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Abstract：
Process Analytical Technologies (PAT) used to monitor and control manufacturing processes are crucial for efficient and automated bioprocessing, which is in congruence with lights-off-manufacturing and Industry 4.0 initiatives. As biomanufacturing seeks to realize more high-throughput and automated operation, an increasing need for multimodal analysis of process metrics becomes essential. Herein, we detail a series of methods for analyzing product yield from a bioreactor and how to conduct cross-method comparisons. We employ a model system of Escherichia coli (E. coli) expression of green fluorescent protein (GFP), which is a simple, cost effective model for students and educators to replicate at different scales. GFP is an ideal analytical marker as it is easy to visualize due to its fluorescence which indicates cellular protein expression, cell localization and physiological changes of the cell population. In this study, samples from a 300 L bioreactor with GFP-expressing E. coli are analyzed to improve product yield and bioprocessing efficiency. Utilizing a fed-batch process for enhanced cell density and product titer, this bioreactor runs on a 24-hour schedule from inoculation to GFP induction and final harvest. To reliably quantify relative GFP expression and E. coli proliferation, we provide simple protocols and example results for comparing three different analytical methods: (1) in-line bioreactor measurements, (2) plate reader assays, and (3) microscopy. The GFP and cell density results follow similar trends based on the various inline and offline analytical methods and show a peak of GFP expression and cell density between 12.5 and 18 hours post inoculation.
摘要：
用于监测和控制制造过程的过程分析技术（PAT）对于高效和自动化的生物处理至关重要。这与关灯制造和工业4.0计划是一致的。随着生物制造寻求实现更多的高通量和自动化操作，对过程度量的多模态分析的需求日益增加。在这里，我们详细介绍了一系列分析生物反应器产品产量的方法，以及如何进行交叉方法比较。我们采用大肠杆菌（E.coli）表达绿色荧光蛋白（GFP）的模型系统，这是一个简单的，学生和教育工作者在不同尺度上复制的成本效益模型。GFP是一种理想的分析标记，因为它很容易可视化，因为它的荧光表明细胞蛋白表达。细胞定位和细胞群的生理变化。在这项研究中,分析来自具有表达GFP的大肠杆菌的300L生物反应器的样品以提高产物产量和生物加工效率。利用补料分批工艺提高细胞密度和产品滴度，该生物反应器以从接种到GFP诱导和最终收获的24小时时间表运行。为了可靠地定量相对GFP表达和大肠杆菌增殖，我们提供了简单的协议和示例结果，用于比较三种不同的分析方法：（1）在线生物反应器测量，（2）读板器测定，（3）显微镜。基于各种在线和离线分析方法,GFP和细胞密度结果遵循类似的趋势,并且在接种后12.5和18小时之间显示GFP表达和细胞密度的峰值。
