Vapour-phase Hydrogen Peroxide Systems

Hydrogen Peroxide Vapour (HPV) or Vapour-phase Hydrogen Peroxide Systems (VHP) as it’s more commonly known, has been used for many years as a contactless method of decontaminating cleanrooms and hospitals. It has also seen use most recently in indoor public spaces of known virus or bacteria contaminated sites such as the recent outbreaks of Covid-19 and even Anthrax bacteria. VHP systems also often built into isolators of aseptically prepared pharmaceuticals and pass-through chambers, as part of the equipment cleaning regime.

In addition to facility and equipment decontamination, the process has been developed as a terminal sterilisation technique for components and medical devices.

In these examples, there are several advantages that hydrogen peroxide has over other chemicals for these applications:

  1. Following the exposure phase, hydrogen peroxide breaks down to oxygen and water during the aeration/purging phase over a relatively short period leaving no harmful residues and is therefore a convenient an environmentally friendly choice.
  2. The VHP process uses much lower quantities of chemicals to achieve an equivalent decontamination or sterilisation than for other chemicals.
  3. Hydrogen peroxide vapour has little corrosive impact on most materials by virtue of the very low quantities needed, however you should risk assess this with your equipment or service provider with respect to any potentially sensitive materials or equipment. For such cases, the equipment maybe simply protected or removed from the area as necessary.

Process Considerations

Whether VHP is used for decontamination or for terminal sterilisation, the processes follow similar phases:

Figure 1: Typical Vapour-phase Hydrogen Peroxide Cycle

Phase 1: Pre-conditions the space. This maybe to establish a particular humidity and/or temperature. In many cases this phase can be eliminated.

Phase 2: Hydrogen peroxide vapour is generated and injected into the space until the required concentration is achieved.

Phase 3: Exposure hold time to achieve the required kill rate.

Phase 4: Aeration either by natural or forced ventilation

Typically 35% hydrogen peroxide solution is used to generate a vapour concentration of around 150 to 400ppm.

A related process used for room decontamination known as Ionised Hydrogen Peroxide (IHP) uses a lower concentration of around 7% hydrogen peroxide solution. This has the advantage that it can be utilised in facilities where sensitive electronics are in use that would otherwise be unsuitable for exposure to VHP concentrations. The cold plasma process produces a finer mist than that of VHP that disperses easily throughout facilities.

Humidity is a debated subject in combination with H2O2 concentration and temperature for the pre-conditioning step. Some suggest that low humidity atmospheres are better because they perform closer towards a “dry cycle” where the hydrogen peroxide remains predominantly in the vapour/gaseous state and may therefore have better penetration effectiveness. Others suggest higher humidity conditions favour micro-condensation of the sterilant on surfaces which provides the most effective microbial inactivation. The debate is complex because the interrelationship between relative humidity and sterilant concentration can yield similar results i.e. a high sterilant concentration in a low humidity environment may achieve a similar inactivation performance as a low sterilant concentration in a higher humidity environment.

One technical paper[1] published by the ISPE in 2008, presented results of experiments performed in a typical barrier isolator with varying combinations of humidity and sterilant concentrations using Biological Indicators to assess inactivation efficacy. On the basis of results from this paper, it shows that higher humidity environments showed the most efficacious inactivation at all sterilant concentrations based on D-value (time in minutes to achieve a 1 log / 90% inactivation). At low humidity levels with high sterilant concentrations, similar D-values were achieved but under the same low humidity conditions in combination with lower sterilant concentrations, the D-values were significantly longer. This suggests that the higher humidity environments are preferable for the majority of cases which promotes micro-condensation and allows more flexibility in the H2O2 concentrations necessary to achieve effective microbial inactivation. However, if a “dry process” is deemed necessary for a given situation, a high sterilant concentration in combination with low humidity can provide equally efficacious conditions.

Validation & Process Qualification

Whether VHP is used for a facility / equipment decontamination or for terminal sterilisation, the methods used for validating and qualifying the effectiveness of the VHP process are similar.

A risk assessment should be performed to determine the “worst-case” locations such as furthest from the VHP generator or injection point and recesses that can be foreseen to have low penetration of the vapour. These locations should be mapped in the qualification documentation and used as labelled sites for indicators.

