FIELD OF THE DISCLOSURE

The disclosure presented herein provides methods for validating the functioning of an apparatus that produces nanoparticles by precipitating a substance from a supersaturated solution. The validation methods are based on the use of reference substances, such as itraconazole. When these reference substances are precipitated in the apparatus, the size of the nanoparticles formed is affected by a parameter of the apparatus.

BACKGROUND

Nanoparticles have aroused attention due to their potential application for safe and effective drug delivery. These nanoparticles have been employed to improve the efficacy, safety, physicochemical properties, pharmacokinetics, and pharmacodynamics of pharmaceutical substances. Among others, nanoparticle formulations can improve the solubility and bioavailability of the active ingredient, increase in the absorption of the active substance, stabilize the active ingredients, target the active ingredient to specific cells and organs, and control release rate of the active ingredient To date, various nanodrug systems have been developed for different applications, which include dendrimers, nanocrystals, nanoemulsions, liposomes, solid lipid nanoparticles, micelles, and polymeric nanoparticles.

Solvent/nonsolvent precipitation methods were found useful for the production of nanoparticles, in particular lipid-based nanoparticles. Solvent/nonsolvent precipitation means that a substance is dissolved in a solvent and collides as a liquid jet with a second liquid jet that contains a nonsolvent, whereby the dissolved substance is precipitated. Microjet reactor technology can be used for automated, continuous nanoparticle synthesis by solvent/nonsolvent precipitation methods. Microjet reactor technology creates a turbulent mixing zone of solvent and nonsolvent and is therefore particularly suitable for the precipitation of particles in the nanometer range.

EP 2395978 B1 describes the solvent/nonsolvent precipitation in the presence of surface- active molecules using a microjet reactor according to EP 1 165 224 Bl. Such a microjet reactor has at least two nozzles located opposite one another, each with an associated pump and feed line for spraying a liquid medium at a common collision point in a reactor chamber enclosed by a reactor housing. Another opening is provided in the reactor housing through which a gas, an evaporating liquid, a cooling liquid, or a cooling gas can be passed to maintain the gas atmosphere in the reactor chamber or for cooling. A further opening is provided for removing the resulting products and excess gas from the reactor chamber. If a solvent/nonsolvent precipitation is carried out in such a microjet reactor a dispersion of precipitated particles is obtained.

It is critical to provide a simple and reliable validation method for verifying the accuracy of the critical parameters of the manufacturing reaction. The validation method used to date consists in precipitating hydroxypropyl methylcellulose phthalate (5 mg/ml HPMCP HP-50 dissolved in a mixture of 85% ethanol and 15% water] from a solvent stream, by mixing it with an aqueous stream. This produces nanoparticles of the substance in the range of 170 nm to 200 nm, with a broad and multimodal distribution. However, the size of the particles generated in this way does not depend on the reaction conditions. In particular the size of the particles does not depend on volume ratio of the ethanolic stream and the water stream injected to the reactor.

Thus, it is not possible to test experimentally in a fast and easy-to-perform precipitation procedure whether the target process parameters correspond to the actual process parameters. It would be desirable, for example, if the volume ratio of solvent stream to antisolvent stream could be experimentally checked in a simple and fast procedure.

Thus, there is a need for a suitable and improved method for validating reactors. The present disclosure provides a validation method that leads to reliable results with respect to the operability of a system for the production of nanoparticles by targeted precipitation from supersaturated solutions.

SUMMARY OF THE DISCLOSURE

In one aspect, disclosed herein is a method for validating the functioning of an apparatus for producing nanoparticles, said apparatus comprising a reactor having a reaction chamber, the method comprising the steps of: a] injecting a first volume of a first stream and a second volume of a second stream into the reaction chamber of the reactor, wherein the first and second streams are injected in a predetermined volume ratio; wherein the first stream comprises an organic solvent and a reference substance, and the second stream comprises water and a surfactant; wherein, the frontal collision of the first and the second streams induces the reference substance and the surfactant to precipitate as nanoparticles; and wherein the size of the nanoparticles obtained by precipitating the reference substance is affected by the volume ratio of the first and the second streams; b] determining the particle size of the precipitated nanoparticles of step (a); c] repeating steps (a] and (b] one or more times, using different predetermined volume ratios; d] providing a function that associates the expected size of the precipitated nanoparticles with the volume ratio of the first and the second streams; e] comparing the observed size of the precipitated nanoparticles of step (b] with the expected nanoparticle size according to the function of step (d); wherein a difference above a predetermined threshold between the observed size and the expected size is indicative of improper functioning of the apparatus.

In some related aspects, the reference substance comprises itraconazole.

In some related aspects, the predetermined threshold is 5%, 10%, 20%, 30%, 40%, or 50%. In some related aspects, validating the functioning of an apparatus comprises validating that the ratio of the volumes it injects to the reactor corresponds to the desired volume ratio.

