medical containers from 50mL up to 10L, yet the material has a number of significant limitations. For example, PVC includes high levels of plasticizers that limit the types of fluids suitable for storage. Additionally, low moisture and gas barrier properties preclude long term storage without suffering significant water loss and degradation from gas (O 2 and CO 2 ) ingress and egress. EVA Mono-Layer Ethylene vinyl acetate (EVA) film was developed as an alternative to PVC. EVA is a flexible, tough material that is capable of absorbing high levels of energy without loss of integrity. Additionally, EVA films do not contain plasticizers. The absence of plasticizers reduces the amount of extractables (leachables) in the contained product. EVA, like PVC, lacks the ability to protect against gas and moisture exchange. For long-term storage of aqueous solutions, both container materials require secondary barrier pouches to minimize water loss and degradation from gas ingress or egress. EVA-Based Single-Web Multi-Layer Films The development of multi-layer films addressed the problems of gas and moisture exchange. Materials with a high gas barrier, such as ethylene vinyl alcohol (EVOH), serve as the core of a multi-layer film. For EVA-based films, the EVOH resin is physically bonded between two layers of EVA film, resulting in a single-web film. This is strong, flexible film, with good gas barrier properties and moderate water vapor properties. EVA-based single-web multi-layer films find common use in container systems of up to 50L in 2D pillow-style geometry. 4 Aegis5-14 Film Characterization Thermo Scientific

5 Multi-Web Containers with Polyethylene Films for Larger Volumes The bioprocess market often requires large (50L to 10,000L) containers. At first, these large containers utilized a 2D, independent multi-web film construction with polyethylene (PE) as the fluid-contacting inner film, and a laminate or co-extrusion of various resins, including EVOH as the outer barrier film. Compared to EVA, PEs are inherently cleaner with lower extractable/leachable levels as compared to EVA. Moreover, PE is inert to a broader range of chemicals. The large volume 2D pillow-style geometry, however, presents challenges with mixing and with shipping reliability. In addition, these large flexible containers prove hard to fill when inserted into rigid support containers, which are required at large volumes. Three-Dimensional Geometry A second generation of large flexible containers alleviated the mixing, shipping and filling shortcomings of 2D containers by designing a three-dimensional (3D) gusseted container. Single-Web Multi-Layer Polyethylene Films A further enhancement combined 3D construction and co-extruded or laminated film composed of a PE fluid-contact layer for a good moisture barrier, EVOH as the gas-barrier layer, and a durable skin layer on the outside of the film. In these, the PE fluid-contact layer is physically bonded to the gas-barrier layer, forming a single-web multi-layer structure. Thus, a single-web flexible container construction has become possible for large volume container systems of 50L to 10,000L with good gas barrier and good water barrier properties, and are inert to the broadest range of chemicals (Figure 2 and 4). Figure 2. Typical single-web multi-layer 3D Thermo Scientific BPC System 5 Aegis5-14 Film Characterization Thermo Scientific

6 An example of this type of film is Thermo Scientific Aegis 5-14 Film, which was developed specifically for liquid handling, production, storage and transportation in the biopharmaceutical industry. The film structure is shown in Figure 3. Polyester Tie EVOH Tie PE Dimensions in mil 10.4 Figure 3. Schematic cross-section and 3D view Performance Requirements Selecting the optimal container for an intended application requires first deciding on some design parameters: Container Geometry: Define the volume and shape (2D vs. 3D) of the container Operating Environment: Define the temperature, humidity and storage time for the containers Permeability: Define level of protection required for the product. Products that are sensitive to oxidation, ph shifts, or concentration changes due to O 2 ingress, CO 2 exchange or water loss, respectively, require higher barrier properties Materials Compatibility: Determine the type of chemicals that will be stored in the container Product contact materials should also be free of animal derived components Transportation: Determine the container size and conditions for shipping and handling filled containers 6 Aegis5-14 Film Characterization Thermo Scientific

