PRINCIPLE, ADVANTAGES,

DISADVANTAGES, APPLICATIONS
OF DIFFERENT STERILISATION
METHODS AND IN PROCESS
CONTROL

PREPARED BY
Haroon Rahim

WHAT IS STERILIZATION:
Sterilization can be defined as any
process that effectively kills or
eliminates
transmissible
agents
(such as fungi, bacteria, viruses)
from a surface, equipment, foods,
medications, or biological culture
medium.

PHYSICAL METHODS:

1. HEAT STERILIZATION:
Heat sterilization is the most widely used and
reliable method of sterilization, involving
destruction of enzymes and other essential cell
constituents.
This method of sterilization can be applied only to
the THERMO STABLE PRODUCTS, but it can be
used for MOISTURE-SENSITIVE MATERIALS.
i) Dry Heat (160-1800C) Sterilization for
thermo stable products
ii) moist heat (121-1340 C) sterilization is
used for moisture- resistant materials.

The efficiency with which heat is able to

inactivate microorganisms is dependent upon
i) the degree of heat, the exposure time and
ii) the presence of water.
The action of heat will be due to induction of
lethal chemical events mediated through the
action of water and oxygen.
In the presence of water much lower
temperature time exposures are required to kill
microbe than in the absence of water.

Dry Heat Sterilization

It employs higher temperatures in the range

of 160-180C and requires exposures time up
to
2 hours, depending upon
the temperature employed.
The benefit of dry heat includes good
penetrability and non-corrosive nature which
makes it applicable for sterilizing glass wares
and metal surgical instruments. It is also
used for sterilizing non-aqueous thermo
stable liquids and thermo stable powders.

Dry heat destroys bacterial endotoxins

(or pyrogens) which are difficult to
eliminate by other means and this
property makes it applicable for
sterilizing glass bottles which are to be
filled aseptically

Moist Heat Sterilization

Moist heat sterilization involves the use

of steam in the range of 121-134C.
Steam under pressure is used to
generate high temperature needed for
sterilization. Saturated steam acts as an
effective sterilizing agent.

Autoclave
Autoclaves use pressurized steam to destroy
microorganisms, and are the most dependable
systems available for the decontamination of
laboratory waste and the sterilization of
laboratory glassware, media, and reagents. For
efficient heat transfer, steam must flush the air
out of the autoclave chamber.
Generally the conditions employed are
Temperature upto121-134C for 15-20 min under
15 lbs pressure,based on type of metiral used.

Radiation Sterilization

Many types of radiation are used for

sterilization like electromagnetic
radiation (e.g. gamma rays and UV
light), particulate radiation (e.g.
accelerated electrons).The major target
for these radiation is microbial DNA.
Radiation sterilization with high energy
gamma rays or accelerated electrons
has proven to be a useful method for the
industrial sterilization of heat sensitive
products.

Radiation sterilization is generally applied to

articles in the dry state; including surgical
instruments, sutures, prostheses, unit dose
ointments, plastic syringes and dry
pharmaceutical products.
UV light, with its much lower energy, and poor
penetrability finds uses in the sterilization of air,
for surface sterilization of aseptic work areas,
for treatment of manufacturing grade water, but
is not suitable for sterilization of
pharmaceutical dosage forms.

Filtration Sterilization

Filtration process does not destroy but

removes the microorganisms. It is used for
both the clarification and sterilization of
liquids and gases as it is capable of
preventing the passage of both viable and
non viable particles.
The major mechanisms of filtration are
sieving, adsorption and trapping within the
matrix of the filter material.
Ex:HEPA FILTERS

Sterilizing grade filters are used in the

treatment of heat sensitive injections and
ophthalmic solutions, biological products
and air and other gases for supply to
aseptic areas. They are also used in
industry as part of the venting systems on
fermentors, centrifuges, autoclaves and
freeze driers. Membrane filters are used
for sterility testing

There are two types of filters used in

filtration sterilization:
(a) Depth filters:
(b) Membrane filters: These are porous
membrane about 0.1 mm thick, made of
cellulose acetate, cellulose nitrate,
polycarbonate, and polyvinylidene
fluoride, or some other synthetic
material.

