pDNA质粒在一次性生物反应器中的放大生产研究

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

摘要/Abstract

摘要：

【目的】探索大肠杆菌（E.coli）菌种复苏后跳过三角摇瓶直接接种一次性WAVE生物反应器进行菌种扩增，并放大至一次性XDR生物反应器进行质粒放大生产的可行性，建立GMP级质粒在全一次性上游平台进行工业化放大生产的工艺。【方法】通过碳氮比优化，筛选并获得不含任何动物源成分的基础培养基和补料培养基；菌种冻存管室温融化后以低密度接种三角摇瓶和WAVE反应器，考查菌种低密度接种的可行性，比较三角摇瓶和WAVE反应器进行菌种扩增的差异，建立菌种在WAVE反应器中的扩增工艺；随后将菌种扩增至50 L XDR生物反应器进行质粒的放大生产。【结果】与LB培养基相比，优化的不含任何动物源成分的基础培养基使菌体最高密度和质粒产量分别提高43%和77%；以1:1 000-1:8 000低密度接种WAVE反应器，菌种比生长速率达到（0.65±0.065）/h，WAVE反应器展现了更好的过程参数控制，通过一级WAVE种子罐可直接为50-200 L生产罐提供种子细胞；质粒在一次性50 L XDR反应器中的放大生产，最高菌体密度和质粒产量分别达到53 OD和340 mg/L，质粒比生产速率达到6.42 mg/L/OD₆₀₀，比常规质粒比生产速率提高2倍以上，超螺旋质粒比例达到90%，较高的上游收获超螺旋比例为下游质粒两步层析纯化提供了可能。【结论】建立了大肠杆菌通过WAVE反应器进行菌种扩增，通过一级种子罐直接接种生产罐进行质粒放大生产的工艺，为GMP级别质粒在50-200 L一次性生产平台中的放大生产建立了生产工艺。

关键词: 大肠杆菌, 一次性反应器, 工艺放大, pDNA质粒生产

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

杨红艳, 韩筱, 杨建军. pDNA质粒在一次性生物反应器中的放大生产研究[J]. 生物技术通报, 2024, 40(1): 168-175.

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.

图/表 10

图1 传统菌种扩增方法（A）及新型菌种扩增方法（B）

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

图2 LB Basic Medium和Opti Basic Medium对菌体密度和质粒产量的影响

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

表1 LB Basic Medium和Opti Basic Medium的培养性能比较

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

图3 极低密度接种菌种扩增研究

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

表2 三角摇瓶及WAVE25 生物反应器菌体扩增比较

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

图4 WAVE25一次性生物反应器关键参数运行曲线

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

图5 XDR50 MO一次性生物反应器关键工艺参数曲线

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

图6 XDR50 MO生物反应器累加补料体积及累加氨水添加量曲线

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

图7 XDR50 MO一次性生物反应器菌体OD600及比生长速率曲线

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

图8 质粒拓扑异构体分析

Fig. 8 Analysis of plasmid topo isomers

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