See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/342883006

Best practices in environmental monitoring

Article · July 2020

CITATIONS READS

0 13,506

1 author:

Tim Sandle
The University of Manchester
836 PUBLICATIONS 1,886 CITATIONS

SEE PROFILE

All content following this page was uploaded by Tim Sandle on 12 July 2020.

The user has requested enhancement of the downloaded file.

Best Practices In
Environmental Monitoring

White Paper

Author:
Dr Tim Sandle

1
Contents
Introduction 3

What environmental monitoring is and what it is not 4

Cleanroom environments 4

Sources of microbial contamination 5

The environmental monitoring programme 6

Summary 17

References and further reading 18

2
Introduction
The purpose of microbiological environmental monitoring is to assess the cleanliness of pharmaceutical (sterile and non-
sterile) and medical device manufacturing environments. Environmental monitoring involves the collection of data relating
to the numbers or incidents of microorganisms present on surfaces, in the air and from people. In addition, non-viable
particle counting, a physical test, is undertaken in conjunction with viable monitoring because of the relationship between
high numbers of airborne particles and microorganisms (Sandle, 2011a).

This white paper has been put together to consider the practical application of environmental monitoring for both sterile
and non-sterile products, together with some best practice ideas. The white paper outlines the important components of
an environmental monitoring programme and provides practical advice for those tasked with setting up a programme or
who wish to review an established programme (an activity which should be undertaken on a periodic basis). Whilst many
of the points discussed will be of interest to microbiologists and quality personnel, no single document can provide a
definitive programme. This is because no two facilities are the same; they differ in terms of products, procedures, people,
design, and environments (Schneider, 1995).

Consequently, each microbiologist will need to develop a monitoring programme appropriate to their facility, drawing on
the different points outlined below.

3
What environmental • When action levels are exceeded or adverse trends
are detected appropriate investigations must
monitoring is and what it be performed using documented procedures to
determine the contamination source, the impact upon
is not the product and to set corrective or preventative
Environmental monitoring is not the same as actions. For this risk based methodologies can be
environmental control (which is outlined in regulations deployed
such as 21 CFR Part 820.70). Environmental control • To allow the effectiveness of the cleaning and
is concerned with the design measures necessary to sanitisation programme to be assessed
maintain environments within the required operating • To understand the performance of the people and
parameters. Such parameters include: temperature, equipment, and the suitability of operating protocols
relative humidity, air velocity, unidirectional air flow, HEPA
• To provide information about environmental control
filtration, and pressure differentials between rooms of
In constructing an environmental monitoring programme,
different classification (Ramstorp, 2000). Environmental
it is important to be aware of the limitations of monitoring.
monitoring does, however, relate to environmental control
The methods deployed, for example, are highly variable in
in that monitoring can indicate a failure of a control and
terms of collection efficiency (Boschi, 2006). Furthermore,
it is sensible to, from a risk-based perspective, target
the culture media selected and the incubation parameters
monitoring where control is weakest.
chosen will only detect those microorganisms which
The implementation of environmental monitoring in will grow under the set of conditions adopted. A further
an organisation should be through the construction of limitation is the time of sampling. Monitoring only provides
a planned environmental monitoring programme. The a ‘snapshot’ of one moment in time and this may or may
requirement to perform monitoring is described in the not reflect conditions throughout the process. It follows
following standards and guidelines: that individual environmental monitoring results are rarely
• EU GMP Guide significant (with the exception of the continual monitoring
of aseptic processing batches where some samples can
• USP <1116>
relate to specific events). Thus, the most important aspect
• FDA Code of Federal Regulations 21 CFR 211 of environmental monitoring is the examination of trends
• ISO 14698 Part 1 over time (Reich et al, 2003).
• PDA Technical Report Number 13
The essential factors of any well-designed environmental Cleanroom environments
monitoring programme are similar (Moldenhauer, 2008). The environments in which processing occurs are
However, the way in which different factors are weighted either classified (to a cleanroom standard, such as ISO
and the decisions made by facilities will vary in terms of 14644) or controlled. Cleanrooms and clean zones are
how programmes are carried out. typically classified according to their use (the main
The objectives of an environmental monitoring activity within each room or zone), controlled through
programme are: the physical operation of HVAC (Heating Ventilation
and Air Conditioning), with the classification confirmed
• To monitor cleanrooms, collect data and to examine
by the cleanliness of the air by the measurement of
trends to show the state of microbiological control of
particles. In addition, recommended limits are applied for
an environment. Monitoring is more meaningful when
microorganisms.
the environment is assessed under representative
conditions (normally when cleanrooms are occupied, With control of cleanliness, the pharmaceutical
and processing is taking place) manufacturing environment is based around a series of
o Data collection may relate to either numbers of rooms with specially controlled environments. These
microorganisms or to the incidence of detection, or to are termed ‘cleanrooms’. A cleanroom, on one level, is
both (using pre-defined monitoring limits). In addition, simply a room that is clean. The key aspect, however, is
some of the microorganisms recovered should be that the level of cleanliness is controlled. The definition
characterised and trended of cleanliness, according to the international cleanroom
standard, ISO 14644-1 is (ISO 14644-1, 2015):
• To show that contamination levels do not increase
through manufacturing as the process is designed to “A room with control of particulates and set
become cleaner (that is contamination levels should environmental parameters. Construction and use of
decrease through ISO class 9, 8 and 7 areas) the room is in a manner to minimise the generation
• To assess the risk to the environment and most and retention of particles. The classification is set
importantly to the product. This is achieved by by the cleanliness of the air.”
selecting monitoring locations which are meaningful While control of airborne particles is important, the
and by monitoring at frequencies will allow the trend ISO 14644 standard does not differentiate whether
to be discerned these particles are inert or biologic. Reference to

4
Figure 1: Working in a cleanroom

biocontamination in relation to cleanrooms is detailed Once a room has been assigned a classification, certain
in ISO 14698, although this latter standard is not, in this environmental parameters (physical and microbiological)
author’s experience, widely followed in drug manufacture are to be met on a routine basis. This is assigned by
(although it does contain some useful advice relating to collecting data and examining the results of monitoring
the qualification of environmental monitoring methods). against pre-set criteria. Part of this assessment is through
a microbiological environmental monitoring programme.
A controlled environment is similar, in that airborne
The extent of monitoring and the monitoring limits
particulate and microorganism levels are controlled,
assigned will relate to the class of cleanroom, the activities
but the rooms are not classified against a national
taking place and the relative risks. Thus, an important
or international standard (Whyte, 2001). These are
question to answer before launching a monitoring
sometimes referred to as ‘controlled but not classified’
programme is “what cleanliness levels are expected?”
(CNC) areas.

