title: Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production.
abstract: OBJECTIVE
  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.
  
  
  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 102-103CFU per ml in a sample containing ~106mammalian 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Microbiological Quality Control Unit, Novartis Pharma Stein AG, Stein, Switzerland.
authors.forename: K
authors.initials: K
authors.lastname: Kleinschmidt
authors.affiliation: Microbiological Quality Control Unit, Novartis Pharma Stein AG, Stein, Switzerland.
authors.forename: E
authors.initials: E
authors.lastname: Wilkens
authors.affiliation: Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Gießen, Giessen, Germany.
authors.forename: S P
authors.initials: SP
authors.lastname: Glaeser
authors.affiliation: Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Gießen, Giessen, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Kaempfer
authors.affiliation: Microbiological Quality Control Unit, Novartis Pharma Stein AG, Stein, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Staerk
authors.affiliation: Microbiological Quality Control Unit, Novartis Pharma Stein AG, Stein, Switzerland.
authors.forename: D
authors.initials: D
authors.lastname: Roesti
mesh_terms: D001419:Bacteria / Q000302:isolation & purification*; D002176:Candida albicans / Q000235:genetics / Q000302:isolation & purification; D018929:Cell Culture Techniques / Q000592:standards*; D002478:Cells, Cultured; D004247:DNA / Q000302:isolation & purification; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005658:Fungi / Q000302:isolation & purification*; D006801:Humans; D057230:Limit of Detection; D008828:Microbiological Techniques / Q000592:standards; D011786:Quality Control; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D012337:RNA, Ribosomal, 18S; D004247:DNA
keywords: 
  PCR
  ; biopharmaceuticals; microbial contamination; quality control; rapid methods