Calibration for quantitative Fc-glycosylation analysis of therapeutic IgG1-type monoclonal antibodies by using glycopeptide standards
Calibration for quantitative Fc-glycosylation analysis of therapeutic IgG1-type monoclonal antibodies by using glycopeptide standards
复制标题
使用糖肽标准品对治疗性 IgG1 型单克隆抗体进行定量 Fc 糖基化分析的校准
DOI：
10.1016/j.aca.2021.338306
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发表时间：
2021
影响因子：
6.2
通讯作者：
Liang Xinmiao
中科院分区：
文献类型：
--
作者：
Cao Cuiyan;Yu Long;Zhang Xiuli;Dong Xuefang;Yuan Jingli;Liang Xinmiao
Fc-糖基化对IgG型治疗性单克隆抗体（mAb）的有效性和安全性具有至关重要的影响。为了提高基于MS的自下而上定量策略的性能，通过改进的二维亲水相互作用液相色谱（HILIC）纯化构建了含有26种常见IgG 1型Fc-糖型的糖肽标准品库。利用获得的糖肽标准品，建立了单克隆抗体Fc-糖基化分析的校准定量策略，并基于3种基于LC-MS的方法进行了评价，包括HILIC-MRM（多反应监测）、HILIC-SIM（选择离子监测）和RPLC-SIM。在6种IgG 1型mAb中，获得了11种单个Fc-糖型的摩尔浓度（0.03 ± 0.001-13.77 ± 0.64 nmol mg−1）以及岩藻糖基化程度（75.44-97.04%）、半乳糖基化程度（3.39-49.47%）和甘露糖基化程度（1.12-21.22%）。此外，Fc-糖基化位点占有率也确定为98.05%至99.83%。与传统的质谱峰面积归一化定量方法相比，该方法的定量准确度和精密度均得到了显著提高，尤其是与HILIC分离相结合。此外，使用基于MRM的方法评估的校准定量在不同实验室的不同仪器上的可转移性也得到了显著增强。这种使用糖肽标准品作为校准品的校准定量策略将用于具有多个糖基化位点的IgG 1型mAb的Fc-糖基化分析。
Fc-glycosylation has crucial impact on the efficacy and safety of IgG-type therapeutic monoclonal antibodies (mAbs). In order to enhance the performance of MS-based bottom-up quantitation strategy, a library of glycopeptide standards containing 26 common IgG1-type Fc-glycoforms has been constructed via modified two-dimensional hydrophilic interaction liquid chromatography (HILIC) purification. Taking advantage of the acquired glycopeptide standards, calibrated quantitation strategy for Fc-glycosylation analysis of mAbs was established and evaluated on the basis of three LC-MS-based methods, including HILIC-MRM (multiple reaction monitoring), HILIC-SIM (selected ion monitor) and RPLC-SIM. Molar concentrations of eleven individual Fc-glycoforms (0.03 ± 0.001–13.77 ± 0.64 nmol mg−1) as well as degree of fucosylation (75.44–97.04%), galactosylation (3.39–49.47%) and mannosylation (1.12–21.22%) in six IgG1-type mAbs were achieved. In addition, Fc-glycosylation site occupancy was also determined from 98.05% to 99.83%. Compared with traditional MS-based quantitation via peak area normalization, the quantitation accuracy and precision of the calibrated strategy had been remarkably improved, especially when combining with HILIC separation. In addition, the transferability of calibrated quantitation as assessed by using MRM-based method had also been significantly enhanced on different instruments from different laboratories. This calibrated quantitation strategy using glycopeptide standards as calibrators will be useful for Fc-glycosylation analysis of IgG1-type mAbs with multiple glycosylation sites.