TABLE OF

CONTENTS
ENVIRONMENTAL
MONITORING
H A N D B O O K

Environmental Monitoring Risk Assessment . . . . . . . . . . . 4

By Tim Sandle

Section One: Determining The Frequency

of Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Section Two: Risk Assessment Tools . . . . . . . . . . . . . 8
Section Three: Numerical Approaches . . . . . . . . . . . 15
Section Four: Criticality Scoring . . . . . . . . . . . . . . . 24
Section Five: Conclusion . . . . . . . . . . . . . . . . . . . . . 26

Issues That can Affect The Accuracy

of Environmental Monitoring Data . . . . . . . . . . . . . . . 30
By Freancesco Boschi, Ph.D.

Section One: Issues Related to

EM Sampling Technique . . . . . . . . . . . . . . . . . . 33
Section Two: Issues Related to EM Sampling
Instrumentation and Incubation Equipment . . . . 32
Section Three: Issues Related to Culture Media . . . 34
Section Four: Issues Related to Isolation
And Recovery of Microorganisms . . . . . . . . . . . . 36
Section Five: Issues Related To
Documentation Practices . . . . . . . . . . . . . . . . . 37
Section Six: Conclusion . . . . . . . . . . . . . . . . . . . 38

Environmental Monitoring 3
 Environmental
Monitoring
Risk Assessment
By Tim Sandle

INTRODUCTION

Environmental Monitoring describes the microbiological testing under-

taken in order to detect changing trends of microbial counts and micro-
flora growth within cleanroom or controlled environments. The results
obtained provide information about the physical construction of the room,
the performance of the Heating, Ventilation, and Air-Conditioning (HVAC)
system, personnel cleanliness, gowning practices, the equipment, and
cleaning operations.
Over the past decade, environmental monitoring has become more
sophisticated in moving from random sampling, using an imaginary grid
over the room and testing in each grid, to the current focus on risk assess-
ment and the use of risk assessment tools to determine the most appro-
priate methods for environmental monitoring.
This paper explores current trends in the application of risk assessment
to the practice of environmental monitoring by examining the following key
areas:

Determining the Frequency of Monitoring: Using the concept of

risk assessment to decide how often to monitor different types of
cleanrooms
Risk Assessment Tools: Applying risk assessment tools to estab-
lish methods for environmental monitoring
Numerical Approaches: Considering a numerical approach to
assess risk data using a case study of an aseptic filling operation

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The examples used are from a sterile drug manufacturing facility and
focus mostly on aseptic filling; however, the concepts and tools are appli-
cable to the environmental monitoring of other types of manufacturing and
packaging operations.

DETERMINING THE FREQUENCY

OF MONITORING
In developing an adequate environmental monitoring programme, there
should be a balance between using resources efficiently and monitoring at
sufficiently frequent intervals so that a meaningful picture can be obtained.
Sources of guidance with respect to monitoring frequencies are very limit-
ed within Europe, and the monitoring frequencies specified within the
United States Pharmacopoeia (USP) <1116> may not be suitable for all
facilities. Some guidance can be obtained from the International
Organization for Standardizations (ISO) standards: principally ISO 14644
and ISO 14698. However, these do not always fit with regulatory guidance
documents because they apply to controlled environments across a range
of industries other than pharmaceuticals, where standards can be higher
(Jahnke, 2001).
When establishing an environmental control programme, the frequency
of monitoring different controlled areas can be determined based on criti-
cality factors relevant to each specific area.

Criticality Factors
The establishment of a criticality scheme on which to base monitoring
frequencies is designed to target monitoring of critical process steps.
Therefore, the final formulation process would receive more monitoring
than an early manufacturing stage with a relatively closed process.
Using a criticality factor is a means of assigning a monitoring frequency
based on the risk assessment of each critical area. The risk assessment
relates to the potential product impact from any risk. For example, an area
of open processing at an ambient temperature, a long exposure time, and
the presence of water, would constitute a high risk and would attract a
higher risk rating. In contrast, an area of closed processing, in a cold area,
would carry a substantially lower risk and associated risk rating.
Using a range of 1 to 6, with 1 being the most critical and 6 the least
critical, a score of 1 would be assigned to an aseptic filling operation; a
score of 2 to final formulation, a score of 3 to open processing, and so on.
Each user must adapt such a scheme to his or her particular area and
defend it by way of supportable rationale. An example of monitoring fre-

Environmental Monitoring 5
Tim Sandle

quencies under such a Figure 1

scheme can be seen in Criticality Factors of Monitoring Frequencies
Figure 1, and an exam-
ple of its application is Criticality Factor Frequency of Monitoring
seen in Figure 2.
1 Daily or Each Batch

Each controlled 2 Weekly

area would be evaluat- 3 Fortnightly or Bi-weekly
ed against set criteria 4 Monthly
and, with the use of a
series of guiding ques- 5 Three-monthly or Quarterly
tions, the monitoring 6 Six-monthly or Semi-annually
frequency would be
determined. Decision criteria include considerations in two category areas:
areas of higher weighting and areas of higher monitoring frequency.
Examples of these categories follow:

Giving Higher Weighting to

Dirtier activity performed in a room adjacent to a clean activity,

even if the clean activity represents later processing
Areas that have a higher level of personnel transit (given that peo-
ple are the main microbiological contamination source). This may
include corridors and changing rooms.
Routes of transfer
Areas that receive in-coming goods
Component preparation activities and sites
Duration of activity (such as a lower criticality for a 30-minute
process compared to a six-hour operation)

Having Higher Monitoring Frequencies for

Warm or ambient areas as opposed to cold rooms

Areas with water or sinks as opposed to dry, ambient areas
Open processing or open plant assembly compared to processing
that is open momentarily or to closed processing (where product
risk exposure time is examined)
Final formulation, purification, secondary packaging, product
filling, etc.
Once the monitoring frequency for each controlled area is determined, it
should be reviewed at regular intervals. This review may invoke changes to a

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rooms status, and hence, its monitoring frequency, or to changes for different
sample types within the room. For example, it may be that after reviewing
data for one year, surface samples produce higher results than air samples
for a series of rooms. In this event, the microbiologist may opt to vary the fre-
quency of monitoring and take surface samples more often than air samples.
There would also be an increased focus on cleaning and disinfection prac-
tices, and their frequencies, based on such data (Sandle, 2004b).
When both types of monitoring are producing low level counts, the bal-

Figure 2
Application of Criticality Factors

Likelihood of
Environmental
Environmental Impact on
Criticality Factor Finished Product Definition Monitoring Frequency

1 Highly Likely Aseptic filling where no further Daily or Each Batch

processing takes place. Here the
risk of contamination would have
a considerable product impact
because contaminants could not
be reduced or removed by
further processing.

2 Likely Area of final formulation. This Weekly

may apply to an area where the
final process is a sterilizing
grade filter.

3 Moderately Likely Direct or indirect exposure of Fortnightly or Bi-Weekly

the product to the environment
is somewhat likely to introduce
contaminants.

This may also apply to an area

that is at ambient temperature
and where there is a high water
presence.

4 Unlikely This may apply to cold areas Monthly

where little or no open processing
takes place.

5 Very Unlikely Indirect exposure to the environment Every 3 Months or Quarterly

is highly unlikely to introduce
contaminates that could affect the
finished product. If a contaminant
were to be introduced, sufficient
downstream controls and/or the use
of preservative agents are highly
likely to remove and significantly
reduce contaminants.

