James E. Akers is the president of Akers Kennedy & Associates, PO Box 22562, Kansas City, MO 64113, akainckc@aol.com.

James E. Akers

With the publicity surrounding the recovery of mold from pharmacy compounding facilities and formulated products connected to patient fungal infections and deaths, there has been a heightened awareness and concern for fungal recoveries in all manufacturing facilities. One comment often heard is that the presence of bacteria in a drug or biological manufacturing facility is expected, because these microorganisms are constituents of normal human flora, but the presence of mold in such facilities is presumed to be atypical as fungi are not considered constituents of human flora. Molds, however, are often found on healthy human skin and associated with hair. At any given point in time, humans may carry dermatophytic molds typically of the genera   

. Although mold are not present in or on humans in the numbers that bacteria are, they certainly can be found.

Humans cannot live in a world that contains at all times substantial concentrations of airborne mold and not be expected to carry some mold along with them as they move from point to point. Mold will be present on clothes, skin, and human hair as a direct consequence of the level of airborne mold that typical individuals inevitably encounter on a daily basis. Reasonable precautions upon moving to sections of facilities where products are aseptically manufactured should include changing into captive shoes and clothing and donning of hair and shoe covers. These precautions, however, cannot be reasonably expected to completely obviate the possibility of random, low-level mold recovery during routine monitoring. It is reasonable to expect that recovery rates for bacteria should be higher than for mold, but it is unreasonable to think that mold should never be recovered. An enforced zero recovery target level for mold is unreasonable, unscientific, and unnecessary.


The presence of mold has become a key component in efforts to achieve appropriate indoor air quality in all environments inhabited by humans. Mold contamination in residential environments or a workplace can represent a significant health risk if conditions are such that mold infection of the building has occurred.

To this end, general classifications have been established regarding acceptable levels of mold that can be present in residential buildings. Baxter, et al. (1) suggests levels for two mold genera that are those most likely to produce respiratory allergies, which is the principle concern with airborne mold. 

 are associated, in sensitive individuals, with allergic rhinitis, bronchial asthma, and alveolitis. Generally, total spore counts of 1200 spores/m

 are considered safe or “clean”, while counts above 1300 spores/m

 are indicative of a “moldy” environment. Concentrations of Aspergillus and Penicillium below 750 spores/m

 are considered generally clean, and counts above 900 spores/m

 are considered moldy. Concentrations of ascospores or basidiospores below 1200 spores/m

 are considered generally clean, and counts above 1300 spores/m

It should be noted that the devices and methods used to recover molds in residential and commercial buildings, including those with water damage, are a gross measure of the relative types and amount of mold in those environments. They are generally performed by environmental engineers, not microbiologists. The quantitation is typically performed using a compound light or phase contrast microscope physcically counting a sample trace on a tape at 300x or 600x power. The identifications are performed based on “spore type.”

The measure of airborne mold spores per unit volume determined by such methods, therefore, will not precisely correlate with recovery and growth on microbiological media sampled with a calibrated instrument, with results expressed as colony forming units (CFU). Generally, plating efficiency would be substantially less than mold spores/m

 as the latter will reflect spores that are not viable or not recoverable on a given growth medium. It is well documented that mold spores are ubiquitous, and those mold spores present at low levels in uncontrolled indoor environments pose an insubstantial risk to humans or products made in those environments.

Mold contamination risk in drug production


Fungi produce substances, in the form of secondary metabolites, which can be toxic to humans and animals. The concern in drug and biological manufacturing most often mentioned relates to aflatoxins and other mycotoxins, some of which may be tumorigenic in animals. Historically, these mycotoxins have found their way into animal feed and have had, at times, a devastating effect on the commercial poultry industry. There are concerns that mycotoxin-related disease may be under-reported in human populations particularly in less developed regions of the world. There is little doubt that vigilance is required to ensure that mycotoxins do not present a risk in human and animal food and food ingredients.

