Phos-tag-based micropipette-tip method for analysis of phosphomonoester-type impurities in synthetic oligonucleotides

Masaya Tsunehiro; Kenji Sasaki; Emiko Kinoshita-Kikuta; Eiji Kinoshita; Tohru Koike

doi:10.1016/j.jchromb.2020.122198

Phos-tag-based micropipette-tip method for analysis of phosphomonoester-type impurities in synthetic oligonucleotides

J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 30:1151:122198. doi: 10.1016/j.jchromb.2020.122198. Online ahead of print.

Authors

Masaya Tsunehiro¹, Kenji Sasaki², Emiko Kinoshita-Kikuta³, Eiji Kinoshita⁴, Tohru Koike⁵

Affiliations

¹ Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Analytical Research Department, Production Technology and Supply Chain Management Division, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan.
² Analytical Research Department, Production Technology and Supply Chain Management Division, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan.
³ Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
⁴ Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Electronic address: kinoeiji@hiroshima-u.ac.jp.
⁵ Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Electronic address: tkoike@hiroshima-u.ac.jp.

PMID: 32512534
DOI: 10.1016/j.jchromb.2020.122198

Abstract

Various chromatographic techniques, combined with mass spectrometry, have been developed for the analysis of impurities in oligonucleotide drugs, but those methods have generally been less focused on possible phosphomonoester-type compounds. Here, we introduce a simple method for separating terminally phosphorylated impurities from parent oligonucleotides by using a phosphate-affinity micropipette tip (Phos-tag tip). All steps for the phosphate-affinity separation (binding, washing, and elution) are conducted in aqueous buffers at neutral pH. The entire separation protocol requires less than 30 min per sample. In practical examples, we demonstrated that phosphorylated impurities in natural-type and chemically modified oligonucleotides can be efficiently separated by the Phos-tag tip method and subsequently characterized by using ion-pairing reversed-phase liquid chromatography mass spectrometry (IP-RPLC-MS). Thus, a combination of the Phos-tag tip method and IP-RPLC-MS is useful for characterizing and identifying phosphomonoester-type impurities in oligonucleotide drugs.

Keywords: Liquid chromatography; Mass spectrometry; Micropipette-tip method; Oligonucleotide therapeutics; Phos-tag technology.