(includes combination products)
By Steven G. Richter, Ph.D

P. ENVIRONMENTAL CONCERNS RELATED TO STERILITY TESTING The sterility test environment is described in USP General Informational Chapter <1211>.E.3 For combination products. This paper presents the general concepts and problems associated with sterility testing as well as the various testing methodologies. the USP (Volume 29) recommends testing forty units per production lot. The room is maintained under positive pressure and has specifications for room air changes per hour. False positive results are generally due to laboratory contamination from the testing environment or technician error. Richter Sterility testing of pharmaceutical articles is required during the sterilization validation process as well as for routine release testing.) filters. The environment should be as stringently controlled as an aseptic processing environment. The need to provide adequate and reliable sterility test data is an important quality assurance issue. An aseptic processing environment (clean room) is used to dispense sterile pharmaceuticals into presterilized containers. testing. there should be an anteroom for gowning and a separate area for the actual sterility testing. Most USP <71> sections are harmonized with the EP/JP. A clean room is generally a room that delivers laminar flow air which has been filtered through microbial retentive High Efficiency Particulate Air (H. the sampling size depends on lot size (see chart). and follow-up must be defined in the validation procedures. 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. Along with particulate testing in the environment. SAMPLING PLANS The official test.A WHITE PAPER PHARMACEUTICAL STERILITY TESTING — ESSENTIAL THINGS YOU MUST KNOW (includes combination products) ABSTRACT Steven G. Growth media used in sterility testing must be meticulously prepared and tested to ensure its ability to support microbial growth. . the laboratory must test for viable 1. Sterility testing should not be carried out under a laminar flow hood located within a room that is not maintained as ISO Class 5. 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. The testing area should meet ISO Class 5 particulate control requirements (specified in USP chapter 1116). An understanding of sterility testing is beneficial in terms of designing a validation process. Sterility testing is an extremely difficult process that must be designed and executed so as to eliminate false positive results. USP1 requirements employ sterility testing as an official test to determine suitability of a lot. Some of the quantities are not harmonized with the EP/JP volumes. INTRODUCTION Sterility testing is an essential part of every sterilization validation. Procedures for sampling.A. Sterility testing is a very tedious and artful process that must be performed by trained and qualified laboratory personnel. In cases where small lots (>1000) are manufactured. the ISO 11137/111354 standards recommend various sterilization validation sampling plans based on lot size and validation method. An environment used for sterility testing should be similar in design to an aseptic processing environment. A reprint of Table 2 “Minimum Quantity to be Used for Each Medium2” is attached.

a drug coated stent.bacterial and fungal organisms ubiquitous to it. The Sterility Test (USP Section <71>) is categorized under General Requirements and is therefore a legal requirement. Please check back. Qualifying and maintaining an isolator system for sterility testing may require extensive work. the ISO radiation sterilization microbial methods (11737-2 1998)6 describes a sterility test which is a modification for the USP method. an isolator may not be the best cost approach to the environmental concerns. This test is specific for the detection of aerobic organisms which have been exposed to sub-lethal sterilization cycles.e. we will see more biologics that are combination products. A combination product is defined as one that has a drug component with medical device. This number is used in gamma and electron beam dose setting methods. The laboratory must have a validation and training program for gowning and sterility testing. This ISO sterility test method is recommended for the validation of both gamma and electron beam sterilization processes. A future white paper explaining radiation and EO validation methods is planned. The isolators are generally sterilized using chemical sterilization. In testing pharmaceutical articles in a closed system such as SteritestTM. The drug product component applied aseptically creates the largest challenge to laboratory personnel. The SIP portion should be validated by sterility testing. Combination products sterilized by radiation are generally handled as medical devices following the ISO 11137 standard. Isolator technology is utilized to create a sterile environment for one to test pharmaceutical articles. PROCESSES Prior to actual sterility testing. 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. For the most part. They are the “Suitability Test” (Growth Promotion Test) and the “Validation Test” (Bacteriostasis and Fungistasis Test). For combination products. pharmaceutical GMPs would take precedent over 820 QSR8 requirements with all combination products. 0.5 METHODOLOGIES The United States Pharmacopeia is a compilation of validated methods and official monographs for pharmaceuticals and medical devices.1 for 10 percent of the sample). Since medical devices come in all shapes and sizes. the test laboratory will determine a Sample Item Portion which is a portion of the sample expressed in fractional terms (i. Official USP sterility testing of combination products is required for all sterile drug products. 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. General Informational Chapters. 2. and General Requirements. Each product should have a unique procedural specification for testing. . Combination products have unique challenges. The sterility test technician must be suitably gowned in sterile garments that prevent microbial shedding into the room. The procedure should be very specific in terms of which items (or vials/syringes) to test. The agency’s Office of Combination Products (OCP) would determine which regulatory branch (CDRH. The USP is broken down into the following sections: Monographs. In the short future. it is very difficult to test large and cumbersome medical devices in their entirety. The Sample Item Portion is the percentage of the complete product tested. CDER or CBER) is officiating the product. The procedure must indicate the Sample Item Portion (SIP). The room should be validated in terms of particulate and microbial levels. Most environmental concerns can be obviated by standard aseptic processing GMP’s. it is prudent to send an example sample to the testing laboratory so the laboratory can determine the appropriate testing procedure. Biologics must be aseptically processed and cannot be terminally sterilized. The validation required to qualify an isolator is extensive. For example. Therefore. Many issues surround the robustness of the sterilization process. General Informational Chapters <1000> series are not legal requirements. The method of choice for EO7 sterilized products is the official USP <71> procedure.

