Mycoplasma Quality Control of Cell Substrates and Biopharmaceuticals

• ATCC

Mycoplasma contamination constitutes a serious concern for cell culturists as these bacterial strains are a common cause of cell line contamination affecting roughly 15-35% of cell cultures and endangering almost all aspects of cell physiology^1,2. This is of particular concern for research laboratories and commercial facilities that employ cell lines in the development and manufacture of cell-derived biopharmaceutical products for medical use^3-7. Contamination of cell substrates used in the production of biopharmaceuticals poses a potential safety risk for patients and presents a serious economic risk for manufacturers in the event of batch adulteration or a product recall. To minimize these risks, routine testing for mycoplasma is performed throughout the product manufacturing and development process. In this article, we will discuss the effects of mycoplasma contamination, how this form of adulteration can affect cell-based drug development, and quality control techniques used to detect the presence of mycoplasma contamination.

Mycoplasma are a distinct group of bacterial strains taxonomically ordered under the class Mollicutes. Presently, over 190 species of mycoplasma distributed among humans, animals, insects, and plants are known; of these, only eight are responsible for approximately 95% of cell culture contamination events^8-10. These microorganisms are distinguishable from other bacterial species by their complete lack of a cell wall, which contributes to the accession of nutrients from a host cell via cytoplasmic exchange. Mycoplasma are also known to be one of the smallest free-living forms of bacteria, ranging in size from 0.15 to 0.3 micrometers¹¹. This small size enables mycoplasma strains to escape a number of filtration systems as well as grow to a high concentration in cell culture without resulting in media turbidity or other obvious symptoms. In addition to their small size and lack of a cell wall, mycoplasma strains are characterized by a small genome, which drastically reduces their biosynthetic capabilities, causing them to heavily rely on an exogenous source of cholesterol, amino acids, fatty acids, vitamins, and other catabolites provided by their host or environment. If undetected, mycoplasma contamination can lead to a number of deleterious effects that affect the physiology of cell lines. These include the induction of chromosomal abnormalities, the disruption of DNA and RNA synthesis, changes in membrane antigenicity, the inhibition of cell proliferation and metabolism due to nutrient withdrawal, decreased transfection rates, changes in gene expression profi les, and cell death^1,8,12.

Figure 1. Costs of Mycoplasma Contamination

For years, cell lines have been used to produce a number of biological products for therapeutic or medicinal use, including cytokines, viral vaccines, monoclonal antibodies, growth factors, and immunologic modulators. Because of the extensive effects mycoplasma strains have on cell line physiology, metabolism, and gene expression, mycoplasma contamination of cell lines used in the biopharmaceutical industry presents a significant safety and economic concern. For the production of biopharmaceuticals, mycoplasma contamination can result in decreased production and may aff ect the quality of the product. Further, if the mycoplasma contaminant is present in the final biological product, it can directly affect patient safety by potentially causing disease^11,13. Once mycoplasma contamination is discovered, it can result in significant costs in the time and expense associated with the loss of impacted batches, investigation into the source of contamination, and decontamination of the facility¹³. In turn, this can result in the loss of months of invaluable production time and tens or hundreds of thousands of dollars in associated expenses¹⁴.

To mitigate the risk of mycoplasma contamination, microbiological monitoring is required throughout the manufacture of biologicals produced in cell substrates^13,15-18. The most frequently used detection methods include direct culture, Hoechst DNA staining, and PCR-based testing. For the production of biopharmaceuticals, the recommended protocols for mycoplasma testing typically rely on the use of culturebased approaches that assess the presence of viable cells. However, this form of testing is fairly laborious and necessitates up to 28 days for completion. This extensive time commitment presents a problem in that some products may have short half-lives whereas other intermediate products may need to be processed quickly. Further, the sensitivity of the culture assay may be aff ected by the quality of the media components, inconsistencies in media preparation, handling of the mycoplasma culture, interpretation of results, or the lack of mycoplasma reference standards¹³.

An alternative approach to mycoplasma detection is through PCR-based testing, which has proven to be a rapid and reliable alternative when validated as a comparable method of detection. This molecular-based method is ideal for research laboratories as it is easy and quick to set-up and analyze. Further, it is highly sensitive, specific, reliable, and fairly cost-effective¹³. However, it must be noted that the primary drawback to this approach is the inability to distinguish viable and nonviable mycoplasma. Generally, this particular method is based on the detection of the 16S ribosomal RNA for common mycoplasma contaminants. To ensure the specificity of this method, primers that are broad enough to recognize Mycoplasma, Ureaplasma, Spiroplasma, and Acholeplasma species, as well as specific enough to prevent the amplification of 16S sequences belonging to other non-mycoplasma bacterial contaminants, are needed. To achieve detection over a wide range of mycoplasma species, a touchdown PCR approach can be used. This approach employs a high annealing temperature in the initial cycle that decreases with subsequent cycles to increase the likelihood of primers binding to the specific targets, and reducing the likelihood that non-specific targets will be amplified. Using this method, mycoplasma contamination is easily recognized as a distinct PCR product. Alternatively, quantitative PCR based methods have also been developed for the detection and identification of mycoplasma^13,19-21. This latter approach allows for the quantitative, high-throughput detection and identification of mycoplasma species, without post-amplification handling¹³.

Because the recommended “gold-standard” mycoplasma testing protocol for biopharmaceutical companies requires the use of a culturebased approach to detect viable mycoplasma, the implementation of an alternative method, such as the PCR-based approach, must show equivalency or superiority to the approved testing procedures, particularly with regard to the limit of detection²². However, comparing the limit of detection of a nucleic acid-based testing method to a culture-based approach poses signifi cant challenges with regard to a direct comparison of differing biological measurements. For an impartial assessment of these methods, the use of well-characterized mycoplasma reference materials that represent common cell culture contaminants while demonstrating a high percentage of viable cells and a low degree of aggregation has been recommended^13,22. Because molecular approaches typically rely on the detection of genomic identifiers from mycoplasma strains, regardless of cell viability, the presence of excessive amounts of dead or aggregated material may result in the overestimation of sensitivity^13,22. Thus, the presence of a signifi cant amount of dead or aggregated cellular material can skew the estimated limit of detection of a nucleic acid-based approach. These features can be assessed for reference strains though evaluating the ratio of genomic copies (GC) to colony forming units (CFU). Here, mycoplasma reference strains exhibiting the lowest possible GC/CFU ratio is indicative of high cell viability and a low degree of aggregation¹³. In addition to the use of titered mycoplasma reference standards in the direct comparison of cultureand molecular-based methods, quantitative mycoplasma DNA reference materials can be used as external controls in inclusivity/ exclusivity testing and establishing limits of detection. These latter reference materials provide an eff ective approach toward the development and evaluation of novel mycoplasma detection assays as well as directly comparing the sensitivity of different molecular-based detection systems.

Figure 2. Advantages and disadvantages of common microbial monitoring techniques.

Overall, mycoplasma contamination is a major concern for biopharmaceutical producers as it represents a potential safety hazard and economic risk. Routinely testing for contamination throughout the manufacturing process through culture- or molecular-based detection methods can help minimize these risks and may pinpoint any potential sources of contamination. To aid in the development or evaluation of mycoplasma quality control procedures, validated titered mycoplasma reference strains and quantitative mycoplasma DNA reference materials can be used. These products are ideal for comparing test methods and can be used as external controls in evaluating method sensitivity and specificity.

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