3/30/2016 Successful Sterility Test Failure Investigations—A Practical Approach | American Pharmaceutical Review - The Review of American Pharmaceutical…

Welcome to American Pharmaceutical Review - Read Articles, find new products & companies, browse Industry news,
tell us what you think on Twitter and Facebook. Keep up to date by subscribing to the magazine or newsletter!

Welcome Guest Sign In Register Subscribe

Search American Pharmaceutical Review

Home Bioprocessing Chromatography Excipients Drug Delivery Formulation Development Instrumentation Microbiology Spectroscopy
Company Profiles Articles News Events Videos White Papers / Application Notes Featured Products Posters Research Issue Archives

Webinars

Articles Article Archives Successful Sterility Test Failure Investigations—A Practical Approach

Successful Sterility Test Failure Investigations—A Practical

Approach
Posted: April 06, 2015 Tw eet Like 0 Share Email Print Comments (0)

Kenneth H. Muhvich, PhD

Micro-Reliance LLC

Introduction
The intent of this article is to provide practical advice based upon gaps that the author has observed during
Connect with Us
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.
Related Categories

Sterility Testing Requirements Microbiology »

Under most circumstances, a sterility test must be performed to demonstrate that pharmaceutical products labeled as Environmental Monitoring and Testing
“STERILE” are not grossly contaminated with viable microorganisms (that are detectable using the media employed). It Equipment »
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. Environmental Monitoring and Testing »
There are only 2 circumstances in which sterility testing is not required for a sterile pharmaceutical product.
Pharmaceutical Clean Room Supplies /
The FDA and other regulatory authorities allow Cleanroom Equipment »
parametric release instead of sterility testing for
products that are terminally sterilized in the final Pharmaceutical Manufacturing »
container. In fact, the FDA Review Microbiologists
prefer parametric release to sterility testing for
release of terminally sterilized products to the Follow APR
marketplace. In addition, sterility testing does not
Keep up with our latest articles, news and events. Plus,
need to be performed on stability test samples at
get special offers and more delivered to your inbox.
time points (test stations) on the commercial stability
protocol. Product is released using the sterility test, Your Email Address
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 one’s 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

http://www.americanpharmaceuticalreview.com/Featured-Articles/173160-Successful-Sterility-Test-Failure-Investigations-A-Practical-Approach/ 1/5
3/30/2016 Successful Sterility Test Failure Investigations—A Practical Approach | American Pharmaceutical Review - The Review of American Pharmaceutical…
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 author’s 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 FDA’s 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 author’s 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
Selected Products
microbial ingress into the testing area. Also, sterility test samples are typically brought to the micro lab and stored until
testing,
You creating
haven't the any
selected opportunity
products.for superficial
Find products.contamination with viable microbes. Get Quote Compare

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.

http://www.americanpharmaceuticalreview.com/Featured-Articles/173160-Successful-Sterility-Test-Failure-Investigations-A-Practical-Approach/ 2/5
3/30/2016 Successful Sterility Test Failure Investigations—A Practical Approach | American Pharmaceutical Review - The Review of American Pharmaceutical…
In the author’s 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 author’s 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 author’s 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 areas—one does not have to exceed alert or action levels

http://www.americanpharmaceuticalreview.com/Featured-Articles/173160-Successful-Sterility-Test-Failure-Investigations-A-Practical-Approach/ 3/5
3/30/2016 Successful Sterility Test Failure Investigations—A Practical Approach | American Pharmaceutical Review - The Review of American Pharmaceutical…
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 FDA’s 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 author’s 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 author’s 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

Citations
1. 2004 FDA Guidance: “Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing— Current
Good Manufacturing Practice.”
2. 2008 FDA Guidance: “Container and Closure System Integrity Testing in Lieu of Sterility Testing as a Component
of the Stability Protocol for Sterile Products.”
3. 2010 FDA Guidance: “Submission of Documentation in Applications for Parametric Release of Human and
Veterinary Drug Products Terminally Sterilized by Moist Heat Processes.”

Author Biography

Dr. Ken Muhvich is the Principal Consultant for Micro-Reliance LLC, which specializes in Sterility Assurance and
Regulatory Compliance Consulting. He has conducted numerous mock Prior-approval audits of sterile
manufacturing facilities, including their microbiology laboratories. He is frequently involved in guiding companies
in sterile process design and validation. Ken is often called upon to help lead investigations into batch sterility
failures and/or to review completed sterility failure investigations to look for possible gaps. From 1992 to 1997 he
was a Review Microbiologist at the U.S. Food & Drug Administration’s Office of Generic Drugs. Ken is a recognized
expert in aseptic processing of sterile drug products. He holds a Master’s degree in Medical Microbiology from West
Virginia University and a Doctor of Philosophy degree in Experimental Pathology from the University of Maryland.

Comments

http://www.americanpharmaceuticalreview.com/Featured-Articles/173160-Successful-Sterility-Test-Failure-Investigations-A-Practical-Approach/ 4/5
3/30/2016 Successful Sterility Test Failure Investigations—A Practical Approach | American Pharmaceutical Review - The Review of American Pharmaceutical…

2 Comments American Pharmaceutical Review 

1 Login

 Recommend ⤤ Share Sort by Best

Join the discussion…

sabita • 8 months ago

I was doing sterility test daily for about 10 years for steam sterilized or aseptically filled
drug product (manufactured under GMP conditions and in a strict controlled environment).
I found that failure was only due to external controls (the maintenance of sterility test area
was not as per the schedule, or operator’s was not capable to do the
sterility test).
△ ▽ • Reply • Share ›

Victor Grayson • a year ago

This covers similar ground to Tim Sandle's 2012 paper on investigating sterility test
failures: http://www.ivtnetwork.com/arti...
△ ▽ • Reply • Share ›

ALSO ON AMERICAN PHARMACEUTICAL REVIEW

FDA Approves Two New Drugs for
Diabetes | American Pharmaceutical …
2 comments • 6 months ago
Gabriela Salisbury — Yes I read this news.
However, every diabetes treatment goal is
to control the blood sugar (glucose) …
Johnson & Johnson Announces Open
Translational Science Project in …
1 comment • 10 months ago
Kylie Downs — Yes, I read about it.
Sharing data of clinical trial progresses the
science and can be the foundation of …

Utilizing Social Media in Clinical
Research: How Understanding the …
1 comment • a year ago
Steve — As it pertains to clinical trials,
minimizing exposure of sensitive study-
related information is going to be …
FDA Grants Priority Review For Daklinza
(daclatasvir) sNDAs | Pharmaceutical …
1 comment • 6 months ago
Christian — Hey ideas - I Appreciate the
information - Does someone know if my
assistant could get a fillable CA MC 706 …

✉ Subscribe d Add Disqus to your site Add Disqus Add Privacy

About APR Resources Subscription

About Us Issue Archive Subscribe eNewsletters

Contact Us Newsletter Archive Subscribe Magazine
Press Room Excipient Search Manage Your Print Subscription American Pharmaceutical Review is the leading
Privacy Policy Bioprocessing review of business and technology for the
Disclaimer Chromatography pharmaceutical industry throughout North America.
Connect with Us
Excipients
Drug Delivery Write for Us
Advertise with Us
Formulation Development Facebook
See our other sites »
Advertise With Us Instrumentation Twitter
Media Kit Microbiology LinkedIn Copyright © 2016CompareNetworks, Inc. All rights reserved.
Spectroscopy YouTube

http://www.americanpharmaceuticalreview.com/Featured-Articles/173160-Successful-Sterility-Test-Failure-Investigations-A-Practical-Approach/ 5/5