Simplifying Validation of Disposable Technologies with Presterilization by Gamma Irradiation

From the elimination of cleaning and cleaning validation to a reduction in capital costs, labor, and space requirements, the myriad benefits of using disposable systems are well-established. However, presterilization is one area that deserves more attention. Presterilization goes to the heart of the disposables concept, which is to minimize opportunities for contamination, whether through operator error, the process, or the product.

As the size and complexity of disposable systems continue to increase, presterilization will become increasingly advantageous and, in some cases, necessary. For example, conventional sterilization techniques such as autoclaving do not always fit a manufacturer's timeline, especially in the case of campaign-based vaccine production. Steam used during autoclaving is also known to damage paper, some plastics, and other heat-labile products and does not always ensure complete sterilization.

In addition to providing validation strategies for presterilization using gamma irradiation, this article addresses the overall benefits of disposables and explains how to minimize revalidation during scale-up.

Validation: An Important Early Process Consideration

Regulatory agencies around the world are focusing on validation, and it is becoming an increasingly important topic to consider early in the drug development process. For example, Annex 13 in Eudralex, the European Union's source for governing medical products, affirms that for investigational medicinal products, the manufacturing process should be validated in its entirety; it also focuses on critical steps, such as sterilization. Annex 13 further defines the process in the following manner:

Figure 1. A fully integrated, single-use filtration system

The different production operations shall be carried out in accordance with GMP; manufacturing facilities must be laid out to allow effective cleaning and reduce cross-contamination risk; equipment used for manufacturing operation, which are critical to the quality of the product, shall be subjected to appropriate qualification and validation.

In North America, FDA's January 2006 draft guidance INDs — Approaches to Complying with CGMP During Phase I — states that there are a number of technologies and resources available for use that can facilitate conformance with current good manufacturing practices (cGMPs) and help streamline product development. More specifically, these include the use of disposable equipment and process aids, prepackaged Water for Injection (WFI) and presterilized containers, and process equipment that is not exposed to the environment during processing.

Example: Virus Particle Filtration

Presterilized Filtration Systems

Filtration systems exemplify disposable technologies that can be presterilized. Disposable filtration systems are offered in a range of sizes, so that corresponding filters and bags are available for every stage of development, ensuring that the most appropriate and economical disposable filter scheme is used. By using the same materials of construction, these systems also generate reproducible results during scale up. Also, like presterilized filter cartridges, the entire single-use filtration system can be gamma irradiated before delivery to the biopharmaceutical manufacturing site. These systems, which generally comprise disposable bags, capsule filters, tubing, clamps, adaptors, and connection devices, also simplify many biopharmaceutical processes, such as preuse filter integrity testing.

Simplifying Scale-Up

To make certain that the process is scaled up with minimal material revalidation, biotechnology and pharmaceutical companies should use components that are made from the same materials from small scale-up to manufacturing scale.

A variety of capsule filter configurations are available to support processes as they are scaled up. Ideally, these differently-sized capsules contain identical filter media and hardware materials. This helps ensure that scale-up and scale-down studies yield relevant information and minimum requalification for various batch sizes.

The availability of a variety of capsules for small-scale operations is especially beneficial for new product development. Since all new biopharmaceutical products may not become commercialized, capital investment can be a concern. Single-use capsules and systems make it possible to produce new products during the early development stages without a large capital investment. Together, these factors can streamline drug development to increase manufacturing capacity, while meeting validation requirements.

Gamma Irradiation

The gamma irradiation process can be used for a variety of filtration applications spanning discovery to production. In use for more than 15 years, gamma irradiation uses well-defined operating parameters to ensure accurate validation methods and dose settings. The relatively simple process works by emitting gamma rays (electromagnetic energy) from a cobalt-60 isotope to penetrate deep into materials, thus destroying microbial bioburden. In a well-designed irradiation facility, for any given density of material, the only variable determining the amount of radiation the product receives is the time the material spends within the radiation field. As a constant and predictable sterilization method, gamma irradiation provides a safe and efficient start to the drug development process.

In addition to eliminating sterilization and sterilization validation procedures, presterilization using gamma irradiation provides cost, safety, and time benefits. Gamma irradiation's ability to demonstrate reproducible results helps biopharmaceutical companies improve the predictability of their applications. Because products are not exposed to heat, humidity, pressure, or vacuum, gamma irradiation presents fewer opportunities for product degradation. It also produces minimal waste byproducts, and does not require quarantine for out-gassing or biological testing. At $0.50–$4.00 per cubic ft., gamma irradiation's comparable cost to other forms of sterilization make it a very viable option.

