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Characterization of Single-UseBioProcess Container™ SystemsBased on HyQ® CX5-14 Film

Technical paper

Technical Paper 001/ Rev 1/ Page 1 of 20

Table of Contents: Introduction...............................................................................................................................................................2 Two-Dimensional Geometry..........................................................................................................................2 PVC Mono-Layer...........................................................................................................................................2 EVA Mono-Layer...........................................................................................................................................2 EVA-Based Single-Web Multi-Layer Films....................................................................................................2 Multi-Web Containers with Polyethylene Films for Larger Volumes..............................................................2 Three-Dimensional Geometry .......................................................................................................................2 Single-Web Multi-Layer Polyethylene Films..................................................................................................3 Performance Requirements ..........................................................................................................................3 Ten Important Characteristics of Container Systems ...............................................................................................3 Discussion and Results ............................................................................................................................................3 1. Biological Compatibility ............................................................................................................................4 2. Tensile Properties ....................................................................................................................................5 3. Puncture Resistance and Puncture Strength ...........................................................................................6 4. Glass Transition Temperature..................................................................................................................7 5. Transportability.........................................................................................................................................8 6. Clarity .......................................................................................................................................................9 7. Permeability ...........................................................................................................................................10 8. pH Stability .............................................................................................................................................11 9. Extractable Compounds.........................................................................................................................12 10. Protein Adsorption.................................................................................................................................18 Overall Conclusions ...............................................................................................................................................20


Introduction The trend towards single-use containers and systems in the biopharmaceutical process 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 HyClone’s 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.

The volume capacity of flexible containers available on the market can range from 50 mL to 10,000 L. The large volume flexible container systems can be used as alternatives to traditional stainless steel processing systems. The small volume flexible containers may replace glass or rigid plastic containers used for applications such as sampling and storage. Two-Dimensional Geometry Historically, small volume flexible

containers first appeared as two-dimensional (2-D) 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

of fluids 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 containers typically used poly(vinyl) chloride (PVC) films. PVC, by itself a brittle material not suitable for flexible containers, becomes a soft and compliant material through the addition of plasticizers such as di-octyl phthalate (DOP). Flexible PVC was used to make a myriad of small volume flexible medical containers from 50 mL up to 10 L, 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-period storage without suffering significant water loss and degradation from gas (O2 and CO2) 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. However, 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 a 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 50 L in 2-D pillow-style geometry. M u l t i - W e b C o n t a i n e r s w i t h Polyethylene Films for Larger Volumes The bioprocess market often requires large (50 L to 10,000 L) containers. At first, these large containers utilized a 2-D, 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, PE’s are inherently cleaner having lower extractable/leachable levels as compared to EVA. Moreover, PE is inert to a broader range of chemicals. The large volume 2-D pi l low-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, a requirement at large volumes. Three-Dimensional Geometry A second generation of large flexible containers alleviated the mixing, shipping and filling shortcomings of 2-D containers by designing a three-dimensional (3-D) gusseted container.

▲Figure 1. A Powdertainer™ Feeding Into the Single-Use Mixer


systems for bioprocess applications. A system’s performance in the listed areas 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. Protein Adsorption Discussion and Results This section discusses each of the important characteristics listed above and describes tests and results for HyQ®CX5-14 film.

Single-Web Multi-Layer Polyethylene Films A further enhancement combined 3-D construction and co-extruded or laminated film. These films normally have 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 s i n g l e - w e b f l e x i b l e c o n t a i n e r construction has become possible for large volume container systems of 50 L to 3000 L. These systems have good gas barrier and good water barrier properties, and are inert to the broadest range of chemicals.

An example of this type of film is HyClone’s HyQ®CX5-14 which was developed specifically for liquid handling, storage and transportation in the biopharmaceutical industry. The film structure is shown in Figure 4. Performance Requirements Selecting the optimal container for an intended application requires first deciding on some design parameters: • Container Geometry: Define the

volume and shape (2-D vs. 3-D) of the container.

• Operating Environment: Define the temperature, humidity and storage time for the containers.

