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Sterile Area

a disinfection stage if the product is a liquid or semisolid
preparation. Plastic bottles that are either blow- or
injection-moulded have a very low microbial count and
may not require disinfection. They may, however, become
contaminated with mould spores if they are transported
in a non-sanitary packaging material such as unlined
cardboard. Packaging materials that have a smooth,
impervious surface, free from crevices or interstices, e.g.
cellulose acetate, polyethylene, polypropylene, polyvinyl
chloride (PVC), and metal foils and laminates, all have a
low surface microbial count.
Closure liners of pulpboard or cork, unless specially
treated with a preservative, foil or wax coating, are often a
source of mould contamination for liquid or semisolid
products. A closure with a plastic flowed-in liner is less
prone to introduce or support microbial growth than one
stuck in with an adhesive, particularly if the latter is based
on a natural product such as casein. Closures can be sterilized by either formaldehyde or ethylene oxide gas if required.
In the case of injectables and ophthalmic preparations
which are manufactured aseptically but do not receive a
sterilization treatment in their final container the packaging has to be sterilized (Figure 23.2b). Dry heat at 170 °C
is often used for vials and ampoules. Containers and closures may also be sterilized by moist heat, chemicals and
irradiation, but consideration of the destruction or
removal of bacterial pyrogens may be necessary (see
Chapter 22). Regardless of the type of sterilization, the
process must be validated and critical control points or
other risk assessment parameters (section 3.1) must be
4 Manufacture of sterile products
Methods of sterilization are discussed in Chapter 21 and
the various types of sterile product are described in
Chapter 22. For production purposes an important distinction exists between sterile products which have been
terminally sterilized (Figure 23.2a) and those which have
not. Terminal sterilization involves the product being
sealed in its container and then sterilized, usually by heat,
but ionizing radiation or, less commonly, ethylene oxide
may be employed. Such a product must be manufactured
in a clean area (sections 4.1.1–4.1.8). A product which
cannot be terminally sterilized is prepared aseptically
(Figure 23.2b) from previously sterilized materials or by
sterile filtration; in either case, aseptic filling is a poststerilization step. Strict aseptic conditions are required
throughout (section 4.1).
Vaccines, consisting of dead microorganisms, microbial extracts or inactivated viruses (see Chapter 24) may
be filled in the same premises as other sterile medicinal
products, so the completeness of killing or removal of
live organisms must be validated before processing.
Separate premises are needed for the filling of live or
attenuated vaccines and for the preparation of other
products derived from live organisms. Non-sterile products and sterile products must not be processed in the
same area.
4.1 Clean and aseptic areas: general
4.1.1 Design of premises
Sterile production should be carried out in a purpose built unit separated from other manufacturing areas and
thoroughfares. The unit should be designed to encourage
separation of each stage of production but should ensure
a safe and organized workflow. A plan of such a facility
is shown in Figure 23.3. Sterilized products held in quarantine pending sterility test results (Chapter 21: Sharp,
1997) must be kept separate from those awaiting
Figure 23.2 A comparison of (a)
terminal sterilization and (b) aseptic
processing in sterile manufacturing.
Aseptic area
Figure 23.3 Example of a diagrammatic
representation of the layout and workflow
of a sterile products manufacturing unit. 1,
the changing area in this example is built
on the black (A)–grey (B)–white (C)
principle; passage into the clean area is
through A and B (see section 3.1.6) whereas
entry to the aseptic area is first through A
and B followed by C (see section 3.2.2). 2,
Dividing step-over sill. 3, For details of
aseptic area requirements, see text; a
laminar airflow work station would be
included in this area. 4i–4iv, These areas are
clean areas. In filling rooms for terminally
sterilized products, care should be exercised
to protect containers from airborne
contamination. The final rinse point (i.e.
where the containers are finally washed)
should be sited as near as possible to the
filling point. 5, Articles which are to be
transferred directly to the aseptic area from
elsewhere must be sterilized by passage
through a double-ended sterilizer. Solutions
manufactured in the clean area may be
brought into the aseptic area through a
sterilizing-grade membrane filter. 6,
Double-doored hatchway through which
presterilized articles may be passed into the
aseptic area (see section 3.2.3). Note:
inspection, holding and final packaging
areas have been omitted. Direction of
workflow: –––––
–––––– , for
terminally sterilized products; ⋅ ⋅ ⋅
⋅ , for aseptically prepared products;
- -, shared stages of preparation.
