Determination of lipid content and stability in lipid nanoparticles using ultra high‐performance liquid chromatography in combination with a Corona Charged Aerosol Detector

Abstract

For many years, lipid nanoparticles (LNPs) have been used as delivery vehicles for various payloads (especially various oligonucleotides and mRNA), finding numerous applications in drug and vaccine development. LNP stability and bilayer fluidity are determined by the identities and the amounts of the various lipids employed in the formulation and LNP efficacy is determined in large part by the lipid composition which usually contains a cationic lipid, a PEG‐lipid conjugate, cholesterol, and a zwitterionic helper phospholipid. Analytical methods developed for LNP characterization must be able to determine not only the identity and content of each individual lipid component (i.e., the parent lipids), but also the associated impurities and degradants. In this work, we describe an efficient and sensitive reversed‐phase chromatographic method with charged aerosol detection (CAD) suitable for this purpose. Sample preparation diluent and mobile phase pH conditions are critical and have been optimized for the lipids of interest. This method was validated for its linearity, accuracy, precision, and specificity for lipid analysis to support process and formulation development for new drugs and vaccines. This article is protected by copyright. All rights reserved

The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case

“…For determination of the purity and stability of lipids in the LNP, methods based on LC with CAD (charged aerosol detection) are most common, as the lipid components tend to lack a UV chromophore. Chromatographic conditions can be chosen such that the four main lipid components and their degradation products are separated 87 . The work by Kinsey et al also includes an example of how stress testing, or a forced degradation study could be conducted for the lipid components.…”

Challenges and emerging trends in liquid chromatography-based analyses of mRNA pharmaceuticals

“…Reversed phase stationary phases including C8, C18, and C30 alkyl chains have been successfully applied to separate lipid species under gradient conditions. Both weak and strong organic solvents have been used for elution; solvent choices have ranged from methanol and acetonitrile to isopropanol and tetrahydrofuran [97] , [98] , [99] . Method optimization has included the use of ion-pairing agents such as trifluoroacetic acid or triethyl amine as well as acetate or formate buffers as a means to control pH and selectivity of lipid species for improved chromatographic resolution [98] , [100] .…”

“…Both weak and strong organic solvents have been used for elution; solvent choices have ranged from methanol and acetonitrile to isopropanol and tetrahydrofuran [97] , [98] , [99] . Method optimization has included the use of ion-pairing agents such as trifluoroacetic acid or triethyl amine as well as acetate or formate buffers as a means to control pH and selectivity of lipid species for improved chromatographic resolution [98] , [100] . While successful, some method development considerations are necessary in these approaches.…”

“…Due to their low UV-activity, alternative detection techniques such as Charged Aerosol Detection (CAD) and Evaporative Light Scattering Detection (ELSD) have been needed for lipid analysis. [98] , [99] , [101] , [100] , [102] , [103] , [104] , [105] , [108] , [109] , [110] , [111] , [112] , [113] , [114] , [115] . From a method development perspective, both detectors offer user-controlled parameters to assist in the optimization process.…”

“…Mobile phase composition has been shown to impact response factors in both detector types where an inverse gradient may be needed to facilitate uniform detector response [101] , [109] , [110] , [111] . Both detector types have been found to offer sufficient sensitivity and dynamic range for most analytical needs and are well suited to impurity profiling, compositional analysis and stability indicating assays [98] , [99] , [104] , [108] , [113] .…”