Theoretical calculation of the amounts of nucleotide deletion impurities in oligonucleotides

Therapeutic oligonucleotides are generally synthesized by solid-phase methods via repetitive nucleotide addition. Nucleotide deletion impurities, a class of impurities specific to oligonucleotide therapeutics, arise from incomplete nucleotide addition and lack one or more nucleotides. Owing to their similar physical and chemical properties as the desired product, they are difficult to remove during the purification processes. In addition, nucleotide deletion impurities may hybridize with off-target RNAs, potentially causing hybridization-dependent off-target effects, such as unintended changes in gene expression. Therefore, controlling their levels appropriately is crucial to ensure product quality and safety. However, owing to technical limitations in chromatographic separation and mass spectrometry in analysing oligonucleotides, accurate quantification of individual nucleotide deletion impurities remains challenging. Under these circumstances, it is beneficial to gain knowledge that would supplement experimental analyses from a theoretical approach. In this study, we first presented experimental data showing the difficulty of quantifying each deletion impurity and then calculated their theoretical amounts to capture overall trends in the proportions of nucleotide deletion impurities. Our results indicate that even when single-nucleotide deletion impurities (n - 1 impurities) are abundant, the proportion of individual impurities lacking two or more nucleotides is much lower, with levels theoretically not exceeding 0.05 %. These results provide novel insights into impurity control strategies for oligonucleotide therapeutics and contribute to their efficient development.