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. A method for lyophilizing a composition, wherein the method comprises the following steps: a) providing a liquid having a glass transition temperature comprising: at least one mRNA, said mRNA comprising from 200 to 15,000 nucleotides, a 5′ cap and at least one coding region; and at least one lyoprotectant comprising a carbohydrate compound, wherein the glass transition temperature of the liquid is in a range from −15° C. to −50° C.; 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 in a range from 0.1° C./min to 2° C./min; d) freezing the liquid having the glass transition temperature at the freezing temperature in order to obtain a frozen liquid, wherein the freezing temperature is in a range from 0.5° C. to 25° C. below the glass transition temperature of the liquid provided in step a); 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 mRNA and at least one lyoprotectant, wherein drying comprises heating the frozen liquid obtained in step d) to a drying temperature; and g) equilibrating the pressure in the freeze drying chamber to atmospheric pressure and removing the lyophilized composition comprising the at least one mRNA and the at least one lyoprotectant obtained in step f) from the freeze drying chamber to provide a lyophilized mRNA composition.

2. The method of claim 1, wherein the at least one mRNA comprises from 300 to 10,000 nucleotides.

3. The method of claim 1, wherein the drying temperature is lower than the glass transition temperature of the liquid.

4. The method of claim 1, wherein the drying temperature is in a range from −40° C. to 40° C.

5. The method of claim 1, wherein the heating of step f) is performed at a defined heating rate, wherein the defined heating rate is 30° C./h or less.

6. The method of claim 5, wherein the heating of step f) is performed at a defined heating rate, wherein the defined heating rate is in a range from 0.1° C./h to 20° C./h.

7. The method of claim 1, wherein the liquid further comprises at least one cationic or polycationic compound.

8. The method of claim 7, wherein the cationic or polycationic compound is a cationic or polycationic peptide or protein.

9. The method of claim 7, wherein the cationic or polycationic compound is a cationic or polycationic lipid.

10. The method of claim 7, wherein the at least one mRNA and the at least one cationic or polycationic compound are present in a complex.

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

12. The method of claim 1, wherein the mRNA comprises a poly(A) sequence of about 50 to about 100 adenine nucleotides.

13. The method of claim 12, wherein the mRNA comprises at least one modified nucleotide.

14. The method of claim 13, wherein the modified nucleotide is pseudouridine or 1-methyl-pseudouridine.

15. The method of claim 14, wherein the modified nucleotide is 1-methyl-pseudouridine.

16. The method of claim 1, wherein the lyoprotectant comprises mannitol, sucrose, glucose, mannose and/or trehalose.

17. The method of claim 1, wherein the concentration of the lyoprotectant in the liquid provided in step a) is in a range from 1 to 20% (w/w).

18. The method of claim 1, wherein the concentration of the at least one mRNA in the liquid provided in step a) is in a range from 0.1 to 10 g/l.

19. The method of claim 1, wherein the glass transition temperature of the liquid is in a range from −25° C. to −40° C.

20. The method of claim 1, wherein the freezing temperature is in a range from −50° C. to −35° C.

21. The method of claim 1, wherein the cooling rate in step c) is in a range from 0.5° C./min to 1.5° C./min.

22. A lyophilized composition comprising at least one mRNA and at least one lyoprotectant, which is produced by a method which comprises the following steps: a) providing a liquid having a glass transition temperature comprising at least one mRNA, said mRNA comprising from 200 to 15,000 nucleotides, a 5′ cap and at least one coding region; and at least one lyoprotectant, wherein the glass transition temperature of the liquid is in a range from −15° C. to −50° C.; 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 in a range from 0.1° C./min to 2° C./min; d) freezing the liquid having the glass transition temperature at the freezing temperature in order to obtain a frozen liquid, wherein the freezing temperature is in a range from 0.5° C. to 25° C. below the glass transition temperature of the liquid provided in step a); 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 mRNA and at least one lyoprotectant, wherein drying comprises heating the frozen liquid obtained in step d) to a drying temperature; and g) equilibrating the pressure in the freeze drying chamber to atmospheric pressure and removing the lyophilized composition comprising the at least one mRNA and the at least one lyoprotectant obtained in step f) from the freeze drying chamber to provide a lyophilized mRNA composition.

23. The composition of claim 22, wherein the liquid further comprises at least one cationic or polycationic compound.

24. The composition of claim 23, wherein the cationic or polycationic compound is a cationic or polycationic peptide or protein.

25. The composition of claim 23, wherein the cationic or polycationic compound is a cationic or polycationic lipid.

26. The composition of claim 23, wherein the at least one mRNA and the at least one cationic or polycationic compound are present in a complex.

