Designing Disinfection Systems Chapt 8

8
IMPLEMENTING A CLEANING AND

DISINFECTION PROGRAM IN PHARMACEUTICAL
AND BIOTECHNOLOGY CLEAN ROOM
ENVIRONMENTS
Arthur L. Vellutato, Jr.
Veltek Associates, Inc.
INTRODUCTION
The implementation of an effective cleaning and disinfection program in a GMP
facility is a multi-phased process that requires the harnessing of a multitude of critical
disciplines in order to achieve success. Like many compliant GMP systems, the design
of a cleaning and disinfection regime utilizes the talents of the quality assurance,
production and validation departments. This compilation of skills is mandatory to
completely address a system that will net continually acceptable environmental
conditions and prove competence during regulatory inspections.
In looking at the basics surrounding the formation of a compliant system, the

ultimate goal is to clean and disinfect the surface. This seems to be very simple;
however, it is slightly more complex than just cleaning and disinfecting surfaces.
By definition, cleaning a surface would require the removal of any contaminant

that is not part of the material surface itself. Such contaminants may be in the form of
spilled product from a previous batch, chemicals used in manufacturing product,
disinfectants, particulates and residues. When cleaning surfaces in the clean room, the
removal of all particulates and residues is virtually impossible. Thus, the clean room
will, once cleaned, have an existent level of particulates and residues that may or may
not affect the next batch of product. Likewise, disinfecting the surface would require
one to ensure that the bioburden of the surface has been eliminated or reduced to a safe
1
2 Laboratory Validation
and contained level which will not affect final product. However, one could not render
such surfaces “sterile”, as technically defined. Sterilizing a surface would require it to
be exposed to a terminal sterilization process such as autoclaving, heat, gamma
radiation, ethylene oxide or extended chemical sterilization.
The only fitting method for use in a clean room from the above list would be
chemical sterilization. Chemical sterilization of a clean room surface using an EPA
registered sporicidal agent may take up to five hours of soaking of all surfaces. This is
not possible in the clean room environment. Thus, an existent bioburden, even after
disinfection, may exist in the area.
Comprehending that the surfaces may not be perfectly clean and sterilized is
critical to an understanding of what may and may not be accomplished. In many
phases of life, “perfect” performance is impossible to achieve, and cleaning and
disinfection are no exception. If a clean room were cleaned, filled with ultra-clean
water, then the water drained and its chemical content evaluated for non-viable matter,
one would find a multitude of spikes in an IR spectrum analysis; and, complete
identification of all contaminants would be unlikely. In a viable sense, if a clean room
was cleaned and disinfected, then subsequently filled with a growth medium like
trypticase soy broth (TSB), and a reasonable growth period allowed, one would see
growth. These two examples, at extreme ends of the spectrum, show that cleaning and
disinfecting of a controlled environment will not be perfect. But despite the
complexity, the goal is to near as close to perfection as is humanly possible.
DESIGNING A COMPLIANT GMP SYSTEM
The first step in developing a compliant cleaning and disinfection system is to evaluate
what is presently being done in the operation. The author's personal experience as a
consultant involved in cleaning and disinfection in this industry for many years is that
Quality Assurance (QA) departments may not know absolutely everything that is done
in a production department; likewise, production may not completely understand what
quality assurance's recommendation may be. This is understandable, as QA and
production are two separate functions with differing agendas but their combination of
skills and disciplines makes for the development of a very intricate and effective
system. But first, QA and production professionals need to understand what is done on
a daily basis, and what should be done on a daily basis.
The next step to achieving near-perfection in our cleaning and disinfection

systems is to put the basics into perspective. The criteria for implementing a cleaning
and disinfection program within a GMP facility require companies to tailor their
system to address their operations' specialized needs. Development of a systematic
approach to addressing and controlling of contamination will serve organizations for
Implementing a Cleaning and Disinfection Program 3
years to come, with attention to detail the key to success. And success is measured by
consistently attaining acceptable environmental conditions, making use of the
available disinfectants and application devices, and utilizing environmental
monitoring data to address the specifics of the system. In turn, regulatory auditors will
routinely scrutinize the system with a fine-tooth comb, and dissatisfaction can result
in possible 483 observations or the shut-down of manufacturing processes. For this
reason, systems need to be complete in their content, and assumptions that situations
may not arise could be fatal. And most importantly, the design must be documented
within the constraints of a quality system.
To complicate things further, there is no common methodology in the industry;

nor will companies receive a specific requirement list of expectations from the
regulatory agencies. It requires the use of a multitude of separate scientifically-based
methodologies and disciplines, combined to render the final infrastructure.
For years, the pharmaceutical and biotechnology industries have been two of the
most regulated in the world. However, within the spectrum of manufacturing a product
that will enter or come in contact with the body, there are several critical areas where
there is a lack of written directive from regulatory agencies. Two of these critical areas
are environmental monitoring, and disinfection of a controlled area. Together these
two critical capacities serve as the basis for determining and maintaining the
environmental conditions during the time of manufacture of a drug product. Initially,
one would assume that there is a specific need for regulatory specifications in these
critical areas. However, after careful evaluation, it appears that these functions require
a very unique design for each manufacturing setting and for each product that is
produced. Specifications from regulatory agencies do, in certain circumstances,
standardize the function or testing requirements throughout the industry. However, in
the areas of environmental monitoring and disinfection, they may place possibly
unattainable criteria to very process-specific functions.
Despite the lack of written regulatory specifications, this does not mean that
regulatory agencies do not have specific expectations. In fact, their expectations may
be greater than if specifications were in place. In the present day, regulatory agencies
have placed the responsibility for system design, implementation, documentation and
justification on the manufacturers themselves. The overall theme is for a drug
manufacturer to "figure out how they are going do it, do it, document it, and be
prepared to justify their methodology upon request." An example of this can be found
in the 1987 FDA Guideline on Sterile Drug Products Produced by Aseptic Processing,
2
(page 9) . At that time, regulatory specified what they expected for viable airborne
conditions: "Air should also be of a high microbial quality. An incidence of no more
than one colony forming unit per 10 cubic feet is considered as attainable and
desirable." Within this text, regulatory said "here is what we expect; now you figure
out how you need to accomplish and document this capacity." However, within this
short statement, technical problems arise.
The first is that humans, by nature, try to interpret hidden meanings in what is
written. Personnel in pharmaceutical and biotechnology varied in their interpretation
of the guidance. At the same time, regulatory inspectors and reviewers also varied
amongst each other understanding and enforcement policies. In short, the guidance
text was not complete. It did not specify many details such as the required total volume
to be sampled, the number of locations to be sampled, and the frequency in which
sampling should be done. This causes confusion as all then develop and implement
their opinions and commonality of task to be performed and the method by which it
will be inspected varies from firm to firm and regulatory inspector to regulatory
inspector.
This is just one example of problematic situations that arise with certain guidance
documents. Earlier in the chapter, the probable imperfection of cleaning and
disinfecting was discussed. In terms of guidance documents, the same is also true. In
terms of environmental monitoring, cleaning and disinfection, no one organization
will ever be able to create the perfect guideline. Guidelines will always lack the critical
uniqueness that exists for implementation in each and every manufacturing and testing
operation. This concept must be understood.
Thus, the problem with developing an environmental monitoring and disinfection

program is one is not able to pick up a book and follow its content. In reality, one may
need to view many books, many guidelines or more effectively, rely on direct
experience. Direct experience is this venue is invaluable and without it in one's arsenal,
there is less of a chance for success.
Experienced professionals design environmental monitoring and disinfection

systems that work. The utilization of inexperienced personnel to design the systems is
a way of ensuring failure and possibly future regulatory complications.
It is necessary to look at a few guideline documents that may assist in understanding

what needs to incorporate in the design of a system. First and foremost, one needs to look
as the specific tone from the US FDA. Presently the 1987 FDA Guideline on Sterile Drug
Products Produced by Aseptic Processing is the current document. However a revision
3
from FDA, entitled Sterile Drug Products Produced by Aseptic Processing Draft has
4
been recently released. A second source is the PDA's Technical Report #13 . the third
source would be the United States Pharmacopeia (USP), Chapter <1116> (Environmental
5 6
Monitoring) and Chapter <1072> (Disinfectants and Antiseptics) .
8
A fourth source looks to review ISO 146447 and 14698 , and the fifth source
reviews current and past industry publications listed in the bibliography of this chapter.
The last, and most important, source relays on what has been learned internally as an
organization and externally from colleagues in the industry. While this list of written
guidance is not complete, coupled together they provide us with the basic concepts
needed to design our systems.
While we review the existent guidelines, publications and past experience data,
we must understand that all differ in their content. The design of a complete and
compliant cleaning and disinfection system uses these critical documents only as tools
to write a master plan; they could never be as complete for one's operations as a system
one could develop ourselves.
So where does one start? Cognizant of the multitude of issues that will require
review, the GMP facility needs to look at the entire picture in a full circle approach.
Prior to design or re-design we need to put some basic perspectives into context. While
incomplete in lengthy discussion for each item noted, this short guide will hopefully
identify the important areas where firms will need to focus their attention.
MAINTAINING CONTROL OF THE ENVIRONMENT
Maintaining control of the environment is critical. It is the most important aspect

surrounding a cleaning a disinfection program. We need to understanding that control
of the environment does not come from using a chemical agent that will destroy all
microorganisms. Rather, control of the environment is the assurance level we establish
to reduce or possibly eliminate the contamination from ever entering our controlled
areas. Once a chemical agent has been applied to the surface and subsequently dries,
its destructive capabilities to a microorganism are complete. Disinfection (or
sporicidal) characteristics of a chemical agent require the organism to be wetted by the
agent. While certain residues from chemical agents may have some remaining
antimicrobial properties, the destructive capabilities of the residue are minimal.
Control over what enters the controlled area now takes over. In reality, control of the
environment has nothing to do with disinfection. Disinfection is complete when
control takes over. Manufacturing operations will not disinfect surfaces while
production is occurring. Disinfection is done prior to manufacturing and the
environment released to produce a product. Our success or failure in control is
measured each and every time as environmental monitoring is conducted during our
filling operations.
Addressing contamination prior to its entry will ensure that we will not have to
contend with its presence. While disinfection of surfaces always take the lead in
structuring our procedures, control over the environment should remain one of the
main focuses.
Criteria for reducing the bioburden that enters the controlled area require us to
evaluate the entry of personnel, components, water, tanks, carts, and even disinfectants
to name a few. This is done to ensure that one do not undermine disinfection efforts by
introducing high levels of contamination after disinfection is complete. We must
ensure each item's appropriateness in the room classification to which it enters. In
Class 100 aseptic operations, one needs to carefully evaluate each item as clean and
sterile prior to entry, as well as assuring the cleanliness, sterility and appropriate fit of
garments for personnel. In Class 10,000–100,000, while our concern is less, we must
ensure that such environmental conditions do not adversely affect the Class 100 area.
In short, a room can be monitored as having very little if any bioburden at rest;
however, in a static condition we can easily corrupt our efforts previously achieve
through poor control.
CLEANING THE ENVIRONMENT
Too often, cleaning is confused with disinfection. They are not the same. Cleaning
characterizes the removal of particulates, microbes and possibly existent residues from
surfaces. Cleaning requires a non-destructive mechanical action be applied that
loosens and removes contaminants from the area. Procedurally, contaminates and
residues are loosened and rinsed to the floor. Subsequently, the dirtied solution on the
floor is collected and removed from the area (normally by a squeegee). By lessening
the level of particulates, microbes and residues on the surface, disinfection efforts
become simpler. First there are fewer organisms to destroy as most have been removed
from the area. And second, as bioburden and residues are lower, the possible
obstructions blocking the chemical agent from contacting the organism are minimized.
In short, cleaning prepares the surface for disinfection.
Disinfection relates to the saturation and penetration of the cell wall of an

organism by a chemical agent. It further requires that an organism remain wetted for
a specified contact time with a chemical agent capable of killing the organism in
question. Disinfection depends upon temperature, saturation and penetration of the
cell wall, contact time, surface and bioburden of the surface, existent soil load,
concentration of the chemical agent and pH. Provided the appropriate chemical agent
is utilized, the key to disinfection in the clean room is keeping the surface wetted for
5–10 minutes. This is difficult, as the movement of air via laminar flow tends to dry
surfaces quicker.
As discussed, there are significant difference between cleaning and disinfection.

