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feng1619

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[交流] USP中关于沉降菌检验频率

各位专家，我国GMP规定沉降菌与浮游菌只检一样即可，请问USP是如何规定的，浮游菌在各种环境级别的检测频率是多少？100000级的标准是多少？
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1楼2008-07-25 08:25:42
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feng1619

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奇怪，怎么只有人看，没有人回答呢？
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2楼2008-07-25 15:24:28
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huigenghao

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feng1619(金币+10,VIP+0):非常感谢你的热心和你提供的资料！
再usp 116节有明确的说明，下面是29版的内容，请你参考

U.S. PHARMACOPEIA         USP29                                 
1116 MICROBIOLOGICAL EVALUATION OF CLEAN ROOMS AND OTHER CONTROLLED ENVIRONMENTS                               
The purpose of this informational chapter is to review the various issues that relate to aseptic processing of bulk drug substances, dosage forms, and in certain cases, medical devices; and to the establishment, maintenance, and control of the microbiological quality of controlled environments.                               
This chapter includes discussions on (1) the classification of a clean room based on particulate count limits; (2) microbiological evaluation programs for controlled environments; (3) training of personnel; (4) critical factors in design and implementation of a microbiological evaluation program; (5) development of a sampling plan; (6) establishment of microbiological Alert and Action levels; (7) methodologies and instrumentation used for microbiological sampling; (8) media and diluents used; (9) identification of microbial isolates; (10) operational evaluation via media fills; and (11) a glossary of terms. Excluded from this chapter is a discussion of controlled environments for use by licensed pharmacies in the preparation of sterile products for home use, which is covered under Pharmaceutical Compounding—Sterile Preparations 797.                               
There are alternative methods to assess and control the microbiological status of controlled environments for aseptic processing. Numerical values included in this chapter are not intended to represent absolute values or specifications, but are informational. Given the variety of microbiological sampling equipment and methods, one cannot reasonably suggest that the attainment of these values guarantees the needed level of microbial control or that excursions beyond values in this chapter indicate a loss of control. The improper application of microbiological sampling and analysis may cause significant variability and the potential for inadvertent contamination. Sampling media and devices, and methods indicated in this chapter, are not specifications but only informational.                               
A large proportion of sterile products are manufactured by aseptic processing. Because aseptic processing relies on the exclusion of microorganisms from the process stream and the prevention of microorganisms from entering open containers during filling, product bioburden as well as microbial bioburden of the manufacturing environment are important factors relating to the level of sterility assurance of these products.                               
                               
Establishment of Clean Room Classifications                               
The design and construction of clean rooms and controlled environments are covered in Federal Standard 209E. This standard of air cleanliness is defined by the absolute concentration of airborne particles. Methods used for the assignment of air classification of controlled environments and for monitoring of airborne particulates are included. This federal document only applies to airborne particulates within a controlled environment and is not intended to characterize the viable or nonviable nature of the particles.                               
The application of Federal Standard 209E to clean rooms and other controlled environments in the pharmaceutical industry has been used by manufacturers of clean rooms to provide a specification for building, commissioning, and maintaining these facilities. However, data available in the pharmaceutical industry provide no scientific agreement on a relationship between the number of nonviable particulates and the concentration of viable microorganisms.                               
The criticality of the number of nonviable particulates in the electronic industry makes the application of Federal Standard 209E a necessity, while the pharmaceutical industry has a greater concern for viable particulates (i.e., microorganisms) rather than total particulates as specified in Federal Standard 209E. A definite concern for counts of total particulates in injectable products exists in the pharmaceutical industry (see Particulate Matter in Injections 788).                               
The rationale that the fewer particulates present in a clean room, the less likely it is that airborne microorganisms will be present is accepted and can provide pharmaceutical manufacturers and builders of clean rooms and other controlled environments with engineering standards in establishing a properly functioning facility.                               
Federal Standard 209E, as applied in the pharmaceutical industry is based on limits of all particles with sizes equal to or larger than 0.5 µm. Table 1 describes Airborne Particulate Cleanliness Classes in Federal Standard 209E as adapted to the pharmaceutical industry. The pharmaceutical industry deals with Class M3.5 and above. Class M1 and M3 relate to the electronic industry and are shown in Table 1 for comparison purposes. It is generally accepted that if fewer particulates are present in an operational clean room or other controlled environment, the microbial count under operational conditions will be less, provided that there are no changes in airflow, temperature, and humidity. Clean rooms are maintained under a state of operational control on the basis of dynamic (operational) data.                               
Table 1. Airborne Particulate Cleanliness Classes*                               
Class Name        Particles equal to and larger than 0.5 µm                       
SI        U.S.        (m3)        (ft3)       
        Customary                       
M1        —        10        0.283       
M1.5        1        35.3        1       
M2        —        100        2.8       
M2.5        10        353        10       
M3        —        1,000        28.3       
M3.5        100        3,530        100       
M4        —        10,000        283       
M4.5        1,000        35,300        1,000       
M5        —        100,000        2,830       
M5.5        10,000        353,000        10,000       
M6                1,000,000        28,300       
M6.5        100,000        3,530,000        100,000       
M7        —        10,000,000        283,000       
*  Adapted from U.S. Federal Standard 209E, September 11, 1992—“Airborne Particulate Cleanliness Classes in Clean Rooms and Clean Zones.”
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Importance of a Microbiological Evaluation Program for Controlled Environments                               
Monitoring of total particulate count in controlled environments, even with the use of electronic instrumentation on a continuous basis, does not provide information on the microbiological content of the environment. The basic limitation of particulate counters is that they measure particles of 0.5 µm or larger. While airborne microorganisms are not free-floating or single cells, they frequently associate with particles of 10 to 20 µm. Particulate counts as well as microbial counts within controlled environments vary with the sampling location and the activities being conducted during sampling. Monitoring the environment for nonviable particulates and microorganisms is an important control function because they both are important in achieving product compendial requirements for Particulate Matter and Sterility under Injections 1.                               
Microbial monitoring programs for controlled environments should assess the effectiveness of cleaning and sanitization practices by and of personnel that could have an impact on the bioburden of the controlled environment. Microbial monitoring, regardless of how sophisticated the system may be, will not and need not identify and quantitate all microbial contaminants present in these controlled environments. However, routine microbial monitoring should provide sufficient information to ascertain that the controlled environment is operating within an adequate state of control.                               
Environmental microbial monitoring and analysis of data by qualified personnel will permit the status of control to be maintained in clean rooms and other controlled environments. The environment should be sampled during normal operations to allow for the collection of meaningful data. Microbial sampling should occur when materials are in the area, processing activities are ongoing, and a full complement of operating personnel is on site.                               
Microbial monitoring of clean rooms and some other controlled environments, when appropriate, should include quantitation of the microbial content of room air, compressor air that enters the critical area, surfaces, equipment, sanitization containers, floors, walls, and personnel garments (e.g., gowns and gloves). The objective of the microbial monitoring program is to obtain representative estimates of bioburden of the environment. When data are compiled and analyzed, any trends should be evaluated by trained personnel. While it is important to review environmental results on the basis of recommended and specified frequency, it is also critical to review results over extended periods to determine whether trends are present. Trends can be visualized through the construction of statistical control charts that include alert and action levels. The microbial control of controlled environments can be assessed, in part, on the basis of these trend data. Periodic reports or summaries should be issued to alert the responsible manager.                               
When the specified microbial level of a controlled environment is exceeded, a documentation review and investigation should occur. There may be differences in the details of the investigation, depending on the type and processing of the product manufactured in the room. Investigation should include a review of area maintenance documentation; sanitization documentation; the inherent physical or operational parameters, such as changes in environmental temperature and relative humidity; and the training status of personnel involved. Following the investigation, actions taken may include reinforcement of training of personnel to emphasize the microbial control of the environment; additional sampling at increased frequency; additional sanitization; additional product testing; identification of the microbial contaminant and its possible source; and an evaluation of the need to reassess the current standard operating procedures and to revalidate them, if necessary.                               
Based on the review of the investigation and testing results, the significance of the microbial level being exceeded and the acceptability of the operations or products processed under that condition may be ascertained. Any investigation and the rationale for the course of action should be documented and included as part of the overall quality management system.                               
A controlled environment such as a clean zone or clean room is defined by certification according to a relevant clean room operational standard. Parameters that are evaluated include filter integrity, air velocity, air patterns, air changes, and pressure differentials. These parameters can affect the microbiological bioburden of the clean room operation. The design, construction, and operation of clean rooms varies greatly, making it difficult to generalize requirements for these parameters. An example of a method for conducting a particulate challenge test to the system by increasing the ambient particle concentration in the vicinity of critical work areas and equipment has been developed by Ljungquist and Reinmuller.1 First, smoke generation allows the air movements to be visualized throughout a clean room or a controlled environment. The presence of vortices or turbulent zones can be visualized, and the airflow pattern may be fine-tuned to eliminate or minimize undesirable effects. Then, particulate matter is generated close to the critical zone and sterile field. This evaluation is done under simulated production conditions, but with equipment and personnel in place.                               
Proper testing and optimization of the physical characteristics of the clean room or controlled environment is essential prior to completion of the validation of the microbiological monitoring program. Assurance that the controlled environment is operating adequately and according to its engineering specifications will give a higher assurance that the bioburden of the environment will be appropriate for aseptic processing. These tests should be repeated during routine certification of the clean room or controlled environment and whenever changes made to the operation, such as personnel flow, processing, operation, material flow, air-handling systems, or equipment layout, are determined to be significant.                               
                               
