IMPROVED DEPYROGENATION PROCESS

TECHNICAL FIELD

The present invention relates to a washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field.

In particular, the products to be washed and/or depyrogenated are preferably containers of various types, made of glass (for example glass bottles and vials, dry or wet), metal or other material, indicatively for pharmaceutical, chemical and/or medical use, caps, water for injection, glucose solutions, medical devices, and finished pharmaceutical and cosmetic products, including cross-linked sodium hyaluronate.

The subject matter of the invention further relates to a method for reducing native endotoxins (depyrogenation) in objects and substances in the medical and/or chemical-pharmaceutical field, characterised by using the washing and/or depyrogenation machine of the invention.

STATE OF THE ART

Bacterial endotoxins, commonly known by the name of pyrogens, are structural components of the outer membrane of Gram-negative bacteria and they are mainly released upon the death of the latter, but also during their growth and division. In terms of chemical structure, endotoxins are lipopolysaccharides composed of a hydrophilic polysaccharide (O- antigen), a central sugar chain (R nucleus) and lipid A (responsible for the toxic effects) composed of an amino sugar and a fatty acid.

Endotoxins are the antigenic parts of Gram-negative bacteria, i.e. the parts on the basis of which the antibodies of the host organism develop.

Endotoxins are complex, diversified bioactive substances: however, antibodies are formed only on the basis of the matrix of the O- polysaccharide chain, whilst the lipid part (the toxic one) varies its structure in the various types of endotoxins, thus reducing the organism’s possibility of defending itself. Endotoxins are mainly responsible for the clinical consequences of infections caused by Gram-negative bacteria, as the effects triggered by endotoxins in the human body are many: in fact, they have a pyrogenic activity, and induce endotoxic shock, the Schwartzman reaction, adrenaline reaction, bone marrow reaction, blood coagulation, a decrease and increase in leukocytes, thrombocytopaenia, hypoglycaemia, a reduction in serum iron, activation of the complement system, adjuvant action, immunostimulatory action, immunosuppressive action, antitumor action, protection against radiation-induced damage, and tolerance to or release of cortico-adrenal hormones. Among these, the exothermic activity is characteristic.

Endotoxins are extremely resistant to heat and to physical agents; for this reason, they are frequent environmental contaminants.

Therefore, especially in the process for the manufacture of pharmaceutical products, parenteral preparations in particular, endotoxins, or pyrogens, must be detected and removed to control quality (depyrogenation). Endotoxins are amphiphilic and form micelles; hence, their molecular morphology is not uniform, and their activity is manifested even when their molecular weight decreases and the chemical treatment is relatively stable. Therefore, it is difficult to remove or completely eliminate endotoxins from instruments and samples. For example, in order to remove endotoxins from heat-resistant devices such as glass and stainless-steel bottles and vials and inorganic substances such as sodium chloride, it is necessary to use intense heating that carbonises the organic matter, such as conditions of dry heat at 250° C for 30 minutes or more. Depyrogenation thus proves more difficult than sterilisation.

Depyrogenation conditions with dry heat, however, might not be suitable for pharmaceutical and/or cosmetic and/or nutraceutical products, in which, by law, harmful quantities of endotoxins must not be present.

Other methods for reducing endotoxins envisage treatment with gamma rays, treatment with an electron beam, treatment with acetylene gas, treatment with low-temperature hydrogen peroxide gas plasma, and treatment with low-temperature hydrogen peroxide/peracetic acid gas plasma. In these depyrogenation methods, 70-90% of the endotoxins can be inactivated in an early phase, but removing the remaining part is very difficult.

Other methods for reducing endotoxins envisage a heat treatment with fatty emulsions, a treatment with quaternary ammonium fat, the use of an acid or a base, the use of an organic solvent, the use of a surfactant, or removal by means of an apparatus for treatment with reverse-osmosis membranes.

