title: Application of a High-Throughput Microfermentation System for the Screening of Protein Refolding
Creators: name: Rubus, Viktor
Publisher: name: TU Wien
Year: 2021
Subjects: subject: Protein Refolding
subject: Solubilization
subject: Inclusion Body Processing
subject: High-Throughput
subject: Green Fluorescent Protein
subject: Horseradish Peroxidase
subject: Batch Refolding
subject: Fed-Batch Refolding
subject: At-Line RPLC

description: Despite of manifold advantages of recombinant protein production in form of inclusion bodies (IBs), the two additionally required unit operations for IB processing, solubilization and refolding, present a bottleneck in the downstream process (DSP) due to their high complexity and low yields. To date, these unit operations are highly empirical and dependent on the protein of interest (POI), opposing the Quality-by-Design (QbD) paradigm. In this thesis, process analytical tools (PATs) for the in-process monitoring (IPM) and in-process control (IPC) of both unit operation steps are presented. Furthermore, a novel technique for the screening of protein refolding was developed that enables the control of critical process parameters (CPPs), which cannot be controlled by prevalent techniques.The current analytical method of choice for the analysis of the solubilization process is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) but a low selectivity, a low accuracy and a time-consuming application are making it unsuitable for the IPM and IPC of the solubilization process. An at-line reversed-phase liquid chromatography (RPLC) method for the monitoring of the target protein concentration during solubilization is presented. Compared to SDS-PAGE, the method exhibits a considerably shorter analysis time (8.1 min), a higher accuracy and meets all crucial criteria for the application as an IPM tool.Versatile high throughput small-scale techniques for the screening of protein refolding parameters using a design of experiments (DoE) approach were reported recently. However, these techniques are incapable of controlling the CPPs refolding temperature and oxygen input. Additionally, the associated analytical tools for the characterization of the refolding process and the capabilities for the realization of fed-batch refolding are severely limited. To solve these problems, the feasibility of the application of the BioLector Pro microfermentation system for the screening of protein refolding parameters was assessed for two proteins: green fluorescent protein (GFP) and horseradish peroxidase (HRP). The device has capabilities for the in-line monitoring of pH, dO2 and fluorescence, while its microfluidic technology enables continuous feeding and pH regulation. The comparison to a conventional screening approach revealed that the screening outcome is fundamentally different, but the approach utilizing the BioLector system is hypothesized to be more accurate and useful for screening experiments. Furthermore, the in line analytics exhibit profound problems, and it was found that the microfluidic technology is not functional at an ambient temperature of 4 °C. Based on the findings of this feasibility study, six proposals are formulated with the goal to improve the application of the BioLector Pro for the screening of protein refolding.