Cleaning tries to remove contamination from the surface while disinfection attempts to
destroy what viable cells exist on the surface. In a realistic example, we can use a
toothbrush and mouthwash as examples. If we were to discuss the options of not using
a toothbrush anymore and only utilizing a daily mouthwash rinse with our dentist,
he/she would inform us that we would soon have no teeth. Residues, particulates and
microbials will build up on the surface of the teeth and the teeth would eventually
deteriorate. This scenario depicts what occurs too often in the pharmaceutical and
biotechnology industry. We forget to brush and just try to kill anything that exists on the
surface. Eventually our surfaces become residue-laden and more difficult to disinfect
and eventually deteriorates. Within our note to technical brilliance we sometimes forget
simple common sense. And unfortunately, the phrase, “simple common sense” is not
the title of any GMP, CFR or guidance document. The effect of the buildup of residues,
particulates and possibly microbials is also aided by the surface itself. Clean room
surfaces are irregular in nature as depicted in the Scanning Electron Microscope (SEM)
photographs (Figures 1–8) seen later in this chapter. Such surfaces trap residues and
other contaminants and make the surface more difficult to disinfect.
Sooner or later we have to clean. The frequency of cleaning can vary from a daily
function to a monthly function. Normality is to clean surfaces either bi-weekly or on
a monthly basis. Some may say, "But that's an additional cleaning operation we need
to do!" The correct response is "That is correct, it needs to be done."
Within the healthcare setting and most commonly reported in the hospital setting,
test reports have shown the effect that cleaning the surface has on the level of
microbial levels in controlled areas. Many publications purport this concept. In the
pharmaceutical and biotechnology setting, a test report conducted and published in
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1989 by A. Vellutato, Sr and A. Vellutato, Jr. of Veltek Associates, Inc. demonstrated
what affect cleaning the surfaces has on the level of microbial contamination found
on the surface. The study focused on the concept that cleaning alone would remove
most of the existent microbial contamination. In the report, all surfaces were cleaned
with a sodium lauryl sulfate detergent and mechanical cleaning action on a daily
basis. Such cleaning and was conducted in a Class 100, Grade A, ISO 5 area.
Environmental Monitoring was routinely conducted on air, surface and personnel.
The manufacturing operations filled an average of 4,000 units of 500 mL. bags of
USP Water for Injection. Manufacturing operations ran for four hours per day and
upon completion of manufacturing, the clean room was completely cleaned.
The cleaning mechanism utilized a mop and two-bucket system, a sprayer and a
squeegee. The procedure for cleaning was to apply the sodium lauryl sulfate detergent
(DECON-Clean®) to the surface utilizing a top to bottom approach. Upon completion
of the mopping, the chemical was then sprayed on to the surface and all excess liquid
on the surfaces pulled downward by squeegee to the floor. The remaining liquid was
then collected on the floor and removed. For 30 days, the results met industry limits for
Class 100, Grade A, ISO 5. At 45 days, control was lost, results exceeded limits and a
sporicidal agent was used once on day 45. The limits returned to acceptable levels for
31 days (day 76 of the study). The final conclusion was one to two uses of a sporicidal
agent with cleaning regime controlled the environment.
Cleaning is based on a few physical factors:
• the surface to be cleaned

• the contamination vested on the surface
• the chemical used to clean the surface
• the effect of the chemical agent on the surface to be cleaned
• the level of surfactants in the chemical agent
• the effect of the chemical on the contamination, moreover residue, that exists on
the surface
Knowledge of the type of surface to be cleaned is very important. As depicted

in Figures 1–5 (further in the chapter), surface irregularity varies as composite
materials change. The surface irregularity may compromise cleaning and
disinfection efforts for two basic reasons. First, cleaning becomes more difficult.
Second, disinfection becomes harder as the chemical agent cannot contact all the
surfaces for the required wetted time period (as obstructions may exist). Cleaning
becomes more difficult as the surface's irregular nature may allow particulates,
residues and even microbial contamination to vest itself within the rutted or porous
areas of the surface. These nooks and crannies are very hard to clean with most clean
room apparatus designed for cleaning. Most clean room mops and wipes and flat and
do not allow the penetration of the fibers or surfaces of the cleaning mechanism to
reach into the crevices. The lack of such an abrasive cleaning action bypasses the
opportunity to loosen and remove these contaminants. Most irregular surfaces are
commonly so across the span of the material, so a multitude of contamination
sources exist on the surface to be cleaned. Cleaning an irregular surface requires one
to use a cleaning device that can penetrate into the nooks and crannies and make
contact with the existent contaminants and residues. For example, we would not use
a wiper or a pad to try to clean our teeth, but a toothbrush. Unfortunately, a clean
room brush is not a commonly-available item in the industry. Nor would requiring a
controlled area to be cleaned with it be a reasonable request to make of production
personnel. This is a subject that cleaning apparatus manufacturers need to address
more closely.
The lack of available products forces the clean room professional to adapt a non-
clean room product to address this specific need.
The second basic complication is that if particulates and residues exist in such
nooks and crannies, the possibility for a disinfecting agent to contact the surface
within the nooks and crannies is improbable. Thus, we are disinfecting the surface of
the particulates and residues and never disinfecting the actual surface. Without such
assurance for cleaning and disinfecting the actual surface, the possibility for
contamination to be vested underneath the particulate and residue may be probable.
In other sections of this chapter, antimicrobial effectiveness testing will be

discussed, giving an understanding that, during such validation testing we look to
inoculate a surface with a known enumeration of microorganisms and soak such
surfaces in a disinfecting agent for a predetermined time period. Upon completion of
the soaking, we will then test the surface to account for the possibly remaining viable
contamination.
When we attempt to use such data in the field, we will find our testing skewed for
two basic factors. First, our testing inoculates a variety of surfaces with a multitude of
microorganisms. Some surfaces are more irregular than others. In our test, after we
have soaked the surfaces, we need to rinse clean the surfaces of any possible
contamination to a filter which is then plated to a growth medium. However, we find
the rinsing of the irregular surfaces more difficult as microorganisms may vest and
cling within the irregular areas of the surface. This means the possibility to rinse free
a microorganism from a smooth surface is easier than from an irregular surface. What
we may learn from this testing is that it may seem that our disinfecting agent is more
effective on the irregular or porous surface but this is due to our inability to rinse free
all of the existent microorganisms. The smooth surfaces are more easily rinsed and
existent microorganisms removed and can grow in our growth medium. However,
microorganisms from the irregular or porous surface may have never been removed
from the surface itself. This means they were never rinsed to the filter and plated for
growth. Thus, we could conclude smooth surfaces are harder to disinfect than irregular
or porous surfaces in our manufacturing and testing areas. The effectiveness report
may show higher remaining colony forming units (CFUs) for the smooth surfaces than
the irregular or porous surfaces. This assumption would be incorrect.
In the manufacturing or testing areas, the opposite occurs and we find it more
difficult to disinfect the irregular or porous surfaces. Understanding this concept is
critical to successful disinfection. We will have the same trouble, if not a more
complicated problem, in disinfecting and rinsing the microorganisms from the
irregular or porous surfaces in our manufacturing and testing areas, and due to this,
we may leave viable contamination on such surfaces. Care needs to be taken when
disinfecting irregular or porous surfaces, as they are harder to clean and disinfect than
smoother surfaces.
In general, clean room material grades can be separated into six basic categories:
• aluminum
• stainless steel
• epoxy-coated finishes
• plastics
Figure 1: Aluminum surface
• vinyl
• glass
In Figure 1, we see an aluminum surface. This surface is a metal grade that is soft
and easily scratches and is deteriorated by chlorine solutions, gulteraldehyde and
peroxide and peracetic acid and hydrogen peroxide solutions (from a list of basic
disinfecting agents). While aluminum is a commonly-used metal, deterioration of the
surface from chemical exposure normally shows as a turquoise bluish gray bubbling
on the metal. Aluminum is also easily stained or discolored from the residues from by
phenol, quaternary ammonium, and iodine to name a few. However, of most clean
room surfaces, aluminum is normally easy to clean and disinfect.
Figure 2: 316 L Stainless Steel surface

Figure 3: Epoxy-oated surface
In Figure 2, we see a 316L stainless steel surface. This surface is very smooth and
contains few impurities in the metal that may be deteriorated by a disinfecting agent.
Most impurities in the metal grade are in the carbon family and are deteriorated by
chlorine solutions, gulteraldehyde and peroxide and peracetic acid and hydrogen
peroxide solutions (from a list of basic disinfecting agents). Stainless comes in grade
based on the level of impurities in the metal. Normally, deterioration of the surface is
in the form of a rusting that pits the surface or surface rust. Rusting of the stainless
itself is from chemicals or water oxidizing and/or reacting with impurities in the metal.
Surface rust is the rusting of airborne heavy metals that deposit atop the surface.
Within the clean room operation, many metal grades exist from of 302 to 402 stainless.
Many in the industry demand the use of 316L stainless; however this metal grade
cannot be used for every component due to its brittle nature. Some perfect examples
of this would be a spring or a solenoid mechanism. Utilizing too hard a metal will
cause the spring or moving part to routinely break as friction of movement on the
metal will cause stress and fracture. Stainless is also easily stained or discolored from
the residues from by phenol, quaternary ammonium, and iodine to name a few.
However, of most clean room surfaces, stainless steel is normally one of the easiest
surfaces to clean and disinfect.
In Figure 3, we see an epoxy-coated surface. Epoxy-type surfaces are very

numerous in types and materials. They are a coating that is applied in a liquid form
and then hardens. This is where disinfection becomes complicated. The irregular
surface appearance of epoxy-coated surfaces challenges the craft of the disinfection
professional. Most clean room walls and floors are made of an epoxy material or
similar material so understanding cleaning and disinfection of this surface is critical.
Common build up of particulates and moreover residues in the crevices complicate
cleaning and disinfection. Problematic situation can arise in the crevices of the
surfaces as air pockets can form and once disinfection is compete, be broken an
Figure 4: Clean Room Curtains #1
possibly release existent contamination to the environment. The most difficult task
with the epoxy-coated surface is to clean the contaminants from the surface so that the
disinfectant can be applied and have the ability to address the surface itself. Normally
epoxy-coated surfaces are deteriorated by chlorine solutions, peroxide, isopropyl
alcohol, ethanol and peracetic acid and hydrogen peroxide solutions (from a list of
basic disinfecting agents). The normal deterioration occurs in the form of over drying
and cracking of the material finish. The material becomes powder-like and orange to
blue in color (dependent upon the material).
Epoxy-coated surfaces need routine replacement or refinishing and as such

remain continuously on a preventative maintenance schedule. The epoxy-coated finish
is also easily stained or discolored from the residues from by phenol, quaternary
ammonium, and iodine to name a few.
Figure 5: Clean Room Curtains #2
Figure 6: Phenolic Residue
The next basic clean room surface type is a collage or porous material such as
plastic, vinyl, plexiglass, delron, and other similar type products. In Figures 4, 5 and
7 we can see what these surfaces may look like when magnified through an SEM
microscope. Characteristically they have pore openings that are rather deep. Cleaning
and disinfection are very difficult with these materials. Normally these type of
surfaces need to be replaced over time. These materials need replacing every two or
three years on average. Replacement is costly but necessary, and should be viewed as
a cost of doing business. Normally, porous surfaces are deteriorated by chlorine
solutions, peroxide, isopropyl alcohol, ethanol and peracetic acid and hydrogen
peroxide solutions (from a list of basic disinfecting agents). The normal deterioration
occurs in the form of over-drying and cracking of the material itself. A yellowing, or
change of color to an orange or brown, are common symptoms of the drying process.
The porous material is very easily stained or discolored from the residues of phenol,
Figure 7: Vinyl surface
Figure 8: Sodium Hypochlorite Residue
quaternary ammonium, and iodine to name a few; such residues and/or stains only
increase the deterioration process. Most notably these materials are use as curtains
that separate process control areas. Items such as plastic curtains are one of the most
widely used materials in the clean room environment. Cleaning these surfaces is
normally done frequently, if not daily, as they represent the second-closest non-
product contact surface next to the filling line itself. This over-cleaning shortens the
life of the material.
The last category is glass. Glass is a relatively smooth which does not deteriorate.
Glass is very difficult to clean, and provides a constant example of how difficult all
surfaces are to clean. The reason glass seems harder to clean is that one can see through
it. All other materials discussed to this point are not clear. If such materials were as clear
as glass, the appearance of these surfaces would look horrifically dirty in comparison
to glass. Glass is very easily dirtied, and is a very difficult material to keep clean.
However, this surface is used more and more in clean room operations as it allows
viewing of the operation by supervisors and visitors. In general, glass does not stain
easily from disinfectants or sporicides. However, residues build up and it is discolored
from the residues of phenol, quaternary ammonium, and iodine to name a few.
We have seen from this section the importance of cleaning. Simply, the effect of
cleaning surfaces ensures the best possible opportunity to disinfect the surface as the
microbial levels and the possibly existent residues and particulates will be lower. Later
in this chapter we will discuss the mechanisms to clean such surfaces. However, first
we must understand the chemical agents that cause the main problem associated with
a dirtied surface, the residue.
TESTING THE ENVIRONMENT AND DEVELOPING DATA

Environmental monitoring is a subject warranting its own presentation. In the world
today, the existent guidelines are numerous. The guidelines, while having a common
theme, are not harmonized. Each guideline's numerical values and recommendations
slightly differ from each other. Some contradict each other's content. Some of the
existing guidelines include, USP Chapter <1116> (in revision), ISO TC209, ISO
14644, ISO 14698, PDA Technical Report # 13, the FDA's 1987 Aseptic Processing
Guidelines (in revision), the revision to the EU Annex I and several others. While there
is no commonality in the guidelines, one should cautiously approach the claim of
adherence to one particular guideline. Doing so would be a claim, and specifically
following the guideline's parameters may be required. Rather, one should use the
guidelines as informational sources. Gain what knowledge can be gleaned from the
writings, then design an environmental monitoring program based on what
information is applicable for one's specific operations.
While structuring one's environmental monitoring program, one will find it

necessary to test for viable cells both in the air and on the surface. Through one's
environmental monitoring program, one can develop a list of environmental isolates
that have been noticed in their operations. By review of one's environmental isolate
list, one can determine what contamination exists in the facility and appropriately
design their cleaning and disinfection program surrounding this scientific knowledge.
Once developed, the key is to successfully integrate and document a plan for the
assured demise of these organisms.
CHOOSING THE APPROPRIATE DISINFECTANTS AND

SPORICIDES
The utilization of environmental monitoring data to implement a cleaning and

disinfection program in controlled environments uses science to justify our ends. We
test our environment and subsequently address the contamination noticed with a
proven and validated efficacious disinfection solution. This ensures that we use a
scientific rationale to control contamination in our operations.
In choosing the appropriate chemical agents, we need to review what the

difference between a sanitizer, a disinfectant and a sporicide. In very simple terms, a
sanitizer has the lowest ability to destroy vegetative cells of the three listed. The
disinfectant destroys a broader spectrum of vegetative cells than the does a sanitizer
and incorporates some log reduction of spores. And the sporicide destroys all
vegetative cells and spores. Specifically, the United States Environmental Protection
Agency (EPA) under FIFRA, CFR Title 40 governs and registers by United States of
America law antimicrobial effectiveness claims for hard surfaces disinfection.
Table 1 gives is a basic overview of what is required by a disinfectant

manufacturing company by the US EPA to make the following claims on marketed
products.
Understanding disinfectant categories is essential. A multitude of categorizations

of chemical agents exist in the world. In Table 1, we identified the EPA's
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Table 1: EPA-Approved Disinfectant Test Methods (for hard surfaces)
Types of Claims Test organism(s)

