Cycle development for hydrogen peroxide clean room decontamination

As part of a major project to design and build a new bulk vaccine antigen plant, the authors were asked to investigate and implement a suitable fumigation system for clean room decontamination. The facility was designed to handle and contain live influenza virus, and has clean room suites designed to containment levels CL2 and CL3 according to the Approved Code Of Practice and Guidance (ACOP, Control of Substances Hazardous to Health 4th Edition). From the outset, specific areas within the facility were identified as requiring fumigation and this formed part of the initial design brief.

Vapourized hydrogen peroxide (VHP) was selected as the fumigant of choice for a variety of reasons:

• The existing manufacturing site already has experience of using VHP within filling/finish and sterility testing isolators.

• It readily degrades into water and oxygen.

• It is effective as a surface sanitant and residue-free agent.

• It is compatible with a wide range of clean room materials.

• It is effective against a wide range of bacteria and viruses, including methicillin-resistant Staphylococcus aureus (MRSA), moulds and Mycoplasma.²

The design criteria used for selecting the fumigation system were that it should be effective, robust, easy to operate and cost-effective.

HVAC system design

Each manufacturing suite that required fumigation was designed to be served by a dedicated heating ventilation and air conditioning (HVAC) system so the suites could be fumigated independently of each other. The uniqueness of the system is that the high efficiency particulate air (HEPA) filters are on the extract system and can be safely changed.

These were located on the extract ductwork in the service area approximately 10 m from the manufacturing laboratory ceiling along with a bypass fan (B) as detailed in Figure 1.

Figure 1

To set the HVAC system for fumigation, a key switch can be activated, which shuts down the supply fan (A), turns off the extract fan (F), starts the bypass fan (B), opens the isolation damper (D), and closes the dampers (C and E), thus allowing fumigant to be circulated from the manufacturing suite up through the ductwork and safe-change HEPA filters, and return into the manufacturing area through the terminal supply HEPA filters, located in the ceiling. This is known as a closed loop recirculation system.

The generator

VHP was introduced into the manufacturing suite using a proprietary mobile generator located in an adjacent corridor (Figure 2). A false door was fabricated to fit into a pass-through hatch. This false door was fitted with camlock fittings to accept supply and return hose connections from the VHP generator. A sealable flap was included to allow sampling of the air in the room during fumigation using a Draegar tube.

Figure 2

The vapour in the return hose (from the room back to the VHP generator) circulates through a catalytic converter that converts hydrogen peroxide gas into water vapour and oxygen. In this way, fumigant is continually circulated from the generator into the room, through the extract ductwork and the safe-change HEPA filters back down the ductwork and into the room via the terminal HEPA filter. Finally returning into the generator, where the VHP is broken down into water and oxygen.

Cycle development studies

Figure 3 shows the layout of the manufacturing suite and associated lobbies. The room contained a Class II microbiological safety cabinet (MBSC), and unidirectional airflow (UDAF) cabinet, two incubators, a fridge, two cupboards and two stainless steel tables. All material and surface finishes in the room were compatible with VHP. The total room volume including lobbies was 122 m³ . This was well within the capacity of the generator (capable of fumigating a volume of approximately 200 m³ ).

Figure 3

In total, six trials were performed on the manufacturing suite, three of which were used for optimizing the cycle parameters and the other three for confirmation to demonstrate reproducibility of the selected cycle.

The VHP generator has four distinct phases for a typical fumigation cycle, each of which can be adjusted on the generator.

Dehumidification. This is the first phase in the fumigation cycle. During this phase HEPA-filtered air is circulated from the room through the generator and dried over a silica gel bed to a preset humidity. This phase takes approximately 20 min.

Conditioning. Sometimes called the 'gassing' phase. During this part of the cycle, VHP was injected into the room at a preset injection rate (10.1 g/min). This allows the concentration of VHP to be rapidly increased within the room. This phase normally takes 30 min.

Decontamination. Sometimes referred to as the 'dwell' phase. During this phase a constant flow of VHP is maintained at a specific injection rate and for a predetermined time (7.9 g/min for 3 h). During this phase, biological indicators (BIs) are used to demonstrate microbiological kill.

