Method for producing nanoparticles in the form of a powder containing a bioabsorbable polyester

Abstract

The present invention provides emulsions with a Z-average particle size D in the range of 1 to 450 nm, with a polydispersity index PDI in the range of 0.01 to 0.5, by emulsion solvent extraction or emulsion solvent evaporation and application of ultrasound. z The present invention relates to a method for producing nanoparticles comprising at least one bioabsorbable polyester in the form of a powder having the formula:

Description

Technical Field

[0001] Technical field to which the invention belongs The present invention lies in the field of a method for producing nanoparticles containing at least one biodegradable polyester in powder form by extraction of an emulsion solvent or evaporation of an emulsion solvent and application of ultrasound.

[0002] Background WO 2004 / 026452 describes a method and a flow-through cell for continuously treating a free-flowing composition by ultrasound. In this method, an ultrasonic flow-through cell is used that includes a cylindrical glass tube capable of transporting an emulsion fluid. The cylindrical glass tube is inserted into a steel mantle, and the empty space is filled with pressurized water in the range of 2 to 20 bar as an ultrasonic transducer. The ultrasonic transducer is excited by a high-frequency generator (sonotrode) to indirectly transmit sound waves to the passing fluid through the ultrasonic transducer. In Example 1, a solution of 5 wt% poly(lactide-co-glycolide) (PLGA) in dichloromethane (DCM) and 10 wt% bovine serum albumin (BSA) in a phosphate buffered aqueous solution are combined and merged, and pass through a flow-through cell having a glass tube with a length of 50 cm and an inner diameter of 2 mm under ultrasonic sound. An oil-in-water emulsion is produced with an average droplet diameter of 0.62 - 1.37 μm. The emulsion is stated to be stable for over 30 minutes and thus generally suitable for further processing into microspheres. The high-frequency generator has a 100% power output of 30 - 35 W, preferably 32 W. WO 2004 / 026452 uses an ultrasonic flow-through cell including a cylindrical glass tube with a sonication region using a power input to the ultrasonic transducer calculated to be 51.4 W per cm of the confluence of the dispersed and continuous phases. 3 An ultrasonic flow-through cell including a cylindrical glass tube with a sonication region using a power input to the ultrasonic transducer calculated to be 51.4 W per cm of the confluence of the dispersed and continuous phases is used.

[0003] International Publication No. 2015 / 181138 describes multilayer calcium phosphate nanoparticles having a diameter in the range of 10 to 300 nm and a method for producing the nanoparticles. Sonication (ultrasonic treatment) was performed for 20 seconds at an amplitude of 70% and a pulse of 0.7 using a Hielscher UP50H device, sonotrode MS2 to form water-in-oil and water-in-oil-in-water emulsions.

[0004] Freitas S. et al. (European Journal of Pharmaceutics and Biopharmaceutics 61 (2005) pp. 181 - 187) describe flow-through sonication combined with static micromixing for the aseptic production of microspheres by solvent extraction. PLGA (RESOMER® RG 503 H) particles were produced from a water-in-oil-in-water emulsion (W1 / O / W2) in a solvent extraction / evaporation process. The droplet size in the primary emulsion (W1 / O) was 0.63 + / - 0.03 μm, and the droplet size in the double emulsion (W1 / O / W2) was 14.8 μm or more. In Freitas, the calculated power input of the ultrasonic transducer, which was calculated to be 48.2 W per 1 cm of the confluence of the dispersed phase and the continuous phase, was used, and an ultrasonic flow-through cell (Dmini250, Dr. Hielscher, Teltow, Germany) including a cylindrical glass tube having a sonication region was used. 3 An ultrasonic flow-through cell (Dmini250, Dr. Hielscher, Teltow, Germany) including a cylindrical glass tube having a sonication region using the calculated power input of the ultrasonic transducer, which was calculated to be 48.2 W per 1 cm of the confluence of the dispersed phase and the continuous phase, was used.

[0005] Freitas S. et al. (Ultrasonics Sonochemistry 13 (2006) pp. 76 - 85) describe continuous contact and contamination-free ultrasonic emulsification (a tool useful for drug development and production). PLGA (RESOMER® RG 503H) particles were produced from a water-in-oil emulsion (O / W) in a solvent extraction / evaporation process. The average PLGA particle size produced under various conditions was in the range of 0.49 to 0.60 μm + / - 0.02 μm. In Freitas, the confluence of the dispersed phase and the continuous phase was 1 cm. 3An ultrasonic flow-through cell is used that includes a cylindrical glass tube (inner diameter 2 mm) with a sonication region using the power input of a sonotrode (24 kHz, UIP250, Dr. Hielscher) calculated to be 51.4 W per hit.

[0006] Doerdelmann G. (Dissertation (2015) University Duisburg, Faculty of Chemistry, Essen, Germany) describes calcium phosphate nanoparticles combined with biodegradable polymers as composite materials for active ingredient transport and bone substitute materials. In Doerdelmann, the confluence of the dispersed phase and the continuous phase is 1 cm 3 An ultrasonic flow-through cell is used that includes a cylindrical glass tube with a sonication region using the power input of a sonotrode calculated to be 51.4 W per hit. In Doerdelmann, an ultrasonic device is used for the homogenization of a pre-emulsion W / O of calcium phosphate nanoparticles in an aqueous suspension and a biodegradable polymer in an organic solution. The dispersed phase did not contain a biodegradable polymer material. Homogenized nanodroplets of an aqueous suspension of calcium phosphate in an organic PLGA solution were collected, and subsequently the nanoparticles were produced by rapid precipitation in an ethanol phase. The maximum flow rate is described as 3.3 ml / min, and the maximum amount of nanoparticles obtained is 50 mg / min.

[0007] Summary of the Invention There is a technical need for a method of providing nanoparticles containing a bioabsorbable polyester in the form of a fine powder. Accordingly, the present invention provides, as claimed and described herein, from a bioabsorbable polyester by emulsion solvent extraction or emulsion solvent evaporation and the application of ultrasound, a polydispersity index (PDI) in the range of 0.01 to 0.5, preferably 0.01 to 0.4, most preferably 0.05 to 0.38, and a Z-average particle size D in the range of 1 to 450 nm, preferably 10 to 300 nm, most preferably 50 to 200 nm. zRelates to a method for producing nanoparticles comprising a bioabsorbable polyester in the form of a powder having. One of the important elements of the present invention for obtaining nanoparticles of relatively small size is that the confluence of the dispersed polymer phase and the continuous phase is 1 m of confluence 3 It was found that it is to be passed through an ultrasonic flow-through cell under sonication using a power input of 20 to 50 W per hit, preferably 27 to 45 W. In contrast to this discovery, in the teaching of Freitas et al. (2006) using a power input calculated to be 51.4 W / cm 3 It would have been expected that lower power inputs would result in rather large particles with diameters of 500 nm or more. Surprisingly, according to the present invention, smaller bioabsorbable polyester particles, optionally containing an active pharmaceutical ingredient, as desired for many types of applications such as parenteral and oral delivery of the active ingredient, are obtained.

