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.

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FTIR 光谱作为比较治疗性单克隆抗体糖基化的分析工具

糖基化是最常见的蛋白质翻译后修饰 (PTM)，尤其是在生物制药中。它是一个关键的质量属性，因为它会影响产品的溶解度、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，当前的聚糖分析方法涉及多个且冗长的样品制备步骤，这会影响分析的稳健性。因此，开发正交、直接和简单的方法是可取的。在本研究中，我们建议使用 FTIR 光谱来应对这一挑战。使用这种技术，结合统计工具，在糖基化或单糖谱方面比较样品或批次，具有三个潜在的应用：比较生物仿制药和原始（创新者）分子的糖基化，用于监控批次间的一致性，以及用于过程控制。十四种治疗性单克隆抗体 (mAb)、一种 Fc 融合蛋白和其他几种常见的糖蛋白已被用于证明糖蛋白的 FTIR 光谱根据其聚糖和单糖组成显示光谱变化。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。该指纹不仅对较大的差异（例如几种单糖的存在与否）敏感，而且对聚糖和单糖含量的较小修改也很敏感。一种 Fc 融合蛋白和其他几种常见的糖蛋白已被用于证明糖蛋白的 FTIR 光谱根据其聚糖和单糖组成显示光谱变化。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。该指纹不仅对较大的差异（例如几种单糖的存在与否）敏感，而且对聚糖和单糖含量的较小修改也很敏感。一种 Fc 融合蛋白和其他几种常见的糖蛋白已被用于证明糖蛋白的 FTIR 光谱根据其聚糖和单糖组成显示光谱变化。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。该指纹不仅对较大的差异（例如几种单糖的存在与否）敏感，而且对聚糖和单糖含量的较小修改也很敏感。