bp tp d mycoplasma testing
Mycoplasma/Spiroplasma Testing Mollicutes, including Mycoplasma and Spiroplasma, are parasitic organisms that can be found intracellularly and on the external surfaces of a wide range of eukaryotic host cells. Their size and lack of a rigid cell wall allow them to pass through 0.2 μm filters and they are not easily detected due to lack of turbidity or cytopathic effects. The Biologics Testing Solutions group at Charles River can help you find out in the critical early phases of product development whether the cell line to be used for your production is contaminated with mollicutes.
Test Methods Available • Testing for Mycoplasma according to: -- Points to Consider (PTC) -- European Pharmacopoeia (EP) -- Japanese Pharmacopoeia (JP)
Mollicute Contamination in Biopharmaceutical Products
-- United States Pharmacopoeia (USP)
Cell substrates used in the manufacture of biologics must be shown to be free of adventitious agents including mycoplasma. Mycoplasma contamination of cell lines can affect cell growth in culture and also influence the production yields of biomolecules. In addition, they contribute to a potential direct or indirect source of human diseases. Potential sources of contamination include culture medium components, such as serum or trypsin, as well as contamination from the laboratory personnel themselves. Mycoplasma are difficult to detect and almost impossible to remove due to their size and flexibility. Testing for mycoplasma must be performed at various phases of product development, including:
-- Combined EP/USP/JP Protocol
• Raw material testing • Cell banks • Viral seed stocks • Unprocessed bulk harvest material
• Testing for the presence of Mycoplasma according to the large-volume method • Quantitative PCR (Q-PCR) methods • Mycoplasma clearance studies • Spiroplasma testing
Positive Control Species: • M. pneumoniae • M. orale • M. gallisepticum • M. hyorhinis
Spiroplasma testing is also suggested for biopharmaceutical products that have been in contact with plant or insect materials.
• M. synoviae
Description of Test Methods
• S. clarkii
• A. laidlawii
There are various methods that can be used to test for a variety of mycoplasma species including the culture method, indicator cell culture method and Q-PCR methods.
[email protected] www.criver.com
© 2014, Charles River Laboratories International, Inc.
Culture Method
Q-PCR Methods
To detect cultivable mycoplasma species, the test items are inoculated onto agar plates and into broth. These are maintained under atmospheric conditions as specified in the various guidelines. The broth is subsequently subcultured onto agar plates and observed for evidence of mycoplasma contamination.
We have many different Q-PCR methods available to test for mycoplasma contamination. They include the following:
Indicator Cell Culture Method For non-cultivable mycoplasma species, the test material is cultured in the presence of an indicator cell line, like Vero cells. The cells are subsequently stained with a DNA binding fluorochrome stain (DAPI) and evaluated microscopically for the presence of a mycoplasma infection. Some test materials may be cytotoxic to the indicator cells and must be diluted before inoculation. Another thing to be considered is the possibility that the test item may interfere with the growth of mycoplasma in the culture or indicator cell culture assay. The European Pharmacopoeia therefore requests qualification of the method by a mycoplasmastasis study testing for inhibitory substances via spiking positive controls into the test material. This procedure is normally performed only once for each test matrix. If the test item interferes with the growth of mycoplasma, the inhibitory substance must be neutralized or the test item must be further diluted.
The testing guidelines that address mycoplasma testing for biopharmaceutical products include the following: • European Pharmacopoeia section 2.6.7, supplement 6.1 • United States Pharmacopeia • ICH Q5D, Quality of Biotechnological Product (1997) • CBER/FDA, Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
• Mycoplasma and Acholeplasma Species Detection by Amplification of Specific Nucleic Acids and Fluorescent Probe Technology: This assay detects the presence of a particular nucleic acid sequence but does not necessarily indicate the presence of viable mycoplasma. • Testing for the Presence of Mycoplasma Using Broth Culture Growth Enrichment and RT-PCR Detection: This assay includes a growth enrichment step prior to the nucleic acid test in order to delineate viable organisms from non-viable organisms and residual environmental sources. Samples are tested at day 0 and day 7. • Testing for the Presence of Mycoplasma Using Growth Enrichment by Cell Co-Cultivation with RT-PCR Detection: This assay includes a growth enrichment step prior to the nucleic acid test to distinguish viable organisms from non-viable organisms and residual environmental sources. Samples are tested from a cell co-cultivation at day 0 and day 5.
Spiroplasma Testing This assay includes a growth enrichment step prior to the nucleic acid test in order to delineate viable organisms from non-viable organisms and residual environmental sources. Samples are tested at day 0 and day 7.
