工艺验证方案模板

年4月19日工艺验证方案模板文档仅供参考，不当之处，请联系改正。ProcessValidation(PV)

ProtocolXXXXXX工艺验证方案Writtenby

起草人Writtendate起草日期年月日Reviewedby审核人Revieweddate审核日期年月日Approvedby批准人Approveddate批准日期年月日Writtendepartment起草部门\o"查找:effectivedate"Effectivedate生效日期年月日Issuedby:

颁发部门：[]Copynumber:

拷贝号：[]Changerecord:Revisionnumber

变更记载：\o"查找:Dateofapproval"Dateofapproval\o"查找:effectivedate"Effectivedate修订号批准日期生效日期Changereasonandgoal:

变更原因及目的：Distributiondepartment:分发部门:所长办公室[]人力资源部[]后勤保卫部[]物资供应部[]工程服务部[]生产管理部[]质量保证部[]销售部[]仓储部[]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[]细胞因子室[]血清制品室[]实验动物室[]分包装室[]培养基室[]Verificationdepartment(department):验证科室（部门）：Verify

completiondate:验证完成日期：Validity:有效期：Index目录1. Purpose目的 7286\h52. Scope范围 7287\h53. Responsibility职责 7288\h53.1 AAAresponsibilitiesAAA的职责 7289\h53.2 XXXresponsibilitiesXXX的职责 7290\h64. RegulationandGuidance法规和指南 7291\h85. Abbreviations缩略语 7292\h96. ProtocolDescription方案说明 7293\h117. ProductAndProcessDescription产品和工艺描述 7294\h147.1 Productspecification 7295\h147.2 DrugapprovalNo. 7296\h147.3 StandardFollowed: 7297\h147.4 RawMaterialsandAdjuvants原辅料情况 7298\h147.5 Processdescription: 7299\h167.6 ProcessRecipe工艺处方： 7300\h217.7 ListofProductionPlants生产车间一览 7301\h267.8 ListofProductionEquipment生产设备一览 7302\h297.9 ProcessFlowDiagram工艺流程图 7303\h337.10 CPPIdentificationCPP确认 7304\h347.11 ValidationPlan验证计划 7305\h498. Validationprerequisite验证先决条件 7306\h518.1 PersonalIdentification人员确认 7307\h518.2 ProcessDocumentationVerification生产文件确认 7308\h518.3 TrainingVerification培训确认 7309\h528.4 SystemandEquipmentInstrumentsCalibrationVerification系统和设备仪表校验确认 7310\h528.5 SystemandEquipmentInstrumentsCalibrationVerification生产相关验证状态确认 7311\h538.6 TestInstrumentsCalibrationVerification测试仪器和仪表校验确认 7312\h538.7 TestingMethodValidationVerification检验方法验证的确认 7313\h548.8 RawMaterialandExcipientVerification原辅料确认 7314\h549. SamplingPlanandEvaluationCriteria取样计划及评估标准 7315\h5610. SampleCodingMethod样品编码原则 7316\h7011. ProcessValidationTestExecution工艺验证执行 7317\h7211.1 LiquidPreparation液体配制 7318\h7211.2 CellResurrectionandPassage细胞复苏及传代 7319\h7411.3 Harvestliquidtest收获液检测 7320\h8111.4 ClarificationFiltrationEfficacyTest澄清过滤效果检测 7321\h8311.5 UFConcentration1EfficacyTest超滤浓缩1效果检测 7322\h8311.6 Ammoniumsulfateprecipitationefficacytest硫酸铵沉淀效果检测 7323\h8511.7 Potassiumbromideextractionefficacytest溴化钾提取效果检测 7324\h8611.8 Firstdensitygradientcentrifugationefficacytest一次密度梯度离心效果检测 7325\h8711.9 Seconddensitygradientcentrifugationefficacytest二次密度梯度离心效果检测 7326\h8811.10 UFConcentration2EfficacyTest超滤浓缩2效果检测 7327\h8911.11 Chromatographypurificationproducttest层析纯化产物检测 7328\h9012. BulkDetermination原液检验 7329\h9213. ProcessValidationSummarization工艺验证总结 7330\h9314. PVReport工艺验证报告 7331\h9414.1 ProcessValidationReport工艺验证报告 7332\h9414.2 ProcessValidationAnalysisandEvaluation工艺验证分析与评价 7333\h9415. RequirementsonExecutionRecords执行记录要求 7334\h9516. DeviationHandling偏差处理 7335\h9517. ChangeHandling变更处理 7336\h9518. TestForm测试表 7337\h96TOC\h\z\c"表格"表格1缩略语 9表格2原辅料质量标准表 11表格3物料消耗表1 21表格4物料消耗表2 21表格5所用液体配料表 22表格6生产车间一览表 26表格7主要生产设备一览表 29表格8工艺风险分析表 33表格9工艺时间表 49表格10取样计划表 56表格11样品编码原则 70表格12配液取样检测信息表 72表格13复苏和传代取样表 74表格14复苏和传代样品编码 80表格15收获液标准 82表格16澄清过滤标准 83表格17超滤浓缩1标准 84表格18硫酸铵沉淀标准 85表格19溴化钾提取标准 86表格20一次密度梯度离心标准 87表格21二次密度梯度离心标准 88表格22超滤浓缩2标准 89表格23纯化产物标准 90表格24原液标准 92表格25测试表列表 96

