Contents
1 概述Summary (2)
2 制剂湿热灭菌工艺Moist heat sterile process (3)
2.1 湿热灭菌工艺的研究Study on moist heat sterile process (3)
2.2 湿热灭菌工艺的验证Moist heat sterilization process validation (6)
3 制剂无菌生产工艺Preparation aseptic production process (10)
3.1 无菌生产工艺的研究Research of aseptic production process (10)
3.2 无菌生产工艺的验证Aseptic production process validation (11)
4 原料药无菌生产工艺API aseptic production process (16)
4.1 无菌原料药生产工艺特点Sterile API production process characteristics (17)
4.2 无菌原料药工艺验证sterile API process validation (19)
1 概述
Summary
无菌药品是指法定药品标准中列有无菌检查项目的制剂和原料药，一般包括注射剂、无菌原料药及滴眼剂等。从严格意义上讲，无菌药品应完全不含有任何活的微生物，但由于目前检验手段的局限性，绝对无菌的概念不能适用于对整批产品的无菌性评价，因此目前所使用的“无菌”概念，是概率意义上的“无菌”。一批药品的无菌特性只能通过该批药品中活微生物存在的概率低至某个可接受的水平，即无菌保证水平（Sterility Assurance Level, SAL）来表征。而这种概率意义上的无菌保证取决于合理且经过验证的灭菌工艺过程、良好的无菌保证体系以及生产过程中严格的GMP管理。Sterile drug means the preparations and API which legal drug standards list of asepsis check, generally, sterile drug including injection, sterile APIs and eye drops, etc. Strictly, sterile drug shouldn’t have any live microorganisms, but in current situation, it can’t be achieved. So current the sterile use a probability concept: SAL.
无菌药品通常的灭菌方式可分为：1）湿热灭菌；2）干热灭菌；3）辐射灭菌；4）气体灭菌；5）除菌过滤。按工艺的不同分为最终灭菌工艺（sterilizing process）和无菌生产工艺（aseptic processing）。其中最终灭菌工艺系指将完成最终密封的产品进行适当灭菌的工艺，由此生产的无
菌制剂称为最终灭菌无菌药品，湿热灭菌和辐射灭菌均属于此范畴。无菌生产工艺系指在无菌环境条件
下，通过无菌操作来生产无菌药品的方法，除菌过滤和无菌生产均属于无菌生产工艺。部分或全部工序采用无菌生产工艺的药品称为非最终灭菌无菌药品。基于无菌药品灭菌/除菌生产工艺的
现状，本指导原则主要对在注射剂与无菌原料药的生产中比较常用的湿热灭菌与无菌生产工艺进行讨论。本指导原则中的湿热灭菌工艺验证主要包括灭菌条件的筛选和研究，湿热灭菌的物理确认，生物指示剂确认等内容；无菌生产工艺验证主要包括无菌分装、除菌过滤、培养基模拟灌装、过滤系统的验证等验证内容。
Sterile method include: moist heat sterilization; dry heat sterilization; Radiation sterilization; gas sterilization; aseptic filtration. Sterile process can be divided into: sterilizing process and aseptic processing. Final sterile product use sterilizing process, moist heat sterilization and radiation sterilization belong to this category. Aseptic production process means: product sterile product in aseptic environment and use aseptic operation, both sterile filtration and aseptic production belong to sterile production processes. Drug that part or the whole process use aseptic process was called non-final sterile products. This guidance focuses on discussions with moist heat sterilization and aseptic processing. Moist sterilization process validation major include select and research on sterilization conditions, physical confirmation, biological indicators confirmation etc. aseptic production process validation bacteria mainly include sterile packaging, sterilization filtration, media simulation filling, filtrati
on system validation etc.
最终灭菌工艺和无菌生产工艺实现产品无菌的方法有本质上的差异，从而决定了由这两类工艺生产的产品应该达到的最低无菌保证水平的巨大差异。最终灭菌无菌产品的无菌保证水平为残存微生物污染概率≤10-6，非最终灭菌无菌产品的无菌保证水平至少应达到95%置信限下的污染概率
<0.1%。由此可见，非最终灭菌无菌产品存在微生物污染的概率远远高于最终灭菌无菌产品，为尽量减少非最终灭菌无菌产品污染微生物的概率，鼓励企业在生产中采用隔离舱等先进技术设备。The lowest SAL between sterilizing process and aseptic process has great differences. Sterilizing process SAL≤10-6, aseptic processing SAL is: limit of pollution probability under the 95% confidence is < 0.1%. Thus it can be seen, probability of microbial contamination of aseptic process is higher than the sterilizing process. To reduce the probability of microbial contamination of aseptic process, we inspire that the company use isolator in production process.
