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Special Types of Environmental Monitoring
Chapter
11
Special Types of Environmental Monitoring CHAPTER OUTLINE
Introduction 179 Monitoring to Support Parametric Release 180 The Need for Anaerobic Monitoring? 182 The Need for Psychrophilic Monitoring? 183 The Need for Thermophilic Monitoring? 186 Compressed Gas Monitoring 187 Microbial Survival in Compressed Gases 189 Microbiological Requirements 190 Bacterial Endotoxin and Compressed Gases 192
Monitoring Sterility Test Environments 192 Monitoring Microbiology Laboratories 194 Test Controls 194 Summary 196 References 197
INTRODUCTION The purpose of this chapter is to look at environmental monitoring, in support of biocontamination control, from a more applied perspective. This includes the use of parametric release, which can be used for terminally sterilized products. Parametric release is a sterility assurance release program where demonstrated control of the sterilization process enables an organization to use defined critical process controls, in lieu of the sterility test. The chapter considers how the inclusion of test controls as part of the environmental monitoring regime can provide robustness as well as useful information should an out-of-limits result be obtained as to whether that result was or was not the consequence of laboratory error. Biocontamination Control for Pharmaceuticals and Healthcare. https://doi.org/10.1016/B978-0-12-814911-9.00011-0 # 2019 Elsevier Inc. All rights reserved.
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Among the “special” types of environmental monitoring performed the chapter looks at the end for (and more strongly the need against) monitoring for thermophilic and psychrophilic organisms. The case for monitoring (or otherwise) for anaerobic bacteria is also discussed. The chapter also discusses the particular requirements for the monitoring of compressed gases (including compressed air). In assessing each of these “special” monitoring requirements the extent to which they are needed depends upon the particular facility and an understanding of any risks. If open processing is conducted in a cold room, for example, then a case might be made for some form of testing in the event that psychrophilic rather than psychrotolerant organisms are recovered. This is a key point with this chapter: the types of assessments discussed need to be considered to determine if they have a place in the contamination control strategy.
MONITORING TO SUPPORT PARAMETRIC RELEASE A detailed examination of parametric release is outside the scope of this book. However, many pharmaceutical manufacturers will use environmental monitoring data as data of their parametric release procedures as a means to either assess the overall bioburden or as a means of considering the probable risk, in relation to terminally sterilized products. The approach is not suitable, in this author’s opinion, for aseptically processed products. European Organization for Quality defines parametric release as: “A system of release that gives the assurance that the product is of the intended quality based on information collected during the manufacturing process and on the compliance with specific GMP requirements related to Parametric Release.” Parametric release is based on evidence of successful validation of the manufacturing process and review of the documentation on process monitoring carried out during manufacturing to provide the desired assurance of quality of the product. It is a system of release that gives the assurance that the product is of the intended quality based on the information collected during the manufacturing process and on the compliance with specific requirements related to parametric release resulting in the elimination of certain specific tests of the finished product (Sandle, 2012). Some of the types of things that need to be considered as part of parametric release are displayed in Fig. 1: This means for products that are intended to be sterile that the microbiological quality of the batch of a medicinal product is stated by using the data
Monitoring to Support Parametric Release 181
Bioburden control
Utility control e.g., water
Materials control
Review of sterilization records
Cleaning and disinfection Contamination control
Cleanroom control
Environmental monitoring
Equipment e.g., maintenance
People and procedures
n FIG. 1 Key factors to consider for parametric release.
from environmental monitoring and process data (which includes other microbial test results from the examination of raw materials, water samples, and intermediate product). In particular, the procedures for quality control of starting materials, packaging materials, process water, and environmental monitoring are checked. Other aspects of importance are, for example, filtration procedures, equipment cleaning/sterilization procedures, maximum holding times for bulk solutions. In this context, a sterility test is not required in batch release. For assessing these sterile products, environmental
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monitoring data, especially when trended, provides a very useful picture concerning the probability and possibility of product contamination.
THE NEED FOR ANAEROBIC MONITORING? Anaerobic environmental monitoring is rarely performed by pharmaceutical manufacturers and there is rarely any requirement for it to be performed. For nonsterile manufacturing there is little scientific reason whatsoever (except where certain products have been identified as at risk from certain toxins produced from organisms that can survive in the production process); for aseptic manufacturing there may be a requirement if particular gases are used with specialized filling machine equipment such as blow-fill technology ( Jimenez, 2004). Blow-fill technology tends to use insert gases. When using inert gases (like nitrogen) or pressurized air in routine aseptic manufacturing process this has to be simulated during media fill. As the usage of nitrogen represents anaerobic conditions then it is arguable that some form of anaerobic monitoring should take place. The sterility of the inert gas has to be separately demonstrated by filtration procedure or by introduction of gas into the nutrient. Some manufacturers adopt to perform limited anaerobic monitoring during media simulation trials only. An anaerobic organism or anaerobe is considered, in this context, to be any microorganism that does not require oxygen for growth. Different types of “anaerobes” can be considered thus:
• •
• •
•
Obligate anaerobes (microorganisms that live and grow in the absence of molecular oxygen) will die when exposed to atmospheric levels of oxygen. Facultative anaerobes can use oxygen when it is present. Some examples of facultative anaerobic bacteria are Staphylococcus (Gram positive), Escherichia coli and Shewanella oneidensis (Gram negative), and Listeria (Gram positive). Aerotolerant organisms can survive in the presence of oxygen, but they are anaerobic because they do not use oxygen as a terminal electron acceptor; Microaerophiles are organisms that may use oxygen, but only at low concentrations (low micromolar range (20% concentration)); their growth is inhibited by the normal oxygen concentration of air (approximately 200 micromolar) (e.g., Campylobacter); Nanaerobes are organisms that cannot grow in the presence of micromolar concentrations of oxygen, but can grow with and benefit from nanomolar concentrations of oxygen (e.g., Bacteroides fragilis).
The Need for Psychrophilic Monitoring? 183
Obligate anaerobes may use fermentation or anaerobic respiration. In the presence of oxygen, facultative anaerobes use aerobic respiration; without oxygen some of them ferment, some use anaerobic respiration. Aerotolerant organisms are strictly fermentative. Microaerophiles carry out aerobic respiration, and some of them can also do anaerobic respiration. Where anaerobic monitoring is required the environmental monitoring samples are taken using standard agar (such as TSA). However, the samples are incubated using specialized cabinets or in gas jars. These systems are designed to cultivate anaerobic and microaerophilic organisms. Most gas jars are cylindrical jar and are composed of transparent and very thick, strong polycarbonate plastic. The jars typically hold anaerobic indicators and a gas-generation pack or even a second low-temperature catalyst. The catalysts are often palladium-coated ceramic beads which provide a large surface area for catalysis. And the object is to enable the rapid generation of an anaerobic atmosphere. Some catalysts require regeneration by dry heat; there will be a limit upon the number of cycles that a catalyst can be regenerated. The catalysts function to remove any residual oxygen in the jar to create a strictly anaerobic environment. The frequency of anaerobic monitoring should be established by the microflora recovered or based on a consideration of probable risk. The level of risk is best established through an examination of historical data (Sandle, 2011).
THE NEED FOR PSYCHROPHILIC MONITORING? Outside of the production of highly specialized medicinal products, most pharmaceutical cleanrooms, used for the preparation of tablets, creams, inhalers, and so on, are operated at temperatures suitable for personnel to work in (typically 18–25°C). In addition, common to many pharmaceutical facilities there are some areas which may become slightly warmer (such as where autoclaves are operated) and there are some areas which function as cold rooms. Cold rooms are used for product storage and conditioning and have become more widespread with the advent and growth of biotechnology products, where cold conditions are required for purification steps. To minimize the risk of contamination, cold rooms are required to be designed and operated as certifiable cleanrooms. Pharmaceutical process area cold rooms vary in their temperature of operation, with 1°C as the potential minima and the maxima, which is set anywhere between 10°C and 14°C. The cold rooms to which the case study outlined in this paper refers where operated between 2°C and 8°C.