Of interest to readers of this article, however, is the risk that accrues to human healthcare product manufacturing as a result of mycotoxin production. It has proven difficult to determine a level of mold spore inhalation or of mold in or on materials that can be associated with clear medical risk relating to mycotoxins. There is no medical or toxicological evidence that a low-level environmental mold recovery in a production environment would result in greater risk to a product or end-user than low-level bacterial contamination. Certainly, airborne mold found intermittently and at low frequency is not indicative of mold colonization. Low-level mold likely finds its way into facility environments as a passenger on personnel entering facilities as well as on materials coming into and warehoused within facilities.

It is reasonable to expect firms to take strong countermeasures to minimize the entry of mold into a facility.  The ubiquity of mold in the external environment and the characteristically high levels of mold spores in air make it impossible to prevent mold entry into any indoor environment with absolute certainty. Thus, it is unreasonable that low-level mold recoveries should result in regulatory comment or enforcement. Also, there is no microbiological or medical reason to react strongly to the recovery of mold at intermittent frequencies and at low levels.

Identifying, remediating,   and preventing facility infection


To reach spore levels, to say nothing of toxin levels relevant as health risks, mold proliferation (i.e., growth) is necessary. This condition would require a production facility to be infected by mold. Conditions that allow a production facility to become infected are always objectionable. A reasonable question is: How would one diagnose the condition of facility infection?

 A facility infection is indicated by the ongoing and consistent presence of mold. Given the microbiological assessments used in drug and biological production facilities, this means high viable recovery levels. There have been reports of building infection even in aseptic manufacturing areas, but fortunately these have occurred infrequently. Those that have been reported were due to water leaks from defective pipes or roofs that soiled wall materials made of sheetrock or drywall panels, which are an excellent food source for mold. The normal course of this type of facility infection is that high counts are recovered over a discrete period of time; the microbiological control team then recommends disinfection of the facility. Disinfection succeeds in a temporary diminution in frequency of recovery and count, but within typically 7-10 days a subsequent bloom is observed, manifested again by atypically frequent recoveries. Additional cycles of disinfection produce similar outcomes, with high counts again appearing within typically one to two weeks post disinfectant treatment. If a sheetrock wall is infected with mold, the only effective remediation is to demolish the walls and reconstruct them with a mold-inhibiting material, such as Plascore pharmaceutical cleanroom panels or fiberglass.

The recovery levels observed in residential and uncontrolled commercial building infection are unusually high and far exceed the total microbial recovery levels recommended in 

 for classified manufacturing environments (2, 3). Should such conditions be observed in controlled pharmaceutical facilities, an extensive diagnostic program and further steps, including mold remediation, may be necessary. In the event that remediation is necessary, the work that must be done is often extensive and may require considerable demolition and reconstruction. A mold remediation will be disruptive to operations, and may have impact upon manufactured product or work in progress.

Limited facility infection. Mold infections within manufacturing facilities and laboratories may be limited to equipment such as refrigerators or incubators. Again, these are not transient, low-level contamination recoveries, but instead are findings reflective of active mold growth. Fortunately, this is typically an easier condition to treat than widespread facility-level mold infection.

A removal of all materials from the equipment followed by careful cleaning and disinfection will, in most cases, eliminate the infection. Preventive actions include avoiding spillage of organic materials such as media or test materials and, where a spill does occur, cleaning effectively and at a suitable frequency. Also, to prevent mold infection, maintaining dry conditions is key, water spills should be removed quickly, and conditions that favor the formation of condensate should be avoided. In most cases, limited mold infection in support areas, such as laboratories, should not pose significant manufacturing risk, particularly when treated promptly and effectively.


The key risk factor regarding mold in drug and biological operations is proliferation-driven infection. Infection of facilities or equipment is easily distinguished from transient low-level contamination. Transient low-level contamination will occur infrequently and randomly. There will be no evidence of high-count levels being reached and no indication of persistence. Given the ubiquity of mold in nature and the levels with which they are present in outdoor air or in office complexes, the recovery of an occasional mold does not merit any particular concern.

