Oligonucleotide Characterization by Agilent 1290 Infinity II Bio LC and 6545XT AdvanceBio LC/Q-TOF

Importance of the Topic

Synthetic oligonucleotide therapeutics are emerging as powerful tools for treating a wide range of diseases. Ensuring accurate characterization of their mass, purity, impurity profile, and sequence is critical for product safety, efficacy, and regulatory compliance. Robust analytical workflows help accelerate development and quality control in pharmaceutical and biopharmaceutical laboratories.

Objectives and Study Overview

This study evaluates two integrated workflows—Target Plus Impurities (TPI) and Sequence Confirmation (SC)—implemented in Agilent MassHunter BioConfirm 12.0 software. Using an Agilent 1290 Infinity II Bio LC system coupled to a 6545XT AdvanceBio LC/Q-TOF, the study characterizes an 18-mer antisense oligonucleotide (ASO) and a 28-mer aptamer. Key goals include confirming oligonucleotide identity, quantifying related impurities, and determining exact sequences in a streamlined manner.

Methodology and Instrumentation

The analytical platform combines:

• Agilent 1290 Infinity II Bio LC system with biocompatible flow path
• AdvanceBio Oligonucleotide column (2.1 × 50 mm, 2.7 μm) at 65 °C
• Mobile phases of 15 mM triethylamine and 400 mM HFIP in water (A) and methanol (B)
• Gradient methods: 10 min for TPI workflow and 5 min for SC workflow at 0.5 mL/min
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Agilent Jet Stream source in negative ion mode
• MassHunter BioConfirm 12.0 software using Max Entropy deconvolution and Find-by-Formula algorithms

Main Results and Discussion

Chromatographic separation of RNA length standards demonstrated baseline resolution of four different oligonucleotide lengths. In the TPI workflow, high-resolution MS enabled accurate deconvolution of the full-length ASO (7,127.2001 Da) with mass accuracy below 1 ppm and detection of low-level truncation and extension impurities. Targeted quantitation with Find-by-Formula achieved relative abundance measurements down to 0.4% with RSD values below 5%.

The SC workflow used a rapid 5-minute gradient and targeted MS/MS acquisition. Single-injection analysis of the ASO and aptamer provided 100% sequence coverage. Optimized charge-state selection and collision energies generated rich fragment ion ladders, facilitating unambiguous sequence confirmation of chemically modified oligonucleotides.

Benefits and Practical Applications

• Integrated software workflows reduce manual data processing time
• High mass accuracy and resolution improve confidence in identity and impurity analysis
• Low-level impurity quantitation supports quality control and regulatory documentation
• Rapid sequence confirmation accelerates method development and release testing

Future Trends and Opportunities

Advances may include further automation of data interpretation, expansion to longer and more heavily modified oligonucleotides, integration with AI-driven analytics, and enhanced profiling of complex impurity structures. Continued improvements will support regulatory compliance and accelerate therapeutic development.

Conclusion

The combination of the Agilent 1290 Infinity II Bio LC, 6545XT AdvanceBio LC/Q-TOF, and BioConfirm 12.0 software delivers a robust platform for comprehensive oligonucleotide analysis. Integrated TPI and SC workflows enable precise identity confirmation, impurity quantitation, and full sequence determination in high-throughput biopharma environments.

Used Instrumentation

• Agilent 1290 Infinity II Bio LC System
• Agilent AdvanceBio Oligonucleotide column, 2.1 × 50 mm, 2.7 μm
• Agilent 6545XT AdvanceBio LC/Q-TOF System with Dual AJS source
• Agilent MassHunter BioConfirm 12.0 software

References

1. Wong D., Rye P. An Integrated Workflow for the Analysis of Oligonucleotides and Their Impurities by Agilent High-Resolution LC/Q-TOF Mass Spectrometry. Agilent Technologies Application Note, 5994-4817EN (2022).
2. Wong D., Rye P. An Integrated Comprehensive and Integrated Workflow for Oligonucleotide Sequence Confirmation by Agilent High-Resolution LC/Q-TOF. Agilent Technologies Application Note, 5994-5071EN (2022).
3. Capaldi D. et al. Impurities in Oligonucleotide Drug Substances and Drug Products. Nucleic Acid Therapeutics, 27, 309–322 (2017).