WO2004002540A1 - Method for inactivating micro-organisms by applying high pressures - Google Patents

Method for inactivating micro-organisms by applying high pressures Download PDF

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Publication number
WO2004002540A1
WO2004002540A1 PCT/FR2003/001821 FR0301821W WO2004002540A1 WO 2004002540 A1 WO2004002540 A1 WO 2004002540A1 FR 0301821 W FR0301821 W FR 0301821W WO 2004002540 A1 WO2004002540 A1 WO 2004002540A1
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phase
pressure
temperature
mpa
approximately
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PCT/FR2003/001821
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French (fr)
Inventor
Luc Grislain
Gérard Demazeau
Alain Largeteau
Yohan Rigaldie
Catherine Lacaze-Saint-Jean
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Ellipse Pharmaceuticals
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Priority to AU2003258807A priority Critical patent/AU2003258807A1/en
Publication of WO2004002540A1 publication Critical patent/WO2004002540A1/en

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    • 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—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
    • 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—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • 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—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods

Definitions

  • the invention relates to a method for inactivating microorganisms by applying high pressures to products, in particular pharmaceutical products.
  • the bacterial spores can be inactivated by a combined and continuous application of heat and high pressure, for example for 3 hours at 60 ° C. under a pressure of 200 MPa.
  • Treatments have also been proposed comprising pressurization cycles, with a first phase at medium pressure and at medium temperature and a second phase at high pressure, which are repeated for periods of a few hours.
  • the first phase at medium pressure and medium temperature for example 60 MPa and 50 - 70 ° C, induces germination of the spores which makes them more sensitive to high pressures.
  • a drawback of these techniques is that they cannot be applied to pharmaceutical or medicinal products which are sensitive to heat and whose active principles are destroyed in whole or in large part by exposure to temperatures greater than or equal to approximately 50 ° C. .
  • the object of the invention is in particular to provide a simple, effective and economical solution to this problem.
  • the temperature of the first phase is between 30 and 40 ° C approximately and, even more preferably, this temperature is approximately 37 ° C.
  • the pressure applied to the microorganisms during this first phase is preferably between 10 and 30 MPa approximately.
  • This range of temperatures and pressures of the first phase of the cyclic pressurization treatment best promotes the germination of bacterial spores and their inactivation by exposure to the high pressures applied during the second phase.
  • Tests have shown that the destructive efficacy of this treatment, defined by the logarithm based on the ratio of the number of viable microorganisms in the product before treatment and after treatment respectively, could reach very high values compatible with the required values by the Pharmacopoeia.
  • the temperature of the first phase of the cyclic pressurization treatment is set at a value below about 40 ° C for a period of less than Approximately 10 minutes, while the known technique provides for higher temperatures and much longer durations, the selection of the temperatures and durations according to the invention making it possible to inactivate in a very satisfactory manner the microorganisms and bacterial spores of a pharmaceutical product or without significantly modifying the fragile therapeutic molecules contained in these products.
  • the duration of the first phase is preferably around 5 minutes
  • the pressure of the second phase is between approximately 300 and 600 MPa and is preferably of the order of 500 MPa
  • the duration of the second phase is substantially equal to that of the first phase, the number of treatment cycles is between approximately 25 and 35 and is preferably at least equal to 30.
  • FIG. 1 schematically shows a survival curve for Bacillus subtilis spores as a function of the number of treatment cycles according to the invention
  • - Figure 2 shows survival curves of Bacillus subtilis spores after treatment according to the invention and after treatment of the same type, the first phase of which is carried out at ambient pressure; - Figure 3 also shows survival curves for Bacillus subtilis spores after cyclic treatments in which the duration and pressure of the first phase have been modified.
  • the effectiveness of the treatment according to the invention was checked on Bacillus subtilis spores suspended in a liquid medium such as water or tryptone salt.
  • the treatment according to the invention included a first phase of application of a pressure of 20 MPa for a period of 5 minutes at a temperature of 37 ° C, this first phase being intended to cause germination of the bacterial spores.