Sterilizer Bacillus subtilis and Clostridium sporogenes
AOAC Sporicidal Test

Tuberculocide Mycobacterium tuberculosis
AOAC Tuberculocidal Activity Method

Modified AOAC Tuberculocidal Activity
Method
Quantitative Tuberculocidal Activity Test
Method
Hospital Disinfectant Salmonella cholerasuis, Staphylococcus
aureus, and Pseudomonas aeruginosa
AOAC Use-Dilution Method
AOAC Germicidal Spray Products Test
General Disinfectant Salmonella cholerasuis and Staphylococcus
aureus
Limited Disinfectant Same as Above
Fungicide Trichophyton mentagrophytes
AOAC Fungicidal Test

Virucide Specific Virus Claimed
EPA Virucidal Test Parameters

Sanitizing Rinse (food contact surfaces) Escherlchia coli and Salmonella typhi or
Staphylococcus aureus
AOAC-available chlorine germicidal

equivalent concentration method
AOAC germicidal detergent sanitizer
method
Sanitizer (inanimate, nonfood contact Staphylococcus aureus and Klebsiella
surfaces) pneumoniae or Enterobacteraer aerogenes
EPA Sanitizer Test Parameters

classifications for antimicrobial effectiveness claims that based on their passing of

particular AOAC test parameters 10. Others have categorized disinfectants as
sanitizers, disinfectants, high level disinfectants, intermediate level disinfectants, low
level disinfectants, sporicides and sterilants. In development and validating of a
cleaning and disinfection system, we need to identify three categories;
Sanitizer: a germicidal product that reduces the population of viable microorganisms
Disinfectant: a chemical agent that completely destroys pathogenic organisms but

does not destroy all microbal forms such as bacterial endospores.
Sterilants/Sporicides: a chemical agent that is capable of sterilizing. Such chemical

can render a product free of viable organisms including all bacterial endospores.
Simplifying this list to three basic categories allows us to determine what we will
require for our operations and subsequently be required to validate. Defined in the
categories below are chemicals agents normally used in pharmaceutical and
biotechnology operations. We need to choose one sanitizer, one or two disinfectants
(depending on our belief in resistance) and one sporicide/sterilant.
Sanitizers:
Isopropyl Alcohol 70%
Ethanol at 70%
Ethyl Alcohol at 70%
Iodine (not normally used in clean room operations)
Disinfectants:
Phenols
Quarternary Ammonium
Hydrogen Peroxide at 3% or below
Sodium hypochlorite below 0.10%
Sporicides/Sterilants:
Sodium Hypochlorite above 0.25%
Hydrogen peroxide above 6% (sporicidal reduction at 6%, a sterilant at 10–35%
depending on the wetted time)
Peracetic Acid & Hydrogen Peroxide
Glutaraldehyde
Formaldehyde
In reviewing the basic types of chemical agents used in pharmaceutical and

biotechnology operations we can come to understand their basic differences and
applicability in our operations.
To follow is a brief description of each of the most used chemical agents in the
industry. As a complete chapter could be written on each, the summaries are brief so
we can develop a basic understanding of each chemical.
ISOPROPYL ALCOHOL, ETHYL ALCOHOL, ETHANOL

SOLUTIONS:
Alcohols have been used for years in pharmaceutical and biotechnology operations for
three basic purposes:
• as a sanitizing spray for gloves, surfaces, carts, etc.
• as a cleaning or wipe down agent to remove possible existent residues from

critical non-product contact surfaces
• as a product contact cleaning agent (ethyl alcohol only)
In testing antimicrobial products' effectiveness, a 70% solution has far superior

efficacy performance than the higher or lower concentrations. Alcohols come in a
variety of forms. The most used forms in the clean room operation are isopropyl
alcohol and ethyl alcohol (ethanol and alcohol). More than 90% of industry operation
will utilize a 70% isopropyl alcohol solution to address clean room organisms as it has
been proven more efficacious than ethyl alcohol (190–200 proof diluted to 70%) or
ethanol 9a mixture with the base as ethyl alcohol [63%] and spiked with methyl [3%]
and isopropyl [4%]) at a small percentage (rendering it undrinkable). Alcohols
demonstrate rapid broad-spectrum antimicrobial activity against vegetative bacteria
11
(including mycobacteria), viruses, and fungi but are not sporicidal . They are however,
12 13
known to inhibit sporulation and spore germination , but this effect is reversible . As
alcohols are not sporicidal, they are not recommended for sterilization and are widely
used for hard surface disinfection and skin antisepsis. As has been previously stated,
of the existent alcohols, isopropyl alcohols demonstrate superior effectiveness against
clean room organisms. However, for viruses, ethyl alcohol or ethanol seems to be
slightly more effective and is used basically within the confines of the laboratory
environment.
Published alcohol effectiveness or results of alcohol effectiveness are presented

when sprayed to the surface and allowed to air dry. This should not be confused in any
way with the product's effectiveness if used in a saturated wipe. A saturated wipe
contains a limited amount of the chemical agent. When used its dry times are
significantly less, and thus, its destructive power substandard to that of the liquid itself
on the surface. While the mechanism of wiping destroys cells in its action, the dry time
of the alcohol is significantly faster.
Isopropyl alcohol wipes carry few if any claims. Normally, they are used to wipe
a surface in a cleaning operation (IPA wipe down) to remove existent disinfectant
residues. Here also such products pose a problem. As they are saturated with isopropyl
alcohol, their ability to soak up residues and clean the surface is minimal. Most of the
time these products just move around the contamination of the surface. A superior
methodology would be the use of an isopropyl alcohol and a dry wipe. Like cleaning
a window, we would spray the solution to the surface and wipe. This will remove the
majority of contaminants on the surface. This can be proven in a home experiment by
using a saturated wipe to clean a window. When completed, the window will have
streaks or swirls on it, and look as though the dirt was just moved around. If one were
to conduct the same experiment with a dry wipe and spray isopropyl alcohol, the
surface would appear much cleaner. The soaking of the liquid with contaminants in
suspension from the surface into a dry wipe is a superior methodology. During
isopropyl alcohol wipe downs of non-product contact but critical surfaces, we need to
employ the cleaner of the two methodologies. Rendering of the products as sterile is a
must prior to use in a Class 100 (Grade A and B, ISO 5) and adjacent Class 10,000
(Grade C, ISO 7) areas. Sterilization of disinfection agents is discussed in depth in the
section on Sterility of Disinfecting Agents.
PHENOLS
Phenolics have been used for years as a disinfecting agent. Phenolics are effective
against gram-positive and gram-negative organisms. However, phenols exhibit better
antimicrobial effectiveness against gram-positive organisms than they do against
gram-negative organisms. They have limited activity against fungi and certain virus
strains such as HIV-1 (AIDS virus) and Herpes Simplex, Type 2. Phenolics normally
are available in an alkaline and acid base version. The theory surrounding the rotation
of these two compounds is described later in this section of this chapter. Overall,
phenolics demonstrate superior antimicrobial effectiveness in an acidic base as
opposed to an alkaline base. Some of the most common chemical compounds in
phenolic germicidal detergents are in a low pH phenolic: an ortho-phenyl-phenol and
ortho-benzyl-benzyl-para-chlorophenol and in a high pH phenolic: a sodium ortho-
benzyl-para-chlorophenate, sodium ortho-phenylphenate, or sodium para-tertiary-
amylphenate. While phenols provide good broad-spectrum disinfection they are not
sporicidal and have major drawbacks in their use. One drawback is the horrific
residues that are noticed from long-term use of the products.
Phenolics are normally an amber (light tan) or light tan color when manufactured.
This color darkens with age and exposure to light (especially fluorescent). Residues
start as a “dripping droplet” that is not easily removed. The use of 70% isopropyl
alcohol or certain residue removers can remove such residues in their early existence
on surfaces. While somewhat effective, both residue-cleaning products eventually give
way to the darkening phenolic that stains the surface with a dark brown color. Transfer
of such residues to an unwanted location is a concern and precautionary measures need
to be implemented to ensure minimization of this scenario. Compatibility with most
chemicals is normally very good, however, the effect of anionic characteristic of the
chemical in relation to applications in conjunction with a cationic surfactants such as
a quarternary ammonium have been reported as problematic. Expiration of a
formulated phenol also carries some concern. The formulation in a closed container
should remain stable for a period of 7–30 days (dependent upon storage). Formulated
solutions should be marked accordingly. The normality in the industry is 7 days, and
less if the solution is in an open container (such as a bucket) and should be discarded
each day. Rendering of these products as sterile is a must prior to use in a Class 100
(Grade A and B, ISO 5) and adjacent Class 10,000 (Grade C, ISO 7) areas.
Sterilization of disinfection agents is discussed in depth in the section on Sterility of
Disinfecting Agents.
QUARTERNARY AMMONIUM COMPOUNDS
Quarternary ammonium products are used in more disinfection applications than

phenolic germicidal detergents. Their spectrum of use is very broad and ranges
throughout the industrial world, through the hospital and institutional setting and even
can be found in home use. Quarternary ammoniums have excellent detergency. They
are one of the best cleaners among the spectrum of disinfecting agents. These cationic
solutions also have excellent deodorizing capabilities. Quarternary ammonium
compounds are effective against gram-positive and gram-negative organisms.
However, phenols exhibit better antimicrobial effectiveness against gram-positive
organisms than they do against gram-negative organisms. They have limited activity
against fungi and certain virus strains such as HIV-1 (AIDS virus) and Herpes
Simplex, Type 2. In fact and due to competition in this arena, quarternary ammonium
compounds have the most organisms registered as label claims with the US EPA. Their
mechanism of antimicrobial effectiveness is related to their positively charged
molecule. Simply, the positively charged molecule attracts to the negatively charged
microorganism's cell wall. The cycle of kill is complete when the chemical agent is
absorbed into the cell and spread throughout the organism. Quarternary ammoniums
come are available in both alkaline and acid based compounds. Quaternary ammonium
compounds normally utilize an alkyl dimethyl benzyl ammonium chloride or a
dimethyl ethyl benzyl ammonium chloride. While a multitude of formulations exist in
the industry, these are two of the most popular components. As with phenols,
quaternary ammoniums do leave sticky residues that become problematic over time.
However, they are not of the scale of phenolic residues. Expiration of a formulated
(from concentrate) quarternary ammonium also carries some concern. The formulation
in a closed container is relatively stable and should remain stable for a 30-day period
in a closed container. Formulated solutions should be marked accordingly. The
normality in the industry is 7–30 days and less if the solution is in an open container
(such as a bucket) and should be discarded each day. However, in recent years,
quarternary ammoniums have been made in ready-to-use formulas that are very stable.
Rendering of these products as sterile is a must prior to use in a Class 100 (Grade A
and B, ISO 5) and adjacent Class 10,000 (Grade C, ISO 7) areas. Sterilization of
disinfection agents is discussed in depth in this chapter in section, Sterility of
Disinfecting Agents.
SODIUM HYPOCHLORITE
Sodium Hypochlorite solutions are one of the oldest known disinfectants and
14
sporicidal agents . The product is available in many forms including ready to use
0.25% and 0,52% solution, to concentrate 5.25% and 10% solutions, to powders that
are mixed with water to formulate a variety of solutions. Bleach as we know it is
normally found in a 5.25% concentration, however, in recent years such formulations
from the Clorox Corporation® have been increase to a near 7% solution to ensure
continued stability of the active percentage.
One of the main problems with the use of sodium hypochlorite is the industry is
that it is used at too strong an active percentage. Sodium hypochlorite is used
throughout the heath care setting and normally diluted to concentrations of 0.25 or
0.52%. One of the problems with sodium hypochlorite formulations is the method of
formulation designation which varies from firm to firm. Some formulate to parts per
million, some to a percentage of a solution and some to a dilution such as 1–10. At a
use-dilution of 0.25% (or a 1-20 dilution or 250 ppm) sodium hypochlorite is effective
against gram positive and gram negative organisms, viruses, fungi and bacterial
endospores. At a slightly increase use-dilution of 0.52% (or a 1-10 dilution or 500
ppm) sodium hypochlorite is effective against gram positive and gram negative
organisms, viruses, fungi and more effective against a wider range of bacterial
endospores. Both formulations are used throughout the pharmaceutical and
biotechnology industry.
In formulation of a sodium hypochlorite solution, many choose to acidify the

solution, which makes it a more potent mixture when focusing on bacterial
endospores. However, acidification causes rapid degradation of the active elements in
the solution and use of the product is limited to approximately two hours. In reality,
acidification may not be necessary as the product demonstrates excellent sporicidal
characteristics in its neutral state and bacterial endospore levels in clean rooms are not
exuberant in numbers (above 1.0 x 106 ) and there is no soil load. A formulation of
250 or 500 ppm has excellent sporicidal activity. However, expiration of the solution
can become a problem. A normal 5.25% concentrate sodium hypochlorite normally
carries a one-year expiration for an unopened container. Some companies have
validated and increased this expiration with applicable assay data over time to 18
months. Once opened, whether in a ready to use formula or a concentrate product, the
product needs to be used within a 30-day period. Open containers (such as buckets,
and open bottles) have a shorter expiration as the chlorine in the solution begins to
burn off leaving only the sodium chloride. Open containers should be formulated and
used within the same day.
Some drawbacks with sodium hypochlorite focus mainly on the residue and
corrosiveness of the product. As previously stated, the chlorine in the solution begins
to burn off leaving only the sodium chloride. This white crystal-like residue attacks the
impurities in stainless steel (as an example) over a longer time frame. When using a
sodium hypochlorite solution it is imperative to remove the sodium chloride residues
frequently to minimize this corrosive action. Rendering of these products as sterile is
a “must” prior to use in a Class 100 (Grade A and B, ISO 5) and adjacent Class 10,000
(Grade C, ISO 7) areas. Sterilization of disinfection agents is discussed in depth in the
section, Sterility of Disinfecting Agents.
Hydrogen Peroxide
Hydrogen peroxide is one of the most common disinfectants in the industrial
marketplace. In hospital and consumer use at a 3% solution the product is commonly
used as an antiseptic. In the pharmaceutical and biotechnology industry, hydrogen
peroxide is used at 35% as a sterilant in isolators and at 3–10% for surface
disinfection. Depending upon the concentration used, hydrogen peroxide is effective
against bacteria, yeasts, viruses, and bacterial spores. Destruction of spores is greatly
15
increased both with a rise in temperature and increase in concentration . Hydrogen
peroxide is a clear, colorless liquid that is environmentally friendly as it can rapidly
degrade into water and oxygen. While generally stable, most hydrogen peroxide
formulations contain a preservative to prevent decomposition. At lower concentrations
(3–10%) the chemical is effective against gram-positive and gram-negative bacteria,
viruses and bacterial endospores in lower enumerations. However, at higher
concentrations and longer contact times the product exhibits superior sporicial
reduction of bacterial spores. Some of the positive features of the product are its mild
(if any) odor and its low residue characteristics. However, the product also has some
setbacks in exceeding OSHA exposure limits if used in confinement in too large a
quantity. Precautions should be vested in this arena. Rendering of these products as
sterile is a must prior to use in a Class 100 (Grade A and B, ISO 5) and adjacent Class
10,000 (Grade C, ISO 7) areas. Sterilization of disinfection agents is discussed in
depth in this chapter in section, Sterility of Disinfecting Agents.
]PERACETIC ACID AND HYDROGEN PEROXIDE

