Optimizing Oligonucleotide Analysis

Importance of the Topic

Therapeutic oligonucleotides represent an innovative class of small synthetic nucleic acids designed to modulate gene expression or protein function through sequence-specific binding. Their capacity to act on previously undruggable targets has made them fundamental to the advancement of personalized medicine.

Objectives and Overview of the Article

This article aims to present the main categories of therapeutic oligonucleotides, summarize recent market growth, identify analytical challenges during manufacturing and quality control, and introduce an ultrahigh performance liquid chromatography (UHPLC) solution to eliminate metal-induced adsorption issues.

Methodology and Instrumentation Used

The workflow for manufacturing and analysis encompasses:

• Automated solid-phase phosphoramidite synthesis on a resin support
• Cleavage and deprotection in aqueous ammonia to release crude oligonucleotides
• Purification strategies including reversed-phase HPLC, anion exchange chromatography (AEXC) and hydrophobic interaction chromatography (HILIC)
• Buffer exchange by precipitation, tangential flow filtration and lyophilization
• Analytical measurement of purity, recovery and identity using HPLC/UHPLC and liquid chromatography-mass spectrometry (LC-MS)

To address metal adsorption, all wetted surfaces in the Nexera XS inert UHPLC system are PEEK or inert polymers, paired with metal-free Shim-pack columns.

Main Results and Discussion

Key findings include:

• Global market growth for oligonucleotide therapeutics at a CAGR of 16.7%, rising from USD 3.68 billion in 2021 to an anticipated USD 10.97 billion by 2028
• Classification of therapeutic oligonucleotides:
  
  • Decoy oligodeoxynucleotides that sequester transcription factors to inhibit gene transcription
  • Antisense oligonucleotides that obstruct translation via RNase H-mediated mRNA degradation, steric blocks, or splice modulation
  • Small interfering RNAs that guide the RISC complex to cleave target mRNA
  • Aptamers that bind protein targets to block interactions or deliver payloads
• Metal adsorption on stainless steel tubing causes peak tailing, reduced sensitivity and poor precision
• The Nexera XS inert system demonstrated a 1.7-fold increase in detection sensitivity and reduced peak tailing, achieving an RSD of 0.56 % versus 10.1 % for standard stainless steel-based UHPLC

Benefits and Practical Applications

The metal-free UHPLC solution enables reliable quantitation of low-concentration oligonucleotide samples, enhances reproducibility in quality control, and reduces the risk of sample loss or degradation during analysis. This supports robust QA/QC in pharmaceutical development and manufacturing.

Future Trends and Possibilities for Application

Emerging directions include integration of fully inert, high-throughput UHPLC platforms, development of novel column chemistries tailored for diverse oligonucleotide constructs, automation of end-to-end workflows, and expansion of personalized therapeutic pipelines driven by rapid analytical feedback.

Conclusion

Therapeutic oligonucleotides continue to drive a transformative shift in drug development. Overcoming analytical challenges such as metal adsorption through inert UHPLC technology enhances data reliability and supports accelerated, quality-driven manufacture of these next-generation therapeutics.

References

1. Moumné L, Marie AC, Crouvezier N. Oligonucleotide therapeutics: from discovery and development to patentability. Pharmaceutics. 2022;14:260. doi:10.3390/pharmaceutics14020260
2. Keefe A, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discov. 2010;9:537–550. doi:10.1038/nrd3141
3. Roehr B. Fomivirsen approved for CMV retinitis. J Int Assoc Physicians AIDS Care. 1998;4:14–16.
4. Market Growth Reports. Global oligonucleotide therapeutics market insights and forecast to 2028.
5. Xiong H, Veedu RN, Diermeier SD. Recent advances in oligonucleotide therapeutics in oncology. Int J Mol Sci. 2021;22:3295. doi:10.3390/ijms22073295
6. Studzińska S, Buszewski B. Evaluation of ultra high-performance liquid chromatography columns for the analysis of unmodified and antisense oligonucleotides. Anal Bioanal Chem. 2014;406:7127–7136. doi:10.1007/s00216-014-7959-5
7. Andrews BI, Antia FD, Brueggemeier SB, et al. Sustainability challenges and opportunities in oligonucleotide manufacturing. J Org Chem. 2021;86:49–61. doi:10.1021/acs.joc.0c02291