  • This first phase was repeated alternately with a second phase of applying a pressure of 500 MPa for a period of 5 minutes at a temperature of 37 ° C, the number of treatment cycles being designated by n.
  • Curve I in FIG. 1 represents the destructive efficiency defined by the logarithm based on the ratio 10 of the number No of viable spores in the suspension before treatment and the number N of viable spores in the suspension after treatment, as a function of the number of cycles n. We see in Figure 1 that this efficiency is close to 5 after 15 cycles and that it reaches the value of 6.5 after 30 cycles.
  • the treatment of Curve II therefore comprises a first phase of germination at ambient pressure at a temperature of 37 ° C for a period of 5 minutes, alternating with a second phase of application of a pressure of 500 MPa at a temperature of 37 ° C for 5 minutes.
  • the destructive efficiency is about 5.4 and is therefore lower than that obtained after the treatment corresponding to the Curve I.
  • the curves in FIG. 3 represent the destructive efficiencies as a function of the number of cycles n, of variants of the treatment according to the invention in which: the temperature of the two phases is 28 ° C. (Curve III),
  • the duration of the first phase is 15 minutes and the temperature of 28 ° C (Curve IV),
  • the pressure of the first phase is 120 MPa and the temperature of 28 ° C (Curve V), the other conditions being the same as those of the treatment corresponding to Curve I.
  • Hydrostatic Pressure An Alternative Method of Sterilization
  • insulin was subjected to a treatment corresponding to Curve I, comprising a first phase at a pressure of 20 MPa, lasting 5 minutes at a temperature of 37 ° C and a second phase at a pressure of 500 MPa lasting 5 minutes at a temperature of 37 ° C, the number of treatment cycles being 30.
  • the physico-chemical integrity of the insulin before and after treatment was measured on a liquid chromotography (HPLC) equipped with an ultraviolet detector. After 6 hours of treatment (30 pressurization-depressurization cycles of 5 minutes each), the insulin content of the solution was reduced by 2%.
  • the destructive efficacy of the treatment on Baccillus subtilis spores contained in the treated solution was 6.5.

Abstract

The invention concerns a method for inactivating micro-organisms, in particular in pharmaceutical products, which consists in subjecting said products to repeated cycles including a medium pressure and medium temperature phase and another phase at high temperature, the first phase being carried out at a temperature ranging between about 30 and 40 °C at a pressure of the order of 10 to 30 MPa for about five minutes, the second phase being carried out at the same temperature and at a pressure of about 500 MPa, there being about 30 cycles. The invention enables inactivation of bacterial spores with a performance higher than 6 on a base 10 logarithmic scale.

Description

PROCEDE D'INACTIVATION DE MICROORGANISMES PAR APPLICATION DE HAUTES PRESSIONS PROCESS FOR INACTIVATION OF MICROORGANISMS BY APPLICATION OF HIGH PRESSURES
L'invention concerne un procédé d'inactivation de microorganismes par application de hautes pressions à des produits, en particulier des produits pharmaceutiques.The invention relates to a method for inactivating microorganisms by applying high pressures to products, in particular pharmaceutical products.
Les effets de la haute pression sur les microorganismes sont bien connus de l'homme du métier et cette technique de stérilisation est appliquée dans de nombreux domaines, notamment alimentaires, pharmaceutiques et médicaux. On a montré que des pressions de 300 MPa ou plus permettent de détruire ou d'inactiver des virus, des levures, des moisissures et des bactéries. Les formes bactériennes sporulées se montrent toutefois très résistantes à la chaleur, à la pression et à l'irradiation.The effects of high pressure on microorganisms are well known to those skilled in the art and this sterilization technique is applied in many fields, notably food, pharmaceutical and medical. Pressures of 300 MPa or more have been shown to destroy or inactivate viruses, yeasts, molds and bacteria. Sporulated bacterial forms are however very resistant to heat, pressure and irradiation.
