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Data Sheet

Biocontamination control in
pharmaceutical production
and compliance to ISO 14698 with
validated active microbial air samplers
Tim Sandle, Ph.D., Head of Microbiology at Bio Products Laboratory (UK), [email protected]
Tony Ancrum, Global Product Manager for Viable Air Monitoring, Merck Millipore, [email protected]
Anne Connors, Field Marketing Manager, Merck Millipore, [email protected]

Introduction Although often overlooked by some laboratories, the

Biocontamination refers to biological contamination of international standard on biocontamination control, ISO
products by bacteria and/or fungi, as well as the toxic 14698, is an important resource for the development of
by-products of these microorganisms, such as endotoxin a biocontamination strategy. There are two parts to this
and mycotoxins from Gram-negative bacteria and fungi, standard: Part 1 covers general principles and methods
respectively. When designing a biocontamination control of biocontamination control; Part 2 covers the evaluation
strategy, which is based on the manufacturing process, and interpretation of biocontamination data. Both parts
there are three components to take into consideration, of ISO 14698 are currently undergoing revision.
each of which requires risk assessment: 1) designing
It is important to clarify the distinction between the
process systems to avoid contamination; 2) monitoring
cleanroom standard ISO 14644 from ISO 14698. ISO
process systems to detect contamination; and 3) reacting
14644 is a 12-part cleanroom certificate standard that
to contamination events and putting proactive measures
is focused on airborne particulates. This standard covers
in place. The design of process systems is where maximal
cleanroom design, HEPA filter specification, pressures,
effort should be placed.
and how to monitor a cleanroom in order to assess the
cleanroom class. ISO 14698 focuses on the ongoing
assessment of cleanrooms for viable contamination.

EMD Millipore Corp. is a subsidiary of Merck KGaA, Darmstadt, Germany

Despite good design and following available ISO 14698 Part 1 Monitoring
guidances, cleanrooms are at risk for several sources
of contamination, of which people are the greatest
Program
The international standard ISO 14698-1 can assist
source. Some studies estimate that people can
in the development of an environmental monitoring
contribute up to 70% of microorganisms found
program. Program development involves asking a
within a standard cleanroom. Second to people, water
series of questions each of which requires a risk-based
is a key source of contamination. The challenge with
response. Not everything can be monitored and
water is that it not only allows contamination to
therefore consideration must be given to when and
spread, but it also helps microorganisms to grow.
why monitoring should occur.
Microorganisms are carried in air streams until they
When developing a program, the sampling plan
are deposited on a surface. Unless they have recently
should take into account the cleanliness level required
been disinfected, most surfaces will have contamination
at each site to be sampled as well as which types of
on them. The risk arises when the contamination moves
samples are appropriate (air and/or surface samples).
from a less critical to a critical location, so it follows
The plan should also define whether this is a quality
that using clean utensils and having clean gloves is
control activity or an activity delegated to production.
very important to minimize contamination transfer.
Additionally, it must be determined how to trend the
data and how to set limits.

