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Understanding how to write a superb autoclave validation protocol is a mandatory requirement for all personnel involved in the use of  biological sterilization processes in the biomedical and pharmaceutical industries within the FDA, MHRA, WHO & EU controlled areas.

Sterilization can be accomplished by either physical or chemical means. The principal physical means is autoclaving, using a Steam sterilizer. Other physical methods include boiling and dry heat. Chemicals used for sterilization include the gases ethylene oxide and formaldehyde, and liquids such as glutaraldehyde.F

Of all these forms of sterilization, steam sterilization by way of autoclaving is the fastest, most reliable and hence; most commonly used within the FDA, MHRA, WHO and EU zones of influence. It must always be remembered that it is also one of the easiest processes to get wrong. This is why regulators will nearly always scrutinize methods used and ask to review past validation records to ensure conformity with acceptable practices and conformance with existing regulatory expectations.

Autoclave validation / Qualification must follow the routine validation / Qualification document string of;

VP – URS – DQ – VRA – IQ – OQ – PQ

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Autoclaving is the most effective and most efficient means of sterilization. All autoclaves must go through the GMP process of autoclave validation / Qualification 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 115ºC/10 p.s.i., 121ºC/15 p.s.i., and 132ºC/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 “D”–values 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 1-log 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° (250°F).

It is not unusual to find people thinking; why is 121° C the temperature for sterilization? In the early days of autoclave validation, steam for sterilization at standard temperature was used in order that studies could be accurately compared, the temperature chosen was a nice round figure of 250deg F (121.1° C). 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 121.1°C 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 121.1°C (250°F) = (9.08 log)(1.6 min/log) = 14.53 minutes.

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Moist heat 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.

The effectiveness of steam sterilization in the form of moist heat sterilization process increases considerably when air is removed before adding steam to the chamber. Obtaining a vacuum can be difficult, resulting in limited capability of the steam to penetrate into cavities of instruments etc. The use of biological indicators during autoclave validation / Qualification is therefore recommended for monitoring allowing the conditions at different points in the sterilized goods to be assessed.

Biological indicators, used during autoclave validation include preparations of selected microorganisms (bacterial spores) with high resistance towards specific sterilization methods. The bacterial spores are deposited on a carrier, e.g. filter paper, which is wrapped in a suitable package making the system ready for use and with defined resistance characteristics. The inactivation of the biological indicator indicates an effective sterilization process. Whether inactivation has been obtained is determined by cultivation after exposure.

Autoclave validation Sequence

To ensure and to test that an acceptable amount of moisture is present in the steam supply for autoclave validation. 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.

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Positioning of the thermocouples (t/c's) during autoclave validation / Qualification 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 / Qualification, 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. In addition to this understanding how to write a superb autoclave validation protocol is of paramount importance

Let me say at this stage if you want to be pedantic and put t/c’s 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.  It also becomes important to understand how to write a superb autoclave validation protocol that is compliant with the current legislation.

If this is a new installation, then get hold of the Factory Acceptance Test (FAT). and follow the instructions in the; How to write a superb autoclave qualification protocol section.

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/c’s)

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 validation is in normal use and another placed alongside the cabinet product temperature probe. This gives us an additional 2 t/c’s, bringing the total for a standard sized autoclave validation task to 11 t/c’s.

This is normally considered sufficient for 1.5 to 2.5 m3 autoclave validation tasks. Any bigger and I would concentrate on heat loses i.e. add t/c’s 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 / Qualification to be successfully executed while using none validated steam.

Your steam must be validated for – 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. Understanding how to write a superb Autoclave Validation protocol is all explained in the template..