1、1,1,Sterilization 滅菌,A General Discussion from CGMP Perspective 從CGMP角度的綜合討論,2,2,Outline 討論綱要,PART I Terminology 專業(yè)用詞的定義 GMP Requirements in The Manufacture of Sterile Pharmaceutical Products無菌藥品生產(chǎn)過程中GMP的基本要求 PART II Facility Design 生產(chǎn)設施的設計 HVAC System 空調系統(tǒng) Environmental Monitoring (EM) 環(huán)境的監(jiān)視 Pharma

2、ceutical Water 制藥用水 Cleaning/Sanitation 清潔消毒 Personnel 無菌區(qū)操作人員 PART III Methods of sterilization 滅菌方法 Sterile Production and validation 無菌生產(chǎn)和驗證 The Trend of Sterile Production 無菌產(chǎn)品生產(chǎn)趨勢,3,Presentation Outline概要,US Regulations 美國法規(guī) Moist Heat Sterilization 濕熱滅菌 Dry Heat/Depyrogenation 干熱/去熱原法 Steriliz

3、ation Process Validation 滅菌工藝驗證 Other Sterilization Methods 其他滅菌方法,4,Code Federal Regulation美國聯(lián)邦法規(guī),211.84(c)(3) sterile equipment 滅菌設備 Sterile equipment and aseptic sampling techniques shall be used when necessary 必要時應使用滅菌設備和無菌取樣技術。 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. 藥品容器和密封系統(tǒng)應清潔并根據(jù)藥品的性質和要求，進行滅菌，除熱原過程以確保預期的用途。 (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. 應建立并執(zhí)行對藥品容器和密封系統(tǒng)的規(guī)格或質量標準，測試方法，清潔方法，滅菌和除熱原過程的相關書面程序。,5,Code Federal Regulation美國聯(lián)邦法規(guī),211.113(a) and (b) sterilization 滅菌 (a) Appropriate written proced

6、ures, designed to prevent objectionable microorganisms in drug products not required to be sterile, shall be established and followed. 應該建立并執(zhí)行用于防止非無菌藥品被致病菌污染的相關書面程序。 (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. 應該建立并執(zhí)行用于阻止無菌藥品被致病菌污染的相關書面程序。這些程序應包括任何無菌工藝的驗證。,6,Code Federal Regulation美國聯(lián)邦法規(guī),211.167(a) testing （a）測試 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. 對無菌和/或無熱原的每批藥品應進行相應的實驗室測試以確定其與符合要求。測試程序應有書面文件并遵照執(zhí)行。,7,Sterilization Methods滅菌方法,Moist Heat: 濕熱 For drugs and devices. The mode of action is protein denaturation.

9、用于藥品和設備。作用方式是使蛋白質變性 Dry Heat: 干熱 For depyrogenation and equipment sterilization. The mode of action is protein denaturation 用于去熱原和設備滅菌。作用方式是使蛋白質變性 Ethylene Oxide: 氧化乙烯 Primarily for devices. The mode of action is alkylation of nucleic acids. 主要用于設備。作用方式是使核酸烷基化 Radiation: 輻射 Primarily for devices. Th

10、e mode of action is DNA strand breakage 主要用于設備。作用方式是使DNA鏈破壞 Other methods? 其他方法？,8,Bioburden生物負荷,Definition: 定義 Population of viable microorganisms on or in a product and/or a package 產(chǎn)品和/包裝上的活性微生物的數(shù)量和類型 Mixture of organisms 有機物混合 Indigenous microflora 地方微植物群 Needs to be inactivated by sterilization

11、 需滅菌失活,9,Bioburden生物負荷,Bioburden Sources 生物負荷來源 Environment 環(huán)境 Product contact surface, personnel, air 產(chǎn)品接觸表面，人員，空氣 Materials 材料 Water, raw materials. plastic, paper 水，原材料，塑料，紙張 Characteristics of Bioburden 生物負荷特點 Types of microorganisms 微生物類型 Resistance to sterilization process 對無菌工藝的耐受 Number of o

