World Health Organization GMP 基本原则 2017年3月10日星期五 无菌药品的生产 This module deals with the important topic of the production of sterile pharmaceutical products. It is a full day session module divided into three roughly equal parts as follows: General points: Premises, equipment, sanitation, personnel Processing Methods of sterilization and Quality Control In each case, there will be 15-40 minutes of presentation, 45 minutes discussion in groups and 30 minutes feedback to the whole group. There will be two tests covering the whole module that will be taken at the end of the day (or at the start of the next day as appropriate). All that can be achieved with this module is a very basic introduction to the topic. Separate courses lasting several days are needed to cover properly such issues as moist heat sterilization. Annex 6. TRS 902, 2002

World Health Organization 无菌药品的生产 World Health Organization 2017年3月10日星期五 目 的 回顾 GMP 对无菌药品生产的基本要求 回顾与无菌药品的生产活动相适应的级区划分情况 回顾不同的灭菌方法 回顾无菌产品在生产、控制过程中的质量保证 探讨你们国家当前的问题 The first objective of this module is to identify and understand the key issues and GMP requirements relating to sterile product manufacture. This type of manufacturing is one of the most complex in the industry. The critical nature of the products in question make this a very important subject indeed. The second objective is to review air classifications for activities related to the manufacture of sterile products. The third is to review the different types of sterilisation methods and the forth is to review quality assurance aspects in the manufacture and control of sterile products We shall also consider current issues applicable in your country.

World Health Organization 无菌药品的生产 World Health Organization 2017年3月10日星期五 GMP 对无菌产品的要求 是额外增加而不是取代 尽可能减少污染风险的具体要素 微生物 空气悬浮物 热原 The first point to be emphasized is that GMP requirements for sterile products are additional to the usual requirements for pharmaceutical manufacture, rather than a replacement for them. The WHO GMP text deals with this subject in a supplementary guideline. The emphasis of all the extra requirements for sterile production is to minimize the risks of contamination by particulates, microorganisms or pyrogens. This is because sterile products are administered to particularly sensitive parts of the body, whether intravenously or intramuscularly as an injection, as an eye ointment or as a wound dressing.

World Health Organization 无菌药品的生产 2017年3月10日星期五 一般要求 在洁净区内生产 合适的洁净标准 经过滤的空气 通过气锁进入洁区 人流、设备 物流 操作隔离区 备料 (容器和包装) 产品生产 灌装、灭菌等 A general requirement for the manufacture of sterile products, is that production must be done in clean areas. The manufacture of the products should take place in areas of appropriate standards of cleanliness. We will go into more details on this later. The areas should have air supplied through appropriate filters e.g. HEPA filters. Entry to these areas are through airlocks for personnel and/or equipment, and airlock for goods or materials. There are different operations to be carried out. This includes component preparation, product preparation and filling. Separate areas for these operations are needed. 1.1 – 1-2

World Health Organization 无菌药品的生产 2017年3月10日星期五 厂房 设计 避免不必要的参观者，控制人员进入 从外面观察操作 洁净区域所有裸露表面 表面光滑、无渗透、无破损 尽量减少脱落、颗粒累积和微生物 便于清洁和消毒 不存在无法清洁的坑凹、边缘、架子、橱柜和设备 无移门 天花板 Let us start by looking at some of the recommendations for premises. There are a number of specific requirements for premises that are used for the manufacture of sterile products. Unnecessary entry to all processing areas should be avoided. The design of the premises should support this. It should be possible to observe operations from the outside. Processing takes place in suites of rooms with different classifications, depending on the activities carried out in them. The classifications relate primarily to the supply of air to the rooms. We will be looking at this topic in more detail in the second part of this session. Assess whether the rooms are designed to reduce the accumulation of dust, with all exposed surfaces being smooth, impervious and unbroken. (The trainer may want to show the slides of photographs of suitable and unsuitable premises and finishing). There should also not be excess equipment, cupboards or tools in the area. Doors should also be suitably designed and sliding doors should be avoided in sterile product manufacturing areas, as these cause areas where it is difficult to clean. Ideally there should be false ceilings, which are sealed so that no dirt can fall from the void above. This should also permit access to light fittings from above allowing maintenance without stopping production. Wherever possible, pipes and ductwork should be outside the area, or boxed in. 9.1 – 9.6

World Health Organization 无菌药品的生产 2017年3月10日星期五 厂房（接上页） 洁净区域所有裸露表面(2) 管道安装合理，无坑凹及未密封开口 减少水池和地漏，A、B级区不得设置水池和地漏 安装在哪里, 设计, 位置, 维护 能够有效清洁 空气间隙，防止逆流 地板明沟敞开且便于清洁 Wherever possible, pipes and ductwork should be outside the area, or boxed in. Sinks and drains should be avoided if possible and must not be installed in aseptic areas. Drains should have cleanable traps and air breaks to prevent back flow. Floor channels must be open and easy to clean. 9.6.

World Health Organization 无菌药品的生产 2017年3月10日星期五 厂房（接上页） 更衣间 设计同气锁 送入过滤过的空气 进出缓冲间最好分开 洗手设施 联锁系统 可视或可听的警报系统 使用过滤过的空气，保持压差 压差保持在10-15帕 风险最大区域实行即时环境检测 Entry to all processing areas should be through airlocks. For personnel, these airlocks generally take the form of changing rooms that have a variable number of interconnecting rooms, depending on the grade of the area. Ideally, separate airlocks should ideally be provided for the entry of materials into the area. Airlocks should be flushed with filtered air. In some facilities, there are different airlocks for entry to and exit from manufacturing areas. This can promote unidirectional flow of personnel and material. Hand washing facilities should only be provided in change rooms, and not in production areas. Filtered air should be supplied to the areas and in such a manner, that the pressure cascade is maintained. There should normally be a pressure differential of 10 to 15 pascal between areas of lower and higher risk. The supply of suitable quality air to sterile manufacturing areas, is very important. Filtered air under positive pressure should be supplied to production areas of sterile products. Verify whether the manufacturer has validation data of aspects relating to airflow patterns, and warning systems indicating failure of air supply (e.g. manometers measuring pressure differentials, or an audible alarm).Check the configuration and maintenance of HVAC and filters. The pressure differentials between areas should be monitored and recorded in accordance with written SOPs. (Note: Trainers should explain with the aid of a flip chart and drawings, suitable layout of premises, indicating air supply and return to areas, desired air flow patterns, design and purpose of air locks, and the concept of pressure differentials between different areas). 9.7 – 9.9

