Microbiological Evaluation of Clean and Controlled Areas

Microbiological Evaluation of Clean and Controlled Areas

Cleanroom is a controlled environment for manufacturing of different products and they may be from simple to complicated multilevel design with heavy equipment. Due to the movement of labor, premises, processes and machinery used there are high chances of contamination which should be eliminated in accordance with standards laid down by ISO.

Sources for the contamination are by various bacteria and various processes where the personnel tend to carry during their movement. Air flow should be checked for its velocity and also filtration through filters.

 Rapid microbiological methods can be considered instead of traditional methods to obtain quick results. But still it has to be evaluated against the standard pharmacopoeial methods before putting in practice.

 Disinfectants used for cleaning rooms should be checked for contamination and stored for finite periods only.

 In order to reduce contamination due to microbes clean areas should be fumigated.

 According to the standards of ISO cleanroom is divided into four zones

Classification of cleanroom

Cleanroom has been divided into four zones. They are

Local zone or A – highly risked operations are performed in this zone. E.g. Opening of bottle , filling, ampoule and closing.

B zone – circled around zone A used for aseptic filling and preparation

C, D zone – few stages of manufacturing sterilized products.

The design and construction of cleanrooms and controlled environments is given by Federal Standard 209E which is defined by absolute concentration of airborne particles. Various methods for classification of air and to monitor airborne particulates are also included. The document given only applies to airborne particulates and is given by manufacturers of clean rooms to provide specifications for building, commissioning and maintaining of facility.

Pharma industry has a greater concern for viable particulates rather than total particulates as specified in 209E. The rationale that fewer particulates in cleanroom which likely may be microorganisms due to air is acceptable and provides pharmaceutical manufacturers and builders with proper standards and helps in establishing proper functional facility.

In pharmaceutical industry Federal Standard 209E is based on limits of particles with size equal to or larger than 0.5µm. Airborne particulate cleanliness classes are given in Table 1 which are followed in pharmaceuticals. The pharma industry includes Class M3.5 and above. Class M1 and M3 relate to electronic industry and are depicted in Table 1 for the purpose of comparison. It is generally accepted that if fewer particulates are present in any clean room or controlled environment where operations are performed, the microbial count under operational conditions will be fewer, only if airflow, temperature and humidity don’t change. Clean rooms are maintained under a state of operational control on the basis of dynamic data

Table 1 – Airborne Particulate Cleanliness Classes*

* Adapted from U.S. Federal Standard 209E, September 11, 1992 – “Airborne Particulate Cleanliness Classes in Clean Rooms and Clean Zones”

Importance of Microbiological Evaluation Program for Controlled Environments

Monitoring of total particulate count in controlled environments even with electronic instrumentation on a continuous basis doesn’t provide information on microbiological content of environment. The limitation of particulate counters is that they measure particles of 0.5 µm or more. Airborne microorganisms are not single cells or free floating frequently they associate with particles of 10 to 20 µm. Particulate and microbial counts within a controlled environment tend to change with the sampling location and the activities being conducted during sampling procedure. Observing and checking the environment for particulates and microorganisms which are non-viable is an important control activity as both are crucial in attaining product compendial requirements for particulate matter, sterility under injections.

Programs for monitoring microbial activity in controlled environments should evaluate the effectiveness of cleaning and sanitization activities by and due to staff who could have an effect on the bioburden of the controlled environment. Monitoring the microbial activity, despite how advanced the system may be will not and need not identify and quantitate all contaminants in controlled environments. However, routine microbial monitoring should provide sufficient information to establish that the controlled environment is operating within an adequate state of control.

Environmental microbial monitoring and data analysis by qualified staff will permit the status of control to be maintained in clean rooms and controlled environments. The area should be sampled during normal operations to allow for the collection of useful data. Microbial sampling should be done when materials are in the area, processing activities are ongoing and a full complement of operating staff is present on site.

