Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns
Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns
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DOI：
10.1016/j.jchromb.2016.02.006
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发表时间：
2016-10-01
影响因子：
3
通讯作者：
Guttman, Andras
中科院分区：
文献类型：
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作者：
Szigeti, Marton;Bondar, Judit;Guttman, Andras
糖蛋白生物治疗药物的N-糖基化分析是生物制药行业产品开发每个阶段的重要步骤。例如，在克隆选择过程中，应该从有限数量的样品中快速分析数百个克隆。另一方面，疾病相关糖基化改变的鉴定可以作为生物医学领域中各种病理状况的早期指标（糖生物标志物）。因此，对于用于N-糖基化分析的快速且易于自动化的样品制备方法的需求日益增长。在本文中，我们报告的设计和实施的固定化重组谷胱甘肽-S-转移酶（GST）标记的PNGase F酶微柱快速，有效地去除N-连接的糖蛋白的碳水化合物。消化速度和效率进行了比较，以传统的基于解决方案。使用PNGase F功能化微柱导致在10分钟内从所有主要N-连接糖蛋白类型中有效去除N-聚糖：（i）中性（IgG），（ii）高度唾液酸化（胎球蛋白）和（iii）高甘露糖（核糖核酸酶B）含糖蛋白标准品的碳水化合物。该方法可以很容易地应用于自动化样品制备系统，如液体处理机器人。(C)2016爱思唯尔B. V.保留所有权利。
N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots. (C) 2016 Elsevier B.V. All rights reserved.