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Some medicines inspectors request monitoring data concerning microorganisms with optimal growth rates that fall outside of mesophilic conditions (what might be called extremophiles: microorganism requiring severe conditions for growth as defined by extremes of temperature, pH, chemical oxidizing agents, hypersalinity, or certain types of ultraviolet light) (Dutta & Paul, 2012). This regulatory approach has been noted with both US Food and Drug Administration (FDA) and European Medicines Agency (EMA), in relation to regulatory assessments of pharmaceutical process area cold rooms. Here, regulatory agencies have inquired about the possibility of microorganisms which can tolerate cold conditions (psychrotolerant) or which will only grow in cold conditions (psychrophilic) being present within cold room environments. Psychrophiles or cryophiles are extremophilic organisms that are capable of growth and reproduction in cold temperatures. Such microorganisms can grow at temperatures lower than 15°C and most are found in the Arctic or in the sea (typically Tmin < 0°C, to, Tmax > 15°C). There are generally considered to be two groups of bacteria which can tolerate cold temperatures: obligate psychrophiles and facultative psychrophiles or psychrotrophs with relatively broad temperature ranges for growth. Obligate psychrophiles are those organisms having a growth temperature optimum of 15°C or lower and which cannot grow in a climate beyond a maximum temperature of 20°C. Psychrophiles are more often isolated from permanently cold habitats, whereas psychrotolerant microorganisms tend to dominate those environments that undergo thermal fluctuations (Thamdrup & Fleischer, 1998). Obligate psychrophiles are adapted to their cold environment by having largely unsaturated fatty acids in their plasma membranes. Psychrophiles possess enzymes that continue to function, albeit at a reduced rate, at temperatures at or near 0°C. Psychrophile proteins do not function at the body temperatures of warm-blooded animals and they are unable to grow at even moderate temperatures. On the basis of risks in pharmaceutical processing, given most intended routes of administration, a psychrophilic contaminant would present only a low risk to the patient. Facultative psychrophiles can survive at temperatures as low as 0°C up through approximately 40°C. These organisms exist in much larger numbers than obligate psychrophiles. They are generally not able to grow at cold temperatures (under 15°C), although they often maintain basic functioning. These organisms have evolved to tolerate cold conditions, where adaptation has required a vast array of sequence, structural, and physiological adjustments. Nonetheless, they are not as physiologically specialized as obligate psychrophiles and are usually not found in the very coldest of environments. Examples of such facultative psychrophiles are microorganisms that include
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Listeria monocytogenes, Vibrio marinus, Pseudomonas fluorescens, and Pseudomonas maltophilia. These “cold tolerant” organisms are more often referred to as psychrotolerant microorganisms. The basis of the regulatory concern is that such microorganisms may not, through their adaption to cold conditions, be detected due to an inability to grow, or only growing in reduced numbers, where mesophilic environmental monitoring is undertaken. Regulators have argued that under conditions of mesophilic monitoring, this leads to an underestimation of the numbers of microorganisms present in cleanrooms designed as cold rooms. Regulators further argue, perhaps more importantly, that the consequential failure to speciate those microorganisms which may present in cold areas means that the risk to the product cannot be fully understood (in that the microbial population in the cold area cannot be surveyed to determine if any of the microorganisms are objectionable in relation to the particular product and potential patient population that the product is intended for). The counterargument to concerns about such psychrophilic microorganisms relates to their metabolic characteristics, that is, such organisms do not pose a risk to pharmaceutical processing because they cannot grow or reproduce in the product. A further argument is that “cold loving” microorganisms may be present, but they are found in such low numbers that do not pose a risk. From these two counterpositions, it can be contended that the focus should be upon risk assessment where any recovered objectionable microorganisms in pharmaceutical processing should be considered in relation to the question: does the particular microorganism, at a certain level, pose a risk to the patient should it contaminated a particular product? To this question, it can be argued that although many pharmaceutical products are stored in cold areas, and assuming that product ingress could occur (however unlikely given product barrier safeguards), then the typical microorganisms found in pharmaceutical process cleanrooms would, given the environmental conditions of cold storage (10°C or lower), either not survive for long periods or would be in stasis and would therefore not proliferate. The exception to this would be if there was a microorganism that was specifically cold loving and was able to reproduce under such conditions. Here, the risk is compounded if the pharmaceutical manufacturer is not aware that such microorganisms are present. The potential risk arises, then, if there are some microorganisms present which will only reproduce at low temperatures and would not be detected by the standard environmental monitoring program (i.e., such organisms would not grow at the incubation regime currently applied and which is designed to capture “mesophilic” microorganisms) (Sandle & Skinner, 2013).
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The way to approach this issue is either to develop a defensible rationale and state the case not to perform such monitoring, or to carry out regular monitoring, or to perform a one-off study. The answer should form part of the contamination control rationale.
THE NEED FOR THERMOPHILIC MONITORING? There is no real case to be made for thermophilic monitoring, either in the cleanroom environment or within pharmaceutical water systems. A thermophile is a microorganism that lives at temperatures >45°C (and generally up 70°C). Hyperthermophiles will grow at temperatures in excess of 70°C (Martinez, 2004). With water systems, pharmaceutical water environments are extremely hostile environments. They are kept at high temperatures and have very low levels of organic materials, minerals, and salts and have a narrow pH range. It is therefore, scientifically, extremely unlikely that thermophilic (or any) microorganisms could live in hot water systems. Most objectionable microorganisms and human pathogens will not grow at temperatures >45°C. With cleanrooms, some pharmaceutical manufacturers will examine prepared autoclave loads in using contact plates and swabs, or for other activities where heat is used (such as blow–fill–seal processes) (Poisson, Sinclair, & Tallentire, 2006). The purpose for performing the monitoring is to:
• •
Build up a library of the type of challenges posed to loads going into sterilizers; To react to any results that exceed preset action levels, either based on the counts obtained or the species identified.
Samples from the autoclave loads are commonly incubated at two temperature regimes:
• •
20–25°C for a minimum of 3 days followed by 30–35°C for a minimum of 6 days, and 55–60°C for a minimum of 2 days.
The reason for performing the work at two different temperatures is to detect both mesophilic and thermophilic contamination. The reason for the thermophilic temperature is the autoclaves are superheat sterilization devices and thermophilic microorganisms may be, although this is theoretically unlikely, a specific cause for concern. Moreover, the chance of detecting thermophiles is low.
Compressed Gas Monitoring 187
In both water systems and with cleanrooms, most extremophiles are anaerobic or microaerophilic chemolithoautotrophs. Because of their unique habitats, metabolism, and nutritional requirements, such organisms are not known to be or opportunistic pathogens, and they are not capable of colonizing a pharmaceutical area. Therefore there is little value in monitoring for such organisms.
COMPRESSED GAS MONITORING Compressed gas is a general term for gas stored or held under pressure that is greater than atmosphere. Compressed gases are used at different stages of the pharmaceutical manufacturing process. Applications include weighing stations process line; use of gas to maintain an inert atmosphere above a liquid or powdered product inside a storage tank, silo, reactor, process equipment, or other vessel; use of liquid nitrogen for the preservation of biological samples; use of inert gas to pressurize new, repaired, or modified tanks, pipelines, and vessels; and use of inert gas to displace air and contaminants from storage tanks. Furthermore, compressed gases such as air, nitrogen, and carbon dioxide are deployed in operations involving purging or overlaying. Compressed gas sampling for microorganisms is an important part of contamination control assessment (Sandle, 2013). While sampling is important, the method of sampling can be hindered by the design of the gas system, where sampling is not easily conducted in an aseptic manner, or by the design of the air sampling instrument. This section reviews the important aspects of compressed air sampling for microbiological assessment and looks at possible sources of contamination, should microorganisms be recovered. Purity is a factor that needs to be maintained with compressed gas; hence the gas should be supplied oil free. Purity overall is achieved through a combination of filtration, purification, and separation. The process of creating the compressed gas can additionally introduce water vapor; thus a process must be in place to remove water vapor before the gas is expelled into a critical zone like a cleanroom. Compressed gas is typically discharged from the compressor hot and it will contain water vapor. Temperature is reduced by using a postcompressor cooler and, as the gas condenses, the water vapor and other impurities can be removed. The risk of water vapor is particularly high with compressed air, which is drawn into a compressor via the atmosphere. Atmospheric air contains a high proportion of water vapor (i.e., water in a gaseous form). Water removal is achieved through a combination of filtration and dehumidification.
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Where air is drawn in from the outside, the process of drawing in air also introduces microorganisms, which require filtering out. The level of filtering depends upon whether “sterile” air is required (absence of viable microorganisms) or air with a low bioburden. Compressed gas can be supplied at source either sterile or nonsterile. Sterility, where required such as with an inhalation product, is achieved through the use of a bacterial retentive membrane filter (0.2 μm pore size). Where a sterilefiltered gas is required it is important that the sterilizing grade filter is maintained dry for condensate in a gas filter that will most probably cause blockage or lead to microbial contamination. Risks of condensate are controlled by heating and use of hydrophobic filters (to prevent moisture residues in a gas supply system). Filters should also be changed periodically. As part of ongoing quality control, filters must be integrity tested at installation and at end of use. Although national standards bodies have guidance documents for compressed air sampling, and reference is made within FDA and EU GMPs, the general approach and requirements for compressed gases are set out in a multipart ISO standard: ISO 8573. This standard consists of the following parts (ISO, n.d.):
• • • • • • • • •
Part Part Part Part Part Part Part Part Part
1: Contaminants and purity classes, 2: Test methods for aerosol oil content, 3: Test methods for measurement of humidity, 4: Test methods for solid particle content, 5: Test methods for oil vapor and organic solvent content, 6: Test methods for gaseous contaminant content, 7: Test method for viable microbiological contaminant content, 8: Test methods for solid particle content by mass concentration, 9: Test methods for liquid water content.
