Process for producing nanocarriers and / or nanoformulations

Abstract

The invention relates to a process in a plant 100 for producing nanocarriers and / or nanoformulations having an average diameter of 20 nm to 300 nm comprising: • a) Inserting a first reactant to a mixing unit 200 by a first feed pump 120, • b) Inserting at least a second reactant to the mixing unit by a second feed pump 122, wherein the mixing unit comprising • - one inner mixing chamber 220 with a diameter (De) and a height (H) in the direction of the longitudinal axis (Ac) comprising • -- at least one circumferential side wall 222, one feed plate 224 and one outlet plate 226, • -- one inner feed orifice 202 being connected to a first feed line 110 and a second feed line 112 and • - at least one inner outlet orifice 204, and wherein the inner feed orifice and the inner outlet orifice are peripherally orientated channel segments, • c) propelling one mixing wheel in one rotation direction (R) with a given rotation speed (V), wherein the mixing wheel having an outer diameter (Dw) mounted on a drive shaft which defines a longitudinal axis (B) eccentrically placed inside the mixing chamber and oriented parallel to longitudinal axis (A) of the mixing unit, wherein the mixing wheel comprises at least two outer extrusions, wherein the mixing chamber having an inner a ring volume defined by the radially free space (gap) between the eccentric propelling mixing wheel and the side wall, wherein the smallest clearance between one rotating extrusion and the side wall in radial direction is factor 0.005 to 0.125 of the diameter (D) of the mixing chamber, and wherein the clearance B is between 15 μm to 400 μm.

Description

[0001] Process for producing nanocarriers and / or nanoformulations

[0002] The present invention provides a process for producing nanocarriers and / or nanoformulations having an average diameter of 20 nm to 300 nm with adjustable output and / or particle size.

[0003] In the context of the present invention a nanocarrier is a composition for carrying pharmaceutical, cosmetic or nutraceutical active ingredients. The nanocarrier may consist of a pure substance or may be a mixture of two or more substances. The substances may be solid, semi-fluid or liquid. The substances may be single-phase or multi-phase substances. In all cases nanocarriers are in particulate form, wherein the average particle size is smaller than 300 nm.

[0004] Examples of nanocarriers are particles derived from natural or synthetic polymers or lipids to form lipid nanoparticles (LNP), liposomes and micelles and nanoemulsions.

[0005] Nanocarriers composed of two or more substances are produced by providing the individual substances or precursors thereof as dispersions or as solutions in liquid media and mixing these with one another. The mixing results in physical interactions between the individual substances or precursors to form the nanocarrier. The nanocarrier may optionally be subjected to subsequent workup, for example by separation from the liquid medium. The obtained nanocarrier may then either be used as a placebo or is laden with an active ingredient to form a nanoformulation.

[0006] In the context of the present invention a nanoformulation is a dosage form of a pharmaceutical, cosmetic or nutraceutical active ingredient carried by a nanocarrier. The active ingredient may be on the surface of the nanocarrier, may be inside the nanocarrier or may build a complex with the nanocarrier.

[0007] Examples of nanoformulations include inter alia so-called lipoplexes or polyplexes, i.e. complexes of polymers or lipids with for example DNA, RNA, proteins, peptides etc. Within these complexes the polymers or lipids form the nanocarrier while the DNA, RNA form the active ingredient.

[0008] As described above the production of nanoformulations may be conducted by loading a nanocarrier with active ingredient.

[0009] However, in pharmaceutical technology it is in the interest of process economy that the production of the nanocarrier and the loading thereof with active ingredient is conducted in an integrated process:

[0010] In such an integrated process the nanocarrier is produced first and loaded with active ingredient in the nascent state. This is generally conducted by providing and mixing the individual components of the nanoformulation as a dispersion or solution in liquid media. The components include the substances forming the nanocarrier / precursors to these substances and also the active ingredient or its precursors. The mixing results in physical interactions between the individual components to form the nanoformulation. If required, the nanoformulation is subsequently subjected to further workup, such as but not limited to separation from the liquid medium, a shift of pH value of the liquid medium. In case of a pH shift to be performed during preparation of nanocarriers or nanoformulations, this is preferably accomplished by means of third liquid phase defining the final pH of the secondary mixture. The pH of the third liquid phase is defined by selecting a suitable buffer in suitable amount. Examples of suitable buffers are phosphate buffer pH 7.4, phosphate buffer saline (pH 7.4) and 2- (4-(2-hydroxyethyl)-1-piperazinyl)ethane sulfonic acid (HEPES buffer).

[0011] Especially nanoformulations using LNP or polyplexes as the carrier are always produced in an integrated process where the loading of the carrier with active ingredient is conducted in the nascent state. If LNPs are to be produced, artificial or natural lipids are preferred precursors of nanocarriers. A prominent example for a natural lipid suitable as a LNP precursor is cholesterol. Examples of artificial lipids usable for the same purpose are 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1 ,2-Dimyristoyl-rac-glycero-3-methoxypolyoxyethylene (PEG-DMG), dilinoleylmethyl-4-dimethylam- inobutyrate (Dlin-MC3-DMA) and 1 ,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0012] An essential apparatus engineering aspect in the production of nanocarriers / nanoformulations is the design of the mixing units with which the components dispersed or dissolved in the liquid media are mixed.

[0013] In the know use of mixing units the problem is the mixing itself. The mixers are therefore made small, so-called micromixers. In such a micromixer the mass flow is divided into numerous small substreams and these substreams are mixed. These small structures are extremely sensitive to fouling (deposits on the wall) and small gas bubbles. The reactants for mixing units must therefore be conditioned, e.g. particle free and / or high-purity. This means that especially dissolved gases such as air or nitrogen must be laboriously removed from the liquid.

