Introduction to Autoclave Use in Pharmaceutical Sterility Assurance and common approaches and considerations used in autoclave validation.
Introduction
One of the pharmaceutical industry's most commonly used sterilisation methods is steam (moist heat) sterilisation. The majority of steam sterilisation will take place in autoclaves. Autoclaves range in scale from benchtop units found in operating theatres or laboratories with a chamber capacity of a few litres to industrial-scale transfer units that can be built into a cleanroom wall with a chamber big enough to walk into.
While the operating principle behind moist heat sterilisation is relatively simple and very well understood as a science, there are significant nuances and intricacies associated with successfully using an autoclave to perform a validated sterilisation process consistently.
This paper merely touches on the fundamental aspects of autoclave operations, considerations in autoclave validation and how control of an autoclave process should integrate with the broader pharmaceutical quality system and contamination control strategy. I am not a subject matter expert, and further reading and relevant guidance is provided at the end.
What is an autoclave?
An autoclave is essentially a computer-controlled pressure cooker. In the same manner that a pressure cooker allows food to heat up and cook faster than an unsealed pot, an autoclave provides a sealed environment where items can be exposed to steam at a high temperature at elevated pressure. The elevated pressure inside the chamber raises the temperature at which water boils, allowing steam to interact with the load at elevated temperatures.
Moist heat and Dry Heat Sterilisation
Heat will deactivate or kill small biological structures by denaturing cellular proteins, permitting biological kill of the offending contamination at a relatively low temperature. The temperature will vary depending on the relative complexity and structure of the microorganism. For example egg white albumin will coagulate at a relatively low temperature whilst some specialised bacteria (thermophiles) with adapted proteins can survive over 100oC.
Steam is a really efficient heat transfer medium. As steam condenses onto a surface its high level of latent heat energy is almost instantaneously transferred to that surface. The high rate of heat transfer achieved is one reason moist heat sterilisation is more effective than dry heat in killing microorganisms – thermal expansion can be fast enough to lyse the cell directly – think hwo quickly a kettle can scald!
Most items can be demonstrated to be “sterilised” from all but the hardiest thermophiles if held for a significant period of time (hours) above 110oC. It is also possible to sterilise most things by dropping them into a cauldron at 2000oC for a second. However in the common sense driven world driven by practical considerations a sterilisation temperature of 121oC to 121.5oC is preferred for pharmaceutical sterilisation purposes. The reason for this standardisation is driven by the following considerations.
Normal steam is not really hot enough to guarantee sterilisation with enough sterility assurance for pharmaceutical purposes. At normal atmospheric pressure, water will boil to steam at 100oC, therefore steam injected into an unsealed environment at atmospheric pressure will not heat the environment beyond 100oC. At a higher pressure equal to 1bar above atmospheric pressure at sea level) water will boil at 121oC. Therefore a sealed autoclave controlled to 1bar above normal atmospheric pressure will allow steam to maintain a temperature of 121oC.
Items held at 121oC for over 15 minutes have proven time over to meet or exceed the required sterility assurance level. The minimum sterilisation criteria everyone in sterility assurance understands is that items must meet a time and pressure criteria of 121oC for 15 minutes. This criteria itself will provide overkill of most common contaminants, permitting an inherent margin for safety in validated sterilisation cycles.
Driving steam to a higher temperature could reduce the time required to sterilise items significantly but building a pressure vessel to hold steam over 1bar becomes a non trivial exercise from a safety and cost standpoint very quickly. As it stands autoclaves require specialised pressure and vacuum routine safety inspections and insurance.
Most materials used in pharmaceutical practise can withstand 121oC with significant ill effect, at the same time these conditions permit a fast enough sterilisation cycle to be practicable.
Historical note: Bar is not a SI unit for pressure but in general autoclave parameters as a technology were largely standardised before SI units became “the norm” in engineering.
How do autoclaves work?
Autoclave cycles get complex quickly but the following basics will still apply. To achieve sterilisation and safe use of the items afterwards in general three things need to occur
Everything within the autoclave need brought up to temperature – Heating Phase.
Items must be held at sterilising conditions (121oC, 1bar) for a minimum period of time – Sterilisation Phase
Items are cooled back down to room temperature – Cooling Phase.
To start the heating phase, steam in injected into the chamber (it is possible to heat water directly in the chamber with a heating element but that is more common in smaller benchtop systems) and the surrounding jacket to heat the chamber and its walls. The system will increase in pressure and temperature until 121oC at 1bar is reached (the exact heating profile depend on when the system is sealed to bring it to pressure).
When the system reaches these conditions the sterilisation phase starts, in a sealed system a drop in temperature will lead to a drop in pressure so a pressure sensor is commonly used to control the system via a computer controlled feedback loop. If the pressure drops then a small quantity of steam is injected to the chamber to maintain temperature and pressure above the required lower limit.
