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AUTOCLAVE VALIDATIONAUTOCLAVE VALIDATION

CONTENT Sterilization Principle Of Autoclave Definitions Types Of Autoclave Sterilization Cycle Load Types Basic Validation Approach Conclusion References

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Sterilization: Sterilization is a process by which an article, surface or

medium is free of all microorganism either in the vegetative or spore state.

Methods:1. Physical method: (a) dry heat sterilization: eg. incineration, red heat. (b) moist heat sterilization: Eg. Pasteurization,

tyndalization, autoclave (c) radiation/cold sterilization: eg. Uv ray, x ray (d) filtration method: eg. Asbestos, membrane filter etc.2. Chemical metod: (a) gaseous sterilization: eg. Formaldehyde etc (b) by using disinfectant: eg. Cresol, phenol.

Preparation of items for Autoclaving

In preparing items for autoclaving, containers should be unsealed and articles should be wrapped in materials that allow steam penetration.

Large packages of dressings and large flasks of media require extra time for heat to penetrate them.

Wrapping objects in aluminum foil is not recommended because it may interfere with steam penetration.

Importance:

Moist heat in the form of pressurized steam is regarded as the most dependable method for the destruction of all forms of life, including bacterial spores. This method is incorporated into a device called the autoclave.

Need of autoclaving:

Reliable sterilization with moist heat requires temperatures above that of boiling water. These high temperatures are most commonly achieved by steam under pressure in an autoclave. Autoclaving is the preferred method of sterilization, unless the material to be sterilized can be damaged by heat or moisture.

Effectiveness of Autoclave Sterilization in an autoclave is most effective

when the organisms are either contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under these conditions, steam at a pressure about 15 psi; attaining temperature (121oC) will kill all organisms and their endospores in about 15 minutes.

Principle of Autoclaving

A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the gas increase proportionally.

For example, when free flowing steam at a temperature of 100oC is placed under a pressure of 1 atmosphere above sea level pressure – that is, about 15 pounds of pressure per square inch (Psi) --- the temperature rises to 121oC. Increasing the pressure to 20 psi raises the temperature to 126oC. The relationship between temperature and pressure is shown in table.

In this way steam is a gas, increasing its pressure in a closed system increases its temperature.

As the water molecules in steam become more energized, their penetration increases substantially.

It is important to note that the sterilizing agent is the moist heat, not the pressure.

Relationship between temperature & pressure:

Table The Relationship Between the Pressure and Temperature of

Steam at Sea Level

Pressure (psi in excess of atmospheric pressure)

Temperature (oC)

0 psi 1005 psi 110

10 psi 11615 psi 12120 psi 12630 psi 135

Working of Autoclave

Most autoclaves contain a sterilizing chamber into which articles are place and a steam jacket where steam is maintained.

As steam flows from the steam jacket into the sterilizing chamber, cool air is forced out and a special valve increases the pressure to 15 pounds/square inch above normal atmospheric pressure.

The temperature rises to 121.5oC, and the superheated water molecules rapidly conduct heat into microorganisms.

The time for destruction of the most resistant bacterial spore is now reduced to about 15 minutes.

For denser objects, up to 30 minutes of exposure may be required.

Mechanisum: denaturation of critical proteins & nucleic acid within the cell.

PRINCIPLE

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HORIZONTAL STEAM AUTOCLAVE

Uses of Autoclave:

Sterilize culture media, instruments, dressings, intravenous equipment, solutions, syringes, transfusion equipment, and numerous other items that can withstand high temperatures and pressures.

The laboratory technician uses it to sterilize bacteriological media and destroy pathogenic cultures.

The autoclave is equally valuable for glassware and metalware,

Limitations and Disadvantages of Autoclave

The autoclave also has certain limitations.For example, some plasticware melts in the high heat, and

sharp instruments often become dull.many chemicals breakdown during the sterilization process

and oily substances cannot be treated because they do not mix with water.

Heat requires extra time to reach the center of solid materials, such as caned meats, because such materials do not develop the efficient heat-distributing convection currents that occur in liquids.

Heating large containers also requires extra time

Different time requirements for sterilizing liquids in various container sizes

Table The effect of Container Size on Autoclve Sterilization Times for

Liquid SolutionsContainer Size Liquid Volume Sterilization Time

(min)

Test Tube:

18×150 mm10 ml 15

Erlenmeyer Flask:

125 ml 95 ml 15

Erlenmeyer Flask:

2000 ml1500 ml 30

Fermentation Bottle:

9000 ml6750 ml 70

Definations: Sterility assurance level: The probability of a single viable microorganism

being present on a sterilized product is one in one million after completion of sterilization process.

