TOPICS

1. Introduction
2. General requirements for the design of aseptic areas
3. Personnel and protective clothing
4. Cleaning and disinfection
5. Containers, labelling & protection of products
 INTRODUCTION
What are sterile products?
Sterile products means any dosage form of a drug devoid/lack of viable
microorganisms

What are pharmaceutical products that must be sterile?

 Category of Sterile Products

1. Those which are prepared under aseptic conditions from previously

sterilized materials
2. Those which are sterilized in their final containers (terminally sterilized)
3. The system where sterility for manufacturing is of prime consideration
but the product being manufactured is highly potent and can cause
adverse reactions in operators.
 Terminally Sterilized Product
● Should be manufactured in a clean room.
● The object should be prepared a pyrogen-free product with low
microbial and particulate counts.
● The efficiency of a sterilization process depends on keeping the
microbial load of the product to a minimum

What is a clean room?

A room with defined environmental control of particulate and
microbial contamination constructed and used so as to reduce
the introduction, generation, and retention of contaminants within
the area.
 Non-terminally Sterilized Product
● Those prepared under aseptic conditions from previously sterilized
materials, can be processed in clean areas until they have been
sterilized but must subsequently be filled into the final sterile
conditions in aseptic areas.

What is an aseptic area?

A room, suite of rooms or, more usually, a special area within a
clean area designed, constructed, and with process utilities
engineered and used with the intention of preventing microbial
contamination of the product.
Methods of Sterilization
GENERAL REQUIREMENTS FOR THE
DESIGN OF ASEPTIC AREAS
 General requirements for the design of aseptic areas

● Sterile medicinal products must be prepared in a specially designed

and constructed manufacturing departments which are separated from
other manufacturing areas

● Different types of operation such as component preparation, solution

preparation, filling and sterilization are effectively segregated one from
another.
 General requirements for the design of aseptic areas
● Production of sterile products should be carried out in a clean
environment with a limit for the environmental quality of microbial and
dust particle contamination.
● This limit for contamination is necessary to reduce the product
contamination.
● The production area is normally divided into
i. The clean-up area,
ii. The compounding area,
iii. The aseptic area,
iv. The quarantine area and
v. The packaging/labelling area.
 Flow Diagram of an Aseptic Area

Airlock
Aseptic filling Storage and
Office Compounding shipping
area area Quarantine

Storeroom
Packaging
Clean-up area Sterilization and
Quality
finishing
Contol
 Clean-Up Area
● The cleaning area has walls and ceilings made up of film coating
materials.
● Air inside the clean area should be free from dust and
microorganisms.
● Thisis ensured through high efficiency (95%) filters.
● Air existing in the clean area should be frequently replace (10-15 air
changes per hour).
 Compounding Area
● This area contains stainless steel cabinets and counters and is
involved in the actual compounding.

● Unlike aseptic area maintenance of sterile conditions is not essential,

but necessary measures should be adopted to control the dust
generated from raw material during weighing and compounding.
 Quarantine area
● This area consists of a store where the in-process batches as well as
approved batches are stored separately.

● This area has limited access and is under the control of a responsible
person.

