Frequently Asked Questions for Isolators and Bio-decontamination

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 Vapor (HPV) and fog lies in their physical states and application methods. HPV is a true vapor, 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 minimize 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.

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 minimizes 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 customized 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 minimize 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, optimized 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.

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 Vapor (HPV) is a validated process which involves vaporizing a 35% hydrogen peroxide solution and introducing it into and throughout an enclosure. The process will deliver the required amount of vapor 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 use of Bioquell Hydrogen Peroxide Sterilant-AQ means that as a sterilant it “destroys or eliminates all forms of microbial life in the inanimate environment, including all forms of vegetative bacteria, bacterial spores, fungi, fungal spores, and viruses" per the code of Federal Regulations Title 40 Chapter I Subchapter E Part 158 Subpart W § 158.2203. A 6-log reduction is achieved when Bioquell system is used according to the label use instructions with Bioquell Hydrogen Peroxide Sterilant-AQ (EPA Registration Number:1677-277).

The process is validated with Geobacillus stearothermophilus biological indicators. After achieving the target 6-log sporicidal kill, the hydrogen peroxide vapor is broken down into water vapor and oxygen, ensuring thorough bio-decontamination without requiring any additional cleaning steps.  In the United States, Bioquell Hydrogen Peroxide Sterilant-AQ is a registered sterilant (Bioquell Hydrogen Peroxide Sterilant-AQ (EPA Registration Number:1677-277). 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.

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 customization 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 vapor. 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 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.

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 sterilization process. It is important that the biological indicator is designed for the process it is being used with, e.g. Autoclaves, vapor 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 vapor. CIs provide an instant, visual confirmation that the bio-decontamination cycle was effectively delivered.