工藝驗(yàn)證方案模板

1、精選優(yōu)質(zhì)文檔-傾情為你奉上Process Validation (PV) Protocol XXXXXX工藝驗(yàn)證方案Written by起 草 人Written date起草日期年 月 日Reviewed by審 核 人Reviewed date審核日期年 月 日Approved by批 準(zhǔn) 人Approved date批準(zhǔn)日期年 月 日Written department起草部門生效日期年 月 日Issued by:頒發(fā)部門： Copy number:拷 貝 號(hào)： Change record:Revision number變更記載： 修訂號(hào) 批準(zhǔn)日期 生效日期 Change rea

2、son and goal: 變更原因及目的：Distribution department:分發(fā)部門:所長辦公室 人力資源部 后勤保衛(wèi)部 物資供應(yīng)部 工程服務(wù)部 生產(chǎn)管理部 質(zhì)量保證部 銷 售 部 倉 儲(chǔ) 部 疫苗一室 疫苗二室 疫苗三室 疫苗五室 疫苗六室 生化制品室 細(xì)胞因子室 血清制品室 實(shí)驗(yàn)動(dòng)物室 分包裝室 培養(yǎng)基室 Verification department (department): 驗(yàn)證科室（部門）： Verify completion date: 驗(yàn)證完成日期： Validity: 有 效 期： Index 目錄1. Purpose目的This purpose o

3、f this process validation is to provide documented evidence to show that the combination of the personnel, materials, equipment, methods, environmental conditions and the other related utilities XXX Co., LTD (XXX) can ensure the consistent production of products conforming to the enterprise internal

4、 standards and the national standards, and the processes are reliable and conforming to the GMP requirements. The process validation and the test results are to be documented based on this validation protocol.本工藝驗(yàn)證用于提供文件化的證據(jù)，證明XXX疫苗一室生產(chǎn)XXX原液的人員、材料、設(shè)備、方法、環(huán)境條件以及其它有關(guān)公用設(shè)施的組合可以始終如一的生產(chǎn)出符合企業(yè)內(nèi)控標(biāo)準(zhǔn)及國家法定標(biāo)準(zhǔn)的產(chǎn)品，

5、工藝穩(wěn)定可靠，符合GMP要求，工藝驗(yàn)證的過程和檢查的結(jié)果將按照該驗(yàn)證方案進(jìn)行記錄。2. Scope范圍This protocol is applicable to the process validation of XXX produced by the equipment located XXX.本方案適用于XXX原液生產(chǎn)的工藝驗(yàn)證。由于工作細(xì)胞庫制備的過程并非常規(guī)生產(chǎn)過程，因此，不在本次驗(yàn)證中進(jìn)行考察。The labeling and packaging processes are not within the scope of this document since these proc

6、esses have been validated per PQ protocol (labeling) and PQ protocol (packaging), and these processes have minimal impact on finished product quality. 貼標(biāo)簽和包裝也不在此文件范圍內(nèi)，因?yàn)檫@些工藝已經(jīng)按照PQ方案（貼標(biāo)簽）和PQ方案(包裝)驗(yàn)證過，并且這些工藝對(duì)產(chǎn)品質(zhì)量極少有影響。3. Responsibility職責(zé)3.1 AAA responsibilities AAA的職責(zé) ü Compile Protocol方案的編寫ü

7、; Guide Protocol execution, data collection/ Provide test data for review upon request by customer指導(dǎo)方案的實(shí)施，數(shù)據(jù)的收集/在客戶要求時(shí)提供測(cè)試數(shù)據(jù)供其審核ü Final report Review/ Analyze and compile data into the summary report最終報(bào)告的審核/對(duì)數(shù)據(jù)進(jìn)行分析并將其編輯入總結(jié)報(bào)告之中3.2 XXX responsibilities XXX的職責(zé)n Production department 生產(chǎn)部ü Revi

8、ew and approve protocol審核并批準(zhǔn)驗(yàn)證方案ü Assure all the equipment used for validation can be maintained timely保證驗(yàn)證用所有設(shè)備均能按SOP進(jìn)行及時(shí)的維護(hù)和保養(yǎng)ü Assure all the raw materials used for validation can be supplied timely and comply with domestic quality criteria of enterprise保證驗(yàn)證用原輔包裝材料能按時(shí)到貨，并符合企業(yè)內(nèi)控標(biāo)準(zhǔn)n Resea

