Optimization of orthogonal separations for the analysis of oligonucleotides using 2D-LC

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B Pub Date : 2023-07-01 DOI:10.1016/j.jchromb.2023.123812

Christina Vanhinsbergh , Elliot C. Hook , Nicola Oxby , Mark J. Dickman

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引用次数: 0

Abstract

Oligonucleotides are commonly analysed using one dimensional chromatography (1D-LC) to resolve and characterise manufacturing impurities, structural isomers and (in respect to emerging oligonucleotide therapeutics) drug substance and drug product. Due to low selectivity and co-elution of closely related oligonucleotides using 1D-LC, analyte resolution is challenged. This leads to the requirement for improved analytical methods. Multidimensional chromatography has demonstrated utility in a range of applications as it increases peak capacity using orthogonal separations, however there are limited studies demonstrating the 2D-LC analysis of closely related oligonucleotides. In this study we optimised OGN size and sequence based separations using a variety of 1D-LC methods and coupled these orthogonal modes of chromatography within a 2D-LC workflow. Theoretical 2D-LC workflows were evaluated for optimal orthogonality using the minimum convex hull metric. The most orthogonal workflow identified in this study was ion-pair reversed phase using tributylammonium acetate (IP-RP-TBuAA) coupled with strong anion exchange in conjunction with sodium perchlorate (SAX-NaClO₄) at high mobile phase pH. We developed a heart-cut (IP-RP-TBuAA)-(SAX-NaClO₄) 2D-LC method for analysis of closely related size and sequence variant OGNs and OGN manufacturing impurities. The 2D-LC method resulted in an increased orthogonality and a reduction in co-elution (or close elution). Application of a UV based reference mapping strategy in conjunction with the 2D-LC method demonstrated a reduction in analytical complexity by reducing the reliance on mass based detection methods.

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2D-LC分析寡核苷酸的正交分离优化

寡核苷酸通常使用一维色谱(1D-LC)来分析和表征制造杂质、结构异构体和(关于新兴的寡核苷酸疗法)原料药和药品。由于低选择性和密切相关的寡核苷酸的共洗脱使用1D-LC，分析物的分辨率受到挑战。这就要求改进分析方法。多维色谱法已经在一系列应用中证明了实用性，因为它使用正交分离增加了峰值容量，然而，证明密切相关的寡核苷酸的2D-LC分析的研究有限。在本研究中，我们使用多种1D-LC方法优化了OGN大小和基于序列的分离，并将这些正交色谱模式耦合到2D-LC工作流程中。使用最小凸壳度量来评估理论2D-LC工作流的最佳正交性。本研究确定的最正交工作流程是在高流动相ph下使用乙酸三丁铵(IP-RP-TBuAA)与强阴离子交换结合高氯酸钠(SAX-NaClO4)进行离子对反相。我们开发了一种心脏切割(IP-RP-TBuAA)-(SAX-NaClO4) 2D-LC方法，用于分析密切相关的大小和序列变异的OGN和OGN制造杂质。2D-LC方法增加了正交性，减少了共洗脱(或近洗脱)。将基于UV的参考映射策略与2D-LC方法相结合，通过减少对基于质量的检测方法的依赖，证明了分析复杂性的降低。

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

Journal of Chromatography B 医学-分析化学

CiteScore

5.60

自引率

3.30%

发文量

306

审稿时长

44 days

期刊介绍： The Journal of Chromatography B publishes papers on developments in separation science relevant to biology and biomedical research including both fundamental advances and applications. Analytical techniques which may be considered include the various facets of chromatography, electrophoresis and related methods, affinity and immunoaffinity-based methodologies, hyphenated and other multi-dimensional techniques, and microanalytical approaches. The journal also considers articles reporting developments in sample preparation, detection techniques including mass spectrometry, and data handling and analysis. Developments related to preparative separations for the isolation and purification of components of biological systems may be published, including chromatographic and electrophoretic methods, affinity separations, field flow fractionation and other preparative approaches. Applications to the analysis of biological systems and samples will be considered when the analytical science contains a significant element of novelty, e.g. a new approach to the separation of a compound, novel combination of analytical techniques, or significantly improved analytical performance.