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非对称垂直流场流分级技术应用于病毒样颗粒稳定性的研究
陈毅
学位类型博士
导师苏志国
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词乙肝表面抗原 病毒样颗粒 非对称垂直流场流技术 多角度激光散射技术 聚集 抗原性
摘要

病毒样颗粒(virus-like particle)是含有某种病毒的一个或多个结构蛋白,但不包含核酸的自组装颗粒,其形态与真正病毒粒子相同或相似,可作为疫苗的抗原成分。乙肝表面抗原(HBsAg)是一种重要的病毒样颗粒,可以通过注射来使机体产生抗体,达到免疫和阻断乙肝传播的目的。在HBsAg的生产和流通过程中,需要面对许多苛刻的环境因素,比如盐浓度、温度变化,反复冻融等,这些环境因素可能会导致HBsAg出现聚集失活现象,从而影响了疫苗的安全性和有效性。但遗憾的是,目前尚无系统的乙肝疫苗稳定性研究报道。在本文中,我们首次采用无填充介质的非对称垂直流场流分级技术偶联多角度激光散射技术(AF4-MALLS)应用于乙肝疫苗病毒样颗粒的表征,并对两种常见的乙肝疫苗产品(分别由中国仓鼠卵CHO细胞和汉逊酵母细胞表达)在其纯化、生产、运输以及储存过程中所经历的不同环境条件下的颗粒稳定性进行了研究,主要内容包括:1)优化得到了适于分离HBsAg及其聚集体的场流分级条件:①进样和聚焦阶段:进样流速0.2 ml/min,聚焦流速1.8 ml/min,外场流速1.0 ml/min,时间4 min,聚焦阶段和分级阶段的过渡时间是1 min,腔室出口流速保持1.0 ml/min恒定;②分级阶段:外场流速1.0 ml/min保持40 min,然后在5 min内线性衰减到0;③再生阶段:腔室流速1ml/min,保持5min。间隔物厚度 350 μm,分离膜为再生纤维素膜,截留分子量为10 kDa。采用AF4-MALLS表征得到中国仓鼠卵CHO细胞表达的CHO-HBsAg和汉逊酵母细胞表达的Hans-HBsAg的重均分子量分别为4742 kDa和3039 kDa;水力学半径分别为29.4 nm和 22.8 nm。2) 通过AF4-MALLS对CHO-HBsAg和Hans-HBsAg在不同溶液环境下的聚集行为进行了研究,并使用抗原诊断试剂盒检测了相应的抗原活性。在2M硫酸铵条件下,CHO-HBsAg比较稳定,活性保持在95%,而Hans-HBsAg明显发生聚集形成分子量105-106 kDa的聚集体,其活性仅保留25%;在反复冻融操作之后,CHO-HBsAg的结构和活性几乎没有什么变化,而Hans-HBsAg发生了明显的聚集,活性仅剩余26.9%;在高温(60°C)处理之后,CHO-HBsAg依然稳定,而Hans-HBsAg的活性已经不足5%。3) 系统考察了氯化钠和硫酸铵对Hans-HBsAg稳定性的影响,并对Hans-HBsAg在盐溶液中的聚集行为进行了分析,在此基础上建立了Hans-HBsAg在盐溶液中的聚集模型。Hans-HBsAg在NaCl溶液中发生可逆聚集形成寡聚体(OP),伴随着生物活性的轻微损失。通过透析除去NaCl之后,寡聚体可重新解聚为正常组装体,同时抗原活性随之恢复。在硫酸铵溶液中的聚集情况较为复杂,当硫酸铵浓度较低(< 0.5M)且作用时间较短时(< 1h),大部分HBsAg聚集成寡聚体形式,当硫酸铵去除后,寡聚体可以重新解聚;而高浓度硫酸铵条件下,OP会进一步聚集形成高聚体(PP),PP的尺寸很大,且该聚集过程会导致抗原活性大幅度降低。即使通过透析去除硫酸铵,也无法使得PP重新解聚成正常组装体,抗原活性也无法恢复。4) 考察了几种常见的小分子添加剂对Hans-HBsAg稳定性以及抗原活性的影响。脲和盐酸胍对乙肝疫苗聚集没有明显抑制作用,相反它们作为变性剂,可使蛋白发生聚集;精氨酸对乙肝聚集方面发挥了比较明显的抑制作用,精氨酸对乙肝疫苗聚集的抑制效果与其浓度相关,当精氨酸浓度达到1M时,抑制效果较好;聚乙二醇对乙肝疫苗的抗原活性似乎没有明显影响。而表面活性剂对乙肝疫苗抗原活性的影响表现出不同的效果,吐温80可以提高乙肝疫苗的活性,而Triton100则会使乙肝疫苗的活性降低。

