​1208 STERILITY TESTING—VALIDATION OF ISOLATOR SYSTEMS

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, “decontaminated” 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 supplied with air through a HEPA or better air filter and is able to be reproducibly decontaminated. Closed isolators, which are systems with no direct opening to the external environment, are normally used for sterility testing, although open isolators which allow the egress of materials through a defined opening that precludes the entry of contamination by means of air overpressure may be used. Closed isolators use only decontaminated interfaces or a rapid-transfer port for the transfer of materials. 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 and out of the isolator are accomplished in an aseptic fashion 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, although a pair of sterile gloves is frequently worn under the isolator gloves as an added precaution against contamination entering the isolator enclosure and for hygiene purposes. The interior of the isolator is treated with sporicidal chemicals that result in the elimination of all viable bioburden on exposed surfaces.

这部分是用于无菌检验隔离器系统的简明验证指南（注意：在这个章 节中，“已灭菌”指的是物品或者表面的微生物被清除的状态）

在 19 世纪 80 年代中期，建立一个无菌检验环境的隔离器就已经开始 使用。隔离器可以通过密封的方法或者采用过滤除菌空气保持正压的方法， 创造一个无菌的环境。当隔离器处于密闭状态时，仅仅能够在隔离器内部 和快速传递仓传递物品；当隔离器打开时，允许通过一个特殊设计的并经 过验证可以避免污染物进入的开口递出物品。隔离器采用柔软的塑料（例 如聚氯乙烯）、硬塑料、玻璃或不锈钢制造。

由于隔离器系统从根本上避免了分析人员与物品的直接接触，因此在 无菌检验操作时可以避免物品和辅助设备被污染。当隔离器内部与环境完 全隔离时，隔离器内部的物品是无菌条件下的传递。操作者穿着半身衣在 隔离器内部进行无菌操作，半身衣是连接在隔离器墙体上的柔软的部分， 操作者穿着半身衣有足够的范围在隔离器内部进行操作，操作者也可以通 过连接在隔离器墙体上的袖子和手套进行操作。在隔离器中，不要求操作 者穿着特殊的无菌衣进行操作，允许操作者穿着标准的实验室服装进行操 作。为确保隔离器内部无菌。使用杀孢子剂对隔离器内部进行灭菌处理。

ISOLATOR DESIGN AND CONSTRUCTION

Air Handling Systems

An isolator used for sterility testing is equipped with microbial retentive filters (HEPA filters or better are required). At rest, the isolator meets the particulate air-quality requirements for an ISO Class 5 area as defined in ISO 14644-1 through -3* (see Microbiological Evaluation of Clean Rooms and Other Controlled Environments 1116). However, the isolator need not meet Class 5 conditions during an operation that may generate particulates, and no requirements for air velocity or air exchange rate exist. The isolator should be sealed well enough during decontamination that the dissemination of sporicidal vapors or gases into the surrounding environment is kept to appropriately low levels. When direct openings to the outside environment exist, constant air overpressure conditions maintain sterile conditions within the isolator. In general, both open and closed isolators are maintained at positive pressure relative to the surrounding environment, and overpressures of 20 Pa or more are typical. The user should never exceed the maximum pressure recommended by the isolator manufacturer. Airflow within isolators used for sterility testing is either unidirectional or turbulent.

隔离器设计和建造 空气处理系统    

用于无菌检验的隔离器需要配备除菌过滤器（HEPA 过滤器是被要求 的）。静态时，要求隔离器尘埃粒子符合美国联邦标准 209E 的 100 级空气质量要求（看洁净室微生物评价和其他环境控制《1116》）。动态时，不要 求隔离器符合 100 级空气质量要求，不要求隔离器内部的空气流速或者空 气交换频率。隔离器系统是要求防止泄漏的，然而，它不是通常意义上的 防止隔离器与外界环境进行的空气交换。当与外界环境直接相连的门打开 时，隔离器内部的正压保证隔离器内部的无菌环境不被污染。用于无菌检 验的隔离器内部空气流可以是单向流或者湍流。

Transfer Ports and Doors

Isolators may be attached to a “pass-through” decontaminator or transfer isolator to enable the direct transfer of sterile media, sterile dilution fluids, and sterile supplies from the decontaminator into the isolator system. Rapid transfer ports (RTPs) enable two isolators, i.e., the work station and transfer isolator, 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 an RTP-equipped 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 should be routinely disinfected 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 is performed according to the RTP manufacturer's recommendations. The RTP gaskets are changed at the recommended frequency and periodically checked for damage, because cut or torn gaskets cannot make a truly airtight seal.

