Oligonucleotide Therapeutics

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications. Oligonucleotide therapies include short, single- or double-stranded DNA, or RNA molecules that bind via Watson-Crick base pairing to enhance or repress the expression of target RNA, to treat or manage a wide range of diseases. They include antisense oligonucleotides (ASOs) RNA interference (RNAi) and aptamers.

In general, oligonucleotide therapeutics interfere or inhibit the RNA translational process within the cell and promote degradation of proteins associated with disease.

Oligonucleotide therapeutics development and manufacture requires confirmation of the correct nucleic acid sequence and comprehensive characterization and quantification of impurities to ensure therapeutic efficacy. In the later stages of development, this must be done in accordance with strict regulatory guidance. Presently, the guidelines themselves are evolving. The main challenge is presented by the complexity of the oligonucleotide molecules themselves and the modifications that are being applied to them.

Chemical modifications have been utilized extensively to improve the binding energy, stability, and tolerability of oligonucleotide therapeutics, including ASOs, siRNAs, and aptamers. Phosphorothioate modifications, which incorporate sulfur in the oligo backbone, have been effective due to their ability to enhance cellular uptake and evade nuclease-mediated degradation in serum.

Chemical modifications continue to evolve and bring more drug-like properties to oligonucleotide therapeutics. These include sugar and base modification and/or conjugated molecules that improve or help to target delivery.

Oligonucleotide technologies continue to evolve with several avenues being explored to further improve them and create better oligonucleotide-based drugs. All current and future synthetic oligonucleotide therapeutics, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), require thorough analysis to establish purity and quality.

Structural characterization

Novel oligonucleotide chemical modifications increase activity, enhance stability against nuclease degradation, modulate protein binding, and decrease immunogenicity. The increased structural complexity brings numerous analytical challenges for the product/process development and manufacturing groups to develop highly selective, sensitive, reproducible, and robust analytical methods to characterize and determine the impurity profile to ensure drug safety and quality.

Therapeutic oligonucleotides produced by chemical synthesis carry various types of product-related impurities, including deletion sequences (‘shortmers’), addition sequences (‘longmers’), and the modified full-length species. The n–x shortmers, the most common impurities present in synthetic oligonucleotides, are formed due to failed base coupling at the 5’ end followed by incomplete capping, which may also result in n–1 impurities with different single deletions. The longmers are mostly the n+1 or n+2 species, while the modified impurities correspond to the full-length product with modifications on its nucleobases or phosphorothioate linkages. Degradation of synthetic oligonucleotides may introduce additional species in the products.

Advanced analytical tools are indispensable for the characterization of various oligonucleotide impurities and degradation products, some of which are present at a very low level. One popular method for oligonucleotide analysis is ion-pair reversed-phase liquid chromatography coupled with mass spectrometry (IP-RP LC-MS). The MS1-based LC-MS method offers intact mass confirmation for oligonucleotides and their common impurities; however, it does not provide base-by-base sequence information and localization of modifications. Additionally, it is challenging to apply the MS1-based method for the identification of impurities with modifications and degradation products. By comparison, an HRAM-based ddMS2 method allows confident identification and mapping of unmodified and modified oligonucleotides.

Sequence confirmation

Sequence confirmation is essential to determine if your oligonucleotide synthesis was successful. Advanced analytical tools are indispensable for the characterization of various oligonucleotide impurities and degradation products, some of which are present at a very low level.

Impurity analysis

Impurity identification and quantification is essential to ensure that your new therapeutics are safe and efficacious.

Quality control (QC) of oligonucleotide synthesis requires confident confirmation of oligonucleotide mass and rough quantification of yield and impurity levels using a rapid and efficient method. Quantification of yield can easily be performed by UV detection because of the strong DNA absorption at 260 nm. Rough estimation of impurities requires a mass spectrometer, as aborted sequences (N-1) are not usually chromatographically separated from complete sequences (N) during a quick QC method. A mass spectrometer also allows for non-ambiguous confirmation of oligomer identity.

Purification

Oligonucleotide synthesis involves many individual reactions, which leads unavoidably to the accumulation of impurities such as truncated sequences. Therefore, the purification of the desired oligonucleotides is a crucial step. Since the 1970s, several chromatographic approaches have been used to analyze and purify synthetic oligonucleotides. Significant developments in terms of instrument performance and stationary phase have been made in recent years. High-performance liquid chromatography (HPLC) is the method of choice if high-purity products are desired; among the separation techniques commonly used for nucleic acid separation is reversed-phase (RP-HPLC). When developing the methods to separate and purify oligonucleotides, some of the unique features of these molecules need to be considered such as failure sequences of similar length, secondary structures (e.g., hairpin loops formation), pH and salt concentration (ionic strength), as well as other additives and variables. All these factors can additionally affect retention time and alter the interaction with the stationary phase.