[0014] EP1519714B1 discloses a process for producing nanoformulations employing an apparatus comprising a T-shaped mixer. The two fluids to be mixed are passed through coaxial feed conduits to a collision point, mixed there and withdrawn through a discharge offset by 90°. The angle between the two feed conduits is 180°. The volume flow of the two fluids into the active element is the same. Another T-shaped mixer is disclosed by EP1937213B1 . According to this process and apparatus for producing nanoformulations two T-shaped mixers are arranged in series. Thus, three fluids may be mixed in two steps. The disadvantage of both solutions is that T-shaped mixers form flow conditions which change upon increasing the production scale and changing volume flow rate - so-called scaling up (fluid-mechanical similarity problem). EP3711749A1 discloses Y-shaped mixers for producing nanoformulations. The two feed conduits to the collision point are thus not parallel. The Y-mixer may be combined with a T-mixer to produce triphasic mixtures. Y-mixers likewise have complex flow dynamics and can therefore be transferred from pilot scale to production scale only through “numbering up”. The problem of inadequate stability of LNP in the obtained mixture is also encountered in processes with Y-mixers.

[0015] W02001005373A1 discloses a mixing head for producing nanoformulations comprising a plurality of feed pumps having a diameter of less than 2 mm and WO2017223135A1 discloses a mixing head which likewise operates according to the feed pump principle. The feed pump is provided with a metering apparatus which is in the form of a servomotor-actuated pipette.

[0016] However, the altered flow conditions bring about altered production properties. For this reason, it is non-trivial to transfer the operating conditions optimized in terms of product quality in laboratory and pilot operation to a larger production scale. Instead, industrial operation comprises simultaneously operating a multiplicity of small-scale apparatuses with the conditions optimized on the pilot scale (“numbering up”). This increases capital costs. A further disadvantage of this process is that the production of LNP forms a mixture, in whose milieu the nanocarriers are not stable for long period. Consequently, the nanocarriers must either be separated from the mixture particularly quickly or stabilized in an additional step. Both options make process management very laborious.

[0017] The present invention has for its object to specify an apparatus for producing nanocarriers / nanoformulations, whose fluid-mechanical ratios are retained even in the course of a scale up, so that increasing production scale is possible without “numbering up.” Furthermore, the process for producing nanocarriers / nanoformulations operated with the apparatus shall save operating steps.

[0018] The object is achieved by a process according to claim 1 .

[0019] The present invention thus provides a process or producing nanocarriers and / or nanoformulations having an average diameter of 20 nm to 300 nm, preferably for producing lipid nanoparticles (LNP), comprising: a) Inserting a first reactant to a mixing unit by a first feed pump, (i.e. carrier substance) b) Inserting at least a second reactant to the mixing unit by a second feed pump (i.e. active ingredient), wherein the mixing unit extending to a longitudinal axis A, comprising

[0020] - one inner mixing chamber with a diameter De and a height H in the direction of the longitudinal axis Ac comprising

[0021] - at least one circumferential side wall, one feed plate and one outlet plate,

[0022] - one inner feed orifice being connected to the first feed line and second feed line and

[0023] - at least one inner outlet orifice, and wherein the inner feed orifice and the inner outlet orifice are peripherally orientated channel segments, wherein propelling one mixing wheel in one rotation direction with a given rotation speed U, wherein the mixing wheel having an outer diameter Dw mounted on a drive shaft which defines a longitudinal axis Aw eccentrically placed inside the mixing chamber and oriented parallel to longitudinal axis Ac of the mixing unit, wherein the mixing wheel comprises at least two outer extrusions, wherein the mixing chamber having an inner a ring volume defined by the radially free space between the eccentrically propelling mixing wheel and the side wall, wherein the smallest clearance B between one rotating extrusion and the side wall in radial direction is factor 0.005 to 0.125 of the diameter De of the mixing chamber 220, preferably factor 0.005 to 0.1 , and wherein the clearance B is between 15 pm to 400 pm.

[0024] According to a preferred version of the process, wherein the clearance B is between 15 pm to 300 pm, preferably between 15 pm to 200 pm, most preferred between 15 pm to 100 pm.

[0025] The extrusions of the mixing wheels can be formed analogously to mixing wheels of a e.g. (micro) gear pump. The “smallest clearance” B is defined by the dynamic wheel area and the shortest distance in radial direction to the inner (circumferential) wall of the mixing chamber. The “dynamic wheel area” is defined by the circle area needed and covert be the propelling mixing wheel.

[0026] The term “diameter of mixing wheel (Dw)” shall mean the diameter of the shadow area (circle) described by a propelling mixing wheel. Thus, the “diameter of mixing wheel (Dw)” describes the ultimate length of the rotating protrusions round the central axis of the mixing wheel.

[0027] According to an advantageous solution, at the smallest clearance B is within a closed area having an ankle W of at least 10° to 60°, preferable 15° to 45°. The closed area is defined by comprising the smallest clearance and no orifice in the feed plate, the outlet plate or the side wall is located therein. The positive effect of such closed area is forcing a pure circumferential flow rate and avoiding undefined side streams.

[0028] Without intending to be bound to any specific interpretation or theory, it is believed, that this defined area has a major effect on the reproducible diameter DLMP of the particles produced with the mixing unit in dependency of the propelling speed of the mixing wheel and the feed volume flow.

[0029] The following terms are used as synonyms “first reactant” and “reactantl”, also “second reactant” and “reactant2” as well as "mixing unit” and “micro mixer”.

[0030] As a preferred embodiment, the first reactant (reactantl) is the nanocarrier matrix forming substance (non-active ingredient / substance) comprising a dispersion, such as an organic phase (mixture), wherein the organic phase can comprise an alcohol, such as but not limited to ethanol or glycerol, and wherein lipids might be included-The second reactant (reactant2) comprises the active ingredient (active substance or mixture), such as, but not limited to DNA; RNA, and wherein the second reactant is comprising a buffer, preferably at pH 3.5 to 4.5, most preferred at pH 4.0.

[0031] More in detail, the mixing wheel is the sole rotating element of the e.g. mixing unit, whereby the mixing unit is placed on or might comprise a connection block, having at least one inner channel connected to at least one inner orifice, preferably having two inner channels each connected to one inner orifice (feed orifice, outlet orifice).