Microbial monitoring of clean rooms and some other controlled environments, if suitable, should involve quantitation of the microbial content of room air, compressor air that penetrates the critical area, surfaces, machinery, containers for  sanitization, floors, walls, personnel garments (e.g., gloves and gowns). The objective of this monitoring program is to obtain representatives estimates of by trained personnel. It is important to evaluate environmental results to determine presence of trends. Trends can be interpreted through the statistical control charts that indicate alert and action levels. The microbial control of controlled environments can be evaluated, in part, on the basis of data obtained from trend analysis. Periodic summaries or reports should be given so that the responsible manager will be watchful.

When the level of microbes in a controlled environment is exceeded, a documentation review and investigation should be done. There may be difference in the details of the investigation, depending in the type and processing of the product manufactured in the area. Investigation should be done by reviewing area maintenance documentation; sanitization documentation; the inherent operational or physical parameters, like changes in environmental temperature and relative humidity; and the status of training of personnel. After investigation, actions taken may include reinforcement of training of personnel to draw attention to the microbial control of the environment; additional sampling at increased frequency, sanitization and product testing; identifying the microbial contaminant and its possible source and evaluating the requirement to access the current standard operating procedures and to revalidate them, if necessary.

After the results obtained from investigation and testing, the exceeded level of the microbial level and the under that condition acceptability of the  products processed or operations  may be attributed. Every action and changes made during investigation should be documented as part of the overall quality management system.

A controlled environment either a clean room or clean zone is defined by clean room operational standard. Parameters like filter integrity, air changes, air velocity, air patterns, and pressure differentials are evaluated. These parameters tend to affect the microbiological bioburden of the clean room operation. The design and construction of clean rooms and its operation vary widely, thus to generalize requirements for these parameters is difficult. A method has been developed by Reinmuller and Ljungquist for carrying a particulate challenge test to the system by increasing the concentration of ambient particle in the vicinity of critical work areas and equipment.¹ Firstly the smoke that is generated allows the movement of air to be visualized throughout a controlled environment or a clean room. The presence of turbulent zones or vortices can be visualized throughout a controlled environment or a clean room. The presence of turbulent zones or vortices and the pattern of flow of air should be adjusted to eliminate undesirable effects. Then the particulate matter generated is close to the sterile field and critical zone. This evaluation should be done under simulated production conditions along with equipment and personnel in place.

Proper optimization and testing of the physical characteristics of the clean room or controlled environment is essential before validation of the microbiological monitoring program is completed. Assurance that the controlled environment is operating adequately to its engineering specifications will give more assurance that the bioburden of the environment will be suitable for aseptic processing. These tests should be repeated during routine certification of the controlled environment or clean room and if any changes are made to the operation such as personnel flow, processing, operation, and material flow, air handling systems or equipment layout are determined to be significant.

Training of Personnel 

Products which are aseptically processed require manufacturers to pay close attention to detail and to maintain rigorous discipline and supervision of staff so that the level of environmental quality appropriate is maintained for assurance of a sterile final product.

Training of all the employees working in controlled environments is critical and is equally crucial for personnel involved in microbial monitoring program, where contamination of the clean working area could inadvertently occur during sampling. The monitoring personnel may be the employees who are most directly in contact with critical areas in automated operations. Monitoring of personnel should be conducted before or after working in the processing area.

During microbiological sampling there may be chance for microbial contamination because of inappropriate sampling techniques. To minimize the risk of contamination a formal personnel training is required. This training should be documented for all staff entering into controlled environment.

Managements of the facility must assure that all the staff involved in operations in clean rooms and controlled environments should know the basic principles of microbiology. The training should have instructions on the basic principles of manufacturing and handling procedures to potential sources of product contamination in aseptic processing. This training must include instructions on the basic principles of microbial physiology, microbiology, sanitation and disinfection, preparation and media selection, sterilization and taxonomy in requirement to the nature of involvement of personnel in aseptic processing. A specialized training on labor methods is required for staff involved in microbial identification. Additional training on the management of the data collected from environmental must be made to staff. Proficiency and understanding of necessary standard operating procedures is crucial, particularly standard operating procedures relating to measures or crucial steps taken when environmental conditions dictate. Understanding of regulatory yielding policies and responsibility of every individual in compliance to good manufacturing practices (GMPs) should be a basic part of training as well as in investigation and analyzing data.

