Easy Column Screening for Lipid Nanoparticle Component Analysis

Significance of the Topic

Lipid nanoparticles (LNPs) have become essential carriers for RNA-based therapeutics, protecting nucleic acids from degradation and improving delivery efficiency. As the variety of ionizable lipids grows with each new RNA drug, reliable analytical methods are required to characterize LNP composition, identity, and purity to ensure consistent safety and efficacy.

Objectives and Study Overview

This study demonstrates a streamlined approach to screen multiple reversed-phase columns for optimal separation of the four main LNP lipids—cholesterol, DSPC (neutral phospholipid), PEG-lipid, and ionizable cationic lipid—using an Agilent 1260 Infinity II Prime Bio LC System coupled with a biocompatible 6-Column Selector Valve. The goal was to rapidly identify the column chemistry and chromatographic conditions that deliver baseline resolution and sharp peak shapes for all components.

Methodology and Instrumentation

The experimental setup included:

• Agilent 1260 Infinity II Bio Flexible Pump
• Agilent 1290 Infinity II Bio Multisampler with Sample Thermostat
• Agilent 1290 Infinity II Multicolumn Thermostat equipped with Biocompatible 6-Column Selector Valve and capillaries
• Agilent 1290 Infinity II ELSD for lipid-sensitive detection
• Agilent OpenLab CDS software, v2.6

Six Agilent reversed-phase columns were evaluated:

• InfinityLab Poroshell 120 Phenyl-Hexyl (2.1×50 mm, 1.9 µm)
• AdvanceBio RP-mAb Diphenyl (2.1×50 mm, 3.5 µm)
• ZORBAX RRHD 300 Å Diphenyl (2.1×50 mm, 1.8 µm)
• InfinityLab Poroshell 120 CS-C18 (2.1×50 mm, 2.7 µm)
• ZORBAX StableBond 300 Å CN (4.6×50 mm, 3.5 µm)
• AdvanceBio Peptide Plus (2.1×50 mm, 2.7 µm)

Mobile phases comprised methanol, acetonitrile, water, and 500 mM ammonium acetate buffer. Screening employed two general gradients, followed by fine adjustments to maximize separation.

Key Results and Discussion

Initial screening identified ZORBAX RRHD 300 Å Diphenyl and InfinityLab Poroshell 120 Phenyl-Hexyl as top performers. Other columns either eluted all lipids too early or lacked sufficient retention.

Optimization for ZORBAX RRHD 300 Å Diphenyl

A gradient from 60 % to 90 % methanol over 3 min, then to 90 % acetonitrile by 5 min, provided clear baseline separation of cholesterol, DSPC, and ionizable lipid, while improving the peak shape for the PEG-lipid.

Optimization for InfinityLab Poroshell 120 Phenyl-Hexyl

An optimized program with a 3 min isocratic hold at 90 % methanol followed by a linear ramp to 90 % acetonitrile over 2 min achieved excellent resolution and symmetrical peaks for all four lipid components.

Benefits and Practical Applications

• Rapid column screening accelerates method development for LNP characterization.
• Biocompatible selector valve enables high-throughput evaluation without system downtime.
• Optimized methods support quality control of RNA therapeutics by ensuring precise lipid profiling.

Future Trends and Prospects

Advancements may include coupling optimized columns with mass spectrometry for structural elucidation, exploring novel stationary phases tailored to emerging ionizable lipids, and integrating automated solvent selection to further reduce development time.

Conclusion

The combination of the Agilent 1260 Infinity II Prime Bio LC System and biocompatible 6-Column Selector Valve proved highly effective for identifying ideal columns for LNP lipid analysis. The ZORBAX RRHD 300 Å Diphenyl and InfinityLab Poroshell 120 Phenyl-Hexyl columns delivered robust separation under optimized gradients, offering reliable tools for RNA drug development and quality control.

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