Method for sterility check validation of a cell product
By pumping sterile water for injection into the filter cartridge to lyse the cells, and combining this with membrane filtration and culture medium verification, the problem of filter membrane clogging in the sterility test of cell products was solved, and sterility test that meets pharmacopoeia standards was achieved.
Patent Information
- Application Number
- CN202111580135.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-12-22
- Publication Date
- 2025-12-12
- Estimated Expiration
- 2041-12-22
AI Technical Summary
In the prior art, when cell products are subjected to aseptic testing using membrane filtration, the pores of the filter membrane are easily blocked by cells, leading to filtration failure.
The cells were fully lysed by pumping sterile water for injection into the filter cartridge, then filtered using a membrane filtration method. After filtration, a culture medium containing a specific bacterial species was added for incubation, and the growth of the bacterial species was observed to verify the effectiveness of the aseptic testing method.
The problem of filter membrane pore blockage was solved, ensuring the effectiveness of aseptic testing and meeting the quality standards of the 2020 edition of the Chinese Pharmacopoeia.
Smart Images
Figure GDA0003610115570000031
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to a sterile inspection verification method of a cell product. BACKGROUND
[0002] According to the requirements of the Chinese Pharmacopoeia (2020 edition), when a sterile inspection method of a drug is established, method verification should be carried out to prove that the method adopted is suitable for the sterile inspection of the drug.
[0003] In the cell culture process, certain antibiotics such as gentamicin are added to inhibit bacterial growth; avoid cell contamination. Therefore, it is necessary to use membrane filtration method for sterile inspection to eliminate the bacteriostatic property of the cell product. When the membrane filtration method is used for sterile inspection method verification, because the diameter of the cell is much larger than the pore size of the filter membrane, the cell will be accumulated on the filter membrane during filtration, causing the pore size to be blocked and unable to filter.
[0004] The prior art often uses direct inoculation method for sterile inspection of cell products, and the present application uses membrane filtration method for sterile inspection of cell products, which can eliminate the bacteriostatic property of the sample itself and solve the problem of filter membrane pore size blockage during filtration. SUMMARY
[0005] The purpose of the present application is to provide a sterile inspection verification method of a cell product. The present application solves the problem of filter membrane pore size blockage when the cell product is subjected to method verification by using membrane filtration method.
[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:
[0007] A sterile inspection verification method of a cell product, specifically comprising the following steps:
[0008] (1) Test group
[0009] Take the test product, filter 2 tubes of the test product in each filter cartridge, and filter the full amount in each tube; pump 150ml of sterile water into the filter cartridge to make the cell fully lysed and then filtered; because the cell is fully lysed and then filtered, the problem of pore size blockage is solved, a set of triple set bacteria incubator is used for sterile operation, after the filtration is completed, 100ml of thioethanolate fluid medium is added to each of the three filter cartridges, and less than 100cfu of Staphylococcus aureus, Escherichia coli and Clostridium sporogenes are added to each of the three filter cartridges, respectively.
[0010] Another set of triple set bacteria incubator is used for sterile operation, after the filtration is completed, 100ml of tryptone soybean peptone liquid medium is added to each of the three filter cartridges, and less than 100cfu of Bacillus subtilis, Candida albicans and Aspergillus niger is added to each of the three filter cartridges, respectively.
[0011] (2) Control group
[0012] Take two sets of triple set bacteria incubator, one of which is added to the three filter cartridges 100 ml of thioglycolate fluid medium, and in each filter cartridge is added less than 100 cfu of Staphylococcus aureus, Escherichia coli and Clostridium sporogenes respectively;
[0013] Another set of three filter cartridges are added to 100 ml of tryptone soy broth liquid medium, and in each filter cartridge is added less than 100 cfu of Bacillus subtilis, Candida albicans and Aspergillus niger respectively;
[0014] (3) negative control group
[0015] Take 2 filter cartridges, take sterile water with the same method, after filtration, add 100 ml of thioglycolate fluid medium and 100 ml of tryptone soy broth liquid medium respectively;
[0016] (4) bacteria liquid group
[0017] Plate count the number of test bacteria added;
[0018] (5) culture
[0019] The culture medium of the test group and the control group is cultured according to the conditions shown in the following table, and the results are observed and recorded.
