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Background: Downstream processing of therapeutic recombinant proteins expressed as inclusion bodies in Escherichia coli is quite challenging. This study aimed to use the Quality by Design (QbD) approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim, in E. coli. Methods: Romiplostim was expressed in E. coli as IBs. For development of a successful downstream process, risk analysis, and experimental designs used to characterize the most critical quality attributes and effects of process parameters on them. Results: The solubilization of IBs were optimized by DoE on three parameters with focus on solubility yield. Anion exchange chromatography in flowthrough mode was used and the pH load optimized with focus on removal of HCD and HCPs. In refolding step, process parameters were screened by a Plackett-Burman design. The design space for further purification step by hydrophobic interaction chromatography was mapped with focus on high molecular weight impurities. After polishing by gel filtration, the final product's biological activity was determined. Conclusions: This study provides a detailed and comprehensive model of mapping the design space to define and predict the relationship of different downstream process parameters to yield and quality of romiplostim.
Type of Study: Full Length | Subject: Pharmaceutical Biotechnology

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