12、rganisms 有機物數(shù)量,10,Biological Indicator (BI)生物指示劑,Microbiological test system providing a defined resistance to a specific sterilization process 微生物測試系統(tǒng)對指定滅菌工藝有明確抵抗性。 A characterized preparation of specific microorganisms resistant to a particular sterilization process 某一確定的微生物(指示劑)應具有對某一特定滅菌工藝的抵抗性,1

13、1,Typical Biological Indicators典型生物指示劑,Moist heat sterilization 濕熱滅菌 Geobacillus stearothermophilus Bacillus stearothermophilus Dry heat and EO sterilization 干熱和環(huán)氧乙烷滅菌 Bacillus atrophaeus Bacillus subtilis var. niger,12,Forms of Biological Indicators生物指示劑形式,Strips/discs in glassine envelopes 在透明紙信封里

14、的條形板/光盤 Strips/discs 條/光盤 Self-contained 獨立包裝的 Ampoules 安瓶 Strips with medium 中號條形板 Liquid suspension 液體懸浮液 Metal 金屬 Fiberglass 玻璃纖維,13,Choice of an Appropriate BI相關生物指示劑的選擇,Sterilization process 滅菌工藝 Cycle design method 循環(huán)設計方法 Product bioburden 產(chǎn)品生物負荷 Population 數(shù)量 Resistance 抵抗性,14,BIs Prepared by

15、 User生物指示劑的準備,Performance 性能 Resistance 抵抗性 Population 數(shù)量 Purity 純度 D- value D 值 Recovery methods 恢復方法 Storage requirements 儲存要求,15,Biological Indicator Use生物指示劑使用,Place BI within 把BI 放進 Product 產(chǎn)品 Package 包裝 Sterilizer load to monitor process 滅菌器負荷以監(jiān)視滅菌工藝 Expose to sterilizing conditions 暴露在滅菌狀態(tài) Re

16、move BI and test 移除BI和測試 Count survivors 生存數(shù)量的計算 Growth/no growth response 生長/無生長反應,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值是在特定條件下微生物數(shù)量降低的90%（或一對數(shù)）所用的時間

17、，通常是以分鐘為單位的。 Bacillus stearothermophilus has a D value: 嗜熱脂肪芽胞桿菌的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 滅菌驗證中，D值可能是最重要的。不考慮D值的驗證過程多半是無效的且不被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對數(shù)改變需要的溫度數(shù) Z = -1/slope of the thermal resistance curve Z = -1/熱阻力曲線斜率 where Slope = logarithmic change in D-v

19、alue/change in temperature 斜率 = D值上對數(shù)的改變/溫度的改變,18,Impact of Z-value Z值的影響,When z-value is small, considerably less inactivation will result below reference temperature and considerably more above the reference temperature 當Z值較小時，較低程度的失活將導致溫度低于參考溫度，較大程度的失活將導致溫度高于參考溫度。,19,Typical Temperature Profiles典

20、型溫度分布,20,Type of Sterilization滅菌類型,Moist Heat Sterilization 濕熱滅菌,21,Moist Heat Sterilization濕熱滅菌,Characteristics: 特征 Well understood and well characterized process, first validated process in pharm industry 濕熱滅菌是一已被很好地理解并描述滅菌工藝，也是在制藥工業(yè)中第一個被驗證的滅菌工藝 Suitable for a wide variety of applications 適合于較大應用范

21、圍 Equipment is readily available 設備很容易從市場購買到 Cost on a per use basis is low 每次使用基準花費低,22,Moist Heat Sterilization濕熱滅菌,Applications: 應用 Terminal sterilization of parental product 注射劑的終端滅菌 Sterilization of equipment and components for use in aseptic filling 無菌灌裝線上設備和配件的滅菌 Sterilization of laboratory m

22、aterials 實驗室用材料的滅菌 In-situ sterilization of process piping and equipment (SIP)工藝管道和設備在線滅菌,23,Basic Types of Moist Heat Sterilization濕熱滅菌基本類型,Saturated steam 飽和蒸汽 Autoclaves (self-closing) 高壓滅菌柜（半封閉） SIP 在線滅菌 Super heated water 過熱水 Spray 噴霧 Submerged 浸沒的 SIP 在線滅菌 Steam-air-mixture (SAM) 水蒸氣空氣混合物,24,B

