Purification of an oligonucleotide at high column loading by high affinity, low-molecular-mass displacers

The development of efficient techniques for large-scale oligonucleotide pur ification is of great interest due to the increased demand for antisense ol igonucleotides as therapeutics as well as their use for target validation a nd gene functionalization. This paper describes the use of anion-exchange d isplacement chromatography for the purification of 20-mer phosphorothioate oligonucleotide from its closely related impurities using low-molecular-mas s amaranth as the displacer. Experiments were carried out to examine the ef fect of the feed load on the performance of the displacement chromatography . In contrast to prior work, displacement chromatography was successfully s caled-up to high column loadings while maintaining high purity and yields. Experiments carried out on a Source 15Q column indicated that crude oliganu cleotide loading as high as 39.2 mg/ml of column were readily processed. re sulting in product recovery of 86% and purity of 92%. These results demonst rate that anion-exchange displacement chromatography can indeed be employed for large-scale oligonucleotide separations at high column loading. (C) 20 01 Published by Elsevier Science B.V.