From ‘Rogues’ to ?: Be Careful What You Wish For… …You Just Might Get it!

Wednesday, June 24, 2026

By: James Agalloco, Agalloco & Associates

Introduction

A recent blog post and discussion during a technical meeting centered around expectations for environmental monitoring approaching Grade A requirements in the assembly of non-product contact parts prior to ambient pressure H2O2 treatment of aseptic isolators. [1.2] There was a stated belief that this was necessary to ensure the suitability of the system for aseptic usage and was expected in response to EMA’s Annex 1.[3] My personal experience with both isolation technology, sterilization methodologies and especially H2O2 processes compelled me to address what I see as a misguided effort and led to the development of this treatise.

Origins

Isolators emerged in the pharmaceutical industry in the late 1980s when LaCalhene (now Getinge) introduced them as a means of separating personnel from the aseptic environment. [4] Initially they were offered with a peracetic acid mist decontamination, but this was rapidly displaced by AMSCO (now Steris) VHP-1000 with a more sophisticated process that utilized a more environmentally friendly agent - vapor phase hydrogen peroxide. [5] At this early stage, the VHP-1000 was described and accepted as a sterilization system. [6] Initial efforts to qualify the H2O2 process frequently required the complete destruction of multiple biological indicators, each with a 106 population of G. stearothermophilus spores, distributed across the isolator system. This biological indicator choice met or exceeded the expectations of regulators, pharmacopoeia and others for treatment. [7, 8, 9,10] It was commonplace, to expect complete kill of all biological indicators as a part of the system qualification / validation efforts. The author (and others) demonstrated this capability repeatedly in systems of varying design and complexity.[11] Where complete kill was not realized ,system modifications and process changes were implemented to resolve difficulties.i

Sterilization or ???

Recognizing that the process goal was treatment of the isolator internal environment rather than preparation of a sterile drug or device, sterilization of the interior surfaces was never considered the ultimate objective. We were endeavoring to provide a system devoid of microorganisms that could then be used for aseptic operations. Sterile, perhaps, but asepsis was near certain given the absence of recoverable microorganisms afterwards. The resulting environment post-v H2O2 exposure within an isolator is far superior to that realizable in any environment supported by manual decontamination. The absence, then and now, of a D-value for vapor phase H2O2 processes eliminates determination of a sterility assurance level in any case.ii

The expiration of the initial patents on hydrogen peroxide sterilization led to the introduction of many new vaporized H2O2 systems.[4,12,13] Along with these systems came an unforeseen difficulty – complete kill of biological indicators became more elusive. [14,15,16] The complexity of vapor phase H2O2 being what it is resolution of these results is challenging and as an expedient measure, a Most Probable Number (MPN) estimation was applied with multiple biological indicators. The surviving biological indicators were termed ‘rogues’ and identified as the ‘root cause’ of the problem. This practice absolved the system fabricators, cycle development staff and validation providers, thereby placing the blame on inadequacies in BI manufacturing. This was an expedient means of rectifying the positive results rather than undertaking extensive redesigns or modifications to correct inadequate systems. That these systems may be poorly designed adhering to a single-phase process perspective and lacking means to enhance internal uniformity (including the inappropriate use of unidirectional air) has been largely ignored.

Early Regulatory Guidance

Initial regulatory guidance with respect to expectations for isolator enclosure treatment were quite flexible which given the relative novelty of isolation technology was wholly appropriate.

FDA – “The decontamination method should render the inner surfaces of the isolator free of viable microorganisms.” [8]

EMA – “Validation should take into account all critical factors of isolator technology, for example the quality of the air inside and outside (background) the isolator, sanitisation of the isolator, the transfer process and isolator integrity.” [17]

These positions acknowledge that the enclosure is not intended to be sterilized but rather prepared for use in an aseptic process where microbial presence is undesirable. The next revision of Annex 1 raised no specific concerns regarding isolator decontamination.

