Method for testing packaging and sealing integrity of aerosol challenge instrument and multi-dose eye drop bottle and bacterial resistance of physical bacterial resistance system during use

Abstract

The invention relates to the technical field of performance testing of medical instruments, and mainly discloses an aerosol challenge instrument and a method for testing packaging and sealing integrity of a multi-dose eye drop bottle and bacterial resistance of a physical bacterial resistance system when the aerosol challenge instrument is used. The test conditions of vacuumizing and pressurizing can be set, and the possible pressure change of the packaging system in the actual storage and transportation process can be simulated; according to the multi-dose eye drop bottle package sealing integrity test method, the integrity of the package of the eye drop bottle is subjected to a microbiological test on the whole, the sensitivity of the test method is improved, and the reliability of a test result is improved; the invention relates to a method for testing the bacterium resistance of a physical bacterium blocking system when a multi-dose eye drop bottle is used, which actually simulates whether liquid microorganisms pollute internal eye drops when eyes are infected, and carries out an aerosol gas return valve challenge test on a gas return valve of the physical bacterium blocking system. Whether microorganisms in air pollute internal eye drops or not during air return of a physical bacteria blocking system is actually simulated.

Description

Technical Field

[0001] The present invention relates to the technical field of medical device performance testing, in particular to an aerosol challenger, a method for testing the sealing integrity of a multi-dose eye drop bottle package and the bacteriostatic property of a physical bacteriostatic system during use. Background Art

[0002] Due to the large packaging volume, multi-dose eye drops are usually used for 20-30 days. During repeated use, microbial contamination is prone to occur. In order to maintain the sterility of the drug and extend the shelf life of the drug after opening, multi-dose eye drops mostly use preservatives to inhibit the growth of microorganisms in the sample. It is well known that preservatives are cytotoxic to the surface of the eye and may cause eye damage. The side effects of preservatives have always been a problem that manufacturers and users are concerned about. Over the years, the risk of side effects of preservatives in topical medication formulations has been widely concerned, and preservative-free is gradually developing into a trend for topical medication. The physical bacteria-blocking system of multi-dose eye drop bottles uses physical methods to block bacteria without adding preservatives, which can maintain the sterility of multi-dose eye drops without the side effects of preservatives.

[0003] The physical bacteria-blocking system of multi-dose eye drop bottles is a multiple-volume container that uses a physical barrier to block microbial contamination of the contents. It has a liquid channel and a return air channel. The liquid channel uses a loaded spring to control the sealing of the drop hole, so that the liquid and contaminated liquid cannot flow back; the return air channel uses a sterile filter element to filter the air to prevent microorganisms from entering the package through the return air channel.

[0004] The multi-dose eye drop bottle with a physical bacteria barrier system is a new type of eye drop packaging. Although eye drops are external medications, they are directly applied to the eyes and are high-risk local medications. The sterility requirements for them have always been the same as those for injections. The biggest risk of its physical bacteria barrier system is that microorganisms contaminate the liquid channel and the return air channel during use. Therefore, the bacteria barrier performance of the liquid channel and the return air channel of the multi-dose eye drop bottle to ensure that there is no microbial invasion inside the package is the key performance of the physical bacteria barrier system of the multi-dose eye drop bottle.

[0005] The sealing of the packaging system, also known as the integrity of the container seal, refers to the ability of the packaging system to prevent the loss of contents, the invasion of microorganisms, and the entry of gases (oxygen, air, water vapor, etc.) or other substances, and to ensure that the drug continues to meet safety and quality requirements. Leakage of sterile drug and medical device packaging systems and contamination of contents by microorganisms will pose a major risk to the safety of patients' medication. Foreign drug regulatory agencies have successively issued standards and specifications requiring that sterile drug packaging systems should be able to ensure the sterility of products within the validity period, and the sealing of sterile drug packaging systems has increasingly become its key quality attribute. In the prior art, the test of the sealing of the packaging system of multi-dose eye drop bottles is to use an instrument to generate a pressure difference between the inside and outside of the sample by vacuuming, thereby observing the gas escape and shape recovery of the sample, and then judging the sealing performance of the sample. The disadvantage is that it is impossible to directly test the microbial barrier performance of the seal, but only the physical strength of the seal is tested. The test method for the sterility maintenance of sterilized packaging in medical institutions mentioned in the 2002 edition of the "Technical Specifications for Disinfection" is to place it in an accelerated aging box for 7 days and perform sterility inspection on the internal medical devices. The test conditions of this method, at a high temperature of 62°C, limit the survival conditions of the microorganisms themselves, and there are no pressurized or vacuum test conditions. It is impossible to simulate the sterile barrier system under real environmental conditions, such as before and after sterilization, low-temperature storage, and air pressure changes that may occur during high-altitude transportation, resulting in low sensitivity of the method.

[0006] Microbial challenge method plays a key role in the study of the sealing of packaging systems. The container sealing system of sterile products should be able to prevent microbial invasion. When direct evidence of the ability to prevent microbial invasion is needed, microbial challenge testing is most useful. The aerosol challenge method is to place the packaging system in a challenge instrument containing a specific concentration of microbial aerosol for a certain period of time. The challenge instrument is a sealed system that can set vacuum and pressurization test conditions to simulate the pressure changes that the packaging system may encounter during actual storage and transportation. The package contains liquid or solid culture medium. After the challenge, culture and observe whether there is microbial growth in the culture medium. Ordinary aerosol challenge boxes cannot be manually operated during the aerosol challenge process. Once the aerosol challenge test begins, each bottle of sample needs to be replaced after the test, so the aerosol challenge box needs to be equipped with sealed gloves for operation. Existing aerosol challenge instruments cannot realize real-time testing of aerosol concentration and the opening and closing of the sprayer, and cannot guarantee the stability of aerosol concentration, which affects the accuracy and repeatability of the experimental results.

[0007] There is no standard test method for the antibacterial property of multi-dose eye drop bottles during use at home and abroad. The prior art discloses a test method that drops a few drops of eye drops to test the antibacterial property of the physical antibacterial system. Considering that the physical antibacterial system has the risk of microbial infection during the use of each drop of eye drops, only dropping a few drops of eye drops to simulate the use of the test cannot represent that the entire bottle of eye drops can guarantee that there is no microbial invasion inside the eye drop bottle during the entire use process. Moreover, it has not been proven whether the antibacterial property of the physical antibacterial device is guaranteed within the validity period of the unsealed eye drops after the eye drops are opened.

[0008] In summary, there is currently no aerosol challenge device in the prior art that can conduct aerosol challenge experiments on multi-dose eye drop bottles. The testing methods in the prior art for the packaging seal integrity of multi-dose eye drop bottles and the bacteriostatic properties of the physical bacteriostatic system of multi-dose eye drop bottles during use have certain limitations and inaccuracies. Summary of the invention

[0009] The purpose of the present invention is to solve the technical problems existing in the prior art, and to provide an aerosol challenger, a test method for the packaging seal integrity of a multi-dose eye drop bottle, and the bacteriostatic performance of the physical bacteriostatic system during use. The present invention first completes the construction of the test device, and then explores the test method. On the one hand, a test device for testing the physical bacteriostatic system of a multi-dose eye drop bottle can be formed to form material conditions for future product testing. On the other hand, a test method for testing the packaging seal integrity of a multi-dose eye drop bottle and the bacteriostatic performance of the physical bacteriostatic system during use is explored to provide a theoretical basis for the formation of a standard method.