Part 1 outlines the required purity classes based on the concentration of particles and level of impurities. The potential “impure” contaminants for compressed air, that can affect whether a required purity class is met, include:
•
• •
Particles (such as dirt, rust, pipe scale), with particles assessed by size. For example, as result of the mechanical compression process, additional impurities may be introduced into the air system. Generated contaminants include compressor lubricant, wear particles, and vaporized lubricant. Furthermore, fittings and accessories can contribute to particles. Water (in both vapor and liquid forms). Water is typically assessed by vapor pressure dew point. This is the temperature at which the air can no longer “hold” all of the water vapor which is mixed with). Oil (including aerosol, vapor, and liquid forms).
Compressed Gas Monitoring 189
With purity, many parts of the pharmaceutical industry will use Class 1 compressed gas based on the maximum number of permitted particulates.
Microbial Survival in Compressed Gases Although compressed gas and air systems are relatively harsh environments, they can aid microbial survival if there are available nutrients. The availability of nutrients is dependent upon the purity of the gas and airline. Nutrients suitable for metabolizing by microorganisms include water and oil droplets. Another factor that can affect survival is temperature, especially where temperatures are warmer (Stewart et al., 1995). In addition to vegetative cells, bacterial spores are well equipped to survive the harsh environmental conditions. Spores are resistant to the types of temperature ranges and moisture levels found within compress gas lines. Another risk exists with biofilm, where microbial communities can potentially form and develop through attachment to air lines and tubing. Although these risk factors exist, typically no microorganisms would be expected to be recovered from compressed gas lines. Research has shown that many microorganisms can survive and multiply in pressurized systems up to 10 bar and some are at least able to recover after being pressurized. However, at 160 bar pressure upwards, survival rates are very low. Where low level counts are recovered, these require investigation. More often the source is adventitious contamination, although a fault with the compressed air line cannot be ruled out. Although microbial contamination of compressed air or gas is a rare event, incidents can occur. Sources of contamination include:
• • • •
•
Source of the air or gas: Contamination can arise from air intake from the surroundings (which can contain oil, dirt, dust, moisture, or microorganisms). Piping distribution systems: Piping distribution and air storage tanks, more prevalent in older systems, will have contaminant in the form of rust, pipe scale, mineral deposits, in addition to bacteria. Bacterial retentive filter: The filter may become blocked, lose its integrity, or become wet. Compressor failure: The compressor itself can create a contaminated environment. For example, the compressor’s prefilters can become overloaded with dust and lint, causing the filter to cease functioning properly. Sample valve: The point-of-use sample valve may not be designed correctly or become faulty.
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Microbiological Requirements Microbial content itself does not influence the gas purity class assigned, although the standards recommend that microbial levels are assessed. Acceptable microbial numbers are subject to a separate assessment, with such assessment being based on an interpretation of GMP. A consensus is that the microbiological quality of the gas must be at least as good as the cleanroom air quality in which the process is taking place. Note that for EU GMP Grade A/ISO 14644 Class 5 areas, the microbial count would then be <1 cfu/m3 and the particle levels conform to the area at rest 3520 particles per m3. Many companies, however, do not monitor gas or compressed air used in Grade A or B areas since these are sterile filtered as close as possible to the point of use. In such cases a filter integrity test is executed in lieu of microbiological monitoring. Compressed air sampling should form part of an environmental monitoring program, along with cleanroom assessments. The program should take into account air points to be tested. This could be every point, points considered to be of greater risk (such as product contact), or representative points along a loop. The frequency of testing must also be considered, and this too would need to tie into risk. While there is an argument, as set out here, for the testing of compressed air where there is product contact there is less of a consensus over the testing of nitrogen. While nitrogen gas can be used to dispense or transfer most fluids from storage, the ISO standard has no specific microbial testing requirements and very few microorganisms, of the types common to pharmaceutical manufacturing environments, would be likely to survive. On this basis a risk-based justification could be made not to perform nitrogen gas testing. The user will need to determine whether each compressed gas line requires testing and the frequency of testing. Certainly all product contact compressed gases should be assessed. A sampling plan should also consider, and adapt to, the following:
• • • • • • •
Cleanroom grade, Type of product manufactured, Increased or reduced production schedules, Seasonal changes, Equipment changes and modifications, Replacement of hardware or filters and dryers, Inactivity of system.
When sampling compressed air for microorganisms, it is important that the air is depressurized and that the flow rate is controlled. Control of the flow
Compressed Gas Monitoring 191
rate is important to ensure that a cubic meter of air is sampled within the required sampling time (this time will be instrument dependent). If the air sampler takes 36 min to capture a cubic meter of air, then it will be sampling at one cubic foot per minute. An external regulator will be needed to bring the flow rate down to the sampling rate of instrument. This is assessed using a flow meter. Pressure reduction to atmospheric conditions is of great importance and knowing the flow allows the agar exposure time to be assessed, so that one cubic meter of air is sampled (Sandle, 2010). It is also important that isokinetic sampling of the air occurs and that air velocity is reduced until it is within the range of the sampler as identified by the manufacturer. This is not only necessary for obtaining the correct sample size but it also impacts on the possibility of microbial survival. The level of impact stress has been shown to affect microbial recovery on agar and be dependent upon the impaction velocity of the cells into the agar as well as the design and operating parameters. Due to the fact that any microorganisms present are transported under pressure and then suddenly released into atmospheric conditions, they may be damaged by the immediate expansion of the gas and the resulting shearing forces. The head of the instrument and any attachments should ideally be sterile before use, to avoid contamination. The culture medium used with the instrument should have been tested for growth promotion and, as for environmental monitoring, has been validated as suitable for gas viable air monitoring. With most samplers the head will be autoclavable. Some users disinfect the tubes and hoses used to connect the sampler with a disinfectant like 70% isopropyl alcohol. This is mentioned as an option in the ISO standard, although this is erroneously described as “sterilization.” Where a disinfectant is used it is important to run the air through the sampler without any agar plate in place; this is necessary to evaporate the disinfectant and to remove any residues. The presence of disinfectant could potentially lead to a “false negative.” With sampling, the sample inlet is connected to the compressed gas line and air is directed over an agar plate or strip. The method works by compressed gas, under reduced pressure, called “partial flow”, is forced over the surface of an agar plate. Any microorganisms are impinged onto the surface of the agar. The sampling time should be sufficient in order to sample one cubic meter of the agar. After sampling, the agar plate or strip is removed and incubated within a microbiology laboratory. At the end of incubation, the agar is examined for colony-forming units. Incubation can be for aerobic or anaerobic organisms, or both. The extent to which either is present should be based
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on initial validation and by taking into account whether such organisms pose a patient risk, should they end up being transferred into the end product. The type of instrument recommended in the ISO standard is a “slit-sampler, a type of impaction air tester,” although alternative samplers can be used, if justified. With a standard impactor sampler, air is drawn through a sampling head via a pump or fan and accelerated, usually through a perforated plate (sieve samplers), or through a narrow slit (slit samplers). This process creates a laminar flow through the sampler head. Hence, the air sampler should be fitted with a diffuser capable of maintaining laminar flow conditions. This is necessary so that particles pass through the sample head in a controlled flow. An appropriate agar must be selected. An example is tryptone soya agar, which is a generally nutritious medium designed to recover a range of bacteria and fungi. A key factor to take into account is whether the process of sampling leads to undue desiccation of the agar, rendering any recovered microorganisms unable to grow on the agar due to depletion of growth nutrients. This will be affected by the flow rate, type of compressed gas, and the model of air sampler, together with the type of culture medium. A risk will remain that microbial cells will become damaged by mechanical stress during the sampling process and lose viability.
Bacterial Endotoxin and Compressed Gases Some users elect to sample compressed air for bacterial endotoxin. Such testing remains relatively uncommon and it is only necessary should the compressed gas have a direct product contact and where there is a concern with Gram-negative bacteria. In most cases there should be no likelihood of endotoxin being present, especially in the context of nonsterile manufacturing. The sampling method for bacterial endotoxin is tricky and inexact. Either colonies are examined for Gram-negative bacteria, and assessment is made about endotoxin risk; or the compressed is passed through pyrogenfree water.
