LYOPHILIZATION OF RNA

Abstract

The present invention is directed to the field of RNA formulation, in particular to lyophilization of RNA. The invention provides a method for lyophilization of RNA. The present invention further concerns a lyophilized composition obtainable by the inventive method, a pharmaceutical composition, a vaccine and a kit or kit of parts. Moreover, the present invention provides a novel use of a lyoprotectant for lyophilizing RNA, the use of the inventive method in the manufacture of a medicament as well as the first and second medical use of the composition obtainable by the inventive method, the pharmaceutical composition, the vaccine or the kit or kit of parts according to the invention.

Claims

1. Method for lyophilizing RNA, wherein the method comprises the following steps: a) providing a liquid comprising at least one RNA and at least one lyoprotectant; b) introducing the liquid provided 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; d) freezing the liquid at the freezing temperature in order to obtain a frozen liquid; e) reducing the pressure in the freeze drying chamber to a pressure below atmospheric pressure; f) drying the frozen liquid obtained in step d) in order to obtain a lyophilized composition comprising the at least one RNA and at least one lyoprotectant g) equilibrating the pressure in the freeze drying chamber to atmospheric pressure and removing the lyophilized composition comprising the at least one RNA and the at least one lyoprotectant obtained in step f) from the freeze drying chamber.

2. The method according to claim 1, wherein the at least one RNA is a long-chain RNA comprising from 100 to 50000 nucleotides, preferably from 200 to 15000 nucleotides, more preferably from 300 to 10000 nucleotides, and most preferably from 400 to 7000 nucleotides.

3. The method according to claim 1 or 2, wherein the at least one RNA is not a viral RNA or an RNA, which is derived from a viral RNA.

4. The method according to any one of the preceding claims, wherein step f) comprises heating the frozen liquid obtained in step d) to a drying temperature, wherein the drying temperature is preferably a pre-determined temperature.

5. The method according to claim 4, wherein the heating is performed at a defined heating rate.

6. The method according to claim 4 or 5, wherein the heating rate is in a range from from 0.1 C./h to 20 C./h.

7. The method according to any one of claims 4 to 6, wherein the drying temperature does not exceed the glass transition temperature of the frozen liquid obtained in step d).

8. The method according to any one of claims 4 to 7, wherein the drying temperature is in a range from 40 C. to 40 C.

9. The method according to any one of the preceding claims, wherein the liquid further comprises at least one cationic or polycationic compound.

10. The method according to any one of the preceding claims, wherein the cationic or polycationic compound is a cationic or polycationic peptide or protein.

11. The method according to any one of the preceding claims, wherein the at least one RNA and the at least one cationic or polycationic compound are present in a complex.

12. The method according to any one of the preceding claims, wherein the at least one RNA comprises at least one coding region.

13. The method according to any one of the preceding claims, wherein the at least one RNA is an mRNA.

14. The method according to any one of the preceding claims, wherein the lyoprotectant is a carbohydrate compound, preferably selected from the group consisting of mannitol, sucrose, glucose, mannose, trehalose.

15. The method according to any one of the preceding claims, wherein the concentration of the lyoprotectant in the liquid provided in step a) is in a range from 1 to 20% (w/w).

16. The method according to any one of the preceding claims, wherein the concentration of the at least one RNA in the liquid provided in step a) is in a range from 0.1 to 10 g/l.

17. The method according to any one of the preceding claims, wherein the freezing temperature is a pre-determined temperature.

18. The method according to any one of the preceding claims, wherein the freezing temperature is equal to or lower than the glass transition temperature of the liquid provided in step a) or of the frozen liquid obtained in step d), respectively.

19. The method according to any one of the preceding claims, wherein the glass transition temperature of the liquid provided in step a) or of the frozen liquid obtained in step d), respectively is in a range from 25 C. to 40 C.

20. The method according to any one of the preceding claims, wherein the freezing temperature is in a range from 50 C. to 35 C.

21. The method according to any one of the preceding claims, wherein the cooling rate in step c) is in a range from 0.1 C./min to 2 C./min.

22. The method according to any one of the preceding claims, wherein the freezing temperature is maintained for at least 3 hours in step d).

23. The method according to any one of claims 4 to 22, wherein step f) comprises at least two drying steps, preferably a primary drying step f1) and a secondary drying step f2).

24. The method according to claim 23, wherein a primary drying temperature in the primary drying step f1) is lower than a secondary drying temperature in the secondary drying step f2).

25. The method according to claim 23 or 24, wherein the primary drying step f1) comprises heating the frozen liquid obtained in step d) to the primary drying temperature, which is in a range from 40 C. to 20 C.

26. The method according to any one of claims 23 to 25, wherein the frozen liquid obtained in step d) is heated from the freezing temperature to the primary drying temperature at a heating rate in a range from 0.1 C./h to 10 C./h.

27. The method according to any one of claims 23 to 26, wherein the secondary drying step f2) comprises heating the frozen liquid obtained in step d) to the secondary drying temperature, which is in a range from 10 C. to 40 C.

28. The method according to any one of claims 23 to 27, wherein the frozen liquid obtained in step d) is heated from the primary drying temperature to the secondary drying temperature at a heating rate in a range from 0.1 C./h to 20 C./h.