MERITS, DEMERITS AND

APPLICATIONS OF
DIFFERENT METHODS OF
STERILIZATION

S.n
o

MERITS
METHOD

DEMERITS

APPLICATIONS

MECHANIS
M

Heat
Destroys
sterilizati bacterial
on
endo
toxins

Most widely
used and
reliable
method of
sterilization
, involving
destruction
of enzymes
and other
essential
cell
constituent
s

Can be
applied
only to
the
thermo
stable
products

Dry heat is
applicable for
sterilizing glass
wares and metal
surgical
instruments and
moist heat is
the most
dependable
method for
decontamination
of laboratory
waste and the
sterilization of
laboratory
glassware,
media, and
reagents.

S.n
o

METHOD

MECHANIS
M

Gaseous
sterilizatio
n

Alkylation

Radiation
sterilizatio
n

Ionization
of nucleic
acids

MERITS

DEMERITS

Penetrating
ability of
gases.

Gases being
alkylating
agents are
potentially
mutagenic and
carcinogenic.

It is a useful
method for
the industrial
sterilization
of heat
sensitive
products

Undesirable
changes occur
in irradiated
products,an
example is
aqueous
solution where
radiolysis of
water occurs.

APPLICATIONS

Ethylene oxide gas has

been used widely to
process heat-sensitive
devices.

Radiation sterilization is
generally applied to
articles in the dry state;
including surgical
instruments, sutures,
prostheses, unit dose
ointments, plastics

S.
no

MERITS
METHOD

MECHANISM

Filtration
sterilizatio
n

Does not
destroy but
removes the
microorganism
s

It is used for both

the clarification
and sterilization
of liquids and
gases as it is
capable of
preventing the
passage of both
viable and non
viable particles

DEMERITS

Does not
differentiate
between viable
and non viable
particles

APPLICATIONS

This method is
Sterilizing grade
filters are used in the
treatment of heat
sensitive injections
and ophthalmic
solutions, biological
products and air and
other gases for supply
to aseptic areas

Pharmaceutical Importance of
Sterilization

Moist heat sterilization is the most efficient

biocidal agent. In the pharmaceutical industry it is
used for: Sheets, Surgical Containers, Closures,
Aqueous injections, Ophthalmic preparations and
Irrigation fluids etc.
Dry heat sterilization can only be used for
thermo stable, moisture sensitive pharmaceutical
and medicinal. These include products like; Dry
powdered drugs, Suspensions of drug in non
aqueous solvents, Oils, fats, waxes, Oily
injections, ophthalmic ointments etc.

Gaseous sterilization is used for

sterilizing thermolabile substances like;
hormones, proteins, various heat sensitive
drugs etc.
U.V light is perhaps the most lethal
component in ordinary sunlight used in
sanitation of garments or utensils.
Gamma-rays from Cobalt 60 are used to
sterilize antibiotic, hormones, sutures,
plastics and catheters etc.

Filtration sterilizations are used in the

treatment of Heat sensitive injections
and ophthalmic solutions, biological
products, air and other gases for supply
to aseptic areas.
They are also used in industry as part of
the venting systems on fermentors,
centrifuges, autoclaves and freeze
driers. Membrane filters are used for
sterility testing.

PHARMACEUTICAL IN
PROCESS CONTROL

In-process controls (IPC) are checks that are

carried out before the manufacturing process is
completed.

The function of in-process controls is monitoring

and if necessary adaptation of the
manufacturing process in order to comply with
the specifications. This may include control of
equipment and environment, too.

The In-Process Quality Control system

lays emphasis on the responsibility of
manufacturers processors in ensuring
consistency in quality during all stages
of production by adopting quality control
drills and exercising control on raw
materials and bought-out components,
manufacturing process, packing and final
testing.

In-Process Quality Control tests of tablets

SIZE & SHAPE

TABLET THICKNESS
COLOUR
HARDNESS
FRIABILITY
POTENCY & CONTENT UNIFORMITY
WEIGHT / WEIGHT VARIATION
DISINTEGRATION TIME

HARDNESS Stokes (Monsanto), Strong

Cobb, Pfizer, Erweka, Hebelein or
Schleuniger tester, units: kg/sq inch of
kg/sq cm,

FRIABILITY Roche , limit less tan 1%

UNIFORMITY OF CONTAINER CONTENTS.

Tablets comply with the test for contents of
packaged dosage forms.
Select a sample of 10 containers and count the
number of capsules, pessaries, suppositories or
tablets in each container. The average number
of the contents in the 10 containers is not less
than the labelled amount and the number in
any single container is not less than 98 per cent
and not more than 102 per cent of the labelled
amount.

