Separation of deamidated peptides with mixed-mode chromatography using phospholipid-functionalized monolithic stationary phases
Separation of deamidated peptides with mixed-mode chromatography using phospholipid-functionalized monolithic stationary phases
复制标题
使用磷脂功能化整体固定相通过混合模式色谱法分离脱酰胺肽
DOI：
10.1016/j.chroma.2019.05.053
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发表时间：
2019-10-11
影响因子：
4.1
通讯作者：
Jiang, Zhengjin
中科院分区：
文献类型：
--
作者：
Liu, Chusheng;Bults, Peter;Jiang, Zhengjin
单克隆抗体（mab）的天冬酰胺（Asn）残基脱酰胺在生物药物的体内/体外降解或药效丧失中起着关键作用。然而，单克隆抗体中含Asn肽的脱酰胺质谱分析面临的一个主要挑战是，天然形式（Asn残基）和脱酰胺形式（质量相同的n-天冬氨酸（n-Asp）和异天冬氨酸（isoAsp）残基）之间只有1da的质量变化。因此，在质谱分析之前，需要对脱酰胺蛋白和/或由此衍生的肽进行色谱分离。本研究制备了包括反相（RP）、单磷脂功能化和混合磷脂功能化等多种固定相的整体柱，用于分离曲妥珠单抗脱酰胺敏感特征肽（IYPTNGYTR）及其两种脱酰胺产物n-Asp55残基iyptdggyfr和isoAsp55残基iyptisodggytr。与RP单体相比，磷脂功能化单体提供了混合模式相互作用，并表现出更好的峰形和分离选择性。考察了缓冲液种类、浓度、温度、pH值等参数对分离性能的影响。在最佳条件下，由于疏水和静电相互作用的共同作用，三种肽在混合磷脂酰胆碱(PC) /磷脂酸（PA）功能化单体(聚(MDPC（60)PA(40)-co-EDMA）上被完全分离。因此，基于混合磷脂功能化整体柱的新方法在分析脱酰胺肽方面具有良好的潜力，这将有助于深入研究单克隆抗体的翻译后修饰。(C) 2019 Elsevier B.V.版权所有
Deamidation of asparagine (Asn) residues of monoclonal antibodies (mAbs) plays a pivotal role in the in vivo/vitro degradation or efficacy loss of biopharmaceuticals. However, a major challenge for MS analysis of deamidation of Asn-containing peptides in mAbs, is due to the fact that there is only a 1 Da mass shift between the native form (Asn residues) and deamidated forms (n-aspartyl (n-Asp) and isoaspartyl (isoAsp) residues with identical mass). Therefore, a chromatographic separation of the deamidated proteins and/or the peptides derived therefrom is needed prior to MS analysis. In this study, the monolithic column with various stationary phases, including reverse phase (RP), single phospholipid-functionalized and mixed phospholipid-functionalized monoliths, were prepared for the separation of the deamidation-sensitive signature peptide (IYPTNGYTR) of trastuzumab and its two deamidated products, n-Asp55 residue IYPTDGYFR and isoAsp55 residue IYPTisoDGYTR. Compared to the RP monolith, the phospholipid-functionalized monoliths provided mixed-mode interactions and exhibited better peak shape and separation selectivity. The effect of the parameters, including the type and concentration of buffer, temperature and pH value on the separation performance were investigated. Under the optimal conditions, the three peptides were fully separated on a mixed phosphocholine (PC) / phosphatidic acid (PA) functionalized monolith (poly (MDPC(60)PA(40)-co-EDMA)) due to the joint contribution of hydrophobic and electrostatic interactions. Therefore, the novel method based on the mixed phospholipids-functionalized monolithic column exhibited good potential for the analysis of deamidated peptides, which will be useful for the in-depth study of post-translational modifications of mAbs. (C) 2019 Elsevier B.V. All rights reserved.