29. The method according to any one of the preceding claims, wherein step e) comprises reducing the pressure in the freeze drying chamber to a pressure below atmospheric pressure subsequently to the freezing of the liquid provided in step a).

30. The method according to any one of the preceding claims, wherein step e) comprises reducing the pressure in the freeze drying chamber to a pressure below atmospheric pressure, wherein the pressure is reduced before or at the beginning of the drying step (f).

31. The method according to any one of the preceding claims, wherein step e) comprises reducing the pressure in the freeze drying chamber to a pressure in a range from about 1 bar to about 300 bar

32. The method according to any one of claims 24 to 31, wherein step e) comprises reducing the pressure in the freeze drying chamber to a primary drying pressure, which is applied before or concomittantly with the heating from the freezing temperature to the primary drying temperature and which is maintained during the primary drying step f1); and subsequently reducing the pressure in the freeze drying chamber to a secondary drying pressure, which is applied before or concomittantly with the heating from the primary drying temperature to the secondary drying temperature and which is maintained during the secondary drying step f2).

33. The method according to claim 32, wherein the secondary drying pressure is lower than the primary drying pressure.

34. The method according to claim 32 or 33, wherein the primary drying pressure is in a range from 1 bar to 300 bar.

35. The method according to any one of claims 32 to 34, wherein the secondary drying pressure is in a range from 1 bar to 100 bar.

36. The method according to any one of claims 23 to 35, wherein the secondary drying step f2) comprises at least two steps, preferably a first step of secondary drying f2a) and a second step of secondary drying f2b).

37. The method according to claim 36, wherein the first step of secondary drying f2a) comprises reducing the pressure to the secondary drying pressure, without increasing the temperature.

38. The method according to claim 36 or 37, wherein the second step of secondary drying f2b) comprises heating the frozen liquid obtained in step d) to the secondary drying temperature.

39. The method according to any one of claims 36 to 38, wherein the frozen liquid obtained in step d) is heated from the primary drying temperature to the secondary drying temperature at a heating rate in a range from 0.1 C./h to 20 C./h.

40. The method according to any one of the preceding claims, wherein the freeze drying chamber is flooded with an inert gas before step g).

41. The method according to claim 40, wherein the inert gas is selected from the group consisting of nitrogen, carbon dioxide, helium, neon, argon, xenon and krypton.

42. The method according to any one of the preceding claims, wherein the temperature is the temperature in the freeze drying chamber.

43. The method according to any one of the preceding claims, wherein the temperature is the shelf temperature, the temperature of the liquid provided in step a) or the frozen liquid obtained in step d), respectively, or the temperature of a portion thereof.

44. The method according to claim 43, wherein the shelf temperature, the temperature of the liquid provided in step a), and/or the frozen liquid obtained in step d), or the temperature of a portion thereof, is measured via one or more probes.

45. The method according to claim 44, wherein the shelf temperature is measured via a probe, which is in physical contact with the shelf, wherein the probe is preferably positioned on the surface of the shelf.

46. The method according to claim 44 or 45, wherein the temperature of the liquid provided in step a), and/or the frozen liquid obtained in step d), or a portion thereof, is measured via a probe, which is positioned in the liquid or the frozen liquid.

47. The method according to any one of claims 44 to 46, wherein the probe is selected from the group consisting of a PT100 platinum resistance thermometer, a PT1000 platinum resistance thermometer, a CuCuNi thermocouple, a NiCrNiAl thermocouple, a NiCrCuNi thermocouple, and a NiCrNi thermocouple.

48. The method according to claims 42 to 47, wherein the temperatures of the liquid provided in step a) and the frozen liquid obtained in step d), or the temperatures of a portion thereof, correspond to the respective shelf temperatures.

49. The method according to claim 48, wherein the liquid provided in step a) and the frozen liquid obtained in step d), or a portion thereof, reach the respective shelf temperatures before or after the shelf has reached the respective temperatures.

50. Lyophilized composition comprising at least one RNA and at least one lyoprotectant, which is obtainable by the method according to any one of claims 1 to 49.

51. Use of a lyoprotectant for lyophilizing RNA, wherein the use comprises controlled cooling and/or controlled heating of the RNA and the lyoprotectant.

52. The use according to claim 51, wherein the use comprises any one the features or any combination of the features as defined with respect to the method according to claims 1 to 49.

53. Use of the method according to any one of claims 1 to 49 in the manufacture of a medicament or a vaccine.

54. Pharmaceutical composition comprising or consisting of the lyophilized composition according to claim 50.

55. The pharmaceutical composition according to claim 54, which comprises at least one additional pharmaceutically acceptable ingredient.

56. Vaccine comprising or consisting of the lyophilized composition according to claim 50.

57. The vaccine according to claim 56, which comprises at least one further pharmaceutically acceptable ingredient.

58. The vaccine according to claim 56 or 57, wherein the lyophilized composition was reconstituted in a suitable solvent or buffer.

