Peer Review Article | Open Access | Published 4th January 2023
Validation of a Sporicidal Bio-decontamination Process for Use in Pharmaceutical Isolators in an
NHS Aseptic Manufacturing Unit
BrianMcBride | EJPPS | 274 (2022) | Click to download pdf
The recently published revision of EU GMP Annex 1 Manufacture of Sterile Medicinal Products includes a requirement for pharmaceutical isolators to undergo a bio-decontamination process of the interior which should be automated, validated and controlled within defined cycle parameters and include a sporicidal agent. It is widely accepted that the easiest way to achieve this is by converting Hydrogen Peroxide (H₂O₂) liquid into a state that will diffuse readily through the closed isolator chamber leading to bio-decontamination of all surfaces which it comes into contact with.
The technology to do this has been around for years and can be validated to give a high level of assurance of surface bio-decontamination comparable with sterilization when used on surfaces in the isolator critical work zone. This type of activity is routinely used in large-scale sterile manufacturing that takes place in the pharmaceutical industry where the amount of materials and timescales involved make this viable.
In the NHS, aseptic preparation and manufacture of sterile products is carried out in isolators both for immediate use and for stock in anticipation of demand. Automated bio-decontamination systems are available but their use is much less than in the pharmaceutical industry and reliance is still placed on manual spraying and wiping of surfaces with disinfectant agents such as alcohol and hydrogen peroxide. This brings a degree of variability and is more difficult to validate.
For sterile manufacturing units holding an MHRA Specials Manufacturing licence the expectation is that proper consideration is given to using an automated bio-decontamination process or there is justification for not doing it. For units preparing products for immediate use there is not the same expectation but as standards are improved, it is reasonable to assume it may become the case in the future. To use this type of process requires it to be integrated into the isolator, meaning either buying a new isolator or retrofitting to an existing one. The relatively high cost of this is a barrier to pharmacy department aseptic units using the technology.
There is a range of bio-decontamination systems on the market from Devea (www.deveaenvironnement.com) which are claimed to be suitable for use in hospital pharmacy clean room facilities. One model from this range, the Phileas Genius is a simple, stand-alone battery powered unit that generates vapourized hydrogen peroxide of particle size 5-10 µm from 7.4% solution using novel spinning disc technology. This generator can be transferred into and out of any closed isolator work zone through the transfer hatch (as long as the hatch door is wide enough) and can be used to do pre-programmed cycles (called zones) to routinely decontaminate inner surfaces.
The aim of this study is to validate this Phileas Genius hydrogen peroxide bio-decontamination process for use in pharmaceutical isolators in a hospital aseptic manufacturing unit operating under an MHRA Specials manufacturing licence. The accepted standard for validation of this type is demonstrating 6 spore Log Reduction (SLR) using Geobacillus stearothermophilus impregnated disc Biological Indicators (BI) placed throughout the isolator chamber.
Specifically the validation needs to show:
The H₂O₂ solution used in the process is able to produce a H₂O₂ vapour that is effective at achieving 6 spore log reduction inside the isolator.
The preprogramed parameters in the generator ‘zones’ (volume of H₂O₂ liquid diffused per cycle, delay time between diffusion cycles and contact time) are sufficient for decontamination and that there is a suitable margin for safety from the point at which the cycle would be sublethal.
There is diffusion of H₂O₂ throughout the isolator work zone to give assurance all internal surfaces of the critical work zone are in contact with the H₂O₂.
Two Phileas Genius hydrogen peroxide decontamination generators and associated equipment were bought. These initially underwent installation and operational qualification to make sure they were working correctly before the validation work started.
Devea recommended possible parameters for gassing cycles to be used based on the internal volume of the isolator being decontaminated and their own studies. This information was used as the basis for the cycles developed for routine use. They also supply a 7.4% Hydrogen peroxide solution (O₂SAFE®7.4) but for the purpose of the validation study dilutions of 30% Analar Grade reagent were used. It is the intention to routinely use the 7.4% proprietary solution from Devea for convenience and because it is BPR compliant. This will require additional validation
During the initial OQ and cycle development the temperature and hydrogen peroxide concentration in the isolator work zone were measured with a stand-alone thermometer and hydrogen peroxide sensor. Devea had provided assurances that the system would function satisfactorily above 10 degrees and this was found to be the case. The isolators for routine decontamination are all sited in clean rooms maintained at constant temperature of 18-20°. An issue that needs to be considered further is the maximum temperature of any surface to be decontaminated as above 25° there may not be condensation of the vapour that is required.
A cycle of six pulses of 10ml of 7.5% liquid to H₂O₂ vapour with a 5-minute interval between each (giving a 33 minute vapour production time ) followed by a 3 hour contact time was decided on. This was programmed into the Phileas Genius. The decontamination takes place with the isolator
turned off at ambient pressure and with the air inlet hatches locked closed so it is not relevant
whether it is routinely positive or negative pressure. It is leak tested beforehand to ensure its integrity is maintained throughout. Before any gassing takes place the inner surfaces of the isolator are cleaned following the normal schedule (daily wipe with sterile alcohol or monthly deep clean with detergent and sporicidal wipes).
