工艺验证方案模板

ProcessValidation(PV) ProtocolXXXXXX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HYPERLINK""\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：HYPERLINK""\o"查找:Dateofapproval"DateofapprovalHYPERLINK""\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[]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：IndexTOC\o"1-3"\h\z\uHYPERLINK\l"_Toc6"1.Purpose目的PAGEREF_Toc6\h5HYPERLINK\l"_Toc7"2.Scope范围PAGEREF_Toc7\h5HYPERLINK\l"_Toc8"3.Responsibility职责PAGEREF_Toc8\h5HYPERLINK\l"_Toc9"AAAresponsibilitiesAAA的职责PAGEREF_Toc9\h5HYPERLINK\l"_Toc0"XXXresponsibilitiesXXX的职责PAGEREF_Toc0\h6HYPERLINK\l"_Toc1"4.RegulationandGuidance法规和指南PAGEREF_Toc1\h8HYPERLINK\l"_Toc2"5.Abbreviations缩略语PAGEREF_Toc2\h9HYPERLINK\l"_Toc3"6.ProtocolDescription方案说明PAGEREF_Toc3\h11HYPERLINK\l"_Toc4"7.ProductAndProcessDescription产品和工艺描述PAGEREF_Toc4\h14HYPERLINK\l"_Toc5"ProductspecificationPAGEREF_Toc5\h14HYPERLINK\l"_Toc6"DrugapprovalNo.PAGEREF_Toc6\h14HYPERLINK\l"_Toc7"StandardFollowed:PAGEREF_Toc7\h14HYPERLINK\l"_Toc8"RawMaterialsandAdjuvants原辅料情况PAGEREF_Toc8\h14HYPERLINK\l"_Toc9"Processdescription:PAGEREF_Toc9\h16HYPERLINK\l"_Toc0"ProcessRecipe工艺处方：PAGEREF_Toc0\h21HYPERLINK\l"_Toc1"ListofProductionPlants生产车间一览PAGEREF_Toc1\h26HYPERLINK\l"_Toc2"ListofProductionEquipment生产设备一览PAGEREF_Toc2\h29HYPERLINK\l"_Toc3"ProcessFlowDiagram工艺流程图PAGEREF_Toc3\h33HYPERLINK\l"_Toc4"CPPIdentificationCPP确认PAGEREF_Toc4\h34HYPERLINK\l"_Toc5"ValidationPlan验证计划PAGEREF_Toc5\h49HYPERLINK\l"_Toc6"8.Validationprerequisite验证先决条件PAGEREF_Toc6\h51HYPERLINK\l"_Toc7"PersonalIdentification人员确认PAGEREF_Toc7\h51HYPERLINK\l"_Toc8"ProcessDocumentationVerification生产文件确认PAGEREF_Toc8\h51HYPERLINK\l"_Toc9"TrainingVerification培训确认PAGEREF_Toc9\h52HYPERLINK\l"_Toc0"SystemandEquipmentInstrumentsCalibrationVerification系统和设备仪表校验确认PAGEREF_Toc0\h52HYPERLINK\l"_Toc1"SystemandEquipmentInstrumentsCalibrationVerification生产相关验证状态确认PAGEREF_Toc1\h53HYPERLINK\l"_Toc2"TestInstrumentsCalibrationVerification测试仪器和仪表校验确认PAGEREF_Toc2\h53HYPERLINK\l"_Toc3"TestingMethodValidationVerification检验方法验证的确认PAGEREF_Toc3\h54HYPERLINK\l"_Toc4"RawMaterialandExcipientVerification原辅料确认PAGEREF_Toc4\h54HYPERLINK\l"_Toc5"9.SamplingPlanandEvaluationCriteria取样计划及评估标准PAGEREF_Toc5\h56HYPERLINK\l"_Toc6"10.SampleCodingMethod样品编码原则PAGEREF_Toc6\h70HYPERLINK\l"_Toc7"11.ProcessValidationTestExecution工艺验证执行PAGEREF_Toc7\h72HYPERLINK\l"_Toc8"LiquidPreparation液体配制PAGEREF_Toc8\h72HYPERLINK\l"_Toc9"CellResurrectionandPassage细胞复苏及传代PAGEREF_Toc9\h74HYPERLINK\l"_Toc0"Harvestliquidtest收获液检测PAGEREF_Toc0\h81HYPERLINK\l"_Toc1"ClarificationFiltrationEfficacyTest澄清过滤效果检测PAGEREF_Toc1\h83HYPERLINK\l"_Toc2"UFConcentration1EfficacyTest超滤浓缩1效果检测PAGEREF_Toc2\h83HYPERLINK\l"_Toc3"Ammoniumsulfateprecipitationefficacytest硫酸铵沉淀效果检测PAGEREF_Toc3\h85HYPERLINK\l"_Toc4"Potassiumbromideextractionefficacytest溴化钾提取效果检测PAGEREF_Toc4\h86HYPERLINK\l"_Toc5"Firstdensitygradientcentrifugationefficacytest一次密度梯度离心效果检测PAGEREF