在线灭菌（SIP）相关法规与执行要点

背景

最近的GMP检查缺陷：

最近发现很多药厂关于SIP（Steam In Place：在线灭菌）方面的缺陷，这些缺陷主要由于对科学技术的不了解导致。

1、SIP工艺管道坡度不恰当（辉瑞HOSPIRA），检查机构：英国药监局

2、药液除菌过滤系统除菌过滤器采用离线方式灭菌后安装，未采取单向流保护措施，未对循环除菌过滤时的最大过滤容量进行验证确认（安徽艾珂尔制药有限公司）。检查机构：国家食品药品监督管理总局食品药品审核查验中心。

对于无菌产品的生产， SIP 经常是无菌处理的关键环节。无菌产品的生产过程中，对药液管道，储罐，呼吸过滤器，无菌气体过滤器、阀门等进行SIP是十分重要的。值得注意的是，用于对无菌生产部件、无菌药品内包材（如胶塞）灭菌的灭菌柜，其安装的除菌级过滤器或呼吸阀也是需要进行在线灭菌的。离线灭菌无法避免他们在完成灭菌后，在安装过程中导致新的微生物污染。现在，新的冻干机、灭菌柜、胶塞清洗灭菌设备也已具备在线灭菌功能。

以下是美国注射剂协会PDA技术报告中关于在线灭菌设计和验证方面的指南，供大家参考。

SIP程序设计的考虑点

Consider the following points:

要考虑以下几点：

The SIP cycle must provide delivery and penetration of saturated steam at predefined temperatures to all internal surfaces. This requirement incorporates design aspects associated with heat transfer, steam supply, and air and condensate removal.

SIP 循环必须在预先定义的温度下对所有的内表面提供饱和蒸汽的交换和穿透。这一需求由热传递、蒸汽供应、以及空气和冷凝水的去除各相关方面的设计组成。
The system should be designed to ensure adequate heat transfer to the system so the heat delivered by steam to the system’s internal surfaces (conduction) is greater than the heat lost from the system to the environment (convection). Steam system delivery capacity must match or exceed the system requirement for attaining and maintaining sterilization conditions.

系统设计应使系统得到足够的热交换以确保蒸汽传递到系统内表面的热量（热传导）大于系统传递到外界环境的热损失（热对流）。蒸汽系统的输送能力必须符合或超过系统实现及保持灭菌条件的需求。
The system should be designed so air and condensate is not trapped in any location where steam is intended to penetrate. Saturated steam must be in contact with the targeted surface for the duration of the exposure phase, or the efficacy of the cycle will be compromised. Ideally, piping systems and vessels are designed so air and condensate is easily purged out of areas such as deadlegs and filter housings through bleed valves or steam traps. Manually operated SIP cycles will need detailed instructions within procedures to ensure this is accomplished each time in a repeatable manner.

系统设计应使空气和冷凝水不会在任何需要进行蒸汽渗透的位置累积。饱和蒸汽必须在接触阶段期间与目标表面持续接触，否则灭菌的效果会受到影响。理想情况下，管道系统和容器应被设计为空气和冷凝水在一些区域，如管道分支和过滤器等处通过排气阀和疏水阀可以很容易的去除。人工操作的 SIP 循环需要详细的程序指令，以确保每次的操作具备可重现性。
The SIP cycle must provide measurement, control, and monitoring of system temperature and pressure during the cycle hot phases (heat-up and exposure), and of pressure during the cool-down and hold phases. To enhance condensate removal, pipe slopes should be maximized wherever possible. A typical piping design specification would reference a minimum slope of a 1/8” per foot of pipe.

SIP 程序必须提供测量、控制、供热阶段（升温和灭菌）系统温度和压力的监测，以及冷却和保持阶段期间压力的监测。为了加强去除冷凝水，管路的坡度应尽可能最大化。典型的管路设计参考参数为每英尺管路最小坡度为 1/8”。
Appropriate instrumentation, controls, and monitoring systems must be installed to enable and ensure the delivery and control of saturated steam.

