Preparation of reference strains for validation and comparison of mycoplasma testing methods

Authors


  • Disclaimer: 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.

Alena Dabrazhynetskaya, Laboratory of Methods Development, Division of Viral Products, Office of Vaccine Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, HFM-470, 1401 Rockville Pike, Rockville, MD 20852, USA. E-mail: alena.dabrazhynetskaya@fda.hhs.gov

Abstract

Aims:  To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)-based methods in comparison to the conventional microbiological methods.

Methods and Results:  A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co-cultured with suspension of Chinese hamster ovary (CHO) cells.

Conclusions:  Tested mycoplasma strains harvested at the exponential-early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study.

Significance and Impact of the Study:  This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT-based mycoplasma testing methods.

Introduction

Mycoplasmas (trivial name assigned for the micro-organisms of the class Mollicutes) are free-living micro-organisms, which lack the cell wall and have one of the smallest genomes (Razin et al. 1998). Because of their broad distribution in nature, mycoplasmas are frequently found to contaminate cell substrates and other starting materials used for manufacture of cell-derived biologics and pharmaceutical products (Rottem and Barile 1993). To mitigate a potential risk of contamination and thus to ensure the quality and safety of the biologics, regulatory agencies worldwide require manufacturers to demonstrate the absence of mycoplasma contamination at specified production stages of cell-derived biologics (FDA 1993, 2010; US Federal Government 2010). According to the recommendations of the regulatory documents, the mycoplasma testing is primarily based on the use of the agar/broth culture test (for detection of cultivable mycoplasma strains) and the indicator cell culture assay (for detection of fastidious strains that are noncultivable in published media formulations). Despite the demonstrated efficiency of aforementioned assays, the overall recommended mycoplasma testing procedure is time consuming and requires specific expertise. The long testing time frame (as minimum as 28 days) required for assay completion may not be acceptable for the testing of some novel biological and therapeutic products with short shelf-lives not exceeding the overall testing time (e.g. products used for tissue regenerative therapy) and may also create an inconvenience with respect to the testing of intermediate products (e.g. bioreactor harvests), which in some cases need to be rapidly processed. Because of these limitations of the conventional mycoplasma testing methods, there has been increasing interest in applying novel emerging molecular technologies for the development of rapid and sensitive alternative mycoplasma testing methods (PDA 2010; Volokhov et al. 2011). However, prior to the implementation in testing programme, alternative methods must go through extensive evaluation and validation to demonstrate their capability to provide a limit of detection (LOD) comparable or superior to that of the conventional culture methods. This comparison may face serious methodological and technical challenges if compared methods are based on readouts of innately different biological features. For example, alternative NAT-based methods measure the amount of nucleic acid (i.e. DNA or RNA) regardless of viability of bacterial cells, whereas culture-based methods are able to detect only viable cells. As result, the LOD of NAT-based methods is generally expressed as the lowest number of genomic copies (GC) of target organism that can be confidently detected in specimen, while the LOD of conventional methods is measured as the lowest number of viable micro-organisms generating the colony forming units (CFU) on the surface of a solid medium. It is prudent to expect that the ratio between GC and CFU in mycoplasma samples may vary in wide range depending on several factors including an inherent propensity of specific mycoplasma strain to form filamentous structures or aggregates (Maniloff and Morowitz 1972; Windsor et al. 2010), cultivation conditions and phase of mycoplasma culture growth (Anderson et al. 1965; Razin and Cosenza 1966; Maniloff 1970; Rosengarten and Kirchhoff 1989; Folmsbee et al. 2010), protocols for processing of mycoplasma reference samples during testing (e.g. cooling/warming rates for freezing/thawing procedures, etc.) (Kim et al. 1966; Raccach et al. 1975) and other factors. It is reasonable to expect that lower values of GC/CFU ratios in bacterial cultures are likely to reflect a higher viability of bacterial cells and a lower extent of their aggregation.

Thus, the use of mycoplasma culture samples with low GC/CFU ratios for methods comparability study is a necessary requirement when mimicking a situation of potential mycoplasma contamination during manufacture and can enable an adequate assessment of relative LOD of NAT-based methods compared against that of the conventional microbiological methods. Therefore, the development and preparation of a panel of viable mycoplasma reference strains with low CG/CFU ratios is of high importance for evaluation and validation of alternative NAT-based mycoplasma testing methods.

