European Journal of Parenteral & Pharmaceutical Sciences 2014; 20(4): 117-126

2015 Pharmaceutical and Healthcare Sciences Society

Assessment of degree of risk from sources of

microbial contamination in cleanrooms; 2:
Surfaces and liquids
W Whyte1 and T Eaton2*
1
James Watt Building South, University of Glasgow, UK
2
AstraZeneca, Macclesfield, UK

The degree of risk from microbial contamination of manufactured products in healthcare

cleanrooms has been assessed in a series of three articles. The first article discussed airborne sources,
and this second article considers surface contact and liquid sources. A final article will consider all
sources and give further information on the application of the risk method.
The degree of risk to products from micro-organisms transferred from sources by surface
contact, or by liquids, has been assessed by the means of fundamental equations used to calculate the
likely number of microbes deposited (NMD) onto, or into, a product. The method calculates the likely
product contamination rate from each source and gives a more accurate risk assessment than those
presently available. It also allows a direct comparison to be made between microbial transfer by
different routes, i.e. surface, liquid and air.

Key words: Risk assessment, degree of risk, source, surface contact, contamination, micro-organisms,
microbes, MCPs.

Introduction assessment carried out in this manner may not be accurate, for
the following reasons.
The requirements for minimising microbial contamination in
pharmaceutical cleanrooms are outlined in regulatory • Assigning risk descriptors and risk scores is subjective.
documents published by authorities that include the European • The way the risk scores are combined may not reflect the
Commission1 and the Food and Drug Administration in the actual mechanism of contamination.
USA2. These authorities also suggest the use of risk • Differences between the transfer mechanisms of air,
management and assessment techniques to identify and surface contact and liquid make it difficult for these types
control sources of microbial contamination3,4. Risk of risks to be compared.
assessment and management methods have been investigated
by the authors of this article5–9 and other approaches are It would be beneficial if a risk assessment method was
discussed by Mollah et al10. available that avoided these short comings, and could calculate
Risk assessment methods are used to calculate the degree the contamination rate of products from the various sources in
of risk to the product from microbial sources in a cleanroom. a cleanroom. A previous article by Whyte and Eaton11
Factors that influence risk are determined and assigned discussed the application of such a method to airborne sources
descriptors of risk, which are of the ‘high’, ‘medium’, and of microbe-carrying particles (MCPs). This article considers
‘low’ type that act as surrogates for actual numerical values. the application of the method to surface and liquid sources.
Numerical scores are assigned to these descriptors and the
scores combined, usually by multiplication, to obtain a risk
assessment for each source of contamination. However, a risk Calculation of microbial deposition onto a
product
Risk is defined12 as the product of the ‘severity’ (also known
as ‘criticality’) of harm and the ‘probability’ of occurrence,
*Corresponding author: Tim Eaton, Sterile Manufacturing Specialist,
AstraZeneca, UK Operations, Silk Road Business Park, Macclesfield, and its magnitude can be determined by multiplying together
Cheshire, SK10 2NA; Email: tim.eaton@astrazeneca.com; Tel: +44(0)1625 values assigned to these two variables.
514916.
117
118 W WHYTE, T EATON

Equation 1 measured. The proportion of microbes on the donating

surface, which are transferred to a receiving surface or
Degree of risk = severity of harm × probability of harm product, is known as the transfer coefficient. Whyte and
Eaton16 have carried out experiments using skin-derived
In the context of microbial contamination of products in a MCPs to obtain transfer coefficients and the following
cleanroom, ‘severity’ can be considered as the product of average values were obtained: gloves to stainless steel =
the concentration of microbes in, or on, a source of 0.19, stainless steel to stainless steel = 0.10, and clothing to
contamination, and the likelihood that these microbes will stainless steel = 0.06. The contact between stainless steel
be transferred to a product. The ‘probability’ can usually and glass is between hard surfaces and assumed to have a
be considered in (a) airborne contamination as the time the similar value to that between stainless steel and stainless
product is exposed to contamination, (b) surface steel. As these coefficients have similar values, and for
contamination as the number of contacts, and (c) liquid simplification, a worst case transfer coefficient of 0.2 was
contamination as continuous. used in all surface transfers considered in this paper.
Fundamental risk factor equations have been derived by Equation 4 can be used to calculate the NMDL from
Whyte and Eaton7,11 to calculate the number of microbes that liquid sources.
deposit onto, or into, a product from air, surface contact, or
liquids. These equations calculate the number of microbes Equation 4; Liquid
deposited (NMD) onto, or into, one product unit, and
typically give a numerical value well below one. The NMD NMDL = c*p*v
in this article uses the format 1 x 10-6 but it can be
alternatively given as a product contamination rate of 1 in Where, NMDL = number of liquid-borne microbes
106, or 1 in a million units. It is important to make sure that deposited into a single product, c = concentration of
the units of measurement are consistent in the risk equations, microbes in a liquid source, p = transfer coefficient of
and those mainly used in this article are centimetres and microbes from source to product, and v = volume of liquid
seconds, although metres and seconds are also used. deposited into product.
Equation 2 has been derived by Whyte and Eaton7, 11 to
calculate the NMDA from air sources.
Description of cleanroom studied
Equation 2; Airborne In the first article of this series, Whyte and Eaton11
described a method of calculating the NMDA from
NMDA = c*p*a*t*sv airborne sources of microbes, and illustrated it with a
pharmaceutical cleanroom used to aseptically fill batches
Where, NMDA = number of airborne MCPs deposited onto a of pharmaceutical products in a unidirectional air flow
single product, c = concentration of microbes in the airborne (UDAF) workstation. The same example will again be
source, p = transfer coefficient of MCPs transmitted from used to calculate the NMD from surfaces and liquids.
source to product, a = area of product exposed to microbial Cleanrooms that control microbial contamination use a
deposition, t = time of exposure to airborne deposition, and variety of designs and manufacturing methods. Increasing
sv = settling velocity of MCPs through air. regulatory expectations are leading to designs of
pharmaceutical cleanrooms for aseptic filling that include
The settling velocity (sv) is the rate that MCPs fall through an isolator or restricted access barrier system (RABS).
the air, and has been discussed and used in the previous However, to illustrate the wider application of the risk
article11. Microbes do not normally exist in the air as assessment method to more traditional cleanroom designs
single cells. They are mainly dispersed on skin particles found in other types of healthcare rooms, the following
by personnel and have an average aerodynamic diameter cleanroom and manufacturing method is used as an
of about 12 µm13,14, with an average deposition velocity of example.
about 0.46 cm/s15.
Equation 3 can be used to calculate the NMDSC from 1 Vials are aseptically filled with 2 cm3 of aqueous
surfaces. solution, and sealed with sterile closures. This is
carried out in batches of 4000, which take about 4
Equation 3; Surface contact hours to process.
2 Eight litres of an aqueous solution of the active
NMDSC = c*p*a*n ingredient is prepared in an adjacent preparation
cleanroom and piped from the preparation vessel
Where, NMDSC = number of MCPs deposited onto a through a sterilised, sterilising-grade filter, and into
single product by surface contact, c = concentration of the filling workstation. An aseptic connection is made
MCPs on the surface of a source, p = transfer coefficient in the workstation with the product-filling equipment
of MCPs from donating to receiving surface, a = area of before filling starts.
contact, and n = number of contacts. 3 The vials are sterilised in a depyrogenation tunnel
Equation 3 is used to calculate the NMDSC onto a from which they exit, and are conveyed through a
product by surface contact. Much of the information UDAF workstation (EU Guidelines to Good
required to solve Equation 3 will be known, or can be Manufacturing Practice (GGMP) Grade A), which is
ASSESSMENT OF DEGREE OF RISK FROM SOURCES OF MICROBIAL CONTAMINATION IN CLEANROOMS; 2: SURFACES AND LIQUIDS 119

