Offline Coupling of Hydrophobic Interaction Chromatography–Capillary Zone Electrophoresis for Monitoring Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies

IF 3 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Deepika Sarin, Sunil Kumar, Anurag S. Rathore
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引用次数: 0

Abstract

A holistic understanding of the charge heterogeneity in monoclonal antibodies (mAbs) is paramount for ensuring acceptable product quality. Hence, biotherapeutic manufacturers are expected to thoroughly characterize their products via advanced analytical techniques. Recently, two-dimensional liquid chromatography (2DLC) methods have gained popularity for resolving complex charged species. Capillary electrophoresis (CE) is regarded as a sensitive and faster tool for charged species estimation in biotherapeutics. In this study, we aim to combine the separation power of chromatographic and electrophoretic tools (liquid chromatography [LC]–CE) so as to achieve maximum resolution of mAb charge variants. Hydrophobic interaction chromatography (HIC) has been used as the preferred LC mode with CE for achieving successful separation of both charge and hydrophobic variants for two of the mAbs (trastuzumab and rituximab). The standalone HIC and capillary zone electrophoresis (CZE) methods separated 4 hydrophobic variants and 7 charge variants for each mAb, whereas the 2DLC method separated 10 and 11 variants for mAbs A and B. On the other hand, the HIC–CZE–UV method resolved 29 variants in mAb A and 23 variants in mAb B. The reproducibility of the HIC–CZE–UV method was demonstrated by % change in values of retention time (RT) and peak area as <5% (mAb A), <3% (mAb B), and <12% (for both mAbs), respectively. Thus, the utility of the proposed LC–CE method for characterization of mAb charge variants has been displayed.

离线耦合疏水相互作用色谱-毛细管区电泳,用于监测重组单克隆抗体的电荷异质性。
全面了解单克隆抗体(mAbs)中的电荷异质性对于确保产品质量合格至关重要。因此,生物制药生产商需要通过先进的分析技术对其产品进行彻底表征。最近,二维液相色谱(2DLC)方法在分辨复杂带电物种方面越来越受欢迎。毛细管电泳(CE)被认为是估算生物治疗药物中带电物种的灵敏而快速的工具。在本研究中,我们旨在结合色谱和电泳工具(液相色谱 [LC]- CE)的分离能力,从而最大限度地分辨 mAb 的电荷变体。疏水相互作用色谱(HIC)已被用作首选的液相色谱模式,并配合 CE 成功分离了两种 mAb(曲妥珠单抗和利妥昔单抗)的电荷变体和疏水变体。独立的 HIC 和毛细管区带电泳 (CZE) 方法分离了每种 mAb 的 4 种疏水变体和 7 种电荷变体,而 2DLC 方法则分离了 mAb A 和 B 的 10 种和 11 种变体。
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
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.
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