必须安装适当的仪表、控制和检测系统以确保能够供应和控制饱和蒸汽。
SIP cycle design should ensure that temperature and/or pressure limitations of the process are not exceeded (such as for filters or elastomers).

SIP 循环的设计应确保温度和/或压力不超过工艺限度（例如对于过滤器或弹性部件）
Access points should be provided for insertion of validation biological indicators and temperature sensors at potential worst-case locations (e.g. low points, high points, filter housings).

应在潜在的最差位置为生物指示剂和温度传感器提供验证接口。（例如：低点、高点、过滤器外壳。）

Consideration should be given to steam supply load to ensure adequate capacity for all uses (e.g., SIP, autoclave, and humidification).

应考虑蒸汽供应的负载以确保有足够的能力供应所有使用点（例如:SIP、高压灭菌柜、加湿）。
Good engineering practices for steam system design must be followed when selecting the pressure reduction valve. Pressure control should be sufficiently robust to minimize fluctuations in steam pressure delivered to the target SIP system(s).

当选择减压阀时必须遵循蒸汽系统设计的良好工程规范（ GEP）。应有足够的压力控制能力使蒸汽压力传递到目标 SIP 系统时波动最小化。

SIP程序验证要点

Performance qualification may include the following:

性能确认包括以下内容：

Physical qualification: will inc lude temperature mapping runs to confirm that the temperature range requirements are met and that the minimum F0, or time and temperature, is consistently achieved in the system.

物理确认：将包括温度测绘以确认符合温度范围要求,以及最小 F0、时间或温度在系统中一致性实现。
Biological qualification: conducted with appropriate microbiological challenges to confirm that the minimum FBIO is consistently achieved in the system (24).

生物确认：用适当的微生物挑战实验以确认最小 FBio 始终在系统中一致性实现
Bioburden control or sterile hold time studies onducted as required to demonstrate that the sanitized or sterilized system can be maintained in that condition for the desired length of time before use.

按要求进行的生物负载控制或无菌保存时间研究表明消毒或灭菌系统可在使用前在所需条件保持期望的时间
Documented assessment and rationale for the selection of locations for the following:

经书面评估和原理阐述对以下位置进行选择：

Permanent temperature monitoring locations

永久性温度监测点
Validation temperature monitoring locations

验证温度监测点
Biological indicator locations

生物指示剂点

Measurements of time, temperature, and pressure may be sufficient for SIP sanitization qualification. For SIP sterilization qualification, measurements of time, temperature, pressure, and biological indicator destruction are required to demonstrate consistency between physical and microbiological results. A safety margin of higher temperatures and/or extended exposure times may be built in for routine operational cycles to account for process, biological, and instrument variability. Temperature and time considerations should be included for heat labile items (e.g., filters, gaskets, tubing).

测量的时间,温度和压力需满足在线灭菌的消毒确认。对于在线灭菌的灭菌确认，测量的时间、温度、压力和生物指示剂的破坏需要证明物理和微生物结果之间的一致性。建立高温和/或延长的曝露时间的安全临界范围用于常规操作周期来说明工艺的、生物的和仪器的可变性。温度和时间应该包括对热不稳定的项目(如：过滤器、垫圈、管道)。

Multiple runs (typically 3) should be conducted to confirm reproducibility of the steam in place process for initial qualification.