Mixing and preparation
4.1.2 Internal surfaces, fittings and floors
Particulate, as well as microbial, contamination must be
prevented. To this end all surfaces must be smooth and
impervious in order to: (1) prevent accumulation of dust
or other particulate matter; and (2) permit easily repeated
cleaning and disinfection. Smooth rounded coving
should be used where the wall meets the floor and the
Suitable flooring may be provided by welded sheets of
PVC; cracks and open joints which might harbour dirt
and microorganisms must be avoided. The preferred surfaces for walls are plastic, epoxy-coated plaster, plastic
fibreglass or glass-reinforced polyester. Often the final
finish for the floor, wall and ceiling is achieved using
continuous welded PVC sheeting. False ceilings should be
adequately sealed to prevent contamination from the
space above. Use should be made of well-sealed glass
panels, especially in dividing walls, to ensure good visibility and allow satisfactory supervision. Doors and windows
should be flush with the walls. Windows should not be
Internal fittings such as cupboards, drawers and shelves
should be kept to a minimum. They must be sited where
they do not interfere with the laminar flow of the filtered
air supply. Stainless steel or laminated plastic are the
preferred materials for such fittings. Stainless steel
trolleys may be used to transport equipment and materials within the clean and aseptic areas but must remain
confined to their respective units. Equipment must be
designed so that it may be easily cleaned and sterilized or
Table 23.3 Operations carried out in the various grades
of air
Examples of operations
For terminally sterilized products
4.1.3 Services
Clean and aseptic areas must be adequately illuminated;
lights are best housed in translucent panels set in a false
ceiling. Electrical switches and sockets must be flush with
the wall or fitted outside. When required, gases should be
pumped in from outside the unit. Pipes and ducts, if they
must be brought into the clean area, must be sealed
through the walls. Additionally, in order to prevent dust
accumulation, pipes and ducts must be boxed in or
readily cleanable. Alternatively, they may be sited above
false ceilings.
Sinks should be of stainless steel with no overfl ow, and
water must be of at least potable quality. Wherever possible, drains should be avoided. If installed they must be
fitted with effective, readily cleanable traps and with air
breaks to prevent backflow. Any floor channels should be
open, shallow and cleanable and connected to drains
outside the area; they should be monitored microbiologically. Sinks and drains should be excluded from aseptic
areas except where radiopharmaceuticals are being processed when sinks are a requirement.
4.1.4 Air supply
Areas for sterile manufacture are classified according to
the required characteristics of the environment. Each
operation requires an appropriate level of microbial and
particulate cleanliness; four grades (Table 23.2) are specified in The Rules and Guidance for Pharmaceutical
Manufacturers and Distributors (2007). Environmental
quality is substantially influenced by the air supplied to
the manufacturing environment. The grades of air
required for specific manufacturing activities are listed in
Table 23.3.
Filtered air (Chapter 22) is used to achieve the necessary standards; this should be maintained at positive
pressure throughout a clean or aseptic area, with the
highest pressure in the most critical rooms (aseptic or
clean filling rooms) and a progressive reduction through
the preparation and changing rooms (Figure 23.4); a
minimum pressure differential of 10 kPa is normally
required between each class of room. A minimum of
20 changes of air per hour is usual in clean and aseptic
rooms. The air inlet points should be situated in or
near the ceiling, with the final filters placed as close as
Filling of products, when unusually at risk
Background environment to grade A
preparation areas
Preparation of solutions, when unusually
at risk
Filling of products
Preparation of solutions and components
for subsequent filling
For aseptic processes
Aseptic preparation and filling
Background environment to grade A
preparation areas
Preparation of solutions to be filtered
Handling of components after washing
Background for an isolator
Entry to aseptic areas
Remove outer shoes, clothing,
swing legs over sill, don slippers
Wash and dry hands and forearms.
Put on sterile hood and mask.
Rewash and dry hands, forearms
Put on suit, overboots, gloves.
Rinse gloved hands in antiseptic
Commence work
Figure 23.4 Entry into aseptic area.
possible to the point of input to the room. Equipment or
furnishings must be sited so as not to interfere with
laminar flow.