基于质量源于设计的药品研发与质量控制的理念，为保证无菌药品的无菌保证水平符合要求，研发者在产品的研发过程中应根据药品的特性选择合适的灭菌方式，并系统地评估生产的各环节及各种因素对无菌保证水平的影响，根据风险的高低与风险发生的可能性等来针对性地验证灭菌工艺的可靠性，验证的内容、范围与批数等取决于工艺与产品的复杂性以及生产企业对类似工艺的经验多少等因素。只有在研
发中经过系统而深入的研究与验证，获得可靠的灭菌工艺，并在日常的生产过程中严格执行该工艺，才能真正保证每批药品的无菌保证水平符合预期的要求。当然，在药品的整个生命周期内，随着对所生产的药品的特性和生产工艺等的了解越来越全面和深入，灭菌工艺也在不断的完善，此时就会涉及到对变更后的工艺如何进行验证的问题，本指导原则也适用于此种情况。由于灭菌/除菌工艺验证的工作在我国开展的时间不长，基础还不牢靠，因此必然在实际工作中会
遇到很多难以预料的问题，故本指导原则只是一个一般性原则，药物研发者应从药物研发的客观规律出发，具体问题具体分析，必要时根据实际情况采用其他有效的方法和手段。同时，本指导原则作为阶段性产物，必将随着药物研究者与评价者对灭菌工艺研究与验证的认知加深，而不断进行修订与完善。
To comply quality by design concept, the develop process should select suitable sterile method according
to drug features and systematic assess the impact of all the segment and factors in production to SAL, and validate the sterile process according to risk level and probability, the validation content and batches were decided on the process complexity and experience. In the whole product life cycle, the sterile process will be more and more perfect, as the knowledge of the product and the process become more and more comprehensive and in-depth, thus we encounter the problem: how to validate
the changed sterile process. This guideline will also suitable for this situation. This guideline only provided a general principle, the company need analysis combined with specific case. Meanwhile, this guideline is a periodic product and need to be modified to become more and more perfect.
2 制剂湿热灭菌工艺
Moist heat sterile process
2.1 湿热灭菌工艺的研究
Study on moist heat sterile processactive下载
2.1.1 湿热灭菌工艺的确定依据
Determination basis of moist heat sterile process
灭菌工艺的选择一般按照灭菌工艺的决策树（详见附件1）进行，湿热灭菌工艺是决策树中首先考虑的灭菌工艺。湿热灭菌法是利用高压饱和蒸汽、过热水喷淋等手段使微生物菌体中的蛋白质、核酸发生变性而杀灭微生物的方法。高温在杀灭微生物的同时，可能对药品的质量也有所影响。如果产品不能耐受湿热灭菌，则需要考虑采用无菌生产工艺。所以，对于药品的灭菌工艺的考察和确定，首先是考察其能
否采用湿热灭菌工艺，能否耐受湿热灭菌的高温。目前湿热灭菌方法主要有两种：过度杀灭法（F0≥12）和残存概率法（8≤F0<12）。用其它F0 值小于8 的终端灭菌条件的工艺，
则应该按照无菌生产工艺要求。以上两种湿热灭菌方法都可以在实际生产中使用，具体选择哪种灭菌方法，在很大程度上取决于被灭菌产品的热稳定性。药物是否能耐受湿热灭菌工艺的高温，除了与药物活性成分的化学性质相关外，还与活性成分存在的环境密切相关，所以在初期的工艺设计过程中需要通过对药物热稳定性进行综合分析，以确定能否采用湿热灭菌工艺。
Sterile process was decided by sterilization process of decision tree. Moist heat sterile process is preferred. The product should consider about to use aseptic process if it can’t use moist heat sterile process. So, the first work is to decide whether the product can use moist heat sterile process, whether the product can tolerate high temperature. Current, there are mainly two moist heat sterile methods: Overkill method
(F0≥12) and probability of survival (8≤F0<12). Use aseptic process if sterile method F0<8. Whether the product can tolerate the high temperature of the moist sterile process is decided both on the chemistry of
the drug active ingredient and active ingredients presence environment, so a comprehensive analysis
of drug thermal stability should be taken and decide whether can use moist heat sterile process in initial process design process.
