Effect of thin-film freezing process parameters and excipient composition on mRNA lipid nanoparticles in inhalable powders

Abstract

Inhalable messenger RNA (mRNA) therapeutics provide direct access to the respiratory tract but liquid mRNA-lipid nanoparticle (LNP) formulations often require frozen storage and can be destabilized during nebulization. Thin-film freeze-drying (TFFD) offers a viable manufacturing approach to convert mRNA-LNP suspensions into stable dry powders with desirable aerosol performance.

Our prior work focused on how LNP composition and excipients affect mRNA-LNPs when they are subject to TFFD. Herein, we evaluated how thin-film freezing (TFF) process parameters in addition to formulation composition influence mRNA-LNPs and the aerosol performance of the resultant powders, using influenza virus Cal07 hemagglutinin (Cal07 HA mRNA)-LNPs. Increasing the lipid-to-mRNA ratio (LPR) improved HA mRNA encapsulation and enabled the selection of two lead HA mRNA-LNP formulation candidates. A 2-level factorial design of experiments (DOE) showed that Tris buffer, HA mRNA-LNP type, excipients, and drum temperature affected mRNA encapsulation efficiency and mRNA-LNP particle size differently after TFFD and reconstitution.

Notably, processing at −30 °C better preserved HA mRNA encapsulation efficiency than at −80 °C and this effect was formulation-dependent. The selected mRNA-LNP powders were amorphous, highly porous, and exhibited desirable aerosol performance. The immunogenicity of the HA mRNA-LNP was maintained after TFFD and reconstitution. Importantly, HA mRNA-LNPs that were destabilized by nebulization did not show significant changes in their particle size and mRNA encapsulation efficiency when actuated as TFFD powders using a dry powder inhaler. These findings highlight the importance of TFF process parameters in developing inhalable mRNA-LNP dry powders using TFFD.

Continue reading here

Materials

Cal07 HA mRNA was synthesized as previously described (Baiersdörfer et al., 2019, Pardi et al., 2012). High purity, low-endotoxin D-(+)-trehalose dihydrate (trehalose), Tris base and Tris HCl were obtained from Pfanstiehl (Waukegan, IL). Compendial grade L-Leucine, poloxamer P188 (P188), and sodium citrate dihydrate were from Spectrum Laboratories (Rancho Dominguez, CA). Multi-compendial citric acid monohydrate was from J.T Baker/Avantor (Radnor, PA). Hyclone™ RNase-free, molecular-grade water.

Michael A. Sandoval, Qin Li, Ruiqi Shi, Bhupendra R. Giri, Sorawee Yanwinitchai, Benjamin Southard, Houping Ni, Donald E. Owens, Robert O. Williams, Drew Weissman, Zhengrong Cui, Effect of thin-film freezing process parameters and excipient composition on mRNA lipid nanoparticles in inhalable powders, International Journal of Pharmaceutics, 2026, 127318, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2026.127318.