Essential things to know
Sterility testing of pharmaceutical articles is required during the sterilization validation process as well as for routine release testing. USP1 requirements employ sterility testing as an official test to determine suitability of a lot. An understanding of sterility testing is beneficial in terms of designing a validation process. The need to provide adequate and reliable sterility test data is an important quality assurance issue. Sterility testing is a very tedious and artful process that must be performed by trained and qualified laboratory personnel. The investigation of sterility test failures is a process that requires attention to environmental data as well as many other factors including training and sample difficulty.
This paper presents the general concepts and problems associated with sterility testing as well as the various testing methodologies. Most USP <71> sections are harmonized with the EP/JP.
Sterility testing is an essential part of every sterilization validation. Sterility testing is an extremely difficult process that must be designed and executed so as to eliminate false positive results. False positive results are generally due to laboratory contamination from the testing environment or technician error. The testing environment must be designed to meet the requirements of the United States Pharmacopeia (USP) in terms of viable microbial air and surface counts. Growth media used in sterility testing must be meticulously prepared and tested to ensure its ability to support microbial growth. Procedures for sampling, testing, and follow-up must be defined in the validation procedures.
The official test, the USP (Volume 30) recommends testing 40 units per production lot. A reprint of Table 2 "Minimum Quantity to be Used for Each Medium2" is on the next page. Some of the quantities are not harmonized with the EP/JP volumes.3
For combination products, the ISO 11137/111354 standards recommend various sterilization validation sampling plans based on lot size and validation method. In cases where small lots (>1000) are manufactured, the sampling size depends on lot size.
Environmental Concerns Related to Sterility Testing
The sterility test environment is described in USP General Informational Chapter <1211>. The environment should be as stringently controlled as an aseptic processing environment. An aseptic processing environment (clean room) is used to dispense sterile pharmaceuticals into presterilized containers. A clean room is generally a room that delivers laminar flow air which has been filtered through microbial retentive High Efficiency Particulate Air (HEPA) filters. The room is maintained under positive pressure and has specifications for room air changes per hour. An environment used for sterility testing should be similar in design to an aseptic processing environment; there should be an anteroom for gowning and a separate area for the actual sterility testing. The testing area should meet ISO Class 5 particulate control requirements (specified in USP chapter (1116)). Sterility testing should not be carried out under a laminar flow hood located within a room that is not maintained as ISO Class 5. Along with particulate testing in the environment, the laboratory must test for viable bacterial and fungal organisms ubiquitous to it. The sterility test technician must be suitably gowned in sterile garments that prevent microbial shedding into the room. The room should be validated in terms of particulate and microbial levels. The laboratory must have a validation and training program for gowning and sterility testing.
Our validation programs require that technicians consecutively test 40 simulated samples for both membrane filtration and direct immersion methods without a false positive test result under less than ideal environmental conditions. Isolator technology is utilized to create a sterile environment for one to test pharmaceutical articles. The validation required to qualify an isolator is extensive. The isolators are generally sterilized using chemical sterilization.
Many issues surround the robustness of the sterilization process. Qualifying and maintaining an isolator system for sterility testing may require extensive work. In testing pharmaceutical articles in a closed system such as SteritestTM, an isolator may not be the best cost approach to the environmental concerns. Most environmental concerns can be obviated by standard aseptic processing GMP's.5
The United States Pharmacopeia is a compilation of validated methods and official monographs for pharmaceuticals and medical devices. IT is broken down into the following sections: Monographs, General Informational Chapters, and General Requirements. General Informational Chapters <1000> series are not legal requirements. The Sterility Test (USP Section <71>) is categorized under General Requirements and is therefore a legal requirement.
For combination products, the ISO radiation sterilization microbial methods (11737-2 1998)6 describes a sterility test which is a modification for the USP method. This test is specific for the detection of aerobic organisms that have been exposed to sub-lethal sterilization cycles. This ISO sterility test method is recommended for the validation of both gamma and electron beam sterilization processes.
The method of choice for EO7 sterilized products is the official USP <71> procedure.
Prior to actual sterility testing, it is prudent to send an example sample to the testing laboratory so the laboratory can determine the appropriate testing procedure. Each product should have a unique procedural specification for testing. The procedure should be very specific in terms of which items (or vials/syringes) to test. The procedure must indicate the Sample Item Portion (SIP). The Sample Item Portion is the percentage of the complete product tested. Since medical devices come in all shapes and sizes, it is very difficult to test large and cumbersome medical devices in their entirety. Therefore, the test laboratory will determine a Sample Item Portion which is a portion of the sample expressed in fractional terms (i.e. 0.1 for 10% of the sample).
