BY DR TIM SANDLE | 22 JUNE 2023
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For isolators to be used effectively, they need to be operating to EU GMP Grade A / ISO 14644 class 5. This is achieved through the barrier concept, the maintenance of a positive pressure, the supply of HEPA filtered air and its extraction, and by subjecting the isolator to an automated decontamination cycle. Isolators are most commonly sanitised using surface contact disinfectants: hydrogen peroxide vapour (an alternative is ionised hydrogen peroxide), chlorine dioxide, ozone or peracetic acid. Of the different chemicals, hydrogen peroxide is the most widely used (1).
What is hydrogen peroxide?
Hydrogen peroxide (H2O2) is a very pale blue liquid, slightly more viscous than water that appears colourless in dilute solution. It is a weak acid which has strong oxidising properties (2). Typically, a 35% (weight/weight) liquid H2O2 concentration is used for isolator system decontamination.
The oxidising capacity means that the hydrogen peroxide vapour disinfection process employs a free radical reaction mechanism to facilitate microbicidal action (Block, 2000). Free radicals are highly energetic atoms or molecules that possess an unpaired electron (the unpaired electron is compelled to exist in a paired state and will physically strip an electron from another compound to promote pairing). This ability makes the compound extremely effective against microbiological organisms. This is because there is no target site specificity on or within the microbial cell, which makes it very difficult for microorganisms to develop resistance to free radical attack (3).
An advantage of hydrogen peroxide is that it breaks down into relatively harmless components. Hydrogen peroxide decomposes (‘disproportionates’) exothermically into water and oxygen gas spontaneously, through the following equation (4):
2 H2O2 → 2 H2O + O2
Hydrogen peroxide vapour has proven biological efficacy against a wide range of micro-organisms including bacterial endospores, mycobacteria, vegetative bacteria, viruses and fungi (5).
What is a vapour?
The term vapour is used because they are evaporated by flash evaporation and are therefore condensable vapours - not a true gases. The process whereby the vapour is distributed to all surfaces within the isolator is called 'micro-condensation'. This is a form of molecule distribution and relates to the gas-to-liquid chemical transition phase (6).
Sterilisation or disinfection?
Isolators are said to ‘disinfect’, ‘decontaminate’, or 'sanitise' rather than 'sterilise' because absolute sterility cannot be demonstrated. Disinfection, in this context, is the reduction of a number of microorganisms within the clean environment, as demonstrated through the use of biological indicators in validation studies.
The decontamination process
To sanitise an isolator, several essential steps are required. We’ll illustrate using hydrogen peroxide as the starting disinfectant.
Preparing the isolator or gassing port
Isolator systems either consist of an isolator or an isolator linked to a gassing port. The advantage of the gassing port is that the sanitisation time will be faster as the surface area of the port is considerably smaller than that of the isolator. The objective with either is to sanitise the load.
To produce the vapour for the sanitisation process, a gas generator is required. Because hydrogen peroxide in the passive state is very poor at diffusion, the function of the gas generator is to heat the vapour and then for the port to actively distribute the vapour via a distribution nozzle. The function of the nozzle is to prevent hot spots arising from an uneven distribution of the gas. The dosage of the disinfectant is controlled by the gas generator, providing a controlled volume of the disinfectant.
When loading an isolator or the port, the presentation of surfaces to the disinfection process is extremely important. Material loads often rely on point contact support, via wire racking or hangers, to assure exposure to the disinfection agent.
The sanitisation process occurs by an aqueous solution of hydrogen peroxide being evaporated in such a way as to produce the same weight ratio in the vapour phase as in the source liquid (starting with 35% w/v of hydrogen peroxide, which is evaporated into the heated carrier gas stream to produce hydrogen peroxide concentrations at level above that established at the Operational Qualification and Performance Qualification stages). This vapour is transported to the chamber to be bio-decontaminated in a heated carrier gas (initially sterile compressed air). Vapours from the chamber are returned to the gas generator where further quantities of the aqueous solution are evaporated.