Training of Personnel                               
Aseptically processed products require manufacturers to pay close attention to detail and to maintain rigorous discipline and strict supervision of personnel in order to maintain the level of environmental quality appropriate for the sterility assurance of the final product.                               
Training of all personnel working in controlled environments is critical. This training is equally important for personnel responsible for the microbial monitoring program, where contamination of the clean working area could inadvertently occur during microbial sampling. In highly automated operations, the monitoring personnel may be the employees who have the most direct contact with the critical zones within the processing area. Monitoring of personnel should be conducted before or after working in the processing area.                               
Microbiological sampling has the potential to contribute to microbial contamination due to inappropriate sampling techniques. A formal personnel training program is required to minimize this risk. This formal training should be documented for all personnel entering controlled environments.                               
Management of the facility must assure that all personnel involved in operations in clean rooms and controlled environments are well versed in relevant microbiological principles. The training should include instruction on the basic principles of aseptic processing and the relationship of manufacturing and handling procedures to potential sources of product contamination. This training should include instruction on the basic principles of microbiology, microbial physiology, disinfection and sanitation, media selection and preparation, taxonomy, and sterilization as required by the nature of personnel involvement in aseptic processing. Personnel involved in microbial identification will require specialized training on required laboratory methods. Additional training on the management of the environmental data collected must be provided to personnel. Knowledge and understanding of applicable standard operating procedures is critical, especially those standard operating procedures relating to corrective measures that are taken when environmental conditions so dictate. Understanding of regulatory compliance policies and each individual's responsibilities with respect to good manufacturing practices (GMPs) should be an integral part of the training program as well as training in conducting investigations and in analyzing data.                               
The major source of microbial contamination of controlled environments is the personnel. Contamination can occur from the spreading of microorganisms by individuals, particularly those with active infections. Only healthy individuals should be permitted access to controlled environments.
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4楼2008-07-27 11:18:30
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huigenghao

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These facts underscore the importance of good personal hygiene and a careful attention to detail in the aseptic gowning procedure used by personnel entering the controlled environment. Once these employees are properly gowned—including complete facial coverage—they must be careful to maintain the integrity of their gloves and suits at all times. Since the major threat of contamination of product being aseptically processed comes from the operating personnel, the control of microbial contamination associated with these personnel is one of the most important elements of the environmental control program.                               
The importance of thorough training of personnel working in controlled environments, including aseptic techniques, cannot be overemphasized. The environmental monitoring program, by itself, will not be able to detect all events in aseptic processing that could compromise the microbiological quality of the environment. Therefore, periodic media-fill or process simulation studies to revalidate the process are necessary to assure that the appropriate operating controls and training are effectively maintained.                       

                               
Critical Factors Involved in the Design and Implementation of a Microbiological Environmental Control Program                               
An environmental control program should be capable of detecting an adverse drift in microbiological conditions in a timely manner that would allow for meaningful and effective corrective actions. It is the responsibility of the manufacturer to develop, initiate, implement, and document such a microbial environmental monitoring program.                               
Although general recommendations for an environmental control program will be discussed, it is imperative that such a program be tailored to specific facilities and conditions. A general microbiological growth medium such as Soybean Casein Digest Medium should be suitable in most cases. This medium may be supplemented with additives to overcome or to minimize the effects of sanitizing agents, or of antibiotics if used or processed in these environments. The detection and quantitation of yeasts and molds should be considered. General mycological media, such as Sabouraud's, Modified Sabouraud's, or Inhibitory Mold Agar are acceptable. Other media that have been validated for promoting the growth of fungi, such as Soybean–Casein Digest Agar, can be used. In general, testing for obligatory anaerobes is not performed routinely. However, should conditions or investigations warrant, such as the identification of these organisms in sterility testing facilities, more frequent testing is indicated. The ability of the selected media to detect and quantitate these anaerobes or microaerophilic microorganisms should be evaluated.                               
The selection of time and incubation temperatures is made once the appropriate media have been selected. Typically, incubation temperatures in the 22.5 ± 2.5 and 32.5 ± 2.5 ranges have been used with an incubation time of 72 and 48 hours, respectively. Sterilization processes used to prepare growth media for the environmental program should be validated and, in addition, media should be examined for sterility and for growth promotion as indicated under Sterility Tests 71. In addition, for the Growth Promotion test, representative microflora isolated from the controlled environment or ATCC strain preparations of these isolates may also be used to test media. Media must be able to support growth when inoculated with less than 100 colony-forming units (cfu) of the challenge organisms.                               
An appropriate environmental control program should include identification and evaluation of sampling sites and validation of methods for microbiological sampling of the environment.                               
The methods used for identification of isolates should be verified using indicator microorganisms (see Microbial Limit Tests 61).                               
                               
Establishment of Sampling Plan and Sites                               
During initial start-up or commissioning of a clean room or other controlled environment, specific locations for air and surface sampling should be determined. Consideration should be given to the proximity to the product and whether air and surfaces might be in contact with a product or sensitive surfaces of container-closure systems. Such areas should be considered critical areas requiring more monitoring than non-product-contact areas. In a parenteral vial filling operation, areas of operation would typically include the container-closure supply, paths of opened containers, and other inanimate objects (e.g., fomites) that personnel routinely handle.                               
The frequency of sampling will depend on the criticality of specified sites and the subsequent treatment received by the product after it has been aseptically processed. 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                               
Sampling Area        Frequency of                       
        Sampling                       
Class 100 or better room designations        Each operating shift                       
Supporting areas immediately adjacent        Each operating shift                       
to Class 100 (e.g., Class 10,000)                               
Other support areas (Class 100,000)        Twice/week                       
Potential product/container contact areas        Twice/week                       
Other support areas to aseptic        Once/week                       
processing areas but non-product con-                               
tact (Class 100,000 or lower)                               
As manual interventions during operation increase, and as the potential for personnel contact with the product increases, the relative importance of an environmental monitoring program increases. Environmental monitoring is more critical for products that are aseptically processed than for products that are processed and then terminally sterilized. The determination and quantitation of microorganisms resistant to the subsequent sterilization treatment is more critical than the microbiological environmental monitoring of the surrounding manufacturing environments. If the terminal sterilization cycle is not based on the overkill cycle concept but on the bioburden prior to sterilization, the value of the bioburden program is critical.                               
The sampling plans should be dynamic with monitoring frequencies and sample plan locations adjusted based on trending performance. It is appropriate to increase or decrease sampling based on this performance.                               
                               
