Instituto de Engenharia de Sistemas e Computadores - Microsistemas e Nanotecnologias (INESC-MN) and IN - Institute of Nanoscience and Nanotechnology, Lisbon, Portugal; IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
J Chromatogr A. 2020 Jan 11;1610:460539. doi: 10.1016/j.chroma.2019.460539. Epub 2019 Sep 10.
Over the past decade significant progress has been found in the upstream production processes, shifting the main bottlenecks in current manufacturing platforms for biopharmaceuticals towards the downstream processing. Challenges in the purification process include reducing the production costs, developing robust and efficient purification processes as well as integrating both upstream and downstream processes. Microfluidic technologies have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner, since a large number of variables can be evaluated in a small time frame, using reduced volumes and manpower. Their modularity also allows to integrate different unit operations into a single chip, and consequently to evaluate the effect of each stage on the overall process efficiency. This paper describes the development of a diffusion-based microfluidic device for the rapid screening of continuous chemical lysis conditions. The release of a recombinant green fluorescent protein (GFP) expressed in Escherichia coli (E. coli) was used as model system due to the simple evaluation of cell growth and product concentration by fluorescence. The concept can be further applied to any biopharmaceutical production platform. The microfluidic device was successfully used to test the lytic effect of both enzymatic and chemical lysis solutions, with lysis efficiency of about 60% and close to 100%, respectively, achieved. The microfluidic technology also demonstrated the ability to detect potential process issues, such as the increased viscosity related with the rapid release of genomic material, that can arise for specific lysis conditions and hinder the performance of a bioprocess. Finally, given the continuous operation of the lysis chip, the microfluidic technology has the potential to be integrated with other microfluidic modules in order to model a fully continuous biomanufacturing process on a chip.
在过去的十年中，在上游生产工艺方面取得了重大进展，将当前生物制药制造平台的主要瓶颈转移到下游加工。纯化过程中的挑战包括降低生产成本、开发稳健高效的纯化工艺以及整合上下游工艺。微流控技术最近已成为加速生物工艺设计的有效工具，因为可以在短时间内评估大量变量，使用减少的体积和人力。它们的模块化还允许将不同的单元操作集成到单个芯片中，从而评估每个阶段对整体工艺效率的影响。本文介绍了一种基于扩散的微流控装置的开发，用于快速筛选连续化学裂解条件。由于通过荧光可以简单地评估细胞生长和产物浓度，因此使用在大肠杆菌 (E. coli) 中表达的重组绿色荧光蛋白 (GFP) 的释放作为模型系统。该概念可以进一步应用于任何生物制药生产平台。微流控装置成功地用于测试酶和化学裂解溶液的裂解效果，分别达到约 60%和接近 100%的裂解效率。微流控技术还展示了检测潜在工艺问题的能力，例如与基因组物质快速释放相关的粘度增加，这可能会出现在特定的裂解条件下，并阻碍生物工艺的性能。最后，鉴于裂解芯片的连续运行，微流控技术有可能与其他微流控模块集成，以便在芯片上模拟完全连续的生物制造过程。