Materials and methods

Mycoplasma strains

The following mycoplasma strains (all from ATCC, Manassas, VA) were used in the study: A. laidlawii (ATCC 14089), Myc. gallisepticum (ATCC 19610), Myc. arginini (ATCC 23838), Myc. fermentans (ATCC 19989), Myc. pneumoniae (ATCC 29085) and Myc. orale (ATCC 23714).

Depending on species, mycoplasmas were grown using ATCC 243 or Hayflick medium (broth or agar), supplemented with either 0·5% (w/v) d-glucose or l-arginine (Sigma-Aldrich Inc., St Louis, MO, USA) at 37°C for 6–14 days. Except for Myc. fermentans, which was grown under anaerobic conditions (GasPak EZ anaerobe pouch system; BD Biosciences, Franklin Lakes, NJ, USA), all other mycoplasma species were grown under aerobic conditions in the presence of 5% CO2.

Chinese hamster ovary (CHO) cell line

CHO cells (cell line hCBE11; ATCC PTA-3357, Manassas, VA, USA) were maintained at 37°C and 5% CO2 in HyClone PF CHO liquid soy broth (HyClone Laboratories Inc., Logan, UT, USA), supplemented with L-glutamine and 10% heat-inactivated foetal bovine serum (Gibco, Invitrogen Inc., Frederick, MD, USA).

Preparation of mycoplasma inoculum cultures (IC)

Starting mycoplasma cultures were prepared from stocks frozen at −80°C in broth containing 15% (w/v) of glycerol as a cryoprotectant. The optical density (turbidity) of mycoplasma cultures was monitored at 600 nm by using UltaSpec3100pro spectrophotometer (Amersham Biosciences, UK). The titre of mycoplasma cell cultures was determined by plating 10-fold serial dilutions of culture onto ATCC 243 solid medium.

The starting cultures of mycoplasma species (except for Myc. pneumoniae and Myc. orale) for obtaining of ICs were subcultured in broth from the frozen stocks (at split ratio 1 : 100) and incubated for c. 24 h (Fig. 1). The procedure was repeated for three consecutive times to ensure a high viability of ICs used in kinetics experiments. Obtained IC titres were between 108 and 109 CFU ml−1 for all selected strains.

Figure 1.

 The strategy of kinetics experiment for assessment of GC/CFU ratios during mycoplasmas incubation in broth: step I – preparation of mycoplasma inoculum culture (IC); step II – time point samples collection, step III – analyses of culture samples on GC and CFU content.

For Myc. pneumoniae and Myc. orale experiments, low initial inoculation titre from the starting mycoplasma cultures were used to minimize the carry-over of significant amounts of nonviable cells that could affect the GC/CFU ratio assessment.

Mycoplasma growth kinetics experiments

The kinetics experiments for A. laidlawii, Myc. gallisepticum, Myc. arginini and Myc. fermentans were started at an initial mycoplasma titre c. 104 CFU ml−1. In case of Myc. orale and Myc. pneumoniae, growth media were inoculated with initial titre c. 102 CFU ml−1. Time point samples were collected from mycoplasma cultures within 5–22 days of incubation, depending on species growth characteristics, and then used for analyses (see Fig. 1). All culture samples were collected in duplicates to ensure the reproducibility of the DNA isolation procedure.

Co-cultivation of mycoplasmas with a suspension culture of CHO cells

The CHO cells of high viability (about 98%), assessed using trypan blue staining, were transferred from 75-cm2 flasks (Corning Inc., Corning, NY, USA) into ProCulture spinner flasks (Corning Inc.) with HyClone medium to obtain the initial density of 1–3 × 105 cells ml−1. CHO cells were cultured for 24 h at 37°C in the presence of 5% CO2 with a constant magnetic spinner agitation at 80 rev min−1. After the culture cell density reached the level of 2–6 × 105 cells ml−1, CHO cells were infected with mycoplasmas at the initial infection titres of ≥1 × 103 CFU ml−1. The co-cultures of CHO cells and mycoplasmas were incubated for up to 8 days.