_Toc5\h87HYPERLINK\l"_Toc6"Seconddensitygradientcentrifugationefficacytest二次密度梯度离心效果检测PAGEREF_Toc6\h88HYPERLINK\l"_Toc7"UFConcentration2EfficacyTest超滤浓缩2效果检测PAGEREF_Toc7\h89HYPERLINK\l"_Toc8"Chromatographypurificationproducttest层析纯化产物检测PAGEREF_Toc8\h90HYPERLINK\l"_Toc9"12.BulkDetermination原液检验PAGEREF_Toc9\h92HYPERLINK\l"_Toc0"13.ProcessValidationSummarization工艺验证总结PAGEREF_Toc0\h93HYPERLINK\l"_Toc1"14.PVReport工艺验证PAGEREF_Toc1\h94HYPERLINK\l"_Toc2"ProcessValidationReport工艺验证报告PAGEREF_Toc2\h94HYPERLINK\l"_Toc3"ProcessValidationAnalysisandEvaluation工艺验证分析与评价PAGEREF_Toc3\h94HYPERLINK\l"_Toc4"15.RequirementsonExecutionRecords执行记录要求PAGEREF_Toc4\h95HYPERLINK\l"_Toc5"16.DeviationHandling偏差处理PAGEREF_Toc5\h95HYPERLINK\l"_Toc6"17.ChangeHandling变更处理PAGEREF_Toc6\h95HYPERLINK\l"_Toc7"18.TestForm测试表PAGEREF_Toc7\h96TOC\h\z\c"表格"HYPERLINK\l"_Toc8"表格1缩略语PAGEREF_Toc8\h9HYPERLINK\l"_Toc9"表格2原辅料质量标准表PAGEREF_Toc9\h11HYPERLINK\l"_Toc0"表格3物料消耗表1PAGEREF_Toc0\h21HYPERLINK\l"_Toc1"表格4物料消耗表2PAGEREF_Toc1\h21HYPERLINK\l"_Toc2"表格5所用液体配料表PAGEREF_Toc2\h22HYPERLINK\l"_Toc3"表格6生产车间一览表PAGEREF_Toc3\h26HYPERLINK\l"_Toc4"表格7主要生产设备一览表PAGEREF_Toc4\h29HYPERLINK\l"_Toc5"表格8工艺风险分析表33HYPERLINK\l"_Toc6"表格9工艺时间表PAGEREF_Toc6\h49HYPERLINK\l"_Toc7"表格10取样计划表PAGEREF_Toc7\h56HYPERLINK\l"_Toc8"表格11样品编码原则PAGEREF_Toc8\h70HYPERLINK\l"_Toc9"表格12配液取样检测信息表PAGEREF_Toc9\h72HYPERLINK\l"_Toc0"表格13复苏和传代取样表PAGEREF_Toc0\h74HYPERLINK\l"_Toc1"表格14复苏和传代样品编码PAGEREF_Toc1\h80HYPERLINK\l"_Toc2"表格15收获液标准PAGEREF_Toc2\h82HYPERLINK\l"_Toc3"表格16澄清过滤标准PAGEREF_Toc3\h83HYPERLINK\l"_Toc4"表格17超滤浓缩1标准PAGEREF_Toc4\h84HYPERLINK\l"_Toc5"表格18硫酸铵沉淀标准PAGEREF_Toc5\h85HYPERLINK\l"_Toc6"表格19溴化钾提取标准PAGEREF_Toc6\h86HYPERLINK\l"_Toc7"表格20一次密度梯度离心标准PAGEREF_Toc7\h87HYPERLINK\l"_Toc8"表格21二次密度梯度离心标准PAGEREF_Toc8\h88HYPERLINK\l"_Toc9"表格22超滤浓缩2标准PAGEREF_Toc9\h89HYPERLINK\l"_Toc0"表格23纯化产物标准PAGEREF_Toc0\h90HYPERLINK\l"_Toc1"表格24原液标准PAGEREF_Toc1\h92HYPERLINK\l"_Toc2"表格25测试表列表PAGEREF_Toc2\h96Purpose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可以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，并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,2008EudraLex第四卷欧盟药品生产质量管理指南医药产品人用及兽用，2008年11月FinalVersionofAnnex15totheEUGuidetoGoodManufacturingPractice:QualificationandValidation,September2001EudraLex第四卷药品生产质量管理规范，附录15验证与确认，2001年9月EMEA-Noteforguidanceonprocessvalidation,September2001欧洲药品管理局–工艺验证指南，2001年9月EMEA-AnnexIItonoteforguidanceonprocessvalidation,January2005欧洲药品管理局–附录II工艺验证指南FDA21CodeofFederalRegulations,Part211,currentGoodManufacturingPracticeforFinishedPharmaceuticals,April2011FDA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，2011年4月FDAProcessValidation:GeneralPrinciplesandPractices,January2011FDA工艺验证：一般原则和实践，2011年01月SFDAGoodManufacturingPractice(2010Revision),March,2011SFDA药品生产质量管理规范（2010年修订），2011年03月PICPIC/SPharmaceuticalInspectionConvention,PharmaceuticalInspectionCo-operationScheme,“RecommendationsonValidationMasterPlan,InstallationandOperationalQualification,Non-sterileProcessValidation,CleaningValidation”,July2004PIC/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”，2004年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)2010Edition中国药典（CP）2010年版ProcessValidationofProteinManufacturing(TechnicalReportNo.42SupplementVol.59,No.S-4September/October2005©2005byPDA)蛋白生产的工艺验证（技术报告42，PDA，2005年版）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”inChinesePharmacopoeia2010Edition.