• Permeabil ity: Define level of protection required for the product. Products that are sensitive to oxidation, pH shifts, or concentration changes due to O2 ingress, CO2 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 handl ing f i l led containers.

In addit ion to determining the per formance requirements, the manufacturing and quality systems of the manufacturer should be considered. Flexible containers for bioprocess applications should be manufactured in a clean-controlled 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 poss ib l y o the r qua l i t y - r e l a ted manufacturing protocols.

Ten Important Characteristics of Container Systems The list below includes 10 important characteristics for evaluating container

▲Figure 2. 3-D BioProcess Container™

▲Figure 3. 2-D BioProcess Containers

▲Figure 4. HyQ®CX5-14 Film Structure

1. Polyester 2. Tie

3. EVOH

4. Tie

5. PE


1. Biological Compatibility Standards Several test standards are available to show biocompatibility, including: United S ta tes Pharmacopoe ia (USP) , I n t e r n a t i o n a l O r g a n i z a t i o n o f Standardization (ISO) and European Pharmacopoeia (EP). These are reported by container manufacturers in the literature associated with their product. Results Table 1 shows the various biological tests performed for the film along with the results and requirements. Discussion Established test standards from several organizations exist, including USP, ISO and EP. 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 10993-5. 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, in t racu taneous reac t i v i t y , and implantation. These tests can be done according to USP <88> Class VI Biological Reactivity. Alternatively, equivalent ISO test methods are available under 10993-10 (Irritation & Sensitization—equivalent to USP <88> Intracutaneous Reactivity), 10993-11 (Systemic Toxicity) and 10993-6 (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>. 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 10993-4. In vitro and in vivo methods are available. Other: The EP <3.2.2.1> is another set of physicochemical tests, which includes the following: Appearance, Initial Color of Solut ion, Acidi ty, Alkal ini ty, Absorbance, Reducing Substances and Transparency.

Biological Test Summary

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 <0.006 EU/ml <0.25 EU/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 10993-4 In-Vitro Hemolysis Study Non-hemolytic Non-hemolytic

EP<3.2.2.1>—Plastic Containers for Aqueous Solutions for Parenteral Infusion Pass Pass

Irradiation Dosage 56.8—61.7 kGy (Run ID15411A/B)

▼Table 1. Biological Test Results Summary


2. Tensile Properties Overview Film tensile properties predict the ability of a container to maintain integrity of liquids in a desired application (i.e. liquid storage, shipping, and handling). Five tensile properties were evaluated: secant modulus, yield strength, ultimate tensile strength, maximum percent elongation and toughness. Container seam strength was also evaluated and compared to material ultimate tensile 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 modu lus cor responds to h igh 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. Ultimate tensile strength (UTS) of a material is the maximum engineering stress, in tension, sustained without fracture. The UTS 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-zero 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 peel 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 peel strength to the material UTS provides data on the influence of the sealing process to the parent material. Experimental Film tensile properties were measured using an Instron 4411 tensile test machine according to D882-02 Standard Test Method for Tensile Properties of Thin Plastic Sheeting. The Instron has a load and displacement accuracy of ± 2-lbf and ± 0.05-inch, respectively. Data was collected from three lots for a total of 100 samples.

Discussion Test results indicated HyQ®CX5-14 film met or exceeded all average tensile requirements.

Property Secant Modulus ksi

Yield Strength lbf (psi)

Ultimate Tensile Strength lbf (psi)

Maximum Percent Elongation

%

Toughness lbf

Seam Strength lbf

Average 35.1 17.9 (1279) 41 (2928) 710 414.1 29

Requirement Average >33

Average >16.8 (1200)

Average >39.2 (2800)

Average >700%

Average >222

Average >18

Tensile Summary—ASTM D882

▼Table 2. HyQ®CX5-14 Tensile Results Summary for ASTM D882


3. 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 4411 tensile test machine in compression mode. Testing was performed on a total of 90 samples from three lots of film at a crosshead speed of 20-in/min with a 1.0-inch diameter probe (Series 9 Method 2) with a film area of 28.3-in2. Discussion Test results indicated that HyQ®CX5-14 film met or exceeded the 118-lbf a v e r a g e p u n c t u r e r e s i s t a n c e requirement. 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 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 (lbf)

