Method for producing mixed lipid particles

Abstract

A method of making an aqueous suspension of mixed lipid particles comprising the steps of: mixing one or more lipids in a solvent or solvent mixture and a suspension of particles of an inorganic material in a solvent or solvent mixture into an aqueous medium; the mixture obtained in step A is then passed through the pores of the extruded film, wherein the average diameter of the mixed lipid particles is at least twice larger than the average diameter of the inorganic material particles. Related products, methods of treatment and medical uses.

Description

TECHNICAL FIELD

[0001] The present invention relates to hybrid lipid particles and methods of manufacturing hybrid lipid particles. In particular, the present invention relates to methods of manufacturing hybrid lipid particles which involve the use of hydrolysable silicon particles to stabilise the hybrid lipid particles in an intermediate state into which an active compound such as an API can be added to form a hybrid lipid particle carrier for that active compound. In particular, the present invention relates to a scalable method of manufacturing hybrid lipid particles which are particularly suitable for use in the delivery of nucleic acid compounds. The present invention also relates to products manufactured by the methods of the present invention or other methods related to the methods of the present invention.

[0002] In certain aspects, the present invention also relates to a convergent manufacturing method for manufacturing hybrid lipid particles for use as active compound carriers, in which the empty carrier is first manufactured and purified, and then loaded with an active compound such as a nucleic acid in a separate step, typically as part of a fill and finish operation. BACKGROUND

[0003] Lipid nanoparticles (LNP) have revolutionised the field of nucleic acid therapeutics by overcoming the major challenges of intracellular delivery of DNA and RNA. They have particularly come to prominence with the mRNA delivery technology in the Moderna and Pfizer / BioNTech COVID-19 vaccines (Wang et al., 2021), and are now the primary delivery mechanism in over 200 ongoing clinical trials of other RNA-based drugs (Curreri et al., 2023). Despite these remarkable achievements, current LNP and general liposomal vesicle formulations still face some recognised deficiencies which must be overcome in order to enable improvements in the clinical translation of RNA-based therapies (Moss et al., 2019; Verma et al., 2023).

[0004] A key limiting factor is the intrinsic chemical and metabolic instability of RNA, especially its hydrolytic instability (Schoenmaker et al., 2021), which poses a major challenge for larger mRNA constructs, which represent the majority of products in clinical development (Curreri et al., 2023). Therefore, most marketed nucleic acid therapeutics contain a large number of chemical modifications to enhance stability and efficacy (Bege & Borbás, 2022; Egli & Manoharan, 2023). Another issue that arises when using LNP as a delivery vehicle is the need to introduce the RNA payload early in the manufacturing process. In practice, since there is no efficient way to encapsulate LNP later, LNP must be formed around the RNA (Nag et al., 2022; Cameau et al., 2022). Due to the instability of RNA, this limits the batch size for commercial manufacturing (Catignol & Lim, 2022) and also impacts the product quality. It is estimated that the mRNA-based Pfizer / BioNTech COVID-19 vaccine Tozinameran retains only 70% of the initial mRNA integrity at the end of the manufacturing process (Daniel et al., 2022). Furthermore, the currently marketed mRNA products (Tozinameran, elasomeran and their derivatives) require cold chain distribution and storage to be clinically and commercially viable. In the long term, this is not ideal for sustainability or global accessibility (Andoh & Yu, 2023; Khairi et al., 2022).

[0005] SiSaf has recently developed silicon-stabilised lipid nanoparticles as an alternative nanocarrier for therapeutic RNA. This vehicle addresses the challenges described above. The addition of hydrolysable silicon modifies the properties of the resulting nanoparticles in a unique and beneficial way, notably conferring superior long-term stability (Saffie-Siebert et al., 2023), and the formulation can be easily adjusted to achieve targeted nucleic acid delivery. The present invention is based on the recognition that silicon-stabilised hybrid lipid nanoparticles can be manufactured in an “empty” form, shipped at room temperature (e.g. as a liquid suspension or a lyophilised powder), and loaded with the desired therapeutic RNA at the point of use.

[0006] RNA is able to penetrate the lipid bilayer of vesicles at room temperature, whereas prior art lipid nanoparticles require higher temperatures (~70-95°C, which depends on the system). Silicon nanostructures facilitate loading at room temperature (RT), which is likely because the lipid bilayer is discontinuous and silicon crosses the vesicle surface. Silicon particles can act as Trojan horses to help RNA enter the lipid bilayer.

[0007] Successful clinical translation technology requires reliable manufacturing processes that can be developed on a large scale. It is well known that subtle changes in the manufacture of active compound carriers can affect the physical and chemical properties of both the active compound carrier and the active compound itself, which in turn can affect the clinical properties of the active compound. Lipid particles, sometimes also referred to as lipid nanoparticles, especially liposomal lipid particles, are of interest, especially for the delivery of active pharmaceutical ingredients (APIs). They are also of interest for the delivery of cosmetics, nutraceuticals, health products, animal and plant health products, and other uses.

[0008] Micellar particles are approximately spherical, formed from lipids (especially lipids with hydrophilic "head" groups, such as phospholipids, and mixtures including lipids with hydrophobic head groups) packed together into supramolecular assemblies, with the hydrophilic tails attracting each other and the hydrophilic heads facing outwards. Micelles have been used to encapsulate delicate APIs, protecting them from the harsher external aqueous environment. For example, US 2018 / 022151 discloses particular lipid particles suitable for encapsulating APIs. Liposomal lipid particles are also approximately spherical. They are composed of a lipid bilayer, which can be thought of as a lipid "balloon" enclosing an internal space, which can be a hydrophilic environment. Alternatively, an aqueous environment can be present inside the liposomal lipid particle. Liposomal lipid particles are particularly suitable for encapsulating active compounds.

[0009] Small lipid particles, especially small liposomal lipid particles, have been found to be useful for protecting active compounds, such as APIs, and for efficiently delivering active compounds, such as APIs. Small particles in this context means particles with a diameter of less than about 200, 150, 100, or 50 nm. Liposomes are especially useful for delivering hydrophilic active compounds, since the internal space of a liposome can provide an aqueous environment for the active compound, protecting it from the external aqueous environment. This can be particularly beneficial, for example, if the external environment contains enzymes or other entities that are capable of destroying the active compound or API.

[0010] Invention discussion

[0011] As discussed in GB2210794.0, both small lipid particles with liposome properties and small lipid particles with micelle properties tend to coalesce into larger particles. The inventors have found that inorganic solid particles, and in particular particles of a hydrolysable silicon-containing material which are themselves smaller than the lipid particles, can "sprinkle" onto the lipid surface and / or "enter" the lipid particle surface (i.e. partially penetrate into the lipid particle, but with some of the inorganic material particles still accessible on the surface), thereby inhibiting the tendency of the lipid particles to coalesce with each other and also situate with any charged lipid components in the lipid particles to protect them and provide the lipid particles with superior ability to complex with certain APIs.

[0012] The inventors have also found that inorganic solid particles, and in particular particles of a hydrolysable silicon-containing material which are themselves smaller than the lipid particles, can penetrate into the lipid particles, thereby providing a route for APIs, in particular nucleic acids, to enter the interior of the lipid particles from the exterior. When the inorganic solid particles are hydrolysable silicon particles, they can optionally be present in the form of aggregates (e.g. chains). These aggregates can extend from the exterior of the lipid particle to the interior of the lipid particle, thereby providing a route for APIs to enter the lipid particle, as further described herein.

[0013] The lipid particles of the present application are characterised as being lipid particles which are different from conventional liposomal lipid particles, with the interior space of the particles remaining accessible. They are referred to herein as "hybrid lipid particles", the term "hybrid" being used to refer to both its lipid properties and components and its non-lipid properties and components (inorganic material particles, e.g. particles of a silicon-containing material). Thus, they contrast with prior art lipid particles in which particles of a silicon-containing material are located in the interior of a lipid coating or liposome. Such prior art products tend to encapsulate the silicon-containing material particles in the lipid. Whilst the silicon-containing particles in such products can provide a beneficial environment inside the encapsulation for the protection of active ingredients or APIs, there is generally no access to the interior of the lipid encapsulation without breaking and subsequently reforming the encapsulation, which presents a significant disadvantage, and there is also no apparent exposure of the silicon-containing particles on the surface of the prior art particles. This means that any interaction between the active ingredients and the silicon-containing particles takes place primarily inside the encapsulation. In some embodiments, the structure of the hybrid lipid particles of the present application can be understood as being an incomplete liposome, with a route for active compounds to pass from the exterior to the interior environment.

[0014] Lipid particles (LPs) similar to those of the present application are generically disclosed in GB 2210794.0 (which is incorporated herein by reference, as it can be obtained at the time of publication or from the disclosure document of other patent applications at the time of their publication, such as international patent applications claiming priority therefrom). They can be manufactured by conventional lipid particle manufacturing techniques. In essence, such techniques can be broadly understood to involve preparing a suitable lipid mixture in a solvent, then evaporating the solvent (e.g. in a rotary evaporator) to form a thin film of lipid material, which is then hydrated to form a lipid particle into which inorganic material particles and active compounds (especially APIs) can be added to form the mixed lipid particles of the present application. Although this manufacturing method can successfully produce the desired product, it can not be efficiently scalable as the evaporation process is limited by the surface area of the evaporation apparatus used, and the surface area of the evaporation apparatus used in a scaled-up process does not increase at the same rate as the volume.

[0015] The present application also relates to improvements to the particles disclosed in GB 2210794.0, which primarily relate to the discovery of the relative sizes of the lipid particles and inorganic material particles, and the fact that they can be made into mixed lipid particles. The present application also relates to improvements in the manufacturing process for these particles, and also discloses beneficial properties of the particles of the present application relating to their mixed nature as mixed lipid particles.

[0016] The mixed lipid particles according to the present application, due to the use of inorganic material particles to stabilize them, are also referred to as "stabilized mixed lipid particles" (shLP), in certain preferred embodiments, these inorganic material particles are particles of hydrolysable silicon, such that the lipid particles are stabilized (especially silicon-stabilized) mixed lipid particles (shLP; especially silicon-stabilized mixed lipid particles sshLP). Such particles are novel lipid nanoparticles (LNP) according to the present application, especially suitable for the delivery of nucleic acids. From a manufacturing perspective, the key difference between the manufacturing process of shLP and prior art LPs is the stage at which the active compound, e.g. nucleic acid, is incorporated into the particle. This difference arises due to the presence of inorganic material particles that stabilize the essentially incompletely formed liposome. From a product perspective, the key difference between shLP and prior art LPs is their mixed nature (as described herein), and the difference in relative size between the lipid particle and the inorganic material particle. From a user perspective, the key difference between shLP and prior art LPs is that shLP can be manufactured "empty", and then loaded with active compound or API, whereas for prior art liposomal lipid particles, the particle needs to be formed "around" the active compound or API to achieve encapsulation of the active compound / API, or harsh conditions such as acids, solvents, or detergents or high temperatures need to be used to disrupt the lipid layer of a preformed liposomal lipid particle to allow the active compound or API to enter the lipid layer, and then reform the lipid layer.

[0017] Prior art methods typically use a sequential manufacturing process, where the active compound, e.g. nucleic acid, is loaded in an initial particle formation step, and then the loaded LP is purified before the filling and final operations are performed. An additional alternative prior art method can involve forming "empty LPs", and then reforming the LP around the active ingredient. In contrast, shLP according to the present application are produced using a fused manufacturing process, where empty shLP are first produced and purified, and then the active compound (especially API, more particularly nucleic acid, e.g. RNA) is loaded in a separate step, typically as part of the filling and final operations. There can be considerable temporal and spatial distance between the formation step and the loading step. This brings a number of practical advantages.

[0018] Mixed lipid particles

[0019] The use of a fusion manufacturing process, where a blank mixed lipid carrier is first prepared and then an active compound (especially an API) is added to it, has advantages but also challenges, which are alleviated by the mixed lipid particles and the manufacturing process of mixed lipid particles provided according to the present application. A major challenge in the prior art is that once a liposome or other lipid carrier is formed, it presents a continuous, unbroken hydrophilic barrier into its interior space. This means that active compounds, especially hydrophilic compounds, such as nucleic acids, cannot easily enter the interior space of the particle (i.e. it cannot easily be encapsulated inside the particle). Methods in the prior art to allow active compounds to enter the interior space of a pre-formed liposomal lipid particle include, for example, using a solvent or a low pH or high temperature to disrupt the liposomal lipid particle, or using a non-fusion manufacturing process where the liposomal carrier is initially formed around the active compound. The first method has the disadvantage that the solvent can damage the carrier or the active compound and adds complexity to the process. The second method has the disadvantage that the formation of the liposomal carrier can require the use of certain conditions, such as the presence of a solvent, a reduced pH and an elevated temperature, which can damage sensitive active compounds and lead to poor control and reduced yield, as the liposomes will need to be disrupted and reformed, and some of the disrupted liposomes can not reform, meaning that the process is not only difficult to control but also leads to reduced yield and loss of lipid components. Thus, the process of the present application includes a method for the active ingredient / API to enter the interior space of a pre-formed mixed lipid particle without the need to use a solvent, a low pH or an elevated temperature to disrupt the lipid barrier for the active ingredient / API to enter. The present application takes a different approach, which uses a mixed lipid particle that has a shell that is predominantly liposomal, due to the presence of inorganic material particles, such as hydrolysable silicon, and the extrusion process used in the manufacture, which does not completely seal the interior of the particle in the absence of the active compound (especially the API). Furthermore, once the active compound or part of it has entered the interior of the particle, the particle "condenses" or "contracts" around the active compound, thereby sealing the active compound inside the particle. This has been found to be particularly effective if the active compound is hydrophilic, and more especially if it is negatively charged, such as if the active compound is a nucleic acid. The mixed lipid particles of the present application can also optionally include inorganic solid particles, such as particles (or aggregates, such as chains) of hydrolysable silicon, which extend from the interior of the mixed lipid particle to the interior of the mixed lipid particle, thereby providing a path for the active compound (especially the API) from the exterior of the mixed lipid particle to the interior of the mixed lipid particle.

[0020] The remote loading method of the present invention also solves the problem of low drug concentration relative to the carrier level, which becomes a greater problem when the drug needs to be tested clinically and eventually marketed as a product.

[0021] The empty shLP according to the present invention can be manufactured using any of the well-established LP manufacturing methods, including evaporation, microfluidics, supercritical fluid or flow injection techniques. Many of these techniques can be optimized to manufacture particles with the desired particle size and low polydispersity. If the initial particles manufactured are too large or have a non-uniform size distribution, membrane extrusion can be used to modify or reform the particles. It has been found that inorganic material particles, such as particles of hydrolysable silicon, which are up to half the size of the mixed lipid particles of the present invention, in combination with manufacturing methods that include membrane extrusion, are able to manufacture particles according to the present invention that have predominantly liposome characteristics, however, the internal space of the mixed particles is not completely sealed from the surrounding medium, whereas once the particles have been loaded with active compounds, in particular APIs, they can preferably become completely sealed liposome particles. In terms of terminology, once the active compounds have been added and the internal space has been completely sealed, the lipid particles can be strictly referred to as liposome lipid particles, rather than mixed lipid particles.

[0022] Once particles with the correct physical characteristics have been obtained, they are purified and concentrated, for example using tangential flow filtration, before being loaded with active compounds, such as nucleic acids.

[0023] This fusion approach to manufacturing shLPs has many advantages over the manufacturing processes of prior art LP products.

[0024] Firstly, the shLPs are cost-effective to manufacture.

[0025] • Degradation of the active compound during particle formation and purification is avoided. This is particularly important if the active compound is a sensitive compound, such as RNA, for example mRNA, saRNA or siRNA.

[0026] • The loading of the active compound into the shLPs is performed under mild conditions; less than 1 hour at a temperature of about 20°C. This is particularly important if the active compound is heat sensitive, such as nucleic acids, in particular RNA, more particularly single-stranded mRNA.

[0027] Secondly, the shLP manufacturing process is scalable and very flexible, capable of operating at production volumes from a few millilitres to several thousand litres, and is therefore suitable for both low and high demand products. The mixed lipid particle formation technology can be adapted according to the production scale required, for example, using microfluidic technology for small batches and flow injection technology for larger batches. The process is also robust and reproducible, enabling materials to be manufactured with consistent composition and physical properties, including lipid content, particle size (e.g. low polydispersity) and zeta potential. Waste of lipid compounds is reduced by eliminating the reforming step which reduces yield, and the amount of initial API used is less by retaining the activity of the API, and is therefore cost effective.

[0028] Finally, shLPs can increase the accessibility of both high and low demand products.

[0029] • High demand: Current LP products typically require ultra-cold chain transport and storage to maintain the activity of sensitive APIs such as nucleic acids, whereas empty shLPs can be stored and transported under refrigerated conditions. Empty shLPs can be easily transported globally, with the loading of active compounds to be carried out as part of local filling and finalisation operations.

[0030] • Low demand: for example, personalised medicines - small quantities of empty shLPs can be supplied to clinics for loading with patient-specific active compounds, for example, patient-specific nucleic acids.

[0031] The present application therefore provides a process for the manufacture of mixed lipid particles, as described in more detail below, which uses a so-called "extrusion" technique (and preferably a process which does not require a solvent evaporation step), in which the solvent used to prepare the lipid mixture and, optionally, to activate the inorganic material particles, is removed from the product after particle formation by a non-evaporative method. The extrusion technique involves forcing the lipid and aqueous components in a suspension of mixed lipid particles through the pores of an extrusion membrane, typically multiple passes, to help form a multiplicity of uniform mixed lipid particles. The present application is based on the discovery that the inclusion of inorganic material particles, especially hydrolysable silicon-containing particles, according to the present application in the mixture to be passed through the extrusion membrane not only enables mixed lipid particles to be formed with similar beneficial properties to those described in GB2210794.0 when loaded with active compounds, especially APIs, but the presence of the inorganic material particles also facilitates the formation of mixed lipid particles with desirable properties.

[0032] In such extrusion processes, the coordination of the extrusion force and the numerical value of the lipid membrane tensile strength is of particular importance for the successful manufacture of lipid particles. In the prior art, it is known that cholesterol can increase the stability of the lipid membrane. However, the problem with cholesterol is that it makes the lipid bilayer more hydrophobic. This changes the kinetics of the formation of the lipid particles, in particular the amount of water available to the extruded membrane pores during the formation of the lipid particles can be insufficient, unless the extrusion force is increased significantly, which leads to the premature rupture of the nascent lipid particles within the extrusion pores, in particular in the case of small pore diameters of the extruded membrane. Therefore, the products of the present application typically employ reduced cholesterol levels, up to complete absence of cholesterol, and include the case of complete absence of cholesterol. In some embodiments, the cholesterol is below 10%, below 5%, or below 1% of the total lipid content by weight. In other embodiments, the products of the present application will not contain significant levels of cholesterol.

[0033] The replacement of cholesterol with particles of inorganic material, in particular particles of hydrolysable silicon, leads to unexpected results. The introduction of particles of inorganic material, in particular particles of hydrolysable silicon, in the membrane extrusion process increases the amount of water available to the extruded membrane pores during the formation of the lipid particles. Therefore, the lipid particles formed at the opening of the pores do not show signs of premature rupture of the nascent lipid particles within the extrusion pores.

[0034] This finding is surprising, as one might have expected that, in order to have a beneficial effect of the particles of inorganic material, for example particles of hydrolysable silicon, on the stability and performance of the mixed lipid particles, a relatively high content of the particles of inorganic material, for example particles of hydrolysable silicon, would need to be retained as part of the lipid particles. This is indeed what is found when lipid particles are prepared by conventional evaporation methods as described in GB2210794.0. Surprisingly, this is not the case when mixed lipid particles are prepared using the extrusion technique according to the present application. The use of the extrusion technique allows for a significant reduction of the level of particles of inorganic material retained as part of the lipid particles, and it has been found that, if a relatively high content of particles of inorganic material is used when mixed lipid particles are manufactured by the extrusion technique, the content of particles of inorganic material in the final mixed lipid particle product can be reduced to a relatively low level during the extrusion process and still exhibit the beneficial properties typically expected of lipid particles prepared by an "evaporation" method in which a relatively high content of particles of inorganic material must be retained in the final lipid particles in order to exert their advantages. Therefore, the present application also relates to mixed lipid particles with a relatively low content of particles of inorganic material, which are prepared by an extrusion technique that includes a relatively high content of particles of inorganic material. The present application also encompasses the corresponding methods.

[0035] Theoretical basis

[0036] Applicants do not wish to be bound by any particular theory relating to the present invention. Two non-mutually exclusive mechanisms can contribute to the inventor's unexpected finding that a relatively low content of inorganic material particles can be used to make a mixed lipid particle with beneficial properties, only to help the understanding that:

[0037] A first theoretical explanation is that for the mixed lipid particles to have beneficial properties, it is most important that the inorganic material particles are present in specific domains or locations on or within the mixed lipid particle where they are tightly bound to the lipid molecules. In this case, it is believed that hydrogen bonds reduce the barrier to active compound (especially API) transport, i.e. increase the permeability of the lipid particle to the active compound. Relatively more inorganic material particles can initially be needed to ensure that all or most of the specific domains are filled, however, once they are filled, the overall level of inorganic material particles can be reduced without harm.

[0038] An alternative or additional explanation can be that the inorganic material (e.g. hydrolysable silicon) can initially need to be present in a relatively high content to assist in dehydrating the mixed lipid particle (i.e. by the silicon hydrolysis reaction consuming water molecules trapped in the mixed structure), and once this initial dehydration is complete, a high content of inorganic material is no longer needed. SUMMARY

[0039] According to a first aspect of the present invention, there is provided a method of making a suspension of mixed lipid particles, comprising the steps of:

[0040] A. mixing a suspension of one or more lipids in a solvent or solvent mixture and inorganic material particles in a solvent or solvent mixture into an aqueous medium; and then

[0041] B. passing the mixture obtained in step A through the pores of an extrusion membrane,

[0042] wherein the average diameter of the mixed lipid particles is at least twice the average diameter of the inorganic material particles.

[0043] According to a second aspect of the present invention, there is provided an aqueous suspension of mixed lipid particles, the average diameter of the mixed lipid particles being from 50 to 150 nm. The mixed lipid particles preferably comprise one or more lipids and inorganic material particles, wherein the average diameter of the mixed lipid particles is at least twice the average diameter of the inorganic material particles.

[0044] According to a third aspect of the present application, there is provided an aqueous suspension of liposomal lipid particles having an average diameter of 50 to 150 nm. The liposomal lipid particles comprise an active compound (in particular an API), one or more lipids and particles of an inorganic material, wherein the average diameter of the mixed lipid particles is at least twice the average diameter of the particles of the inorganic material.

[0045] According to a fourth aspect of the present application, there is provided a method of preparing an aqueous suspension of liposomal lipid particles according to the third aspect of the present application from an aqueous suspension of mixed lipid particles according to the second aspect of the present application, the method comprising the step of contacting the aqueous suspension of mixed lipid particles with an active compound (in particular an API).

[0046] According to a fifth aspect of the present application, there is provided a lyophilized powder of liposomal lipid particles having an average diameter of 50 to 150 nm, the liposomal lipid particles comprising a mixture of one or more cationic lipids or ionizable lipids and one or more further lipids selected from neutral and polar lipids, optionally comprising one or more additional lipid components, the liposomal lipid particles comprising particles of an inorganic material (preferably particles of hydrolysable silicon), the average diameter of the particles of the inorganic material being at most half the average diameter of the liposomal lipid particles, wherein the weight ratio of the particles of the inorganic material to the lipids is 1 :2 to 1 : 100 (preferably 1 : 10 to 1 : 100, more preferably 1 :20 to 1 : 10), and wherein the liposomal lipid particles further comprise one or more active compounds, such as one or more active pharmaceutical ingredients (APIs), at least a portion of which is encapsulated within the interior of the liposomal particles.

[0047] According to a sixth aspect of the present application, there is provided a lyophilized powder of mixed lipid particles having an average diameter of 50 to 150 nm, the mixed lipid particles comprising a mixture of one or more cationic lipids or ionizable lipids and one or more further lipids selected from neutral and polar lipids, optionally comprising one or more additional lipid components, the mixed lipid particles comprising particles of an inorganic material (preferably particles of hydrolysable silicon), the average diameter of the particles of the inorganic material being at most half the average diameter of the mixed lipid particles, wherein the weight ratio of the particles of the inorganic material to the lipids is 1 :2 to 1 : 100 (preferably 1 : 10 to 1 : 100, more preferably 1 :20 to 1 : 10).

[0048] According to a seventh aspect of the present application, there is provided a pharmaceutical composition comprising an aqueous suspension of mixed lipid particles of the present application or a lyophilized powder of mixed lipid particles of the present application, or an aqueous suspension of liposomal lipid particles of the present application or a lyophilized powder of liposomal lipid particles of the present application.

[0049] According to other aspects of the present application, there are provided the use of the pharmaceutical composition of the present application as a medicament, and related methods of medical treatment.

[0050] The present application also provides an aqueous suspension of mixed lipid particles comprising a pharmaceutically active ingredient (API) manufactured by a process comprising the steps of:

[0051] (a) obtaining an aqueous suspension of mixed lipid particles manufactured by the steps of:

[0052] (i) mixing one or more lipids in a solvent or solvent mixture and a suspension of inorganic material particles in a solvent or solvent mixture into an aqueous medium;

[0053] (ii) passing the mixture obtained in step (i) through the pores of an extrusion membrane; and

[0054] (iii) optionally, purifying and / or sterilizing the suspension by tangential flow filtration; and

[0055] (b) contacting the mixed lipid particles with an active compound, in particular a pharmaceutically active ingredient (API).

[0056] The present application also provides an aqueous suspension of mixed lipid particles comprising a pharmaceutically active ingredient (API) manufactured by a process comprising the steps of:

[0057] (a) obtaining an aqueous suspension of mixed lipid particles manufactured by the steps of:

[0058] (i) mixing one or more lipids in a solvent or solvent mixture and a suspension of inorganic material particles in a solvent or solvent mixture into an aqueous medium;

[0059] (ii) passing the mixture obtained in step (i) through the pores of an extrusion membrane; and

[0060] (b) purifying and / or sterilizing the suspension by tangential flow filtration. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1A Schematic illustration of the manufacturing process for certain embodiments of the present application.

[0062] Figure 1B Illustrative showing of the more detailed manufacturing process according to specific embodiments.

[0063] Figure 2 Illustration of the manufacturing strategy overview for generating scalable processes for manufacturing micellar lipid particles.

[0064] Figure 3 Illustration of the micellar lipid particle manufacturing process by evaporation methods.

[0065] Figure 4 Visual observation of the solution obtained with sample MVI0001 is shown.

[0066] Figure 5 DLS and zeta potential analysis results of sample MVI0001 during the extrusion step are shown.

[0067] Figure 6 General procedure to generate sample MVI0002 is shown.

[0068] Figure 7 Visual observation of the solution obtained with sample MVI0002 is shown.

[0069] Figure 8 DLS and zeta potential analysis results of sample MVI0002 during the extrusion step are shown.

[0070] Figure 9 General procedure to generate sample MVI0003 is shown.

[0071] Figure 10 Visual observation of the solution obtained with sample MVI0003 is shown.

[0072] Figure 11 DLS and zeta potential analysis results of sample MVI0003 during the extrusion step are shown.

[0073] Figure 12 Visual observation of the solution obtained with sample MVI0004 is shown.

[0074] Figure 13 DLS and zeta potential analysis results of sample MVI0004 during the extrusion step are shown.

[0075] Figure 14 Representative pictures of the visual observation of the solution obtained with sample MVI0005 are shown.

[0076] Figure 15 DLS and zeta potential analysis results of sample MVI0005 during the extrusion step are shown.

[0077] Figure 16 DLS and zeta potential analysis results of sample MVI0006 during the extrusion step are shown.

[0078] Figure 17 Experimental setup of sample MVI0007 using a 0.8 pm membrane is shown.

[0079] Figure 18 Visual observation of the solution obtained with sample MVI0007 during the experiment is shown.

[0080] Figure 19A Pictures of 0.4 μιη and 0.1 μιη films after extrusion without using 0.8 μιη film are shown.

[0081] Figure 19B Pictures of 0.8 μιη, 0.4 μιη and 0.1 μιη films after extrusion are shown.

[0082] Figure 20 DLS and zeta potential analysis results of MVI0007 during the extrusion step are shown.

[0083] Figure 21 Investigation results showing the effect of dilution on DLS and zeta potential measurements Figure 21 A: 20x dilution factor; Figure 21 B: 5x dilution factor).

[0084] Figure 22 Visual observation results of the solutions obtained in each step of experiment MVI0008 are shown.

[0085] Figure 23 DLS and zeta potential analysis results of MVI0008 during the extrusion step are shown.

[0086] Figure 24 Experimental setup of MVI0011 is shown.

[0087] Figure 25 Visual observation results of the solutions obtained in each step of experiment MVI0011 are shown.

[0088] Figure 26 DLS and zeta potential investigation results of MVI001 are shown.

[0089] Figure 27 Visual observation results of the solutions obtained in each step of experiment MVI0012 are shown.

[0090] Figure 28 Film after extrusion is shown.

[0091] Figure 29 DLS and zeta potential investigation results of MVI0012 are shown.

[0092] Figure 30 Pressure during extrusion in experiment MVI0007 using a 25 mm diameter film and an extrusion volume of 50 ml is shown.