Peracetic acid and hydrogen peroxide mixtures have received much recent attention in
the pharmaceutical and biotechnology industry in recent years. The chemical is
considered a more potent biocide than hydrogen peroxide being bactericidal, virucidal,
16
fungicidal and sporicidal at very low concentrations (<0.3%) . The product as
originally designed for the sterilization of medial devices. The chemical destroys the
cell by destroying vital membrane lipids, DNA and denatures proteins and enzymes.
Peracetic acid and hydrogen peroxide mixtures decompose to acetic acid and oxygen.
Active percentages of marketed products range from 0.3% to 1.3% as a sterilant in
both ready to use and concentrate solutions. Ready to use solutions require a very high
level of acetic acid as a stabilizer nearing 5.2%. While concentrate products need
smaller amounts of acetic acid in the formulation. Concentrate solutions incorporate
approximately 8% acetic acid and upon formulation to a use dilution, this value drops
to near 0.4%.
One of the main misconceptions with the use of peracetic acid and hydrogen
peroxide mixtures as well as most registered sporicides is that the product needs to
be used at the sterilant label claim active percentage. First we must understand the
requirements set for by the US EPA. The US EPA requires all registrants making
label claims to do so by following test methods outlined in by AOAC protocol. The
sterilant claim on products/labels follows the AOAC sporicidal test. This test
requires the complete reduction of 106 of B. subtilis and C. cporogones in a 60-
carrier test, at 20°C, in a soil load. In the clean room, bioburden is significantly
lower. Thus, the active percentage needed to destroy the flora normally seen is
significantly less. Simply, registered sterilant label claims are too strong for what is
noticed in a clean room. Coupled with the high enumeration of the AOAC test
parameters is also a soil load, not present in clean rooms. Thus end-users should
look to validate a concentration of peracetic acid and hydrogen peroxide mixtures
as well as other registered sporicides at realistic bioburden values as discussed later
in this chapter in the section entitled Determining Antimicrobial Effectiveness. This
will significantly reduce odors, deterioration of surfaces, problematic user
situations and residues.
Peracetic acid and hydrogen peroxide mixtures have pungent vinegar smell that
is offensive, if not intolerable to many users. Due to the horrific smell and the
characteristic drying of mucosal membranes, peracetic acid and hydrogen peroxide
mixtures have caused dissatisfaction among end-users. Facilities that have used
sodium hypochlorite (bleach) for years find the transition to peracetic acid and
hydrogen peroxide very difficult to handle in terms of worker satisfaction. The
product, if used in a clean room environment that may have 15–20% fresh air, may
easily exceed required levels when industrial hygiene testing is performed. Safety
precautions for end users should be ensured prior to its use.
While reports of the product deem it non-corrosive to metals, industry reports have
shown this product react adversely with most stainless steels, aluminums, plastics,
epoxies and most clean room surfaces. After application, a white cloudy residue is
normally left that requires either an IPA wipe down or a WFI rinse to remove.
GLUTARALDEHYDE
Glutaraldehyde has been used for some time as a disinfectant and sterilant for
endoscopes and surgical equipment. Glutaraldehyde is normally sold in a 2.0%
solution. The product is usually supplied as an amber solution with an acid pH.
Glutaraldehyde is a powerful biocidal agent having the advantage of continued activity
17
in the presence of organic material. Glutaraldehyde has broad spectrum activity
against bacteria, bacterial spores, viruses, and fungi. The mechanism of action
involves the destruction of the outer layers of the cell. Glutaraldehyde is the only
aldehyde to exhibit excellent sporicidal activity. In recent years, glutaraldehyde's use
has been focused mainly on the hospital environment. Many pharmaceutical and
biotechnology organizations do not use a glutaraldehyde product in their operations.
The product is very toxic and specific handling precautions must be employed prior to
its use. Especially noted are the gaseous fumes and the possible absorption through
human tissue (skin).
FORMALDEHYDE
Formaldehyde is widely known as a fumigant for rooms and buildings. It has been
18
shown to be effective against bacteria and bacteria spores and vegetative bacteria .
Acklund et al. (1980) showed that at 20°C and a relative humidity of approximately
100%, a 6-log reduction of B. subtilis spores was obtained after 1.5 hours exposure to
300 µ/L whereas at 250 µg/L only a 4-log reduction was obtained after six hours of
exposure. The mechanism of action of formaldehyde is assumed to be due to the
reaction with cell protein and DNA or RNA (Russell, 1976).
Formaldehyde is normally used in the pharmaceutical and the biotechnology

industry as a means to bring back an area after shut down or major maintenance.
During its implementation very stringent safety precautions are assured for personnel
protection that include areas and building clearance and hold times of areas prior to
release.
While formaldehyde is effective, this chapter focuses on routine methods of

cleaning and disinfection. Formaldehyde does not fit appropriately as a choice in this
venue and would be used as a mechanism in opening of a new area or as a method
when coming back from a shutdown period.
As previously discussed, antimicrobial effectiveness depends on a variety of

factors. The two most notable are the time period afforded to the disinfectant to
saturate and penetrate the cell wall of the organism, and whether the chemical is
capable of killing the organism in question. The key to selection is answered in one's
list of environmental isolates. The decision is based on the correlation between the
organisms on the list versus the chemical agent that can be proven efficacious against
such organisms. Process constraints may also affect one's choice of disinfectants, as
some may be harmful to final product or incompatible with clean room surfaces.
Varying applications require various solutions to be in place. he choice to use a phenol,
quaternary ammonium or hydrogen peroxide based solution on a daily basis (in a Class
100, ISO 5, Grade A or B area) is normality for the pharmaceutical and biotechnology
industry. The choice delineates the rotation parameters. The choice of one disinfectant
and a sporicide is completely appropriate; however, some may decide to rotate similar
disinfectants while also utilizing a sporicide.
Rotation systems are designed to address known or possibly existent

contamination with proven efficacious disinfectant. The basis for the rotation of
disinfecting or sporicidal agents is to address an organism that may not be destroyed
by a particular disinfectant with another that has proven efficacy performance against
such organism. An example would be a phenol may not kill a b.subtilis spore in a 5–10
minute contact time and thus, the rotation to a more efficacious product such as
sodium hypochloride at 0.52% (a sporicide) may be warranted to destroy this
organism.
However, organisms do not develop and immunity or resistance to a chemical

agent over time. This theory is exactly that, a theory that has never been proven in the
clean room environment. This confusion arises by the multiple meaning of the word
“resistance”. Resistance is used to describe "not destroyed by" and incorrectly used to
describe "the building of an immunity by the organism to the chemical agent". The
theory was originally borrowed from the medical industry where it was applicable to
noticed resistance in the human body. Subsequently and without proven, published
data, such theory was implemented into the clean room industry. However, antibiotic
resistant microorganisms are susceptible (or killed) to chemical germicides. The
mechanisms by which chemical germicides and antibiotics work are completely
different and there does not seem to be a relationship between antibiotic resistance and
chemical germicide effectiveness. We need to also realize that “natural selection” is a
slow process that requires high populations of cells, constant selective pressure and a
rich growth environment. These are not found in the clean room environment.
Resistance to antibiotics is usually acquired through modification of a single gene (or
acquisition of a single gene) that blocks the very specific action of the antibiotic. The
antimicrobials we deal with do not act on a single enzyme, but have numerous effects
on almost every aspect of cellular physiology. This meaning that multiple mutations
would be required to overcome their detrimental effects. Again, this is not likely, given
the environmental conditions and low cell numbers within the clean room
19
environment . Therefore, the basis for rotation is to address an organism that is not
destroyed by, nor ever was destroyed by, one chemical agent with another that has
proven efficacy performance against such organism. And, the basis for implementing
a rotation system may not be to address the building of immunity by an organism to a
chemical germicide.
On the contrary, one such study that reported supported the resistance theory was
published in article form in 1992, entitled Rotation of Phenolic Disinfectants (Connor
20
and Eckman ). In this paper they examined whether it was possible for a Pseudomonas
6
aeruginosa (ATCC 15442) at a population of cells equal to 10 CFU/ml. could develop
a resistance to a phenolic germicidal detergent in a laboratory experiment. From the
paper: "The testing method consisted of treating P. aeruginosa on agar surfaces,
subculturing (transferring) survival cells, and then repeating the process. A total of 40
transfers per treatment per trial and two trials ere performed. Data indicated that the P.
aeruginosa (or resistant sub populations) became adapt or resistant to the alkaline
phenolic disinfectant. However, when this compound was applied to the culture on a
rotational basis with the acid phenolic disinfectant during the same time period, no
adaptation or selection occurred."
However, this test report was conducted in a laboratory setting. The published
article attempted to prove that if such an occurrence existed that the "resistant
microorganism" would be destroyed by either varying pH phenolic.
The pharmaceutical and biotechnology industry demands thorough testing in all

aspects of our operations. During this time period, many jumped to what may have
been thought to be some applicable data that should be implemented across the board
with all organisms. But in reality as an industry, we needed to investigate the subject
further. During this time period, the data as it was presented was bastardized
throughout the industry. It turned from "in this test we found that P. aeruginosa was
not completely killed by a low pH phenolic and was subsequently destroyed by a low
pH phenolic" to "all organisms can develop a resistance to disinfectants". This was not
factually proven. And if it were proven so, the industry would have a horrific problem.
If organisms could develop a resistance to disinfectants at the low levels that they are
seen in the clean room, the first question that would scientifically be asked is "when
does this occur?" As this question could not be answered, the industry would be forced
into reproducing antimicrobial effectiveness testing on a routine basis. An
subsequently, such testing would be done for an unscientific reason that would burden
firms with an extremely high overhead cost. The average time contact kill study may
cost a firm from $1,500 to $2,000 per test of one organism against one disinfecting
agent (in solution or on carrier surfaces). Added to the cost of routinely reproducing
antimicrobial effectiveness would be the question of how often should this data be
reproduced. Simply an unanswered question that would force firms into a greater
frequency as time continued. The Conner and Eckman article tested one organism, P.
aeruginosa. Obviously the scale of flora in the clean room setting would be much
broader. A multitude of microorganisms would be noticed in normal clean room
operation. For the industry to base all disinfection systems on the testing of one
organism would represent an incomplete analysis of pertinent data surrounding this
subject. Thus, coupled with the question of how frequently should we test would be
the question, "What organisms do we test?" In completeness, if one believed that an
organism could become resistant to the disinfectant used, all environmental isolates
could be suspect. Would one species of Staphylococcus be affected like another? All
of these questions could be pushed to the foreground when one utilizes an unproven
beginning basis as our starting point. Simply stated, conclusive evidence that relates to
our specific scenario should be the basis for us determining our starting ground.
In the above paragraph, we discussed the possible need to reproduce antimicrobial

effectiveness on a routine basis if we believed the theory of resistance. However, if we
believe in resistance then the list of questions continues. If we use a disinfecting agent
that an organism may become resistant to in this month, and one that it is not resistant
to the next month, are we not destroying the cell during the first month time period?
Could it compromise our product?
Further, if we do not rotate a high pH phenolic with a low pH phenolic, what do

we rotate with a quarternary ammonium? We cannot rotate a quarternary ammonium
monthly with a phenol as they are not compatible and create a sticky residue on the
surface. Likewise, what do we rotate other chemical compounds with? Rotation of two
disinfecting agents may limit us to a phenol rotation that may or may not be the best
choice to choose.
The rotation of disinfecting agents to combat microbial resistance incorporates yet