On the other hand, evidence of mold proliferation indicative of infection of facilities or equipment must be taken seriously and requires the prompt implementation of corrective and preventive actions. In such cases, the potential effect of mold contamination on product should be carefully assessed.  
In the authors’ experience, mold recoveries typically occur in industry at levels far lower than bacterial recovery. Certainly, microbiologists should keep a watchful eye on patterns to ensure that they have the means to take note of changes in pattern. It is important that significant microbial changes not be missed or ignored.

2 (1) 8-18 (2005).
  2.	USP, General Chapter <1116>, Microbiological Control and Monitoring of Aseptic Processing Environments, (US Pharmacopeial Convention, Rockville, MD, 2012). 
3.	Eudralex, 

Volume 4: EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use: Annex 1: Manufacture of Sterile Medicinal Products


Jim E. Akers is president of Akers Kennedy & Associates,

.
John M. Lindsay is president of Aseptic Solutions and of the Aseptic Training Institute,

Articles

The recovery of an occasional mold does not merit any particular concern. On the other hand, evidence of mold proliferation indicative of infection of facilities or equipment must be taken seriously and requires the prompt implementation of corrective and preventive actions.

Determining Facility Mold Infection

With the publicity surrounding the recovery of mold from pharmacy compounding facilities and formulated products connected to patient fungal infections and deaths, there has been a heightened awareness and concern for fungal recoveries in all manufacturing facilities. Humans cannot live in a world that contains at all times substantial concentrations of airborne mold, however, and not be expected to carry some mold along with them as they move from point to point. Mold will be present on clothes, skin, and human hair as a direct consequence of the level of airborne mold that typical individuals inevitably encounter on a daily basis. Reasonable precautions upon moving to sections of facilities where products are aseptically manufactured should include changing into captive shoes and clothing and donning of hair and shoe covers. These precautions, however, cannot be reasonably expected to completely obviate the possibility of random, low-level mold recovery during routine monitoring. There is no medical or toxicological evidence that a low-level environmental mold recovery in a production environment would result in greater risk to a product or end-user than low-level bacterial contamination. Certainly, airborne mold found intermittently and at low frequency is not indicative of mold colonization. Low-level mold likely finds its way into facility environments as a passenger on personnel entering facilities as well as on materials coming into and warehoused within facilities.

To reach spore levels, to say nothing of toxin levels relevant as health risks, mold proliferation (i.e., growth) is necessary. This condition would require a production facility to be infected by mold. Conditions that allow a production facility to become infected are always objectionable. A reasonable question is how would one diagnose the condition of facility infection?


 A facility is infected by the ongoing and consistent presence of mold. Given the microbiological assessments used in drug and biological production facilities, this means high viable recovery levels. There have been reports of building infection even in aseptic manufacturing areas, but fortunately these have occurred infrequently. Those that have been reported were due to water leaks from defective pipes or roofs that soiled wall materials made of sheetrock or drywall panels, which are an excellent food source for mold. The normal course of this type of facility infection is that high counts are recovered over a discrete period of time; the microbiological control team then recommends disinfection of the facility. Disinfection succeeds in a temporary diminution in frequency of recovery and count, but within typically 7–10 days a subsequent bloom is observed, manifested again by atypically frequent recoveries. Additional cycles of disinfection produce similar outcomes, with high counts again appearing within typically one to two weeks post disinfectant treatment. If a sheetrock wall is infected with mold, the only effective remediation is to demolish the walls and reconstruct them with a mold-inhibiting material, such as Plascore pharmaceutical cleanroom panels or fiberglass. 
The recovery levels observed in residential and uncontrolled commercial building infection are unusually high and far exceed the total microbial recovery levels recommended in 

 for classified manufacturing environments (1, 2). Should such conditions be observed in controlled pharmaceutical facilities, an extensive diagnostic program and further steps, including mold remediation, may be necessary. In the event that remediation is necessary, the work that must be done is often extensive, and may require considerable demolition and reconstruction. A mold remediation will be disruptive to operations and may have impact upon manufactured product or work in progress.    