Sources of microbial contamination

There are different sources of microbiological contamination within clean environments: water, air, surfaces (both within
the room and from equipment) and personnel. These hazards should be evaluated by the microbiologist in terms of the
relative risks to the product, and the environmental monitoring programme should be orientated towards the points
of greatest risk. The sampling methods should be appropriate in relation to the types of contamination sources (and a
comprehensive monitoring programme should assess each of the main contamination sources). The greatest risks are
those which could lead to product contamination. This is illustrated in the diagram below:

Secondary
sources

• Personnel • Surfaces • Product

• Water • Air
• Equipment

Primary Risk of
sources Ingress

Figure 2: Diagram showing sources of microbial contamination within a cleanroom

5
The primary sources of contamination are people and settling. Therefore, what often matters most is not the
water. This is because both are vectors of contamination. microorganisms in the air but their potential for settling.
People are the most significant source of contamination, A well-designed cleanroom will filter air (to dilute the
although they are a highly variable and unpredictable number of microorganisms) and have a pressure cascade
source. Microorganisms are shed from hair, skin, eyes and to prevent re-contamination of a clean area from a less
mucous membranes. Microorganisms are either deposited clean area (since microorganisms cannot move against an
into the air stream or can spread through contact. Water air current) (Whyte and Eaton, 2004).
is a common feature in pharmaceutical processing (as an
The other secondary contamination source is materials and
ingredient, a cleaning agent, a diluent for disinfectants,
surfaces. Here, the key risks are the transfer of items in and
steam supply, and so on). The concern with water in
out of a clean area, where materials are more at risk if they
cleanrooms is that it not only provides a means for
are of a design that cannot be easily cleaned or disinfected;
microorganisms to survive, it provides the opportunity
and from personnel touching surfaces. Another risk is the
for the numbers of microorganisms to increase and
contamination of surfaces through deposition (such as
microorganisms are invariably found in all residues of
settling from the air) (Sandle et al, 2010a).
water (some bacteria, especially Gram-negative rods, can
grow and multiply in low nutrient states). Based on these contamination sources certain factors
will lead to contamination risks being more likely. These
The secondary sources of contamination are air and
factors include:
surfaces. The air in most areas contains microorganisms.
However, the number of microorganisms will vary • Poorly designed cleanrooms
according to the cleanroom grade. Air is a secondary • Water remaining on surfaces for prolonged periods
contamination source because air is a vector for • Inadequate cleaning and sanitisation
microorganisms, but it is not a nutritive environment
• Inadequate personnel gowning
and whilst some bacteria can survive in air streams
they cannot multiply. Generally Gram-positive bacteria • Poor aseptic practices such as direct surface-to-
are more commonly found in air (typically Bacillus spp, surface transfer (such as by personnel directly
Staphylococcus spp, and Micrococcus spp) (Ackers touching the product or contaminated water entering
and Agallaco, 2001). Bacteria in air are normally in the process)
association with dust particles or skin flakes, rather than as • Airborne transfer, often arising from personnel
individual microorganisms (for which the term ‘microbial shedding microorganisms. Shedding increases with
carrying particle’ is sometimes used). This makes the increased personnel movement and fast movement
microorganisms heavier and more prone to gravitational also increases the potential for microbial dispersion

The environmental monitoring programme

The assessment of environments for primary and secondary contamination sources is undertaken through a defined
environmental monitoring programme. The programme should be documented and detailed in a policy or rationale
together with accompanying standard operating procedures. Although many regulatory guidance documents indicate that
environmental monitoring is required there is little in the way of regulatory guidance about the contents of the programme
or how often monitoring should be conducted. To an extent this depends upon the type of facility, its design and nature of
operations; as well as depending upon how much data needs to be collected in order for the microbiologist to be satisfied
that an area is or is not in control. Thus, the microbiologist has considerable scope in designing a monitoring programme,
although the finalised form the programme needs to be justifiable to the regulatory authorities.

As a minimum, the programme should address the following elements:

• Types of monitoring methods • Clear responsibilities describing who can take the
• Culture media and incubation conditions samples

• Frequency of environmental monitoring • Processing and incubation of samples

• Selection of sample sites (where monitoring will take • Alert and action levels
place) • Data analysis, including trending
• Maps showing sample locations • Investigative responses to action levels excursions,
• Duration of monitoring • Appropriate corrective and preventative actions for
• When and where the samples are taken (i.e. during or action level excursions
at the conclusion of operations) • Consideration if special types of environmental
• Method statements describing how samples are taken monitoring are required (such as the use of selective
and methods describing how samples are handled agars for objectionable microorganisms or anaerobic
monitoring)

The important elements of the environmental monitoring programme are examined below together with the practical aspects.

6
Figure 3: Working in a cleanroom (Image: Tim Sandle) activities on-going in the area at the time of sampling.
Settle plates, if located in a meaningful place, can provide
Monitoring methods
a further indication of the chance of microorganisms
Monitoring methods are divided into viable monitoring in the air-stream settling. It is sensible to have settle
and non-viable particle monitoring. This distinction plates positioned close to air samplers and to have both
may become less relevant in the future with the advent monitoring methods located close to the critical activity
of rapid methods which, through the application of within the cleanroom.
fluorescence counting technologies, can detect both non-
viable and viable particles from the same sample of air.
In the meantime the classic sampling techniques remain
commonplace in most facilities.

Viable monitoring

The objective of viable environmental monitoring is to

enumerate the numbers of microorganisms present at a
location within a cleanroom. This is undertaken using a
range of different air and surface counting methods:

a. Active air-sampling: volumetric air-sampler

b. Passive air-sampling: settle plates
c. Surface samples: contact (RODAC) plates and swabs
d. Personnel samples: finger plates and gown plates
Although these methods are well established there are
several practical aspects to consider when using each of
these methods. These are considered below.

Active air-samples

An active (or volumetric) air sampler collects a proportion

Figure 4: Active air-sampler (Image: Tim Sandle)
of the microorganisms present in a given volume of air.
The volume of air sampled is normally one cubic meter There are two main types of active air-sampler: impaction
(with results expressed as x CFU / m3). or centrifugal, although samplers of a filter type design
are also used. An impaction air-sampler functions by
When assessing the results from active air sampling it accelerating air, at an angle of 90o, through holes in the
is not often known if the numbers of microorganisms head of an air sampler (often a ‘sieve like’ design). The
recovered reflects those which may have settled onto force impacts the microorganisms onto an agar strip or
a critical surface. For aseptic filling operations an plate. A centrifugal air-sampler draws air into the sampler
indication of this likelihood is provided from the analysis head through a rotating vane mechanism. The vane causes
of airflow visualisation patterns (‘smoke studies). For microorganisms to be thrown out of the air and onto the
other operations the relative risk is assessed from the agar through the effect of the centrifugal force. Effective
location of the sampler and an understanding of the air-samplers must be able to precipitate particle sizes of

