Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides.

The complementary nature of anion exchange chromatography and capillary gel electrophoresis for oligonucleotide analysis is demonstrated by evaluating a comprehensive series of authentic deletion sequences and partial phosphodiester analogs of five phosphorothioate oligonucleotides of different base composition and sequence. While anion exchange HPLC is sensitive to differences in backbone length of phosphorothioate oligonucleotides, oligomers with length difference of one base unit are not resolved. Capillary gel electrophoresis, on the other hand, has excellent single-base resolution while being relatively insensitive to phosphate in the phosphorothioate backbone. The data definitively establish the necessity of employing both separation techniques for adequate characterization of lower order process-related impurities potentially found in synthetic phosphorothioate oligonucleotides.