Completion of proving runs
A 4 Glove positive pressure isolator was used for the validation runs. It was empty during the validation. 6Log Geobacillus stearothermophilus Biological Indicator (BI ) discs in Tyvek envelopes (figure 2) were placed in 7 positions in the isolator. This was carried out on two occasions, on the first 2 x BI were placed at each position and on the second 1 x BI was placed at each, meaning a total of 3 x BI were used at each position.
The Phileas Genius was aseptically transferred into the isolator via the hatch and placed in the centre of the isolator work zone. The isolator was prepared for decontamination and the cycles were
started. The complete cycles which included a 3 hour contact time at the end lasted approximately 4 hours. 2 x additional BI were restricted to a 1 hour and 2 hour contact time to try to show the cycles are robust and will still achieve 6Log decontamination even with a shorter contact time.
Equipment and materials and used:
Devea Phileas Genius Serial No: 02107240
Isomat 3 positive pressure isolator Serial No: 083041
100ml bag Tryptone Soya Broth, Cherwell Laboratories, Bn: 22070136 Exp: 01/10/22. Hydrogen Peroxide Analar Grade 30% Fisher Scientific Bn 2208788 Exp 24/08/24.
Sterile Water 500ml Baxter Bn (10) 22C07BA1B Exp 28/02/25 (For dilution of 30% Hydrogen Peroxide)
Geobacillus stearothermophilus Apex Biological Indicators, Mesa Labs Bn AH-156 Exp 31/03/23
Fig 2 Geobacillus stearothermophillus Biological Indicator (BI)
At the end of the contact time the BI and Phileas Genius were removed from the isolator via the hatch and isolator fans restarted to remove any residual Hydrogen Peroxide. A two hour aeration time is required before the isolator is used again (isolator has approximately 1750 Total air changes per hour)
The BI were aseptically transferred into sterile broth. These were incubated at 55° for 7 days. Cloudiness in the broth indicates failure in decontamination. (Figure 3)
A positive control was carried out by putting a BI disc that had not been exposed to the H₂O₂ into sterile broth. Cloudiness in this broth indicated that the broth and incubation conditions support the growth of viable Geobacillus stearothermophilus spores. This broth was used to inoculate the broth for the 7 isolator positions that had reached the end of its incubation time to confirm that these would have supported growth had there been viable Geobacillus stearothermophilus present.
Proving runs 1 and 2
Proving run 3
All of the validation aims listed have been achieved successfully.
The results confirm that the Devea Phileas Genius hydrogen peroxide decontamination system can achieve 6 Log decontamination in a pharmaceutical isolator when challenged with Geobacillus stearothermophilus BI.
When compared with the current well established alternative systems the Phileas is not as
sophisticated and advanced and requires more operator input during the process. The main feature lacking is a Hydrogen Peroxide sensor to record concentration in the isolator during the cycle. Not having this means relying on procedural checks at the filling of the liquid hydrogen peroxide reservoir before starting the cycle and that all the liquid has been used at the end. Chemical indicator strips are used to indicate presence of Hydrogen peroxide in the isolator but this is qualitative rather than quantitative. Bioquell chemical indicators have been suggested following the completion of the validation and will be tried for any further work. A standalone Hydrogen peroxide sensor has been tried with limited success. The USP suggests that H₂O₂ concentration in the isolator during a cycle does not necessarily relate to kill rate. Use of Enzyme Indicators to show cycle validity is the latest technology but the cost to validate this makes it prohibitive at this time.
A question also arises from the BIs that were used for validation. As indicated, Mesa Labs brand was used successfully. During the cycle development 2 other brands were tried but it was not possible to achieve SLR with these. It was observed that these were only killed when the steel disc was removed from its Tyvek envelope leading to the conclusion that the vapour produced by the Phileas Genius could not penetrate the particular Tyvek envelope these were in. It was only possible to investigate this because an isolator was available for use in a QA microbiology laboratory far removed from production areas. It would not be appropriate to remove BIs from their Tyvek envelopes in production isolators due to risk of contamination.
The Phileas Genius is portable and stand alone so can be used in any type of isolator provided the access is large enough, without any modifications to the isolator. This makes it a viable option for a lot of hospital pharmacy aseptic units. Potentially it could be put forward as a solution to the requirement in Annex 1 and could be used in existing isolators in a facility.
The Phileas Genius Hydrogen Peroxide decontamination system is suitable for use as part of the routine decontamination schedule in the isolators within the facility. It is now being used in different types of isolators without any physical modification to them and will be used in any future isolator being purchased.
Devea has provided technical advice and support on developing cycles for this study. They continue to be available for advice. All equipment and materials used were purchased by Pharmacy Dept WHSCT.
USP, Supplement 1, <1229.11>, “Vapor Phase Sterilization” USP 38 (US Pharmacopeial Convention, Rockville, MD, 2015)
Brian McBride QualityAssurance Pharmacist, Altnagelvin Hospital, Western Health and Social Care Trust, Derry, Northern Ireland