Average 132.27

Requirement Average >118

▼Table 3. Puncture Resistance Results Summary


4 . G l a s s T r a n s i t i o n 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 flex-cracking during shipping or handling, and demonstrate superior impact resistance and other mechanical properties. Tg also indicates the material capabil i ty for low temperature applications. Experimental The glass transition temperature was measured using a Dynamic Mechanical Analyzer (DMA) according to E1640-04 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 damping (energy dissipat ion) propert ies. Polymeric materials are viscoelastic; 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 rad/sec, 0.15 percent strain, 50g initial static force, 3°C/min temperature ramp rate, and at a temperature range of -90° to +80°C. Tg was calculated at the extrapolated onset to the sigmoidal change in the storage modulus.

Discussion Test results indicated that HyQ®CX5-14 film met the average Tg requirement of ≤ –31°C. In general, the lower the Tg, the lower the operating temperature of the container.

DMA Testing

Tg °C

Average -34.4

Requirement Average ≤ -31

▼Table 4. Glass Transition Results


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 (see Figures 4). Large volume fluid systems (≥ 50 L) are more difficult to ship and handle as compared to small volume systems (≤ 20 L) due to the mass and size of the filled containers.

Experimental BPC units, 500 L and 200 L, were subjected to simulated transportation testing to determine the durability of the film. Transportation testing is considered t h e m o s t e x t r e m e f u n c t i o n a l environment for a liquid-filled BPC. The 500 L unit is shipped in a suspended flexible caged-pouch, and the 200 L units in a rigid barrel. The 500 L unit allows for excessive movement (wave motion) of the BPC and is considered the worst case shipper. Testing was performed on a LAB vibration unit at a 1-inch vertical displacement at a frequency of 2.5-Hz (150-cpm). ISTA (International Safe Transit Association) requires a 2.63-hr vibration duration at this frequency. Units were filled and packaged according to standard HyClone procedures.

Discussion The HyQ®CX5-14 containers in the 200 L barrel shipper were tested for 160 hours without evidence of integrity fail-ure. Examination of these units indi-cated that there were no flex-cracks in the film. Test results indicated that all 500 L units met the 10-hour HyClone no leak re-quirement, which is 3.8 times greater than the ISTA test requirement.

▲Figure 4. Flex-Cracking in Film

▲Figure 5. LAB Vibration Unit

▲Figure 6. BPC 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 D1003-00 Standard Test Method for Haze and Luminous Transmittance of Transparent Plastics. Testing was performed on one lot of film with a Hunterlab Colorquest CQ1100 haze meter. 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 indicated that HyQ®CX5-14 film met or exceeded the average haze requirement of ≤ 32%.

Haze / Clarity Testing

Average 20.0

Requirement Average ≤ 32%

% Haze

▼Table 5. Haze / Clarity Results


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 D3985-02e1 Standard Test Method for Oxygen Gas Transmission Rate Through Plastic Film and Sheeting Using a Coulometric Sensor on one lot of film. Water vapor permeability testing was performed according to F1249-01 Standard Test Method for Water Vapor Transmission Rate Through Plastic Film and Sheeting Using a Modulated Infrared Sensor on one lot of film. Discussion Test results indicated that HyQ®CX5-14 film met or exceeded the average permeability requirements of ≤ 0.56 cc/m2 /24-hrs and ≤ 1.35 cc/m2/24-hrs for O2TR and CO2TR, respectively.

O2 Transmission 50% / 100% RH

0% / 0% TH CO2 Transmission 50% / 100% RH

WVTR 5°C: 0% / 100% RH

23°C: 0% / 100% TH

0.56 0.62 1.35 0.05 0.29

Oxygen cc/sq.m.day.atm 23°C

Carbon Dioxide cc/sq.m.day.atm 23°C

Water Vapor g/m^2.day.atm 23°C

65% RH IN/OUT 100% RH IN/50% RH OUT

100%RH IN/50%RH OUT 100%RH IN/0%RH OUT

Average 0.330 0.290 0.562 0.3069

Requirement Average ≤ 0.56

Average ≤ 1.35

F-Test = 0.261

T-Test = 0.766

N/A

▼Table 6. Barrier Properties (Values are in units of cc/m2/24 hrs)