其他摘要

Virus-like particles are self-assembled macromolecules consisting of virus capsid proteins but devoiding of genetic materials. They mimic the morphology of the true virus and could act as vaccine. For example, hepatitis B surface antigen (HBsAg), an important virus-like particle, has been used for vaccination to block the transmission of the hepatitis B virus. In the production and transportation processes, HBsAg would face several harsh environmental factors such as change of the salt solution, temperature or repeated freezing-thawing, which could induce aggregation and deactivation of HBsAg and influence badly the safety and validity of the vaccine. Unfortunately, until now, there is no systemic report concerning the stability of HBsAg. In this work, we employed asymmetrical flow field-flow fractionation (AF4) coupled with multi-angle laser light scattering (MALLS), as a first attempt, to characterize the HBsAg virus like particles and studied the stabilities of two important HBsAg VLP products (expressed respectively by Chinese Hamster Ovary (CHO) cell and Hansenula polymorpha (Hans) yeast cell) under different environmental conditions which may occur during their purification, production, transportation and storage processes. The main results are following:1) The AF4 fractionation method for HBsAg and its aggregates was optimized and established as: ① injection and focus step: injection flow was 0.2 ml/min,focus flow was 1.8 ml/min,crossflow was 1.0 ml/min,time was 4 min,the transition time between focus step and elution step was 1 min, detector flow was constant at 1.0 ml/min; ② elution step: crossflow was constant at 1.0 ml/min for 40 min and then decreased to zero linearly in 5 min; ③ rinse step: kept channel flow of 1.0 ml/min for 5 min. The spacer height was 350 μm and the membrane was regenerated cellulose with cutoff Mw of 10 kDa. The molecular weight and hydrodynamic diameter of CHO-HBsAg and Hans-HBsAg characterized by AF4-MALLS was 4727 kDa and 3039 kDa, 29.4 nm and 22.8 nm respectively.2) The aggregation behaviours of CHO-HBsAg and Hans-HBsAg in different solution environment were studied by AF4-MALLS and the antigenicy was analyzed by ELISA kit. Under condition of 2 M ammonium sulfate, CHO-HBsAg was stable with antigenicity keeping above 95%, while Hans-HBsAg aggregated heavily to large particles with molecular weight about 105-106 kDa and kept only 25% antigenicity. During freeze-thaw cycles, CHO-HBsAg had little change in structure and antigenicity. By contrast, Hans-HBsAg aggregated heavily and kept only 26.9% antigenicity. After the experiment of high temperature at 60°C, CHO-HBsAg was still stable but the antigenicity of Hans-HBsAg was below 5%.3) The stability of Hans-HBsAg in NaCl and (NH4)2SO4 was researched and the aggregation behavior was discussed, based on which a model concerning aggregation of Hans-HBsAg was established. Hans-HBsAg aggregated reversibly into oligomeric particle (OP) in NaCl solution, accompanied by a slight loss of antigenicity. After removing NaCl by dialysis, both particle form and antigenicity could recover to original state. The aggregation of HBsAg in (NH4)2SO4 solution was more profound and complicated. At low concentration (<0.5M) and short incubation time (<1 hour), most aggregates of HBsAg VLP were in OP form, and could be disaggregated when the salt was removed. However, as the concentration increased, the OP further aggregated into polymeric particles (PP) with greater size, accompanied by severe loss of antigenicity. Removal of (NH4)2SO4 by dialysis could not restore the PP into original HBsAg form, and the antigenicity was lost irreversibly.4) Effect of several additives on the stability and antigenicity of Hans-HBsAg was studied. Arginine could suppress the aggregation of Hans-HBsAg in salt solution and the suppressing effect was related with the concentration of Arginine. When the concentration of Arginine reached 1M, the suppressing effect was satisfactory. Urea and GdmHCl would not prevent the aggregation of Hans-HBsAg. Conversely, they can promote Hans-HBsAg to aggregate. Polyethylene (PEG) had almost no apparent effect on the antigenicity of Hans-HBsAg. Different surfactant exhibited different effect in antigenicity of Hans-HBsAg, Tween 80 could improve the antigenicity of Hans-HBsAg while Triton 100 could decrease the antigenicity of Hans-HBsAg. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22902
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陈毅. 非对称垂直流场流分级技术应用于病毒样颗粒稳定性的研究[D]. 北京. 中国科学院研究生院,2016.
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