In the pharmaceutical industry, nanobodies show promising properties for its application in serotherapy targeting the highly diffusible scorpion toxins. The production of recombinant nanobodies in Escherichia coli has been widely studied in shake flask cultures in rich medium. However, there are no upstream bioprocess studies of nanobody production in defined minimal medium and the effect of the induction temperature on the production kinetics. In this work, the effect of the temperature during the expression of the chimeric bispecific nanobody CH10-12 form, showing high scorpion antivenom potential, was studied in bioreactor cultures of E. coli. High biomass concentrations (25 g cdw/L) were achieved in fed-batch mode, and the expression of the CH10-12 nanobody was induced at temperatures 28, 29, 30, 33, and 37°C with a constant glucose feed. For the bispecific form NbF12-10, the induction was performed at 29°C. Biomass and carbon dioxide yields were reported for each culture phase, and the maintenance coefficient was obtained for each strain. Nanobody production in the CH10-12 strain was higher at low temperatures (lower than 30°C) and declined with the increase of the temperature. At 29°C, the CH10-12, NbF12-10, and WK6 strains were compared. Strains CH10-12 and NbF12-10 had a productivity of 0.052 and 0.021 mg/L/h of nanobody, respectively, after 13 h of induction. The specific productivity of the nanobodies was modeled as a function of the induction temperature and the specific growth rates. Experimental results confirm that low temperatures increase the productivity of the nanobody.

Key points

• Nanobodies with scorpion antivenom activity produced using two recombinant strains.

• Nanobodies production was achieved in fed-batch cultures at different induction temperatures.

• Low induction temperatures result in high volumetric productivities of the nanobody CH10-12.

在制药工业中，纳米抗体在针对高度扩散性蝎毒素的血清疗法中显示出广阔的应用前景。已经在丰富培养基中的摇瓶培养中广泛研究了在大肠杆菌中重组纳米抗体的生产。然而，目前尚无上游生物工艺研究在限定的基本培养基中进行纳米抗体生产以及诱导温度对生产动力学的影响。在这项工作中，在大肠杆菌的生物反应器培养物中研究了嵌合双特异性纳米抗体CH10-12形式表达过程中温度的影响，显示出高蝎子抗蛇毒的潜力。。在分批补料模式下获得了高生物质浓度（25 g cdw / L），并且在恒定葡萄糖进料的情况下，在温度28、29、30、33和37°C诱导了CH10-12纳米抗体的表达。对于双特异性形式的NbF12-10，诱导在29℃下进行。报告了每个培养阶段的生物量和二氧化碳产量，并获得了每个菌株的维持系数。CH10-12菌株在低温（低于30°C）下产生的纳米抗体产量较高，并且随着温度的升高而下降。在29°C下，比较了CH10-12，NbF12-10和WK6菌株。诱导13小时后，菌株CH10-12和NbF12-10的产率分别为0.052和0.021mg / L / h的纳米抗体。将纳米抗体的比生产率建模为诱导温度和比生长速率的函数。实验结果证实低温可提高纳米体的生产率。

关键点

•使用两种重组菌株生产的具有蝎子抗蛇毒活性的纳米抗体。

•在不同的诱导温度下，分批补料培养实现了纳米抗体的生产。

•较低的感应温度导致纳米抗体CH10-12的高容积生产率。