Stable liquid lipid nanoparticle formulation

Abstract

The present invention provides, inter alia, liquid lipid nanoparticle (LNP) formulations that encapsulate mRNA encoding a peptide or polypeptide, and that are resistant to aggregation and mRNA degradation after multiple freezing and re-thawing at -20°C.

Claims

1. Liquid lipid nanoparticle (LNP) formulation, One or more liquid lipid nanoparticles (LNPs) having lipid components including or consisting of cationic lipids, non-cationic lipids, and PEG-modified lipids; mRNA encapsulated in one or more LNPs and encoding a peptide or polypeptide; disaccharide pH buffer solution with a concentration of approximately 30–150 mM, or pH buffer solution with a concentration of approximately 100–300 mM; and In addition to the ionic strength of the aforementioned pH buffer, the salt provides high ionic strength to the LNP preparation, wherein the LNP preparation is a salt with a total ionic strength of approximately 150 mM to 750 mM and a pH of approximately 6.0 to 8.0; The LNP formulation is resistant to LNP aggregation and / or mRNA degradation, and after at least three freeze-and-thaw cycles at -20°C, the LNP formulation has only the minimum buffer ion strength necessary to provide the same pH as the LNP formulation, otherwise exhibiting less LNP aggregation, degradation of encapsulated mRNA, or both compared to the same LNP formulation; An LNP preparation in which the total ionic strength of the LNP preparation is approximately 150 mM to 750 mM is at least twice the minimum buffered ionic strength of the same LNP preparation otherwise.

2. In other respects, the minimum buffer ion strength of the same LNP formulation is approximately 75 mM to 200 mM, but the total ion strength of the LNP formulation is in the range of approximately 150 mM to 750 mM, which is at least twice the minimum buffer ion strength; The concentration of disaccharides is approximately 2.5% to 3.0%; or The LNP formulation according to claim 1, wherein the molar ratio of disaccharide to pH buffer is 0.2:1 to 0.5:

1.

3. Noncationic lipids include 1,2-dielycoyl-sn-glycero-3-phosphoethanolamine (DEPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), and dipalmitoylphosphatidyl An LNP formulation according to claim 1, selected from glycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, or 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE).

4. The LNP formulation according to claim 3, wherein the noncationic lipid is dioleoylphosphatidylethanolamine (DOPE).

5. The LNP preparation according to claim 4, wherein the proportion of DOPE in the total lipids is 10 mol% or more.

6. An LNP preparation according to any one of claims 1 to 5, wherein the cationic lipid is a lipidoid.

7. An LNP preparation according to any one of claims 1 to 6, wherein the mRNA encodes a protein or vaccine antigen that is deficient in the target.

8. An LNP preparation according to any one of claims 1 to 7, wherein the concentration of the disaccharide is approximately 1% to 20%.

9. An LNP preparation according to any one of claims 1 to 8, wherein the concentration of the disaccharide is approximately 2.5 to 3.0%.

10. The LNP preparation according to claim 6, wherein the lipidoid is 40 mol% to 60 mol% of the lipid component.

11. An LNP preparation according to any one of claims 1 to 10, wherein the disaccharide is trehalose or sucrose.

12. An LNP preparation according to any one of claims 1 to 11, wherein the pH is approximately 7.0 to 8.

0.

13. An LNP formulation according to any one of claims 1 to 12, wherein the pH buffer has a pKa between 6.0 and 8.

2.

14. The LNP formulation according to claim 13, wherein the pH buffer is selected from the group consisting of phosphate buffer, citrate buffer, imidazole buffer, histidine buffer, and Good's buffer.

15. The LNP formulation according to claim 14, wherein the Good buffer is Tris buffer or HEPES buffer.

16. The LNP formulation according to any one of claims 1 to 15, wherein the minimum buffered ion strength of the LNP formulation is approximately 100 mM to 200 mM, and the total ionic strength of the LNP formulation is approximately 200 mM to 750 mM, provided that the total ionic strength is at least twice the minimum buffered ion strength.

