continuous process

Abstract

A continuous process for preparing microparticles, such as nanoparticles or microparticles, is described, the process comprising: (i) feeding a first liquid phase to a first membrane, the first membrane defining a first plurality of openings; (ii) controlling the feeding of a second liquid phase to the first membrane through the first plurality of openings to form a mixture; controlling the feeding of the mixture to a second membrane to form a stabilized particle suspension; concentrating the particle suspension; controlling the feeding of a buffer to a third membrane; and controlling the feeding of the stabilized mixture to a fourth membrane for microfiltration to isolate the microparticles.

Description

[Technical Field]

[0001] The present invention relates to a continuous process for producing microparticles or nanoparticles.

[0002] More particularly, the present invention relates to a continuous process for producing microparticles or nanoparticles, in particular lipid nanoparticles (LNPs). [Background technology]

[0003] Microparticles and nanoparticles have important applications in biomedicine, pharmacology, medicine, cosmetics, the chemical industry, agriculture, veterinary medicine, and the like.

[0004] Many techniques are available for the production of microparticles and nanoparticles, including precipitation or co-precipitation of sparingly soluble products from aqueous or non-aqueous solutions, sol-gel processing, and the use of microemulsions.

[0005] WO 2022 / 018441 (Patent Document 1) describes a method for preparing lipid vesicles, liposomes, or lipid nanoparticles (LNPs) using membrane emulsification technology.

[0006] Membrane emulsification techniques are generally advantageous for preparing lipid vesicles and micro- and nanoparticles.

[0007] However, most commercially available techniques for preparing microparticles and nanoparticles utilize batch processes, which are processes in which material does not enter or leave the system during operation. Typically, material remains in the system, material accumulates, and reaction vessels must be cleaned between each batch.

[0008] Examples of batch process applications include beverage processing, dairy, pharmaceutical and soap manufacturing.

[0009] The use of batch processes tends to limit production scale and is labor intensive. Additionally, plants are typically down for batch changes and cleaning between batches. Product quality is also difficult to maintain because of possible batch-to-batch variations.

[0010] In the case of fed-batch processes, which are often used in biotechnology, the residence time of particles in the process can also vary greatly, which can lead to differences in quality. For example, tangential flow filtration is often used in fed-batch processes. The product is gradually introduced into a circulation loop, concentrated, and then further recycled while diafiltration buffer is gradually introduced. The particles introduced first have a significantly longer residence time than the particles introduced last.

[0011] Therefore, continuous process technology is required for the production of microparticles and nanoparticles. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2022 / 018441 [Patent Document 2] U.S. Patent Application Publication No. 2009 / 211991 [Patent Document 3] International Publication No. 2022 / 023759 [Patent Document 4] U.S. Patent No. 6,506,906 [Non-patent literature]

[0013] [Non-Patent Document 1] Dervan, “Molecular Recognition of DNA by Small Molecules, Bioorganic & Medicinal Chemistry (2001) 9: 2215-2235” Summary of the Invention [Problem to be solved by the invention]

[0014] We have now discovered a novel continuous process for producing small particles (eg, microparticles and nanoparticles) by using membrane emulsifiers and membrane emulsification technology. [Means for solving the problem]

[0015] Thus, according to a first aspect of the present invention, there is provided a continuous process for preparing microparticles (e.g., nanoparticles or microparticles), the process comprising: (i) controlling the feeding of a first liquid phase to a first membrane, the first membrane defining a first plurality of openings; (ii) controlling the feeding of a second liquid phase to the first membrane through the first plurality of openings to form a mixture; (iii) controlling the feeding of the mixture to a second membrane, the second membrane defining a second plurality of openings; and (iv) controlling the feeding of a pH buffer phase to the second membrane defining a second plurality of openings. (v) controlling the feeding of the concentrated suspension phase to a second membrane through the openings in the first membrane to form a stabilized particle suspension; and optionally, (v) controlling the feeding of the concentrated, pH buffered mixture to a third membrane, the third membrane defining a third plurality of openings; (vi) controlling the feeding of the concentrated suspension phase to the third membrane through the third plurality of openings to remove solvent and exchange the particles for an excipient buffer; and (vii) controlling the feeding of the stabilized mixture to a fourth membrane for microfiltration to isolate the microparticles.

[0016] Those skilled in the art will appreciate that one or more of steps (v) and (vi) may be optional.

[0017] Those skilled in the art will also appreciate that it may be possible to use unbuffered solutions, as much of the stabilization is believed to result from the ethanol dilution.

[0018] In step (v), concentration of the particle suspension can be achieved using a single-pass tangential flow filtration cassette equipped with an ultrafiltration or microfiltration membrane of a suitable size to retain the particles. For larger microparticles, an AXF crossflow device (available from Micropore Technologies Limited) equipped with a membrane having a slot aperture may be used. Such a slot aperture membrane is described in U.S. Patent Application Publication No. 2009 / 211991, which is incorporated herein by reference.

