STERILIZATION METHODS

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STERILIZATION METHODS
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.
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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.
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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).
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A. STEAM STERILIZATION (Autoclave)
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 STERLIZATION a. Very efficient
procedure, b. The method of choice for products
and articles that can withstand the
treatment, c. Quick and inexpensive.
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• The mechanism of microbial destruction in moist
  heat is by
• Denaturation and coagulation of some of the
  organism's essential protein.
• b. The presence of the hot moisture within the
  microbial cell permits destruction at relatively
  low temperature.
• "Important factors should be controlled
• Application of pressure
• 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

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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).
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b. Time of application Time is an important
factor in the destruction of microorganisms by
heat. The usual conditions (time/pressure/tempera
ture), 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.
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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).
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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
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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.
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B. DR Y -HEA T STERILIZA TION

• 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.

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• 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

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• 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.

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C. GAS STERILIZATION (ETHYLENE OXIDE)

• 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.

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• 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.

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• 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)

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Sterilization Process
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.
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APPLICATION OF GAS STERILIZATION

• The great penetration qualities of ETO make it a
  useful sterilizing agent in special applications
• 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)

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D. STERILIZATION B Y FILTRATION
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
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• Millipore filters are
• Are thin plastic membranes of cellulosic esters
  with millions of pores/square inch of filter
  surface
• 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

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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
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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
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ADVANTAGES OF BACTERIAL FILTERS
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
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DISADVANTAGES OF BACTERIAL FILTERS

• 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

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RADIO-STERILIZATION
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Advantages of Radiosterilization
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.
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Applications

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.

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• 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.

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• 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.

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Selection of the Sterilizing Process

• The criteria for the selection of the type of
  radiation
• include the following
• 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.
• The radiation should be capable of adequate
  penetration of the material and deliver a
  reasonably uniform dose therein.

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(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 ?-rays and
electron beams. Other radiations have limited
and specialised uses.
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Determination of the Sterilizing Dose

• 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.

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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.
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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.
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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.
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Factors Affecting the Radiation Sensitivity
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.
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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.
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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.
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• 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.

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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.
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• Protective and Sensitizing Agents
• A large variety of compounds affect the radiation
  sensitivity of microorganisms.
• Protective agents include reducing agents
  (cysteine, thiourea, cysteamine,
  ß-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.

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Testing of Sterilization Process
Two 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.

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Indicator Tests For Indication Of The Efficiency
Of The Sterilization Process
Physical indicator tests

• 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.

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• 3) In case of sterilization by radiation A
  measurement of radiation dose i.e. the amount of
  energy absorbed by the material tested.
• In case of sterilization by filtration
• A bubble pressure test is used to determine
  the pore size of filters.

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Chemical indicator tests
Types 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.
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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.
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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.
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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).
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• There are four types of Brownes tubes
• Brownes tube type I Suitable for ordinary
  steam sterilizers
• Brownes tube type II Suitable for high vacuum
  sterilizers
• Brownes tube type III Suitable for hot air oven
• Brownes tube type IV Suitable for I.R conveyer
  oven.

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Biological indicator tests
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.
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.
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• 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.