Mycoplasma Detection in Biotherapeutics – Current and Advanced Technologies

• Amgen Inc.

Mycoplasmas represent microorganisms that belong to the class Mollicutes. They are free-living and self-replicating bacteria that are notable for their small size (0.1 μm to 0.3 μm) and lack of a rigid cell wall.¹ Due to the small size of their genome, mycoplasmas are dependent on complex media or host cell for metabolites. The members of the class mollicutes represent a large group of microorganisms divided in 10 genera based on their nutritional requirements, oxygen usage, metabolic capabilities and their host preference. To date, more than 200 species of Mollicutes have been identified with the number expected to grow over the next few years.² Their widespread distribution makes them a likely cause for contamination of cell substrates and other raw materials such as animal serum used for manufacture of cell-derived biologics and pharmaceutical products.³ The most common cases of mycoplasma contamination are reported to involve M. orale, M. hyorhinis, M. arginini, M. fermentans, M. homonis and A. laidlawii.⁴ Contamination by mycoplasmas can lead to a number of different effects on cell culture ranging from altered levels of protein, RNA and DNA synthesis to total culture degeneration and loss.⁴ Mycoplasma contaminations in biopharmaceutical industry present a risk for the biosafety and quality of cell-derived biotherapeutics. Control of raw materials and tests for the presence of mycoplasma in mammalian cell substrates for production of protein therapeutics and drug substance intermediate (unprocessed bulk harvest) are two pillars of an integrated adventitious agent safety approach which ensures freedom of cell culture processes from mycoplasma contamination and other adventitious agents.

Mycoplasma Testing and Detection Methods

Several sources have provided historical reviews of mycoplasma testing^5,6 and various regulatory guidelines and compendial chapters provide the details of the standard testing method.^7-9 The current “gold standard” testing for protein therapeutics produced in mammalian cells utilizes a direct culture method with broth and agar as well as an indirect indicator cell culture method with subsequent DNA staining. When performing the direct culture method test samples are inoculated in broth and subsequently sub-cultured on agar plates. This direct method allows for the detection of a wide range of mycoplasma species. However, fastidious mycoplasma species may not be detected using this method as they are not able to grow in broth and agar. The indirect indicator cell culture method ensures the detection of fastidious mycoplasma species that are considered non-cultivable in broth and agar and are dependent on mammalian cells for growth. Typically, a test sample is inoculated onto an indicator cell line and following an incubation period the cells are fixed and stained with a DNA binding dye. The presence of mycoplasma contamination is indicated by the pattern of staining which will appear extranuclear in the shape of filamentous strands and aggregated clusters. The direct and indirect detection methods for mycoplasma are deemed sensitive and specific methods because they allow for amplification of contaminating mycoplasma. However, a comprehensive analysis of this culture based method with respect to its breadth of detection using different species of mycoplasma has not been performed. Therefore, an important consideration is the selection of a suitable growth media that will support the growth of a wide range of mycoplasma species. Complex and universal growth media have been developed that will presumably support amplification of a wide range of mycoplasma species. However, on the impact of the composition of the test samples as well as the incubation temperatures and atmospheric conditions should to be optimized as they have a critical impact on the detection of mycoplasma contaminants in the test samples. Further, sample handling and processing procedures should be designed to minimize adverse impact on the viability of any potential mycoplasma contaminants. The most significant drawback of the current “gold standard” testing is its duration of 28-35 days. In recent years, interest has developed for identifying and developing more rapid mycoplasma detection methods. The main focus of such rapid methods would be to provide sensitive alternative methods capable of detection of a wide range of mycoplasma species while allowing for a reduction in cycle time for testing and getting the medicine to patients faster. Additionally, rapid testing methods can be used as a risk mitigation measure to provide data for faster decision making and before downstream processing to limit the spread of contaminated material. Naturally, these rapid detection methods are based on nucleic acid-based technologies (NAT).

The European Pharmacopoeia EP 5.8 (Chapter 2.6.7) provides for nucleic acid-based mycoplasma testing as a replacement for the current culture-based methods, and details the validation and comparability requirements.¹⁰ The data package associated with alternate testing methods should provide assurances that the new method is comparable or better than the current testing method and provides for broad detection of mycoplasma species. It should allow for a fast turnaround time for testing at reduced costs. Lastly, it needs to be easy to use and represent a robust testing system. As stated, the method of choice to fit these criteria for rapid detection of mycoplasma is often based on nucleic acid detection technologies. Conserved regions in the mycoplasma genome provide for amplification targets that can enable the detection of a large number of mycoplasma species in a single platform. Several nucleic acid-based technologies have been developed and assessed for detection of mycoplasma in cell culture.^5,11-17

Mycoplasma detection assays based on qPCR technology are now commercially available. With the employment of modified or multiplexed primers targeted to conserved sequences in the mycoplasma genome such assays can be capable of detecting a broad range of mycoplasma species in one single reaction. When selecting an alternative testing method to the culture-based direct and indirect detection methods the assay of choice has to demonstrate a high sensitivity that is comparable to the conventional testing method and enables objective data interpretation. The most significant factor in the studies to evaluate comparability of nucleic acid-based methods with the culture method is the preparation and characterization of the stocks of mycoplasma species. The quality of mycoplasma stocks used in the comparison studies may significantly impact the outcome and bias the data. Significant research has been performed in establishing criteria for acceptable reference stocks of mycoplasma species.¹⁸ As a part of any comparability study, data should be generated to demonstrate the suitability of the mycoplasma stocks used. Because the standard culture based method allows for amplification of mycoplasma present in the sample, it is important to evaluate the ability of representative mycoplasma species to replicate in relevant cell substrates used in production of protein therapeutics when assessing the suitability of a nucleic acid-based technology.

