Evaluation of novel process indicators for rapid monitoring of hydrogen peroxide decontamination processes

N. P. McLeod*, Melanie Clifford, J. M. Sutton

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)


Geobacillus stearothermophilus spores on stainless steel discs are routinely used as biological indicators for the validation of hydrogen peroxide bio-decontamination processes. Given ongoing concerns about the reliability and response time of biological indicators, we explored the potential for an enzyme-based approach to decontamination process evaluation. Thermostable adenylate kinase enzyme was coated onto a solid support and exposed to hydrogen peroxide vapour, in parallel with standard commercial 6-log biological indicators, during a series of vapour-phase hydrogen peroxide cycles in a flexible film isolator. The exposed biological indicators were enumerated to define the degree of kill at different time intervals and the results compared to the thermostable adenylate kinase values, as determined by measuring adenosine triphosphate produced by residual active enzyme. Both biological indicators and the thermostable adenylate kinase indicators exhibited a biphasic inactivation profile during the process. There was significant variance between individual cycles, with some cycles showing complete inactivation of the biological indicators to the limit of detection of the assay, within 6 min, whereas biological indicators in some cycles were inactivated at a time greater than 12 min. The log-kill of the biological indicators at intermediate time points were plotted and compared to the fully quantifiable measurements derived from the thermostable adenylate kinase indicators at the same time points. The results demonstrated very similar inactivation profiles for the enzyme and for the biological indicators, thus it was possible to define a relationship between relative light units measurement and biological indicator kill. This indicates that it is possible to use thermostable adenylate kinase measurement as a direct measure of vapour-phase hydrogen peroxide bio-decontamination performance, expressed in terms of log reduction. Because thermostable adenylate kinase measurement can be achieved within a few minutes of vapour-phase hydrogen peroxide cycle completion, compared with a minimum of 7 days for the evaluation of biological indicator growth, this offers a potentially valuable tool for rapid vapour-phase hydrogen peroxide bio-decontamination cycle development and subsequent re-qualification.

Original languageEnglish
Pages (from-to)393-404
Number of pages12
JournalPDA Journal of Pharmaceutical Science and Technology
Issue number5
Publication statusPublished - 1 Sept 2017

Bibliographical note

Funding Information:
This work was supported financially by Protak Scientific Ltd. The authors would also like to thank Dr. Tim Coles from Pharminox Isolation Ltd. for his input into the study analysis and for interpretation and guidance on the use of VPHP process monitoring and validation within the pharmaceutical industry.


  • Biological indicator (BI)
  • Decontamination monitoring
  • Geobacillus stearothermophilus
  • Hydrogen peroxide
  • Isolator validation
  • Thermostable adenylate kinase (tAK)


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