Dry powder composition comprising long-chain RNA

Abstract

The present invention is directed to a storage-stable formulation of long-chain RNA. In particular, the invention concerns a dry powder composition comprising a long-chain RNA molecule. The present invention is furthermore directed to methods for preparing a dry powder composition comprising a long-chain RNA molecule by spray-drying. The invention further concerns the use of such a dry powder composition comprising a long-chain RNA molecule in the preparation of pharmaceutical compositions and vaccines, to a method of treating or preventing a disorder or a disease, to first and second medical uses of such a dry powder composition comprising a long-chain RNA molecule and to kits, particularly to kits of parts, comprising such a dry powder composition comprising a long-chain RNA molecule.

Claims

1. A method for expression of a polypeptide in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising a dry powder composition comprising a long-chain RNA molecule comprising at least 200 nucleotides, wherein the dry powder has a residual moisture content of 7% (w/w) or less, wherein the long-chain RNA molecule is in a complex with a cationic or polycationic carrier, wherein the pharmaceutical composition is formulated for mucosal, intranasal, inhalation, or pulmonary delivery, and wherein the long-chain RNA encodes the polypeptide.

2. The method of claim 1, wherein the dry powder composition is prepared by a method comprising the following steps: a) providing a liquid comprising the long-chain RNA molecule, b) spray-drying the liquid of step a) with a spray-drying device having a drying gas inlet and a drying gas outlet, wherein the drying gas has a temperature at the inlet of at least 85° C., wherein the drying gas has a temperature at the outlet of at least 50° C.

3. The method according to claim 2, wherein the liquid of step a) further comprises at least one excipient selected from a cryoprotectant, a lyoprotectant and a bulking agent.

4. The method according to claim 2, wherein the liquid of step a) does not contain a lipid compound.

5. The method according to claim 2, wherein the liquid of step a) comprises a spray-drying compatible solvent.

6. The method according to claim 2, wherein the liquid of step a) is atomized and the droplets resulting from the atomization of the liquid are characterized by a mass median aerodynamic diameter of 300 nm to 200 μm.

7. The method according to claim 1, wherein the subject has a disorder or disease selected from the group consisting of cancer or tumor diseases, infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, cardiovascular diseases and neuronal diseases.

8. The method according to claim 1, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

9. The method according to claim 1, wherein the pharmaceutical composition comprises a plurality of particles.

10. The method according to claim 8, wherein the median particle size in a volume weighted distribution of the resulting dried powder is at least 1 μm.

11. The method according to claim 8, wherein the average sphericity of the particles in the resulting dried powder is in a range from 0.7 to 1.0.

12. The method according to claim 1, wherein the long-chain RNA molecule comprises more than 200 nucleotides.

13. The method according to claim 1, wherein the long-chain RNA molecule comprises at least one modification.

14. The method according to claim 2, wherein the liquid of step a) further comprises a suspending agent and/or an osmolality of about 200 mosmol/1 to about 400 mosmol/1.

15. The method according to claim 6, wherein at least a first pressure nozzle is used as an atomizer, optionally wherein the pressure is less than about 1 bar.

16. The method of claim 1, wherein the dry powder comprises an average particle size of 1 μm to 20 μm.

17. The method of claim 1, wherein the pharmaceutical composition is administered by inhalation.

18. The method of claim 1, wherein the pharmaceutical composition is administered to the lungs of the patient.

19. The method of claim 1, wherein the pharmaceutical composition is administered as an aerosolized formulation.

20. The method of claim 1, wherein the dry powder has a residual moisture content of 2% to 5% (w/w).

21. The method of claim 1, wherein the wherein the cationic or polycationic carrier comprises a cationic or polycationic polypeptide.

22. The method of claim 1, wherein the wherein the cationic or polycationic carrier comprises a cationic or polycationic lipid.

23. The method of claim 1, wherein the wherein the cationic or polycationic carrier comprises a cationic or polycationic polymer.

24. The method of claim 1, wherein the pharmaceutical composition is administered by mucosal, intranasal, inhalation, or pulmonary delivery.

