How sterile products are manufactured

Abstract

The present invention relates to a method for producing a sterile product containing heat labile and / or bioactive ingredients. The method includes a step of filter-sterilizing the product.Preferably, the sterile product obtained by the method of the invention is intended for cosmetic use, more preferably for use in cosmetic mesotherapy, such as anti-aging mesotherapy, or for medical use in aesthetic medicine or dermatology, preferably as an injectable.The method of the invention is particularly preferred for the production of sterile hyaluronic acid or its derivatives.

Description

[Technical field]

[0001] The present invention relates to a method for producing sterile products containing heat labile and / or bioactive ingredients such as vitamins, peptides, polypeptides, sodium deoxycholate, polymers such as hyaluronic acid or its derivatives, other biopolymers with structural protein function such as collagen, keratin, elastin, functional proteins such as enzymes and hormones, cellulose, cellulosic polymers and their derivatives, functional polysaccharides such as chitin. Such a method includes the step of sterile filtration of said product.

[0002] Preferably, said sterile product is intended for cosmetic use, more preferably for use in cosmetic mesotherapy, such as anti-aging mesotherapy, or for medical use, in particular for use in cosmetic medicine or dermatology, preferably as an injectable.A particularly preferred active polymer contained in said sterile product is hyaluronic acid or a derivative thereof. [Background technology]

[0003] Mesotherapy is a technique for administering active substances intraepidermally, into the superficial and deep dermis, as well as subcutaneously or intradermally.

[0004] The advantages of this technique are the ability to provide a lower dose of bioactive ingredient that diffuses into the tissue under inoculation and lasts longer compared to the intramuscular route of administration, with benefits such as a longer effect over time, reduced involvement of other organs, and reduced risk of adverse events or side effects. The main applications of mesotherapy include medical therapies such as the treatment of pain, trauma, arterial disease, venous lymphedema, dermatological treatments, as well as cosmetic treatments.

[0005] In the aesthetic field, anti-aging mesotherapy (MAE) is widely used and consists of applying very small amounts of hyaluronic acid (AI) at the epidermal level, with or without association with a cocktail of further active substances or moisturizers. In this context, MAE is also called bio-rejuvenation, bio-revitalization or mesolift. The aim is to enhance the ability of fibroblasts to synthesize neo-collagen, elastin and AI, resulting in increased firmness, radiance and moisture in the skin.

[0006] Generally, products containing active ingredients, especially polymers, are used in the cosmetic field and are preferably administered by mesotherapy techniques. The terms "active" or "bioactive" with respect to a material such as a polymer indicate that the material has a biological function and / or activity, including the mechanical activity performed by structural polymers, particularly proteins, in the cells or extracellular matrix of an organism.

[0007] By "thermolabile" material is meant a material whose chemical and / or physical characteristics and / or function are altered by heat; in particular, for the purposes of the present invention, a "thermolabile" material is one whose chemical and / or physical characteristics and / or function are altered by temperatures above 120°C, such as the temperatures typically reached during autoclave sterilization.

[0008] The prices of raw materials also used in cosmetology, especially MAE, are very high. It is therefore highly desirable that the manufacture of products containing them does not result in their deterioration. Furthermore, the effectiveness of such ingredients depends on the integrity of their functional, structural and rheological characteristics. For example, the effectiveness of hyaluronic acid or its derivatives in any medical or cosmetic application is closely linked to the integrity of the molecules that form it and the rheological properties of the product.

[0009] However, the manufacturing process of a sterile product requires sterilization procedures that may result in changes in the structure and function of the ingredients contained in the product compared to the original ingredients. Typically, sterilization can be performed by one or more of the methods described below. The procedure selected must always be verified, both for its effectiveness and for the integrity of the product, including its container or its packaging, at the end of sterilization. Furthermore, in all sterilization methods, critical process parameters are controlled to ensure that the pre-determined required conditions are reached within the batch throughout the sterilization process.

[0010] For example, in terminal sterilization, i.e., sterilization of packaged products, it is very important to consider the heterogeneity of the physical and sometimes chemical conditions within the sterilization chamber, which will determine the locations within the sterilization chamber that are least accessible to the sterilant, the minimum lethality produced by the sterilization cycle, and the repeatability of the cycle to ensure that all loads receive the specified treatment. Other combinations of time and temperature may be used for any process, so long as the selected process has been satisfactorily demonstrated to achieve adequate and reproducible lethality levels when routinely performed within established tolerance thresholds. The procedures and precautions employed should be consistent with the 10 -6 This gives the above LAS.

[0011] Sterilization by saturated pressurized steam (autoclave heating), preferably for aqueous formulations, the reference condition is heating at 121°C for 15 minutes. The temperature is usually measured by a temperature sensor inserted in the container together with other elements placed in a pre-set less hot part of the load of the sterilization chamber. The parameters of each cycle are appropriately recorded, for example by temperature / time graphs or other suitable means. Biological evaluation of the sterilized products is performed using suitable biological indicators. For dry heat sterilization, the reference condition is 160°C or higher for at least 2 hours. Dry heat sterilization is carried out in a forced air drying oven or other similar equipment designed for this purpose. The sterilizer is subjected to a load so as to reach a uniform temperature throughout the load. The temperature reached in the sterilizer during the sterilization process is usually measured by a temperature sensor inserted in a representative container together with other elements placed in a pre-set less hot part of the sterilizer. The temperature is appropriately recorded throughout the duration of each cycle. Dry heat at temperatures above 220°C is often used for sterilization and depyrogenation of glassware. In this case, the biological endpoint can be substituted by demonstrating a 3 log reduction in the amount of heat-stable endotoxin.

[0012] Ionizing radiation sterilization is performed by exposing the product to ionizing radiation in the form of gamma rays produced by a suitable radioisotope source (e.g. Cobalt 60) or an electron beam excited by a suitable electron accelerator. During the sterilization process, the radiation absorbed by the product is periodically controlled by well-defined dosimetry procedures that allow the measurement of the dose actually received by the product, regardless of the amount of radiation produced. The dosimeters are calibrated against a standard source of a reference irradiation system upon receipt from the supplier and at appropriate intervals not exceeding one year.