7 In addition to determining the performance requirements, the manufacturing and quality systems of the manufacturer should be considered. Flexible containers for bioprocess applications should be manufactured in a clean-room cgmp environment under a proper quality system. Fabricating flexible containers and their sub-components in classified clean rooms prevents particulate, bio-burden, or other contamination. Product and process validations ensure that the system has an inherent reliability and capability to perform as intended. The quality system ensures lot traceability, vendor qualification, document control, design control, employee training, and possibly other quality-related manufacturing protocols. Figure 4. Various sizes of single-web multi-layer 3D BPC systems Ten Important Characteristics of Container Systems The list below includes 10 important characteristics for evaluating container systems for bioprocess applications. A system s performance can help determine the suitability of that system for a specific application. These characteristics relate directly to the ability of a container system to maintain product integrity and otherwise perform as required. The importance of each item, naturally, varies with the application. 1. Biological Compatibility 2. Tensile Properties 3. Puncture Resistance and Puncture Strength 4. Glass Transition Temperature 5. Transportability 6. Clarity 7. Permeability 8. ph Stability 9. Extractable Compounds 10. Cell Culture Growth Performance 7 Aegis5-14 Film Characterization Thermo Scientific

8 Discussion and Results This section discusses each of the important characteristics listed above and describes tests and results for Aegis5-14 Film. 1. Biological Compatibility Standards Several test standards are available to show biocompatibility, including: United States Pharmacopoeia (USP), International Organization for Standardization (ISO), ASTM International (ASTM) and European Pharmacopoeia (EP). These are reported by container manufacturers in the literature associated with their products. Results Table 1 shows the various biological tests performed for Aegis5-14 Film along with the results and requirements. Discussion Established test standards from several organizations, including USP, ISO and EP exist. There may be other tests done that are not standardized. The tests chosen by each manufacturer depend on targeted market and intended application. Biological Reactivity Tests, In Vitro: This category of tests evaluates biological reactivity of mammalian cell cultures to polymeric materials. To be considered biocompatible, materials should not cause cell lysis or show other evidence of toxicity. USP<87> describes two methods to test for cytotoxicity: the MEM elution test and the agar diffusion test. Alternatively, cytotoxicity can be evaluated by an elution test according to ISO Several mammalian cell lines can be used to test for cytotoxicity and cell growth inhibition. One or more tests may be performed, depending on the specific application of the product. Biological Reactivity Testing, In Vivo: This is a series of three tests that evaluate biological reactivity of animals to polymeric materials: systemic toxicity, intracutaneous reactivity, and implantation. These tests can be done according to USP<88> Class VI Biological Reactivity. Alternatively, equivalent ISO test methods are available under (Irritation & Sensitization equivalent to USP<88> Intracutaneous Reactivity), (Systemic Toxicity) and (Implantation). The USP and ISO tests differ in such details as the number of replicates, time between replicates, testing time, etc. Bacterial Endotoxin Testing: LAL testing is done to evaluate the presence of bacterial endotoxins in or on a sample. Several methods exist to do this test. The USP testing standard is USP<85>. 8 Aegis5-14 Film Characterization Thermo Scientific

9 Physicochemical Testing: This set of tests evaluates the physical and chemical properties of plastics and their extracts. The USP tests for plastics include the following: Buffer Capacity, Non- Volatile Residue, Residue on Ignition and Heavy Metals. USP<661> testing for containers consists of Multiple Internal Reflectance, Thermal Analysis, Light Transmission, Water Vapor Permeation, Heavy Metals and Non-Volatile Residue. Hemolysis: This test assesses the hemolytic properties of materials. There are several test standards: ASTM, EP, and ISO In Vitro and In Vivo methods are available. Other: The EP< > is another set of physicochemical tests, which includes the following: Appearance, Initial Color of Solution, Acidity, Alkalinity, Absorbance, Reducing Substances and Transparency. Test Results Requirement USP<88> Systemic Toxicity Pass Pass USP<88> Intracutaneous Pass Pass USP<88> Implantation Pass Pass USP<87> Cytotoxicity, Agar Diffusion Pass Pass USP<87> Cytotoxicity, Elution Pass Pass USP<85> Kinetic-Chromogenic LAL <0.006EU/mL <0.25EU/mL USP<661> Physicochemical Non Volatile <1mg <15mg USP<661> Physicochemical Residue on Ignition <1mg <5mg USP<661> Physicochemical Heavy Metals <1ppm <1ppm USP<661> Physicochemical Buffering Capacity <1mL <10mL ISO In-Vitro Hemolysis Study Non-hemolytic Non-hemolytic EP< > Plastic Containers for Aqueous Solutions for Parenteral Infusion Pass Pass Irradiation Dosage kGy kGy Table 1. Biological test results summary for Aegis5-14 Film 9 Aegis5-14 Film Characterization Thermo Scientific

