主题：【求助】无菌保证水平如何验证

对于使用灭菌器的，一般是验证灭菌器的物理F0与生物F0，即温度分布和微生物挑战。如果是过滤，则微生物挑战。

原文由 lingzhong 发表:
对于使用灭菌器的，一般是验证灭菌器的物理F0与生物F0，即温度分布和微生物挑战。如果是过滤，则微生物挑战。

好像有些人说F0和无菌保证水平没有必然的相关性，F0只是无菌保证的一个方面，一个条件，但达到F0并不一定就能达到所说的无菌保证水平。我对这个不是很了解，我是想知道如何验证我们的产品达到了无菌保证水平（如所说的10^-6），具体是怎么做的，国内是否可以做，国家有关部门对此是如何考核的！依据是什么？谢谢!

我暂时没做过这个，但我个人理解：

1.产品灭菌前要检验其微生物状况；
2.中间体也要检测；
3.成品还要检测；
4.F0是其必须的一项，而对于整个生产工艺也要进行验证。整个生产工艺当然首先包括所用设备的验证，其次是针对该产品、该方法、该流程、该工艺的一个验证。而这个工艺验证合格，则完全能证明该产品是符合标准的啊。

The basic principles for validation and certification of a sterilizing process are enumerated as follows:
1.    Establish that the process equipment has capability of operating within the required parameters.
2.    Demonstrate that the critical control equipment and instrumentation are capable of operating within the prescribed parameters for the process equipment.
3.    Perform replicate cycles representing the required operational range of the equipment and employing actual or simulated product. Demonstrate that the processes have been carried out within the prescribed protocol limits and finally that the probability of microbial survival in the replicate processes completed is not greater than the prescribed limits.
4.    Monitor the validated process during routine operation. Periodically as needed, requalify and recertify the equipment.
5.    Complete the protocols, and document steps (1) through (4) above.
The principles and implementation of a program to validate an aseptic processing procedure are similar to the validation of a sterilization process. In aseptic processing, the components of the final dosage form are sterilized separately and the finished article is assembled in an aseptic manner.
Proper validation of the sterilization process or the aseptic process requires a high level of knowledge of the field of sterilization and clean room technology. In order to comply with currently acceptable and achievable limits in sterilization parameters, it is necessary to employ appropriate instrumentation and equipment to control the critical parameters such as temperature and time, humidity, and sterilizing gas concentration, or absorbed radiation. An important aspect of the validation program in many sterilization procedures involves the employment of biological indicators (see Biological Indicators for Sterilization  1035 ). The validated and certified process should be revalidated periodically; however, the revalidation program need not necessarily be as extensive as the original program.
A typical validation program, as outlined below, is one designed for the steam autoclave, but the principles are applicable to the other sterilization procedures discussed in this informational chapter. The program comprises several stages.
The installation qualification stage is intended to establish that controls and other instrumentation are properly designed and calibrated. Documentation should be on file demonstrating the quality of the required utilities such as steam, water, and air. The operational qualification stage is intended to confirm that the empty chamber functions within the parameters of temperature at all of the key chamber locations prescribed in the protocol. It is usually appropriate to develop heat profile records, i.e., simultaneous temperatures in the chamber employing multiple temperature-sensing devices. A typical acceptable range of temperature in the empty chamber is ±1  when the chamber temperature is not less than 121 . The confirmatory stage of the validation program is the actual sterilization of materials or articles. This determination requires the employment of temperature-sensing devices inserted into samples of the articles, as well as either samples of the articles to which appropriate concentrations of suitable test microorganisms have been added, or separate BIs in operationally fully loaded autoclave configurations. The effectiveness of heat delivery or penetration into the actual articles and the time of the exposure are the two main factors that determine the lethality of the sterilization process. The final stage of the validation program requires the documentation of the supporting data developed in executing the program.
It is generally accepted that terminally sterilized injectable articles or critical devices purporting to be sterile, when processed in the autoclave, attain a 10–6 microbial survivor probability, i.e., assurance of less than 1 chance in 1 million that viable microorganisms are present in the sterilized article or dosage form. With heat-stable articles, the approach often is to considerably exceed the critical time necessary to achieve the 10–6 microbial survivor probability (overkill). However, with an article where extensive heat exposure may have a damaging effect, it may not be feasible to employ this overkill approach. In this latter instance, the development of the sterilization cycle depends heavily on knowledge of the microbial burden of the product, based on examination, over a suitable time period, of a substantial number of lots of the presterilized product.
The D value is the time (in minutes) required to reduce the microbial population by 90% or 1 log cycle (i.e., to a surviving fraction of 1/10), at a specific temperature. Therefore, where the D value of a BI preparation of, for example, Bacillus stearothermophilus spores is 1.5 minutes under the total process parameters, e.g., at 121 , if it is treated for 12 minutes under the same conditions, it can be stated that the lethality input is 8D. The effect of applying this input to the product would depend on the initial microbial burden. Assuming that its resistance to sterilization is equivalent to that of the BI, if the microbial burden of the product in question is 102 microorganisms, a lethality input of 2D yields a microbial burden of 1 (10  theoretical), and a further 6D yields a calculated microbial survivor probability of 10–6. (Under the same conditions, a lethality input of 12D may be used in a typical “overkill” approach.) Generally, the survivor probability achieved for the article under the validated sterilization cycle is not completely correlated with what may occur with the BI. For valid use, therefore, it is essential that the resistance of the BI be greater than that of the natural microbial burden of the article sterilized. It is then appropriate to make a worst-case assumption and treat the microbial burden as though its heat resistance were equivalent to that of the BI, although it is not likely that the most resistant of a typical microbial burden isolates will demonstrate a heat resistance of the magnitude shown by this species, frequently employed as a BI for steam sterilization. In the above example, a 12-minute cycle is considered adequate for sterilization if the product had a microbial burden of 102 microorganisms. However, if the indicator originally had 106 microorganisms content, actually a 10–2 probability of survival could be expected; i.e., 1 in 100 BIs may yield positive results. This type of situation may be avoided by selection of the appropriate BI. Alternatively, high content indicators may be used on the basis of a predetermined acceptable count reduction.