9、rch and development department 研發(fā)部ü Provide product registration criteria and review whether recipe and process step described in the protocol is complying with registered criteria提供產(chǎn)品的注冊(cè)工藝，審核方案中生產(chǎn)處方、工藝與注冊(cè)資料的一致性ü Review and make assurance the compliance of raw material supplier with regist

10、ered supplier審核原輔料供應(yīng)商和注冊(cè)資料的一致性ü Review relevant quality criteria including raw material, intermedia and finished product審核原輔料標(biāo)準(zhǔn)、中控質(zhì)量標(biāo)準(zhǔn)和成品的質(zhì)量標(biāo)準(zhǔn)n Vaccine one Plant 疫苗一室ü Coordination and implementation of this validation protocol協(xié)調(diào)、實(shí)施本驗(yàn)證方案ü Training of personnel based on this validation

11、 protocol按驗(yàn)證方案對(duì)有關(guān)人員進(jìn)行培訓(xùn)ü Collection, sorting and reviewing of validation data and drafting of validation reports (including the deviation report, if there are any deviations)收集、整理和審核驗(yàn)證數(shù)據(jù)，起草驗(yàn)證報(bào)告（如方案執(zhí)行有偏差，要完成偏差報(bào)告）。n Utility support department 工程服務(wù)部ü Assistance in implementation of this valid

12、ation protocol協(xié)助本驗(yàn)證方案的實(shí)施ü Calibration of instruments related to this validation protocol and providing relevant calibration certificates and quality certificates負(fù)責(zé)校驗(yàn)和本驗(yàn)證方案有關(guān)的儀器、儀表，出具校驗(yàn)報(bào)告并貼合格證ü Assure normal operation of the utility system保證公用系統(tǒng)的正常運(yùn)行ü Final report Review 最終報(bào)告的審核n Quali

13、ty control of quality department 質(zhì)量部質(zhì)量控制ü Determination of test methods used in this validation protocol負(fù)責(zé)確定本驗(yàn)證方案的檢驗(yàn)方法ü Validation of the necessary test methods for XXXXX involved in this validation protocol負(fù)責(zé)本驗(yàn)證方案涉及的重組乙型肝炎疫苗（CHO細(xì)胞）原液生產(chǎn)的必要的檢驗(yàn)方法的驗(yàn)證ü The quality inspections on XXXXX Tab

14、let involved in this validation protocol, and presenting full item certificates of analysis and quality inspection reports and records負(fù)責(zé)本驗(yàn)證方案涉及的重組乙型肝炎疫苗（CHO細(xì)胞）原液生產(chǎn)的質(zhì)量檢驗(yàn)，并出具全項(xiàng)檢驗(yàn)報(bào)告，提供質(zhì)量檢驗(yàn)報(bào)告書及檢驗(yàn)記錄ü Providing technical supports and guidance for tests at each of the process steps負(fù)責(zé)各工序檢驗(yàn)的技術(shù)支持和指導(dǎo)ü

15、; Coordinating the plant in the implementation of this validation protocol協(xié)同車間實(shí)施本驗(yàn)證方案ü Final report Review 最終報(bào)告的審核n Quality assurance of quality department 質(zhì)量部質(zhì)量保證ü Supervision of validation process, review and approval of this protocol, and ensuring the implementation following this proto

16、col負(fù)責(zé)驗(yàn)證過程監(jiān)督，方案的審核與批準(zhǔn)，確保按批準(zhǔn)的方案執(zhí)行ü Sampling at each process steps during the process validation負(fù)責(zé)工藝驗(yàn)證中各工序的取樣ü Coordination among different departments involved in the validation activities負(fù)責(zé)驗(yàn)證涉及到的各部門之間的協(xié)調(diào)ü Providing equipment validation reports related to this validation protocol負(fù)責(zé)提供和本驗(yàn)

17、證方案有關(guān)的設(shè)備驗(yàn)證報(bào)告ü Identification, audit and assessment of suppliers供應(yīng)商的確認(rèn)、審計(jì)及評(píng)價(jià)ü Assessment and handling of changes and deviations occur during this process validation負(fù)責(zé)對(duì)工藝驗(yàn)證實(shí)施過程中出現(xiàn)的變更和偏差的評(píng)價(jià)和處理ü Final report Review 最終報(bào)告的審核4. Regulation and Guidance 法規(guī)和指南ü EudraLex Volume 4 EU Guideli