One of the most common cell substrates used for production of protein therapeutics is Chinese hamster ovary (CHO) derived cells. Our internal data has shown that different species of mycoplasma, including the non-agar cultivable species, can grow to significant levels in cultures of CHO cells. Essentially, this demonstrates that the cell culture process can act as an amplification step for replication of any contaminating mycoplasma and therefore at the point to testing for unprocessed bulk harvest the mycoplasma concentration is significant and further amplification is not necessary. Furthermore, considerations should also be given to factors stated in the European Pharmacopeia chapter 2.6.7.¹⁰ In addition, the alternative testing methods need to be managed throughout product lifecycle and cell culture process changes need to be evaluated for potential impact on assay performance. For example, because these alternate methods are nucleic acid based, the impact of increased cell mass, at the unprocessed bulk harvest stage, or changes to cell culture media composition on assay performance needs to be evaluated. In order to test suitability of these rapid testing methods as an alternative to the agar/broth testing, the comparability of two assays must be demonstrated. As part of this analysis, the following criteria should be tested: limit of detection, specificity, robustness, head-to-head comparison, the correlation of GC/ mL to CFU/mL. Furthermore, it should be demonstrated that the assay is suitable for testing sample volumes of up to 10 mL. Matrix interference may be an obstacle to overcome when a using nucleic acid-based technology. At a high concentration of production cells (up to 1x10⁷cells/mL) inhibition or interference may be observed and could lead to false positive or false negative results. To test for assay interference or inhibition unspiked test samples are assessed during the assay evaluation and compared to negative controls.

A possible solution to circumvent the matrix interference is the introduction of a short sample enrichment step in growth promoting broth prior to using the mycoplasma specific nucleic acid-based assay. The inclusion of the enrichment step reduces the interference and also increases the mycoplasma specific signal by allowing further amplification in broth. The inclusion of an enrichment step as part of an alternative nucleic-acid based assay will not provide a same day turn-around time but it still reduces the testing time from 28 days to approximately 7 days. For assay qualification a variety of mycoplasma species should be tested by spiking them in independent lots of a single commercial protein product harvest. The following mycoplasma species are recommended for assay qualification based on prevalence, significance and growth properties: M. pneumonia, M. orale, A. laidlawii, M. salivarium, M. arginini, M. hyorhinis, M. fermentans and M. hominis. It should be noted that additional species may be used based on specific risk assessments performed on raw materials or other potential sources for a mycoplasma contamination. Furthermore, those species of mycoplasma determined to be slow growers in the specific cell substrate used for production may be considered for this evaluation. For limit of detection comparability, the assessment can be done by spiking different mycoplasma species at multiple dilutions in the test sample. Ideally, the results should empirically demonstrate the limit of mycoplasma detection as determined by the nucleic-acid based method and compared to the standard culture assay. However, it is important to demonstrate that the detection limit of the alternate nucleic acid-based method is well below the expected limit.

Summary

To summarize, the evaluation and selection of a rapid mycoplasma detection method, as an alternative to the standard culture method, should include assessment of comparability, including attributes such as specificity, limit of detection, sample volume, assay performance monitoring. In addition, the suitability of the method for the sample type should be evaluated. A risk assessment for the introduction and detection of a mycoplasma contamination should be performed to ensure replacement of the culture assay does not result in an unforeseen increased risk profile.

Finally, in order to further advance the development of alternative rapid mycoplasma detection methods the biology of relevant mycoplasma species should be continued to be a research topic as well as a better understanding of mycoplasma contaminations. A continuous improvement and development of such rapid detection methods will be of significant benefit to mitigate the risk of contaminations.

References

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7. FDA, Points to Consider in “Characterization of cell lines used to produce biologicals”. Rockville, MD: US Department of Health and Human Services, 1993.
8. US Government, Code of Regulations, Title 21, Subpart D-Mycoplasma 610.30.
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10. EPP, Mycoplasmas. Chapter 2.6.7, 2007. 6th Edition(Strasbourg, France: Council of Europe).
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Author Biographies

Martina Kopp received her PhD in molecular virology from the Friedrich-Löffler-Institut, the Federal Research Institute for Animal Health in Germany, in 2004. She continued her work in the virology field by completing a postdoctoral fellowship at the Center of Hepatitis C Research at the Rockefeller University in New York. Martina Kopp joined the Biosafety Development Group at Amgen in 2011 where she focuses on the risk mitigation of adventitious contaminations in manufacturing processes, the detection and identification of such contaminants as well as the viral clearance assessment for products produced in mammalian cell culture.

Dr. Dehghani’s area of expertise is viral and microbial safety evaluation in biomanufacturing. Currently leads the Cellular Sciences department at Amgen, with groups involved in cell banking, managing critical reagents and development and assessment of new technologies for detection and identification of adventitious contaminants as well as viral clearance assessment studies.