1.

A disinfectant is one of a diverse group of chemicals, which reduces the number

of microorganisms present (normally on an inanimate object). A disinfectant is defined
as: "[a] chemical or physical agent that inactivates vegetative micro-organisms but not
necessarily highly resistant spores.
Disinfectants vary in their spectrum of activity, modes of action and efficacy.
Some are bacteriostatic, where the ability of the bacterial population to grow is halted.
Here the disinfectant can cause selective and reversible changes to cells by interacting
with nucleic acids, inhibiting enzymes or permeating into the cell wall. Once the
disinfectant is removed from contact with bacteria cells, the surviving bacterial
population could potentially grow. Other disinfectants are bactericidal in that they destroy
bacterial cells through different mechanisms, including causing structural damage to the
cell; autolysis; cell lysis and the leakage or coagulation of cytoplasm . Within these
groupings the spectrum of activity varies with some disinfectants being effective against
vegetative Gram positive and Gram-negative micro-organisms only while others are
effective against fungi. Some disinfectants are sporicidal in that they can cause the
destruction of endospore forming bacteria (these are the most difficult forms of
microorganisms to eliminate from cleanroom surfaces). However, a chemical agent does
not have to be sporicidal in order to be classed as a 'disinfectant' or as a iocide'. The
bacteristatic, bactericidal and sporicidal properties of a disinfectant are influenced by
many variables, not least their active ingredients.
There are many different types of disinfectants for use within the pharmaceutical
industry , with different spectrums of activity and modes of action. The mechanisms of
action are not always completely known and continue to be investigated. A range of
different factors needs to be considered as part of the process of selection including the
mode of action, and also efficacy, compatibility, cost and with reference to current health
and safety standards . In examining these criteria further, the main points to consider
when selecting a disinfectant are:
A disinfectant must have a wide spectrum of activity. This refers to the ability of
the disinfectant to kill different types of microorganisms and microorganisms which are
in different physiological states. Whether there is a requirement that the disinfectant is
sporicidal. This requirement influences the type of disinfectant purchased. Sporicidial
disinfectants tend to have greater health and safety considerations and some, particularly
chlorine based disinfectants, are aggressive to certain types of surfaces and will cause
discoloration and abrasion.The disinfectant must be rapid in action with an ideal contact
time of less than ten minutes. The contact time is the time taken for the disinfectant to
bind to the microorganism, traverse the cell wall and membrane and to reach its specific
target site. The longer the contact time, then the longer the surface or article needs to be
left for prior to use.The disinfectants selected must have different modes of action. Some
different types of disinfectants and their different modes of action are discussed below.
The emphasis on different modes of action is also tied to the regulatory expectation that
disinfectants are rotated, which is discussed later.Some disinfectants require certain
temperature and pH ranges in order to function correctly. One type of disinfectant, for
example, may not be effective in a coldroom due to the lower temperature. The reason for
this is because the validation standards for disinfectants measure the bactericidal activity
at 20oC and therefore the disinfectant may not be as effective at higher or lower
temperatures. Prior to the use of disinfectants it is essential that as much dirt and soil is
removed as possible. This requires the application of a detergent. Some disinfectants are
not compatible with certain detergents. In such circumstances detergent residues could
neutralize the active ingredient in the disinfectant. Any disinfectant purchased should be
compatible with the detergent used.Other disinfectants leave residues on surfaces. Whilst
this can mean a continuation of an antimicrobial activity, residues can also lead to sticky
surfaces and or the inactivation of other disinfectants. Different disinfectants are not
compatible with all types of surfaces. The disinfectants must not damage the material to
which they are applied to (although it is recognized that repeated applications over
several years may cause some corrosion). For more aggressive disinfectants a wipe down
using water or a less aggressive disinfectant like an alcohol is sometimes necessary in
order to remove the residues. In addition to some disinfectants having a corrosive affect,
others may be absorbed by fabrics, rubber and so on, which lessens their bactericidal
properties .The disinfectants must meet the requirements of the validation standards to
measure bactericidal, fungicidal and, if appropriate, sporicidal and viriucidal activity.
There are detailed standards which describe how disinfectants should be
validated, parts of which are undertaken by the manufacturer and some by the
pharmaceutical organization which purchases the disinfectants against a range of
different surfaces.The presentation of the disinfectant is an important choice whether as a
pre- diluted preparation in a trigger spray, or as a ready to use concentrate or an
impregnated wipe. Disinfectant suppliers like schlke provide a wide range of different
presentations of disinfectants.The disinfectants must be relatively safe to use, in terms of
health and safety standards. Here the main concern is with operator welfare. A related
concern is the impact upon the environment.The cost of the disinfectant is also a factor to