The sanitisation stage functions within the chamber by depositing an even layer of hydrogen peroxide over all surfaces (micro-condensation). With the micro-condensation process, there has been a long-standing debate as to whether ‘wet’ or ‘dry’ hydrogen peroxide processes are the most effective (with the debate centred on rival technologies). Research has found that more than a few microns of molecule-monolayer deposition (which is invisible micro-condensation) contribute little additional efficacy. The optimum bio-decontamination processes are between the ‘wet’ visible condensation condition (with over injection of vapour) and the invisible gas phase, a ‘dry’ process that is specified to remain just below of dew point. An optimum kill condition requires dew point to be reached. Achieving dew point is based on a certain target volume of gas, which is affected by the starting temperature, relative humidity conditions and the subsequent amount of vapour injected into the target volume (7).
Condensation formation at saturated vapour conditions, past the dew point is the mechanism that delivers the hydrogen peroxide molecules to all exposed surfaces. This is physical chemistry and hence has physical parameters of control. Therefore, isolator and gassing port cycle times should be established and examined for each operation to ensure that the recorded parameters fall within the ranges set during validation.
There are four key steps to ensure an optimal hydrogen peroxide vapour disinfection process:
1. Vaporisation of liquid to small molecules – gas phase delivery to target volume
2. Development of the gas concentration in the target environment to saturated vapour conditions, past dew point and transition into liquid phase. At saturated vapour conditions the gas concentration can hold no more molecules, hence the process of condensation formation and disinfection agent surface deposition starts
3. Micro-condensation formation on surfaces by merging molecules. Initially, nuclei form on any surface contaminants, before full condensation occurs over the entire available surface, eventually forming a disinfectant monolayer (from gas to liquid phase)
4. Re-evaporation of the surface condensate and removal of residual gas to target endpoint.
The cycle operates through the following steps:
- The generator initially dehumidifies the ambient air (conditioning). Here, initial temperature and relative humidity conditioning, before vapour injection. Hot, dry air is first exchanged with the target environment to achieve the required starting humidity conditions
- The generator then produces hydrogen peroxide vapour by passing aqueous hydrogen peroxide over a vaporiser. The gas distribution is an active process controlled through a nozzle for a pre-determined period of time, based on the gassing port pump speed range (gassing or dosage phase). The gassing phase is validated to deliver a disinfection agent dose volume that will reach required micro-condensation conditions in the target area and on target surfaces
- The vapour is then circulated at a programmed concentration in the air and held for a set period of time (dwell or hold phase). The dwell time is optimised to maintain micro-condensation conditions throughout the complete dwell phase for assured disinfectant contact time. Arguably this 'contact time' is the most important part of the sanitisation process
- After the hydrogen peroxide vapour has circulated in the enclosed space for a pre-defined period of time, it is circulated and broken down into water and oxygen by a catalytic converter, until concentrations of hydrogen peroxide vapour fall to safe levels, at 1 ppm (aeration phase) (less than 1 ppm is below the Occupational Exposure Level, and hence deemed safe for personnel). Gas residual removal is typically achieved with integral or supporting HEPA-catalysts and/or supporting dilution via barrier air being vented to the environment
Primary process variables for using hydrogen peroxide vapour include:
- Starting relative humidity (RH)
- Surface and environmental temperatures
- Gas distribution for a homogeneous deposition of vapour and resulting surface condensate
- The amount of evaporated hydrogen peroxide solution dosed into the target volume (g/min) to reach saturated vapour conditions and provide a sufficient monolayer of disinfectant
- Surface area for condensate distribution together with amount of surface absorbency (e.g., packaging)
It is important that these process variables are controlled. Many of these are established at the time of validation (8).
Monitoring the sanitisation cycle
It is particularly important that, following completion of each cycle of the gassing port, the critical parameters are examined. These will be used to verify that the gassing cycle was satisfactory, and, by implication, the result of the sterility test is valid. The parameters normally examined are (9):
• Volume of hydrogen peroxide used
• Gas concentration alert level
• Alarm status during the cycle
• Gas injection phase time
• Humidity at the start of gassing
Selected parameters should also be trended in order to determine if the gassing port is operating as expected and as a means to determine on-going performance.
This article has outlined the key steps involved in the decontamination of an isolator, using an example of hydrogen peroxide in the vapour form. It is important that each load is qualified, and the overall cycle adjusted accordingly. Qualification is achieved through the use of chemical and biological indicators.
For hydrogen peroxide validation, the microorganism Geobacillus stearothermophilus is normally used for the biological indicator (either traceable to ATCC 7953 or ATCC 12980). The biological indicators are spores of Gs. stearothermophilus dried onto stainless steel discs. Alternatively, rapid enzymatic indictors can be used.