Method of determining a microorganism contamination

Abstract

The invention addressed herein relates to a method of detecting a presence or an absence of microorganism contamination inside a container by measuring a concentration change of a first gas species in a container being at least in parts transparent to electromagnetic radiation.

Description

[0001] Method of determining a microorganism contamination

[0002] Technical field

[0003] The invention addressed herein relates to a method of determining a microorganism contamination inside a container .

[0004] Description

[0005] In several applications there are specific requirements to the composition of a gas present in the headspace of a container with sensitive contents . Such sensitive contents may in particular be food or pharmaceuticals . In the case of the latter, an aseptic packaging is especially important and legally required to ensure a high quality of the packed product . This poses a particular challenge to the industrial systems and processes , e . g . , filling systems , sealing systems , etc . which must meet the aseptic condition requirements .

[0006] The obj ect of the present invention is therefore to provide an alternative , easy method of determining a microorganism contamination inside a container, in particular in the headspace of the container . An even further obj ect of the invention is to provide an alternative method for evaluating the aseptic conditions of production processes .

[0007] An aspect of the invention, on its own or in the context of the still to be addressed, addresses a method . In one embodiment of the method according to the invention, which may be combined with any of the embodiments still to be addressed unless in contradiction, the method is a method of determining a microorganism contamination inside a container by measuring a concentration change of a first gas species in the container, in particular a headspace of the container . In the context of the present invention, the headspace of the container describes the gaseous space or room above the actual solid and / or liquid content of the container . In case of a solid content, the headspace may also extend between and around the content of the container and is only present if the container is not completely filled . The container is at least in parts transparent to electromagnetic radiation, such as for example UV-radiation, visible light and / or IR-radiation . In an alternative embodiment , the container is at least in parts transparent to IR-radiation ( infrared radiation) , meaning that at least one area of the container is transparent to IR-radiation . The said method comprises the steps of : a) introducing a gas and a sterile culture medium for microorganisms into the container prior to the following steps ; b) subj ecting the gas atmosphere in the container, in particular the gas atmosphere in the headspace of the container to an input electromagnetic laser radiation; c) receiving and / or recording an output electromagnetic radiation emitted from an interior of the container, wherein the output electromagnetic radiation originates from the input electromagnetic laser radiation; d) generating a first concentration indicative result being indicative for a concentration of said first gas species from the received and / or recorded output electromagnetic radiation; e ) after a time delay, repeating at least once the steps of : f ) subj ecting the gas atmosphere in the container, in particular the gas atmosphere in the headspace of the container to an input electromagnetic laser radiation; g) receiving and / or recording an output electromagnetic radiation from the container, wherein the output electromagnetic radiation originates from the input electromagnetic laser radiation; and h) generating at least one further concentration indicative result being indicative for the concentration of said first gas species from the received electromagnetic radiation; i ) detecting presence or absence of the microorganism contamination based on a comparison of the generated first concentration indicative result and the at least one further concentration indicative result generated after the time delay .

[0008] Herein, introducing the gas and the sterile culture medium for microorganisms into the container prior to the following steps can be performed manually or by a processing system, such as for example an industrial gas and / or solid filling system . In the context of the present invention, the introduced gas can comprise or consist of the first gas species of which the concentration change should be measured . However, in some embodiments of the invention, the introduced gas does not comprise said first gas species . In the latter case , the first gas species is for example generated by a microorganism present inside the container which may grow on the culture medium for microorganisms .

[0009] The further steps of the method comprise subj ecting the gas atmosphere in the container to an input electromagnetic laser radiation, receiving and / or recording an output electromagnetic radiation from the container, for example in form of transmitted and / or reflected and / or diffused input electromagnetic laser radiation and generating from the received and / or recorded output electromagnetic radiation a first gas species concentration indicative result . In one exemplary embodiment , these further steps (apart from introducing the gas and sterile culture medium for organisms ) can be performed by an apparatus , wherein : i . the input electromagnetic laser radiation is diffused outside the container and distant from the container and / or ii . the output electromagnetic radiation is diffused outside the container and distant from the container and / or iii . the container is moved with respect to the input electromagnetic laser radiation .

[0010] All three options , i ) , ii ) and iii ) have the effect of averaging the intensity of the electromagnetic radiation over a multiplicity of various possible radiation paths of the electromagnetic radiation traversing the gas atmosphere in the container . Furthermore , a combination of at least two of the named options i ) , ii ) and iii ) improves the averaging effect , as the averaging mechanisms are independent . The averaging over a multiplicity of radiation paths reduces the dependency of the first concentration indicative result from the individual distribution of the first gas species inside the container .

[0011] After a time delay, at least the steps of : f ) subj ecting the gas atmosphere in the container, in particular the gas atmosphere in the headspace of the container to an input electromagnetic laser radiation; g) receiving and / or recording an output electromagnetic radiation emitted from the interior of the container, wherein the output electromagnetic radiation originates from the input electromagnetic laser radiation; and h) generating at least one further concentration indicative result being indicative for the concentration of said first gas species from the received and / or recorded electromagnetic radiation; are performed . Based on a comparison between the generated first concentration indicative result and the at least one further concentration indicative result generated after the time of delay, it is detected whether a microorganism contamination is present or absent inside the container .

