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.