灭菌的方法及注意事项.pptx

1 1Sterilization 灭菌灭菌A General Discussion from CGMP Perspective从从CGMP角度综合讨论角度综合讨论灭菌的方法及注意事项第1页2 2Outline 讨论纲要讨论纲要nPART InTerminology 专业用词定义专业用词定义nGMP Requirements in The Manufacture of Sterile Pharmaceutical Products无菌药品生产过程中无菌药品生产过程中GMP基本要求基本要求nPART IInFacility Design 生产设施设计生产设施设计nHVAC System 空调系统空调系统nEnvironmental Monitoring(EM)环境监视环境监视nPharmaceutical Water 制药用水制药用水nCleaning/Sanitation 清洁消毒清洁消毒nPersonnel 无菌区操作人员无菌区操作人员nPART IIInMethods of sterilization 灭菌方法灭菌方法 nSterile Production and validation 无菌生产和验证无菌生产和验证nThe Trend of Sterile Production 无菌产品生产趋势无菌产品生产趋势灭菌的方法及注意事项第2页3Presentation Outline概要概要nUS Regulations 美国法规美国法规nMoist Heat Sterilization 湿热灭菌湿热灭菌nDry Heat/Depyrogenation 干热干热/去热原法去热原法nSterilization Process Validation 灭菌工艺验证灭菌工艺验证nOther Sterilization Methods 其它灭菌方法其它灭菌方法灭菌的方法及注意事项第3页4Code Federal Regulation美国联邦法规美国联邦法规211.84(c)(3)sterile equipment 灭菌设备灭菌设备nSterile 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。必要时应使用灭菌设备和无菌取样技术。211.94(c)and(d)sterilizedn(c)Drug product containers and closures 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依据药品性质和要求，进行灭菌，除热药品容器和密封系统应清洁并依据药品性质和要求，进行灭菌，除热原过程以确保预期用途。原过程以确保预期用途。n(d)Standards or specifications,methods of testing,and,where indicated,methods 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规格或质量标准，测试方法，清洁应建立并执行对药品容器和密封系统规格或质量标准，测试方法，清洁方法，灭菌和除热原过程相关书面程序方法，灭菌和除热原过程相关书面程序。灭菌的方法及注意事项第4页5Code Federal Regulation美国联邦法规美国联邦法规211.113(a)and(b)sterilization 灭菌灭菌n(a)Appropriate written procedures,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预防非无菌药品被致病菌污染相关书面程序。应该建立并执行用于预防非无菌药品被致病菌污染相关书面程序。n(b)Appropriate written procedures,designed to prevent microbiological contamination of drug products purporting to be sterile,shall be established and followed.Such procedures shall include validation of any sterilization process.应该应该建立建立并执行用于阻止无菌药品并执行用于阻止无菌药品被致病菌污染被致病菌污染相关书面程序。相关书面程序。这些程序应包含任何无菌工艺验证。这些程序应包含任何无菌工艺验证。灭菌的方法及注意事项第5页6Code Federal Regulation美国联邦法规美国联邦法规211.167(a)testing（a）测试测试nFor each batch of drug product purporting to be sterile and/or pyrogen-free,there shall be appropriate laboratory testing to determine conformance to such requirements.The test procedures shall be in writing and shall be followed.对无菌和对无菌和/或无热原每批药品应进行对应试验室测试以确定其与或无热原每批药品应进行对应试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。符合要求。测试程序应有书面文件并遵照执行。灭菌的方法及注意事项第6页7Sterilization Methods灭菌方法灭菌方法nMoist Heat:湿热湿热For drugs and devices.The mode of action is protein denaturation.用于药品和设备。作用方式是使蛋白质变性用于药品和设备。作用方式是使蛋白质变性nDry Heat:干热干热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用于去热原和设备灭菌。作用方式是使蛋白质变性nEthylene Oxide:氧化乙烯氧化乙烯 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主要用于设备。