2.1.1.1 活性成分的化学结构特点与稳定性
Chemical structure characteristics and stability of the active ingredient
通过对活性成分的化学结构进行分析，可以初步判断活性成分的稳定性，如果活性成分结构中含有一些对热不稳定的结构基团，则提示主成分的热稳定性可能较差。在此基础之上，还应该通过设计一系列的强制降解试验对活性成分的稳定性做进一步研究确认，了解活性成分在各种条件下可能发生的降解反应，以便在处方工艺的研究中采取针对性的措施，保障产品能够采用湿热灭菌工艺。Through the analysis of the chemical structure of active ingredients, can preliminary assessment the stability of the active ingredient, if the structure of the active ingredient contains some thermal instability structure, indicates that the thermal stability of the principal component maybe poor. On this basis, design a series of forced degradation test to qualify the stability of active ingredients to understand the active ingredients in the possibility of degradation reaction under different conditions is needed.
2.1.1.2 处方工艺的研究
Prescription craft research
在对活性成分的结构特点与稳定性进行研究的基础上，可以有针对性的进行处方工艺的优化研究。如活性成分易发生氧化反应，则需要考虑是否需要在工艺中去除氧并采取充氮的生产工艺，或在处方中加入适宜的抗氧剂；如活性成分的稳定性与pH 值相关，则需要通过研究寻最利于主成分稳
定性的pH 值，当然此时需要关注该pH 值在临床时能否接受；如果主成分是因为某些杂质的
存在影响了稳定性，则需要通过适宜的手段去除相关的杂质；如果是主成分在某种溶剂系统中稳定性较差，则需要考虑更换溶剂系统，此时同样需要考虑所选用的溶剂系统在临床应用时能否被接受；湿热灭菌的不同灭菌温度和灭菌时间的组合对产品的稳定性的要求有所不同，可以在保证提供所需的SAL 的基础上，通过灭菌时间和灭菌温度的调整来确定药物可以耐受的湿热灭菌工艺。
The study of the structure characteristics and stability of the active ingredients can directly help prescription process optimization research. For example: if the active ingredient easy to happen oxidation reaction, then the process design will consider about to add deoxidize and fill nitrogen procedures. Different sterile temperature combination different sterile time have different stability requirement, so we can establish the product sterile process by modulation the sterile temperature and sterile time, on the basis of an adequate SAL.
总之，需要通过各个方面的研究，使药物尽可能的可以采用湿热灭菌工艺。只有在理论和实践均证明即
使采用了各种可行的技术方法之后，活性成分依然无法耐受湿热灭菌的工艺时，才能选择无菌保证水平较低的无菌生产工艺。
Overall, moist heat sterile process is the best choice. The aseptic production process is used only when active ingredient is still no tolerance of moist heat sterilization process after try a variety of research.
2. 2.1.3 稳定性研究stability study
无论使用何种设计方法，都需要进行最终灭菌产品的稳定性研究。考察最终灭菌程序对产品性质稳定性影响的试验可包括产品的降解、含量、pH值、颜、缓冲能力以及产品的其它质量特性。灭
菌时，杀灭微生物的效果和活性成分的降解都随着时间和温度而累积。这意味着加热和冷却的变化将影响产品的稳定性，同时影响杀灭效果。因此，稳定性研究用样品最好选取处于最苛刻的灭菌条件的产品，如：可采用在热穿透试验中F0最大的位置上灭菌的产品进行稳定性考察，以确保灭菌产品的质量仍能符合要求。
No matter use what kind of design method, the study on the stability of the final sterilization products is required. The final sterilization process maybe have an influence on stability study, so the test can incl
ude the degradation of products, content, pH value, color, buffering capacity, and other quality attributes.
2.1.2 过度杀灭法的工艺研究process research of overkill method
通常来说，与残存概率法相比，过度灭杀法所需的被灭菌品开始生产阶段和日常监控阶段生物负荷的信息较少，但是过度杀灭要求的热能比较大，其后果是被灭菌品降解的可能性增大。
Overkill method need less information of the bioburden monitor data before sterilization, but it need more energy, so the degradation possibility of sterilized goods increased.
过度杀灭法的目标是确保达到一定程度的无菌保证水平，而不管被灭菌产品初始菌的数量及其耐热性如何。过度杀灭法假设的生物负荷和耐热性都高于实际数，而大多数微生物的耐热性都比较低，很少发现自然生成的微生物的D121℃值大于0.5分钟。因此，过度杀灭的灭菌程序理论上能完全杀
灭微生物，从而能提供很高的无菌保证值。由于该方法已经对生物负荷及耐热性作了最坏的假设，从技术角度看，对被灭菌品进行初始菌监控就没有多大必要了。但这并不意味着生产过程中对污染可以完全不加控制。仅从控制热原的角度，也应当遵循工艺卫生规范，控制产品的微生物污染。如果实际生产中能够严格遵循GMP 的要求，这一点是可以实现的。
As overkill method assumed bioburden and heat resistance are higher than the actual number (most of the microbial heat resistance is low, it is hard to find a naturally occurring microorganisms with
D121℃>0.5min), so, In theory the overkill method can completely kill microorganisms. As this method have assume the worst bioburden and heat resistance, so from a technical point of view, the initial bacteria monitor before sterilization is not necessary. But it doesn’t means the microbe control in production process is not necessary, we should control microbe at least for one reason: to control pyrogen.