[0019] Generally, in the continuous process of the present invention, the sequence of steps may be considered to include: Formation of microparticles Dilute / adjust microparticles Concentrate microparticles Diluting microparticles with diafiltration buffer ·concentrated Dilution with diafiltration buffer

[0020] These steps may be further repeated to achieve the desired composition, stopping after either concentration or dilution.

[0021] When the continuous process of the invention involves lipid vesicles, e.g., lipids or LNPs, the mixture may comprise a lipid vesicle suspension. When the continuous process of the invention involves conventional microparticles, the mixture may comprise an emulsion, e.g., a solid-in-liquid or liquid-liquid emulsion.

[0022] In the case of nanoparticles, the continuous process of the present invention may include an optional final step involving in-line sterile filtration to remove bioburden.

[0023] The continuous process of the present invention may include any number of concentration / dilution steps, usually at least two to achieve a sufficiently low solvent concentration, but may include three or more.

[0024] For continuous processes, steps (i)-(vii) may preferably be carried out consecutively, with or without intervening isolation or purification steps. Additionally, intermediate holding vessels, residence coils, pumps, etc. may be utilized. For example, if the solvent diffusion is slow (e.g., through large particles with fewer pores), additional residence time may be required. Splitting the streams also allows for the use of less expensive, lower-pressure pumps.

[0025] For the avoidance of doubt, the term "microparticles" is intended to encompass nanoparticles, microparticles, microspheres, microcapsules and the like.

[0026] Each of the membranes, such as the first, second, third, and fourth membranes, can be the same or different. Generally, each membrane defines a plurality of pores, can be substantially tubular or cylindrical in shape, and can have a first end defining a first inlet opening and a second outlet opening. Thus, each membrane, which can be the same or different, can comprise a laboratory dispersion cell (LDC) (which uses a precisely designed circular membrane and an agitator to generate the shear forces necessary for droplet formation), or a cross-flow device (AXF).

[0027] Those skilled in the art will appreciate that the number of membranes used in a continuous process may vary depending, inter alia, on the nature of the material being processed. Generally, the number of membranes used in the continuous process of the present invention is from about 1 to about 4. More stages may be used to minimize the amount of buffer exchange / wash solution, with less fresh buffer used at each stage. Capital costs increase, but solution costs decrease. For example, in the case of microparticles, a three-stage diafiltration process would involve one dilution membrane and one filtration membrane at each stage, for a total of seven stages, including the droplet-generating membranes.

[0028] For each tubular or cylindrical membrane, the inner diameter of the membrane may vary. Generally, the inner diameter of the membrane is fairly small.

[0029] Alternatively, one or more of the membranes, e.g., one or more of the first, second, third, and fourth membranes, may comprise a cross-flow membrane device (AXF). According to one embodiment of the present invention, at least one of the membranes comprises a cross-flow device (AXF). According to another embodiment of the present invention, all of the membranes comprise a cross-flow device (AXF). A cross-flow membrane device uses, for example, a continuous phase flow to push, for example, a dispersed phase flow through the pores of the membrane, thereby uniformly mixing the two.

[0030] It will be understood that some or all of the membranes used may comprise cross-flow membranes. A mixture of conventional tubular membranes, such as laboratory dispersion cells (LDCs), and cross-flow membranes may be used in the continuous process of the present invention. In a preferred embodiment, all of the membranes used in the continuous process of the present invention comprise cross-flow membranes.

[0031] In one embodiment of the invention, the continuous process may involve the preparation of small lipid vesicles, such as liposomes or lipid particles, more particularly liposomes or lipid nanoparticles (LNPs).

[0032] Lipid vesicles, such as LNPs, are particularly useful as drug delivery carriers and for encapsulating a wide variety of nucleic acids (RNA and DNA). Lipid vesicles are therefore the most common non-viral gene delivery system, used, for example, for vaccine delivery.

[0033] A method for preparing lipid vesicles using membrane emulsification technology is described in WO 2022 / 018441 (Patent Document 1), which is incorporated herein by reference.

[0034] According to this aspect of the present invention, step (i) of the continuous process may include controlling the supply of a first liquid phase to a first membrane, the first liquid phase comprising a lipid phase, and the second liquid phase comprising an aqueous phase. In particular, the aqueous phase may comprise one or more therapeutically active agents, such as DNA and RNA (e.g., mRNA). According to one aspect of the present invention, the lipid vesicles are liposomes. According to another aspect of the present invention, the lipid vesicles are LNPs.

[0035] The active agent may be dispersed in water droplets within a polymer solution in solvent droplets. Thus, such a dispersion of active agent may comprise a "primary emulsion" (as shown in Figure 2 herein).

[0036] In this embodiment of the present invention, the product of the continuous process for preparing lipid vesicles is a lipid vesicle composition consisting of a lipid bilayer enclosing an aqueous core. The aqueous core may contain one or more active agents, or the lipid vesicles may be prepared in an unloaded state and then loaded (active loading). Loading of the active agent can be achieved either by passive loading, i.e., the active agent is entrapped during the formation of the lipid vesicle, or by active loading, i.e., the active agent is loaded after the formation of the lipid vesicle.