This number is used in gamma and electron beam dose setting methods. The SIP portion should be validated by sterility testing.
Combination products have unique challenges. A combination product is defined as one that has a drug component with medical device. For example, a drug coated stent. The agency's Office of Combination Products (OCP) would determine which regulatory branch (CDRH, CDER or CBER) is officiating the product. Official USP sterility testing of combination products is required for all sterile drug products. The drug product component applied aseptically creates the largest challenge to laboratory personnel. Biologics must be aseptically processed and cannot be terminally sterilized. In the near future, we will see more biologics that are combination products. Combination products sterilized by radiation are generally handled as medical devices following the ISO 11137 standard. For the most part, pharmaceutical GMPs would take precedent over 820 QSR8 requirements with all combination products. The more robust GMP9 requirement would assure reduced bioburden counts and consistent microbial populations during manufacturing.
The USP <71> Sterility Test contains two qualifying assays which must be performed prior to sterility testing. They are the "Suitability Test" (Growth Promotion Test) and the "Validation Test" (Bacteriostasis and Fungistasis Test).
The Suitability Test is used to confirm that each lot of growth media used in the sterility test procedure will support the growth of fewer than 100 viable microorganisms. If the media cannot support the growth of the indicator organisms, then the test fails. Secondly, a portion of each media lot must be incubated and assessed for sterility according to the incubation parameters (time, temperature) established by the method. If the media is found to be non-sterile, then the test fails.
The Validation Test is used to determine if the test sample will inhibit the growth of microorganisms in the test media. Stasis, in terms of microbiology, is defined as the inability of a microorganism to grow and proliferate in microbiological media. Media that is bacteriostatic does not necessarily kill bacteria; it simply may retard bacterial growth and proliferation. The Validation Test must be performed on each product prior to and/or during sterility testing. This test determines if the media volumes are valid for the particular product. Some medical products contain bacteriostatic and fungistatic compounds that may require special procedures and special media for testing. This test is similar to the Suitability Test described above, however, the product sample is placed in the media along with the microorganisms. Microbial growth in the presence of the test samples is compared to controls without test samples. If microbial growth is present in the sample and control containers, then the test is valid. The next step is to proceed to actual sterility testing. Suitability, validation and sterility tests can be performed simultaneously.
The USP describes three general methods for sterility testing: 1) Membrane Filtration, 2) Direct Transfer (Product Immersion); and 3) Product Flush.
Membrane Filtration Sterility Testing
The Membrane Filtration Sterility Test is the method of choice for pharmaceutical products. It is not the method of choice for medical devices; the FDA may question the rationale behind using the membrane filtration test over the direct transfer test for devices. An appropriate use of this test is for devices that contain a preservative and are bacteriostatic and/or fungistatic under the direct transfer method. With membrane filtration, the concept is that the microorganisms will collect onto the surface of a 0.45 micron pore size filter. This filter is segmented and transferred to appropriate media. The test media are fluid thioglycollate medium (FTM) and soybean casein digest medium (SCDM). FTM is selected based upon its ability to support the growth of anaerobic and aerobic microorganisms. SCDM is selected based upon its ability to support a wide range of aerobic bacteria and fungi (i.e. yeasts and molds). The incubation time is 14 days. Since there are many manipulations required for membrane filtration medical device sterility testing, the propensity for laboratory contamination is high. Therefore, in an open system, more sterility failures are expected when using this method. A closed system is recommended for drugs and small devices or combination products. Most pharmaceutical articles are tested using a closed system. In closed systems, the propensity for extrinsic contamination is very low.
Direct Transfer Sterility Testing
Combination products: This method is the method of choice for medical devices because the device is in direct contact with test media throughout the incubation period. Viable microorganisms that may be in or on a product after faulty/inadequate sterilization have an ideal environment within which to grow and proliferate. This is especially true with damaged microorganisms where the damage is due to a sub-lethal sterilization process. All microorganisms have biological repair mechanisms that can take advantage of environmental conditions conducive to growth. The direct transfer method benefits these damaged microorganisms. The entire product should be immersed in test fluid. With large devices, patient contact areas should be immersed. Large catheters can be syringe filled with test media prior to immersion. Cutting catheter samples to allow for complete immersion is the method of choice.
