No, hydrogen peroxide vapour bio-decontamination and sterilisation are not the same. Sterilisation is typically a requirement for medical devices to ensure they are sterile, whereas bio-decontamination targets surfaces of enclosures where aseptic processing is performed.

Although both processes operate on the same mechanism of action i.e. oxidation, the difference is that sterilisation is a process that absolutely eliminates every single microorganism. As it is not possible to prove the absence of something, sterilisation cannot truly be proven so sterility assurance levels (SALs) are used as a means to express the probability that you will have a live organism after the sterilisation process. Typically, a sterilisation process will claim a SAL of 10-6. Bio-Decontamination, on the other hand, reduces microbial contamination to a pre-determined acceptable level. In the case of Ecolab’s Bioquell Hydrogen Peroxide Vapour technology a 6-log sporicidal kill will be achieved and validated with biological indicators.

The borderline between hydrogen peroxide sterilisation and bio-decontamination is often blurred due to both processes commonly using a 6-log Geobacillus stearothermophilus biological indicator as the means of validation. However, in sterilisation the biological indicator usually provides a SAL through a half-cycle overkill approach and with bio-decontamination the biological indicator is used to demonstrate a 6-log spore kill.

Ecolab does not recommend using triplicate BIs for gassing cycle development (GCD) of performance qualification (PQ) as this increases cost and adds little benefit. In a scenario where three BIs are placed at each location and only one were to exhibit growth, this does not necessarily prove that the BI was at fault. The only scenario when using triplicate BIs is recommended by Ecolab is when performing an investigation cycle in response to an unexpected BI growth during GCD or PQ, to determine whether the unexpected growth was a rogue result or a genuine cycle failure.

The primary difference between Hydrogen Peroxide Vapour (HPV) and fog lies in their physical states and application methods. HPV is a true vapour, the Hydrogen Peroxide molecules are mixed with the air and water molecules. As such, it will distribute evenly within an enclosure and when a sufficient amount has been introduced to reach saturation conditions, the hydrogen peroxide will form a fine layer of micro-condensate on exposed surfaces, making it effective for bio-decontaminating enclosed spaces. In contrast, fogging systems work by passing a liquid solution of hydrogen peroxide through a pressurized nozzle or other processes which produce fine aerosol droplets. Crucially, the hydrogen peroxide droplets are still in the liquid phase meaning that they can be affected by gravity, which can result in uneven distribution on surfaces. Some fogging systems also mix other antimicrobial agents with the hydrogen peroxide such as peracetic acid and silver nitrate, which are both hard to measure and can leave residues on surfaces.

It is an isolator that is designed for use in aseptic processing. EU GMP Annex 1 defines an Isolator as “An enclosure capable of being subject to reproducible interior bio-decontamination, with an internal work zone meeting grade A conditions that provides uncompromised, continuous isolation of its interior from the external environment.” Isolators use gloves or robotic arms to manipulate products inside without direct human contact to minimise the risk of contamination entering the critical process/product. In the United States, the term “Aseptic isolator” is often associated with Compounding Aseptic Isolators (CAI) and Compounding Aseptic Containment Isolators (CACI). However, these barrier systems are not isolators, but Restricted Access Barrier Systems (RABS). Further information on the classification of CAI, CACI and Pharmaceutical isolators can be found in USP 797.

Log reduction (“log” is short for logarithm) is a numerical expression of the effectiveness of a bio-decontamination procedure. In the context of sporicidal bio-decontamination a 4-log reduction represents killing 10,000 spores, and a 6-log reduction represents killing 1,000,000 spores. Validation of a log reduction typically involves using biological indicators with known concentrations of microorganisms and demonstrating that the bio-decontamination process consistently achieves the required reduction. Ecolab’s Bioquell hydrogen peroxide vapour technology is validated to achieve a 6-log reduction on every exposed surface which is achieved with 6-log biological indicators. Ecolab has optimised their portfolio to provide rapid and repeatable bio-decontamination cycles.

