In the second article of this series, the development of robust sporicidal gassing cycles is discussed
Tim Coles BSc(Hons) MPhil
Managing Director, Pharminox Isolation Ltd, Cambridge, UK
The first article in this series described the vapour phase hydrogen peroxide sanitising process (VPHP), and the true action of the fast kill effect. The fundamental importance of micro-condensation was established, and the two golden rules of the VPHP process were set out. The next stage is to use that understanding to develop robust sporicidal gassing cycles. This may sound like a daunting venture but cycle development can be relatively simple.
The majority of VPHP users will be seeking the quickest possible cycle time, especially in production applications, where materials need to be brought into isolators for aseptic processing. A good example of this would be in hospital pharmacies.
As detailed in the first article, the measured parts per million (ppm) level of VPHP is not necessarily an indicator of a good gassing cycle. Our best indicator of a good cycle is a biological indicator (BI). If the gassing cycle can demonstrate the inactivation of a large population of resistant micro-organisms, then there is confidence that a gassed enclosure is ready for aseptic work. The spores of Geobacillus stearothermophilus (G. stearothermophilus) are resistant to the VPHP process. They are non-pathogenic and have the advantage of incubating at high temperature (55oC), so that other micro-organisms are unlikely to interfere with results. Furthermore, they are widely available for use with autoclave validation. To form the BI, a suspension of spores is placed on a small stainless steel disc. The volume of suspension deposited and the concentration of spores in that suspension are previously established, and the population of spores on the disc is known. The disc is then dried and placed inside a convenient Tyvek envelope, which allows gas to penetrate to the spores, but does not allow spores to escape.
The surfaces inside the enclosure, which are to be sanitised, vary greatly. They may include stainless steel with differing finishes, glass, and a wide selection of plastics and rubbers. It has been suggested that each surface presents a different challenge to the VPHP process and so each should be evaluated during gassing cycle development (GCD). However, the resulting workload would be prohibitive in most cases, and there is now a convention to use only stainless steel spore carriers.
Ideally, all of the BIs used during a cycle development should be from the same production batch. This is because the resistance of BIs can vary significantly from batch to batch, as seen from the D-value given by the manufacturer for each batch. If the same batch cannot be used throughout, then try to obtain BIs with a similar stated D-value.
Log 6 reduction
The often-stated target for the VPHP process is log 6 reduction. This means reduction by a factor of 106, or by one million-fold. Practical problems begin to arise with spore populations of more than a few million on spore carriers, so it is normal to use BIs with approximately 3–4×106 spores. A log 6 reduction on such a BI would, in theory, yield three or four surviving BIs. In practice, most operators will use BIs with this sort of population, but look for a total kill. In less rigorous applications, log 3 reduction has been used as the standard.
Some large pharma operators have taken the trouble to assess the wild flora remaining on the surfaces of the enclosure after cleaning but prior to gassing. Results suggest that the number of viable micro-organisms remaining is very low if the cleaning has been carried out effectively, and that log 6 reduction generally represents a very large safety margin over the real-world challenge.
Much more detail on the manufacture, selection and use of BIs for sporicidal gassing is available in the PDA monograph TR51 (2010).
VPHP generators and kill time
To sum up thus far, in GCD work, it is conventional to look for the complete kill of G. stearothermophilus spores on stainless steel carriers, inside Tyvek envelopes. The population of such carriers is likely to be between 2×106 and 5×106.
With most VPHP generators, there are a number of parameters that need to be set for each gassing cycle. It is beyond the scope of this article to describe each of these but, for the most part, the following settings are critical:
- The flow rate of the hydrogen peroxide solution during the gas build up or ‘conditioning’ phase
- The time length of the conditioning phase
- The flow rate of the hydrogen peroxide solution during the steady gassing or ‘sterilisation’ phase
- The time length of the sterilisation phase.
The manufacturer of the VPHP generator should provide clear guidelines to establish most of these settings, but, briefly, the logic for each phase is as follows.
The flow rate of peroxide during ‘conditioning’ is set high, in order to raise the concentration of gas as quickly as practical. For a typical 1m3 enclosure, this might be around 8ml/min. How should we establish this? Simply increase the flow rate until condensation becomes clearly visible, and then back off the flow rate by perhaps 1ml/min. The length of this conditioning phase is the time taken to achieve micro-condensation, that is, the time taken to arrive just short of visible, frank condensation. It should be approximately five minutes for a 1m3 enclosure.
