Romiplostim下游工艺开发中的质量设计

Q2 Biochemistry, Genetics and Molecular Biology
Saeedeh Pouri, Fatemeh Torkashvand, Hooman Aghamirza Moghim Aliabadi, Pezhman Fard-Esfahani, Majid Golkar, Behrouz Vaziri
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引用次数: 0

摘要

背景:在大肠杆菌中以包涵体(IBs)表达的治疗性重组蛋白的下游加工是相当具有挑战性的。本研究旨在利用质量设计方法开发结构复杂的治疗性Fc-Peptide融合蛋白romiplostim的多步下游工艺。方法:为了开发成功的下游工艺,采用风险分析和实验设计来表征最关键的质量属性(cqa)以及工艺参数对这些质量属性的影响。结果:以溶解度为重点,通过3个参数的实验设计对IBs的增溶效果进行了优化,使目标蛋白的增溶效果提高了75%以上。在阴离子交换层析中,样品的pH值被鉴定为CQA,这可能对实现>85%的宿主细胞蛋白质去除和>90%的宿主细胞DNA还原有影响。在再折叠步骤中,筛选了工艺参数。通过Box-Behnken分析进一步优化确定为CPPs的半胱氨酸/半胱氨酸比例、pH和孵育时间,目标蛋白的再折叠率>85%。绘制了HIC进一步提纯步骤的设计空间,重点是高分子量杂质。经凝胶过滤抛光后,最终产品的生物活性在以romiplostim和Nplate®作为参比产品的两组之间无统计学差异。结论:本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的收率和质量及其下游工艺参数之间的联系。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Quality by Design in Downstream Process Development of Romiplostim

Quality by Design in Downstream Process Development of Romiplostim

Quality by Design in Downstream Process Development of Romiplostim

Quality by Design in Downstream Process Development of Romiplostim

Background: 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: 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: 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.

Conclusions: 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.

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来源期刊
Iranian Biomedical Journal
Iranian Biomedical Journal Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
3.20
自引率
0.00%
发文量
42
审稿时长
8 weeks
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