A high-resolution measurement of nucleotide sugars by using ion-pair reverse chromatography and tandem columns
A high-resolution measurement of nucleotide sugars by using ion-pair reverse chromatography and tandem columns
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DOI：
10.1007/s00216-020-02608-6
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发表时间：
2020-06-01
影响因子：
4.3
通讯作者：
Yoon, Seongkyu
中科院分区：
文献类型：
--
作者：
Sha, Sha;Handelman, Garry;Yoon, Seongkyu
N-连接糖基化是将糖从糖基供体转移到蛋白质或脂质的细胞过程。通过培养哺乳动物细胞如中国仓鼠卵巢（CHO）细胞广泛产生的生物药物产品通常在生物合成期间糖基化。对于某些生物制剂，N-连接聚糖是药物的关键质量属性。核苷酸糖是聚糖供体并且影响细胞内糖基化过程。在目前的分析方法中，由于核苷酸糖异构体如UDP葡萄糖和UDP半乳糖的结构相似性，它们的稳健分离仍然是一个挑战。在这项研究中，我们开发了一种策略，以解决主要的核苷酸糖，包括具有挑战性的异构体的基础上使用离子对反相（IP-RP）色谱分离。该策略应用核壳柱并串联连接多个柱以增加分离能力，并最终实现细胞提取物中核苷酸糖的高分辨率检测。本文对IP-RP方法中的关键参数，包括温度、移动的相和流速进行了系统的评价，并提出了IP-RP色谱行为的理论机理。
N-Linked glycosylation is a cellular process transferring sugars from glycosyl donors to proteins or lipids. Biopharmaceutical products widely produced by culturing mammalian cells such as Chinese hamster ovary (CHO) cells are typically glycosylated during biosynthesis. For some biologics, the N-linked glycan is a critical quality attribute of the drugs. Nucleotide sugars are the glycan donors and impact the intracellular glycosylation process. In current analytical methods, robust separation of nucleotide sugar isomers such as UDP glucose and UDP galactose remains a challenge because of their structural similarity. In this study, we developed a strategy to resolve the separation of major nucleotide sugars including challenging isomers based on the use of ion-pair reverse phase (IP-RP) chromatography. The strategy applies core-shell columns and connects multiple columns in tandem to increase separation power and ultimately enables high-resolution detection of nucleotide sugars from cell extracts. The key parameters in the IP-RP method, including temperature, mobile phase, and flow rates, have been systematically evaluated in this work and the theoretical mechanisms of the chromatographic behavior were proposed.Graphical abstract