Comparative Approaches for Quantification of Product Yield in the Production of Recombinant Green Fluorescent Protein...

Process Analytical Technologies (PAT) are critical for efficient and automated bioprocessing; moreover, sensor and assay-driven automation will be necessary for the realization of Industry 4.0. Herein, we demonstrate methods for analyzing product yield from a pilot-scale bioreactor (300L) and conduct cross-method comparisons. We used a system of recombinant green fluorescent protein (GFPuv) expressed in Escherichia coli (E. coli), which is a simple, cost-effective model for evaluation at both laboratory and pilot scales. By comparing inline bioreactor measurements, plate reader assays, and a novel image analysis pipeline, we identify optimal harvest timelines and demonstrate the strengths and limitations of each technique. Results indicate peak cell density and GFPuv expression between 12.5h and 18h post-inoculation, with declining viability thereafter. Comparisons across techniques suggest that imaging methods may be more effective in capturing adverse outcomes, such as increased membrane permeability or cell death, which typically occur at later stages of operation. This integrated analysis offers actionable insights for optimizing biomanufacturing workflows and advances the development of scalable PAT applications, bridging the gap between laboratory research and industrial implementation.