Updates of the current strategies of labeling for N-glycan analysis

This mini review summarizes the current methods used for screening N-glycosylation of glycoproteins, with a specific focus on therapeutic proteins and on techniques involving the release of N-glycans. With the continuous development of biopharmaceuticals, particularly monoclonal antibodies (mAbs), which are N-glycosylated proteins, monitoring has gained importance in recent decades. Glycosylation of therapeutic glycoproteins is considered a critical quality attribute because it can impact the efficacy and safety of these therapeutic drugs. The protocols and instrumentation have evolved with the advancement of technologies. Nowadays, methods are becoming increasingly robust, rapid, and sensitive. For the release of N-glycans, the most commonly used method is enzymatic release using PNGase F. The latter is discussed in light of the advent of rapid release that is now possible. The strategy for separating N-glycans using either liquid chromatography (LC) with hydrophilic interaction liquid chromatography (HILIC) chemistry or capillary electrophoresis will be discussed. The selection of the labeling agent is a crucial step in sample preparation for the analysis of released N-glycans. This review also discusses labeling agents that are compatible with and dependent on the separation and detection techniques employed. The emergence of multiplex labeling agents is also summarized. The latter enables the analysis of multiple samples in a single run, but it requires MS analysis.