[0010] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: an aerosol challenge instrument, comprising a sealed cabin, an aerosol generator, a first air source pump, a capsule high-efficiency filter and a second air source pump, the sealed cabin is provided with a first sealed door and / or a second sealed door, the first sealed door is completely sealed, the second sealed door is equipped with safety gloves, and the sealed cabin is used for microbial challenge test of test samples; the aerosol generator is used to generate microbial aerosol, and one end of the aerosol generator is connected to the top of the sealed cabin; one end of the first air source pump is connected to the other end of the aerosol generator, and the other end of the first air source pump is connected to the bottom of the sealed cabin, and gas is taken out from the sealed cabin as the air source for aerosol generation, and the first air source pump transports the aerosol generated by the aerosol generator into the sealed cabin; the capsule high-efficiency filter is used to filter and clean the gas entering and leaving the sealed cabin, and one end of the capsule high-efficiency filter is connected to the sealed cabin. The top interior of the sealed cabin is connected, one end of the second air source pump is connected to the other end of the capsule high efficiency filter, and the other end of the second air source pump is connected to the bottom interior of the sealed cabin, and the second air source pump is used for vacuuming and pressurizing the sealed cabin; the top interior of the sealed cabin is also connected to the third pipeline, and the bottom of the sealed cabin is also connected to the fourth pipeline, and the third pipeline and the fourth pipeline are both provided with a third valve, and the fourth pipeline is connected to a sampling instrument or a particle counter, and the sampling instrument is an air microbial sampler, and the third pipeline is connected to the filter, and the third pipeline is used for sampling the inlet pipeline, and the fourth pipeline is used for sampling the outlet pipeline. Under normal pressure, the running time of the aerosol generator and the generated microbial aerosol particles, as well as the time that the concentration of the microbial aerosol can be maintained are verified, and sampling is performed using the sampling port at this time, and the sampling instrument or the particle counter collects the microbial aerosol from the sealed cabin through the sampling outlet. The sampling inlet, in order to maintain the air pressure balance of the sealed cabin, can open the valve when the instrument is used at normal pressure, and is connected to the pipeline with a filter; a pressure sensor is arranged on the top of the sealed cabin, and the pressure sensor is used to measure the pressure in the sealed cabin; a stacking bracket is also arranged inside the sealed cabin, and the stacking bracket is used to place the test samples, which are stacked according to the sample size and can be applied to samples of different volumes; a fan is arranged at the bottom of the sealed cabin, and a temperature sensor is also arranged in the sealed cabin, and the fan is used to ensure the uniformity of the microbial aerosol and the repeatability and accuracy of the test results; the top of the sealed cabin is also connected to the first safety valve and the second safety valve, the first safety valve is a positive pressure relief valve, and the second safety valve is a negative pressure safety valve, and the first safety valve and the second safety valve are used for safe pressure relief to avoid excessive pressure in the sealed cabin and potential safety hazards. When the pressure exceeds a certain set pressure of the sealed cabin, the first safety valve and the second safety valve are opened to release the pressure.

[0011] A further solution of the present invention is that the aerosol generator is connected to the sealed cabin via a second pipe, and a second valve is provided on the second pipe; the aerosol generator is connected to the first air source pump via an eighth pipe, and a first flow controller is provided on the eighth pipe; the first air source pump is connected to the sealed cabin via a fifth pipe, and a fifth valve is provided on the fifth pipe.

[0012] A further solution of the present invention is that the capsule high efficiency filter is connected to the sealed cabin via a first pipeline, and a first valve is provided on the first pipeline; the capsule high efficiency filter is connected to the sealed cabin via a sixth pipeline, and a second flow controller, a seventh valve and a sixth valve are provided on the sixth pipeline in sequence; the second air source pump is connected in parallel with the seventh valve; a first branch pipe connected to the outside is also provided on the first pipeline, a fourth valve is provided on the first branch pipe, a second branch pipe connected to the outside is provided between the node where the sixth valve is connected in parallel with the seventh valve and the second air source pump on the sixth pipeline, and the The second branch pipe is provided with an eighth valve; when pressurizing, the eighth valve of the second branch pipe is opened, the second air source pump is turned on, the pressurization size is controlled by the second flow controller, and the external air source opens the first valve after passing through the capsule-type high-efficiency filter to enter the sealed cabin; when vacuuming, the sixth valve is opened, the second air source pump is turned on, the vacuuming size is controlled by the second flow controller, and the internal air source passes through the capsule-type high-efficiency filter, and the fourth valve is opened to discharge out of the sealed cabin; when quickly releasing pressure, the sixth valve is locked, the seventh valve is opened, the pressure relief size is controlled by the second flow controller, and the internal air source passes through the capsule-type high-efficiency filter, and the fourth valve is opened to directly discharge out of the sealed cabin.

[0013] A further solution of the present invention is that the first sealing door and the sealing cabin are fixed by a sealing hand wheel.

[0014] A further solution of the present invention is that the second sealing door is fixed to the sealing cabin by screws.

[0015] A further solution of the present invention is that a distribution box is provided on the side of the sealed cabin, and a display screen control system is provided on the top of the distribution box.

[0016] A further solution of the present invention is that the pressure sensor, the first flow controller, the second flow controller, the first valve, the second valve, the fourth valve, the fifth valve, the sixth valve, the seventh valve and the eighth valve are all communicatively connected to the display screen control system, and the display screen control system obtains the pressure data of the pressure sensor, the flow data of the first flow controller and the second flow controller and controls the opening and closing of each valve.

[0017] A further solution of the present invention is that casters are provided at the bottom of the sealed cabin and the distribution box.

[0018] Through the above technical scheme, the sterilized and dried test sample is first placed in a sealed cabin, and the sealed cabin is closed; then a capsule high-efficiency filter is used to flush the sealed cabin; the test parameters are set on the control panel of the sealed cabin, including the operation cycle of the aerosol generator (during the instrument verification, confirm the aerosol concentration generated by the cyclic operation of the aerosol generator and the time the aerosol can maintain this concentration), the test vacuum pressure and maintenance time, the test pressurization and maintenance time, the test normal pressure and maintenance time, then, the test is run, the aerosol generator generates microbial aerosol and transports it to the sealed cabin; the air source pump performs vacuum or pressurization treatment, etc.

[0019] A method for testing the sealing integrity of a multi-dose eye drop bottle package, comprising the following steps:

[0020] A1. Aseptically fill the prepared and sterilized tryptic soy broth (TSB) into an eye drop bottle as a sample bottle, culture it and observe whether it is turbid, and select the sample bottle that is not turbid for the test;

[0021] A2. Divide the non-turbid sample bottles into a test group, a positive group, and a negative group;

[0022] A3. Place the sample bottles of the positive group and the negative group in a biosafety cabinet; place the sample bottles of the test group in the sealed cabin of the above-mentioned aerosol challenge instrument for an aerosol challenge test, use the aerosol challenge instrument to generate an aerosol of the configured Bacillus atrophaeus ATCC 9372 spore suspension and transport it to the inside of the sealed cabin, and set the test parameters of the aerosol challenge instrument, including pressure, temperature, holding time, and periodic operation time of the aerosol generator;

[0023] A4. After the aerosol challenge test, the sample bottles of the test group were repeatedly wiped with 75% alcohol sterile alcohol cotton pads and gently shaken to ensure that the tryptic soy broth (TSB) in the sample bottles of the test group contacted all the inner surfaces of the sample bottles; the sample bottles of the positive group were inoculated with Bacillus atrophaeus ATCC 9372 spore solution; the sample bottles of the negative group were not treated;

[0024] A5. The sample bottles of the test group, positive group and negative group in A4 were cultured at 35°C for 7 days, and the tryptic soy broth (TSB) in the sample bottles was observed to be turbid.