[0032] According to a preferred embodiment of the process, the inner feed orifice and the inner outlet orifice are peripherally orientated channel segments, each having a small end portion (S) and one brought end portion (B), and wherein the brought end portion (B) is at least 1 .1 to 3.0 times of the small end portion (S).

[0033] The broader end portion (B) preferably is 1 .5 times of the small end portion (S). The mixing wheel pushes fluid in circumferential direction from the smaller end portion (S) to the broader end portion (B) and thus adds a certain volume of fluid to the incoming mass flow of the second, broader end portion (B). In case of an identical width as the second end a considerable increase and / or pulsation would appear. The advantage of the asymmetric width of the orifices is a homogenisation pressure due to a gradient in flow velocity of one orifice, and thus a total flow with less pressure pulsation.

[0034] According to another preferred embodiment of the process, the inner feed orifice and the inner outlet orifice are mirrored by a mirror axis (M) toward each other, wherein the small end portions (S) are facing each other in close distance and the brought end portions (B) are facing each other in closer distance.

[0035] According to another preferred embodiment of the process, the inner feed orifice and the inner outlet orifice: a) both located on the feed plate, b) the inner feed orifices is located on the feed plate and the inner outlet orifice is located on the outlet plate, or c) both or at least one orifice is located analogously on the circumferential side wall of the mixing chamber.

[0036] The mirroring and / or the asymmetric geometry of the respective orifices can be beneficial and can be applied to the mixing unit at any variation according to a), b) or c).

[0037] According to an improved or preferred embodiment of the process, the inner and outer orifices are both integrated in the same (feed) plate or on the circumferential side wall, a second pair of peripherally orientated channel segments are located at the opposite side of the mixing chamber, i.e. on the face plate or on the opposite side of the side wall, being equally oriented towards each other but 90° rotated relative to the first pair of peripherally oriented channels, wherein at least a portion of the first and second reactant or a mixture of it is temporary placed inside the second pair of peripherally oriented channel segments while the mixing wheel is propelling.

[0038] Said second pair of peripherally orientated channel segments works as intermediate or temporary flow chambers, receiving and / or storing temporary at least a partial volume of the fluid. In a preferred embodiment said second pair of peripherally orientated channel segments having an identical or analogous dimension and / or shape as the (first) peripherally orientated channels and / or channel segments.

[0039] According to another preferred embodiment of the process, the propelling mixing wheel comprises three to ten outer extrusions, preferably four to eight extrusions, most preferably six extrusions. However, the number of extrusions is generally not limited.

[0040] According to another preferred embodiment of the process, the void volume inside the mixing chamber between mixing wheel and side wall is 3, .5 pl to 8.5 pl, preferably 4.0 pl to 7.5 pl, most preferably 4.5 pl to 4.7 pl.

[0041] According to another preferred embodiment of the process, prior to the production of the nanocarriers and / or nanoformulations, at least one characteristic line, preferably a set of characteristic lines, is preferably determined (Determining) for the nanocarrier and / or nanoformulation produced by a mixture of the first reactant and the at least second reactant as a function (I) of V, U, DLNP, wherein

[0042] - DLNP: diameter of the nanocarrier and / or nanoformulation (nm),

[0043] - V: total volume flow of the reactants (ml / min),

[0044] - U: propelling speed form 500 to 15.000 r / m (rpm), and storing the characteristic line / s as data in a computer readable format at a data storage integrated and / or connected to a control unit, such as but not limited to a processor, computer, CPU, pProcessor of the mixing unit. The determining of characteristic data might be done as one close process step prior to the subsequent septs and / or rendered as a general service or general (stand-alone) process step, independently from a specific production (process).

[0045] This determining step is followed by the following steps: a) Providing characteristic data for a defined mixture of a first reactant and at least one second reactant, i.e. providing on and / or for the control unit of the mixing unit; b) Defining a diameter DLNP of the nanocarriers and / or nanoformulations, a total feed volume flow, and / or the total outlet volume flow; c) Feeding the at least two substances to the mixing chamber; d) Providing by a human machine interface (HMI) and / or the control unit at least the propelling speed (U) according to the function (I) to the mixing wheel; and e) Producing the intended amount of nanocarriers and / or nanoformulations.

[0046] Optionally, f) nanocarriers and / or nanoformulations (mixture) produced might be collected in a collection vessel. As a preferred version or the process, the order a) to e) is followed.

[0047] The term / s “characteristic line” or “set of characteristic lines” are named jointly herein “characteristic data”, meaning digital data usable by a computer, software program, data processor etc. As a simple, alternative solution of the process, the characteristic data might be stored and provided as a list of discrete data, e.g. in form of a look-up-list / sheet, which can be used manually by a human operator, e.g. by tipping in and / or selection this data via HMI as control data for the control unit of the mixing unit.

[0048] According to another preferred embodiment of the process, data any related value, such as but not limited to the substance inherent coefficient (krvi) , the substance inherent intercept (LM) in a data storage device.

[0049] According to another preferred embodiment of the process, providing the characteristic data and / or any related value to a control unit equipped to control the mixing unit, the first feed pump and / or the at least second feed pump.

[0050] Thus, depending on the respective particle diameter DLNP selected by a user via an input device such a computer system (CS), an Enterprise Resource Planning System (ERP) and / or an Manufacturing Execution System (MES) working on a server, computer and / or any kind of computer system / network (CSN), the related feed flow and thus volume of product in a defined range can be automatically and / or semiautomatically be adjusted.

[0051] Analogously, depending on the intended feed flow and thus volume of product, a user via an input device such a computer system (CS), an Enterprise Resource Planning System (ERP) and / or a Manufacturing Execution System (MES) working on a server and / or any kind of computer system network (CSN), the related particle diameter DLNP of the product in a defined range can be automatically and / or semiautomatically be adjusted.

[0052] Therein “computer” shall mean any kind of data processing machine by using at least one software product, such as but not limited to a (personal) computer, notebook, tablet computer, computer network, computer system, microprocessor (pP) and shall be understood without limitation. Thus, the terms “computer system (CS)” or “compute system network (CSN)” can be understood synonymously to “computer”.

