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Transcript of The term sterilization for pharmaceutical preparations, means the complete destruction of all living...
The term sterilization for pharmaceutical
preparations, means the complete destruction of all
living organisms and their spores or their complete
removal from the preparation.
Five sterilization processes are described in the
USP:
a. STEAM
b. DRY-HEAT,
c. FILTRATION,
d. GAS,
e. IONIZING RADIATION.
Sterilization processes are commonly used for
parenteral
products, except gas and ionizing radiation, which
are
widely used for devices and surgical materials.
The selection of the sterilization method depend on:
a. The nature and amount of product,
b. Whether the product and container-closure
system will have a predominately moist or dry
environment during sterilization.
Both of these factors are of great importance in
determining the conditions (time and temperature )
of any
sterilization method chosen.
For sterilization purposes, microorganisms can be
categorized into three general categories:
A. Easy to kill with either dry or moist heat;
B. Susceptible to moist heat, but resistant to dry
heat (Bacillus subtilis);
C. Resistant to moist heat but susceptible to dry
heat (Clostridium sporogenes).
Sterilization of product and equipment by saturated
steam (moist heat ) is one of the most widely used
treatment in the parenteral drug industry.
Is conducted in an autoclave and employs steam
under pressure.
ADVANTAGES OF STEAM STERLIZATIONADVANTAGES OF STEAM STERLIZATION a. Very efficient procedure,
b. The method of choice for products and articles
that
can withstand the treatment,
c. Quick and inexpensive.
A. STEAM STERILIZATION (Autoclave)
The mechanism of microbial destruction in moist heat
is by:
a. Denaturation and coagulation of some of the
organism's essential protein.
b.b. The presence of the hot moisture within the
microbial cell permits destruction at relatively low
temperature.
"Important factors should be controlled :
a. Application of pressure
b.Time of application
c. The velocity of the steam entering the autoclave
d. The efficiency of water separation from incoming
steam
e. The size of the drain,
f. The penetration time of the moist heat
a. Application of pressure:
Because it is not possible to raise the temperature of
the steam above 100C. under atmospheric
conditions, pressure is employed to achieve higher
temperature
(it should be recognized that the temperature, not
the pressure is destructive to the microorganisms
and that the application of pressure only for the
purpose of increasing the temperature of the
system).
b. Time of application:Time is an important factor in the destruction of
microorganisms by heat.
The usual conditions (time/pressure/temperature),
are as follow:
10 pounds pressure (115.50C) for 30 minutes
15 pounds pressure (121.50C) for 20 minutes
20 pounds pressure (126.50C) for 15 minutes
As can seen, the greater the pressure applied, the
higher the temperature obtainable and the less the
time required for sterilization.
The temperature at which most autoclaves are
routinely operated is usually 1210C.
c. The velocity of the steam
entering the autoclave
d. The efficiency of water separation
from incoming steam
e. The size of the drain,
f. The penetration time of the moist heat
into the load may vary with the nature of the load,
and the exposure time must be adjusted to account
for this latent peroid (an estimate of these latent
period must be added to the total time in order to
ensure adequate exposure times).
A. Applicable for pharmaceutical preparations and
materials that can withstand the required
temperature and are penetrated by, but not
adversely affected by, moisture.
B. In sterilizing aqueous solutions, the moisture is
already present, and all that is required is the
elevation of the temperature of the solution for
the prescribed period of time.
Thus solutions packaged in sealed containers as
ampuls, are readily sterilized by this method
C. Also applicable to bulk solutions, glassware and
instruments.
APPLICATION OF AUTOCLAVE
AUTOCLAVE NOT APPLICABLE FOR:
A. The sterilization of oils, fats, oleaginous
preparations
B. Other preparations not penetrated by the moisture
C. Sterilization of exposed powders that may be
damaged by condensed moisture.
Dry heat sterilization is widely used to sterilize
glassware and equipment parts in manufacturing
areas for parenteral products.
Is usually carried out in sterilizing ovens
specifically designed for this purpose.
Because dry heat is less effective in killing
microorganisms than is moist heat, higher
temperature and longer period of exposure are
required. Depending on the size and type of
product and on the container and its heat
distribution characteristics.
B. DR Y -HEA T STERILIZA TION
Individual unit to be sterilized should be as small as
possible, and the sterilizer should be loaded in such
a manner as to permit free circulation of heated air
throughout the chamber.