MONITORING STERILITY TEST ENVIRONMENTS Isolators used for sterility testing should be approached in a similar way as the monitoring of Grade A/ISO Class 5 areas. This section of the unit sets out the approach taken to environmental monitoring inside a half-suit isolator during sterility testing, at the author’s laboratory.
Monitoring Sterility Test Environments 193
The use of a sterility testing isolator in a regulated environment means that having reliable results for its sterility tests is essential, and although there is a history of confidence in the isolator system, having a microbiological environmental monitoring program is imperative. The reason for this is twofold: one, to demonstrate that if a product was to fail a sterility test then the reason for that failure could be attributed to the product alone and not as a factor of the isolator environment, and, two, to show that the overall background environment of the isolator is “in control” through trend analysis. The microbiological environmental monitoring typically consists of air samples, settle plates, finger plates, and contact plates or swabs. A standard culture media is typically used, like tryptone soya agar (TSA), which is a nonselective, highly nutritious media. Preliminary work need to take place in order to confirm that the action of sanitizing the media into the isolator, using hydrogen peroxide or peracetic acid vapor, or alternative method, does not cause inhibition of the TSA’s growth-promoting properties. The microbiological environmental monitoring should be performed for each sterility test by the operator. This provides invaluable information in the event of a sterility test failure. Monitoring typically includes:
•
•
•
•
Settle plates: uncovered TSA plates (9 cm diameter) exposed either side of the testing environment for the duration of the test. The final result should be converted to a number of colony-forming units (cfu) per 4 h. Air sample: there are pros and cons concerning the use of an air sampler inside an isolator. This debate centers on the disruption of the airflow that an air sampler may cause. The view here is that an air sample should be taken at each sterility test since having an assessment of the air quality outweighs any possible the effect on the airflow. Finger plates: at the end of the test, the operator should take finger plates (“dabs”) of each gloved hand by imprinting his/her fingers onto an agar plate. It is important that the gel strength of the agar is suitable for this task. Swabs or contact plates: the last microbiological monitoring activity is the taking of surface samples at set locations.
Once all the monitoring had been completed, the isolator is cleaned with an appropriate disinfectant to remove residues, such as an isopropanol alcoholbased disinfectant. Samples should be appropriately incubated (Chapter 7 discusses incubation regimes). The action limit applied to all samples should be 1 cfu, which
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matches the same environmental standard as a process environment. A result at or exceeding the “action level” implies an “out-of-specification” investigation of aspects like the isolator gloves and the overall integrity; the matter becomes more serious should an upward trend develop.
MONITORING MICROBIOLOGY LABORATORIES Although there is no written regulatory requirement or guidance, it can be prudent to perform some level of environmental monitoring within the microbiology laboratory and associated incubators. The data gathered from such monitoring can be useful in assessing out-of-limits results especially when considering if contamination arose from the laboratory environment. The data gathered from such exercises can be examined and trended in a similar way to that from nonsterile manufacturing facilities. Although there are no prescribed action levels or specifications, most laboratories should have monitoring levels below to EU GMP Grade D/ISO Class 9 specifications (Table 1). Although there is no direct relationship to testing within the microbiology laboratory, the data gathered can be illuminating in terms of hygiene status and it could be useful should any test errors require root cause investigation.
TEST CONTROLS Most microbiological tests should have a test control, in order to assess validity, especially in relation to adventitious contamination. This is normally a negative control, although positive controls are used in some specific laboratory tests. Such controls serve a number of purposes:
• •
To assess the aseptic technique of the tester. To test the quality of the media and reagents. Table 1 Possible alert and action levels for monitoring microbiology laboratories
Sample type
Warning level (cfu)
Action level (cfu)
Active air Settle plate Contact plate
Not defined Not defined Not defined
200 100 50
Test Controls 195
Table 2 Example of viable microbiological environmental monitoring test controls Test Method
Control
Purpose
Microbiological monitoring: swab
Negative
Microbiological monitoring: contact plate Microbiological monitoring: active air sampler Microbiological monitoring: finger plate Microbiological monitoring: settle plate
None
For plain swabs: swab is soaked in Ringer’s solution and streaked out onto an agar plate, under a UDAF. This demonstrates that the Ringer’s solution, swab, and agar are not contaminated. For transport swabs: a swab is tested as presented. This control tests the environment in which the swab was taken, sampled, stored, and transported and the Technician’s technique in preparing and plating out swabs. Performed once during each monitoring session. Sterility of media checked through nutritive properties release procedure. Sterility of media checked through nutritive properties release procedure. Sterility of media checked through nutritive properties release procedure. Sterility of media checked through nutritive properties release procedure.
• •
None None None
To test the “testing” environment. To demonstrate that samples are operating as expected.
Controls should be examined as part of out-of-specification or out-of-limits reporting. A control, which is OOS or OOL, should be recorded onto an OOS/L report form (or an electronic record) and assessed along with samples tested during the test session. For samples that are OOS/L, the control should be examined for contamination or to check that it operated as expected. Controls can also be used as part of training and as a check for ongoing assessment. Where a control is consistently overaction the preventative action can include retraining and assessment. For viable environmental monitoring, test controls are normally negative controls. A possible approach to the use of controls is outlined in Table 2. A second form of control is with growth promotion testing. This is addressed in Chapter 7 of this book. Growth promotion testing can additionally be used if there are doubts about the media (such as stored outside of standard incubation conditions or media that has gone beyond its expiry time, although of course the acceptability of using testing to mitigate poor practices needs to be understood within a given organization’s quality system). An example of the growth promotion test is presented in Table 3. These types of controls can provide useful information about the suitability of the media and its handling.
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Table 3 Example of media growth promotion test controls Test method
Control
Purpose
Growth promotion testing
Positive
To demonstrate that the challenge count to the media on test is within the acceptance range of <100 cfu. To show how the test media compares to a previously released lot of the same media type with respect to the test challenge. Batches of media are incubated for a specified duration and inspected for contamination by the manufacturer. This demonstrates that the media has been prepared in a way that maintains sterility. The strains used to inoculate plates are visually checked for purity. Can assess whether media has become contaminated.
Comparative control Sterility
Negative
SUMMARY Although there are some important fundamentals for the contamination control strategy, as discussed in many of the chapters in this book, there are other aspects that might need to be included depending upon the nature of the pharmaceutical or healthcare organization. This relates to certain types of organisms found in specific locales. Are anaerobes of concern, for example? If nitrogen is used as part of the manufacturing process then this may be so. These questions are best addressed through risk assessment. In doing so, the nature of the likely contaminants needs to be assessed. Undertaking additional monitoring for psychrophiles, for example, serves no purpose if the organisms recovered from process rooms operating at <10°C are all capable of being recovered from standard environmental monitoring conducted using mesophilic incubation temperatures. The risk concept also applies to parametric release. This approach is not suitable for aseptically filled products but it can be applied to terminally sterilized products, with controls in place. Where environmental monitoring can assist is not only with the assessment of the absolute numbers recovered but also with the types of organisms recovered, in terms of their resistance to thermal destruction. Understanding the organisms is also something that needs to be factored into a risk assessment. The chapter has additionally addressed compressed gas monitoring. This is important to have in place since compressed gases are generally viewed as critical utilities when either in direct product contact or directly entered into the cleanroom environment. The chapter has also looked at test controls and the monitoring of sterility test facilities and of microbiology laboratories, areas that are associated with the primary environmental monitoring program but which equally do not form a central part of the program.
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The extent to which each area discussed here should be applied, of if it all, needs to be risk assessed, taking into account the nature of the process and the types of hazards that might occur.
REFERENCES Dutta, C., & Paul, S. (2012). Microbial lifestyle and genome signatures. Current Genomics, 13(2), 153–162. ISO ISO 8573-1:2010, Compressed air contaminants and purity classes, ISO, Geneva, Switzerland. Jimenez, L. (2004). Environmental Monitoring. In L. Jimenez (Ed.), Microbial contamination control in the pharmaceutical industry (pp. 103–132). New York: Marcel Dekker. Martinez, J. (2004): Hyperthermophilic microorganisms and USP hot water systems, Pharmaceutical Technology, Vol. 6, No.7, February, pp 50–65. Poisson, P., Sinclair, C., & Tallentire, A. (2006). Challenges to blow/fill/seal process with airborne microorganisms having different resistances to dry heat. PDA Journal of Pharmaceutical Science and Technology, 60(5), 323–330. Sandle, T. (2010). Selection of active air samplers. European Journal of Parenteral and Pharmaceutical Sciences, 15(4), 119–124. Sandle, T. (2011). Environmental monitoring. In M. R. Saghee, T. Sandle, & E. C. Tidswell (Eds.), Microbiology and sterility assurance in pharmaceuticals and medical devices (pp. 293–326). New Delhi: Business Horizons. Sandle, T. (2012). Environmental Monitoring: a practical approach. In J. Moldenhauer (Ed.), Vol. 6. Environmental monitoring: A comprehensive handbook (pp. 29–54). River Grove, IL: PDA/DHI. Sandle, T. (2013). Contamination control risk assessment. In R. E. Masden, & J. Moldenhauer (Eds.), Vol. 1. Contamination control in healthcare product manufacturing (pp. 423–474). River Grove, IL: DHI Publishing. Sandle, T., & Skinner, K. (2013). Study of psychrophilic and psychrotolerant microorganisms isolated in cold rooms used for pharmaceutical processing. Journal of Applied Microbiology, 114(4), 1166–1174. Stewart, S. L., Grinshpun, S. A., Willeke, K., Terzieva, S., Ulevicius, V., & Donnelly, J. (1995). Effect of impact stress on microbial recovery on an agar surface. Applied and Environmental Microbiology, 61, 1232–1239. Thamdrup, R., & Fleischer, S. (1998). Temperature dependence of oxygen respiration, nitrogen mineralization, and nitrification in Arctic sediments. Aquatic Microbial Ecology, 15, 191–199.