[0012] The microorganism contamination refers herein to the presence of for example microbes , bacteria, archaea, eukaryota, fungi , and / or algae . Generally, the method is suitable and claimed for determining the contamination by any microorganism or microorganisms having a metabolism in which the first gas species , of which the concentration should be determined, is consumed, or produced .

[0013] The method according to the invention and embodiments of the method discussed below has useful application in the context of a so-called media fill test, which is a known test for determining if a packaging has been contaminated with microorganisms . According to the principles of good manufacturing practices (GMP) , pharmaceutical and food manufacturers must regularly carry out media fill tests to confirm the microbiological status of their aseptic production processes . The media fill test is a process simulation test which evaluates the performance of aseptic production processes in which the pharmaceutical or food is replaced by a sterile culture medium . Possible impurities would result in turbidity of the medium or odor development, which can be inspected visually . The visual inspection process is often carried out manually . Since the media fill test must be representative for an entire batch size , this process can be very time consuming . For some specific types of containers , such as amber glass or nontransparent plastic containers which are e . g . , often used for the packaging of pharmaceuticals or food, visual inspection is not easily applicable and requires additional steps , such as e . g . , transferring the culture medium into clear containers in an aseptic environment . This additional step requires even more time and carries the risk of contamination, which can lead to false results . Furthermore , given the fact that the processing system ( in which e . g . , the cleaning, filling, packaging, and / or sealing of the product , optionally the container comprising the product takes place ) requires a long testing time , the production must be stopped for a long time .

[0014] Moreover, infrared laser absorption spectroscopy is a known method, which is suitable for determining the concentration of a specific gas inside a container . This particular method allows a non-invasive concentration determination, i . e . without the need of opening the container or inserting the measurement apparatus inside the space in which the concentration of a gas species should be determined . Only the infrared radiation passes through the walls of the container and through the gas atmosphere to be analyzed . Depending on the gas species and gas concentration, the radiation intensity of the infrared radiation is reduced, and gas specific absorption bands arise .

[0015] For example , the presence of an increased level of carbon dioxide (CO2 ) inside the container may be an indicator for the presence of microorganisms . However, this particular gas species gives especially intense IR absorption bands , meaning that even small impurities and small CO2 concentrations lead to a signal dominating the IR spectrum .

[0016] As discussed in context of embodiments of the method, the first gas species may be different from CO2 thus the above problem can be avoided in the context of the present invention . In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, the method comprises a step of closing, optionally sealing the container comprising the gas atmosphere and the sterile culture medium prior to the other steps , in particular prior to subj ecting the gas atmosphere in the container, in particular the gas atmosphere in the headspace of the container to the input electromagnetic laser radiation .

[0017] Closing the container comprising the gas atmosphere and the sterile culture medium has the advantage that there is no gas exchange between the inside of the container and the external environment . Introducing this step into the method of claim 1 effects in a higher reliability and accuracy of the contamination determination, as no external contaminants , in particular no microorganisms or no nutritional particles can enter from the outside into the container . In an alternative embodiment of the invention, this step is performed prior to subj ecting the gas atmosphere in the container before the time delay in order to avoid any external contamination as soon as possible .

[0018] In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, the generated first concentration indicative result is a gas concentration . In the context of the present invention, the first concentration indicative result is to be understood as a result value being directly the concentration of the gas species or allowing to draw an inference about the concentration of the gas species , for example in the form of the absorption value or values . An embodiment of the invention, in which the gas species concentration indicative result is directly ( i . e . without further interpretation) obtained as the gas species concentration, is claimed by claim 3 .

[0019] In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, said gas comprises said first gas species or consists or said first gas species .

[0020] In a more specific embodiment , the gas atmosphere inside the container consists only of the first gas species prior to subj ecting the gas atmosphere in the headspace of the container to an input electromagnetic laser radiation before the time delay .

[0021] In another embodiment of the method according to the invention, which may be combined with any of the preaddressed embodiments and any of the embodiments still to be addressed unless in contradiction, the introduced gas is selected as one from the group consisting of Oz, CO2 , N2, and Ar . Accordingly to this embodiment, the gas atmosphere inside the container consists only of the first gas species being one of dried air, N2, O2 , CO2 , and noble gas , in particular Ar, prior to subj ecting the gas atmosphere in the headspace of the container to an input electromagnetic laser radiation before the time delay . Under the assumption that the respective container was contaminated by microorganisms under non-aseptic working conditions , the concentration of the first gas species can decrease or increase during the time delay, the decrease or increase depending on what type of gas species has been introduced in the container and what type of microorganism or microorganisms is present . Under the assumption that the gas species O2 was introduced in a container contaminated by aerobic organisms , the O2 concentration indicative result will most likely indicate a decrease of the O2 concentration since aerobes undergo an aerobic respiration and convert carbohydrates and O2 into CO2 and H2O .