consider, especially it is to be used over a large surface area. If the disinfectant is required
for use in an aseptic filling area then it will need to be sterile filtered or supplied sterile in
a suitably wrapped container. Many disinfectant manufacturers like schlke supply
disinfectants which have been sterile filtered (through a 0.2m filter) and are provided in
gamma irradiated containers with outer wrapping.
Any disinfectant will only be effective if it is used at the correct concentration,
applied to relatively clean surfaces using appropriate cleanroom grade mops or cloths and
left for the correct contact time. In addition to surface disinfectants, hand sanitizers are
also required (for cleanroom staff to apply either to skin or to gloved hands) as part of a
comprehensive disinfection program.
Disinfectants have varying modes of action against microbial cells due to their
chemical diversity. Different disinfectants and target different sites within the microbial
cell. These include the cell wall, the cytoplasmic membrane (where the matrix of
phospholipids and enzymes provide various targets) and the cytoplasm. Some
disinfectants, on entering the cell either by disruption of the membrane or through
diffusion, then proceed to act on intracellular components. There are different approaches
to the categorization and sub-division of disinfectants, including grouping by chemical
nature, mode of activity or by bacteristatic and bactericidal effects on micro-organisms.
Some different types of disinfectant are: Non-oxidizing disinfectants; The majority of this
group of disinfectants have specific modes of action against micro-organisms, but
generally they have a narrower spectrum of activity compared to oxidising disinfectants.
This group includes: Alcohols like schlkes perform Alcohol EP (which disrupt the
bacterial cell membrane and has a one minute contact time), aldehydes (which have a
non-specific effect in the denaturing of bacterial cell proteins and can cause coagulation
of cellular protein), amphoterics (which have both anionic and cationic character and
possess a relative wide spectrum of activity), phenolics (some phenols cause bacterial cell
damage through disruption of proton motive force, while others attack the cell wall and
cause leakage of cellular components and protein denaturation) and quaternary
ammonium compounds (QAC), which are among the most commonly used disinfectants
in the pharmaceutical industry and include preparations like schlkes perform
Concentrate QB (available in convenient single-use bottles which reduces the preparation
time). The mode of action of QACs is on the cell membrane leading to cytoplasm
leakage and cytoplasm coagulation through interaction with phospholipids .Oxidizing
disinfectants generally have non-specific modes of action against micro- organisms.
They have a wider spectrum of activity than non-oxidizing disinfectants, with most types
able to damage endospores, but they can pose greater risks to human health and therefore
require greater control. This group includes: halogens like iodine and oxidizing agents
like peracetic acid, such as schlkes sporicidal perform Concentrate PAA, chemical
containing oxygen deposits like perform Concentrate OXY (available in single-use
sachets) and hydrogen peroxide. Concentrate OXY has an excellent material
compatibility and does not damage most surfaces. There are many commercially
available hand sanitizers with the most commonly used types being alcohol-based gels or
alcoholic hand rubs like desderman Pure provided by schlke. With hand sanitizers the
most important factor is the hand rubbing technique for the sanitizers are most effective
through the act of agitation by rubbing the hand sanitizer into the hands.Disinfectant
rotation; In selecting disinfectants many pharmaceutical manufacturers will opt to have
two in-use disinfectants and sometimes to have a third disinfectant as a reserve in case a
major contamination incident arises, such as a bioburden contamination build up, which
appears resistant or difficult to eliminate using the routinely used disinfectants. The
reserve disinfectant will often be more powerful and sporicidal, such as an oxidizing
agent, the routine use of which is restricted because of likely damage to the equipment
and premises. Typically the two primary disinfectants are rotated. This is a requirement of
regulatory bodies and the strongest pressure for it has come from Europe with the EU
GMP Guide stating that where disinfectants are used, more than one type should be
employed. This quotation is normally interpreted as a requirement for two different
types of disinfectant to be rotated. The USP in contrast, is less exacting and poses some
questions about the scientific need for rotation. The argument for rotating two
disinfectants is to reduce the possibility of resistant strains of microorganisms
developing. Whilst the phenomenon of microbial resistance is an issue of major concern
for antibiotics there are few data to support development of resistance to disinfectants.
This is particularly so when applied to dry environments such as cleanrooms where
microbial replication, as a primary process for gaining resistance, is minimal.
Whilst there is limited scientific evidence to support disinfectant resistance, there
is a need to meet regulatory expectations and many pharmaceutical organizations adopt
policies for disinfectant rotation. When using disinfectants with different modes of
activity more often one of the selected disinfectants is sporicidal. With regard to the
frequency of rotation this tends to based on the environmental monitoring data. Given
that environmental monitoring data should be reviewed for trends on a regular basis this
allows the frequency of cleaning and disinfection to be based on risk.