6 High Unlikely An area that is uncontrolled or Every 6 Months

where microbial contamination is or Semi-annually
very unlikely, such as a freezer.

Environmental Monitoring 7
Tim Sandle

ance of risk would be toward air samples. This is because air samples are
direct indicators of the quality of the process and assign a level of control to
the process, whereas surface samples are indicators of cleaning and disin-
fection. If the results of surface samples are generally satisfactory, as indi-
cated by trend analysis, then either the number of samples or the frequen-
cy at which they are taken can be reduced. If subsequent data showed an
increase in counts, the monitoring frequency could easily be restored.
Indeed, all types of monitoring frequencies may increase as part of an
investigation, as appropriate. Therefore, the criticality factor approach not
only sets the requirement for a room, it can also be used to vary the sam-
ple types within a room (Ljungqvist and Reinmuller, 1996).

RISK ASSESSMENT TOOLS

Once the status for each room has been selected, a risk assessment
procedure is required to determine locations for environmental monitoring.
Such risk-based approaches are recommended in ISO 14698 and regulato-
ry authorities are increasingly asking drug manufacturers about this subject.
Risk-based approaches include Failure Mode and Effects Analysis
(FMEA), Fault Tree Analysis (FTA), and Hazard Analysis and Critical
Control Points (HACCP), all of which employ a scoring approach. (Other
approaches include: Failure Mode, Effects, and Criticality Analysis
(FMECA); Hazard Operability Analysis (HAZOP); Quantitative
Microbiological Risk Assessment (QMRA); Modular Process Risk Model
(MPRM); System Risk Analysis (SRA); Method for Limitation of Risks; and
Risk Profiling.)
At present, no definitive method exists, and the various approaches dif-
fer in their process and in the degree of complexity involved. However, the
two most commonly used methods appear to be HACCP, which originated
in the food industry, and FMEA, which was developed for the engineering
industry (Whyte and Eaton, 2004a).
These various analytical tools are similar in that they involve:

Constructing diagrams of work flows

Pin-pointing areas of greatest risk
Examining potential sources of contamination
Deciding on the most appropriate sample methods
Helping to establish alert and action levels
Taking into account changes to the work process and seasonal
activities

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These risk assessment approaches are not only concerned with

selecting environmental monitoring locations. They integrate the environ-
mental monitoring system with a complete review of operations within the
cleanroom to ensure those facilities, operations, and practices are also
satisfactory. The approaches recognise a risk, rate the level of the risk,
and then set out a plan to minimise, control, and monitor the risk. The
monitoring of the risk will help to determine the frequency, locations for,
and level of environmental monitoring (for example, refer to an article by
Sandle [2003a], for a more detailed example).

This paper explores an example from three different techniques:

A simple conceptualisation of risk using a table

HACCP
FMEA

Tabular Approach
An example using a simple table for analyzing risk in environmental
monitoring situations appears in Figure 3.

Figure 3
Tabular Approach to Risk Assessment

Area or Equipment: Sterility Testing Isolator

Risk: Contamination due to build-up of microbial counts in the isolator environment

Failure or Situation: Failure to adequately clean after use

Minimising the Risk

Effect (Mitigations to Reduce Risk) Monitoring

When isolators are not Cleaning surfaces using water to
cleaned regularly, there remove dirt or spillages prior to the
is a possibility of application of a suitable disinfectant.
micro-organisms
remaining in the The disinfectant used must have
environment. a wide spectrum of efficacy, but
not be aggressive to the isolator
material.
The isolator should be designed
so that it is easy to clean.
An environmental monitoring
programme (using settle plates,
air samples, contact plates, swabs,
or finger plates) will show the areas
of greatest risk. This data should be
examined for trends.
For out-of-limits environmental
monitoring results, appropriate
Corrective and Preventive Actions
(CAPA) should be put in place.

Environmental Monitoring 9
Tim Sandle

HACCP
The seven principles behind constructing an HACCP analysis consist of:

1. Identifying hazards or contamination risks and assessing their

severity
2. Determining Critical Control Points (CCPs)
3. Establishing critical limits
4. Establishing a system to monitor and control CCPs
5. Establishing corrective action when a CCP is not under control
6. Establishing procedures for verification to confirm that the
HACCP system is working effectively
7. Establishing documentation and reporting systems for all proce-
dures

Each of these seven key points is a vital step in developing the risk
assessment.

The seven points include:

1. Construct a risk diagram, or diagrams, to identify sources of contami-

nation. Diagrams should show sources and routes of contamination.

Examples include:
Areas adjacent to Cleanroom or Isolator (e.g.: airlocks, chang-
ing rooms)
Air supply and Room air
Surfaces
People
Machines and Equipment

2. Assess the importance of these sources and determine whether

or not they are hazards that should be controlled.

Examples include:
Amounts of contamination on, or in, the source that is avail-
able for transfer
Ease by which the contamination is dispersed or transferred
Proximity of the source to the critical point where the product
is exposed
Ease with which the contamination can pass through the
control method

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The use of a scoring method can greatly help in assessing the

relative importance of these contamination sources.

3. Identify the methods that can be used to control these hazards.

For example:
Air Supply: High Efficiency Particulate Air (HEPA) filters
Dirty Areas adjacent to Cleanroom or
Isolator: differential pressures, airflow movement
Room Air: air change rates, use of barriers
Surfaces: sterilisation, effectiveness of cleaning and
disinfection procedures
People: cleanroom clothing and gloves, room ventilation,
training
Machines and Equipment: sterilisation, effectiveness of
cleaning, exhaust systems

4. Determine valid sampling methods to monitor either the hazards

or their control methods or both.

For example:
HEPA filter integrity tests
Air supply velocity, air change rates
Room pressure differentials
Particle counts
Air samplers, settle plates, contact plates, etc.

5. Establish a monitoring schedule with alert and action levels

and the corrective measures to be taken when these levels
are exceeded.

For example:
The greater the hazard, the greater the amount of monitoring
required
Trend analysis for alert and action levels, in or out of control

Environmental Monitoring 11
Tim Sandle

6. Verify that the contamination control system is working effectively

by reviewing key targets like product rejection rate, sampling
results, control methods, and so on. These may require modifica-
tion over time.
For example:
System for data review
Examine filling trials
Audits
Reassess - hazards, effectiveness of control systems, frequency
of monitoring, appropriateness of alert and action levels

7. Establish and maintain documentation.

For example:
Describe the steps being taken
Describe the monitoring procedures
Describe the reporting and review procedures

Before implementing HACCP, it is important to train all staff involved in

the process and to use a multi-disciplinary team. For example, the team
may be comprised of personnel from Production, Engineering, Quality
Control (QC), Quality Assurance (QA), Validation, and so on.

FMEA
FMEA schemes vary in their approach, scoring, and categorisation. All
methods share a numerical approach. The example presented here,
based on a sterility testing isolator, assigns a score (from 1 to 5) to each
of the following categories:

Severity
Occurrence
Detection

Where:
Severity is the consequence of a failure
Occurrence is the likelihood of the failure happening based on
past experience
Detection is based on the monitoring systems in place and on
how likely a failure can be detected

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By asking a series of questions, each main part of the cleanroom or

isolator system can be grouped or classified into key parts.