Establishment of Microbiological Alert and Action Levels in Controlled Environments                               
The principles and concepts of statistical process control are useful in establishing Alert and Action levels and in reacting to trends.                               
An Alert level in microbiological environmental monitoring is that level of microorganisms that shows a potential drift from normal operating conditions. Exceeding the Alert level is not necessarily grounds for definitive corrective action, but it should at least prompt a documented follow-up investigation that could include sampling plan modifications.                               
An Action level in microbiological environmental monitoring is that level of microorganisms that when exceeded requires immediate follow-up and, if necessary, corrective action.                               
Alert levels are usually based upon historical information gained from the routine operation of the process in a specific controlled environment.                               
In a new facility, these levels are generally based on prior experience from similar facilities and processes; and at least several weeks of data on microbial environmental levels should be evaluated to establish a baseline.                               
These levels are usually re-examined for appropriateness at an established frequency. When the historical data demonstrate improved conditions, these levels can be re-examined and changed to reflect the conditions. Trends that show a deterioration of the environmental quality require attention in determining the assignable cause and in instituting a corrective action plan to bring the conditions back to the expected ranges. However, an investigation should be implemented and an evaluation of the potential impact this has on a product should be made.                               
                               
Microbial Considerations and Action Levels for Controlled Environments                               
Classification of clean rooms and other controlled environments is based on Federal Standard 209E based on total particulate counts for these environments. The pharmaceutical and medical devices industries have generally adopted the classification of Class 100, Class 10,000, and Class 100,000, especially in terms of construction specifications for the facilities.                               
Although there is no direct relationship established between the 209E controlled environment classes and microbiological levels, the pharmaceutical industry has been using microbial levels corresponding to these classes for a number of years; and these levels have been those used for evaluation of current GMP compliance.2 These levels have been shown to be readily achievable with the current technology for controlled environments. There have been reports and concerns about differences in these values obtained using different sampling systems, media variability, and incubation temperatures. It should be recognized that, although no system is absolute, it can help in detecting changes, and thus trends, in environmental quality. The values shown in Tables 3, 4, and 5 represent individual test results and are suggested only as guides. Each manufacturer's data must be evaluated as part of an overall monitoring program.                               
Table 3. Air Cleanliness Guidelines in Colony-Forming Units (cfu) in Controlled Environments (Using a Slit-to-Agar Sampler or Equivalent)                               
Class*                cfu per cubic meter of air**        cfu per cubic feet of air       
SI        U.S. Customary                       
M3.5        100        Less than 3        Less than 0.1       
M5.5        10,000        Less than 20        Less than 0.5       
M6.5        100,000        Less than 100        Less than 2.5       
*  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                               
Class                cfu per Contact Plate*               
SI        U.S. Customary                       
M3.5        100        3 (including floor)               
M5.5        10,000        5               
                10 (floor)               
*  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 24 cm2 but no larger than 30 cm2.                               
Table 5. Surface Cleanliness Guidelines in Controlled Environments of Operating Personnel Gear in cfu                               
Class                        cfu per Contact Plate*       
SI        U.S.                Gloves        Personnel Clothing
        Customary                        & Garb
M3.5        100                3        5
M5.5        10,000                10        20
*  See in Table 4 under (*).                               
                               
Methodology and Instrumentation for Quantitation of Viable Airborne Microorganisms                               
It is generally accepted by scientists that airborne microorganisms in controlled environments can influence the microbiological quality of the intermediate or final products manufactured in these areas. Also, it generally is accepted that estimation of the airborne microorganisms can be affected by instruments and procedures used to perform these assays. Therefore, where alternative methods or equipment is used, the general equivalence of the results obtained should be ascertained. Advances in technology in the future are expected to bring innovations that would offer greater precision and sensitivity than the current available methodology and may justify a change in the absolute numbers of organisms that are detected.                               
Today, the most commonly used samplers in the U.S. pharmaceutical and medical device industry are the impaction and centrifugal samplers. A number of commercially available samplers are listed for informational purposes. The selection, appropriateness, and adequacy of using any particular sampler is the responsibility of the user.                               
Slit-to-Agar Air Sampler (STA)— This sampler is the instrument upon which the microbial guidelines given in Table 3 for the various controlled environments are based. The unit is powered by an attached source of controllable vacuum. 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. A remote air intake is often used to minimize disturbance of the laminar flow field.                               
Sieve Impactor— The apparatus consists of a container designed to accommodate a Petri dish containing a nutrient agar. The cover of the unit is perforated, with the perforations of a predetermined size. A vacuum pump draws a known volume of air through the cover, and the particles in the air containing microorganisms 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 propeller or turbine 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 unit's 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 are available.
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huigenghao

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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 unit's 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.                               
Settling 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 enviroments.                               
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 15 minutes. It may be necessary to use sampling times in excess of 15 minutes 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 selectivity 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 has not 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 defined using a sterile template of appropriate size. In general, it is in the range of 24 to 30 cm2. 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 media, liquid or solid, are listed below.                               
Liquid Media*        Solid Media*                       
Tryptone saline        Soybean-casein digest agar                       
Peptone water        Nutrient agar                       
Buffered saline        Tryptone glucose extract agar                       
Buffered gelatin        Lecithin agar                       
Enriched buffered gelatin        Brain heart infusion agar                       
Brain heart infusion        Contact plate agar                       
Soybean-casein medium                               
*  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 area, 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 (see Critical Factors Involved in the Design and Implementation of Environmental Control Program).                               
                               
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 are 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-fill runs be done to cover all production shifts for line/product/container combinations. This guideline should be considered not only for qualification media-fill runs, 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-fill 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 (see Sterility Tests 71) 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 or at 32.5 ± 2.5. 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 the sources of contamination.
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6楼2008-07-27 11:22:46
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huigenghao

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Critical issues in performing media fills are the number of fills to qualify an aseptic process, the number of units filled per media fill, the interpretation of results, and implementation of corrective actions. Historically, three media-fill runs during initial 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 Number 17,4 “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 clean room 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 isolator 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 of 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 in 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 below 0.1%. 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 principle 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 nonsterile 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 106.                               
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 a 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 system 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 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 106; therefore, isolators contain sterile air, do not exchange air 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 relatively 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.                               
                               
GLOSSARY                               
Airborne Particulate Count (also referred to as Total Particulate Count)—Particles detected are 0.5 µm and larger. When a number of particles is specified, it is the maximum allowable number of particles per cubic meter of air (or per cubic foot of air).                               
Airborne Viable Particulate Count (also referred to as Total Airborne Aerobic Microbial Count)—When a number of microorganisms is specified, it is the maximum number of colony-forming units (cfu) per cubic meter of air (or per cubic foot of air) that is associated with a Cleanliness Class of controlled environment based on the Airborne Particulate Count.
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7楼2008-07-27 11:23:30
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huigenghao

