Observations from a decade of oligonucleotide bioanalysis by LC-MS

Abstract

There is a growing need for efficient bioanalysis of oligonucleotide therapeutics. This broad class of molecules presents numerous challenges relative to traditional small molecule therapeutics. Methodologies including ligand-binding assays or polymerase chain reaction may be fit-for-purpose in many instances, but liquid chromatography coupled to mass spectrometry (LC-MS) often delivers the best balance of sensitivity and selectivity. Over the last decade, we have engaged with many such molecules and derived insights into challenges and solutions. Herein, we provide four case studies illustrating challenges we have encountered. These issues include low or variable analyte recovery, poor resolution from related species, chromatographic abnormalities or challenging sensitivity. We present a summary of considerations, based on these experiences, to assist others working in the area.

Executive summary

• We have validated over 80 chromatographic methods for analysis of oligonucleotides (OGNs) in the past decade, with significant growth in demand noted over recent years.

Case studies

• We present four case studies highlighting the challenges associated to LLE and SPE-based extraction workflows, with recommendations on how best to optimize these. Common pitfalls such as low/variable recovery, dirty extracts and reagent integrity are discussed.

• The choice of triple-quadrupole MS/MS versus HRMS is important. We show case studies highlighting the benefits of each for optimum sensitivity and selectivity, respectively.

• Challenges unique to a fatty acid conjugated oligonucleotide are highlighted.

• The need to consider the potential impact of metabolites is shown.

Conclusion

• A list of considerations for LC-MS methods has been provided from our experience.

• Protease digestion and hybridization-based LC-FD or LC-MS approaches are gaining momentum. However, they will inevitably bring their own unique challenges.

• Hence, LLE/SPE with LC-MS workflows will likely remain popular.

Keywords: :

• bioanalysis
• chromatographic
• HRMS
• LC-MS
• oligonucleotide
• perspective
• siRNA
• triple quadrupole

Author contributions

M Ewles and A Ledvina contributed equally to the manuscript; B Powers also provided significant insights, text and data.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.