18、nes to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use, November, 2008EudraLex 第四卷 歐盟藥品生產(chǎn)質(zhì)量管理規(guī)范指南 醫(yī)藥產(chǎn)品 人用及獸用，2008年11月ü Final Version of Annex 15 to the EU Guide to Good Manufacturing Practice : Qualification and Validation , September 2001EudraLex 第四卷 藥品生產(chǎn)質(zhì)量管理規(guī)范，附錄15

19、 驗(yàn)證與確認(rèn)，2001年9月ü EMEA - Note for guidance on process validation, September 2001 歐洲藥品管理局 工藝驗(yàn)證指南，2001年9月ü EMEA - Annex II to note for guidance on process validation, January 2005歐洲藥品管理局 附錄II 工藝驗(yàn)證指南ü FDA 21 Code of Federal Regulations, Part 211, current Good Manufacturing Practice for Fin

20、ished Pharmaceuticals, April 2011FDA聯(lián)邦法典第21篇第211部分，現(xiàn)行藥物制劑生產(chǎn)質(zhì)量管理規(guī)范，2011年4月ü FDA Process Validation: General Principles and Practices , January 2011FDA工藝驗(yàn)證：一般原則和實(shí)踐，2011年01月ü SFDA Good Manufacturing Practice (2010 Revision), March, 2011SFDA 藥品生產(chǎn)質(zhì)量管理規(guī)范（2010年修訂），2011年03月ü PIC PIC/S Pharmac

21、eutical Inspection Convention, Pharmaceutical Inspection Co-operation Scheme, “Recommendations on Validation Master Plan, Installation and Operational Qualification, Non-sterile Process Validation, Cleaning Validation”, July 2004PIC/S藥品監(jiān)管公約，藥品監(jiān)管合作計(jì)劃，“有關(guān)驗(yàn)證主計(jì)劃、安裝和運(yùn)行確認(rèn)、非無菌工藝驗(yàn)證、清潔驗(yàn)證的建議”，2004年7月ü A

22、WHO Guide to good manufacturing practice(GMP) requirements , Part 2 Validation , 1997 WHO 關(guān)于GMP要求的指南， 第二部分：驗(yàn)證，1997年ü Chinese Pharmacopoeia (CP) 2010 Edition 中國藥典（CP）2010年版ü Process Validation of Protein Manufacturing (Technical Report No. 42 Supplement Vol. 59, No. S-4 September/October 20

23、05 © 2005 by PDA)蛋白生產(chǎn)的工藝驗(yàn)證（技術(shù)報(bào)告42，PDA， 2005年版）5. Abbreviations縮略語The abbreviations which will be used in this protocol are listed in the following form.在下面的表格中規(guī)定了本方案中使用的縮略語。表格 1 縮略語Abbreviations縮略語Definition定義APIActive Pharmaceutical Ingredient (or “Drug Substance”)原料藥（或藥品物質(zhì)）CPPCritical Process

24、 Parameter關(guān)鍵工藝參數(shù)CQACritical Quality attribute關(guān)鍵質(zhì)量屬性CVCleaning Validation清洗驗(yàn)證IQInstallation Qualification安裝確認(rèn)MFVMedia Fill Validation培養(yǎng)基模擬灌裝試驗(yàn)N/ANot applicable不適用OQOperation Qualification運(yùn)行確認(rèn)PLCProgrammable Logic Controller可編程邏輯控制器PQPerformance Qualification性能確認(rèn)PVProcess Validation工藝驗(yàn)證PWPurified Wate

25、r純化水QAQuality Assurance質(zhì)量保證RARisk Assessment風(fēng)險(xiǎn)分析SOPStandard Operation Procedure標(biāo)準(zhǔn)操作程序URSUser Requirement Specification用戶使用要求VMPValidation Master Plan驗(yàn)證主計(jì)劃WFIWater for Injection注射用水6. Protocol Description方案說明n Using this defined process validation shall be performed for the results of the Process Ris