[0037] Thus, according to one aspect of the present invention, lipid vesicles are produced in a loaded state (passive loading).

[0038] According to another aspect of the invention, lipid vesicles are prepared in an unloaded state and subsequently loaded (active loading).

[0039] If the lipid vesicles are liposomes, the solvent phase may comprise an aqueous phase. If the lipid vesicles comprise LNPs, the solvent phase may comprise a non-aqueous solvent phase.

[0040] Lipid vesicles and liposomal particles are typically divided into three groups: multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar vesicles (LUVs). MLVs have multiple bilayers within each vesicle, forming several independent aqueous compartments. SUVs and LUVs have a single bilayer that confines an aqueous core. SUVs typically have a diameter of 100 nm or less, while LUVs have a diameter of >100 nm.

[0041] The lipid vesicles of the present invention may be SUVs or LUVs, preferably having a diameter in the range of 50 to 220 nm. For compositions containing a population of SUVs or LUVs with different diameters, (i) at least 80% of the population should have a diameter in the range of 20 to 220 nm, (ii) the mean diameter of the population should ideally be in the range of 40 to 200 nm, and / or (iii) the diameter should have a polydispersity index (PDI) of ≦0.3, e.g., about 0.02 to about 0.3, preferably 0.02 to 0.2. The lipid vesicles may be substantially spherical.

[0042] According to another aspect of the invention, the microparticles are microparticles or nanoparticles as described herein and find use in biomedicine, pharmaceuticals, medicine, cosmetics, chemical industry, agriculture, veterinary medicine, etc. Such microparticles typically contain a chemically or biologically active substance. As such, a continuous process of the invention can include preparing microparticles containing a chemically or biologically active substance, the method comprising controlling the feeding of a liquid phase (the liquid phase containing a solution of a compound) in a first flow direction to a membrane (the membrane defining a plurality of pores) and controlling the liquid phase after it passes through the membrane through the plurality of pores to form microparticles containing the chemically or biologically active substance.

[0043] According to yet another aspect of the present invention, the microparticles are solidified particles, for example, crystalline particles. Thus, the continuous process of the present invention may include preparing solid particles of a compound, the method comprising controlling the supply of a liquid phase (the liquid phase comprising a solution of the compound) in a first flow direction to a membrane (the membrane defining a plurality of pores), and controlling the supersaturation of the liquid phase after passing through the membrane through the plurality of pores to form solid particles of the compound. Such solid particles are described in co-pending WO 2022 / 023759, which is incorporated herein by reference.

[0044] Step (iv) of the continuous process of the present invention may include forming a pH buffered mixture by controlling the feeding of the mixture to a second membrane and controlling the feeding of a pH adjusted buffer to form a stabilized mixture.

[0045] The choice of loading of the lipid vesicles, i.e., whether liposomes or LNPs are actively or passively loaded, may influence the choice of pH-adjusting buffer for the aqueous phase.

[0046] Batch tangential flow filtration tends to take several hours with circulating pumps and moderate shear. Reducing residence times to a few minutes and a single pass should be much gentler and reduce losses.

[0047] The apparatus for the continuous processes of the present invention may include one or more analytical singularities. Thus, for example, an analytical singularity may be included at or near the end of each step of the continuous process or each pair of steps of the continuous process, e.g., at or near the end of one or more of steps (ii), (iv), and (vi). Those skilled in the art will appreciate that such analytical singularities may be located at or adjacent to other suitable points in the continuous processes of the present invention.

[0048] The technique of preparing suitable lipid vesicles is well known in the prior art.One of such methods involves mixing the ethanol solution of lipid with the aqueous solution of active agent.Because this technique utilizes the miscibility of solvent with water, those skilled in the art will understand that other water-miscible solvents can also be suitably used, such as methanol, ethanol, propanol, butanol, pentanol, hexanol, etc. C1-C6 alkanol.

[0049] The method of the present invention is applicable to large-scale commercial production of nano-scale lipid vesicle preparations, particularly preparations that comprise substantially uniform lipid vesicle particle size, which may be about 220nm or less in diameter.For example, more than 90% of lipid vesicles (volume weighted, for example, measured by dynamic light scattering) are less than about 220nm, or more than 99% are less than about 220nm.Particles of this size can be easily sterilized by filtration according to industry-accepted clinical manufacturing standards and / or GMP (Good Manufacturing Practice).

