Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques☆
Introduction
Mycoplasmas (commonly used trivial name for microorganism of the class Mollicutes) are known to be one of the smallest free-living forms of bacteria that are broadly distributed in nature [1]. Based on their genetic background and molecular features, mycoplasmas were hypothesized to originate evolutionarily from gram-positive eubacteria [1], [2]. As a result of degenerative evolution, mycoplasmas irreversibly lost the bacterial cell wall and some metabolic pathways present in phylogenetically closely related bacteria (Lactobacilli, Clostridia, and Streptococci) [2]. These structural and biochemical deficiencies make mycoplasma replication largely dependent on the nutritional compounds provided by their natural hosts or in the environment they inhabit [1], [3]. Thus, Ureaplasma species colonizing the genitourinary tract are able to efficiently utilize urea as a source of energy [4], whereas the hemotrophic mycoplasmas (hemoplasmas) are able to replicate only in the bloodstream of their hosts [5]. The majority of known mycoplasmas are host-specific commensals colonizing a wide range of plant, insect, reptilian, avian, mammalian, and humans [1]. Nevertheless, a few mycoplasma species are pathogenic for their hosts and can, under certain conditions, cause diseases in the hosts [1], [6].
Due to their broad distribution in the nature, mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are found to frequently be the cause of contaminations of cell substrates as well as raw materials commonly used for manufacture of a variety of biological and therapeutic products [7]. Mycoplasma contamination of cell substrates inevitably results in a dramatic alteration of biological characteristics of the contaminated cells [7]. This in turn causes manufacturing and safety problems for biologics produced from the contaminated cells. Most mycoplasmas from warm-blooded animals have a potential to efficiently grow in cell substrates. Mycoplasma contamination, even at very low levels, at the initial steps of manufacture, generally results in the loss of the whole batch of the product due to poor cell growth or regulatory safety concerns. Regardless of an observable impact during manufacturing, the detection of mycoplasma would necessitate the time and expense of the impacted batches being discarded and the facility being appropriately cleaned. The detection of mycoplasma contamination during manufacture of even one batch of product, therefore, represents a serious risk to the facility and other products manufactured in that facility. To mitigate the risk of mycoplasma contamination of cell substrates and to ensure the safety of cell-derived biologics, all current regulation documents do not permit the use of mycoplasma contaminated cell substrates, starting materials or crude harvests for manufacture of biological and therapeutic products [8], [9], [10], [11]. According to key regulatory guidance documents, the purity of cell substrates should be assured by using well-defined detection methods recommended by regulatory agencies in guidances or in compendia. It is necessary to note that in general the recommended protocols for mycoplasma testing rely on the use of approaches based on culturing of viable mycoplasmas in broth or indicator cell line. Needless to say, these testing protocols are laborious and usually necessitate a long time (up to 28 days) for the test completion. The latter limitation significantly complicates the application of recommended methods for testing of some novel biological and therapeutic products, which possess short shelf-lives not exceeding the overall testing time (e.g., products used for tissue regenerative therapy). This time frame is also inconvenient for the mycoplasma testing of intermediate products such as bioreactor harvests, which in some cases need to be rapidly processed. Thus, development and application of alternative methods based on latest achievements in molecular technologies are important and urgent needs for safety testing of novel cell-derived biologics. The purpose of the current review is to analyze advantages that alternative molecular technologies offer for improving mycoplasma testing and describe their limitations in terms of critical issues that impact their adequate evaluation, validation, and comparison to conventional mycoplasma testing methods.
Section snippets
Brief overview of requirements of mycoplasma testing regulations and methods currently recommended for mycoplasma testing
Methodologies acceptable for mycoplasma testing during manufacture of different biological products are described in detail in several national and international regulatory and compendial documents [8], [9], [10], [12], [13]. In principal, the conventional mycoplasma testing procedure includes two main assays, i.e., the agar/broth media and indicator cell culture assays. The agar/broth media procedure is meant to detect so called “cultivable” mycoplasmal agents capable of growing in currently
Alternative technical approaches for development of mycoplasma testing methods
Recent progress in molecular methods targeting different genetic and biochemical targets of bacteria has resulted in attempts to apply these new technologies that offer more simple and rapid ways of mycoplasma testing. Several different methodological approaches based on the detection of structural or functional elements of mycoplasmal cells have been proposed for development of rapid alternative mycoplasma testing methods. Below we describe some selected techniques that are believed to have a
Nucleic acid testing (NAT) methods