known in this article as the ‘filling workstation’. The solution, and product-contacting surfaces, such as the
vials, which have an inner neck area of 2 cm2, are sterile vial closures, storage hopper, forceps, and
automatically filled in the filling workstation and track-ways, are sterilised.
closed by a stopper. The vials are open in the filling
workstation to airborne contamination for 600 s.
Degree of risk from sources of surface
4 The filling workstation is situated in a non-
and liquid microbial contamination in a
unidirectional airflow cleanroom (EU GGMP Grade
cleanroom
B) which is known as the ‘filling room’. The filling
room has a volume of 300 m3 and an air supply of 3.33 Shown in Figure 1 are the main surface and liquid sources
m3/s of HEPA-filtered air (40 air changes per hour). of microbial contamination of a product, along with
5 Two people work in the filling cleanroom and one of methods of controlling microbial concentrations and
these attends to the filling operation within the transfer. The source of most, if not all, of microbes in a
workstation. Access into the filling workstation is cleanroom is people, who are considered the prime source.
through plastic-strip curtains that hang round the Also included are sources external to the cleanroom that
perimeter and down to just above the floor. may be the cause of contamination in the primary product
Interventions may occur when there are problems with and containers. The sources which directly contact product
the filling line, and these are normally corrected by are within the UDAF workstation and are known as
sterilised long forceps. primary sources. The floor is also included as it is within
6 Vial stoppers are held in a hopper that has a capacity of the workstation, but it is not a primary source, as microbes
1000 stoppers, and replenished every hour. on the floor’s surface are firstly dispersed into the air by
7 Personnel wear cleanroom clothing consisting of a walking, and then transferred by air to product. Also given
one-piece polyester coverall with full hood, overboots in Figure 1, for the sake of completeness, are methods of
and mask. Sterilised, latex, double sets of gloves are controlling airborne transfer of MCPs to surfaces.
worn over disinfected hands. Not shown in Figure 1, or considered in this article, are
8 Hard surfaces, which do not come into contact with the secondary sources, e.g. walls, doors, trolleys, tables,
product containers or closures, are disinfected. Hard disinfectant cans, etc., whose surface microbes do not
surfaces, such as pipework that contacts the product directly contact the product but do so through an

Figure 1. Risk diagram showing sources of surface and liquid microbial contamination along with control methods.
120 W WHYTE, T EATON

intermediate vector. These secondary sources are too Gloves

numerous to be considered, but are usually less important In the cleanroom example, personnel wear disposable
than primary sources as their microbes are subject to an double latex gloves. These gloves have been sterilised
additional transfer step associated with an intermediate by gamma radiation and, as demonstrated in Annex A,
vector. However, the last vector which contacts the the risk from surface microbes on unused gloves can be
product will be one of the primary sources, with gloves the ignored. However, there is a low possibility of skin
main one, and the degree of risk can therefore be microbes being on glove surfaces because of
indirectly ascertained by a risk assessment of the primary punctures17. Glove surfaces may also be contaminated
sources, particularly gloves. when donned, and by touching various surfaces during
Should it be thought necessary, the NMDSC from the cleanroom manufacturing activities, as well as
secondary sources can be calculated. Equation 2 can be deposition of airborne contamination. Gloves are
used to calculate the number of source microbes deposited routinely disinfected during manufacture with sterile
onto an intermediate vector surface, and the surface 70% isopropyl alcohol (IPA) to control the level of
concentration on the vector can then be used to calculate the surface contamination. Personnel are instructed never to
NMDSC onto product. The NMD can also be calculated for contact product with gloves, but it is useful to consider
mixed routes of transfer, such as surface contact and liquid what may occur if the vulnerable inner neck area of
transfer, or surface contact and air transfer, as demonstrated vials is touched by gloves, and the NMDSC can be
in the "Pipework, filling tubes and needles" and the calculated as follows.
"Microbial dispersion from cleanroom floor" sections,
respectively.
The microbial sources shown in Figure 1 are tools,
gloves, cleanroom garments, product solution, solution
Risk factor Assessment

