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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01b2773z97m
Title: Post-Loading: An Alternative Process to Formulate RNA Vaccines
Authors: Amelemah, David
Advisors: Prud'homme, Robert
Department: Chemical and Biological Engineering
Class Year: 2023
Abstract: The use of RNA vaccines in the COVID-19 pandemic was crucial for global health. Lipid nanoparticles, created with the Confined Impinging Jet mixer via Flash NanoPrecipitation, encapsulated and protected the RNA used in the vaccines from degradation via self-hydrolysis or enzymatic degradation. Despite this encapsulation of RNA in a process called co-loading, ultra- cold storage temperatures (-40 °C for Moderna and -70 °C for Pfizer) were required during vaccine distribution, prohibiting accessibility to vaccination in low-income countries lacking the necessary cold storage infrastructure. To eliminate the need for cold-chain handling, empty lipid nanoparticles and lyophilized mRNA can be shipped at higher, inexpensive temperatures (-20°to 4 °C) and combined at the site of vaccination in a process we call post-loading. However, it is unknown if post-loaded lipid particles would have the same characteristics as co-loaded particles. To investigate this, we first determine the typical size of empty LNPs at pH 5 (27-31 nm), pH 7 (55-80 nm 5% EtOH, 70-95 nm 10% EtOH), and pH 8 (40-45 nm). Then, we find ways to control the reproducibility and stability of these empty LNPs via the size of pH 7 empty LNPs at 5% EtOH dependence on CIJ flow rate and the enhanced stability of pH 5 and pH 8 empty LNPs through DMG-PEG2k processing. We show successful empty LNP dialysis pathways between acidic and neutral pHs, demonstrating the flexibility of empty LNP processing and the feasibility of post-loading. Lastly, we exhibit post-loaded LNPs with monodisperse size distributions, an encapsulation efficiency of 91%, and a 6 nm RNA internal packing arrangement similar co-loaded LNPs shown through small angle x-ray scattering. As motivation for future studies, we find that the arrangement does not depend on EtOH content, and show that shifting from neutral to acidic pH is crucial for effective post-loading.
URI: http://arks.princeton.edu/ark:/88435/dsp01b2773z97m
Type of Material: Princeton University Senior Theses
Language: en
Appears in Collections:Chemical and Biological Engineering, 1931-2024

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