Glycoscience Research Laboratory, Genos Ltd., Zagreb, Croatia.
Exp Suppl. 2021;112:29-72. doi: 10.1007/978-3-030-76912-3_2.
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.
免疫球蛋白 (Ig) 的糖基化已被证明会极大地影响其结构和效应功能。Ig 糖基化的变化与不同的疾病有关，并显示出作为疾病诊断和进展预测的有前途的生物标志物潜力。另一方面，基于 Ig 的结构和功能特征的治疗性生物分子在生产过程中需要严格的质量控制，以确保其安全性和有效性。液相色谱 (LC) 和基于凝集素的方法通常与其他分析方法（例如质谱和毛细管电泳）一起用于 Ig 糖基化分析，是互补的。本章涵盖了基于 LC 和凝集素的分析方法，用于低和高通量的 N-和 O-糖基化分析 Ig，重点是 IgG 的应用。描述了用于 Ig 纯化的最常用 LC 方法的一般原理和实际示例，以及游离糖、糖肽、亚基或完整 Ig 上 N-和 O-聚糖分析的典型工作流程。凝集素色谱法是分析凝集素-碳水化合物相互作用和糖蛋白纯化的历史方法，但仍被用作 Ig 纯化和聚糖分析的有价值工具。另一方面，凝集素微阵列已应用于完整蛋白质上糖谱的快速筛选。