Purpose目的Thispurposeofthisprocessvalidationistoprovidedocumentedevidencetoshowthatthecombinationofthepersonnel,materials,equipment,methods,environmentalconditionsandtheotherrelatedutilitiesXXXCo.,LTD(XXX)canensuretheconsistentproductionofproductsconformingtotheenterpriseinternalstandardsandthenationalstandards,andtheprocessesarereliableandconformingtotheGMPrequirements.Theprocessvalidationandthetestresultsaretobedocumentedbasedonthisvalidationprotocol.本工艺验证用于提供文件化的证据，证明XXX疫苗一室生产XXX原液的人员、材料、设备、方法、环境条件以及其它有关公用设施的组合能够始终如一的生产出符合企业内控标准及国家法定标准的产品，工艺稳定可靠，符合GMP要求，工艺验证的过程和检查的结果将按照该验证方案进行记录。Scope范围ThisprotocolisapplicabletotheprocessvalidationofXXXproducedbytheequipmentlocatedXXX.本方案适用于XXX原液生产的工艺验证。由于工作细胞库制备的过程并非常规生产过程，因此，不在本次验证中进行考察。ThelabelingandpackagingprocessesarenotwithinthescopeofthisdocumentsincetheseprocesseshavebeenvalidatedperPQprotocol(labeling)andPQprotocol(packaging),andtheseprocesseshaveminimalimpactonfinishedproductquality.

贴标签和包装也不在此文件范围内，因为这些工艺已经按照PQ方案（贴标签）和PQ方案(包装)验证过，而且这些工艺对产品质量极少有影响。Responsibility职责AAAresponsibilitiesAAA的职责CompileProtocol

方案的编写GuideProtocolexecution,datacollection/Providetestdataforreviewuponrequestbycustomer

指导方案的实施，数据的收集/在客户要求时提供测试数据供其审核FinalreportReview/Analyzeandcompiledataintothesummaryreport

最终报告的审核/对数据进行分析并将其编辑入总结报告之中XXXresponsibilitiesXXX的职责Productiondepartment生产部Reviewandapproveprotocol

审核并批准验证方案Assurealltheequipmentusedforvalidationcanbemaintainedtimely

保证验证用所有设备均能按SOP进行及时的维护和保养Assurealltherawmaterialsusedforvalidationcanbesuppliedtimelyandcomplywithdomesticqualitycriteriaofenterprise

保证验证用原辅包装材料能按时到货，并符合企业内控标准Researchanddevelopmentdepartment研发部Provideproductregistrationcriteriaandreviewwhetherrecipeandprocessstepdescribedintheprotocoliscomplyingwithregisteredcriteria

提供产品的注册工艺，审核方案中生产处方、工艺与注册资料的一致性Reviewandmakeassurancethecomplianceofrawmaterialsupplierwithregisteredsupplier

审核原辅料供应商和注册资料的一致性Reviewrelevantqualitycriteriaincludingrawmaterial,intermediaandfinishedproduct

审核原辅料标准、中控质量标准和成品的质量标准VaccineonePlant疫苗一室Coordinationandimplementationofthisvalidationprotocol