The greatest risk of contamination of a product comes
from its immediate environment. Additional protection
is needed both in the filling area of the cleanroom and in
the aseptic suite. This can be provided by a workstation
supplied with a unidirectional flow of filtered sterile air.
This is known as a laminar flow cabinet. Displacement of
air may be vertical or horizontal with a typical homogeneous air flow of 0.45 m/s at the working position.
Consequently airborne contamination is not added to the
work space, and any generated by manipulation is swept
away by the laminar air currents. A fuller description of
high efficiency particulate air (HEPA) filters in laminar
flow cabinets is given by Gardner and Peel (1998).
The efficacy of the filters through which the air is
passed should be monitored at predetermined intervals.
Air quality may be monitored for bacteria and fungi by
slit sampler or settle plate. Particles are measured using a
discrete airborne particle counter. The latest edition of
The Rules and Guidance for Pharmaceutical Manufacturers
and Distributors (2007) states that particles must be
monitored continuously in a grade A area and recommends it for grade B areas. It should be noted that grade
A air is not the purest that can be obtained; four even
cleaner grades are used in the electronics industry
4.1.5 Clothing
Clothing worn in a clean area must be of non-shedding
fibres; polyester is a suitable fabric. Airborne contamination, both microbial and particulate, is reduced when
trouser suits, close-fitting at the neck, wrists and ankles,
are worn. Clean suits should be provided once a day, but
fresh headwear, overshoes and powder-free gloves are
necessary for each working session. Special laundering
facilities are desirable. Additional requirements for
aseptic rooms are discussed in section 5.1.
4.1.6 Changing facilities
Entry to a clean or aseptic area should be through a
changing room fitted with interlocking doors; this design
acts as an airlock to prevent influx of air from the outside.
This route is for personnel only, not for the transfer of
materials and equipment. Staff entering the changing
room should already be clad in the standard factory
or hospital protective clothing. For entry into a clean
area, passage through the changing room should be from
a ‘black’ to a ‘grey’ area, via a dividing step-over sill
(Figure 23.4). Movement through these areas and finally
into the cleanroom is permitted only when observing a
strict protocol, whereby outer garments are removed in
the ‘black’ area and cleanroom trouser suits donned in
the ‘grey’ area. Only after hand-washing in a sink fitted
with elbow- or foot-operated taps may the operator enter
the cleanroom.
The changing procedure for entry to an aseptic area is
described in section 5.1.2.
4.1.7 Cleaning and disinfection
A strict, validated disinfection policy is necessary if
microbial contamination is to be kept to a minimum.
Cleaning agents used include alkaline detergents and
ionic and non-ionic surfactants. A wide range of chemical disinfectants is available (Chapter 19). Clear, soluble
phenolics are commonly used for interior services and
fittings. Disinfectants for working surfaces are alcohols
(70% ethanol or isopropanol) or, less commonly,
chlorine-based agents such as hypochlorites. Skin may be
disinfected with cationic detergents such as cetrimide or
chlorhexidine, usually formulated with 70% alcohol to
avoid the need for rinsing. Gloved hands may be disinfected with these detergents or 70% alcohol. The former
have the advantage of offering residual activity. Rotation
of different disinfectants reduces the risk of the emergence of resistant strains, but such rotation should be
validated. In-use dilutions must not be used unless sterilized. Disinfectants and detergents for use in grade A/B
areas must be sterile prior to use and formulated with
water for injections. Modern sprays are fitted with devices
to prevent air being sucked back, extending the life of the
disinfectant. As mentioned in section 4.1.2, smooth polished surfaces are more readily cleaned. Floors and horizontal surfaces should be cleaned and disinfected daily,
walls and ceilings as often as required, but the interval
should not exceed 1 month. Regular microbiological
monitoring should be carried out to determine the efficacy of disinfection procedures. Records must be kept
and immediate remedial action taken should normal
levels for that area be exceeded.
4.1.8 Operation
The number of persons involved in sterile manufacture
should be kept to a minimum to avoid the inevitable
turbulence and shedding of particles and organisms associated with the operatives. All operations should be
undertaken in a controlled and methodical manner as
excessive activity may increase turbulence and particle
Containers made from fibrous materials such as paper,
cardboard and sacking are generally heavily contaminated (especially with moulds and bacterial spores)
and should not be taken into clean areas. Ingredients
which must be brought into clean areas must first be
transferred to suitable metal or plastic containers.