Since there are many manipulations required for membrane filtration medical device sterility testing. The USP authors understand that appropriate modifications are required due to the size and shape of the test samples. Secondly. The incubation time is 14 days.e. If the media is found to be non-sterile. or transferred whole. The entire product should be immersed in test fluid. The test article should be completely immersed in the test media. in an open system. and 3) Product Flush. validation and sterility tests can be performed simultaneously. more sterility failures are expected when using this method. All microorganisms have biological repair mechanisms that can take advantage of environmental conditions conducive to growth. Most pharmaceutical articles are tested using a closed system. Large catheters can be syringe filled with test media prior to immersion. This test is similar to the Suitability Test described above. With large devices. The Validation Test is used to determine if the test sample will inhibit the growth of microorganisms in the test media. the propensity for extrinsic contamination is very low. it simply may retard bacterial growth and proliferation. then the test fails. This test determines if the media volumes are valid for the particular product. Viable microorganisms that may be in or on a product after faulty/inadequate sterilization have an ideal environment within which to grow and proliferate. The product is aseptically cut. patient contact areas should be immersed. SCDM is selected based upon its ability to support a wide range of aerobic bacteria and fungi (i. This is especially true with damaged microorganisms where the damage is due to a sub-lethal sterilization process. The USP describes three general methods for sterility testing: 1) Membrane Filtration. After transferring. Microbial growth in the presence of the test samples is compared to controls without test samples. Cutting catheter samples to allow for complete immersion is the method of choice. then the test fails. the FDA may question the rationale behind using the membrane filtration test over the direct transfer test for devices. the concept is that the microorganisms will collect onto the surface of a 0. however. Suitability. a portion of each media lot must be incubated and assessed for sterility according to the incubation parameters (time. 3. FTM is selected based upon its ability to support the growth of anaerobic and aerobic microorganisms.45 micron pore size filter. The test media are fluid thioglycollate medium (FTM) and soybean casein digest medium (SCDM). The USP limits the media volume to 2500 mL. the propensity for laboratory contamination is high. A closed system is recommended for drugs and small devices or combination products. . is defined as the inability of a microorganism to grow and proliferate in microbiological media. The Validation Test must be performed on each product prior and/or during sterility testing. The method requires that the product be transferred to separate containers of both FTM and SCDM. 2) Direct Transfer (Product Immersion). Some medical products contain bacteriostatic and fungistatic compounds that may require special procedures and special media for testing. the samples are incubated for 14 days. the product sample is placed in the media along with the microorganisms. Therefore. If the media cannot support the growth of the indicator organisms. 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. In closed systems. Stasis. With membrane filtration. The next step is to proceed to actual sterility testing. It is not the method of choice for medical 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.The Suitability Test is used to confirm that each lot of growth media used in the sterility test procedure will support the growth of less than 100 viable microorganisms. Media that is bacteriostatic does not necessarily kill bacteria. MEMBRANE FILTRATION STERILITY TESTING The Membrane Filtration Sterility Test is the method of choice for pharmaceutical products. into the media containers. yeasts and molds). The direct transfer method benefits these damaged microorganisms. If microbial growth is present in the sample and control containers then the test is valid. This filter is segmented and transferred to appropriate media. in terms of microbiology. temperature) established by the method.