[0093] Figure 31 Pressure during extrusion in experiment MVI0011 using a 25 mm diameter film and an extrusion volume of 100 ml. The SiNP solution was filtered with a 0.8 μιη syringe filter prior to extrusion is shown.

[0094] Figure 32 The pressure during extrusion is shown for experiment MVI0012, which used a 47 mm diameter film, with an extrusion volume of 1 L.

[0095] Figure 33 The chromatogram of the different lipid components is shown.

[0096] Figure 34 The chromatogram of the lipid mixture is shown.

[0097] Figure 35 The gradient curve for the modified and initial HPLC method is shown.

[0098] Figure 36 The chromatogram of the lipid mixture with final product concentrations of [DOTAP-Cl] = 0.725 mg / ml, [DOPE] = 0.73 mg / ml and [mPEG2000'DSPE] = 0.145 mg / ml is shown.

[0099] Figure 37 The calibration curve for the various lipids in MVI0010 is shown.

[0100] Figure 38 The chromatogram of experiment MVI0010 is shown.

[0101] Figure 39 The calibration curve for experiment MVI0017 is shown.

[0102] Figure 40 The chromatogram measured for undiluted sample MVI0017 is shown.

[0103] Figure 41 The calibration curve for MVI0017 using a diluted standard lipid solution is shown.

[0104] Figure 42 The chromatogram measuring 2x diluted sample MVI0017 is shown.

[0105] Figure 43 The chromatogram of nuclease-free water and THR-GLY solution is shown.

[0106] Figure 44 The chromatogram of the sample stored at RT at different time points is shown.

[0107] Figure 45 The change in lipid concentration over time in the sample analyzed at RT is shown.

[0108] Figure 46 The chromatogram of the sample stored at RT at different time points is shown.

[0109] Figure 47Lipid concentration over time in samples analyzed at 4°C is shown.

[0110] Figure 48 TFF settings used in the examples are shown.

[0111] Figure 49 MeOH calibration curve is shown.

[0112] Figure 50 Pre- and post-TFF MVI0010 is shown. 1 H-NMR signal.

[0113] Figure 51 Experimental setup for MVI0013 using a pre-filtration step is shown.

[0114] Figure 52 Visual observations of the solutions obtained in each step of experiment MVI0013 are shown.

[0115] Figure 53 Pictures of a 0.8 pm hydrophilic polyether sulfone syringe filter (A) and an extruded membrane (B) after filtration and extrusion, respectively, are shown.

[0116] Figure 54 DLC and zeta potential analysis results of MVI0013 in the extrusion step are shown.

[0117] Figure 55 Extrusion pressure during the investigation of experiment MVI0013 is shown.

[0118] Figure 56 Zeta potential analysis results of MVI0013 are shown.

[0119] Figure 57 Experimental setup for MVI0014 is shown.

[0120] Figure 58 Visual observations of the solutions obtained in each step of experiment MVI0014 are shown.

[0121] Figure 59 DLS and zeta potential analysis results of MVI0014 in the extrusion step are shown.

[0122] Figure 60 Extrusion pressure investigation results of MVI0014 are shown.

[0123] Figure 61 Experimental setup for MVI0015 is shown.

[0124] Figure 62 Visual observations of the solutions obtained in each step of experiment MVI0015 are shown.

[0125] Figure 63 DLS and zeta potential analysis results of MVI0015 in the extrusion step are shown.

[0126] Figure 64 The experimental procedure for producing 2 kg batches is shown.

[0127] Figure 65 Pictures of the extruded film after the extrusion step are shown.

[0128] Figure 66 DLS and zeta potential investigation results of MVI0022 are shown.

[0129] Figure 67 Trend of the size increase with the extrusion volume is shown.

[0130] Figure 68 Various possible configurations for fast mixing according to certain embodiments of the application are shown.

[0131] Figure 69 Long-term stability of the mixed lipid particles of the application compared to lipid particles without silicon or other inorganic material particles is shown.

[0132] Figure 70 The size stability of the particles of the application remains after loading of mRNA is shown.

[0133] Figures 71 to 81 Methods and results related to Examples 17 and 18 are shown.

[0134] Figure 71 (A) Flow chart of the original lipid film hydration method (Method 1) where the organic solvent is evaporated from the activated silicon nanoparticles and the improved protocol (Method 2) where the activated silicon nanoparticle suspension is added directly to the aqueous phase is shown. (B) More settling is observed in Method 2 (lower panel) after the lipid film hydration. (C) DLS results show that the mixed lipid particles produced using the improved method have lower PDI and higher zeta potential.

[0135] Figure 72Method to remove the second evaporation step and optimize solvent injection mixing is shown schematically. (A) Flow diagram of the initial direct injection mixing method (Method 3). (B) Reduced sedimentation was observed compared to lipid film hydration. (C) The modified flow diagram incorporates an additional 0.8 pm extrusion step, and other adjustments as shown. (D) The additional extrusion step (Method 4) yields smaller mixed lipid particles with similar PDI and zeta potential. (E) Pre-filtration prior to extrusion (Method 6, right) removes insoluble aggregates that would otherwise accumulate on the 0.8 pm extrusion membrane (Method 4, left), although the 0.4 pm membrane appears to remove additional aggregated material. (F) Incorporation of the pre-filtration step does not affect the properties of the final mixed lipid particles.

[0136] Figure 73 Purification of the mixed lipid particles of the application by TFF is shown. (A) Schematic of the experimental setup illustrating how the second pump (Pump 2) was included to enable ultrafiltration and diafiltration operations. (B) Representative results illustrating how the TFF method was able to remove MeOH from the mixed lipid particles prepared by Method 5, as judged by H NMR. 1 H NMR. (C, D) The TFF method was found to slightly alter the DLS characteristics (C) and lipid content (D) of the particles, but these changes were within the relevant reference ranges and therefore not considered problematic.

[0137] Figure 74 Initial large scale (1 L) run to manufacture mixed lipid particles is shown. (A) Schematic of the flow used; scale up of Method 5. (B) Representative images of the extrusion membranes and sample aliquots (lower right portion) showing that aggregates still accumulated and insoluble material remained after the first extrusion step despite the use of larger filtration membranes. (C) Accumulation of aggregates on the 0.8 pm and 0.4 pm membranes resulted in significantly higher operating pressures than the 50 mL scale (Method 4). (D) The DLS characteristics of the mixed lipid particles were not adversely affected and were within the reference ranges.

[0138] Figure 75 Manufacture of mixed lipid particles in a 2 L batch is shown. (A) Schematic of the flow used. Notably, the TFF step included two stages: ultrafiltration to concentrate the sample from 4 L to 2 L, followed by diafiltration to remove MeOH and free lipid. (B) The extrusion step was compromised by the large accumulation of aggregates on the membrane, which affected its performance. (C) The average particle size and PDI of the finished batch were outside the reference ranges. (D) This was due to the deterioration of the extrusion membrane and excessive accumulation of aggregates, indicating that a pre-filtration step after activating the mixed lipid particles is needed when manufacturing mixed lipid particles at a large scale.

[0139] Figure 76Figure 6 shows that pre-filtration of the activated silicon nanoparticle suspension improves the process. (A) Schematic of the improved process, named Method 7. (B) In this case, there is no visible accumulation of aggregates on the extrusion membrane. (C-E) This is reflected in a significantly lower operating pressure during extrusion compared to the protocol without pre-filtration (C), and in both small scale (D) and 1 L batch size (E) resulting in sshLNPs with DLS characteristics within the reference ranges.

[0140] Figure 77 Figure 7 shows that mixed lipid particles manufactured in small scale (using the lipid film hydration method, Method 2) and in large scale (using Method 7) exhibit similar physical and functional properties. (A) The final particles manufactured in large scale are slightly smaller, but the PDI is comparable. (B) The zeta potential is not affected by the scale-up. (C) Mixed lipid particles manufactured in both methods have equivalent RNA encapsulation efficiency. (D) Large scale manufacturing also preserves the efficiency of transfection of HEK293 cells with mRNA encoding firefly luciferase (fLuc). Fluorescence intensity was measured to determine the expression level of fLuc 24 h after transfection. Lipofectamine 2000 was used as a positive control and untreated cells as a negative control. Graphs (A-C) show the mean ± SD of samples analyzed in triplicate, while graph D shows the mean ± SD of three independent biological replicates.

[0141] Figure 78 Figure 8 shows that inverse mixing after activation of the silicon nanoparticles does not affect the properties of the mixed lipid particles. (A) Schemes of Methods 4 and 5, showing only the difference in mixing order. (B) DLS-derived parameters comparing samples prepared by Methods 4 and 5. While the final zeta potential of this run was lower (right panel), subsequent experiments showed that Method 5 can reliably manufacture mixed lipid particles with zeta potential within the reference range, including in large scale manufacturing.

[0142] Figure 79 Figure 9 shows that the introduction of a pre-filtration step (Method 6) before extrusion significantly reduces the operating pressure of the 0.8 pm and 0.4 pm extrusion membranes compared to the process without pre-filtration (Method 4).

[0143] Figure 80 shows development of HPLC-based lipid recovery assay method. (A) Chromatogram of mixed lipid standard solution, where the molar ratios of DOTAP, DOPE, and mPEG2000-DSPE components are the same as when the mixed lipid particles are prepared. (B) Calibration curve for the lipid reference solution used in the assay of Method 5. (C) Chromatogram of the post-extrusion sample made using Method 5, showing the presence of two unidentified impurity peaks. In this sample, the observed lipid recoveries for DOTAP, DOPE, and mPEG2000-DSPE were 86%, 81%, and 76%, respectively. (D) An alternative UPLC assay method is being developed, especially aimed at improving the peak shape of the PEGylated lipids. The chromatogram shows representative results for a mixed lipid standard solution.

[0144] Figure 81 (A) shows the increase in membrane surface area available for extrusion by using two sets of parallel extrusion membranes.

[0145] Figure 81 (B) shows the combination of pre-filtration of activated silicon nanoparticles, which prevents the accumulation of residuals on the 0.8 pm and 0.4 pm membranes. The 0.1 pm membrane appears yellow, indicating that smaller aggregates and / or oversized particles have been removed from the sample.

[0146] Figure 82 A transmission electron microscope (TEM) image of a mixed lipid particle according to an embodiment of the application. It shows the aggregation of silicon particles as described herein. The particles labeled "lipid" are mixed lipid particles according to an embodiment of the application. The image also shows relatively small silicon particles that are part of an aggregate extending from the outside of a mixed lipid particle to the inside of the mixed lipid particle. DETAILED DESCRIPTION

[0147] According to a first aspect of the application, there is provided a method of manufacturing a suspension of mixed lipid particles, comprising the steps of:

[0148] A. mixing a suspension of one or more lipids in a solvent or solvent mixture and of inorganic material particles in a solvent or solvent mixture into an aqueous medium; and then

[0149] B. passing the mixture obtained in step A through the pores of an extrusion membrane,

[0150] wherein the average diameter of the mixed lipid particles is at least twice as large as the average diameter of the inorganic material particles.

[0151] According to a second aspect of the present application, there is provided an aqueous suspension of mixed lipid particles having an average diameter of 50 to 150 nm. The mixed lipid particles comprise one or more lipids and inorganic material particles, wherein the average diameter of the mixed lipid particles is at least twice the average diameter of the inorganic material particles.

[0152] According to a third aspect of the present application, there is provided an aqueous suspension of liposomal lipid particles having an average diameter of 50 to 150 nm. The liposomal lipid particles comprise an active compound (in particular an API), one or more lipids and inorganic material particles, wherein the average diameter of the mixed lipid particles is at least twice the average diameter of the inorganic material particles.

[0153] According to a fourth aspect of the present application, there is provided a method of preparing an aqueous suspension of liposomal lipid particles according to the third aspect of the present application from an aqueous suspension of mixed lipid particles according to the second aspect of the present application, the method comprising the step of contacting the aqueous suspension of mixed lipid particles with an active compound (in particular an API).

[0154] According to a fifth aspect of the present application, there is provided a lyophilized powder of liposomal lipid particles having an average diameter of 50 to 150 nm, the liposomal lipid particles comprising a mixture of one or more cationic or ionizable lipids and one or more further lipids selected from neutral and polar lipids, optionally comprising one or more additional lipid components, the liposomal lipid particles comprising inorganic material particles (preferably particles of hydrolysable silicon) having an average diameter of at most half the average diameter of the liposomal lipid particles, wherein the weight ratio of inorganic material particles to lipids is 1 :2 to 1 : 100 (preferably 1 : 10 to 1 : 100, more preferably 1 :20 to 1 : 10), and wherein the liposomal lipid particles further comprise one or more active compounds, such as one or more active pharmaceutical ingredients (APIs), at least a portion of which is encapsulated within the interior of the liposomal particles.

[0155] According to a sixth aspect of the present application, there is provided a lyophilized powder of mixed lipid particles having an average diameter of 50 to 150 nm, the mixed lipid particles comprising a mixture of one or more cationic or ionizable lipids and one or more further lipids selected from neutral and polar lipids, optionally comprising one or more additional lipid components, the mixed lipid particles comprising inorganic material particles (preferably particles of hydrolysable silicon) having an average diameter of at most the average diameter of the mixed lipid particles, wherein the weight ratio of inorganic material particles to lipids is 1 :2 to 1 : 100 (preferably 1 : 10 to 1 : 100, more preferably 1 :20 to 1 : 10).

[0156] According to a seventh aspect of the application, there is provided a pharmaceutical composition comprising: an aqueous suspension of mixed lipid particles of the application or a lyophilized powder of mixed lipid particles of the application, or an aqueous suspension of liposomal lipid particles of the application or a lyophilized powder of liposomal lipid particles of the application.

[0157] According to other aspects of the application, there is provided the use of a pharmaceutical composition of the application as a medicament, and related methods of medical treatment.

[0158] The application also provides an aqueous suspension of lipid particles comprising a pharmaceutically active ingredient (API) manufactured by a process comprising the steps of:

[0159] (a) obtaining an aqueous suspension of mixed lipid particles manufactured by the steps of:

[0160] (i) mixing one or more lipids in a solvent or solvent mixture and a suspension of inorganic material particles in a solvent or solvent mixture into an aqueous medium;

[0161] (ii) passing the mixture obtained in step (i) through the pores of an extrusion membrane; and

[0162] (iii) optionally, purifying and / or sterilizing the suspension by tangential flow filtration; and

[0163] (b) contacting the mixed lipid particles with an active compound, in particular a pharmaceutically active ingredient (API).

[0164] The application also provides an aqueous suspension of mixed lipid particles comprising a pharmaceutically active ingredient (API) manufactured by a process comprising the steps of:

[0165] (c) obtaining an aqueous suspension of mixed lipid particles manufactured by the steps of:

[0166] (i) mixing one or more lipids in a solvent or solvent mixture and a suspension of inorganic material particles in a solvent or solvent mixture into an aqueous medium;

[0167] (ii) passing the mixture obtained in step (i) through the pores of an extrusion membrane; and

[0168] (d) purifying and / or sterilizing the suspension by tangential flow filtration.

[0169] Inorganic material particles

[0170] According to all aspects of the application, the inorganic material particles can be of any inorganic material, including a mixture of one or more inorganic materials. In preferred embodiments of all aspects of the application, the inorganic material particles are particles of hydrolysable silicon or include particles of hydrolysable silicon. Preferably, such particles including hydrolysable silicon consist of at least 70%, at least 80%, at least 90%, most preferably at least 98% of hydrolysable silicon (collectively referred to as "particles of hydrolysable silicon" or interchangeably as "particles of hydrolysable silicon").

[0171] Solvents and activating solvents and mixtures thereof

[0172] According to the methods of the application, the inorganic material particles can optionally be activated by exposing them to a solvent or a mixture of solvents. Preferably, the solvent or mixture of solvents is an activating solvent or activating solvent mixture. An activating solvent or activating solvent mixture can be understood as a solvent or mixture of solvents that "activates" the inorganic material particles, preferably particles of hydrolysable silicon. Activation is to be understood as including washing contaminants from the particles to enable the particles to interact fully with other ingredients present in the methods or products of the application. "Activation" can also include removing contaminants from any pores present in the particles. Contaminants include soluble contaminants, such as surface oxides and hydroxides, and solid contaminants, such as surface "dust". Preferably, the solvent is an organic compound, such as a volatile organic compound, such as an alcohol. Preferably, the activating solvent mixture includes methanol. More preferably, the activating solvent is methanol. The activating solvent can optionally be allowed to evaporate from the inorganic material particles before the methods of the application proceed to subsequent steps. Alternatively, the activating solvent is not pre-evaporated. According to certain embodiments, the activating solvent is toxic and / or not approved as a pharmaceutical ingredient. It is to be understood that the solvent or mixture of solvents supplying the one or more lipids can be the same solvent or mixture of solvents as the solvent or mixture of solvents suspending the inorganic material particles or a different solvent or mixture of solvents.

[0173] According to certain preferred embodiments of all aspects of the application, "activation" by a solvent or mixture of solvents includes surface treatment of the inorganic material particles, which are particles of hydrolysable silicon or include particles of hydrolysable silicon. Surface treatment with a solvent or mixture of solvents is to be understood as surface treatment with one or more alcohols. Especially preferred alcohols include methanol, benzyl alcohol and methanol (or mixtures thereof). "Surface treatment" of hydrolysable silicon can optionally include formation of Si-O(CH2) x CH3(wherein x is 0 for methanol and 1 for ethanol, etc.), Si-(CH2) x CH3and / or Si-H moieties, wherein -(CH2) xCH3may be replaced by other carbon-containing groups, such as aromatic rings, for example, the alcohol is benzyl alcohol or includes benzyl alcohol.

[0174] Hydrolysable silicon

[0175] As used herein, "hydrolysable silicon" includes pure elemental silicon. However, complete purity is not required. Rather, the present application is not intended to encompass pure silicon dioxide (including sand, quartz, silica gel). The key requirement is that the material is hydrolysable, that is, it will tend to break down into soluble products, such as orthosilicic acid (OSA), under physiological conditions. According to certain embodiments, the definition of "hydrolysable silicon" is satisfied if at least half of the mass of the material hydrolyzes to soluble products within one month of injection into a subject (e.g., following intramuscular or subcutaneous injection).

[0176] The hydrolysable silicon according to certain embodiments of the application is preferably mesoporous silicon. That is, it contains pores having a diameter of 2 to 50 nm.

[0177] The hydrolysable silicon particles can be commercially available or can be manufactured by any suitable method.

[0178] In certain embodiments, the particles comprising the hydrolysable silicon can be pure silicon or substantially pure silicon.

[0179] Alternatively, the particles can be a material that contains another hydrolysable silicon. If the particles are not pure silicon, they comprise at least about 50% silicon by weight, that is, they comprise at least about 50% silicon atoms by weight based on the total mass of atoms in the particles. For example, the silicon particles can contain at least about 60% by weight, about 70% by weight, about 80% by weight, about 90% by weight, or about 95% by weight silicon. The rate of hydrolysis of these particles (e.g., in PBS buffer at room temperature) can be shown to be at least 10% of the rate of hydrolysis of pure silicon particles of the same size. Determination of the hydrolysis of silicon-containing materials is well known in the art; see, for example, WO 2011 / 001456, which is incorporated herein by reference in its entirety.

[0180] While the particles can contain trace amounts of silicon dioxide, silicon dioxide is not hydrolysable silicon. At least about half of the silicon atoms in the particles can be in the form of elemental silicon (or doped elemental silicon).

[0181] The particles can be, inter alia, nanoparticles. Nanoparticles according to certain embodiments can have a nominal diameter in the range of about 1 to about 500 nm, in particular about 1 to about 250 nm, more in particular about 1 to about 100 nm (e.g., about 30 nm). As used herein, the term "nominal diameter" can refer to the average diameter, and at least about 90% of the total mass of the particles in a sample can fall within the specified size range.

[0182] The particles can be porous, especially mesoporous. Particles comprising hydrolysable doped silicon can be made porous by standard techniques, for example by contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and applying an electric current. By varying the HF concentration, current density and exposure time, the density of the pores and their size can be controlled and can be monitored by scanning electron microscopy and / or nitrogen adsorption-desorption volumetric isotherm measurements. If the particles are porous, their total surface area will increase due to their porosity. For example, their surface area can increase by at least about 50% or at least about 100% compared to the surface area of the corresponding non-porous particles. In many cases, the porous particles will in fact increase their total surface area by more due to their porosity. According to certain embodiments, the porosity is at least about 30%, about 40%, about 50% or about 60%; this means that at least about 30%, about 40%, about 50% or about 60% of the volume of the particle is pore space, respectively. The pore diameter can be in the range of about 1 nm to about 50 nm, for example about 1 nm to about 5 nm.

[0183] doped silicon

[0184] Preferably, the hydrolysable silicon used in all aspects of the application comprises (or consists of) hydrolysable doped silicon.

[0185] As used herein, the term "doped silicon" can refer to silicon that behaves as an extrinsic semiconductor due to the presence of dopant atoms, whether substitutional (replacing Si atoms) or interstitial (located between Si atoms, rather than replacing Si atoms).

[0186] Advantageously, the silicon particles have a doping level of at least about 1 x 1018dopant atoms / cm3, especially at least about 1 x 1019dopant atoms / cm3. 15 3 , especially at least about 1 x 1019dopant atoms / cm3. 16 3 .

[0187] For example, the particles can have a doping level of at least about 1 x 1018dopant atoms / cm3, at least about 1 x 1019dopant atoms / cm3, at least about 1 x 1020dopant atoms / cm3, or at least about 1 x 1021dopant atoms / cm3. 17 3 18 3 19 3 .

[0188] The silicon particles can have a doping level of at most 1 x 1021dopant atoms / cm3. 20 3 .

[0189] ​​​​​​​​The silicon particles can be n-doped or p-doped. The silicon particles can be doped with one or more elements selected from the group consisting of B, P, Mg, Cu, Ga, Al, In, Bi, Ge, Li, Xe, N, Au and Pt. Thus, the dopant can be a p-dopant, in particular boron. The dopant can be an n-dopant, in particular phosphorous.

[0190] When boron is preferably used as dopant, a doping level of 1 x 1019 15 doping atoms / cm 3 and 1 x 1019 20 doping atoms / cm 3 corresponds to a resistivity of 13.6 Ω-cm and 1.3 mΩ-cm, respectively. Embodiments with boron as dopant do not exclude silicon which is doped with boron (e.g. heavily doped), but additionally doped with other elements (preferably, in this case, boron is the main dopant).

[0191] The term "heavily doped" as used herein is understood to mean doped with at least about 1 x 1020 15 doping atoms / cm 3 . In some preferred embodiments, the doping level of the dopant is at least about 1 x 1020 16 doping atoms / cm 3 . Thus, in particularly preferred embodiments, the dopant is boron with a doping level of at least about 1 x 1020 16 boron atoms / cm 3 . For example, the doping level of boron is at least about 1 x 1020 16 boron atoms / cm 3 and at most about 1 x 1021 20 boron atoms / cm 3 .

[0192] "Undoped" silicon as referred to herein (e.g. the particles of composition SIS0012 in Examples 1 and 2) can mean that no or only a small amount of doping atoms are present, e.g. at most about 1 x 1019 2 doping atoms / cm 3 . Additionally, or alternatively, "undoped" silicon can mean that the silicon does not behave as an extrinsic semiconductor.

[0193] Doped silicon particles can improve various functions described herein, in particular the ability to enter the interior of the hybrid lipid particles according to the application. Additionally, or alternatively, doped silicon particles can improve the stability of active compounds, in particular APIs, in the interior of the liposome particles according to the application.

[0194] The semiconductor industry provides ready sources of appropriately doped silicon and a wealth of expertise in silicon doping techniques. The manufacture of doped silicon is well known in the semiconductor industry, including ion implantation and diffusion methods, which makes doped silicon itself readily available. As an example of a diffusion method, silicon powder and a dopant (e.g., B2O3 for boron doping) are mixed in an N2atmosphere at a temperature of 1050 °C - 1175 °C for several minutes to allow diffusion of the dopant (e.g., boron) into the silicon.

[0195] Manufacture of inorganic material particles

[0196] It will be appreciated that the particles can be prepared by a variety of techniques familiar to the skilled person.

[0197] These techniques can include, for example, purely physical (sometimes referred to in the art as "non-wet") methods starting from bulk inorganic solid material (especially silicon wafers), such as pulsed laser ablation, thermal degradation and ball milling. Thus, the particles can be obtained by a method comprising or consisting of one or more of pulsed laser ablation, thermal degradation and ball milling of bulk inorganic material (especially silicon wafers).

[0198] Additionally, or alternatively, the particles can be manufactured by chemical (sometimes referred to in the art as "wet") techniques, including but not limited to electrochemical etching of bulk inorganic material (especially silicon wafers). Such techniques optionally include the HF etching described above. Thus, the particles can be obtained by a method comprising or consisting of electrochemical etching of bulk inorganic material (especially silicon wafers).

[0199] Once the inorganic material particles (e.g., particles of hydrolysable silicon) are formed, they can be sorted by size, for example by air sifting, sieving and / or filtering. Thus, the particles can be obtained by a method comprising one or more of air sifting, sieving and / or filtering.

[0200] Thus, for example, the particles can be obtained by a method comprising: manufacturing inorganic solid material particles from a solid material (especially a silicon wafer), for example by one or more of pulsed laser ablation, thermal degradation, ball milling and electrochemical etching of bulk inorganic material (especially silicon wafers); and then, optionally, sorting by size, for example by air sifting, sieving and / or filtering.

[0201] Optionally, the particles can be washed, for example in methanol or ethanol, prior to use. In the art, this can be referred to as "activation", and is discussed elsewhere herein.

[0202] The inorganic solid material thus obtained can have a narrow size distribution and uniform surface chemistry, enabling reliability from batch to batch and reproducibility of one or more of the advantages described herein.

[0203] Suitable physical and chemical techniques are set out in, for example, WO 2011 / 012867 Al (in the name of SISAF LTD); Tokarska K et al., Facile production of ultra-fine silicon nanoparticles, R. Soc. Open Sci., 2020, 7: 200736; and Kim, T., Lee, J. Silicon nanoparticles: fabrication, characterization, application and perspectives, Micro and Nano Syst. Lett, 2023, 11 : 18, the entire contents of each of which are incorporated herein by reference.

[0204] Size of mixed lipid particles and liposome particles

[0205] According to all aspects of the present application, the average diameter of the mixed lipid particles of the present application and the liposome lipid particles of the present application is from 50 nm to 400 nm, for example from 50 nm to 200 nm, for example from 60 nm to 150 nm, for example from 60 nm to 120 nm, for example from 60 nm to 100 nm. Particles having an average diameter of from 1 nm to 100 nm can be referred to as nanoparticles (NPs) or lipid nanoparticles (LNPs). The mixed lipid particles of the present application are silicon-stabilized mixed lipid nanoparticles, which can be referred to as sshLNPs. According to certain preferred embodiments, where hydrolysable silicon particles are used, the average diameter of the mixed lipid particles is from 50 nm to 200 nm (for example from 70 nm to 160 nm) and the average diameter of the hydrolysable silicon particles is from 4 nm to 20 nm (for example from 6 nm to 16 nm).

[0206] Mixed lipid particles

[0207] The mixed lipid particles of the present application include micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid globules.

[0208] In some embodiments, the hybrid lipid particles according to the present application can comprise a lipid bilayer surrounding an aqueous interior space (or, in lyophilized form, an empty interior space). However, this lipid bilayer does not completely isolate the interior space from the environment outside the particle, rather, the interior space is at least partially accessible. Active compounds, especially hydrophilic compounds, can enter, which otherwise would have difficulty crossing the lipid bilayer, especially negatively charged nucleic acids, a representative example being mRNA. This accessibility can be achieved by discontinuities in the lipid bilayer, but also by the presence of inorganic material particles (e.g. hydrolysable silicon particles) providing a path through the lipid bilayer.

[0209] Liposomal lipid particles

[0210] Liposomal lipid particles according to the present application comprise a lipid bilayer surrounding an aqueous interior space. Unlike the hybrid lipid particles of the present application, the lipid bilayer completely isolates the interior space from the environment outside the particle, thereby protecting any material in the interior space, e.g. an API in the interior space.

[0211] Conversion between hybrid lipid particles and liposomal lipid particles

[0212] Surprisingly, the hybrid lipid particles of the present application are able to remain stable for a long time. One might have thought that the not completely sealed lipid bilayer would seal during storage, thus effectively converting the hybrid lipid particles of the present application into liposomal particles. This is not the case. It has been found that the hybrid lipid particles of the present application are able to remain stable for a long time in the absence of active compound or API. For example, according to some embodiments, this stability can be defined as the interior space of the particles remaining accessible, rather than isolated from the external environment. According to certain embodiments, this stability can be manifested as the interior space of at least 90% of the hybrid lipid particles remaining accessible from the external environment in an aqueous suspension at room temperature (understood in this specification to be 25 °C) for at least 1 week, at least 2 weeks, or at least 4 weeks. According to other embodiments, this stability can be manifested as the interior space of at least 90% of the hybrid lipid particles remaining accessible from the external environment in an aqueous suspension at 4 °C for at least 4 weeks, at least 8 weeks, at least 16 weeks, or at least 32 weeks. According to other embodiments, this stability can be manifested as the interior space of at least 90% of the hybrid lipid particles remaining accessible from the external environment in an aqueous suspension at -20 °C for at least 4 weeks, at least 8 weeks, at least 16 weeks, at least 32 weeks, or at least 64 weeks. According to certain embodiments, this stability can be manifested as the interior space of at least 90% of the hybrid lipid particles remaining accessible from the external environment in a lyophilized powder obtained by lyophilizing an aqueous suspension of the particles for at least 8 weeks, at least 16 weeks, or at least 32 weeks. Accessibility of the interior space can be assessed conveniently using any suitable method. For example, accessibility can be assessed using a reporter mRNA or a label (e.g., a fluorescent label, an immunolabel, or a radiolabel) mRNA. According to certain embodiments, this stability can be manifested as the ability of the hybrid lipid particles of the present application to react with an active compound, especially an API (e.g., mRNA) to manufacture liposomal particles of the present application. The liposomal particles of the present application preferably exhibit good active compound (e.g., API) stability; for example, no more than 50% of the active compound, especially the API such as mRNA, will degrade when stored at 4 °C for 3 months or 6 months.

