University Institute of Pharmaceutical Sciences is a flag bearer of excellence in Pharmaceutical education and research in the country. Here is another initiative to make study material available to everyone worldwide. Based on the new PCI guidelines and syllabus here we have a presentation dealing with "Aseptic requirements for parenteral products". Thank you for reading. Hope it was of help to you. UIPS,PU team

1. Aseptic Requirements for
Parenteral Products
A presentation by –
Sachin Pasricha (1743)
Sanjeevni Dhamija (1744)
University Institute Of Pharmaceutical
Sciences, Panjab University,Chd

2. THE NEED FOR ASEPSIS
• The need for aseptic conditions for parenteral products is
to prevent the access of microorganisms and other
contaminations during their preparation and testing
• These requirements are very necessary as unlike the oral
route where the microbes get killed either by the gastric
acid or the MALT in the intestine , here there is no such
way . So , the parenteral route can easily provide the
microorganisms , an access to the blood and the vital
organs which can cause sepsis and that can even lead to
death.

3. Manufacture of sterile preparations
• Clean areas for the manufacture of sterile products are classified
according to the required characteristics of the environment. Each
manufacturing operation requires an appropriate level of environmental
cleanliness in the operational state to minimize the risks of particulate or
microbial contamination of the product or materials being handled.
• For the manufacture of sterile pharmaceutical preparations, four grades
of clean areas are distinguished as follows:
• Grade A: The local zone for high-risk operations, e.g. filling and making
aseptic connections. Normally such conditions are achieved by using a
unidirectional airflow workstation. Unidirectional airflow systems should
provide a homogeneous air speed of 0.36–0.54 m/s (guidance value) at
a defined test position 15–30 cm below the terminal filter or air
distributor system. The velocity at working level should not be less than
0.36 m/s. The uniformity and effectiveness of the unidirectional airflow
should be demonstrated by undertaking airfl ow visualization tests.

4. Contd
• Grade B: In aseptic preparation and filling, this is the
background environment for the Grade A zone.
• Grades C and D: Clean areas for carrying out less
critical stages in the manufacture of sterile products
or carrying out activities during which the product is
not directly exposed (i.e. aseptic connection with
aseptic connectors and operations in a closed
system).
• A unidirectional airflow and lower velocities may be
used in closed isolators and glove boxes.

5. • In order to reach the B, C and D air grades the number of
air changes should be appropriate for the size of the
room and the equipment and personnel present in it.
• High-efficiency particulate air (HEPA) filters should be
subjected to an installed filter leakage test in accordance
with ISO 14644-3 (3) at a recommended interval of every
6 months, but not exceeding 12 months. The purpose of
performing regular leak tests is to ensure the filter
media, filter frame and filter seal are free from leaks. The
aerosol selected for HEPA leak testing should not support
microbial growth and should be composed of a sufficient
number or mass of particles. HEPA filter patching is
allowed at the filter manufacturer and in situ operation
provided that the patch sizes and procedures follow the
recommendations of ISO 1822-4 (4).

6. AIM OF ASEPTIC TECHNIQUE
• The aim of aseptic technique is to prevent the
access of microorganisms during the preparation and
testing of pharmaceutical products
• PREPARATION
aseptic technique is unnecessary if the last two
stages of the sterile product are –
1. packing of container and sealing to prevent
contamination after sterilization , followed by
2. sterilization by one of the heat sterilization
processes—dry heat , moist heat or heating with a
bactericide

7. • The terminal heat sterilization and reliable packing
ensure the issue of sterile preparations
• However there are several class of which terminal heat
treatment is not possible , for example
(a) thermolabile soluble substances stable in solution
eg: Thiamine Hydrochloride Injection
(b) thermolabile soluble substances unstable in solution
eg: Chorionic Gonadotrophin Injection
(c) thermolabile suspensions stable in vehicle eg:
Propyliodone and Propyliodone oily injections
(d) thermolabile powders that require dilution with
other powders eg: antibiotics dusting powders
(e)thermolabile powders that require incorporation in a
semisolid base eg: eye ointments of the BP
• filtration is impossible in the last three cases and ,
therefore, the medicament must be mixed with
appropriate sterile vehicle aseptically

8. TESTING
Aseptic technique is also necessary in sterility
testing. Growth in nutrient medium inoculated with
a sterile product can be attributed to contamination
of the product only if the manipulations involved in
performing the test are faultless.

