Understanding and Preventing Clean Room Contamination

This presentation is compiled by “ Drug Regulations” a
non profit organization which provides free online
resource to the Pharmaceutical Professional.
Visit http://www.drugregulations.org for latest
information from the world of Pharmaceuticals.
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 This presentation is compiled from freely available resources like
the websites of FDA, EMA ,WHO and research papers published
by experts in this field like
Sandle, T Reinmüller, B , Hyde, W,, Costello, E.K., Lauber, C. L.,
Hamady, M., Fierer, N., Gordon, J.I., Knight, R.
 Paper published by T. Sandle on clean room contamination was
referred extensively for this presentation.
 “Drug Regulations” is a non profit organization which provides
free online resource to the Pharmaceutical Professional.
 Visit http://www.drugregulations.org for latest information from
the world of Pharmaceuticals.
4/1/2016 2
Drug Regulations : Online
Resource for Latest Information

4/1/2016 3
Drug Regulations : Online Resource for Latest
Information

 Staff gowning
 Personnel behavior
 How to minimize cleanroom risk
 Basic training
◦ Cleanroom staff
◦ Cleanroom entry
◦ Gowning practices
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 Cleanroom contamination can arise from a
number of sources.
 Type of cleanroom
 Its geographic location
 The types of products processed
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 Contamination sources can be divided into
following
◦ People
◦ Water
◦ Air and ventilation Surfaces
◦ Transport of items in and out of clean areas
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 Most contamination can be traced to
humans working in cleanrooms.
◦ Conclusion based on organisms associated with
isolates from environmental monitoring in
controlled environments.
 These microorganisms are transient or
residential to the human skin.
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 Human personnel shed high numbers
of skin cells mostly as skin flakes.
 The cleanroom garments worn by
personnel cannot contain all human
detritus.
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 Trillions of microbial cells across the epithelial surface
and within the mouth and gut.
 These microorganisms play a role in human physiology
and organ function including digestion and immunity.
 The microorganisms also affect the outside
environment
 This is due to shedding from the skin or deposited
through different orifices.
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 This latter issue has important implications for
cleanrooms.
 The outer layer of human skin can host up to one
million microorganisms per square centimeter.
 The population, as well as the diversity, varies
according to anatomical locale.
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 Microorganisms within Human Body
◦ Scalp: 1 million
◦ Saliva and nasal fluid: 10 million/gram
◦ Back: 100
◦ Groin: 1 – 20 million
◦ Forehead: 100 – 1000
◦ Hand: 10,000 – 100,000
◦ Armpit: 1 – 10 million
◦ Feet: 1 million
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 Research suggests that a typical person sheds
 1,000,000,000 skin cells per day of a size 33 μm x 44 μm x 4
μm
 This is equivalent to a rate of 30,000 to 40,000 dead skin cells
shed from the surface of the skin every minute.
 Of these approximately 10% of particles carry microorganisms.
 There are, on average, four microorganisms per skin cell.
 A term commonly used to describe skin flakes with adhered
microorganisms is “microbial carrying particles.”
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 People are
 A source of contamination,
 Agents for transferring contamination possibly to locations
that could pose a product risk.
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 Microorganisms are spread from
◦ Sneezing
◦ Coughing
◦ Touching
 Microorganisms suspended in the air are less of a
concern
 When such organisms gravitate towards a product
or critical location, they present a significant risk.
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 The Human Microbiome Project (HMP)
◦ United States National Institutes of Health initiative.
◦ The project goal is to identify and characterize the
microorganisms found in association with both healthy and
diseased humans (the human microbiome).
 Our understanding of the risk the people pose to
cleanrooms has been advanced
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 The human microbiome describes the aggregate of
microorganisms and their genetic interactions that
reside
◦ On the surface and in deep layers of skin
◦ In the saliva and oral mucosa
◦ In the conjunctiva
◦ In the gastrointestinal tract
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 Skin is a complex ecosystem
 Supports a range of microbial communities
 These live in distinct niches
 These niches are affected by
◦ Available nutrients
◦ Several non-nutritional factors such as pH, humidity, and
temperature.