The flow rate of peroxide during ‘sterilisation’ is set to hold the enclosure just short of visible condensation. This rate might be approximately one third of the value during conditioning. It can be established in the same way as the conditioning flow, by increasing by stages until condensation appears.
Determining the length of the sterilisation phase is the real target of sporicidal GCD. We need to determine how long it takes to completely inactivate our chosen BI, termed the ‘kill time’.
And finally, in setting up the gas generator, most users will opt for the maximum air/gas flow rate that the generator can deliver, because this will speed the process as far as possible. All of the above can be applied equally to the ‘conventional’ gas generators with dehumidification, and also to those without dehumidification.
The D-value is a term often used in the discussion of sterilising processes, particularly autoclaving. The D-value is the time taken to reduce the viable population of a BI by one log or, in other words, to reduce the population to one tenth of a given level. It is a useful measure of the resistance of a batch’s BIs, and it should be noted that resistance does vary quite widely from batch to batch. If the D-value is known for the BI, then the time taken to achieve log 6 reduction is simply six-times the D-value. If this were actually the case, then setting the sterilisation time would be a matter of reading the D-value off the manufacturer’s labelling and multiplying by six. Some operators do indeed use this as the basis for cycle development however, this method assumes that the killing process is constant over time and this is probably not the case with the VPHP process. It is perhaps better to use real tests to establish the kill time and use these to establish a robust gassing cycle.
Load patterns and gas penetration
The gassing process has to treat all of the surfaces present in the enclosure, not just the walls and floor, but also the materials, equipment and tools that may be loaded into it. These loads may well vary according to the work being carried out in the enclosure, both in terms of numbers of items and in terms of their materials. For this reason, it may be necessary to develop gassing cycles for each different load pattern. However, this can be quite a laborious process, and some operators take the view that a cycle should be developed for the heaviest load pattern, and then this should then be used for all loads.
Serious thought needs to be given to how loads should be presented during gassing. The VPHP process has limited penetrative properties so that loaded materials need to be as widely spaced as practical. In addition, the gas needs to be actively circulated by some means, such as a distribution nozzle or agitating fans. Ideally, all items should be suspended from hooks with minimum contact area, or at least placed on wire racks. Some operators even go so far as to move items during gassing, to avoid untreated occluded surface areas.
‘Worst case’ BI siting
Having prepared the enclosure and its load for cycle development, we need to consider where to place the BIs (Figure 1). The correct place for BIs is where the VPHP process is likely to be least effective, the ‘worst case’ sites. If challenging sites show total kill, then we may have confidence that a given cycle is effective throughout the enclosure and load. If a single site in the isolator could be shown to be the worst case, then a single BI would suffice for all cycle development, but in reality we can only roughly demonstrate worst case sites, and thus a spread of BIs around the isolator and its load will invariably be used. So, now we have to look for potential worst case BI sites.
Mapping and CIs
Perhaps the first step is to visualise the gas flow in the enclosure and carefully note any areas of poor circulation; this might be done by introducing dispersed oil particulate (DOP) smoke at the gas inlet point of the enclosure, while the gas generator is running with pure water, not peroxide solution. DOP smoke tends to coat the surfaces with oil, so a water mist generator is preferable. The visualisation might be recorded on video as part of the documentation process.
Since the VPHP process relies on the critical micro-condensation effect, local temperature and humidity are going to have an influence. It is sensible to map the temperature and humidity in the enclosure while the gas generator is again running on pure water. It is important to note clearly that, since we are seeking a condensation effect, areas of low humidity would represent worst-case sites, as would any areas of high temperature. This last sentence runs contrary to some earlier accounts of VPHP cycle development, stemming from the earlier misunderstanding of the process.
Small data temperature/humidity loggers are available to buy or hire. These can be set up to run for a given time and then downloaded to computer. The results can then be reviewed and any poor areas noted as challenging sites for BIs. Again, the results can be held as part of the documentation package.