[0008] Detailed description of the invention The present invention provides, by emulsion solvent extraction or emulsion solvent evaporation and application of ultrasound, a polydispersity index PDI in the range of 0.01 to 0.5, preferably 0.01 to 0.4, most preferably 0.05 to 0.38, and a Z-average particle size D in the range of 1 to 450 nm, preferably 10 to 300 nm, most preferably 50 to 200 nm z A method for producing nanoparticles comprising at least one bioabsorbable polyester in the form of a powder having, the method comprising steps a) to f):

[0009] a) Supplying an organic phase (OP) containing a solvent or solvent mixture S1 containing one or more organic solvents containing 0.1 to 55% by weight, preferably 0.4 to 50% by weight, of a bioabsorbable polyester into a first container.

[0010] The first container can be, for example, a beaker, a supply container or a holding tank.

[0011] b) Supplying an aqueous phase (AP) containing a solvent or solvent mixture S2 containing water and an emulsion stabilizer into a second container.

[0012] The second container can be, for example, a beaker, a supply container or a holding tank.

[0013] c) Supplying the flows of the organic phase (OP) and the aqueous phase (AP) and combining these flows into a confluence.

[0014] The separate flows of the organic phase (OP) from the first container and the aqueous phase (AP) from the second container can usually be supplied through a hose or a pipe driven, for example, by a pumping device. The application of other driving forces such as simple gravity, pressure or vacuum is also possible. Subsequently, the separate flows can be combined before or in the inlet region of the ultrasonic flow-through cell. The separate flows can be combined, for example, by a common pipe connection (tee connection or Y-connection), or, for example, in a premixing device cell where the flows are combined and optionally pre-mixed.

[0015] d) Passing the confluence through an ultrasonic flow-through cell under sonication using a power input of 20 to 50 W, preferably 27 to 45 W per centimeter of confluence, to obtain an emulsion emerging at the outlet of the ultrasonic flow-through cell. 3 The power input per centimeter of confluence (W) is calculated as follows:

[0016] per centimeter of confluence 3 where:

Equation

[0017] e) Removing the solvent or solvent mixture S1 and S2 by evaporation, or removing them by mixing the emulsion with an excess of an aqueous extraction phase (EP) to form a combined phase, such that the solvent or solvent mixture S1 is removed from the emulsion and nanoparticles of the bioabsorbable polyester are formed.

[0018] f) Obtaining nanoparticles containing a bioabsorbable polyester by concentration and drying from the evaporated or combined extraction phases, and having a Z-average particle size D in the range of 1 to 450 nm, preferably 10 to 300 nm, most preferably 50 to 200 nm z , and obtaining a polymer powder having a PDI value in the range of 0.01 to 0.5, preferably 0.01 to 0.4, most preferably 0.05 to 0.38.

[0019] Bioabsorbable polyester In step a), an organic phase (OP) containing a solvent or solvent mixture S1 and containing 0.1 to 55% by weight, preferably 0.4 to 50% by weight, most preferably 0.5 to 25% by weight of a bioabsorbable polyester is supplied.

[0020] The term "bioabsorbable" in "bioabsorbable polyester" preferably refers to a lactic acid-based or lactide-based polymer, and means a polyester that is slowly hydrolyzed into oligomers after being implanted or injected into the body of a human or animal in contact with body fluids. Hydrolysis end products such as lactic acid or glycolic acid are metabolized into carbon dioxide and water. Other interchangeable expressions for the commonly used term "bioabsorbable polyester" are "absorbable polyester", "resorbable polyester", "biodegradable polyester" or "adsorbable polyester".

[0021] The bioabsorbable polyester can be selected from polyorthoesters, polylactides, polydioxanones, polycaprolactones, polytrimethyl carbonates, polyglycolides, poly(lactide-co-glycolide) (PLGA), poly(lactide-co-caprolactone), poly(lactide-co-trimethyl carbonate), poly(lactide-co-polyethylene glycol) and any blends thereof. Preferably, the bioabsorbable polyester is selected from polyorthoesters, poly(lactide-co-glycolide) (PLGA) or blends thereof.

[0022] Active pharmaceutical ingredient The organic phase (OP) or the aqueous phase (AP) or both may contain an active pharmaceutical ingredient.

[0023] The active pharmaceutical ingredient may be selected from the group consisting of analgesics, antibiotics or anti-infectives, antibodies, anti-epileptic drugs, plant-derived antigens, anti-rheumatic drugs, benzimidazole derivatives, beta-blockers, cardiovascular drugs, chemotherapeutic drugs, CNS drugs, digitalis glycosides, gastrointestinal drugs such as proton pump inhibitors, combinations of proton pump inhibitors and non-steroidal anti-inflammatory drugs (NSAIDs), enzymes, hormones, liquid or solid natural extracts, oligonucleotides, DNA, RNA, mRNA, siRNA, Protac (protein degradation targeting chimeras), peptide hormones, therapeutic bacteria, peptides, proteins, urinary drugs, and vaccines in the form of salts such as aspartates or hydrochlorides. For example, diclofenac or ritonavir (e.g., diclofenac sodium) in the form of their salts is suitable.

[0024] Ultrasonic flow-through cell The ultrasonic flow-through cell includes a cylindrical glass tube with a sonication region through which the emulsion fluid is transported, which is inserted into a steel mantle, and there is a void space between the steel mantle and the glass tube, and the void space is filled with a pressurized liquid, preferably water at 5 to 15 bar, preferably 8 to 12 bar, as an ultrasonic transducer, and the ultrasonic transducer is excited by an attached high-frequency generator (sonotrode) at 18 to 22 kHz, preferably 20 kHz, for transmitting sound waves, and the high-frequency generator has a power output of 1 to 200 W, preferably 80 to 110 W.