EMA – “Decontamination processes of an isolator or RABS should be validated and controlled in accordance with defined parameters. Evidence should also be available to demonstrate that the agent does not affect any process performed in the isolator or RABS, such as having an adverse impact on product or sterility testing.” [18]

Rogues Take Center Stage

The continued reporting of positive biological indicator results in v H2O2 decontaminated systems became an object of concern. With ‘rogue’ biological indicator results accounting for an estimated 2-5% of all indicators tested a regulatory response emerged. An MHRA blog post addressed their concerns related to vapor phase H2O2 processes.

“However, our concern is that although under ideal conditions, VHP can achieve a reduction of biological Indicator spores of up to 6 logs, the process itself is incredibly fragile.”

“VHP, when well controlled and validated, is a useful method for the decontamination of the surrounding workspace, e.g., an isolator environment. However, given the above concerns, our current stance is that VHP cannot be used to sterilize critical items.” [19]

The fallout from this was not immediate, likely because it does not appear in an official regulatory guidance document, but rather in a blog post. The 2017 draft revision of Annex 1 was comparatively benign.

“For isolators, the decontamination process should be automated and should include a sporicidal agent in a suitable form (e.g. gaseous, aerosolized or vaporized form) to ensure thorough microbial decontamination of its interior. Decontamination methods (cleaning and sporicidal disinfection) should render the interior surfaces and critical zone of the isolator free of viable microorganisms.” [18]

The 2022 final version of Annex 1 introduced some substantial changes suggesting that in the intervening period ‘rogue’ problems had persisted and greater attention was directed towards the subject.

“The bio-decontamination process of the interior should be automated, validated and controlled within defined cycle parameters and should include a sporicidal agent in a suitable form (e.g. gaseous or vaporized form). Gloves should be appropriately extended with fingers separated to ensure contact with the agent. Methods used (cleaning and sporicidal bio-decontamination) should render the interior surfaces and critical zone of the isolator free from viable microorganisms.” [20]

The subject was of sufficient importance that a unique definition was included that mimicked the early expectations of many practitioners. [11]

“Bio-decontamination - A process that eliminates viable bioburden via use of sporicidal chemical agents.” [20]

Two Sides to Every Coin

As EMA offered its new interpretations of the process and its implications better understood, corrective measures for the ‘rogue’ events which had continued to occur were offered. [21, 22, 23] These struck a chord with those practitioners that hadn’t experienced difficulties with their systems and were alarmed by the potential adverse consequences discrediting vapor H2O2 treatments might trigger. At the same time, others were developing a workaround which proposed near aseptic assembly of isolator components prior to bio-decontamination. [1] The expectations included remote sterilization of direct and indirect contact parts installed within the isolator prior to VHP treatment while adhering to first air principles, supportive smoke studies and using near aseptic garb. System modifications may be required in some systems to provide adequate access. This blog post brought forth considerable backlash from those who believed that vapor phase hydrogen peroxide systems had proven their ability to reliably destroy bioburden microorganisms over decades of successful use. [2]

Taking A Broader View

Considering the positive results biological results more broadly, there are aspects that have been ignored. A lack of penetration on the part of vaporized H2O2 is cited as a major limitation of that agent. This ignores the exhaustive requirements for air removal and steam penetration with moist heat. Every sterilization modality except for radiation has issues with penetration depending upon the barrier presented. [24] Fixation on this as a limitation of vapor H2O2 is inappropriate, when the same could be said about other sterilization methods.