[0050] When at least one of the membranes used in the continuous process of the present invention comprises a cross-flow membrane device (AXF), the cross-flow emulsification device comprises: The method may include an outer tubular sleeve having a first inlet at a first end, an outlet for lipid vesicles, and a second inlet distal to the first inlet and inclined relative to the first inlet, a tubular membrane having a plurality of holes and adapted to be disposed within the tubular sleeve, and optionally an insert adapted to be positioned inside the tubular membrane, the insert having an inlet end and an outlet end, each of the inlet end and the outlet end being provided with a chamfered region, the chamfered region being provided with a plurality of orifices and a branching plate, and may include controlling the supply of a first liquid phase to the tubular membrane, and controlling the supply of a second liquid phase to the tubular membrane through the plurality of holes to form a lipid vesicle suspension.

[0051] In the method of the present invention, cross-flow membrane emulsification utilizes the flow of the continuous phase to force the flow of the dispersed phase through the pores of the membrane to achieve uniform mixing. Mixing or micromixing involves controlled mixing of the phases.

[0052] In one embodiment of the invention, a crossflow device comprises an insert as described herein, wherein the first inlet is a first inlet for a continuous phase and the second inlet is an inlet for a dispersed phase, whereby the dispersed phase moves from the outside to the inside of the tubular membrane.

[0053] In another embodiment of the invention, the cross-flow device does not include an insert, and the first inlet is a first inlet for the dispersed phase and the second inlet is an inlet for the continuous phase, whereby the dispersed phase moves from the inside to the outside of the tubular membrane.

[0054] In another embodiment of the invention, the dispersed phase is a solvent phase and the continuous phase is a lipid phase. The solvent phase may optionally contain one or more active agents as defined herein.

[0055] In one embodiment of the invention, the dispersed phase is a lipid phase and the continuous phase is a solvent phase, which may optionally contain one or more active agents as defined herein.

[0056] When an insert is present and a tubular membrane is disposed inside the outer sleeve, the spacing between the insert and the tubular membrane may vary depending on the desired laminar flow conditions, etc. Typically, the insert is centrally disposed within the tubular membrane, so that the spacing between the insert and the membrane forms an annulus of equal or substantially equal dimensions at any point around the insert. Thus, for example, this spacing may be about 0.05 to about 10 mm (the distance between the outer wall of the insert and the inner wall of the membrane), about 0.1 to about 10 mm, about 0.25 to about 10 mm, or about 0.5 to about 8 mm, or about 0.5 to about 6 mm, or about 0.5 to about 5 mm, or about 0.5 to about 4 mm, or about 0.5 to about 3 mm, or about 0.5 to about 2 mm, or about 0.5 to about 1 mm.

[0057] When a tubular membrane is disposed within an outer sleeve, the spacing between the tubular membrane and the outer sleeve may vary. Typically, the tubular membrane is centrally disposed within the outer sleeve, so that the spacing between the membrane and the sleeve defines an annulus of equal or substantially equal dimensions at any point around the circumference of the tubular membrane. Thus, for example, the spacing may be about 0.5 to about 10 mm (the distance between the outer wall of the membrane and the inner wall of the sleeve), or about 0.5 to about 8 mm, or about 0.5 to about 6 mm, or about 0.5 to about 5 mm, or about 0.5 to about 4 mm, or about 0.5 to about 3 mm, or about 0.5 to about 2 mm, or about 0.5 to about 1 mm.

[0058] In an alternative embodiment of the present invention, the insert may be tapered, so that the spacing between the insert and the tubular membrane widens along the length of the membrane. The spacing and magnitude of the widening may vary depending on the slope of the tapered insert, the desired laminar flow conditions / flow rate, size distribution, etc. Those skilled in the art will understand that, depending on the direction of the taper, the spacing between the insert and the tubular membrane may widen or converge along the length of the membrane. The use of a tapered insert may be advantageous in that an appropriate taper can maintain a consistent laminar flow for a particular formulation and set of flow conditions. Thus, a tapered insert may be used to control changing mixing conditions resulting from changes in fluid properties, such as viscosity, as the concentration of ethanol or other solvent and lipid increases through a path along the length of the membrane.

[0059] In an alternative embodiment of the present invention, when a cross-flow device is used, the cross-flow device may include more than one tubular membrane located inside an outer tubular sleeve, i.e., a plurality of tubular membranes. When a plurality of tubular membranes is provided, each membrane may optionally have an insert located therein, as described herein. The plurality of membranes may be grouped together as a group of membranes arranged side by side with one another. It is desirable that the membranes are not in direct contact with one another. It will be understood that the number of membranes may vary depending, inter alia, on the nature of the material being manufactured. Thus, by way of example only, when there is a plurality of tubular membranes, the number of membranes may be between 2 and 100.

[0060] The angled second inlet in the outer tubular sleeve generally comprises a bifurcation of the tubular sleeve and may be perpendicular to the longitudinal axis of the tubular sleeve. The location of the bifurcation or second inlet may vary and may be determined by the plane of the membrane. In one embodiment, the bifurcation or second inlet is located substantially equidistant from the inlet and outlet, although one skilled in the art will appreciate that the location of this second inlet may vary. It is within the scope of the present invention to provide more than one bifurcation inlet. For example, the use of two bifurcations may suitably allow for the outflow of the continuous phase during priming, for flushing for cleaning, or for draining / venting for sterilization.

