Pan Siqi, Zelger Monika, Jungbauer Alois, Hahn Rainer
Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria; Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
J Biotechnol. 2014 Sep 20;185:39-50. doi: 10.1016/j.jbiotec.2014.06.010. Epub 2014 Jun 17.
An integrated continuous tubular reactor system was developed for processing an autoprotease expressed as inclusion bodies. The inclusion bodies were suspended and fed into the tubular reactor system for continuous dissolving, refolding and precipitation. During refolding, the dissolved autoprotease cleaves itself, separating the fusion tag from the target peptide. Subsequently, the cleaved fusion tag and any uncleaved autoprotease were precipitated out in the precipitation step. The processed exiting solution results in the purified soluble target peptide. Refolding and precipitation yields performed in the tubular reactor were similar to batch reactor and process was stable for at least 20 h. The authenticity of purified peptide was also verified by mass spectroscopy. Productivity (in mg/l/h and mg/h) calculated in the tubular process was twice and 1.5 times of the batch process, respectively. Although it is more complex to setup a tubular than a batch reactor, it offers faster mixing, higher productivity and better integration to other bioprocessing steps. With increasing interest of integrated continuous biomanufacturing, the use of tubular reactors in industrial settings offers clear advantages.
开发了一种集成的连续管式反应器系统，用于处理以包涵体形式表达的自蛋白酶。将包涵体悬浮并送入管式反应器系统进行连续溶解、重折叠和沉淀。在重折叠过程中，溶解的自蛋白酶会自我切割，将融合标签与目标肽分离。随后，切割后的融合标签和任何未切割的自蛋白酶在沉淀步骤中沉淀出来。经过处理的流出溶液得到纯化的可溶性目标肽。在管式反应器中进行的重折叠和沉淀产率与间歇反应器相似，并且该过程至少稳定20小时。纯化肽的真实性也通过质谱法进行了验证。在管式过程中计算的生产率（以mg/l/h和mg/h为单位）分别是间歇过程的两倍和1.5倍。尽管设置管式反应器比间歇反应器更复杂，但它提供了更快的混合、更高的生产率以及与其他生物处理步骤更好的集成。随着对集成连续生物制造的兴趣增加，在工业环境中使用管式反应器具有明显的优势。