协调、实施本验证方案Trainingofpersonnelbasedonthisvalidationprotocol

按验证方案对有关人员进行培训Collection,sortingandreviewingofvalidationdataanddraftingofvalidationreports

(includingthedeviationreport,ifthereareanydeviations)

收集、整理和审核验证数据，起草验证报告（如方案执行有偏差，要完成偏差报告）。Utilitysupportdepartment工程服务部Assistanceinimplementationofthisvalidationprotocol

协助本验证方案的实施Calibrationofinstrumentsrelatedtothisvalidationprotocolandprovidingrelevantcalibrationcertificatesandqualitycertificates

负责校验和本验证方案有关的仪器、仪表，出具校验报告并贴合格证Assurenormaloperationoftheutilitysystem

保证公用系统的正常运行FinalreportReview

最终报告的审核Qualitycontrolofqualitydepartment质量部质量控制Determinationoftestmethodsusedinthisvalidationprotocol

负责确定本验证方案的检验方法ValidationofthenecessarytestmethodsforXXXXXinvolvedinthisvalidationprotocol

负责本验证方案涉及的重组乙型肝炎疫苗（CHO细胞）原液生产的必要的检验方法的验证ThequalityinspectionsonXXXXXTabletinvolvedinthisvalidationprotocol,andpresentingfullitemcertificatesofanalysisandqualityinspectionreportsandrecords

负责本验证方案涉及的重组乙型肝炎疫苗（CHO细胞）原液生产的质量检验，并出具全项检验报告，提供质量检验报告书及检验记录Providingtechnicalsupportsandguidancefortestsateachoftheprocesssteps

负责各工序检验的技术支持和指导Coordinatingtheplantintheimplementationofthisvalidationprotocol

协同车间实施本验证方案FinalreportReview

最终报告的审核Qualityassuranceofqualitydepartment质量部质量保证Supervisionofvalidationprocess,reviewandapprovalofthisprotocol,andensuringtheimplementationfollowingthisprotocol

负责验证过程监督，方案的审核与批准，确保按批准的方案执行Samplingateachprocessstepsduringtheprocessvalidation

负责工艺验证中各工序的取样Coordinationamongdifferentdepartmentsinvolvedinthevalidationactivities

负责验证涉及到的各部门之间的协调Providingequipmentvalidationreportsrelatedtothisvalidationprotocol

负责提供和本验证方案有关的设备验证报告Identification,auditandassessmentofsuppliers

供应商的确认、审计及评价Assessmentandhandlingofchangesanddeviationsoccurduringthisprocessvalidation

负责对工艺验证实施过程中出现的变更和偏差的评价和处理FinalreportReview

最终报告的审核RegulationandGuidance法规和指南EudraLexVolume4EUGuidelinestoGoodManufacturingPracticeMedicinalProductsforHumanandVeterinaryUse,November,

EudraLex第四卷欧盟药品生产质量管理规范指南医药产品人用及兽用，11月FinalVersionofAnnex15totheEUGuidetoGoodManufacturingPractice:QualificationandValidation,September

EudraLex第四卷药品生产质量管理规范，附录15验证与确认，9月EMEA-Noteforguidanceonprocessvalidation,September

欧洲药品管理局–工艺验证指南，9月EMEA-AnnexIItonoteforguidanceonprocessvalidation,January

欧洲药品管理局–附录II工艺验证指南FDA21CodeofFederalRegulations,Part211,currentGoodManufacturingPracticeforFinishedPharmaceuticals,April

FDA联邦法典第21篇第211部分，现行药物制剂生产质量管理规范，4月FDAProcessValidation:GeneralPrinciplesandPractices,January

FDA工艺验证：一般原则和实践，01月SFDAGoodManufacturingPractice(Revision),March,

SFDA药品生产质量管理规范（修订），03月PICPIC/SPharmaceuticalInspectionConvention,PharmaceuticalInspectionCo-operationScheme,“RecommendationsonValidationMasterPlan,InstallationandOperationalQualification,Non-sterileProcessValidation,CleaningValidation”,July

PIC/S药品监管公约，药品监管合作计划，“有关验证主计划、安装和运行确认、非无菌工艺验证、清洁验证的建议”，7月AWHOGuidetogoodmanufacturingpractice(GMP)requirements,Part2Validation,1997WHO关于GMP要求的指南，第二部分：验证，1997年ChinesePharmacopoeia(CP)Edition