谢谢您，您的理解是过程控制，这一点很重要
我的意思是，如果这些都做了验证，生产的产品是否可以直接判为达到无菌保证水平？因为我有一点不放心，设备达到了要求，过程检验也合格，产品是否合格？如何检验达到无菌保证水平？据我了解，溶液中的菌数很难到均一、稳定，过程检验能否正真说明问题？我觉得这只能作为一种控制手段，我们尽可能从过程控制，但最终产品质量必须有个报告，要给出这个报告，必须有可行有效有说服力和代表性的方法、方案、而这个方法、方案才是问题的关键，就如考试，有些题只给出答案，就能得分，有些题必须给出过程，如果只有答案，别人不知道我们是懵的还是照抄别人的，还是自己的真才实学？所以我觉得我们不仅要做好（质量是生产出来的），而且也是经得起检验的（有可行有效有说服力和代表性的方法、方案，有充分的检验数据），这样才能得到大家的认可，也就是用自己的结果说明自己的产品质量过硬，我想要的是如何给出这个结果，给出结果的方法为什么能代表我们所有的产品？

过程控制贯穿生产的始终。

我没有明白你所谓的过程检验是指什么？

我理解的是：如同其他制剂要进行三批验证一样，整个无菌工艺要进行验证，其具体批次、具体检验量应该有相关规定。当该验证合格后，日常检验也每批进行，这种情况下，当然要认定产品质量没有问题。现在你有疑虑的关键可能就是这个工艺验证的问题，这需要查找多方面的资料，而这个工艺验证方案的内容，我实在是没有发言权。但我前面说的，你里面都应该有，甚至你产品的包装也是要考虑进去的，即你产品包装的密封性、完整性。