World Health Organization 无菌药品的生产 2017年3月10日星期五 厂房（接上页） 致病、剧毒、放射性物料 压差可能不同 净化措施-空气、设备、服装 认证（包括气流方式） 不对产品产生风险 设置报警系统以指示送风失常 压力计（定期记录结果） 限制性进入（如使用门禁） When highly toxic materials are processed, the pressure differential should be such that there is no risk to the product, and no risk to the operators and external environment. In addition, decontamination procedures should be followed (e.g. penicillins, cytotoxic substances). This could be chemical or a combination of air showers and the use of chemical substances. Validation and qualification are essential in the manufacture and control of sterile products, equipment, and premises. Failure in utilities' performance can result in contamination of the products. Warning/alarm systems can assist in indicating failure of air supply. Pressure indicators such as gauges can should be in place to enable monitoring of the pressure cascade. 9.9 – 9.12

World Health Organization 无菌药品的生产 2017年3月10日星期五 设备 传送带 设备的有效灭菌 在级区外进行保养和修理 如果设备离开级区，使用前要重新消毒 使用清洁的仪器和工具 有计划的保养、验证和监测 设备、空气过滤系统、灭菌器、 水处理系统 You should also check whether the manufacturer uses conveyer belts that pass through a clean and dirty area, to convey components or products. This can only be allowed if the conveyer belt is sterilised before moving into the clean area. Equipment for use in the sterile area should be designed so that it can be operated with the minimum of personnel interference, thus reducing the possibility of contaminating the product. It should also be easily sterilized by moist or dry heat sterilization. Sterilizers should be designed with a door at each end (known as double door autoclaves or double- ended autoclaves) to eliminate the possibility of mixing up sterile and non-sterile materials. This is particularly important for sterilizing components that are going into the filling room. They are loaded in the preparation area and unloaded in the sterile area, although preferably in a buffer room rather than directly in the filling room. It is important that the zone in which the product is to be exposed is protected to the maximum extent possible. This requires the installation of laminar airflow cabinets over the piece of equipment, to ensure a supply of filtered air flowing with positive pressure towards the surrounding areas. It is also necessary to ensure that the locations of the equipment and the operator do not cause a risk to the product by interrupting the flow of filtered air. Where possible, maintenance and repairs of equipment should take place outside the area. However, if this is impossible, it should be done when there is no work going on and should be followed by a complete clean down and disinfection. Tools for such work should be sterilized before being taken into the area. It is even better if a full set of sterilized tools can be stored in the area specifically for this purpose. After maintenance has been completed, there should be a documented procedure for obtaining approval to resume operations in the area. It is permissible to have transport systems to take product from the filling room to the sterilization/finishing area, but there must be a physical barrier across the actual interface between the two areas. 10.1 – 10.5

World Health Organization 无菌药品的生产 2017年3月10日星期五 设备(continued) 水的处理和分配系统 设计、建造、维护 使用和设计能力 检测项目 注射用水 (WFI) 生产、存储、使用 – 防止微生物生长 70摄氏度以上或4摄氏度以下定向循环 Water of appropriate quality should be supplied by a water treatment plant that is suitably designed, constructed and maintained. You should evaluate the water treatment plant in terms of maintenance and qualification, as well as the monitoring of the quality of the water. The production, storage and distribution of water should be done in such a way that microbial growth is prevented. Evaluate the SOP for water sampling and review the results of the water testing. You should also check the deign of the water treatment plant, the distribution and storage of water. If water is stored in a tank, then the temperature should be kept at about 70 degrees Celsius. Refer the participants also to the additional training module on Water systems. 10.6

World Health Organization 无菌药品的生产 World Health Organization 2017年3月10日星期五 环境监测 - I 微生物方面 空气 表面 人员 The first aspect of microbiological monitoring of the environment relates to the air supplied to the rooms. There are a number of methods for taking samples, but the simplest and most widely used is to place open settle plates of growth medium on the floor for around 2 - 4 hours. (The exact time period has to be developed to suit local conditions – a validation protocol and report should be inspected). The exposure time should not be too short as non-representative results will be obtained. If the exposure time is too long, then the plates can dry out. The number of plates required depends on the classification and use of the room, and can be determined from international standards. The location of the plates will have been determined during validation and will be based on the risk to the product and the level of activity in the area. It is not necessary to obtain zero-growth results from these plates, but a validated pattern of likely contamination will be established and significant deviations need to be investigated. If zero growth is observed, then low levels of bacteria are inoculated onto the plate to demonstrate that it will support growth. Monitoring of surfaces is generally carried out using swabs. Emphasis should be placed on the areas that come into contact with the product. In these areas, zero-growth results are expected. This method of monitoring, carried out before and after cleaning and disinfecting can be used to validate the methods being used. Finally, it is necessary to monitor the micro-organisms that could be shed from the personnel in the clean rooms. Personnel can be the greatest source of contamination. Samples are generally taken by swabs from clothing and by “finger-dabs” onto plates. Sampling should be representative of the situation during operations. So, if the operator normally wears gloves and disinfects them before use, the samples should be taken afterwards. Review the SOP and records of results during your inspection. The trainer can also expand on the current recommendations for monitoring, as reflected in other guidance documents.

World Health Organization 无菌药品的生产 World Health Organization 2017年3月10日星期五 环境监测– II 物理要素 粒子 压差 换气次数, 气流型式 自净时间/恢复 过滤器的完整性 温度和相对湿度 气体流速 The ventilation systems used to supply air to clean rooms have already been referred to. We shall now look at the physical monitoring that is carried out on these systems. Particulate matter counts are carried out with a particle counter that measures the number of particles in a given quantity of air. They should be carried out during validation and at regular intervals thereafter. You can consult ISO 14644 for more details on the methods and locations for monitoring particles. The differential pressures between rooms are measured by means of manometers. The manometers should be calibrated and should provide continuous monitoring. Values should be recorded regularly. The number of air changes within a room is calculated from the air volumes supplied to the room. The calculation should be made during validation and regularly thereafter. The HEPA filter integrity is tested by a number of means. An aerosol generator can be used to send an aerosol across the filter and a photometer used to view it. This will show whether there is any damage to the filter. Additionally, a manometer can be used to measure the pressure differential across the filter. These tests should be carried out when filters are installed and repeated at regular intervals (at least annually). Again, you should verify compliance with this by reviewing the SOP and records to assess compliance with the SOP. The temperature and humidity can be measured by a variety of instruments from the very simple to the very complex. Several of the above parameters can be monitored automatically, and new factories often have sophisticated building management systems (BMS) that not only monitor, but also make adjustments if required. If you are inspecting a factory with such a system, it is worth spending some time checking on the understanding of the personnel regarding this system. It can be all too easy to assume that everything is under control and not notice when something goes wrong. Establish whether any validation had been done to ensure that all the controls and monitors are working as intended?