2.
Steam Sterilization
Of all the methods available for sterilization, moist heat in the form of saturated
steam under pressure is the most widely used and the most dependable. Steam
sterilization is nontoxic, inexpensive rapidly microbicidal, sporicidal, and rapidly heats
and penetrates fabrics. Like all sterilization processes, steam sterilization has some
deleterious effects on some materials, including corrosion and combustion of lubricants
associated with dental hand pieces; reduction in ability to transmit light associated with
laryngoscopes; and increased hardening time (5.6 fold) with plaster-cast.

The basic principle of steam sterilization, as accomplished in an autoclave, is to

expose each item to direct steam contact at the required temperature and pressure for the
specified time. Thus, there are four parameters of steam sterilization: steam, pressure,
temperature, and time. The ideal steam for sterilization is dry saturated steam and
entrained water (dryness fraction 97%). Pressure serves as a means to obtain the high
temperatures necessary to quickly kill microorganisms. Specific temperatures must be
obtained to ensure the microbicidal activity. The two common steam-sterilizing
temperatures are 121oC (250oF) and 132oC (270oF). These temperatures (and other high
temperatures) 830 must be maintained for a minimal time to kill microorganisms.
Recognized minimum exposure periods for sterilization of wrapped healthcare supplies
are 30 minutes at 121oC (250oF) in a gravity displacement sterilizer or 4 minutes at 132oC
(270oC) in a pre-vacuum sterilizer. At constant temperatures, sterilization times vary
depending on the type of item (e.g., metal versus rubber, plastic, items with lumens),
whether the item is wrapped or unwrapped, and the sterilizer type.

