Autoclave Validation / Qualification is mandatory for all machines used for biological sterilization, in the biomedical and pharmaceutical industries within the FDA, WHO & EU controlled areas. Sterilization can be accomplished by either physical or chemical means.
The principal physical means is autoclaving; other physical methods include boiling and dry heat. Chemicals used for sterilization include the gases ethylene oxide and formaldehyde, and liquids such as glutaraldehyde .
Of all these sterilants, autoclaving is the fastest, most reliable and hence; most commonly used within the FDA, 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 and ask about validation methods used in autoclave qualification / Qualification activities.
Autoclave validation / Qualification must follow the routine validation / Qualification document string of;
Autoclave validation / Qualification must follow the routine validation / Qualification document string of;
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 comforming 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 121° C is the temperature for sterilization. In the early days of steam sterilization a 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.
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 a 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 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 primary 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.
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.
There are quite a few reasons why superheated steam is not as suitable for use in steam autoclaves. In heat transfer applications, steam with a large degree of superheat is of little use because it:
a) Gives up little heat until it has cooled to saturation temperature.
b) Creates temperature gradients as it cools to sat temperature.
c) Lower rates of heat transfer whilst the steam is superheated.d) Requires larger heat transfer areas.
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.
Lets 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.
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/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 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 to 11 t/c’s.
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/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 – superheat
– 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.
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This document follows our well-developed method of using a generic document and allowing the customer to apply an attached detailed SOP to it, turning the generic document quickly into a first class company bespoke document. This VP details and integrates all validation activities and procedures required for a small to medium sized project, involving production/facility/utility equipment using electronic controls or monitoring.
This document follows our proven practice of supplying a interactive generic document with an automatic method of populating it. Once populated, all you need to do is follow the prompts in the attached SOP. They will take you through the completion process section, by section. At the end of this process your generic document has progressed into a detailed, referenced, bespoke company document. The document follows our three level URS system that ensures functionality traceability from the URS to the various testing protocols. This document interfaces with our Validation Risk Assessment (VRA), Validation Project Plan (VP), User Requirements Specification (URS), giving a seamless flow from your VMP through the VP - IQ - OQ - PQ, while integrating flawlessly with the URS - DQ - VRA.
This combination protocol has been produced in response to several hundred reader suggestions we received in our ‘Suggestions Section’. It has been carefully designed to make it the preferred choice for Process and Laboratory stand alone equipment. It is interactive, easy to use and suitable for all mixes of equipment with and without software.
The IQ section establishes documented verification that key aspects of the equipment adhere to approved design intentions and that the recommendations of the manufacturer have been suitably considered. The OQ section establishes that there is documented verification that the installed system functions as specified and that there is sufficient documentary evidence to demonstrate this. The PQ section gives documented verification that the equipment performance in its normal operating environment is consistently exactly as specified in the URS.
1 CFWhy does something as simple as a spreadsheet figure in so many
regulatory citations? Good question; and at times a difficult one to
answer. When you ask a group of compliance personnel the same question
you will be informed that Excel cannot be validated because it does not
seal the original copy (of the spreadsheet), allows the original to be
modified and has an audit trail that can be disabled. All true, but none
of these problems interfere with your ability to validate that the
spreadsheet is fit for purpose. They only preclude you from using the
spread sheet as a compliant repository for any data that has to be store
in compliance with 21 CFR Part 11.
If the spreadsheet is signed off and dated by the user, their supervisor and QA, it becomes regulatory acceptable data stored in hard-copy, and Part 11 does not apply.
After numerous requests for this, we have launched our brand new SOP for Spreadsheet Creation to cover these and other known target points that the regulators consistently hone into as soon as they find that spreadsheets are being used. Use this Spreadsheet Creation SOP to ensure that you create spreadsheets that are validatable. Then use our spreadsheet validation pack to validate them.
It now appears that the current FDA Guidance rules pertaining to 21 CFR Part 11 may be with us for a longer time than was originally anticipated. So we have incorporated the guidance suggestions in their latest guidance document, into our current Validation Risk Assessment (now issue 10), which is now available for immediate download. This VRA document now comes with a downloadable matrix for registering the justification for all your Part 11 assessments as this is now a mandatory requirement.