Two principal methods of dry-heat sterilization are
infrared and convection hot air)
Infrared rays will sterilize only surfaces
Dry heat sterilization is usually conducted at
temperature of 160-1700C for 2 hrs or 2600C for 45
min.
Higher temperatures permit shorter exposure time.
If a chemical agent melts or decomposed at 170 °C,
but is unaffected at 140 °C, the lower temperature
must be used and the exposure time would be
increased
Dry heat kill microorganisms primarily through
oxidation.
Dry heat sterilization is generally employed for
substances that are not effectively sterilized by
moist heat such as:
a. Fixed oils,
b. Glycerin,
c. Various petroleum products such as petrolatum,
liquid
petrolatum (mineral oil),
d. Various heat-stable powders such as zinc oxide,
kaolin and
sulfur.
Some heat-sensitive and moisture-sensitive
materials can be sterilized by exposure to
ethylene oxide (ETO) or propylene oxide gas than
by other means
(ETO) gas is a colorless gas and widely used as a
sterilant in hospitals and industry
These gases are highly flammable when mixed
with air but can employed safely when properly
diluted with an inert gas such as carbon dioxide or
a suitable fluorinated hydrocarbon.
C. GAS STERILIZATION (ETHYLENE OXIDE)
The mechanism by which ETO kills miroorganisms
is by alkylation of various reactive groups
(interference with the metabolism) in the spore or
vegetative cell.
One of the more resistant organisms to ETO is B.
subtilis. Which can be used as USP biological
indicator for monitoring the effectiveness of ETO
sterilization cycle.
Several factors are important in determining
whether ETO is effective as a sterilizing gas.
a. Gas concentration (500mL/L),
b. Temperature (50-600C),
c. Humidity (60%),
d. Exposure time (4-16 hrs)
The basis of the lethal action of radiations on
microorganisms is the production of ionizations and
excitations when radiation traverses the cell.
However, lethally irradiated cells remain intact,
respiration continues normally, motile cells retain
their motility for some time and, under conditions
where reproduction is possible, death may not occur
until after one or two divisions of a cell.
Sterilization ProcessSterilization Process
The great penetration qualities of ETO make it a
useful sterilizing agent in special applications:
a. Sterilization of medical and surgical supplies such
as
catheters, needles, and plastic disposable
syringes in
their final plastic packaging
b. Sterilize certain heat-labile enzyme preparations
c. Certain antibiotics, and other drugs (with tests to
assure
of the absence of chemical reactions)
APPLICATION OF GAS STERILIZATION
Sterilization by filtration, depends upon the physical
removal of microorganisms by adsorption on the
filter medium or by sieving mechanisms, is used for
sterilization of heat-sensitive solutions
Filters have variety of pore-size specifications; one
of these filters, the millipore filters
D. STERILIZATION B Y FILTRATION
Millipore filters are:
1) Are thin plastic membranes of cellulosic esters
with millions of pores/square inch of filter surface
2) The pores are extremely uniform in size and
occupy approximately 80% of the filter
membrane's volume
3) The remaining 20% being the solid filter material
4) This high degree of porosity permits high flow
rates
5) Millipore filters are made from a variety of
polymers to provide membrane characteristics
required for the filtration of almost any liquid or
gas system
6) Filters are made of various pore size to meet the
selective filtration requirements
7) They are available in pore size from 14-0.025μm
where the smallest bacteria, about 0.2μm, and
viruses about 0.025μm
Factors that affecting the removal of M.O.
A. Pore size of filter
B. Electrical charge of the filter and that of the
M.O.,
C. PH of the solution,
D. Temperature,
E. Pressure or vacuum applied
a. Its speed in the filtration
b. Its ability to sterilize thermolabile materials
c. Inexpensive equipment required
d. The complete removal of living and dead M.O.
as well as other particulate matter from the
solution
ADVANTAGES OF BACTERIAL FILTERSADVANTAGES OF BACTERIAL FILTERS
a. The membrane is fragile thus it is essential to be
sure that
the membrane is not ruptured
b. Filtration of large volumes of liquids would require
more
time (particularly if the liquids were viscous)
c. Are useful when heat cannot be used and small
volumes
of liquids
DISADVANTAGES OF BACTERIAL FILTERSDISADVANTAGES OF BACTERIAL FILTERS
1) Highly effective.
2) Treatment times is very short.
3) It is a continuous process suitable for long
production run.
4) The thermal conductivity of the material is
irrelevant.