[0025] The test sample is a low-density polyethylene multi-dose eye drop bottle with a specification of 5ml. 12 bottles of samples filled with sterile culture medium were used, including 1 bottle of negative control group, 1 bottle of positive control group, and 10 bottles of test group. The test reagents are Bacillus atrophaeus ATCC 9372 spore suspension (MesaLabs, batch number: SSG-682), tryptic soytone liquid culture medium (BD Company, USA, batch number: 0350681). The test instruments are aerosol challenge instrument, biological safety cabinet, vertical pressure steam sterilizer, constant temperature and humidity chamber, etc.

[0026] In A1, the conditions for culturing the sample bottles aseptically filled with TSB broth (TSB) are as follows: the sample bottles filled with TSB are cultured at 25° C. for 7 days and at 35° C. for 7 days.

[0027] In A3, the test parameters of the aerosol challenge test are: first, the temperature is 35°C, then the vacuum is evacuated to -30KPa for 30 minutes, then the pressure is increased to 30KPa for 30 minutes, and the aerosol generator is set to open periodically to maintain the required aerosol concentration of 10 5 CFU / L.

[0028] In A4, the inoculation amount of the spore solution of Bacillus atrophaeus ATCC 9372 was 0.1 ml, and the spore concentration of the spore solution of Bacillus atrophaeus ATCC 9372 was lower than 100 CFU / 0.1 ml.

[0029] A method for testing the bacteriostatic property of a physical bacteriostatic system when a multi-dose eye drop bottle is used, comprising the following steps:

[0030] S1. Aseptically fill the prepared and sterilized tryptic soy broth (TSB) into an eye drop bottle as a sample bottle, culture it and observe whether it is turbid, and select the sample bottle without turbidity for the test;

[0031] S2. Subject the non-turbid sample bottles to the Pseudomonas aeruginosa bacterial solution immersion dropper challenge test:

[0032] S2-1. Prepare a suspension of Pseudomonas aeruginosa ATCC 9027 for later use;

[0033] S2-2. The non-turbid sample bottles were divided into test group, positive group and negative group and placed in a biosafety cabinet;

[0034] S2-3. The sample bottles in the test group in S2-2 were immersed in the bacterial suspension of Pseudomonas aeruginosa ATCC 9027, and the dropper was immersed in the challenge test. The bottle body was pressed and the TSB was squeezed out drop by drop. The challenge bacterial solution on the dropper was wiped with sterile gauze, and the dropper was wiped with a 75% alcohol sterile alcohol cotton pad. The eye drop bottle was gently shaken to ensure that the TSB was in contact with the inner surface of all the sample bottles in the test group; the sample bottles in the positive group in S2-2 were inoculated with the bacterial suspension of Pseudomonas aeruginosa ATCC 9027; the sample bottles in the negative group in S2-2 were not treated;

[0035] S2-4. The sample bottles of the test group, positive group and negative group in the above S2-3 were placed at 35°C for incubation, and the tryptic soy bean broth (TSB) in the eye drop bottle was observed to be turbid;

[0036] S2-5. Repeat the steps in S2-3 to S2-4 in a cycle;

[0037] S2-6. After the submerged dropper challenge test is carried out periodically, the sample bottles of the test group, positive group and negative group after the test are cultured at 35°C for 7 days, and the tryptic soy broth (TSB) is observed to see if it is turbid.

[0038] In S1, the conditions for culturing the sample bottles aseptically filled with tryptic soy broth (TSB) are as follows: the sample bottles filled with tryptic soy broth (TSB) are placed at 25° C. for 7 days and at 35° C. for 7 days.

[0039] In S2-1, the prepared Pseudomonas aeruginosa ATCC 9027 bacterial suspension is prepared by inoculating the standard stock strain of Pseudomonas aeruginosa ATCC 9027 into tryptic soy broth (TSB) medium and culturing at 35°C overnight. The concentration of the Pseudomonas aeruginosa ATCC 9027 bacterial suspension required for the immersion dropper challenge test is 10 6 CFU / ml; in S2-3, the inoculation volume of the Pseudomonas aeruginosa ATCC 9027 bacterial suspension in the sample bottle in the positive group was 0.1 ml, and the concentration of the inoculated Pseudomonas aeruginosa ATCC 9027 bacterial suspension was lower than 100 CFU / 0.1 ml.

[0040] In S2-3, the amount of TSB (tryptic soy bean broth) dropped out by repeated squeezing was 1 ml, and the cycle described in S2-5 was 5 cycles, with an interval of 7 days between each cycle.

[0041] The test sample is a low-density polyethylene multi-dose eye drop bottle with a specification of 5ml. 8ml of tryptic soy broth (TSB) is aseptically filled in the sample bottle to simulate the use of the eye drop bottle. After 5ml of the simulated use is dropped, 3ml of culture medium remains for microbial culture. There are 10 bottles in the test group, 1 bottle in the positive group, and 1 bottle in the negative group.

[0042] S3. Subject the non-turbid sample bottles to the Bacillus atrophaeus ATCC 9372 spore aerosol return valve challenge test:

[0043] S3-1. Prepare the prepared spore suspension of Bacillus atrophaeus ATCC 9372 for use;

[0044] S3-2. The non-turbid sample bottles were divided into a test group, a positive group and a negative group, the sample bottles of the test group were placed in the sealed cabin of the aerosol challenge instrument, and the sample bottles of the positive group and the negative group were placed in the biosafety cabinet;

[0045] S3-3. The prepared Bacillus atrophaeus ATCC 9372 spore suspension is used to generate an aerosol and transported to the inside of the sealed cabin, and the test parameters of the aerosol challenger are set, including pressure, temperature, maintenance time and periodic operation time of the aerosol generator; when the required spore aerosol concentration is reached in the sealed cabin of the aerosol challenger, the sample bottle of the test group in S3-2 is pressed against the bottle body through safety gloves, and the tryptic soy bean broth (TSB) is repeatedly squeezed out drop by drop;

[0046] S3-4. After the aerosol return valve challenge test of the sample bottles of the test group is completed, the sample bottles of the test group are taken out and the eye drop bottles are repeatedly wiped with 75% alcohol sterile alcohol cotton pads, and the eye drop bottles are gently shaken to ensure that the tryptic soy broth (TSB) contacts the inner surface of the sample bottles of all test groups; the sample bottles in the positive group in S3-2 are inoculated with the spore suspension of Bacillus atrophaeus ATCC 9372; the sample bottles in the negative group in S3-2 are not treated;

[0047] S3-5. After the sample bottles of the test group, positive group and negative group in the above S3-4 are capped, they are placed at 35°C for incubation.

[0048] Observe the tryptic soy broth (TSB) in the eye drop bottle to see if it is turbid;

[0049] S3-6. Repeat the steps in S3-3 to S3-5 in a cycle;

[0050] S3-7. After the aerosol return valve challenge test is carried out periodically, the sample bottles of the test group, positive group and negative group after the test are cultured at 35°C for 7 days, and the tryptic soy broth (TSB) is observed to see if it is turbid.

[0051] In S3-3, during the aerosol challenge test, the test parameters were set to a temperature of 35°C, a periodic start of the aerosol generator, and a required aerosol concentration of 10 5 CFU / L; the spore concentration of the Bacillus atrophaeus ATCC 9372 spore solution inoculated in S3-4 was lower than 100 CFU / 0.1 ml.

[0052] In S3-3, the amount of TSB (tryptic soy bean broth) dropped out by repeated squeezing was 1 ml, and the cycle described in S3-6 was 5 cycles, with an interval of 7 days between each cycle.

[0053] In this test method, the test sample is a low-density polyethylene multi-dose eye drop bottle with a specification of 5ml. 8ml of tryptic soy broth (TSB) is aseptically filled in the sample bottle to simulate the use of the eye drop bottle. After 5ml of the simulated use is dropped, 3ml of culture medium remains for microbial culture. There are 10 bottles in the test group, 1 bottle in the positive group, and 1 bottle in the negative group.

