Spray freeze drying formation of dry powder compositions

Abstract

The present invention relates to dry powder compositions, methods, apparatus and systems for manufacture of the same. In particular, the present invention relates to a system for producing a dry powder composition including a vacuum chamber depressurised by a vacuum pump and a spray head assembly configured to introduce a liquid formulation into the vacuum chamber, wherein the spray head assembly is ultrasonically stimulated during introduction of the liquid formulation into the vacuum chamber to initiate breakup of the liquid formulation into liquid droplets within the vacuum chamber. The present invention further relates to dry powder compositions comprising lyophilised aggregates produced with such apparatus.

Description

[0001] SPRAY FREEZE DRYING FORMATION OF DRY POWDER COMPOSITIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to dry powder compositions, methods, apparatus and systems for manufacture of the same. In particular, the present invention relates to spray freeze-drying liquid formulations to form dry powder compositions suitable for long term storage without freezing, and dry powder compositions suitable for drug delivery, including pulmonary delivery.

[0004] BACKGROUND

[0005] The success of messenger RNA (mRNA) vaccines for Covid-19 has shown that not only does mRNA enable rapid product development and manufacturing but also that the resulting vaccines can be more than 90% effective. Once transported into the patient’s cells, usually in lipid nanoparticles (LNP), the mRNA can act as a template to produce an extremely wide range of proteins including vaccine antigens, therapeutic agents to replace missing or mutated proteins, or CRISPR associated proteins for gene editing (Nature Rev. Drug Discovery 2021 , 20, 735, Nature Biotechnology 2022, 40, 1586). This gives a powerful platform for the development of vaccines and personalised drugs, as attested by the large and growing product development pipelines of mRNA companies (Nature Biotechnology 2022, 40, 840).

[0006] However, a key bottleneck for the licensed mRNA-LNP vaccines is the requirement of ultra-cold temperatures (-80C to -20C) for their storage and distribution (Pharmaceutics 2022, 14, 430).

[0007] Systems capable of maintaining these ultra-cold temperatures incur significant costs in their storage and transportation, need for continuous temperature monitoring, and the trained personnel necessary to operate them. Furthermore, poor temperature control and maintenance may lead to reduced vaccine potency or wastage. Such ultra-cold systems also typically incur a large carbon footprint. Thus, there exists a need for formulations of mRNA-LNP vaccines stable above 0C, for example at refrigerated temperatures of around 2C to 8C or preferably up to 25C, and more preferably up to 40C (Nature Biotechnology 2023, https: / / doi.Org / 10.1038 / S41587-023-01774-z).

[0008] Additionally, inhaled pulmonary delivery of mRNA-LNP is highly sought after for treatment of lung diseases such as asthma, idiopathic pulmonary fibrosis, lung cancer and a range of pulmonary infections (Nature Biotechnology 2020, 38, 1110).

[0009] Pulmonary delivery, however, requires particles or aerosols with mass median aerodynamic diameters (MMAD) between 1 pm and 5pm, as these particles exhibit the highest deposition in the central and peripheral airways inside lungs which leads to sedimentation and subsequent absorption of the particles (Curr Opin Biotechnol. 2022, 74:104). However, particles having a greater mass median aerodynamic diameter may nevertheless be suitable for other forms of delivery such as intranasal delivery.

[0010] Thus, it is highly desirable to develop effective methods of making mRNA-LNP formulations stable above 0C, for example at refrigerated temperatures of 2C to 8C or preferably up to 25C, and more preferably up to 40C, wherein these mRNA- LNP formulations preferably have well-controlled particle sizes, which may be suited to direct lung delivery.

[0011] The method used to make room-temperature stable mRNA-LNP formulations is also beneficial for other formulations, including biologies, cells, and fragile molecules.

[0012] Commonly, freeze-drying (lyophilisation) and spray-drying have been employed to make mRNA-LNP formulations stable above 0C. Some deficiencies of these methods, compared to those of the present invention, are set out below.

[0013] Freeze-drying may be carried out by conventional lyophilization in vials or by spray-freeze drying (SFD). In conventional lyophilisation, vials of mRNA-LNP formulations are solidified by freezing. Solvent in this vial is then removed from the solidified formulation by sublimation to generate a dry powder (Mol Ther Nucleic Acids. 2022 13;30:226). This dry powder may be reconstituted to form a liquid mRNA-LNP formulation before administration.

[0014] This conventional approach, however, is a lengthy process (taking between 40 and 100 hours) and is known to deform LNP structures leading to LNP breakage and aggregation, or mRNA damage or leakage and consequently resulting in reduced delivery efficacy (Molecular Therapy 2022, 3, 1941 ; Cell Discovery (2023) 9:9; Bioactive Materials 5 (2020) 358).

[0015] For the reconstituted liquid mRNA-LNP formulation to be suitable for lung delivery, it may be atomised and inhaled, typically using a high cost nebuliser. The nebulisation of mRNA-LNP dispersions is also known to disrupt the structure of the LNPs (Nature Biomedical Engineering 2021 , 5, 1059).

[0016] In spray-freeze drying, liquid mRNA-LNP formulations are sprayed as microdroplets into a cold medium (fluid or mist or cold air) (Int J Pharm 2017516(1- 2): 170-177, PCT Publication No. WO 2023 / 057444A1 ) or on a cold surface (US Application No. US 2022-0023204A1 ) to quickly freeze. These frozen microparticles are then dried under vacuum (via sublimation) to make dry powder compositions. However, these methods typically create powder consisting of large particles (typically with geometric diameters greater than >100 pm) or non- spherical, irregularly shaped particles that are not suitable for efficient inhaled lung delivery.

[0017] Additionally, methods which cool droplets by contact with cold gas, liquid, or solid surfaces provide limited cooling rate due to the time required for heat to diffuse to the liquid droplets being cooled. Faster cooling is beneficial to minimise damage due to ice crystal formation and to ensure freezing occurs before the droplet impacts on a collection surface or chamber wall. Methods which generate sprays using random break-up of the liquid feed solution generate droplets with a broad range of sizes, undesirably generating particles with a broad range of sizes and broad range of requirements for cooling and drying time. Larger droplets will require longer freezing and drying times, which in turn require larger chambers and longer process duration. Smaller droplets may generate particles with undesirable properties, such electrostatic attraction to surfaces, powder handling difficulties, and inhalation risk for particles not intended for inhalation. Certain existing spray technologies generate a broad range of droplet sizes with span > 1. These include pressurised jet nozzles, impinging jet nozzles, two-fluid nozzles, centrifugal atomisers, ultrasonic nozzles (such as described in US patent 4,723,708) and other spray technologies where the liquid droplet formation is not precisely controlled and periodic.

[0018] In addition, the spray-freeze drying process requires a constant supply of cryogenic media to freeze the particles, the separation of the frozen particles from said cryogenic media, and the transfer of particles from the freezing process area to a lyophilisation process area (typically under vacuum).

[0019] Additionally, these methods are in their infancy for use with mRNA formulations and are not proven to scale up effectively, nor is it proven whether they are suitable for deployment in GMP-compliant manufacturing.

[0020] The alternative method of spray drying involves atomisation of a solution or dispersion into droplets followed by rapid solvent evaporation in a drying (heated) gas medium (Journal of Controlled Release 2023, 363, 389-401). This method is similarly deficient in that it often results in a dry powder of non-uniform particles with altered sizes (generally increased) and altered structure of LNPs which is detrimental to the transfection performance of LNPs (Journal of Controlled Release 2023, 363, 389-401 & Drug Deliv. and Transl. Res. 2023, https: / / doi.Org / 10.1007 / s13346-023-01402-y).

[0021] As discussed, previous methods to make mRNA-LNP dry powder formulations stable above 0°C can be unsuitable, as they may damage the LNPs, leading to reduced or diminished efficacy. Further, these methods produce compositions which are unsuitable for pulmonary delivery.

[0022] The deficiencies of these current methods highlight the need for a method and related systems and apparatus for freeze-drying mRNA-LNP to produce a dry powder composition of LNPs that is stable during storage and transportation at temperatures above 0C, for example at refrigerated temperatures of around (2C to 8C or preferably up to 25C, and more preferably up to 40C.

[0023] The temperature stability of LNPs is directly related to their moisture content, with particles of a low moisture content generally being more stable at high temperatures. Therefore, there is a need for a method and related systems and apparatus for freeze-drying LNPs to produce a dry powder composition of mRNA- LNPs with an aggregate moisture content by mass of less than 2%, and preferably less than 1 %, and more preferably less than 0.5%.

[0024] There is a need for a method and related systems and apparatus for freeze-drying mRNA-LNPs which has a reduced negative impact on the LNPs structure or stability.

[0025] There is a need for a method and related systems and apparatus for freeze-drying mRNA-LNPs having a reduced effect on LNPs aggregation and their size distribution.

[0026] There is a need for a method and related systems and apparatus for freeze-drying mRNA-LNP which has a reduced effect on the encapsulation rate of agents encapsulated in LNPs.

[0027] There is a need for a method and related systems and apparatus for freeze-drying mRNA-LNP having no or reduced effect on protein expression from the mRNA.

[0028] There is a need for a method and related systems and apparatus for freeze-drying mRNA-LNPs to produce a dry powder composition of LNPs suitable for inhaled lung delivery. There is a need for a dry powder composition of mRNA-LNP that is suitable for storage up to 6 months, or preferably up to 1 year or more preferably up to 2 years at temperatures above OC, for example refrigerated temperatures of around 2C to 8C) or preferably up to 25C or more preferably up to 40C.

[0029] There is a need for a dry powder composition of mRNA-LNPs and other formulations that consists of substantially spherical particles of appropriate sizes suitable for inhaled delivery into lungs. Preferably, said particles have volume- weighted median geometric diameters between 2pm and 100pm, and more preferably between 2pm and 25pm.

[0030] SUMMARY OF INVENTION

[0031] According to the present invention, there may be a system for producing a dry powder composition, the system comprising: a vacuum chamber configured to be depressurised by a vacuum pump connected thereto; and a spray head assembly sealably mounted to the vacuum chamber and configured to introduce a liquid formulation into the vacuum chamber; wherein the spray head assembly is further configured to be ultrasonically stimulated during introduction of the liquid formulation into the vacuum chamber, whereby to initiate breakup of the liquid formulation into liquid droplets within the vacuum chamber.

[0032] The system may further comprise a tray for receiving frozen droplets thereupon, the tray being disposed inside the vacuum chamber and having a surface that can be temperature controlled to maintain a surface temperature between -50C and 20C, preferably wherein the surface may be controlled to sequentially maintain more than one surface temperature.

[0033] The system may further comprise a cold trap located inside the vacuum chamber, the cold trap being configured to reduce the water vapour partial pressure inside the vacuum chamber.

[0034] In some embodiments, said vacuum chamber is a first vacuum chamber, and the system may further comprise a second vacuum chamber maintained a pressure lower than the first vacuum chamber. The spray head assembly may be configured to produce electrostatically charged liquid droplets.

[0035] The system may further comprise a flexible container disposed in the vacuum chamber for receiving solidified particles therewithin, preferably wherein the container comprises a gas-permeable portion configured to permit gas flow out of the container.

[0036] The container may comprise a thermoplastic material, and the system may further comprise a heat-sealing device disposed in the vacuum chamber, configured to heat-seal the container.

[0037] The cold trap may comprise one or more coils configured to reduce the water vapour partial pressure inside the vacuum chamber via the deposition of water vapour from the vacuum chamber onto one or more surfaces of the cold trap. The one or more surfaces of the cold trap may be configured to be temperature controlled to maintain a surface temperature of -35C and below and preferably between -50C and -80C.

[0038] In some embodiments, the system may comprise a first vacuum chamberfor liquid droplets to freeze within, thereby forming solidified particles; and a second vacuum chamber provided for removal of ice from the solidified particles by sublimation. Preferably, the second vacuum chamber may be maintained at a pressure lower than the first vacuum chamber.

[0039] The system may further comprise a first re-pressurisation valve in fluid communication with the vacuum chamber, the valve being configured to fluidly couple to an external gas supply whereby to repressurise the vacuum chamber.

[0040] In embodiments having two vacuum chambers, the system may further comprise a second re-pressurisation valve in fluid communication with the second vacuum chamber.

[0041] The spray head assembly is preferably configured to receive a pressurised flow of liquid formulation via a controllable valve and may be configured to receive the liquid formulation from a syringe coupled to a syringe pump, preferably via a controllable valve.

[0042] The spray head assembly may be configured to receive a pressurise flow of inert gas and introduce it into the vacuum chamber, for example via a controllable valve and may further be configured to receive a pressurised flow of deionised water and introduce it into the vacuum chamber, for example via a controllable valve.

[0043] The system may comprise an electrostatically charged surface positioned within the vacuum chamber remotely from the spray head assembly, the charged surface having an electrostatic charge opposite to that of the electrostatically charged liquid droplets whereby to attract the charged liquid droplets to it.

[0044] The system may further comprise one or more filtration devices disposed upstream of the spray head assembly such that any fluid being supplied to the spray head assembly passes through the filtration device before entering the spray head assembly.

[0045] The system may further comprise a deflector disposed within the vacuum chamber and arranged to deflect frozen droplets that impinge onto an initial impact zone within the vacuum chamber away from said initial impact zone towards a collection zone within the vacuum chamber.

[0046] The flexible container may further comprise a weakened portion of material formed circumferentially around an outer surface of the container to promote opening of the container at the weakened portion, preferably wherein the heat sealing device is arranged to seal the container below said weakened portion, preferably substantially adjacent to the weakened portion.

[0047] According to another aspect of the invention, there is a method of producing a dry powder composition of lyophilised particles from a liquid formulation, the method comprising the steps of: introducing a liquid formulation into a depressurised vacuum chamber; stimulating the liquid formulation ultrasonically while introducing it into the vacuum chamber so as to initiate breakup of the liquid formulation into liquid droplets; freezing the liquid droplets within the vacuum chamber by means of evaporative cooling to form solidified particles; and sublimating water molecules out of the solidified particles whereby to form the dry powder composition.

[0048] Sublimating the solidified particles may comprise controlling their temperature using a temperature controlled surface inside the vacuum chamber.

[0049] The solidified particles may be sublimated until they have a moisture content by mass of less than 2%, preferably less than 1%, and more preferably less than 0.5%.

[0050] The method may further comprise introducing, via a nozzle, the liquid formulation into the vacuum chamber as a capillary jet having a liquid jet velocity of between 1m / s and 200m / s, such as between 3m / s and 200m / s and preferably between 38m / s m / s and 113m / s. In some embodiments, the capillary jet having a liquid jet velocity of between 1 m / s and 50m / s.

[0051] The method may further comprise ultrasonically stimulating the capillary jet at a frequency which matches a predetermined growth rate instability mode for Rayleigh breakup of the capillary jet.

[0052] The method may further comprise flowing inert gas surrounding the capillary jet whereby to accelerate the capillary jet.

[0053] The method may further comprise moving the nozzle in a cyclic fashion while introducing the liquid formulation such that the solidified particles are received at multiple locations on a surface within the vacuum chamber.

[0054] The method may further comprise using electrostatic potential to disperse the liquid droplets within the vacuum chamber as they are initially introduced.

[0055] The temperature of the particles may be controlled by maintaining the solidified particles at a temperature below a collapse temperature of the liquid formulation under low pressure conditions until an expected amount of sublimation has occurred; and then heating the solidified particles to a temperature above the collapse temperature, whereby to increase the rate of sublimation. Preferably the temperature of the solidified particles may range between -50C and 20C. The liquid formulation may be ultrasonically stimulated at a frequency of between 0.006MHz and 89MHz, preferably at a frequency between 0.28MHz and 8.9MHz. In some embodiments the liquid formulation may be ultrasonically stimulated at a frequency of between 30kHz and 2500kHz, such as between 50kHz and 600kHz.

[0056] Preferably, the ultrasonic stimulation frequency growth rate instability mode which initiates the break-up of the liquid formulation into liquid droplets is the largest growth rate instability mode.

[0057] The vacuum chamber may be maintained at a pressure of less than 25 Pa, preferably less than 10 Pa, and more preferably less than 6 Pa prior to introducing the liquid formulation.

[0058] The pressure of the vacuum chamber is preferably maintained at or below a pressure that causes the liquid droplets with diameters in the range of 1.5pm to 100pm to cool from approximately 20C to -30C with cooling rates in the range of 1 x108C / s to 2*104C / S and preferably to cause liquid droplets with diameters in the range of 10pm to 50pm to cool from approximately 20C to -30C with cooling rates in the range of 2.4x106C / s to 9.5x104C / s.

