Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/05—Living organisms or biological materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/05—Living organisms or biological materials
  • A61L2103/09—Blood or products thereof

Definitions

• 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.
• the human blood plasma obtained is a biologically active plasma in which at least one pathogen is inactivated.
• references in parentheses refer to the list of references presented at the end of the text.
• 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.
• PSL labile blood product preparation
• High pressure treatment processes are known in the food industry to inactivate certain pathogens such as molds, bacteria and sometimes viruses.
• pathogens such as molds, bacteria and sometimes viruses.
• 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.
• 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.
• 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 :
• 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 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.
• 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.
• sample of human blood plasma 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
• inactivation is meant the suppression of the biological activity of at least one pathogen or set of pathogens.
• 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)
• pathogen any biological entity known to those skilled in the art.
• it may be a bacterium, a mold, a virus, a yeast, an infectious protein and / or parasites.
• 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.
• thermodynamics are 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.
• the initial pressure P 0 is for example the pressure at which the sample is located before implementing the method of the invention.
• 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.
• 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.
• 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).
• 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
• the packaging may for example comprise a connector, that is to say means for filling and / or empty packaging.
• 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.
• 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.
• the pressure can be applied isostatically by means of a pressure transmitter liquid.
• any medium known to those skilled in the art may be used in the method of the invention.
• it may be a liquid or gaseous medium.
• 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
• the liquid transmitting medium is selected from the group consisting of a glycol and water mixture, for example Kryo Lauda, and ethanol.
• pressure isostatically allows advantageously to compress and relax uniformly and without pressure gradient at any point in the sample.
• 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.
• the initial temperature may range from -18 ° C to -3 ° C.
• the compression and / or the expansion are adiabatic.
• the pressure Pi may be equal to
• 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.
• 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
• 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.
• 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.
• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• this figure represents the effect of the rate of application of the pressure on the activity of coagulation factors during a cyclic treatment (5 ⁇ 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.
• SSC 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
• mean as the mean value of the fluorescence intensity
• 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 activity of coagulation factors 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
• 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.
• 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.
• 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.
• 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 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.
• 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).
• 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.
• 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.
• 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.
• 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).
• 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.
• Example 1 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 .
• Example 6 Example of inactivation of a pathogen in human blood plasma at high pressures as a function of temperature.
• 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
• ATCC 6538 tryptone-salt broth
• TCS Tryptic Soy Agar
• the contaminated plasma samples were then placed in sterile and sealed tubes, then kept in the freezer awaiting treatment by an inactivation process.
• a count of the samples is made in order to determine the destructive efficiency of this treatment.
• cascade dilutions of the samples are carried out in TS broth.
• 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:
• ⁇ c 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
• N 0 concentration of S. aureus before high pressure treatment
• the inactivation treatment used was that described in Example No. 5.
• 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).
• the samples were placed in a high pressure generator equipment by indirect compression set up internally with ethanol as the transmitting medium.
• Figure 6 shows the effect of temperature during such treatment on its destructive efficacy vis-à-vis S. aureus.
• 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.
• Example 7 Example of Inactivation of a Pathogen in Human Blood Plasma under High Pressures as a Function of Temperature and Pressure Value
• 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 samples were placed in a high pressure generating equipment set up internally with ethanol as the transmitting medium.
• 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.
• Example 8 Example of inactivation of pathogens in human blood plasma under high pressures as a function of temperature.
• the value of the pressure was set at 200 MPa.
• 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 samples were placed in a high pressure generator equipment by indirect compression set up internally with ethanol as the transmitting medium.
• 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 is a function of the sensitivity of each of the microorganisms vis-à-vis in particular the pressure.
• 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
• 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 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
• 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 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) .
• 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).
• the samples were placed in a high pressure generator equipment by indirect compression set up internally with ethanol as the transmitting medium.
• 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) .
• 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.
• 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.
• 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 ⁇ g / ml, detection of PrP Sc by Western Blot (the antibody used being the 3F4 antibody).
• 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 ).
• Staphylococcus aureus is a well-identified pathogen and causes many nosocomial diseases This pathogen was chosen as a model in this example.
• 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.
• FIG. 13 represents the values of the destructive efficiency.
• 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
• 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.
• 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.
• VA speed of application
• 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.
• 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.
• 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)
• example 13 only a continuous application mode was applied.
• 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
• 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.
• 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
• the objective of this example is to evaluate the impact of the treatment temperature on the inactivation of S. aureus.
• 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)
• 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 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)].
• 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).
• 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
• the residual activity of the coagulation factors City and V was determined by the partial thromboplastin method with activator described in Example 1.
• 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).
• 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
• 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.
• 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).
• 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.
• Example 20 Inactivation of strains of S. aureus multi-resistant to antibiotics by implementing an exemplary method of the invention.
• Table 2 below represents the results of the destructive efficiency (ED) obtained according to the strains tested.
• 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.
• 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)).
• 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)
• 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
• 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.
• 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.
• a Lac Z reporter gene whose expression is under the control of a promoter activated by the Tat protein.
• 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.
• 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".
• 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.
• 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.
• 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,
• Each of the samples control or having undergone high pressure treatment, was observed by optical microscopy.
• the microscope used was an OLYMPUS CA20.
• 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.
• 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.
• an example of a method that is the subject of the present invention makes it possible to inactivate all the parasites tested.
• 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.
• monocytes were placed in the presence of plasma treated according to an exemplary method of the invention.
• DCs dendritic cells
• 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.
• 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:
• GMSCF granulocyte-macrophage colony stimulating factor
• IL4 interleukin 4
• 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).
• PBS phosphate buffered saline
• 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.
• 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).
• SSC Sideways Scatter Channel - signal correlated to the cellular complexity
• FSC Forward Scatter Channel - signal correlated with the relative size of the cells.
• 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.
• the monocytes remained in their initial form, the fluorescence associated with the CD14 marker being higher than that of CD40.
• 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.
• the plasma obtained can, for example be used in the therapeutic field.
• Tf is a factor H and plasminogen binding protein. The Journal of Immunology vol.179 (5): 2979-

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