WO2011001402A2

WO2011001402A2 - Method for inactivating at least one pathogen in human blood plasma sample

Info

Publication number: WO2011001402A2
Application number: PCT/IB2010/053017
Authority: WO
Inventors: Gérard Demazeau; Nolwennig Rivalain; Alain Largeteau; Jean Roquain; Jean-Michel Boiron; Jean-Paul Maurel; Zoran Ivanovic
Original assignee: Universite Bordeaux 1; Universite Victor Segalen - Bordeaux 2; Etablissement Francais Du Sang
Priority date: 2009-07-03
Filing date: 2010-07-01
Publication date: 2011-01-06
Also published as: CN102762231A; WO2011001402A3; FR2947456B1; FR2947456A1; BR112012000018A2; EP2448601A2

Abstract

The present invention relates to a method for inactivating at least one pathogen of a human blood plasma sample, including: adiabatically compressing said sample, the temperature of said plasma being, at a pressure P₁, between -10°C and -3°C as a result of the compression; optionally maintaining the pressure P₁ for a time of 110 to 130 seconds; and adiabatically expanding the sample. The present invention also relates to the use of the method of the invention and to the plasma obtained by the method of the invention. The method and the materials obtained by said method are particularly useful in the therapeutic and non-therapeutic fields.

Description

METHOD FOR INACTIVATION OF AT LEAST ONE PATHOGENIC AGENT IN A HUMAN BLOOD PLASMA SAMPLE

DESCRIPTION

Technical area

The present invention relates to a method of inactivating at least one pathogen of a human blood plasma sample, to a plasma obtained by this method and to a device for implementing the method.

[002] The human blood plasma obtained is a biologically active plasma in which at least one pathogen is inactivated.

[003] The method and the products obtained by this method find particular applications in the therapeutic and non-therapeutic areas.

[004] In the description below, references in parentheses (Ref) refer to the list of references presented at the end of the text.

State of the art

[005] Securing human blood plasma, which can be considered as the inactivation of pathogens and the safeguarding of its therapeutic properties, is currently provided by chemical methods. These are processes comprising the addition to the plasma of chemical substances capable of decontaminating it, for example solvents and detergents, etc., processes combining chemical substances and physico-chemical processes, for example photosensitive molecules associated with illumination. by a light radiation of given wavelength (UV for example), or thermal processes, for example pasteurization at 60 ⁰ C after addition of Protein stabilizers, filtration, gamma irradiation ... (Ref.1) C. Naegelen et al. [Evolution of labile blood product preparation (PSL) techniques: inactivation of pathogens in PSL], Clinical and Biological Transfusion 16 (2006) pp.179-189.

[006] These methods are binding because any addition of a foreign substance in the human blood plasma implies at the end of the process a step of elimination and / or purification of the plasma or that they affect the properties of the plasma . This final step does not make it possible to obtain an "identical" blood plasma. In fact, the elimination of chemical substances is never complete and can lead to long-term toxicity, for example by accumulation. Furthermore, the treatment of the plasma at high temperatures, that is to say greater than or equal to 60 ⁰ C denatures the proteins present and therefore requires to add further chemicals to stabilize these proteins.

[007] High pressure treatment processes are known in the food industry to inactivate certain pathogens such as molds, bacteria and sometimes viruses. For example A. JOFRE et al. [LWT Food Science and Technology 42 (5) (2009) p.924-928, entitled "Efficiency of high hydrostatic pressure at 600 MPa against micro-organisms by challenge test convenience") (Ref.2) describe the application of a pressure of 600 MPa to secure food.

[008] Other methods have been used to inactivate pathogens, for example Staphylococcus aureus (S. aureus) in ham (CC TASSOU et al., "Temperature-assisted high hydrostatic pressure inactivation of Staphylococcus aureus in a ham model System: in-selective and non-selective medium evaluation" J. Applied Microbiology 104 (2008), 1764-1773 ( Ref.3) The results obtained showed that it was necessary to apply a pressure of 500 MPa at 20 ⁰ C for 45 minutes in order to reach a reduction of 5 log of the amount of present pathogens. A reduction of 6 log was reached at 400 MPa with heating of the medium at 55 ° C during the first 7 minutes of treatment. The pressures and temperatures used in this article do not permit the maintenance of the biological activity of human blood plasma.

[009] Previous work has also highlighted that it was in particular necessary to achieve high values of the pressure (about 500 MPa) in order to obtain a reduction at ordinary temperature, that is to say at 20 ° C. ⁰ C of 6 log of the population of Staphylococcus aureus (S. aureus) in a model medium such as tryptone-salt broth [Y. RIGALDIE: "On the Impact of High Pressure Treatments in the Decontamination and Sterilization of Pharmaceutical Forms Containing Therapeutic Molecules Sensitive to Energy Processes" Thesis of the University Bordeaux 1 (July 01, 2002) Order No. 2526 (Ref. 4)]. The high pressure values necessary to inactivate certain pathogens including S. aureus are an important constraint for the development of a method of inactivating a pathogen in blood plasma while maintaining the biological activity of said plasma.

[0010] Moreover, in Y. RIGALDIE et al. [Pharmaceuticals perspectives of High Pressures: Defect and Diffusion Forum 208-209 (2002), 55-58 (Ref 5)] has shown that inactivation of S. aureus by continuous high pressure treatment reached in a model medium (for example a tryptone-salt broth) about 2.3 log for a treatment of 10 minutes at 600 MPa and on the other hand that low temperatures (-17 ⁰ C) had little effect on inactivation compared to ambient temperature.

[0011] It is known that human blood plasma has specific biological properties. Indeed, the plasma comprises about three hundred different proteins. The most important proteins in proportion are albumin, antibodies or immunoglobulins, fibrinogen, alpha 1 antitrypsin, alpha 2 macroglobulin, transferrin, lipoproteins (HDL and LDL). It also includes coagulation factors that are taken in most cases as a reference for measuring the biological activity of plasma. Plasma is "biologically active" when the activity of coagulation factors, including the factors City, V and Xl present in the plasma is greater than 50% (European Pharmacopeia 5.6 01/2007: 1646 (Ref 6). may, through some of its proteins, interact with the membranes of certain microorganisms (A. KUNERT et al., The Journal of Immunology 179 (2007), 2979-2988 (Ref.7) or induce sporulation (the introduction into the plasma of a suspension composed of approximately 50% vegetative form of B. subtilis and 50% sporulated form of B. subtilis very rapidly leads to almost complete sporulation).

Various studies have shown that the treatment of blood plasma at pressures between 200 and 500 MPa at a temperature of -10 ⁰ C was not interesting for the preservation of blood products and that this method was not to retain for the inactivation of viruses and microorganisms in blood products (AM MATSER et al., High Pressure Processing for Preservation of Blood Products, High Pressure Research 25 (2005), p.37-41) (Ref.8).

International Application WO 02/056824 (Ref 9) describes a method for sterilizing blood products using pressure values that are incompatible with the preservation of the biological activity of the blood plasma. Different combinations of temperatures and pressures are tested, however these combinations do not maintain the activity of the coagulation factors Ville, V and Xl in order to obtain a biologically active human blood plasma. Moreover, in this document, the methods differ according to the pathogen; which makes these processes difficult to industhalable.

Other tests have been carried out in the state of the art to inactivate S. aureus in blood plasma ("Deciphering

Mechanisms for Infectious Agent Inactivation Using Cyclic High Pressure Gold Pressure Cycling Technology F. TAO et al. (BBI Inc. - Boston Biomedica Inc.) BIOPHYSICS 2003 - Poster 021604 (Ref.10).

In the article by S. DUSING et al. ["Inactivation of viruses in plasma by cycled fields of high pressure", Trends in High Pressure Bioscience and Biotechnology, Ed. R. HAYASHI, Elsevier Science BV (2002)] (Ref.11), only the mode of application of the pressure, in particular 60-second cycles at high pressures, that is to say between about 400 and 500 MPa, separated by 300 seconds at ambient pressure and low temperatures, that is to say about -20 ^° C. C. The pressures used in this document are not compatible with the conservation of the biological activity of human blood plasma.

Also, there is a real need in the state of the art to overcome these defects, disadvantages and obstacles of the prior art, in particular a method for inactivating at least one pathogen in the human blood plasma. and also to keep the plasma protein activity, for example the Ville, V and Xl factors, so that the plasma is biologically active.

There is also a real need in the state of the art to find a method for inactivating at least one pathogen in the human blood plasma and also to keep the biological activity without introduction of external products, by example chemicals, and without subsequent purification step to remove introduced products that may be toxic, for example by accumulation or any other process.

In particular, there is a real need in the state of the art to find a more efficient method to reduce costs, reduce the time required for inactivation and improve the inactivation efficiency. at least one pathogen while maintaining the biological activity of human blood plasma.

Description of the invention The present invention solves such defects, disadvantages and obstacles of the prior art by providing a method of inactivating at least one pathogen of a human blood plasma sample comprising at least 5 cycles each comprising :

compressing said sample of its initial pressure Po at a pressure P _1, P ₁ eta com pri nt sde 1 90-21 0 M Pa, the temperature of said plasma to the pressure P ₁ is between -10 ⁰ C and -3 ° C due to compression,

the possible maintenance of the pressure P ₁ for a time ranging from 1 to 130 seconds, and

the expansion of the sample up to the pressure P ₀ , the rate of increase of the pressure for the said compression and the rate of decrease of the pressure for the said expansion are carried out at a speed V _{1 of} between 47.5 and 52 5

MPa.s ^"1 .

In other words, the present invention relates to a method of inactivating at least one pathogen of a human blood plasma sample comprising at least 5 cycles each comprising:

the expansion of the sample up to the pressure P ₀ , said compression and said expansion being performed at a speed V ₁ of 47.5 to 52.5 MPa.s ^-1 . Surprisingly and completely unexpectedly, the inventors have demonstrated that the combination of the different parameters defined in the process of the invention makes it possible to inactivate at least one pathogenic agent with a much higher efficiency than the processes of the state of the invention. technique, and further allows to maintain the biological activity of the sample of human blood plasma treated.

In addition, the inventors have shown that, surprisingly and unexpectedly, the characteristics of the process of the invention have a synergistic effect on the inactivation of biological targets, for example at least one pathogenic agent, thus allowing inactivate said targets while maintaining the biological activity of the human blood plasma sample.

In addition the method of the invention allows the inactivation of at least one pathogen without introduction of external products and does not require a purification step thus reducing the time and costs of production.

