Abstract
Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding.
Highlights
To date, approximately 20–30% of all approved biopharmaceuticals are produced in microbial hosts [1,2]
In order to determine whether at-line Reversed phase liquid chromatography (RPLC) met the criteria of an in-process monitoring tool, DTT concentration and the solubilization time of HRP inclusion body (IB) were varied in a design of experiments (DoEs)
We developed an at-line RPLC method to monitor the target protein concentration during the solubilization unit operation of IB processing
Summary
Approximately 20–30% of all approved biopharmaceuticals are produced in microbial hosts [1,2]. Better known as inclusion bodies (IBs), produced by the gram-negative bacterium Escherichia coli, present a dominant fraction of the microbial production segment [3]. This is mainly because cultivation with E. coli can be carried out at very low costs in short fermentation run-times and high target protein concentrations at a high purity can be achieved [4,5,6]. Solubilization and refolding strategies are commonly developed empirically with protocols being highly dependent on the target protein [9,10]. For instance, affects the required molarity of the chaotropic agent and pH in solubilization and refolding [11]
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