The major source of microbial contamination in controlled environments is the personnel. Contamination can occur due to microorganisms being spread by individuals and in particular those with active infections. Only healthy individuals should be allowed in controlled environment.

These facts underscore the importance of good personal hygiene and attention to minute detail in the gowning procedure used by personnel entering the controlled environment under aseptic conditions. Once these employees are properly gowned with complete facial coverage they must be careful to maintain the integrity of their suits and gloves at all times as the major threat of contamination of product being aseptically processed comes from the staff in operation activity, microbial contamination control associated with these personnel is one of the crucial elements of the environmental control program.

In controlled environment the importance of thorough training of personnel in aseptic techniques have to be prioritized. The environmental monitoring program can't detect all events in aseptic processing, by itself, which impacts the quality of microbial level in a controlled environment. Hence, media-fill or process simulation studies are to be done periodically to revalidate the process are necessary to assure that the proper training and operating controls are effectively maintained.

Factors to be considered in designing and implementing of a microbiological environmental control program         

An environmental control program should detect any adverse drift in microbiological conditions in a timely manner which helps in producing effective and corrective action. It lies in the hand of manufacturer to initiate, develop, implement and document a microbial environmental monitoring program.

General recommendations for an environmental control program are given by taking into account specified facilities and conditions. Soybean Casein Digest Medium is the most commonly used growth medium microbiologically which is suitable in most sanitizing agents or of antibiotics if used or processed in these environments. Yeasts and molds should be identified and quantified. Generally mycological media such as Sabouraus’s, Modified Sabouraud’s, or Inhibitory mold Agar is acceptable. Soybean-Casein Digest Agar can be used to promote the growth of fungi. Obligatory anaerobes are not tested but still identification of these organisms in sterility testing facilitates, more frequent testing is indicated when investigations warrant. Selected media should be evaluated to detect and quantify these microaerophilic microorganisms or anaerobes.

After appropriate media is selected time and incubation temperature are selected with incubation time of 72 and 48 hours and incubation temperature in 22.5±2.5o and 32.5±2.5o range, respectively. In this environmental program the processes of sterilization for growth media should be validated and also evaluated for sterility and for growth promotion as detailed in sterility tests. Microflora separated from the controlled environment or ATCC strain preparations of these isolates can be used to test media in addition to Growth Promotion test. Media should support growth even when inoculated with 100 colony-forming units (cfu) less than challenge organisms.

An appropriate environmental control program should identify and evaluate sampling sites and validate methods for microbiological sampling of the environment. The methods for identification of isolates should be verified using indicator microorganisms.

Establishment of Site and Sampling Plan

When a clean room or any other controlled environment is set up locations for surface sampling and air should be determined. Consideration should be given to air and surfaces in contact with a product or sensitive surfaces of container-closure systems which is considered as critical areas of operation and to the product details. In a parenteral vial filling operation, areas of operation would typically include the paths of opened containers, container-closure supply and inanimate objects like fomites which are handled routinely by personnel.

Sampling frequency depends on the criticality of specified sites and the subsequent treatment product receives after processing it aseptically. Table 2 shows suggested frequencies of sampling in decreasing order of frequency of sampling and in relation to the criticality of the area of the controlled environment being sampled.

Table 2. Suggested Frequency of Sampling on the basis of Criticality of Controlled Environment

As manual interventions increase during operation then there is increase in the potential for personnel contact with the product which in turn relatively increases the importance of an environmental monitoring program. This program is more critical for products that are processed and then terminally sterilized. Determination and quantitation of resistant microorganisms to the subsequent sterilization treatment is more critical than monitoring of the surrounding microbiological manufacturing environments. The value of the bioburden program becomes critical when terminal sterilization cycle is not based on the overkill cycle concept but on the bioburden before sterilization.

