The production of sterile pharmaceutical products and medical devices usually encompass an approved, validated, and appropriate sterilization method. The most common sterilization methods that are generally used are steam, heat, ethylene oxide, and radiation sterilization. However, there are few sterilization options for sterilizing pharmaceutical or biological products, which may be labile when exposed to heat, chemical treatment or radiation. One of the most common options for sterilizing labile products is filtration sterilization. Sterilizing sensitive pharmaceutical and biological products by filtration is becoming a preferred method and the needs of the industry are increasing in parallel to the growth of the pharmaceutical industry. In September 2004, the U.S. Food and Drug Administration published a guideline recommending that whenever filtration is used in the production process, validation procedures will include microbial retention testing of the filters. These tests need to be conducted using simulated pharmaceutical processing procedures which define worst case production conditions. Parameters to be considered include, but are not limited to: organism size, number of organisms, flow rate, temperature, pH, viscosity, and pressure.
defines validation as:
Furthermore, both the FDA and the Parenteral Drug Association (PDA) have concluded that when it comes to validation, it is the users responsibility to demonstrate the complete microbial removal from each product or product family using a representative challenge. In an attempt to help the user, points to consider when developing a validation plan for filter sterilization by means of filtration are evaluated and summarized in this article.
Selecting a Sterilizing Grade
As the mechanism of retention in membrane filters is not a sieving function, the chemical and physical properties of the product can have a significant influence on the effectiveness of the filter. It is therefore essential that the products effect on the retention function is evaluated before traditional challenge testing. Filter compatibility studies are required and the chemical and physical properties of the product are evaluated prior to design of the validation protocol.
Validating Sterile Filtration
Extractables/leachables: The PDA Technical Report 26 defines an extractable as any chemical component that is removed from a material by the application of an artificial or exaggerated force and a leachable as a chemical component that migrates from a contact surface into a drug product or process fluid during storage or normal use conditions. During filtration, the process fluid is exposed to many components such as the filter membrane, housing, o-rings, piping, etc., all of which can contribute to extractables entering the final product. It is essential in the validation to determine the amount of extractables that might enter the fluid stream. There are many ways to determine this, the most common being the measurements of non-volatile residues. Other methods include measuring total organic carbons (TOC), Fourier transform infrared spectroscopy (FTIR), gas chromatography with mass spectroscopy (GC-MS), etc. Since the drug product may interfere with many of these tests, it is common to perform an extraction using a surrogate solution. The extraction can be performed using static (soaking) or dynamic (movement of fluid through the filter) conditions with special consideration for the sterilization process, flushing procedures, contact time, temperatures and chemical attributes of both the system and the process fluid.
Compatibility: Testing for compatibility can be performed as a separate part of the validation or as part of the microbial retention. Validating the compatibility of the filter and the entire system including hoses, o-rings, pumps, housings, core, etc., ensure that they can withstand the worst-case conditions of the sterilization and production processes. Compatibility tests can include integrity tests, evaluation of flow rates, examining physical aspects of the membrane (shrinking or swelling of the membrane, discoloring, shredding, etc.).
Integrity Testing: During validation, the user needs to assess the integrity of the filter and apparatus in order to verify tight seals, installation, and the effects from sterilization and production. Another aspect to consider and validate is product-wet integrity testing. During manufacturing it is not always feasible to perform a rinse prior to an integrity test; therefore, it becomes necessary to validate the appropriate integrity test method using the product. This can be assessed by comparing and calculating the integrity test results using the filter manufactures recommended wetting agent and the end users product. During this process, it is recommended to perform multiple replicates and/or multiple filter lots due to variation within the membrane lots.
Microbial Retention: The microbial retention test is performed using the industry standard concentration of at least 10 CFU/cm2 of the effective filtration area. The typical challenge organism used is Brevundimonas diminuta ATCC 19146. Other challenge organisms may also be considered with sound justification and consultation with the FDA. The microbial retention test is performed using worst case processing parameters and the goal is to validate that during these conditions a sterile effluent is produced. Below are some points to consider when developing a protocol for filtration sterilization validation:
Product Toxicity: The primary concern in the validation process is the affect of the product on the challenge organism. Many products are designed to inhibit bacterial growth and may even have disinfectant properties. Other products intentionally incorporate antimicrobial ingredients and in some cases, bacterial toxicity may be a consequence of a synergistic activity of two or more ingredients, or a by-product of various physical properties. To a certain extent, product toxicity can be anticipated. Where no overt toxicity is expected after a review of the ingredients and physical properties, a toxicity test against the challenge organism should be performed. If the toxicity causes a two-log reduction or less, the challenge titer can be adjusted up to compensate. If the impact is greater than a two-log reduction, it is considered best practice to use a surrogate that has similar properties to the ingredients found in the product. FDA guidance for an appropriate surrogate is highly recommended. If an appropriate surrogate cannot be formulated, the next best practice is the re-circulating technique. In this case, the filter is exposed to the product using worst-case process conditions and then the product is flushed out of the filter and apparatus, which are subsequently challenged with the organism. This method exposes the filter to the product under worst-case conditions and then challenges the filter under conditions where the challenge organism can survive. These same considerations must be addressed for assay filters used in the validation testing as well. The user may also decide to use a surrogate for the challenge portion of the re-circulation test. If the product and process is found to not be toxic, then the preferred method of directly inoculating the product should be used. In this case, the product is directly inoculated with an appropriate amount of the challenge organism and the conditioning and challenge are performed concurrently using worst case processing parameters.