It is thus known that endotoxins are sensitive to alkalis or acids: under alkaline conditions, the fatty acids bound to lipid A are released. Under acidic conditions, the central polysaccharide is easily split and converted into lipid A, which is poorly soluble in water, and the phosphate bound to glucoside is easily eliminated in the molecule of lipid A. The methods for chemically modifying endotoxins include alkylation reactions such as acetylation, succinylation and phthalylation.

The United States pharmacopeia recommends depyrogenation by means of a dry heat treatment at temperatures above 220° C for a duration long enough to obtain a logarithmic reduction of endotoxin activity greater than or equal to 3 if the value of endotoxin units is greater than or equal to 1000 (EU)/ml. The term EU describes the biological activity of endotoxins. For example, 100 pg of standard endotoxin EC-5, 200 pg of EC-2 and 120 pg of endotoxin from E. coli O111 :B4 all have an activity of 1 EU (Hirayama, C., M. Sakata (2002). “Chromatographic removal of endotoxin from protein solutions by polymer particles”, J. Chromatogr. B 781 :419-432).

Despite the ongoing research on endotoxins, however, no universal method for the targeted reduction and removal of native endotoxins has yet been found.

There is thus a felt need for a method for reducing endotoxins that is versatile and overcomes the disadvantages of the known methods. The use of washing and/or rinsing systems adapted to clean and depyrogenate objects and substances is known in the medical and/or chemical-pharmaceutical field.

The washing and depyrogenation machines of the known type comprise three contiguous thermal chambers through which a conveyor belt passes, its function being to transfer the products/containers arriving from washing machine through the first chamber, called the inlet chamber, through the second chamber, called the depyrogenation chamber, and through a third chamber, called the cooling chamber.

In particular, the products to be washed and depyrogenated are preferably containers of various types, made of glass, (for example glass bottles and vials, dry or wet), metal or other material, indicatively for pharmaceutical, chemical and/or medical use, caps, water for injection, glucose solutions, medical devices, and finished pharmaceutical and cosmetic products, including cross-linked sodium hyaluronate.

In particular, there are known washing and/or rinsing systems of the batch type, based on machines of different sizes where the objects and the substances to be treated are presented to the washing systems after being loaded onto a loading trolley and/or on special loading racks present in the machines themselves. Such machines are commonly called “rack washers” in the sector.

Loading trolleys and loading racks are usually made in such a way as to expose the objects to be treated, the substances and/or the accessory parts thereof to the washing systems present in the machines.

The washing systems can be of various types, for example: with fixed bars, swinging bars, translating bars or rotating arms.

Rack washers have a washing chamber that is typically parallelepipedshaped, where at least either the front wall or the rear wall is used as a loading/unloading zone, whilst the washing system is installed on at least one of the two opposite side walls (typically on both). Some rack washers also have a washing system positioned on the upper surface of the washing chamber, i.e. in proximity to the zone defined as the roof or ceiling of the washing chamber. The trolleys also have spray nozzles.

The trolleys of a known type feature shelves for containing the parts of the objects and/or products to be treated and which enable them to be placed and contained in such a way as to correctly expose the surfaces to be washed (in particular the internal/hidden and dirtiest parts) to the direct washing jets, and so that most of the surfaces of the parts of the objects and/or products to be treated are inclined for drainage in order to reduce to a minimum stagnant buildups of fluid at the end of the cycle and facilitate drying.

It should be noted that in the present context, the term “surface to be washed” is meant to indicate a surface of the object that must be directly struck by pressurised fluid jets in order to be correctly processed, i.e. washed and/or rinsed.

It is thus evident that the effectiveness of the washing and/or rinsing system is measured by the capacity of the system itself to reach most of the surfaces of the objects to be washed/rinsed directly with the jets of the washing/rinsing solution so as to act upon said surfaces with the mechanical action (impact force) of the jets.

It is well known, in fact, that in some types of objects and/or products to be treated, above all on the internal surfaces of the bases, if present, the residual dirt can be removed in part by chemical treatment (with the use of specific detergents and suitable washing and/or rinse temperatures), but a strong mechanical action is also necessary to be able to remove a large part of the residual dirt and give the chemical the possibility of acting only on the residual layer by reducing the presence of organic material on the load.

