Centre for Biologics Evaluation, Biologic and Radiopharmaceutical Drugs Directorate, Health Canada, Ottawa, Canada.
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai, China.
Sci Rep. 2022 Sep 6;12(1):15109. doi: 10.1038/s41598-022-19488-8.
Glycosylation is an important attribute of monoclonal antibodies (mAbs) for assessing manufacturing quality. Analysis of non-human glycans containing terminal galactose-α1,3-galactose and N-glycolylneuraminic acid is essential due to the potential immunogenicity and insufficient efficacy caused by mAb expression in non-human mammalian cells. Using parallel sequencing of isobaric glycopeptides and isomeric glycans that were separated by reversed-phase and porous graphitic carbon LC, we report a highly sensitive LC MS/MS method for the comprehensive characterization of low-abundance non-human glycans and their closely related structural isomers. We demonstrate that the straightforward use of high-abundance diagnostic ions and complementary fragments under the positive ionization low-energy collision-induced dissociation is a universal approach to rapidly discriminate branch-linkage structures of biantennary glycans. Our findings reveal the structural diversity of non-human glycans and sulfation of α-galactosylated glycans, providing both an analytical method and candidate structures that could potentially be used in the crucial quality control of therapeutic mAb products.
糖基化是评估单克隆抗体 (mAb) 制造质量的一个重要属性。由于 mAb 在非人类哺乳动物细胞中的表达可能引起免疫原性和效力不足，因此分析含有末端半乳糖-α1,3-半乳糖和 N-羟乙酰神经氨酸的非人类糖基至关重要。本研究采用等压糖肽和反相及多孔石墨碳 LC 分离的异构糖的平行测序，报告了一种用于全面表征低丰度非人类糖基及其密切相关结构异构体的高灵敏度 LC-MS/MS 方法。我们证明，在正离子低能碰撞诱导解离下，直接使用高丰度诊断离子和互补片段是一种快速区分双天线聚糖分支连接结构的通用方法。我们的研究结果揭示了非人类糖基和α-半乳糖基化糖基的结构多样性，为治疗性 mAb 产品的关键质量控制提供了分析方法和候选结构。
