Quality Assistance SA, Technoparc de Thudinie 2, Donstiennes, Belgium.
University of Bordeaux, INSERM and CNRS, Laboratoires Acides Nucléiques: Régulations Naturelle et Artificielle (ARNA, U1212, UMR5320), IECB, Pessac, France.
Methods Mol Biol. 2021;2271:237-247. doi: 10.1007/978-1-0716-1241-5_17.
The presence of sialic acids is one characteristic of glycosylated therapeutic proteins. The presence of these charged monosaccharides is critical for the immunogenicity properties and structural properties of the proteins. Profiling of the N-glycans and their charge state is a requisite for complete protein characterization. Two analytical methods developed on released N-glycans are described in this chapter, allowing for the determination of the sialoglycosylation with different levels of details. In the first method (AEX-HILIC/FLR), N-glycans are separated based on their charge and the average charge state can be determined from the fluorescence profile. In the second method (AEX-RP-FLR-MS), N-glycans are also separated based on their charge and the sialylation level is determined based on the fluorescence signal. In addition, in this method, the N-glycans are also separated by type and identified with the hyphenated MS. For both methods, an optimized protocol with fast and high-throughput sample preparation and purification is presented.
唾液酸的存在是糖基化治疗蛋白的一个特征。这些带电单糖的存在对于蛋白质的免疫原性和结构性质至关重要。对 N-糖链及其电荷状态的分析是完整蛋白质特性分析的必要条件。本章描述了两种已开发的基于释放 N-糖链的分析方法，允许以不同的详细程度来确定唾液酸化糖基化。在第一种方法（AEX-HILIC/FLR）中，根据电荷分离 N-糖链，并且可以从荧光谱中确定平均电荷状态。在第二种方法（AEX-RP-FLR-MS）中，N-糖链也根据电荷分离，并且根据荧光信号确定唾液酸化水平。此外，在该方法中，还通过连接的 MS 对 N-糖链进行分离并进行鉴定。对于这两种方法，都提出了一种优化的方案，具有快速和高通量的样品制备和纯化。