● Without the consent of the in charge, other personnel cannot enter

into this particular area.
 Packing and labelling area
● In this area, the batches are packed and labelled.
● Packing is carried out by packaging machines, while labels are
obtained by over printing devices.
● At a time, only one product labels are printed.
● Parenteral packing plays a vital role in the production of sterile
preparations.
● Packing should be carried out in such a manner that the sterility of
the product is maintained.
 Floors, walls and ceilings
● All clean surfaces including the floor, walls and ceilings must be
smooth, easy to clean, disinfected and be constructed to minimize
microbial and particulate contamination.
● Flexing and non-flexing types of materials are used for construction
of floor.
● Flexing floor materials are made up of synthetic elastomers of
which most commonly used are polyvinylchloride (PVC).
● PVC flooring is easily repaired, cleaned, relatively cheap and simple.
● Non-flexing floors are made of hard inorganic filler substances in a
matrix material.
 Floors, walls and ceilings
● Walls must be made up of non-inflammable or fire-resistant material
e.g: Stainless steel, glass, enamelled steel, etc.
● Generally, plaster walls are easily damaged by the impact.
● For reduction of fungal growth, 1% of 8-hydroxyquinolone,
pentachlorophenol, etc may be added to the paint.
● Epoxy resin paints and polyurethane paints are also used to avoid
cracking and peeling.
● The ceilings are sealed to prevent the entry of microbial contaminants.
● Internal fittings such as a cupboards, drawers, shelves, and equipment
must be kept to a minimum.
 Doors, windows and services
● Doors and windows should fit flush with the walls. Windows if
required, are solely to provide illumination and are not for ventilation.
● Windows should be non-openable.
● Doors should be well fitted by maintaining the positive pressure air
flow and self closing. Doors must be limited in number.
● All pipes passing through the walls of the room should be effectively
sealed and should be flush fitting and easily cleaned.
● Gas cylinders should be excluded, and all gases should be piped
from outside the area.
 Doors, windows and services
● Sinks and drains must be excluded from the areas where aseptic
procedures are performed in clean room areas.
● Light sources in clean rooms are fitted with the ceilings to reduce
the collection of the dust and to avoid the disturbance of the air
flow pattern within the room.
● Non-essential switches such as room lighting switches should be
installed outside the clean area.
PERSONNEL AND PROTECTIVE
CLOTHING
 Personnel and protective clothing
● The main source of contamination of clean areas arises from skin
scales which are released by the operators.
● Personnel selected to work on the preparation of the parenteral
products must be neat and reliable.
● They should be in good health and free from dermatological
conditions that might increase the microbial load.
● Operator –borne contamination can be controlled by limiting the
number of operators in clean area.
● All personnel should be trained for Good Manufacturing
Practices(GMP) and aseptic techniques.
 Personnel and protective clothing
● The operator should wear sterile protective clothing including head
wear, powder free rubber or plastic gloves, a non-fibre shedding
facemask and footwear.
● All protective clothing is designed to prevent the contamination
from the body.
● All protective clothing must be sterilized by moist heat sterilization
or ethylene oxide sterilization.
● Fresh sterile clothing should normally be provided each time the
person enters the aseptic area.
Personnel and protective clothing
 Changing procedure
● Access to aseptic areas should be through rooms where normal
clothing is exchanged for the protective garments
● Changing rooms are usually divided into:
a) black area—where normal protective garments (e.g. lab coat
are removed, separated by a step-over bench from
b) grey area where hands are scrubbed, and protective garments
donned
c) white area where overboots are put on and, if necessary,
sterile gloves.
CLEANING AND DISINFECTION
 Cleaning and Disinfection
● Cleaning and disinfection procedures are used for the removal of microbial and
particulate contamination.
● Cleaning agents are the alkaline detergents, non-ionic and ionic surfactants.
● Routine daily cleaning (floor, instruments etc.) before work commences.
● Periodic cleaning of the walls, fittings, and working surfaces, and after cleaning
should be tested for acceptability before full working is resumed.
● The control and cleaning of protective garments including periodic checks to
ensure that the cleaning procedures do not produce particulates.
 Cleaning and Disinfection
● Different types of disinfectant should be employed in rotation to discourage the
development of resistant strains of microorganisms.
● Different types of disinfectants should be employed in rotation to prevent the
development of resistant strains of microorganisms.
● Different concentration of quarternary ammonium compounds, sodium
hypochloride, ethanol and formaldehyde solutions are used as disinfectants in
cleaning area.
● Cetrimide or chlorhexidine in 70% alcohol are suitable for use as skin
disinfectants.
 Monitoring
According to the guide to Good Pharmaceutical Manufacturing
Practice:
“Areas should be frequently monitored microbiologically by means of
settle plates, surface sampling, air sampling, or other appropriate
methods. The monitoring should be performed whilst normal
production operations are in progress. Records should be retained, and
immediate remedial action taken as soon as results deviate
significantly from those usually found in the area concerned”.