The USP authors understand that appropriate modifications are required due to the size and shape of the test samples. The method requires that the product be transferred to separate containers of both FTM and SCDM. The product is aseptically cut, or transferred whole, into the media containers. The test article should be completely immersed in the test media. The USP limits the media volume to 2500 ml. After transferring, the samples are incubated for 14 days.
Product Flush Sterility Testing
Combination products: The product flush sterility test is reserved for products that have hollow tubes such as transfusion and infusion assemblies where immersion is impractical and where the fluid pathway is labeled as sterile. This method is easy to perform and requires a modification of the FTM media for small lumen devices. The products are flushed with fluid D and the eluate is membrane filtered and placed into FTM and SCDM. This method is not generally used.
Bulk Drug Products / Biologics and Pharmaceuticals
Bulk Pharmaceuticals (APIs) are tested for sterility per USP 71 prior to release to the manufacturing processes.
Bulk Biologics are tested according to 21 CFR 610.12 for sterility testing. This method requires one media (FTM). The sample test sizes are listed in the document. Volumes are no less than 10 ml.10
Interpretation of Sterility Test Results
The technician must be trained in the method of detecting growth during the incubation period. Growth is determined by viewing the media, which is generally clear and transparent, against a light source. Turbid (cloudy) areas in the media are indicative of microbial growth. Once growth is detected, the suspect vessel is tested to confirm that the turbidity present is due to microorganisms and not due to disintegration of the sample; sometimes samples produce turbidity because of particulate shedding or chemical reactions with the media. Once a suspect container has been tested, it should be returned to the incubator for the remainder of the incubation period. Samples that render the media turbid are transferred on Day 14 of the test and incubated for four days. Growth positive samples require further processing such as identification and storage.
Sterility Test Failure Investigation
For every positive sterility test (OOS), the laboratory should perform an OOS investigation to determine the validity of the positive growth. This investigation encompasses the following items:
- clean room environmental test (EER) data;
- media sterilization records;
- technician training records;
- the relative difficulty of the test procedure;
- control data (open and closed media controls);
- technician sampling data (microbial counts on gloves and/or garments post testing).
The USP allows for a re-test of the product if persuasive evidence exists to show that the cause of the initial sterility failure was induced by the laboratory. Identification and speciation of the isolate(s) is a significant contributing factor to the final decision. If the First Stage sterility test can be invalidated by the laboratory, then the USP allows for Second Stage sterility testing. Second Stage sterility testing requires double the original number of samples tested. The Second Stage test can be repeated if evidence exists invalidating the test due to a laboratory error as above.
A detailed investigation may uncover circumstantial evidence to support a final decision. It is recommended that sterilization cycle data, environmental data, and bioburden data be reviewed prior to making any decision to release product.
It is recommended that medical device manufacturers qualify the test procedure with non-sterile samples.
The probability of a false positive can be calculated using John Lee's formula.11 The formula is based upon sample container diameter, amount of time container is left open and the room particulate count.
Sterility testing requires high levels of control with regards to GMPs, Good Laboratory Practices12, environment (aseptic clean room ISO class 5 or better), and employee practices. It is essential that meticulous technique be employed in the practice of sterility testing. Sterility testing is an integral part of sterilization validation as well as a routine quality control. Generally, false positive results are uncommon in testing drug products using a closed system. Combination products have challenges that should be planned into a robust QA program.
- The United States Pharmacopeia, 30th Revision, The United States Pharmacopeial Convention: 2008
- USP 30 Table 2 Minimum Quantity to be Used for Each Medium
- USP 30 Table 3: Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch
- ISO 11137 Sterilization of health care products – Radiation – Part 2 2006: Establishing the sterilization dose
- FDA Guidelines 2004 "Guidance for Industry Sterile Drug Products by Aseptic Processing, Current Good Manufacturing Practices," September, 2004
- ISO 11737 ANSI/AAMI/ISO 11737-2 1998 – Sterilization of Medical Devices – Microbiological Methods – Part 2, Tests of Sterility Performed in the Validation of a Sterilization Process
- ISO 11135 1994 Medical Devices Validation and Routine Control of Ethylene Oxide Sterilization
- Code of Federal Regulations Title 21/Chapter I/Part 820, "Quality Systems Requirements: General," 2006
- GMPs CFR 201 Title 21 2006
- 21 CFR Part 610.12 Bulk Biologics
- Lee, John Y. "Investigation Sterility Test Failures" Pharmaceutical Technology, February 1990
- Code of Federal Regulations Title 21/Chapter I/Part 58, "Good Laboratory Practice for Nonclinical Laboratory Studies," 2006