GRAM-SCALE PURIFICATION OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING ION-EXCHANGE DISPLACEMENT CHROMATOGRAPHY

The purification of oligonucleotides by ion-exchange displacement chro matography is demonstrated on the gram-scale. Using a 50 mmD x 100 mmL (203 ml) column operated in the displacement mode, 1.2 g of a 24mer p hosphorothioate oligonucleotide was purified. Product yield for this s eparation was 70% (780 mg) at a purity of 96.4% and the mass balance r ecovery of all oligonucleotide was 97.5%. The displacement purificatio n of four additional phosphorothioate oligonucleotides ranging in leng th from 18 to 25 bases is also demonstrated on the semi-preparative (1 0-50 mg) scale. All of these oligonucleotides were purified using simi lar displacement conditions and typical results were 60% yield at 96% purity. The displacement portion of these separations required <15 min and total cycle time including equilibration, feed loading and regene ration can be performed in under 30 min. These results seem to indicat e that displacement chromatography may be amenable to generalizations in separation protocol that would greatly reduce the effort required t o obtain an optimized purification scheme for moderately long oligonuc leotides.