Autoclave Validation

Introduction
Sterile products have several unique dosage form properties, such as Freedom from micro-organisms, Freedom from pyrogens, Freedom from particulates, Extremely high standards of purity and quality; However, the ultimate goal in the manufacture of a sterile product is absolute absence of microbial contamination. Three principles are involved in the validation process for sterile product. 1. To build sterility into a product 2. To demonstrate to a certain maximum level of probability that the processing and sterilization methods have established sterility to all units of a product batch 3. To provide greater assurance and support of the results of the end product sterility test A)Methods of Sterilization of Products 1.Heat Moist heat (autoclave) Dry heat oven or tunnel

2.Gas Ethylene oxide Peracetic acid Vapor phase hydrogen peroxide Chlorine dioxide 3.Radiation Gamma Beta Ultraviolet

Autoclave Principle
Moist sterilization (or autoclaving) is conducted by supplying dry, saturated steam under pressure to an autoclave. The energy (heat) from the condensation of steam on the items in the sterilizer will kill the present microorganisms by irreversible damage of cell components.

Method of Operation
Steam autoclaves generally follow the principles shown in Figure 1. Water is heated and the steam pressure is built up in the jacket. Once sufficient pressure is obtained, the steam is allowed to enter the chamber, thereby driving the air out of the drain port. Once all the air has been replaced by steam, the drain valve closes and steam pressure builds up in the chamber. The resultant pressure causes the temperature in the chamber to rise to the required point, usually 121 or 135 C. Some type of autoclaves have a vacuum function that pumps out the air before the steam is allow to enter. This method is much faster and more efficient.

Cycles Stages
Generally an autoclave cycle consists of 3 stages viz. 1)heating, 2)sterilizing, 3)de-pressure. Vertical autoclaves may have a water fill stage before the heating stage. Autoclaves fitted with vacuum facility may have vacuum stages before the heating stage and also as part of the depressurizing stage.

Load Types
Autoclaves are used to sterilize several different types of loads: _ Solid metal, glass, plastic _ Porous linen, gowns, paper, gauze, complex instruments, hollow tubes _ Liquid water, saline, media

_ Laboratory waste Petri dishes, sample bottles, syringes

Important considerations related to sterilisation

1)D value:It is time required for a 90% reduction in microbial population. Quantitative expression of rate of killing of micro organism. In other words, the D value will be affected by The type of microorganism used as BI, The formulation components and characteristics The surface on which the micro-organism is exposed The temperature, gas concentration, or radiation dose of sterilization process. D value found by 2 methods, 1) Survivor curve method (log number of surviving organism versus time/gas concentration/radiation dose) 2) Fraction negative method 2)Z value Used exclusively in validation of heat sterilization process. Z value is reciprocal of slope of plot of log D verses T at which D value is found i.e. increase in temperature required to reduce D value of organism by 90 % (1 log reduction) 3)F value Used exclusively in validation of heat sterilization process. It is time in min required to kill all spores in suspension at 121oC. Measures equivalent time

TEMPERATURE AND TIME RELATIONSHIP

Autoclaving is the most effective and most efficient means of sterilization. All autoclaves must go through the GMP process of autoclave validation during which, the various programs are verified as conforming to the requirements detailed in the User Requirement Specification (URS). They operate on a time/temperature relationship. These two variables are extremely important. Higher temperatures ensure more rapid killing. Some standard temperature/pressures employed are 115C/10 p.s.i., 121C/15 p.s.i., and 132C/27 p.s.i. Longer times are needed for larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal for sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic media, liquids, and many other things. When proper conditions and time are employed, no living organisms will survive a trip through an autoclave. The thermal resistance of specific microorganisms is characterized by Dvalues and Z values. A D-value is the time in minutes, at a specific temperature, to reduce the surviving microbial population by 1 log. A Z-value is the temperature change required to result in a 1log reduction in D-value. Other time measurement variables pertaining to thermal resistance are F-values and Fo-values. An Fo-value is the number of minutes to kill a specified number of microorganisms with a specified Z-value at a specific temperature. An Fo value is the number of minutes to kill a specified number of microorganisms with a Z-value of 10 C (50 F) at a temperature of 121.1 (250F).

