Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA.
Facility for Biotechnology Resources, United States Food and Drug Administration, Silver Spring, MD 20993, USA.
Cell Rep Methods. 2024 Aug 19;4(8):100834. doi: 10.1016/j.crmeth.2024.100834. Epub 2024 Aug 7.
Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by β-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.
糖基化通常被认为是治疗性糖蛋白的关键质量属性，因为聚糖会影响蛋白药物产品的疗效、半衰期、稳定性和安全性。用于表征 N-聚糖的分析程序已经相对成熟，但由于复杂的工作流程和缺乏酶工具，O-聚糖的表征具有挑战性。在这里，我们提出了一种简化的化学酶法，通过质谱 (MS) 以一锅格式从同一样品中同时分析 N-和 O-聚糖。首先用 PNGase F 释放 N-聚糖，然后用蛋白酶 K 生成 O-糖肽，选择性还原 N-聚糖，并在 N-和 O-聚糖的全甲基化过程中通过 β-消除释放 O-聚糖。通过一锅式质谱获得聚糖结构分配和 N-到 O-聚糖比的确定。这种简化的一锅法是一种可靠的方法，将有助于治疗性糖蛋白的质量评估的高级表征。
