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

Irene Vanni, Elisabetta Ugolotti, Alessandro Raso, Eddi Di Marco, Giovanni Melioli, Roberto Biassoni

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)752-766
Number of pages15
JournalCytotherapy
Volume14
Issue number6
DOIs
Publication statusPublished - Jul 2012

Keywords

  • Acholeplasmas
  • Clinical trials
  • European Pharmacopoeia
  • Food and Drug Administration
  • Good manufacturing practice
  • Mollicutes
  • Mycoplasmas
  • Quantitative polymerase chain reaction
  • Tissue culture contaminants

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy
  • Molecular Medicine

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