[0059] The method may further comprise introducing inert dry gas, then deionised water via the nozzle into the vacuum chamber before introducing the liquid formulation via the same nozzle.

[0060] The deionised water may be introduced via the nozzle as a capillary jet and ultrasonically stimulated to initiate breakup into water droplets before introducing the liquid formulation.

[0061] The method may further comprise introducing deionised water and then inert dry gas through the nozzle after introducing the liquid formulation. The method may further comprise deflecting the lyophilised particles away from an initial contact surface within the vacuum chamber towards a collection zone within the vacuum chamber.

[0062] The method may further comprise repressuring the vacuum chamber using an inert gas prior to retrieving the dry powder composition.

[0063] The method may further comprise introducing the liquid formulation into a flexible container disposed in the vacuum chamber and sealing the container within the vacuum chamber prior to re-pressurising the vacuum chamber.

[0064] The liquid formulation may be introduced into the vacuum chamber via a nozzle having at least one aperture with a diameter between 1 pm and 50pm.

[0065] According to another aspect of the invention, there is a dry powder composition which is obtainable by any of the methods described herein. Said composition may optionally have any of the properties of the dry powder compositions described below.

[0066] According to another aspect, the present invention provides a dry powder composition comprising lyophilised aggregates of nanoparticulate drug particles, wherein the aggregates have a volume-weighted median geometric diameter in the range 2 pm to 100 pm.

[0067] More preferably the aggregates have a volume-weighted median geometric diameter in the range 2 to 50 pm, yet more preferably 2 to 25 pm.

[0068] Said aggregates preferably also have a geometric diameter size distribution with a span of less than 1 , preferably less than 0.2.

[0069] The dry powder composition described above can have a moisture content by mass of less than 2%, preferably less than 1 %, more preferably less than 0.5%. In another aspect, the present invention provides a dry powder composition comprising lyophilised aggregates of nanoparticulate drug particles wherein the particles have a moisture content by mass of less than 0.5%.

[0070] In any of the dry powder compositions described above the nanoparticle drugs particles may be lipid nanoparticles.

[0071] Preferably the lipid nanoparticles comprise encapsulated nucleic acids.

[0072] Examples of suitable nucleic acids include messenger RNA (mRNA), microRNA (miRNA), short (or small) interference RNA (siRNA), small hairpin RNA (shRNA), long non-coding RNA (IncRNA), asymmetrical interfering RNA (aiRNA), selfamplifying RNA (saRNA), a self-replicating RNA (srRNA), a circular RNA (cRNA) or endless RNA (eRNA), a guide RNA (gRNA); or combinations thereof. In preferred embodiments, the RNA is mRNA.

[0073] In any of the dry powder compositions described above the aggregates may be substantially spherical and have an aspect ratio of the largest diameter to the smallest diameter which is less than 1.1. In some embodiments, the aggregates may have aspect ratios of the largest diameter to the smallest diameter which are less than 1.05.

[0074] The dry powder compositions described above may be suitable for pulmonary delivery. The dry powder compositions may be suitable for use in a dry powder inhaler.

[0075] In any of the dry powder compositions described above, the aggregates may have a mass median aerodynamic diameter in the range 1 to 50 pm, such as 1 to 5 pm or 10 to 50 pm.

[0076] In any of the dry powder compositions described above, the aggregates may have a mass median aerodynamic diameter in the range 1 to 5 pm. In any of the dry powder compositions described above the mass aerodynamic diameter distribution of the aggregates in the composition may have a span of less than 1 , preferably less than 0.2.

[0077] In any of the dry powder compositions described above the aggregates may have a porosity of 60% to 95%.

[0078] In any of the dry powder compositions described above the aggregates may have a density between 0.05g / cm3and 0.4g / cm3.

[0079] Dry powder compositions described above having aggregates with a density between 0.05g / cm3and 0.2g / cm3, such as around 0.1g / cm3, may be particularly suitable for pulmonary delivery.

[0080] Dry powder compositions described above having aggregates with a density between 0.2g / cm3and 0.4g / cm3, such as around 0.3g / cm3, may be particularly suitable for nasal delivery.

[0081] In any of the dry powder compositions described above the size distribution of the aggregates may have a span of less than 1 , preferably less than 0.5, more preferably less than 0.2.

[0082] The method used to make, for instance, room-temperature stable mRNA-LNP formulations is also beneficial for other formulations, including biologies, cells, and fragile molecules.

[0083] Thus, lyophilised particles comprising biologies, cells and / or fragile molecules, having any of the properties discussed above in relation to lyophilised aggregates of nanoparticulate drug particles, may also be advantageously produced.

[0084] The dry powder compositions described above may be obtained by the methods described herein.

[0085] Also disclosed herein is a spray head assembly for introducing a liquid formulation into a vacuum chamber, the assembly comprising: a first sub-assembly comprising an ultrasonic drive device; and a second sub-assembly comprising a nozzle configured to introduce fluid into the vacuum chamber; wherein the ultrasonic drive device of the first sub-assembly is configured to stimulate the nozzle of the second sub-assembly, when attached thereto, whereby the ultrasonic stimulation of the nozzle initiates breakup of the liquid formulation into liquid droplets as it is introduced into the vacuum chamber.

[0086] The first sub-assembly may be configured such that it is not in contact with the fluid path of the liquid formulation.

[0087] The nozzle may be configured to be heated whereby to inhibit freezing of any liquid formulation within the spray head assembly.

[0088] The nozzle may be configured to introduce the liquid formulation into the vacuum chamber as a capillary jet having a velocity between 1m / s and 200m / s, such as between 3m / s and 200m / s and preferably between 38m / s and 113m / s. In some embodiments, the capillary jet may have a velocity of between 1m / s and 50m / s, The spray head may further be configured to electrostatically charge the liquid formulation and thereby produce electrostatically charged liquid droplets within the vacuum chamber.

[0089] The nozzle may comprise at least one fluid aperture having a diameter between 1 pm and 50 pm.

[0090] Also disclosed herein are spray caps for use as the second sub-assembly of the spray head disclosed herein.

[0091] In a first aspect, the spray cap may comprise a micronozzle array including: a fluid contact layer having at least one group of apertures formed therein, the fluid contact layer arranged to receive pressurised liquid formulation; and a support layer having at least one aperture that is aligned with and encompasses or surrounds all of the apertures in a group of apertures wherein the support layer is arranged to be exposed to an interior of the vacuum chamber and wherein the fluid contact layer is thinner than the support layer. In a second aspect, the spray cap may comprise a nozzle configured to form a capillary jet of the liquid formulation and further to introduce a flow of gas that substantially surrounds said capillary jet, whereby to accelerate the capillary jet and reduce the diameter of the capillary jet.

[0092] The spray cap may further comprise: a nozzle plate having at least one aperture; a capping plate having at least one aperture; a gas chamber formed between the nozzle plate and capping plate; a skimmer plate having at least one aperture, wherein the apertures of the nozzle plate, capping plate and skimmer plate are all axially aligned; a skimmer chamber formed between the capping plate and the skimmer plate; and a vacuum pump in fluid communication with the skimmer chamber, wherein: the nozzle plate is configured to introduce the capillary jet of liquid formulation into the gas chamber; the gas chamber is configured to introduce the flow of gas substantially surrounding said capillary jet and to accelerate the capillary jet through the aperture in the capping plate, into the skimmer chamber, and towards the aperture in the skimmer plate; and the skimmer chamber is configured to vent via the vacuum pump a substantial amount of the gas in the surrounding gas flow thereby to prevent the surrounding gas flow from entering the vacuum chamber via the at least one aperture in the skimmer plate and re-pressurise the vacuum chamber.

[0093] In a third aspect, a spray cap may comprise: a fluid conduit having an inlet and outlet; a housing having a first opening and a second opening, the second opening configured to receive the outlet of the fluid conduit, wherein the housing is configured to interface mechanically with an ultrasonic drive device; a nozzle plate having at least one aperture, wherein the nozzle plate is disposed in the housing, the aperture being in alignment with the first opening of the housing, a nozzle cap force transfer component configured to apply a force to the nozzle plate, such that contact is maintained between the nozzle plate and a portion of the housing; wherein the spray cap is configured to form a sealed fluid path between the inlet of the fluid conduit and the nozzle plate.

[0094] The fluid conduit may comprise microfluidic tubing with an internal diameter of between 100pm and 2.5mm. The spray cap may further comprise an annular ferrule disposed about the fluid conduit, with a distal end of the fluid conduit extending beyond said ferrule, wherein the ferrule is configured to form a substantially fluid-tight seal about the fluid conduit to inhibit fluid flow along an outer surface of said conduit.

[0095] The force transfer component may have an annular shape with a bore through its centre within which at least a portion of the fluid conduit may be retained, and further comprising first and second annular surfaces, the first annular surface abutting an annular surface of the ferrule and configured to form a fluid-tight seal between the force transfer component and the ferrule.

[0096] The spray cap may further comprise a sealing ring disposed between the force transfer component and the nozzle plate and defining a cylindrical chamber therebetween, the fluid path from the fluid conduit outlet to the nozzle plate being formed through said cylindrical chamber, wherein the sealing ring, when compressed by a set distance, is configured to form a seal between the force transfer component and the nozzle plate. A force may be applied to the nozzle plate to maintain contact between the nozzle plate and the housing.

[0097] The spray cap may further be configured to be separably fastened to a casing via a fastening feature located proximal to the second opening of the housing. For example, the casing may contain an ultrasonic drive device, wherein the fluid path is sealed within the apparatus and does not contact the casing or ultrasonic drive device.

[0098] The fastening feature may comprise a threaded interior surface configured to interface with a threaded exterior surface of the casing, wherein at least a portion of the casing is retained within the housing.

[0099] The ferrule may have a tapered surface, wherein the tapered surface tapers radially inwards in the axial direction towards the second opening of the housing, whereby to abut an opposing tapered surface of the casing when the housing is fastened to the casing. The tapered surface of the ferrule and the tapered surface of the casing may interface to transfer a fastening force to the nozzle plate, thereby maintaining contact between the nozzle plate and the housing.

[0100] It will be understood by a skilled person that any apparatus feature described herein may be provided as a method feature, and vice versa. It will also be understood that particular combinations of the various features described and defined in any aspects described herein can be implemented and / or supplied and / or used independently.

[0101] Moreover, it will be understood that the present invention is described herein purely by way of example, and modifications of detail can be made within the scope of the invention. Furthermore, as used herein, any “means plus function” features may be expressed alternatively in terms of their corresponding structure.

[0102] BRIEF DESCRIPTION OF DRAWINGS

[0103] FIG 1a is a flow diagram detailing an exemplary use-case of the disclosed method and system, with reference to upstream drug manufacturing steps and downstream drug delivery steps.

[0104] FIG 1 b is a flow diagram further detailing processes that the liquids droplets undergo, including formation, solidifying and drying steps.

[0105] FIG 2 is a flow diagram detailing a traditional lyophilisation process with reference to downstream therapeutic steps.

[0106] FIG 3 is a flow diagram detailing the freeze-drying process of Figure 1 , further depicting downstream therapeutic steps.

[0107] FIG 4 shows an embodiment of a freeze-drying system disclosed herein.

[0108] FIG 5a shows another embodiment of a freeze-drying system disclosed herein.

[0109] FIG 5b shows yet another embodiment of a freeze-drying system disclosed herein. FIG 6 shows yet another embodiment of a freeze-drying system disclosed herein, further comprising guiding electrostatic elements.

[0110] FIG 7 shows another embodiment of a freeze-drying system disclosed herein, configured to package the dry powder composition within the system.

[0111] FIG 8a shows an embodiment of a spray head assembly, suitable for use with the system of Figures 4 to 7.

[0112] FIG 8b shows a spray cap suitable for use with the spray head assembly of Figure 8a.

[0113] FIG 9a is a plan view of a microarray nozzle according to another embodiment of the present invention.

[0114] FIG 9b shows a section view of the microarray nozzle of Figure 9a.

[0115] FIG 10 shows a surrounding gas flow focusing nozzle with skimmer according to another embodiment of the present invention.

[0116] FIG 11a is a chart showing the relationship between particle geometric diameter and particle density for inhalable particles having different aerodynamic diameters.

[0117] FIG 11 b is a chart showing the relationship between nozzle diameter and particle density for inhalable particles having different aerodynamic diameters.

[0118] FIG 11c is a chart showing the relationship between particle geometric diameter and particle density for particles suitable for intranasal delivery having different aerodynamic diameters.

[0119] FIG 11 d is a chart showing the relationship between nozzle diameter and particle density for particles suitable for intranasal delivery having different aerodynamic diameters. FIG 12a is a chart showing the relationship between the minimum ejection velocity from a nozzle required to form stable droplets and the aerodynamic diameter (1- 5 pm) of solidified particles for solidified particles formed from liquid formulations having different solid contents.

[0120] FIG 12b is a chart showing the relationship between the minimum ejection velocity from a nozzle required to form stable droplets and the aerodynamic diameter (1 - 100 pm) of solidified particles formed from liquid formulations having different solid contents.

[0121] FIG 13 is a chart showing the expected relationship between droplet temperature and time for various droplet diameters using a diffusion-controlled evaporation model.

[0122] FIG 14a is a chart showing droplet cooling times against droplet diameter using a thermal conduction model.

[0123] FIG 14b is table showing cooling times and cooling rates for droplets with diameters in the range of 1.5pm to 100pm.

[0124] FIG 15 is a chart showing saturation vapour pressures against water or ice temperature.

[0125] FIG 16a is a chart showing exemplar jet velocity lower limits, upper limits and preferred values for the generation of particles having aerodynamic diameters in the range of 1 pm to 100pm from liquid formulations having a solid content of 5% by weight(g) / volume(cm3) in order to form solid particles with a corresponding density of 0.05 g / cm3.

[0126] FIG 16b is a chart showing the same as FIG 16a for liquid formulations having a solid content of 10%.

[0127] FIG 16c is a chart showing the same as FIG 16a for liquid formulations having a solid content of 20%. FIG 16d is a chart showing the same as FIG 16a for liquid formulations having a solid content of 40%.

[0128] FIG 16e is a table showing some exemplar operating conditions for the generation of particles having aerodynamic diameters in the range of 1 pm to 100pm from liquid formulations having solid contents in the range of 0.05g / cm3to 0.4g / cm3.

[0129] FIG 16f is table showing example operating conditions for production of particles for inhaled and nasal delivery.

[0130] FIG 17a is an image of droplets of water generated using a 25pm diameter nozzle.

[0131] FIG 17b is a chart showing measured relationship between droplet velocity and nozzle vibration frequencies used to generate stable periodic droplets for different feed flow rates of water through a 25pm nozzle.

[0132] FIG 17c is a chart showing measured relationship between droplet velocity and feed flow rates of water through a 25pm nozzle.

[0133] FIG 17d is a chart showing measured relationship between droplet diameter and nozzle vibration frequency for different feed flow rates of water through a 25pm nozzle.

[0134] FIG 17e is a chart showing the measured relationship between droplet velocity and feed flow rates of water through a 20pm nozzle.

[0135] FIG 17f is a chart showing the measured relationship between droplet diameter and nozzle vibration frequency for different feed flow rates of water through a 20pm nozzle.

[0136] FIG 17g is a chart showing measured relationship between droplet velocity and feed flow rates of 10% trehalose solution through a 20pm nozzle.

[0137] FIG 17h is a chart showing measured relationship between droplet diameter and nozzle vibration frequency for different feed flow rates of 10% trehalose solution through a 20pm nozzle.

[0138] FIG 18 is a diagram of an exemplary dry particle composition, including a representation of a lyophilised particle. FIG 19a is a light microscope image of particles in a dry powder sample obtained after drying of 10% trehalose solution in 50mM Tris-HCI buffer pH 7.4.

[0139] FIG 19b is a line drawing representation of the microscope image of Figure 19a.

[0140] FIG 19c is a chart showing particle size distribution measured by analysing the sizes of 50 randomly chosen particles from the image in Figure 19a.

[0141] FIG 19d is a table showing various calculated particle characteristics for the 50 randomly chosen particles in Figure 19b.

[0142] FIG 20a shows some exemplar nozzle geometry used for fluidic simulation shear stress calculations.

