Concept
1.The drug injections/infusions that are manufactured in the pharmaceuticals are tested for sterility for every lot/batch. Sterility testing is performed to check the drug injections/infusions that are released to the market as “STERILE” i.e., they are free from microorganisms. Any drug injections/infusions that reach the blood system should be sterile or else the contaminated drug or infusion will become deadly to the recipient/patient.
2.In practice, the sterility of a product is defined by the absence of viable and actively multiplying micro-organisms when tested in specified culture media. Turbidity in the broth media usually indicates contamination. This test is performed on the end-product and is one of the quality control tests specified for release of a batch of sterile product.
3.Sterility test cannot be used to demonstrate the sterility of the entire batch but it may assist in identifying a non-sterile batch of product.
4.It is acknowledged that the sterility test suffers from significant statistical limitations and this contributes to the low probability of detecting anything less than gross contamination. However, these limitations can be reduced considerably by performance of the test under conditions that optimize the recovery of micro-organisms.
Training
1.Sterility testing should only be performed by personnel who have been trained, qualified and certified to perform the various tasks and procedures related to sterility testing. The examination of test and control containers during and at the end of the incubation period should be included as part of the analyst training program.
2.Supervisors should ensure that all personnel are monitored and follow Standard Operating Procedures [SOPs]. Personnel should undergo periodic re-certification, particularly when problems are detected during the course of routine environmental or when analysts perform the test infrequently.
3.Personnel training should be documented and records maintained.
Sterility test facilities
- Clean room design
Sterility testing should be performed under aseptic conditions, which are preferably consistent with the standard of clean room required for the aseptic manufacture of pharmaceutical products. Premises and equipment should be qualified according to the relevant principles of Installation Qualification and Operational Qualification [IQ/OQ].
- Classification
1.The sterility test should be conducted within a ISO-5 laminar airflow cabinet located within a class B clean room, or in an isolator that need not be located within a controlled environment. The test may also be performed within a class A clean room, if available. Sterility testing should be carried out in a work zone that offers sufficient space and material should be placed in such a way that it does not disrupt the laminar airflow.
2.The test environment, which includes the laminar airflow cabinet or isolator, should be certified by a competent person for compliance with the specified clean room standards.
- Air supply
1.Air supplied to the environment should be provided through terminal HEPA filters, which should be fitted with audible and/or visual alarms to indicate any sustained, out of specification pressure differentials across the HEPA filters.
2.There should be a pressure differential of not less than 10 to 15 Pascals [Guidance value] between each of the areas, i.e., ambient / airlock and airlock/test room.
3.Pressure readings should be taken and recorded from externally mounted gauges unless a validated continuous monitoring system is installed. As a minimum, readings should be taken prior to analyst entry to the test suite.
4.Pressure gauges should be labelled to indicate the area served, the acceptable specification, and whether or not the reading is absolute or differential.
- Airlock / change room conditions
1.Entry to the clean room should be via an airlock in which analysts are required to change into clean room garments.
2.The airlock should be designed to facilitate movement of the analyst between the relatively unclean and clean areas of the room without compromising the aseptic gowning procedure. A step-over bench is a suitable division between these areas.
3.The airlock should contain: a full-length wall mirror; gowning instructions; hand washing, disinfection and drying facilities. If clean room garments are stored in the airlock, then adequate and appropriate storage facilities should be provided.
Aseptic gowning
1.The sterility test analyst should change into sterile / sterilized clean room garments consisting of a one-piece coverall suit, head cover, overshoes, gloves.
2.Protective garments should be changed for each work session and usage justified from the results of microbiological monitoring and validation studies.
3.Records should be kept for the sterilization of the garments. This record may be in the form of a certification from an external supplier of sterile clean room garments provided that they have been approved by the sterility testing laboratory.
4.Each analyst should be trained and certified in gowning procedures with training records maintained.
Clean room fittings and surfaces
1.All fittings, such as power outlets and light fittings should be flushed with the wall or ceiling surfaces and sealed to prevent entrainment of unclean air. Surfaces should be smooth and impervious to the cleaning agents used.
2.The joints between ceiling/walls/floor should be coved to facilitate cleaning.
3.If supplied, intercom or communication systems should be designed to allow handsfree use, or their design should facilitate disinfection.