9. SOURCES OF CONTAMINATION
1. The atmosphere
2. The breath
3. The hands
4. Clothing
5. The hair
6. The working space
7. Equipment

10. THE ATMOSPHERE
• it has no flora of its own and cannot support the growth
of microorganisms. But , it is highly contaminated with
particles. Microorganisms are associated with many of
these.
• Types of contamination
a. Dust
a high percentage of dust particles come from the soil
and therefore , may carry soil bacteria which are
chiefly saprophytes and include cocci and sporing rods
but pathogenic sporing rods are also quite common

11. b. Droplets
large no. of droplets are expelled from the
respiratory tract by coughing and sneezing and
may contain nose , mouth , throat and lungs which
may or may not be pathogenic
c. Droplet Nuclei
the smaller droplets evaporate quickly , and as they
contain saliva or mucus the residue consists of tiny
protein flakes carrying any organism previously
suspension
d. Free Microorganisms
bacteria and virus free from droplets are
uncommon but naked yeast and mould spores are
often abundant

12. THE BREATH
• Coughing , sneezing and spitting can cause
contamination at considerable distances by expelling
droplets of fluids lining the respiratory tract
• Staphylococcus aureus and haemolytic steptococci
are present in the noses and throats of a high
percentage of population

13. THE HANDS
• Major means of transmitting infection
• It has been estimated that there are 10000 organism
per sq cm of normal hand skin. These organism scan
be of two types
• A. resident flora : which normally resides , grows and
multiplies on skin , glands and hair follicles
• B. transient flora : consist of organisms collected
from other parts of the body or the environment

14. CLOTHING
• Part of the atmospheric dust that becomes
entangled in the fibres of the fabrics is dislodged by
body movements and can considerably raise the
levels of contamination around a person wwho is
working carelessly eg: a handkerchief dried after
previous use

15. THE HAIR
• Constantly exposed to atmospheric dust which it
entangles and may liberate during shaking the head
and brushing a lock from the face

16. THE WORKING SURFACE
• This is a potential source of contamination because
organisms will sediment on it from air

17. EQUIPMENTS
• In aseptic technique , no source of contamination is
more serious than unsterile equipment

18. THE DESIGN OF AN ASEPTIC LABORATORY
• Important factors are
• A. site
• B. size
• C . Windows
• D. doors
• E. surfacing materials
1. Floors
2. Walls and ceilings
3. bench tops
• F. Services
1. ventilation
2. electricity
3. gas
4. compressed air or vacuum
5. nitrogen
6. water
7. a method of dealing with waste

19. • SITE
away from lifts , rooms of non pharmaceutical staff ,
stairs , corridors .
no storage facilities
access to laboratory should be through one or more
rooms with changing and washing facilities
• SIZE
depends upon max. no. of people using it.
large and high ceiling rooms are pleasant to work in but
are difficult to maintain hence sometimes small rooms
with lower ceilings are also preferred.
• WINDOWS
although due to their dust collecting and distributing
properties, one is tempted to completely do without
them but then it will not be pleasant to work which will
cause decrease in efficiency plus sunshine is the best
detector of dust

20. Large windows of clear glass which should not get
opened . Glass should be double glazed
• DOORS
there should be an air lock with double doors 1m
apart to prevent sudden inrush of air when it is
opened.
swing doors are preferred over sliding doors
a wall hatch between store and laboratory with an
airlock is necessary for transferring equipments.

