灭菌的方法及注意事项课件

1、11Sterilization 灭菌A General Discussion from CGMP Perspective从CGMP角度的综合讨论11Sterilization 灭菌22Outline 讨论纲要PART ITerminology 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 IIFacility Design 生产设施的设计HVAC System 空调系统Environmental Monitoring (EM) 环境的监视Pha

2、rmaceutical Water 制药用水Cleaning/Sanitation 清洁消毒Personnel 无菌区操作人员PART IIIMethods 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22Outline 讨论纲要PART I3Presentation Outline概要US Regulations 美国法规Moist Heat Sterilization 湿热灭菌Dry Heat/Depyrogenation 干热/

3、去热原法Sterilization Process Validation 灭菌工艺验证Other Sterilization Methods 其他灭菌方法3Presentation Outline概要US Reg4Code 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)

4、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

5、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。4Code Federal Regulation美国联邦法5Code Federal Regulation美国联邦法规211.113

6、(a) and (b) sterilization 灭菌(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 conta

7、mination of drug products purporting to be sterile, shall 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。5Code Federal Regulation美国联邦法6Code Federal Regulation美国联邦法规211.167(a) testing （a）测试For each batc

8、h 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/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。6Code Federal Regulation美国联邦法

9、7Sterilization Methods灭菌方法Moist Heat: 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 m

10、ode 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. The 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？7Sterilization Methods灭菌方法Moi8Bioburden生物负荷Definition: 定义Population of viable microorganisms on or in a product and/or

11、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 需灭菌失活8Bioburden生物负荷Definition: 定义9Bioburden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. pla

12、stic, 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 organisms 有机物数量9Bioburden生物负荷Bioburden Sourc10Biological Indicator (BI)生物指示剂Microbiological test system providing a defined resistance to a specific sterilizat

13、ion 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10Biological Indicator (BI)生物11Typical Biological Indicators典型生物指示剂Moist heat sterilization 湿热灭菌Geobacillus stearothermophilusBacillus s

14、tearothermophilusDry heat and EO sterilization 干热和环氧乙烷灭菌Bacillus atrophaeusBacillus subtilis var. niger11Typical Biological Indicator12Forms of Biological Indicators生物指示剂形式Strips/discs in glassine envelopes 在透明纸信封里的条形板/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

15、suspension 液体悬浮液Metal 金属Fiberglass 玻璃纤维12Forms of Biological Indicato13Choice 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13Choice of an Appropriate BI14BIs Prepared by User生物指示剂的准备Performance 性能Resistance 抵抗性Po

16、pulation 数量Purity 纯度D- value D 值Recovery methods 恢复方法Storage requirements 储存要求14BIs Prepared by User生物指示剂的准15Biological 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Remove BI and test 移除BI和测试Count

17、 survivors 生存数量的计算Growth/no growth response 生长/无生长反应15Biological Indicator Use生物指16D-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，通常是以

18、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 130oCOf 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 acce

19、ptable 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。16D-value D值The D value is th17Z-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 whereSlope = logarith

20、mic change in D-value/change in temperature 斜率 = D值上对数的改变/温度的改变17Z-value Z值Z-value: number o18Impact 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

21、致温度高于参考温度。18Impact of Z-value Z值的影响When19Typical Temperature Profiles典型温度分布19Typical Temperature Profiles20Type of Sterilization灭菌类型Moist Heat Sterilization湿热灭菌20Type of Sterilization灭菌类型Mo21Moist Heat Sterilization湿热灭菌Characteristics: 特征Well understood and well characterized process, first validate

22、d 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21Moist Heat Sterilization湿热灭22Moist Heat Sterilization湿热灭菌Applications: 应用Terminal sterilization

23、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 materials 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 22Moist Heat Sterilization湿热灭23Basic Types of Moist Heat Sterilization湿

24、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23Basic Types of Moist Heat S24Basic 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 distributio

