Retention behavior of short double-stranded oligonucleotide and its potential impurities by anion-exchange chromatography under non-denaturing conditions

•AEX for quality control of siRNA as non-denatured forms under alkaline conditions.•Optimized conditions showed potential to separate excess single strands adequately.•Non-optimal duplexes with truncated ends were eluted at different retention times.•Non-optimal duplexes with bulge structures were eluted at different retention times.•A retention model of short double-strand with overhang bases was proposed. Small interfering RNA (siRNA), consisting of two complementary single-stranded RNAs with overhanging bases, is being adopted as a potent and specific inhibitor of target gene expression. However, non-duplexed single strands and undesired double strands composed of impurities (e.g., n-1 mer) could be produced in addition to the target double strand in the siRNA manufacturing process. Compared to the liquid chromatography analysis of single strands, the analysis of the duplexes under non-denaturing conditions is challenging, since restricted chromatographic conditions are required to maintain the Watson-Crick hydrogen bonds. This study reports the analysis of double-stranded oligomers having approximately 20 base pairs with some overhanging bases as non-denatured forms by anion-exchange chromatography (AEX). Optimization of the chromatographic conditions could potentially achieve the adequate separation of excess single strands from the double strand. Non-optimal duplexes, such as duplexes with long overhangs or bulge structures, were prepared by intentionally deleting terminal or middle nucleotide(s) of either the sense or the antisense strand, and these non-optimal duplexes were eluted at different retention times in most of the cases. Interestingly, under alkaline chromatographic conditions (pH 9.0), non-optimal duplexes containing a shortmer tended to exhibit a stronger retention than their parent duplexes, although they possessed a less negative charge. This study demonstrated some retention behavior of double strands with overhangs by AEX under non-denaturing conditions.