[0025] In d), the part of the cylindrical glass tube where the confluence of the organic phase (OP) and the aqueous phase (AP) is ultrasonically treated through the glass mantle and the ultrasonic transducer from the sonotrode is called the sonication region. The cylindrical glass tube is usually longer than the sonication region.

[0026] The sonication region of the cylindrical glass tube can have a length of 150 - 250 mm, preferably 180 - 220 mm and an inner diameter of 2.0 - 6.5 mm, preferably 3.5 - 5.5 mm. The thickness of the glass wall can be 0.5 - 2 mm, preferably 0.8 - 1.5 mm.

[0027] The cylindrical tube can also be made of rigid plastic, metal, or any other material capable of transmitting energy.

[0028] The residence time of the confluence within the sonication region of the ultrasonic flow-through cell can be 0.5 - 80 seconds, preferably 2 - 50 seconds.

[0029] The flow rate of the confluence within the ultrasonic flow-through cell can be 2 - 200 ml / min, preferably 4 - 40 ml / min.

[0030] Oil-in-water (O / W) emulsion In d), an (ultimate) oil-in-water (O / W) emulsion can be formed.

[0031] In this case, in step c), the flows of the organic phase (OP) and the aqueous phase (AP) can be supplied at a flow rate of the organic phase (OP) of 0.5 - 50 ml / min, preferably 1 - 10 ml / min and a flow rate of the aqueous phase (AP) of 1.5 - 150 ml / min, preferably 3 - 30 ml / min, provided that the flow rate of the aqueous phase (AP) must be higher than the flow rate of the organic phase (OP), and as a result, an oil-in-water emulsion (O / W) occurs in d).

[0032] In the case of an oil-in-water (O / W) emulsion, the organic phase (OP) is the dispersed phase and the aqueous phase (AP) is the continuous phase.

[0033] Water-in-oil-in-water emulsion (W1 / O / W2) Alternatively, in step d), a primary (intermediate) water-in-oil (W1 / O) emulsion may be formed, which is then preferably mixed and emulsified with an additional aqueous phase (W2) by a static mixer or a further sonication flow-through cell before step e), to obtain a final water-in-oil-in-water emulsion (W1 / O / W2). The aqueous phase W2 is usually added to the water-in-oil (W1 / O) emulsion in an excess volume.

[0034] In this case, in step c), the flows of the organic phase (OP) and the aqueous phase (AP) can be supplied at a flow rate of the organic phase (OP) of 1.5 to 150 ml / min, preferably 3 to 30 ml / min and a flow rate of the aqueous phase (AP) of 0.5 to 50 ml / min, preferably 1 to 10 ml / min, provided that the flow rate of the organic phase (OP) is higher than the flow rate of the aqueous phase (AP), resulting in the formation of a primary water-in-oil (W1 / O) emulsion in d). When using a further sonication flow-through cell to produce the (W1 / O / W2) emulsion, the flow rate of the aqueous phase (W2) must be higher than the combined flow rate of the (W1 / O) emulsion.

[0035] In the case of the primary (intermediate) water-in-oil (W1 / O) emulsion, the organic phase (OP) is the continuous phase and the aqueous phase (AP) is the dispersed phase.

[0036] Organic phase (OP) In step a), an organic phase (OP) is supplied, which contains a solvent or solvent mixture S1 containing one or more organic solvents. The organic phase further contains 0.1 to 55 wt%, preferably 0.4 to 50 wt% of a bioabsorbable polyester and is supplied to a first container.

[0037] The organic phase (OP) may contain a solvent or solvent mixture S1 that is immiscible or only partially miscible with the solvent or solvent mixture of the aqueous phase (AP).

[0038] The term "immiscible" shall mean that separate phases (OP) and (AP) are formed at, for example, 25 °C, at any mixing ratio.

[0039] The term "partially miscible" means, for example, that at 25 °C, a part of the solvent or solvent mixture S1, usually less than 25% by weight or less than 10% by weight, can dissociate or migrate into the solvent or solvent mixture S2 of the aqueous phase (AP).

[0040] The solvent or solvent mixture S1 may include one or more organic solvents selected from, for example, dichloromethane (DCM), ethyl acetate (EtOAc), chloroform, benzyl alcohol, diethyl carbonate (DMC), dimethyl sulfoxide (DMSO), methanol, propylene carbonate, isopropyl acetate, methyl acetate, methyl ethyl ketone, butyl lactate, and isovaleric acid or any mixture thereof. Preferred solvent or solvent mixture S1 may include EtOAc, DCM, EtOAc and DMSO or DCM and DMSO.

[0041] For example, when the aqueous phase (AP) can be water or contains 90% to 100% by weight of water as the solvent S2, a suitable solvent mixture S1 can be selected from, for example, dichloromethane (DCM), ethyl acetate (EtOAc) or dimethyl sulfoxide (DMSO) and methanol in a ratio of 1:9 to 9:1.

[0042] For example, when the aqueous phase (AP) can be water or contains 90% to 100% by weight of water as the solvent S2, a suitable solvent mixture S1 can be, for example, a mixture of ethyl acetate (EtOAc) and dimethyl sulfoxide (DMSO) in a ratio of 1:9 to 9:1.

[0043] For example, when the aqueous phase (AP) can be water or contains 90% to 100% by weight of water as the solvent S2, another suitable solvent mixture S1 can be, for example, a mixture of dichloromethane (DCM) and dimethyl sulfoxide (DMSO) in a ratio of 1:9 to 9:1.

[0044] The organic phase (OP) may contain an active pharmaceutical ingredient. The organic phase (OP) may contain up to 25% by weight, preferably 0.1 - 15% by weight of the active pharmaceutical ingredient.

[0045] aqueous phase (AP) The aqueous phase (AP) contains a solvent or solvent mixture S2 that contains water, preferably 75% by weight or more of water. The solvent or solvent mixture S2 is immiscible or only partially miscible with the (organic) solvent or solvent mixture S1, and the aqueous phase (AP) and the organic phase (OP) preferably form separate phases after mixing at any ratio from 1:9 to 9:1, between about 5 - 35°C, preferably 20 - 25°C.

[0046] The term "immiscible" shall mean that separate phases (OP) and (AP) are formed at, for example, 25°C, at any mixing ratio.

[0047] The term "partially miscible" shall mean that, for example, at 25°C, a part of the solvent or solvent mixture S1, usually less than 25% by weight or less than 10% by weight, can dissociate or migrate into the solvent or solvent mixture S2 of the aqueous phase (AP).

