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Introduction

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.

Sterility Testing Requirements


Under most circumstances, a sterility test must be performed to demonstrate that pharmaceutical

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.

Investigating Sterility Test Failures


If sterility testing is performed for ones products, the odds are that sooner or later one will be called

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

provide sufficient guidance on conducting sterility test failure investigations.

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

viable microbial contamination).

Testing Laboratory Environments


Section XI Sterility Testing of the FDAs 2004 Aseptic Processing Guidance states that the testing

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

recovered the same bacterium from multiple sample sites.


In the authors experience, performance of sterility testing in clean room suites is problematic for

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

superficial contamination with viable microbes.

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

are not the best.


In the authors opinion, the following 3 simultaneous investigations should be conducted whenever a

sterility test failure occurs:

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

look for changes in production cycles or loads.

Investigative (Extraordinary) Environmental


Monitoring (EM)
Review! Review! Review! When a sterility test growth-positive is discovered, most firms do a great job

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

of the microbial contaminant.

The following statement assumed that all aspects of the cleaning and sanitization program were

properly performed, which may not have been the case:

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)

and Cladosporium species (dematiaceous mold).

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

Sterility Test Isolators


Section XI of the FDAs 2004 guidance also states, The use of isolators for sterility testing minimizes

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:

How often is the isolator cleaned and sanitized?


How often is the room in which the sterility test isolator is located cleaned and sanitized? Is a sporicidal
disinfectant used?
Who performs cleaning and sanitization of the sterility test isolator? Do they know what they are doing?
How often is leak testing of the isolator and isolator gloves performed?
Is EM performed for the room surrounding the isolator? How often is that done?

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

effective in preventing false-positive sterility test results.

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.

or

Person who observe activity which is carried out by another person.

Verified by: to make sure the activity is correct and accurate