James Agalloco

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Since the beginnings of aseptic processing, personnel participating in the process were recognized as a major source of contamination. Most of the improvements can be linked to the desire to mitigate the negative consequences of the operator’s presence. Remotely operable filling machines, automated component delivery, aseptic gowning upgrades, unidirectional air, substantially increased physical separation, and more have helped improve the performance of the aseptic process. In most systems, including many of the more advanced designs of restricted access barriers (RABs) and isolators, the operator plays an essential role amidst the many supportive elements needed. The potential for contamination ingress and dispersion is exacerbated by the activities the operator is needed to perform. This situation persists because 

the perfect intervention is the one that is not required” (1,2).

The author initially used the terms ‘routine’ and ‘non-routine’ to distinguish between those activities that are required as part of the process and those that would be needed to rectify the occasional failure. The need to reduce the incidence rate of interventions was included, with the primary focus expected to be on the ‘non-routine’ activities, as their elimination should be the goal in every aseptic process. Unfortunately, multiple firms have tolerated extensive ‘non-routine” interventions to correct repetitive faults in their operations as ‘routine’ because they happened frequently. That was completely contrary to the intent and was addressed in a subsequent publication where the now prevalent terms of ‘inherent’ and ‘corrective’ were introduced (3). Inherent interventions are those operator manipulations necessary to initially prepare the equipment and maintain operation throughout its execution. In an ideal world, no other operator activity would be needed in the critical environment. Corrective interventions would address the actions needed to return the line to operation after a fault or stoppage. This terminology is now generally accepted, but the desired goal is still somewhat distant.

Recommendations for detailed descriptions of each intervention included in operating procedures accompanied with still images, video, and hands-on training were recommended to ensure uniformity of execution and adherence to best practices. These would be employed to support media fill execution and improved consistency in daily execution. Later publications endeavored to make further distinctions in corrective interventions (4,5). The simpler corrective interventions require removal of jammed or broken items, adjustments to equipment, and similar tasks which are typically short duration. A new term was added, ‘critical corrective’, to describe interventions that entail the replacement of sterilized product contact equipment that often require increased manipulation and longer time. While ‘critical corrective’ interventions are common at some firms, consideration should be given to their elimination by ceasing production when these are encountered rather than accept the added risk associated with their execution.

In parallel with these intervention focused publications, James Akers and the author have published on aseptic processing risk assessment that focused on interventional activity (6,7,8). The initial risk assessments published in 2005–2006 arbitrarily assigned greater risk to ‘corrective’ interventions relative to ‘inherent interventions.’ The authors also used a linear scale for potential contamination dispersion. After working with multiple clients applying the method in a variety of different settings, it was revised substantially in 2017. Rather than an arbitrary categorization, the authors shifted to a time-based risk factor for interventions within the critical zone. This resulted in a major shift as a ‘corrective intervention’ of three seconds duration should be considered less risky than an ‘inherent intervention’ requiring 45 seconds to complete. The simple removal of a downed vial would thus be considered less risky than the lengthy addition of stoppers to a feed hopper. The other major change was changing the ‘proximity’ factor from linear to an ‘inverse-square’ approach, thereby eliminating an arbitrary (and hence potentially misleading) metric in the method. The recommendations included within this publication were made with the 2017 revision to the A-A risk method in mind.

The publications summarized above only go so far. Redefinition of terms and refinement of risk evaluation methodology only point the general direction to take as they do not detail the specific actions needed. To establish those, it’s useful to consider the “perfect intervention” objective somewhat differently. It could be restated as—as it is not possible to perform an intervention without adding to the risk of contamination ingress, every effort should be made to eliminate interventions entirely in aseptic processing. If they cannot be completely eliminated, they need to be made simpler and/or less frequent.

Some basic principles for reduction in intervention and contamination risk can be used to guide the practitioner (see 

Table I: Strategies for reduction of intervention risk.

Strategies to tactics–inherent interventions

Inherent interventions may not appear to provide opportunities for reducing the contamination risk, because the necessary activities are explicitly required in operating procedures or manufacturing/filling records. This presupposes that those activities have already embraced the goal of minimizing operator interaction with sterile materials. In the author’s experience, the guiding principles are rarely considered sufficiently. The following real-life examples are unfortunately typical:

In reviewing the sterilization validation for an aseptic filling system there were more than 60 separate pieces of the filling manifold being individually wrapped and sterilized. The firm indicated that these would be aseptically assembled after sterilization to avoid difficulties with steam sterilization equilibration time. A custom-fabricated replacement was substituted and sterilized as a single item.