[0048] By way of example, when the solvent S1 of the organic phase (OP) is, for example, ethyl acetate and the solvent S2 of the aqueous phase (AP) is water, a small amount of up to about 8% by weight of ethyl acetate can dissociate or migrate into the aqueous phase (AP) after mixing. However, the remaining mixed phases remain separate and can be processed as disclosed herein. If the migration of such a partially water-miscible organic solvent S1 into the aqueous phase (AP) must be avoided, the solvent S2 of the aqueous phase (AP), water, can be saturated from the start by the addition of the corresponding organic solvent (using about 8% by weight of ethyl acetate in this example) before contacting the organic phase (OP).

[0049] The aqueous phase (AP) contains the solvent or solvent mixture S2.

[0050] The solvent or solvent mixture S2 may contain from 60% to 100% by weight, preferably from 80% to 100% by weight, of water.

[0051] The aqueous phase (AP) contains a solvent or solvent mixture S2 containing water that is immiscible or only partially miscible with the solvent or solvent mixture S1 of the organic phase (OP). The aqueous phase (AP) and the organic phase (OP) form separate phases after mixing. The separate phases can be processed as disclosed herein.

[0052] The solvent or solvent mixture S2 contains water and may optionally contain a solvent that is completely miscible with water and insoluble or only partially soluble in the solvent or solvent mixture S1.

[0053] The solvent or solvent mixture S2 contains at least 60% by weight (60% by weight or more), preferably at least 80% by weight (80% by weight or more), of water, and optionally up to 40% by weight, preferably up to 20% by weight, of, for example, ethanol, acetone, isopropanol, dichloromethane (DCM), ethyl acetate (EtOAc), chloroform, benzyl alcohol, diethyl carbonate (DMC), dimethyl sulfoxide (DMSO), methanol, propylene carbonate, isopropyl acetate, methyl acetate, methyl ethyl ketone, butyl lactate, isovaleric acid, or any mixture thereof.

[0054] The aqueous phase (AP) may contain water and an emulsion stabilizer.

[0055] The aqueous phase (AP) may contain water and 0.1 to 10% by weight, preferably 1 to 8% by weight, of an emulsion stabilizer, preferably polyvinyl alcohol (PVA) or polysorbate.

[0056] The aqueous phase (AP) may optionally contain an active pharmaceutical ingredient. The aqueous phase (AP) may contain up to 25% by weight, preferably 0.1 to 15% by weight, of the active pharmaceutical ingredient.

[0057] Emulsion - solvent extraction / aqueous extraction phase (EP) The nanoparticles can be obtained from the emulsion by emulsion - solvent extraction in step e).

[0058] The aqueous extraction phase (EP) can contain 80% by weight or more, at least 80% by weight up to 100% by weight of water.

[0059] The aqueous extraction phase (EP) can contain at least 80% by weight of water and optionally up to 20% by weight of a water - miscible solvent, such as ethanol, acetone, isopropanol or any mixture thereof. The extraction phase (EP) can further contain 0 - 10% by weight, preferably 1 - 8% by weight, of an emulsion stabilizer such as polyvinyl alcohol (PVA) or polysorbate.

[0060] In step e), the emulsion can be mixed with an excess amount of the aqueous extraction phase (EP) to form a combined phase, and as a result, the solvent S1 is removed from the emulsion, and nanoparticles of the biodegradable polyester or a mixture of the active pharmaceutical ingredient and the biodegradable polyester are formed. The excess amount of the aqueous extraction phase is 2 - 150 times, preferably 5 - 70 times, the volume of the emulsion.

[0061] Emulsion - solvent evaporation The nanoparticles can be obtained from the emulsion by emulsion - solvent evaporation in step e). The method of emulsion - solvent evaporation is well - known to those skilled in the art.

[0062] The solvent S1 and / or S2 can be removed by evaporation, and as a result, nanoparticles of the biodegradable polyester or a mixture of the active pharmaceutical ingredient and the biodegradable polyester are formed.

[0063] Nanoparticles The nanoparticles obtained by the disclosed method exhibit a homogeneous surface structure. The homogeneous structure can be shown, for example, by electron microscope imaging.

[0064] Polymer powder: Z - average size Dz and PDI The polymer powder containing a bioabsorbable polyester and optionally an active pharmaceutical ingredient has a Z-average size D in the range of 1 to 450 nm, preferably 10 to 300 nm, more preferably 50 to 200 nm z (Z-average particle size D z ) and the PDI value is in the range of 0.01 to 0.5, preferably 0.01 to 0.4, more preferably 0.05 to 0.38. The Z-average size D z can be measured by dynamic light scattering (DLS) in accordance with ISO 22412:2017 (Publication date 2017-02) “Particle size analysis - Dynamic light scattering (DLS)”.

[0065] Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), is a widely available method used for the routine analysis of the hydrodynamic size of particles in solution. This method relies on the measurement of the scattering intensity of nanoparticles in Brownian motion when illuminated by a monochromatic beam of light. This scattering intensity fluctuates on a microsecond time scale, and this fluctuation corresponds to the diffusion rate of the particles.

[0066] The polydispersity index (PDI) is determined from a two-parameter fit (cumulant analysis) to the correlation data. The calculations used for the determination of PDI are defined in the ISO standard document 22412:2017.