Such questions include:

What is the function of the equipment? What are its perform-
ance requirements?
How can it fail to fulfil these functions?
What can cause each failure?
What happens when each failure occurs?
How much does each failure matter? What are its conse-
quences?
What can be done to predict or prevent each failure?
What should be done if a suitable proactive task cannot be
found?

The scoring is 1 (very good) to 5 (very bad). Therefore, a likelihood of

high severity would be rated 5; high occurrence rated 5; but a good detec-
tion system would be rated 1.

Using these criteria, a final FMEA score is produced from:

Severity score
x
Occurrence score
x
Detection score

Decisions on further action will depend upon the score produced.

There is no published guidance on what the score that dictates some form
of action should be. However, 27 is the suggested score for the cut-off
value at which action is required. This is based on 27 being the score
derived when the mid-score is applied to all three categories (i.e., the
numerical value '3' for severity 3 x occurrence 3 x detection 3) and the
supposition that if the mid-rating (or a higher number) is scored for all
three categories, then at a minimum, the system should be examined in
greater detail.

Environmental Monitoring 13
Tim Sandle

Figure 4
Isolator Operation Example

An example of one area of an isolator operation, and the risks associated with the
room in which the isolator is housed, is examined below.

Description of the Critical Area: The isolator is situated in an unclassified

room. There is no requirement to place a sterility testing isolator in a classified room.

FMEA Schematic:

Severity of
Significance Consequence
Process Step Failure Mode of Failure (score)
Loading isolators That contamination from the Reduced efficiency of 3
pre-sanitisation, room could enter transfer or transfer isolator
performing sterility main isolators sanitisation, contamination
testing inside main isolator

Measures to Detection
Detect Failure Occurrence (score) Detection Systems (score)

Would be shown from 1 Isolator room is monitored 1

reduced evaporation rate monthly for viables
for isolator sanitisation, and particles, staff wear
poor environmental over-shoes on entry, Dycem
monitoring results in mat in place, entry to room
main isolator, potential has controlled
sterility test failures. access, environmental
Sanitisation cycle has monitoring performed
been validated using inside main isolator.
biological indicators Isolators are at positive
of 106 spores. pressure to the
room, and air is
HEPA filtered.

FMEA score: 3 x 1 x 1 = 3

Analysis: There is no problem to be considered from the room environment

described. Entry to the room is controlled; the sanitisation cycle has been challenged
with a level of micro-organisms far greater than would ever be found in the environ-
ment (spores of Geobacillus stearothermophilus); all items entering the isolator are
sanitised (using a chlorine dioxide-based sporicidal disinfectant); and the isolator itself
is an effective, positive pressure barrier to the outside (at >15 pascals).

As detailed earlier, environmental monitoring is performed inside the isolator during

testing. This monitoring, which has an action level of 1 CFU (Colony Forming Unit), is
designed to detect any potential contamination inside the isolator environment.

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NUMERICAL APPROACHES
A third component of the risk assessment approach is to evaluate a
risk once an activity has taken place. Then, by using a largely numerically-
driven set of tools, repeatability and reproducibility can be ensured.
Examples of individual out-of-limits results and data-sets relating to an
operation are examined below using examples from an aseptic filling
process. Following this, an example of an overall assessment of different
processes over time is explored. Numerical approaches are useful in
applying a level of consistency between one decision and another.

Individual Assessments
The section below details some methods that can be used to quantify
the risk of contamination in pharmaceutical cleanrooms. The models out-
lined are based on the work performed by Whyte and Eaton (2003a and b).

Estimating the Risk to Product Using Settle Plate Counts

The method applies to the assessment of settle plates at the point-

of-fill, under the Grade A zone. It allows an estimate of the proba-
ble contamination rate to the product as derived from the following
equation:

Contamination rate (%) =

Settle plate count x

Area of product x
Area of petri dish

Time product exposed x 100

Time settle plates exposed

Environmental Monitoring 15
Tim Sandle

The fixed value is the area of the petri dish, which for a 90mm plate,
is 64 cm2.

Settle Plate Count Worked Example:

Area of petri dish = 64 cm2

Settle plate count = 1 cfu
Neck area of product = 1 cm 2
Exposure time of product = 1 minute
Exposure time of settle plate = 240 minutes
By inserting these example values into the equation:

1 x 1 x 1 = 0.000065 x 100 = 0.0065%

64 240

The formula can also be applied to the monitoring of product filtration

activities when 1 is entered as a constant for neck area of product.
There is no available guide as to what percentage constitutes which
level of risk. The 0.03% figure has been used by some practitioners. This
is based on the Parenteral Drug Association Survey of Aseptic Filling
Practices (2002), where it is common in the pharmaceutical industry to
allow 0.03% of broth bottles in a media simulation trial to exhibit growth at
a warning level (where 0.03% = 1/3,000, with 3,000 being the average
size of a media fill). An action level is often set as 3/3,000 bottles or 0.1%.
This would constitute a high risk. Logically, the range between 0.03 and
0.1 would be a medium risk (Whyte and Eaton, 2004c).
Therefore, where the risk is that of micro-organisms detected on a set-
tle plate, with a probability of <0.1% depositing in the neck of a bottle
when bottles are exposed in a unidirectional air flow, risk categories would
be as shown in Figure 5.

Figure 5
Micro-Organism Risk Categories

Percentage Risk
<0.03% Low
>0.03 0.09% Medium
0.1% High

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Finger Plate Assessment

The formula can readily be applied to operations that relate to Grade A
operations, for example: filtration connection, vessel to filling machine con-
nection, the filling activity, and loading a freeze-dryer. Where the operator
is only present in the Grade B room and has no impact on the Grade A
operation, this is automatically considered to be low risk if there are no
other special factors. (Low risk does not imply lack of action or assess-
ment. However, it aims conceptualisation of the result in terms of probable
risk to the batch.)

The following formula can be used:

Microbial count x Location x Method of intervention x Duration of operation

Where:

Microbial Count = Count in cfu for the plate

Location = Area of the filling machine, or
other location to which the
plate relates
Activity = Whether the hand directly
touched part of the filling
machine or if utensils were
used
Duration = Length of the activity
in seconds

In this example of a finger plate assessment, the location, activities,

and duration require weighting. Examples of logic that apply to the rating
of the location, activities, and duration categories can be seen in Figures
6, 7, and 8, respectively

Environmental Monitoring 17
Tim Sandle

Figure 6
Weighting Location Example

Location Rating* Reason

General part of machine 0.5 Data from air flow patterns suggests very low risk of contamination
not close to filling zone movement into the unidirectional air stream over the filling zone

Off-load 0.5 Off-load areas are present for all filling machines. The bottles and
vials are partially stoppered and utensils are normally used. The
likelihood of contamination is considered to be low.

On-load 1.0 On-load areas are present for all filling machines. The bottles and
vials are not stoppered, although utensils are normally used. The
likelihood of contamination is higher than for off-load.

Stopper bowl 1.5 Stopper bowls are present for filling machines. A direct intervention
into the bowl could result in micro-organisms being deposited onto
stoppers. The risk of this is considered higher than the risk with
on-load or off-load activities, although such an intervention is rare.

Freeze-dryer loading 1.5 This is a direct intervention Grade A activity. However, vials and
bottles are partially stoppered and are contained with cassettes.

Point-of-fill: air sample 2.0 The placement of an air sampler does not involve the touching of
placement any filling equipment (such as needles, balances etc.). However, as
a direct intervention into the Grade A zone, it is a higher risk than
those parts of the filling machine previously examined.