▼Table 7. Permeability Results


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. Experimental Controls were typical rigid containers made o f po l yp ropy lene (PP) , polystyrene (PS), and polyethylene terephthalate glycol (PETG). The containers were filled aseptically to half volume with Dulbecco’s Modified Eagle’s Medium (DME), a typical cell culture medium. The experiment shows how well DME is protected from pH shifts when stored in various materials under typical storage conditions. Data was generated for three temperatures (4°C, 23°C, and 37°C) and for two storage periods (four weeks and three months). Since DME is buffered with bicarbonate, it undergoes substantial pH changes as a f u n c t i o n o f a m b i e n t C O 2 concentration. The DME medium used in th is exper iment contains a b icarbonate concentra t ion that equilibrates in a 10 percent CO2 environment, such as found in a CO2 incubator. When this media is exposed to normal atmospheric CO2 (much less than 10 percent), off-gassing of CO2 occurs, causing an increase in pH. The pH of a typical bicarbonate buffered-medium increases 1.8 or more pH units upon the loss of CO2. Media stored in

containers with higher permeability rates are expected to be more susceptible to pH shifts. The pH was measured with a Beckman o|34 pH meter. Accuracy is ±0.05 pH units. Results The attempt was made to create samples with equal surface-area-to-volume ratios (SA:V). However, rigid container volumes are fixed, therefore it was not possible to create equal SA:V values. Discussion DME media stored in flexible containers show good pH stability even when container size and volume (contact sur face area) are taken in to consideration. The pH stability in the flexible containers are comparable or better than the rigid PS and PETG containers. The PP container shows a dramatic increase in pH compared to the other containers and demonstrates the potential pH shift of the media if the gas barrier properties of the container are insufficient. Users are encouraged to conduct tests for unique conditions (e.g., buffering, type of solution, acceptable pH). Container size may also affect pH since it impacts surface-area-to-volume ratio, headspace volume, and other factors that can have a secondary impact on pH.

HyQ®CX5-14

4°C 0.02

23°C 0.03

37°C 0.05

PP PS PETG HyQ®CX5-14

cm2/mL 0.50 9.08 1.03 2.90

4°C 1.90 0.02 0.04 -0.01

23°C 2.62 0.05 0.14 0.02

37°C 2.94 0.11 0.23 0.01

▼Table 8. pH Stability After Three Months. Data is normalized to surface-area-to-volume. Change in pH for DME stored in flexible containers for 3 months

▼Table 9. Changes in pH in Rigid and Flexible Container Systems. Data is normalized to surface-area-to-volume. Change in pH Units/cm2/mL at four weeks


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 each manufactured from plastic resins. All plastic resins 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 “st icking” together during processing. Lubricants have historically contained stearate compounds that have an animal derived source. Other classes of plastic additives are: adhesives, anti-stats, colorants, light stabilizers and plasticizers. Plastic resins used in flexible bioprocess containers should contain minimal additives and avoid those that contain animal derived products.

Containers used to store solutions utilized in a drug manufacturing process, 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 extractable or leachable 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 solut ion. 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 of the containers is a significant source of extractable c o m p o u n d s . S o m e e x t r a c t e d 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 or drug. However, an extractables analysis, utilizing extreme so lu t ion concent ra t ions 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,

HyQ®CX5-14 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. Study Summary The purpose of this study is to identify and quantify compounds that may migrate from the test article, 500 mL HyQ®CX5-14 Labtainer BPCs, into extraction solutions. A total of 6 extraction solutions were evaluated under two different conditions: 1. Phase I: 55 ± 2°C Day 0 (for 30

minutes) and 7 Days 2. Phase II: 40 +/- 2°C for 90 days The test solutions used to separately extract the test articles include: 1. Water for Injection (WFI) 2. 3 M Sodium hydroxide (NaOH) 3. 4 M Sodium Chloride (NaCl) 4. 2 M Hydrochloric acid (HCl) 5. 20 percent Ethanol (EtOH) 6. 10 percent dimethyl sulfoxide