[0213] Location of inorganic material

[0214] The hybrid lipid particles of the present application and the liposomal lipid particles of the present application comprise inorganic material particles (e.g., hydrolysable silicon particles). Preferably, some of these particles are exposed at the surface of the particles, some are located within the lipid bilayer, and some are located within the interior of the lipid particles. According to certain embodiments, at least 10% of the total inorganic material particles are exposed at the surface of the particles, at least 10% of the total inorganic material particles are located within the lipid bilayer, and at least 10% of the total inorganic material particles are located within the interior of the lipid particles.

[0215] Arrangement of aggregates of inorganic material particles

[0216] According to certain embodiments, inorganic material particles, such as particles of hydrolysable silicon, are present in the mixed lipid particles of the application in the form of one or more aggregates. Such aggregates can optionally consist of 10 to 200 particles. They can optionally comprise or consist of chains of particles, especially chains extending into the interior of one or more lipid structures, such as into the interior of one or more of the following: micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid spheres, especially liposomes and / or lipid spheres.

[0217] In embodiments using hydrolysable silicon, particles of hydrolysable silicon can spontaneously aggregate into aggregates of particles, such as shown in the transmission electron microscopy (TEM) image of Figure 82 Spontaneous aggregation is observed especially pronounced when the average diameter of the particles of hydrolysable doped silicon is about 1 nm to about 50 nm, especially about 1 nm to about 30 nm, more especially about 5 nm to about 20 nm, such as about 10 nm. Such particle diameters can optionally be significantly lower than the typical diameter of the mixed lipid particles of the application.

[0218] Thus, preferably, the mixed lipid particles of the application can comprise aggregates of particles of hydrolysable doped silicon, especially chains of particles of hydrolysable doped silicon, wherein the average diameter of the particles of hydrolysable doped silicon is about 1 nm to about 50 nm, especially about 1 nm to about 30 nm, more especially about 5 nm to about 20 nm, such as about 10 nm.

[0219] In manufacturing processes comprising extrusion, such as the methods of the application, aggregation can especially easily occur. The extrusion can optionally be or comprise extrusion through a porous membrane having an average pore diameter of about 0.01 pm to about 1 pm, such as about 0.05 pm to about 0.6 pm. Preferably, the extrusion is performed prior to the addition of the API. Thus, the mixed lipid particles can be formed prior to the optional addition of the API.

[0220] The term "aggregate of particles of hydrolysable silicon" as used herein can refer to a cluster of silicon particles, wherein the closest neighboring silicon particles are in contact with each other. Such clusters can have different configurations, such as a generally spherical cluster of particles and / or a chain of particles. Especially preferred are configurations comprising or consisting of a chain of particles.

[0221] Thus, the mixed lipid particles of the application can comprise one or more aggregates of particles of hydrolysable silicon. Preferably, the aggregates comprise one or more chains of particles.

[0222] The aggregate or aggregates of particles of hydrolysable silicon can be associated with one or more lipids, such as embedded in a lipid and / or attached to the surface of a lipid.

[0223] The one or more aggregates of hydrolysable silicon particles can be associated with, for example embedded in and / or attached to, one or more lipid structures described herein, including micelles, incomplete micelles, liposomes, incomplete liposomes, and lipid spherules. Thus, the one or more aggregates of hydrolysable silicon particles can be associated with, for example embedded in and / or attached to the surface of, one or more of a lipid micelle, an incomplete lipid micelle, a liposome, an incomplete liposome, and a lipid spherule. In particular, the one or more aggregates of hydrolysable silicon particles can be embedded in or attached to the surface of one or more of a liposome, an incomplete liposome, and a lipid spherule.

[0224] The ratio of the longest dimension of the aggregate to the longest dimension of the lipid structure can be, on average, about 1 :5 to 5: 1, especially about 1 :3 to 3: 1; especially when the lipid structure is or includes a liposome and / or a lipid spherule and the one or more aggregates are embedded in or attached to the surface thereof. This can be measured by, for example, transmission electron microscopy (TEM) as shown in Figure 82 .

[0225] The average longest dimension of the aggregate can be about 50 nm to about 500 nm, especially about 50 nm to about 200 nm, for example about 50 nm to about 150 nm, for example as measured by TEM. In particular, the one or more aggregates can be or include one or more chains of particles, wherein the average length of the chains is about 50 nm to about 500 nm, especially about 50 nm to about 200 nm, for example about 50 nm to about 150 nm. The average cross-sectional diameter of the chains can be about 5 nm to about 50 nm, for example about 5 nm to about 30 nm.

[0226] The ratio of the longest dimension of the individual hydrolysable silicon particles to the longest dimension of the lipid structure can be, on average, about 1 : 100 to about 1 :2, especially about 1 : 100 to about 1 :5, more especially about 1 : 100 to about 1 :9; especially when the lipid structure is or includes a liposome and / or a lipid spherule and the one or more aggregates are embedded in or attached to the surface thereof. This can be measured by, for example, transmission electron microscopy (TEM) as shown in Figure 82 .

[0227] When the one or more aggregates are present, the average (e.g. mean) diameter of the particles in the aggregate can preferably be about 1 nm to about 50 nm, especially about 1 nm to about 30 nm, more especially about 5 nm to about 20 nm, for example about 10 nm. Additionally or alternatively, the particles can be porous, having an average (e.g. mean) pore diameter of about 0.1 nm to about 5 nm, for example about 2 nm.

[0228] Activation of inorganic materials

[0229] According to certain preferred embodiments of all aspects of the application, the particles of inorganic material are particles of hydrolysable silicon material. Preferably, such particles are activated by use of a solvent. "Activation" by a solvent or solvent mixture includes surface treatment of the particles comprising hydrolysable silicon. Surface treatment with a solvent or solvent mixture is understood to mean surface treatment with one or more alcohols. Especially preferred alcohols include methanol, benzyl alcohol and methanol (or mixtures thereof). The "surface treatment" of hydrolysable silicon can include the formation of Si-0(CH2)x x CH3(wherein x is 0 for methanol, x is 1 for ethanol, etc.), Si-(CH2) x CH3and / or Si-H moieties, wherein -(CH2) x CH3may be substituted by other carbon-containing groups (e.g. aromatic rings), for example when the alcohol is benzyl alcohol or comprises benzyl alcohol). According to certain embodiments of the method of the application, the silicon nanoparticles are filtered immediately after activation to reduce their aggregation.

[0230] Disposition of API in liposomal particles of the application

[0231] The liposomal particles of the application comprise an active compound, especially an API. The API can be located in the internal space of the particle (i.e. encapsulated) and / or non-covalently associated with the external surface of the particle. According to certain embodiments, at least 10% of the API is located in the internal space of the particle (i.e. encapsulated) and / or at least 10% of the API is non-covalently associated with the external surface of the liposomal particle. According to certain embodiments, at least 10%, 20%, 30%, 40% or 50% of the API is completely encapsulated within the internal space of the particle, the remainder being optionally non-covalently associated with the external surface of the liposomal particle.

[0232] Accordingly, the hybrid lipid particles of the application can comprise particles of hydrolysable silicon associated (especially bound) with one or more liposomes and / or one or more incomplete liposomes, wherein the API is associated (especially bound) with the particles of hydrolysable silicon. Amino acids (especially glycine, arginine and / or tyrosine, for example glycine) can also be associated with the particles of hydrolysable silicon.

[0233] The average diameter of the optionally present liposomes or incomplete liposomes can be in the range of about 50 nm to about 400 nm, especially in the range of about 50 nm to about 200 nm, more especially in the range of about 60 nm to about 100 nm.

[0234] The API can be non-covalently bound to the hydrolysable silicon particles, which are bound to the surface of one or more liposomes and / or one or more incomplete liposomes. Up to about 10 or 20% of the API can be non-covalently bound to the hydrolysable silicon particles, which are in turn bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0235] The API can be non-covalently bound to the hydrolysable silicon particles, which are located inside one or more liposomes and / or one or more incomplete liposomes. Preferably, at least about 50%, 60% or 70% of the API can be non-covalently bound to the hydrolysable silicon particles, which are located inside one or more liposomes and / or one or more incomplete liposomes.

[0236] Preferably, the API (especially RNA, most especially mRNA) is non-covalently bound to the hydrolysable silicon particles, which are located inside one or more liposomes and / or one or more incomplete liposomes; and the API (especially RNA, most especially mRNA) is non-covalently bound to the hydrolysable silicon particles, which are bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0237] In some embodiments, the hybrid lipid particles of the application can be free or substantially free of liposomes; and / or can be free or substantially free of incomplete liposomes.

[0238] Optionally, the one or more lipids can form or comprise one or more lipid monolayers. Optionally, the one or more lipids can be or comprise one or more micelles or incomplete micelles. It will be appreciated that micelles have similar properties to liposomes, except that the wall of a micelle is formed from a lipid monolayer; whereas the wall of a liposome is formed from a lipid bilayer. Thus, a micelle can refer to a vesicle having at least one lipid monolayer, which can approximate a spherical shape. Like a liposome, a micelle can be considered as a lipid "bubble" surrounding an internal space. The internal space can be a hydrophilic environment.

[0239] Thus, the hybrid lipid particles of the application can comprise hydrolysable silicon particles associated with one or more micelles and / or one or more incomplete micelles, wherein the API is associated (especially bound) with the hydrolysable silicon particles.

[0240] Optionally present micelles can have an average diameter in the range of about 50 nm to about 400 nm, especially in the range of about 50 nm to about 200 nm, more especially in the range of about 60 nm to about 100 nm.

[0241] The API can be non-covalently bound to particles of hydrolysable silicon, which are bound to the surface of one or more micelles and / or one or more incomplete micelles. Up to about 10 or 20% of the API can be non-covalently bound to particles of hydrolysable silicon, which are in turn bound to the surface of one or more micelles and / or one or more incomplete micelles.

[0242] The API can be non-covalently bound to particles of hydrolysable silicon, which are located within one or more micelles and / or one or more incomplete micelles. Preferably, at least about 50%, 60% or 70% of the API can be non-covalently bound to particles of hydrolysable silicon, which are located within one or more micelles and / or one or more incomplete micelles.

[0243] Preferably, the API (especially RNA, most especially mRNA) is non-covalently bound to particles of hydrolysable silicon, which are located within one or more micelles and / or one or more incomplete micelles; and the API (especially RNA, most especially mRNA) is non-covalently bound to particles of hydrolysable silicon, which are bound to the surface of one or more micelles and / or one or more incomplete micelles.

[0244] In some embodiments, the hybrid lipid particles of the application can be free or substantially free of micelles; and / or can be free or substantially free of incomplete micelles.

[0245] The one or more lipids can form, or can comprise, one or more lipid spheres, each lipid sphere optionally surrounded by a layer of surfactant. The lipid spheres do not enclose an internal space or cavity. Rather, they are solid or substantially solid with other components (e.g. particles of hydrolysable silicon bound to API molecules) being dispersed. Thus, the interior of the lipid spheres can be interspersed with particles of hydrolysable silicon; in turn, API molecules are (non-covalently) bound to the particles of hydrolysable silicon. Additionally or alternatively (preferably additionally), the particles of hydrolysable silicon (non-covalently) bound to the API molecules can be bound to the surface of the one or more lipid spheres.

[0246] Thus, the hybrid lipid particles of the application can comprise particles of hydrolysable silicon associated (especially dispersed within and / or bound to the surface of) one or more (solid or substantially solid; i.e. not hollow) lipid spheres, with API associated (especially bound) to the particles of hydrolysable silicon.

[0247] The average diameter of the lipid spheres, which are optionally present, can be in the range of about 50 nm to about 400 nm, especially in the range of about 50 nm to about 200 nm, more especially in the range of about 60 nm to about 100 nm.

[0248] The API can be non-covalently bound to the hydrolysable silicon-containing particles, which are bound to the surface of the one or more lipospheres. Up to about 10 or 20% of the API can be non-covalently bound to the hydrolysable silicon-containing particles, which are in turn bound to the surface of the one or more lipospheres.

[0249] The API can be non-covalently bound to the hydrolysable silicon-containing particles, which are in turn located within the one or more lipospheres. Preferably, at least about 50%, 60% or 70% of the API can be non-covalently bound to the hydrolysable silicon-containing particles located within the one or more lipospheres.

[0250] Preferably, the API (especially RNA, most especially mRNA) is non-covalently bound to the hydrolysable silicon-containing particles located within the one or more lipospheres; and the API (especially RNA, most especially mRNA) is non-covalently bound to the hydrolysable silicon-containing particles bound to the surface of the one or more lipospheres.

[0251] Relative size

[0252] According to all aspects of the application, the mixed lipid particles or liposome lipid particles have an average diameter that is at least twice the average diameter of the inorganic material particles. In some embodiments, the difference in relative size can be greater than this, for example, the average diameter of the liposome lipid particles can be at least 3, 4, 5, 6, 7, 8, 10, 12, 15 or 20 times the average diameter of the inorganic material particles. In some embodiments, the average diameter of the liposome lipid particles can be at least 3 to 10 times the average diameter of the inorganic material particles, or at least 5 to 20 times the average diameter of the inorganic material particles. In some embodiments, the average diameter of the mixed lipid particles or liposome lipid particles is between 50 nm and 400 nm, while the average diameter of the inorganic material particles (e.g. hydrolysable silicon-containing particles) is between 10 nm and 60 nm (while the relative diameter is still within the above ranges).

[0253] Explanation of average diameter

[0254] The particle sizes described in this specification are average diameters. Particle sizes can be measured by any suitable method, including dynamic light scattering, electron microscopy and size exclusion. Preferably, the distribution of diameters of the particles is around the average diameter, such that 80% of the particles have a diameter within ±25% of the average diameter. This is known to improve monodispersity, especially after filtration and extrusion.

[0255] Extruded films

[0256] According to the present application, any suitable extrusion membrane can be used. Preferably, the extrusion membrane is used as part of an in-flow extrusion system comprising the extrusion membrane and a flow pump (e.g. an HPLC pump). Preferred extrusion membranes include extrusion membranes comprising polycarbonate. Preferred pore sizes include pore sizes of 0.05 to 1.2 pm, e.g. 0.08 to 1.0 pm, e.g. 0.8 pm, 0.4 pm or 0.1 pm.

[0257] Extrusion method

[0258] The flow rate can depend on the total volume to be extruded and the area of the extrusion membrane used. For example, a flow rate of 10 to 100 ml / min can be used. The extrusion pressure can be selected to achieve a sufficient flow rate. The extrusion is typically carried out at elevated temperature. This is necessary in order to increase the flowability of the lipids used. The exact optimum temperature can depend on the exact lipid formulation used, but the extrusion is typically preferably carried out at 50 to 70 °C, e.g. at 55 to 65 °C, e.g. at about 60 °C.

[0259] According to certain preferred embodiments of the present application, multiple extrusions are typically carried out. For example, at least 6 or at least 8 extrusions can be carried out on the same material. According to certain embodiments, it is preferred to use membranes with decreasing pore sizes for the multiple extrusions. For example, a membrane with a larger pore size can be used for multiple extrusions, then a membrane with an intermediate pore size can be used for multiple extrusions, then again a membrane with a smaller pore size can be used for multiple extrusions. In certain preferred embodiments, a membrane with a pore size of 0.6 to 1.0 pm (e.g. 0.8 pm) is used for multiple extrusions (e.g. at least 2 extrusions, or at least 3 extrusions), then a membrane with a pore size of 0.3 to 0.5 pm is used for multiple extrusions (e.g. at least 2 extrusions, or at least 3 extrusions), then again a membrane with a pore size of 0.08 to 0.2 pm (e.g. 0.1 pm) is used for multiple extrusions (e.g. at least 2 extrusions, or at least 3 extrusions).

[0260] Reduction of inorganic material by extrusion

[0261] As mentioned above, the present application is based in part on the finding that inorganic material particles (e.g. silicon-containing material particles) have a tendency to be removed from the mixed lipid particles of the present application during extrusion, but this does not matter for the continued stability and beneficial properties of the mixed lipid particles. It appears that if the initial concentration of inorganic material particles is relatively high, this concentration can be reduced during extrusion without causing harm. Thus, in certain embodiments of the first aspect of the present application, it is preferred that in the first aspect of the present application, the extrusion step (step B) results in at least 50%, at least 60% or at least 70% or 80% (by weight) of the inorganic material particles present in step A being retained.

[0262] Lipid component

[0263] According to all aspects of the application, the lipid mixture used to form the inventive mixed lipid particles or the liposomal lipid particles or mixed lipid particles comprising the inventive liposomes preferably consists of one or more cationic or ionizable lipids and one or more further lipids selected from neutral and polar lipids, and optionally one or more additional lipid components.

[0264] The charge of the lipids (and thus their classification as e.g. cationic, anionic or zwitterionic) is preferably assessed at pH 7.4, e.g. can be assessed in a physiologically compatible phosphate buffer at pH 7.4.

[0265] According to all aspects of the application, the mixture of one or more cationic or ionizable lipids and one or more neutral or polar lipids comprises at least one cationic or ionizable lipid. Preferably, the total cationic or ionizable lipid (in molar ratio) is 20% to 70%, e.g. 30% to 60%, or 40% to 60% of the total lipid.

[0266] According to certain embodiments, the cationic or ionizable lipid is a cationic lipid. The cationic lipid can be selected from DOTAP (dioleoyl-3-trimethylammonium propane, 18:1 TAP); DODAC (dioctadecyldimethylammonium chloride): SA (stearylamine, octadecylamine) and DOTMA (9-(trimethyl[2,3-(dioleyloxy)propyl]ammonium chloride) and any mixture thereof. Especially preferred are mixtures comprising DOTAP. According to certain embodiments, at least half or all of the cationic lipid is DOTAP.

[0267] According to other embodiments, the cationic or ionizable lipid is an ionizable lipid. The ionizable lipid can be selected from [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, 7-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl 2-octyldecanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, and DODMA and any mixture thereof.

[0268] According to certain embodiments, the cationic or ionizable lipid component can be a mixture of one or more cationic lipids (e.g. one or more of the above listed cationic lipids) and one or more ionizable lipids (e.g. one or more of the above listed ionizable lipids).

[0269] The lipid mixture can also optionally include one or more neutral or polar lipids. The neutral phospholipids DOPE (dioleoylphosphatidylethanolamine), PC (phosphatidylcholine) and egg phosphatidylcholine (a mixture predominantly of PC) are all examples of non-cationic phospholipids which can be used as the neutral or polar lipid according to the application.

[0270] According to certain preferred embodiments, the lipid mixture is entirely or predominantly cationic lipid and phospholipid. For example, the lipid mixture can consist of about equal amounts of DOTAP and DOPE.

[0271] The lipid mixture can also optionally or additionally include a conjugated lipid (e.g. a PEGylated lipid) and / or a sterol / solid component (e.g. cholesterol) as an additional lipid component. According to certain preferred embodiments, the lipid mixture does not contain a substantial amount of conjugated lipid (e.g. PEGylated lipid) or a substantial amount of sterol / solid. In all aspects, the particles of the application preferably do not include cholesterol. In other embodiments, a small amount of cholesterol can be present, for example the cholesterol can comprise less than 10%, less than 8%, less than 5%, less than 2%, less than 1% or less than 0.5% of the total lipid present (by weight).

[0272] Lipids are generally understood to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, glyco lipids and polyketides. As used in this application, the term "lipid" can encompass lipidated oligopeptides (which term is used herein interchangeably with the term lipopeptide), in which a short peptide sequence (e.g. a peptide sequence having 3 to 20 amino acid residues, e.g. 5 to 15 amino acid residues, especially 3, 4 or 5 amino acid residues, most especially 5 amino acid residues) is conjugated to one or more fatty acid chains (especially carbon chain lengths of 10 to 24, preferably carbon chain lengths of 12 to 18; e.g. fatty acid chains of carbon chain lengths of 14, 15 or 16; for example, the peptide moiety can optionally be lipidated by a palmitoyl, cetyl or myristoyl moiety).

[0273] Accordingly, the one or more lipids can include one or more lipidated oligopeptides. Preferably, each of the one or more lipidated oligopeptides includes a fatty acid chain having about 12 to about 18 carbon atoms.

[0274] Preferably, each of the one or more lipidated oligopeptides includes 3 to 20 amino acid residues. Accordingly, the lipidated oligopeptide can be a lipidated tetrapeptide, a lipidated pentapeptide or a lipidated hexapeptide.

[0275] Preferably, the amino acid residues include at least one amino acid residue which is cationic at a pH of about 7.4 (physiological pH) (e.g. about 2 or about 3 amino acid residues), e.g. lysine or arginine. For example, the lipidated oligopeptide can include one or more (e.g. about 2) lysine residues.

[0276] A specific example of a lipidated oligopeptide ("lipo-peptide") is palmitoyl pentapeptide-4 (CAS No. 214047-00-4; abbreviated as PAL-KTTKS).

[0277] Thus, preferably, the one or more lipids can comprise or be one or more lipidated oligopeptides, especially those having one or more amino acid residues that are positively charged at a pH of about 7.4 (i.e. about physiological pH), such as one or both of lysine and arginine.

[0278] The lipidated oligopeptide can be used in combination with one or more phospholipids (e.g. DOPE or DPPC), for example. The alkyl chains of the lipidated oligopeptide molecule can be absorbed by the phospholipid bilayer, while the surface of the bilayer is modified by the peptide portion. Without being bound by theory, it is believed that the peptide portion of the lipidated oligopeptide can enable targeting to one or more specific tissues and / or cells. At the same time, when the peptide portion is positively charged at a pH of about 7.4 (i.e. about physiological pH), it can stabilize a negatively charged API (e.g. a nucleic acid, especially mRNA or siRNA).

[0279] The one or more lipids can be or comprise one or more of: one or more cationic lipids (e.g. DOTAP); one or more phospholipids (e.g. DOPE); and one or more polyethylene glycol (PEG) lipids (e.g. DSPE-PEG 2000 ).

[0280] The one or more lipids can be or comprise one or more structural lipids (e.g. a cholesteroid lipid). However, the one or more lipids can optionally not comprise a structural lipid. Thus, the one or more lipids can not comprise a solid sterol; especially, they can not comprise cholesterol. It has been found that the compositions disclosed herein do not necessarily rely on these types of lipids, which are typically relied upon by conventional API delivery systems. Thus, the compositions disclosed herein have the potential to provide an alternative to API delivery systems that rely on these types of lipids, especially cholesterol. This can be advantageous when cholesterol is unavailable or cannot be used (e.g. due to its effects in the body).

[0281] The one or more lipids can optionally comprise one or more of: phosphatidylcholine (PC); hydrogenated PC; stearylamine (SA); dioleoyl phosphatidyl ethanolamine (DOPE); cholesteryl 3beta-N-(dimethylaminoethyl)carbamate hydrochloride (DC-chol); 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP); PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), such as DSPE-PEG 2000 .

[0282] In certain embodiments, the lipid is selected from phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), or any combination thereof.

[0283] In some embodiments, the lipid or lipid component can be or include a cationic lipid. The term "cationic lipid" refers to a molecule that has a net positive charge at pH 7.4 (physiological pH), with a cationic head group connected to a hydrophobic tail by some spacer. Examples include DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleoyloxypropyl-l-trimethylammonium), DHDTMA (dihexadecyltrimethylammonium); dioleoyl-3-trimethylammonium propane (DOTAP); and stearylamine (SA). The positive charge can be stabilized by a counterion, typically negative.

[0284] Accordingly, the one or more lipids can optionally be or include DOTAP. DOTAP exists in S and R enantiomeric forms, and can exist in the S form, the R form, or as a racemate. Optionally, the R form and the S form can be present in approximately equal amounts (i.e., no more than 60% of the total DOTAP present by weight) of the total DOTAP present. In other embodiments, at least about 80%, 90%, 95%, 98%, or 99% of the total DOTAP is in the R form. In other embodiments, at least about 80%, 90%, 95%, 98%, or 99% of the total DOTAP is in the S form.

[0285] Nonetheless, as described herein, silicon doping can enable the use of less cationic lipid (e.g., DOTAP) compared to conventional compositions for API delivery (e.g., lipid nanoparticles including cationic lipids).

[0286] Accordingly, the one or more lipids can optionally exclude cationic lipids. As described herein, cationic lipids can be unnecessary when using doped silicon, particularly p-doped silicon.

[0287] Accordingly, the one or more lipids can be or include one or more of: one or more phospholipids (e.g., DOPE); and one or more polyethylene glycol (PEG) lipids (e.g., DSPE-PEG 2000 ).

[0288] Overall, the inorganic material particles disclosed herein can provide the potential to use less lipid, especially less cationic lipid, e.g. less DOTAP, in the API delivery vehicle, compared to conventional API delivery vehicles that do not contain inorganic material particles (e.g. conventional liposomal nucleic acid delivery vehicles, such as those typically used for in vivo mRNA delivery). Additionally, or alternatively, the inorganic material particles can provide the potential for the API delivery vehicle to be formulated with a wider range of lipids, while still providing the potential for transfection efficiency, storage stability and / or targeted delivery to particular types of tissue or to particular types of cells. In turn, this can reduce the dependency on particular lipids, especially cationic lipids, in the field, especially cationic lipids that are specific to API delivery purposes and therefore can not be cost effective or readily available.

[0289] The average molecular weight of the one or more lipids can be in the range of about 500 to about 1000.

[0290] The ratio of the one or more lipids (which refers to all lipid components in the composition) to silicon can be in the range of about 40: 1 to about 1 : 1, especially in the range of about 20: 1 to about 1 : 1; for example, the ratio is about 16: 1 when the components are assembled to make the delivery system, i.e. prior to any further processing.

[0291] As described herein, the one or more lipids can especially include or be a phospholipid. The term "phospholipid" as used herein can refer to a lipid that includes a fatty acid chain and a phosphate group. Phospholipids can be negatively charged, unlike cationic lipids which are positively charged. However, phospholipids are typically zwitterionic compounds, including both positively and negatively charged components, and thus are generally not charged. As such, phospholipids are typically classified as neutral lipids.

[0292] A suitable phospholipid can be or include a glycerophospholipid. An especially suitable phospholipid can be or include a phospholipid with a polar head group attached to a quaternary ammonium salt moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. The phospholipid can be or be derived from lecithin. A preferred phospholipid is DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

[0293] Preferably, the side chain of the phospholipid can be an aliphatic side chain having about 15 or more carbon atoms, or an ether side chain having about 6 or more repeating ether units, such as a polyethylene glycol or polypropylene glycol chain.

[0294] Lipids having ether side chains can be referred to as "PEG-lipids" or "PEGylated" lipids. Thus, as used in this application, the term "lipid" can encompass PEG lipids. Thus, according to preferred embodiments, the one or more lipids can comprise or be one or more polyethylene glycol (PEG) lipids, particularly PEGylated DSPE, such as DSPE-PEG 2000 .

[0295] The one or more lipids can optionally comprise or consist essentially of phosphatidylcholine (PC), hydrogenated phosphatidylcholine, stearylamine (SA), or a combination thereof.

[0296] The one or more lipids can optionally comprise at least about 5% (e.g., at least about 30% or at least about 50%) by weight of PC, based on the total weight of the one or more lipids.

[0297] The one or more lipids can optionally comprise at least about 5% (e.g., at least about 30% or at least about 50%) by weight of hydrogenated PC, based on the total weight of the one or more lipids.

[0298] The one or more lipids can optionally comprise at least about 5% (e.g., at least about 30% or at least about 50%) by weight of SA, based on the total weight of the one or more lipids.

[0299] The one or more lipids can optionally comprise or consist essentially of PC and SA, in a weight ratio of PC to SA optionally ranging from about 1 : 1 to about 20: 1.

[0300] The one or more lipids can optionally comprise or consist essentially of a combination of DOPE, SA, and DC.

[0301] In certain preferred embodiments, the one or more lipids can optionally comprise or consist essentially of a combination of DOTAP, DOPE, and PEG-lipid (particularly DSPE-PEG 2000 ). The weight ratio of DOTAP:DOPE can range from about 1 :2 to about 2: 1; e.g., about 1 : 1. The weight ratio of DOTAP:PEG-lipid can range from about 10: 1 to about 5: 1; e.g., about 7: 1. The weight ratio of DOPE:PEG-lipid can range from about 10: 1 to about 5: 1; e.g., about 7: 1.

[0302] Additional Components

[0303] It has been found that the stability of the mixed lipid particles and liposomal lipid particles of the application can be further improved in the presence of one or more amino acids and / or one or more non-reducing disaccharides; thus, the stability of the active compound (especially the API) in the liposomal lipid particles of the application can be further improved. Thus, the method of the application can optionally be performed in the presence of one or more amino acids and / or one or more non-reducing disaccharides. The lipid particles of all aspects of the application can also comprise a non-reducing disaccharide and / or an amino acid. A preferred non-reducing disaccharide is trehalose. A preferred amino acid is glycine. In certain preferred embodiments, both glycine and trehalose are preferably used. These components are able to electrostatically coordinate with inorganic materials (e.g. silicon) and improve the overall stability of the system. Glycine is especially preferred because of its hydrophilic, moderately non-polar nature, and because it exists in the form of a zwitterion in aqueous solution at physiological pH (about pH 7.4). Glycine binds to the surface of inorganic materials (e.g. silicon) and, because of its zwitterionic nature, is able to electrostatically coordinate with positive and negative charges as well as partial charges, thereby enhancing the stability of the particles.