Once a prescribed period of time has elapsed the cooling phase will commence. In simple cycles the drain valve of the autoclave will open at the bottom of the chamber and release pressure. The heat of the chamber walls and the load itself will dry the items inside from residual condensation.
But wait, it’s never that simple…
In normal use all items that are sterilised (such as surgical instruments, cleanroom gowning, and filling machine parts) will be wrapped in porous paper or cloth. This is to protect the items from being re-contaminated by the environment once they are removed from the autoclave. This wrapping no matter how well executed, will leave an air gap between some surfaces and the wrapping. Air is excellent insulator and of left within the autoclave chamber at the start of the sterilisation phase result in some areas or items not achieving the desired time/pressure/temperature combination.
In pharmaceutical applications some items, such as clothing or porous sterilising filters are excellent at retaining air unless we take it out by force.
This is where the use of vacuum pumps come in. Where a simple autoclave cycle will use steam to displace air from the chamber (gravity displacement autoclaves) a vacuum displacement autoclave will use a dedicated vacuum pump to forcibly suck air out of the chamber before injecting steam. This effectively removes the air insulation from within porous items. This is referred to as a pre-vacuum phase. A vacuum used to quickly remove steam from the autoclave can also be used during the cooling phase to ensure items and there packaging is completely dry. Wet material existing an autoclave will present an ideal environment for contamination to develop and should be avoided.
How do automated vacuum autoclaves work?
At the start of a vacuum cycle, the vacuum pump pulls the air out of the closed chamber reducing the pressure below normal atmospheric pressure. Once a set point is reached the vacuum is tuned off and steam is injected into the chamber. This vacuum-steam cycle is repeated several times, as it is impossible to pull a perfect vacuum and remove all air in one cycle. This is sometimes referred to a vacuum pulsing. Eventually the system stops pulling vacuum and the system is brought to temperature and pressure with steam. One advantage of this approach is that it may provide a longer warm up period which allows items with a large thermal mass to reach temperature with the rest of the autoclave contents.
The sterilisation phase is unchanged. During the cooling phase the vacuum pump supports the drying process by sucking the steam out of the chamber. In most modern autoclaves the chamber jacket is kept heated during the initial drying phase to help drying via radiate heat.
Common components in a pharmaceutical autoclave.
Pharmaceutical autoclaves, especially when built into a factory are run by steam from an external pure steam generator. Steam quality is important as steam that is too dry will not effectively transfer heat to the autoclave load. The jacket may be heated by plant grade steam but designs differ with some autoclaves passing steam into the vessel via the jacket itself. A supply of enough quality steam is vital to ensure a high throughput sterilisation system.
In general the following components are common.
The chamber 1 is surrounded by a heated jacket 2 and sealed with a large door 3. The load to be sterilised is generally placed onto a removable trolley or rack 4. When the jacket inlet valve 5 is open, hot steam from the steam generator enters the jacket 2 of the autoclave. The chamber gets preheated. The pressure gauge 6 shows the jacket pressure. When valve 7 is opened, the steam gets into the chamber. The colder air escapes through the open drain valve 8. The valve is a temperature controlled one-way valve. The valve closes when the steam in the chamber is hot enough. Now the pressure rises until it is as high as the desired pressure and sterilisation can commence. The chamber pressure is shown by the pressure gauge 9 and the temperature by the thermometer at the drain 10. The drain temperature probe 10 is placed at the bottom of the chamber, where the lowest temperature is. When the sterilisation time is over, the inlet valve has to be closed and the drain valve opened. Steam escapes through the drain. The pressure and the temperature in the chamber decrease but the jacket is still under hot steam pressure. This supports drying the load. After a while the steam from the jacket can also be released. In many cleanroom installations where an autoclave is used to prepare components an autoclave may have a door at each end of the chamber.
Autoclave use in dry heat sterilisation.
When sterilising sealed items such as finished vials of an injectable solution an autoclave may be used as a dry heat steriliser. In this mode of operation two things are changed.
Temperature control of the autoclave will switch from a drain valve temperature sensor to a load probe – this is a thermocouple inserted into a reference material such as a glass vial filled with water at the same volume of the items to be sterilised. As these items will take longer to heat than the autoclave itself the load probe is placed in a simulation of a worst case scenario. It is assumed (and backed up by validation) that the temperature response of the reference material closely matches that of the sealed product.
Vacuum pulsing may not be possible due to the formulation or packaging. As the autoclave chamber cools the chamber will be topped up with compressed air to maintain pressure above atmospheric.
Run times will generally be longer due to the time taken to reach an internal temperature sufficient to sterilise the contents. As the interior of the items do not experience steam the principles of dry heat sterilisation apply.
Autoclave control in routine operation.
In normal operation a validated and auditable computer system will control the autoclave functions, following its programming. The following control measures are common
A validated “recipe”. The recipe is a series of pre-programmed instructions for the control software to follow. It may include one, three or no pre vacuum cycles. A porous load may require maintenance at 121oC for 20 minutes but a dry heat sterilisation of finished goods may prescribe a sterilisation hold of one hour.