The sampling plans should be in correlation with frequencies and sample plan locations adjusted based on trending performance. Based on the performance sampling can be increased or decreased.

Establishment of microbiological alert and action levels in controlled environments

The principles and concepts laid down by statistical process control are useful in indicating Alert and Action levels and depicting trends.

An Alert level in microbiological environmental monitoring is that level of microorganisms that shows a potential drift from normal operating conditions. When the Alert level is exceeded, even if no action is taken but it should be documented following an investigation which includes sampling plan modifications.

An Action level in microbiological environmental monitoring is that level of microorganisms that when exceeded requires immediate follow-up and corrective action.

Alert levels are based upon historical information gained from the routine operation of the process in a specified controlled environment.

In a new facility, these levels are generally based on historical experiences from similar facilities and processes; and to establish a baseline, several weeks of data should be evaluated on microbial environmental levels.

If data from previous experiences demonstrate improved conditions then these levels can be re-examined and altered to reflect the conditions. Trends that depict a deterioration of the environmental quality should be evaluated for assignable cause and corrective action plan to bring the condition back to the expected level. However, its potential impact on the product should be evaluated after implementation.

Microbial Action Levels and Considerations for Controlled Environments

Classification of clean rooms and other controlled environments is based on Federal Standard 209E which is based on total particulate counts for these environments. The pharmaceutical and medical devices industries have adopted the classification of Class 100, Class 10,000, and Class 100,000, in terms of specifications for the construction of facilities. Although there is no direct relationship between the 209E controlled environment classes and microbiological levels, the pharma industry has been using microbial levels corresponding to these classes for a number of years; and these levels have been those used for evaluation in compliance with current GMP.² These levels were readily achievable with the current technology for controlled environments. There have been concerns about differences in the values obtained by different sampling systems, incubation temperatures, and media variability that have been reported. Although there is no absolute system but still changes and trends can be detected in environmental quality. Tables 3,4, and 5 represent values of individual test results and are only for guidance. Data from each manufacturer’s must be evaluated as part of an pilot monitoring program.

Table 3. Air Cleanliness Guidelines in Colony- Forming Units (cfu) in Controlled Environments (Using a Slit-to-Agar Sampler or Equivalent)

* As defined in Federal Standard 209E, September 1992

** A sufficient volume of air should be sampled to detect excursions above the limits specified.

Table 4. Surface Cleanliness Guidelines of Equipment and Facilities in cfu in Controlled Environments

* Contact plate areas vary from 24 to 30 cm². When swabbing is used in sampling, the area covered should be greater than or equal to 24cm² but no larger than 30cm².

Table 5. Surface cleanliness Guidelines in Controlled Environments of Operating Personnel Gear in cfu

* Contact plate areas vary from 24 to 30 cm². When swabbing is used in sampling, the area covered should be greater than or equal to 24cm² but no larger than 30cm².

Methodology and Instrumentation for Quantitation of Viable Airborne Microorganisms

Scientists accept microorganisms that are airborne tend to impact the microbiological quality of intermediate or final products manufactured in controlled environments. Estimation of the microorganisms that are airborne can be affected by procedures and instruments used to perform the assays. Therefore the results obtained should be ascertained by alternative methods or equipment whenever they are available. In the future advanced technology brings innovations which offer greater precision and sensitivity than the currently available methodology by justifying the change in the absolute numbers of organisms that are detected.

Impaction and centrifugal samplers are the most commonly used samplers in the U.S. medical and pharmaceutical device industry but still commercially available samplers are cited for the purpose of information. It is the user’s responsibility to select a particular sampler based on its appropriateness.

Slit-to-Agar Air Sampler (STA) – This instrument is based upon the microbial guidelines indicated in Table 3 for the various controlled environments. An attached source of controllable vacuum powers the unit. The air intake is obtained through a standardized slit below which is placed a slowly revolving Petri dish containing a nutrient agar. Particles in the air that have sufficient mass impact on the agar surface and viable organisms are allowed to grow out. To minimize disturbance of the laminar flow field a remote air intake is used.