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Aseptic Processing—A mode of processing pharmaceutical and medical products that involves the separate sterilization of the product and of the package (containers/closures or packaging material for medical devices) and the transfer of the product into the container and its closure under microbiologic critically controlled conditions.                               
Air Sampler—Devices or equipment used to sample a measured amount of air in a specified time to quantitate the particulate or microbiological status of air in the controlled environment.                               
Air Changes—The frequency per unit of time (minutes, hours, etc.) that the air within a controlled environment is replaced. The air can be recirculated partially or totally replaced.                               
Action Levels—Microbiological levels in the controlled environment, specified in the standard operating procedures, which when exceeded should trigger an investigation and a corrective action based on the investigation.                               
Alert Levels—Microbial levels, specified in the standard operating procedures, which when exceeded should result in an investigation to ensure that the process is still within control. Alert levels are specific for a given facility and are established on the basis of a baseline developed under an environmental monitoring program. These Alert levels can be modified depending on the trend analysis done in the monitoring program. Alert levels are always lower than Action levels.                               
Bioburden—Total number of microorganisms detected in or on an article.                               
Clean Room—A room in which the concentration of airborne particles is controlled to meet a specified airborne particulate Cleanliness Class. In addition, the concentration of microorganisms in the environment is monitored; each Cleanliness Class defined is also assigned a microbial level for air, surface, and personnel gear.                               
Clean Zone—A defined space in which the concentration of airborne particles and microorganisms are controlled to meet specific Cleanliness Class levels.                               
Controlled Environment—Any area in an aseptic process system for which airborne particulate and microorganism levels are controlled to specific levels, appropriate to the activities conducted within that environment.                               
Commissioning of a Controlled Environment—Certification by engineering and quality control that the environment has been built according to the specifications of the desired cleanliness class and that, under conditions likely to be encountered under normal operating conditions (or worst-case conditions), it is capable of delivering an aseptic process. Commissioning includes media-fill runs and results of the environmental monitoring program.                               
Corrective Action—Actions to be performed that are in standard operating procedures and that are triggered when certain conditions are exceeded.                               
Environmental Isolates—Microorganisms that have been isolated from the environmental monitoring program.                               
Environmental Monitoring Program—Documented program, implemented through standard operating procedures, that describes in detail the procedures and methods used for monitoring particulates as well as microorganisms in controlled environments (air, surface, personnel gear). The program includes sampling sites, frequency of sampling, and investigative and corrective actions that should be followed if Alert or Action levels are exceeded. The methodology used for trend analysis is also described.                               
Equipment Layout—Graphical representation of an aseptic processing system that denotes the relationship between and among equipment and personnel. This layout is used in the Risk Assessment Analysis to determine sampling site and frequency of sampling based on potential for microbiological contamination of the product/container/closure system. Changes must be assessed by responsible managers, since unauthorized changes in the layout for equipment or personnel stations could result in increase in the potential for contamination of the product/container/closure system.                               
Federal Standard 209E—“Airborne Particulate Cleanliness Classes in Clean Rooms and Clean Zones” is a standard approved by the Commissioner, Federal Supply Services, General Service Administration, for the use of “All Federal Agencies.” The Standard establishes classes of air cleanliness based on specified concentration of airborne particulates. These classes of air cleanliness have been developed, in general, for the electronic industry “super-clean” controlled environments. In the pharmaceutical industry, the Federal Standard 209E is used to specify the construction of controlled environment. Class 100, Class 10,000, and Class 100,000 are generally represented in an aseptic processing system. If the classification system is applied on the basis of particles equal to or greater than 0.5 µm, these classes are now represented in the SI system by Class M3.5, M5.5, and M6.5, respectively.                               
Filter Integrity—Testing that ensures that a filter functional performance is satisfactory [e.g., dioctyl phthalate (DOP) and bubble point test].                               
Material Flow—The flow of material and personnel entering controlled environments should follow a specified and documented pathway that has been chosen to reduce or minimize the potential for microbial contamination of the product/closure/container systems. Deviation from the prescribed flow could result in increase in potential for microbial contamination. Material/personnel flow can be changed, but the consequences of the changes from a microbiological point of view should be assessed by responsible managers and must be authorized and documented.                               
Media Growth Promotion—Procedure that references Growth Promotion under Sterility Tests 71 to demonstrate that media used in the microbiological environmental monitoring program, or in media-fill runs, are capable of supporting growth of indicator microorganisms and of environmental isolates from samples obtained through the monitoring program or their corresponding ATCC strains.                               
Media Fill—Microbiological simulation of an aseptic process by the use of growth media processed in a manner similar to the processing of the product and with the same container/closure system being used.                               
Out-of-Specification Event—Temporary or continuous event when one or more of the requirements included in standard operating procedures for controlled environments are not fulfilled.                               
Product Contact Areas—Areas and surfaces in a controlled environment that are in direct contact with either products, containers, or closures and the microbiological status of which can result in potential microbial contamination of the product/container/closure system. Once identified, these areas should be tested more frequently than non-product-contact areas or surfaces.                               
Risk Assessment Analysis—Analysis of the identification of contamination potentials in controlled environments that establish priorities in terms of severity and frequency and that will develop methods and procedures that will eliminate, reduce, minimize, or mitigate their potential for microbial contamination of the product/container/closure system.                               
Sampling Plan—A documented plan that describes the procedures and methods for sampling a controlled environment; identifies the sampling sites, the sampling frequency, and number of samples; and describes the method of analysis and how to interpret the results.                               
Sampling Sites—Documented geographical location, within a controlled environment, where sampling for microbiological evaluation is taken. In general, sampling sites are selected because of their potential for product/container/closure contacts.                               
Standard Operating Procedures—Written procedures describing operations, testing, sampling, interpretation of results, and corrective actions that relate to the operations that are taking place in a controlled environment and auxiliary environments. Deviations from standard operating procedures should be noted and approved by responsible managers.                               
Sterile Field—In aseptic processing or in other controlled environments, it is the space at the level of or above open product containers, closures, or product itself, where the potential for microbial contamination is highest.                               
Sterility—Within the strictest definition of sterility, an article is deemed sterile when there is complete absence of viable microorganisms. Absolute sterility cannot be practically demonstrated without testing every article in a batch. Sterility is defined in probabilistic terms, where the likelihood of a contaminated article is acceptably remote.                               
Swabs—Devices provided that are used to sample irregular as well as regular surfaces for determination of microbial status. The swab, generally composed of a stick with an absorbent extremity, is moistened before sampling and used to sample a specified unit area of a surface. The swab is then rinsed in sterile saline or other suitable menstruum and the contents plated on nutrient agar plates to obtain an estimate of the viable microbial load on that surface.                               
Trend Analysis—Data from a routine microbial environmental monitoring program that can be related to time, shift, facility, etc. This information is periodically evaluated to establish the status or pattern of that program to ascertain whether it is under adequate control. A trend analysis is used to facilitate decision-making for requalification of a controlled environment or for maintenance and sanitization schedules.                               
                               
                               
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.                               
                               
Auxiliary Information— Staff Liaison : Radhakrishna S Tirumalai, Scientist       
Expert Committee : (MSA05) Microbiology and Sterility Assurance               
USP29–NF24 Page 2969                               
Pharmacopeial Forum : Volume No. 31(2) Page 524                       
Phone Number : 1-301-816-8339       

如果你有31版的话，就参考31版，不过这个没有什么改变的
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8楼2008-07-27 11:24:28
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huigenghao

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刚才找到USP29版的1116的中文稿，你参考的看一下，应该对你有帮助的