Aeration. The longest phase in the cycle (approximately 5 h). At the beginning of this phase the injection of VHP into the room is stopped, air is circulated back through the generator where the catalyst breaks down the VHP into water and oxygen. The aeration phase reduces the concentration of VHP to below 1 part per million (ppm).

The study was executed under a technical protocol, which outlined the scope and purpose of the study, the cycle parameters, acceptance criteria, testing requirements, and detailed instructions and methodology for operation of the VHP generator.

Trial prerequisites

Some key documents were implemented before the first trial began:

• A risk assessment document to detail all the health and safety controls that needed to be in place before starting the cycle.

• A draft operating procedure (OP) for using the VHP generator. This was amended based on the experience gained during the trials to produce a confirmed workable procedure.

• A control of substances hazardous to health (COSHH) assessment document. This detailed each operation that may result in operator exposure to VHP, either in the liquid or gaseous form, the likely duration of exposure, the frequency of exposure and the current exposure control measures, and relevant personal protective equipment (PPE) to be used.

In addition, leak testing of ductwork was performed on the HVAC system to ensure no leakage of VHP from ductwork into the service area, and finally room fabric leak testing was performed to ensure integrity of the rooms.

Chemical and biological mapping

Studies by Rickloff and Orelski have shown that GeoBacillus stearothermophilus spores are the most resistant to VHP.³ Figure 3 shows the layout of the manufacturing suite to be fumigated. The area consists of five clean rooms, two room pass-through hatches, and an MBSC/UDAF cabinet with associated pass-through hatch.

The manufacturing suite and adjacent lobbies were pre-assessed for placement of biological indicators (BIs) and chemical indicators (CIs). CIs were used to evaluate the distribution of gas in the rooms and ductwork, and BIs were used to indicate kill of the microorganism GeoBacillus stearothermophilus.

The ceiling, floors, room corners, doorways, MBSC/UDAF, inside cupboards, incubators and refrigerator, pre- and post-extract HEPA filter were selected for BI and CI locations. On average, 280 of both chemical and biological indicators were used for each trial.

Using such a large number of BIs and CIs allowed the worst case positions to be assessed and to establish the effectiveness of VHP distribution within the rooms.

Specific training was given on the handling of BIs to avoid contamination of BIs if not handled correctly.

An important feature to note was the configuration of the BI. The organism is actually located on a stainless steel concave disc, which is sealed inside a Tyvec pouch. It is, therefore, possible that false positive results could be obtained if part of the pouch was not exposed correctly; for example, if they are placed flat against a wall. Therefore, it was essential that the BIs were placed with the concave side facing the direction of flow of the fumigant, that the tape used to hang the BIs did not cover the stainless steel disc and that space was left as far as possible for the fumigant to get behind the spore strip.

Collection and testing of the BIs proved to be a time consuming and laborious task. First, BIs had to be handled using latex gloves, then each one was placed carefully inside a screw-cap jar, which was then placed into a labelled plastic bag.

All BIs had to be retrieved, transferred to the microbiology department and tested within 4 h.

Logistically, this was difficult because of the number and location of BIs used and presented a challenge to get this number of BIs on test within the 4 h. In hindsight, the number of BIs could have been reduced to a more manageable number and for future trials the number of BIs has been reduced whilst ensuring critical areas are mapped.

Collection and visual inspection of CIs were less challenging. No issues were observed with handling CIs. They were transferred to a plastic bag, labelled and sealed. Colour change from dark blue to light blue/beige was observed on all CIs, indicating good gas distribution throughout the room.

Key points

Results

Table 1 summarizes the cycle parameters and BI/CI results for all six trials performed during this investigation. Initial operational parameters were selected after consultation with the generator equipment supplier, and review of the relevant equipment operation and maintenance manuals. For the first trial, the room was not fully equipped, the MBSC and UDAF were not running and the inlet hose was placed in the centre of the room suite.

Table 1

The outlet hose was placed in the change-in lobby because this is the furthest point from the generator, thus ensuring maximum distribution of gas.