Parenteral Preparations

Parenteral Preparations

1
2
3
4
5
6
7
8

Particulate matter
Uniformity of content
Extractable volume
Sterility
Pyrogens
Uniformity of weight
Clarity of solution
Leakage

Particulate matter

Parenteral preparations including solutions

constituted from
. sterile solids are
expected to be free from particles of
approximately 50 m or more that can be
observed byinspection with the unaided
eye

Particulate matter

Sterility
Culture Media

MEMBRANE FILTRATION.
After transferring the contents of the container or
containers to be tested to the membrane add an inoculum of
a small number of viable micro-organisms (not more than
100 CFU) to the final portion of sterile diluent used to rinse
the filter.
After filtration, aseptically remove the membrane(s) from the
holder, transfer the whole membrane or cut it aseptically
into 2 equal parts. Transfer one half to each of two suitable
media.

Direct Inoculation. After transferring the contents of

the container or containers to be tested to the culture
medium add an inoculum of a small number of viable microorganisms (not more than 100 CFU) to the medium

Quantity in each
container
Less than 1 ml

Minimum quantity to
be used
Total contents of a
container
40 ml or more but less 1 ml or more but less
than 100 ml
20 ml than 40 ml
Half
the contents of a
container
100 ml or more

10 per cent of the

contents of a
container but not less
than 20 ml

Incubate the inoculated media for not less

than 14 days.

Observe the cultures several times during the incubation

period. Observe the containers of media periodically during
the 14 days of incubation. If the test specimen is positive
before 14 days of incubation, further incubation is not
necessary.
For products terminally sterilised by a validated moist heat
process, incubate the test specimen for not less than 7 days.
Observation and Interpretation of Results
If no evidence of microbial growth is found, the preparation
under examination complies with the test for sterility. If
evidence of microbial growth is found, the preparation under
examination does not comply with the test for sterility

Pyrogens

The test involves measurement of the rise in body temperature

of rabbits following the intravenous injection of a sterile solution
of the substance under examination. It is designed for products
that can be tolerated by the test rabbit in a dose not exceeding
10 ml per kg injected intravenously within a period of not more
than 10 minutes

Test Animals:

Use healthy, adult rabbits of either sex, preferably of the same variety, weighing
not less than 1.5 kg

MAIN TEST:

Carry out the test using a group of three rabbits.

PREPARATION OF THE SAMPLE:
Dissolve the substance under examination in, or dilute with, pyrogen-free saline
solution orother solution prescribed in the monograph. Warm the liquid under
examination to approximately 38.5 before injection.

Pyrogens

INTERPRETATION OF RESULTS:
If the sum of the responses of the group of three
rabbits does not exceed 1.4 and if the response of
any individual rabbit is less than 0.6, the
preparation under examination passes the test. If
the response of any rabbit is 0.6 or more, or if the
sum of the response of the three rabbits exceeds
1.4, continue the test using five other rabbits.
If not more than three of the eight rabbits show
individual responses of 0.6 or more, and if the sum
of responses of the group of eight rabbits does not
exceed 3.7, the preparation under examination
passes the test

Uniformity of content

Determine the content of active ingredient(s) of each of

10 containers taken at random, using the method given
in the monograph or by any other suitable analytical
method of equivalent accuracy and precision.

The preparation under examination complies with the

test if the individual values thus obtained are all
between 85 and 115 per cent of the average value.
The preparation under examination fails to comply with
the test if more than one individual value is outside the
limits 85 to 115 per cent of the average value or if any
one individual value is outside the limits 75 to 125 per
cent of the average value

If one individual value is outside the limits 85 to

115 per cent but within the limits 75 to 125 per
cent of the average value, repeat the
determination using another 20 containers taken
at random.
The preparation under examination complies with
the test if in the total sample of 30 containers not
more than one individual value is outside the
limits 85 to 115 per cent and none is outside the
limits 75 to 125 per cent of the average value

Clarity of solution
Constitute the injection as directed on the
label.
a) The solid dissolves completely, leaving no
visible residueas undissolved matter.
b) The constituted injection is not significantly
less clear
than an equal volume of the diluent or of
water for
injections contained in a similar container and
examined in the same manner.

Leakage

A LEAKERS TEST :
It is a useful method for evaluating the
efficiency of the sealing process.
The test consists of immersing completely
the sterile sealed ampoules in an aqueous
dye bath (0.5 to 1.0% of methylene blue)
within a vacuum chamber.
Negative pressure of 27 inches Hg or more
is created, a tiny drop of dye solution can
penetrate an opening of an incompletely
sealed ampoule.
the colored ampoule are sorted out during
washing and 100% inspection that follows
after.