中国药典（CP）ProcessValidationofProteinManufacturing(TechnicalReportNo.42SupplementVol.59,No.S-4September/October©byPDA)

蛋白生产的工艺验证（技术报告42，PDA，）Abbreviations缩略语Theabbreviationswhichwillbeusedinthisprotocolarelistedinthefollowingform.在下面的表格中规定了本方案中使用的缩略语。表格SEQ表格\*ARABIC1缩略语Abbreviations缩略语Definition定义APIActivePharmaceuticalIngredient(or“DrugSubstance”)

原料药（或药品物质）CPPCriticalProcessParameter

关键工艺参数CQACriticalQualityattribute

关键质量属性CVCleaningValidation

清洗验证IQInstallationQualification

安装确认MFVMediaFillValidation

培养基模拟灌装试验N/ANotapplicable

不适用OQOperationQualification

运行确认PLCProgrammableLogicController

可编程逻辑控制器PQPerformanceQualification

性能确认PVProcessValidation

工艺验证PWPurifiedWater

纯化水QAQualityAssurance

质量保证RARiskAssessment

风险分析SOPStandardOperationProcedure

标准操作程序URSUserRequirementSpecification

用户使用要求VMPValidationMasterPlan

验证主计划WFIWaterforInjection

注射用水

ProtocolDescription方案说明UsingthisdefinedprocessvalidationshallbeperformedfortheresultsoftheProcessRiskAssessment.本方案实施的工艺验证，是基于工艺风险评估结果的。UsingthisdefinedprocessvalidationshallbeperformedforthreeconsecutivesuccessfulrunsbasedonGMPrequirementstodemonstratetheprocesscontrolreliabilityandtherepeatability.鉴于XXX的生产已经有近20年历史，本次验证仅是对生产地址发生变更后且主要设备未发生变化的再验证，考虑到本产品生产周期长（总生产周期为158天）的原因，本方案实施的工艺验证将生产1批成功的产品以证明其可靠性。Thedeterminationoftheintermediateandfinalproductwillimplemententerpriseinternalstandardbutreleasefinishedproductshouldbeinaccordancewithcriteriaoftheitem“XXX”inChinesePharmacopoeiaEdition.半成品、成品按企业内控标准检验，成品发放按中国药典三部“XXX”标准执行Rawmaterialinternalstandard原辅料内控标准：表格SEQ表格\*ARABIC2原辅料质量标准表QualityStandardDoc.质量控制标准文件Doc.Number文件编号SodiumChloride(BP)QualitySpecification

氯化钠（药用级）检验质量标准SodiumDihydrogenPhosphate(BP)QualitySpecification

磷酸二氢钠（药用级）检验质量标准CrystalAluminumChloride结晶氯化铝AR检验质量标准Glucose(BP)QualitySpecification葡萄糖（药用级）检验质量标准GlutamineQualitySpecification谷氨酰胺质量标准PotassiumBromideQualitySpecification溴化钾质量标准DisodiumEdetateQualitySpecification乙二胺四乙酸二钠质量标准SodiumHydroxide(BP)QualitySpecification氢氧化钠（药用级）质量标准DisodiumHydrogenPhosphate(BP)QualitySpecification磷酸氢二钠(药用级)质量标准Merthiolate(BP)QualitySpecification硫柳汞（药用）质量标准HydrochloricAcid(BP)QualitySpecification盐酸（药用级）检验质量标准Proline(BP)QualitySpecification脯氨酸（药用）质量标准SodiumBicarbonate(BP)QualitySpecification碳酸氢钠（药用级）检验质量标准DMEMQualitySpecificationDMEM质量标准FormaldehydeSolution(BP)QualitySpecification甲醛溶液(药用)质量标准AmmoniumSulfate硫酸铵AR质量标准Glycine(BP)QualitySpecification甘氨酸（药用）质量标准Trypsin(BP)QualitySpecification胰蛋白酶(药用)质量标准NewBornBovineSerumQualitySpecification新生牛血清质量标准Methotrexate氨甲喋呤DimethylSulphoxide二甲基亚砜Semi-productofXXXstandard,includetheHarvestliquid,thePurificationliquidandtheBulk.

