Overcoming Limitations of Vaporized Hydrogen Peroxide

The use of hydrogen peroxide (H₂O₂) in the global healthcare industry and other industries that require high levels of contamination control has grown steadily. This growth is attributable to the chemical’s ability to kill spores and sterilize materials, which has been demonstrated in a variety of practical applications. Properly used, H₂O₂ is an effective sterilant capable of efficient and rapid elimination of contaminating microbes. Some difficulties have  been associated with the implementation of H₂O₂ processes in the healthcare field although these issues appear to have been avoided in commercially sterile food and beverage manufacture. Specifically, persistent problems regarding the development of H₂O₂ processes and their subsequent validation have been reported. The author discusses the technical issues associated with achieving lethal concentrations of H₂O₂ delivered in vaporous form on decontamination targets, explores the core scientific principles behind H₂O₂‘s use in decontamination and sterilization, and provides experience-based solutions to frequently encountered operational issues.

Hydrogen peroxide (H₂O₂) is an extremely powerful oxidant that is capable of effectively killing resistant spore-forming bacteria over a wide range of concentrations; at concentrations of 3% or less, it is suitable for use as a topical antiseptic (1). H₂O₂ has been accepted by both FDA and the US Environmental Protection Agency (EPA) as a sterilizing agent for many years (2, 3). In the food industry, H₂O₂ is widely used to sterilize containers, closures, and aseptic chambers (i.e., isolators) used for manufacturing low-acid and dairy-based beverages as well as other applications (4).

The potency of H₂O₂ as a sterilant and its usefulness in a broad range of antimicrobial applications are beyond dispute. The problems associated with vaporized H₂O₂ processes in the healthcare industry lie in fundamental misunderstandings concerning physicochemical characteristics of H₂O₂ sterilization. These errors profoundly influence real-world H₂O₂applications.  

What is Vapor?
There are three primary states of matter--solid, liquid, and gas. The term “vapor” is defined in several ways. Scientifically, a vapor is a gas at a temperature lower than its critical point; a vapor is a gas phase where the same substance can also exist as a liquid. An example is atmospheric water vapor. At temperatures above the dew point, water in the atmosphere is a gas. As the temperature is lowered through the dew point, the gaseous water condenses to form a fog or mist, or it can condense and form liquid water on a cold surface. Another definition of vapor is visible moisture in the air, as in fog or steam—a system in which a liquid is suspended in a gas.

Figure 1 shows water in various phases: the lake, the dense fog at the foot of the mountain, the wisps of cloud, and the blue sky above. The lake is certainly liquid water; the blue sky is just as clearly a gas which contains water in the gaseous state. The fog or cloud in the center is a mixture of a gas phase (comprised of nitrogen, oxygen, water, carbon dioxide, and trace amounts of inert gases) and a suspended liquid phase (small droplets of water). The density of the fog or cloud varies with its temperature. It is thickest (i.e., suspending the most liquid) near the base of the mountain where it is coldest. It is clearly less dense, with less suspended water droplets near the top of the image where the temperature is higher.

One of the major difficulties with hydrogen-peroxide (H₂O₂) processes is the use of a vapor for delivery of H₂O₂ and water (H₂O) to the target chamber. It must be understood that a vapor is a mixture of air and liquid that is present within the chamber. In decontamination or sterilization using H₂O₂, the liquid phase is comprised of both H₂O₂ and H₂O, and the concentration of each in the gas and suspended liquid state can vary across the system.

James P. Agalloco and James P. Akers

Understanding vapors
To fully understand the physical factors that affect the distribution of H₂O₂ in the vapor phase, one must consider the factors that affect vapors in general and the factors that allow them to exist in air, which is the medium in which H₂O₂ in the vapor phase is distributed within a decontamination target. Air contains varying, but small, amounts of water in the vapor phase, which is described using the term relative humidity (RH). An important factor in the distribution of a chemical is the dew point. The dew point is, in simplest terms, a function of both concentration and temperature. When the concentration of water exceeds the saturation point at a particular temperature, condensation occurs. The gaseous water converts to the liquid phase, and droplets of liquid water may appear. On the other hand, if the water concentration is below the saturation point, it will remain in the gas phase. When the temperature of the air is actively lowered (or simply drops as a function of thermodynamics) below the dew point, some portion of the water (H₂O) present as a gas mixed with air condenses and forms liquid droplets. We observe this as clouds, dew, fog, or frost.