Filtration transfer
2.0 The connection of a vessel for the purpose of transferring a product
into the Aseptic Filling Suite requires human intervention and aseptic
technique. If this process becomes contaminated, this could affect the
product. The time taken to perform the connection is normally very
short (under 30 seconds), which reduces the risk.

Machine connection 2.5 The connection of a vessel to the filling machine requires human
intervention and aseptic technique. If the transfer line is contaminated,
this could cause contamination to the product.

Point-of-fill: 2.5 A direct intervention, where for example, filling needles are re-adjusted,
intervention is the highest risk rating. Counts associated with such activities require
detailed examination.

Figure 7
Weighting Activities Example

Activity Method Rating Reason

Using forceps 0.5 The operative does not directly touch the machine and the
utensils used are sterile.

Hand 1.0 The operative directly touches the machine, thereby creating
a greater risk. However, it is procedure to sanitise hands prior
to undertaking the operation.

* Based on the average time taken for media simulation trials, based on data from a
UK pharmaceutical facility.

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Figure 8
Weighting Duration Example

Duration Rating Reason

Less than 30 seconds 0.5 The length of the intervention is considered minimal.

30 seconds - 120 seconds 1.0 The intervention is at the average* time taken.

Plus 120 seconds 1.5 The intervention has taken longer than average*.

Finger Plate Assessment Worked Example

A finger plate with a count of 1 cfu for an activity at point-of-fill, using

forceps, that lasts for one minute.

Microbial count x Location x Method of intervention x Duration of operation

1 x 2.5 x 0.5 x 1 = 1.25

The score produced would be rated Score Risk

according to standard risk assessment 1-3 Low
categories:
4-8 Medium
These risk ratings are based, in part, on
9+ High
the worked example. Based on historical data
over the past six-months, the highest record
example of a Grade A intervention finger plate is a count of 2 cfu: using
forceps to retrieve a fallen vial and lasting for more than 120 seconds. This
would have given a score of 7.5, which falls within the medium risk cate-
gory. The user should develop a scheme that fits his or her facility (Whyte
and Eaton, 2004b).

Surface Sample Assessment

The following formula can be applied to filling and filtration activities:

Microbial count x Risk Factor A x Risk Factor B x Risk Factor C

Where:
Risk Factor A = Proximity to critical area
Risk Factor B = Ease of dispersion of micro-organisms
Risk Factor C = Effectiveness of control measure

Environmental Monitoring 19
Tim Sandle

Samples are taken using contact plates and swabs and are all
post-operation.

The following approach can be used in setting the risk factors:

The first step is to assign the risk (A) factor based on proximity of
location to the critical area (filled product). The logic demonstrated
in Figure 9 may be used to determine risk factor A.

Figure 9
Determining Risk Factor A

Manufacturing Risk Factor Reason

Stage/ Location (A)

Filtration room 2.0 Samples of the transfer line may indicate potential for
product contact contamination to affect product.

General filling room 0.5 The samples reflect room cleanliness and general trends only.
or filtration room area The impact upon the Grade A activity is low - unless the same
(Grade B) micro-organism has been detected from a Grade B action level
sample. In this event, the risk factor increases to 1.

Machine general 1.0 Samples indicate state of general machine cleanliness but,
(non-product contact) the risk of exposure of product to contaminant is low - unless
the same micro-organism has been detected from a Grade B
action level sample. In this event, the risk factor increases to 2.5.

Machine product 2.5 Sites include utensils, filling needles, and stopper bowls.
contact site Direct contact with product: highest risk.

The second step is to assign a risk factor (B) based on ease of

dispersion or transfer of micro-organisms. See Figure 10 for an
example of the reasoning that would support Risk Factor B.

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Figure 10
Determining Risk Factor B

Manufacturing Risk Factor Reason

Stage/ Location (B)

Filtration room 0.5 Connection is a short activity (less than thirty seconds)
product contact performed under Grade A Uni-Directional Air Flow (UDAF)
protection; operator wears sanitised gloves.

General filling room or 1.0 Periphery to the Grade A zone. Risk of transfer is low.
filtration room area Risk rating would increase to 1.5 if a Grade A and a Grade B
(Grade B) sample exceeded action level and was characterised as the
same microbial species.

Machine general 1.5 Location is within the critical area, but not directly in product
(non-product contact) contact area. Some risk of transfer exists, but protective
measures should prevent this.

Machine product 2.5 Sites include utensils, filling needles, and stopper bowls that
contact site are directly within the critical zone. Direct contact with
product: highest risk.

The third step is to weight the risk factor C by assessing the

effectiveness of the control measure. See Figure 11 for an
example of this assessment.

Figure 11
Determining Risk Factor C

Manufacturing Risk Factor Reason

Stage/ Location (C)

Filtration room 0.5 Grade A UDAF and sterilised components;

product contact operator wears two pairs of gloves and sanitises hands.

General filling room 0.5 Floor is sanitised. Barrier exists by way of filling
or filtration room area machine doors and UDAF.
(Grade B)

Machine general 1.0 Sterilised machine components; lines are wiped with
(non-product contact) disinfectants; UDAF protection.

Machine product 1.5 Sterilised machine components; no direct intervention; UDAF

contact site protection; however, site is in direct contact with product.

Environmental Monitoring 21
Tim Sandle

Surface Sample Worked Example:

Where a count of 2 is detected from a conveyor belt (a filling
machine non-product contact location)

Using the formula:

Microbial count x Risk Factor A x Risk Factor B x Risk Factor C

2 x 1 x 1.5 x 1 = 3

Risks can be scored Score Risk

against standard risk 1-3 Low
assessment categories:
4-8 Medium
9+ High

This scoring scheme is based on contamination of a product contact

site being high risk by virtue of its direct proximity to the critical area or the
product.
A count of 1 cfu on one of these product contact site locations would
give a score of 9.4. In most filling zones and clean zones, sample results
from product contact sites would be expected to record zero counts for
999 samples out of every 1000. Whereas, a count of 3 from a non-product
contact site would result in a medium risk category.

Air Sample Assessment

Approaches are available for the risk assessment of active air samples
that use a numerical system. However, the formulae associated with these
are difficult to calculate in practice because often all information is not
available and assessment of variables, such as impaction speed, are not
readily calculable. Therefore, a qualitative assessment, such as the one
included in the example of the numerical approach, may be more suitable.

An example of the numerical approach:

Airborne microbial count (cfu /m3) x deposition velocity of micro-

organisms from air (cm/s) x area of product exposed (cm2) x time
of exposure (s)

Alternatively, non-numerical risk assessment can be used based on

the proximity and the operation. See Figure 12 for this example.

22 Institute of Validation Technology

 Tim Sandle

Figure 12
Non-Numerical Risk Assessment Example

Activity, Area, Risk Reason

Proximity

General room Low Provided there are no Grade A interventions and there are
environment during no counts recorded at Grade A, where counts are recorded
filling or filtration, at Grade A and there is a micro-organism match, then the
away from Grade A zone category is re-defined as medium risk

Grade A, near critical Medium Close, but not at the point-of-fill

zone

Grade A point-of-fill at High At point-of-fill

critical zone

Note: Where growth is detected on the operator who placed the

air sampler at Grade A, and this is shown to be the same
micro-organism, the category of risk is increased by one
(i.e.: low becomes medium and medium becomes high).