隔离器有一个附属的“传递通路”杀菌器，通过传输通道杀菌器可以 直接将无菌的培养基、无菌的稀释液、无菌的装备等传递进隔离器系统。 一般设计成快速传递仓或门（RTPs），通过快速传递仓或门可以把两个隔离 器彼此相连，无菌的物品就能够从一个隔离器传递到另一个隔离器。通过 快速传递仓，两个隔离器或者一个隔离器和一个容器就可以在普通环境中 连接。通过密封圈或法兰，将传递仓的非无菌表面连接。用垫圈压紧来保 证气密性，避免微生物进入。

当两个传递仓法兰连接形成一个密封通道时，存在一个狭长的垫圈带， 这个部位可能存在微生物污染。因此一旦连接完，在使用传递仓传递物品 之前，必须立即用杀孢子剂对垫圈暴露部分进行处理。并且在传递物品时， 应当注意无菌技巧的使用，避免物品或手套接触垫圈。

将垫圈装配在法兰上时，应当按照传递仓生产商的建议进行预防性的 维护和润滑。传递仓垫圈应当按照（生产商的）的要求定期更换并且定期 检查，破损的垫圈不能够保证真正的密封。

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 utilization of certain decontamination technologies. Uniform temperature conditions in the room are desirable when temperature-sensitive decontamination methods are employed. Care should be taken in locating the isolator so that cold spots are avoided that might result in excessive condensation when condensing vapors are used for decontamination.

用于无菌检验的隔离器不需要安装在洁净区，但是安装在一个限制非 授权人员进入的区域仍然是重要的。安装时，应当使隔离器周围有足够的 范围，以便移动隔离器，传递物料，以及通常的维护。隔离器所在的房间 不要求进行环境监测。 隔离器房间温度和湿度对于操作者的安全和舒适是重要的，温湿度对 于除菌和净化技术的影响效果也很关键。如果隔离器位于空气补给窗的气 流通道中，当隔离器采用蒸汽灭菌时，空气气流会使隔离器个别部位温度 较低形成冷凝水。当采用对温度敏感的灭菌方法时，隔离器房间的温度应 当是均一的。

VALIDATION OF THE ISOLATOR SYSTEM

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.

无菌检验合格与否是产品放行的前提时，在进行无菌检验之前，隔离 器系统的验证必须完成。为了核实隔离器及其辅助设备能够用于进行无菌 检验，隔离器系统的验证可以分成三个部分：安装验证、性能验证、操作 验证。在进行无菌检验隔离器系统验证过程中，应当考虑到下面的观点。 在验证程序的特定阶段（例如 IQ、OQ、PQ），各个阶段试验的任务是不被确 定的，在隔离器用于确定的检测前，隔离器的功能应当被验证并且有文字 记录。

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, 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 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 decontamination 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 summary. 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 stored securely.

IQ 阶段包括隔离器系统详细的外观描述，例如隔离器尺寸、内部结构、 所用的材料。关于接触面和传递系统，要清晰的画图并标注尺寸。设计的 隔离器是否符合使用的规格需要被核实，例如空气补给、真空、外部排气、温湿度控制等。其他与隔离器系统一起使用的设备也要详细描述；任何设 计规格的修订都应当详细描述。设备指南和复印件应当编成目录并保存， 在需要的时候，操作者可以得到并重新查阅。一旦设计规格符合性被核实， 所有的图纸、方法和设备图要编成目录，保存并能够重新得到。

所有的文档应重新被审阅，便于核实能够反映出安装系统的关键属性要 求。这样就建立了符合隔离器系统设计规格和安装要求通用的基准。

在故障模式下或高风险情况下进行分析时，可能导致试验失败的潜在的 过程控制问题和设备问题可能被发现，这些问题应当进行鉴定并记录在文 档中。如果必要，系统可以被修改，以便将失败的风险降到最低点，并且 关键点的控制方法被建立。

