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English

High-throughput chromatographic separation of oligonucleotides: a proof of concept using ultra-short columns

Contributors

Lardeux, Honorine; Fekete, Szabolcs; Lauber, Matthew; D'Atri, Valentina; Guillarme, Davy

Published in

Analytical chemistry, vol. 95, no. 27, p. 10448-10456

Publication date

2023-06-29

First online date

2023-06-29

Abstract

Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an "on-off" elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.

Keywords

  • Oligonucleotides
  • Biopolymers
  • Mixtures
  • Molecular modeling
  • Phase transitions
  • Solution chemistry
  • Proteins

UNIGE affiliation entities

UNIGE research groups

Citation (ISO format)

LARDEUX, Honorine et al. High-throughput chromatographic separation of oligonucleotides: a proof of concept using ultra-short columns. In: Analytical chemistry, 2023, vol. 95, n° 27, p. 10448–10456. doi: 10.1021/acs.analchem.3c01934

Main files (1)

Article (Published version)
CC BY-4.0

Secondary files (1)

Supplemental data

Identifiers

Additional URL for this publication

https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934

Journal ISSN

0003-2700
98views
210downloads

Technical informations

Creation

13/07/2023 08:20:28

First validation

14/08/2023 08:26:33

Update

14/08/2023 08:26:33

Status update

14/08/2023 08:26:33

Last indexation

01/11/2024 05:47:36
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