7
at least 2μm. The ability of a sampler to capture particles The length of time a settle plate can be exposed must be
sized 10μm or larger is more efficient because most validated in order to determine if the weight loss of the
airborne microorganisms are attached to larger particles agar in the plate affects the growth promotion properties
like skin detritus (Mwier and Zingre, 2000). The choice of the plate post-exposure. This can be assessed by
of active air sampler is not straightforward as different designing an experiment where settle plates are pre-
models of air-sampler vary in their efficiency (Kaye, 1986). weighed, exposed for a pre-defined time under the
Furthermore, in order to maintain reliability samplers worst-case conditions (normally within a unidirectional
should be calibrated at a minimum of annual intervals. airflow (UDAF) cabinet since this environment will cause
the greatest desiccation). After exposure the plates can
Another consideration for air-samplers is that the devices
be challenged with a suitable range of microorganisms to
themselves can generate a relatively high level of non-
determine if the amount of ‘drying out’ has affected the
viable particle counts, and this should be considered in
ability of the plates to support microbial growth.
the design qualification of such devices especially when
used in ISO class 5 / Grade A environments. Other design Surface samples: contact plates and swabs
considerations include the suitability of the sampler to the
The two primary surface sampling techniques: contact
sanitisation agents used for cleanroom equipment. For
plates and swabs. Contact plates are agar plates, typically
samplers used in UDAF cabinets the impact of this should
of 25cm2 diameter, with a raised (domed) surface
be assessed through airflow mapping studies.
designed such that when the plate is inverted the agar will
Settle plates press against a surface. The reproducibility of the contact
plate can be strengthened by setting the sample time and
Settle plates are agar plates, typically of either 9cm
by controlling the pressure applied. Commercial devices
or 14cm diameter. They are designed to detect any
are available for this purpose. Variations to the contact
microorganisms carried in the air-stream which directly
plate are flexible plastic strips with raised agar surfaces.
settle within a particular area. If settle plates are located in
appropriate locations then they can provide an indication Swabs are typically made up of sterile cotton (or more
of how often and, possibly, how many microorganisms commonly superior synthetic materials) tips. They are
may have been deposited onto a critical surface or into either contained within a transport medium or require
any exposed product. pre-wetting with a suitable recovery medium (such as
Phosphate Buffered Saline or sterile water). Swabs are
The results from settle plates can either be assessed
either designed to be sub-cultured on agar or dissolved
as the number of microbial colonies per plate or semi-
and membrane filtered. Swabs are useful for sampling
quantified by calculating the number of microorganisms
small areas or curved surfaces.
per unit of time (the time being the length of time that the
plate has been exposed for). Guidelines such as EU GMP The microbial recovery from a contact plate is superior to
express action levels as CFU (colony forming unit) per four that of the swab and contact plate should preferably be
hours. Nevertheless, the use of settle plates as a semi- used (with swabs used where contact plates cannot, such
quantitative measure is contested. One problem is where as on narrow or irregular surfaces). Swabs that are have a
a microbial carrying particle settles and forms a colony flocked tip have been shown to give better recoveries than
forming unit for it is unknown if the particle consisted swabs with ‘plain’ tips (Goverde et al, 2016).
of one or more microorganisms. This is why some
There are some practical considerations for surface
microbiologists elect to use settle plates as a measure of
sampling. Importantly after using either contact plates
what may have settled (incident “hits”) rather than as a
or swabs the residue (agar or diluent) should always
quantifiable measure (Andon, 2006).
be wiped clean with a suitable sanitiser to avoid
providing residual material for microbial growth. Another
consideration is that for monitoring critical activities this
is often carried out at the end of the activity due to the
invasive nature of the act of sampling.

Both contact plates and swabs can be quantified. The

typical surface area of a contact plate is 25cm2 and when
sampling using a swab a template can be used so that a
known surface area is sampled, although such templates
are not always practical for the types and shapes of
surfaces that swabs are typically used to sample. Thus,
both methods allow the number of microorganisms
detected in a given area to be estimated and such data
can be extrapolated for a larger surface.

Surface monitoring is further differentiated between

Figure 5: Microbial colony on a settle plate (Image: Tim floor surfaces and surface at working height. Surfaces
Sandle) at working height generally have tighter limits and

8
are considered to be more critical as they relate to a as, pressure differentials, HEPA filtration and so on. A
preparation step or product contact area. Samples from high level of particles may imply corresponding levels of
floors normally have lower limits, since no product touches microorganisms (De Abreu et al, 2004). Research has
the floor, and the monitoring of these locations acts as a shown the relationship to be that for every 105 particles
check of cleaning and sanitisation practices. recorded, one particle is a microorganism (Ljungqvist and
Reinmuller 1996).
Personnel samples
Particle counting is used for either classification purposes
For some operations, most notably aseptic filling, samples
(to assess a cleanroom under different operating
are taken from the personnel working in the cleanrooms.
conditions: as built, at rest or in operation); for routine
These samples are finger plates (or finger dabs) and
spot checks; for the continuous monitoring of aseptic
samples of the gowns worn by the operators (where the
filling; or as an investigation tool when examining out of
sleeves are often the areas of greatest risk area in relation
limits events.
to personnel manipulations of product or equipment).
An important consideration for spot checking and
Gloves are sampled by finger plates where each finger of
continuous monitoring is the location of the particle
the gloved hand is pressed onto the surface of an agar
counter within the cleanroom. For classification the ISO
plate (of 9cm size or larger). Sleeves are sampled by
14644 standard provides a formula for the location of each
using contact plates. Therefore, both sample types can
particle counter (where the square root of the surface area
be quantified as CFU per five fingers per hand or as CFU
of the cleanroom is calculated and counters are situated
per 25cm2. Additionally, gloveport gauntlets and sleeves
at equidistant locations). The location of a particle counter
of isolators and RABS (Rapid Access Barrier Systems) are
for routine monitoring requires a rationale and could be
also sampled at the end of batch campaigns (although
determined from:
these samples do not avoid the need for the leak testing of
gloves and sleeves in situ). • The results of the particle count classification study, if
one location was ‘worst case’
Samples of personnel hands are taken at different intervals
during batch campaigns and should be taken after each • By means of examining the process flow and selecting
critical activity (such as an intervention into a UDAF representative tests of the operation
device). To be representative of the activity hands must • By means of risk assessment. This could include
not be sprayed before sampling. However, after sampling, considering which parts of the process are most at risk
hands must be disinfected using a glove sanitiser. Samples from ingress (for example, with aseptic filling the point
from gowns and face masks are taken at the end of the of fill is a point where product vials are vulnerable).
test session because the act of sampling will compromise An alternative consideration is the risk to clean area
the integrity of the material. from a less clean adjacent area. Here it might be better
to particle count the less clean area to note for any
Particle monitoring
upward build upo in particles which might present at
Particle counting is performed using a discrete particle risk to the cleaner area.
counter. This is a device that measures the size and Other important considerations when using particle
number of air-borne particles (some of which will be counters are that the counters must be located close to
viable and many more which will be non-viable). The the activity being measured so that the sampling tubing
counter functions by having a photodiode which detects is no longer than three meters (any longer then the risk
light scattered by single particles passing through a laser, of ‘drop out’ or particle loss, rendering the sample results
sited within the sensing zone. The scattered light from the inaccurate. This risk is greater of 5.0μm sized particles). In
laser is then concentrated by a lens system and converted addition, where counters are used in UDAF devices they
into electrical pulses by the photodiode. The amplitude of should be fitted with isokinetic probes in order to ensure
the pulses is proportional to the particle size. The particle that the air velocity entering the counter is the same as
counter will also count the number of pulses produced the speed of the air within the clean zone (which also
by the photodiode, which gives the number of particle minimised particle loss).
counts. Like any electronic system particle counters
can be prone to electrical interference which can create Locations for viable monitoring
‘background noise’ and the occasional ‘false’ particles. Having reviewed sampling methods (above) the next
The key particle sizes for pharmaceutical manufacturing important consideration is where, within the cleanroom,
and testing are 0.5μm (required by the 2004 FDA to take the samples. The number of environmental
guide to aseptic filling, EU GMP and ISO 14644) and monitoring locations will depend upon the size of the
5.0μm (required by EU GMP). These guidelines provided cleanroom and the activities taking place. To determine
recommended limits for one cubic meter of air. Particle this, a study of the room (layout and equipment) and the
counting is an important part of the environmental process (understanding what happens, what equipment
monitoring programme as sampling can dynamically is used and what the people working in the area do) is
indicate the quality of the air continuously over time and required. It is important that the types and locations for
hence the effectiveness of cleanroom parameters, such monitoring have relevance to the process; that the data