 Epithelial cells are continually being shed,
 Many microbial communities on the external surface are
rarely stable.
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 Outer layer of Skin
 Has High population
 Considerable diversity of microbial species
across
 There are approximately 1000 species of
bacteria from 19 phyla on human skin.
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 Of these, most bacteria can be categorized
into following phyla:
◦ Actinobacteria (51.8%).
◦ Actinobacteria are a group of Gram-positive
bacteria with high guanine and cytosine content,
 Such as Micrococcus, Corynebacteria and
Propionibacteria.
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 Of these, most bacteria can be categorized
into following phyla:
◦ Firmicutes (24.4%).
◦ This includes the genera Clostridia and Bacillus.
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 Of these, most bacteria can be categorized into
following phyla:
 Proteobacteria (16.5%).
◦ This is a major phylum of bacteria
◦ Includes a wide variety of pathogens such as
 Escherichia,
 Salmonella,
 Vibrio,
 Helicobacter, and
 many other notable genera.
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 Of these, most bacteria can be categorized into
following phyla:
 Bacteroidetes (6.3%).
 The phylum Bacteroidetes is composed of three
large classes of
◦ Gram-negative
◦ Nonspore-forming
◦ Anaerobic
◦ Rod-shaped bacteria
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 Reasons for the topographical variations relate
to the physicochemical properties of the skin.
 This is especially so for
◦ Temperature
◦ pH
◦ Amounts of oil
◦ Moisture
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 From this, there are three main
ecological areas of the skin:
◦ Sebaceous
◦ Moist
◦ Dry
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 Examples of microbial divergence include
◦ Propionibacteria and Staphylococci species dominating the Dry, calloused areas
(arms and legs)
◦ Gram-positive cocci (primarily the Micrococcaceae) are found on the arms and
legs
◦ Gram-positive rods are found in high numbers on the torso.
◦ Staphylococci and Corynebacteria are found together with some Gram-negative
bacteria in moist areas.
 These types of microorganisms generally reflect the types recovered
from cleanrooms
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 Gram-positive bacteria predominate across the
skin as
◦ The skin is generally a dry environment
◦ Any fluids present on the surface generally have a high osmotic
pressure
 Thus Gram-positive bacteria are better adapted for
such environments, not least to being resistant to
desiccation.
◦ (especially the Staphylococci and Micrococci)
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 Where other species occur this is due to
◦ Variations in temperature and
◦ With areas of higher sweat production
 For example, this can lead to higher levels of fungi
on the feet
 In relation to pharmaceutical manufacturing, the
presence of any such organisms remains
problematic.
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 The ratio of the microorganisms recovered from
the skin is relatively evenly divided between the
aerobic and the anaerobic.
 The aerobic microorganisms tend to live on the
outermost layers of the skin
 The anaerobic microorganisms live in the deeper
layers of the skin and hair follicles
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 The information from HMP introduces several
implications for cleanroom environmental monitoring.
◦ Whether gowning practices are adequate to exclude all
microorganisms from the richest areas of the skin microbiome.
 Most bacteria free-floating in cleanroom air current are
not free-living
 They are instead the result of
◦ Direct particle shedding of desquamated skin cells and
◦ Subsequent re-suspension of skin detritus in the air stream.
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 The answer to earlier question should lead to a
consideration of:
◦ The types of cleanroom undergarments used
◦ An examination as to whether these provide an effective
barrier, especially for the more moist parts of the body.
◦ The importance of the outer gown covering all parts of the
body, including the forehead.
◦ The quality of cleanroom certified undergarments.
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consideration of:
◦ The level of training required for operators in relation to
gowning and the way that gowning qualification was
conducted.
◦ How long a cleanroom suit should be worn for in relation to
material integrity against operator perspiration.
