The Hidden Influence: Impacts of Residual Dimethylformamide in NDSB-211 on icIEF Separation for Monoclonal Antibodies.

IF 2.5 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

ELECTROPHORESIS Pub Date : 2025-09-08 DOI:10.1002/elps.70024

Delaney Doran, Matthew Myers, Katarina Moravcevic, Steven LaBrenz, Pinger Wang, Xiaoying Ji

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引用次数: 0

Abstract

Monoclonal antibodies (mAbs) present analytical challenges due to their inherent heterogeneity and susceptibility to post-translational modifications (PTMs) during production and storage. Monitoring of charge heterogeneity profiles by imaged capillary isoelectric focusing (icIEF) has been aided by the use of non-detergent sulfobetaines (NDSBs), particularly NDSB-211, to enhance protein solubility and stability. When used in a quality control laboratory setting, NDSB-211 has shown performance variability over time due to residual manufacturing impurities that impact the capillary isoelectric focusing separation. Dimethylformamide (DMF) was detected using ¹H nuclear magnetic resonance (NMR) in several lots of NDSB-211 that simultaneously produced poor resolution of basic variants of a mAb on icIEF. A parallel spiking study using DMF at 1%-10% (v/v) on ¹H NMR and icIEF illustrates the interference in charge heterogeneity separation when a large amount of DMF is present in the NDSB-211 reagent. As DMF is not found on the NDSB-211 certificate of analysis from any vendor, more traditional additives for icIEF are suggested to limit the potential interference for analytical methods.

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隐性影响：NDSB-211中残留二甲基甲酰胺对单克隆抗体icIEF分离的影响。

单克隆抗体（mab）由于其固有的异质性和在生产和储存过程中对翻译后修饰（PTMs）的易感性，给分析带来了挑战。使用非洗涤剂磺胺甜菜碱（NDSB-211），特别是NDSB-211，有助于通过成像毛细管等电聚焦（icIEF）监测电荷异质性，以提高蛋白质的溶解度和稳定性。当在质量控制实验室环境中使用时，由于残余的制造杂质会影响毛细管等电聚焦分离，NDSB-211表现出性能随时间的变化。二甲基甲酰胺（DMF）采用1H核磁共振（NMR）在几批NDSB-211中检测，同时在icIEF上产生单抗的基本变体的低分辨率。用DMF在1% ~ 10% （v/v）时在1H NMR和icIEF上的平行峰研究表明，当大量DMF存在于NDSB-211试剂中时，会干扰电荷非均质性分离。由于没有在任何供应商的NDSB-211分析证书上找到DMF，因此建议使用更传统的icIEF添加剂来限制分析方法的潜在干扰。

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

ELECTROPHORESIS 生物-分析化学

CiteScore

6.30

自引率

13.80%

发文量

244

审稿时长

1.9 months

期刊介绍： ELECTROPHORESIS is an international journal that publishes original manuscripts on all aspects of electrophoresis, and liquid phase separations (e.g., HPLC, micro- and nano-LC, UHPLC, micro- and nano-fluidics, liquid-phase micro-extractions, etc.). Topics include new or improved analytical and preparative methods, sample preparation, development of theory, and innovative applications of electrophoretic and liquid phase separations methods in the study of nucleic acids, proteins, carbohydrates natural products, pharmaceuticals, food analysis, environmental species and other compounds of importance to the life sciences. Papers in the areas of microfluidics and proteomics, which are not limited to electrophoresis-based methods, will also be accepted for publication. Contributions focused on hyphenated and omics techniques are also of interest. Proteomics is within the scope, if related to its fundamentals and new technical approaches. Proteomics applications are only considered in particular cases. Papers describing the application of standard electrophoretic methods will not be considered. Papers on nanoanalysis intended for publication in ELECTROPHORESIS should focus on one or more of the following topics: • Nanoscale electrokinetics and phenomena related to electric double layer and/or confinement in nano-sized geometry • Single cell and subcellular analysis • Nanosensors and ultrasensitive detection aspects (e.g., involving quantum dots, "nanoelectrodes" or nanospray MS) • Nanoscale/nanopore DNA sequencing (next generation sequencing) • Micro- and nanoscale sample preparation • Nanoparticles and cells analyses by dielectrophoresis • Separation-based analysis using nanoparticles, nanotubes and nanowires.