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This chapter provides guidelines for the validation of isolator systems for use in sterility testing of compendial articles. [NOTE—In the context of this chapter, “sterilized” refers to an item or surface that has been subjected to a process that eliminates viable bioburden.]
Isolators—devices that create controlled environments in which to conduct Pharmacopeial sterility tests—have been used since the mid-1980s. An isolator is either sealed or supplied with air through a microbial retentive filter and is able to be reproducibly sterilized. When closed, it uses only sterilized interfaces or a specialized rapid-transfer port for the transfer of materials. When open, it allows the egress of materials through a defined opening that has been designed and validated to preclude the entry of contamination. Isolators are constructed of flexible plastics (such as polyvinyl chloride), rigid plastics, glass, or stainless steel.
Isolator systems protect the test article and supplies from contamination during handling by essentially eliminating direct contact between the analyst and the test articles. All transfers of material into the isolator are accomplished aseptic while maintaining complete environmental separation. Aseptic manipulations within the isolator are made with half-suits, which are flexible components of the isolator wall that allow the operator a full range of motion within the isolator, or by gloves and sleeves. Operators are not required to wear special clean-room clothing for conducting sterility tests within isolators; standard laboratory clothing is adequate. The interior of the isolator is treated with sporicidal chemicals that result in the elimination of all viable bioburden.

Air Handling Systems
An isolator used for sterility testing is equipped with microbial retentive filters (HEPA filters are required). At rest, the isolator meets the particulate air-quality requirements for Class 100 area as defined in U.S. Federal Standard 209E (see Microbiological Evaluation of Clean Rooms and Other Controlled Environments 1116). However, the isolator need not meet Class 100 conditions during operation, and no requirements for air velocity or air exchange rate exist. The isolator system is leakproof; however, it is not generally impermeable to gas exchange with the surrounding environment. When direct openings to the outside environment exist, constant air overpressure conditions maintain sterile conditions within the isolator. Airflow within isolators used for sterility testing is unidirectional or turbulent.
Transfer Ports and Doors
Isolators are attached to a “pass-through” sterilizer to enable the direct transfer of sterile media, sterile dilution fluids, and sterile supplies from the sterilizer into the isolator system. Specially designed rapid transfer ports or doors (RTPs) enable two isolators to be connected to one another so that supplies can be moved aseptically from one isolator to another. Aseptic connections between two isolators or an isolator and a container can be made in unclassified environments using RTPs. The nonsterile surfaces of the RTP are connected using locking rings or flanges. A compressed gasket assembly provides an airtight seal, thereby preventing the ingress of microorganisms.
When the two RTP flanges are linked to form an airtight passage, a narrow band of gasket remains that could harbor microbial contamination. This exposed gasket is treated with a sporicidal agent immediately after the connection is made and before materials are transferred through the RTP. Good aseptic technique is used when transferring materials and care is taken not to touch the gasket with the materials, being transferred or with the gloved hands.
Preventive maintenance and lubrication of the gasket assemblies on the flanges are performed according to the RTP manufacturer's recommendations. The RTP gaskets are changed at the recommended frequency and periodically checked for damage, since cut or torn gaskets cannot make a truly airtight seal.
Selection of a Location for the Isolator
Isolators for sterility testing need not be installed in a classified clean room, but it is important to place the isolator in an area that provides limited access to nonessential staff. The appropriate location provides adequate space around the isolator for moving transfer isolators, staging of materials, and general maintenance. No environmental monitoring of the surrounding room is required.
Temperature and humidity control in the room is important to operator safety and comfort and is critical for the effective use of certain sterilization or decontamination technologies. If an isolator is directly in the flow path of an air supply grille, it could cool sections of the isolator's walls and result in condensation during vapor sterilization. Uniform temperature conditions in the room are desirable when temperature-sensitive sterilization methods are employed.

