Exploring New Methods for Purifying Therapeutic Oligonucleotides

In a recent study published in the Journal of Chromatography A, researchers have made strides in enhancing the purification of therapeutic oligonucleotides (1). These molecules, including antisense oligonucleotides (ASOs), small interfering ribonucleic acids (siRNAs), and conjugates, have garnered attention for their potential to target currently undruggable disease-associated targets. As the demand for oligonucleotide-based therapies grows, the need for efficient purification protocols and analytical methods becomes increasingly critical.

Oligonucleotide synthesis and storage have long posed a challenge due to the generation of numerous compound-related impurities. These impurities can impact the safety and efficacy of oligonucleotide-based therapies, making the development of effective purification methods paramount. Among the existing purification techniques, ion-pair chromatography stands out as the standard method for separating and analyzing therapeutic oligonucleotides.

However, optimizing ion-pair chromatography has not been without its challenges. Mathematical modeling has the potential to enhance the accuracy and efficiency of this purification process, but its application has proven to be complex. While simple models may fall short when dealing with advanced single molecules, complex models remain inefficient for the industrial-scale optimization of oligonucleotide purification.

The experimental system employed in the study involved a traditional C18 column using (dibutyl)amine as the ion-pair reagent and acetonitrile as an organic modifier. The models were constructed and tested using three crude 16-mer oligonucleotides, each with varying degrees of phosphorothioation, along with their respective n – 1 and (P = O)1 impurities.

Phosphorothioation can be relevant in the context of C18 chromatography columns as it allows for the attachment of thiol-modified DNA or RNA molecules to these columns, enabling selective separation and purification of nucleic acids based on their affinity for the C18 stationary phase, making it a useful technique for therapeutic oligonucleotides isolation in analytical and preparative chromatography.

The results of the study demonstrated the effectiveness of the proposed models in predicting overloaded concentration profiles for different gradients of the organic modifier and column load. This development marks a significant step forward in optimizing the purification of therapeutic oligonucleotides, a crucial step in their development as potential treatments for various diseases.

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Reference

Leśko, M.; Kaczmarski, K.; Jora, M.; Stavenhagen, K.; Leek, T.; Czechtizky, W.; Fornstedt, T.; Samuelsson, J. Strategies for Predictive Modeling of Overloaded Oligonucleotide Elution Profiles in Ion-Pair Chromatography. Journal of Chromatography A 2023, 1711, 464446. DOI:10.1016/j.chroma.2023.464446.