10 2. Tensile Properties Overview Film tensile properties predict the ability of a container to maintain integrity of liquids in a desired application (e.g., liquid storage, shipping, and handling). Six tensile properties were evaluated: secant modulus, yield strength, tensile strength, maximum percent elongation, toughness, and seam strength. Secant Modulus, measured at 2 percent strain, is the stiffness of a material measured in tension over the elastic region of the material. A low secant modulus corresponds to high compliance or flexibility. Yield Strength is the maximum engineering stress, applied in tension, within the elastic region. The yield strength is a measure of the maximum load that may be applied to a material before it permanently deforms. Tensile Strength (TS) of a material is the maximum engineering stress, in tension, sustained without fracture. The TS provides information related to a material s resistance to deformation and overall strength. Percent Elongation (%EL) is the maximum strain, in tension, sustained without fracture. Films that exhibit high elongation and low modulus values correspond to a system that is more resistant to flex-cracking and damage in sub-freezing temperature environments. Flex-Cracking is a failure mode in the flexible container where the film forms and propagates a crack through cyclic fatigue. Fatigue occurs due to the stress cycling of the film associated with the wave action of the contained sterile fluid during shipping and handling. Tensile Strength and Elongation both describe material strength and ductility. Toughness is a tensile property that indicates the amount of energy absorbed by a material as it fractures. The total area under the tensile stress-strain curve indicates toughness. Films with high toughness are able to absorb high levels of energy during service. Seam Strength: To fabricate flexible containers, panels of film are sealed together. The strength of these seals is important to the physical integrity of the container. A comparison of seam strength to the material TS provides data on the influence of the sealing process to the parent material. 10 Aegis5-14 Film Characterization Thermo Scientific

11 Experimental Film tensile properties were measured using an Instron 5965 tensile test machine according to ASTM D882 Standard Test Method for Tensile Properties of Thin Plastic Sheeting. The Instron machine has a load accuracy of ±0.12lbf and displacement accuracy of ± Data was collected from an average of 60 samples. Discussion Table 2 test results indicate that Aegis5-14 Film has superior tensile characteristics, making it an ideal choice for single-use film bioproduction applications. Property 2% Secant Modulus Yield Strength Tensile Strength Percent Elongation Toughness Seam Strength Average 43ksi 1494psi 2276psi 507% 252in-lb 30lbf/in Table 2. Aegis5-14 Film tensile results summary for ASTM D Puncture Resistance and Puncture Strength Overview Puncture resistance predicts the durability of a flexible container film in use. Since the film has a large surface area, it is most susceptible to damage by impact with another object. Films with high puncture resistance correspond to materials that can absorb the energy of an impact by both resistance to deformation and increased elongation. Films with high puncture strength, on the other hand, correspond to materials that inhibit deformation during puncture. A film with high puncture resistance offers superior resistance to damage, thereby providing increased protection to the container s contents. Experimental Film puncture resistance was measured using an Instron 5965 tensile test machine in compression mode. Testing was performed on a total of 30 samples at a crosshead speed of 20in/min with a 1 diameter probe (Series 9 Method 2) with a film area of 28.3in 2. Discussion Table 3 test results indicate that Aegis5-14 Film performed as expected during the puncture resistance testing. Puncture strength is similar to tensile strength in that both properties indicate the material s resistance to deformation. Puncture resistance, measured in energy units, evaluates the film strength and extensibility 11 Aegis5-14 Film Characterization Thermo Scientific

