1/7/23, 9:11 AM An Introduction To Environmental Monitoring Cleaning For Aseptic Environments

Guest Column | August 21, 2017

An Introduction To Environmental Monitoring & Cleaning For Aseptic

Environments
By Crystal M. Booth, PSC Biotech

Microbial control is critical in cleanroom environments. Contaminated environments can lead to product
recalls, regulatory observations, fines, or even consumer deaths. To prevent, destroy, and monitor
microbial contamination in cleanrooms, several aspects of cleanroom microbiology must be understood.
This foundational introduction to cleanroom microbiology article series discusses some of those aspects.
Parts 1 and 2 introduced cleanroom microbiology, discussed guidance documents and FDA observations,
and summarized common sources of microbial contamination in cleanrooms. Part 3 provided an overview
of cleanroom gowning procedures. This final article will address concepts of environmental monitoring
and the importance of disinfectant efficacy and proper cleaning.

Environmental Monitoring

According to 21 CFR 211.113(b): “Firms must follow appropriate written procedures designed to prevent microbiological contamination
of drug products claiming to be sterile.” In order to control microbial contamination, the sources must first be understood.
Environmental monitoring, utility monitoring, and product testing are used to gain an understanding of these sources. For
environmental monitoring, the air is sampled for viable and nonviable particulates. In addition, personnel and surfaces are sampled for
microbial contamination. If microorganisms are recovered, their identification can be used to help identify the potential sources of the
contamination and any corrective and preventive actions that may be required.

Monitoring trends in the environment and facilities assists in ensuring that procedures for preventing contamination are effective and
that the facility is in a state of control. Other benefits include allowing the facility to be proactive by identifying areas of concern before
an out-of-limits event occurs, helping to establish relevant alert levels, helping to establish a list of predominant microorganisms and
resident flora of the facility, and providing historical data so that abnormalities in the data can be detected.

It is important to provide a clear definition of what a “trend” looks like. This definition should be included in procedures to provide
consistent interpretation across the facility and over time. In general, a trend is understood to be a shift from historical data or a shift in
a specified direction.1

Alert levels should be used as an early warning system for when processes are drifting from their established state of control. Utilizing
50 percent of the action level without proper justification is not scientifically sound and can be frowned upon during regulatory audits.
Statistical analysis can be performed on trend data to establish alert limits based on the process capabilities of the facility. For example,
one can utilize historical data and establish a 95 percent confidence interval, where 95 percent of samples taken will be expected to be
within the proposed alert limits.

The majority of expected action levels have been established in multiple regulatory documents. In cases where action limits are not
described by regulatory bodies, such as personnel gowning qualifications (other than gloved hands), risk assessments and statistical
analysis may provide justification for establishing action limits.

Contamination recovery rates are described in USP <1116>. It is a useful tool for trending results that frequently contain zero colony
forming units (CFU), like in aseptic processing environmental monitoring. USP <1116> states that “Because of the inherent variability of
microbial sampling methods, contamination recovery rates are a more useful measure of trending results than is focusing on the
number of colonies recovered from a given sample.”2 The contamination recovery rate is calculated by taking the total number of plates
containing growth of any kind, divided by the total number of plates, times 100 to give a percentage. USP <1116> also provides a table
for suggested initial contamination recovery rates in aseptic environments.

Disinfectant Efficacy And The Importance Of Proper Cleaning

Control of the introduction of contamination into the cleanroom is the most critical concern for the entire cleaning and disinfection
process.3 The cleanliness of components, personnel, carts, tanks, tools, and instruments that are transferred into the clean area is of
upmost importance. If these items are not properly disinfected or cleaned, or if personnel are not properly gowned, microorganisms can
be easily transported into the cleanroom. Multiple guidance documents and articles are available that discuss disinfectant efficacy
testing. Two very good sources include USP <1072> and the PDA technical report 70.

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Get expert advice straight from Crystal in her webinar:

Cleanroom Microbiology – A Foundational Introduction

Life Science Training Institute

Items must be cleaned before they can be disinfected or sterilized. Cleaning is the process of removing foreign material from objects,
and it is necessary for disinfectants to work as intended.3 Foreign material that is not removed may block the disinfectant from coming
into contact with microorganisms on the surface. Foreign materials may also negatively react with disinfectants and inactivate them.3

Difficult to clean areas are governed by 21 CFR 211.42. The regulation requires smooth, hard surfaces that are easily cleanable. Porous
surfaces can harbor microorganisms, making it difficult to recover them for environmental monitoring. Porous surfaces can also make it
difficult to destroy microorganisms during cleaning and disinfection.3 Hard to reach surfaces pose the risk of not being cleaned
properly.3

Rushing the cleaning process, being sloppy, or doing it incorrectly are dangerous practices. Even worse is skipping the cleaning process
entirely. Dirt, oil, fingerprints, and disinfectant residues that are left behind create hospitable habitats for microorganisms or even
protect underlying microorganisms from applied disinfectants.3 It is extremely important to follow SOPs and clean properly to remove
dirt sources, even if they are not visible to the naked eye. This will help to ensure that the disinfectants can work properly.