  Mold infections within manufacturing facilities and laboratories may be limited to equipment such as refrigerators or incubators. Again, these are not transient low-level contamination recoveries, but instead are findings reflective of active mold growth. Mold infection of incubators and/or refrigerators has not been uncommon in microbiology laboratories. Fortunately, this is typically an easier condition to treat than wider-spread facility level mold infection.  
A removal of all materials from the equipment followed by careful cleaning and disinfection will in most cases eliminate the infection. Preventive actions include avoiding spillage of organic materials such as media or test materials and where a spill does occur cleaning effectively and at a suitable frequency. Also, to prevent mold infection maintaining dry conditions is key, water spills should be removed quickly, and conditions that favor the formation of condensate should be avoided. In most cases, limited mold infection in support areas, such as laboratories, should not pose significant manufacturing risk, particularly when treated promptly and effectively.


  The key risk factor regarding mold in drug and biological operations is proliferation-driven infection. Infection of facilities or equipment is easily distinguished from transient low-level contamination. Transient low-level contamination will occur infrequently and randomly. There will be no evidence of high-count levels being reached and no indication of persistence. Given the ubiquity of mold in nature and the levels with which they are present in outdoor air or in office complexes, the recovery of an occasional mold does not merit any particular concern.  

On the other hand, evidence of mold proliferation indicative of infection of facilities or equipment must be taken seriously and requires the prompt implementation of corrective and preventive actions. In such cases, the potential effect of mold contamination on product should be carefully assessed.  

In our experience, mold recoveries are typically occurring in industry at levels far lower than bacterial recovery. Certainly, microbiologists should keep a watchful eye on patterns to ensure that they have the means to take note of changes in pattern. It is important that significant microbial changes not be missed or ignored. 


  1. USP, General Chapter <1116>, “Microbiological Control and Monitoring of Aseptic Processing Environments,” (US Pharmacopeial Convention, Rockville, MD, 2012.)
  2. Eudralex, 

For further information, read the full article, 

Understanding how to identify, remediate, and prevent facility infection is crucial for product quality.

Diagnosing Facility Mold Infection

) 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, H

 is an effective sterilant capable of efficient and rapid elimination of contaminating microbes. Some difficulties have  been associated with the implementation of H

 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 H

 processes and their subsequent validation have been reported. The author discusses the technical issues associated with achieving lethal concentrations of H

 delivered in vaporous form on decontamination targets, explores the core scientific principles behind H

‘s use in decontamination and sterilization, and provides experience-based solutions to frequently encountered operational issues.

) 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). H

 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, H

 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).

 as a sterilant and its usefulness in a broad range of antimicrobial applications are beyond dispute. The problems associated with vaporized H

 processes in the healthcare industry lie in fundamental misunderstandings concerning physicochemical characteristics of H

 sterilization. These errors profoundly influence real-world H


 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.

 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 (H

) processes is the use of a vapor for delivery of H

O) 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 H

, the liquid phase is comprised of both H

O, and the concentration of each in the gas and suspended liquid state can vary across the system.


  To fully understand the physical factors that affect the distribution of H

 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 H

 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 (H

O) present as a gas mixed with air condenses and forms liquid droplets. We observe this as clouds, dew, fog, or frost. 

 generator and isolator manufacturers use for H

 introduction is one in which a hot air stream is used to introduce a heated H

O gas into the target environment, which may be an aseptic chamber or isolator. Within the generator, the temperature of the air/H

O 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., H

 = 150.2 °C, and a 30-35% aqueous solution of H

 = approximately 108 °C). At these temperatures, both H

O are present as gases and are carried into the target vessel with the hot air. The H

O is supplied as an aqueous solution of H

in varying percentages typically ranging from 31% to 50% H

. 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 H

O that is in equilibrium with the liquid. 