59. Kit comprising the lyophilized composition according to claim 50, the pharmaceutical composition according to claim 54 or 55, or the vaccine according to any one of claims 56 to 58, a solvent or buffer for resuspending the lyophilized composition, the pharmaceutical composition or the vaccine, and optionally technical instructions comprising information regarding the administration and/or dosage of the lyophilized composition, the pharmaceutical composition or the vaccine.

60. Kit of parts comprising in one or more parts of kit the lyophilized composition according to claim 50, the pharmaceutical composition according to claim 54 or 55, or the vaccine according to any one of claims 56 to 58, a solvent or buffer for resuspending the lyophilized composition, the pharmaceutical composition or the vaccine, and optionally technical instructions comprising information regarding the administration and/or dosage of the lyophilized composition, the pharmaceutical composition or the vaccine.

61. The lyophilized composition according to claim 50, the pharmaceutical composition according to claim 54 or 55, or the vaccine according to any one of claims 56 to 58 for use in the treatment or prophylaxis of a disorder or a disease.

62. The lyophilized composition according to claim 50, the pharmaceutical composition according to claim 54 or 55, or the vaccine according to any one of claims 56 to 58 for use according to claim 61, wherein the disorder or the disease is selected from neoplasms (e.g. cancer or tumor diseases), infectious and parasitic diseases, preferably viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, chromosomal abnormalities, cardiovascular diseases, diseases of the blood and blood-forming organs, endocrine, nutritional and metabolic diseases, mental and behavioural disorders, diseases of the nervous system, diseases of the eye and adnexa, diseases of the ear and mastoid process, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system.

63. Method for treating or preventing a disorder or a disease, wherein the method comprises administering to a subject in need thereof a pharmaceutically effective amount of the lyophilized composition according to claim 50, the pharmaceutical composition according to claim 54 or 55, or the vaccine according to any one of claims 56 to 58.

64. The method for treating or preventing a disorder or disease according to claim 63, wherein the disorder or the disease is selected from neoplasms (e.g. cancer or tumor diseases), infectious and parasitic diseases, preferably viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, chromosomal abnormalities, cardiovascular diseases, diseases of the blood and blood-forming organs, endocrine, nutritional and metabolic diseases, mental and behavioural disorders, diseases of the nervous system, diseases of the eye and adnexa, diseases of the ear and mastoid process, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0275] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

[0276] FIG. 1: SEQ ID NO: 1, which is the mRNA sequence corresponding to PpLuc(GC)-muag-A64-C30.

[0277] FIG. 2: SEQ ID NO: 2, which is the mRNA sequence corresponding to HA(GC)-muag-A64-C30-histone stem-loop.

[0278] FIG. 3: SEQ ID NO: 3, which is the mRNA sequence corresponding to HsFOLH1(GC)-muag-A64-C30-histone stem-loop.

[0279] FIG. 4: SEQ ID NO: 9, which is the mRNA sequence corresponding to RAV-G(GC)-muag-A64-C30-histone stem-loop.

[0280] FIG. 5: A. Temperature profile and pressure profile of a standard lyophilization cycle. B. Relative integrity of mRNA lyophilized under standard conditions stored at 25 C./60% r.H. or at 40 C./75% r.H., respectively, for 40 weeks (Example 3).

[0281] FIG. 6: Optimization of a lyophilization cycle under controlled freezing conditions (Example 4) [0282] A. Residual moisture content of compositions lyophilized under controlled freezing conditions in the presence of different amounts of trehalose, wherein the residual moisture content was determined after storage of the lyophilized compositions at 50 C. (without controlling the relative humidity) for 3 months. [0283] B. Relative integrity of mRNA lyophilized under controlled freezing conditions in the presence of different amounts of trehalose, wherein the lyophilized samples were stored for 3 months at 50 C. (without controlling the relative humidity).

[0284] FIG. 7: Optimization of a lyophilization cycle under controlled freezing and controlled drying conditions (Example 5) [0285] A. Temperature profile and pressure profile of the lyophilization cycle. [0286] B. Residual moisture content of compositions lyophilized under controlled freezing and controlled drying conditions, wherein the residual moisture content was determined after storage of the lyophilized compositions at 40 C./75% rH for 24 weeks. [0287] C. Relative integrity of mRNA lyophilized under controlled freezing and controlled drying conditions, wherein the lyophilized samples were stored at 40 C./75% rH for 24 weeks.

[0288] FIG. 8: Optimization of a lyophilization cycle under controlled freezing and controlled drying conditions; biological activity of lyophilized mRNA (Example 6) [0289] A. Residual moisture content of compositions lyophilized under controlled freezing and controlled drying conditions after storage of the compositions at 80 C., at +5 C., at +25 C./60% r.H. or at +40 C./75% r.H., wherein the residual moisture content was determined after 1, 2, 3, 6, 9 or 12 months of storage. [0290] B. Relative integrity of mRNA lyophilized under controlled freezing and controlled drying conditions, wherein the lyophilized samples were stored at 80 C., at +5 C., at +25 C./60% r.H. or at +40 C./75% r.H. for 1, 2, 3, 6, 9 or 12 months. [0291] C. HI titers measured after injection of mRNA that was lyophilized under controlled freezing and controlled drying conditions and stored at 80 C. or at +25 C./60% r.H. for 6 months before reconstitution and injection. [0292] D. Virus-neutralizing titers determined after injection of mRNA that was lyophilized under controlled freezing and controlled drying conditions and stored at 80 C. or at +25 C./60% r.H. for 6 months before reconstitution and injection.