Trial 1. BI results for Trial 1 showed 30 BI failures, of which 10 were located in the MBSC/UDAF assembly. The other 20 were located around the room itself. All CIs changed colour during this trial (i.e., all were positive results).

Trial 2. The MBSC/UDAF were switched on with the sashes in the operating position to obtain greater airflow through the units. To ensure a greater amount of hydrogen peroxide in the room, the injection rate during conditioning phase was increased from 9.4 to 10.1 g/min, and during the decontamination phase from 7.2 to 7.9 g/min.

BI results for Trial 2 showed 18 BI failures, 16 of which were located in the MBSC/UDAF assembly, despite the units being turned on. This meant the increased injection rate was having an effect on the BIs in the room, but not in the MBSC/UDAF units.

Trial 3. Cycle parameters were the same as for Trial 2 except the sashes were in the 'up' position to further increase the airflow to the MBSC/UDAF units. Results showed 23 BI failures, 16 of which were in the MBSC/UDAF, one on the laboratory ceiling, three in lobby 1 and three in change out room. All CIs changed colour. During this trial, condensation was observed on the floor by the VHP inlet pipe, which was on the floor.

Trial 4. It was evident from Trials 2 and 3 results that while getting good distribution of gas (seen from CI results), adequate BI kill was not achieved in the MBSC/UDAF unit. Thus, for Trial 4 we decided to increase the cycle decontamination hold time from 2 to 3 h, and decrease the velocity in the UDAF from 0.45 to 0.02 m/s, thus increasing the residence time for VHP in the unit.

The unwanted condensation observed in Trial 3 was a result of the length of pipe used and the fact that it was resting on the cold floor. Therefore, for Trial 4 the configuration of the inlet and supply hose was adjusted to ensure maximum gas distribution. The inlet hose was shortened to about 0.5 m from the generator outlet and raised slightly from the surface to ensure gas was immediately injected into the room. This solution overcame the problem of condensation and was not observed after Trial 3.

Results from Trial 4 were much better with only one BI failure observed out of a total of 283. This was located in the UDAF, position 58, which was on the back of the UDAF at the top right hand side. No CI failures were observed.

Trial 5. This was a repeat of Trial 4. Again, only one BI out of 283 failed, this being located in position 41, which was on the base of the MBSC at the front right hand side. No CI failures were observed.

Trial 6. Trial 6 was a repeat of Trials 4 and 5, but with a reduced number of BIs. The same number of BIs in the MBSC and UDAF were used, but with a reduced number used in the manufacturing suite. All BIs were inactivated during this trial. Again there were no CI failures.

Conclusions

The investigation outlined in this article has met the original protocol design criteria which was to establish an effective, robust, easy to operate and cost-effective system. The VHP fumigation procedure is simple to operate and the generator easily transferred to other clean rooms. BI and CI results have shown the effectiveness of the VHP as a fumigant, with repeatable results obtained over three consecutive trials. PTE

References

1. Control of Substances Hazardous to Health Regulations 2002, Approved Code of Practice Guidlines schedule 3 Part II, www.opsi.gov.uk

2. N.A. Klapes, "New Applications of Chemical germicides: hydrogen peroxide", in Program and Abstracts of the ASM International Symposium on Chemical Germicides. (American Society for MicroBiology, Washington, DC, USA, 1990) , abstract 20, p 14–15.

3. J. Rickloff and P. Orelski, "Resistance of various microorganisms to vapour phase hydrogen peroxide in a prototype dental hand piece/general instrument sterilizer", in Abstracts of the 89th Annual Meeting of the American Society for Microbiology (American Society for Microbiology, Washington, DC, USA, 1989), abstract Q-59, p 339.

Carmel Clare is manager documentation Site 4 at Novartis Vaccines Diagnostics (UK). She has spent more than 15 years in pharmaceutical research and manufacturing including drug delevopment and validation, quality and technical support functions within pharmaceutical manufacturing.

Howard Preece is a validation contractor currently working at Novartis Vaccines Diagnostics (UK). He has spent more than 30 years in pharmaceutical manufacturing including validation, technical and commissioning activities in both API and finished product manufacture.