[0143] FIG 20b shows the axisymmetric fluidic simulation domain in the context of the nozzle geometry of Figure 20a.

[0144] FIGs 20c, 20d and 20e represent simulations of shear stress due to flow through nozzles having diameters of 2pm, 5pm and 10pm respectively, with a jet velocity of 40 m / s.

[0145] FIG 20f represents simulations of shear stress due to flow through nozzle diameters in the range of 2 to 20pm with jet velocities equal to the upper velocity limit corresponding to a shear stress limit of 100 kPa.

[0146] FIG 20g is a chart showing the relationship between the velocity limits and the nozzle diameters shown in Figure 20f.

[0147] FIG 21 shows characterisation results of Fluc-mRNA-LNP (a firefly luciferase (Flue) encoding mRNA encapsulated inside LNP made of SM102 ionisable lipids) before (dotted bars) and after (solid bars) spray through the droplet generator at atmospheric pressure.

[0148] FIG 21a is a dynamic light scattering (DLS) size measurement of LNPs dispersed in 2% trehalose solution in 50mM Tris-HCI buffer pH 7.4.

[0149] FIG 21 b is a DLS polydispersity index (PDI) measurement of LNPs dispersed in 2% trehalose solution in 50mM Tris-HCI buffer pH 7.4.

[0150] FIG 21c is a mRNA encapsulation efficiency measurement of LNPs using Ribogreen assay. FIG 21 d is a protein (Flue) expression measurement (bars) in HeLa cells. % cell viability is a control that indicates number of viable cells

[0151] DETAILED DESCRIPTION

[0152] FIG 1a is a flow diagram detailing an exemplary use case of the apparatus and method disclosed herein, with reference to upstream and downstream steps. Dashed lines mark out steps which may comprise the essential components of the ultra-fast freeze-drying (UFFD) process 113 according to the present invention but it will be appreciated that the UFFD process 113 may comprise further steps which lie outside the dashed region or which are not depicted in Figure 1 at all.

[0153] According to this example, a nano-particulate formulation of an active drug substance 100 may be provided as an input to the UFFD process 113 and combined with excipients 101 to form a liquid formulation 102.

[0154] A nano-particle formulation of an active drug substance 100 may refer to an active therapeutic substance supplied in a nanoparticle format. For example, lipid nanoparticle encapsulated mRNA.

[0155] The combination of the nano-particulate formulation 100 with one or more excipients 101 may happen within the UFFD process 113 or it may occur upstream, with the resulting liquid formulation 102 being provided as the input to the UFFD process 113.

[0156] In one stage of the UFFD process 113, the liquid formulation 102 undergoes droplet formation 109 to form liquid droplets 103 of the liquid formulation 102.

[0157] The liquid droplets 103 then undergo droplet solidification 110 by means of evaporative cooling within a vacuum chamber to form solidified particles 104.

[0158] These solidified particles 104 then dry via particle sublimation 111 , the water content of the solidified particles 104 subliming out as water vapour, thereby resulting in a dry powder composition 105 consisting of lyophilised microparticles. The dry powder composition 105 undergoes a packaging 112 step, which may occur as a part of the UFFD process 113 or may form a step downstream of said process 113.

[0159] As a result of its low moisture content, the packaged dry powder composition may be suitable for storage or transportation at temperatures above 0C, such as refrigerated temperatures of between 2C and 8C, or preferably up to 25C, or up to 40C.

[0160] In some embodiments, the dry powder composition 105 may be suitable for administration via, for example, nasal delivery 122, pulmonary delivery 106, transdermal delivery 107, or liquid reconstitution 108.

[0161] Pulmonary delivery 106 and nasal delivery 122 generally refer to delivery of the drug product directly as a dry powder composition 105. Pulmonary delivery is typically administered directly to the lungs, for example, by using the dry powder composition 105 to fill a dry powder inhaler. Nasal delivery 122 is administered into the nasal cavity, which also might be as a dry powder. Nasal delivery 122 might also be performed by reconstituting the dry powder composition 105 as a liquid formulation and administering this liquid with a nasal spray, for example. Transdermal delivery 107 may refer to administering the dry powder composition 105 by way of micro-needle fabrication. Liquid reconstitution 108 refers to any method of administering a liquid drug product, by injection 315, nasal delivery 322 or otherwise.

[0162] FIG 1 b is a flow diagram further detailing processes that the liquid droplets 103 undergo, from their formation through to a final drying step, as marked out by the dashed line 120.

[0163] Liquid formulation 102 is passed through a spray head assembly and may therein form a liquid jet 114. This liquid jet formation 114 may occur within the spray head assembly, or as the liquid formulation 102 is ejected from the spray head assembly (thereby forming the jet upon ejection).

[0164] This liquid jet subsequently undergoes a breakup into a stream of droplets 115. Liquid droplets 103 undergo evaporative cooling 116 within the vacuum chamber and subsequently freeze 117 to form solidified particles 104. These steps of cooling 116 and freezing 117 may form the general process of droplet solidification 110.

[0165] Said solidified particles 104 undergo particle sublimation 111. This may comprise a first stage 118, wherein the particles undergo sublimation at a temperature below the collapse temperature of solutes (mainly excipient) in the liquid formulation 102. This first stage may comprise maintaining a surface in thermal contact with the solidified particles 104 at a temperature below the relevant collapse temperature. For typical excipients this may be in the range of around -50C to -5C. For example, for a sucrose solution, this is around -35C, for lactose around -28C, for trehalose around -29C, for dextrose around -44C, and for gelatine around -9C. Solidified particles may be allowed to dry in the first stage 118 for up to around 240 minutes. For example, the drying time of the first stage 118 may be around 60 to 180 minutes, such as around 120 minutes. In some examples the solidified particles may be allowed to dry in the first stage 118 for up to 20 minutes.

[0166] Particle sublimation 111 may further comprise a second stage 119, wherein the particles may be dried at a temperature above the relevant collapse temperature. Solidified particles may be allowed to dry in the second stage 119 for up to around 240 minutes. For example, the drying time of the second stage 119 may be around 30 to 90 minutes, such as around 60 minutes.

[0167] Dry, lyophilised particles obtained by step 119 may then be subsequently collected and packaged 121.

[0168] FIG 2 is a flow diagram detailing a traditional lyophilisation process 213 with reference to downstream therapeutic steps. A nano-particle dispersion 200 is provided as an input to a traditional lyophilisation process 213. After the nanoparticle dispersion 200 is lyophilised to form a dry powder, the dry powder generally must undergo liquid reconstitution 208 before delivery to a patient. A nano-particle dispersion 200 may refer to any liquid formulation comprising nanoparticles of an active drug substance, of which the liquid formulation 100 of Figure 1 is an example.

[0169] Common delivery methods of the reconstituted nano-particle dispersion include inhalation via a nebuliser 214 or injection 215.

[0170] Traditional lyophilisation 213 is a lengthy process, generally taking approximately 40 to 100 hours to complete. Reconstitution 208 is necessary as traditional lyophilisation 213 fails to produce particles which are suitable for inhalation. This inherently limits the drug delivery routes available, with inhalation from a nebuliser 214 and injection 215 as the primary routes.

[0171] The reconstitution step 208 and the slower lyophilisation itself may damage the drug product as ice crystal formation introduces mechanical forces which may detrimentally deform the structure of the active substance.

[0172] FIG 3 is a flow diagram detailing the ultra-fast freeze-drying (UFFD) process 313 pertaining to this invention, in contrast to the traditional method of Figure 2.

[0173] The main input to the UFFD process herein 313 is a nano-particle dispersion 300, synonymous with the liquid formulation 100 of Figure 1 . After the UFFD process 313 is used to produce a dry powder composition from the nano-particle dispersion 300, the dry powder may either be reconstituted as a liquid 308 or administered directly as a dry powder. The same means of delivery for the reconstituted nanoparticles are available as with traditional lyophilisation 213, namely inhalation via nebuliser 314 and injection 315. Liquid reconstitution 322 may also be followed by nasal delivery 322 by way of a nasal spray, for example.

[0174] The direct dry powder delivery methods may include nasal delivery 322 and pulmonary delivery 306 via, for example, a dry powder inhaler and transdermal delivery 307 with, for example, a micro needle patch.

[0175] These alternative routes, compared to the traditional lyophilisation process 213, are possible due to the controlled nature of the dry powder composition formed from the UFFD process 313. Herein, particle properties such as the excipient used, excipient to nanoparticle ratio, the density, size, shape, and mass median aerodynamic diameter (MMAD) of the particles and the distribution and variance of these properties in aggregate may be well controlled in order to produce particles with a high yield which are suitable for use in nasal delivery 322, pulmonary delivery 306 or transdermal delivery 307, while particles produced via traditional lyophilisation methods 213 may not be.

[0176] Further advantages of the UFFD process 313 include its speed. The first stage 118 of drying may take under 240 minutes. For example, the first stage 118 of drying may take between 60 and 180 minutes, such as around 120 minutes. In some embodiments, the first stage 118 of drying may take up to 20 minutes, and the combined first 118 and second stages 119 may take up to 240 minutes. This speed involves a rapid droplet freezing step, in which droplets may cool from 20C to -5C in under 0.1 s, for example.

[0177] This rapid freezing advantageously may aid in the avoidance or significant minimisation of crystallisation of water in the liquid droplet as it solidifies. This may result in the drug product incurring less damage as it is trapped in low shear, largely amorphous ice particles.

[0178] This rapid freezing is, in part, enabled by the small diameters of the liquid droplets. Smaller droplets have greater surface area to volume ratios, therefore will experience increased rates of evaporative cooling compared to larger droplets.

[0179] FIG 4 is a diagram of a freeze-drying system 417 pertaining to one embodiment of the present invention. The system 417 comprises a vacuum chamber 400 and a spray head assembly 401 which may be mounted to the vacuum chamber 400. The spray head assembly 401 may be sealably mounted in such a way as to prevent gases external to the vacuum chamber 400 from leaking into the vacuum chamber 400 at the interface between the vacuum chamber 400 and the spray head assembly 401.

[0180] According to one method suitable for use with the system 417 of Figure 4, liquid formulation 102 is provided to the spray head assembly 401 and introduced into the vacuum chamber 400 via the spray head assembly 401. The spray head assembly 400 is configured to ultrasonically stimulate the liquid formulation 102 as it is introduced, initiating the breakup of the liquid formulation into liquid droplets 103, thereby promoting droplet formation 109.

[0181] The liquid formulation 102 may initially form a capillary jet and the breakup of this capillary jet into liquid droplets 102 may be driven by Rayleigh breakup. This may comprise ultrasonically stimulating the capillary jet at a frequency which matches a growth rate instability mode for the capillary jet, preferably wherein the growth rate instability mode is the largest growth rate instability mode.

[0182] In some embodiments where the liquid formulation 102 is introduced into the vacuum chamber as a capillary jet, via the spray head assembly, through a nozzle having a diameter D, the jet may be ejected from the spray head assembly with a jetting velocity v and ultrasonically stimulated at a frequency f in the range: and preferably at an optimal stimulation frequency f close to: v = 4.5

[0183] In some embodiments, this stimulation frequency may be in the range of 0.006MHz to 89MHz, and preferably in the range of 0.28MHz to 8.9MHz. In some embodiments the liquid formulation may be ultrasonically stimulated at a frequency of between 30kHz and 2500kHz, such as between 50kHz and 600kHz. In this way, the break-up distance, which refers to the distance covered by a portion of liquid formulation 102 in the vacuum chamber 400 before it breaks up into liquid droplets 103, may be well-controlled. In this embodiment, droplet formation 109 occurs within a region of the vacuum chamber 400 referred to as the droplet formation zone 412. However, in some embodiments droplet formation 109 may occur within the spray head assembly 401 such that the liquid formulation 102 is introduced directly into the vacuum chamber 400 as liquid droplets 103. Preferably the capillary jet may also be, in this manner, encouraged to break up into droplets having well-defined shapes and substantially consistent diameters. The consistent sizing of droplets is advantageous as it results in more homogenous freezing and drying times between droplets and therefore a dry powder composition which, in aggregate, has lower variance in properties between different lyophilised particles.

[0184] This low variance is advantageous as, for example, anomalously large particles may fail to sufficiently dry in a given period and therefore introduce unwanted moisture into the dry powder composition. Anomalously large particles may also be unsuitable for inhalation and, in some cases, they may be unsuitable for nasal delivery also, thus reducing the efficacy of a drug product delivered in this manner.

[0185] It will be appreciated that the ultrasonic stimulation of the liquid formulation 202 may be performed my means other than the mechanical stimulation of the spray head assembly 401 , for example, via pressure wave propagation in the liquid formulation 102.

[0186] The system 417 may comprise a vacuum pump 416 in fluid communication with the vacuum chamber 400 and configured to depressurise the vacuum chamber 400 to pressures as low as 25 Pa and preferably below 10 Pa and more preferably below 6 Pa.

[0187] The pressure of the vacuum chamber 400 will be sufficient to cause the liquid droplets 103 to evaporatively cool 116 and freeze 117. It will therefore be appreciated that the suitable pressure may vary for liquid droplets having different diameters, or for different compositions of liquid formulation 102.

[0188] When the vacuum chamber 400 is in its depressurised state, the liquid droplets 103 will freeze 117 by means of evaporative cooling 116 as water vapour leaves the liquid droplets 103, thereby undergoing droplet solidification 110 to form solidified particles 104. The region of the vacuum chamber 400 in which this occurs is referred to as the droplet freezing zone 413. Preferably the droplet freezing zone 413 is positioned downstream of the droplet formation zone 412, as shown in Figure 4, such that the liquid formulation 102 is broken up into droplets before the onset of freezing. This promotes the formation of substantially spherical droplets (e.g. droplets having well defined diameters with low variance) as their shape is well formed by surface tension before they begin to freeze. The downstream position of the droplet freezing zone 413 also inhibits freezing of the liquid formulation 102 close to, or inside the spray head assembly 401 and thereby blocking the spray head assembly 401 .

[0189] In some embodiments, the time between ejection from the spray head assembly 401 and solidifying, herein referred to as the flight time of a liquid droplet, may be between 2ms and 500ms, and preferably in the range of 20ms to 200ms. The corresponding a flight distance may be in the range of 20cm to 2m and preferably 50cm to 1 m.

[0190] The vacuum chamber 400 may be maintained at or below a pressure sufficient to cause liquid droplets with diameters in the range of 1 pm to 100pm, 2pm to 100pm, or 1.5pm to 100pm (which form lyophilised particles having approximately the same geometric diameters) to cool from approximately 20C to -30C with cooling rates in the range of 1 *108C / s to 2*104C / S and preferably to cause liquid droplets with diameters in the range of 10pm to 50pm to cool from approximately 20C to - 30C with cooling rates in the range of 2.4*106C / s to 9.5*104C / s.

[0191] After freezing, the solidified particles 104 travel through the vacuum chamber 400 and are received at a collection zone. In the embodiment depicted in Figure 4, the collection zone is the same area as the sublimation zone 415, as solidified particles are received at the same location as they are allowed to sublimate in. However, it will be appreciated that this is not necessarily true of all embodiments. Particles may be deflected or moved from their initial collection zone and allowed to sublimate elsewhere.

[0192] The spray head assembly 401 may be mounted to a vertical side of the vacuum chamber 400 such that the liquid droplets 103 and solidified particles 104 have a ballistic trajectory (i.e. a trajectory having motion in two planes wherein the downwards motion is substantially governed by gravity) from the spray head assembly 401 to the sublimation zone 415. The spray head assembly 401 may also be mounted in alternative locations or orientations, such as on the uppermost surface of the vacuum chamber 400 so that the liquid droplets 103 and solidified particles 104 fall directly downwards from the spray head assembly 401 to the sublimation zone 415.

[0193] Throughout their flight from the droplet freezing zone 413 to the sublimation zone 415, the solidified particles 104 may begin to dry via particle sublimation 111 whereby frozen water content within a solidified particle 104 sublimes out as water vapor into the vacuum chamber 400.

[0194] Once the solidified particles 104 are received at the collection and sublimation zone 415 within the vacuum chamber 400 they may remain there until a substantial amount of theirwater content has sublimed out. In some embodiments they may remain there until they form a dry powder composition 105 having an aggregate water content by mass of around or less than 2%, preferably less than 1 %, and more preferably 0.5%.

[0195] The aggregate water content may be determined via Karl-Fischer moisture analysis or NIR spectroscopy and also may be determined approximately by comparing the mass of the dry powder composition 105 to the mass of the introduced liquid formulation 102, which may have a known water content.