In the final stages of making a small-scale sterile suspension, a sample was taken immediately after transfer from the mixing vessel to the dilution vessel. Leaving a sample size amount in the mixing vessel eliminated the sampling intervention in the materials to be further processed.

Following the subdivision of a sterile powder blend into sterile containers in a closed system, samples were aseptically taken from selected containers. Introduction of sample size containers fillable using the closed system eliminated exposing materials intended for filling.

After automatic loading of a lyophilizer through a “pizza-door”, the entire door would be opened to place thermocouples in containers on each shelf. The process would be reversed at the completion of the process to remove the thermocouples before unloading. Awareness that the process was not controlled or adjusted in any way due to the thermocouples in the containers resulted in their removal after completion of lyophilization process validation.

It is important to look at the aseptic process holistically to lessen process vulnerabilities. This may entail changes at the detailed level, including equipment configurations, sequence of activities, sampling requirements, and more. An activity or configuration that might be entirely satisfactory from a process perspective may include easily avoidable microbial contamination risks that can be reduced or eliminated.

. To the casual observer, the aseptic process begins when formulated sterile product and components are first exposed within the critical environment. The preparation of equipment for use within the aseptic environment is the true starting point. This can require direct contact with sterilized items very differently from what is necessary during the aseptic process itself. Adherence to core aseptic technique principles such as ‘first air’, ‘slow and deliberate’ movement, avoidance of direct contact, etc. can be difficult given the unique nature of the manipulations. Useful tactics for aseptic set-up include the following:

To the maximum extent possible, product contact equipment should be pre-assembled prior to sterilization. Alternatively, a one-piece custom fabrication could replace multiple individual pieces. Sterilization processes can be adapted as necessary.

A pre-sterilized single-use disposable system can replace complex piping configurations. This is increasingly commonplace. It introduces other factors for consideration such as material compatibility, system integrity, etc., but the elimination of numerous set-up interventions is clearly advantageous.

 of the filling parts after assembly eliminating remote sterilization and aseptic assembly.

Components for the set-up should be prepared and sterilized separately (and cleared from the line prior to the start of the fill) from those used for the process to avoid exposing components for extended time periods.

Process equipment, especially that intended for aseptic filling and sealing, should be selected for ease of set-up and preferably remote adjustment, initially and during use.

Sterilizing filters that are included in the set-up should be integrity tested prior to sterilization. The availability of confirming validation on filter integrity following sterilization avoids uncertainties that have resulted in requests for PUPSIT (Pre-Use Post-Sterilization Integrity Tests) as a routine requirement. 

Use of filling machines able to remotely adjust for fill weight, container height, rail spacing, and other parameters to allow for minimal interventions for product, fill, and container changeover.

Tactics for aseptic filling/processing inherent activities. 

The aseptic process is the focus of attention as interventional activities can be frequent during its execution. Inherent activities for aseptic filling processes are either material handling or monitoring related, such as:

Component introduction linked to the speed of filling/processing, and items sterilized in sealed containers are introduced as needed.

The use of larger component containers to reduce the frequency of interventions.

The repetitive actions associated with component addition make them well suited to the use of robotics.

Robots and automation are also effective in the transfer of containers to/from and, loading/unloading of lyophilizers.

Rather than an arbitrary time interval as the basis for weight checks, define the interval based upon filling system performance which can allow for a longer-periods without sampling.

The use of robotics for sampling throughout the aseptic process.

Remote monitoring and adjustment of fill weights eliminating interventions. An investment in newer, more sophisticated, and safer equipment is needed.

Elimination of thermocouples from product containers within lyophilizers.

Less frequent environmental monitoring based upon operational trends.

Elimination of sample points remote from operator activity due to their limited utility.

Elimination of mid-process monitoring of personnel as an avoidable and overly risky intervention.

The use of passive (settle plates) rather than active air samples to eliminate the more difficult interventions needed for loading and unloading of active air samplers.

The selection of viable monitoring systems that simplify the introduction and removal of test samples.

Greater emphasis on post-process surface and personnel monitoring to avoid interventional activity mid-process in the critical environment.

Strategies to tactics for corrective interventions. 

