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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (4): 25-33    DOI: 10.13523/j.cb.1909065
技术与方法     
狂犬病疫苗蔗糖密度梯度离心纯化工艺开发 *
刘珍珍,田大勇()
上海青赛生物科技有限公司 上海 201506
Development of Sucrose Density Gradient Centrifugation Purification Process for Rabies Vaccine
LIU Zhen-zhen,TIAN Da-yong()
Shanghai King-cell Biotechnology Co., Ltd., Shanghai 201506, China
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摘要：

目的：开发冻干人用狂犬病疫苗(鸡胚成纤维细胞)的连续流蔗糖密度梯度离心纯化工艺。方法：分别比较两种不同初始蔗糖浓度和不同上样速度对纯化效果的影响,初步确定纯化工艺;通过多批次实验确定样品收集范围;比较不同浓缩倍数条件下杂质去除和抗原回收情况,确定合适的收获液浓缩比例;比较不同批次样品纯化后的杂质去除率和重复性,判定本纯化工艺的稳定性。结果：选取60%作为初始蔗糖浓度,在上样速度为150~200ml/min时,可以有效地对10倍浓缩的病毒收获液进行纯化;卵清蛋白、牛血清白蛋白和庆大霉素去除率分别达到99%,95%和95%,且工艺具有极好的稳定性。结论：开发的连续流蔗糖密度梯度离心技术可以作为冻干人用狂犬病疫苗(鸡胚成纤维细胞)的产业化纯化工艺。
关键词： 狂犬病病毒疫苗;  纯化工艺;  连续流蔗糖密度梯度离心    
Abstract:

Objective: To develop a continuous flow sucrose density gradient centrifugation purification process for freeze-dried human rabies vaccine (chicken embryo fibroblasts). Methods: The effects of concentration of initial sucrose solution and loading speeds on the purification of Rabies virus (RABV) were compared to determine the purification process. The sample collection range was determined by multi-batch experiments. The impurity removal and antigen recovery were compared under different concentration multiples to determine the appropriate concentration ratio of the harvest liquid. The impurity removal rate and repeatability of the different batch samples after purification were compared to determine the stability of the purification process. Results: When the initial sucrose concentration is 60%, the 10-fold concentrated virus could be effectively purified at 150-200 ml/min. The removal rates of ovalbumin, bovine serum albumin and gentamicin reached respectively 99%, 95% and 95%. Conclusions: The continuous flow sucrose density gradient centrifugation technique developed in this study can be used as an industrial purification process for freeze-dried human rabies vaccine (chicken embryo fibroblasts).
Key words: Rabies virus vaccine    Purification process    Continuous flow sucrose density gradient centrifugation
收稿日期: 2019-09-27 出版日期: 2020-05-18
ZTFLH:  Q81  
基金资助: * 国家自然科学基金资助项目(31702248)
通讯作者: 田大勇     E-mail: tiandayong@king-cell.com
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引用本文:

刘珍珍,田大勇. 狂犬病疫苗蔗糖密度梯度离心纯化工艺开发 *[J]. 中国生物工程杂志, 2020, 40(4): 25-33.

LIU Zhen-zhen,TIAN Da-yong. Development of Sucrose Density Gradient Centrifugation Purification Process for Rabies Vaccine. China Biotechnology, 2020, 40(4): 25-33.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1909065    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I4/25

图1  RABV分布的蔗糖溶液的浓度范围
图2  基于不同初始蔗糖浓度的纯化效果的比较
图3  不同上样速度的纯化效果比较
批号 主峰蔗糖浓度范围(%) 主峰混合蔗糖浓度(%)
001 30~43 36.5
002 34~43.5 39.4
003 32~48.5 40.6
004 32~48.5 39.7
005 30~47.5 39.4
006 28.5~46 39
007 30~48 37
008 30~47 39
表1  RABV分布的蔗糖浓度范围
图4  病毒收获液浓缩倍数的选择
批号 卵清蛋白去除率
(%)		 牛血清蛋白去
除率(%)		 庆大霉素去除率
(%)
009 99.95 97.98 95.04
010 99.96 99.22 99.39
011 99.09 99.09 98.93
表2  纯化后样品杂质去除率
图5  连续流蔗糖密度梯度离心纯化工艺稳定性分析
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