The two basic types of steam sterilizers (autoclaves) are the gravity displacement
autoclave and the high-speed prevacuum sterilizer. In the former, steam is admitted at the
top or the sides of the sterilizing chamber and, because the steam is lighter than air, forces
air out the bottom of the chamber through the drain vent. The gravity displacement
autoclaves are primarily used to process laboratory media, water, pharmaceutical
products, regulated medical waste, and nonporous articles whose surfaces have direct
steam contact. For gravity displacement sterilizers the penetration time into porous items
is prolonged because of incomplete air elimination. This point is illustrated with the
decontamination of 10 lbs of microbiological waste, which requires at least 45 minutes at
121oC because the entrapped air remaining in a load of waste greatly retards steam
permeation and heating efficiency . The high-speed prevacuum sterilizers are similar to
the gravity displacement sterilizers except they are fitted with a vacuum pump (or
ejector) to ensure air removal from the sterilizing chamber and load before the steam is
admitted. The advantage of using a vacuum pump is that there is nearly instantaneous
steam penetration even into porous loads. The Bowie-Dick test is used to detect air leaks
and inadequate air removal and consists of folded 100% cotton surgical towels that are
clean and preconditioned. A commercially available Bowie-Dick-type test sheet should
be placed in the center of the pack. The test pack should be placed horizontally in the
front, bottom section of the sterilizer rack, near the door and over the drain, in an
otherwise empty chamber and run at 134oC for 3.5 minutes 813, 819. The test is used each
day the vacuum-type steam sterilizer is used, before the first processed load. Air that is
not removed from the chamber will interfere with steam contact. Smaller disposable test
packs (or process challenge devices) have been devised to replace the stack of folded
surgical towels for testing the efficacy of the vacuum system in a prevacuum
sterilizer.833 These devices are "designed to simulate product to be sterilized and to
constitute a defined challenge to the sterilization process" 819, 834. They should be
representative of the load and simulate the greatest challenge to the load 835. Sterilizer
vacuum performance is acceptable if the sheet inside the test pack shows a uniform color
change. Entrapped air will cause a spot to appear on the test sheet, due to the inability of
the steam to reach the chemical indicator. If the sterilizer fails the Bowie-Dick test, do not
use the sterilizer until it is inspected by the sterilizer maintenance personnel and passes
the Bowie-Dick test.
Another design in steam sterilization is a steam flush-pressure pulsing process,
which removes air rapidly by repeatedly alternating a steam flush and a pressure pulse
above atmospheric pressure. Air is rapidly removed from the load as with the prevacuum
sterilizer, but air leaks do not affect this process because the steam in the sterilizing
chamber is always above atmospheric pressure. Typical sterilization temperatures and
times are 132oC to 135oC with 3 to 4 minutes exposure time for porous loads and
instruments.
Like other sterilization systems, mechanical, chemical, and biological monitors
monitor the steam cycle. Steam sterilizers usually are monitored using a printout (or
graphically) by measuring temperature, the time at the temperature, and pressure.
Typically, chemical indicators are affixed to the outside and incorporated into the pack to
monitor the temperature or time and temperature. The effectiveness of steam sterilization
is monitored with a biological indicator containing spores of Geobacillus
stearothermophilus (formerly Bacillus stearothermophilus). Positive spore test results are
a relatively rare event and can be attributed to operator error, inadequate steam
delivery or equipment malfunctions.
Portable (table-top) steam sterilizers are used in outpatient, dental, and rural
clinics. These sterilizers are designed for small instruments, such as hypodermic syringes
and needles and dental instruments. The ability of the sterilizer to reach physical
parameters necessary to achieve sterilization should be monitored by mechanical,
chemical, and biological indicators.

The oldest and most recognized agent for inactivation of microorganisms is heat.
D-values (time to reduce the surviving population by 90% or 1 log10) allow a direct
comparison of the heat resistance of microorganisms. Because a D-value can be
determined at various temperatures, a subscript is used to designate the exposure
temperature (i.e., D121C). D121C-values for Geobacillus stearothermophilus used to monitor
the steam sterilization process range from 1 to 2 minutes. Heat-resistant non spore-
forming bacteria, yeasts, and fungi have such low D121C values that they cannot be
experimentally measured

Moist heat destroys microorganisms by the irreversible coagulation and

denaturation of enzymes and structural proteins. In support of this fact, it has been found
that the presence of moisture significantly affects the coagulation temperature of proteins
and the temperature at which microorganisms are destroyed.

