At-line N-linked glycan profiling for monoclonal antibodies with advanced sample preparation and high-performance liquid chromatography
At-line N-linked glycan profiling for monoclonal antibodies with advanced sample preparation and high-performance liquid chromatography
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DOI：
10.1016/j.jbiosc.2020.04.009
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发表时间：
2020-09-01
影响因子：
2.8
通讯作者：
Yoon, Seongkyu
中科院分区：
文献类型：
--
作者：
Sha, Sha;Handelman, Garry;Yoon, Seongkyu
N-糖基化是一种翻译后修饰，发生在许多蛋白质的生物合成过程中。不同糖链在蛋白质上的分布是包括单抗(MAbs)在内的一些重组生物药物蛋白质的关键质量属性。分析葡聚糖的方法应该是健壮、快速和敏感的。将多聚糖从蛋白质中分离出来，并在多聚糖上贴上标签是多聚糖图谱最常用的技术。目前，现有的样品制备方法复杂、耗时和昂贵，这限制了葡聚糖图谱方法的广泛应用。作为使用的另一个障碍，通常需要昂贵的超高压液相色谱(UHPLC)系统来进行剖面分析。在这篇文章中，一个低成本和易于使用的样品制备工作流程与标准的高效液相色谱(HPLC)系统相结合，以获得与UHPLC相当的结果。在保持稳健的分析性能的同时，大大减少了方案中所需的步骤和时间，以及与样品制备相关的成本。我们描述了作为校准标准的人血清免疫球蛋白糖链文库的创建和验证，并成功地分析了各种单抗的糖型。(C)2020年，日本生物技术学会。版权所有。
N-linked glycosylation is a post-translational modification that occurs on many proteins during biosynthesis. The profile of different glycans on the protein is a critical quality attribute of some recombinant biopharmaceutical proteins including monoclonal antibodies (mAbs). Methods for profiling glycan should be robust, fast, and sensitive. Isolating glycans from proteins and tagging a label on glycans is the most commonly used technique for glycan profiling. Currently, existing protocols for sample preparation can be complicated, time-consuming, and expensive, which can limit the wide adaptation of glycan profiling methods. As a further barrier to use, an expensive ultra-high-pressure liquid chromatography (UHPLC) system is frequently required for the profile. In this article, a low cost and easily-used workflow of sample preparation is coupled with a standard high-performance liquid chromatography (HPLC) systemto achieve comparable results to UHPLC. The number of steps required in the protocol and the time, as well as the cost associated with the sample preparation, is significantly reduced, while maintaining robust analytical performance. We describe the creation and validation of a human serum IgG glycan library to be used as the calibration standard, and successful profiling of glycoforms from a variety of mAbs. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.