The Promise and the Problem
Oligonucleotide therapeutics represent one of modern medicine’s most exciting frontiers. These short nucleic acid sequences show remarkable potential to treat diseases previously considered undruggable, targeting conditions at the genetic level with unprecedented specificity.
As this therapeutic class expands rapidly from research through clinical development to commercialization, the need for effective and efficient analytical techniques grows proportionally. Comprehensive characterization of oligonucleotides and their related impurities is essential for quality control, regulatory compliance, and patient safety.
The pharmaceutical industry has largely relied on ion-pair reversed-phase liquid chromatography coupled with mass spectrometry (IP-RP-LC-MS) as the dominant separation approach for oligonucleotide analysis. The technique works well for separation, achieving good peak shape and resolution.
But it creates a persistent operational problem: ion-pairing reagents contaminate LC-MS systems, reducing sensitivity for other applications and requiring extensive cleaning protocols. For laboratories running diverse sample types, this cross-contamination risk and sensitivity loss create significant workflow challenges.
Watch: Ion-Pair Free Method Development Using Design of Experiments
In this video, Resolian analytical scientist Emily How presents systematic development of a native size exclusion chromatography-mass spectrometry (nSEC-MS) method that eliminates ion-pairing reagents while delivering comprehensive oligonucleotide characterization in just 7.5 minutes.
Emily’s research project, performed during her final university year and continued at Resolian, demonstrates how Design of Experiments methodology enables efficient optimization of complex separation techniques for emerging therapeutic modalities.
What You’ll Learn in This Video:
- The growing importance of oligonucleotide therapeutics in pharmaceutical development
- Why ion-pair reagents create system contamination and sensitivity challenges
- Native size exclusion chromatography as an alternative separation mechanism
- Design of Experiments approach to method optimization
- Parameters investigated: flow rate, temperature, salt concentration, injection volume
- Statistical modeling for determining optimal conditions
- Performance comparison: 7.5-minute runtime for oligonucleotide characterization
- Life cycle assessment comparing nSEC-MS to high-throughput IP-RP methods
- Environmental benefits: reduced mobile phase consumption and waste ecotoxicity
- How the method characterizes stressed oligonucleotides and related substances
Emily’s journey from unfamiliarity with oligonucleotides and SEC to successful method development demonstrates how systematic approaches and collaborative learning enable rapid capability development.
Understanding the Ion-Pair Contamination Challenge
Ion-pairing reagents work by forming complexes with charged oligonucleotide molecules, enabling their retention on reversed-phase columns. Common ion-pairing agents include alkylamines like triethylammonium acetate (TEAA) or hexafluoroisopropanol (HFIP).
These reagents are effective but problematic:
System contamination: Ion-pairing agents adsorb onto column stationary phases, tubing, and MS source components, persisting even after method changeover
Sensitivity reduction: Contamination from ion-pairing reagents suppresses ionization efficiency for subsequent samples, particularly for applications requiring high sensitivity
Cleaning requirements: Removing ion-pairing contamination requires extensive washing protocols with specific solvent sequences, consuming time and resources
Cross-contamination risk: Traces of ion-pairing reagents can interfere with unrelated analyses run on the same system
For laboratories analyzing diverse compound classes, dedicating instruments solely to oligonucleotide work is expensive and inefficient. A separation approach that doesn’t require ion-pairing agents solves these operational challenges.
Native Size Exclusion Chromatography: The Alternative Mechanism
Size exclusion chromatography (SEC) separates molecules based on their hydrodynamic size rather than chemical interactions with the stationary phase. Larger molecules elute first, unable to enter the pores of the stationary phase packing material. Smaller molecules penetrate the pores and elute later.
For oligonucleotides, native SEC operates under aqueous conditions using volatile buffers compatible with mass spectrometry, without requiring ion-pairing reagents. The “native” designation means oligonucleotides remain in their natural charged state rather than being paired with counter-ions.
