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1、1,1,Sterilization 灭菌,A General Discussion from CGMP Perspective 从CGMP角度的综合讨论,2,2,Outline 讨论纲要,PART I Terminology 专业用词的定义 GMP Requirements in The Manufacture of Sterile Pharmaceutical Products无菌药品生产过程中GMP的基本要求 PART II Facility Design 生产设施的设计 HVAC System 空调系统 Environmental Monitoring (EM) 环境的监视 Pharma
2、ceutical Water 制药用水 Cleaning/Sanitation 清洁消毒 Personnel 无菌区操作人员 PART III Methods of sterilization 灭菌方法 Sterile Production and validation 无菌生产和验证 The Trend of Sterile Production 无菌产品生产趋势,3,Presentation Outline概要,US Regulations 美国法规 Moist Heat Sterilization 湿热灭菌 Dry Heat/Depyrogenation 干热/去热原法 Steriliz
3、ation Process Validation 灭菌工艺验证 Other Sterilization Methods 其他灭菌方法,4,Code Federal Regulation美国联邦法规,211.84(c)(3) sterile equipment 灭菌设备 Sterile equipment and aseptic sampling techniques shall be used when necessary 必要时应使用灭菌设备和无菌取样技术。 211.94(c) and (d) sterilized (c) Drug product containers and closur
4、es shall be clean and, where indicated by the nature of the drug, sterilized and processed to remove pyrogenic properties to assure that they are suitable for their intended use. 药品容器和密封系统应清洁并根据药品的性质和要求，进行灭菌，除热原过程以确保预期的用途。 (d) Standards or specifications, methods of testing, and, where indicated, me
5、thods of cleaning, sterilizing, and processing to remove pyrogenic properties shall be written and followed for drug product containers and closures. 应建立并执行对药品容器和密封系统的规格或质量标准，测试方法，清洁方法，灭菌和除热原过程的相关书面程序。,5,Code Federal Regulation美国联邦法规,211.113(a) and (b) sterilization 灭菌 (a) Appropriate written proced
6、ures, designed to prevent objectionable microorganisms in drug products not required to be sterile, shall be established and followed. 应该建立并执行用于防止非无菌药品被致病菌污染的相关书面程序。 (b) Appropriate written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, shall
7、 be established and followed. Such procedures shall include validation of any sterilization process. 应该建立并执行用于阻止无菌药品被致病菌污染的相关书面程序。这些程序应包括任何无菌工艺的验证。,6,Code Federal Regulation美国联邦法规,211.167(a) testing （a）测试 For each batch of drug product purporting to be sterile and/or pyrogen-free, there shall be app
8、ropriate laboratory testing to determine conformance to such requirements. The test procedures shall be in writing and shall be followed. 对无菌和/或无热原的每批药品应进行相应的实验室测试以确定其与符合要求。测试程序应有书面文件并遵照执行。,7,Sterilization Methods灭菌方法,Moist Heat: 湿热 For drugs and devices. The mode of action is protein denaturation.
