High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns.
Anal Chem
; 95(27): 10448-10456, 2023 07 11.
Article
em En
| MEDLINE
| ID: mdl-37384898
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligonucleotídeos
/
Proteínas
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Suíça