Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production.

ABSTRACT

AIMS: The aim of this study was to develop and evaluate a real-time PCR technology for microbiological control methods to examine individualized cell therapeutics, an emerging class of pharmaceutical formulations. METHODS AND RESULTS: Oligonucleotide primers and hybridization probe for bacterial detection targeting the 16SrRNA gene were adapted based on Nadkarni et al. [Microbiology148 (2002) 257]. For detection of yeast and moulds, primers and probe were designed from conserved sequences of the 18SrRNA gene in this study. The real-time PCR assays were tested on genomic DNA of Escherichia coli and Candida albicans to assess efficiency and linear dynamic range. After successful establishment of robust real-time PCRs, applicability of the assays was evaluated by extracting microbial target DNA from cell-based preparations. Different commercial DNA extraction methods were compared identifying the MagNA Pure DNA Isolation Kit III as the method of choice. Sensitivity was examined for different strains and a detection limit of 102 -103 CFU per ml in a sample containing ~106 mammalian cells per ml was achieved. CONCLUSIONS: This study reports the successful establishment of two qualitative real-time PCR assays, enabling in general the broad-range detection of microbial contaminants in a cell-based sample matrix. SIGNIFICANCE AND IMPACT OF THE STUDY Individualized cell therapeutics tend to have a short shelf life. Due to lengthy incubation periods, compendial testing according to current pharmacopoeial guidelines may not be applicable. We report a suitable alternative method upon which future microbiological quality control methods for such products could be based on. However, to implement valid rapid microbiological testing methods using real-time PCR technology, further challenges need to be addressed.