In some related aspects, the organic solvent comprises acetone. In some related aspects, the surfactant comprises hydroxypropyl methylcellulose acetate succinate (HPMCAS] or naphthalenesulfonic acid and formaldehyde. In some related aspects, the predetermined volume ratio of the first and the second streams is in the range from 1:3 to 1:20, respectively. In some related aspects, the expected nanoparticle size is in the range from 160 to 500 nm, , or in the range of 180 to 500 nm, or from 200 to 320 nm.

In some related aspects, the ratio of the hydrodynamic pressure between the first and second streams is in the range from 1:0.5 to 1:2, respectively. In some related aspects, the first and the second streams are injected to the reaction chamber through a first and a second nozzle, respectively. In some related aspects, the diameter of the first nozzle is in the range from 50 to 400 pm, and the diameter of the second nozzle is in the range from 200 to 1200 pm.

In some related aspects, the first stream and the second streams collide frontally in the reaction chamber. In some related aspects, the function comprises any of the functions of table 1.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows the observed sizes of the precipitated nanoparticles as a function of the volume ratio of the first and the second stream. The first stream comprised an acetone solution, and the second stream comprised a demineralized water solution. The concentration of acetone therefore represents the concentration of the first volume in the total volume.

Figure 2 shows the observed polydispersity index (PDI] of the precipitated nanoparticles as a function of volume ratio of the first and the second stream. The first stream comprised an acetone solution, and the second stream comprised a demineralized water solution. The concentration of acetone therefore represents the concentration of the first volume in the total volume.

Figures 3 shows the observed sizes of the precipitated nanoparticles as a function of the volume ratio of the first and the second stream. Five different apparatuses were tested and particle size was measured by a Malvern Nano ZS device. The given volume ratio is based on the second stream.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to a method for validating the functioning of an apparatus for producing nanoparticles, said apparatus comprising a reactor having a reaction chamber, the method comprising the steps of: a] injecting a first volume of a first stream and a second volume of a second stream into the reaction chamber of the reactor, wherein the first and second streams are injected in a predetermined volume ratio; wherein the first stream comprises an organic solvent and a reference substance, and the second stream comprises water and a surfactant; wherein, the frontal collision of the first and the second streams induces the reference substance and the surfactant to precipitate as nanoparticles; and wherein the size of the nanoparticles obtained by precipitating the reference substance is affected by the volume ratio of the first and the second streams; b] determining the particle size of the precipitated nanoparticles of step (a); c] repeating steps (a] and (b] one or more times, using different predetermined volume ratios; d] providing a function that associates the expected size of the precipitated nanoparticles with the volume ratio of the first and the second streams; e] comparing the observed size of the precipitated nanoparticles of step (b] with the expected nanoparticle size according to the function of step (d); wherein a difference above a predetermined threshold between the observed size and the expected size is indicative of improper functioning of the apparatus.

The present subject matter may be understood more readily by reference to the following detailed description, which forms a part of this disclosure. It is to be understood that this disclosure is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

In the present disclosure the singular forms "a," "an," and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. The term "plurality", as used herein, means more than one. When values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In some embodiments, the term "about”, refers to a deviance of between 0.0001-10% from the indicated number or range of numbers. In some embodiments, the term "about”, refers to a deviance of up to 25% from the indicated number or range of numbers. The term "comprises” means encompasses all the elements listed, but may also include additional, unnamed elements, and it may be used interchangeably with the terms "encompasses”, "includes”, or "contains” having all the same qualities and meanings. The term "consisting of means being composed of the recited elements or steps, and it may be used interchangeably with the terms "composed of” having all the same qualities and meanings.

The present disclosure provides methods for validating the proper functioning of an apparatus for producing nanoparticles, said apparatus comprising a reactor. In some embodiments, the apparatus is configured for injecting a first stream through a first nozzle, and a second stream through a second nozzle, into the reaction chamber of the reactor. The first nozzle is located at an angle of about 180° from the second nozzle, and the two streams collide, or impinge, frontally in the reaction chamber. Such frontal collision of two streams is also referred to as jet impingement. As used herein, a stream means a stream of a fluid material, such as a liquid. In some embodiments, the apparatus disclosed comprises an impinging jet reactor as disclosed in EP1165224, EP 1352682, US2017/0361299, and EP2395978.

In some embodiments, the nozzles comprise an opening in the range of 5-900 pm in diameter. The nozzles, which are herein termed also pinholes, are located one opposite to the other, i.e. their pinholes are arranged such as to point at one another at an angle of about 180°. Thus, the injected streams collide frontally, i.e. at about 180° as well, and at high velocities in the mixing chamber of the reactor, which produces an intensive and turbulent mix generating a micro- and/or nanoparticle suspension.

