Li Xiang, Wang Wentao, Luo Ji, Guo Lihai, Zhou Yong, Li Yan, Chen Hong-Xu
Division of Recombinant Biological Products, National Institutes for Food and Drug Control (NIFDC), Beijing, 100501, People's Republic of China.
SCIEX, Beijing, 100015, People's Republic of China.
Appl Biochem Biotechnol. 2024 Nov;196(11):8317-8331. doi: 10.1007/s12010-024-04954-y. Epub 2024 May 14.
Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy, which has three N-glycosylation sites and one O-glycosylation site. It contains a variety of different glycosylation modifications, such as sialyation, O-acetylation on sialic acids, etc., which causes a big challenge for the glycosylation analysis of rhEPO. In this study, a liquid chromatography-mass spectrometry (LC-MS) method combined with electron-activated dissociation (EAD) technology was used in qualitative and quantitative characterization of rhEPO N-glycosylation and O-glycosylation in just one injection. The usage of EAD not only generated abundant MS/MS fragment ions of glycopeptides and improved the MS/MS sequence coverage but also preserved the glycan structures in the MS/MS fragment ions and the integrity of the glycosidic bond between the glycans and peptides. Three N-glycosylation sites (N24, N38, and N83) and one O-glycosylation site (S126) of rhEPO samples were successfully identified. Among them, the glycosylation ratios of N24, N38, and N83 sites were 82.7%, 100%, and 100% respectively, and 15, 10, and 12 different N-glycans could be identified at the glycopeptide level. The total average number of sialic acids, N-hydroxyacetylneuraminoic acid, and O-acetylation on sialic acid were 7.28, 4.21, and 0.66 at the intact protein level, respectively. For O-glycosylation site S126, O-glycosylation ratios analyzed at the intact protein level and the glycopeptide level were 80.2% and 80.3%, respectively, and two O-glycans were identified, including Core1_S1 and Core1_S2. This study also compared the difference of the glycans and their relative contents in batch-to-batch rhEPO samples. The results proved that the workflow using EAD fragmentation in LC-MS method could be effectively applied for characterizing the glycosylation analysis of rhEPO samples and batch-to-batch consistency analysis, which would help to reasonably guide the optimization of rhEPO production process.
重组人促红细胞生成素（rhEPO）是一种糖蛋白，是参与调节红细胞生成以治疗慢性肾病或化疗所致贫血的主要激素，它有三个N-糖基化位点和一个O-糖基化位点。它含有多种不同的糖基化修饰，如唾液酸化、唾液酸上的O-乙酰化等，这给rhEPO的糖基化分析带来了巨大挑战。在本研究中，一种液相色谱-质谱（LC-MS）方法结合电子激活解离（EAD）技术被用于在一次进样中对rhEPO的N-糖基化和O-糖基化进行定性和定量表征。EAD的使用不仅产生了丰富的糖肽MS/MS碎片离子并提高了MS/MS序列覆盖率，还在MS/MS碎片离子中保留了聚糖结构以及聚糖与肽之间糖苷键的完整性。成功鉴定出rhEPO样品的三个N-糖基化位点（N24、N38和N83）和一个O-糖基化位点（S126）。其中，N24、N38和N83位点的糖基化比率分别为82.7%、100%和100%，在糖肽水平可鉴定出15种、10种和12种不同的N-聚糖。在完整蛋白水平，唾液酸、N-羟乙酰神经氨酸和唾液酸上O-乙酰化的总平均数分别为7.28、4.21和0.66。对于O-糖基化位点S126，在完整蛋白水平和糖肽水平分析的O-糖基化比率分别为80.2%和80.3%，鉴定出两种O-聚糖，包括Core1_S1和Core1_S2。本研究还比较了批次间rhEPO样品中聚糖及其相对含量的差异。结果证明，在LC-MS方法中使用EAD碎裂的工作流程可有效应用于rhEPO样品的糖基化分析表征和批次间一致性分析，这将有助于合理指导rhEPO生产工艺的优化。