12 properties. Puncture resistance is similar to tensile toughness, which measures the amount of energy absorbed by a material under loading. High puncture resistance improves the durability and reliability of the flexible container by enabling it to resist damage during handling, storage and shipping of product. Puncture Resistance Average 173in-lb Table 3. Puncture resistance results summary for Aegis5-14 Film 4. Glass Transition Temperature Overview The glass transition temperature (Tg) is the temperature at which a polymeric material changes from a viscous or rubbery state to a brittle or glassy state. The lower the Tg of a film, the greater its ability to absorb and dissipate energy imparted to the flexible container. The capability of a flexible container to maintain fluid integrity is dependent upon the amount of energy the container can absorb or dissipate during use. Materials that exhibit a low Tg are more resistant to flexcracking during shipping or handling and demonstrate superior impact resistance and other mechanical properties. Tg also indicates the material capability for low temperature applications. Experimental The glass transition temperature was measured using a Dynamic Mechanical Analyzer (DMA) according to ASTM E1640 Standard Test Method for Assignment of the Glass Transition Temperature By Dynamic Mechanical Analysis. The DMA operates by applying a periodic or oscillatory stress to a material over a temperature range while measuring the modulus (stiffness) and dampening (energy dissipation) properties. Polymeric materials are viscoelastic; i.e., they exhibit a combination of elastic recovery and viscous flow under stress. The DMA measures the material s ability to store energy in the form of a storage modulus (elastic behavior), and a material s ability to dissipate energy in the form of a loss modulus (viscous behavior). Testing was performed on one lot of film in tension mode at a fixed frequency of 1 Hz, 0.08% strain, 3 C/min temperature ramp rate, and at a temperature range of -90 to +70 C. Tg was calculated at the extrapolated onset to the sigmoidal change in the storage modulus. Discussion Test results indicated that Aegis5-14 Film performed as expected and exhibited an average Tg of -42 C. In general, the lower the Tg, the lower the operating temperature of the container. DMA Testing Average -42Tg C Table 4. Glass transition results for Aegis5-14 Film 12 Aegis5-14 Film Characterization Thermo Scientific

13 5. Transportability Overview Transportation (shipping) testing provides information on the durability of a fluid-filled flexible container. During shipping, wave action in the container imparts energy into the plastic film, which leads to cyclic fatigue of the material. Cyclic fatigue creates and propagates cracks (flex-cracking) in the film that result in fluid integrity failures (Figure 5). Large volume fluid systems ( 50L) are more difficult to ship and handle as compared to small volume systems ( 20L) due to the mass and size of the filled containers. Figure 5. Flex-cracking in film Experimental BPC units of various sizes were subjected to simulated transportation testing to determine the durability of the film. Transportation testing is considered the most extreme functional environment for a liquid-filled BPC. The larger unit shipped in a rigid plastic container, and the smaller units shipped in a rigid barrel. The larger unit allows for excessive movement (wave motion) of the BPC system and is considered the worst-case shipper. Testing was performed to ISTA (International Safe Transit Association) and other applicable guidelines. Units were filled and packaged according to standard Thermo Fisher Scientific procedures. Discussion The containers made of Aegis5-14 Film were tested for applicable guideline time frames without evidence of integrity failure. Examination of these units indicated that there were no flex-cracks in the film. Test results indicated that all units met the Thermo Fisher Scientific no-leak requirement, which is several times greater than the ISTA test requirement. 13 Aegis5-14 Film Characterization Thermo Scientific

14 Figure 6. LAB vibration unit Figure 7. BPC shipping units 6. Clarity Overview The clarity of the plastic film allows for the viewing of the contents within the flexible container. The contents may be examined for sterility breach as evidenced by turbidity in the solution or for particulate contamination. The clarity of a film depends on the resin properties, the presence of chemical additives, physical surface treatments and film structure. Experimental The film clarity was evaluated according to ASTM D1003 Standard Test Method for Haze and Luminous Transmittance of Transparent Plastics. Testing was performed on one lot of film with industry-accepted equipment. 14 Aegis5-14 Film Characterization Thermo Scientific