(DMSO) The extracts were characterized as appropriate for pH, conductivity and Total Organic Carbon (TOC). Additional aliquots of extraction solutions were acid digested and analyzed for metals. Aliquots of extracts were purged and a n a l y z e d d i r e c t l y b y g a s chromatography/mass spectrometry for volatile organic compounds. Aliquots of the extracts were solvent extracted in methylene chloride and analyzed by gas chromatography/mass spectrometry for semi-volatile organic compounds. Aliquots of the extracts were solvent extracted in methylene chloride, reconstituted in methanol and analyzed by l iqu id chromatography/mass spectrometry for non-volatile organic compounds.


Methods pH: Procedure: Samples were equilibrated at room temperature prior to analysis. An aliquot was analyzed for pH. Following calibration, a pH probe was immersed in the solution and the pH recorded. Conductivity: Procedure: Samples were equilibrated at room temperature prior to analysis. A solution was analyzed for conductivity. The probe was calibrated based on standard potassium chloride solutions. The probe was immersed in the sufficient volume of sample. The conductance reading was obtained directly off the meter and the result was recorded. Total Organic Carbon (TOC): Procedure: Samples were equilibrated at room temperature prior to analysis. The test article extract was analyzed for total organic carbon by converting TOC to carbon dioxide (CO2) by acidification and chemical wet oxidation with sodium persulfate. The CO2 liberated from the test article extract was measured using an infrared detector. Solution blanks of the extraction medium were prepared. The TOC level was automatically calculated by the instrument. Metals Analysis by ICP: Procedure: An aliquot of extract was acid digested in order to reduce interference by organic matter and to convert metals associated with particulates to a form that can be measured by inductively-coupled plasma (ICP) spectroscopy. The solution was digested with nitric acid at 105 ± 2°C for 20 minutes. The digestate was brought to a final volume of 50 mL with WFI. The digestate was analyzed for the following metals: Aluminum, Antimony, Arsenic, Barium, Bery l l i um, Cadmium, Ca lc ium, Chromium, Cobalt, Copper, Iron, Lead, Magnesium, Manganese, Nickel, Potassium, Selenium, Silver, Sodium, Thallium, Vanadium and Zinc. Tungsten was separa te l y eva lua ted as methodology is being investigated.

Justification: A range of polymer additives such as fillers, pigments and catalyst residues are metal base complexes or contain certain metals in their molecular structure. Migration of these polymer additives into a solution may be seen via the use of ICP spectroscopy. Volatile Organic Compounds (VOC) by Purge-and-Trap GC/MS: Procedure: Extract was purged with an ultra pure helium flow. All volatile and purgeable compounds were purged from the solution and adsorbed onto an activated carbon trap. The carbon trap was heated to desorb all compounds that were purged from the solution. Subsequently, these compounds were delivered to the GC/MS injection system for analysis and ident i f icat ion. Identification of the analytes was based on the chromatographic retention time as well as the mass spectrum of the analyte under the instrument conditions specified in the method. Dilutions of solution may be required to address instrumental requirements. Volatile organic compounds present in the target analyte library were positively identified. For other compounds that are detectable by the method but are not present in the target analyte library, the compound was identified via a best-fit analysis of the mass spectrum of the unknown to the NIST reference library of mass spectra. The compound was reported as a tentatively identified compound. A semi-quantitative analysis was carried out by comparing the signals of the unknown compounds with signals of internal standards added to the sample prior to purging. Semi-volatile Organic Compounds (SVOC) by solvent extraction GC/MS: Procedure: Aliquots of extraction so lu t ions were ex t rac ted wi th dichloromethane (DCM). The DCM extract was concentrated to 1 mL final volume. The DCM extract was injected into the GC/MS for analysis and identification. Identification of the

a n a l y t e s w a s b a s e d o n t h e chromatographic retention time as well as the mass spectrum of the analyte under the instrument conditions specified in the method. Dilutions of solution may be required to address instrumental requirements. Semi-volatile organic compounds present in the target analyte library can be positively identified. For other compounds that are detectable by the method but are not present in the target analyte library, the compound was identified via a best-fit analysis of the mass spectrum of the unknown to the NIST reference library of mass spectra. The compound was reported as a tentatively identified compound. A semi-quantitative analysis was carried out by comparing the signals of the unknown compounds with signals of internal standards to the sample extract.