[0022] In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, the gas introduced in the container is sterilized, in particular pre-sterilized . Suitable gas sterili zation means are known to those skilled in the art and comprise for example an aseptic filtration, application of heat, and / or chemical sterilization by contacting the gas with a sterilizing chemical such as e . g . H2O2 or isopropanol .

[0023] In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, a step of incubating the container is performed during the time delay and after obtaining the first concentration indicative result .

[0024] According to the invention, the step of incubating the container describes a step, in which the container is subj ected to an incubation temperature , which incubation temperature is higher than 19 °C . In this step, the container can be subj ected to the incubation temperature once and kept at this temperature until the end of the incubation, or it can be subj ected to a first incubation temperature in a first incubation step, followed by a second incubation temperature in a second incubation step . The beneficial effect of the incubation is that the potentially contaminating organisms grow and multiply faster . Due to the faster growth and / or metabolism of the microorganism or microorganisms , the concentration of the first gas species changes faster compared to a method in which no incubation is performed during the said time delay . Accordingly, conducting this method embodiment allows a quicker decision as to whether the container was / is contaminated by microorganisms . In an alternative embodiment the incubation is performed at a temperature range of 20 ° C to 40 °C, in particular 20 ° C to 35 ° C, further in particular 20 ° C to 30 ° C . In a further alternative embodiment, the step of incubating comprises further means for enhancing the growth speed of the microorganisms , such as for example a shaking of the container, and / or regulating the light intensity in the container . In another embodiment of the method according to the invention, which may be combined with any of the preaddressed embodiments and any of the embodiments still to be addressed unless in contradiction, the step of incubating the container takes 1 hour to 1 year, in particular 1 day to 1 month, further in particular 1 week to 2 weeks .

[0025] In an alternative embodiment of the method, the container comprising the gas atmosphere and the culture medium is incubated at two temperatures, particularly at 20°C to 25°C for 7 days, followed by 30°C to 35°C for another 7 days. Also, a single incubation temperature in the range of 20 °C to 35°C may be used for 14 days.

[0026] In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, the container subjected to the method is made of plastic, optionally made of a CO2-permeable plastic.

[0027] A big advantage of the method is the fact, that it is not in any way limited to CO2 gas species. Quite the opposite, also gas concentration indicative results of gas species different from CO2 can be conveniently obtained. As will be described later, the gas species can be in particular O2.

[0028] Suitable plastic containers are for example made of polyethylene, polystyrene, polypropylene, polyvinyl chloride, bio-based plastics (e.g., PHAs, PLA, starch blends, cellulose-based plastics, lignin-based polymer composites) , and / or petroleum-based plastics (e.g., polyglycolic acid ( PGA) , polybutylene succinate ( PBS ) , polycaprolactone ( PCL) , polyvinyl alcohol ( PVA) , polybutylene adipate terephthalate ( PBAT) ) . Plastic bottles , j ars and blisters offer several advantages , including their light-weight nature , durability, safe handling, and resistance to breakage .

[0029] In particular, determining an increase or a decrease of the CO2 concentration in a container made of a C02-permeable plastic (e . g . , PET ) would suffer a great susceptibility to errors , as a part or the total of the CO2 gas could escape from the container before the CO2 concentration indicative result has been generated . The present invention addresses this problem by an embodiment in which the concentration indicative result is generated for a first gas species being different from CO2 . In this embodiment , the container is made of a CO2 permeable plastic and the method still gives reliable results . In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, the method comprises a step of determining the container as contaminated with microorganisms if the value of the first concentration indicative result generated prior to the time delay is higher than the value of the at least one further concentration indicative result generated after the time delay and / or determining the container as not contaminated with microorganisms if the value of the first concentration indicative result generated prior to the time delay essentially equals the value of the at least one further concentration indicative result generated after the time delay .

[0030] In one embodiment of the method, the evaluation whether the container is contaminated with microorganisms is carried out by a comparison of the values of the first concentration indicative result with the at least one further concentration indicative result generated after the time delay . If no essential change between these at least two indicative results has been determined, the container is determined as not contaminated with microorganisms and e . g . , the processing conditions or the production conditions are determined as aseptic . No essential change between the concentration indicative results is considered when the difference between or the quotient of the at least two concentration indicative results lie within the limits of measurement error which are known to those skilled in the art .

[0031] In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, the gas species is oxygen (O2 ) .

