Offline comprehensive liquid chromatography in combination with a Deoxyribonuclease I immobilized enzymatic reactor for selective screening of oligonucleotide mixtures

• An enzymatic reactor was integrated into an offline comprehensive LC × LC platform.
• Qualitative degradation profiles of mixtures of oligonucleotides can be generated.
• DNase I reactors were used to differentiate stable from labile oligomers.
• The methodology allows stability assessment of oligonucleotides mixtures.

The development of a comprehensive ion-pair chromatography-immobilized enzyme reactor × ion-pair chromatography (IPC-IMER × IPC) methodology for the advanced characterization of DNA/RNA oligonucleotides (ONs) mixtures has been carried out. More in detail, a DNase I IMER has been coupled to IPC in the post column configuration, followed by the collection of the eluting fractions and reanalysis by IPC. The effect of the mobile phase over the IMER activity was qualitatively evaluated. The methodology proved to generate relevant ON degradation profiles that might be correlated with the ON stability towards nucleases. Moreover, this platform shows potential for its further implementation in selective analysis of ON mixtures and in mapping studies.