作用方式是使核酸烷基化nRadiation:辐射辐射Primarily for devices.The mode of action is DNA strand breakage 主要用于设备。作用方式是使主要用于设备。作用方式是使DNA链破坏链破坏nOther methods?其它方法？其它方法？灭菌的方法及注意事项第7页8Bioburden生物负荷生物负荷nDefinition:定义定义Population of viable microorganisms on or in a product and/or a package 产品和产品和/包装上活性微生物数量和类型包装上活性微生物数量和类型nMixture of organisms 有机物混合有机物混合 nIndigenous microflora 地方微植物群地方微植物群 nNeeds to be inactivated by sterilization 需灭菌失活需灭菌失活灭菌的方法及注意事项第8页9Bioburden生物负荷生物负荷Bioburden Sources 生物负荷起源生物负荷起源nEnvironment 环境环境Product contact surface,personnel,air 产品接触表面，人员，空气产品接触表面，人员，空气nMaterials 材料材料Water,raw materials.plastic,paper 水，原材料，塑料，纸张水，原材料，塑料，纸张Characteristics of Bioburden 生物负荷特点生物负荷特点nTypes of microorganisms 微生物类型微生物类型nResistance to sterilization process 对无菌工艺耐受对无菌工艺耐受nNumber of organisms 有机物数量有机物数量灭菌的方法及注意事项第9页10Biological Indicator(BI)生物指示剂生物指示剂nMicrobiological 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微生物测试系统对指定灭菌工艺有明确抵抗性。nA characterized preparation of specific microorganisms resistant to a particular sterilization process某一确定微生物某一确定微生物(指示剂指示剂)应含有对某一特定灭菌工艺抵抗性应含有对某一特定灭菌工艺抵抗性灭菌的方法及注意事项第10页11Typical Biological Indicators经典生物指示剂经典生物指示剂nMoist heat sterilization 湿热灭菌湿热灭菌nGeobacillus stearothermophilusnBacillus stearothermophilusnDry heat and EO sterilization 干热和环氧乙烷灭菌干热和环氧乙烷灭菌nBacillus atrophaeusnBacillus subtilis var.niger灭菌的方法及注意事项第11页12Forms of Biological Indicators生物指示剂形式生物指示剂形式nStrips/discs in glassine envelopes 在透明纸信封里条形板在透明纸信封里条形板/光盘光盘nStrips/discs 条条/光盘光盘nSelf-contained 独立包装独立包装nAmpoules 安瓶安瓶nStrips with medium 中号条形板中号条形板nLiquid suspension 液体悬浮液液体悬浮液nMetal 金属金属nFiberglass 玻璃纤维玻璃纤维灭菌的方法及注意事项第12页13Choice of an Appropriate BI相关生物指示剂选择相关生物指示剂选择nSterilization process 灭菌工艺灭菌工艺nCycle design method 循环设计方法循环设计方法nProduct bioburden 产品生物负荷产品生物负荷nPopulation 数量数量nResistance 抵抗性抵抗性灭菌的方法及注意事项第13页14BIs Prepared by User生物指示剂准备生物指示剂准备nPerformance 性能性能nResistance 抵抗性抵抗性nPopulation 数量数量nPurity 纯度纯度nD-value D 值值nRecovery methods 恢复方法恢复方法nStorage requirements 储存要求储存要求灭菌的方法及注意事项第14页15Biological Indicator Use生物指示剂使用生物指示剂使用nPlace BI within 把把BI 放进放进nProduct 产品产品nPackage 包装包装nSterilizer load to monitor process 灭菌器负荷以监视灭菌工艺灭菌器负荷以监视灭菌工艺nExpose to sterilizing conditions 暴露在灭菌状态暴露在灭菌状态nRemove BI and test 移除移除BI和测试和测试nCount survivors 生存数量计算生存数量计算nGrowth/no growth response 生长生长/无生长反应无生长反应灭菌的方法及注意事项第15页16D-value D值值nThe 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值是在特定条件下微生物数量降低90%（或一对数）所用时间，通常是（或一对数）所用时间，通常是以分钟为单位。以分钟为单位。nBacillus stearothermophilus has a D value:嗜热脂肪芽胞杆菌D值2 min at 121oC 20 min at 110oC 0.2-0.3 min at 130oC nOf 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 not acceptable from CGMP perspective 灭菌验证中，灭菌验证中，D值可能是最主要。不考虑值可能是最主要。不考虑D值验证过程多半是无效且不被值验证过程多半是无效且不被CGMP接收。