CN114574331A - Sterility test and verification method for cell product - Google Patents
Sterility test and verification method for cell product Download PDFInfo
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- CN114574331A CN114574331A CN202111580135.6A CN202111580135A CN114574331A CN 114574331 A CN114574331 A CN 114574331A CN 202111580135 A CN202111580135 A CN 202111580135A CN 114574331 A CN114574331 A CN 114574331A
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The application discloses a sterility test verification method of a cell product, which belongs to the field of biomedicine and specifically comprises the following steps: (1) test groups: taking a test sample, filtering 2 tubes of the test sample by each filter cylinder, and filtering the whole amount of each tube; pumping 150ml of sterilized water for injection into the filter cartridge to fully lyse the cells and then filtering; (2) a control group; (3) a negative control group; (4) a bacterial liquid group: counting the number of added test bacteria on a plate; (5) culturing: the culture media of the test group and the control group were cultured under the conditions shown in the following table, and the results were observed and recorded. The judgment basis is as follows: if the test bacteria in each container containing the test sample grow well in the specified culture period compared with the control tube, the test amount of the test sample has no or negligible bacteriostatic action under the test condition, and the sterility test of the test sample can be carried out according to the test method and the test condition. The invention solves the problem that the aperture of the filter membrane is blocked when the cell product is verified by adopting a membrane filtration method.
Description
Technical Field
The application belongs to the technical field of biological medicine, and particularly relates to a sterility test verification method for cell products.
Background
When a method for sterility testing of a pharmaceutical product is established, a method verification should be performed to prove that the method employed is suitable for sterility testing of the pharmaceutical product, according to the requirements of the "chinese pharmacopoeia" (2020 edition).
During the cell culture process, certain antibiotics such as gentamicin and the like are added to inhibit the growth of bacteria; avoiding cell contamination. Therefore, there is a need to perform sterility testing using membrane filtration to eliminate bacterial inhibition in cell products. When the sterility test method is verified by using a membrane filtration method, the diameter of cells is far larger than the aperture of the filter membrane, so the cells can be accumulated on the filter membrane during filtration, the aperture is blocked, and the filtration cannot be performed.
The prior art often carries out the aseptic examination of cell product through direct inoculation method, and this patent carries out the aseptic examination of cell product through the membrane filtration method, can eliminate the bacteriostasis of sample itself to the problem of filter membrane aperture jam when having solved the filtration.
Disclosure of Invention
The invention aims to provide a sterility check and verification method for cell products. The invention solves the problem that the aperture of the filter membrane is blocked when the cell product is verified by adopting a membrane filtration method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a sterility check verification method for cell products specifically comprises the following steps:
(1) test group
Taking a test sample, filtering 2 tubes of the test sample (3 ml/tube) by each filter cylinder, and filtering the whole amount of each tube; pumping 150ml of sterilized water for injection into the filter cartridge to fully lyse the cells and then filtering; because the problem of pore diameter blockage is solved by filtration after the cells are fully lysed, a triple bacteria collecting incubator is operated in an aseptic mode, after filtration is finished, 100ml of thioglycolate fluid culture medium is respectively added into 3 filter cylinders, and staphylococcus aureus, escherichia coli and clostridium sporogenes which are less than 100cfu are respectively added into the filter cylinders;
and (3) taking another triple bacteria collection incubator, respectively adding 100ml of tryptone soy peptone liquid culture medium into 3 filter cylinders after filtering, and respectively adding bacillus subtilis, candida albicans and aspergillus niger which are less than 100 cfu.
(2) Control group
Taking two sets of triple bacteria-collecting incubators, wherein one set of triple bacteria-collecting incubators respectively adds 100ml thioglycolate fluid culture medium into 3 filter cartridges, and respectively adds staphylococcus aureus, escherichia coli and clostridium sporogenes which are less than 100cfu into each filter cartridge;
adding 100ml trypticase soy peptone liquid culture medium into 3 filter cartridges respectively, and adding bacillus subtilis, candida albicans and aspergillus niger which are less than 100cfu into each filter cartridge respectively;
(3) negative control group
Taking 2 filter cartridges, taking sterilized water, operating by the same method, and respectively adding 100ml of thioglycolate fluid culture medium and 100ml of tryptone soy peptone liquid culture medium after filtering;
(4) bacterial liquid set
Counting the number of added test bacteria on a plate;
(5) culturing
The culture media of the test group and the control group were cultured under the conditions shown in the following table, and the results were observed and recorded.
