Hydrophilic interaction liquid chromatography with mass spectrometry for the separation and identification of antisense oligonucleotides impurities and nusinersen metabolites.

Autor: Vosáhlová Z; Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 12800, Prague, Czech Republic., Kalíková K; Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 12800, Prague, Czech Republic. Electronic address: kveta.kalikova@natur.cuni.cz., Gilar M; Waters Corporation, 34 Maple Street, Milford, MA 01757, USA., Szymarek J; Department of Developmental Neurology, Medical University of Gdansk, 7 Dębinki Str., PL-80-952, Gdańsk, Poland., Mazurkiewicz-Bełdzińska M; Department of Developmental Neurology, Medical University of Gdansk, 7 Dębinki Str., PL-80-952, Gdańsk, Poland., Studzińska S; Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 12800, Prague, Czech Republic; Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, 7 Gagarin Str., PL-87-100 Toruń, Poland; Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University in Toruń, 4 Wilenska St., 87-100 Toruń, Poland. Electronic address: kowalska@umk.pl.
Jazyk: angličtina
Zdroj: Journal of chromatography. A [J Chromatogr A] 2024 Jan 04; Vol. 1713, pp. 464535. Date of Electronic Publication: 2023 Nov 26.
DOI: 10.1016/j.chroma.2023.464535
Abstrakt: With the development of therapeutic oligonucleotides for antisense and gene therapies, the demand for analytical methods also increases. For the analysis of complex samples, for example plasma samples, where the use of mass detection is essential, hydrophilic interaction liquid chromatography is a suitable choice. The aim of the present work was to develop a method for separation and identification of the oligonucleotide impurities and metabolites by hydrophilic interaction liquid chromatography. First of all, the effects of different chromatographic conditions (e.g. pH of the aqueous part of the mobile phase, buffer concentration, column temperature) on the retention and separation of phosphorothioate oligonucleotides standards on the amide stationary phase were investigated. A set of model oligonucleotides containing a fully modified 21mer and its typical impurities (shortmers and oligonucleotides with different number of thiophosphate modifications) was used. The results showed that the concentration of the salt in the mobile phase as well as its pH, are the most influential parameters with regard to peak shape and separation. The knowledge gained was applied to the analysis of an unpurified 18mer oligonucleotides, analogues of the drug nusinersen used for the treatment of spinal muscular atrophy. The successful separation and identification of twenty-six and twenty-eight impurities was performed with the developed HILIC method. The method was applied to analysis of nusinersen metabolites of serum samples of patients treated with Spinraza.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023. Published by Elsevier B.V.)
Databáze: MEDLINE
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