EXAMPLES

[0293] The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

Example 1: Preparation of DNA and RNA Constructs

[0294] Vectors for in vitro transcription were constructed, which contain a T7 promoter followed by a GC-enriched coding sequence.

[0295] A vector (PpLuc(GC)-muag-A64-C30) was constructed, which contains a T7 promoter followed by a GC-enriched sequence encoding the luciferase reporter gene, a sequence derived from the albumin-3-UTR (muag), a stretch of 64 adenosines (poly(A)-sequence) and a stretch of 30 cytosines (poly(C)-sequence). The sequence of the corresponding mRNA is shown in SEQ ID NO: 1.

[0296] Another vector (HA(GC)-muag-A64-C30-histone stem-loop) was prepared, which contains a T7 promoter followed by a GC-enriched sequence encoding the hemagglutinin (HA) protein of influenza A virus (A/Netherlands/602/09), a sequence derived from the albumin-3-UTR (muag), a stretch of 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence) and a histone stem-loop. The sequence of the corresponding mRNA is shown in SEQ ID NO: 2.

[0297] A further vector (HsFOLH1(GC)-muag-A64-C30-histone stem-loop) was constructed which contains a T7 promoter followed by a GC-enriched sequence encoding the FOLH1 protein from Homo sapiens, a sequence derived from the albumin-3-UTR (muag), a stretch of 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence) and a histone stem-loop. The sequence of the corresponding mRNA is shown in SEQ ID NO: 3.

[0298] A further vector (RAV-G(GC)-muag-A64-C30-histone stem-loop) was constructed which contains a T7 promoter followed by a GC-enriched sequence encoding the RAV-G protein from Rabies virus, a sequence derived from the albumin-3-UTR (muag), a stretch of 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence) and a histone stem-loop. The sequence of the corresponding Rav-G mRNA sequence is provided in SEQ ID NO: 9.

[0299] The obtained vectors were linearized and subsequently in vitro transcribed by using T7 RNA polymerase. The DNA template was then degraded by DNAseI digestion. The mRNA was recovered by LiCl precipitation and further cleaned by HPLC extraction (PUREMessenger, CureVac GmbH, Tbingen, Germany).

Example 2: Complexation of RNA

[0300] mRNA obtained by in vitro transcription as described in Example 1 was complexed with protamine and trehalose. mRNA was diluted (0.87 g/L mRNA final concentration) and a protamine/trehalose mixture was prepared (0.43 g/L protamine; 10.87% trehalose in water for injection). Both solutions were mixed in an mRNA:protamine ratio of 2:1 (w/w).

[0301] The solution of RNA/protamine complexes was subsequently supplemented with free mRNA to yield final concentrations of 0.4 g/L mRNA complexed with 0.2 g/L protamine, 0.4 g/L free mRNA and 5% trehalose (w/w).

[0302] Alternatively, the concentration of trehalose in the protamine/trehalose mixture was adapted in order to obtain a final trehalose concentration (in the final solution) of 2.5% or 10% (w/w).

[0303] Such formulated RNA was used for lyophilization experiments.

Example 3: Standard Lyophilization Process

[0304] mRNA encoding luciferase according to SEQ ID NO: 1 formulated according to Example 2 with a final mRNA concentration of 0.8 g/l and a final trehalose concentration of 5% (w/w). Aliquots of 75 l were dispensed into sterile 2R glass vials (Type 1). The vials were half-closed with a freeze drying rubber stopper. The vials were frozen by using liquid nitrogen and loaded into a freeze dryer Alpha 2-4 (Martin Christ Gefriertrocknungsanlagen) and dried under the following conditions.

TABLE-US-00007 TABLE 1 Pressure Duration Step Description Temperature (shelves) Pirani (hh:mm) 1 Loading <70 C. atm 00:00 4 Evacuation <50 C. (shelves cooled 63 bar ~00:20 with liquid nitrogen) 5 Primary <50 C. .fwdarw. 40 C. 63 bar 18:30 drying heating only controlled by final temperature 9 Secondary 40 C. shelves results in 45 bar 06:00 drying approximately 20 C. in the product 10 Nitrogen 40 C. n.a. back-fill 11 Vial closure 40 C. n.a. 12 Aeration 40 C. atm

[0305] The vials were sealed by crimping an aluminum cap over the stopper and the neck of the vial. Afterwards, the samples were stored at 25 C./60% relative humidity (r.H.) and 40 C./75% r.H. and analyzed for relative integrity after 5, 8, 13, 24 and 40 weeks (3 samples each). The relative integrity of the mRNA comprised in the lyophilized compositions was determined via agarose gel electrophoresis. Specifically, the relative integrity was determined by measuring the signal intensities corresponding to full-length mRNA and all other signals, respectively, in a lane of the agarose gel (i.e. in a given sample) and calculating the ratio of the signal intensity for full-length mRNA related to all other signals in that lane.