This approach offers several advantages:
- No system contamination from ion-pairing agents
- Maintained sensitivity for diverse applications
- Simpler mobile phase composition
- Faster method changeover between applications
- Reduced cross-contamination risk
The challenge: optimizing SEC conditions to achieve adequate separation, sensitivity, and throughput for oligonucleotide characterization.
Design of Experiments: Systematic Optimization
Rather than traditional one-factor-at-a-time optimization that would require dozens of experimental runs, Emily used Design of Experiments (DoE) software to systematically investigate multiple parameters simultaneously.
The DoE approach creates a statistical model predicting how different parameter combinations affect method performance, enabling identification of optimal conditions from a minimum number of carefully designed experiments.
Parameters investigated:
- Mobile phase flow rate: Affecting separation efficiency and analysis time
- Column temperature: Influencing diffusion rates and peak shape
- Mobile phase salt concentration: Impacting oligonucleotide conformation and retention
- Injection volume: Balancing sensitivity against column loading
Optimization goals:
- Maximize theoretical plates (separation efficiency)
- Maximize MS sensitivity
- Minimize reagent consumption
The statistical model generated response surfaces showing how parameter combinations affect each goal, enabling identification of conditions that optimize all objectives simultaneously.
This systematic approach delivered optimized method performance in far fewer experiments than traditional optimization would require, demonstrating the efficiency advantage of DoE methodology.
The Performance Achievement
The optimized nSEC-MS method achieved comprehensive oligonucleotide characterization including related substances in just 7.5 minutes, without any ion-pairing reagents. This runtime is competitive with ion-pair RP methods while eliminating contamination concerns.
The method successfully characterized stressed oligonucleotide samples on a time-of-flight mass spectrometer (ToF-MS) coupled with UPLC-UV, demonstrating its capability for impurity profiling and degradation assessment, critical applications for quality control and stability studies.
The Sustainability Advantage: Life Cycle Assessment
Beyond solving the ion-pair contamination problem, Emily evaluated whether the developed method offered genuine sustainability benefits compared to existing high-throughput IP-RP approaches.
Life cycle assessment (LCA) compared environmental impacts across the method lifecycle, revealing significant improvements:
Reduced mobile phase consumption: Native SEC requires less total mobile phase volume per analysis, reducing both solvent usage and waste generation
Lower waste ecotoxicity: The aqueous volatile buffers used in native SEC have much lower environmental impact than the organic solvent-heavy mobile phases typical in IP-RP methods, plus elimination of toxic ion-pairing reagents
These improvements mean the method delivers better environmental performance throughout routine use, not just as a one-time achievement but compounding across hundreds or thousands of analyses.
For pharmaceutical companies with sustainability commitments, this demonstrates that green chemistry alternatives can simultaneously solve operational problems (contamination) and environmental concerns.
Implications for the Oligonucleotide Market
As oligonucleotide therapeutics transition from niche applications to mainstream pharmaceutical development, analytical laboratories face increasing demands for oligonucleotide characterization. Methods that eliminate ion-pair contamination while maintaining or improving performance enable more flexible laboratory operations.
The approach also facilitates multi-product laboratories where oligonucleotide analysis shares instruments with other therapeutic modalities, reducing capital equipment requirements and operational costs.
Expert Capability Development for Emerging Modalities
Emily’s journey from unfamiliarity with oligonucleotides and SEC to successful method development exemplifies Resolian’s approach to emerging analytical challenges: systematic methodology, collaborative learning, and rigorous optimization.
Our analytical sciences team brings:
- Advanced separation techniques including native SEC for oligonucleotides
- Design of Experiments expertise for efficient method optimization
- Life cycle assessment capabilities for sustainability evaluation
- Experience across therapeutic modalities from small molecules to biologics
- Flexible method development responsive to operational constraints
Whether you’re developing oligonucleotide therapeutics and need ion-pair free analytical approaches, seeking to reduce environmental impact of routine testing, or exploring emerging separation techniques for challenging molecules, we bring the systematic problem-solving and technical depth that enables successful method development.
Ready to discuss oligonucleotide analysis or other emerging analytical challenges?
Contact our analytical sciences team to explore how advanced separation techniques and systematic optimization can address your specific needs.
