Analysis of Lipid Nanoparticle Composition

Importance of the Topic

Lipid nanoparticles LNPs serve as critical carriers for nucleic acid therapies, offering protection and efficient delivery of siRNA and mRNA. Precise characterization of lipid composition and ratios is vital to ensure safety, efficacy and regulatory compliance in biopharma applications.

Study Objectives and Overview

• Develop a robust chromatographic method for four key LNP lipids
• Optimize solvent combinations in a quaternary pump configuration
• Implement evaporative light scattering detection ELSD for universal lipid detection
• Validate dynamic range precision and sensitivity in patisiran like samples

Methodology and Instrumentation

• Agilent 1290 Infinity II Bio LC system with four channel flexible pumps and multicolumn thermostat
• Agilent 1290 Infinity II ELSD for detection of non UV absorbing lipids
• InfinityLab Poroshell 120 Phenyl Hexyl column 2.1 × 50 mm 1.9 µm
• Mobile phases: water with 10 mM ammonium acetate methanol acetonitrile with continuous buffer supply
• Quaternary gradient combining MeOH and ACN from 82 to 90 then to 90 ACN with controlled temperatures
• Sample dissolution in methanol and serial dilutions for calibration and linearity studies

Main Results and Discussion

• Binary gradients using only methanol or acetonitrile resulted in incomplete elution or broad peaks
• Quaternary method with dual organic solvents achieved baseline separation of all four lipids
• Retention time RSD consistently below 0.15 and peak area RSD below 3.3 ensured method precision
• Limits of detection ranged from 0.46 to 8.1 pmol and limits of quantification from 1.6 to 27 pmol on column
• Shortened 3 minute gradient on a binary pump maintained resolution and improved throughput

Benefits and Practical Applications

This ELSD based method delivers universal detection for lipids lacking chromophores and provides a wide dynamic range for quantification. It supports formulation development quality control and batch release testing of lipid based RNA therapeutics.

Future Trends and Potential Applications

• Integration with mass spectrometry for combined lipid and nucleic acid profiling
• Automation and high throughput workflows in vaccine and gene therapy production
• Emergence of new stationary phase chemistries to further reduce run times
• Real time monitoring of LNP stability and degradation during manufacture and storage

Conclusion

The described quaternary LC ELSD approach offers superior flexibility high resolution and reliable quantification of LNP components. Combining methanol and acetonitrile under controlled buffer conditions yields sharp peaks excellent precision and adaptability to both quaternary and binary systems. This method is well suited for both research and regulatory environments in biopharma.

Reference

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5. NDA 210922 Onpattro Patisiran Lipid Complex Injection Addendum to Drug Product Quality Review 2018
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7. Agilent Technologies A Sensitive Detection Technique for Lipids in Liposomal Formulations Application Note 2019