26、k Assessment.本方案實(shí)施的工藝驗(yàn)證，是基于工藝風(fēng)險(xiǎn)評(píng)估結(jié)果的。n Using this defined process validation shall be performed for three consecutive successful runs based on GMP requirements to demonstrate the process control reliability and the repeatability.鑒于XXX的生產(chǎn)已經(jīng)有近20年歷史，本次驗(yàn)證僅是對(duì)生產(chǎn)地址發(fā)生變更后且主要設(shè)備未發(fā)生變化的再驗(yàn)證，考慮到本產(chǎn)品生產(chǎn)周期長（總生產(chǎn)周期為158天

27、）的原因，本方案實(shí)施的工藝驗(yàn)證將生產(chǎn)1批成功的產(chǎn)品以證明其可靠性。n The determination of the intermediate and final product will implement enterprise internal standard but release finished product should be in accordance with criteria of the item “XXX” in Chinese Pharmacopoeia 2010 Edition.n 半成品、成品按企業(yè)內(nèi)控標(biāo)準(zhǔn)檢驗(yàn)，成品發(fā)放按中國藥典2010版三部“XXX ”標(biāo)準(zhǔn)

28、執(zhí)行Raw material internal standard原輔料內(nèi)控標(biāo)準(zhǔn)：表格 2原輔料質(zhì)量標(biāo)準(zhǔn)表Quality Standard Doc.質(zhì)量控制標(biāo)準(zhǔn)文件Doc. Number文件編號(hào)Sodium Chloride (BP) Quality Specification氯化鈉（藥用級(jí)）檢驗(yàn)質(zhì)量標(biāo)準(zhǔn)Sodium Dihydrogen Phosphate (BP) Quality Specification磷酸二氫鈉（藥用級(jí)）檢驗(yàn)質(zhì)量標(biāo)準(zhǔn)Crystal Aluminum Chloride AR Quality Specification 結(jié)晶氯化鋁AR檢驗(yàn)質(zhì)量標(biāo)準(zhǔn)Glucose (BP) Q

29、uality Specification 葡萄糖（藥用級(jí)）檢驗(yàn)質(zhì)量標(biāo)準(zhǔn)Glutamine Quality Specification 谷氨酰胺質(zhì)量標(biāo)準(zhǔn)Potassium Bromide Quality Specification 溴化鉀質(zhì)量標(biāo)準(zhǔn)Disodium Edetate Quality Specification 乙二胺四乙酸二鈉質(zhì)量標(biāo)準(zhǔn)Sodium Hydroxide (BP) Quality Specification 氫氧化鈉（藥用級(jí)）質(zhì)量標(biāo)準(zhǔn) Disodium Hydrogen Phosphate (BP) Quality Specification 磷酸氫二鈉(藥用級(jí))質(zhì)量標(biāo)準(zhǔn)

30、Merthiolate (BP) Quality Specification 硫柳汞（藥用）質(zhì)量標(biāo)準(zhǔn)Hydrochloric Acid (BP) Quality Specification 鹽酸（藥用級(jí)）檢驗(yàn)質(zhì)量標(biāo)準(zhǔn)Proline (BP) Quality Specification 脯氨酸（藥用）質(zhì)量標(biāo)準(zhǔn)Sodium Bicarbonate (BP) Quality Specification 碳酸氫鈉（藥用級(jí)）檢驗(yàn)質(zhì)量標(biāo)準(zhǔn)DMEM Quality Specification DMEM質(zhì)量標(biāo)準(zhǔn)Formaldehyde Solution (BP) Quality Specification 甲

31、醛溶液(藥用)質(zhì)量標(biāo)準(zhǔn)Ammonium Sulfate AR Quality Specification 硫酸銨AR質(zhì)量標(biāo)準(zhǔn) Glycine (BP) Quality Specification 甘氨酸（藥用）質(zhì)量標(biāo)準(zhǔn)Trypsin (BP) Quality Specification 胰蛋白酶(藥用)質(zhì)量標(biāo)準(zhǔn) New Born Bovine Serum Quality Specification 新生牛血清質(zhì)量標(biāo)準(zhǔn)Methotrexate氨甲喋呤Dimethyl Sulphoxide二甲基亞砜Semi-product of XXX standard, include the Harvest

