在大肠杆菌中表达为IB的治疗性重组蛋白的下游加工是相当具有挑战性的。本研究旨在使用QbD方法开发结构复杂的治疗性Fc-肽融合蛋白的多步骤下游过程,romiplostim.
为了开发成功的下游工艺,风险分析,和实验设计用于表征大多数CQAs以及工艺参数对它们的影响。
通过DoE在三个参数上优化了IBs的增溶,重点是溶解度产率,并且>75%的靶蛋白被溶解。样品的pH在阴离子交换色谱中被鉴定为(CQA),其可能对实现>85%宿主细胞蛋白质去除和>90%宿主细胞DNA减少具有影响。在重新折叠步骤中,对工艺参数进行了筛选。半胱氨酸/半胱氨酸比例,pH值,和孵育时间被确定为CPPs进一步优化使用Box-Behnken分析,并且>85%的靶蛋白被重折叠。通过HIC的进一步纯化步骤的设计空间被映射为聚焦于高分子量杂质。通过凝胶过滤抛光后,在接受romiplostim和Nplate作为参比产品的组中,最终产品的生物活性无统计学显著性差异.
这项研究提出了一个精确而详尽的模型,用于绘制设计空间,以描述和预测romiplostim的产量和质量与其下游工艺参数之间的联系。
Background: Downstream processing of therapeutic recombinant proteins expressed as the inclusion bodies (IBs) in E. coli is quite challenging. This study aimed to use the quality by design 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 critical quality attributes (CQAs) and effects of process parameters on these quality attributes.
Results: The solubilization of IBs was optimized by design of experiment 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 anion exchange chromatography 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 high molecular weight 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.