9
produced must is meaningful (Lowry, 2001). This is often could cause contamination (such as with an aseptic filling
achieved by mapping the process and the flow of people line). In such cases this should be detailed in a rationale.
and materials, by applying risk assessment tool such as Instead locations close by should be assessed and post-
HACCP (Hazard Analysis and Critical Control Points) activity sampling carried out.
(Jahnke and Kuhn, 2003) or FMEA (Failure Modes and
Some facilities elect not to fix monitoring locations and
Effects Analysis) (Sandle, 2003a).
instead rotate monitoring positions. The argument in
These risk tools are similar in that they identify potential favour of rotation is that in doing so more areas of a
hazards (WHO, 2003). The key aspects are: cleanroom will be monitored and that the personnel
tasked with sanitisation will not be able to ‘guess’ where
• Constructing a route map (where the facility is drawn
monitoring will occur therefore ensuing a better standard
and the process flow indicated).
of cleaning. The argument against rotation is that
• Identification of hazards (which can be divided having fixed locations allows for long term trends to be
into biological, physical, equipment, transport and examined. Random sampling can be useful when assessing
chemical). The hazards of greatest risk (severity) can newly built facilities where the determining the most
be distinguished from those of lower risk. representative locations is not straightforward. However, if
• An assessment of existing control measures. the locations have been selected by risk assessment they
• Consideration of measures to improve control, are most meaningful and there would appear to be no
which will minimise the probability (likelihood) of a need for sample rotation.
contamination event happening.
• Pinpointing areas of greatest risk (where control is
weakest).
• Orientating monitoring towards areas where control
remains weak.
• Deciding on the most appropriate monitoring
methods to use.
Thus, when setting locations for monitoring, the main
areas of risk are considered. This often centres on areas
where personnel activity is the greatest. This will bias
monitoring to areas like routes of human traffic; primary
items of equipment in the room, such as open processing;
areas which might become more heavily contaminated,
such as door handles; where contamination is likely to
spread or proliferate such as near water outlets and
product contact sites. In addition, some focus of the
environmental monitoring programme should also be
Figure 6: Pharmaceutical processing
towards areas which could be neglected by cleaning
regimes or which are generally inaccessible. Other areas In determining the final number of sample locations, a
can be chosen based on where there is potential for balance needs to be struck between the objective of
direct product impact; where microbial contamination generating sufficient monitoring data in order to show
would affect product quality and where contamination adequate control, on one side, and interference with the
could spread through movement of samples, equipment production process and the costs of monitoring on the
or personnel. As room usages differ, the environmental other side. The use of a documented risk based approach,
monitoring locations will vary from room to another. as outlined, is a good way to achieve this (Baird, 2015).

After completing the mapping exercise each selected Sampling responsibilities

site should be justified as to why it has been selected The individuals who take the environmental monitoring
and described on a sampling map so that sampling is samples should be clearly defined in the written
consistent and reproducible. Here, each location can be programme. Normally trained microbiologists take
given a risk rating (such as high, medium, or low risk) samples in lower class cleanrooms (ISO 8 and 9). For
based upon proximity to the critical area (such as exposed sampling aseptic filling areas there are two different
product or vials) and taking into account the ease of approaches. One approach is that all environmental
transfer of any contamination towards product, vials, monitoring samples should be taken by independent QC
or other critical areas. Documenting this will show the staff. The other approach is that the presence of additional
regulators that attention has gone onto site selection. staff in a cleanroom increases the risk and to avoid this
Although the optimal sites for monitoring should be risk process staff should undertake monitoring. A ‘middle
selected this is not always possible. Sometimes what way’ approach is for process staff to take the majority of
appear to be most appropriate locations for activities the samples for aseptic filling but in addition having QC
should not be sampled because the act of sampling itself staff monitor filling runs periodically (such as on a basis

10
of 1 in 10) or to take finger dab samples at unannounced frequent monitoring whereas rooms in which automated
intervals. processing takes place or are used infrequently tend to
require a lower level of monitoring.
Where process staff do take samples, it is important that
they go through the same rigorous training programme How often different categories or sets of rooms are
as QC staff and are trained in microbiological awareness monitored can only be established by an historical review
(Sandle, 2010b). of data and by looking at the frequency interval between
excursions. Suitable monitoring frequencies for non-
Frequency of monitoring
sterile processing are twice-weekly, weekly, fortnightly
The frequency of environmental monitoring for sterile and monthly. With these sets of frequencies different
products (aseptically filled or terminally sterilised) is rooms will be monitored at different times based on risk
set out in regulatory guidance: it should be continuous assessment.
throughout the fill and at times where product or product
components are exposed. Environmental monitoring
for other activities requires the microbiologist to set the
monitoring frequency. The frequency should be based on
a risk assessment of the activities in the cleanroom and
should be often enough to enable meaningful trends to be
assessed (Gordon et al, 2015).

The risk assessment should examine the different

parameters of the cleanroom and weight these according
to the severity of the contamination, should it occur,
and the likelihood that contamination will occur. For
this different frequency categories can be drawn up or
numerical scoring processes used. Some of the risks to be
considered are:

• Room temperature (cold rooms are a lower risk than

ambient rooms due to the microstatic effect on most
bacterial from the lower temperature. Based on this
ambient rooms would be monitored more often than
cold rooms);
• Whether the room is normally ‘wet’ (such as a wash
bay) or dry. Wet areas present a greater contamination
risk and could be monitored more often;
• Whether a drain is present (where there is a drain the
risk may be higher due to back-flow);
• The environmental monitoring history of the
cleanroom. A room with a poor history will require
Figure 7: Preparing an active air-sampler for monitoring
more assessment;
(Image: Tim Sandle)
• Whether equipment is cleaned-in-place or whether it
In addition to the established programme other sampling
is mobile and is cleaned elsewhere (fixed equipment is
sessions may be performed, such as immediately after
easier to control);
sanitisation so that the effectiveness of the cleaning and
• Whether equipment cleaning is manual or automated sanitisation can be assessed (so termed ‘field trials’).
(automated cleaning is easier to validate and thus Another example of targeted monitoring is following
more reliable); maintenance and after process area shutdowns to that the
• Whether open or closed processing occurs (with open suitability to commence manufacturing can be assessed.
processing at the greatest risk); Other types of monitoring may be undertaken less often.
• The duration of processing (where the longer the This might include examinations using selective agars
process then the greater the possibility of something (such as during seasons where fungi may be a concern) or
affecting the product); for special incubation conditions (such as monitoring for
anaerobic bacteria where nitrogen gas lines are used).
• The room occupancy (where higher occupancies
present a greater risk because personnel are the Room conditions for monitoring
primary contamination source within cleanrooms).
Microbiological monitoring should ordinarily be performed
When such factors are examined, weighted and in the in operation (or ‘dynamic’) state, as opposed to the
monitoring frequencies set then a pattern tends to emerge at rest (or ‘static’) state, because the former represents
where rooms in which open processing occurs or which the “worst case” scenario (that is the cleanroom with
have a high personnel involvement tend to require more people present, equipment operating and processing