Surface Tension: The presence of surface active ingredients may interfere with the bacterial retention mechanism of the filter, especially if the bacteria are smaller than the smallest pore in the filter. Recent research has shown that sieving action may not be the primary retention mechanism in membrane filtration. Any phenomenon that might interfere with other filtration mechanisms must be considered in the validation process. The change in the bubble point for the filter by the drug product does not, by itself, indicate a reduction in bacterial retention, but might be an indicator of the presence of surface-active ingredients.
Pressure and Flow Rate: Scientific literature confirms that pressure can have an effect on bacterial retention. Log reduction values decreased as the pressure increased in research by Leahy. The FDA therefore recommends validation under worst case processing conditions (i.e., highest differential pressure) to demonstrate bacterial retention. In the traditional test system, the interconnecting tubing diameter is somewhat non-variable so that in scale down procedures, pressure and flow rate become interdependent. We have constructed our test apparatus with oversized tubing in order to eliminate as well as possible any flow rate limiting. In most validations, rate of flow per filter surface area is adjusted to match that of the actual process. We recommend validations at the highest flow rate on the assumption that these conditions will also result in the highest pressure differential. The recorded differential pressure may be higher or lower than the actual process pressure.
Temperature: From a bacterial challenge perspective, temperature can have a significant effect on the viability and recoverability of the microorganisms selected for the challenge. The challenge organism (Brevundimonas diminuta) has a relatively narrow temperature range for maintaining viability. Thermal shock to the challenge organisms could reduce the recovery process. A re-circulating test can be used in cases where the process temperature does not permit the challenge organisms to survive. The drug product is re-circulated through the filter at the elevated temperature for the prescribed duration to simulate temperature exposure. Retention testing is then conducted on the filter and cooled down to a temperature which will preserve the organisms viability.
Duration: It has been demonstrated that bacteria can migrate through filters in use for extended periods of time. While the mechanism for this migration is not clear, it is suggested that filtration processes do not exceed 24 hours in length. Validations for longer processes usually involve procedures for replenishment of the challenge and interim samples to ascertain bacterial breakthrough.
Filter Selection and Integrity Testing: The microbial retention is normally performed on at least three filters, each from different manufacturing lots. It is recommended that at least one of those filters be at or near the filter manufacturers recommended minimum bubble point as the user is limited to using filters that match or exceed the lowest bubble point in the validation. Normally the integrity test is performed before and after the microbial challenge. There should not be a significant change in the integrity test results after the microbial challenge has taken place. Significant changes in integrity test results before and after the microbial challenge could indicate occlusion, swelling, caking, precipitate, etc.
The test procedure/validation consists of testing at least three filters from different manufacturing lots. Each test filter is challenged with a suspension of Brevundimonas diminuta ATCC #19146 that has been cultured to maximize the percentage of organisms capable of passing through a typical 0.45 µm filter. Sufficient volume of this challenge suspension, equivalent to at least 1 x 107 CFU/cm2 of effective filtration area (EFA) is prepared for each filter.
The challenge is conducted at a high flow rate and/or at a maximum differential pressure consistent with worst case processing conditions. The effluent is collected and assayed quantitatively by membrane filtration. Integrity testing is performed before and after the bacterial challenge procedure. The selection of Brevundimonas diminuta as the challenge organism is based on literature reports that the organism attains a very small size when grown under stress or minimal nutritional conditions. The test procedure complies in intent and content with the ASTM F838-83 Standard Test Method Determining Bacterial Retention of Membrane Filters Utilized For Liquid Filtration and the Health Industry Manufacturers Association (HIMA) Test Method Microbiological Evaluation of Filters For Sterilizing Liquids. Saline lactose broth is selected as the growth media because the literature reports that use of this media in concert with carefully controlled growth conditions, results in a minimally aggregated population of Brevundimonas diminuta cells, a significant percentage of which will pass through 0.45 µm membranes. The test objective of a most severe bacterial challenge to the filter is met by the challenge conditions, which include high pressure, high flow rates and a high bacterial concentration per cm2 of effective filtration area. The growth parameters, temperatures and media are adapted from the ASTM and HIMA methods.
Inoculated Product Challenge