21. Large windows of clear glass which should not get opened .
Glass should be double glazed
• DOORS
there should be an air lock with double doors 1m apart to
prevent sudden inrush of air when it is opened.
swing doors are preferred over sliding doors
a wall hatch between store and laboratory with an airlock is
necessary for transferring equipments.
• SURFACING MATERIALS
should be smooth , impervious , easy to clean and resistant
to chemicals
the most suitable materials used for floors are Terrazzo ,
linoleum and plastics for finishing
The possible surfaces for walls and ceilings are tiles , hard gloss
paint on smooth plaster and plastic laminated boards
The most popular surfaces for bench tops used for asepsis work
are stainless steel and plastic laminates

22. • F. Services
An asepsis laboratory will require many if not all of the
following services–
a. Ventilation- includes removal of microorganisms ,
control of humidity and temperature , and provision of
fresh air
b. Electricity- for lighting and , sometimes , for a hot plate
, ultraviolet lamp , aerosol producer or vacuum pump
c. Gas – for the bunsen burner
d. compressed air and/or vacuum - for clarification and
bacterial filtration.
e. nitrogen – for replacement of air by an inert gas
f. water – not of importance for laboratory itselfbut
necessary in the washing room
g. a method of dealing with waste

23. FURNITURE
• 1. benches
to reduce dust collection and facilitate its detection and removal,
conventional benching may be replaced by tables and wall mounted work
shelves.
2. Seats
must be adjustable and comfortable. Discomfort is likely to cause
distractions , unnecessary and inefficient movements. Chairs are
preferred over stools
3. Trolleys
trolleys with removable trays , preferably of stainless steel are better than
tables , benches or cuboards for holding spare equipments.
4. Doormat
it is useful to have, in the airlock , a mat part immersed in a detergent –
disinfectant solution to prevent contamination
5. Screens
can be a. shack type
b. fume cupboard type

24. CLEANING METHODS
• DRY CLEANING
floors- brooms are not preferred as these disperse large amounts
of dust into the air. Vacuum cleaners are very satisfactory if the bag
has paper insert to prevent tiny particles from passing through the
fabric.
• WET CLEANING
floors- moping with hot detergent water is adequate if the mop is
washed daily
walls and furniture- wiping with slightly damp clean cloth is
sufficient
• DISINFECTION
need not to be done very frequently
the wet technique can be used but the mop should be soaked
in a separate quantity of solution to saturate it with disinfectant
and to prevent reduction in strength

25. CLOTHING
• Emphasis on clothing is essential for-
a. maintenance of highest standards of asepsis
b. comfort and appearance since either can affect
quality of work
c. ease of changing- because the impatience caused
by complicated regimen may lead to neglect of
important features

26. Gowns and trousers
• Gown must completely cover the clothes
underneath, even when the wearer is seated.
Therefore it must be long. Should be made up of
closely woven synthetic fibres like terylene . Cotton
and linen are not preferred as these shed tiny fibres .
• Long trousers are not often worn. The advantages
appear to be slight and the additional changing
requires extra time and possibly , privacy

27. Head dress
• The hairs , particularly if long and loose are very
likely to scatter dust and skin fragments
• Long hoods are preferred over caps due to comfort.

28. masks
• Covering the nose and mouth will not necessarily
prevent the microbiological contamination of
working area.
• So , the material , design , fit and period of use of the
masks are important
• Double layered masks can give best results regarding
the prevention of sterilization.
• Masks should be sterile and changed frequently

29. Gloves
• The wearing of sterile rubber gloves for aseptic work
presents more complications than advantages
• They get easily contaminated , costly , difficult to
sterilize and uncomfortable
• Non touch technique is much more preferred. Hands
even though clean and disinfected are considered to
be bacteriologically dirty
• Though gloves should be used in case of chemicals
like penicillin and close derivatives as exposure may
cause dermatitis

30. footwear
• Tennis and rubber shoes and surgeon’s boots are
uncomfortable if worn for long
• Leather shoes with low heels and rubber soles are
satisfactory and are easy to be kept clean if never
used outside suite.
• Overshoes of terylene may be used in industrial
asepsis laboratories

31. ASEPSIS ROOM VENTILATION
The processes involved in a typical ventilation system are
in order –
1. Intake of fresh air
2. Admixture of air exhausted from the room
3. Cooling if necessary
4. Dehumidification or humidification if necessary
5. Heating if necessary
6. Removal of microorganisms
7. Admission to the room at a rate that will maintain a
satisfactory temperature and relative humidity and ,
particularly , a low level of bacterial contamination
even when work is in progress.