There should be no tolerance for corrective interventions in aseptic processing. Their continued presence reflects a willingness to accept the consequences of defects in a variety of areas: process design, equipment selection, component cost, and operating procedures. While not all corrective interventions are difficult to perform or time-consuming to execute, they each represent avoidable faults in the process. Measures to prevent their reoccurrence should be taken to eliminate the need for the interventional activity. The following real-life examples are representative of the problems they present, and potential for their elimination and remediation:

Shortly after successful factory acceptance testing (FAT) and site acceptance testing (SAT), and initial media fills without adverse incident and meeting all requirements, the firm experienced serious problems with vial stoppering with frequent broken containers requiring line clearances. Investigation in conjunction with the equipment supplier revealed that the firm used vials of the same configuration with less restrictive acceptable quality levels (AQLs) on vial dimensions concurrent with the start of production with the expectation that it would have no adverse impact. Reverting to the equipment manufacturer’s more restrictive AQLs for the vials quickly resolved the problem.

Media fills for lyophilized vials on a multi-use line resulted in multiple contaminated units. Solution fills with the same vial were consistently free of any positives. Review of the lyophilization media fill video revealed frequent operator interventions into the stopper bowl to remove clumps of stoppers. Corrections to the stopper preparation and sterilization processes eliminated both the clumping and the subsequent corrective operator intervention.

During media fills performed as part of the validation of a substantially modified filling line, the firm was unable to successfully perform stopper bowl replacement without contamination. In reviewing their operational history, the firm realized that that intervention had only been required once in the previous eight years of operation. It was decided that eliminating the intervention entirely and re-starting with a fresh set-up in the event a mid-process replacement was necessary, was more appropriate.

The examples included two distinct types of corrective interventions. The easiest corrective interventions to perform are those that entail removal of an object or adjustment of an already installed piece of equipment. These are generally easy to perform and are ordinarily of comparatively brief duration. The more challenging are more complex entailing replacement of a sterilized equipment item that is a part of the initial set-up (e.g., a filling needle, stopper bowl, etc.) should be considered critical correctives and are generally more complicated and of longer duration

Tactics for aseptic filling/processing corrective activities.

 Ideally an aseptic process can be completed without requiring a corrective intervention. This should be the goal for all processes; regrettably, it is only rarely achievable. Firms should collect data on their corrective intervention experience and use the data constructively to identify means for reducing their frequency. Some ofthe more frequently encountered corrective interventions include:

Stopper misfeeds or clumping can be a consequence of hidden variations in their preparation and sterilization processes.

The use of more restrictive AQLs for component dimension/configuration can reduce the rate of misfeeds, jams, breakage, etc.

The use of neck-hold vial handling avoids misalignment of the closure with container seal surfaces as well as elimination of container mis-feeds at conveyor transitions. This can also serve to reduce spillage.

Equipment set-up that is more automated/reproducible can result in better performance during the fill.

Remote adjustment of conveyor rails, fill needle height, and other equipment provides for less invasive adjustment and more precision in set-up and vial change-over.

Equipment designs that accommodate self-clearance for mis-fed components can eliminate their manual removal.

Relocation of controls, and adjustment locations outside the critical zone.

Tactics for aseptic filling/processing critical corrective activities. 

The invasive nature of critical corrective activities coupled with the time required for their execution should make these a rare occurrence in any operation. The contamination risk from these activities is substantially greater than that associated with inherent or ordinary corrective interventions. Tolerance for their routine execution in any process or line should be extremely limited. The more common critical correctives include product filter change, filling needle replacement; fill-pump replacement; and stopper bowl changes. The execution of these may entail significant human manipulation within the critical zone and, while certainly possible, may present extraordinary contamination risks. As such, they should be closely scrutinized to ensure they should be maintained as a continued practice. The contamination potential from these due to the product contact nature, proximity and execution time is greatest, and their elimination is highly recommended.

The attention focused by regulators on aseptic processing activities brought attention to all of the interventional activities performed on a filling line. Firms were asked to include interventions in process simulations to support their ability to execute them with contamination ingress. In their eagerness to be compliant, some firms identified activities that fall outside the realm of aseptic processing. There are several categories of these:

Operator activity outside the critical zone that does not involve aseptic handling of exposed sterile materials or equipment. These have no impact on the aseptic process and need not be included in a firm’s media fill program.

An activity that compromises the integrity or performance of the system or air supply to the critical zone should be understood as unacceptable during an aseptic process. It should result in immediate cessation of operation, and rejection of all exposed sterile items. There is no defendable rationale for continuing aseptic operations when interventions that interfere with the system’s primary protection (e.g., barriers, gloves, partitions, etc.) occur.

Any intervention that cannot be performed with proper aseptic technique while maintaining the critical environment within the required limits should not be tolerated.