In these machines of a known type, it is in fact possible to provide for specific pre-washing phases where the action on the load is only of a mechanical nature in order to remove most of the dirt and carry it towards the machine drainage outlet.

The Applicant has thus observed that the washing of objects and/or products to be treated involves important critical aspects tied mainly to the efficiency and effectiveness of the jets of washing fluid.

Directing the washing fluid correctly is in fact fundamental to obtain an effective washing of the surfaces to be washed of the objects present in the washing chamber.

The coverage capacity of the washing system, understood as the ratio between the total surface to be washed of the objects and/or products to be treated and the actual surface of the objects that can be washed by the machine (i.e. the surface that can actually be reached directly by the fluid jets coming out of the nozzles), should be as close as possible to 1 .

The washing devices installed in the washing chambers typically comprise hollow bars or tubing connected to a pressurised fluid source and provided with nozzles from which the washing fluid is delivered.

Such solutions generally use jets directed perpendicularly to the banks and which strike the inside of the objects to be treated.

It is evident, however, that this solution has a major functional limit, which is that of not being able to adequately cover the internal corners of the exposed objects, which thus remain unexposed to a direct impact.

Typically, in fact, the bases of the objects, such as for example containers, are positioned inclined to facilitate the drainage of the washing fluid; however, this positioning makes it difficult for the fluid jets to reach some internal corners because they are covered by the side walls of the bases These areas of the objects that cannot be struck directly by the jets are defined as “blind zones”.

The presence of “blind zones” leads to the presence and incomplete elimination of any bacterial endotoxins.

This inevitably means the presence of bacterial endotoxins at the end of every washing cycle. As the removal of endotoxins takes place by mechanical removal, dependent on jets of water on the parts to be washed, the presence of blind zones could represent an obstacle to an adequate removal of the residual endotoxins.

In this context, the object of the present invention is thus to propose a washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field that makes it possible to overcome the drawbacks mentioned above with reference to the prior art.

In particular, the general object of the present invention is to propose a washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field, the use of which allows for carrying out an optimal, rapid, efficient washing and depyrogenation of objects and/or substances.

A further object of the present invention consists in the fact of providing a washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field which prevents the spread of unwanted contaminations through the system of conveyance of the containers to be treated.

Another object of the present invention is to provide a washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field which assures high standards of reliability under every operating condition.

A further object of the present invention is to enable a rapid diffusion and uniform distribution of steam inside the washing and depyrogenation chamber.

Another object of the present invention is to enable a rapid and uniform distribution of temperature inside the washing and depyrogenation chamber.

Another object of the present invention is to prevent or minimise the formation of zones of steam stagnation inside the washing and depyrogenation chamber. A further object of the present invention is to prevent or minimise the formation of zones of condensed steam around the products to be treated. Another object of the present invention is to enable thermal uniformity throughout the internal volume of the washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field.

Another object of the present invention is to provide a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field by hydrothermal depyrogenation (heat inactivation) at a temperature below 200° C, preferably below 150°C and above 120°C, preferably comprised in a temperature range comprised between 121 °C and 140°C (where the endpoints of the range are included), with a treatment duration varying in a range between 30 and 60 minutes, preferably of 30 minutes.

According to a preferred aspect, the method of the invention for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field comprises the use of the washing and/or depyrogenation machine of the invention.

According to a further object of the present invention, the method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field comprises the use of the washing and/or depyrogenation machine of the invention with a temperature of the depyrogenation fluid below 200°C, preferably below 150°C and above 120°C, preferably comprised in a temperature range comprised between 121 °C and 140°C (where the endpoints of the range are included), with a treatment duration varying in a range between 30 and 60 minutes, preferably 30 minutes.

DEFINITIONS

Unless otherwise defined, all the terms of the art, notations and other scientific terms used herein are intended to have the meanings commonly understood by those who are skilled in the art to which this description pertains. In some cases, terms with commonly understood meanings are defined herein for the sake of clarity and/or for ease of reference; the inclusion of such definitions in the present description should thus not be interpreted as representing a substantial difference from what is generally understood in the art.