World Health Organization 无菌药品的生产 2017年3月10日星期五 卫生 相关区域需要进行经常的、彻底的清洁 书面规程 定期检测，以发现微生物的耐消毒菌株 化学试剂消毒 消毒剂和洗涤剂管理 稀释 使用清洁容器，在规定期限内存放 在进入A、B级区使用前灭菌 A high level of cleanliness is required in clean areas. Frequent, thorough cleaning of areas necessary according to a written program should be done. Regular monitoring to detect resistant strains of micro-organisms should be done. A program for chemical disinfection and corresponding records should be available. Manufacturers also have to monitor disinfectants and detergents for effectiveness. Preparation of dilutions should be done in accordance with a program and SOP - and records should be available. Disinfectants should be kept in clean containers and stored only for defined periods of time. These should be sterilized before use, when used in Grade A or B areas 3.1 – 3.2

World Health Organization 无菌药品的生产 2017年3月10日星期五 卫生 (接上页) 洁净区监测 在关键操作后要进行人员和表面检测 无菌操作区要经常进行监测 沉降碟, 定量空气样品监测, 表面取样 (擦拭和接触碟) 取样不得污染环境 批次放行时要参照该结果 Monitoring of clean areas, of personnel and surfaces after critical operations should be done. Frequent monitoring should be done in areas where aseptic operations are carried out. Monitoring further includes the use of settle plates, volumetric air samples, surface sampling (swabs and contact plates). Review the SOP to see how the samples are taken, as the sampling methods should not result in contamination of the area. Results if the monitoring should be considered when batch release is done 3.3

World Health Organization 无菌药品的生产 2017年3月10日星期五 卫生 (接上页) 制定检测限度 警戒和纠偏限度，监测空气质量趋势 表 1.微生物污染限度 (仅供参考) 3.4 The manufacturer should have alert and action limits, and should monitor the trends of results of air quality. The table gives some guidance of limits of microbial contamination when samples are taken.

World Health Organization 无菌药品的生产 2017年3月10日星期五 人员 洁区内人员数应尽可能少 尤其是无菌生产 从外部进行监测和控制 对所有员工进行培训，包括清洁工和修理工 初级培训和常规培训 生产、卫生、微生物 特殊情况 外部人员的管理 消除污染的措施 (如处理动物的员工) Personnel play an important part in ensuring the quality of manufacture. It is also relevant (perhaps in particular) in the manufacture of sterile products. Only a minimum number of personnel should work in clean areas, especially during aseptic processing. As far as possible, all inspections and controls should be done from outside the production rooms. Training should be given to all including cleaning and maintenance staff, and should include initial and regular training on manufacturing, hygiene, and microbiology. Look at the procedure for training, training program, training material and assessment of the personnel. In special cases, when outside staff have to enter the clean areas, they should be supervised. Remember also the previous discussion on decontamination procedures (e.g. staff who worked with animal tissue materials). 8.1 – 8.3

World Health Organization 无菌药品的生产 2017年3月10日星期五 人员（接上页） 卫生、清洁标准高 定期健康检查 无粒子脱落 不带入微生物 不穿着便装 更衣和洗衣 不戴手表、首饰，不使用化妆品 Personnel working in clean areas should maintain high standards of hygiene and cleanliness. They should undergo periodic health checks, wear clothing that do not shed particles, and should take care not to introduce microbiological contaminants in the areas. No outdoor clothing should be brought into clean change rooms. Personnel should follow changing and washing procedures, wear no watches, jewellery and cosmetics 8.4 – 8.6

World Health Organization 无菌药品的生产 2017年3月10日星期五 人员（接上页） 着装要求: D级区 包住头发和胡须 防护服和鞋子 C级区 单层或双层外套 (能包住手腕和脖子), 鞋子 无纤维脱落 A级区、B级区 戴帽子、包住胡须、戴口罩和手套 无纤维脱落，防止操作者身上粒子脱落 The WHO GMP text specifies the type of clothing that is appropriate for the different grades of rooms. 8.7

World Health Organization 无菌药品的生产 2017年3月10日星期五 人员（接上页） 便装不得进入B、C级区更衣室 每班更换服装，或每天更换一次 (如果有数据支持) 每班更换手套和口罩 操作过程中手套要消毒 洗衣 – 使用单独的洗衣机 无损坏，符合经验证的程序 Garments should be changed at every working session, or once a day (if supportive data exist through validation studies). Gloves and masks should be changes at every working session Personnel should disinfect their gloves frequently during operations to prevent possible introduction of contaminants (micro) into the areas where they work or touch. Arrangements must be in place for the laundering and sterilization of clean-room clothing. This should be carried out in a controlled environment. If fibres are damaged due to inappropriate cleaning or sterilization, an increased risk for contamination may develop as clothing could shed particles. The use of contract laundries for this purpose, requires an audit by the company to ensure that appropriate procedures are in place. 8.8 – 8.9

World Health Organization 无菌药品的生产 2017年3月10日星期五 分组讨论-1 你被邀请去参观一个生产以下产品的工厂： 安瓿或西林瓶装的注射剂，包括胰岛素、疫苗和耐热药品。 无菌眼用软膏 描述你希望看到的设施配备情况 列出一些代表性房间以及它们的用途和洁净级别

World Health Organization 无菌药品的生产 2017年3月10日星期五 可能出现的问题 不合理的厂房设计 不合理的介质供应系统设计，如：水系统和空调系统 人流 物流 无验证或确认 不能满足当前要求的旧厂房