Il a été constaté que l'on pouvait inactiver les spores bactériennes par une application combinée et continue de la chaleur et de la haute pression, par exemple pendant 3 heures à 60°C sous une pression de 200 MPa. On a également proposé des traitements comprenant des cycles de pressurisation, avec une première phase à pression moyenne et à température moyenne et une seconde phase à pression élevée, qui sont répétés pendant des durées de quelques heures. La première phase à pression moyenne et température moyenne, par exemple 60 MPa et 50 - 70°C, induit une germination des spores qui les rend plus sensibles aux hautes pressions. Les travaux réalisés, décrits par exemple dans l'articleIt has been found that the bacterial spores can be inactivated by a combined and continuous application of heat and high pressure, for example for 3 hours at 60 ° C. under a pressure of 200 MPa. Treatments have also been proposed comprising pressurization cycles, with a first phase at medium pressure and at medium temperature and a second phase at high pressure, which are repeated for periods of a few hours. The first phase at medium pressure and medium temperature, for example 60 MPa and 50 - 70 ° C, induces germination of the spores which makes them more sensitive to high pressures. The work carried out, described for example in the article
« Inactivation des spores bactériennes par les hautes pressions hydrostatiques » de H. Delacour, C. Cléry, P. Masson et D. R. Vidal, Ann. Pharm. FR 2002, 60, pages 38 - 43, montrent également que l'élévation de la température, par exemple à 60 - 80°C, favorise l'inactivation des spores bactériennes par les hautes pressions qui sont par exemple de 500 MPa, de très bons résultats étant obtenus avec des cycles de pressurisation de 60 MPa et 500 MPa à une température de 50 ou 70°C."Inactivation of bacterial spores by high hydrostatic pressures" by H. Delacour, C. Cléry, P. Masson and DR Vidal, Ann. Pharm. FR 2002, 60, pages 38 - 43, also show that raising the temperature, for example to 60 - 80 ° C, promotes the inactivation of bacterial spores by high pressures which are for example 500 MPa, very good results being obtained with pressurization cycles of 60 MPa and 500 MPa at a temperature of 50 or 70 ° C.
L'article de B. SOJKA et H. LUDWIG : "Pressure-induced germination and inactivation of Bacillus subtilis spores", Pharm. Ind. vol. 56, n°7, 1994, pages 660-663, décrit un procédé comprenant une combinaison d'étapes à moyenne pression à 60 MPa et à haute pression à 500 MPa sur des intervalles de 30 minutes répétés pendant 240 minutes, à une température de 40°C pour réduire d'un facteur 106 le nombre de spores variables, et à 50°C pour obtenir une solution stérile.The article by B. SOJKA and H. LUDWIG: "Pressure-induced germination and inactivation of Bacillus subtilis spores", Pharm. Ind. flight. 56, no. 7, 1994, pages 660-663, describes a process comprising a combination of medium pressure steps at 60 MPa and high pressure steps at 500 MPa at 30 minute intervals repeated for 240 minutes, at a temperature of 40 ° C to reduce the number of variable spores by a factor of 10 6 , and at 50 ° C to obtain a sterile solution.
Un inconvénient de ces techniques est qu'elles ne peuvent être appliquées à des produits pharmaceutiques ou médicamenteux sensibles à la chaleur et dont les principes actifs seraient détruits en totalité ou en majeure partie par une exposition à des températures supérieures ou égales à 50°C environ.A drawback of these techniques is that they cannot be applied to pharmaceutical or medicinal products which are sensitive to heat and whose active principles are destroyed in whole or in large part by exposure to temperatures greater than or equal to approximately 50 ° C. .
L'invention a notamment pour but d'apporter une solution simple, efficace et économique à ce problème.The object of the invention is in particular to provide a simple, effective and economical solution to this problem.