安装确认的结果被总结成为一个安装确认报告，建议包括下列文档。

设备---设备应符合设计规格并被列表记录。IQ 报告应核实：符合设计 规格的设备被接收，并且按照生产厂商的要求进行安装。

结构材质---隔离器系统关键部位的结构材质经检测是符合设计要求

的。对隔离器材质的杀菌方法兼容性应当被核实。

仪表---仪表被核实符合其精度要求并列表。

功能规格---所有操作功能，例如在操作手册、流程、仪器图表中指明 的功能，都应当被核实能够执行并且符合规格要求。隔离器系统和其他系 统的所有连接部分应进行审核，其功能应被核实符合设计要求。

过滤器证明---HEPA 过滤器和微生物截留过滤器的测试和证书；测试结 果和证书复印件应包括在 IQ 报告中。核对采购合同，核实空气过滤系统符 合规格要求。

计算机软件---所有的和隔离器系统有关系的计算机软件列出名称、规格、文档编号。计算机系统盘被检查贴签并安全保存。

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 the compliance with the manufacturer's functional specifications and to ensure safety prior to charging the isolator with a decontaminating sporicidal chemical. To safeguard against adventitious contamination, isolators are operated at a suitable positive pressure during normal operation. Validation studies must show that the air pressure set point can be maintained and controlled during operation.

Decontamination Cycle Verification— A decontamination cycle that is the function of the decontamination equipment in concert with the isolator(s) is verified.

Different decontamination 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 decontamination method and to describe them in the functional specifications. The temperature inside the isolator is also important, particularly for hydrogen peroxide vapor decontamination, where it is critical to maintain the concentration relative to the condensation point. Some sterilization chemicals, such as chlorine dioxide and ozone, require the addition of moisture to the isolator prior to decontamination. 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 decontaminating chemical. When applied in gaseous or vapor form, the distribution may be evaluated using chemical indicators, spectroscopic methods, or electronic sensors.

Gas and vapor decontamination methods may require fans in the isolator to distribute the chemical evenly. The location and orientation of these fans are adjusted to ensure optimum air distribution. If the isolator utilizes a recirculating unidirectional airflow system, distribution fans may not be required, but this should be evaluated on a case-by-case basis. Because shelving units, equipment, glove-and-sleeve assemblies, and half-suits have an impact 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 decontamination 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 decontaminating 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. Special precautions should be in place for treating surfaces known to be occluded with a sporicide if such surfaces may be revealed during the conduct of sterility tests.]

Decontamination 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 decontamination equipment or by utilizing the isolator air handling system. 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 decontamination equipment. The aeration system is checked; if an open-loop configuration is used, the external exhaust system's flow and safety are checked.

Decontamination Cycle Development— When the OQ is completed, decontamination cycle development is performed to establish the parameters necessary for process control during routine decontamination cycles. Any of the methods generally used in the industry for the validation of decontamination processes—including bioburden-based, fractional cycle, and overkill methods—are adequate. The decontamination process is challenged with biological indicators (BIs). The spore population and resistance of the BIs to the decontamination conditions being applied are known. Wherever possible, a D value estimate is done for each B1 system or, alternatively, a survivor curve for the BI system is obtained (see Biological Indicators—Resistance Performance Tests 55); it is acceptable to obtain the D value from the BI vendor.

OQ 阶段核实隔离器系统的操作能够符合说明书的功能。

操作性能检查---这个试验核实所有的警报和警报的功能符合说明书要 求。系统所有设置点和可调整的参数都应当被核实符合规定的要求。

隔离器完整性检测---在通常的操作条件下，隔离器应保持完整性。在 隔离器载物并使用化学杀菌剂灭菌前，一个泄露试验可以证明隔离器符合 厂家说明书要求并且是安全的。为了避免外界污染，隔离器通常在 20-50Pa 正压下进行操作。如果隔离器系统要求一个恒定的正压，验证过程中必须 核实：操作时的压力设置值能够维持并且可以控制。