2.1.3 残存概率法的工艺研究Survival probability method technology research.
与过度杀灭法相比，残存概率法方法所需的信息量要大得多，包括被灭菌品生产开始阶段及常规生产阶段的信息、指示菌（对灭菌程序呈现强耐热性的试验菌）以及生物负荷的信息。只有积累了这类有价值的信息后，才能制定比过度杀灭法F0值低的热力灭菌程序，同时产品的无菌保证水平不会降低。使用热力较低灭菌程序更有利于药品的稳定性，使产品的有效期延长。正是因为这个原因，残存概率法更适合那些处方耐热性较差的最终灭菌产品。
Compared with overkill method, survival probability method needs more information, include: beginning and routine production phase information, indicator and bioburden information. survival prob
ability method needs less energy and this is good for product stability, also can prolong product expiry date. For this reason, the survival probability method is more suitable for the final sterilization products with poor heat resistance.
通常说来，不耐热药品的灭菌可能不能使用过度杀灭法，需要设计一个灭菌程序能够恰当地杀灭生物负荷，同时不导致产品不可接受的降解。这种情况下，灭菌程序的确认就需研究产品的生物负荷和耐热性。根据以下公式可以比较清楚的说明这一点：
In general, Thermolabile drug can’t use overkill method and need design a appropriate sterile process to both kill bioburden and promise an accepted degradation. Bioburden and heat resistance research of the product is needed. See the table below:
无菌保证值sterility assurance value= F0 / D - lgN0
其中，无菌保证值是SAL的负对数，N0 为灭菌开始时产品中的污染微生物总数，D 为污染微生物
的耐热参数。所以，灭菌工艺的无菌保证值与F0、N0、D密切相关。
Sterility assurance value means the negative logarithm of SAL, N0 means the initial bioburden before sterilization. D means the thermal parameters of pollution microorganisms.
2.1.
3.1 灭菌前生物负荷的控制bioburden control before sterilization
采用残存概率法进行终端灭菌的产品，除了需要关注灭菌过程本身，还需要在生产过程中采用一些适当的手段来监测和控制药品灭菌前的生物负荷。具体的措施通常包括灭菌前微生物数量与耐热性的监测、药液过滤、工艺参数的控制等等。灭菌前微生物污染水平的监测将在下面的章节详细阐述。产品过滤在终端灭菌的产品中仅仅作为辅助的控制手段，但是在工艺确定的过程中，也应该对滤膜的孔径、材质、滤器的使用周期进行必要的筛选。在工艺参数控制方面，由于微生物的特性，通常在药液放置期间也会逐渐繁殖，尤其一些营养型的注射液，如葡萄糖注射液、复方氨基酸注射液等，其环境更有利于微生物的生长和繁殖，因此应通过工艺筛选和验证来确定溶液配制至过滤前、以及过滤后至灭菌前能够放置的最长时限，并相应确定产品的批量、生产周期等关键工艺参数。
If use survival probability method, bioburden monitor and control before sterilization is needed, include: The monitoring of microbial quantity and heat resistance before sterilization, drug filtration, process parameter control. Microbial contamination level monitoring before sterilization will be in the following chapters in detail. For final sterile product, the drug filtration is only an auxiliary control method, but a selection of membrane aperture, material and use cycle are needed. Liquid will gradually breed microb
e during the placement, especially for nutritional injection product, so the interval between liquid preparation and before filtration as well as after filtration and before sterilization should be selected and validated, And corresponding determine the key process parameters, such as product batch, production cycle etc.
2.1.
