A novel ion-exchange stationary phase will be developed for displacement chromatography of synthetic phosphorothioate oligonucleotide. To date, this class of compounds are purified using ion-exchange and reversed phase-chromatography with non-linear elution techniques. These methods suffer from low recovery, non-predictable selectivity and very un- economical use of the stationary phases. The proposed research will focus on the optimization of the surface chemistry with the use of minimal amount of oligonucleotides. The base material will be made of non-porous polystyrene divinylbenzene with high chemical and physical stability. The surface of the polystyrene support will be coated first with a covalent, non-charged, hydroxyl rich, hydrophilic layer. The hydroxyl groups are then reacted with amine functionalities and, secondary interactions are controlled by derivatizing the resulting amines with activated glycols. The optimized surface will separate phosphorothioate oligonucleotides based exclusively on their length even at overloaded conditions. The separation power of displacement chromatography on the new columns will be demonstrated by micropreparative purification of crude synthetic phosphorothioate oligonucleotides. The proposed research will provide new test methods for analyzing chromatographic surfaces for displacement chromatography as well.
Commercialization will start with providing services for the separation of synthetic oligonucleotide for researchers and separation engineers at national and private laboratories. The new ion-exchange chromatography products will potentially be used by the oligonucleotide therapeutics industry whose first generation products will enter to the market early in the next century.
