UNIT IV
4.1: Designing of aseptic area, Laminar flow equipment’s
4.2: Study of different sources of contamination in an aseptic area and methods of prevention
4.3: Clean area classification
4.4: Principles and methods of different microbiological assay
4.5: Methods for standardization of antibiotics, vitamins and amino acids
4.6: Assessment of a new antibiotic
4.1. ASEPTIC AREA
4.1.1. Introduction
Aseptic techniques are employed to provide protection to ophthalmic and parenteral products
by preventing the entry of microbial and particulate contamination. Prevention of microbial
contamination is also required to remove pyrogens and toxic bacterial products. The terminally
sterilised products (product sealed in container and then sterilised) are prepared in clean areas:
while products not terminally sterilised are prepared under aseptic conditions using sterile
materials or are sterilised by filtration before being packed in sterile containers. Such aseptic
products are formulated or prepared in an aseptic area, which is a room within a clean area
designed, constructed, serviced, and used for controlling and preventing microbial
contamination of the product.
Like sterile medicinal products, vaccines containing dead microorganisms, microbial extracts,
or inactivated viruses are also filled in aseptic areas; while live or attenuated vaccines are filled
in separate areas.
The goal of any facility is to design such an aseptic or sterile environment which provides a
controlled environment so that the entrance of viable (microbial) and non-viable (particles)
contaminants can be minimised. A controlled environment prevents cross- contamination of
Compounded Sterile Preparations (CSPs). To reduce the contamination-related risk, all
compounding should be done within primary engineering controls, like a Laminar Airflow
Hood (LAFH), also known as workbenches, Biological Safety Cabinet (BSC), or a
Compounding Aseptic Isolator (CA).
4.1.2. Designing of Aseptic Area
The sterile production unit and the general manufacturing area within the hospital pharmacy or
factory should be located separately. The sterile production unit should be buffered, i.e.,
unauthorised personnel should not gain access to this unit.
, Figure 4.1: Flow Diagram of Aseptic Area
The aseptic unit is designed to carry out each stage of production separately. The unit should
also ensure a safe and organised workflow so that the need for personnel to move around the
clean rooms is minimised. The unit is built and the equipment is positioned in such a manner
that the product remains protected from contamination.
The layout of aseptic area should be such that cleaning can be done easily and dust
accumulation can also be reduced. Arrangement should be such that the risk of cross-
contamination (contamination of one product or material with another) is reduced.
The filling area should be adjacent to the compounding area where the personnel assemble and
prepare materials utilised by the staff in the filling area. In figure 4.1 the layout of areas and
rooms for preparing terminally sterilised products (such as small or large volume injections) is
shown.
4.1.2.1. Design and Construction
Only authorised personnel can gain access to the clean and aseptic filling areas. The personnel
enter the clean rooms by passing through the changing rooms where they put on and remove
their clean room uniform.
A pass-Over (or cross-over) bench extends across the changing room to form a physical barrier
for separating the different areas for changing by the personnel.
Special precautions are taken for preventing clean and aseptic filling areas from getting
contaminated while materials are being passed through airlocks or hatchways. Thus, sterilisers
and entry ports are fitted with double-sided doors, which are interlocked to prevent
simultaneous opening of both the doors.
,4.1.2.2. Surfacing Materials
The floor, wall, and ceiling surface of clean rooms should be smooth, impervious, and
unbroken to reduce the release and accumulation of contaminating particles and organisms.
The surface material should be such that they can withstand the effects of cleaning agents and
disinfectants. The ceilings are scaled so that the contaminants do not enter from the space above
them.
Uncleanable recesses should be avoided within the clean rooms to minimise the accumulation
of contaminating particles. Thus, the wall and floor junction should be covered. Minimum
shelves, ledges, cupboards, and equipment should be present. Non opening and sealed windows
should be present to prevent the entry of contaminants.
4.1.2.3. Services
The piped liquids and gases entering the clean rooms should be filtered first, thus ensuring that
the liquid or gas at the work position is as clean as the air in the clean room. The position of
pipes and ducts should be such that they can be easily cleaned. Other fittings, like fuse boxes
and switch panels should be placed outside the clean rooms.
Sinks and drains should not be present in areas where aseptic procedures are carried out within
the clean room areas. They should nowhere be present in the complete unit. The areas having
sinks and drains should be designed, positioned, and maintained such that the risk of microbial
contamination is reduced; therefore, they are fitted with easily cleanable traps, installed with
electrically heated disinfection devices.
A limited number of doors and ports should be present for entry of personnel and materials,
respectively. The entry doors should be self-closing to allow easy movement of the personnel.
Airlock doors, wall ports, through-the-wall autoclaves, and dry heat sterilisers should have
interlocked doors to prevent simultaneous opening of both the doors. All the doors should have
an alarm system which should ring when more than one door are being opened.
