Section of Pharmaceutical Sciences, School of Pharmacy Geneva-Lausanne , University of Geneva , Centre Médical Universitaire, Rue Michel Servet 1 , 1211 Geneva , Switzerland.
Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové , Charles University , Hradec Králové , Czech Republic.
Anal Chem. 2019 Jan 2;91(1):873-880. doi: 10.1021/acs.analchem.8b03584. Epub 2018 Dec 13.
Etanercept is a recombinant Fc fusion protein widely used to treat rheumatic diseases. This protein is highly glycosylated and contains numerous O- and N-glycosylation sites. Since glycosylation is recognized as an important critical quality attribute (CQA) that can affect immunogenicity, solubility, and stability of Fc fusion proteins, it should be thoroughly characterized. In this work, hydrophilic interaction chromatography (HILIC) was combined with high-resolution mass spectrometry (HRMS) by using a quadrupole time-of flight mass spectrometer to assess glycosylation of etanercept at the middle-up level of analysis (fragments of ca. 25-30 kDa). In addition, a combination of different enzymatic digestion procedures (i.e., glycosidase, sialidase, and protease) was systematically employed to facilitate spectra deconvolution. With the developed procedure, the main post-translational modifications (PTMs) of etanercept were assessed, and a global overview of the subunit-specific distribution of the glycosylation pattern was obtained at a middle-up level of analysis.
依那西普是一种广泛用于治疗风湿性疾病的重组 Fc 融合蛋白。该蛋白高度糖基化，含有许多 O-和 N-糖基化位点。由于糖基化被认为是一个重要的关键质量属性（CQA），可以影响 Fc 融合蛋白的免疫原性、溶解性和稳定性，因此应该对其进行彻底的表征。在这项工作中，亲水相互作用色谱（HILIC）与高分辨率质谱（HRMS）相结合，使用四极杆飞行时间质谱仪在中分析水平（约 25-30 kDa 的片段）评估依那西普的糖基化。此外，还系统地采用了不同的酶切处理（即糖苷酶、唾液酸酶和蛋白酶）组合来促进谱图解析。采用所开发的方法，可以评估依那西普的主要翻译后修饰（PTMs），并在中分析水平获得糖基化模式的亚基特异性分布的全面概述。