Elle propose, à cet effet, un procédé d'inactivation de microorganismes par application de hautes pressions à des produits, en particulier des produits pharmaceutiques, consistant à soumettre ces produits à des cycles répétés comprenant une première phase à pression moyenne et température moyenne et une seconde phase à pression élevée, caractérisé en ce que la première phase est réalisée à une température inférieure à 45°C environ, à une pression comprise entre 1 et 50 MPa environ, et a une durée comprise entre 1 et 10 minutes environ.To this end, it proposes a process for the inactivation of microorganisms by applying high pressures to products, in particular pharmaceutical products, consisting in subjecting these products to repeated cycles comprising a first phase at medium pressure and average temperature and a second phase at high pressure, characterized in that the first phase is carried out at a temperature below approximately 45 ° C, at a pressure between 1 and 50 MPa approximately, and has a duration between 1 and 10 minutes approximately.
De préférence, la température de la première phase est comprise entre 30 et 40°C environ et, de façon encore plus préférée, cette température est de 37°C environ.Preferably, the temperature of the first phase is between 30 and 40 ° C approximately and, even more preferably, this temperature is approximately 37 ° C.
La pression appliquée aux microorganismes pendant cette première phase est comprise, de façon préférée, entre 10 et 30 MPa environ. Cette gamme de températures et de pressions de la première phase du traitement cyclique de pressurisation favorise au mieux la germination des spores bactériennes et leur inactivation par exposition aux hautes pressions appliquées pendant la seconde phase. Des essais ont permis de constater que l'efficacité destructrice de ce traitement, définie par le logarithme à base 10 du rapport des nombres de microorganismes viables dans le produit avant traitement et après traitement respectivement, pouvait atteindre des valeurs très élevées compatibles avec les valeurs requises par la Pharmacopée. Contrairement à ce qui ressort des travaux antérieurs publiés sur ce sujet, il est, de façon surprenante, plus efficace que la température de la première phase du traitement cyclique de pressurisation soit réglée à une valeur inférieure à 40°C environ pendant une durée inférieure à 10 minutes environ, alors que la technique connue prévoit des températures supérieures et des durées beaucoup plus longues, la sélection des températures et des durées selon l'invention permettant d'inactiver de façon très satisfaisante les microorganismes et spores bactériennes d'un produit pharmaceutique ou médicamenteux sans modifier de façon sensible les molécules thérapeutiques fragiles contenues dans ces produits.The pressure applied to the microorganisms during this first phase is preferably between 10 and 30 MPa approximately. This range of temperatures and pressures of the first phase of the cyclic pressurization treatment best promotes the germination of bacterial spores and their inactivation by exposure to the high pressures applied during the second phase. Tests have shown that the destructive efficacy of this treatment, defined by the logarithm based on the ratio of the number of viable microorganisms in the product before treatment and after treatment respectively, could reach very high values compatible with the required values by the Pharmacopoeia. Contrary to what emerges from previous works published on this subject, it is surprisingly more effective for the temperature of the first phase of the cyclic pressurization treatment to be set at a value below about 40 ° C for a period of less than Approximately 10 minutes, while the known technique provides for higher temperatures and much longer durations, the selection of the temperatures and durations according to the invention making it possible to inactivate in a very satisfactory manner the microorganisms and bacterial spores of a pharmaceutical product or without significantly modifying the fragile therapeutic molecules contained in these products.
Selon d'autres caractéristiques de l'invention :According to other characteristics of the invention:
- la durée de la première phase est de préférence d'environ 5 minutes,the duration of the first phase is preferably around 5 minutes,
- la pression de la seconde phase est comprise entre 300 et 600 MPa environ et est de préférence de l'ordre de 500 MPa,the pressure of the second phase is between approximately 300 and 600 MPa and is preferably of the order of 500 MPa,
- la durée de la seconde phase est sensiblement égale à celle de la première phase, - le nombre de cycles du traitement est compris entre 25 et 35 environ et est de préférence au moins égal à 30.the duration of the second phase is substantially equal to that of the first phase, the number of treatment cycles is between approximately 25 and 35 and is preferably at least equal to 30.