Contamination Control
To minimize contamination from people, proper Sampling Types
gowning is essential to curtail the amount of shedding The measurement of airborne particle counts is a key
of skin matter and microorganisms that a person can part of environmental control. Airborne particles are
deposit within a cleanroom. Localized protection, such measured using optical particle counters, whereby air
as isolators and unidirectional airflow cabinets, should is drawn through the instrument and moves through
also be established around the product to minimize a laser. The laser calculates the number and size of
contact with people. Good cleanroom design includes airborne particles present. In pharmaceuticals, there is
high-efficiency particulate air filters (HEPA), pressure either one or two particle sizes to look at, depending
cascade, and air distribution. Cleanrooms must also upon the region of the world. The US Food and Drug
be cleaned and disinfected regularly, and transfer of Administration requires particles that are of equal or
items in and out of the cleanroom must be controlled. greater size than 0.5 micron from one cubic meter of
air to be counted. In addition to this requirement, EU
Once good design principles are in place, an environ- GMP also requires the monitoring of particles equal
mental monitoring program should be designed in and greater to 5 micron. The reason for the 5 micron
order to provide information about the state of size requirement is that this is closer to the size of skin
control of the facility. It is important to note that cells, which is the most common type of contamination
environmental monitoring does not replace good floating within a cleanroom. The limits will be according
environmental control (the design of cleanrooms to the class of cleanroom, and out-of-limits particles
and operational practices); environmental monitoring may indicate a problem, for example, with an air
only provides a ‘snapshot’ of time. Individually counts handling system.
are rarely significant, but it is the trends over time
that are important: as counts, as frequency of incidents,
and as microflora. The presence of microflora, such as
waterborne bacteria or organisms that are hard to kill
with disinfectants, may indicate the breakdown
of control.
Viable monitoring methods all use either a general Sampling Locations
purpose culture medium like trypotone soya agar (TSA), The monitoring plan should also note the choice of
at a dual incubation regimen of 20-25˚C followed by sample locations based on the nature of the work to be
30-35˚C, or two different culture media that are used at carried out and the impact that cleanroom operators
two different temperatures, of which one of the media is and equipment (both fixed and portable) will have.
selective for fungi (e.g. Sabouraud Dextrose Agar, SDA). Using a risk assessment tool such as Hazard Analysis
Importantly, the choice of culture media, incubation Critical Control Points (HACCP) helps to construct
times, and temperatures all require assessment and workflows. Through these workflows, the areas of
validation. There is no clear regulatory guidance on greatest risk can be pinpointed, the appropriate sample
which agar to use, and for how long to incubate. It is types and locations can be selected, and the basis of a
also important that before use, each lot of culture media sample map can be formed.
must be verified and compared to the manufacturer’s
certificate of analysis.
Sampling Number, Time and Frequency
There is a requirement to monitor different surfaces,
Next, the number of samples to be taken during a
either those close to the product, which may indicate
production run can be determined. The time of
contamination, or other samples to indicate cleaning
sampling should also take account of testing after a
and disinfection efficacy. Surface monitoring includes
‘cleandown’ testing at the end of a shift, testing at
testing these various surfaces, including product contact
times of highest operator activity or high levels of
surfaces, floors, walls and ceilings, for microorganisms.
materials in the area. The frequency of sampling should
The two common methods used are contact plates and
be assessed based on a risk management approach.
swabs. In general, contact plates get better recovery;
Factors to consider may include room activities,
however, swabs are frequently used for curved and thin
product exposure risk, room temperature, process stage,
surfaces, such as a window frame. The culture media
duration of process activities, and water exposure.
should ideally contain a suitable disinfectant neutralizer