Bioburden: The number/type of viable microorganism contaminating a product.

D-value: Time in minutes at any defined temperature to destroy 90% viable microorganism is called D value.

it indicate the efficiency of sterilization process. Z-value: is a no. of degrees of temp. change required to produce a 10 fold

change in D value. it relates the heat resistance of a microorganism to change in temp. F-value: The number of minutes to kill a specified number of microorganisms

with a specified z-value at a specific temperature. used to calculate the probable no. of survivors remaining in a load as, F = D (log N̥ - log N) where, D = D value at 121°C of organism. N˳= initial population number/ unit volume . N = final population number/ unit volume.

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Autoclave types

There are several types of autoclave that we can find on the market. 1.Positive Pressure Displacement : This type is the improvement of downward displacement type. The steam is created within a second in separate unit (steam

generator). The steam is released to the sterilization chamber to start the

sterilization progress.2. Negative Pressure Displacement : most recommended types of autoclave . very accurate & achieve high sterility assurance level. disappointing fact is that too expensive .3. Downward Displacement : also called as the gravity displacement autoclave. heats water that becomes steam. steam then forces the air in the chamber to go through the drain hole. drain hole is closed once the temperature is sufficient for the serialization

process to begin.

4. Triple Vacuum Displacement : similar to the negative pressure displacement type. unit is named "triple vacuum" because there are three process of removing the air and the steam's pulse .5. Type "N" or type "B“ Type "N" does not use vacuum to remove air from the chamber.

Contrary, type "B" uses vacuum pump in operation.

Sterilization Cycle

Steam enters chamber & conditions load.

Air displaced through chamber drain.

Steam processes load at selected time& temp.

Steam is removed from chamber & pressure released.

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Sterilization Cycle A steam sterilization cycle consists of three basic phases:

Heating (come-up) phase - Steam enters the sterilizer jacket and air is removed from the sterilizer chamber, either by gravity displacement or mechanically (pre vacuum).

Sterilization (exposure) phase - Load is exposed to steam at a set temperature (measured and controlled by a temperature sensor in the drain line) for a set time.

Cool-down (drying) phase - Sterilizer chamber is exhausted to

atmospheric pressure followed by circulating air through the chamber or by drawing a deep vacuum. Jacket heat is maintained during the drying phase.

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Sterilizer DesignThe validation of a steam sterilization cycle is dependent on

the equipment chosen. The sterilizer & its support systems must be designed & constructed to deliver the effective cycles repeatedly & consistently.

Qualification of the autoclave consist of proper design, installation according to design & operational testing to ensure design criteria & specification are met.

All steam sterilization cycles are based on direct contact with saturated steam. saturated steam is water vapour in equilibrium with liquid water.

Saturated steam can exist only along the phase boundry i.e. relation between its temperature & pressure is fixed.

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Characteristics of modern steam sterilizer

Pressure vesselSteam jacket & insulationSafety door Thermostatic steam trapTemperature control systemCycle timerMicrobial retentive vent filterChamber pressure indicator

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Load types:

Autoclaves are used to sterilize severaldifferent types of loads:

Solid – metal, glass, plastic Porous – gowns, paper ,gauze, complex instruments, hollow tubes Liquid – water, saline, media Laboratory waste – Petri dishes, sample bottles, syringes

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VALIDATIONWHO validation definition

The documented act of proving that any procedure, process, equipment, material, activity, or system actually leads to the expected results. 

A system must be qualified to operate in a validated process Qualify a system and/or equipment Validate a process Qualification versus validation, e.g. you

qualify an autoclave, whereas you validate a sterilization process.

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BASIC VALIDATION APPROACH

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INSTALLATION QUALIFICATIONInstallation tests should include checking:• That the electrical supply is suitable for the autoclave.

• That the temperature and humidity are sufficiently well controlled.

• That there is no interference to or from other equipment.

• The accuracy of indicators has been certified to national standards.

• The temperature and pressure of the chamber during a sterilization cycle are within specifications.

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INSTALLATION QUALIFICATION

The leakage into the chamber during a vacuum cycle does not exceed the specified maximum.

That there is no leakage of steam, water or effluent at any point during the sterilization cycle.

That both water quality and steam quality comply with the sterilizer specification

The chamber is capable of withstanding its specified maximum pressure. This test may be a legal requirement and should be performed by a suitably certified organization.