[0054] If the culture medium is turbid, it means that bacteria are growing, that is, the microorganisms have contaminated the culture medium through the antibacterial system of the eye drop bottle. After the culture medium is turbid, it can also be further verified whether the challenge microorganism has contaminated the culture medium in the eye drop bottle. The specific operation is to compare and observe the morphology of the isolated representative single colony and the challenge bacteria colony for the positive control group and the turbid test sample. In addition, the representative colony needs to be Gram-stained, and then the morphology comparison and Gram microscopy are used to determine whether it is positive.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] 1. The present invention provides an aerosol challenge instrument. By designing a first sealing door and a second sealing door, the first sealing door can realize an aerosol challenge test in a highly sealed state of vacuum or pressurization, and the second sealing door can realize an aerosol challenge test in a relatively good sealed state; the generation and transportation of aerosols are realized by the first air source pump and its pipeline, the severity of the test conditions is increased, and the sensitivity of the test method is increased; the vacuuming, pressurization and rapid pressure relief functions in the sealed cabin are realized by the second air source pump and its pipeline, and the vacuuming and pressurization test conditions can be set to simulate the pressure changes that may be encountered by the packaging system during actual storage and transportation. By setting a pressure sensor, when the pressure value deviates from the set value, the central control system controls the air source pump to maintain the stability of the pressure; the sampling inlet and the sampling outlet are connected to the sampling instrument or the particle counter to realize the operation time of the aerosol generator, the microbial aerosol particles generated, and the time that the concentration of the microbial aerosol can be maintained under normal pressure. The aerosol challenge instrument can be used for the integrity test of the sterile barrier system of sterile breathable packaging such as medicines, foods, and sterilization wraps of medical institutions.

[0057] 2. The present invention provides a testing method for the sealing integrity of a multi-dose eye drop bottle packaging. The testing method performs a microbial test on the packaging integrity of the eye drop bottle as a whole, rather than only testing the materials and seals of the eye drop bottle. The method generates an aerosol through an aerosol challenger to increase the severity of the test conditions, increase the sensitivity of the test method, and set the positive and negative pressure states in the sealed cabin to simulate the pressure difference that may be formed during actual sterilization, storage and transportation of the package. By using smaller and mobile microorganisms, it is possible to penetrate tiny leaks, increase the sensitivity of the method, set a suitable temperature for the survival of the challenge bacteria, ensure the activity of the microorganisms, and increase the reliability of the test results.

[0058] 3. The present invention provides a method for testing the bacteriostatic properties of a physical bacteriostatic system when a multi-dose eye drop bottle is used. The test method conducts a bacterial liquid immersion dropper challenge test on the dropper of the physical bacteriostatic system, which actually simulates whether the microorganisms in the liquid will contaminate the internal eye drops when an eye infection occurs; the test method conducts an aerosol return valve challenge test on the return valve of the physical bacteriostatic system, which actually simulates whether the microorganisms in the air will contaminate the internal eye drops when the physical bacteriostatic system returns air; within the unsealed validity period of the multi-dose eye drop bottle, the test is conducted every week to prove the bacteriostatic effectiveness of the physical bacteriostatic system within the unsealed validity period of the multi-dose eye drop bottle; within the experimental period, all the culture media in the specifications of the eye drops are dripped, which proves that the bacteriostatic system can ensure effective bacteriostasis during each use process. The test method can also be applied to bacteriostatic performance testing of other multi-dose drug delivery systems, such as beverages, nasal drops and other physical bacteriostatic systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0060] Figure 1 The overall structure of the aerosol challenger of the present invention is three-dimensional Figure 1 ;

[0061] Figure 2 The overall structure of the aerosol challenger of the present invention is three-dimensional Figure 2 ;

[0062] Figure 3 The internal structure of the aerosol challenger of the present invention is shown in FIG. Figure 1 ;

[0063] Figure 4 The internal structure of the aerosol challenger of the present invention is shown in FIG. Figure 2 ;

[0064] Figure 5 The internal structure of the aerosol challenger of the present invention is shown in FIG. Figure 3 ;

[0065] Figure 6 It is a schematic diagram of installing the second sealing door of the aerosol challenger of the present invention;

[0066] Figure 7 A schematic diagram of pipeline connection of the testing device of the present invention;

[0067] Figure 8 The sample appearance of the eye drop bottle used in the test method of the present invention;

[0068] Fig. 9 The sterility confirmation test result in the test method of the present invention;

[0069] Fig.10 The result of the Pseudomonas aeruginosa bacterial liquid immersion dripper challenge test in the test method of the present invention;

[0070] Fig.11 The results of the aerosol challenge test of Bacillus atrophaeus (ATCC 9372) spores in the test method of the present invention are shown in FIG.

[0071] In the figure: 1, sealed cabin, 2, distribution box, 3, casters, 4, first sealed door, 5, sealed handwheel, 6, display screen control system, 7, aerosol generator, 8, pressure sensor, 9, first pipeline, 10, second pipeline, 11, third pipeline, 12, fourth pipeline, 13, first safety valve, 14, second safety valve, 15, third valve, 16, second valve, 17, first valve, 18, first branch pipeline, 19, fourth valve, 20, capsule high efficiency filter, 21, first air source pump, 22, second air source pump, 23, fifth pipeline, 24, sixth pipeline, 25, fifth valve, 26, sixth valve, 27, seventh pipeline, 28, eighth pipeline, 29, seventh valve, 30, second branch pipe, 31, eighth valve, 32, second flow controller, 33, first flow controller, 34, stacking bracket, 35, temperature sensor, 36, fan, 37, second sealed door, 38, safety gloves. DETAILED DESCRIPTION

[0072] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0073] Example 1

[0074] like Figure 1-7 As shown, an aerosol challenge instrument includes a sealed cabin 1, an aerosol generator 7, a first air source pump 21, a capsule high efficiency filter 20 and a second air source pump 22.

[0075] The sealed cabin 1 is provided with a first sealed door 4. Specifically, the first sealed door 4 and the sealed cabin 1 are fixed by a sealing handwheel. The first sealed door 4 is completely sealed. The sealed cabin 1 is used for microbial challenge tests of test samples.

[0076] The aerosol generator 7 is used to generate microbial aerosol, and one end of the aerosol generator 7 is connected to the top of the sealed cabin 1; one end of the first air source pump 21 is connected to the other end of the aerosol generator 7, and the other end of the first air source pump 21 is connected to the bottom of the sealed cabin 1, and the gas is taken out from the sealed cabin 1, which is used as the gas source for aerosol generation. The first air source pump 21 transports the aerosol generated by the aerosol generator 7 into the sealed cabin 1. A further solution of the present invention is that the aerosol generator 7 is connected to the sealed cabin 1 through a second pipeline 10, and a second valve 16 is provided on the second pipeline 10; the aerosol generator 7 is connected to the first air source pump 21 through an eighth pipeline 28, and a first flow controller 33 is provided on the eighth pipeline 28; the first air source pump 21 is connected to the sealed cabin 1 through a fifth pipeline 23, and a fifth valve 25 is provided on the fifth pipeline 23.