Assigning a Risk Factor to Areas of the Filling Room

The location where a high bio-burden is isolated within the filling area
is arguably of greater consequence than the actual count. The location
can be given a risk rating in relation to its proximity to the critical zone,
ease of dispersion or transfer, and effectiveness of control methods.
The table shown in Figure 13 is proposed as a tool for risk assess-
ment and to aid investigations. It supplements the risk assessment tools
that have been previously examined.

Figure 13
Filling Room Risk Assessment Example

Ease of Dispersion Proximity or Location Effectiveness of

or Transfer of of Source from Control Method
Micro-organisms Critical Area

None None None

Very low, e.g.: fixed place Low, e.g.: at extreme limit Low, e.g.: barrier control; UDAF
on sterilised area of room away from filling zone

Medium, e.g.: product Medium, e.g.: general Medium, e.g.: sanitisation

contact device area of cleanroom near
filling machine or at edges
of Grade A zone

High, e.g.: gloved hands High, e.g.: within the critical area High, e.g.: no effective controls
of operators with direct
contact with product

Environmental Monitoring 23
Tim Sandle

The type of product and whether further processing occurs can also
influence the risk factors. See Figure 14 for an example. In considering
batches with a high risk rating, further processing of the product can be
considered and ranked (1 = lowest risk, 4 = highest risk).

Figure 14
Product-related Risk Assessment Factors

Product Rank Reason

Freeze-dried 1 Freeze-drying will destroy most micro-organisms

Liquid product, heat treated 2 Undergoes pasteurisation - effective against most non spore-forming
micro-organisms

Intra-muscular product 3 Small volume; intra-muscular route

Intravenous product, 4 Intravenous route; no further processing

no further treatment

An Overall Assessment
The approach taken for an overall assessment involves the historical
examination of a number of operations and assigning a value above which
the operation is considered to be atypical. A 95% cut-off is considered to
be the most suitable cut-off point.

CRITICALITY SCORING
Criticality scoring is a way of assessing the totality of results from an
environmental monitoring session. This may be, for example, a batch fill.
The data from the session is examined and points are awarded for each
result above a pre-set warning or action level. The total score is then
summed and the results obtained are compared to a set level at which
atypical sessions are indicated.
The pre-set level would be assessed from historical data over a
reasonable time period (such as one year). An example of such a
scheme follows:

For Grade A
The results from a filling operation are examined (for individual viable
counts and for the mean particle counts taken during the fill). Each result,
which equals or exceeds a warning or action level, is scored according to

24 Institute of Validation Technology

 Tim Sandle

Figure 15
Viable Counts Criteria for Grade A

Count (cfu)

Sample Warning Level Action Level

Active Air Sample 5 points 10 points
Settle Plate 5 points 10 points
Contact Plate 5 points 10 points
Swab 5 points 10 points
Finger Plate 5 points 10 points

Figure 16
Particle Count Criteria for Grade A

Count (cfu)

Particle Size Mean Counts from fill Mean Counts from fill
at Warning Level at Action Level
0.5 m 5 points 10 points
5.0 m 5 points 10 points

the criteria in Figure 15 and Figure 16. Using the criteria presented in
Figures 15 and 16 produces the Grade A score.

For Grade B
The results from a filling operation are examined (for the individual
viable counts and the mean particle counts taken during the fill). Each
result that equals or exceeds a warning level is scored according to the
criteria in Figure 17 and Figure 18.

Figure 17
Viable Counts Criteria for Grade B

Count

Sample 1 cfu 2-3 cfu 4-5 cfu Warning Level Action Level
Active Air Sample 1 point 2 points 3 points 3 points 5 points
Settle Plate 1 point 2 points 3 points 3 points 5 points
Contact Plate 1 point 2 points 3 points 3 points 5 points
Swab 1 point 2 points 3 points 3 points 5 points
Finger Plate 1 point 2 points 3 points 3 points 5 points

Environmental Monitoring 25
Tim Sandle

Figure 18
Particle Count Criteria for Grade B

Count (c/cf)

Particle Size Mean Counts from fill Mean Counts from fill
at Warning Level at Action Level
0.5 m 4 points 5 points
5.0 m 4 points 5 points

If a warning level or action level is of the same count (cfu) as a value in

the count (cfu) column, the warning or action level score should be select-
ed. This produces the Grade B score. To produce the total criticality score,
the two scores are added together (Grade A + Grade B).
Once the data has been generated, the score at which approximately
95% of the fills would be below (and 5% would be above) can be calculat-
ed. That figure would then be used as the cut-off value with which to
assess the atypical filling operations.
Figure 19 displays a simple representation of this assessment. For the
data set, the criticality score was calculated at 25, which corresponded
with the 95th percentile for a set of data from the filling of an example
drug.
The graph in Figure 19 indicates that some fills exceeded the cut-off
criticality value during a particular time period (see fill numbers 12 through
15). After some corrective action, the scores for the fills were reduced (see
fill numbers 16 through 29) and the situation returned to a state of control.

CONCLUSION

The use of risk assessment approaches is an important current Good

Manufacturing Practice (cGMP) topic in microbiological environmental
monitoring. This paper has outlined some possible tools for such a risk
assessment approach; however, each suite of cleanrooms or isolator will
be subtly different. The microbiologist must consider each aspect of the
environment and decide what level of monitoring best suits his or her sys-
tem, and then must justify the techniques used and the locations selected.
The approach adopted should be detailed in a written rationale and
approved by senior management. After this, a rigorous and defensible sys-
tem will be in place to satisfy regulatory expectations, and to aid the user
in assessing the risk of problematic environmental monitoring situations or
results.

26 Institute of Validation Technology

 Tim Sandle

Figure 19
Graph of Criticality Scores

Criticality cut-off
value = 25 Criticality Scoring
45
40
35
30
25
20
15
10
5
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Fill Number

REFERENCES
BS EN ISO 14698 1:2003: Cleanrooms and Associated Controlled Environments
Bio-Contamination Control Part 1: General Principles and Methods.
Code of Federal Regulations, 1998, Title 21, Part 210, Current Good Manufacturing
Practice in Manufacturing, Processing, Packaging, or Holding of Drugs General, 210:3.
'Guidelines on Sterile Drug Products Produced by Aseptic Processing,' FDA, 1987 (draft
produced for review in August 2003).
ISO 14644-1 Cleanrooms and Associated Controlled Environments Classification of
Air Cleanliness.
ISO 14644-2 Cleanrooms and Associated Controlled Environments Specifications for
Testing and Monitoring to prove continued compliance with ISO 14644-1.
ISO 14698-1 Cleanrooms and Associated Controlled Environments Biocontamination
Control Part 1: General Principles.
Jahnke, M. Introduction to Environmental Monitoring in Pharmaceutical Areas, PDA, 2001.
Ljungqvist, B. and Reinmuller, B. Some Observations on Environmental Monitoring of
Cleanrooms, European Journal of Parenteral Science, 1996 1: 9 13.
PDA Technical Report No. 13 (revised): 'Fundamentals of an Environmental Monitoring
Programme,' September/October 2001.
Sandle, T. 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. Selection and Use of Cleaning and Disinfection Agents in Pharmaceutical
Manufacturing in Hodges, N and Hanlon, G. (2003): Industrial Pharmaceutical
Microbiology Standards and Controls, Euromed Communications, England.
Sandle, T. General Considerations for the Risk Assessment of Isolators Used for Aseptic
Processes, Pharmaceutical Manufacturing and Packaging Sourcer, Samedan Ltd,
Winter 2004, pp: 43-47.