pathways (pipework, filling tubes and needles, etc.),

1. Microbial The post-manufacture measurement of

containers, and floor, and their degree of risk is now

concentration on the microbial concentration of five finger

assessed.
glove surface tips gives an average of 3.9 x 10-3/cm2.
(number/cm2) The five finger tips have a total surface
area of approximately 7.5 cm2, and so

Tools
the glove surface concentration is

Sterilised tools include items such as long-length forceps

5.2 x 10-4 /cm2

used to correct product vials that are displaced or fall

2. Transfer As discussed in the “Calculation of

over, and the forceps may contact the product. The tools
coefficient microbial deposition onto a product”

will be sterilised and, as demonstrated in Annex A, the

section, the transfer coefficient between
gloves and vials is assumed to be 0.2,

surface concentration of microbes following sterilisation

and all contamination transferred enters

and prior to use is negligible, and can be ignored.

the product

However, the forceps may be contaminated by airborne

3. Area of contact The area of contact between a single

deposition, or by touching other contaminated surfaces

(cm2) glove tip and the inner neck of the vial

and, if they contact the vulnerable inner neck of the vial,

was measured and estimated to be
0.5 cm2

microbes may be transferred to product. The NMDSC can

be calculated as follows.
4. Number of At worst, the glove tip might accidently
contacts contact the internal neck area 1 per
4000 containers, which is a frequency
of 2.5 x 10-4
Using Equation 2, the NMDSC can be calculated;
Risk factor Assessment
NMDSC = c*p*a*n = 5.3 x 10-4 * 0.2 * 0.5 * 2.5 x 10-4 = 1.3 x 10-8
1. Microbial The microbial concentration on sterile
concentration on surfaces was determined by sampling
forceps surface after completion of manufacturing and

Cleanroom garments
(number/cm2) will, therefore, represent the worst case

Garments are sterilised by radiation prior to use and, as

concentrations. From 38,062 samples,

shown in Annex A, they will be effectively free of

one microbe was recovered. The
forceps had a contact area with the

microbes when unused. However, their surface may be

sampling media of 1.2 cm2, which

contaminated when donned, touched by contaminated

gives a surface concentration of

surfaces during manufacturing, or from microbes

2.2 x 10-5/cm2

depositing from the air. It is expected that the use of

2. Transfer As discussed in the “Calculation of

long-length sterilised tools and good aseptic practices

coefficient microbial deposition onto a product”
section, the transfer coefficient

will prevent garments contacting product. However, if

between stainless steel and glass

they accidently contact the vial, it is useful to know the

surfaces is assumed to be 0.2

degree of risk, and the NMDSC can be calculated as

3. Area of contact The area of the forceps that makes

follows.
(cm2) contact with the inner neck of the vial
was measured and found to be 0.3 cm2
4. Number of At worst, the internal neck area is
contacts contacted 10 times per 4000 containers,
which is a frequency of 2.5 x 10-3
Using Equation 2, the NMDSC is:
NMDSC = c*p*a*n = 2.2 x 10-5 * 0.2 * 0.3 * 2.5 x 10-3 = 3.3 x 10-9
ASSESSMENT OF DEGREE OF RISK FROM SOURCES OF MICROBIAL CONTAMINATION IN CLEANROOMS; 2: SURFACES AND LIQUIDS 121

and needles are decontaminated and steam sterilised at

121°C. The internal surface of all these items prior to
Risk factor Assessment

sterilisation has been determined experimentally to have

14 microbes and, as calculated in Annex A1, the number
1. Microbial The forearms and chest of garments are
concentration on sampled after manufacturing using

of microbes likely to survive steam sterilisation is 10-19. If

garment surface RODAC plates and an average

all of these microbes are washed off the pipework by the

(number/cm2) concentration is 2.7 x 10-2 per 24 cm2,

passage of 8000 cm3 of product solution, the concentration

which is a concentration of
1.1 x 10-3/cm2

of microbes in the product solution will be 1.3 x 10-23 per

2. Transfer As discussed in the “Calculation of

cm3. Such a low concentration can be ignored.

coefficient microbial deposition onto a product”

When the flexible pipe is connected to the filter, or

section, the transfer coefficient between

needles fitted into the filling machinery, the opening of a

the garment and the vulnerable inner
neck area of the vial is assumed to be

pipe or needle surface may touch a glove, and microbial

0.2. All contaminants were assumed to

transfer may occur. Any microbes transferred are assumed

enter the product

to mix with product solution and be subsequently

3. Area of contact The area of contact between a garment

dispensed into the vials. The area of the glove that contacts
(cm2) and the vulnerable neck area of the

with the pipework is likely to be different from that of a

container was measured and estimate
to be about 0.5 cm2

needle opening. However, to avoid multiple calculations,

the area of 0.5 cm2, previously used in the “Gloves” section
4. Number of At worst, the contact of the garment with
contact the internal neck area of the container is

when a glove touches vials, is again used.