[0067] Item The present invention can be summarized by the following items: 1. A method for producing nanoparticles in the form of a powder having a Z-average particle size D in the range of 1 to 450 nm and having a polydispersity index PDI in the range of 0.01 to 0.5, the powder containing at least one bioabsorbable polyester, by emulsion solvent extraction or emulsion solvent evaporation and application of ultrasound, steps a) to f): z comprising the steps a) to f): a) Supplying an organic phase (OP) containing one or more organic solvents and including a solvent or solvent mixture S1 containing 0.1 to 55% by weight of a bioabsorbable polyester into a first container; b) Supplying an aqueous phase (AP) containing water and an emulsion stabilizer and including a solvent or solvent mixture S2 into a second container; c) Supplying the flows of the organic phase (OP) and the aqueous phase (AP) and combining these flows into a confluence; d) Passing the confluence through an ultrasonic flow-through cell using a power input of 20 to 50 W per 1 cm of the confluence under sonication to obtain an emulsion emerging at the outlet of the ultrasonic flow-through cell; 3 e) Removing the solvent or solvent mixtures S1 and S2 by evaporation or by mixing the emulsion with an excess amount of an aqueous extraction phase (EP) to form a combined phase, such that as a result, the solvent or solvent mixture S1 is removed from the emulsion and nanoparticles are formed; f) Obtaining nanoparticles containing a bioabsorbable polyester by concentration and drying from the evaporated extraction phase or the combined extraction phase, to obtain a polymer powder having a Z-average particle size D in the range of 1 to 450 nm and a polydispersity index PDI in the range of 0.01 to 0.5 z The method comprising. 17. The method according to item 1, wherein the organic phase (OP) contains a solvent or solvent mixture S1 that is immiscible or only partially miscible with the solvent or solvent mixture of the aqueous phase (AP). 18. The method according to item 1 or 2, wherein in d), an oil-in-water (O / W) emulsion is formed. 19. The method according to item 1 or 2, wherein in d), a water-in-oil (W1 / O) emulsion is formed, and before e), the emulsion is preferably mixed and emulsified with a further aqueous phase (W2) by a static mixer or a further sonication flow-through cell to obtain a water-in-oil-in-water emulsion (W1 / O / W2). 20. The method according to item 1 or 2, wherein in d), a water-in-oil (W1 / O) emulsion is formed, and before e), the emulsion is preferably mixed and emulsified with a further aqueous phase (W2) by a static mixer or a further sonication flow-through cell to obtain a water-in-oil-in-water emulsion (W1 / O / W2). 5. The method according to any one of items 1 to 4, wherein the solvent or solvent mixture S1 comprises dichloromethane, ethyl acetate, chloroform, benzyl alcohol, diethyl carbonate, dimethyl sulfoxide, methanol, propylene carbonate, isopropyl acetate, methyl acetate, methyl ethyl ketone, butyl lactate, isovaleric acid, or any mixture thereof. 6. The method according to any one of items 1 to 5, wherein the solvent or solvent mixture S2 comprises water in an amount of 60% to 100% by weight. 7. The method according to item 6, wherein the solvent or solvent mixture S2 is immiscible or only partially miscible with the (organic) solvent or solvent mixture S1, and the aqueous phase (AP) and the organic phase (OP) form separate phases after mixing. 8. The method according to any one of items 1 to 7, wherein the aqueous phase (AP) comprises 0.1 to 10% by weight of an emulsion stabilizer, preferably polyvinyl alcohol or polysorbate. 9. The method according to any one of items 1 to 8, wherein the aqueous extraction phase (EP) comprises 80% by weight or more of water. 10. The method according to any one of items 1 to 9, wherein the bioabsorbable polyester is selected from polyorthoesters, polylactide, polydioxanone, polycaprolactone, polytrimethyl carbonate, polyglycolide, poly(lactide-co-glycolide), poly(lactide-co-caprolactone), poly(lactide-co-trimethyl carbonate), poly(lactide-co-polyethylene glycol), and any blend thereof. 11. The method according to any one of items 1 to 9, wherein the organic phase (OP) comprises an active pharmaceutical ingredient. 12. The method according to any one of items 1 to 9, wherein the aqueous phase (AP) comprises an active pharmaceutical ingredient. 13. In c), the flow rates of the organic phase (OP) and the aqueous phase (AP) are supplied at a flow rate of the organic phase (OP) of 0.5 to 50 ml / min and a flow rate of the aqueous phase (AP) of 1.5 to 150 ml / min, provided that the flow rate of the aqueous phase (AP) is higher than the flow rate of the organic phase (OP), and as a result, an oil-in-water emulsion (O / W) is obtained in d). The method according to any one of items 1 to 3 and items 5 to 12. In 14.c), the flows of the organic phase (OP) and the aqueous phase (AP) are supplied at a flow rate of the organic phase (OP) of 1.5 to 150 ml / min and a flow rate of the aqueous phase (AP) of 0.5 to 50 ml / min, provided that the flow rate of the organic phase (OP) is higher than the flow rate of the aqueous phase (AP), and as a result, in d), a water-in-oil emulsion (W1 / O) is obtained. The method according to any one of item 1 or 2 and items 4 to 12. 15. The method according to any one of items 1 to 14, wherein the residence time of the confluence in the ultrasonic flow-through cell is 0.5 to 80 seconds. 16. The method according to any one of items 1 to 15, wherein the flow rate of the confluence in the ultrasonic flow-through cell is 2 to 200 ml / min. 17. The ultrasonic flow-through cell includes a cylindrical glass tube, metal tube or rigid plastic tube having a sonication region through which the emulsion fluid is transported, which is inserted into a steel mantle, and there is a free space between the steel mantle and the tube. The free space is filled with a pressurized liquid, preferably water at 5 to 15 bar as an ultrasonic transducer. The ultrasonic transducer is excited by an attached high-frequency generator (sonotrode) of 18 to 22 kHz, preferably 20 kHz, for transmitting sound waves. The high-frequency generator has a power output in the range of an amplitude of 10 to 100%, preferably 60 to 100%, most preferably 80 to 100% and 1 to 200 W, preferably 80 to 110 W. The method according to any one of items 1 to 16. 18. The method according to any one of items 1 to 17, wherein the sonication region of the cylindrical tube has a length of 150 to 250 mm, preferably 180 to 220 mm and an inner diameter of 2.0 to 6.5 mm, preferably 3.5 to 5.5 mm. 19. The method according to any one of items 1 to 18, wherein the solvent or solvent mixture S2 comprises at least 60% by weight, preferably at least 80% by weight of water, and optionally up to 40% by weight, preferably up to 20% by weight of ethanol, acetone, isopropanol, dichloromethane (DCM), ethyl acetate (EtOAc), chloroform, benzyl alcohol, diethyl carbonate (DMC), dimethyl sulfoxide (DMSO), methanol, propylene carbonate, isopropyl acetate, methyl acetate, methyl ethyl ketone, butyl lactate, isovaleric acid or any mixture thereof. 20. The method according to any one of items 1 to 19, wherein the organic phase (OP) comprises up to 25% by weight, preferably 0.1 to 15% by weight of the active pharmaceutical ingredient. 21. The method according to any one of items 1 to 20, wherein the aqueous phase (AP) comprises up to 25% by weight, preferably 0.1 to 15% by weight of the active pharmaceutical ingredient.