Collection of cell culture samples

Depending on the growth phase and expected titre of mycoplasmas in the cultures, the time point sample volumes varied from 1 to 20 ml. To achieve the equal medium background, each sample was adjusted (if necessary) to 20 ml with plain broth. Mycoplasma cells were pelleted at 15 700 g for 30 min. The speed and the time of centrifugation were optimized experimentally to ensure an efficient collection of mycoplasma cells from a 20-ml sample volume. The preliminary experiments with the culture A. laidlawii showed that the use of the aforementioned centrifugation conditions resulted in a complete (>99·9%) removal of mycoplasma cells from the supernatant. The efficiency of pelleting was monitored by measuring the titre of culture before and after centrifugation by plating. It was also shown that the centrifugation did not affect the viability of pelleted A. laidlawii cells. Pellets were re-suspended in 1·5 ml of 1 × PBS buffer and spun down at 15700 g for 30 min. Final mycoplasma pellets were stored at −20°C until genomic DNA extraction.

Species-specific mycoplasma reference DNAs used for the preparation of qPCR standard curves and DNA copy number calculation were prepared from 1–2-ml aliquots of each mycoplasma species culture harvested at early stationary growth phase. To reduce the amount of nonmycoplasmal DNA background in the culture samples, each aliquot was adjusted to 20 ml using PBS buffer. The cells were then pelleted, washed once with PBS and collected as described earlier. Concentration of mycoplasma genomic DNA in reference DNA samples was measured by NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA).

The medium (background) DNA isolated from 20 ml of plain broth using the same protocol was used as an additional control in each experiment and also was spiked into the serial dilutions of standard mycoplasma DNA used to prepare a qPCR standard curve.

Similar procedures were used for processing of co-cultured samples. Briefly, the samples adjusted to 20 ml with CHO growth medium were vigorously vortexed for a few seconds to breakdown cell aggregates; and mammalian cells were removed by the low-speed centrifugation at 100 g for 1 min. Mycoplasma cells were pelleted from the clarified supernatant by the second step of centrifugation at 15 700 g for 30 min, followed by re-pelleting of mycoplasma cells from PBS at 15 700 g for 30 min. The mycoplasma pellets were stored at −20°C until their use for DNA isolation.

Isolation of mycoplasma genomic DNA

A. laidlawii, Myc. gallisepticum, Myc. arginini and Myc. fermentans genomic DNAs were isolated from the frozen pellets using DNeasy blood and tissue kit (Qiagen, Valencia, CA), according to the manufacturer’s protocol with some modifications. Briefly, a pellet was re-suspended in 180 μl of ATL buffer and treated subsequently with proteinase K and RNase A for 5 min at room temperature. After addition of 200 μl of lysis buffer AL, the mixture was incubated in thermomixer (Eppendorf, Inc., Edison, NJ, USA) at 72°C with agitation at 850 rev min−1 for an hour. Two hundred microlitres of 99·5% ethanol was added to each lysate prior to loading onto the QIAmp DNA mini column. The columns were washed consecutively with AW1 and AW2 washing buffers as recommended by manufacturer. DNA was eluted from the column by using three consecutive elutions with 100 μl of AE buffer with 10–15 min incubation prior to the subsequent column centrifugation. The amounts of DNA in final samples were assessed using NanoDrop 2000 (Thermo Fisher Scientific). DNA samples were stored at −20°C before use.

The extraction of Myc. pneumoniae and Myc. orale genomic DNA was performed using Qiagen MagAttract DNA Blood Midi Kit M48 (Qiagen, Valencia, CA, USA), according to the manufacturer’s recommendations.