[0032] In this embodiment of the invention, a concentration change of O2 is measured in the container, in particular in the headspace of the container . Both the first concentration indicative result and the at least one further concentration result according to claim 1 are O2 concentration indicative results which are compared in order to determine whether the container is contaminated by microorganisms . This embodiment is advantageous in cases where the container is made from a CO2 permeable material and determining a CO2 concentration indicative result could lead to error-retained values . Due to the chemical and physical properties of O2 , e . g . , an IR signal obtained from this gas species is very specific and does not dominate the spectrum . For example , electromagnetic radiation in the range of approx . 760 nm is especially suitable for detecting oxygen, which has an absorption maximum close to 760 nm . A wavelength range of about + / - 60 pm around the absorption maximum may be sufficient for measuring the absorption line of O2 .

[0033] The obj ective of the present invention is further solved by a method of processing at least one first container and at least one second container, wherein at least one first container is subj ected to the method according to any one of claims 1 to 11 .

[0034] According to this embodiment , at least one first container and at least one second container, are processed e . g . by a mechanical processing system . The processing is for example selected from a group consisting of a cleaning, filling, drying, sterilizing, closing, and sealing of the container .

[0035] In one embodiment , the first and the second container have an identical appearance , or they are different , the latter meaning that they have a different appearance (e . g . different shape , different size , are made from different materials , etc . ) . Some examples for the first and the second container are : a medication packaging in the form of an eye drops packaging, or a pill blister, or a syringe . The obj ective of the present invention is further solved by a method according to claim 12 comprising one or more than one processing steps performed by a processing system, wherein the at least one first container and the at least one second container are processed under the same conditions , at least one of the processing steps being introducing a material into the containers , wherein for the first container the material is the sterile culture medium of step a) of claim 1 , and for the second container the material is different from said culture medium, in particular wherein the material is a medication or food, wherein the at least one first container and the at least one second container are processed by the processing system simultaneously or one after the other .

[0036] In an aspect of the invention, the first container is subj ected to one or more than one processing steps , which processing steps are conducted by a processing system such as for example an industrial bottling or packaging line , reprocessing line , or testing line .

[0037] At least one of said steps is introducing a material into the at least one first container, which in the case of the container subj ected to the method according to any of claims 1 to 11 ( first container) is a culture medium for microorganisms of step a) of claim 1 , such as for example a nutrient agar, a trypticase soy agar, or any other suitable growth medium, in particular excluding food and / or pharmaceuticals .

[0038] At least one of said steps is introducing a material into the at least one second container, the material being chemically different from the culture medium for microorganisms of step a) of claim 1 and is for example a pharmaceutical or food . It is important to mention that processing the containers , and in particular introducing the material into the first container or containers and into the second container or containers should be performed under essentially the same conditions , meaning that the same processing system is used, the containers are sealed in the same way, etc . Before the at least one first container is subj ected to the steps 102 , 103 , 104 , 105 , at least one repetition of the steps 102 , 103 and 104 , and step 107 of claims 1 to 11 , the first and the second container or containers are subj ected to the same processing steps , additional to introducing the material into the containers , which processing steps are performed by a processing system, such as for example a cleaning, drying, sterilizing, closing, and / or sealing .

[0039] For example : the first container is cleaned, sterilized, filled with a gas and a sterile culture medium ( corresponding to step 101 ) of claims 1 to 11 , sealed, and subj ected to the steps of the method steps according to claim 1 to 11 (apart step 101 , which was already done ) . In this case the second container is processed by cleaning, sterilizing, filling with the same , or optionally a different gas as the first container and with a material different from the culture medium for microorganisms and subsequently sealed .

[0040] In another embodiment of the method according to the invention, which may be combined with the embodiment of claim 12 or 13 and / or any of the embodiments still to be addressed unless in contradiction, a presence of a microorganism contamination is determined in the second container if a presence of a microorganism contamination is detected in the first container .

[0041] A big advantage becomes evident if a microorganism contamination should be detected or determined inside a container having a housing which does not allow a visual evaluation of its content . According to one aspect of the method, assuming that the first container has been determined as contaminated with microorganisms , the second container will also be determined as contaminated by the same or different microorganisms without the need for visual inspection inside the container or packaging . In a more specific embodiment of the invention, at least one of the second containers and preferably all of the second containers determined as contaminated with microorganisms are sorted out from a charge to be brought to market and subsequently to the consumer .

[0042] In another embodiment of the method according to the invention, which may be combined with any embodiment of claims 12 to 14 and / or any of the embodiments still to be addressed unless in contradiction, the first container is a container for a pharmaceutical or food and / or the second container is a container for a pharmaceutical or food .

[0043] In one embodiment , the first container is made of glass polymer material , plastic, composite materials and / or a mixture thereof . In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, the method is a part of a media fill test .

[0044] The media fill test is a method being used to quantify the aseptic technique of compounding personnel or processes and to ensure that the products produced in the processes are sterile and without microbiological contamination . During this test, a culture medium for microorganisms , such as for example a soybean-casein digest medium ( SCDM) , is substituted for the actual product , in particular medication product , to simulate processing or admixture compounding . After simulating a sterile preparation on the container comprising the culture medium for microorganisms , the container is incubated and visually checked for turbidity, which indicates the presence of microbial contaminants . The visual checking is however not always easy or even possible , as containers having a dark or rough surface are used for some special applications , such as for example pharmaceuticals packaging or food packaging . In these or similar cases , the visual inspection process is often carried out manually and requires additional steps , such as e . g . , transferring the culture medium into clear containers in an aseptic environment . This additional step requires even more time and carries the risk of contamination, which can lead to false results . Furthermore , given the fact that the processing system ( in which e . g . , the cleaning, filling, packaging, and / or sealing of the container comprising or not comprising the product takes place ) requires a long testing time , the production must be stopped for a long time .

[0045] The present method addresses this problem by detecting the presence or absence of the microorganism contamination by the means of determining gas species concentration indicative results performed instead of the time-consuming manual inspection . The required steps are described in claim 1 and comprise introducing a gas into the container, the container comprising a culture medium, followed by :

[0046] - subj ecting the gas atmosphere of the container, in particular the gas atmosphere in the headspace of the container to an input electromagnetic laser radiation;

[0047] - receiving and / or recording an output electromagnetic radiation emitted from an interior of the container, wherein the output electromagnetic radiation originates from the input electromagnetic laser radiation;

[0048] - generating a first concentration indicative result being indicative for a concentration of said first gas species from the received and / or recorded output electromagnetic radiation;

[0049] - after a time delay, performing at least once the steps of :

[0050] • subj ecting the gas atmosphere in the container, in particular the gas atmosphere in the headspace of the container to an input electromagnetic laser radiation;

[0051] • receiving and / or recording an output electromagnetic radiation emitted from the interior of the container, wherein the output electromagnetic radiation originates from the input electromagnetic laser radiation; and

[0052] • generating at least one further concentration indicative result being indicative for the concentration of the first gas species from the received and / or recorded output electromagnetic radiation; detecting a presence or absence of the microorganism contamination based on a comparison of the generated first concentration indicative result and the at least one further concentration indicative result generated after the time delay .

[0053] At this place it should be noted that in one embodiment of the method, in particular the method used in the context of the media fill test , the method is in no way limited to varying only the material contained by the first and the second container or containers , as also the gases introduced in the containers may vary . For example, the first container may be subj ected to the method according to any of the preceding embodiments , in particular the media fill test , and be filled with a gas such as O2 . The second container, which will either be determined contaminated or not contaminated depending on the result obtained from the first container, may however be filled with a gas different from the gas that has been filled in the first container (here a gas different from O2 ) . This allows for determining a microorganism contamination in containers with a material sensitive to O2 and which have been filled by the same processing system as the first container . In one embodiment of the method, the steps of subj ecting the gas atmosphere of the container to an input electromagnetic laser radiation, receiving and / or recording an output electromagnetic radiation emitted from the interior of the container, and / or generating a concentration indicative result from the received and / or recorded output electromagnetic radiation are performed by an apparatus according to the disclosure of WO 2018 / 141752 Al . This apparatus comprises :

[0054] - a transmitter configured to direct input electromagnetic laser radiation towards a measuring zone ;

[0055] - a holder configured to position the container, in particular the headspace of the container, in the measuring zone ;

[0056] - a receiver configured to receive output electromagnetic radiation emitted from the measuring zone ; and

[0057] - an evaluation unit operably connected to the receiver and configured to generate the concentration indicative result based on the output electromagnetic radiation received by the receiver .

[0058] In another embodiment of the method according to the invention, which may be combined with any one of the preaddressed embodiments and / or any of the embodiments still to be addressed unless in contradiction, subj ecting the gas atmosphere in the container, in particular the gas atmosphere in the headspace of the container to an input electromagnetic laser radiation comprises the steps of : transmitting a first electromagnetic radiation of a first wavelength along a first radiation path across said container, in particular the headspace of the container, wherein said first wavelength corresponds to the wavelength of an absorption line of the first gas species ; and transmitting a second electromagnetic radiation of a second wavelength along a second radiation path across said container, in particular the headspace of the container, wherein said second wavelength corresponds to the wavelength of an absorption line of a second gas species different from the first gas species , wherein said second radiation path is collinear with said first radiation path in a section of said first radiation path and wherein said section extends at least across said container, in particular the headspace of the container; and wherein receiving and / or recording an output electromagnetic radiation from the container comprises the steps of

[0059] - receiving transmitted first electromagnetic radiation; and

[0060] - receiving transmitted second electromagnetic radiation; and wherein generating the first concentration indicative result from the received and / or recorded output electromagnetic radiation comprises determining a first characteristic of the received first electromagnetic radiation and determining a second characteristic of the received second radiation and generating the first concentration indicative result in function of said first and second characteristics .

[0061] In an alternative embodiment, the first characteristic is a peak height of an absorption line of said first gas species and the second characteristic is a width of an absorption line of said second gas species .