[0068] Examples Materials For Examples 1 to 3 PLGA = poly(D,L-lactide-co-glycolide) 50:50 (RESOMER® RG 502H, M w = 7,000 - 17,000 g / mol -1 , Evonik Industries AG (Darmstadt). Polyvinyl alcohol (PVA, M w = about 31,000 g / mol -1 , 86 - 90% hydrolysis) was purchased from Sigma-Aldrich. Both ethyl acetate and dichloromethane (both J.T.Baker-Avantor® Performance Materials, LLC) were used as solvents for the polymers. For the encapsulation experiments, ritonavir (M w = 720.95, Angene international limited) and diclofenac sodium (Sigma Aldrich) were used, first dissolved in dimethyl sulfoxide (DMSO, WAK-Chemie Medical GmbH).

[0069] For Examples 4 to 7 The polymer film was composed of 70 wt% polyorthoester (POE) and 30 wt% RESOMER® Select 7525 DLG 8E ((PLGA) = obtained from 75 mol% DL-lactide and 25 mol% glycolide (poly(D,L-lactide-co-glycolide), Evonik Industries AG (Darmstadt)). RESOMER® Select 7525 DLG 8E (Evonik Industries AG (Darmstadt)) was additionally used as a single substance. Polyvinyl alcohol (PVA, M w = ca. 31,000 g mol -1 , 86 - 90% hydrolysis, Sigma-Aldrich), ethyl acetate (J.T. Baker-Avantor® Performance Materials, LLC) and MilliQ water were used as solvents.

[0070] All other chemicals were of analytical grade and used without further purification.

[0071] Equipment For the formation of water-in-oil, oil-in-water and water-in-oil-in-water emulsions, sonication (ultrasonic treatment) was carried out with a GDmini2 (Hielscher Ultrasonics GmbH). A cylindrical glass tube with a length of 198 mm and an inner diameter of 4.00 mm was used. The thickness of the glass wall was 1 mm. Dynamic light scattering was applied to the determination of the particle size using a Zetasizer nanoseries instrument (Malvern Nano-ZS, laser: λ = 532 nm). Data were obtained from the Malvern software without further correction.

[0072] The particle size data refer to the scattering intensity distribution (z-average). Diafiltration was carried out at ambient temperature in Examples 1 to 3 using a KrosFlo® Kr2i and a 750 kD modified polyethersulfone filtration module (Spectrum Labs).

[0073] The drug loading and encapsulation efficiency were determined by HPLC.

[0074] Calculation of encapsulation efficiency and drug loading:

Number

[0075] Example 1 : Synthesis of PLGA - ritonavir nanoparticles Example 2 : Synthesis of PLGA - diclofenac sodium nanoparticles Example 3 : Synthesis of PLGA nanoparticles Example 4: Synthesis of 6% POE - PLGA nanoparticles Example 5: Synthesis of 15% POE - PLGA nanoparticles Example 6: Synthesis of 30% POE - PLGA nanoparticles Example 7: Synthesis of 50% POE - PLGA nanoparticles

[0076] Example 1 : Synthesis of PLGA - ritonavir nanoparticles For the synthesis of particles containing ritonavir, which is a water - insoluble active ingredient, an O / W emulsion was prepared. For this purpose, two solutions (A and B) were continuously supplied to an indirect sonication device. Aqueous solution A containing the stabilizer PVA was emulsified by sonication using organic solution B containing the polymer and the active ingredient. The emulsion was combined with a further aqueous extraction phase and added continuously after emulsification to extract the solvent.

[0077] As a result, when the partially water - miscible solvent was extracted into an excess amount of water, solid polymer nanoparticles were obtained. Thereby, a nearly transparent, white - turbid dispersion was created. 1.8 g of PLGA was dissolved in ethyl acetate (PLGA: 225.5 mg mL -1 ; 7.98 mL), and 0.2 g of ritonavir was dissolved in dimethyl sulfoxide (DMSO) (ritonavir: 57.9 mg mL -1, 3.45 mL). The two phases were combined and mixed to obtain Solution B, the dispersed phase (= organic phase (OP)). This phase was combined with Solution A (= aqueous phase (AP)), an aqueous phase containing polyvinyl alcohol (PVA; 20 mg / mL -1 ; 34.31 mL), using a T-junction immediately before the sonication device at flow rates of 8 mL / min and 24 mL / min, respectively. Next, this mixture was continuously pumped into the sonication device and emulsified to form a white, turbid O / W emulsion. The exposure time to the sonication region was 4.7 seconds at 100% amplitude, 14 °C, and 9 bar pressure in the permeating fluid. Immediately after the sonication device, deionized water (= aqueous extraction phase (EP)) was introduced into the emulsion at a flow rate of 162 mL / min through a second T-junction to dilute the emulsion. During this process, ethyl acetate was extracted, PLGA particles were solidified, and ritonavir was incorporated into the PLGA matrix.

[0078] Tangential flow filtration was used to remove excess PVA, ethyl acetate, DMSO, and free ritonavir, and the particle suspension was further concentrated. Next, the concentrated nanoparticle suspension was lyophilized with 3% trehalose added as a cryoprotectant. The particles were easily redispersible in water by gentle shaking.

[0079] To determine the encapsulation efficiency of ritonavir, the particles were dissolved in acetonitrile and analyzed by HPLC after prior calibration with the dissolved ritonavir.

[0080] The ritonavir-PLGA nanoparticles contained 53.0 mg / g of ritonavir, corresponding to an encapsulation efficiency of 53.0%.

Table 1

[0081] Example 2 : Synthesis of PLGA-diclofenac sodium nanoparticles An O / W emulsion was produced in a first step. For this purpose, two solutions (A and B) were continuously fed into an indirect sonication device. An aqueous solution A containing the stabilizer PVA was emulsified by sonication with an organic solution B containing a polymer and an active ingredient. The emulsion was combined with a further aqueous extraction phase and added continuously after emulsification to extract the solvent.