Appropriate concentrations of disinfectants are established during the disinfectant efficacy study. If disinfectants are not properly
prepared, handled, or used, their effectiveness to destroy microorganisms can be compromised.3 This can lead to the survival of
microorganisms. The effectiveness of disinfectants can be compromised if they are not applied and utilized correctly. For example,
shortcutting the established disinfectant contact time can lead to microbial survival.3 Even if disinfectant solutions are properly made
and changed frequently, dirty equipment, such as mop heads or buckets, can impact the effectiveness of the disinfectant and lead to
microbial survival.3 The contamination may be unknowingly spread throughout the facility. It is also important to ensure the cleaning
equipment is clean and properly maintained.

Disinfectants must be qualified for use in the facility. When qualifying the disinfectants, the testing should include a variety of surfaces
found in the facility that represent the worst-case porosity and the most difficult to clean surfaces.3 The microorganisms chosen for the
study should include environmental isolates from the facility, when possible.3 During the study, the suggested contact times will also be
established. The term “contact time” refers to the amount of time that disinfectants will remain wet and in contact with the surface
being disinfected.3

The performance of the disinfectant should be evaluated throughout the in-use period to establish the in-use expiration dates. PDA
technical report 70 recommends using disinfectants that are close to or slightly beyond their expiration date for the study. It is
important to note that disinfectants should be sterile-filtered or sterilized prior to use in Grades A/B to prevent the disinfectant from
being a source of contamination that can be spread throughout the cleanroom.3

Disinfection efficacy testing and disinfection programs are usually hot topics in regulatory audits. To make the process move along
smoothly, be prepared and have all the data summarized and neatly organized in clean reports.

Conclusion

Cleanroom microbiology encompasses a variety of subjects. Multiple guidance documents and regulations are available referencing
cleanroom microbiology concepts. To avoid potential regulatory findings, be proactive and research warning letters published online,
and correct any noted procedural gaps as soon as possible.

To help regulate microbial contamination, all activities that happen in the cleanroom must be controlled, including processes,
procedures, people, raw materials, excipients, components, APIs, the facility, equipment, machines, environment, surfaces, air supply,
utilities, water sources, material transfer, adjacent areas, and disinfectant and cleaning regimens. Employees must follow procedures,
use proper aseptic technique, pay attention to details, and use utmost care each time they deal with aseptic products. A consumer’s life
could depend on the employee’s behavior, their integrity, properly controlled cleanrooms, and cleanroom microbiology principles.

References:

1. Trend. (n.d.) American Heritage® Dictionary of the English Language, Fifth Edition. (2011). Retrieved May 11, 2017 from
http://www.thefreedictionary.com/trend
2. USP <1116> Microbiological Control and Monitoring of Aseptic Processing Environments
3. PDA Technical Report No. 70 – Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities,
Parenteral Drug Association, Bethesda, MD (2015)

About The Author:

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Crystal M. Booth is president of Azzur Labs, LLC. She has over 19 years of experience in pharmaceutical microbiology, working in
quality assurance, CDMOs, R&D, and quality control laboratories, including startup companies. During her career, she has developed
and validated methods for antibiotics, otic products, topical creams, topical ointments, oral solid dose products, oral liquid dose
products, veterinary products, human parenterals, vaccines, biologics, aseptically filled products, and terminally sterilized products.
Those methods include microbial limits testing, bacterial endotoxins testing, particulate testing, sterility testing, pharmaceutical water
system validations, EM programs, surface recovery validations, disinfectant efficacy studies, minimum inhibitory concentration testing,
antimicrobial effectiveness testing, hold time studies, and various equipment validations. Booth earned her bachelor’s degree in biology
from Old Dominion University and her master’s in microbiology from North Carolina State University.

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Pavan Joshi − ⚑
PJ ⏲ 3 years ago
Well written. Thanks
0 0 • Reply • Share ›

Sandeep Sura − ⚑
SS ⏲ 3 years ago
Very informative article. I want to know more about surface recovery validation and any correlation
of this with % CRR.
0 0 • Reply • Share ›

Crystal Booth > Sandeep Sura − ⚑

CB ⏲ 3 years ago
Hi Sandeep, I actually haven't heard of anyone trying to correlate surface recovery
validation and CCR... yet. :-)
0 0 • Reply • Share ›