[0053] According to another preferred embodiment of the process, the first reactant is a mixture contains a precursor to the nanocarrier and a dispersion medium, and the second reactant is a mixture comprising at least one active ingredient, such as, but not limited to DNA; RNA, and preferably containing at least a buffer.

[0054] According to another preferred embodiment of the process, the first reactant contains a precursor to the nanocarrier in an organic phase, preferably an alcoholic phase, wherein the precursor is a LNP lipid selected from the group consisting of a) 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA) or dilinoleylmethyl-4-dimethylaminobutyrate (Dlin-MC3-DMA); and b) cholesterol, 1 ,2-Dimyristoyl-rac-glycero-3-methoxypolyoxyethylene (PEG-DMG), and 1 ,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0055] Due to an advantageous solution, the alcoholic phase may comprise ethanol or is basically formed of ethanol.

[0056] According to another preferred embodiment of the process, the first reactant contains a precursor to the nanocarrier in an organic phase, preferably an alcoholic phase, wherein the precursor is a natural or artificial lipid, and / or a mixture of cholesterol and phosphatidylcholine, in particular a phosphatidylcholine obtained from soy, from sunflower or from egg.

[0057] Due to an advantageous solution, the alcoholic phase may comprise ethanol or is basically formed of ethanol. According to an alternative solution, the alcohol is a diol, such as, but not limited to glycerol.

[0058] According to another preferred embodiment of the process, the first reactant contains a precursor to the nanocarrier in a liquid organic phase, wherein the precursor is a polymer, preferably the polymer is selected from the group of a non- pegylated Poly(D,L-lactide-co-glycolide), and a pegylated Poly(D,L-lactide-co-glycolide).

[0059] The liquid organic phase containing a polymer as precursor to the nanocarrier, is preferably containing or made of acetone, or dimethyl sulfoxide (DMSO).

[0060] In the given context, "Pegylated" means the process of attaching polyethylene glycol (PEG) molecules to Poly(D,L-lactide-co-glycolide). This modification can enhance the solubility, stability, and bioavailability of the nanocarrier or nanoformulation, as well as reduce immunogenicity and clearance from the body. Pegylation is commonly used in biopharmaceuticals to improve their therapeutic efficacy and duration of action. According to another preferred embodiment of the process, the first reactant contains as precursor to the nanocarrier a LNP lipid in an organic dispersion medium, and the second reactant contains a buffer, wherein the buffer is selected from the group consisting of acetate, and citrate.

[0061] The buffer may have a pH value from pH 3.5 to 4.5, preferred of pH 3.8 to 4.2, and most preferred of pH 4.0. According to an advantageous solution, the alcoholic phase may comprise ethanol or is basically formed of ethanol.

[0062] According to another preferred embodiment of the process, the first reactant contains as precursor to the nanocarrier a natural or artificial lipid, and / or a mixture of cholesterol and phosphatidylcholine in an organic dispersion medium, and the second reactant contains a buffer, wherein the buffer is selected from the group consisting of ammonium sulphate, and 2-(4-(2-hydroxyethyl)-1- piperazinyl)-ethanesulfonic acid (HEPES). The organic dispersion medium may be in a preferred solution an alcoholic dispersion, most preferred an ethanol.

[0063] According to another preferred embodiment of the process, the first reactant contains as precursor to the nanocarrier a polymer in an organic dispersion medium and the second reactant contains a buffer, wherein the buffer is polyvinyl alcohol.

[0064] The buffer can be preferably present in an aqueous solution having 1 to 5 wt.% polyvinyl alcohol in H2O, preferred 1 .5 to 2.5 wt.%, most preferred 1 .8 to 2 wt.%.

[0065] According to another preferred embodiment of the process, releasing the mixture via the inner outlet orifice into a liquid reservoir. The reservoir might be any kind of reservoir, such as but not limited to a vessel, tank and / or tube.

[0066] Products of this inventive process are micro carriers (MC) with a particle range from 1 nm to 10000 nm and / or micro formulations (MF) based thereon. Preferably, the particle size ranges from 1 nm to 100 nm or from 20 nm to 99 nm.

[0067] The preferred embodiments of inventive process for preparing microcarriers and / or microformulations are analogous to preferred embodiments of inventive process for preparing nano carriers (NC) and / or nano formulations (NF).

[0068] The processes according to the invention shall now be elucidated with reference to exemplary embodiments. To this end:

[0069] Fig. 1 : shows an embodiment of the process line related to the process;

[0070] Fig. 2: shows in two vertical sectional views details of a first embodiment of the micro mixer;

[0071] Fig. 3: shows an alternative embodiment of the micro mixer and Fig. 4: shows a graph composed by a number / set of characteristic lines.

[0072] Figure 1 shows an example of the inventive process and the related process line 100. The process line 100 for producing nanocarriers and / or nanoformulations as product having an average diameter of 20 nm to 300 nm, comprises a micro mixier 200, a first feed pump 120 connected to a first vessel 102, a second feed pump 122 connected to a second vessel 104, a control unit 130 connected via data and / or power line 132 with at least one of the feed pumps 120, 122 and / or the micro mixer 200. Both feed pumps 120, 122 are downstream connected via a respective feed line 110, 112 with the micro mixer 200 and the product generated within the micro mixer 200 is released via line 114 to a product vessel 106.

[0073] First vessel 102 contains reactantl (first reactant) and second vessel 104 contains reactant2 (second reactant). The preparation, such as, but not limited to preparing, mixing, and / or pretreatment of first and / or second reactant can be necessary, but is neither shown nor discussed herein. The reactantl is fed via line 110 and pump 120 to the micro mixer 200, and analogously reactant2 is fed via line 112 and pump 122 to the micro mixer 200.

