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

アクセス数:381件（2025-07-19 20:49 集計）

固定URL: https://hdl.handle.net/11094/100579

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タイトル	Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography
著者	著者名 Hayashida, Momoka
	著者名 Suzuki, Risa
	著者名 Horie, Shinnosuke
	著者名 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 著者名 Yamaguchi, Takao
	NRID 1000080243252 1000080243252 ORCID 0000-0002-6842-6812 0000-0002-6842-6812 researchmap permalink read0014368 read0014368 Scopus Author ID 7003800550 7003800550 著者名 Obika, Satoshi
キーワード等	Deamination
	Oligonucleotide
	Supercritical fluid chromatography
内容	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.
抄録	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.
出版者	Elsevier B.V.
収録物名	Journal of Chromatography A
巻(号)	1744
ページ	465731
刊行年月	2025-03-15
言語	英語
ハンドルURL	https://hdl.handle.net/11094/100579
PISSN	00219673
eISSN	18733778
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1016/j.chroma.2025.465731
アクセス権	open access
権利情報	This article is licensed under a Creative Commons Attribution 4.0 International License.
出版タイプ	Version of Record
カテゴリ	学術雑誌論文 Journal Article

資源タイプ	学術雑誌論文
ローカル資源タイプ	学術雑誌論文
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	英語
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	英語
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

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