25. A method for expression of a polypeptide in a subject comprising: a) providing a dry powder composition comprising a long-chain RNA molecule comprising at least 200 nucleotides, wherein the dry powder has a residual moisture content of 7% (w/w) or less, wherein the long-chain RNA molecule is in a complex with a cationic or polycationic carrier and wherein the long-chain RNA encodes the polypeptide; b) reconstituting the dry powder composition in a pharmaceutically acceptable aqueous solvent to provide a liquid pharmaceutical composition; and c) administering an effective amount of a pharmaceutical composition to the subject.

26. The method of claim 24, wherein the pharmaceutical composition is administered by injection.

27. The method of claim 24, wherein the pharmaceutical composition is administered as an aerosolized formulation for mucosal, intranasal, inhalation, or pulmonary delivery.

28. The method of claim 26, wherein the RNA encodes an antigen, said method further defined as a method for stimulating an immune response.

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: Scheme of a co-current spray-drying apparatus A: solution or suspension to be dried inlet, B: atomization gas (e.g. nitrogen) inlet, 1: drying gas (e.g. nitrogen) inlet, 2: heating of drying gas, 3: spraying of solution or suspension, 4: drying chamber, 5: part between drying chamber and cyclone, 6: cyclone, 7: drying gas outlet, 8: product collection vessel.

(3) FIG. 2: Sequence of the mRNA used in this study (R2564; SEQ ID NO: 1).

(4) FIG. 3: Photograph of powder of protamine-formulated RNA (T-SD1, T-SD2, T-SD3) spray-dried with outlet temperatures of 47, 69 and 87° C. respectively and of placebo sample (T-SD-P) spray-dried at 84° C.

(5) FIG. 4: Residual water content of dry powder formulations obtained by spray-drying using different outlet temperatures.

(6) FIG. 5: X-ray powder diffraction analysis of protamine-formulated RNA (T-SD1, T-SD2, T-SD3) spray-dried with outlet temperatures of 47, 69 and 87° C. respectively and placebo sample (T-SD-P) spray-dried at 84° C.

(7) FIG. 6: Particle size distributions of protamine-formulated RNA (T-SD1, T-SD2, T-SD3) spray-dried with outlet temperatures of 47, 69 and 87° C. respectively and placebo sample (T-SD-P) spray-dried at 84° C. as determined by laser diffraction analysis.

(8) FIGS. 7A-D: Scanning electron microscope (SEM) images of protamine-formulated RNA powder particles (T-SD-1 (FIG. 7A, 5100x), T-SD-2 (FIG. 7B, 4950x), T-SD-3 (FIG. 7C, 2080x) and placebo powder particles (T-SD-P, FIG. 7D, 2660x).

(9) FIG. 8: Particle size distribution of protamine-formulated RNA before (T0) and after (T-SD 1, T-SD 2, T-SD 3) spray-drying with outlet temperatures of 47, 69 and 87° C. respectively and particle size of spray-dried placebo sample (T-SD-P) with an outlet temperature of 84° C. The particle size was determined by dynamic light scattering (DLS).

(10) FIG. 9: Particle size distribution of protamine-formulated RNA before (T0) and after (T-SD 1, T-SD 2, T-SD 3) spray-drying with outlet temperatures of 47, 69 and 87° C. respectively and particle size of spray-dried placebo sample (T-SD-P) with an outlet temperature of 84° C. The particle size was determined by nanoparticle tracking analysis (NTA).

EXAMPLES

(11) 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

(12) A vector for in vitro transcription was constructed containing a T7 promoter followed by a GC-enriched sequence encoding the hemagglutinin (HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)) and used for subsequent in vitro transcription reactions. According to a first preparation, the DNA sequence coding for the above mentioned mRNA was prepared. The constructs R2564 (SEQ ID NO: 1) was prepared by introducing a 5′-TOP-UTR derived from the ribosomal protein 32L4, modifying the wild type coding sequence by introducing a GC-optimized sequence for stabilization, followed by a stabilizing sequence derived from the albumin-3′-UTR, a stretch of 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence), and a histone stem loop. In SEQ ID NO: 1 (see FIG. 2) and the sequence of the corresponding mRNA is shown.