Isolators offer several benefits over traditional cleanrooms and biological safety cabinets, including enhanced sterility assurance, reduced risk of contamination, and lower operational costs. Isolators provide a controlled environment that minimises human intervention, which is a significant source of contamination in cleanrooms. Additionally, they can be situated in a lower grade cleanroom than an open biosafety cabinet, leading to cost savings in facility maintenance and operation. According to EU and PIC/s GMP Annex 1 guidance the background grade of a cleanroom used for the manufacture and preparation of aseptic medicinal products must be grade B when operating in a biological safety cabinet but can be grade C or D when operating in an isolator. The Bioquell Qube is designed to meet and exceed industry standards for isolators. With its ability to be customised to meet the end user’s needs, the Bioquell Qube impacts multiple key customer drivers: product and operator safety, easy installation, operational cost reductions and productivity improvements.

EU and PIC/s GMP Annex 1 guidance (section 4.3) are both very clear that Restricted Access Barrier Systems (RABS) or Isolators are needed to minimise contamination from human interventions and that any alternative approaches should be justified. Therefore, isolators play a key role in ensuring compliance with Annex 1. There are many requirements within EU GMP Annex 1 concerning the function, design, and servicing of isolators, which relate to operating pressure, air quality, airflow, separation of the environment, material transfer, glove systems, bio-decontamination as well as other elements. The Bioquell Qube has undergone extensive testing and modifications to ensure compliance with Annex 1 standards and Ecolab can provide a detailed document explaining how it complies.

Isolators support sterility testing by creating a highly controlled, enclosed environment that prevents contamination from external sources and which prevents false positives. They use HEPA filters to maintain air quality and positive pressure to keep contaminants out. The use of glove ports allows operators to handle materials without direct contact, ensuring that the aseptic environment is maintained throughout the testing process. This is equally important for traditional sterility test processes as well as new rapid methods where the product being tested is exposed to the atmosphere.  With its integrated Merck Millipore sterility pump, optimised bio-decontamination cycles and modular configurations to allow a high throughput of tests, the Bioquell Qube is an ideal isolator for sterility testing.

EU GMP Annex 1 provides guidance on the manufacture of sterile medicinal products in the European Union. It impacts pharmaceutical and biotechnology companies involved in the production of sterile products supplied to the European market, including those imported into the EU. Annex 1 provides general guidance that should be used in the design and control of facilities used for the manufacture of sterile products to ensure that microbial, particulate and endotoxin/pyrogen contamination is not present in the final product. The Pharmaceutical Inspection Co-operation Scheme (PIC/s) which is adopted in 53 countries including almost all of the main global medicine regulators has also adopted Annex 1 guidance, meaning that it has a global impact far beyond Europe. The Bioquell Qube has undergone extensive testing and modifications to ensure compliance with Annex 1 standards.

According to EU GMP Annex 1, when manufacturing sterile medicinal products in an open laminar flow unit/biological safety cabinet, the background grade of cleanroom must be grade B, but when processing in a closed isolator system such as an isolator, the cleanroom can be downgraded to grade C or D as the risk of contaminating the process is substantially lower.

Downgrading cleanrooms can save on Environmental Monitoring Performance Qualification (EMPQ) and other operating costs such as ongoing maintenance, operator gowning, cleaning and disinfection products, ongoing environmental monitoring consumables and energy costs due to the HVAC system running at a lower capacity. One study found that running an isolator in a grade C/D cleanroom resulted in 30% lower running costs than a RABS in a grade B cleanroom¹.

1. Répartition aseptique sous Isolateur ou open RABS — La Vague n°33 février 2012 (calameo.com)

• HEPA filtration and sufficient airflow for maintaining grade A/ISO 5 air quality.
• Positive pressure to prevent contamination ingress from the external environment.
• Lightweight and easy to use glove ports for safe manipulation of materials.
• Leak tightness as proven by a validated leak test of the isolator and gloves separately.
• An automated, validated, gaseous, sporicidal bio-decontamination system like hydrogen peroxide vapor.
• Robust construction to ensure durability and ease of cleaning.
• Integrated environmental monitoring systems for continuous environmental control.

