ANALYTICAL TOOL FOR CHARACTERIZATION OF LIPID NANOPARTICLES
Described herein are methods of separating lipid nanoparticles (LNPs) according to their isoelectic points, the methods comprising applying a separating voltage to a separation matrix comprising carrier ampholytes and the LNPs for a sufficient time to separate the LNPs according to their isoelectic points.
1 . A method of separating lipid nanoparticles (LNPs) according to their isoelectic points, the method comprising applying a separating voltage to a separation matrix comprising carrier ampholytes and the LNPs for a sufficient time to separate the LNPs according to their isoelectic points.
2 . The method of claim 1 , wherein the separation matrix further comprises a stabilizer.
3 . The method of claim 2 , wherein the stabilizer is glycerol.
4 . The method of claim 2 , wherein the stabilizer is present in the separation matrix at 5-15% (w/v or v/v).
5 . The method of claim 1 , wherein the separation matrix further comprises methylcellulose.
6 . The method of claim 5 , wherein the methylcellulose is present in the separation matrix at 0.2%-0.25% w/v.
7 . The method of claim 1 , wherein the carrier ampholytes form a linear pH gradient when the voltage is applied.
8 . The method of claim 1 , wherein the carrier ampholytes form a sigmoidal pH gradient capable of higher resolution separation in a pH sub-range that includes the isoelectric points of the LNPs when the voltage is applied.
9 . The method of claim 1 , wherein the separation matrix is in a capillary.
10 . The method of claim 9 , wherein the capillary is a silica capillary coated with fluorocarbon.
11 . The method of claim 1 , wherein the LNPs comprise, individually, one or more cationic lipid species, one or more non-cationic lipid species, cholesterol, one or more PEG-lipids, or a combination thereof.
12 . The method of claim 1 , wherein the LNPs comprise, individually, one or more encapsulated nucleic acids.
13 . The method of claim 1 , wherein the LNPs comprise, individually, one or more encapsulated mRNA species.
14 . The method of claim 1 , further comprising imaging the separation matrix after the sufficient time to produce an electropherogram.
15 . The method of claim 14 , wherein imaging the separation matrix comprises detecting UV absorbance at 280 nm.
16 . The method of claim 14 , further comprising measuring a peak area corresponding to LNPs having a selected pI in the electropherogram and comparing the peak area to a calibration curve to calculate a total lipids concentration.
17 . The method of claim 14 , further comprising comparing the electropherogram to a reference electropherogram produced under identical conditions.
18 . The method of claim 17 , wherein the reference electropherogram was produced from the same batch of LNPs as the LNPs in the electropherogram, and wherein an acidic shift or altered peaks in the electropherogram indicate a change in LNP stability.
19 . The method of claim 17 , wherein the reference electropherogram was produced from a reference batch of LNPs having the same lipid composition as the LNPs in the electropherogram, and wherein a significant difference between the electropherograms indicates a manufacturing problem for the LNPs.
20 . The method of claim 1 , wherein the LNPs in the separation matrix comprise a level of cationic lipids that is normalized to a level of cationic lipids used to produce the reference electropherogram.