Quantitative PCR

The amplification of the 16SrRNA gene fragments of A. laidlawii, Myc. gallisepticum, Myc. arginini and Myc. fermentans using universal mycoplasma primers: 5′-GGCGAATGGGTGAGTAACACG-3′ (forward) and 5′-GGATAACGCTTGCGACCTATG-3′ (reverse) was performed as described previously (Eldering et al. 2004). The use of universal mycoplasma-specific primers complementary to the conserved regions within the mycoplasma 16S rRNA genes sequences allowed us to efficiently amplify the target regions of all tested species and observe the syntheses of amplicons of 438–470 bp in length (depending on species). The qPCR was performed using a QuantiTect SYBR Green PCR kit (Qiagen, GmbH) with HotStar Taq® DNA polymerase and SYBR Green I fluorescent dye according to the manufacturer’s protocol with minor modifications. A standard qPCR mixture (50 μl) contained 25 μl QuantiTect SYBR Green PCR Master Mix, 0·3 μmol l−1 of each forward and reverse primers, ≤0·5 μg per reaction template DNA and RNase-free water up to 50 μl. qPCR was performed using 7900HT Fast Real-time PCR unit (Applied Biosystems Inc., Foster City, CA, USA) with Sequence Detection System (SDS) at the following conditions: 1st step – DNA denaturation and Taq polymerase activation at 95°C for 15 min; 2nd step – 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 s and elongation at 72°C for 1 min; 3rd step – final elongation at 72°C for 5 min; and 4th step – dissociation curve analyses for obtained PCR products. The number of mycoplasma genomic copies in each sample was calculated based on comparison of obtained Ct value with the standard curve. All qPCRs, including standards and their dilutions, were performed in duplicates. qPCR data were analysed using 7900HT SDS 2.3 software. The copy numbers were determined as an average from two PCRs.

The synthesis of amplicons with expected sizes during qPCR was additionally confirmed by 1% agarose gel electrophoresis in 1 × TBE buffer. DNA bands were visualized using a UV light after staining of the gel with ethidium bromide.

Quantification of Myc. pneumoniae and Myc. orale was conducted by qPCR utilizing the TaqMan® technology (Applied Biosystems, Inc., Foster City, CA) that also targeted a conserved 16S rRNA region of the mycoplasma genome. The in-house developed assay used six forward primers (BREL196 5′-TGACGGTACCTTGTTAGAAAGC;

  • BREL198 5′-TTGACGGTACCTTATTAGAAAGC;

  • BREL241 5′-TTGACTGTACCATTTGAATAAGTA;

  • BREL243 5′-TGACGGTACCTTGTCAGAAAGC;

  • BREL245 5′-TTTGACTGTACCATTTTGAATAAGT;

BREL301 5′-GTTTGACGGTACCATATGAATAAG), three reverse primers (BREL200 5′-GATAACGCTCGCCCCCTA; BREL246 5′-AATCCGGATAACGCTTGCG;

BREL300 5′-AATCCGGATAACGCTTGCA) and one sequence-specific probe [BREL032 5′-ACTATGTGCCAGCAGYCGCGG labelled with FAM (fluorophore) and TAMRA (quencher)]. The qPCR profile included: 1st step – UNG (Uracil-N-glycosylase) incubation at 50°C for 1 min; 2nd step – DNA denature and Taq polymerase activation at 95°C for 10 min; 3rd step – 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 1 min and elongation at 72°C for 1 min. The fluorescence was detected using the ABI Prism 7700/7900 instrument using the SDS software. The mycoplasma copy equivalent was calculated by comparing the sample to the standard curve. Included in the PCR were the negative controls, negative extraction controls, six-log dynamic range standard curves and extraction recovery controls. The copy numbers were determined as an average of three PCRs.

Results

Selection of mycoplasma reference strains for methods evaluation

All mycoplasma species used in this study and their characteristics are presented in Table 1. Selected mycoplasma strains were isolated from different host origins (human, avian, animal) and represent phylogenetically distant species (Razin et al. 1998). Some of those species are reported as major contaminants of cell banks, production cell lines and the other cell substrates (Rottem and Barile 1993; Stormer et al. 2009). All these species are recommended by the EP as reference strains for validation of novel NAT-based methods (EP.6.1 2008). The US Pharmacopeia also recommends five species from this list as suitable quality control strains to monitor the performance of mycoplasma testing procedure (USP 2010).

