Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography

Hayashida, Momoka; Suzuki, Risa; Horie, Shinnosuke; Masuda, Junichi; Yamaguchi, Takao; Obika, Satoshi

doi:https://doi.org/10.1016/j.chroma.2025.465731

Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography

Hayashida, Momoka; Suzuki, Risa; Horie, Shinnosuke et al.

Journal of Chromatography A, 2025, 1744, 465731

Number of Access:406（2025-08-15 01:09 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/100579

Link to primary information

File	Format	Terms of use	Size	Views	Date.Available	Description	information
JChromatogrA_1744_465731	pdf	None	3.85 MB	71	2025.03.14

Creative Commons : Attribution

Item Information

Output File Export EndNote Basic Export Mendeley

Title	Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography
Creator	CreatorName Hayashida, Momoka
	CreatorName Suzuki, Risa
	CreatorName Horie, Shinnosuke
	CreatorName Masuda, Junichi
	NRID 1000080596601 1000080596601 ORCID 0000-0003-3180-0257 0000-0003-3180-0257 researchmap permalink takao-yamaguchi takao-yamaguchi Scopus Author ID 56322895500 56322895500 CreatorName Yamaguchi, Takao
	NRID 1000080243252 1000080243252 ORCID 0000-0002-6842-6812 0000-0002-6842-6812 researchmap permalink read0014368 read0014368 Scopus Author ID 7003800550 7003800550 CreatorName Obika, Satoshi
Subject	Deamination
	Oligonucleotide
	Supercritical fluid chromatography
Description	Hayashida M., Suzuki R., Horie S., et al. Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography. Journal of Chromatography A 1744, 465731 (2025); https://doi.org/10.1016/j.chroma.2025.465731.
Abstract	With recent advancements concerning the optimization of the analytical conditions, it is feasible to analyze polar molecules using supercritical fluid chromatography (SFC). In this study, the applicability of SFC is evaluated for analyzing 5-, 10-, 15-, and 18-mer oligonucleotides, and SFC is then applied to analyze deaminated products, which are side products generated during oligonucleotide synthesis. These side products are difficult to separate from the target oligonucleotide, with the difficulty varying depending on the deamination position and sequences, even when using ion-pair reversed-phase liquid chromatography (IP-RPLC), a common method for oligonucleotide analysis. Our results demonstrate that SFC, with octylamine as a modifier additive, can achieve sharp chromatographic peaks for 5-, 10-, 15-, and 18-mer oligonucleotides modified with 2′-O-methoxyethyl RNA (2′-MOE), regardless of the presence of the hydrophobic 4,4′-dimethoxytrityl (DMTr) group on the sequence. After optimization of the column oven temperature, modifier additive, and stationary phase, SFC successfully separated oligonucleotides with various numbers and positions of deamination from the target oligonucleotide. SFC exhibited different selectivities for DMTr-on and DMTr-off oligonucleotides compared with those for IP-RPLC, which indicates that SFC can serve as a valuable alternative tool for the purification and analysis of oligonucleotides.
Publisher	Elsevier B.V.
Source Title	Journal of Chromatography A
Volume (Issue)	1744
Page	465731
Date of Issued	2025-03-15
Language	English
Handle URL	https://hdl.handle.net/11094/100579
PISSN	00219673
eISSN	18733778
Relation.isIdenticalTo	DOI (Publisher Version) https://doi.org/10.1016/j.chroma.2025.465731
Access Rights	open access
CopyRight	This article is licensed under a Creative Commons Attribution 4.0 International License.
oaire:version	Version of Record
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCTERMS.bibliographicCitation	Journal of Chromatography A. 2025, 1744, p. 465731
DC.title	Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography
DC.creator	Hayashida, Momoka
	Suzuki, Risa
	Horie, Shinnosuke
	Masuda, Junichi
	Yamaguchi, Takao
	Obika, Satoshi
DC.publisher	Elsevier B.V.
DC.language' scheme='DCTERMS.RFC1766	English
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2025-03-15
DC.identifier	https://doi.org/10.1016/j.chroma.2025.465731
DC.subject	Deamination
	Oligonucleotide
	Supercritical fluid chromatography
DC.description	Hayashida M., Suzuki R., Horie S., et al. Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography. Journal of Chromatography A 1744, 465731 (2025); https://doi.org/10.1016/j.chroma.2025.465731.
DCTERMS.abstract	With recent advancements concerning the optimization of the analytical conditions, it is feasible to analyze polar molecules using supercritical fluid chromatography (SFC). In this study, the applicability of SFC is evaluated for analyzing 5-, 10-, 15-, and 18-mer oligonucleotides, and SFC is then applied to analyze deaminated products, which are side products generated during oligonucleotide synthesis. These side products are difficult to separate from the target oligonucleotide, with the difficulty varying depending on the deamination position and sequences, even when using ion-pair reversed-phase liquid chromatography (IP-RPLC), a common method for oligonucleotide analysis. Our results demonstrate that SFC, with octylamine as a modifier additive, can achieve sharp chromatographic peaks for 5-, 10-, 15-, and 18-mer oligonucleotides modified with 2′-O-methoxyethyl RNA (2′-MOE), regardless of the presence of the hydrophobic 4,4′-dimethoxytrityl (DMTr) group on the sequence. After optimization of the column oven temperature, modifier additive, and stationary phase, SFC successfully separated oligonucleotides with various numbers and positions of deamination from the target oligonucleotide. SFC exhibited different selectivities for DMTr-on and DMTr-off oligonucleotides compared with those for IP-RPLC, which indicates that SFC can serve as a valuable alternative tool for the purification and analysis of oligonucleotides.
DC.rights	This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.format	100579
citation_title	Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography
citation_author	Hayashida, Momoka
	Suzuki, Risa
	Horie, Shinnosuke
	Masuda, Junichi
	Yamaguchi, Takao
	Obika, Satoshi
citation_publisher	Elsevier B.V.
citation_language	English
citation_date	2025-03-15
citation_journal_title	Journal of Chromatography A
citation_volume	1744
citation_firstpage	465731
citation_doi	https://doi.org/10.1016/j.chroma.2025.465731
citation_public_url	https://hdl.handle.net/11094/100579

Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography

Link to primary information

Item Information

Show details