GMP-konforme Matrixvalidierungen und die Besonderheit von Clostridium sporogenes bei der mirkobiologischen Kontrolle
Nicole Arlt (Cottbus / DE), Remo Rothe (Cottbus / DE), Barbara Ludwig (Dresden / DE), Thomas Juretzek (Cottbus / DE), Heidrun Peltroche (Cottbus / DE), Irene Sopivnik (Cottbus / DE)
In manufacturing ATMP treatments, for example like pancreatic islet cell transplants for type 1 diabetes mellitus therapy, sterility is an indisputable necessity and need a good manufacturing practice (GMP) admission performance. Using the microbes indicated by the Paul-Ehrlich-Institut with regard to Ph. Eur., General Chapter 2.6.1/2.6.27 it is essential to inoculate Clostridium sporogenes. Repeated validation of matrices with fresh isolated rat islet cells raised the question of possible non-growth of Clostridia. The present study should answer this unsolved issue.
Clostridium sporogenes were spiked two or threefold < 100 colony forming units (CFU) in several validations and matrices, in: fresh isolated rat islet cells, NK-92-wildtype cell line, buffy coat, stem cells, red blood cells, T-Reg cells, culture medium and bottle culture medium while using different blood culture bottle types (SA/SN, iAST/iNST, iFAplus/iFNplus) in the blood culture device Bact/Alert®3D (bioMérieux, Nürtingen, Germany). The anaerobic culture bottles were incubated for 14 days at 35°C in a Bact/Alert®3D 240 incubator and species identification was performed in the Institute of microbiology by mass spectronomy.
The growing characteristics of Clostridium sporogenes can vary significantly during growth phases, as well as in the different matrices and bottle types, up to non-growth. Growing time differs from an average of 0.91 d in buffy coats, stem cells, some bottle culture media to an average of 4.14 d in NK-92 wild-type cell lines, culture medium and red blood cells, more than the respective growing time of a slow-growing bacteria like Cutibacterium acnes. However, non-growth results were also observed in fresh isolated rat islet cells, T-Reg cells, culture medium, and some bottle culture media. Initial results show that the use of iFNplus blood culture bottles can lead to stable, reproducible results.
Our analysis shows that Clostridium sporogenes needs special growth conditions. Due to the different growing characteristics in the different matrices a detailed matrix validation is essential and indispensable.
No conflicts of interest.