In some embodiments, the first stream comprises an organic solvent. In some embodiments, the second stream comprises an aqueous solvent. In some embodiments, the apparatus disclosed herein comprises a first pump, a second pump, and a reactor. In some embodiments, said first and second pumps inject a first and a second stream, respectively, to a reactor chamber in said reactor. Said first and second streams can be injected in a raised pressure. In some embodiments, the apparatus comprises the pipelines connecting said first and second pump to said reactor. A skilled artisan would appreciate that the apparatus may further comprise additional pieces needed for the proper functioning of the reactor, such as valves, and/or measuring devices for measuring pressure, flow, weight, or any other desired parameter. The first stream and the second stream are pumped by the apparatus into a reaction chamber where the two streams collide frontally. The reaction chamber, or reactor chamber, as used in parallel herein, provides a cavity for the two streams to form a highly turbulent mixing zone.

A skilled artisan would appreciate that validating the functioning of an apparatus refers to checking its accurate functioning. In some embodiments, validating the functioning of an apparatus comprises validating the accuracy of all parameters relevant for the production of the desired product, e.g. nanoparticles comprising the desired features. In some embodiments, validating an apparatus comprises validating that the volume of the first stream and the volume of the second stream, are injected into the reactor chamber in the desired ratio.

In some embodiments, the methods disclosed herein validate that the first and/or the second stream is/are injected in the desired volume. In some embodiments, the methods disclosed herein validate that the first and/or the second streams are injected at the desired pressure(s). In some embodiments, the methods disclosed herein validate that the first and/or the second stream is injected in the desired velocity.

In some embodiments, the methods disclosed herein can be used for predicting that the nanoparticles produced by the apparatus will be of a desired size. In some embodiments, the methods disclosed herein validate that the nanoparticles produced by the apparatus will be of a desired polydispersity index (PDI]

The present disclosure teaches the use of certain reference substances for validating the functioning of an apparatus for producing nanoparticles, said apparatus comprising a reactor. In some embodiments, the reactor produces nanoparticles by precipitating substances from supersaturated solutions. Such a solvent/antisolvent precipitation are known in the art, for example, from EP2550092 and EP2395978B1. In some embodiments, the apparatus that can be validated by the methods disclosed herein comprises any of the reactors disclosed in EP1165224B1, EP1165224A2 or WO2018/234217A1. In some embodiments, an apparatus injects a first and a second stream that are mixed, or collide with each other, in a reaction chamber.

For most substances, the ratio of the volumes of the first and second streams do not have a noticeable effect on the size of the precipitated nanoparticles. I.e., a small change to the volume ratio of the first to second stream does not have a noticeable effect The present disclosure teaches that, for certain substances, the volume ratio of the first and the second stream affect the size of the precipitated nanoparticles. Thus, the reference substances disclosed herein, can be any substance characterized by the fact that when precipitated in a reactor, the size of the nanoparticles formed is precisely and reproducibly affected by a parameter of the reactor.

In some embodiments, the size of the nanoparticles is affected by the ratio of the volumes of the first and the second streams. In some embodiments, the reference substance is characterized by the fact that the PDI of the precipitated nanoparticles is low. In some embodiments, the reference substance is not water-soluble or is only slightly water-soluble. In some embodiments, the reference substance is a pharmaceutical excipient or an active pharmaceutical ingredient

A skilled artisan would appreciate that any substance, for which the size of its precipitated nanoparticles is affected by at least one of the reaction conditions, can be used as the reference substance in the validation methods disclosed herein. For example, a substance for which the size of its precipitated nanoparticles is affected by the volume ratio of the first and the second stream, can be used as the reference substance in the methods disclosed herein.

In some embodiments, the reference substance is itraconazole. This substance has proven to be suitable, since the particle size of precipitated particles can be adjusted very precisely by the volume ratio of first to second stream and the precipitated particles have a small size distribution. Itraconazole (ITZ, C35H38CI2N8O4, MW 705.6 g/mol] is an active ingredient from the triazole antifungal group for the treatment of fungal infections with various pathogens. It is effective against yeasts, dermatophytes and molds, among others. The effects are based on inhibition of ergosterol biosynthesis in the fungi. ITZ was approved as early as 1991, and the substance is readily and inexpensively available. ITZ is a white powder that is practically insoluble in water and dissolves in organic solvents such as acetone, dimethylformamide and tetrahydrofuran.

In some embodiments, the reference substance is furosemide. Furosemide was found to be suitable for the method of the invention since its the particle size of precipitated particles can be adjusted very precisely by the volume ratio of first to second stream and the precipitated particles have a small size distribution. Furosemide or frusemide (C12H11CIN2O5S, MW 330.74 g/mol], also known under the brand name Lasix among others, is a loop diuretic medication used to treat fluid build-up due to heart failure, liver scarring, or kidney disease. It may also be used for the treatment of high blood pressure.