Each set of items to be sterilised will be placed in a validated load pattern. Every item should reside in a dedicated location within the chamber and in a specific orientation. In practice operatives will use preference photographs or diagrams to achieve this. It is possible to validate a load to use a random patter of components but this time consuming to validate.
A standard operating procedure for selection of the correct autoclave function/recipe
A paper copy or electronic record of the time/temperature and pressure condition within the autoclave is commonly kept with the associated batch record and separately backed up.
A series of routine performance tests may be carried out daily or weekly. Examples include a leak rate test to verify the chamber integrity (particularly the doors ability to seal fully), and a prescribed warm up cycle to pre heat the autoclave prior to use.
According to the international standards a specialised Bowie-Dick-test has to be done every day at minimum. For this purpose a test pack of folded material is placed in the chamber at a prescribed location and the sterilisation cycle is started. After sterilisation a colour change indicates whether the steriliser works correctly or not. A failed test can be the result of a defective autoclave.
Autoclave validation.
As with any sterilisation process, validation is a procedure for obtaining, recording and analysing the results required to establish that the sterilisation process yields reliable, repeatable load sterilisation complying with a predetermined specifications for sterility.
Autoclave validation is significantly impacted by the load configuration and composition itself as this will determine the autoclave recipe to be validated. Any significant change to a load pattern managed within a change control process will result in revalidation of the autoclave cycle.
Validation will be performed at both initial installation of the system and at a prescribed frequency (typically bi-annually) provided that the autoclave is regularly calibrated and maintained.
IQ/OQ/PQ
As the performance of the autoclave is critically important, a fully documented and recorded purchasing and approval process is used. A URS is developed based on the items requiring sterilisation to which the autoclave supplier can respond with the expected performance of the system.
Installation qualification (IQ) is similar to most pieces of automated equipment and will focus on a series of tests to ensure the autoclaves components are present and function and the autoclave is of the right capacity and construction.
An autoclave Operational Qualification (OQ) will test or verify items such as cycle operation and programming instructions, safety and alarm testing, error reporting, empty chamber temperature profiling and chamber temperature limits/specifications, air removal testing, leak testing, temperature control anomalies, full cycle completion), and determination of any hot or cold spots within the chamber.
Process qualification (PQ) will include a series of performance tests which establish that the installed and properly operating autoclave will process efficiently and effectively the user’s desired chamber loads to the specified sterility assurance level (SAL). These fall into two brad categories
Thermometric tests will independently measure and recorded temperature and pressure within several locations of each load pattern over multiple cycles. These independent data logging systems must be subject to calibration and routine verification. These verify that the autoclaves control system is operating off correct and representative temperature and pressure data as well as demonstrate that the load contents are meeting the required conditions. This data will also be used to determine worst case locations for the inclusion of any load probe used to control a specific cycle
Biological indicator strips are used to demonstrate functional biological kill. Biological indicators, or spore tests, are the most accepted means of monitoring sterilization because they assess the sterilization process directly by killing known highly resistant microorganisms (e.g., Geobacillus or Bacillus species).
Analysis of these results may point to problems in the set-up of the autoclave or in the way the load is contained. Significant differences can be seen, for instance, between glass and plastic bottles or plastic and metal discard containers, or single and double bagged discard loads.
The primary goal for the commonly employed “overkill” validation is that the autoclave validation needs to complete three consecutive successful half cycles to qualify their proposed full-cycle exposure for routine processing of sterilisation loads. If, for example, there was no BI growth for the three test cycles at 10 minutes exposure at 121°C, then a 20 minute exposure at the same temperature would be adequate for routine daily processing, assuming all other aspects or requirements of the IQ/OQ/PQ are successful, documented, reviewed and approved.
Calibration and maintenance
Calibration determines the accuracy of the autoclaves own measurement systems by measuring the actual temperature inside when a given temperature is set. The electronics may be wrong, the temperature probe may be damaged, the pressure gauge (a useful backup) may also be wrong, etc. The accuracy of the timer should also be checked. Without regular calibration any performance testing or validation work will become invalid.
The intervals between ongoing calibration and validation should be determined based on any specific regulatory requirement, documented experience and the performance of the autoclave. Regular maintenance such as gasket inspection, and cleaning of the chamber should be performed at the manufacturers recommended intervals and a full service should be performed at least annually, as should the safety check of the pressure vessel.
Additional reading
This is a very basic overview of how autoclaves operate common consideration in validation. Some very helpful additional reading can be found at https://www.pharmout.net/downloads/white-paper-autoclave-validation.pdf.
The key relevant legislation for EU based colleagues is the newly revised Annex 1 found here - 20220825_gmp-an1_en_0.pdf (europa.eu) – in particular section 8.