4.Chairs, storage cabinets and trolleys should be designed to facilitate cleaning and be suitable for use in a clean room environment.
5.There should be no extraneous equipment within the clean room environment.
6.Ultraviolet lamps may be fitted within pass-through cabinets only. Where there is more than one parallel tube, they should be shielded from each other and checked at least annually for performance, or whenever new lamps are fitted.
Cleaning, sanitization and disinfection
1.Outer surfaces of samples and equipment entering the testing suite should be disinfected, preferably with a sporicidal agent. Disinfection of surfaces and sample containers should be carried out in such a way as to avoid adventitious contamination of the samples by the chemical agent. Therefore, disinfectants should be free of microbiological contamination, which may be achieved through aseptic filtration or use of a product-compatible terminal sterilization method.
2.Surfaces and analyst gloved hands should be disinfected regularly during the test session.
3. There should be approved protocols / procedures to cover all daily, weekly, and periodic cleaning, sanitization, disinfection and fogging procedures used in the testing environment.
4.If an isolator is used, the method of disinfection or sterilization should be specified. Prior to implementation, all cleaning, sanitizing and disinfecting procedures should be validated from a microbiological perspective with respect to minimum disinfectant contact times and efficacy. Cleaning and disinfecting agents should be purchased to agreed and documented specifications.
5.Records should be retained in respect of routine preparation of cleaning and disinfecting agents, directions for their use, and validation of their efficacy.
Environmental monitoring
1.Environmental microbiological monitoring should include a combination of air and surface sampling methods, such as:
– active air sampling;
– settle [exposure] plates;
– surface contact [RODAC] plates, swabs.
– analyst’s gloved hand plates.
2.Environmental monitoring should be performed under operational [dynamic] conditions either within the isolator or in the laminar airflow and associated background areas.
3.Location maps, exposure duration, and frequency of all types of microbiological environmental monitoring should be specified in written procedures.
4.The media used for each type of monitoring should be specified and the recovery of micro-organisms on the chosen media should be validated.
5.Suitable inactivators of disinfectants and cleaning solutions may need to be incorporated into recovery media used for samples taken from surfaces.
6.There should be written specifications, including appropriate alert and action levels for microbial contamination.
7.Records should be maintained of the numbers and type of organisms isolated and results presented in a format that facilitates early detection of trends.
8.Routine identification of environmental micro-organisms to at least the genus level should assist in detecting trends. Sensitive techniques such as molecular typing techniques will be required for identification of micro-organisms if equivalence of identity of environmental and test isolates is the sole rationale used to invalidate the original sterility test.
Sterility Test
Sampling
1.The number of containers tested per batch and quantity tested from each container should be, as a minimum, in accordance with the Pharmacopoeial method followed.
2.Samples from aseptic fills should be selected from at least the beginning, middle and end of the batch fill. Additionally, SOPs should define criteria for inclusion and collection of samples immediately after interruptions and operator interventions during the filling process.
3.If an original test is declared invalid, then any samples used for the repeat sterility test should reflect the original samples in terms of sampling locations or aseptic processing times.
Test methodology
1.The test methodology should be in accordance with the Pharmacopoeial method used. Membrane filtration of the product, with either an open or a closed system, is the preferred sterility test methodology.
2.The filter should be pre-wetted, particularly when small volumes and antibiotics are tested. Filtration of the product should be followed by the minimum number of washes of the membrane with a suitable rinsing fluid established during validation studies. The membrane should not be permitted to dry out between filtration steps.
Media types and manufacture
1.The media used should be in accordance with the Pharmacopoeial method followed. Soya-bean casein digest medium [SCDM] and fluid Thioglycolate media [FTGM] should normally be used.
2.Alternative media are permitted and may be appropriate if the nature of the product or method of manufacture could result in the presence of fastidious organisms [e.g. vaccines, blood products, etc.].
3.Validation studies should demonstrate that alternative media are capable of supporting the growth of a wide range of micro-organisms.
4.Inactivators of antimicrobials may be incorporated into growth media or rinse solutions as indicated by validation studies.
5.Media should be purchased from an approved supplier, or prepared in-house according to standard operating procedures that are based on validated sterilization processes.