In some embodiments, the methods disclosed herein comprise providing a function that associates the size of the precipitated nanoparticles with the volume ratio of the injected streams. Such a function can be represented by any tool suitable for expressing the relation between the size of the precipitated nanoparticles with the volume ratio of the injected streams. The function can comprise a mathematical function, a graph, a table of corresponding values, one or more pairs of corresponding values or any other suitable tool known to skilled artisan. A skilled artisan would appreciate that such a function can be easily obtained for any substance of interest, for example by precipitating the substance in any of the reactors disclosed herein under different conditions, and measuring the obtained sizes. In some embodiments, the function is obtained from an apparatus which functioning is independently validated by other means, e.g. by using scales, flow meters, or any other device for measuring the volume of the injected streams.

In some embodiments, the function provided under step d] of the method of the invention is established by determining the size of the precipitated nanoparticles of the reference substance at different volume ratios of the first and the second streams under defined conditions. These conditions can relate to other parameters that can be set at the apparatus, or when carrying out the method, like e.g. nozzle size, hydrodynamic pressure, the kind of the reactor, or the composition of first or the second stream. In a preferred embodiment, the establishing of the function is carried out under the same conditions as the method for validation itself. In other words, the function provided under step d] of the method of the invention was established under the same conditions as the method for validating the functioning of an apparatus. In a preferred embodiment, the establishment of the function is carried out with an apparatus that can provide standardized conditions.

Thus, a skilled artisan can find whether a substance can or cannot be used as a reference substance in the methods disclosed herein. Further, by precipitating a substance under different reaction conditions, for example under different volume ratios, an artisan can construct a function that associates the size of the precipitated nanoparticles of said substance with the volume ratios of the streams. A skilled artisan would appreciate that the apparatus disclosed herein injects a first and a second volume of a fluid into the reactor chamber during a period of time. Therefore, the volume ratio can be interpreted as the ratio of the volume injected per time unit, or the flow rate ratio (FRR] Thus, the terms "volume ratio” and "flow rate ratio” are used herein interchangeably. Therefore, in some embodiments, disclosed herein are methods for validating the flow rate ratio in an apparatus.

In some embodiments, the function for itraconazole is described in Table 1.

Table 1 - Function associating the size and PDI of precipitated itraconazole nanoparticles and volume ratios.

As disclosed herein, the ratio of the volumes between the first and the second stream affects the properties of the precipitated nanoparticles. In some embodiments, the volume ratio between the first and the second stream is in the range from 1:4 to 1:13. In some embodiments, the volume ratio between the first and the second stream is in the range from 1:5 to 1:16, form 1:5 to 1:11, from 1:3 to 1:20, from 1:1.5 to 1:4.5, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:1.5 to

1:3, from 1:1.5 to 1:2.5, from 1:1.5 to 1:2. In some embodiments, the volume ratio between the first and the second stream is in the range from 1:3 to 1:20. In some embodiments, the volume ratio between the first and the second stream is about 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, or 1:10. In some embodiments, the precipitated nanoparticles have an average particle size in the range from 1 to 500 nm, from 5 to 400 nm, from 10 to 200 nm, from 20 to 100 nm, from 30 to 90 nm, or from 40 to 80 nm. In some embodiments, a nanoparticle comprises an average particle size below 100 nm, or below 90 nm, or below 80 nm, or below 70 nm. In some embodiments, a nanoparticle comprises an average particle size in the range of 10 to 25, 25 to 50, 50 to 75, 75 to 100, 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 225, 225 to 250, 250 to 275, 275 to 300, 300 to 325, 325 to 350, 350 to 375, 375 to 400, 400 to 425, 425 to 450, 450 to 475, 475 to 500 nm.

In some embodiments, a nanoparticle comprises an average particle size in the range of 160 to 500 nm, or in the range of 180 to 500 nm. In some embodiments, a nanoparticle comprises an average particle size in the range of 200 to 320 nm.

In some embodiments, the precipitated nanoparticles have an average particle size of about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nm. In some embodiments, the precipitated nanoparticles have an average particle size of 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 nm. In some embodiments, a nanoparticle comprises an average particle size of about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nm.

In some embodiments, the nanoparticles of the dispersion have a polydispersity index (PDI] below 0.4, or below 0.3, or below 0.25, or below 0.2, or below 0.1. In some embodiments, the nanoparticles PDI is in the range from 0.01 to 0.025, 0.025 to 0.05, 0.05 to 0.075, 0.075 to 0.1, 0.01 to 0.05, 0.05 to 0.1, 0.1 to 0.15, 0.15 to 0.2, 0.2 to 0.25, or 0.25 to 0.3.

A skilled artisan would appreciate that the terms "raised pressure”, "high pressure”, "overpressure", "hydrodynamic pressure”, or "pressure”, which are used herein interchangeably, refer to any pressure above the atmospheric pressure. Further, as used herein in some embodiments, the pressure of the first and second stream is measured in comparison to the atmospheric pressure. Therefore, a pressure of 0.1 bar is to be understood as 0.1 bar above the atmospheric pressure.