灭菌周期确认---执行一个无菌周期，以便核实操作的实际数值与无菌

周期的各个步骤情况以及设置数值一致。

可以采用几种不同的灭菌方法来除去隔离器和辅助系统的微生物。可以 用于隔离器灭菌的化学试剂包括过氧乙酸、二氧化氯、臭氧、过氧化氢， 不同的试剂有不同的暴露条件和过程控制。选定的灭菌方法应符合厂家的 操作要求，并且在实用规程中对这些方法进行描述是十分关键的。使用过 氧化氢蒸汽灭菌时，隔离器房间的温度和湿度的控制是关键的。隔离器内 部的温度也是重要的，尤其在使用过氧化氢蒸汽灭菌过程中的影响是非常 重要的，关键在于保持过氧化氢的浓度，避免低于过氧化氢的冷凝点。一 些化学试剂，例如二氧化氯和臭氧，要求在灭菌前预先增加隔离器的湿度。 如果采用对湿度有较高的要求的灭菌方法时，在 OQ 中，控制湿度的能力必 须被核实。

灭菌剂的浓度和浓度分布情况也是重要的。当灭菌剂以气态或蒸汽状态 存在时，它的浓度可以通过化学指示剂、光谱方法或电极检测。灭菌剂分 布情况也可以使用化学指示剂测量（注意：化学指示剂仅能够提供定性的 而不是定量的信息）。

采用灭菌剂在气态或蒸汽的状态下进行灭菌时，要求隔离器内部使用 风机来均匀分布灭菌剂。风机的位置和方向可以进行调节以保证适宜的气 流分布。由于架子、设备、手套、袖子装置和半身衣，能够影响气流分配 模式，必须对隔离器满载状态下的气流分布进行检测，物品的摆放情况进 行详细描述并以文件形式记录。

有时使用更小的传递隔离器作为便携式表面灭菌单元，待验物品和辅助 用具通过传递仓进入隔离器之前，这些物品和用具在便携式表面隔离器中 进行化学灭菌。在 OQ 过程中，应制定出便携式表面灭菌器中物品和用具的 摆放情况，审核摆放图并记录在 OQ 上。（注意：化学灭菌剂作用在设备表 面，因此，一些被遮盖的设备表面能够残留微生物并存活）。

当达到杀菌剂作用时间后，可以使用通过除菌装置的新鲜空气或其他 方式处理过的新鲜空气将杀菌剂从隔离器中移走。通过一个向外开放的通 道，气体通过排风口排入大气；或者通过一个密闭的通道，在通道中使用 一个杀菌装置移走并消除灭菌剂。如果使用一个向外开放的排风通道，排 放系统的气流和安全性应当被检测。

灭菌周期的研究---当 OQ 完成后，进行灭菌周期的研究以便建立日常灭 菌控制过程必要的参数。一些普遍使用在工业中的无菌工艺验证的方法是 可行的，例如生物负荷量和过度杀灭。灭菌工艺进行生物指示剂挑战试验。 生物指示剂的孢子数和耐热性是已知的。无论如何，应该得到生物指示剂真实的 D 值；（看生物指示剂---耐热性能试验《55》）；从生物指示剂供应 商处获得 D 值是可以接受的。如果不可能获得准确的 D 值，同时无法证明 灭菌剂浓度的有效性，可以使用半个周期（循环）的方法对整个周期（循 环）进行确认。

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 decontamination cycle and, if appropriate, the adequacy of decontaminating 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, because 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, because 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.

Decontamination 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 decontamination methods used to treat isolators, test articles, and sterility testing supplies are capable of reproducibly yielding greater than a three-log reduction against highly resistant biological indicators (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 103 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 decontaminating agent. Particular attention is given to areas that pose problems relative to the concentration of the agent. A larger number of BIs may be required in isolators that are heavily loaded with equipment and materials. The ability of the process to reproducibly deliver a greater than three-log kill is confirmed in three consecutive validation studies.

The operator establishes a frequency for re-decontamination 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).