Settle plates
 Air Supply
● The air supplied to a clean room must be filtered through High
● Efficiency Particulate Air (HEPA) filters.
● The HEPA filter must be positioned at the inlet of the clean room and
the pre-filter may be fitted upstream of the HEPA filters to prolong the
life of final filter.
● HEPA filters are used in the construction of vertical and horizontal
laminar air flow bench.
● The air velocity at all parts of the filter area has to be about 0.54 m/sec
or (90+20feet/min).
 Air Supply
● Removes the Dioctyphthalate (DOP) particles of size-0.3μm
● The air filtered from the laminar air flow is claimed to be 99.97% free
from the microbial contamination.
● These filters are supported to provide class 100 air and they should be
certified every 6 to 12 months.
● Air quality is evaluated using settle plates, microbial air sampler or by
particle counters.
 Laminar flow equipment
1. Vertical laminar air flow bench
2. Horizontal laminar air flow bench
Vertical laminar air flow bench
Horizontal laminar air flow bench
Direction of air flow in horizontal laminar air flow
 Air flow pattern
● The air flow pattern within the clean room must be carefully
regulated
● To avoid generating particles from the clean room floor, walls and
operators.
● The general airflow patterns in in clean rooms are
• 1. Unidirectional airflow
• 2. Non-unidirectional airflow
• 3. Combined airflow
CONTAINERS, LABELLING &
PROTECTION OF PRODUCTS
 Containers for Sterile Products
• The purposes of sterile packaging is to identify, contain, protect
and deliver a dosage form in a sterile conditions to a patient.
• The requirements for containers are:
o They are chemically compatible with the product.
o They withstand sterilization.
o They maintain the sterility of the product.
o The permit safe withdrawal of the product.
• According to European Pharmacopeia (EP):
“ A container of pharmaceutical use is an article which contains
or intended to contain a product and is, or maybe in direct
contact with it. The closure is part of the container”
 Containers for Sterile Products (GLASS)
• Small volume parenteral (SVP) solutions
are usually 100 ml or less and are
packaged in different ways depending on
the intended use.

• Large volume parenterals (LVPs)

(sometimes called large volume
injections) are aqueous solutions usually
supplied in volumes of at least 100 ml
with sizes of 250 ml, 500 ml, 1000 ml,
3000 ml, and 5000 ml most common.
Directions usually recommend that large
quantities be administered.
 Containers for Sterile Products (GLASS)
Advantages Disadvantages
• Breakage during sterilization – soda-lime
• Good chemical resistance
glasses
• Neither adsorbs or release organic
• Attacked by alkaline solution
ingredients
• Impermeable • Development of hairline cracks
• Easily cleaned • Heavier than plastics
• Transparent • Requires venting during administration

• When involved with other materials (esp. for

• Rigid, strong and dimensionally stable
sealing) – autoclaving can be problematic

• Resists puncture • Requires inspection and washing before use

• Will hold a vacuum
• Can be autoclaved
 Containers for Sterile Products (PLASTIC)
• Plastic have become alternative to glass for LVPs and also used on a limited scale
for SVPs.
• Each polymer has its own characteristics and limitations.
• The limitation can be reduced by adding additives to improve the polymer
performance.
Advantages Disadvantages
• Relatively unbreakable • Reduced clarity
• The tolerance to oxygen and moisture is lesser
• Light
than glass
• Additive (if present) can be leached into
• Easily fabricated
medication
• Cheap • Sterilization is more difficult
• Single use
• Small filling ports – less chance of
contamination during filling
• Possible to completely seal
 Containers for Sterile Products (PLASTIC)
• Additives are usually added to improve a particular polymer’s performance.
• The general classes of additives commonly found in plastic for LVP packaging:
 Closures: Elastomeric Components
• Elastomers (rubbers) – can be molded into almost a variety of shapes to
meet specific needs.
• Desirable properties: compressibility and resealability.
• Seal small voids in mating surfaces such as vial necks. They are easily
penetrated by hypodermic needles and will reseal after withdrawal.
• The materials used to make elastomers should not include any
substance that can be extracted by the contents.
 Depyrogenation

