Compendial Requirements for the Detection of Mycoplasma Contamination in Cell Cultures

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1 PDA Journal of GMP and Validation in Japan Vol. 11, No. 2 (2009) Compendial Requirements for the Detection of Mycoplasma Contamination in Cell Cultures Tsuguo Sasaki Pharmaceuticals and Medical Devices Agency Kasumigaseki, Chiyoda-ku, Tokyo sasaki-tsuguo@pmda.go.jp 培養細胞からのマイコプラズマ検出法局方要件 佐々木次雄 独立行政法人医薬品医療機器総合機構 1. Mycoplasma species isolated from cell cultures 1.1 Worldwide In the 1970s, the most frequently isolated mycoplasmas were M. arginini, M. orale and M. hyorhinis (Table 1). M. arginini is commonly found in bovine sera and M. hyorhinis is found in the porcine upper respiratory tract. The IOM (International Organization for Mycoplasmology) IRPCM (International Research Programme on Comparative Mycoplasmology) Team has summarized Mycoplasma contamination in cell cultures reported from Sweden, France, the Netherlands, and the UK (Table 2). In recent years, M. hyorhinis, M. orale, M. fermentans and M. arginini have remained the predominant isolates from cell cultures. The original source of M. hyorhinis, M. arginini and A. laidlawii is likely to have been bovine serum contaminated with mycoplasmas, while contamination with M. orale might have originated from laboratory staš. M. fermentans is a This paper was presented at PDA's 4 th Annual Global Conference on Pharmaceutical Microbiology, October 7, major isolate from human-derived lymphocytes, so the original source materials might have been contaminated with M. fermentans. 1.2 In Japan Large-scale surveillance of Mycoplasma contamination of cell cultures was carried by Kohara et al. 3) in Japan in The rate of Mycoplasma contamination was found to be 22 of 1,470 cell cultures collected from 17 institutions (Table 3). Kohara et al. used MycoAlert, which allows detection of Mycoplasma contamination in cell cultures within half an hour, with high speciˆcity and sensitivity, based on the formation of ATP from ADP by using substrates of Mycoplasma-speciˆc enzymes. Thus, Mycoplasma contamination of cell cultures clearly remains a major concern in Japan, and throughout the world. 2. Compendial Requirements for Detection of Mycoplasmas in Cell Cultures 2.1 Culture methods In 2000, Japanese Pharmacopoeia (JP) introduced a new chapter entitled, ``Mycoplasma Testing for Cell Substrates 49

2 PDA Journal of GMP and Validation in Japan Table 1 Mycoplasmas isolated from cell cultures in the 1970s 1) Table 2 Mycoplasmas isolated from cell cultures in recent years (IOM IRPCM Team Report; 2008) 2) Used for Production of Biotechnological/Biological Products''. The new chapter was prepared in response to the issue of ICH/Q5D guidance on ``Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products''. Table 4 shows the requirements for Mycoplasma testing in 5 compendia, i.e., WHO 4), JP 5),EP 6),USP 7) and VICH (ICH of Technical Requirements for Registration of Veterinary Medical Products) 8). Common testing methods in these compendia are a culture method and an indicator cell culture method. The culture method takes 35 days, while the indicator cell culture method takes 3 to 6 days. On the other hand, PCR can be done in only 1 to 2 days, but is described only in EP and JP. In the JP, Mycoplasma testing is applied to cell cultures such as master cell bank (MCB) cultures, working cell bank (WCB) cultures, and cell cultures used in processing, but the 50

3 Vol. 11, No. 2 (2009) Table 3 Mycoplasma Contamination of Cell Cultures in Japan 3) Table 4 Testing samples & methods other compendia also target vaccines and any other materials in which Mycoplasma contamination is suspected. In the case of live viral vaccines, in general, the culture method and the indicator cell culture method are not applicable to products after ˆltration. However, PCR can be applied to any kind of sample. Major discussion points on the culture method are summarized in Table Indicator cell culture method Vero cells are widely used as the indicator cell substrate. Vero cells were established from African green monkey kidney in Japan. Vero means ``truth'' in Esperanto. Other e- quivalent cell substrates, such as vaccine-production cell lines, may also be used as indicator cell substrates. Mycoplasma strains commonly used for validation of the cell substrate are M. hyorhinis and M. orale. The WHO and JP specify the strain number of these two mycoplasmas. The inoculum size of these mycoplasmas to be used as a positive control is not more than 100 CFU. Sampling volume is almost the same among the 5 compedia, that is 1 ml or more. Subculture of indicator cells after inoculation of sam- 51

