Mycoplasma Screening in Biopharmaceutical Manufacturing
Cell substrates used in the manufacture of biologics must be shown to be free of adventitious agents, including mycoplasma. Mycoplasma testing must be performed at various phases of product development, including raw materials, cell banks, viral seed stocks, unprocessed bulk harvest, and final products. Our clients have access to rapid and compendial mycoplasma test methods, which are compliant with the following regulatory guidelines:
- United States Pharmacopeia (USP) General Chapter <63>
- European Pharmacopoeia (Ph. Eur.) General Chapter 2.6.7
- Japanese Pharmacopoeia (JP)
- Combination protocols
Compendial Mycoplasma Screening Test Methods
Culture Method
- Test samples are inoculated directly onto agar plates as well as into liquid medium.
- Agar and liquid media chosen are shown to have satisfactory nutritive properties to support the growth of a broad range of mycoplasma species.
- Negative and positive controls are included in each mycoplasma detection assay to check the system suitability.
- Tests for the absence of inhibitory properties of the sample are included, if needed.
- During the incubation period the liquid cultures are subcultured at multiple time points onto agar plates to visualize mycoplasma growth.
- At the end of the incubation period, the agar plates are examined microscopically for the presence of mycoplasma colonies.
- Total test time is 28 days.
Indicator Cell Culture Method for Mycoplasma Contamination Testing
There are fastidious mycoplasma strains that are called “non-cultivable” because they do not grow in the standard media used for the culture method. Cultures of Vero or NIH3T3 cells are used to grow mycoplasmas. After an incubation period of one week, the cells are fixed and stained with a DNA-binding fluorescent dye and evaluated microscopically.
In the case of a mycoplasma infection there is a characteristic pattern of bright fluorescence on the surfaces of the cells and in surrounding areas when the contamination is heavy.
From a regulatory standpoint, both tests, the culture method and the indicator cell culture method, are needed to for cultivable and non-cultivable mycoplasma detection.
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Rapid Mycoplasma Screening and Testing Services
In some cases, culture-based procedures cannot be used, necessitating the use of nucleic acid amplification techniques (NAT). This could be the case if the product is cytotoxic or if fast turnaround times are required (e.g. when testing cell therapy products for mycoplasma contamination). NAT-based assays indicate the presence of a particular nucleic acid sequence and may be used as an alternative to the culture method, and/or indicator cell culture method.
Our rapid NAT-based mycoplasma assay utilizes Real-Time polymerase chain reaction technology with sequence-specific primers and dual-labeled fluorescent probes. Using this rapid mycoplasma testing method affords clients rapid results in as little as 5 days with a full COA provided within 10-14 business days. In urgent cases, the Fast-Track options deliver GMP mycoplasma testing results within 5 days.
Enrichment Nucleic Acid Amplification Techniques (NAT) Testing Methods
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Broth culture growth enrichment and RT-PCR
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. -
Growth enrichment by cell co-cultivation with RT-PCR
This mycoplasma detection 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.
Advantages of Rapid Mycoplasma Testing
- Increased sensitivity
- Reduced turnaround time
- Faster response to contamination and shorter investigations
- Broad detection range of mycoplasma species
- Ideal for cell and gene therapies and those with short shelf lives
Spiroplasma Testing
For those biopharmaceuticals that have been in contact with plant or insect materials, spiroplasma testing should be included. Spiroplasma testing is offered with three options:
- Spiroplasma can be determined in the traditional culture assay, due to dedicated primer sets in the reaction mix.
- Spiroplasma can be detected by the RT-qPCR method.
- Hybrid approach: Pre-cultivation followed by qPCR analysis. Samples are tested at day 0 and day 7.
Avoid Costly Delays With The Optimal Mycoplasma Contamination Testing Strategy
Switching mycoplasma testing methods is costly and time-consuming, therefore selecting the optimal method the first time helps avoid delays in the development, manufacturing, and release of your products. Charles River offers the traditional mycoplasma testing and the rapid mycoplasma detection testing strategies as well as detection of spiroplasmas. Our scientific and regulatory experts test thousands of samples every year so they can help strategize and select the best method for your project.
Frequently Asked Questions (FAQs) about Mycoplasma Testing
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What mycoplasma testing methods are available at Charles River?
We perform mycoplasma testing of biologic products according to worldwide regulatory guidance documents, including those from the US, EU, and Japanese Pharmacopoeia (USP, EP, JP) and Points to Consider (PTC), as well as combination protocols covering multiple regulations:
- Combined EP/USP/JP Protocol
- Combined EP/USP/PTC Protocol
- Combined EP/USP/JP/PTC Protocol
Additional mycoplasma testing includes:
- Large volume and quantitative PCR (qPCR) assays
- Mycoplasma clearance studies
- Spiroplasma testing
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In which cases is mycoplasmastasis testing method utilized?
Mycoplasmastasis testing methods or tests for inhibitory properties are used to demonstrate that a test sample does not contain inhibitory substances. These test samples must demonstrate the ability of different mycoplasma species to grow. The test for mycoplasmastasis should be performed at least once per individual sample matrix and must be repeated whenever there is a change in the composition that may affect the detection of mycoplasmas. For rapid mycoplasma testing an internal alternative target control is included in the reaction setup to monitor PCR inhibition.
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What regulatory requirements are recommended for mycoplasma testing?
The testing guidelines that address mycoplasma testing for biopharmaceutical products include the following:
- European Pharmacopoeia 2.6.7 (Mycoplasmas)
- European Pharmacopoeia 5.1.6 (Alternative Methods for Control of Microbiological Quality)
- United States Pharmacopeia <63> (Mycoplasma Tests)
- Japanese Pharmacopeia G3-14-170 (Mycoplasma Testing for Cell Substrates used for the Production of Biotechnological/Biological Products)
- ICH Q5D, Quality of Biotechnological Product (1997)
- CBER/FDA, Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
- CBER/FDA, Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997)
- 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)
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Do regulatory agencies accept rapid mycoplasma detection by PCR for unprocessed bulk harvest samples?
Yes, mycoplasma by is accepted by major regulatory agencies for bulk harvest testing. Nucleic acid amplification techniques (NATs), like qPCR or ddPCR, are mentioned as a suitable alternative by the European Pharmacopoeia (chapter 2.6.7), the United States Pharmacopeia (chapter 63) and the Japanese Pharmacopeia (G3-14-170). The applied NAT needs to be validated and able to detect at least 10 CFU/mL or less to be used as a replacement for both traditional methods (culture method and indicator cell method). Generic method validation should include the strains mentioned by the applicable guideline. Product-specific validation should include strains which are relevant for the cell line used and for the production process.
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Why is mycoplasma testing necessary for the bulk harvest stage?
Mycoplasma testing at the bulk harvest stage is a regulatory and scientific requirement because this is the point where all upstream biological material—cells, media etc.—has accumulated. Mycoplasmas will grow best at this stage, and thus the likelihood to detect an infection is highest at this point. Furthermore, since the product is produced by the cells at this point, a mycoplasma infection will alter the product quality at this point.
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How does mycoplasma contamination occur?
Mycoplasma contamination can be introduced by contaminated production cells, contaminated medium, and contaminated raw materials from the production process, especially animal-derived products or from operators carrying a mycoplasma infection.