[0306] Results

TABLE-US-00008 TABLE 2 Storage +25 C./60% r.H. +40 C./75% r.H. time Rel. integrity Rel. integrity (weeks) (%) (%) 0 75 75 5 84 71 8 79 52 13 79 40 24 76 1 40 80 1

[0307] The storage of the mRNA/trehalose formulations dried under standard lyophilization conditions resulted in decreased integrity (relative integrity <80%) at 40 C./75% rH over time (see FIG. 5B), indicating that the compositions were not storage-stable.

Example 4: Optimization of a Lyophilization Cycle Under Controlled Freezing Conditions

[0308] mRNA encoding hemagglutinin (HA) of A/Netherlands/602/09 (SEQ ID NO: 2) was formulated with protamine according to Example 2 in order to obtain a final mRNA concentration of 0.8 g/l and different trehalose concentrations of 2.5%, 5% and 10% (w/w), respectively. The glass transition temperature Tg was determined by DSC (differential scanning calorimetry) and formulations were grouped into two classes on the basis of their Tg. Group I comprises formulations having a Tg between 30 and 32.5 C. (i.e. the mRNA formulation containing 5% and 10% trehalose); group II comprises formulations having a Tg between 32.5 and 35 C. (i.e. the mRNA formulation containing 2.5% trehalose).

[0309] 500 l of each formulation were transferred into sterile glass vials (Type 1). The vials were half-closed with a freeze drying rubber stopper and were loaded onto the shelves of a freeze drier (Alpha 2-4; Christ Gefriertrocknungsanlagen) at 15 C. The controlled freezing to 40 C. was performed under controlled conditions for 02:55 h at a linear cooling rate of 0.31 C./min.

[0310] Lyophilization Cycle

TABLE-US-00009 TABLE 3 Group I Cooling Temperature rate/ (shelf temp. if not Heating Pressure Duration Step Description indicated otherwise) rate Pirani (hh:mm) 1 Loading 15 C. atm 00:00 1a Pre-cooling 15 C. atm 02:05 2 Cooling 15 C. .fwdarw. 40 C. 0.31 C./ atm 02:55 down/ (shelf) min Freezing 3 Freezing 40 C. atm 07:20 4 Evacuation 40 C. 100 bar 00:20 5 Primary 40 C. .fwdarw. 15 C. 4.2 C./h 100 bar 06:00 drying (1) (shelf) 6 Primary 15 C. 100 bar 11:00 drying (2) 7 Secondary 15 C. 45 bar 00:20 drying (1) 8 Secondary 15 C. .fwdarw. 20 C. Not 45 bar 03:00 drying (2) (product) controlled 40 C. (shelf) 9 Secondary 20 C. (product) 45 bar 07:00 drying (3) 40 C. (shelf) 10 Nitrogen 20 C. (product) n.a back-fill 40 C. (shelf) 11 Vial closure 20 C. (product) n.a. 40 C. (shelf) 12 Aeration 20 C. (product) atm 40 C. (shelf)

TABLE-US-00010 TABLE 4 Group II Cooling Temperature rate/ (shelf temp. if not Heating Pressure Duration Step Description indicated otherwise) rate Pirani (hh:mm) 1 Loading 15 C. atm 00:00 1a Pre-cooling 15 C. atm 02:05 2 Freezing 15 C. .fwdarw. 40 C. 0.31 C./ atm 02:55 (shelf) min 3 Freezing 40 C. atm 07:20 4 Evacuation 40 C. 100 bar 00:20 5 Primary 40 C. .fwdarw. 17 C. 2.6 C./h 100 bar 09:00 drying (shelf) 6 Primary 17 C. 100 bar 16:00 drying 7 Secondary 17 C. 45 bar 00:20 drying 8 Secondary 17 C. .fwdarw. 20 C. Not 45 bar 03:00 drying (product) controlled 40 C. (shelf) 9 Secondary 20 C. (product) 45 bar 07:00 drying 40 C. (shelf) 10 Nitrogen 20 C. (product) n.a. back-fill 40 C. (shelf) 11 Vial closure 20 C. (product) n.a. 40 C. (shelf) 12 Aeration 20 C. (product) atm 40 C. (shelf)

[0311] Due to the functionality of the freeze dryer only shelf temperatures between about 40 C. and 0 C. can be controlled. Higher temperatures can be reached, but are not associated with a constant heating rate. Therefore, only a heating rate for the primary drying can be specified.

[0312] After secondary drying (Step 9), the freeze drying chamber was flooded with nitrogen (step 10) and the vials were manually closed by lowering the shelves above (step 11). The chamber was finally vented to atmospheric pressure (atm, step 12) and the vials were removed from the freeze dryer.

[0313] The vials were sealed by crimping an aluminum cap over the stopper and the neck of the vial. The residual moisture content was determined by Karl-Fischer titration. Afterwards, the samples were stored at +50 C. (without controlling the relative humidity) and analyzed with respect to residual moisture content of the formulation and the relative integrity of the RNA (see Example 3) after 2 weeks (relative integrity only), 1, 2 and 3 months.

