TY - JOUR
T1 - Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines
AU - Vanni, Irene
AU - Ugolotti, Elisabetta
AU - Raso, Alessandro
AU - Marco, Eddi Di
AU - Melioli, Giovanni
AU - Biassoni, Roberto
PY - 2012/7
Y1 - 2012/7
N2 - Background aims. The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. Methods. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. Results. Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. Conclusions. We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.
AB - Background aims. The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. Methods. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. Results. Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. Conclusions. We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.
KW - Acholeplasmas
KW - Clinical trials
KW - European Pharmacopoeia
KW - Food and Drug Administration
KW - Good manufacturing practice
KW - Mollicutes
KW - Mycoplasmas
KW - Quantitative polymerase chain reaction
KW - Tissue culture contaminants
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U2 - 10.3109/14653249.2012.671517
DO - 10.3109/14653249.2012.671517
M3 - Article
C2 - 22458958
AN - SCOPUS:84862183862
VL - 14
SP - 752
EP - 766
JO - Cytotherapy
JF - Cytotherapy
SN - 1465-3249
IS - 6
ER -