another stumbling block when we review the subject of a routine sporicidal application
to our controlled environments. The rotation of two disinfectants does not address the
possible content of spore populations in our clean room environments. While some
disinfectants may have a very small log reduction of spores, we will still incorporate,
on a frequency, a sporicidal agent into our disinfection regime. Thus, how will this
resistant microorganism that was not killed by one disinfectant but destroyed by
another survive through the application of a sporicide? There is no scientific theory or
case study that would support the cell's ability to live through this scenario.
As an industry, the push for the rotation of two disinfecting agents is becoming
less and less supported. We can see this in review of some of the current guidelines
published in the industry such as USP 1072, Disinfectants and Antiseptics which
states: "The development of microbial resistance to antibiotics is a well-described
phenomenon. The development of microbial resistance is less likely, as disinfectants
are more powerful biocidal agents than antibiotics and are applied in high
concentrations against low populations of microorganisms, so the selective pressure
for the development of resistance is less profound."
On September 27, 2002, the FDA released the Sterile Drug Products Produced by
Aseptic Processing Draft. While in the preliminary stages and not for implementation,
the writings make no mention of any concern that an organism may develop a
resistance to a chemical agent over time. However, in several instances the guideline
does mention that one should continually evaluate their systems. While this document
may change, we should consider the basis for their thought process. An excerpt from
the guideline is as follows.
Sanitization Efficacy
"The suitability, efficacy, and limitations of sanitization agents should be
assessed with their implementation for use in clean areas. The
effectiveness of these sanitization procedures should be measured by
their ability to ensure that potential contaminants are adequately removed
from surfaces (i.e., via obtaining samples before and after sanitization)."
"Upon preparation, disinfectants should be rendered sterile, and used for

a limited time, as specified by written procedures. Disinfectants should
retain efficacy against the normal microbial flora and be effective against
spore-forming microorganisms. Many common sanitizers are ineffective
against spores, for example, 70% isopropyl alcohol is not effective
against Bacillus, spp. spores. A sporicidal agent should be used regularly
to prevent contamination of the manufacturing environment with
otherwise difficult to eradicate spore forming bacteria or fungi."
"After the initial assessment of sanitization procedures, ongoing

sanitization efficacy should be frequently monitored through specific
provisions in the environmental monitoring program, with a defined
course of action in the event samples are found to exceed limits."
Resistance of microorganisms to disinfecting agents is a subject that is based on

the theory that it could occur. Rarely in our internal scientific evaluations and our
presentation of our validation to regulatory inspectors do we use the justification that
"in theory, we think it could occur." The use of theory may guide us to proof, but never
is a replacement for proof.
If we define rotation of chemical agents as the "desire to assure a more broad-

spectrum kill in our disinfecting regime" then we have identified the key element for
the reason why we would scientifically consider this subject. Rotation is an applicable
and a scientifically proven term. However, the rotation of two disinfectants is not
applicable to address microorganisms we may see. Destroying contamination in a
clean room operation requires addressing the known level of vegetative cells and the
known level of spores. In design of a rotation system, there are three types:
• a single disinfectant rotated with a sporicide (Table 2)
• a two disinfectant system (rotated monthly) plus a sporicide (Table 3)
• the use of a sporicide alone (Table 4)
A few suggested rotations of the chemical agents with frequency are presented
here for use in a Class 100 and the adjacent Class 10,000 environment.
Table 2: Rotating One Disinfectant and a Sporicide
DAY(S) Phenolic Quaternary Hydrogen

Ammonium Peroxide
Day 1–13 Low pH Phenol Quaternary Hydrogen
Ammonium Peroxide
Day 14 (if warranted Clean the surfaces, Clean the surfaces, Clean the surfaces,
by EM data) allow to dry, then allow to dry, then allow to dry, then
apply the Sporicide apply the Sporicide apply the Sporicide
Day 15–29 Low pH Phenol Quaternary Hydrogen
Ammonium Peroxide
Day 30 Clean the surfaces, Clean the surfaces, Clean the surfaces,
allow to dry, then allow to dry, then allow to dry, then
After disinfection, when all surfaces are dry, all critical surfaces should be rinsed with hot
WFI or an IPA wipe down performed.
Table 3: Rotating Two Disinfectants and a Sporicide
DAY(S) Phenolic Quaternary Hydrogen

Ammonium Peroxide
Day 1–13 High pH Phenol Quaternary Hydrogen
Ammonium Peroxide
Day 15–29 Low pH Phenol Hydrogen Quaternary
Peroxide Ammonium
Table 4: Use of a Sporicide
DAY(S) Sodium Hypochlorite Peracetic Acid and Hydrogen

at 0.52% Hydrogen Peroxide Peroxide
Day 1–13 Sodium Hypochlorite Peracetic Acid and Hydrogen
at 0.52% Hydrogen Peroxide Peroxide 6%
Day 15-29 Sodium Hypochlorite Peracetic Acid and Hydrogen
at 0.52% Hydrogen Peroxide Peroxide 6%
The first two systems (Tables 2 and 3) require, at minimum, a monthly sporicidal
application. This may be increased or decrease in time frames and will be determined
by the environmental conditions. Industry standards look to a weekly application at the
beginning and such application becoming lessened as control of the environment is
consistently attained. The third uses a sporicide daily and is the most effective but is not
recommended, as it is overkill for the known contamination level and will deteriorate
surfaces over time. Within our cleaning and disinfection regime, we also need to
address the cleaning aspect. As we have discussed previously, cleaning and disinfecting
are not the same. The use of cleaner is considered an optional step in controlling
existent residues and should be done at least once a month. After disinfection all critical
surfaces should be rinsed with hot WFI or an IPA wipe down performed. These subjects
will be completely discussed later in this chapter in the section entitled, Assuring
Application of Our Disinfectants and Sporicides.
DETERMINING ANTIMICROBIAL EFFECTIVENESS
Determining what chemical agents will destroy a known level of one's environmental
isolates or ATCC cultures is the next step. Prior to conducting either a Time Contact Kill
Study (Tube Dilution), or a Time Contact Kill Study (On User Surfaces) or an AOAC
Protocol Study, one needs to review the available disinfecting agents and determine
which is initially appropriate for their operations. Upon choosing 1 or 2 disinfecting
agents and a sporicide, one can continue with the antimicrobial effectiveness studies.
Validating one's sanitizing, disinfecting and sporicidal agents requires them to

delineate the organisms to be tested. One could use a list of ATCC cultures, however,
utilization of one's environmental isolates nets a more exacting test for each unique
manufacturing operations. Testing ATCC cultures such as Bacillus subtilis, Aspergillus
niger, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and
Escherichia coli is acceptable, however not as exacting as testing a plane of one's
known environmental isolates. The use of one's environmental isolates in a preferred
methodology by most regulatory agencies.
Antimicrobial effectiveness studies need to be based on realistic bioburdens that

may be noticed in the controlled areas. Normality is to test an enumeration greater
4
than or equal to 1.0 x 10 CFUs. Our goal is to prove a 3-log reduction. However,
21
some guidelines like the British Standard BS EN 13697:2001 calls for a 4-log
reduction as proof.
In determining which test to conduct, one needs to review how one will address
an organism in the clean room. As the organism will be on the surface, a time contact
kill study that confirms the destruction of a known enumeration of cells on an end-
user's surface is more depictive than a time contact kill study done in a tube dilution
(in suspension). The reasoning for this is organisms dried on a surface better depict the
situation of disinfecting an organism in the clean room. The available surface area of
the organism that can be contacted by the disinfectant that rests on the surface is 270°.
The available surface area of the organism that can be contacted by the disinfectant in
the tube dilution study is 360°. Obviously, the surface test presents a more realistic
scenario. AOAC protocol testing is required by the US Environmental Protection
Agency (EPA) to register a claim for a disinfecting agent. It utilizes 60 carriers (a
ceramic penicylinders) and requires a high enumeration value of equal to or greater
6
than 1.0 x 10 . Protocols use either AOAC Use-Dilution or AOAC Sporicidal tests
procedures. While this is the method used for registration, it may be too involved and
expensive for pharmaceutical and biotechnology firms to utilize as a method for
testing antimicrobial effectiveness.
The EPA supports the use carrier methods for the evaluation of a disinfectant
product's efficacy. This test requires the microorganism is to be dried on a non-porous
carrier. The rationales behind the choice of a carrier method are the beliefs that:
• microorganisms that are dried are more difficult to chemically inactivate than
those microorganisms in suspension
• that in the health care setting microorganisms are more often found in the dried
22
state than in suspension
Within the framework of antimicrobial effectiveness testing, we also need to

incorporate realistic contact times to depict representative dry times of the disinfectant
on clean room surfaces. Due to the significant movement of air from laminar flow in
the clean room, normal dry time is 5 minutes at best. While our floors may remain
wetted longer (possibly up to 10 minutes), the vertical surfaces will dry faster (3–5
minutes). Thus antimicrobial effectiveness testing should incorporate a worse case
scenario and utilize a 3–5 minute dry time. An exception to this rule would be
sanitizing agents such as isopropyl alcohol, ethyl alcohol or AAA ethanol at a
concentration of 70%. These products dry faster nearing 1–2 minutes and testing
should be altered as such. In recent years, many firms have begun to test three time
frames to prove the disinfectant's activity over varying time periods. The author
supports this practice and would suggest the time period of 3, 5 and 10 minutes be
tested consecutively. Normally in 3 minutes the disinfectant shows average activity, in
5 minutes good activity, and in 10 minutes excellent activity. Testing of a variety of
times ensures one has data in the file to support a complete range of dry times (contact
times) that may be noticed in their manufacturing or testing operations. Upon
completion, this testing will provide the justification for utilizing the chemical agents
to destroy the known and possibly existent contamination in the facility. As time
progresses, we need to continually updating our profile of organisms versus our
chemical agents.
While the type of test, the enumeration level of microorganisms, and the contact
time are some of the most critical factors to assess, other critical factors also need to
be determined. One of these is expiration of the disinfectant. Expiration for
effectiveness can be determined by incorporating a simple variable in our test called
aging of the disinfecting agent. Simply done, one would open a bottle of disinfectant
and age it for the time period that they plan to use it, say 30 days (concentrate and
ready to use). A ready to use solution is tested at the 30-day period. For a concentrate,
the aged concentrate solution is diluted to the prescribed use-dilution. The use-dilution
is then aged for the time period that it will be used (for example, for 7 days). At the
expiration of the use-dilution it is then tested for antimicrobial effectiveness. This
system of expiration proves that a solution, in use for “X” time period has the ability
to destroy an acceptable level of microorganisms that we have determined to be
present in our operations.
Expiration dating of disinfectant effectiveness can also be tested by conducting an

antimicrobial effectiveness test on a newly open bottle of disinfectant (ready to use or
concentrate to use dilution) and then subsequently aging the solution for the expiry
period and testing the active ingredients. The correlation between the active
ingredients at time of opening and their satisfactory stability at end of expiry provides
the needed data to support the use of the agent for the time period. However, in this
test, there must be the understanding and explanation of the relationship between the
tested solution and the expiry active data that followed.
In performing antimicrobial effectives testing some other factors come to the fore
that warrant attention. The first is soil load. Normally, clean room operations do not
have a soil load present and the most soil that exists would be that of disinfectant
residues. In the case of an existing soil load, one should conduct their testing in a
similar situation. Commonly used as a soil load or an increase level of protein (a fetal
bovine serum) at 5% v/v is added to the organism challenge to test the ability of the
disinfectant or sporicide under the circumstances of a soil load (dirtied) condition.
Another factor that may surface is hardness and temperature of water. Required
by all registrants of disinfecting agents must state in their labeling their ability or
inability to achieve antimicrobial effectiveness in the presence of hard water (400 ppm
as CaCO3). At the same time the temperature of the solution may vary in its effect
against microorganisms as elevated temperatures tend to increase the ability of the
chemical agents performance.
While disinfectant validations are expensive and time consuming they are
foremost in most regulatory agency's minds. And thus, are a required to assuring the
effectiveness of a cleaning and disinfection system that will prove an acceptable
environment during the manufacture of product.
ASSAY OF DISINFECTING AGENTS
Analytical validation of our disinfecting agents tests that the required percentage of
the chemical agent is present to ensure antimicrobial effectiveness. If the appropriate
use instructions are followed, a ready-to-use product or a formulation from
concentrate is normally easy to prove as having a sufficient amount of the active
ingredients to reconfirm the required percentage that was validated in our
antimicrobial effectiveness studies. Upon the use of this product past the first use is
where we start to see the possibility that the percentage of the active ingredients may
begin to slowly become too low to warrant continued use. Varying products have
varying in-use time periods. Time periods range from 7–30 days. This scenario needs
to be validated for each chemical agent and each container type. As an example of the
same chemical having varying in-use time periods, an isopropyl alcohol solution in
an aerosol for can be used for a longer time period than a trigger spray bottle. The
reasoning for this difference is the aerosol container is sealed in a pressure vessel.
The reduction in percentage of the active ingredient due to evaporation or the
question of sterility over time is not founded in this container type. On the other hand,
the trigger spray container may slowly aspirate room air to the master reservoir that
may compromise the level of active ingredient and the sterility of the container over
time. The basic validation question is "what is the time frame that we can prove the
chemical's active percentage and sterility to be valid for, once opened?"
STERILITY OF DISINFECTING AGENTS

