(W1230-07-39) Lipid Nanoparticles Particle Size Control: Effect of Buffer and Linear vs Circular mRNA

Purpose: Recently mRNA delivery through LNPs has become a hot topic in drug development. Although most LNPs reported tend to control particle size below 100 nm, for some applications such as for vaccine, larger particles may prove to be beneficial for immune cell uptake after administration, at least as previously seen in mice (1). Manufacturing larger LNPs using microfluidic systems can be challenging due to changes in process conditions such as alteration of aqueous vs lipid phase ratio. Decreasing aqueous phase ratio relative to lipid phase (lipids in ethanol) ratio may lead to an increase in particle size (PSD) but at an expense of lowered encapsulation efficiency (%EE) and particle stability. In this study, we explored the effect of aqueous to lipid phase ratio in particle size, encapsulation efficiency changes. In addition, we explored the effect of buffer system changes in improving %EE. We also used circular and linear mRNAs and investigated if this change effect particle size and encapsulation.

Methods: LNPs were manufactured using NanoAssemblr Ignite microfluidic system. Different aqueous to lipid phase ratios were used for manufacturing LNPs. Buffer strengths were changed as well as mRNAs (circular vs linear). mRNA concentration, ionizable lipid to mRNA ratio, lipid molar ratios were kept constant in all formulations. LNPs were dialyzed against before characterization. Malvern zetasizer was used for measuring particles size and ribogreen assay was used for encapsulation efficiency measurement.

Results: Decreasing aqueous phase ratio relative to ethanol phase ratio led to larger particle sizes. Incubating larger particles, prepared at aqueous: lipid ratio of 1.5:1 before dialyzing lead to further increase in particle sizes which was not the case for smaller particles prepared at aqueous: lipid ratio of 3:1. Further decreasing aqueous phase to 1:1 (aqueous: lipid) lead to higher polydispersity (figure 1). Circular mRNA showed better encapsulation efficiency in larger particles compared to when linear mRNA was used at different buffer strengths. This phenomenon was observed with different sized circular mRNA where size varied by ~1.8x kb (figure 1, 2 and 3) Changing buffer strength led to higher encapsulation efficiency in larger particles noticeably matching encapsulation of mRNA in larger particles to the smaller particles (figure 1). 8x scaled up LNPs showed similar properties as small scale LNPs. Particle size and encapsulation efficiency were preserved throughout further processing of particles i.e. dialysis and concentration (figure 3).

Conclusion: In the study, we developed a method for LNP particle size control. Circular mRNA seemed to have more robustness in terms of particle size control, showed higher encapsulation efficiency and was less susceptible to buffer changes.

References: [1] K. J. Hassett et al., “Impact of lipid nanoparticle size on mRNA vaccine immunogenicity,” Journal of Controlled Release, vol. 335, pp. 237–246, Jul. 2021, doi: 10.1016/j.jconrel.2021.05.021.

Figure 2: Comparison between linear vs circular mRNA at higher buffer strength. Target PSD was achieved with high encapsulation efficiency.

Figure 3: Large circular mRNA showed similar particle size control with great encapsulation efficiency at scale up. PSD and %EE was maintained throughout the processing steps.