23、asic Elements of Sterilization Process Validation 滅菌工藝驗證的基本元素,Empty vessel heat distribution 空容器熱分布 Heat distribution and penetration 熱分布和滲透 3.Microbiological challenges 微生物挑戰(zhàn),25,Steam Sterilization Validation: Prerequisites 蒸汽滅菌驗證：前提,OQ for an autoclave: 高壓滅菌柜運行確認 Empty chamber temperature mapping

24、within 1.0oC of the mean 空腔體溫度分布圖在平均值的1.0oC 內 Chamber integrity test (no leaking) 腔體完整性測試 Certification of HEPA filtration on the air used to break vacuum or integrity testing of the vent filter 用于隔斷真空或通氣過濾器完整性測試的空氣HEPA過濾認證 Requirements for SIP 在線滅菌要求 Temperature mapping 溫度分布圖 An integrity test, whe

25、re appropriate 相關完整性測試 Use of BI 生物指示劑的使用 All critical instruments must be calibrated 所有關鍵儀器需校驗,26,Steam Sterilization Validation: Prerequisites 蒸汽滅菌驗證：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 應獲得用于高壓滅菌柜/SIP的潔凈蒸汽中的不凝氣體，過熱蒸汽及干燥度的可接受測試結果 5. Tools for the conduct of the PQ study: 進行PQ研究的工具 BI with 106 spores and known D and Z values BI 有106個孢子，已知D值和Z值 Temperature sensors 溫度傳感器 Recording device capable of supporting 12 temp sensors with an accuracy of 0.5 oC, recording data every minute or

27、 less 記錄設備能支撐 12溫度傳感器，精度 0.5 oC，每分鐘或間隔更短時間記錄數(shù)據(jù) Means of introducing temp sensors into the autoclave/SIP 將溫度傳感器導入高壓滅菌柜/SIP的方法,27,Steam Sterilization Validation: Preparation of PQ protocol 蒸汽滅菌驗證：PQ方案的準備,A protocol shall be prepared for : 對于下列各項應建立方案： New autoclave/SIP 新高壓滅菌柜/SIP New loading patterns

28、or product configurations 新裝料方式或產(chǎn)品配置 Changes to existing patterns 對現(xiàn)有裝料方式的變更 Changes to operation cycle parameters 對運行周期參數(shù)的變更 Major change to equipment as directed by change control 變更控制要求的設備主要變更,28,Steam Sterilization Validation: Preparation of PQ protocol 蒸汽滅菌驗證：PQ方案的準備,The protocol may include: 方

29、案可能包括： Objectives of the validation study 驗證研究的目的 Identification and description of the sterilizer and its process controls 滅菌器的識別和說明及工藝控制 Identification of SOPs for the process equipment 工藝設備SOP的識別 Description of or SOP reference for instrument calibration procedures 儀器校驗程序的說明或SOP參考 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 溫度監(jiān)測設備校驗程序的識別，包括一個兩點預運行校驗和每次運行后的確認 Process parameter acceptance criteria 工藝參數(shù)的驗收標準,29,Steam Sterilization Validation: Preparation of PQ protocol 蒸汽滅菌驗證：PQ方案

31、的準備,A description of the following:以下說明 Biodurden determination studies 生物負荷確認研究 Empty chamber heat distribution studies ( 1 oC) 空腔體熱分布研究（ 1 oC ） Loaded chamber (including Load configuration, max lading and min loading) heat penetration studies 滿載腔體（包括裝載配置, 最大和最少）熱穿透研究 Container mapping studies (may

32、 not needed if 100mL) 容器分布圖研究（如果容量100mL ，可能不需要） Microbiological challenge studies 微生物挑戰(zhàn)研究 Evaluation of drug product cooling water (where applicable) 藥品冷卻水評估（若適用） Evaluation of vent filter membranes associated with the sterilizer 與滅菌器相關的通氣過濾膜的評估,30,Steam Sterilization Validation: Preparation of PQ p