The advantages of steam sterilization is that it is nontoxic to patient, staff,

environment, cycle easy to control and monitor, Rapidly microbicidal, least affected by
organic/inorganic soils among sterilization processes listed, rapid cycle time and
penetrates medical packing, device lumens. The major disadvantage of this system is that
it is Deleterious for heat-sensitive instruments. Microsurgical instruments might be
damaged by repeated exposure. It may leave instruments wet; causing them to rust
moreover there is potential for burns.

Steam sterilization should be used whenever possible on all critical and semi
critical items that are heat and moisture resistant (e.g., steam sterilizable respiratory
therapy and anesthesia equipment), even when not essential to prevent pathogen
transmission. Steam sterilizers also are used in healthcare facilities to decontaminate
microbiological waste and sharps containers but additional exposure time is required in
the gravity displacement sterilizer for these items.

3.

Microbiological testing of sterile products in the pharmaceutical industry remains

a regulatory requirement, despite the limitations of sterility tests. Absence of evidence
does not equal evidence of absence. Sampling of sterile products must be representative
and must not allow any opportunities for accidental contamination and false positive
results. Compendial sterility test methods require 14-day incubation times, but rapid
methods have the potential to reduce that to five days or less.

SterileProducts The term microbiologically sterile is an absolute, meaning a

complete absence of viable microorganisms. Unfortunately, for all practical purposes it is
impossible to be certain that a given product is sterile without testing, and therefore
destroying, all of it. Since such an approach would be of little value in a manufacturing
environment, it is sterility assurance that is the important concept for industry. Sterility
assurance is a probabilistic function, and refers to the probability of an item containing
viable microorganisms after the application of a validated sterilisation process. If that
probability can be reduced to a sufficiently low level, the product can be referred to as
sterile.

Sterilisation processes are applied to products in a number of industries, including

food and beverage manufacture, but it is mainly in the pharmaceutical and medical
sectors where the sampling of sterile products for testing remains an important routine
task for microbiologists. Sterility assurance is critical in the manufacture of many drugs
and other medicinal products and is closely regulated worldwide. Products carrying a
claim of sterility typically require some form of sterility test to be carried out before
release in order to verify that claim. Ideally, a sterilisation process (e.g. heat, or ionising
radiation) would be applied to the product in the final container at the end of the
manufacturing process. Such terminal sterilisation can give sterility assurance levels of
10-6 (one non-sterile unit in a lot of one million) or better. However, some products
contain heat-, or radiation-sensitive components, which cannot be terminally sterilised in
pack. These products may need to be sterilised by filtration, then filled aseptically.
Sterility assurance levels for such products are necessarily lower.

While sterility testing may be required or recommended by regulations governing

the pharmaceutical industry, it plays a relatively minor role in sterility assurance. By far
the greatest contribution to sterility comes from the validation and control of the
sterilisation process, and/or of aseptic processing procedures. Sterility testing is only
capable of detecting relatively high levels of contamination in a given lot of product. For
example, suppose a 10,000-unit lot with a contamination level of 0.1% were sterility
tested by sampling 20 units. There is a 98% probability that the contamination would not
be detected and that the lot would be passed as sterile. These limitations have lead to the
almost complete abandonment of sterility testing in industries other than pharmaceuticals.
The canning of food products, for instance, requires a very high level of sterility
assurance, which is achieved entirely by validation and control of the sterilisation
process, and by careful control of other processing factors post sterilisation. Nevertheless,
the sterility test remains an important tool for pharmaceutical microbiology laboratories
to determine conformance where there is a claim that a product is sterile.