◦ The environment in which gowns are donned, where higher
air-change rates might prove effective.
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consideration of:
◦ How often gowns should be recycled which involves
washing and irradiation.
◦ What types of testing is conducted on recycled gowns
◦ What the procedures are for rejecting gowns where a
loss of integrity is detected.
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 Cleanroom microbiologists may wish to consider how
concerned they are with each of the items listed in
excluding microorganisms found on all regions of the
skin.
 There must be good understanding of the environmental
monitoring methods used to assess cleanrooms.
 These may not show how good or bad gown changing and
gown wearing is.
 These concerns are best addressed through good gowning
practices
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 Despite some advances with automation and robotics, in
most situations people cannot be eliminated from
cleanrooms.
 Control of contamination from people in cleanrooms is
achieved by application of two principles:
◦ We “wrap” the people to minimize the amount of “shedding” of
microorganisms.
◦ We put localized protection around the product to minimize the amount of
contact with the people.
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 The localized protection is achieved through local air
protection, such as
◦ Unidirectional airflow cabinets and
◦ Isolators.
 With clothing, personnel working in cleanrooms are required
to wear special clothing designed for the clean environments.
 Such clothing is necessary because the human body creates
its own microenvironment of potentially damaging particulate
contamination.
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 To be effective, cleanroom clothing must:
◦ Form a particulate barrier for the human micro-
environment.
◦ Allow freedom of movement and be comfortable.
◦ Address any specialist requirement,
 e.g. static dissipation.
 Avoid being a significant particulate contributor in itself.
◦ Cleanroom garments must meet specific protection criteria.
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 Not all cleanroom garments are of the same quality.
 This involves manufacturing the garments from special
materials, following particular construction methods, and
then tailored for individual styling.
 The gowns must be comfortable, easy to apply and practical
in use.
 Some gowns are disposable and others are made to be re-
laundered and sterilized depending on the cleanroom grade.
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 Fabrics used in the manufacture of cleanroom garments must
have the following features:
◦ Be low in particulate shedding.
◦ Permit the body to breathe while trapping particles within the garment.
◦ The contaminant should be retained within the garment and not released
into the surrounding atmosphere.
◦ Be flexible enough for comfortable wearing.
◦ Withstand repeated cleaning and sterilization cycles.
◦ Meet any specific requirements such as control of static.
◦ Meet opacity requirements.
◦ Look and feel as good as possible. Be cost-effective.
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 There are three broad categories of fabric used in the
construction of cleanroom garments:
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 Woven fabrics.
◦ Woven or re-usable fabrics are the most commonly used fabrics in
cleanroom environments.
◦ Such garments are woven on sophisticated looms from yarns of
continuous filaments of polyester.
◦ The thickness of the yarn and filaments is important –
 the finer the yarn, the tighter the weave can be made, and the better the filtration.
◦ The pattern and tightness of the weave is important to reduce the pore
size to a minimum.
◦ The use of continuous filament polyester means that there are few loose
ends from which particles may be shed.
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 Laminated or membrane fabrics.
◦ Laminated fabrics are favored for some high-
grade microelectronic environments.
◦ These types of garments are not commonly used
in the pharmaceutical sector.
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 Disposable or limited life materials.
◦ The most common of these non-woven fabrics are from spun
bonded olefin and polypropylene.
◦ Comprising a densely interlinked matt of fibers, these fabrics can
provide good results for a limited period.
◦ Garments from such materials need to be processed and
decontaminated before use in the cleanroom.
◦ Disposable or limited use garments are mainly used in those
environments where protection of the wearer against potentially
hazardous products is required.
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 Garments are designed to provide protection for the head, body,
hands and feet.
 In establishing a system for garment selection, it is important to
consider the broader aspects of cleanroom use:
◦ Suitability of fabric,
◦ Garment style,
◦ Layers,
◦ The nature of the tasks involved,
◦ Costs,
◦ Regulatory requirements, and
◦ Any specific customer requirements.