Example 2

In Vitro Transcription and Purification of RNA

(13) The respective DNA plasmids prepared according to section 1 above were transcribed in vitro using T7 polymerase. The in vitro transcription of influenza HA encoding R2564 was performed in the presence of a CAP analog (m7GpppG). Subsequently the RNA was purified using PureMessenger® (CureVac, Tubingen, Germany; WO2008/077592A1).

Example 3

Preparation of Protamine-Formulated RNA

(14) RNA was diluted (0.87 g/L RNA final concentration) and a protamine/trehalose mixture was prepared (43000 anti-heparin IU/L protamine; 10.87% trehalose in water for injection). One volume unit of each solution was mixed to yield a ratio of protamine to RNA of 50 anti-heparin IU per mg RNA.

(15) The solution of RNA/protamine complexes were supplemented with R2564 to yield final concentrations of 0.4 g/L RNA complexed with 20000 anti-heparin IU/L of protamine (corresponding to a protamine concentration of about 1.5 g/L), 0.4 g/L free RNA and 5% trehalose (w/w).

(16) Such formulated RNA was used for spray-drying experiments.

(17) As a placebo, 5% trehalose was prepared in water for injection.

Example 4

Spray-Drying of Protamine-Formulated RNA and Placebo Formulation

(18) The objective of the experiments presented in this section was to test the feasibility of large scale production of the inventive dry powder composition by spray-drying. In summary, three spray-drying experiments were performed at different outlet temperatures using the protamine-formulated RNA prepared according to Example 3. As a control, the placebo formulation as described in Example 3 was processed in parallel.

(19) Protamine-formulated RNA (Example 3) or placebo sample was thawed and each aliquot was homogenized by gentle mixing using a magnetic stirrer before spray-drying.

(20) Spray-drying of protamine-formulated RNA and placebo formulation was carried out using a Büchi Mini Spray-Dryer B-290 equipped with a two-fluid nozzle and a high performance cyclone. The spray-dryer was operated using nitrogen as drying gas in a closed cycle mode. The spray-drying experiments were carried out under the process parameters listed in Table 1.

(21) TABLE-US-00001 TABLE 1 Process parameters spray-drying Process T-SD1 T-SD2 T-SD3 parameter (verum) (verum) (verum) T-SP-P Nozzle type two-fluid two-fluid two-fluid two-fluid nozzle nozzle nozzle nozzle Atomization 30 mm ± 30 mm ± 30 mm ± 30 mm ± gas flow 5 mm 5 mm 5 mm 5 mm setting* (~357 l/h) (~357 l/h) (~357 l/h) (~357 l/h) (theoretical volume flow) Inlet 65° C. 100° C. 128° C. 128° C. temperature Outlet 47° C. 69° C. 87° C. 84° C. temperature Drying gas nitrogen nitrogen nitrogen nitrogen Drying gas 100% 100% 100% 100% rate/aspirator (~35 m.sup.3/h) (~35 m.sup.3/h) (~35 m.sup.3/h) (~35 m.sup.3/h) Pump speed 3% 3% 3% 3% (~1 ml/min) (~1 ml/min) (~1 ml/min) (~1 ml/min) Yield [g] 0.788 0.915 1.017 0.909* Calculated 44.2 51.4 56.5 48.3* relative yield [%] *Atomization gas flow setting of 30 mm correlates with a pressure drop at the nozzle of 0.23 bar

(22) Following spray-drying, the produced powders were filled into vials (see section 5) and characterized (see section 6).

(23) 5. Powder Filling

(24) The powder obtained after spray-drying was collected in a glass container at the product outlet of the cyclone. For storage, shipment and further analysis the powder was divided into aliquots and transferred into 10 R vials under controlled humidity conditions (<15% RH) in a glove box. Vials were stoppered inside the glove box. The exact weight of the dry powder was documented during filling. FIG. 3 shows a photograph of glass vials containing the spray-dried powder.

(25) 6. Analytical Characterization

(26) A sampling scheme, including the sampling time points and analytical methods is shown in Table 2.