25、n and penetration 热分布和渗透3.Microbiological challenges 微生物挑战24Basic Elements of Sterilizat25Steam 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 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 Ce

26、rtification 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, where 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25

27、Steam Sterilization Validati26Steam 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/SIP 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

28、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 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 se

29、nsors into the autoclave/SIP 将温度传感器导入高压灭菌柜/SIP的方法26Steam Sterilization Validati27Steam 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/SIPNew loading patterns or product configurations 新装料方式或产品配置Changes to exi

30、sting 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27Steam Sterilization Validati28Steam Sterilization Validation: Preparation of PQ protocol 蒸汽灭菌验证：PQ方案的准备The protocol may include: 方案可能包括：Objectives of the va

31、lidation 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 of calibration procedures for t

32、emp-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28Steam Sterilization Validati29Steam Sterilization Validation: Preparation of PQ protocol 蒸汽灭菌验证：PQ方案的准备A

33、 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 not need

34、ed 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29Steam Sterilization Validati30Steam Sterilization Validation: Pre

35、paration of PQ protocol 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

36、not in the 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 s

37、ensor 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。30Steam Sterilization Validati31Steam Sterilization Validation: Preparation of PQ protocol 蒸汽灭菌验证：PQ方

38、案的准备5.Acceptance criteria for temp. pressure, 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 c

39、hamber temperature and not subject 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

40、在稳定状态下要求均一性31Steam Sterilization Validati32Steam Sterilization 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

41、品中For 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 n

42、umber of the appropriate temperature sensor of the BI 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32Steam Sterilization Validati33Steam 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

43、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-value, 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 crit

44、eria shall require cycle parameter modifications, load pattern modification 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33Steam Sterilization Validati34Case 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

45、 空腔体热分布验收标准No.Acceptance Criteria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 phase. 整个灭菌阶段，稳定后，每分钟分布在腔体内的12个 TC必须在平均热电偶温度的1C 2Dwell time not less than the pre-se

46、t sterilization time of 40 minutes. 停留时间不少于预设定的灭菌时间40分钟3The TCs monitoring the drain and chamber RTDs may not fail post check calibration. Ten of the twelve TCs must be functional at the end of the cycle. 监测排水和腔体RTD的TC不应在实验后校验失败。循环结束时，12个TC中的10个必须在实验后校验时功能完好34Case Study 实例分析Autoclave St35Case Study

47、实例分析Autoclave Sterilization Validation 高压蒸汽灭菌验证Table 2Acceptance Criteria for Heat Distribution表2 热分布验收标准No.Acceptance Criteria验收标准1Dwell time must be not less than the pre-set sterilization time of 15 minutes 停留时间不得少于预设定的灭菌时间15分钟2The drain PT-100 and TC in the drain must be within 1.0C of each othe

48、r during the stabilized sterilization period. 在稳定灭菌期间，排水管PT-100和排水管中TC之间温差必须在1.0oC 之内3The temperature deviation during holding times does not exceed -1C and +2C from the set point (during the stabilization period) 停留时间的温度偏差不超过-1oC及设定点的 +2oC （在稳定阶段）4At least 10 TCs of 12 thermocouples must be functio

49、nal at the end of the run循环结束时，12个TC中的10个必须功能完好35Case Study 实例分析Autoclave St36Case 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No.Acceptance Criteria 验收标准1F0 must be not less than preset time of 15 minutes F0不得低于预设定的15分钟2The drain PT-1

50、00 and TC in the drain must be within 1.0C of each other during the stabilized sterilization period. 在稳定灭菌期间，排水管PT-100和排水管中TC之间温差必须在1.0C 之内3The temperature sensors remained in position at the end of cycle 循环结束时温度传感器仍然在位4The items containing sensors are intact; bottles have not leaked, burst or broke