而所谓的是否达到10^-6这个无菌水平，目前应该是拿生物指示剂进行验证，即用你的灭菌工艺处理后，其培养阴性即可，但这个东西哪种适合你用需要你进一步核实。

MANUFACTURE OF STERILE MEDICINAL PRODUCTS
无菌医药产品的生产
Principle 原则
The manufacture of sterile products is subject to special requirements in order to minimise risks of microbiological contamination, and of particulate and pyrogen contamination. Much depends on the skill, training and attitudes of the personnel involved. Quality Assurance is particularly important, and this type of manufacture must strictly follow carefully established and validated methods of preparation and procedure. Sole reliance for sterility or other quality aspects must not be placed on any terminal process or finished product test.
无菌药品的生产，必须符合一些特殊的要求，以防止微生物、微粒和热源的污染。这很大程度上要依赖工作人员的技术水平、培训和工作态度。在这方面质量保证显得特别重要，这种类型的生产，必须严格按照完善的和经过验证的生产方法和工作程序。仅靠产品的最终灭菌和某一方面的质量控制是不允许的。
Note:
This guidance does not lay down detailed methods for determining the microbiological and particulate cleanliness of air, surfaces etc. Reference should be made to other documents such as the EN/ISO Standards.
注：本规范没有详述测定空气、表面等微生物和微粒洁净度的详细方法，请参阅EN/ISO中相关标准。
General 一般要求
1. The manufacture of sterile products should be carried out in clean areas entry to which should be through airlocks for personnel and/or for equipment and materials. Clean areas should be maintained to an appropriate cleanliness standard and supplied with air which has passed through filters of an appropriate efficiency.
无菌产品的生产要在洁净区域内进行，进入这些区域内的人员、设备或原料，必须通过气闸室。洁净区必须保持一定的洁净级别，空气必须通过规定的过滤器。
2. The various operations of component preparation, product preparation and filling should be carried out in separate areas within the clean area. Manufacturing operations are divided into two categories; firstly those where the product is terminally sterilised, and secondly those which are conducted aseptically at some or all stages.
各种原料的准备、产品的准备和灌装，必须在洁净区的不同区域进行，生产操作分为两类，一是最终灭菌型，二是部分过程或全过程的无菌操作。
3. Clean areas for the manufacture of sterile products are classified according to the required characteristics of the environment. Each manufacturing operation requires an appropriate environmental cleanliness level in the operational state in order to minimise the risks of particulate or microbial contamination of the product or materials being handled.
In order to meet “in operation” conditions these areas should be designed to reach certain specified air-cleanliness levels in the “at rest” occupancy state. The “at-rest” state is the condition where the installation is installed and operating, complete with production equipment but with no operating personnel present. The “in operation” state is the condition where the installation is functioning in the defined operating mode with the specified number of personnel working.
The “in operation” and “at rest” states should be defined for each clean room or suite of clean rooms. For the manufacture of sterile medicinal products 4 grades can be distinguished.
无菌生产的洁净区，按照产品对环境的要求分级，每一步生产操作，在操作状态，对环境有相应的洁净级别的要求，以防止对所处理的材料或产品造成粉尘或微生物的污染。
为达到“动态”的条件，这些区域在设计上要达到“静态”的洁净标准。“静态”指设备已经安装并运行，生产设备就位但是没有操作人员在场。“动态”是指在设备正常运转状态下和有规定的工作人员在场的情况下。每个或每套房间都要分别进行“静态”和“动态”的确定。无菌产品的生产有4个环境级别：
Grade A : The local zone for high risk operations, e.g. filling zone, stopper bowls, open ampoules and vials, making aseptic connections. Normally such conditions are provided by a laminar air flow work station. Laminar air flow systems should provide a homogeneous air speed in a range of 0.36 – 0.54 m/s (guidance value) at the working position in open clean room applications.
The maintenance of laminarity should be demonstrated and validated.
A uni-directional air flow and lower velocities may be used in closed isolators and glove boxes.
A级：用于高风险的生产操作，如灌装区、加盖区、容器开口区、和进行无菌连接的地方。通常这种情况是带有层流罩的工作点。在开放的洁净区内的工作点上，层流罩应该能产生风速为0.36 – 0.54米/秒的均匀气流。层流罩的维护，必须有充分的证明和经过验证。
密封隔离箱和手套箱内，可采用单向低速气流。
Grade B : For aseptic preparation and filling, this is the background environment for the grade A zone.
B级：对于无菌制备和灌装，B级区域是A级区域的背景环境。
Grade C and D: Clean areas for carrying out less critical stages in the manufacture of sterile products.
C级和D级：无菌产品非关键生产步骤的洁净区。