The typical H₂O₂ process
The process that most H₂O₂ generator and isolator manufacturers use for H₂O₂ introduction is one in which a hot air stream is used to introduce a heated H₂O₂/H₂O gas into the target environment, which may be an aseptic chamber or isolator. Within the generator, the temperature of the air/H₂O₂/H₂O mixture is sufficiently high that all three materials are in a gaseous state. The hot air is conventionally at temperatures in excess of 100 °C, which takes advantage of the respective boiling points of the pure components (i.e., H₂O = 100 °C, H₂O₂ = 150.2 °C, and a 30-35% aqueous solution of H₂O₂ = approximately 108 °C). At these temperatures, both H₂O₂ and H₂O are present as gases and are carried into the target vessel with the hot air. The H₂O₂/H₂O is supplied as an aqueous solution of H₂O₂in varying percentages typically ranging from 31% to 50% H₂O₂. At typical room temperatures, each of these solutions is predominantly liquid, and the headspace air within the closed containers has a small amount of gas phase H₂O₂/H₂O that is in equilibrium with the liquid.

If the concentration remains below the saturation point upon introduction into the target environment, then both the H₂O₂ and H₂O will remain in the gas phase. When the hot and relatively humid gas mixture from a H₂O₂ generator is introduced to the target chamber, it will encounter colder air as well as ambient temperature surfaces of the chamber and materials inside it. As the hot gas mixture cools to the temperature of the chamber, it will fall below the dew-point temperature of both H₂O₂/H₂O, and some portion of these materials will condense on the surfaces as liquids. In effect, the H₂O₂/H₂O are returning to their initial equilibrium state of liquids in equilibrium with the adjacent gas, which they possessed before being converted to a gas in the generator.

Condensation that forms on the surfaces will tend to be nonuniform in concentration across the chamber for several reasons:

• The H₂O₂ will condense first due to its lower equilibrium vapor pressure (i.e., lower dew point) relative to H₂O.

• The temperature in the system may be non-uniform across the chamber and is generally hottest near the inlet where the hot gas mixture is introduced; for the purposes of vapor-phase hydrogen peroxide (VPHP) technology, ± 2.5 °C can be considered effectively uniform.

• The continued introduction of the hot gas mixture into the chamber, in which VPHP generators rely on continuous replenishment of mixture vapor, results in a slow increase in temperature within the chamber. This effect is more pronounced in smaller enclosures and those with relatively low mass.

• In larger enclosures, the amount of heat added by the hot air stream laden with H₂O₂/H₂O will have little impact on temperatures remote from the injection port.  

• Where the localized temperature within the enclosure is low enough and concentrations of H₂O₂ and H₂O are high enough, they will condense. Many present-day H₂O₂ generator systems are designed such that the process relies on the presence of condensation. In these cases, one should recognize that the heated gas or vapor is used only as a convenient delivery system for the H₂O₂/H₂O to the target environment. The sterilization or decontamination is accomplished by H₂O₂ in the form of liquid condensate on surfaces.

• Depending upon the decontamination approach used, H₂O₂/H₂O introduction during the process dwell period can be continuous, intermittent, or absent entirely. In cases where the hot air/vapor stream is present only during a comparatively short initial introduction period, the effects of the hot air stream on target chamber temperatures will be less profound.  

• Chambers with a large number of objects to be decontaminated have added surfaces upon which condensate may accumulate. As the load size increases, the amount of H₂O₂ added and/or the process dwell period may need to be increased to ensure condensation on all target surfaces.

The extent of condensation that occurs depends upon the temperature (i.e., colder locations will have more condensation), the concentration or amount of H₂O₂/H₂O introduced (and removed if a circulating process is used), the size of the enclosure (i.e., affects the surface/volume ratio), and the quantity of material within the chamber (i.e., adds to the surface area).

Phase states in the enclosure
In must be understood that the enclosure will contain a mixture of air/H₂O₂/H₂O internally, with some of the H₂O₂/H₂O in a liquid state on surfaces and the remainder in the gas phase. There is no simple means to establish how much H₂O₂/H₂O is in each phase or where in the chamber a particular phase is present. Additionally one cannot know the percentage of H₂O₂ or H₂O at any single location, and certainly not at every location within the enclosure. The Gibbs Phase rule makes it clear that conditions can vary across the system (see Equation 1).