[0074] The micro mixer 200 comprises an inner mixing chamber 220, wherein the mixing chamber 220 having a cylindrical shape enclosed by a circumferential side wall 222, a feed plate 224 and an outlet plate 226. The mixing chamber 220 has a central, longitudinal axis AC, a diameter De and a height H in the direction of the longitudinal axis AC. A feed orifice 202 is provided in the feed plate 224 and an outlet orifice 204 is provided in the outlet plate 226, shown more in detail in figure 2. In the mixing chamber 220 a mixing wheel 210 is placed, rotatably mounted on a drive shaft 214 having a longitudinal axis AW which is oriented parallel to the longitudinal axis AC of the mixing chamber 220. As shown in more details in figure 2, the longitudinal axis AC and AW are oriented parallel eccentrically to each other. Thus, the mixing wheel 210 when activated is eccentrically propelling within the mixing chamber 220.

[0075] Via line 116 product is released form product vessel 106. The micro mixer 200 further comprises an inner feed channel 206 at the side of the feed plate 224 and an outlet channel 208 at the opposite end, i.e. at the side of the outlet plate 226, wherein the feed lines 110, 112 are connected to the feed orifice 202 via the single feed channel 206 and the product is released through the outlet orifice 204 in the outlet plate 204 into the outlet channel 208 an downstream into the line 114.

[0076] As an option, the feed lines 110, 112 are in thermal connection with an indirect heat exchanger 124, with might be controlled by the control unit 134. The needed motors or drives of the feed pumps 120, 122 or of the micro mixer 200 are not shown, as other ordinary equipment, such as but not limited to sensors, power lines, actors, mounting devices etc. In Figure 2, the inner of the mixing chamber 200 of the micro mixer 200 is shown. Partial view I shows the inner side of the feed plate 224 having a feed orifice 202 at the left side. On the right side the outlet orifice 204 located in the opposite outlet plate 226 is indicated be the dotted line. The feed orifice 202 is connected directly or via an inner channel (not shown in figure 2) with the feed lines 110, 112 and the outlet orifice 204 is connect directly or via an inner channel (not shown in figure 2) with the (outlet) line 114. The orifices 202, 204 do have identical shapes, formed as ring segments mirrored to the vertical dot-and-dash-line 228 (mirror line). The second dot-and-dash-line 230 (cross line 230) is perpendicular to the mirror line 228 and crossing at the centre point of the of the mixing chamber 220. In the embodiment according to figure 2 the ring segments have a smaller opening width in radial direction at the lower end and a broader opening width in radial direction at the upper end. The orifices 202, 204 are connected and correlates to peripherally orientated channel 206, 208 or related channel segments thereof.

[0077] The mixing wheel 210 is formed as a star wheel and comprising a number of outer protrusions 216 and being mounted to drive shaft 214. According to the shown embodiment, the mixing wheel 210 comprises six protrusions 216.

[0078] The direction of the rotation R of the mixing wheel 210 is clockwise indicated by the arrow, wherein the mixing wheel 210 is propelling round the axis Aw being eccentrically (length C, also eccentricity C) to the central axis Ac of the cylindrical mixing chamber 220 and / or the plates 224, 226. The clockwise rotation of the mixing wheel correlates with the asymmetric shape of the feed orifice 202 and outlet orifice 204, and connected channels 206, 208 such, that the smaller opening width (high pressure side) at the lower end of the feed orifice 202 is traversed first and incoming fluid is directed to the broader opening width (low pressure side) at the upper end. Subsequently the fluids are directed to the outlet orifice 204 from the broader end (low pressure side) to the smaller end (high pressure side) of the outlet orifice 204.

[0079] The propelling mixing wheel 210 covers a wheel area 212 with the diameter Dw, shown in the partial II as circle with vertical hatching, wherein an outer ring volume 218, shown in partial view II as area with horizontal hatching, wherein the fluids flow. The wheel area 212 multiplied with the hight H of the mixing chamber 220 defines the dynamic wheel volume 213. The mixing chamber 218 comprises the outer ring volume 218 and the dynamic wheel volume 213. The wheel area 212 having a smallest clearance B between one rotating protrusion 216 and the side wall 222 in radial direction and the largest clearance A on the opposite side along the dot-and-dashed mirror line 228, wherein the axis Ac and axis Aw are perpendicularly cutting this vertical line, and also the plates 224, 226. Thus, a partial volume of the fed fluids via feed orifice 202 is not directed outwards through outlet orifice 204 but is forced in the (lower) outer ring volume 218 to flow through the smallest clearance B back to the feed orifice 202. The clearance B is factor 0.005 to 0.125 of the diameter De of the mixing chamber 220, e.g. with diameter De of 3 mm, B is between 15 pm to 400 pm. Not intending to be bound by any theory we believe, beside the inner turbulence in the entire ring volume 218, this inner recycling stream of the fluids through the smallest clearance B in the square size of view micro meter (pm) leads to the surprising effect of predictive particle size due to the propelling speed of the mixing wheel 210.

[0080] It is understood by a skilled person, due to the inner pressure flow, the micro mixer 200 might have any orientation relative to gravity, meaning the flow direction might be vertically, up-side-down, horizontal and / or in any tilted way.

[0081] The embodiment of the micro mixer 200 shown in Figure 3 differs from the one of Figure 2, by a feed orifice 202 and the outlet orifice 204 located in the same (feed) plate 202, and wherein the opposite (outlet) plate 204 preferably has no orifice and / or is not in fluid connection to any line. Figure 4 is discussed in detail together with the experiments.

[0082] Generally spoken, the process and any variation and / or embodiments thereof can preferably be performed with a process line 100 and / or the micro mixer 200 as shown in the figures 1 to 3.

[0083] Experiments

[0084] The micro mixer 200 used for the experiments is based on a microannular gear pump TYP of HNP- M. At the TYP of HNPM microannular gear pump, an outer ring room 218 was provided by removing the external gear to allow certain quantity of contained fluid to circulate and / or at least once flow back inside the mixing chamber 220 from the outlet orifice 204 to the feed orifice 202.

[0085] The micro mixer 200 as used for the experiments was built as shown in figure 2 or 3, wherein both orifices 202, 204 are located on the same (feed) plate 224.

