Development of integrated continuous flow systems for transient transfection, cultivation and monitoring of mammalian cells

近年来，各种制造业广泛证明了连续种植和生产过程的好处。随着对生物制药的需求不断增长，生产率提高以及降低制造成本的压力不断增加，生物技术行业似乎对连续生物制造系统的开发表现出持续的兴趣。尽管市场上出现了新兴的连续生物加工技术，但几乎没有成功实施生物制品完全连续工艺的具体实例。为了加速工艺优化，有利的是利用适当的技术实施生物反应器，以连续监测和调节关键培养参数。此外，市场正在寻求快速基因递送技术，该技术将允许灵活生产单个靶蛋白和生产过程的并行化。为了进一步加强这一发展过程中，构建一个新的控制和连续的细胞转染和培养系统的重组蛋白生产和分析物的同时监测不同功能的微流控芯片实验室（lab-on-a-chip）设备的集成提出。生物传感器设备为生物过程提供了许多优势，例如小样品体积，定义和可重现的工作条件，便携性，即时（POC）诊断以及降低过程成本的潜力。连续生物反应器培养的主要挑战是功能单元和监测系统的开发和集成，这些单元和监测系统适用于上游和下游工艺。在此，该项目将专注于上游工艺和新传感器的开发，以监测细胞培养系统。作为上游工艺的重要组成部分，本项目旨在设计、制造和整合一种用于连续和受控瞬时基因转移到宿主细胞以生产重组蛋白的装置。此外，一种新的机电适配体为基础的生物传感器的目标蛋白质的快速监测以及早期检测潜在的微生物污染的建议。

Benefits of continuous cultivation and production processes have been widely demonstrated by a variety of manufacturing industries in recent years. With growing demand for biopharmaceuticals, higher productivity and ever-increasing pressure to reduce manufacturing costs, the biotech industry seems to exhibit a persisting interest in the development of continuous bio-manufacturing systems. Although there are emerging continuous bioprocessing technologies available in the market, there are almost no concrete examples for successful implementation of a fully continuous process for biological products. To accelerate the process optimization it is advantageous to implement the bioreactor with appropriate technology for continuous monitoring and regulation of critical cultivation parameters. In addition, the market is seeking for rapid gene delivery technologies that would allow flexible production of individual target proteins and parallelization of production processes. To further enhance this development process, the construction of a novel controlled and continuous cell transfection and cultivation system for recombinant protein production and simultaneous analyte monitoring by integration of different functional microfluidic lab-on-a-chip (LOC) devices is proposed. LOC devices offer many advantages for bioprocesses such as small sample volume, defined and reproducible working conditions, portability, point-of-care (POC) diagnosis, and the potential of reducing process costs. Main challenges for continuous bioreactor cultivation are the development and integration of functional units and monitoring systems, which are suitable for both upstream and downstream processes. Herein, this project will focus on the upstream process and development of new sensors to monitor cell culture systems. As an important part of the upstream process, this project aims to design, fabricate and integrate a LOC for continuous and controlled transient gene transfer to the host cells for recombinant protein production. Furthermore, a novel electromechanical aptamer-based biosensor for rapid monitoring of target proteins as well as for early detection of potential microbial contamination is proposed.