32. METHODS OF PRODUCING
BACTERIOLOGICALLY CLEAN AIR
These are
• Mechanical filtration
• Electrostatic precipitation
• Heat
• Ultra violet light
• Chemicals

33. MECHANICAL FILTRATION
• MECHANISMS
a. straining
b. direct interception
c. diffusion
d. impingement
e. electrostatic attraction

34. • TYPES OF FILTERS
due to the requirement of high efficiency , choice of
filters becomes quite limited
1. Coarse filters– used if the concentration of one or
two bacteria carrying particles/cubic dm does not
constitute a hazard to aseptic processing
2. High Efficiency Filters– used where high efficiency
is desired like large rooms accommodating many
workers
3. Absolute filters – they are used in nuclear energy ,
for filters to retain even miniscule amount of
radioactive dust

35. c. Filter Beds
• Filter beds may consist of granules. Loosely
packed fibres or filter papers.

36. 1. Granules
• This type of bed has been used in the antibiotic
industry. The most successful material was
activated charcoal.

37. 2. Pads of loosely packed fibres.
• These may be animals , vegetables or mineral and
except for some slag – wool types are loosely packed.

38. A) Animal Fibres.
• One example is merino wool treated with resin to increase
electrostatic attraction but this type of medium is relatively
uncommon.
B) Vegetable Fibres
• Cotton wool. The use of non absorbent cotton wool for air filteration
naturally followed its success as a closure for containers of sterile
liquids in bacteriology and aseptic techninique.

39. c) Mineral Fibres.
• i) Slag Wool. Slag is a vitreous residue from the
smelting of ores. It is disintegrated into fibres by
allowing a molten stream to drop into a
horizontal blast of steam.
• ii) Asbestos. This must be blended with cotton or
wool because, when used alone, it compacts
badly and, therefore, offers high resistance.

40. 3) Filter Papers.
• A) cellulose-asbestos: The cellulose (from esparto
grass) and asbestos fibres are compressed into a
sheet only about 0.5mm thick.
• B) Glass: Fine glass fibres of 1 to 2 micrometer in
diameter are compressed and the sheet is mounted
as above except that the spacers are of expanded
aluminium to make the whole unit non
inflammable.

41. II. ELECTROSTATIC PRECIPITATION
•An electrostatic precipitator is
essentially a type of filter because
it removes solid matter from the
air, unlike mechanical filters, it is
not less efficient at removing
small particles.
The precipitron is an example.

42. III) ULTRA VIOLET LIGHT.
• The main points relevant to its use for purifying
atmospheres are-
a) Its penetrating power is poor and, therefore, it
does not reach organisms inside dust particles and
large droplets.
b) continuous irradiation of the atmosphere is
desirable,therefore workers must be protected
from harmful effects of radiation on skin and eyes
c) it is less effective at low humidities.
d) installation and replacement costs are heavy but
the method is clean and the equipment is easy to
maintain.

43. IV) CHEMICALS.
• For many years there has been considerable interest in the killing of
the air borne pathogens by methods suitable for occupied public
buildings such as schools, offices etc. The main is to prevent cross-
infection , so it is better to use a method that begins to act on the
organisms as soon as they are liberated from the respiratory tract
and clothes. Therefore, investigators have concentrated on ultra-
violet and chemical sprays or vapours.
• A good aerial bactericide should be:
a) Effective at normal temperatures and relative humidities
b) Easily dispersed into and persistent in the air.
c) Harmless to the occupants
d) Non wetting
e) Invisible. Inodorous , non-inflammable and nin-corrosive to
fittings.

44. >>MODE OF ACTION:
• Lidwell (1948) has pictured the action of an aerial
bactericide in the following way-
a) Vapour molecules of the germicide diffuse through
the atmosphere to the particles at a rate
determined by Langmuir formula.
b) The molecules condense on the surface of the
particles where they dissolve in the water present.
c) the bactericide diffuses into the particles and
aparent death of the associated organism results if
a sufficiently high concentration is reached to
inhibit their growth when the particle is collected
on a culture medium.

45. >>METHODS OF DISPERSAL.
1) spraying:
This is suitable for bactericidal liquids and
solutions.The aim is to produce a cloud of minute
liquid droplets that are mostly within the aerosol
range (0.1 to 50 micrometer in diameter)
2) Vaporisation:
This is considered as the more logical method. If
the theory of the bactericidal action through the
vapor pressure is correct. It is applicable to mant
arial bactericides. It cannot be used for
thermolabile substances sucg as hypochlorites.

46. EXAMPLES OF AERIAL BACTERICIDES:
1) Sodium Hypochlorite
2) Phenols:-
-Resorcinol
- Hexylresorcinol
3) Glycols
- propylene Glycol
- Triethylene Glycol

47. INVESTIGATING THE EFFICIENCY OF AIR
STERILLISATION METHODS:
A) Air Sampling: the efficiancy of an air sterlisation method is estimated
by comparing the number of bacteria before, after and sometimes
during the process.
some reliable methods or air sampling are:
1) Settling Plates: open petri dishes of nutrient agar are exposed for a
suitable time. Incubated and the colonies counted.
2) Impingement Methods:
a) on th solid media: the particles from a known volume or air are
made to impinge on the surface of a solid culture medium.
b) In to liquid media: The capillary impinger:
Air, at known flow rate, is drawn by vaccum
through a glass capillary into a suitable collecting fluid.
3) Bubblers: In these the air id bubbled into a liquid through a glass tube
ending in a bulb containing small holes.
4) Filters: A microfilter containing a pad of merino wool is mixed with
asbestos or treated with acacia. After filteration it was shaken with a
liquid.

48. B) THE EVALUATION OF FILTERS:
1) BACTERIOLOGICAL TESTING: Essentially it
involves mixing bacteria with the upstream air and
sampling before and after passage through the
filters.
2) THE METHYLENE BLUE TEST: This test is
primarily used for high efficiancy and absolute
filters.
3) THE SODIUM FLAME TEST: NaCl is sprayed
and atomized. The sodium content of the
unfiltered and filtered air is estimated by the
intensity of its yellow line in the hydrogen flame.

49. OTHER ASPECTS OF ASEPSIS ROOM
VENTILATION:
1)Control of contaminated Air
• A good ventilation system does not merely admit a steady flow
of clean air; it also prevents the accumulation of contaminants.
>>Accumulation of contaminants: This is prevented by
admitting clean air ata rate sufficient to replace the atmosphere
frequently.
There are two main types of ventilation system. In the first the
turbulent system , air is admitted turbulently through suitably
shaped louvres.
The aternative is the displacement method in which thr air is
introduced through carefully placed diffusers on the ceiling.
>>Entry of unsterile Air:
Dirty air is prevented from entering the door cracks and openings
by pressurisation.

50. 2) MAINTENANCE OF THE TRUNKING:
The trunking of the
ventilation system must
be accessible and contain
doors through which it
can be cleaned regularly.

51. 3) LAMINAR FLOW CABINETS:
• They consist of a bank of absolute filters through
which air is forced by a fan to give a flow rate of
about 0.45m/s across the working space.
• Usually the absolute filters are capable of
removing 99.97 to 99.99% of particles and
bateria of abput 0.3 micrometer and above.

52. SIMPLE EXERCISES IN ASEPTIC
TECHNIQUE:
• Reducing contamination: During the training period the
following are the adequate methods of reducing
contamination :
1) Atmosphere: Protect the area with a screen. Also
reduce the air movements.
2) Breath: Raise the chair seat so that the face is well
above the screen opening
3) Hair: it is frequently necessary to brush or shake hair
from the face. A suitable cap should be worn.
4) Clothes: the clothing must be long and kept flly
buttoned up.
5) Working space: swab the bench top with disinfectants
6) Hands: wash and disinfectant

53. REFERENCES
• Carter , S.J.; Cooper and Gunn’s Dispensing for
Pharmaceutical Students ; CBS Publishers Pvt. Ltd. ,
New Delhi ; 12th edition ; pp 494-520

54. THANK
YOU!!!