Huhn Carolin, Selman Maurice H J, Ruhaak L Renee, Deelder André M, Wuhrer Manfred
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands.
Proteomics. 2009 Feb;9(4):882-913. doi: 10.1002/pmic.200800715.
A multitude of monoclonal IgG antibodies directed against a variety of therapeutic targets is currently being developed and produced by biotechnological companies. The biological activity of IgGs is modulated by the N-glycans attached to the fragment crystallizable (Fc) part. For example, lack of core-fucoses on these N-glycans may lead to a drastic enhancement of antibody-mediated cellular cytotoxicity. Moreover, sialylation of Fc N-glycans determines the immunosuppressive properties of polyclonal IgG from human blood, which stimulates research into Fc glycosylation of human plasma IgG in various disease settings. This review presents and evaluates the different approaches which are used for IgG glycosylation analysis: N-glycans may be enzymatically or chemically released from purified IgG, prior to chromatographic or mass spectrometric analysis. Moreover, IgGs may be treated with endoproteinases such as trypsin, followed by glycosylation analysis at the glycopeptide level, which is generally accomplished by HPLC with ESI-MS. Alternatively, intact IgGs or fragments thereof obtained by enzymatic cleavages in the hinge region and by reduction may be analyzed by a large number of analytical techniques, including MS and chromatography or CE.
目前，生物技术公司正在研发和生产多种针对各种治疗靶点的单克隆IgG抗体。IgG的生物活性受附着于可结晶片段（Fc）部分的N-聚糖调节。例如，这些N-聚糖上缺乏核心岩藻糖可能会导致抗体介导的细胞毒性急剧增强。此外，Fc N-聚糖的唾液酸化决定了人血中多克隆IgG的免疫抑制特性，这激发了对各种疾病环境下人血浆IgG的Fc糖基化的研究。本文综述并评估了用于IgG糖基化分析的不同方法：在进行色谱或质谱分析之前，可通过酶法或化学法从纯化的IgG中释放N-聚糖。此外，IgG可用诸如胰蛋白酶等内切蛋白酶处理，随后在糖肽水平进行糖基化分析，这通常通过HPLC与ESI-MS来完成。或者，通过铰链区的酶切和还原获得的完整IgG或其片段可通过大量分析技术进行分析，包括MS、色谱法或CE。
