Enhanced N-Glycan Profiling of Therapeutic Monoclonal Antibodies through the Application of Upper-Hinge Middle-Up Level LC-HRMS Analysis.

IF 3 Q3 IMMUNOLOGY

Antibodies Pub Date : 2024-08-06 DOI:10.3390/antib13030066

Natalia Mesonzhnik, Anton Belushenko, Polina Novikova, Alexey Kukharenko, Mikhail Afonin

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引用次数: 0

Abstract

Therapeutic monoclonal antibodies (mAbs) are crucial in modern medicine due to their effectiveness in treating various diseases. However, the structural complexity of mAbs, particularly their glycosylation patterns, presents challenges for quality control and biosimilarity assessment. This study explores the use of upper-hinge middle-up (UHMU)-level ultra-high-performance liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis to improve N-glycan profiling of mAbs. Two specific enzymes, known as IgG degradation enzymes (IGDEs), were used to selectively cleave therapeutic mAbs above the hinge region to separate antibody subunits for further Fc glycan analysis by means of the UHMU/LC-HRMS workflow. The complexity of the mass spectra of IGDEs-digested mAbs was significantly reduced compared to the intact MS level, enabling reliable assignment and relative quantitation of paired Fc glycoforms. The results of the UHMU/LC-HRMS analysis of nine approved therapeutics highlight the significance of this approach for in-depth glycoform profiling.

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通过应用上铰链中上水平 LC-HRMS 分析增强治疗性单克隆抗体的 N-糖谱分析。

治疗性单克隆抗体（mAbs）因其治疗各种疾病的有效性而成为现代医学的关键。然而，mAbs 结构复杂，尤其是其糖基化模式，给质量控制和生物相似性评估带来了挑战。本研究探索了利用上铰链中间向上（UHMU）级超高效液相色谱-高分辨质谱（LC-HRMS）分析来改进 mAbs 的 N-糖谱分析。通过UHMU/LC-HRMS工作流程，两种被称为IgG降解酶（IGDEs）的特异性酶被用来选择性地裂解铰链区以上的治疗用mAbs，以分离抗体亚基，进一步进行Fc糖分析。与完整的质谱水平相比，IGDEs 消化的 mAbs 质谱的复杂性大大降低，因此可以对配对的 Fc 糖型进行可靠的分配和相对定量。对九种获批治疗药物进行的 UHMU/LC-HRMS 分析结果突显了这种方法对深入分析糖形的重要意义。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Antibodies IMMUNOLOGY-

CiteScore

7.10

自引率

6.40%

发文量

审稿时长

11 weeks

期刊介绍： Antibodies (ISSN 2073-4468), an international, peer-reviewed open access journal which provides an advanced forum for studies related to antibodies and antigens. It publishes reviews, research articles, communications and short notes. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. There is no restriction on the length of the papers. Full experimental and/or methodical details must be provided. Electronic files or software regarding the full details of the calculation and experimental procedure - if unable to be published in a normal way - can be deposited as supplementary material. This journal covers all topics related to antibodies and antigens, topics of interest include (but are not limited to): antibody-producing cells (including B cells), antibody structure and function, antibody-antigen interactions, Fc receptors, antibody manufacturing antibody engineering, antibody therapy, immunoassays, antibody diagnosis, tissue antigens, exogenous antigens, endogenous antigens, autoantigens, monoclonal antibodies, natural antibodies, humoral immune responses, immunoregulatory molecules.

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