一种用于单克隆抗体自聚集和稳定性测定的简易凝胶电泳-本征荧光成像方法

IF 6.7 1区 化学 Q1 CHEMISTRY, ANALYTICAL
Zixian Yu, Yiren Cao, Lei Wang, Youli Tian, Rui Zou, Keer Chen, Weiwen Liu, Qiang Zhang, Qingjiang Wang, Baohong Zhang, Chengxi Cao
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引用次数: 0

摘要

单克隆抗体(mab)广泛应用于肿瘤和自身免疫性疾病的治疗和临床诊断,但单克隆抗体易自聚集,特别是二聚体的形成,影响其稳定性和疗效,并可能引起严重的过敏。传统的检测mAb二聚体的方法,如TEM、AUC、DLS、SEC和CE,受低通量、高成本和定性数据的限制,不适合生物制药行业的大规模样品分析和抗体研究。为了解决这些问题,我们开发了一种简单的蛋白质交联凝胶电泳在线本禀荧光成像(PC-GE-IFI)方法,用于单克隆抗体的自聚集和稳定性分析。该方法能够实时定量检测单克隆抗体(mAb)单体和二聚体,灵敏度高,检出限低(单体:0.9 nM;二聚体:0.45 nM)和较宽的动态范围(单体:2.50-2500 nM;二聚体:1.25-1250 nM)。此外,样品孔可以作为窗口,用于测定mAb聚集形成的沉淀。该方法支持在各种应力条件下准确评估mAb二聚化和单体纯度,包括热应力、机械搅拌和冻融循环。此外,该方法允许在不同浓度的多个样品中同时分析二聚体和沉淀物,而非线性拟合提供了单体-二聚体相互作用的解离常数(Kd),这是一个有助于评估聚集倾向的关键参数,并为单抗产品的设计和开发提供信息。PC-GE-IFI方法在单抗、双特异性抗体、adc和蛋白质药物的开发、质量控制和安全性评估方面具有巨大潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

A Facile Method for Gel Electrophoresis with Intrinsic Fluorescence Imaging for Self-Aggregation and Stability Assay of Monoclonal Antibody

A Facile Method for Gel Electrophoresis with Intrinsic Fluorescence Imaging for Self-Aggregation and Stability Assay of Monoclonal Antibody
Monoclonal antibodies (mAbs) are widely used in tumor and autoimmune disease therapy and clinical diagnosis, but they suffer from self-aggregation, particularly dimer formation, impacting their stability, efficacy, and potentially causing severe allergies. Traditional methods for detecting mAb dimers, such as TEM, AUC, DLS, SEC, and CE, are limited by low throughput, high costs, and qualitative data, making them unsuitable for large-scale sample assays in biopharmaceuticals industry as well as antibody research. To address these issues, we developed a facile method of protein cross-linking gel electrophoresis with online intrinsic fluorescence imaging (PC-GE-IFI) for self-aggregation and stability assays of mAbs. This method enables the real-time quantification of monoclonal antibody (mAb) monomers and dimers with exceptional sensitivity, characterized by low detection limits (monomer: 0.9 nM; dimer: 0.45 nM) and a broad dynamic range (monomer: 2.50–2500 nM; dimer: 1.25–1250 nM). Furthermore, the sample wells can be used as windows for the assay of precipitates formed by mAb aggregation. The method supports accurate assessment of mAb dimerization and monomer purity under various stress conditions, including thermal stress, mechanical agitation, and freeze–thaw cycles. Moreover, the method allows concurrent analysis of dimers and precipitates across multiple samples at different concentrations, while nonlinear fitting provides the dissociation constant (Kd) for monomer–dimer interactions, a critical parameter that aids in assessing aggregation propensity, and informs the design and development of mAb products The PC-GE-IFI method has great potential for development, quality control, and safety assessment of mAbs, bispecific antibodies, ADCs, and protein drugs.
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来源期刊
Analytical Chemistry
Analytical Chemistry 化学-分析化学
CiteScore
12.10
自引率
12.20%
发文量
1949
审稿时长
1.4 months
期刊介绍: Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.
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