Properties of Materials Used in Single-Use Flexible Containers: Requirements and Analysis

The adoption of single-use containers in the biopharmaceutical industry is becoming more frequent as the popularity and availability of the technologies increase. The choice of a solution for storage in single-use containers clearly depends on the application and the inherent risks associated with the application. A "one fits all" single-use system cannot respond to all the requirements of a particular step in a biopharmaceutical process, much less to all the steps of a process. The needs of an application will lead to very specific single-use solutions.

The physical properties of the film, such as the gas barrier, water loss, tensile strength, and temperature resistance, depend not only on the fluid contact surface, but on the overall composition of the film. A critical analysis of these physical properties is fundamental and cannot be taken at face value. And in all cases, the film is just one component of a single-use system, and all the components must be evaluated as a whole. This evaluation must include compatibility between the container and its contents (chemical resistance, extractables, and leachables) and an evaluation of the physical properties of the film under the appropriate operating conditions, based on a pertinent risk analysis of the manufacturing process. The type and amount of tests required are directly dependent on this risk analysis.

Overview of single-use technology

Advantages

Single-use technology eliminates the risk of cross contamination, which is of growing concern because the industry increasingly is moving from dedicated single-product plants to multiproduct facilities. Eliminating cleaning and cleaning validation is another key reason for moving to single-use technology; cleaning is not a perfect science and the number of FDA warning letters containing remarks about cleaning procedures, analytical methods used, or indeed lack of validation of cleaning procedures, has grown in recent years.¹ Single-use systems also can help companies achieve their manufacturing improvement goals, by offering faster turnaround and thus higher throughput, as well as high flexibility, which facilitates the implementation of process improvements. Several studies in recent years have demonstrated that significant savings in investment and cost of goods can be achieved as a result of implementing single-use technology.² Disposables also can shorten the time needed to validate new facilities by several months by reducing cleaning and steaming validation requirements.

Table 1 lists the classical applications in which single-use technology has been adopted and has shown to benefit the companies that have implemented it.

Table 1. The classical applications of single-use technologies

Material properties of films used in single-use technologies

General description of a multilayer film structure

Monolayer film structures such as PVC and EVA have been widely used for many years for blood storage and parenteral nutrition. The properties that are required in film structures today, however, cannot always be achieved by a monolayer structure. As a result, polymeric structures are now more common. The minimum barrier structure features at least three layers:

• A structural layer (e.g., PA, PET, LDPE) that determines the overall mechanical behavior of the film

• A barrier layer (e.g., EVOH, PVDC, PA) that determines the structure's permeability behavior

• The fluid contact layer (e.g, ULDPE, EVA, PP), which must combine inertness and good sealing properties.

The interaction of the layers is important in the overall performance of the film. For example, even though PE has better barrier properties than EVA, a film with an EVA contact layer may have better barrier properties than a film with a PE contact layer if the EVA-based film contains an EVOH layer and the PE-based film does not.

The film structure can be laminated, co-extruded, or a combination of the two. In laminated films, all film layers are extruded separately and are bonded together by a thermoset adhesive. In co-extruded films, the film is manufactured in a single-step operation in which the film layers are bonded together by a tacky thermoplastic polymer. These two manufacturing techniques can be combined. Figure 1 shows the structure of a sample polymeric film.

Figure 1. An example of a polymeric film. The outer layer (1) is made of PET, a clear protective polymer; the PA layer (2) gives mechanical strength; the EVOH layer (4) reduces gas transmission; and the fluid contact layer (5) is made of medical grade ULDPE. The four layers are laminated and the adhesion strength between layers is provided by a thermoset polymer layer (3).

Required testing and validation

Testing and validation requirements for films used in disposable equipment should be established by carrying out a risk analysis for each application. Product specifications, both technical and regulatory, depend on the level of safety required for each step in which the single-use products will be used. Applications such as final drug packaging obviously need more guarantees than initial formulation applications, after which further purification and sterilization steps are performed. For every application, an appropriate level of requirements and testing must be chosen.

Test conditions often are treated as variable parameters in the standards and are left to the convenience of the users. The choice of testing parameters must be driven by the application. Organizations such as the Bio-Process Systems Alliance (BPSA) are currently working on publishing a set of guidelines to help define these test conditions.³

Regulatory Requirements

The regulatory requirements that apply to films used in disposable equipment fall into several broad categories:

Biocompatibility requirements.^4–9 The biocompatibility requirements of a material depend on how it will be used within the process (e.g., media kitchen versus purification) and vicinity to the human body.

Sterility requirements.¹⁰ The sterility requirements of a film clearly depend on the sterility requirements of the application. If a sterilization step is carried out after the use of a disposable container, that container may not need to be sterile.

Contamination requirements.^11,12 The contamination requirements of a film depend on the cleanliness requirements of the application. If a filtration step is carried out after the use of a disposable container, the actual particle contamination level in the disposable container may not need to be that low.

Endotoxins.^13,14 Regardless of the application, endotoxin requirements must be met, although the levels at which the actual limits are set can vary. It is important to note that the traditional methods for removing pyrogens cannot be performed in disposable equipment, because the high temperatures required would be detrimental to the polymeric nature of the materials used.

