Fast and Ultrasensitive Glycoform Analysis by Supercritical Fluid Chromatography–Tandem Mass Spectrometry

The glycoform of
a therapeutic monoclonal antibody (mAb) has a
significant impact on its effector function as well as its safety
and pharmacokinetics. Glycoform heterogeneity is influenced by various
factors, including the producing cells and cell culture processes.
Therefore, accurate glycoform characterization is essential for drug
design, process optimization, manufacturing, and quality control of
therapeutic mAbs. In this study, we developed a fast, quantitative,
and highly sensitive analytical platform for glycan profiling by supercritical
fluid chromatography–tandem mass spectrometry (SFC-MS/MS) and
applied the technique to the glycan structural analysis of mAbs. To
achieve both the highest sensitivity and the most comprehensive glycan
profiling, we integrated our energy-resolved oxonium ion monitoring
(Erexim) method with SFC-MS to construct a new SFC-Erexim technology.
An 8 min analysis of bevacizumab, nivolumab, ramucirumab, rituximab,
and trastuzumab by SFC-Erexim detected a total of 102 glycoforms,
with a detection limit of 5 attomoles. The dynamic range of glycan
abundance was over 6 orders of magnitude for bevacizumab analysis
by SFC-Erexim compared to 3 orders of magnitude for conventional fluorescence
HPLC analysis. This method revealed the glycan profile characteristics
and lot-to-lot heterogeneity of various therapeutic mAbs. We were
also able to detect a series of structural variations in pharmacologically
important glycan structures. The SFC-MS-based glycoform profiling
method will provide an ideal platform for the in-depth analysis of
precise glycan structure and abundance.

治疗性单克隆抗体（monoclonal antibody，mAb）的糖型对其效应子功能、安全性以及药代动力学具有显著影响。糖型异质性受多种因素影响，包括生产细胞及细胞培养工艺。因此，精准的糖型表征对于治疗性单克隆抗体的药物设计、工艺优化、生产制造以及质量控制至关重要。本研究开发了一种基于超临界流体色谱-串联质谱（supercritical fluid chromatography–tandem mass spectrometry，SFC-MS/MS）的快速、定量且高灵敏度的糖谱分析平台，并将该技术应用于单克隆抗体的糖基结构分析。为同时实现最高灵敏度与最全面的糖谱分析，我们将本研究建立的能量分辨氧鎓离子监测（energy-resolved oxonium ion monitoring，Erexim）方法与超临界流体色谱-质谱（supercritical fluid chromatography-mass spectrometry，SFC-MS）相结合，构建了全新的SFC-Erexim技术。通过SFC-Erexim技术在8分钟内对贝伐珠单抗、纳武利尤单抗、雷莫芦单抗、利妥昔单抗以及曲妥珠单抗进行分析，共检测到102种糖型，检测限达5阿托摩尔（attomoles）。相较于传统荧光高效液相色谱（high performance liquid chromatography，HPLC）分析的3个数量级，基于SFC-Erexim的贝伐珠单抗分析中，糖基丰度的动态范围超过6个数量级。该方法揭示了多种治疗性单克隆抗体的糖谱特征及批次间异质性。本研究还成功检出一系列具有药理学重要性的糖基结构变异形式。这种基于超临界流体色谱-质谱的糖型分析方法，将为精准解析糖基结构与丰度提供理想的研究平台。