F = C - P + 2 = 3 - 2 + 2 = 3  (Eq. 1)

where F = number of degrees of freedom (i.e., concentration, temperature, pressure), C = number of components in the system, and P = number of phases in the system.

Almost nothing is known with certainty with respect to concentration and location. There is, however, one constant in the process: H₂O₂ is lethal to microorganisms in both the gas and liquid phases. It is reasonable to assume that liquid-phase kill will be somewhat faster than the gas-phase kill for two important reasons as further outlined:

• The concentration of H₂O₂ in the liquid phase will always be higher. A 35% H₂O₂ mixture will have equilibrium concentrations of H₂O₂ of ~2% in the gas phase and ~79% in the liquid phase (5).

• The presence of adequate moisture at the point of sterilization is certain in liquids, as H₂O is the other component of the liquid phase.

An older reference describes more rapid kill occurring with H₂O₂ in a gas-phase process compared to a liquid-phase process (6). This reference identifies a gas-phase process at 25 °C, with no mention of any liquid H₂O₂ present. At that temperature, however, H₂O₂ is a liquid, so there must be some liquid H₂O₂ in equilibrium with the gas. There is no means to establish that the kill in this “gas” process was actually accomplished in that phase. It is more likely that the cited kill was accomplished in the liquid phase. Misinterpreting what is actually “vapor” as a “gas” has led to the erroneous belief that gaseous-phase kill is more rapid than liquid-phase kill.

The expected microbial kill rates in the system might appear as shown in Figure 1, which visualizes H₂O₂ sterilization as a process that occurs within a band, bounded by the extremes of liquid and gas-phase kill. Figure 1 represents what is believed to occur and does not reflect any specific H₂O₂ process. The absolute slopes of the death curves are unknown. Given that the localized concentrations in both phases are variable due to temperature differences and proximity to the inlet with its heated air supply, it must be recognized that there will be different kill rates in different locations in both the liquid and gas phases. Figure 1 represents what might occur at a single point within the chamber; similar appearing death curves with differing slopes can be considered for other locations where the local conditions are different. These variations are the underlying cause of the variable performance experienced when using vapor-phase H₂O₂ as a lethal process.

Figure 1: Estimated relative kill rates in liquid and gas phases; the exact kill rates and their differences are unknown. (Figure courtesy of the authors.)

D-values for H₂O₂  decontamination
The death curves in Figure 1 seem to show that a D-value (or an approximation of one) could be established against a challenge microorganism for the combined processes. That assumption is faulty because there is no way of establishing what conditions (e.g., phase, concentration, or humidity) are present in the system at the point where the microorganism is killed. D-value determination requires knowledge of the specific lethal conditions to which a microorganism is exposed. In a single-phase sterilization process, gas or liquid, information on concentration of the agent, humidity (assumed at 100% for liquid processes), and temperature is readily determined. In the context of H₂O₂, this is easiest for liquids, and published D-values for Geobacillus stearothermophilus in various H₂O₂/H₂O liquid solutions are available (1). These liquid phase D-values demonstrate extremely rapid kill (in seconds) at even modest H₂O₂ concentrations (7). At the estimated concentrations where condensation first occurs in vapor H₂O₂ processes, the D-values should be lower as the concentration will be substantially higher than that published in the literature. Unfortunately, no comparable data are available on H₂O₂, where a strictly gas-phase process is present. Thus, any labeled “D-values” for vapor H₂O₂ biological indicators must be considered nothing more than an approximation as the killing conditions are unknown. The conditions of kill may be consistent enough that they could be replicated in an independent study in the same test system. What cannot be established from these labeled “D-values“ is how that same biological indicator will respond in a different environment where the conditions are also unknown and most likely substantially different.

In the 20-plus years that this industry has been using H₂O₂ decontamination, a BIER (biological indicator evaluation resistometer) vessel for H₂O₂ has not been developed as a standard for compendial or routine use. The same conundrum faced with respect to variable and unknown biphasic conditions in a larger system has prevented the development of a H₂O₂ BIER. The absence of a BIER vessel and, thus, a fully useable “D-value” for H₂O₂ biological indicators has caused some difficulties. What can be established from the vendor “D-value” is the relative resistance of one lot to another from the same vendor. How any individual lot will perform under different conditions is something the user must determine for each application.

