WHO对HPV疫苗质量、安全性及有效性指导原则

1、GUIDELINES TO ASSURE THE QUALITY, SAFETY ANDEFFICACY OF RECOMBINANT HUMANPAPILLOMAVIRUS VIRUS?LIKE PARTICLE VACCINESHPV二、 Special consideration section: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

2、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 L1 VLP 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

3、（ Part A. Guidelines on manufacturing）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16 L1 蛋白）3.1.2 定义描述重组 HPV VLP 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 TerminologyThe definitions given below

4、 apply to this document only.HPV L1 protein : The major structural protein of human papillomavirus, of which 360 molecules are found in the native virion associated in 72 pentameric capsomers.L1 virus? like particle : A non?infectious, non?enveloped, icosahedral capsid particle which does not contai

5、n viral DNA and which is composed of regular arrays of L1 pentameric capsomers.Parental yeast cell: Yeast host cell to be manipulated for the expression of protein(s) to give rise to a recombinant yeast production strain.Inoculum intermediate: A quantity of recombinant baculovirus of uniform composi

6、tion, derived from the working seed lot. The inoculum intermediate has a defined shelf?life. It is intended to be used to initiate the production of recombinant L1 proteins.Cell bank: A collection of ampoules containing aliquots of a suspension of cells from a single pool of cells of uniform composi

7、tion,stored frozen under defined conditions (typically - 60 C for yeast, and in liquid nitrogen for insect or mammalian cell lines).Master cell bank (MCB): A collection of containers containing aliquots of a suspension of cells from a single pool of cells of uniform composition, stored frozen under

8、defined conditions (typically- 60 C for yeast, and in liquid nitrogen for insect or mammalian cell lines). The MCB is used to derive all working cell banks for the anticipated lifetime of the vaccine product.Working cell bank (WCB): A collection of containers containing aliquots of a suspension of c

9、ells from a single pool of cells of uniform composition, derived from the MCB, stored frozen underdefined conditions (typically - 60 C for yeast, and in liquid nitrogen for insect or mammalian cell lines). One or more aliquots of the WCBare used for routine production of the vaccine. Multiple WCBs a

10、re made and used during the lifetime of vaccine productProduction cell culture: A cell culture derived from one or more containers of the WCB used for the production of vaccines.End of production cells: A cell suspension containing the cells harvested at the end of culture/fermentation.Adventitious

11、agents: Contaminating microorganisms of the virus, or cell substrate or materials used in their cultures, that may include bacteria, fungi, mycoplasmas, and endogenous and exogenous viruses that have been unintentionally introduced.Fermentation cell paste:A suspension of cells harvested at theend of

12、 the yeast fermentation stored frozen (?60C).Single antigen harvest: A cell?suspension containing the intended HPV antigens of one virus type harvested from cell cultures prepared from a single production runSingle harvest pool: A homogenous pool of multiple single harvests of the intended HPV antig

13、ens of one virus type, collected into a single vessel before clarification.Purified monovalent antigen bulk: A batch of purified antigen of the same HPV type. Different batches of purified monovalent antigen bulks may be pooled before collection into a single vessel.Adsorbedmonovalent antigenbulk:Ab

14、atch ofpurifiedmonovalent antigen bulk adsorbed on an aluminium containing adjuvant. Different batches of adsorbed monovalent antigen bulks may be pooled before collection into a single vessel.Adjuvant: A vaccine adjuvant is a component that potentiates the immune response to an antigen and/or modul

15、ates it towards the desired immune responses.Final vaccine bulk: The formulated bulk present in the container from which the final containers are filled. The final bulk may be prepared from one or more adsorbed monovalent antigen bulks and may contain VLP antigens from one or multiple HPV virus type

16、s.Filling lot (final vaccine lot): A collection of sealed final containers of vaccine that is homogeneous with respect to the risk of contamination during the filling process. A filling lot must therefore have been filled or prepared in one working session.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 HPV L1

17、VLP 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HPV VLP 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 SDSPAGE，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

18、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Part B and C）3.3 原材料控制3.3.1抗原生产细胞培养物Cell cultures for antigen productionThe use of any cell line should be based on a cell bank system. Only cells that

19、 have been approved and registered with the national regulatory authority should be used to produce HPV L1 protein. The national regulatory authority should be responsible for approving the cell bank. Appropriate history of the cell bank should be provided.3.3.1.1 Yeast cellsThe characteristics of t

20、he recombinant production strain (host cell in combination with the expression vector system) should be fully describedand information given on the absence of adventitious agents and on gene homogeneity for the master and working cell banks. A full description of the biological characteristics of th

21、e host cell and expression vectors should be given. The physiological measures used to promote and control the expression of the cloned gene in the host cell should be described in detail. This should include genetic markers of the host cell, the construction, genetics and structure of the expression vector and the origin and identification of the gene that is being cloned.The nucleotide sequence of the gene insert and of adjacent segments of the