In some embodiments, the pressure of the first and the second streams are similar. In some embodiments, the pressure of the first stream is higher than that of the second stream. In some embodiments, the pressure of the first stream is lower than or equal to that of the second stream. In some embodiments, the pressure of the first stream is lower than about 30 bar, lower than about 25 bar, lower than about 12 bar, or lower than about < 5 bar. In some embodiments, the pressure in the first stream is from about 0.0001 bar to about 30 bar, from about 0.001 bar to about 25 bar, from about 0.01 bar to about 12 bar, or from about 0.01 bar to about 5 bar.

In some embodiments, the pressure of the second stream is lower than about 30 bar, lower than about 25 bar, lower than about 12 bar, or lower than about < 5 bar. In some embodiments, the pressure in the second stream is from about 0.0001 bar to about 30 bar, from about 0.001 bar to about 25 bar, from about 0.01 bar to about 12 bar, or from about 0.01 bar to about 5 bar.

In some embodiments, the pressure of said first or second stream is lower than 0,1 bar. In some embodiments, the pressure of first of second streams is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 bar. In some embodiments, the pressure of first of second streams is about 2, 3, 4, 5, 6, 7, 8, 9, or 10 bar. In some embodiments, the pressure of first of second streams is about 10, 15, 20, 25, or 30 bar. In some embodiments, the pressure of first of second streams is higher than 30 bar.

In some embodiments, the ratio of the hydrodynamic pressures between the first and the second stream is in the range of 1:0.1 to 1:05, 1:0.5 to 1:1, 1:1 to 1:1.5, 1:1.5 to 1:2, 1:2 to 1:5. In some embodiments, the ratio of the hydrodynamic pressures is in the range from 1:0.5 to 1:2.

In some embodiments, the stream pressure is regulated, among others, by the size of the nozzles’ openings. In some embodiments, the size of the first and/or second nozzle opening is smaller than 100 pm in diameter. In some embodiments, the size of a nozzle opening is between 100 and 200 pm radius. In some embodiments, the size of a nozzle opening is between 200 and 300 pm radius. In some embodiments, the size of a nozzle opening is between 300 and 400 pm radius. In some embodiments, the size of the nozzle opening is between 400 and 500 pm radius. In some embodiments, the size of the nozzle opening is between 500 and 600 pm radius. In some embodiments, the size of the nozzle opening is between 600 and 700 pm radius. In some embodiments, the size of the nozzle opening is larger than 700 pm radius.

In some embodiments, the size of the first and/or second nozzle opening is about 100 pm radius. In some embodiments, the size of a nozzle opening is about 200 pm radius. In some embodiments, the size of a nozzle opening is about 300 pm radius. In some embodiments, the size of a nozzle opening is about 400 pm radius. In some embodiments, the size of a nozzle opening is about 500 pm radius. In some embodiments, the radius of the first and second openings are the same. In some embodiments, the radius of the first and the second openings are different, or asymmetric. In some embodiments, the radius of said first opening is 100 pm and the radius of said second opening is 100, 200, 300, 400, or 500 pm. In some embodiments, the radius of the second opening is 200 pm and the radius of said first opening is 100, 200, 300, 400, or 500 pm. In some embodiments, the radius of said second opening is 300 pm and the radius of said first opening is 100, 200, 300, 400, or 500 pm.

In some embodiments, the first stream comprises an organic solvent In some embodiments, the organic solvent is a solvent for the reference substance. A skilled artisan would appreciate that many organic solvents are used in the pharmaceutical industry. Any of them can be used in the methods disclosed herein. In some embodiments, the organic solvent comprises an alcohol, a ketone, a halogenated solvent, an amide, or an ether. In some embodiments, an organic solvent is selected from the group comprising ethanol, ethylene, bromide, butanol, acetone, chloroform, 2-ethylhexanol methylethylketone, ethylene chloride, isobutanol, methylisobutylketone, dichloromethane, isopropanol, methylisopropylketone, tetrachloroethylene methanol, mesityl oxide, carbon tetrachloride, propanol, trichloroethylene, propylene glycol, 1,4-dioxane butyl ether, ethyl ether, dimethylformamide, diisopropyl ether, dimethyl sulfoxide, tetrahydrofuran, tert-butyl methyl ether, hydrocarbons, aromatic hydrocarbons, cyclohexane, toluene, hexane, and xylene.

In some embodiments, the organic solvent is miscible with water. In some embodiments, the organic solvent comprises a mixture of 2 or more organic solvents. In some embodiments, the organic solvent comprises ethanol. In some embodiments, the organic solvent comprises acetone. In some embodiments, the organic solvent is acetone when the reference substance is itraconazole.

In some embodiments, the second stream comprises an aqueous solvent In some embodiments, the aqueous solvent is a non-solvent for the reference substance. A skilled artisan would appreciate that many aqueous solvents are used in the pharmaceutical industry. Any of them can be used in the methods disclosed herein. In some embodiments, the aqueous solvent comprises water. In some embodiments, the aqueous solvent comprises phosphate buffer saline (PBS], or Dulbecco's phosphate-buffered saline (DPBS] In some embodiments, the pH of the aqueous solvent is modified to any desired value.

