Laboratoire de Chimie Physique Moléculaire, École Polytechnique Fédérale de Lausanne, EPFL SB ISIC LCPM, Station 6, CH-1015 Lausanne, Switzerland.
Analyst. 2020 Oct 21;145(20):6493-6499. doi: 10.1039/d0an01206h. Epub 2020 Aug 4.
Glycans covalently attached to protein biotherapeutics have a significant impact on their biological activity, clearance, and safety. As a result, glycosylation is categorized as a critical quality attribute that needs an adequate analytical approach to guarantee product quality. However, the isomeric complexity and branched structure of glycans makes their analysis a significant challenge. In this work, we propose a multidimensional approach for monitoring released glycans that combines ultrahigh-resolution ion mobility spectrometry (IMS) and cryogenic vibrational spectroscopy, and we demonstrate this technique by characterizing four N-glycans cleaved from the therapeutic fusion protein etanercept that range in abundance from 1% to 22% of the total N-glycan content. The recorded vibrational spectra exhibit well-resolved transitions that can be used as a fingerprint to identify a particular glycan. This work represents an important advance in the analysis of N-linked glycans cleaved from biopharmaceutical proteins that could eventually be used as tool for monitoring biopharmaceutical glycoforms.
糖基共价结合到蛋白质生物治疗剂上会对其生物活性、清除率和安全性产生重大影响。因此，糖基化被归类为关键质量属性，需要采用适当的分析方法来保证产品质量。然而，糖基的异构复杂性和支链结构使其分析成为一项重大挑战。在这项工作中，我们提出了一种用于监测释放糖基的多维方法，该方法结合了超高分辨率离子淌度谱（IMS）和低温振动光谱，并通过对从治疗性融合蛋白依那西普中切割得到的四种 N-糖基进行表征来证明该技术，这四种 N-糖基的丰度范围占总 N-糖基含量的 1%至 22%。所记录的振动光谱表现出良好分辨的跃迁，可以用作鉴定特定糖基的指纹。这项工作代表了从生物制药蛋白质中切割的 N-连接糖基分析的重要进展，最终可能被用作监测生物制药糖型的工具。