The aseptic operator is universally recognized as the primary source of microbial contamination in the critical environment (9). There is also definitive evidence that increased activity levels result in increases in viable and non-viable particles (10,11). This supports the original premise that there are no truly safe interventions with respect to aseptic processing regardless of the underlying aseptic technology (1,2). Nevertheless, as system perfection is still an elusive goal in manned aseptic processing and system automation is not universal, there will be instances where operators will be required to manipulate sterile items in either preparation for or during the aseptic process. It behooves the firm to review every expected interventional activity regardless of type in detail to identify the preferred process for its execution. Adherence to good aseptic technique is required and review by an experienced microbiologist is recommended. Once the intervention is defined, it should be well documented and used as the basis for operator training. Documentation should incorporate written instructions, drawings, still and video images detailing the practice in detail such that all operators can execute it in a near identical manner. Confirmation of acceptability should be confirmed by incorporation within the media fill program at a frequency that meets or exceeds its use in routine production.

There are two guiding principles to adhere to for the improvement of intervention practices in aseptic processing:

A useful principle to follow to is to make every intervention as simple as possible to execute such that all operators can successfully execute it at all times. This holds true regardless of whether the operators are highly proficient or new to the job. Extraordinary skill should never be necessary to execute any intervention. The objective should be to make any intervention so safe to perform that the operator can perform it on their worst day.

Just because an intervention can be done doesn’t mean that it should. Interventions always increase the risk of contamination ingress. Whether they are corrective or inherent, firms should be committed to their eventual elimination. The pursuit of the “perfect intervention” should be continuous in all aseptic operations.

Agalloco, J. Management of Aseptic Interventions. 

Agalloco, J.; Akers, J. The Truth about Interventions in Aseptic Processing. 

Agalloco, J.; Akers, J. Revisiting Interventions in Aseptic Processing. 

Agalloco, J. Uncommon Sense in Execution of Process Simulations. 

Agalloco. J. Complications in Process Simulation Execution. 

Biologics and Sterile Drug Manufacturing 

Agalloco, J.; Akers, J. Risk Analysis for Aseptic Processing: The Akers-Agalloco Method. 

Agalloco, J.; Akers, J. The Simplified Akers-Agalloco Method for Aseptic Processing Risk Analysis. 

Agalloco, J.; Akers, J. A Revised Aseptic Risk Assessment and Mitigation Methodology. 

Avallone, H. Current Regulatory Issues Regarding Parenteral Inspections. 

Journal of Parenteral Science & Technology,

, 2nd Edition, John Wiley & Sons, Chichester, 1999

Ljundvist, B. and Rheinmuller, B. Modern Cleanroom Clothing Systems: People as a Contamination Source. 

PDA Journal of Pharmaceutical Science & Technology

Articles

Aseptic processing has evolved to minimize contamination risks, primarily from human operators. Despite advancements like automated systems and improved gowning, operators remain crucial, often necessitating interventions that increase contamination risk. The distinction between 'inherent' and 'corrective' interventions is now standard, with efforts focused on reducing the latter. Risk assessments have shifted to time-based evaluations, emphasizing the need to simplify or eliminate interventions. Strategies include pre-sterilization assembly, robotics, and remote monitoring. Ultimately, interventions should be minimized, with a focus on making them as simple and infrequent as possible.

Addressing human interventions to reduce their impact on contamination requires addressing broader considerations than limiting operator activities. Equipment, automation, procedural and component changes can be utilized to make the aseptic activities safer. This article offers suggestions for reducing the adverse impact of intervention throughout the aseptic process.

Interfering with Interventions in Aseptic Processing

In the late 1970s, media fill studies began to be used in aseptic processing for the expressed purpose of evaluating sterility assurance levels in filling activities. These initial studies were simplistic. In response to regulatory expectations, the activity grew in sophistication to address perceived vulnerabilities (1). 

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	When referring to this article, please cite it as J. Agalloco,  “Complications in Process Simulation Execution,"

Regulators have exaggerated expectations for simulated media fills. 


Complications in Process Simulation Execution

The introduction of isolation technology in the pharmaceutical industry by the French firm La Calhene in the 1980s required a means for the reliable microbial decontamination of the isolator interior. This was initially performed using a mist of peracetic acid/water; however, this was considered undesirable for a variety of reasons, with objections over its use primarily aimed at the resultant corrosion of surfaces, wet surfaces, and lengthy aeration times.  In the late 1980s, AMSCO (now Steris) offered the first widely available alternative with their VHP-1000 generator, which delivered vapor phase hydrogen peroxide (VHP). With the introduction of the VHP-1000, this technology and its derivatives became the dominant means for isolator decontamination. 