PQ 是核实隔离器系统的功能是符合操作者的要求。PQ 过程完成后，灭 菌周期是适合的，排出口的灭菌剂被检测是适合的，所有的 PQ 过程的数据 应该被统计，分析，存档。

清洗确认---，在通常情况下，对于无菌检验来说，清洁不是关键的， 但是，残留的产品可能会对检验有影响，有杀菌效果产品的残留对检验结 果的影响尤其显著，在检测低水平污染时，这些残留的物质会对检测产生 影响。由于粉末容易扩散，杀菌性粉末产品的残留对检验结果的影响是重 要的。对于无菌检验隔离器系统和操作者的要求来说，没有肉眼可见污染 这种清洗水平是合适的。清洗方法、周期、设备、清洗隔离器中所用的材 料记录在文件中。

无菌验证---隔离器内表面、隔离器内的设备、拿进隔离器的各种物料 都应当去除微生物。隔离器表面、无菌检验设备、检验物品的灭菌和产品 接触部位或者产品药物成分接触部位的灭菌是不同的。隔离器灭菌可以用 过度灭菌的 log 降低值来评估实现。如果超过灭菌周期，无菌保证水平不 再被确定。隔离器灭菌操作完成之后，通过空气过滤系统的维持，好的物 品传递技术的采用，更重要的是：经过无菌处理的隔离器手套完整性的保 持，隔离器的无菌性被保证。

通过阴性分数法或者过度杀灭法可以证实，用于隔离器、试验物品、 无菌检验用设备的灭菌方法可以使某种合适的、高抗性的生物指示剂的微 生物数下降 6 个 log 降。（看用于灭菌的生物指示剂《1035》）。过度杀灭 分析法适用于每单位 104 孢子数的生物指示剂，而阴性分数法适用于每单 位孢子数大于等于 105 的生物指示剂。使用足够数量的生物指示剂进行试 验可以从统计学上证明灭菌效果是可以再现的以及灭菌剂的分布是合适 的。隔离器内设备和物品满载时，需要用更多的指示剂进行试验。因此， 没有一个或者更多的足够量的传感器来测量杀菌剂的浓度时，增加生物指 示剂的放置是可以考虑的。在连续三次灭菌试验中，六个 log 降的杀菌能 力是证明可以灭菌的。

操作者建立隔离器再次灭菌的周期。这个周期可以是短短的几天，也 可以是几周的时间，建立的依据是无菌保持能力（看隔离器内部环境无菌 保持）

PACKAGE INTEGRITY VERIFICATION

Some materials are adversely affected by decontaminating agents, which can result in inhibition of microbial growth. Of concern are the penetration of decontaminating 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 decontamination process. Screw-capped tubes, bottles, or vials sealed with rubber stoppers and crimp overseals have proven very resistant to the penetration of commonly used decontaminating agents. Wrapping materials in metal foil or placing them in a sealed container will prevent contact with the decontaminating agent; however, these procedures may also result in some surfaces not being decontaminated. In some cases, the use of shorter duration decontamination cycles and reduced concentrations may be necessary to minimize penetration of decontaminating agents into the package or container. Cycles that provide a less than three-log kill of resistant BIs may be acceptable provided microbiological analysis of the environment proves that the isolator(s) are free of recoverable bioburden.

In many cases, the operator will choose to treat the surfaces of product containers under test with the decontaminating 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 decontaminating 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 decontamination 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.

一些材料对杀菌剂有影响，这些材料能抑制微生物的生长。我们关注的 是杀菌剂在产品包装上的穿透（能力），在一些辅助设施例如过滤装置和管 道的穿透力、在一些能够和产品、培养基、无菌试验的稀释剂接触的材料 上的穿透力。在灭菌过程中，操作者有责任核实包装物、培养基、辅助物 品在被批准使用的灭菌过程中没有受到影响。带旋转帽的管状容器、胶塞 密封的小瓶、卷曲的褶皱已经被证明能够降低杀菌剂的穿透力。金属箔包 装并且密封的容器能够防止杀菌剂的进入；然而，这些情况也能够导致一 些表面不被灭菌。在多数情况下，在进行无菌检验之前，操作者使用杀菌剂处理产品包装 表面来减少进入隔离器物品的生物负荷量。操作者有责任通过验证证明： 使用杀菌剂处理产品包装这个过程，存在于产品内部的低水平的微生物污 染没有受到杀菌剂的影响，不会影响到检验的检出限度。建议用化学和微 生物试验来检测包装物对污染的抵制能力。经过隔离器全部的灭菌过程后， 杀菌剂对于细菌和真菌的抑制情况的验证需要进行（参照无菌检验《71》）。 对于医疗装置的包装、药品的包装和密封系统包装的要求都是一致的。

不论无菌检验用培养基还是环境控制用的培养基，在无菌检验时，都必 须符合需氧菌、厌氧菌、真菌的生长促进试验（无菌检验《71》）。 
MAINTENANCE OF ASEPSIS WITHIN THE ISOLATOR ENVIRONMENT

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 decontamination on the first day of operation and sampling on the last day of the projected maintenance of asepsis period. Periodic sampling throughout the use period can be 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, because 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, and that those surfaces are carefully cleaned and disinfected. Active air samples and settling plates may be used, but they may not be sufficiently sensitive to detect the very low levels of contamination present within the isolator enclosure.