11
on-going) (Sutton, 2010). Practically, in large facilities, two media one medium is designed to encourage the
there may be occasions when areas are not used very growth of bacteria and the other medium is designed to
often however attempts should be made to sample under select fungi) (Sandle, 2014).
operational conditions. There may be occasions when
Where one medium is used this is often soya-bean casein
at rest monitoring is useful. For example, to establish a
digest medium (tryptic soya agar (TSA)). This is a non-
baseline with which to compare rooms in the operation
selective, highly nutritious medium and has a long history
state against. Such comparisons are particularly useful
of being used within cleanrooms. Where two media
when examining particle count excursions.
are used, the medium for recovering bacteria is often
Duration of monitoring TSA. For fungi, a selective medium is used. The actual
medium will depend upon the types of environmental
For the filling of sterile products, the duration of
fungi most prevalent. Common media include Sabouraud
monitoring should be for the entire length of the filling
Dextrose Agar, Malt Extract Agar, Potato Dextrose Agar
operation. For other activities and in lower classes of
or Rose Bengal Agar. These media may have an altered
cleanrooms, monitoring sessions are normally between
pH or contain antibiotics to inhibit bacterial growth. If it
one and four hours (which is based on the time for the
can be shown that the majority of microorganisms can
settle plate exposure). This is normally sufficient for no
be recovered from one culture medium then this offers
microbiological monitoring programme can (or needs to)
considerable time and cost savings. To adopt one medium
assess all of the microbiological contamination in a clean
requires confirmatory studies using fungi isolated from the
area at times of use. Programmes are designed to provide
cleanroom environment or from adjacent cleanrooms.
“snap shots” of a cleanroom at a particular time and
provided that the rooms are in use at the time of sampling Before use, all culture medium must be validated.
and that the frequencies of monitoring are often enough Validation of culture media normally consists of testing
so that data trends can be assessed then a short duration articles of the medium at all of the applicable incubation
of monitoring is normally sufficient. Unless there is temperatures and against a range of microorganisms.
something very specific about the process monitored then The microorganisms used include those recommended
no specific time represents “worst case” and therefore any by the pharmacopeia (typed cultures from ATCC or
given monitoring time is “equal case” (Cundell et al, 1998). a similar culture collection including Bacillus subtilis,
Staphylococcus aureus, Candida albicans, Pseudomonas
For sterile filling, where continuous monitoring is required
aeruginosa and Aspergillus niger) and a number of
by GMPs, a typical sampling regimen is:
environmental isolates from the cleanroom environments.
• Settle plates are exposed for the duration of a fill The challenge inoculum is to be less than 100 CFU. The
(additional settle plates may need to be used if the fill use of a low challenge is of particular importance because
exceeds the validated plate exposure time). high numbers of microorganisms will not be present
• Active air samples will be taken at the (near) start and in the cleanroom environment. Following validation,
(near) end of the fill. routine growth promotion testing should be performed
• Finger plates will be taken immediately after a on each lot as part of the release of the culture media (in
connection activity, for any persons present during the additional to physical tests, such as pH, appearance and
fill at a random time during the fill and after a gel strength) at a representative incubation temperature.
Grade A / ISO Class 5 zone intervention. This is necessary, partly due to the variations with
media manufacture and partly due to the risks involved
• Surface monitoring will take place immediately at the
with shipping media where media can be subjected to
end of the fill. This is not performed during filling due
extremes of temperature.
to the invasive and disruptive nature of the techniques.
• Contact plates of gowns will be taken from all
personnel immediately before they exit the Grade B
area (Aseptic Filling Suite).
For batch campaigns the monitoring of shift changes
should be monitored.

Culture media

The objective of most environmental monitoring

programmes is to detect mesophilic bacteria and fungi
(organisms which will grow between 20 and 35°C) for
these microorganisms are most likely to be present and
pose the greatest risk to most products. To monitor for
such microorganisms, it is typical to use either one culture
medium, which is subject to a two-step incubation regime;
or to use two microbiological culture media which are
incubated at different temperatures. With both regimes Figure 8: Contact plate growing an actinomycete from
the intention is to recover both bacteria and fungi (with environmental sampling (Image: Tim Sandle)

12
Some practical aspects relating to the use of culture media
include the importance of performing an assessment
of the method of transferring the culture media into
cleanroom from an adjacent clean area or into an isolator
in order to check that the method does not cause
inhibition of microbial growth.

It may be, for example, that the hydrogen peroxide or

peracetic acid used to sanitise loads into an isolator or
the fumigant used to sanitise a cleanroom could cause
inhibition if the sanitiser penetrated the bags containing
the media. To counter this the media can have neutralisers
added to it (such as pyruvate to neutralise hydrogen
peroxide (Ohresser et al, 2004)).

A further consideration is that where disinfectant residues

are likely, the media used for surface samples (like RODAC
plates) or for sampling personnel hands (finger plates) Figure 9: Plates used for environmental monitoring (Image:
should also contain a suitable disinfectant neutraliser to Tim Sandle)
counteract disinfectant residues on surfaces or remnants All test samples are read at the end of the final incubation
of hand sanitisation agents on gloved hands (Russell, period. It is important that samples are not left in the
1997). incubator for too long a period because desiccation of
Another issue is that media should be sterile. The agar occurs. This could render the plate unreadable and
sterilisation of culture media is normally by gamma mean that any microbial growth cannot be characterised.
irradiation. Studies should be conducted to show that the Reading samples
method of sterilisation does not cause then inhibition of
microbial growth. When reading environmental monitoring samples post-
incubation an artificial light source should be used so that
Incubation of test samples small colonies, or those of a colour similar to the surface
Samples from environmental monitoring should be agar, can be discerned. Colonies should be expressed
incubated at a pre-defined time and temperature regime. as CFU and reported against the units of measurements
In terms of incubation time this can be assessed by applicable to the monitoring method. Where samples
removing samples at set intervals, counting them (taking not of a standard size, results may need adjusting (by
care not to cause adventitious contamination) and the re- extrapolation) so that they can be compared against
incubating over a period of time to establish a point where action limits (for example, if one cubic meter of air was not
the growth of visible colonies no longer occurs. taken for an air-sampler or where a settle plate was not
exposed for four hours). In relation to air-samplers, some
In terms of temperature requirements, this depends
samples require the use of a correction factor.
whether one or two culture media are used (as described
above). Where two culture media are used, the regime is Alert and action levels
typically: Results from environmental monitoring are assessed either
• For bacteria: 30-35°C as incidents or are compared against alert and action
levels (Caputo and Huffman, 2004). Importantly, these
• For fungi: 20-25°C
levels are not specifications and should not be used for
The important choice is the order of incubation. An
pass or fail decisions in relation to the product. They are
argument for the lower temperature first is that fungi
used as indicators of drift from the operating norm and are
are more likely to be in a stressed state and should be
best assessed by looking at the data trend over a period of
encouraged to grow first.
time.
An argument for the higher temperature first is that,
Alert and action levels should be defined prior to
should a filamentous fungus be present, this could grow
undertaking the monitoring programme. For sterile
in a way which obscures the bacterial colonies (Sandle,
manufacturing regulatory guidance or compendial
2014). The decision taken should be justified and based on
informational levels can be used. For other operations, and
sample data (Marshall et al, 1998).
for alert levels, monitoring limits should be selected based
on an historical review of data.