4 PDA Journal of GMP and Validation in Japan ples is not required in the WHO or JP. The incubation period after sample inoculation or subculture is the same among EP, USP and VICH. The uorescent dye most widely used in Mycoplasma testing is DAPI (4,6-diamidino-2- phenylindole, also known as bisbenzimide). Observation is carried out microscopically at 100X (fold) 400X magniˆcation. In the USP, greater magniˆcation than 400x is recommended. In the case of the indicator cell culture method, several problems arise. The ˆrst is that M. hyorhinis DBS 1050 cannot form colonies on an agar plate. So, it is impossible to ad- Table 5 Major discussion points in culture methods 1. Which samples are more appropriate for mycoplasma testing (supernatant or cell suspension)? 2. If the test cannot be done immediately after sampling, what storage conditions of the samples are appropriate for mycoplasma testing? 3. What culture media should be employed for mycoplasma testing? 4. Which species or strains are suitable for evaluating nutritive properties of media? 5. What volume of sample should be used in testing? 6. What are the best incubation conditions for agar plate and/or liquid broth? 7. What is the most appropriate subculture method? 8. How should cell-derived constituents appearing on agar plate be distinguished from mycoplasma colonies? just cell density on the basis of CFU. The second problem arises from the use of M. orale as a positive control. Figure 1 shows DNA staining of M. hyorhinis DBS 1050 and M. orale CH These Mycoplasma strains were inoculated onto Vero cells at less than 100 cfu and incubated for 6 days. M. hyorhinis showed strong uorescent precipitates, but M. orale did not. In the case of M. orale, the result was the same in the case of inoculation at a high concentration, such as 1,000 cfu. Thus, if mycoplasmas contaminating a cell culture show weak cytadsorption, false-negative judgment may occur. The third problem is that judgement of results obtained with the indicator cell culture method is not easy in laboratories where Mycoplasma testing is not routinely carried out. Furthermore, the DNA staining method is not speciˆc for mycoplasmas, because DAPI binds to DNAs of nonmycoplasmal prokaryotic organisms, as well as to cell debris derived from indicator cells and/or test cells. 2.3 PCR EP and JP allow PCR for the detection of mycoplasmas. PCR is a rapid and sensitive assay that can be used routinely to screen Mycoplasma contamination in cell culture samples. In the JP, Mycoplasma testing is recommended to be performed using both the culture method and the indicator cell culture method. When a positive result is obtained only with the indicator cell culture method, the PCR method may be Fig 1 DNA staining with DAPI 52

5 Vol. 11, No. 2 (2009) Table 6 Validation of NAT and Mycoplasma species to be used in comparability study. Validation of NAT/EP Speciˆcity: demonstrate absence of cross-detection of phylogenetically close bacteria including Clostridium, Lactobacillus and Streptococcus. Detection limit: determine a positive cut-oš point for species selected in terms of frequent contaminants and phylogenetic relationships. Robustness: variations in method parameters/reliability of the analytical procedure Comparability study/ep A. laidlawii, M. fermentans, M. hyorhinis, M. orale, M. pneumoniae (or M. gallisepticum), M.synoviae,M.arginini,S.citri,etc. Acceptability criteria for NAT, for each test species culture method: 10 CFU/mL indicator cell culture method: 100 CFU/mL used. The main reason why PCR is not recommended as the test method of ˆrst choice is that a positive result in PCR does not always indicate the presence of viable mycoplasmas. However, if PCR is well controlled, it is not so di cult to identify Mycoplasma contamination with only PCR. In the EP, nucleic acid testing (NAT) (PCR) is not an o cial method. EP says that NAT may be used 1) as a complementary test (for example, for cytotoxic viral suspensions) or for in-process control purposes, and 2) as an alternative method to replace an o cial method (indicator cell culture method or culture method). IntheEP,validation methods of NAT are described in detail. In validation, it is necessary to consider speciˆcity, detection limit, and robustness. EP describes validation of NAT and deˆnes Mycoplasma speciestobeusedinacomparabilitystudy(table 6). In the EP, acceptability criteria for NAT for each test species should be established. If NAT is proposed as the alternative method to replace the culture method, the NAT system must be shown to detect 10 cfu/ml for each Mycoplasma species. If the NAT system is proposed to replace the indicator cell culture method, the detection limit is 100 cfu/ml. 2.4 Sensitivity and speciˆcity of PCR primers JP primers are targeted to the spacer region between the 16S and 23S rrna genes (Table 7). The primers of Kuppeveld are targeted to speciˆc regions of the 16S rrna gene. Figure 2 shows patterns of PCR products from representa- Table 7 PCR primers 1) JP primers 1 st PCR F1: 5 -ACACCATGGGAG(C/T)TGGTAAT-3 R1: 5 -CTTC(A/T)TCGACTT(C/T)CAGACCCAAG- GCAT-3 2 nd PCR F2: 5 -GTG(G/C)GG(A/C)TGGATCACCTCCT-3 R2: 5 -GCATCCACCA(A/T)A(A/T)AC(C/T)CTT-3 2) Kuppeveld et al. primers (265 bp) GPO-3: 5 -GGGAGCAAACAGGATTAGATACCCT-3 MGSO: 5 -TGCACCATCTGTCACTCTGTTAACCTC-3 tive Mycoplasma species that have been reported as contaminants in cell cultures. The 1 st PCR of JP did not show positive reaction to some tested species, but the 2 nd PCR of JP showed positive results in all species, with two bands. On the other hand, the primers of Kuppeveld showed positive results at the 1 st PCR. The JP primers had a tendency to give false-positive reactions. However, Kuppeveld's primers have not given false-positive reactions in PCR for biological samples in my experience over more than 10 years. At present, some commercial PCR kits are available for the detection of mycoplasmas. However, Kuppeveld's primers are excellent candidate PCR primers for detecting mycoplasmas in cell cultures. The sensitivity of these PCR primers was compared by using puriˆed DNAs from individual Mycoplasma species. Table 8 showsasummaryoftheresultsofthecomparison study of the primer sets of JP and Kuppeveld et al.. The sensitivity is signiˆcantly dišerent among Mycoplasma species, especially in case of the JP primers. For the 1 st PCR, the primers of Kuppeveld et al. showed higher sensitivity than those of JP. The JP primers gave a negative result with A. laidlawii. TheJP2 nd PCR showed similar or one order of magnitude higher response to the other Mycoplasma species, except M. arginini (two orders of magnitude higher response). 2.5 Uncultivatable mycoplasmas contaminating Vero cells. Several years ago, I applied PCR to examine Vero cells provided by vaccine manufacturers in Japan. In Japan, Vero cells are used for quality control testing of vaccines. Except for Vero cells from one manufacturer, all the Vero cell samples were contaminated with mycoplasmas (Fig 3). The contaminatant in Lane No. 6 was M. arginini, and the others 53

