ENCAPSULATION OF MESSENGER RNA
The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature.
1 . A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature.
2 . The process of claim 1 , wherein the pre-determined temperature is or is greater than about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C.
3 . The process of claim 1 , wherein the pre-determined temperature ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C.
4 . (canceled)
5 . The process of claim 1 , wherein the mRNA solution and the lipid solution are heated to the pre-determined temperature separately prior to the mixing.
6 - 8 . (canceled)
9 . The process of claim 1 , wherein the mRNA solution and the lipid solution are mixed by a pulse-less flow pump.
10 . The process of claim 9 , wherein the pump is a gear pump.
11 . The process of claim 9 , wherein the pump is a centrifugal pump.
12 . The process of claim 1 , wherein the mRNA solution is mixed at a flow rate ranging from about 150-250 ml/minute, 250-500 ml/minute, 500-1000 ml/minute, 1000-2000 ml/minute, 2000-3000 ml/minute, 3000-4000 ml/minute, or 4000-5000 ml/minute.
13 . (canceled)
14 . The process of claim 1 , wherein the lipid solution is mixed at a flow rate ranging from about 25-75 ml/minute, about 75-200 ml/minute, about 200-350 ml/minute, about 350-500 ml/minute, about 500-650 ml/minute, about 650-850 ml/minute, or about 850-1000 ml/minute.
15 - 22 . (canceled)
23 . The process of claim 1 , wherein the lipid solution comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids in ethanol.
24 . The process of claim 1 , wherein the mRNA solution and the lipid solution are mixed into a 20% ethanol, resulting in a suspension of lipid nanoparticles.
25 . The process of claim 24 , wherein the lipid nanoparticles are further purified by Tangential Flow Filtration.
26 . The process of claim 25 , wherein greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size less than 100 nm.
27 - 29 . (canceled)
30 . The process of claim 25 , wherein the purified nanoparticles have an encapsulation rate of greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
31 . (canceled)
32 . A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising
a. Separately heating a mRNA solution and/or a lipid solution to a pre-determined temperature greater than ambient temperature;
b. Mixing the heated mRNA solution and/or the heated lipid solution to generate a suspension of lipid nanoparticles; and
c. Purifying the lipid nanoparticles.
34 . The process of claim 32 , wherein a composition of lipid nanoparticles is generated.
35 . A composition comprising purified lipid nanoparticles, wherein greater than about 90% of the purified lipid nanoparticles have an individual particle size of less than about 100 nm and greater than about 70% of the purified lipid nanoparticles encapsulate a mRNA within each individual particle.
36 - 41 . (canceled)
42 . The process of claim 1 , wherein the one or more cationic lipids are selected from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (Imidazol-based), HGT5000, HGT5001, 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, HGT4003, and combinations thereof.
43 . The process of claim 1 , wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)).
44 - 45 . (canceled)
46 . The process of claim 32 , wherein the mRNA comprises one or more modified nucleotides.
47 . (canceled)