The Bioquell Qube offers these and many more features.

Operator safety is ensured by using isolators that are equipped with robust containment features, such as sealed glove ports, glove leak testers and an in-built pressure integrity test to verify that the enclosure is airtight. Regular maintenance and leak testing are essential to ensure the integrity of an isolator. Isolators where cytotoxic compounds are handled are often run at negative pressure and with the exhaust air ducted to the outside of the building to avoid it being exhausted into the room and coming into contact with operators. Additionally, operators should be trained in proper handling techniques and use of appropriate personal protective equipment (PPE). The Bioquell Qube is designed with safety in mind, incorporating all these features to protect operators during the bio-decontamination process and while in production.

Hydrogen Peroxide Vapour (HPV) is a validated process which involves vaporising a 35% hydrogen peroxide solution and introducing it into and throughout an enclosure. The process will deliver the required amount of vapour to reach saturation conditions which will result in a fine layer of micro-condensate being laid down on all exposed surfaces within the target enclosure. This micro-condensation layer ensures effective bio-decontamination by oxidizing vital cellular components of microorganisms on surfaces, including proteins, lipids and DNA.

The process is validated with Geobacillus stearothermophilus biological indicators. After achieving the target 6-log sporicidal kill, the hydrogen peroxide vapour is broken down into water vapour and oxygen, ensuring thorough bio-decontamination without requiring any additional cleaning steps. In the EU, the Bioquell hydrogen peroxide and bio-decontamination process are approved under the Biocide Product Regulation (BPR) for use against bacteria (vegetative and spores), mycobacteria, viruses, yeasts and fungi.*

*Bioquell HPV-AQ 35% hydrogen peroxide approval written into European law – Biocidal Products Regulation (BPR, Regulation (EU) 528/2012)

Isolators offer several benefits for cell and gene therapy production, including enhanced sterility assurance, reduced risk of contamination and improved operator safety. They provide a controlled environment for conducting aseptic processes therefore minimizing human contact, which is a significant source of contamination in cleanrooms. Ecolab’s modular Bioquell Qube isolator allows for more flexible and scalable manufacturing processes, which is crucial for the complex, variable and often bespoke nature of cell and gene therapies. The Bioquell Qube provides a high level of customisation making it an ideal solution for production of both cell and gene therapy products. Its 6-log sporicidal bio-decontamination cycles, ease of installation and use and Annex 1 compliance are just some of the reasons why the Bioquell Qube is in high demand for cell and gene therapy production.

The main difference between an isolator and a RABS lies in their level of containment and bio-decontamination/disinfection methods. Isolators provide a sealed environment with automated bio-decontamination cycles, typically using hydrogen peroxide vapour. Operators always remain separated from the internal environment by a solid physical barrier.  RABS, on the other hand, provide an enclosed but directly accessible environment which is typically disinfected manually with a sporicidal agent. Both enclosures achieve defined air quality conditions (grade A for aseptic processing) and use integrated gloves to separate the interior from the surrounding cleanroom environment. However, isolators generally offer higher levels of protection and are used in more critical applications.  The Bioquell Qube is an isolator system with integrated Hydrogen Peroxide Vapour (HPV) bio-decontamination technology; providing product and operator safety when used as directed.

The integrity of isolator gloves is validated through regular leak testing, which can include pressure decay tests and visual inspections. These tests ensure that the gloves maintain their integrity and do not have any holes or defects that could compromise the aseptic environment. As an option, the Bioquell Qube has an integrated glove leak tester, which inflates the glove to 1,000Pa in line with ISO14644-7 and then measures the pressure decay over a 120 second period, with a decrease of more than 35Pa being registered as a failure. Ecolab have proven that this test criteria will detect a hole of 50 microns and larger in the latex gloves supplied with the Bioquell Qube. It’s important to perform glove leak tests at defined intervals and after any manipulation that might tear or damage the gloves.