In some embodiments, the second stream comprises an aqueous solvent and a surfactant In some embodiments, the surfactant facilitates the precipitation of the reference substance into nanoparticles, and allows more accurate control of the nanoparticle size. In some embodiments, the surfactant comprises a polymer of naphthalenesulfonic acid and formaldehyde. In some embodiments, the surfactant comprises hydroxypropyl methylcellulose acetate succinate (HPMCAS).

In some embodiments, the surfactants is selected from the group comprising Pluronic F- 127; D-a-tocopherol polyethylene glycol 1000 succinate (TPGS); polyethylene glycol dimethyl ether 500 (DMPEG); and Tamol NN9401 (polymer of naphthalenesulfonic acid and formaldehyde]

HPMCAS is an excipient for gastroresistant or enteric oral drug formulations, for the formation of solid dispersions, as a matrix-forming carrier, and in some cases to inhibit crystallization of pharmaceutical ingredients. Examples of commercially available HPMCAS in different grades for use according to the invention are AFFINSIOL™ HPMCAS, for example HPMCAS 716G, 912G, and 126G, from company Dow chemical, AquaSolve HPMCAS in different grades from company Ashland, or AQOAT Hypromellose Acetate Succinate from company ShinEtsu.

In some embodiments, the surfactant is provided in the first stream. In some embodiments, the concentration of the surfactant is from 0.01 to 1 wt%. In some embodiments, the concentration of the surfactant is about 0.1 wt%. In some embodiments, 10 to 20 mg/mL, preferably 15 mg/mL HPMC-AS is used as the surfactant

A skilled artisan would appreciate that a "nanoparticle”, also referred to as an ultrafine particle, is usually defined as a particle of matter smaller than about 1000 nm. In a nanoparticle dispersion, there is a variance in the nanoparticles’ particle size, and therefore it is useful to refer to the nanoparticles "average particle size”, as well as to the "polydispersity index” or "PDF’. The term "average particle size", when used herein to describe the size of nanoparticles, refers to the z-average diameter.

A skilled artisan would appreciate that a number of methods for measuring nanoparticle size are available in the art Any of these methods can be applied for measuring the nanoparticle sizes as taught in the present disclosure. In some embodiments, the z-average diameter is measured by Dynamic Light Scattering. Dynamic light scattering (DLS] is a technique in physics commonly known to be used to determine the size distribution profile of small particles in suspension. For the measurement Zetasizer devices from Malvern Panalytical Ltd., e.g. Malvern Zetasizer Advance Range or Zetasizer AT or Zetasizer ZS or ZS90, Anton Paar Litesizer 500, or other suitable devices, like e.g. DLS technology (e.g. Nanophox] from Wyatt Technology Corporation can be used. In some embodiments, the z-average diameter, together with the polydispersity index (PDI], are calculated from the cumulants analysis of the DLS measured intensity autocorrelation function as defined in ISO 13321:1996 and ISO 22412:2017. PDI is a dimensionless estimate of the width of the particle size distribution, scaled from 0 to 1.

In some embodiments, the reference substance is precipitated under different reaction conditions a number of times. In some embodiments, the different reaction conditions comprise different injected volumes of the first stream, the second stream, or both. In some embodiments, the different reaction conditions comprise different volume ratios between the first and the second stream. In some embodiments, the different reaction conditions comprise different hydrodynamic pressure of the first stream, the second stream, or both. In some embodiments, the different reaction conditions comprise different flow velocity of the first stream, the second stream, or both.

In some embodiments, the reference substance is precipitated under predetermined reaction conditions once. In some embodiments, the reference substance is precipitated in at least 2 different reaction conditions. In some embodiments, the reference substance is precipitated under 2, 3, 4, 5, 6, 7, 8, 9, or 10 different reaction conditions.

In some embodiments, the sizes of the precipitated nanoparticles are compared with the expected sizes of the nanoparticles, as predicted by a function that associated between nanoparticle size and reaction conditions. In some embodiments, a difference above a predetermined threshold between the observed size and the expected size is indicative of improper functioning of the apparatus.

In some embodiments, a difference of more than 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% between the observed size and the expected size is indicative of improper functioning of the apparatus. In some embodiments, a difference of more than 10% between the observed size and the expected size is indicative of improper functioning of the apparatus.

In some embodiments, step e] of the method of the invention is repeated one or more times for different predetermined volume ratios. When different predetermined volume ratios are used for the validation, the threshold indicative of the improper functioning of the apparatus is optionally the average of the thresholds for each of the observed differences between the observed size and the expected size. EXAMPLES

The following example is presented in order to more fully illustrate some embodiments of the technology disclosed herein. They should in no way be construed, however, as limiting the scope of the invention.