9、用于药品和设备。作用方式是使蛋白质变性 Dry Heat: 干热 For depyrogenation and equipment sterilization. The mode of action is protein denaturation 用于去热原和设备灭菌。作用方式是使蛋白质变性 Ethylene Oxide: 氧化乙烯 Primarily for devices. The mode of action is alkylation of nucleic acids. 主要用于设备。作用方式是使核酸烷基化 Radiation: 辐射 Primarily for devices. Th
10、e mode of action is DNA strand breakage 主要用于设备。作用方式是使DNA链破坏 Other methods? 其他方法？,8,Bioburden生物负荷,Definition: 定义 Population of viable microorganisms on or in a product and/or a package 产品和/包装上的活性微生物的数量和类型 Mixture of organisms 有机物混合 Indigenous microflora 地方微植物群 Needs to be inactivated by sterilization
11、 需灭菌失活,9,Bioburden生物负荷,Bioburden Sources 生物负荷来源 Environment 环境 Product contact surface, personnel, air 产品接触表面，人员，空气 Materials 材料 Water, raw materials. plastic, paper 水，原材料，塑料，纸张 Characteristics of Bioburden 生物负荷特点 Types of microorganisms 微生物类型 Resistance to sterilization process 对无菌工艺的耐受 Number of o
12、rganisms 有机物数量,10,Biological Indicator (BI)生物指示剂,Microbiological test system providing a defined resistance to a specific sterilization process 微生物测试系统对指定灭菌工艺有明确抵抗性。 A characterized preparation of specific microorganisms resistant to a particular sterilization process 某一确定的微生物(指示剂)应具有对某一特定灭菌工艺的抵抗性,1
13、1,Typical Biological Indicators典型生物指示剂,Moist heat sterilization 湿热灭菌 Geobacillus stearothermophilus Bacillus stearothermophilus Dry heat and EO sterilization 干热和环氧乙烷灭菌 Bacillus atrophaeus Bacillus subtilis var. niger,12,Forms of Biological Indicators生物指示剂形式,Strips/discs in glassine envelopes 在透明纸信封里
14、的条形板/光盘 Strips/discs 条/光盘 Self-contained 独立包装的 Ampoules 安瓶 Strips with medium 中号条形板 Liquid suspension 液体悬浮液 Metal 金属 Fiberglass 玻璃纤维,13,Choice of an Appropriate BI相关生物指示剂的选择,Sterilization process 灭菌工艺 Cycle design method 循环设计方法 Product bioburden 产品生物负荷 Population 数量 Resistance 抵抗性,14,BIs Prepared by
15、 User生物指示剂的准备,Performance 性能 Resistance 抵抗性 Population 数量 Purity 纯度 D- value D 值 Recovery methods 恢复方法 Storage requirements 储存要求,15,Biological Indicator Use生物指示剂使用,Place BI within 把BI 放进 Product 产品 Package 包装 Sterilizer load to monitor process 灭菌器负荷以监视灭菌工艺 Expose to sterilizing conditions 暴露在灭菌状态 Re
16、move BI and test 移除BI和测试 Count survivors 生存数量的计算 Growth/no growth response 生长/无生长反应,16,D-value D值,The D value is the time, usually in minutes, required to achieve inactivation of 90% (or one logarithm) of a population of the test microorganism at specified conditions. D值是在特定条件下微生物数量降低的90%（或一对数）所用的时间
17、，通常是以分钟为单位的。 Bacillus stearothermophilus has a D value: 嗜热脂肪芽胞杆菌的D值 2 min at 121oC 20 min at 110oC 0.2-0.3 min at 130oC Of all the aspects of sterilization validation, the D value is perhaps the most important. Validating a process without consideration of the D value is largely ineffective and is n
18、ot acceptable from CGMP perspective 灭菌验证中，D值可能是最重要的。不考虑D值的验证过程多半是无效的且不被CGMP接受。,17,Z-value Z值,Z-value: number of degrees of temperature required for a 1 logarithm change in the D-value Z值：D值1对数改变需要的温度数 Z = -1/slope of the thermal resistance curve Z = -1/热阻力曲线斜率 where Slope = logarithmic change in D-v