	Initially, the performance expectations for the decontamination process varied according to the end user’s protocol requirements. The process target (decontamination or sterilization) and the means to establish them (the biological indicator [BI] population to use and the selection of cycle duration) varied widely. Firms with near identical systems and practices considered them differently.  

	The first definition of process expectations was provided in 

Sterility Testing-Validation of Isolator Systems

 (2000), with an expectation for sterilization, “The sterilization methods used to treat isolators, test articles, and sterility testing supplies are capable of reproducibly yielding a six log kill against an appropriate, highly resistant biological indicator” (see 

	The inclusion of a “six-log kill” requirement is problematic in that this terminology is uncommon in sterilization; there being no generally accepted definition for it in sterilization practice. It could be interpreted as either a six-log reduction or complete kill of a six-log population (a nine-log reduction, see 

). Stating that and expecting “sterilization” in the first version of 

<1208> was clearly problematic, as sterilization is more widely accepted as a minimum 12-log reduction of a BI.

	The Parenteral Drug Association issued its publication on isolators in 2001. It included several different means to establish its decontamination requirement, “for the purposes of isolator decontamination a Total Kill Analysis study of a suitable bioindicator with a population of 10

 or greater is considered an overkill cycle. Such a cycle indicates a spore log reduction value of >7 logs” (2). 

	FDA formally addressed isolator decontamination first in 2002, with the initial draft of its revised aseptic processing guidance (3). The stated expectation was, “For most production applications, demonstration of a six-log reduction of the challenge BI is recommended.” The draft was also explicit in stating decontamination, as opposed to sterilization, of the interior was expected. Sterilization was correctly identified as the requirement for product contact surfaces.

	The European Medicines Agency, through the assembly of inspectors with the Pharmaceutical Inspection Co-Operation Scheme (PIC/S), issued its first isolator-related guidance in 2004 and aligned closely with FDA, “…, but a target of six log reductions is often applied” (4). This document was explicit in defining a six-log reduction, as not requiring the complete destruction of all microorganisms on a 10

 population BI. This document adds confusion of a different kind by referring to the decontamination processes primarily as a gaseous process, which is a serious error (see below).

	The final version of the FDA guidance published in September 2004 relaxed the log reduction position; “Normally, a four- to six-log reduction can be justified depending on the application” (5). This guidance in conjunction with the PIC/S six-log definition has largely shaped global industry decontamination practice to this date.

revised chapter <1208> to align more closely with the regulatory and industry guidance documents that had been issued, “The ability of the process to reproducibly deliver a greater than three-log kill is confirmed in three consecutive validation studies” (6). In this revision, the process is now termed decontamination consistent with the other standards.

significantly revised its sterilization content in 2013–2018 and added vapor phase sterilization in those changes. The new content includes two related subchapters that directly impact VHP processes (7,8). The 

content changes expectations in many ways. First, vapors are considered two-phase systems, the presence of which substantially complicates process design and execution. Second, because of the dual-phase nature of the process, D-value estimation is not possible because the conditions of microbial death are indeterminate. Lastly, if the D-value is indeterminate, the log reduction expectations may be inadequate. The 

’s chapters disrupt many of the commonly held misconceptions regarding VHP validation.

	A regulatory perspective was provided by the United Kingdom’s Medicines and Healthcare products Regulatory Agency (MHRA) in an April 2018 blog on their website. “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” (9). This position creates substantial difficulties for many current installations. The 

 sterilization of stopper bowl, feed tracks, and many other surfaces in direct contact with sterilized items (and presumably considered “critical”) is called into question by this MHRA position. Industry response has been one of alarm, as it potentially invalidates many systems current in use. 

	A shift in perspective is essential to succeed in the changing landscape. The existing log reduction requirements are poorly suited to the emerging perspectives of decontamination using a vapor treatment. Increasing usage of vaporized hydrogen peroxide has resulted in numerous publications describing applications of various types, from small pass-through chambers to entire suites of rooms. Unfortunately, past mistakes and misstatements have been incorporated which have adversely influenced industry practices. This document endeavors to clarify the process relying on the core science underlying it. Many of the problematic statements regarding vapor decontamination are provided in this paper followed by the scientific reality.

 Vapor phase hydrogen peroxide decontamination is a single phase gas process.