In view of the limitations of sterility testing it is crucial that a representative

sample of the product is tested. What constitutes a representative sample depends on a
variety of factors, but it must be based on rational criteria, such as random sampling
procedures, so that the sample accurately reflects the material to be tested
Where sterility testing is carried out for quality control purposes as a pre-release
test there is guidance on the minimum number of samples that should be tested in the
largely harmonised compendial test methods published in pharmacopoeias (USP Chapter
71, EP 2.6.1 & JP 4.06). These provide guidance on the minimum number of units to be
tested for different types of products and for different batch sizes and also the minimum
quantity of product from each unit that should be sampled. It is important to note that the
guidance covers only the minimum sampling rate required. In practice, the number of
samples tested may be determined by other factors, such as the desired sterility assurance
level (the acceptable contamination rate) for the product. The sampling plan also needs to
take into account the nature of the sterilisation process. For example, a sampling plan for
a product that is subject to an aseptic filling process should include samples from the
beginning, middle and end of each fill and should also include samples taken after
significant process interventions. Whereas, for a product terminally sterilised by heat, the
sampling plan should ensure that samples are taken from the coolest part of the load.

Sterility testing may also be carried out for quality assurance purposes as a means
of continuously monitoring the process, rather than as a pre-release test. The development
of a statistically valid sampling plan is equally important, since it must be able to detect
any deviations from the acceptable contamination rate. Compendial sterility test methods
do not usually contain guidance for the development of this type of sampling plan, but the
statistical principles involved are well documented. The key is to decide exactly what the
sampling plan should be able to detect and then design a plan that will achieve the desired
detection level while taking into account any other relevant characteristics of the
manufacturing and sterilisation processes.

Sterility testing methods

Compendial methods for sterility testing of pharmaceutical products are based on
culturing any viable microorganisms in the sample, but there are important considerations
with regard to the laboratory environment.
It is absolutely vital that the possibility of accidental contamination being
introduced during testing is minimised. False positive results inevitably mean that the
batch or lot under test will be condemned as non-sterile. Re-testing is not a practical
option, since the chances of detecting low level contamination are even less once a
contaminated unit has been removed from the lot. For this reason the testing laboratory
must be able to provide a level of contamination control at least equivalent to that of an
aseptic filling facility. This usually means an ISO Class 5 cleanroom, or an isolator to
provide a barrier between the laboratory environment and the product. The use of
isolators for aseptic operations, including sterility testing, is reported to be growing in the
pharmaceutical industry.

Compendial methods for sterility testing require that a sample be cultured using
two separate media. These are usually fluid thioglycollate medium (FTM), to culture both
anaerobic and some aerobic bacteria, and soybean casein digest medium (SCDM) to
culture fungi and aerobic bacteria. The cultures are incubated for 14 days at 32.5oC and
22.5oC and then examined. Any turbidity in the culture may indicate growth and must be
investigated.

There are two recommended methods for carrying out the test. The first is by
direct inoculation, whereby a small volume of sample is removed aseptically from the
sample unit and inoculated directly into a suitable volume of growth medium prior to
incubation. This method has some significant disadvantages. Firstly, only small volumes
of product can be inoculated into the culture medium, limiting the sensitivity of the test.
Secondly, if the sample appears milky or turbid, it can be very difficult to detect turbidity
caused by microbial growth at the end of the incubation period.
 To overcome these drawbacks the recommended method wherever possible is
membrane filtration. Here the sample is passed through a 0.45 m membrane filter and
the filter is then transferred to the culture medium for incubation. Membrane filtration
allows the whole sample, or a composite sample, to be passed through a single filter and
is therefore potentially much more sensitive than direct inoculation. Filtration also
provides an opportunity to rinse away components in the sample that may cause turbidity
and any growth inhibitors, such as antibiotics or preservatives, which may be present.

The membrane filtration method may be carried out using a traditional open
filtration system, or by using one of the commercially available closed systems, where
the sample is never exposed to the test environment, thus minimising the opportunities
for contamination and false positive results. A widely used example of a closed
membrane filtration system is the Millipore Steritest filtration unit, which includes
connection devices and tubing so that samples can be withdrawn aseptically from
ampoules, collapsible bags and other containers without being exposed to the external
environment. The sample is then pumped through two individual filter canisters each
containing a 47 mm, 0.45 m membrane filter. Once filtration is complete each canister is
filled with 100 ml of medium (FTM and SCDM) and incubated. A range of filters is
available to suit products with different characteristics and the canisters are colour coded.
The system can also be used to sterility test medical devices.