11
Special Types of Environmental Monitoring CHAPTER OUTLINE
Introduction 179 Monitoring to Support Parametric Release 180 The Need for Anaerobic Monitoring? 182 The Need for Psychrophilic Monitoring? 183 The Need for Thermophilic Monitoring? 186 Compressed Gas Monitoring 187 Microbial Survival in Compressed Gases 189 Microbiological Requirements 190 Bacterial Endotoxin and Compressed Gases 192
Monitoring Sterility Test Environments 192 Monitoring Microbiology Laboratories 194 Test Controls 194 Summary 196 References 197
INTRODUCTION The purpose of this chapter is to look at environmental monitoring, in support of biocontamination control, from a more applied perspective. This includes the use of parametric release, which can be used for terminally sterilized products. Parametric release is a sterility assurance release program where demonstrated control of the sterilization process enables an organization to use defined critical process controls, in lieu of the sterility test. The chapter considers how the inclusion of test controls as part of the environmental monitoring regime can provide robustness as well as useful information should an out-of-limits result be obtained as to whether that result was or was not the consequence of laboratory error. Biocontamination Control for Pharmaceuticals and Healthcare. https://doi.org/10.1016/B978-0-12-814911-9.00011-0 # 2019 Elsevier Inc. All rights reserved.
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Among the “special” types of environmental monitoring performed the chapter looks at the end for (and more strongly the need against) monitoring for thermophilic and psychrophilic organisms. The case for monitoring (or otherwise) for anaerobic bacteria is also discussed. The chapter also discusses the particular requirements for the monitoring of compressed gases (including compressed air). In assessing each of these “special” monitoring requirements the extent to which they are needed depends upon the particular facility and an understanding of any risks. If open processing is conducted in a cold room, for example, then a case might be made for some form of testing in the event that psychrophilic rather than psychrotolerant organisms are recovered. This is a key point with this chapter: the types of assessments discussed need to be considered to determine if they have a place in the contamination control strategy.
MONITORING TO SUPPORT PARAMETRIC RELEASE A detailed examination of parametric release is outside the scope of this book. However, many pharmaceutical manufacturers will use environmental monitoring data as data of their parametric release procedures as a means to either assess the overall bioburden or as a means of considering the probable risk, in relation to terminally sterilized products. The approach is not suitable, in this author’s opinion, for aseptically processed products. European Organization for Quality defines parametric release as: “A system of release that gives the assurance that the product is of the intended quality based on information collected during the manufacturing process and on the compliance with specific GMP requirements related to Parametric Release.” Parametric release is based on evidence of successful validation of the manufacturing process and review of the documentation on process monitoring carried out during manufacturing to provide the desired assurance of quality of the product. It is a system of release that gives the assurance that the product is of the intended quality based on the information collected during the manufacturing process and on the compliance with specific requirements related to parametric release resulting in the elimination of certain specific tests of the finished product (Sandle, 2012). Some of the types of things that need to be considered as part of parametric release are displayed in Fig. 1: This means for products that are intended to be sterile that the microbiological quality of the batch of a medicinal product is stated by using the data
Monitoring to Support Parametric Release 181
Bioburden control
Utility control e.g., water
Materials control
Review of sterilization records
Cleaning and disinfection Contamination control
Cleanroom control
Environmental monitoring
Equipment e.g., maintenance
People and procedures
n FIG. 1 Key factors to consider for parametric release.
from environmental monitoring and process data (which includes other microbial test results from the examination of raw materials, water samples, and intermediate product). In particular, the procedures for quality control of starting materials, packaging materials, process water, and environmental monitoring are checked. Other aspects of importance are, for example, filtration procedures, equipment cleaning/sterilization procedures, maximum holding times for bulk solutions. In this context, a sterility test is not required in batch release. For assessing these sterile products, environmental
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monitoring data, especially when trended, provides a very useful picture concerning the probability and possibility of product contamination.
THE NEED FOR ANAEROBIC MONITORING? Anaerobic environmental monitoring is rarely performed by pharmaceutical manufacturers and there is rarely any requirement for it to be performed. For nonsterile manufacturing there is little scientific reason whatsoever (except where certain products have been identified as at risk from certain toxins produced from organisms that can survive in the production process); for aseptic manufacturing there may be a requirement if particular gases are used with specialized filling machine equipment such as blow-fill technology ( Jimenez, 2004). Blow-fill technology tends to use insert gases. When using inert gases (like nitrogen) or pressurized air in routine aseptic manufacturing process this has to be simulated during media fill. As the usage of nitrogen represents anaerobic conditions then it is arguable that some form of anaerobic monitoring should take place. The sterility of the inert gas has to be separately demonstrated by filtration procedure or by introduction of gas into the nutrient. Some manufacturers adopt to perform limited anaerobic monitoring during media simulation trials only. An anaerobic organism or anaerobe is considered, in this context, to be any microorganism that does not require oxygen for growth. Different types of “anaerobes” can be considered thus:
• •
• •
•
Obligate anaerobes (microorganisms that live and grow in the absence of molecular oxygen) will die when exposed to atmospheric levels of oxygen. Facultative anaerobes can use oxygen when it is present. Some examples of facultative anaerobic bacteria are Staphylococcus (Gram positive), Escherichia coli and Shewanella oneidensis (Gram negative), and Listeria (Gram positive). Aerotolerant organisms can survive in the presence of oxygen, but they are anaerobic because they do not use oxygen as a terminal electron acceptor; Microaerophiles are organisms that may use oxygen, but only at low concentrations (low micromolar range (20% concentration)); their growth is inhibited by the normal oxygen concentration of air (approximately 200 micromolar) (e.g., Campylobacter); Nanaerobes are organisms that cannot grow in the presence of micromolar concentrations of oxygen, but can grow with and benefit from nanomolar concentrations of oxygen (e.g., Bacteroides fragilis).
The Need for Psychrophilic Monitoring? 183
Obligate anaerobes may use fermentation or anaerobic respiration. In the presence of oxygen, facultative anaerobes use aerobic respiration; without oxygen some of them ferment, some use anaerobic respiration. Aerotolerant organisms are strictly fermentative. Microaerophiles carry out aerobic respiration, and some of them can also do anaerobic respiration. Where anaerobic monitoring is required the environmental monitoring samples are taken using standard agar (such as TSA). However, the samples are incubated using specialized cabinets or in gas jars. These systems are designed to cultivate anaerobic and microaerophilic organisms. Most gas jars are cylindrical jar and are composed of transparent and very thick, strong polycarbonate plastic. The jars typically hold anaerobic indicators and a gas-generation pack or even a second low-temperature catalyst. The catalysts are often palladium-coated ceramic beads which provide a large surface area for catalysis. And the object is to enable the rapid generation of an anaerobic atmosphere. Some catalysts require regeneration by dry heat; there will be a limit upon the number of cycles that a catalyst can be regenerated. The catalysts function to remove any residual oxygen in the jar to create a strictly anaerobic environment. The frequency of anaerobic monitoring should be established by the microflora recovered or based on a consideration of probable risk. The level of risk is best established through an examination of historical data (Sandle, 2011).
THE NEED FOR PSYCHROPHILIC MONITORING? Outside of the production of highly specialized medicinal products, most pharmaceutical cleanrooms, used for the preparation of tablets, creams, inhalers, and so on, are operated at temperatures suitable for personnel to work in (typically 18–25°C). In addition, common to many pharmaceutical facilities there are some areas which may become slightly warmer (such as where autoclaves are operated) and there are some areas which function as cold rooms. Cold rooms are used for product storage and conditioning and have become more widespread with the advent and growth of biotechnology products, where cold conditions are required for purification steps. To minimize the risk of contamination, cold rooms are required to be designed and operated as certifiable cleanrooms. Pharmaceutical process area cold rooms vary in their temperature of operation, with 1°C as the potential minima and the maxima, which is set anywhere between 10°C and 14°C. The cold rooms to which the case study outlined in this paper refers where operated between 2°C and 8°C.
