Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/02, Université Libre de Bruxelles, Bld Du Triomphe 2, CP206/2, B1050, Brussels, Belgium.
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, United Kingdom.
Anal Chim Acta. 2020 May 22;1112:62-71. doi: 10.1016/j.aca.2020.03.038. Epub 2020 Mar 31.
Glycosylation is the most common protein post-translational modification (PTM), especially in biopharmaceuticals. It is a critical quality attribute as it impacts product solubility, stability, half-life, pharmacokinetics and pharmacodynamics (PK/PD), bioactivity and safety (e.g. immunogenicity). Yet, current glycan analysis methods involve multiple and lengthy sample preparation steps which can affect the robustness of the analyses. The development of orthogonal, direct and simple method is therefore desirable. In this study, we suggest use of FTIR spectroscopy to address this challenge. Use of this technique, combined with statistical tools, to compare samples or batches in terms of glycosylation or monosaccharide profile, has three potential applications: to compare glycosylation of a biosimilar and the original (innovator) molecule, for monitoring of batch-to-batch consistency, and for in-process control. Fourteen therapeutic monoclonal antibodies (mAbs), one Fc-fusion protein and several other common glycoproteins have been used to demonstrate that FTIR spectra of glycoproteins display spectral variations according to their glycan and monosaccharide compositions. We show that FTIR spectra of glycoproteins provide a global but accurate fingerprint of the glycosylation profile. This fingerprint is not only sensitive to large differences such as the presence or absence of several monosaccharides but also to smaller modifications of the glycan and monosaccharide content.
糖基化是最常见的蛋白质翻译后修饰（PTM），特别是在生物制药中。它是一个关键的质量属性，因为它影响产品的溶解度、稳定性、半衰期、药代动力学和药效学（PK/PD）、生物活性和安全性（例如免疫原性）。然而，目前的聚糖分析方法涉及多个冗长的样品制备步骤，这可能会影响分析的稳健性。因此，需要开发正交、直接和简单的方法。在本研究中，我们建议使用傅里叶变换红外（FTIR）光谱来解决这一挑战。该技术与统计工具结合使用，可根据糖基化或单糖图谱比较样品或批次，具有三个潜在应用：比较生物类似药和原始（创新）分子的糖基化，监测批间一致性，以及用于过程控制。本研究使用了 14 种治疗性单克隆抗体（mAb）、一种 Fc 融合蛋白和几种其他常见糖蛋白，证明了糖蛋白的 FTIR 光谱根据其聚糖和单糖组成显示出光谱变化。我们表明，糖蛋白的 FTIR 光谱提供了糖基化谱的全局但准确的指纹。该指纹不仅对存在或不存在几种单糖等较大差异敏感，而且对聚糖和单糖含量的较小变化也敏感。