[0077] The capsule high-efficiency filter 20 is used for filtering and cleaning the gas entering and leaving the sealed cabin 1. One end of the capsule high-efficiency filter 20 is connected to the top interior of the sealed cabin 1. One end of the second air source pump 22 is connected to the other end of the capsule high-efficiency filter 20. The other end of the second air source pump 22 is connected to the bottom interior of the sealed cabin 1. The second air source pump 22 is used for vacuuming and pressurizing the sealed cabin 1. A further solution of the present invention is that the capsule high efficiency filter 20 is connected to the sealed cabin 1 through a first pipeline 9, and a first valve 17 is provided on the first pipeline 9; the capsule high efficiency filter 20 is connected to the sealed cabin 1 through a sixth pipeline 24, and a second flow controller 32, a seventh valve 29 and a sixth valve 26 are provided on the sixth pipeline 24 in sequence; the second air source pump 22 is connected in parallel with the seventh valve 29; the first pipeline 9 is also provided with a first branch pipe 18 connected to the outside, and a fourth valve 19 is provided on the first branch pipe 18; a second branch pipe 30 connected to the outside is provided between the node where the sixth valve 26 is connected in parallel with the seventh valve 29 and the second air source pump 22 on the sixth pipeline 24, and the An eighth valve 31 is provided on the second branch pipe 30; when pressurizing, the eighth valve 31 of the second branch pipe 30 is opened, the second air source pump 22 is turned on, and the pressurization size is controlled by the second flow controller 32. The external air source opens the first valve 17 after passing through the capsule high efficiency filter 20 to enter the sealed cabin 1; when vacuuming, the sixth valve 26 is opened, the second air source pump 22 is turned on, and the vacuuming size is controlled by the second flow controller 32. After the internal air source passes through the capsule high efficiency filter 20, the fourth valve 19 is opened to discharge the air out of the sealed cabin 1; when quickly releasing pressure, the sixth valve 26 is locked, the seventh valve 29 is opened, and the pressure relief size is controlled by the second flow controller 32. After the internal air source passes through the capsule high efficiency filter 20, the fourth valve 19 is opened to discharge the air directly out of the sealed cabin 1.

[0078] like Figure 2As shown, the top of the sealed cabin 1 is also connected to the third pipe 11, and the bottom of the sealed cabin 1 is also connected to the fourth pipe 12. The third pipe 11 and the fourth pipe 12 are both provided with a third valve 15. The fourth pipe 12 is connected to a sampling instrument or a particle counter, and the third pipe 11 is connected to a filter. The sampling instrument is an air microbial sampler, and the filter is a disposable consumable. The air microbial sampler, the filter and the particle counter are all prior art. The third pipe 11 is used for sampling the inlet pipe, and the fourth pipe 12 is used for sampling the outlet pipe. Under normal pressure, the operation time of the aerosol generator 7 and the generated microbial aerosol particles, as well as the time that the concentration of the microbial aerosol can be maintained are verified. At this time, sampling is performed using the sampling port, and the sampling instrument or the particle counter collects the microbial aerosol from the sealed cabin 1 through the sampling outlet. The sampling inlet, in order to maintain the air pressure balance of the sealed cabin 1, can open the valve when the instrument is used at normal pressure, and the pipeline with the filter is connected.

[0079] like Figure 3 As shown, a pressure sensor 8 is provided on the top of the sealed cabin 1, and the pressure sensor 8 is used to measure the pressure in the sealed cabin 1; a stacking bracket 34 is also provided inside the sealed cabin 1, and the stacking bracket 34 is used to place test samples, which are stacked according to the size of the samples and can be suitable for samples of different volumes. A fan 36 is provided at the bottom of the sealed cabin 1, and a temperature sensor 35 is also provided in the sealed cabin 1. A heater is also provided to adjust the temperature in the sealed cabin, and the fan 36 is used to ensure the uniformity of the microbial aerosol and the repeatability and accuracy of the test results.

[0080] like Figure 2 As shown, the top of the sealed cabin 1 is also connected to a first safety valve 13 and a second safety valve 14, wherein the first safety valve 13 is a positive pressure relief valve and the second safety valve 14 is a negative pressure relief valve. The first safety valve 13 and the second safety valve 14 are used for safe pressure relief to avoid excessive pressure in the sealed cabin 1 and potential safety hazards. When the pressure exceeds a certain set pressure of the sealed cabin 1, the first safety valve 13 and the second safety valve 14 are opened to relieve pressure.

[0081] A further solution of the present invention is that a distribution box 2 is provided on the side of the sealed cabin 1 , and a display screen control system 6 is provided on the top of the distribution box 2 .

[0082] The pressure sensor 8, the first flow controller 33, the second flow controller 32, the first valve 17, the second valve 16, the fourth valve 19, the fifth valve 25, the sixth valve 26, the seventh valve 29 and the eighth valve 31 are all connected to the display screen control system 6 for communication. The display screen control system 6 obtains the pressure data of the pressure sensor 8, the flow data of the first flow controller 33 and the second flow controller 32 and controls the opening and closing of each valve.

[0083] A further solution of the present invention is that casters 3 are provided at the bottom of the sealed cabin 1 and the distribution box 2.

[0084] Example 2

[0085] This embodiment is based on embodiment 1, and a further solution is as follows: Figure 6 As shown, the sealed cabin 1 is provided with a second sealed door 37 , and the second sealed door 37 is equipped with safety gloves 38 .

[0086] The second sealing door 37 is fixed to the sealed cabin 1 by screws.

[0087] According to the above technical scheme, the sterilized and dried test sample is first placed in the sealed cabin 1, the appropriate first sealed door 4 or second sealed door 37 is selected, and the sealed cabin 1 is closed; then the capsule high efficiency filter 20 is used to flush the sealed cabin 1; the test parameters are set on the control panel of the sealed cabin 1, including the operation cycle of the aerosol generator 7 (during the instrument verification, confirm the aerosol concentration generated by the cyclic operation of the aerosol generator 7 and the time the aerosol can maintain this concentration), the test vacuum pressure and maintenance time, the test pressurization and maintenance time, the test normal pressure and maintenance time, then, the test is run, the aerosol generator 7 generates microbial aerosol and transports it to the sealed cabin 1; the air source pump performs vacuum or pressurization treatment, etc.

[0088] Experimental Example 1

[0089] The eye drop bottles were tested using the aerosol challenge instrument described in Example 1 and the method for testing the sealing integrity of a multi-dose eye drop bottle provided by the present invention.

[0090] In this experimental example, the test sample is a low-density polyethylene multi-dose eye drop bottle with a specification of 5ml. 12 bottles of samples filled with 5ml sterile culture medium were used, including 1 bottle of negative control group, 1 bottle of positive control group, and 10 bottles of test group. The test reagents are Bacillus atrophaeus ATCC 9372 spore suspension (MesaLabs, batch number: SSG-682), tryptic soy bean liquid medium (BD Company, USA, batch number: 0350681). The test instruments are aerosol challenge instrument, biological safety cabinet, vertical pressure steam sterilizer, constant temperature and humidity chamber, etc.

[0091] The specific method comprises the following steps: A1. Aseptically filling the prepared and sterilized tryptic soy broth (TSB) into an eye drop bottle as a sample bottle, placing the sample bottle filled with tryptic soy broth (TSB) in a constant temperature and humidity chamber at 25°C for 7 days, and in a constant temperature and humidity chamber at 35°C for 7 days and observing whether it is turbid, and selecting the sample bottle without turbidity for the test;

[0092] A2. Divide the non-turbid sample bottles into a test group, a positive group, and a negative group;

[0093] A3. Place the sample bottles of the positive group and the negative group in a biosafety cabinet; place the sample bottles of the test group in the sealed cabin 1 of the above-mentioned aerosol challenge instrument for aerosol challenge test, use the aerosol challenge instrument to generate aerosol with the configured Bacillus atrophaeus ATCC 9372 spore suspension and transport it to the inside of the sealed cabin 1, set the test parameters of the aerosol challenge instrument, including pressure, temperature, holding time and periodic operation time of the aerosol generator, and the test parameters of the aerosol challenge test are: first, the temperature is 35°C, then vacuum -30Kpa, holding time 30min, then pressurize 30Kpa, holding time 30min, set the aerosol generator 7 to open periodically to maintain the required aerosol concentration for 10 5 CFU / L;

[0094] A4. After the aerosol challenge test, the sample bottles of the test group were repeatedly wiped with 75% alcohol sterile alcohol cotton pads and gently shaken to ensure that the tryptic soy broth (TSB) in the sample bottles of the test group contacted all the inner surfaces of the sample bottles; the sample bottles of the positive group were inoculated with Bacillus atrophaeus ATCC 9372 spore solution, the inoculation volume of Bacillus atrophaeus ATCC 9372 spore solution was 0.1ml, and the spore concentration was less than 100CFU / 0.1ml; the sample bottles of the negative group were not treated;

[0095] A5. After capping the sample bottles of the test group, positive group and negative group in A4 above, place them in a constant temperature and humidity chamber at 35°C for 7 days and observe whether the tryptic soy broth (TSB) in the sample bottles is turbid.