One suggested approach to get beyond this lack of a definitive D-value for a biological indicator is to establish a process or system “D-value” for a biological indicator within a large enclosure and rely upon that as the basis for destruction in the system rather than the vendor’s reported value. This approach presumes that the conditions used to establish the process/system “D-value” are representative of the entire system. That assumption is decidedly not the case, nor is it known whether the location(s) chosen for the process “D-value” determination are best case or worst case with respect to kill across the chamber. A number, which is not a D-value in the strict sense, can be calculated, but the utility of that number in any estimation kill rate across the chamber is essentially nil.  

Reports of vapor-phase “D-value” variations as a consequence of different substrates must also be recognized as uncertain (8, 9). Because there is no objective biological indicator evaluation method available, published “D-values” are not standardized and thus of very limited use. Unless the concentration on the individual surfaces tested can be known and demonstrated to be constant, any hint that the substrate variations are meaningful must be viewed with some skepticism. There is also some published evidence that “D-values” may vary with spore concentration applied to the carrier material, which means kill may not be linear with concentration. That represents a serious flaw in the use of any biological indicator.

Is safety a concern with H₂O₂?
Given the rapid kill observed in the H₂O₂ liquid phase, the difficulties in attaining consistent kill with H₂O₂ vapor processes can only be explained by a lack of adequate condensation, for there is little doubt then when condensation does occur, kill will be quite rapid (10). Many of the newer generator designs, either freestanding or integrated into enclosures, rely on condensation to decontaminate/sterilize extremely rapidly.

Since the rapid kill provided by liquid H₂O₂ is well documented, why has industry been cautioned to avoid condensation in vapor H₂O₂ processes? The answer lies in the early teachings of AMSCO (now Steris) when the first H₂O₂ generator was introduced in the late 1980s. Caution was routinely raised regarding the potential hazards of high concentrations of liquid H₂O₂. (The H₂O₂ concentration in the gas phase at ambient temperature will always be substantially lower than its equilibrium concentration in the liquid phase.) The relevant safety issues with the use of H₂O₂ vapors are:

• Explosive vapors. The caution here relates to concentrations of > 70% H₂O₂ giving off explosive vapors at temperatures greater than 70 °C (11). If this situation were to occur anywhere in vapor processes, the generators themselves would represent the greatest risk. Temperatures inside enclosures rarely exceed 30 °C, and thus the likelihood of this presenting a real-world problem during a sterilization process is unlikely.

• Hazardous reactions. There are reports of H₂O₂ reacting with greases, alcohols, ketones, carboxylic acids (particularly acetic acid), amines, and phosphorus. Small amounts of other materials that contain catalysts (e.g., silver, lead, copper, chromium, mercury, and iron oxide rust) can cause rapid decomposition and an explosive pressure rupture of the containing vessel if it is not properly vented (12). None of these compounds and materials is typically present in pharmaceutical enclosures.  

• Corrosivity. This is possible with some materials, but the typical stainless steel, glass, and other materials exposed to H₂O₂ are known to be compatible and are chosen explicitly for that purpose. The chemical compatibility of H₂O₂/H₂O solutions is well documented.

• Worker safety. The US Occupational Safety and Health Administration has established an 8-hour, time-weighted average for exposure to H₂O₂ of 1 ppm, with an immediate hazard in the presence of concentrations greater than 75 ppm (13, 14). This limit is managed in pharmaceutical facilities through external alarms in the surrounding areas and requirements for aeration before personnel or material exposure.

While there is a need for caution with respect to the use of vapor phase H₂O₂, undue concern is unwarranted. In more than 20 years of use in the global industry, there have been no reported incidents of personal injury or equipment damage associated with this process.

Claims that vapor-phase H₂O₂ processes do not result in condensation are speculative. The laws of physics and temperature within enclosures are such that some measure of condensation will always occur, and in many recent equipment and process designs the creation of condensation is intentional. Thus, within the context of real-world experience, the safety issues associated with vapor H₂O₂ systems where condensation is present appear to be adequately managed, assuming appropriate worker-safety precautions are maintained.