17. The LNP preparation according to any one of claims 1 to 15, wherein the ionic strength of the pH buffer is between approximately 40 mM and 250 mM, the salt increases the ionic strength, and the total ionic strength of the LNP preparation is between approximately 150 mM and 500 mM.

18. The salts are NaCl, KCl and CaCl 2 An LNP preparation according to any one of claims 1 to 17, selected from the group consisting of the following.

19. The LNP formulation according to claim 18, wherein the salt is selected from NaCl and the disaccharide is trehalose or sucrose.

20. The LNP preparation according to any one of claims 1 to 15, wherein the salt has a concentration of approximately 50 to 300 mM, increases the ionic strength, and the total ionic strength of the LNP preparation becomes approximately 150 mM to 500 mM.

21. An LNP formulation according to any one of claims 1 to 15, wherein the concentration of the pH buffer is between approximately 100 and 300 mM.

22. An LNP formulation according to any one of claims 1 to 15, wherein the total concentration of the pH buffer providing ionic strength and one or more additional agents is selected from about 50 to 200 mM NaCl as the salt and about 40 mM Tris buffer, about 50 mM Tris buffer, about 100 mM Tris buffer, about 40 mM imidazole buffer, about 50 mM imidazole buffer, about 100 mM imidazole buffer, about 40 mM phosphate buffer, about 50 mM phosphate buffer, or about 100 mM phosphate buffer.

23. An LNP preparation according to any one of claims 1 to 22, wherein the LNPs further contain cholesterol.

24. The LNP preparation according to any one of claims 1 to 15, wherein the ionic strength of the LNP preparation is between approximately 150 mM and 400 mM.

25. The LNP formulation according to any one of claims 1 to 24, wherein turbidity analysis determines that there is little aggregation of LNPs and / or little degradation of encapsulated mRNA.

26. An LNP formulation according to any one of claims 1 to 25, wherein the LNPs have a diameter of less than approximately 100 nm.

27. The LNP formulation according to claim 26, wherein the LNPs have a diameter between approximately 70 nm and 90 nm.

28. An LNP preparation according to any one of claims 1 to 27, wherein the lipid component comprises or consists of DMG-PEG-2000, cKK-E10, cholesterol, and DOPE.

29. An LNP formulation according to any one of claims 1 to 28, wherein the N / P ratio is between approximately 3 and 5, or approximately 4.

30. An LNP preparation according to any one of claims 1 to 29, wherein the mRNA concentration is between approximately 0.05 mg / mL and 1.0 mg / mL, or between approximately 0.2 mg / mL and 0.5 mg / mL.

31. LNPs are stable at -20°C for at least 3 months, and / or The LNP preparation according to any one of claims 1 to 30, wherein the LNP preparation is stable after dilution.

32. An LNP formulation according to any one of claims 1 to 31, wherein subcutaneous or intramuscular delivery of the formulation is accompanied by a reduction in pain compared to an LNP formulation that does not contain a buffer having a concentration of 300 mM or less and a pH between approximately 7.0 and 7.5, the reduction in pain being assessed by a 10 cm visual analog scale (VAS) or a 6-item verbal rating scale (VRS).

33. A method for reducing LNP aggregation and / or mRNA degradation, comprising storing one or more LNPs in an LNP formulation according to any one of claims 1 to 32.

34. The molar ratio of disaccharide to pH buffer is between 0.2:1 and 0.5:1; and Contains salts at a concentration of approximately 50–150 mM; An LNP preparation according to any one of claims 1 to 15.

35. The disaccharide is trehalose or sucrose, and the salts are NaCl, KCl, and CaCl 2 An LNP preparation according to any one of claims 1 to 15, selected from the group consisting of the above, wherein the total ionic strength of the LNP preparation is between approximately 150 mM and 400 mM.

36. The LNP formulation according to any one of claims 1 to 15, wherein the LNP formulation does not contain an amphiphilic polymer.

Images