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Some medicines inspectors request monitoring data concerning microorganisms with optimal growth rates that fall outside of mesophilic conditions (what might be called extremophiles: microorganism requiring severe conditions for growth as defined by extremes of temperature, pH, chemical oxidizing agents, hypersalinity, or certain types of ultraviolet light) (Dutta & Paul, 2012). This regulatory approach has been noted with both US Food and Drug Administration (FDA) and European Medicines Agency (EMA), in relation to regulatory assessments of pharmaceutical process area cold rooms. Here, regulatory agencies have inquired about the possibility of microorganisms which can tolerate cold conditions (psychrotolerant) or which will only grow in cold conditions (psychrophilic) being present within cold room environments. Psychrophiles or cryophiles are extremophilic organisms that are capable of growth and reproduction in cold temperatures. Such microorganisms can grow at temperatures lower than 15°C and most are found in the Arctic or in the sea (typically Tmin < 0°C, to, Tmax > 15°C). There are generally considered to be two groups of bacteria which can tolerate cold temperatures: obligate psychrophiles and facultative psychrophiles or psychrotrophs with relatively broad temperature ranges for growth. Obligate psychrophiles are those organisms having a growth temperature optimum of 15°C or lower and which cannot grow in a climate beyond a maximum temperature of 20°C. Psychrophiles are more often isolated from permanently cold habitats, whereas psychrotolerant microorganisms tend to dominate those environments that undergo thermal fluctuations (Thamdrup & Fleischer, 1998). Obligate psychrophiles are adapted to their cold environment by having largely unsaturated fatty acids in their plasma membranes. Psychrophiles possess enzymes that continue to function, albeit at a reduced rate, at temperatures at or near 0°C. Psychrophile proteins do not function at the body temperatures of warm-blooded animals and they are unable to grow at even moderate temperatures. On the basis of risks in pharmaceutical processing, given most intended routes of administration, a psychrophilic contaminant would present only a low risk to the patient. Facultative psychrophiles can survive at temperatures as low as 0°C up through approximately 40°C. These organisms exist in much larger numbers than obligate psychrophiles. They are generally not able to grow at cold temperatures (under 15°C), although they often maintain basic functioning. These organisms have evolved to tolerate cold conditions, where adaptation has required a vast array of sequence, structural, and physiological adjustments. Nonetheless, they are not as physiologically specialized as obligate psychrophiles and are usually not found in the very coldest of environments. Examples of such facultative psychrophiles are microorganisms that include
The Need for Psychrophilic Monitoring? 185
Listeria monocytogenes, Vibrio marinus, Pseudomonas fluorescens, and Pseudomonas maltophilia. These “cold tolerant” organisms are more often referred to as psychrotolerant microorganisms. The basis of the regulatory concern is that such microorganisms may not, through their adaption to cold conditions, be detected due to an inability to grow, or only growing in reduced numbers, where mesophilic environmental monitoring is undertaken. Regulators have argued that under conditions of mesophilic monitoring, this leads to an underestimation of the numbers of microorganisms present in cleanrooms designed as cold rooms. Regulators further argue, perhaps more importantly, that the consequential failure to speciate those microorganisms which may present in cold areas means that the risk to the product cannot be fully understood (in that the microbial population in the cold area cannot be surveyed to determine if any of the microorganisms are objectionable in relation to the particular product and potential patient population that the product is intended for). The counterargument to concerns about such psychrophilic microorganisms relates to their metabolic characteristics, that is, such organisms do not pose a risk to pharmaceutical processing because they cannot grow or reproduce in the product. A further argument is that “cold loving” microorganisms may be present, but they are found in such low numbers that do not pose a risk. From these two counterpositions, it can be contended that the focus should be upon risk assessment where any recovered objectionable microorganisms in pharmaceutical processing should be considered in relation to the question: does the particular microorganism, at a certain level, pose a risk to the patient should it contaminated a particular product? To this question, it can be argued that although many pharmaceutical products are stored in cold areas, and assuming that product ingress could occur (however unlikely given product barrier safeguards), then the typical microorganisms found in pharmaceutical process cleanrooms would, given the environmental conditions of cold storage (10°C or lower), either not survive for long periods or would be in stasis and would therefore not proliferate. The exception to this would be if there was a microorganism that was specifically cold loving and was able to reproduce under such conditions. Here, the risk is compounded if the pharmaceutical manufacturer is not aware that such microorganisms are present. The potential risk arises, then, if there are some microorganisms present which will only reproduce at low temperatures and would not be detected by the standard environmental monitoring program (i.e., such organisms would not grow at the incubation regime currently applied and which is designed to capture “mesophilic” microorganisms) (Sandle & Skinner, 2013).
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The way to approach this issue is either to develop a defensible rationale and state the case not to perform such monitoring, or to carry out regular monitoring, or to perform a one-off study. The answer should form part of the contamination control rationale.
THE NEED FOR THERMOPHILIC MONITORING? There is no real case to be made for thermophilic monitoring, either in the cleanroom environment or within pharmaceutical water systems. A thermophile is a microorganism that lives at temperatures >45°C (and generally up 70°C). Hyperthermophiles will grow at temperatures in excess of 70°C (Martinez, 2004). With water systems, pharmaceutical water environments are extremely hostile environments. They are kept at high temperatures and have very low levels of organic materials, minerals, and salts and have a narrow pH range. It is therefore, scientifically, extremely unlikely that thermophilic (or any) microorganisms could live in hot water systems. Most objectionable microorganisms and human pathogens will not grow at temperatures >45°C. With cleanrooms, some pharmaceutical manufacturers will examine prepared autoclave loads in using contact plates and swabs, or for other activities where heat is used (such as blow–fill–seal processes) (Poisson, Sinclair, & Tallentire, 2006). The purpose for performing the monitoring is to:
• •
Build up a library of the type of challenges posed to loads going into sterilizers; To react to any results that exceed preset action levels, either based on the counts obtained or the species identified.
Samples from the autoclave loads are commonly incubated at two temperature regimes:
• •
20–25°C for a minimum of 3 days followed by 30–35°C for a minimum of 6 days, and 55–60°C for a minimum of 2 days.
The reason for performing the work at two different temperatures is to detect both mesophilic and thermophilic contamination. The reason for the thermophilic temperature is the autoclaves are superheat sterilization devices and thermophilic microorganisms may be, although this is theoretically unlikely, a specific cause for concern. Moreover, the chance of detecting thermophiles is low.
Compressed Gas Monitoring 187
In both water systems and with cleanrooms, most extremophiles are anaerobic or microaerophilic chemolithoautotrophs. Because of their unique habitats, metabolism, and nutritional requirements, such organisms are not known to be or opportunistic pathogens, and they are not capable of colonizing a pharmaceutical area. Therefore there is little value in monitoring for such organisms.
COMPRESSED GAS MONITORING Compressed gas is a general term for gas stored or held under pressure that is greater than atmosphere. Compressed gases are used at different stages of the pharmaceutical manufacturing process. Applications include weighing stations process line; use of gas to maintain an inert atmosphere above a liquid or powdered product inside a storage tank, silo, reactor, process equipment, or other vessel; use of liquid nitrogen for the preservation of biological samples; use of inert gas to pressurize new, repaired, or modified tanks, pipelines, and vessels; and use of inert gas to displace air and contaminants from storage tanks. Furthermore, compressed gases such as air, nitrogen, and carbon dioxide are deployed in operations involving purging or overlaying. Compressed gas sampling for microorganisms is an important part of contamination control assessment (Sandle, 2013). While sampling is important, the method of sampling can be hindered by the design of the gas system, where sampling is not easily conducted in an aseptic manner, or by the design of the air sampling instrument. This section reviews the important aspects of compressed air sampling for microbiological assessment and looks at possible sources of contamination, should microorganisms be recovered. Purity is a factor that needs to be maintained with compressed gas; hence the gas should be supplied oil free. Purity overall is achieved through a combination of filtration, purification, and separation. The process of creating the compressed gas can additionally introduce water vapor; thus a process must be in place to remove water vapor before the gas is expelled into a critical zone like a cleanroom. Compressed gas is typically discharged from the compressor hot and it will contain water vapor. Temperature is reduced by using a postcompressor cooler and, as the gas condenses, the water vapor and other impurities can be removed. The risk of water vapor is particularly high with compressed air, which is drawn into a compressor via the atmosphere. Atmospheric air contains a high proportion of water vapor (i.e., water in a gaseous form). Water removal is achieved through a combination of filtration and dehumidification.