[0013] Gas sterilization is only used when there are no suitable alternatives. It is very important that the penetration of gas and moisture into the material to be sterilized is guaranteed and is followed by a gas removal process under pre-defined conditions to ensure that residual gas or its transformation products in the product are below potentially toxic concentrations during the use of the product. Where possible, gas concentrations, relative humidity, temperature and process duration are measured and recorded. Measurements are made at the least likely location for achieving sterilization conditions, determined during validation. The effectiveness of the process applied to each sterilization load is verified using appropriate biological indicators.

[0014] Raw materials and products that cannot be terminally sterilized may be subjected to a filtration procedure through filters whose effectiveness has been demonstrated by a sample microbial infectivity test carried out with a suitable test microorganism, e.g. a reduced Pseudomonas suspension (ATCC 19146, NCIMB 11091 or CIP 103020). In such cases, the cm of active filtration surface 2 At least 10 per 7 CFU are used to prepare a suspension in soy tryptone broth. After passing through a filter, the suspension is aseptically collected and incubated under aerobic conditions at 32°C.

[0015] These products require special care. Manufacturing processes and environments are selected and routinely subjected to appropriate control procedures to minimize microbial contamination. Equipment, containers and closures, and raw materials, where possible, are subjected to appropriate sterilization processes.

[0016] Filtration should be performed as soon as possible after preparation of the product, and post-filtration operations should be performed under sterile conditions. Typically, filtration is performed through a bacteria-retaining membrane with pores nominally 0.22 μm in diameter or less, or through other filters with the same bacteria-retaining properties. Appropriate measures are taken to prevent loss of solutes by adsorption to the filter and to avoid release of contaminants from the latter. It is necessary to take into account microbial contamination before filtration, filter capacity, batch size and filtration period. Filters must not be used for longer than the period approved after the combined validation of the filter itself and the product to be filtered. The integrity of the assembled sterile filter is checked before use and confirmed after use by tests appropriate to the type of filter used and the stage of testing at which validation is performed, e.g. bubble point, pressure seal or diffusion rate tests. Due to other potential disadvantages of the filtration method compared to other sterilization processes, prefiltration is generally performed through a filter that retains bacteria and other particulate contaminants, when the reduction of the sterile prefiltration bioburden cannot be guaranteed by other processes.

[0017] The process of filtering heat-labile substances in order to sterilize said substances has been known for a long time, see for example the document Soelkner P et al., "Cartridge Filters", Filtration in the biopharmaceutical industry, 1988, pages 145-168, which represents the common general knowledge in the art. In this text, different types of filters are described: inter alia pleated cartridge filters made of different materials, in particular filters made of polyethersulfone, with pore sizes in the range 0.05-5 μm.

[0018] However, products containing active polymers such as hyaluronic acid or its derivatives that have heat sensitive ingredients and have relatively high viscosities can cause filterability problems.

[0019] "Filterability" refers to the filtering capacity of a filter in terms of flow rate and saturation, below and above which, respectively, the filter is not suitable for processing the product to be sterilized.

[0020] Furthermore, filtration of such products must not result in undesirable changes in the filtered product compared to the pre-filtered product, both in the functional and structural characteristics of the individual ingredients and the filtered product, since the filtered product must maintain the functionality of the pre-filtered product.

[0021] Thus, the filtered product must maintain the integrity of the individual ingredients and the viscosity of the starting product; moreover, the same pre-filtered product must have characteristics such that filterability is not adversely affected. [Prior art documents] [Non-patent literature]

[0022] [Non-Patent Document 1] Soelkner P et al., "Cartridge Filters", Filtration in the biopharmaceutical industry, 1988, pp. 145-168 Summary of the Invention [Problem to be solved by the invention]

[0023] It is therefore an object of the present invention to provide a method for the preparation of a sterile product containing heat-labile and / or bioactive ingredients, in particular polymers, which is efficient and provides a filtered product that maintains the chemical and physical characteristics and functional effectiveness of the product before filtration. Furthermore, it is an object of the present invention to provide a method that makes it possible to maintain a high filterability even in the presence of viscous products. [Means for solving the problem]

[0024] 1. A method for producing a sterile product, comprising: i) providing an aqueous solution containing a thermolabile and / or bioactive ingredient; ii) pretreating the aqueous solution by maintaining it at 40-70° C. under stirring, preferably at a speed of 100-5,000 rpm, for 1-6 hours; iii) filtering said aqueous solution through at least one filter at the same temperature as in pretreatment step ii); In order, The flow of the aqueous solution through the filter defines an outer surface of the filter (a) where the aqueous solution enters the filter and an inner surface of the filter (b) where the sterile product exits; The method, wherein the filter is a pleated filter (1, 2) comprising a layer of at least one polyethersulfone (PES) filtration membrane (11, 21) having pores with a nominal diameter of 0.25 μm or less, preferably about 0.22 μm. Effect of the Invention

[0025] Said object is achieved by the method of the present invention, the characteristics and advantages of which will become apparent from the following detailed description, from embodiments given by way of illustrative and non-limiting examples, and from the accompanying drawings, in which: [Brief description of the drawings]

