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.

Keywords: Cationic lipid; Charged aerosol detection (CAD); Lipid degradation; Lipid nanoparticles (LNP); Reverse-phase chromatography.

Figure 1

Recovery of the lipids (PEG‐DMG, Cholesterol, Cationic Lipid 1 and DSPC) from mRNA‐LNP samples at four‐, six‐, eight‐, and tenfold dilutions with (A) 1:1 Ethanol: DMSO and four‐ and tenfold dilutions with (B) 85:15 ethanol:formamide.

Figure 2

Chromatographic profile of (A) PEG‐DMG, cholesterol, Cationic Lipid 1, and DSPC with the mobile phases of water and methanol containing formic acid and Triethylamine (pH 3.5); (B) PEG Lipid 2, cholesterol, Cationic Lipid 2, and DSPC with the mobile phases of water and methanol containing acetic acid and triethylamine (pH 5.4).

Figure 3

Mobile phase pH effects on cationic lipid separation (A) PEG Lipid 2, cholesterol, Cationic Lipid 2, and DSPC separation at pH 3.5 (B) PEG‐DMG, cholesterol, DSPC, and Cationic Lipid 1 separation at pH 5.4.

Figure 4

Selectivity of lipid degradation products of (A) myristic acid, lyso‐PC‐1, and lyso‐PC‐2, stearic acid, and a known degradant of Cationic Lipid 1 with mobile phase at pH 3.5 (B) oxidized Cationic Lipid 2 and Cationic Lipid 2 with mobile phase at pH 5.4 (C) hydrolyzed product 1, 2, 3, and 4 of PEG Lipid 2, and PEG Lipid 2 with mobile phase at pH 5.4.

Figure 5

Stability data of a 10‐month stability study for lipids PEG‐DMG, Cholesterol, Cationic Lipid 1, and DSPC at –70°C, –20°C, 4°C, and 25°C storage conditions.