重组人促红细胞生成素(rhEPO)是一种糖蛋白,可作为调节红细胞生成的主要激素,用于治疗由慢性肾脏疾病或化疗引起的贫血,具有三个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,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。在这项研究中,使用液相色谱-质谱(LC-MS)方法结合电子激活解离(EAD)技术对一次注射中的rhEPON-糖基化和O-糖基化进行定性和定量表征。EAD的使用不仅产生了糖肽的丰富的MS/MS片段离子并改善了MS/MS序列覆盖率,而且还保留了MS/MS片段离子中的聚糖结构以及聚糖与肽之间的糖苷键的完整性。成功鉴定了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位点的糖基化率为82.7%,100%,分别为100%,可以在糖肽水平鉴定15、10和12种不同的N-聚糖。唾液酸的总平均数,N-羟基乙酰神经氨酸,在完整的蛋白质水平上,唾液酸的O-乙酰化分别为7.28、4.21和0.66,分别。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%,分别,并鉴定了两种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片段的工作流程可以有效地用于表征rhEPO样品的糖基化分析和批次间一致性分析。有助于合理指导rhEPO生产工艺的优化。
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.