Yes, hydrogen peroxide vapor (HPV) is generally compatible with electronics, which has been proven in extensive testing by the EPA¹. Ecolab’s technology is specifically designed to be acceptable on sensitive electronic equipment while achieving a high efficacy bio-decontamination on surfaces. This makes it a preferred method for bio-decontaminating areas with complex and delicate electronic devices. In fact, one study showed that regular use of Ecolab’s Bioquell technology on a hospital ICU ward over an 8-year period resulted in a reduction in the rate that physiology monitors break down², demonstrating that it is compatible with sensitive electronic devices. Ecolab can provide a full material compatibility data package and other reports upon request. Customized material compatibility testing for specific equipment of concern is also available.

1. Compatibility of Material and Electronic Equipment With Hydrogen Peroxide and Chlorine Dioxide Fumigation – cfpub.epa.gov/si/si_public_file_download.cfm?p_download_id=500259&Lab=NHSRC
2. Boyce et al, “Compatibility of Hydrogen Peroxide Vapor Room Decontamination with Physiological Monitors” Infection Control and Hospital Epidemiology, Vol. 35, No. 1 (January 2014), pp 92-93

The time required to bio-decontaminate an isolator can vary depending on many factors including the size of the isolator, environmental conditions (temperature and humidity), the concentration of the biocide being used, the efficiency of the distribution system, the absorbency and amount of material being decontaminated, and the air changes available during the aeration phase. In the Bioquell Qube, empty system cycles typically take anywhere from 40-90 minutes depending on the configuration size, and material loaded cycles are typically in the region of 25-35 minutes. The Bioquell Qube is designed to optimise cycle time length whilst ensuring a repeatable, validated 6-log sporicidal bio-decontamination is achieved on every cycle. Additionally, the Bioquell Qube modular design allows for continuous processing with an independent, dedicated HPV chamber to bio-decontaminate the next materials into the system, while the previous load is being processed in the adjacent working chamber.

Hydrogen peroxide vapour (HPV) bio-decontamination is a low-risk process when properly managed. The 8-hour exposure level is 1ppm in the majority of the world. In the EU the limit is 0.9ppm with country variation. Levels during a bio-decontamination cycle will rise into the hundreds of ppm, however Ecolab’s Bioquell HPV systems are designed to ensure operator safety through extensive alarms and lockouts. The bio-decontamination process is uniquely developed to ensure the concentrations have returned to an acceptable level before use or entry into the system by the operator. Additionally, hand-held sensors are provided with the Bioquell Qube to help ensure there are no leakages during the bio-decontamination process and to ensure safe operator re-entry into the enclosure after completion of the process. Safety training and hydrogen peroxide concentration testing protocols are provided. In addition hydrogen peroxide does not diffuse readily so it tends not to leak out of the enclosure being decontaminated unless being forced out.

A Bioquell biological indicator consists of a small stainless-steel disk inoculated with a 6-log spore culture of Geobacillus stearothermophilus, a microorganism that is resistant to disinfection, that is then placed inside a Tyvek pouch. Biological indicators are used to prove the effectiveness of a bio-decontamination or sterilisation process. It is important that the biological indicator is designed for the process it is being used with, e.g. Autoclaves, vapour phase hydrogen peroxide. Biological indicators are placed at strategic locations inside the enclosure being bio-decontaminated, exposed to the bio-decontamination process, recovered and each disc is then transferred into a vial of tryptone soya broth and incubated. If the vial shows no signs of growth after 7-days then the bio-decontamination process is considered a success, and if showing signs of growth, it is a failure.

A Bioquell chemical indicator (CI) is a small card impregnated with an ink that undergoes a visible change when exposed to hydrogen peroxide vapour. CIs provide an instant, visual confirmation that the bio-decontamination cycle was effectively delivered.