The method of the invention therefore advantageously to inactivate at least one pathogen present in the human blood plasma but also to maintain the biological activity of said sample of human blood plasma.

In the present "sample of human blood plasma", it is understood any plasma sample from a human regardless of its origin, age, sex, size and / or weight. The sample may also have been previously treated by any technique known to those skilled in the art for, for example, its preservation, for example by freezing

In the present "inactivation" is meant the suppression of the biological activity of at least one pathogen or set of pathogens. For example, inactivation does not allow recovery of the biological activity of the pathogen. Inactivation can be observed by any method known to those skilled in the art. By for example, it may be the absence of growth of a pathogen in a medium adapted for its growth and / or to transmit its pathogenic activity ["Pathogen inactivation techniques", JPR PELLETIER, S. TRANSUE, EL SNYDER, Best Practice & Research Clinic Haematology vol.19 (1) (2006), pp.205-242] (Ref.12)

In the present "pathogen" is meant any biological entity known to those skilled in the art. For example, it may be a bacterium, a mold, a virus, a yeast, an infectious protein and / or parasites. For example, the pathogen may be selected from the group comprising the pathogens of the European Pharmacopoeia described in European Pharmacopoeia 6.0, 5.1 General Texts on Microbiology 01/2008: 50101 (Ref.13), for example it may be act as disease-causing pathogens, for example Candida albicans, Aspergillus Niger, Bacillus subtilis or responsible for nocosomal diseases, for example Staphylococcus aureus, Pseudomonas aeruginosa.

In the present text, "adiabatic" is understood to mean the increase or decrease in temperature resulting from compression or decompression, in particular of a liquid or gaseous component in a closed system because of the first principle of thermodynamics. [E. HECHT «Physics» Publisher: De Boech (1999)] (Ref 14)

In the present the initial pressure P ₀ is for example the pressure at which the sample is located before implementing the method of the invention. For example, the pressure P ₀ may be equal to the atmospheric pressure, or any blood plasma storage pressure less than Pi. The initial pressure may be, for example equal to about 0.1 MPa.

In the present "rate of increase and / or decrease of the pressure", it is understood a speed expressed in Mega Pascal per second (MPa, s ^"1 ) It can be linear and / or variable, in the latter case we speak rather of kinetics.

According to the invention, the human blood plasma sample to which the process of the invention is subject is preferably contained in any container known to those skilled in the art to be resistant to the pressures of the process of the invention. For example, in hermetic and / or deformable containers, for example a sampling tube, a plastic bag for transfusion, a sterile container, a specific packaging, for example packages described in Y. LAMBERT, G. DEMAZEAU, A. LARGETEAU, S. LABORDE-CROUBIT, M. CABANNES and JM BOUVIER, ["New packaging solutions for high pressure treatments of foods". High Pressure Research. 2000, vol 19, 207-212] (Ref 15).

For example, the package may comprise an inner surface in contact with the plasma chemically and biologically compatible with the plasma and an outer surface in contact with the transmitting medium chemically compatible with the latter, for example in terms of permeability, etc. as described in the Pharmacopoeia

European 5.0 01/2005: 30203 (Ref 16)

The packaging may for example comprise a connector, that is to say means for filling and / or empty packaging. For example, a connector may allow the filling and / or the use, for example therapeutic human blood plasma after implementation of a method according to the invention.

For example, the hermetic package may be any package known to those skilled in the art preferably responding to the following 4 criteria: (i) the preservation of hermeticity of said packaging and its connectors, (ii) the chemical compatibility between the inner surface of the packaging and its connection with the plasma, and with a pressure transmitting medium for its external part, (iii) the maintenance of the barrier properties in particular in order to avoid any gas transfer or chemical molecules through the wall, (iv) safeguarding the chemical integrity of the package, that is to say the non-release of chemical molecules to the plasma container.

In the present, the pressure can be applied isostatically by means of a pressure transmitter liquid.

In the present "application of pressure isostatically", it is understood the uniform application of pressure at any point of the sample.

In the present "transmitting medium", it is understood any medium known to those skilled in the art may be used in the method of the invention. For example, it may be a liquid or gaseous medium. For example, it may be a liquid medium that is easy to implement and often chosen as a function of its melting temperature, especially when it is used at low temperature, for example a medium whose melting point is less than the treatment temperature of the sample, for example an apolar liquid medium, for example hexane, glycol or a polar medium, for example propanol, n-butanol, ethanol, acetic acid, isopropanol, preferably the liquid transmitting medium is selected from the group consisting of a glycol and water mixture, for example Kryo Lauda, and ethanol.

The application of pressure isostatically allows advantageously to compress and relax uniformly and without pressure gradient at any point in the sample.

In the present "biological activity of a sample of human blood plasma", it is understood an activity of factors City, V and Xl present in the plasma greater than 50% as indicated by the standard of the Pharmacopoeia European Union for Viro-inactivated Human Plasma (European Pharmacopoeia 5.6 01/2007: 1646 (Ref.6) and / or greater than 70% for the City factor and greater than 50% for the V and Xl factors as required by the Legislation French (Ref.17). In the present example, the initial temperature T ₀ of the sample before compression can be chosen, for example, as a function of the rate of rise in pressure Vi, of the chemical nature of the millet and pressure transmitter, and of the value pressure. For example, the initial temperature may range from -18 ° C to -3 ° C.

According to a particular embodiment of the method of the invention, the compression and / or the expansion are adiabatic.

In the present invention, the pressure Pi may be equal to

200 MPa.

In the present temperature of said plasma pressure

Pi may be from -10 ⁰ C to -3 ° C, preferably from -8 ° C to -4 ° C, even more preferably equal to about -5 ° C.

In the present invention, the pressure Pi can be maintained for a period of 110 to 130 seconds per cycle, for example for a duration of 120 seconds per cycle.

The present invention also relates to the use of the method defined above for the inactivation of at least one pathogen and / or a set of pathogens in human blood plasma.

The present invention also relates to the human blood plasma obtained by the method defined above.

The human blood plasma obtained by the method of the invention can be advantageously used in the medical field, for example for the treatment of various pathologies, for transfusions as described in the document of the French Agency of

Health Safety of Health Products "Transfusion of fresh frozen plasma: products, indications" (2002) (Ref 18) and / or any other pathology.

The plasma obtained by the process of the invention advantageously has the biological properties of the blood plasma. according to the standards of the European Pharmacopoeia and also of the French Legislation.

Other advantages may still appear to those skilled in the art on reading the examples below, illustrated by the accompanying figures, given for illustrative purposes.

Brief description of the figures

FIGS. 1A to C are histograms representing the percentage of plasma protein activity (ordinate) as a function of pressure in Mega Pascal (MPa) (abscissa). In particular, they respectively represent the effect of the value of the pressure on the activity of coagulation factor VIIIIc (FIG. 1A), coagulation factor V (FIG. 1B) and coagulation factor X1 (FIG. 1C). the application rate is 3.33 MPa. s ^"1 , the application time, also referred to as maintaining the pressure, for 10 minutes at room temperature.

FIGS. 2A-C are histograms representing the percentage of plasma protein (ordinate) activity as a function of pressure in MPa (abscissa) for three different rates of application of pressure. In particular, they respectively represent the effect of the rate of application of the pressure on the activity of the coagulation factor Ville (FIG. 2A), the coagulation factor V (FIG. 2B), the coagulation factor X1 (FIG. 2C). ) for different pressure values (100, 150, 200 and 250 MPa), the pressure being applied for 10 minutes at room temperature.

FIGS. 3A to C are histograms representing the percentage of plasma protein activity (ordinate) as a function of the pressure in MPa (abscissa) and of different modes of application of the pressure and for the same total duration of treatment. The application rate of the pressure is 3.33 MPa. s ^"1 at room temperature. In particular, Figures 3A, 3B and 3C respectively show the effect of the pressure application mode on the activity of coagulation factors VIIIIc, V and XI in continuous mode (10 minutes), cyclic modes (5 cycles of 2 minutes and 20 cycles of 30 seconds).

FIGS. 4A to C are histograms representing the percentage of plasma protein activity (ordinate) as a function of the pressure in MPa (abscissa) for two values of the temperature: ambient and

-15 ° C. The application rate of the pressure is greater than or equal to 50 MPa. s ^"1 and the time of application of the pressure 10 minutes. In particular, Figures 4A, 4B and 4C respectively show the effect of temperature on the activity of coagulation factors depending on the value of the pressure .

FIGS. 5A-C are histograms representing the percentage of plasma protein (ordinate) activity as a function of the pressure in MPa (abscissa), the rate of application of the pressure and the temperature. In particular, FIGS. 5A-C respectively show the effect of the combination of a fast application rate, a cyclic application of pressure and temperatures on the activity of coagulation factors VIIIIc, V and Xl , the application speed being greater than or equal to 50 MPa. s ^"1 , the application time of 2 minutes repeated 5 times and the temperature of -15 ° C.

Fig. 6 is a diagram illustrating the inactivation of Staphylococcus aureus in human blood plasma (ordinate) as a function of temperature (abscissa) for a combination of parameters characteristic of the treatment: a rate of application of the pressure of 50 MPa. s ^"1 , a cyclic mode of application of the pressure of 5 cycles of 2 min and for a pressure of 200 MPa.In particular, this figure represents the drastic effect of the temperature on the inactivation by high pressures of Staphylococcus aureus suspended in human blood plasma.

Fig. 7 is a diagram illustrating the inactivation of Staphylococcus aureus (ordinate) as a function of temperature. (abscissa) in human blood plasma for increasing pressure values ranging from 200 to 300 MPa.

FIG. 8 is a diagram showing the inactivation of S. aureus, P. aeruginosa, A. niger and C. albicans (ordinate) as a function of temperature (abscissa). In particular, this figure represents the effect of temperature on the inactivation of 4 microorganisms suspended in a sample of human blood plasma.

FIG. 9 is a histogram showing the inactivation of Staphylococcus aureus (ordinate) as a function of the pressure (abscissa) and the rate of application of the pressure. In particular, this figure represents the effect of the rate of application of the pressure on the inactivation of Staphylococcus aureus suspended in human blood plasma by selecting as mode of application of the pressure 5 cycles of 2 minutes and a temperature about -5 ° C.

FIG. 10 is a histogram showing the percentage of coagulation factor activity (ordinate) versus pressure (abscissa) for various values of the rate of pressure application. Figure 10A shows the percentage of City factor activity (FVIIIc) and Figure 10B the percentage of factor V (FV) activity. In particular, this figure represents the effect of the rate of application of the pressure on the activity of coagulation factors during a cyclic treatment (5 ^{χ 2} min) at low temperature (temperature of about -5 ° C. ).