[0082] Thereby, when a partially water-miscible solvent was extracted into an excess amount of water, solid polymer nanoparticles were obtained. Thereby, a nearly transparent white turbid dispersion was created. To produce solution A, 0.3 g of polyvinyl alcohol (PVA) was dissolved in 30 mL of deionized water. The dispersed phase solution B contained 1.67 g of PLGA in ethyl acetate (225.5 mg mL -1 , 7.39 mL) and contained 0.33 g of diclofenac in DMSO (400.1 mg mL -1 , 1.21 mL). Solutions A and B were continuously combined at a ratio of 3:1 at flow rates of 2 mL / min and 6 mL / min at a T-junction immediately before the sonication device. Next, this mixture was emulsified in a sonication device cooled to 14 °C at 100% amplitude to form a white turbid O / W emulsion. The pressure of the permeate fluid was 9 bar and the residence time of the mixture in the sonication region was 18.7 seconds. Immediately after the sonication device, deionized water was introduced into the emulsion at a flow rate of 52 mL / min through a second T-junction to dilute the emulsion. During this process, ethyl acetate was extracted, the PLGA particles were solidified, and sodium diclofenac was incorporated into the PLGA matrix.

[0083] Tangential flow filtration was used to remove excess PVA, ethyl acetate, DMSO, and free ritonavir and to further concentrate the particle suspension. Next, the concentrated nanoparticle suspension was freeze-dried with 3% trehalose added as a cryoprotectant. The particles were redispersed in water for 1 minute using a vortex.

Table 2

[0084] Example 3 :Synthesis of PLGA Nanoparticles For the synthesis of placebo particles, two solutions (A and B) were continuously supplied to an indirect sonication device. Aqueous solution A containing the stabilizer PVA was emulsified with organic solution B containing the polymer by sonication. The emulsion was combined with a further aqueous extraction phase and added continuously after emulsification to extract the solvent.

[0085] Thus, when a partially water-miscible solvent was extracted into an excess amount of water, solid polymer nanoparticles were obtained, thereby creating a nearly transparent white turbid dispersion. 2 g of PLGA was dissolved in ethyl acetate (PLGA: 200 mg mL -1 ; 4 mL) to obtain solution B as the dispersed phase. This phase was combined with solution A, an aqueous phase containing polyvinyl alcohol (PVA; 20 mg mL -1 ; 12 mL), using a T-junction just before the sonication device at flow rates of 2 mL / min and 6 mL / min, 5 mL / min and 15 mL / min, and 8 mL / min and 24 mL / min, respectively. Next, this mixture was continuously pumped into the sonication device and emulsified to form a white turbid O / W emulsion. The exposure time in the sonication region was 18.7 s, 7.5 s, or 4.7 s, respectively, at 100% amplitude, 14 °C temperature, and 9 bar pressure in the permeating fluid. Immediately after the sonication device, deionized water was introduced into the emulsion through a second T-junction at flow rates of 63 mL / min, 121 mL / min, and 193 mL / min, respectively, to dilute the emulsion. During this time, ethyl acetate was extracted and the PLGA particles were solidified.

Table 3

[0086] Examples 4 to 7: For the synthesis of POE-PLGA particles, an O / W emulsion was prepared. Four different percentages of POE / PLGA were utilized to create different formulations:

Table 4

[0087] For all of the following examples, the final polymer content (POE + RESOMER®) percentage was set to 5% w / w in ethyl acetate.

[0088] Example 4: Synthesis of 6% POE-PLGA nanoparticles 0.05 g of a film made of 70 wt% POE and 30 wt% RESOMER® Select 7525 DLG 8E was dissolved in ethyl acetate together with 0.50 g of RESOMER® Select 7525 DLG 8E (52.22 mg mL -1 ; 10.53 mL (= organic phase (OP)). This phase was combined with an aqueous phase (AP) containing polyvinyl alcohol (PVA; 20 mg mL -1 ; 31.60 mL) using a T-junction immediately before the sonication device at flow rates of 2 mL / min and 6 mL / min, respectively. Next, this mixture was emulsified in a sonication device cooled to 14 °C at 100% amplitude to form a white, turbid O / W emulsion. The pressure of the permeate fluid was 9 bar, and the residence time of the mixture in the sonication region was 18.7 seconds. Immediately after the sonication device, deionized water was introduced into the emulsion at a flow rate of 61 mL / min through a second T-junction to dilute the emulsion. During this process, ethyl acetate was extracted and the POE-PLG particles were solidified. Next, the nanoparticle suspension was lyophilized with 3% trehalose added as a cryoprotectant. The particles were easily redispersible in water by gentle shaking.

Table 5

[0089] Example 5: Synthesis of 15% POE-PLGA nanoparticles 0.1 g of a film made of 70 wt% POE and 30 wt% 7525 DLG 8E was dissolved in ethyl acetate together with 0.37 g of RESOMER® Select 7525 DLG 8E (47.47 mg mL -1 ; 9.83 mL (= organic phase (OP)). This phase was combined with an aqueous phase (AP) containing polyvinyl alcohol (PVA; 20 mg mL -1 ; 29.49 mL) at flow rates of 2 mL / min and 6 mL / min, respectively, using a T-junction just before the sonication device. Next, this mixture was emulsified in a sonication device cooled to 14 °C at 100% amplitude to form a white, turbid O / W emulsion. The pressure of the permeating fluid was 9 bar, and the residence time of the mixture in the sonication zone was 18.7 s. Immediately after the sonication device, deionized water was introduced into the emulsion at a flow rate of 61 mL / min through a second T-junction to dilute the emulsion. During this process, ethyl acetate was extracted and the POE-PLG particles solidified. Next, the nanoparticle suspension was lyophilized with 3% trehalose added as a cryoprotectant. The particles were easily redispersible in water by gentle shaking.

Table 6

[0090] Example 6: Synthesis of 30% POE-PLGA Nanoparticles 0.2 g of a film made of 70 wt% POE and 30 wt% 7525 DLG 8E was dissolved in ethyl acetate together with 0.27 g of RESOMER® Select 7525 DLG 8E (47.47 mg mL -1 ; 9.83 mL (= organic phase (OP)). This phase was combined with an aqueous phase (AP) containing polyvinyl alcohol (PVA; 20 mg mL -1The aqueous phase (AP) containing (29.49 mL) was combined at flow rates of 2 mL / min and 6 mL / min, respectively, using a T-junction immediately before the sonication device. Next, this mixture was emulsified in a sonication device cooled to 14 °C at 100% amplitude to form a white, turbid O / W emulsion. The pressure of the permeate fluid was 9 bar, and the residence time of the mixture in the sonication region was 18.7 seconds. Immediately after the sonication device, deionized water was introduced into the emulsion at a flow rate of 61 mL / min through a second T-junction to dilute the emulsion. During this process, ethyl acetate was extracted and the POE-PLG particles solidified. Next, the nanoparticle suspension was lyophilized with 3% trehalose added as a cryoprotectant. The particles were easily redispersible in water by gentle shaking.