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Where air is drawn in from the outside, the process of drawing in air also introduces microorganisms, which require filtering out. The level of filtering depends upon whether “sterile” air is required (absence of viable microorganisms) or air with a low bioburden. Compressed gas can be supplied at source either sterile or nonsterile. Sterility, where required such as with an inhalation product, is achieved through the use of a bacterial retentive membrane filter (0.2 μm pore size). Where a sterilefiltered gas is required it is important that the sterilizing grade filter is maintained dry for condensate in a gas filter that will most probably cause blockage or lead to microbial contamination. Risks of condensate are controlled by heating and use of hydrophobic filters (to prevent moisture residues in a gas supply system). Filters should also be changed periodically. As part of ongoing quality control, filters must be integrity tested at installation and at end of use. Although national standards bodies have guidance documents for compressed air sampling, and reference is made within FDA and EU GMPs, the general approach and requirements for compressed gases are set out in a multipart ISO standard: ISO 8573. This standard consists of the following parts (ISO, n.d.):
• • • • • • • • •
Part Part Part Part Part Part Part Part Part
1: Contaminants and purity classes, 2: Test methods for aerosol oil content, 3: Test methods for measurement of humidity, 4: Test methods for solid particle content, 5: Test methods for oil vapor and organic solvent content, 6: Test methods for gaseous contaminant content, 7: Test method for viable microbiological contaminant content, 8: Test methods for solid particle content by mass concentration, 9: Test methods for liquid water content.
Part 1 outlines the required purity classes based on the concentration of particles and level of impurities. The potential “impure” contaminants for compressed air, that can affect whether a required purity class is met, include:
•
• •
Particles (such as dirt, rust, pipe scale), with particles assessed by size. For example, as result of the mechanical compression process, additional impurities may be introduced into the air system. Generated contaminants include compressor lubricant, wear particles, and vaporized lubricant. Furthermore, fittings and accessories can contribute to particles. Water (in both vapor and liquid forms). Water is typically assessed by vapor pressure dew point. This is the temperature at which the air can no longer “hold” all of the water vapor which is mixed with). Oil (including aerosol, vapor, and liquid forms).
Compressed Gas Monitoring 189
With purity, many parts of the pharmaceutical industry will use Class 1 compressed gas based on the maximum number of permitted particulates.
Microbial Survival in Compressed Gases Although compressed gas and air systems are relatively harsh environments, they can aid microbial survival if there are available nutrients. The availability of nutrients is dependent upon the purity of the gas and airline. Nutrients suitable for metabolizing by microorganisms include water and oil droplets. Another factor that can affect survival is temperature, especially where temperatures are warmer (Stewart et al., 1995). In addition to vegetative cells, bacterial spores are well equipped to survive the harsh environmental conditions. Spores are resistant to the types of temperature ranges and moisture levels found within compress gas lines. Another risk exists with biofilm, where microbial communities can potentially form and develop through attachment to air lines and tubing. Although these risk factors exist, typically no microorganisms would be expected to be recovered from compressed gas lines. Research has shown that many microorganisms can survive and multiply in pressurized systems up to 10 bar and some are at least able to recover after being pressurized. However, at 160 bar pressure upwards, survival rates are very low. Where low level counts are recovered, these require investigation. More often the source is adventitious contamination, although a fault with the compressed air line cannot be ruled out. Although microbial contamination of compressed air or gas is a rare event, incidents can occur. Sources of contamination include:
• • • •
•
Source of the air or gas: Contamination can arise from air intake from the surroundings (which can contain oil, dirt, dust, moisture, or microorganisms). Piping distribution systems: Piping distribution and air storage tanks, more prevalent in older systems, will have contaminant in the form of rust, pipe scale, mineral deposits, in addition to bacteria. Bacterial retentive filter: The filter may become blocked, lose its integrity, or become wet. Compressor failure: The compressor itself can create a contaminated environment. For example, the compressor’s prefilters can become overloaded with dust and lint, causing the filter to cease functioning properly. Sample valve: The point-of-use sample valve may not be designed correctly or become faulty.
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Microbiological Requirements Microbial content itself does not influence the gas purity class assigned, although the standards recommend that microbial levels are assessed. Acceptable microbial numbers are subject to a separate assessment, with such assessment being based on an interpretation of GMP. A consensus is that the microbiological quality of the gas must be at least as good as the cleanroom air quality in which the process is taking place. Note that for EU GMP Grade A/ISO 14644 Class 5 areas, the microbial count would then be <1 cfu/m3 and the particle levels conform to the area at rest 3520 particles per m3. Many companies, however, do not monitor gas or compressed air used in Grade A or B areas since these are sterile filtered as close as possible to the point of use. In such cases a filter integrity test is executed in lieu of microbiological monitoring. Compressed air sampling should form part of an environmental monitoring program, along with cleanroom assessments. The program should take into account air points to be tested. This could be every point, points considered to be of greater risk (such as product contact), or representative points along a loop. The frequency of testing must also be considered, and this too would need to tie into risk. While there is an argument, as set out here, for the testing of compressed air where there is product contact there is less of a consensus over the testing of nitrogen. While nitrogen gas can be used to dispense or transfer most fluids from storage, the ISO standard has no specific microbial testing requirements and very few microorganisms, of the types common to pharmaceutical manufacturing environments, would be likely to survive. On this basis a risk-based justification could be made not to perform nitrogen gas testing. The user will need to determine whether each compressed gas line requires testing and the frequency of testing. Certainly all product contact compressed gases should be assessed. A sampling plan should also consider, and adapt to, the following:
• • • • • • •
Cleanroom grade, Type of product manufactured, Increased or reduced production schedules, Seasonal changes, Equipment changes and modifications, Replacement of hardware or filters and dryers, Inactivity of system.
When sampling compressed air for microorganisms, it is important that the air is depressurized and that the flow rate is controlled. Control of the flow
Compressed Gas Monitoring 191
rate is important to ensure that a cubic meter of air is sampled within the required sampling time (this time will be instrument dependent). If the air sampler takes 36 min to capture a cubic meter of air, then it will be sampling at one cubic foot per minute. An external regulator will be needed to bring the flow rate down to the sampling rate of instrument. This is assessed using a flow meter. Pressure reduction to atmospheric conditions is of great importance and knowing the flow allows the agar exposure time to be assessed, so that one cubic meter of air is sampled (Sandle, 2010). It is also important that isokinetic sampling of the air occurs and that air velocity is reduced until it is within the range of the sampler as identified by the manufacturer. This is not only necessary for obtaining the correct sample size but it also impacts on the possibility of microbial survival. The level of impact stress has been shown to affect microbial recovery on agar and be dependent upon the impaction velocity of the cells into the agar as well as the design and operating parameters. Due to the fact that any microorganisms present are transported under pressure and then suddenly released into atmospheric conditions, they may be damaged by the immediate expansion of the gas and the resulting shearing forces. The head of the instrument and any attachments should ideally be sterile before use, to avoid contamination. The culture medium used with the instrument should have been tested for growth promotion and, as for environmental monitoring, has been validated as suitable for gas viable air monitoring. With most samplers the head will be autoclavable. Some users disinfect the tubes and hoses used to connect the sampler with a disinfectant like 70% isopropyl alcohol. This is mentioned as an option in the ISO standard, although this is erroneously described as “sterilization.” Where a disinfectant is used it is important to run the air through the sampler without any agar plate in place; this is necessary to evaporate the disinfectant and to remove any residues. The presence of disinfectant could potentially lead to a “false negative.” With sampling, the sample inlet is connected to the compressed gas line and air is directed over an agar plate or strip. The method works by compressed gas, under reduced pressure, called “partial flow”, is forced over the surface of an agar plate. Any microorganisms are impinged onto the surface of the agar. The sampling time should be sufficient in order to sample one cubic meter of the agar. After sampling, the agar plate or strip is removed and incubated within a microbiology laboratory. At the end of incubation, the agar is examined for colony-forming units. Incubation can be for aerobic or anaerobic organisms, or both. The extent to which either is present should be based
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on initial validation and by taking into account whether such organisms pose a patient risk, should they end up being transferred into the end product. The type of instrument recommended in the ISO standard is a “slit-sampler, a type of impaction air tester,” although alternative samplers can be used, if justified. With a standard impactor sampler, air is drawn through a sampling head via a pump or fan and accelerated, usually through a perforated plate (sieve samplers), or through a narrow slit (slit samplers). This process creates a laminar flow through the sampler head. Hence, the air sampler should be fitted with a diffuser capable of maintaining laminar flow conditions. This is necessary so that particles pass through the sample head in a controlled flow. An appropriate agar must be selected. An example is tryptone soya agar, which is a generally nutritious medium designed to recover a range of bacteria and fungi. A key factor to take into account is whether the process of sampling leads to undue desiccation of the agar, rendering any recovered microorganisms unable to grow on the agar due to depletion of growth nutrients. This will be affected by the flow rate, type of compressed gas, and the model of air sampler, together with the type of culture medium. A risk will remain that microbial cells will become damaged by mechanical stress during the sampling process and lose viability.