6 PDA Journal of GMP and Validation in Japan Fig 2 PCR products from representative Mycoplasma species by JP primers or Kuppeveld's primers Table 8 Detection limit by PCR were considered to be M. orale. I tried to isolate this Mycoplasma from Vero cell cultures, but failed even when the many types of culture media recommended in EP were used. The growth of Mycoplasma wasveryfastinverocell culture, but no growth was seen in fresh or cultured medium of Vero cells. Contamination with this microorganism was 54

7 Vol. 11, No. 2 (2009) Fig 3 PCR for Vero cells collected from different vaccine manufacturers in Japan also conˆrmed by means of direct DNA staining with DAPI (data not shown). Therefore, I sequenced the full length of the 16S rrna gene of the Mycoplasma and registered the sequence in DDBJ (DNA Data Bank of Japan) as AB The sequence of arginine deiminase of the Mycoplasma was exactly the same as that of M. orale (E02256). TheISLE (insertion sequence-like element) sequence showed more than 99 homology with that of M. orale (AY084048). Thus, I concluded that the main contaminating Mycoplasma in Vero cell culture is M. orale or a very similar microorganism. CONCLUSION In process control of the production of biological products, rapid conˆrmation of non-contamination with mycoplasmas is very important. In the case of the microbiological culture method, it takes several weeks to get a ˆnal result, and in the case of the DNA staining method, about one week. However, with PCR, it is possible to get a result within only 2 days. PCR has clear advantages for detecting mycoplasmas in cell cultures. In the JP, test methods are presented in the section of General Tests or General Information. The test for mycoplasmas is described in the General Information section. The PCR primers shown in the JP are useful, but other PCR primer sets are also applicable to the detection of mycoplasmas. I suggest that 1) PCR seems to be a reliable and practical method for detecting Mycoplasma contamination in cell cultures, and 2) the combination of the culture method and PCR is recommended as the most appropriate method for detecting mycoplasmas in cell cultures. REFERENCES 1) M. Barile, Mycoplasma-tissue cell interactions. The Mycoplasmas. Vol.II.(ed.,J.G.TullyandR.F.Whitcomb), Academic Press, p. 452 (1979). 2) Prevention and control of mycoplasma contamination in cell cultures. IOM IRPCM, ) A. Kohara et al., Tiss. Cult. Res. Commun. 26: (2007). 4) General requirements for the sterility of biological substances. WHO Tech Rep Ser, No. 872, (1995). 5) 11. Mycoplasma testing for cell substrates used for the production of biotechnological/biological products. JP (2009). 6) Mycoplasmas. EP (2008). 7) Mycoplasma tests. USP (2009). 8) Testing for the detection of Mycoplasma contamination. VICH (2002). 55