Containers and closures for terminally sterilized products
must be thoroughly cleaned before use and should
undergo a final washing and rinsing process in apyrogenic distilled water (which has been passed through a
bacteria-proof membrane filter) immediately prior to
filling. Containers and closures for use in aseptic manufacture must, in addition, be sterilized after washing and
rinsing in preparation for aseptic filling (Figure 23.2).
5 Aseptic areas
5.1 Additional requirements
Additional requirements for aseptic areas, over and above
those discussed in sections 4.1.1–4.1.8, are discussed
5.1.1 Clothing
Requirements in addition to those in section 4.1.5 are
needed for aseptic areas. The operative is a potential
source of microorganisms and it is imperative that steps
are taken to prevent this contamination. The operative
must wear sterile protective headwear totally enclosing
hair and beard, spectacles, powder-free rubber or plastic
gloves (often two pairs are worn), a non-fibre-shedding
facemask (to prevent the release of droplets) and footwear. A suitable garment is a one- or two-piece trouser
suit. Fresh sterile clothing should be provided each time
a person enters an aseptic area.
5.1.2 Entry to aseptic areas
Entry to an aseptic suite is usually through a ‘black–grey–
white’ changing procedure (Figure 23.4), where white represents the highest level of cleanliness. Movement from
‘black’ to ‘white’ is via two changing rooms, the ‘grey’ area
also serving as an entry to the cleanroom (Figure 23.4 and
section 4.1.6). There are several types of entry system in
use. More details may be found in Whyte (2010).
5.1.3 Equipment and operation
Any articles entering the aseptic area should ideally be
sterilized, but may be disinfected. In order to achieve this,
articles should be transferred via a double-ended sterilizer or hatch (i.e. with a door at each end). If they are
not to be discharged directly to the aseptic area, they
should be (1) double-wrapped before sterilization; (2)
transferred immediately after sterilizing into a clean environment until required; (3) transferred from this clean
environment via a double-doored hatch (where the outer
wrapping is removed) to the aseptic area (where the inner
wrapper is removed at the workbench). Hatches and
sterilizers must be designed so that only one door may be
opened at any one time. Solutions manufactured in the
cleanroom may be brought into the aseptic area through
a sterile 0.22 μm membrane filter.
Workbenches, including laminar flow units, and
equipment, should be disinfected immediately before and
after each work session. Equipment must be of the simplest design possible for the operation being performed.
Aseptic manipulations must be carried out in the
grade A air of a laminar flow cabinet or isolator. Speed,
accuracy and economy of movement are essential features of good aseptic technique. It is therefore essential
that workers are well trained and motivated and familiar
with the task in hand. Observation and microbiological
monitoring of the operator and of the environment are
very important. Under no circumstances must living
microorganisms, including those used for vaccine preparation (Chapter 24) and for biological monitoring be
introduced into the aseptic area.
5.2 Environmental monitoring
Monitoring of the environment is essential during manufacturing. It ensures that environmental requirements are
being met and also helps spot trends.
Air is monitored for particles (section 4.1.4) and
microorganisms. Microorganisms are usually sought
using settle plates or active samplers, such as the slit-toagar sampler. Settle plates rely on organisms falling from
the atmosphere and settling onto an exposed agar plate.
After a specified time (usually 4 hours) the plate will be
covered and incubated. A slit-to-agar sampler draws in a
specified volume of air, forcing organisms onto the
surface of an agar plate. This latter method is able to give
a viable count per volume, but organisms may be damaged
and hence rendered non-viable by the capture process.
Limits of viable counts for different grades of air are
shown in Table 23.4. One of the limitations of traditional
microbial detection is the time taken to culture bacteria
and fungi. There is a great deal of interest in developing
rapid (Denyer, 2007) or instantaneous (Jiang, 2009)
methods of microbial detection.
The nature of contamination can be informative. For
example, the presence of Staphylococcus spp. suggests
human-borne contamination. The adequacy of changing
facilities and gowning would then be checked. In contrast, Bacillus spores would suggest environmental contamination and the entry of equipment into the
cleanroom would be scrutinized.