15 Discussion The clarity of a film is influenced by a number of factors, including chemical additives, physical surface treatment, inherent component resin properties, and film properties such as thickness, structure, and solution contact. Physical surface treatments generally take the form of imprinting or texturing to prevent internal or external sticking. Though such mechanical surface finishing does not impact the chemical composition of the film, it can increase the film haze due to the increased light scattering. Solution contact also impacts film clarity in that wet film has higher clarity (exhibits less haze) than dry film. Improved clarity allows for better visual observation of flexible container contents. Test results are described in Table 5. Haze/Clarity Testing Average 52% Haze Table 5. Haze/clarity results for Aegis5-14 Film 7. Permeability Overview Gas and water permeability are predictors of the flexible container s ability to maintain the chemical stability, ph and concentration of its fluid contents over time. Permeability rates of a multilayer film are dependent on a number of factors. These include the composition of film layers, the order of layers, temperature, and the film s moisture content as determined by relative humidity. Experimental Oxygen and carbon dioxide permeability testing was performed according to ASTM D3985 Standard Test Method for Oxygen Gas Transmission Rate Through Plastic Film and Sheeting Using a Coulometric Sensor on one lot of film. Moisture vapor permeability testing was performed according to ASTM F1249 Standard Test Method for Moisture Vapor Transmission Rate Through Plastic Film and Sheeting Using a Modulated Infrared Sensor on one lot of film. Discussion Table 6 test results indicate that Aegis5-14 Film exhibits the following permeability results for O 2 transmission, CO 2 transmission, and moisture vapor transmission, respectively. Oxygen Carbon Dioxide Moisture Vapor 100% RH IN/0% RH OUT 23 C 100% RH IN/0% RH OUT 23 C 100% RH IN/0% RH OUT 0.491cc/m 2 /24 hrs 2.737cc/m 2 /24 hrs 0.34g/m 2 /24 hrs Table 6. Permeability/transmission rate results for Aegis5-14 Film 15 Aegis5-14 Film Characterization Thermo Scientific

16 8. ph Stability Overview The importance of ph stability in cell culture and other applications is well documented. Changes in ph can affect protein structure and function. The ph stability is, among other factors, a function of container properties (such as permeability), the buffering system of the contained solution, and the environmental (storage) conditions. ph Stability Values ph values for the Aegis5-14 Film extraction solvents are shown in Table 7 below. The ph of water dropped when stored in bags made from Aegis5-14 Film. This is consistent with industryaccepted polyethylene films, and is most likely due to the formation of Acetic Acid from Acetate. Solvent Control ph Aegis5-14 Film ph 1-day 30-days 90-days 1-day 30-days 90-days WFI % EtOH M NaCl M NaOH M HCl Table 7. ph measurements of various solutions after 1-, 30-, and 90-day storage time points in Aegis5-14 Film 9. Extractable Compounds Overview Flexible bioprocess containers are manufactured from a variety of plastic films. These films are composed of several distinct layers co-extruded into a single sheet. The distinct layers are manufactured from plastic resins. All plastic resins may contain chemical additives, which are required to process or convert the plastic resin into an end product. Anti-oxidants or heat stabilizers are common additives that prevent hydrolysis or the breakdown of polymer chains during processing. Lubricants (anti-block or slip-agents), primarily used in plastic films, prevent the film from sticking together during processing. Lubricants have historically contained stearate compounds that have an animal derived source. Other classes of plastic additives are: adhesives, anti-statics, colorants, light stabilizers and plasticizers. Plastic resins used in flexible bioprocess containers should contain minimal additives and are free of animal derived products. 16 Aegis5-14 Film Characterization Thermo Scientific