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 The classification of the cleanroom will inevitably be the
major factor in determining the degree of personnel
protection required and the fundamental choice of garments.
 One important issue with gowns is the maximum length of
time that a gown can be worn.
 As people perspire, the integrity of the gown will weaken.
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 Complicating factors are the temperature and humidity
of the cleanroom and the variations between people.
 The length of time will also depend upon the grade of
the cleanroom.
 In aseptic areas, such as ISO 14644 class 7 / EU GMP
Grade B areas, gowns are typically worn only for the
length of the shift
◦ (normally four hour periods to enable operators to take breaks).
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 In lower grade cleanrooms, a gown might be worn
for several sessions during the course of the
working day.
 Other factors affecting the lifespan of the gowns
that are subject to recycling are repairs and the
number of permitted washing cycles.
 With repairs, it is prudent to have a repair policy.
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 This will vary across facilities, and again, it will be affected by
the cleanroom class.
 With aseptic areas, if a gown becomes torn it is normally
discarded.
 In other grades of cleanroom, a gown can be repaired
depending upon the size of the hole and the impact on the
material.
 Some organizations set a maximum size for any hole or tear
and for the number of times a gown can be repaired.
 Gowns that are recycled are subject to laundering.
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 Gowns are washed by special washing machines with suitable
detergents,
 They are then dried, folded, and then wrapped in cleanroom packaging.
 For gowns that are to be used in aseptic areas, such gowns are
irradiated.
 A policy should be in place outlining how often a gown can be processed
-- typical times range between 20 and 40 times.
 To make the tracking task easier, many gowns sterilized by irradiation or
gassing are fitted with barcodes and scanned.
 It is further important to establish the extent that the sterilization
process affects the integrity of the gown material.
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 In order to assess the contamination risks from re-
laundering, gowns are subject to particle counting.
 There are different ways to do this
 Most common means is the Helmke Drum particle
emission test.
 With this, the test method simulates particle
shedding of clothing under movement.
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 The garment under test is tumbled in a rotating drum
(approximately 10 revolutions per minute)
 This releases particles from the surface of the
cleanroom garment in a controlled manner.
 An automatic particle counter is used to sample the air
within the drum
 Determination of the average particle concentration of
the air during the initial ten minutes of the test is
performed.
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 The common standard is the IEST "Recommended Practice RP-CC003.3:
Garment System Considerations for Cleanrooms and Other Controlled
Environments".
 An alternative measure is the Body Box test.
 This method simulates particle filtration and release under real wear
conditions.
 As a consequence it measures the contamination of the cleanroom by
the clothing/wearer.
 For this, particle counters determine the quantity of particles generated
by the wearer/garment that are emitted into the chamber.
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 Cosmetics, such as powder, rouge, eye liner, mascara, and lipstick
must be banned in cleanroom environments.
 Jewelry, such as rings, watches, necklaces, bracelets, earrings and
other items, together with all forms of visible piercing, are
commonly not allowed in cleanrooms.
 The best method of changing into cleanroom garments is one that
minimizes contamination getting onto the outside of the garments.
 Change areas can vary in design, but it is common to find them
divided into three zones:
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 Pre-change zone.
 Outside of changing rooms 'tacky mats' or polymeric flooring can be
positioned to help reduce the level of particles carried on footwear.
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 Changing zone.
 The changing room design contributes to the assurance of
appropriate personnel access and microbial contamination control.
 The changing room should be provided with filtered air.
 Intermediate (bag) filters will typically be suitable for this purpose,
though High Efficiency Particulate Air (HEPA) filtration may be used.
 The air pressure should be negative with regards to the
manufacturing area corridor, but positive relative to external
adjacent areas.
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 Cleanroom entrance zone.
 This must be of the same grade or class as the main cleanroom into
which the area leads.
 Ideally there should be separate routes through airlocks for material
required in cleanrooms.
 Taking items through personnel change areas should be
discouraged.