The terms “comprising”, “having”, “including” and “containing” are to be understood as open-ended terms (i.e. the meaning of “comprising, but not limited to”) and are to be considered as a support also for terms like “consist essentially of”, “consisting essentially of”, “consist of” or “consisting of”.

“Depyrogenation” means a process of eliminating/reducing pyrogenic substances.

“Pyrogen” means any substance able to cause an increase in body temperature (fever). Pyrogens are distinguished into endogenous, i.e. present in the organism, and exogenous, i.e. introduced from the outside. The most common exogenous pyrogens are represented by bacterial endotoxins.

“Endotoxin load” means the quantity of pyrogens present on a given sample/product, either solid or liquid.

“Logarithmic reduction value” (LRV) means the logarithm of the result of the difference between the endotoxin concentration before treatment of the sample and the endotoxin concentration after treatment of the sample to reduce the endotoxin load.

“Reduction of the endotoxin load” means a reduction in the quantity of endotoxins in a sample. Historically, the regulatory requirement for the logarithmic reduction value was to observe, preferably, a logarithmic reduction of at least 3. Today the United States pharmacopeia mentions that a logarithmic reduction of at least 3 may be an excessive requirement, i.e. a requirement for a logarithmic reduction to an excessively reduced endotoxin load. “Hydrothermal depyrogenation” or “heat inactivation” means a depyrogenation process with moist heat at temperatures below 200°C, preferably below 150°C and above 120°C, preferably comprised between 121 °C and 140°C, i.e. temperatures below the ones indicated in the United States pharmacopeia for dry heat depyrogenation. Some synonyms are: moist heat thermal inactivation or moist heat inactivation, or moist heat depyrogenation, or reduction with moist heat.

The acronym “CSE” (control standard endotoxin) means the standard secondary endotoxin.

The acronym “RSE” means the reference standard endotoxin.

The acronym “NOE” (naturally occurring endotoxin) means the native endotoxin.

The acronym “LPS” means lipopolysaccharides.

The term “reduce” means decrease.

“Native endotoxin(s)” or “autochthonous endotoxin(s)” means endotoxins (pyrogens) naturally present in a sample and/or deriving from possible contaminations in the production chain.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field.

In particular, the present invention regards a washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field, comprising: a closed washing and/or depyrogenation chamber 2 intended to contain the products to be depyrogenated, said chamber 2 identifying an internal compartment and being provided with an opening that identifies a passage for inserting/removing the products to be sterilised; a removable door associated in closing with said opening so as to pass from an open position, away from said opening, to a closed position, in which it assures a hermetic sealing of said passage of said depyrogenation chamber 2; a means for the introduction, into said depyrogenation chamber (2), of a depyrogenation fluid under pressure for a predetermined time interval, wherein the depyrogenation fluid has a temperature below 200°C.

In a second aspect, the invention relates to a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field, comprising at least one hydrothermal depyrogenation (heat inactivation) cycle carried out at a temperature below 200°C, preferably below 150°C and above 120°C, preferably comprised in a temperature range comprised between 121 °C and 140°C (where the endpoints of the range are included), with a treatment duration varying in a range between 30 and 60 minutes, preferably 30 minutes.

In a third aspect, the invention relates to a method for reducing the native endotoxin load (depyrogenation) in objects and substances in the medical and/or chemical-pharmaceutical field, characterised by using a washing and/or depyrogenation machine to obtain said reduction (depyrogenation). In particular, the products to be washed and depyrogenated are preferably containers of various types, made of glass (for example glass bottles and vials, dry or wet), metals or other materials, indicatively for pharmaceutical, chemical and/or medical use, caps, water for injection, glucose solutions, medical devices, and finished pharmaceutical and cosmetic products, including cross-linked sodium hyaluronate.