[0096] Experimental Example 2

[0097] The aerosol challenge instrument provided in Example 2 and the method for testing the bacteriostatic property of the physical bacteriostatic system of a multi-dose eye drop bottle provided by the present invention are used to test the eye drop bottle.

[0098] In this experimental example, the test sample is a low-density polyethylene multi-dose eye drop bottle with a specification of 5ml. 8ml of tryptic soy bean liquid medium (TSB) is aseptically filled in the sample bottle to simulate the use of the eye drop bottle. After 5ml is dropped, 3ml of medium remains for microbial culture. The test group is 10 bottles, the positive group is 1 bottle, and the negative group is 1 bottle. The test reagents are Bacillus atrophaeus ATCC 9372 spore suspension (MesaLabs, batch number: SSG-682), tryptic soy bean liquid medium (American BD Company, batch number: 0350681). The test instruments are aerosol challenge instrument, biological safety cabinet, vertical pressure steam sterilizer, constant temperature and humidity chamber, etc.

[0099] The specific method includes the following steps: S1. Aseptically fill the prepared and sterilized tryptic soy bean liquid medium (TSB) into an eye drop bottle as a sample bottle, and place the sample bottle filled with tryptic soy bean liquid medium (TSB) in a 25℃ constant temperature and humidity chamber for 7 days and in a 35℃ constant temperature and humidity chamber for 7 days. Observe whether it is turbid, and select the sample bottle without turbidity for the test; the culture medium does not become turbid, and the sterility confirmation test results are shown in Fig. 9 .

[0100] S2. Subject the non-turbid sample bottles to the Pseudomonas aeruginosa bacterial solution immersion dropper challenge test:

[0101] S2-1. Prepare a suspension of Pseudomonas aeruginosa ATCC 9027 for use. The prepared suspension of Pseudomonas aeruginosa ATCC 9027 is inoculated into a tryptic soy broth (TSB) medium with a standard stock strain of Pseudomonas aeruginosa ATCC 9027 and cultured overnight in a constant temperature and humidity chamber at 35°C. The concentration of the suspension of Pseudomonas aeruginosa ATCC 9027 required for the immersion dropper challenge test is 10 6 CFU / ml;

[0102] S2-2. Divide the non-turbid sample bottles into test group, positive group and negative group and place them in a biosafety cabinet.

[0103] S2-3. The sample bottle in the test group in S2-2 is kept completely immersed in the drop hole of Pseudomonas aeruginosa ATCC 9027 bacterial suspension for immersion dropper challenge test, the bottle body is pressed, and the trypsin casein soytone liquid medium (TSB) is repeatedly squeezed and dripped out drop by drop. The amount of the trypsin casein soytone liquid medium (TSB) is 1ml, 10 bottles of test group eye drop bottles, 1 bottle of positive control sample bottle and 1 bottle of negative control sample bottle are taken, and the bottle cover is opened and placed in a biosafety cabinet. Take one bottle of test group eye drop bottle, keep the drop hole completely immersed in the challenge bacterial suspension, press the bottle body, squeeze out 1 drop of culture medium, return air with the drop hole facing up, press repeatedly drop by drop, and drip out about 5ml culture medium in total, about 1ml each time. Repeat the above operation for the remaining 9 bottles of test group eye drop bottles. Use sterile gauze to wipe the challenge bacteria solution on the drip tip, use 75% alcohol sterile alcohol cotton pad to wipe the drip tip, and gently shake the eye drop bottle to ensure that the tryptic soy bean broth (TSB) contacts the inner surface of all test group sample bottles; inoculate the sample bottles in the positive group in S2-2 with Pseudomonas aeruginosa ATCC 9027 suspension, and in S2-3, the inoculation volume of Pseudomonas aeruginosa ATCC 9027 suspension in the sample bottles in the positive group is 0.1 ml, and the concentration of the inoculated Pseudomonas aeruginosa ATCC 9027 suspension is less than 100 CFU / 0.1 ml; the sample bottles in the negative group in S2-2 are not treated;

[0104] S2-4. After the sample bottles of the test group, positive group and negative group in the above S2-3 are capped, they are placed in a constant temperature and humidity chamber at 35°C and cultured to observe whether the tryptic soy bean broth (TSB) in the eye drop bottle is turbid;

[0105] S2-5. Repeat steps S2-3 to S2-4 according to the cycle in Table 1; the cycle described in S2-5 is 5 cycles, and the interval between each cycle is 7d.

[0106]

[0107] Table 1. Pseudomonas aeruginosa bacterial liquid immersion dripper challenge test cycle

[0108] S2-6. After the immersion dropper challenge test is carried out periodically, the sample bottles of the test group, positive group and negative group after the test are placed in a constant temperature and humidity chamber at 35°C for 7 days to observe whether the tryptic soy broth (TSB) is turbid.

[0109] At the end of the test, after 7 days of culture at 35°C, the positive control medium became turbid, the negative control medium was clear, and none of the 10 sample mediums became turbid. Fig.10 .

[0110] S3. Subject the non-turbid sample bottles to the Bacillus atrophaeus ATCC 9372 spore aerosol return valve challenge test: In this test method, the test sample is a low-density polyethylene multi-dose eye drop bottle with a specification of 5ml. 8ml of tryptic soy broth (TSB) is aseptically filled in the sample bottle to simulate the use of the eye drop bottle. After 5ml of the simulated use is dropped, 3ml of the culture medium remains for the cultivation of microorganisms. There are 10 bottles in the test group, 1 bottle in the positive group, and 1 bottle in the negative group.

[0111] S3-1. Prepare the prepared spore suspension of Bacillus atrophaeus ATCC 9372 for use;

[0112] S3-2. The non-turbid sample bottles were divided into a test group, a positive group and a negative group, the sample bottles of the test group were placed in the sealed cabin 1 of the aerosol challenge instrument, and the sample bottles of the positive group and the negative group were placed in a biosafety cabinet;

[0113] S3-3. The configured Bacillus atrophaeus ATCC 9372 spore suspension is used to generate an aerosol and transported to the inside of the sealed cabin 1, and the test parameters of the aerosol challenge instrument are set, including pressure, temperature, and maintenance time; in S3-3, during the aerosol challenge test, the test parameters are set to a temperature of 35°C, the aerosol generator 7 is set to be opened periodically, and the required aerosol concentration is 10 5 CFU / L; the spore concentration of Bacillus atrophaeus ATCC 9372 spore liquid inoculated in S3-4 is lower than 100CFU / 0.1ml. When the required spore aerosol concentration is reached in the sealed cabin 1 of the aerosol challenger, press the bottle body of the sample bottle of the test group in S3-2 through the safety gloves 38, and squeeze out the trypsin soy bean broth (TSB) drop by drop repeatedly. The amount of trypsin soy bean broth (TSB) repeatedly squeezed out is 1ml; take 10 bottles of eye drop bottles in the test group, open the outer cover of the bottle, and place them in the microbial aerosol tester. One positive sample bottle and one negative sample bottle are placed in the biological safety cabinet. When the required spore concentration of 10 is reached in the sealed cabin of the microbial aerosol challenger, the sample bottle of the test group in S3-2 is pressed against the bottle body, and the trypsin soy bean broth (TSB) is repeatedly squeezed out. 5 When the number of CFU / L is 2, take one of the eye drop bottles in the test group, press the bottle body, squeeze out one drop of culture medium, press repeatedly drop by drop, and drop out about 5 ml of culture medium. Repeat the above operation for the remaining 9 eye drop bottles in the test group.