to reduce the risk of disinfectant residues that remain
on surfaces.
ISO 14698 Part 2 Trending Data
Air monitoring methods include active air samplers
In order to identify patterns and possible reasons
and settle plates. Active air samplers generally fall
for a given trend, it is useful to include appropriate
into three designs, which affects how they collect
information with tables and graphs. Such information
airborne particles: still to agar, membrane filtration,
includes locations, dates, times, identification results,
and Anderson impaction. The standard requirement
changes to room design, operation of new equipment,
is to sample one cubic meter of air and capture the
shift or personnel changes, seasons and HVAC problems
microorganisms onto an agar surface. It is important
(e.g., an increase in temperature).
to know the particle collection size efficiency of an
air sampler, known as the D50 value, as well as the It is also important to regularly profile the
biological collection efficiency. microorganisms found during environmental monitoring,
some of which must be speciated using identification
Settle plates, in contrast, detect microorganisms that
techniques. The species should be profiled regularly
fall out of the air due to gravity. They are useful when
for changes in profile, the presence of unexpected or
placed in the right location, especially within the
‘objectionable’ microorganisms, and cross-comparison
unidirectional airflow cabinet, and smoke studies are
(e.g. comparing surfaces to people or differently
useful for helping to select the best locations. Settle
graded cleanrooms).
plate results are typically expressed as the number
of microorganisms collected per time of exposure.
Care must be taken when using settle plates because
they are quite easy to cross-contaminate, especially
in grade A or ISO class V conditions.
 Planned Revision to ISO 14698 Air Sampler Validation
There are some issues with the existing standard, The MAS-100® family of air samplers (Merck KGaA
particularly how it fits in with EU GMP and FDA guidance Darmstadt, Germany) is specially designed for monitoring
documents (e.g. guidance on aseptic filling). There is microbiological contamination in aseptic production
also a lack of working examples for constructing an areas and isolators with the rigorous requirements of
environmental monitoring program. Additionally, rapid the pharmaceutical industry. All MAS-100® instruments
microbiological methods, such as spectrophotometric are tested and designed to ISO 14698-1 annex B
counters, are not included. Therefore, there is clearly a (guidance in validating air samplers). The MAS-100®
need for a revised standard, and this process has been instrument family was recently independently validated
ongoing since January 2013. It is important for industry according to this international standard.
to contribute towards this process.
The test facility incorporated a cleanroom with a
volume of 20 cubic meters, and a 10-minute flush of
A revised standard is expected to provide clean, horizontal flow of clean air after sampling. The
setup allows for quick turnover of the room between
the following:
bacterial aerosol challenges.
•D
 etail on how to classify airborne biocontamination in
cleanrooms, including methods of measurement and There are two critical factors in air sample validation:
the validation of sampling methods physical efficiency and biological efficiency. Suspensions
•D
 etail concerning approaches to classify and monitor of washed Bacillus atrophaeus spores (NCTC 10073) in
cleanroom surfaces distilled water were prepared and used as the source in
• S etting of microbial monitoring limits physical and biological efficiency testing. Suspensions
of spores (1 x 105 colony forming units (cfu) per ml) in
•R
 isk management and assessment techniques
0%, 0.007%, 0.07%, 0.7% and 7% of potassium iodide
•C
 onsideration of the relationship between
(KI) in 80% aqueous ethanol were prepared for use
enumeration and the types of isolates detected
(objectionable microorganism) in the physical efficiency testing. The B. atrophaeus
solution was then dispensed using a spinning top
•C
 onsideration of some of the weaknesses of
environmental monitoring sampling methods, aerosol generator to produce an aerosol of controlled
such as active (volumetric) air-samplers, which particle size. This calibrated instrument can determine
could be outlined the actual amount of particles captured by the
• Introduce a possible classification scheme for instrument with high accuracy.
maximum permitted viable counts in a similar way
to ISO 14644’s tables for particle counts