[0026] [Figure 1a] Figures 1a, 1b and 1c show the effect of temperature on the viscosity of a product containing 0.5% w / w 1.6 MDa hyaluronic acid and 0.8% w / w sodium chloride in water. Figure 1a shows the viscosity of the product after different heating and stirring treatments, measured at room temperature (about 20°C). [Figure 1b] Figure 1b shows the viscosity of the products after different heat treatments, measured at the treatment temperature or up to 10°C lower than the previous treatment. [Figure 1c] FIG. 1c summarizes the results shown in FIGS. 1a and 1b (histograms of the same color or texture in FIGS. 1a, 1b and 1c represent the same heat treatment). [Diagram 2] FIG. 2 shows the effect of the same heat treatment and stirring on the viscosity of different products with increasing hyaluronic acid content according to Example 2. [Diagram 3]FIG. 3 shows the change in viscosity of two products (D and E in Example 2) at different steps of the process before and after filtration compared to the same unfiltered product according to Example 4. [Figure 4] FIG. 4 shows a comparison of a sample of MESO HAIR REVOl from Example 7 that was sterile filtered according to the method of the present invention (left image) with a sample of the same formulation that was sterilized by autoclave (right image). [Diagram 5] FIG. 5 shows a comparison of a sample of MESO WHITE REVOl from Example 7 that was sterile filtered according to the method of the present invention (left image) with a sample of the same formulation that was sterilized by autoclave (right image). [Figure 6] FIG. 6 shows a sample of SODIUM DEOXYCHOLATE REV02 from Example 7 after sterilization in an autoclave. [Figure 7] FIG. 7 shows a cartridge filter (1) according to a preferred embodiment of the invention; two images of the filter (1) are shown in cross section in A: on the left, the filter is assembled in a cylindrical container (3) and the central duct (4) of the cartridge is visible; on the right, the filter is disassembled from the container (3); in B, the filter (1) is shown in side view and removed from the container (3); in C, the layers of the multi-layer cartridge filter (1) are shown in cross section: on the left, a pleat detail is visible, showing the upstream external (10a) and internal (10b) pleats; on the right, the filter layers are separated from each other and the central filtration membrane (11) and the external (12a) and internal (12b) support membranes on either side of the filtration membrane (11) are visible; the dashed arrows indicate the flow direction of the filtered solution from the outside (a) to the inside (b) of the filter. [Figure 8]FIG. 8 shows a cartridge filter (2) according to a particularly preferred embodiment of the invention; two images of the filter (2) are shown in A in cross section: on the left, the filter is assembled in a cylindrical container (3) and the central duct (4) is visible; on the right, the filter is disassembled from the container; in B, the filter (2) is shown in side view and removed from the container (3); in C, the layers of the multilayer cartridge filter (2) are shown in cross section: on the left, a pleat detail is visible, showing the upstream external (20a) and internal (20b) pleats; on the right, the filter layers are separated from each other and the filtration membrane (21), the external (22a) and internal (22b) support membranes on either side of the filtration membrane (21), as well as the two outermost (23a and 23′a) and two innermost (23b and 23′b) rigid grids are visible; the dashed arrows indicate the flow direction of the filtered solution from the outside (a) to the inside (b) of the filter. [Figure 9] FIG. 9 shows all the layers of the filter of FIG. 8: from left to right, the second and first external rigid grids (23′a and 23a), the external support membrane (22a), the filtration membrane (21), the internal support membrane (22b), the first and second internal rigid grids (23b and 23′b) are visible; the arrows indicate the filtration flow direction from the outer surface (a) to the inner surface (b) of the filter. [Figure 10] FIG. 10 shows a diagram of the filter (2) of FIG. 9: the filtration membrane (21), the external (22a) and internal (22b) support membranes, and the external (23a, 23'a) and internal (23b, 23'b) rigid grids are visible; the flow direction through the filter of the filtered solution from the outside (a) to the inside (b) of the filter is indicated by the arrows; the external interpleat space (a') between the two external upstream pleats (20a) and the internal interpleat space (b') between the two internal upstream pleats (20b) are also shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention relates to a method for producing a sterile product, the method comprising the steps of: i) providing an aqueous solution containing a thermolabile and / or bioactive ingredient; ii) pretreating the aqueous solution by maintaining it at 40-70° C. and preferably with stirring at a speed of 100-5,000 rpm (rotations per minute of the stirring bar) for 1-6 hours; iii) filtering said aqueous solution through at least one filter at the same temperature as in pretreatment step ii); In order, The flow of the aqueous solution through the filter defines an outer surface (a) where the aqueous solution enters the filter and an inner surface (b) where the sterile product exits; The filter is a pleated filter comprising at least one polyethersulfone (PES) filtration membrane having pores of size 0.25 μm or less.

[0028] Single and multi-layer PES filters are typically used for the sterilization of aqueous liquids; the pore size varies depending on the purpose. Such filters have a low affinity for proteins and other molecules that may bind to the membrane, provide high filtration flow rates, and are compatible with a wide pH range. In addition, they can be washed to remove organic and inorganic matter that is retained by the filter.

[0029] The flow direction through the filter is indicated by the orientation of the arrows in Figures 7C, 8C and the left-hand images of Figures 9 and 10, which show the outer surface where the aqueous solution enters the filter (a) and the inner surface of the filter where the sterile product exits (b).

[0030] Preferably, the pleated filter is a multi-layer pleated filter. Preferably, the pleated filter is a cartridge filter (1, 2), as shown in Figures 7A and 8A, whose outer surface (a) faces the inner wall of the container (3) in which it is housed, while its inner surface (b) defines a central duct (4). "Pleated filter" means a filter that is pleated or folded.

[0031] In a preferred embodiment, in which said pleated filter is a cartridge multi-layer pleated filter (1, 2), the upstream pleats (10a, 20a) alternate on the outer surface (a) of the filter with the pleat lines on the inside of the filter (b), and in the downstream pleats, the pleat lines are on the outside of the filter (a); said downstream pleats on the outside of the filter correspond to the upstream pleats (10b, 20b) on the inside of the filter, as can be seen in the left side of Figures 7C and 8C and in the view of Figure 10.

[0032] As shown in FIG. 10, on the exterior of the filter, adjacent upstream pleats define an exterior inter-pleat space (a') therebetween; on the interior of the filter, adjacent upstream pleats define an exterior inter-pleat space (b') therebetween. Preferably, the multi-layer filter comprises overlapping and cross-layered membranes.