 There are varying definitions of vapor, some of which suggest that vapors are equivalent to single phase gas mixtures. However, at the ambient temperatures used for VHP processes condensation of hydrogen peroxide (H

O), is unavoidable (10,11). Vapor decontamination processes operate below the boiling point of both materials, and, while some of each will remain in the gas phase due to their vapor pressure, they are both liquids at ambient temperature. The amount of H

O maintained in the gas phase decreases as the temperature decreases below that used to introduce them into the system. Thus, VHP must be understood to be a two-phase process in which both gas and liquid are present. Depending on the amount of liquid phase present, it may or may not be visible. The original characterization of VHP as a completely gas phase process originates in the originating patent; however, the inventors did not consider or determine what phase was actually present at the point of kill (12). 

is more rapid in the gas phase than in the liquid phase.

 The experiments that support this claim can be interpreted differently to assert that the exact opposite is true (13). No determination of phase or concentration measurement at the point of kill was performed in the experiments. Condensation is highest at the lowest operating temperature, and these demonstrated the fastest kill rate. Published data on H

solutions indicate that spores are killed extremely rapidly (14–16). This suggests that liquid-phase kill will be more rapid than gas-phase kill because the concentration of H

in the liquid will always be higher than in the gas phase (17). This has been independently confirmed in challenge studies conducted using various decontamination systems (18–20).

 VHP decontamination is a single phase gas process that resembles ethylene oxide (EtO) and other sterilizing gas processes.

 Considering gases and vapors as identical goes to the very origins of hydrogen peroxide decontamination (12,21). The confusion in terminology may originate in the means to introduce a 35% aqueous solution into a hot air stream where high heat rapidly converts the H

aqueous solution into a multi-component gas at elevated temperature. Above their boiling points, both liquids are converted to gases. The hot gas mixture is introduced into the ambient temperature target system where it loses heat to its surrounding surfaces. As this occurs, the H

O concentrations in the gas phase are above their saturation vapor pressure and condensation must occur (22). The presence of two phases in an ambient-temperature vapor hydrogen peroxide process is unavoidable. Hydrogen peroxide having a higher boiling point and lower vapor pressure than water condenses first and concentrations of H

in the liquid phase will be higher than the original solution percentage. 

 Because VHP decontamination is a gas phase process, temperature is not an important influence on process lethality.

 VHP is a multi-phase process with both liquid and gas phases present (23). The amount of condensation on the surface is directly related to the local temperature. As the H

O solution is first vaporized and then introduced with hot air, there will be temperature differences across the system. The hottest areas, typically closest to the vapor inlet and any operating equipment, are potentially “worst case” because of reduced condensation. Changes in temperature during the process duration are somewhat unavoidable, especially in smaller chambers, which will alter the amount of condensation and, thus, process lethality. It is important to maintain near-constant room conditions to minimize process variability during the individual VHP process and between multiple VHP cycles over time. 

gas concentration can be used to reliably control VHP processes. 

 The gas- and liquid-phase concentrations at any location is dependent upon the temperature of the system at that location. Gas-phase measurement cannot be used to estimate concentrations on the surface unless the system reaches equilibrium. Variations in temperature across location and process duration are unavoidable with most VHP processes, which minimizes the utility of concentration measurements taken in the gas phase. 

 Condensation during VHP decontamination is to be avoided, as it slows kill and extends aeration times. Thus, dehumidification prior to processing helps prevent condensation.

 Condensation is unavoidable, and, while not always visible, it actually supports more rapid kill (see previous scientific explanations), condensation being necessary to assure rapid kill is actually desirable. Dehumidification prior to the introduction of H

is unnecessary because it delays condensation, thus, increasing process time and system cost. Kill is quicker in condensing systems such that the process dwell can be shortened substantially. 

on surfaces is a corrosion and explosion hazard and is to be avoided.

 Corrosion is not a concern when appropriate materials are used for construction of the system. Condensation of H

has always been present, even if not always readily visible. Nevertheless, in more than 30 years of vapor phase hydrogen peroxide use, there has never been a reported explosion. This includes the many newer systems where H

is intentionally condensed to expedite the process.

on surfaces can result in greater adsorption of H

than a gas phase process without condensation. This can result in lengthy aeration.

 Gases can permeate Tyvek wrapping and be readily absorbed by polymers, while condensed liquids cannot permeate the hydrophobic material and remain on the exterior surface. It might seem counter intuitive, but a “wet” process can result in less adsorption than a “dry” process. Note that the distinction between “wet” and “dry” processes is an artificial one. As mentioned, all VHP processes have some condensation present, so the “wet” vs. “dry” distinction refers to the differing beliefs of whether kill occurs best when the BI is contacted with a liquid or a gas. With rapid kill, diffusive adsorption of H

from the gas phase is reduced and overall process duration may be shorter in “wet” processes than in “dry” processes. The longest segment of many VHP cycles can be the aeration period, which is limited by slow desorption of H

from polymeric materials and permeable packaging.