In general, gamma irradiation is outsourced to specialty radiation facilities, such as Isotron Steris, SteriGenics, and RTI in the US. These facilities are subject to quarterly radiation dose audits. They are ISO-certified, maintain thorough records, and provide information, as needed, for submission to FDA. They also are equipped with safety containment walls that form a concrete shield around the facility to protect against the irradiation source (cobalt-60). To ensure the safety of the operators and the process, radiation facilities are monitored and regulated by a number of organizations.

Material Suitability

As with any sterilization method, the material to be sterilized must be compatible with the type of method used. Materials that are suitable for gamma irradiation include: polyethersulfone (PES), polyvinylidiene fluoride (PVDF), nylon and stabilized polypropylene. Materials that are not suitable include polytetrafluoroethylene (PTFE) and unstabilized polypropylene.

Setting the Parameters for Radiation

The objective of sterilization by gamma irradiation is to ensure that there is sufficient energy to kill microorganisms without compromising the integrity of the device materials.

The degree of sterilization or the Sterility Assurance Level (SAL) provides a benchmark for determining what the minimum radiation dose setting should be for a given material. SAL is a measurement of the theoretical probability of there being a viable microorganism present on the device. The industry accepted SAL for terminally sterilized products is expressed as 10^-6. This means the theoretical probability of there being a viable microorganism present on the device shall be equal or less than one in a million.

In setting the radiation dose, it is important to understand that not all parts of a device receive the same dose because of density, thickness, and geometric differences within the device. Some parts may need to receive a 30%–50% higher dose than the minimum dose needed to ensure that the devices are effectively sterilized.

The membrane filters used in disposable capsules can consist of different materials (e.g., PVDF, PES, or nylon) and thus, these materials need to be tested to ensure that the appropriate dose is established for sterilization. The method described below can be used to establish a suitable gamma irradiation dose for presterilization of capsule filters.

Establishing the Minimum Dose for Disposable Capsules

Methods set forth by the Association for Advancement of Medical Instrumentation (AAMI) are typically used to establish the minimum radiation dose setting that can routinely meet the preselected SAL requirements. Specifically, Method One of ANSI/AAMI/ISO 11137 has been used for filter capsules.

Method One, commonly called the Bioburden Method, is based on an assumed number of organisms residing on the product prior to sterilization. A database of typical industry bioburden levels is used to determine this assumed number of organisms, and represents a broader, more stringent challenge than the natural bioburden found on a device would. Ten samples from each of three lots are tested for a total of 30 samples. The bioburden results of these samples are used to calculate an experimental radiation dose called the verification dose, which is anticipated to yield a SAL of 10^-2. An additional 100 samples from a single production lot are exposed to this dose and sterility tested. If there are no more than two nonsterile cultures in the 100 sterility test samples, the validation is considered successful, and a routine SAL sterilization dose is calculated based on the original bioburden data.

A bacteriostasis or fungistasis test is also conducted with selected microorganisms to examine whether the presence or absence of various other substances inhibits their growth. Additional samples are required for this test.

By following the above method, a minimum radiation dose that will routinely provide the appropriate SAL can be established for a filter capsule. When the filters are irradiated at a suitable facility, a dose range will typically be used. For example, if the minimum dose for sterilization has been established at 25 kGy, the actual range used may be 25–35 kGy.

Dosimetric Release

Dosimetric release is used to verify that the dose absorbed by the product matches that of the validated specifications. During the irradiation process, dosimeters are placed in the irradiation chamber to measure the absorbed radiation and to perform a dose mapping inside the chamber. The product is determined to be sterile based on the physical irradiation process data rather than sterility testing.

Validation of Product Performance After Irradiation

After the proper dosage has been established for sterilization of a capsule filter by gamma irradiation, validation tests need to be conducted to ensure product integrity following gamma irradiation. These tests include: filter or capsule seal testing, physical tests (burst testing, creep-rupture testing and pressure-fatigue testing), real-time shelf-life testing, extractables testing, and biological safety testing.

The Kleenpak Nova capsule filter validation study is used as an example of a presterilized filter validation strategy. Kleenpak Nova capsule filters incorporate either a 10, 20, or 30 in. length standard cartridge filter in a gamma-stable polypropylene capsule. The filters used in the capsule are also used in stainless steel filter housings.

In many irradiation facilities, the maximum dose may be as high as 50 kGy. The filter capsules used in this study were exposed to the maximum radiation dose of 50 kGy to provide the worst case conditions in terms of gamma irradiation exposure to the filter materials.

Filter Capsule Seal Integrity

The purpose of filter seal integrity tests is to confirm that the seal between the filter and the outer capsule is integral. The testing of the seal can be achieved by performing integrity and bacterial challenge tests on typical filter capsules after they have been irradiated.