[0314] Results

TABLE-US-00011 TABLE 5 2.5% Trehalose 5% Trehalose 10% Trehalose Rel. Rel. Rel. Storage Residual integ- Residual integ- Residual integ- Time moisture rity moisture rity moisture rity (months) (%) (%) (%) (%) (%) (%) 0 2.7 96 1.3 98 1.5 98 0.5 94 95 95 1 3.9 95 2.4 93 1.8 96 2 89 90 93 3 2.7 80 2.0 83 1.9 89

[0315] All samples have a residual moisture content of <4%. Nevertheless, higher concentrations of lyoprotectant result in a reduced residual moisture content (see FIG. 6A). All samples show an integrity >80% after storage at +50 C. for 3 months (see FIG. 6B). Lyophilization of RNA under controlled freezing conditions thus results in increased integrity of the lyophilized RNA product over time and outstanding storage stability.

Example 5: Optimization of a Lyophilization Cycle Under Controlled Freezing and Controlled Drying Conditions

[0316] The integrity of lyophilized compositions, in particular the integrity of the mRNA comprised in those compositions, after storage under conditions with controlled relative humidity (40 C./75% r.H.) was analyzed. To this end, an mRNA encoding HsFOHL1 (SEQ ID NO: 3) was formulated with protamine according to Example 2 with a final mRNA concentration of 0.8 g/l in the presence of 5% (w/w) trehalose and filled into sterile glass (Type1) vials (600 l per vial). The vials were half-closed with a freeze drying rubber stopper and loaded onto the shelves of the freeze drier at 20 C. Lyophilization was performed using the freeze-drier Epsilon 2-12D (Martin Christ, Osterrode, Germany). The vacuum during the freeze-drying process was controlled by a MKS Capacitance Manometer. The process parameters of the cycle are detailed in the table below.

TABLE-US-00012 TABLE 6 Cooling/ Pressure Descrip- Shelf heating MKS Duration Step tion temperature rate (mbar) (hh:mm) 1 Load 20 C. 1000 00:00 2 Freezing 20 C. .fwdarw. 40 C. 0.5 C./ 1000 02:00 3 Freezing 40 C. min 1000 02:00 4 Evacuation 40 C. 0.1 00:33 5 Primary 40 C. .fwdarw. 10 C. 5 C./h 0.1 06:00 drying 6 Primary 10 C. 0.1 11:00 drying 7 Secondary 10 C. 0.045 00:33 drying 8 Secondary 10 C. .fwdarw. 20 C. 10 C./h 0.045 03:00 drying 9 Secondary 20 C. 0.045 07:00 drying 10 Nitrogen 20 C. n.a back-fill 11 Vial 20 C. n.a. closure 12 Aeration 20 C. atm

[0317] The residual moisture content of the obtained samples was determined by Karl-Fischer titration. The samples were stored at 40 C./75% r.H. and analyzed after 2, 4, 6, 12 and 24 weeks. The relative integrity (see Example 3) of the lyophilized mRNA was used as a measure of storage stability of the lyophilized composition under the specific storage conditions in this experiment, i.e. at 40 C./75% r.H.

[0318] Results

[0319] The residual moisture content of the lyophilized mRNA/trehalose formulations increased over time if stored at +40 C./75% r.H., but remained well below 2.5% (see FIG. 7B). Nevertheless, a relative integrity of the lyophilized RNA of above 80% could be obtained for the samples stored for up to 12 weeks (see FIG. 7C). After storage for 24 weeks, the relative integrity of the RNA was still 79.1%. These results demonstrate that lyophilization under controlled freezing and controlled drying conditions results in increased relative integrity of the lyophilized RNA product and further results in improved storage stability of the lyophilized RNA as compared to the relative integrity of an RNA lyophilized under non-controlled conditions and its storage stability, respectively (Example 3).

Example 6: Optimization of a Lyophilization Cycle Under Controlled Freezing and Controlled

[0320] drying conditions; biological activity of lyophilized mRNA mRNA encoding hemagglutinin (HA) of A/Netherlands/602/09 (SEQ ID NO: 2) was formulated with protamine in a weight ratio of 4:1 according to Example 2 with a final mRNA concentration of 0.8 g/l in the presence of 5% (w/w) trehalose. The formulation was cooled to 80 C. Prior to filling of 600 l formulation per sterile 2R glass (type 1) vials, the formulation was allowed to thaw at room temperature (20-25 C.). The vials were half-closed with freeze drying rubber stoppers and loaded onto the shelves of the freeze drier at 20 C. Lyophilization was performed on a BOC Edwards Lyoflex 04 freeze-drier and included a freeze drying cycle with the following conditions:

TABLE-US-00013 TABLE 7 Cooling/ Shelf heating Pressure Duration Step Description Temperature rate Pirani (hh:mm) 1 Load 20 C. atm 00:00 2 Freezing 20 C. .fwdarw. 40 C. 0.5 C./ atm 02:00 3 Freezing 40 C. min atm 02:00 4 Evacuation 40 C. 160 bar 00:20 5 Primary 40 C. .fwdarw. 10 C. 5 C./h 160 bar 06:00 drying 6 Primary 10 C. 160 bar 11:00 drying 7 Secondary 10 C. 68 bar 00:20 drying 8 Secondary 10 C. .fwdarw. 20 C. 10 C./h 68 bar 03:00 drying 9 Secondary 20 C. 68 bar 07:00 drying 10 Nitrogen 20 C. 0.8 bar back-fill 11 Vial closure 20 C. 0.8 bar 12 Aeration 20 C. atm