Jlseagullclean − ⚑
J ⏲ 3 years ago
Ms. Crystal Booth: Another subject matter I am interested in is getting myself more familiar with
which regulatory codes and bodies regulate which areas I am charged with cleaning and which
trade/professional oversight associations recommend best practices for the areas I am overseeing.
Sometimes it gets confusing and I almost wish I had a flow chart of some type. If one does not exist, I
may have to create my own. I have skimmed a few of your articles already and I am amazed at the
thoroughness and depth which they articulate. I am horrified at what I see happening in
environmental services, commercial janitorial, the cleaning of critical areas and basically all things
related to cleaning in all commercial environments is generally speaking the bastard child of the
commercial world. Almost nobody is interested, few know much about it, there is very little
regulation or oversight and what is happening from the highest level leadership on down to the
person doing the cleaning is often scary. Infection rates are out of control too. I feel like I am
navigating a mine field and need to get my directional bearings straight to make decisions with which
I can gain leverage moving forward to hopefully make a difference, save a couple of lives and get
people to see the value of infection control, quality control, risk management and all around value
based systems that deliver excellent results measured in empirical terms understandable to a wide
spectrum of professionals from the presidents of organizations down to the folks on the ground.
Recently, a very high ranking state of California executive who is a consultant, discovered doctors
using the same bone drills on multiple ortho surgery patients without sterilization as a matter of
regular practice day in and day out. The infection rates were out of control and the housekeepers were
being blamed! My friend observed and finally realized sterile processing was not cleaning the drills
"because there was not enough time." People die for this! I catch nurses throwing dripping blood in
cups in the trash.....the list goes on forever and it just drives me crazy because 1. I care for innocent
vulnerable people and 2. Safety is my top priority and 3. I can't stand sloppiness. My energy is
endless. I am offering the hospital to spend $60. per month to make up on the cost difference for
sterile coveralls. All of which I speak is happening in major, well known healthcare hospitals and
facilities in California. I am guessing that this is normal throughout the country. I need to push for
change, at least in my immediate sphere of influence, and then take it from there. Also, the Centers
for Medicare and Medicaid come around once every three years, everyone get's written up in
ambulatory surgery centers (doctors not wearing masks for endoscopies where fecal matter spatters
the computer and EVS does not clean it up), then people and leadership lie, cheat and steal to barely
pass a CMS inspection to limp for another three years until they come around again.......I feel lost in a
rabbit hole. Any list of resources would be helpful along with consulting advice or a referral to a
consultant or a quote if you do consulting. Thanks, Dave Aria
0 0 • Reply • Share ›

Crystal Booth > Jlseagullclean − ⚑

CB ⏲
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CB ⏲ 3 years ago
Thank you for the comments Dave. I'll reach out to you.
0 0 • Reply • Share ›

Pradip M Patel − ⚑
PM ⏲ 4 years ago
Excellent article and good learning session for EM microbiologist.
0 0 • Reply • Share ›

Crystal Booth > Pradip M Patel − ⚑

CB ⏲ 4 years ago
Thank you Pradip!
0 0 • Reply • Share ›

Ravinder Singh − ⚑
RS ⏲ 5 years ago
Its very good article for aseptic processing of DP.Can you expand your scope with respect to class C
also. Its in this class DS is produced before going for terminal sterilization through filtration or any
other methods suitable for product. For Biological product , environment is important to produce
Endo free DS. I will appreciate if you can articulate on this aspects also. rgds Ravinder
0 0 • Reply • Share ›

crystalbooth > Ravinder Singh − ⚑

C ⏲ 5 years ago
Hi Ravinder, according to Annex 1, the requirements are to fill in Grade C unless the
product is at risk and then you would need Grade A. Formulation and preparation of
components can occur in Grade D. If the product is at unusual risk, they recommend
formulation in Grade C.__In my experience with terminal sterilized products, we
formulated the product in a Grade D environment. The product was then filtered through
ultrafiltration units on its way to the filling room. We tested bioburden and endotoxin pre
and post filtration. The adjacent supporting rooms in the cleanroom suite were all Grade C
rooms, but the filling line itself was encased into an environment that passed Grade A
testing (this is was NOT required). The capping areas outside the cleanroom also produced
Grade A air from the source. Most components were prepared in a Grade D environment
(some were processed in a Grade C). Autoclaving occurred rather quickly after filling. After
cooling, we tested sterility, endotoxin, and particulates. Gowning in the cleanroom
consisted of double-sterile gloves, safety glasses, sterile hoods (over hair net), sterile body
suites (over sterile scrubs), sterile boot covers (over booties and safety shoes) and sterile
face masks. As this was technically Grade C, skin was exposed around the eye areas and
googles were not used.__I recommend you set limits for bioburden based on your process
and filter validation. When thinking of your controlled environments, consider your
facility capabilities, manufacturing process, product needs, and the regulations.
Remember, you can always go above and beyond (i.e. stricter requirements) from what the
regulators require of you to ensure your product is safe. Just don't short cut the
regulations.
0 0 • Reply • Share ›

Wilson Nazareth − ⚑
WN ⏲ 5 years ago
Excellent articles, kindly post us articles related to Qualification of non sterile environments by
microbiological methods, and risk based approach for environmental monitoring locations.
0 0 • Reply • Share ›

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