[0062] The invention shall now be further exemplified with the help of figures , which should provide a better understanding of the invention and are not intended to limit its scope . The figures show :

[0063] Fig . 1 a flow chart of the method according to the invention of determining a microorganism contamination inside a container by measuring a concentration change of a gas species inside the container;

[0064] Fig . 2 a flow chart of the method according to the invention of determining a microorganism contamination inside a container and a packaging different from the container by measuring a concentration change of a gas species inside the container;

[0065] Fig . 3 schematic depiction of the container;

[0066] Fig . 4 schematic depiction of an embodiment of the inventive method; Fig . 5 schematic depiction of an embodiment of the inventive method .

[0067] Figure 1 shows an exemplary flow chart of the method, the method starting with the step of introducing a gas and a sterile culture medium into the container 101 . According to some embodiments , the container is closed and / or sealed 108 and the gas atmosphere in the container is subj ected to an input electromagnetic laser radiation 102 , for example near infrared (NIR) and / or middle infrared (MIR) laser radiation . The input electromagnetic laser radiation interacts with the gaseous molecules in the headspace of the container and results in an output electromagnetic radiation which is received and / or recorded by a suitable receiver and / or detector 103 . This step is followed by a step of generating a first concentration indicative result based on the received and / or recorded data from the output electromagnetic radiation 104 and is alternatively conducted by a control unit such as for example a computer with a suitable software . A time delay is performed as the next step 105 during which an additional processing of the container may take place , for example an incubation, shaking on a vial shaker, optical inspection, etc . After the time delay 105 , the container is subj ected to a repetition of the steps 102 , 103 and 104 in order to obtain at least one further concentration indicative result . If more than only one repetition of these steps is performed and more than one further concentration indicative result is obtained, further time delay steps 105 between the blocks of the steps 102 , 103 and 104 can be incorporated . In the last step of this method embodiment, it is determined whether the container is contaminated by microorganisms , meaning whether the presence of microorganisms is detected inside the container, based on a comparison of the first concentration indicative result and at least one further concentration indicative result which was generated after at least one time delay and at least one repetition of the steps 102 , 103 and 104 .

[0068] Figure 2 shows an exemplary flow chart of a method embodiment being a modification of the method embodiment in Fig . 1 , wherein at least two containers (at least one first container and at least one second container) are processed by a processing system, for example by an industrial filling device . Herein, the first container is subj ected to the method described and shown in Fig . 1 . The method comprises a step of determining a microorganism contamination in the second container if a presence of a microorganism contamination was detected in the first container subj ected to the method of Fig . 1 . Herein, the method does not end after step 107 , but it is evaluated if the detected presence of microorganism contamination (derived from the concentration change of the gas species ) is in an admissible range , or if there is any contamination at all . Given that the first container is detected as contaminated, and the contamination exceeds an admissible range , at least one of the second container or containers which was processed (e . g . assembled, filled, cleaned, closed and / or sealed) under the same conditions and by the same processing system as the first container, is determined as contaminated 109 . The latter step ends this pathway of the method . Of course , the method can be repeated by further processing other first and / or second container or containers by the processing system and further subj ecting another first container to the method of Fig . 1 . If in step 107 no contamination of the first container was determined, meaning no presence of a microorganism contamination was detected, or the contamination lies in an admissible range , said second container is determined as not contaminated by microorganisms 110 .

[0069] Fig . 3 shows a schematic container 2 having the shape of an eye drop container such as a blow-fill-container . Inside the container 2 and marked by 5 is the culture medium for microorganisms . Around the culture medium 5 and inside the container 2 , in particular in the top part of the container, extends the headspace of the container, marked by 0 . The gas atoms and molecules inside the container 2 form the gas atmosphere 6 in said container .

[0070] Figure 4 shows schematically an embodiment of the method . A gas 4 and the culture medium for microorganisms 5 are introduced into the container 2 . In an alternative embodiment, they are introduced by a processing system such as for example an industrial packaging system, however also other means of introduction, such as for example manually, i . e . by hands , are possible . Subsequently, the inside of the container comprises the culture medium for microorganisms 5 and a gas atmosphere 6 , which gas atmosphere consists of the introduced gas 4 or consists of the introduced gas 4 and at least one further gas species . Optionally, the container is closed or sealed and the method steps 102 , 103 and 104 are performed to obtain a first gas concentration indicative result 9 of the gas species , or any other gas inside the container . After a time delay 105 , during which the container 2 was for example incubated, the number of microorganism cells increased ( if the container was contaminated by microorganisms ) or remained unchanged ( if there was no microorganism contamination, there were no cells to multiply) . Figure 4 shows an embodiment in which the container 2 was contaminated by microorganisms as can be seen by the increase of microorganism contamination 1 after the time delay 105 . The method steps 102 , 103 and 104 are repeated in order to obtain at least one further gas concentration indicative result 10 being indicative for the concentration of the same gas species as the first gas concentration indicative result 9 . According to the invention, the step of time delay 105 and the steps 102 , 103 , 104 to obtain a further gas concentration indicative result 10 can be repeated once , twice , or more than twice to obtain even more further gas concentration indicative results . This embodiment ends with a step of detecting the presence or absence ( in the depicted case the presence ) of the microorganism contamination 107 by comparing the at least one further gas concentration indicative result 10 with the first gas concentration indicative result 9 . For example , if the container was incubated twice , two further gas concentration indicative results were obtained . Therefore , the first further gas concentration indicative result 10 can be compared with the first gas concentration indicative result 9 (obtained before any incubating) , and / or the second further gas concentration indicative result can be compared with the first gas concentration indicative result 9 (obtained before any incubating) .

