Background:
The increasing importance of viral vectors for gene therapy purposes has brought into question the efficiency of the manufacturing process. A higher space/time yield is one of the key factors for an increase in productivity and thus a broader adaption of a cell culture-based process to industrial scale. Recently, a suspension-based process operated in batch mode using HEK293T17SF cells was developed. High infectious viral particle titers of 4-5 x 10⁷ TU/mL and a total of 7 x 10⁸ transducing units were achieved. However, production was thought to be limited due to a lack of nutrients and aggregation of toxic metabolites. One potential solution to reduce metabolites is the switch to perfusion mode. A semi-continuous production procedure was developed in ambr®15 as a multi-bioreactor platform.
Results:
As part of a DoE, we focused on enhancer concentration, stirring speed and perfusion rate as representatives of volumetric productivity. Both process modes, batch and perfusion, were compared in terms of the resulting responses. These included physiological cell parameters, metabolic analysis, and a total and infectious virus titer. Increasing the cell concentration at time of transfection from 1 x 10⁶ to 5 x 10⁶ and setting a 50% perfusion rate leads to a titer of 6-7 x 10⁷ TU/ml, corresponding to a total production of 2 x 10⁹ transducing units. Compared to the batch mode, this is a 5- fold increase in viral vector production.
Conclusions:
These results confirm that the perfusion mode increases the space/time yield and the relevance for industrial scale production. However, the production process still shows potential for optimization. Further investigations relate to the establishment of a cell-specific perfusion rate and factors influencing up-scaling to several liters.

Alexander Tappe

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