Improving Recovery and Quantitation of Oligonucleotide Impurities Using ACQUITY PREMIER with MaxPeak HPS Technology

Importance of the Topic

Oligonucleotide analysis plays a central role in the development and manufacturing of nucleic acid therapeutics and molecular biology tools. Reliable separation and quantitation of synthetic oligonucleotides and their impurities is challenged by strong non specific adsorption to metal surfaces under typical ion pair reversed phase conditions. Such interactions reduce recovery and sensitivity, complicating impurity profiling, method robustness, and trace level quantitation.

Objectives and Study Overview

This application note evaluates the performance of the ACQUITY PREMIER Solution featuring MaxPeak High Performance Surfaces Technology in comparison with conventional stainless steel columns and LC systems. Key aims include reduction of conditioning requirements, improvement in recovery of n minus one impurities, extension of dynamic range, enhancement of linearity, and increased reproducibility of trace level oligonucleotide quantitation.

Methodology and Instrumentation

A series of oligonucleotide analytes was prepared, including a MassPREP oligodeoxythymidine standard, a fluorescein labeled 25 mer, a cyanine labeled 25 mer, and a fully phosphorothioated antisense oligonucleotide (GEM91). Ion pair reversed phase separations were performed at pH 7 (TEAA buffer) and pH 8.25 (TEA HFIP buffer) under gradient elution. Three setups were compared: stainless steel column with conventional system, ACQUITY PREMIER Column with conventional system, and ACQUITY PREMIER Column with ACQUITY PREMIER System. Data were acquired by UV detection at 260 nm and processed in Empower 3 software.

Used Instrumentation

• ACQUITY UPLC H Class PLUS Bio Binary System
• ACQUITY PREMIER System
• Oligonucleotide BEH C18 Column 1.7 µm 2.1×50 mm (stainless steel)
• PREMIER Oligonucleotide C18 Column 1.7 µm 2.1×50 mm
• Tunable UV detector at 260 nm
• Empower 3 Chromatography Data Software

Main Results and Discussion

The ACQUITY PREMIER Column demonstrated immediate out of the box performance, resolving and quantitating a 0.1 percent n minus one impurity of a FAM labeled 25 mer with 2.4 percent RSD over six injections, without any conditioning. In contrast, stainless steel columns required extensive conditioning and acid passivation to achieve comparable detection, yet still suffered from higher RSD. A two fold dilution series of GEM91 showed that the full PREMIER Solution delivered up to fourfold higher peak area, extended linear range over three orders of magnitude, lowered detection limits, and maintained RSDs below 10 percent at low mass loads. Similar improvements of 1.5 to twofold in recovery were observed for unmodified oligodeoxythymidine standards and fluorescently labeled oligonucleotides.

Benefits and Practical Applications

• Elimination of time consuming conditioning and passivation workflows
• Improved recovery of metal sensitive oligonucleotides and their impurities
• Extended dynamic range and enhanced linearity for trace level quantitation
• Increased sensitivity and lower limits of detection
• Greater reproducibility of impurity profiles and percent RSD reduction

Future Trends and Opportunities

Continued innovation in surface technologies will enable broader application to other metal sensitive biomolecules such as proteins, polar metabolites, and complex conjugates. Integration with mass spectrometry platforms promises enhanced structural characterization. Miniaturized flow paths and high throughput screening formats will benefit from surface inertness. Emerging therapeutic modalities including modified RNA and peptide nucleic acids may particularly gain from improved surface coatings to ensure robust analytics.

Conclusion

The ACQUITY PREMIER Solution with MaxPeak HPS Technology effectively addresses non specific adsorption of oligonucleotides to metal surfaces, delivering immediate high recovery, extended dynamic range, superior linearity, and reliable trace level quantitation without conditioning. This streamlined workflow enhances confidence in impurity analysis and supports rigorous quality control during oligonucleotide development and production.

References

1. DeLano M, Walter TH, Lauber MA, Gilar M, Jung MC, Nguyen JM, et al. Using Hybrid Organic Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Anal Chem. 2021.
2. Zhou W, Saran R, Liu J. Metal Sensing by DNA. Chem Rev. 2017;117(12):8272–8325.