World Health Organization 无菌药品的生产 2017年3月10日星期五 可能出现的问题 （ 续前页 ） 空气悬浮粒子/微生物水平 压差 换气次数 温度/湿度

World Health Organization 无菌药品的生产 2017年3月10日星期五 两种生产方式 最终灭菌 配制，灌装和灭菌 无菌工艺 生产的部分阶段或全过程 There are two main categories of manufacturing operations, relating to the methods of sterilization: The nature of the product determines the manufacturing and sterilization requirements, as we will see later on. The types of sterilization are listed on this slide. The first type is terminally sterilized products. These are products that are tolerant to sterilization in their final containers. This usually means they are process tolerant; for example, they are stable when exposed to heat or gamma-irradiation, so they can be manufactured and filled under clean rather than aseptic conditions. The key task here is to reduce the bioburden to a minimum so that the challenge to the sterilization process will be as low as possible. This is the method of choice for sterile manufacture where possible. Some materials and products cannot be terminally sterilized by exposing them to heat. They are manufactured under clean conditions, and then filtered into containers in the filling room where they are filled under aseptic conditions. All components, such as primary containers, must be sterilized before they are introduced into the filling area. Sterile filtered production should only be considered if all methods of terminal sterilization are impossible. Some products are produced from sterile starting materials. In this case, manufacture and filling are both carried out under aseptic conditions, with components sterilized before use. 1.3

World Health Organization 无菌药品的生产 2017年3月10日星期五 无菌制剂的生产 洁净区域划分 在合适洁净等级的环境中生产 将风险降至最低 – 粒子和微生物污染 – 产品和物料 符合静态洁净等级 (即所有设备均安装并运行，但无操作人员在场) During the inspection, you have to verify that the operations for the manufacture of sterile products are carried out in the correct grade or class of air. Precautions should be taken to prevent possible contamination with particulate matter and microorganisms. This applies to products, materials including primary packaging materials. The manufacturer should demonstrate that the areas meet the required classification, This must be done through monitoring including particles and microorganisms. Normally, this is done while the area is "at rest". Monitoring should also be done during operations. 4.1

World Health Organization 无菌药品的生产 2017年3月10日星期五 无菌制剂的生产 对于无菌药品的生产: A级区 局部区域, 进行高风险操作, 如灌封、无菌连接 常使用 UDAF 系统 B级区 A级区的背景环境 (在无菌配料和灌封时) C、D级区 操作要求稍低的洁净区域 Operations should be done in areas as follows: Grade: This is the critical zone, and high risk operations should be carried out in Class A areas including filling and aseptic connections. Usually, Unidirectional Air Flow is used to achieve the required class with appropriate filtration of air (e.g. HEPA). Grade B is normally the background to Grade A areas. 4.1

World Health Organization 无菌药品的生产 2017年3月10日星期五 空气分级系统 This table is taken from the WHO GMP text and describes four classifications A - D. The different grades are defined in terms of the maximum permitted number of particles per cubic metre of air (at two different sizes). The WHO text also contains values for microbial contamination of the various grades. They have been included in the text for information and are not intended to represent specifications. We will talk later about how these characteristics are measured. They are attained by means of a ventilation system in which air is passed through a series of filters. The generally accepted design is for two pre-filters and a HEPA (high efficiency particulate air) filter at the outlet into the room. The air inlet is usually located at a high level in the room, whereas the extract is at a low level. The filtered air supply must be maintained at positive pressure to the surrounding areas. Airflow patterns must be designed so that they do not distribute particles into the area where the product is exposed. An alarm system, in the case of air supply failures, should be installed. During the inspection, you have to evaluate records and results from the manufacturer in which it is established and proven, that a claimed grade is achieved. 3.1

World Health Organization 无菌药品的生产 2017年3月10日星期五 无菌制剂的生产 要达到B、C、D级区的要求，换气次数要须根据空间大小、人员数量和设备情况来确定 每小时最低换气20次 自净时间15-20分钟 级区内气流型式适当 使用高效微粒空气过滤器过滤空气 采用适当的方法测定颗粒物质和微生物 如 欧盟药典、ISO标准、日本药典、美国药典 To reach Grade B, C and D, the number of air changes should be appropriate to the size of the area, number of personnel, and number and type of equipment present. A minimum number of 20 air changes per hour and a clean up time of about 15 – 20 minutes is recommended. A good air flow pattern in the area is necessary and should eb proven e.g. by means of a smoke test. HEPA filtered air should be supplied to the areas. Suitable methods to determine particulate matter and micro should be used as described in various guidelines E.g. EU, ISO, Japan, USA 4.1 – 4.2.

World Health Organization 无菌药品的生产 2017年3月10日星期五 无菌制剂的生产 生产过程要控制粒子 对生产过程进行监控 粒子和微生物警戒限度、纠偏限度 超标时的措施 级区需经过验证 (如验证运行、培养基灌封、环境、时间限制 – 以 微生物污染、含菌量为基础) The manufacturer should control particulate matter during operation to prevent contamination of the product. Therefore, to assist and ensure control, monitoring during operation is necessary. As there are alert and action limits for particulate and micro defined by the manufacturer, the operators have to stop working in case the condition is out of limits. Action should be taken when the limits are exceeded, e.g. cleaning and/or sanitization. Area grades should be proven through qualification and validation (e.g. validation runs, media fills, environment, time limits). Limits should be based on microbiological contamination/bioburden found. 4.3 – 4.5

World Health Organization 无菌药品的生产 2017年3月10日星期五 悬浮粒子划分系统 This table gives a comparison of the various classifications for areas in terms of airborne particulate classification 4.1

