甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的N-糖基化表征

采用甲胺化衍生结合基于硅氢化物固定相的正相色谱（SiH-NPC）分析单抗的N-糖基化。样品经酶切、甲胺化衍生、纯化后由液相色谱-质谱进行分析。结果表明，相较于亲水相互作用色谱（HILIC），SiH-NPC分离机制不同，使用常规的无盐流动相即可实现高分离度，避免污染质谱，色谱柱结构稳定，使用寿命长，更适合快速分析。结合唾液酸衍生方法，SiH-NPC在液相色谱-质谱联用鉴定酸性糖和糖异构体方面呈现显著优势，在生物制药行业中具有重要的应用潜力。

Methylamidation and normal phase chromatography with silica-hydride-based stationary phase for N-glycosylation characterization of monoclonal antibody drugs

Monoclonal antibodies (mAbs) are efficacious therapeutic agents against various complex diseases. N-glycosylation characterization of mAbs is based primarily on fluorescence derivatization coupled with hydrophilic interaction liquid chromatography (HILIC). However, this method has some drawbacks. Presently, the N-glycosylation of mAbs was analyzed by methylamidation and silica-hydride-based normal phase chromatography (SiH-NPC). Samples were analyzed by liquid chromatography-mass spectrometry after enzymatic cleavage, methylamidation, and purification. SiH-NPC had some advantages compared to HILIC. It offered a novel separation mechanism that allowed for high resolution using salt-free mobile phases, avoiding contamination of the mass spectrometer. SiH-NPC may be applicable for rapid analysis. Its structure was more stable than that of HILIC, and it provided a long service life. In combination with the sialic acid derivatization, SiH-NPC presented a significant advantage in the analysis of sialylated glycans as well as isomeric oligosaccharides by liquid chromatography-mass spectrometry. The approach has potential applications in the biopharmaceutical industry.