Lipid nanoparticle formulations for mRNA delivery

Abstract

The present invention provides, inter alia, methods for encapsulating messenger RNA in lipid nanoparticles without the use of flammable solvents for mRNA delivery in therapeutic applications, and compositions produced by these methods. The present invention is based, in part, on the surprising discovery that mRNA can be encapsulated with high efficiency in the presence of an amphiphilic polymer without the use of an ethanol solvent. Thus, the present invention provides a safe, cost-effective, and efficient method for producing LNP formulations from large-scale manufacturing processes as well as in low-volume formulations for therapeutic applications.

Claims

1. A method for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs), comprising the steps of (a) mixing an mRNA solution containing one or more mRNAs with (b) a lipid solution containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, The step of mixing the mRNA solution and the lipid solution includes mixing them in the presence of an amphiphilic polymer solution containing triethylene glycol monomethyl ether (mTEG) to form mRNA encapsulated within LNPs (mRNA-LNPs) in the LNP formulation solution, and The method described above, wherein the method does not contain ethanol.

2. The method according to claim 1, wherein the step of mixing the mRNA solution and the lipid solution yields mTEG at a concentration greater than 25% by volume / volume.

3. The method according to claim 1, wherein the step of mixing the mRNA solution and the lipid solution yields mTEG at a concentration of approximately 50% volume / volume.

4. The method according to any one of claims 1 to 3, wherein the mRNA solution contains less than 5 mM citrate, and the mRNA-LNPs have an inclusion efficiency of more than 60%.

5. The method according to any one of claims 1 to 4, wherein the mRNA solution and / or lipid solution are at approximately ambient temperature.

6. The method according to claim 5, wherein the ambient temperature is less than approximately 35°C, less than approximately 30°C, less than approximately 26°C, less than approximately 23°C, less than approximately 21°C, less than approximately 20°C, or less than approximately 18°C.

7. The method according to claim 6, wherein the ambient temperature is in the range of approximately 18 to 32°C, approximately 21 to 26°C, or approximately 23 to 25°C.

8. One or more noncationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), and dipalmitoylphosphatidylcholine. Zylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl The method according to any one of claims 1 to 7, selected from phosphophosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof.

9. The method according to any one of claims 1 to 8, wherein one or more noncationic lipids are distearoylphosphatidylcholine (DSPC).

10. The method according to any one of claims 1 to 9, wherein the mRNA solution further comprises trehalose.

11. The method according to any one of claims 1 to 10, which does not require a step of heating the mRNA solution and the lipid solution before the mixing step.

12. The method according to any one of claims 1 to 11, wherein the mRNA solution contains more than approximately 1 g of mRNA per 12 L of mRNA solution.

13. The method according to claim 12, wherein the mRNA solution contains approximately 1 g of mRNA per 8 L of mRNA solution.

14. The method according to claim 12, wherein the mRNA solution contains approximately 1 g of mRNA per 4 L of mRNA solution.

15. The method according to claim 12, wherein the mRNA solution contains approximately 1 g of mRNA per 2 L of mRNA solution.

16. The method according to claim 12, wherein the concentration of mRNA in the mRNA solution is greater than approximately 0.125 mg / mL, greater than approximately 0.25 mg / mL, greater than approximately 0.5 mg / mL, or greater than approximately 1.0 mg / mL.

17. The method according to any one of claims 1 to 16, wherein the mRNA solution and the lipid solution are mixed in a ratio (v / v) between 2:1 and 6:

1.

18. The method according to claim 17, wherein the mRNA solution and the lipid solution are mixed in a ratio of approximately 4:1 (v / v).

19. The method according to any one of claims 1 to 18, wherein the mRNA solution has a pH between 3.0 and 5.

0.

20. The method according to claim 19, wherein the mRNA solution has a pH of approximately 3.5, 4.0, or 4.

5.

21. The method according to any one of claims 1 to 20, wherein the mixing step is carried out with a total volume between approximately 3 and 10 mL.

