Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2020, Vol. 40 Issue (4): 25-33    DOI: 10.13523/j.cb.1909065
    
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
Download: HTML   PDF(2350KB) HTML
Export: BibTeX | EndNote (RIS)      

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     
Received: 27 September 2019      Published: 18 May 2020
ZTFLH:  Q81  
Corresponding Authors: Da-yong TIAN     E-mail: tiandayong@king-cell.com
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
Zhen-zhen LIU
Da-yong TIAN
Cite this article:

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

URL:

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

Fig.1 Concentration range of sucrose solution which RABV was distributed in (a) Distribution of RABV in sucrose solution (b) The levels of total protein in sucrose solution layers were tested by SDS-PAGE (c) The levels of G protein and P protein in sucrose solution layers were detected by Western blot
Fig.2 Comparison of purification effects based on the sucrose solution with different initial concentration (a) Density range formed by different initial sucrose solution (b) Analysis of purification effectiveness base on sucrose solution with initial concentration 60% (c) Analysis of purification effectiveness base on sucrose solution with initial concentration 30%+60% (d) The total gray value of G protein after purification
Fig.3 Comparison of purification efficiency based on different sampling speed (a) The purification effects based on different sampling speed by SDS-PAGE and Western blot analysis (b) Quantitative analysis of the total gray value of G protein after purification
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
Table.1 Concentration range of sucrose solution which RABV were distributed in
Fig.4 The determination of concentration rate of virus harvest (a) Western blot analysis of G protein under different concentration rate (b) Analysis of total protein and viral titers under different concentration rate (c) The purification effects of virus harvest (d) The purification effects of 10-time concentrated virus harvest
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
Table 2 Removal rate of non-target component in purified samples
Fig.5 Repeatability analysis of continuous flow sucrose density gradient centrifugal purification process (a)(b)(c) Purification effects of the same batch of harvest (d) Merged UV map of purification of the same batch harvest (e)(f)(g) Purification effects of three different batches of harvest (h) Merged UV map of purification of the three different batches of harvest
[1]   Fontana D, Kratje R, Etcheverrigaray M , et al. Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate. Vaccine, 2015,33(35):4238-4246.
doi: 10.1016/j.vaccine.2015.03.088 pmid: 25869890
[2]   Balsamo G A, Ratard R, Claudet A . The epidemiology of animal bite, scratch, and other potential rabies exposures, Louisiana. J La State Med Soc, 2009,161(5):260-265.
pmid: 19927939
[3]   Coleman P G, Dye C . Immunization coverage required to prevent outbreaks of dog rabies. Vaccine, 1996,14(3):185-186.
doi: 10.1016/0264-410x(95)00197-9 pmid: 8920697
[4]   Hampson K, Coudeville L, Lembo T , et al. Estimating the global burden of endemic canine rabies. PLoS Neglected Tropical Diseases, 2015,9(4):e0003709.
doi: 10.1371/journal.pntd.0003709 pmid: 25881058
[5]   周航, 李昱, 陈瑞丰 , 等. 狂犬病预防控制技术指南(2016版). 中华流行病学杂志, 2016,37(2):139-163.
doi: 10.3760/cma.j.issn.0254-6450.2016.02.001 pmid: 26917506
[5]   Zhou H, Li Y, Chen R F , et al. Technical guideline for human rabies prevention and control (2016), Chinese Journal of Epidemiology, 2016,37(2):139-163.
doi: 10.3760/cma.j.issn.0254-6450.2016.02.001 pmid: 26917506
[6]   巩薇, 付瑞, 王吉 , 等. 动物源性生物制品病毒灭活/去除工艺的验证. 中国生物制品学杂志, 2007,12:920-923.
[6]   Gong W, Fu R, Wang J , et al. Verification of inactivation/removal procedure of viruses in biologics of animal origins. Chinese Journal of Biologicals, 2007,12:920-923.
[7]   朱俊颖, 孙晔, 蒋丽华 , 等. 疫苗规模化分离纯化研究进展. 生物技术进展, 2015,5(6):405-413.
[7]   Zhu J Y, Sun Y, Jiang L H , et al. Overview of current scalable methods for vaccine purification. Progress in Biotechnology, 2015,5(6):405-413.
[8]   廖玉宜, 张吉凯, 游志毅 , 等. 国产纯化Vero细胞狂犬病疫苗的发展现状. 中国疫苗和免疫, 2015,5:580-588.
[8]   Liao Y Y, Zhang J K, You Z Y , et al. Current situation of chinese purified vero cell rabies vaccine. Chinese Journal of Vaccines and Immunization, 2015,5:580-588.
[9]   李建平, 杨屹, 王宇 , 等. 纯化乙型肝炎病毒表面抗原新层析工艺的建立. 中国生物制品学杂志, 2006,6:641-643.
[9]   Li J P, Yang Y, Wang Y , et al. Establishment of a new chromatographic process for purification of hepatitis B virus surface antigen. Chinese Journal of Biologicals, 2006,6:641-643.
[10]   解红艳, 王济源, 刘静 , 等. 层析法去除A群流脑多糖疫苗粗糖中杂蛋白. 中国生物工程杂志, 2010,30(8):88-92.
doi: Q819
[10]   Xie H Y, Wang J Y, Liu J , et al. Chromatography for removal of heteroproteins in crude sugar of group A meningococcal polysaccharide vaccine. China Biotechnology, 2010,30(8):88-92.
doi: Q819
[11]   Lee K C, Lim D, Wong S M , et al. Purification, crystallization and X-ray analysis of Hibiscus chlorotic ringspot virus. Acta Crystallographica Section D, Biological Crystallography, 2003,59(8):1481-1483.
doi: 10.1107/s0907444903011648 pmid: 12876359
[12]   Pyke A T, Phillips D A, Chuan T F , et al. Sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine. BMC Microbiology, 2004,4(3):1471-2180.
doi: 10.1186/1471-2180-4-3 pmid: 14720306
[13]   Lodmell D L, Esposito J J, Ewalt L C . Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro. Journal of Virology, 1993,67(10):6080-6086.
pmid: 8371354
[14]   Takita-Sonoda Y, Fujii H, Mifune K , et al. Resistance of mice vaccinated with rabies virus internal structural proteins to lethal infection. Archives of Virology, 1993,132(1-2):51-65.
doi: 10.1007/BF01309843 pmid: 8352659
[15]   焦洋, 黄平, 唐青 , 等. 狂犬病病毒糖蛋白的性质及其应用. 慢性病学杂志, 2013,8:600-604.
[15]   Jiao Y, Hang P, Tang Q , et al. Properties and application of rabies virus glycoprotein. Chronic Pathematology Journal, 2013,8:600-604.
[16]   俞永新 . 狂犬病和狂犬病疫苗. 中国医药科技出版社, 2009.
[16]   Yu Y X. Rabies and rabies vaccine. China Medical Science Press, 2009.
[17]   赵永强, 杨丽, 白春杰 , 等. 不同截留分子量超滤膜包浓缩狂犬病病毒的效果. 中国生物制品学杂志, 2013,26(12):1848-1850.
[17]   Zhao Y Q, Yang L, Bai C J , et al. Concentration of rabies virus by ultrafiltration membranes with various molecular weight cutoff values. Chinese Journal of Biologicals, 2013,26(12):1848-1850.
[1] DONG Yue, SU Cai-xia, YANG Yan, ZHU Nai-shuo. The Study on Fermentation and Purification Process of HBsAg Binding Protein(SBP) in Pichia pastoris[J]. China Biotechnology, 2014, 34(12): 59-68.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会