World Health Organization 无菌药品的生产 2017年3月10日星期五 生产加工 将污染风险降至最低- 包括灭菌前和生产过程的所有阶段 无不当的物质存在，如活的微生物 尽量减少活动 操作人员限制性、有秩序的活动 避免颗粒脱落 温度和湿度适宜 级区中的容器和物料 At all times during processing, there should be measures to ensure that contamination of product, material and components, is minimized. No unsuitable materials should be used in the areas. All furniture and fittings should be of metal or plastic rather than wood. Paper may need to be used in the area, e.g. for batch documentation, but this should be kept to a minimum. Bonded paper or lint-free paper is available. Paper should not be used in a grade A area at all. Alternatives include plastic sheets and permanent markers. It goes without saying that extras such as calendars and notices should be excluded. The processing of preparations containing live micro-organisms is not allowed in the same facility as other pharmaceuticals. Products with dead organisms can be processed in the same facility providing validated procedures for inactivation and cleaning are used. During processing in sterile areas, it is important that the amount of activity is kept to a minimum. This is one area where you will have to spend time to observe the activities of the personnel.The greatest source of contamination in a sterile area is the personnel. Validated automization of processes with fewer people in the area, could minimise the risk of contamination. Processing areas should be built with plenty of inspection (glass) windows to limit the number of persons who need to go into the room during processing. The temperature and humidity in the areas should be controlled to ensure that the integrity of materials is mainteianed, and the operators are also comfortable (considering the nature of the garmetns they should wear in the areas). No unsuitable materials should be used in the areas. All furniture and fittings should be of metal or plastic rather than wood. Paper may need to be used in the area, e.g. for batch documentation, but this should be kept to a minimum. Bonded paper or lint-free paper is available. Paper should not be used in a grade A area at all. Alternatives include plastic sheets and permanent markers. It goes without saying that extras such as calendars and notices should be excluded. The microbiological contamination load or bioburden for starting materials prior to sterilization should be kept to a minimum. There should be limits when monitoring has shown that they are needed. Extreme care must be taken with materials that have been sterilized in the area for use in aseptic production, such as primary containers and filling machine parts. After removal from the sterilizer, they should be stored in a way which maintains their sterility (examples: in laminair airflow, triple wrapping etc.). All packs should be marked with the date of sterilization and there should be a procedure setting out how long an item can remain in the area before it needs to be resterilized. There must also be a validated maximum storage period between the preparation of a bulk solution and its sterilization or filtration through a bacteria-retaining filter. 4.15 – 4.16, 4.20 – 4.21

World Health Organization 无菌药品的生产 2017年3月10日星期五 生产加工 验证不能影响生产 无菌工艺的验证: 无菌培养基灌封 (“肉汤灌装”) 尽量模拟实际操作过程 模拟可能达到的最差状态 使用 适宜的培养基 足够数量的灌装单位, 如等于批量（小批次） 合格标准 调 查 再验证：定期和变更之后 新工艺验证 重大变更后再验证 定期 Validation is an essential part of GMP. Validation is a very important part of sterile product manufacture. Validation is required for new processes, equipment, premises and personnel. Re-validation is also required, periodically and after change of processes, equipment or maintenance. Let’s look at some GMP and validation requirements specifically for aseptic processing. Sterile media fill (“broth fills”) with nutrient media supporting microbial growth is a valuable part of the validation process. It simulates the actual operation. During the inspection of the micro laboratory, you should establish whether appropriate media are used. When reviewing the records and results of the broth fill, establish whether a sufficient number of units e.g. at least 3000 had been filled, whether acceptable limits had been set (not more than 0,1% contaminated units) and whether any investigations are performed when there is contamination. Revalidation should be performed at periodic intervals, and after any significant change to equipment, processes or materials. 4.17, 4.18, 4.28

World Health Organization 无菌药品的生产 2017年3月10日星期五 生产加工 水源和水处理系统及处理过的水 定期监测 化学物质 微生物污染 内毒素 水的标准 结果记录及采取的措施 Treated water and the equipment used to produce it should be monitored regularly for biological and chemical contamination and for the presence of endotoxins. Evaluate the SOP and recorded results of monitoring the water. Determine whether there is provision made for any corrective action should the results indicate problems with the quality of the water. 4.19

World Health Organization 无菌药品的生产 2017年3月10日星期五 生产加工 组分、内包装材料和设备 产生纤维 清洁后不产生再次污染 标明状态 在无菌区域内使用的一切物料都经过灭菌 在洁净区使用的物料，要经过双扉灭菌设备或三层包装 用于排空溶液或覆盖产品的气体，须经过无菌过滤 Components, bulk product containers and equipment used should not generate any fibres and should not re-contaminate the area after final cleaning, or become contaminated after cleaning. They should be sterilised when used in aseptic areas. When sterilized, these should be passed through double ended sterilizers or use triple wrapping should be used to prevent recontamination. Gas used to purge solution or blanket a product has to be passed through a sterilising filter 4.22 – 4.23

World Health Organization 无菌药品的生产 2017年3月10日星期五 生产加工 含菌量测定 产品: 灭菌前 确定内控限度 溶液灌封前经过滤 (特别是LVP) 压力解除出口– 气体经除菌过滤器过滤 原料的微生物污染降至最低 根据各自标准进行监测 The microbiological contamination load or bioburden for starting materials prior to sterilization should be determined and kept to a minimum. There should be limits specified in specifications and evidence of testing. The microbiological contamination of products should be kept to a minimum. Large volume parenterals should be passed through a micro-organism retaining filter immediately before sterilisation. Where solutions are stored in sealed vessels, make sure that the pressure release outlets are protected with hydrophobic air filters. There should be a minimum or no containers or other materials in the area, liable to generate fibres due to the risk of contamination. Extreme care must be taken with materials that have been sterilized in the area for use in aseptic production, such as primary containers and filling machine parts. After removal from the sterilizer, they should be stored in a way which maintains their sterility (examples: in laminar airflow, triple wrapping etc.). All packs should be marked with the date of sterilization and there should be a procedure setting out how long an item can remain in the area before it needs to be re-sterilized. The stage of processing should thus be identified (e.g. proper labelling). 4.26, 5.3

World Health Organization 无菌药品的生产 2017年3月10日星期五 生产加工 时间限制：内包装材料、容器、设备 清洗、干燥和灭菌;灭菌和使用 时间应尽可能短 时间限度经验证 时间限制: 产品制备 开始配制溶液和灭菌(过滤） 时间尽可能短 每种产品的最长制备时间 When reviewing the batch manufacturing documents and other relevant documentation, establish whether the manufacturer has validated the time intervals between washing, drying and sterilisation for components, containers, and equipment. The time interval between sterilisation and use as well as the storage conditions must have been validated. As far as production is concerned, the time intervals between preparation and sterilisation should be as short as possible and a maximum time for each product must be set by the manufacturer. You could verify this for each individual product, by requesting the validation report for the product. The batch manufacturing document should reflect this time limit, based on the validation data. 4.23 - 4.24

World Health Organization 无菌药品的生产 World Health Organization 2017年3月10日星期五 分组讨论2 就前一组参观的同一工厂，讨论灭菌工艺 列出所有需要灭菌的物品名称(说明灭菌工艺的选择) 在每次灭菌中，什么是关键因素？ 哪些方面应经过验证？ We are now going to move into our second group session. Considering the same factory as in the previous group session, discuss the process of sterilization. List all the items that will need to be sterilized (and indicate the choice of sterilization process). What are the key features you should find in each sterilization situation? Discuss the relevance, need, and the extent of qualification and validation required.