The isolator system must be validated before its use in sterility testing as part of a batch release procedure. To verify that the isolator system and all associated equipment are suitable for sterility tests, validation studies are performed in three phases: installation qualification (IQ), operational qualification (OQ), and performance qualification (PQ). The following sections contain points to consider in the validation of isolator systems for sterility testing. The assignment of test functions to a particular phase of the validation program (i.e., IQ, OQ, and PQ) is not critical, as long as proper function of the isolator is demonstrated and documented before its use in compendial Assays.
Installation Qualification (IQ)
The IQ phase includes a detailed description of the physical aspects of the system, such as the dimensions, internal configuration, and materials of construction. The unit layout is diagrammed with interfaces and transfer systems clearly and dimensionally indicated. Compliance with design specifications for utility services, such as air supply, vacuum, external exhaust, and temperature and humidity control, is verified. Other equipment used with the isolator system is also described in detail; if any revisions to design specifications are made, these are included. Equipment manuals and copies are catalogued and stored where they can be retrieved and reviewed. Compliance of drawings to design specifications is verified. All drawings and process and instrumentation diagrams are catalogued, and stored and are retrievable.
All documentation is reviewed to verify that it precisely reflects the key attributes of the installed system. This establishes a general benchmark for the isolator system's compliance with design specifications and installation requirements.
Potential process-control or equipment problems that could cause system failure during operation are identified and documented during failure-mode analysis and hazard analysis. The system is modified, if necessary, to minimize the risk of failure, and critical control point methods are established.
The results of the IQ are summarized in an Installation Qualification Report. The following documentation is suggested.
Equipment— The equipment is listed with its relevant design specifications. The IQ Report verifies that equipment meeting the appropriate design specifications was received and that it was installed according to the manufacturer's requirements.
Construction Materials— The construction materials of critical system components are checked for compliance with design specifications. The compatibility of the intended sterilization method with the construction materials is verified.
Instruments— System instruments are listed with their calibration status.
Utility Specifications— All utilities required for operation—as defined in the operating manuals and process and instrumentation diagrams—are checked for availability and compliance with design specifications. Any connection between utility systems and the isolator system is inspected, and conformance of these connections to specifications is verified.
Filter Certification— HEPA filters and other microbial retentive filters are tested and certified; copies of test results and certificates are included in the IQ Report. Purchase orders are reviewed, and conformance of the air filtration system to specifications is verified.
Computer Software— All computer software associated with the isolator system is listed with its name, size, and file revision number. The master computer disks are checked for proper labeling and are stored securely.
Operational Qualification (OQ)
The OQ phase verifies that the isolator system operates in conformance to functional specifications.
Operational Performance Check— This test verifies that all alert and alarm functions comply with their functional specifications. The system's ability to comply with all set points and adjustable parameters is verified.
Isolator Integrity Check— The integrity of the isolator is maintained during all normal operating conditions. A leak test is performed to verify compliance with the manufacturer's functional specifications and to ensure safety prior to charging the isolator with a sterilizing chemical. To safeguard against adventitious contamination, isolators are operated at a positive pressure differential of about 20–50 Pa during normal operation. If constant overpressure is needed, validation studies must show that the set point can be maintained and controlled during operation.
Sterilization Cycle Verification— A sterilization cycle is performed to verify that all actual values conform to cycle steps and set points.
Different sterilization methods can be used to eliminate bioburden from isolator systems and supplies. Among the chemicals that have been used to treat isolators are peracetic acid, chlorine dioxide, ozone, and hydrogen peroxide; each has different requirements for exposure conditions and process control. It is critical to comply with the manufacturer's operational requirements for the selected sterilization method and to describe them in the functional specifications. Temperature and humidity control within the room is critical when hydrogen peroxide vapor is used in sterilization. The temperature inside the isolator is also important, particularly for hydrogen peroxide vapor sterilization, where it is critical to maintain the concentration below the condensation point. Some sterilization chemicals, such as chlorine dioxide and ozone, require the addition of moisture to the isolator prior to sterilization. When elevated relative humidity is required, the ability to control it must be verified during OQ.
It is also important to verify the concentration and distribution of the sterilizing chemical. When applied in gaseous or vapor form, the concentration is measured using chemical indicators, spectroscopic methods, or electronic sensors. Distribution may also be tested using chemical indicators. [NOTE—Chemical indicators provide qualitative, but not quantitative, information.]
Gas and vapor sterilization methods require fans in the isolator to distribute the chemical evenly. The location and orientation of these fans are adjusted to ensure optimum air distribution. Since shelving units, equipment, glove-and-sleeve assemblies, and half-suits have an effect on distribution patterns, distribution checks are done with the isolator fully loaded with equipment and supplies, and the setup of these units is defined and documented.
Many installations use smaller transfer isolators as portable surface sterilization units. In these transfer isolators, test articles and supplies are treated chemically to eliminate bioburden before transfer through an RTP into the testing isolator. Its loading configuration is defined, and configuration drawings are reviewed and verified during the OQ. [NOTE—The sterilizing chemicals used in isolators work on the surfaces of materials; therefore, any surface that is occluded will not be treated and could contain viable bioburden.]
Sterilization agents need to be removed from the isolator after the exposure period, which is accomplished by a current of fresh air provided either by the sterilization equipment or by some other means. Aeration is accomplished either in an open loop, in which the gas is exhausted through a vent to the atmosphere, or in a closed loop, in which the chemical is removed and destroyed by the sterilization equipment. The aeration system is checked; if an open-loop configuration is used, the external exhaust system's flow and safety are checked.
Sterilization Cycle Development— When the OQ is completed, sterilization cycle development is performed to establish the parameters necessary for process control during routine sterilization cycles. Any of the methods generally used in the industry for the validation of sterilization processes—including bioburden-based and overkill methods—are adequate. The sterilization process is challenged with biological indicators (BIs). The spore population and resistance of the BIs to the sterilization conditions being applied is known. Wherever possible, a true D value is obtained for the BI system (see Biological Indicators—Resistance Performance Tests 55); it is acceptable to obtain the D value from the BI vendor. When it is impossible to determine an accurate D value and no means to verify the concentration of the sterilizing agent is available, the half-cycle approach to cycle development and verification is employed.
Performance Qualifications (PQ)
The PQ phase verifies that the system is functioning in compliance with its operator requirement specifications. At the completion of the PQ phase, the efficacy of the sterilization cycle and, if appropriate, the adequacy of sterilizing chemical venting are verified. All PQ data are adequately summarized, reviewed, and archived.
Cleaning Verification— In general, cleaning is not critical for sterility testing applications. However, residual products are a concern in multiproduct testing, particularly for aggressive antimicrobial agents, since these materials could interfere with the ability of subsequent tests to detect low levels of contamination in the product. Concerns about contamination with the product are heightened when it is an inherently antimicrobial powder, since powders are more readily disseminated. Cleaning to a level at which no visible contamination is present is adequate for sterility test isolator systems and is a suitable operator requirement specification. The cleaning method, frequency, equipment, and materials used to clean the isolator are documented.
Sterilization Validation— The interior surfaces of the isolator, the equipment within the isolator, and the materials brought into the isolator are treated to eliminate all bioburden. The sterilization of isolator surfaces, sterility testing supplies, and test articles is different from the sterilization of product contact parts or drug components used in product manufacturing. The methods used to sterilize an isolator may be able to achieve log reduction values typical of production overkill processes. This level of insurance of sterility cannot be guaranteed over time. Upon completion of the sterilization process, asepsis within the isolator is maintained primarily by the air filtration system, by the appropriate materials transfer operations, and, most importantly, by the integrity of gloves used to conduct aseptic manipulations.
The sterilization methods used to treat isolators, test articles, and sterility testing supplies are capable of reproducibly yielding a six-log kill against an appropriate, highly resistant biological indicator (BI; see Biological Indicators for Sterilization 1035), as verified by the fraction negative or total kill analysis methods. Total kill analysis studies are suitable for BIs with a population of 104 spores per unit, while fraction negative studies are suitable for BIs with a population of 105 or greater. A sufficient number of BIs are used to prove statistical reproducibility and adequate distribution of the sterilizing agent. Particular attention is given to areas that pose problems relative to the concentration of the agent. A larger number of BIs are used in isolators that are heavily loaded with equipment and materials. Also, when it is not possible to use one or more calibrated sensors to directly measure the concentration of the sterilizing agent, the placement of additional BIs is considered. The ability of the process to reproducibly deliver a six-log kill is confirmed in three consecutive validation studies.
The operator establishes a frequency for resterilization of the isolator. The frequency may be as short as a few days or as long as several weeks, depending on the sterility maintenance effort (see Maintenance of Asepsis within the Isolator Environment).