多次运行(通常 3 次)应在初始确认时确认过程中蒸汽的重现性。

The following activities should be completed and documented in accordance with company policy and current regulatory expectations prior to performance qualification:

以下活动应优先于性能确认按照公司政策和现行法规预期完成并以文件形式记录:

• Procedures for the operation of the system(s) being qualified

系统(s)操作规程是合格的

• Qualification of utilities as needed for the steam in place process (e.g., steam and compressed gases with

appropriate quality and capacity testing)

公用工程的确认同样需要工艺中的蒸汽(如具有合适质量与容量测试的蒸汽与压缩空气)

• Qualification of the system (e.g., design qualification, commissioning and/or installation qualification, and operational qualification) and calibration of critical instrumentation (e.g., control systems, monitoring devices, and alarms)

系统确认（例如，设计确认，调试和安装确认，运行确认）以及关键仪器的校准（例如，控制系统，监控设备，和报警设备）

• Development of the SIP cycle parameters, including preliminary temperature mapping

在线灭菌循环参数的制定，包括初始温度测绘

• Training of personnel involved in performance qualification

包含在性能确认中的人员培训

Physical Qualification物理确认

The primary objective of the physical qualification component of SIP qualification is to obtain physical data confirming that the developed cycle consistently delivers the desired minimum lethality throughout the SIP system. The minimum lethality depends on the kind of SIP cycle used and on the desired degree of sanitization or sterility assurance.

在线灭菌确认中的物理确认部分的主要目标是获取物理数据以确认开发周期持续在在线灭菌系统中提供所需的最低杀伤力。最低杀伤力取决于在线灭菌的循环使用种类和期望的消毒灭菌保证。

Sanitization vs. Sterilization

消毒和灭菌

The extent of qualification depends on the intent of the SIP cycle. SIP performed for the purpose of system sanitization may only require physical qualification. For systems that are sanitized, bioburden testing (via applicable sampling methods such as rinse water samples and swab sampling) should be performed as part of the physical qualification of the SIP process as applicable.

确认的程度取决于在线灭菌的周期的目的。若在线灭菌操作只为进行系统消毒，那么只需物理确认。对于需要进行消毒和生物负载监测(通过适用的取样方法如冲淋水取样和擦拭取样)的系统，物理确认是在线灭菌过程中的一部分。

SIP performed for the purpose of system sterilization will require both physical and biological qualification. Physical and biological qualification should be performed simultaneously. The destruction of a biological challenge alone is not sufficient evidence of the suitability of a cycle. The biological challenge data should support the physical data and vice versa.

若在线灭菌的目的是进行系统灭菌，则同时需要物理确认和生物确认。物理确认和生物确认应同时进行。仅有生物挑战并不足以证明循环的适用性。生物挑战数据应当支持的物理数据,反之亦然。

Initial qualification runs are typically performed in triplicate to ensure consistency. Fewer runs may be performed if a technical rationale is provided.

初始确认通常运行三次,以确保一致性。如果有技术依据支持，可进行更少的运行次数.

Temperature Mapping

温度分布

The primary purpose of temperature mapping is to verify steam distribution throughout the system. Significant variation of temperatures during this study could indicate the presence of air or condensate at an insuffic ient temperature.

温度分布的主要目的是验证整个系统的蒸汽分配。在此项研究中充足的温度变化可能表明在温度不足时有空气或冷凝水存在。

Temperature probes should be positioned in the cold spots identified during cycle development. The number and location of the temperature sensors depend on the size, layout, and complexity of the SIP system (e.g., a simple SIP system, such as a transfer line, may only have temperature mapping probes adjacent to the controlling temperature device). Placement of the temperature probes and rationale should be documented.

温度探头在冷点的位置在周期开发中确定。温度传感器的数量和位置取决于 SIP 系统大小,布局,和复杂性 (如,一个简单的 SIP 系统,如输送管线,可能只有温度探头邻近温度控制设备)。温度探头的布置位置和原理应以文件形式记录。

Probe insertion should be performed in a manner that does not impact the SIP process or impair any of the safety equipment/features of the process. For example, care should be taken to ensure that introduction of the probes into the system does not hinder steam flow or removal of condensate nor facilitate air removal. Typical temperature probe locations for an SIP system include:

探头的插入方式不应影响 SIP 流程或损害任过程中的任何安全设备或安全性能。例如,应该小心以确保探头的插入不妨碍蒸汽流或阻碍冷凝水或空气的去除。 SIP 系统中典型的温度探头位置包括:

• Downstream and/or within filter core

下游和/或内部的过滤器

• Possible condensate accumulation locations (e.g., low points and the upstream side of filter housings)

冷凝水可能聚集的位置。 (例如,低点和过滤器外壳上游侧

• Upstream of steam traps

上游的蒸汽疏水阀

• Nozzles and high points where air may be difficult to displace

喷嘴和高点,这些位置空气可能难以置换

• Surfaces of large mass items (e.g., lyophilization shelves)

大体积物体表面（如冻干机装架）

• Surfaces of uninsulated portions of the system being steamed

蒸汽经过的不保温部分的表面

• System boundaries

系统的边缘

• Deadlegs (condensate/air entrapment)

盲管（冷凝水/空气冷井）

• Adjacent to temperature and/or pressure instruments

邻近温度或压力设备的位置

Care should be taken to ensure that the measurements accurately represent the system being measured when installing thermocouples. One consideration is that probe tips should be oriented to avoid erroneous measurements due to condensate droplet accumulation at the tip of the probe. Figure above shows two adjacent probes with different orientations. The figure shows that condensate does not accumulate on the tip of TC 1 but does accumulate on the tip of TC 2. This insulates the probe and affects the temperature readings. Figure below depicts data resulting from installing thermocouples oriented as illustrated in Figure above.

应注意确保测量值准确反映在安装热电偶时系统中被测到的情况。其中一个考虑因素是,应调整探头的位置以避免由于冷凝水在探头的聚集而导致的错误测量。上图显示两个不同位置的相邻探针。表明,冷凝水不聚集在探头 TC1 上但是聚集在探头 TC2 上。隔离探头发现影响温度读数。下图描述了上图所示安装热电偶时的数据结果。

Depending on the system being steamed and the intent of the SIP cycle, the study may require that the temperature locations reach the minimum desired temperature at the end of the heat-up phase. The temperature achieved at the

end of the heat-up phase, as measured by a permanent probe, may be used as a routine check (automated or manual) to confirm satisfactory heat up for routine production cycles.

根据产生蒸汽的系统和 SIP 循环的目的,这项研究要求在升温阶段结束时温度位点达到所需的最低温度。在升温阶段结束时,可用一个永久的探头作为日常检查(自动或手动)来确认日常生产周期的令人满意的升温效果。

Temperature data should also be collected from permanent temperature probes that are designed into the system to control and/or monitor temperature during the SIP process. This data should be compared to the data from the temperature probes installed during validation to provide a link between the routine production monitoring and the validation study.

温度数据也应收集从永久温度探针设计到系统的控制和/或监测 SIP 过程中温度。这个数据应该比温度探测器安装在验证的数据提供一个联系日常生产监控和验证研究。

Biological Qualification生物学确认

The objective of the biological component of cycle qualification is to obtain microbiological data confirming that the developed cycle achieves lethality requirements established during cycle design. Not all SIP systems require biological qualification. If sterility of the SIP system is not claimed, biological qualification may not be required.

灭菌确认的生物学部分的目的是获得微生物数据来确认灭菌方法达到了设计时确立的致死率要求。不是所有 SIP 系统都需要生物学确认。如果 SIP 不要求无菌性，则不要求生物学确认。

Microbial Challenge

微生物挑战

In order to assess whether the cycle delivers sufficient lethality to meet design requirements, an appropriate microbiological challenge should be selected to give meaningful results. The microbiological challenge system should have a resistance and challenge level appropriate for its purpose. The biological qualification data is used to calculate the FBIO for the cycle.

为了评估灭菌过程实现的致死率满足设计要求，需要采用合适的微生物挑战来给出有意义的结果。微生物挑战系统应该具有适合其目的的抵抗性和挑战性。生物学确认数据用于计算灭菌过程的 FBIO。

Each microbiological challenge system is individually incubated in appropriate media and conditions for growth of survivors. Directions for use, including data about conditions to be used for recovery of test organisms after exposure to the sterilization process should be obtained from the Biological Indicator manufacturer. The length of time that the exposed Biological Indicator is held before incubation should be validated.

