Linda Thunberg works in the separation science team at AstraZeneca, where she serves as Associate Director. Her team purifies around 1500 compounds every year including oligonucleotides, peptides, and conjugates – a challenging and resource-intensive task. This presentation highlights the work of the separation science team in purifying a wide range of oligonucleotides, from naked oligos to those with various linkers and conjugates.
Thunberg explained that there are many different types of modifications that can be made to these molecules. For example, the bases, the sugars, and the backbone, including fully or partly thioated backbones, can all be modified which could create numerous diastereomers and complex impurity patterns.
There are two common types of purification techniques used for oligos: ion exchange chromatography and ion pair chromatography. Thunberg said that of these two techniques, the latter is preferred for AstraZeneca’s early drug discovery efforts due to its efficiency and quick turnaround times.
Traditionally, when purifying phosphodiesters, triethyl amyl acetate is used as the ion pair for the purification, which can create a many diastereomers, making purification challenging. Thunberg explained how using dibutyl amine as an ion pair can suppress diastereomer separation, but it is toxic and must be removed before in vivo studies.
For the separation science team at AstraZeneca, large scale purification involves balancing yield and purity, often requiring multiple chromatographic steps and careful handling of toxic amines. New analytical methods have improved the resolution and quantification of impurities, leading to potential changes in purity guidelines.
Thunberg highlighted that optimising their purification workflow, the team have managed to reduce the time from synthesis to purified oligo from five to three days, improving yield and purity.
The presentation also details how purifying conjugates, such as peptide and GalNAc conjugates, is complex and often requires multiple types of chromatographic techniques.
Thunberg finished off her talk by discussing guide RNAs, and how they present their own synthesis and purification challenges due to their length and closely related impurities, necessitating ongoing method development.