19、alue/change in temperature 斜率 = D值上对数的改变/温度的改变,18,Impact of Z-value Z值的影响,When z-value is small, considerably less inactivation will result below reference temperature and considerably more above the reference temperature 当Z值较小时，较低程度的失活将导致温度低于参考温度，较大程度的失活将导致温度高于参考温度。,19,Typical Temperature Profiles典
20、型温度分布,20,Type of Sterilization灭菌类型,Moist Heat Sterilization 湿热灭菌,21,Moist Heat Sterilization湿热灭菌,Characteristics: 特征 Well understood and well characterized process, first validated process in pharm industry 湿热灭菌是一已被很好地理解并描述灭菌工艺，也是在制药工业中第一个被验证的灭菌工艺 Suitable for a wide variety of applications 适合于较大应用范
21、围 Equipment is readily available 设备很容易从市场购买到 Cost on a per use basis is low 每次使用基准花费低,22,Moist Heat Sterilization湿热灭菌,Applications: 应用 Terminal sterilization of parental product 注射剂的终端灭菌 Sterilization of equipment and components for use in aseptic filling 无菌灌装线上设备和配件的灭菌 Sterilization of laboratory m
22、aterials 实验室用材料的灭菌 In-situ sterilization of process piping and equipment (SIP)工艺管道和设备在线灭菌,23,Basic Types of Moist Heat Sterilization湿热灭菌基本类型,Saturated steam 饱和蒸汽 Autoclaves (self-closing) 高压灭菌柜（半封闭） SIP 在线灭菌 Super heated water 过热水 Spray 喷雾 Submerged 浸没的 SIP 在线灭菌 Steam-air-mixture (SAM) 水蒸气空气混合物,24,B
23、asic Elements of Sterilization Process Validation 灭菌工艺验证的基本元素,Empty vessel heat distribution 空容器热分布 Heat distribution and penetration 热分布和渗透 3.Microbiological challenges 微生物挑战,25,Steam Sterilization Validation: Prerequisites 蒸汽灭菌验证：前提,OQ for an autoclave: 高压灭菌柜运行确认 Empty chamber temperature mapping
24、within 1.0oC of the mean 空腔体温度分布图在平均值的1.0oC 内 Chamber integrity test (no leaking) 腔体完整性测试 Certification of HEPA filtration on the air used to break vacuum or integrity testing of the vent filter 用于隔断真空或通气过滤器完整性测试的空气HEPA过滤认证 Requirements for SIP 在线灭菌要求 Temperature mapping 温度分布图 An integrity test, whe
25、re appropriate 相关完整性测试 Use of BI 生物指示剂的使用 All critical instruments must be calibrated 所有关键仪器需校验,26,Steam Sterilization Validation: Prerequisites 蒸汽灭菌验证：前提,Acceptable test results for non-condensable gases, super-heated steam and dryness should be obtained for the clean steam used for the autoclave/S
26、IP 应获得用于高压灭菌柜/SIP的洁净蒸汽中的不凝气体，过热蒸汽及干燥度的可接受测试结果 5. Tools for the conduct of the PQ study: 进行PQ研究的工具 BI with 106 spores and known D and Z values BI 有106个孢子，已知D值和Z值 Temperature sensors 温度传感器 Recording device capable of supporting 12 temp sensors with an accuracy of 0.5 oC, recording data every minute or
27、 less 记录设备能支撑 12温度传感器，精度 0.5 oC，每分钟或间隔更短时间记录数据 Means of introducing temp sensors into the autoclave/SIP 将温度传感器导入高压灭菌柜/SIP的方法,27,Steam Sterilization Validation: Preparation of PQ protocol 蒸汽灭菌验证：PQ方案的准备,A protocol shall be prepared for : 对于下列各项应建立方案： New autoclave/SIP 新高压灭菌柜/SIP New loading patterns
28、or product configurations 新装料方式或产品配置 Changes to existing patterns 对现有装料方式的变更 Changes to operation cycle parameters 对运行周期参数的变更 Major change to equipment as directed by change control 变更控制要求的设备主要变更,28,Steam Sterilization Validation: Preparation of PQ protocol 蒸汽灭菌验证：PQ方案的准备,The protocol may include: 方
29、案可能包括： Objectives of the validation study 验证研究的目的 Identification and description of the sterilizer and its process controls 灭菌器的识别和说明及工艺控制 Identification of SOPs for the process equipment 工艺设备SOP的识别 Description of or SOP reference for instrument calibration procedures 仪器校验程序的说明或SOP参考 Identification