If the concentration remains below the saturation point upon introduction into the target environment, then both the H

O will remain in the gas phase. When the hot and relatively humid gas mixture from a H

 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 H

O, and some portion of these materials will condense on the surfaces as liquids. In effect, the H

O 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:

 will condense first due to its lower equilibrium vapor pressure (i.e., lower dew point) relative to H

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 H

O will have little impact on temperatures remote from the injection port.  

Where the localized temperature within the enclosure is low enough and concentrations of H

O are high enough, they will condense. Many present-day H

 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 H

O to the target environment. The sterilization or decontamination is accomplished by H

 in the form of liquid condensate on surfaces.

Depending upon the decontamination approach used, H

O 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 H

 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 H

O 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).


  In must be understood that the enclosure will contain a mixture of air/H

O in a liquid state on surfaces and the remainder in the gas phase. There is no simple means to establish how much H

O is in each phase or where in the chamber a particular phase is present. Additionally one cannot know the percentage of H

O 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 

).
  
  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: H

 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:

 in the liquid phase will always be higher. A 35% H

 mixture will have equilibrium concentrations of H

 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 H

O is the other component of the liquid phase.

An older reference describes more rapid kill occurring with H

 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 H

 present. At that temperature, however, H

 is a liquid, so there must be some liquid H

 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 

 sterilization as a process that occurs within a band, bounded by the extremes of liquid and gas-phase kill. 

 represents what is believed to occur and does not reflect any specific H

 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. 

 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 H

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

 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 H

, this is easiest for liquids, and published D-values for Geobacillus stearothermophilus in various H

O liquid solutions are available (1). These liquid phase D-values demonstrate extremely rapid kill (in seconds) at even modest H

 concentrations (7). At the estimated concentrations where condensation first occurs in vapor H

 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 H

, where a strictly gas-phase process is present. Thus, any labeled “D-values” for vapor H

 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 H

 decontamination, a BIER (biological indicator evaluation resistometer) vessel for H

 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 H

 BIER. The absence of a BIER vessel and, thus, a fully useable “D-value” for H

 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. 


  Given the rapid kill observed in the H

 liquid phase, the difficulties in attaining consistent kill with H

 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 H

 is well documented, why has industry been cautioned to avoid condensation in vapor H

 processes? The answer lies in the early teachings of AMSCO (now Steris) when the first H

 generator was introduced in the late 1980s. Caution was routinely raised regarding the potential hazards of high concentrations of liquid H

 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 H

The caution here relates to concentrations of > 70% H

 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.

 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.  

 This is possible with some materials, but the typical stainless steel, glass, and other materials exposed to H

 are known to be compatible and are chosen explicitly for that purpose. The chemical compatibility of H

 The US Occupational Safety and Health Administration has established an 8-hour, time-weighted average for exposure to H

 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 H

, 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. 

 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 H

 systems where condensation is present appear to be adequately managed, assuming appropriate worker-safety precautions are maintained. 

Limitations of multipoint process-control measurements

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 H

 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 H

 is, or was, present at a specific location. 

 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 H

 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 H

 processes are unavoidable in recirculating systems. The only means to establish a consistent process is to use enough H

 that even the warmest locations attain some measure of condensation. This solution is more easily accomplished in the non-circulating systems.


  Years of experience with vapor-phase H

 processes have shown how best to address the adverse impact of its adsorption as further explained: 

 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.

 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 H

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

Aeration periods can ordinarily be improved by additional air changes.

 penetration through Tyvek has not been documented.

Some biological materials have demonstrated extreme sensitivity to H

 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-phaseH

 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 H

 penetration/absorption/desorption is a problem, the situation might be worse with alternative materials.


  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 H

 as a sterilant. These problems are multifaceted but resolvable when the sterilization process is properly established.

decontamination 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 H

 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 H

 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.

 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 H

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 H

 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 H

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 H

Vaporous hydrogen peroxide, used for sterilization and decontamination, is highly potent but presents implementation challenges.