[0033] Said multi-layer pleated filter (1, 2) comprises at least three layers: as shown in the right image of FIG. 7C, a central layer consisting of said at least one polyethersulfone filtration membrane (11, 21) sandwiched between two additional layers consisting of an outer support membrane (12a, 22a) on one side of the filtration membrane and an inner support membrane (12b, 22b) on the other side of the filtration membrane, said support membranes being suitable to separate the outer upstream pleats (10a, 20a) of the filter from each other to define an interpleat space (a') of preferably 0.1 to 10 mm, more preferably 0.5 to 5 mm, even more preferably 0.5 to 2 mm. The distance between two pleats (interpleat space) is preferably measured between the apexes of two adjacent pleats. Preferably, the support membrane is also made of polyethersulfone, or nylon, cellulose or derivatives thereof.

[0034] Preferably, the filtration membranes (11, 21) have pores of 0.22 μm; preferably, the support membranes (12, 22) have pores with dimensions greater than 0.22 μm, for example pores with dimensions of 0.25 μm to 500 μm, 0.45 to 500 μm, 0.65 to 500 μm, and more preferably 100 to 500 μm. Advantageously, by preventing the pleats from approaching each other, the support membranes (12, 22) improve filtration in the process of the present invention.

[0035] More preferably, the filter further comprises at least one, and even more preferably at least two, rigid grids (23a&b, 23'a&b) after each support membrane (22a&22b), as shown in the right image of FIG. 8C, said rigid grids being adapted to form together with the support membrane a physical thickness of preferably 0.5-10 mm, more preferably 1-5 mm, such that both the outer upstream pleats (20a) and the inner upstream pleats (20b) are further widened compared to a filter not comprising a rigid grid.

[0036] Indeed, advantageously, the rigid lattice is suitable for spacing both the inner pleats (20b) increasing the inner inter-pleat spacing (b') by preferably at least 500 μm, and the outer pleats (20a) increasing the outer inter-pleat spacing (a') by at least 1000 μm, as seen in Figures 8A and B and illustrated diagrammatically in Figure 10, compared to a filter not including a rigid lattice.

[0037] The term "rigid lattice" preferably means a mesh lattice, more preferably an oval mesh of 0.5 to 1.5 mm, characterized by a flexibility lower than that of the filter membrane and the support membrane; said rigid lattice thereby helps to keep the pleats open and apart from each other. Suitable materials for forming said rigid lattice are polypropylene or high density polyethylene.

[0038] Thus, in a particularly preferred embodiment, the pleated multi-layer filter (2) comprises, from the outer surface (a) to the inner surface (b): a second outer rigid lattice (23'a) and a first outer rigid lattice (23a), a first outer support membrane (22a), a filtration membrane (21), a second inner support membrane (22b), a first inner rigid lattice (23b) and a second inner rigid lattice (23'b), as shown in Figures 8 and 9. The flow of the aqueous solution during filtration proceeds from the outside (a) to the inside (b) of the cartridges (1, 2), as shown in Figure 9, passing through all layers of the filter (2) from the outermost to the innermost. More preferably, the pleats (20a, 20b) are conical pleats. The filters employed in the process of the present invention have a higher efficiency in terms of filterability, both in terms of the filtered volume and in terms of less interaction with the raw materials used.

[0039] Preferably, the sterile product obtained by the process of the present invention is a product intended for medical and / or cosmetic use, more preferably for use in cosmetic mesotherapy, such as anti-ageing mesotherapy.

[0040] The sterile product preferably comprises as ingredients hyaluronic acid or a derivative thereof, and / or a heat labile ingredient. The term "hyaluronic acid derivatives" is meant to include: - esters of hyaluronic acid, the carboxyl groups of which are esterified in part or in whole with alcohols of the aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic series, as also described in EP 0 216 453; - self-crosslinking esters of hyaluronic acid, some or all of whose carboxyl groups are esterified with alcohol groups of the same polysaccharide chain or of other chains, as also described in EP 0 341 745; - crosslinked hyaluronic acid compounds, also described in EP 0 265 116, in which the carboxyl groups are esterified in part or in whole with polyalcohols of the aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic series and which form crosslinks via spacer chains; - half esters of succinic acid or heavy metal salts of hyaluronic acid or partial or complete esters of hyaluronic acid and succinic acid, which are also described in WO 96 / 357207, - O-sulphated derivatives (also described in WO 95 / 25751) or N-sulphated derivatives (also described in WO 1998 / 045335), and mixtures thereof.

[0041] In a particularly preferred embodiment, the hyaluronic acid or derivative thereof has a molecular weight of 500 Da to 3 MDa.

[0042] Unless otherwise specified, concentrations of one or more ingredients in an aqueous solution or sterile product are referred to herein as weight concentration (w / w) relative to the total weight of the aqueous solution or sterile product, respectively.

[0043] Preferably, the concentration of the hyaluronic acid or a derivative thereof in the aqueous solution is 0.05 to 10% w / w, more preferably 0.1 to 10% w / w, and even more preferably 1 to 10% w / w, based on the total weight of the aqueous solution.

[0044] Indeed, advantageously, the method of the invention also makes it possible to sterilize solutions containing high concentrations of polymer. Preferably, the viscosity of the aqueous solution containing heat labile and / or bioactive ingredients and the sterile product is between 100 cP and 10,000 cP.

[0045] The method of the present invention is particularly advantageous for sterilizing aqueous solutions having a viscosity of 100 cP to 10,000 cP, more preferably 1000 cP to 10,000 cP, resulting in a sterile product whose viscosity at room temperature does not substantially change from the viscosity of the solution at room temperature before sterilization (viscosity change after sterilization is maximum ±15%, preferably maximum ±10%).

[0046] Preferably, step ii) of maintaining said aqueous solution under stirring at a speed of 100-5,000 rpm is carried out at a temperature of about 60° C. for a period of 1-4 hours, more preferably for about 2 hours, or overnight at room temperature. The term "overnight" indicates that the specified conditions are maintained for 10-18 hours, typically about 12 hours. For purposes of this invention, a temperature being "about" a particular value means that the temperature is equal to that value plus or minus 2°C. "Room temperature" herein means a temperature of 20°C ± 2°C.