World Health Organization 无菌药品的生产 World Health Organization 2017年3月10日星期五 可能出现的问题 灭菌釜-无压力表 灭菌釜 –无温度记录仪 灭菌釜 –蒸汽过热 洁净室-压差 沉降碟的暴露时间 联锁系统关闭 层流台生锈 没有定期检查高效过滤器 There are a number of areas where you might expect to find problems: Autoclave - no pressure gauge Autoclave - no temperature recorder Autoclave - superheated steam Clean room - pressure differentials Exposure for settle plates Interlocks turned off Rusty Laminar airflow cabinets HEPA filters not checked regularly

World Health Organization 无菌药品的生产 2017年3月10日星期五 灭菌 灭菌方法 湿热灭菌、干热灭菌 辐射灭菌 杀菌气体灭菌 (如环氧乙烷气体) 过滤灭菌 最终灭菌尽可能使用热力灭菌-方法选择 We are now going to talk about sterilization in more detail, with particular reference to the different methods of sterilization. There are a number of available methods, each of which has advantages and disadvantages. Heat sterilization should always be the preferred method if it can be used. Methods of sterilization include: Moist or dry heat Irradiation (ionizing radiation) Sterilising gaseous agents (e.g. ethylene oxide) Filtration with subsequent aseptic filling 5.1 – 5. 2

World Health Organization 无菌药品的生产 2017年3月10日星期五 灭菌 验证 所有灭菌程序 使用非药典方法时要特别注意 非水性或油性溶液 方法使用前，应在目的状态下验证适宜性和有效性 所有装载部位 每种装载方式 物理测定 和生物指示剂 (适当选用) 至少每年或变更之后进行再验证 记录 Validation of all processes and the method of sterilization is essential, particularly as sterility testing is always a destructive test and can only be carried out on a sample of the batch. You should look very carefully at validation results for any methods that are not in accordance with national standards or pharmacopoeia, or for materials and products that are not solutions. If there are changes in the sterilization method, they must be validated. The manufacturer must have data to support its decision for the sterilisation process. The suitability and efficacy in achieving the desired sterilising conditions in each part of load, and each type of load must have been validated. This validation is done initially and repeated at least annually and after change. 5.4 – 5.5

World Health Organization 无菌药品的生产 2017年3月10日星期五 灭菌 有效灭菌 所有物料都需经过灭菌 生物指示剂 其他监测方法 贮存和使用，通过阳性对照检查质量 污染风险 To ensure that the sterilization is effective, the whole load of material has to be subjected to the treatment. Biological indicators (BIs) can be considered as part of the monitoring of the sterilization process. Their use should always be controlled to prevent contamination of the facility and product with live micro-organisms. The manufacturer should have proper control over the storage and use of BIs. Their quality should also be checked through positive control. Don't forget that there is a risk of contamination in case of uncontrolled handling, breakage etc. 5.6 - 5.7

World Health Organization 无菌药品的生产 2017年3月10日星期五 灭菌 已灭菌和没有灭菌产品之间的区分 每个容器（框、盘或其他容器）都应贴上适当的标签 物料名称 批号 灭菌情况 高压蒸汽灭菌釜的使用 每次的灭菌记录 – 批次放行 It is very important that a company has effective methods for separation of sterilized and unsterilized materials. Ideally, sterilizers should be double-ended, so that there is no cross-flow of products or materials. Containers should be clearly labelled with relevant information, and indicators such as autoclave tape or irradiation discs can be used. However, it is important to remember that these indicators only show that a load of material has passed through the sterilizer. They are not in themselves proof of sterility. I am sure that some of have have seen examples where sterilized and not-sterilized products were stored next to each other where there had been a possibility of a mix-up, or where batch documentation had been completed and signed in advance, indicating that products had been sterilised (but in fact, had not yet been through the sterilising process). Always verify the stage in the production process against the batch documentation. 5.8 - 5.9

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌 热力灭菌 湿热灭菌 干热灭菌 辐射灭菌 气体或熏剂灭菌 It is not possible to deal with all the aspects and requirements for sterilisation in the basic module. The information provided here is only a brief introduction. It is recommended that the novice inspector should have an experienced inspector with him/her or an expert, when performing an inspection of sterile product manufacture. An expert adviser should be considered for an in-depth assessment. Let us now look at the different methods of sterilisation. We will first look at the basic principles of heat sterilisation, and then review the different methods. Methods of Terminal Sterilization Sterilization by heat Sterilization by moist heat Sterilization by dry heat Sterilization by radiation Sterilization by gases and fumigants 6

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌 热力灭菌 记录每一灭菌过程，如，时间和温度 T温度: 最冷点经过验证 另一支独立探头 化学或生物指示剂 加热阶段 :充足的时间使整个装载受热 测定每个装载过程 冷却阶段: 灭菌环节后 防止污染的措施 灭菌冷却用水/气 Sterilization by heat There should be recording of each cycle, e.g. time and temperature chart as part of the monitoring process of the sterilization cycle. The temperature should be monitored from the point that was validated as the coolest part of the sterilizer. It is advisable that a check is done from second independent probe. Additional chemical or biological indicators should be used. There is a heating phase – and sufficient time for the whole load to reach the required temperature should be allowed. This should be determined for each load. After sterilization, there is a cooling phase. What precautions can a manufacturer take to prevent contamination during this cooling phase? Use of sterilized cooling fluid/gas? Verify that all sterilization cycles are monitored using appropriate recording equipment. The accuracy and precision of the equipment should have been validated. This is applicable to at least monitors for temperature and time. This must provide a record of all the cycle parameters. The probes for determining temperature must be situated at the coolest part of the loaded chamber so that they are recording the worst case situation. A second independent probe should also be placed in the same position. The charts from these recorders must form part of the batch processing records. The recording of the cycle time should not commence until this heating period has been completed.The manufacturer should also show that any leaking container would not be approved for release or use. 6.2 – 6.3

