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Biotech-Catalog Mycoplasma/Endotoxin/Sterility Testing |
Mycoplasma, Bacterial Endotoxin &
Sterility Testing
| In this section | | Mycoplasma Testing Back to Top | Mycoplasma contamination of cell lines and biotherapeutics represents a significant biosafety concern. The ubiquitous nature of mycoplasma in man, animals, and the environment increases the likelihood of the introduction of these organisms into cell lines or a manufacturing process. AppTec offers a variety of mycoplasma detection protocols to address your research and regulated testing needs.
The testing protocols employed to ensure product quality and safety during different stages of pharmaceutical development, i.e., pre-IND, phase I, II, III, BLA/NDA, are summarized below. Research / Pre-IND
At this stage of development the focus is mainly on ensuring that the research-grade material that you are working with is free from mycoplasma contamination. Protocol 30016 "Research Test for Mycoplasma" or Protocol 39083 "Mycoplasma PCR (Research)" — see Molecular Biology section – is appropriate for most applications. These protocols are research-based tests and are not suitable for regulatory submissions. Protocol 30016 employs both the DNA fluorochrome staining and microbiological detection methods for determining the presence of mycoplasma. Protocol 39083 is a PCR test that employs primers to highly conserved regions of the mycoplasma genome thus enabling the detection of a wide variety of mycoplasma species. IND through Market Approval
For the testing of cell banks and lot release of clinical trial or marketed bulk product material, Protocol 30055 "Mycoplasma Detection: Points to Consider" is appropriate for nearly all types of products. Protocol 30252 "Mycoplasma Detection: 21 CFR 610.30" or Protocol 30002 "Mycoplasma Detection: 21 CFR 610.30 and DNA Fluorochrome Staining" are appropriate for live-attenuated virus vaccine products. The 9 CFR mycoplasma test (Protocol 30266) is most appropriate for animal-derived veterinary products. Protocol 30227 is the fluorochrome method of mycoplasma detection and is offered as a quick screen for the detection of some infectious mycoplasma species. GLP and GMP PCR assays for the detection of mycoplasma DNA are also available. (See Molecular Biology section.) The PCR and DNA fluorochrome assays can be used as lot release tests for products with very short "shelf lives," such as autologous cell therapies. Typically, the full "Points to Consider" assay (Protocol 30055) will also have to be performed in a "look back" mode on these products as well.
| 30016 | This assay is used to detect the presence of mycoplasma by both indirect (cell culture) and direct (broth and agar) assays. This procedure is an abbreviated, informal screening test for research purposes only. It is not intended to support regulatory submissions for licensure of biological products. The test article is incubated with monkey kidney cells or mouse embryo fibroblasts and is then fixed, stained with a DNA-binding fluorochrome (Hoechst Stain), and evaluated microscopically by epifluorescence for the presence of mycoplasma. Mycoplasma agar plates and broth flasks are inoculated with the test article and are incubated anaerobically and aerobically, respectively. Samples from the broth flasks are subcultured on day 7 onto one or more agar plates, which are examined no sooner than 14 days post-inoculation. Three species of mycoplasma serve as positive controls. Notes
· If the test article includes cells, passage twice in media free of antibiotics and methotrexate before sample submission.
· Cell cultures should be confluent at the time of harvest. It is recommended that it be at least 3-4 days since the last feed/medium change. The cells should be harvested by scraping into conditioned media. Trypsin should not be used. (Culture medium without cells can also be tested; however, this is not recommended.)