Thanks to the washing and/or depyrogenation machine it is possible to carry out a heat inactivation of the endotoxins by means of a moist process at a lower temperature (light hydrothermal process) compared to a dry treatment: by controlling the parameters of the steam/air percentage (in the case of a counter-flow treatment), temperature and duration of the treatment (time) it is possible to decrease, preferably by at least 3 times, the logarithmic reduction value of the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field, preferably containers of various types, made of glass (for example glass bottles and vials, dry or wet), metals or other materials, indicatively for pharmaceutical, chemical and/or medical use, caps, water for injection, glucose solutions, medical devices, and finished pharmaceutical and cosmetic products, including cross-linked sodium hyaluronate.

BRIEF DESCRIPTION OF THE FIGURES

Additional features and advantages of the present invention will become more apparent from the approximate, and thus non-limiting, description of a preferred but not exclusive embodiment of a washing and/or depyrogenation machine for the depyrogenation of objects and substances, as illustrated in the accompanying drawings, in which:

- figure 1 shows an exploded perspective view of a machine for washing and/or depyrogenation of objects and substances according to the present invention;

- figure 2 shows a section of figure 1 in a perspective view;

- figure 3 shows a front sectional view of the machine in figure 1 .

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention regards a washing and/or depyrogenation machine for the depyrogenation of objects and substances in the medical and/or chemical-pharmaceutical field.

With reference to figure 1 , it shows a schematic view of a washing and/or depyrogenation machine containing a washing chamber 2, inside which the objects and the substances to be washed and depyrogenated are arranged.

Preferably, the objects and the substances to be washed and depyrogenated are arranged on one or more shelves 22 of a trolley 20, which during use is inserted inside the washing and depyrogenation chamber 2.

The washing and depyrogenation chamber 2 is closed and is intended to contain therein the products to be washed and depyrogenated. The chamber 2 defines an internal compartment and is provided with at least one opening that identifies a passage for inserting/removing the products to be sterilised - by way of non-limiting example, by means of the trolley 20.

A removable door is associated in closing with said opening so as to pass from an open position, away from said opening, to a closed position, in which it assures a hermetic sealing of said passage of said washing and depyrogenation chamber 2.

During use, the chamber 2 is hermetically sealed by the door and a depyrogenation agent (e.g. steam and/or a mixture of air and steam) is fed inside it as described further below.

The washing and/or depyrogenation machine 1 can have a cylindrical or quadrangular section or one of any other shape.

The washing and depyrogenation chamber 2 can extend on a horizontal axis or a vertical axis (such as, for example, in the case of small mechanical components used in laboratories).

In the present description, vertical axis of the washing and/or depyrogenation machine 1 means an axis perpendicular to the floor on which the machine rests during its operation.

The washing process is based on a series of consecutive steps defined on the basis of the dirt to be removed and adapted for cleaning the load.

The removal could take place with or without the use of detergent, with an initial step of injecting steam to soften the adherent dirt, at a high or low temperature, according to the product to be washed and the detergent used.

The step that follows takes place according to the rules of saturated steam or counterpressure sterilisation, depending on the load in question.

In a second aspect, the invention relates to a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field by hydrothermal depyrogenation (heat inactivation) at a temperature below 200°C, preferably below 150°C and above 120°C, preferably comprised in a temperature range comprised between 121 °C and 140°C (where the endpoints of the range are included), with a treatment duration varying in a range between 30 and 60 minutes, preferably 30 minutes.

The method of the present invention envisages reducing the native endotoxins (depyrogenation) by using the above-described washing and/or depyrogenation machine to obtain said reduction. The process can comprise a washing step, which has the purpose of mechanically removing chemical contaminants from the surfaces to be depyrogenated and can likewise contribute to removing contaminants of a biological natura, such as endotoxins.