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 Personnel training in gowning is an important function.
 Gowning practices must be assessed periodically and monitored
frequently.
 Training programs should ideally include visual assessment and
microbiological assessment.
 The microbiological assessment varies, but can include
◦ The exposure of settle plates during the change process and
◦ The assessment of gown cleanliness through post-use suit contact plates.
 The results of the cleanroom sampling should not exceed those of the
room class.
 If results are exceeded, the individual may be an unusually high shedder
of skin particles.
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 Training required for staff who work in cleanrooms should
include:
◦ Introduction to micro-organisms and microbiological contamination
control.
◦ Entry and exit of production facilities (including gowning)
◦ Personal hygiene training
◦ Microbiological risks associated with specific production tasks.
◦ Training must be documented and regularly reviewed. Training must be
effective.
◦ Actual performance of personnel competency in gowning should be
demonstrated on a regular basis.
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 Working in clean environments demands
 Knowledge,
 Discipline,
 Motivation as well as
 A thorough understanding of contamination risks among all
personnel involved.
 Each individual cleanroom should have its own documented rules
and procedures.
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 Training includes reminding personnel that they must not be
allowed to touch critical products and equipment with their naked
hands.
 All critical work must be undertaken wearing gloves.
 Critical activities requiring personnel contact such as aseptic
processing or sampling must be done through the use of clean
utensils such as tweezers, forceps, and the equivalent.
 All devices and gloves used must fully comply with the cleanliness
demands of the cleanroom and work undertaken in the cleanroom.
 They must be cleaned, disinfected, or sterilized as appropriate for
the criticality or activity and risk of contamination.
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 Another aspect of best practice is in instructing personnel in the
appropriate behaviors within the cleanroom.
 The generation of contamination is proportional to activity
conducted.
 A person with head, arms, and body moving can generate about
1,000,000 particles ≥ 0.5 μm/min.
 A person who is walking can generate about 5,000,000 particles ≥
0.5 μm/min.
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 However a person in motionless position can generate only
100,000 particles ≥ 0.5 μm/min.
 In addition, personnel should reduce activities like talking,
singing, whistling, coughing, sneezing etc., especially when
being close to the handled products and production
equipment.
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 People working in cleanrooms and other forms of controlled
environments must be physically healthy.
 Diseases in the upper respiratory tract as well as stomach disorders can
create problem in hygienic applications.
 Another factor that can impact upon the environment is the number of
people in the cleanroom.
 Only necessary and limited number of persons should be allowed in a
cleanroom at the same time.
 The more persons simultaneously present in a cleanroom, then the
higher the contamination level will be,
◦ i.e., the higher concentration of particles in the air
 This is particularly important in relation to changing rooms.
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 Good personal hygiene is a requirement of all
pharmaceutical cleanroom activities.
 However, studies show poor compliance is
common in relation to basic hand washing
technique.
 Hand hygiene and glove hygiene are important
given the high numbers of microorganisms found
on the human body
 They also pose risk of contamination by transfer
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 Microorganisms associated with hands are found mainly
on the surface of the skin and under the superficial cells
of the stratum corneum.
 The dominant species is Staphylococcus epidermidis
 This is found on almost every hand, together with other
species of Staphylococcus and species of the genera
Micrococcus.
 Hands must be washed with soap and water prior to
entry to the cleanroom.
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 Hand washing facilities should not be located in an
actual cleanroom, but rather in an area leading to
the cleanroom changing room.
 As an alternative, a hygienic handrub can be used.
 Where gloves are required these should be put on
using a method designed to prevent the ungloved
hand from touching the clean or sterile outer part
of the glove.
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 Once in the cleanroom, gloved hands should be
subject to periodic hand sanitization
 When decontaminating hands with an alcohol-
based antiseptic hand rub,
◦ Apply product to palm of one hand and rub hands together,
◦ Covering all surfaces of hands, fingers and wrists,
◦ Until hands are dry
 (alcohol-based hand rubs are not to be used with water).