· Freeze in polypropylene screw-capped vial(s), allowing some air space for expansion. (Cells are frozen in conditioned medium without cryopreservatives.) | Research Test for the Presence of Mycoplasma
Turnaround Time:
5 weeks Container/Volume:
1 x 5 ml Shipment Temperature:
Frozen on dry ice
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| 30055 | This assay is used to detect the presence of mycoplasma by both indirect (cell culture) and direct (broth and agar) assays. The test article is incubated with monkey kidney cells and is then fixed, stained with a DNA-binding fluorochrome (Hoechst Stain), and evaluated microscopically by epifluorescence for the presence of mycoplasma. Agar plates and broth flasks are inoculated with the test article and are incubated anaerobically and aerobically, respectively. Samples from the broth flasks are subcultured on days 3, 7 and 14 onto agar plates; all plates are examined no sooner than 14 days post-inoculation. Three species of mycoplasma serve as positive controls. Notes
· If the test article includes cells, passage twice in media free of antibiotics and methotrexate before sample submission.
· Cell cultures should be confluent at the time of harvest. It is recommended that it be at least 3-4 days since the last feed/medium change. The cells should be harvested by scraping into conditioned media. Trypsin should not be used. (Culture medium without cells can also be tested; however, this is not recommended.)
· Freeze in polypropylene screw-capped vial(s), allowing some air space for expansion. (Cells are frozen in the conditioned medium without cryopreservatives.) | Mycoplasma Detection:
"Points to Consider"
Turnaround Time:
6 weeks Container/Volume:
1 x 15 ml Shipment Temperature:
Frozen on dry ice |
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| 30200 | This assay is used to detect the presence of mycoplasma by both indirect (cell culture) and direct (broth and agar) assays. The test article is incubated with either monkey kidney cells or NIH/3T3 cells. Optionally, an antiserum supplied by the sponsor can be used to prevent adverse effects of the virus on the indicator cells. The cell layer is then fixed, stained with a DNA-binding fluorochrome (Hoechst Stain), and evaluated microscopically by epifluorescence for the presence of mycoplasma. Agar plates and broth flasks are inoculated directly with the test article. Agar plates are incubated anaerobically and broth flasks aerobically. Samples from the broth flasks are subcultured on days 3, 7 and 14 onto two or more agar plates. The agar plates are examined for the presence of mycoplasma colonies after 14 or more days of incubation. The broth flasks are examined daily for 14 days (normal working days only) for color change. Three species of mycoplasma serve as positive controls. Notes
· The amount of test article required may vary from one sample to another. Please consult with the study director.
· If possible, submit back-up for this assay.
· Include instructions on how to handle the test article and the sample concentration (i.e., dilute up to volume needed for assay or instructions for use of antiserum). | | Mycoplasma Detection: "Points to Consider" (1993) for Virus Stocks
Turnaround Time:
6 weeks Container/Volume:
Equivalent of 1 patient dose
See Notes. Shipment Temperature:
Frozen on dry ice |
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| 30266 | This assay is used to detect the presence of mycoplasma in vaccines, cell lines, or samples of primary cells according to 9 CFR 113.28. The test article is inoculated into broth using the formulation recommended by 9 CFR. The broth flasks are incubated for 14 days at 33° to 37°C, with subcultures being performed on days 3, 7, 10, and 14. The agar plates are incubated for 10–14 days at 33° to 37°C with high humidity and 4-6% CO2. At the end of the incubation period the plates are examined for mycoplasma colonies. Notes
· If the test article includes cells, passage twice in media free of antibiotics and methotrexate before sample submission.
· Cell cultures should be confluent at the time of harvest. It is recommended that it be at least 3-4 days since the last feed/medium change. The cells should be harvested by scraping; trypsin should not be used. (Culture medium without cells can also be tested; however, this is not recommended.)