FIG. 11 is a photograph showing the optical microscope observation (Objective * 100) of blood contaminated with Plasmodium berghei before (A) and after treatment with the "High Pressure Plasma Safety Pressing Method" (B). (N: Core, C: Cytoplasm, V: Vacuole)

FIG. 12 represents the result of a Western Blot analysis of human blood plasma samples infected or not by the infectious protein of the hamster prion (263 K), TIx: Infected control not treated with the method, HPIx: Sample infected treated by the method, TNx: Witness Negative untreated by the process, HPNx: Negative control processed by the process.

FIG. 13 is a histogram showing the destructive efficiency (ED) (ordinate) as a function of the pressure (abscissa). The application rate of the pressure was 3.33 MPa. s ^"1 , the continuous mode of application (MA), the treatment time of 10 minutes and the treatment temperature of 25 ° C.

FIG. 14 is a histogram showing the destructive efficiency (ED) (ordinate) as a function of the pressure (abscissa). The application rate of the pressure was 3.33 MPa. s ^"1 (full sticks) or 50 MPa. s ^{" 1} (hatched sticks).

FIG. 15 is a histogram showing the destructive efficiency (ED) (ordinate) as a function of the pressure (abscissa). The application rate of the pressure was 3.33 MPa. s ^"1. The application of the pressure mode was 5 cycles of 2 minutes (hatched bars), 20 cycles of 30 seconds (open bars) or a 10-minute cycle (solid bars)

FIG. 16 is a histogram showing the destructive efficiency (ED) (ordinate) as a function of the pressure (abscissa). The temperature was -5 ° C (hatched sticks) or 25 ° C (full sticks)

FIG. 17 is a diagram showing the inactivation of S. aureus, P. aeruginosa, A. niger, C. albicans and B. subtilis (ordinate) as a function of temperature (abscissa).

FIGS. 18A and B are histograms representing the percentage of plasma protein activity (% act) (ordinate) as a function of pressure (P) in Mega Pascal (MPa) (abscissa). In particular, they respectively represent the effect of the value of the pressure on the activity of coagulation factor VIIIIc (FIG. 18A), coagulation factor V (FIG. 18B).

FIG. 19 represents the intensity of the fluorescence (ordinate) as a function of the dilution factor (abscissa). The crosses correspond to the results obtained with the method (HP), the black squares to the results obtained with a control "T".

FIG. 20 represents the intensity of the fluorescence (ordinate) as a function of the dilution factor (abscissa). The crosses correspond to the results obtained with the method (HP), the black squares to the results obtained with a control "T".

Figure 21 shows the FACS diagrams. On the SSC diagrams corresponds to the signal correlated to the cell complexity, FSC: signal correlated to the relative size of the cells, "event" to the total number of events (cell) in the defined population,% Parent to the number of events in the population defined by the total number of events expressed as a percentage and "mean" as the mean value of the fluorescence intensity.

EXAMPLES

Example 1 Measurement of the biological activity of human blood plasma after treatment under high pressures

Samples of about 4 mL of fresh human blood plasma were collected and placed in sealed tubes, and then frozen at -30 ⁰ C waiting to be treated under high pressure.

The treatment conditions were as follows: a pressure application rate of 100 MPa in 30 seconds (3.33 MPa.s ^-1 ), a treatment time of 10 minutes continuously at the desired pressure. that is to say from 100 MPa to 250 MPa and an ambient temperature of the order of 20 ^° C.

The samples were placed in a high pressure liquid phase direct compression type generator device. "Framatome", the transmitting medium was the glycol / water mixture (Kryo 30

Lauda).

By continuous application of the pressure, it is understood a constant application of pressure to a given value.

The activity of coagulation factors: factor Ville, factor V and factor Xl was chosen as a reference in order to determine the biological activity of human blood plasma. This activity was determined before and after treatment with the activated partial thromboplastin time method described in the STA CK instructions. PREST (Diagnostica Stago) (Ref 19). This assay consists in measuring, in the presence of cephalin and activator, the coagulation time of an immuno-depleted plasma factor to be assayed, the missing coagulation factor being provided by the sample to be tested. For each coagulation factor, the percentage of residual activity is then determined by the ratio between the activity value after high pressure treatment and the activity value before high pressure treatment. The effect of the value of the pressure on the percentage of residual activity for these three coagulation factors is shown in FIGS. 1A to C.

Analysis of the residual activity of coagulation factors vis-à-vis the pressure, shown in Figure 1, showed that the activity of factor Xl was little affected after treatment at pressures of 100 MPa and 250 MPa (1000 bar and 2500 bar), however the factors City and V were, in this pressure range, very sensitive to the pressure causing a strong loss of activity, that is to say of the order of 80 to 100% for a pressure of about 250 MPa.

As demonstrated in this example, the treatment of blood plasma at increasing pressures of 100 MPa to 250 MPa leads to a sharp decrease in the activity of the factors City and V which leads to consider that any method of securing the plasma human blood, that is to say, any process allowing the inactivation of pathogenic micro-organisms by application of high pressures, is accompanied by a significant loss of activity of clotting factor activity and thus loss of biological activity of blood plasma.

Example 2 Measurement of the biological activity of human blood plasma after treatment under high pressures as a function of the rate of application of the pressure

Fresh human blood plasma was prepared according to the method described in Example 1. The treatment conditions were: a treatment time of 10 minutes continuously at a given pressure ranging from 100 to 250 MPa, at a temperature of the order of 20 ^° C.

The rate of application of the pressure was chosen as a variable. In this example, three speeds have been chosen with sufficiently different values on the one hand and values accessible for an industrial equipment produced by the skilled person on the other hand, that is to say 100 MPa in 30 seconds (or 3.33 MPa s ^-1 ) (value identical to the rate of application of the pressure used in Example 1), 100 MPa in 12 seconds (8.33 MPa.s ^-1 ) and 100 MPa in 2 seconds (50 MPa.s ^"1 ).

The samples were placed in a high-pressure liquid-phase direct compression generator device of the "Framatome" type and the transmitting medium was the glycol / water mixture (Kryo Lauda) for the speeds of 3.33 MPa. s ^"1 and 8.33 MPa.s ^{" 1} . Indirect compression high pressure generating equipment set up internally with ethanol transmission medium was used for the speed of 50 MPa.s ^"1

The biological activity of the human blood plasma was measured after treatment with the percentage of activity of the coagulation factors as described in Example 1 (City factor, factor V and factor Xl) measured by the method of time. The percentage of residual activity relative to the activity before high pressure treatment of each of the coagulation factors for the same pressure values but with application rates. variable pressure: 3.33 MPa. s ^"1 , 8.33 MPa s ^{" 1} and 50 MPa. s ^"1 are shown in Figure 2.

The analysis of the results presented in FIG. 2 showed that the rate of application of the pressure in the range of 3.33 MPa. ¹ to 50 MPa.s ^-1 , regardless of the value of the treatment pressure and regardless of the nature of the clotting factor (City factor, factor V, factor X1) has little effect (to experimental errors near) on the activity of coagulation factors, at least for pressures between 100 MPa and 250 MPa.

The lack of effect of the rate of application of the pressure on the biological activity of the plasma is surprising considering the technical teaching described in the literature, for example in the international application WO 00/48641 ( Ref 20) which recommends very fast pressure increases up to 1000 MPa. s ^"1. Furthermore, the application of these very rapid increases in pressure are very difficult to implement with industrial equipment.

Example 3 Measurement of the biological activity of human blood plasma after treatment under high pressures depending on the mode of application of the pressure

Fresh human blood plasma was prepared according to the method described in Example 1. The treatment conditions under high pressures were: a rate of application of the constant pressure, equal to 100 MPa in 30 seconds (ie 3 , 33 MPa. S ^"1 ), the temperature of the order of 20 ⁰ C, a treatment time of 10 minutes at a pressure ranging from 100 to 250 MPa.

The samples were placed in a high-pressure liquid phase direct compression generator device of the "Framatome" type and the transmitting medium was the glycol / water mixture (Kryo Lauda). The mode of application of the pressure varies according to a continuous mode, that is to say the maintenance of the constant pressure for a time of 10 minutes, or a cyclic mode, that is to say 5 cycles of 2 minutes at the selected pressure or 20 cycles of 30 seconds at the selected pressure. The cycles correspond, for example to compression of the sample, the maintenance of the pressure for a suitable time and the expansion of the sample. The duration of the cycles has been chosen in such a way that it can be transposed on an industrial scale.

The biological activity of the human blood plasma was measured by the percentage of residual activity of the coagulation factors as described in Example 1 (City factor, factor V and factor Xl). The activity of these coagulation factors was measured by the activated partial thromboplastin time method described in the STA CK Instructions for Use. PREST (Diagnostica Stago) (Ref 19).

The values obtained for the residual activity of the three coagulation factors taken as references at the end of these three types of treatment (one in continuous mode and two in cyclic mode) for a corresponding pressure application speed. at 3.33 MPa. s ^"1 and a temperature of the order of 20 ^° C. are given in FIG.

The comparison of the application of a pressure in cyclic mode (5 cycles of 2 minutes) compared to an application in continuous mode for the same duration of 10 minutes and for the same value of the applied pressure (the speed application and temperature being constant) shows a difference in behavior between the City factor and the factor V.

Factor X1 always had a particular behavior compared to the other two coagulation factors insofar as its activity was little or not (with errors of experience) disturbed by the pressure.

As demonstrated in this example, the factor V is more sensitive than the factor City to the cyclic mode of application of the pressure, that is, its residual activity appears to be more saved for cyclic application than for continuous application for the same value of pressure.

In addition unexpectedly and surprisingly, this phenomenon is more pronounced for a cyclic mode corresponding to 5 cycles of 2 minutes compared to 20 cycles of 30 seconds.

Example 4: Measurement of the biological activity of human blood plasma after treatment under high pressures as a function of temperature

Fresh human blood plasma was prepared according to the method described in Example 1.

The treatment conditions were: a rate of application of the constant pressure and equal to 50 MPa. s ^"1 , a continuous treatment time of 10 minutes at a pressure of between 100 and 300 MPa.

The samples were placed in a high pressure generator equipment by indirect compression set up internally with ethanol as the transmitting medium.

In this example, two different treatment temperatures were tested, either the ambient temperature (approximately 20 ^° C.), or a negative temperature close to -15 ° C.

