Comparative Approaches for Quantification of Product Yield in a Model Recombinant Green Fluorescent Protein Expressed in E. coli.

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.

Advances and opportunities in process analytical technologies for viral vector manufacturing.

Viral vectors are an emerging, exciting class of biologics whose application in vaccines, oncology, and gene therapy has grown exponentially in recent years. Following first regulatory approval, this class of therapeutics has been vigorously pursued to treat monogenic disorders including orphan diseases, entering hundreds of new products into pipelines. Viral vector manufacturing supporting clinical efforts has spurred the introduction of a broad swath of analytical techniques dedicated to assessing the diverse and evolving panel of Critical Quality Attributes (CQAs) of these products. Herein, we provide an overview of the current state of analytics enabling measurement of CQAs such as capsid and vector identities, product titer, transduction efficiency, impurity clearance etc. We highlight orthogonal methods and discuss the advantages and limitations of these techniques while evaluating their adaptation as process analytical technologies. Finally, we identify gaps and propose opportunities in enabling existing technologies for real-time monitoring from hardware, software, and data analysis viewpoints for technology development within viral vector biomanufacturing.