· Freeze in polypropylene screw-capped vial(s), allowing some air space for expansion. (Cells are frozen in conditioned medium without cryopreservatives.) | | Mycoplasma Detection:
9 CFR 113.28
Turnaround Time:
8 weeks Container/Volume:
2 x 15 ml Shipment Temperature:
Frozen on dry ice |
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| 30252 | Mycoplasma detection according to 21 CFR 610.30 is recommended for human vaccine production and injectables. This assay is used to detect the presence of mycoplasma by using the agar and broth media procedure as specified in 21 CFR 610.30. The test article is inoculated onto agar plates of at least two different agar media and into semi-solid broth medium. Half of each group of plates and broth are incubated anaerobically and half aerobically. The broth flasks are subinoculated onto agar plates on days 3 and 14. All plates are examined after at least 14 days of incubation for the presence of mycoplasma colonies. At least two different strains of mycoplasma serve as the positive controls. Notes
· If the test article includes cells, passage twice in media free of antibiotics and methotrexate before sample submission.
· Cell cultures should be confluent at the time of harvest, it is recommended that it be at least 3-4 days since the last feed/medium change. The cells should be harvested by scraping, trypsin should not be used. (Culture medium without cells can also be tested; however, this is not recommended.)
· Freeze in polypropylene screw-capped vial(s), allowing some air space for expansion. (Cells are frozen in conditioned medium without cryopreservatives.) | Mycoplasma Detection:
21 CFR 610.30
Turnaround Time:
8 weeks Container/Volume:
2 x 10 ml S hipment Temperature:
Frozen on dry ice |
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| 30002 | Mycoplasma detection according to 21 CFR 610.30 is recommended for vaccine production. In addition to the direct test recommended by the 21 CFR 610.30, this also includes the indirect assay to detect non-cultivable mycoplasma. This assay is used to detect the presence of mycoplasma by both indirect (cell culture) and direct (broth and agar) assays. The test article is incubated with monkey kidney cells and is then fixed, stained with a DNA-binding fluorochrome (Hoechst Stain), and evaluated microscopically by epifluorescence for the presence of mycoplasma. Mycoplasma agar plates (at least 2 different types) and semi-solid broth tubes are inoculated with the test article. Half of each group of plates and broth are incubated anaerobically and half aerobically. The broth tubes are subcultured onto agar plates at days 3 and 14 during the 28-day incubation period. All agar plates are examined no sooner than 14 days post-inoculation for the presence of mycoplasma colonies. Three different strains of mycoplasma serve as positive controls. Notes
· If the test article includes cells, passage twice in media that is free of antibiotics and methotrexate before sample submission.
· Cell cultures should be confluent at the time of harvest. It is recommended that it be at least 3-4 days since the last feed/medium change. The cells should be harvested by scraping; trypsin should not be used. (Culture medium without cells can also be tested; however, this is not recommended.)
· Freeze in polypropylene screw-capped vial(s), allowing some air space for expansion. (Cells are frozen in conditioned medium without cryopreservatives.) | Mycoplasma Detection: 21 CFR 610.30 and DNA Fluorochrome Staining
Turnaround Time:
8 weeks Container/Volume:
2 x 13 ml Shipment Temperature:
Frozen on dry ice |
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| 30227 | This assay is used to indirectly detect the presence of mycoplasma. The test article is incubated with monkey kidney cells and is then fixed, stained with a DNA-binding fluorochrome (Hoechst Stain), and evaluated microscopically for epifluorescence. This method of detection can be used as a screen since it has a short duration (7 days) in comparison to the direct assay (28 days). Notes
· If test article includes cells, passage twice in media free of antibiotics and methotrexate before sample submission.
· Cell cultures should be confluent at the time of harvest. It is recommended that it be at least 3-4 days since the last feed/medium change. The cells should be harvested by scraping. Trypsin should not be used. (Culture medium without cells can also be tested; however, this is not recommended.)
