Formulation, characterisation and lyophilisation of mRNA loaded lipid nanoparticles
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Full Text E-thesis
Date
2025
Authors
Walsh, Emma
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Publisher
University College Cork
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Abstract
Over the past number of years, the use of lipid nanoparticles (LNPs) to deliver a nucleic acid such as mRNA has gained great attention with a large number of products in the clinical pipeline. The manufacturing of LNPs can prove to be complex and have its challenges. Microfluidics is often applied as a processing method to produce LNPs due to its advantages such as fast producibility, ease of scale up and reproducibility. LNPs are often produced in aqueous buffering systems whereby their stability is often compromised. Therefore, lyophilisation is often employed, as a process that involves dehydration of a liquid formulation converting it to a solid form. The parameters used within a lyophilisation cycle must be chosen carefully as they can have a significant impact on the stability and biological activity of the active pharmaceutical ingredient (API), particularly for nucleic acids like mRNA.
The aim of this project was to develop stable lipid nanoparticles encapsulating mRNA as a solid dosage form through lyophilization. This involved two key objectives: (i) optimizing LNP production using microfluidics by examining parameters such as total flow rate, flow rate ratio, and N/P ratio, and (ii) enhancing LNP stability through lyophilization by selecting a suitable buffer assessing its impact on mRNA-loaded LNPs. These efforts aimed to identify key factors influencing the production of stable, lyophilized mRNA-containing LNPs for therapeutic applications.
The methods used to achieve the project aims consisted of a range of physiochemical characterisation techniques consisting of Dynamic Light Scattering (DLS) to determine Z-Average and polydispersity index (PDI), determination of encapsulation efficiency through a Ribogreen assay and agarose gel electrophoresis to assess presence of mRNA, as well as a visual assessment of lyophilized samples appearance.
In conclusion the impact of the microfluidic operating parameters and formulation components on the physiochemical characteristics of lipid nanoparticles does have an impact on the final LNP product. Varying the total flow rate did not impact the particle size whereas varying the flow rate ratio (FRR) had an impact on the particle size. It was determined that LNPs with encapsulated mRNA had an increased particle size when compared to empty LNPs. The N/P ratio was varied, and while no significant changes were observed in particle size or PDI, the encapsulation efficiency showed the most notable difference, with the highest efficiency achieved at an N/P ratio of 8. An assessment to identify a suitable lyophilisation buffer for the LNP determined mannitol to be the most suitable buffer for lyophilisation. The impact of lyophilisation on a LNP both empty and encapsulated with mRNA was then assessed. Lyophilisation did not impact the pH of the lipid mix with mRNA. However, the size and PDI increased, and the encapsulation efficiency was significantly decreased post the lyophilisation cycle.
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Keywords
mRNA , Lyophilisation
Citation
Walsh, E. 2025. Formulation, characterisation and lyophilisation of mRNA loaded lipid nanoparticles. MSc Thesis, University College Cork.