The measurement of the biological activity of the human blood plasma was measured by the percentage of residual activity of the coagulation factors as described in Example 1 (City factor, factor V and factor Xl) measured by the method. activated partial thromboplastin time described in the STA CK instructions. PREST (Diagnostica Stago) (Ref 19).

The percentages of residual activity (relative to the activity before high pressure treatment) of the three coagulation factors taken as references at the end of these treatments (for a speed application of the pressure of 100 MPa in 2 seconds (ie 50 MPa.s s ^-1 ), a duration of treatment of 10 minutes in continuous mode) but with two different temperatures (20 ^° C. and -15 ° C.) are data in Figure 4.

It appears surprisingly and unexpectedly that the three coagulation factors have different behavior vis-à-vis the temperature variation (between 20 ⁰ C and -15 ° C approximately). Factor X1 remains insensitive to temperature, within the limits of experimental errors, and retains an activity close to 90%, whatever the conditions. On the other hand, the saving of activity of the factors Ville and V is favored at low temperatures, in particular it is for the factor V that this phenomenon seems most amplified. For example, at 220 MPa at -15 ° C., the factor V activity is greater than 50% (and thus meets the standards of the European Pharmacopoeia), whereas under the same pressure conditions but at ambient temperature, it is to say at about 20 ^° C, it is very low (about 10%).

As demonstrated in this example, a significant synergistic effect between low temperature and rate of application of the pressure exists for the conservation of the activity of factors City and V, clearly amplified for the factor V.

EXAMPLE 5 Measurement of the biological activity of the human blood plasma after treatment under high pressures by combining three parameters that are characteristic of the treatment: the rate of application of the pressure, the mode of application of the pressure and a low temperature

Fresh human blood plasma was prepared according to the procedure detailed in Example 1.

The samples were placed in a high pressure generator equipment by indirect compression set up internally with ethanol as the transmitting medium. The treatment process consisted in associating a set of parameters associated with the pressure: rate of application of the pressure, mode of application of the pressure and temperature, in particular low temperature, ie a temperature about -15 ° C.

Different pressure values between 100 MPa and 250 MPa were tested. The values of the associated parameters were 50 MPa. s ^"1 for the application rate of the pressure, 5 cycles of 2 minutes for the application mode and a temperature of -15 ° C.

The percentage of residual activity of the coagulation factors already taken as references in Example 1 (City factor, factor V and factor Xl) was as in Example 1 measured by the partial thromboplastin time method with activator .

The percentages of residual activity (relative to the activity before high pressure treatment) of the three coagulation factors, at the end of a treatment under the conditions described above, are shown in FIG. 5.

As demonstrated in this example, surprisingly and unexpectedly, a very large synergistic effect is observed through the combination of the parameters associated with the pressure parameter for the factors Ville and V (the most important effect being observed for the factor V). For example, at 240 MPa (2400 bar) the activity of the factor V is of the order of 90% and that of the neighboring City factor of 70%. These values of activities are compatible with the French legislation (OJ n ° 123 of May 28, 2003, Order of April 29, 2003 fixing the list and the characteristics of labile blood products) (Ref 17) and allows, in addition the therapeutic use obtained human blood plasma.

If we refer to the example No. 1 where only the value of the pressure was taken into account, we observed at 250 MPa a value of the activity close to 10% for the factor City and close to zero for factor V. As demonstrated in this example, the combination of the 3 parameters associated with the pressure: rate of application of the pressure, mode of application of the pressure and temperature makes it possible, surprisingly, to maintain the activity of the pressure factors. coagulation and therefore the maintenance of the biological activity of human blood plasma.

Example 6: Example of inactivation of a pathogen in human blood plasma at high pressures as a function of temperature.

[0090] Staphylococcus aureus is a well-identified pathogen and causes many nosocomial diseases. This pathogen was chosen as a model in this example.

A dense suspension, ie greater than 10 ⁸ CFU / mL

(CFU: Colony Forming Unit) of S. aureus (ATCC 6538) was prepared in tryptone-salt broth (TS) (AES Chemunex) from a 48h culture on Tryptic Soy Agar (TCS) (AES Chemunex) . This suspension was used to contaminate human blood plasma at the rate of 10 ml of suspension per 300 ml of plasma.

The contaminated plasma samples were then placed in sterile and sealed tubes, then kept in the freezer awaiting treatment by an inactivation process.

Before and after the inactivation process, a count of the samples is made in order to determine the destructive efficiency of this treatment. For this purpose, cascade dilutions of the samples are carried out in TS broth. For each dilution, two petri dishes are inoculated as follows: 1 ml of the dilution is placed at the bottom of the dish and then supercooled TCS agar is poured. After homogenization and gelation, the petri dishes are put in an oven at 35 ° C for 48h. Boxes containing between 15 and 300 isolated colonies are then selected for enumeration, and, after counting colonies, the concentration of S. aureus is determined by the following calculation:

N = ≥

(H ₁ + 0.1 X n ₂ ) xd

with N: concentration of S. aureus (in CFU / mL)

Σc: sum of the colonies counted on all the boxes retained nor: number of boxes retained at the first dilution

n ₂ : number of boxes retained at the second dilution

d: dilution rate of the first dilution [0094] The destructive efficiency of the treatment is then calculated as follows:

with ED: destructive efficiency

N: concentration of S. aureus after high pressure treatment

N ₀ : concentration of S. aureus before high pressure treatment

The inactivation treatment used was that described in Example No. 5. For a constant value of the pressure (200 MPa), the values of the associated parameters were a speed of 50 MPa. After ¹ , 5 cycles of 2 minutes for the mode of application, only the temperature was taken into account as a variable (from -25 ° C to + 35 ° C).

Figure 6 shows the effect of temperature during such treatment on its destructive efficacy vis-à-vis S. aureus.

Quite unexpectedly and surprisingly, it appears a narrow temperature range between -10 ⁰ C and -5 ° C where the effectiveness destructive S. aureus is amplified. This same phenomenon is observed by changing with the same apparatus the nature of the transmitting medium: the glycol / water mixture (Kryo Lauda) instead of ethanol. For very low temperatures (temperature below -10 ⁰ C), the role of the temperature seems critical since the sensitivity of S. aureus vis-à-vis the imposed treatment decreases very rapidly (destructive efficiency ED decreasing drastically) then the variation is more gradual for temperatures above -5 ° C.

As demonstrated in this example, an example of a method of the invention via the appropriate combination of the 3 parameters associated with the value of the pressure: rate of application of the pressure, mode of application of the pressure and temperature has a synergistic effect on the inactivation of S. aureus.

In addition and very surprisingly, it appears as shown in Figure 6 that there is a temperature range where the inactivation of S. aureus is exacerbated. For example, between -15 ° C. and -10 ^° C. the destructive efficiency goes from 1.5 to substantially 5 which constitutes an entirely unexpected increase. Example 7: Example of Inactivation of a Pathogen in Human Blood Plasma under High Pressures as a Function of Temperature and Pressure Value

In example 6, the value of the pressure was set at 200 MPa. While retaining the values of the parameters associated with the pressure (application speed of 50 MPa · sec ^-1 and cyclic application mode of 5 cycles of 2 minutes), the inventors sought to evaluate whether the surprising effect related to amplification of the destructive efficacy of S. aureus for a narrow range of temperature could be observed for various pressure values (200 MPa, 250 MPa, 300 MPa). In other words, this example aims to investigate whether the inactivation of S. aureus observed at 200 MPa can also be observed at other pressures, in particular at 250 MPa and 300 MPa.

The methods of preparation and counting of the samples are identical to those described in Example No. 6.

The samples were placed in a high pressure generating equipment set up internally with ethanol as the transmitting medium.

The analysis of FIG. 7 gives the evolution of the destructive efficiency of S. aureus as a function of temperature for three values of the pressure, all the other parameters associated with the treatment remaining constant (speed of application of 50 MPa.s s ^-1 and cyclic application mode of 5 cycles of 2 minutes) shows a very particular phenomenon.

As demonstrated in this example, in a pressure range of between 200 and 300 MPa, it appears that inactivation of the pathogen is maximum in a particular and narrow temperature range of -5 ° C. ± 20 ^° C.

In addition, it is surprising that the amplification phenomenon of S. aureus inactivation can be observed in a pressure range, the destructive inactivation increasing when the pressure increases.

Example 8: Example of inactivation of pathogens in human blood plasma under high pressures as a function of temperature.

In example 6, the value of the pressure was set at 200 MPa. In this example, the values of the parameters associated with the pressure were as follows: application speed of 50 MPa. s ^"1 and cyclical enforcement mode 5 cycles of 2 minutes. The inventors have investigated whether the surprising effect related to an amplification of the destructive efficiency of S. aureus in a particular temperature range was observed for other pathogens of the European Pharmacopoeia such as Pseudomonas aeruginosa (ATCC 9027), Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404) by maintaining 200 MPa as the pressure value (Figure 8).

The methods for preparation and enumeration of plasma samples contaminated with S. aureus and P. aeruginosa are identical to those described in Example 6. For C. albicans and A. niger, the protocol is identical to that of exception of the culture medium which is Sabouraud agar (AES Chemunex) and the incubation temperature which is 28 ° C. Spores of A. niger were obtained by incubating the strain for 28 days in Roux flasks containing Sabouraud agar and recovered by washing these flasks with a mixture of water and water. Tween 80 (0.5 g / L) in the presence of glass beads.

The analysis of FIG. 8 demonstrates quite surprisingly and unexpectedly, despite very important differences in the structure of the pathogenic agents: two bacteria, including a Gram-positive shell (S. aureus) and a Gram bacillus. Negative (P. aeruginosa), spore-shaped mold (A. niger), and yeast (C. albicans), amplifying the destructive efficacy for a particular area or particular temperature range. This temperature range covered (with errors of experience) that of S. aureus (Temperature equal to -6 ° C ± 5 ° C).

The difference observed at very low temperature, that is to say temperatures below -5 ° C, is a function of the sensitivity of each of the microorganisms vis-à-vis in particular the pressure.

As demonstrated in this example, the method of the invention via the particular combination of the parameters associated with the value of the pressure (speed of application of the pressure, mode of application of the pressure and temperature) has a synergistic effect on the inactivation of a set of biological targets.

Example 9 Example of inactivation of a pathogen at high pressures as a function of the speed of application of the pressure

[00114] In order to evaluate the contribution of the speed of application of the pressure in the synergy phenomenon between the three parameters associated with the inactivation process (rate of application of the pressure, mode of application of the pressure and temperature), human blood plasma contaminated with S. aureus (prepared according to the protocol described in Example 6) was treated under the following conditions: a cyclic application mode of 5 cycles of 2 minutes, a temperature of about -5 ° C, and a pressure of 200 MPa, 250 MPa or 300 MPa. For each of these treatments, two pressure application rates were used, namely 3.33 MPa. s ^"1 and 50 MPa s ^{" 1} . The results obtained are shown in FIG. 9.