51、n 装传感器的器具完整；瓶子不漏，爆裂或破碎5No more than 1% of the bottles has burst or broken 爆裂或破碎的瓶不超过1%6The temperature measured in any fluid containers is not greater than 80 C at the end of the cycle 循环结束时，任何液体容器中测量的温度不超过80 C 7The temperature deviation during holding time does not exceed -1C and +2C from the setpo

52、int during the stabilized sterilization period 停留时间的温度偏差不超过-1C及稳定灭菌阶段设定点的 +2C 8No spore survival of the biological indicators 生物指示器上无孢子生存9At least 10 TCs of 12 thermocouples are functional at the end of the run for themaximum & minimum load configurations, respectively 每次最大&最小装载配置结束时，12个热电偶中至少10个TC起

53、作用10Autoclave and validation timers must be synchronized 高压灭菌柜和确认计时器必须同步36Case Study 实例分析Autoclave St37Dry Heat Sterilization 干热灭菌 Depyrogenation去热原37Dry Heat Sterilization 干热灭菌 38Terminology and Definitions 术语和定义Pyrogen: Fever producing substance: endotoxin, viruses, fungi, toxin from gram(+) and g

54、ram(-) 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LPSEnvironmental: complex of protein, carbohydrate & lipid. 环境：复合蛋白质， 碳水化合物&脂类 Purified: lipopolysac

55、chride, used in endotoxin standards 提纯后：脂多糖，用于内毒素标准38Terminology and Definitions 39Sources of Pyrogen in Parenterals注射用药热原的来源Water systems and resin columns 水系统和树脂交换柱APIs produced without endotoxin removal 生产API而未除内毒素API & excipients from natural sources 来自自然源的API&赋形剂Manufacturing equipment 生产设备Anyw

56、here gram(-) microbes are growing or have been growing 革兰氏阴性细菌生长或曾生长的任何地方39Sources of Pyrogen in Parent40Depyrogenation去热原A process to remove or destroy Endotoxin 除去或破坏内毒素的一种工艺 3 log reduction per USP 按照USP ， 3对数减少值Depyrogenation requires: 去热原要求：Distillation or ultrafiltration of water 对水进行蒸馏或超滤Dry

57、heat incineration 干热焚烧Destruction by strong alkali or oxidation with peroxide 用强碱或过氧化物进行氧化来破坏40Depyrogenation去热原A process 41Types of Dry Heat Sterilizers干热灭菌器的种类Common 常用Forced-convection batch sterilizers: utensils, glassware, stainless steel equipment, product 强制对流批灭菌器：器具，玻璃器皿，不锈钢设备，产品Tunnel: glas

58、sware 烘箱：玻璃器皿Flame: ampoule sealing 火焰：安瓿封口Less common 不常用Microwave 微波Laser plasma 激光等离子体41Types of Dry Heat Sterilizer42Components for Aseptic Processing 无菌工艺元件Component部件Endotoxin内毒素Method方法LRV*Water 水Most likely 很可能Distillation, RO 蒸馏，RO4Lg. Containers 容器Possible 可能Chemicals, CIP 化学品，CIP2Excipien

59、ts 赋形剂Possible 可能Purification 提纯 NDGlass vials 玻璃瓶Never seen 未见过Dry heat 干热4Closures 塞子Unlikely 不可能Wash/sterilize清洗/灭菌3Lubricant 润滑油Never seen 未见过Dry heat 干热ND42Components for Aseptic Proce43Dry Heat Depyrogenation: Glass干热除热原：玻璃Temp. range: 200-350 oC 温度范围：200-350 oC Heat tolerant materials, glassw

60、are, metal parts, scissors, oils, inorganic salts 耐热材料，玻璃器皿，金属部件，剪刀，油，无机盐Process: heated, HEPA filtered air, uniformly distributed by convection & fan 工艺：加热，HEPA 过滤空气，通过对流&风扇进行均匀分布Lethal effect: oxidation of proteins and denaturation 致命效应：蛋白质氧化和变性USP : 3 log reduction 大于3个对数减少值43Dry Heat Depyrogenat