At this point, we may note that cycle development is only valid for any length of time if the enclosure is stable in terms of temperature and humidity, which means the room housing the enclosure must be reasonably stable. In practice, most enclosures, such as pharmaceutical isolators, will be placed in controlled environments such as ISO 7 (Class C) cleanrooms. These are generally stable enough for cycles to be valid between re-validation exercises, described later.
Chemical indicators (CIs) are available for the VPHP process. These take the form of slips of paper that change colour when exposed to appropriate levels of hydrogen peroxide vapour (Figure 2). They are relatively inexpensive and are used to map the interior of the enclosure and the load. The strips are taped at a number of locations on the walls of the enclosure, in the load and under the gauntlets or sleeves, at sites that are visible when the isolator is closed down. Gas is then applied and the CIs observed, noting any that change most slowly as worst-case sites for BIs. The gas generator settings at this stage will, out of necessity, be ‘guesstimates’. Many operators will add the exposed CIs to the documentation file to demonstrate that the work was carried out effectively.
Data gathered from visualisation, temperature and relative humidity
(T/RH) mapping and CIs can be reviewed and any worst-case sites identified as places to position the BIs. Experience, however, indicates that there tend to be few obvious worst-case sites and sites are chosen intuitively. These would include apparently inaccessible corners, deep in racks of loaded materials, underneath gauntlets, or at the end of RTP containers.
How many sites?
How many BI sites are needed to demonstrate a robust cycle? Some developers will place large numbers of BIs but just how much do conditions vary over short distances within an enclosure? As a rough guide for a 1m3 four-glove isolator, approximately 12 sites in the top and bottom corners, in the middle of the walls and floor, and under the gauntlets or sleeves should be sufficient. A further five or so placed within the load would
How many BIs?
The next decision is the number of BIs to place at each chosen site. Every BI costs approximately £6 (€7) and when several hundred might be used for the development, costs can mount considerably. However, there is a distinct advantage in placing more than one BI at each site.
Put quite simply, more data are generated to support the developed cycle. To use an analogy from electronic transmission, there is a better signal-to-noise ratio. In purely statistical terms, if two out of three BIs at one site are killed, then log 6 reduction has been achieved. A good compromise is to use two BIs at each site (Figure 3).
It is simple to number each pair of BIs using an indelible marker, writing on the end of the tyvek envelope that is away from the suspension hook hole. If hooks are available in the enclosure, the BIs should be hung, but in most cases they will need to be attached, ideally with autoclave tape.
After each trial gassing cycle, the enclosure must be purged down to safe VPHP level. If the enclosure is to be opened up for harvesting the BIs, then 1 ppm (eight-hour occupational exposure limit; OEL) is the safe level (see later). The may be taken to a Class 2 safety cabinet and opened up to drop the stainless steel coupon down into a numbered media tube, preferably within one hour of harvest.
If the BIs can be harvested without opening the enclosure, then 8ppm (one-hour OEL) is suitable. In this case, we may choose to place the media tubes in the enclosure and open the envelopes via the gauntlets or glove-sleeves, but this makes for slow work.
The BIs should be incubated according to the manufacturer’s instructions, probably at 55oC. If growth is going to take place, it is usually apparent as clouding of the media after just 24 hours; however, the tubes should be finally read at seven days. Positive and negative controls should be run with each cycle.
Finding the kill time and the developed cycle
There are a number of ways in which the kill time can be established.
In one method, BIs are removed at regular intervals during a long cycle and incubated. From the growth/no growth data, it is possible to deduce the time after which all the spores were killed. However, the time interval will be quite short in smaller enclosures, making harvesting difficult, and a way has to be devised of getting the BIs out of exposure during the cycle. A skilled operator is required to enable this to be carried
Another method is to remove BIs during the cycle and enumerate them to arrive at the D-value. Then the indicated time for log 6 reduction is six times this value, as discussed previously. This again requires skilled operators.
Perhaps the simplest method, the safest, and the one that requires least training, is as follows:
- Seek the advice of the gas generator manufacturer and/or other workers in this field such as isolator manufacturers or consultants. They will certainly be able to suggest a sterilisation phase time for your application, based on extensive experience. This might be approximately ten minutes for a 1m3 enclosure
- Run this cycle with BIs and incubate
- If none grow, run a cycle with half the previous sterilisation phase time
- If some grow, run a cycle with 50% more sterilisation phase time
- Repeat cycles with more or less sterilisation time until the kill time is established to within perhaps five minutes accuracy.