Table 1. Mycoplasma species used in the study
SpeciesATCC no.Host originPrevalence of contamination (%)References
Mycoplasma orale23714Human20–40Uphoff et al. (1992)
Mycoplasma arginini23838Bovine20–30Rottem and Barile (1993)
Mycoplasma fermentans19989Human10–20Uphoff and Drexler (2001)
Mycoplasma gallisepticum19610Avian  0·2Uphoff and Drexler (2002)
Mycoplasma pneumoniae29085Human2Stormer et al. (2009)
Acholeplasma laidlawii14089Bovine5–20Uphoff et al. (1992)

Growth of mycoplasma reference strains in broth

The titres of A. laidlawii, Myc. fermentans, Myc. arginini and Myc. gallisepticum cultures were monitored within 144 h of incubation in broth. The obtained growth curves of four species are shown in Fig. 2. Myc. fermentans, Myc. arginini, A. laidlawii and Myc. gallisepticum showed typical shape of bacterial growth curve.

Figure 2.

 Growth curves of Acholeplasma laidlawii (○), Mycoplasma fermentans (bsl00066), Mycoplasma arginini (bsl00001) and Mycoplasma gallisepticum (◊) cultures incubated in broth within 144 h.

Although the starting titre of these tested mycoplasmas was c. 104 CFU ml−1, the growth kinetics of individual species differed significantly (see Fig. 2). Thus, Myc. arginini and Myc. fermentans showed a very rapid culture growth under used conditions and mounted to the stationary phase 24 h postinoculation (p.i.), whereas A. laidlawii and Myc. gallisepticum reached maximum titres only 36 and 48 h p.i., respectively (Fig. 2). We observed a rapid titre reduction (death phase) for Myc. arginini and Myc. fermentans after 48 h of cultivation, while A. laidlawii and Myc. gallisepticum showed slower titre decline during the late time points (Fig. 2).

Quantification of mycoplasma genomic copy number by qPCR

The numbers of genomic copies (genomic equivalents) in mycoplasma culture samples collected at different time points were accurately assessed using the quantitative PCR as described in details in Materials and methods. The analysis of qPCR products, amplified using control DNA isolated from a plain medium used for mycoplasma growth (medium-related DNA), revealed the synthesis of amplicons with the melting temperature close to the mycoplasma-specific products after 22 cycles of qPCR (Fig. 3). The data presented in Fig. 3 show the effect of the medium-related DNA on resulting Ct values obtained for serial dilutions of A. laidlawii standard DNA. The use of serial dilutions of standard mycoplasma DNA (see Materials and methods) demonstrated a good linearity of qPCR assay in the range of eight logarithms of mycoplasmal DNA concentrations. However, the addition to reaction mixtures of medium-related DNA in quantities equal to that we had in all tested time point DNA samples resulted in a substantial loss of the curve linearity after the 22nd cycle of qPCR, and thus reduction of the linearity range from eight to five logarithms (Fig. 3). Therefore, to ensure the accuracy of GC quantification of analysed DNA samples and monitor the dynamic range of the standard curve, all qPCRs, including a standard curve, were carried out in the presence of the same amount of medium-related DNA extract. qPCR assays for Myc. orale and Myc. pneumoniae were performed according to the protocols developed by BioReliance, which ensured a six-log dynamic range of DNA quantification (data not shown).

Figure 3.

 The effect of medium-related DNA on standard curve linearity in qPCR. The standard curve obtained for serial dilutions of Acholeplasma laidlawii gemonic DNA in the absence (bsl00001) and in the presence (Δ) of medium-related DNA. The total number of genomic equivalents in qPCR is shown on x-axis, and qPCR cycle thresholds are shown on y-axis.

Assessment of GC/CFU ratios for mycoplasmas cultured in broth

To assess changes in GC/CFU ratios during the mycoplasma culture growth, we monitored both, GC and CFU, values in culture samples, collected at different time points. The general scheme of kinetics experiment is shown in Fig. 1. The protocol (see Materials and methods) consisted of three subsequent steps: (I) mycoplasma IC preparation, (II) time point samples collection within the different phases of bacterial growth and (III) samples analyses. Samples, collected in duplicates from Myc.  fermentans, Myc. arginini, Myc. gallisepticum, A. laidlawii, Myc. orale and Myc. pneumoniae cell cultures, were analysed to determine the cell titres (CFU ml−1) and the genomic copy numbers (GC ml−1) using agar plating and qPCR, respectively.

