Data for: "Production of mRNA Lipid nanoparticles using advanced crossflow mixing"

This data set supports work we we look at Lipid nanoparticles (LNPs) production. LNPs play a crucial role in RNA-based therapies, and their production is generally based on nanoprecipitation and coalescence of lipids around an RNA core. This process is primarily controlled via the rate of ethanol-lipid to mRNA aqueous phase mixing. When considering a manufacturing process for LNPs, choosing between single-use and reusable systems influences production efficiency, cost, and sustainability. Single-use systems offer advantages in minimising contamination and downtime, while reusable systems reduce plastic use, avoid the problem of leachables, especially in solvents and present long-term cost savings. Our results demonstrate the reproducible production of mRNA-LNPs with controlled Critical Quality Attributes, including high mRNA encapsulation from the initial screening scale through to GMP-scale production, where the same mixing ratio can be adopted across all product speeds from 30 to 500 mL/min used. These mRNA-LNPs were confirmed to be effective as therapeutics (protein expression) and for vaccination (antibody and cytokine responses).

Within the data is the following.
Figure 2. The effect of the flow rate ratio and total flow rate on LNPs z-average size, PDI, zeta potential, and Encapsulation Efficiency (%). PolyA was used as the payload in these studies. LNPs were produced using Micropore Pathfinder™. DOTAP, SM102 or C12-200 LNPs (see Table 1 for full compositions) were prepared at a flow rate ratio of 1:1, 3:1 or 5:1 and a production speed of 10, 55 or 100 mL/min to a final polyA concentration of 0.0213 mg/mL before ethanol removal. LNPs were purified to remove ethanol via dialysis. Results represent the mean ± SD of 3 independent studies.

Figure 3. LNPs produced using AXFTMmini in vitro efficacy. SM102 LNPs were prepared using the AXFTMmini at a flow rate ratio of 3:1 (citrate buffer 50 mM pH4: ethanol) at a production speed of 30 mL/min to a final mRNA concentration of 0.0213 mg/mL before ethanol removal. LNPs were purified to remove ethanol and concentrated to the required mRNA/polyA concentration via spin-column purification. These LNPs were then tested in terms of a) mRNA integrity (RNA Millenium Marker hyperladder, n1 to 3 are replicate samples, Pos: Fluc mRNA positive control, NTC: No template control), b) HEK293 cell viability (polyA as the payload) and c) protein (luciferase) expression. Results represent mean ± SEM of 3 independent studies

Figure 4. In vivo expression profile of LNPs encapsulated Fluc mRNA prepared with SM-102, ALC-0315 and C12-200 as ionisable lipids. a) representative IVIS images of 3 female BALB/c mice injected with 5 μg Fluc mRNA-LNP intramuscularly. b) Quantification of the bioluminescent signal at the injection (dark blue) site and liver (light blue) 6 hours after the LNPs injection. LNPs were produced using AXFTMmini at a flow rate ratio of 3:1 at a 30 mL/min production speed. Data is expressed by mean ± SEM (6 mice per formulation split over 2 independent studies). followed by conducting ANOVA combined with non-parametric tests using GraphPad Prism (*P< 0.05).

Figure 5. Physiochemical properties of LNPs produced using AXF™one. DDAB LNPs were prepared using the AXF™one at a flow rate ratio of 3:1 (citrate buffer 50 mM pH6: ethanol) to a final polyA concentration of 0.0213 mg/mL before ethanol removal. PolyA was used as a surrogate for mRNA in this study. These LNPs were then studied for the effect of total flow rate (200, 300 or 500 mL/min); a) LNPs z-average size, PDI, b) zeta potential and c) Encapsulation Efficiency (%). Consistency of the product produced at a flow rate of 500 mL/min on LNP production was then tested by collecting samples at different time points (20, 40, 60 and 80 seconds); d) LNP z-average size, PDI, e) zeta potential and f) Encapsulation Efficiency (%). Results represent the mean ± SD of 3 independent studies.

Figure 6. LNPs produced using AXF™one in vitro efficacy. SM102 LNPs were prepared at a flow rate ratio of 3:1 (citrate buffer 50 mM pH4: ethanol) at a production speed of 500 mL/min to a final mRNA (encoding luciferase) concentration of 0.0213 mg/mL before ethanol removal. LNPs were purified to remove ethanol and concentrated to the required concentration via spin-column purification. These LNPs were then tested in terms of a) LNPs entrapping mRNA CQAs (z-average size, PDI, zeta potential and Encapsulation Efficiency (%). b) HEK293 LNPs cell uptake % (using polyA as a surrogate), c) cell viability (using polyA as a surrogate) and d) protein (luciferase) expression. Results represent the mean ± SD of 3 independent studies.

Figure 7. In vivo biodistribution and expression profile of LNPs encapsulated F-Luc mRNA prepared with 50 mM citrate buffer using the AXF™one. a) IVIS images of groups of 5 female BALB/c mice injected intramuscularly with 5 μg DiR labelled Fluc mRNA-LNP and imaged at 0, 0.25, 1, 2, 3, 6 and 9 days. b) Quantitative analysis of the fluorescence intensity in the injection site and c) Quantification of the bioluminescent signal at the injection site. Results represent mean ± SEM of 10 mice per formulation split over 2 independent studies (5 mice per study).

Figure 8. Vaccine potency of SM-102 LNPs encapsulated mRNA encoding OVA prepared with 50 mM of citrate buffer using the AXF™one. Groups of 5 female mice BALB/c were immunised intramuscularly on days 0 and 28 with 5 μg OVA mRNA-LNP, OVA mRNA or nothing (control). The mice serum was then used to study the effect of vaccination on a) the specific Total IgG, b) the specific IgG1, and c) the specific IgG2a antibody titres. Mean d) IL-5 and e) IFN-g production, splenocytes (1× 106/mL) from the same mice were incubated with medium alone (controls), ConA (10 μg/mL) or OVA soluble antigen (5 μg/mL) for 72 h. Results represent mean ± SEM of a total of 5 mice per formulation followed by conducting ANOVA combined with non-parametric tests using GraphPad Prism (*P< 0.05).