[0196] In some embodiments, the system 417 may comprise a second vacuum chamber 400b, maintained a pressure lower than the first vacuum chamber 400a. Liquid droplets 103 may undergo droplet freezing 110 in the first vacuum chamber 400a, but particle sublimation 111 in the second chamber 400b. These two distinct chambers may be separated by a dividing boundary 418 having an aperture 420, large enough to allow solidified particles 104 to pass from the first chamber 400a to the second 400b therethrough, but small enough to maintain a pressure differential between the two chambers. Solidified particles 104 may pass through the aperture 420 directly in flight, as depicted in Figure 4, or may pass through the aperture 420 after being deflected by a different surface within the vacuum chamber 400.

[0197] Advantageously, having two chambers maintained at different pressures may reduce the need for extremely low pressures to be maintained in the first vacuum chamber 400a. Such low pressures are beneficial in increasing the rate of sublimation, but arduous to maintain in large volumes, and may also increase the likelihood of the spray head assembly 401 becoming blocked with frozen liquid.

[0198] The system 417 may further comprise a second vacuum pump 419, in fluid communication with the second chamber 400b, in addition to the first vacuum pump 416 which is in fluid communication with the first chamber 400a. The pumps may be configured to depressurise their respective chambers to different pressures.

[0199] In some embodiments, there may be disposed in the vacuum chamber 400 a collection tray 403 for receiving the solidified particles 104 thereupon, the collection tray 403 preferably being horizontal. The collection tray 403 may be heated by an internal heating element (not shown) and configured to heat the solidified particles 104. It may also be configured to have a well-controlled temperature, such that temperature control of the tray may also comprise cooling the tray.

[0200] It is preferable that, in the initial stages of drying, (for example, the first stage 118) the collection tray is maintained at a temperature below the collapse temperature of excipients 101 in the liquid formulation 102. For typical excipients this may be in the range of around -50C to -5C. For a sucrose solution, this is around -35C, for lactose around -28C, and for trehalose, around -29C.

[0201] These low temperatures are desirable in order to inhibit the collapse of the structure of frozen excipients 101 within the solidified particles 104 during the drying process. Advantageously, this may protect drug product from mechanical damage, and promote the porosity of the lyophilised particles which form the dry powder composition 105. It is advantageous to promote the porosity of lyophilised particles as particles with high porosity may have an aerodynamic diameter lower than their geometric diameter. In this way, particles which may otherwise not be suitable for inhaled pulmonary delivery due to their large size, can be suitable for inhalation by virtue of this porosity.

[0202] In later stages of drying, such as the second stage 119, the temperature of the collection tray 403 may be increased. When the water content remains significant, the temperature is preferably maintained at below 0C in order to inhibit the melting of any water content remaining in the solidified particles.

[0203] The inhibition of melting is advantageous as it limits the mechanical forces on or deformation of the solidified particles 104 and the sublimation of ice as water vapour directly from the solidified particles 104 leaves behind vacated space, promoting the porosity of the dry lyophilised particles.

[0204] If the water content is sufficiently low, the temperature of the collection tray 103 may be raised to as high as 20C. In some examples, the solidified particles 104 may be allowed to dry in stage 119 for up to 240 minutes. For example, the solidified particles 104 may be allowed to dry in stage 119 for between 30 to 90 minutes, such as around 60 minutes.

[0205] Raising the temperature of the collection tray 103 advantageously raises the temperature of the landed solidified particles 414, thereby increasing the rate and effectiveness of particle sublimation 111.

[0206] Additionally, there may be a moisture cold trap 402 disposed inside the vacuum chamber 400, preferably proximal to the droplet sublimation zone 415. The moisture cold trap 402 may comprise a plurality of coils (not shown), the surfaces of which are preferably held at a temperature below -35C and more preferably in the range of approximately -80C to -50C, and is configured to, in use, remove water vapour from the vacuum chamber 400, thereby reducing the water vapour partial pressure inside the vacuum chamber 400 by promoting the deposition of water vapour from the vacuum chamber 400 onto the surface of the coils as ice. This inhibits the accumulation of water vapour within the vacuum chamber 400. Doing so, particularly in the vicinity of the sublimation zone 415, promotes the sublimation of ice out of the solidified particles 104, thereby increasing the rate of particle sublimation 111.

[0207] Low water vapour partial pressures may also be maintained by use of a vacuum pump, configured to continuously pump out water vapour from the vacuum chamber 400. This continuous pumping action may be performed by either or both of the vacuum pumps 416, 419, or it may be performed by a different pump (not shown) which is preferably disposed proximal to the sublimation zone 415.

[0208] The system 417 may comprise a re-pressurisation valve 405 configured to, upon use, re-pressurise the vacuum chamber 400. Preferably the system 417 further comprises an external gas supply 404 (preferably having inert gas) fluidly coupled to the re-pressurisation valve 405 such that the vacuum chamber 400 may be repressurised with inert gas rather than with atmosphere. Advantageously, the introduction of inert gas from the external gas supply 404 into the vacuum chamber 400 ahead of collecting the dry powder composition 105 may inhibit the wetted atmosphere from introducing any moisture back into the dry powder composition 105.

[0209] In some embodiments the system 417 may further comprise an inert gas supply 406, a deionised water supply 407 and a liquid formulation supply 408, in addition to a set of supply pumps 409 and supply valves 410, said pumps 409 and valves 410 being configured to controllably supply the spray head assembly 401 with the fluids of one or more of the an inert gas supply 406, deionised water supply 407 and liquid formulation supply 408.

[0210] The system 417 may further comprise a filtration device 411 disposed in the fluid path upstream of the spray head assembly 401 such that any fluid being supplied to the spray head assembly 401 passes through the filtration device 411 before entering the spray head assembly 401. The filtration device 411 is configured to inhibit particulate contaminants such as aggregates or organisms like bacteria entering the spray head assembly 401. In some embodiments, the filtration device 411 comprises a 0.2pm filter.

[0211] The spray head assembly 401 may be configured to introduce a jet of inert gas from the inert gas supply 406 into a vacuum chamber 400 having an atmospheric or vacuum environment. Deionised water from the deionised water supply 407 and liquid formulation 102 may be introduced as capillary jets or liquid droplets into atmospheric or vacuum environments, but the conditions of the vacuum chamber 400 may determine whether proper formation of said capillary jets or liquid droplets is achieved.

[0212] The purpose of the spray head assembly 401 being configured to introduce the fluids of the inert gas supply 406 and deionised water supply 407 in addition to the liquid formulation 102 into the vacuum chamber 400 is the suppression of the blocking of the spray head assembly 401 (by frozen liquids or otherwise) and the promotion of proper droplet formation 109 in the droplet formation zone 412. Therefore, it is advantageous for the inert gas supply 406 and deionised water supply 407 to be introduced into the vacuum chamber 400 via the same nozzle in the spray head assembly 401 as the liquid formulation 102 is.

[0213] Such exemplar nozzles include the nozzle plate 802 of Figure 8b, the nozzle apertures 1201 of Figure 9a and the nozzle plate 1300 of Figure 10.

[0214] These nozzles or apertures must be the same to benefit from the inhibition of said nozzle or apertures being blocked, and also for the purpose of continuity between the deionised water and liquid formulation 102 in the promotion of proper droplet formation 109.

[0215] In some embodiments, to obtain these advantages, the following process may be employed. Inert gas is introduced into the vacuum chamber 400 via the spray head assembly 401 from the inert gas supply 406 as the vacuum pump 416 reduces the pressure in the vacuum chamber. The pressure may initially be atmospheric. Then, by using the fluid pumps 409 and valves 410, the supply to the spray head assembly 401 is switched, and the spray head assembly 401 now introduces deionised water from the deionised water supply 407 into the vacuum chamber 400. A stable capillary jet of deionised water may be allowed to develop over time.

[0216] Once a deionised water capillary jet is formed in the droplet formation zone 412, the spray head assembly 401 may start ultrasonically stimulating the deionised water, initiating the breakup of the capillary jet to form water droplets in the droplet formation zone 412. By using the fluid pumps 409 and valves 410, the supply to the spray head assembly 401 is switched again such that the spray head assembly 401 now introduces liquid formulation 102 from the liquid formulation supply 408 into the vacuum chamber 400.

[0217] This switch is performed in such a manner that the flow of fluid into the spray head assembly 401 smoothly transitions from deionised water to the liquid formulation 102 so as to not disrupt the capillary jet or droplet formation 109 in the droplet formation zone 412.

[0218] Advantageously, this process ensures that the liquid formulation 102 is introduced into an already depressurised vacuum chamber 400 and may immediately form liquid droplets 103 and subsequently solidified particles 104, thereby wasting minimal drug product.

[0219] In order to cease introducing the liquid formulation 102, these steps may be reversed. By using the fluid pumps 409 and valves 410, the supply to the spray head assembly 401 is switched from liquid formulation 102 to deionised water which is, in turn, switched to inert gas. The ultrasonic stimulation by the spray head assembly 401 may cease during any of these stages, but preferably after the supply has been switched away from liquid composition 102, again, to minimise wastage of the liquid formulation and maximise yield.

[0220] The liquid capillary jet (or either liquid formulation 102 or deionised water) will disappear during this halting process, after which the re-pressurisation valve 405 may be utilised to prepare the vacuum chamber 400 to be opened to atmosphere by re-pressurising the vacuum chamber 400, preferably with an inert gas from the external gas supply 404.

[0221] FIG 5a is a diagram of another embodiment of a freeze-drying system, differing from the arrangements of Figure 4 by the addition of features to further make the system suitable for manufacturing medicaments, according to Good Manufacturing Practice (GMP) guidelines.

[0222] Herein, the liquid formulation 102 is supplied in a syringe pump 500 format. The syringe pump 500 may be single use to avoid contamination between batches.

[0223] In some embodiments, the system 417 may comprise a container disposed within the vacuum chamber 400 for receiving the solidified particles therewithin. Preferably, the container is a flexible container.

[0224] In some embodiments, this container may comprise a single use disposable bag 501 which interfaces with the spray head assembly 401 to allow the spray head assembly 401 to introduce the liquid formulation directly into the disposable bag 501. In the embodiment depicted in Figure 5a, this is achieved by forming the bag directly around the spray head assembly such that the majority of the interior of the vacuum chamber 400 is encompassed by the disposable bag 501 .

[0225] In other embodiments, for example, as shown in Figure 5b, the bag 501 may not be directly formed about the spray head assembly 401 , instead having an opening 503 elsewhere which is configured to receive liquid droplets 103 or solidified particles 104 therethrough as they travel from the spray head assembly 401 to a collection zone within the bag 501 .

[0226] To inhibit the accumulation of sublimed water vapour and other gases and to allow a vacuum to be formed inside the disposable bag 501 , the disposable bag 501 may include a vent 502 formed of filtration material. Preferably, the vent 502 should permit gas flow, be hydrophobic and have a pore size suitable to prevent landed solidified particles 414 from leaving the disposable bag 501. Preferably the vent comprises a gas-permeable portion of the disposable bag 501 . In some embodiments, the vent 502 may be either a surface or depth filter. Preferably the vent 502 is located proximal to the sublimation zone 415. The container or bag 501 may be otherwise foil-lined to prevent moisture ingress.

[0227] FIG 6 is a diagram of an alternate embodiment of a freeze-drying system 417 with guiding electrostatic elements. Herein the spray head assembly 600 may be configured to negatively or positively charge the liquid droplets 103 and thus form electrostatically charged solidified particles 601.

[0228] The spray head assembly 600 itself may be charged and the act of introducing liquid formulation 102 from the syringe pump 500, or deionised water from the deionised water supply 407, through the spray head assembly 600 produces electrostatically charged liquid droplets and thus electrostatically charged solidified particles 601 which inherit the charge nature of the charged spray head assembly 600.

[0229] Acting under electrical field forces, charged solidified particles 601 may be guided to land on an oppositely charged landing platform 603. The charged landing platform 603 may be heated in the same manner and for the same purpose as the collection tray 403 in Figure 5.

[0230] The opposite charges of electrodes at the spray head assembly 600 and charged landing platform 603 may be maintained by a power supply 604.

[0231] In some embodiments, the act of employing electric fields to guide the charged lyophilised particles 601 to the landing platform 603 may aid in the collection of the dry powder composition and removal of the dry powder composition 603 from the vacuum chamber 400 and the disposable bag 501 .

[0232] Furthermore, the act of employing electric fields to guide the charged solidified particles 601 may result in the dispersal of said charged particles 601 as they have like charges. Particles may therefore have deflected flight paths 605, thereby causing a given particle to have a different landing site on the landing platform 603 as a previous particle. This dispersal may inhibit the formation of an icicle nucleating from the landing platform 603. It will be appreciated by the skilled person that the use of guiding electrostatic elements in this embodiment requires that the flight of the droplets and particles is no longer strictly ‘ballistic’ (i.e. under the force of gravity only). However, any such references to ballistic flight in alternate embodiments is not meant to preclude the possibility that electrostatic guiding may be used in conjunction with those embodiments.

[0233] Other methods may be employed to disperse the droplets or particles, thereby inhibiting the formation of an icicle. For example, a deflector or a shaped feature disposed within the vacuum chamber may be arranged to deflect frozen droplets or solidified particles that impinge onto an initial impact zone within the vacuum chamber away from said initial impact zone towards a different zone within the vacuum chamber.

[0234] A method of operating the system 417 may further comprise moving a nozzle or other portion of the spray head assembly 401 in, for example, a cyclic fashion, such that solidified particles 104 are received at different locations on a surface within the vacuum chamber 400.

[0235] FIG 7 is a diagram of an alternate embodiment of a freeze-drying system 417 comprising a bag 700 and bag sealing device 701 disposed within the vacuum chamber.

[0236] To ensure that the dry powder composition 105 remains dry when removed from the vacuum chamber 400, the landed solidified particles 414 may be received within a single use thermoplastic bag 700 which is disposed inside the vacuum chamber 400 and interfaces with the spray head assembly 401 such that the spray head assembly 401 may introduce liquid formulation 102 and thus the solidified particles 104 into the thermoplastic bag 700 while the thermoplastic bag already contains a portion of dry powder composition 105 having been formed from earlier introduced liquid formulation 102.

[0237] The thermoplastic bag 700 may be sealed to form an air-tight seal by use of an internal bag sealer 701 which is placed within the vacuum chamber 400. Preferably the internal bag sealer 701 is a heat-sealing device. The position of the internal bag sealing device 701 is such that the seal is preferably made below the vent 502 but above a portion of the bag 700 containing the landed solidified particles 414.

[0238] In some embodiments, above the seal, a weakened portion 702 of material may be formed circumferentially around the bag 700 to promote separation of the sealed bag from the remaining portion of the bag 700 at the weakened portion 702. This weakened portion 702 may be made circumferentially in a surface of the thermoplastic bag 700 to allow convenient separation of the part of the thermoplastic bag 700 containing the dry powder composition 105.

[0239] Preferably the vacuum chamber 400 is re-pressurised with inert gas from the external gas supply 404 prior to use of the bag sealing device 701 on the thermoplastic bag 700, and the bag 700 is sealed before the vacuum chamber 400 is vented to atmosphere. Advantageously, this ensures that the majority of the gas within the sealed thermoplastic bag 700 containing the dry powder composition 105 is inert gas and not wetted atmosphere. This promotes the low moisture content of the dry powder composition 414, thus maintaining the quality and room temperature stability of the drug product.

[0240] FIG 8a is a diagram of an embodiment of a spray head assembly 401 , suitable for use with the freeze-drying system 417 of Figures 4 to 7.

[0241] The spray head assembly 401 may comprise a first subassembly 801 that does not contact the fluid path, comprising a casing 810 and an ultrasonic drive 809 disposed therein. The spray head assembly also comprises a second subassembly 800, which is in direct contact with the fluid path, comprising the spray cap 803 and tubing 806.

[0242] To support product quality and ease of use in a GMP-compliant environment, the spray head assembly 401 can be designed such that the second subassembly 800 (and thus the components which are in contact with the liquid formulation 102) are detachable from the first subassembly 801 (and thus the components which generate the ultrasonic stimulation and / or the components which mechanically interface the spray head assembly 401 to the freeze drying system 417).

[0243] For deployment in a GMP workflow, the liquid formulation contacting second subassembly 800 might preferably be packaged with a single-use fluidic set which supports upstream processes (i.e. the production of the liquid formulation) and could be pre-sterilised. This single-use fluid set, for example, may comprise the syringe pump 500 of liquid formulation 102.

