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| **          | Two of the most common concerns with products derived  |
| Abstract:** | from banked cell lines are cross-contamination with    |
|             | unrelated cells and contamination of cell lines with   |
|             | adventitious agents microorganisms (predominantly      |
|             | Mycoplasma). Here, we propose to investigate the use   |
|             | microarray technology to develop rapid and highly      |
|             | accurate tests for cell line authentication as well as |
|             | the detection of contamination with Mycoplasma and     |
|             | cells of other origins.                                |
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| **Specific  | The aim of the study is to develop simple, sensitive,  |
| aims:**     | and reliable methods for assessing of the quality,     |
|             | consistency and safety of cell lines used for vaccine  |
|             | production and manufacturing of other biological       |
|             | products. Monitoring of cell line quality will be      |
|             | conducted on the basis of the analysis of several      |
|             | genetic markers, potentially allowing detection of     |
|             | Mycoplasma contamination and the presence of unrelated |
|             | cells. Where necessary, required sequences of genetic  |
|             | markers for some Mycoplasma and vertebrate species     |
|             | will be determined. The feasibility of the method for  |
|             | detection and discrimination between different         |
|             | Mycoplasma species and cell line clones originated     |
|             | from one species will be evaluated.                    |
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| **Ba        | Cross-contamination and misidentification of cell      |
| ckground:** | lines are major concerns that can potentially affect   |
|             | reliability of scientific experiments and the quality  |
|             | of biological products prepared using these cell       |
|             | lines. The main causes of contamination are            |
|             | contamination of cell lines (in particular with        |
|             | Mycoplasma) as well as the cell culture overgrowth by  |
|             | unrelated cells. Recently, several warnings regarding  |
|             | cell line contaminations were published *(Stancey,     |
|             | G.N. et al, 2000, Drexler, H.G. et al, 2002, Drexler,  |
|             | H.G. et al, 2003)*, claiming that the number of        |
|             | reported cases of cross contamination continue to      |
|             | increase despite numerous preventive efforts. Analysis |
|             | of the quality of cell lines in main cell banks        |
|             | worldwide indicates that about one-third of the cell   |
|             | lines are either cross-contaminated or Mycoplasma      |
|             | infected or both *(Drexler, H.G. et al, 2002)*. The    |
|             | most reasonable explanation of this is the low         |
|             | reliability, specificity, and sensitivity of current   |
|             | tests used for the assessment of the quality of cell   |
|             | lines. Accurate diagnosis of Mycoplasma is complicated |
|             | by the difficulty to cultivate it in vitro whereas     |
|             | authentication of cell lines is obscured by the cost   |
|             | and complexity of isoenzyme analysis that is           |
|             | considered as the standard assay for cell line quality |
|             | control. In addition, this method has limited          |
|             | sensitivity and can be used only when the              |
|             | contamination exceeds 10% of the total cell            |
|             | population.                                            |
|             |                                                        |
|             | Molecular methods based on detection and analysis of   |
|             | Mycoplasma DNA or DNA of unrelated cells (e.g. PCR     |
|             | amplification) were shown to be much more sensitive,   |
|             | accurate, rapid, and less tedious than current         |
|             | methods. However, the use of gel-electrophoresis for   |
|             | separation of PCR products followed by Mycoplasma or   |
|             | vertebrate species identification on the basis of the  |
|             | size of the PCR product is the main cause of false     |
|             | identification. This problem can be easily resolved by |
|             | replacement of gel-electrophoresis analysis of PCR     |
|             | products by hybridization with microarrays containing  |
|             | multiple species-specific probes. In contrast to       |
|             | gel-electrophoresis, the microarray method determines  |
|             | identity of amplified DNA on the basis of sequences of |
|             | PCR products. The use of microarray techniques enables |
|             | simultaneous monitoring of several genetic parameters  |
|             | of different organisms in one analysis and is capable  |
|             | to detect and discriminate between closely related     |
|             | species. It makes this approach suitable for accurate  |
|             | quality control of cell banks used for production of   |
|             | vaccines and other biological products.                |
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| **Study     | To reach the goal of the study we plan to conduct      |
| des         | analysis of Mycoplasma ribosomal genes (16S RNA,       |
| ign/Plan:** | 16-23S intergenic spacer, and 23S RNA) as well as      |
|             | vertebrate mitochondrial genes (cytochrome B, cox1,    |
|             | and highly variable D-loop), which were previously     |
|             | shown to be appropriate genetic markers for detection  |
|             | of cell line contamination. The most conserved regions |
|             | of these genes will be used to design a set of         |
|             | universal PCR primers enabling simultaneous            |
|             | amplification of the target genes from most widely     |
|             | distributed Mycoplasma species and from different      |
|             | origins of cell lines including human, rat, African    |
|             | green monkey, dog, cat, rabbit, horse, hamster,        |
|             | chicken etc. species. In cases, when sequences of the  |
|             | target genes are not available, additional             |
|             | amplification and sequencing of required genes will be |
|             | conducted. In contrary, species-specific sequences     |
|             | within amplified regions of the target genetic markers |
|             | will be used to design individual oligonucleotide      |
|             | microarray probes. A single microarray for             |
|             | simultaneous cell line authentication and detection of |
|             | contamination of cell lines with Mycoplasma or cells   |
|             | of unrelated origin will contain multiple oligoprobes  |
|             | specific to Mycoplasma ribosomal DNA and vertebrate    |
|             | genetic markers. Preliminary experiments that have     |
|             | recently been conducted in our laboratory using        |
|             | microarray for analysis of mammalian genetic markers   |
|             | showed the high potential of the approach. Theoretical |
|             | analysis of Mycoplasma ribosomal genes also showed the |
|             | presence of regions suitable for Mycoplasma species    |
|             | identification.                                        |
|             |                                                        |
|             | Evaluation of the microarray and validation of the     |
|             | proposed method will be conducted using different cell |
|             | lines from the ATCC and other sources. The sensitivity |
|             | of the method will be estimated using the spiking of   |
|             | cell lines with cells of another origin, or            |
|             | Mycoplasma, or both. We expect that development of     |
|             | such test might become a useful instrument for         |
|             | monitoring of the quality and consistency of cell      |
|             | lines used for production of vaccines and other        |
|             | biological products.                                   |
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