Degradation of an Fc-Fusion Recombinant Protein by Host Cell Proteases: Identification of a CHO Cathepsin D Protease
Degradation of an Fc-Fusion Recombinant Protein by Host Cell Proteases: Identification of a CHO Cathepsin D Protease
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DOI：
10.1002/bit.22494
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发表时间：
2009-12-15
影响因子：
3.8
通讯作者：
Mitchell-Logean, Christine
中科院分区：
文献类型：
--
作者：
Robert, Flavie;Bierau, Horst;Mitchell-Logean, Christine
在重组 Fc 融合蛋白生产过程中鉴定出宿主细胞相关的蛋白水解活性。本报告描述了通过结合细胞培养研究和专用分析工具来表征和分离导致这种降解的酶的策略。对捕获后材料中生成的剪切片段进行分离和测序后，在不同的培养条件下追踪酶活性，从而将活的 CHO 细胞鉴定为蛋白酶的来源。抑制剂和 pH 值筛选表明该酶属于天冬氨酸蛋白酶家族，并且在酸性 pH 值下具有较好的活性。该蛋白酶通过胃酶抑素 A 柱纯化而分离，并被鉴定为与组织蛋白酶 D 相关的蛋白质。另一种受 EDTA 抑制的金属蛋白酶经鉴定在中性 pH 下具有最佳活性。这项研究是细胞培养参数如何强烈影响治疗性重组分子的质量和稳定性的一个例子。生物技术。生物工程。 2009年； 104：1132-1141。 (C) 2009 年 Wiley 期刊公司。
A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters. Biotechnol. Bioeng. 2009; 104: 1132-1141. (C) 2009 Wiley Periodicals, Inc.