Non-volatile Organic Compounds (NVOC) by solvent extraction LC/MS: Procedure: An aliquot of extract s o l u t i o n w a s e x t r a c t e d w i t h dichloromethane (DCM). The DCM extract was reconstituted in methanol and injected into the LC/MS for analysis and identification. Identification of the a n a l y t e s w a s b a s e d o n t h e chromatographic retention time as well as the mass spectrum of the analyte under the instrument conditions specified in the method. The list below indicates the target analyte library evaluated during the conduct of this study. For other compounds that are detectable by the method but are not present in the target analyte library, additional methodologies may be required to identify and quantify these a n a l y t e s . T h e s e a d d i t i o n a l methodologies would be examined in follow up studies.


Results of pH Analysis

Day 0 Day 7 Day 90

WFI Sample Average 8.28 6.12 6.55

Control 7.98 6.46 6.77

Sample Average 10.80 6.04 6.37

Control 9.80 5.77 7.59 4M NaCl

Results

◄Table 10. Results of pH Analysis

Results of Conductivity Analysis—(uS/cm)

Samples Equilibrated to RT Prior to Analysis

Day O Day 7 Day 90

Test Temperature 55°C 55°C 40°C

WFI Sample Average 0.0040 20.87 8.51

Control 0.0040 25.20 6.65

3M NaOH Sample Average 621 684 817

Control 621 627 805

4M NaCl Sample Average 243 245 251

Control 247 246 251

2M HCl Sample Average 767 753 867

Control 779 725 750

20% ETOH Sample Average 2.70 2.97 7.19

Control 3.00 2.60 7.17


Control 6.00 4.20 6.22 10% DMSO

Results of TOC Analysis—(ppm)

Day 0 Day 7 Day 90


Control 0.42 0.35 0.54

◄Table 11. Results of conductivity testing of all extraction solvents

◄Table 12. Results of Total Organic Carbon (TOC) Analysis


Metals Limits of Detection by ICP

Metal Detection Limit

Calcium 1.00 ppm

Potassium, Sodium 0.50 ppm

Thallium 0.30 ppm

Selenium 0.25 ppm

Antimony 0.15 ppm

Iron 0.10 ppm

Aluminum, Arsenic, Lead, and Magnesium 0.05 ppm

Nickel, Zinc 0.02 ppm

Barium, Copper, Chromium, Cobalt, Manganese, Vanadium 0.01 ppm

Cadmium, Silver 0.005 ppm

Beryllium 0.004 ppm

Results

None Detected

None Detected

None Detected

None Detected

None Detected

None Detected

None Detected

None Detected

None Detected

None Detected

None Detected

▼Table 13. Metals Limits of Detection and Results


Volatile Organic Compounds From Extracts

Average Concentrations (ppb)

