Analyzing Raw Material for Oligonucleotide Synthesis

Importance of the Topic

The quality of nucleoside phosphoramidites is critical for reliable oligonucleotide synthesis in biopharmaceutical production. Robust analytical methods ensure minimal sequence errors and high coupling efficiency across research, process development, and quality control laboratories.

Objectives and Study Overview

This study aimed to develop and validate flexible liquid chromatography methods for the analysis of 5'-DMT-deoxy adenosine 3'-phosphoramidite raw material using the Agilent 1290 Infinity II Bio LC System and to demonstrate seamless method transfer from a conventional LC platform.

Methodology and Instrumentation

Methods were implemented on the Agilent 1290 Infinity II Bio LC System, equipped with a four-channel flexible pump, multisampler with thermostat, multicolumn thermostat with heat exchangers, and variable wavelength detector with a micro flow cell. Mass confirmation was performed on the Agilent 6545XT AdvanceBio LC/Q-TOF. The column used was Agilent ZORBAX RRHD Eclipse Plus C18 (2.1×100 mm, 1.8 µm). Two gradient methods were developed: Method A with a ternary solvent system (acetonitrile, water, 500 mM ammonium acetate pH 5.5) at 0.2 mL/min and 25 min runtime; Method B optimized to 8.33 min with 0.6 mL/min flow and constant gradient volume.

Main Results and Discussion

On-the-fly gradient mixing enabled identification of the main product (two epimer peaks) and multiple impurities, including critical reverse amidite isomers, by accurate mass and UV detection. Ten replicate injections with Method A showed retention time RSD between 0.015%–0.050% and area RSD averaging 0.27%. Comparison to the conventional LC system yielded an average retention time deviation of 0.686%, confirming robust method transfer. Method B delivered a 66% reduction in runtime while maintaining high precision (retention time RSD ≤ 0.115%, area RSD ≤ 0.99%).

Benefits and Practical Applications

The high-pressure, biocompatible flow path and flexible pump of the 1290 Infinity II Bio LC System enable rapid method development, reliable impurity profiling, and easy transfer across laboratories. Reduced runtimes increase sample throughput, supporting quality control in oligonucleotide manufacturing.

Future Trends and Opportunities

Advances in biocompatible LC platforms will drive further integration of high-resolution MS for impurity elucidation and real-time process monitoring. Continued optimization of ultrafast LC methods and data analytics will support automated, high-throughput workflows for emerging nucleic acid therapeutics.

Conclusion

The Agilent 1290 Infinity II Bio LC System with Flexible Pump demonstrated robust, precise, and versatile analysis of nucleoside phosphoramidites. Seamless method transfer and significant runtime reduction affirm its value for oligonucleotide raw material qualification and routine quality control.

References

• Beaucage SL, Caruthers MH. Deoxynucleoside Phosphoramidites—A New Class of Key Intermediates for Deoxypolynucleotide Synthesis. Tetrahedron Lett. 1981;22:1859–1862.
• Hughes RA, Ellington AD. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb Perspect Biol. 2017;9:a023812.
• Roberts TC, Langer R, Wood MJA. Advances in Oligonucleotide Drug Delivery. Nat Rev Drug Discov. 2020;19:673–694.