Mycoplasma testing
Do you have any requests or concerns as listed below in your R&D and manufacturing of Biopharmaceuticals and regenerative medicinal products?
- To rapidly perform mycoplasma testing for launching a product in Japan in compliance with the Japan Pharmacopeia.
- To speedily perform mycoplasma testing for launching a product in the US or Europe in compliance with the guidelines.
- To perform mycoplasma testing in drugs manufacturing or cell culturing processes.
Eurofins Analytical Science Laboratories, Inc. offers comprehensive services covering validation, measurement, and the method transfer of mycoplasma testing (NAT-based assays).
Cell culture technology using animal cells has been widely utilized in antibodies and vaccines, and also in the fields of regenerative medicine and gene therapy.
According to the Research paper series No.71 by Office of Pharmaceutical Industry Research, the number of development items such as antibodies, cell therapy, gene therapy after Phase I increased from 300 in 1995 to over 2,000 in 2018 and it is increasing year by year. These biopharmaceutical items must be completely free of mycoplasma and the regulatory authorities request to do release testing as part of the manufacturing process.
Mycoplasma is a naturally occurring microorganism. And it adversely affects cell growth and metabolism when infected.
Mycoplasma is too small so it can pass through the removal filter and contaminates drugs. To avoid this risk of contamination, a detection of mycoplasma is required.
About Mycoplasma testing
"Mycoplasma Testing for Cell Substrates used for the Production of Biotechnological/Biological Products", the Japan Pharmacopoeia (17th Edition), describes the following three testing methods:
- A. Culture method
- B. DNA staining method (The indicator cell culture method)
- C. Nucleic acid amplification technique : NAT-based assays
Master cell back, working cell bank, cell lots in processes of pharmaceutical manufacturing shall be tested by the culture method (A) or the DNA staining method (B). Appropriately validated NAT techniques may be used as an alternative to these two methods.
In Method A, samples are cultured in ager plate medium at 35-37°C for at least 14 days. Then, the samples (cell suspension) are inoculated into a vessel containing liquid medium and incubated at 35-37°C.
After the start of incubation, observe every 2 or 3 days, and on the day when the color of the liquid medium is changed, or if there is no change in color, on days 3, 7, and 14, collect from the liquid medium and inoculate on the agar plate medium.
Incubate at 35 -37℃ for at least 14 days and the plates are then observed on the 7th and the 14th days under a microscope to enumerate the mycoplasma colonies.
In Method B (the indicator cell culture method), samples are cultured on Vero cells at 35-38℃ for 3-6 days, then stained with the fluorescent dye, which binds specifically to DNA.
The negative control and two types of mycoplasma reference standards shall be prepared.
The dye facilitates the detection of mycoplasma, as there is a characteristic particulate or filamentous pattern of fluorescence on the cell surface, and in surrounding areas too, if the contamination is particularly heavy. Compare the microscopic appearance of the test cultures with that of the negative and reference controls, examining for extranuclear fluorescence.
Method C (Nucleic acid amplification: NAT) is a highly sensitive method for detecting the presence of nucleic acids originating from mycoplasma by amplifying nucleic acids extracted from samples using primers and probes specific to mycoplasma.
NAT indicates the presence of the target sequence and does not necessarily mean the presence of viable mycoplasma organisms.
NAT does not have a specific method, and any method can be used as long as it is validated for specificity, detection sensitivity, and robustness.
In general, when performing the Method C, a positive control (viable bacteria) and a negative control are used, and the test is qualified when the mycoplasma gene is not amplified in the sample.
The method A and B are time consuming. Those are requiring at least 28 days for the A method and 4 days for the B method.
However, this time requirement is not amenable for obtaining the rapid lot release testing results needed for biopharmaceutical products that have short half-lives or those that are in high market demand.
The lengthy assay period is also not conducive to the rapid screening of raw materials intended for use in future production, nor to the rapid in-process screening of intermediates for the purpose of detecting and containing contamination events.
So many regulatory agencies now accept rapid nucleic acid amplification technique for mycoplasma testing.
Eurofins Analytical Science Laboratories, Inc. offers this rapid mycoplasma testing services.
Workflow of Mycoplasma testing (Method C: NAT)
We receive samples from clients and perform necessary validations such as specificity, detection sensitivity, and robustness according to the worldwide guidelines and also characteristics of client's samples.
Correlation data between mycoplasma (live bacteria) and control DNA, which will be used as a positive control for the actual measurement, will also be obtained.
Venor®GEM qEP (Minerva biolabs) is used in combination with a real-time PCR cycler.
This kit enables to detect Mollicutes (Mycoplasma, Acholeplasma, Spiroplasma) contaminants of cell cultures and media components by amplifying a highly conserved rRNA operon, or more specifically, the 16S rRNA coding region in the mycoplasma genome. The mycoplasma-specific amplification is detected at 520 nm (FAM™ channel).
An internal control is added to the sample to verify if it contains substances that inhibit the PCR reaction.
This kit complies the Japan Pharmacopoeia (17th Edition) can detect all mycoplasma filed in EP2.6.7.
Sample preparation and manipulation are quite simple, and results can be obtained in approximately 4.5 to 6.5 hours.
In the actual measurement, a validated test method is used with no viable bacteria and control DNA is measured as a positive control.
Amplification plot
[Red: Mycoplasma 520 nm, Blue: Internal control 610 nm]
In the method transfer, we ensure your smooth measurement works in laboratories and manufacturing sites.
Depending on the scale and need of your R&D, we offer the following three Mycoplasma Testing Services.
- Validation and Actual measurement
- Validation, Actual measurement, and Method transfer
- Validation and Method transfer
For example, "validation + actual measurement" is suitable if your plan is for clinical research or small-scale clinical trials with less than 10 runs. Or if you have enough time before the release of the drug and you want to perform testing in-house, you can choose "validation + technology transfer".
Why choose Eurofins Analytical Science Laboratories?
- Eurofins companies have over 20 years of experience performing Mycoplasma testing.
- After the method transfer, we can perform the testing without using viable bacteria. This enables us to provide the services with low-risk and low-cost.
- You can order only Feasibility testing.
- We have various analytical equipment under GMP control.
Eurofins companies have solid knowledges and experiences of Mycoplasma testing for regulatory filing in several countries.
We can offer optimal testing proposal of Mycoplasma testing by utilizing these knowledges.
We obtain correlation data between viable mycoplasma and control DNA during the method validation and provide it to our clients.
When using viable mycoplasma in your laboratory or manufacturing site, you need to take measures to avoid contamination of cells. But these measures are not necessary if using control DNA.
Therefore, after the method transfer, you can perform mycoplasma testing at low risk and low cost without using live bacteria in your site.
We also perform a feasibility study only, which is a preliminary step to validation. We can verify if we can perform mycoplasma testing without having any issues.
Some samples may not be measurable due to their characteristics or contaminants. By investigating these issues in the early stages of development, we can utilize them to improve manufacturing methods.
Eurofins Analytical Science Laboratories, Inc. is an organization compliant with GMP Ministerial Ordinance. Under its control and management, we create protocols, perform testing, store samples, and manage facilities, equipment, and systems.
Eurofins Analytical Science Laboratories, Inc. offers comprehensive Mycoplasma testing services according to the JP/EP/USP guidelines with fully cGMP-compliant analytical equipment.
We support your Mycoplasma testing. Please feel free to contact us if you have any questions and requests.