Through our antimicrobial effectiveness studies, we realize that disinfectants do not
kill all organisms. As all chemical agents may have an inherent bioburden (normally
spores), we must ensure that such bioburden is removed prior to their entry to our
controlled areas. The transfer of such organisms through our disinfectants to our
controlled areas, especially our aseptic filling areas, should be viewed as a catastrophic
event. To even further reinforce the issue, we spread the disinfecting agents all over
our walls, ceiling and floors. Controlling the contamination from ever entering is much
easier that subsequently removing it from the controlled area. If we review regulatory
expectations in this area we will find the requirement to sterilize all disinfectants and
sporicides prior to entry to the controlled environment.
The FDA has stated in its Sterile Drug Products Produced by Aseptic Processing
Draft that, "Upon preparation, disinfectants should be rendered sterile, and used for a
limited time, as specified by written procedures."
Purchasing a disinfectant or sporicidal product as sterile from an audited vendor

requires one to review the following critical items as a quality control measure to
assure what one is using is sterile prior to use:
• Assessment of the bioburden of the solution
• Assessment of the bioburden of the container that the solution is to be filled into
• Pre-washing of containers (cleanliness level)
• Requires a filter validation proving microorganisms are retained by the filter
• Assay of the solution (RTU or concentrate) to an acceptable active percentage
• Filtering the solution at 0.2 microns
• Aseptically filling the product into pre-sterilized containers or exposing the entire
contents to a terminal sterilization process such as gamma irradiation.
• Requires a lot-by-lot sterility test per current USP or EP compendium (conducting

sterility testing requires the completion of bacteriostasis and fungistasis (B/F
testing. This proves that the sterility test is capable of growing organisms in the
presence of the chemical agent.)
• Requires the assessment for expiration dating of an unopened container

If the disinfectant or sporicide is to be processed sterile in-house, then the review

of the following critical items as a quality control measure is suggested to assure what
one is using is sterile prior to use.
• Assessment of the bioburden of the solution
• Assessment of the bioburden of the container that the solution is to be filled into
• Pre-washing of containers (cleanliness level)
• Requires a filter validation providing microorganisms are retained by the filter
• Assay of the solution (RTU or concentrate) to an acceptable active percentage
• Filtering the solution at 0.2 microns
• Aseptically filling the product into pre-sterilized containers or exposing the entire
contents to a terminal sterilization process such as gamma irradiation.
• If the product is aseptically filtered, then the assurance that all that comes in
contact with the product after the filter is rendered sterile.
• Requires validation to be performed using a lot by lot sterility test per current USP
or EP compendium or a bioburden analysis for at least three lots at the beginning
and subsequently routinely tested as a quality control check. Sterility testing on a
lot-by-lot basis need not be performed if sufficient validation testing is conducted.
• Conducting sterility testing requires the completion of bacteriostasis and

fungistasis (B/F) testing. This proves that the sterility test is capable of growing
organisms in the presence of the chemical agent.
• Requires the assessment for expiration dating of an unopened container
Validation of a sterility claim for disinfectants should be a focus of one’s

validation efforts. Normally the testing of three processed lots for sterility provides
sufficient data. The pre-sterilization of our chemical agents prior to entry to the
controlled area is simple common sense and common practice in the industry.
Container type is critical when using a sterile disinfectant. Container types

include aerosol, trigger spray, squeeze bottle, and larger closed containers (1–5 gallon
and larger). Aerosol container is the most lucrative type, as the vessel does not aspirate
the room air to the master reservoir. However, the container and its contents must be
sterilized via gamma radiation or all the components pre-sterilized and subsequently
filled via a validated aseptic filling operation. For obvious reasons, gamma
sterilization of the entire contents is considered far superior methodology for

achieving a sterile product. In 1992, the first sterile disinfectant, DECON-AHOL® (a
23
sterile USP IPA), was marketed under US patent 6,123,900, Method of Sterilization .
This patent and product showed the industry the effectiveness of this type of container.
Thus, no viable or particulate contamination is returned to the solution contained. A
pre-sterilized aerosol or pressurized vessel assures assay of the active ingredients (for
the time period they remain stable) and sterility for the expiration period designated by
the manufacturer.
Other smaller containers include the squeeze bottle and trigger spray bottles.
While acceptable containers for all disinfectants and sporicides, the container itself
aspirates the room air back to the master reservoir. Thus, assay and sterility are
compromised after the initial use. The same is true for larger 1–5 gallon containers and
larger. Once opened sterility is compromised.
For varying disinfecting and sporicidal agents a variety of containers need to be

utilized. For ready to use mixtures we have to decide how the product will be used.
If in a smaller aerosol, trigger spray or squeeze bottle, we may want to utilize a
product that is pre-sterilized in this smaller form rather than attempting to pour or
filter such solutions to a empty pre-sterilized container. We may also want to limit
that the capping of product “to be used later” as our assay and sterility may be
compromised over time. For concentrate products that need to be diluted with a
quality water grade, we may want to look to implement unit dose bottles that are
incorporate a pre-measured dose and are sterile. This system is superior to that of
pouring a concentrate disinfectant or sporicide into a measuring cup and capping the
remainder for later use. Questions may arise as to the assay and sterility of this
remaining solution over time.
Thus, no viable or particulate contamination is returned to the solution contained.

A pre-sterilized aerosol or pressurized vessel assures assay of the active ingredients
(for the time period they remain stable) and sterility for the expiration period
designated by the manufacturer.
TYPES AND STERILITY OF CLEANING COMPONENTS
The sterility of disinfectants and sporicides is essential to assure the non-transfer of

organisms to the Class 100–10,000 areas. Of equal importance is the transfer of
organisms to the Class 100–10,000 areas via our cleaning components. Our cleaning
components are defines as spray-mop-fog pressure devices, buckets, mops sprayers,
mop handles, mop frames, mop heads and other components used during the cleaning
and disinfection operation.
In review of this subject we see problematic quality control situations that may
arise from these items not being rendered sterile. At the same time, manufacturing
operational difficulties may exist in implementing these items as sterile.
Let's review the quality assurance side of the subject: the utilization of a sterile
disinfectant that is diluted in a sterile quality water grade (such as sterile water for
injection) and that is subsequently poured into a non-sterile bucket compromises our
solution's sterility and our environment. Moreover, if we then dip a non-sterile mop
head into the solution, we again corrupt our solution and possibly the surfaces that we
touch with the mop head. If our handles and mop frames are not sterile they too can
bring in unwanted contamination to the area. A typical quality assurance line may be
"You need to sterilize each component prior to use to reduce the ingress of
contamination." However, quality assurance may not be completely abreast of what it
will take to accomplish this scenario.
On the production side of the subject if we utilize sterile components we may

possibly need to purchase two to three times the numbers we presently have (due to
sterilization schedules) which is very costly. Production will have to transfer the
wrapped sterile components to the controlled areas, which can be awkward, and will
be required to decontaminate the exterior of the wrappings prior to entry to the Class
100–10,000 area. Dependent upon the location of quality water that will be used to
dilute our concentrate disinfectants, we may need to open such components outside the
controlled area. If this occurs, they will not be sterile any more. Incorporating a
purchased or pre-made sterile water grade available in the area will also add significant
costs. And finally, of the components that enter the area during manufacturing, these
items should be of little consequence, as they will have disinfectants in and on them
during the disinfecting operation. A typical statement from production may be "Well,
we wheel in big tanks during manufacturing and they are not sterile."
Both sides have their valid points. All considerations need to be reviewed. The
goal in the sterilization of the cleaning components is to reduce bioburden to the area.
If this is done, chances are lessened for contamination entering. While not every item
can be sterilized prior to entry to the aseptic manufacturing area, as much as can be
sterilized should be sterilized. Our cleaning components and our disinfectants
represent a common place where we can start anew each day with control. Prior to
sterilization we must review the subject from the end of the cleaning operation and
assure that these items are cleaned prior to sterilization. Otherwise, bioburden on the
items and deterioration to the items will increase over time.
But the real question is frequency. How often do we clean and sterilize these
components? Spray-mop-fog devices, buckets, mop frames and mop handles need to
be cleaned after use. This includes the inside, the outside, underneath and the wheels.
We can then start with a clean component. At this juncture we need to determine where
the component will be taken and stored. If it is stored outside the Class 100 area, then
the item needs to be disinfected or sterilized again prior to entry. Most of the time, this
is the case. While we understand the need to clean the components after use,
disinfection or sterilization prior to entry still looms as a unique question that needs to
be answered by each operation. An important factor in consideration is where the
water will be obtained to create the dilution. Of enormous expense would be the
establishment of new WFI water drop lines to serve this operation. And so the story
encounters more complication.
As an industry smaller firms or firms with only a few areas seem to be able to
sterilize these components before use. As the scale increases to larger operations this
becomes more difficult. As scientists we know that sterilizing the buckets is the
superior methodology and we should make arrangements to move towards such a
scenario. Sterilization of buckets on a weekly, monthly or quarterly basis does very
little to control bioburden to the area on a daily basis and should not be considered. At
the minimum level, we need to clean these devices each day and disinfect these items
prior to entry to a controlled area. Disinfection should be done using the chemical
agent used in the cleaning operation. Science may say that we need to use a sporicidal
on the items prior to entry; however, if a disinfectant is used in, say, the bucket during
that cleaning shift, we may have a mixture of chemical agents. This should be avoided.
Mop handles and mop frames are a different story. They are small enough and
inexpensive enough to have a variety on available, pre-sterilized and used per cleaning
operation. The mop handles and frames should also be cleaned prior to sterilization.
The same is true for mop heads. Pre-sterilized mop heads (foam or string) should be
used and changed daily in a class 100 and 10,000 operation. The frequency of
changing a mop head used in class 100,000 could move forward to weekly, however,
a better scenario is also to use this mop head and then discard. The reasoning for
routine discarding of the mop in Class 100,000 is it is dirtied and is normally air-dried.
Bioburden levels build up on the mop head and may cause potential problems. These
problems may not be worth the cost of the mop head.
IN-USE EXPIRATION OF DISINFECTANTS AND SPORICIDES
Questions surrounding the expiration of an in-use disinfectant and sporicide will be

questioned by all involved internally and externally by regulatory agencies. Several
methodologies were previously discussed in this chapter (in the Antimicrobial
Effectiveness section) as to how to develop this data. The two main questions that arise
are:
• How long does the disinfectant retain its active percentage enabling it to destroy
microorganisms?
Table 5: In-Use Disinfectant Expiration
Approved Concentrate Ready to Use Formulations Formulations in

Disinfectant Bottle Unit Dose in Open Closed Containers
or Sporicide Containers such as Squeeze,
(Buckets and Trigger Spray or
Tanks) closed bottles
Chemical 30 days after 30 days after 1 day after 7 days after
agent opening opening formulation formulation
• How long does the container and the contents held within the container remain
sterile?
Both are very important questions and, from the author's standpoint, two of the
most frequently-asked questions in the industry surrounding disinfection. Simply an
assay over time of use and an accompanying sterility or bioburden test will delineate
this time frame. The importance of reporting this information to cover all potential
situations is critical. As an example, SOP's should look to address the various type of
use situations as depicted in Table 5 and identified as concentrate bottles, ready to use
bottles, unit dose concentrate bottles, open formulations (in tanks, etc.) and closed
containers (such as squeeze bottles, trigger sprays and closed bottles).
CONDUCTING IN SITU FIELD STUDIES
Once a disinfection system has been chosen and antimicrobial effectiveness testing has
been completed, conducting an "in situation field study" is important to prove the
effectiveness of the combination of our cleaning SOPs (standard operating procedures)
and our antimicrobial effectiveness testing. Simply, environmental monitoring, both air
and surface, is conducted in a dirtied room. Upon completion of the monitoring, the
room is cleaned and disinfected per the current operating procedures. Upon completion
and drying of all surfaces, the room is monitored again. Satisfactory results need to be
obtained in three different and separate in situation field studies prior to acceptance of
the disinfection system. This testing is the movement of lab knowledge to the real life
situation. It tests our overall effectiveness in conducting a disinfection operation.
Coordination between the quality assurance group and the production services or
cleaning group is sometimes a very difficult task. However, the magnitude of this
testing should outweigh the coordination of scheduling. As an alternative method,
some organizations have taken cultures enumerated at 10–100 CFUs and inoculated a
similar production surface. Likewise, some have just monitored a dirtied surface
similarly used in production. After inoculation the surface is cleaned using a multitude
of methods that may be employed. Such methods are:
• mopping
• spraying
• wiping
• fogging
The surface is then monitored again after cleaning as a test for effectiveness of the
cleaning operation in conjunction with the disinfecting agents. Inoculation of actual
production services in controlled areas should never be done, as one would never
introduce any microorganisms knowingly to the area that may compromise
manufacturing of product later in the scope. While the in situ or field study conducted
on similar surfaces nets good information, it is still not a test of the actual environment
and the cleaning that will be performed. Thus, it behooves organizations to conduct the
actual in situ or field study in their manufacturing areas.
Regulatory agencies such as the FDA, consider the in situ or field study extremely
important. In an excerpt from the FDA's Sterile Drug Products Produced by Aseptic
18
Processing Draft 9/27/02 guideline is as follows:
Sanitization Efficacy
"The suitability, efficacy, and limitations of sanitization agents should be
assessed with their implementation for use in clean areas. The
effectiveness of these sanitization procedures should be measured by
their ability to ensure that potential contaminants are adequately removed
from surfaces (i.e., via obtaining samples before and after sanitization)."
One concern that is vested throughout the industry from regulatory guidance is
the need to routinely perform this task. In support of the industry, such routine
testing is a revalidation of a validated system. Once the system is in place and the
testing conducted thereof, the system, if performed to accompanying SOPs, should
prove effective each time it is performed. As an industry, the concern needs to be
vested in the absolute assurance that such cleaning and disinfection procedures are
followed on a daily basis. If this is done, the system should satisfy the need for an
acceptable environment. If the system fails, this will be noticed in our
environmental monitoring program and such corrective action must then be taken
on an immediate basis.
The in situ or field study is a realistic test of the ability of our cleaning and
disinfection systems, as a system (chemical and method) to render a tested “dirtied
surface” as free of microorganisms. Our lab work in our antimicrobial effectiveness
testing renders us the understanding of what our disinfectants and sporicides are
capable of destroying in the presence of high bioburden levels. While important, it is
still a laboratory test and never ventures to a real manufacturing setting. The in situ or
field study is a critical test of our systems.
ASSURING APPLICATION OF OUR DISINFECTANTS AND

