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Investigation on a Freeze-Drying Process for Long-Term Stability of mRNA-LNPs.

M D Faizul Hussain Khan1, Ayyappasamy Sudalaiyadum Perumal2, Amine A Kamen1

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Optimizing mRNA vaccine formulations for thermostability is crucial. Freeze-drying with specific sugars and buffers preserves mRNA-LNPs (lipid nanoparticles) in solid form, reducing cold-chain needs.

Keywords:
bufferscold chaincryoprotectantsformulation optimizationfreeze-dryinglipid nanoparticles (LNPs)long-term storagelyophilizationmRNA vaccinesmRNA-LNPsphysicochemical stabilityrefrigerated storagestabilizersvaccine delivery

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Area of Science:

  • Pharmaceutical Sciences
  • Biotechnology
  • Vaccine Development

Background:

  • Thermostability of mRNA-LNPs (messenger ribonucleic acid-lipid nanoparticles) is a major challenge for vaccine accessibility.
  • Current cold-chain requirements limit the widespread distribution and use of mRNA vaccines.

Purpose of the Study:

  • To optimize freeze-drying formulations for mRNA-LNPs to enhance thermostability.
  • To evaluate the impact of various excipients and buffers on the stability of solid mRNA-LNPs.
  • To assess the functional integrity of preserved mRNA-LNPs during long-term storage.

Main Methods:

  • Screening of various excipients including sugars (sucrose, trehalose), sugar alcohols (mannitol), and proteins (gelatin).
  • Evaluation of different buffer systems (Tris, histidine, PBS).
  • Assessment of physicochemical properties (particle size, PDI, EE) and in vitro transfection efficiency after storage.

Main Results:

  • Optimal freeze-drying formulations included 20% sucrose or trehalose with Tris or histidine buffers, maintaining high EE (>90%), particle size (~200 nm), and low PDI (<0.2).
  • Formulations stored at 4°C showed stability for up to four months, retaining physicochemical properties and transfection activity.
  • Storage at 20°C led to progressive destabilization, with significant loss of encapsulation efficiency by six months.

Conclusions:

  • Sugars are superior cryoprotectants for mRNA-LNPs compared to sugar alcohols and gelatin.
  • Solid mRNA-LNPs can be stored at refrigerated temperatures (4°C) for at least four months while maintaining functionality.
  • A sucrose/Tris formulation demonstrated promising stability at 20°C, indicating potential for storage at non-freezing temperatures.