Table 7

[0091] Example 7: Synthesis of 50% POE-PLGA Nanoparticles 0.31 g of a film made of 70 wt% POE and 30 wt% 7525 DLG 8E was dissolved in ethyl acetate together with 0.12 g of RESOMER® Select 7525 DLG 8E (47.47 mg mL -1 ; 9.14 mL (= organic phase (OP)). This phase was mixed with polyvinyl alcohol (PVA; 20 mg mL -1The aqueous phase (AP) containing (; 27.43 mL) was combined at flow rates of 2 mL / min and 6 mL / min, respectively, using a T-junction immediately before the sonication device. Next, this mixture was emulsified in a sonication device cooled to 14 °C at 100% amplitude to form a white, turbid O / W emulsion. The pressure of the permeating fluid was 9 bar, and the residence time of the mixture in the sonication zone was 18.7 seconds. Immediately after the sonication device, deionized water was introduced into the emulsion at a flow rate of 61 mL / min through a second T-junction to dilute the emulsion. During this step, ethyl acetate was extracted and the POE-PLG particles solidified. Next, the nanoparticle suspension was freeze-dried with 3% trehalose added as a cryoprotectant. The particles were easily redispersible in water by gentle shaking.

Table 8

Claims

1. By emulsion solvent extraction or emulsion solvent evaporation and application of ultrasound, a powder having a Z-average particle size D in the range of 50 to 200 nm and having a polydispersity index PDI in the range of 0.01 to 0.5 ｚ A method for producing nanoparticles, in the form of a powder, comprising at least one biodegradable polyester, comprising steps a) to f): a) Supplying an organic phase (OP) containing one or more organic solvents and a solvent or solvent mixture S1 containing 0.1 to 55% by weight of said at least one biodegradable polyester into a first container; b) Supplying an aqueous phase (AP) containing water and an emulsion stabilizer and a solvent or solvent mixture S2 into a second container; c) Supplying the flows of said organic phase (OP) and said aqueous phase (AP), and combining these flows using a T-shaped junction to make them merge; d) passing the merging through an ultrasonic flow-through cell under sonication using a power input of 27 to 45 W per 1 cm of merging to obtain an emulsion emerging at the outlet of the ultrasonic flow-through cell; ３ ​ e) Removing said solvent or solvent mixtures S1 and S2 by evaporation or by mixing said emulsion with an excess of an aqueous extraction phase (EP) to form a combined phase, such that as a result, said solvent or solvent mixture S1 is removed from said emulsion and nanoparticles are formed; f) Obtaining nanoparticles containing the at least one bioabsorbable polyester by concentration and drying from the evaporated extraction phase or combined extraction phases, to obtain a polymer powder having a Z-average particle size D in the range of 50 to 200 nm ｚ and a polydispersity index PDI in the range of 0.01 to 0.5 A method comprising the above steps.

2. The method according to claim 1, wherein the organic phase (OP) contains a solvent or solvent mixture S1 that is immiscible or only partially miscible with the solvent or solvent mixture S2 of the aqueous phase (AP).

3. The method according to claim 1 or 2, wherein in step d), an oil-in-water (O / W) emulsion is formed.

4. d) form a water-in-oil (W １ / O) emulsion, and before step e), preferably by a static mixer or a further sonication flow-through cell, mix the emulsion with a further aqueous phase (W ２ ) and emulsify to obtain a water-in-oil-in-water emulsion (W １ / O / W ２ ), the method according to claim 1 or 2.

5. The method according to any one of claims 1 to 4, wherein the solvent or solvent mixture S1 contains dichloromethane, ethyl acetate, chloroform, benzyl alcohol, diethyl carbonate, dimethyl sulfoxide, methanol, propylene carbonate, isopropyl acetate, methyl acetate, methyl ethyl ketone, butyl lactate, isovaleric acid, or any mixture thereof.

6. The method according to any one of claims 1 to 5, wherein the solvent or solvent mixture S2 contains 60% to 100% by weight of water.

7. The method according to any one of claims 1 to 6, wherein the solvent or solvent mixture S2 is immiscible or only partially miscible with the solvent or solvent mixture S1, and the aqueous phase (AP) and the organic phase (OP) form separate phases after mixing.

8. The method according to any one of claims 1 to 7, wherein the aqueous phase (AP) contains 0.1 to 10% by weight of an emulsion stabilizer, preferably polyvinyl alcohol or polysorbate.

9. The method according to any one of claims 1 to 8, wherein the aqueous extraction phase (EP) contains 80% or more by weight of water.

10. The method according to any one of claims 1 to 9, wherein the biodegradable polyester is selected from polyorthoesters, polylactides, polydioxanones, polycaprolactones, polytrimethyl carbonates, polyglycolides, poly(lactide-co-glycolide), poly(lactide-co-caprolactone), poly(lactide-co-trimethyl carbonate), poly(lactide-co-polyethylene glycol), and any blends thereof.

11. The method according to any one of claims 1 to 10, wherein the organic phase (OP) or the aqueous phase (AP) or both contain an active pharmaceutical ingredient.

12. In c), the flow of the organic phase (OP) and the aqueous phase (AP) is supplied at a flow rate of the organic phase (OP) of 0.5 to 50 ml / min and a flow rate of the aqueous phase (AP) of 1.5 to 150 ml / min, provided that the flow rate of the aqueous phase (AP) is higher than the flow rate of the organic phase (OP), and as a result, an oil-in-water emulsion (O / W) is obtained in d). The method according to any one of claims 1 to 3 and claims 5 to 11.

13. c) wherein the flows of the organic phase (OP) and the aqueous phase (AP) are supplied at a flow rate of the organic phase (OP) of 1.5 to 150 ml / min and a flow rate of the aqueous phase (AP) of 0.5 to 50 ml / min, provided that the flow rate of the organic phase (OP) is higher than the flow rate of the aqueous phase (AP), and as a result, in d) a water-in-oil emulsion (W １ / O) is obtained, the method according to any one of claims 1 or 2 and claims 4 to 11.

14. The method according to any one of claims 1 to 13, wherein the residence time of the confluence in the ultrasonic flow-through cell is 0.5 to 80 seconds.

15. The method according to any one of claims 1 to 14, wherein the flow rate of the confluence in the ultrasonic flow-through cell is 2 to 200 ml / min.

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