Granted Patent B2

US 12,194,148 · App. 17/822,511 · Granted Jan 14, 2025

Inventors: Thomas Ketterer (Gomaringen, DE); Thorsten Mutzke (Reutlingen, DE); Michael Wiggenhorn (Munich, DE); Frank Schaubhut (Germering, DE); Florian Von Der Mülbe (Stuttgart, DE)

Assignee: CureVac Manufacturing GmbH

A61K9/19A61K31/7105A61K47/549A61K47/64C12Q1/6806C12N15/1003

Abstract

Claims (26)

1. A method for producing a lyophilized RNA composition, wherein the method comprises the following steps:

a) providing a liquid having a glass transition temperature of from −15° C. to −50° C., said liquid comprising: (i) a RNA molecule comprising from 200 to 15,000 nucleotides, a 5′ cap, a Poly (A) sequence and a coding region, encoding an antigen from a pathogen; and (ii) at least one carbohydrate compound, wherein said RNA molecule is associated with a cationic or polycationic compound;

b) introducing the liquid into a freeze drying chamber of a freeze dryer;

c) cooling the liquid to a freezing temperature, wherein the cooling is performed at a defined cooling rate in a range from 0.1° C./min to 2° C./min and wherein the freezing temperature is in a range from 0.5° C. to 25° C. below the glass transition temperature of the liquid to provide a frozen liquid;

d) reducing the pressure in the freeze drying chamber to a pressure below atmospheric pressure;

e) drying the frozen liquid by heating the frozen liquid obtained in step d) to a drying temperature; and

f) equilibrating the pressure in the freeze drying chamber to atmospheric pressure to provide a lyophilized RNA composition, wherein said lyophilized RNA composition has a water content of 0.1% to 10% (w/w).

2. The method of claim 1 , wherein the 5′ cap is a m7GpppN cap.

3. The method of claim 2 , wherein the 5′ cap is a Cap1.

4. The method of claim 2 , wherein the Poly (A) sequence is 50 to 100 adenine nucleotides.

5. The method of claim 4 , wherein the RNA comprises at least one modified nucleotide.

6. The method of claim 5 , wherein the at least one modified nucleotide is 1-methyl-pseudouridine.

7. The method of claim 1 , wherein said liquid further comprises a cationic or polycationic peptide or protein.

8. The method of claim 1 , wherein said liquid further comprises a cationic or polycationic lipid.

9. The method of claim 8 , wherein the at least one carbohydrate compound is present in the liquid of step a) in a range of 1 to 20% (w/w).

10. The method of claim 9 , wherein the at least one carbohydrate compound comprises mannitol, sucrose, glucose, mannose and/or trehalose.

11. The method of claim 10 , wherein the at least one carbohydrate compound comprises mannose and/or sucrose.

12. The method of claim 11 , wherein the at least one carbohydrate compound comprises mannose and sucrose.

13. The method of claim 9 , wherein the RNA in the liquid provided in step a) is present in a concentration of from 0.1 to 10 g/l.

14. The method of claim 13 , wherein the glass transition temperature of the liquid provided in step a) is in a range from −25° C. to −40° C.

15. The method of claim 13 , wherein the freezing temperature is in a range from −50° C. to −35° C.

16. The method of claim 1 , wherein the lyophilized RNA composition has a water content of equal to or less than 5% (w/w).

17. The method of claim 16 , wherein the lyophilized RNA composition has a water content of equal to or less than 4% (w/w).

18. The method of claim 1 , wherein the lyophilized RNA composition has a water content of 3%±2% (w/w).

19. The method of claim 18 , wherein the RNA molecule is a mRNA molecule.

20. The method of claim 19 , wherein the mRNA molecule comprises, from 5′ to 3′, the 5′ cap, a 5′ untranslated region (UTR), the coding region, a 3′UTR, and the Poly (A) sequence, said poly (A) sequence having 50 to 100 adenine nucleotides.

Priority Claims (1)

Continuity (4)

References Cited (274)