Control and Monitoring of Microbiological Quality in Nonsterile Manufacturing Facilities
Amy Jo Karren
In the health care industry, there is minimal guidance and, in fact, standards do not exist for establishing a microbiological monitoring program in a nonsterile environment. As a result, there are inconsistent practices, and in some cases, inadequate monitoring programs exist. There is a regulatory expectation that an environmental monitoring program is established that includes the monitoring of the microbial burden in the manufacturing area. This requirement applies to nonsterile, terminally sterilized, and aseptically manufactured products. Although the required state of control will vary depending on the product being manufactured, the requirement for microbiological environmental monitoring is universal across all health care manufacturing.
In the manufacturing of health care products, manufacturing controls provide assurance that the process is in a state of control and is the appropriate for the protection of product. A comprehensive environmental control and monitoring program typically includes the control and monitoring of air, water, and compressed gases (including compressed air). Microbiological monitoring provides a means to assess the current state of cleanliness and the stability of the cleanliness over time. During the course of the manufacturing process, the microbiological challenge on the environment will change due to the activity level of personnel, materials, components, packaging, etc. Throughout the year, seasonal changes may also impact the potential microbial burden on the environmental control systems depending on the geographical location. The culmination of these and other environmental stresses can impact the ability of the manufacturing control systems to maintain the required level of cleanliness, therefore establishing a robust sampling plan, based on risk, is prudent so that excursions or loss of control can be quickly identified, contained, and remediated.
For the purposes of microbial control, the controlled environment (a defined zone in which sources of viable and nonviable particulates are controlled by specified means) does not necessarily have to meet the requirements as defined by International Organization for Standardization (ISO) 14644-1.1 Particles (or particulates) are minute pieces of matter with defined physical boundaries and, for purposes of classification of air cleanliness, they fall within a cumulative distribution that is based on a lower limit size in the range from 0.1 to 5 µm. Viable particulates are those that consist of, or support, one or more viable microorganisms. In addition, the ISO standard does not specify the level of environmental control that is required in a manufacturing area. There are no regulatory requirements defining the level of environmental control to be used for the manufacture of nonsterile health care products based product classification. The only exception is that the People’s Republic of China requires ISO-classified cleanrooms for the manufacture of all types of nonsterile products. A cleanroom is a room within which the number of airborne particles is controlled and classified and which is designed, constructed, and operated in a manner to control the introduction, generation, and retention of particles inside the room.1 Additionally, the ISO 14644 standards address particle counts and do not discern between viable and nonviable particles. It is recognized that the levels of microorganisms and particulates are not necessarily directly related, but it is understood that particles could carry microorganisms and are frequently associated with particles of 10 to 20 µm in size.2,3 To provide some guidance, the international pharmaceutical community has recommended minimal cleanroom classification according to the following scheme4:
ISO 5: Aseptic processing zone, sterile product, and/or packaging component is exposed.
ISO 7: Area immediately surrounding the aseptic processing zone (ISO 6 may be employed but is neither required nor recommended.)
ISO 8: Nonsterile manufacturing, manufacturing area for terminally sterilized products
A classification in this case describes a method of assessing the level of cleanliness against a specification for a cleanroom, clean zone, controlled zone, or a defined location therein. Levels are expressed in terms of an ISO class, which represents maximum allowable concentrations of contaminants in a unit volume of air.1 A contaminant is any particulate, molecular, nonparticulate, and biological entity that can adversely affect the product or process. The decision on the level of control and cleanroom classification required for the manufacturing process is made by the manufacturer, with the approval of the regulatory or advisory body. The majority of terminally sterilized products are not manufactured using an aseptic process, so the presence of microorganisms in the environment is expected. The sampling strategies and responses to excursions in a nonsterile manufacturing environment should be significantly different than an aseptic process (see chapter 58). The program should be representative of the risk to the process and product. For nonsterile products and environments, the emphasis should be placed on the long-term trends including any seasonal variations, if applicable, rather than individual events.
RISK ASSESSMENTThe risk assessment for environmental control of the product is typically a separate activity performed prior to conducting the risk assessment for the environmental monitoring program. Both assessments are critical and provide complimentary information.
Environmental Control Risk
An appropriate and practical environmental control program requires an in-depth analysis of the manufacturing process to determine the impact of the environment on the product.5,6 A risk assessment to determine the potential for the production environment to affect product quality, including the presence of viable and nonviable particles, should be performed to determine the level of control that is needed to assure product quality.7,8 The program should be designed to monitor selected environmental control parameters in order to detect adverse conditions and be an integral part of the quality management system.9 The advantage to performing a thorough risk assessment is that the level of control required, and in turn, the investment for maintenance of that control can be established based on risk to the product and not on the expectation for absolute environmental control. If an appropriate environmental control risk assessment is not conducted, the facilities and environmental controls in manufacturing environment may be over- or underengineered. By performing a risk assessment, the level of environmental control implemented can be addressed with a pragmatic approach and appropriate for the product to be manufactured.