XXX中间品质量标准，包括细胞收获液、纯化产物、原液（文件号：XXX）FinalproductofXXXstandardXXX成品质量标准：（文件号：XXX）Thevalidationprocedures,itemsandcontentsaredividedbasedonthesystemprocesssteps.Theprocesssteps,validationitems,samplingmethods,testingmethodsandacceptancecriteriaaredescribedforeachoftheprocesssteps.验证的程序、项目和内容中以系统工序为单位，对各工序中的所涉及的工艺步骤、验证项目、取样方法、检验方法和接受标准进行了规定和描述。TheprocessrecipeofBulkofXXX(RegistrationbatchNo.:for20μg:GUOYAOZHUNZIXXX,andfor10μgGUOYAOZHUNZIXXX)isbrieflydescribedinthisprotocol.Thecontentsoftherecipearedescribedandtheprocessflowdiagramislisted.Theprocessprocedureisconformedtotheregistrationstandard.本方案对XXX原液的工艺处方（注册批号：20μg：国药准字XXX、10μg：国药准字XXX）进行了简介，对处方内容进行描述，而且列出直观化的工艺流程图。工艺规程与注册标准一致。Thisprotocolisusedtospecifythepurposeandthescopeofthisvalidationandtheresponsibilitiesofeachofthedepartmentsduringtheimplementationofthisvalidationprotocol.本方案旨在指明验证的目的、范围，并规定了该验证方案在实施过程中的各部门的职责。Summarizeandevaluatetheentireprocessvalidationbasedonthevalidationofeachoftheprocessstepsandrecordtheevaluationresultstothereport.在各工序验证的基础上，对整个工艺验证进行了总结评价，评价结果记录到验证报告中。

ProductAndProcessDescription产品和工艺描述Productspecification(1)Ampoule,onehumandoseof1.0ml,containing10μgofHBsAg(1)安瓿瓶，每1次人用剂量为1.0ml，含HBsAg10μg；

(2)Ampoule,onehumandoseof1.0ml,containing20μgofHBsAg(2)安瓿瓶，每1次人用剂量为1.0ml，含HBsAg20μg；

(3)Vial,onehumandoseof1.0ml,containing20μgofHBsAg(3)西林瓶，每1次人用剂量为1.0ml，含HBsAg20μg；

(4)Vial,onehumandoseof0.5ml,containing10μgofHBsAg(4)西林瓶，每1次人用剂量为0.5ml，含HBsAg10μg。DrugapprovalNo.20μg：GUOYAOZHUNZIS10960047