Historical reviews of data should take into account the

fact that environmental monitoring data is not normally
distributed and is typically positively skewed. Such data
does not lend itself to common statistical techniques
like standard deviation. PDA Technical Report #13 offers
some different approaches for setting limits, such as the

13
percentile cut-off approach where alert levels can be set at investigation and they may indicate a breakdown of
the 95th or 97th percentile and the action level set at the environmental control (Sandle, 2011b).
97th or 99th percentile (Moldenhauer et al, 2001). Once
Investigating microbiological data deviations
set, levels should be reviewed periodically with an annual
review recommended. There should be a documented Environmental monitoring results which exceed the
rationale describing how alert and action levels are action level; or where there is an upward trend relating
established. to excursions of the alert level; or where the frequency of
incidents exceeds a predetermined cut-off value, represent
Trending microorganisms
scenarios which should be investigated.
A selection of microorganisms identified from the
Care should be taken when reacting to individual results
monitoring programme should be examined and
for microbiological results are often difficult to interpret.
trended. For this assessment all microorganisms from the
This is for several reasons:
environmental monitoring samples taken in the Grade A /
ISO 5 area should be identified to species level. For lower • Microorganisms are ubiquitous in nature and are
class clean areas, it is recommended that all sample results common environmental contaminants
which exceed the action level should be identified. • A single result does not by itself indicate a breakdown
of environmental control
The most important reasons for examining microflora
are to look for objectionable microorganisms which • More meaningful data is often assessed from trends
might pose a product risk or in relation to cleaning and (see below)
disinfection practices, and to assist with investigations into • The microbiologist has the potential to introduce
out of limits results. contaminating microorganisms during sampling and/
or testing
Objectionable microorganisms are organisms which would
cause harm to patients if they survive in the product, thus • Microbiological sampling methods are subject to
objectionable microorganisms are of most concern in considerable inherent variability
relation to non-sterile products. Whether a microorganism Therefore, the most important consideration when
is objectionable is assessed by risk assessment based on interpreting environmental monitoring data is the trend.
the type of product and the risk of product contamination
The investigation of an environmental monitoring level
(Moldenhaurer, 2010). Some species of microorganism are
excursion should be covered by a Standard Operation
more likely to proliferate in certain types of products than
Procedure (SOP) and be formally documented. The
others.
SOP should contain decision trees to ensure that, where
Regular recoveries of microorganisms which are possible, the conclusions reached are consistent. Before
theoretically more resistant to disinfectants may indicate proceeding with a formal investigation, a check should
concerns with the cleaning and sanitisation regime. EU be made in the microbiology laboratory to ensure that
GMP places a particular emphasis upon “resistant strains” the result is not due to “laboratory error”. Laboratory
and implies that any microorganisms resistant to the in- error investigations should be undertaken independently,
use disinfectants or an isolator sanitisation process should that is by someone not directly involved with conducting
be characterised. If detected, the expectation is that these the test. Such investigations should be carried out in a
microorganisms are challenged against the disinfectants timely manner. Laboratory error investigations include
through microbial disinfectant efficacy tests (Sandle, whether the sample was taken and handled correctly;
2003b). if the correct culture media was used; if the equipment
was within calibration date (such as the active air-
A third reason for trending microorganisms is as part of
sampler); if the sample was incubated for the correct
investigations into out of limits events. Characterising
time and temperature; if there is any possibility of sample
the microflora allows the microbiologist to determine
contamination (issues of aseptic technique); and whether
whether two or more microorganisms may be related; this
the result was read correctly and reported to the correct
information can provide an indication as to the possible
units of measurement. In the event of a laboratory error,
origin of the contamination.
measures should be taken to prevent the error from
With environmental monitoring it is expected that a large reoccurring.
proportion of the microflora will be primarily of human
Once a data deviation is confirmed or an adverse trend
origin (such as Staphylococci or Micrococci). Some
established an investigation should be conducted. The
Gram-positive rods will be recovered from lower class
scope of the investigation will depend upon the nature
cleanrooms (such as Bacillus spp. and Corynebacterium
of the trend or the risk to the product (growth from a
spp.), possibly transferred into the area from equipment.
swab taken of a filling needle from an aseptic fill is of a
Where water is present, such as wash-bays, Gram-
substantially higher risk than a floor contact plate from an
negative rods will additionally be isolated. Where unusual
ISO class 8 preparation area). Assessing trends requires
microorganisms are detected, such as, Gram-negative
some experience in order to determine if the datum is part
bacteria from dry areas or unusually high numbers of
of an adverse trend, whether is signals an unusual problem
endospore forming bacteria, these should warrant special
or loss of control; or if it is an isolated event. Several alert

14
level hits are often indicative of a gradual drift towards an
action level event. To do this requires a look-back at past
data, such as a three to six-month period.
Contamination
There are many different approaches for examining out- Source
of-limits events. In general, approaches include (Sandle,
2006):

a. A description of the problem or event

b. Examination of trends
c. Data collection Direct
Air
Transfer
d. Investigation
e. Risk assessment (focusing on the product)
f. Determination of the most probable root cause
g. Consideration of corrective and preventative actions
h. Summary
Product
Completed investigations should be copied to local
managers and discussed at a regular review meeting,
where senior managers are present.

The purpose of the investigation is to determine, where

possible, the root cause of the incident (why the action Figure 10: Relationship of contamination sources
level was exceeded and why the contamination event Corrective actions relate to either things that can be done
has occurred) and to consider if it can be prevented from at the time or if any additional testing or monitoring can
reoccurring. Often, the process of assigning a root cause be performed (to determine the extent of the problem).
involves a process of elimination, by working through an This may include a particle count being noticed during an
investigation checklist to determine what “is” or “is not” a operation (from an alarm or beacon on a counter) and an
potential problem. activity being halted and corrective measures taken. Or,
For determining the source of the contamination the impact of particle counts may be re-assessed through
invaluable data can come from understanding the the conducting of an airflow (or smoke) pattern to note air
microflora. The range of microorganisms found in the direction and turbulence.
cleanroom environment can be subcategorised according Preventative actions can be difficult to set, especially
to the source (such as a skin commensurable linking the where isolated action level excursions have occurred.
organism to personnel) or their probable location (such as As a minimum, preventative actions include additional
from an item of equipment) (Hussong and Madsen, 2004). cleaning sanitisation, additional sampling, and briefing
Investigations should focus on ‘cause and effect’: how did and retraining of operators. If a particular microorganism
the event occur and what is the impact upon the product is isolated on several occasions consideration should
or environment? This will involve an understanding of what be given to the effectiveness of the sanitisation
has happened with the process and an examination of programme or the resistance of the organism to the
both routine events, like cleaning and sanitisation regimes, in-use disinfectants. Other preventative actions can
and any unusual occurrences. include re-designing an activity (such as situations where
cross contamination has occurred); changes to process
Here risk assessment tools can again be useful particularly
times; modifications to the HVAC system of a cleanroom;
in relation to the concepts of severity and probability. With
or major repairs to equipment and machinery. Senior
severity, this is a consideration of the maximum impact
management should endorse the conclusions. For aseptic
of the event upon the product or process where the most
filling a conclusion relating to the batch disposition will be
severe outcome is product contamination. Probability is
required.
concerned with the likelihood of contamination affecting
the product or reoccurring in the environment. In weighing It is important that all investigations have a clear and
up probability an investigation will need to consider succinct summary, which briefly describes the origin of
the time that event occurred and the frequency of the contamination, the risk upon the product and process,
contamination events over a given time period. and measures to prevent reoccurrence. Such summaries
are useful for senior management and for showing to
Consideration of likelihood will also need to assess the
regulators.
contamination source and the if transfer to product could
occur, through either direct transfer or through the air. This
is illustrated in Figure 10 above, right.

