Development of an imaged capillary isoelectric focusing method for characterizing the surface charge of mRNA lipid nanoparticle vaccines

Abstract

Lipid nanoparticles (LNPs) have been employed for drug delivery in small molecules, siRNA, mRNA, and pDNA for both therapeutics and vaccines. Characterization of LNPs is challenging because they are heterogeneous mixtures of large complex particles. Many tools for particle size characterization, such as dynamic and static light scattering, have been applied as well as morphology analysis using electron microscopy. CE has been applied for the characterization of many different large particles such as liposomes, polymer, and viruses. However, there have been limited efforts to characterize the surface charge of LNPs and CIEF has not been explored for this type of particle. Typically, LNPs for delivery of oligonucleotides contain at least four different lipids, with at least one being an ionizable cationic lipid. Here, we describe the development of an imaged capillary isoelectric focusing method used to measure the surface charge (i.e., pI) of an LNP-based mRNA vaccine. This method is capable of distinguishing the pI of LNPs manufactured with one or more different ionizable lipids for the purpose of confirming LNP identity in a manufacturing setting. Additionally, the method is quantitative and stability-indicating making it suitable for both process and formulation development.

Keywords: Cationic lipid; Imaged capillary isoelectric focusing; Isoelectric point (pI); Lipid nanoparticles; Maurice; mRNA vaccine.

Figure 1

(A) Electropherograms of an LNP using various ampholytes and additives. Traces A and B show high background or precipitation and LNP containing sharp peaks using the broad range Servalyt pH 2–9 and Pharmalyte ampholytes pH 3–10, respectively. Trace C shows a focused LNP with an apparent pI of approximately 7.3 using pH 3–10 Pharmalyte ampholytes containing 10% glycerol. Trace D uses a mixture of 33.3% ampholyte pH 5–8 and 66.6% ampholytes pH 3–10 with 10% glycerol. Trace E uses a 66.6% ampholyte pH 5–8 and 33.3% ampholytes pH 3–10 with 10% glycerol. Trace F uses ampholyte pH 5–8 containing 10% glycerol. The pI of the LNP shifts to approximately 7.6–7.8 in traces D, E, and F. Two pI markers are 5.85 and 8.40. (B) Electropherogram of an LNP prepared in triplicate. An LNP sample was prepared in triplicate for the icIEF experiment. The LNP has an apparent pI of approximately 7.89 and peak shape was consistent for the three replicates. (C) Calibration curve of LNP, which ranges from 7.2–115 µg/mL of total lipids. LNP samples were diluted in icIEF ampholyte mixtures from 0.56 to 9.0 µg/mL of mRNA (equivalent to 7.2 to 115 µg/mL of total lipid). This linear range has a coefficient of determination (R ²) ≥ 0.997.

Figure 2

LNP pI plotted by cationic lipids and mRNA concentration: different LNP batches were formulated to contain different cationic lipid to mRNA ratios. These LNPs were then diluted to five different lipid concentrations and subjected to icIEF. The pI (y‐axis) was plotted against cationic lipid concentration (Fig. 2A) and the mRNA concentration (Fig. 2B). A strong correlation of pI to cationic lipid concentration was observed (R ² = 0.956; logarithmic fit, left graph) compared to a weaker correlation of pI to mRNA concentration (R ² = 0.653; logarithmic fit, right graph).

Figure 3

(A) UV absorbance of LNPs in both aqueous and icIEF ampholyte mixtures. LNPs containing mRNA were tested in Tris buffer (blue trace) and icIEF ampholyte mixture (black dash trace). LNPs without mRNA were tested in Tris buffer (green trace) and icIEF ampholyte mixture (red dash trace). LNPs containing mRNA show an absorbance max at 260 nm compared to LNP without mRNA, which lack a peak at 260 nm. Both aqueous and icIEF ampholyte mixtures absorbance traces were identical when comparing the wavelengths at 260 nm demonstrating that with or without mRNA, LNPs are stable and intact in the final cIEF ampholyte mixture. (B) Electropherogram of LNP formulated with and without mRNA. LNPs without mRNA (red dashed trace) have a similar pI to LNPs formulated with mRNA (black solid trace). The pI of both LNPs is approximately 7.6–7.7. Two pI markers were 5.85 and 8.40.

Figure 4

LNP with different cationic lipids have unique pIs. Trace A shows an LNP containing Cationic Lipid‐1with a lower pKa value has a pI of 7.6–7.7. (B) LNP containing Cationic Lipid‐2 with a higher pKa value has a pI of 8.1. (C) Separation of a mixture of LNP containing different cationic lipids. Two pI markers were 5.85 and 8.40.

Figure 5

(A) Stability of LNPs containing mRNA. The LNPs containing mRNA were exposed to elevated temperatures for 24 h. The LNP stored at 2–8°C showed a symmetrical peak shape with a pI of approximately 7.7. As the temperature increased, the LNP with mRNA peaks became more acidic and split into two distinct peaks. (B) Stability of empty LNPs. LNPs without mRNA stored at 2–8°C showed a symmetrical peak shape with a pI of approximately 8.0. As the temperature increased, the LNP without mRNA showed a different degradation pattern compared to the LNPs containing mRNA. Two pI markers were 5.85 and 8.40.