The protocols for implementation, preparation and enumeration are identical to those of Example 6.

The samples were placed in a high-pressure liquid-phase direct compression generator device of the "Framatome" type and the transmitting medium was the glycol / water mixture (Kryo Lauda) for the speed of 3.33 MPa. s ^"1. A generator equipment of high pressure indirect compression implemented internally as transmitting medium with ethanol was used to the speed of 50 MPa · ^{s" 1}

The results presented in FIG. 9 demonstrate that it is necessary to combine both a fast application rate (50 MPa.s ^-1 ) with a cyclic application mode (5 cycles of 2 minutes) and a negative temperature (about -5 ° C) to obtain the synergic effect so that the inactivation of S. aureus is the most effective. In particular at 200 MPa, this synergistic effect is well demonstrated because using an application speed of 3.33 MPa. s ^"1 , the destructive efficiency ED (inactivation of the pathogen) of the treatment was very low, even nil, whereas using an application speed of 50 MPa.s- ¹ , it is close to 5.

In addition, it appears that the rate of application of the pressure (VA) associated with a mode (MA) cyclic application of pressure, 5 cycles of 2 minutes, and at a temperature of -5 ° C. plays a key role in the inactivation of S. aureus.

The comparison between the results observed in FIG. 14 of example 14 below and those of FIG. 9 demonstrates that the parameters associated with the pressure, the particular choice of the speed of application of the pressure, the The application of pressure and temperature leads to very surprising and unexplained synergistic effects on the inactivation of S. aureus.

The method of the invention thus allows the inactivation of pathogens in human plasma with a synergistic effect.

Example 10: Example of Implementation of a Method for Inactivating at Least One Pathogen in Human Blood Plasma

[00122] In order to evaluate the effect of the pressure application rate during a cyclic treatment (5 cycles of 2 minutes) at a temperature of about -5 ° C. on the backing of the activity coagulation factors (F City and FV), two application speeds were tested (3.33 MPa s ^-1 and 50 MPa s ^-1 ) for increasing pressure values

(150 MPa to 250 MPa).

The samples were placed in a high pressure liquid phase generator device by direct compression type "Framatome" and the transmitter medium was the glycol / water mixture (Kryo Lauda) for the speed of 3.33 MPa. s ^"1. High pressure indirect compression generating equipment set up internally with as the transmitting medium ethanol was used for the speed of 50 MPa.s ^"1

The percentages of residual activity (relative to the activity before the implementation of an inactivation process) of the coagulation factors City and V, at the end of the implementation of a process inactivation under the conditions described above, are shown in FIG.

Examination of FIG. 10 demonstrates that the value of the speed of application has little influence on the safeguarding of the activity of the factors Ville and V. It has been shown, in example 9, that a rapid application rate (50 MPa.s ^-1 ) significantly increased (synergistic effect) the destructive efficiency (inactivation) of the pathogen inactivation process when this parameter is suitably combined with a cyclic treatment (5 cycles of 2 minutes) and a low temperature (T = -6 ° C ± 5 ° C).

In addition, it is important to note that by choosing a pressure of 200 MPa, a rate of application of the pressure of 50 MPa.s ^"1 , a cycle of 5 cycles of 2 minutes and a temperature of 1 hour. order of -5 ° C, the City factor activity safeguard is greater than 70%, and that of factor V greater than 50%, which meets both the requirements of the European Pharmacopoeia (activity of coagulation factors greater than 50%) (Ref 6) but also to those of the French Legislation (coagulation factor activity higher than 50% except for the City factor for which the activity must be greater than 70%) (Ref 17) .

As demonstrated in this example, the present invention thus makes it possible to inactivate at least one pathogen in human blood plasma while retaining its biological activity.

Example 11 Inactivation of Plasmodium berghei (hematozoan) and Trypanosoma brucei brucei (extracellular protozoan) by implementing a method of the invention An example of inactivation process of a pathogen according to the invention was then tested on parasite models likely to contaminate the blood products of Plasmodium berghei part (hematozoan) and secondly Trypanosoma brucei brucei (extracellular protozoan).

For each of the two parasites, 10 samples of contaminated mouse (Mus musculus) blood were treated. The parameter values associated with the pressure were: a pressure application rate of 50 MPa. s ^"1 , a mode of application of the pressure of 5 cycles of 2 minutes, a temperature of about -5 ° C, an initial pressure of 0.1 MPa and a pressure of 200 MPa.

In parallel, an untreated control sample was stored and a temperature control (sample equilibrated at -15 ° C for 30 minutes). Each sample was then observed by light microscopy and injected into a mouse. The conclusions of the microscopic observation on the one hand and the results concerning the number of surviving mice one month after the injection are presented in Table 1 below.

[00132] Table 1: Microscopic Results and Number of Surviving Mice

The results of the microscopic observation are further illustrated in FIG. 11.

As demonstrated in this example, an example of inactivation process according to the invention is effective for the inactivation of one part Plasmodium berghei (hematozoan) and secondly Trypanosoma brucei brucei (extracellular protozoan) .

As demonstrated in this example, the method of the invention can inactivate pathogens such as parasites while maintaining the biological activity of human blood plasma.

Example 12: Implementation of an Example Method According to the Invention on the Infectious Prion Protein Present in a Sample of Human Blood Plasma

The Prion infectious protein is currently one of the objectives of securing biological products. The hamster prion was chosen as a model.

[00137] Human blood plasma contaminated with hamster brain broth infected with the infectious prion protein (263 K) (EFS Pyrenees-Mediterranean) was used.

From said pieces of hamster brain infected with strain 263 K (IBH), samples of human blood plasma (1 mL) were contaminated and placed in sealed tubing on the one hand and resistant to pressure on the other hand.

In parallel negative samples (uncontaminated) were prepared under the same conditions, then adding healthy hamster brain (NBH). The tubings were placed immediately after preparation in the dry ice.

[00140] The samples were treated in pairs, a tubing containing plasma contaminated with the infected brain meal and a tubing containing plasma contaminated with the non-brain meal. infected, using an example pathogen inactivation method according to the invention combining the appropriate combination of parameters associated with the pressure that is to say a rate of application of the pressure of 100 MPa in 2 seconds, or a rate of increase of the pressure and decrease of the pressure of 50 MPa. s ^{"1 of} a user applying pressure of 5 cycles of 2 minutes, the compression of the sample, maintaining the pressure for 120 seconds and the pressure decrease, the compression being equal to a pressure P of 200 MPa, the temperature of the sample at the pressure Pi was about -5 ° C. The samples were placed in a high-pressure generating equipment by indirect compression set up internally with the ethanol-transmitting medium .

The analysis of the samples was carried out using the following protocol: extraction of the prion protein by precipitation with NaPTA 4%, proteinase K digestion at 200 μg / ml, detection of PrP ^Sc by Western Blot (the antibody used being the 3F4 antibody).

Comparison 2 to 2 of the signals obtained, the prion migrating at about 27-29 kDa for the infected samples, showed a very sharp decrease in the amount of PrP ^Sc for those having been subjected to an example of a method according to US Pat. compared to untreated (Figure 12 comparison of samples treated HPM to HPI4 versus untreated samples TM to TI4). These results were further reproducible for the 4 pairs of infected samples (untreated control T and treated sample by the method of the invention). It thus clearly appears that the infectious PrP ^Sc protein has been modified by the implementation of an exemplary method according to the invention; which suggests a negative impact on its infectivity.

Example 13: Evaluation of the value of the pressure on the inactivation of a pathogen (S. aureus) in a human blood plasma sample by treatment at high pressures for 10 minutes minutes continuously at room temperature (25 ° C) and with a relatively moderate and conventional pressure application rate (VA) (3.33 MPa s ^-1 ). [00143] Staphylococcus aureus is a well-identified pathogen and causes many nosocomial diseases This pathogen was chosen as a model in this example.

[00145] The samples were placed in an indirect high pressure generator equipment set up internally with the ethylene glycol transmitter medium associated with Kryo lauda water (60% monoethylene glycol and 40% by weight). water).

The temperature was maintained at 25 ° C. The rate of application of the pressure (VA) was 3.33 MPa. s ^"1 The duration of treatment was 10 minutes keeping constant pressure value. Several pressure values were used to evaluate the impact on the inactivation of the pathogen S. aureus agent 200 MPa 250 MPa, 300 MPa, 400 MPa.

[00147] FIG. 13 represents the values of the destructive efficiency.

(ED) observed for each of these pressures.

The results obtained show that the pressure, at least up to 400 MPa, has little effect on the inactivation of S. aureus because of the relatively low values of the destructive efficiency (ED).

Example 14: Evaluation of inactivation of a pathogen (S. aureus) in a blood plasma sample through treatment at high pressures at room temperature (25 ° C.), a mode of application of pressure continuous, a duration of 10 min with different application speeds VA of the pressure In example 13 above, the mode of application of the pressure MA was continuous and the rate of application of the pressure was relatively "conventional" (ie 3.33 MPa.s ^-1 ). the treatment time was 10 minutes and the treatment temperature was 25 ° C.

[00150] The purpose of this example is to have an evaluation of the effect of the speed of application (VA) of the pressure on the inactivation of S. aureus, the values of the other parameters remaining constant: a mode of continuous MA application of pressure, a treatment temperature of 25 ° C and a treatment time of 10 minutes.

[00151] In order to have an evaluation of the impact of the VA application rate on the inactivation of S. aureus, two very different rates of application of the pressure were chosen: a conventional speed of 3.33 MPa . s ^"1 and a fast speed 50 MPa s ^{" 1} .

[00152] The pressure values were 200 MPa, 250 MPa and 300 MPa. Higher values were not selected as the biological activity of human blood plasma was significantly impaired above 300 MPa.

[00154] FIG. 14 represents the destructive efficiency ED with respect to S. aureus according to the value of the pressure for different values of the application speed of the pressure VA.

The analysis of the results illustrated in FIG. 14 shows that, to the errors of the experiments, no effect of the application speed VA of the pressure on the value of the destructive efficiency of S is observed. aureus. Indeed, the destructive efficiency regardless of the pressure and / or speed is less than 1.