In practice, the kill time can usually be established with approximately eight test cycles. Indeed, operators may choose to run a series of back-to-back cycles with say four, six, eight and ten minutes of sterilisation time, and then incubate. Having checked the results of these cycles, a further back-to-back series of similar cycles, perhaps five, seven and nine minutes, can be run to get closer to the kill time. There is clearly no need to wait for one cycle to incubate before running another.
Having established the kill time, it is suggested that a further safety margin be added for the actual cycle that will be used in production – the ‘developed cycle’. A suitable margin might be a further 25–50% to be added onto the sterilisation phase time.
Aeration or purge time
As part of development of any gassing cycle, it is prudent to establish the time needed to aerate or purge the enclosure down to a safe level. If the enclosure is to be opened up, perhaps to a clean room following a materials’ transfer cycle, then 1ppm is the safe level. This is the acknowledged eight-hour OEL for VPHP.
If, on the other hand, processing is to take place after gassing in the enclosure, then a higher level may be acceptable. A suggested higher level is 8ppm, this being the one-hour OEL for VPHP.
The concentration of VPHP can be measured with indicator tubes (for example, Draeger tubes) or with an electronic instrument, such as an ATI IsoMon device.
The aeration phase is invariably the longest phase of VPHP gassing because the peroxide absorbs more-or-less into the materials of the enclosure and its load. Thus, a high airflow rate is desirable to minimise the purge time. Many isolators will have an airflow rate greater than that of the gas generator and so it is a good plan to switch over to isolator-driven ventilation as soon as it is practical.
Having established the aeration time need to achieve the defined safe level, a further margin, maybe 50%, might be added for extra personnel security in production.
As a final test of the robustness of the developed gassing cycle, it is suggested that three complete back-to-back gassing cycles be carried out. This might be termed the performance qualification (PQ) of the development programme and documented accordingly.
We would expect the three cycles to show no growth of any BI; however, there is a potential problem for which those concerned with the VPHP process should be aware. This is the problem of the so-called ‘rogue’ BI. It is quite difficult to produce BIs with an even spread of spores and no residual material. The spores can tend to form clumps and aggregates in even the best-produced BI. In this case, clumps of spores may occlude the centre and some might survive even the longest gassing cycle. Such BIs are termed ‘rogues’. There is evidence to suggest that 0.3% of BIs are rogues. If your PQ cycles total, for example 300 BIs, then statistically at least one will grow, however robust the cycle.
This means that a clear policy needs to be put in place to deal with rogues before the PQ process starts. A suitable rogue policy might state:
“A single BI failure at any one location across three repeat cycles will be considered to be a ‘rogue’ and, provided that the remaining five BIs from the same location across the three repeat cycles do not fail, the three cycles shall be deemed valid. BIs will be used in duplicate at all site locations. (All BI failures may be subject to review by the validation team).”
Given this kind of policy, the regulatory authorities should accept the validity of the gassing cycles in the face of the occasional positive BI.
From time to time, many users will re-qualify the gassing cycle to maintain confidence. The simplest solution is to re-run the PQ protocol, ideally obtaining BIs with similar D-values to those used during the previous PQ.
If BIs with longer D-value times have to be used, it may be prudent to re-run part of the development process with a set of suitable sterilisation phase times around those of the developed cycle. This should be followed by the suggested three-cycle PQ for confirmation.
Developing robust gassing cycles for the VPHP process does not need to be very complex, or very lengthy, and can be carried out, at least in part, by lab technicians, if required.
- VPHP gassing cycles need to be verified, and this is carried out by a development process using resistant spores or biological indicators (BIs) as a challenge.
- The first step of the cycle development process is to establish the ’worst-case’ sites for the BIs in the isolator, and its load, using visualisation, temperature and humidity mapping and chemical indicators.
- Other methods are noted but the ‘kill time’ method is described, this being a simple and safe way to develop a robust gassing cycle.
- The appropriate number of BIs for cycle development and the issue of duplicate and triplicate BIs is discussed.
- The safe aeration or purging of the isolator after gassing should also be established as part of the gassing cycle development process.