· Freeze in polypropylene screw-capped vial(s), allowing some air space for expansion. (Cells are frozen in conditioned medium without cryopreservatives.) | Mycoplasma Screening of Cell Culture by DNA Fluorochrome Staining
Turnaround Time:
3 weeks Container/Volume:
2 x 3 ml cells Shipment Temperature:
Frozen on dry ice |
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| 30110 | This assay is used for microoganism identification including bacteria, mycoplasma and fungi. This is a nucleic acid-based method for microorganism identification using the 16S ribosomal RNA or smaller subunit ribosomal RNA or DNA sequence analysis. |
Microbial Identification using
16S Ribosomal RNA Gene Sequencing (Research)
Turnaround Time:
2 weeks Container/Volume:
Plate containing colonies Shipment Temperature:
Cold on cold packs |
| | f | | Bacterial Endotoxin Testing Back to Top | PROTOCOLS
30735 Gel Clot Method: USP/FDA
30037 Gel Clot Method: USP/FDA – GLP
37653 Kinetic-Chromogenic Method: USP/FDA
37651 Kinetic-Chromogenic Method: USP/FDA – GLP This assay is used to determine if bacterial endotoxins are present in the test article. A quantitative procedure is performed, either by the gel-clot method or the kinetic-chromogenic method. Known amounts of endotoxin are tested in parallel with the test article for an accurate determination of the level of bacterial endotoxin. In order to use an LAL procedure, the FDA requires a validation study be performed which includes inhibition/enhancement (I/E) testing and the determination of endotoxin levels in at least three product lots. Either method can be performed as a standard USP/FDA test or a GLP study. Notes
· Sample must be sent in pyrogen-free containers.
· Sample volume of 1 ml is suggested. (If sample can be diluted, smaller volume may be adquate.) |
Bacterial Endotoxin Testing using Limulus Amebocyte Lysate (LAL)
Turnaround Time:
4 days (USP/FDA)
1-2 weeks (GLP) Container/Volume:
See Notes. Shipment Temperature:
Per client’s discretion |
PROTOCOLS
30743 Gel Clot Method – I/E Assay: USP/FDA
30746 Gel Clot Method – I/E Assay: USP/FDA – GLP
30739 Kinetic-Chromogenic Method – I/E Assay: USP/FDA
30518 Kinetic-Chromogenic Method – I/E Assay: USP/FDA – GLP This assay validates the use of the LAL gel-clot or kinetic-chromogenic method for the detection of endotoxins. Validation requires inhibition/enhancement (I/E) testing and determination of endotoxin levels in at least three product lots. Testing must be performed on the undiluted product or on an appropriate dilution of the final product, and results must be within the prescribed endotoxin limits. The potential for interference by the test article is examined by spiking the test article plus LAL reagent with specified levels of endotoxin. Following the inhibition/enhancement test, the endotoxin content of the test article is then determined. Either validation can be performed as a standard USP/FDA test or a GLP study. Notes
· Three lots required for validation.
· Submit samples in pyrogen-free containers.
(Note: Polypropylene tubes may cause inhibition in the LAL assay.)
· Submit maximum patient dose for final product testing.
· Sample volume of 1 ml is suggested. (If sample can be diluted, smaller volume may be adquate.) | Bacterial Endotoxin Testing using Limulus Amebocyte Lysate (LAL)
I/E Assay/Validation
Turnaround Time:
4 days (USP/FDA)
1-2 weeks (GLP) Container/Volume:
See Notes. Shipment Temperature:
Per client’s discretion |
| | f | | Sterility Testing Back to Top |
30744 <20 samples
30737 20 samples | This procedure is used to determine if the test article is free from viable bacterial and fungal contamination. The test article is aseptically transferred into Soybean-Casein Digest Medium (SCDM) and Fluid Thioglycollate Medium (FTM). These broths are incubated for 14 days and inspected for evidence of bacterial and fungal growth. This test methodology is essentially identical to 30045 and 30093, and is performed according to 21 CFR 610.12 requirements except that a B/F test is not performed with each test (but may be performed separately if desired). Notes
· Product must be submitted in unopened final containers containing total fill volume.
· Number of samples required for testing:
– Lots of 20 containers or less: 2 containers.
– Lots of 21 - 200 containers: 10% of the final number of containers.
– Lots of > 200 containers: 20 final containers.