This result suggests that the rate of application of the pressure is not a parameter likely to play an important role in the inactivation of a pathogen such as S. aureus, hence the importance of the parameters associated with the pressure since it has been shown that the VA was a primary factor when a cyclic MA and negative temperatures (-5 ^° C.) were used (see Example 9). Example 15: Evaluation of inactivation of a pathogen (S. aureus) in a sample of human blood plasma through treatment at high pressures at room temperature (25 ° C.), with a rate of application of the pressure VA 3.33 MPa. s ^"1 depending on the application of pressure (MA), continuous (10 min), or cyclic (5 cycles of 2 min or 20 cycles of 30 seconds)

In example 13, only a continuous application mode was applied. In this example, the objective was to evaluate the role of the pressure application mode (MA), for pressure values of 200 MPa, 250 MPa and 300 MPa - the biological activity of the plasma being significantly altered above 300 MPa. The other parameters were kept constant: application speed of 3.33 MPa. s ^"1 and treatment temperature of 25 ° C.

FIG. 15 is a histogram representing the destructive efficiency (ED) (ordinate) as a function of the pressure (abscissa). The application rate of the pressure was 3.33 MPa. s ^"1. The application of the pressure mode was 5 cycles of 2 minutes (hatched bars), 20 cycles of 30 seconds (open bars) or a cycle of

10 minutes (full sticks).

[00160] The analysis of FIG. 15 shows that the mode of application of the pressure is continuous or cyclically has no effect (with errors of experience) on the value of the efficiency. destructive (ED) of S. aureus at both 200 MPa and 250 MPa. For the pressure value of 300 MPa, there is a slight increase in the value of destructive efficiency for a cyclic mode of application. However, it should be noted that the improvement made, of the order of 1, 5, is not significant.

This example clearly demonstrates that the mode of application of the pressure does not achieve significant values of the inactivation of a pathogen such as S. aureus.

EXAMPLE 16 Evaluation of the Inactivation of a Pathogen (S. aureus) in a Human Plasma Sample by High-Pressure Treatment with a Continuous MA Pressure Application Mode, a Time of 10 Minutes . a VA application speed of 3.33

MPa. s ^"1 depending on the treatment temperature: 25 ° C and -5 ° C.

In example 13, the application mode of the pressure MA was continuous and the rate of application of the pressure was relatively "conventional", that is to say equal to 3.33 MPa. s ^"1. The ree of treatment was 10 minutes and the treatment temperature was

25 ° C.

The objective of this example is to evaluate the impact of the treatment temperature on the inactivation of S. aureus.

Two treatment temperatures were selected: on the one hand + 25 ° C as in Example 13 and on the other hand a negative temperature: -5 ° C.

The other values of the parameters were kept constant and equal to those used in Example 13, namely continuous MA, treatment time of 10 minutes and application speed (VA) of

3.33 MPa.s ^"1 .

The results obtained are given in FIG. 16. FIG. 16 is a histogram representing the destructive efficiency (ED) (ordinate) as a function of the pressure (abscissa). The temperature was -5 ° C (hatched sticks) or 25 ° C (full sticks)

The analysis of FIG. 16 shows a marked increase in the inactivation of S. aureus when the treatment temperature goes from + 25 ° C. to the negative temperature of -5 ° C. (hatched sticks). However, it appears that this effect is capped when the pressure increases from 250 MPa to 300 MPa, no increase in the value of the destructive efficiency being observed between these two values of the pressure. Example 17: Study of the synergistic effect of inactivation of pathogens other than S aureus

The same values of the parameters as those used in Example 6, ie a pressure of 200 MPa, a mode of cyclic application of the pressure MA of 5 cycles of 2 minutes and a speed of application of the pressure VA 50 MPa. s ^"1 have been implemented.

[00170] The temperature domain explored was between -

15 ° C and + 30 ° C.

[00171] The microorganisms of the European Pharmacopoeia were chosen as pathogens [S. aureus (ATCC 6538), P. aeruginosa (ATCC 9027), C. albicans (ATCC10231), A. niger (ATCC 16404), B. subtilis in the vegetative state (ATCC 9372)].

The methods of preparation and enumeration of plasma samples contaminated with S. aureus and P. aeruginosa were identical to those described in Example 6, with the exception of the culture medium for C. albicans and A. niger , for which the agar used is Sabouraud agar (AES Chemunex)

In the case of B. subtilis the method of preparation consisted in transplanting the strain ATCC 9372 into a heart-brain broth incubated 24h at 35 ° C. This culture is then centrifuged at 12,000 g for 4 minutes and the pellet is resuspended in 10 ml of broth. TS. This suspension was then used to contaminate 300 ml of human blood plasma

The results observed are given in FIG. 17. FIG. 17 is a diagram representing the destructive efficacy with respect to S. aureus, P. aeruginosa, A. niger, C. albicans and B. subtilis (ordinate) as a function of temperature (abscissa).

[00175] Surprisingly, it appears that whatever the nature of the microorganism studied (S. aureus, P. aeruginosa, C. albicans, A. niger, B. subtilis), an effect is observed. amplification of the destructive efficiency in a certain temperature range.

[00176] Unexpectedly, the temperature domains where this amplification of the inactivation for all the microorganisms of the European Pharmacopoeia are observed overlap. Thus, it is possible to define a common temperature range where all pathogenic microorganisms are inactivated.

This example clearly demonstrates that an example of a process of the invention makes it possible to inactivate all the microorganisms of the European pharmacopoeia with a synergistic effect by the particular choice of the pressure, the speed of compression. and expansion and the mode of application (MA) of the pressure, for example a pressure of 200 MPa, a speed VA = 50 MPa. s ^"1 , a mode of application of the pressure of 5 cycles of 2 minutes and a temperature (T) ranging from -10 ° C to -3 ° C.

In addition, this example clearly demonstrates that an example of a method of the invention makes it possible to inactivate all the pathogens present in a plasma sample.

Example 18: Evaluation of the biological activity of human blood plasma by applying the parameter values characterizing an example of pathogen inactivation method of Example 17 A method of treating human blood plasma with a pressure of P = 150 MPa, 200 MPa, 250 MPa and 300 MPa, an application rate of VA = 50 MPa. s ^"1 , an application mode MA = 5 cycles of 2 minutes and -10 ° C <T <-3 ° C was applied to human blood plasma.

A median value of the temperature (-5 ⁰ C) was chosen for this evaluation.

[00181] Samples of about 4 ml of fresh human blood plasma were taken and placed in sealed tubes, and then frozen at -30 ^° C. waiting to be treated under high pressures.

The residual activity of the coagulation factors City and V was determined by the partial thromboplastin method with activator described in Example 1.

[00183] The residual activities of the City and V coagulation factor carrier proteins are given in the general figure 18. FIGS. 18A and B are histograms representing the percentage of plasma protein activity (% act) (ordinate) as a function of the pressure (P) in Mega Pascal (MPa) (abscissa).

The analysis of FIG. 18 shows that at 200 MPa a relative activity of the City Factor of about 75% is observed and that of the Factor V about 58%. These values are higher than those required by the European Legislation (50% for the City factor and for the Factor V) and the French Legislation (70% for the City Factor and 50% for the Factor V

V).

[00185] Thus, an example of a method of the invention makes it possible to obtain a significant destructive efficiency of the most baroresistant pathogenic agent: S aureus, that is to say a destructive efficiency of the order of 5 maintaining the biological activity of the City and V Factors.

In other words, an exemplary method of the invention makes it possible to obtain both a synergy of inactivation of the pathogens present in the human blood plasma while retaining the biological activity of the proteins present in the blood. blood plasma. Example 19: Evaluation of the irreversibility of the inactivation of a pathogen through the "Method of securing at least one pathogen in a blood plasma sample" in a sample of human blood plasma.

The pathogen selected was S. aureus (ATCC 6538) because of its high resistance to pressure effects (also called baroresistance).

A suspension of S. aureus in the blood plasma was prepared according to the method described in Example 6 so as to obtain a titration of the order of N = 1. 10 ⁵ CFU / ml.

This suspension was used to prepare, on the one hand, a No. 1 tube of a volume of 4 ml serving as a control and, on the other hand, a No. 2 tube of a volume also of 4 ml intended to be submitted. a treatment comprising the application of a pressure of 200 MPa at a speed of 50 MPa. s ^"1 , according to a mode of application of 5 cycles of 2 minutes at a temperature of -5 ° C (condition of an exemplary method of the invention).

The tubes No. 1 and No. 2 were kept in the freezer for 14 days.

Then the contents of these 2 tubes were used to seed broths heart-brain culture.

After 48 hours of incubation at 35 ⁰ C, if we observe a significant growth recovery for the culture medium inoculated with the control tube No. 1 not treated by an example of the method of the invention, on the other hand no growth recovery could be detected for the culture medium seeded by the No. 2 tube treated according to the conditions of an exemplary method of the invention.

[00193] Another observation was conducted after 7 days. For the culture medium inoculated by the No. 2 tube treated with an example of the method of the invention, it has still not been possible to demonstrate growth recovery. Given the nutritive values of the heart-brain broth, well known to those skilled in the art, the lack of growth after a relatively long incubation time of 7 days demonstrates that the inactivation of A pathogen according to the method such as S. aureus, considered very baroresistant, is irreversible.

As demonstrated in this example, an example of the method of the invention thus allows irreversible inactivation of pathogens such as S. aureus. Example 20: Inactivation of strains of S. aureus multi-resistant to antibiotics by implementing an exemplary method of the invention.

The inactivation by an exemplary method of the invention of two strains of S. aureus with multiple antibiotic resistance: ATBR1 (P ⁺ ) and ATBR2 (P ^" ) was tested and compared to the reference strain. S. aureus (ATCC 6358).

The protocols used in the tests relating to the two strains ATBR1 (P ⁺ ) and ATBR2 (P ^" ) are identical to those described in Example 6.

The table below gives the values of the destructive efficiency (ED) observed for the 3 strains: ATBR1 (P ⁺ ) and ATBR2 (P ^" ) and that of reference (ATCC 6358) after treatment according to the conditions of pressure, rate of application, mode of application and temperature described in Example 19 (VA = 50 MPa.s ^-1 , MA = 5 cycles of 2 minutes, T = -5 ° C., P = 200 MPa).

[00199] Table 2 below represents the results of the destructive efficiency (ED) obtained according to the strains tested.

ATCC 6538

Strain

ATBRi (P ⁺ ) ATBR ₂ (P-)

"Of

reference »

ED ^« 4.9 ^« 4 ^« 6.2 It appears that for strains of S. aureus antibiotic resistance on the one hand and the behavior vis-à-vis an example of the method of the invention on the other hand are not comparable.