[0321] The vacuum was controlled by a Pirani manometer. After the secondary drying (Step 9), the freeze drying chamber was flooded with nitrogen up to a pressure of 0.8 bar (Step 10) and the vials were automatically closed by lowering the shelves above (Step 11). The chamber was finally vented to atmospheric pressure (Step 12) and the vials were removed from the freeze dryer. The vials were sealed by crimping an aluminum cap over the rubber stopper and the neck of the vial. After determination of the residual moisture content of each sample, the samples were stored at 80 C., +5 C., +25 C./60% r.H. or +40 C./75% r.H. for 1, 2, 3, 6, 9 and 12 months, respectively. After that storage period, the samples were analyzed with respect to their residual moisture content and with respect to the relative integrity of the lyophilized mRNA.

[0322] Results

TABLE-US-00014 TABLE 8 +25 C./ +40 C./ Storage 80 C. +5 C. 60% r.H. 75% r.H. Time r.M. r.I. r.M. r.I. r.M. r.I. r.M. r.I. (months) (%) (%) (%) (%) (%) (%) (%) (%) 0 0.875 99 0.875 99 0.875 99 0.875 99 1 0.93 97 1.015 97 1.185 96 1.55 95 2 0.975 97 0.995 99 1.275 97 2.005 95 3 1.015 98 1.08 99 1.62 97 2.43 92 6 1.25 97 1.555 95 1.92 96 4.36 86.2 9 0.415 98 0.605 97 1.605 96 5.595 83 12 0.18 99 0.525 n.d. 1.53 97 6.41 80 (r.M.: residual moisture content; r.I.: residual integrity)

[0323] The residual moisture content of the lyophilized mRNA/trehalose formulations increased over time if stored at +40 C./75% r.H. (see FIG. 8A). The increase of residual moisture above 4% after 6 months in mRNA/trehalose formulations correlated with a decreased relative integrity of the lyophilized RNA of below 90% (see FIG. 8B).

[0324] Nevertheless, a relative integrity of the lyophilized RNA of above 80% was obtained for all samples, even in the samples stored at +40 C./75% r.H. over 12 months. Lyophilization under controlled freezing and controlled drying conditions results in improved stability of the lyophilized RNA compared to lyophilization under non-controlled conditions (Example 3).

[0325] Biological Activity

[0326] The biological activity of the mRNA was measured after storage of the lyophilized samples for 6 months at 80 C. and +25 C./60% r.H. To this end, the lyophilized mRNA was reconstituted subsequent to the storage period and used for vaccination of mice. The presence of functional antibodies was subsequently determined by using a hemagglutinin inhibition assay and a virus neutralization assay.

[0327] Vaccination

[0328] Lyophilized mRNA was reconstituted in Ringer-Lactate solution. Female BALB/c mice were immunized in a prime/boost scenario using 80 g mRNA coding for hemagglutinin (HA) of A/Netherlands/602/09 (SEQ ID NO: 2) complexed with protamine prepared according to Example 2, lyophilized as described above and stored at +25 C. or at 80 C. Blood was collected 34 days after last vaccination and analyzed for the presence of functional antibodies by hemagglutinin inhibition assay and virus neutralization assay.

[0329] Hemamlutination Inhibition (HI) Assay

[0330] For the hemagglutination inhibition (HI) assays, mouse sera was heat inactivated (56 C., 30 min), incubated with kaolin and pre-adsorbed to chicken red blood cells (CRBC) (both Labor Dr. Merck & Kollegen, Ochsenhausen, Germany). For the HI assay, 50 l each of two-fold dilutions of pre-treated sera were incubated for 45 min with 4 HAU (units of HA) of inactivated A/California/07/2009 virus and 50 l 0.5% CRBC were added. The highest dilution of serum that prevents hemagglutination is referred to as the HI titer of the serum.

[0331] Virus-Neutralizing Titers

[0332] Virus-neutralizing titers were determined in sera pre-treated by heat inactivation (56 C., 30 min). Serially diluted sera were incubated for 2 hours with 100TCID50 (tissue culture 50% infectious dose) of virus and subsequently transferred to monolayers of MDCK cells. Presence or absence of virus was determined after 3 days by performing a hemagglutination assay of supernatants using inactivated A/California/04/09 virus.

