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K. Kleinschmidt, K. Kleinschmidt Microbiological Quality Control Unit Novartis Pharma Stein AG Stein Switzerland Correspondence Kerstin Kleinschmidt, Rifeldweg 8, CH 4322 Mumpf AG, Switzerland. E‐mail: k-kleinschmidt@gmx.de Search for other works by this author on: Oxford Academic E. Wilkens Microbiological Quality Control Unit Novartis Pharma Stein AG Stein Switzerland Search for other works by this author on: Oxford Academic S.P. Glaeser Institut für Angewandte Mikrobiologie Justus‐Liebig‐Universität Gießen Giessen Germany Search for other works by this author on: Oxford Academic P. Kaempfer Institut für Angewandte Mikrobiologie Justus‐Liebig‐Universität Gießen Giessen Germany Search for other works by this author on: Oxford Academic A. Staerk Microbiological Quality Control Unit Novartis Pharma Stein AG Stein Switzerland Search for other works by this author on: Oxford Academic D. Roesti Microbiological Quality Control Unit Novartis Pharma Stein AG Stein Switzerland Search for other works by this author on: Oxford Academic
Journal of Applied Microbiology, Volume 122, Issue 4, 1 April 2017, Pages 997–1008, https://doi.org/10.1111/jam.13387
Published:
01 April 2017
Article history
Received:
15 June 2016
Revision received:
12 November 2016
Accepted:
22 December 2016
Published:
01 April 2017
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K. Kleinschmidt and others, Development of a qualitative real‐time PCR for microbiological quality control testing in mammalian cell culture production, Journal of Applied Microbiology, Volume 122, Issue 4, 1 April 2017, Pages 997–1008, https://doi.org/10.1111/jam.13387
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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.
biopharmaceuticals, microbial contamination, PCR, quality control, rapid methods
© 2017 The Society for Applied Microbiology
This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Issue Section:
Food/Beverage Microbiology Original Articles
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