5) It is a cold process resulting in a temperature rise of
only a few degrees and so is suitable for
thermolabile materials.
6) Materials may be irradiated in the dry or frozen
state
7) Products are processed in the final container after
packaging with no risk of subsequent contamination
until used.
8) The process may be controlled and monitored
accurately.
Advantages of Radiosterilization
1.Sterilization of materials for which conventional
methods are unsatisfactory (catgut, rubber, certain
dressings, oils) or to disposable plastic materials
which cannot be heat sterilized.
2.Medical applications as the sterilization of tissue
graft materials, such as freeze dried bone or aorta,
the production of vaccines and the elimination of
the virus of serum hepatitis by the irradiation of
freeze-dried plasma.
3. In food sterilization using low radiation doses to
eliminate insect infestations of stored foods.
Applications
4. A list of medical products which are known to have
been radiosterilized is as following:
Antibiotics of the tetracycline group.
Arterial prostheses.
Cardiac valve prostheses.
Endotracheal tubes.
Cannulae.
Atropine eye drops.
Chloramphenicol eye ointment.
Dialysis units.
Plastic catheters, gloves, hypodermic syringes (with
needles), Petri dishes, tubing.
Rubber catheters and gloves.
Surgical blades
Surgical dressings: bandages, gauze, eye pads,
swabs.
Sutures: catgut', collagen, silk, polyester, nylon.
Transfusion giving and taking sets.
The criteria for the selection of the type of
radiation
include the following:
(1)The specific ionization produced should be
relatively low. A relatively small number of
ionizations within a microorganism is sufficient to
cause inactivation so radiations which produce a
high specific ionization are less efficient and are
more likely to damage the product.
(2) The radiation should be capable of adequate
penetration of the material and deliver a
reasonably uniform dose therein.
Selection of the Sterilizing Process
(3) The radiation source should be capable of
delivering high doses economically, at the same
order of cost as conventional sterilization
techniques.
(4) It should be possible to irradiate materials in any
physical state.
(5) The dose must be accurately reproducible and
capable of accurate measurement and control.
(6) The safety of operators should be ensured and
radioactivity should not be induced in any
irradiated material.
The only types of radiation to meet the above
criteria are
γ-rays and electron beams. Other radiations have
limited
and specialised uses.
The selection of the radiation dose required to
sterilize various materials depends on several factors:
The nature of the species present and their
radiation resistance in the particular environment and
under the conditions of processing used,
The margin of safety required.
Determination of the Sterilizing Dose
The wide variations in sensitivity between species has
already been referred to and the most resistant
species likely to occur as a contaminant in the
material and the degree of such contamination, are
the two most important considerations.
Since the principal type of material sterilized by
radiation comprises individual solid items,
nonuniformity of contamination is the normal
situation and results in a higher dose requirement
than if contamination were uniform, since all items
have to be treated according to the highest
contamination level which occurs.
The margin of safety required depends on the risk of
the occurrence of non-sterile articles which is
considered to be acceptable for the intended
purpose, thus the sterilization process is a probability
function.
An increase in the dose, and so in the inactivation
factor, gives a lower probability of residual
contamination.
Thus selection of an appropriate sterilizing dose may
be difficult and in the absence of precise information
about the initial conditions large doses are used, in
the range 2.5 to 4.5 Mrad. The 2.5 Mrad dose has
been widely accepted but it has been suggested that
this may be inadequate. With these high doses,
especially at 4.5 Mrad, the effects of irradiation on
the material may be important and each type of
product requires careful evaluation as to stability,
activity, physical properties, storage life and acute
and chronic toxicity.
The Species:
The variation in radio-sensitivity between species is
the most important factor in the determination of
the sterilizing effect of a given dose.
The radio-resistance of microorganisms generally
increases in the order vegetative bacteria, fungi,
bacterial spores, viruses.
Factors Affecting the Radiation Sensitivity
Inoculum level:
The higher the initial population the greater the
dose required to achieve inactivation.
This population effect causes the need to keep the
initial degree of contamination as low as possible so
that the sterilization of articles is achieved with
minimum dose.
Gaseous environment:
The presence or absence of oxygen during and after
irradiation influences the sensitivity to radiation
damage.
Degree of Hydration:
Microorganisms are less radiation sensitive under
dry conditions than in the presence of water due to
the absence of indirect effects arising from the
radiolysis of water.
There are a 30 % change in the inactivation rate
over a range of about 20 to 40 per cent relative
humidity.