Units are placed in semi-circle

at the same distance from STAG

Units are placed at the same

height as each other, a small
fan is placed below the STAG
and a larger fan above to
ensure even distribution

Figure 1.
Test Installation According to ISO 14698
Physical Efficiency Biological Efficiency
For the physical efficiency testing procedure, membrane For biological efficiency testing, a three-jet Collison
filtration (diameter of 80 mm, pores of 0.8 microns) was nebulizer (manufactured by Mesa Labs) operating at a
used as a reference method. The filtration system was pressure of 26 pounds per square inch (psi) was used
connected to a vacuum pump and flow was regulated to generate the mixed microbial aerosol. The spray
by a mass flow meter. The reference method membranes suspensions were made up using a stock 105 sterile
were then transferred to gross medium and incubated aqueous dilution of the B. atrophaeus spore suspension
for at least 18 hours at 37 ˚C before examination. The and suitably diluted recent liquid culture of Staph
MAS-100® air samplers and the reference method epidermidis. After the bacterial solution was prepared,
equipment were equidistant from the aerosol generator the nebuliser was placed in the controlled test chamber.
to ensure consistency in sampling, and appropriate
distribution of the bioaerosol within the environment The biological efficiency testing was carried out in a Class
chamber (Figure 1). III microbiological safety cabinet that allows for greater
aerosol control during testing. Because of the nature of
When evaluating physical efficiency, the impaction cultured microorganisms, biological efficiency results are
speed of the air sampler is an important parameter. typically lower than physical efficiency results, leading to
The speed should be high enough to aspirate particles extra control measures being put in place.
down to one micron, and should be adapted in a way
that viable particles are not damaged when they are Biological ratio Se/Ba sampled by the air sampler
= x 100
Efficiency (%) ratio Se/Ba sampled by reference method
impacted on the agar plate. Also, physical efficiency
is the same whether the particle is a microorganism,
The biological efficiency of the MAS-100® range was
carries a microorganism, or is an inanimate particle.
compared to a commonly used reference Instrument
Physical viable particles aspirated by air sampler method. Figure 3 contains the results of each type
= of MAS-100® instrument that was tested, and their
Efficiency (%) viable particles aspirated by reference method
corresponding biological efficiency results. These
All validated MAS-100® instruments perform similarly results support the ISO statement that there should be
across the range of the portfolio (Figure 2). The a compromise between the ability to collect particles
MAS-100® family performs well above the d50 cutoff (physical efficiency) and recover viable microorganisms
value according to ISO, and becomes even more accurate (biological efficiency). These results indicate great
as the particle sizes increase. Since all the air samplers recovery, even at an accelerated impaction rate over
have a standard flow of 100L per minute, these data the reference method.
support using a variety of MAS-100® systems in various
Instrument % Efficient
applications while maintaining consistency in results.
MAS 100 ISO MH 1 meter 76.74
MAS 100 ISO MH 9 meter 73.47
MAS 100 ISO NT 78.08
120 MAS 100 NT 82.62
Average % Physical Efficiency

100 MAS 100 VF 76.48

80
Figure 3.
60 MAS-100® Family ISO 14698 Microbiological Efficiency results

40

20

0
0 1 2 3 4 5 6
Particle Size (Mass mean diameter, Microns)
MAS-100 ISO MAS-100 VF MAS-100 ISO MH 9 m
MAS-100 NT MAS-100 ISO MH 1 m

Figure 2.
MAS-100® Family ISO 14698 Physical Efficiency results
Selecting an Agar Plate Conclusion
According to ISO 14698, certain criteria should be The international standard ISO 14698 can serve
considered when selecting an appropriate growth as a resource for laboratories when developing a
medium. It should be a broad, non-selective medium, biocontamination control strategy, which should
with appropriate additives or neutralizers to minimize focus on product quality, efficacy and patient safety.
antimicrobial activity of disinfectants or antibiotics. It is Environmental control, or the design of process
also important to evaluate the packaging of the plates systems to avoid contamination, is the most important
used, depending on the level of cleanliness expected component of any biocontamination strategy.
in the sample area. Using double- or triple-wrapped Environmental monitoring is a useful program to verify
plates can help. environmental control. A successful environmental
monitoring program must identify and encompass all
Additionally, it is recommended to use internally
the possible sources of contamination including
sterilized media, such as gamma-irradiated media,
people, air, surfaces and water spillages. Environmental
in aseptic areas. The agar should contain the right
monitoring data should be reproducible, taking both
neutralizers for their intended application, whether
physical and biological efficiency into account. The
the objective is to neutralize VHP, disinfectants, or
selection of high quality media also has an impact on
overcome antibiotic activity from residual products.
the quality of environmental monitoring results. The
Following incubation of the sample plates, the colonies combination of instruments and media chosen must
of the reference media and ICR media (Merck Millipore), be designed for different clean environments, and the
in combination with the MAS-100® air samplers, were instruments selected for each area should be matched
evaluated. A third-party laboratory reported both to give the same result using the same media so easy
larger colonies and good morphology, and also noted baseline measurements can be achieved.
that the strains grew slightly faster using ICR media.

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