Automated fed-batch fermentation with feed-back controls based on dissolved oxygen (DO) and pH for production of DNA vaccines
Automated fed-batch fermentation with feed-back controls based on dissolved oxygen (DO) and pH for production of DNA vaccines
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DOI：
10.1038/sj.jim.2900355
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发表时间：
1997-01-01
影响因子：
3.4
通讯作者：
Ciccarelli, RB
中科院分区：
文献类型：
--
作者：
Chen, W;Graham, C;Ciccarelli, RB
利用基于溶解氧（DO）和pH的自动反馈控制营养物进料策略，开发了用于生产用作DNA疫苗的超螺旋质粒DNA的大肠杆菌发酵过程。该过程通过计算机辅助数据处理系统进一步自动化，以基于DO的交互控制营养物进料速率和搅拌速度来调节细胞生长速率，与人工补料分批培养相比，该方法使质粒DNA的总产量增加了约10倍，自动化方法的最终细胞产量达到60 g/L（-1）细胞干重（OD 600 = 120），24 h内，A质粒DNA产量为100 mg/L（-1）（1.7mg/g（-1）细胞重量）。使用高效液相色谱（HPLC）分析确认质粒产率，由于细胞在自动化过程中在碳限制条件下生长，乙酸的产量很小（低于0.01 g L（-1））。相反，在手动过程中，观察到高达0.36 g L（-1）的酸积累速率，可能是由于用于维持最大生长速率的高营养补料速率，人工补料分批过程产生平均10-12 g L（-1）的低细胞密度。（OD 600 = 25-30）和质粒产量为5-8 mg/L（-1）（约0.7 mg g（-1）细胞），在基于DO和pH的补料中提高的质粒DNA产率后控工艺的改变被认为是细胞密度增加、生长速率（CL）从0.69 h（-1）降低到0.13 h（-1）以及补料分批阶段中的碳/氮限制的组合的结果。基于DO和pH的反馈控制、补料分批工艺已经证明其本身在调节细胞生长速率以实现高细胞密度和质粒产率两者而不必使用纯氧方面是有利的。该工艺在7-L和80-L规模的三次发酵中是可重复的。
A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO, The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture, The final cell yield from the automated process reached 60 g L(-1) of dry cell weight (OD600 = 120) within 24 h, A plasmid DNA yield of 100 mg L(-1) (1,7 mg g(-1) cell weight) was achieved by using an alkaline cell lysis method, Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis, Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L(-1)) throughout the fed-batch stage, In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L(-1) was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate, The manual fed-batch process produced a low cell density averaging 10-12 g L(-1) (OD600 = 25-30) and plasmid yields of 5-8 mg L(-1) (approximately 0.7 mg g(-1) cells), The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (CL) from 0.69 h(-1) to 0.13 h(-1) and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen, The process was reproducible in triplicate fermentations at both 7-L and 80-L scales.