Sieve Impactor – The apparatus has a container which is designed to accommodate a petri dish containing a nutrient agar. The unit's cover is perforated with perforations of a predetermined size. A vacuum pump draws known volume of air through the cover, and the particles in the air containing microorganism’s impact on the agar medium in the petri dish. Some samplers are available with a cascaded series of containers containing perforations of decreasing size. These units allow for the determination of the distribution of the size ranges of particulates containing viable microorganisms, based on which size perforations admit the particles onto the agar plates.

Centrifugal Sampler – The unit consists of a turbine or propeller that pulls a known volume of air into the unit and then propels the air outward to impact on a tangentially placed nutrient agar strip set on a flexible plastic base.

Sterilizable Microbiological Atrium- The unit is a variant of the single-stage sieve impactor. The units cover contains uniformly spaced orifices approximately 0.25 inch in size. The base of the unit accommodates one petri dish containing a nutrient agar. A vacuum pump controls the movement of air through the unit, and a multiple-unit control center as well as a remote sampling probe is available.

Surface Air System Sampler – This integrated unit consists of an entry section that accommodates an agar contact plate. Immediately behind the contact plate is a motor and turbine that pulls air through the units perforated cover over the agar contact plate and beyond the motor, where it is exhausted. Multiple mounted assemblies are also available.

Gelatin Filter Sampler – The unit consists of a vacuum pump with an extension hose terminating in a filter holder that can be located remotely in the critical space. The filter consists of random fibers of gelatin capable of retaining airborne microorganisms. After a specified exposure time, the filter is aseptically removed and dissolved in an appropriate diluent and then plated on an appropriate agar medium to estimate its microbial content.

Setting plates – This method is still widely used as a simple and inexpensive way to qualitatively assess the environments over prolonged exposure times. The exposure of open agar- filled petri dishes or settling plates is not to be used for quantitative estimations of the microbial contamination levels of critical environments.

One of the major limitations of mechanical air samplers is the limitation in sample size of air being sampled. Where the microbial level in the air of a controlled environment is expected to contain not more than three cfu per cubic meter, several cubic meters of air should be tested if results are to be assigned a reasonable level of precision and accuracy. Often this is not practical. To show that microbial counts present in the environment are not increasing over time, it might be necessary to extend the time of sampling to determine if the time of sampling is a limiting factor or not. Typically, slit-to-agar samplers have an 80-liter-per-minute sampling capacity (the capacity of the surface air system is somewhat higher). If one cubic meter of air is tested, then it would require an exposure time of 15minutes. It may be necessary to use sampling times in excess of 15minutes to obtain a representative environmental sample. Although there are samplers reported to be capable of very high sampling volume rates, consideration in these situations should be given to the potential for disruption of the airflow patterns in any critical area or to the creation of a turbulence that could increase the probability of contamination.

For centrifugal air samplers, a number of earlier studies showed that the samples demonstrated a selectively for larger particles. The use of this type of sampler may have resulted in higher airborne counts than the other types of air samplers because of that inherent selectivity.

When selecting a centrifugal sampler, the effect of the sampler on the linearity of the airflow in the controlled zone where it is placed for sampling should be taken into consideration. Regardless of the type of sampler used, the use of a remote probe requires determining that the extra tubing does not have an adverse effect on the viable airborne count. This effect should either be eliminated or if this is not possible, a correction factor should be introduced in the reporting of results.

