灭菌的方法和注意事项.ppt

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,灭菌的方法和注意事项,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,Pharmaceutical 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,Sterilization 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 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 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,。,5,Code Federal Regulation,美国联邦法规,211.113(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 contamination 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。,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 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。,7,Sterilization 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 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.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？,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,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 organisms,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,11,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 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,Remove 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，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 121,o,C,20 min at 110,o,C,0.2-0.3 min at 130,o,C,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 not 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,-valuenumber 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,-value/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,22Moist 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 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,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,Basic 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 within,1.0,o,C,of the mean,空腔体温度分布图在平均值的,1.0,o,C,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,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,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/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 of the PQ study:,进行,PQ,研究的工具,BI with 10,6,spores and known D and Z values,BI,有,10,6,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,o,C,recording data every minute or less,记录设备能支撑,12,温度传感器，精度,0.5,o,C,，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 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 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,o,C),空腔体热分布研究（,1,o,C,）,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 needed if 100mL),容器分布图研究（如果容量,4,Lg.Containers,容器,Possible,可能Chemicals,CIP,化学品，,CIP,2,Excipients,赋形剂,Possible,可能,Purification,提纯,ND,Glass vials,玻璃瓶,Never seen,未见过,Dry heat,干热,4,Closures,塞子,Unlikely,不可能,Wash/sterilize,清洗,/,灭菌,3,Lubricant,润滑油,Never seen,未见过,Dry heat,干热,ND,43,Dry Heat Depyrogenation:Glass,干热除热原：玻璃,Temp.range:200-350,o,C,温度范围：,200-350,o,C,Heat tolerant materials,glassware,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,44,Advantages of Dry-heat Depyrogenation,干热去热原的优点,Inactivates pyrogens while sterilization,灭菌时使热原失活,Materials dry at the end of cycle,循环结束时物料干燥,Corrosive effects are minimal,腐蚀性最小,Conveyor systems allow for higher temps and shorter dwell time,传送系统允许更高温度和更短的停留时间,45,Disadvantages of Dry-heat Depyrogenation,干热去热原的缺点,Slow process(air is a poor conductor),降低工艺速度（空气是不良导体）,Heat penetration slower than steam,热穿透比蒸汽慢,Rate varies(slow for glass,rapid for stainless steel),速度各异（玻璃慢，不锈钢快）,Heat must penetrate to inner surface via conduction,热必须通过传导穿透到内表面,Layering can occur due to differences in air density with temperature;mechanical circulation 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 laminar 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 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 15,o,C 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,15,o,C,内,A minimum cumulative FH value of twelve minutes must be demonstrated for each penetration thermocouple at the end of each cycle.,每次循环结束时，必须证明每个渗透热电偶,12,分钟内的最少累计,F,H值,A minimum 3-log reduction of endotoxin must be demonstrated for each endotoxin spiked vial exposed to the depyrogenation cycle.,必须证明暴露于去热原循环的每个内毒素长颈瓶至少有,3,个内毒素对数减少值,49,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,The controlling RTD(hot zone Entrance RTD)and the thermocouple adjacent to the controlling RTD must be within 10,o,C of each other during the exposure/dwell phase after stabilization.,稳定后，暴露,/,停留阶段控制,RTD,（高温区入口,RTD,）和靠近控制,RTD,的热电偶之间温差必须在,10C,A minimum of 20 of 26 thermocouples must be functional and meet post cycle calibration check criteria per XXXXX SOP.,26,个热电偶中至少有,20,个能起作用，满足,XXXXX SOP,后循环校验检查标准,。,50,Other Sterilization Methods,其他灭菌方法,Ethylene Oxide Sterilization,氧化乙烯 灭菌,A gaseous chemical agent,一种气态化学剂,Mode of action is alkylation of nucleic acid,作用模式为烷化核酸,Used primarily for devices,主要用于医疗器材,(,或设备,),Effective against most microorganisms including viruses,对大多数微生物,(,包括病毒,),都有效,Inexpensive,便宜,Compatible with most materials,与大多数物料兼容,Disadvantages:toxic,carcinogenic;explosive with oxygen,缺点：有毒，可致癌；与氧接触可能爆炸,51,Other Sterilization Methods,其他灭菌方法,Radiation,辐射,Used primarily for devices,主要用于医疗器材,(,或设备,),Mode of action is breakage of DNA strands,行动模式为,DNA,链断裂,Gamma(photons),-,射线（光子）,Electron beam(electrons),电子束（电子）,X-ray,X-,射线,Filtration,过滤,To sterilize heat sensitive products and remove sub visible particles by size exclusion and/or adsorption,对热敏感产品进行灭菌并通过体积排斥和,/,或吸附法除去可见颗粒,Others:EO bag systems,hydrogen peroxide,plasma,ozone,chlorine dioxide,UV,infrared,microwave,其他,:EO,包装系统，过氧化氢，等离子，臭氧，二氧化氯，,UV,，红外线，微波,52,Summary,总结,Sterile product posses high risk.CGMPs have to be in place all the time,无菌产品有高风险，,CGMP,应一直在线。,Sterilization equipment needs to be qualified.Sterilization process has to be validated with microbiological challenge,灭菌设备应合格。灭菌工艺应通过微生物挑战来验证,SAL should be 1 in 10,6,or less,SAL,应,1/10,6,或更少,D,Z,and F,0,are required to determine SAL,要求用,D,Z,和,F,0,值来确定,SAL,Bioburden has to be characterized.,应说明生物负荷,53,53,Important Components in Sterile Product Manufacturing,无菌生产中的重要组成部分,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,