Un tel traitement appliqué à la variété sporulée de Bacillus subtilis permet d'obtenir une efficacité destructrice de ce microorganisme supérieure à 6 dans l'échelle logarithmique à base 10. Les formes sporulées de Bacillus subtilis sont parmi les plus résistantes à ce type de traitement et peuvent servir de référence pour la vérification de l'efficacité d'un traitement de stérilisation, comme indiqué dans la note intitulée « Kinetics of microbial inactivation for alternative food processing technologies - High pressure processing » US Food and Drug Administration, Center of Food Safety and Applied Nutrition, Juin 2000. L'invention sera mieux comprise et d'autres caractéristiques, détails et avantages de celle-ci apparaîtront plus clairement à la lecture de la description qui suit, faite à titre d'exemple en référence aux dessins annexés dans lesquels :Such a treatment applied to the sporulated variety of Bacillus subtilis makes it possible to obtain a destructive efficiency of this microorganism greater than 6 in the logarithmic scale based on 10. The sporulated forms of Bacillus subtilis are among the most resistant to this type of treatment and can be used as a reference for verifying effectiveness sterilization treatment, as indicated in the note entitled "Kinetics of microbial inactivation for alternative food processing technologies - High pressure processing" US Food and Drug Administration, Center of Food Safety and Applied Nutrition, June 2000. The invention will be better included and other characteristics, details and advantages thereof will appear more clearly on reading the description which follows, given by way of example with reference to the appended drawings in which:
- La Figure 1 représente schématiquement une courbe de survie des spores de Bacillus subtilis en fonction du nombre de cycles du traitement selon l'invention ;- Figure 1 schematically shows a survival curve for Bacillus subtilis spores as a function of the number of treatment cycles according to the invention;
- La Figure 2 représente des courbes de survie des spores de Bacillus subtilis après traitement selon l'invention et après un traitement du même type dont la première phase est réalisée à pression ambiante ; - La Figure 3 représente également des courbes de survie des spores de Bacillus subtilis après des traitements cycliques dans lesquels on a modifié la durée et la pression de la première phase.- Figure 2 shows survival curves of Bacillus subtilis spores after treatment according to the invention and after treatment of the same type, the first phase of which is carried out at ambient pressure; - Figure 3 also shows survival curves for Bacillus subtilis spores after cyclic treatments in which the duration and pressure of the first phase have been modified.
L'efficacité du traitement selon l'invention a été vérifiée sur des spores de Bacillus subtilis en suspension dans un milieu liquide tel que de l'eau ou du tryptone sel. Le traitement selon l'invention comprenait une première phase d'application d'une pression de 20 MPa pendant une durée de 5 minutes à une température de 37°C, cette première phase étant destinée à provoquer une germination des spores bactériennes. Cette première phase était répétée en alternance avec une seconde phase d'application d'une pression de 500 MPa pendant une durée de 5 minutes à une température de 37°C, le nombre de cycles du traitement étant désigné par n.The effectiveness of the treatment according to the invention was checked on Bacillus subtilis spores suspended in a liquid medium such as water or tryptone salt. The treatment according to the invention included a first phase of application of a pressure of 20 MPa for a period of 5 minutes at a temperature of 37 ° C, this first phase being intended to cause germination of the bacterial spores. This first phase was repeated alternately with a second phase of applying a pressure of 500 MPa for a period of 5 minutes at a temperature of 37 ° C, the number of treatment cycles being designated by n.
La Courbe I dans la Figure 1 représente l'efficacité destructrice définie par le logarithme à base 10 du rapport du nombre No de spores viables dans la suspension avant traitement et du nombre N de spores viables dans la suspension après traitement, en fonction du nombre de cycles n. On voit en Figure 1 que cette efficacité est voisine de 5 après 15 cycles et qu'elle atteint la valeur de 6,5 après 30 cycles.Curve I in FIG. 1 represents the destructive efficiency defined by the logarithm based on the ratio 10 of the number No of viable spores in the suspension before treatment and the number N of viable spores in the suspension after treatment, as a function of the number of cycles n. We see in Figure 1 that this efficiency is close to 5 after 15 cycles and that it reaches the value of 6.5 after 30 cycles.