17 Containers used to store solutions utilized in a drug manufacturing process, such as process intermediates, or final product formulations are a potential source of contaminants in the final drug product. Container interactions with drug solutions may result in either extracted or leached substances, or both. Extractables are substances that can be extracted from a bioprocessing containment system using solvents that are expected to be more aggressive than the conditions of contact between the containment system and a relevant drug solution. Leachables are substances that are present in the finished drug product because of its interaction with a bioprocessing containment system during normal use. The presence of extractable compounds may not be a concern if they are not present as a leachable, are inert, or are present at low levels. Extractables that are removed or inactivated during normal processing of a drug product are not a concern. If extractables remain in the final drug product, they are considered leachables. Extractable and leachable compounds are related to the chemical composition of the plastic resins that make up the container. Degradation of the container material that occurs during sterilization by gamma irradiation is a significant source of extractable compounds. Some extracted compounds can alter or affect the product stored in the container by interacting or reacting with the fluid contents. The presence of extractable compounds can cause changes in overall solution characteristics, including ph shifts and an increase in total organic carbon (TOC) levels. In the worst case, an extractable compound may be biologically reactive. Container materials, in general, should be inherently clean. They all must pass biological and toxicological testing to ensure that they are biocompatible, which minimizes the risk of harmful extractable compounds. Appropriate functional testing must be done to assess the effects of extractables for each particular application. Finished drug product solutions must be analyzed for the presence of leachable substances. The presence of leachable substances is dependent upon the exact processing conditions employed in the drug manufacturing process. Since a wide variety of drug products and processing solutions are used, it is not possible for a supplier of Flexible Bioprocess Containers to provide a leachables analysis for a particular process liquid or drug. However, an extractables analysis, utilizing extreme solution concentrations and/or conditions may be performed. The results of this extractables analysis can be considered a worst-case extreme for any possible leachables compounds derived from a Flexible Bioprocess Container. Extractable types and levels depend on the nature of the solution and the storage conditions. The factors of storage time and temperature, and container surface-area-to-fluid-volume ratio are important in determining the suitability of a container with a particular extractable profile. In this study, Aegis5-14 Film was analyzed for extractable compounds in a variety of extreme solutions. The main purpose of the testing was to compare the level of selected extractable compounds from these containers. The acceptable types and levels of extractable compounds must be assessed for each application. 17 Aegis5-14 Film Characterization Thermo Scientific

18 Extractables Testing/Screening Bioprocess containers made from Aegis5-14 Film were irradiated to kGy and sent to NSF Pharmalytica for extractables screening. The parameters of the test design are described in Table 8. The methods used for identifying and quantifying the extractants are commonly accepted by the industry. The results from the Aegis5-14 Film are shown in Table It is important to note that the concentration values are only semi-quantitative since the detected compounds responses are compared to reference standards which may or may not exactly match. Summaries of the results are presented in following sections. Parameter Description Parameter Value Surface Area-to-Volume Ratio (SA/V) 2200cm 2 /L Sample Quantity Storage Temperatures Storage Time Model Solvents Analyses Sample Sterilization Triplicate 60 C All Tests 1-, 30-, and 90-days Water for Injection (WFI) 20% EtOH 4M NaCl 3M NaOH 2M HCl Volatile Organic Compounds (VOC) Semi-Volatile Organic Compounds (SVOC) Non-Volatile Organic Compounds (NVOC) Inorganics (i.g., Metals) Total Organic Carbon (TOC) (WFI only) Gamma Irradiation: kGy Table 8. Aegis5-14 Film extractables test parameter outline 18 Aegis5-14 Film Characterization Thermo Scientific