[0061] The inlet and outlet ends of the outer sleeve are typically equipped with seal assemblies. The seal assemblies may be the same or different at the inlet and outlet ends of the outer sleeve, but each seal assembly is preferably the same. Typical O-ring seals, in which an O-ring is compressed between two surfaces that must be sealed, come in a variety of shapes. Commercially available O-ring seals are optionally equipped with a variety of standard groove sizes. Each seal assembly includes a tubular ferrule with flanges at each end. The first flange, located at the end adjacent to the outer sleeve (when connected), may have an internal circumferential recess that serves as a seat for the O-ring seal.

[0062] However, those skilled in the art will appreciate that other means of providing a seal may be used as appropriate, such as using threaded joints tightened to a specific torque to avoid the need for close tolerances, or tightening parts to a specific force followed by welding, which may be particularly suitable when using plastic cross-flow equipment.

[0063] The inner diameter of each tubular membrane may vary. In particular, the inner diameter of the tubular membrane may vary depending on the presence or absence of an insert and at which stage of a continuous process the membrane is used. Generally, the inner diameter of the tubular membrane is fairly small. Without an insert, the inner diameter of the tubular membrane may be about 1 mm to about 10 mm, or about 2 mm to about 8 mm, or about 4 mm to about 6 mm. If the tubular membrane is intended to be used with an insert, the inner diameter of the tubular membrane may be about 5 mm to about 50 mm, or about 10 mm to about 50 mm, or about 20 mm to about 40 mm, or about 25 mm to about 35 mm. Larger inner diameter tubular membranes may be able to withstand lower injection pressures. The upper limit of the inner diameter of the tubular membrane may depend, among other things, on the thickness of the membrane tube, since the cylinder must be able to withstand the external injection pressure and is dependent on the ability to consistently puncture its thickness. The inner chamber of the cylindrical membrane typically contains a continuous phase liquid.

[0064] The perforations in the membrane may be uniformly spaced or may have a variable pitch, or the membrane perforations may have a uniform pitch in a row or circumference but a different pitch in another direction.

[0065] The pores in the membrane may vary. By way of example only, the pores in the membrane may have a pore diameter of about 1 μm to about 200 μm, or about 1 μm to about 100 μm, or about 10 μm to about 100 μm, or about 20 μm to about 100 μm, or about 30 μm to about 100 μm, or about 40 μm to about 100 μm, or about 50 μm to about 100 μm, or about 60 μm to about 100 μm, or about 70 μm to about 100 μm, or about 80 μm to about 100 μm, or about 90 μm to about 100 μm. In yet another embodiment of the invention, the pores in the membrane may have a pore diameter of about 1 μm to about 40 μm, e.g., about 3 μm, or about 5 μm to about 20 μm, or about 5 μm to about 15 μm.

[0066] The pore shape of the membrane can be substantially tubular or linear, e.g., slotted. Slotted pores are sometimes preferred for microparticle continuous process filtration. The aspect ratio of the slots can be such that they retain product particles but do not "suck" the particles into the pores, thereby blocking them and allowing the particles to continue flowing. When using membranes with slotted pores, the feed flows between the insert (e.g., a tapered insert) and the membrane, and the permeate flows through the pores and exits through a side port (second inlet).

[0067] However, it is within the scope of the present invention to provide membranes with uniformly tapered pores. Such uniformly tapered pores can be advantageous in that their use can reduce pressure drop across the membrane, potentially increasing throughput / flux. It is also within the scope of the present invention to provide membranes with essentially constant diameter but with internal pores that are non-circular (e.g., rectangular slots) or convoluted (e.g., tapered or stepped in diameter to minimize pressure drop) to provide pores with high aspect ratios.

[0068] The distance or pitch between the holes may vary depending, inter alia, on the size of the holes, and may be from about 1 μm to about 5,000 μm, or from about 1 μm to about 1,000 μm, or from about 2 μm to about 800 μm, or from about 5 μm to about 600 μm, or from about 10 μm to about 500 μm, or from about 20 μm to about 400 μm, or from about 30 μm to about 300 μm, or from about 40 μm to about 200 μm, or from about 50 μm to about 100 μm, for example, about 75 μm.

[0069] The surface porosity of the membrane may be determined by the pore size and may be from about 0.001% to about 20%, or from about 0.01% to about 20%, or from about 0.1% to about 20%, or from about 1% to about 20%, or from about 2% to about 20%, or from about 3% to about 20%, or from about 4% to about 20%, or from about 5% to about 20%, or from about 5% to about 10% of the surface area of the membrane.

[0070] The arrangement of the holes may vary depending on, among other things, hole size, throughput, etc. In general, the holes may be in a patterned arrangement and may be square, triangular, linear, circular, rectangular, or other arrangement. In one embodiment, the holes are in a square arrangement.

