High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor.
High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor.
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DOI：
10.1186/1475-2859-9-42
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发表时间：
2010-05-30
影响因子：
6.4
通讯作者：
Neubauer P
中科院分区：
文献类型：
--
作者：
Glazyrina J;Materne EM;Dreher T;Storm D;Junne S;Adams T;Greller G;Neubauer P
与标准搅拌罐生物反应器相比，一次性摇摆运动型袋式生物反应器具有以下优点：降低污染风险、减少清洁和灭菌时间、降低投资成本以及简单且更便宜的验证。目前，它们被广泛用于细胞培养，尽管它们用于用微生物宿主小规模和中等规模生产重组蛋白可能非常有吸引力。然而，摇摆或波浪诱导的运动型生物反应器用于快速生长的好氧微生物是有限的，因为它们较低的氧传质速率。降低培养物需氧量的常规方法是补料分批技术。BIOSTAT® CultiBag RM系统等新开发为在摇摆式生物反应器中应用先进的补料分批控制策略铺平了道路。或者，内部底物递送系统如EnBase® Flo提供了采用易于使用的分批补料型策略来振荡培养的机会。在这里，我们调查的可能性，这两种策略提供的高细胞密度培养的E。大肠杆菌和重组蛋白生产。E.在BIOSTAT® CultiBag RM系统中以常规分批模式在没有对照的情况下对大肠杆菌进行的培养产生了3至4的光密度（OD 600），这与摇瓶相当。文化运行到氧气限制。在具有指数进料和氧脉冲的葡萄糖限制的补料分批培养中，培养物完全有氧生长至OD 600为60（20 g L-1细胞干重）。通过使用内部控制的葡萄糖递送系统，EnBase® Flo，获得30的OD 600（10 g L-1细胞干重），而不需要计算机控制的外部营养供应。EnBase® Flo在CultiBag RM系统中与重组E. coli RB 791菌株表达异源醇脱氢酶（ADH）至非常高的水平，表明基于酶的补料供应策略在摇摆运动型生物反应器中也对重组蛋白生产起良好作用。摇摆运动型生物反应器可以提供一个有趣的替代标准培养的生物反应器中的细菌培养和重组蛋白生产。配备一次性传感器和先进控制系统的BIOSTAT® Cultibag RM系统为分批补料技术和摇摆式生物反应器铺平了道路。有可能达到远高于摇瓶的细胞密度，并且对于具有改进的氧转移速率的搅拌釜反应器是典型的。对于更简单的应用，EnBase® Flo方法为没有这种先进控制可能性的摇摆运动系统提供了简单而强大的解决方案。
Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production. Cultivation of E. coli in the BIOSTAT® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD600) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD600 of 60 (20 g L-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase® Flo, OD600 of 30 (10 g L-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor. Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.
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影响因子：
4.1
作者：
RIESENBERG, D;SCHULZ, V;DECKWER, WD
通讯作者：
DECKWER, WD
影响因子：
6.4
作者：
Soini, Jaakko;Ukkonen, Kaisa;Neubauer, Peter
通讯作者：
Neubauer, Peter
影响因子：
3.8
作者：
Mikola, Mark;Seto, Jennifer;Amanullah, Ashraf
通讯作者：
Amanullah, Ashraf
影响因子：
3.8
作者：
NEUBAUER, P;HAGGSTROM, L;ENFORS, SO
通讯作者：
ENFORS, SO
DOI：
10.1007/bf01575849
发表时间：
1991-10-01
期刊：
JOURNAL OF INDUSTRIAL MICROBIOLOGY
影响因子：
--
作者：
NANCIB, N;BRANLANT, C;BOUDRANT, J
通讯作者：
BOUDRANT, J