Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA, 94080, United States.
Thermo Fisher Scientific, Tudor Road, Manor Park, Runcorn, Cheshire, WA7 1TA, United Kingdom.
Anal Chim Acta. 2020 Feb 22;1099:155-164. doi: 10.1016/j.aca.2019.11.055. Epub 2019 Nov 26.
Comparative glycosylation analysis of biopharmaceuticals requires the development of methods that deliver the necessary throughput, support structural elucidation and relative quantitation of glycans released from therapeutics. The current study presents the development and applicability assessment of a twoplex approach using light and heavy isotopolouges of 3-aminobenzenesulfonic acid (3-ASA) under wet labeling conditions followed by UHPLC-MS analysis in data dependent acquisition mode. Excellent labelling efficiency, >90%, was achieved for both the light and heavy variants of the reagent. Glycan distributions of two human IgG lots labeled by light and heavy isotopolouges were identical, demonstrating no labeling bias introduced by either of the isotopologues. Peak area distributions of glycan profiles of two human IgG lots were compared to 2-aminobenzamide (2-AB) and RapiFluor-MS protocols. The comparison led to identical results in peak area distribution across the three dyes, but differences in chromatographic selectivity attributed to the different tags. MS based relative quantitation was further validated by releasing glycans from the same lot of human IgG, with glycan pools obtained labeled with light and heavy isotopologues separately, followed by mixing and clean-up of the same amount of light and heavy labeled glycan pools. MS analyses of each glycan resulted in a ratio of light and heavy XIC in the range of 0.97 ≤ x ≤ 1.05, demonstrating the method is amenable for the relative quantitation of glycans. Excellent correlation between the relative quantitation data of N-glycans from two human IgG N-glycan pools using the twoplex approach and ratios from peak area distribution calculated from the fluorescent chromatogram was observed (r = 0.986), further corroborating the reliability of the method and its potential applicability in the biopharmaceutical industry. Highly informative HCD-MS spectra dominated mostly by Y- and Z-type single and double glycosidic fragment ions facilitate structural interpretation of the oligosaccharides.
生物制药的比较糖基化分析需要开发能够提供必要通量的方法，支持从治疗剂中释放的聚糖的结构阐明和相对定量。本研究提出了一种使用 3-氨基苯磺酸（3-ASA）的轻和重同位素在湿标记条件下的双plex 方法的开发和适用性评估，随后在数据依赖采集模式下进行 UHPLC-MS 分析。两种试剂的轻变体和重变体都实现了 >90%的优异标记效率。用轻和重同位素标记的两种人 IgG 批次的聚糖分布相同，证明两种同位素都没有引入标记偏倚。两种人 IgG 批次的聚糖谱的峰面积分布与 2-氨基苯甲酰胺（2-AB）和 RapiFluor-MS 方案进行了比较。比较导致三种染料的峰面积分布完全一致，但由于不同的标签，色谱选择性存在差异。基于 MS 的相对定量通过从同一批人 IgG 释放聚糖进一步验证，用轻和重同位素分别标记聚糖池，然后混合并清洗相同量的轻和重标记聚糖池。对每个聚糖的 MS 分析导致轻和重 XIC 的比值在 0.97≤x≤1.05 的范围内，表明该方法适用于聚糖的相对定量。使用双plex 方法对两种人 IgG N-聚糖池的 N-聚糖的相对定量数据与从荧光色谱图计算的峰面积分布的比值之间观察到极好的相关性（r=0.986），进一步证实了该方法的可靠性及其在生物制药行业中的潜在适用性。主要由 Y-和 Z-型单和双糖基片段离子主导的高度信息丰富的 HCD-MS 谱有助于寡糖的结构解释。