Environmental Monitoring 27
Tim Sandle

USPNF#25 <1116>
Whyte, W. Cleanroom Technology: Fundamentals of Design, Testing and Operation,
2001.
Whyte, W. and Eaton, T. Microbiological Contamination Models for Use in Risk
Assessment during Pharmaceutical Production, European Journal of Parenteral and
Pharmaceutical Sciences, 2004 Vol. 9, No.1, pp: 11-15.
Whyte, W. and Eaton, T. Microbiological Risk Assessment in Pharmaceutical
Cleanrooms, European Journal of Parenteral and Pharmaceutical Sciences, 2004, Vol. 9,
No.1, pp: 16-23.
Whyte, W. and Eaton, T. Assessing Microbial Risk to Patients from Aseptically
Manufactured Pharmaceuticals, European Journal of Parenteral and Pharmaceutical
Sciences, 2004, Vol. 9, No.3, pp: 71-79.

Article Acronym Listing

CAPA Corrective and Preventive Action
CCPCritical Control Point
CFU Colony Forming Unit
cGMP Current Good Manufacturing
Practice
FMEA Failure Mode and Effects Analysis
FMECA Failure Mode, Effects, and Criticality Analysis
FTA Fault Tree Analysis
HACCP Hazard Analysis and Critical
Control Point
HAZOP Hazard Operability Analysis
HEPA High Efficiency Particulate Air
HVAC Heating, Ventilation, and
Air-Conditioning
ISO International Organization for
Standardization
MPRM Modular Process Risk Model
QA Quality Assurance
QC Quality Control
QMRA Quantitative Microbiological Risk Assessment
SRA System Risk Analysis
UDAF Uni-Directional Air Flow
USP United States Pharmacopoeia

28 Institute of Validation Technology

 Tim Sandle

SUGGESTED READING
Anon. (1998), The Gold Sheet, Vol. 32, No.10, October 1998.
De Abreu, C., Pinto, T. and Oliveira, D. Environmental Monitoring: A Correlation Study
between Viable and Nonviable Particles in Cleanrooms, Journal of Pharmaceutical
Science and Technology, Vol. 58, No.1, January-February 2004, pp: 45-53.
Kaye, S. Efficiency of Biotest RCS as a Sampler of Airborne Bacteria, Journal of
Parenteral Science and Technology, Vol. 42, No.5, September-October 1986, pp: 147-
152.
Meir, R. and Zingre, H. Qualification of Air Sampler Systems: MAS-100, Swiss Pharma,
22 (2000), pp: 15 21.
Ohresser, S., Griveau, S. and Schann, C. Validation of Microbial Recovery from
Hydrogen Peroxide-Sterilised Air, Journal of Pharmaceutical Science and Technology,
Vol. 58, No. 2, March-April 2004, pp: 75-80.
PhRMA Environmental Monitoring Work Group Microbiological Monitoring of
Environmental Conditions for Nonsterile Pharmaceutical Manufacturing, Pharm.
Technol., March 1997, pp: 58-74.
Reich, et al. Developing a Viable Microbiological Environmental Monitoring Program for
Nonsterile Pharmaceutical Operations. Pharm. Technol., March 2003, pp: 92-100.
'Rules and Guidance for Pharmaceutical Manufacturers and Distributors' ('EU GMP
Guide'), MHRA, 2002.
Sandle, T. Environmental Monitoring in a Sterility Testing Isolator, PharMIG News No. 1,
March 2000.
Sandle, T. Microbiological Culture Media: Designing a Testing Scheme, PharMIG News
No. 2, August 2000.

ABOUT THE AUTHOR

Tim Sandle is the company Microbiologist at Bio Products Laboratory (BPL).
BPL is the manufacturing unit of the UK National Health Service - Blood and
Transplant.
Prior to his current role, Tim has worked on a number of different microbiological
projects within the Pharmaceutical Industry, including: developments in the test-
ing of endotoxins and pyrogens for protein-based products, establishing the
environmental monitoring regime for a network of over two-hundred cleanrooms,
and validating a sterility testing isolator system.

Tim has written more than forty articles relating to microbiology and pharmaceu-
tical operations, including: LAL testing, operation of isolators,
cleanrooms, and environmental monitoring. Tim may be contacted by email at
his BPL address, tim.sandle@bpl.co.uk or at his home address of:
timsandle@aol.com

Originally published in the January 2006 issue of the Journal of GXP Compliance

Environmental Monitoring 29
 Issues That Can Affect
The Accuracy Of
Environmental
Monitoring Data
By Francesco Boschi, Ph.D.

INTRODUCTION

In the last decades, the importance of implementing and maintaining

an Environmental Monitoring (EM) Program that is compliant with current
Good Manufacturing Practice (cGMP) has increased significantly, espe-
cially in manufacturing plants dedicated to the production of injectable
drugs. Numerous guidelines have been issued on this topic and the inter-
est regulatory authorities have shown concerning EM data, and how these
data are generated, has been growing continually.
EM is generally recognized as a fundamental part of the sterility assur-
ance system related to the manufacture of sterile drugs, especially by
aseptic processing. Nevertheless, it is generally assumed that monitoring
environmental conditions from a microbiological point of view can never
lead to complete and objective knowledge of the quantity and quality of
microbial flora in the manufacturing area. The sensitivity of traditional moni-
toring techniques, such as air sampling, swabs, or Rodac , is well known to
be very low, and, from a time and space perspective, there is no practical
possibility of covering all the manufacturing processes with a monitoring
program.
Although monitoring is similar to photographing the manufacturing envi-
ronment using an old camera (with a delay of five to seven days in obtain-

30 Institute of Validation Technology

 Francesco Boschi. Ph.D.

ing the image due to incubation time), the resulting pictures have great
importance in maintaining the process under control and should reflect
reality as closely as possible. Regarding EM data, it is essential to com-
pare data obtained in a medium-long timeframe against expected perform-
ance to detect any drift from the normal status of the system(as it was
assessed during the initial validation phase),and simply to provide evi-
dence that things are still going as expected.
The ability of generating accurate, reliable, and consistent EM data is,
therefore, a key aspect of laboratory compliance and a fundamental tool in
sterility assurance, especially for aseptically filled products. Moreover, a
lack of reliability in EM data could lead to serious business issues, since
contamination caused by improper sampling techniques or non sterile
media in a critical location within grade A areas may have a significant
impact, at least in delaying batch release and in allocating time and
resources for the necessary investigation. On the other hand, any question
regarding the ability of the EM program to effectively detect environmental
contamination, especially in critical areas, could raise serious doubts con-
cerning the credibility of the aseptic process, of all sterility assurance lev-
els, and could potentially lead to dramatic consequences such as the
recall of batches already released.
Recent inspection focus has been moving from a simple revision of EM
data to an accurate assessment concerning data generation in order to
evaluate whether the data are really meaningful and representative of the
actual situation.