[0047] Without wishing to be bound by theory, it is believed that stirring the solution at the indicated speeds for the indicated periods of time, followed by filtration, results in stabilization of the formulation that promotes alignment of the polymer chains of hyaluronic acid or its derivatives, while maintaining the viscosity of the solution at a desired level and remaining constant over time. Preferably, the aqueous solution contains a salt, more preferably sodium chloride (NaCl), at a concentration of 0.01-5% w / w based on the total weight of the aqueous solution. Preferably, the filtration step iii) is carried out at a pressure between 1 and 8 bar, more preferably between 1.5 and 6 bar, even more preferably at about 5 bar.

[0048] Preferably, the method of the invention is carried out in a closed circuit plant, in which a filtration system comprising a filter is connected to an upstream thermostatically jacketed mixing tank and a downstream filling machine, said tank being adapted to carry out a pre-treatment of the aqueous solution. The invention therefore further relates to such a plant.

[0049] In a preferred embodiment, the method for producing the sterile product further comprises the steps of: iv) packaging the filtered product in vials, filling said vials with the filtered product to a desired volume and capping said vials with caps, preferably made of butyl elastomer with an aluminum ring.

[0050] In a preferred embodiment, the product obtained by the method according to the invention is a cosmetic or medical device, in particular used as an adjuvant to enhance and promote hair growth, to revitalize dull and lifeless skin, to combat the formation of expression lines and deep wrinkles, to improve the appearance of age spots, to biostimulate the regeneration of connective tissue, to increase the volume of soft tissues or to aid the action of lipolytic treatments. In another aspect, the present invention also relates to the cosmetic or medical use of the sterile product obtained by the method described above, as an adjunct in cosmetic mesotherapy or medicine.

[0051] The sterile product obtained according to the invention preferably comprises water-soluble bioactive ingredients such as hyaluronic acid, amino acids, vitamins, oligopeptides, sh-polypeptides, chelating agents, and further water-soluble ingredients such as basic and / or acidic pH adjusters, etc. The product preferably further comprises preservatives and bactericides, and optionally glycol or glycerol soluble ingredients such as terpenes and essential oils. Particularly preferred is a sterile product having the following composition: (CI = water soluble components; C = preservatives; CG = glycerin soluble components).

[0052] I. Sterile mesotherapy bio-revitalizing products suitable for treating the scalp, enhancing and promoting hair growth through improved microcirculation and stimulation of endogenous processes: [Table 1]

[0053] II. A sterile mesotherapy product that can be used as an adjunct bio-revitalizing agent in the treatment of dull skin and expression lines through the improvement of microcirculation and stimulation of endogenous processes: [Table 2]

[0054] III. Sterile mesotherapy bio-revitalizing products that can be used as adjuncts in the prevention and treatment of facial lines and deep wrinkles through the improvement of microcirculation and stimulation of endogenous processes: [Table 3]

[0055] IV. Sterile mesotherapy bio-revitalizing products that can be used as adjuncts in the treatment of age spots of various etiologies through the improvement of microcirculation and stimulation of endogenous processes. [Table 4]

[0056] V. A sterile mesotherapy bio-revitalizing product adapted to aid in the treatment of dull, lifeless skin through the improvement of microcirculation and stimulation of endogenous processes. [Table 5]

[0057] VI. Sterile mesotherapy bio-activating products used as adjuncts in the prevention and treatment of localized steatosis through the improvement of microcirculation and stimulation of endogenous processes. [Table 6]

[0058] VII. A sterile preparation containing 0.5% w / w hyaluronic acid of molecular weight 1.6 MDa. [Table 7]

[0059] Here, "% w / w" stands for "weight % of the weight of the product" and "WFI" stands for water for injection. Preferably, step i) of the method of the invention of preparing the aqueous solution comprises the following substeps: ia) a substep of mixing an aqueous solution of a water-soluble compound in water to obtain a first solution (A); ib) optionally a sub-step of mixing a preservative and / or a compound soluble in glycol or glycerol to obtain a second solution (B) and adding the second solution (B) to the first solution (A) to obtain a third solution (C); ic) adding a thermolabile and / or bioactive ingredient, if present, to the third solution (C) or to the first solution (A) to obtain an aqueous solution comprising the thermolabile and / or bioactive ingredient of step i); Here, substeps ia) to ic) are carried out at room temperature.

[0060] It is to be understood that all possible combinations of preferred aspects of the steps and sub-steps of the method, as well as the raw materials of the sterile product, as described above, are also described and therefore similarly preferred.

[0061] It should further be understood that all aspects identified as preferred and advantageous for the present method should also be considered as preferred and advantageous for the sterile product and its uses. The following examples of embodiments of the present invention are presented below by way of illustration and not by way of limitation. EXAMPLES

[0062] Example 1 A solution containing 0.5% w / w hyaluronic acid (molecular weight: 1.6 MDa) and 0.8% w / w NaCl was subjected to several heat treatments to monitor the effect of heat on the weight loss of the solution due to water evaporation. Six samples were treated at elevated temperature for 4 hours with stirring. Viscosity (Brookfield DV2T viscometer) and weight loss were measured at room temperature (20°C) or at the treatment temperature. The results are shown in Table 1a (viscosity measured at room temperature) and Table 1b (viscosity measured at approximately the same temperature as the processing temperature). Figures 1a and 1b show graphical representations of the results shown in Tables 1a and 1b, respectively.

[0063] [Table 8]

[0064] [Table 9]

[0065] The viscosity reduction in Table 1b was calculated relative to the viscosity of sample (1) kept at room temperature with stirring. It is known that the viscosity of a solution containing hyaluronic acid decreases as the temperature increases. For example, it is known that exposure of a solution containing hyaluronic acid to temperatures up to 70° C. does not result in degradation of the linear and lateral polymer chains, but rather reduces the viscosity so that it can be filtered through 0.22 μm pores. However, such a decrease in viscosity is temporary and at room temperature, if the solvent (water) has not evaporated, the solution will return to its pre-heating viscosity value. Indeed, Table 1a and Figure 1a show a higher viscosity (measured at room temperature of 20°C) after treatment at higher temperatures, which is due to the evaporation of more solvent. Conversely, Table 1b and Figure 1b show lower viscosities (measured at temperatures close to the processing temperature) at higher temperatures.

