通过无盐亲水相互作用液相色谱串联质谱法扩大治疗蛋白质糖基化微异质性的结构解析。

IF 5.6 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL
mAbs Pub Date : 2024-01-01 Epub Date: 2024-08-27 DOI:10.1080/19420862.2024.2395503
Yutian Gan, Steffen Lippold, John Stobaugh, Christian Schöneich, Feng Yang
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引用次数: 0

摘要

糖基化会影响治疗蛋白质的安全性和有效性,通常被认为是关键质量属性(CQA)。因此,在药物开发过程中识别和量化聚糖非常重要。糖基化是一种高度复杂的翻译后修饰 (PTM),因为其结构具有异质性,即糖基化位点占有率、糖组成、修饰和异构体。目前的分析工具在结构分辨率或位点特异性方面都不尽如人意。亲水作用液相色谱-荧光质谱法(HILIC-FLR-MS)是对释放的聚糖进行结构分析的黄金标准,但缺乏关于位点特异性和占据情况的信息。然而,HILIC-FLR-MS 通常在溶剂中使用盐,这会影响分析的稳健性和灵敏度。通常采用反相液相色谱-串联质谱(RPLC-MS/MS)对蛋白酶消化后的糖肽进行位点特异性糖基化分析,但只能提供组成和有限的结构聚糖信息。在本研究中,我们介绍了一种无盐、基于糖肽的 HILIC-串联质谱(HILIC-MS/MS)方法,该方法可同时提供聚糖鉴定、聚糖异构体分离和特定位点信息。此外,HILIC-MS/MS 与释放糖 HILIC-FLR-MS 的相对定量结果相当。此外,我们的新方法还提高了亲水肽的保留率,可同时分析重要的 CQAs,如抗体中的脱酰胺。所开发的方法为简化糖蛋白的特异位点糖基化分析提供了宝贵的工具,这对于生物制药行业中不断扩大的新型治疗模式尤为重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry.

Glycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its structural heterogeneity, i.e. glycosylation site occupancy, glycan compositions, modifications, and isomers. Current analytical tools compromise either structural resolution or site specificity. Hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry (HILIC-FLR-MS) is the gold standard for structural analysis of released glycans, but lacks information on site specificity and occupation. However, HILIC-FLR-MS often uses salt in the solvent, which impairs analysis robustness and sensitivity. Site-specific glycosylation analysis via glycopeptides, upon proteolytic digestion, is commonly performed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), but provides only compositional and limited structural glycan information. In this study, we introduce a salt-free, glycopeptide-based HILIC-tandem mass spectrometry (HILIC-MS/MS) method that provides glycan identification, glycan isomer separation and site-specific information simultaneously. Moreover, HILIC-MS/MS demonstrated comparable relative quantification results as released glycan HILIC-FLR-MS. Further, our new method improves the retention of hydrophilic peptides, allowing simultaneous analysis of important CQAs such as deamidation in antibodies. The developed method offers a valuable tool to streamline the site-specific glycosylation analysis of glycoproteins, which is particularly important for the expanding landscape of novel therapeutic formats in the biopharmaceutical industry.

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来源期刊
mAbs
mAbs 工程技术-仪器仪表
CiteScore
10.70
自引率
11.30%
发文量
77
审稿时长
6-12 weeks
期刊介绍: mAbs is a multi-disciplinary journal dedicated to the art and science of antibody research and development. The journal has a strong scientific and medical focus, but also strives to serve a broader readership. The articles are thus of interest to scientists, clinical researchers, and physicians, as well as the wider mAb community, including our readers involved in technology transfer, legal issues, investment, strategic planning and the regulation of therapeutics.
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