半成品、成品按企业内控标准检验，成品发放按中国药典2010版三部“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CrystalAluminumChlorideARQualitySpecification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AmmoniumSulfateARQualitySpecification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，并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,onehumandoseof,containing10μgofHBsAg(1)安瓿瓶，每1次人用剂量为，含HBsAg10μg；(2)Ampoule,onehumandoseof,containing20μgofHBsAg(2)安瓿瓶，每1次人用剂量为，含HBsAg20μg；(3)Vial,onehumandoseof,containing20μgofHBsAg(3)西林瓶，每1次人用剂量为，含HBsAg20μg；(4)Vial,onehumandoseof,containing10μgofHBsAg(4)西林瓶，每1次人用剂量为，含HBsAg10μg。DrugapprovalNo.20μg：GUOYAOZHUNZIS20μg：国药准字S10μg：GUOYAOZHUNZIS10μg：国药准字SStandardFollowed:ChinesePharmacopoeia2010Edition执行标准：中国药典2010版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≥%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≥%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12UFconcentration超滤浓缩13Ammoniumsulfateprecipitation硫酸铵沉淀4~514Potassiumbromideextraction溴化钾提取115Firstdensitygradientcentrifugation一次密度梯度离心16Seconddensitygradientcentrifugation二次密度梯度离心17UFconcentration超滤浓缩118Chromatographypurification层析纯化119Bulksolutioncombination原液合并Preparationofproductioncells生产用细胞制备Take1to2tubesofcellsfromtheworkingcellbank(ofasamebatchNo.),resurrect,mixandinoculatethemintoa100mlcellcultivationbottleinaratioof1:1.Afterthecellshavegrownfullandadheringtothewalltoasinglelayer,performthe%trypsindigestionandpassage.Andtheninoculatethemintoa100mlcellcultivationbottleinaratioof1:4.Passageandproliferatefollowingthesequenceof100mlcellcultivationbottle,Kolleflaskandtherotarybottle.Theratioisdeterminedaccordingtodifferentcellcultivationbottlespecifications.Two100mlcellcultivationbottlesareusedforthepassageto1Kolleflask.OneKolleflaskisusedforthepassageto13Lrotarybottle.One3Lrotarybottleisusedforthepassageto7to83Lrotarybottles.One3Lrotarybottleisusedforthepassageto115Lrotarybottle.One15Lrotarybottleisusedforthepassageto7to815Lrotarybottles.Incubatethemwhilethebottlesarekeptstillorrotatedat36±1℃(the100mlcellcultivationbottlesarekeptstillduringincubationandrotarybottlesarerotated).Thedurationofeachexpansionincubationstepis5to7days.AfterproliferatedtogenerationNo.32,thechangesolutionismaintained.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，%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℃条件下，采用静止或旋转培养（100ml细胞培养瓶和克氏瓶采用静止培养，转瓶采用旋转培养法）。每次扩增培养时间为5～7天，传代扩增至32代，开始维持换液。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,discardtheincubationliquor48hou