[0066] Figure 1c summarizes the results from Figures 1a and 1b and shows an overview of the results. Pretreatment at a temperature of 60°C is particularly advantageous in that at such a temperature the viscosity is reduced to a sufficient level, with a weight loss of less than 5%. The viscosity of the product maintained at 60°C is therefore favorable to facilitate filtration of the solution through 0.22 μm pores; moreover, at 60°C there is no evaporation of water, which would increase the concentration of the polymer in the solution, and there is no decomposition of the hyaluronic acid chains. Thus, by subjecting the solution to pretreatment and filtration at a temperature of about 60°C, there are particularly great advantages in terms of filterability, product shelf life, and stability.

[0067] Example 2 According to a preferred embodiment of the method of the invention, the effect of the same heating and stirring pretreatment (step ii) on the viscosity of different products with increasing hyaluronic acid (HA) content and therefore increasing viscosity was tested. The solutions tested were: sample A with 1% w / w HA 0.5MDa and 0.1% w / w HA 1MDa; sample C with 1.5% w / w HA 0.5MDa and 0.1% w / w HA 1MDa; sample D with 1.75% w / w HA 0.5MDa and 0.1% w / w HA 1MDa; sample E with 2% w / w HA 0.5MDa and 0.1% w / w HA 1MDa; sample G with 2.5% w / w HA 0.5MDa and 0.1% w / w HA 1MDa. The sample was stirred at room temperature overnight, then allowed to stand at room temperature overnight, and finally heated to approximately 60° C. and stirred for 2 hours. After each treatment, the viscosity of the different samples was measured at the same temperature as the treatment. The results (viscosity η and sample temperature at the time of viscosity measurement) are shown in Table 2 below and in Figure 2.

[0068] [Table 10]

[0069] The percentage change in viscosity measured during the three stages of the analysis (room temperature and overnight stirring, room temperature after standing overnight, and 60 °C after 2 h stirring) is consistent for the different samples: after standing overnight the viscosity of the solution increases by 14%-35% compared to the samples left stirring overnight; after 2 h of heat treatment the viscosity decreases by 50%-56% compared to the samples left standing overnight. A reproducible and predictable exponential correlation is evident between samples of increasing hyaluronic acid content at the three stages of analysis.

[0070] Example 3 The filterability of cosmetic grade hyaluronic acid with a molecular weight of 1.6 MDa was tested through a 0.22 μm classical filtration cartridge at room temperature. Filter clogging was observed on the outer surface, as if a gel-like coating had formed that did not allow the solution to pass through the pleats of the cartridge. Furthermore, the viscosity of the small amount of filtered product was much lower than that of the initial sample. This indicates that without treatment of the sample before filtration and the use of an appropriate filter, the solution cannot be filtered and therefore cannot be sterilized by filtration.

[0071] Example 4 The filterability of solutions D and E of Example 2, which contain HA of a molecular weight and concentration similar to that of a sterile product that can be used in mesotherapy, was tested using a filter (1) (see filter in Figure 7) comprising support membranes (12a, 12b) on both sides of a polyethersulfone filtration membrane (11) according to a preferred embodiment of the present invention. The viscosity of the analyzed solutions was measured at various stages of the test: - Initial viscosity of the unfiltered solution - Viscosity after stirring for 2 hours at about 60°C - Viscosity immediately after filtration - Viscosity after filtration and overnight standing - Viscosity of unfiltered solution after standing overnight - Viscosity after filtration and leaving it to stand for two nights - Viscosity of unfiltered solution after standing for two nights The viscosity values ​​of the filtered and unfiltered comparative samples measured at each stage are shown in Table 3 below (the sample temperatures at the time of viscosity measurement are shown in brackets).

[0072] The unfiltered solution (comparative solution) is observed to increase in viscosity over time. Surprisingly, the solution pretreated and filtered using a multi-layer pleated filter at the same pretreatment temperature according to the method of the present invention does not show a significant change in viscosity over time at room temperature, and maintains substantially the same viscosity as the unfiltered sample at room temperature for several days after testing (see "Initial viscosity of unfiltered solution" and "Viscosity after overnight settling after filtration" in Figure 3).

[0073] [Table 11]

[0074] The heated unfiltered solutions exhibit higher viscosity values ​​at room temperature compared to the initial solutions and tend to increase over time (see viscosity at days +1 and +2 of the unfiltered comparative solutions), which is probably due to undesirable inter-chain interactions stimulated by the heat treatment; on the other hand, the filtered solutions exhibit very similar viscosity values ​​to the initial unfiltered solutions at room temperature and such values ​​tend to remain constant over time, indicating a higher stability of the product.

[0075] Figure 3 compares the viscosity at various stages of the test, before and after filtration. In particular, it can be seen that the initial viscosity and the viscosity after filtration are almost the same. Meanwhile, the viscosity of the unfiltered solution is higher after 2 days compared to the initial solution, while the viscosity of the filtered solution is more stable after 2 days. After filtration, not only are the rheological properties of the initial solution maintained, but the compound is more stable over time compared to the unfiltered solution.

[0076] Example 5 Further tests were carried out using a multi-layer pleated cartridge filter (2) according to a preferred embodiment of the present invention (see filter (2) in Figures 8 and 9), which has a similar structure to the filter (1) used in Example 4, but is further strengthened by the presence of two rigid grids (23a and b, 23'a and b) on both sides of the filtration membrane (21) after the support membrane (22a and 22b), thus separating the pleats (20a, 20b) and reducing clogging of the cartridge. In fact, the filtration rate through these filters was even higher than that through the filter (1) of Example 4. The filtered solutions were sterile. The filters used in this example are particularly suitable for filtering solutions with high viscosity.