Whichever method is used, it must be properly validated for the product being
tested to ensure that it does not increase the probability of recording a false negative
result. A number of different test microorganisms, including Staphylococcus aureus,
Bacillus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes and Candia albicans,
should be used to demonstrate that the method is able to detect the required range of
organisms.

Rapid Methods for Sterility Testing

Current compendial methods for sterility testing in the pharmaceutical industry
remain culture-based and include an incubation period of 14 days. Clearly, this is a delay
that is becoming less and less acceptable in a modern manufacturing operation. However,
there are signs that the situation is changing. For example, initiatives such as Process
Analytical Technology (PAT) and parametric product release are challenging the need for
sterility tests to be completed before product can be released and both the FDA and the
EMEA are encouraging the adoption of new analytical technologies to help ensure final
product quality. In the USA the FDA Center for Biologics Evaluation and Research
(CBER) is proposing radical changes to the sterility test requirements for biological
products, though not so far for all pharmaceuticals, promoting the use of rapid
microbiological methods (RMM) as alternatives to the compendial sterility test methods.
Such changes in the regulatory climate are creating renewed interest in RMM for sterility
testing and several technologies are already available commercially.

Adenosine triphosphate (ATP) bioluminescence is a well established rapid method

utilising a specific substrate and enzyme combination, luciferin/luciferase, to break down
microbial ATP from growing cells and produce visible light, which can be measured
using a luminometer. Several commercial systems have been developed for a range of
pharmaceutical test applications, including sterility testing, especially for filterable
samples where non-microbial ATP in the sample is less of a concern. The test time can be
reduced considerably because detection of microbial growth in culture media is
accomplished by ATP-bioluminescence, rather than by visible turbidity. Typically, results
equivalent to those of compendial tests are available within 7 days or less. An example is
the Celsis Rapid Detection System, combining the companys Advance luminometer and
AMPiScreen ATP-bioluminescence assay reagents, which use proprietary enzyme
technology to increase the quantity of microbial ATP produced and reduce detection
times by 25-50%.

The Milliflex Rapid Microbiology Detection and Enumeration system from

Millipore also uses ATP-bioluminescence to detect microbial cells and is designed
specifically for monitoring microbial contamination in filterable samples. It is automated,
employing image analysis technology to detect microcolonies growing directly on the
surface of a membrane filter after the addition of bioluminescence reagents. The system
is designed to be quantitative, but a method has been developed and validated to use it for
a rapid sterility test with an incubation time of just five days.
 Colorimetric growth detection methods rely on a colour change being produced in
a growth medium as a result of microbial metabolism during growth, often as a result of
CO2 production. The best example of a commercial colorimetric assay system, which can
be used for sterility testing is the BacT/ALERT 3D Dual-T Microbial Detection System
from bioMerieux. The system is automated and employs sensitive colour detection and
analysis technology to produce a result in as little as three days. It can detect both aerobic
and anaerobic bacteria, as well as yeasts and moulds.

All living cells produce a small amount of fluorescence (autofluorescence) and

this can be used to detect microbial colonies growing on a solid surface long before they
are visible to the naked eye. This technique is particularly useful for filterable samples,
where a membrane filter can be incubated on a conventional nutrient medium and
scanned using highly sensitive imaging systems to detect microcolonies, sometimes
several days earlier than using traditional colony counting methods.

Autofluorescence detection has been commercialised by Rapid Micro Biosystems

as Growth Direct, which uses a large area CCD imaging system without magnification
to detect developing microcolonies. Although not yet validated for testing sterile
products, proof of concept has been establish can be used for sterility testing is the
BacT/ALERT 3D Dual-T Microbial Detection System from bioMerieux. The system is
automated and employs sensitive colour detection and analysis technology to produce a
result in as little as three days. It can detect both aerobic and anaerobic bacteria, as well
as yeasts and moulds.