[0333] Results

[0334] No difference in biological activity of the lyophilized mRNA could be seen after storage of the formulations at 80 C. and +25 C./60% r.H. It can be concluded that storage at higher temperatures does not affect the biological activity of mRNA lyophilized by an optimized lyophilization cycle. (see FIG. 8C)

Example 7: Optimization of a Lyophilization Cycle Under Controlled Freezing and Controlled

[0335] drying conditions; Long-term stability and safety of lyophilized mRNA mRNA encoding RAV-G (SEQ ID NO: 9) was formulated with protamine in a weight ratio of 4:1 according to Example 2 with a final mRNA concentration of 0.8 g/l in the presence of 5% (w/w) trehalose. The formulation was cooled to 80 C. Prior to filling of 600 l formulation per sterile 2R glass (type 1) vials, the formulation was allowed to thaw at room temperature (20-25 C.). The vials were half-closed with freeze drying rubber stoppers and loaded onto the shelves of the freeze drier at 20 C. Lyophilization was performed on a BOC Edwards Lyoflex 04 freeze-drier and included a freeze drying cycle with the conditions provided in Table 9.

TABLE-US-00015 TABLE 9 Pressure Cooling/heating MKS Duration Step Description Shelf temperature rate (mbar) (hh:mm) 1 Load 20 C. 1000 00:00 2 Freezing 20 C. .fwdarw. 40 C. 0.5 C./min 1000 02:00 3 Freezing 40 C. 1000 02:00 4 Evacuation 40 C. 0.1 00:33 5 Primary 40 C. .fwdarw. 10 C. 5 C./h 0.1 06:00 drying 6 Primary 10 C. 0.1 11:00 drying 7 Secondary 10 C. 0.045 00:20 drying 8 Secondary 10 C. .fwdarw. 20 C. 10 C./h 0.045 03:00 drying 9 Secondary 20 C. 0.045 07:00 drying 10 Nitrogen 20 C. n.a back-fill 11 Vial closure 20 C. n.a. 12 Aeration 20 C. atm

[0336] Long-Term Stability and Safety of Lyophilized mRNA

[0337] In order to assess long term stability and safety of the lyophilized RAV-G mRNA under different temperature conditions, certain quality attributes of stored RAV-G RNA were analyzed including appearance, RNA integrity, RNA content, pH value, and osmolarity. These quality attributes are discussed in further detail below.

[0338] Appearance:

[0339] The visual appearance of the lyophilisate cake is an indicator for the stability of the RNA. RNA lyophilisates should be white to yellowish in colour to meet that stability specification.

[0340] RNA Integrity:

[0341] Degradation of RNA over time leads to a loss of RNA integrity. The integrity of the RNA was analyzed after re-constitution of the RNA in water via RNA gelelectrophoresis. RNA gelelectrophoresis was performed according to methods commonly known in the art. Band sharpness was analyzed to determine the integrity of the RNA. Moreover, the gel was analyzed for the presence of additional undesired bands or artefacts.

[0342] RNA Content:

[0343] Increasing RNA content over time is an indicator for an evaporation of solvent. Therefore, the RNA content of the stored RNA lyophilisate was analyzed. A dried RNA sample was re-suspended in 10 ml WFI. The RNA concentration of the sample was determined photometrically.

[0344] pH Value:

[0345] A change in pH over time may be an indicator for undesired chemical reactions of the product components. Potentiometric determination of the pH content was performed using a commercially available volt-meter according to the European pharmacopedia (PhEur) 2.2.3.

[0346] Osmolarity

[0347] Changes in osmolarity over time may be an indicator for undesired chemical reactions of the product components. The measurement of the osmolality was performed according to European pharmacopedia (PhEur) 2.2.35, using a commercially available osmometer.

[0348] One stability study was conducted that analyzed long term stability (up to 36 months) under controlled conditions at 5 C. (results are shown in Table 10). Moreover, one stability study at higher temperatures (25 C.) over 36 months has been performed (see Table 11).

[0349] Results:

TABLE-US-00016 TABLE 10 Results of the stability analysis; up to 36 months; 5 C. Analysis of time points [months] Attribute 0 3 6 9 12 18 24 36 Appearance conform conform conform conform conform conform conform conform Integrity [%] 100 100 97 88 95 82 87 87 Content [g/l] 0.69 0.72 0.71 0.70 0.72 0.75 0.65 0.74 pH value 6.6 6.6 6.5 6.3 6.6 6.5 6.2 6.3 Osmolarity 150 145 148 142 154 141 144 144 [mOsmol/kg]

TABLE-US-00017 TABLE 11 Results of the stability analysis; up to 36 months; 25 C. Analysis of time points [months] Attribute 0 3 6 9 12 18 24 36 Appearance conform conform conform conform conform conform conform conform Integrity [%] 100 95 93 83 84 77 80 75 Content [g/l] 0.69 0.70 0.67 0.70 0.67 0.75 0.69 0.72 pH value 6.6 6.6 6.5 6.3 6.6 6.5 6.0 6.4 Osmolarity 150 150 150 144 150 145 143 144 [mOsmol/kg]

[0350] The results show that the inventive lyophilisation method according to the present invention is particularly suitable to produce stable RNA lyophilisates for long-term storage. The results shown in Table 10 and 11 show that all quality attributes analysed during the experimental period (up to 36 months) meet the stability specifications of a stable and safe RNA medicament. Notably, even at higher temperatures (25 C., see Table 11) these stability specifications were met, showing that the inventive lyophilisation method is particularly suitable to produce long term stable and temperature resistant RNA lyophilisates.