The resulting GC ml−1, CFU ml−1 and GC/CFU values obtained for different mycoplasma cultures are presented in Fig. 4 and summarized in Table 2. It is noteworthy that all tested strains, except for A. laidlawii, showed low (≤10) GC/CFU ratios during the logarithmic and early stationary growth phases (Fig. 4a–f). Only A. laidlawii demonstrated GC/CFU ratios of slightly higher values (≤ 100) during the aforementioned culture growth phases (Fig. 4a).

Figure 4.

 The GC ml−1, CFU ml−1 and GC/CFU profiles obtained during Acholeplasma laidlawii (a), Mycoplasma gallisepticum (b), Mycoplasma arginini (c), Myc. fermentans (d), Mycoplasma orale (e) and Myc. pneumoniae (f) culturing in broth. GC ml−1 (bsl00001, □) and GC/CFU (bsl00066, Δ) values of A. laidlawii, Myc. gallisepticum, Myc. arginini and Myc. fermentans are presented as two independent sets of samples; in case of Myc. orale and Myc. pneumoniae indicated values (bsl00001, bsl00066) are presented as an average of three independent experiments; ○– CFU ml−1.

Table 2.   Summary of GC/CFU ratios measured at different phases of mycoplasma culture growth
SpeciesGC/CFU ratios at specific time point of incubation
0 h24 h48 h72 h96 h120 h144 h168 h240 h360 h480 h
  1. –, not measured.

Acholeplasma laidlawii1010215 3 × 1021033 × 104
Mycoplasma gallisepticum410 812102104
Mycoplasma arginini4 52·3 × 102 2 × 106
Mycoplasma fermentans8101·3 × 102 4 × 1034 × 104107
Mycoplasma orale0 210132·3 × 102
Mycoplasma pneumoniae03250

As Myc. orale and Myc. pneumoniae had significantly lower starting titres (≤102 CFU ml−1), it resulted in a longer time course (500h vs 140 h) required to monitor their growth phases (Fig. 4e,f). However, despite the difference in the growth time, the results of GC/CFU ratio assessment showed that the lowest (<10) GC/CFU ratio values were observed also during the log and early stationary phases of their growth (Fig. 4e,f), similar to that observed for other four species tested. A remarkable increase in GC/CFU ratios (up to three logarithms), observed on the late growth phases of some tested strains, could be attributed to the decline in mycoplasma titre because of the cell death (Fig. 4a,f). These results have indicated that all tested mycoplasma cultures at logarithmic and early stationary phases of their growth have the lowest GC/CFU ratios and, thus, could be valuable culture samples for preparation of mycoplasma reference stocks suitable for a comparison of NAT-based and conventional mycoplasma testing methods.

Assessment of mycoplasma GC/CFU ratios in co-culture with CHO cells

In the previous experiment, we demonstrated that mycoplasmas grown in broth under optimized conditions possess the lowest CG/CFU ratios during the early phases of culture growth. It was interesting to explore whether the GC/CFU ratio alters when mycoplasma was co-cultured with mammalian cells. This study was conducted using the experimental approach previously described for the axenic mycoplasma experiments (see Fig. 1). The mycoplasma genomic copy numbers and mycoplasma titres were monitored within 192-h postinfection of CHO cell suspensions with two mycoplasmas (A. laidlawii and Myc. gallisepticum). CHO cells were selected because of their common use for the production of different biologics (Eldering et al. 2004; Deutschmann et al. 2010; Lawrence et al. 2010). We used ProCulture spinner flasks for co-cultivation of mycoplasmas with suspension of CHO cells as a down-scaled model of a mycoplasma contamination of bioreactors during the manufacture of cell-derived biologics. Our preliminary experiments indicated that the main pool of mycoplasma cells for both strains, co-cultured with CHO cells, was not strongly associated with the surface of CHO cells under described conditions (data not shown). Thus, mycoplasma titres in supernatant fraction, obtained after removal of CHO cells by low-speed centrifugation, were close to that measured for samples of entire co-culture suspension.