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌 湿热灭菌（灭菌釜） 仅用于可被水润湿的材料及水溶性配方 监测温度、时间和压力 记录仪表和控制装置彼此独立 独立的温度指示剂 排水 – 从这个位置开始记录温度 如果真空是整个环节的一部分，则要定期进行泄露试验 物料允许空气排除和蒸汽穿透 所有装载部位接触蒸汽 蒸汽质量-无污染 Sterilization by moist heat (heating in an autoclave) The method can be used for water-wettable materials only, and aqueous formulations. During sterilization, the temperature, time and pressure should be monitored. The temperature recorder should be independent of the controller. An additional independent temperature indicator can be used. If the autoclave is fitted with a drain, then the temperature should be recorded from this position. A regular leak test should be done when vacuum is part of the cycle Material used to pack materials / product should allow for the removal of air and penetration of steam All parts of the load has to come in contact with steam to ensure sterilization. You have to check that the manufacturer has procedures, specifications, and test methods to ensure that the quality of the steam used is acceptable and that it cannot be an accidental source of contamination 6.4 – 6.6

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌 干热灭菌 仅适用于非水溶液、干粉 腔室内保持空气循环 腔室内保持正压以防止含菌空气进入 过滤过的洁净空气 去热原，挑战试验 验证（使用内毒素） Sterilization by dry heat This method is used mainly for non-aqueous liquids and dry powders. Air is circulated in the chamber and there should be a positive pressure in chamber to prevent entry of non-sterile air. The air should be filtered through a HEPA filter When removing pyrogens (e.g. sterilization and depyrogenation of glass ampoules), challenge tests have to be done. The validation includes the use of endotoxins. 6.7

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌 辐射灭菌 适用于对热敏感的原料和产品的灭菌 不得采用紫外灭菌 确认方法对物料的适合性 委托服务 – 确保验证状态、责任 剂量测定 放射剂量测定仪 定量测定 数量、位置和校验时间限度 生物指示剂仅作为附加手段 辐射敏感性变色指示片 Sterilization by radiation Suitable for heat sensitive materials and products. The manufacturer has to confirm the suitability of this method for material. Ultraviolet irradiation not acceptable. When the manufacturer is contracting the service out to a contract acceptor, the manufacturer still has to ensure the validation status and specify the responsibilities. Measurement of dose during procedure should be done. The dosimeters should be independent of dose rate quantitative measurement number, location and calibration time-limit Biological indicators can be used only as additional control measure. Radiation sensitive colour discs can be used 6.8 – 6.10

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌 辐射灭菌 (2) 灭菌情况记入批记录 进行验证，以掌握包装材料密度变化的作用 采取措施防止已灭菌和未灭菌物料混料 每份灭菌物品上都应贴有辐射敏感性指示片 预定时间段内的总放射剂量 All the information of the sterilization should form part of the batch record Validation should cover the effects of variation in density of packages, as the packaging material can have an influence on the effectiveness of the sterilization. Handling procedures to prevent misidentification of irradiated and non-irradiated materials should be in place. Each package should have a radiation-sensitive indicator. The total radiation dose should be administered within a predetermined period of time. 6.10 – 6.13

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌 气体灭菌、熏蒸灭菌 仅适用于无其它可行的灭菌方法时 如 环氧乙烷, 过氧化氢蒸汽 验证：证明气体对产品无危害 排气时间和条件 (规定限度) - 残留 直接作用于微生物细胞 包装材料的性质和数量 湿度和温度的平衡 Sterilization by gases and fumigants Gas and fumigants should only be used when no other method is suitable. Materials used include ethylene oxide, hydrogen peroxide vapour. Validation here is essential, and the manufacturer also has to prove that the gas has no damaging effect on product Time and conditions for degassing (specified limits) should be specified, as there should be no residue remaining on the product. In this process, direct contact with microbial cells is essential and therefore here also the nature and quantity of packaging materials play an important role in ensuring effectiveness of the sterilization method. Humidity and temperature equilibrium should be reached and each cycle should be monitored with biological indicators. Other parameters to be monitored include time, pressure, temperature, humidity and the gas concentration. 6.14 – 6.20

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌 对每一灭菌过程都应监控 时间、压力 温度、湿度 气体浓度 气体灭菌、熏剂灭菌 (2) 灭菌后的贮存– 规定方式 通风 残留气体的规定限度 验证过的工艺 安全和毒性事宜 One of the problems with ETO is the residues that are left behind at the end of the cycle. The processing cycle must include a validated degassing period, where the load must be stored in a suitably ventilated room under quarantine. The gas is explosive in air at relatively low concentrations and results in significant residues in the product that need to be removed before the batch can be passed. The load should be stored in a ventilated area after sterilization. The product should not be released until the residual gas has fallen to the defined level. There should be validation data for this process. This method is used for plastic items such as medical devices that are both heat and radiation-sensitive. The cycle is a combination of time, temperature, humidity and gas concentration. The first three parameters are generally recorded directly, while the last is recorded indirectly. The volume of gas used is also calculated by weighing the cylinders before and after the cycle to cross-check that the amount used is as expected. If you are going to inspect a facility using ETO gas, then specialist support should be considered 6.21

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌产品 Trainer to discuss the tabel 4.6 – 4.7

World Health Organization 无菌药品的生产 2017年3月10日星期五 最终灭菌产品 Trainer to discuss the tabel 4.8 – 4.9

World Health Organization 无菌药品的生产 2017年3月10日星期五 无菌生产工艺和过滤灭菌 无菌生产工艺 由无菌原料制备无菌产品 防止微生物污染的操作条件 你认为哪些方面需要特别注意？ Aseptic processing and sterilization by filtration Aseptic processing We have already discussed the fact that terminal sterilization of a product is preferable as it reduces the risk of and provides more assurance of sterility. However, for some types of products, this is not possible. Another method to prepare a sterile products then is to maintain the sterility of a product, assembled from sterile components. All operating conditions should be such to prevent microbial contamination. What do you think are the aspects that require careful attention? 7.1 – 7.2

