Please wait a minute...

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  2019, Vol. 39 Issue (4): 101-105    DOI: 10.13523/j.cb.20190413
综述     
工程细胞单克隆筛选及单克隆源性验证 *
江一帆(),董静,魏敬双
华北制药新药研究开发有限责任公司 抗体药物研究国家重点实验室 石家庄 050015
Monoclone Selection and Monoclonal Verification of Engineering Cell Lines
Yi-fan JIANG(),Jing DONG,Jing-shuang WEI
New Drug Research and Development Company Limited, North China Pharmaceutical Corporation, State Key Laboratory of Antibody Drug Development, Shijiazhuang 050015, China
 全文: PDF(359 KB)   HTML
摘要：

工程细胞的单克隆源性是保证产品质量的重要因素之一,因此得到了越来越多的重视。当前,药物审评机构要求报批单位采用合适的实验手段证明所使用细胞的单克隆源性。综述通过介绍生产用工程细胞株单克隆的挑选过程及诸如有限稀释法、ClonePix、流式细胞术等常用方法,讨论如何确保工程细胞的单克隆源性,以及在生物药品生产过程中保证工程细胞单克隆源性的意义。
关键词： 工程细胞;  单克隆源性;  单克隆成像系统;  有限稀释法    
Abstract:

The monoclonality of recombinant cell lines is one of the important factors to ensure the quality of the product, so it has received more and more attention. Currently, some drug regulatory agencies require the application organization to demonstrate the monoclonality of the cells via appropriate experimental. The process and common methods (such as limiting dilution cloning, ClonePix, and fluorescent-activated cell sorting) for the clone selection of engineering cell lines for production were introduced.How to ensure the monoclonal of engineering cell, and the significance of engineering monoclonal in the production of the biological drug were discussed.
Key words: Engineering cell line    Monoclonality    Single-cell cloning imaging system    Limiting dilution method
收稿日期: 2018-10-11 出版日期: 2019-05-08
ZTFLH:  Q819  
基金资助: * 国家重大新药创制项目资助项目(2017ZX09306010)
通讯作者: 江一帆     E-mail: jyifan@126.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
江一帆
董静
魏敬双

引用本文:

江一帆,董静,魏敬双. 工程细胞单克隆筛选及单克隆源性验证 *[J]. 中国生物工程杂志, 2019, 39(4): 101-105.

Yi-fan JIANG,Jing DONG,Jing-shuang WEI. Monoclone Selection and Monoclonal Verification of Engineering Cell Lines. China Biotechnology, 2019, 39(4): 101-105.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190413    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I4/101

稀释比例 单克隆数 克隆数 单克隆率(%) 二轮单克隆率*
二轮0.3稀释(%) 二轮0.6稀释(%) 二轮0.8稀释(%)
0.3 12.6 14.0 89.3 98.9 97.5 96.9
0.6 18.8 24.6 76.4 97.5 94.4 93.2
0.8 21.8 30.6 71.2 96.9 89.2 88.0
表1  有限稀释法联合单克隆成像系统计算单克隆率
Proximity(mm) P(clonality)(%)
0.50.7a 44
0.80.9 61
0.91.0 71
1.01.2 93
>1.25 >99
表2  ClonePix挑选过程中克隆距离和单克隆率的关系
[1] Quiroz J, Tsao Y S . Statistical analysis of data from limiting dilution cloning to assess monoclonality in generating manufacturing cell lines. Biotechnology Progress, 2016,32(4):1061-1068.
doi: 10.1002/btpr.2290 pmid: 27111698
[2] Evans K, Albanetti T, Venkat R , et al. Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging. Biotechnology Progress, 2015,31(5):1172-1178.
doi: 10.1002/btpr.2145 pmid: 26195345
[3] Zingaro K, Shaw D, Carson J , et al. Implementation of plate imaging for demonstration of monoclonality in biologics manufacturing development. PDA J Pharm Sci Technol, 2018,81(10):3835-3842.
doi: 10.5731/pdajpst.2018.008789 pmid: 29669815
[4] Gargi R, Guillermo M Q, Li Z , et al. Sequential screening by ClonePix FL and intracellular staining facilitate isolation of high producer cell lines for monoclonal antibody manufacturing. Journal of Immunological Methods, 2017,415:100-110.
doi: 10.1016/j.jim.2017.08.012 pmid: 28890364
[5] Mateus D L, Mariana L S, Angelica G , et al. A new CHO (Chinese hamster ovary)-derived cell line expressing anti-TNFα monoclonal antibody with biosimilar potential. Immunogic Research, 2018,66(1):1-14.
doi: 10.1007/s12026-017-8964-5 pmid: 29098527
[6] Hou J J C, Hughes B S, Smede M , et al. High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system. New Biotechnology, 2014,31(3):214-220.
doi: 10.1016/j.nbt.2014.02.002 pmid: 24518824
[7] Ahmed O, Burke J, Mann C , et al. Using ClonePix FL to assess monoclonality. Genrtic Engineering & Biotechnology News, 2009,29(19):38-39.
[8] Sivia C, Mohamed A R . The selection of high-producing cell lines using flow cytometry and cell sorting. Expert Opinion on Biological Therapy, 2004,4(11):1821-1829.
doi: 10.1517/14712598.4.11.1821
[9] Basu S, Campbell H M, Dittel B N , et al. Purification of specific cell population by fluorescence activated cell sorting. Journal of Visualized Experiments, 2010,41(e1546):1-4.
doi: 10.3791/1546 pmid: 3144656
[10] Lee C J, Seth G, Tsukuda J , et al. A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies. Biotechnology and Bioengineering, 2008,102(4):1107-1118.
doi: 10.1002/bit.22126 pmid: 18985612
[11] Gross A, Schondube J, Niekrawitz S , et al. Single-cell printer: automated, on demand, and label free. Journal of Laboratory Automation, 2013,18(6):504-518.
doi: 10.1177/2211068213497204
[12] Stumpf F, Schoendube J, Gross A , et al. Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation. Biosensors & Bioelectronic, 2015,69:301-306.
doi: 10.1016/j.bios.2015.03.008 pmid: 25771302
[13] Trask B J . Fluorescence in situ hybridization: applications in cytogenetics and gene mapping. Trends in Genetics, 1991,7(5):149.
doi: 10.1016/0168-9525(91)90378-4 pmid: 2068787
[14] Levsky J M, Singer R H . Fluorescence in situ hybridization: past, present and future. Journal of Cell Science, 2003,116(14):2833-2838.
doi: 10.1242/jcs.00633 pmid: 12808017
[15] Scarcelli J J, Hone M, Beal K , et al. Analytical subcloning of a clonal cell line demonstrates sequence heterogeneity that does not impact process consistency or robustness. Cell Culture and Tissue Engineering, 2018,34(3):602-612.
doi: 10.1002/btpr.2646
[16] Nakamura T, Omasa T . Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system. Journal of Bioscience and Bioengineering, 2015,120(3):323-329.
doi: 10.1016/j.jbiosc.2015.01.002 pmid: 25792187
[1] 徐秀英, 陈琳, 卢柏松, 黄培堂, 邓继先. 人红细胞生成素(EPO)工程细胞建立及特性分析[J]. 中国生物工程杂志, 2000, 20(3): 72-75.
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