Preconcentration and separation of antisense oligonucleotides by on-column isotachophoresis and capillary electrophoresis in polymer-filled capillaries

Small, single-stranded, chemically modified oligonucleotides, complementary to a specific gene section, commonly referred to as antisense compounds, are being investigated as potential therapeutic drugs. A number of modified oligonucleotides, in particular phosphorothioates, are in clinical development. Shorter fragments are found as metabolic products. Isotachophoresis (ITP) allows the introduction of large, diluted sample plugs into the separation capillary. In this work, ITP and capillary electrophoresis (CE) in polymer solutions were successfully coupled in a single capillary in a commercial instrument to increase sensitivity with UV detection and to shorten the time for sample pretreatment. It was shown that ITP-CE can be used as a preconcentration and clean-up method for phosphodiester- and phosphorothioate-containing samples. Up to 3 microL sample could be injected into the capillary without significantly disturbing the separation performance. ITP-CE of phosphodiesters directly out of salt- and protein-containing samples could be demonstrated. For phosphorothioates in serum samples an additional sample clean-up was necessary, due to oligonucleotide-protein binding. An optimized replaceable polymer solution was developed to increase the separation performance for heterogeneous phosphorothioates. A dextran-based sieving medium showed a good separation performance in ITP-CE of phosphorothioates. A concentration detection limit of 8.10(-9) mol/L for the 20-mer phosphorothioate ISIS5132, isolated from rat serum, was found.