<1116> 洁净室和其他受控环境的微生物学评估
本章信息旨在综述与散装原料药、剂型以及某些医疗用具无菌操作相关的各种问题；并对控制环境进行制定、保持和控制其微生物学特性。
本章包括的讨论有关以下方面：(1) 基于颗粒计数限制上的洁净室分级；(2)控制环境的微生物学评估程序；(3) 人员培训；(4) 设计和实施微生物学评估程序中的关键因素；(5) 取样策略的制定；(6) 建立微生物警告和行动标准；(7) 应用于微生物取样的方法学和仪器操作；                                                                                                                                                                                                                           (8) 培养基和稀释剂；(9) 微生物隔离群的鉴别；(10) 经培养基填充的操作评估；以及 (11) 词汇表。本章以外的讨论是有执照药房制备家用无菌产品应用控制环境的问题，其包含在药用配方—无菌制备（Pharmaceutical Compounding—Sterile Preparations）á797ñ 中。
对于无菌操作的控制环境中微生物状况的控制和评估存在方法选择性。本章中的数字评估并非有意代表绝对的评估或规范，而仅是一种信息上的表示。考虑到微生物取样设备和方法的多样性，不能说达到这些值就能保证微生物控制的需求标准，或者与本章这些值有偏差、高于本章的值就认为是失去控制。微生物取样和分析应用不当，可能会引起显著的差异和不慎污染的潜在性。本章标明的取样培养基、设备和方法，不是特定的规范而只是一种信息参考。                                                                                                                                                                                                                通过无菌操作可产生高比率的无菌产品。因为无菌操作依赖的是流程中微生物的排除和阻止包装过程中微生物进入敞口容器，制造环境中的微生物负担和产品的生物负担对于保证这些产品的无菌水平来说，是相关的重要因素。

洁净室分级的确立
洁净室和控制环境的设计和构建，在联邦标准209E中被提及。此空气洁净标准通过空气播散颗粒绝对浓度定义。其中包括用于控制环境空气级别分配和监测空气播散颗粒的方法。这一联邦文件仅适用于控制环境内的空气播散颗粒，且并非有意的强调颗粒的活性与非活性属性特征。
制药工业中的洁净室和其他控制环境的联邦标准209E已经被洁净室制造商所采用，以提供这些设施的组装、试车和维护的规范。然而，制药工业中的有效数据并不能给非活性颗粒数量和活性微生物浓度之间的关系提供科学的一致性。
电子工业的非活性颗粒数量的关键程度使得应用联邦标准209E成为必需，虽然制药工业在与联邦标准中特定的总颗粒相比较而言，更关心的是活性颗粒(即微生物) 。制药行业中对注射制剂中的总颗粒数有明确规定。(见 注射品中的颗粒物质（Particulate Matter in Injections）á788ñ。
洁净室的颗粒数越少，空气播散微生物的可能性越小。这一理论是经认可的，且能够为制药生产商和洁净室及受控环境的建造者提供功能设施建造方面的工程学标准。
应用在制药工业的连邦标准209E，是以所有颗粒大小的限定为依据的，即，大小等于或大于0.5 µm的。 表1 描述了联邦标准209E适用于制药工业的空气播散颗粒洁净级别。制药工业适用 M3.5以及更高级别。M1 级别和M3级别相关于电子工业，表 1 所示仅为出于比较目的。通常认为，假设不存在气流、温度和湿度的改变，则使用中的洁净室或其他控制环境中存在的颗粒越少，操作条件下的微生物计数将会越少。 洁净室被维持在以动态的 (运作的)数据为根据的操作控制状态下。
表 1. 空气播散颗粒洁净级别*
级别名        等于或大于0.5 µm的颗粒
SI        U.S.习惯        (m3)        (ft3)
M1        —         10.0        0.283
M1.5        1         35.3        1.00
M2        —         100        2.8
M2.5        10         353        10.0
M3        —         1,000        28.3
M3.5        100         3,530        100
M4        —         10,000        283
M4.5        1,000         35,300        1,000
M5        —         100,000        2,830
M5.5        10,000         353,000        10,000
M6                1,000,000        28,300
M6.5        100,000         3,530,000        100,000
M7        —         10,000,000        283,000
*  来自 U.S. 联邦标准 209E, 1992年9月11日—“洁净室及洁净区内空气播散颗粒洁净级别。”

控制环境中微生物学评估程序的重要性
监测控制环境中总颗粒计数，甚至应用连续电子仪器计数，仍不能提供关于环境中微生物含量的信息。颗粒计数器的基本限制在于它们测量颗粒为0.5 µm 或者更大的。而空气播散微生物并不是自由浮动的或是单细胞的，它们经常联合成10到20 µm的颗粒。控制环境中的颗粒计数和微生物计数是随着取样位置和取样期间采取的活动而改变的。监测环境中的非活性颗粒和微生物是一项重要的控制措施，因为它们对达到颗粒物质（Particulate Matter）和注射（Injections）á1ñ 条件下无菌（Sterility）的产品要求来说，都是重要的。
控制环境的微生物监测程序应当对人员的以及由人员执行的可能对控制环境造成生物负担影响的清洁卫生处理的实行以及洁净效应予以评估。不管系统多么复杂，微生物监测将不再、也不需要对这些控制环境中存在的所有微生物污染进行鉴别和定量。然而，常规的微生物监测应该提供用以确定控制环境是在适当的控制状态下运作的有效信息。
具备资格人员对环境微生物监测和数据分析，应该在容许洁净室和其他受控环境保持在控制状态下进行。考虑到有意义数据的收集，则应当在环境正常运行过程中取样。微生物取样应当发生在原料在场、加工处理在进行中且操作人员在岗时。
适当的时候，洁净室和一些其他控制环境的微生物监测应当包括室内空气以及进入关键区域、表面、设备、清洁卫生处理容器、地板、墙壁和人员着装(如工作服和手套)的压缩空气的微生物含量定量。微生物监测程序的目的对环境生物负载来说具有代表性的评估。当数据被收集和分析时，受过训练的人员应对任何趋势进行评估。尽管以推荐和特定的频率为根据来考查环境的结果是重要的，可是考查扩展时期的结果对于确认趋势是否为目前的也同样的关键。趋势可以通过构建包括警告和行动标准的统计检验图表而形象化。控制环境的微生物控制可以以这些趋势数据为根据，进行部分的评估。应当做定期报告或总结借以警示责任管理人员。
当受控环境中特定微生物水平超标时，应当进行文件性的综述和调查。在调查的细节上可能会有所不同，这要根据室内制造产品的类型和加工过程的不同来确定。调查应当包括区域维护文件考查；清洁卫生处理文件；固有物理的或操作的参数，如环境温度和相对湿度的改变；以及相关人员的培训状况。调查之后，采取的行动可能会包括加强人员培训以强调微生物控制的重要性；增加频率添加取样；添加清洁卫生处理；添加产品测试；鉴定微生物污染及其可能来源；如果必要的话，需对现有标准化操作规程进行再评价并使其重新生效。
根据调查综述和结果测试，微生物水平超标的显著性和在那种条件下操作或产品处理的可接受性可能是需要查明的。任何对于行动过程的调查和理论都应当通过文件证明，且应作为整个质量管理系统的一部分。
控制环境，如洁净区或洁净室，是依照相关洁净室运行标准证明确定的。评估参数包括过滤的完整性、空气流速、通风模式、换气以及压力差别。这些参数能够影响洁净室运行中的微生物生物负载。洁净室的设计、构建以及运行存在多样性，这使得对这些参数的泛化变得困难。 Ljungquist和Reinmuller1发展了通过增加邻近关键工作区域和设备的周围粒子浓度对系统进行颗粒激发测试的方法。首先，烟的产生使得洁净室或一个控制环境的各处空气流动成为可见。旋涡或紊流区段的存在能够完全可见，且气流模式可以微调到消除或最小化不良影响的状态。然后，颗粒物质就产生于关键区和无菌区的附近了。此评估是在模拟生产条件下进行的，但是设备和人员均在场。
在微生物监测程序生效之前，完成适当的测试和优化洁净室或控制环境的物理特征是必要的。确保适当的控制环境并依照其工程规范运作，将会更高效的确保控制环境中对于无菌操作而言能有适当的生物负载。在洁净室或受控环境的日常确认过程中，应当重复进行这些测试；在进行如人员流动、加工过程、操作过程、原料流程、空气处理系统或设备布局等显著性的操作改变时，也应当重复进行这些测试。