It is not unusual to find people thinking 121 C is the temperature for sterilisation. The Fo-value can be determined as per the following Fo = 10 (T 121.1)/10 Where T = temperature ( C) and Fo = equivalent sterilization time (minutes) So given a Bioburden of 1215 CFU, with a D-value of 1.6 min/log at 121.1C and a required SAL of 10-6. Then: Log (1215) = 3.08 Loge reduction = 3.08 log + 6 log = 9.08 log. Ideal Cycle at 121.1C (250F) = (9.08 log)(1.6 min/log) = 14.53 minutes.

HOW MANY THERMOCOUPLES?

Positioning of the thermocouples (t/c's) during autoclave validation or indeed in any GMP temperature mapping exercise is all about appreciating what is adding or subtracting heat from the room or cabinet being qualified. In the case of temperature mapping during autoclave validation, heat is added in the form of pressurized wet steam, anything that can affect the distribution of the incoming steam, can affect uniformity of temperature. Conversely anything that can take heat away from the chamber can affect temperature uniformity. Lets me say at this stage if you want to be pedantic and put t/cs down the drain, the mapping exercise will probable fail. However you are there to verify that product will be sterilized, and product is never placed down the drain. Only the designated product containment area has to be verified. If this is new installation, then get hold of the Factory Acceptance Test (FAT). In the FAT the chamber is subjected to detailed temperature transfer studies. Even distribution of the in coming steam can be verified by placing a thermocouple sensor (t/c) in each of the eight corners in the autoclave and one in the cabinet centre. (9 t/cs) Cooling due to heat loss will be maximum the further away you are from the steam inlet and the closer you are to metal that will conduct heat out of the chamber. That is usually, the door, or doors if double sided. The drain is also a heat sink that conducts heat out of the chamber. One t/c should be placed as close to the drain as product would be, when the autoclave is in normal use and another placed alongside the cabinet product temperature probe. This gives us an additional 2 t/cs, bringing the total for a standard sized autoclave to 11 t/cs. This is normally considered sufficient for 1.5 to 2.5 m3 autoclaves. Any bigger and I would concentrate on heat loses i.e. add t/cs to the top and bottom of the doors and or end wall. It is most important to understand that it is impossible for autoclave validation to be successfully executed while using none validated steam. Your steam must be validated for superheat dryness none condensable gases. Another GMP essential is to carry out pre and post mapping, calibration of your thermocouples. These should be calibrated against test standard instruments whose calibration is traceable to national standards, and for which you have valid current calibration certification.

Lethality Calculation

Even if it is not possible to attain absolute sterility, it is possible to sterilize food products for extended storage, so that they are safe for human consumption. This degree of sterility is referred to as practical or commercial sterility. The F-Value is introduced as a standard on which to base the sterilization of food products. The F-Value is defined as the number of minutes which it takes to reduce the initial spore count of a certain microorganism to a desired safety level at a defined lethal reference temperature. To determine the time period required for a certain sterilization process the following data is needed: -The characteristic decimal reduction value of the endospores in the environment of the product -Initial spore count per weight or volume unit (grams or ml) multiplied by the weight or volume of the product quantity in the container -Desired maximum probable survival of spores in the thermal processed product The technology to determine the required heat treatment beforehand in order to achieve commercial sterility is available for not only heat sterilized food but for pasteurized products as well. The parameters of the heat process can be tailored using the determined treatment. An important role in this case is the heat resistance of those organisms, which may be destroyed between 60C and 100C. As in the case of sterilization, there is a symbol indicating the pasteurization value necessary for the sterility of the processed product which is the P-Value.