(27) TABLE-US-00002 TABLE 2 sampling time points for samples from spray-drying Time point Description analytical methods T0 Liquid verum sample before DLS, NTA, MFI, turbidity, (verum) spray-drying. ZP T0-P Liquid placebo formulation before DLS, NTA, MFI, turbidity spray-drying T-SDX Spray-dried verum sample obtained DLS, ZP, NTA, MFI, from experiment T-SD1, T-SD2, turbidity Karl-Fischer T-SD3, T-SD4 titration, DSC, XRD, laser diffraction analysis, SEM T-SD-P Spray-dried placebo sample DLS, NTA, MFI, turbidity obtained from experiment T-SD-P

(28) 6.1 Methods for Physico-Chemical Characterization of Spray-Dried Powder

(29) Spray-dried powders were characterized with respect to physico-chemical properties of the spray-dried formulation using various methods (Table 3).

(30) TABLE-US-00003 TABLE 3 Analytical methods for physico-chemical characterization of spray-dried powder. Sample Abbreviation Full term Dry powder DSC Differential scanning calorimetry KF Karl Fischer titration XRD X-ray powder diffraction Laser diffraction Laser diffraction analysis SEM Scanning electron microscopy

(31) 6.1.1 Karl Fischer Titration

(32) The residual moisture content of the dried powders were determined using the coulometric Karl Fischer titrator Aqua 40.00 (Analytik Jena GmbH, Jena, Germany), which is equipped with a headspace module.

(33) As a system suitability check, the residual moisture content of a Pure Water Standard (Apura 1 water standard oven 1.0, Merck KGaA) was analyzed prior to sample measurement. The residual moisture content of the standard had to be within 1.00±0.03% in order to comply with the manufacturer specifications.

(34) For the measurement, about 20 mg of sample were weighed into 2 R glass vials and heated to a measurement temperature of 120° C. in the oven connected to the reaction vessel via a tubing system. The evaporated water was transferred into the titration solution and the amount of water was determined. The measurement was performed until no more water evaporation was detectable (actual drift comparable to drift at the beginning of the measurement). Ambient moisture was determined by measurement of three blanks (empty vials prepared in the preparation environment). Results obtained for samples were corrected for the determined ambient moisture by blank subtraction. Samples were measured in duplicates. The results are shown in FIG. 4 and Table 4.

(35) TABLE-US-00004 TABLE 4 Water content of spray-dried formulation Sample Water content [%] T-SD1 4.37 T-SD2 2.81 T-SD3 1.78 T-SD-P 1.70

(36) 6.1.2 Differential Scanning Calorimetry (DSC)

(37) Differential scanning calorimetry (DSC) in a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) was used to determine thermal events of the spray-dried samples (e.g. glass transition temperature (Tg), crystallization or endothermal melting). Approximately 10 mg of the spray-dried samples were analyzed in crimped Al-crucibles (Mettler Toledo, Giessen, Germany). The samples were cooled to 0° C. at a cooling rate of 10 K/min and reheated to 120° C. with a rate of 10 K/min. The measurement of the temperature profile was repeated in a second cycle in order to evaluate reversibility of thermal events. The Tg was determined as the midpoint of the endothermic shift of the baseline during the heating scan (see Table 5). The maximum of exothermic/endothermic peaks were reported as Tcryst/Tm.

(38) TABLE-US-00005 TABLE 5 Tg values of spray-dried formulation Sample Tg (1.sup.st scan) [° C.] T-SD1 59.0 T-SD2 72.1 T-SD3 87.9 T-SD-P 87.8

(39) Tg values correlate with the water contents of the samples. No relaxation phenomenon (exothermic event) was detectable in spray-dried samples, in contrast to thermograms obtained for lyophilized samples.

(40) 6.1.3 X-Ray Powder Diffraction (XRD)

(41) Wide angle X-ray powder diffraction (XRD) was used to study the morphology of lyophilized products. The X-ray diffractometer Empyrean (Panalytical, Almelo, The Netherlands) equipped with a copper anode (45 kV, 40 mA, Kα1 emission at a wavelength of 0.154 nm) and a PIXcel3D detector was used. Approximately 100 mg of the spray-dried samples were analyzed in reflection mode in the angular range from 5-45° 2θ, with a step size of 0.04° 2θ and a counting time of 100 seconds per step. The respective XRD diagrams are shown in FIG. 5.

(42) All samples showed an amorphous pattern and no indication of crystalline phases.