[0077] Example 6 Filtration tests were carried out at different operating pressures. At an operating pressure of 1.5 bar there is a loss of filtered product of 64% at 800g. However, at 5 bar there is an advantage in both increasing the filtrate flow rate and minimizing waste which amounts to 2% at 800g.

[0078] Example 7 The effect of sterilization by filtration according to the present invention was compared to the effect of sterilization by autoclave for the following formulations, where "% w / w" stands for "weight % of weight of product" and "WFI" stands for water for injection.

[0079] - MESO HAIR REV01: A sterile mesotherapy bio-activating product suitable for the treatment of the scalp, enhancing and promoting hair growth through the improvement of microcirculation and the stimulation of endogenous processes. The qualitative and quantitative composition of the product is shown in Table 4. [Table 12]

[0080] - MESO SUCCINIC+HA+AA REV00: A sterile mesotherapy product used as a general bio-revitalizing agent in the treatment of lifeless skin and facial wrinkles through the improvement of microcirculation and stimulation of endogenous processes. The qualitative and quantitative composition of the product is shown in Table 5. [Table 13]

[0081] - MESO ANTIAGE-C REV02: A sterile mesotherapy bio-revitalizing product used as an adjunct in the prevention and treatment of facial lines and deep wrinkles through the improvement of microcirculation and stimulation of endogenous processes. The qualitative and quantitative composition of the product is shown in Table 6. [Table 14]

[0082] - MESO WHITE REV01: A cosmetic mesotherapy bio-activating product used as an adjunct in the treatment of age spots of various etiologies through the improvement of microcirculation and stimulation of endogenous processes. The qualitative and quantitative composition of the product is shown in Table 7. [Table 15]

[0083] - MESO ANTIOX+HA REV01: A sterile mesotherapy bio-revitalizing product intended to help treat dull and lifeless skin through the improvement of microcirculation and stimulation of endogenous processes. The qualitative and quantitative composition of the product is shown in Table 8. [Table 16]

[0084] -SODIUM DEOXYCHOLATE REV02: sterile mesotherapy bio-activating product used as an adjunct in the prevention and treatment of localized steatosis through the improvement of microcirculation and stimulation of endogenous processes. The qualitative and quantitative composition of the product is shown in Table 9. [Table 17]

[0085] -HA1.6MDa0.5%: A cosmetic grade formulation containing 0.5% w / w hyaluronic acid with a molecular weight of 1.6MDa. The qualitative and quantitative composition of the product is shown in Table 10. [Table 18]

[0086] To assess the advantages of sterilization by filtration according to the invention compared to sterilization by autoclave, the following effects related to the changes in the qualitative and quantitative composition are taken into account: - Color change due to decomposition of thermolabile substances. Other thermolabile substances, such as preservatives, do not produce visible results but lose their effectiveness and original structure; - unacceptable pH changes due to the release of nitrogen compounds, oxidation of alcohols or cleavage of intramolecular bonds (7.30 ± 1 for mesotherapy preparations, except those intended for the eye area or other specific applications); - Decrease in viscosity due to degradation of hyaluronic acid polymer chains, which lose their viscous power and resistance to hyaluronidase even with the same initial concentration (the tolerance range of viscosity is provided by the device itself); - Unknown undesirable interactions between products and by-products; - Release of irritants such as nitrogen compounds; - Decomposition of heat-labile ingredients that lose their effectiveness (i.e. preservatives, peptides, vitamins, etc.); - Formation of flocs or sediments due to changes in pH and / or decomposition of raw materials.

[0087] In particular, the effect of autoclave sterilization compared to filtration sterilization according to the present invention is shown for each formulation on the following parameters: - pH before and after sterilization - Solution color - Viscosity before and after sterilization - Presence or absence of sediment

[0088] After sterilization by filtration according to the invention, the solution does not undergo significant chemical or physical changes compared to the starting solution.

[0089] The change in pH is shown in Table 11 and the change in viscosity in Table 12, the latter measured for the HA1.6MDa 0.5% formulation and is representative of the change in viscosity for all other formulations.

[0090] [Table 19]

[0091] [Table 20]

[0092] MESO HAIR REV01 contains thermolabile substances which, after exposure to heat in an autoclave, may result in color changes, fragmentation of the hyaluronic acid chains, inactivation of the preservative system and other ingredients, and release of by-products and / or irritants.

[0093] FIG. 4 shows a colorimetric comparison of a sample of MESO HAIR REVOl that was sterilized by filtration according to the method of the present invention (left) and a sample of the same formulation that was sterilized by autoclave (right).

[0094] MESO SUCCINIC+HA+AA REV00 contains thermolabile substances which, after exposure to heat in an autoclave, may result in fragmentation of the hyaluronic acid chains, inactivation of the preservative system and other ingredients, and release of by-products and / or irritants.

[0095] MESO ANTIAGE-C REV02 contains thermolabile substances which, after exposure to heat in an autoclave, may increase the pH, fragment the hyaluronic acid chains, inactivate the preservative system and other ingredients, and potentially release by-products and / or irritants.

[0096] MESO WHITE REV01 contains thermolabile substances which, after exposure to heat in an autoclave, may result in a decrease in pH, a change in color, fragmentation of the hyaluronic acid chains, inactivation of the preservative system and other ingredients, and the possible release of by-products and / or irritants.

[0097] FIG. 5 shows a colorimetric comparison of a sample of MESO WHITE REVOl that was sterile filtered according to the methods of the present invention (left) and a sample of the same formulation that was sterilized by autoclave (right).

[0098] MESO ANTIOX+HA REV01 contains thermolabile substances which, after exposure to heat in an autoclave, may result in a change in color, fragmentation of the hyaluronic acid chains, inactivation of the preservative system and other ingredients, and the release of by-products and / or irritants.

[0099] SODIUM DEOXYCHOLATE REV02 contains thermolabile substances that, after exposure to heat in an autoclave, may result in the formation of a white precipitate, fragmentation of the hyaluronic acid chains, inactivation of the preservative system and other ingredients, and the release of by-products and / or irritants (see Figure 6).