 Labeled “D-values” for VHP BIs are definitive and accurate (author’s note: throughout the document, the term “D-values” in quotes denotes instances where it is believed that the values are improperly identified as such, and the term D-values without quotes is used elsewhere).

 A D-value can only be established when the conditions (concentration, relative humidity, and temperature) to which the microorganisms are exposed are known (8). The presence of two phases in VHP systems makes D-value determination impossible. BIs labeled for VHP processes do not state the conditions of kill, but typically cite the H

injection rate in the BI manufacturers test system. The injection rate cannot be correlated to precise and reproducible destruction of microorganisms in a different system. None of the BI labels report accurate “D-values” because the conditions at the point of kill are unknown and, thus, the labels themselves are misleading. 

 Vendors selling VHP BIs provide reliable “D-values” on their certificates of analysis.

 BI vendors have responded to customer expectations and the “D-value” information they provide does assure consistent resistance for their products, but only in the BI vendors test system (24,25). The performance of those same indicators in users’ systems cannot be predicted from the certificates provided because of the differing conditions in the users’ systems.

 Publications showing the effect of varying materials on the “D-values” of BIs in VHP processes are useful because the BIs used in those studies are positioned on the surface (26–29).

 As stated earlier, D-value can only be established when the conditions (concentration, relative humidity, and temperature) to which the microorganisms are exposed are known (8). The various studies do not provide that information, however. Further, because the internal conditions inside the various systems must be understood to vary with location and time, the relative resistance associated with different materials and finishes cannot be established.

 BIs for VHP can demonstrate anomalous behavior (30,31). These are often termed “rogues”, and their survival is an indication of defective BIs, not flaws in the process being evaluated. 

 BIs are produced for a variety of sterilization processes using well-established and consistent methods. There is no comparable problem with “rogue” BIs associated with any sterilization or decontamination process other than VHP. The “rogue” BI is more likely the result of poorly defined decontamination processes where the lethality delivered is insufficient to kill all of the BI microorganisms present.

 Because there are “rogue” BIs, multiple BIs should be used with all VHP decontamination processes.

 Properly developed VHP processes that use sufficient H

to create modest amounts of condensation have been proven reliable without resorting to multiple BIs in an attempt to compensate for either a minimally lethal or excessively variable decontamination process.

 A “system D-value” can be used to establish reliable VHP processes; “D-value of a BI measured in a specified gas generator/separative enclosure combined with a defined sporicidal vapor-phase (decontamination cycle)” (31).

 As indicated previously, D-value determination requires maintenance of specified conditions (concentration, relative humidity, and temperature). This is not currently possible for two-phase systems using a BIER type design (5,8). Suggestions that it could be achievable and useful in the operation of a large system with potentially greater variation in the critical parameters are poorly founded. 

	Putting aside the unusual MHRA blog position, there are principles and practices to follow with respect to VHP decontamination that include: 

 colony-forming unit (CFU)/unit where decontamination is the process expectation.

		BI positioned on product contact surfaces should have a population of 10

 CFU/unit where sterilization must be demonstrated.

		All BIs should be fully inactivated. The use of multiple indicators at each location is unnecessary, especially if the intent of multiple BIs is to explain away positive results.

		VHP is most effective when provided using a “wet” process, and this may entail the use of greater quantities of H

		Labeled “D-values” for VHP processes are inaccurate and should never be used to determine cycle duration. They may be useful in comparing different BI lots from the same manufacturer.

		Log-kill times cannot be estimated from “D-values” and can only be established using BIs in the system being evaluated.

	The preceding could be misinterpreted as denigrating the utility and efficacy of VHP as a decontaminating or sterilizing agent. There are certainly difficulties with its use; however, properly delivered, its many advantages outweigh the negative aspects and assure its continued and expanded usage (22). It lacks the simplicity of a gas process, and adaptation to the unique requirements of its two-phase nature is essential to success. Reliable decontamination/sterilization cycles and systems require the following:

		Acknowledgement that condensation is a necessary element of reliable kill

		Awareness that BIs provide prima facie evidence of lethality

		Abandonment of gas concentration as a useable metric  

		Acceptance of empirical evidence based on BI destruction as the definitive indicator of process suitability.