每个挑战系统但与培养在适合存活微生物生长的培养基和条件下。需要生物指示剂供应商提供使用说明，包括有关经过灭菌工艺的测试微生物的回收的条件的数据。需要验证培养前灭菌后的生物指示剂的存放时间。

Growth of the microbiological challenge organism is required in positive controls.

微生物挑战用的微生物的生长要有阳性对照。

Use and Placement of Biological Indicators

生物指示剂的使用和替换

Biological indicators are typically obtained from commercial sources. The BI challenge system is typically spores of Geobacillus stearothermophilus; however, other certified BIs may be used. The use of the semi-logarithmic model to determine the inactivation characteristics of the BI challenge system may also be used to calculate the appropriate challenge to biologically qualify a cycle, regardless of the resistance of the challenge organism selected.

生物指示剂一般都是购买的。生物指示剂挑战系统一般是嗜热脂肪芽孢杆菌的孢子，然而也可用其它合格的生物指示剂。用于确定生物指示剂挑战系统失活性质的半对数模型也可用于生物学角度确认灭菌周期，不考虑选择的菌种。

There are several types of biological indicator challenge systems. The different types of BI systems appropriate for SIP validation are discussed in Table below. The table gives the description of the different BI types and the pros and cons of using them to qualify an SIP cycle.

有几种 BI 挑战系统。下表讨论了适合 SIP 验证的集中 BI。表格中给出了 BI 种类的描述，和使用它们来确认 SIP 灭菌的优缺点。

BI Type

BI 类型

BI Descrip tion

BI 描述

Pros

优点

Cons

缺点

Spore

Suspension

孢子混悬液

Suspension of spores ofknown D-value, population, and z-value inoculated onto an item or coupon

已知 D 值，浓度， Z 值的孢子混悬液接种到条上或器具上

• Allows direct inoculation of components (e.g., filters,

tubing) being sterilized

可以直接接种到被灭菌部件上（例如滤芯、管道）

• Does not obstruct steam, air or

condensate flow

不会阻止蒸汽、空气或者冷凝物流动

• Inoculation and recovery method

more difficult

接种和回收率方法比较难

• Surviving spores can cause contamination

存活的孢子可能导致污染

• D-value needs to be measured

with the coupon or item

D 值需要协同条或物件计算

• Introduction of open spore suspensions into a manufacturing facility may present significant regulatory, logistical or product

safety issues

开放的孢子混悬液接种到生产厂房设施上可能导致严重的法规、逻辑或产品安全问题

Self-contained BI*

自含式生物指示剂

Growth medium contained inside the primary packaging for the indicator

指示剂内包材里面有培养基

• Convenient packaging

包装方便

• Eliminates aseptic manipulation of the indicator strip, which can lead to indicator contamination(i.e., false positives)

省去了去除指示剂的无菌操作，这种操作可能引起指示剂污染（例如，假阳性）

• Simple recovery method

回收率方法简单

• Recovery method and D-value are typically supplied by vendor

供应商提供回收率方法和 D值

• Potentially eliminates exposure of the area to the spores

潜在的排除了洁净区暴露向孢子的可能指示剂

• Indicator is bulky and not suitable

for monitoring small diameter systems

比较大不适合检测小体积的系统

• Glass media container can break

when not anchored properly and

exposed to a turbulent steam flow

玻璃容器可能碎掉如果没有放好或者暴露在强烈的蒸汽流中

BI Carrier

BI 载体

Spores added on a carrier (e.g., stainless, paper,plastic, glass, wire) individually packaged to maintain Integrity

孢子添加在单个载体上（例如不锈钢，纸，塑料，玻璃，金属线）并包装好以保持完整

• Allows versatility in size and rigidity based on the selection of the carrier

根据选择载体而有多样硬度和尺寸

• Minimizes exposure of the area to the spores

减少洁净区暴露向孢子的可能

• Widely recognized and used for SIP sterilization validation

广泛认可并用于 SIP 灭菌验证

• Recovery method and D-value are typically supplied by vendor

回收率方法和 D值由供应商提供

• Depending on the selection of the

carrier, may not be suitable for small-diameter systems

取决于载体特点，可能不适用于

小体积系统

• In the case of non-packaged/ bare

BI ＇ s, the BI ＇ s should be aseptically handled.