and bioburden data be reviewed prior to making any decision to release product. amount of time container is left open and the room particulate count. Turbid (cloudy) areas in the media are indicative of microbial growth. This investigation encompasses the following items: 1) clean room environmental test (EER) data. . The sample test sizes are listed in the document. 5) control data (open and closed media controls). STERILITY TEST FAILURE INVESTIGATION For every positive sterility test (OOS). which is generally clear and transparent. It is recommended that sterilization cycle data. This method is easy to perform and requires a modification of the FTM media for small lumen devices. against a light source. This method is not generally used. Identification and speciation of the isolate(s) is a significant contributing factor to the final decision.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. Once growth is detected. 4) the relative difficulty of the test procedure.12 for sterility testing. the suspect vessel is tested to confirm that the turbidity present is due to microorganisms and not due to disintegration of the sample.10 INTERPRETATION OF STERILITY TEST RESULTS The technician must be trained as to how to detect growth during the incubation period. Second Stage sterility testing requires double the original number of samples tested. This method requires one media (FTM). 4. Volumes are no less than 10 ml. The probability of a false positive can be calculated using John Lee’s formula. Growth positive samples require further processing such as identification and storage. A detailed investigation may uncover circumstantial evidence to support a final decision. environmental data. the laboratory should perform an OOS investigation to determine the validity of the positive growth. 3) technician training records. 6) technician sampling data (microbial counts on gloves and/or garments post testing). Once a suspect container has been tested. BULK DRUG PRODUCTS/BIOLOGICS AND PHARMACEUTICALS Bulk Pharmaceuticals (API’s) are tested for sterility per USP 71 prior to release to the manufacturing processes. Growth is determined by viewing the media. Samples that render the media turbid are transferred on Day 14 of the test and incubated for 4 days. then the USP allows for Second Stage sterility testing.11 The formula is based upon sample container diameter. it should be returned to the incubator for the remainder of the incubation period. If the First Stage sterility test cannot be invalidated by the laboratory. Sometimes samples produce turbidity because of particulate shedding or chemical reactions with the media. 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. The Second Stage test can be repeated if evidence exists invalidating the test due to a laboratory error as above. Bulk Biologics are tested according to 21 CFR 610. 2) media sterilization records. It is recommended that medical device manufacturers qualify the test procedure with non-sterile samples. The products are flushed with fluid D and the eluate is membrane filtered and placed into FTM and SCDM.

” 2006 9 GMPs CFR 201 Title 21 2006 10 21 CFR Part 610. Good Laboratory Practices12.CONCLUSION Sterility testing requires high levels of control with regards to GMP’s. “Good Laboratory Practice for Nonclinical Laboratory Studies. “Investigation Sterility Test Failures” Pharmaceutical Technology. February 1990 12 Code of Federal Regulations Title 21/Chapter I/Part 58. and employee practices. “Quality Systems Requirements: General.” 2006 5. Combination products have challenges that should be planned into a robust QA program. Sterility testing is an integral part of sterilization validation as well as a routine quality control. The United States Pharmacopeial Convention: 2005 2 USP 29 Table 2 Minimum Quantity to be Used for Each Medium 3 USP 29 Table 3: Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch 4 ISO 11137 Sterilization of health care products – Radiation – Part 2 2006: Establishing the sterilization dose 5 FDA Guidelines 2004 “Guidance for Industry Sterile Drug Products by Aseptic Processing. false positive results are uncommon in testing drug products using a closed system. 2004 6 ISO 11737 ANSI/AAMI/ISO 11737-2 1998 – Sterilization of Medical Devices – Microbiological Methods – Part 2. .12 Bulk Biologics 11 Lee. Tests of Sterility Performed in the Validation of a Sterilization Process 7 ISO 11135 1994 Medical Devices Validation and Routine Control of Ethylene Oxide Sterilization 8 Code of Federal Regulations Title 21/Chapter I/Part 820. environment (aseptic clean room ISO class 5 or better). It is essential that meticulous technique be employed in the practice of sterility testing. 29th Revision. John Y.” September. Current Good Manufacturing Practices. REFERENCES 1 The United States Pharmacopeia. Generally.