Environmental Monitoring Program Risk
Once the environmental control risk for the product is established, the environmental monitoring program can use this information. Microbial levels in controlled environments are not typically uniform (normally distributed) in a given area or over time. Because of this, during the initial risk assessment of the manufacturing area, it is important to establish and document a rationale when selecting the criteria for the monitoring program. To design a comprehensive environmental monitoring program, the following should be included in the assessment10:
Requirement for product microbial quality (eg, nonsterile, terminally sterilized, aseptically processed)
Requirements of applicable standards and regulations (eg, ISO 14644)
The product sterilization claim, if applicable (eg, sterile, sterile fluid path, sterile contents)
The validated method for terminal sterilization, if applicable (eg, moist heat, radiation, ethylene oxide, dry heat)
Environmental control parameters that should be monitored, as deemed critical for patient safety (eg, particulates, microbial levels and types)
Factors that might affect the environmental control (eg, number of personnel, raw material flow, relative humidity, disinfectant and method of application, seasonal changes)
Particulate and microbial monitoring method selection (eg, passive or active air sampling, surface sampling method)
Monitoring of water (eg, microbial, bacterial endotoxin, pH, total organic carbon, conductivity)
Monitoring of compressed air or gasses (eg, microbial, moisture, particulates)
Sampling plans (eg, number of sites sampled and frequency)
Microbial characterization (eg, Gram staining, colony morphology, phenotypic identification, genotypic identification)
Alert and action levels (eg, data required to set levels, statistical model, frequency of reassessment)
Trending requirements (eg, frequency, method of analysis, microbial characterization)
Process for handling excursions (eg, investigation procedure and documentation, evaluation of risk of an undetected excursion over time)
Maintenance of Risk Assessment
A review of the environmental monitoring risk assessment should be performed periodically as it might indicate that the parameters for the routine monitoring need to be adjusted. Additionally, there should be established requirements when there are planned or unplanned interruptions to a controlled environment. The monitoring
program should define the activities required to demonstrate that the controlled environment has returned to a qualified state, including demonstration of acceptable data following the interruption.
program should define the activities required to demonstrate that the controlled environment has returned to a qualified state, including demonstration of acceptable data following the interruption.
ENVIRONMENTAL MONITORING PROGRAM CONSIDERATIONSParticulate and microbial contamination is not usually uniformly distributed throughout a controlled environment and will fluctuate over time. Individual monitoring events and the derived data represent only a single point in time and may not reflect overall environmental conditions that are evident only by trending. The controlled environment and the respective monitoring results are influenced by a wide variety of factors and reflect the state of control at the time of sampling. Significance should be placed on trends and consistency of results rather than individual data points. The trending of the data over time provides evidence of a continued state of control of the environment. While alert and action levels should be established, the impact of an excursion (in well-controlled process) may not correlate with an increase in the product bioburden. Common factors that can influence the controlled environment include facility design, cleaning and disinfection processes, personnel, and monitoring methods.
Facility Design
Design factors such as air flow pattern, the number of air exchanges, differential pressures, materials of construction, traffic flow, doorways, and equipment placement all have the potential to impact the controlled environment. The initial design and construction of the cleanroom should use materials of construction that reduce the potential for particle shedding and facilitate cleaning. The layout of the room should consider the movement of the product and the personnel throughout the area to minimize disruption in the air flow. Equipment should be placed appropriately, to work effectively with the location of air returns and vents to ensure the air flow pattern is effective at maintaining the controlled environment.
Water or excessive moisture in the area can promote microbial growth as well as geographical areas with high humidity. The use or presence of water in a controlled environment elevates the microbial contamination risk, therefore, highlights the need for control measures and monitoring in the area of use.
The occupancy level of the area should also be a consideration in the design and control of the environment. At-rest monitoring data provides useful information related to facility design and performance, but operational conditions create greater stresses on the environmental controls. The occupancy of the room should be considered during qualification of the area, so that when routine monitoring is employed, the impact of the occupancy level is understood. At-rest sampling is useful to determine if there are changes to the baseline environment, such as after a major cleaning or maintenance events, but should not be used for routine monitoring. The facility design, layout, and occupancy should influence the selection of the sampling sites. Areas that have a greater potential for turbulent air (eg, doorways or equipment) or particle shedding (eg, equipment or known materials) or the presence of water should be considered in the sample site selection.
Cleaning and Disinfection Processes
There are many processes that facilitate microbiological control that should be considered when establishing and maintaining a controlled environment. One critical element of the overall control process is cleaning and disinfection of the room, equipment, and materials brought into the room. The effectiveness of the cleaning methods and frequency of cleaning can have a direct impact on level of control and resulting monitoring data. The disinfectants used should be effective for the expected microbial challenge in the area and associated risk to the product from specific types of organisms. The type of disinfectant, concentration used, application method, surface type, temperature, and contact time should all be considered when establishing a cleaning and disinfection program.11 The cleaning frequency should be adequate to maintain the required level of cleanliness. When performing microbial monitoring, consideration should be given to the residues from cleaners and disinfectants. There is potential that residues may influence the results of the monitoring. To overcome potential inhibition, media that contains neutralizers should be used in surface sampling to ensure accurate microbial monitoring results.
