High-throughput and sensitive HPLC analysis of lipids used in LNP formulations with evaporative light scattering detection

Lipid nanoparticles (LNPs) are used to administer mRNA vaccines against Covid-19
and are considered as a crucial factor in ending the pandemic. Moreover, these
nanoparticles are being researched as a potential delivery system for active
pharmaceutical ingredients (API) that can combat infectious diseases, cancer, and
genetic disorders. KNAUER is the global leader in the production of impingement
jet mixing systems for the formulation of LNPs and also provides HPLC systems which
can be used for the analysis and quality control of LNP formulations. Here, we show
the development of several analytical methods for high-throughput analysis of the
lipid composition of LNPs. The lipids can be analyzed with a fully porous or a coreshell
phase in gradient or isocratic mode. The separation was speed up to a 2 min
method. KNAUER AZURA® UHPLC system coupled to evaporative light scattering
detection provided high sensitivity, with LODs in the range of 0.8–3 μg/ml (4–15 ng)
and LOQs of 0.9–4 μg/ml (5–32 ng).

Fig. 1 Separation of LNP-0315 mix using core-shell phenyl-hexyl column and a gradient with acetonitrile (70 % → 90 % in 4 min). CT = 50°C; ET = 45°C; 0.4 ml/min; 10 mM ammonium acetate as modifier. 1: Cholesterol; 2: ALC- 0159; 3: DSPC; 4: ALC-0315.
Fig. 2 Separation of LNP-0315 mix using core-shell phenyl-hexyl column and a gradient with methanol (80 % → 95 % in 4 min). CT = 50°C; ET = 45°C; 0.4 ml/min; 10 mM ammonium acetate as modifier. 1: Cholesterol; 2: DSPC; 3: ALC-0315.
Fig. 3 Separation of LNP-0315 mix using core-shell phenyl-hexyl column and gradient M2 (90 % methanol → 90 % acetonitrile in 4 min), CT = 50°C; ET = 45°C. 1: Cholesterol; 2: DSPC; 3: ALC-0315; 4: ALC-0159.
Fig. 4 Separation of LNP-SM102 mix using core-shell phenyl-hexyl column and gradient M2 (90 % methanol → 90 % acetonitrile in 4 min) CT = 50°C; ET = 45°C. 1: Cholesterol; 2: DSPC; 3: SM-102; 4: DMG-PEG(2000).
Fig. 5 Separation of LNP-0315 mix using core-shell phenyl-hexyl column and a gradient with acetonitrile:methanol 65:35 (70 % → 90 % in 4 min). CT = 50°C; ET = 45°C; 0.4 ml/min; 10 mM ammonium acetate as modifier. 1: Cholesterol; 2: ALC-0159; 3: DSPC; 4: A
Fig. 6 Separation of LNP-0315 mix using core-shell phenyl-hexyl column and a gradient with acetonitrile:methanol 65:35 (70 % → 90 % in 4 min). CT = 50°C; ET = 45°C; 0.4 ml/min; dark blue: 0.01 % formic acid as modifier; red: no modifier. 1: Cholesterol; 2
Fig. 7 Separation of LNP-0315 mix using core-shell phenyl-hexyl column and isocratic eluent composition M1. CT = 55°C; ET = 45°C; 0.4 ml/min. 1: Cholesterol; 2: ALC-0159; 3: DSPC; 4: ALC-0315.
Fig. 8 Separation of LNP-SM102 mix using core-shell phenyl-hexyl column and isocratic eluent composition M1. CT = 55°C; ET = 45°C. 1: Cholesterol; 2: DMG-PEG(2000); 3: DSPC; 4: SM-102.
Fig. 9 Optimization of the ELSD evaporation temperature demonstrated with isocratic method M1 for the core-shell phenyl-hexyl column. Blue: ET = 45°C; Red: ET = 35°C. Sample: LNP-0315 mix (dilution 1:2). 1: Cholesterol; 2: ALC0159; 3: DSPC; 4: ALC-0315.
Fig. 10 Comparison of core-shell column (dark blue) and fully porous column (red) with method M2. Sample: LNP-0315 mix (dilution 1:2). 1: Cholesterol; 2: DSPC; 3: ALC-0315; 4: ALC-0159.
Fig. 11 Separation of LNP-0315 mix (dilution 1:2) with fully porous phenylhexyl column and method M4. 1: Cholesterol; 2: ALC-0159; 3: DSPC; 4: ALC-0315.
Fig. 12 Separation of LNP-SM102 mix (dilution 1:2) with fully porous phenylhexyl column and method M4. 1: Cholesterol; 2: DMG-PEG(2000); 3: DSPC; 4: SM-102.
Fig. 13 Separation of of LNP-0315 mix (dilution 1:2) with fully porous phenylhexyl column and isocratic method M3. 1: Cholesterol; 2: ALC-0159; 3: DSPC; 4: ALC-0315.
Fig. 14 Separation of lipids LNP-SM102 mix (dilution 1:2) with fully porous phenyl-hexyl column isocratic method M3. 1: Cholesterol; 2: DM-PEG 2000; 3: DSPC; 4: SM-102.