assumed to be 1 contact per 4000

The NMD is calculated in two stages, namely, glove to

containers, which is a frequency of

pipework or needles, and then from pipework or needles

2.5 x 10-4

to product.
Using Equation 2, the NMDSC can be calculated;
NMDSC = c*p*a*n = 1.1 x 10-3 * 0.2 * 0.5 * 2.5 x 10-4 = 2.8 x 10-8

Filtered aqueous product solution

The solution of primary product is a potential source of
Risk factor Assessment

microbial contamination and is filtered through a sterilised,

1. Microbial The post-manufacture measurement of

sterilising-grade filter of the membrane type. Pre- and post-

concentration on the microbial concentration of five finger
glove surface tips gives an average of 3.9 x 10-3/cm2.

use integrity testing of the filter is carried out using an

(number/cm2) The five finger tips have a total surface

automated test unit that measures the rate of diffusive gas

area of approximately 7.5 cm2, and so

flow. This measurement is directly related to a bacterial

the glove surface concentration is
5.2 x 10-4 /cm2

challenge test performed by the filter manufacturer, where

the filter is challenged with Brevundimonas diminuta, with
2. Transfer As discussed in the “Calculation of
coefficient microbial deposition onto a product”

a size of approximately 0.3 µm. Filters are required2 to

section, the transfer coefficient between

retain a challenge of 1 x 107 bacteria per cm2 of filter area.

gloves and pipework or needles is

The number of microbes deposited (NMDL) in product can

assumed to be 0.2

be calculated as follows.
3. Area of contact An area of 0.5 cm2 was assumed
(cm2)
4. Number of The frequency of contact is unlikely to
Risk factor Assessment contacts exceed 1 contact per filling batch of
4000 vials, which is a frequency of
1. Microbial The maximum concentration, prior to 2.5 x 10-4
concentration in sterile filtration, is determined
the product experimentally to be 10/cm3 Using Equation 2, the NMDSC onto the pipe or needle opening
solution can be calculated as follows:
(number/cm3)
NMDSC = c*p*a*n = 5.2 x 10-4* 0.2 * 0.5 * 2.5 x 10-4 = 1.3 x 10-8
2. Transfer The filter has a total filtration area of
coefficient 1000 cm2, and required to retain a The calculation in the row above shows that 1.3 x 10-8 MCPs
challenge of 1010 bacteria. The transfer are transferred to the pipe and needle openings and these are
coefficient across the filter is, therefore, assumed to enter the product solution. The number that will
1 x10-10. Although there may be enter a product by liquid transfer can now be calculated
deposition of microbes throughout the 5. Microbial The product solution passes through the
pipework from filter to filling point, this concentration in internal areas of pipework and needles,
will be very small compared to the product solution all microbes are assumed to be washed
removal efficiency of the filter, and is (number/cm3) and mixed into the product solution, and
ignored the concentration in the solution of
3. Volume of product 2 cm3 4000 cm3 is 1.3 x 10-8 ÷ 4000 =
solution dispensed 1.6 x 10-12/cm2
into vial (cm3) 6. Transfer All microbes introduced by contact are
Using Equation 4, the NMDL can be calculated; coefficient assumed to be swept by product
solution into the containers and the
NMDL = c*p*vc = 10 * 1 x 10-10 * 2 = 2.0 x 10-9 transfer coefficient is 1
7 Volume of product 2 cm3

Pipework, filling tubes and needles

solution dispensed

The product solution is transferred from the sterilising-

into vial (cm3)

grade filter to the filling point through a flexible transfer

Using Equation 4, the NMDL can be calculated;

pipe that is connected to filling needles. The flexible pipe

NMDL = c*p*v = 1.6 x 10-12 * 1 * 2 = 3.3 x 10-12
122 W WHYTE, T EATON

Product vials
Following decontamination in an automated washing unit,
Risk factor Assessment

the vials are transferred to the filling workstation through

a depyrogenation tunnel, where they are sterilised. The
1. Concentration of The airborne concentration of MCPs in
airborne MCPs the filling cleanroom derived from the

possibility that microbes can survive within the vial after

dispersed from the floor is 6.3 x 10-12/cm3 (see calculation

sterilisation can be calculated using the method given in

filling cleanroom in Annex B)

Annex A.
floor (no/cm3)

The maximum microbial concentration on the inner,

2. Transfer The transfer coefficient is assumed to
coefficient of be 1 x 10-4

product-contacting surface of each vial, following

MCP from filling

decontamination and prior to depyrogenation, was

cleanroom to

determined experimentally to be 10. As calculated in

product

Annex A2, a dry heat sterilisation cycle of 170°C for 2

3. Area of product The inner neck area of vial is 2 cm2

hours would reduce this to a concentration of about

exposed (cm2)

1 x 10-119. However, the depyrogenation cycle uses a

4. Time of The proportion of time that a person

temperature of 250°C for 30 minutes and this additional

deposition (s) works in the filling workstation is 0.1 of
the total time, and therefore the time

heat will decrease the microbial concentration to about 1

for the transfer of contamination from

x10-300000 per vial. As these microbes are within the vial,

the filling room (normally 600 s) is

and there is no transfer coefficient to be considered, the

reduced to 60 s

NMDSC will remain at about 1 x 10-300000.

5. Deposition The average setting velocity of MCPs
velocity through through the air and into the vial is
air of MCPs assumed to be 0.46 cm/s (see the

Microbial dispersion from cleanroom floor

(cm/s) “Calculation of microbial deposition

The transfer of microbes from cleanroom floor to product

onto a product” section).

occurs in two stages. MCPs are dispersed into the air by

Using Equation 1, the NMDA can be calculated to be as follows:

contact of shoes with the floor, and then transmitted

NMDA = c*p*a*t*s = 6.3 x 10-12 * 1 x 10-4 * 2 * 60 * 0.46 =

through the air to the product, where they may deposit.