Biological Indicators (BI’s)

For any sterilisation process, the most resistant indicator organism (spore) should be used to validate the lethality of the process. For hydrogen peroxide processes, Geobacillus Stearothermophilus is used. The BI should be positioned in such a way that the spore coated surface is facing outwards and not shadowed.

Biological Indicators must be carefully selected for compatibility with VHP processes. Cellulosic BI strips are known to absorb hydrogen peroxide and are therefore unsuitable. Also, physical attributes of the BI may steer the selection e.g. for small intricate spaces, it may not be possible to fit a disc type BI, so a ribbon type of BI maybe needed.

As mentioned earlier, VHP is a contactless surface process and cannot penetrate surface particulates or residues. If viable organisms exist under these contaminants, the VHP will not be capable of sterilising these areas. The same applies to the BIs themselves. If the spores on the BI are “clumped” (agglomerated) there maybe survivors under the exposed spores that later show growth in the culture media giving a “false positive”. A similar result might be seen if a BI is incorrectly handled i.e. oils from the skin provide a temporary protective barrier to the hydrogen peroxide vapour which later breaks down in the growth media revealing the active spores.

The UK’s GMP inspectorate; the MHRA have publicly[2] raised concerns over the “fragility” of VHP for use as a sterilisation process of Direct and Indirect surfaces with particular respect to the risk that BIs can give a false indications sometime referred to as “rogue BIs”. PIC/S PI 014-3 (Isolators Used for Aseptic Processing and Sterility Testing) also makes the point that with single BIs, we do not know if a positive result represents one surviving spore or a much larger number. For these reason, the accepted practice is to place replicate BIs (typically three) in each location to allow us to calculate the most probable number (MPN) of surviving spores[3]. This number can be used along with the initial spore population on a non-exposed BI to determine the spore log reduction (SLR).

For guidance [4]:

A log 6 reduction is considered ‘sterilising’ (kills/inactivates all microorganisms and spores).

A log 4 reduction is considered ‘disinfecting’ (kills/inactivates all microorganisms but not spores).

A log 2 reduction is deemed to be ‘sanitising’ (reduces but does not eliminate microorganisms).

Chemical Indicators (CI’s)

Chemical Indicators provide an instantaneous visual indication of exposure to hydrogen peroxide vapour by way of a colour change. This allows the validation and Quality Assurance personnel an immediate assurance and high degree of confidence that the process has performed as expected and the opportunity to release the facility for use (“at-risk”) pending incubation of BI’s.

Since Chemical Indicators are very cost effective and provide immediate feedback, they are very useful in preliminary studies for cycle development in ensuring good distribution of vapour prior to validation with BIs.

It should be noted that interpretation of CI results is based on colour change. Therefore care must be taken to be sure that a partial colour change is not interpreted as a successful result.

Enzyme Indicators (EI’s)

Enzyme Indicators are a relative new form of biological test but in contrast to BIs, they provide a better assessment of cycle performance because they produce a quantitative response rather than a binary response.

EIs work on the basis of luminescence of a protein called thermostable Adenylate Kynase (tAK) and utilise a reader to give an instantaneous quantitative response (log reduction reading) without the need to incubate the indicator coupon in growth media as required with BIs. This alone makes EIs highly desirable for routine production use such as isolator VHP cycles or VHP sterilisation by allowing immediate assessment of cycles without the delay of a 7-day incubation period.

When used for cycle validation, EIs used alongside BIs will give immediate results and can be used to rule out the “rogue BI” scenario discussed above.


Facility Decontamination

Contactless facility decontamination using vapour-phase hydrogen peroxide can be used in a number of scenarios:

  • as an initial decontamination of a new or refurbished facility following traditional manual surface cleans
  • used between campaigns of different products to ensure inactivation of any active products
  • routinely performed as a contamination control measure most commonly at the end of an annual shutdown period.

It is important to understand that the VHP process is a microbial decontamination/inactivation process and not a particulate clean. This may seem an obvious statement but if used in conjunction with manual cleans, the VHP decontamination should be the final step performed on particle-free, residue-free surfaces and cannot penetrate any such contaminants.

An advantage VHP decontamination has over traditional manual sporicidal cleans is that the vapour can be targeted at hard or impossible to reach locations providing the vapour can be reach these locations. Furthermore, infrastructure can be installed in facilities and HVAC systems to make VHP decontamination a simple plug and play procedure that maybe configured to incorporate the entire HVAC systems. This is a highly effective safeguard against the potential growth of mould spores that may form over time in ductwork and onwards into cleanrooms as a result of condensation carry-over downstream of cooling coils.


