STERILITY TESTING OF PHARMACEUTICAL PRODUCTS
21 CFR 210.3(b)(21) states that “Representative sample means a sample that consists of a number of units that are drawn based on rational criteria such as random sampling and intended to assure that the sample accurately portrays the material being sampled.”
21 CFR 211.110(a) states, in part, that “To assure batch uniformity and integrity of pharmaceutical drug products, written procedures shall be established and followed that describe the in-process controls, and tests, or examinations to be conducted on appropriate samples of in-process materials of each batch. Such control procedures shall be established to monitor the output and to validate the performance of those manufacturing processes that may be responsible for causing variability in the characteristics of in-process material and the pharmaceutical drug product.”
21 CFR 211.160(b) states that “Laboratory controls shall include the establishment of scientifically sound and appropriate specifications, standards, sampling plans, and test procedures designed to assure that components, pharmaceutical drug product containers, closures, in process materials, labeling, and drug products conform to appropriate standards of identity, strength, quality, and purity. Laboratory controls shall include: (1) Determination of conformance to appropriate written specifications for the acceptance of each lot within each shipment of components, drug product containers, closures, and labeling used in the manufacture, processing, packing, or holding of pharmaceutical drug products. The specifications shall include a description of the sampling and testing procedures used. Samples shall be representative and adequately identified. Such procedures shall also require appropriate retesting of any component, drug product container, or closure that is subject to deterioration. (2) Determination of conformance to written specifications and a description of sampling and testing procedures for in-process materials. Such samples shall be representative and properly identified. (3) Determination of conformance to written descriptions of sampling procedures and appropriate specifications for pharmaceutical drug products. Such samples shall be representative and properly identified. (4) The calibration of instruments, apparatus, gauges, and recording devices at suitable intervals in accordance with an established written program containing specific directions, schedules, limits for accuracy and precision, and provisions for remedial action in the event accuracy and/or precision limits are not met. Instruments, apparatus, gauges, and recording devices not meeting established specifications shall not be used.”
21 CFR 211.165(a) states, in part, that “For each batch of drug product, there shall be appropriate laboratory determination of satisfactory conformance to final specifications for the drug product, including the identity and strength of each active ingredient, prior to release * * *.”
21 CFR 211.165(e) states that “The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented. Such validation and documentation may be accomplished in accordance with § 211.194(a)(2).”
21 CFR 211.167(a) states that “For each batch of drug product purporting to be sterile and/or pyrogen free, there shall be appropriate laboratory testing to determine conformance to such requirements. The test procedures shall be in writing and shall be followed.”
21 CFR 211.180(e) states, in part, that “Written records required by this part shall be maintained so that data therein can be used for evaluating, at least annually, the quality standards of each drug product to determine the need for changes in drug product specifications or manufacturing or control procedures * * *.”
21 CFR 211.192 states that “All drug product production and control records, including those for packaging and labeling, shall be reviewed and approved by the quality control unit to determine compliance with all established, approved written procedures before a batch is released or distributed. Any unexplained discrepancy (including a percentage of theoretical yield exceeding the maximum or minimum percentages established in master production and control records) or the failure of a batch or any of its components to meet any of its specifications shall be thoroughly investigated, whether or not the batch has already been distributed. The investigation shall extend to other batches of the same drug product and other drug products that may have been associated with the specific failure or discrepancy. A written record of the investigation shall be made and shall include the conclusions and followup.”
Certain aspects of sterility testing are of particular importance, including control of the testing environment, understanding the test limitations, and investigating manufacturing systems following a positive test.
The testing laboratory environment should employ facilities and controls comparable to those used for aseptic filling operations. Poor or deficient sterility test facilities or controls can result in test failure. If production facilities and controls are significantly better than those for sterility testing, the danger exists of mistakenly attributing a positive sterility test result to a faulty laboratory even when the product tested could have, in fact, been nonsterile. Therefore, a manufacturing deficiency may go undetected. The use of isolators for sterility testing minimizes the chance of a false positive test result.
A. Microbiological Laboratory Controls
Sterility testing methods are required to be accurate and reproducible, in accordance with 211.194 and 211.165. USP “Sterility Tests” is the principal source used for sterility testing methods, including information on test procedures and media.
As a part of methods validation, appropriate microbiological challenge testing will demonstrate reproducibility of the method to reliably recover representative microorganisms. If growth is inhibited, modifications (e.g., increased dilution, additional membrane filter washes, addition of inactivating agents) to the test method should be implemented to optimize recovery. Ultimately, methods validation studies should demonstrate that the method does not provide an opportunity for false negatives.
It is essential that the media used to perform sterility testing be rendered sterile and demonstrated as growth promoting. Personnel performing sterility testing should be qualified and trained for the task. A written program should be in place to maintain updated training of personnel and confirm acceptable sterility testing practices.