3.5 x 10-14

The concentration of airborne microbes in the air of the

filling cleanroom and filling workstation that have been Filling workstation
dispersed from a floor is calculated in Annex B, and can In the filling workstation, the mechanism of dispersion of
now be used to calculate the NMDA. MCPs from floor to air by walking is the same as the
filling cleanroom. However, the walking activity is
Filling cleanroom reduced, as is the microbial concentration on the floor, and
The concentration of MCPs dispersed into the cleanroom this information is used in Annex B to calculate the
air from the floor is calculated in Annex B. Assuming a dispersion rate from the floor. Because of the downward
microbial concentration on the floor of 1.2 x 10-4/cm2, and flow of UDAF, the MCPs dispersed from the floor will not
two people walking about for half of the total mix with all of the air in the filling workstation, but only
manufacturing time, the number of MCPs dispersed into with air close to the floor. The airborne concentration
the air in the filling cleanroom is calculated. These MCPs above the floor has been calculated in Annex B to be 2.3 x
mix with room air, and the airborne concentration of 10-7/m3 (2.3 x 10-13/cm3).
microbes in the filling cleanroom that is derived from the It is now necessary to consider the transfer of the
floor has been calculated to be 6.3 x 10-6/m3 (6.3 x airborne MCPs from the area near to the floor to vials at
10-12/cm3). the filling location. The experiments carried out by
For the airborne MCPs in the filling cleanroom to reach Ljungqvist and Reinmuller18 did not investigate this exact
a product, they have to be transmitted across the curtains situation but found that the proportion that reached a
and the UDAF within the filling workstation, and closures hopper from the floor area was about 1 x 10-3. It
deposited into a container. People may work through the may seem surprising that the proportion of MCPs that
curtain, or enter the workstation to attend to containers reaches the closures or vials against the downflow of air
and machinery. Movement through the curtain and within may be greater than that transmitted across the airflow
the UDAF allows airborne MCPs from the filling (found to be about 1 x 10-4). However, machinery can
cleanroom to be transmitted to product. Experiments disrupt the downward airflow and produce a turbulent
carried out by Ljungqvist and Reinmuller18 have shown wake where particles can flow in the opposite direction to
the proportion of airborne particles released outside the the overall flow. The transfer of contamination in such
workstation that reached the product when personnel were conditions can be complicated, and it is best determined
working, was about 1 x 10-4; this proportion is the transfer experimentally in the individual situation. However, it has
coefficient. The NMDA dispersed from the floor of the been assumed that in the worst condition, the transfer
filling cleanroom and deposited into the vial by the coefficient is 1 x 10-3.
airborne route can be calculated as follows.
ASSESSMENT OF DEGREE OF RISK FROM SOURCES OF MICROBIAL CONTAMINATION IN CLEANROOMS; 2: SURFACES AND LIQUIDS 123

To illustrate the method, a pharmaceutical cleanroom is

used in which batches of vials are aseptically filled in a
Risk factor Assessment

UDAF workstation. Cleanrooms used for aseptic filling are

1. Concentration of The airborne concentration of MCPs

now being designed with isolators and RABS but the

airborne MCPs just above the floor that is derived from

cleanroom used in the example allows the demonstration

derived from filling the workstation floor by walking is

of the risk assessment in a wider spectrum of cleanroom

workstation floor 2.3 x 10-13 /cm3
(no/cm3)

design and manufacturing methods. However, if a different

cleanroom or manufacturing process is to be considered,
2. Transfer The transfer coefficient is assumed to
coefficient of be 1 x 10-3

the risk assessment must be carried out for that cleanroom.

MCP from around

The equations used to calculate the NMD from

the floor to vial

surface contact or liquids are fundamental, and if the

3. Area of product The inner neck area of vial is 2 cm2

input into the equations is correct then the result will be

exposed (cm2)

exact. Some of the equations variables (risk factors) will

4. Time of airborne The proportion of time that a person

be known, e.g. the horizontal area of product exposed to

deposition (s) works in the filling workstation is 0.1 of
the total time, and therefore the time for

airborne contamination, and others may need an

the transfer of contamination from the

additional collection of information, such as the

filling room (normally 600 s) is reduced

concentration of MCPs on surfaces and in liquids. The

to 60 s

transfer coefficient is a more difficult variable to

5. Settling velocity The settling velocity through air and into

ascertain, as information is not readily available. The

of MCPs through a vial is 0.46 cm/s
air (cm/s)

values of the airborne transfer coefficient used in this

article are obtained from the results of Ljungvist and
Using Equation 1, the NMDA can be calculated to be as follows:

Reinmuller18, but we recommend further experiments to

NMDA = c*p*a*t*s = 2.3 x 10-13 * 1 x 10-3 * 2 * 60 * 0.46 =
1.3 x 10-14

extend this knowledge. The surface transfer coefficients

are based on our experimental results16, which
Relative importance of sources of determined that in the worst case situation the surface
contamination in a typical transfer coefficient was unlikely to be greater than 0.2.
The values of the transfer coefficients are, therefore,
pharmaceutical cleanroom
reasonable estimates. However, if the required risk
The NMDs from surface contact and liquid routes found variables cannot be obtained, and estimates based on an
in the example cleanroom are given in Table 1. informed estimate, the resulting risk assessment is
almost certain to be more accurate than a risk
assessment based on descriptors and risk scores.
Discussion and conclusions
It can be seen in Table 1 that the highest degree of
The method of ascertaining the degree of risk from risk from surface contact and liquid sources in the
sources of microbial contamination in a cleanroom is cleanroom example occurs if the vulnerable area of the
carried out by calculating the number of MCPs deposited product is touched by the gloves or garments worn by
(NMD) into, or onto, a product by means of equations the cleanroom personnel. Personnel are trained to avoid
presented in the introduction. A previous article11 has such contact but the calculation shows what can occur if
considered the degree of risk from airborne sources and mistakes are made, and the NMDSC is in the region of
this article ascertains the risk from surface and liquid 10-8, i.e. one product in every 108 may be contaminated
sources. by microbes. However, if contact is made with an

Table 1. Importance of sources of surface contact and liquid contamination in a pharmaceutical cleanroom.