Temperature:
The radiation sensitivity of microorganisms is
reduced at,
or below, the freezing point of water, due to the
immobilization of free radicals and reactive species
produced in the water, thus reducing indirect
effects.
At temperatures used for sterilization there is a
synergistic effect of temperature and irradiation
that sterility may be achieved by a combination of
milder treatments of both sterilizing agents.
This phenomenon is of potential practical
importance in the sterilization of products such as
foodstuffs which are both heat and radiation
labile.
Stage of Cell Division:
The sensitivity of vegetative bacteria varies during
the growth cycle that the growth phase affect the
dose required if there is the possibility of growth in
the material to be sterilized.
Since bacterial spores are more resistant than
vegetative cells of the same species there is an
sudden increase in radiosensitivity as spore
germination occurs.
Protective and Sensitizing Agents: A large variety of compounds affect the radiation
sensitivity of microorganisms.
Protective agents include reducing agents
(cysteine, thiourea, cysteamine, β-
mercaptoethanol), alcohols and polyhydric
compounds (ethanol, glycerol, mannitol) and
proteins or complex foodstuffs.
The effect of free radical scavengers abolish part of
the oxygen effect.
Sensitization has been reported with oxidizing
agents (nitrate, nitrite), water soluble stable free
radicals.
Vitamin K5 has been reported both as a sensitizer and
as a protective agent with different bacterial
species.
Testing of Sterilization ProcessTwo types are performed to test the efficiency of sterilization:
1.Testing the sterility of the final product
2.Testing the sterilization process (by physical,
chemical and biological methods) to confirm that
the equipment is working satisfactory.
Indicator Tests For Indication Of The Efficiency Of The Sterilization
ProcessPhysical indicator tests
1) The performance of steam and gas sterilizers can
be tested by observing the reading of
temperature, pressure, vacuum gauges and stage
timers throughout an automatically controlled
sterilization cycle.
Recorded charts should be examined carefully.
2) Thermocouples test: is used to measure the
temperature at selected sites in the chamber or
within the load of a dry heat, steam or gas
sterilizer.
3) In case of sterilization by radiation: A
measurement of radiation dose i.e. the amount of
energy absorbed by the material tested.
4) In case of sterilization by filtration:
A bubble pressure test is used to determine the
pore size of filters.
Chemical indicator testsTypes that cannot indicate time of exposure:
A) Klintex papers:
These are paper strips or stickers
attach to each object to be sterilized.
The word (sterile) is written on the
strip (colorless) but after exposure to
the sterilizing agent as steam the
word (sterile) will be cleared.
B) Klintex test tablets:
These contain 75% lactose, 24% starch and 1%
magnesium trisilicate.
They are hard and white but after steam sterilization
they become brown and gelatinous.
They should be examined soon after removal from the
sterilizer.
C) Autoclave test tape (Bowie – Dick test):
This is a valuable test for confirming
that the steam has displaced all the air
from a porous load (i.e. air removal
test).
The tape carries heat sensitive bars
which become colored if steam has full
penetrated the pack.
If air remains, the bars in the centre
are lighter in color.
Types that indicate time of exposure:
Browne’ tubes:Each tube consists of a sealed glass tube which
contains a red fluid (an ester and acid - base
indicator) that changes to yellow, brown and finally
green on heating
(the ester undergoes heat hydrolysis to form an acid +
alcohol. The acid will change the color of the
indicator).
There are four types of Browne’s tubes:
Browne’s tube type I: Suitable for ordinary steam
sterilizers
Browne’s tube type II: Suitable for high vacuum
sterilizers
Browne’s tube type III: Suitable for hot air oven
Browne’s tube type IV: Suitable for I.R conveyer oven.
Biological indicator tests
At the end of the process, the
bacteria are transferred to a
nutrient medium which is
incubated and the presence or
absence of growth is noted.
Biological indicators consist of bacterial cultures
which are usually used in the form of impregnated
strips e.g. paper and metal foil and are placed in
different sites in the sterilizer.
The bacterial species to be used must be carefully
selected, since it must possess high resistance to the
particular process.
The following types of bacteria are commonly used in
the
different sterilization process.
Moist heat (autoclave):
Standardized preparation of Bacillus
strearothermophilus spores.
Hot air oven & Ethylene oxide sterilizer:
Standardized preparation of Bacillus globigii spores
Gamma radiation:
Standardized preparation of Bacillus pumilus spores.
Filtration: Serratia marcescens is used.