• CBER/FDA, Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997) • US FDA 21 CFR 610.30, Test for Mycoplasma (1998) • FDA, Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (February 2010)
[email protected] www.criver.com
© 2014, Charles River Laboratories International, Inc.
Test Methods Available • Testing for Mycoplasma according to: -- Points to Consider (PTC) -- European Pharmacopoeia (EP) -- Japanese Pharmacopoeia (JP)
Mollicute Contamination in Biopharmaceutical Products
-- United States Pharmacopoeia (USP)
Cell substrates used in the manufacture of biologics must be shown to be free of adventitious agents including mycoplasma. Mycoplasma contamination of cell lines can affect cell growth in culture and also influence the production yields of biomolecules. In addition, they contribute to a potential direct or indirect source of human diseases. Potential sources of contamination include culture medium components, such as serum or trypsin, as well as contamination from the laboratory personnel themselves. Mycoplasma are difficult to detect and almost impossible to remove due to their size and flexibility. Testing for mycoplasma must be performed at various phases of product development, including:
-- Combined EP/USP/JP Protocol
• Raw material testing • Cell banks • Viral seed stocks • Unprocessed bulk harvest material
• Testing for the presence of Mycoplasma according to the large-volume method • Quantitative PCR (Q-PCR) methods • Mycoplasma clearance studies • Spiroplasma testing
Positive Control Species: • M. pneumoniae • M. orale • M. gallisepticum • M. hyorhinis
Spiroplasma testing is also suggested for biopharmaceutical products that have been in contact with plant or insect materials.
• M. synoviae
Description of Test Methods
• S. clarkii
• A. laidlawii
There are various methods that can be used to test for a variety of mycoplasma species including the culture method, indicator cell culture method and Q-PCR methods.
[email protected] www.criver.com
© 2014, Charles River Laboratories International, Inc.
Culture Method
Q-PCR Methods
To detect cultivable mycoplasma species, the test items are inoculated onto agar plates and into broth. These are maintained under atmospheric conditions as specified in the various guidelines. The broth is subsequently subcultured onto agar plates and observed for evidence of mycoplasma contamination.
We have many different Q-PCR methods available to test for mycoplasma contamination. They include the following:
Indicator Cell Culture Method For non-cultivable mycoplasma species, the test material is cultured in the presence of an indicator cell line, like Vero cells. The cells are subsequently stained with a DNA binding fluorochrome stain (DAPI) and evaluated microscopically for the presence of a mycoplasma infection. Some test materials may be cytotoxic to the indicator cells and must be diluted before inoculation. Another thing to be considered is the possibility that the test item may interfere with the growth of mycoplasma in the culture or indicator cell culture assay. The European Pharmacopoeia therefore requests qualification of the method by a mycoplasmastasis study testing for inhibitory substances via spiking positive controls into the test material. This procedure is normally performed only once for each test matrix. If the test item interferes with the growth of mycoplasma, the inhibitory substance must be neutralized or the test item must be further diluted.
The testing guidelines that address mycoplasma testing for biopharmaceutical products include the following: • European Pharmacopoeia section 2.6.7, supplement 6.1 • United States Pharmacopeia • ICH Q5D, Quality of Biotechnological Product (1997) • CBER/FDA, Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
• Mycoplasma and Acholeplasma Species Detection by Amplification of Specific Nucleic Acids and Fluorescent Probe Technology: This assay detects the presence of a particular nucleic acid sequence but does not necessarily indicate the presence of viable mycoplasma. • Testing for the Presence of Mycoplasma Using Broth Culture Growth Enrichment and RT-PCR Detection: This assay includes a growth enrichment step prior to the nucleic acid test in order to delineate viable organisms from non-viable organisms and residual environmental sources. Samples are tested at day 0 and day 7. • Testing for the Presence of Mycoplasma Using Growth Enrichment by Cell Co-Cultivation with RT-PCR Detection: This assay includes a growth enrichment step prior to the nucleic acid test to distinguish viable organisms from non-viable organisms and residual environmental sources. Samples are tested from a cell co-cultivation at day 0 and day 5.
Spiroplasma Testing This assay includes a growth enrichment step prior to the nucleic acid test in order to delineate viable organisms from non-viable organisms and residual environmental sources. Samples are tested at day 0 and day 7.
• CBER/FDA, Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997) • US FDA 21 CFR 610.30, Test for Mycoplasma (1998) • FDA, Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (February 2010)
[email protected] www.criver.com
© 2014, Charles River Laboratories International, Inc.
