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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  2018, Vol. 38 Issue (6): 63-69    DOI: 10.13523/j.cb.20180609
技术与方法     
单克隆抗体Mab-A生产细胞Sp2/0缩小模型培养优化研究
汪国强1,2,*,刘建平1,周航2,**()
1 复旦大学生命科学学院 上海 200438
2 上海药明生物技术有限公司 上海 200131
Study on the Optimal Scale-down Model for Cell Growth and Mab-A Production of Sp2/0 Cells
Guo-qiang WANG1,2,*,Jian-ping LIU1,Hang ZHOU2,**()
1 School of Life Sciences Fudan University, Shanghai 200438, China
2 WuXi Biologics (Shanghai) Co., Ltd., Shanghai 200131, China
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摘要：

Sp2/0是一种生产单克隆抗体的常用细胞株。本研究首先在批次培养模式中对适合Sp2/0细胞生长的5种基础培养基、摇床转速、培养温度、二氧化碳浓度、微量元素和GlutaMAX TM替换谷氨酰胺等影响因素进行了筛选研究。结果显示Sp2/0细胞在批次培养中细胞密度最高值达到13.12×10 6 cells/ml,培养时间为7天。除培养温度会导致不同的细胞生长密度和活率、进而影响培养时间外,其它因素不能导致明显的细胞生长差异。随后在流加培养模式下就14种补料组合进行了筛选,Sp2/0在流加培养模式下细胞的峰值密度可达20~30×10 6 cells/ml,培养时间9天,单克隆抗体Mab-A日产量最高达到27.20mg/L。最后应用批次-反复流加培养模式培养Sp2/0细胞,该条件下峰值细胞数为50.42×10 6 cells/ml,培养时间14天,每天单抗产量(141.10mg/L)是流加培养的5.19倍。这些研究结果为Sp2/0细胞规模化生产单克隆抗体奠定了一定基础。

关键词： Sp2/0;  缩小模型;  批次-反复流加    
Abstract:

Five different kinds of basal media were screened in batch mode to find out the optimal basal medium for cell growth of Sp2/0. The peak viable cell density in batch mode was 13.12×10 6cells/ml, and the culture duration was 7 days. Various cell culture conditions including different shaking speeds, concentrations of carbon dioxide, glutamine replaced by GlutaMAX TM, addition of trace element and different culture temperatures were studied in batch mode as well. Few different cell growth were found with these conditions except for culture temperatures, which leaded to different peak viable cell concentrations, different viabilites and then different culture durations. Fourteen kinds of combinations of feed media were screened in fed-batch mode. The peak viable cell densities were up to 30×10 6cells/ml, the culture duration were around 9 days, and the highest daily Mab-A production was 27.20mg/L. Batch re-feed mode was used in the third part of the study. The peak viable cell density was 50.42×10 6cells/ml, the culture duration was 14 days, and the highest daily Mab-A production (141.10mg/L) was 5.19 fold greater than in fed-batch mode. These studies suggest batch re-feed mode is the optimal scale-down mode for cell growth and Mab-A production of Sp2/0 cells.

Key words: Sp2/0    Scale-down mode    Batch re-feed
收稿日期: 2017-12-24 出版日期: 2018-07-06
ZTFLH:  Q256  
通讯作者: 汪国强,周航     E-mail: zhou_hang@wuxiapptec.com
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引用本文:

汪国强,刘建平,周航. 单克隆抗体Mab-A生产细胞Sp2/0缩小模型培养优化研究[J]. 中国生物工程杂志, 2018, 38(6): 63-69.

Guo-qiang WANG,Jian-ping LIU,Hang ZHOU. Study on the Optimal Scale-down Model for Cell Growth and Mab-A Production of Sp2/0 Cells. China Biotechnology, 2018, 38(6): 63-69.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180609    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I6/63

Medium code Vendor Category
A,B ThermoFisher Basal medium
C,D,E GE
F,G ThermoFisher Feed medium
H,I,J,K GE
L Merck
M Kerry
N,O Lonza
P,Q Kerry Hydrolysate
R Merck Additive
Dexamethasone Merck
GlutaMAXTM ThermoFisher
表1  培养基信息表
Culture No. Medium Code Condition
SF1,5 A+C(50%+50%) Control
SF2,6 D
SF3,7 B
SF4,8 E
SF9 A+C(50%+50%) 160 RPM
SF10 D
SF11 B
SF12 E
SF13 A+C(50%+50%) 10% CO2
SF14 D
SF15 B
SF16 E
SF17 A+C(50%+50%) 33℃
SF19 E
SF18 A+C(50%+50%) 35℃
SF20 E
SF21 A+C(50%+50%) Trace element
SF22 E
SF23 A+C(50%+50%) GlutaMAXTM
SF24 D
SF25 B
SF26 E
表2  批次培养条件
Culture No. Medium Code Conditon
SF27 99% F+1% L Feed medium screen
SF28 99% G+1% L
SF29 49.5% F+49.5% G+1% L
SF30 99% H+1% L
SF31 49.5% H+49.5% I+1% L
SF32 99% J+1% L
SF33 80% J+1% L+19% water
SF34 99% K+1% L
SF35 99% J (with 15 g/L P)+1% L
SF36 99% J (with 15 g/L Q)+1% L
SF37 99% N+1% L
SF38 99% M+1% L
SF39 99% O+1% L
SF40 99% J+1% L 36.5℃ shift to
33℃ on day 4
表3  流加培养条件
图1  Sp2/0在不同基础培养基中的生长及活率曲线
SF9 SF10 SF11 SF12 SF13 SF14 SF15 SF16 SF21 SF22 SF23 SF24 SF25 SF26
Peak VCC (106 cells/ml) 7.81 12.44 3.56 12.69 8.50 12.39 4.02 9.90 6.71 12.45 8.77 13.44 3.46 12.85
Culture duration (days) 5 7 4 7 5 7 4 7 5 7 5 7 4 7
表4  批次培养条件(摇床转速、CO2浓度、微量元素、GlutaMAXTM运用)对Sp2/0细胞生长影响
图2  温度对Sp2/0细胞生长和活率的影响
Culture
No.		 Peak VCC
(106
cells/ml)		 Culture
duration
(days)		 Titer
(mg/L)		 qMab-A,
(pg/cell/
day)
SF27 25.65 7 73.73 0.99
SF28 27.56 7 35.40 0.46
SF29 26.57 7 46.93 0.63
SF30 20.58 7 39.69 0.68
SF31 21.49 7 41.39 0.74
SF32 23.26 7 138.58 1.97
SF33 20.48 8 99.14 1.28
SF34 22.96 6 41.74 0.87
SF35 37.08 9 165.92 1.23
SF36 20.29 8 157.20 1.83
SF37 25.63 6 42.94 0.83
SF38 22.57 6 47.85 1.02
SF39 22.91 6 56.72 1.13
SF40 20.52 8 217.59 2.48
表5  Sp2/0在不同补料培养基中的表现
Culture
No.		 Peak VCC
(106
cells/ml)		 Culture
duration
(days)		 Titer
(mg/L)1)		 qMab-A,
(pg/cell/
day)
ST1 50.42 14 141.10 2.98
ST2 42.23 14 115.38 2.77
ST3 53.01 14 162.56 3.19
表6  Sp2/0在批次-反复流加培养中的表现
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