20μg：国药准字S10960047

10μg：GUOYAOZHUNZIS1096008010μg：国药准字S10960080StandardFollowed:ChinesePharmacopoeiaEdition执行标准：中国药典RawMaterialsandAdjuvants原辅料情况TableSEQ表格\*ARABIC3RawMaterialsandAdjuvants表格SEQ表格\*ARABIC4原辅料信息表NameSpecificationsManufacturerPackagingSpecificationsSodiumChlorideBPTianjinHaiguangPharmaceuticalIndustrialCo.,Ltd.1000g/bagDisodiumhydrogenphosphateBPHunanJiudianPharmaceuticalCo.,Ltd.0.5kg/bottleCrystalAluminumChlorideAPBeijingYiliFineChemicalCo.,Ltd.500g/bottleGlucoseAPBaitaikeBioltechnologyCo.,Ltd.(filling)500g/bottleGlutamineuspGradeinvitrogencorporation(Gibco)1kg/drumPotassiumBromideAPBeijingYiliFineChemicalCo.,Ltd.500g/bottleDisodiumEdetateAPSinopharmChemicalReagentCo.,Ltd250g/bottleSodiumHydroxideAPBeijingChemicalFactory500g/bottleMono-waterdisodiumhydrogenphosphateBPHunanJiudianPharmaceuticalCo.,Ltd.0.5kg/bottleMerthiolateBP(USP/BP/EP)GIHON1kg/drumHydrochloricacidAPBeijingChemicalFactory2500ml/bottleProline-ShanghaiXieheAminoAcidCo.,Ltd.500g/bagSodiumbicarbonateAPBeijingChemicalFactory500g/bottleDMEM-Gibco6687g/drumFormaldehydeAPSinopharmChemicalReagentCo.,Ltd500ml/bottleAmmoniumsulfateAPBeijingYiliFineChemicalCo.,Ltd.25kg/drumGlycineAPSinopharmChemicalReagentCo.,Ltd100g/bottlePancreatin-BD500g/bottleGibco100g/bottleMethotrexate≥99.0%sigma-aldrich（fluka-analytical）100mg/tubeDimethylSulphoxide-SERVAElectrophoresisGmbH1L/bottleNewbornbovineserumTopgradeWuhanSanliBiotechnologyCo.,Ltd.1000ml/bottle-InnerMongoliaJinyuankangBiotechnologyCo.,Ltd.500ml/bottle原辅料名称规格生产厂家包装规格氯化钠药用级天津海光药业有限公司1000g/袋磷酸氢二钠药用级湖南九典制药有限公司0.5kg/瓶结晶氯化铝分析纯北京益利精细化学品有限公司500g/瓶葡萄糖分析纯宝泰克生物科技公司（分装）500g/瓶谷氨酰胺uspGradeinvitrogencorporation（Gibco）1kg/桶溴化钾分析纯北京益利精细化学品有限公司500g/瓶乙二胺四乙酸二钠分析纯国药集团化学试剂有限公司250g/瓶氢氧化钠分析纯北京化工厂500g/瓶一水磷酸二氢钠药用级湖南九典制药有限公司0.5kg/瓶硫柳汞药用级（USP/BP/EP）GIHON1Kg/桶盐酸分析纯北京化工厂2500ml/瓶脯氨酸-上海协和氨基酸有限公司500g/袋碳酸氢钠分析纯北京化工厂500g/瓶DMEM-Gibco6687g/桶甲醛分析纯国药集团化学试剂有限公司500ml/瓶硫酸铵分析纯北京益利精细化学品有限公司25kg/桶甘氨酸分析纯国药集团化学试剂有限公司100g/瓶胰酶-BD公司500g/瓶Gibco100g/瓶氨甲喋呤≥99.0%sigma-aldrich（fluka-analytical）100mg/管二甲基亚砜-SERVAElectrophoresisGmbH1L/瓶新生牛血清优级武汉三利生物技术有限公司1000ml/瓶-内蒙古金源康生物工程有限公司500ml/瓶Processdescription:工艺简述：ProcessCycle工艺周期TableSEQ表格\*ARABIC5ProcessSchedule表格SEQ表格\*ARABIC6工艺时间表No.序号ProcessSteps工艺步骤Time(Day)时间（天）1Resurrection复苏12C25-FirstsquarebottleincubationC25-方瓶第一次培养5~73C26-secondsquarebottleincubationC26-方瓶第二次培养5~74C27-KolleflaskincubationC27-克氏瓶培养5~75C28-First3LrotarybottleincubationC28-3L转瓶第一次培养5~76C29-second3LrotarybottleincubationC29-3L转瓶第二次培养5~77C30-third3LrotarybottleincubationC30-3L转瓶第三次培养5~78C31-first15LbottleincubationC31-15L瓶第一次培养5~79C32-second15LbottleincubationC32-15L瓶第二次培养5~71020consecutiveharvest连续20次收获液4011Filtrationforcellresidueremoval去细胞残渣过滤0.512UFconcentration超滤浓缩0.513Ammoniumsulfateprecipitation硫酸铵沉淀4~514Potassiumbromideextraction溴化钾提取115Firstdensitygradientcentrifugation一次密度梯度离