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 The process typically takes between thirty seconds
and one minute.
 Follow the manufacturer’s recommendations
regarding the volume of product to use.
 The technique for applying alcohol to gloved hands
is similar to applying a handrub to skin.
 It is important to ensure that all surfaces are
covered.
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 With glove sanitization, there are two alcohols of
choice:
 Ethyl alcohol (ethanol) and isopropyl alcohol (IPA)
 Other alcohols, such as methyl alcohol (methanol)
are unsuitable.
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 IPA is slightly more bactericidal than ethanol
 Ethanol has better viricidal properties
 Another factor is application to the skin;
 IPA can be quite harsh.
 Thus, ethanol is more often applied to bare skin
(often in a denatured form)
 Whereas IPA is more often applied to gloves.
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 These sanitizers have bactericidal action against vegetative
cells but not spores.
 The concentration of alcohol to water varies, although the
optimal range is 60 to 90% (volume/volume).
 Below 60%, bactericidal action drops,
 Above 90% there is insufficient water for the bacterial cell to
absorb water.
 The alcohol does not enter the cell and is unable to denature
the bacterial proteins.
 Most preparatory concentrations are 70%.
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 While most bacteria are killed after ten seconds of
contact with alcohol contact times in practice are
longer due to the variability of hand rubbing.
 Typical contact times are thirty seconds.
 It is important that the selection of a hand sanitizer
is qualified.
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 There are different approaches that can be taken
for qualification.
 Most of these require
◦ Individuals to wear gloves
◦ Place their hands into broth containing a high concentration
of a non-pathogenic microorganism.
◦ Disinfectant is then applied
◦ Bacterial reduction is assessed through placing the treated
hands into broth and performing dilutions
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 Assess the risk of personnel to the process by taking suit
contact plates of the gown as worn by the person as they
leave the aseptic area.
 The gown must be discarded after the plates have been taken
 This is due to the potential effect on the gown integrity when
an agar plate contacts the gown.
 It is good practice to begin suit sampling with a higher
number of samples.
 These can then be reduced over time.
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 Some facilities perform more samples from the
gown during gowning test qualifications compared
with routine sampling.
 Sites considered for selection include
◦ The top of the head
◦ The facemask
◦ Both arms
◦ Middle torso
◦ Both legs
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 In terms of limits, for EU GMP Grade B/ISO class 7 areas, the aim is
often to adopt the same limits as per the limits applied to
fingerplates.
 The action level for gowns is ordinarily 5 CU/25cm
 Experience has shown that higher counts are obtained from the top
of the head,
 This is perhaps because this is the warmest region of the body.
 Care must be undertaken when sampling as so not to break the
integrity of the gown.
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 In addition to gowning control, a procedure should be in
place for the notification of health conditions by staff.
 Staff who are ill (coughs, colds, and so on) should not enter
cleanrooms.
 This is because the illness may affect product quality.
 It is important to control the potential risks from personnel
carrying Infectious disease.
 Open lesions on any exposed part of body.
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 Shedding skin conditions, such as eczema or psoriasis,
dermatitis, and dandruff
◦ (skin scales may harbor objectionable micro-organisms that may impact
pharmaceutical products and patients).
 Gastric upsets.
 Personnel with any of the above conditions must be excluded
from working within cleanrooms for the duration of their
illness.
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 This presentation is compiled from freely available resources like
the websites of FDA, EMA ,WHO and research papers published
by experts in this field like
Sandle, T Reinmüller, B , Hyde, W,, Costello, E.K., Lauber, C. L.,
Hamady, M., Fierer, N., Gordon, J.I., Knight, R.
 Paper published by T. Sandle on clean room contamination was
referred extensively for this presentation.
 “Drug Regulations” is a non profit organization which provides
free online resource to the Pharmaceutical Professional.
 Visit http://www.drugregulations.org for latest information from
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Understanding and Preventing Clean Room Contamination

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