Separation of phosphorothioate oligonucleotide impurities by WAX HPLC under high organic content elution conditions

The separation of impurities in phosphorothioate diester (PS) oligonucleotides is complicated by (1) the presence of a very large number of diastereoisomers, e.g., 2¹⁹ for a 20-mer oligonucleotide, (2) peak broadening due to the hydrophobic character of the sulfur atom, and (3) the chemical similarity of the impurities to the parent oligonucleotide and each other. Further difficulties arise due to the chemical nature of oligonucleotides, which display a complex mixture of ionic, hydrophobic, H-bonding, and other functionalities. To minimize hydrophobic interactions and peak broadening due to the PS modification, we have developed a novel method that combines a weak anion exchange (WAX) column with a mobile phase elution system designed to maximize separation by a single ionic/electrostatic interaction. We found that although chaotropes are helpful, the most significant beneficial effect of the hydrophilic WAX column is that high-organic, low-salt mobile phase is required for product elution. Separations are also benefitted by pH gradient effects on stationary phase electrostatic potential and analyte ionization. An extraordinary degree of separation is achieved by the new WAX method in comparison to SAX (strong anion exchange) chromatography. For the first time, the extent of deamination of PS oligonucleotides is directly determined by a chromatography-only method. The approach, representative results, and the mechanisms of separation are discussed.