Lights in clean rooms should be fitted with the ceiling so that dust accumulation reduces and
also the airflow pattern within the room is not disturbed. The equipment in clean rooms should
be positioned such that accumulation of particles and microbial contaminants does not Occur.
4.1.3. Laminar Flow Equipment (Laminar Airflow Hood)
A Laminar Airflow Hood (LAFH)/ Laminar Aseptic Hoods, or a workbench, is a primary
engineering control device which provides the following services during aseptic compounding
1) Clean air to the critical sites (immediate aseptic compounding area),
2) Constant flow of air out of the work area to prevent the entry of room air, and
3) Outward flow of air from the hood that suspends and removes contaminants which have
been introduced in the work area by personnel.
, Figure 4.2: Section through a Mini-Pleat High-Efficiency Filter
A High Efficiency Particulate Air (HEPA) Filter is the most important part of a LAFH. The air
within the room is taken into this filter and passed through a pre-filter which removes the gross
contaminants (lint, dust, etc.). The air is then blown at a uniform velocity through the hood and
HEPA filter in a unidirectional (laminar flow) manner over the critical sites (immediate aseptic
compounding area). HEPA filter is a particulate filter which traps the airborne particles and
microbes; but allows the gases to pass through.
HEPA filter should be fitted either at or near to the clean room inlet. A pre-filter is fitted
upstream of the HEPA filter, thus extending the final filter's life. A fan is also fitted which
pumps the air through the filter.
The filter medium used in HEPA filters 1S made up of pleated fiberglass paper. Parallelly
arranged pleats not only increase the filter surface area but also the air flow through the filter
(figure 10.2). This parallel arrangement of filter medium also allows the filter to retain a
compact volume. In the traditional type of HEPA filters, aluminium foil was used as spacers,
which are no longer used in the modern mini-pleat type of filter (now widely used). The mini-
pleat filters have a shallower depth in construction than the traditional HEPA filter. The filter
material is sealed to an aluminium frame within the filter (figure l0.2). One side of the filter is
protected with a coated mild steel mesh. HEPA filters provide:
1) A high air flow rate,
2) A high particulate holding capacity, and
3) A low-pressure drop across the filter.
HEPA filters remove larger, medium, and smaller particles from the air by inertial impaction,
direct interception, and by Brownian diffusion, respectively. The HEPA filters are least
4.1: Designing of aseptic area, Laminar flow equipment’s
4.2: Study of different sources of contamination in an aseptic area and methods of prevention
4.3: Clean area classification
4.4: Principles and methods of different microbiological assay
4.5: Methods for standardization of antibiotics, vitamins and amino acids
4.6: Assessment of a new antibiotic
4.1. ASEPTIC AREA
4.1.1. Introduction
Aseptic techniques are employed to provide protection to ophthalmic and parenteral products
by preventing the entry of microbial and particulate contamination. Prevention of microbial
contamination is also required to remove pyrogens and toxic bacterial products. The terminally
sterilised products (product sealed in container and then sterilised) are prepared in clean areas:
while products not terminally sterilised are prepared under aseptic conditions using sterile
materials or are sterilised by filtration before being packed in sterile containers. Such aseptic
products are formulated or prepared in an aseptic area, which is a room within a clean area
designed, constructed, serviced, and used for controlling and preventing microbial
contamination of the product.
Like sterile medicinal products, vaccines containing dead microorganisms, microbial extracts,
or inactivated viruses are also filled in aseptic areas; while live or attenuated vaccines are filled
in separate areas.
The goal of any facility is to design such an aseptic or sterile environment which provides a
controlled environment so that the entrance of viable (microbial) and non-viable (particles)
contaminants can be minimised. A controlled environment prevents cross- contamination of
Compounded Sterile Preparations (CSPs). To reduce the contamination-related risk, all
compounding should be done within primary engineering controls, like a Laminar Airflow
Hood (LAFH), also known as workbenches, Biological Safety Cabinet (BSC), or a
Compounding Aseptic Isolator (CA).
4.1.2. Designing of Aseptic Area
The sterile production unit and the general manufacturing area within the hospital pharmacy or
factory should be located separately. The sterile production unit should be buffered, i.e.,
unauthorised personnel should not gain access to this unit.
, Figure 4.1: Flow Diagram of Aseptic Area
The aseptic unit is designed to carry out each stage of production separately. The unit should
also ensure a safe and organised workflow so that the need for personnel to move around the
clean rooms is minimised. The unit is built and the equipment is positioned in such a manner
that the product remains protected from contamination.
The layout of aseptic area should be such that cleaning can be done easily and dust
accumulation can also be reduced. Arrangement should be such that the risk of cross-
contamination (contamination of one product or material with another) is reduced.