[0100] Table 13 below summarizes the effects seen after autoclave sterilization of different formulations. [Table 21]

[0101] After sterilization by filtration according to the invention, the undesirable effects reported after autoclave sterilization were not detected. Therefore, the method of the present invention provides a high quality, safe and sterile product and ensures that the qualitative and quantitative composition of the product after filtration is identical to that of the initial one.

[0102] Example 8 For manufacturing scale-up, 2 kg of MESO ANTIAGE-C REV02 (see composition in Table 6), which represents the most viscous and most important product among the formulations studied, was filtered through a 5-inch PES 66 filter; the product was allowed to run continuously for approximately 2 minutes to completely filter. Thus, the same 30-inch filter (1.8 m 2 ) is estimated to be able to filter 20-30 kg of similar product without clogging.

[0103] Example 9 Sterile products, including but not limited to the formulations analyzed in the previous examples, are obtained by the following manufacturing process: 1. Mix the water-soluble components in water at approximately 20°C (Solution A). 2. Adding premixed solution B, which contains preservatives and glycol and / or glycerol soluble ingredients, to solution A (solution C); 3. Add hyaluronic acid to solution C at approximately 20°C (solution D). 4. Measure the pH of solution D. 5. Stir at 100-5,000 rpm at 20°C overnight. 6. Measure the viscosity. 7. Pretreat by stirring at 30-70°C and 100-5,000 rpm for 1-3 hours; 8. Sterilize by filtration at the same pretreatment temperature at a pressure of 1.5-6 bar. 9. The final filtered product is packaged in vials, filled to the desired volume, and closed with butyl elastomer caps and aluminum rings to obtain a product similar to that shown in Figures 4 and 5; 10. Perform necessary tests to demonstrate sterility of the product. [Explanation of symbols]

[0104] 1: Cartridge filter 2: Cartridge filter 10a: External upstream pleats 10b: Internal upstream pleats 11: Polyethersulfone filtration membrane 12a: External support membrane 12b: Internal support membrane 20a: External upstream pleats 20b: Internal upstream pleats 21: Polyethersulfone filtration membrane 22a: External support membrane 22b: Internal support membrane 23a: External rigid grid 23'a: External rigid grid 23b: Internal rigid grid 23'b: Internal rigid grid A: First solution B: Second solution C: The third solution

Claims

1. 1. A method for producing a sterile product, comprising: i) providing an aqueous solution containing a thermolabile and / or bioactive ingredient; ii) pretreating the aqueous solution by maintaining it at 40-70°C, preferably with stirring at a speed of 100-5,000 rpm for 1-6 hours; iii) filtering said aqueous solution through at least one filter at the same temperature as in pretreatment step ii); in order, The flow of the aqueous solution through the filter defines an outer surface of the filter (a) where the aqueous solution enters the filter and an inner surface of the filter (b) where the sterile product exits; The method, wherein the filter is a pleated filter (1, 2) comprising a layer of at least one polyethersulfone (PES) filtration membrane (11, 21) having pores with a nominal diameter of 0.25 μm or less, preferably about 0.22 μm.

2. 2. The method of claim 1, wherein the pleated filter (1, 2) is a multi-layer filter comprising at least three layers, the polyethersulfone filtration membrane (11, 21) forming a central layer sandwiched between two further layers consisting of an outer support membrane (12a, 22a) and an inner support membrane (12b, 22b) adapted to space the pleats (10a, 20a) of the filter, preferably by 0.1 to 10 mm.

3. 3. The method of claim 2, wherein the pleated filter (2) further comprises at least one or at least two outer rigid grids (23a, 23'a) arranged more outwardly relative to the outer support membrane (22a) and at least one or at least two inner rigid grids (23b, 23'b) arranged more inwardly relative to the inner support membrane (22b), the rigid grids being adapted to space the pleats (20a, 20b) of the filter apart, preferably by 0.5 to 10 mm, more preferably by 1 to 10 mm.

4. 2. The method according to claim 1, wherein the thermolabile and / or bioactive ingredient is hyaluronic acid or a derivative thereof having a molecular weight of 500 Da to 3 MDa and a weight concentration in the aqueous solution of 0.05 to 10%, preferably 1 to 10%, relative to the total weight of the aqueous solution.

5. 2. The method of claim 1, wherein step ii) of pretreating the aqueous solution is carried out at a temperature of 50 to 70°C, preferably at about 60°C.

6. 2. The method of claim 1, wherein the filtering step iii) is carried out at a pressure of 1 to 8 bar, preferably 1.5 to 6 bar, more preferably about 5 bar.

7. Step i) of providing the aqueous solution comprises: i. a) a substep of mixing a water-soluble compound in an aqueous solution with water to obtain a first solution (A); i. b) optionally mixing a preservative and / or a compound soluble in glycol or glycerol to obtain a second solution (B) and adding the second solution (B) to the first solution (A) to obtain a third solution (C); i. c) adding a thermolabile and / or bioactive ingredient, if present, to the third solution (C) or to the first solution (A) to obtain an aqueous solution comprising the thermolabile and / or bioactive ingredient of step i); in order, 10. The method of claim 1, wherein substeps i.a) to i.c) are carried out at room temperature.

8. 8. A closed-circuit plant for carrying out the method according to any one of claims 1 to 7, comprising a filtration system comprising at least one of said pleated filters (1, 2) for filtering said aqueous solution, said at least one filter being connected to at least one upstream thermostatically jacketed mixing tank and a downstream filling and capping machine, said at least one tank being configured to carry out a pre-treatment of said aqueous solution.

9. Cosmetic use of a sterile product obtained by the method according to any one of claims 1 to 7 in cosmetic mesotherapy or aesthetic medicine, said sterile product preferably being in the form of an injectable solution.

10. A sterile product obtainable by the method according to any one of claims 1 to 7, preferably in the form of an injection, intended for use in dermatology.

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