Challenges and Solutions in the Purification of Oligonucleotides

This presentation noted the diverse range of purification techniques and strategies AstraZeneca employ for peptides, oligonucleotides, and oligonucleotide conjugates. 

Their portfolio of peptides encompasses linear, cyclic, and bicyclic types. Most of these are produced by solid phase synthesis and in most cases are purified by reverse phase liquid chromatography (RP-LC). In vitro samples use TFA as an additive for their purification while in vivo samples are TFA-free, instead using acidified ammonium acetates or acetonitrile in the mobile phase. 

Oligonucleotides are similarly purified on solid supports and can be released with their five prime protecting group on or off. This would be left on because the DMT or MMT provides extra hydrophobicity to the oligo. If the protecting group is left off, the oligo needs to be purified using ion pairing RP-LC. In the mobile phase, they use either ammonium bicarbonate buffers or alkylamine acetates. 

For oligonucleotides which are conjugated to other molecules like dyes, GalNAcs, lipids, or peptides, these can either be synthesised in solution or directly on solid supports. because usually the conjugated part will offer the extra hydrophobicity, they are purified by the reverse phase HPLC. The mobile phase uses ammonium bicarbonate, ammonium acetate or acetonitrile buffers. 

Wu then discussed the challenge of purifying guanine (G)-rich oligonucleotides. These particular oligos have a tendency to form heterogeneous aggregates which lead to poor chromatographic behaviour and low solubility. This can make the purification process time-consuming and challenging. 

The team investigated how this aggregation is stabilised. They found that the aggregate structure is stabilised by the presence of a cation which sits in a central channel between each pair of tetrads. Guanine tetrads often feature Hoogsteen-style hydrogen bonding and are more stable when stacked as intermolecular forces between each layer helps stabilise them. Therefore, by reducing cations during synthesis and adjusting mobile phase conditions, the team successfully mitigated aggregation issues, improving chromatography outcomes.