Böttinger Katharina, Regl Christof, Schäpertöns Veronika, Rapp Erdmann, Wohlschlager Therese, Huber Christian G
Department of Biosciences and Medical Biology, Bioanalytical Research Labs, University of Salzburg, Salzburg, 5020, Austria.
Center for Tumorbiology and Immunology (CTBI), University of Salzburg, Salzburg, 5020, Austria.
J Pharm Anal. 2024 Oct;14(10):100982. doi: 10.1016/j.jpha.2024.100982. Epub 2024 Apr 26.
Glycans associated with biopharmaceutical drugs play crucial roles in drug safety and efficacy, and therefore, their reliable detection and quantification is essential. Our study introduces a multi-level quantification approach for glycosylation analysis in monoclonal antibodies (mAbs), focusing on minor abundant glycovariants. Mass spectrometric data is evaluated mainly employing open-source software tools. Released glycan and glycopeptide data form the basis for integrating information across different structural levels up to intact glycoproteins. Comprehensive comparison showed that indeed, variations across structural levels were observed especially for minor abundant species. Utilizing modification finder (MoFi), a tool for annotating mass spectra of intact proteins, we quantify isobaric glycosylation variants at the intact protein level. Our workflow's utility is demonstrated on NISTmAb, rituximab and adalimumab, profiling their minor abundant variants for the first time across diverse structural levels. This study enhances understanding and accessibility in glycosylation analysis, spotlighting minor abundant glycovariants in therapeutic antibodies.
与生物制药药物相关的聚糖在药物安全性和有效性方面发挥着关键作用，因此，对其进行可靠的检测和定量至关重要。我们的研究引入了一种用于单克隆抗体（mAb）糖基化分析的多层次定量方法，重点关注低丰度糖型变体。质谱数据主要使用开源软件工具进行评估。释放的聚糖和糖肽数据构成了整合不同结构水平直至完整糖蛋白信息的基础。综合比较表明，确实在不同结构水平上观察到了差异，尤其是对于低丰度物种。利用修饰查找器（MoFi）这一用于完整蛋白质质谱注释的工具，我们在完整蛋白质水平上对同量异位糖基化变体进行定量。我们的工作流程在NISTmAb、利妥昔单抗和阿达木单抗上得到了验证，首次在不同结构水平上对它们的低丰度变体进行了分析。这项研究增进了对糖基化分析的理解并提高了其可及性，突出了治疗性抗体中的低丰度糖型变体。
