Identification and Impact of On-Column Higher-Order Structure Formation during Anion Exchange Purification of Oligonucleotides

During large-scale anion exchange purification of a guanosine-rich oligonucleotide sequence, a 10% yield loss was observed. The elution profile showed an unwanted peak in the wash step of the gradient and was suspected as the cause of the specific loss. The primary oligonucleotide structure and the high-salt conditions hinted toward the formation of a higher-order structure, which, due to its higher charge, would be retained more than the oligonucleotide product. Higher-order structures comprise the secondary and tertiary structures that oligonucleotides can form and are driven by noncovalent interactions which depend on the oligonucleotide primary structure and various environmental factors. Analytical experiments focusing on secondary and tertiary structure characterization, including circular dichroism and melting temperature, identified an unstable higher-order structure which was specifically stabilized on the column and was not stable once eluted in solution. We herein describe how the anion exchange purification of this guanosine-rich oligonucleotide sequence was redeveloped with support of analytical characterizations to limit the higher-order structure formation and increase final product recovery. To this end, different denaturing conditions were screened by circular dichroism experiments, providing input for purification conditions at a semipreparative scale. Different factors including pH, organic modifiers, denaturing agents, and salt were hereby evaluated. A selection of conditions was further upscaled to additionally evaluate the impact of concentration on the higher-order structure formation. Two conditions were identified that resolved the on-column higher-order structure formation and solved the initial observed yield loss.