National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin A94X099, Ireland.
Glycobiology. 2022 Mar 31;32(4):289-303. doi: 10.1093/glycob/cwab128.
The glycosylation profile of biotherapeutic glycoproteins is a critical quality attribute that is routinely monitored to ensure desired product quality, safety and efficacy. Additionally, as one of the most prominent and complex post-translational modifications, glycosylation plays a key role in disease manifestation. Changes in glycosylation may serve as a specific and sensitive biomarker for disease diagnostics and prognostics. However, the conventional 2-aminobenzamide-based N-glycosylation analysis procedure is time-consuming and insensitive with poor reproducibility. We have evaluated an innovative streamlined 96-well-plate-based platform utilizing InstantPC label for high-throughput, high-sensitivity glycan profiling, which is user-friendly, robust and ready for automation. However, the limited availability of InstantPC-labeled glycan standards has significantly hampered the applicability and transferability of this platform for expedited glycan structural profiling. To address this challenge, we have constructed a detailed InstantPC-labeled glycan glucose unit (GU) database through analysis of human serum and a variety of other glycoproteins from various sources. Following preliminary hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection separation and analysis, glycoproteins with complex glycan profiles were subjected to further fractionation by weak anion exchange HILIC and exoglycosidase sequential digestion for cross-validation of the glycan assignment. Hydrophilic interaction ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry was subsequently utilized for glycan fragmentation and accurate glycan mass confirmation. The constructed InstantPC glycan GU database is accurate and robust. It is believed that this database will enhance the application of the developed platform for high-throughput, high-sensitivity glycan profiling and that it will eventually advance glycan-based biopharmaceutical production and disease biomarker discovery.
治疗性糖蛋白的糖基化谱是一个关键的质量属性，通常需要进行监测，以确保产品的质量、安全性和疗效达到预期。此外，作为最突出和复杂的翻译后修饰之一，糖基化在疾病表现中起着关键作用。糖基化的变化可能成为疾病诊断和预后的特异性和敏感生物标志物。然而，传统的基于 2-氨基苯甲酰胺的 N-糖基化分析程序既耗时又不灵敏，重现性差。我们评估了一种创新的基于 96 孔板的简化平台，该平台利用即时 PC 标签进行高通量、高灵敏度的聚糖分析，具有用户友好、稳健且易于自动化的特点。然而，即时 PC 标记的聚糖标准品的有限可用性极大地限制了该平台在加速聚糖结构分析中的适用性和可转移性。为了解决这一挑战，我们通过分析人血清和各种来源的其他糖蛋白，构建了一个详细的即时 PC 标记的聚糖葡萄糖单位 (GU) 数据库。在初步进行亲水相互作用液相色谱 (HILIC) 分离和荧光检测分析后，对具有复杂聚糖谱的糖蛋白进行进一步的阴离子交换 HILIC 弱分级和外切糖苷酶顺序消化，以验证聚糖的归属。随后，利用亲水作用超高效液相色谱与电喷雾电离质谱联用进行聚糖片段化和准确的聚糖质量确认。构建的即时 PC 聚糖 GU 数据库准确可靠。相信该数据库将增强开发平台在高通量、高灵敏度聚糖分析中的应用，并最终推进基于聚糖的生物制药生产和疾病生物标志物的发现。