Limitations of multipoint process-control measurements
FDA’s Guideline on Sterile Drug Products Produced by Aseptic Processing recommends: “The uniform distribution of a defined concentration of decontaminating agent should also be evaluated as part of these studies” (15). This suggestion is made without reference to a specific methodology that could be employed. There is no technology that could address this expectation throughout a two-phase environment. Nor would the resulting data on concentration in the gas phase be useful in correlating to microbial kill on surfaces. When appropriate amounts of H₂O₂ are used for decontamination or sterilization, some of the available instruments, such as those that rely on near-infrared transmission, are unusable due to condensation on the lenses.  Because accurate measurement is not possible, chemical indicators provide the only widely available means to confirm that H₂O₂ is, or was, present at a specific location.

Problems in an unsteady-state process
The introduction of H₂O₂ into a room-temperature enclosure uses vapor-process heating to convert the liquid solution into a gas for mixing and distribution in hot air. The temperatures in vaporizers are in the range of 105-150 °C. This high temperature results in some localized heating of the enclosure, primarily in locations close to the entry point of the heated materials. The effects of this heat input are multiple:

• Temperatures during the process will change over its duration with the greatest impact found in locations nearest the infeed locations. This heating is more pronounced in smaller, flexible-wall and lightly loaded enclosures where there is less overall mass.

• The resulting changes in temperature will result in varying amounts of condensation (and thus kill) across the enclosure (and also varying over the duration of the process dwell period at a single location).

• The conditions close to the infeed are more likely to remain in the gas phase throughout the process, which can result in less condensation (if any) and potentially slower kill rates in those locales. In one project, the authors observed that a biological indicator location directly beneath the supply port was repeatedly found to be the only location where the biological indicator could not be killed.

These phenomena are more problematic in those generators where H₂O₂ is fed and removed throughout the process. Systems that operate in a fill-and-soak mode may attain equilibrium conditions within the targeted volume.

The negative consequences of the unsteady-state nature of vapor-phase H₂O₂ processes are unavoidable in recirculating systems. The only means to establish a consistent process is to use enough H₂O₂ that even the warmest locations attain some measure of condensation. This solution is more easily accomplished in the non-circulating systems.

Penetration and adsorption by H₂O₂
Years of experience with vapor-phase H₂O₂ processes have shown how best to address the adverse impact of its adsorption as further explained:

• H₂O₂ can penetrate high-density polyethylene fiber materials (Tyvek, Dupont), which are primary packaging for many presterilized items. Tyvek-wrapped materials of larger dimension may prove difficult to aerate because there is no internal turbulence to aid in aeration.

• Some polymeric materials will adsorb H₂O₂ readily and desorb it very slowly. A small (1 ft3), empty isolator manufactured from polycarbonate (Lexan, SABIC Innovative Plastics) was found to require more than 24 h of aeration (16). Careful attention to materials of construction is important to reduce any unintended adsorption.

• Typical sterile-product container materials (e.g., glass vial, elastomeric closure, aluminum crimp) and many polymeric materials are largely impervious to H₂O₂.

• Shorter cycle dwell times allowing less overall time for adsorption are generally preferable.

• Aeration periods can ordinarily be improved by additional air changes.

• Liquid H₂O₂ penetration through Tyvek has not been documented.

• Some biological materials have demonstrated extreme sensitivity to H₂O₂ requiring aeration to levels in the parts-per-billion range (17).

The adverse consequences of decontamination and sterilization processes should be considered in the development and control of every process. Vapor-phaseH₂O₂ processes, because of their dual-phase nature, present new challenges. Were other gases to be used, similar, but different, concerns would present themselves and appropriate solutions would be identified. A more penetrating agent would only increase the penetration/aeration difficulties encountered, so while H₂O₂ penetration/absorption/desorption is a problem, the situation might be worse with alternative materials.

Biological indicator issues
Difficulties encountered in the destruction of biological indicators have been commonly reported and are so well known that there are some who doubt the efficacy of H₂O₂ as a sterilant. These problems are multifaceted but resolvable when the sterilization process is properly established.

First, H₂O₂decontamination and sterilization must be understood as a two-phase system. Considering it as a single, gas-phase process has caused more difficulties than anything else. The variability demonstrated in lethality is the direct result of applying process constraints that are suitable for a gas process but inadequate for two-phase H₂O₂ processes. Adapting process models and approaches from the most common gas sterilant, ethylene oxide (EO), to a vapor process created much of the problem. The largest flaw in this thinking is the deliberate avoidance of condensation in endeavoring to make what must be a two-phase vapor process into one that operates in a single phase. Some wrong assumptions are:

• Process conditions (e.g., temperature, relative humidity, and H₂O₂ concentration) throughout the enclosure can be made uniform.