World Health Organization 无菌药品的生产 2017年3月10日星期五 无菌生产工艺和过滤灭菌 无菌生产工艺 (2) 特别注意: 环境 人员 关键表面 容器、包装灭菌 转移操作 灌装前最长保持时间 Careful attention should be paid to ensure that there is no risk of contamination due to: Environment Personnel Critical surfaces Container/closure sterilization Transfer procedures Maximum holding period before filling The trainer can facilitate discussion around these points 7.3

World Health Organization 无菌药品的生产 2017年3月10日星期五 无菌生产 Discuss the table 4.10, 4.11, 4.14

World Health Organization 无菌药品的生产 2017年3月10日星期五 无菌生产 Discuss the table 4.10 – 4.13

World Health Organization 无菌药品的生产 2017年3月10日星期五 过滤灭菌 通过孔径≤ 0.22 µm的无菌过滤器，过滤进已灭过菌的容器 去除细菌和霉菌 不能滤除病毒和支原体 以一些热处理为辅助 灌装前双层膜过滤或二次过滤 - 无纤维脱落或不含石棉 过滤器使用后及时进行完整性测试 可能的话，使用前也测试 For some types of products, such as vaccines and insulin, sterilization by heat is not possible. In this case, sterilization by filtration into a previously sterilized container can be used. The filter should have a nominal pore size of no more than 0.22µm. However, it should be remembered that although these filters can remove bacteria and moulds, viruses and mycoplasmas might not be removed by this method. In order to reduce the risks associated with the filtration method, double filtration may be advisable. There is usually a pre-filter before the main one anyway, but in addition, a final filter, just prior to filling, should also be used where possible. Fibre shedding filters and asbestos filters may not be used. There should be documented evidence of filters integrity tests having been performed after use and in some cases also before use. This requires the use of equipment such as a bubble-point tester. In addition, validation of the method will have produced standard times and pressure differentials for a given volume of liquid. Any variations from this should be noted and investigated. 7.4 – 7.7

World Health Organization 无菌药品的生产 2017年3月10日星期五 过滤灭菌 (2) 验证包括 已知体积的过滤时间 过滤器的压差 注意并调查重大变化，记载在批记录上 过滤器使用后应检查滤膜完整性，其他过滤器隔一定时间也要检 查 Validation should include the time taken to filter a known volume of product and the pressure difference to be used across the filter. If during routine production, any significant differences are noted, these should be investigated, and recorded in batch records. Integrity of gas and air vent filters should be checked after use, and other filters at appropriate intervals 7.7

World Health Organization 无菌药品的生产 2017年3月10日星期五 过滤灭菌 (3) 过滤器使用不超过一个工作日，除非经过验证 过滤器和产品无相互作用，如 吸附产品组份 有脱落物 Filters should not be used for more than one working day, unless longer use has been validated. Filters should further not interact with the products, including removal of ingredients or releasing of substances into the product. 7.8 – 7.9

World Health Organization 无菌药品的生产 2017年3月10日星期五 质量控制 进行无菌检测的样品应有代表性 批产品中风险最大的 无菌灌装 - 每批生产开始、结束时，或有偏差发生时 热力灭菌 – 装载中的最冷点 通过验证确保批产品无菌 验证灭菌环节 培养基灌封 根据药典进行无菌检查，针对每种产品验证 核查批加工记录、无菌检查记录、环境监测记录 In this final part of the module, we are also going to look at the quality control. Testing for sterility is a destructive test, and one cannot test every ampoule that comes from the batch. It is therefore necessary to ensure that samples for sterility testing are representing the batch, and where possible, the worst case scenario. Samples should be taken from parts of the batch that are most at risk. These are: Aseptic filling - at beginning and end of batch filling, and after interruptions Heat sterilized – coolest part of the load. As sterility of the batch can not be ensured through testing, it has to be ensured through validation. This includes qualification of the equipment, supporting systems, process and sterilization cycle. For aseptic processes, media fills have to be done at regular intervals. The sterility test procedure should be as per pharmacopoeia, and validated for each product. Batch processing records, sterility testing records, environmental records should be reviewed as part of the batch evaluation and release procedure. 2.1 -2.2

World Health Organization 无菌药品的生产 2017年3月10日星期五 质量控制 注射剂的内毒素试验 水，半成品，成品 总是针对大输液 产品的检查方法为经过验证的药典方法 实验失败– 调查 纠偏措施 Injectable products should also be tested for Endotoxin levels. This includes tests on Water for injection, intermediate and finished product. It should always be done for large volume infusion solutions. The Pharmacopoeia method should be used and it should be validated for each product In case of failure of the test, a thorough investigation has to be done, the cause identified and recorded, and corrective action taken to prevent possible recurrence. 2.3

World Health Organization 无菌药品的生产 2017年3月10日星期五 成品 已验证的密封工艺 完整性检查 真空（适宜的地方）保持情况检查 肠外用药品逐个检查 在适合的控制条件下进行肉眼检查 照明和背景 操作者视力检查 常休息 其他方法 经验证的、不定期的设备运行检查 结果记录 The closing and sealing of containers of sterile products, should be done in accordance with validated methods. Review the validation protocols and reports for the sealing of the containers such as ampoules and vials. You should further evaluate how the manufacturer takes samples of products to check the integrity of the seals. Evaluate the SOP and compliance with the SOP for this process. When containers are sealed under vacuum – samples should be taken and tested at regular intervals as specified in the SOP or batch document. Parenteral products should be individually inspected for foreign particles, pieces of glass, cracks and other contaminants. You should inspect the area where the inspection is performed to assess whether the inspection is done visually or by using automated equipment. When visual inspection is done, assess whether this is done under illumination and background, whether operators have regular eyesight checks, regular breaks are given to rest their eyes, and whether their performance had been validated. (Trainer can elaborate). If automated equipment is used, then the equipment should have been subjected to validation/qualification. 11.1 – 11.3

World Health Organization 无菌药品的生产 World Health Organization 2017年3月10日星期五 分组讨论 3 就前组讨论的同一工厂，制订该厂的监测计划 列出需要测试的参数、试验项目、合格标准和试验频率 We now move into the final group session. Using the same hypothetical factory as before, review all the monitoring that will be required. List the parameters that need to be tested, the tests that should be used and the acceptance criteria. Finally, propose a programme for the monitoring that covers the frequency for each different test. (Refer to the supplementary notes giving an outline of the sort of responses that may be expected)