[0114] S3-4. After the aerosol return valve challenge test of the sample bottles of the test group is completed, the sample bottles of the test group are taken out and the eye drop bottles are repeatedly wiped with 75% alcohol sterile alcohol cotton pads, and the eye drop bottles are gently shaken to ensure that the tryptic soy broth (TSB) contacts the inner surface of the sample bottles of all test groups; the sample bottles in the positive group in S3-2 are inoculated with the spore suspension of Bacillus atrophaeus ATCC 9372; the sample bottles in the negative group in S3-2 are not treated;

[0115] S3-5. After the sample bottles of the test group, positive group and negative group in the above S3-4 are capped, they are placed at 35°C for incubation.

[0116] Observe the tryptic soy broth (TSB) in the eye drop bottle to see if it is turbid;

[0117] S3-6. Repeat the steps S3-3 to S3-5 according to Table 2 for 5 cycles, with an interval of 7 days between each cycle;

[0118]

[0119]

[0120] Table 2. Bacillus atrophaeus ATCC 9372 spore aerosol return valve challenge test cycle

[0121] S3-7. After the aerosol return valve challenge test is carried out periodically, the sample bottles of the test group, positive group and negative group after the test are placed in a constant temperature and humidity chamber at 35°C for incubation for 7 days, and the tryptic soy broth (TSB) is observed to see if it is turbid.

[0122] At the end of the test, after 7 days of culture at 35°C, the positive control medium became turbid, the negative control medium was clear, and none of the 10 sample mediums became turbid. Fig.11 .

[0123] If the culture medium is turbid, it means that bacteria are growing, that is, the microorganisms have contaminated the culture medium through the antibacterial system of the eye drop bottle. After the culture medium is turbid, it can also be further verified whether the challenge microorganism has contaminated the culture medium in the eye drop bottle. The specific operation is to compare and observe the morphology of the isolated representative single colony and the challenge bacteria colony for the positive control group and the turbid test sample. In addition, the representative colony needs to be Gram-stained, and then the morphology comparison and Gram microscopy are used to determine whether it is positive.

[0124] The test results show that the challenge test of the Pseudomonas aeruginosa liquid immersion dropper showed sterile growth, proving that the physical bacteria barrier system droplet channel is effective in blocking microbial suspension; the challenge test result of Bacillus atrophaeus (ATCC 9372) spore aerosol showed sterile growth, proving that the physical bacteria barrier system return air channel and liquid medicine channel are effective in blocking microbial aerosol. Through simulated use under extreme test conditions, the physical bacteria barrier system liquid medicine channel and return air channel of the low-density polyethylene bacteria barrier multi-dose medicinal eye drop bottle are effective in blocking bacteria after the nominal capacity of the eye drop bottle is used up, which can ensure the sterility of the eye drops.

[0125] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims be included in the specification.

[0126] Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. An aerosol challenge instrument, characterized in that: The invention comprises a sealed cabin (1), an aerosol generator (7), a first air source pump (21), a capsule high efficiency filter (20) and a second air source pump (22); the sealed cabin (1) is provided with a first sealed door (4) and / or a second sealed door (37); the first sealed door (4) is completely sealed, and the second sealed door (37) is provided with a safety glove (38); one end of the aerosol generator (7) is connected to the top of the sealed cabin (1); one end of the first air source pump (21) is connected to the top of the sealed cabin (1); one end of the first air source pump (21) is connected to the top of the sealed cabin (1); and one end of the second air source pump (37) is connected to the top of the sealed cabin (1). The other end of the aerosol generator (7) is connected, and the other end of the first air source pump (21) is connected to the bottom interior of the sealed cabin (1); one end of the capsule high-efficiency filter (20) is connected to the top interior of the sealed cabin (1), one end of the second air source pump (22) is connected to the other end of the capsule high-efficiency filter (20), and the other end of the second air source pump (22) is connected to the bottom interior of the sealed cabin (1); the top interior of the sealed cabin (1) is also connected to the third pipeline (11), and the sealed cabin ( The bottom of the sealed cabin (1) is also connected to a fourth pipe (12), the third pipe (11) and the fourth pipe (12) are both provided with a third valve (15), the fourth pipe (12) is connected to a sampling instrument or a particle counter, and the third pipe (11) is connected to a filter; a pressure sensor (8) is provided at the top of the sealed cabin (1), a stacking bracket (34) is also provided inside the sealed cabin (1), a fan (36) is provided at the bottom of the sealed cabin (1), and a temperature sensor (35) is also provided inside the sealed cabin (1); a first safety valve (13) and a second safety valve (14) are also connected to the top of the sealed cabin (1), the first safety valve (13) is a positive pressure relief valve, and the second safety valve (14) is a negative pressure safety valve; a distribution box (2) is provided on the side of the sealed cabin (1), a display screen control system (6) is provided on the top of the distribution box (2), and casters (3) are provided at the bottom of the sealed cabin (1) and the distribution box (2).

2. An aerosol challenge instrument according to claim 1, characterized in that: The aerosol generator (7) is connected to the sealed cabin (1) via a second pipeline (10), and a second valve (16) is provided on the second pipeline (10); the aerosol generator (7) is connected to the first air source pump (21) via an eighth pipeline (28), and a first flow controller (33) is provided on the eighth pipeline (28); the first air source pump (21) is connected to the sealed cabin (1) via a fifth pipeline (23), and a fifth valve (25) is provided on the fifth pipeline (23).

3. An aerosol challenge instrument according to claim 2, characterized in that: The capsule high-efficiency filter (20) is connected to the sealed cabin (1) via a first pipeline (9), and a first valve (17) is provided on the first pipeline (9); the capsule high-efficiency filter (20) is connected to the sealed cabin (1) via a sixth pipeline (24), and a second flow controller (32), a seventh valve (29) and a sixth valve (26) are provided on the sixth pipeline (24) in sequence; the second air source pump (22) is connected in parallel with the seventh valve (29); the first pipeline (9) is also provided with a first branch pipe (18) connected to the outside, and a fourth valve (19) is provided on the first branch pipe (18); a second branch pipe (30) connected to the outside is provided between the node where the sixth valve (26) and the seventh valve (29) are connected in parallel with the second air source pump (22) on the sixth pipeline (24), and an eighth valve (31) is provided on the second branch pipe (30).

4. An aerosol challenge instrument according to claim 3, characterized in that: The first sealing door (4) and the sealing cabin (1) are fixed via a sealing hand wheel, and the second sealing door (37) and the sealing cabin (1) are fixed via screws.

5. An aerosol challenge instrument according to claim 4, characterized in that: The pressure sensor (8), the first flow controller (33), the second flow controller (32), the first valve (17), the second valve (16), the fourth valve (19), the fifth valve (25), the sixth valve (26), the seventh valve (29) and the eighth valve (31) are all connected to the display screen control system (6) for communication. The display screen control system (6) obtains the pressure data of the pressure sensor (8), the flow data of the first flow controller (33) and the second flow controller (32) and controls the opening and closing of each valve.