[0071] It will be appreciated that the device, and in particular the membrane, of the present invention may comprise known materials such as glass, ceramic, metal, e.g., stainless steel or nickel, polymer / plastic, such as fluoropolymer, or silicon. The use of metal, e.g., stainless steel or nickel, or polymer / plastic, such as fluoropolymer, is advantageous, among other things, in that the device and / or membrane may be sterilized using conventional sterilization techniques known in the art, including gamma irradiation, if desired. The use of polymer / plastic materials, such as fluoropolymer, is advantageous, among other things, in that the device and / or membrane may be manufactured using injection molding techniques known in the art.

[0072] As described herein, one of the membranes may include an insert to facilitate even flow distribution. However, the absence of an insert is within the scope of the crossflow device of the present invention. When an insert is present, the branching plate may be adapted to split the continuous or dispersed phase flow into several branches. Whether the branching plate splits the continuous or dispersed phase depends on the direction of the continuous phase flow, i.e., whether the continuous phase flows through the first inlet or the second inlet. The number of branching plates may vary, but the number selected must be appropriate to provide even flow distribution and avoid excessive shear (at the lipid vesicle outlet end). Preferably, when an insert is present, the branching plate is a bifurcated or trifurcated plate to provide a uniform continuous phase flow within the annular region between the insert and the membrane. Most preferably, the branching plate is a trifurcated plate.

[0073] The number of orifices provided in the insert may vary depending on the injection speed, etc. Generally, the number of orifices may be 2 to 6. Preferably, the number of orifices is 3.

[0074] The chamfered area of the insert is advantageous in that it allows the insert to be centered when positioned within the membrane. The outer periphery of the insert's end has minimal tolerance to the inner diameter of the tubular membrane. This allows the insert to be precisely centered, resulting in a consistent annulus and consistent laminar flow. The chamfered area, typically a shallow chamfer, has the advantage of evenly distributing the flow and allowing the insert orifice to have a larger cross-sectional area than would be achievable if the flow simply entered through an orifice parallel to the insert axis. This keeps the fluid velocity low, thereby minimizing unwanted pressure losses and outlet shear. The distance between the beginning of the orifice and the beginning of the porous region of the tubular membrane allows for the establishment of an even velocity distribution. The radial dimension of the insert is determined to achieve an annulus depth that provides a specific laminar flow for a given flow rate. The axial dimension is typically designed to provide a combined orifice area, which is larger than both the annulus area and the inlet / outlet tubing area.

[0075] The use of membrane emulsification techniques in the continuous preparation of lipid vesicles described herein may include the use of turbulent flow, e.g., by stirring, or the use of laminar flow. In certain aspects of the invention, continuous membrane emulsification techniques involve the use of laminar flow, i.e., while generally avoiding or minimizing turbulence.

[0076] The use of membrane emulsification technology in preparing lipid vesicles as described herein may include the use of one or more pump systems.It will be understood that any conventionally known pump system for membrane emulsification may be used appropriately.However, in certain embodiments of the present invention, the pump system may be comprised of a gear pump or a peristaltic pump, or a combination thereof.

[0077] The lipid vesicles thus obtained are highly reproducible in both encapsulation rate and particle size distribution (polydispersity). The lipid vesicles may have a polydispersity index of ≦0.3, for example, from about 0.05 to about 0.3.

[0078] Using the continuous methods of the present invention, the distribution of chemical conditions and mechanical forces can be precisely controlled so that both are consistent over length scales comparable to the lipid vesicles, and therefore the resulting lipid vesicle population is more uniform in size and therefore less polydisperse.

[0079] In one embodiment of the invention, when at least one cross-flow device is used, the cross-flow device may include an insert as described herein, wherein the first inlet is a first inlet for the continuous phase and the second inlet is an inlet for the dispersed phase, and the dispersed phase moves from the outside to the inside of the tubular membrane.

[0080] In another embodiment of the invention, when at least one cross-flow device is used, the cross-flow device does not include an insert, the first inlet is a first inlet for the dispersed phase, the second inlet is an inlet for the continuous phase, and the dispersed phase moves from the inside to the outside of the tubular membrane.

[0081] Separation, purification and / or dilution of lipid vesicles can also be carried out by any suitable method.Preferably, in the continuous process of the present invention, lipid vesicles are filtered, more preferably, lipid vesicles are separated or purified by filtration through a sterile filter.In the case of active loading and / or RNA loading, dilution may be carried out to reduce solvent concentration or to exchange buffer solution, followed by concentration by ultrafiltration.

[0082] According to yet another aspect of the present invention, there is provided an apparatus for a continuous process for preparing the microparticles described herein, said apparatus comprising an array of membranes.