In main experiments, CHO cell cultures were infected with mycoplasmas at an initial titre c. 104 CFU ml−1 that corresponded to multiplicity of infection (MOI) equal to one mycoplasma cell per 10 CHO cells. The growth kinetics of mycoplasmas in co-cultures with CHO cells (supernatant fraction) is shown in Fig. 5. We observed a rapid increase in Myc. gallisepticum titre in co-culture from 104 to 108 CFU ml−1 in 72 h p.i. (Fig. 5b). An apparent decline in Myc. gallisepticum titre was also observed after 5 days p.i. The analysis of GC/CFU for Myc. gallisepticum culture at different time points showed that the lowest GC/CFU ratios were observed at the logarithmic and early stationary phases of culture growth. Thus, in general, the growth characteristics of Myc. gallisepticum in co-culture with CHO cells (supernatant fraction) did not differ significantly from that observed for this species cultured in broth (see Figs 4b and 5a).

Figure 5.

 The GC ml−1, CFU ml−1 and GC/CFU profiles of Mycoplasma gallisepticum (a) and Acholeplasma laidlawii (b), co-cultured with Chinese hamster ovary (CHO) cells. Data are representative of two independent sets of samples for: bsl00001– GC ml−1; ○– CFU ml−1; bsl00066– GC/CFU.

In contrast to Myc. gallisepticum, A. laidlawii growth kinetics in co-culture with CHO cells was significantly different. Thus, the A. laidlawii titre increased only for one logarithm 24 h p.i. and sustained at the level of 105 CFU ml−1 for the following 7 days (Fig. 5a). In addition, the GC/CFU ratio sustained at the relatively low level (≥10, in average) throughout the duration of experiment. Thus, the growth kinetics exhibited by A. laidlawii in co-culture with CHO cells was quite unique, and additional experiments will be required to clarify the reasons of such growth behaviour.

The low GC/CFU ratio values (Fig. 5a,b) observed for A. laidlawii and Myc. gallisepticum at logarithmic and early stationary phases of their growth in co-culture with CHO cells indicated the possibility to use a supernatant fraction, represented by highly viable and dispersed mycoplasma cells, for the preparation of mycoplasma reference stocks suitable for methods comparison.

The analysis of GC ml−1 and CFU ml−1 values in A. laidlawii samples collected from co-culture and processed by a low-speed centrifugation showed that the mycoplasma GC/CFU ratio in CHO-pellet fraction was at least one logarithm higher than that measured in CHO-free supernatant fraction (Fig. 6). It is likely to be caused by minor adherence of mycoplasma cells (not more than 100 per cell) to the surface of CHO cells.

Figure 6.

 The GC ml−1, CFU ml−1 and GC/CFU profiles obtained for Acholeplasma laidlawii in the supernatant (♦– GC ml−1; •– CFU ml−1; bsl00066– GC/CFU) and the Chinese hamster ovary (CHO) cells pellet (◊– GC ml−1; ○– CFU ml−1; Δ– GC/CFU) fractions.

Discussion

Application of novel molecular technologies holds great promise to provide more rapid, efficient and simple ways for detecting mycoplasma contamination in cell substrates and cell-derived biologics. However, before the alternative methods can be implemented in testing programme, they need to go through extensive evaluation and validation procedures to demonstrate their equivalency in terms of LOD and specificity to the conventional methods, currently recommended by regulatory and compendial documents (EP.6.1 2008; USP 2010). As mentioned earlier, the comparison of alternative and conventional methods may face a significant challenge, because of the difference in the nature of measured biological features (parameters) (e.g. DNA or RNA content vs viability). Because of their high intrinsic sensitivity, specificity and short turnaround time, NAT-based methods are considered as one of the most promising approaches for development of alternative mycoplasma testing methods. However, NAT methods rely on detection of mycoplasmal nucleic acid (NA) regardless the mycoplasma cell viability, while the conventional methods detect only viable contaminants able to grow in artificial media or indicator cell culture.