30、of calibration procedures for temp-monitoring equipment, which include a two point pre-run calibration and a post-run verification for each run 温度监测设备校验程序的识别，包括一个两点预运行校验和每次运行后的确认 Process parameter acceptance criteria 工艺参数的验收标准,29,Steam Sterilization Validation: Preparation of PQ protocol 蒸汽灭菌验证：PQ方案
31、的准备,A description of the following:以下说明 Biodurden determination studies 生物负荷确认研究 Empty chamber heat distribution studies ( 1 oC) 空腔体热分布研究（ 1 oC ） Loaded chamber (including Load configuration, max lading and min loading) heat penetration studies 满载腔体（包括装载配置, 最大和最少）热穿透研究 Container mapping studies (may
32、 not needed if 100mL) 容器分布图研究（如果容量100mL ，可能不需要） Microbiological challenge studies 微生物挑战研究 Evaluation of drug product cooling water (where applicable) 药品冷却水评估（若适用） Evaluation of vent filter membranes associated with the sterilizer 与灭菌器相关的通气过滤膜的评估,30,Steam Sterilization Validation: Preparation of PQ p
33、rotocol 蒸汽灭菌验证：PQ方案的准备,4.Description of the temp sensor placement within the load 在负荷范围内温度传感器位置的说明 One next to the temp controlling sensor for autoclave. 一1个接近高压灭菌柜的温度控制传感器 NLT 12 sensors for autoclave 不少于12 个传感器用于高压灭菌柜 NLT 3 sensors for SIP不少于3传感器用于在线灭菌 If the temp controlling sensor is not in the
34、drain, an additional sensor shall be placed in the drain 如果温度控制传感器不在排水口中，应置另一个传感器于排水口中 A minimum # of sensors meeting the calibration and verification shall be established. Any sensor that fail at the end of the study shall be investigated. Any sensor located next to the temp controlling sensor or i
35、n the drain shall meet the pre and post calibration as part of the acceptance criteria for a successful run 应建立符合校验和验证的最少#传感器。最终任何传感器的失败应调查研究。任何临近温度控制传感器或在排水口的传感器应符合预校验和后校验，这是成功运行的验收标准的一部分。,31,Steam Sterilization Validation: Preparation of PQ protocol 蒸汽灭菌验证：PQ方案的准备,5.Acceptance criteria for temp. p
36、ressure, Fo where appropriate, vacuum and destructions of BIs 温度压力，相关Fo，真空及生物指示剂灭活的验收标准 6.A requirement for 3 consecutive, successful runs 3次连续成功运行的要求 The drain is the coldest point and is outside of the sterilization zone, not included in the calculation of mean chamber temperature and not subject
37、to chamber distribution requirement. 排水口是最冷点，在灭菌区外，且不包括在平均腔体温度计算内，不受限于腔体分布要求 Thermocouples should be placed at the same location for empty chamber and loaded chamber heat distribution studies. 热电偶应置于相同位置，进行空腔体和满载腔体热分布研究 Uniformity is expected only at the steady state. 仅在稳定状态下要求均一性,32,Steam Steriliza
38、tion Validation: Execution of PQ 蒸汽灭菌验证：PQ执行,1.Temperature sensor shall: 温度传感器应： For heat penetration data, be located such that they penetrate the equipment, or the product container and are immersed in the product 对于热穿透数据，应置于能穿透设备或产品容器处，并包含在产品中 For heat distribution data, be evenly distributed wit
39、hin the load or system and shall not contact metal surface 对于热分布数据，平均分布在负荷范围内或系统中，且不能接触金属面 2.The BI shall:生物指示剂应： Be placed near the tip of the temperature sensor used for penetration 置于靠近用于穿透的温度传感器的顶端 Include the identification that links to the number of the appropriate temperature sensor of the B