对于无包装或裸露的 BI， BI 需要进行无菌操作。

Biological indicators should have direct contact with steam. Some BIs are in sealed ampoules that contain spore suspension in liquid media and therefore may not represent actual system conditions. Therefore, these types of indicators should not be used.

备注：生物指示剂需要直接与蒸汽接触。一些生物指示剂是含有孢子混悬液密封安瓶，因此不代表实际的系统条件。因此不能使用这类指示剂。

BI challenge systems are placed adjacent to temperature sensors in the cold spots/and hardest-tosterilize locations within the SIP system. For example, they may be located within cartridge filters, in nozzles, in the highest point in

the tanks, in deadlegs where it may be difficult for steam to access or in low point drain/condensate valves. For large filter housings (e.g., > 20 inches or > 1 cartridge), BIs may be placed in the top and bottom of the filter cartridge(s) to evaluate the areas within the housing that could have excessive trapped air and/or excessive condensate pooling. In addition, BIs should be placed in low locations, especially in distal lines where condensate flows to a drain or pools.

生物指示剂挑战系统放在灭菌系统最冷点/和最难灭菌位置的温度探头旁边。例如，他们可能安放在滤芯里、针头、罐子最高点，蒸汽难以达到的死角或者低的排水点/冷凝水阀门。对于大的滤壳（例如，＞20 英寸或者＞1 个滤壳），生物指示剂放在滤壳顶部或者底部来评价可能有多余空气或者/和过多冷凝物的位置。另外， BI 要放于低的位置，尤其是末端有冷凝物流向排水处或蓄水处。

Typical placement of thermocouples (TC) and biological indicators (BI) on a product filter system. TC and BI placement is intended to bracket both the upstream high point and the downstream low point in order to demonstrate air removal, steam penetration, and biological kill throughout the system. The TC and BI location (TC3/BI2) proximal to the controlling temperature element (TE) also demonstrates fitness of that element as a process indicator.

一般放置在产品滤芯系统中的热电偶和生物指示剂的位置。 TC和BI放置位置要包括上游蒸汽的高点和下游蒸汽的低点来证明气体移出，蒸汽的穿透和生物灭菌。 TC和BI的位置接近控制温度也证明作为指示剂的适用性。

To evaluate the correlation between FPHYS and FBIO, biological indicators should be placed near temperature probes. Biological indicators, TCs, and probes should not block the steam path nor hinder the removal of condensate. Materials used for placement or attachment of the biological indicators inside the system should not hinder the inactivation of the BI or operation of steam traps and sanitary valves. Attachment method must be robust enough to prevent the BI being carried away with the turbulent flow and condensate. For paper strip BIs it is recommended that they be held in place with wire mesh or other suitable method to allow steam penetration while containing the wet BI. Placement and location rationale of biological indicators should be documented.

为了评价FPHYS和FBIO的联系，生物指示剂应放置在温度探头附近。生物指示剂， TCs和探头不能阻止蒸汽流动或冷凝物的排出。用于生物指示剂放置或粘贴的材料不应妨碍生物指示剂的灭活或者疏水阀和洁净阀的操作。粘贴方式要足够结实以防止强烈气流和冷凝水把BI冲走。对于纸条生物指示剂，最好使用金属网或者其它合适方法固定，以便于蒸汽穿透，那时纸条会变湿。放置位置的合理性需要文件化说明。

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