33、rotocol 蒸汽滅菌驗證：PQ方案的準備,4.Description of the temp sensor placement within the load 在負荷范圍內溫度傳感器位置的說明 One next to the temp controlling sensor for autoclave. 一1個接近高壓滅菌柜的溫度控制傳感器 NLT 12 sensors for autoclave 不少于12 個傳感器用于高壓滅菌柜 NLT 3 sensors for SIP不少于3傳感器用于在線滅菌 If the temp controlling sensor is not in the

34、drain, an additional sensor shall be placed in the drain 如果溫度控制傳感器不在排水口中，應置另一個傳感器于排水口中 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 應建立符合校驗和驗證的最少#傳感器。最終任何傳感器的失敗應調查研究。任何臨近溫度控制傳感器或在排水口的傳感器應符合預校驗和后校驗，這是成功運行的驗收標準的一部分。,31,Steam Sterilization Validation: Preparation of PQ protocol 蒸汽滅菌驗證：PQ方案的準備,5.Acceptance criteria for temp. p

36、ressure, Fo where appropriate, vacuum and destructions of BIs 溫度壓力，相關Fo，真空及生物指示劑滅活的驗收標準 6.A requirement for 3 consecutive, successful runs 3次連續(xù)成功運行的要求 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. 排水口是最冷點，在滅菌區(qū)外，且不包括在平均腔體溫度計算內，不受限于腔體分布要求 Thermocouples should be placed at the same location for empty chamber and loaded chamber heat distribution studies. 熱電偶應置于相同位置，進行空腔體和滿載腔體熱分布研究 Uniformity is expected only at the steady state. 僅在穩(wěn)定狀態(tài)下要求均一性,32,Steam Steriliza

38、tion Validation: Execution of PQ 蒸汽滅菌驗證：PQ執(zhí)行,1.Temperature sensor shall: 溫度傳感器應： For heat penetration data, be located such that they penetrate the equipment, or the product container and are immersed in the product 對于熱穿透數(shù)據(jù)，應置于能穿透設備或產(chǎn)品容器處，并包含在產(chǎn)品中 For heat distribution data, be evenly distributed wit

39、hin the load or system and shall not contact metal surface 對于熱分布數(shù)據(jù)，平均分布在負荷范圍內或系統(tǒng)中，且不能接觸金屬面 2.The BI shall:生物指示劑應： Be placed near the tip of the temperature sensor used for penetration 置于靠近用于穿透的溫度傳感器的頂端 Include the identification that links to the number of the appropriate temperature sensor of the B

40、I 包括與相關BI溫度傳感器數(shù)量有關的識別 The D value of the BI shall be determined in the product BI的D值應在生產(chǎn)中確定,33,Steam Sterilization Validation: Execution of PQ 蒸汽滅菌驗證：PQ執(zhí)行,The autoclave shall be loaded and operated in accordance with SOP 高壓滅菌器應按照SOP進行裝載并運行 Using 121.1 oC as the base temperature and 10 oC as the Z-va

41、lue, Fo shall be calculated 121.1 oC 作為基底溫度， 10 oC 作為Z值， Fo應為計算值 Acceptance criteria shall be based on no growth on exposed BIs and growth on the positive control BI 驗收標準應依據(jù)暴露的BI上未生長和陽性控制BI上的生長 Failure to meet acceptance criteria shall require cycle parameter modifications, load pattern modification

42、 and/or equipment repairs/corrections 未達到驗收標準應要求周期參數(shù)變更，負荷曲線圖變更和/或設備修理/修正,34,Case Study 實例分析Autoclave Sterilization Validation 高壓蒸汽滅菌驗證,Table 1Acceptance Criteria of Empty Chamber Heat Distribution 表1 空腔體熱分布驗收標準,35,Case Study 實例分析Autoclave Sterilization Validation 高壓蒸汽滅菌驗證,Table 2Acceptance Criteria