[0304] The term "amino acid" broadly includes any artificially or naturally occurring organic compound containing an amine group (-NH2) and a carboxyl group (-COOH) functional group. It includes alpha, beta, gamma and delta amino acids. It includes amino acids in any chiral configuration. The amino acid can be especially a naturally occurring alpha amino acid. It can be a proteinogenic amino acid or a non-proteinogenic amino acid (e.g. carnitine, levothyroxine, hydroxyproline, ornithine or citrulline).

[0305] The one or more amino acids can help to stabilise the inorganic material particles themselves, especially the silicon. In vivo, the one or more amino acids can help to modulate the rate of hydrolysis of the silicon particles to hydrolyse the silicon to biologically utilizable orthosilicic acid (OSA) degradation products; rather than insoluble polymeric hydrolysis products. Controlling the rate of hydrolysis of the silicon in vivo can influence the rate of release of the active compound (especially the API).

[0306] Controlling the rate of release of the active compound (especially the API) can modulate the length of the period of time for which the active compound (especially the API) is protected, especially in vivo in the presence of various bodily fluids. Thus, more active compound (especially the API) can be delivered to the target cells in a given period of time compared to other compositions with the same ingredients.

[0307] In preferred embodiments, the amino acid can comprise, or consist essentially of, glycine.

[0308] Additionally, or alternatively, amino acids that exhibit neutrality or a positive charge at physiological pH (about pH 7.4), such as tyrosine or arginine, can help stabilize a negatively charged API (e.g., a nucleic acid, such as mRNA). Meanwhile, amino acids that exhibit neutrality or a negative charge at physiological pH can help stabilize a positively charged API. However, the charge-based interactions and / or other interactions (e.g., steric hindrance) resulting from the combination of inorganic materials, lipids, and amino acids can make it so that an amino acid that is positively charged at physiological pH can help stabilize a positively charged active compound (especially an API), or an amino acid that is negatively charged at physiological pH can help stabilize a negatively charged active compound, especially a negatively charged API.

[0309] The weight ratio of the one or more lipids (i.e., total lipid component) to the amino acid(s) can range from about 40: 1 to about 1 : 1; for example, about 32: 1.

[0310] Optionally, in addition to the amino acids described above, the composition can specifically include the amino acid tyrosine. Optionally, the composition can specifically include tyrosine in place of the amino acids described above. Thus, it should be understood that, while tyrosine is an amino acid, for the purposes of the present disclosure it can optionally be present as a separate other component, distinct from the amino acids described above. Thus, when tyrosine is present as an other component distinct from and in addition to the amino acids described above, it should be understood that the ranges of about 40: 1 to about 1 : 1 of the one or more lipids to the amino acid(s) disclosed above (e.g., about 32: 1) do not include the amount of the additional, distinct tyrosine.

[0311] Additionally, or alternatively, one or more non-reducing disaccharides, especially trehalose, can be included. The weight ratio of the one or more lipids (i.e., total lipid component) to the non-reducing disaccharide(s) can range from about 20: 1 to about 1 : 1; for example, about 16: 1.

[0312] Particle stability

[0313] The mixed lipid particles of the present application and the liposome particles of the present application exhibit enhanced size stability compared to corresponding particles that do not contain inorganic material (e.g., hydrolysable silicon material) particles according to the present application. This enhanced size stability is manifested in the particles' resistance to coalescence into larger particles. According to certain embodiments, the coalescence rate at 5°C is at most half that of a corresponding particle of identical composition but without the inorganic material particles, e.g., hydrolysable silicon particles according to the present application. According to certain embodiments, at least 90% of the particles do not coalesce and retain their original size after storage in an aqueous solution at physiological pH (about 7.4) at 5°C for 3 months.

[0314] Charge stability

[0315] The surface charge of the lipid particles (including the mixed lipid particles and the liposome lipid particles of the application) can be estimated using the zeta potential (electrokinetic potential) parameter. Generally, a suspension of particles in nuclease-free water with a low zeta potential (0 to ±5 mV) is unstable and will quickly coalesce. Values of ±30 mV to ±40 mV correspond to reasonable stability, values of ±40 mV to ±60 mV correspond to good stability, and values of ±60 mV or more correspond to excellent stability.

[0316] According to certain embodiments, the above values for the mixed lipid particles of the application and the liposome lipid particles of the application are greater than ±40 mV, more preferably greater than ±45 mV, greater than ±50 mV or greater than ±60 mV. Preferably, the presence of the hydrolysable silicon increases the zeta potential by at least ±10 mV (that is, the zeta potential is at least ±10 mV higher than an equivalent lipid particle which is identical to the application but does not contain particles of inorganic material (for example, the hydrolysable silicon particles according to the application)).

[0317] The presence of inorganic material (for example, hydrolysable silicon) also inhibits the loss of positive charge of the cationic lipids. This is known as lipid ageing, and in the lipid particles of the application, the loss is preferably slowed (at 5°C) by at least 2, 4, 8 or 16 fold.

[0318] Nucleic acid stability

[0319] The liposomal lipid particles of the present invention serve to protect active compounds and APIs. The liposomal lipid particles of the present invention are particularly useful for protecting active compounds and APIs, especially nucleic acids, especially RNA, more particularly mRNA, complexed electrostatically inside and on the surface of the liposomal lipid particles. The present invention makes therapeutic formulations, e.g. vaccines, easier to store; for example, at 5°C or room temperature, rather than subzero temperatures. They also improve stability and reduce degradation of nucleic acids during lyophilisation, rehydration, transport and storage. According to certain embodiments of the present invention, the half-life of the active compound or API, especially when the active compound or API is mRNA, is extended at least 100-fold, at least 1000-fold or at least 10000-fold compared to the corresponding mRNA not complexed with the liposomal lipid particles of the present invention. According to certain embodiments, the half-life of the mRNA is extended at least 10-fold, at least 100-fold or at least 1000-fold compared to the corresponding mRNA complexed with a liposomal lipid particle lacking a particulate component of inorganic material, e.g. hydrolysable silicon, according to the present invention. The half-life can be measured in a physiologically compatible aqueous solution at pH 7.4 at 5°C. According to certain embodiments, especially when the active compound or API is a nucleic acid, e.g. RNA, the half-life can be measured in a natural physiological lipid. For example, it can be measured in vivo, ex vivo or in vitro in blood or a blood constituent, e.g. plasma. For example, it can be measured in vitro in human plasma. The requirement for such a measurement is especially high for active ingredients or APIs that are RNA, because human plasma is known to contain substances, e.g. enzymes, that are known to degrade RNA.

[0320] Method of the present invention

[0321] The method of manufacturing a mixed lipid particle aqueous suspension comprises a step A of mixing a suspension of one or more lipids in a solvent or solvent mixture and particulate inorganic material in a solvent or solvent mixture into an aqueous medium. This step can optionally be carried out using one or more different devices and methods, including bulk mixing methods or devices and microfluidic mixing methods or devices.

[0322] Optional step

[0323] The method of the present invention can optionally comprise an additional step D after step B and step C, if present, of contacting the mixed lipid particles of the present invention with an active compound, especially a pharmaceutically active ingredient (API). This method optionally results in the production of a liposomal lipid particle of the present invention. According to certain embodiments of the method of the present invention, there is an interval of at least 1 week, at least 1 month or at least 6 months between step B and step C, if present, and step D. Optionally, during this interval, the mixed lipid particles are kept at 4°C or 20°C.

[0324] Optional filtration

[0325] The process of the application can optionally comprise an additional step C, purification, concentration and / or sterilization of the suspension by tangential flow filtration, after step B.

[0326] Accordingly, in a first aspect of the application, an additional step C can be inserted between steps B and D. Preferably, step C is performed using tangential flow filtration, although any suitable filtration method can be used. One purpose of the filtration is to improve the size homogeneity of the mixed lipid particles by filtration through a membrane having a cut-off size at the desired particle size, e.g. 100 nm. Surprisingly, it was also found that filtration removes many particles of inorganic material (e.g. particles of hydrolysable silicon) that are not tightly associated with the mixed lipid particles, i.e. particles of inorganic material that are not bound to the surface or inside of the lipid particles, from the solution. The filtration is effective despite the fact that the particles of inorganic material (e.g. particles of hydrolysable silicon) are smaller than the exclusion size of the filtration membrane and the mixed lipid particles. Accordingly, in the first aspect of the application, an optional filtration step can be performed between steps B and C.

[0327] After filtration, the total weight proportion of mixed lipid particles formed by particles of inorganic material (e.g. particles of hydrolysable silicon) can be less than 20%, e.g. less than 10% or less than 5% or 1%. By keeping the amount of inorganic material (e.g. silicon) at a low level, concerns about side effects of the inorganic material (e.g. silicon) are mitigated.

[0328] According to certain embodiments, the optional filtration is diafiltration. In certain embodiments, the diafiltration can use a diafiltration solution containing a non-reducing disaccharide (e.g. trehalose) and an amino acid (e.g. glycine) to maintain the concentration of these substances in the product.

[0329] No solvent evaporation step

[0330] The process of the application does not necessarily require a solvent evaporation step. According to a preferred embodiment of the process of the application, such a process does not contain a solvent evaporation step. In particular, such a process does not contain a step of evaporation of the solvent used for activation of the particles of inorganic material (e.g. particles of hydrolysable silicon) nor a step of evaporation of the solvent used for mixing of the one or more lipids. Preferably, almost all of the solvent (in particular all of the alcohol, e.g. methanol) is removed by the filtration method described herein without evaporation of any material.

[0331] Process control step

[0332] According to preferred embodiments of the method of the application, such method additionally comprises one or more optional process quality control steps. The method can optionally comprise a visual check of whether the lipid is completely dissolved prior to mixing in step A. Additionally alternatively or additionally, the method can optionally comprise measuring the dynamic light scattering (DLS) parameters mean hydrodynamic size parameter, measuring the polydispersity (PDI) and / or measuring the zeta potential after step B. Optionally, the measurement results are compared to product specifications, and if they do not comply with the product specifications, the extrusion step B is optionally repeated, followed by optionally repeating the measurements and repeating the comparison to product specifications.

[0333] Pharmaceutical compositions and uses thereof

[0334] The application also encompasses the use of the mixed lipid particles of the application and the liposomal lipid particles of the application in the formulation of pharmaceutical products which are also within the scope of the application. Such pharmaceutical products include injectable formulations (e.g. injectable vaccines), topical creams, capsules, tablets and ointments. They also include pharmaceutical precursors or products, such as dehydrated (lyophilised) products and concentrated products, which must be diluted and / or rehydrated before use.

[0335] Temperature

[0336] The temperature employed in the extrusion process is typically about 60°C, for example 50°C to 70°C. Such temperatures can compromise the integrity of certain APIs, including when the API is a nucleic acid such as RNA (siRNA, saRNA, mRNA), as is preferred in certain embodiments. As an example of the method of the application, the step of manufacturing the mixed lipid particles of the application is first performed in the absence of the API. This is followed by the optional steps of transporting the mixed lipid particles for further use, lyophilising, freezing or storing them prior to further use; and then the step of contacting the mixed lipid particles with the API to manufacture the liposomal lipid particle suspension of the application. This final step is preferably performed at a relatively lower, and thus milder, temperature. For example, it can be performed at room temperature (25°C) or at a temperature slightly above freezing (0°C). According to certain embodiments, it can be performed at 0°C to 30°C, for example 0°C to 25°C, or 0°C to 10°C.

[0337] Other method features

[0338] According to certain preferred embodiments, step A of the method of the application, mixing one or more lipids and inorganic material particles into an aqueous medium, comprises mixing one or more lipids into an aqueous medium, wherein the one or more lipids are provided in a solvent (e.g. an alcohol, such as methanol). The use of such a solvent advantageously ensures thorough mixing between the one or more lipids. It can be thought that the solvent would need to be evaporated, particularly if the solvent is a toxic solvent (e.g. methanol). It can be thought that the presence of the solvent would be detrimental to the formation of the lipid particles. The present inventors have surprisingly found that good lipid particle formation can be achieved even if the solvent is not evaporated and the one or more lipids and particles are mixed into an aqueous medium according to step A of the method of the application, wherein the one or more lipids are provided in a solvent (e.g. an alcohol, such as methanol). Such a method preferably comprises a downstream step of purifying the suspension by tangential flow filtration (e.g. the method listed in step C of the method of the application). This step has been found to be suitable for removing the solvent from the suspension. If an activating solvent or solvent mixture is used to activate the inorganic material particles (such a solvent can be the same solvent or the same solvent mixture as the solvent in which the lipids are provided), then the activating solvent can be removed in a subsequent step using TFF. TFF can optionally be used in a diafiltration process. In certain embodiments, this diafiltration process can use a diafiltration solution containing a non-reducing disaccharide (e.g. trehalose) and an amino acid (e.g. glycine) to maintain the concentration of these substances in the product.

[0339] Mixing methods

[0340] Lipid particles can be manufactured by a variety of methods. Prior art methods often use methods involving hydration of a lipid film. Whilst such methods can be effective, they are prone to produce lipid particles of inconsistent size and low encapsulation efficiency. The present application, for example the method according to the first aspect of the application, mixes the suspension of one or more lipids in a solvent or solvent mixture and inorganic material particles in a solvent or solvent mixture, and then mixes the solvent or solvent mixture containing the suspension of one or more lipids and inorganic material particles into an aqueous medium.

[0341] According to certain embodiments, this final mixing can be performed by injection of the solvent or solvent mixture into the aqueous medium or any other method of mixing lipids and inorganic material particles that is effective and efficient, including bulk or microfluidic device methods.

[0342] According to certain preferred embodiments, it has been found that production efficiency and product quality can be further improved by mixing the solvent or solvent mixture with the aqueous medium in a rapid mixing method.

[0343] The rapid mixing methods according to embodiments of the application include cross-flow injection or T-junction mixing. Such methods are adaptations of solvent injection methods that enable rapid mixing of organic and aqueous solutions / suspensions. These methods have been found to reliably produce mixed lipid microparticles, and the particle size can be conveniently controlled by varying the injection flow rate and / or pressure. Figure 68 Examples of various rapid mixing methods according to certain embodiments of the application, in particular of the first aspect thereof, are shown. These methods include cross-flow injection, T-junction mixing, microfluidic hydrodynamic focusing, staggered herringbone mixing (SHM), baffle mixing, segmented flow micro-mixing, and annular / diverging micro-mixing.

[0344] Active pharmaceutical ingredients and other active compounds

[0345] In certain embodiments, the active pharmaceutical ingredient of the application can be any pharmaceutically active compound. Preferably, the compound is a hydrophilic compound, such as a negatively charged compound, e.g. a nucleic acid. In other embodiments, the methods and products of the application include other types of "active compounds". Such other active compounds are not necessarily pharmaceutically active compounds. For example, the active compound can be a cosmetically useful compound, a research tool, or a plant protection compound. Preferably, the compound is a hydrophilic compound, such as a negatively charged compound, e.g. a nucleic acid.

[0346] The API or other active compound according to the application can be, for example, a fragile compound. The terms "reactive compound" and "fragile compound" are used interchangeably herein and can each refer to a compound that (i) is susceptible to complete degradation upon storage at about 25°C for more than about one week; and / or (ii) has an in vivo half-life of less than about 1 hour.

[0347] The API can be any pharmaceutically active compound; thus, for example, it is understood that the term "API" encompasses prodrugs. In particular, the API can be a nucleic acid, more in particular an siRNA or an mRNA. Also, in other preferred embodiments, the API can be a protein.

[0348] Nucleic acids used in the application

[0349] The present application is particularly suitable for any nucleic acid, more particularly RNA, as RNA is particularly susceptible to degradation in the absence of the protection provided by the present application. Thus, according to certain preferred embodiments of all aspects of the present application, the nucleic acid is RNA. The RNA can optionally be siRNA. It can also optionally be mRNA. For example, it can be mRNA encoding a vaccine antigen. The RNA can optionally be chemically modified or sequence modified to improve its stability and prevent its degradation. According to certain embodiments of the present application, the RNA is chemically modified to improve its stability or prevent its degradation. However, in certain preferred embodiments, the RNA is not chemically modified as such treatment has been found to be unnecessary as the liposomal lipid particles of the present application can provide sufficient protection to prevent RNA degradation such that, for example, RNA modification is not necessary.

[0350] According to certain embodiments of all aspects of the present application, the nucleic acid is DNA. According to other preferred embodiments, the nucleic acid is RNA. It can be siRNA, mRNA, saRNA or shRNA. It can be of any suitable length, but typically siRNA, saRNA or shRNA is 10 to 30 nucleotides in length, or mRNA is 200 to 2000 nucleotides in length. It can be double stranded or single stranded, or, especially in the case of siRNA, saRNA or shRNA, it can be chemically single stranded but have one or more base-paired regions (and optionally unpaired overhangs). It can optionally be chemically modified (for example by substitution with N1-methyl pseudouridine) or its sequence modified (for example by UTR shortening). Preferably, the nucleic acid (i.e. RNA) can be unmodified (especially not chemically modified) as this can not be necessary to provide stability.

[0351] According to certain embodiments, the nucleic acid can be RNA which can optionally include a 5-prime cap and / or a poly-A tail, but wherein no other modifications are present.

[0352] The RNA according to the present application can be small interfering RNA (siRNA), small activating RNA (saRNA), short hairpin RNA (shRNA) or messenger RNA (mRNA), especially mRNA (e.g. mRNA encoding a protein of a pathogenic organism).

[0353] Other nucleic acids for use according to the present disclosure include: double stranded and single stranded DNA; DNA:RNA hybrids; peptide:DNA complexes; and peptide:RNA complexes.

[0354] The RNA and DNA can be naturally occurring, or can be chemically modified to enhance its therapeutic properties, such as to enhance activity, increase serum stability, decrease off-target effects, and decrease immune activation. Chemical modifications of the RNA and DNA can include any modification known in the art.

[0355] Accordingly, the terms nucleic acid, DNA, and RNA as used herein also include known types of modifications, such as labels, methylation, "capping," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, amino phosphonates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkyl phosphonates, aminoalkyl phosphotriesters), with intercalators, with chelators, with alkylators, with modified linkages between the nucleotides of a polynucleotide, or oligonucleotide, and the like, as known in the art.

[0356] Likewise, the terms "nucleoside" and "nucleotide" as used herein will include moieties that contain not only the known purine and pyrimidine bases, but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocyclic bases. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., where one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are otherwise functionalized as ethers, amines, etc. Other modifications to the nucleotide or polynucleotide include rearrangements, additions, substitutions, or otherwise altering the functional groups on the purine or pyrimidine bases that form hydrogen bonds with the corresponding complementary pyrimidine or purine, such as isoguanine, isocytosine, etc. In some embodiments, the oligonucleotide and / or probe includes at least one, two, three, or four modified nucleotides.

[0357] In some embodiments, the nucleic acids (e.g., RNA) disclosed herein include one or more universal bases. The term "universal base" as used herein refers to a nucleotide analog that can hybridize to more than one nucleotide selected from A, U / T, C, and G. In some embodiments, the universal base can be selected from deoxyinosine, 3-nitropyrrole, 4-nitroindole, 6-nitroindole, and 5-nitroindole.

[0358] The term“saRNA” broadly encompasses small activating RNA, including RNA molecules that operate in the RNA activation (RNAa) pathway. The saRNA can be double stranded. The saRNA can have a length in the range of about 5 to about 50 base pairs, especially about 10 to about 40 base pairs, more especially about 10 to about 30 base pairs.

[0359] The term“shRNA” broadly encompasses short hairpin RNA, including RNA molecules that operate in the RNA interference (RNAi) pathway. The shRNA can be single stranded, while also having base pairing, thereby forming a hairpin loop. The single stranded length of the shRNA can be in the range of about 10 to about 100 base pairs, especially about 25 to about 75 base pairs, more especially about 40 to about 70 base pairs; which can then form a hairpin loop.

[0360] The term“siRNA” broadly encompasses small interfering RNA, including RNA molecules that operate in the RNA interference (RNAi) pathway. The siRNA is sometimes also referred to as short interfering RNA or silencing RNA. The siRNA can be double stranded. The siRNA can have a length in the range of about 5 to about 50 base pairs, especially about 10 to about 40 base pairs, more especially about 15 to about 30 base pairs.

[0361] The mRNA can encode an antigen, thereby providing a composition as a vaccine. The antigen can be a viral antigen, especially of one of the viral diseases described below; more especially an antigen of a respiratory virus, such as an antigen of SARS-CoV-2, such as an antigen derived from the SARS-CoV-2 spike protein.

[0362] The mRNA can encode a plurality of proteins, thereby providing more effective pharmacological activity. The mRNA can encode a plurality of antigens, especially a plurality of viral antigens.

[0363] The mRNA can also encode an adjuvant protein. The adjuvant can additionally or alternatively be provided as a further component of the composition other than the API.

[0364] Complexing of components, especially particle / lipid / API complexing

[0365] Preferably, the inorganic material particles are complexed with one or more lipids, thereby forming a delivery vehicle for the delivery of the active compound or API. Thus, when the active compound or API is added, it also becomes complexed with the particles and / or the lipids. In other words, the particles and the lipids are organized into a delivery vehicle that is loaded with the active compound or API. Advantageously, this can make the active compound or API less susceptible to reaction with one or more external reactive species. The active compound or API can be less susceptible to be at risk of being catalytically degraded by enzymes outside the complex, especially in vivo, e.g. in the cytoplasm of cells in circulation and / or in the body; this is especially the case when the active compound or API is a nucleic acid, especially an mRNA.

[0366] In a broad sense, the term "complexed with" as used herein can encompass ionic and / or covalent and / or physical interactions, especially charge-charge interactions, e.g. interactions caused by the zeta potential of the particles.

[0367] Thus, preferably, the zeta potential of the particles, especially as modulated by the one or more lipids and any other components present, can e.g. attract the active compound or API and facilitate its binding.

[0368] In preferred embodiments where amino acids are present, the amino acids can also be complexed with the particles, the lipids and / or the active compound / API. The amino acids, especially when charged, can modulate the zeta potential of the particles, thereby modulating the complexation of the active compound / API and / or the lipids with the particles.

[0369] Tangential flow filtration

[0370] The methods of the present application optionally comprise one or more tangential flow filtration (TFF) steps. TFF can be used to concentrate the aqueous suspension according to the present application. Additionally, or alternatively, tangential flow filtration can be used to remove solvents, e.g. methanol, used in earlier stages of the method.

[0371] In certain preferred embodiments of the methods of the present application, the use of tangential flow filtration is especially preferred due to the lack of solvent evaporation steps in these methods.

[0372] Beneficial properties and product parameters

[0373] The particles of the present application - mixed lipid particles and liposome lipid particles - preferably have a size of 60 nm to 120 nm, a polydispersity index (PDI) of 0.100 to 0.200 and a zeta potential of 50 mV to 70 mV.

[0374] Construction of mixed lipid particles and liposome lipid particles

[0375] The mixed and liposomal lipid particles of the application, in all aspects thereof, preferably have the following configuration, in addition to the lipid structure. The lipid particles have inorganic material particles (e.g., hydrolysable silica particles) that can be integrated into the lipid bilayer or that can be partially or fully exposed on the surface of the mixed or liposomal particle, so as to be available for interaction with active compounds (especially APIs, and more especially nucleic acids). Preferably, at least 10%, at least 20%, at least 30%, or at least 50% of the total inorganic material particles (e.g., hydrolysable silica particles) are accessible on the surface of the lipid particle and are not fully encapsulated within the lipid structure. When active compounds (especially nucleic acids or other APIs) are present, they are located on the surface of the lipid particle primarily by means of electrostatic binding. For example, according to certain embodiments, more than 90% of the total active compounds (especially nucleic acids or other APIs) will be bound to the surface of the lipid particle, and less than 10% of the total active compounds will be encapsulated within the lipid structure. In some embodiments, zero or almost zero (e.g., less than 0.5%) of the total active compounds (especially nucleic acids or other APIs) will be encapsulated within the lipid structure. In other embodiments, the ratio between active compounds or APIs that are encapsulated and active compounds or APIs that are associated with the surface of the lipid particle will be more balanced. For example, according to some embodiments, at least 10% of the active compounds or APIs will be associated with the surface of the particle, and at least 10% of the active compounds or APIs will be encapsulated.

[0376] Therapeutic methods and related products

[0377] The products of the application can be used in a therapeutic method, or can be products for use in a therapeutic method. The methods of the application can also include subsequent steps that constitute a therapeutic method.

[0378] The therapeutic method comprises treating or preventing a disease or condition. In some embodiments, the therapeutic method can comprise downregulating gene expression by siRNA. In other embodiments, the therapeutic method can comprise vaccination, for example, vaccination against cancer or vaccination against an infectious disease by delivering mRNA encoding an antigen (or fragment thereof) or an infectious disease pathogen (e.g., the spike protein of SARS-CoV-2).

[0379] The disease or condition can be an infectious disease. The term “infectious” as used herein can be used to refer to a disease that is susceptible to transmission from one organism to another, especially from one human to another.

[0380] The infectious disease can be a viral, bacterial, fungal, or parasitic disease; especially a viral disease.

[0381] If the disease is a viral disease, it can be a respiratory virus, such as respiratory syncytial virus (RSV), parainfluenza virus (HPIV), metapneumovirus (HMPV), rhinovirus (HRV), a coronavirus, such as SARS-CoV (especially SARS-CoV-1, more especially SARS-CoV-2), adenovirus (HAdV), enterovirus (EV), bocavirus (HBoV), parvovirus (HPeV), or influenza virus.

[0382] The viral disease can be a dengue virus, an Ebola virus, an encephalomyocarditis virus, a hepatitis virus, a herpes virus, a human immunodeficiency virus, a human papillomavirus, a human T-lymphotrophic virus, a measles virus, a monkeypox virus, a mumps virus, a poliovirus, a rabies virus, a rotavirus, a rubella virus, a varicella-zoster virus, a West Nile virus, a yellow fever virus, or a Zika virus.

[0383] The disease or condition can be a genetic disease or condition.

[0384] In some embodiments, the genetic condition can be characterized by a deficiency in the expression of one or more proteins, especially one or more enzymes.

[0385] The genetic condition can be a polygenic disease, i.e. not limited to any particular single-gene inheritance pattern, but can be related to the effects of multiple genes as well as environmental factors; for example, schizophrenia, diabetes, asthma, depression, epilepsy, heart disease, or hypothyroidism.

[0386] The genetic condition can involve one or more mutations in one or more genes.

[0387] Thus, the genetic condition can be a monogenic disease, which is liable to occur if at least one mutation occurs in a single gene. If the genetic condition is a monogenic disease, it can involve one mutation or more than one mutation in a single gene. Examples of monogenic diseases include sickle cell anemia, cystic fibrosis, Huntington’s disease, or Duchenne muscular dystrophy.

[0388] The genetic condition can involve one or more mutations in more than one gene. By way of non-limiting example, the genetic condition can involve more than one mutation in a first gene and one mutation in a second gene.

[0389] The genetic condition can be a disease that is liable to occur if at least one mutation occurs in at least one gene of a group of genes; especially, such genetic condition can be osteopetrosis.

[0390] The genetic disorder can be Angelman syndrome; Canavan disease; Charcot-Marie-Tooth disease; color blindness; Cri du chat syndrome; cystic fibrosis; DiGeorge syndrome; Down syndrome; Duchenne muscular dystrophy; familial hypercholesterolemia; hemochromatosis type 1; hemophilia; Klinefelter syndrome; neurofibromatosis; phenylketonuria; polycystic kidney disease; Prader-Willi syndrome; Scheuermann’s disease; sickle cell disease; spinal muscular atrophy; Tay-Sachs disease; or Turner syndrome.

[0391] In a broad sense, a “genetic disorder” as used herein can encompass a cancer. The cancer can be or include a blood cancer (e.g., leukemia, lymphoma, or myeloma) or a solid tumor (e.g., sarcoma; carcinoma; carcinosarcoma; or lymphoma).

[0392] Thus, in particular, the cancer can be a cancer of the blood, skin, brain, prostate, breast, lung, esophagus, stomach, small intestine, pancreas, colon, and / or rectum, central nervous system, bladder, thyroid, kidney, uterus, mouth, or ovary.

[0393] In more detail, the cancer can be or include a cancer of the pulmonary system, brain, gastrointestinal tract, skin, genitourinary system, pancreas, lung, medulloblastoma, basal cell carcinoma, glioma, breast, prostate, testis, esophagus, hepatocellular, stomach, gastrointestinal stromal tumor (GIST), colon, colorectum, ovary, melanoma, neuroectodermal tumor, head and neck, sarcoma, soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, leiomyosarcoma, cervical, uterine, endometrial, carcinoma, bladder, epithelial carcinoma, squamous cell carcinoma, adenocarcinoma, bronchopulmonary carcinoma, renal cell carcinoma, liver carcinoma, biliary duct carcinoma, neuroendocrine carcinoma, carcinoid, diffuse large cell carcinoma, or glioblastoma.