[0071] Figure 5 shows schematically an embodiment of the method, wherein two containers are processed ( first container 11 at the top and second container 12 at the bottom) . In this case only two containers are shown for simplification, however also 100 , 1000 , 1 . 000 . 000 or 1 . 000 . 000 . 000 containers can be processed under the condition that at least one of the containers ( referred to as the first container 11 ) is subj ected to the method according to any one of claims 1 to 11 . The containers which are not subj ected to the method steps 102 , 103 , 104 and 105 according to claim 1 are referred to as the second container or containers 12 .

[0072] The processing of the first container 11 and the processing of the second container 12 are alternatively conducted by exactly the same processing system . Herein, the processing of the first and the second container or containers can be performed simultaneously or one after the other, i . e . first the processing of the first container or containers 11 and then the processing of the second container or containers 12 , or first the processing of the second container or containers 12 and then the processing of the first container or containers 11 . In the depicted embodiment, in first step, the first container 11 and the second container 12 are washed and / or sterilized . In the next step, a gas 4 and a material (depicted as rectangles below the gas 4 ) are introduced into the containers 11 and 12 , in the case of the first container 11 always the culture medium for microorganisms 5 is introduced as the material and in the case of the second container 12 any material , such as for example eye or ear drops , can be introduced . In this embodiment, the gas 4 introduced in the first container 11 is the same as the gas 4 introduced in the second container 12 . In an alternative embodiment , the containers are subsequently closed and / or sealed 108 and the first container is subj ected to the method steps 102 , 103 , and 104 to obtain the first gas concentration indicative result 9 , followed by a time delay 105 , e . g . for incubation, and a repetition of the steps 102 , 103 and 104 to obtain at least one further gas concentration indicative result 10 . As explained in Figure 4 , a comparison of these results allows to determine whether microorganisms are present inside the container, or if they are absent . I f the gas concentration indicative results ( 9 and 10 ) are essentially the same , it is determined that no living microorganisms are / were present inside the first container 11 during the processing by the processing system (no microorganism contamination 1 , represented in the figure by a smiling face ) . This applies also to the second container 12 , since both containers ( 11 and 12 ) were processed by the same processing system and under the same conditions - the second container 12 is herein determined as not contaminated by microorganisms ( represented in the figure by a smiling face ) .

[0073] If the presence of microorganisms was detected in the first container (microorganism contamination 1 ) , the second container would also be determined as contaminated by microorganisms . List of reference signs

[0074] 0 headspace

[0075] 1 microorganism contamination

[0076] 2 container

[0077] 4 gas

[0078] 5 culture medium

[0079] 6 gas atmosphere

[0080] 9 first concentration indicative result

[0081] 10 further concentration indicative result

[0082] 11 first container or containers

[0083] 12 second container or containers

[0084] 101 step of introducing a gas and a sterile culture medium into the container

[0085] 102 step of subj ecting the gas atmosphere in the container to an input electromagnetic laser radiation

[0086] 103 step of receiving and / or recording an output electromagnetic radiation from the container

[0087] 104 step of generating a first gas species concentration indicative result

[0088] 105 step of time delay

[0089] 106 step of repeating the steps 102 , 103 and 104 at least once to obtain at least one further gas species concentration indicative result

[0090] 107 detecting presence or absence of a microorganism contamination step of closing, optionally sealing the container step of determining the at least one further packaging as contaminated step of determining the at least one further packaging as not contaminated

Claims

Claims1. A method of detecting a presence or an absence of a microorganism contamination (1) inside a container (2) by measuring a concentration change of a first gas species in the container (2) , in particular in a headspace (0) of the container (2) , wherein the container (2) is at least in parts transparent to electromagnetic radiation, wherein the container (2) is in particular a container for food and / or a pharmaceutical, the method comprising the steps of: a) introducing (101) a gas (4) and a sterile culture medium (5) for microorganisms into the container (2) prior to the following steps; b) subjecting (102) the gas atmosphere (6) in the container (2) , in particular the gas atmosphere (6) in the headspace (0) of the container (2) to an input electromagnetic laser radiation; c) receiving and / or recording (103) an output electromagnetic radiation emitted from an interior of the container (2) , wherein the output electromagnetic radiation originates from the input electromagnetic laser radiation; d) generating (104) a first concentration indicative result (9) being indicative for a concentration of said first gas species from the received and / or recorded output electromagnetic radiation; e) after a time delay (105) , performing at least once the steps of:f) subjecting (102) the gas atmosphere (6) in the container (2) , in particular the gas atmosphere (6) in the headspace (0) of the container (2) to an input electromagnetic laser radiation; g) receiving and / or recording (103) an output electromagnetic radiation emitted from the interior of the container (2) , wherein the output electromagnetic radiation originates from the input electromagnetic laser radiation; and h) generating (104) at least one further concentration indicative result (10) being indicative for the concentration of said first gas species from the received and / or recorded output electromagnetic radiation; i) detecting a presence or absence (107) of the microorganism contamination (1) based on a comparison of the generated first concentration indicative result (9) and the at least one further concentration indicative result (10) generated after the time delay.