[0086] The following dimensions of the micro mixer 200 were given:

[0087] Clearance B: app. 450 pm

[0088] Clearance C (largest) app. 1200 pm

[0089] Eccentricity C: app. 300 pm

[0090] Diameter of mixing chamber De: 3 mm Diameter of mixing wheel Dw: 1 .5 mm Height of mixing chamber H: 1 .25 mm

[0091] The micro mixer 200 used was a dynaMix-29-1-hs-fb from HNP Mikrosysteme GmbH, which based on a modified micro-annular gear pump mzr-2521X1 of HNP Mikrosysteme GmbH, whereby the outer gear ring was removed, and the peripheral inner ring room has the smallest clearance of app. 450 pm in the lower centre between the inner feed and outlet orifices 202, 204 as best shown in figure 2 or figure 3. The outer ring room 224 with former app. 3.4 mm diameter was limited in its diameter by a foil ring having app. 200 pm thickness, thus limiting the diameter down to app. 3.0 mm. As feed pumps 120, 122 two Harvard Instrument syringe pumps are used.

[0092] The term “app.” meaning “approximately” shall indicate the abbreviations by tolerances of any kind, such as but not limited to manufacturing tolerances, measuring tolerances.

[0093] The mixing intensity was directly controlled via the propelling speed. The propelling speed was set manually in the HMI of the control unit 130 of the micro mixer 200.

[0094] "Particle size”, “PDI” (Polydispersity Index), a measure of the distribution of molecular mass in the respective sample, was calculated as weight average molecular weight divided by the number average molecular weight.

[0095] “Zav” (averaged particle size) were determined according to former ISO 13321 now DIN ISO 22412:2017 and omni trust software according to CFR Part 1 1 , by using the equipment of Zetasizer Ultra with Malvern Panalyticals Compliance Solution from Malvern Panalytical Ltd.

[0096] PDI and Zav were evaluated according to DIN 66161 of 2010 and DIN ISO 9276-2.

[0097] “Encapsulation efficiency” (EE) was determined according to pharmaceutical method, as e.g. described in Pharm Res. 18, p. 878-885 (Jiang W, Schwendemann SP. 2001 . Stabilization and controlled release of bovine serum albumin encapsulated in poly(D, L-lactide) and poly(ethylene glycol) microsphere blends. Pharm Res. 18: 878-885).

[0098] The following abbreviations are used:

[0099] Dlin-MC3-DMA: dilinoleylmethyl-4-dimethylaminobutyrate

[0100] DODMA: 1 .2-dioleyloxy-3-dimethylaminopropane

[0101] DSPC: 1 .2-Distearoyl-sn-glycero-3-phosphocholine

[0102] PEG-DMG: 1 .2-Dimyristoyl-rac-glycero-3-methoxypolyoxyethylene, also named Polyethylenglycol dimethacrylate

[0103] Poly A: polyadenosine monophosphate

[0104] Experiment 1 :

[0105] The experiments had the following process parameters:

[0106] Reactantl : Dlin-MC3-DMA / DSPC / cholesterol / PEG-DMG (50 / 10 / 38.5 / 1.5 mol%) as ethanolic phase at 15 mMol total lipids, e.g. in accordance with ONPATTRO® (Fa. Alnylam Pharmaceuticals) formulation.

[0107] Reactant2: Acetic acid buffer 50 mMol, pH 4.0, Poly A (Cytiva) (Fa. Sigma-Aldrich), as aqueous phase. Ratio (N / P) of 6:1 (molar ratio of positive charged amine functions from ionizable lipid to negative charged phosphate group from Poly A (polyadenosin monophosphate) Mixing at a fix ratio of 3:1 (aqueous: organic)

[0108] Flow rate aqueous phase from 3 ml / min to 30 ml / min at given mixing ratio with organic phase. Propelling speed of the mixing wheel 210 from as given in figure 4 from 1000 to 12000 rpm Temperature of reactants were at ambient temperature.

[0109] As shown in figure 4, surprisingly it was found that there was a reproducible and predictable dependency between particle size, propelling speed of the mixing wheel 210 and the total reactant flow into the mixing chamber of the micromixer and a set of characteristic lines can be generated for each set of reactants, as shown in figure 4 for the reactants according experiment 1 . The fields F1 to F7 bordered by the dashed lines indicate the respective particle sizes as listed in the explanation on the right. Thus, an intended increase of NLP output of a defined particle size can be calculated within given limits as a function of reactant flow rate and propelling speed, or an intended particle size can be calculated within given limits as a function of reactant flow rate and propelling speed of the mixing wheel.

[0110] As standard in the technical field in question, purification of the particles is done by dialysis processes and / or storage of the particles takes place in a frozen stage. Thus, the behavior of the particles produced with the micro mixer were evaluated, and the results are presented in table 2 (experiment 1) and table 3 (experiment 2).

[0111] The results of experiment 1 following the dialysis treatment of the product are shown in table 2, where in the dialysis conditions were as follows:

[0112] Cassette: Slide-A-Lyzer™, 10 K MWCO (Fa. ThermoFisher Science), 3 ml Buffer: Phosphate Buffer Saline pH 7.4 with 5% sucrose Duration: 180 min at continuous stirring Buffer exchange: 60 min, 120 min Tabel 2: Particle size and size distribution results before and following dialysis

[0113] There was an expected increase in the diameter (Zav) 29.8 % at particles produced at 3000 rpm and 53.5 % for particles produced at 6000 rpm. However, this increase is at an expected level and complies to similar effects in particle stabilization with other manufacturing methods (e.g. impingement jet mixing). The Polydispersity Index (PDI), a measure of the distribution of molecular mass in the respective sample, was calculated as weight average molecular weight divided by the number average molecular weight. Also, the influence and deviation following the dialysis is within tolerable ranges.

[0114] Experiment 2:

[0115] The experiments had the following process parameters:

[0116] Reactantl : Dlin-MC3-DMA / DSPC / cholesterol / PEG-DMG (50 / 10 / 38.5 / 1.5 mol%) as ethanolic phase at 15 mMol total lipids, e.g. in accordance to ONPATTRO® (Fa. Alnylam Pharmaceuticals) formulation.