[0244] The non-liquid formulation contacting first subassembly 801 might be installed within the freeze-drying system 417 in a manner which allows for easy attachment and detachment of the second subassembly 800 from the first subassembly 801 and with a fastening method which would support the transmission of ultrasonic stimulation between the ultrasonic drive 809 and liquid formulation 102.

[0245] In the embodiment depicted in Figure 8a this fastening feature comprises a threaded interior surface 811 of the spray cap 808 configured to interface with a threaded exterior surface 812 of the casing 810, wherein at least a portion of the casing 810 extends into an interior portion of the spray cap 808.

[0246] This principle of fluid-contacting components being easy detachable, and thus preferably single-use is well suited to the GMP instrumentation space. It minimises the amount of cleaning or sterilisation apparatus or systems must undergo between batches and thus allows for cheaper and quicker processes which are better able to have sterility verified.

[0247] FIG 8b is a diagram of a spray cap 808 and tubing 806 suitable for use as a second subassembly 800 with the spray head assembly 401 of Figure 8a.

[0248] In this embodiment, the spray cap 808 comprises a nozzle cap 803, the nozzle cap 803 having a first opening and a second opening, the second opening configured to receive the outlet of the fluid conduit, and the first opening configured to face the interior of the vacuum chamber 400 and introduce fluid into the vacuum chamber 400 therethrough. The spray cap further comprises a nozzle plate 802 disposed inside the nozzle cap 803 and in alignment with the first opening of the nozzle cap 803. Preferably the nozzle plate extends beyond the boundaries of the first opening and abuts an internal surface of the nozzle cap 803 about the first opening, such that a force may be applied to the nozzle plate 802 in the direction of this internal surface of the nozzle cap 803, thereby holding the nozzle plate 802 against the nozzle cap 803.

[0249] The tubing 806 is configured to fluidically interface with at least one of the liquid formulation supply 408, the inert gas supply 406 and deionised water supply 407 at its distal (inlet) end, and at the proximal (outlet) end it is configured to introduce fluid from these supplies to the nozzle plate 802, thereby forming a fluid conduit from a least one of the liquid formulation supply 408, the inert gas supply 406 and deionised water supply 407 to the nozzle plate 802.

[0250] The nozzle plate 802 preferably comprises at least one aperture having a diameter between 0.8pm and 240pm, preferably between 1 pm and 60pm, and more preferably between 1 pm and 50pm, through which fluid may be introduced into the vacuum chamber 400. The positioning of the nozzle plate 802 is such that the at least one aperture aligns with the first opening of the nozzle cap 803 and is not occluded by the nozzle cap 803.

[0251] These small aperture diameters may preferably allow the production of liquid droplets in the preferred size range of 2pm to 100pm, or in any of the other preferred ranges disclosed herein.

[0252] Smaller aperture diameters may be more suitable for the production of particles which are suited to pulmonary delivery, while larger diameters may be more suited for the production of particles which are suited to nasal delivery. For example, a nozzle (such as the nozzle plate 802) having aperture diameters between 1 pm and 15pm, such as between 2pm and 12pm, and preferably around 5pm may be suitable for the pulmonary delivery case. A nozzle having aperture diameters between 5pm and 140pm, 8pm and 60pm, preferably between 20pm and 30pm, and more preferably around 25pm may be suitable for the nasal delivery case. The design of the first subassembly 801 is preferably such that liquid formulation (or other fluids) can be pressurised at the nozzle plate 802 from an external pumping source (such as the syringe pump 500 or the supply pumps 409), whilst minimising dead-volume within the fluid path.

[0253] Dead-volume generally refers to the volume of active substance wasted in a process. A low dead-volume fluid path is desirable to minimise the amount of liquid formulation 102 lost at the end of each product batch produced from the freeze dry system. To reduce dead volume in the tubing 806, a Low Internal Diameter (Low-ID) microfluidic tubing can be used to transmit the liquid formulation 102 from the pump source to the spray head assembly 401 . This low- ID microfluidic tubing may have a diameter between 0.1 mm and 2.5mm, for example.

[0254] Smaller diameters may have undesirably high flow resistance, while larger diameters may result in undesirably large dead volumes and also experience more significant effects due to gravity (which typically may be neglected when using microfluidic tubing). Non-negligible gravitational effects detrimentally make the control of any liquid-gas interfaces in the tube less predictable.

[0255] To ensure the fluid path between the inlet of the tubing 806 and the nozzle plate 802 is sealed when the first assembly 801 is connected to the second subassembly 800, GMP-compliant components may be used at the junctions between components to seal those junctions, thereby inhibiting fluid from escaping from the fluid path at these junctions.

[0256] For at least this purpose, in this embodiment, the spray cap 808 may further comprise a ferrule 807, a force transfer component 805, and a nozzle plate compression 0-ring 804. These components may be housed within the nozzle cap 803.

[0257] Herein, the ferrule 807 is disposed annularly about the tubing 806, with the distal end of the tubing 806 extending beyond said ferrule 807. The ferrule 807 may be configured to form a substantially fluid-tight seal about the tubing 806 to inhibit fluid flow along an outer surface of said tubing 806. Preferably, the ferrule 807 is formed of a material having a high Shore hardness.

[0258] The outlet of the tubing 806, may be retained within the bore through the ferrule 807, may extend beyond it, or may lie flush with a lower annular surface of the ferrule 807.

[0259] The ferrule 807 may comprise an upper tapered surface, wherein the tapered surface tapers radially inwards in the axial direction towards the second opening of the nozzle cap 803, and is configured to abut an opposing tapered surface of the casing 810 when the spray cap 808 is fastened to the casing 810 via, for example, the threaded interior 811 and exterior 812 surfaces.

[0260] A further purpose of the ferrule 807 is to form a seal between the tubing 806 and the force transfer component 805. The force transfer component 805 has an annular shape with a bore through its centre within which at least a portion of the tubing 806 may be retained.

[0261] The force transfer component 805 has a first, upper annular surface which abuts the lower annual surface of the ferrule 807 in a manner which does not occlude the outlet of the tubing 806. A substantially fluid-tight seal may be formed between the ferrule 807 and the force transfer component 805 at these abutting surfaces.

[0262] The force transfer component 805 is sealed to the nozzle plate 802 with the nozzle plate compression O-ring 804, which is compressed by a set distance based on a machined step in the nozzle cap 803.

[0263] These seals are, in part, achieved by transmitting a high spray head fastening force from the ferrule 807, through the force transfer component 805 and O-ring 804, to the nozzle plate 802, thereby maintaining contact between the nozzle plate 802 and the nozzle cap 803.

[0264] A sealed fluid path is therefore defined from the outlet of the tubing 806, through a central bore in the force transfer component 805 and through the open centre of the O-ring 804, to the nozzle plate 802. The contact between the nozzle plate 802 and the nozzle cap 803 allows ultrasonic motion to be transmitted from the ultrasonic drive 809, through the casing 810, to the nozzle cap 803, and thus the nozzle plate 802. Via this stimulation of the nozzle plate 802, the liquid formulation 102 may be ultrasonically stimulated as it is introduced into the vacuum chamber 400 to initiate the breakup of the liquid formulation 102 into liquid droplets 103.

[0265] This high spray head fastening force may be applied by the fastening of the first subassembly 801 to the second subassembly 800 and transmitted, for example, between the interfacing tapered surfaces of the casing 810 and the ferrule 807.

[0266] FIG 9a shows a plan view of a microarray nozzle 1200 with a plurality of nozzle apertures 1201. The plurality of apertures 1201 sit in alignment with a clearance hole 1202 in a vacuum layer 1204 of the micronozzle array.

[0267] The nozzle apertures 1201 may be arranged in a plurality of groups, as depicted in Figure 9a, and each group may sit within its own clearance hole 1202.

[0268] A plurality of apertures 1201 is advantageous as the apertures 1201 preferably have extremely small diameters between 0.8pm and 240pm, and preferably between 1 pm and 60pm, and more preferably between 1 pm and 50pm.

[0269] Smaller aperture diameters may be more suitable for the production of particles which are suited to pulmonary delivery, while larger diameters may be more suited for the production of particles which are suited to nasal delivery. For example, a nozzle having aperture diameters between 1 pm and 15pm, such as between 2pm and 12pm, and preferably around 5pm may be suitable for the pulmonary delivery case. A nozzle having aperture diameters between 8pm and 60pm, preferably between 20pm and 30pm, and more preferably around 25pm may be suitable for the nasal delivery case.

[0270] Aperture diameters this small promote the production of liquid droplets 103 and thus lyophilised particles 104 in the desired size range of 2 pm to 100 pm. However, they also have a reduced volume throughput compared to larger apertures. In order for the present apparatuses and methods to scale effectively, an array or plurality of apertures may be preferable in order to maintain high volume throughput. Throughput may also be increased by way of larger nozzle apertures and increased ejection velocities of the capillary jet.

[0271] A section view of the micronozzle array 1200 is shown in Figure 9b. The apertures 1201 are positioned in alignment within a clearance hole 1202 within a vacuum layer 1204, such that the apertures 1201 may be in fluid communication with the vacuum chamber 400 via the clearance hole 1202.

[0272] The microarray nozzle 1200 may comprise two layers: a fluid contacting layer 1203 in which the apertures 1201 are formed, and a vacuum contacting layer 1204 in which the clearance hole 1202 is formed. The microarray nozzle 1200 is preferably made of two layers because to manufacture the apertures 1201 at micron scales, it is preferable that the liquid formulation contacting layer 1203 is thin enough such that its thickness is not too great to make the hole forming method impractical. Furthermore, a thinner formulation contacting layer 1203 results in the apertures 1201 necessarily being shorter, thus reducing the pressure required to pump liquids through the apertures 1201 in the nozzle 1200. Such manufacturing methods may include etching, laser drilling, and 3D printing.

[0273] If the fluid contacting layer 1203 is then thin, a thicker vacuum contacting layer 1204 may be included to ensure the microarray nozzle 1200 is sufficiently stiff to be handled, to sustain the back pressure necessary to form capillary jets through the micron sized apertures 1201 , and to sustain low pressures within the vacuum chamber. This layer may therefore be referred to as a support layer. To not occlude the micron sized apertures 1201 , an aligning clearance hole 1202 may be included in the vacuum contacting layer 1204, said clearance hole 1202 encompassing all the micron sized nozzle apertures 1201.

[0274] The micron sized apertures 1201 may also be made with larger diameters using the same structure.

[0275] The microarray nozzle 1200 may be used in conjunction with the first subassembly 801 of Figures 8a and 8b. Herein, in some embodiments, the nozzle plate 802 may be a micronozzle array 1200. In other embodiments, the nozzle plate 802 may form the fluid contacting layer 1203 of the micronozzle array and the nozzle cap 803 may form the support, or vacuum contacting layer 1204.

[0276] FIG 10 is a diagram of an alternative embodiment of the first subassembly 801 configured to use a surrounding gas flow to focus a liquid jet in a nozzle assembly 1312 with a skimmer 1307.

[0277] In this embodiment, the nozzle assembly 1312 comprises a fluid contacting reservoir nozzle plate 1300 and a skimmer 1307. The fluid contacting reservoir nozzle plate 1300 interfaces with a fluid reservoir 1301 such that the reservoir nozzle plate 1300 forms a fluid outlet in the reservoir 1301 . The reservoir nozzle plate 1300 may be mechanically coupled to, such that it may be ultrasonically stimulated by, an ultrasonic drive device in a similar manner as to the nozzle plate 802.

[0278] A headspace pressure may be applied to the fluid reservoir 1301 such that a capillary fluid jet 1302 may be formed at an exit aperture of the reservoir nozzle plate 1300. The exit aperture may be substantially analogous to the apertures of the nozzle plate 802 and the apertures 1202 and thus may have a similar diameter as disclosed in connection with these features.

[0279] The fluid jet 1302 exits the reservoir nozzle plate 1300 into a gas chamber 1303 which is defined by a surface of the reservoir nozzle plate 1300 and an adjacent surface of a gas chamber capping plate 1304. Said surface of the reservoir nozzle plate 1300 opposes the surface of the reservoir nozzle plate 1300 which contacts the fluid reservoir 1301 .

[0280] Into the gas chamber 1303 is supplied a gas to form a gas flow 1305 surrounding the fluid jet 1302, for example, as a coaxial jacket of gas, and configured to accelerate the fluid in the fluid jet 1302 such that the neck of the fluid jet 1302 reduces to a diameter smaller than the diameter of the exit aperture of the reservoir nozzle plate 1300. Both the fluid jet 1302 and the gas flow 1305 exit the gas chamber 1303 through an aperture in the gas chamber capping plate 1304 and enter into a skimmer chamber 1306 which is defined by two adjacent surfaces of the gas chamber capping plate 1304 and the skimmer 1307.

[0281] The purpose of the skimmer chamber 1306 is to vent the majority of the gas flow 1305 (via a vacuum pump in fluid communication with the skimmer chamber 1306), before it enters the low vacuum chamber 1311. This may reduce the load on a vacuum pump which is configured to depressurise the low vacuum chamber 1311.

[0282] The low pressure in the skimmer chamber 1306 may be sufficient to cause gas to expand to form a bow shock 1308 at the entrance to the skimmer chamber 1306 on the gas chamber capping plate 1304.

[0283] Depending upon fluid and gas flow conditions, the fluid jet 1302, which, in some embodiments, may extend into the vacuum chamber 1311 , may break-up into liquid droplets 1310. This breakup may be initiated by ultrasonic stimulation of the reservoir nozzle plate 1300 or via pressure waves applied to the fluid reservoir 1301.

[0284] The gas flow 1305 surrounding the fluid jet 1302 will increase the pressure in the vicinity of the surface of the fluid jet 1302, thus reducing the rate of evaporative cooling and delaying the freezing of the fluid jet 1302 to preferably be after the formation of the liquid droplets 1310.

[0285] This configuration may aid in the inhibition of the blocking of the spray head assembly 400 and may allow the vacuum chamber 1311 , where sublimation occurs, to be maintained at a lower water vapour pressure, reducing drying time and allowing lower terminal moisture content to be achieved.

[0286] In this embodiment, the fluid jet 1302 preferably breaks up into droplets 1310 in the skimmer chamber 1306, such that liquid from the reservoir 1301 may be introduced into the vacuum chamber 1311 directly as well-formed spherical droplets. Therefore, the vacuum chamber 1311 may be held at a lower pressure than if the liquid was introduced as a fluid jet 1302 and, thereby, the rate of freezing of the liquid droplets 1309 may be increased.

[0287] FIG 11a is chart showing the relationship between the geometric diameter, DGof the lyophilised particles forming the dry powder composition 105, assuming said particles have a perfectly spherical shape, and the density, pPof the lyophilised particles for particles which have aerodynamic diameters, DA of 1 pm and 5pm.

[0288] Herein, aerodynamic diameter is determined using the following relationship:

[0289] DA= DG / ? ’ AJ PO where po has a value of 1 g / cm3. The lyophilised particles (i.e. the dried solidified particles 104) have a particle porosity related to a particle density pPby the following relationship:

[0290] The lyophilised particles of the present invention (e.g. the aggregates of nanoparticulate drug particles) typically have volume-weighted median geometric diameters in the range 2 to 100pm, preferably in the range 2 to 50pm, more preferably in the range 2 to 25pm. Preferably, the particles have volume-weighted median geometric diameters in the range 2 to 22.4pm, more preferably in the range 2 to 20pm. In some embodiments, the particles may have volume-weighted median geometric diameters in the range 4 to 18pm.

[0291] Particles having volume-weighted median geometric diameters in the range 2 to 25pm, such as 2 to 20pm, or 5 to 15pm may be particularly suitable for delivery by inhalation (i.e. pulmonary delivery). In some embodiments, particles suitable for delivery by inhalation (i.e. pulmonary delivery) may have volume-weighted median geometric diameters in the range 4 to 20pm. FIG 11 b is a chart showing the relationship between nozzle diameter and particle density for inhalable particles having different aerodynamic diameters of 1 pm and 5pm.

[0292] A preferred diameter of a nozzle aperture is between 1 pm and 50pm, and more preferably, for the generation of particles with an aerodynamic diameter suitable for lung (pulmonary) delivery, the diameter of a nozzle aperture may be between 1 pm and 25pm, preferably between 1 pm and 15pm, and more preferably between 2pm and 12pm, such as around 5pm.