Risk importance Source of microbial contamination NMD from surface

contact and liquids

1 Contact of product with cleanroom garments* 2.8 x 10-8

2 Contact of product with double gloves* 1.3 x 10-8

3 Contact of product with ‘sterile’ tools, e.g. forceps with container neck 3.3 x 10-9

4 Filtered aqueous product solution 2 x 10-9

5 Liquid contamination through contact of gloves with pipework and filling needles* 3.3 x 10-12

6 Floor in the UDAF filling workstation EU GGMP grade A 1.3 x 10-14

7 Floor in the non-unidirectional airflow filling room EU GGMP grade B 3.5 x 10-14

8 Sterilised product containers 1 x 10-300000

*Under normal control conditions, the risk will be much smaller. However, it is useful to determine the degree of risk when normal
control measures have been breached and these contamination rates relate to this.
124 W WHYTE, T EATON

inanimate item, such as a sterilised tool or ancillary 8 Whyte W and Eaton T. Risk Management of Contamination During
item, e.g. forceps, the NMDSC will be about 10-9. If the Manufacturing Operations in Cleanrooms. Parenteral Society
Technical Monograph No 14. Swindon, UK: The Parenteral Society
personnel’s gloves make contact with vulnerable areas and The Scottish Society for Contamination Control; 2005. ISBN
of pipework or needle assembly, during the set-up of the No. 1-905271-12-3.
filling equipment, the NMDSC is likely to be about 10-12. 9 Whyte W. Operating a Cleanroom: Managing the Risk from
Contamination. In: Cleanroom Technology: Fundamentals of
When the primary solution of product is filtered by a Design, Testing and Operation, 2nd Edition. Chichester, UK: John
single sterilised sterilising grade filter, the NMDL is Wiley & Sons; 2010, Chapter 16. ISBN 978-0-470-74806-0.
likely to be less than about 10-9. The NMD from the 10 Mollah H, Baseman H and Long M (editors). Risk Management
Applications in Pharmaceutical and Biopharmaceutical
floor in both the filling cleanroom and the filling Manufacturing. Chichester, UK: John Wiley & Sons; 2013. ISBN
workstation is negligible and about 10-14. The risk from 978-0-470-55234-6.
sterilised (depyrogenation cycle) containers is infinitely 11 Whyte W and Eaton T. Assessment of degree of risk from sources of
microbial contamination in cleanrooms; 1: airborne. European
low (1 x 10-300000). Journal of Parenteral and Pharmaceutical Science 2015;20(2):52–
In a previous article, Whyte and Eaton11 discussed and 62.
calculated the NMDA from airborne sources. A further 12 International Standards Organization. ISO/IEC Guide 51:2014.
Safety Aspect – Guidelines for their Inclusion in Standards. Geneva,
article will consider all sources of microbiological Switzerland: ISO; 2014.
contamination in various types of cleanrooms, i.e. those 13 Noble WC, Lidwell OM and Kingston D. The size distribution of
transferred by air, surface contact, and liquid routes. Also airborne particles carrying micro-organisms. Journal of Hygiene
1963;61:385–391.
discussed will be methods used to reduce the degree of 14 Whyte W and Hejab M. Particle and microbial airborne dispersion
risk, where it is considered too high. from people. European Journal of Parenteral and Pharmaceutical
Science 2007;12(2):39–46.
15 Whyte W. Sterility assurance and models for assessing airborne
References bacterial contamination. Journal of Parenteral Science and
Technology 1986;40:188–197.
1 European Commission. EudraLex. The Rules Governing Medicinal 16 Whyte W and Eaton T. Microbial transfer by surface contact in
Products in the European Union. Volume 4: EU Guidelines to Good cleanrooms. European Journal of Parenteral and Pharmaceutical
Manufacturing Practice – Medicinal Products for Human and Sciences 2015;20(4):127-131.
Veterinary Use. Annex 1 – Manufacture of Sterile Medicinal 17 Eaton T. A safe pair of hands – how secure are your gloves used for
Products. Brussels, Belgium: European Commission; 2008. aseptically prepared pharmaceutical products? European Journal of
2 Food and Drug Administration. Guidance for Industry: Sterile Drug Parenteral and Pharmaceutical Sciences 2005;10(3):35–42.
Products Produced by Aseptic Processing – Current Good 18 Ljungqvist B and Reinmuller B. Chapter 8: Risk assessment with the
Manufacturing Practice. Silver Spring, MD, USA: FDA; 2004. LR-method. In: Practical Safety Ventilation in Pharmaceutical and
3 European Commission. EudraLex. The Rules Governing Medicinal Biotech Cleanrooms. Bethesda, MD, USA: PDA; 2006. ISSN: 1-
Products in the European Union. Volume 4: EU Guidelines to Good 930114-89-3.
Manufacturing Practice – Medicinal Products for Human and 19 Parenteral Drug Association. Validation of Moist Heat Sterilisation
Veterinary Use. Annex 20 – Quality Risk Management. Brussels, Processes. PDA Technical Report No. 1 (Revised 2007). Bethesda,
Belgium: European Commission; 2009. MD, USA: Parenteral Drug Association.
4 Food and Drug Administration. Pharmaceutical cGMPs for the 21st 20 Parenteral Drug Association. Validation of Dry Heat Processes Used
Century – a Risk-Based Approach. Silver Spring, MD, USA: FDA; for Depyrogenation and Sterilization. PDA Technical Report No. 3
September 2004. (Revised 2013). Bethesda, MD, USA: Parenteral Drug Association.
5 Whyte W. A cleanroom contamination control system. European 21 International Organization for Standardization. ISO 11137-2: 2012.
Journal of Parenteral Sciences 2002;7(2):55–61. Sterilisation of Health Care Products – Radiation – Part 2:
6 Whyte W and Eaton T. Microbial risk assessment in pharmaceutical Establishing the Sterilization Dose. Geneva, Switzerland: ISO.
cleanrooms. European Journal of Parenteral and Pharmaceutical 22 Whyte W, Whyte WM, Blake S and Green G. Dispersion of
Sciences 2004;9(1):16–23. microbes from floors when walking in ventilated rooms.
7 Whyte W and Eaton T. Microbiological contamination models for use International Journal of Ventilation 2013;12(3):271–284.
in risk assessment during pharmaceutical production. European
Journal of Parenteral and Pharmaceutical Sciences 2004;9(1):11–15.

