Improved process of preparing mrna-loaded lipid nanoparticles
OwnerShrirang Karve, Frank DeRosa +7inventors
InventorsShrirang Karve, Frank DeRosa, Michael Heartlein, Ashish Sarode, Zarna Patel, Rebecca L. Ball, Natalia Vargas Montoya, Priyal Patel, Asad Khanmohammed
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Abstract
The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process for enhanced encapsulation of messenger RNA (mRNA) in lipid nanoparticles comprising a step of heating the mRNA-encapsulated lipid nanoparticles in a drug product formulation solution.
Claims27
A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs) comprising the steps of:
- (a) mixing one or more lipids in a lipid solution with one or more mRNAs in an mRNA solution to form mRNA encapsulated within the LNPs (mRNA-LNPs) in a lipid nanoparticle (LNP) formation solution;
- (b) exchanging the LNP formation solution for a drug product formulation solution to provide mRNA-LNP in a drug product formulation solution;
- and
- (c) heating the mRNA-LNP in the drug product formulation solution;
- wherein the encapsulation efficiency of the mRNA-LNPs resulting from step (c) is greater than the encapsulation efficiency of the mRNA-LNPs resulting from step (b).
The process according to any one of the preceding claims, wherein in step (a) the one or more cationic lipids are selected from cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE (Imidazol-based), HGT5000, HGT5001, HGT4001, HGT4002, HGT4003, HGT4004, HGT4005, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl) (2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target 23), 3-(5-(bis(2-hydroxydodecyl)amino) pentan-2-yl)-6-(5-((2-hydroxydodecyl) (2-hydroxyundecyl)amino) pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24), N1GL, N2GL, V1GL, and combinations thereof.
The process according to any one of the preceding claims, wherein the lipid component of the lipid solution consists of:
- (a) a cationic lipid,
- (b) a helper lipid,
- (c) a cholesterol-based lipid, and
- (d) a PEG-modified lipid.
The process according to claim 8, wherein the molar ratio of the cationic lipid to helper lipid to cholesterol-based lipid to PEG-modified lipid is about 20-50:25-35:20-50:1-5.
The process according to any one of the preceding claims, wherein the mRNA encodes for a protein or peptide.
The process according to any one of the preceding claims, wherein in step (c) the drug product formulation solution is heated by applying heat from a heat source to the solution and the solution is maintained at a temperature greater than ambient temperature for between 10 and 20 minutes.
The process according to any one of the preceding claims, wherein the encapsulation efficiency following step (c) provides at least 5% or more over the encapsulation efficiency following step (b).
The process according to any one of the preceding claims, wherein the encapsulation efficiency following step (c) is improved by at least 10% or more from the encapsulation efficiency following step (b).
The process according to any one of the preceding claims, wherein in step (a) the lipid solution comprises lipids dissolved in ethanol.
The process according to any one of the preceding claims, wherein in step (a) the mRNA solution comprises mRNA dissolved in citrate buffer.
The process according to any one of the preceding claims, wherein the drug product formulation solution is an aqueous solution comprising pharmaceutically acceptable excipients comprising a cryoprotectant.
The process according to any one of the preceding claims, wherein the drug product formulation solution is an aqueous solution comprising sugar.
The process according to any one of the preceding claims, wherein in step (b) the drug product formulation solution is an aqueous solution comprising about 10% weight to volume of trehalose
The process according to any one of the preceding claims, wherein both ethanol and citrate are absent from the drug product formulation solution.
The process according to any one of the preceding claims, wherein the lipid solution comprises ethanol, the mRNA solution comprises citrate, and both ethanol and citrate are absent from the drug product formulation solution.
The process according to any one of the preceding claims, wherein the mRNA solution has a pH less than pH 5.0.
The process according to any one of the preceding claims, wherein the drug product formulation solution has a pH between pH 5.0 and pH 7.0.
Dependencies are inferred from the claim text.