3.2 灭菌前微生物污染的监测Microbial contamination level monitoring before sterilization
灭菌前微生物污染水平的监测应在正常生产过程中取样并覆盖整个生产过程，取样设计应选取生产过程中污染最大，最有代表性的样品，且要充分考虑到产品从灌封到灭菌前的放置时间。一般而言，如果灌装持续一段时间，可从每批产品灌装开始、中间及结束时分别取样。污染水平检查可以采用如下的方法：先用灭菌的5%吐温充分湿润0.45um 的滤膜，然后定量过滤药液，将此滤膜移至营养
琼脂平板上，在30~35℃下培养3~7 天，计数。分离获得的污染菌需要进行耐热性的检查。污染菌
的耐热性检查可以采用以下的测定方法：先用灭菌的5%吐温充分润湿0.45um 的滤膜，然后过滤污
染水平监测所取的药液样品，再将此膜移至装有无菌的待监测产品的试管中，在沸水浴上煮沸约30 分钟，然后在30-35℃下在硫乙醇酸盐肉汤中培养，观察是否有耐热菌生长。
Microbial contamination level monitoring before sterilization should cover all the production process of the routine production, sampling design should select the biggest pollution and most representative samples, and consider the interval between filling and before sterilization. Generally, if the filling process will last for some time, we can sampling at the beginning, middle and end of filling process. Microbial contamination level test method can use this method: With 5% twain fully wet sterilization 0.45 um filter membrane, and then quantitative filter liquid, move the filter membrane on nutrient AGAR plate, in 30 ~ 35 ℃ for 3 ~ 7 days, counting. The isolated pollution of bacteria need heat resistance tests, the test method is: With 5% sterilization twain fully wetting 0.45 um filter membrane, and then filter pollution samples from the liquid level monitoring, the film to be moved to the test tube containing sterile products, boil about 30 minutes on a boiling water bath, and then culture in sulfur glycolic acid salt culture broth in 30-35℃, observe whether there is a heat resistant bacteria to grow.
当耐热性检查发现药液存在耐热污染菌污染时，可采用定时煮沸法将它和已知的生物指示剂的耐热性加以比较，必要时，可再测试耐热污染菌的D 值（D值的具体检测方法详见附件2），然后根据灭菌的F0 值及污染菌的数量与耐热性对产品的无菌做出评价。当产品微生物污染水平超标准时，应对污染菌进行鉴别、调查污染菌的来源并采用相应的纠正措施。
When heat resistance test found that there is a heat resistant bacteria pollution, we can compare its he
at resistance with known biological indicator by timing boiling method, and also can test its D value if is necessary. Then give a production aseptic assessment according to the F0 value, pollution bacteria number and heat resistant. If the microbial contamination level exceed the standard, identify pollution bacteria, investigate the source of the pollution bacteria and adopts the corresponding corrective measures is necessary.
2.2 湿热灭菌工艺的验证Moist heat sterilization process validation
湿热灭菌工艺的验证一般分为物理验证和生物学验证两部分，物理验证包括热分布、热穿透试验，生物学验证主要是微生物挑战试验。物理验证是证实灭菌效果的间接方式，而微生物挑战试验则直接反映灭菌的效果，两者不能相互替代。
It includes two parts: Physical and biological validation. Physical qualification include: heat distribution, heat penetration test. Biological qualification mainly means microbial challenge test. Physical qualification can indirect indicate the sterilizing effect, while biological qualification can indirect indicate the sterilizing effect, both these two method can't replace each other.
2.2.1 物理确认Physical qualification
2.2.1.1 空载热分布试验empty heat distribution test
空载热分布的目的是主要是了解整个灭菌设备的运行情况，确认灭菌室内的温度均匀性，测定灭菌腔内不同位置的温差状况，确定可能存在的冷点。空载热分布试验通常采用足够数量的热电偶或热电阻作温度探头，进行编号后将它们固定在灭菌柜腔室的不同位置。温度探头的安放位置需要根据设备类型和不同位置下的灭菌风险评估而定，应包括可能的高温点、低温点，灭菌柜温度控制探头处、靠近温度记录探头处，其他的探头可以均匀地分布于灭菌柜腔室内，以使温度的检测具有较好的代表性。温度探头在试验前后至少需要两个温度点进行校正。温度探头安放结束后，即可以按照设定的灭菌程序进行灭菌。
Empty heat distribution test is mainly is to understand the operation of the entire sterilization equipment, confirm the sterilization chamber temperature distribution uniformity, test temperature difference of different position, and determine cold spots. Empty heat distribution test usually adopt a sufficient number of thermocouple or heat resistance as temperature probe, numbered them and fixed at different positions of the sterilization cabinet chamber. Temperature probe position is decided by the device type and risk assessment. The possible highest, lowest temperature position, temperature control probe of sterilization cabinet, temperature record probe of sterilization cabinet should put the temperature probe, the other temperature probe can evenly distributed in the sterilization cabinet chamber. Temperature probe need at least two temperature points calibration before and after the test.
2.2.1.2 装载热分布试验on-load heat distribution test

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