19 Volatile Organic Compounds (VOC) Volatile Organic Compound extractants from Aegis5-14 Film were tested for using Headspace Gas Chromatography/Mass Spectrometry (GC/MS). No volatile extractants were detected above the reporting thresholds in any of the model solvents at 1, 30, and 90 days. Solvent Film Identification WFI EtOH HCl NaOH NaCl Aegis5-14 Film Table 9. Volatile extractables by headspace GC/MS for 1-, 30-, and 90-day samples in Aegis5-14 Film Semi-Volatile Organic Compounds (SVOC), Acetate, and Formate Semi-Volatile Organic Compound extractants from Aegis5-14 Film were tested for using Direct Inject GC/MS. No semi-volatile extractants were detected above the reporting thresholds in any of the model solvents at 1-, 30-, or at 90-day time intervals. In addition to the lab s standard reference library, specific reference standard of Sodium Acetate and Sodium Formate were used to quantify the amount of Acetate and Formate extractables using Direct Inject GC/MS. The Formate concentrations for the 1-day samples were all very similar in magnitude to the control, while the concentrations in the 90-day samples seemed to increase slightly over the control. The Acetate concentrations for the 1-, 30-, and 90-day samples were significantly higher than the control across the board. It is important to note that the concentrations of Acetate and Formate in the Aegis5-14 Film are very similar to the concentrations measured in industry-acceptable polyethylene films. Due to the high variability amongst the three samples, each sample s concentration along with the control concentration is shown in Table 11. Solvent Film Identification WFI EtOH HCl NaOH NaCl Aegis5-14 Film Table 10. Semi-volatile extractables for 1-, 30-, and 90-day samples in Aegis5-14 Film by direct inject GC/MS 19 Aegis5-14 Film Characterization Thermo Scientific

20 Solvent WFI Sample Formate (µg/ml) Acetate (µg/ml) 1-day 30-day 90-day 1-day 30-day 90-day Control Aegis5-14 Film Table 11. Acetate and Formate extractables from Aegis5-14 Film treated with kGy gamma irradiation at 1-, 3-, and 90-day time points Non-Volatile Organic Compounds (NVOC) Non-Volatile Organic Compound extractants from Aegis5-14 Film were tested for using Liquid Chromatography/Mass Spectrometry (LC/MS). There were no non-volatile compounds detected above the reporting thresholds. Solvent Film Identification WFI EtOH HCl NaOH NaCl Aegis5-14 Film Table 12. Non-volatile extractables for 1-, 30-, and 90-day samples by direct inject LC/MS Inorganics (i.e., Metals) Inorganic extractables were analyzed using Inductively Coupled Plasma/Mass Spectrometry (ICP/ MS). There were two inorganic extractants found (Sodium and Potassium) at levels higher than the controls. Sodium was found only in the 1-day EtOH test, and Potassium was found in the NaCl and NaOH tests at 1- and 90-days. Metal Detection Limit Results Calcium 1.00 ppm None Detected Potassium, Sodium 0.50 ppm 0-10* Thallium 0.30 ppm None Detected Selenium 0.25 ppm None Detected Antimony 0.15 ppm None Detected Iron 0.10 ppm None Detected Aluminum, Arsenic, Lead, and Magnesium 0.05 ppm None Detected Nickel, Zinc 0.02 ppm None Detected Barium, Copper, Chromium, Cobalt, Manganese, Vanadium 0.01 ppm None Detected Cadmium, Silver ppm None Detected Beryllium ppm None Detected Table 13. Metals limits of detection and results at 1-, 30-, and 90-day time points *Typical results for polyethylene film structures 20 Aegis5-14 Film Characterization Thermo Scientific