SPORICIDES
The most important subject is the arena of disinfection is application of the chemical
agent to the surface. As an industry, we do not spend enough time on this extremely
important function. Throughout the industry, we hear tales of this chemical is not
effective against this species of an organism. In fact, we may spend too much time
trying to figure out which chemical agent is most appropriate to destroy all the
possible organisms that we may encounter. At the same time we are focusing on
which chemical kills what organism, we need to assure that the chemical agent is
appropriately applied to the surface. To do so, we need to view more than just a
wetted surface. We need to identify the factors, aside from the chemical agent, that
will assist our disinfection efforts. Putting the basics into perspective can simplify
our understanding of this subject and allow us to refocus our efforts appropriately.
First and foremost, we need to assure the saturation and penetration of the cell wall
over a lengthened contact time. We need to reproduce the contact times from our
antimicrobial effectives testing on all surfaces in the clean room. Second, we need to make
its existence in the area very difficult. One way we can do this is by removing the
organism's required food source, such as particulates and water. This requires routine
cleaning of the area. Another way is to change the temperature it is exposed to (Hot WFI,
if permitted by our safety group and by the characteristics of the disinfectant) or by
assuring it must exist in an undesirable environment. In reality, the undesirable
environment is the clean room itself. Sufficient support conditions for sustaining an
organism's existence are lacking in the clean room environment. In short, it's a difficult
place to survive. Some consider the disinfectant or sporicidal residue as an undesirable
environment. This is true to some degree, and for a short time period. However, it is also
true that a residue on the surface can complicate our manufacturing for a variety of
reasons. This will be discussed in detail later in the chapter. And third, we can remove
organism from the area by our cleaning practices. We can wipe it, mop it or rinse it from
the area; we can also fatally damage the cell wall by an abrasive friction type action as is
common by wiping or mopping. All of these factors will assist our disinfection efforts.
Our design then relies on the cleaning and disinfecting procedures that we have in
place. Our procedures need to include elements such as: the cleaning of a surface, the
saturating of a surface, routine change of dirtied solutions, prior sterilization and
cleaning of our buckets and mop handles, routine changing of our dirtied mop heads,
cleaning and sterilization of our sprayers and foggers, and our procedures relating to
training of our personnel to accomplish these critical tasks.
Application is normally done by four methods: spraying, mopping, wiping and

fogging.
Spray nets the best coverage of a surface and proves to be the best form for
assuring efficacy against microorganisms on the surface as the dry time or contact time
is longer. Simply, the surface remains wetted for a longer time frame. Effective
spraying is done from a light spray and not a power or pressure spray. While pressure
spraying does clean the surface better, the key to assuring saturation and penetration
of the cell wall for the specified contact time is though a light spray with a larger
droplet size. Spraying should be done from the top surfaces to the bottom of the walls.
Spraying should be done by applying to the ceilings first (without contact to filters)
and then be done to the walls. Spraying should not be done on floors and the correct
option would be to apply the chemical via mopping. While spraying is effective in
efficacy performance, it does not clean the surface as well as mopping. Types of spray
devices include canister sprayers, spray-mop-fog devices (Core2Clean®), pressurized
canisters, and piped facility pressure systems. Cleaning and sterilization of these
components has been previously discussed, and arrangements need to be made to
assure the reduction of bioburden prior to entry.
Mopping a disinfectant to the surface creates a non-destructive abrasive type

action that loosens particulates, residues and microbial contamination. Mopping
should be done in order: ceilings, then walls then floors. The mopping action should
be done from the top of the wall to the bottom with overlapping stokes. Similarly, the
ceiling should incorporate the same method over overlapping stokes and start from the
furthest corner of the room. Mopping the floor is done by starting at the furthest corner
and working towards the exit door (similar to painting a floor). While mopping creates
an excellent scenario for cleaning, it is less effective in addressing microorganisms as
the mop head and the wall are less wetted. Thus, the surface dries faster. However, it
is a very critical step that needs to be accomplished on a routine basis. Methodology
for cleaning a surface (not disinfecting) using a mop is enhanced by a quality water
rinse such as WFI. During the cleaning operation the non-destructive abrasive type
action loosens particulates, residues and microbial contamination. After this has been
done, it is important to rinse the surface to remove the contaminants from the surface.
Upon rinsing, the dirtied solution on the floor is collected and removed. Once all
surfaces dry, then a disinfectant or sporicide is applied.
Mopping requires a variety of components that include buckets, spray-mop-fog

devices, handles, mop frames and mop heads. Buckets come in a variety of makes and
models. Some include the single, double and triple bucket systems. In the single
bucket system, the disinfectant or sporicide is placed into a bucket and the mopping
procedure calls for the dipping of the mop into the bucket, then to the wringer, then to
the surface where the chemical is to be applied. In this system, the wringer and
application solutions are mixed in one chamber. Increase in soil load and bioburden
occurs in the bucket and the solution is dirtied in a short time. The double and triple
bucket system attempts to separate the wringer solution from the application solution
by creating separate chambers. Procedurally (in a two-bucket system), the mop enters
the front chamber to be wetted and is wrung into the back chamber. This system is
suppose to keep the solution cleaner as the rinse is collected in the back bucket and
never contacts the application solution in the front chambers. The three-bucket system
exemplifies the two-bucket system and adds one additional bucket as middle rinse
bucket for the mop. While these systems attempt to reduce the soil and bioburden load
in each chamber, the mop head is the contaminating factor of the solutions and
eventually corrupts or soils all cavities in the system. Material grades of buckets are
normally made of stainless steel, galvanized steel or some form of plastics or
autoclavable plastics. Compatibility with the disinfecting agent and capability of
sterilization should be tested prior to use. Buckets should also remain chemical
specific as the mixture of chemical agents and their accompanying residues should be
avoided (especially in a liquid state).
In recent years spray-mop-fog systems have been designed and patented for use
in controlled areas. These devices are made of stainless steel and completely
autoclavable. They provide a chamber for the disinfectant or sporicide and once
introduced, the canister is pressurized with compressed air (ASME rated to 100 psi).
Off the tank exists the ability to connect a trigger spray activated power sprayer or a
trigger activated mop (sending liquid automatically to the mop head or a fogger
(allowing 2–3 hours of fog time). These systems dispense a clean liquid continuously
from the canister. Superior wetting to the surface occurs as the and cleaning time is
24
reduced by nearly 50% .
The single, double and triple bucket system and the spray-mop-fog systems all
use mop heads. Mop heads come in disposable and reusable forms. Mop heads also
come in foam flat mops for wall and string mops for floors. A single use principle
should be applied in all classified areas. The question of disposable or reusable is an
operation specific concern. Disposable means use once and throw away and the cost
of doing so is approximately $8.00 for each sterile mop head. Washing and re-
sterilizing each mop head is an involved process and an operation should review the
total costs and possible impact to the environmental conditions prior to implementing
this scenario. A mop is changed:
• after use in each class 100 room. The same mop head may be sequentially used in
Class 10,000 or 100,000 areas. A mop is never used in a Class 10,000 and then
used in a class 100
• when it is noticeable dirtied
• when the mop head is damaged.

Mop heads for class 100 and 10,000 should be rendered sterile prior to use.
Wiping is the fourth way of application. As was expressed earlier in the chapter,
a very wetted wipe is excellent for disinfection but very poor for cleaning. To clean
with a wipe, one should use a chemical agent and a cleaner. The key is to wet and
loosen the contaminants from the surface and then wipe them away with a dry wipe.
Consider cleaning a window at home as an example of the action needing to be
performed in a cleaning operation in controlled environments. Likewise, for
disinfection, it is necessary to saturate the surface.
Fogging is the last method of application. While fogging a room can be very
effective, there are many important aspects that need consideration. First is coverage
and droplet size. The droplet size required to effectively fog an area and saturate all
19
surfaces is 25.0 µm . This droplet size accompanied with the ability of the fog or mist
to be circulated to all surfaces is the key to success. Fogging can produce very good
results in destroying levels of vegetative bacteria and spores on the surface but is
dependent upon the chemical agent that is used and the time frame of the fogging
5
operation. Carrier surface tests inoculated with b. subtilis at 1.6 x 10 (11 carriers)
show the destruction of such spores using a peracetic acid and hydrogen peroxide
mixture from a concentrate (DECON-SPORE 200 Plus® at a 0.4% use dilution) in a 12
25
foot x 15 foot room, using two two-jet foggers for two hours . While this study shows
the ability of the method to destroy microorganisms on the surface, fogging does not
clean the surface: it only disinfects the surface. Fogging also requires room release
times that may near 3–4 hours. This release time is to assure harmful vapors have
subsided to a safe level for personnel to enter per OSHA standards.
One of the most confusing subjects surrounding application is frequency.

Frequency of application is dependent upon a variety of factors that include the
bioburden of the area, contamination control capabilities, the classification of the area,
the process or manufacturing operation, personnel, product changeover, and the type
of facility and its associated controls. While many other factors come into play, these
are the main culprits for contamination of a manufacturing facility.
Normality is frequency is dependent upon the classification of the environment. A

class 100, 10,000 and 100,000 environment will all be cleaned and disinfected with
varying frequencies. Coupled with the classification is the review of the environmental
test data from each area. The use of the environmental test data to classify the
frequency of an area is superior to any other methodology. In understanding how to
accomplish this we must understand that if we had an area that showed very low
bioburden, if any, and invoked a daily sporicidal application, we would destroy no
more microorganisms than existed in the area. Once disinfectants and sporicides have
dried their ability to kill is complete. It is true that some chemical agent residues create
an undesirable environment for an organism to live, however, such residue kill is short-
term and should not be a part of our system design. By applying a disinfectant and/or
sporicide, we do not create a preventative measure for organisms that may enter the
environment. From the experience of this author, the following charts cultivate a
guideline structure to base our disinfection and sporicidal frequencies.
In discerning frequencies, we need to first identify the type of areas that may exist
in our facility. The can be classified as:
• Class 100 stand-alone (gowning room to a Class 100)
• Class 100 surrounded by a Class 10,000
• Class 100 surrounded by Class 100,000
• Class 10,000
• Class 100,000
• Not Classified Areas that may effect classified areas
Table 6: Frequency of Disinfectant Application
Classification Walls Floors Ceilings Curtains

Class 100 Daily spray Daily mop Weekly spray Daily or per shift
or mop or mop application followed
by 70% IPA wipe
surrounded by or mop or mop application followed
Class 10,000 by 70% IPA wipe
surrounded by or mop or mop application followed
Class 100,000 by 70% IPA wipe
Class 10,000 Daily spray Daily mop Weekly spray If applicable, daily or
or mop or mop per shift application
followedby 70% IPA
wipe
Class 100,000 Weekly spray Daily mop Weekly spray If applicable, daily or
or mop or mop per shift application
followedby 70% IPA
wipe
Not classified Monthly or Weekly mop Monthly or Not applicable
quarterly spray quarterly spray
or mop or mop
Table 7: Frequency of Sporicidal Application
Classification Walls Floors Ceilings Curtains

Class 100 Weekly or bi- Weekly or bi- Weekly or bi- Weekly or bi-weekly
weekly spray or weekly mop or weekly spray or spray or mop or
mop or following EM mop or following EM at
following EM at at action following EM at action level. Followed
action level level action level by 70% IPA wipe
surrounded by weekly spray or weekly mop or weekly spray or spray or mop or
Class 10,000 mop or following EM mop or following EM at
surrounded by weekly spray or weekly mop or weekly spray or spray or mop or
Class 100,000 mop or following EM mop or following EM at
Class 10,000 Bi-weekly or Bi-weekly or Bi-weekly or If applicable, weekly
monthly spray monthly mop monthly spray spray or mop or
or mop or or following or mop or following EM at
following EM at EM at action following EM at action level. Followed
Class 100,000 Monthly spray Monthly mop Monthly spray If applicable, weekly
or mop or or following or mop or spray or mop or
following EM at EM at action following EM at following EM at
action level level action level action level. Followed
by 70% IPA wipe
Not classified Upon Action EM Upon Action EM Upon Action EM Not applicable
determination determination determination
of problem of problem of problem
RESIDUES
With any application of a disinfecting agent comes the accompanying residue
problem. Only a few disinfectants do not leave a residue. Isopropyl alcohol and ethyl
(ethanol) alcohol are two of these disinfectants. In fact, both isopropyl and ethyl
alcohol can assist in the removal of disinfectant and sporicidal residues. In review of
the level of residues attained from each chemical agent we see that if not addressed on
a routine basis, such residue can develop into a critical problem in our operations.
26
Table 8 : Residues from Disinfectants
Chemical Tested When Applied After Rinse

High pH Phenol 759 ppm 61 ppm
Low pH Phenol 731 ppm 41 ppm
Quaternary Ammonium 133 ppm 11 ppm
Bleach @ 5.25% 929 ppm 66 ppm
Bleach @ 0.52% 144 ppm 14 ppm
Hydrogen Peroxide 0.067 ppm 0 ppm
Peracetic Acid/H202 RTU 123 ppm 16 ppm
Peracetic Acid/H202 Concentrate 44 ppm 6 ppm
Some problematic situations with residues relate to the complication of the

surface to be disinfected. The surface, if laden with chemical residues compromises
the disinfectant and/or sporicide's ability to fully contact an organism. This is coupled
with the ability for air pockets to form underneath residues that may possibly break
and release contaminants to the environment. Most residues are unwanted. Personnel
can transfer them to a critical site. Such transfer may occur, as personnel would touch
a surface and then make a critical aseptic connection. This may compromise the
integrity of the clean system in place.
Residues are most notably the cause of deterioration of surfaces in the clean room.
Most notably is stainless steel. Deterioration occurs for two basic reasons:
• the chemical agent reacts adversely, or is incompatible with the surface and
attacks the material composite material or impurities in the surface, or
• the chemical agent stains or discolors the surface
It is first important to conduct compatibility testing to assure the chemical agent

does not harm the surface. Once this has been completed, routine cleaning can remove
or reduce most residues.
DEVELOPING PROCEDURES AND TRAINING
Developing SOPs is critical in design and assuring correct implementation of our

systems. Likewise training our personnel on such procedures is critically important. In
developing the required applicable SOPs, a GMP facility needs to write a very
complete “Use and Rotation” operating procedure. All other departmental operating
procedures can then reference this particular operating procedure in their content.
Critical “Use and Rotation” SOP's should contain the following pertinent data. While
SOP's will vary from firm to firm, the following is a general guideline list of the
important areas.
Without the ability to prepare this critical standard operating procedure, we

cannot assure personnel in varying departments will all follow a general company
scheme of disinfection. Standardizing disinfection plant-wide should be a main
objective of the company. Only in specific instances where EM data warrants the
alteration from the general master plan should such systems change in specific areas.
Once we have established our SOPs, our next step is to assure that all is done to what
has been written. This is not an easy task and the main method of assuring this occurs
is through our training.
Training is without doubt one of the most critical elements in our spectrum of
disinfection. Too often we train only our general workers and forget to train
Table 9: Standard Operating Procedures
Standard Operating Procedure Description