[0117] Reactant2: Acetic acid buffer 50 mMol, pH 4.0, CleanCap Flue (Fa. Tri Link Bio Technologies) mRNA, as aqueous phase.

[0118] Ratio (N / P) of reactantl to reactant2 of 6:1.

[0119] Mixing at a fix ratio of 3:1 (aqueous: organic).

[0120] Flow rate 9:3, i.e. aqueous phase from 9 ml / min (aqueous phase) and 3 ml / min (organic phase). Propelling speed of the mixing wheel 3000 rpm.

[0121] Temperature of reactants were at ambient temperature.

[0122] The results of experiment 2 following the dialysis treatment of the product, are shown in table 3, where in the dialysis conditions were identical to experiment 1 . The sample named “After Dialysis and freeze storage” means storage at minus 80°C and freeze-thaw-cycle.

[0123] Tabel 3: Particle size, size distribution and encapsulation efficiency results before and following dialysis, and following dialysis and one freeze thaw cycle. There was an expected increase in the diameter (Zav) 28.24 % at particles produced at 3000 rpm; which was also at an acceptable level. Also, the increase of the PDI of 21.63 % through the influence of the dialysis is within tolerable ranges.

[0124] As very positive effect, it was observed that the encapsulation efficiency remains at highest level after dialysis as well as after freezing followed by thawing (minus 6.3 %). Encapsulation efficiency was evaluated based on Nucleic Acid Quantitation using a Quant-IT RiboGreen assay-kit (Thermo- Scientific). The read out was performed by using an Infinite 200 Pro (Tecan).

[0125] Finally, it is now possible generating for each combination of reactants as stated herein as at least one characteristic line or a set of characteristic lines as shown in figure 4 and / or providing it as control data in the related control unit and / or data storages (Generation phase / step). The data storage might be part of the control unit or connected to the control unit of the micromixer to make use of the saved data when needed (Control step or production phase).

[0126] The term “characteristic lines” and / or “set of characteristic line” shall mean any mathematical equation, mathematical dependency, function and / or lock-up list of defined control data usable as digital data for the control of the micromixer and / or at least temporary storage of said data.

[0127] Experiment 3:

[0128] Reactantl : Dlin-MC3-DMA / DSPC / cholesterol / PEG-DMG (50 / 10 / 38.5 / 1.50 mol%) as ethanolic phase (in accordance to ONPATTRO® (Fa. Alnylam Pharmaceuticals) formulation) at 15 mMol total lipids

[0129] Reactant2: Acetic acid buffer 50 mMol, pH 4.0, Poly A (Cytiva) (Fa. Sigma-Aldrich), as aqueous phase.

[0130] Ratio (N / P) of 6:1. Results are shown in Table 4.

[0131] Table 4: Results of experiment 3 Experiment 4:

[0132] The model formulations of experiment 4 consist of (reactantl) an ethanolic lipid mixture containing DODMA, DSPC, Cholesterol and PEG-DMG at 50 / 10 / 38.5 / 1.5 mol%. As a suitable surrogate (reactant2) for mRNA a polyadenylic acid, namely polyadenosine monophosphate (Poly A, Cytiva) was used in citric buffer pH 4.0. The N / P ratio of the final product was 3:1 . Results are shown in Table 5.

[0133] Table 5: Results of experiment 4 As by the experiments 1 and 2, it could be demonstrated that also in the experiments 3, 4 by the use of reactant as named, that there is a reproducible dependency of the Zav as a function of propelling speed and reactant flow rate.

[0134] List of reference numerals:

[0135] 100 process line

[0136] 102 vessel (reactantl)

[0137] 104 vessel (reactant2)

[0138] 106 vessel (product)

[0139] 110 feed line, first

[0140] 112 feed line, second

[0141] 114 line

[0142] 120 feed pump (pumpl)

[0143] 122 feed pump (pump2)

[0144] 124 heat exchanger

[0145] 130 control unit

[0146] 132 data line

[0147] 200 mixier, micro

[0148] 202 feed orifice, inner

[0149] 204 outlet orifice, inner

[0150] 206 feed channel, inner

[0151] 208 outlet channel, inner

[0152] 210 mixing wheel

[0153] 212 wheel area (dynamic)

[0154] 213 wheel volume (dynamic)

[0155] 214 drive shaft

[0156] 216 extrusion

[0157] 218 ring volume

[0158] 220 mixing chamber

[0159] 222 side wall

[0160] 224 feed plate

[0161] 226 outlet plate

[0162] 228 mirror line

[0163] 230 cross line

[0164] A clearance, biggest (length)

[0165] Ac axis, longitudinal

[0166] Aw axis, longitudinal

[0167] B Clearance, smallest (length)

[0168] C Eccentricity (length)

[0169] De diameter, inner (220)

[0170] Dw diameter, outer (210)

[0171] H height

[0172] R rotation direction

[0173] W ankle

Claims

Claims1. Process in a plant (100) for producing nanocarriers and / or nanoformulations having an average diameter (DLNP) of 20 nm to 300 nm, preferably for producing lipid nanoparticles (LNP), comprising: a) Inserting a liquid first reactant to a mixing unit (200) by a first feed pump (120), b) Inserting at least a liquid second reactant to the mixing unit (200) by a second feed pump (122), wherein the mixing unit (200) extending to a longitudinal axis Ac, comprising- one inner mixing chamber (220) with a diameter (De) and a height (H) in the direction of the longitudinal axis (Ac) comprising- at least one circumferential side wall (222), one feed plate (224) and one outlet plate (226),- one inner feed orifice (202) being connected to the first feed line (110) and second feed line (112) and- at least one inner outlet orifice (204), and wherein the inner feed orifice (202) and the inner outlet orifice (204) are peripherally orientated channel segments, wherein propelling one mixing wheel (210) in one rotation direction (R) with a given rotation speed (v), wherein the mixing wheel (210) having an outer diameter (Dw) mounted on a drive shaft (214) which defines a longitudinal axis (Aw) eccentrically placed inside the mixing chamber (220) and oriented parallel to longitudinal axis (Ac) of the mixer chamber (220), wherein the mixing wheel (210) comprises at least two outer extrusions (216), wherein- the mixing chamber (220) having an inner a ring volume (218) defined by the radially free space between the eccentrically propelling mixing wheel (210) and the side wall (222), characterized in that the smallest clearance (B) between one rotating extrusion (216) and the side wall (222) in radial direction is factor 0.005 to 0.125 of the diameter (De) of the mixing chamber 220), preferably factor 0.005 to 0.1 , and wherein clearance (B) is between 15 pm to 400 pm.