The apparatus used in these experiments included a first pump and a first nozzle to inject a first stream into a reaction chamber, and a second pump and a second nozzle to inject a second stream into the reaction chamber. The first nozzle had an opening diameter of 100 pm, and the second nozzle had an opening of 300 pm diameter. The nozzles of the reactor were positioned at a 180° angle from each other, so that the streams collided frontally. The reaction chamber was at room temperature (25 °C] The hydrodynamic pressures were 10 bar and 8 bar, for the first and the second streams, respectively. The flow rates were initially 28.3 mL/min and 148.4 mL/min, for the first and second streams, respectively. This resulted in a target volume ratio of approximately 1:5 between the first and the second streams.

During the experiments, the accuracy of the injected volumes, and consequently of the volume ratios, was confirmed by using a scale under the supply vessels of the first and second stream. Thus, the flow rate and the injected volumes could be verified gravimetrically. The consumption of the 0.1% solution of Tamol NH 9401 and the 0.2% solution of itraconazole in acetone was read from the balances to check the actual volume ratio of the first stream and the second stream. The volume ratio was calculated according to: mass of first stream [g] ^c p of the first stream [0.792 g/mL] / mass of the second stream [g] ^c p of the second stream [1 g/mL]

Alternatively, two flow meters can be installed in the system to continuously measure the injected volumes of the two streams and display the measurement result continuously.

In order to check whether the actual volume ratio of the first stream to the second stream corresponds to the target volume ratio, and whether the pumps are properly functioning, a precipitation process was carried out under the conditions described above.

The particle size (z-average] and size distribution or polydispersity index (PDI] of the precipitated particles was determined at 25 °C by dynamic light scattering (DLS], specifically by using a Zetasizer Nanoseries from Malvern Panalytical Ltd. Size and size distribution were calculated according to ISO 13321:1996 and ISO 22412:2017. Particle size is reported as z- average, which is obtained by cumulant analysis of the autocorrelation function is obtained. The polydispersity index is considered a measure of the width of the distribution and is a dimensionless quantity between 0 and 1. The smaller this number, the narrower the distribution. Values less than 0.4 generally indicate a somewhat homogeneous particle size distribution, while values greater than 0.7 indicate that the sample has a very inhomogeneous size distribution. The software Zetasizer V7.13 was used with the following parameters indicated in Table 2:

Table 2 - LS parameters used for measuring particle size

The DLS instrument was validated before the measurement with a dispersion of a particle size standard (about 100 nm] from the manufacturer. Particle sizes were determined immediately (within 30 minutes] after precipitation or after a fixed time (for example, 2 minutes] had elapsed.

EXAMPLE 1

In the first step, the first stream consisted of 0.2 wt% itraconazole in acetone, and it was injected at a flow rate of 28 mL/min. The second stream consisted of demineralized water, and it was injected at a flow rate of 150 mL/min velocity. In the second step, the second stream was changed to 0.1 wt% Tamol NH 9401 in demineralized water.

Acetone was heated to 35 °C to facilitate dissolving the itraconazole, and later used at room temperature (25°C]

Flow rates were changed at short time intervals as disclosed in Table 3, and a sample of the obtained suspension was taken for each flow rate. In each of these conditions, hydrodynamic pressures were measured, the solvent content of the mixture was calculated, and nanoparticle size and size distribution (polydispersity index, PDI] were determined using a Zetasizer Nanoseries from Malvern Panalytical Ltd. 1.5 minutes after the mixture was collected:

Table 3 - Particle sizes and PDI obtained at different volume ratios (Experiment 1]

The experiment was validated on two additional days. The data are summarized in Tables

4 and 5.

Table 4 - Particle sizes and PDI obtained at different volume ratios (Experiment 2]

Table 5 - Particle sizes and PDI obtained at different volume ratios (Experiment 3]

The results obtained in experiments 2 and 3, which are shown in Tables 4 and 5, confirmed the accuracy of the method for predicting volume ratios according to observed particle size. In all experiments and conditions, the hydrodynamic pressure of the first and second stream was in the range of 6 and 23 bar. The determination of the particle sizes and size distribution was carried out as described in the general section.

The scales verified that the observed volume ratios were indeed equal to the target volume ratios. The precipitation of itraconazole from acetone against water in the presence of Tamol NH9401 resulted in high quality particles with PDI. The size of the particles was successfully adjusted from 180 nm to 320 nm by varying the volume ratios (Figure 1] The size distribution of the nanoparticles remained constant and small (Figure 2] for all conditions. Other substances, such as hydroxypropyl methylcellulose phthalate, would have precipitated in indistinguishable particle size despite different mixing ratios of solvent and antisolvent Figures 1 and 2 show particle size as a function of the percentage of acetone in the suspension, i.e. as a function of percentage of the volume of the first stream out of the volume of the first and second streams. Thus, an acetone concentration of 6%, for example, would correspond approximately to a ratio of 1:15,5 between the first and the second streams; an acetone concentration of 8% would correspond approximately to a ratio of 1:11,5. These experiments indicate that the methods disclosed herein reproducibly yield nanoparticles with sizes varying according to the ratio of the injected volumes, and with a small size distribution. If no deviation from the expected values is detected, proof of the accurate injection of the desired volume ratio is thus provided.