[0394] Preparation, storage, stability, and administration of the disclosed pharmaceutical compositions (also simply “compositions”)

[0395] Treatment or prevention of a disease or condition according to the present application can include administering to a subject (especially a human subject) a prophylactically effective amount of a pharmaceutical composition disclosed herein, wherein the subject is in need thereof; e.g., a subject in need thereof as determined by a physician or other healthcare practitioner. Also, treatment of a human subject for a disease or condition can include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition disclosed herein.

[0396] The dosage of the pharmaceutical composition disclosed herein can vary depending on the amount of API effective to achieve the desired prophylactic and / or therapeutic response for a given subject and not cause toxicity to the subject. A suitable dosage of the composition can be the lowest effective dose of API to produce a therapeutic and / or prophylactic effect.

[0397] The selected dosage, dosage form, and regimen will depend on a variety of factors. These factors can include, for example, the activity of the API, the route of administration, the time of administration, the rate and extent of excretion or metabolism of the API, the duration of the treatment, the presence of other drugs, compounds, and / or materials used in combination with the API, the age, sex, weight, condition, general health, and prior medical history of the subject being treated, and other such factors well known in the medical arts.

[0398] The composition can be administered by intramuscular injection or intravenous injection (encompassing transdermal delivery by patch), orally (encompassing sublingual administration), intranasally, or by any other suitable route.

[0399] Preferably, the composition can be administered by injection, e.g., intravenous injection or intramuscular injection. Optionally, when the composition is administered by injection, the subject is monitored for hypersensitivity symptoms or signs, e.g., vaccine-related hypersensitivity reactions.

[0400] Also preferably, the composition can be administered orally or intranasally. Compositions suitable for oral administration can be presented in discrete dosage forms, especially liquid or aerosol spray, each containing a predetermined amount of the composition. Such dosage forms can be prepared by any of the known pharmaceutical methods.

[0401] According to conventional pharmaceutical compounding techniques, the compositions can be combined with a pharmaceutical carrier in intimate admixture. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. Any of the well-known pharmaceutical media can be used as a carrier, such as, for example, water, oil and alcohol (including glycols). The disclosed pharmaceutical compositions can incorporate for administration forms, especially when formulated for injection, oral or intranasal administration, can include aqueous solutions of physiological saline. The compositions can also include one or more pharmaceutically acceptable additives and excipients, such as, for example, one or more of the following: anti-adherents, antifoams, buffers, polymers, antioxidants, chelating agents, viscosity-adjusting agents, tonicifiers, palatants, opacifiers, suspending agents, fillers, plasticizers, flavorings, preservatives, colorants, diluents, binders, disintegrants, and mixtures thereof.

[0402] The effects of microorganisms can be prevented or diminished by the addition of various antibacterial and antifungal agents; for example, one or more of the following: parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0403] The compositions disclosed herein can be provided in sterile solution, i.e., by adding the required amount of the composition to an appropriate solvent (with the appropriate addition of various other ingredients) by any known pharmaceutical method. The compositions disclosed herein can be provided in sterile dispersion, i.e., by adding the required amount of the composition to an appropriate sterile carrier (with the appropriate addition of various other ingredients). The compositions disclosed herein can be provided in sterile powder (e.g., for subsequent preparation of a sterile injectable solution), such as by vacuum drying and lyophilization (lyophilization) techniques to obtain a powder of the composition.

[0404] Optionally, the compositions can be stored prior to administration to a subject. The compositions can be stored at a temperature in the range of above 0 °C, especially at a temperature of above 4 °C, for at least 1 week (optionally, up to 6 months, especially up to 1 year) prior to administration of the compositions to a subject.

[0405] In some embodiments, the liposomal particles can improve the stability of the API during circulation in the body, especially where the active compound (e.g., API) is or includes a nucleic acid (e.g., mRNA). In some embodiments, the particles can protect the API from degradation, especially enzymatic degradation, especially where the API is or includes a nucleic acid (e.g., mRNA). Thus, the compositions disclosed herein can alleviate or address the problem of how to ensure that the API reaches the cells once administered to a patient, including how to stabilize the API during circulation in the body.

[0406] At the same time, the disclosed compositions can alleviate or meet the need for tissue or cell targeting, so that the API can be delivered to the correct cells.

[0407] Additionally, or alternatively, once at the target cell, the disclosed compositions can alleviate or address the problem of how to ensure that the API is taken up by the cell efficiently. For example, the disclosed compositions can aid in the transport of the API from outside the cell to the cytoplasm.

[0408] After the API has been taken up by the cell, the disclosed compositions can alleviate or address the problem of how to prevent the API from being degraded too rapidly in the cytoplasm of the cell. It is believed that the liposomal particles can increase the stability of the API in the cytoplasm of the cell, especially against enzymatic degradation, especially where the API is or includes a nucleic acid (e.g. mRNA).

[0409] Relationships between aspects of the invention

[0410] It will be appreciated that the method according to the first aspect of the invention can optionally be used to manufacture an aqueous suspension of mixed liposomal particles according to the second aspect of the invention, and that such a suspension can optionally be used to manufacture a lyophilised powder of mixed liposomal particles according to the invention. Such a product can optionally be used to manufacture an aqueous suspension of liposomal liposomal particles according to the invention. Such an aqueous suspension can optionally be used to manufacture a lyophilised powder liposomal liposomal particle of the invention. The advantage of the methods and products of the invention over the above-mentioned liposomal particles which attempt to encapsulate the API or other active ingredient during the initial liposomal particle formation process is that the above-mentioned encapsulation process typically needs to be performed at high temperatures (typically 50 to 70°C) necessary for the formation of the liposomal particles. Such high temperatures can degrade heat-sensitive active ingredients, such as RNA molecules. The invention allows the mixed liposomal particles to be prepared prior to the introduction of the active ingredient to be delivered by the liposomal particles. It also allows the mixed liposomal particles to be stored as an intermediate stock or commodity for long periods of time (e.g. in an aqueous suspension or lyophilised powder) for subsequent production of the final product, which can be a liposomal liposomal particle.

[0411] The lyophilised powder of liposomal particles of the invention can be used to manufacture an aqueous suspension of liposomal particles of the invention.

[0412] The lyophilised powder of liposomal particles of the invention and / or the aqueous suspension of liposomal particles of the invention can be used to manufacture a pharmaceutical composition of the invention.

[0413] The mixed liposomal particles of the invention and the liposomal liposomal particles of the invention can optionally be prepared using the methods of the invention.

[0414] It will be appreciated that optional features described or claimed herein as part of one aspect of the invention are to be understood, where appropriate, as optional features of other aspects of the invention.

[0415] Example

[0416] The following non-limiting examples describe various aspects and embodiments of the application.

[0417] Embodiments can also describe subject matter that is not within the scope of the application, but which is included in order to help understand the application or to provide a comparison to the application.

[0418] Method

[0419] A manufacturing process for silicon stabilized hybrid lipid nanoparticles (sshLNP) has been developed that combines flow extrusion to form sshLNP with desired particle size and surface charge characteristics, followed by concentration and purification of the solution by tangential flow filtration (TFF). To target different tissues, two sshLNP formulations have been developed, containing and not containing polyethylene glycolated lipids, respectively.

[0420] The process manufactures an intermediate hybrid lipid particle to which the nucleic acid is loaded immediately prior to the load-finish operation. The addition of the nucleic acid at this stage can minimize degradation. It also provides an opportunity for late customization of the sshLNP.

[0421] The combination of flow extrusion and TFF provides a highly scalable manufacturing process that can reliably manufacture sshLNP at scales from less than 1 L to more than 100 L, suitable for a range of products from personalized medicine to large volume products.

[0422] Figure 1A An embodiment of the process of the application is shown. Silicon nanoparticles (SiNPs) are activated by dispersion in methanol as the activating solvent. An optional filtration step can be employed to reduce aggregation of the silicon nanoparticles. Mixing is performed by dissolving the lipids DOTAP-Cl, DOPE and optionally mPEG2000-DSPE in methanol. An aqueous solution supplemented with trehalose and glycine is first mixed with the SiNP suspension. The dissolved lipid / methanol stream is injected into it. The resulting mixture is extruded through a membrane of reduced pore size, followed by TFF to remove excess trehalose, glycine, methanol and lipids. IPC1 and IPC2 are quality controls in which the product is inspected according to specifications.

[0423] sshLNP manufacturing

[0424] 1.1 Composition of biological messenger sshLNP

[0425]

[0426] 1. DOTAP-Cl

[0427] 1,2-dioleoyloxy-3-trimethylpropylammonium chloride,

[0428] (UNII: 3R78UC794Z, CAS No. 132172-61-3)

[0429]

[0430] 2. DOPE

[0431] 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine

[0432] (UNII: JNP6V6AI0U, CAS No. 4004-05-1)

[0433]

[0434] 3. mPEG2000-DSPE

[0435] N-(Carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt

[0436] (UNII: 3L6NN8ZZKU, CAS No. 147867-65-0)

[0437]

[0438] Description of the manufacturing process of methanol injection in combination with TF

[0439] Silicon nanoparticles (500 mg) were dispersed in methanol (25 mL) and left to stir for 30 minutes to activate them. The dispersion was filtered through a 0.8 pm hydrophilic polyether sulfone filter. Trehalose (400 mg) and glycine (200 mg) were dissolved in nuclease-free water (380 ml) and mixed with the activated silicon nanoparticles dispersion (20 mL) by stirring at 50 °C for 60 minutes.

[0440] Alternatively, for pegylated sshLNPs,

[0441] DOTAP-Cl (3500 mg) was dissolved in methanol (350 mL) by stirring at 40 °C for 30 minutes. DOPE (3500 mg) was dissolved in methanol (350 mL) by stirring at 40 °C for 30 minutes. mPEG2000-DSPE (650 mg) was dissolved in methanol (65 mL) by stirring at 40 °C for 30 minutes (IPC 1).

[0442] The DOTAP-Cl solution (297 mL), DOPE solution (299 mL), and mPEG2000-DSPE solution (59 mL) were combined. The combined lipid solution (640 mL) was slowly infused into the mixture of silicon nanoparticles, trehalose, and glycine at a flow rate of 10 mL / min. After the addition was complete, nuclease-free water was added to bring the volume to 4 L.

[0443] Alternatively, for non-polyethylene glycolated sshLNPs,

[0444] DOTAP-Cl (3500 mg) was dissolved in methanol (350 mL) by stirring at 40 °C for 30 minutes. DOPE (3500 mg) was dissolved in methanol (350 mL) by stirring at 40 °C for 30 minutes (IPC 1).

[0445] The DOTAP-Cl solution (320 mL) and DOPE solution (340 mL) were combined. The combined lipid solution (640 mL) was slowly infused into the mixture of silicon nanoparticles, trehalose, and glycine at a flow rate of 10 mL / min. After the addition was complete, nuclease-free water was added to bring the volume to 4 L.

[0446] The resulting mixture was extruded through two sets of 47 mm polycarbonate extrusion membranes of decreasing pore size (arranged in parallel to increase the effective surface area); each set of extrusion membranes was 3 x 0.8 pm, 3 x 0.4 pm, and 3 x 0.1 pm, respectively. A second extrusion through the 3 x 0.1 pm membranes can be required to achieve the desired physical properties (IPC 2).

[0447] A tangential flow filtration technique was employed using 0.5 m 2 The extruded mixture was ultrafiltered using a polyethersulfone membrane (100 KDa molecular weight cut-off). After the mixture was concentrated to a volume of 2 L, diafiltration was performed using 10 volumes (20 L) of a solution containing trehalose (0.1 mg / mL) and glycine (0.05 mg / mL).

[0448] After diafiltration, the solution was filtered through a 0.2 pm polyethersulfone membrane.

[0449] Unlike conventional processes where product composition is directly proportional to the amount of material added, flow extrusion / TFF is a dynamic method of changing material content. As developed, this method produces sshLNPs with key chemical and physical properties that enable effective delivery of nucleic acids.

[0450] The method has four key unit operations:

[0451] a. Solution preparation and mixing

[0452] Trehalose and glycine were dissolved in nuclease-free water, and lipids were dissolved in methanol.

[0453] The silicon nanoparticles are dispersed in methanol and activated. Activation changes the contact angle of the silicon nanoparticle surface, increasing its dispersibility. Activation also promotes the expression of hydroxyl groups on the surface of the silicon nanoparticles, facilitating electrostatic interactions and the physical adsorption of other excipients on the surface. The initial silicon nanoparticle dispersion contains 20 mg / mL of silicon, and using lower concentrations of silicon can affect the physical attributes of the resulting LNP, such as mean particle size, polydispersity index, and zeta potential.

[0454] The dispersion is filtered through a 0.8 pm hydrophilic polyether sulfone (PES) membrane filter to remove silicon agglomerates and oversized particles. If filtration is not performed, there is a risk of the extrusion membrane becoming blind, which can affect the physical attributes of the resulting LNP, such as mean particle size, polydispersity index, and zeta potential.

[0455] The trehalose and glycine solutions are mixed with the silicon dispersion, and then the lipid solution is slowly added to form lipid nanoparticles. However, at this stage, the mean particle size and polydispersity index of the LNP are greater than expected.

[0456] Lipid particles are preferably formed by flow injection, as opposed to forming a thin film by evaporation and subsequent rehydration. The thin film / rehydration method has some limitations, including difficulty in obtaining a uniform dispersion. The thin film / rehydration method also presents challenges to manufacturability beyond a certain manufacturing scale.

[0457] b. Flow extrusion

[0458] To reduce the mean particle size and polydispersity to the desired range, the mixture is then extruded through a series of polycarbonate membranes with decreasing pore size: 3 x 0.8 pm, 3 x 0.4 pm, and 3 x 0.1 pm.

[0459] Gradually decreasing the pore size of the membranes reduces the extrusion pressure and associated mechanical stress during the process. Using 3 membranes at each stage provides multiple extrusion cycles. This method of decreasing pore size and multiple extrusion cycles enables the manufacture of LNP with controlled and reproducible physical characteristics (mean particle size, polydispersity index, and zeta potential).

[0460] During this operation, silicon is further removed, and some lipid remains on the membranes.

[0461] c. Tangential flow filtration (TFF)

[0462] Two functions are performed using TFF with polyethersulfone membranes (100 KDa molecular weight cut-off): ultrafiltration (UF) and diafiltration (DF). First, the LNP solution is concentrated by 50% in volume by UF, followed by purification by DF using 10x diafiltration volumes to remove methanol and free (unbound) lipid. Since the molecular weight of trehalose and glycine is low, these molecules also pass through the membrane. To compensate for the loss of these molecules, a diafiltration solution containing trehalose and glycine is used to maintain the concentration of these substances in the product. TFF has no significant effect on the physical properties of the LNP and does not result in the removal of silicon.

[0463] d. Final filtration

[0464] After TFF is complete, the resulting solution is filtered through a 0.2 pm polyethersulfone filter to remove microbial contamination and to manufacture a low-bioburden sshLNP biocourier.

[0465] The product can then be complexed with mRNA at room temperature.

[0466] In summary, critical product attributes are controlled by the following process operations.

[0467]

[0468] • DOPE

[0469] • DOTAP-Cl

[0470] • mPEG2000-DSPE

[0471]

[0472]

[0473] Method

[0474] Analytical methods

[0475] Flow extrusion

[0476] Flow extrusion was performed using an HPLC pump Knauer K-501 equipped with a 50 ml / min stainless steel pump head. The flow rate was set in the range of 10 ml / min to 50 ml / min depending on the extrusion volume. Extrusion membranes of 0.8 pm, 0.4 pm and 0.1 pm were used at high temperature of 60 °C and performed in a hot bath. The clamp containing the membrane was immersed in water to reach the desired temperature. We investigated the extrusion process by DLS, measuring the particle size and particle size distribution as well as zeta potential analysis. The extrusion pressure on the membrane was also monitored over time and extrusion volume. Other flow extrusion investigation parameters include: extrusion flow rate, number of extrusion membranes and a step of pre-filtration using a syringe filter before extrusion.

[0477] Tangential flow filtration

[0478] Tangential flow filtration (TFF) was used to remove methanol and other impurities, such as unbound lipids as well as any potential degradation products.

[0479] For 50 ml and 100 ml volumes, TFF used TangenX SIUS PD membrane cassettes with standard groove offset. The specifications of the used membrane cassettes are:

[0480] • Part number: XP100LP2L

[0481] • Membrane type: HyStream

[0482] • MWCO: 100 kD

[0483] • Membrane area: (m 2 ) 0.02 m 2

[0484] • Flow channel type: LP screen mesh flow channel

[0485] • Clamp structure: L

[0486] The micellar lipid particles were concentrated two-fold and washed with 10-fold diafiltration volume (DV) of THR (trehalose) and GLY (glycine) solutions, aiming at removing the organic solvents. In this case it was noted that diafiltration solutions containing 0.1 mg / ml of THR and 0.05 mg / ml of GLY were used in order to prevent the loss of these components during the TFF process. The in-process samples were then analyzed after TFF.

[0487] For 2 kg batches, the specifications of the used membrane cassettes are as follows (standard groove offset):

[0488] • Part number: XP100G05L

[0489] • Membrane type: HyStream

[0490] • MWCO: 100 kD

[0491] • Membrane area: (m 2 ) 0.5 m 2

[0492] • Flow channel type: LP mesh flow channel

[0493] • Clamp configuration: L

[0494] The only difference between the two membrane packs was the membrane area. The larger the membrane, the larger the filtration volume.

[0495] The surface charge, particle size and particle size distribution of all extrusion steps and post-TFF samples were evaluated by DLS and zeta potential measurements. For the described experiments, the zeta potential, Z-average diameter and PDI were reported. The feasibility of the currently available HPLC method for the quantification of lipids post-extrusion and post-TFF was investigated with the starting material as a standard for the calibration curve. By 1 The removal of methanol was investigated by H-NMR analysis and headspace gas chromatography.

[0496] Manufacturing and feasibility studies of flow extrusion and TFF were performed under non-GMP conditions without additional low-bio- load handling measures. The TFF membrane packs were reusable for different batch manufacturing after cleaning.

[0497] Additional measures were taken during manufacturing when using nuclease-free water, such as wearing gloves disinfected with 70% v / v ethanol, cleaning the workstations and instruments regularly with 70% v / v ethanol, and using sterile laboratory equipment, sampling in a laminar flow cabinet.

[0498] During extrusion, the solution was filtered through Whatman 25 mm polycarbonate membrane filters with a defined pore size of 0.4 pm and 0.8 pm. Three membranes were used in a row for each extrusion step. To complete the extrusion and push the fluid through the membrane, a Knauer K501 HPLC pump equipped with a 50 ml / min stainless steel pump head was used in combination with a water bath to maintain a working temperature of 60 °C. Before each extrusion step, the membranes were washed with MeOH (20 ml) and rinsed with MilliQ water (20 ml). The extrusion flow rate was 10 ml / min. Finally, to evaluate the feasibility of adding SiNPs directly to an aqueous solution and the extrusion process, and to monitor the properties of SiNPs during all experimental steps, DLS and zeta potential analysis were performed for all steps.

[0499] Example 1 - Description of prior art "evaporation" manufacturing method for the production of reference values for comparison

[0500] The "evaporation" manufacturing process of the micellar lipid particles in Figure 1 first dissolves selected lipids DOTAP, DOPE, and mPEG2000-DSPE in methanol. At the same time, mesoporous hydrolysable silicon nanoparticles (SiNPs) are exposed to methanol for activation. Subsequently, the methanol solvent is evaporated in a slow evaporation manner to generate activated SiNPs. This activation step is designed to make SiNPs readily dispersed in water. Then, the activated SiNPs are dispersed in nuclease-free water containing trehalose (THR) and glycine (GLY). Appropriate amounts of lipids are transferred to round bottom flasks sequentially, and methanol is evaporated by rotary evaporation. As a result of this evaporation step, a lipid film is generated on the wall of the flask. The suspension containing dispersed silicon, THR, and GLY is then added to the flask containing the lipid film for lipid rehydration.

[0501] Example 2 - Application of membrane extrusion

[0502] Validation of the concept of membrane extrusion method effectiveness

[0503] Micellar lipid particles were prepared using a general protocol. Activated SiNPs were mixed with THR and GLY in water, and the resulting solution was sonicated at 50 °C for 60 minutes. Appropriate amounts of the aqueous solution containing NPs-THR-GLY were exposed to lipid films sequentially for lipid film hydration. Lipid films were generated by mixing different methanolic lipid solutions at the concentrations specified in the general protocol and evaporating the solvent by rotary evaporation. Pre-activated SiNPs were used without further exposure to MeOH. The general steps for sample MVI0001 are shown in Figure 3

[0504] Table 1 SiNP-THR-GLY stock solution concentration

[0505] [SiNP] fin 1 mg / ml

[0506] [THR] fin 1 mg / ml

[0507] [GLY] fin 0.5 mg / ml

[0508] 25 mg of DOTAP-Cl, DOPE, and mPEG2000-DSPE lipids were dissolved in 5 ml of MeOH, respectively, to prepare lipid solutions with a concentration of 5 mg / ml. The lipid solutions were then sonicated thoroughly at 40 °C for 30 minutes. After sonication, appropriate amounts of each lipid solution were transferred to 10 ml round bottom glass flasks, as shown in Table 2.

[0509] Table 2 Lipid solution volume to be transferred for 10 ml batch

[0510] ​

[0511] The lipid film was generated by evaporation of the solvent using rotary evaporation with the following settings: P = 300 mbar, T = 40 °C, t = 30 min. Next, 1 ml of the aqueous solution containing the SiNP-THR-GLY was exposed to the lipid film for 5 min lipid film hydration at 60 °C. Finally, MilliQ water was added until 10 ml. After the preparation of the crude sample, flow extrusion was performed as described in the first part of this section and reported in Figure 1. In short, the sample was extruded at room temperature using a Knauer K501 HPLC pump at a speed of 10 ml / min through a three-in-a-row 25 mm extrusion membrane with a pore size of 0.4 pm and a three-in-a-row 25 mm extrusion membrane with a pore size of 0.1 pm. Before each extrusion step, the membranes were washed with 20 ml MeOH and rinsed with 20 ml MilliQ water.

[0512] Figure 4 The visual observation of the resulting solutions of each step is reported.

[0513] A fine sediment was observed in the SiNP dispersion with THR and GLY. It was confirmed that the lipid film had been generated correctly, as indicated by the presence of an opaque film on the walls of the round bottom flask. Upon addition of the SiNP solution to the lipid film, a larger sediment was observed to form. When extrusion was performed, the sediment disappeared. After extrusion, the solution became clear. Before extrusion and after each extrusion step, DLS and zeta potential analysis was performed on the resulting MVI0001 sample. The resulting values were compared to the reference values and reported in Table 3.

[0514] Table 3. DLS and zeta potential investigation results of MVI0001 before and after each extrusion step. SiSaf reference values are also reported. Figure 5

[0515]

[0516] Example 3 The results of the analysis of the Z-average, PDI and zeta potential of MVI0001 in the extrusion steps are reported.

[0517] The experimental observations show that extrusion improves the monodispersity of the sample. The larger sediment was captured by the 0.4 pm membrane and the solution after the 0.1 pm extrusion looked completely clear. The zeta potential remained essentially constant throughout the extrusion steps. The Z-average, PDI and zeta potential values were similar to the reference values shown for SiSaf reported in Table 3.

[0518] Figure 6

[0519] In this experiment, the direct addition of unactivated SiNPs to a lipid membrane was investigated. First, the unactivated SiNPs were exposed to MeOH for 30 minutes for activation. After activation, the SiNPs were directly added to a THR-GLY aqueous solution and sonicated at 50°C for 60 minutes. Finally, the aqueous solution containing SiNP-THR-GLY was exposed to a lipid membrane formed by evaporating methanol from a methanol-based lipid solution. The general procedure for sample MVI0002 is as follows: Table 3 SiNP stock solution concentration in MeOH As shown.

[0520] To prepare this sample, 20 mg of unactivated SiNP was first soaked in 1 ml of MeOH (Table 3).

[0521] Figure 6

[0522] [SiNP] 20mg / ml

[0523] Simultaneously, 20 mg THR and 10 mg GLY were weighed into the same test tube, and 19 ml of MilliQ water was added to the test tube. After 30 minutes of MeOH exposure, SiNP was considered to be activated, so it was added to the THR-GLY solution, and the solution was sonicated at 50°C for 60 minutes. The final concentration of the SiNP-THR-GLY stock solution was the same as reported in Experiment MVI0001 in Table 1.

[0524] As described in MVI0001, lipid solutions of DOTAP-Cl, DOPE, and mPEG2000-DSPE with a concentration of 5 mg / ml were prepared, and appropriate amounts of each lipid solution were transferred to 10 ml round-bottom glass flasks, as shown in Table 2. Next, the solvents were evaporated by rotary evaporation to generate a lipid membrane. The rotary evaporation was performed using the following settings: P = 300 mbar, T = 40 °C, t = 30 min. Subsequently, 1 ml of an aqueous solution containing NP-THR-GLY was exposed to the lipid membrane to hydrate it at 60 °C for 5 min. Finally, MilliQ water was added to a final volume of 10 ml.

[0525] After preparing the crude sample, such as Figure 7 As shown, flow extrusion was performed using a Knauer K501 HPLC pump at a flow rate of 10 ml / min at 60 °C through 0.4 μm and 0.1 μm extrusion membranes. Before each extrusion step, the membranes were washed with 20 ml of MeOH and rinsed with 20 ml of MilliQ water.

[0526] Figure 7 The results of visual examination of the solutions obtained at each step were reported.

[0527] In the experimental sample MVI0002, the SiNPs were first exposed to MeOH for activation. As shown in Table 5. DLS and zeta potential investigation results of MVI0002 before and after each extrusion step. SiSaf reference values are also reported. a large amount of SiNPs was deposited at the bottom of the container. In addition, it was observed that a precipitate was formed in the SiNP dispersion with THR and GLY. The lipid membrane was correctly generated as indicated by the presence of an opaque film on the walls of the round flask. Upon addition of the SiNP solution to the lipid membrane, a very large precipitate and deposit was observed at the bottom of the round flask. The precipitate disappeared when extrusion was performed. After extrusion, the solution became clear. DLS and zeta potential analysis were performed on the generated MVI0002 sample before extrusion and after each extrusion step. The obtained values were compared to the SiSaf reference values and reported in Table 5.

[0528] Figure 8 Example 4 - Elimination of the evaporation step in sample MVI0003

[0529]

[0530] Figure 9 The results of the analysis of the Z-average, PDI and zeta potential of sample MVI0002 in the extrusion steps are reported.

[0531] In agreement with the experimental observations of sample MVI0001, the extrusion improved the monodispersity of the analyzed sample. The large precipitate was captured by the 0.4 pm membrane and the solution after the 0.1 pm extrusion looked completely clear. The zeta potential remained essentially unchanged throughout the extrusion steps. The PDI and zeta potential values were similar to the reference values (Table 5), while the Z-average was higher than the reference values.

[0532] Figure 9

[0533] The injection of the lipid solution directly into the aqueous solution containing SiNPs-THR-GLY was investigated. The SiNPs were first exposed to MeOH for 30 minutes for activation. After activation, the SiNPs were directly added to the aqueous solution of THR-GLY and sonicated for 60 minutes at 50 °C. Finally, the lipid solution was prepared and slowly injected into the aqueous solution containing SiNPs-THR-GLY. The general procedure for sample MVI0003 is shown in Figure 10

[0534] ​To prepare this sample, a SiNP-THR-GLY solution was prepared as described for sample MVI0002. Briefly, 20 mg SiNP were exposed to 1 ml MeOH for 30 minutes to activate (see Table 4). 20 mg of THR and 10 mg of GLY were weighed in the same test tube and 19 ml MilliQ water was added to the test tube. After activation, the SiNP were added to the THR-GLY solution and the solution was sonicated for 60 minutes at 50°C. The final concentration of the SiNP-THR-GLY stock solution was the same as reported in the experiments of samples MVI0001 and MVI0002 and in Table 1.

[0535] A lipid solution of DOTAP-Cl, DOPE and mPEG2000-DSPE at a concentration of 5 mg / ml was prepared as described for sample MVI0001 and an appropriate amount of each lipid solution was transferred to a 10 ml round bottom glass flask as indicated in Table 2. Subsequently, 1 ml of the aqueous solution containing SiNP-THR-GLY was transferred to a glass vial with a stirrer. Next, the lipids were collected using a 10 ml sterile syringe and slowly injected into the SiNP solution using a ProSense NE1000 syringe pump. After setting the correct diameter of the syringe used, the flow rate of the syringe pump was set to 3.2 ml / min. Subsequently, MilliQ water was added until a final volume of 10 ml was reached. After dilution, the solution was stirred for 30 minutes to homogenize the solution.

[0536] After preparation of the crude sample, similar to the experiment of sample MVI0002, a Figure 10 flow extrusion was performed using a Knauer K501 HPLC pump at a flow rate of 10 ml / min at a temperature of 60°C through a 0.4 μιη and a 0.1 μιη extrusion membrane. Again, before each extrusion step, the membrane was washed with 20 ml MeOH and rinsed with 20 ml MilliQ water.

[0537] Figure 7 The results of the visual inspection of the solutions obtained at each step are reported.