P-Value

Even if it is not possible to attain absolute sterility, it is possible to sterilize food products for extended storage so that they are safe for human consumption. This degree of sterility is referred to as practical or commerical sterility. The P-Value is introduced as a standard on which to base the pasteurization of food products. The P-value is defined as the number of minutes which it takes to reduce the initial spore count of a certain microorganism to a desired safety level at a defined lethal reference temperature.

To determine the time period required for a certain pasteurization process the following data is needed: -The characteristic decimal reduction value of the endospores in the environment of the product -Initial spore count per weight or volume unit (grams or ml) multiplied by the weight or volume of the product quantity in the container -Desired maximum probable survival of spores in the thermal processed product. An important role in this case is the heat resistance of those organisms, which may be destroyed between 60C and 100C.

A.Qualification and Calibration 1) Mechanically Checking, Upgrading, and Qualifying the Sterilizer Unit The main concern with steam sterilization is the complete removal of air from the chamber and replacement with saturated steam. Autoclaves can also involve airsteam mixtures for Sterilizing flexible packaging systems and syringes. When autoclave system is used, the unit must be installed properly and all operations qualified through installation qualification and operation qualification (IQ/OQ). 2) Selection and Calibration of Thermocouples Thermocouples must be durable for repeated use as temperature indicators in steam sterilization validation and monitoring. Copper constantan wires coated with Teflon are a popular choice as thermocouple monitors.

 Accuracy of thermocouples should be 0.5C. Temperature accuracy is especially important in steam sterilization validation. Thermocouple accuracy is determined using National Bureau of Standards (NBS). 3) Selection and Calibration of BI Sr. No 1. Sterilization process Biological Indicator(BI) B. steriothermophillus spores B. subtilis var. niger spores B. subtilis, 5230 spores B. coagulance spores Clostridium sporogenes spores 2. Dry heat B. subtilis var. niger spores B. subtilis, 5230 spores 3. Ethylene Oxide B. subtilis var. niger spores

Autoclave

4.

Radiation

B. pumilus spores Micrococcus radiodurans vegetative cells

3)STEAM QUALITY IN AUTOCLAVE VALIDATION.

In a mixture of air and steam, the presence of air will cause the temperature to be lower than expected. The total pressure of a mixture of gases is made up of the sum of the partial pressures of the components in the mixture. Example Consider a steam/air mixture made up of steam and air by volume. The total pressure is 4 bar. Therefore the steam only has an effective pressure of 3 bar as opposed to its apparent pressure of 4 bar. The mixture would only have a temperature of 134C rather than the expected saturation temperature of 144C. This could render autoclaving ineffective where a minimum temperature is essential in order to kill bacteria. It is therefore of paramount importance during the autoclave validation task to validate that all air has been removed from the chamber None Condensable Gasses The Non-Condensable Gas Test demonstrates that the attainment of sterilization conditions in all parts of a steriliser load (particularly for porous load items) is not impaired by the presence of non-condensable gases. The measurement of non-condensable gases is made by cooling a steam sample with an efficient condenser, using water siphoned from a tank at 200ml per minute. Minimum requirements are: one metre head and water temperature below 28 degrees centigrade. Pressurised water is not required. When the sampled steam is condensed any non-condensable gases present are released and separated from the cooled condensate into sight glass columns.

Dryness Value test: To ensure and to test that an acceptable amount of moisture is present in the steam supply. For little amount of moisture there is a chance of superheating may occur. Even too little moisture may prevent sterilizing conditions in the chamber. Steam with a dryness fraction of 0.99 consists of 99% steam and 1% water. Similarly, steam with a dryness fraction of 0.95 consists of 95%

steam and 5% water. The dryness value of the steam should be equal to or greater than 0.9 for porous loads or 0.95 where metal loads are processed.