Annex A: Calculation of the reduction of Equation A1

surface microbial concentrations by
sterilisation Log B = Log A – (F0 /D)
In the main body of this article, it has been assumed that Where, A = number of microbes at the start of sterilisation,
surfaces of microbial sources, such as gloves, tools and B = number of microbes at the end of sterilisation, F0 =
garments, which are unused and sterilised by steam, dry equivalent exposure time, and D = D-value
heat and radiation, have no surface micro-organisms, or
such an extremely small number that it will make no The values of F0 and D are ascertained as follows.
significant contribution to microbial contamination of
product. The justification of this assumption is contained Fo: For steam sterilisation, 121°C is the reference
in this annex. With knowledge of sterilisation kinetics, the temperature used to calculate the effectiveness of
number of microbes likely to survive sterilisation can be sterilisation at other temperatures, and calculated by
calculated for the three sterilisation processes. Equation A2:
Steam sterilisation Equation A2
The number of microbes that survive steam sterilisation
can be calculated by means of the following equation19. F0 = L x t
ASSESSMENT OF DEGREE OF RISK FROM SOURCES OF MICROBIAL CONTAMINATION IN CLEANROOMS; 2: SURFACES AND LIQUIDS 125

Where, L = lethal rate, and t = sterilisation time. Where, TO = sterilisation temperature utilised, Tb = base
temperature, and Z = z value.
At 121°C, the lethal rate (L) has a value of 1 and,
therefore, for sterilisation at 121°C for 20 minutes, the F0 The z-value is the temperature coefficient of microbial
value is 20 minutes. destruction and is the number of degrees Centigrade
required to cause a 10-fold increase in the sterilisation
D value: The D-value is the time required, at a specified rate, and is assumed to be 20°C20. Utilising a Tb value of
temperature, to reduce the microbial population by one 170°C, the lethal rate at 250°C is calculated to be 104. The
logarithmic value (90% reduction). The D-value varies FH value for a 30 minute cycle at this temperature is then
according to the type of micro-organism but at 121°C, calculated by Equation A4 and found to be 3 x105. Under
most microbes die instantly. However, bacterial spores these conditions, the number of surviving microbes in
have a much greater thermal resistance, and a D-value of 1 each container can then be calculated using Equation A3.
minute is often assumed19. This is a reasonable value, as As an example, if the D-value at a dry heat temperature of
spores isolated in cleanrooms are likely to be the 250°C for 1 minute is considered with a microbial
mesophilic type that is more susceptible to heat treatment concentration on the internal surface of a vial of 10, the
and are likely to be less than 5% of the microflora found in number of surviving organisms is 10-299999.
cleanrooms.
If appropriate values of F0 and D are used in Equation Radiation sterilisation
A1, the number of surviving organisms can be calculated. Cleanroom garments are normally sterilised by gamma
For example, if the number of microbes in the internal radiation, using a minimum radiation dose of 25 kGy. The
surfaces of pipework and needles is 14, the number of number of microbes on a cleanroom garments prior to
surviving microbes is 10-19. sterilisation can be determined by immersing and
agitating the garment in liquid, filtering the liquid, and
Dry heat sterilisation incubating the filter. A one-piece coverall is the item of
The number of microbes remaining after dry heat cleanroom clothing with the largest area and, therefore,
sterilisation can be calculated by means of the following the highest bioburden, and shown to have a bioburden
equation20. prior to sterilisation of 190 microbes.
The radiation dose required to achieve a given
Equation A3 sterility assurance level up to 1 x 10-6, for a range of
average bioburdens of microbes with a standard
Log B = Log A – (FH /D) distribution of resistance against radiation, is given in
table 5 of ISO 11137-221. For a bioburden of 190
Where, FH = equivalent exposure time in dry heat. microbes, this is represented graphically in Figure A1.
By extrapolation of the graph, it can be seen that the
For dry heat sterilisation, 170°C is the reference number of surviving microbes after exposure to 25 kGy
temperature from which the effectiveness of sterilisation is approximately 1 x 10-7. As the one-piece coverall has
at other temperatures can be calculated by means of an external surface area of about 16,000 cm2, and hence a
Equation A4.