[0293] It will be appreciated, however, that the particles may additionally, or alternatively be administered via nasal delivery. While particles for pulmonary delivery may ideally have mass median aerodynamic diameters between 1 pm and 5pm, larger particles may be suitable for nasal (intranasal) delivery. For example, particles suitable for nasal delivery may have mass median aerodynamic diameters between 10pm and 50pm and may preferably have mass median aerodynamic diameters between 20pm and 35pm, or between 20pm and 30pm. In some examples, particles with an aerodynamic diameter of approximately 15pm or 14pm may be particularly preferred for intranasal delivery.

[0294] Therefore, a preferred MMAD, suitable for at least one of pulmonary or nasal delivery may be between 1 pm and 50pm.

[0295] FIG 11c is a chart showing the relationship between particle geometric diameter and particle density for particles suitable for intranasal delivery having different aerodynamic diameters of 10pm and 50pm.

[0296] Particles having volume-weighted median geometric diameters in the range 20 to 100pm, such as 40 to 70pm may be particularly suitable for nasal delivery. In some embodiments, particles suitable for nasal delivery may have volume- weighted median geometric diameters in the range 15 to 115pm and densities between 0.2 and 0.4g / cm3, more preferably they may have volume-weighted median geometric diameters in the range of 15 to 100pm, such as 40 to 70pm, and densities between 0.25 and 0.4g / cm3. FIG 11 d is a chart showing the relationship between nozzle diameter and particle density for particles suitable for intranasal delivery having different aerodynamic diameters of 10pm and 50pm.

[0297] The diameter of a nozzle aperture for the production of particles for intranasal delivery may be between 5pm and 140pm, preferably between 8pm and 60pm, and more preferably between 20pm and 30pm, such as around 25pm.

[0298] FIG 12a is a chart showing the relationship between the minimum ejection velocity from a nozzle required to form stable droplets and the aerodynamic diameter of those particles for inhalable particles having different solidities generated from liquid formulations with solid content in the range of 5% to 40% in weight (g) by volume (cm3).

[0299] This range of liquid formulation solid content substantially corresponds to particle densities of about 0.05 g / cm3to 0.4 g / cm3.

[0300] It is preferable to keep the percentage of excipient 101 below that of the eutectic, to ensure that the excipient does not crystallise out of solution. For aqueous-sugar formulations, the eutectic point is generally below 70% excipient by weight.

[0301] The percentage of the solid content dissolved or suspended in the liquid formulation (i.e. the non-solvent components of the liquid formulation) is preferably in the range 5% to 40% of weight (g) by volume (cm3) in order to achieve a preferred range of particle densities which may provide inhalable aerodynamic diameters.

[0302] Solid content will be understood to refer to the components of the liquid formulation which substantially do not evaporate under the lyophilisation of a droplet of liquid formulation. When expressed as a percentage, or a percentage of weight (g) by volume (cm3), it is understood that this refers to the percentage, by mass, of the liquid formulation which the solid content comprises.

[0303] The resulting dried, lyophilised particle has a geometric diameter close to the liquid droplet diameter and has a particle density close to density of the solid content in the liquid formulation and is therefore preferably in the range 0.05 g / cm3to 0.4 g / cm3.

[0304] Solid content higher than 40% in the liquid formulation would result in undesirably high liquid formulation viscosity and, in order to produce particles having the same MMAD, would require use of smaller nozzles, which can be blocked more easily and require high pressure delivery of the liquid formulation. Solid content lower than 5% would require an undesirably large quantity of water to be removed, reducing throughput, increasing sublimation times, and would result in lower density particles, which would require an undesirably large volume when packaged.

[0305] In some embodiments, the ejection velocity of a liquid jet may preferably be in the range of 5m / s to 25m / s.

[0306] In some embodiments the minimum ejection velocity of a liquid jet may preferably be in the range of 5m / s to 25m / s for the production of particles having aerodynamic diameters between 1 pm and 5pm.

[0307] FIG 12b is a chart showing the relationship between the minimum ejection velocity from a nozzle required to form stable droplets and the aerodynamic diameter of those particles over a wider diameter range (1 pm to 100 pm), thereby including particles suitable for pulmonary delivery (i.e. inhalable particles) and those suitable for nasal delivery. The relationship between the minimum ejection velocity and the aerodynamic diameter of the solidified particles produced is shown at different solid contents, over a range of 5% to 40% of weight (g) by volume (cm3).

[0308] In some embodiments, the ejection velocity of a liquid jet may preferably be greater than 1 m / s, for the formation of particles with aerodynamic diameters of up to of 100pm, or greater than 25m / s, for formation of particles with an aerodynamic diameter of at least 1 pm. In some embodiments the minimum ejection velocity of a liquid jet may preferably be in the range of 2m / s to 5m / s for the production of particles having aerodynamic diameters between 10pm and 50pm. FIG 13 is a chart showing the expected liquid droplet temperature transient to freezing for a range of droplet geometric diameters. The temperature is calculated based on formulas from “Spherical-shaped ice particle production by spraying water in a vacuum chamber” by H.T. Shin et al, Applied Thermal Engineering 20 (2000) 439-454.

[0309] FIG 14a is a chart showing cooling times against droplet diameter using a simplified thermal conduction model in which evaporation cools the surface of the droplet instantaneously to a temperature for which the saturation vapour pressure of water is equal to the water vapour partial pressure in the vacuum chamber. The model uses a starting droplet temperature of 20C, a value of 1.34x1 O'7m2 / s for the thermal diffusivity of water, and a vacuum chamber water vapour partial pressure of 6 Pa, corresponding to a saturation vapour pressure of water at -48C. Cooling times are calculated as the time for the centre of the droplet to cool from 20C to a temperature of -5C, -30C and -40C, and are plotted against droplet diameter. The cooling times are proportional to the square of the droplet diameter.

[0310] FIG 14b is a table showing the cooling times, as plotted in Fig 14a, and the average cooling rates in cooling from 20C to -5C, -30C and -40C, respectively, in a vacuum chamber with water vapour pressure of 6Pa, equal to the saturated vapour pressure of water at -48°C.

[0311] FIG 15 is a chart showing saturation vapour pressure against water / ice temperature. The rate of evaporation of water from the liquid formulation is determined by the saturation vapour pressure of the liquid and by the water vapour partial pressure in the vacuum chamber 400. The cold trap 402 primarily controls the water vapour partial pressure by freezing water molecules onto the coils of 402. Literature suggests that water droplets may freeze between -5C and -35C depending upon the purity of the sample. Therefore, the temperature of the coils is preferably below -35C, and more preferably between -80C and -50C.

[0312] FIG 16a is a chart showing preferred operating conditions for particle generation using a liquid formulation with solute concentration of 5% by weight (g) / volume (cm3), calculated for the case where the solute is sucrose.

[0313] Similar plots can be generated for other solutes such as trehalose or lactose.

[0314] In some embodiments the solute concentration of the liquid formulation may be understood to mean the solid contents of the liquid formulation.

[0315] The plot shows jet velocity limits and referred values against nozzle diameter for generation of particles with aerodynamic diameters from 1 pm to 100pm. The lower jet velocity limits are defined by a minimum velocity for jetting (Weber number = 4) and by a droplet break-up time being equal to the time for the droplet to cool to a temperature that will cause the onset of freezing. The preferred jet velocity is defined by an operating condition where the liquid drive pressure is 100 bar. The upper jet velocity limits are defined by the need to avoid turbulence (Reynolds number = 5000), stress limits on the nozzle due to the liquid formulation supply pressure (peak stress = 200 MPa), and the requirement for the droplet to cool to a freezing temperature, taken as -40°C, before impacting a chamber wall, taken to be at a distance of 1 m from the nozzle. For each nozzle diameter, the possible range of jet velocity values is given by the region between the highest of the lower limit lines and the lowest of the upper limit lines.

[0316] FIG 16b, FIG 16c, and FIG 16d are charts similar to FIG 16a, showing preferred operating conditions for particle generation using a liquid formulation with solute concentration of 10%, 20% and 40% by weight (g) I volume (cm3), respectively.

[0317] FIG 16e is a table showing example operating conditions for generation of particles with aerodynamic diameters from 1 pm to 100pm. The table is divided into four parts, showing operating conditions for use with liquid formulations having a solid content of 0.05g / cm3, 0.1 g / cm3, 0.2g / cm3and 0.4g / cm3. The table shows a minimum, preferred and maximum ejection velocity for each combination of aerodynamic particle diameter and formulation solid content. The preferred ejection velocity is chosen to require a liquid formulation pressure of 100 bar to provide a preferred trade-off between high throughput and conveniently low pump pressure. Preferred operating conditions are enclosed in dashed boxes and indicate conditions where the preferred ejection velocity is greater than the minimum ejection velocity and is lower than the maximum ejection velocity.

[0318] The minimum ejection velocity is determined by the combination of two requirements, first that the liquid must emerge from the nozzle as a jet rather than by dripping, and second that the liquid jet must break up into droplets before freezing.

[0319] The jetting requirement is satisfied if the Weber number We > 4, where the Weber number, We is defined as: where the liquid formulation has a density ptand a surface tension / and the nozzle has a radius r, operating with an ejection velocity v (Wm van Hoeve et al, “Break-up of diminutive liquid jets”, Physics of Fluids 22, 122003 (2010)).

[0320] The minimum velocity for break-up before freezing (BBF in FIG 16d) is given by an ejection velocity for which a break-up time is equal to a time for the centre of the droplet to cool to a temperature at which ice nucleation will cause freezing to start. The minimum ejection velocity is the greater of the velocity that satisfies We > 4 and the break-up before freezing velocity.

[0321] The breakup time tb for an ultrasonically stimulated jet is calculated from the breakup length lband jet velocity v: where 10.5A. is an empirically determined minimum break-up distance and = 4.5D is a preferred wavelength for stable jet break-up into droplets and D is a nozzle diameter and v is a liquid jet velocity. The stimulation frequency f is given by the relationship:

[0322] An alternative the breakup timescale may be estimated as the capillary time tcgiven by the equation: where p is the density of the liquid, r is the radius of the jet and y is the surface tension of the liquid (Wim van Hoeve et al, “Break-up of diminutive liquid jets”, Physics of Fluids 22, 122003 (2010)).

[0323] The time for freezing tf is calculated using the thermal conduction model shown in FIG 14a and FIG 14b. The minimum jet velocity required to enable break-up before freezing is found by equating the break-up time tb with a time for freezing tf in listed in FIG 14b.

[0324] The upper limit on jet velocity is set by the lower of the following constraints: the Reynolds number is less than 5000 to avoid the onset of turbulence in the flow through the nozzle; the stress in the nozzle plate is below 200 MPa to avoid mechanical failure of the nozzle plate; the freezing time is less than the time for a droplet to impact a chamber wall at a distance of 1 m, freezing before impact (FBI) in FIG 16d.

[0325] The Reynolds Re number is calculated as:

[0326] Re = ^, where the liquid formulation has a density ptand a viscosity and the nozzle has a radius D, operating with an ejection velocity v.

[0327] The maximum stress in the nozzle plate crmaxis calculated as the stress at the centre of a clamped circular plate subject to a uniform pressure load p on one face: 3(l+v)prp max ~ > where rpis the radius of the nozzle plate, taken to be 5D, h is the thickness of the nozzle plate, taken to be 2D, with a minimum value of 5 m and a maximum value of 100 pm, where D is the nozzle diameter and where v> is the Poisson’s ratio of the nozzle plate material, taken to be 0.33.

[0328] The time for impact ti is calculated as: where v is a jet velocity and L is a distance to a chamber wall, taken to be 1 m. The maximum jet velocity for freezing before impact is determined by equating an impact time ti with a time for freezing Misted in FIG 14b.

[0329] FIG 16f is a table showing example operating conditions for production of particles for inhaled (i.e. pulmonary) and nasal delivery. In some embodiments, for producing particles particularly suitable for inhaled delivery, a nozzle diameter of between 1 pm and 25pm, such as between 1 pm and 15pm, or 2pm and 12pm, and such as 5pm may be used. The liquid formulation may have a 10% solid content, although it will be appreciated that other solid contents might be suitable. Droplet velocity (which may be substantially the same as the velocity of the capillary jet) may be between 35m / s and 50m / s, such as around 40m / s. A drive (stimulation) frequency of between 1000kHz and 2500kHz, such as around 1780kHz might be used to break up the liquid jet into droplets. The generated particles may have a geometric diameter of between 4pm and 20pm, such as around 9.5pm and an aerodynamic diameter of between 1 pm and 5pm, such as around 3pm.

[0330] The evaporation capacity of the vacuum chamber 400 may be limited. Therefore, the maximum throughput of the nozzle or nozzle array might be limited by the evaporation capacity of the vacuum chamber 400. In the exemplar embodiment set out in Figure 16f, a nozzle plate (which may be analogous to the nozzle plate 802) containing an array of 18 nozzles was used, thus increasing throughput, within a 50g / hr evaporation capacity of a benchtop freeze dryer (one example of the vacuum chamber 400 the drying stages 118 and 119 might be performed in).

[0331] For nasal delivery, a nozzle diameter of between 20pm and 30pm, such as 25pm might be used. The liquid formulation may have a 30% solid content although it will be appreciated that other solid contents might be suitable. Droplet velocity may be between 1 m / s and 20m / s, such as between 1 m / s and 5m / s, and such as around 5m / s. A drive (stimulation) frequency of between 30kHz and 60kHz, such as around 44kHz might be used to break up the liquid jet into droplets. The generated particles may have a geometric diameter of between 40pm and 70pm, such as around 47.2pm and an aerodynamic diameter of between 10pm and 50pm, such as around 25.9pm.

[0332] As this example utilises a large nozzle, a nozzle plate containing an array of 7 nozzles was used to increase throughput, within the 50g / hr evaporation capacity of a benchtop freeze dryer.

[0333] Preferably, a lower droplet velocity might be used for the production of particles for nasal delivery, as this allows a longer freezing time which is required by larger diameter droplets before impact occurs. Preferably a higher droplet velocity might be used for the production of particles for inhaled delivery, in order to increase throughput per nozzle, as the smaller diameter droplets have a shorter freezing time and therefore require less time before impact.

[0334] Figure 17a is an image of liquid droplets generated through a 25pm nozzle. Water was fed to the nozzle, with ultrasonic stimulation of the nozzle to drive nozzle vibration at a frequency of 120 kHz (nozzle drive frequency), at a flow rate of 6 pl / s. The image was captured using a stroboscopic imaging system synchronised with the ultrasonic drive applied to the nozzle. The droplets are spherical, have uniform size 45.0pm ± 1.0pm, and are ejected at the same frequency as the ultrasonic stimulation. Ultrasonic stimulation can be made by vibrating the nozzle or by driving pressure oscillations in the liquid feed upstream of the nozzle. Figure 17b is a chart showing measured relationship between droplet velocity and nozzle drive frequency used to generate stable periodic droplets for different feed flow rates of water through a 25 pm nozzle. The nozzle drive frequency does not affect the droplet velocity.

[0335] Figure 17c is a chart showing measured relationship between droplet velocity and feed flow rates of water through a 25 pm nozzle. Droplet velocity increases linearly with the flow rate.

[0336] Figure 17d is a chart showing measured relationship between droplet diameter and nozzle drive frequency for different feed flow rates of water through a 25 pm nozzle. The frequency range for stable droplet generation depends on the flow rate and this range increasingly shifts to the higher frequency values with increasing flow rates. Droplet size depends on the nozzle drive frequency. For example, as shown in Figure 17d, droplets having diameters of between around 40pm and 70pm, such as between 40pm and 60pm may be produced through a nozzle having a diameter between 20pm and 30pm, such as around 25pm. The flow rate through the nozzle might be between around 4pl / s and 12pl / s. The frequency of the ultrasonic stimulation might be between around 50kHz and 300kHz. As shown in Figure 17c, flow rates of between around 4pl / s and 12pl / s through a nozzle having a diameter between 20pm and 30pm, such as around 25pm may result in a ejection velocity (which is substantially the same as droplet velocity).

[0337] Figure 17f is a chart showing measured relationship between droplet diameter and nozzle drive frequency for different feed flow rates of water through a 20 pm nozzle. The frequency range for stable droplet generation depends on the flow rate and this range increasingly shifts to the higher frequency values with increasing flow rates. Droplet size depends on the nozzle drive frequency, for a given flow rate droplet size decreases with increasing frequency.