15
Trend analysis

There is little value in undertaking environmental monitoring unless the data is reviewed. Environmental monitoring data
should be studied for trends. Trending provides valuable information about environmental control (Tetzlaff, 1992). For
example, data from the environmental monitoring programme is important for measuring the effectiveness of cleaning and
sanitisation procedures such as the rotation of disinfectants.

Trending is particularly important for low count data where trends cannot be readily discerned. For example, the data
displayed below (in Table 1) is the mean of settle plates taken at the periphery of a filling zone within an isolator.

Day Mean Count Day Mean Count

1 0 26 0.25
2 0.25 27 0.25
3 0 28 0.75
4 0 29 0
5 0 30 0
6 0 31 0
7 0 32 0.5
8 0 33 0.5
9 0 34 1
10 0 35 1.25
11 1 36 0.25
12 0.25 37 0.5
13 0.25 38 0.75
14 0 39 0.5
15 0 40 0.25
16 0 41 1
17 0 42 1.25
18 0 43 0.25
19 0 44 0.25
20 0 45 0
21 0.25 46 0.5
22 0.25 47 1
23 0 48 0.25
24 0 49 1.25
25 0.5 50 0.25

Table 1: Example data from the environmental monitoring of an isolator using settle plates

From the table it is not immediately clear as to the direction that the trend is taking. However, when this data is plotted in a
simple chart in MS Excel, with the use of a three-point moving average, the trend becomes much clearer.

Mean settle plate count

Mean count (cfu / plate)

Number of days
Figure 11: Plot of data from the environmental monitoring of an isolator using settle plates
With the use of the graph, an upward trend is much clearer to visualize.

16
Environmental monitoring documentation • Location map showing monitoring sites

This white paper has placed a strong emphasis on the • Frequencies of monitoring
need for a documented environmental monitoring • Incubation parameters
programme. The salient points should be capture in a • Results reading and reporting
rationale. To support this, a well constricted Standard • Alert and action limits
Operating Procedure (SOP) is required. The SOP should
• Method of data analysis
cover such aspects of the programme as:
• Actions required for out of limits events
• Types of culture media
In addition, recording when samples were taken, the
• Methods for transferring culture media into duration of monitoring, who took the samples is important
cleanrooms and should be captured. This, together with the SOP,
• Responsibilities for monitoring ensures that data is traceable.
• Monitoring techniques

Summary
From the first look environmental monitoring appears relatively straightforward. The methods are long established and
the process generates large quantities of data. However, an environmental monitoring programme will be inadequate if
it is not thought out. The wrong types of samples maybe taken in the wrong locations; the times of monitoring maybe
too infrequent; data maybe be generated but not analysed and data deviation investigations maybe imperfect. To guard
against this a practical, well-structured and thought out monitoring programme is required. For this a programme based
on risk assessment is required.

This white paper has outlined many of the important factors which require consideration when an environmental
monitoring programme is designed and later reviewed. If such a practical approach is adopted then a rigorous and
defensible system will emerge and be able to satisfy regulatory expectations. Furthermore, some of the best practice tips
presented will help to make the monitoring programme more meaningful in terms of what the results are telling you as well
as having something that is easier to explain to auditors.

17
References
Ackers, J. and Agallaco, J. (2001). Environmental Monitoring: Myths and Misapplications. PDA Journal of Pharmaceutical
Science and Technology, 55 (3): 176-184

Andon, B.M. (2006). Active Air vs. Passive Air (Settle Plate) Monitoring in Routine Environmental Monitoring Programmes,
PDA Journal of Pharmaceutical Science and Technology, Vol. 60, No. 6, pp350-355

Baird, R. (2015) Microbiological Environmental Monitoring. In Hanlon, G. and Sandle, T. (Eds.) Industrial Pharmaceutical
Microbiology: Standards and Controls, Euromed Publications, Passfield, UK, pp8.1-8.28

Boschi, F. (2006). Issues That Can Affect The Accuracy Of Environmental Monitoring Data in Sandle, T. and Boschi, F.
Environmental Monitoring: A Handbook, Institute of Validation Technology: Duluth, MN, USA.

Caputo, R.A. and Huffman, A. (2004): Environmental Monitoring: Data Trending Using a Frequency Model, PDA Journal of
Pharmaceutical Science and Technology, Vol. 58, No. 5, pp254-260

Cundell, A., Bean, R., Massimor, L. and Maier, C. (1998). Statistical Analysis of Environmental Monitoring Data: Does a Worst
Case Time for Monitoring Clean Rooms Exist?, PDA Journal of Pharmaceutical Science and Technology, 52 (6): 326-330

De Abreu, C., Pinto, T. and Oliveira, D. (2004): Environmental Monitoring: A Correlation Study Between Viable and Nonviable
Particles in Cleanrooms, PDA Journal of Pharmaceutical Science and Technology, Vol. 58, No. 1, pp45-53

Hussong, D. and Masden, R. E. (2004). Analysis of Environmental Microbiology Data from Cleanroom Samples.
Pharmaceutical Technology Aseptic Processing 2004, pp10-15

Gordon, O., Goverde, M., Pazdan, J., Staerk, A and Roesti, D. (2015) Comparison of Different Calculation Approaches for
Defining Microbiological Control Levels Based on Historical Data, PDA J Pharm Sci Technol.;69(3):383-98

Goverde, M., Willrodt, J. and Staerk, A. (2016) Evaluation of the recovery rate of different swabs for microbial environmental
monitoring, PDA J Pharm Sci Technol. 2016. pii: pdajpst.2016.006783

International Standards Organization (2003) ISO 14698-1. Cleanrooms and associated controlled environments–
Biocontamination Control’: Part 1: ‘General principles and methods. ISO, Geneva, Switzerland

International Standards Organization (2003) ISO 14698-2. Cleanrooms and associated controlled environments.
Biocontamination control - Part 2. Evaluation and interpretation of biocontamination data

International Standards Organization (2015) ISO 14644 - Cleanrooms and associated controlled environments - Part 1:
Classification of air cleanliness, ISO, Geneva, Switzerland

International Standards Organization (2015) ISO 14644 - Cleanrooms and associated controlled environments - Part 2:
Monitoring to provide evidence of cleanroom performance related to air cleanliness by particle concentration, ISO, Geneva,
Switzerland

Jahnke, M and K-D Kuhn. 2003. Use of the Hazard Analysis and Critical Control Points (HACCP) Risk Assessment on a
Medical Device for Parenteral Application. PDA Journal of Pharmaceutical Science and Technology, Vol. 57, No.1, pp32-42.

Kaye, S. (1986): Efficiency of Biotest RCS as a Sampler of Airborne Bacteria, PDA Journal of Pharmaceutical Science and
Technology, Vol. 42, No. 5, pp147-152

Ljungqvist, B. and Reinmuller, B. (1996): Cleanroom Design: Minimizing Contamination Through Proper Design, Interpharm
Press: Colorado, USA

Lowry, S. 2001. Designing a Contamination Control Programme. In Prince, R. (Ed.) Microbiology in Pharmaceutical
Manufacturing. DHI/PDA Publ., pp203-266.