.USP STERILITY TESTING Test Media For Growth Promotion No Pass? Yes Perform Bacteriostasis Fungistasis Test No Pass? Yes Test Sample Perform Method Development Test Invalidate Test No Growth? Yes Perform Sterility Test Failure Investigation No Meets Requirements of Test Valid Growth? Yes Perform Stage (2) Test 2 X Samples No Growth? Yes Perform Sterility Test Failure Investigation Meets Requirements Valid Growth? Yes Sample Does Not Meet Requirements No 6.

STERILITY TESTING SAMPLING PLAN MATRIX Combination Products USP Official Requirement Batch Size Normal Lots ≤ 100 ≥ 1000 Sample Size 40 Articles 0% or 8 (whichever is greater) 20 Articles 4% or 40 (whichever is less) 100-500 500-1000 Sterilization ISO Standards ISO 11135 Ethylene Oxide Sterilization (Biological Indicator Qualification Study) Follow USP sterility method 40 product sterility Radiation Sterilization Procedure Method 1: 100 VDA 100 SDA Single Batch Sterility Test 100 Samples VDA (only) VDMax: 25 kGy Method 10 VDA and CVDA Single and Multiple Batch Test ISO 11137 ISO 15843 Alternate Sampling Schemes for 11137 A: 52 VDA B: 60 VDA 50/70/140 SDA 35 SDA VDA – Verification Dose Audit SDA – Sterility Dose Audit SCDM – Soybean Casein Digest Media USP FTM – Fluid Thio Glycollate Media USP CVDA – Conformatory Dose Exp. VDMax 7. .

(Water validation issues) Aseptic fill GMP’s out of compliance. 6. This is similar to the efficiency of an aseptic processing area and comparable to the microbial efficiency of aseptically processed pharmaceutical. Gowning validations. 10. Cleaning and disinfectant validations. Oligotrophic organisms pass 0. A validation of the sterility suite is recommended.5% false positive rate. Very difficult to disinfect the outer packaging. Frequency of air/surface sampling in the sterility suite. TM 3. takes 10 minutes to cut it and place it into a test vessel). Trend analysis of false positives: FDA guideline requires less than 0.e. Direct Transfer (Product Immersion) Method of choice for medical devices Complete immersion recommended: 2500 mL Max. 5. 9. The USP indicates that a 10-3 level of non-product contamination is required. Identification of bacterial and fungal isolates. This relates to one non-sterile out of 1000 processed units. Volume 14 Day Incubation 3.2 µm filter during aseptic fill.USP STERILITY TESTING Methods 1. (EM compliance issues) STERILITY TESTING ENVIRONMENTAL ISSUES 1. Solutions? Laboratory error due to extrinsic contamination: Solutions? EM issues. 8. (See MDM article SGR) 2. Certification of the room as ISO Class 5 or better. What do you do when a sterility test is positive? Combination products: Contaminated outer pouch prior to sterility test: Failures can be attributed to gross outer package contamination. Membrane Filtration First choice for pharmaceutical products or medical devices with Bacteriostatic/Fungistatic properties 14 Day Incubation 2. Validation of garment sterilization. (Micro seq ) 4. 7. Analyst contamination: Solutions? (Training issues) Difficult Combination Product Samples (i. Product Flush Recommended for transfusion and infusion assemblies that indicate a sterile fluid pathway that cannot be cut without contamination sample. Drugs Vial integrity compromised (Solution: Leak test using methylene blue dye) High level disinfectant compromised by bioburden organisms. . Action and alert levels for surface and air viable contaminates.

