1.1 本文件中概述的微生物测试方法适用于一次性使用系统（SUS）或其单个组件的微生物进入风险评估，该系统或其单个组件需要由组件供应商或组件的最终用户基于产品或制造过程违约的潜在风险进行完整性测试。 1.2 生物制药制造中使用的无菌SUS的微生物进入测试有两个目的: 1.2.1 首先，它用于评估SUS流体通道在受到微生物暴露挑战后保持无菌的能力。微生物暴露可以通过直接将SUS放置在微生物激发溶液的容器中，或通过将雾化微生物激发物输送到SUS上来实现，SUS放置在设计用于产生和输送气溶胶的测试室内。 应根据SU的风险评估和使用条件，证明测试挑战生物体的选择是合理的。 1.2.2 此外，微生物进入测试可用于确定在特定测试条件下不允许微生物进入的最大允许泄漏极限（MALL）。可以通过特定的物理完整性测试方法检测到的缺陷大小可以与该MALL相关，以便声称微生物完整性。可测试在一系列尺寸范围内含有校准缺陷的供试品，包括预期始终允许微生物进入作为阳性对照（基于缺陷的阳性对照）的缺陷大小，以确定MALL。 1.3 微生物进入测试的两个目的如所述 1.2.1 和 1.2.2 可以通过液体浸泡或气溶胶暴露进行。用于中所述目的 1.2.2 暴露类型应根据SUS的用例条件和风险评估确定。 1.4 用于在一次性使用的薄膜或SUS测试品中产生破裂、孔洞或缺陷的方法，以及用于物理表征缺陷尺寸的分析方法不在本文件的范围内。给定供试品的取样计划应根据取样量的基本原理进行证明，以获得具有统计意义的效果（实践 E3244 ). 确定适当数量的SUS供试品将取决于SUS的风险评估及其使用条件，也不在本文件的范围内。 1.5 以国际单位制表示的数值应视为标准值。本标准不包括其他计量单位。 1.6 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的用户有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.7 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认标准化原则制定的。 ====意义和用途====== 4.1 用于生物制药制造的一次性使用系统（SUSs）必须保持内部液体的无菌和产品质量。因此，应验证此类物品或系统是否能有效防止微生物进入。可以使用与微生物完整性相关的确定性物理测试来证明SUS的微生物屏障特性。描述了两种测试方法（气溶胶暴露和浸没暴露），可用于证明SUS的微生物完整性或确定MALL（不允许微生物进入SUS的最大缺陷尺寸）。 4.2 需要注意的是，微生物侵入试验的结果在很大程度上取决于进行试验的条件，并且由于试验的破坏性，不适合对SUS进行常规检查。 4.2.1 在通常不反映正常使用条件的足够侵蚀性条件下（例如，包括足够大的样品尺寸、高差压或高静水压），任何尺寸缺陷都可能被迫失效。因此，有必要通过对实际SUS索赔及其最终使用（实践）的风险评估，明确定义测试的相关条件 E3244 ). 一旦确定，可以使用定义的缺陷确定在这些条件下可以检测到的缺陷大小（如果需要）。 4.2.2 “相关条件”是指最坏的实际使用条件，但并不意味着必须在理论绝对（极端）“最坏”条件下测试SUS- 案例”条件。 4.2.3 测试可以在单个组件或整个系统上进行。定义“相关条件”和测试设计的考虑因素应基于SUS预期用途的风险评估，并应包括: 4.2.3.1 通过薄膜厚度或接缝或连接的缺陷或裂口形成的通道，必须填充液体以允许微生物通过。 5. , 6. 4.2.3.2 可能影响通道液体填充的因素，包括液体的粘度、缺陷尺寸和类型、塑料材料和施加在SUS内的压力。 4.2.3.3 选择缺陷类型的理由应基于SUS生命周期内可能出现的缺陷类型 4.2.3.4 选择缺陷尺寸的基本原理应基于确定性物理测试方法（检测极限） 4.2.3.5 考虑测试期间施加的压差，以模拟SUS在实际使用条件下可能受到的条件（实践 E3244 ). 4.3 激发微生物和最低目标激发浓度的选择应基于风险评估，必要时应证明并验证检测限。至少10 6. CFU/cm 2. 表面积（气溶胶）或10 6. 通常使用CFU/mL（液体浸泡）（ISO 15747和Aliaskarisohi 7. ). 4.4 可以生产和测试带有校准缺陷的SUS测试品，以确定在给定条件下（例如，微生物进入）可通过微生物测试方法检测到的最小缺陷尺寸，或确定使用中的SUS数量- 案例条件（例如，气溶胶测试）。 4.4.1 如果测试目标是确定MALL并证明物理完整性测试灵敏度和微生物入侵之间的相关性，则应根据SUS生命周期内可能出现的最可能的缺陷类型选择人工缺陷（激光钻孔、毛细管、铜线）。 4.4.2 缺陷尺寸的选择应基于在SUS的预期用例条件下从进入到无进入的预期过渡，或者，可以选择最坏的情况。如实践中所述 E3244 ，典型范围为1µm至100µm。应通过定义的方法校准缺陷尺寸。 4.4.3 确定SUS薄膜材料最大厚度的一种方法是在支架中测试具有校准缺陷的一次性薄膜试样。这可以实现更高的吞吐量测试；然而，使用试样作为测试品可能并不代表整个SUS的缩小模型。 4.4.4 另一种方法是在替代容器（如小瓶）上验证测试方法。原则不变。替代容器必须能够容纳最小尺寸的缺陷。 4.5 这些程序应由经过培训的人员在微生物实验室中进行。假设用于执行常规微生物操作的基本微生物设备和用品（例如，标准平板计数、高压灭菌器灭菌等）。 )都可用。

1.1 The microbial test method outlined in this document applies to microbial ingress risk assessment of a single-use system (SUS) or its individual components that require integrity testing either by the assembly supplier or the end user of the assembly based on a potential risk of a breach to the product or manufacturing process. 1.2 The aim of microbial ingress testing of sterile SUSs used in biopharmaceutical manufacturing is two-fold: 1.2.1 Firstly, it is used to evaluate the ability of a SUS fluid path to remain sterile after a SUS has been challenged by microbial exposure. Microbial exposure is achieved either by directly placing a SUS into a container of microbial challenge solution, or by delivering an aerosolized microbial challenge onto a SUS that is placed inside a test chamber designed to generate and deliver the aerosol. The choice of the test challenge organism should be justified based on a risk assessment of the SUS and conditions of use. 1.2.2 Additionally, microbial ingress testing can be used to determine the maximum allowable leakage limit (MALL) that does not allow microbial ingress under specific test conditions. The defect size that can be detected by specific physical integrity testing methods can be correlated to this MALL in order to claim microbial integrity. Test articles bearing calibrated defects over a range of dimensions, including up to a defect size expected to consistently allow microbial ingress as a positive control (defect-based positive control), may be tested to determine the MALL. 1.3 Both purposes for microbial ingress testing as described in 1.2.1 and 1.2.2 can either be conducted by liquid immersion or aerosol exposure. For the purpose described in 1.2.2 , the type of exposure should be determined according to the SUS’s use-case conditions and a risk assessment. 1.4 The method used to create a breach, hole or defect in single-use film or in a SUS test article, as well as the analytical method used to physically characterize the defect size is outside of the scope of this document. The sampling plan for a given test article should be justified with the rationale of sampling size to obtain a statistically meaningful effect (Practice E3244 ). Determining the appropriate number of SUS test articles will depend on a risk assessment of the SUS and the conditions of its use and is also outside of this document’s scope. 