接收。灭菌的方法及注意事项第16页17Z-value Z值值nZ-value:number of degrees of temperature required for a 1 logarithm change in the D-value Z值：值：D值值1对数改变需要温度数对数改变需要温度数nZ=-1/slope of the thermal resistance curve Z=-1/热阻力曲线斜率热阻力曲线斜率 whereSlope=logarithmic change in D-value/change in temperature 斜率斜率=D值上对数改变值上对数改变/温度改变温度改变灭菌的方法及注意事项第17页18Impact of Z-value Z值影响值影响nWhen z-value is small,considerably less inactivation will result below reference temperature and considerably more above the reference temperature 当当Z值较小时，较低程度失活将造成温度低于参考温度，较大程度失活将造成温值较小时，较低程度失活将造成温度低于参考温度，较大程度失活将造成温度高于参考温度度高于参考温度。灭菌的方法及注意事项第18页19Typical Temperature Profiles经典温度分布经典温度分布灭菌的方法及注意事项第19页20Type of Sterilization灭菌类型灭菌类型Moist Heat Sterilization湿热灭菌湿热灭菌灭菌的方法及注意事项第20页21Moist Heat Sterilization湿热灭菌湿热灭菌Characteristics:特征特征nWell understood and well characterized process,first validated process in pharm industry湿热灭菌是一已被很好地了解并描述灭菌工艺，也是在制药工业中第一个湿热灭菌是一已被很好地了解并描述灭菌工艺，也是在制药工业中第一个被验证灭菌工艺被验证灭菌工艺nSuitable for a wide variety of applications 适合于较大应用范围适合于较大应用范围nEquipment is readily available 设备很轻易从市场购置到设备很轻易从市场购置到nCost on a per use basis is low 每次使用基准花费低每次使用基准花费低灭菌的方法及注意事项第21页22Moist Heat Sterilization湿热灭菌湿热灭菌Applications:应用应用nTerminal sterilization of parental product 注射剂终端灭菌注射剂终端灭菌nSterilization of equipment and components for use in aseptic filling 无菌灌装线上设备和配件灭菌无菌灌装线上设备和配件灭菌nSterilization of laboratory materials 试验室用材料灭菌试验室用材料灭菌nIn-situ sterilization of process piping and equipment(SIP)工艺管道和设备在线灭菌工艺管道和设备在线灭菌 灭菌的方法及注意事项第22页23Basic Types of Moist Heat Sterilization湿热灭菌基本类型湿热灭菌基本类型nSaturated steam 饱和蒸汽饱和蒸汽nAutoclaves(self-closing)高压灭菌柜（半封闭）高压灭菌柜（半封闭）nSIP 在线灭菌在线灭菌nSuper heated water 过热水过热水nSpray 喷雾喷雾nSubmerged 浸没浸没nSIP 在线灭菌在线灭菌nSteam-air-mixture(SAM)水蒸气空气混合物水蒸气空气混合物灭菌的方法及注意事项第23页24Basic Elements of Sterilization Process Validation 灭菌工艺验证基本元素灭菌工艺验证基本元素1.Empty vessel heat distribution 空容器热分布空容器热分布2.Heat distribution and penetration 热分布和渗透热分布和渗透3.Microbiological challenges 微生物挑战微生物挑战灭菌的方法及注意事项第24页25Steam Sterilization Validation:Prerequisites 蒸汽灭菌验证：前提蒸汽灭菌验证：前提1.OQ for an autoclave:高压灭菌柜运行确认高压灭菌柜运行确认nEmpty chamber temperature mapping within 1.0oC of the mean 空腔体温度分布图在平均值空腔体温度分布图在平均值1.0oC 内内nChamber integrity test(no leaking)腔体完整性测试腔体完整性测试 nCertification 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空气用于隔断真空或通气过滤器完整性测试空气HEPA过滤认证过滤认证2.Requirements for SIP 在线灭菌要求在线灭菌要求nTemperature mapping 温度分布图温度分布图nAn integrity test,where appropriate 相关完整性测试相关完整性测试nUse of BI 生物指示剂使用生物指示剂使用3.All critical instruments must be calibrated 所相关键仪器需校验所相关键仪器需校验灭菌的方法及注意事项第25页26Steam Sterilization Validation:Prerequisites 蒸蒸汽灭菌验证：前提汽灭菌验证：前提4.Acceptable test results for non-condensable gases,super-heated steam and dryness should be obtained