Methodology and Equipment for Sampling of surfaces for Quantitation of Viable Microbial Contaminants in Controlled Environments

Another component of the microbial environmental control program in controlled environments is surface sampling of equipment, facilities, and personnel gear used in these environments. The standardization of surface sampling methods and procedures hasn’t been as widely addressed in the pharmaceutical industry as the standardization of air sampling procedures.³ To minimize disruptions to critical operations, surface sampling is performed at the conclusion of operations. Surface sampling may be accomplished by the use of contact plates or by the swabbing method. Surface monitoring is generally performed on areas that come in contact with the product and on areas adjacent to those contact areas. Contact plates filled with nutrient agar are used when sampling regular or flat surfaces and are directly incubated at the appropriate time for a given incubation temperature for quantitation of viable counts. Specialized agar can be used for specific quantitation of fungi, spores, etc.

The swabbing method may be used for sampling of irregular surfaces, especially for equipment. Swabbing is used to supplement contact plates for regular surfaces. The swab is then placed in an appropriate diluent and the estimate of microbial count is done by plating of an appropriate aliquot on or in specified nutrient agar. The area to be swabbed is denied using a sterile template of appropriate size. In general, it is in the range of 24-30cm². The microbial estimates are reported per contact plate or per swab.

Culture media and diluents used for sampling or quantitation of microorganisms

 The type of medium, liquid or solid that is used for sampling or quantitation of microorganisms in controlled environments will depend on the procedure and equipment used. A commonly used all-purpose medium is Soybean-casein Digest Agar when a solid medium is needed. Other medium, liquid or solid are listed below

*  Liquid and solid media are sterilized using a validated process.

These media are commercially available in dehydrated form. They are also available in ready-to-use form. When disinfectants or antibiotics are used in the controlled environment, consideration should be given to using media with appropriate inactivating agents.

Alternative media to those listed can be used provided that they are validated for the purpose intended.

Identification of Microbial Isolates from the Environmental Control Program

The environmental control program includes an appropriate level of identification of the flora obtained from sampling. A knowledge of the normal flora in controlled environments aids in determining the usual microbial flora anticipated for the facility being monitored; evaluating the effectiveness of the cleaning and sanitization procedures, methods and agents; and recovery methods. The information gathered by an identification program can also be useful in the investigation of the source of contamination, especially when the Action levels are exceeded.

Identification of isolates from critical areas and areas immediate to these critical areas should take precedence over identification of microorganisms from noncritical areas. Identification methods should be verified and ready to use kits should be qualified for their intended purpose.

Operational Evaluation of the Microbiological Status of Aseptically Filled Products in Clean Rooms and Other Controlled Environments

The controlled environment is monitored through an appropriate environmental monitoring program. To assure that minimal bioburden is achieved, additional information on the evaluation of the microbiological status of the controlled environment can be obtained by the use of media fills. An acceptable media fill shows that a successful simulated product run can be conducted on the manufacturing line at that point in time. However, other factors are important, such as appropriate construction of facilities, environmental monitoring and training of personnel.

When an aseptic process is developed and installed it is generally necessary to qualify the microbiological status of the process by running at least three successful consecutive media fills. A media fill utilizes growth medium in lieu of products to detect the growth of microorganisms. Issues in the development of a media fill program that should be considered are the following: media-fill procedures, media selection, fill volume, incubation, time and temperature, inspection of filled units, documentation, interpretation of results, and possible corrective actions required.

Since a media fill is designed to simulate aseptic processing of a specified product, it is important that conditions during a normal product run in effect during the media fill. This includes the full complement of personnel and all the processing steps and materials that constitute a normal production run. During the conduct of media fill, various predocumented interventions that are known to occur during actual product runs should be planned (e.g., changing filling needles, fixing component jams).

Alternatively, in order to add a safety margin, a combination of possible conditions can be used. Examples may include frequent start and stop sequences, unexpected repair of processing system, replacement of filters, etc. The qualification of an aseptic process need not be done for every product, but should be done for each processing line. Since the geometry of the container (size as well as opening of the container) and the speed of the line are factors that are variable in the use of an aseptic processing line, appropriate combination of these factors, preferably at the extremes, should be used in the qualification of the line. A rationale for products used should be documented.