To demonstrate seal integrity, standard (10 in.) Kleenpak Nova filter capsules (incorporating 0.2 μm-rated Posidyne filter membranes) were used for the tests (Pall Corp., part number NP6NFZP1G). Before challenge testing, six samples were each subjected to a gamma irradiation dose of approximately 50 kGy. The six filters were subjected to a liquid bacterial challenge using Brevundimonas diminuta (ATCC 19146) at a minimum challenge level of 10⁷ CFU/cm² of effective filtration area.

Each of the six filters tested passed the forward flow integrity test and gave sterile effluent when challenge tested with an aqueous suspension of B. diminuta. These results demonstrated the integrity of the seal for the presterilized capsule filters.

Physical Tests

Physical tests are used to ensure that filter capsules can withstand maximum pressures and maximum temperatures during operation after they have been subjected to gamma irradiation. The Kleenpak Nova capsule filters were designed for a maximum pressure of 43.5 psig (3 bar) at 40 °C for continuous operation for one week (168 h).

Burst pressure and creep rupture tests are used to demonstrate maximum pressure thresholds for the Kleenpak Nova capsules. In all cases the capsules were subjected to a gamma irradiation dose of approximately 50 kGy.

Burst pressure tests are conducted to ensure that there is a suitable safety margin for the maximum operating pressure and temperature. This test is performed by filling a sealed capsule housing (without the filter) with water and placing the capsule in a 40 °C water bath. A pressure source is connected to the inlet of the capsule and the pressure is gradually increased until the capsule bursts.

Five (20 in.) Kleenpak Nova capsules were tested for burst pressure after pre-treatment with a gamma irradiation dose of approximately 50 kGy. The capsules all had burst pressures over 200 psi at 40 °C, which provides a considerable safety margin over the maximum operating pressure of 40 psi.

A creep rupture test is performed to determine how long a filter can withstand a given pressure before failure occurs. During the test, the capsules were immersed in a water bath held at 40 °C. The capsules were filled with the warm water and connected to a creep-rupture rig designed to maintain set pressures within the inside of the capsule until failure of the capsule occurs.

For the creep rupture test, 20 in. empty filter capsule housings were used. All were pretreated with a gamma irradiation dose of approximately 50 kGy. Creep rupture data were graphed on semilogarithmic plots and average values were used for each point. Figure 3 shows the results for irradiated 20 in. Kleenpak Nova filter capsules and indicates that these capsules will withstand approximately 100 psig (6.9 barg) for 500 hours.

Figure 3. Creep rupture test results

Real-Time Shelf-Life Tests

Real-time shelf-life tests are used to determine the length of time a filter capsule will remain sterile while in storage. Additionally, filters are tested to ensure that an adequate safety margin is maintained for burst pressure of gamma-irradiated capsules following storage at room temperature. In this case, filters were subjected to burst pressure and bacterial challenge tests at 0, 6, 12, 24, and 36 months to establish interim shelf-life claims. The test results led to establishing a three-year shelf-life for these capsule filters.

Biological Safety Tests

Biopharmaceutical companies also must consider the biological safety of the materials with which filter capsules are constructed. For example, single-use filter capsule housings made out of polypropylene need to meet the requirements of the biological reactivity tests in vivo for Class VI Plastics (121 °C) as described in the current United States Pharmacopoeia. (For the Kleenpak Nova capsule example, the filter membrane is validated separately.)

Extractables Tests

Filter capsules should demonstrate low extractables. For example, extractables were measured for an empty 20 in. Kleenpak Nova capsule and were found to be less than one mg in water and less than 15 mg in a 96% ethanol solution. The extractables for the capsule housing can be added to the filter cartridges used in the application for total filter extractables. In turn, the total filter capsule extractables can be added to the extractables for other system components for a total extractables quantity.

The Role of Disposables

Disposables: A Path to Reduced Validation

As the $65 billion disposables industry continues to mature, manufacturers are looking to simplify disposables implementation with plug-and-play systems. They are also paying closer attention to disposable materials and product configurations to minimize revalidation as processes are scaled up. Echoed in the myriad validation guidelines of global regulatory agencies, companies that use these strategies to simplify validation and reduce contamination risks will find themselves at a regulatory advantage as they navigate the course to drug approval.

Hélène Pora, PhD, is the marketing director of Pall Life Sciences, Pall France, Pall BioPharmaceuticals Division 3, rue des Gaudines - B.P. 5253, 78175 Saint Germain-en-Laye

Cedex - France

Tel +33 1 3061 32 20

Fax +33 1 30 61 57 08

Email: helene_pora@europe.pall.com

Reference:

1. Haughney H, Aranha H. Disposable Processing Gains You a Competitive Edge: Enhancing Manufacturing Capacity with Disposable Filters, Connectors, and Membrane Chromatography, Biopharm Int. 2003;16(10): 50-8.