Testing of Vaccine ,In Vitro Tests for Non-Viral Agents Mycoplasma/Spiroplasma
All licensed viral vaccines produced in cell substrates (and similar products) should be tested to ensure the absence of mycoplasma contamination. Testing is usually performed on the virus seed and/or MCBs, cell substrate, and a representative portion of each working cell stock used for manufacture of the product. Each lot of product harvest concentrate should be sampled for testing prior to further processing, e.g., prior to clarification, filtration, purification, and inactivation, unless testing at this stage of the manufacturing process is not feasible. Under 21 CFR 610.30, for virus vaccines produced from in vitro living cell cultures, you must test each virus harvest pool and control fluid pool (when control cells are used) for mycoplasma prior to clarification or filtration in the case of live virus vaccines and prior to inactivation in the case of inactivated virus vaccines. Prior to testing, the product harvest concentrate sample must be stored either:
1) between 2 and 8°C for no longer than 24 hours, or 2) at -20°C or lower if stored for more than 24 hours. If stored for longer than 24 hours, we recommend that you store the sample at -60°C or lower to allow for greater recovery of mycoplasma.
Although 21 CFR 610.30 specifies agar and broth media culture procedures, recent data suggest that some fastidious strains of mycoplasma, especially Mycoplasma hyorhinis, might not be detected by these agar and broth media procedures. Therefore, you should include an indicator cell culture procedure to detect such strains. We consider the testing described below to be acceptable alternatives to the method specified in 21 CFR 610.30. However, you must seek and obtain FDA approval for these modifications in accordance with 21 CFR 610.9(b).
For Spiroplasma testing by the culture method and by DNA staining, you should use media that supports the growth of Spiroplasma, Entomoplasma, and Mesoplasma
Testing for Mycoplasma using both Alternative Agar and Broth Media, and Indicator Cell Culture Procedures
Testing for Mycoplasma using Agar and Broth Media Procedure
(1) Each lot of agar and broth medium should be free of antibiotics except for penicillin, and each lot of medium should be examined for mycoplasma growth-promoting properties. To demonstrate the capability of the media to detect known mycoplasma contaminants, use the mycoplasma cultures specified below in (3)(i) as positive controls.
(2) (i) Inoculate no less than 0.2 mL of the test sample (e.g., product harvest concentrate sample) in evenly distributed amounts over the surface of 2 or more agar plates of 1 medium formulation.
(ii) Inoculate no less than 10 mL of the product harvest concentrate sample into a flask containing 50 ml of broth medium which is incubated at 36 ± 1° C.
(iii) Test 0.2 mL of the broth culture on the 3rd, 7th, and 14th days of incubation by subculture onto 2 or more agar plates of the same medium formulation as that used above in (i).
(iv) Incubate 2 of the initial isolation plates and 2 each of the three subculture plates in a 5 to 10 % carbon dioxide in nitrogen and/or hydrogen atmosphere containing less than 0.5% oxygen during the test incubation period.
(v) Incubate all culture agar plates for no fewer than 14 days at 36 ± 1°C and observe them microscopically at 100 times magnification or greater for growth of mycoplasma colonies.
(3) (i) Include in each test at least 2 known mycoplasma species or strains as positive controls, 1 of which should be a dextrose
fermenter (i.e., M. pneumoniae strain FH or equivalent species or strains) and 1 of which should be an arginine hydrolyzer (i.e., M. orale strain CH19299 or equivalent species or strain). Positive control cultures should be not more than 15 passages from isolation and should be used in a standard inoculum of 100 colony-forming units (CFU) or 100 color-changing units (CCU) or less.
(ii) Include uninoculated agar medium as a negative control.
(4) Interpret the results of the procedure according to the specification detailed in this section under Interpretation of Results.
Testing for Mycoplasma using Indicator Cell Culture Procedure
(1) Using a Vero cell culture substrate, pre-test the procedure by using the mycoplasma cultures specified below in (3)(i) as positive controls to demonstrate the capability of the cell substrate to detect known fastidious mycoplasmal contaminants. An equivalent indicator cell substrate might be acceptable if data demonstrate at least equal sensitivity for the detection of known mycoplasmal contaminants.
(2) (i) Inoculate no less than 1 mL of the test sample (e.g., product harvest concentrate sample) to 2 or more indicator cell cultures grown on cover slips in dishes or equivalent containers.
(ii) Incubate the cell cultures for 3 to 5 days at 36 ± 1°C in a 5% carbon dioxide atmosphere. Examine the cell cultures for the presence of mycoplasmas by epifluorescence microscopy using a DNA-binding fluorochrome, such as bisbenzimidazole or an equivalent stain.
(3) (i) Include in each test two known mycoplasma species or strains as positive controls (i.e., M. hyorhinis strain DBS 1050, M. orale strain CH19299, or equivalent species and strains), using an inoculum of 100 CFU or 100 CCU or fewer.
(ii) Include as a negative control a non-infected indicator cell culture.
(4) Interpret the results of the procedure according to the specifications detailed in this section under Interpretation of Results.
Interpretation of Results
(1) For the agar and broth media procedure, compare the appearance of the media inoculated with the product to that of the positive and negative controls.
(2) For the indicator cell culture procedure, using 600 times magnifications or greater, compare the microscopic appearance of the cultures inoculated with the product to that of the positive and negative cell controls.
(3) Marked cytopathic effects or nuclear chromatin fragmentation caused by vaccine virus infection that affect the interpretation of the results can be minimized by using a specific neutralizing viral antiserum or a nonpermissive cell culture substrate. The antisera should also be added to the positive and negative controls.
(4) The product is considered satisfactory for manufacture if both the agar and/or broth media procedure and the indicator cell culture procedure show no evidence of mycoplasma contamination (i.e., no growth) and thus resemble the negative control(s) for agar and broth and indicator cell procedures.
(5) If mycoplasmas are recovered, you should perform confirmatory testing to establish the species.
Testing for Mycoplasma Using PCR-based Assays
PCR-based assays may be used to detect mycoplasma, provided that such an assay can be shown to be comparable to the agar and broth media procedure and the indicator cell culture procedure. Such assays may combine culture and PCR. In some cases, culture-based procedures cannot be used due to an inability to completely neutralize vaccine viruses, thus necessitating the use of PCR-based assays to test for mycoplasma in these products.
b.Bacterial and Fungal Sterility
You must perform the test for bacterial and fungal sterility described in 21 CFR 610.12 or an equivalent method that fulfills the requirements of 21 CFR 610.9.
A 2.0 mL sample of the filtrate in its most concentrated form should be inoculated onto Lowenstein – Jensen’s egg medium or other media demonstrated to be equally capable of supporting growth. An appropriate positive-control test should be conducted simultaneously with the sample under test, and the test should be shown to be capable of supporting the growth of small numbers of the mycobacteria. All the test vessels should be incubated at a suitable temperature for a period of 6 weeks under conditions that will prevent drying of the medium, after which the cultures should be examined for evidence of mycobacterial colonies. The filtrate is satisfactory for use if the test shows no evidence of mycobacteria
Guinea pig tests have also been used to detect mycobacteria. If you use alternative tests, including PCR assays, they should be shown to be sufficiently sensitive to test for the presence of mycobacteria.
Terminologies In vaccine Production
TESTING FOR ADVENTITIOUS AGENTS CELL PROPERTIES IN VIRAL VACCINE PRODUCTION
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