Technical University of Munich, Institute of Biochemical Engineering, Boltzmannstr. 15, 85748, Garching, Germany.
Technical University of Munich, Institute of Biochemical Engineering, Boltzmannstr. 15, 85748, Garching, Germany.
J Biotechnol. 2021 May 20;332:103-113. doi: 10.1016/j.jbiotec.2021.03.021. Epub 2021 Apr 15.
Automation, parallelization and autonomous operation of standard lab equipment, usually applied for manual bioprocess development, is considered as the key for reduction of bioprocess development time and costs. An automated bioreactor system with 4 stirred-tank bioreactors on a L-scale was combined with a custom-made biomass transfer system to distribute the cell suspensions produced on the L-scale into 48 parallel stirred-tank bioreactors on a mL-scale. Afterwards parallel protein expression studies automated by a liquid handling system with integrated fluorescence reader were performed. Isopropyl β-D-1-thiogalactopyranoside-induced (IPTG) expression of the red fluorescence protein mCherry was studied as an example of using fed-batch processes with recombinant Escherichia coli. In a first automated study, IPTG concentrations were varied in 48 parallel fed-batch processes with E. coli cells produced at a growth rate of 0.1 h on an L-scale and transferred automatically to the mL-scale. The mCherry expression rate increased with increasing inducer concentration until the highest protein expression rate was observed at > 9 μM IPTG. In a second automated study, the growth rate of E. coli was varied between 0.1-0.2 h in parallelly-operated stirred-tank bioreactors on a L-scale. The cells were automatically transferred and distributed into the stirred-tank bioreactors on a mL-scale and the concentration of the inducer IPTG was varied as before in parallel fed-batch processes. An increased growth rate during the production of the recombinant E. coli cells and/or higher cell densities during protein expression resulted in the increased IPTG concentrations necessary to achieve identical expression rates compared to a growth rate of 0.1 h with the exception of very low inducer concentrations and inducer concentrations in excess. The new automated multi-scale cascade of parallel stirred-tank bioreactors should easily be applicable for performing fast optimisation studies with other microbial production systems and will have the potential to reduce bioprocess development time and staff assignment considerably.
自动化、并行化和标准实验室设备的自主操作，通常应用于手动生物工艺开发，被认为是减少生物工艺开发时间和成本的关键。一个具有 4 个搅拌罐生物反应器的 L 规模自动化生物反应器系统与一个定制的生物质转移系统相结合，将在 L 规模上生产的细胞悬浮液分配到 48 个 mL 规模的平行搅拌罐生物反应器中。然后，通过集成荧光读取器的液体处理系统进行自动并行蛋白质表达研究。以异丙基 β-D-1-硫代半乳糖吡喃糖苷（IPTG）诱导的红色荧光蛋白 mCherry 表达为例，研究了重组大肠杆菌的分批补料过程。在第一次自动化研究中，在 48 个平行的分批补料过程中，通过在 L 规模上以 0.1 h 的生长速率生产的大肠杆菌细胞，自动将 IPTG 浓度变化转移到 mL 规模。随着诱导剂浓度的增加，mCherry 的表达率增加，直到在 > 9 μM IPTG 时观察到最高的蛋白质表达率。在第二次自动化研究中，在 L 规模上的平行搅拌罐生物反应器中，大肠杆菌的生长速率在 0.1-0.2 h 之间变化。细胞自动转移并分布到 mL 规模的搅拌罐生物反应器中，与之前一样，在平行分批补料过程中，诱导剂 IPTG 的浓度变化。与生长速率为 0.1 h 相比，在重组大肠杆菌细胞生产过程中增加的生长速率和/或表达蛋白时更高的细胞密度导致需要增加 IPTG 浓度以达到相同的表达率，除了非常低的诱导剂浓度和过量的诱导剂浓度。新的自动化多尺度平行搅拌罐生物反应器级联系统应该很容易适用于与其他微生物生产系统进行快速优化研究，并有可能大大缩短生物工艺开发时间和人员分配。