When considering VHP decontamination of a facility for the first time, a thorough safety risk assessment should be performed with the service provider or experienced personnel to ensure the process is performed in a safe and controlled manner.

The risk assessment should consider:

  • Containment of the facility during the procedure with regards to sealing doorways (gaps around doors), room penetrations etc.
  • HVAC systems – should they be switched off and grilles/vents sealed. If left open or running, fully understand whether the systems recirculate are common to other areas. Also consider the air pressure regime – if the area is positive pressure, consider whether adjacent rooms should be unoccupied during the procedure e.g. performed out of hours.
  • Signage and communication are essential to ensure that all personnel are aware that the procedure is taking place, the hazard it presents and who they can contact if they have any questions or concerns.
  • Any personal protective equipment (PPE) requirements for personnel performing the procedure
  • Gas detection methods at the boundary at the start of the procedure to verify that effective containment has been achieved
  • How will the space be aerated following exposure; either by natural aeration or forced ventilation (any special modes of operation needed on the HVAC system to facilitate this i.e. extract only, once-through or normal recirculation). Verification of the end-point for aeration with gas detection e.g. based on an in-situ gas monitoring system or using hand-held gas monitoring devices with personnel wearing breathing apparatus.

Process Equipment Decontamination

VHP decontamination systems can be built into process equipment enclosures for aseptic processes and laboratory applications such as barrier isolators for fill-finish lines or sterility testing as well as pass-through chambers. For such systems the four phases of the cycle must be qualified for both efficacy and safety.

It is recommended that any facility that has processes with integral VHP system installed has an independent safety system to monitor the ambient air in the workspace surrounding the equipment for personnel safety.

Terminal Sterilisation of Medical Devices

There are several chemical-based sterilants that can be used to terminally sterilise products and medical devices. Ethylene oxide, nitrogen dioxide, chlorine dioxide and formaldehyde. These other chemicals have more onerous environmental, toxic (carcinogenic) and even explosion hazards associated with them. Furthermore, much lower quantities of hydrogen peroxide are required to achieve microbial inactivation compared with these more hazardous alternatives.

Terminal sterilisation with vapour-phase hydrogen peroxide uses a chamber similar to that of an autoclave. The process follows the same basic four-phase cycle as shown in Figure. 1 above. In this process, the pre-conditioning step draws deep vacuum in the chamber before introducing the hydrogen peroxide vapour. With the absence of air (and therefore humidity), under full vacuum conditions, the mode of microbial inactivation is entirely through exposure to the sterilant in its vapour/gaseous state and can therefore be considered a “dry process”. This is particularly important for terminal sterilisation of medical devices. Penetration of the sterilant maybe challenging with more intricate device geometries, therefore maintaining the sterilant in its vapour/gaseous phase improves the mobility of the sterilant to contact these surfaces. Nevertheless, it is still vitally important that the worst-case locations are qualified through correct placement of appropriate indicators during cycle validation.


The versatility and relative safety of vapour-phase hydrogen peroxide make it an attractive application for both facility and equipment decontamination as well as a viable (pun intended) alternative to heat sterilisation methods where products, components or devices may not be thermally tolerant.

The challenge to the application of VHP has been in qualification with opportunity for error in results from biological indicators. The recent advent of Enzyme Indicators shows a great deal of promise in making the qualification process more robust and effectively provides results in real-time.


[1] The Influence of Humidity, Hydrogen Peroxide Concentration, and Condensation on the Inactivation of Geobacillus stearothermophilus Spores with Hydrogen Peroxide Vapor, by Beatriz Unger-Bimczok, Volker Kottke, Christian Hertel & Johannes Rauschnabel; published 08 May 2008, International Society for Pharmaceutical Engineering
[2] VHP (Vapour Hydrogen Peroxide) Fragility, posted by:  Andrew Hopkins, 20 April 2018;
[3] Using replicate BIs to evaluate biodecontamination cycles in isolators, by Garrett Krushefski; MesaLabs Spore News Vol 9, No. 4.
[4] Rapid Decontamination Systems, by Brett Cole, Bio-Safety Pty Ltd, presented at the PharmOut National GMP & Validation Forum, 11 July 2016

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