Scaling up Production of pDNA Plasmids in Disposable Bioreactors

doi:10.13560/j.cnki.biotech.bull.1985.2023-0691

Abstract

Abstract:

【Objective】The objective is to explore the feasibility of using WAVE bioreactor for Escherichia coli strain amplification after the strain recovered without using shake flask and expanding the seed cells into a disposable XDR bioreactor for plasmid production, as well as to establish a process for GMP grade plasmids production on a fully disposable upstream platform. 【Method】Optimizing the carbon to nitrogen ratio for E.coli growth, basic culture medium and supplementary culture media that do not contain any animal derived components were screened.Directly inoculating E.coli strain to shake flasks and WAVE bioreactor after strain thawing, the feasibility of low-density innoculation was investigated, and the differences of E.coli strain amplification in shake flask and in WAVE bioreactor was compared. The expansion process of E.coli strains in WAVE bioreactor was established, and then the seed cells were amplified into a 50L XDR bioreactor for plasmid production. 【Result】Compared with traditional LB medium, the optimized basic medium without any animal origin ingredients increased the maximum OD₆₀₀ and plasmid yield by 43% and 77% respectively. After E.coli strain thawing, WAVE25 bioreactor was directly inoculated with a low density of 1:1 000-1:8 000. The specific growth rate of the E.coli strain in WAVE25 bioreactor reached (0.65±0.065)/h. WAVE bioreactor showed better capability for controlling process parameters, and the seed cells from one WAVE bioreactor directly inoculated 50-200 L production fermentor for plasmid production. The maximum OD₆₀₀ and plasmid titer reached 53 OD and 340 mg/L respectively, the specific plasmid yield reached 6.42 mg/L/OD₆₀₀, which was more than 2 times higher than that of conventional plasmid process. The proportion of supercoiled plasmids reached 90% at upstream harvest, which made it possible for downstream two-step chromatography purification of plasmids. 【Conclusion】In conclusion, a full disposable production platform has been established to directly amplify E. coli strain through WAVE bioreactor, and the seed cells from one WAVE bioreactor can be used to inoculate 50-200 L production fermentor for GMP grade plasmid production.

Key words: Escherichia coli, disposable bioreactor process, scaleup, pDNA plasmid production

YANG Hong-yan, HAN Xiao, YANG Jian-jun. Scaling up Production of pDNA Plasmids in Disposable Bioreactors[J]. Biotechnology Bulletin, 2024, 40(1): 168-175.

Figures/Tables 10

Fig. 1 Traditional seeding expansion method（A）and novel seeding expansion method（B）for E. coli

Fig. 2 Effects of LB Basic Medium and Opti Basic Medium on the E. coli density and plasmid production

Table 1 Performance comparison between LB Basic Medium and Opti Basic Medium

基础培养基 Medium type	最高菌体密度 Peak OD₆₀₀	质粒产量 Plasmid titer/（mg·L^-1）	单位菌体密度质粒产量 Specific plasmid yield /（mg·L^-1·OD₆₀₀^-1）
LB培养基 LB basic medium	6.07	11.29	1.86
Opti培养基 Opti basic medium	8.66	19.98	2.31

Fig. 3 Study on the amplification of E. coli strains using extremely low density inoculum

Table 2 Comparison of E. coli seed expansion between shake flask and WAVE25 bioreactor

培养装置 Seed expansion vessel	培养时间 Culture time/h	菌体密度OD₆₀₀	平均比生长速率Average specific growth rate /（h^-1）	关键工艺参数是否可控和可记录 Parameters controllable and recordable
三角摇瓶Shake flask	10.2	1.01	0.72	No
WAVE25生物反应器WAVE25 bioreactor	11.0	1.20	0.66	Yes

Fig. 4 Key parameter curves of E. coli cultured in WAVE25 bioreactor

Fig. 5 Key process parameter curves of E. coli cultured in XDR50 MO bioreactor

Fig. 6 Accumulated feeding volume and accumulated ammonia volume of E. coli cultured in XDR50 MO bioreactor

Fig. 7 OD600 and specific growth rate curves of E.coli cultured in XDR50 MO bioreactor