When a test sample is prepared, cells are dissolved in sterilized injection water, and the cells are quickly swelled due to different osmotic pressures inside and outside the cells, so that the cells are quickly cracked, and the filter pore diameter is prevented from being blocked.
Preferably, in step (1), 2 tubes of the test sample are filtered per cartridge using a membrane filtration method.
Preferably, in step (1-2), the triple bacteria-collecting incubator aseptically operated is fixed to the bacteria collector.
Preferably, in the step (5), the culture media of the test group and the control group are cultured for not more than 5 days under the conditions shown in the following table.
Preferably, in steps (1) to (3), the culture temperature of the thioglycolate fluid medium is 30 to 35 ℃.
Preferably, in the steps (1) to (3), the culture temperature of the tryptone soy peptone liquid medium is 20 to 25 ℃.
Preferably, the judgment is based on: if the test bacteria in each container containing the test sample grow well in comparison with the control tube during the specified culture period, the test amount of the test sample has no bacteriostasis or the bacteriostasis is negligible under the test condition, and the sterility test of the test sample can be carried out according to the test method and the test condition.
The invention has the beneficial effects that:
the prior art often carries out the sterility test of cell product through direct inoculation method, and this patent carries out the sterility test of cell product through the membrane filtration method, can eliminate the bacteriostasis of sample itself to the problem of filter membrane aperture jam when having solved the filtration.
Detailed Description
The technical solutions in the embodiments of the present application are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
As introduced in the background section, certain antibiotics such as gentamicin and the like can be added in the cell culture process to inhibit the growth of bacteria; avoiding cell contamination. Therefore, there is a need to perform sterility testing using membrane filtration to eliminate bacterial inhibition in cell products. When the sterility test method is verified by using a membrane filtration method, the diameter of cells is far larger than the aperture of the filter membrane, so the cells can be accumulated on the filter membrane during filtration, the aperture is blocked, and the filtration cannot be performed. Based on the above analysis, the embodiments of the present application provide a method for verifying sterility test of cell products.
The method for verifying sterility test of cell product provided in the embodiments of the present application is described in detail below.
Embodiment 1, a method for verifying sterility test of cell products, comprising the following steps:
1. test group
Sterile operation 1 set of triple bacteria-collecting culture device, fix it on bacteria-collecting instrument. Taking a test sample, and filtering 2 tubes of the test sample by each filter cylinder by adopting a membrane filtration method, wherein each tube is filtered in full. Pumping 150ml of sterilized water for injection into the filter cartridge to fully lyse the cells and then filtering; after the filtration is finished, respectively adding 100ml of thioglycolate fluid culture medium into 3 filter cylinders, and respectively adding staphylococcus aureus, escherichia coli and clostridium sporogenes which are less than 100cfu into the filter cylinders;
and (3) another triple bacteria collection incubator is adopted, the operation is carried out according to the steps, after the filtration is finished, 100ml of trypticase soy peptone liquid culture medium is respectively added into 3 filter cylinders, and bacillus subtilis, candida albicans and aspergillus niger which are less than 100cfu are respectively added into the filter cylinders.
2. Control group
Taking 2 sets of triple bacteria collecting culture devices, adding 100ml of thioglycollate fluid culture medium into 3 filter cartridges, and respectively adding staphylococcus aureus, escherichia coli and clostridium sporogenes with the volume less than 100cfu into each filter cartridge;
adding 100ml trypticase soy peptone liquid culture medium into the other set of 3 filter cartridges, and respectively adding bacillus subtilis, candida albicans and aspergillus niger with the volume less than 100cfu into each filter cartridge;
3. negative control group
Taking 2 filter cartridges, taking sterilized water, operating by the same method, filtering, and respectively adding 100ml of thioglycollate fluid medium and 100ml of tryptone soy peptone liquid medium.
4. Bacterial liquid set
The plates were counted for the number of test bacteria added.
5. Culturing: the culture media of the test group and the control group were cultured for not more than 5 days under the conditions shown in the following table, and the results were observed and recorded.
Table verification test bacterium and culture condition thereof
Conclusion 6
Through the sterility test method applicability test of the product, compared with a control group, the test bacteria in each container containing the test sample grow well, which shows that the test sample with the test amount has no bacteriostasis or the bacteriostasis of the test sample is negligible under the test condition, and the sterility test of the test sample can be carried out according to the test method and the test condition.