The filling area should be adjacent to the compounding area where the personnel assemble and
prepare materials utilised by the staff in the filling area. In figure 4.1 the layout of areas and
rooms for preparing terminally sterilised products (such as small or large volume injections) is
shown.
4.1.2.1. Design and Construction
Only authorised personnel can gain access to the clean and aseptic filling areas. The personnel
enter the clean rooms by passing through the changing rooms where they put on and remove
their clean room uniform.
A pass-Over (or cross-over) bench extends across the changing room to form a physical barrier
for separating the different areas for changing by the personnel.
Special precautions are taken for preventing clean and aseptic filling areas from getting
contaminated while materials are being passed through airlocks or hatchways. Thus, sterilisers
and entry ports are fitted with double-sided doors, which are interlocked to prevent
simultaneous opening of both the doors.
,4.1.2.2. Surfacing Materials
The floor, wall, and ceiling surface of clean rooms should be smooth, impervious, and
unbroken to reduce the release and accumulation of contaminating particles and organisms.
The surface material should be such that they can withstand the effects of cleaning agents and
disinfectants. The ceilings are scaled so that the contaminants do not enter from the space above
them.
Uncleanable recesses should be avoided within the clean rooms to minimise the accumulation
of contaminating particles. Thus, the wall and floor junction should be covered. Minimum
shelves, ledges, cupboards, and equipment should be present. Non opening and sealed windows
should be present to prevent the entry of contaminants.
4.1.2.3. Services
The piped liquids and gases entering the clean rooms should be filtered first, thus ensuring that
the liquid or gas at the work position is as clean as the air in the clean room. The position of
pipes and ducts should be such that they can be easily cleaned. Other fittings, like fuse boxes
and switch panels should be placed outside the clean rooms.
Sinks and drains should not be present in areas where aseptic procedures are carried out within
the clean room areas. They should nowhere be present in the complete unit. The areas having
sinks and drains should be designed, positioned, and maintained such that the risk of microbial
contamination is reduced; therefore, they are fitted with easily cleanable traps, installed with
electrically heated disinfection devices.
A limited number of doors and ports should be present for entry of personnel and materials,
respectively. The entry doors should be self-closing to allow easy movement of the personnel.
Airlock doors, wall ports, through-the-wall autoclaves, and dry heat sterilisers should have
interlocked doors to prevent simultaneous opening of both the doors. All the doors should have
an alarm system which should ring when more than one door are being opened.
Lights in clean rooms should be fitted with the ceiling so that dust accumulation reduces and
also the airflow pattern within the room is not disturbed. The equipment in clean rooms should
be positioned such that accumulation of particles and microbial contaminants does not Occur.
4.1.3. Laminar Flow Equipment (Laminar Airflow Hood)
A Laminar Airflow Hood (LAFH)/ Laminar Aseptic Hoods, or a workbench, is a primary
engineering control device which provides the following services during aseptic compounding
1) Clean air to the critical sites (immediate aseptic compounding area),
2) Constant flow of air out of the work area to prevent the entry of room air, and
3) Outward flow of air from the hood that suspends and removes contaminants which have
been introduced in the work area by personnel.
, Figure 4.2: Section through a Mini-Pleat High-Efficiency Filter
A High Efficiency Particulate Air (HEPA) Filter is the most important part of a LAFH. The air
within the room is taken into this filter and passed through a pre-filter which removes the gross
contaminants (lint, dust, etc.). The air is then blown at a uniform velocity through the hood and
HEPA filter in a unidirectional (laminar flow) manner over the critical sites (immediate aseptic
compounding area). HEPA filter is a particulate filter which traps the airborne particles and
microbes; but allows the gases to pass through.
HEPA filter should be fitted either at or near to the clean room inlet. A pre-filter is fitted
upstream of the HEPA filter, thus extending the final filter's life. A fan is also fitted which
pumps the air through the filter.
The filter medium used in HEPA filters 1S made up of pleated fiberglass paper. Parallelly
arranged pleats not only increase the filter surface area but also the air flow through the filter
(figure 10.2). This parallel arrangement of filter medium also allows the filter to retain a
compact volume. In the traditional type of HEPA filters, aluminium foil was used as spacers,
which are no longer used in the modern mini-pleat type of filter (now widely used). The mini-
pleat filters have a shallower depth in construction than the traditional HEPA filter. The filter
material is sealed to an aluminium frame within the filter (figure l0.2). One side of the filter is
protected with a coated mild steel mesh. HEPA filters provide:
1) A high air flow rate,
2) A high particulate holding capacity, and
3) A low-pressure drop across the filter.
HEPA filters remove larger, medium, and smaller particles from the air by inertial impaction,
direct interception, and by Brownian diffusion, respectively. The HEPA filters are least
