Kleinschmidt K, Wilkens E, Glaeser S P, Kaempfer P, Staerk A, Roesti D
Microbiological Quality Control Unit, Novartis Pharma Stein AG, Stein, Switzerland.
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Gießen, Giessen, Germany.
J Appl Microbiol. 2017 Apr;122(4):997-1008. doi: 10.1111/jam.13387. Epub 2017 Feb 16.
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.
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 10 -10 CFU per ml in a sample containing ~10 mammalian cells per ml was achieved.
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.
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.
本研究旨在开发并评估一种用于微生物控制方法的实时聚合酶链反应（PCR）技术，以检测个体化细胞疗法（这是一类新兴的药物制剂）。
基于Nadkarni等人[《微生物学》148（2002）257]的研究，针对细菌检测，对靶向16S核糖体RNA（rRNA）基因的寡核苷酸引物和杂交探针进行了调整。在本研究中，针对酵母和霉菌的检测，从18S rRNA基因的保守序列设计了引物和探针。在大肠杆菌和白色念珠菌的基因组DNA上对实时PCR检测进行测试，以评估其效率和线性动态范围。在成功建立稳健的实时PCR后，通过从基于细胞的制剂中提取微生物靶DNA来评估该检测的适用性。比较了不同的商业DNA提取方法，确定MagNA Pure DNA分离试剂盒III为首选方法。检测了不同菌株的灵敏度，在每毫升含有约10个哺乳动物细胞的样品中，实现了每毫升10-10菌落形成单位（CFU）的检测限。
本研究报告成功建立了两种定性实时PCR检测方法，总体上能够广泛检测基于细胞的样品基质中的微生物污染物。
个体化细胞疗法往往保质期较短。由于培养周期长，按照现行药典指南进行的药典检测可能不适用。我们报告了一种合适的替代方法，未来此类产品的微生物质量控制方法可基于此。然而，要使用实时PCR技术实施有效的快速微生物检测方法，还需要应对进一步的挑战。