Day O Day 7 Day 90

Test Temperature 55°C 55°C 40°C

WFI

Acetone 8.23 36.70 34.80

t-Butyl Alcohol 58.40 60.00 110.30

Iso-Propyl Alcohol 20.00 83.30 184.00

Pentanal N/A 16.00 N/A

Hexanal N/A 14.00 N/A

3M NaOH

Acetone 11.53 185.56 29.33

t-Butyl Alcohol 100.43 263.63 N/A


2-Butanone N/A 40.26 N/A

2-Hexanone N/A 11.25 N/A

3,3-dimethyl 2 Butanone N/A 61.66 50.33

Hexanal N/A 10.50 N/A

4M NaCl

Acetone 23.35 N/A 42.43

t-Butyl Alcohol 186.20 316.26 151.13


2-Butanone N/A 20.00 N/A

Pentanal 15.00 14.00 N/A

Acetaldehyde N/A N/A 13.00

2M HCl

Acetone N/A 11.90 10.20

t-Butyl Alcohol 56.40 188.43 335.00


Pentanal 16.00 13.00 11.00

Hexanal 21.00 11.33 N/A

2-Octanone 10.00 N/A N/A

Butanol N/A N/A 11.00

20% ETOH

10% DMSO

Acetone N/A N/A 351.60

t-Butyl Alcohol 51.00 N/A N/A


No target compounds detected

◄Table 14. Results of Volatile Organic Compound Testing


Discussion The model solvents used in this study represent a range of conditions, most of which are substantially more extreme than normal usage conditions for Flexible Bioprocess Containers. WFI is considered a very aggressive solvent, NaOH and HCl represent pH extremes. 4M NaCl represents an extreme salt concentration. ETOH and DMSO are typical solvents used in purification of drug products. Extractables were uniformly less than 1 ppm (1 mg/L), with most being less than 0.3 ppm. Several extractables were identified in the range of 10-20 ppb. This concentration range is near the limits of detectability of the instrumentation and is outside the range o f accuracy for concent ra t ion measurement. These extractables are not likely to be present as a measurable leachable, or have any effect on a final drug product if they are detectable. The only exception to this was Iso-Propyl Alcohol (IPA), which reached a maximum average concentration of 4.6 ppm in the 90 day study. IPA is a solvent used during the manufacturing process for Bioprocess Containers and would be expected to be present at some level in an extractables analysis.

Tables 15 and 16 present the surface to volume ratios for several standard Bioprocess Containers. The surface to volume ratio of the test articles (500 ml) was 1432 cm2/L, which is high. As seen in these tables, as the container size increases, the surface to volume ratio dec reases rap id l y . Thus , t he concentration of extractables in ppb or ppm will be proportionately reduced. Since the concentration of most extractables are at extremely low concentration at high surface to volume ratios, use of larger containers will result in extractables concentrations that are e i t h e r n o t d e t e c t a b l e o r a t concentrations well below 100 ppb. 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 i s poss ib le . Ex t rac tab le 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, surface area to volume ratio, and solution characteristics. It is important to be aware of any interaction

or react ion 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.

3-D Square—Large Volume

Chamber Size Total Surface Area (in2)

cm2 (m2)

20 L 517.00 3334.65 0.33

50 L 1845.16 11901.28 1.19

SA/V Ratio (cm2/L)

167

238

100 L 1541.00 9939.45 0.99 99

200 L 3697.90 23851.46 2.39 119

500 L 6978.94 45014.16 4.50 90

1000 L 11523.51 74326.64 7.43 74

2-D Pillow—Small Volume

Chamber Size (L) Total Surface Area

in2 cm2

0.05 29 187 3733

0.1 41 266 2661

0.2 63 406 2032

0.5 111 716 1432

1 149 959 959

SA/V Ratio(cm2/L)

2 219 1411 705

5 298 1921 384

10 489 3154 315

20 549 3543 177

▼Table 15. 3-D Surface Area to Volume Ratios (SA/V)

▼Table 16. 2-D Surface Area to Volume Ratios (SA/V)


10. Protein Adsorption Overview Whether in research or industry, containers of various sizes are required to store, transfer, or collect products and raw materials used in the production of many types of solutions. These solutions may contain proteins. Many cell culture media and pharmaceutical products are produced to contain specific levels of protein. In this study, containers with a variety of product contact surfaces were used to compare adsorptive properties of containers. Experimental High concentration protein loss was evaluated using bovine serum albumin (BSA) with PBS at a 100 mg/L concentration. The units were stored for 8-weeks at 4°C, and then evaluated for protein loss using a BSA assay. The results from this assay (see Figure 7) indicated that the containers did not sorb or show evidence of protein adsorption. Samples were assayed in triplicate. Low concentration protein loss was evaluated by using insulin growth factor 1 (IGF-1) in SFM4CHO™ medium. The units were stored for 11 weeks at 4°C, and then evaluated using an IGF-1 ELISA, a specific assay used to detect antibodies/antigens or other cell signaling proteins. Results, see Figure 3, of this ELISA showed no loss in IGF-1 concentration indicating that none of the materials have sorption affinity under these conditions.