(43) 6.1.4 Laser Diffraction Analysis

(44) Size distribution of spray-dried powders were measured by laser diffraction. Laser diffraction measurements were performed using a Partica LA-950 Laser Diffraction Particle Size Distribution Analyzer (Horiba, Kyoto, Japan) equipped with a 605 nm laser diode for detecting particles >500 nm and 405 nm blue light emitting diode (LED) for detecting particles <500 nm. The powder samples were dispersed in Miglyol 812 by ultra sonication for up to 5 min. Prior to measurement, the system was blanked with Miglyol 812. Each sample dispersion was measured 3 times. Measurement results were analyzed using Horiba LA-950 Software.

(45) The results were reported as

(46) D10: particle diameter corresponding to 10% of the cumulative undersize distribution;

(47) D50: particle diameter corresponding to 50% of the cumulative undersize distribution;

(48) D90: particle diameter corresponding to 90% of the cumulative undersize distribution.

(49) The results are summarized in Table 6 and FIG. 6.

(50) TABLE-US-00006 TABLE 6 Particle size distribution of spray-dried powders as measured by laser diffraction Median Modal Mean Sample Absorbance Diameter Diameter Value St. Dev. D10 D50 D90 T-SDP 0.126 4.21 6.75 4.032 0.394 1.204 4.21 12.516 T-SD3 0.217 4.132 6.75 3.615 0.408 0.938 4.132 11.151 T-SD2 0.187 4.31 6.75 3.728 0.418 0.933 4.31 11.588 T-SD1 0.051 4.608 6.75 3.927 0.375 1.075 4.608 10.854 (sizes are indicated in μm)

(51) 6.1.5 Scanning Electron Microscopy (SEM)

(52) Images of spray-dried powder particles were generated by using the bench top scanning electron microscope Phenom (Phenom-World B.V., Eindhoven, Netherlands). The instrument is equipped with a CCD camera and a diaphragm vacuum pump. Each sample was prepared in a glove box under controlled humidity conditions (<20% relative humidity) by using the following method: a small amount of the powder was carefully put on a self-adhesive carbon foil placed on a sample holder. The sample was analyzed under vacuum with a light optical magnification of 24× and 5 kV acceleration voltage. The electron optical magnification was adjusted between 1160× and 1700× and images were made from representative sections of each sample.

(53) The obtained images (see FIG. 7) demonstrate that the obtained powder particles have spherical shape and that the size of the powder particles is in the range from less than 1 μm to approximately 20 μm.

(54) 6.2 Reconstitution of Spray-Dried Samples

(55) For reconstitution of the spray-dried samples, the reconstitution volume was calculated for each sample individually based on the amount of powder weighed into the vial. The calculation was based on the method for reconstitution of lyophilized samples (addition of 600 μl water for injection to 30.6 mg powder per vial).

(56) The reconstitution volume for varying amounts of spray-dried powder was calculated according to the following equation:
V.sub.reconst.=m.sub.powder*1000 μl/51 mg

(57) V.sub.reconst.: reconstitution volume in ml

(58) m.sub.powder: mass of powder to be reconstituted in mg

(59) (based on a theoretical solid content of 51 mg per ml (50 mg/ml trehalose, 0.8 mg/ml RNA (free+complexed), 20 anti-heparin IU/mL protamine))

(60) The spray-dried samples were reconstituted under laminar flow conditions using a procedure comparable to the procedure for the lyophilized product: cap and stopper were removed from the vial and the calculated volume of water for injection was added to the dry powder (into the center of the vial) by using a multipette with 10 ml combitip. The vial was carefully slewed (shaking was avoided), until all powder particles were dissolved. The reconstitution time was measured as the time required in order to achieve full reconstitution of the dry powder after the liquid has been added. The reconstitution behavior was judged, mainly with respect to foaming, and recorded (see Table 7).

(61) TABLE-US-00007 TABLE 7 Reconstitution behaviour Sample Reconstitution time [mm:ss] Foam formation T-SD1 01:27 0 T-SD2 01:20 0 T-SD3 01:37 0 T-SD-P 01:04 0

(62) In conclusion, reconstitution times for the spray-dried formulations were below 2 minutes. A slightly shorter resolution time was observed for the placebo formulation (T-SD-P).