Indeed the strain particularly resistant to antibiotics ATBR2 (P ^" ) appears very sensitive to the effects of an exemplary method of the invention (ED of the order of 6.2) and the value of the destructive efficiency is even greater than that observed under the same conditions for the reference strain (ATCC 6358).

It is also noted that whatever S. aureus strain particularly resistant to antibiotics [ATBR1 (P ⁺ ) and ATBR2 (P ^" )], is observed - after application of an example of a method of the invention a important value of destructive efficiency.

Example 21 Inactivation of the Measles Virus Taken as a Virus Contaminating Human Blood Plasma by an Example of a Method of the Invention

This example aims to evaluate the effectiveness of an exemplary method of the invention vis-à-vis a virus.

In a first step, the inventors have selected as virus that of measles.

The infectivity of the measles virus before and after treatment with an example of a method of the invention was evaluated by infecting vero cells (S. ROZENBLATT, T. KOCH, O. PINHASI, S. BRATOSIN, " Infective substructures, Measuring viruses from Acutely and Persistently Infected CeIIs, J. of Virology (1979), 32, pp. 329-333 (Ref 21)).

The protocol implemented for the evaluation of infectivity is described in R. RUSTIGIAN "Persistent Infection of CeIIs in Culture by Measles Virus, I. Development and Characteristics of HeLa Sublines Persistently Infected with Complete Virus", J. of Bacteriology (1966), 92, p.1792-1804 (Ref 22).

The initial viral titer was about 2.10 ³ units / mL. This viral suspension was then divided into two tubes: the tube No. 1 which served as untreated control by the method and the tube No. 2 which has undergone the following conditions: pressure P = 200 MPa, speed of compression compression VA = 50 MPa. s ^"1 , mode of application of pressure MA = 5 cycles of 2 minutes and temperature T = -5 ° C.

After this treatment, the viral titre was evaluated in tube No. 2 and compared to that of tube No. 1 taken as a control (Table below).

Table 3: Title of the virus before application of an example of the method of the invention (before HP) or after application of an example of the method of the invention (after HP)

As demonstrated in this example, an example of a method of the invention makes it possible to inactivate the measles virus in human blood plasma.

Example 22 Inactivation of the Human Immunodeficiency Virus (HIV) taken as a virus contaminating human blood plasma by an example of the method of the invention

[00212] HIV is a retrovirus that infects CD4 + T cells. At the beginning of the infection, it binds to these receptors and then fuses with the membrane of the host cell. Its two RNA strands are then retranslated into double-stranded DNA thanks to its reverse transcriptase. Integrase then allows this DNA to integrate into the genome of the host cell.

A. Principle of the HIV test

The inactivation of HIV by an exemplary method of the invention was evaluated by the 4-MUG test described in the document. VR SOULTRAIT, A. CAUMONT, V. PARISSI, N. MORELLET, M. VENTURA, C. LENOIR, S. LITVAK, M. FOURNIER, B. ROQUES "A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase J. Mol. Biol. (2002), 31J3, p.45-58 (Ref 23). This test is based on the expression of the viral protein Tat, which is an activator for transcription of HIV-1 genes. For this test, the infection is carried out on HeLa P4 cells, which have a Lac Z reporter gene whose expression is under the control of a promoter activated by the Tat protein. Thus, if HIV is not inactivated, it will penetrate into these cells, integrate into the genome, express the Tat protein which will then activate the transcription of the Lac Z gene. This leads to the synthesis of β-galactosidase, which catalyzes the degradation of 4-MUG into a fluorescent product. The intensity of the fluorescence is then measured and is proportional to the viral infectivity.

The experiments were carried out directly on viral supernatants prepared by co-cultivation of MT ₄ and H ₉ Lai cells according to the protocol described in P. CHARNEAU, G. MIRAMBEAU, P. ROUX, S. PAULOUS, H. BUC, F. CLAVEL, "HIV-1 Reverse Transcription - A Genomic Termination at the Center of the Genome", J. Mol. Biol. (1994), 241, p. 651-662 (Ref 24).

A first sample was subjected to an exemplary method of the invention, namely a pressure of 200 MPa, an application speed of 50 MPa. s ^"1 , a mode of application of the pressure of 5 cycles of 2 minutes and a temperature of -5 ° C while the second was placed in the refrigerated bath in which plunged the experiment chamber (" witness of At the end of these treatments, the viral supernatants were recovered to infect 96-well plates in which HeLa P4 cells were cultured according to the protocol described in P. CHARNEAU, G. MIRAMBEAU, P. ROUX, S. PAULOUS, H. BUC, F. CLAVEL, "HIV-1 Reverse Transcription - A termination step at the center of the genome", J. Mol Biol (1994), 241, P-651-662 (Ref 24). . After the first infection, the plates were incubated for 72 h at 37 ° C and 5% CO2, and then they were revealed by the 4-MUG method. In parallel, the viral supernatants were recovered to infect a new 96-well plate, which was placed in an oven for 2 hours, while the viruses possibly present could infect the cells. The volume of viral supernatants was 100 μl. It was then washed with 1 X PBS solution to remove all elements that could have been transported with the viral supernatants, and then returned to the oven for 72 hours. A revelation at 4-MUG was then realized.

B Results

The results were obtained by the method described in V.R.

SOULTRAIT, A. CAUMONT, V. PARISSI, N. MORELLET, M. VENTURA, C. LENOIR, S. LITVAK, M. FOURNIER, B. ROQUES "A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase »J. Mol. Biol. (2002), 318, p.45-58 (Ref 23).

For each plate, the average of the fluorescence intensities measured in the wells containing the uninfected cells (background) was subtracted from the values detected in each other well. The results obtained after 72 hours of incubation are shown in FIG. 19. FIG. 19 represents the intensity of fluorescence (ordinate) as a function of the dilution factor (abscissa). The crosses correspond to the results obtained with (HP), the black squares to the results obtained with a control "T".

The results reported in FIG. 19 show a decrease in the signal detected in the wells having been infected by the viral supernatants subjected to an example of the method of the invention compared to those having received the viral supernatants of the "temperature control ". There has therefore been an inactivation of HIV-1 by an exemplary method of the invention. The viral supernatants of this plate were still recovered to infect new cells. The infection process is identical to the aforementioned infection method. The results observed 72 h after this reinfection are shown in FIG.

The results of reinfection (FIG. 20) reveal that no signal could be detected in the wells having received the samples subjected to the pressure. Thus, this example demonstrates that the entire initial viral population has been inactivated by an exemplary method of the invention. Example 22: inactivation of various parasites by an example of the method of the invention.

[00222] Blood parasites are a problem for transfusions and injections of blood products. Indeed, post-injection parasitosis of these products has been reported, including malaria, more rarely leishmaniasis, and in South America, Chagas disease.

[00223] For P. berghei, T.b.b. and T. Feo, the set of experiments consisted in taking blood from mice contaminated with the parasite to be tested. For T. cruzi and L. donovani, the parasites were prepared by cell culture in LIT (Liver Infusion Tryptose) medium for T. cruzi and RPMI 1640 plus SVF for L. donovani.

The samples were then separated into four batches:

- the first sample corresponded to the "untreated control",

- the second consisted of a "transport control", that is to say, it followed the same route as the treated samples but remained all day at room temperature,

the third, the "temperature control", was immersed in the refrigerated bath in which the high-pressure chamber was placed in order to evaluate the effect of freezing alone on the parasites,

the last batch was treated under high pressures, using the parameters following pressure P = 200 MPa, speed of application and of relaxation VA = 50 MPa. s ^"1 , mode of application of pressure MA = 5 cycles of 2min, temperature T≈ -5 ° C.

Each of the samples: control or having undergone high pressure treatment, was observed by optical microscopy. The microscope used was an OLYMPUS CA20. For P. berghei, Tbb and T. Feo, the samples were then injected into healthy mice. The number of surviving mice one month after the injection then made it possible to conclude on the inactivating effect of the application of this treatment. For T. cruzi and L. donovani, the samples were put back into contact with the culture media specified above and were placed in the incubator for 5 weeks at 35 ° C. for T. cruzi and at 28 ^° C. without CO2. for L.donovani to observe a possible multiplication of parasites. The sample volume was 1 mL of suspension of high pressure treated parasites for 9 mL of culture medium.

The "transport" controls correspond to the samples having followed the same experimental conditions with the exception of the treatment with an example of the process of the invention and remained all day at room temperature.

The "temperature" controls correspond to the samples having been immersed in the refrigerated bath in which the high pressure chamber was placed in order to evaluate the effect of freezing alone on the parasites.

[00228] Table 4 below summarizes the results obtained.

As demonstrated in this example, an example of a method that is the subject of the present invention makes it possible to inactivate all the parasites tested.

Example 23 Evaluation of the Non-Toxicity of the Plasma Processed by an Example of a Process of the Invention

The objective of the present invention is to verify that the plasma having been subjected to a process which is the subject of the invention is not immunogenic.

In this example, monocytes were placed in the presence of plasma treated according to an exemplary method of the invention. A verification that the treated plasma does not induce the formation of dendritic cells (DCs), cells involved in the presentation of the antigen during the immune response was then performed.

Monocytes were prepared from a whole blood bag according to the method described hereinafter.

The blood was distributed in Falcon tubes (registered trademark) at 50 mL per tube and the tubes were centrifuged for 15 min at 900 rpm without brake. The plasma was then removed and the pellet is taken up in PBS (qs 50 ml). After homogenization, the tubes were again centrifuged for 15 min at 2500 rpm without brake. The "buffy coat" present between the "plasma-PBS" and "red blood cells" layers is removed and then supplemented with RPMI (qs 35 mL). After homogenization, this suspension was poured very gently into a tube containing 15 ml of Ficoll. The tubes were centrifuged for 20 min at 2000 rpm without brake. The serum was then aspirated and the monocyte-containing ring was removed and transferred to a new Falcon tube. The tubes were supplemented with RPMI + L-Glutamine + SVF 8% medium (qs 50 mL) and then centrifuged. for 10 minutes at 1500 revolutions per minute. The supernatants were removed and the pellets were taken up in 5 ml of medium before being pooled in a single Falcon tube, which was supplemented to 50 ml with medium. After centrifugation for 10 min at 900 rpm, the supernatants were removed, and the pellets were taken up in 20 ml of medium. The cells were counted in Malassez cell to determine the concentration. After centrifugation at 1500 rpm for 10 min, the pellets were resuspended in a medium volume defined to have a monocyte concentration of about 4.10 ⁶ cells / mL.