人员的培训
无菌处理产品需要生产商密切关注人员管理的细节，保持严格的纪律以及对其进行严格的监督，以确保维持适当的环境质量水平，并确保最终产品的无菌性。
对控制环境中工作人员进行培训是个关键。这种培训对微生物监测程序的责任人员同样也是重要的，因为微生物取样过程如不注意操作，很容易引起洁净工作区域污染的发生。 在高度自动化的操作中，监测人员可能是加工处理区域内与关键区带有最直接联系的人员。在加工处理区域内工作前和工作后，都应当对人员进行监督检查。
微生物取样是导致微生物污染的潜在因素，如果取样方法不当，会导致微生物污染的发生。正式有效的人员培训计划会使这种危险性因素减到最小。这一正式有效培训应当是针对所有进入受控环境的人员的，并形成文件性规定。
对于设备的管理必须保证洁净室和控制环境内所有的相关操作人员能够很好的精通相关微生物学原理。培训应当包括无菌处理基本原则以及对产品潜在污染源处理过程的关联训练。此培训应当包括微生物基本原则、微生物生理学、消毒和卫生、培养基选择和制备、分类学以及灭菌等指导，这些对涉及无菌操作的人员本身都是必需的。涉及微生物鉴别的人员，还需要进行实验室所需方法的专业培训。此外，还必须给相关人员提供所收集环境数据的管理方面的附加培训。对应用标准化操作规程的认识和理解是很关键的，尤其是那些与当环境条件这样要求时采用的纠偏措施相关的标准操作规程。了解关于生产质量管理规范 (GMPs) 中每一个体责任以及顺应性调整政策，同指导调研和分析数据的培训一样，都应该作为整体培训计划的一部分来实行。
控制环境中微生物污染的主要污染源是工作人员。微生物可以通过人体传播而引起污染的发生，尤其是那些处于活动传染期个体。因此控制环境只能准许健康的个体进入。
由这些事实来看，良好的个人卫生以及对个人进入控制环境所用的无菌衣处理过程细节上的注意都显得十分重要。这些人员一旦穿戴整齐，包括完全的面部覆盖处理，就必须在整个期间内小心的保持他们手套和制服的完整性。由于无菌处理过程中产品的主要污染威胁来自操作人员，使得控制与这些人员相关联的微生物污染成为环境控制程序中一个重要的因素。
包括无菌操作在内，对控制环境的工作人员进行彻底的培训是重要的，但是也不能去过分的强调。因为环境监测程序仅通过自身是无法检测无菌操作的所有对环境微生物品质可能的妥协事件的。因此，周期性的培养基填充或过程模拟研究，对于使方法重新生效以确保适当的操纵管理和保持有效培训来说是必需的。

微生物学环境控制程序设计和实施中的关键因素
一项环境控制程序应当具有实时检测微生物状态中逆变的能力，这样才能保证采取有意义并有效的改善行动。厂商有责任制定、启动、执行这样的环境控制程序并使其成文件性规定。
尽管对于环境控制程序的一般性建议将被讨论，但对于特定设备和特殊条件，仍急需制定这样的程序。一般的微生物菌株生长培养基，如大豆酪蛋白消化培养基，在大多数情况是适用的。如果这些环境中使用或应用了消毒剂或抗生素，这种培养基可以通过补充添加剂来克服消毒剂或抗生素的效应或使其效应最小化。酵母菌和霉菌的检测和定量应当给以考虑。一般的真菌培养基，如萨布罗氏培养基、改良萨布罗氏培养基或抑制霉菌琼脂，都是可以接受的。 其它已经确证的促进真菌生长的培养基，如大豆-酪蛋白消化琼脂，也可以应用。一般而言，对于专性厌氧菌，不进行常规的检测。然而，应当有适当的条件或研究做保证，比如无菌试验装置中这些微生物的频繁检测证明的鉴别等。应当对选择性培养基检测和定量这些厌氧或微需氧微生物的能力进行评估。
一旦选定适当的培养基，就要进行时间和孵育温度的选择。典型的孵育温度为22.5 ± 2.5 和32.5 ± 2.5 ，孵育时间分别为72和48小时。为环境程序所制备的生长培养基的灭菌过程应当是有效的，且应当对其进行无菌检测和生长促进检测，详述可见无菌检查（SterilityTests）á71ñ。另外，对于生长促进检测，从控制环境中分离得到具代表性的微生物群或从ATCC菌株制备这些分离菌株也可能用来检测培养基。培养基在接种量小于100个菌落形成单位（cfu）时，必须能保证支持菌种生长。
适当的环境控制程序应当包括确认和评估取样点以及确认环境的微生物取样方法。
鉴别被分离菌株的方法应当用微生物指示剂(见 微生物限度检查（Microbial Limit Tests）á61ñ验证。

取样策略和位置的确立
在洁净室或其它控制环境启动或试车初始阶段，就应当确定空气和表面取样的特定位置。 应当考虑到与产品的接近度、空气和表面是否会与产品或者容器闭包系统的敏感表面有接触的可能性。这样的区域应当作为关键区域，需要进行比非产品接触区更多的监测。在安泰乐装瓶操作中，典型的操作区域包括容器闭包配备、容器开启路径以及其它工作人员常规处理的无机物体(如污染物)。
取样频率要依据特定位点的重要性和对于产品无菌操作后的后继处理来定。表 2显示的为与被取样的控制环境中区域重要性相关的降序排列的建议取样频率。
表 2. 基于控制环境重要性的建议取样频率
样地面积        取样频率
洁净级100或更好的设计室        每个操作交接
刚靠近洁净级100的支持区 (如10,000洁净级)        每个操作交接
其它支持区(100,000洁净级)        每周两次
潜在的产品/容器接触区        每周两次
其它无菌操作区但非产品接触区支持区(100,000洁净级或较低)        每周一次
由于操作过程中手工介入的增加以及人员接触产品潜在性的增加，使得环境监测程序的相对重要性增加。环境监测对于无菌操作生产的产品来说，远比生产后进行终端灭菌的产品要重要得多。确定和定量对后继灭菌处理具有抗性的微生物，远比在制造环境周围进行微生物环境监测要重要得多。如果终端灭菌循环不是基于过度杀伤循环，而是基于生物负担优先灭菌，那么生物负担的评估就是关键性的。
取样策略应当是动态性的，即监测频率和取样策略位置的动态性。其动态调整是以趋势特征为根据的。根据这一趋势可以进行适当的增加或减少取样。

建立控制系统的微生物警告和行动标准
统计过程控制的概念和原理，对于建立警告和行动标准以及趋势应答是有用的。
微生物环境监测的警告标准，即微生物水平呈现源自标准操作条件的潜在变化。超过警告标准并不一定需要最后的改善行动，但是它至少应该提示进行继而的书面调查，其内容包括取样策略的修正。
微生物环境监测的行动标准，是微生物水平超过此标准时，如需要则应立即采取改善行动。
行动标准通常是根据特定控制环境常规运转期间所得历史信息来确定的。
对于新设备，这些标准通常是根据从相似设备和操作过程所得的先前经验来确定的，并且至少应依据几个星期的微生物环境水平数据的评估来建立基线。
在确定频率的情况下，为了确保适当性，对这些标准通常要进行再考查。当历史数据表明条件改善时，对这些标准可以进行再考查并做能反映实际条件的改变。如果有趋势表明环境质量恶化，则需要注意检测直接原因并建立改善行动计划以使条件回到原期望值范围。无论如何，应实行调查，且对产品造成影响的潜在性因素做出评估。

