不同肿瘤坏死因子受体-Fc融合蛋白N-糖基化修饰糖链的初步分析

N-Glycosylation on different tumor necrosis factor receptor-Fc fusion protein: a preliminary analysis

目的建立基于LC-MS的N-糖基化修饰糖链的分析方法,并比较不同肿瘤坏死因子受体(TNFR)-Fc融合蛋白N-糖基化修饰差异。方法用肽-N-糖苷酶F释放糖蛋白中的修饰糖链,有机溶剂沉淀蛋白后,利用还原胺化反应将2-氨基苯甲酰胺标记至糖链上,并通过亲水作用萃取柱除去过量标记试剂,所得糖链采用ZIC-HILIC亲水作用色谱柱结合离子阱质谱进行分析。以市售TNFR-Fc融合蛋白为标准品,优化影响标记效率的各种因素后,对A和B两种TNFR-Fc融合蛋白制品的N-糖基化修饰糖链进行了分析和比较。结果 TNFR-Fc融合蛋白A和B中的修饰糖链主要含有岩藻糖、末端唾液酸、末端半乳糖、末端N-乙酰葡糖胺等糖型,且末端唾液酸含量相近,但融合蛋白A的岩藻糖和末端N-乙酰基葡萄糖的含量显著高于融合蛋白B,而融合蛋白B末端半乳糖含量高于融合蛋白A。结论成功建立了基于LC-MS的N-糖基化修饰糖链的分析方法,该方法可有效分析糖蛋白中的修饰糖链类型和含量比例,对于详细表征糖蛋白药物结构及性质具有参考价值,并为进一步研究糖基化对糖蛋白药物药理活性以及药代动力学研究奠定了良好基础。

Objective To develop a method for N-glycans analysis based on LC-MS,and compare the N-glycans of different tumor necrosis factor receptor（TNFR）-Fc fusion proteins.Methods Glycans were released from TNFR-Fc fusion glycoproteins by PNGase F.Proteins were removed by precipitation with organic solvent and the oligosaccharides were evaporated to dryness and then labeled with 2-aminobenzamide.After excess 2-aminobenzamide was removed from the labeling samples by hydrophilic solid phase extraction column,the glycans were separated on ZIC-HILIC columns and then analyzed with linear ion trap mass spectrometer.The Nglycans analysis method was developed with the commercially available TNFR-Fc fusion glycoprotein,the factors affecting labeling and separating efficiency were optimized.And glycans samples from two different TNFR-Fc products,A and B were analyzed with the method.Results The results showed that glycans from TNFR-Fc products A and B were similar,especially the sialic acid content,but the fucose and terminal N-acetyl glucosamine contents in product A were significantly higher than that in product B;while the glycans with terminal galactose in product B were higher than those in product A.Conclusion The N-glycans analysis method based on LC-MS is developed and it is effective to analyze the glycans type and contents ratio of samples released from glycoproteins.This method could provide structural,functional and pharmaceutical information of glycoproteins,and may be helpful for further pharmacological,especially for pharmacokinetic studies.