(63) 6.3 Particle Characterization

(64) The particles comprised in liquid samples of the protamine-formulated RNA as described above were characterized before spray-drying, and after reconstitution of the spray-dried samples (Table 8).

(65) TABLE-US-00008 TABLE 8 Analytical methods for characterization of liquid samples (before and after spray-drying and reconstitution). Sample Abbreviation Full term Liquid sample Visual Visual inspection DLS Dynamic light scattering MFI Micro-Flow Imaging Turbidity Turbidity NTA Nanoparticle tracking analysis ZP Zeta potential

(66) 6.3.1 Visual Inspection

(67) For visual inspection, the reconstituted vials were inspected for the presence or absence of visible particles under gentle, manual, radial agitation for 5 sec in front of a white background and for 5 sec in front of a black background according to the European Pharmacopoeia. The inspection was performed by two independent examiners. To further classify the particle content, the method described in the “Deutscher Arzneimittel-Codex” (DAC) was used.

(68) The classification can be described as follows:

(69) No particles visible within 5 sec: 0 point

(70) Few particles visible within 5 sec: 1 point

(71) Medium number of particles visible within 5 sec: 2 points

(72) Large number of particles directly visible: 10 points

(73) (Particles that were on the limit of being visible as distinct particles (cloudiness, schlieren) were rated with 2 points.)

(74) The results of the visual inspection of the liquid samples are recorded in Table 9.

(75) TABLE-US-00009 TABLE 9 Results of visual inspection Sample Score [Exp 1/2] T-SD1 2*/2* T-SD2 1*/2  T-SD3 1/1 T-SD-P 2*/2* *fiber-like particle(s)

(76) As a result, visible particles were observed in all of the analyzed samples. The majority of the observed particles were fiber-like particles that were likely due to a contamination.

(77) 6.3.2 Turbidity

(78) The NEPHLA turbidimeter (Dr. Lange, Dusseldorf, Germany), operating at 860 nm and detecting at 90° angle, was used for turbidity measurements. The system was calibrated with formazin as a standard and the results were given in formazin nephelometric units (FNU).

(79) For the measurement, 2.0 ml solution were analyzed in a clean glass cuvette. The turbidity of the individual samples is indicated in Table 10. After analysis, the sample material was used for further analysis (e.g. DLS, MFI, etc.).

(80) TABLE-US-00010 TABLE 10 Turbidity of the liquid samples Sample Turbidity [FNU] T0 20.1 T-SD1 26.1 T-SD2 17.7 T-SD3 18.0 T-SD-P 3.5

(81) In summary, the turbidity of the spray-dried protamine-formulated RNA after reconstitution varied from 18 to 26 FNU.

(82) 6.3.3 Dynamic Light Scattering (DLS)

(83) DLS measurements were carried out by using a Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK) instrument. 150 μl of the sample were analyzed in small volume disposable cuvettes (UVette) by using an automated mode for each sample. As a control (T0), the protamine-formulated RNA before spray-drying was used.

(84) The Malvern Zetasizer Software was used to calculate Z-average diameter, polydispersity index (PDI) and an intensity size distribution (refractive index and viscosity of water was selected in the software). The results are shown in Table 11 and FIG. 8.

(85) TABLE-US-00011 TABLE 11 Z-average diameter, polydispersity index, main peak diameter and main peak intensity as determined by Dynamic light scattering Z-average Main peak Main peak diameter diameter intensity Derived Count Sample [nm] PdI [nm] [%] Rate T0 218.7 ± 0.5 0.183 ± 0.012 263.1 ± 3.6  100 ± 0 55383 ± 219 T-SD1 282.9 ± 8.7 0.128 ± 0.103 325.6 ± 19.4 100 ± 0 54725 ± 363 T-SD2 267.3 ± 4.9 0.208 ± 0.021 328.3 ± 4.6  100 ± 0 48428 ± 394 T-SD3 292.7 ± 4.9 0.252 ± 0.012 380.4 ± 3.6  100 ± 0 39503 ± 42  T-SD-P  425.2 ± 36.5 0.418 ± 0.050 252.3 ± 15.3  98.3 ± 3.0  1989 ± 197

(86) As a result, slightly increased Z-average and main peak diameters were determined for spray dried samples.

