Lectin and Liquid Chromatography-Based Methods for Immunoglobulin (G) Glycosylation Analysis.

Abstract

Immunoglobulin (Ig) glycosylation has been shown to dramatically affect its structure and effector functions. Ig glycosylation changes have been associated with different diseases and show a promising biomarker potential for diagnosis and prognosis of disease advancement. On the other hand, therapeutic biomolecules based on structural and functional features of Igs demand stringent quality control during the production process to ensure their safety and efficacy. Liquid chromatography (LC) and lectin-based methods are routinely used in Ig glycosylation analysis complementary to other analytical methods, e.g., mass spectrometry and capillary electrophoresis. This chapter covers analytical approaches based on LC and lectins used in low- and high-throughput N- and O-glycosylation analysis of Igs, with the focus on immunoglobulin G (IgG) applications. General principles and practical examples of the most often used LC methods for Ig purification are described, together with typical workflows for N- and O-glycan analysis on the level of free glycans, glycopeptides, subunits, or intact Igs. Lectin chromatography is a historical approach for the analysis of lectin-carbohydrate interactions and glycoprotein purification but is still being used as a valuable tool in Igs purification and glycan analysis. On the other hand, lectin microarrays have found their application in the rapid screening of glycan profiles on intact proteins.