Purification of oligonucleotides by high affinity, low molecular weight displacers

High affinity, low molecular weight anionic displacers were successfully em ployed for the purification of antisense oligonucleotides. Several importan t structural characteristics were identified that contribute to the affinit y of low molecular weight displacers to a hydrophilized polystyrene divinyl benzene anion exchanger. Sulfonic acid groups were found to possess higher affinity than carboxylic acid and phosphate functionalities, and nonspecif ic interactions (particularly hydrophobic interactions) were shown to play a major role in the retention process on this stationary phase material. Us ing this information, two high affinity, low molecular weight displacers we re identified. These molecules are relatively inexpensive organic dyes that possess multiple sulfonic acid moieties, as well as aromatic functionaliti es, which increase nonspecific interactions with the stationary phase. Thes e high affinity displacers, which can be readily detected, were then employ ed to displace several strongly retained antisense oligonucleotides that co uld not be displaced by previously established low molecular weight displac ers. The displacement process resulted in very high purities of the antisen se oligonucleotides. The results presented in this paper are significant in that they demonstrate that low molecular weight displacers for ion-exchang e chromatography can possess equal to or greater affinities than their high er molecular weight counterparts, when nonspecific interactions with the st ationary phase are exploited. In addition, the results illustrate the high resolutions possible with displacement chromatography and demonstrate an at tractive technology for the process scale purification of oligonucleotides.