22. The method according to claim 21, wherein the mixing step is carried out with a total volume of approximately 3 mL.

23. The method according to any one of claims 1 to 22, further comprising the step of incubating mRNA-LNPs.

24. The method according to claim 23, wherein the mRNA-LNP is incubated at a temperature between 21°C and 65°C.

25. The method according to claim 24, wherein the mRNA-LNP is incubated at a temperature of approximately 26°C, approximately 30°C, or approximately 65°C.

26. The method according to any one of claims 23 to 25, wherein the mRNA-LNP is super-incubated for approximately 20 minutes, approximately 30 minutes, approximately 60 minutes, approximately 90 minutes, or approximately 120 minutes.

27. The method according to claim 26, wherein the mRNA-LNP is incubated for approximately 60 minutes.

28. The method according to any one of claims 1 to 27, wherein the lipid solution further comprises one or more cholesterol-based lipids.

29. The method according to any one of claims 1 to 28, wherein mRNA-LNPs are purified by tangential flow filtration.

30. The method according to any one of claims 1 to 29, wherein the mRNA-LNP has an average diameter of less than 150 nm, less than 100 nm, less than 80 nm, less than 60 nm, or less than 40 nm.

31. The method according to claim 30, wherein the mRNA-LNP has an average diameter in the range of 40 to 70 nm.

32. The method according to any one of claims 1 to 31, wherein the lipid nanoparticles have a PDI of less than approximately 0.3, less than approximately 0.2, less than approximately 0.18, less than approximately 0.15, and less than approximately 0.

1.

33. The method according to any one of claims 1 to 32, wherein the mRNA-LNP encapsulation efficiency is approximately 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99%.

34. The method according to any one of claims 1 to 33, wherein the mRNA-LNP has an N / P ratio between 1 and 10.

35. The method according to claim 34, wherein the mRNA-LNP has an N / P ratio between 2 and 6.

36. The method according to claim 35, wherein the mRNA-LNP has an N / P ratio of approximately 4.

37. The method according to any one of claims 1 to 36, wherein 5 g or more, 10 g or more, 20 g or more, 50 g or more, 100 g or more, or 1 kg or more of mRNA is encapsulated in lipid nanoparticles in a single batch.

38. The mRNA solution and the lipid solution are mixed by a pulseless flow pump, claims 1-3. The method described in any one of item 7.

39. The method according to claim 38, wherein the pump is a gear pump.

40. The method according to claim 39, wherein the pump is a centrifugal pump.

41. The method according to any one of claims 1 to 40, wherein the mRNA solution is mixed at a flow rate in the range of approximately 150-250 ml / min, 250-500 ml / min, 500-1000 ml / min, 1000-2000 ml / min, 2000-3000 ml / min, 3000-4000 ml / min, or 4000-5000 ml / min.

42. The method according to claim 41, wherein the mRNA solution is mixed at a flow rate of approximately 800 ml / min, approximately 1000 ml / min, or approximately 12000 ml / min.

43. The method according to any one of claims 1 to 42, wherein the lipid solution is mixed at a flow rate in the range of approximately 25 to 75 ml / min, approximately 75 to 200 ml / min, approximately 200 to 350 ml / min, approximately 350 to 500 ml / min, approximately 500 to 650 ml / min, approximately 650 to 850 ml / min, or approximately 850 to 1000 ml / min.

44. The method according to claim 43, wherein the lipid solution is mixed at a flow rate of approximately 100 ml / min, approximately 150 ml / min, approximately 200 ml / min, approximately 250 ml / min, approximately 300 ml / min, and approximately 350 ml / min.

45. The method according to any one of claims 41 to 45, wherein the flow rate of the mRNA solution is 2 times, 4 times, or 6 times greater than the flow rate of the lipid solution.

46. The method according to any one of claims 1 to 45, wherein mRNA is purified by a method that does not contain volatile organic compounds.

47. The method according to claim 46, wherein the mRNA is purified by an alcohol-free method.

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