Fig. 8 Analysis of plasmid topo isomers

References 20

[1]	Ginn SL, Amaya AK, Alexander IE, et al. Gene therapy clinical trials worldwide to 2017: an update[J]. J Gene Med, 2018, 20(5): e3015. doi: 10.1002/jgm.v20.5 URL
[2]	Pardi N, Hogan MJ, Weissman D. Recent advances in mRNA vaccine technology[J]. Curr Opin Immunol, 2020, 65: 14-20. doi: S0952-7915(20)30010-8 pmid: 32244193
[3]	Mohanty R, Chowdhury CR, Arega S, et al. CAR T cell therapy: A new era for cancer treatment[J]. Oncol Rep, 2019, 42(6):2183-2195. doi: 10.3892/or.2019.7335
[4]	Schmeer M, Buchholz T, Schleef M. Plasmid DNA manufacturing for indirect and direct clinical applications[J]. Hum Gene Ther, 2017, 28(10): 856-861. doi: 10.1089/hum.2017.159 pmid: 28826233
[5]	Schmeer M, Schleef M. Production of plasmid DNA as pharmaceutical[J]. Methods Mol Biol, 2015, 1317: 315-326. doi: 10.1007/978-1-4939-2727-2_17 pmid: 26072414
[6]	Grijalva-Hernández F, Vega-Estrada J, Escobar-Rosales M, et al. High kanamycin concentration as another stress factor additional to temperature to increase pDNA production in E. coli DH5α batch and fed-batch cultures[J]. Microorganisms, 2019, 7(12): 711. doi: 10.3390/microorganisms7120711 URL
[7]	de la Cruz M, Ramírez EA, Sigala JC, et al. Plasmid DNA production in proteome-reduced Escherichia coli[J]. Microorganisms, 2020, 8(9): 1444. doi: 10.3390/microorganisms8091444 URL
[8]	Ruiz O, Miladys, Jorge, et al. Scalable technology to produce pharmaceutical grade plasmid DNA for gene therapy[M]//Gene Therapy - Developments and Future Perspectives. InTech, 2011
[9]	Ganjave SD, Dodia H, Sunder AV, et al. High cell density cultivation of E. coli in shake flasks for the production of recombinant proteins[J]. Biotechnol Rep, 2021, 33: e00694.
[10]	Ali S, Perez-Pardo MA, Aucamp JP, et al. Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed-batch fermentations[J]. Biotechnol Prog, 2012, 28(1): 66-75. doi: 10.1002/btpr.v28.1 URL
[11]	Ramamoorthy S. High cell density fermentation for production of flavonoids using recombinant E. coli in a bioreactor[J]. Dissertations & Theses - Gradworks, 2011
[12]	Yang T, Huang Y, Han ZQ, et al. Novel disposable flexible bioreactor for Escherichia coli culture in orbital shaking incubator[J]. J Biosci Bioeng, 2013, 116(4): 452-459. doi: 10.1016/j.jbiosc.2013.04.004 URL
[13]	Diekmann S, Dürr C, Herrmann A, et al. Single use bioreactors for the clinical production of monoclonal antibodies - a study to analyze the performance of a CHO cell line and the quality of the produced monoclonal antibody[J]. BMC Proc, 2011, 5(Suppl 8): P103.
[14]	Shukla AA, Gottschalk U. Single-use disposable technologies for biopharmaceutical manufacturing[J]. Trends Biotechnol, 2013, 31(3): 147-154. doi: 10.1016/j.tibtech.2012.10.004 pmid: 23178074
[15]	杨建军, 隋礼丽. WAVE^TM生物反应器灌注培养生产种子细胞的开发和应用[J]. 生物工程学报, 2012, 28(3): 358-367.
	Yang JJ, Sui LL. Development and application of perfusion culture producing seed cells in WAVE^TM bioreactor[J]. Chin J Biotechnol, 2012, 28(3): 358-367.
[16]	国家药典委员会. 生物制品病毒安全性控制.《中国药典》2020三部[M]. 北京: 中国医药科技出版社, 2022.
	Chinese Pharmacopoeia Commission. Control of Viral Safety in Biological Products. Chinese Pharmacopoeia(2020 Edition Volume III)[M]. Beijing: China Medical Science Press, 2022.
[17]	Manzke A, Kleindienst B. Virus risk mitigation in cell culture media[J]. Biopharm Int, 2016, 29(10): 20, 22, 24-25.
[18]	Djemal L, Fournier C, von Hagen J, et al. Review: High temperature short time treatment of cell culture media and feed solutions to mitigate adventitious viral contamination in the biopharmaceutical industry[J]. Biotechnol Prog, 2021, 37(3): e3117. doi: 10.1002/btpr.3117 URL
[19]	Mahajan E, Matthews T, Hamilton R, et al. Use of disposable reactors to generate inoculum cultures for E. coli production fermentations[J]. Biotechnol Prog, 2010, 26(4): 1200-1203. doi: 10.1002/btpr.v26:4 URL
[20]	Aurora G, Angelica G, Roberto G, et al. Substrate-source flexibility of an exponential-fed perfusion process to produce plasmid DNA for use as leishmaniasis vaccine[J]. Biotechnol Biotechnol Equip, 2019, 33(1): 195-203. doi: 10.1080/13102818.2018.1560232 URL