La valeur de 6,5 obtenue après 30 cycles de traitement à 37°C est remarquablement élevée et est comparable aux meilleurs résultats obtenus dans la technique antérieure par exécution d'un traitement à des températures supérieures à 50°C, qui aurait pour effet de détruire complètement ou au moins en majeure partie les principes actifs de produits pharmaceutiques ou médicamenteux.The value of 6.5 obtained after 30 treatment cycles at 37 ° C is remarkably high and is comparable to the best results obtained in the prior art by carrying out a treatment at temperatures above 50 ° C, which would have the effect of destroy all or at least most of the active ingredients in pharmaceutical or medicinal products.
La résistance élevée des spores de Bacillus subtilis au traitement par des hautes pressions et l'intérêt du traitement selon l'invention peuvent être bien appréciés si l'on rappelle que lorsqu'on soumet des spores de Bacillus subtilis en suspension à une pression de 500 Mpa pendant 6 heures à température ambiante, l'efficacité destructrice mesurée de ce traitement est inférieure à 1. En Figure 2, la Courbe I est identique à celle de la Figure 1 et la Courbe II représente l'efficacité destructrice d'une variante du traitement selon l'invention, dans laquelle la première phase est réalisée à pression ambiante, les autres conditions de température, de pression et de durée n'étant pas modifiées. Le traitement de la Courbe II comprend donc une première phase de germination à pression ambiante à une température de 37°C pendant une durée de 5 minutes, alternant avec une seconde phase d'application d'une pression de 500 MPa à une température de 37°C pendant 5 minutes. On voit qu'après 30 cycles, l'efficacité destructrice est de 5,4 environ et est donc inférieure à celle obtenue après le traitement correspondant à la Courbe I.The high resistance of Bacillus subtilis spores to treatment by high pressures and the advantage of the treatment according to the invention can be well appreciated if it is recalled that when spores of Bacillus subtilis in suspension are subjected to a pressure of 500 Mpa for 6 hours at room temperature, the measured destructive efficiency of this treatment is less than 1. In Figure 2, Curve I is identical to that of Figure 1 and Curve II represents the destructive efficiency of a variant of the treatment according to the invention, in which the first phase is carried out at ambient pressure, the other conditions of temperature, pressure and duration not being modified. The treatment of Curve II therefore comprises a first phase of germination at ambient pressure at a temperature of 37 ° C for a period of 5 minutes, alternating with a second phase of application of a pressure of 500 MPa at a temperature of 37 ° C for 5 minutes. We see that after 30 cycles, the destructive efficiency is about 5.4 and is therefore lower than that obtained after the treatment corresponding to the Curve I.
Les courbes de la Figure 3 représentent les efficacités destructrices en fonction du nombre de cycles n, de variantes du traitement selon l'invention dans lesquelles : - la température des deux phases est de 28°C (Courbe III),The curves in FIG. 3 represent the destructive efficiencies as a function of the number of cycles n, of variants of the treatment according to the invention in which: the temperature of the two phases is 28 ° C. (Curve III),
- la durée de la première phase est de 15 minutes et la température de 28°C (Courbe IV),- the duration of the first phase is 15 minutes and the temperature of 28 ° C (Curve IV),
- la pression de la première phase est de 120 MPa et la température de 28°C (Courbe V), les autres conditions étant les mêmes que celles du traitement correspondant à la Courbe I.- the pressure of the first phase is 120 MPa and the temperature of 28 ° C (Curve V), the other conditions being the same as those of the treatment corresponding to Curve I.
On voit en Figure 3 que la diminution de la température se traduit par une diminution de l'efficacité destructrice, que l'augmentation de la durée de la première phase se traduit par une nouvelle diminution de cette efficacité destructrice, et que l'augmentation de la pression de la première phase a également un effet négatif sur l'efficacité destructrice.We see in Figure 3 that the decrease in temperature results in a decrease in destructive efficiency, that the increase in the duration of the first phase results in a further decrease in this destructive efficiency, and that the increase in the pressure of the first phase also has a negative effect on the destructive efficiency.
Les essais qui ont permis de tracer les courbes des Figures 1 à 3 ont été réalisés avec un appareillage du type décrit dans l'article « HighThe tests which made it possible to draw the curves of Figures 1 to 3 were carried out with an apparatus of the type described in the article "High
Hydrostatic Pressure (HHP) : An Alternative Method of Sterilization andHydrostatic Pressure (HHP): An Alternative Method of Sterilization and
Decontamination of Fragile Drugs ? » de Rigaldie et al., publié dans European Journal of Parenteral Sciences 2001 , 6 (3), pages 73 à 77.Decontamination of Fragile Drugs? By Rigaldie et al., Published in European Journal of Parenteral Sciences 2001, 6 (3), pages 73 to 77.
D'autres essais ont permis de vérifier que le traitement selon l'invention n'altérait pas de façon sensible les principes actifs des produits pharmaceutiques ou médicamenteux.Other tests have made it possible to verify that the treatment according to the invention does not significantly alter the active principles of pharmaceutical or medicinal products.
En particulier, on a soumis de l'insuline à un traitement correspondant à la Courbe I, comprenant une première phase à une pression de 20 MPa, d'une durée de 5 minutes à une température de 37°C et une seconde phase à une pression de 500 MPa d'une durée de 5 minutes à la température de 37°C, le nombre de cycles du traitement étant de 30. L'intégrité physico-chimique de l'insuline avant et après traitement a été mesurée sur un appareil de chromotographie liquide (HPLC) équipé d'un détecteur à ultra-violet. Après 6 heures de traitement (30 cycles de pressurisation-dépressurisation de 5 minutes chacun), la teneur en insuline de la solution était diminuée de 2%.In particular, insulin was subjected to a treatment corresponding to Curve I, comprising a first phase at a pressure of 20 MPa, lasting 5 minutes at a temperature of 37 ° C and a second phase at a pressure of 500 MPa lasting 5 minutes at a temperature of 37 ° C, the number of treatment cycles being 30. The physico-chemical integrity of the insulin before and after treatment was measured on a liquid chromotography (HPLC) equipped with an ultraviolet detector. After 6 hours of treatment (30 pressurization-depressurization cycles of 5 minutes each), the insulin content of the solution was reduced by 2%.
L'efficacité destructrice du traitement sur des spores de Baccillus subtilis contenues dans la solution traitée a été de 6,5.The destructive efficacy of the treatment on Baccillus subtilis spores contained in the treated solution was 6.5.
Des essais analogues ont été réalisés sur une solution d'héparine dont l'intégrité physico-chimique a été mesurée avant et après traitement par electrophorese capillaire. Les mesures effectuées sur l'héparine ne permettent pas de mettre en évidence une modification de sa structure physico-chimique à la suite du traitement. L'efficacité destructrice mesurée sur les spores de Baccilus subtilis contenues dans la solution d'héparine a été de 6,62. A titre de comparaison, lorsque ces solutions d'insuline et d'héparine sont soumises à un traitement de stérilisation par un autoclavage de 20 minutes à 120°C, l'insuline est détruite à 98% et l'héparine est totalement détruite. Similar tests have been carried out on a solution heparin whose physicochemical integrity was measured before and after treatment by capillary electrophoresis. The measurements carried out on heparin do not make it possible to highlight a modification of its physico-chemical structure following the treatment. The destructive efficacy measured on the spores of Baccilus subtilis contained in the heparin solution was 6.62. By way of comparison, when these insulin and heparin solutions are subjected to a sterilization treatment by autoclaving for 20 minutes at 120 ° C., the insulin is 98% destroyed and the heparin is completely destroyed.

Claims

REVENDICATIONS
1/ Procédé d'inactivation de microorganismes par application de hautes pressions à des produits, en particulier pharmaceutiques, consistant à soumettre ces produits à des cycles répétés comprenant une première phase à pression moyenne et température moyenne et une seconde phase à pression élevée, caractérisé en ce que la première phase est réalisée à une température inférieure à 45°C environ, à une pression comprise entre 1 et 50 MPa environ, et a une durée comprise entre 1 et 10 minutes environ.1 / Method for inactivating microorganisms by applying high pressures to products, in particular pharmaceuticals, consisting in subjecting these products to repeated cycles comprising a first phase at medium pressure and medium temperature and a second phase at high pressure, characterized in that the first phase is carried out at a temperature below about 45 ° C., at a pressure of between 1 and 50 MPa approximately, and has a duration of between 1 and 10 minutes approximately.
2/ Procédé selon la revendication 1 , caractérisé en ce que la température de la première phase est comprise entre 30 et 40°C environ.2 / A method according to claim 1, characterized in that the temperature of the first phase is between 30 and 40 ° C. approximately.
3/ Procédé selon la revendication 1 ou 2, caractérisé en ce que la température de la première phase est de 37°C environ.3 / A method according to claim 1 or 2, characterized in that the temperature of the first phase is about 37 ° C.
4/ Procédé selon l'une des revendications précédentes, caractérisé en ce que les températures des deux phases sont identiques ou sensiblement identiques.4 / Method according to one of the preceding claims, characterized in that the temperatures of the two phases are identical or substantially identical.
5/ Procédé selon l'une des revendications précédentes, caractérisé en ce que la pression de la première phase est comprise entre5 / Method according to one of the preceding claims, characterized in that the pressure of the first phase is between
10 et 30 MPa environ.10 and 30 MPa approximately.
6/ Procédé selon l'une des revendications 1 à 5, caractérisé en ce que la durée de la première phase est d'environ 5 minutes.6 / Method according to one of claims 1 to 5, characterized in that the duration of the first phase is approximately 5 minutes.
Il Procédé selon l'une des revendications précédentes, caractérisé en ce que la pression de la seconde phase est comprise entreIl Method according to one of the preceding claims, characterized in that the pressure of the second phase is between
300 et 600 MPa environ.300 and 600 MPa approximately.
8/ Procédé selon la revendication 7, caractérisé en ce que la pression de la seconde phase est de 500 MPa environ.8 / A method according to claim 7, characterized in that the pressure of the second phase is approximately 500 MPa.
9/ Procédé selon l'une des revendications précédentes, caractérisé en ce que la durée de la seconde phase est comprise entre 1 et9 / Method according to one of the preceding claims, characterized in that the duration of the second phase is between 1 and
10 minutes environ. 10/ Procédé selon la revendication 9, caractérisé en ce que la durée de la seconde phase est de 5 minutes environ.About 10 minutes. 10 / A method according to claim 9, characterized in that the duration of the second phase is approximately 5 minutes.
11/ Procédé selon l'une des revendications précédentes, caractérisé en ce que le nombre de cycles auxquels sont soumis les produits est compris entre 25 et 35 environ.11 / Method according to one of the preceding claims, characterized in that the number of cycles to which the products are subjected is between 25 and 35 approximately.
12/ Procédé selon l'une des revendications précédentes, caractérisé en ce que le nombre de cycles auxquels sont soumis les produits est de 30 environ. 12 / Method according to one of the preceding claims, characterized in that the number of cycles to which the products are subjected is approximately 30.
PCT/FR2003/001821 2002-06-26 2003-06-16 Method for inactivating micro-organisms by applying high pressures WO2004002540A1 (en)

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Publication number Priority date Publication date Assignee Title
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