用聚亚胺接枝阴离子交换层析树脂选择性去除密切相关的剪切蛋白杂质

Ganesh T. Sivanathan, H. Mallubhotla, Satyanarayana V. Suggala
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引用次数: 1

摘要

摘要蛋白水解降解是一个严重的问题,使重组治疗蛋白生产过程中的下游加工复杂化。它会导致产品产量下降,生物活性降低,产品质量次优。蛋白水解降解或蛋白截断在不同的表达宿主中都有发生,这主要归因于宿主细胞释放的蛋白酶的活性。由于这些剪切蛋白除了效力外还会影响药代动力学和免疫原性,因此需要对其进行适当控制,以确保产品质量和患者安全的一致性。本研究开发了一种从完整蛋白中选择性去除剪切蛋白的色谱步骤。对聚乙亚胺接枝阴离子交换树脂(PolyQUAT和PolyPEI)进行了评价,并与传统的大孔阴离子交换树脂和触角阴离子交换树脂进行了比较。通过等压保留实验确定了保留因子k′,并通过经典的化学计量位移模型确定了电荷因子Z。PolyQUAT树脂对密切相关的剪切蛋白具有很高的选择性。通过基于实验设计(design - of - experiments, DoE)的工艺优化,为PolyQUAT色谱建立了稳健的设计空间。结果表明,产品回收率可达63%,纯度可达99.0%。大约,宿主细胞蛋白的一对数清除率和宿主细胞DNA的两对数清除率也得到了。新开发的PolyQUAT工艺与现有工艺进行了比较,结果表明,在工艺步骤数量、工艺时间、工艺收率和产品质量方面,PolyQUAT工艺具有优越性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Selective removal of closely related clipped protein impurities using poly(ethylenimine)- grafted anion-exchange chromatography resin
Abstract Proteolytic degradation is a serious problem that complicates downstream processing during production of recombinant therapeutic proteins. It can lead to decreased product yield, diminished biological activity, and suboptimal product quality. Proteolytic degradation or protein truncation is observed in various expression hosts and is mostly attributed to the activity of proteases released by host cells. Since these clipped proteins can impact pharmacokinetics and immunogenicity in addition to potency, they need to be appropriately controlled to ensure consistency of product quality and patient safety. A chromatography step for the selective removal of clipped proteins from an intact protein was developed in this study. Poly(ethylenimine)-grafted anion- exchange resins (PolyQUAT and PolyPEI) were evaluated and compared to traditional macroporous anion-exchange and tentacled anion-exchange resins. Isocratic retention experiments were conducted to determine the retention factors (k′) and charge factors (Z) were determined through the classical stoichiometric displacement model. High selectivity in separation of closely related clipped proteins was obtained with the PolyQUAT resin. A robust design space was established for the PolyQUAT chromatography through Design-Of-Experiments (DoE) based process optimization. Results showed a product recovery of up to 63% with purity levels >99.0%. Approximately, one-log clearance of host cell protein and two-logs clearance of host cell DNA were also obtained. The newly developed PolyQUAT process was compared with an existing process and shown to be superior with respect to the number of process steps, process time, process yield, and product quality.
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