Asia-Pacific Glycomics Reference Site, Chungnam National University, Daejeon, South Korea.
Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, South Korea.
Methods Mol Biol. 2021;2271:121-131. doi: 10.1007/978-1-0716-1241-5_9.
Glycosylation of biologics, an important factor in pharmacological functions such as efficacy, safety, and biological activity, is easily affected by subtle changes in the cellular environment. Therefore, comprehensive and in-depth glycan characterization of therapeutic glycoproteins should be performed to ensure product quality and process consistency, but it is analytically challenging due to glycan microheterogeneity occurring in the glycan biosynthesis pathway. LC-based chromatographic separation combined with mass spectrometry (MS) has been widely used as a prominent tool for the qualitative and quantitative analysis of glycosylation of therapeutic glycoproteins. However, prior to LC/MS analysis, glycans are selectively captured and fractionated by solid-phase extraction (SPE) utilizing physicochemical characteristics for comprehensive characterization of a wide range of glycan heterogeneity on glycoengineered therapeutic proteins. In particular, porous graphitized carbon (PGC) SPE has been employed as a useful technique for the fractionation of native glycans having different sizes and polarities. Here, we describe a systematic method for comprehensive glycan characterization of therapeutic proteins using stepwise PGC SPE and LC/MS.
糖基化是生物制品的一个重要因素，它影响着药物的疗效、安全性和生物活性等药理学功能，容易受到细胞环境中细微变化的影响。因此，为了确保产品质量和工艺一致性，应对治疗性糖蛋白进行全面深入的聚糖表征，但由于糖基化生物合成途径中存在聚糖微异质性，这在分析上具有挑战性。基于液相色谱（LC）的色谱分离技术结合质谱（MS）已广泛应用于治疗性糖蛋白糖基化的定性和定量分析，是一种重要的工具。然而，在 LC/MS 分析之前，利用物理化学特性，通过固相萃取（SPE）选择性地捕获和分离糖基，以对糖基化治疗蛋白的广泛糖基异质性进行全面表征。特别是，多孔石墨化碳（PGC）SPE 已被用作分离具有不同大小和极性的天然聚糖的有用技术。在这里，我们描述了一种使用逐步 PGC SPE 和 LC/MS 对治疗性蛋白进行全面糖基化表征的系统方法。