In the method of the invention, to reduce the endotoxin load of objects and substances in the medical and/or chemical-pharmaceutical field, such as, for example, containers of various types, made of glass (for example glass bottles and vials, dry or wet), metals or other materials, indicatively for pharmaceutical, chemical and/or medical use, caps, water for injection, glucose solutions, medical devices, and finished pharmaceutical and cosmetic products, including cross-linked sodium hyaluronate, at least one heat inactivation cycle (process) is carried out with moist heat at temperatures below 200°C, preferably below 150°C and above 120°C, preferably comprised in a temperature range comprised between 121 °C and 140°C (where the endpoints of the range are included), with a treatment duration varying in a range between 30 and 60 minutes.

The pressures used are preferably comprised in the range of 2 to 6 bar absolutes.

According to the product, cycles with saturated steam or cycles with an air/steam mixture, under counterpressure, are carried out. The choice of one process or the other is closely correlated with the type of load: the saturated steam cycle will be suitable for empty containers, the one under counterpressure for hermetically sealed containers with an aqueous solution inside. It is possible to carry out the at least one heat inactivation cycle under the above-described conditions of temperature, pressure, and duration, working with the washing and/or depyrogenation machine using pure saturated steam or an air/steam mixture under counterpressure.

Advantageously, the particular treatment conditions in terms of duration, temperature and pressure combined with the particular washing and/or depyrogenation machine make it possible to obtain a reduction in the endotoxin load with a logarithmic reduction value preferably of at least 3, also on native endotoxins, not only purified standards.

In fact, the contaminant used to assess the depyrogenation under the operating conditions and the washing and/or depyrogenation machine of the invention was, alternatively:

- a commercial secondary standard, that is, a secondary standard endotoxin (CSE - control standard endotoxin);

- a NOE (naturally occurring endotoxin - native endotoxin);

- autochthonous endotoxins present in the tested products and the result of a probable contamination in the production chain.

The secondary standard endotoxin CSE, which is an extract of E. coli O55:B5, is widely used as a standard for endotoxin testing, as an alternative to RSE.

CSE is standardised against RSE, so the results can be expressed in Endotoxin Units (EU) and International Units (IU).

The differences between purified lipopolysaccharides (LPS) and native endotoxins (NOE) are shown in table 1 below:

Table 1

In view of the differences between purified LPS (CSE) and the native endotoxins (NOE) shown in table 1 , it is not foreseeable that the conditions of temperature, duration and pressure used with the washing and/or depyrogenation machine of the invention would be capable of reducing the endotoxin load of products such as medical devices, containers made of glass, (for example glass bottles and vials), water for injection, and finished pharmaceutical and cosmetic products, including cross-linked sodium hyaluronate, preferably with a logarithmic endotoxin load reduction value of at least 3.

The subject matter of the invention thus relates to a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field, comprising at least one hydrothermal depyrogenation (heat inactivation) cycle carried out at a temperature below 200°C, preferably below 150°C and above 120°C, preferably comprised in a temperature range comprised between 121 °C and 140° C (where the endpoints of the range are included), with a treatment duration varying in a range comprised between 30 and 60 minutes, preferably 30 minutes. According to a preferred aspect, the subject matter of the invention relates to a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field that comprises the use of the washing and/or depyrogenation machine of the invention.

In particular, the subject matter of the invention relates to a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field comprising the use of the washing and/or depyrogenation machine of the invention with a temperature of the depyrogenation fluid below 200°C, preferably below 150°C and above 120°C, preferably comprised in a temperature range comprised between 121 °C and 140°C, and with a treatment duration varying in a range between 30 and 60 minutes.

According to a preferred aspect, the invention relates to a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field comprising the use of the washing and/or depyrogenation machine of the invention, wherein the temperature of the depyrogenation fluid is 130°C with a treatment duration varying in a range between 30 and 60 minutes, preferably 30 minutes. According to another preferred aspect, the invention relates to a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field comprising the use of the washing and/or depyrogenation machine of the invention, wherein the temperature of the depyrogenation fluid is 140 °C with a treatment duration varying in a range between 30 and 60 minutes, preferably 30 minutes.