 The 1987 FDA Guideline on sterile Drug Products produced by Aseptic processing indicates that media fills run be done to cover all production shifts for line/product/container combinations. This guideline should be considered not only for qualification media fills run, but also for periodic reevaluation or revalidation. Media fill programs should also simulate production practices over extended runs. This can be accomplished by doing media fills runs at the end of production runs.

In general, an all-purpose, rich medium such as Soybean Casein Broth that has been checked for growth promotion with a battery of indicator organisms at a level of below 100 cfu/unit, can be used. Isolates from the controlled environment where aseptic processing is to be conducted may also be used. Following the aseptic processing of the medium, the filled containers are incubated at 22.5± 2.5^o or at 32.5± 2.5^o. All media filled containers should be incubated for a minimum of 14 days. If two temperatures are used for incubation of media filled samples, then these filled containers should be incubated for at least 7 days at each temperature. Following incubation, the medium-filled containers should be inspected for growth. Media filled isolates are identified by genus and, when possible, by species in order to investigate to the source of contamination.

Critical issues in performing media fills are the number of fills to qualify as aseptic process, the number of units filled per media fill, the interpretation of results, and implementation of corrective actions. Historically, three media-fill run during intial qualification or start-up of a facility are conducted to demonstrate consistency of the aseptic processing line. The minimum number of units to demonstrate a contamination rate of not more than 0.1% which is the criterion for acceptance of a successful media-fill run, is at least 3,000. It should be emphasized that many firms in the United states and other countries are filling more than 3,000 units in a single media-fill run.⁴ Pilot plant facilities used for preparing small clinical lots may use smaller media fills. .

  A number of international documents (i.e., ISO and EU-GMP) have also cited an expectation of zero positives out of 3,000 media filled units at the 95% confidence level. However, it is recognized that repeated media runs are required in order to confirm the statistical validity of the observed contamination rate for the process.

PDA Technical Monograph Number17,⁴ “A Survey of Current Sterile Manufacturing practices” indicated that many manufacturers believe that their aseptic processes are capable of contamination rates below 0.1%.

Since the most critical source of contamination in the cleanroom is the personnel, visual documentation that can be helpful in correlating production activities to contamination events during media fills is encouraged. The widespread use of isolator systems for sterility testing has demonstrated that elimination of personnel does reduce contamination in aseptic handling.

An Overview of the Emerging technologies for Advanced Aseptic Processing

Because of the strong correlation between human involvement and intervention and the potential for product contamination in aseptic processing, production systems in which personnel are removed from critical zones have been designed and implemented. Methods developed to reduce the likelihood of contamination include equipment automation, barriers, and isolated systems. Facilities that employ these advanced aseptic processing strategies are already in operation. In facilities where personnel have been completely excluded from the critical zone, the necessity for room classification based on particulate and environmental microbiological monitoring requirements may be significantly reduced.

The following are definitions of some the systems currently in place to reduce the contamination rate in aseptic processing:

Barriers – In the context of aseptic processing systems, a barrier is a device that restricts contact between operators and the aseptic field enclosed within the barrier. These systems are used un hospital pharmacies, laboratories, and animal care facilities, as well as in aseptic filling. Barriers may not be sterilized and do not always have transfer systems that allow passage of materials into or out of the system without exposure to the surrounding environment. Barriers range from plastic curtains around the critical production zones to rigid enclosures found on modern aseptic-filling equipment. Barriers may also incorporate such elements as glove ports, half-suits, and rapid- transfer ports.

Blow/Fill/Seal – This type of system combines the blow-molding of container with the filling of product and a sealing operation in one piece of equipment. From a microbiological point of view, the sequence of forming the container, filling with sterile product, and formation and application of the seal are achieved aseptically in an uninterrupted operation with minimal exposure to the environment. These systems have been in existence for about 30 years and have demonstrated the capability of achieving contamination rates below0.1%. Contamination rates of 0.001% have been cited for blow/fill/seal systems when combined media-fill data are summarized and analyzed.

Isolator – This technology is used for a dual purpose. One is to protect the product from contamination from the environment, including personnel, during filling and closing, and the other is to protect personnel from deleterious or toxic products that are being manufactured.