Equation A4

FH = L x t

For dry heat sterilisation at 170°C, the lethal

rate (L) is 1. Therefore, using Equation A4,
the FH value for a cycle of 120 minutes at
170°C is 120. At a dry heat temperature of
170°C, a D-value of 1 minute is assumed20.
Using Equation A3, the number of
surviving micro-organisms, when the
maximum number on the internal surface of
an object such as a container is 10, can be
calculated to be 10-119. However, containers
that are subjected to the depyrogenation
conditions of 250°C for 30 minutes will have
an increased lethal rate at this temperature that
can be calculated from use of Equation A5.

Equation A5
Figure A1. Radiation dose required to achieve a sterility assurance level for an
L = 10 [(To-Tb)/Z] average bio-burden of 190, extrapolated for a dose of 25 kGy.
126 W WHYTE, T EATON

total internal and external area of about 32,000 cm2, the is assumed: two people walk about the filling cleanroom
concentration of surviving micro-organisms on the for a proportion of 0.5 of the time, at a rate of 1.5 steps per
garment surface can be assumed to be 1 x 10-7 ÷ 32,000 = second, and have shoes with a contact area of 110 cm2
3.1 x 10-12/cm2. (0.011 m2). The redispersion fraction is 0.0012, the air
supply rate is 3.33 m3/s, and the floor area is 100 m2 with a
microbial surface concentration of 1.2/m2. The airborne
Annex B: Number of MCPs dispersed
concentration of MCPs in the filling cleanroom in the
from cleanroom floor
steady-state condition during manufacturing (C) is,
To calculate the risk to product from MCPs dispersed by therefore, as follows.
personnel walking on a floor, it is necessary to know the
concentration of MCPs in the air of a clean zone that are 1.2*0.011*0.0012*2*1.5*0.5
derived from the floor. These are calculated in this annex. C = 111111111111 = 6.3x10-6/m3 = 6.3x10-12/cm3
The number of MCPs dispersed from a floor by 3.33+(0.0046×100)
walking has been investigated by Whyte et al22 who
showed it to be dependent on the total number of steps per This concentration is used in the “Filling cleanroom”
second taken by all of the personnel in the room, the shoe subsection of the “Microbial dispersion from cleanroom
area, and the ‘redispersion fraction’ (RF), which is the floor” section to calculate the NMDA when the source is
fraction of MCPs on the floor surface that is dispersed by the filling cleanroom floor.
one step. The dispersion rate can be calculated as follows.
Filling workstation
Equation B1 In the filling workstation, the mechanism of dispersion of
MCPs from the floor into air is the same as the filling
DF = CF x AS x RF x N x W x P cleanroom, and the number of MCPs dispersed per second
can also be calculated by Equation B1. However, only one
Where, DF = microbial dispersion rate, CF = concentration person attends to the filling line, and spends a smaller
of microbes on floor surface, AS = area of shoe in contact proportion of their total time (0.1) working and walking in
with floor, RF = redispersion fraction, N = number of the filling workstation. Their walking rate is again 1.5/s
people in room, W = walking rate (number of steps/s), and with a shoe area of 0.011 m2. The microbial concentration
P = proportion of time spent walking. on the floor of the filling workstation is lower than the
filling room, and 0.42/m2. The microbial dispersion rate
Knowing the dispersion rate of MCPs from the floor by (DF) is therefore as follows.
walking, the airborne concentration of MCPs in both the
filling cleanroom and filling workstation can be calculated DF = CF x AS x RF x N x W x P =
as follows. 0.42*0.011*0.0012*1*1.5*0.1 = 8.3 x 10-7/s

Filling cleanroom Because of the downward UDAF in the filling

When MCPs are dispersed from the cleanroom floor, they workstation, the MCPs dispersed from the floor will not
will mix with the air in the non-unidirectional airflow mix with all of the air in the workstation but only with the
filling cleanroom to give a reasonably constant air above the floor. The area of the air supply filters is 3 m
concentration across the room. The airborne concentration x 3 m, and air is discharged from the filters at a velocity of
of MCPs can be calculated by Equation B2 derived by 0.4 m/s; there is, therefore, an air supply volume of
Whyte et al22 to take account of dilution by the air supply 3.6 m3/s. As the UDAF does not pass through the floor, the
to the cleanroom and the loss by gravitational re- UDAF will change to non-unidirectional airflow above
deposition onto the floor. the floor and turbulently mix with the dispersed MCPs.
The concentration of MCPs close to the floor can,
Equation B2 therefore, be calculated in the steady-state condition by
use of Equation B2 but, as the floor area is so small, the
Airborne 𝐶𝐹∗𝐴𝑆∗𝑅𝐹∗𝑁∗𝑊∗𝑃 MCP deposition on the floor area is ignored.
concentration of = 1111 = 11111111
𝐷𝐹

floor-derived MCPs/m3 𝑄+(𝑉𝐷∗𝐴) 𝑄+(𝑉𝐷∗𝐴) Airborne concentration of floor-derived MCPs close to floor =
microbial dispersion rate from floor/s 8.3 x 10−7/s
Where, Q is the rate of air supply volume (m3/s), VD is the =
deposition velocity of MCPs (0.0046 m/s), and A is the air supply volume rate (m3/s) 3.6m3/s
deposition area (m2) in the room (normally the floor).
111111111111111 11111

= 2.3 x 10-7/m3 = 2.3 x 10-13/cm3

The meaning and the value of the deposition velocity of
MCPs, which is 0.0046 m/s, is discussed in Part 1 of these The NMDA is calculated in the “Filling workstation”
articles11 and the experiment to determine the redispersion subsection of the “Microbial dispersion from cleanroom
factor, which was 0.0012, is described by Whyte et al22. floor” section from this concentration.
In the cleanroom example being studied, the following