Biomedical Engineering and Biotechnology, University of Massachusetts Lowell, Lowell, MA, 01854, USA.
Biomedical & Nutritional Sciences, University of Massachusetts Lowell, Lowell, MA, 01854, USA.
Anal Bioanal Chem. 2020 Jun;412(15):3683-3693. doi: 10.1007/s00216-020-02608-6. Epub 2020 Apr 16.
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.
N-连接糖基化是一种将糖从糖供体转移到蛋白质或脂质的细胞过程。生物制药产品通常在生物合成过程中发生糖基化，这些产品是通过培养哺乳动物细胞（如中国仓鼠卵巢（CHO）细胞）广泛生产的。对于一些生物制剂而言，N-连接聚糖是药物的关键质量属性。核苷酸糖是糖供体，并影响细胞内糖基化过程。在当前的分析方法中，由于结构相似，仍难以分离 UDP 葡萄糖和 UDP 半乳糖等核苷酸糖异构体。在这项研究中，我们开发了一种策略，基于离子对反相（IP-RP）色谱法来解决包括具有挑战性的异构体在内的主要核苷酸糖的分离问题。该策略应用核壳柱并串联连接多个柱以增加分离能力，最终能够从细胞提取物中进行高分辨率检测核苷酸糖。本工作系统地评估了 IP-RP 方法中的关键参数，包括温度、流动相和流速，并提出了色谱行为的理论机制。
