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.

Development and validation of online SPE purification coupled to HILIC-fluorescence-MS analysis for the characterization of N-glycans.

N-glycans of therapeutic glycoproteins is a critical quality attribute to be addressed. We developed a sensitive method for N-glycan characterization using procainamide (ProcA) labelling and online solid phase extraction (online SPE). N-glycans were enzymatically released, then labeled with ProcA and cleaned up via the online SPE using HILIC chemistry (online HILIC SPE). Two preparation protocols were optimized: a short one (1 h 30) and a long one (18 h). Furthermore, the developed approach was compared to RapiFluor-MS (RFMS) kit (from Waters) and to InstantPC kit (from Agilent) which both include a classical HILIC µElution plate SPE purification. Samples were analyzed using HILIC separation coupled to fluorescence and MS detection (HILIC-FLD-MS) with or without the online HILIC SPE. During the validation, repeatability, intermediate precision, stability, response function and injection volume were tested. Human IgG mix (Multigam®) and NIST mAb standard were used as references as their glycoprofiles are well described. A comparison of three batches of a rituximab biosimilar (Truxima®) and one batch of its originator (MabThera®) was also performed. Online HILIC SPE sample cleanup shows a higher sensitivity and repeatability compared to the classical HILIC µElution SPE. Our online HILIC SPE approach also offers the highest MS signal compared to both commercial kits. However, InstantPC shows the highest FLD signal. The analyses of rituximab samples were in line with the literature showing the efficiency of the method for N-glycan monitoring of biotherapeutics. In conclusion, the results demonstrated the usefulness and ease of application of the developed protocol with the online HILIC SPE purification.

Fc Glycosylation Characterization of Human Immunoglobulins G Using Immunocapture and LC-MS.

Immunoglobulins G (IgG) are proteins produced by the immune system of higher life forms that play a central role in the defense against microbial pathogens. IgG bind pathogens with the hypervariable Fab component and mediate a diversity of effector functions by binding to immune effector cells via their crystallizable (Fc) component. All IgG Fc carry a polymorphic N-glycan that regulates its binding properties and thereby its effector functions. The glycosylation profile of IgG Fc is modulated by physiological and pathological conditions, including infectious diseases and inflammatory disorders. Characterization of IgG Fc glycosylation profiles is a promising approach to understand the pathogenesis of diseases involving the immune system and to develop novel biomarkers of disease activity. Measuring the proportion of the different IgG Fc glycoforms remains an analytical challenge, that requires a sensitive and reproducible analytical approach.This chapter describes an optimized approach for the preparation and the analysis of Fc N-glycans from total serum or plasma IgG using magnetic beads, RapiFluor MS label©, and LC-MS.