6.pH checks of media should be included in these procedures to ensure that the pH is within specifications at the time of use.
7.A batch number and a shelf-life should be assigned to all media and batch manufacturing documentation should be maintained.
Incubation period
1.All test containers should be incubated at temperatures specified by the Pharmacopoeial method for each test media for at least 14 days.
2.The temperature of incubators should be monitored and there should be records of calibration of the temperature monitoring devices.
3.Test containers should be inspected at intervals during the incubation period and these observations recorded.
Negative test controls
1.All media should be pre-incubated for 14 days at appropriate test temperatures to demonstrate sterility prior to use. Alternatively, this control test may be conducted concurrently with the product sterility test.
2.Media sterility testing may involve either a representative portion or 100 percent of the batch.
Positive test controls
All positive control tests use viable challenge micro-organisms. These tests should be conducted in a laboratory environment separate from either the isolator or aseptic area where the product is tested.
Growth promotion test
1.Challenge organism strains that are used to verify the fertility of each batch of standard test media should be selected from those reference strains specified by the Pharmacopoeial method.
2.Environmental isolates to be used.
3.Media purchased from external vendors should be accompanied by certification of the growth promotion test performed on each batch of media. The test need not be repeated by the sterility testing laboratory provided there is documented control over the conditions used to transport media between the media manufacturer and the sterility testing laboratory.
4.The media should be inoculated with 10-100 CFU of challenge organisms. The challenge inoculum should be verified by concurrent viable plate counts.
5.Growth promotion challenge organisms should show clearly visible growth in the test media within 3 days for bacteria and 5 days for fungi.
6.There should be written instructions and protocols covering all procedures for the preparation, maintenance and cultivation of test organism strains.
7.The identity[morphological and physiological properties] of the strains should be checked periodically.
8.At the time of use, cultures maintained by seed lot culture techniques should be no more than five passages from the original type culture strain which was obtained from a recognized reference culture supplier.
Validation parameters
1.Selection of canisters/membranes.
2.Equal splitting.
3.GPT in canisters.
4.Bacteriostasis and Fungistasis.
5.Sterility testing.
6. Post Bacteriostasis and Post Fungistasis.
Validation of Sterility testing method [Bacteriostasis and Fungistasis]
1.The test methodology should be validated by inoculation with 10-100 CFU of challenge organism strains to the media/product container at the beginning of the test incubation period. The challenge inoculum should be verified by concurrent viable plate Counts.
2.The preferred validation method involves addition of challenge organisms directly to the product prior to direct inoculation or membrane filtration.
3.However, where this is not practical due to inhibition or irreversible binding by the product, the challenge organisms should be added directly to the media containing the product in the case of direct test methodology, or to the last rinse solution if membrane filtration methodology is used.
4.The test is declared invalid if validation challenge organisms do not show clearly visible growth of bacteria within 3 days, and fungi within 5 days in the test media containing product.
5.In most cases, unless the sterile product causes turbidity in the media, visual recovery times should be comparable to those of the growth promotion test.
6.Validation should be performed on all new products, and repeated whenever there is a change in the experimental conditions.
7.Records of validation and/or re-validation tests should be maintained in the change control procedures.
Post-stasis test
1.It is not mandatory, but it is recommended that a stasis test be performed when antibiotics, inherently antimicrobial or preserved products are tested. is a requirement of TGA.
2.This test is sometimes referred to as an inhibition test by veterinary testing laboratories. The test is performed by inoculation of 10-100 CFU of challenge organisms directly to representative test containers of media containing product, which do not display any signs of contamination at the end of the test incubation period.
3.This test is particularly important for antibiotics, slow release sterile products and for direct inoculation methods where validity of the test method depends on the use of an exact amount of product (i.e. marginal methodology).
4.The stasis test has also identified problems with dehydrated commercial media, which were not apparent when a validation test was conducted at the beginning of the incubation period.
5.Stasis test challenge organisms should show clearly visible growth in the test medium within 3 days for bacteria and 5 days for fungi, otherwise the test is invalid.
6.Clostridium sporogenes ATCC 11437 for Fluid Thioglycolate Medium canister and Candida albicans ATCC 10231 for Soybean Casein Digest Medium shall be chosen as the challenge organism.