[0338] For example, as shown in Figure 17f, droplets having diameters of between around 25pm and 45pm may be produced through a nozzle having a diameter between 15pm and 25pm, such as around 20pm, at flow rates of between around 4 p l / s and 1 Opl / s and frequencies between around 100kHz and 600 kHz. As shown in Figure 17e, flow rates of between around 4pl / s and 1 Opl / s through a nozzle having a diameter between 15pm and 25pm, such as around 20pm may result in an ejection velocity of between 10m / s and 35m / s.

[0339] Such parameters may produce droplets, and thus lyophilised particles having geometric diameters of between around 25pm and 45pm. These droplets may be suitable for nasal delivery or pulmonary delivery.

[0340] Figure 17g is a chart showing measured relationship between droplet velocity and feed flow rates of 10% trehalose solution in 50mM Tris-HCI buffer pH 7.4 through a 20 pm nozzle. Droplet velocity increases linearly with the flow rate.

[0341] Figure 17h is a chart showing measured relationship between droplet diameter and nozzle drive frequency for different feed flow rates of 10% trehalose solution in 50mM Tris-HCI buffer pH 7.4 through a 20 pm nozzle. The frequency range for stable droplet generation depends on the flow rate and this range increasingly shifts to the higher frequency values with increasing flow rates. Droplet size depends on the nozzle drive frequency, for a given flow rate droplet size decreases with increasing frequency.

[0342] Together, these results help to identify appropriate operating conditions, flow rate and nozzle drive frequency, for a given nozzle orifice size for stably generating droplets of an identified size.

[0343] FIG 18 is a diagram of an exemplary dry powder composition according to the present invention 105. The diagram includes a representation of one of the freeze-dried aggregates of nanoparticulate drug particles 1700.

[0344] The aggregates contain the nanoparticulate drug particles, such as LNPs 1701. With the remainder of the aggregate comprising excipient. The aggregates may contain pores 1702.

[0345] FIG. 19 is a light microscope image of particles in the dry powder sample obtained after drying of 10% trehalose solution in 50mM Tris-HCI buffer pH 7.4. For drying, 10% trehalose solution in 50mM Tris-HCI buffer pH 7.4 was fed to a 25pm nozzle, with ultrasonic stimulation of the nozzle to drive nozzle vibration at a frequency of 120 kHz (nozzle drive frequency), at a flow rate of 6 pl / s. The nozzle was housed in a spray head assembly (401) attached to a vacuum chamber. 45.0 ± 1.0 pm droplets were ejected inside vacuum (30 mTorr). Droplets froze during the flight and landed on a sample collection tray (403) maintained at -35C. After 2 hours, the collection tray temperature was raised to 4C and maintained there for 1 hr. Subsequently, vacuum was released using dry nitrogen gas and the powder was collected in a sealed container. Powder was then imaged using a Digital Microscope VHX light microscope (Keyence VHX-7000). The particle size and size distribution were calculated manually by analysing size of 50 randomly chosen particles from the microscope image.

[0346] FIG 19a is the light microscope image of particles in the dry powder sample described above. The particles are highly spherical in shape and uniform in size.

[0347] FIG 19b is a line drawing representation of the microscope image of Figure 19a.

[0348] FIG 19c is a chart showing particle size distribution measured by analysing size of 50 randomly chosen particles from the image in FIG 19a. The size distribution is narrow with a span of 0.06 (FIG 19c).

[0349] FIG 19d is a table showing various calculated particle size characteristics for the 50 randomly chosen particles in figure 19b. These particles have 10% solidity and a mass median aerodynamic diameter (MMAD) of 14.0 pm, suggesting their suitability for intranasal delivery.

[0350] The geometric diameter of the particles is 44.4 pm which is close to the size of droplets 45.0 pm and particles are highly uniform. This could be attributed to formation of uniform size droplets and their instant freezing not allowing for size reduction.

[0351] The dry powder compositions of the present invention are suitable for storage and transportation at above 0C temperatures for example refrigerated temperature (between 2C and 8C) or preferably up to 25C or more preferably up to 40C. Due to the advantageous methods used herein, the dry powder compositions may have an extremely low moisture content by mass such as less than 2%, preferably less than 1 %, most preferably less than 0.5%. This low moisture content improves stability. Thus, the active drug substance in powder compositions (e.g. aggregates of nanoparticulate drug particles) according to the present invention can retain its activity upon storage at temperatures such as those mentioned above. In the case of LNPs containing nucleic acids, the nucleic acids such as mRNA retain their biological activity upon storage.

[0352] Thus, in some embodiments the present invention provides dry powder compositions comprising aggregates of nanoparticulate drug particles such as mRNA-LNPs in which the active drug substance, e.g. mRNA, retains biological activity after storage at temperatures at above 0C temperatures for example refrigerated temperature (between 2C and 8C) or preferably up to 25C or more preferably up to 40C for at least a week. More preferably, it may be suitable for storage for up to 6 months, more preferably up to 1 year and yet more preferably up to 2 years

[0353] Such powder compositions typically comprise aggregates of nanoparticulate drug particles, wherein the aggregates have volume-weighted median geometric diameters in the range 2pm to 100pm, preferably 2pm to 50pm, more preferably in the range 2pm to 25pm. Said aggregates preferably have a geometric diameter size distribution with a span of less than 1 or preferably less than 0.5 or more preferably less than 0.2.

[0354] In some embodiments the dry powder compositions of the present invention are suitable for pulmonary delivery. Specifically, the advantageous uniformity of shape, sphericalness, size and porosity allow for the production of particles which are well suited for pulmonary delivery without the need for further processing. Thus, such powder compositions may be suitable for use in dry powder inhalers. In particular the dry powder compositions according to the present invention may have mass median aerodynamic diameters in ranges particularly suitable for inhalation such as those described below. The nanoparticulate drug particles described herein may be lipid nanoparticles which encapsulate one or more pharmaceutically active substances.

[0355] Nanoparticulate, e.g. nanoparticles, refers to particles having at least one dimension in the order of nanometres, for instance 1 -900nm, preferably 10- 500nm.

[0356] Lipid nanoparticles (LNPs) are nanoparticles comprising one or more lipids. LNPs may be used as drug delivery systems for the delivery of an active / therapeutic agent to a site of interest such as a cell, tissue, or organ.

[0357] In some embodiments, the lipids may comprise lipid components such as cationic ionizable lipids, neutral lipids, steroid alcohols and / or esters thereof.

[0358] Said LNPs may be functionalised, for instance with polyethylene glycol (PEG), or a targeting moiety such as proteins (e.g. antibodies and their fragments), peptides, nucleic acids (e.g. aptamers), small molecules, or others (e.g. vitamins or carbohydrates).

[0359] Suitable LNPs which may be used in the present invention include those described in WO2023057444A1 , for instance on pages 29 to 50.

[0360] Alternative nanoparticles which may be used according to the present invention, include lipidoid nanoparticles, oil-in-water emulsion-based cationic nanocarriers (LION), exosomes, chitosan nanoparticles, peptide nanoparticles, polymeric nanoparticles, liposomes, Adeno-associated virus (AAV), and polymer-based polyplex nanoparticles; or nanoparticles made from their combinations.

[0361] The lipid nanoparticles may encapsulate any suitable pharmaceutically active components such as cytotoxic chemotherapy agents, antibiotics or nucleic acids. In preferred embodiments the LNPs encapsulate nucleic acids.

[0362] Suitable nucleic acids include messenger RNA (mRNA), microRNA (miRNA), short (or small) interference RNA (siRNA), small hairpin RNA (shRNA), long noncoding RNA (IncRNA), asymmetrical interfering RNA (aiRNA), self-amplifying RNA (saRNA), a self-replicating RNA (srRNA), a circular RNA (cRNA) or endless RNA (eRNA), a guide RNA (gRNA); or combinations thereof.

[0363] Suitable nucleic acids which may be encapsulated in the LNPs of the present invention include those described in WO2023057444A, for instance on pages 50- 62.

[0364] The encapsulated nucleic acids may be or encode a therapeutic agent. Suitable therapeutic agents include genome-editing polypeptides, chemokines, cytokines, growth factors, antibodies, enzymes, structural proteins, blood proteins, hormones, transcription factors, and antigens.

[0365] Suitable encoded therapeutic agents further include those described in WO2023057444A, for instance on pages 63-68.

[0366] In preferred embodiments, the nanoparticulate drug particles are mRNA containing LNPs (mRNA-LNP).

[0367] The compositions of the present invention are particularly advantageous, as they allow effective delivery of the aggregates of nanoparticulate drug particles to the lungs. In some embodiments, the nanoparticulate drug particles contain therapeutic agents intended for administration to the lung, for instance those targeting respiratory conditions. Due to the direct delivery of such compositions, e.g. mRNA-LNP targeting respiratory conditions, to the lung tissue it can be possible to reduce the dosage required compared with other forms of administration such as injection.

[0368] The aggregates in the dry powder compositions of the present invention typically have volume-weighted median geometric diameters in the range 2 to 100 pm, preferably in the range 2 to 50 pm, more preferably in the range 2 to 25 pm. Preferably, the aggregates have volume-weighted median geometric diameters in the range 2 to 22.4 pm, more preferably in the range 2 to 20 pm. In some embodiments, the aggregates may have volume-weighted median geometric diameters in the range 4 to 18 pm. Aggregate particles having volume-weighted median geometric diameters in the range 2 to 25 pm, such as 2 to 20 pm or 5 to 15 pm may be particularly suitable for delivery by inhalation (i.e. pulmonary delivery). In some embodiments, aggregate particles particularly suitable for delivery by inhalation may have volume-weighted median geometric diameters in the range 4 to 20pm.

[0369] Aggregate particles having volume-weighted median geometric diameters in the range 20 to 100 pm, such as 40 to 70 pm may be particularly suitable for nasal delivery. In some embodiments, aggregate particles suitable for nasal delivery may have volume-weighted median geometric diameters in the range 15 to 115pm and densities between 0.2 and 0.4g / cm3, more preferably they may have volume- weighted median geometric diameters in the range of 15 to 100pm, such as 40 to 70pm, and densities between 0.25 and 0.4g / cm3.

[0370] Where a collection of particles (such as the aggregates of the present invention) is said to have a geometric diameter within a certain range, geometric diameter may be understood to refer to the volume-weighted median geometric diameter of the relevant particles, for instance the aggregates of nanoparticulate drug particles.

[0371] Volume-weighted median geometric diameter, which may be considered to correspond with mass median geometric diameter in the present application, (for instance of the aggregates particles), Dv50, may be measured by a suitable means, such as laser scattering, or may be calculated from dimensions obtained from a microscope image (e.g. light microscope image) of a sample of the aggregates. Unless stated otherwise, volume-weighted median geometric particle sizes and diameters discussed herein are measured by means of laser scattering methods.

[0372] The geometric diameters of individual particles / aggregates may be measured by suitable means such as strobe imaging of the droplets during production, or by imaging with scanning electron microscopy. The geometric diameter size distribution span is calculated from the volume- weighted cumulative particle size distribution: where Dv10, Dv50and Dv90represent cumulative particle size distribution of 10%, 50% and 90% of the powder particle volume, respectively. Preferably, the dry powder aggregates have a substantially spherical shape.

[0373] The size distribution (volume-weighted geometric diameter size distribution) of the aggregates preferably has a span of less than 1 , more preferably less than 0.5, yet more preferably less than 0.2.

[0374] The present invention advantageously provides novel particles of this size, which are particularly advantageous for pulmonary administration. Further, the present invention allows for the production of particles which are of more uniform size than prior art compositions, and which therefore have a low geometric diameter distribution span. This ensures that the aggregates will have more uniform properties and will substantially all be suitable for pulmonary administration. Uniform particle sizes are similarly useful for particles suitable for nasal administration.

[0375] The small particles provided by the present invention are further advantageous, as they allow for fast freezing times, which can avoid crystallisation damage to the nanoparticulate drug particles such as LNPs.

[0376] Additionally, the small particle size may advantageously allow for faster drying due to the high surface area to volume ratio. This allows for a higher level of moisture reduction (providing greater stability and improved efficacy for the nanoparticulate drug particles), and shorter production times. This can also avoid the need for lengthy and complex drying steps needed for larger particles.

[0377] Using methods of the present invention allows for the production of aggregate particles having a high porosity. As discussed, this allows for particles with larger geometric diameters to have lower aerodynamic diameters which may therefore be suitable for pulmonary administration. This porous structure, may also contribute to the advantageous fast freezing and low moisture content. The aggregates of the dry powder compositions of the present invention preferably have a porosity of greater than 50%, more preferably greater than 60%, yet more preferably greater than 85%. In a preferred embodiment, the porosity is between 60% and 95%.

[0378] Particle porosity may be calculated from particle density pPas set out above. When expressed as a percentage, porosity refers to 100 x , i.e.

[0379] 100 where density is specified in g / cm3.

[0380] The aggregates of the dry powder compositions of the present invention preferably have a density of between 0.05g / cm3and 0.4g / cm3.

[0381] Dry lyophilised particles substantially retain the same diameter as the liquid droplets from which they are formed. On account of their low moisture content, the density of lyophilised particles, and thus the aggregates of the dry powder compositions, may therefore be approximately determined as the same as the solid content of the liquid formulation.

[0382] Aggregates having this porosity are advantageous, as this allows for more effective pulmonary delivery. In particular, aggregate particles according to the present invention can have mass median aerodynamic diameters of 1 to 5 pm, more preferably 2 to 5 pm. This allows for effective delivery of the formulations to the lungs without the need for further processing. Such formulations are therefore inherently suitable for inhaler applications.

[0383] Aggregate particles of the invention may be useful for a variety of applications and may have mass median aerodynamic diameters of 1 to 50 pm. In some embodiments, which are particularly suitable for nasal delivery, the aggregate particles may have a mass median aerodynamic diameters of 10 to 50 pm, preferably 20 to 30 pm.

[0384] The aggregates of the dry powder compositions of the present invention preferably have a fine particle fraction of greater than 50%, or preferably 80%, or more preferably 90%.

[0385] The mass aerodynamic diameter distribution of the aggregates preferably has a span of less than 1 , and preferably less than 0.5, and more preferably less than 0.2. Mass aerodynamic diameter span is calculated analogously as for geometric diameter particle size distribution span above.

[0386] The mass median aerodynamic diameter (MMAD) may be measured using methods including time-of-flight spectrometry (such as with an aerodynamic particle sizer, APS), light scattering, or inertial impaction methods. Alternatively, it may be calculated based on the mass median geometric diameter and the density using the equation for DAset out above. Unless stated otherwise, it should be understood that the preferred method of measurement is inertial impaction.

[0387] The aggregates in the dry powder composition are preferably substantially spherical in shape. Specifically, the aggregate particles preferably have an aspect ratio of the largest diameter to the smallest diameter which is less than 1.1 , more preferably less than 1.05. Most preferably, the particles have an aspect ratio of the largest diameter to the smallest diameter which is less than 1.03. As specified herein, a particle (e.g. an aggregate particle) is said to be substantially spherical if it is round and has an aspect ratio of its largest diameter to its smallest diameter of less than 1 .1 as determined by image analysis. As stated herein, when particles such as aggregates are stated as being spherical it is to be understood that at least 90% of the particles are substantially spherical. Preferably at least 95%, more preferably at least 99% of the aggregates are substantially spherical.

[0388] Particles which are substantially spherical are particularly advantageous, as they are more readily entrained in a free flow of gas, and therefore are well suited to pulmonary delivery. The processes described herein advantageously allow the production of such spherical particles compared with other processes, as the surface tension causes the droplets to adopt a spherical shape, and rapid freezing fixes them in this shape. Prior methods typically result in more distorted shapes, for instance due to drag on particles when injected into a fluid, irregular breaking when grinded, or flattening when sprayed onto a surface prior to freezing.

[0389] Having particle sizes in this range is particularly advantageous as it allows for improved delivery of the formulation to the lungs, at least partially due to their reduced mass median aerodynamic diameters.

[0390] The aggregates in the dry powder compositions of the present invention preferably have a moisture content by mass of less than 2%, and preferably less than 1 %. In particularly preferred embodiments, the aggregates have a moisture content below 0.5%.

[0391] Moisture content may be measured by Karl-Fischer moisture analysis or NIR spectroscopy. Unless stated otherwise, moisture content is measured via Karl- Fischer (moisture analysis) Titration.

