Moreover, the capacity to continue cell growth at the moment of virus infection may be important as the applied MOI was 0.01 which means 99% of the cells will not be infected during the first virus replication cycle and can potentially grow further. These topics are currently under investigation to be able to further optimize the virus culture at increased cell densities. The highest virus yields, based on d-antigen concentrations, were observed using the recirculation mode for cell culture. At the first glance, to maximize bioreactor capacity, this seems to be the best choice. However, it should be Pictilisib solubility dmso mentioned that a larger pre-culture needs to be prepared

as here the cell culture is started at 0.6 × 106 instead of 0.1 × 106 cells mL−1 used for the other cell culture strategies. Hence, extending the overall process throughput time. Further, considering the cell specific d-antigen productivity, the semi-batch cell culture strategy appeared to be a good alternative. In addition, this method can be applied in existing manufacturing equipment without large

investments. At present, we are optimizing this method with respect to microcarrier concentration, feed frequency and feed/bioreactor volume Lumacaftor clinical trial ratio. In addition, adaptation of downstream processing to concentrate and purify the poliovirus obtained from increased cell density cultures is studied. Focus points are the filter load with cell debris during clarification and concentration and the removal of the increased concentrations of host cell proteins and host cell DNA during column chromatography. Also, product quality and immunogenicity after purification remains to be assessed. In that way, discrimination between intact virus particles and virus progeny, which may have attributed to the observed increased d-antigen levels, can be made. This study shows that adherent Vero cell culture using different methods of medium refreshment allows higher cell densities. Increased cell densities allowed up to 3 times higher d-antigen levels when compared with that

obtained from batch-wise Vero cell culture. The cell specific d-antigen production was lower when cells were medroxyprogesterone infected at higher cell densities. Application of a semi-batch mode of operations allowed the highest cell specific d-antigen production, while 2 fold lower cell specific d-antigen yields were found using perfusion or recirculation cultures. This reduction may be related to the presence of multilayers of cells on the microcarriers, which were observed at higher cell densities that were reached using perfusion or recirculation mode. In our view, the most promising concept for polio d-antigen yield optimization would be semi-batch cultivations. This strategy has potential to be further improved and can be implemented in current manufacturing facilities. Using the here presented method for semi-batch cell culture and subsequent virus culture, d-antigen yields per run can be doubled.