As the field of oligonucleotide therapeutics continues to grow and mature, with applications expanding in areas such as gene therapy, RNA interference, and antisense technologies, the analytical landscape becomes increasingly complex. To address these complexities, it is essential to integrate traditional chromatographic and mass spectrometry techniques into cohesive workflows that ensure both maximum efficiency and analytical accuracy.
Oligonucleotide therapeutics pose particularly unique analytical challenges due to their complexity and regulatory scrutiny. The Boost Efficiency in Oligonucleotide QC webinar, hosted by Separation Science, was designed to address these challenges and demonstrate how integrated chromatographic and mass spectrometry workflows can simplify oligonucleotide analysis.
Below, we explore highlights presented during the webinar by leading expert Brian Rivera, Product Manager at Agilent—including key challenges, technical solutions, and real-world data.
Analytical Needs: Why Oligonucleotide LC-MS Workflows Must Address Complexity
Oligonucleotides often contain synthesis-related modifications and impurities that require highly sensitive detection methods. Rivera also notes that as liquid chromatography (LC) and mass spectrometry (MS) increasingly converge in biopharma, the real challenge for analysts is no longer just generating data—it’s confidently interpreting data to meet stringent regulatory standards.
Given these demands, workflows must effectively separate and detect impurities while integrating seamlessly with advanced mass spectrometry methods. This understanding forms the foundation for optimizing chromatographic strategies.
Optimizing Oligonucleotide HPLC Analysis for Seamless LC-MS Integration
Rivera emphasizes that successful oligonucleotide LC-MS analysis starts with carefully tailoring chromatographic conditions to ensure clean separation and accurate detection of impurities:
- Choosing the right mobile phase and column for oligonucleotide LC-MS analysis: Selecting a mobile phase such as tri-butyl ammonium acetate (TBAA) simplifies spectra and improves MS compatibility, as opposed to traditional options such as triethylamine (TEA) and hexafluoroisopropanol (HFIP).
- Minimizing adsorption and metal interactions with bio-inert LC: Using metal-free LC systems prevents analyte adsorption and adduct formation, preserving signal quality and ensuring consistent separations.
- Optimizing sample injection and detection: Controlling method parameters, such as flow rates, temperature, and solvent choice, ensures high-quality, reproducible results and smooth sample transition to MS detection.
Each of these elements supports a seamless and effective integration of HPLC and LC-MS analysis for oligonucleotides.
Real-World Application: Oligonucleotide LC-MS Analysis Using Oligo Analysis Accelerator (OAA)
While method optimization is crucial, real-world performance ultimately determines the success of any analytical workflow. To demonstrate this, Rivera presented the following case study.
Experimental Setup: Assessing Degradation with Integrated LC-MS
In this case study, both a standard and a stressed oligonucleotide sample were analyzed using LC-MS. Initially, the stressed sample underwent harsh conditions, such as elevated temperature and acidic pH, to promote degradation and generate impurities typically encountered during stability studies.
The following integrated LC-MS workflow was used:
- Separation: Agilent InfinityLab Bio LC, a bio-inert liquid chromatography system designed to prevent metal interactions and maintain analyte integrity, was selected to minimize metal interactions and avoid peak tailing, which could obscure impurities.
- Chromatographic resolution: An Agilent RP-18 bio-compatible column, engineered for biomolecule separations, ensured effective resolution of the parent oligo and degradation products.
- Ion pairing and MS compatibility: Tri-butyl ammonium acetate (TBAA) was used as the mobile phase additive to simplify MS spectra by concentrating ions into fewer charge states and improving compatibility with mass spectrometry.
- Detection: The Agilent InfinityLab LCMSD XT single quadrupole MS, a robust and routine mass spectrometer, was used to show that advanced oligonucleotide impurity analysis can be achieved even with widely accessible instrumentation.
- Automation: The Oligo Analysis Accelerator (OAA), a dedicated software module within the Agilent OpenLab Chromatography Data System (CDS), automated peak integration, impurity detection, and reporting, reducing manual review and enhancing data confidence.