Bacterial Endotoxin and Compressed Gases Some users elect to sample compressed air for bacterial endotoxin. Such testing remains relatively uncommon and it is only necessary should the compressed gas have a direct product contact and where there is a concern with Gram-negative bacteria. In most cases there should be no likelihood of endotoxin being present, especially in the context of nonsterile manufacturing. The sampling method for bacterial endotoxin is tricky and inexact. Either colonies are examined for Gram-negative bacteria, and assessment is made about endotoxin risk; or the compressed is passed through pyrogenfree water.
MONITORING STERILITY TEST ENVIRONMENTS Isolators used for sterility testing should be approached in a similar way as the monitoring of Grade A/ISO Class 5 areas. This section of the unit sets out the approach taken to environmental monitoring inside a half-suit isolator during sterility testing, at the author’s laboratory.
Monitoring Sterility Test Environments 193
The use of a sterility testing isolator in a regulated environment means that having reliable results for its sterility tests is essential, and although there is a history of confidence in the isolator system, having a microbiological environmental monitoring program is imperative. The reason for this is twofold: one, to demonstrate that if a product was to fail a sterility test then the reason for that failure could be attributed to the product alone and not as a factor of the isolator environment, and, two, to show that the overall background environment of the isolator is “in control” through trend analysis. The microbiological environmental monitoring typically consists of air samples, settle plates, finger plates, and contact plates or swabs. A standard culture media is typically used, like tryptone soya agar (TSA), which is a nonselective, highly nutritious media. Preliminary work need to take place in order to confirm that the action of sanitizing the media into the isolator, using hydrogen peroxide or peracetic acid vapor, or alternative method, does not cause inhibition of the TSA’s growth-promoting properties. The microbiological environmental monitoring should be performed for each sterility test by the operator. This provides invaluable information in the event of a sterility test failure. Monitoring typically includes:
•
•
•
•
Settle plates: uncovered TSA plates (9 cm diameter) exposed either side of the testing environment for the duration of the test. The final result should be converted to a number of colony-forming units (cfu) per 4 h. Air sample: there are pros and cons concerning the use of an air sampler inside an isolator. This debate centers on the disruption of the airflow that an air sampler may cause. The view here is that an air sample should be taken at each sterility test since having an assessment of the air quality outweighs any possible the effect on the airflow. Finger plates: at the end of the test, the operator should take finger plates (“dabs”) of each gloved hand by imprinting his/her fingers onto an agar plate. It is important that the gel strength of the agar is suitable for this task. Swabs or contact plates: the last microbiological monitoring activity is the taking of surface samples at set locations.
Once all the monitoring had been completed, the isolator is cleaned with an appropriate disinfectant to remove residues, such as an isopropanol alcoholbased disinfectant. Samples should be appropriately incubated (Chapter 7 discusses incubation regimes). The action limit applied to all samples should be 1 cfu, which
194 CHAPTER 11 Special Types of Environmental Monitoring
matches the same environmental standard as a process environment. A result at or exceeding the “action level” implies an “out-of-specification” investigation of aspects like the isolator gloves and the overall integrity; the matter becomes more serious should an upward trend develop.
MONITORING MICROBIOLOGY LABORATORIES Although there is no written regulatory requirement or guidance, it can be prudent to perform some level of environmental monitoring within the microbiology laboratory and associated incubators. The data gathered from such monitoring can be useful in assessing out-of-limits results especially when considering if contamination arose from the laboratory environment. The data gathered from such exercises can be examined and trended in a similar way to that from nonsterile manufacturing facilities. Although there are no prescribed action levels or specifications, most laboratories should have monitoring levels below to EU GMP Grade D/ISO Class 9 specifications (Table 1). Although there is no direct relationship to testing within the microbiology laboratory, the data gathered can be illuminating in terms of hygiene status and it could be useful should any test errors require root cause investigation.
TEST CONTROLS Most microbiological tests should have a test control, in order to assess validity, especially in relation to adventitious contamination. This is normally a negative control, although positive controls are used in some specific laboratory tests. Such controls serve a number of purposes:
• •
To assess the aseptic technique of the tester. To test the quality of the media and reagents. Table 1 Possible alert and action levels for monitoring microbiology laboratories
Sample type
Warning level (cfu)
Action level (cfu)
Active air Settle plate Contact plate
Not defined Not defined Not defined
200 100 50
Test Controls 195
Table 2 Example of viable microbiological environmental monitoring test controls Test Method
Control
Purpose
Microbiological monitoring: swab
Negative
Microbiological monitoring: contact plate Microbiological monitoring: active air sampler Microbiological monitoring: finger plate Microbiological monitoring: settle plate
None
For plain swabs: swab is soaked in Ringer’s solution and streaked out onto an agar plate, under a UDAF. This demonstrates that the Ringer’s solution, swab, and agar are not contaminated. For transport swabs: a swab is tested as presented. This control tests the environment in which the swab was taken, sampled, stored, and transported and the Technician’s technique in preparing and plating out swabs. Performed once during each monitoring session. Sterility of media checked through nutritive properties release procedure. Sterility of media checked through nutritive properties release procedure. Sterility of media checked through nutritive properties release procedure. Sterility of media checked through nutritive properties release procedure.
• •
None None None
To test the “testing” environment. To demonstrate that samples are operating as expected.
Controls should be examined as part of out-of-specification or out-of-limits reporting. A control, which is OOS or OOL, should be recorded onto an OOS/L report form (or an electronic record) and assessed along with samples tested during the test session. For samples that are OOS/L, the control should be examined for contamination or to check that it operated as expected. Controls can also be used as part of training and as a check for ongoing assessment. Where a control is consistently overaction the preventative action can include retraining and assessment. For viable environmental monitoring, test controls are normally negative controls. A possible approach to the use of controls is outlined in Table 2. A second form of control is with growth promotion testing. This is addressed in Chapter 7 of this book. Growth promotion testing can additionally be used if there are doubts about the media (such as stored outside of standard incubation conditions or media that has gone beyond its expiry time, although of course the acceptability of using testing to mitigate poor practices needs to be understood within a given organization’s quality system). An example of the growth promotion test is presented in Table 3. These types of controls can provide useful information about the suitability of the media and its handling.
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Table 3 Example of media growth promotion test controls Test method
Control
Purpose
Growth promotion testing
Positive
To demonstrate that the challenge count to the media on test is within the acceptance range of <100 cfu. To show how the test media compares to a previously released lot of the same media type with respect to the test challenge. Batches of media are incubated for a specified duration and inspected for contamination by the manufacturer. This demonstrates that the media has been prepared in a way that maintains sterility. The strains used to inoculate plates are visually checked for purity. Can assess whether media has become contaminated.
Comparative control Sterility
Negative
SUMMARY Although there are some important fundamentals for the contamination control strategy, as discussed in many of the chapters in this book, there are other aspects that might need to be included depending upon the nature of the pharmaceutical or healthcare organization. This relates to certain types of organisms found in specific locales. Are anaerobes of concern, for example? If nitrogen is used as part of the manufacturing process then this may be so. These questions are best addressed through risk assessment. In doing so, the nature of the likely contaminants needs to be assessed. Undertaking additional monitoring for psychrophiles, for example, serves no purpose if the organisms recovered from process rooms operating at <10°C are all capable of being recovered from standard environmental monitoring conducted using mesophilic incubation temperatures. The risk concept also applies to parametric release. This approach is not suitable for aseptically filled products but it can be applied to terminally sterilized products, with controls in place. Where environmental monitoring can assist is not only with the assessment of the absolute numbers recovered but also with the types of organisms recovered, in terms of their resistance to thermal destruction. Understanding the organisms is also something that needs to be factored into a risk assessment. The chapter has additionally addressed compressed gas monitoring. This is important to have in place since compressed gases are generally viewed as critical utilities when either in direct product contact or directly entered into the cleanroom environment. The chapter has also looked at test controls and the monitoring of sterility test facilities and of microbiology laboratories, areas that are associated with the primary environmental monitoring program but which equally do not form a central part of the program.
References 197
The extent to which each area discussed here should be applied, of if it all, needs to be risk assessed, taking into account the nature of the process and the types of hazards that might occur.
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