[0538] In experiment MVI0003, similar to experiment MVI0002, the SiNP were first exposed to MeOH to activate. As shown in Table 6. DLS and zeta potential investigation results of MVI0003 before and after each extrusion step. SiSaf reference values are also reported. It was observed that the SiNP were deposited at the bottom of the container. In addition, it was observed that large precipitates were formed in the SiNP dispersion with THR and GLY. The formation of precipitates was also observed when the lipids were added to the SiNP solution. However, the slow direct injection of the lipids into the SiNP solution using a syringe seemed to improve the solubility of the SiNP and the formation of precipitates was less compared to MVI0002 Figure 11), about the precipitation, less precipitate was observed. When extruded, the precipitate disappeared. After extrusion, the solution became clear. DLS and zeta potential analysis was performed on MVI0003 samples before and after each extrusion step. The obtained values were compared to SiSaf reference values and reported in Table 6.

[0539] Table 7 Comparison of DLS characteristics of the three investigated experimental steps Example 5 - Optimization of flow extrusion

[0540]

[0541]

[0542] Investigation of the use of an additional 0.8 pm extrusion membrane to capture large aggregates (MVI0007) The results of the analysis of the Z-average, PDI and zeta potential of MVI0003 in the extrusion steps are reported.

[0543] Again, extrusion improved the monodispersity of the MVI0003 sample. Larger precipitates were captured by the 0.4 pm membrane and the solution after 0.1 pm extrusion looked completely clear. The zeta potential remained essentially unchanged throughout the extrusion steps. The Z-average, PDI and zeta potential values were similar to the reference values, as shown in Table 6.

[0544] In Table 7, the DLS characteristics of all three analyzed samples after 0.1 pm extrusion are reported.

[0545] Figure 17

[0546]

[0547] The observations on sample MVI0001 and sample MVI0003 showed that the DLS characteristics of the samples after extrusion were consistent with the reference values. Sample MVI0001 was also visually similar to the expected outcome, while sample MVI0003 was characterized by the presence of large aggregates in the SiNP solution. These observations have shown that the evaporation step has been successfully reproduced in trial MVI0001 and that it is feasible to inject lipids directly into a solution of unpreactivated SiNPs. It is worth noting that the last experimental approach is the most suitable for scaling up the micellar lipidic particle manufacturing process.

[0548] Figure 17

[0549] The flow extrusion method was further investigated by evaluating:

[0550] 1. Use of an additional 0.8 pm membrane to capture large silicon aggregates (MVI0007)

[0551] 2. Reproduction of the experimental results using nuclease-free water (MVI0008)

[0552] 3. Pre-filtering using 0.8 pm syringe filter prior to flow extrusion (MVI0011)

[0553] 4. Extruding large (1 L) volumes of micellar lipid particle solution - (MVI0012)

[0554] 5. Extrusion pressures generated by different experimental setups

[0555] Representative results from each experiment are reported.

[0556] Figure 18

[0557] To remove large SiNP aggregates as a target, the use of an additional extrusion membrane with a pore size of 0.8 pm was investigated. Those aggregates can indeed generate the experimental variability observed in the reported experiments. Figure 19A The experimental setup for sample MVI0007 using an additional 0.8 pm membrane is reported.

[0558] Figure 19B Micellar lipid particles with a final volume of 50 ml were prepared. Flow extrusion was performed at 60 °C through a three-stage consecutive 25 mm extrusion membrane with a pore size of 0.8 pm, a three-stage consecutive 25 mm extrusion membrane with a pore size of 0.4 pm and a three-stage consecutive 25 mm extrusion membrane with a pore size of 0.1 pm. Figure 19A The visual observation results of the solutions obtained at each step in experiment MVI0007 are reported.

[0559] In experiment MVI0007, a precipitate was observed to form in the SiNP dispersion with THR and GLY. A precipitate was also observed to form when the lipids were added to the SiNP solution. The precipitate disappeared when the extrusion was performed. After extrusion, the solution became clear.

[0560] For comparison, in Figure 19B images of the 0.4 pm and 0.1 pm membranes after extrusion without the use of a 0.8 pm membrane are reported. Figure 20 Images of the 0.8 pm, 0.4 pm and 0.1 pm membranes after extrusion are reported.

[0561] In Table 8. DLS and zeta potential investigation results of sample MVI0007 before and after each extrusion step. SiSaf reference values are also reported. , the 0.4 pm extrusion membrane appears to have been completely covered by the brown layer of SiNPs, which indicates that in the absence of the 0.8 pm membrane, this membrane captured most of the aggregates. Conversely, as shown in Figure 21 , the 0.8 pm extrusion filter membrane present captured most of the aggregates in the solution as it appears to be covered by the brown layer of SiNPs.

[0562] DLS and zeta potential analysis was performed on MVI0007 samples before and after each extrusion step. DLS investigation results for the samples are reported in Table 8 and Figure 21

[0563] Example 6 - Reduction of process steps Figure 17

[0564]

[0565]

[0566] DLS investigation results confirmed that the 0.8 μιη extruded film removed most of the SiNP aggregates, as indicated by the decrease in the Z-average value after 0.8 μιη extrusion.

[0567] Zeta potential measurements after 0.1 μιη extrusion showed relatively low values. For this reason, samples with higher concentration were analyzed to obtain good zeta potential values. The dilution factors investigated were 20x (value indicated for SiSaf) and 5x. Investigation results are shown in Figure 22

[0568] Figure 22 It is important to note that the reference dilution value was 20x. Interestingly, the Z-average diameter obtained using a 20x dilution factor was 72 nm, while the Z-average diameter obtained using a 5x dilution factor was slightly higher (80 nm). The reference value was 108 nm.

[0569] Figure 23

[0570] In this example, the use of nuclease-free water to generate 50 ml batches of micellar lipid particles was investigated. The experimental setup used in this experiment was the same as in Table 9: DLS and zeta potential investigation results of sample 08 before and after each extrusion step. SiSaf reference values are also reported. for sample MVI0007. It is important to note that in this case, the experimental steps were slightly changed to reduce the time of exposure of the SiNPs to non-sterile environment and to avoid unnecessary steps that could lead to microbial contamination. Thus, after activation, the SiNPs were diluted by adding the THR-GLY aqueous solution directly into the same test tube in which the SiNPs were activated, instead of transferring the SiNPs in the test tube to the THR-GLY aqueous solution. Flow extrusion of 0.8 μιη, 0.4 μιη and 0.1 μιη membranes was performed at 60 °C.

[0571] Example 7 - Use of an optional pre-filtration step Pictures of the solutions obtained for labeled sample MVI0008 are reported in

[0572] As Figure 24 ​​As shown, a large amount of precipitate was still observed in experimental MVI0008 before extrusion. DLS analysis was performed on the MVI0008 sample before extrusion and after each extrusion step. The DLS results of the three samples are shown in Table 9 and... Figure 24 Report from the Central Committee.

[0573] Figure 26 Table 10. DLS and zeta potential investigation results of MVI0011 before and after each extrusion step. SiSaf reference values are also reported.

[0574]

[0575]

[0576] In this experiment, sample MVI0007 was used as a reference, and nuclease-free water was used to prepare micellar lipid particles. Experimental observations show that sample MVI0008 successfully reproduced the properties of MVI0007, thus achieving the expected characteristics using nuclease-free water. The precipitate was largely captured by the 0.8 μm membrane. Extrusion improved the monodispersity of all analyzed samples. The solution after 0.1 μm extrusion appeared completely clear. Importantly, changing the order of addition had no effect on the experimental results.

[0577] Figure 25

[0578] In this embodiment, the removal of large aggregates from the SiNP solution using a pre-filtration step prior to flow extrusion was investigated. The final volume of this experiment was increased to 100 ml. Furthermore, a MeOH lipid stock solution with a concentration increased to 10 mg / ml (instead of 5 mg / ml) was prepared to reduce the volume of MeOH injected with the lipids in the final solution. For pre-filtration, a 0.8 μm hydrophilic polyethersulfone injection filter was used. Example 8 - Demonstration on large batches. The strategy used in this experiment is shown and named MVI0011.

[0579] Before flow extrusion, micelle lipid particles were filtered using a 0.8 μm hydrophilic polyethersulfone injection filter. Figure 17 The images of the injection filter reported in the report have shown that the pre-filtration step captured most of the large aggregates in the SiNP solution, as illustrated by the brown layer in the injection filter and the absence of the same layer on the 0.8 μm extruded membrane. Conversely, in experiments without this pre-filtration step, the 0.8 μm membrane appeared to be completely covered by the SiNP brown layer (see Figure 19).

[0580] DLS analysis was performed on the MVI0011 samples after each pre-filtration and extrusion step. The results of the DLS and zeta potential analyses of the samples are shown in Table 10 and... Table 11 Lipid solution volume to be transferred for 1000 ml batch Report from the Central Committee.

[0581] Figure 27Figure 28

[0582]

[0583]

[0584] After filtration, the Z-average improved, indicating that large aggregates were removed from the SiNP solution, which is in line with the reported visual observations. The DLS measurements of sample MVI0011 were in line with the expected values after extrusion. Figure 29 The visual observations are in line with the reported results. The DLS measurements of sample MVI0011 were in line with the expected values after extrusion.

[0585] Finally, the use of a higher concentration of the lipid stock solution was also investigated. The increase in methanol concentration had no effect on the DLS and zeta potential characteristics of the micellar lipid particles.

[0586] Table 12 DLS and zeta potential investigation results of MVI0012 In this example, the manufacturing of micellar lipid particles with a final volume of 1 L was explored to demonstrate the feasibility of the extrusion method for large batch sizes. This was considered as an intermediate step before manufacturing. In the experiment named MVI0012, the use of an extrusion membrane with a diameter of 47 mm was explored.

[0587] To prepare the micellar lipid particles, the experimental setup shown in Fig. 1 was used and the volume was adjusted to a final batch size of 1 L. In short, a SiNP-THR-GLY aqueous solution was prepared using the standard procedure: SiNPs were activated in MeOH and then diluted in the THR-GLY aqueous solution. For this, 100 mg of non-pre-activated SiNPs were weighed into a 200 ml sterile Falcon tube, 5 ml MeOH was added for activation. 100 mg of THR and 50 mg of GLY were weighed and added to 95 ml MilliQ water to prepare the THR-GLY aqueous solution. After activation of the SiNPs for 30 min at room temperature, the THR-GLY solution was added to the SiNPs and sonicated for 60 min at 50 °C. Example 9 - HPLC analysis

[0588] ​Next, 750 mg of DOTAP-CI, 750 mg of DOPE and 200 mg of mPEG2000-DSPE were weighed and 75 ml of MeOH were added to dissolve the DOTAP-CI and DOPE, and 20 ml of MeOH were added to dissolve the mPEG2000-DSPE to prepare a 10 mg / ml stock solution of DOTAP-CI, DOPE and mPEG2000-DSPE. Next, the lipid solution was sonicated at 40 °C for 30 minutes. 100 ml of SiNP-THR-GLY solution was transferred to a 1 L sterile bottle. Table 11 reports the volume of each lipid solution injected into the SiNP solution at this scale. Due to the large volume of mixed lipid solution, a HPLC pump was used to inject slowly. The flow rate was 6 ml / min. Finally, MilliQ water was added until 1 L.

[0589] ​

[0590]

[0591] The flow extrusion experiment was performed at 60 °C through 0.8 pm, 0.4 pm and 0.1 pm membranes. ​ Images of the obtained solutions are reported.

[0592] Interestingly, unlike other experiments, the solution after 0.8 pm extrusion was yellow, indicating that some silicon had passed through the membrane. ​ This is confirmed by the visual observation of the membranes after extrusion reported in Table 13, where a brown layer appeared on the 0.4 pm membrane, which was not observed in other experiments.

[0593] The DLS investigation results of the samples are reported in Table 12 and ​ Figure 6.

[0594] ​

[0595]

[0596] The experimental observation of experiment MVI0012 shows that the DLS and zeta potential measurements after extrusion are consistent with the reference values. Moreover, the use of a 47 mm membrane reduced the extrusion pressure, while increasing the lipid concentration in the initial MeOH lipid solution reduced the amount of MeOH in the final solution.

[0597] ​

[0598] In this section, the development of a method for the HPLC-CAD quantification of the lipid content in the solution was investigated. To this end, we investigated the use of a Waters Xbridge Phenyl column (SKU: 186003352):

[0599] • Particle size: 5 μm

[0600] • Pore size:

[0601] • Inner diameter: 4.6 mm

[0602] • Length: 150 mm

[0603] We used ammonium acetate at a concentration of 40 mM and methanol as elution buffer, with a gradient profile as reported in Table 13.

[0604] Table 13 Gradient profile of eluents used

[0605]

[0606] Then, 1 mg / mL solutions of each lipid were prepared and injected separately into the phenyl column.

[0607] Also, a 1 : 1 : 1 mixture of all three lipid solutions was prepared and investigated using the same method. The chromatogram of the mixed solution is reported in Figure 31 .

[0608] For the phenyl column, the elution peak of the lipid DOPE appeared at 15.95 min. DSPE-mPEG2k appeared as a broad peak between 16 and 23 min, which is mainly related to the polydispersity of the PEG chain. Finally, DOTAP-Cl eluted at about 24 min. For DOTAP-Cl, we observed a slight overlap of the peaks, which was due to the sudden change of the solvent at the end of the method. For this reason, the method used initially was optimized to elute the lipids earlier, as reported in Figure 33 .

[0609]

[0610]

[0611] Finally, a mixture of lipids was prepared and analyzed using the improved method, with a final product concentration of

[0612] [DOTAP-Cl] fin 0.725 mg / ml

[0613] [DOPE] fin 0.73 mg / ml

[0614] [mPEG2000'DSPE] fin 0.145 mg / ml

[0615] The chromatogram of the lipid mixture is reported in Figure 34 .

[0616] Experimental observations showed that the developed HPLC method separated the lipid peaks well. Therefore, the method was used to evaluate the lipid content in the extruded solutions of all samples.

[0617] The determination of the lipid content in the extruded samples was based on the preparation of a calibration curve for the three lipids, followed by HPLC sample analysis using the method described above. As a representative experiment, the detailed procedure and results obtained for experiment MVI0010 are reported here. A table summarizing the lipid recovery for all analyzed samples is also reported at the end of the example.

[0618] To perform the lipid quantification of the extruded samples, standard lipid solutions were prepared using the prepared lipid stock solutions as starting material. The standards used to build the calibration curves are reported in Table 14.

[0619] Table 14 Standard solutions for lipid quantification

[0620]

[0621] The chromatograms of each standard solution were determined and analyzed, and the calibration curves for each lipid were built by recording the peak area (area) versus the lipid concentration. Figure 37 The calibration curves for each lipid obtained for sample MVI0010 using the standards reported in Table 14 are shown.

[0622] The calibration curves for the three lipids were used to determine the lipid recovery after extrusion. Sample 10 was generated following the routine experimental procedure: SiNPs were exposed to methanol for activation and lipids were injected into the SiNP solution (see Figure 17 ). Then, the solution was extruded through 0.8 pm, 0.4 pm and 0.1 pm membranes at 60 °C, as previously described. Next, the sample was analyzed by HPLC and the chromatogram is reported in Figure 38 .

[0623] The area of each peak was used to determine the lipid concentration in the sample after extrusion. Table 15 shows the lipid recovery after extrusion for experiment MVI0010.

[0624] Table 15 Lipid recovery after extrusion of experiment MVI0010

[0625]

[0626] Finally, Table 16 summarizes the HPLC content of all analyzed samples after extrusion following the described procedure.

[0627] Table 16 HPLC content of analyzed samples

[0628]

[0629]

[0630] The experimental results show that the average recovery after extrusion is:

[0631] ■ DOPE: 79.2%

[0632] ■ DSPE-mPE: 76.6%

[0633] ■ DOTAP-Cl: 70.4%

[0634] Example 10 - Stability over time

[0635] In the chromatograms of the extruded samples, we noticed the appearance of unknown peaks at 7 min, 8 min and 9.5 min (see representative examples in Figure 36 ). To investigate possible sources of contamination, the chromatograms of samples of nuclease-free water and THR-GLY solution were first analyzed, and the results are reported in Figure 41

[0636] As shown in Figure 41 , GLY and THR elute directly at injection. Therefore, the peaks at 7 min, 8 min and 9.5 min do not seem to be related to GLY and THR.

[0637] To evaluate the evolution of the unknown peaks, the HPLC and DLS characteristics of the samples were analyzed over time. The samples were stored at room temperature (RT) and at 4°C, and the HPLC content and DLS characteristics were measured at 0 h (at manufacturing), after 1 week and after 2 weeks.

[0638] (i) Investigation of samples stored at room temperature

[0639] The evolution of the lipid content of the samples stored at RT was determined at different time points, specifically at 0 h, after 1 week and after 2 weeks. To this end, calibration curves at different time points were constructed. Then, for each lipid, the evolution of the lipid concentration was determined by comparing the lipid concentration measured at 0 h and the lipid concentration measured at different time points. In Figure 44 , the chromatogram of the sample stored at RT for 2 weeks is reported.

[0640] To further investigate the stability of the samples over time, the DLS characteristics were measured, and the results are reported in Table 17.

[0641] Table 17 DLS characteristics of analyzed samples over time at RT

[0642]

[0643] Table 17 shows that the DLS characteristics of the samples stored at RT are essentially maintained over time.

[0644] ​(II) Samples stored at 4°C were investigated

[0645] The lipid content of samples stored at 4°C was determined at different time points using the same procedure as for samples stored at RT. Therefore, calibration curves were constructed at each time point and the lipid concentration was determined by comparing the concentration measured at time 0 and over time. In Figure 44 The chromatograms of the analyzed samples at different time points at 4°C are reported in

[0646] DLS and zeta potential characteristics were measured over time and the results are reported in Table 18.

[0647] Table 18 Characteristics of analyzed samples over time at RT

[0648]

[0649] As reported in Table 18, also for samples stored at 4°C, DLS characteristics were substantially maintained over time.

[0650] The experimental observations on the investigated samples show that the lipid concentration decreases over time in samples stored at 4°C and at RT. This decrease can also be attributed to instrumental differences, as the measured concentration: peak area for each lipid can lead to greater experimental variability. Interestingly, increasing the injection volume leads to an increase in the recovery of DSPE-mPEG lipids (114%). This observation also indicates instrumental variability of the results obtained.

[0651] Example 11 - Evaluation of flow rate of tangential flow

[0652] Tangential flow filtration (TFF) is a fast and efficient method for separating and purifying biomolecules. In this study, TFF was used to purify the samples by diafiltration and to finally concentrate them to match the desired component concentration range. By diafiltration, smaller molecules are washed through a membrane with a defined pore size, while larger molecules are retained in the retentate. For this reason, diafiltration was used to remove methanol. In this study, we performed diafiltration by adding the diafiltration solution to the sample feed reservoir at the same rate as the filtrate was generated, using a second pump. In this way, the volume in the sample reservoir remained constant, but MeOH freely permeated through the membrane and was washed away. Since THR and GLY have a small molecular weight, they can be removed during TFF. For this reason, we used a THR-GLY solution with the same final concentration (THR 0.1 mg / ml, GLY 0.05 mg / ml) as the diafiltration solution.

[0653] The same volume of diafiltration solution as the product in the system was added to the feed tank, then concentrated to the starting volume, constituting a diafiltration volume (DV). Thus, for a 100 mL starting sample, 1 DV = 100 mL. It was previously observed that using 10 DVs, the MeOH content was drastically reduced to values in the 100 ppm range by continuous diafiltration (European standard acceptable MeOH content < 3000 ppm).

[0654] To perform a feasibility study of TFF, we used a membrane with the following characteristics:

[0655] ■Surface area 0.02 m 2

[0656] ■MWCO 100 kDa

[0657] ■HyStream membrane. The HyStream membrane has a very strong hydrophilicity, which resists fouling by hydrophobic substances (e.g., lipids). The membrane has good chemical resistance.

[0658] ■Structure LP-sieve. This type of flow channel is best suited for clarified feed streams with a wide viscosity range.

[0659] The general TFF setup used in this study is shown in Figure 46 .

[0660] The samples were generated following this procedure: NPs were exposed to methanol for activation, then diluted in a THR-GLY aqueous solution. Subsequently, a mixed lipid solution was prepared and injected into the NP-THR-GLY dispersion. Then, the solution was first extruded through 0.8 pm, 0.4 pm and 0.1 pm membranes at 60 °C, then purified by TFF using the experimental setup described in Figure 46 To study the effect of TFF on the system, it was necessary to evaluate the DLS characteristics, HPLC lipid content and MeOH content before TFF and after TFF. The DLS characteristics and HPLC content analysis were performed as described above. The investigation of the MeOH content was performed by 1 H-NMR.

[0661] The general method to determine the MeOH content in the samples was based on making a MeOH calibration curve. The detailed procedure and results obtained for experiment MVI0010 are shown here. As a representative experiment, the detailed procedure and results obtained for experiment MVI0010 are reported here. A table including the lipid recovery for all the analyzed samples is also reported at the end of the example.

[0662] MeOH standard solutions of different MeOH concentrations were prepared, with a final volume of 1 ml (Table 21).

[0663] Table 19 Concentration data used to construct MeOH calibration curve

[0664]

[0665] Ten percent volume of D2O was added to the standards (100 μL D2O). 600 μL of each standard was added to an NMR sample tube and analyzed using 1 H-NMR was used to analyze all standards. Figure 47 The calibration curve reported in the results was constructed by recording the peak area associated with the MeOH signal at different MeOH concentrations.

[0666] After the standard curve was made, samples were analyzed to determine the MeOH content. Samples before TFF were prepared by diluting the original sample appropriately to match the range of the calibration curve. For sample 11, a 1000x dilution was employed because the expected methanol content in the sample was approximately 30% of the sample volume (see Table 2 for an example). Samples after TFF were analyzed for concentration. The final volume of each sample was 1000 μL. Therefore, ten percent volume of D2O was added to the samples (100 μL D2O). 600 μL of each sample was added to an NMR sample tube and analyzed using 1 H-NMR. The area of the MeOH peak in the NMR chromatogram was then used to determine the concentration of MeOH in the samples before and after TFF.

[0667] Figure 48 The NMR signal of sample 10 before and after TFF is shown as a representative result.

[0668] Table 20 shows the MeOH quantification results of sample 10 before and after TFF.

[0669] Table 20 MeOH quantification results in sample MVI0010 before and after TFF

[0670]

[0671]

[0672] An estimation of the MeOH content was made taking into account the density of the MeOH-H2O mixture, which is 0.988 kg / L [2].

[0673] Using a similar method to the MeOH determination in sample 10, we also determined the MeOH content of the other analyzed samples (i.e. sample 08 and sample 09). The MeOH content of all analyzed samples before and after TFF is reported in Table 21.

[0674] Table 21 MeOH quantification results in analyzed samples before and after TFF

[0675]

[0676] Experimental observations show that the MeOH concentration of all analyzed samples was reduced by three orders of magnitude from 10 5 to 10 2 .

[0677] As already mentioned, the HPLC and DLS characteristics of the samples have to be evaluated after TFF in order to determine the feasibility of the process. Tables 35 and 36 report the DLS characteristics of all analyzed samples before and after TFF.

[0678] Table 35 DLS characteristics of analyzed samples before and after TFF

[0679]

[0680] Table 24 HPLC content of samples before and after TFF

[0681]

[0682] Experimental observations show that the DLS characteristics are essentially maintained after TFF. Finally, the lipid recovery is also maintained after TFF.

[0683] It is important to note that sample MVI0010 was 2x concentrated by TFF. This was necessary to increase the silicon content to match the required silicon concentration in the final product.

[0684] Example 12 - Further optimization and reduction of silicon content

[0685] In this example, further optimization of the process for developing micellar lipid particles is reported. We investigated the following three different experimental setups:

[0686] i. Filtration of the SiNP solution after activation, preparation of micellar lipid particle batches.

[0687] ii. Preparation of micellar lipid particle batches using 1 / 5 of the usual amount of Si (4 mg).

[0688] iii. Preparation of micellar lipid particle batches in the absence of DSPE-mPEG2000.

[0689] Filtration of the SiNP solution after activation, preparation of micellar lipid particle batches (MVI0013)

[0690] Figure 51 The experimental steps for the preparation of micellar lipid particles after filtration of the SiNP solution after activation are reported. This experiment is named MVI0013.

[0691] The preparation of micellar lipid particles is as Figure 52The final volume reported was 50 ml. After the SiNP solution was activated with MeOH, it was manually filtered using a 0.8 pm hydrophilic polyether sulfone syringe filter. The solution was flow-extruded through 25 mm extrusion membranes with pore sizes of 0.8 pm, 0.4 pm and 0.1 pm at 60 °C. Figure 52 The visual observations of the solutions obtained at each step of experiment MVI0007 are reported. Figure 53 Pictures of the 0.8 pm hydrophilic polyether sulfone syringe filter and the extrusion membranes after the filtration and extrusion steps, respectively, are reported.

[0692] In Figure 52 , the filtered solution was light yellow and no large aggregates were present, which already indicated that most of the aggregated SiNPs were removed from the solution. Figure 53 This observation was also confirmed as indicated by the presence of a brown layer of NPs on the 0.8 pm syringe filter shown in Figure 53 A. Moreover, the 0.8 pm and 0.4 pm extrusion membranes were completely clean as indicated by the absence of any brown areas, while the 0.1 pm membrane still captured some smaller aggregates of SiNPs.

[0693] DLS and zeta potential analyses were performed on the MVI0013 sample before the extrusion and after each extrusion step. The obtained values were compared to the SiSaf reference values. The DLS investigation results of the sample are reported in Table 37 and Figure 54 .

[0694] Table 37 DLS and zeta potential investigation results of MVI0013 before extrusion and after each extrusion step. SiSaf reference values are also reported. Figure 55

[0695]

[0696] The DLS investigation results indicated that the 0.8 pm syringe filter removed most of the aggregates of SiNPs as indicated by the initial value (value before extrusion) of the Z-average. The Z-average was substantially maintained until the 0.4 pm extrusion step, which also confirmed that the aggregates were removed by the syringe filter. The extrusion pressure is also reported in Table 38 HPLC content investigation results of MVI0013 and showed similar indications: the pressure on the 0.8 pm and 0.4 pm membranes was almost zero, confirming the absence of large aggregates in the extruded solution, while some pressure was reported on the 0.1 pm pore size membrane. Finally, the PDI and zeta potential values were substantially maintained in all steps.

[0697] Figure 56

[0698]

[0699] The experimental observations in experiment MVI0013 indicated that the DLS characteristics were in line with the expected values. However, we noticed the absence of a clear peak in the zeta potential analysis, which is not the case for the SiSaf reference.Figure 57 ). Finally, HPLC quantification results showed a lipid recovery of 90%.

[0700] (ii) a micellar lipid particle batch was prepared using 1 / 5 of the silicon content (MVI0014).

[0701] Figure 57 The experimental procedure for the preparation of a micellar lipid particle batch filtering the SiNP solution after activation is reported. This experiment is named MVI0014.

[0702] The preparation of the micellar lipid particle was as follows Table 39 Final theoretical concentrations of each component for the preparation of MVI0014 micellar lipid particle batch. It is reported that the final volume was 50 ml. The SiNP solution was generated by reducing the silicon content usually used in all the other experiments, as shown in Table 39.

[0703] Figure 58

[0704]

[0705] To this end, 4 mg of non-activated SiNPs were weighed and exposed to 1 ml of MeOH, reducing the concentration of the SiNP solution to 4 mg / ml. The visual observation results of the resulting solution are reported in Figure 59 .

[0706] DLS and zeta potential analyses were performed on the MVI0014 sample before extrusion and after each extrusion step. The resulting values were compared with the SiSaf reference values. The DLS investigation results of the sample are reported in Table 40 and Table 40 DLS and zeta potential investigation results of MVI0014 before extrusion and after each extrusion step. SiSaf reference values are also reported. .

[0707] Figure 60 Figure 60 .

[0708]

[0709] Table 41 HPLC content investigation results of MVI0014 The extrusion pressure investigation results of MVI0013 are reported. As shown in Figure 61 , the extrusion pressure values were relatively low, which can be related to the lower silicon content used in this experiment.

[0710] Finally, the HPLC content was investigated, and the results are reported in Table 41.

[0711] Figure 61

[0712]

[0713] The experimental observation results in the experiment MVI0014 showed that the DLS characteristics were consistent with the expected values. HPLC quantification results showed a lipid recovery of 80%.

[0714] (iii) Preparation of a batch of micellar lipid particles in the absence of DSPE-mPEG2000 (MVI0015)

[0715] Table 42 Final theoretical concentrations of each component for the preparation of MVI0015 micellar lipid particle batch. The experimental procedure for the preparation of a batch of micellar lipid particles from a filtered SiNP solution after activation is reported. This experiment is named MVI0015. It is important to note that after extrusion, TFF was performed to remove MeOH and concentrate the sample by a factor of 2.

[0716] The preparation of micellar lipid particles was as described in Figure 62 It is reported that the final volume was 200 ml (before TFF). The concentration of lipids in the final solution was adjusted as reported in Table 42.

[0717] Figure 63

[0718]

[0719] The results of the visual observation of the resulting solution are reported in Table 43 DLS and zeta potential investigation results of MVI0015 before extrusion and after each extrusion step. SiSaf reference values are also reported. .

[0720] After extrusion, TFF was performed as explained in section 2.6 "Feasibility of tangential flow filtration" and the MVI0015 sample was purified and 2x concentrated. DLS and zeta potential characteristics were investigated and the results are reported in Table 43 and Figure 60 .

[0721] Table 44 HPLC content of MVI0015 before and after TFF Table 45 Methanol content in MVI0015 .

[0722]

[0723] Additionally, HPLC content was investigated and the results are reported in Table 44.