Notes注:
a)    Particle measurement based on the use of a discrete airborne particle counter to measure the concentration of particles at designated sizes equal to or greater than the threshold stated. A continuous measurement system should be used for monitoring the concentration of particles in the grade A zone, and is recommended for the surrounding grade B areas. For routine testing the total sample volume should not be less than 1 m3 for grade A and B areas and preferably also in grade C areas.
尘埃粒子检查是用不连续尘埃粒子记数器测量一定量空气中等于或大于一定粒子大小的尘埃粒子的浓度。在A区必须有持续监控的装置，来检测该区域内尘埃粒子的浓度，建议在A区周围的B区也安装这样的装置。在A级和B级区常规的检查取样量，应不少于1 m3，在C级区的取样量最好也达到该要求。
b)    The particulate conditions given in the table for the “at rest” state should be achieved after a short “clean up” period of 15-20 minutes (guidance value) in an unmanned state after completion of operations. The particulate conditions for grade A “in operation” given in the table should be maintained in the zone immediately surrounding the product whenever the product or open container is exposed to the environment. It is accepted that it may not always be possible to demonstrate conformity with particulate standards at the point of fill when filling is in progress, due to the generation of particles or droplets from the product itself.
上表中的“静态”下的微粒情况，必须在操作完成后，“自净”运行15-20分钟后达到。当产品和开口容器接触环境时，在紧接产品的的周围区域，尘埃情况必须保持在A级“动态”标准。灌装时，在灌装点由于产品本身产生的微粒和液滴，有时达不到微粒标准是可以接受的。
c)    In order to reach the B, C and D air grades, the number of air changes should be related to the size of the room and the equipment and personnel present in the room. The air system should be provided with appropriate terminal filters such as HEPA for grades A, B and C.
为达到B、C、D级洁净，要结合房间的大小、设备和相关人员的情况确定换气次数。在A、B、C级洁净区，必须在空气系统中使用合适的终端过滤器，如HEPA。
d)    The guidance given for the maximum permitted number of particles in the "at rest" and “in operation” conditions correspond approximately to the cleanliness classes in the EN/ISO 14644-1 at a particle size of 0.5 µm.
对粒径为0.5μm的粒子，规范规定的“静态”和“动态”下允许的最多尘埃粒子数，与EN/ISO14644-1中洁净区的规定相当。
e)    These areas are expected to be completely free from particles of size greater than or equal to 5 µm. As it is impossible to demonstrate the absence of particles with any statistical significance the limits are set to 1 particle / m3. During the clean room qualification it should be shown that the areas can be maintained within the defined limits.
这些区域不允许有大于或等于5μm的粒子，由于不可能用任何统计学意义证明没有尘埃粒子，这里将限度定为1个/m3。在洁净房间的验证中，必须表明在规定的限度内。
f)    The requirements and limits will depend on the nature of the operations carried out.
要求和限度，将根据操作的性质而定。
Other characteristics such as temperature and relative humidity depend on the product and nature of the operations carried out. These parameters should not interfere with the defined cleanliness standard.
其他特征参数，如温度和相对湿度，必须根据操作的性质和相应的产品而定。这些参数必须不影响规定的洁净度。

