Standardized characterization of therapeutic oligonucleotides through in-house related substance calculator by LC-MS.

Synthetic oligonucleotides (OGN) are a new and emerging class of drugs because of their potential as therapeutic molecules for a wide range of indications and diseases. In particular, both antisense oligonucleotides (ASO) and small interference (si)RNA have gained a lot of interest. Besides several OGN drugs are already approved by the FDA, numerous clinical trials are ongoing or planned for a large variety of RNA drugs designed to treat or prevent various cancers, cardiovascular diseases, and infectious diseases.

Due to the complex multistep synthesis process of therapeutic oligonucleotides (OGN), the final product contains several related substances, including deletion sequences (shortmers) and addition sequences (longmers). Moreover, full-length products (FLPs) are often modified (to improve distribution and potency of the OGN) on the base, on the sugar, or at the phosphate backbone. Although synthetic oligonucleotides are often quite stable, degradation may introduce additional species in the products. All of these potential chemical alterations result in a significant degree of complexity of the final oligonucleotide structures and have impact on their impurity profile. Therefore, advanced analytical tools are critical for the characterization of various oligonucleotide impurities and degradation products, some of which are present at a very low level.

A very powerful method for oligonucleotide analysis is ion-pair reversed-phase liquid chromatography (IP-RP) combined with UV detection and mass spectrometry (LC-MS). Using an ion-pair reagent is essential to retain oligonucleotides (which are large polar, acidic molecules) onto reversed phase columns. The detection orthogonality of both UV and MS allows identification and quantification even when structurally closely related impurities co-elute. Although HR-MS(/MS) analysis of synthetic oligonucleotides can be challenging, due to the relatively large molecular weight resulting in several multiply charged distributions, it offers accurate mass determination and confident identification of oligonucleotides and their related substances.

At AnaBioTec, we developed a screening platform consisting of an in-house developed calculator that predicts masses of potential related substances in combination with an ion-pair reversed phase LC separation coupled to UV detection and high resolution mass spectrometry (IP-RP-UV-HRMS).