WHO对HPV疫苗质量安全性及有效性指导原则

GUIDELINESTOASSURETHEQUALITY,SAFETYANDEFFICACYOFRECOMBINANTHUMANPAPILLOMAVIRUSVIRUS¬LIKEPARTICLEVACCINESHPV二、Specialconsiderationsection:在生产、非临床及临床中过程中的考虑因素：1、生产方面：VLP是复杂的生物产物，必须在不同水平下对其进行检测分析。因而在其生产过程及质量控制上必须考虑以下几个因素：1)新的表达体系如杆状病毒（GSK），新的特殊要求。但我们用的毕赤酵母，相对比较常见。2)新佐剂（略）3)天然的L1蛋白是没有被糖基化修饰，目前的两种表达体系，在糖基化修饰上不存大问题,但要对糖基化及其位点进行分析。4)L1衣壳蛋白亚单位的解聚与再聚，可能有利于纯化，并得到更稳定的VLP。目前，我们的路线可能是不经解聚，直接纯化获得VLP。个人感觉，到后期可以兵分两路，一路直接获得VLP，而另一路则将VLP解聚后，再进行纯化与重组。5)纯化后的L1VLP要进行生化及免疫上的鉴定，并测定L1的浓度、纯度及组聚情况。6)如加入了防腐剂，应对其免疫性进行验证，并确认不会有负作用2、非临床方面：关键就是要证明其免疫原性，并能否产生免疫中和抗体。3、临床方面：（略）三、生产指导（PartA.Guidelinesonmanufacturing）3.1定义definitions3.1.1国际名称和专有名称国际名称：重组人乳头瘤类病毒颗粒疫苗（基因型16L1蛋白）3.1.2定义描述重组HPVVLP疫苗为无菌的液态疫苗，里面含纯化后由一种或多种HPV基因型重组的主要的衣壳蛋白，并与相应佐剂混合。3.1.3国际标准品在此指导原则编写时，市场上暂无国际标准品提供。但有相应试剂在实验室水平上，在进行注射后进行生物效价方面的评价如抗体滴度和病毒DNA检测。3.2术语TerminologyThedefinitionsgivenbelowapplytothisdocumentonly.HPVL1protein:Themajorstructuralproteinofhumanpapillomavirus,ofwhich360moleculesarefoundinthenativevirionassociatedin72pentamericcapsomers.L1virus¬likeparticle:Anon¬infectious,non¬enveloped,icosahedralcapsidparticlewhichdoesnotcontainviralDNAandwhichiscomposedofregulararraysofL1pentamericcapsomers.Parentalyeastcell:Yeasthostcelltobemanipulatedfortheexpressionofprotein(s)togiverisetoarecombinantyeastproductionstrain.Inoculumintermediate:Aquantityofrecombinantbaculovirusofuniformcomposition,derivedfromtheworkingseedlot.Theinoculumintermediatehasadefinedshelf¬life.ItisintendedtobeusedtoinitiatetheproductionofrecombinantL1proteins.Cellbank:Acollectionofampoulescontainingaliquotsofasuspensionofcellsfromasinglepoolofcellsofuniformcomposition,storedfrozenunderdefinedconditions(typically−60°Cforyeast,andinliquidnitrogenforinsectormammaliancelllines).Mastercellbank(MCB):Acollectionofcontainerscontainingaliquotsofasuspensionofcellsfromasinglepoolofcellsofuniformcomposition,storedfrozenunderdefinedconditions(typically−60°Cforyeast,andinliquidnitrogenforinsectormammaliancelllines).TheMCBisusedtoderiveallworkingcellbanksfortheanticipatedlifetimeofthevaccineproduct.Workingcellbank(WCB):Acollectionofcontainerscontainingaliquotsofasuspensionofcellsfromasinglepoolofcellsofuniformcomposition,derivedfromtheMCB,storedfrozenunderdefinedconditions(typically−60°Cforyeast,andinliquidnitrogenforinsectormammaliancelllines).OneormorealiquotsoftheWCBareusedforroutineproductionofthevaccine.MultipleWCBsaremadeandusedduringthelifetimeofvaccineproductProductioncellculture:AcellculturederivedfromoneormorecontainersoftheWCBusedfortheproductionofvaccines.Endofproductioncells:Acellsuspensioncontainingthecellsharvestedattheendofculture/fermentation.Adventitiousagents:Contaminatingmicroorganismsofthevirus,orcellsubstrateormaterialsusedintheircultures,thatmayincludebacteria,fungi,mycoplasmas,andendogenousandexogenousvirusesthathavebeenunintentionallyintroduced.Fermentationcellpaste:Asuspensionofcellsharvestedattheendoftheyeastfermentationstoredfrozen(¬60°C).Singleantigenharvest:Acell¬suspensioncontainingtheintendedHPVantigensofonevirustypeharvestedfromcellculturespreparedfromasingleproductionrunSingleharvestpool:AhomogenouspoolofmultiplesingleharvestsoftheintendedHPVantigensofonevirustype,collectedintoasinglevesselbeforeclarification.Purifiedmonovalentantigenbulk:AbatchofpurifiedantigenofthesameHPVtype.Differentbatchesofpurifiedmonovalentantigenbulksmaybepooledbeforecollectionintoasinglevessel.Adsorbedmonovalentantigenbulk:Abatchofpurifiedmonovalentantigenbulkadsorbedonanaluminiumcontainingadjuvant.Differentbatchesofadsorbedmonovalentantigenbulksmaybepooledbeforecollectionintoasinglevessel.Adjuvant:Avaccineadjuvantisacomponentthatpotentiatestheimmuneresponsetoanantigenand/ormodulatesittowardsthedesiredimmuneresponses.Finalvaccinebulk:Theformulatedbulkpresentinthecontainerfromwhichthefinalcontainersarefilled.ThefinalbulkmaybepreparedfromoneormoreadsorbedmonovalentantigenbulksandmaycontainVLPantigensfromoneormultipleHPVvirustypes.Fillinglot(finalvaccinelot):Acollectionofsealedfinalcontainersofvaccinethatishomogeneouswithrespecttotheriskofcontaminationduringthefillingprocess.Afillinglotmustthereforehavebeenfilledorpreparedinoneworkingsession.3.3生产建议生产必须符合GMP要求，生物安全上要求无菌。不同类型HPVL1VLP应分别生产，同时，还要有充分的清洁验证。抗原生产过程，须验证以证明生产的稳定性，至少要连续三批。但如两种蛋白的纯化步骤一样，可只用验证一种。生产一致性评估应包括关键质量参数评估及它们相应的特征，如宿主DNA与HCP清除、工艺过程系数如柱负荷。不同批次抗原蛋白生产过程验证应与之前HPVVLP生产的规格保持一致如抗原特性和纯度。3.3.1鉴定HPV的鉴定应进行多批次疫苗生产中进行，包括过程验证中的批次。蛋白质组成验证：还原SDS－PAGE，并对条带进行染色，如有可能最好用相应抗体或质谱技术，确认L1目的蛋白存在。蛋白完整性（identity）验证：肽谱图和末端氨基酸序列分析。空间表位对有效性有着重大的影响，因而必须检测分析VLP的形态特征及聚合程度。此外，在合适情况下，应检测蛋白质，脂质、核酸和碳水化合物等。VLP特征参数可通过下面这些技术获得：原子力和透射电子显微镜、动态散光、表位作图、单抗中和反应。宿主蛋白质残留应满足非临床和临床要求（参见PartBandC）3.3原材料控制3.3.1抗原生产细胞培养物CellculturesforantigenproductionTheuseofanycelllineshouldbebasedonacellbanksystem.OnlycellsthathavebeenapprovedandregisteredwiththenationalregulatoryauthorityshouldbeusedtoproduceHPVL1protein.Thenationalregulatoryauthorityshouldberesponsibleforapprovingthecellbank.Appropriatehistoryofthecellbankshouldbeprovided.3.3.1.1YeastcellsThecharacteristicsoftherecombinantproductionstrain(h