控制环境微生物的考察及行动标准
洁净室及其它控制环境的分级依据联邦标准209E，此标准是基于对这些环境总颗粒计数来确定的。药用和医疗用具企业通常采用分级为100级、10,000级和100,000级，特别的针对设备的建设规范。
虽然在209E控制环境级别和微生物水平之间没有建立直接的联系，可制药工业采用与这些级别相关的微生物标准已经有许多年了。并且这些标准当前已用于GMP认证的评估。2在控制环境中应用当前的技术来达到这些标准是很容易的。
有相关报道显示，由于取样系统的不同、培养基的差异以及孵育温度的不同，可导致这些测量值的不同。应当认识到，虽然没有绝对完美的检测系统，但它的存在确实能够帮助我们检测环境质量的变化，进而了解其趋势。表 3, 4,和5 中所示的值代表个别测验的结果，仅作为指导性建议。每一厂家的数据必须作为整个监测程序的一部分来进行评估。

表 3. 以菌落形成单位（cfu）表示的控制环境中空气洁净指导方针
(用Slit-to-Agar 取样器或同等物)
级别*
每立方米空气的cfu **
每立方英尺空气的cfu
SI        U.S. 习惯               
M3.5        100        少于 3        少于0.1
M5.5        10,000        少于 20        少于 0.5
M6.5        100,000        少于 100        少于2.5
*  定义于联邦标准 209E, 1992年9月.
** 应该取足够体积的空气样品，以检测超特定限度的偏差。

表 4. 以cfu表示的控制环境中仪器和设备表面洁净指导方针
级别        每接触板的cfu *

SI        U.S. 习惯        
M3.5        100        3 (包括底面)
M5.5        10,000        5
                10 (底面)
*  接触板区域从24到30 cm2不同。当取样过程用到冲洗时，区域面积应大于或等于24 cm2 ，但是不能超过30 cm2。

[ Last edited by huigenghao on 2008-7-27 at 12:03 ]
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表 5. 以cfu表示的控制环境中工作人员活动表面洁净指导方针。
级别                每接触板的cfu*

SI         U.S. 习惯                手套         工作人员衣着
& 装束
M3.5        100                3        5
M5.5        10,000                10        20
*  见表 4 下注 (*)。


活性空气播散微生物定量的方法学和仪器操作
控制环境中空气播散微生物能够对这些区域内半成品或最终产品的微生物质量起到影响。这一观点已被科学家们所普遍接受。另外人们也普遍接受这种观点，即空气播散微生物的估测也会受到执行这些分析所用仪器和相关操作的影响。因此，当方法和仪器有所变化时，应当确定其所获结果的一般等价转换。我们期待将来的技术进步可以带来革新，以提供比现有可用方法更高的精密度和敏感度，并且可用确定微生物数量检测中的变化绝对值。
目前，美国制药和医用器材工业最常用的取样器为嵌入式和离心取样器。以下所列取样器均可通过商业购买得到，仅作为信息提供。对特定取样器的选择、购买和充分利用则由使用者负责。
Slit-to-Agar空气取样器 (STA)— 这一取样器是依据多种控制环境表 3的微生物指导方针的一种仪器。其动力来源为附属可控真空装置。空气通过一个标准化的缝隙进入缝隙下面设置的缓慢回转式有盖培养皿，皿内含有营养琼脂。空气中的颗粒物质具有足够的质量撞击琼脂表面，使得活菌能够在其表面生长。通常用吸入稀疏空气的方法来减小空气层流区域的干扰。
滤网取样器（Sieve Impactor）— 此装置包括一个容器，可以调节含营养琼脂的有盖培养皿。装置的盖子有穿孔，穿孔的大小是预先设计好的。真空泵通过盖子的穿孔抽取已知体积的空气，空气中含有微生物的颗粒就与有盖培养皿内的琼脂培养基碰撞。一些取样器具有一系列串联级有孔容器，其穿孔按大小降序排列。这些装置能够依照尺寸范围分级测量含活性微生物的颗粒样品，因为颗粒是经过有大小尺寸的穿孔而到达琼脂平板表面的。
离心取样器（Centrifugal Sampler）— 此装置包括一个推进器或涡轮，以推动已知体积的空气进入装置内部，然后向外推进空气使其与独立设置在一个柔韧塑料基板上的营养琼脂条碰撞。
无菌微生物房（Sterilizable Microbiological Atrium）— 此装置是单孔滤网打入器的一种变型。装置的盖子含有均匀分布的约0.25英寸的小口。装置的基底适合于一个含营养琼脂的有盖培养皿。由真空泵来控制通过装置的空气运动，并可以应用多重装置控制中心和远距离取样探针。
表面空气系统取样器（Surface Air System Sampler）— 这是一种整合装置，包括一个适合琼脂接触板的进入部分。紧接着接触板的后面，是一个传动器和涡轮，可以推动空气穿过装置中琼脂接触板的有孔盖子，以及穿过传动器远端。也可以有多重包埋组合。
凝胶薄膜过滤取样器（Gelatin Filter Sampler）— 这一装置包括一个带有延长管的真空泵，延长管的终端是一个滤纸夹，此末端可以被放置于遥远的关键性空间。滤纸含有无规则明胶纤维，对空气播散微生物起到防卫作用。经过特定的暴露时间后，滤纸被无菌的移开，并用适当的稀释剂溶解，然后涂于适当的琼脂培养基上进行微生物含量的评价。
沉降皿（Settling Plates）— 作为一种简单和廉价的方法，仍被广泛用于延长暴露时间的环境品质评估。但是开放性琼脂填充培养皿或沉降皿的方法，不能用于对关键环境进行微生物污染的品质评估。
机械空气取样器的一个主要的缺陷是对被取样空气样本量的限制。如果一个控制环境的空气微生物水平期望值为不高于3 cfu 每立方米，那么就需要对几个立方米的空气进行测试，进而得到准确度和精确度合乎给定标准的结果。通常这是不切实际的。为了显示环境中存在的微生物数量不随时间而增加，可能就需要通过延长取样时间来确定取样时间是否是一个限制性因素。具代表性的有，slit-to-agar取样器具有每分钟80升的取样能力(对于表面空气系统，此能力有时可能会更高)。如果检测1立方米的空气，接着将需要暴露时间为15分钟。用这过多的15分钟取样时间，对于获得代表性的环境样品，可能是必需的。尽管报导有一些取样器，其取样体积比率能达到很高，可是这些情形中存在的对任何关键区域气流模式的潜在破坏性或是能够增加污染可能性的紊流形成，都应该给予考虑。
对于离心空气取样器，许多前期的研究表明，所取样品显示了对较大颗粒的选择性。这种类型取样器的应用，可能会由于其固有的选择性，引起比其它类型空气取样器更高的空气播散计数。
当选择离心取样器时，应当考虑到取样器对取样位置的控制区域气流线性的影响。如果忽略所用取样器的类型，则远距离探针的应用需要检测其额外的配管对活性空气播散计数没有不良影响。 如有影响，则应设法消除；如果无法消除，则应当在报告结果时引进校正系数。

测量控制环境内活性微生物污染表面取样的仪器和方法学
微生物环境控制程序的另一组成部分是这些环境中仪器、设备以及工作人员着装的表面取样。在制药工业中，表面取样方法和程序的标准化程度还不及空气取样程序的标准化程度广泛。3 为使对关键性操作的影响最小化，表面取样应该在该操作的结尾进行。表面取样可以通过应用接触平板或者水刷方法来完成。表面监测通常在于产品有关联的区域或与这些区域相邻近的区域表面进行。装有营养琼脂的接触平板通常应用于规则的或平坦的表面取样，并且在给定温度下直接孵育适当时间以对活菌进行定量检测。对于真菌、孢子等的特殊定量检测应当用专门的琼脂。
水刷方法可以用于不规则表面的取样，尤其是仪器表面。在规则表面，水刷法可作为接触平板方法的补充。取样后，将水刷置于适当的稀释剂冲之中，然后在适当的营养琼脂上或其中接种适当量以进行微生物计数评估。将要被刷的区域用适当大小的无菌模板确定。通常其大小在 24 到30 cm2范围内。对报告的每接触板或每刷进行微生物学评估。

