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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  2023, Vol. 43 Issue (10): 20-31    DOI: 10.13523/j.cb.2305046
技术与方法     
CHO细胞强化流加培养过程理性设计与建立
苏影1,张炜坚1,万宇翔2,沈天放2,张欣然1,张如悦1,谭文松1,3,赵亮1,3,*()
1 华东理工大学生物反应器工程国家重点实验室 上海 200237
2 海正药业杭州有限公司 杭州 311404
3 上海倍谙基生物科技有限公司 上海 201203
Rational Design and Establishment of CHO Cell Intensified Fed-batch Culture Process
SU Ying1,ZHANG Wei-jian1,WAN Yu-xiang2,SHEN Tian-fang2,ZHANG Xin-ran1,ZHANG Ru-yue1,TAN Wen-song1,3,ZHAO Liang1,3,*()
1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
2 Zhejiang Hisun Pharmaceutical Co., Ltd., Hangzhou 311404, China
3 Shanghai Bioengine Sci-Tech Co., Ltd., Shanghai 201203, China
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摘要：

目的:由于强化流加培养工艺(intensified fed batch of ultra-high seeding density,uHSD-IFB)的接种密度与运行密度较高,传统低密度培养工艺的流加策略往往不能提供充足的营养物质用于该过程的细胞维持与产物表达,最终导致过程产率低、经济性下降;通过优化流加培养基以及补料方案,成功建立CHO细胞强化流加培养过程,从而提高目的蛋白产量。方法:以一株表达单克隆抗体的CHO-K1细胞株为研究对象,通过代谢动力学与化学计量学分析,设计出以葡萄糖为控制模型的两阶段动态反馈流加策略,并结合实验设计(design of experiment,DoE)筛选并优化流加培养基中关键微量元素的营养浓度。结果:优化设计后的uHSD-IFB过程有效缓解了uHSD-IFB过程营养物质的耗竭与代谢副产物累积之间的矛盾,实现了超高接种密度培养工艺的细胞生长与产物合成的目的;累积产量相较于优化前提高了95%,日体积产量提高了约97%。结论:该补料策略有助于快速建立高细胞密度、高产物表达的高接种密度强化流加培养过程。
关键词： CHO细胞;  过程强化;  动态反馈流加;  理性设计;  葡萄糖控制模型    
Abstract:

Objective: Due to the higher seeding density and running density of the intensified fed-batch of ultra-high seeding density (uHSD-IFB) process, the feeding strategy of the traditional low-density culture process often does not provide sufficient nutrients for cell maintenance and product expression of the process, which ultimately leads to low process yield and lower production efficiency. By optimizing the feed medium and feeding strategy, the uHSD-IFB process will be successfully established and the target protein yield will be increased. Methods: A monoclonal antibody-expressing CHO-K1 cell line was studied in this paper, a two-stage dynamic feedback feeding strategy with glucose as the control indicator was designed by metabolic kinetics and stoichiometry analysis, and the nutrient concentrations for key trace elements in the feed medium were screened and optimized in combination with design of experiment (DoE). Results: The optimized process effectively alleviated the contradiction between nutrient depletion and accumulation of metabolic by-products in the uHSD-IFB process and achieved the purpose of cell growth and product synthesis in the ultra-high inoculation density culture process. The cumulative titer of the optimized design of the uHSD-IFB process was increased by 95% and the daily yield was increased by about 97% compared to that before optimization. Conclusion: This proposed feeding strategy can provide help to rapidly establish an enhanced fed-batch culture process of ultra-high seeding density with high cell density and high product expression.
Key words: CHO cell    Process intensification    Dynamic feedback fed-batch    Rational design    Glucose control model
收稿日期: 2023-05-30 出版日期: 2023-11-02
ZTFLH:  Q813  
通讯作者: *电子信箱:zhaoliang@ecust.edu.cn   
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苏影
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引用本文:

苏影, 张炜坚, 万宇翔, 沈天放, 张欣然, 张如悦, 谭文松, 赵亮. CHO细胞强化流加培养过程理性设计与建立[J]. 中国生物工程杂志, 2023, 43(10): 20-31.

SU Ying, ZHANG Wei-jian, WAN Yu-xiang, SHEN Tian-fang, ZHANG Xin-ran, ZHANG Ru-yue, TAN Wen-song, ZHAO Liang. Rational Design and Establishment of CHO Cell Intensified Fed-batch Culture Process. China Biotechnology, 2023, 43(10): 20-31.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2305046    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I10/20