[0392] By drying the particles to this extent the stability of the compositions is greatly improved. In particular, for LNPs such as mRNA-LNP, such dry compositions may be stable even at above 0C temperatures, thus reducing the costs of storage and distribution of the compositions.

[0393] The dry powder compositions of the present invention may be obtainable by the methods of the invention described hereon.

[0394] Thus, the dry powder compositions of the present invention may be obtainable from aqueous dispersion of nanoparticular drug particles as described herein.

[0395] The aqueous dispersion of nanoparticular drug particles used to form the dry powder via the above method preferably further comprises excipients. Such excipients may be a buffer solution, bulking agents, pH stabilizers, pH adjusters, thermal stabilizers, cryoprotectants, lyoprotectants, antioxidants. Excipients such as cryoprotectants may contribute to stabilize the LNPs during the freeze-drying process.

[0396] The cryoprotectant or lyoprotectants may be selected from disaccharides (such as lactose, trehalose, sucrose, maltose, and mannos), sorbitol, amino acids, peptides, polymers and proteins such as albumins (bovine serum albumin, human serum albumin) or gelatins.

[0397] In some embodiments, trehalose may be used in an amount of 5 to 40 weight (in grams) by volume (in cm3) percent (% w / v) relative to the total volume of the composition. In further embodiments, trehalose may be used in an amount of 10 to 20 weight (in grams) by volume (in cm3) percent (% w / v) relative to the total volume of the composition.

[0398] Using an amount of excipient in the aqueous solution of 5% by mass or more may provide aggregates with good mechanical strength.

[0399] The resultant aggregates take the form of freeze-dried porous particles which are preferably composed primarily of dehydrated excipient, for instance sugars such as trehalose in which the nanoparticulate drug particles are contained. The nanoparticulate drug particles such as mRNA-LNP typically make up 0.1 to 10%, for instance 1 to 5%, of the mass of the aggregate particles.

[0400] Suitable excipients for use in the present invention may be any pharmaceutically acceptable excipient which is compatible with the nanoparticulate drug particles.

[0401] Examples of pharmaceutically acceptable excipients which may be used in the present invention include diluents such as water; physiological salt solutions, such as amino acids buffers (e.g. including histidine, arginine, glycine, proline, glycylglycine or combinations thereof), saline buffers (e.g. including inorganic salts such as NaCI and / or calcium chloride), phosphate buffers, acetate buffers, citrate buffers, succinate buffers; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, lactose, maltose, mannitol; surfactants such as Polysorbate 80, polysorbate 20, poloxamer 188 and the like, as well as combination thereof. Examples of pharmaceutically acceptable excipients which may be used in the present invention include diluents such as water; physiological salt solutions, such as amino acids buffers (e.g. including histidine, arginine, glycine, proline, glycylglycine or combinations thereof), saline buffers (e.g. including inorganic salts such as NaCI and / or calcium chloride), phosphate buffers, acetate buffers, citrate buffers, succinate buffers; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, lactose, maltose, mannitol; surfactants such as Polysorbate 80, polysorbate 20, poloxamer 188 and the like, as well as combination thereof.

[0402] The excipients may include isotonic agents, such as sugars, polyalcohols, or sodium chloride. The excipients may optionally also contain an anti-oxidant such as tryptamine and a stabilizing agent such as Tween 20 or 80, other solvents such as monohydric alcohols, e.g. ethanol or isopropanol, and polyhydric alcohols such as glycols and edible oils such as soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate; binders, adjuvants, solubilizers, thickening agents, stabilizers, disintegrants, lubricating agents, buffering agents, emulsifiers, wetting agents, suspending agents, sweetening agents, colourants, flavours, preservatives, anti-oxidants, processing agents, drug delivery modifiers and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-p-cyclodextrin, polyvinylpyrrolidone and / or polyethylene glycol. Pharmaceutically acceptable excipients may also include any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, and the like that are physiologically compatible.

[0403] The aggregates of the present invention may comprise excipients, such as lyophilised (dehydrated) versions of the above compositions.

[0404] In some embodiments the aqueous dispersion may contain 5 to 40%, for instance 10 to 20%, weight (in grams) by volume (in cm3) percent (% w / v) of sugars, e.g. trehalose, relative to the total volume of the composition. In particular embodiments, the aqueous dispersion may be based on a 50mM Tris-HCI buffer with a pH 7.4 around. More particularly, the aqueous dispersion may be a 10% trehalose solution in 50mM Tris-HCI buffer pH 7.4. The above dispersions may contain additional components, including the nanoparticulate drug particles.

[0405] FIG 20 shows nozzle geometry and shear stress simulation results calculated for flow through nozzle diameters in the range 2 pm to 10 pm, ejecting water at 40m / s.

[0406] FIG 20a shows a nozzle geometry, where the inlet diameter 2012 is 50 pm, the nozzle support thickness 2010 is 50 pm, and the nozzle membrane thickness 2011 is 0.8 pm, although it will be appreciated that there may exist many other suitable geometries.

[0407] This nozzle geometry may be used in conjunction with any of the nozzle arrangements depicts in Figures 8 to 10. For example, the nozzle plate 802 of Figure 8 may be configured as the multi-layered structure of Figure 2. For example, the nozzle plate 802 may include a nozzle support 2010 having the nozzle inlet 2012 of Figure 20a defined therethrough. The nozzle inlet 2012 may be configured to receive a pressurised flow of liquid formulation. The nozzle plate 802 may further comprise a nozzle membrane 2011 adjacent the nozzle support 2010. The nozzle membrane 2011 may include the nozzle outlet 2011 defined therethrough, in line with the nozzle inlet 2021 .

[0408] In this way, the nozzle outlet 2009 may be analogous to the aforementioned apertures in the nozzle plate 802. In a similar manner, the nozzle outlet 2009 may be analogous to the nozzle apertures 1201 of Figure 9 of the fluid outlet of the nozzle plate 1300 of Figure 10.

[0409] The nozzle outlet diameter may be between 0.8pm and 240pm, preferably between 1 pm and 60pm, more preferably between 1 pm and 50pm, and even more preferably between 2pm and 20pm.

[0410] FIG 20b shows the axisymmetric fluidic simulation domain, with inlet 2001 defined to have a flow rate to give an average velocity (i.e. average ejection velocity) of 40m / s at the outlet 2009. The dashed box indicates the region of the enlarged images shown in FIGs 20c, 20d and 20e, which represent simulations of flow through nozzle diameters 2pm, 5pm and 10pm, respectively. The inlet pressures and flow rates for each simulation are as indicated in the Figure titles. Shear stress contours at levels of 100 kPa (2004), 10 kPa (2005) and 1 kPa (2006) are shown. Streamlines 2003 mark the flow paths containing 90% of the flow volume, which just touches the 100 kPa shear stress contour in Figure 20d.

[0411] FIG 20f shows the streamline 2003 and 100 kPa shear stress contour 2004 for nozzle diameters ranging from 2 pm to 20 pm. In each case, the velocity is close to the limit where the streamline 2003 crosses the 100kPa shear stress contour 2004.

[0412] Figure 20g plots the relationship between the limiting velocity and nozzle diameter, where the limiting velocity indicates the ejection velocity above which shear stress damage to LNPs may occur. The relationship is approximately linear with the formula: velocity limit = 3.7 (m / s) / pm x nozzle diameter + 18.7 m / s. Above this velocity limit, shear stress damage to LNPs may occur, as above this limit, the streamline 2003 will cross the 10OkPa contour. Therefore, the preferred operating conditions are below the velocity limit. In some embodiments, the liquid formulation may advantageously be sprayed (e.g. introduced into the vacuum chamber) as a capillary jet having a liquid jet velocity of less than (3.7 x nozzle diameter in pm + 18.7) m / s.

[0413] The threshold for LNP shear stress damage is estimated to be approximately 100 kPa based on a reduction of protein expression of approximately 30% after spraying LNP formulations under conditions similar to Figure 20c, reported in Low energy nebulization preserves integrity of SARS-CoV-2 mRNA vaccines for respiratory delivery, Nature Scientific Reports (2023) 13:8851. Other LNPs may have a higher or lower damage threshold, leading to a different characteristic curve for velocity limit as a function of nozzle diameter.

[0414] FIG 21 shows characterisation results of FLuc-mRNA-LNP (a firefly luciferase (Flue) encoding mRNA encapsulated inside LNP made of SM102 ionisable lipids) before (dotted bars) and after (solid bars) they are sprayed through the droplet generator at atmospheric pressure. FLuc-mRNA-LNPs were prepared by mixing FLuc-mRNA (TriLink™ Biotechnologies, L-7602) in citrate buffer (pH 4.0) with a lipid solution in ethanol at a volume ration of 3:1 using a NanoAssemblR (Cytiva) equipment. The lipid solution in ethanol consisted of four lipids: SM102, DSPC (1 ,2-distearoyl-sn- glycero-3-phosphocholine), cholesterol, and DMG-PEG2000 (1 ,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000) in a molar ration of 50:10:38.5:1.5. The N / P ratio was 6.

[0415] 0.1 mg / ml FLuc-mRNA-LNP dispersion was prepared in 10% (w / v) trehalose solution in 50 mM tris-HCI buffer pH 7.4 for the spray test. The spray dispersion was then fed to a platinum-iridium nozzle with 25 pm aperture vibrating at 120kHz, at a flow rate of 6 pL / s. The droplets generated were collected and characterised for LNP size and size distribution using dynamic light scattering (DLS - Zetasizer Nano ZS, Malvern Panalytics) and for mRNA encapsulation efficiency using Quant-iT RiboGreen Assay kit from Invitrogen using manufacturer’s protocol. For the DLS analysis, the sprayed LNP samples were diluted 5x in the buffer (50 mM tris-HCI buffer pH 7.4).

[0416] FLuc expression in HeLa cells were measured for FLuc-mRNA-LNP samples. 10,000 Cells / well in a 96-well plate, were treated with 200 ng / well (2 pg / mL) FLuc- mRNA-LNPs for 6 hours, then incubated with fresh media for 24 hours. After incubation cells were harvested and bioluminescence, an indication of FLuc expression, was measured using Promega’s ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay according to manufacturer’s protocol. The cell viability assay uses a fluorescence readout and is not technically related to the FLuc assay, although it is performed in the same well on the same samples.

[0417] FIG 21a and FIG 21 b show DLS size and polydispersity index (PDI) measurements for FLuc-mRNA-LNPs before and after spraying. The size of LNPs remain unaltered. The slight increase in PDI is not statistically significant and is due to the limited precision of the measurement. These results indicate that spraying does not cause LNPs to aggregate or change their size or their size distribution. FIG 21c shows that the mRNA encapsulation efficiency is unaffected by the spraying process and the mRNA remains encapsulated inside the LNPs.

[0418] FIG 21 d shows that FLuc expression in HeLa cells does not decrease after spraying indicating that the transfection efficiency of LNPs and the integrity of cargo FLuc-mRNA, remain intact after spraying. The cell viability in is an indicator of number of viable cells expressing FLuc protein and is used as a control for cell numbers.

[0419] Taken together, these results indicate that FLuc-mRNA-LNP remain unaffected by the spraying process establishing gentle droplet generation. Thus, nanoparticulate drug particles, e.g. mRNA LNPs, may be successfully sprayed whilst maintaining their dimensions and biological activity.

[0420] While the foregoing is directed to exemplary embodiments of the present invention, it will be understood that the present invention is described herein purely by way of example, and modifications of detail can be made within the scope of the invention. Indeed, other and further embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and may be devised without departing from the basic scope thereof, which is determined by the claims that follow. All documents referred to herein are hereby incorporated by reference.

Claims

CLAIMS1 . A system for producing a dry powder composition, the system comprising: a vacuum chamber configured to be depressurised by a vacuum pump connected thereto; and a spray head assembly sealably mounted to the vacuum chamber and configured to introduce a liquid formulation into the vacuum chamber; wherein the spray head assembly is further configured to be ultrasonically stimulated during introduction of the liquid formulation into the vacuum chamber, whereby to initiate breakup of the liquid formulation into liquid droplets within the vacuum chamber.

2. The system of claim 1 , further comprising a tray for receiving frozen droplets thereupon, the tray being disposed inside the vacuum chamber and having a surface that can be temperature controlled to maintain a surface temperature between -50C and 20C.

3. The system of claim 1 or 2, further comprising a cold trap located inside the vacuum chamber, the cold trap being configured to reduce the water vapour partial pressure inside the vacuum chamber.

4. The system of any of claims 1 to 3, wherein said vacuum chamber is a first vacuum chamber, the system further comprising a second vacuum chamber maintained a pressure lower than the first vacuum chamber.

5. The system of any preceding claim, wherein the spray head assembly is configured to produce electrostatically charged liquid droplets.

6. The system of any preceding claim, further comprising a flexible container disposed in the vacuum chamber for receiving solidified particles therewithin.

7. The system of claim 6, wherein the container comprises a gas-permeable portion configured to permit gas flow out of the container.

8. The system of 6 or 7, wherein the container comprises a thermoplastic material, the system further comprising a heat-sealing device disposed in the vacuum chamber and configured to heat-seal the container.

9. A method of producing a dry powder composition of lyophilised particles from a liquid formulation, the method comprising the steps of: introducing a liquid formulation into a depressurised vacuum chamber; stimulating the liquid formulation ultrasonically while introducing it into the vacuum chamber so as to initiate breakup of the liquid formulation into liquid droplets; freezing the liquid droplets within the vacuum chamber by means of evaporative cooling to form solidified particles; and sublimating water molecules out of the solidified particles whereby to form the dry powder composition.

10. The method of claim 9, wherein sublimating the solidified particles comprises controlling their temperature using a temperature controlled surface inside the vacuum chamber.

11. The method of claim 9 or 10, wherein the solidified particles are sublimated until they have a moisture content by mass of less than 2%, preferably less than 1 %, and more preferably less than 0.5%.

12. The method of any of claims 9 to 11 , further comprising introducing, via a nozzle, the liquid formulation into the vacuum chamber as a capillary jet having a liquid jet velocity of between 38m / s and 113m / s.

13. The method of claim 12, further comprising ultrasonically stimulating the capillary jet at a frequency which matches a predetermined growth rate instability mode for Rayleigh breakup of the capillary jet.

14. The method of claim 12 or 13, further comprising flowing inert gas surrounding the capillary jet whereby to accelerate the capillary jet.

15. The method of any of claims 11 to 14, further comprising moving the nozzle in a cyclic fashion while introducing the liquid formulation such that the solidified particles are received at multiple locations on a surface within the vacuum chamber.

16. The method of any of claims 9 to 15, further comprising using electrostatic potential to disperse the liquid droplets within the vacuum chamber as they are initially introduced.

17. A dry powder composition which is obtainable by the methods of any one of claims 9 to 16.

18. A dry powder composition, comprising lyophilised aggregates of nanoparticulate drug particles, wherein the aggregates have a volume-weighted median geometric diameter in the range 2 to 100 pm, preferably in the range 2 to 25 pm, and a geometric diameter size distribution with a span of less than 1 , preferably less than 0.2.

19. A dry powder composition according to claim 18, wherein the aggregates have a moisture content by mass of less than 2%, preferably less than 1%, more preferably less than 0.5%.

20. A dry powder composition comprising lyophilised aggregates of nanoparticulate drug particles wherein the particles have a moisture content by mass of less than 0.5%.

21. A dry powder composition according to any of claims 17 to 20, wherein the nanoparticle drugs particles are lipid nanoparticles, preferably wherein the lipid nanoparticles comprise encapsulated nucleic acids, such as messenger ribonucleic acid (mRNA).

22. A dry powder composition according to any of claims 17 to 21 , wherein the aggregates are substantially spherical and have an aspect ratio of the largest diameter to the smallest diameter which is less than 1.1.

23. A dry powder composition according to any of claims 17 to 22, wherein the aggregates have a mass median aerodynamic diameter in the range 1 to 50 pm.

24. A dry powder composition according to claim 23, wherein the aggregates have a mass median aerodynamic diameter in the range 1 to 5 pm.

25. A dry powder composition according to claim 23, wherein the aggregates have a mass median aerodynamic diameter in the range 10 to 50 pm.

26. A dry powder composition according to any of claims 17 to 25, wherein the mass median aerodynamic diameter distribution of the aggregates in the composition has a span of less than 1 , preferably less than 0.2.

27. A dry powder composition according to any of claims 17 to 26, wherein the aggregates have a density of between 0.05g / cm3and 0.4g / cm3.

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