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Mechanical equipment specification (Chamber, valves, traps, stainers, filters, regulators, vacuum pump, heat exchanger, condenser, etc.)

Control & instrumentation specifications (programmable logic controller, operator interface, printer/recorder, control valves, transducers, pressure & temperature transmitters, resistance temperature devices, switches, level sensors, interlocks, photocells.

Site specifications/utilities(power, grounding, surge protector, uninterruptible power supply, breakers, water, clean steam, plant steam, drain, isolation valves)

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Approval documentation (e.g. pressure vessel, electrical) ,bill of materials, vendor specification sheets, purchase orders, preventive maintenance program, piping installation verification, operating & maintenance manuals.

Control system documentation (system configuration/block diagram, flow sheets, display layouts, general process limits, data monitoring, software inventory, electronic application code listing, system security.

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OPERATIONAL QUALIFICATION The OQ process is intended to demonstrate that the components of the

autoclave operate properly & that the autoclave is deemed ready for performance or load testing.

An OQ may include the following checks: Operational tests(operator/supervisory/maintenance modes, doors,

abort & emergency stop, programmable parameter, menu navigation, power-up & shut down, procedure select/start control switch & interlock tests.

Leak/air removal/steam penetration/vacuum hold test Jacket mapping Saturated steam check Empty chamber tests

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Empty chamber heat distribution studies

Intent of this study to demonstrate the temperature uniformity & stability of the sterilizing medium throughout sterilizer.

Temperature uniformity may be influenced by type, size, design & installation of sterilizer.

In production size sterilizer, 15-20 thermocouples are used & distributed geometrically throughout sterilizer.

Thermocouple should be placed in the exhaust drain which is adjacent to the sensor that control vessel temperature.

Temperature deviation should not greater than +2.5 degree celsius of the mean chamber temperature.

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Thermocouple locations

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Container Mapping Intent of this study to determine coolest point within liquid filled

containers.A sufficient no of thermocouples should be positioned in the upper, middle

& lower portions of the containers.The profile point having the lowest temperature is designated as the cold

spot.The no. of thermocouples positioned within the container will be dependent

upon the container volume.

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PERFORMANCE QUALIFICATIONLoaded chamber steam penetration tests:

intent of this study is to determine the coolest point within a specified load.

Load cool points are dependent on the type of objects that compose the loads i.e. liquid filled containers, process equipment.etc.

The penetration thermocouples are positioned within liquid filled containers at the cool point previously determined by the container mapping studies.

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Loaded chamber microbiological challenge studies

The microorganisms most frequently used to challenge moist heat sterilization cycles are Bacillus stearothermophilus & Clostridium sporogenes.

These studies are typically conducted concurrently with the heat penetration studies.

After the sterilization cycle is complete, the spore strips are recovered & subjected to microbiological test procedures.

Strips are aseptically immersed in a suitable growth medium ( soyabean digest medium is typical) & incubated for up to 7 days.

Incubation temperature far B. stearothermophilus is 50-55 degree celsius.

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Conclusion:

If the temperature in an autoclave does not reach 121°c, not all spore will destroyed in the typical cycle time of 20min.

Validation is therefore required to ensure that the autoclave is operating correctly and that complete sterilization is being achieved.

To ensure that qualification and validation are performed correctly, the test should only be carried out by suitably qualified & experienced personnel & organization.

References1) Textbook of microbiological methods, by Collins & Lyne’s,7th

edition, K.M. Varghese Company, Mumbai.2) Indian Pharmacopoeia 1996 vol , Govt. of India , appendix-

12,monograph a-140.3) Pharmaceutical process validation, Robert A. Nash, Alfred

H.Wachter. Marcel, 3rd edition,volume-129,Marcel Dekker, page no 176-190.

4) Validation of pharmaceutical processes, sterile products, II edition, by Frederick J.Carleton, James P. Agalloco, Marcel Dekker. Page no.413-449.

5) The theory & practice of industrial pharmacy, by Leon Lachnan. Herbert A. Lieberman, Joseph L. Kanig. 3rd edition,page no.625.

6) Pharmaceutical Quality Assurance, M.A. Potdar, Nirali Prakashan, Pune.

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Web referenceswww.pharmtech.comwww.validation-online.netwww.pda.orgwww.askaboutvalidation.comwww.intercal.co.zawww.fda.comwww.google.comwww.pharmaweb.netwww.sterilizers.com

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