There’s a more relevant factor that’s been minimally explored. In any analysis of samples from a validation study, one must consider that the results might reveal something undesirable – the underlying process might be inadequate. In the various documents describing anomalous BI results, there’s been significantly less attention being given to the potential weaknesses in the process. Admittedly, due to the presence of multiple phases, vapor delivery of H2O2 is more complex than other sterilization processes. Concentration, temperature and humidity variations within a system are inherently present. Delivering uniform conditions across the system and process is the goal in every sterilization process. Vapor phase processes are unforgiving and that may be the ‘fragility’ of which MHRA spoke. Belief in ‘vapors’ as true gases, relying on unidirectional air to distribute the vapor, and a general lack of appreciation for the importance of mixing (something that even sterilizing gas processes benefit from) can result in systems and processes that undermine the proven lethality of H2O2 in both gas and liquid phases. What appears to be a consequence of overly resistant ‘rogue’ biological indicators may be the direct result of inferior system designs. [25]

A False Fix

Adding near-aseptic assembly and pre-process environmental monitoring is not the answer. Knowledge of the bioburden prior to a known effective H2O2 decontamination has minimal benefits, adds no truly useful information and increases cost. Moreover, sampling and testing are not process control measures and do not provide additional protection of the product or enclosure.

Concluding Remarks

The introduction vapor phase H2O2 processes assisted in the adoption of isolation technology as the preferred means of aseptic manufacture.[26,27] The superior performance of isolators without added set-up accoutrements is widely accepted and well documented.[28,29] Isolation technology represents the best available technology for aseptic activities. This has been established without the addition of extraordinary measures prior to system decontamination and imposition of added monitoring measures practice is unwarranted.

The means to resolve the perceived difficulties some have encountered are known.[25] The following measures and others can prove beneficial.

  • Changes in the biological indicator to eliminate Tyvek® and use of a permeable substrate material to remove excessive barriers.
  • Refinement of isolator decontamination systems to assure uniformity across the system.
  • Refinement of component and equipment cleaning and preparation practices to better control pre-v H2O2 treatment microbial content.[30]

Isolation technology has significantly improved our ability to manufacture sterile drug products aseptically. Its performance far exceeds that of other aseptic technologies and has been successfully demonstrated for more than two decades in numerous installations worldwide. Implementation of aseptic-like monitoring and assembly methods is unwarranted when the root-cause for these is correctable by more direct means.[31]