for the clean steam used for the autoclave/SIP 应取得用于高压灭菌柜应取得用于高压灭菌柜/SIP洁净蒸汽中不凝气体，过热蒸汽洁净蒸汽中不凝气体，过热蒸汽及干燥度可接收测试结果及干燥度可接收测试结果5.Tools for the conduct of the PQ study:进行进行PQ研究工具研究工具oBI with 106 spores and known D and Z values BI 有有106个孢子，已知个孢子，已知D值和值和Z值值oTemperature sensors 温度传感器温度传感器oRecording device capable of supporting 12 temp sensors with an accuracy of 0.5 oC,recording data every minute or less 统计设备能支撑统计设备能支撑 12温度传感器，精度温度传感器，精度 0.5 oC，每分，每分钟或间隔更短时间统计数据钟或间隔更短时间统计数据oMeans of introducing temp sensors into the autoclave/SIP 将温度传将温度传感器导入高压灭菌柜感器导入高压灭菌柜/SIP方法方法灭菌的方法及注意事项第26页27Steam Sterilization Validation:Preparation of PQ protocol 蒸汽灭菌验证：蒸汽灭菌验证：PQ方案准备方案准备1.A protocol shall be prepared for:对于以下各项应建立方案：对于以下各项应建立方案：nNew autoclave/SIP 新高压灭菌柜新高压灭菌柜/SIPnNew loading patterns or product configurations 新装料方式或产品配置新装料方式或产品配置nChanges to existing patterns 对现有装料方式变更对现有装料方式变更nChanges to operation cycle parameters 对运行周期参数变更对运行周期参数变更nMajor change to equipment as directed by change control 变更控制要求设备主要变更变更控制要求设备主要变更灭菌的方法及注意事项第27页28Steam Sterilization Validation:Preparation of PQ protocol 蒸汽灭菌验证：蒸汽灭菌验证：PQ方案准备方案准备2.The protocol may include:方案可能包含：方案可能包含：nObjectives of the validation study 验证研究目标验证研究目标nIdentification and description of the sterilizer and its process controls 灭菌器识别和说明及工艺控制灭菌器识别和说明及工艺控制nIdentification of SOPs for the process equipment 工艺设备工艺设备SOP识别识别nDescription of or SOP reference for instrument calibration procedures 仪器校验程序说明或仪器校验程序说明或SOP参考参考nIdentification 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识别，包含一个两点预运行校验和每次运行温度监测设备校验程序识别，包含一个两点预运行校验和每次运行后确实认后确实认nProcess parameter acceptance criteria 工艺参数验收标准工艺参数验收标准灭菌的方法及注意事项第28页29Steam Sterilization Validation:Preparation of PQ protocol 蒸汽灭菌验证：蒸汽灭菌验证：PQ方案准备方案准备3.A description of the following:以下说明以下说明nBiodurden determination studies 生物负荷确认研究生物负荷确认研究nEmpty chamber heat distribution studies(1 oC)空腔体热分布研究（空腔体热分布研究（1 oC）nLoaded chamber(including Load configuration,max lading and min loading)heat penetration studies 满载腔体（包含装载配置满载腔体（包含装载配置,最大和最少）热穿透研究最大和最少）热穿透研究nContainer mapping studies(may not needed if 100mL)容器分布图研究（假如容量容器分布图研究（假如容量100mL，可能不需要），可能不需要）nMicrobiological challenge studies 微生物挑战研究微生物挑战研究nEvaluation of drug product cooling water(where applicable)药品冷却水评定（若适用）药品冷却水评定（若适用）nEvaluation of vent filter membranes associated with the sterilizer 与灭菌器相关通气过滤膜评定与灭菌器相关通气过滤膜评定灭菌的方法及注意事项第29页30Steam Sterilization Validation:Preparation of PQ protocol 蒸汽灭菌验证：蒸汽灭菌验证：PQ方案准备方案准备4.Description of the temp sensor placement within the load 在负荷范围内温度传感器位置说明在负荷范围内温度传感器位置说明nOne next to the temp controlling sensor for autoclave.