· If material in final container is >1 ml, the volume tested should be the largest single dose of 1 ml, whichever is greater. (No more than 10 ml or the entire contents of a final container need be tested.) |
Sterility Test - USP
Turnaround Time:
2-3 weeks Container/Volume:
See Notes. Shipment Temperature:
Per client's discretion |
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30045 <20 samples
30093 20 samples | This procedure is used to determine if the test article is free from viable bacterial and fungal contamination. The test article is aseptically transferred into Soybean-Casein Digest Medium (SCDM) and Fluid Thioglycollate Medium (FTM). These broths are incubated for 14 days and inspected for evidence of bacterial and fungal growth. In addition, broths containing the test article are spiked with specified microorganisms to show that microbial contamination, if present, would be apparent (bacteriostasis/
fungistasis test). This test is performed according to GLP requirements. Notes
· Product must be submitted in unopened final containers containing total fill volume.
· Number of samples required for testing:
– Lots of 20 containers or less: 2 containers.
– Lots of 21 - 200 containers: 10% of the final number of containers.
– Lots of > 200 containers: 20 final containers.
· If material in final container is >1 ml, the volume tested should be the largest single dose of 1 ml, whichever is greater. (No more than 10 ml or the entire contents of a final container need be tested.) |
Sterility Test + Bacteriostasis/Fungistasis (B/F): 21 CFR 610.12
Turnaround Time:
4-5 weeks Container/Volume:
See Notes. Shipment Temperature:
Per client's discretion |
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30736 USP
30738 USP - GLP | The B/F test is a validation of the sterility test and is performed to ensure that if viable bacteria or fungi were present, they would be apparent. Sterility test broths containing the test article are inoculated with low levels of specified microorganisms and then inspected for evidence of microbial growth. Growth indicates no bacteriostatic or fungistatic activity and means the sterility test parameters are valid. The B/F test does not need to be repeated as long as there is no change in the test article or test parameters. This test can be performed as a standard USP test or a GLP study. Notes
· Product can be less than the total fill volume since the test is based on the test article-to-media ratio. |
Bacteriostasis/Fungistasis (B/F)
Test
Turnaround Time:
7 days (USP)
2-3 weeks (GLP) Container/Volume:
See Notes. Shipment Temperature:
Per client's discretion |
| | f | | Bioburden Testing Back to Top | PROTOCOLS
30750 Aerobe Count
30755 Aerobe Count – GLP
30751 Aerobe and Fungi Count
30758 Aerobe & Fungi Count – GLP
30752 Aerobe / Fungi / Spore Count
30759 Aerobe / Fungi / Spore Count – GLP
30753 Aerobe / Fungi / Spore / Anaerobe Count
30760 Aerobe / Fungi / Spore / Anaerobe Count – GLP This procedure is used to determine the number and types of viable organisms that are present in a non-sterile test article. The test article is plated on various test media under specific incubation conditions to determine the number of organisms in each test category. Recovered organisms may be further identified to genus and species if desired. This test can be performed as a standard USP test or a GLP study. Notes
· Product should be submitted in unopened final containers containing total fill volume. |
Bioburden Determination
Turnaround Time:
7-10 days
2-3 weeks (GLP) Container/Volume:
See Notes. Shipment Temperature:
Per client’s discretion |
| 30754
30756 GLP | A recovery efficiency test (bioburden validation) is performed to determine approximately what percent of organisms can be detected using the specified bioburden test method. A specified number of bacterial spores are inoculated into the test article and the bioburden test established for that test article is performed. The recovery efficiency is the number of organisms recovered divided by the number inoculated. This test can be performed as a standard USP test or a GLP study. Notes
· Product should be submitted in unopened final containers containing total fill volume. |
Bioburden Recovery Efficiency (Validation) - Inoculation Method
Turnaround Time:
7-10 days
2-3 weeks (GLP) Container/Volume:
See Notes. Shipment Temperature:
Per client's discretion |
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