B. Sampling and Incubation
Sterility tests are limited in their ability to detect contamination because of the small sample size typically used. For example, as described by USP, statistical evaluations indicate that the sterility test sampling plan “only enables the detection of contamination in a lot in which 10% of the units are contaminated about nine times out of ten in making the test” . To further illustrate, if a 10,000-unit lot with a 0.1 percent contamination level was sterility tested using 20 units, there is a 98 percent chance that the batch would pass the test.
It is important that the samples represent the entire batch and processing conditions. Samples should be taken:
•at the beginning, middle, and end of the aseptic processing operation
•in conjunction with processing interventions or excursions
Because of the limited sensitivity of the test, any positive result is considered a serious CGMP issue that should be thoroughly investigated.
C. Investigation of Sterility Positives
Care should be taken in the performance of the sterility test to preclude any activity that allows for possible sample contamination. When microbial growth is observed, the lot should be considered nonsterile and an investigation conducted. An initial positive test would be invalid only in an instance in which microbial growth can be unequivocally ascribed to laboratory error.
Only if conclusive and documented evidence clearly shows that the contamination occurred as part of testing should a new test be performed. When available evidence is inconclusive, batches should be rejected as not conforming to sterility requirements.
After considering all relevant factors concerning the manufacture of the product and testing of the samples, the comprehensive written investigation should include specific conclusions and identify corrective actions. The investigation’s persuasive evidence of the origin of the contamination should be based on at least the following:
1. Identification (speciation) of the organism in the sterility test
Sterility test isolates should be identified to the species level. Microbiological monitoring data should be reviewed to determine if the organism is also found in laboratory and production environments, personnel, or product bioburden. Advanced identification methods (e.g., nucleic-acid based) are valuable for investigational purposes. When comparing results from environmental monitoring and sterility positives, both identifications should be performed using the same methodology.
2. Record of laboratory tests and deviations
Review of laboratory deviation and investigation findings can help to eliminate or implicate the laboratory as the source of contamination. For example, if the organism is seldom found in the laboratory environment, product contamination is more likely than laboratory error. If the organism is found in laboratory and production environments, it can still indicate product contamination.
The proper handling of deviations is an essential aspect of laboratory control. When a deviation occurs during sterility testing, it should be documented, investigated, and remedied. If any deviation is considered to have compromised the integrity of the sterility test, the test should be invalidated immediately without incubation.
A sterility positive result can be viewed as indicative of production or laboratory problems, and the entire manufacturing process should be comprehensively investigated since such problems often can extend beyond a single batch. To more accurately monitor potential contamination sources, US fda recommend keeping separate trends by appropriate categories such as product, container type, filling line, sampling, and testing personnel. Where the degree of sterility test sample manipulation is similar for a terminally sterilized product and an aseptically processed product, a higher rate of initial sterility failures for the latter should be taken as indicative of aseptic processing production problems.
Microbial monitoring of the aseptic area of the laboratory and personnel can also reveal trends that are informative. Upward trends in the microbial load in the aseptic area of the laboratory should be promptly investigated as to cause, and corrected. In some instances, such trends can appear to be more indicative of laboratory error as a possible source of a sterility test failure.
Where a laboratory has a good track record with respect to errors, this history can lower suspicion of the lab as a source of contamination since chances are higher that the contamination arose from production. However, the converse is not true. Specifically, where a laboratory has a poor track record, firms should not assume that the contamination is automatically more attributable to the laboratory and consequently overlook a genuine production problem. Accordingly, it is essential that all sterility positives be thoroughly investigated.
3. Monitoring of production area environment
Trend analysis of microorganisms in the critical and immediately adjacent areas is especially helpful in determining the source of contamination in a sterility failure investigation. Consideration of environmental microbial data should not be limited to results of monitoring the production environment for the lot, day, or shift associated with the suspect lot. For example, results showing little or no recovery of microorganisms can be misleading, especially when preceded or followed by a finding of an adverse trend or atypically high microbial counts. It is therefore important to look at both short- and long-term environmental trend analyses.
4. Monitoring Personnel
The review of data and associated trends from daily monitoring of personnel can provide important information indicating a route of contamination. The adequacy of personnel practices and training also merit significant review and consideration.
5. Product Presterilization Bioburden
US FDA recommend review of trends in sterile pharmaceutical product bioburden and consideration of whether adverse bioburden trends have occurred.
6. Production record review
Complete batch and production control records should be reviewed to detect any signs of failures or anomalies that could have a bearing on product sterility. For example, the investigation should include elements such as:
• Events that could have impacted on the critical zone
• Batch and trending data that indicate whether utility and/or support systems are functioning properly. For instance, records of air quality monitoring for filling lines could show a time at which there was improper air balance or an unusually high particle count.
• Whether construction or maintenance activities could have had an adverse impact
7. Manufacturing history
The manufacturing history of a product or similar products should be reviewed as part of the investigation. Past deviations, problems, or changes (e.g., process, components, equipment) are among the factors that can provide an indication of the origin of the problem.
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This Guidance is published by US FDA on September 2004
We also recommend our readers to visit US FDA’S website for undated guidance on sterile drug products