References

  1. Ispeak DACH Blog Post, https://ispe.org/pharmaceutical-engineering/ispeak/smart-strategies-aseptic-filling-line-setup, accessed June 22, 2026.
  2. Marsh Steed – LinkedIn post – https://www.linkedin.com/posts/marsha-steed_our-industry-has-already-caved-em – Accessed June 8, 2026.
  3. EMA, Annex 1, Manufacture of Sterile Medicinal Products, 2022.
  4. Agalloco, J., Akers, J., “Introduction to Advanced Aseptic Processing”, chapter in Advanced Aseptic Processing Technology, edited by J. Agalloco, & J. Akers, InformaUSA, New York, 2010.
  5. Meyer, D., “Design and Engineering of Isolators”, chapter in Advanced Aseptic Processing Technology, edited by J. Agalloco, & J. Akers, InformaUSA, New York, 2010.
  6. AMSCO, VHP-1000 Equipment Manual, P-129363-064, September 1991
  7. FDA, Guidance for Industry- Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice, 2004.
  8. Pharmaceutical Inspection Co-Operation Scheme, Recommendation on Isolators Used for Aseptic Processing and Sterility Testing, PI-014-2, July 2004.
  9. PDA, TR 34: Design and Validation of Isolator Systems for the Manufacturing and Testing of Health Care Products, Bethesda, MD, 2001.
  10. USP, <1208>, “Sterility Testing—Validation of Isolator Systems” USP 30, US Pharmacopeial Convention, Rockville, MD, 2008.
  11. Agalloco, J., client experience 1992 – present
  12. US Patent 4,169,123, Hydrogen Peroxide Vapor Sterilization Method, September 25, 1979.
  13. US Patent 4,169,124, Cold Gas Sterilization Process. September 25, 1979.
  14. Sigwarth. V., & Moirandat, C., Development and Quantification of H2O2 Decontamination Cycles. PDA Journal of Pharmaceutical Science and Technology, Vol. 54, No. 4, pp 286-304, 2000.
  15. PDA, TR 51, Biological Indicators for Gas and Vapor-Phase Decontamination Processes: Specification, Manufacture, Control and Use. PDA, Bethesda, Md, 2010.
  16. Agalloco, J., “Thoughts on the Misuse of Most-Probable Number in Rogue Bioindicator Remediation”. accepted for publication in submitted to American Pharmaceutical Review, Vol, May 2026.
  17. EMA, Annex 1, Manufacture of Sterile Medicinal Products, 2001.
  18. EMA, Annex 1, Manufacture of Sterile Medicinal Products, 2017 draft revision
  19. Medicines and Healthcare Products Regulatory Agency. MHRA Inspectorate Blog: VHP (Vapour Hydrogen Peroxide) Fragility. https://mhrainspectorate.blog.gov.uk/2018/04/20/vhp-vapour-hydrogen-peroxidefragility/, 2018.
  20. EM EMA, Annex 1, Manufacture of Sterile Medicinal Products, 2022.
  21. Coles, T., Nieskes, R, & Agalloco, J., “Where did it all go so wrong? An account of the turbulent history of Vapour Phase Hydrogen Peroxide (VPHP) bio-decontamination”, Clean Air and Containment Review, Issue 49, No, 1, pp. 22-24, 2023.
  22. Agalloco, J., “Ridding the World of ‘Rogues’: Improving Vapor Phase H2O2 Sterilization and Decontamination Processes”, PDA Journal of Pharmaceutical Science & Technology, Vol 77, No. 5., pp 412-419, 2023.
  23. Agalloco, J., & DeSantis, P., “The Science Behind Hydrogen Peroxide Decontamination and Sterilization”, published on-line on LinkedIn.com, January 2024. and American Pharmaceutical Review, Vol. 28, No. 1, pp. 30-36, 2025.
  24. Agalloco, J., “Penetrating Sterilization”, American Pharmaceutical Review, Vol 29, No. 1, pp 30-32, 2026.
  25. Agalloco, J., “Biological Indicators, Process Lethality and Vapor Phase Hydrogen Peroxide Processes”, PDA Journal of Pharmaceutical Science & Technology, Vol 79, No. 5, pp 556-563, 2025.
  26. Agalloco, J., & DeSantis, P., “Technologies for Aseptic Filling: The Choice is Clear”, American Pharmaceutical Review, Vol. 27, No. 2, pp. 36-39, 2024.
  27. Dorn, E., Frantz, J., & Valerio, P., “Tracking the Journey of Barrier Technology”. Pharmaceutical Engineering, July/August 2020, pp. 14-23.
  28. McCall, J., et al, “Gloveless isolator performance Environmental Monitoring for Closed Robotic Workcells Used in Aseptic Processing: Data to Support Advanced Environmental Monitoring Strategies, AAPS PharmSciTech 23: 215, 2022.
  29. PDA, 2017 Aseptic Processing Survey, 2017.
  30. Von Esch, M., & Sandler, s., “Regulatory Panel Session Explores Key Aseptic Topics, Pharmaceutical Engineering, July/August 2020, pp. 24-30.
  31. Agalloco, J., “Vaporized H2O2 Decontamination: Failure is Not an Option”, on-line at https://www.americanpharmaceuticalreview.com.featured-articles/622937/vaporized-H2O2-Decontamination-Failure-is-not-an-Option, November 2025

iIncreases in internal mixing to improve uniformity across the entire enclosure were often sufficient to ensure complete kill of the biological indicators,

iiThis is true for all sterilization methods. Estimation of PNSU / SAL requires knowledge of pre-sterilization bioburden population and resistance at the specified lethal conditions. Absent that specific knowledge there is no proof of ‘overkill’ beyond complete kill of the biological indicator.

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