[0724] Example 13 - Determination of silicon content

[0725]

[0726] Finally, the MeOH content was investigated by 1 H-NMR and the results are reported in Table 45.

[0727] Table 46 Overview of analyzed samples in silicon content determination

[0728]

[0729] In MVI0015, the MeOH content measured after TFF showed a value lower than the lowest concentration point in the calibration curve, 50 ppm. This value can not be accurate. However, this indicates that MeOH has been almost completely removed from the sample.

[0730] Experimental observations in Experiment MVI0015 (absence of DSPE-mPEG2000) showed that DLS characteristics were consistent with reference values and that DLS characteristics were substantially maintained after TFF. HPLC quantification results showed a lipid recovery of 90% prior to TFF, but we observed a decrease in lipid recovery after TFF (90% to 60%).

[0731] Table 47 Silicon content investigation results of all analyzed samples using different experimental methods.

[0732] The silicon content was determined by ICP-OES at SiSaf for samples prepared using the different experimental methods described above. Specifically, the silicon content was determined in samples prepared by the original SiSaf experimental procedure of exposing pre-activated SiNPs to a lipid membrane (Experiment MVI0004) and compared to the silicon content measured using the modified experimental procedure of injecting lipids into a solution of non-pre-activated SiNPs (i.e. non-pre-hydrated SiNPs + lipid injection). For convenience, Table 46 reports an overview of the samples analyzed.

[0733] Example 14 - Manufacturing of 2 kg batch

[0734] MVI0004 Pre-hydration + Membrane hydration

[0735] MVI0005C Non-pre-hydration + Injection

[0736] MVI0009 Non-pre-hydration + Injection + TFF

[0737] MVI0010 Non-pre-hydration + Injection + TFF 2x Concentration

[0738] MVI0011 Non-pre-hydration + Injection + 0.8 pm filtration of SNiP-lipid solution before extrusion

[0739] MVI0013 Non-pre-hydration + Injection + 0.8 pm NP filtration after activation

[0740] MVI0014 Non-pre-hydration + Injection using 1 / 5 of silicon content

[0741] MVI0015 Non-pre-hydration + Injection, no DSPE-mPEG 2000 + TFF 2x Concentration

[0742] MVI0022 Non-pre-hydration + Injection + TFF 2x Concentration - 2 Kg batch

[0743] Table 47 summarizes the ICP-OES investigation results for all analyzed samples.

[0744] Figure 64

[0745]

[0746] The reference value for the silicon content in the final product was 2.1 mg / L. This value was also found in the experiment MVI0004, where the original SiSaf procedure was repeated. On the other hand, using the experimental procedure reported in MVI0005C and MVI0009 (non-prehydrated SiNP + lipid injection), a silicon content of 1.2 mg / L was found. By applying the 2x concentration method by TFF to MVI0010 (as reported in section 2.6 “Feasibility of tangential flow filtration”), we recovered the silicon content, finding a silicon content of 2.18 mg / L in the sample after TFF. Interestingly, some variability in the results was observed in the samples MVI0011 to MVI0015, which were modified as shown in Table 46.

[0747] Table 48 Theoretical concentrations before and after TFF

[0748] In this example, the results of manufacturing a 2 kg batch of micellar lipid particles are reported. Table 49 Stock solutions and volumes used to generate 4 L solution The experimental procedure to produce a 2 kg batch is shown in Table 46. The experiment was named MVI0022.

[0749] The obtained batch was characterized by DLS and zeta potential, the lipid content was determined by HPLC, and MeOH quantification was performed by H-NMR. 1 H-NMR.

[0750] For the calculation, the density of the SiNP-lipid dispersion was assumed to be 1 g / cm3. 3 Therefore, the 4 L SNiP-lipid solution was 2x concentrated by TFF. The final volume was 2 L, which corresponds to 2 kg. Table 48 reports the theoretical concentrations of all components before and after concentration. It is important to note that the concentrations of THR and GLY were maintained after TFF, since the final concentration of the THR-GLY solution we used (0.1 mg / ml THR and 0.05 mg / ml GLY) was the same as the diafiltration solution.

[0751] Table 50 Visual observations of solutions obtained at each step in experiment MVI0022

[0752]

[0753] To prepare a 2 kg batch of micellar lipid particles, the SiNP-THR-GLY aqueous solution was prepared using the standard procedure: SiNP were activated in MeOH (MeOH dispersion + sonication), and then SiNP were diluted in the THR-GLY aqueous solution.

[0754] For this purpose, 400 mg of unactivated SiNP was weighed and transferred to a 50 ml sterile Falcon tube, and 20 ml of MeOH was added for activation. A THR-GLY aqueous solution was prepared by dissolving 400 mg THR and 200 mg GLY in 380 ml of nuclease-free water (in a Nalgene sterile bottle). After activating SiNP at RT for 30 minutes, SiNP was added to the THR-GLY solution, and the resulting dispersion was sonicated at 50 °C for 60 minutes. A 10 mg / ml stock solution of DOTAP-Cl, DOPE, and mPEG2000-DSPE was prepared by dissolving 3000 mg DOTAP-Cl, 3000 mg DOPE, and 600 mg mPEG2000-DSPE in 60 ml MeOH. The lipid solution was then sonicated at 40 °C for 30 minutes. Transfer 400 ml of SiNP-THR-GLY solution to a 5 L sterile vial. Slowly inject the lipids using a Knauer K501 HPLC pump at a flow rate of 6 ml / min. After adding the lipids, add nuclease-free water to a final volume of 4 L, and then stir to homogenize the batch. Table 49 reports the prepared stock solution and the volume used to produce the 4 L solution.

[0755] Figure 65

[0756]

[0757] The preparation of the 4L crude product was not particularly difficult, and it was stored at 4°C overnight. As usual, large SiNP aggregates were observed.

[0758] Then, flow extrusion was performed at 60°C using three consecutive 47mm extrusion films (pore sizes of 0.8μm, 0.4μm, and 0.1μm). Extrusion was performed using a Knauer K501 pump equipped with a pressure sensor and a 50ml / min stainless steel pump head. The flow rate was set to 50ml / min.

[0759] The visual observation results of MVI0022 are reported in Table 50.

[0760] Figure 66

[0761]

[0762] It is worth noting that, such as Table 52 HPLC investigation results of MVI0022 The report states that SiNP aggregates saturate the membrane. The membrane undergoes changes during extrusion due to pressure fluctuations.

[0763] TFF was performed according to MVI0010 setup. For MVI0022, we increased the surface area of the TFF membrane package to 0.5 m 2 TFF membrane, while preserving other characteristics of the membrane package for small scale batches of extruded (HyStream, MWCO 100 kD, LP screen flow channel). Importantly, the entire system was flushed with 0.2 M NaOH solution for 1 h prior to TFF to remove pyrogens. This approach was introduced at this stage for 2 kg batch manufacturing. The removal of pyrogens is widely used to remove pyrogens from contact materials.

[0764] DLS investigation results for MVI0022 are reported in Table 51 and Figure 67 Table 52.

[0765] Table 51 DLS and zeta potential investigation results for MVI0022

[0766]

[0767] HPLC investigation results in MVI0022 are reported in Table 52. It is important to note that samples were diluted with MeOH as indicated in section 2.4.3, page 32: 2x dilution for samples before TFF and 4x dilution for samples after TFF treatment to match the range of the calibration curve.

[0768] Example summary

[0769]

[0770] Investigation results for MeOH quantification in MVI0022 1 H-NMR investigation results are reported in 53.

[0771] Table 53 MVI0022 1 H-NMR investigation results

[0772]

[0773] Finally, the silicon recovery of the sample after TFF was determined by ICP-OES to be 2.9 mg / L.

[0774] The experimental observation results of experiment MVI0022 show that:

[0775] DLS measurement results

[0776] o The zeta mean is slightly higher than expected

[0777] • MVI0022 is 132 nm, reference value is 108 nm, small scale batch is mean 78 nm

[0778] • The zeta mean is essentially maintained after TFF

[0779] o PDI was consistent with expected value before TFF (0.175) and slightly increased after TFF (0.248)

[0780] o Zeta potential was consistent with expected value before and after TFF (64.5 mV)

[0781] HPLC measurement

[0782] o Analysis before TFF showed high lipid recovery relative to expected concentration (95%)

[0783] o Analysis after TFF showed high lipid recovery relative to expected concentration (100%)

[0784] 1 H-NMR measurement

[0785] o No MeOH was detected in the sample after TFF

[0786] ICP-OES measurement

[0787] o Silicon recovery was consistent with expected value

[0788] It is worth noting that we observed a general trend of increasing size with increasing extrusion volume, as Table 55 HPLC content and lipid recovery of all analyzed samples reported in the literature.

[0789] The clogging of the extrusion membrane during flow extrusion due to the presence of SiNP aggregates can be the source of the Z-average experiment variability. A pre-filtration step (0.8 pm or larger filter) can be performed right after SiNP activation to avoid filter clogging.

[0790] Due to pressure fluctuations, the extrusion membrane was changed during the extrusion process. In the next experiments, flow extrusion can be considered by using a parallel extrusion jig that houses two extrusion membranes of the same pore size to increase the extrusion surface area.

[0791] Time is a critical parameter for the extrusion process. Increasing the extrusion flow rate by changing the pump / pump head will have a positive impact on the extrusion time (the intrinsic limit of the pump / pump head we are currently using is 30 ml / min, which decreases over time due to membrane clogging).

[0792] Finally, based on this experimental observation, 0.5 m 2 TFF membranes can be used for 5 kg batch size production.

[0793] Example 15 - Storage stability

[0794] These examples investigate the feasibility of a scalable process to manufacture the micellar lipid particles of the present application. The feasibility of adding activated SiNPs directly from methanol into an aqueous solution, and the feasibility of adding lipids directly into SiNPs, followed by flow extrusion and tangential flow filtration workup, to produce a scalable process to manufacture micellar lipid particles was investigated. The experimental results confirm the feasibility of these steps. Both DLS and zeta potential characteristics are in line with expected characteristics. The recovery of lipids after extrusion is 70-80% of the initial lipid concentration. (DOPE: 79.2%, DSPE-mPEG: 76.6%, DOTAP-Cl: 70.4%). All these characteristics are essentially maintained after TFF. In addition, TFF shows successful removal of methanol from all investigated samples, and also demonstrates the feasibility of 2x concentration of the solution to match the optimal concentration of components in the final product.

[0795] HPLC investigation of the lipid content shows the presence of unknown peaks. HPLC investigation of the samples over time indicates a decrease in lipid concentration. However, the decrease in concentration shown by the HPLC measurement can be attributed to instrument error due to the low concentrations measured. Supporting this hypothesis is the increase in recovery of DSPE-mPEG lipids with increased injection volume. This characteristic can be further investigated by LC-MS, for example. Silicon recovery shows large variations. Concentration by TFF workup is currently required to match the reference value for silicon recovery.

[0796] Tables 54 and 55 summarize the characteristics of all analyzed samples, reported in the following pages.

[0797] Table 54 DLS and zeta potential characteristics of all analyzed samples

[0798]

[0799]

[0800] Figure 69

[0801]

[0802]

[0803] Figure 69

[0804] The present study compares the storage stability of the mixed lipid particles of the present application formulated with silicon versus lipid nanoparticles formulated without silicon or other inorganic material particles. Particles were prepared according to the above described method for Biocourier MVI0012 and MVI0012. The comparative particles (LNP) of the prior art were also prepared using the same method as MVI0012 and had the same composition as MVI0012 but with silicon particles or other inorganic material particles. The particles were then stored at 4°C or room temperature for several weeks. The size of the particles and their surface charge were determined periodically and the results are shown in Example 16 - Size stability after RNA loading It can be seen that the positive surface charge of the particles of the present application is maintained at room temperature for at least 24 weeks and at 4°C for at least 80 weeks. It is hypothesized that this positive surface charge (zeta potential > 30 mV) inhibits particle coalescence, from Figure 70 It can be seen that the particle size remains constant throughout the study compared to the LNP control.

[0805] Materials and methods for examples 17 and 18

[0806] To demonstrate the stability of the "empty" mixed lipid particles of the present application, the particles of Example 15 were prepared in the absence of active API. This example shows that the addition of mRNA to the particles of the present application does not compromise their stability. Empty Biocourier MVI0012 and LNP particles were mixed with mRNA at different ratios and their size and size monodispersity (PDI) were measured. The results are shown in Figure 71 It can be seen that the particles of the present application maintain their small size upon complexation with mRNA at all tested ratios, allowing for higher mRNA loading without compromising the particles, unlike the case of the LNP particles of the prior art.

[0807] Figure 72

[0808] Silicon nanoparticles were purchased from commercially available electrochemically etched powders (purity >98%; from American Elements, Inc., Los Angeles, CA; or The Porous Silicon Company, Salzburg, Austria). Trehalose and glycine (USP / PhEur specifications) were purchased from Merck. The following lipids were provided by Lipoid GmbH, Ludwigshafen, Germany: 1,2-dioleoyl-3- trimethylammoniumpropane chloride (DOTAP chloride; CAS number: 132172-61-3); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; CAS number: 4004-05-1); and N- (carbonyl-methoxypolyethylene glycol-2000)-1,2-distearyl-sn-glycero-3-phosphoethanolamine sodium salt (mPEG2000 DSPE; CAS number: 147867-65-0). All manipulations were performed using in-house prepared nuclease-free water. Measurements of mean hydrodynamic size, PDI, and zeta potential were performed using a Zetasizer Nano (Malvern Instruments, UK) as reported in previous studies (Baran-Rachwalska et al., 2020; Maurizi et al., 2023).

[0809] Formation of sshLNPs by lipid film hydration Figure 72). In Method 1, the silica nanoparticles were suspended in methanol (20 mg / mL), stirred for 0.5 h and then slowly evaporated to activate them, as previously described (Baran-Rachwalska et al., 2020). Then, they were added to an aqueous solution of trehalose (1 mg / mL) and glycine (0.5 mg / mL) at a concentration of 1 mg / mL and sonicated at 50 °C for 1 h. Separately, a 5 mg / mL lipid solution of DOTAP chloride, DOPE and mPEG2000-DSPE (if required) in MeOH was prepared and sonicated at 40 °C for 0.5 h. Aliquots of these solutions (1.44 mL, 1.46 mL and 0.29 mL, respectively) were transferred to 10 mL round bottom flasks and mixed. The solvent was removed by rotary evaporation at 40 °C for 0.5 h to generate a lipid film, which was subsequently hydrated by adding 1 mL of the aqueous solution described above and incubating at 60 °C for 5 min. Water was added to a final volume of 10 mL (dehydration-rehydration method), and the mixture was then extruded through a series of 5 x 0.4 pm and 5 x 0.1 pm polycarbonate hydrophilic membranes (Whatman Nucleopore) using an Avanti Polar Lipid Extruder in combination with manual injection through a gas-tight Hamilton syringe. The membranes were pre-washed with MeOH and water (20 mL each) before use. In Method 2, the MeOH in the activated silica nanoparticles was not evaporated before further use. Instead, an aliquot of the activated suspension was added directly to the aqueous trehalose / glycine solution to achieve the same final concentrations as described above: 1 mg / mL SiNP, 1 mg / mL trehalose and 0.5 mg / mL glycine. Then, the mixed-lipid particles were formed and extruded as described above.

[0810] Formation of mixed-lipid particles by direct slow injection of lipids Figure 72 ). In Method 3, the SiNP (20 mg) were activated in MeOH (1 mL) as described above and then combined with a solution of trehalose (20 mg) and glycine (10 mg) in water (19 mL). The mixture was sonicated at 50 °C for 1 h. Separately, the lipid stock solutions were prepared and combined as in Method 1. The combined lipid solution was added to the 1 mL aqueous solution described above using a PSNE100 syringe pump (ProSense, Munich, Germany) at a flow rate of 3.2 mL / min. The volume was brought to 10 mL with water and the solution was stirred for 0.5 h before extrusion, which was performed as described above.

[0811] Improved method with an additional 0.8 pm extrusion step Figure 73D). In Method 4, the same procedure as Method 3 was followed, but with the exception that the procedure was scaled up 5x (i.e. final volume was 50 mL) and in the extrusion step, a continuous 3x 0.8 pm polycarbonate hydrophilic membrane (Whatman Nucleopore) was added prior to the 3x 0.4 pm and 3x 0.1 pm filters.

[0812] Other modifications to the small scale protocol Figure 73 E, 78). In Method 5, the order of addition of the aqueous solutions was changed. The pre-prepared aqueous trehalose / glycine solution was added directly to the activated SiNP methanol suspension, rather than the other way around as before (in Method 4). The final concentrations of the SiNPs (1 mg / mL), trehalose (1 mg / mL) and glycine (0.5 mg / mL) in the intermediate aqueous solution were unchanged, and the remaining steps were followed as before. Method 6 was scaled up by a factor of two from Method 4 (i.e. final volume was 100 mL), and two new changes were introduced. First, the lipid stock was prepared at 10 mg / mL rather than 5 mg / mL, and then equal portions of DOTAP chloride (7.20 mL), DOPE (7.30 mL) and mPEG2000-DSPE (1.45 mL) were combined and added to the pre-prepared aqueous solution described above. After adjusting the volume to 100 mL with water, the mixture was pre-filtered through a 0.8 pm hydrophilic polyethersulfone (PES) syringe filter, and then extruded as per Method 4.

[0813] Diafiltration using TFF setup Figure 73 ) SIUS PD 0.02 m 2 (LP) HyS 100 kD membrane cassettes (XP100LP2L; Repligen, Waltham, MA) for volumes up to 100 mL, and SIUS 0.5 m 2 100 kD TFF membrane cassettes (NC1095082; Fisher Scientific, Waltham, MA) for larger volumes. After the indicated initial ultrafiltration step (typically to 0.5x input volume), the sample was diafiltered with 10 volumes of an aqueous solution containing 0.1 mg / mL trehalose and 0.05 mg / mL glycine to maintain the concentrations of these two excipients while removing MeOH and unbound lipids. This was achieved by using a second pump to introduce the trehalose / glycine solution into the flow path at the same flow rate as the main system pump Figure 73 A). Figure 73 C and Figure 74 D show the results of repeating Method 5, followed by TFF with diafiltration only, or followed by TFF with diafiltration after an ultrafiltration step (to half the initial volume).

[0814] Methanol content analysis. This was achieved by measuring the concentrations of 10% D₂O in standard aqueous solutions with known MeOH concentrations: 50, 100, 300, and 500 ppm (v / v). Figure 6 B, illustration) 1 The 1H NMR peak area (δ = 3.34 ppm; Gottlieb et al., 1997) was used to construct a calibration curve. Samples before and after TFF were analyzed in H₂O / D₂O (90:10), and the MeOH content was estimated using the calibration curve. Due to the high MeOH content, samples before TFF were diluted 1:1,000 before obtaining NMR spectra.

[0815] Lipid recovery analysis. Lipid quantification relied on HPLC analysis using a Waters XBridge BEH Phenyl column incorporating an electro-fogging detector (CAD). (5 μm, 4.6 × 150 mm). Buffer A was 40 mM NH4OAc, and buffer B was 100% MeOH. The following elution gradient was used: 25% A for 1 min, gradually increasing to 5% A over 6 min, holding at 5% A for 18 min, gradually increasing to 25% A over 0.1 min, and then holding at 25% A for 4.9 min (flow rate 1 mL / min). For quantitative analysis, calibration curves were constructed for DOTAP chloride, DOPE, and mPEG2000-DSPE (showing retention times of 12.8 min, 15.1 min, and 20.8 min, respectively; see Figure 80). For DOTAP chloride and DOPE, standard solutions were prepared at concentrations of 0.1, 0.2, 0.5, 0.7, and 0.9 mg / mL; for mPEG2000-DSPE, reference solutions were prepared at concentrations of 0.01, 0.05, 0.15, 0.2, and 0.3 mg / mL. Plot the peak area versus concentration to determine the lipid concentration in the test sample. Lipid recovery is calculated as the ratio of the measured concentration to the theoretical concentration assuming all lipids are incorporated into sshLNP, expressed as a percentage.

[0816] The first large-scale trial operation of manufacturing SSHLNP (1L scale) Figure 73). SiNPs (100 mg) were suspended in MeOH (5 mL) in a sterile Falcon tube and incubated at room temperature for 0.5 h for activation. Then, a solution of trehalose (100 mg) and glycine (50 mg) in water (95 mL) was added directly to the SiNP suspension and the mixture was sonicated at 50 °C for 1 h. A stock solution of lipids was prepared by dissolving DOTAP chloride (750 mg), DOPE (750 mg) and mPEG2000-DSPE (200 mg) in MeOH at 10 mg / mL and sonicating the resulting mixture at 40 °C for 0.5 h. Then, equal portions of the DOTAP chloride (72.5 mL), DOPE (73.0 mL) and mPEG2000-DSPE (14.5 mL) solutions were combined (i.e. final concentrations of 0.725, 0.730 and 0.145 mg / mL, respectively). The pre-prepared aqueous solution (100 mL) was transferred to a 1 L sterile bottle and the mixed lipid methanolic solution (160 mL) was injected using a Knauer K501 HPLC pump at a flow rate of 6 mL / min. Upon completion of the addition, the volume was brought to 1 L with water and the flow-extrusion was performed at 60 °C with a flow rate of 85 mL / min using 3 x 0.8 pm, 3 x 0.4 pm and 3 x 0.1 pm polycarbonate hydrophilic membranes (47 mm diameter, Whatman Nucleopore) arranged in series as described above.

[0817] A second large scale pilot manufacturing of mixed lipid particles (2 L scale, Figure 76 ). SiNPs (400 mg) were activated in MeOH (20 mL) as described above. A solution of trehalose (400 mg) and glycine (200 mg) in water (380 mL) was prepared in a sterile bottle and then added directly to the activated SiNP suspension and the resulting dispersion was sonicated at 50 °C for 1 h. A stock solution of 10 mg / mL of DOTAP chloride (3.00 g), DOPE (3.00 g) and mPEG2000-DSPE (600 mg) in MeOH was prepared as described above. The aqueous solution (400 mL) was transferred to a 5 L sterile bottle and the combined lipid mixture (290 mL, 292 mL and 58 mL of the DOTAP chloride, DOPE and mPEG2000-DSPE stock solutions, respectively; total volume 640 mL) was injected into the bottle using a HPLC pump at a flow rate of 6 mL / min. Water was added to a final volume of 4 L and stirring was continued until the mixture was homogeneous. The crude mixture was stored at 4 °C overnight and then flow-extruded as described in previous examples. The TFF setup (as Figure 76 A second large scale pilot manufacturing of mixed lipid particles (2 L scale, 2100 kD membrane bags) were first decontaminated with 0.2 M NaOH for 1 h, then concentrated to 2 L from 4 L by ultrafiltration, and diafiltrated with 10 volumes (i.e., 20 L) of a 0.1 mg / mL trehalose and 0.05 mg / mL glycine aqueous solution. Final and intermediate mixed lipid particle samples were analyzed using the methods described above, with the exception that for lipid content analysis, samples before and after TFF were diluted with MeOH at 1 :2 and 1 :4, respectively, to match the range of the calibration curve.

[0818] Improved method of mixed lipid particles, including pre-filtration of aggregates Figure 77 D). Method 7 was identical to Method 5 (50 mL scale) with the exception of an additional pre-filtration step of the activated silica nanoparticles. Specifically, silica nanoparticles (20 mg) were suspended in MeOH (1 mL) and stirred at room temperature for 0.5 h for activation, then the mixture was manually filtered through a 0.8 pm hydrophilic PES syringe filter. A previously prepared solution of trehalose (20 mg) and glycine (10 mg) in water (19 mL) was added directly to the silica nanoparticle suspension, and the dispersion was sonicated at 50 °C for 1 h. Separately, stock solutions of 10 mg / mL of DOTAP chloride, DOPE, and mPEG2000-DSPE in MeOH were prepared and sonicated at 40 °C for 0.5 h. Aliquots (3.63 mL, 3.65 mL, and 0.73 mL, respectively) of these solutions were combined, and the mixed lipid solution was injected into a 5 mL aliquot of the aqueous solution using a syringe pump at a flow rate of 3.2 mL / min. The volume was brought to 50 mL with water, and stirred at room temperature for 0.5 h before extrusion, which was performed as described in Method 4.

[0819] Demonstration of large batch with aggregate filtration (1 L scale, Example 17 - Removal of evaporation step E).

[0820] Silicon nanoparticles (100 mg) were stirred in MeOH (5 mL) at room temperature for 0.5 h to activate. To remove large aggregates, the suspension was manually filtered through three consecutive 0.8 pm hydrophilic PES syringe filters. The resulting sample was combined with a previously prepared solution of trehalose (100 mg) and glycine (50 mg) in water (95 mL) and stirred vigorously (500 rpm) at 50 °C for 1 h. Separately, stock solutions of DOTAP chloride (750 mg), DOPE (750 mg) and mPEG2000-DSPE (160 mg) in MeOH were prepared and sonicated at 40 °C for 0.5 h. Aliquots (72.5 mL, 73.0 mL and 14.5 mL, respectively) of these solutions were combined and the mixed lipid solution was injected into the aqueous solution (in a 1 L polycarbonate vessel) using a Knauer BlueShadow80P HPLC pump at a flow rate of 6 mL / min. The volume was brought to 1 L with water (using the same pump at a flow rate of 100 mL / min and stirring) and stirring was continued at room temperature for 0.5 h to homogenize. Flow-fractionation was performed at 60 °C using two sets of parallel arranged extrusion membranes (three pores of 0.8 pm, 0.4 pm and 0.1 pm, respectively, for each set) at a flow rate of 85 mL / min. After TFF setup was de-sterilized with 0.2 M NaOH for 1 h and rinsed with water, methanol was removed by diafiltration as described above.

[0821] Evaluation of RNA encapsulation efficiency Figure 71 C) As previously reported (Maurizi et al., 2023), the Quant-iT RiboGreen assay kit (Fisher Scientific, UK) was used according to the manufacturer’s instructions. For analysis, a Varioskan LUX microplate reader (Fisher Scientific) was used (excitation wavelength 480 nm, emission wavelength 520 nm).

[0822] Figure 71

[0823] A conventional method of manufacturing lipid nanoparticles can include at least 2 solvent evaporation steps. First, the inorganic particles can be activated with a solvent, which can be evaporated in a first evaporation step. Second, the inorganic particles and lipids can be mixed in a solvent, which is evaporated to form a lipid film. It was previously believed that the first evaporation step was necessary for sufficient activation of the inorganic particles. It was previously believed that the second evaporation step resulted in the formation of a lipid film, and subsequent hydration of the lipid film was necessary for proper hydration and formation of the lipid nanoparticles. This example shows that one or both of the evaporation steps can be omitted.

[0824] As mentioned above, small scale manufacturing of lipid nanoparticles can be performed using a workflow based on lipid film hydration followed by flow extrusion. Such a method is schematically illustrated in Figure 71 A, labelled "Method 1". Two successive flow extrusion steps through membranes of decreasing pore size (0.4 pm and 0.1 pm) were performed to reduce the operating pressure on the membranes and to ensure that the final particles had the desired size. Trehalose (1 mg / mL) and glycine (0.5 mg / mL) were also included in the aqueous phase as excipients, as it has been found that they further stabilise the hybrid lipid particles, presumably by complexation with the silicon nanoparticles (SiNPs).

[0825] The presence of trehalose and glycine facilitates the dispersibility of the silicon particles in the mixed hydroalcoholic environment if the proportions are appropriate. Amino acid excipients are also included to modulate the rate of hydrolysis of the silicon, promoting the formation of orthosilicic acid rather than insoluble polymeric silicon species (see WO2011012867A1 for more details).

[0826] To develop the method for large scale applications, the two solvent evaporation steps need to be eliminated; i.e. the removal of MeOH from the activated silicon nanoparticle suspension, and from the dissolved lipids used to generate the film. The first of these steps is removed by mixing the activated silicon nanoparticle suspension directly into the aqueous trehalose / glycine solution prior to hydration of the lipid film. Such a method is schematically illustrated in Figure 71 A, labelled "Method 2". This method produces an initial hybrid lipid nanoparticle suspension with significantly more precipitate than previously (see Figure 72 B, lower graph; upper graph shows Method 1), but produces particles with a lower polydispersity index (PDI) and slightly higher zeta potential after extrusion, eliminating this difference (see ​ C). Preferably, the zeta potential of the finished hybrid lipid nanoparticle should be +50 to +70 mV, which indicates good to excellent colloidal stability, and minimal tendency to aggregate (due to charge repulsion). It can be seen that the removal of the first evaporation step does not preclude the production of hybrid lipid particles with acceptable zeta potential and polydispersity.

[0827] To demonstrate that the lipid film evaporation and subsequent hydration steps can be removed without causing harm, an attempt was made to inject the lipid methanolic solution directly and slowly into the aqueous silicon nanoparticle suspension (schematically illustrated in ​ A, labelled "Method 3"). Less precipitate was observed using this method ( Figure 72 B), and the average size (102 ± 0.17 nm), PDI (0.135 ± 0.01) and zeta potential (56.0 ± 0.81 mV) of the resulting hybrid lipid particle sample were comparable to previous batches ( Figure 72B). This proved the feasibility of scaling up the direct injection mixing technique to larger scale. To ensure reproducibility, we introduced two in-process checks (IPCs) in the process Figure 72 A): visual check of complete lipid dissolution before mixing (IPC 1), and a reference range of specified dynamic light scattering (DLS) parameters after extrusion: average hydrodynamic size 75-140 nm, PDI 0.1-0.2, and zeta potential +50 to +70 mV (IPC 2). If needed, the 0.1 pm extrusion step was repeated after IPC 2.