	In spite of it being misrepresented as a single-phase gas process, hydrogen peroxide usage has increased steadily since its introduction. The cited difficulties have been overcome by many practitioners. Implementing vapor H

processes following sound scientific principles of chemistry, physics, and engineering can establish designs for the delivery of a complex, but lethal, chemical agent in the most effective manner. Numerous vendors and end users have realized success with vapor hydrogen peroxide, and, despite the difficulties, some have experienced, increased future use is near certain.

	1. USP, <1208>, “Sterility Testing-Validation of Isolator Systems” 

 (US Pharmacopeial Convention, Rockville, MD, 2000).
	2. PDA, 

TR 34: Design and Validation of Isolator Systems for the Manufacturing and Testing of Health Care Products

Draft Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing, September 

(Rockville, MD, 2002).
	4. Pharmaceutical Inspection Co-Operation Scheme, 

Recommendation on Isolators Used for Aseptic Processing and Sterility Testing

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing 

(Rockville, MD, September 2004).
	6. USP, <1208> “Sterility Testing-Validation of Isolator Systems” 

(US Pharmacopeial Convention, Rockville, MD, 2008).
	7. USP, Supplement 1, <1229.11>, “Vapor Phase Sterilization” 

(US Pharmacopeial Convention, Rockville, MD, 2015).
	8. USP, Supplement 1, <1229.5>, “Biological Indicators for Sterilization”

(US Pharmacopeial Convention, Rockville, MD, 2016).
	9. MHRA, “

, April 20, 2018.
	10. W.M. Haynes, Ed., 

 (CRC Press LLC, Boca Raton, FL, 95th ed., 2014) pp. 4–67.
	11. T.E. Daubert, and R.P. Danner, 

Physical and Thermodynamic Properties of Pure Chemicals Data Compilation

(Taylor and Francis, Washington, DC, 1989).
	12. USPTO, “Hydrogen Peroxide Vapor Sterilization Method,” US Patent 4,169,123, Sept. 25, 1979. 
	13. D. Eddington, et al, “Chemical and Biological Aspects of Hydrogen Peroxide Vapor,” Presentation at ISPE Barrier Isolation Technology Conference (Washington, DC, 2000).
	14. R. Toledo, et al., 

, 26 (4) 592–597 (1973).
	15. S.S. Block, “Peroxygen Compounds,” in 

Disinfection, Sterilization and Preservation

(Lippincott Williams and Wilkins, Philadelphia, PA, 5th ed., 2001), pp. 185–204.
	16. M. Ahlgren, and Z. Bjorkland, “Robustness of a Biological Indicator Resistance Test Rig-a Study of Different Parameters, Methodologies, and Their Impact on the Measured D-value of the Biological Indicator,” Lund University, LTH School of Engineering, 2005.
	17. C. Hultman, et al., 

27, 22–32 (2007).
	18. J.E. Akers, et al., “A New Approach to VPHP Decontamination,” presentation to ISPE, 2006.
	19. D. Watling, “Is H

a wet or dry process?” presentation at PDA Isolator Technology Conference, April 29-30, 2002.
	20. B. Unger-Bimczok, et al., 

 3, 123–133 (2008).
	21. G.S. Graham, et al., “Sterilization of Isolators and Lyophilizers with Hydrogen Peroxide in the Vapor Phase,” proceedings from the International Congress of the Parenteral Drug Association (Basel, Switzerland, 1992), pp. 32–51.
	22. J. Agalloco and J. Akers, 

37 (9) 46-56 (2013).
	23. D. Watling and M. Parks, 

 9 (4) (2009).
	25. J. Drinkwater, et al., 

 14(1) 5–11 (2009).
	26. V. Sigwarth and C. Moirandat, 

54 (4) 286–304 (2000).
	27. V. Sigwarth and A. Stark, 

57 (1) 3–11 (2003).
	28. T. Mau, et al., 

 58 (3) 130–146 (2004).
	29. B. Unger, et al., 

 61 (4) 255–275 (2007).
	30. P. Templeton and J. Hillebrand, “Case Study: Isolator Sanitisation Cycle Development, Validation and Revalidation,” presentation at ISPE Isolation Barrier Forums (Washington, DC, and Prague, 2005).
	31. PDA, 

TR 51: Biological Indicators for Gas and Vapor-Phase Decontamination Processes: Specification, Manufacture, Control and Use 

	James Agalloco, jagalloco@aol.com, is principal of Agalloco & Associates.


	Vol. 44, No. 1
	January 2020
	Pages: 53–57

Past mistakes and misstatements have adversely influenced industry decontamination practices with vapor phase hydrogen peroxide, and this article endeavors to clarify the process.