[0083] According to this aspect of the invention, the array of membranes comprises: (i) a first membrane for forming a mixture by controlling the feeding of a first liquid phase to the first membrane and controlling the feeding of a second liquid phase to the first membrane; (ii) a second membrane for controlling the feeding of the mixture to the second membrane, the second membrane defining a second plurality of openings, and for controlling the feeding of a pH buffer phase to the second membrane through the second plurality of openings to form a pH buffered mixture; (iii) a third membrane for controlling the feeding of the pH buffered mixture to a third membrane, the third membrane defining a third plurality of openings, and for controlling the feeding of a diafiltration buffer phase to the third membrane through the third plurality of openings to form a stabilized mixture; and (vii) a fourth membrane for isolating microparticles by controlling the feeding of the stabilized mixture to the fourth membrane for microfiltration; may include:

[0084] Lipid vesicles, such as liposomes and lipid nanoparticles (LNPs), prepared by the methods of the present invention are useful as components of pharmaceutical compositions for immunizing subjects against various diseases. These compositions typically include, in addition to the lipid vesicles, a pharmaceutically acceptable carrier.

[0085] Therefore, according to yet another aspect of the present invention, there is provided a lipid vesicle prepared by the continuous process described herein. According to one aspect of the present invention, the lipid vesicle prepared by the continuous process described herein is a liposome. According to another aspect of the present invention, the lipid vesicle prepared by the method described herein is an LNP.

[0086] According to this aspect of the invention, the lipid vesicles may further comprise an active agent.

[0087] For example, the active agent used in the lipid vesicles of the present invention includes, but is not limited to, biologically active agents such as pharmaceutical active agents, vaccines and insecticides.Biologically active compounds can also include, for example, plant nutrients or plant growth regulators.Alternatively, the active agent can be a non-biologically active agent such as plant nutrients, food flavors, and fragrances.

[0088] A pharmaceutical active agent refers to a naturally occurring, synthetic, or semi-synthetic substance (e.g., a compound, ferment, extract, or cell structure) capable of inducing one or more physical, chemical, and / or biological effects directly or indirectly in vitro and / or in vivo. Such an active agent may be capable of preventing, alleviating, treating, and / or curing an abnormal and / or pathological condition in an organism, such as by destroying a parasite or substantially altering the physiology of a host or parasite to reduce the effects of the disease or disorder. Such an active agent may be capable of maintaining, enhancing, reducing, inhibiting, or disrupting a physiological bodily function. An active agent may be capable of diagnosing a physiological condition or state by in vitro and / or in vivo testing. An active agent may be capable of controlling or protecting the environment or an organism by attracting, disabling, inhibiting, killing, modifying, repelling, and / or preventing the growth of animals or microorganisms. Active agents may also have the ability to address the body in other ways (such as deodorizing, protecting, decorating, or caring for the body). Active agents may also be referred to as bioactive agents, pharmaceuticals (such as preventative or therapeutic agents), diagnostic agents, dietary supplements, and / or cosmetics, depending on their effect and / or application, and include, but are not limited to, prodrugs, affinity molecules, synthetic organic molecules, polymers, molecules with a molecular weight of 2 kD or less (such as 1.5 kD or less, or 1 kD or less), macromolecules (such as those with a molecular weight of 2 kD or more, preferably 5 kD or more), protein compounds, peptides, vitamins, steroids, steroid analogs, nucleic acids, carbohydrates, precursors thereof, and derivatives thereof. Active agents may be ionic, nonionic, neutral, positively charged, negatively charged, or zwitterionic, and may be used alone or in combination of two or more thereof. Active agents may be water-insoluble or water-soluble.

[0089] As used herein, the term "polymer" refers to a material capable of achieving three-dimensional (eg, tertiary and / or quaternary) structure.

[0090] A wide variety of pharmaceutically active agents may be used in the continuous processes of the present invention, and thus may include one or more of polynucleotides, peptides, proteins, small organic active agents, small inorganic active agents, and mixtures thereof.

[0091] The polynucleotide active agent may comprise one or more of oligonucleotides, antisense constructs, siRNA, enzymatic RNA, recombinant DNA constructs, expression vectors, and mixtures thereof. The lipid vesicle delivery system of the present invention may be useful for in vivo or in vitro delivery of active agents such as amino acids, peptides, and proteins. Peptides may be signaling molecules such as hormones, neurotransmitters, and neuromodulators, or may be active fragments of larger molecules such as receptors, enzymes, or nucleic acid-binding proteins. Proteins may be enzymes, structural proteins, signaling proteins, or nucleic acid-binding proteins such as transcription factors.

[0092] When the pharmaceutically active agent comprises a small organic active agent, the pharmaceutically active agent may comprise a therapeutic agent or a diagnostic agent. In certain embodiments, the small organic active agent may comprise a sequence-specific DNA-binding oligomer, an oligomer of a heterocyclic polyamide, such as those disclosed in U.S. Pat. No. 6,506,906, which is incorporated herein by reference. Other small organic active agents may include those disclosed by Dervan in "Molecular Recognition of DNA by Small Molecules, Bioorganic & Medicinal Chemistry (2001) 9:2215-2235," which is incorporated herein by reference. In certain embodiments, the oligomer may comprise a monomer subunit selected from the group consisting of N-methylimidazolecarboxamide, N-methylpyrrolecarboxamide, beta-alanine, and dimethylaminopropylamide.

