Improvement of Oligonucleotide Peak Shape Using Automatic Pretreatment Function (Co-injection)

Significance of the topic

The analysis of oligonucleotides by reversed-phase ion-pair chromatography (RP-IP) plays a crucial role in biopharmaceutical research and quality control. High salt concentrations in purified fractions can interfere with ion-pair formation, causing poor peak shape and split peaks. Developing automated strategies to mitigate these matrix effects improves throughput and data reliability in oligonucleotide therapeutic development.

Objectives and overview

This study demonstrates the use of an automated co-injection function to improve chromatographic peak shape for oligonucleotides in high-salt sample solvents. By simultaneously introducing a diluent into the sample stream, the method aims to suppress salt-induced retention issues and eliminate manually intensive desalting steps.

Methodology

Sample solvents containing 3 mol/L NaCl or 3 mol/L NaBr were prepared to simulate anion exchange–purified oligonucleotide fractions. Co-injection solvents included water and aqueous solutions of 100 mmol/L HFIP combined with 10, 30, or 50 mmol/L hexylamine (HA). Injection volumes of 1, 3, and 5 µL were evaluated. Chromatographic performance was assessed based on full-length product (FLP) peak shape and normalized peak area.

Instrumentation used

• UHPLC system: Nexera XS inert with autosampler co-injection capability
• Column: Accura Triart Bio C18, 100 mm × 2.1 mm I.D., 1.9 µm
• Mobile phases: Pump A – 100 mmol/L HFIP + 10 mmol/L HA in water; Pump B – 100 mmol/L HFIP + 10 mmol/L HA in methanol
• Temperature: 60 °C; Flow rate: 0.2 mL/min; Detection: UV at 260 nm

Results and discussion

Without co-injection, high salt concentrations suppressed ion-pair formation, producing split or broadened peaks and poor retention. Introducing co-injection significantly restored peak shape and retention. A minimum co-injection volume of 3 µL—regardless of solvent composition—eliminated split peaks and yielded stable normalized FLP peak areas. Both pure water and HFIP/HA mixtures effectively suppressed salt effects when delivered via co-injection.

Benefits and practical applications

• Automates sample dilution or desalting directly in the injection sequence, saving labor.
• Reduces manual pretreatment steps for multiple fractions.
• Maintains robust peak shape and retention for oligonucleotides in high-salt matrices.
• Facilitates high-throughput quality control in therapeutic oligonucleotide development.

Future trends and potential applications

Further optimization of co-injection conditions—such as tailored solvent composition or dynamic volume adjustment—could extend applicability to a broader range of biopolymers. Integration with method scouting and automated workflows may enable real-time adaptation to varying sample matrices. Advances in autosampler design could combine co-injection with inline desalting or trapping cartridges for enhanced sample cleanup.

Conclusion

The co-injection function on the Nexera XS inert UHPLC system provides a straightforward, automated approach to counteract high-salt matrix effects in oligonucleotide analysis. By delivering a diluent or ion-pair enhancer directly at injection, it restores peak integrity without manual pretreatment, significantly improving efficiency in biopharmaceutical analytics.