Issues Related to EM Sampling Technique

The accuracy of EM data can be affected by many factors, such as the
training of Quality Control (QC) operators or, generally speaking, the train-
ing of anyone responsible for the execution of monitoring activities; the cal-
ibration of monitoring equipment, including those dedicated to incubation
and processing of samples; the choice and validation of culture media; the
quality control of media; and the choice and validation of incubation condi-
tions and documentation.
Regarding operator training, people conducting EM should understand
the scientific basis of their activity including the factors that can affect
microorganism proliferation. The educational background and experience of
all operators should be adequate to support the understanding required by
their duties.
More specifically, people conducting EM should be trained properly on
the subjects of gowning procedures and aseptic techniques, in the use of

Environmental Monitoring 31
Francesco Boschi. Ph.D.

sampling devices and equipment, and in all relevant cGMP documentation

skills. Management of sampling activities is another critical issue that
requires specific skills and training, since routine EM monitoring activities
in an average-size clean room typically imply a huge number of sam-
plings, with many different locations and frequencies.
While operating in grades A and B, people conducting EM may experi-
ence problems in communicating with their supervisors or colleagues who
work outside the clean room. Therefore, it is important for EM technicians
to be able to manage monitoring activities properly, according to defined
Standard Operating Procedures (SOPs) and sampling plans. They must
also react in appropriate ways to unexpected events in the clean room,
performing extra monitoring, as necessary. An EM technician's witnessing
of manufacturing activities and aseptic behaviors can often be useful in
the development of investigations due to excursions from alert or action
levels. Since monitoring activities are usually complicated, good skills in
documentation are also required to be able to track all the performed
activities correctly and in a timely manner.
Working with culture media in the clean room is critical, since these
materials have been designed to promote the proliferation of exactly what
we do not want in the clean room itself! Ideally, monitoring activities in criti-
cal areas should not be conducted by humans, who are by far the biggest
source of contamination, but, since the outcome of automation in aseptic
operations, including environmental monitoring, is still far away, we must
rely on effective personnel training, the development of adequate skills,
and the personal, professional commitment of the technicians.
EM operators must be aware of the significant potential impact of their
activities on product sterility assurance. They should consistently apply
proper clean room procedures. The personnel monitoring data of EM oper-
ators should be carefully reviewed and, at a minimum, their annual partici-
pation in successful media fills, with actual involvement in monitoring activ-
ities, is required. (The debate concerning the delegation of all or some
monitoring to production staff, instead of dedicated people from the
Quality Unit, is still open.)
In case you have production staff performing some or all EM activities,
training and qualification issues become even more important. At mini-
mum, the Quality Unit should have a system in place to monitor the per-
formance of the production staff and their adherence to cGMP and inter-
nal SOPs.

32 Institute of Validation Technology

 Francesco Boschi. Ph.D.

Issues Related to EM Sampling

Instrumentation and Incubation Equipment
Calibration of sampling instrumentation involved in EM is another key
requirement in achieving a compliant EM program. Dynamic air samplers
should be consistently able to sample the required volume of air (usually 1
m3). Air quality is ensured by a semi-annual calibration, usually performed
by the supplier following a validated procedure. The most relevant sampling
parameter to be verified is the vol-
umetric flow rate. Verification is
usually performed by putting the In the case of the
sampler in series with a refer- recovery of molds or
enced flow meter.
It is well known that each kind
anaerobes, monitoring
of commercially available air sam- should be enforced in
pler (slit to Agar samplers, surface terms of frequency and
air samplers, centrifugal air sam- number of locations in
plers, sterilizable microbiological
order to check whether
atrium, and gelatin filter samplers)
has its own characteristics and the episode is an isolat-
specific performance in terms of ed incident or it is part
1
sampling and microbial recovery. of an adverse trend.
It is, therefore, difficult to say
which is the best one in absolute
terms.
Again, the most meaningful exercise in terms of air sampling is the
continuous comparison of data to expected results over a medium-long
timeframe, in order to detect any deviation from the standard levels of
environmental contamination. Comparison of data from different time peri-
ods is obviously possible only when the sampling parameters are consis-
tently the same over a long timeframe. Therefore, it is essential (1) not to
change the particular sampler you have selected and used to qualify your
EM program and (2) to verify the sampling parameters with an adequate
frequency in order to be assured that your equipment is still working as it
did in the initial qualification phase.
Incubators used to process EM plates are obviously a key part of your
data accuracy assurance. It is a sound practice to have dedicated incubators
for EM samples that have been qualified and temperature mapped at the
desired temperature ranges. Temperature mapping should be performed
with the chamber empty and fully loaded. The selection of temperature
probe locations should be scientifically defensible. Additional challenges to
verifying the effect of some routine activities in the use of the equipment

Environmental Monitoring 33
Francesco Boschi. Ph.D.

could be useful, such as the determination of the time required to return to

the desired temperature range after the opening of the incubator door and
the subsequent temperature variation. As usual, data gathered during the
validation activities should be used in writing the related SOP in a way that
will guarantee reliable and consistent performance.
Temperature should be continuously monitored and recorded by a quali-
fied system; any alarm (significant excursion from temperature range longer
than a pre-defined time period) should be tracked and managed according
to a specific SOP. Impact on the related samples should be formally
assessed. The same consideration should be applied to other laboratory
equipment, such as refrigerators used to store EM plates or other reagents,
whose criticality is comparable to incubators.

Issues Related to Culture Media

Culture media is obviously the most critical material in EM activities.
A robust quality system should be in place to guarantee that culture
media are effectively able to support the growth of the microorganisms
that are expected to be recovered in the clean room. Media are to per-
form as expected in actual operational conditions, from storage through
final incubation.
It is quite uncommon, nowadays, to prepare culture media for clean
rooms, internally, since the need for materials sterilized by ionizing radia-
tion and wrapped in special clean room packaging has forced compa-
nies to widely utilize ready-to-use media for these applications. For
ready-to-use media, choice and qualification of the supplier are obvious-
ly critical; many parameters should be taken into account in selecting an
EM media supplier.
Suppliers should have a cGMP compliant quality system in place in
order to guarantee consistency in product performance and quality.
Particular care should be applied to guarantee the sterility of the media
to avoid the accidental introduction of microbial contamination into the
clean room and to minimize the differences in composition and quality
between different batches (media are made from natural source raw
material, which have great inherent variability). To achieve these goals,
an adequate classified production environment should be ensured and
many checks at different production phases, starting from raw materials,
should be performed.
Another particularly critical step in the supply chain is the transporta-
tion of media from production site to client site. It is essential, but also
difficult in practice, to maintain validated storage conditions for every

34 Institute of Validation Technology

 Francesco Boschi. Ph.D.

plate throughout the journey to the final destination. Therefore, the sup-
plier must consider particular worst-case situations that can actually
occur in everyday activities, such as occasional heat-shock, during the
shipping validation.
The biggest criticality inherent in the shipping of media, is usually
one of the reasons that forces users to verify growth promotion proper-
ties, and all other QC tests, on each batch and each shipment of media.
Typical tests confirmed internally in the users lab include: visual inspec-
tion, pH, pre-incubation (with the same incubation conditions as in rou-
tine monitoring), and Growth Promotion Testing (GPT). It is also a sound
practice to include representative environmental isolates together with
the standard American Type Culture Collection (ATCC) strains, among
the ones used to challenge media in GPT. The scientific rationale in iso-
late selection should be clear, periodically reviewed, and properly docu-
mented.
Another good practice is to take into account the particular conditions
of storage, exposure, and incubation present on site, since these param-
eters could have a dramatic impact on the actual performance of media.
It is, therefore, good practice to validate microbial recovery on particular-
ly stressed plates and to periodically perform GPT on plates that have
been exposed and incubated in the clean room.
For air monitoring, since drying is a key factor for recovery, plate
exposure is the most critical condition (i.e., aspiration of 1 m3 or more
under Laminar Flow (LAF)), while surface monitoring inactivation of dis-
infectant residues should be assessed by GPT data (data collected from
literature may also be supportive). The effect of decontamination proce-
dures, such as introducing UltraViolet (UV) light or Hydrogen Peroxide
into clean room pass-boxes, could also have an impact on media per-
formances and should be included in these validation QC activities.
Minimum recovery expected in GPT is a debated issue. Usually a
50% recovery in respect to control plates can be acceptable, because it
is the one guaranteed by most media suppliers, and it is in accordance
with the variability inherent in all traditional microbiological testing. On
the other hand, a higher recovery (70%) could be requested, based on
the requirement of the microbial recovery test set in the United States
2
Pharmacopoeia (USP) Chapter 1227.
Incubation conditions have a great influence on the growth of the
microorganism that can be recovered from a controlled environment.
Although some standard temperature ranges do exist, incubation condi-
tions (i.e., time and temperatures) may vary from site to site. Incubation
conditions should be able to promote growth of the normal flora (the kind

