Analysis of antisense oligonucleotides by capillary electrophoresis, gel-slab electrophoresis, and HPLC: a comparison

Three techniques are evaluated for assessing the purity of synthetic oligodeoxyribonucleotides: reversed phase high-performance liquid chromatography (RP-HPLC), polyacrylamide gel-slab electrophoresis (PAGE), and capillary zone electrophoresis highly concentrated (18% T) entangled polymer networks (CZE). RP-HPLC does not seem to be able to discriminate and resolve the spectrum of failed sequences expected to accompany an oligonucleotide of a given length. The purity data, as given by the manufacturer, are most often close to 100%. PAGE in 20% T matrices, followed by ethidium bromide staining, gives a good resolution of failure sequences and purity assessments decidedly more realistic. CZE in 18% liquid polyacrylamide is able to resolve to baseline all shorter fragments and to give a precise evaluation of the amount of impurities, based on the intrinsic DNA absorbance at 254 nm. Most of the 18 mer oligonucleotides (and of their phosphorothioate derivatives) analyzed by us, as supplied by three different manufacturers, were found to be contaminated by a spectrum of failed and truncated sequences, ranging in size from 7 mer to 17 mer. The purity data rarely exceeded 80% and most often were of the order of 60%-70%. Conditions for a good routine performance of the CZE technique are described.