(87) 6.3.4 Nanoparticle Tracking Analysis (NTA)

(88) NTA experiments were carried out with a NanoSight LM20 (NanoSight, Amesbury, UK). The instrument is equipped with a 405 nm blue laser, a sample chamber and a Viton fluoroelastomer O-Ring. The samples were diluted with ultra-pure water in order to achieve suitable concentrations for NTA measurement. After the measurement, all results were normalized to the original concentration.

(89) Samples were loaded into the measurement cell using a 1 ml syringe. The results of the NTA analysis are shown in Table 12 and FIG. 11. Movements of the particles in the samples were recorded as videos for 60 seconds at room temperature using the NTA 2.0 Software. The recorded videos were analyzed with the NTA 2.0 Software.

(90) TABLE-US-00012 TABLE 12 Results from NTA analysis Mean size Mode size D10 size D50 size D90 size Total Concentration Sample [nm] [nm] [nm] [nm] [nm] [#/ml] T0 108 ± 2 96 ± 7 71 ± 1 102 ± 3 150 ± 5 4.74 (±0.16) E+11 T-SD1 109 ± 6 92 ± 9 73 ± 5 102 ± 9 149 ± 5 3.96 (±0.89) E+11 T-SD2 120 ± 3 107 ± 6  83 ± 3 114 ± 5 161 ± 2 5.71 (±0.42) E+11 T-SD3 136 ± 5 116 ± 10 86 ± 4 126 ± 3  197 ± 13 6.04 (±0.59) E+11 T-SD-P  139 ± 30 131 ± 40 117 ± 41  136 ± 33  161 ± 19 8.31 (±9.65) E+09

(91) The particle size values determined for the spray-dried samples were comparable or slightly increased with respect to the control (T0).

(92) 6.3.5 Zeta Potential Measurements

(93) Zeta potential measurements were carried out with a Zetasizer Nano Series instrument (Malvern Instruments, Worcestershire, UK). 750 μl of each formulation were analyzed in disposable folded capillary cells. For each sample, 3 zeta potential measurements consisting of 100 sub-runs were performed and the mean value for zeta potential was calculated. For all samples, a negative zeta potential was determined (see Table 13).

(94) TABLE-US-00013 TABLE 13 Results of Zeta potential measurements Sample Zeta potential [mV] T0 −32.4 T-SD1 −35.8 T-SD2 −36.7 T-SD3 −41.2 T-SD-P −22.5

(95) 6.3.6 Micro-Flow Imaging (MFI)

(96) Micro-Flow Imaging measurements were conducted by using a DPA-5200 particle analyzer system (ProteinSimple, Santa Clara, Calif., USA) equipped with a silane coated high resolution 100 μm flow cell. Samples were analyzed undiluted. In case of excess of the MFI concentrations limits (≥2.5 μm: 900,000 particles/ml, ≥5 μm: 400,000 particles/ml and ≥10 μm: 250,000 particles/ml), samples were diluted before analysis by adding ultrapure water (e.g. Milli-Q water).

(97) For the analysis of the liquid samples, a pre-run volume of 0.17 ml was followed by a sample run of 0.26 ml. Approximately 1100 images were taken per sample. Between measurements, the flow cell was cleaned with water and the background illumination was optimized by using ultrapure water. The MFI View System Software (MVSS) version 2-R2-6.1.20.1915 was used to perform the measurements and the MFI View Analysis Suite (MVAS) software version 1.3.0.1007 was used to analyze the data. The particle counts of the diluted samples were normalized to the original concentration. Significantly increased particle concentrations were determined for the spray-dried samples including placebo (see Table 14), pointing towards particle contamination.

(98) TABLE-US-00014 TABLE 14 Particle concentration as determined by MFI Particle concentration [#/ml] Sample ≥1 μm ≥2 μm ≥10 μm ≥25 μm T0 12,509 4,115 203 11 T-SD1 174,199 32,426 218 4 T-SD2 128,351 27,151 289 18 T-SD3 139,397 28,440 225 7 T-SD-P 183,275 35,053 86 0