2. Process according to claim 1 , characterized in that the inner feed orifice (202) and the inner outlet orifice (204) are peripherally orientated channel segments, each having a small end portion (S) and one brought end portion (B), and wherein the brought end portion (B) is at least 1 .1 to 3.0 times of the small end portion (S).

3. Process according to the preceding claims, characterized in that the inner feed orifice (202) and the inner outlet orifice (204) are mirrored by a mirror axis (228) toward each other, wherein the small end portions (S) are facing each other in close distance and the brought end portions (B) are facing each other in closer distance.

4. Process according to the preceding claims, characterized in that the inner feed orifice (202) and the inner outlet orifice (204): a) both located on the feed plate (224), b) the inner feed orifices (202) is located on the feed plate (224) and the inner outlet orifice (224) is located on the outlet plate (246), or c) both or at least one orifice is located on the circumferential side wall (222) of the mixing chamber (220).

5. Process according to claim 4, characterized in that the inner feed orifice (202) and the inner outlet orifices (204) are both on the same feed plate (224, 226) or on the circumferential side wall (222), a second pair of peripherally orientated channel segments are located at the opposite side of the mixing chamber (220), i.e. on the face plate (224) or on the opposite side of the side wall (222), being equally oriented towards each other but 90° rotated relative to the first pair of peripherally oriented channels, wherein at least a portion of the first and second reactant or a mixture of it is temporary placed inside the second pair of peripherally oriented channel segments while the mixing wheel (210) is propelling.

6. Process according to the preceding claims, characterized in that the propelling mixing wheel (210) comprises three to ten outer extrusions (216), preferably four to eight extrusions (216), most preferably six extrusions (216).

7. Process according to the preceding claims, characterized in that the void volume inside the mixing chamber (220) between mixing wheel (210) and side wall (222) is 3.5 pl to 8.5 pl, preferably 4.0 pl to 7.5 pl, most preferably 4.5 pl to 4.7 pl.

8. Process according to the preceding claims, characterized in a) Providing characteristic data for a defined mixture of a first reactant and at least one second reactant, i.e. providing on and / or for the control unit of the micromixer; b) Defining a diameter DLNP of the nanocarriers and / or nanoformulations, a total feed volume flow, and / or the total outlet volume flow; c) Feeding the at least two substances to the mixing chamber (220); d) Providing by a Human Machine Interface (HMI) and / or the control unit (130) at least the propelling speed (U) according to the function of V, U to the mixing wheel (210); e) Producing the intended amount of nanocarriers and / or nanoformulations.

9. Process according to claim 8, characterised in storing the at least one characteristic data any related value, such as but not limited to the substance inherent coefficient (krvi) , the substance inherent intercept (LM) in a data storage device.

10. Process according to any of the claims 8 or 9, characterised in providing the characteristic data and / or any related value to a control unit (130) equipped to control the mixing unit (200), the first feed pump (120) and / or the at least second feed pump (122).

11. Process according to any one of the preceding claims, characterised in that the first reactant is a mixture contains a precursor to the nanocarrier and a dispersion medium, and the second reactant is a mixture comprising at least one active ingredient, such as, but not limited to DNA; RNA, and preferably containing at least a buffer.

12. Process according to according to claim 11 , characterised in that the first reactant contains a precursor to the nanocarrier in an organic phase, preferably an alcoholic phase, wherein the precursor is a LNP lipid selected from the group consisting of a) 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA) or dilinoleylmethyl-4-dimethylamino- butyrate (Dlin-MC3-DMA); and b) cholesterol, 1 ,2-Dimyristoyl-rac-glycero-3-methoxypolyoxyethylene (PEG-DMG), and 1 ,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC).

13. Process according to claim 11 , characterised in that the first reactant contains a precursor to the nanocarrier in an organic phase, preferably an alcoholic phase, wherein the precursor is a natural or artificial lipid, and / or a mixture of cholesterol and phosphatidylcholine, in particular a phosphatidylcholine obtained from soy, from sunflower or from egg.

14. Process according to claim 11 , characterised in that the first reactant contains a precursor to the nanocarrier in a liquid organic phase containing acetone, or dimethyl sulfoxide (DMSO), wherein the precursor is a polymer, preferably the polymer is selected from the group of Poly(D,L-lactide-co-glycolide), and pegylated Poly(D,L-lactide-co-glycolide).

15. Process according to claims12, characterised in that the first reactant contains an organic dispersion medium, preferably ethanol, and the second reactant contains a buffer, wherein the buffer is selected from the group consisting of acetate, and citrate.

16. Process according to claim 13, characterised in that the first reactant contains an organic dispersion medium, preferred an alcoholic dispersion medium, and the second reactant contains a buffer, wherein the buffer is selected from the group consisting of ammonium sulphate, and 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid (HEPES).

17. Process according to any one of the preceding claims 14, characterised in that in that the first reactant contains an organic dispersion medium and the second reactant contains a buffer, wherein the buffer is polyvinyl alcohol.

18. Process according to any one of the preceding claims 1 to 11 , characterised in that the first reactant contains two precursors to a nanocarrier, and wherein the two precursors to the nanocarrier are lipoid E PC and cholesterol HP.

19. Process according to any one of the preceding claims, characterised in releasing the mixture via the inner outlet orifice (204) into a liquid reservoir or vessel (106).

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