6. A method for testing the sealing integrity of a multi-dose eye drop bottle package using the aerosol challenger according to any one of claims 1 to 5, characterized in that: The following steps are involved: A1. Aseptically fill the prepared and sterilized tryptic soy broth (TSB) into an eye drop bottle as a sample bottle, culture it and observe whether it is turbid, and select the sample bottle that is not turbid for the test; A2. Divide the non-turbid sample bottles into a test group, a positive group, and a negative group; A3. Place the sample bottles of the positive group and the negative group in a biosafety cabinet; place the sample bottles of the test group in the sealed cabin (1) of the above-mentioned aerosol challenge instrument for an aerosol challenge test, generate an aerosol using the aerosol challenge instrument for the prepared Bacillus atrophaeus ATCC 9372 spore suspension and transport it to the inside of the sealed cabin (1), and set the test parameters of the aerosol challenge instrument, including pressure, temperature, holding time, and periodic operation time of the aerosol generator; A4. After the aerosol challenge test, the sample bottles of the test group were repeatedly wiped with 75% alcohol sterile alcohol cotton pads and gently shaken to ensure that the tryptic soy broth (TSB) in the sample bottles of the test group contacted all the inner surfaces of the sample bottles; the sample bottles of the positive group were inoculated with Bacillus atrophaeus ATCC 9372 spore solution; the sample bottles of the negative group were not treated; A5. After capping the sample bottles of the test group, positive group and negative group in A4 above, incubate at 35°C for 7 days and observe whether the TSB broth (TSB) in the sample bottles is turbid.

7. The testing method according to claim 6, characterized in that: In the above A1, the conditions for culturing the sample bottles aseptically filled with tryptic soy broth (TSB) are as follows: the sample bottles filled with tryptic soy broth (TSB) are placed at 25° C. for 7 days and 35° C. for 7 days; In the above A3, the test parameters of the aerosol challenge test are as follows: first, the temperature is 35°C, then the vacuum is evacuated to -30KPa for 30 minutes, then the pressure is increased to 30KPa for 30 minutes, and the aerosol generator (7) is set to be turned on periodically to maintain the required aerosol concentration of 10 5 CFU / L; In the A4, the inoculation amount of the Bacillus atrophaeus ATCC 9372 spore solution is 0.1 ml, and the spore concentration of the Bacillus atrophaeus ATCC 9372 spore solution is lower than 100 CFU / 0.1 ml.

8. A method for testing the bacteriostatic property of a physical bacteriostatic system of a multi-dose eye drop bottle using the aerosol challenger according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Aseptically fill the prepared and sterilized tryptic soy broth (TSB) into an eye drop bottle as a sample bottle, culture it and observe whether it is turbid, and select the sample bottle without turbidity for the test; S2. Subject the non-turbid sample bottles to the Pseudomonas aeruginosa bacterial solution immersion dropper challenge test: S2-1. Prepare a suspension of Pseudomonas aeruginosa ATCC 9027 for later use; S2-2. The non-turbid sample bottles were divided into test group, positive group and negative group and placed in a biosafety cabinet; S2-3. The sample bottles in the test group in S2-2 were immersed in the bacterial suspension of Pseudomonas aeruginosa ATCC 9027, and the dropper was immersed in the challenge test. The bottle body was pressed and the TSB was squeezed out drop by drop. The challenge bacterial solution on the dropper was wiped with sterile gauze, and the dropper was wiped with a 75% alcohol sterile alcohol cotton pad. The eye drop bottle was gently shaken to ensure that the TSB was in contact with the inner surface of all the sample bottles in the test group; the sample bottles in the positive group in S2-2 were inoculated with the bacterial suspension of Pseudomonas aeruginosa ATCC 9027; the sample bottles in the negative group in S2-2 were not treated; S2-4. The sample bottles of the test group, positive group and negative group in the above S2-3 were placed at 35°C for incubation, and the tryptic soy bean broth (TSB) in the eye drop bottle was observed to be turbid; S2-5. Repeat the steps in S2-3 to S2-4 periodically; S2-6. After the submerged dropper challenge test was performed periodically, the sample bottles of the test group, positive group, and negative group were cultured at 35°C for 7 days to observe whether the tryptic soy broth (TSB) was turbid; S3. Subject the non-turbid sample bottles to the Bacillus atrophaeus ATCC 9372 spore aerosol return valve challenge test: S3-1. Prepare the prepared spore suspension of Bacillus atrophaeus ATCC 9372 for use; S3-2. The non-turbid sample bottles were divided into a test group, a positive group and a negative group, the sample bottles of the test group were placed in the sealed cabin (1) of the aerosol challenge instrument, and the sample bottles of the positive group and the negative group were placed in a biosafety cabinet; S3-3. The prepared Bacillus atrophaeus ATCC 9372 spore suspension is used to generate an aerosol and transported to the inside of the sealed cabin (1). The test parameters of the aerosol challenger are set, including pressure, temperature, maintenance time and periodic operation time of the aerosol generator; when the desired spore aerosol concentration is reached in the sealed cabin (1) of the aerosol challenger, the sample bottle of the test group in S3-2 is pressed against the bottle body through the safety gloves (38), and the tryptic soy bean broth (TSB) is repeatedly squeezed out drop by drop; S3-4. After the aerosol return valve challenge test of the sample bottles of the test group is completed, the sample bottles of the test group are taken out and the eye drop bottles are repeatedly wiped with 75% alcohol sterile alcohol cotton pads, the bottle caps are covered, and the eye drop bottles are gently shaken to ensure that the tryptic soy broth (TSB) contacts the inner surface of the sample bottles of all test groups; the sample bottles in the positive group in S3-2 are inoculated with the spore suspension of Bacillus atrophaeus ATCC 9372; the sample bottles in the negative group in S3-2 are not treated; S3-5. The sample bottles of the test group, positive group and negative group in the above S3-4 were placed at 35 ° C for culture, and the tryptic soy bean broth (TSB) in the eye drop bottle was observed to be turbid; S3-6. Repeat the steps in S3-3 to S3-5 in a cycle; S3-7. After the aerosol return valve challenge test is carried out periodically, the sample bottles of the test group, positive group and negative group after the test are cultured at 35°C for 7 days, and the tryptic soy broth (TSB) is observed to see if it is turbid.

9. The testing method according to claim 8, characterized in that: In S1, the conditions for culturing the sample bottle aseptically filled with tryptic soy broth (TSB) are as follows: the sample bottle filled with tryptic soy broth (TSB) is placed at 25° C. for 7 days and 35° C. for 7 days; In S2-1, the prepared Pseudomonas aeruginosa ATCC 9027 bacterial suspension is prepared by inoculating the standard stock strain of Pseudomonas aeruginosa ATCC 9027 into a tryptic soy broth (TSB) medium and culturing at 35°C overnight. The concentration of the Pseudomonas aeruginosa ATCC 9027 bacterial suspension required for the immersion dropper challenge test is 10 6 CFU / ml; in S2-3, the inoculation volume of the Pseudomonas aeruginosa ATCC 9027 bacterial suspension in the sample bottle in the positive group was 0.1 ml, and the concentration of the inoculated Pseudomonas aeruginosa ATCC 9027 bacterial suspension was less than 100 CFU / 0.1 ml; In the S2-3, the amount of the TSB medium (TSB) repeatedly squeezed out was 1 ml, and the cycle described in S2-5 was 5 cycles, with an interval of 7 days between each cycle.

10. The testing method according to claim 8, characterized in that: In S3-3, during the aerosol challenge test, the test parameters are set to a temperature of 35°C, the aerosol generator 7 is periodically turned on, and the required aerosol concentration is 10 5 CFU / L; The spore concentration of the Bacillus atrophaeus ATCC 9372 spore solution inoculated in S3-4 is lower than 100 CFU / 0.1 ml; In the S3-3, the amount of TSB (tryptic soy bean broth) that is repeatedly squeezed and dripped out is 1 ml, and the cycle described in S3-6 is 5 cycles, and the interval time between each cycle is 7 days.

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