氨基酸 0~4 d的浓度
/(mmol·L-1)		 5~8 d的浓度
/(mmol·L-1)		 氨基酸 0~4 d的浓度
/(mmol·L-1)		 5~8 d的浓度
/(mmol·L-1)
天冬氨酸 72.02 29.22 酪氨酸 39.17 23.88
谷氨酸 81.12 81.7 胱氨酸 49.32 31.45
天冬酰胺 100.43 91.03 缬氨酸 114.1 69.02
丝氨酸 136.98 93.59 甲硫氨酸 29.31 14.25
脯氨酸 43.2 43.2 色氨酸 16.93 17.39
组氨酸 41.47 19.59 苯丙氨酸 26.21 37.6
苏氨酸 64.09 50.11 异亮氨酸 61.1 40.16
精氨酸 46.85 19.25 亮氨酸 78.2 65.01
丙氨酸 23.6 23.6 赖氨酸 46.76 69.02
表1  葡萄糖控制模型的uHSD-IFB过程中流加培养基的氨基酸浓度
图1  Optimized uHSD-IFB过程的理性设计流程
图2  传统与强化流加培养过程中细胞生长与产物合成比较
图3  uHSD-IFB过程中氨基酸浓度变化
图4  uHSD-IFB(AA)过程中氨基酸的比消耗(生成)速率
图5  uHSD-IFB(AA)过程中细胞生长阶段与产物合成阶段氨基酸的累计消耗与葡萄糖的累积消耗之间的关系
图6  葡萄糖控制模型的uHSD-IFB过程与优化前的uHSD-IFB过程比较
编号 物质 高水平质量浓度/(mg·L-1) 编号 物质 高水平质量浓度/(mg·L-1)
A D-泛酸钙 95.306 N 柠檬酸钠 103.228
B 氯化胆碱 55.852 O ZnSO4·7H2O 3.22
C 抗坏血酸钠 39.603 P 亚硒酸钠 0.346
D 叶酸 44.1 Q 腐胺 17.63
E 肌醇 84 R 乙醇胺盐酸盐 9.75
F 烟酰胺 12.213 S 胰岛素 11.6
G 钴胺素 13.55 T 谷胱甘肽 12.29
H 生物素 0.673 U 胸苷 9.69
J 核黄素 1.882 V α-酮戊二酸 59.44
K 盐酸吡哆醇 6.169 W 虚拟因子1 -
L CuSO4·5H2O 0.1 X 虚拟因子2 -
M FeSO4·7H2O 55.6
表2  Plackett-Burman试验设计因素及水平
因素 平方和 均方 F值 P值 重要性
Model 396.84 18.9 878.62 0.001 1 Significant
A-D-泛酸钙 0.055 7 0.055 7 2.59 0.248 9 15
B-氯化胆碱 2.22 2.22 103.17 0.009 6 2
C-抗坏血酸钠 2.05 2.05 95.38 0.010 3 3
D-叶酸 0.005 3 0.005 3 0.248 3 0.667 7 21
E-I-肌醇 0.081 3 0.081 3 3.78 0.191 3 14
F-烟酰胺 0.344 2 0.344 2 16.01 0.057 2 12
G-钴胺素 1.11 1.11 51.55 0.018 9 5
H-生物素 0.661 9 0.661 9 30.78 0.031 0 7
J-核黄素 0.891 8 0.891 8 41.47 0.023 3 6
K-盐酸吡哆醇 0.040 3 0.040 3 1.88 0.304 4 16
L-五水硫酸铜 0.005 6 0.005 6 0.26 0.660 8 20
M-七水硫酸亚铁 0.008 0.008 0.371 9 0.604 0 18
N-柠檬酸钠 0.271 1 0.271 1 12.6 0.071 0 13
O-七水硫酸锌 0.353 5 0.353 5 16.43 0.055 8 11
P-亚硒酸钠 0.489 8 0.489 8 22.77 0.041 2 10
Q-腐胺 0.521 8 0.521 8 24.26 0.038 8 9
R-乙醇胺 1.33 1.33 61.99 0.015 8 4
S-胰岛素 385.74 385.74 17 935.24 < 0.000 1 1
T-谷胱甘肽 0.602 8 0.602 8 28.03 0.033 9 8
U-胸苷 0.039 6 0.039 6 1.84 0.307 6 17
V-α-酮戊二酸 0.006 0.006 0.279 4 0.649 9 19
表3  Plackett-Burman试验结果方差分析
Parameters Traditional FB uHSD-IFB Optimized uHSD-IFB
Seeding density (106 cells/mL) 0.5 15 15
μ-Growth phase (1/day) 0.44 0.43 0.34
Peak VCD (106 cells/mL) 19.10 37.35 33.33
IVCC (109cells·day/L) 173 246 214
Feeding strategy Constant ratio Constant ratio Glucose control + DoE
Culture time (day) 14 8 8
Titer (g/L)% - 11.79±0.60 117.89±0.79
QP (pg/cell/day)% - 17.26±0.57 156.94±1.45
Vp (g/L/day)% - 95.93±4.88 255.56±1.72
Peak lactate concentration (mmol/L) 30.87 30.65 24.26
Peak osmolality (mOsm/kg) 452 586 520
表4  几种流加培养过程的主要结果比较
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