How AstraZeneca Optimized Vapor Phase Hydrogen Peroxide Gassing Cycle Development With Enzyme Indicators
By Stephen Dawson and Miriam Guest, AstraZeneca
Vaporized hydrogen peroxide (vH2O2) is widely used as a surface decontamination tool in the pharmaceutical industry. Vaporized hydrogen peroxide is safe to use, has good material compatibility and low toxicity, and is active at ambient temperatures; it is scientifically proven to have broad, non-specific, and rapid microbial activity. Within the pharmaceutical industry, it is extensively used to support aseptic manufacturing and sterility testing environments as well as a tool for decontamination of cleanrooms.1 Standard procedures include the preparation of the location to be decontaminated prior to a decontamination cycle being performed. As with any GMP procedure, the process must be understood, verified, and validated.
Demonstration of efficacious decontamination is a critical aspect of aseptic processing and sterility testing. This process currently takes a large data set and the use of biological indicators (BIs). Enzyme indicators provide a mechanism to further understand the process and therefore enhance cycle robustness and sterility assurance. By adopting enzyme indicators in the cycle development phases, greater understanding of efficacy of the gassing process can be achieved by providing quantitative results in a faster time frame. This can lead to efficiency benefits through cycle design being performed with data driven decisions and by demonstrating a substantial margin quantitatively (rather than a simple pass/fail criteria).
The application of vH2O2 is widely adopted and recognized in the both the European2 and United States3 pharmacopoeias for sterilizing primary packaging, equipment, and some pharmaceuticals. Different gases may be used, including ethylene oxide, and the typical process involves exposure to the agent within a leak-proof chamber. In the case of production RABs (restrictive access barrier systems) and isolators, equipment to be sterilized is cleaned prior to the application of the gas cycle. It is essential to monitor any cycle for temperature, humidity, and gas concentration in routine use (as well as throughout cycle optimization and validation).
Cycle efficacy, in line with sterilization techniques, is an assessment of the lethality of the cycle; traditionally, biological indicators are used to demonstrate this. There is an expectation that they are placed at locations where decontamination conditions are most difficult to achieve.
Understanding decontamination and sterilization cycles is a responsibility that industry should take seriously. With the development of overkill cycles, establishing worst-case conditions can be challenging, with biological indicators providing a binary answer on cycle efficacy. Enzyme indicators can provide a quantifiable result, which enables safety margins to be built into cycle design based on data.
Learnings From Our Enzyme Indicator Study
A sterility testing isolator with well-established decontamination cycles was used to perform a study to assess the application of enzyme indicators to cycle optimization (see Figure 1). Vaporized hydrogen peroxide (vH2O2) is used to decontaminate surfaces within isolators prior to use. The validation and cycle development of vH2O2 bio-decontamination processes is routinely undertaken by using BIs consisting of Geobacillus stearothermophilus spores carried on a vehicle such as a stainless-steel disc. BIs can be deactivated/killed by vH2O2 if the decontamination cycle conditions are appropriate and repeatable deactivation of BIs under defined parameters allows a validated cycle to be determined. However, BIs have limitations in that they are prone to false positives, only give a qualitative positive or negative result, and they require seven days’ incubation to provide a result. Thermostable adenylate kinase (tAK) as an enzyme indicator (EI) takes an enzyme-based approach as a process indicator4 alternative to the bacterial spore-based BIs. The enzyme tAK has been shown to be inactivated by vH2O2, and a rapid 1-minute test has been developed using a luciferin-luciferase based assay for the immediate quantification of oxidized tAK values, determined by measuring ATP (adenosine triphosphate) produced by residual active tAK enzyme remaining after vH2O2 dosing. EI inactivation by vH2O2 is dose- and time-dependent, and when exposed to vH2O2 alongside BIs, the EI activity from RLU (relative light unit) obtained can be compared with the BI inactivation to establish a quantitative estimate of achieved log reduction (ALR) in RLU values rather than the qualitative growth/no growth outcome of a BI.
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