According to another preferred aspect, the invention relates to a method for reducing the native endotoxin load in objects and substances in the medical and/or chemical-pharmaceutical field comprising the use of the washing and/or depyrogenation machine of the invention for the depyrogenation of objects and substances in the medical and/or chemicalpharmaceutical field. Said objects and substances in the medical and/or chemical-pharmaceutical field are selected from containers of various types, made of glass (for example glass bottles and vials, dry or wet), metals or other materials, indicatively for pharmaceutical, chemical and medical use, caps, water for injection, glucose solutions, medical devices, pharmaceutical and cosmetic finished products, including cross-linked sodium hyaluronate.

The invention is illustrated below by means of experimental examples, which are not to be considered as limiting for the object of the invention.

EXAMPLES

Example 1 - reduction of endotoxin load of glucose solutions.

Use was made of the washing and/or depyrogenation machine of the invention in a condition of calibration performed with the LAL method, i.e. with the kinetic chromogenic method. The depyrogenation was carried out with an air-steam mixture to reduce the endotoxin load of a test tube containing 1 ml of a 10% glucose solution (product which can be terminally depyrogenated) and 1 ml of a solution containing NOE (“raw” endotoxins, such as to represent, better than a standard endotoxin (CSE), the normal contamination of the product). The negative control consisted of a test tube with 1 ml of 10% glucose solution.

The machine of the invention was used with counterpressure, at a depyrogenation temperature of 140°C for a duration of 30 minutes, at a pressure of 4.8 bar absolute.

As shown in table 2 below, a logarithmic reduction value greater than 3 was obtained.

Positive control: test tube with 1 ml NOE + 1 ml of 10% glucose solution.

Table 2 - results of the cycle for reducing native endotoxins in a washing and/or depyrogenation machine, carried out under counterpressure at 140°C for 30 minutes; Glucose 10%; NOE: CRF02100.

Example 2 - reduction of endotoxin load on dry glass.

Along the same lines as in example 1 , dry glass vials were inoculated with endotoxins, CSE or NOE, and depyrogenated

CSE: from E. Coli 055:B5, Endosafe, potency: 2.5 million EU - the endotoxin indicator was rehydrated with 1 ml of LAL aqueous reagent. The dry vials were contaminated with 0.1 ml of the endotoxin indicator and allowed to dry.

NOE: from Enterobacter Cloacae ATCC 7526, potency 9465 EU/ml - the vials were contaminated with 0.1 ml of the endotoxin indicator and allowed to dry.

Two hydrothermal (depyrogenation) treatments were carried out: one treatment was performed at 140°C (treatment A) for 30 minutes and one at 121 °C for 30 minutes (treatment B). The results obtained are shown in table 3 below.

Table 3

Example 3 - reduction of endotoxin load on wet glass. Along the same lines as in example 1 , wet glass vials were inoculated with endotoxins, CSE or NOE, and depyrogenated

CSE: from E. Coli 055:B5, Endosafe, potency: 1 million EU - the endotoxin indicator was rehydrated with 10 ml of LAL aqueous reagent; this solution was diluted 1 :2 with WFI. The vials were contaminated with 1 ml of the endotoxin indicator.

NOE: from Enterobacter Cloacae ATCC 7526, potency 9465 EU/ml - the vials were contaminated with 1 ml of the endotoxin indicator.

Two hydrothermal (depyrogenation) treatments were carried out: one treatment was performed at 140°C (treatment A) for 30 minutes and one at 121 °C for 30 minutes (treatment B). The results obtained are shown in table 4 below.

Table 4

Example 4 - reduction of endotoxin load on cross-linked sodium hyaluronate.

3 samples (syringes) containing 2 ml of cosmetic sodium hyaluronate (filler, gel) were tested with the kinetic chromogenic method to determine the endotoxin load thereof, which proved to be 41.85 EU/ml in the first sample, 37.14 EU/ml in the second sample and 18.49 EU/ml in the third sample.

The endotoxins contained in the samples of cross-linked sodium hyaluronate were autochthonous toxins resulting from the production process for obtaining the gel.

One hydrothermal treatment (depyrogenation cycle) was carried out at 130° C for 30 minutes. The results obtained are shown in table 5 below.

Table 5