43、for Heat Distribution 表2 熱分布驗收標準,36,Case Study 實例分析Autoclave Sterilization Validation 高壓蒸汽滅菌驗證,Table 3Acceptance Criteria for Heat Penetration 表3熱分布驗收標準,37,Dry Heat Sterilization 干熱滅菌,Depyrogenation 去熱原,38,Terminology and Definitions 術語和定義,Pyrogen: Fever producing substance: endotoxin, viruses, fung

44、i, toxin from gram(+) and gram(-) bacterials, peptiglycan 熱原：熱產(chǎn)生的物質：內毒素，病毒，真菌，革蘭氏陽性和陰性細菌產(chǎn)生的毒素，肽糖 Endotoxin: Component from outer membrane of gram(-) bacterial, most potent pyrogens, LPS in structure 內毒素：革蘭氏陰性細菌外膜的成分，大多數(shù)有效熱原，結構中的LPS Environmental: complex of protein, carbohydrate mechanical circulati

45、on needed 空氣密度，溫度不同可能導致分層，需要機械循環(huán) Heat degradation limits materials 熱降解限制材料 Contraction during cooling may draw contaminants 冷卻過程中接觸可能引起污染,46,Depyrogenation by Tunnels 通過烘箱去熱原,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 在加熱和冷卻區(qū)經(jīng)HEPA過濾的垂直層流氣流，或加熱區(qū)的輻射加熱器和冷卻區(qū)的垂直層流氣流 Conveyor belt to provide in-line continuous flow of sterile glassware to aseptic area 傳送帶提供連續(xù)在線的無菌玻璃器皿到無菌區(qū)的流動 Limited to one type of load at a time 一次限用一種裝載方式 Problem to control speed match other line equipment (filler) 控制速度以匹配其他

47、線設備（灌裝機）的問題 Difficult to achieve uniform heating, and heat source may generate particles 很難達到均勻加熱，熱原可能產(chǎn)生顆粒 Large product volume needed to justify tunnel 調整烘箱要求大的產(chǎn)品容量,47,Validation of Depyrogenation Cycles去熱原循環(huán)驗證,Run engineering trials to find cold spots 進行試運行以找到冷點 Run trials to determine worse case

48、vial, e.g., thickest glass, packing effects 試車以確定最差狀況的玻璃瓶，如最厚的玻璃，緊束效應 Procure or prepare endotoxin indicators 獲得或制備內毒素指示劑 Run 3 full loads with endotoxin indicators in place at cold spots and random sites 進行3次滿載運行，內毒素指示劑置于冷點和隨機位置 Acceptance criteria 驗收標準 min. 3 log reduction in EU for spiked vials 帶

49、孔玻璃瓶3個對數(shù)減少值，單位為EU 3 successful runs with controls and documentation 受控并記錄的3次成功運行,48,Case Study 實例分析Dry Heat Depyrogenation Tunnel 干熱去熱原隧道烘箱,Validation Acceptance Criteria 驗證驗收標準 A minimum of three successful validation runs must be performed. 必須至少進行三次成功的驗證 Distribution thermocouple temperatures must

50、 be within 15oC of the set point temperature, after stabilization. 穩(wěn)定后，分配熱電偶溫度必須在設定溫度的 15oC 內 A minimum cumulative FH value of twelve minutes must be demonstrated for each penetration thermocouple at the end of each cycle. 每次循環(huán)結束時，必須證明每個滲透熱電偶12分鐘內的最少累計FH值 A minimum 3-log reduction of endotoxin must

51、be demonstrated for each endotoxin spiked vial exposed to the depyrogenation cycle. 必須證明暴露于去熱原循環(huán)的每個內毒素長頸瓶至少有3個內毒素對數(shù)減少值,49,Case Study 實例分析Dry Heat Depyrogenation Tunnel 干熱去熱原隧道烘箱,Validation Acceptance Criteria 驗證驗收標準 The controlling RTD (hot zone Entrance RTD) and the thermocouple adjacent to the controlling RTD must be within 10oC of each other during the exposure/dwell phase after stabilization. 穩(wěn)定