Messenger RNA-lipid nanoparticles (mRNA-LNPs) are commonly stored frozen, but the effects of freezer conditions on post-thaw quality remain insufficiently understood. Two mRNA-LNP formulations, without or with 10% sucrose, underwent 10 freeze-thaw cycles in freezers set at - 10, -30, or - 80 °C. Particle size, zeta potential, apparent entrapment efficiency (EE), mRNA integrity, and functional activity were assessed; negative-stain transmission electron microscopy provided morphological context. For the first time, electron paramagnetic resonance (EPR), differential scanning calorimetry (DSC), and in-vial Pt100 profiling were integrated to relate the quality outcomes of the formulation containing 10% sucrose (F2) to freeze-concentrated-phase mobility and thermal residence. The freezer conditions produced distinct thermal trajectories and quality profiles. The - 80 °C condition showed the fastest cooling and better preserved particle size, mRNA integrity, and functional activity than - 10 and - 30 °C, despite lower apparent EE. The - 10 and - 30 °C conditions caused greater losses of mRNA integrity and functional activity, with more extensive particle association and coalescence. In F2, EPR identified marked mobility restriction near - 20 °C and DSC measured the glass transition temperature of the maximally freeze-concentrated phase (Tg') at - 33.4 °C. The - 10 °C profiles remained above the mobility-transition interval, the - 30 °C profiles entered this interval but remained above Tg' throughout monitoring, whereas the - 80 °C profiles traversed the interval rapidly. Thus, post-thaw quality reflects coupled thermal-history components rather than cooling rate alone. Rapid solidification may contribute to apparent EE loss, whereas prolonged exposure to a mobile, non-vitrified freeze-concentrated phase may contribute to cumulative molecular and functional damage. Freeze-concentrated-phase residence is therefore a process parameter for optimizing freezing conditions for liquid mRNA-LNPs.
mRNA Technology