Vanhinsbergh, C., Hook, E.C., Oxby, N. et al. (1 more author) (2023) Optimization of orthogonal separations for the analysis of oligonucleotides using 2D-LC. Journal of Chromatography B, 1227. 123812. ISSN 1570-0232
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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Copyright, Publisher and Additional Information: | © 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). | ||||||
Keywords: | 2D HPLC; Anion exchange HPLC; HPLC; Ion pair reversed phase HPLC; Oligonucleotides | ||||||
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Institution: | The University of Sheffield | ||||||
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) | ||||||
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Depositing User: | Symplectic Sheffield | ||||||
Date Deposited: | 23 Aug 2023 11:30 | ||||||
Last Modified: | 23 Aug 2023 11:30 | ||||||
Status: | Published | ||||||
Publisher: | Elsevier BV | ||||||
Refereed: | Yes | ||||||
Identification Number: | https://doi.org/10.1016/j.jchromb.2023.123812 | ||||||
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