These monocytes were then distributed in 6-well plates at a concentration of 4.10 ⁶ cells / mL (2 mL / well). Plates were incubated for 2 hours to allow monocytes to adhere to the bottom of puffs. The monocyte culture medium comprising RPM I (marketed by Sigma), Fetal Calf Serum (FBS) and penicillin was subsequently replaced by:

→ either culture medium alone (negative control: Mono),

→ or culture medium supplemented with 25 ng of granulocyte-macrophage colony stimulating factor (GMSCF) and

10 μg of interleukin 4 (IL4) (cocktail necessary for the generation of DC: positive control: GMIL4),

→ either culture medium supplemented with 10% of plasma not subjected to the process (untreated control: Tc),

→ or culture medium supplemented with 10% of plasma subjected to the process (HP: HP sample).

The plates were then incubated for 3 to 5 days at 37 ° C / 5% CO 2. Supernatants containing dendritic cells (DC) were recovered and centrifuged. After washing with phosphate buffered saline (Phosphate Buffer Saline (PBS)) containing the cells, they were resuspended in phosphate buffered saline (PBS). The suspensions thus prepared were divided and placed in the presence of markers coupled to a fluorochrome. These markers are generally IgGs capable of binding specifically to markers expressed on the cell surface. The following markers have been selected:

→ CD14: expressed on the surface of monocytes but not DC, → CD40 and HLA-Dr: over-expressed on the surface of DC,

→ CD80: expressed on the surface of mature DCs.

After incubation for 15 min at room temperature and in the dark, the suspensions are centrifuged, the pellets containing the cells are washed before being resuspended in 200 .mu.l of PBS. The reading was then performed in flow cytometry with the BD FACS Diva device.

The results obtained are shown in FIG. 21.

A population of P1 cells was defined according to the size of the expected cells (SSC: Sideways Scatter Channel - signal correlated to the cellular complexity, FSC: Forward Scatter Channel - signal correlated with the relative size of the cells). The data taken into account were as follows:

- #Events: total number of events (cells) in the defined population,

-% Parent: number of events in the defined population divided by the total number of events, expressed as a percentage,

- Mean: mean value (in this case of the fluorescence intensity) for the events of the defined population.

The results obtained are shown in Table 5 below:

The most significant results are those obtained with the CD14 and CD40 markings. Indeed, the CD14 marker is expressed only on the surface of monocytes and not DC while the CD40 marker is over-expressed on DC.

It was observed while for the positive control (GMIL4), there was indeed DC production, the fluorescence associated with the CD14 marker is very low while that associated with the CD40 marker is very high.

As for the negative control (Mono), the monocytes remained in their initial form, the fluorescence associated with the CD14 marker being higher than that of CD40.

As regards the plasma samples, it is necessary to compare them two by two (Tem1 / HP1 and Tm2 / HP2). For each pair of samples, no difference could be detected at the level of the fluorescence intensities for each marker, which means that the method which is the subject of the invention does not induce a change in the immunogenic power of the plasma. As demonstrated in the previous examples, the method of the present invention makes it possible to inactivate at least one pathogen, for example a pathogen in human blood plasma, while maintaining the biological activity of said plasma. Thus the plasma obtained can, for example be used in the therapeutic field. List of references

(Ref 1) C. Naegelen, H. Isola, D. Denis, J-P. Maurel, R. Tardivel, S.

Wood, C. Vignoli, J-P. Cazenave. 2009. Evolution of labile blood product preparation (PSL) techniques: inactivation of pathogens in PSL. Clinical and Biological Transfusion vol.16: 179-189

(Ref 2) A. Jofré, T. Aymerich, N. Grèbol, M. Garriga. Efficiency of high hydrostatic pressure at 600 MPa against food-borne microorganisms by challenge tests on convenient meat products. LWT - Food Science and Technology Vol.42 (5): 924-

928

(Ref 3) CC. Tassou, E.Z. Panagou, FJ. Samaras, P. Galiatsatou, CG.

Mallidis. 2008. Temperature-assisted high hydrostatic pressure inactivation of Staphylococcus aureus in a ham model System: selective and non-selective evaluation medium J. Applied

Microbiology vol.104: 1764-1773

(Ref 4) Y. Rigaldie. 2002. On the impact of treatments under Hautes

Pressures in the decontamination and sterilization of pharmaceutical forms containing therapeutic molecules sensitive to energy processes. Thesis of the University

Bordeaux 1 (01/07/2002) order number 2526

(Ref. 5) Y. Rigaldie, G. Lemagnen, A. Largeteau, D. Larrouture, M.

Abba, R. Haller, L. Grislain, G. Demazeau. 2002. Pharmaceuticals perspectives of high pressure: a soft tool for sterilization of fragile drugs. Defect and Diffusion Forum vol.208-209: 55-58

(Ref 6) European Pharmacopoeia 5.6. Human plasma (mixture of) treated for viro-inactivation. 01/2007: 1646. pp 2439-2441 (Ref. 7) A. Kunert, J. Losse, C. Gruszin, M. Hϋhn, K. Kaendler, S.

Mikkat, D. Flight ke, R. Hoffmann, T. Sakari Jokiranta, H.

Seeberger, U. Moellmann, J. Hellwage, P. F. Zipfel. 2007.

Immune evasion of the human pathogen Pseudomonas aeruginosa: Tf is a factor H and plasminogen binding protein. The Journal of Immunology vol.179 (5): 2979-

2988

(Ref 8) A. M. Matser, C. Van Der Ven, C. W. N. Gouwerok, D. De Korte.

2OO5.High-pressure processing for preservation of blood products. High Pressure Research vol.25 (1): 37-41

(Ref 9) R. A. Hess, M. Manak, S. K. Dusing. 2002. Pressure cycling inactivation of pathogens in biological materials used for therapeutics or vaccines. WO 02/056824

(Ref 10) F. Tao, N. Lawrence, C. Li, J. Behnke, J. Lathey, M. Manak, R.

T. Schumacher. 2003. Deciphering mechanisms for infectious agent inactivation using cyclic high pressure or pressure cycling technology (PCT). Boston Biomedica Inc. Biophysics Poster

021,604.

(Ref 11) S. Dusing, C. Li, J. Behnke, M. Manak, R. Schumacher. 2002.

Inactivation of viruses in plasma by cycles of high pressure. Trends in High Pressure Bioscience

Biotechnology, R. Hayashi, ed. Elsevier Science B. V. pp 355-

359

(Ref 12) J. P. R. Pelletier, S. Transue, E. L. Snyder. Pathogen Inactivation Techniques Best Practice and Research Clinic

Haematology, vol 19 (1) 205-242

(Ref 13) European Pharmacopoeia 6.0, 5.1 General Texts on Microbiology 01/2008: 50101

(Ref 14) E. Hecht, "Physics", Publisher De Boech (1999) (Ref. 15) Y. Lambert, G. Demazeau, A. Largeteau, S. Laborde-Croubit, M.

Cabannes and J. M. Bouvier. 2000. New packaging solutions for high pressure. High Pressure Research., Flight

19, 207-212.

(Ref 16) European Pharmacopoeia. 5.0 01/2005: 30203

(Ref 17) J.O. n ° 123 of May 28, 2003, Order of April 29, 2003 fixing the list and characteristics of labile blood products.

(Ref 18) Transfusion of fresh frozen plasma: products, indications.

2002. French Agency for Sanitary Safety of Health Products.

(Ref 19) Operating instructions. STA - CK. PREST ^® . Diagnosis Stago

(Ref 20) J.A. Laugharn, D.W.Bradley, R.A. Hess, 2000. Rapid Cryobaric Sterilization and Vaccination Preparation, WO 00/48641

(Ref 21) S. ROZENBLATT, T. KOCH, O. PINHASI, S. BRATOSIN,

"Infective substructures od Measles viruses from Acutely and

Persistently Infected Ceites, J. of Virology (1979), 32, pp. 329-333 (Ref 22) R. RUSTIGIAN "Persistent Infection of CeIIs in Culture"

Measles Virus, I.Development and Charactehstics of HeLa

Sublines Persistently Infected with Complete Virus, J. of Bacteriology (1966), 92, p.1792-1804.

(Ref 23) V.R. From SOULTRAIT, A. CAUMONT, V. PARISSI, N.

MORELLET, M. VENTURA, C. LENOIR, S. LITVAK, M.

FOURNIER, B. ROQUES "A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase" J. Mol. Biol. (2002), 318, p.45-58)

(Ref. 24) P. CHARNEAU, G. MIRAMBEAU, P. ROUX, S. PAULOUS, H.

BUC, F. CLAVEL, "HIV-1 Reverse Transcription - A step-stop at the center of the genome", J. Mol. Biol.

(1994), 241, p. 651-662

Claims

claims

1. A method of inactivating at least one pathogen of a human blood plasma sample characterized in that it comprises at least 5 cycles each comprising:

the compression of said sample of its initial pressure Po at a pressure Pi, Pi being between 1 90 and 210 MPa, the compression of Po at Pi, the temperature of said plasma at the pressure Pi being from -10 ^° C. to -3 ° C. C due to compression,

the possible maintenance of the pressure Pi for a time of 110 to 130 seconds, and

the expansion of the sample up to the pressure P ₀ ,

the rate of increase of the pressure for said compression and the rate of decrease of the pressure for said expansion being carried out at a speed Vi of 47.5 to 52.5 MPa / s ^-1 .

2. The method of claim 1 wherein the compression and / or expansion is adiabatic.

The method of claim 1 or 2, wherein the pathogen is selected from the group consisting of bacteria, molds, yeasts, viruses, infectious proteins and / or parasites.

The method of any one of claims 1 to 3, wherein the sample is at an initial compressional temperature of from -18 ° C to -3 ° C.

5. Process according to any one of claims 1 to 4, wherein the initial pressure Po is 0.1 MPa.

The method of any one of claims 1 to 5, wherein the pressure Pi is maintained for a period of 120 seconds.

7. Process according to any one of claims 1 to 6, wherein the pressure Pi is equal to 200 MPa.

The method of any one of claims 1 to 7, wherein the sample is in an airtight package.

The method of any one of claims 1 to 8, wherein the pressure is isostatically applied by means of a pressure transmitting medium.

The method of claim 9, wherein the pressure transmitting medium is a polar or apolar liquid medium.

11. The method of claim 9 or 10, wherein the transmitting medium is ethanol and / or a glycol and water mixture.

12. Use of the method according to claim 1 for the selective removal of at least one pathogen in human blood plasma.

13. Human blood plasma obtained by the method according to any one of claims 1 to 12.