Effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein release in Chinese hamster ovary cells.
Effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein release in Chinese hamster ovary cells.
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DOI：
10.1002/btpr.2455
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发表时间：
2017-05
影响因子：
2.9
通讯作者：
Bracewell DG
中科院分区：
文献类型：
--
作者：
Migani D;Smales CM;Bracewell DG
重组人酸性α葡萄糖苷酶（GAA）是用于治疗庞贝氏症的治疗酶，庞贝氏症是一种罕见的遗传性疾病，其特征是细胞溶酶体中GAA缺乏（拉本等人，Curr Mol Med.2002; 2：145-166）。GAA的生产工艺可能具有挑战性，部分原因是蛋白酶降解。本研究的总体目标是了解GAA过表达对细胞溶酶体表型和宿主细胞蛋白（HCP）释放的影响，以及对蛋白酶水平和易于生产的任何结果。为此，我们首先生成了人重组GAA生产稳定的CHO细胞系，并设计了捕获色谱步骤阴离子交换（IEX）。然后，我们通过透射电子显微镜（TEM）收集细胞溶酶体的图像，并将所得数据与来自空CHO细胞系的数据进行比较。TEM成像显示GAA细胞系中72%的所有溶酶体充血，表明广泛的细胞应激;相比之下，无效CHO中仅8%的溶酶体具有相似的表型。此外，细胞系（GAA、mAb和mAb）捕获洗脱液之间HCP谱的比较表明，虽然释放的大多数HCP在它们之间是常见的，但有些是GAA生产者特有的，这表明GAA过表达引起的细胞应激对HCP释放具有分子特异性影响。通过酶谱进行的蛋白酶分析显示，捕获步骤后蛋白水解活性总体降低，但也显示存在约80 KDa的共洗脱蛋白酶，MS分析鉴定为二肽基肽酶3和脯氨酰内肽酶。© 2017美国化学工程师学会生物技术。程序：33：666-676，2017
Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterized by GAA deficiency in the cell lysosomes (Raben et al., Curr Mol Med. 2002; 2:145–166). The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this study was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO cell line and designed the capture chromatographic step anion exchange (IEX). We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison only 8% of lysosomes in the null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines (GAA, mAb, and Null) capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co‐eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:666–676, 2017