7.It is recommended that the stasis test is repeated at least every 12 months on the relevant categories of products or when product is reformulated or media is changed.
8.Records of stasis tests should be maintained.
Results
1.Apparent or suspected growth in media should be verified by subculture to solid microbiological media and micro-organisms identified at least to genus but preferably to species level.
2.Automated or semi-automated biochemical organism identification systems should be subjected to periodic verification using reference strains of organisms that can be traced to a recognized reference culture collection, such as the American Type Culture Collection [ATCC], Maryland, USA or the National Collection of Type Cultures [NCTC], London, UK.
3.Results of sterility testing for test samples and negative controls should be presented in a format that allows easy recognition of trends.
Interpretation and repeat tests
1.A test may be repeated only when it can be demonstrated that the test was invalid for causes unrelated to the product being examined.
(a) the data of the microbiological monitoring of the sterility testing facility show a fault;
(b) a review of the testing procedure used during the test in question reveals a fault;
(c) microbial growth is found in the negative controls;
(d) after determination of the identity of the micro-organisms isolated from the test the growth of this species or these species may be ascribed unequivocally to faults with respect to the material and/or the technique used in conducting the sterility test procedure.
Note-01:When conditions (a), (b) or (c) apply then the test should be aborted prior to the completion of the incubation period.
Note-02: If a stasis test is performed at the end of the test incubation period, failure of challenge micro-organisms to grow in this test also invalidates the test.
2.For condition (d) to apply as the sole criterion used to invalidate a test, it is necessary to demonstrate that a micro-organism isolated from the product is identical to an isolate from the materials and/or the environment. This determination entails the use of a sensitive typing technique such as a molecular typing technique or other techniques similar to those used for epidemiological studies.
3.However, if tests are performed competently in a clean room environment the chance of simultaneous adventitious contamination occurring in the environment, test sample and negative controls is negligible.
4.Provisions that allow repeat testing based on morphological or biochemical characterization of environmental and/or product contaminants should not be permitted. It is possible for the environment to become contaminated by the samples under test, which may contain multiple micro-organisms that are difficult to differentiate without employing sensitive typing techniques.
5.If contamination, which is established to be unrelated to the product, occurs in the original test, the test may be repeated with the same number of test samples as used in the original test, with negative product controls tested concurrently.
6.If contamination is detected in the repeat test performed on the same number of test samples, the product does not comply with the test for sterility and the entire batch should be rejected.
483’s
The form FDA 483 Inspectional observations is intended for use in notifying the inspected establishment’s top management in writing of significant objectionable conditions, relating to products and/or processes, which were observed during the inspection.
1.Improper disinfections of samples were done, in that alcohol was sprayed on the bottle and not allowed to dry prior to placing the needle. “A technician was observed spraying isopropyl alcohol onto a sample without letting the sample dry.“
2.”The firm failed to follow its own SOP entitled ‘Sterility Test: Membrane Filtration Method.’ The firm performed sterility retest prior to determining contamination was from an outside source
3.Sterility complaint investigation reports failed to identify and discuss any possible correlation of the sterility test isolate with microorganisms found in your firm’s environmental and personal monitoring.
4.Employee conducting sterility testing on 11/4/99 was observed spraying a cloth and disinfecting both hands prior to RODAC fingertip dab.
5.Individuals conducting sterility tests wear a single glove instead of a double glove.
6.“An analyst performed an sterility testing for XXX samples and there was no record of the training in the analyst’s training file.”
7.Personnel performing sterility testing were observed with exposed skin.
8.An analyst was observed adjusting cleanroom clothing while performing sterility.
9.Twelve out of a total of 96 bottles from media lot #__________ remained after the sterility failure finding in October 2006. All 12 remaining bottles of media lot #_______ were discarded without subjecting the media to sterility testing to determine the extent of the contamination.
10.Environmental and personnel monitoring microbial sample results were not addressed by the sterility failure investigation reports. We note that your firm collects numerous samples with results from personnel, equipment, and air, from within the sterile API production area, and identifies these microbes. However, these data were not assessed or reported and the failure investigation reports are missing this testing.
11.The investigation failed to confirm the root cause conclusion that microbes found in water samples were the cause of the contamination, in that these isolates were not shown [characterized to their genus and species level] to be related to the batch sterility failure isolate.
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