4.  The areas should be monitored during operation, in order to control the particulate cleanliness of the various grades.
这些区域内必须有运行过程中的监控，以控制各个级别下的尘埃粒子洁净度。
5.  Where aseptic operations are performed monitoring should be frequent using methods such as settle plates, volumetric air and surface sampling (e.g. swabs and contact plates). Sampling methods used in operation should not interfere with zone protection. Results from monitoring should be considered when reviewing batch documentation for finished product release. Surfaces and personnel should be monitored after critical operations.
对无菌操作区，必须经常使用沉降皿、空气定量法和表面取样（棉签或接触皿）等方法进行监控。生产过程中所用的取样方法，必须不影响洁净区的保护，当审核最终产品放行的批记录时，也要考虑环境监控的结果。在关键操作后，要对人员和设施的表面进行监控。
Additional microbiological monitoring is also required outside production operations, e.g. after validation of systems, cleaning and sanitisation.
在非生产状态下，要进行微生物的监控，包括在系统的验证、清洁或消毒后。
6. Appropriate alert and action limits should be set for the results of particulate and microbiological monitoring. If these limits are exceeded operating procedures should prescribe corrective action.
对尘埃粒子和微生物的监控结果，要设置适当的警戒限度和措施限度。当超出这些限度时，操作规程应说明需要采取的措施。

Blow/fill/seal technology吹/灌/封技术
10. Blow/fill/seal units are purpose built machines in which, in one continuous operation, containers are formed from a thermoplastic granulate, filled and then sealed, all by the one automatic machine. Blow/fill/seal equipment used for aseptic production which is fitted with an effective grade A air shower may be installed in at least a grade C environment, provided that grade A/B clothing is used. The environment should comply with the viable and non viable limits at rest and the viable limit only when in operation. Blow/fill/seal equipment used for the production of products which are terminally sterilised should be installed in at least a grade D environment.
Because of this special technology particular attention should be paid to, at least the following: equipment design and qualification, validation and reproducibility of cleaning-in-place and sterilisation-in-place, background cleanroom environment in which the equipment is located, operator training and clothing, and interventions in the critical zone of the equipment including any aseptic assembly prior to the commencement of filling.
吹/灌/封单元都安装在一台专用的设备上，连续运转完成从热塑材料吹成容器、然后灌装、密封一次自动完成。吹/灌/封设备用于配备了有效的A级空气流的无菌生产时，假如使用A/B级的工作服，设备可以安装在最低C级的环境里。静态时环境必须符合可行限度和非可行限度，动态时只要求符合可行限度。用于生产终端灭菌产品时，吹/灌/封设备必须安置在最低D级环境中。
对此设备，要特别注意如下几点：
设备设计和验证，现场清洁消毒的验证和可重复性，设备安装环境，人员的培训和服装，
设备关键区域的设备连接，包括灌装前的无菌装置。
Terminally sterilised products 最终灭菌产品
11. Preparation of components and most products should be done in at least a grade D environment in order to give low risk of microbial and particulate contamination, suitable for filtration and sterilisation. Where the product is at a high or unusual risk of microbial contamination, (for example, because the product actively supports microbial growth or must be held for a long period before sterilisation or is necessarily processed not mainly in closed vessels), then preparation should be carried out in a grade C environment.
Filling of products for terminal sterilisation should be carried out in at least a grade C environment.
Where the product is at unusual risk of contamination from the environment, for example because the filling operation is slow or the containers are wide-necked or are necessarily exposed for more than a few seconds before sealing, the filling should be done in a grade A zone with at least a grade C background. Preparation and filling of ointments, creams, suspensions and emulsions should generally be carried out in a grade C environment before terminal sterilisation.
为了降低微生物和微粒污染的风险，原材料和大部分产品的处理，应在最低D级的环境里进行，适用于过滤和灭菌。如果产品的微生物污染的风险很高（如产品易生菌、或灭菌前要长时间存放，生产过程要在开口容器中进行），生产应在C级环境进行。最终灭菌产品的灌装最低要在C级环境进行。
如果产品受到环境污染的风险高，例如：灌装速度慢，使用大口容器，或密封前要暴露几秒钟，灌装要在C级环境中的A级区域进行，背景环境最低为C级。在灭菌前，软膏、霜剂、混悬剂、栓剂的制备和灌装应在C级环境进行。