A potential 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 potential source of microbial contamination. Gloves are of particular concern because they are used to handle both sterility testing materials and test articles. Resistance to puncture and abrasion should be considered in the selection of gloves and sleeves. Hypalon materials are resistant to both chemical sporicides used in the decontamination of isolators and to punctures and are available in several thicknesses to provide adequate tactile feel through the gloves while maintaining their integrity.

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, because 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.

在一个无菌周期内，隔离器系统保持内部环境无菌的能力必须被验证。 隔离器系统出现故障或者隔离器内部的偶然污染情况下的微生物必须被检 测。微生物检测通常包括一个例行的取样程序，它可以包括，例如，灭菌 后的第一天和无菌保持期的最后一天。放置在隔离器中的培养基被证明是 无菌的。

隔离器内部可以使用平面接触双碟或者棉签搽拭监控。然而，因为培 养基残留会使隔离器产生染菌的风险，因此，最好在检验完成后进行检测。 如果试验中有培养基残留，细心地从隔离器内部清理干净。空气取样和培 养基检测双碟可以使用，但它们在检测隔离器内部非常低水平的污染时不 是很灵敏。

最可能污染的途径是检验用具和样品进入隔离器的过程。验证所有进 入隔离器内部的物品是无菌的这一点是关键的，也应当定期检查垫圈，确保其完整，避免微生物的进入。手套和半身衣是另一个可能产生污染的来 源。因为操作者戴着手套处理无菌检验物品，所以手套是应当特别关注的， 手套上微小的破损很难检查出来，手套在使用时，在拉伸情况下，手套上 微小的破损可以体现出来（注意：培养基接触法检测手指菌落的方法，在 检测低水平的污染时灵敏度是不足的。用 0.1%的蛋白胨水溶液浸泡手套， 将溶液过滤，然后将滤膜放在生长培养基碟子上检测，这个方法可以检测 出其他方法检测不出的泄露。）

隔离器内部进行连续的尘埃粒子检测是理想的，这样可以快速检测到 过滤器的泄露。第二个选择是使用便携式的尘埃粒子检测器进行周期检测。 尘埃粒子检测取样不能对隔离器内部的无菌环境产生风险。

INTERPRETATION OF STERILITY TEST RESULTS

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 gloves, sleeves, and half-suits are free of leaks; and if the RTPs are functioning properly. 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.

如果人员没有直接接触工作区，验证过的传输通道没有破损，隔离器内 部微生物被去除并有一个高的无菌保证水平，那么，无菌检验中的假阳性 是很难出现的。然而，隔离器是一种机械装置，好的无菌操作技术仍然是 必须的。只有充分地符合“无菌检验检测和调查《71》的情况下，才能下 阳性结果无效这个结论。

TRAINING AND SAFETY

As with sterility testing conducted in conventional clean rooms, operators are trained in procedures that are specific to their isolator. Use of proper aseptic techniques is vital to the conduct of sterility tests in isolators, just as it is in clean rooms. Therefore, training in proper aseptic techniques is required for all sterility testing technicians. 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 decontaminating agent must be assessed. Material Safety Data Sheets, or equivalent documents, are available in the immediate area where the decontaminating 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.

因为无菌检验操作在洁净室进行，操作者针对隔离器专门程序进行培 训。所有的培训记录和操作者操作的评估应有一个单独的记录并且存档。 对所有进行操作或者维护隔离器的人员进行必要的安全培训是必须的。

人员使用杀菌剂的安全性必须被评估。使用杀菌剂的直接区原材料的安全数据或者一个类似安全数据文件是必要的。贮藏和安全方面的预防措施 应当符合规定。在隔离器使用之前，隔离器和所有连接的通道安全检查应 当被执行。

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