In the event of a defect in one of the two feed pumps for the fluids to the inlet nozzle, the particle size produced deviates significantly from expected values and the functionality of the apparatus is not validated. Thus, the method disclosed herein provides a fast and reliable method for checking the functioning of an apparatus for the production of nanoparticles. EXAMPLE 2

Experiments 1, 2, and 3 of EXAMPLE 1 indicated that the size of itraconazole precipitated nanoparticles is affected by the volume ratio between the first and second streams. However, the itraconazole nanoparticles were unstable over a longer period of time and should be measured immediately after precipitation.

Further experiments were done with the first stream consisting of 5 mg/mL ITZ and 15 mg/mL hydroxypropyl methylcellulose acetate succinate (HPMCAS] in acetone, and the second stream consisting of 50 mM acetate buffer pH 5. Particle size was measured with Malvern Nano ZS or Ultra. Further, the experiments were realized using different type of apparatuses, termed herein Apparatus 1, Apparatus 2, Apparatus 3.

A smaller particle size was observed for higher volumes ratios for all apparatuses tested, confirming the previous results (Figure 3; the given volume ratio is based on the second stream and sould be read as for example "4” is a volume ration of 1:4 (first strear second stream]].

ITEM LIST

In some embodiments, disclosed here is a method of inspecting a plant for the production of nanoparticles, wherein the plant comprises a first pump for generating a first flow, a second pump for generating a second stream, and a chamber for mixing the first stream and the second stream, the method comprising the steps of:

(a] mixing said first stream and said second stream, wherein said first stream is generated by the first pump and the second flow is generated by the second pump, wherein by setting a target pumping volume for the first pump and a desired pumping volume for the second pump, a desired volume ratio of the first to the second second stream is determined, wherein the first stream contains an organic solvent and a reference substance and the second stream contains water and a surfactant, wherein the target volume ratio of first to second stream has a value of between 1:3 and 1:20, and wherein, by mixing the first stream and the second stream, at least part of the reference substance and at least part of the surfactant are precipitated as particles;

(b] determining the particle size and optionally the size distribution of the precipitated particles;

(c] repeating step a] and step b] one or more times, wherein the nominal volume ratio assumes a different value between 1:3 and 1:20 in each repetition; (d] comparing the particle sizes and optionally the size distributions of the precipitated particles with the values of the target volume ratios, wherein the reference substance is a substance defined in that the size of precipitated particles is adjusted by the volume ratio of first to second stream.

In some embodiments, the reference substance is an active pharmaceutical ingredient, preferably itraconazole. In some embodiments, the organic solvent contains acetone, preferably is acetone. In some embodiments, the surfactant is a polymer of naphthalenesulfonic acid and formaldehyde.

In some embodiments, the first stream consists of the organic solvent and the reference substance and the second stream consists of water and the surfactant In some embodiments, in step a] and in step b] the particle size and the size distribution of the precipitated particles is determined and in step d] the particle sizes and size distributions of the precipitated particles as a function of the value of the nominal volume ratio of the first to the second stream is compared.

In some embodiments, the reference substance crystallizes during precipitation from the organic solvent. In some embodiments, the particle size of precipitated particles at each volume ratio of first to second stream between 1:3 and 1:20 is between 160 and 500 nm, preferably between 200 and 320 nm.

In some embodiments, the equipment for feeding the first stream and the second stream into the chamber comprises at least one nozzle each, wherein the diameter of the at least one nozzle of the first stream and the diameter of the at least one nozzle of the second stream.

In some embodiments, the at least one nozzle of the second stream is designed in such a manner that a mixing of the first stream and the second stream is ensured and wherein the first stream and the second stream are each passed through the at least one nozzle for mixing into the chamber.

In some embodiments, the diameter of the at least one nozzle of the first stream and the diameter of the at least one nozzle of the second stream is dimensioned in such a way that in the first stream and in the second stream similar hydrodynamic pressures occur in the first stream and in the second stream, it being preferred that the ratio of the hydrodynamic pressure in the first stream to the hydrodynamic pressure in the second stream is between 1:0.5 and 1:2.

In some embodiments, the diameter of the at least one nozzle of the first stream is 50 to 400 pm, preferably about 100 pm, and the diameter of the at least one nozzle of the second stream is 200 to 1200 pm, preferably about 300 pm. In some embodiments, the method is a method for checking whether an actual-volume ratio of the first stream to the second stream corresponds to the desired volume ratio. In some embodiments, the method is a method for verifying the actual pump volume of the first pump and the actual pump volume of the second pump. In some embodiments, disclosed herein is the use of itraconazole for checking a plant for the production of nanoparticles, according to any of the methods disclosed above.