Through the result research of 3 batches of samples, referring to the 'Chinese pharmacopoeia' of 2020 edition, the quality standard is established, and the sterility test of the test sample is in accordance with the regulations. The method for checking the sterility of the cell bank comprises the following steps:
the sterile operation is connected with a set of two-joint bacteria collecting incubator, and the two-joint bacteria collecting incubator is fixed on the bacteria collecting instrument. Taking a test sample, filtering 1/4 test samples in each filter cylinder by adopting a membrane filtration method, pumping 100ml of sterilized injection water into the filter cylinders, fully cracking cells and then filtering; after filtration was complete, 100ml of thioglycolate fluid medium was added to each of the 2 cartridges.
And another two-combined bacterium collecting incubator is taken, the operation is carried out according to the steps, after the filtration is finished, 100ml of thioglycollate fluid medium and 100ml of trypticase soytone liquid medium are respectively added into 2 filter cylinders, and staphylococcus aureus with the volume less than 100cfu is added into 100ml of thioglycollate fluid medium to serve as a positive control.
Wherein 1 thioglycollate fluid medium filter cartridge is placed at 30-35 ℃ for culture, the other 1 thioglycollate fluid medium filter cartridge and the trypticase soytone fluid medium filter cartridge are placed at 20-25 ℃ for culture, the positive control filter cartridge is placed at 30-35 ℃ for culture for no more than 5 days, and the test tube is cultured for no less than 14 days.
The present application has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to limit the present application. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the presently disclosed embodiments and implementations thereof without departing from the spirit and scope of the present disclosure, and these fall within the scope of the present disclosure. The protection scope of this application is subject to the appended claims.
Claims (7)
1. A sterility test verification method for cell products is characterized by comprising the following steps:
(1) test group
Taking a test sample, filtering 2 tubes of the test sample (3 ml/tube) by each filter cylinder, and filtering the whole amount of each tube; pumping 150ml of sterilized water for injection into the filter cartridge to fully lyse the cells and then filtering;
aseptically operating a triple bacteria-collecting incubator, respectively adding 100ml of thioglycolate fluid culture medium into 3 filter cartridges after filtration, and respectively adding staphylococcus aureus, escherichia coli and clostridium sporogenes which are less than 100cfu into the filter cartridges;
and (3) after the filtration of another set of triple bacteria collecting incubator is finished, respectively adding 100ml of trypticase soy peptone liquid culture medium into 3 filter cylinders, and respectively adding bacillus subtilis, candida albicans and aspergillus niger which are less than 100 cfu.
(2) Control group
Taking two sets of triple bacteria collecting incubators, wherein 100ml of thioglycolate fluid culture medium is respectively added into 3 filter cartridges in one set, and staphylococcus aureus, escherichia coli and clostridium sporogenes which are less than 100cfu are respectively added into each filter cartridge;
adding 100ml trypticase soytone liquid culture medium into 3 filter cartridges respectively, and adding bacillus subtilis, candida albicans and aspergillus niger with the volume less than 100cfu into each filter cartridge respectively;
(3) negative control group
Taking 2 filter cartridges, taking sterilized water, operating by the same method, filtering, and respectively adding 100ml of thioglycolate fluid culture medium and 100ml of trypticase soytone liquid culture medium;
(4) bacterial liquid set
Counting the number of added test bacteria on a plate;
(5) culturing
The culture media of the test group and the control group were cultured under the conditions shown in the following table, and the results were observed and recorded.
2. The method of claim 1, wherein in step (1), 2 tubes of the test article are filtered per cartridge using a membrane filtration process.
3. The method of claim 1, wherein, in steps (1) to (2), the aseptically operated triple-stack bacteria-collecting incubator is fixed to the bacteria-collecting instrument.
4. The method according to claim 1, wherein in the step (5), the culture media of the test group and the control group are cultured for not more than 5 days under the conditions shown in the following table.
5. The method according to claim 1, wherein the culture temperature of the thioglycolate fluid medium in steps (1) to (3) is 30 to 35 ℃.
6. The method according to claim 1, wherein the culture temperature of trypticase Soy peptone broth in steps (1) to (3) is 20 to 25 ℃.
7. The method of claim 1, wherein the determining is based on: if the test bacteria in each container containing the test sample grow well in the specified culture period compared with the control tube, the test amount of the test sample has no or negligible bacteriostatic action under the test condition, and the sterility test of the test sample can be carried out according to the test method and the test condition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111580135.6A CN114574331A (en) | 2021-12-22 | 2021-12-22 | Sterility test and verification method for cell product |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111580135.6A CN114574331A (en) | 2021-12-22 | 2021-12-22 | Sterility test and verification method for cell product |
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| CN114574331A true CN114574331A (en) | 2022-06-03 |
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| CN202111580135.6A Pending CN114574331A (en) | 2021-12-22 | 2021-12-22 | Sterility test and verification method for cell product |
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