Environmental Monitoring 35
Francesco Boschi. Ph.D.

of bacteria and fungi expected to be found in the environment). Growth

promotion data and scientific literature should support the established
conditions.
Usually, if you use a generic media such as Tryptic Soy Agar (TSA) for
both bacteria and fungi, you are expected to incubate both at 30-35C
(ideal for mesophilic bacteria growth) and at 20-25C (to support the
growth of most yeast and molds species), but again, this is only one of the
possible combinations of time and temperature.
Although a single media is accepted for routine monitoring, the per-
formance, at least periodically, of some monitoring with a specific media
for yeast and molds (i.e., Sabouraud Dextrose Agar typically incubated not
less than 5 days at 20-25C) is recommended, if data or environmental
conditions e.g., high humidity) indicate the potential presence of molds. It
is also recommended to use this specific media during the EM validation
activity, together with TSA, to get more data on clean room normal flora
levels and then to establish the most suitable conditions for routine moni-
toring.
Monitoring for obligatory anaerobes may be performed occasionally,
again, depending upon historical data and on the process to be moni-
tored. This activity is required especially when your process is at risk from
these organisms (e.g., the product is oxygen sensitive and therefore filled
under nitrogen) or when you have recovered them in sterility test media for
anaerobes (Fluid Thioglicollate Media).
In the case of the recovery of molds or anaerobes, monitoring should
be enforced in terms of frequency and number of locations in order to
check whether the episode is an isolated incident or it is part of an
adverse trend. If a substantial change in your normal flora occurs, relevant
changes in the EM program may be necessary.

Issues Related to Isolation and Recovery

of Microorganisms
Generally speaking, information about the quality of microorganisms
recovered in the facility is essential, but as a matter of fact, you cannot
identify each isolate, taking into account that in a standard size clean
room hundreds of colonies are recovered every week (most of them from
non-critical and ancillary areas).
Therefore, an identification (ID) program should be defined in your
SOPs and should be rationally defensible. Practically, you must state what,
how, and when you want to identify and up to which level (gram staining,
genus, species).

36 Institute of Validation Technology

 Francesco Boschi. Ph.D.

What we should never forget in dimensioning an ID program is that the

aim is to identify the normal flora related to the manufacturing environment
and to detect any deviation from it. This because significant drifts from the
normal flora could be interpreted as adverse trends, as well as a relevant
increase in the number of organisms recovered.
Moreover, assessing the normal flora can help in identifying the most
significant and representative strains to be used for: growth promotion
testing of media for EM and media fills, validation of disinfectants, and the
validation of sterility tests and other lab methods. In other words, an effi-
cient and reliable ID program can help you in improving the quality of your
data and, therefore, the control you have on the manufacturing area.

Issues Related to Documentation Practices

Documentation practices are another crucial issue in a compliant EM
program. Since documentation provides the only track of the whole work,
the more you document, the more you will be able to conduct an effective
investigation in case of failure. Moreover, the more you document, the bet-
ter you can assess the accuracy and meaning of your data.
Appropriate sampling data sheets can be a great help to operators in
properly documenting their activity. Minimum data should include: the iden-
tification of sampling points, the number of samples to be taken for each
location, and the sampling frequencies. Indicate the sampling method,
sampling times or intervals, the method to be used in completing the
forms, and any other relevant comments that will standardize the proce-
dure and clarify what is expected of the operator(s).
Sampling locations should be clearly indicated in approved drawings,
which are usually attached to the sampling SOP. A controlled copy of
these drawings should be present in the clean room to help EM techni-
cians in their activity.
Incubation results should be recorded in laboratory notebooks accord-
ing to procedure. These records should be easily correlated with sampling
sheets to catch potential omissions and to standardize documentation per-
formed by different operators. An exhaustive list of all information that
should be documented in sampling sheets and in result notebooks can be
found in the Parenteral Drug Association (PDA) Technical Report about
3
EM. Raw data must be signed by technicians and reviewed by a supervi-
sor as a basic GMP practice.

Environmental Monitoring 37
Francesco Boschi. Ph.D.

CONCLUSION
As briefly reviewed in this article, accuracy of EM data can be influ-
enced by many different factors each of which should be continually
checked and taken into account in the management of an EM program.
Because media is the primary material utilized in generating EM data,
their selection, management, and control, is fundamental.
On the other hand, since EM - as with most clean room activities - is
still a manual activity, we must rely almost exclusively on human behavior
and commitment. Even the best-designed and validated EM program can-
not be useful and effective if operators and supervisors do not apply it
accurately and consistently day after day. Adequate time and resources
should be spent in the training, qualification, control, and supervision of
peoples work, whose inherent and natural variability should never be
underestimated.

REFERENCES
1. USP 27 <1116> Microbiological Evaluation of Clean Rooms and Other Controlled
Environments
2. USP 27 <1227> Validation of Microbial Recovery from Pharmacopoeial Articles
3. PDA Technical Report No. 13 Fundamentals of an Environmental Monitoring Program,
September 2001.

38 Institute of Validation Technology

 Francesco Boschi. Ph.D.

ABOUT THE AUTHOR

Dr. Francesco Boschi studied Biology at the University of Milan and
obtained his doctorate in Applied Biotechnology in 1997. From 1993 to
1998 he joined a Research and Development Group at Milan University
performing research activities of industrial interest focused on the devel-
opment of fermentation processes of genetically engineered yeast
strains. After some working experience in the field of industrial microbi-
ology, and prior to joining his current company, he was in charge of the
Q.A. Environmental Microbiological Laboratory of Pharmacia & Upjohn,
Nerviano (Italy) Site. His current position (since 2001) in Patheon Italia
(Monza Operations) is Laboratory Manager in the Microbiology Quality
Control Unit, responsible for testing of raw materials and finished prod-
ucts, sterility testing, environmental monitoring, process and equipment
validation, microbial identification, and endotoxin testing.

Dr. Boschi has been involved in various aspects of industrial microbiolo-

gy applied to the manufacture of sterile drugs, including the design and
start-up of new drug manufacturing facilities supplying European and
U.S. markets. He has presented training seminars at both national and
international events in the field of microbiology. Dr. Boschi can be
reached by email at: francesco.boschi@patheon.com.

Originally published in the October 2006 issue of the Journal of GXP Compliance

Environmental Monitoring 39