A comparison of NAT-based methods against conventional methods necessitate the use of mycoplasma standard materials (usually, calibrated stocks of mycoplasma reference strains). Therefore, the results of methods comparison depend in a large extent on the selection of adequate mycoplasma reference strains and proper preparation of their calibrated stocks. As we showed earlier, a remarkable increase in GC/CFU ratios (up to 3 logarithms) can be observed in the mycoplasma culture at the late stationary (death) phase (Fig. 4a–f). To minimize the risk of accidental overestimation of the LOD of NAT methods, it is prudent to use mycoplasma strains with innately low GC/CFU ratios that may ensure high viability and dispersal of their stocks (‘worst case scenario’ situation). It is apparent that mycoplasma stocks containing a large amount of filamentous structures, aggregates or merely dead cells will exclusively benefit the LOD of NAT methods.

The goal of the current study was to assess the variation range of GC/CFU ratios for six different mycoplasma species: A. laidlawii, Myc. gallisepticum, Myc. arginini, Myc. fermentans, Myc. orale and Myc. pneumoniae at different phases of their growth in broth and in co-culture with mammalian cells (suspension of CHO cells). This study was inspired by the unmet need to establish the innate limits of GC/CFU for different mycoplasma species and develop conditions for preparation of highly viable stocks of mycoplasma reference strains. The results of the study showed that lowest GC/CFU ratios (<10) can be obtained at logarithmic and early stationary phases of mycoplasma growth in broth and in the supernatant fraction of the co-culture with mammalian cells, with the two mycoplasma organisms tested. The GC/CFU ratios observed in this study for several mycoplasmas are in good concordance with the GC/CFU and GC/colour changing units (CCU) previously determined for Ureaplasma urealyticum and Myc. hominis (Stemke and Robertson 1982). The results of that study showed the ratios between genome equivalents (calculated on basis of the amount of isolated genomic DNA) and CFU determined for U. urealyticum and Myc. hominis cultures at the end of their exponential growth were in the range 3·5–7·1 and 3·7, respectively. We also observed the lowest GC/CFU ratios for five species i.e. Myc. gallisepticum, Myc. arginini, Myc. fermentans, Myc. orale and Myc. pneumoniae at log and early stationary phases of their growth. Slightly higher GC/CFU ratios (more than 100) observed for A. laidlawii could likely be explained by the propensity of this A. laidlawii strain (ATCC14089) to form larger aggregates or filamentous structures (S. Razin et al. 1966; Brian Beck, ATCC, personal communication).

As we showed in our study, the GC/CFU ratio of mycoplasma cell culture strongly depends on the growth phase. Therefore, the use of overgrown culture at the late stationary or death phases (when culture titre began to decline) cannot be recommended for a comparability study. Another important factor, not considered in this study, which potentially can cause the uncontrolled reduction of the mycoplasma viability and increase GC/CFU ratio of mycoplasma reference sample, is the use of not-optimized freezing/thawing conditions during the reference strain stock preparation and utilization. It was shown that because of the specific composition of lipid membrane, mycoplasma cells are very susceptible to freezing damage and long-term storage at −70 to −80°C (Addey et al. 1970; Raccach et al. 1975; Cheng et al. 2007). The viability of mycoplasma cells can be stabilized with different cryoprotective additives and chemicals, such as 12% sucrose, 10% DMSO or 10–30% glycerol (Raccach et al. 1975; Boonyayatra et al. 2010). Moreover, the viability of mycoplasma stocks strongly depends on utilized thawing procedure (Biddle et al. 2004; Boonyayatra et al. 2010). In view of these findings, accurately assessed prefreezing viabilities should be performed when frozen materials are used for the comparison of NAT-based methods against conventional mycoplasma testing methods.

In conclusion, the proper preparation, preservation and use of optimized protocols for characterization and utilization of mycoplasma reference samples become a critical issue to adequately evaluate and compare new rapid methods against the conventional mycoplasma testing methods, with the long-term goal of being able to implement rapid mycoplasma testing assays for in-process and lot release testing.

Acknowledgements

We thank Dr Rajesh Gupta and Dr Hyesuk Kong from the FDA and Dr Brian Beck from ATCC for the discussion of mycoplasma sample preparation and Dr Selwyn Wilson for the help with the optimization of experimental conditions.

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