WHO对HPV疫苗质量、安全性及有效性指导原则

GUIDELINES TO ASSURE THE QUALITY, SAFETY AND EFFICACY OF RECOMBINANT HUMAN PAPILLOMA VIRUS VIRUS?LIKE PARTICLE V ACCINES
HPV
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(Part A. Guidelines on manufacturing)
3.1定义definitions
3.1.1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Terminology
The definitions given below 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 contain viral DNA and which is composed of regular arrays of L1 pentameric capsomers.
Parental yeast c ell: 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 composition, 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 composition, 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 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 cells from a single pool of cells of uniform composition, derived from the MCB, stored frozen under defined conditions (typically <?60 °C for yeast, and in liquid nitrogen for insect or mammalian cell lines). One or more aliquots of the WCB
are used for routine production of the vaccine. Multiple WCBs are made and used during the lifetime of vaccine product
Production 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 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 the end of the yeast fermentation stored frozen (<?60°C).
Single antigen harvest: A cell?suspension containing the intended HPV antigens of one virus type harvested from cell cultures prepared from a single production run
Single harvest pool: A homogenous pool of multiple single harvests of the intended HPV antigens 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.
Adsorbed monovalent antigen bulk: A batch of purified
monovalent 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 modulates 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 types.
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 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 production
The use of any cell line should be based on a cell bank system. Only cells that 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 cells
The characteristics of the recombinant production strain (host cell in combination with the expression vector system) should be fully described
and 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 the 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 vector and restriction?enzyme mapping of the vector containing the gene insert should be provided as required by the national control authority.
其他略
3.3.2 细胞培养基Cell culture medium
如添加血清,应验证无抗生素。

略
3.3.3主细胞库和工作细胞验证Tests on master and working cell banks
略
3.4 HPV疫苗生产控制
3.4.1酵母表达体系中单一表达HPV抗原生产控制
3.4.1.1微生物纯度
略
略
3.5纯化后单价抗原质量控制
3.5.1 鉴定:免疫学测定
3.3.2 纯度:还原SDS-PAGE,考染染色后,对条带进行灰度计算,分析目的条带纯度
3.3.3蛋白含量:总蛋白的量可用凯氏定氮法,或Lowry法,及Brandford
3.3.4 抗原含量:用专一性强的方法,对纯化各环节的抗原进行定量分析。

此外,在纯化环节中,还应对抗原含量与总蛋白质的比值进行计算。

3.3.5 无菌检查
3.3.6 L1单体完整率
生产稳定后,采用合适的方法(什么方法?)对L1单体完整率进行测定
3.3.7 VLP大小和结构:电镜等
3.3.8工艺相关杂质测定
在生产中,所使用的任何有潜在负作用的试剂,都应对其残留进行检测。

在工艺稳定后,可以不必进行日常检测。

宿主DNA与宿主蛋白残留检测,方法应为专一性好、灵敏度高的分析方法。

DNA残留水平不得超过规定水平。

工艺稳定后,可以不必日常检测。

3.3.9 血清蛋白含量与病毒清除
略。