• Condensation is to be avoided at all times.

• Comparatively gentle mixing of the enclosure is adequate.

• D-values for challenge microorganisms can be established.

In the actual two-phase H₂O₂ process, none of these assumptions is correct or attainable at the present time. These assumptions led to the establishment of vapor processes that are inadequate for their intended purpose. They do not adequately induce condensation or use sufficient mixing and thus fail to deliver reasonably consistent conditions throughout the enclosure. The experienced difficulties are a consequence of poor cycle design and not problems with the lethality of H₂O₂.  

Second, biological indicators must be specifically designed for the intended process. While there have been attempts at this design, what has been accomplished is largely empirical. The methods used for manufacturing H₂O₂ biological indicators may be identical to those used for other sterilization processes, but because correlation to actual process resistance is lacking, the process suggestions inferred from labeled resistance values are essentially unusable. In the absence of a BIER (and thus truly reproducible biological indicator resistance), the typical biological indicator process response can not be expected for vapor-phase H₂O₂ processes.  

The most important attribute of any biological indicator is its reproducible resistance to the intended process. There is no established D-value method, which severely limits the certainty of process understanding and biological-indicator design and selection. Variable results with biological indicators could be attributable to either variations in the biological-indicator resistance or variation in the conditions resulting from poorly conceived controls for a complex process. Lacking a biological indicator whose response to the process is precise, vapor-phase decontamination and sterilization becomes a more challenging process to control.    

Third, there is a demonstrated biological indicator concentration effect associated with the H₂O₂ processes unlike that seen in other sterilization processes. Biological indicators with a higher initial population have proven more difficult to kill with H₂O₂ than would be expected based upon the results of the same lot at a lower concentration (18). This phenomenon contradicts the core principle in all sterilization processes that microorganisms die at a constant logarithmic rate regardless of population. Occurrence of this phenomenon in H₂O₂ processes can be attributed to several possible causes:

• Excess cellular debris and perhaps both organic and inorganic salts provide a protective layer of spores. This problem is somewhat exacerbated by the use of stainless-steel coupons that allow these materials to remain on the surface adjacent to the spores.

• The use of biological-indicator populations above what is necessary for process certainty creates potential for clumping of spores through which H₂O₂ penetration may not readily occur. FDA, US Pharmacopeia, EMA, and the Parenteral Drug Association all accept biological indicator log reductions of 4-6 logs, where surface sterilization is not the objective (15, 19-21).  

• Some users adhere to an incorrect belief that a 106 spore population of the resistant biological indicator must be used to demonstrate a probability of nonsterile unit (PNSU) of 1x10-6.

• Inadequate processes that rely more on gas-phase kill than the substantially more lethal liquid-phase kill only serve to exacerbate all of the above problems.

All of these are correctable. Using a lower population biological indicator eliminates the first two of these difficulties. A hundred-fold reduction in spore population reduces the amount of debris present at the edge of the biological indicator drop and eliminates spore clumping significantly. This single change would result in more linear death curves than what has been evidenced. The third difficulty is a common mistake that is all too prevalent in the healthcare industry and has no basis in fact (22). The food industry has used H₂O₂ successfully for sterilization for many years and operates without this artificial and erroneous expectation. The last issue is an artifact of the limited process understanding still prevalent on many existing H₂O₂ processes. In cases for which condensation is actively promoted in the process, fewer problems with sterilization are encountered.

Much has been made recently of so-called “rogue” biological indicators. These rogues (i.e., outliers) are presumably biological indicators that failed to conform to the user’s expectations of their demise. There is little doubt that the production of spore crops, substrate selection, and the manufacture of biological indicators could result in clumping and encapsulation in contaminants that could result in a lack of uniform performance (23). Properly manufactured biological indicators should be largely free of outliers. Greater frequency of outliers detected in vapor H₂O₂ processes seems to be the result of poor understanding of vapor-phase H₂O₂ that results in marginally lethal processes and the creation of biofilms and clumps of spores on stainless steel at 106 concentrations, which result in what are effectively false-positive biological indicators that do not represent the elimination of normal flora at more diffuse concentrations.