Review component sterilization data b. . Identification and speciation of the isolate is a significant contributing factor to the final decision of an action plan (1). REFERENCES: 1 Lee. Sample procedure (level of difficulty) e. Environmental testing (ID’s) b. “Investigation Sterility Test Failures” Pharmaceutical Technology. Equipment malfunction c. Review environmental monitoring data c. Employee practices 4. John Y. Review pertinent records a. 9. Controls CONCLUSION: A detailed investigation may uncover circumstantial evidence to support a final decision. February 1990. 1. Trends in sterility tests d. Training d.INVESTIGATING STERILITY TEST FAILURES (OOS) Sterility retests are valid only if “persuasive evidence” exists to show that the cause of the initial sterility test failure was induced in the laboratory(1). Laboratory procedures f. Manufacturing processes 5. Validated cycle parameters b. 2. Media sterilization c. Follow-up investigations before making any final decisions for product release have been recommended (1). Review of package integrity 3. Equipment and Components a. Review Bioburden data e. Laboratory Investigation a.

cut into pieces or disassembled NH Sutures and other individually packaged single-use material Other medical devices NH – Not Harmonized 10. creams. but not less than 1 mL 20 mL 10% of the contents of the container. and not greater than 100 mL Greater than 100 mL Antibiotic liquids Other preparations soluble in water or in isopropyl myristate Insoluble preparations. . but not less than 20 mL 1 mL The whole contents of each container to provide not less than 200 mg Use the contents of each container to provide not less to than 200 mg The whole contents of each container Half the contents of each container. but not less than 50 mg 150 mg 500 mg Surgical dressing/cotton/gauze (in packages) 100 mg per package The whole device The whole device. and ointments be suspended or emulsified Solids Less than 50 mg 50 mg or more. but less than 300 mg 300 mg-5 g Greater than 5 g Devices Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30-cm long) NH Minimum Quantity to be Used (unless otherwise justified and authorized) The whole contents of each container Half the contents of each container. Minimum Quantity to be Used for Each Medium Quantity per Container Liquids (other than antibiotics) Less than 1 mL 1-40 mL Greater than 40 mL.Table 2.

whichever is the greater. but not more than 50 containers More than 50 containers Each container 20% or 4 containers. whichever is greater 10 articles 2% or 20 articles. . Devices Catgut and other surgical sutures for veterinary use 2% or 5 packages. whichever is the greater 10 containers Not more than 100 articles 10% or 4 articles. whichever is less For large-volume parenterals Antibiotic solids Pharmacy bulk packages Pharmacy bulk packages (>5 g) Bulks and blends Ophthalmic and other noninjectable preparations Not more than 200 containers More than 200 containers If the product is presented in the form of singledose containers.Table 3: Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch Number of Items in the Batch (unless otherwise justified and authorized) Parenteral preparations Not more than 100 containers More than 100 but not more than 500 containers More than 500 containers NH Minimum Number of Items to be Tested for Each Medium 10% or 4 containers. whichever is the greater 10 containers 2% or 20 containers. whichever is greater 2% or 10 containers. this column gives the number of containers needed for both the media together. whichever is less 2% or 10 containers. NH – Not Harmonized 11. whichever is greater * If the contents of one container are enough to inoculate the two media. but not more than 500 articles More than 500 articles Bulk solid products Up to 4 containers More than 4 containers. up to a maximum total of 20 packages NH (<5 g) 20 containers 6 containers See Bulk solid productsNH 5% or 2 containers. whichever is lessNH More than 100. apply the scheme shown above for preparations for parenteral use.

contact: info@microtestlabs. Inc. Ph.About the Author Steven Richter.com (800) 631-1680 www. For more information.com 12. Richter founded Microtest in 1984 after a distinguished career at the U. With his expertise in Sterilization Sciences. and biotechnology industries with premier testing and manufacturing support. Food & Drug Administration. Dr. .D. pharmaceutical.microtestlabs.S. Under his leadership. is President and Chief Scientific Officer of Microtest Laboratories. Microtest has provided the medical device.

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