Section Title
Purpose The purpose of the SOP or why it is being done
Scope A scope of what is covered in the SOP
Responsibilities A listing of whom is responsible for each
capacity in the SOP
Safety Precautions Internal corporate and product specific safety
precautions for each product
Approved Disinfectants/Sporicides A listing of the approved disinfectants,
and Cleaning Products sporicides, wipers, and saturated wipers
General Guidance in Preparing Specific guidance as to the general corporate
Disinfectants and Sporicides issues surrounding the preparation of
disinfectants and sporicides such as approved
dilution water types, dating of containers,
storage, etc.
Specific Approved Disinfectant and Specific approved usage instructions for each
Sporicide Usages disinfectant and sporicide. Including dilution
mixtures, container types, use instructions,
expiration dating, storage of unopened
containers, storage of opened containers
Method of Application How are the disinfectants to be applied in
varying classifications and on varying surfaces
Frequency Frequency of application of disinfectants and
sporicides
Approved Equipment and Materials Approved mop heads, mop handles, mop frames,
buckets, sprayers and foggers
Procedure How each specific are is to be cleaned and
disinfected
management. An unknowledgeable management person in the area of disinfection can

harm our efforts. They may lack the skills to train or supervise this important function.
At the same time, they may lack the ability to notice a shift away from acceptability.
It is important to train all involved in the disinfection process on a routine basis.
But “what is training and what content should be included” is a commonly asked
question. First, people learn by doing. People learn very little in a classroom. Their
attention span is short within a classroom setting and "learning by doing" is a far
superior method.
Second, know what you teach to be correct. The worst thing that can happen to an
organization is that the trainees learn and prove that the methods in place are not
scientifically justified or flawed. Trust then becomes a concern.
Third, sometimes inside trainers become boring to trainees. An outside source of

training may provide a fresh approach that one's employees may enjoy. It breaks up
the daily monotony.
Fourth, training must place in the foreground what is expected and if not
accomplished, what the organization will be required to do to assure the function's
completion. People have to know what is expected.
And last, there are countless year 2000 techniques available to the trainer of today.
Utilization of past training methods should be reevaluated to see if implementation of
a better methodology exists.
A FINAL SUMMARY AND CONCLUSION
Too often we just want to kill things. We forget the multitude of critical areas that need
to be addressed to assure the continued and consistent attainment of acceptable
environmental conditions in our controlled areas. If we wait for regulatory agencies to
comment before we change or if we wait for an excursion to exist before assuring a
complete system, we place ourselves into the situation of “hurried quality
implementation”. This will cause us to fail. Hysteria during a crisis situation will cause
us to place a band aide on the situation and not create a strong infrastructure that will
assure our success for years to come. We will forget many critical aspects. Decisions
for design are always made better without emotional turmoil.
As an industry we need to review the required time to adequately clean and

disinfect manufacturing areas. Too often time period are too short to complete this task
and shortcuts may be taken in order to run product sooner. However, when the product
is in jeopardy as environmental levels have exceeded prescribed action levels, the first
scenario that is blamed is cleaning and disinfection. It must not have been done
correctly. On the same note cleaning and disinfection is also the last word as too many
times we use it as the corrective action procedure instead of delving more deeply into
the root cause.
We must also remember that the painting we have created will not gain value over
time as is common for artistic impressions. Each day our design becomes outdated and
we need to routinely reevaluate our systems to assure they include updates for process
changes, changes in technology and changes in our personnel structure.
BIBLIOGRAPHY
Vellutato, A. L., Utilizing environmental monitoring data to implement a cleaning and

disinfection program. CleanRooms Magazine, January 2001, p. S10–S20.
Center for Drugs and Biologics and Office of Regulatory Affairs, Food and Drug
Administration, Guidelines on Sterile Drug Products produced by Aseptic
Processing, June 1987, p. 9.
Center for Drugs and Biologics and Office of Regulatory Affairs, Food and Drug
Administration, Sterile Drug Products Produced by Aseptic Processing Draft,
Concept paper (Not for Implementation), September 27, 2002.
PDA TR #13
United States Pharmacopoeia, The National Formulary, USP 25 NF 20, Chapter 1116,
United States Pharmacopeial Convention, Inc., National Publishing, Philadelphia,
1999
United States Pharmacopoeia, The National Formulary, USP 25 NF 20, Chapter 1072,
United States Pharmacopeial Convention, Inc., National Publishing, Philadelphia,
1999
ISO 14644
ISO 14698
A. Vellutato Sr. and A. Vellutato, Jr., 1989. The Use of Cleaners and Disinfectants in
GMP Controlled Environments, Technical update Notice Monthly. Veltek
Associates, Inc. p. 1–14
Official Methods of Analysis (1998) 16th Ed., 5th Revision, 1998, AOAC
INTERNATIONAL, Gaithersburg, MD
McDonnell, G. and R. Denver. Antiseptics and Disinfectants: Activity, Action, and

Resistance, Clinical Microbiological Reviews, Jan 1999, p. 151.

Resistance, Clinical Microbiological Reviews, Jan 1999, p. 151 (545)

Resistance, Clinical Microbiological Reviews, Jan 1999, p. 151 (513)
Chlorine and Chlorine Compounds. In Disinfection,, Sterilization and Preservation,

4th Edition, edited by S.S. Block. Philadelphia: Lea and Febiger,
Peroxygen Compounds. In Disinfection, Sterilization and Preservation, 4th Edition,

edited by S.S. Block. Philadelphia: Lea and Febiger,

Resistance, Clinical Microbiological Reviews, Jan 1999, Jan 1999, p. 156
Gluteraldehyde. In Disinfection, Sterilization and Preservation, 4th Edition, edited by

S.S. Block. Philadelphia: Lea and Febiger.
Formaldehyde. In Disinfection, Sterilization and Preservation, 4th Edition, edited by

S.S. Block. Philadelphia: Lea and Febiger.
Casey, W.M., Comment, The Pharmaceutical Microbiology Mail List, Rotation of

Disinfectants, Nov 17, 2000.
Connor, D.E. and M.K. Eckman, Rotation of Phenolic Disinfectants, Pharmaceutical

Technology, September 1992, 148–158.
European Committee for Standardization, Chemical Disinfectants and Antisepics –

Quanitative non-porous surface test for the evaluation of bactericidal and/or
fungicidal activity of chemical disinfectants used in food, industrial, domestic and
institutional areas – Test methods and requirements without mechanical action
(phase 2/step2), EN 13697 (2001) E, ICS 11.080.20; 71.100.35 Management
center: ue de Stassart 36, B-1050 Brussels .
Brady, C. S., A.T. Snyder and B.L. Baskin, 2001. Disinfectants Efficacy Testing. In
Microbiology in Pharmaceutical Manufacturing, edited by R.Prince. Godalming:
Davis Horwood International Publishing.
Vellutato, Arthur, Sr., United States Patent 6, 123,900, Untites States patent Office,
1992
Vellutato, Arthur, Jr., Validation of the Core2Clean Spray Mop Fog Systems, Internal
Validation Report, Veltek Associates, Inc. January 2000
Vellutato, Arthur, Jr., Validation of the Core2Clean Fog System, Internal Validation
Report, Veltek Associates, Inc. April 2000
Code of Federal Regulations. 1992. Title 21, Part 211, Good Manufacturing Practices
for Finished Pharmaceuticals, 160, Washington, DC: US Government Printing
Office
ADDITIONAL READING
Proceedings of the Second International Kilmer Memorial Conference on the

Sterilization of Medical Products, Washington, DC, 1980.
Sterilization of healthcare products – Requirements for validation and routine control

– Radiation sterilization, ANSI/AAMI/ISO 11137–1994, Association for the
Advancement of Medical Instrumentation, Arlingto, VA, 1995.
Veltek Associates, Inc., DECON-AHOL Sterile Spray Validation Report, Validation

Report VL-101and VL 101B, Exton, PA, 1989.
Veltek Associates, Inc., DECON-AHOL WFI Sterile Spray Validation Report,

Validation Report VL-201 and VL201B, Exton, PA, 1991.
Veltek Associates, Inc., Sterile Disinfectant and Sporicide Validation Reports,

Validation Reports VL-301, VL401, VL-501, VL601, VL-701, VL-801, VL901,
VL1011, VL2000, VL2001, Phoenixville, PA, 1993
ABOUT THE AUTHOR
Art Vellutato, Jr. is the vice-president of Technical Support Operations, vice-president

of Sales & Marketing and one of the two founders of Veltek Associates, Inc. (an EPA
and FDA registered facility) in 1981.
He is a frequent industry speaker with 21 industry publications. He lends over 18

years of valuable experience that include his tenure as the Director of Quality
Assurance at VAI for nine years and the Director of manufacturing for six years.
Art Vellutato, Jr. co-owns US Patent 6,123,900, Method of Sterilization, that was
used to market the first sterile disinfectant in the industry, DECON-AHOL®. He has
also conducted disinfectant training for FDA's CDER and CBER divisions.
He is the President of the Delaware Valley Chapter of the Parenteral Drug

Association and a faculty member teaching the Disinfection segment at PDA's
Training and Research Institute for the Aseptic Processing Course. Art Vellutato, Jr. is
also the chairman of the PDA Special Interest Group on Cleaning and Disinfection
(PDA Delaware Valley Chapter)
Combining experience of production, quality assurance and validation, Art

Vellutato, Jr. has assisted many pharmaceutical manufacturing operations in
implementing their environmental monitoring and disinfection programs, worldwide.

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8

IMPLEMENTING A CLEANING AND

Arthur L. Vellutato, Jr.

Veltek Associates, Inc.

In looking at the basics surrounding the formation of a compliant system, the

By definition, cleaning a surface would require the removal of any contaminant

DESIGNING A COMPLIANT GMP SYSTEM

The next step to achieving near-perfection in our cleaning and disinfection

To complicate things further, there is no common methodology in the industry;

Thus, the problem with developing an environmental monitoring and disinfection

Experienced professionals design environmental monitoring and disinfection

It is necessary to look at a few guideline documents that may assist in understanding

MAINTAINING CONTROL OF THE ENVIRONMENT

Maintaining control of the environment is critical. It is the most important aspect

CLEANING THE ENVIRONMENT

Disinfection relates to the saturation and penetration of the cell wall of an

As discussed, there are significant difference between cleaning and disinfection.

Cleaning is based on a few physical factors:

• the surface to be cleaned

• the contamination vested on the surface

• the chemical used to clean the surface

• the effect of the chemical agent on the surface to be cleaned

• the level of surfactants in the chemical agent

Knowledge of the type of surface to be cleaned is very important. As depicted

In other sections of this chapter, antimicrobial effectiveness testing will be

Figure 1: Aluminum surface

Figure 2: 316 L Stainless Steel surface

Figure 3: Epoxy-oated surface

In Figure 3, we see an epoxy-coated surface. Epoxy-type surfaces are very

Figure 4: Clean Room Curtains #1

Epoxy-coated surfaces need routine replacement or refinishing and as such

Figure 6: Phenolic Residue

Figure 8: Sodium Hypochlorite Residue

TESTING THE ENVIRONMENT AND DEVELOPING DATA

While structuring one's environmental monitoring program, one will find it

CHOOSING THE APPROPRIATE DISINFECTANTS AND

The utilization of environmental monitoring data to implement a cleaning and

In choosing the appropriate chemical agents, we need to review what the

Table 1 gives is a basic overview of what is required by a disinfectant

Understanding disinfectant categories is essential. A multitude of categorizations

Types of Claims Test organism(s)

AOAC Sporicidal Test

AOAC Tuberculocidal Activity Method

AOAC Fungicidal Test

EPA Virucidal Test Parameters

AOAC-available chlorine germicidal

EPA Sanitizer Test Parameters

classifications for antimicrobial effectiveness claims that based on their passing of

Sanitizer: a germicidal product that reduces the population of viable microorganisms

Disinfectant: a chemical agent that completely destroys pathogenic organisms but

Sterilants/Sporicides: a chemical agent that is capable of sterilizing. Such chemical

In reviewing the basic types of chemical agents used in pharmaceutical and

ISOPROPYL ALCOHOL, ETHYL ALCOHOL, ETHANOL

• as a sanitizing spray for gloves, surfaces, carts, etc.

• as a cleaning or wipe down agent to remove possible existent residues from

• as a product contact cleaning agent (ethyl alcohol only)

In testing antimicrobial products' effectiveness, a 70% solution has far superior

Published alcohol effectiveness or results of alcohol effectiveness are presented

QUARTERNARY AMMONIUM COMPOUNDS

Quarternary ammonium products are used in more disinfection applications than

In formulation of a sodium hypochlorite solution, many choose to acidify the

]PERACETIC ACID AND HYDROGEN PEROXIDE