Quality by Design in Downstream Process Development of Romiplostim

BACKGROUND: Downstream processing of therapeutic recombinant proteins expressed as the IBs in E. coli is quite challenging. This study aimed to use the QbD approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim. METHODS: For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most CQAs and effects of process parameters on these quality attributes. RESULTS: The solubilization of IBs was optimized by DoE on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in AEX that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on HMW impurities. After polishing by gel filtration, the final product's biological activity showed no statistically significant differences among the groups received romiplostim and Nplate(®), as the reference product. CONCLUSION: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters.