METHOD FOR VALIDATING A STERILIZATION PROCESS INVOLVING TWO SUCCESSIVE CONTAMINATIONS

Abstract

The invention relates to a method for validating a sterilization process for an article, enabling validation of the level of sterility assurance achieved with this sterilization process. The method according to the invention is characterized in that it consists of first contaminating a container (12) receiving the article with more than 10⁵ live microorganism cells, then performing a first sterilization cycle with the selected process, then opening the container (12) to contaminate it again with more than 10⁵ live microorganism cells, then performing a second sterilization cycle with the same process, and finally verifying the sterility of the container (12) after the first cycle and after the second sterilization cycle. This method is particularly applicable to products and devices intended for healthcare.<!--endfragment-->

Description

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a method for validating a sterilization process for a product, in particular a health product (medicine, medical device, cosmetic product, biotechnological product, etc.) packaged in a container that can be opened and then resealed. TECHNOLOGICAL BACKGROUND OF THE INVENTION

[0002] The sterility of certain items, and more specifically the sterility of health products, particularly injectable products and their containers, is a constant concern for both medical professionals and health authorities. Sterility is defined in various standards and regulations as a level of sterility assurance, designated by the acronym "NAS," which represents the probability of non-sterility for each sterilized item—product or container.

[0003] The NAS sterility assurance level generally requires a statistical concentration below a threshold of 10⁻⁶ live colony-forming units, designated by the acronym "CFU", which corresponds to a maximum of one live cell of microorganism per million items, or a probability of non-sterility of one "chance" in a million for each sterilized item.

[0004] Sterilization processes can be classified into two categories based on the curves obtained: a) a first category of sterilization processes for which, according to some authors and a number of regulatory texts, it is possible to deduce an exponential decay curve of the number of living microorganism cells, as a function of a parameter such as the exposure time or an applied dose of irradiation; these processes consist in particular of sterilization by moist heat, by dry heat, by ethylene oxide, as well as sterilization by beta or gamma irradiation, b) a second category of sterilization processes for which it is not possible to deduce an exponential decay curve of the number of living microorganism cells as a function of a parameter such as the exposure time or an applied dosage, which second category includes in particular sterilization by hydrogen peroxide, by peracetic acid, or by hydrostatic hyperpressure.

[0005] One method for assessing the sterility assurance level (NAS) for Category 1 sterilization processes uses biological indicators prepared with the defined microorganism or, if possible, one proven to be the most difficult to destroy with the chosen sterilization process. These biological indicators are inoculated into samples of the article—product or container—to be sterilized. After sterilization, the number of remaining live microorganism cells is measured by varying certain parameters of the sterilization process, most often the sterilization time or dose, in order to assess the interactions between the article and the process.

[0006] This method yields a decay curve of the number of living microorganism cells as a function of several sterilization parameters, generally including exposure time for sterilization processes using moist heat, or dry heat, or ethylene oxide, and including the dose received for sterilization processes by beta or gamma irradiation.

[0007] For this first evaluation method, the curve is generally extended for very low levels of contamination, statistically containing less than one surviving microorganism cell, in order to deduce the characteristics of the sterilization process that allows the desired NAS sterility assurance level to be reached.

[0008] This method, however, presents a problem because extrapolating the curve for very low levels of contamination relies on an assumption that experience cannot always prove. Some standards require achieving a minimum correlation coefficient for high contamination levels through measurements that are indeed difficult to perform.

[0009] A second method for evaluating the NAS sterility assurance level for second category sterilization processes involves inoculating the articles to be sterilized with biological indicators presenting a desired number of live cells of the microorganism, often between 103 and 106 cells, and then, after the sterilization treatment, checking the destruction of all the live cells of these indicators.

[0010] With this method, a decay curve in the number of live microorganism cells as a function of a parameter, such as exposure time, sterilization dose, or the number of cells inoculated, cannot be calculated. Therefore, the probability of non-sterility cannot be assessed either, which raises issues regarding the NAS (Natural Adequacy Score) guarantee. In some cases, both regulations and experts consider this to be decontamination without the requirement of achieving sterility. GENERAL DESCRIPTION OF THE INVENTION

[0011] The present invention is specifically designed to avoid all the problems of the prior art described above.

[0012] To this end, it proposes a method for validating a sterilization process for any item, for example, a container or a product contained in a container suitable for the sterilization process in question. This method is applicable to all sterilization processes and is primarily, but not exclusively, aimed at products and devices intended for healthcare.

[0013] US patent 6,428,746, published on August 6, 2002, describes a method for validating a sterilization process for an article, particularly a health product, allowing validation of the Sterility Assurance Level achieved with this sterilization process. This method consists of carrying out one and only one step of contaminating the article with more than 105 live cells of microorganisms, then carrying out a sterilization cycle with the selected process, and finally verifying the sterility of the article after the sterilization cycle.

[0014] According to the invention, the method for validating a sterilization process for any article, allowing validation of the level of sterility assurance achieved with this sterilization process, is characterized by the fact that it consists of carrying out a first step of contaminating a container receiving the article with more than 10⁵< live cells of microorganism, then carrying out a first sterilization cycle with the selected sterilization process, then opening the container to contaminate it again with more than 10⁵< live cells of microorganism, then carrying out a second sterilization cycle with the same process, and finally verifying the sterility of the container after the first cycle and after the second sterilization cycle.

[0015] The validation method according to the invention may further include one or more of the following features, which may be combined with each other.

[0016] Thus, advantageously, each contamination is carried out with at least 10 6< living cells of microorganism.

[0017] Advantageously, the contaminations are made with the living cells of the microorganism proven to be the most difficult to eliminate with the chosen sterilization process.

[0018] Advantageously, the verification of the sterility of the container involves the revelation of biological indicators demonstrating that none of these indicators have achieved growth.

[0019] Advantageously, both contaminations are carried out under the same conditions, and both sterilization cycles are also carried out under the same conditions, unless required by the process and in particular if the second cycle does not allow the destruction of at least 5 logs of live cells of the microorganism most difficult to destroy by the sterilization process in question.

[0020] Advantageously, the validation method according to the invention makes it possible to modify the second sterilization cycle to ensure that the actual destruction of at least 5 logs of live cells of the microorganism most difficult to destroy by the sterilization process considered has been achieved.

[0021] Advantageously, the invention allows the opening, re-contamination, and closing of containers to be carried out very quickly in order to optimize the time required for routine cycles, consisting of the first cycle, a waiting time equal to these phases of opening, re-contamination, and closing of these containers under the same environmental conditions, and the second cycle.

[0022] The objective of this method is to demonstrate a Sterility Assurance Level (SAL) of at least 10⁻⁵ for an initial contamination of 10⁵ live cells of the microorganism proven to be the most difficult to destroy by the sterilization process under consideration. This method is entirely independent of the mode of action of the sterilization process, and in particular of whether the sterilization process belongs to the first or second category mentioned above.

[0023] Achieving such a NAS is based on obtaining a contamination reduction of at least 10 logs.

[0024] The present method is remarkable in that it actually proves these 10 log reductions in contamination, which no other method can achieve today. It consists of inoculating the item to be sterilized with at least 10⁵< live cells of the microorganism advantageously proven to be the most difficult to destroy by the sterilization process in question, carrying out a first sterilization cycle that succeeds in destroying these at least 10⁵< live cells of this microorganism, then opening the containers, re-inoculating them, and resealing them with at least 10⁵< live cells of the microorganism proven to be the most difficult to destroy by the sterilization process in question, then carrying out a second sterilization cycle identical to the first, which again succeeds in destroying at least 10⁵< live cells of the microorganism most difficult to destroy by this sterilization process.

[0025] The operations of opening the containers, re-inoculating, and re-sealing are carried out using one of the devices conforming to the invention.

[0026] At the end of the first sterilization cycle, samples contaminated with 10⁵< CFU of the microorganism most difficult to destroy by the sterilization process in question are taken and subjected to a test known to those skilled in the art and / or described in regulations to demonstrate the destruction of at least 10⁵< live cells of the microorganism. It is therefore proven at the end of this first cycle that the microorganism is capable of destroying 5 logs of contamination.

[0027] At the end of the second sterilization cycle, samples contaminated with 105 live cells of the microorganism most difficult to destroy by the sterilization process in question are also taken and subjected to the same test known to those skilled in the art and / or described in the regulatory texts to prove the destruction of these at least 105 live cells of the microorganism most difficult to destroy by the sterilization process in question.

[0028] It is therefore proven at the end of this second cycle that the latter is capable of destroying at least 5 additional logs of living cells of the microorganism mentioned above.

[0029] Performing this second cycle is essential because the container and the product or article it contains have already undergone a sterilization cycle, which may have affected their characteristics and behavior during a subsequent sterilization cycle. Without testing, it is not possible to confirm that a second cycle identical to the first actually destroys at least 5 additional logs of live cells of the microorganism most difficult to destroy by the microorganism in question.

[0030] In the event that the second cycle, for the reasons mentioned above, does not allow the reduction of at least 5 additional logs of living cells of this microorganism, a person skilled in the art may well modify the characteristics of this second cycle to achieve this reduction of at least 5 logs.

[0031] The present method is remarkable in that it makes it possible to prove the destruction of at least 5 logs for the first cycle and at least 5 additional logs for the second cycle, which no method can achieve to date.

[0032] The present method is remarkable in that it demonstrates that the routine cycle consisting of the first cycle, a waiting period under the same conditions as those tested to open the containers, re-inoculate them, and close them according to the invention, and then a second cycle, makes it possible to destroy at least 10 logs of the microorganism most difficult to destroy by the sterilization process considered.

[0033] As demonstrated above, a SSN of at least 10⁻⁵ is achieved if the initial contamination before sterilization of the containers to be sterilized is less than or equal to 10⁵ CFU of the microorganism most difficult to destroy by the sterilization process considered, and the sterilization process allows at least 10 logs of this microorganism to be destroyed.

[0034] The present method is remarkable in that it allows us to demonstrate this NAS of at least 10 -5< if the initial contamination is less than 10 5< cells of the microorganism most difficult to destroy by the sterilization process studied.

[0035] An installation designed to implement such a validation method is described. It includes any one of the preceding characteristics, which installation includes means for the successive movement of containers on a workstation comprising at least one opening system, means for inoculation and at least one closing system for these containers.

[0036] According to a first embodiment of this installation, at least one opening system, the inoculation means and at least one closing system are arranged on the same container location.

[0037] According to another embodiment of the installation, at least one opening system, the inoculation means and at least one closing system are arranged on container locations which are offset. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The invention will be better understood and other features and advantages will become more apparent upon reading the following description, given by way of example and without limitation, with reference to the accompanying drawings in which: there figure 1 is a graph showing a curve measuring the number of live microorganism cells as a function of exposure time or sterilization dose, for a sterilization process that establishes an exponential decay curve; the figure 2 is a graph showing the logarithmic conversion of this decay curve; the figure 3 is a graph showing a curve measuring the number of live microorganism cells as a function of exposure time or sterilization dose, for a sterilization process that does not produce an exponential decay curve; the figure 4presents in cross-section, along a vertical plane perpendicular to the direction of scrolling, the workstation of an installation implementing a sterilization validation process according to the invention; the figure 5 is a top view of this installation; and the figure 6 is a top view of an installation following a variant. DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

[0039] There figure 1 represents, for a sterilization process of the first category, a curve 2 giving the number N of live cells of microorganisms measured after an exposure time T of the process or after a dose D in the case of treatment by irradiation, after having introduced a biological indicator comprising a quantity N 0 of live cells of microorganism equal to 10 6< in a closed container containing an article.

[0040] We obtain an exponential curve which tends towards zero for a sufficiently long time T, or for a sufficiently high dose D.

[0041] Alternatively, the same type of curve can be produced for a sterilization process by inoculating the closed container with a quantity N of live cells of microorganisms less than 106, similarly giving an exponential decay curve as a function of the quantity inoculated at the start within this product.

[0042] There figure 2 represents, as a function of the exposure time T, or the applied dose D, a straight line 4 calculated for positive log(N) values, by the logarithmic conversion log(N) of the number N of living cells of microorganisms presented to the figure 1 . Line 4 starts at time T = 0 with the value log (N) = 6 corresponding to contamination by 10 6< cells.

[0043] Note that with log (1) = 0 the logarithmic conversion of an exponential curve thus gives a straight line which passes through 0 at time T1 (or at dose D1) corresponding to the number N = 1 representing a single living cell of measured microorganism.

[0044] Various studies propose, following a validation method known for these sterilization processes, the possibility of extrapolating the line in negative log (N) values ​​by continuing it below 0. These negative values ​​correspond to a number N less than 1, that is to say a probability of less than one surviving microorganism cell per treated container.

[0045] In particular, the European standard "NF EN556" concerning the sterilization of medical devices requires a probability of presence of a microorganism cell of less than 10⁻⁶, meaning a maximum risk of one in a million that a sterilized item still contains surviving microorganisms. This is interpreted by some authors as equivalent to the probability of one live microorganism cell per million sterilized items. Starting from a contamination level of log(N) = 6, a reduction of 12 logs is therefore required, resulting in a value of log(N) = -6. This value is generally achieved at the required treatment time T2 or at dose D2.

[0046] In the case of an article initially containing a higher or lower number of live microorganism cells, the slope of the line given by the sterilization process remaining the same, a longer or shorter exposure time (or a higher or lower dose) is required respectively to reach the value log (N) = -6.

[0047] As a general rule, commonly used biological indicators contain a number of live microorganism cells between 10⁶ and 10⁷.

[0048] To avoid going down into negative log (N) values, one could start from a biological indicator with a very high level of contamination, for example equal to 12, to reach the final value equal to 0. However, this type of biological indicator is still not available on the market today, at least for the reference microorganisms used for sterilization processes.

[0049] It should be noted that the average initial contamination of a batch of medical devices or injectable products prepared for sterilization is generally low, often less than 10 live cells of microorganisms. However, this level of contamination can be random, and an exceptional initial contamination, for example, approaching the maximum level of 106, cannot be ruled out.

[0050] One problem with this validation method is that, since experience shows that some standards impose a minimum correlation coefficient, it is sometimes difficult to establish the logarithmic decay line. Consequently, negative logarithmic values ​​cannot be determined, and therefore the required exposure time T2 or the dose D2 cannot be calculated.

[0051] Another problem posed by this validation method is that the extension of line 4 towards strongly negative values ​​cannot be experimentally verified, and the margin of error remains unknown.

[0052] In particular, for the value log(N) = -6, one million units would need to be tested under identical conditions, which is not physically feasible. This would notably raise a problem of the stability of the biological indicator, which would need to be included in all articles within a very short period of time to avoid its own evolution, which would skew the test.

[0053] Furthermore, it cannot be ruled out that, for negative values ​​of log(N), the law governing the evolution of the quantity of living microorganism cells is considerably more complex, as the mechanisms of destruction of these microorganisms are not yet fully understood. These mechanisms depend in particular on the nature of the microorganisms, the sterilization process studied, and the interactions between the product to be sterilized and the types of microorganisms, which are often significant.

[0054] There figure 3 represents, for a sterilization process of the second category, a curve 6 giving the number N of live microorganism cells measured after an exposure time T of the process, after introducing a biological indicator comprising a quantity of live microorganism cells equal to 10 6< into a closed container containing an article.

[0055] We obtain a curve 6 descending continuously, which does not have a particular shape.

[0056] The texts presenting these sterilization processes of the second category provide for inoculating the articles with biological indicators containing a defined number of live cells of microorganisms, often between 10 3< and 10 6< , and then verifying after sterilization that all the indicators have been destroyed.

[0057] Since it is impossible to establish a regular curve for measuring cell count from a given parameter, it is therefore impossible to calculate the probability of non-sterility for these processes. Furthermore, in some cases, regulations and experts consider this a decontamination process without the requirement of achieving sterility.

[0058] Furthermore, another problem with all the validation methods presented above, with the notable exception of the method using very high hydrostatic pressure, is that a sterilization load cannot be treated homogeneously. In particular, for processes using dry heat, moist heat, or ethylene oxide, there are consistently temperature variations within the load, and for processes involving irradiation, there are dose variations between different points within the load.

[0059] We then have significant variations in sterility assurance which are frequently estimated at several logs of contamination reduction.

[0060] THE Figures 4 and 5 present an installation capable of implementing the validation method according to the invention concerning any sterilization process, which may belong to either the first or the second category.

[0061] The installation comprises an elongated platform 10 receiving a series of containers 12 aligned longitudinally, each containing a product to be sterilized. The container may be of various shapes and sizes. In particular, the container may be a bag, a sachet, a rigid or flexible bottle, a syringe, a rigid or flexible ampoule, a thermoformed tray, or any other container intended to hold any product to be sterilized. Among these products to be sterilized, we can mention, but not exclusively, health products, including, in particular, pharmaceuticals (chemical or biotechnological), medical devices, cosmetic products, gene therapy products, advanced therapy medicinal products, tissues and organs for transplantation, food products, etc.

[0062] For simplicity in the description, the example cited below will use a traditional type of container, namely the flexible pouch, containing a liquid product.

[0063] The advancement of the pockets 12 in the longitudinal direction is done step by step, by means of a manual or automated system.

[0064] The 12 bags underwent a first sterilization cycle beforehand, after a first contamination carried out on one or more samples of articles to be sterilized, with a number of live microorganism CFU cells of 10< 6< preferentially using a microorganism proven to be the most difficult to sterilize by the sterilization process studied.

[0065] The flat pockets 12, held on the platform 10 by a mechanical clamping device if necessary, have necks 14 arranged parallel in the transverse direction, necks which are closed by a plug 16. The platform 10 includes a means for longitudinally translating the pockets 12 in a forward direction, like a conveyor.

[0066] The platform 10 includes a central workstation 30 which is located between upstream bags to be treated 32 and downstream bags already treated 34. The workstation 30 maintains the bag 12 being treated in the same position during the successive operations of opening, first or second contamination, and then closing of this bag.

[0067] It should be noted that it is advantageous to carry out the two contaminations under similar conditions, in particular at the same workstation keeping bag 12 in the same position, with an identical opening time, in order not to modify the parameters allowing comparable effects for these inoculations.

[0068] The tray 10 with its equipment fixed to it can be arranged horizontally, or inclined in the transverse direction, in particular along a vertical plane, by raising the neck 14 in order to avoid leakage of products when opening the bags 12.

[0069] The plate 10 supports a cutting blade 18 fixed to a vertical guide 20, cutting the neck 14 transversely as close as possible to its cap 16 in order to alter the internal volume of the container as little as possible. The cutting blade 18 can be operated either manually, by pressing on an upper support 22, or automatically with, for example, an electric actuator, including an electromagnet.

[0070] After the opening of the bag 12 by cutting its neck 14, the process includes a contamination step which can be carried out manually or automatically, for example with a syringe or a single or multiple pipette in the case where several bags have been opened simultaneously.

[0071] The plate 10 supports a welding device adapted for each type of pocket 12 after the cutting of its neck 14, such a device comprising a matrix 24 mounted below a descent system 26 which welds this neck as close as possible to the cut, and this also in order to limit the modification of the internal volume and the loss of microorganisms found inside.

[0072] In particular, welding can be carried out by resistance with continuous or discontinuous heating, by high-frequency welding, by a high-inductance electromagnet or by any other system adapted to closing the pocket.

[0073] The lowering system 26 can be operated manually or automatically, for example with an electric actuator using an electromagnet. The lowering system 26 is retracted after welding to release the pocket 12, which can then move in the longitudinal direction.

[0074] After, if necessary, opening the mechanical clamping device of bag 12 on tray 10, all the bags are moved longitudinally to place a new bag on workstation 30, and the bags are then moved downstream to the sterilizer using either manual or automated handling. Advantageously, for the second sterilization, the same bags are placed in the sterilizer in the same location as for the first, in order to obtain identical sterilization conditions.

[0075] After placing (for the first cycle) or replacing (for the second cycle) all the 12 bags in the sterilizer, the sterilization cycle is started. After the first cycle, a biological indicator test is performed to verify that no indicator has grown, and the same procedure is followed after the second cycle.

[0076] The absence of growth following the first cycle proves that this cycle has destroyed at least 10< 6< spores of the microorganism most difficult to destroy by the sterilization process considered or, equivalently, that it has reduced the contamination of this microorganism by at least 6 logs.

[0077] The absence of growth following the second cycle also proves that this cycle reduces contamination by at least 6 logs of the same microorganism.

[0078] Advantageously, a person skilled in the art can perform a second cycle different from the first if the second cycle does not completely destroy the inoculated biological indicators, and therefore does not reduce contamination by at least 6 logs. This can be explained, in particular, by changes in the product or container resulting from the first sterilization cycle.

[0079] Since the two cycles were carried out consecutively, the sum of their effects demonstrates a 6 + 6 = 12 log reduction in contamination of this microorganism, as shown in the curves above. figure 2 If the initial contamination of the item to be sterilized is at most equal to 10⁶ spores, the sterility assurance level (NAS) of 10⁻⁶ is reached after 12 logs of contamination reduction as indicated by the figure 2 .

[0080] A biological indicator consists of the microorganism most difficult to destroy by the sterilization process under consideration, and an initial contamination of 10⁶ spores of this microorganism corresponds to a very unfavorable case. Achieving a sterility assurance level (SAL) of 10⁻⁶ for an initial contamination of 10⁶ spores of the biological indicator corresponds to a situation described in reference texts as conditions of over-destruction. The present validation tests described in the method according to the invention therefore guarantee the achievement of an SAL of 10⁻⁶ in all cases where the microbial contamination of the article to be sterilized is controlled at a reasonable level, consistent with that described in the literature and regulatory texts, i.e., significantly lower than 10⁶ live cells of microorganisms, for a cocktail of microorganisms less resistant than the spores of the biological indicators.

[0081] In routine practice, it is sufficient to replicate the validation conditions exactly, consisting of a first sterilization cycle, a waiting period under the conditions described during the validation tests (time, temperature, pressure, etc.), and a second sterilization cycle also identical to that performed during the validation tests. This entire sequence guarantees with certainty and proven accuracy the achievement of a SAL of 10⁻⁶ if the initial contamination is less than or equal to 10⁶ spores. Therefore, this entire sequence guarantees with certainty and proven accuracy the achievement of this SAL of 10⁻⁶ if the initial contamination of the products to be sterilized is less than 10⁶ live cells of microorganisms, regardless of the type of microorganism, since they are less resistant to the sterilization process than spores.

[0082] In routine practice, the bags will not be contaminated for obvious health and safety reasons. The following cycle will be used: first sterilization, waiting time corresponding to the time required for opening, second inoculation and closure of the bags under conditions identical to those used in validation (time, temperature, pressure, etc.), second sterilization.

[0083] There figure 6 presents a platform 10 comprising a central workstation 30 receiving three bags 12a, 12b, 12c offset longitudinally. The cutting operation is performed simultaneously on the first bag 12a with the cutting blade 18, on the second bag 12b the inoculation, and on the third bag 12c the neck sealing.

[0084] After this series of simultaneous operations, the entire set of 12 pockets advances one step in the longitudinal direction, in order to repeat the same operations on the following pockets. This results in a productivity gain for the installation, particularly with automated cutting and welding operations, as all three operations are performed simultaneously.

[0085] As can be expected, the invention is not limited to the preferred embodiments described above.

[0086] On the contrary, it embraces all possible embodiments, provided that these do not fall outside the scope defined by the attached claims which define the scope of the present invention.

[0087] Therefore, the products or articles to be sterilized may be other than health products or articles, provided that sterilization is recommended. Similarly, the containers may be other than bags, provided that such containers are designed to be opened by a cutting system and then immediately resealed, all within a short time.

[0088] In any case, it is of course necessary that the installation described allows for rapid inoculation and closure of all containers in a sterilization load, in order to minimize the overall time that elapses between the two sterilization cycles carried out in validation.

Claims

1. Method for validating a sterilisation process of an item, in particular a product or a device intended for healthcare, allowing one to validate the level of assurance of the sterility reached with said sterilisation process, characterised in that it consists in carrying out a first step of contaminating a container (12) receiving the item with more than 105 living cells from micro-organisms, then carrying out a first sterilisation cycle using the selected process, then opening the container (12) to contaminate it again with more than 105 living cells from micro-organisms, then carrying out a second sterilisation cycle using the same process and, finally, in verifying the sterility of the container (12) after the first cycle and after the second cycle of sterilisation.

2. Validation method according to claim 1, characterised in that each contamination is carried out with at least 106 living cells from micro-organisms.

3. Validation method according to claim 1 or 2, characterised in that the contaminations are carried out using living cells from the micro-organism that has been proven to be the most difficult to eliminate by using the sterilisation process selected.

4. Validation method according to any of claims 1 to 3, characterised in that the verification of the sterility of the container (12) comprises the revelation of biological indicators demonstrating that none of these indicators has increased.

5. Validation method according to any of claims 1 to 4, characterised in that the two contaminations are carried out under the same conditions.

6. Validation method according to any of claims 1 to 5, characterised in that the two sterilisation cycles are carried out under the same conditions.

7. Validation method according to any of claims 1 to 6, characterised in that the routine cycle comprises a first sterilisation cycle, a waiting time and a second sterilisation cycle.

8. Validation method according to claim 7, characterised in that the waiting time for the routine cycles comprised between the two sterilisation cycles equals the time required to contaminate the load between the first and second cycles during validation.

9. Validation method according to any of claims 7 and 8, characterised in that the environment conditions (time, temperature and pressure, in particular) for the routine cycles are identical to or as close as possible to the environment conditions during the contamination of the load between the first and second cycles.