Analytical Tools for Profiling Future-Oriented Oligonucleotide Therapeutics

Oligonucleotides represent a rapidly growing class of biological therapeutics, poised to revolutionize the treatment landscape for a myriad of diseases. The characterization of these complex preparations necessitates a sophisticated arsenal of analytical tools. Oligonucleotides, or oligos, are nucleic acid sequences based on RNA or DNA that specifically bind to genes, messenger RNA (mRNA) or proteins, thereby influencing gene expression or protein function. This unique capability positions oligos as potential game-changers in treating conditions ranging from cancer and viral infections to cardiovascular diseases and rare genetic disorders like spinal muscular atrophy (SMA). The success of antisense oligonucleotide (ASO) therapies, such as nusinersen (Spinraza), underscores the transformative impact these molecules can have on patient outcomes.

The high specificity of oligos aligns with the global vision of personalized medicine.¹ With over 20 pharmaceutical products approved in the USA and Europe, and the remarkable success of mRNA vaccines, the potential of oligonucleotide therapeutics is undeniable.

Each nucleotide in an oligonucleotide consists of a phosphate group, a pentose (ribose or deoxyribose), and a nucleobase. These nucleotides form linear molecules through diester bonds between sugars and phosphate groups. Oligos can be single-stranded (ss) or double-stranded (ds), with double strands forming through interactions between complementary base pairs. Examples include small interfering RNA (siRNA) and antisense oligonucleotides. Oligo therapeutics are often chemically modified to enhance their pharmacokinetic properties such as resorption, efficacy, the specificity towards a target or their half-life.²

Analyzing oligo products and impurities

The synthesis of oligos is a complex, multi-step process where nucleotides are sequentially added to form a predefined sequence. Producing a 25-mer, for instance, can involve more than a hundred consecutive chemical reactions, creating ample opportunities for impurities to form. Common impurities include shortmers (where nucleotides are missing), longmers (where additional unintended nucleotides have been added), base-associated impurities, incomplete modifications and by-products of undesired reactions.³

Characterizing oligo preparations is crucial in both development and production. Regulatory authorities demand confirmation of the nucleotide sequence and the identification of impurities that exceed certain thresholds.⁴ Analytical tasks encompass quality checks of raw materials and products, as well as exploring potential degradation pathways. Liquid chromatography (LC) and mass spectrometry (MS) are pivotal techniques in this regard, with reversed-phase LC with ion pairing (IP-RPLC) and anion-exchange chromatography (AEX) being gold standards. Other methods use mixed mode procedures (combination of RP and AEX interactions), hydrophilic interaction and size exclusion chromatography (HILIC and SEC) or a combination of such modes employing two-dimensional LC (2D-LC).

Separating with LC

Before starting measurements, it is essential to ensure the system’s condition is in good shape to receive oligos, whose phosphate groups can interact with stainless steel in the sample path, leading to losses in recovery and separation performance.⁵ Saturating polar surface groups, for instance, by repetitive injection of oligos or acid flushes, or the use of stainless steel-free equipment, can mitigate these issues.

IP-RPLC has been demonstrated to separate many impurities besides the main peaks and is widely regarded as the separation method of choice for oligos. The principle behind it is that ion-pairing reagents like triethylamine (TEA) pair with the anionic, hydrophilic oligo backbone and thus form more hydrophobic molecules that are retained better in RP chromatography. Other IP agents such as hexyl- or dibutylamine can be considered during method development and for counterions, acetic acid is often preferred for ultraviolet-based detection, and hexafluoro-2-propanol (HFIP) for improved compatibility with mass spectrometry (MS).⁶

Identifying with MS

MS is indispensable for obtaining mass and structural information about oligos and their impurities, and is usually operated with electrospray ionization (ESI) in negative mode. High-resolution MS data are invaluable for confirming the expected mass of the main component and identifying less common impurities. The interplay between LC and MS is crucial, as good LC separation enhances spectral purity, facilitating the work of deconvolution algorithms and automated tandem MS (MS/MS) experiments.

In MS/MS mode, specific ions are selectively fragmented to elucidate the structure or confirm the sequence of oligos. While interpreting MS/MS spectra can be complex and time-consuming, modern software tools significantly streamline this process.⁷

Coupling AEX and MS via 2D-LC

The anionic backbone of oligos makes AEX a preferred separation technique. AEX separates molecules based on their charges, with salt gradients commonly used to displace oligos adsorbed on the anion exchange materials. However, the use of non-volatile salts poses challenges for MS detection.

2D-LC can address this issue by transferring fractions from the first dimension (1D) to an online-coupled second dimension (2D) for further analysis under complementary separation conditions.⁸ This approach, such as using a desalting HILIC method in 2D, makes oligo separations with AEX in 1D accessible to MS analysis in near real-time, and in addition can reveal further heterogeneities.⁹

The flexibility of 2D-LC in oligo analytics is immense, with various couplings like AEX/HILIC, HILIC/IPRP and SEC/IPRP reported in the literature.¹⁰

Conclusion

In summary, precise analytics, particularly through advanced techniques like 2D-LC coupled with MS, are pivotal in the development and production of future-oriented therapeutics such as oligonucleotides. These methodologies not only enhance our ability to characterize complex preparations but also ensure the detection and quantification of impurities with high specificity and sensitivity. As the field of oligonucleotide therapeutics continues to evolve, the integration of robust analytical tools will be essential in advancing personalized medicine and improving patient outcomes across a wide range of diseases.