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

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.