In the last two decades, NAT-based methods have become a powerful analytical tool used in many fields of molecular biology, diagnostics, and medicine. Recently, NAT methods have been seriously considered for mycoplasma detection during biologics manufacture. The main advantage of the NAT-based methods is that they allow for rapid, sequence-specific, and highly sensitive detection of the presence of target DNA or RNA in complex biological samples. Different modifications of NAT assays have also
Endpoint PCR detection methods (regular PCR, touchdown PCR, reverse transcription-PCR)
The NAT methods most broadly used for detecting mycoplasmas (as well as other bacterial and viral agents) are, to a large extent, endpoint PCR assays. This approach relies on an instrumental detection of the final DNA products (amplicons) obtained after the completion of PCR reaction. This can be accomplished using different DNA detecting methods (e.g., fluorescent staining of the DNA after agarose/acrylamide gel electrophoresis, chromatographic or capillary electrophoresis DNA detection
Real time quantitative PCR
Quantitative PCR (qPCR) approaches have been applied for detecting multiple adventitious agents in biologics [38], including mycoplasma contamination in cell cultures [32], [39]. The qPCR technique is based on measurements, in a real time manner, of the fluorescence increase during a PCR reaction due to either direct intercalation of fluorescent dyes (e.g., SYBR Green I and II) into PCR-synthesized DNA amplicons [40], or from quencher-linked fluorogenic probes that are cleaved during the PCR
Touchdown PCR assays
The efficient use of PCR for detection of mycoplasma contamination can, in some cases, be compromised by mispriming (non-specific binding of PCR primers to DNA templates) during PCR reactions. This mispriming can result in spurious DNA bands which often complicate an interpretation of the PCR results. The risk of mispriming significantly increases with the use of universal degenerate primers, which are designed to have broad specificity to cover all possible genetic variations among targeted
Reverse transcription-PCR (RT-PCR) assays
RT-PCR methods based on detection of bacterial 16S rRNA and other RNA markers have been shown to be useful rapid and sensitive detection of mycoplasmas in biological samples [48], [49]. In contrast to the 16S rRNA genes present at 1 to 2 copies per mycoplasma cell [1], 16S rRNA transcripts are present at by 102–103 copies and above [33], [50]. Direct side-by-side comparison of RT-PCR and PCR targeting the 16S rRNA and the 16S rRNA gene, respectively, has demonstrated that RT-PCR was able to
Microarray based methods for detection and identification
Microarray technology permits not only the detection, but also the identification of mycoplasma contaminants to the species level. This is possible due to the ability of microarray oligonucleotide species-specific probes, which are attached to or synthesized on a solid microarray surface, to specifically recognize targeted unique sequences in mycoplasma genomes [54], [55]. The extensive analysis of the 16S–23S rRNA intergenic transcribed spacer (ITS) region between the 16S rRNA and 23S rRNA
Massively parallel sequencing (MPS)
MPS is a powerful alternative method for high-throughput sequencing. It can provide a great opportunity for the simultaneous detection and identification of multiple adventitious agents in complex biological samples even without prior knowledge of the nature of the agents [58], [59]. MPS combined with the use of random priming PCR has been shown to be able to detect adventitious agents in cell substrates or in virus seed stocks [58]. While most MPS platforms are at the research and development
Immunologic tests
Immunologic (serological) tests have been developed for the use mainly for the diagnosis of mycoplasmal infections in human and animals as well as for the identification and characterization of novel Mycoplasma species [62], [63], [64]. The application of serological tests for the detection of mycoplasmas in cell substrates and biologics is very limited due to the lack of commercial antibodies capable of recognizing all possible mycoplasma contaminants. Universal antibodies are difficult to
Enzyme-based methods
Biochemical tests that exploit mycoplasma-specific enzymatic activity are also considered as a potential alternative approach for mycoplasma testing. It is important to note that to provide suitable sensitivity of detection, the target enzyme should be present in significant quantity in mycoplasma cells and have a long half-life period. Enzymes from the mycoplasmas’ energy-generating systems meet both aforementioned criteria. Mycoplasmas have truncated respiratory pathway, lacking cytochromes,
Mycoplasma testing using a recombinant cell line
Recombinant cell-based approaches have also been utilized for designing mycoplasma testing methods. The assay relies on the ability of the innate immune system, in particular Toll-like receptor 2 (TLR2), to recognize mycoplasmal lipoproteins (LP) [72]. Upon recognition of mycoplasmal LP, TLR2s induce a signaling cascade that leads to the activation of the cellular transcription factor NF-κB. The recently developed PlasmoTest assay is based on the use of genetically engineered mammalian cell
Biological enrichment of mycoplasmal contaminants
Biological enrichment is a promising approach to enhance NAT assay sensitivity due to its ability to significantly increase the amount of viable mycoplasma cells and consequently their DNA/RNA material to levels readily for reliable detection by routine NAT or other mycoplasma testing methods. The Mycoplasma species known to be common cell line contaminants can be easily enriched using either cultivation in appropriate broth media or by co-cultivation with permissive mammalian cell lines.