Personal gowning requirements are also critical to control the number and types of particulates from skin and clothing that may be introduced into the controlled environment. These (gloves, gowns, etc) may be sourced as sterile, single-use items but may also be reused and, in the latter case, are required to be periodically laundered (cleaned and disinfected) to reduce contamination risks. ISO 14644-5 provides considerations for a gowning process, based on the criticality of the associated environment.11 Additionally, raw materials and equipment may introduce microorganisms if not prepared or cleaned, as appropriate, prior to entering into the controlled environment. The following items are typically not recommended for entry into the cleanroom: cardboard, exposed paper, pallets, and unsealed wood. Items of this nature can be significant sources of microorganisms, are difficult to adequately clean or disinfect, and have a high potential to shed particles.
Personnel
Data indicate that people are the greatest contributors to microbial contamination in the cleanroom.12 The number of people and the behaviors in the cleanroom can influence the ability to control the environment. The majority of microorganisms related to personnel originate from skin and clothing. Proper gowning procedures provide measures to minimize the transfer of microorganisms from personnel into the controlled environment. Personnel also have the potential to add microorganisms to the environment by sneezing, coughing, talking, and touching surfaces with exposed hands. The process of hand washing, hand sanitization, and the use of gloves can reduce the transfer of microorganisms by touch. Practices such as use of cell phones, use of cosmetics, exposed body piercing, ear buds, or exposed wires should be avoided. There should also be no eating, drinking, or the consumption of chewing gum, lozenges, etc, in the cleanroom. No hair, beards or mustaches, jewelry, or clothing should be exposed with the use of proper gowning practices.
The training of personnel on acceptable behavior in the controlled environment can have a significant impact on microbial control. An example of training for behavior is teaching personnel that even the speed of their movements can impact the environment, whereas rapid movements can generate higher levels of particulates than deliberate and methodical movements. If there is an understanding of the criticality of proper behavior and the need for compliance to procedures to iterate and influence personnel, there is generally compliance. Personnel need to know that what they do impacts product quality and the patients we all serve. Established procedures and training for proper hygiene, gowning, and behaviors are required.
Monitoring Methods
Microbial monitoring results can be highly variable due to the methods used for measurement. Sources of variation might include the sampling technique, sample volume or duration of exposure, timing of sampling during routine manufacturing, distribution of microorganisms in the environment, and the methods used to culture and quantify the microorganisms. The microbial sampling methods used for routine monitoring should be the same sampling methods used during qualification of the controlled environment. If the sampling method is changed, an evaluation of the change should take into consideration the following impact to the routine monitoring:
Applicability of established alert and action levels (eg, relationship with historical data)
Ability to detect similar microflora (eg, fastidious or vegetative organisms)
Ability to neutralize potential disinfectant residuals
Ability to detect organisms at a similar frequency (eg, impact of active versus passive sampling methods)
In controlled environments, the potential microbiological contamination is most often from human skin, human mucosal membranes, sources of water, and soil. The types of microbiological media and incubation conditions selected should be appropriate for the types of microorganisms present in the environment, with additional consideration regarding the ability to detect organisms that are a known risk to the product. A nonselective growth media, such as Soybean Casein Digest agar, incubated for greater than 3 days at 30°C to 35°C is generally acceptable to detect most heterotrophic environmental organisms. For the culturing of fungal isolates, a selective media that promotes the cultivation of fungi and suppresses aerobic bacterial growth, such as Sabouraud Dextrose agar or Potato Dextrose agar, is appropriate. The incubation conditions for fungal plates are generally greater than 5 days at 20°C to 25°C. The standard for product bioburden testing, ISO 11737-1, is a good source of information on appropriate media and incubation conditions for cultivation of environmental isolates.13
Methods for viable sampling are not equivalent. For example, when sampling air, the data generated from a sieve impactor sampler is not equivalent to that of a settling plate. The impactor provides data based on a volume of air sampled, whereas the settling plate results represent the time of exposure. This is true also for methods for viable surface sampling. Changes in culture media and incubation conditions may also impact the detection of the organisms. If the established method for monitoring or the media used in the test system changes, the alert and action levels of the sampling sites must be reassessed and, if appropriate, trend data must be reestablished.
The timing of sampling in the manufacturing area can also influence the data. Periods of time such as shift change or when there is the high level of activity in the controlled environment tend to be worst case for monitoring results. Conversely, monitoring during an at-rest state may falsely provide confidence in the effectiveness of the controls. The time of the monitoring should be understood and defined so that the monitoring provides relevant and consistent data to demonstrate the state of control. Facilities with no history should perform more extensive qualification testing at a greater frequency (eg, daily, weekly, monthly) when establishing the environmental monitoring program. Once baseline testing has been completed, routine monitoring with a reduced sampling plan might be appropriate. The sampling site selection, frequency, and method of monitoring should be designed to provide long-term historical data regarding the microbiological characterization and to provide early indication of changes to the environment.
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