Summary and recommendations
The successful use of any decontamination or sterilization process requires a thorough understanding of the underlying principles of the process with particular attention to those aspects that differentiate it from other methods because these represent potential new learning. The two-phase nature of the vapor-phase H₂O₂ process introduces complexities that, if not well understood, can prevent successful use. The healthcare industry has experienced considerable difficulty in the implementation of this process.

The greatest improvements in operating these processes can be obtained through the use of conditions that force some measure of condensation and by recognition that the desired log reduction of these processes need not be excessive given the end use of the enclosure. Only product contact parts must be sterilized, and shifting attention to those locales within the enclosure alone would result in substantial improvements in process outcomes.

References

1. S. Block, “Peroxygen Compounds,” in Disinfection, Sterilization & Preservation, S. Block, Ed. (Lippincott, Williams, and Wilkins, Philadelphia, PA, 5th ed., 2001), pp. 185-204.
2. CFR Title 21, Part 178.1005, Hydrogen Peroxide Solution (Government Printing Office, Washington, DC), pp. 363-364.
3. EPA, List A: Antimicrobial Products Registered with the EPA as Sterilizers, (Arlington, VA, Dec. 2011).
4. M. Ansari and A. Datta, Trans. IChemE 81 (C) 57-65 (2003).
5. C. Hultman, A. Hill, and G. McDonnell, Pharm. Eng. 27 (1) 23-30 (2007).
6. S. Block, “Peroxygen Compounds,” in Disinfection, Sterilization & Preservation, S. Block, Ed. (Lea & Febiger, Philadelphia, PA, 4th ed., 1991), pp. 167-181.
7. J. L. Sagripanti and A. Bonifacino, Appl. Environ. Microbiol. 65 (9) 4255-4260 (1999).
8. G. B. McNassor, Proceedings of the ISPE Barrier Isolation Technology Conference (Arlington, VA, 1999).
9. S. M. Davenport, Proceedings of the ISPE Barrier Isolation Technology Conference (Arlington, VA, 1999).
10. J. Rauschnabel, “Vapour Phase Hydrogen Peroxide-Process Development, Qualification and Validation,” presentation at the PDA Europe meeting (Berlin, Germany, 2008).
11. Arkema, “Hydrogen Peroxide 70% (all grades), Product code 04504,” MSDS, Oct., 2010.
12. US Peroxide, “Hydrogen Peroxide Safety and Handling Guidelines,” www.H₂O₂.com/technical-library/default.aspx?pid=66&name=Safety-amp-Handling, accessed July 29, 2013.
13. CFR Title 29, Part 1910.1000, Table Z-1 (Government Printing Office, Washington, DC), pp 7-19.
14. CFR Title 29, Standard 1910.103, “Hazardous Materials, Hydrogen” (Government Printing Office, Washington, DC), www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=9749, accessed Aug. 8, 2013.
15. FDA, Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing, (Rockville, MD, Sept., 2004).
16. J. Agalloco, private communication (1995).
17. A. Hui, S. Yim, K. Eppler, and X. Lam, Proceedings of ISPE Brussels Conference on Risk-based Control Strategies in Pharmaceutical Industries (Brussels, Sept. 2010).
18. E. Gillitzer, Spore News, Mesa Laboratories, 9 (3) 1-3 (2012).
19. USP General Chapter <1208>, “Sterility Testing--Validation Of Isolator Systems“  (US Pharmacopeial Convention, Rockville, MD, 2011).
20. PIC/S, “Isolators Used For Aseptic Processing And Sterility Testing,” PI 014-2 (Geneva, Switzerland, 2004).
21. PDA, “TR #34, Design and Validation of Isolator Systems for the Manufacturing and Testing of Health Care Products,” (Bethesda, MD, 2001).
22. J. Agalloco, J. Akers, and R. Madsen, PDA J. of Pharm. Sci. Technol. 63 (2) 89-102 (2009).
23. K. McCauley and J. Gillis, “Aseptic Processing” supplement to Pharm. Technol. 31 s26-31 (2007).

About the Authors
James P. Agalloco is president of Agalloco & Associates, P.O. Box 899, Belle Mead, NJ 08502, tel. 908.874.7558, jagalloco@aol.com. He is also a member of Pharmaceutical Technology’s editorial advisory board.

James E. Akers, PhD is president of Akers Kennedy and Associates.  

Submitted: Feb. 1, 2013. Accepted: Mar. 1, 2013.