基于N-聚糖和完整糖肽的单克隆抗体药物糖基化修饰表征

N-Glycans and intact glycopeptide-based characterization of N-glycosylation of monoclonal antibody drugs

近年来生物制药产业快速增长,其中单克隆抗体药物的市场规模增速显著,对其进行精确的结构表征和质量控制的需求日益突出。作为抗体药非常重要的翻译后修饰,糖基化对单抗药的疗效、稳定性、免疫原性都具有重要的影响。对糖基化修饰进行表征的主要方式之一是液相色谱串联质谱技术,但该方法目前主要集中在中高丰度的聚糖表征,对低丰度的糖基化修饰研究较少。本研究建立了基于RapiFluor-MS试剂标记的单克隆抗体药物的N-聚糖定性和定量分析技术,该方法样品处理时间短,灵敏度高,不仅能够表征3种单抗药阿达木单抗、贝伐珠单抗和曲妥珠单抗的主要糖型,而且对低丰度糖基化修饰能够准确的表征并定量分析。基于该方法,作者研究了上述3种单抗药主要糖型,并对不同批次的样品中N-聚糖的相对丰度进行了对比。同时在完整糖肽水平对N-聚糖的连接位点和糖型进行解析,进一步丰富了单抗药的N-聚糖结构信息。基于RapiFluor-MS试剂标记的单克隆抗体药物的N-聚糖定性和定量分析技术能够实现对单抗药糖基化修饰的深度表征和控制。

In recent years, the biopharmaceutical industry has grown rapidly, and the market size of monoclonal antibody drugs has increased significantly. Accurate structural characterization and quality control are the supporting technologies for the development of monoclonal antibody drugs. As a significant post-translational modification of antibody drugs, glycosylation has an important influence on its efficacy, stability, and immunogenicity. The existing literature usually uses liquid chromatography-mass spectrometry to perform major glycosylation modifications of monoclonal antibody drugs. Characterization, there are few studies on low-abundance glycosylation, but the characterization and control of low-abundance glycosylation cannot be ignored. In this study, we have established a qualitative and quantitative analysis technology for N-glycans based on RapiFluor-MS reagent-labeled monoclonal antibody drugs. This method has a short sample processing time and high sensitivity. It can not only characterize the main glycoforms of three monoclonal antibody drugs(adalimumab, bevacizumab, and trastuzumab) but also can quantify low-abundance N-glycans. The results of the study showed that the main glycoforms specified in the Pharmacopoeia could be detected in different batches of monoclonal antibody drugs, but the content of N-glycans in different batches of samples is not identical. After that, we analyzed the N-glycans connection sites and glycoforms at the intact glycopeptide level, further enriching the N-glycans structure information of the monoclonal antibody. The qualitative and quantitative analysis technology of N-glycans based on RapiFluor-MS reagent-labeled monoclonal antibody drugs can realize the in-depth characterization and control of glycosylation modification of monoclonal antibody drugs.