40、I 包括与相关BI温度传感器数量有关的识别 The D value of the BI shall be determined in the product BI的D值应在生产中确定,33,Steam Sterilization Validation: Execution of PQ 蒸汽灭菌验证：PQ执行,The autoclave shall be loaded and operated in accordance with SOP 高压灭菌器应按照SOP进行装载并运行 Using 121.1 oC as the base temperature and 10 oC as the Z-va
41、lue, Fo shall be calculated 121.1 oC 作为基底温度， 10 oC 作为Z值， Fo应为计算值 Acceptance criteria shall be based on no growth on exposed BIs and growth on the positive control BI 验收标准应依据暴露的BI上未生长和阳性控制BI上的生长 Failure to meet acceptance criteria shall require cycle parameter modifications, load pattern modification
42、 and/or equipment repairs/corrections 未达到验收标准应要求周期参数变更，负荷曲线图变更和/或设备修理/修正,34,Case Study 实例分析Autoclave Sterilization Validation 高压蒸汽灭菌验证,Table 1Acceptance Criteria of Empty Chamber Heat Distribution 表1 空腔体热分布验收标准,35,Case Study 实例分析Autoclave Sterilization Validation 高压蒸汽灭菌验证,Table 2Acceptance Criteria
43、for Heat Distribution 表2 热分布验收标准,36,Case Study 实例分析Autoclave Sterilization Validation 高压蒸汽灭菌验证,Table 3Acceptance Criteria for Heat Penetration 表3热分布验收标准,37,Dry Heat Sterilization 干热灭菌,Depyrogenation 去热原,38,Terminology and Definitions 术语和定义,Pyrogen: Fever producing substance: endotoxin, viruses, fung
44、i, toxin from gram(+) and gram(-) bacterials, peptiglycan 热原：热产生的物质：内毒素，病毒，真菌，革兰氏阳性和阴性细菌产生的毒素，肽糖 Endotoxin: Component from outer membrane of gram(-) bacterial, most potent pyrogens, LPS in structure 内毒素：革兰氏阴性细菌外膜的成分，大多数有效热原，结构中的LPS Environmental: complex of protein, carbohydrate mechanical circulati
45、on needed 空气密度，温度不同可能导致分层，需要机械循环 Heat degradation limits materials 热降解限制材料 Contraction during cooling may draw contaminants 冷却过程中接触可能引起污染,46,Depyrogenation by Tunnels 通过烘箱去热原,HEPA-filtered, vertical laminar air flow in heating and cooling zones, or radiant heaters in heating zone and vertical lamina
46、r air flow in cooling zone 在加热和冷却区经HEPA过滤的垂直层流气流，或加热区的辐射加热器和冷却区的垂直层流气流 Conveyor belt to provide in-line continuous flow of sterile glassware to aseptic area 传送带提供连续在线的无菌玻璃器皿到无菌区的流动 Limited to one type of load at a time 一次限用一种装载方式 Problem to control speed match other line equipment (filler) 控制速度以匹配其他
47、线设备（灌装机）的问题 Difficult to achieve uniform heating, and heat source may generate particles 很难达到均匀加热，热原可能产生颗粒 Large product volume needed to justify tunnel 调整烘箱要求大的产品容量,47,Validation of Depyrogenation Cycles去热原循环验证,Run engineering trials to find cold spots 进行试运行以找到冷点 Run trials to determine worse case
48、vial, e.g., thickest glass, packing effects 试车以确定最差状况的玻璃瓶，如最厚的玻璃，紧束效应 Procure or prepare endotoxin indicators 获得或制备内毒素指示剂 Run 3 full loads with endotoxin indicators in place at cold spots and random sites 进行3次满载运行，内毒素指示剂置于冷点和随机位置 Acceptance criteria 验收标准 min. 3 log reduction in EU for spiked vials 带
49、孔玻璃瓶3个对数减少值，单位为EU 3 successful runs with controls and documentation 受控并记录的3次成功运行,48,Case Study 实例分析Dry Heat Depyrogenation Tunnel 干热去热原隧道烘箱,Validation Acceptance Criteria 验证验收标准 A minimum of three successful validation runs must be performed. 必须至少进行三次成功的验证 Distribution thermocouple temperatures must be within 15oC of the set point temperature, after stabilization. 稳定后，分配热电偶温度必须在设定温度的 15oC 内 A minimum cumulative FH value of twelve minutes mus