Thermo Scientific DNAPac Family of Columns

Importance of the Topic

Oligonucleotide analysis plays a pivotal role in the development and quality control of nucleic acid therapeutics, diagnostics, and research. Ensuring accurate separation, characterization, and quantitation of full-length sequences, truncated products, and chemically modified variants is essential for regulatory compliance and reliable data interpretation in both academic and industrial laboratories.

Objectives and Study Overview

The Thermo Scientific DNAPac column family has been engineered to deliver high-resolution anion-exchange and reversed-phase liquid chromatography solutions for oligonucleotide separation. This overview describes the evolution from legacy PA100 columns to modern PA200, PA200RS, RP, and DNASwift SAX-1 formats, highlighting their performance in purity assessment, sequence confirmation, and coupling to mass spectrometry.

Methodology and Study Design

The column chemistries and formats were evaluated using model oligonucleotides (e.g., 22-mer DNA and 21-mer truncated sequences) under optimized gradient conditions, varying mobile-phase composition, temperature, and flow rate. Key experiments included:

• Anion-exchange separation on PA200 and PA200RS columns demonstrating single-base resolution in UV detection.
• Reversed-phase separation on DNAPac RP columns at elevated temperature (up to 100 °C) for direct LC-MS compatibility.
• LC-UV and LC-MS analysis of failure sequences and therapeutic longer RNAs, including antiviral NAPs.
• Preparative purification using DNASwift SAX-1 monolithic columns for milligram scale fraction collection.

Instrumentation used

The following systems and detectors were employed:

• UHPLC/HPLC systems configured for anion-exchange and reversed-phase modes.
• UV detectors set at 260 nm or 295 nm for oligonucleotide absorbance.
• High-resolution mass spectrometer (Q Exactive Plus) operating in negative-ion mode for accurate mass determination.
• Chromatographic columns: DNAPac PA200 (8 µm, 4.6×250 mm), DNAPac PA200RS (4 µm, 4.6×150 mm), DNAPac RP (2.1×50 mm and 3.0×100 mm), and DNASwift SAX-1 (5×150 mm monolith).

Main Results and Discussion

Anion-exchange columns achieved baseline separation of full-length oligonucleotides from n-1 truncated forms and resolved diastereomeric phosphorothioate RNA species. Reversed-phase RP columns provided high-temperature stability and direct coupling to LC-MS for accurate mass analysis, successfully identifying minor failure sequences at the –4 charge state. The DNASwift SAX-1 column delivered high-capacity preparative separations with >85 % purity in target fractions.

Benefits and Practical Applications

The DNAPac column portfolio offers:

• Single-base resolution up to 100 mers and compatibility with a broad pH range (1–14).
• Robust performance at elevated temperatures (up to 100 °C) with minimal carryover.
• Seamless integration with UV and mass spectrometric detection for structural confirmation and quantitation.
• High throughput capability in both analytical and preparative workflows.

Future Trends and Opportunities

Advances in oligonucleotide therapeutics, including mRNA vaccines and gene editing tools, will drive demand for even higher throughput and resolution. Emerging applications may include automated UHPLC-MS workflows, expanded pore-size media for large biomolecules, and integration with bioinformatics for real-time data analysis.

Conclusion

The Thermo Scientific DNAPac and DNASwift column families provide versatile, high-performance solutions for comprehensive oligonucleotide analysis and purification. Their robust chemistries, broad pH and temperature tolerances, and compatibility with mass spectrometry make them indispensable tools in modern nucleic acid research and quality control.