• The most significant pyrogen for the pharmaceutical industry is

endotoxins associated with the outer membrane of gram-negative
bacteria.
• These toxins are constantly shed into organism’s environment .
• They are heat stable and can survive ordinary steam sterilization
cycles.
 Depyrogenation
Two most common methods for depyrogenation:

Dry heat treatment

• Method of choice for depyrogenation of metal and
glass.
• Requires not less than 250 °C for not less than 30 min.
• This method of endotoxin inactivation is known as
incineration.
Rinsing
• The oldest and simplest method to remove endotoxin
from solid surfaces.
• Involve rinsing with non pyrogenic solvent: sterile
water for injection BP* through wash-rinse procedure.
• Limulus amebocyte lysate (LAL) test is used to
validate endotoxin removal.
Sources of contamination in aseptic area and method to prevention
• Contamination, in broad sense, is the presence of minor unwanted
particulate matter called contaminants in atmosphere, physical body, etc.

• Right from production to packaging almost every sector of

pharmaceutical industry comes across contamination.

• The most common sources of contamination fall into the following three
main categories:

• Atmospheric contamination
• Fluid contamination
• Transfer contaminants
 1. Atmospheric contamination
• Atmospheric conditions during manufacturing as well as during storage
affects the quality of final preparation.

• Atmosphere in and around the industrial area contains potential

contaminants like dust, silica, etc and gases like CO2 , water vapour, etc.

• Besides the above mentioned contaminants, microorganisms like P.

aeruginosa, A. niger, etc.

• These contaminants may get incorporated into the end product either
during the process of manufacturing or during purification.
 Prevention:

• Prior to its entry into the working area, the air should be initially
passed through a suitable pre-filter then treated with an
electrostatic precipitator and finally through HEPA filters.

• Periodic removal of air-borne dust settled on walls, floors and

ceilings is essential.
 2. Fluid contamination

• Besides serving as the most common solvent in pharmaceutical

industry, water also serves as the greatest solvent in pharmaceutical
industry.
• Although, it is deprived of most of the contaminants yet it contains
pyrogens and traces of sulphates, chlorides and carbonates of Ca, Mg
and Na.
• Therefore, usage of water for washing the machineries and working
areas may leave traces of these contaminants.
 Prevention:

• Almost all of the pharmaceutical operations should be carried out

using purified water obtained upon deionization, distillation, ion
exchange, reverse osmosis, filtration or other similar processes.
• For the preparation of parenterals, water for injection, sterile water
for injection or bacteriostatic water for injection must be employed.
 3. Transfer contaminants

• Transfer contaminants refer to the contaminants sourced from

personnel and wheels of trolleys used for transport of goods.
• Personnel working in aseptic areas, if suffering from cold, allergies,
dermatological conditions or any similar illness carry multiple
microorganisms which upon expulsion into atmosphere via
sneezing, coughing, talking etc., can lead to contamination.
• For example, atmospheric dust particles may get entangled with the
fibres of the clothes which can get dislodged due to body
movements and lead to contamination.
 Prevention:

• Personnel should be well trained and periodically evaluated in the

principles of aseptic processing and techniques to be employed
before participating in the preparation of sterile products.
• Apart from gown, the personnel area also required to put on face
mask, head cap, gloves, foot covers and even goggle to ensure
complete coverage of all skin areas.
• The entrance of most of the working areas is equipped with air
blowers that aid in removing any loose dirt, lint from uniform of the
operators.
 Prevention:
● Those mechanical parts of the equipments that come in
contact with the parenteral products should be
demountable(removed from its setting) which enables their
easy cleaning and sterilization.
● All the apparatus and their carriers being carried to the aseptic
areas should be sterilized by suitable methods.
Thank you