Strategies for the evaluation and characterization of higher-order structures, supramolecular higher-order...

Synthetic oligonucleotides have emerged as a promising therapeutic class, holding significant potential for the treatment of a wide range of indications, including previously undruggable targets. Due to specific primary structure motifs, oligonucleotide therapeutics may form higher-order structures (HOS), supramolecular higher-order structures (sHOS) and/or aggregates, which are influenced by factors such as dissolution medium and oligonucleotide concentration. Although rarely observed, unintended (s)HOS and/or aggregates might influence various stages of drug substance (DS) or drug product (DP) manufacturing. There is no published guidance on how to best approach the characterization of oligonucleotide therapeutics (s)HOS and aggregation in a scientific and systematic manner. In this article, we provide an overview of oligonucleotide therapeutics (s)HOS formation and aggregation behavior. Based on industry experience and available literature, we also propose strategies for their evaluation and characterization under relevant conditions. The recommended approach involves conducting appropriate scientific assessments during product development with the support of designed workflows, which can help anticipate and/or mitigate the formation of unintended species.