灭活病毒及病毒样颗粒的结构表征和稳定性研究

发布时间：2018-12-13 16:22

【摘要】：灭活病毒及病毒样颗粒(Virus like particles, VLPs)是两类重要的疫苗抗原,都具有多聚亚基的大颗粒组装体结构,在生产和储存过程中易于发生结构变化,影响疫苗的安全性和有效性。如何稳定这种大颗粒组装体结构是疫苗工程研究领域的一个重要课题。本论文以灭活口蹄疫病毒(Foot and mouth disease virus, FMDV)及乙肝病毒表面抗原病毒样颗粒(Hepatitis B virus surface antigen VLPs, HBsAg-VLPs)为对象,研究了其溶液中和固/液界面上颗粒结构变化的检测方法,分析了层析纯化中颗粒结构变化的机理,探索了影响结构稳定性的因素,主要内容如下：1)建立了高效液相凝胶过滤色谱法(HPSEC)快速检测和分析抗原颗粒组装体结构的技术,应用于FMDV、HBcAg-VLPs、HBsAg-VLPs在不同生产阶段以及储存溶液中颗粒完整性的检测,与传统的超高速离心分析法相比,具有简单、快速、可靠等优点。该方法经中国兽医药品监察所推荐,已被疫苗生产企业使用。2)分析了溶液中FMDV和HBsAg-VLPs的颗粒性质,采用场流分级、圆二色光谱、荧光光谱、动态光散射技术对两种抗原进行了表征,进一步证实了HPSEC方法的可靠性,同时提供了更为全面的结构变化的信息。3)探索了双偏极化干涉(DPI)分析抗原颗粒在液固相界面的变化,发现HBsAg-VLPs在DEAE离子交换表面发生吸附,其厚度随pH不同而变化。在pH5.0下,平均吸附层厚度高于HBsAg-VLPs的颗粒直径；在pH值7.0或9.0下,平均吸附层厚度则小于HBsAg-VLPs的颗粒直径,说明病毒样颗粒在离子交换中可能出现复杂的结构变化。通过对比DPI分析和离子交换层析纯化的实验结果,证明了HBsAg-VLPs在离子交换层析中的活性损失与界面失活密切相关。4)建立了提高颗粒型抗原在生产过程中稳定性的策略。对于FMDV,最适pH范围在7.5-8.0,最适的盐类为铵盐。通过HPSEC及差示扫描量热法(DSC)证实了添加多羟基化合物对于疫苗抗原的颗粒结构具有稳定作用。当溶液中存在多羟基化合物时,超滤过程中的FMDV收率提高了10.4%,离子交换层析的收率从17.1%提高到了84.0%。整个工艺获得的FMDV完整颗粒(146S)的纯度达到98%以上,整体收率达到56.7%。
[Abstract]:Inactivated virus and virus-like particle (Virus like particles, VLPs) are two kinds of important vaccine antigens, both of which have a large particle assembly structure with poly-subunits, and are prone to structural changes during production and storage, which will affect the safety and effectiveness of the vaccine. How to stabilize the structure of this large particle assembly is an important subject in vaccine engineering. In this paper, inactivated foot-and-mouth disease virus (Foot and mouth disease virus, FMDV) and hepatitis B virus surface antigen virus like particle (Hepatitis B virus surface antigen VLPs, HBsAg-VLPs) were used to detect the changes of particle structure at the solid / liquid interface. The mechanism of particle structure change in chromatographic purification was analyzed, and the factors influencing the structure stability were explored. The main contents are as follows: 1) High performance liquid phase gel filtration chromatography (HPSEC) was established for rapid detection and analysis of antigen particle assembly structure, which was applied to FMDV,HBcAg-VLPs,. Compared with the traditional ultra-high speed centrifugation, HBsAg-VLPs has the advantages of simplicity, rapidity, reliability and so on. The method has been recommended by the China Veterinary Drug Administration and has been used by vaccine manufacturers. 2) the particle properties of FMDV and HBsAg-VLPs in the solution were analyzed by field flow classification, circular dichroism spectrum and fluorescence spectrum. The dynamic light scattering technique was used to characterize the two antigens, which further confirmed the reliability of the HPSEC method. At the same time, it provides more comprehensive information on the structural changes. 3) the changes of antigenic particles at the liquid-solid interface are analyzed by dual-polarization interference (DPI). It is found that HBsAg-VLPs adsorbs on the DEAE ion exchange surface and its thickness varies with pH. Under pH5.0, the average thickness of adsorption layer is higher than that of HBsAg-VLPs particles. At the pH value of 7.0 or 9.0, the average thickness of the adsorbed layer is smaller than the diameter of the HBsAg-VLPs particles, which indicates that the virus like particles may exhibit complex structural changes in ion exchange. By comparing the experimental results of DPI analysis and ion exchange chromatography purification, it is proved that the loss of HBsAg-VLPs activity in ion exchange chromatography is closely related to the interfacial inactivation. 4) A strategy to improve the stability of granular antigen in the process of production is established. For FMDV, the optimum range of pH is 7.5-8.0, and the most suitable salt is ammonium salt. HPSEC and differential scanning calorimetry (DSC) showed that the addition of polyhydroxyl compounds could stabilize the particle structure of vaccine antigen. When polyhydroxyl compounds were present in the solution, the yield of FMDV in ultrafiltration process was increased by 10.4%, and the yield of ion exchange chromatography was increased from 17.1% to 84.0%. The purity of the whole FMDV particles (146S) was above 98% and the overall yield was 56.7%.
【学位授予单位】：中国科学院研究生院(过程工程研究所)
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392

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