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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (8): 34-40    DOI: 10.13523/j.cb.20180805
    
Anti-CD20rh MAb Quality Evaluation and Monoclonal Cell Line Screening
Hai-jiao JI1,Wen-lei LI2,Rui-jing Huang2,Jian LI2,Han-mei XU1,*()
1. China Pharmaceutical University, Nanjing 211100, China
2. Shanghai Tasly Pharmaceutical Co. LTD., Shanghai 201203, China
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Abstract  

Objective: To screen high expression monoclonal cell lines and optimize the production and quality of Anti-CD20rh MAb. Method: Screen high expression monoclonal cell lines by finite dilution method, and evaluated the yield of monoclonal cell lines by double-sandwich ELISA. In order to screen the best cell culture program, two to three cells were picked based on the cells’ growth state, yield, viability, and so on. The result were evaluated by the sugar type, the isoelectric point, the purity and the distribution of the acid and alkali base peak. Results: The production of CHOS cells tripled after a series of optimization, increased from nearly 500mg/L to 2 290mg/L. After the optimization of culture program, the purity of the target protein reached up to 97.48%, and the distribution of the acid and alkali base peak looks more close to the ideal state. Conclusion: The production and quality of the target antibody were optimized by cloning and culture optimization, these results are of great significance to the later experimental research and industrial production.

Key words： Monoclonal      Medium optimization      Quality evaluation     
Received: 01 March 2018      Published: 11 September 2018
ZTFLH:  Q813  
Corresponding Authors: Han-mei XU     E-mail: 1037714870@qq.com
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Hai-jiao JI
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Han-mei XU
Cite this article:

Hai-jiao JI,Wen-lei LI,Rui-jing Huang,Jian LI,Han-mei XU. Anti-CD20rh MAb Quality Evaluation and Monoclonal Cell Line Screening. China Biotechnology, 2018, 38(8): 34-40.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180805     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I8/34

Fig.1 Growth status and antibody expression of monoclonal cell lines (a) Growth status of monoclonal cell lines (b) Antibody expression of monoclonal cell lines; A-S are the 19 monoclonal cell lines that has been screened
Fig.2 Growth status and antibody expression of monoclonal cell lines after optimization (a) Growth status of monoclonal cell lines after optimization (b) Antibody expression of monoclonal cell lines after optimization; A、B are different cells; 1~11 are different cell culture and flow plus scheme
样品编号 发酵液体积(ml) UV(OD) 浓度(mg/ml) 捕获体积(ml) 抗体蛋白产量(mg)
A1 35 0.6707 5.047 6.45 32.55
A4 30 0.8178 6.224 5.7 35.48
B1 35 1.2092 9.674 6.25 60.46
B4 30 0.9489 7.273 5.00 36.37
Table 1 The concentration of ProteinA purified intermediate
Fig.3 SDS-PAGE electrophoresis map (a) SDS-PAGE reduction electrophoresis map (b) SDS-PAGE non-reduction electrophoresis map; A、B are different cells; 1、4 are different cell culture and flow plus schemes
Fig.4 The SEC-HPLC chromatograms (a) SEC-HPLC chromatograms of Sample A1 (b) SEC-HPLC chromatograms of Sample A4 (c) SEC-HPLC chromatograms of Sample B1 (d) SEC-HPLC chromatograms of Sample B4; A、B are different cells; 1、4 are different cell culture and flow plus schemes
样品 酸峰
(%)		 主峰
(%)		 碱峰
(%)		 主峰+碱
峰(%)		 等电点
A1 44.83 21.01 34.16 55.17 8.274
A4 68.57 18.79 12.63 31.42 8.332
B1 48.04 20.48 31.48 51.96 8.354
B4 55.14 28.65 16.22 44.87 8.377
Table2 Results of CIEF
Fig.5 Chromatogram of glycosylation analysis of HPLC-FLR
峰4 峰5 峰7 峰8 峰9,
10		 峰17
样品 Man5 G0 G0F G1 G1F G2F
A1 1.37 1.84 61.24 2.48 25.42 3.32
A4 2.71 1.89 51.93 4.7 28.28 6.97
B1 0.73 1.47 57.39 2.3 30.04 4.82
B4 1.79 2.52 56.79 3.56 26.46 6.42
Mean 1.65 1.93 56.84 3.26 27.55 5.38
Std.Dev 0.83 0.44 3.82 1.11 2.04 1.65
CV(%) 50.32 22.57 6.72 34.04 7.40 30.65
Table 3 Results of glycosylation analysis of HPLC-FLR
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