Table 17 describes container types, sizes, volumes, surface areas, and surface-area-to-volume ratios. As in the pH stability study, ratios were kept as close as possible for this study within the limitations of available containers. Results Any change in protein concentration is assumed the result of adsorption to the contact layer of the container. Discussion As expected, polypropylene containers caused l i t t l e change in BSA concentration in any of the three temperature conditions after eight weeks. The change in concentration in the polypropylene containers was near the lowest detectable concentration change for the assay when surface area a n d v o l u m e a r e t a k e n i n t o cons idera t ion . The g lass and polystyrene conta iners showed s ign i f i can t change in p ro te in concentration. Test results indicated that there was no evidence of significant protein loss in HyQ®CX5-14 or PETG containers regardless of temperature. Protein loss due to adsorption in the flexible containers is very low, in fact l e s s t h a n o r c o m p a r a b l e t o polypropylene. Many variables can affect the concentration of a protein in solution. Protein adsorption varies with factors such as protein type, protein concent ra t ion , sur face contac t properties of the container, storage conditions such as temperature, solution pH and other components in solution.

In this study, BSA at 200 µg/mL in PBS at pH 7.2 was used as a typical protein solution. Users are encouraged to conduct tests for specific conditions (i.e., type of protein stored, type of buffer solution, desired pH range, and protein concentration). • F l e x i b l e c o n t a i n e r s p r o v e d

comparable to rigid polypropylene containers for storing and collecting solutions containing protein. No significant protein loss due to adsorption occurs in either type of container.

• The flexible containers used in this experiment are equivalent to or better than typical biotechnology industry containers made with PETG or glass with respect to protein adsorption.

• Container size may also affect the extent of protein loss due to adsorption. Surface-area-to-volume ratios are much greater for small volume containers.

• Protein concentration may have an effect on protein adsorption.

• Protein adsorption will be a more significant factor in situations where there are low protein concentrations and high surface area to volume ratios.

Container Size Volume Surface Area SA:V (cm2/mL)

HyQ®CX5-14 200 mL 100 mL 290 cm2 2.90

Polypropylene 50 mL 50 mL 109 cm2 2.18

Glass bottle 500 mL 250 mL 174 cm2 0.70

PETG bottle 125 mL 125 mL 129 cm2 1.03

Polystyrene T-flask T-25 12 mL 25 cm2 2.08

▼Table 17. Container Type, Size, Volume, Surface Area, and Surface-Area-To-Volume (SA:V)


▲Figure 8. Low concentration protein sorption results in various containers. The data indicate that no sorption was observed at the 11 week storage time interval.

BSA Concentration @ 8 weeks

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

Time 0 8Weeks

Time

ug/m

L

HyQ®CX5-14

PETG

Polypropylene

PVDF1

PVDF2

FEP

Relative IGF-1 Levels

0

100

200

300

400

500

600

700

800

900

Time 0 5 Weeks 11 Weeks

pg/m

L

PETG

FEP

PP

PVDF1

PVDF2

HyQ®CX5-14

No IGF1

▲Figure 7. High concentration protein sorption results in various containers. The data indicate that no sorption was observed for the given storage period of 8 weeks.


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Overall Conclusions The results presented in this paper show that single-use flexible container systems constructed from HyQ®CX5-14 are excellent alternatives to conventional rigid containers. T h e f i l m m e e t s t h e 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 bu f fe r app l ica t ions the properties of HyQ®CX5-14 result in pH stability on storage plus the mechanical properties to allow secure storage and transport of large unit volumes of liquid. The freezing and low protein binding characteristics of the film make it suitable for low t e m p e r a t u r e s t o r a g e applications. 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 BPC Technical Support at 1-800-HyClone (US) or 32 53 85 71 80 (EU).

Americas: Telephone: 1 (435) 792-8000 Toll-free: 1 (800) 492-5663 Fax: 1 (435) 792-8001 Email: [email protected] Europe: Telephone: +32 53 83 44 04 Fax: +32 53 83 76 38 Email: [email protected] Asia: Telephone: 1 (435) 792-8000 Fax: 1 (435) 792-8001 Email: [email protected] Web sites: www.thermo.com www.hyclone.com ©2007 Thermo Fisher Scientific Inc. All rights reserved. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details

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