Isolator technology is based on the principal of placing previously sterilized components \ (containers/products/closures) into a sterile environment. These components remain sterile during the whole processing operation, since no personnel or non-sterile components are brought into the isolator. The isolator barrier is an absolute barrier that does not allow for interchanges between the protected and unprotected environments. Isolators either may be physically sealed against the entry of external contamination or may be effectively sealed by the application of continuous overpressure. Manipulations of materials by personnel are done via use of gloves, half-suits or full suits. All air entering the isolator passes through either an HEPA or UPLA filter and exhaust air typically exits through an HEPA-grade filter. Peracetic acid and hydrogen peroxide vapor are commonly used for the surface sterilization of the isolator unit’s internal environment. The sterilization of the interior of isolators and all contents are usually validated to a sterility assurance level of 10^-6. Equipment, components, and materials are introduced into the isolator through a number of different procedures: use of a double – door autoclave, continuous introduction of components via a conveyor belt passing through sterilizing tunnel; use of a transfer container system through a docking system in the isolator enclosure. It is also necessary to monitor closely an isolator unit’s integrity, calibration, and maintenance.

The requirements for controlled environments surrounding these newer technologies for aseptic processing depend on the type of technology used.

Blow/fill/seal equipment that restricts employee contact with the product may be placed in a controlled environment, especially if some form of employee intervention is possible during production.

Barrier systems will require some form of controlled environment. Because of the numerous barrier systems types and applications, the requirements for the environment surrounding the barrier system will vary. The design and operating strategies for the environment around these systems will have to be developed by the manufacturers in a logical and rational fashion. Regardless of these strategies, the capability of the system to produce sterile products sterile products must be validated to operate in accordance with pre-established criteria.

In isolators, the air enters the isolator through integral filters of HEPA quality or better and their interiors are sterilized typically to a sterility assurance level of 10^-6; therefore, isolators contain sterile air, do not exchange sir with the surrounding environment, and are free of human operators. However, it has been suggested that when the isolator is in a controlled environment, the potential for contaminated product is reduced in the event of a pinhole leak in the suit or glove.

The extent and scope of an environmental microbiological monitoring of these advanced systems for aseptic processing depends on the type of system used. Manufacturers should balance the frequency of environmental sampling systems that require human intervention with the benefit accrued by the results of that monitoring. Since barrier systems are designed to reduce human intervention to a minimum, remote sampling systems should be used in lieu of personnel intervention. In general, once the validation establishes the effectiveness of the barrier system, the frequency of sampling to monitor the microbiological status of the aseptic processing area could be reduced, as compared to the frequency of sampling of classical aseptic processing systems.

Isolator systems require infrequent microbiological monitoring. Continuous total particulate monitoring can provide assurance that the air filtration system within the isolator is working properly. The methods for quantitative microbiological air sampling described in this chapter may not have sufficient sensitivity to test the environment inside an isolator. Experience with isolators indicates that under normal operations pinhole leaks or tears in gloves represent the major potential for microbiological contamination; therefore frequent testing of the gloves for integrity and surface monitoring of the gloves is essential. Surface monitoring within the isolator may also be beneficial on an infrequent basis.

¹ Interaction Between Air Movements and the Dispersion of Contaminants: Clean Zones with Unidirectional Air Flow, Journal of Parenteral Science and Technology, 47(2), 1993.

² NASA, 1967- Microbiology of Clean Rooms

³ The Sixteenth Edition of Standard Methods for the Examination of Dairy Products (the American Health Association) provides a section on surface sampling

⁴ A Parenteral Drug Association Survey (Technical Monograph 17) showed that out of 27 respondents, 50% were filling more than 3,000 units per run.

Microbial, cleanroom, controlled environment, sampler

• microbiological evaluation of clean rooms and other controlled environments. u.s. pharmacopeia. http://www.pharmacopeia.cn/v29240/usp29nf24s0_c1116.html. accessed 03 june 2016.