Workflow Execution and Analytical Results
Rivera notes that upon sample injection, the RP-HPLC method with TBAA enabled effective separation of the parent oligo from degradation products. The workflow provided sharp peaks and simplified MS spectra, with more than 90% of ions in the -4 charge state. This simplification improved sensitivity and streamlined impurity identification, even on a single quadrupole MS.
He emphasizes this point in his presentation: "Total degradation products must also be monitored... For antisense oligonucleotides (ASOs), ion pair LCMS is key." Following data acquisition, OAA automated peak detection, integrated UV and MS signals, classified oligonucleotide-related species, and flagged potential impurities, eliminating manual processing.
This case study demonstrates that by combining optimized chromatographic methods, MS-friendly ion pairing, and advanced automation, analysts can confidently identify and quantify impurities, even under stressed sample conditions, without relying on high-end MS platforms.
Impact and Implications
The streamlined workflow, as shown in the webinar, delivered significant benefits:
- Simplified MS data via charge state control
- Automated post-run analysis through OAA
- Reduced review time and improved confidence in impurity identification
"You don’t want your QC analysts doing investigative work — OAA streamlines this," Rivera noted during the session, underscoring automation's importance in regulated environments. Ultimately, this workflow supports rapid decision-making and ensures data integrity. Full data, including chromatograms and impurity profiles, can be found in the webinar.
GMP-Ready Oligonucleotide LC-MS Workflows: Ensuring Data Integrity and Flexibility
While optimizing analytical performance is essential, workflows must do more than simply deliver results — they need to ensure data traceability, meet regulatory requirements, and remain adaptable as therapeutic formats evolve.
Rivera addresses these challenges, emphasizing two critical aspects of a GMP-ready workflow:
- Software integration: "The web application maintains data integrity through OpenLab audit trails, enabling 21 CFR part 11 readiness," Rivera explains, highlighting how OAA supports secure data handling and compliance.
- Supporting QC and R&D flexibility: "Applying this workflow to duplex oligonucleotides may create challenges... we’re interested in exploring this," adds Rivera, referring to the need to extend optimized methods beyond single-stranded oligos. While single-stranded oligonucleotides are well supported, workflows must remain adaptable as therapeutic formats become more complex.
Expert Tips and Trends for Future Oligonucleotide LC-MS Analysis
To help laboratories prepare for what lies ahead, Rivera also shares valuable perspectives on emerging trends and strategies in oligonucleotide analysis:
- Simplify charge states for more effective LC-MS analysis: Rivera notes that using optimized mobile phases such as TBAA helps to concentrate ion distribution, with over 90% of ions in the -4 charge state. This significantly simplifies the spectra, making it easier for routine single quadrupole mass spectrometry to detect oligonucleotide impurities.
- Prepare for future trends and emerging complexity: “AI-based tools, native mass spectrometry, and top-down analytical approaches are going to be essential for analyzing more complex oligonucleotide formats,” Rivera explains.
These capabilities ensure that laboratories are not only achieving accurate analytical results but also meeting stringent regulatory standards and staying agile in the face of evolving oligonucleotide formats.
Final Thoughts on Oligonucleotide Analysis by LC-MS
The Boost Efficiency in Oligonucleotide QC webinar offers valuable real-world strategies for overcoming the analytical challenges facing today’s biopharmaceutical laboratories. By highlighting case studies and workflow examples, Rivera demonstrates how integrated LC-MS workflows offer much more than technical performance—they deliver tangible benefits that improve daily laboratory operations.
Through simplified MS data, reduced manual intervention, automated analysis, and regulatory-ready reporting, these workflows alleviate common burdens faced by QC teams.
Rivera’s insights ultimately highlight a critical takeaway: intelligent, integrated chromatographic and MS solutions empower laboratories to operate more efficiently, reduce costs, and tackle complex oligonucleotide analysis with confidence.
Meet the Expert
Brian Rivera, Product Manager, Agilent Technologies
Brian Rivera is a Product Manager at Agilent Technologies specializing in LC and LC-MS solutions for biopharmaceutical applications. With deep expertise in oligonucleotide analysis, Rivera works closely with scientists in regulated environments to develop workflows that meet the unique challenges of therapeutic oligo characterization and impurity profiling. His focus is on simplifying complex analyses, enhancing data integrity, and ensuring regulatory compliance through intelligent, user-friendly analytical solutions.