Some materials are adversely affected by sterilizing agents, which can result in inhibition of microbial growth. Of concern are the penetration of sterilizing agents into product containers; accessory supplies such as filter sets and tubing; or any material that could come in contact with product, media, or dilution fluids used in the sterility test. It is the responsibility of the operator to verify that containers, media, and supplies are unaffected by the recommended sterilization process. Screw-capped tubes, bottles, or vials sealed with rubber stoppers and crimp overseals have proved very resistant to the penetration of commonly used sterilizing agents. Wrapping materials in metal foil or placing them in a sealed container will prevent contact with the sterilizing agent; however, these procedures may also result in some surfaces not being sterilized.
In many cases, the operator will choose to treat the surfaces of product containers under test with the sterilizing agent in order to minimize the likelihood of bioburden entering the isolator. It is the responsibility of the operator to demonstrate, via validation studies, that exposure of product containers to the sterilizing agent does not adversely affect the ability of the sterility test to detect low levels of contamination within these test articles. It is suggested that the ability of the package to resist contamination be examined using both chemical and microbiological test procedures. Bacteriostasis and fungistasis validation tests must be performed using actual test articles that have been exposed to all phases of the sterilization process (see Sterility Tests 71). This applies to medicinal device packages as well as pharmaceutical container and closure systems.
Validation studies determine whether both sterility test media and environmental control media meet the requirements for Growth Promotion Test of Aerobes, Anaerobes, and Fungi under Sterility Tests 71.