Marshall, V., Poulson-Cook, S. and Moldenhauer, J. (1998). Comparative Mold and Yeast Recovery Analysis (The Effect of
Differing Incubation Temperature Ranges and Growth Media), PDA Journal of Pharmaceutical Science and Technology, Vol.
52, No.4, pp165-169

Meir, R. and Zingre, H. (2000): Qualification of air-sampler systems: MAS-100, Swiss Pharma, 22 (1-2): 15- 21

Moldenhauer, J. et al. (2001). Fundamentals of an Environmental Monitoring Programme,” PDA Technical Report No. 13, PDA
J. Pharma. Sci. Technol. 55 (5), supplement (2001).

Moldenhauer, J. (2008). Environmental Monitoring. In Prince, R. (Ed.). Microbiology in Pharmaceutical Manufacturing,

Parenetral Drug Association, Bethesda, MD, USA, pp19-92

Moldenhaurer, J. (2010). Passing Cleanroom Inspections. Pharmaceutical Technology Europe. 22 (12): 7-8

18
Ohresser, S., Griveau, S. and Schann, C. (2004): Validation of Microbial Recovery from Hydrogen Peroxide-Sterilised Air, PDA J
Pharm Sci Tech. 58 (2): 75-80

Ramstorp, M. (2000). Introduction to Contamination Control and Cleanroom Technology, Wiley-VCH: Weinheim

Reich, R., Miller, M., and Patterson, H. (2003). Developing a viable environmental monitoring programme for nonsterile
pharmaceutical operations. Pharmaceutical Technology. March 2003: 92-100

Russell, A.D., Furr J R and Maillard J-Y (1997). Microbial Susceptibility and Resistance to Biocides, ASM News, Vol. 9, pp. 481-
487

Sandle, T. (2003a): The use of a risk assessment in the pharmaceutical industry – the application of FMEA to a sterility testing
isolator: a case study, European Journal of Parenteral and Pharmaceutical Sciences, 2003; 8(2): 43-49

Sandle, T (2003b).: Selection and use of cleaning and disinfection agents in pharmaceutical manufacturing. In Hodges, N
and Hanlon, G. (2003 edition): Industrial Pharmaceutical Microbiology Standards and Controls, Euromed Communications:
Basingstoke, UK

Sandle, T. (2006). Environmental Monitoring Risk Assessment, Journal of GXP Compliance, Volume 10, Number 2, 2006, pp.
54-73.

Sandle, T., Saghee, M.R. and Ramstrop, M. (2010): Environmental Monitoring and Cleanrooms, IDMA-APA Guideline, Technical
Monograph No.5, Indian Drug Manufacturers Association, Mumbai.

Sandle, T. (2010b): Best practices in microbiology laboratory training in Hodges, N. and Hanlon, G.(2010 edition): Industrial
Pharmaceutical Microbiology: Standards and Controls, Supplement 11, ppS11.1 to S11.16

Sandle, T. (2011a): Environmental Monitoring. In Saghee, M.R., Sandle, T. and Tidswell, E.C. (Eds.) (2011): Microbiology and
Sterility Assurance in Pharmaceuticals and Medical Devices, New Delhi: Business Horizons, pp293-326

Sandle, T. (2011b): A Review of Cleanroom Microflora: Types, Trends, and Patterns, PDA Journal of Pharmaceutical Science and
Technology, Vol. 65, No. 4, pp392-403

Sandle, T. (2014) Examination of the Order of Incubation for the Recovery of Bacteria and Fungi from Pharmaceutical
Cleanrooms, International Journal of Pharmaceutical Compounding, 18 (3): 242 – 247

Schneider, P.K. (1995). Practical Cleanroom Design, Business News Publishing Company: Troy, MI, USA.

Suton, S. (2007). Surface Sampling. In Dixon, A.M. (Editor) Environmental monitoring for cleanrooms and controlled
environments. CRC Press: New York.

Sutton, S. (2010). The Environmental Monitoring Programme in a GMP Environment, Journal of GXP Compliance, 14 (3): 22-30

Tetzlaff, R.F. (1992). Investigational Trends: Clean Room Environmental Monitoring. PDA Journal of Pharmaceutical Science
and Technology, Vol. 46, No. 6, pp206214.

Whyte, W. (2001). Cleanroom Technology: Fundamentals of Design, Testing and Operation. Wiley-Blackwell: Oxford, UK

Whyte, W. and Eaton, T. (2004). Assessing Microbial Risk to Patients from Aseptically Manufactured Pharmaceuticals.
European Journal of Parenteral and Pharmaceutical Science. 9 (3): 71-79

World Health Organization (2003): Application of Hazard Analysis and Critical Control Point (HACCP) Methodology to
Pharmaceuticals, WHO Technical Report Series No. 908, Annex 7, World Health Organization, Geneva, 2003
(http://www.who.int/medicines/library/qsm/trs908/trs908-7.pdf).

About the author

Dr Tim Sandle
Dr. Tim Sandle has over twenty-five years’ experience of microbiological research and
biopharmaceutical processing. He is a member of several editorials boards and he has
written over six-hundred book chapters, peer reviewed papers and technical articles
relating to microbiology. Dr. Sandle works for a pharmaceutical manufacturer in the UK,
and is a visiting tutor at both the University of Manchester and UCL.

19
Excellence in Science and Service

Reading Scientific Services Limited (RSSL), is an award-winning Contract Research Organisation (CRO) and winner of Best
CRO at 2019 OBN awards. We pride ourselves on our excellence in science, quality and service.
For over 30 years, we have been providing support to
the Pharmaceutical Sterile Manufacturing Industry and
recently launched Sterility Testing (membrane filtration
and direct inoculation), with Mycoplasma Testing to be
offered soon. Our expert team can also support with raw
material, vial and stopper testing to microbial analysis such
as TAMC/TYMC and endotoxin (LAL).

We work in partnership with our clients to ensure that

they meet the regulatory requirements both with routine
testing as well as more complex projects such as cleaning
validation and environmental monitoring, using the wealth
of experience from our multi-disciplinary team of technical
experts and consultants.

Sterile Manufacturing Support Services:

• Sterility Testing
• Endotoxin Testing
• Environmental Monitoring
• Raw Materials
• Vial and Stopper Testing
• Mycoplasma
• Investigative Problem Solving
• 24/7 Emergency Response
Service
• Training and Consultancy

To find out more about how we can support your Sterile Manufacturing or to discuss your needs further, please contact us on:
+44 (0)118 918 4076, email [email protected], or visit www.rssl.com

20
 About Reading Scientific Services Ltd (RSSL)
RSSL is an award-winning Contract Research and UKAS which ensures that our analytical
Organisation (CRO), providing analytical, services meet the needs of industry. We are trusted
investigational, consultancy and training by industry to provide a solution with scientific
services to clients in the global pharmaceutical, excellence, outstanding customer service and
biopharmaceutical and healthcare sectors. professionalism.

Our chemical, physical, biochemical, biological

and microbiological services are wide ranging, and
provide support through the full product lifecycle.

RSSL is routinely inspected by the MHRA, FDA

Contact us to find out more about our

expertise and how we can support you:
Tel: +44 (0)118 918 4076 Email: [email protected] Web: www.rssl.com

© 2020 RSSL. All rights reserved.

Reading Scientific Services Ltd

The Reading Science Centre, Whiteknights Campus,
Pepper Lane, Reading, Berkshire RG6 6LA

21
View publication stats