B. Heat-Distribution Studies Heat-distribution studies include two phases: 1) Heat distribution in an empty autoclave chamber 2) Heat distribution in a loaded autoclave chamber. The trips where the wires are soldered should not make contact with the autoclave interior walls or any metal surface. Heat-distribution studies may employ thermocouples as the cool spot in the chamber. The principle is the location of the cool spot and the effect of the load size and/or configuration on the cool spot location. The difference in temperature between the coolest spot and the mean chamber temperature should be not greater than 2.5C . Greater temperature differences may be indicative of equipment malfunction. c)Heat-Penetration Studies This is the most critical component of the entire validation process. The main purpose is to determine the F0 value of the cold spot inside the commodity. The container cold spot for containers 100 ml is determined using container-mapping studies.

 Thermocouple probes are inserted within a container and repeat cycles are run to establish the point inside the container Thermocouples will be placed both inside and outside the container at the cool spot location(s), in the steam exhaust line, and in constant-temperature baths outside the chamber. F0 value will be calculated based on the temperature recorded by the thermocouple inside the container at the coolest area of the load. F0 value will indicate whether the cycle is adequate or alterations are needed Three critical parameter associated with all wet heat sterilization Processes: 1. A minimum F value 2. A design F value 3. A sterilization process time Any changes in the load size, load configuration, or container characteristics must be accompanied; To prove that the cool spot location has not changed or, If it has, that it receives the design F0 time exposure from the sterilization cycle used.

d. Equipment Qualification Prior to the initiation of process, it is important that the sterilizer be suitably qualified to perform its function.

 Typical critical requirements that are considered to affect the sterilization process (e.g.quality requirements) are: 1. Accurate temperature and pressure measurement 2. Air removal to some predefined level of vacuum 3. Temperature distribution and uniformity in the chamber. 4. The qualification of a sterilizer should include the following : 1.Calibration of temperature and pressure sensors (traceable to national or international standard) 2.Air removal (usually measured by vacuum level achieved vs. defined requirement) 3.Demonstration of the sequence of operations, 4.Confirmation of alarms and interlocks 5.Precision of temperature control 6.Temperature distribution and uniformity

e. Microbiological Challenge Studies Microbiological challenges studies are employed to provide additional necessary assurance that adequate lethality has been delivered to all parts of the load. Calibrated BIs used as bioburden models providing data that can be employed to calculate Fo. The microorganisms used to challenge moist heat sterilization cycles are G. stearothermophilus and Clostridium sporogenes.

 After the sterilization cycle is complete, the inoculated items or spore strips are recovered and subjected to microbiological test procedures. Strips are immersed in a suitable growth medium (soybean casein digest medium is typical) and incubated for up to seven days. G. Sterilizer Filter Evaluation Microbial filters are employed on most parts of sterilizers to ensure that loads are not contaminated by air used to vent the chamber as it cools or dries. Product loads are protected from such contamination by their primary containers (vials, bags) and many nonproduct loads are protected by wraps to provide a microbial barrier. For filters, two issues are of concern: Sterility and Integrity. If the load will undergo a bioburden cycle, it may be necessary to sterilize the filter in a separate phase of the cycle. To ensure that filters will remain functional under all expected conditions, the integrity tests should be done following the maximum cycle time and temperature. Triplicate studies are recommended

Routine Checking
Routine checking may be broken down into daily, weekly, quarterly and annual checks. Daily checks should be performed by the operator and should include the observation of all indicators for normal values. On a weekly basis safety checks should be performed such as inspection for leaks and electrical wiring faults, inspection of door seals, correct of operation of door interlocks and high temperature and low water alarms. In the case of vacuum autoclaves, a vacuum leak test should also be performed. Quarterly checks may include verification of the calibration of the instrumentation, a check of the automatic control functions, a check of the vacuum leak sensor and a thermometric test on a small load. Annually, a full re-commissioning may be warranted. Thermometric testing should be performed. Load testing should be performed as well, but different loads may be scheduled into bi- or tri-annual cycles. [1]

REERENCE
Validation of steam sterilization cycle: PDA technical monograph NO.1 (1978) Sterilization of medical device validation and routine control of sterilization by moist heat : Europiun standard EN 554 (1994) www.validation-online.com nash