The ability of the isolator system to maintain an aseptic environment throughout the defined operational period must be validated. In addition, a microbiological monitoring program must be implemented to detect malfunctions of the isolator system or the presence of adventitious contamination within the isolator. Microbiological monitoring usually involves a routine sampling program, which may include, for instance, sampling following sterilization on the first day of operation and sampling on the last day of the projected maintenance of asepsis period. Intermediate sampling is performed to demonstrate maintenance of asepsis within the isolator.
The surfaces within the isolator can be monitored using either contact plates for flat surfaces or swabs for irregular surfaces. However, since media residues could impose a risk on isolator asepsis, these tests are generally best done at the end of the test period. If performed concurrently with testing, care is used to ensure that any residual medium is removed from isolator surfaces. Active air samples and settling plates may be used, but they might not be sufficiently sensitive to detect the very low levels of contamination present within the isolator enclosure.
The most likely route for contamination to enter the isolator is during the introduction of supplies and samples into the enclosure. Validating that all materials taken into the isolator enclosure are free of microbial contamination is critical, as is periodic inspection of gaskets to detect imperfections that could allow ingress of microorganisms. Gloves and half-suit assemblies are another likely source of microbial contamination. Gloves are of particular concern, since they are used to handle both sterility testing materials and test articles. Very small leaks in gloves are difficult to detect until the glove is stretched during use. There are several commercially available glove leak detectors; the operator ensures that the detectors test the glove under conditions as close as possible to actual use conditions. Microbiological tests are used to supplement or substitute physical tests. [NOTE—Standard “finger dab plates” may not be sensitive enough to detect low levels of contamination. Submersion of the gloves in 0.1% peptone water followed by filtration of the diluent and plating on growth media can detect loss of integrity in the gloves that would otherwise go unnoticed.]
Continuous nonviable particulate monitoring within the isolator's enclosure is ideal, since it can quickly detect filter failure. A second choice is periodic monitoring using a portable particle counter. Sampling for particles must be done in a manner that poses no risk to the maintenance of asepsis within the isolator.

A sterility test resulting in a false positive in a properly functioning and validated isolator is very unlikely if bioburden is eliminated from the isolator interior with a high degree of assurance, if personnel are not in direct contact with the work area, and if the integrity of the transfer ports is validated. Nevertheless, isolators are mechanical devices, and good aseptic techniques are still required. A decision to invalidate a false positive is made only after fully complying with the requirements of Observation and Interpretation of Results under Sterility Tests 71.

As with sterility testing conducted in conventional clean rooms, operators are trained in procedures specific to their isolator. All training sessions and the evaluation of the operator's performance are documented in the individual's training record. Training of all personnel in the appropriate safety procedures necessary for the operation and maintenance of the isolation system is imperative.
Personnel safety in the use of a sterilizing agent must be assessed. Material Safety Data Sheets, or equivalent documents, are available in the immediate area where the sterilizing agent is being used. All storage and safety precautions are followed. An operational readiness inspection of the safety of the isolator and all associated equipment is performed and documented prior to placing the unit in service.

Auxiliary Information—
Staff Liaison : Radhakrishna S Tirumalai, Scientist
Expert Committee : (MSA05) Microbiology and Sterility Assurance
USP29–NF24 Page 3037
Pharmacopeial Forum : Volume No. 30(6) Page 2162
Phone Number : 1-301-816-8339