32、liquid, the Purification liquid and the Bulk.XXX中間品質(zhì)量標(biāo)準(zhǔn)，包括細(xì)胞收獲液、純化產(chǎn)物、原液（文件號(hào)：XXX）Final product of XXX standard XXX成品質(zhì)量標(biāo)準(zhǔn)：（文件號(hào)：XXX）n The validation procedures, items and contents are divided based on the system process steps. The process steps, validation items, sampling methods, testing methods and a

33、cceptance criteria are described for each of the process steps.驗(yàn)證的程序、項(xiàng)目和內(nèi)容中以系統(tǒng)工序?yàn)閱挝?，?duì)各工序中的所涉及的工藝步驟、驗(yàn)證項(xiàng)目、取樣方法、檢驗(yàn)方法和接受標(biāo)準(zhǔn)進(jìn)行了規(guī)定和描述。n The process recipe of Bulk of XXX (Registration batch No.: for 20g: GUOYAOZHUNZI XXX, and for 10g GUOYAOZHUNZI XXX) is briefly described in this protocol. The contents of

34、the recipe are described and the process flow diagram is listed. The process procedure is conformed to the registration standard.本方案對(duì)XXX原液的工藝處方（注冊(cè)批號(hào)：20g：國藥準(zhǔn)字 XXX、10g：國藥準(zhǔn)字 XXX）進(jìn)行了簡介，對(duì)處方內(nèi)容進(jìn)行描述，并且列出直觀化的工藝流程圖。工藝規(guī)程與注冊(cè)標(biāo)準(zhǔn)一致。n This protocol is used to specify the purpose and the scope of this validation and

35、 the responsibilities of each of the departments during the implementation of this validation protocol.本方案旨在指明驗(yàn)證的目的、范圍，并規(guī)定了該驗(yàn)證方案在實(shí)施過程中的各部門的職責(zé)。n Summarize and evaluate the entire process validation based on the validation of each of the process steps and record the evaluation results to the report.在各

36、工序驗(yàn)證的基礎(chǔ)上，對(duì)整個(gè)工藝驗(yàn)證進(jìn)行了總結(jié)評(píng)價(jià)，評(píng)價(jià)結(jié)果記錄到驗(yàn)證報(bào)告中。7. Product And Process Description產(chǎn)品和工藝描述7.1 Product specification(1) Ampoule, one human dose of 1.0ml, containing 10g of HBsAg (1) 安瓿瓶，每1次人用劑量為1.0ml，含HBsAg 10g；(2) Ampoule, one human dose of 1.0ml, containing 20g of HBsAg (2) 安瓿瓶，每1次人用劑量為1.0ml，含HBsAg 20g；(3) Via

37、l, one human dose of 1.0ml, containing 20g of HBsAg (3) 西林瓶，每1次人用劑量為1.0ml，含HBsAg 20g；(4) Vial, one human dose of 0.5ml, containing 10g of HBsAg (4) 西林瓶，每1次人用劑量為0.5ml，含HBsAg 10g。7.2 Drug approval No. 20g：GUOYAOZHUNZI S20g：國藥準(zhǔn)字 S10g：GUOYAOZHUNZI S10g：國藥準(zhǔn)字 S7.3 Standard Followed: Chinese Pharmacopoeia

38、2010 Edition執(zhí)行標(biāo)準(zhǔn)：中國藥典2010版7.4 Raw Materials and Adjuvants原輔料情況Table 3 Raw Materials and Adjuvants表格 4原輔料信息表Name SpecificationsManufacturerPackaging SpecificationsSodium Chloride BP Tianjin Haiguang Pharmaceutical Industrial Co., Ltd. 1000g/bagDisodium hydrogen phosphate BP Hunan Jiudian Pharmaceutic

39、al Co., Ltd. 0.5kg/bottleCrystal Aluminum Chloride AP Beijing Yili Fine Chemical Co., Ltd. 500g/bottleGlucose AP Baitaike Bioltechnology Co., Ltd. (filling)500g/bottleGlutamineusp Gradeinvitrogen corporation (Gibco)1kg /drum Potassium Bromide AP Beijing Yili Fine Chemical Co., Ltd. 500g/bottleDisodi

40、um Edetate AP Sinopharm Chemical Reagent Co., Ltd 250g/bottleSodium Hydroxide AP Beijing Chemical Factory 500g/bottleMono-water disodium hydrogen phosphate BP Hunan Jiudian Pharmaceutical Co., Ltd. 0.5kg/bottleMerthiolate BP (USP/BP/EP)GIHON1kg /drum Hydrochloric acid AP Beijing Chemical Factory 250