心1.516Seconddensitygradientcentrifugation二次密度梯度离心1.517UFconcentration超滤浓缩118Chromatographypurification层析纯化119Bulksolutioncombination原液合并0.5Preparationofproductioncells生产用细胞制备Take1to2tubesofcellsfromtheworkingcellbank(ofasamebatchNo.),resurrect,mixandinoculatethemintoa100mlcellcultivationbottleinaratioof1:1.Afterthecellshavegrownfullandadheringtothewalltoasinglelayer,performthe0.25%trypsindigestionandpassage.Andtheninoculatethemintoa100mlcellcultivationbottleinaratioof1:4.Passageandproliferatefollowingthesequenceof100mlcellcultivationbottle,Kolleflaskandtherotarybottle.Theratioisdeterminedaccordingtodifferentcellcultivationbottlespecifications.Two100mlcellcultivationbottlesareusedforthepassageto1Kolleflask.OneKolleflaskisusedforthepassageto13Lrotarybottle.One3Lrotarybottleisusedforthepassageto7to83Lrotarybottles.One3Lrotarybottleisusedforthepassageto115Lrotarybottle.One15Lrotarybottleisusedforthepassageto7to815Lrotarybottles.Incubatethemwhilethebottlesarekeptstillorrotatedat36±1℃取工作细胞库细胞1～2支（同一批号），复苏混合后按1：1比例接种100ml细胞培养瓶，待细胞贴壁长满至单层后，0.25%胰蛋白酶消化传代，按1：4比例接种100ml细胞培养瓶。按100ml细胞培养瓶→克氏瓶→转瓶传代扩增，比例根据不同规格细胞培养瓶而定，2个100ml细胞培养瓶传1个克氏瓶；1个克氏瓶传1个3L转瓶；一个3L转瓶传7～8个3L转瓶；一个3L转瓶传1个15L转瓶；1个15L转瓶传7～8个15L转瓶。置36±1℃Incubationmedia培养液ThegrowthmediausedforcellpassageandproliferationisDMEMsolutioncontaining9%to10%inactivatednewbornbovineserumwithafinalconcentrationofmethotrexateof10-6mol/LandL-glutamineof2mmol/L.ThemaintenancesolutionisDMEMsolutioncontaining3%to5%inactivatednewbornbovineserum.细胞传代扩增用生长液为含有9%～10%灭能新生牛血清的DMEM液，其中含终浓度为10-6mol/L氨甲喋呤、2mmol/LL-谷氨酰胺。维持液为含有3%～5%灭能新生牛血清的DMEM液。Harvesting收获AfterpassagetotheNo.32generationandthecellshavegrownintoacompactsinglelayeradheringtothewall(Day5to7),discardthegrowthliquor,addthemaintenancesolution,discardtheincubationliquor48hourslaterandaddthemaintenancesolution.Replacethemaintenancesolutionevery48hoursthereafterandharvesttheincubationliquor.MeasuretheHBsAgcontentintheharvestedsupernatantwithELISA(orreversehemagglutinationmethod),theresultshallbeabove1.0mg/L(RPHAtiter1:128).Atthesametimeasterilitytestshallbeperformedfortheharvestedsolutionofeachbottle.Thenumberofharvestingforasamebatchofproductioncellsshallbe19to21(20±1)andtheharvestsolutionof1to2batchescanbecombinedforpurification.传代扩增至32代的细胞贴壁长成致密单层后（5～7天），弃去生长液，加入维持液，48小时后弃去培养液，加入维持液。此后，每48小时更换一次维持液，并收获培养液。收获的培养上清液采用ELISA法(或反向血凝法)检测HBsAg含量，应在1.0mg/L(RPHA滴度1：128)以上，同时每瓶细胞培养收获液应进行无菌检查，同一批生产细胞收液次数应达到19～21（20±1）次，每1～2批收获液可合并纯化。Purification纯化Filtration过滤Theharvestedculturemediagofirstthrougha0.8μmfilterandthena0.2μmfiltertoremoveanycellresidues.收获的培养液经过0.8μm，然后经过0.2μm滤柱，过滤去除细胞碎片。Thefiltrategoesthroughafilterthatcanretainmoleculesof100KDandabove.Thenitisconcentrated3to5timesthroughUF.滤液经过截留分子量100KD的滤柱，超滤浓缩至3～5倍。