[0093] In another embodiment of the present invention, two or more polynucleotides can be encapsulated in the lipid vesicle delivery system. Such polynucleotides can express multiple gene products under control, and in certain embodiments, at least one expressible gene product is a membrane protein, such as a membrane receptor, most preferably a membrane-bound receptor of a signaling molecule. In some embodiments, at least one expressible gene product is a soluble protein, such as a secreted protein, such as a signaling protein or peptide. [Brief explanation of the drawings]

[0094] [Figure 1] FIG. 1 is a schematic diagram of a continuous process for LNP production using single-pass tangential flow filtration (SPTFF) membranes. [Figure 2] FIG. 1 is a schematic diagram of a continuous process for manufacturing using slotted AXF membranes for high flux microfiltration. Detailed Description of the Invention

[0095] The present invention will now be described, by way of example only, with reference to the accompanying embodiments and drawings.

Claims

1. A continuous process for preparing fine particles, such as nanoparticles or microparticles, wherein the process is: (i) A step of controlling the supply of a first liquid phase to a first membrane, wherein the first membrane comprises the step of defining a first plurality of openings, (ii) A step of forming a mixture by controlling the supply of a second liquid phase to the first membrane through the first plurality of openings, Optional, (iii) A step of controlling the supply of the mixture to a second membrane, wherein the second membrane comprises the step of defining a second plurality of openings, (iv) A step of controlling the supply of the pH buffer phase to the second membrane through the second plurality of openings, comprising the step of forming a stabilized particle suspension, (v) A step of controlling the supply of a mixture obtained by concentrating the particle suspension and treating it with a pH buffer to a third membrane, wherein the third membrane defines a third plurality of openings, (vi) A step of controlling the supply of the diafiltration buffer phase to the third membrane through the third plurality of openings to remove the solvent and replace the particles with the excipient buffer, (vii) A step of isolating the fine particles by controlling the supply of the stabilized mixture to a fourth membrane and microfiltration. Includes, Contains one or more analytical singularities, Continuous process.

2. The continuous process according to claim 1, characterized in that the fine particles are nanoparticles or microparticles.

3. The continuous process according to claim 1, wherein the aforementioned membranes may be the same or different, and the process is characterized by comprising a laboratory dispersion cell (LDC) or a cross-flow apparatus (AXF®).

4. The continuous process according to claim 3, characterized in that at least one of the membranes is equipped with a cross-flow membrane apparatus (AXF).

5. The continuous process according to any one of claims 1 to 4, characterized in that in step (v), the concentration of the particle suspension is achieved using a single-pass tangential flow filtration cassette equipped with an ultrafiltration membrane or microfiltration membrane of a size suitable for holding the particles.

6. The continuous process according to claim 1, characterized in that the process includes the preparation of minute lipid vesicles.

7. The continuous process according to claim 6, characterized in that the lipid vesicles include liposomes or lipid nanoparticles (LNPs).

8. The continuous process according to claim 6, characterized in that the process comprises preparing a minute lipid vesicle containing a lipid bilayer enclosing an aqueous core, or preparing lipid nanoparticles (LNPs) containing an aqueous solvent phase core or a non-aqueous solvent phase core.

9. The continuous process according to claim 8, characterized in that the aqueous core contains one or more activators.

10. The aforementioned lipid vesicles are manufactured in an unfilled state and then filled (active filling) or manufactured in a filled state (passive filling). The continuous process according to claim 6, characterized in that

11. The continuous process according to any one of claims 1 to 10, characterized in that step (iv) comprises forming a pH buffered mixture by controlling the supply of the mixture to a second membrane and controlling the supply of a pH adjusting buffer to form a stabilized mixture.

12. The continuous process according to any one of claims 1 to 11, characterized in that the fine particles are microparticles or nanoparticles containing a chemically or biologically effective substance.

13. The continuous process according to any one of claims 1 to 12, characterized in that the fine particles are solidified particles, for example, crystalline particles.

14. The continuous process according to claim 1, characterized in that the analytical singularity is located at or near the end of each step or each pair of steps in the continuous process.

15. A continuous process according to claim 4, comprising at least one cross-flow membrane apparatus (AXF), wherein the cross-flow emulsifier is An outer tubular sleeve having a first inlet at a first end, an outlet for lipid vesicles, and a second inlet distal to the first inlet and inclined with respect to the first inlet, A tubular membrane having multiple holes and adapted to be positioned inside the tubular sleeve, An optional insert adapted to be located inside the tubular membrane, wherein the insert has an inlet end and an outlet end, each of the inlet end and the outlet end is provided with a chamfered region, and the chamfered region is provided with a plurality of orifices and branching plates. It is equipped with, and Controlling the supply of the first liquid phase to the tubular membrane and controlling the supply of the second liquid phase to the tubular membrane through the plurality of pores to form a lipid vesicle suspension. A continuous process that includes this.

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