[0828] Additional process adjustments were also investigated before scaling up Figure 72 C, labeled “Methods 4 to 6”). Most notably, an additional flow extrusion step (pore size 0.8 pm) was tested to remove larger silica nanoparticle aggregates and successfully reduced the average size of the final mixed-lipid particles Figure 72 D, “Method 4”). Adding the trehalose / glycine solution to the methanolic suspension of activated silica nanoparticles, instead of the other way around Figure 72 C “Method 5”, Figure 78 ), had no impact on the results. Including a 0.8 pm pre-filtration step after the initial mixed-lipid particle formation reduced the accumulation of aggregates on the extrusion membrane Figure 72 E, “Method 6”), significantly reduced the operating pressure of the 0.8 pm membrane Figure 79 ), and essentially obtained sshLNPs with unchanged properties Figure 72 F).

[0829] For commercial manufacturing of pharmaceuticals, it is preferable that the organic solvent (i.e., MeOH) must be removed from the final product to have residual levels below regulatory limits (e.g., <3,000 ppm in European pharmaceuticals; European Medicines Agency, 2022). For this purpose, tangential flow filtration (TFF) can be used, as Figure 73 A is schematically shown. The preliminary feasibility study (up to 100 mL scale) used a TFF membrane pack with a MWCO of 100 kDa and a membrane surface area of 0.02 m 2 Since trehalose and glycine were also removed from the solution under these conditions, a second pump was used to inject a 0.1 mg / mL trehalose and 0.05 mg / mL glycine aqueous solution for diafiltration at a constant flow rate matching the filtrate generation. Diafiltration was performed with 10 volumes of this solution, reducing the MeOH content from ~20% v / v after extrusion to within acceptable regulatory limits (~200 ppm, based on 1H NMR) while maintaining the expected levels of trehalose / glycine. The final process included an initial ultrafiltration step to reduce the input volume by 50% before performing diafiltration to produce a product with the required silicon concentration (see below) and low residual MeOH levels (~450 ppm; Figure 73 B) of the finished mixed-lipid nanoparticle suspension.

[0830] After TFF, the DLS characteristics of the extruded sshLNPs were essentially maintained. Lipid recovery was determined using the HPLC method (Figure 80). The results show that with diafiltration alone, there was a significant drop in lipid content, with a recovery of around 10% for each lipid. However, this impact was greatly mitigated by employing the improved TFF step combining ultrafiltration and diafiltration. In addition, the silicon content of the final mixed-lipid nanoparticle samples was also determined using inductively coupled plasma optical emission spectrometry (ICP-OES). For the batch where TFF with diafiltration alone was performed, the silicon content was 1.19 mg / L, compared to the target value of 2.1 mg / L (2.0 mg / L for the product based on the original lipid film hydration method. Figure 71 A). The above analytical methods were used to guide further optimisation work to advance towards scale-up production. They can also be used for periodic quality control in a commercial manufacturing process. As an alternative to the H NMR method for determining residual MeOH content, headspace GC analysis can be used (Maurizi et al., 2023). 1 H NMR method for determining residual MeOH content, headspace GC analysis can be used (Maurizi et al., 2023).

[0831] Example 18 - Large scale pilot run

[0832] The first large-scale pilot run (schematically shown in Figure 74 A) employed a protocol comprising three flow extrusion steps (i.e. Method 4 of Example 17 was scaled up using the modifications introduced in Method 5 of Example 17), but TFF was omitted at this stage. The lipid stock was now formulated at 10 mg / mL instead of 5 mg / mL as before, to limit the volume of MeOH in the system. At the 1 L scale, the main issue encountered was an increase in the accumulation of insoluble aggregates on the extrusion membrane, even with a larger filter membrane (47 mm diameter, previously 25 mm) Figure 74 B). This resulted in higher operating pressures during extrusion Figure 74 C), although the DLS characteristics of the samples were not adversely affected Figure 74 D). A two-stage TFF protocol Figure 75 A) was demonstrated for the manufacture of a 2 L batch, but in this case a membrane pack with a larger filter membrane surface area (0.5 m 2 ) was used. As before, the main issue encountered was saturation of the filter membrane with insoluble aggregates during extrusion Figure 75B), and the filtration membrane had to be changed mid-process due to pressure fluctuations. The sample after extrusion exhibited suboptimal visual and DLS characteristics Figure 75 C), but closer process monitoring revealed that the average particle size gradually increased with extrusion volume Figure 75 D), and only after processing ~1.5 L out of 4 L total volume. After TFF, residual MeOH was below the limit of detection. When an aliquot of the sample before 100 mL TFF was subjected to an additional extrusion step through a 3x 0.1 pm filtration membrane, the average particle size decreased from 129 ± 0.44 nm to 105 ± 0.38 nm, and the PDI decreased from 0.17 ± 0.02 to 0.11 ± 0.01 (both within the reference range), confirming that extrusion membrane overload was an issue during large batch processing, and suggesting that such issues could be easily resolved by using additional extrusion.

[0833] Given these results, we modified the procedure to reduce sedimentation during sshLNP formation. In a small-scale pilot run omitting TFF, when the activated SiNPs suspension was filtered before mixing with the trehalose / glycine solution Figure 7 A, method 7), aggregates were successfully removed, and no significant insoluble material deposition was observed on the extrusion membrane Figure 7 B). The operating pressure during extrusion was lower Figure 7 C), and the DLS characteristics of the resulting sshLNP were within the reference range Figure 7 D). To implement this method at a large scale, two larger 47 mm extrusion membranes were used, arranged in parallel Figure 76 A and Figure 81 ), and a 1 L batch of sshLNP was successfully manufactured by the optimized protocol Figure 76 E). In the current implementation, an additional 0.2 pm filtration step was also used at the end to reduce bioburden.

[0834] After developing a kilogram-scale manufacturing protocol, it was also crucial to establish equivalency between sshLNP manufactured by the original lipid film hydration technique (method 2) and the optimized large-scale process (method 7). We found that scale-up resulted in slightly smaller particles on average Figure 77 A), but essentially identical PDI and zeta potential Figure 77 A and 77B). Both batches exhibited excellent mRNA encapsulation efficiency as determined by RiboGreen detection Figure 77C). In cell transfection experiments, sshLNPs made by either route were effective at introducing mRNA encoding firefly luciferase (fLuc) into HEK293 cells, and significantly higher transgene expression was observed after 24 hours compared to Lipofectamine 3000, which served as a reference Figure 77 D). Thus, sshLNPs made by the large-scale process developed herein are identical in physical properties and function to sshLNPs made by the original dehydration-rehydration process.

[0835] Discussion, especially Examples 17 and 18

[0836] Realizing the full potential of lipid nanoparticles for clinical applications requires a reliable manufacturing process that can make particles with relevant properties at kilogram scale. Examples 17 and 18 demonstrate such a process.

[0837] The convergent manufacturing process makes a platform intermediate (i.e., “empty”) hybrid silicon nanoparticle that is amenable to subsequent nucleic acid loading prior to filling / finalization operations, which can be separated in time and distance from the original manufacturing. This capability is a consequence of the presence of silicon nanoparticles in some of the above-described silicon nanoparticles that remain surface-accessible and stabilize the lipid membrane. Their presence mitigates the well-known tendency of LNPs to aggregate or fuse over time (i.e., Ostwald ripening), which is a limiting factor for current formulation shelf life (Gindy et al., 2014; Nag et al., 2022). The addition of SiNPs also allows for optional omission of the cholesterol component of conventional LNPs, which accounts for ~40 mol% of the lipid content in commercial RNA-LNP products (Sun et al., 2023). In the hybrid lipid particles of the present invention, the interaction of the phospholipids with the silicon enhances the structural integrity, reducing the risk of breakage during the extrusion process. Interestingly, it also appears to result in a final particle with an incompletely closed lipid bilayer, with the interior remaining accessible to nucleic acid loading.

[0838] Another advantage of the silicon component is that it allows greater formulation flexibility, which is significant in that the lipid composition can be easily modified without major adjustments to the manufacturing process. For example, mixed lipid particles can be formulated without the use of PEGylated lipids, which are a necessary component of conventional LNPs but are sometimes associated with safety and efficacy concerns. It is well known that PEGylation can induce anti-PEG antibodies, potentially stimulating premature release of RNA payloads through antibody binding (Shi et al., 2022; Senti et al., 2022). Therefore, for individuals with a history of PEG hypersensitivity, non-PEGylated mixed lipid particles can be a clinically useful alternative (Chen et al., 2023; Ibrahim et al., 2022).

[0839] Furthermore, the compatibility of the mixed lipid particles of the present invention with point-of-care RNA loading minimizes the degradation of nucleic acid drugs while offering opportunities for late-stage customization and personalization of therapeutic formulations. In fact, this “post-hoc loading” concept has recently been highlighted as a potential strategy to overcome the remaining challenges in the field of RNA drugs (Li et al., 2023). Low delivery efficiency, short shelf life, and high market access barriers (i.e., the cost of developing and manufacturing optimized LNP formulations) are particularly considered as limiting factors currently hindering the development of the field (Verma et al., 2023), and sshLNP is expected to address all of these challenges.

[0840] Since 2020, there has been a priority to address the cold chain requirements and limited shelf life issues faced by COVID-19 vaccines. Recent noteworthy innovations include improved lyophilized mRNA-LNP formulations that maintain stability and transfection efficiency for weeks at 4°C (or even at room temperature) (Ai et al., 2023; Meulewaeter et al., 2023; Shirane et al., 2023). However, the commercial scalability of these workflows remains to be established, and they also do not address the problem of having to incorporate the RNA component early in the manufacturing process. Therefore, extensive optimization of LNP formulations is still required on a case-by-case basis. For example, the identification of the optimal formulation for patisiran (the first FDA-approved RNA therapy delivered by LNPs) required the screening of more than 300 individual ionizable lipids (Kulkarni et al., 2019). In contrast, our studies with ADO2 mice only required the preliminary screening of 7 sshLNP formulations to identify promising lead candidates for the delivery of siRNAs to the skeleton (Maurizi et al., 2022). Therefore, the Bio-Courier platform is expected to accelerate the clinical translation of RNA therapies.

[0841] Based on this, the stabilizing effect of the mixed lipid particles of the present application is also strong enough to make it unnecessary to chemically modify the RNA, as confirmed by the aforementioned in vivo studies using unmodified siRNA (Baran-Rachwalska et al., 2020; Maurizi et al., 2023). In contrast, all currently FDA-approved oligonucleotide therapies contain chemical modifications (Bost et al., 2021), including the five siRNA-based products (Friedrich & Aigner, 2022). By reducing the substantial time and cost of developing constructs that often require a series of complex chemical modifications, the use of unmodified (or minimally modified) RNA with sshLNPs is another possible factor that could facilitate an increase in translation.

[0842] In summary, sshLNPs show great promise as a platform technology for improving RNA delivery, but the manufacturing methods reported previously are not easily scalable.

[0843] Bibliography

[0844] Mikhail (1961), "Densities and Viscosities of Methanol-Water Mixtures," (Journal of Chemical & Engineering Data, vol. 6, no. 4, p. 533-537, 1961.

[0845] Wang, C., Zhang, Y., and Dong, Y. (2021). Lipid nanoparticle-mRNA formulations for therapeutic applications. Acc. Chem. Res. 54, 4283-4293.

[0846] Curreri, A., Sankholkar, D., Mitragotri, S., and Zhao, Z. (2023). RNA therapeutics in the clinic. Bioeng. Transl. Med. 8, e10374.

[0847] Moss, K. H., Popova, P., Hadrup S. R., Astakhova, K., and Taskova, M. (2019). Lipid nanoparticles for delivery of therapeutic RNA oligonucleotides. Mol. Pharm. 16, 2265-2277.

[0848] Verma, M., Ozer, I., Xie, W., Gallagher, R., Teixeira, A., and Choy, M. (2023). The landscape for lipid-nanoparticle-based genomic medicines. Nat. Rev. Drug Discov. 22, 349-350.

[0849] Schoenmaker, L., Witzigmann, D., Kulkarni, J. A., Verbeke, R., Kersten, G., Jiskoot, W., and Crommelin, D. J. A. (2021). mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 601, 120586.

[0850] Bege, M., and Borbás, A. (2022). The medicinal chemistry of artificial nucleic acids and therapeutic oligonucleotides. Pharmaceuticals (Basel) 15, 909.

[0851] Egli, M., and Manoharan, M. (2019). Re-engineering RNA molecules into therapeutic agents. Acc. Chem. Res. 52, 1036-1047.

[0852] Nag, K., Sarker, M. E. H., Kumar, S., Khan, H., Chakraborty, S., Islam, M. J., Baray, J. C., Khan, M. K., Mahmud, A., Barman, U., et al. (2022). DoE-derived continuous and robust process for manufacturing of pharmaceutical-grade wide-range LNPs for RNA-vaccine / drug delivery. Sci. Rep. 12, 9394.

[0853] Cameau, E., Zhang, P., Ip, S., Mathiasson, L., and Stenklo, K. (2022). Process & analytical insights for GMP manufacturing of mRNA lipid nanoparticles. Cell Gene Ther. Insights 8, 621-635.

[0854] Catignol, P., and Lim, H. (2022). Overcoming bottlenecks in mRNA manufacturing (Pharma’s Almanac). https: / / www.pharmasalmanac.com / articles / overcoming-bottlenecks-in-mrna-manufacturing.

[0855] Daniel, S., Kis, Z., Kontoravdi, C., and Shah, N. (2022). Quality by Design for enabling RNA platform production processes. Trends Biotechnol. 40, 1213-1228.

[0856] Andoh, E. A., and Yu, H. (2023). A two-stage decision-support approach for improving sustainable last-mile cold chain logistics operations of COVID-19 vaccines. Ann. Oper. Res. 328, 75-105.

[0857] Khairi, L. N. H. M., Fahrni, M. L., and Lazzarino, A. I. (2022). The race for global equitable access to COVID-19 vaccines. Vaccines (Basel) 10, 1306.

[0858] Saffie-Siebert, S., Gibson, A., and Dehsorkhi, A. (2023). Bio-Courier: A new delivery technology to increase the global accessibility of RNA therapeutics (Manufacturing Chemist). https: / / www.manufacturingchemist.com / news / article_page / Bio-Courier_a_new_delivery_technology_to_increase_the_global_accessibility_of_RNA_therapeutics / 212116.

[0859] Baran-Rachwalska, P., Torabi-Pour, N., Sutera, F. M., Ahmed, M., Thomas, K., Nesbit, M. A., Welsh, M., Moore, C. B. T., and Saffie-Siebert, S. R. (2020). Topical siRNA delivery to the cornea and anterior eye by hybrid silicon-lipid nanoparticles. J. Control. Release 326, 192-202.

[0860] Maurizi, A., Patrizii, P., Teti, A., Sutera, F. M., Baran-Rachwalska, P., Burns, C., Nandi, U., Welsh, M., Torabi-Pour, N., et al. (2023). Novel hybrid silicon-lipid nanoparticles deliver a siRNA to cure autosomal dominant osteopetrosis in mice. Implications for gene therapy in humans. Mol. Ther. Nucleic Acids 33, 925-937.

[0861] Saffie-Siebert, R. S., Ahmed, M., and Sutera, F. (2023). Compositions comprising doped silicon particles, and related methods (WIPO Patent No. WO 2023 / 002222 A1).

[0862] Saffie-Siebert, R. S., and Sutera, F. (2023). Protection of biological species from degradation (WIPO Patent No. WO 2023 / 002224 A1).

[0863] Hou, X., Zaks, T., Langer, R., and Dong, Y. (2021). Lipid nanoparticles for RNA delivery. Nat. Rev. Mater. 6, 1078-1094.

[0864] Kulkarni, J. A., Witzigmann, D., Chen, S., Cullis, P. R., and van der Meel, R. (2019). Lipid nanoparticle technology for clinical translation of siRNA therapeutics. Acc. Chem. Res. 9, 2435-2444.

[0865] Maurizi, A., Patrizii, P., Teti, A., Sutera, F. M., Hadainamrei, R., Dehsorkhi, A., and Saffie-Siebert, S. (2022). Development of a potential clinical product for treating CLCN7-dependent autosomal dominant osteopetrosis type 2 (ADO2). Bone Rep. 16, 101199.

[0866] Li, D., Liu, C., Li, Y., Tenchov, R., Sasso, J. M., Zhang, D., Li, D., Zou, L., Wang, X., and Zhou, Q. (2023). Messenger RNA-based therapeutics and vaccines: What’s beyond COVID-19? ACS Pharmacol. Transl. Sci. 6, 943-969.

[0867] Ai, L., Li, Y., Zhou, L., Yao, W., Zhang, H., Hu, Z., Han, J., Wang, W., Wu, J., Xu, P., et al. (2023). Lyophilized mRNA-lipid nanoparticle vaccines with long-term stability and high antigenicity against SARS-CoV-2. Cell Discov. 9, 9.

[0868] Meulewaeter, S., Nuytten, G., Cheng, M. H. Y., De Smedt, S. C., Cullis, P. R., De Beer, T., Lentacker, I., and Verbeke, R. (2023). Continuous freeze-drying of messenger RNA lipid nanoparticles enables storage at higher temperatures. J. Control. Release 357, 149-160.

[0869] Shirane, D., Tanaka, H., Sakurai, Y., Taneichi, S., Nakai, Y., Tange, K., Ishii, I., and Akita, H. (2023). Development of an alcohol dilution-lyophilization method for the preparation of mRNA-LNPs with improved storage stability. Pharmaceutics 15, 1819.

[0870] Gindy, M. E., Feuston, B., Glass, A., Arrington, L., Haas, R. M., Schariter, J., and Stirdivant, S. M. (2014). Stabilization of Ostwald ripening in low molecular weight amino lipid nanoparticles for systemic delivery of siRNA therapeutics. Mol. Pharm. 11, 4143-4153.

[0871] Sun, H., Zhang, Y., Wang, G., Yang, W., and Xu, Y. (2023). mRNA-based therapeutics in cancer treatment. Pharmaceutics 15, 622.

[0872] Bost, J. P., Barriga, H., Holme, M. N., Gallud, A., Maugeri, M., Gupta, D., Lehto, T., Valadi, H., E. K., et al. (2021). Delivery of oligonucleotide therapeutics: Chemical modifications, lipid nanoparticles, and extracellular vesicles. ACS Nano 15, 13993-14021.

[0873] Friedrich, M., and Aigner, A. (2022). Therapeutic siRNA: State-of-the-art and future perspectives. BioDrugs 36, 549-571.

[0874] Gottlieb, H.E., Kotlyar, V., and Nudelman, A. (1997). NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 62, 7512-7515.

[0875] Shi, D., Beasock, D., Fessler, A., Szebeni, J., Ljubimova, J.Y., Afonin, K.A., and Dobrovolskaia, M.A. (2022). To PEGylate or not to PEGylate: Immunological properties of nanomedicine’s most popular component, polyethylene glycol and its alternatives. Adv. Drug. Deliv. Rev. 180, 114079.

[0876] Senti, M.E., de Jongh, C.A., Dijkxhoorn, K., Verhoef, J.J.F., Szebeni, J., Storm, G., Hack, C.E., Schiffelers, R.M., Fens, M.H., and Boross, P. (2022). Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement. J. Control. Release 341, 475-486.

[0877] Chen, W.-A., Chang, D.-Y., Chen, B.-M., Lin, Y.-C., Barenholz, Y., and Roffler, S. R. (2023). Antibodies against poly(ethylene glycol) activate innate immune cells and induce hypersensitivity reactions to PEGylated nanomedicines. ACS Nano 17, 5757-5772.

[0878] Ibrahim, M., Ramadan, E., Elsadek, M. E., Emam, S. E., Shimizu, T., Ando, H., Ishima, Y., Elgarhy, O. H., Sarhan, H. A., Hussein, A. K., et al. (2022). Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products. J. Control. Release 351, 215-230.

Claims

1. A method of manufacturing an aqueous suspension of mixed lipid particles comprising the steps of: A. mixing a suspension of one or more lipids in a solvent or solvent mixture and inorganic material particles in a solvent or solvent mixture into an aqueous medium; and then B. passing the mixture obtained in step A through the pores of an extrusion membrane, wherein the average diameter of the mixed lipid particles is at least two times larger than the average diameter of the inorganic material particles.

2. The method according to claim 1, wherein the inorganic material particles comprise or consist of particles comprising or consisting of hydrolysable silicon.

3. The method of claim 1 or 2, wherein, Step B is carried out at 50 to 70 °C, optionally step A is carried out at 50 to 70 °C.

4. The method according to claim 1 or 2 or 3, wherein the inorganic material particles are configured to impart tensile strength to one or more lipid membranes in the mixed lipid particles.

5. The method according to any one of claims 1 to 4, wherein the mixture is passed through the pores of the extrusion membrane at least three times, the membrane having a pore size cut-off diameter of 0.05 to 1 pm, optionally, the size, PDI and charge of the particles do not substantially change.

6. The method according to any one of claims 1 to 5, wherein after step B there is an additional step C of purifying and / or sterilizing the suspension by tangential flow filtration, wherein step C optionally removes activating solvent and / or solvent of step A, and wherein the tangential flow filtration is optionally diafiltration using a diafiltration solution containing amino acids, such as glycine, and disaccharides, such as trehalose.

7. The method according to any one of claims 1 to 6, wherein after step B (and step C, if present) there is an additional step D of contacting the micellar lipid particles with an active compound, in particular a pharmaceutically active ingredient (API).

8. The method according to claim 7, wherein step D is carried out at a temperature below 20 °C (e.g., below 10 °C, 5 °C or 3 °C), and the API is an RNA molecule, in particular an mRNA.

9. The method according to any one of claims 1 to 8, further comprising freeze-drying the suspension after all required steps or between steps C and D to manufacture a lyophilized powder comprising the micellar lipid particles.

10. The method according to any one of claims 1 to 9, wherein the one or more lipids comprise at least one cationic lipid and at least one polar lipid.

11. The method according to claim 10, wherein the one or more lipids consist of at least DOTAP, DOPE and mPEG2000-DSPE, optionally in a molecular ratio of 2-7:2-7:1-2.

12. The method according to any one of claims 1 to 11, wherein the inorganic material particles are particles of hydrolysable silicon that have been activated by exposure to an alcohol, such as methanol, ethanol or benzyl alcohol.

13. The method according to any one of claims 1 to 12, which does not comprise a solvent evaporation step.

14. The method according to any one of claims 1 to 13, comprising one or more quality control steps selected from measuring a dynamic light scattering (DLS) parameter, measuring an average hydrodynamic size parameter, measuring a polydispersity (PDI) and / or measuring a zeta potential, and optionally comparing the measurement to product specifications, and optionally repeating the extrusion step B if the measurement does not comply with the product specifications.

15. An aqueous suspension of mixed lipid particles having an average diameter of 50 to 150 nm, the mixed lipid particles comprising a mixture of one or more cationic or ionizable lipids and one or more other lipids selected from neutral and polar lipids, optionally comprising one or more additional lipid components, the mixed lipid particles comprising inorganic material particles (e.g. hydrolysable silica particles) having an average diameter of at least half of the average diameter of the mixed lipid particles, wherein the weight ratio of inorganic material particles to lipids is 1 :2 to 1 : 100 (preferably 1 : 10 to 1 : 100, more preferably 1 :20 to 1 : 10).

16. The aqueous suspension of mixed lipid particles according to claim 15, having an average diameter of 80 to 200 nm, wherein the inorganic material particles are hydrolysable silica particles having an average diameter of 2 to 20 nm and are present in one or more aggregation chains of the hydrolysable lipid particles, the aggregation chains extending from the outside to the inside of the mixed lipid particles.

17. The aqueous suspension of mixed lipid particles according to claim 15 or 16, further comprising one or more active compounds, such as one or more active pharmaceutical ingredients (APIs).

18. An aqueous suspension of liposomal lipid particles having an average diameter of 50 to 150 nm, the liposomal lipid particles comprising a mixture of one or more cationic or ionizable lipids and one or more other lipids selected from neutral and polar lipids, optionally comprising one or more additional lipid components, the liposomal lipid particles comprising inorganic material particles (e.g. hydrolysable silica particles) having an average diameter of at least half of the average diameter of the liposomal lipid particles, wherein the weight ratio of inorganic material particles to lipids is 1 :2 to 1 : 100 (preferably 1 : 10 to 1 : 100, more preferably 1 :20 to 1 : 10), and wherein the liposomal lipid particles further comprise one or more active compounds, such as one or more active pharmaceutical ingredients (APIs), at least a portion of which is encapsulated inside the liposomal particles.

19. A method of manufacturing a liposomal lipid particle comprising an active compound (e.g. API), comprising performing the method according to any one of claims 1 to 14, and then contacting the active compound with the mixed lipid particle in an aqueous suspension under conditions that cause the mixed lipid particle to transform into a liposomal lipid particle.

20. A lyophilized powder of mixed or liposomal lipid particles having an average diameter of 50 to 150 nm, the mixed or liposomal lipid particles comprising a mixture of one or more cationic or ionizable lipids and one or more further lipids selected from neutral and polar lipids, optionally comprising one or more additional lipid components, the mixed or liposomal lipid particles comprising particles of inorganic material (e.g. hydrolysable silica particles) having an average diameter of at least half the average diameter of the liposomal lipid particles, wherein the weight ratio of inorganic material particles to lipids is 1 :2 to 1 : 100 (preferably 1 : 10 to 1 : 100, more preferably 1 :20 to 1 : 10), and wherein the mixed or liposomal lipid particles further comprise one or more active compounds, e.g. one or more active pharmaceutical ingredients (APIs), at least a portion of which is encapsulated inside the mixed or liposomal particles.

21. A lyophilized powder of mixed lipid particles having an average diameter of 50 to 150 nm, the mixed lipid particles comprising a mixture of one or more cationic or ionizable lipids and one or more further lipids selected from neutral and polar lipids, optionally comprising one or more additional lipid components, the mixed lipid particles comprising particles of inorganic material (e.g. hydrolysable silica particles) having an average diameter of at least half the average diameter of the mixed lipid particles, wherein the weight ratio of inorganic material particles to lipids is 1 :2 to 1 : 100 (preferably 1 : 10 to 1 : 100, more preferably 1 :20 to 1 : 10).

22. An aqueous suspension of mixed lipid particles according to claim 15, 16 or 17, or a lyophilized powder according to claim 20, 21 or 22, wherein the active compound (e.g. API) is a nucleic acid, e.g. mRNA.

23. A pharmaceutical composition comprising an aqueous suspension of mixed lipid particles according to claim 15, 16 or 17, an aqueous suspension of liposomal lipid particles according to claim 18, or a lyophilized powder according to claim 20 or 21.

24. A pharmaceutical composition according to claim 23, which is a vaccine composition, optionally loaded in a vaccine delivery device, e.g. a syringe.

25. Use of a pharmaceutical composition according to claim 23 or 24 as a medicament.

26. Use of a pharmaceutical composition according to claim 25 as a medicament for treating or preventing a disease or condition in a subject.

27. Use of a pharmaceutical composition according to claim 25 as a vaccine against an infectious disease.

28. A method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to any one of claims 23 to 27.

29. A method of providing prophylactic vaccination to a subject, comprising administering to the subject a pharmaceutical composition according to any one of claims 23 to 27.

30. A method of manufacturing an aqueous suspension of lipid particles comprising a pharmaceutically active ingredient (API), the method comprising: (a) obtaining an aqueous suspension of mixed lipid particles manufactured by: (i) mixing one or more lipids in a solvent or solvent mixture and a suspension of inorganic material particles in a solvent or solvent mixture into an aqueous medium; (ii) passing the mixture obtained in step (i) through the pores of an extruded membrane; and (iii) optionally, purifying and / or sterilizing the suspension by tangential flow filtration; and (b) contacting the mixed lipid particles with an active compound, in particular a pharmaceutically active ingredient (API).

31. A method of manufacturing an aqueous suspension of mixed lipid particles comprising a pharmaceutically active ingredient (API), the method comprising: (a) obtaining an aqueous suspension of mixed lipid particles manufactured by: (i) mixing one or more lipids in a solvent or solvent mixture and a suspension of inorganic material particles in a solvent or solvent mixture into an aqueous medium; (ii) passing the mixture obtained in step (i) through the pores of an extruded membrane; and (b) purifying and / or sterilizing the suspension by tangential flow filtration.

32. An aqueous suspension of lipid particles comprising a pharmaceutically active ingredient (API) prepared by a method comprising: (a) obtaining an aqueous suspension of mixed lipid particles manufactured by: (i) mixing one or more lipids in a solvent or solvent mixture and a suspension of inorganic material particles in a solvent or solvent mixture into an aqueous medium; (ii) passing the mixture obtained in step (i) through the pores of an extruded membrane; and (iii) optionally, purifying and / or sterilizing the suspension by tangential flow filtration, and (b) contacting the mixed lipid particles with an active compound, in particular a pharmaceutically active ingredient (API).

33. An aqueous suspension of mixed lipid particles comprising a pharmaceutically active ingredient (API) prepared by a method comprising: (a) obtaining an aqueous suspension of mixed lipid particles manufactured by: (i) mixing one or more lipids in a solvent or solvent mixture and a suspension of inorganic material particles in a solvent or solvent mixture into an aqueous medium; (ii) passing the mixture obtained in step (i) through the pores of an extruded membrane; and (b) purifying and / or sterilizing the suspension by tangential flow filtration. ​

Images