Container requirements.^15–18 Few current standards for disposables address the containers themselves, and those that exist mainly deal with the use of disposable bags for drug delivery. Only some of the existing standards are relevant for bioprocessing applications, and must be adapted to the bag configurations (e.g., sizes, tubes, connectors) used in processing. Risk analysis is necessary to identify the relevant tests, methods, and specifications for any container under study.

Technical requirements of the film

Mechanical properties

Tensile properties.^19,20 A tensile test is conducted by applying stress to the sample (i.e., elongating it) and measuring the resulting strength. Raw data consist of a stress–strain curve. From this raw data, mechanical properties can be defined. The most useful tensile tests for plastic films are the ultimate tensile strength test, which demonstrates the maximum stress a material can withstand, and the elongation at break test. Elongation at break is the elongation recorded at the moment of specimen rupture, often expressed as a percentage of the original length. Materials with a high elongation at break withstand a high degree of deformation before rupturing. A high elongation at break means a high level of flexibility.

Toughness: Toughness is a material's resistance to fracture when stressed. It is defined as the amount of energy that a material can absorb before rupturing, and can be found by calculating the area underneath the stress–strain curve. A material can be strong but not tough; in that case it is said to be brittle.

Elastic modulus or secant modulus at 2%: The elastic modulus (also called Young's Modulus) is a measure of a material's stiffness. This can be experimentally determined from the slope of the stress–strain curve. For some materials, however, the elastic portion of the stress–strain curve may not be straight enough to measure the elastic modulus. In such cases, the elastic modulus can be approximated from the secant modulus at 2% (stress at 2% divided by 0.02). The higher the modulus, the higher the rigidity of the material. The lower the modulus, the higher the ductility, and therefore, the greater the flexibility of the material.

Puncture resistance.^21,22 Puncture resistance testing predicts the durability of a film while in use, especially its resistance to damage when impacted by another object. Films with high puncture resistance are made of materials that can absorb the energy of an impact by both resistance to deformation and increased elongation. Puncture resistance, measured in energy units, evaluates the film's strength and extensibility properties. Puncture resistance is similar to tensile toughness.

Tear resistance: Tear resistance is a complex result of other basic properties, such as modulus and tensile strength. It is a measure of the film's ability to resist tearing. Various standard methods are available for determining the tear resistance of plastic films. Certain tests also can determine tear resistance at low rates of loading. Other tests measure the force required to propagate a pre-cut slit across a sheet specimen.^23–26

Flex durability.²⁷ In the Gelbo Flex test, failure is determined by measuring the pinholes formed in a barrier structure after it has been subjected to the test. This test combines a twisting motion with a horizontal pushing motion, thus repeatedly twisting and crushing the film. The duration of the tests (number of cycles) and the amplitude of the deformation must be defined according to the application.

Glass transition temperature (T_g) and brittle temperature

The glass transition temperature (T_g) of a polymer is the temperature at which the polymer goes from a hard, rigid state to a rubbery, flexible state.^28–31 The brittle temperature is defined as the temperature at which the material becomes brittle.^32–34 The correlation between the brittle temperature, the glass transition temperature, and mechanical behavior at cold temperatures is not obvious, especially in the case of multilayer structures. In one study, it was shown that failure of a disposable bag in a frozen state did not correlate to the value of the bag material's glass transition temperature.³⁵ As shown in Figure 2, a low T_g does not always mean a good resistance at cold temperatures.

Figure 2. Relationship between bag failure and Tg in a drop test at frozen state (–70°C). There is no direct relationship between a low Tg and a low failure rate (Reference 1).

Barrier properties

Water loss.^36–39 Water loss is important when the concentration of the products inside a container is critical. For drug delivery containers, acceptable losses are described in the ICH Q1A guideline.⁴⁰

Gas Permeability.^41–43 Permeability to gas, particularly O₂ and CO₂, is important when the product inside the container is sensitive to oxidation. (If the solution is not oxidizable, the oxygen contained in the headspace above the solution will not be consumed.) Because the permeation phenomenon is driven by the partial pressure equilibrium on both sides of the film,⁴⁴ no oxygen will enter the container if oxygen is not consumed by the solution. In such a case, a low barrier film could be sufficient. Regarding the barrier to CO₂, if the quantity of carbon dioxide contained in the air is below 380 ppm, the main reason to examine this property is if the product will be shipped in dry ice.

Transparency / Haze^{45, 46}

Transparency, also known as haze, is evaluated by measuring the specific light transmitting and wide-angle light-scattering properties of the materials, and is performed on a planar section of film. This criterion applies to essentially transparent plastic materials. This property can depend on whether the film is wet or dry.