21 Total Organic Carbon (TOC) Total organic carbon concentrations were measured in the WFI solutions only. The results are shown in Table 14. The TOC levels in the Aegis5-14 Film were as expected. The TOC levels agree with the Acetate concentrations in the WFI extracts, there are no other organic extractables in water. Sample Total Organic Carbon (ppm) 1-day 30-day 90-day Control Aegis5-14 Film Table 14. Total organic carbon in WFI extracts stored in Aegis5-14 Film at 1-, 30-, and 90-day time points Conclusions About Extractable Compounds This study did not generate a full extractable profile for each of the container materials. Rather, the study quantified a set of prominent target compounds identified through extraction using a range of solvents. This work shows typical values for extractable compound levels, not the full range of what is possible. Extractable compounds and levels can vary due to factors such as material manufacturing lot, empty bag storage conditions, time and level of irradiation. The acceptable type and level of extractables depends on the usage conditions such as storage temperature, storage time, and surface area to volume ratio and solution characteristics. It is important to be aware of any interaction or reaction between extracted compounds and the particular solution that is stored in the container. Well defined and characterized materials that minimize chemical additives and have good lot traceability reduce the risk associated with extractable compounds. Chamber Size Total Surface Area SA/V Ratio 0.05L 187cm 2 (29in 2 ) 3733cm 2 /L 0.1L 266cm 2 (41in 2 ) 2661cm 2 /L 0.2L 406cm 2 (63in 2 ) 2032cm 2 /L 0.5L 716cm 2 (111in 2 ) 1432cm 2 /L 1L 959cm 2 (149in 2 ) 959cm 2 /L 2L 1411cm 2 (219in 2 ) 705cm 2 /L 5L 1921cm 2 (298in 2 ) 384cm 2 /L 10L 3154cm 2 (489in 2 ) 315cm 2 /L 20L 3543cm 2 (549in 2 ) 117cm 2 /L Table 15. 2D surface area to volume ratios (SA/V) 21 Aegis5-14 Film Characterization Thermo Scientific

22 10. Cell Culture Growth Performance Cell culture testing was done to ensure that the Aegis5-14 Film would be suitable for storing growth media at warm temperatures. Bags measuring 7 x 10 with a single ½ port were used to store 420mL of CDM4CHO media under various time and temperature conditions. This equates to a 2.2/1cm surface area-to-volume ratio, which is severe. The media was then transferred into shake flasks and used for culturing two CHO Clones. Three passages and a terminal growth curve were done for each configuration. Three cell cultures were done for each media treatment. PETE bottles were used as a negative control. Table 16 shows the parameters for the cell culture tests. All cell cultures using media stored in Aegis5-14 Film and the PETE bottles grew very well. Cell Type CHO Clone 1 CHO Clone 2 Media Storage 4 C Media Storage 37 C Replicates 0hrs 48hrs 3 24hrs 24hrs 3 48hrs 0hrs 3 0hrs 48hrs 3 24hrs 24hrs 3 48hrs 0hrs 3 Table 16. Cell culture test design for growth media stored in Aegis5-14 Film bags at warm temperatures Viable Cell Density (cells/ml) Viability % Process Time (Days) Process Time (Days) Aegis5-14 Film warm 48 Aegis5-14 Film warm 24 Aegis5-14 Film unwarm PETE warm 48 PETE warm 24 PETE unwarm PETE unwarm w/o antib Figure 8. CHO growth and viability with media stored in Aegis5-14 Film bags 22 Aegis5-14 Film Characterization Thermo Scientific

23 8 Viable Cell Density (cells/ml) Viability % Process Time (Days) Process Time (Days) Aegis5-14 Film warm 48 Aegis5-14 Film warm 24 Aegis5-14 Film unwarm PETE warm 48 PETE warm 24 PETE unwarm PETE unwarm w/o antib Figure 9. CHO growth and viability with media stored in Aegis5-14 Film bags Overall Conclusions The results presented in this paper show that single-use flexible container systems constructed from Aegis5-14 Film are excellent alternatives to conventional rigid containers. The film meets the biocompatibility requirements of the biopharmaceutical industry and extractables should not be a concern in a wide range of applications in bioprocessing. For cell culture and media and buffer applications the properties of Aegis5-14 Film result in ph stability on storage plus the mechanical properties to allow secure storage and transport of large unit volumes of liquid. Cell culture growth performance data clearly demonstrates extractable levels are acceptable for cell lines sensitive to extremely low levels of extractants. The cold storage and cell culture performance levels of the film make it suitable for low temperature storage applications as well as elevated temperature holding situations routinely required for mammalian cell line cultivation. The range of applications for flexible containers continues to increase. A review of the design requirements and the relative importance of each of the 10 characteristics should precede selection of a single-use flexible container system for each application. For further information contact Technical Support at (US) or (EU). 23 Aegis5-14 Film Characterization Thermo Scientific

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