27. The composition of claim 22, wherein the 5′ cap is a m7GpppN cap.

28. The composition of claim 22, wherein the mRNA comprises a poly(A) sequence of about 50 to about 100 adenine nucleotides.

29. The composition of claim 28, wherein the mRNA comprises at least one modified nucleotide.

30. The composition of claim 29, wherein the modified nucleotide is pseudouridine or 1-methyl-pseudouridine.

31. The composition of claim 30, wherein the modified nucleotide is 1-methyl-pseudouridine.

32. The composition of claim 22, wherein the lyoprotectant comprises a carbohydrate compound.

33. The composition of claim 22, wherein the lyoprotectant comprises mannitol, sucrose, glucose, mannose and/or trehalose.

34. The composition of claim 33, wherein the concentration of the lyoprotectant in the liquid provided in step a) is in a range from 1 to 20% (w/w).

35. The composition of claim 33, wherein the lyoprotectant comprises mannose and/or sucrose.

36. The composition of claim 35, wherein the lyoprotectant comprises mannose and sucrose.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) 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.

(2) FIG. 1: SEQ ID NO: 1, which is the mRNA sequence corresponding to PpLuc(GC)-muag-A64-030.

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

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

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

(6) 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).

(7) FIG. 6: Optimization of a lyophilization cycle under controlled freezing conditions (Example 4) 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. 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).

(8) FIG. 7: Optimization of a lyophilization cycle under controlled freezing and controlled drying conditions (Example 5) A. Temperature profile and pressure profile of the lyophilization cycle. 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. 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.

(9) FIG. 8: Optimization of a lyophilization cycle under controlled freezing and controlled drying conditions; biological activity of lyophilized mRNA (Example 6) 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. 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. 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. 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

(10) 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

(11) Vectors for in vitro transcription were constructed, which contain a T7 promoter followed by a GC-enriched coding sequence.

(12) 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.

(13) 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.

(14) 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.

(15) 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.

(16) 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, Tübingen, Germany).

Example 2: Complexation of RNA

(17) 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).

(18) 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).

(19) 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).

(20) Such formulated RNA was used for lyophilization experiments.

Example 3: Standard Lyophilization Process

(21) 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 μl 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.

(22) TABLE-US-00006 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 μbar ~00:20 with liquid nitrogen)  5 Primary <−50° C. .fwdarw. 40° C. 63 μbar   18:30 drying heating only controlled by final temperature  9 Secondary 40° C. shelves results in 45 μbar   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 —

(23) 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.

(24) Results

(25) TABLE-US-00007 TABLE 2 +25° C./60% r.H. +40° C./75% r.H. Storage time (weeks) Rel. integrity (%) Rel. integrity (%) 0 75 75 5 84 71 8 79 52 13 79 40 24 76 1 40 80 1

(26) 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

(27) 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).

(28) 500 μl 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.

(29) Lyophilization Cycle

(30) TABLE-US-00008 TABLE 3 Group I Temperature (shelf temp. if not Coolingrate/ Pressure Duration Step Description indicated otherwise) Heating rate Pirani (hh:mm)  1 Loading   15° C. atm 00:00  1a Pre-cooling   15° C. atm 02:05  2 Cooling down/   15° C. .fwdarw. −40° C. 0.31° C./min atm 02:55 Freezing (shelf)  3 Freezing −40° C. atm 07:20  4 Evacuation −40° C. 100 μbar 00:20  5 Primary drying (1) −40° C. .fwdarw. −15° C. 4.2 ° C./h 100 μbar 06:00 (shelf)  6 Primary drying (2) −15° C. 100 μbar 11:00  7 Secondary drying (1) −15° C.  45 μbar 00:20  8 Secondary drying (2) −15° C. .fwdarw. 20° C. (product) Not controlled  45 μbar 03:00   40° C. (shelf)  9 Secondary drying (3)   20° C. (product)  45 μbar 07:00   40° C. (shelf) 10 Nitrogen back-fill   20° C. (product) n.a —   40° C. (shelf) 11 Vial closure   20° C. (product) n.a. —   40° C. (shelf) 12 Aeration   20° C. (product) atm —   40° C. (shelf)

(31) TABLE-US-00009 TABLE 4 Group II Temperature (shelf temp. if not Coolingrate/ Pressure Duration Step Description indicated otherwise) Heating 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. (shelf) 0.31° C./min atm 02:55  3 Freezing −40° C. atm 07:20  4 Evacuation −40° C. 100 μbar 00:20  5 Primary drying −40° C. .fwdarw. −17° C. (shelf)  2.6° C./h 100 μbar 09:00  6 Primary drying −17° C. 100 μbar 16:00  7 Secondary drying −17° C.  45 μbar 00:20  8 Secondary drying −17° C. .fwdarw. 20° C. (product) Not controlled  45 μbar 03:00   40° C. (shelf)  9 Secondary drying   20° C. (product)  45 μbar 07:00   40° C. (shelf) 10 Nitrogen back-fill   20° C. (product) n.a. —   40° C. (shelf) 11 Vial closure   20° C. (product) n.a. —   40° C. (shelf) 12 Aeration   20° C. (product) atm —   40° C. (shelf)

(32) 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.