41、0ml/ bottleProline -Shanghai Xiehe Amino Acid Co., Ltd. 500g/bagSodium bicarbonate AP Beijing Chemical Factory 500g/bottleDMEM-Gibco6687g/drum Formaldehyde AP Sinopharm Chemical Reagent Co., Ltd 500ml/ bottleAmmonium sulfate AP Beijing Yili Fine Chemical Co., Ltd. 25kg /drum GlycineAP Sinopharm Chem

42、ical Reagent Co., Ltd 100g/ bottle Pancreatin -BD 500g/bottleGibco100g/ bottle Methotrexate99.0%sigma-aldrich（fluka-analytical）100mg/ tube Dimethyl Sulphoxide-SERVA Electrophoresis GmbH1L/ bottle New born bovine serum Top grade Wuhan Sanli Biotechnology Co., Ltd. 1000ml/ bottle-Inner Mongolia Jinyua

43、nkang Biotechnology Co., Ltd. 500ml/ bottle原輔料名稱規(guī)格生產(chǎn)廠家包裝規(guī)格氯化鈉藥用級(jí)天津海光藥業(yè)有限公司1000g/袋磷酸氫二鈉藥用級(jí)湖南九典制藥有限公司0.5kg/瓶結(jié)晶氯化鋁分析純北京益利精細(xì)化學(xué)品有限公司500g/瓶葡萄糖分析純寶泰克生物科技公司（分裝）500g/瓶谷氨酰胺usp Gradeinvitrogen corporation（Gibco）1kg/桶溴化鉀分析純北京益利精細(xì)化學(xué)品有限公司500g/瓶乙二胺四乙酸二鈉分析純國藥集團(tuán)化學(xué)試劑有限公司250g/瓶氫氧化鈉分析純北京化工廠500g/瓶一水磷酸二氫鈉藥用級(jí)湖南九典制藥有限公司0.5

44、kg/瓶硫柳汞藥用級(jí)（USP/BP/EP）GIHON1Kg/桶鹽酸分析純北京化工廠2500ml/瓶脯氨酸-上海協(xié)和氨基酸有限公司500g/袋碳酸氫鈉分析純北京化工廠500g/瓶DMEM-Gibco6687g/桶甲醛分析純國藥集團(tuán)化學(xué)試劑有限公司500ml/瓶硫酸銨分析純北京益利精細(xì)化學(xué)品有限公司25kg/桶甘氨酸分析純國藥集團(tuán)化學(xué)試劑有限公司100g/瓶胰酶-BD公司500g/瓶Gibco100g/瓶氨甲喋呤99.0%sigma-aldrich（fluka-analytical）100mg/管二甲基亞砜-SERVA Electrophoresis GmbH1L/瓶新生牛血清優(yōu)級(jí)武漢三利生物技術(shù)

45、有限公司1000ml/瓶-內(nèi)蒙古金源康生物工程有限公司500ml/瓶7.5 Process description:工藝簡述：Ø Process Cycle 工藝周期Table 5 Process Schedule表格 6工藝時(shí)間表No.序號(hào)Process Steps 工藝步驟Time (Day)時(shí)間（天）1Resurrection 復(fù)蘇12C25 - First square bottle incubation C25-方瓶第一次培養(yǎng)573C26 - second square bottle incubation C26-方瓶第二次培養(yǎng)574C27 - Kolle flask in

46、cubation C27-克氏瓶培養(yǎng)575C28 - First 3L rotary bottle incubation C28-3L轉(zhuǎn)瓶第一次培養(yǎng)576C29 - second 3L rotary bottle incubation C29-3L轉(zhuǎn)瓶第二次培養(yǎng)577C30 - third 3L rotary bottle incubation C30-3L轉(zhuǎn)瓶第三次培養(yǎng)578C31 - first 15L bottle incubation C31-15L瓶第一次培養(yǎng)579C32 - second 15L bottle incubation C32-15L瓶第二次培養(yǎng)571020 conse