微生物取样或定量用培养基及稀释剂
控制环境中用于微生物取样或定量的培养基类型是液体还是固体，取决于所用仪器和操作的过程。当需要固体培养基时，一般选用通用的大豆-酪蛋白消化琼脂培养基。其它固体和液体培养基见下表。
液体培养基*
固体培养基*

胰蛋白胨盐        大豆-酪蛋白消化琼脂
胨水        营养琼脂
缓冲盐水        胰蛋白胨葡萄糖浸液琼脂
缓冲明胶        卵磷脂琼脂
浓缩缓冲明胶        脑心浸液琼脂
脑心浸液        琼脂平板
大豆-酪蛋白培养基       
*  液体和固体培养基已用有效方法进行过灭菌处理.
这些培养基的脱水型都可以在市场上购买到。它们的即用型也能够买得到。当控制区域用了消毒剂或抗生素时，应当考虑应用具有适当灭活剂的培养基。
可以从上表中选择培养基，如果确证其能够有效到达预期目的。

来自环境控制程序的微生物隔离群的鉴别
环境控制程序包括对从样品中获得的菌丛进行适当标准的鉴别。具有控制环境中一般菌株的知识，能帮助我们预先确定被监测设备通常的微生物菌株；评估洁净和清洁卫生处理程序、方法及试剂的有效性；并恢复一些方法。通过鉴别程序收集的信息，在对污染源的调查中也是有用的，尤其是当其超过行动标准时。
鉴别从关键区域以及紧邻关键区域得到的隔离群，应当优先于对非关键区域微生物的鉴别。鉴别方法应当校验，而且即用试剂盒的质量与其预期目的相当。(见环境控制程序设计和实施中的相关关键因素)。

洁净室和其他控制环境中无菌填充产品微生物状况的操作评估
控制环境的监测是通过适当的环境监测程序进行的。为了确保得到最小生物负载，控制环境中微生物状况评估的额外信息，可以通过应用培养基填充来获得。一个可接受的培养基填充可以在生产线上对点及时成功模拟出产品的流程。然而，其它的因素也是重要的，比如适当的设备构建、环境监测以及人员培训。
当一套无菌操作系统被制定和安装时，通过至少连续三次成功的培养基填充以检测微生物状况的合格性通常是必需的。培养基填充是用生长培养基替代原产品，以检测微生物的生长。在培养基填充程序的发展过程中值得注意的问题如下：培养基填充过程、培养基的选择、填充体积、孵育时间和温度、填充装置的检查、文件证明、结果解释以及需要的可能改善行动。
由于设计培养基填充是为了模拟特定产品的无菌过程，使得培养基填充过程中，正常产品生产流程条件对其的影响显得重要了。这包括整个的工作人员、所有的加工处理步骤以及构成一般产品流程的原料。培养基填充生产期间，应当计划发生在实际产品生产流程期间的多种初始文件性干涉(如改变填充针、固定组件塞)。
为了增加安全域，可以选择性的进行可能条件的联合应用。实例可能包括频繁启动和停止顺序、非预期的处理系统修复、滤纸的替换等等。取得无菌操作资格不需要每一产品的检测，而应当对每一生产线进行检测。容器的几何形状(容器的大小和其开启)和流水线的速度都是无菌操作线上的可变因素，因此应当在生产线质控上对这些因素进行适当的结合，最好是在终端结合。产品应用的原则应当形成文件性指导。
1987年 FDA关于通过无菌操作生产无菌药物制剂的指导方针指出，培养基填充流程应覆盖对于生产线/产品/容器结合的所有生产换岗。这一指导方针应不仅仅被认为是培养基填充流程的质控，而且应看成是周期性再评估或再生效的指导。培养基填充程序还应当模拟扩展运行的生产实践。这可以通过在产品生产运作末端进行培养基填充运作来实现。
通常，像大豆酪蛋白牛肉汤这种已经用一系列水平低于100 cfu/unit的指示微生物 (见 无菌检查（Sterility Tests）á71ñ进行过促生长检验的通用丰富培养基，可以被应用。来自将要进行无菌操作生产的控制环境中的隔离群也可以被应用。培养基的无菌操作之后，填充容器被置于22.5 ± 2.5 或32.5 ± 2.5 进行孵育。所以填充培养基的容器至少应当孵育14天。如果培养基样品的孵育温度有两个，那么这些填充容器在每个温度下至少要孵育7天。孵育过后，应当对培养基填充容器进行生长检查。培养基填充隔离群以属进行鉴别，如果出于调查污染源为目的的需要，可能的话要鉴别到种。
在进行培养基填充时关键性问题有：使无菌操作质量合格的填充数量，每一培养基填充的单位数量，对结果的解释以及改善行动的实行。据以往经验，常在质控初始或设备初启时采取3个培养基填充运行来证明无菌操作线程的连贯性。0.1%污染率是一个成功培养基填充运行的可接受标准，用于证明污染率不超过0.1%的最小单位数至少应为3,000。应当强调指出， 美国以及其它国家的的许多生产商在单次培养基填充运行时填充单位超过了3,000。4 中试工厂用于制备临床批产品的设备，可以用较小的培养基填充。
许多国际性文件(即ISO 和EU-GMP)也已经引用了3,000培养基填充单位中期望零阳性检出在95%置信度。可是，公认的为了确证过程中实测污染率的统计真实性，就需要进行重复的培养基填充运行。
PDA 技术专题论文17期4的“当前无菌制造业实践的调查”表明，许多生产商认为他们的无菌操作过程能够使污染率低于0.1%
既然洁净室最关键的污染源是工作人员，那么在培养基填充过程中能够对相关生产活动中污染事件起到帮助的可视性文件就应得到支持。无菌测试隔离系统的广泛应用，已经表明无菌操作中去除人员确实能减少污染的发生。

目前出现的先进无菌操作技术综述
由于无菌操作中人员的参与和介入与产品污染潜在性之间存在密切的联系，所以设计并运行了可以使人员和关键区域相隔绝的生产系统。研究减少污染可能性的方法包括自动化装备、阻挡层以及隔离系统。采用这些先进无菌操作策略的设备现已投入运行。在应用这些设备的地方，人员已经被完全排除在关键区域之外，那么基于颗粒和环境微生物监测需求的洁净室分类的必需性可能就会明显的减少了。
以下是关于一些目前应用于无菌操作用以减少污染的系统的定义：
阻挡层（Barriers）— 在无菌操作系统里，阻挡层是一种装置，用来限制操作人员与阻挡层内被隔离无菌区域之间的接触。这些系统用于无菌填充，同时也用于医院药房、实验室以及照管动物设备。阻挡层可以是没有灭过菌的，并且不能总是有准许没有暴露在周围环境中的原料进出/出通行的传递系统。阻挡层可以是围住关键生产区域的塑料屏障，也可以是在现代无菌填充设备外建立的坚硬的外壳。阻挡层也可以结合一些元素，如手套孔、不完全工作服以及快速传递通道等。
Blow/Fill/Seal— 这种类型的系统将容器吹成型、产品填充以及单片设备加封操作相结合。从微生物学角度来看，使容器成型、装入无菌产品以及封盖的形成和应用这系列连续事件，达到了连续的无菌操作并使其于环境中的暴露最小化。这些系统的存在已有大约30年的历史，并且显示出使污染率到达低于0.1%的能力。当综合和分析组和培养基填充数据时，blow/fill/seal系统的污染率能够达到0.001%。
隔离器（Isolator）— 这一技术可有双重目的应用。一个是保护产品免受来自环境中的污染，包括装入和封闭过程中的人员方面污染；另一个是保护人员免受正在制造的有毒或有害产品的影响。
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