Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography.
The purification of oligonucleotides by ion-exchange displacement chromatography 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 phosphorothioate oligonucleotide was purified. Product yield for this separation was 70% (780 mg) at a purity of 96.4% and the mass balance recovery of all oligonucleotide was 97.5%. The displacement purification of four additional phosphorothioate oligonucleotides ranging in length from 18 to 25 bases is also demonstrated on the semi-preparative (10-50 mg) scale. All of these oligonucleotides were purified using similar 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 regeneration can be performed in under 30 min. These results seem to indicate that displacement chromatography may be amenable to generalizations in separation protocol that would greatly reduce the effort required to obtain an optimized purification scheme for moderately long oligonucleotides.
Gerstner
J A
JA
PerSeptive Biosystems, Inc., Framingham, MA 01701, USA.
Pedroso
P
P
Morris
J
J
Bergot
B J
BJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004229:Dithiothreitol / Q000494:pharmacology; D009841:Oligonucleotides / Q000302:isolation & purification*; D013873:Thionucleotides / Q000302:isolation & purification*; D013997:Time Factors
D009841:Oligonucleotides; D013873:Thionucleotides; D004229:Dithiothreitol