Technical requirements of the final product configuration

Container–solution interactions

Chemical compatibility. The purpose of container–solution interaction studies is to determine the possible chemical attack the solution might have on the film. Very few standard tests exist for this purpose. An adaptation of the ASTM D543 test⁴⁷ can be used to "grade" the compatibility of the film. The signs of chemical attack on the film can be evaluated by measuring the following aspects:

• Change in thickness

• Change in the film's physical properties

• Change in color (absorption, extraction, chemical reaction)

• Change in weight (absorption, extraction)

• Change in surface quality (cracking, transparency)

A standard matrix with a large number of solvents usually can be referenced for the initial evaluation of the compatibility of a film with a solution. If a container is to be used with a solution that is not on this list, two options are available, depending on the process risk analysis. The first option is to compare the new solution with a solution that has been tested already. If the solutions are similar, then the compatibility can be considered similar. The second option is to conduct a study with the specific solution and under the specific conditions of use.

Extractable and leachables. The purpose of extractables and leachables studies is to determine the possible impurities (extractables or leachables) that can come out of the films. A model solvent approach is commonly used to determine the amount and type of extractables under conditions that are more aggressive than normal operating parameters. A leachables study is normally carried out with a specific solution and conditions (time and storage) that are similar to operating parameters. The types of tests that are usually carried out will make it possible to identify and quantify volatile, semi-volatile, and nonvolatile products as well as heavy metals that can be leached from the film. Once again, a process risk analysis will determine the extent of these studies, mainly depending on the proximity of the process to the final drug delivery.

Abbreviations and Definitions

Protein adsorption. Protein adsorption can be approached in two general ways. The first approach is to conduct classical potency testing at various stages of product storage and then determine if any adsorption has taken place. The second approach can be used as a basis to estimate the potential loss of proteins upon contact with the film's surface. This second approach quantitatively evaluates the amount of "model" proteins that can adhere to a polymeric surface and compares that to the amount that can adhere to a glass surface.⁴⁸

Product integrity

Regardless of the production step in which a single-use container is used, the physical and microbial integrity of the complete product must be guaranteed. Depending on the production stage, this guarantee can be achieved in various ways. A 100% leak test conducted before use can be considered the ultimate guarantee. In some cases, however, this type of testing can be destructive or weaken the product. The highest level of integrity guarantee is achieved by having a capable and validated manufacturing process. Common tests used to conduct process validation on single-use equipment include seal testing, burst testing, drop testing, air leak testing, ship testing, and microbial ingress testing. These tests are generally described in well known standards (e.g. ASTM, ISO).

Most importantly, it is the final product that is validated and not its individual components (films, connectors, tubing, etc.). The integrity of the product is determined by its weakest link; it is not necessary to have bag seals that are twice as strong as the connection of the tubing to a bag, because the connection will fail long before the seals are stressed.

Expiry dating

Product aging needs to be considered for most of the tests and qualifications mentioned above. Polymeric materials are known to age and their physical and compatibility properties may change with time. These changes can be increased by gamma irradiation and need to be measured either through natural ageing or under accelerated ageing conditions.⁴⁹

Examples of Polymer Choices for Single-Use Applications

Example 1:

Input data: Need to store 200 L of product for two to four weeks at 2 to 8 °C. Solution is at pH 5, needs mixing, and is sensitive to oxygen. Container needs to be aseptically filled.

Analysis: The film must be a barrier to oxygen at 8 °C (since the solution is oxidizable); it must be sterilized before use (because it will be used in aseptic filling); and it must be resistant to acidic solutions (pH 5). The system needs to be able to contain large volumes (200 L), needs to have a non-invasive mixing system (since the solution must be mixed, and will be filled under aseptic conditions) and needs to resist a slightly acidic solution during storage for two to four weeks.

Proposed solution: A gamma-sterilized product composed of a 200-L bag made of a multilayer gas barrier film with a PE contact layer and a closed loop mixing system associated with a containment system.

Example 2:

Input data: Need to store a monoclonal antibody at –40 °C for two months. The product is sensitive to bulk-scale freeze-concentration, and long-distance transportation is required.

Analysis: The container needs to withstand low temperatures. The system needs to offer protection to the frozen product during freezing, thawing, and transport. The contact layer of the product needs to have low protein adsorption properties to maintain yield. The freezing and thawing processes must be controlled to maximize the heat transfer and thus minimize freeze-concentration.

Proposed solution: A complete system with a controlled freeze–thaw unit able to process flexible containers made of a multilayer EVA-based material. Each individual disposable unit is assembled into a rigid frame protecting the product at every process step.

Conclusions

Single-use products have been on the market for quite a number of years and have become familiar items for most of the manufacturers in this field. This familiarity has the significant drawback that the technologies behind the single-use systems are taken for granted.

The choice of a solution for storage in single-use containers clearly depends on the application and the inherent risks associated with the application. Beyond the minimum requirements (biocompatibility, basic container–content fit, appropriate mechanical properties, etc.), a number of specific tests can facilitate the decision-making process to select the right single-use system. The two theoretical examples described in this article illustrate how the needs of an application will lead to very specific single-use solutions. A "one fits all" single-use system cannot respond to all the requirements that are specific to a particular step of a biopharmaceutical process, much less to all the steps of this process.

Magali Barbaroux is an R&D development leader, and Andrew Sette is the vice president of quality and regulatory affairs, both at Stedim Biosystems, Z.I. des Paluds–BP 1051, 13781 Aubagne Cedex, France, tel. +33 4 4284 5600, fax +33 4 4284 5619, m-barbaroux@stedim.fr

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