(W1230-11-65) Effect of Lyophilization Process Parameters on the Stability of mRNA Lipid Nanoparticles (LNP)

Purpose: Lipid nanoparticle (LNP) systems have enabled the development of mRNA-based vaccines by acting as a delivery device that also provides some stabilization of the mRNA payload. However, there are efforts to improve the storage conditions of mRNA-containing LNPs beyond the typical shipping and storage at -20 to -80°C, for the first generation of commercially available RNA-based COVID vaccines. Lyophilization of mRNA LNPs may reduce or eliminate the need for a cold-chain by providing additional stability for inherently fragile mRNA molecules and enabling refrigerated storage. The studies described here explored the effects of lyophilized formulation matrix composition and freeze-drying process parameters on critical drug product quality attributes of mRNA LNP, including particle size and polydispersity, % encapsulation efficiency, and mRNA integrity.

Methods: The effect of (i) annealing (no annealing vs. annealing at -10°C), (ii) primary drying at or above the collapse temperature (T_shelf between -38°C to 0°C, P<sub>chamber: 30-100 mTorr), secondary drying at or above the lipids transition temperature (T_shelf between 35°C to 65°C) was investigated.  The following formulation matrices were lyophilized in 10 mM Tris (pH 7.4): (1) 8% w/v mannitol and 2% w/v sucrose; (2) 8% w/v mannitol and 2% w/v trehalose; (3) 10% w/v trehalose; (4) 10% w/v trehalose and 6 mg/mL NaCl; (5) 5% w/v trehalose and 5% w/v sucrose; and (6) 10% w/v sucrose.  The moisture content was measured using a Mettler-Toledo Titrator Compact C10SX.  The particle size and polydispersity index (PDI) were measured by Dynamic Light Scattering using a Zetasizer Red Label Ultra^®.  %Encapsulation was determined using a Quant-iT^TM RNA RiboGreen Assay Kit from (Thermo-Fisher Scientific) and a BioTek Synergy neo 2 plate reader.  The percentage of intact mRNA was measured using an Agilent 5300 Fragment Analyzer with a 96-capillary array for a time of 40 minutes.

Results: The moisture content of the dried mRNA LNP formulations ranged from 0.3% to 0.8% w/w.  The Z-average, polydispersity, %Encapsulation, and RNA integrity remaining for each formulation were determined post reconstitution in saline (Tables 1-3).  The inclusion of a crystallizable excipient such as mannitol led to substantial changes in morphological properties (LNP size attributes, and %Encapsulation) highlighting the need to maintain an amorphous formulation matrix during processing and storage.  As expected, no significant changes in RNA integrity were observed.

Conclusion: The quality attributes of mRNA-containing LNPs were affected by both formulation and lyophilization process parameters. In mannitol containing formulations, annealing facilitated solute crystallization which further exacerbated the effects of lyophilization and reconstitution stresses on LNP attributes with no impact on mRNA integrity.  Sucrose containing formulations afforded greater protection during freeze-drying than in trehalose-based formulations.

Acknowledgements: The authors declare the following competing financial interest: EJM is a partial owner of Kansas Analytical Services, a company that provides solid-state NMR services to the pharmaceutical industry. The results presented here are from academic work at Purdue University, and no data from Kansas Analytical Services are presented.

Z-average, PDI, encapsulation efficiency, and percent intact mRNA remaining for frozen controls of Formulations 1 - 6.

Z-average, PDI, encapsulation efficiency, and percent intact mRNA remaining for Formulations 1 – 6 lyophilized without an annealing step during freezing.

Z-average, PDI, encapsulation efficiency, and percent intact mRNA remaining for Formulations 1 – 6 lyophilized with an annealing step during freezing.