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The intent of this article is to provide practical advice based upon gaps that the author has observed
during investigations into numerous sterility test failures. The approach described will also apply to
other types of viable microbial contamination events, such as process simulation test (media fill)
failures. The author hopes that the reader will be able to avoid some of the common pitfalls that
prevent solving the puzzle, ie, arriving at the root cause of the microbial contamination observed.
products labeled as STERILE are not grossly contaminated with viable microorganisms (that are
detectable using the media employed). It is possible to avoid performance of sterility testing
altogether. If one does not have to perform sterility tests, then, to state the obvious, there is no
possibility of having a test failure (contamination event) that will have to be investigated. There are
only 2 circumstances in which sterility testing is not required for a sterile pharmaceutical product.
The FDA and other regulatory authorities allow parametric release instead of sterility testing for
products that are terminally sterilized in the final container. In fact, the FDA Review Microbiologists
prefer parametric release to sterility testing for release of terminally sterilized products to the
marketplace. In addition, sterility testing does not need to be performed on stability test samples at
time points (test stations) on the commercial stability protocol. Product is released using the sterility
test, which is the time zero test station on the stability protocol. Thereafter, container/closure integrity
testing (CCIT) can be performed in lieu of sterility testing. Maintenance of sterility is then
demonstrated using CCIT (physical method) over the shelf life of the product. This makes perfect
sense, because some sort of seal integrity failure would be the only way that a product can become
non-sterile over its shelf life. The author has helped investigate >70 sterility test failures for stability
samples. Those investigations revealed that all results were probable or proven false-positives.
upon to investigate a sterility test failure (either real or false-positive). One is faced with the following
question: How should a sterility test failure be investigated? Many facilities have no real idea how to
begin an investigation into a sterility test growth-positive (failure) because they do not have prior
experience. There is no formal plan, ie, a specific SOP with a decision tree that describes how a
sterility test failure should be investigated. Many facilities use their QC OOS SOP that describes what
to do for testing deviations. But that SOP is typically chemistry test oriented and usually does not
In the authors experience, sterility test failure investigations are typically flawed to some extent. For
example, only negative findings are documented. Often the scope (breadth and depth) of the
investigation is not sufficient to detect the root cause. Documentation does not reflect all of the efforts
expended, so one cannot tell which areas were investigated by reading the investigation summary
report. Often assumptions are made that preclude finding the root cause of the contaminated sterility
test. Also, conclusions regarding the root cause for the sterility test contamination are made that are
not supported by data. So, those conclusions are really speculation that could lead to performance
of corrective and preventive measures that do not solve the problem (mitigate the root cause for
laboratory environment should employ facilities and controls comparable to those used for aseptic
filling operations. Poor or deficient sterility test facilities can result in test failure [false-positive
results]. Yet many facilities still employ laminar flow hoods located in clean room suites for sterility
testing of products manufactured using advanced aseptic processing, such as isolator technology.
The same can be said for products that are terminally sterilized using a qualified steam cycle. In both
scenarios, there is a much higher possibility of having a false-positive sterility test result than of
detecting a real sterile batch failure (non-sterility). The author observed one scenario recently in which
a product was manufactured using aseptic processing into glass ampules in an isolator followed by
steam sterilization using a qualified cycle. The product was then tested for sterility in a laminar flow
hood and the test was growth-positive for a Paenibacillus species. The investigation demonstrated that
the result was a false positive due to inadequate decontamination of sterility test samples and testing
materials. Environmental monitoring of the sterility-testing suite during that particular testing session
many reasons. Sterility testing suites are often located directly adjacent to micro testing labs where
numerous cultures of viable microbes are manipulated. Some clean room suites do not have an
adequate air cleanliness cascade to prevent microbial ingress into the testing area. Also, sterility test
samples are typically brought to the micro lab and stored until testing, creating the opportunity for
Section XI of the FDA 2004 guidance also states that 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 non-
sterile. Therefore, a manufacturing deficiency may go undetected. The use of isolators for sterility
testing minimizes the chance of a false positive test result. The author is in total agreement with this
statement.