Ammoniumsulfateprecipitation硫酸铵沉淀AfterUFconcentration,312gofammoniumsulfateisaddedtoeachliteroftheobtainedculturemedia.Itisthenallowedtostandatroomtemperaturefor4to5daysafterstirringtoallowHBsAgtoprecipitate.超滤浓缩后的培养液每升加入312g硫酸铵，搅拌均匀后，室温放置4～5天，使HBsAg沉淀。Potassiumbromideextraction溴化钾提取Theprecipitationsolutioniscentrifugedfor30minat4000r/min.Thesupernatantisdiscarded.Theprecipitatesaresufficientlydissolvedafteraddingpotassiumbromidepowder(4.5to5.5gofpotassiumbromideisaddedtoeachliteroftheharvestsolution).Then50%potassiumbromideisusedfortheextractionofHBsAg.沉淀液4000r/min离心30min，弃上清，沉淀中加入溴化钾固体粉末充分溶解（每升收获液加4.5～5.5g溴化钾），再用50%溴化钾溶液提取HBsAg。Potassiumbromidedensitygradientcentrifugation(twice)溴化钾密度梯度离心（两次）HBsAgisseparatedwithtwopotassiumbromidedensitygradientcentrifugations.Thedensityratiosofthetwodensitygradientcentrifugationwereboth:采用两次溴化钾密度梯度离心HBsAg，两次离心时密度梯度液的比重均为：SolutionA:1.04g/mlsodiumchloridesolutionA液:1.04g/ml的氯化钠溶液；SolutionB:1.28g/mlsodiumbromidesolutionB液:1.28g/ml的溴化钾溶液；SolutionC:1.32g/mltestsubstanceC液:1.32g/ml的供试品；SolutionD:1.34g/mlsodiumbromidesolutionD液:1.34g/ml的溴化钾溶液。InjectthesamplesfollowingthesequenceofsolutionA,solutionB,solutionCandsolutionD.Thespeedofthecentrifugeisrpmbothatthetimeofsampleinjectionandatthetimeofsamplecollection.Thecentrifugationisperformedataspeedo25000rpmfor22hours.HBsAgpeaksarecollectedaccordingtotheUVabsorbancevalueat280nm.TheHBsAgpeakispeakB.ThesamemethodisusedforthecollectionofHBsAgforthetwopotassiumbromidedensitygradientcentrifugation.按A液、B液、C液、D液的顺序依次上样，上样和收样时离心机的转速均为每分钟转。以每分钟25000转的转速离心22小时，根据波长为280nm的紫外吸光度值收集HBsAg峰。HBsAg峰为B峰。两次溴化钾密度梯度离心收集HBsAg的方法相同。Columnchromatography柱色谱ThesamplescollectedthroughthetwodensitygradientcentrifugationarefilteredthroughaUFmembranethatcanretainmoleculesof100KDtoconcentrateit(for4to5times).And20mmol/LphosphatebuffersolutionwithapHvalueof7.0isusedforwashing.Sepharose4FFgelfiltrationandchromatographyareperformedfortheconcentratedsamples.Thesampleinjectionamountis4%to6%ofthecolumnvolume.Themobilephaseis20mmol/LphosphatebuffersolutionwithapHvalueof7.0,theelutionspeedis480ml/handHBsAgpeaksarecollectedaccordingtoaUVabsorbancevalueat280nm.TheHBsAgpeakispeakB.Theliquidcollectedaftersterilefiltrationisthepurifiedproduct.二次密度梯度离心收集样品经过截留分子量100KD的超滤膜超滤浓缩（4～5倍），并用pH7.0的20mmol/L磷酸盐缓冲液进行冲洗。浓缩样品进行Sepharose4FF凝胶过滤层析，上样量为柱体积的4%～6%，流动相为pH7.0的20mmol/L磷酸盐缓冲液，洗脱流速为480ml/h，根据波长为280nm的紫外吸光度值收集HBsAg峰。HBsAg峰为B峰。经除菌过滤后收集的液体即为纯化产物。Formaldehydetreatment甲醛处理Add5%formaldehydesolutiontotheHBsAgpurifiedproduct.Thefinalconcentrationofformaldehydeis200μg/ml.Maintainthetemperatureat36℃±1向HBsAg纯化产物中加入5%甲醛溶液，甲醛终浓度为200μg/ml，置36℃±1℃SterilizationofPurifiedProduct纯化产物除菌Afteracceptableresultsareobtained,HBsAgpurifiedproductsfromasamecelldigestionbatcharecombined.AndthebulkisobtainedafterUF,concentrationand0.2μmsterilefiltration.同一细胞消化批来源的HBsAg纯化产物检定合格后合并，经超滤、浓缩、0.2μm除菌过滤后即为原液。ProcessRecipe工艺处方：MajorRecipe主处方：Materialsconsumed