[0020] When preparing the test sample, the cells are lysed in sterile water for injection, and the different osmotic pressures inside and outside the cells cause the cells to swell quickly, which can cause the cells to lyse quickly, thereby preventing the filter aperture from being blocked.
[0021] As a preferred, in step (1), thin film filtration method is used to filter 2 tubes of test sample in each filter cartridge.
[0022] As a preferred, in step (1-2), the sterile operation of the triple set bacteria incubator is fixed on the bacteria collector.
[0023] As a preferred, in step (5), the culture medium of the test group and the control group is cultured according to the conditions shown in the following table for not more than 5 days.
[0024] As a preferred, in steps (1)-(3), the culture temperature of thioglycolate fluid medium is 30-35℃.
[0025] As a preferred, in steps (1)-(3), the culture temperature of tryptone soy broth liquid medium is 20-25℃.
[0026] As a preferred, the judgment basis: within the specified culture time, compared with the control tube, if the test bacteria in each container containing the test sample grow well, it means that the test amount of the test sample has no or negligible antibacterial effect under the test conditions, and the sterile test of the test sample can be carried out according to the test method and test conditions.
[0027] The present application has the following advantages:
[0028] The prior art usually performs the sterile inspection of the cell product by direct inoculation method, and the present application performs the sterile inspection of the cell product by membrane filtration method, which can eliminate the bacteriostasis of the sample itself and solve the problem of filter membrane aperture blockage during filtration. DETAILED DESCRIPTION
[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.
[0030] As introduced in the background section, in the cell culture process, certain antibiotics such as gentamicin are added to inhibit bacterial growth; and cell contamination is avoided. Therefore, it is necessary to use the membrane filtration method to perform the sterile inspection to eliminate the bacteriostasis in the cell product. When the sterile inspection method is verified by using the membrane filtration method, since the diameter of the cell is much larger than the aperture of the filter membrane, the cell will be accumulated on the filter membrane during filtration, causing the aperture blockage and failing to filter. Based on the above analysis, the present application provides a sterile inspection verification method for a cell product.
[0031] The sterile inspection verification method for a cell product provided by the embodiments of the present application will be described in detail below.
[0032] Embodiment 1: a sterile inspection verification method for a cell product, specifically comprising the following steps:
[0033] 1. Test group
[0034] One set of sterile culture incubator is fixed on the sterilization instrument. The test product is taken, and the membrane filtration method is used to filter 2 tubes of test product in each filter cartridge, and each tube is filtered with full amount. 150 ml of sterile water for injection is pumped into the filter cartridge to fully lyse the cell and then filter; after the filtration is completed, 100 ml of thioethanolate fluid medium is added into each of the three filter cartridges, and less than 100 cfu of Staphylococcus aureus, Escherichia coli and Clostridium sporogenes is added into each of the three filter cartridges, respectively.
[0035] Another set of sterile culture incubator is fixed on the sterilization instrument. The above steps are performed, and after the filtration is completed, 100 ml of tryptone soybean peptone liquid medium is added into each of the three filter cartridges, and less than 100 cfu of Bacillus subtilis, Candida albicans and Aspergillus niger is added into each of the three filter cartridges, respectively.
[0036] 2. Control group
[0037] Take two sets of triple set bacteria culture device, one set of 3 filter cartridges, add 100 ml of thioethanolate fluid medium to each filter cartridge, and add less than 100 cfu of Staphylococcus aureus, Escherichia coli and Clostridium sporogenes to each filter cartridge respectively;
[0038] Another set of 3 filter cartridges, add 100 ml of pancreatic tryptone soybean peptone liquid medium to each filter cartridge, and add less than 100 cfu of Bacillus subtilis, Candida albicans and Aspergillus niger to each filter cartridge respectively;
[0039] 3, negative control group
[0040] Take 2 filter cartridges, take sterile water and operate in the same way, add 100 ml of thioethanolate fluid medium and 100 ml of pancreatic tryptone soybean peptone liquid medium to each filter cartridge after filtration.
[0041] 4, bacteria liquid group
[0042] Plate count the number of test bacteria added.
[0043] 5, culture: the culture medium of test group and control group is cultured according to the conditions shown in the following table for not more than 5 days, and the results are observed and recorded.