Investigation Approach
It is difficult to support invalidation of a positive sterility test. One must have conclusive and
documented evidence that clearly shows that the contamination occurred due to the testing that was
performed. Key Elements of the Investigation as described in Section XI.C.1 are as follows:
Identification (speciation) of the organism isolated from the sterility test (a strain level ID is desirable for such
investigations)
Record of laboratory tests and deviations
Monitoring of production area environment
Personnel monitoring
Product pre-sterilization bioburden
Production record review
Manufacturing history
When performing any investigation, including one for a sterility test contamination, one must have an
open mind to all possible causes for that failure. One cannot jump to conclusions, because that
typically leads the investigation in the wrong direction. All evidence should be evaluated equally. One
needs to document everything that was reviewed and its status, good or bad. Investigations should be
viewed as opportunities to discover what is being done correctly as well as procedures/practices that
Microbiology investigation
Manufacturing investigation
Validation investigation
Often firms wait until the microbiology investigation has been performed to a great extent before
commencing an investigation into the manufacturing areas. People are convinced that a laboratory
error of some sort has occurred and that there is no issue with the aseptic filling operation. After all,
the most recent media fill passed. Delaying the manufacturing area investigation can result in
spread of viable microbes to other areas, including aseptic filling lines. Therefore, it is imperative to
begin the manufacturing investigation without delay once a sterility test growth-positive has been
identified. Most facilities include cursory review of re-validation experiments in the manufacturing
investigation. The author recommends a separate targeted in-depth review instead. Firms should
review all recent sterilization, depyrogenation, and decontamination cycle re-validations and compare
them to the original qualification experiments performed. It is the authors experience that loading
patterns for sterility test isolators change over time and typically become more crowded. The
increased material load within the isolator becomes a barrier to the flow of vaporized hydrogen
peroxide (VHP). Also, the possibility of mated (touching) surfaces of testing materials is increased due
to the crowding of materials when the load size increased. The author has seen such a scenario be
the root cause for false-positive sterility tests several times around the world. So, it is very important to
of reviewing historical EM data that they have collected for a particular area. However, in the authors
experience, they almost never perform any meaningful investigative EM to look for the source of the
contaminating microbe. Without knowing the source(s) of the contaminating microbe it is almost
impossible to arrive at a probable or definitive root cause. Furthermore, many people confuse the
source of the microbe with the root cause. The root cause is how the microbe got into the product; it is
not the source of the microbe. But, you really cannot have one without the other.
Investigative EM is defined as additional environmental monitoring performed during sterility test
failure or other microbial contamination investigations. Samples are typically taken using swabs,
because irregular surfaces and hard-to-get-to sites (nooks and crannies) need to be sampled.
Samples are taken at non-routine sites, which may not have been cleaned and/or sanitized
effectively. An increased sampling frequency at the non-routine sites is also required. A one-off
sampling is not sufficient! In most cases one needs to perform aggressive sampling multiple times to
have a realistic chance of finding the source of the sterility test contaminant. A check-the-box
investigative EM of 20 samples is unlikely to be helpful and may send the wrong message to
regulatory authorities; they may conclude that sufficient due diligence has not been exerted to find the
source of the microbial contamination. In reality it may take hundreds of samples to locate the source
The following statement assumed that all aspects of the cleaning and sanitization program were
But, the area has been cleaned and sanitized multiple times since the batch was filled. So, we have
no chance of isolating anything with the extraordinary EM that you propose. (They were wrong; a
filamentous mold was isolated within the guts of the filling machine where no one thought to look.)
The rate of growth of sterility test contaminants may be very slow and some types of microorganisms
will never be seen during routine EM, eg, Propionibacterium acnes (microaerophillic or anaerobic)
Trending of EM data should help prevent product contamination. The following are negative EM
trends that can contribute to a batch sterility failure if they are ignored:
Increased numbers of viable microorganisms in critical areasone does not have to exceed alert or action levels
to have a batch failure
New or unusual isolate(s) in the facility
Increase in baseline microbial load over time
Increase in bioburden of raw materials
Presence of a microorganism resistant to disinfectant used in the facility
the chance of a false positive test result. In principle the author agrees with that statement, but
isolators should not be viewed as foolproof. The operating principles of isolators can be bypassed and
lead to false-positive sterility test results. Once sterility test isolators are qualified, they are often
largely ignored in terms of cleaning and maintenance. Each of the following questions should be
answered during an investigation into a failure for a sterility test performed in an isolator:
Proper decontamination and transfer of sterility test samples into the isolator is essential to avoid
false-positive test results. Often not much thought is given to decontamination required for test
samples and testing materials before they are placed into the sterility test isolator. The assumption is
made that VHP by itself is adequate for decontamination of test samples. Typically 70% IPA is used
for decontamination of test samples prior to placing them in isolators. However, in the authors
opinion, it is necessary to use a sporicidal agent for that purpose instead. Seventy percent IPA is not
sporicidal, ie, will not destroy Bacillus spp. or filamentous mold spores. Many of the isolates from
false-positive sterility tests performed in isolators were identified as spore-forming microbes that were
not destroyed by 70% IPA. It is fine to use 70% IPA as a final decontamination step as the samples
and materials are passed into the isolator, if those have first been decontaminated with a sporicide. In
the authors experience a 2-step procedure, ie, samples are decontaminated using a sporicide outside
the isolator and are then exposed to the sterilant used for isolator decontamination, eg, VHP, is very
Summary
For successful investigation of a failed sterility test result, one should:
Perform aggressive extraordinary (investigative) environmental monitoring to find the source of the sterility test
failure isolates
Make no assumptions and keep an open mind
Document everything
Witness by will be used who o the activity which is carried out by another person.
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