1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 4.1 Single-use systems (SUSs) used for biopharmaceutical manufacturing must maintain sterility and product quality of the fluid inside. Such articles or systems should therefore be validated as providing an effective barrier against microbial ingress. The microbial barrier properties of a SUS may be demonstrated using deterministic physical tests that have been correlated to microbial integrity. Two test methods (aerosol exposure and immersion exposure) are described that can be used to demonstrate microbial integrity of a SUS or determine the MALL, the maximum defect size that does not allow microbial ingress, into a SUS. 4.2 It is important to note that the results of microbial ingress tests are heavily dependent on the conditions under which the test is performed and are not suitable for routine checking of a SUS due to the test’s destructive nature. 4.2.1 Any size defect may be forced to fail under sufficiently aggressive conditions (including a large enough sample size, high differential pressure, or high hydrostatic pressure, for example) that would not ordinarily reflect normal use conditions. Thus, it is necessary to clearly define the relevant conditions for a test through a risk assessment of both the actual SUS claims and its final use (Practice E3244 ). Once that is established, the size of defect that can be detected under those conditions can be determined, if required, using defined defects. 4.2.2 “Relevant conditions” refers to worse-case actual use conditions but does not mean that a SUS must be tested under theoretically absolute (extreme) “worst-case” conditions. 4.2.3 Testing may be performed on individual components or entire systems. Considerations for defining “relevant conditions” and testing design should be based on a risk assessment for the SUS intended use and should include: 4.2.3.1 A channel created by a defect or breach through the film thickness or through a seam or connection which must be filled with liquid to allow microbial passage. 5 , 6 4.2.3.2 Factors that could influence liquid filling of a channel, including a liquid’s viscosity, defect size and type, plastic materials and pressure applied inside the SUS. 4.2.3.3 Rationale for selecting a defect type should be based on the probable type of defect(s) that could occur during the SUS life cycle 4.2.3.4 Rationale for selection of defect sizes should be based on a deterministic physical testing method (detection limit) 4.2.3.5 Consideration of pressure(s) differential applied during testing to simulate conditions that a SUS may be subjected to during actual use conditions (Practice E3244 ). 4.3 The selection of challenge microorganism and minimum target challenge concentration should be based on a risk assessment, justified, and validated, as necessary, for the limit of detection. A minimum of 10 6 CFU/cm 2 surface area (aerosol) or 10 6 CFU/mL (liquid immersion) is typically used (ISO 15747 and Aliaskarisohi 7 ). 4.4 SUS test articles bearing calibrated defects may be produced and tested to allow either the determination of the minimum defect size that can be detected by a microbial test method under given conditions (for example, microbial ingress) or to determine the MALL of SUSs under use-case conditions (for example, aerosol test). 4.4.1 If the test objective is to determine the MALL and demonstrate correlation between physical integrity test sensitivity and microbial ingress, selection of the artificial defect (laser-drilled hole, capillary, copper wire) should be based on the most probable type of defect that could occur during the SUS’s life cycle. 4.4.2 The selection of defect sizes should be based on the expected transition from ingress to no ingress under the SUS’s intended use-case conditions, alternatively, worst-case conditions can be selected. As described in the Practice E3244 , a typical range is from 1 µm to 100 µm. The defect sizes should be calibrated by a defined method. 4.4.3 One approach for determining the MALL of a SUS film material is to test single-use film coupons with calibrated defects, in holders. This enables higher throughput testing; however, using coupons as test articles may not represent a scale-down model of an entire SUS. 4.4.4 Another approach is to validate the test method on alternative container-like vials. The principle remains the same. The alternative container must be able to hold the minimum size defect. 4.5 These procedures should be conducted in a microbiological laboratory by trained personnel. It is assumed that basic microbiological equipment and supplies for conducting routine microbiological manipulations (for example, standard plate counts, autoclave sterilization, etc.) are available.