Cytometry does not rely on microbial growth to detect contamination, but instead
uses cell labelling techniques to detect viable microorganisms. This approach has the
potential to detect a wide range of organisms, including yeasts and moulds, within
minutes. Commercial systems utilise combined fluorescent cell labelling and flow
cytometry or solid phase cytometry to detect viable microbial cells. Typically, the cells
are labelled using a fluorescent dye or a non-fluorescent substrate, which is converted to
a fluorochrome in viable cells. Detection of the labelled cells occurs by laser scanning in
either a flow cell (flow cytometry), or on a solid phase platform such as a membrane
filter (solid phase cytometry). AES Chemunex has developed solid phase cytometry
detection systems. The companys ScanRDI (also known as ChemScan RDI) system is
capable of detecting 1 CFU per sample and has been evaluated as a possible RMM for
sterility testing. The technology has been developed for the Stereal-T sterility testing
system.

4.
Clean rooms and clean air devices should be routinely monitored in operation and
the monitoring locations based on a formal risk analysis study and the results obtained
during the classification of rooms and/or clean air devices. For Grade A zones, particle
monitoring should be undertaken for the full duration of critical processing, including
equipment assembly, except where justified by contaminants in the process that would
damage the particle counter or present a hazard, e.g. live organisms and radiological
hazards. In such cases monitoring during routine equipment set up operations should be
undertaken prior to exposure to the risk. Monitoring during simulated operations should
also be performed. The Grade A zone should be monitored at such a frequency and with
suitable sample size that all interventions, transient events and any system deterioration
would be captured and alarms triggered if alert limits are exceeded. It is accepted that it
may not always be possible to demonstrate low levels of 5.0 m particles at the point of
fill when filling is in progress, due to the generation of particles or droplets from the
product itself. (c) It is recommended that a similar system be used for Grade B zones
although the sample frequency may be decreased. The importance of the particle
monitoring system should be determined by the effectiveness of the segregation between
the adjacent Grade A and B zones. The Grade B zone should be monitored at such a
frequency and with suitable sample size that changes in levels of contamination and any
system deterioration would be captured and alarms triggered if alert limits are exceeded.
(d) Airborne particle monitoring systems may consist of independent particle counters; a
network of sequentially accessed sampling points connected by manifold to a single
particle Working document QAS/09.295 Rev.1 page 8 counter; or multiple small particle
counters located near to monitoring points and networked to a data acquisition system.
Combinations of systems can also be used. The system selected should be appropriate for
the particle size considered. Where remote sampling systems are used, the length of
tubing and the radii of any bends in the tubing should be considered in the context of
particle losses in the tubing. The selection of the monitoring system should take account
of any risk presented by the materials used in the manufacturing operation, for example
those involving live organisms or radiopharmaceuticals. The sample sizes taken for
monitoring purposes using automated systems will usually be a function of the sampling
rate of the system used. It is not necessary for the sample volume to be the same as that
used for formal classification of clean rooms and clean air devices. The airborne particle
conditions given in Table 2 for the at rest state should be achieved in the absence of the
operating personnel after a short clean-up or recovery period of about 1520 minutes
(guidance value), after completion of the operations. The particulate conditions given in
Table 2 for grade A in operation should be maintained in the zone immediately
surrounding the product whenever the product or open container is exposed to the
environment. In order to demonstrate control of the cleanliness of the various clean areas
during operation, they should be monitored for airborne particles and microbiological
contamination. In addition to at rest and in operation classification, airborne particles
should be monitored periodically in operation at critical locations.
The sampling plan need not be the same as that used for classification. Locations
and sample sizes should be determined based on assessment of the process and
contamination risk. The monitoring of Grade C and D areas in operation should be
performed in accordance with the principles of quality risk management. The
requirements and alert/action limits will depend on the nature of the operations carried
out, but the recommended clean up period should be attained. Other characteristics
such as temperature and relative humidity depend on the product and nature of the
operations carried out. These parameters should not interfere with the defined cleanliness
standard.