(33) 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.

(34) 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.

(35) Results

(36) TABLE-US-00010 TABLE 5 2.5% Trehalose 5% Trehalose 10% Trehalose Storage Residual Rel. Residual Rel. Residual Rel. Time moisture integrity moisture integrity moisture integrity (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

(37) 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

(38) 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 μl 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.

(39) TABLE-US-00011 TABLE 6 Cooling/ Pressure MKS Duration Step Description Shelf temperature heating 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 drying −40° C. .fwdarw. −10° C.   5° C./h   0.1 06:00  6 Primary drying −10° C.   0.1 11:00  7 Secondary drying −10° C.   0.045 00:33  8 Secondary drying −10° C. .fwdarw. 20° C.  10° C./h   0.045 03:00  9 Secondary drying   20° C.   0.045 07:00 10 Nitrogen back-fill   20° C. n.a — 11 Vial closure   20° C. n.a. — 12 Aeration   20° C. atm —

(40) 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.

(41) Results

(42) 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 Drying Conditions; Biological Activity of Lyophilized mRNA

(43) 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 μl 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:

(44) TABLE-US-00012 TABLE 7 Cooling/ Pressure Duration Step Description Shelf Temperature heating rate Pirani (hh:mm)  1 Load   20° C. atm 00:00  2 Freezing   20° C. .fwdarw. −40° C. 0.5° C./min atm 02:00  3 Freezing −40° C. atm 02:00  4 Evacuation −40° C. 160 μbar 00:20  5 Primary drying −40° C. .fwdarw. −10° C.   5° C./h 160 μbar 06:00  6 Primary drying −10° C. 160 μbar 11:00  7 Secondary drying −10° C.  68 μbar 00:20  8 Secondary drying −10° C. .fwdarw. 20° C.  10° C./h  68 μbar 03:00  9 Secondary drying   20° C.  68 μbar 07:00 10 Nitrogen back-fill   20° C. 0,8 bar — 11 Vial closure   20° C. 0,8 bar — 12 Aeration   20° C. atm —

(45) 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.

(46) Results

(47) TABLE-US-00013 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)

(48) 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).

(49) 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).

(50) Biological Activity

(51) 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.

(52) Vaccination

(53) Lyophilized mRNA was reconstituted in Ringer-Lactate solution. Female BALB/c mice were immunized in a prime/boost scenario using 80 μg 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.

(54) Hemagglutination Inhibition (HI) Assay

(55) 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.

(56) Merck & Kollegen, Ochsenhausen, Germany). For the HI assay, 50 μl 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 μl 0.5% CRBC were added. The highest dilution of serum that prevents hemagglutination is referred to as the HI titer of the serum.

(57) Virus-Neutralizing Titers

(58) 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 100×TCID50 (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.

(59) Results

(60) 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 Drying Conditions; Long-Term Stability and Safety of Lyophilized mRNA

(61) 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 μl 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.

(62) TABLE-US-00014 TABLE 9 Cooling/ Pressure MKS Duration Step Description Shelf temperature heating 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 drying −40° C..fwdarw. −10° C.   5° C./h   0.1 06:00  6 Primary drying −10° C.   0.1 11:00  7 Secondary drying −10° C.   0.045 00:20  8 Secondary drying −10° C..fwdarw. 20° C.  10° C./h   0.045 03:00  9 Secondary drying   20° C.   0.045 07:00 10 Nitrogen back-fill   20° C. n.a — 11 Vial closure   20° C. n.a. — 12 Aeration   20° C. atm —

(63) Long-Term Stability and Safety of Lyophilized mRNA

(64) 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.

(65) Appearance:

(66) 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.

(67) RNA Integrity:

(68) 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.

(69) RNA Content:

(70) 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.

(71) pH Value:

(72) 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 pharmacopeia (PhEur) 2.2.3.

(73) Osmolarity:

(74) 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 pharmacopeia (PhEur) 2.2.35, using a commercially available osmometer.

(75) 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).

(76) Results:

(77) TABLE-US-00015 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 [mOsmol/kg] 150 145 148 142 154 141 144 144

(78) TABLE-US-00016 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 [mOsmol/kg] 150 150 150 144 150 145 143 144

(79) 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.