Physical concentration of mycoplasmal contaminants prior their detection
Another possible approach to enhance the sensitivity of alternative methods is based on physical concentration of the targeted mycoplasma material (either mycoplasma cells or mycoplasma-specific molecules such as bacterial proteins, DNA or RNA). The concentration of mycoplasma cells from culture supernatant via centrifugation is a straightforward approach that may be able to increase the sensitivity of detection of mycoplasma contamination. Concentration of Mycoplasma spp. from supernatant by
Evaluation of alternative methods: some important issues for consideration
The development and implementation of rapid mycoplasma testing methods are an important technical and regulatory issue. Many people believe that there is an unmet need in the biological, therapeutic, and cell therapy products for particular applications that can be addressed by rapid mycoplasma testing protocols. It is important to note that the application of alternative methods and acceptance criteria for the methods’ results should always be balanced against a consideration for the quality
Sample volume
The inherent advantage of culture methods is their ability to analyze relatively large sample volumes which helps to ensure efficient and sensitive detection of mycoplasma contamination. Thus, the methods described in the EP, USP, JP and FDA guidances permit the analysis of up to a 10 mL sample volume. In general, the methods described in the aforementioned guidances are very similar with only minor differences as described previously [77]. Therefore, the culture based methods, in general,
The viability of the mycoplasma reference standards (GC/CFU ratio): a critical factor affecting method comparison
Because NAT methods measure the presence of mycoplasmal nucleic acid (NA), not live mycoplasma cells, their intrinsic sensitivity can be expressed in the number of genomic copies (or genomic equivalents) that are required to provide confident mycoplasma NA detection in the test reaction. It is important to remember that the LOD of the mycoplasma testing assay (including the sample preparation step) should also be expressed in the concentration of genomic copies (DNA or RNA) in the original
Preparation and characterization of mycoplasma reference strains for comparability study
To ensure the performance of mycoplasma culture testing methods, the current regulatory and compendial documents recommend using preparations of defined mycoplasma species with well established titer values. In general species that represent strains with different nutritional preferences and that reflect potential contaminants during manufacture of biological products. Another important requirement for reference strains is their low passage (less than 15) history after strain isolation [20].
Mycoplasma testing using alternative methods: the use of a risk-based approach
As described above, the presence of mycoplasma in biological therapeutics and process intermediates represents a serious safety and manufacturing risk, and it is, therefore, essential that mycoplasma be tested for at appropriate stages in the manufacturing process, such as in the cell substrates and crude harvests. Conventional mycoplasma testing procedures, such as the agar/broth media and indicator cell culture assays, while considered to be the “gold standard” are not without their
References (78)
- et al.
Beware of mycoplasmas
Trends Biotechnol
(1993) - et al.
Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction
Methods Enzymol
(1987) DNA probes and PCR in diagnosis of mycoplasma infections
Mol Cell Probes
(1994)- et al.
Validation and international regulatory experience for a mycoplasma touchdown PCR assay
Biologicals
(2010) - et al.
Validation of a NAT-based Mycoplasma assay according European Pharmacopoeia
Biologicals
(2010) - et al.
Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins
Biologicals
(2004) - et al.
Validation of a PCR method for the detection of mycoplasmas according to European Pharmacopoeia section 2.6.7
Biologicals
(2010) - et al.
Molecular methods for the assessment of bacterial viability
J Microbiol Methods
(2003) - et al.
Broad-range real-time PCR assay for the rapid identification of cell-line contaminants and clinically important mollicute species
Int J Med Microbiol
(2009) - et al.
Typing of Mycoplasma pneumoniae by nucleic acid sequence-based amplification, NASBA
Mol Cell Probes
(1996)
Evaluation of the LightCycler 1536 Instrument for high-throughput quantitative real-time PCR
Methods
Cross comparison of rapid mycoplasma detection platforms
Biologicals
Type-specific amplification of viral DNA using touchdown and hot start PCR.
J Virol Methods
Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction
J Microbiol Methods
A reverse transcription-PCR assay to detect viable Mycoplasma synoviae in poultry environmental samples
Vet Microbiol
Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition
J Struct Biol
Massively parallel sequencing, a new method for detecting adventitious agents
Biologicals
Rapid detection of mycoplasma in continuous cell lines using a selective biochemical test
Leuk Res
Short communication: the effect of centrifugation and resuspension on the recovery of Mycoplasma species from milk
J Dairy Sci
Evaluation of cell substrates for the production of biologicals: revision of WHO recommendations
Biologicals
Molecular biology and pathogenicity of mycoplasmas
Microbiol Mol Biol Rev
Phylogeny of mycoplasmas
Structure and function in mycoplasma
Annu Rev Microbiol
Catabolism in mollicutes
J Gen Microbiol
Hemotrophic mycoplasmas (hemoplasmas): a review and new insights into pathogenic potential
Vet Clin Pathol
Identification and treatment of pathogenic mycoplasmas
Quality control of individual components used in Middlebrook 7H10 medium for mycobacterial susceptibility testing
J Clin Microbiol
Quality-control testing of mycoplasma medium
Methods Mol Biol
Comparison of various atmospheric conditions for isolation and subcultivation of Mycoplasma hyorhinis from cell cultures
Antonie Van Leeuwenhoek
Factors influencing microbiological assay of cell-culture mycoplasma
In Vitro
Survival of frozen mycoplasmas
Appl Microbiol
Effect of storage conditions on detection of mycoplasma in biopharmaceutical products
In Vitro Cell Dev Biol Anim
Establishment of European pharmacopoeia Mycoplasma reference strains
Pharmeuropa Bio
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The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.