47、cutive harvest 連續(xù)20次收獲液4011Filtration for cell residue removal 去細(xì)胞殘?jiān)^濾0.512UF concentration 超濾濃縮0.513Ammonium sulfate precipitation 硫酸銨沉淀4514Potassium bromide extraction 溴化鉀提取115First density gradient centrifugation 一次密度梯度離心1.516Second density gradient centrifugation 二次密度梯度離心1.517UF concentration 超濾

48、濃縮118Chromatography purification 層析純化119Bulk solution combination 原液合并0.5Ø Preparation of production cells生產(chǎn)用細(xì)胞制備Take 1 to 2 tubes of cells from the working cell bank (of a same batch No.), resurrect, mix and inoculate them into a 100ml cell cultivation bottle in a ratio of 1:1. After the cells

49、 have grown full and adhering to the wall to a single layer, perform the 0.25% trypsin digestion and passage. And then inoculate them into a 100ml cell cultivation bottle in a ratio of 1:4. Passage and proliferate following the sequence of 100ml cell cultivation bottle, Kolle flask and the rotary bo

50、ttle. The ratio is determined according to different cell cultivation bottle specifications. Two 100ml cell cultivation bottles are used for the passage to 1 Kolle flask. One Kolle flask is used for the passage to 1 3L rotary bottle. One 3L rotary bottle is used for the passage to 7 to 8 3L rotary b

51、ottles. One 3L rotary bottle is used for the passage to 1 15L rotary bottle. One 15L rotary bottle is used for the passage to 7 to 8 15L rotary bottles. Incubate them while the bottles are kept still or rotated at 36±1 (the 100ml cell cultivation bottles are kept still during incubation and rot

52、ary bottles are rotated). The duration of each expansion incubation step is 5 to 7 days. After proliferated to generation No. 32, the change solution is maintained. 取工作細(xì)胞庫細(xì)胞12支（同一批號(hào)），復(fù)蘇混合后按1：1比例接種100ml細(xì)胞培養(yǎng)瓶，待細(xì)胞貼壁長滿至單層后，0.25%胰蛋白酶消化傳代，按1：4比例接種100ml細(xì)胞培養(yǎng)瓶。按100ml細(xì)胞培養(yǎng)瓶克氏瓶轉(zhuǎn)瓶傳代擴(kuò)增，比例根據(jù)不同規(guī)格細(xì)胞培養(yǎng)瓶而定，2個(gè)100ml細(xì)胞培養(yǎng)

53、瓶傳1個(gè)克氏瓶；1個(gè)克氏瓶傳1個(gè)3L轉(zhuǎn)瓶；一個(gè)3L轉(zhuǎn)瓶傳78個(gè)3L轉(zhuǎn)瓶；一個(gè)3L轉(zhuǎn)瓶傳1個(gè)15L轉(zhuǎn)瓶；1個(gè)15L轉(zhuǎn)瓶傳78個(gè)15L轉(zhuǎn)瓶。置36±1條件下，采用靜止或旋轉(zhuǎn)培養(yǎng)（100ml細(xì)胞培養(yǎng)瓶和克氏瓶采用靜止培養(yǎng)，轉(zhuǎn)瓶采用旋轉(zhuǎn)培養(yǎng)法）。每次擴(kuò)增培養(yǎng)時(shí)間為57天，傳代擴(kuò)增至32代，開始維持換液。 Ø Incubation media培養(yǎng)液The growth media used for cell passage and proliferation is DMEM solution containing 9% to 10% inactivated new born bovi

54、ne serum with a final concentration of methotrexate of 10-6mol/L and L-glutamine of 2mmol/L. The maintenance solution is DMEM solution containing 3% to 5% inactivated new born bovine serum. 細(xì)胞傳代擴(kuò)增用生長液為含有9%10%滅能新生牛血清的DMEM液，其中含終濃度為10-6mol/L氨甲喋呤、2mmol/L L-谷氨酰胺。維持液為含有3%5%滅能新生牛血清的DMEM液。Ø Harvesting收

55、獲After passage to the No. 32 generation and the cells have grown into a compact single layer adhering to the wall (Day 5 to 7), discard the growth liquor, add the maintenance solution, discard the incubation liquor 48 hours later and add the maintenance solution. Replace the maintenance solution every 48 hours thereafter and harvest the incubation liquor. Measure the HBsAg content in the harvested supernatant with ELISA (or reverse hemagglutination method), the result shall be above 1.0mg/L