[0044] Table verification test bacteria and culture conditions
[0045]
[0046] 6 Conclusion
[0047] Through the sterility test method applicability test of the product, compared with the control group, the test bacteria in the containers containing the test product grow well, which shows that the test product under the test conditions has no or negligible antibacterial effect, and the sterility test of the test product can be carried out according to the test method and test conditions.
[0048] Through the results of 3 batches of samples, reference to the 2020 edition of Chinese Pharmacopoeia, the sterility test of the test product should meet the requirements. The sterility test method of cell bank is confirmed as follows:
[0049] Connect a set of two set bacteria culture device with sterile operation, and fix it on the bacteria culture instrument. Take the test product, filter 1 / 4 amount of test product in each filter cartridge by membrane filtration method, pump 100 ml of sterile water into the filter cartridge to make the cells fully lysed and then filter; after filtration, add 100 ml of thioethanolate fluid medium to each filter cartridge respectively.
[0050] Another set of two sets of bacteria culture device, according to the above steps, after filtration, respectively to 2 filter cartridges into 100 ml of thioethanolate fluid medium and 100 ml of tryptone soy broth liquid medium, and to 100 ml of thioethanolate fluid medium to add less than 100 cfu of staphylococcus aureus, as a positive control.
[0051] One of the thioethanolate fluid medium filter cartridge is placed in 30-35℃ culture, another thioethanolate fluid medium filter cartridge and tryptone soy broth liquid medium filter cartridge is placed in 20-25℃ culture, positive control filter cartridge is placed in 30-35℃ culture for no more than 5 days, test product tube culture for no less than 14 days.
[0052] The above detailed description of the present application is made in conjunction with specific embodiments and exemplary examples, but these descriptions cannot be understood as limiting the present application. Those skilled in the art understand that various equivalent substitutions, modifications or improvements can be made to the technical solutions and embodiments of the present application without departing from the spirit and scope of the present application, and these all fall within the scope of the present application. The scope of protection of the present application is subject to the appended claims.
Claims
1. A method of sterility check validation of a cell product, characterized in that, Specifically comprising the following steps: (1) Test group Take the test product, filter 2 tubes of test product per filter cartridge, filter the full amount in each tube; pump 150 ml of sterile water for injection into the filter cartridge, and filter after the cells are fully lysed; Sterile operation of a set of triple set bacteria incubator, after filtration, add 100 ml of thioethanolate fluid medium to the three filter cartridges respectively, and add less than 100 cfu of Staphylococcus aureus, Escherichia coli and Clostridium sporogenes to each of them; Take another set of triple set bacteria incubator, after filtration, add 100 ml of tryptone soy peptone liquid medium to the three filter cartridges respectively, and add less than 100 cfu of Bacillus subtilis, Candida albicans and Aspergillus niger to each of them; (2) Control group Take two sets of triple set bacteria incubator, one set of which adds 100 ml of thioethanolate fluid medium to the three filter cartridges respectively, and adds less than 100 cfu of Staphylococcus aureus, Escherichia coli and Clostridium sporogenes to each of them; The other set adds 100 ml of tryptone soy peptone liquid medium to the three filter cartridges respectively, and adds less than 100 cfu of Bacillus subtilis, Candida albicans and Aspergillus niger to each of them; (3) Negative control group Take 2 filter cartridges, take sterile water and operate in the same way, after filtration, add 100 ml of thioethanolate fluid medium and 100 ml of tryptone soy peptone liquid medium respectively; (4) Bacterial liquid group Plate count the number of test bacteria added; (5) Culture Culture the culture medium of the test group and the control group, observe and record the results.
2. The method according to claim 1, characterized in that, In step (1), thin film filtration method is used to filter 2 tubes of test product per filter cartridge.
3. The method according to claim 1, characterized in that, In steps (1)-(2), the triple set bacteria incubator for sterile operation is fixed on the bacteria collector.
4. The method according to claim 1, characterized by, In step (5), the culture medium of the test group and the control group is cultured for no more than 5 days.
5. The method of claim 1, characterized in that, In steps (1)-(3), the culture temperature of thioethanolate fluid medium is 30-35℃.
6. The method of claim 1, characterized in that, In steps (1)-(3), the culture temperature of tryptone soy peptone liquid medium is 20-25℃.
Citation Information
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