2. The method according to claim 1 comprising a step of closing, optionally sealing the container (2) comprising the gas atmosphere (6) and the sterile culture medium (5) prior to the other steps, in particular prior to subjecting the gas atmosphere (6) in the container (2) before the time delay, in particular the gas atmosphere (6) in the headspace (0) of the container (2) to the input electromagnetic laser radiation.

3. The method according to claim 1 or 2, wherein the first concentration indicative result (9) is a gas concentration .

4. The method according to any one of claims 1 to 3, wherein said gas (4) comprises said first gas species or consists of said first gas species.

5. The method according to any one of claims 1 to 4, wherein said gas (4) is selected from the group consisting of dried air, N2, O2, CO2 and noble gas, in particular Ar.

6. The method according to any one of claims 1 to 5, wherein said gas (4) is sterilized.

7. The method according to any one of claims 1 to 6, wherein a step of incubating the container (2) is performed during the time delay and after obtaining the first concentration indicative result (9) .

8. The method according to claim 7, wherein the step of incubating the container (2) takes 1 hour to 1 year, in particular 1 day to 1 month, further in particular 1 week to 2 weeks .

9. The method according to any one of claims 1 to 8, wherein the container (2) is made of plastic, optionally made of a CO2-permeable plastic.

10. The method according to any one of claims 1 to 9 comprising a step of determining the container (2) as contaminated with microorganisms if the value of the first concentration indicative result (9) generated prior to the time delay is higher than the value of the at least one further concentration indicative result (10) generated after the time delay and / or determining the container (2) as not contaminated with microorganisms if the value of the first concentration indicative result (9) generated prior to the time delay essentially equals the value of the at least one further concentration indicative result (10) generated after the time delay.

11. The method according to any one of the claims 1 to 10, wherein the gas species is oxygen (O2) .

12. A method of processing at least one first container (11) and at least one second container (12) , wherein the at least one first container (11) is subjected to the method according to any one of claims 1 to 11.

13. A method according to claim 12 comprising one or more than one processing steps performed by a processing system, wherein the at least one first container (11) and the atleast one second container (12) are processed under the same conditions, at least one of the processing steps being introducing a material into the containers, wherein for the first container (11) the material is the sterile culture medium (5) of step a) of claim 1, and for the second container the material is different from the culture medium (5) , in particular wherein the material is a medication or food, wherein the at least one first container (11) and the at least one second container (12) are processed by the processing system simultaneously or one after the other.

14. The method according to claim 12 or 13, wherein a presence of a microorganism contamination is determined in the second container (12) if a presence of a microorganism contamination is detected in the first container (11) .

15. The method according to any one of claims 12 to 14, wherein the first and / or the second container are a container for a pharmaceutical or food.The method according to any one of claims 1 to 15, wherein the method is a part of a media fill test.

17. The method according to any one of the claims 1 to 16, wherein the gas species is different from O2 and / or CO2.

18. The method according to any one of the claims 1 to 17, wherein subjecting the gas atmosphere (6) in the container (2) , in particular the gas atmosphere (6) in the headspace (0) of the container (2) to an input electromagnetic laser radiation comprises the steps of: transmitting a first electromagnetic radiation of a first wavelength along a first radiation path across said container (2) , in particular the headspace (0) of the container, wherein said first wavelength corresponds to the wavelength of an absorption line of the first gas species; and transmitting a second electromagnetic radiation of a second wavelength along a second radiation path across said container (2) , in particular the headspace (0) of the container, wherein said second wavelength corresponds to the wavelength of an absorption line of a second gas species different from the first species, wherein said second radiation path is collinear with said first radiation path in a section of said first radiation path and wherein said section extends at least across said container (2) , in particular the headspace (0) of the container (2) ; and wherein receiving and / or recording (103) an output electromagnetic radiation from the container (2) comprises the steps of- receiving transmitted first electromagnetic radiation; and- receiving transmitted second electromagnetic radiation;and wherein generating ( 104 ) the first concentration indicative result ( 9 ) from the received and / or recorded output electromagnetic radiation comprises determining a first characteristic of the received first electromagnetic radiation and determining a second characteristic of the received second radiation and generating ( 104 ) the first concentration indicative result ( 9 ) in function of said first and second characteristics .

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