一一1个靠近高压灭菌柜温度控制传感器个靠近高压灭菌柜温度控制传感器nNLT 12 sensors for autoclave 不少于不少于12 个传感器用于高压灭菌柜个传感器用于高压灭菌柜nNLT 3 sensors for SIP不少于不少于3传感器用于在线灭菌传感器用于在线灭菌i.If the temp controlling sensor is not in the drain,an additional sensor shall be placed in the drain 假如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假如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ii.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 in 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最少应建立符合校验和验证最少#传感器。最终任何传感器失败应调查研究。传感器。最终任何传感器失败应调查研究。任何临近温度控制传感器或在排水口传感器应符合预校验和后校验，这是任何临近温度控制传感器或在排水口传感器应符合预校验和后校验，这是成功运行验收标准一部分。成功运行验收标准一部分。灭菌的方法及注意事项第30页31Steam Sterilization Validation:Preparation of PQ protocol 蒸汽灭菌验证：蒸汽灭菌验证：PQ方案准备方案准备5.Acceptance criteria for temp.pressure,Fo where appropriate,vacuum and destructions of BIs 温度压力，相关温度压力，相关Fo，真空及生物指示剂灭活验收标准，真空及生物指示剂灭活验收标准6.A requirement for 3 consecutive,successful runs 3次连续成功运行要求次连续成功运行要求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 to chamber distribution requirement.排水口是最冷点，在灭菌区外，且不包含在平均腔体温度计算内，不受限于腔体排水口是最冷点，在灭菌区外，且不包含在平均腔体温度计算内，不受限于腔体分布要求分布要求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仅在稳定状态下要求均一性灭菌的方法及注意事项第31页32Steam Sterilization Validation:Execution of PQ 蒸汽灭菌验证：蒸汽灭菌验证：PQ执行执行1.Temperature sensor shall:温度传感器应温度传感器应：nFor 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，并包含在产品中含在产品中nFor heat distribution data,be evenly distributed within 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:生物指示剂应生物指示剂应：nBe placed near the tip of the temperature sensor used for penetration 置于靠近用于穿透温度传感器顶端置于靠近用于穿透温度传感器顶端nInclude the identification that links to the number of the appropriate temperature sensor of the BI 包含与相关包含与相关BI温度传感器数量相关识别温度传感器数量相关识别i.The D value of the BI shall be determined in the product BID值应在生产中确定值应在生产中确定灭菌的方法及注意事项第32页33Steam Sterilization Validation:Execution of PQ 蒸汽灭菌验证：蒸汽灭菌验证：PQ执行执行3.The autoclave shall be loaded and operated in accordance with SOP 高压灭菌器应按照高压灭菌器应按照SOP进行装载并运行进行装载并运行4.Using 121.1 oC as the base temperature and 10 oC as the Z-value,Fo shall be calculated 121.1 oC 作为基底温度，作为基底温度，10 oC 作为作为Z值，值，Fo应为计算值应为计算值5.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验收标准应依据暴露BI上未生长和阳性控制上未生长和阳性控制BI上生长上生长nFailure to meet acceptance criteria shall require cycle parameter modifications,load pattern modification and/or equipment repairs/corrections 未到达验收标准应要求周期参数变更，负荷曲线图变更和未到达验收标准应要求周期参数变更，负荷曲线图变更和/或设备修或设备修理理/修正修正灭菌的方法及注意事项第33页34Case Study 实例分析实例分析Autoclave Sterilization Validation 高压蒸汽灭菌验证高压蒸汽灭菌验证Table 1 Acceptance Criteria of Empty Chamber Heat Distribution表表1 空腔体热分布验收标准空腔体热分布验收标准No.Acceptance Criteria验收标准验收标准1The 12 TCs distributed throughout the chamber must be within 1C of the mean thermocouple temperature each minute after stabilization throughout the duration of the sterilization