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Kultivierung von felinen Lungenfibroblasten und Vermehrung von Nerz Enteritus Virus in Microcarriersystemen bei Batch- und Perfusions-Betrieb


Straube,  S.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;
Impfstoffwerk Dessau-Tornau, Roßlau;

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Straube, S. (2005). Kultivierung von felinen Lungenfibroblasten und Vermehrung von Nerz Enteritus Virus in Microcarriersystemen bei Batch- und Perfusions-Betrieb. Diploma Thesis, Hochschule Anhalt (FH), Magdeburg.

In this work, an industrial process for producing a veterinary vaccine against mink enteritis was characterized and further optimized with respect to virus yield and process design. Adherent embryonic feline lung fibroblasts (E-FL) are used as substrate for mink enteritis virus (MEV) progagation in roller bottles and in Wave® Bioreactor microcarrier culture. MEV replicates only in proliferating cells. Therefore the infection starts together with cell inoculation. The industrial process is performed as "repeated batch" resulting in 3-6 virus-containing harvests. In this diploma thesis the use of perfusion systems in a 0.4 L working volume (wv) stirred-tank reactor and in a 1 L wv Wave® Bioreactor microcarrier culture to replace the repeated batch process were be characterized and evaluated. For this purpose a number several perfusion systems were tested, a self-designed perfusion module showed the best results with respect to reliability and idling cycle. Perfusion led to 20 % higher cell densities in the standard process with 2 g·L-1 microcarrier in comparison to the "repeated batch" process. An increase in microcarrier concentration to 5 g·L-1 resulted in high cell densities of > 5.0 · 106 cells·mL-1 for over three to four days cultivation time. The use of perfusion for virus propagation resulted in high virus titers (107.5 TCID50·mL-1) in the bioreactor and a titer of 105.7 TCID50·mL-1 in the harvest pool, which exceeds the necessary amount for vaccine production. The results of this work show that perfusion strategies are suitable to simplify and improve this production technology significantly. Now, the whole process can be operated as a closed system, thus fulfilling GMP-requirements in an ideal manner.