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Using Continuous Integrated Micro Filtration for the Production of Pseudotype Vectors in a Fixed Bed Reactor

Dirk Nehring1, Ralf Poertner2, and Peter Czermak1. (1) Department of Biotechnology, University of Applied Sciences Giessen-Friedberg, Wiesenstrasse 14, Giessen, 35390, Germany, (2) Department of Biotechnology I, Technical University Hamburg-Harburg, Denickestrasse 15, Hamburg, 21071, Germany

Retroviral pseudotype vector, derived from the murine leukaemia virus carrying the HIV-1 envelop protein MLV (HIV-1) were produced using a integrated, continuous cultivation and harvest process. A 200 ml fix bed reactor was used to cultivate the anchorage dependent packaging cell line on macro-porous carriers until the maximum glucose uptake was reached. After starting the cultivation in batch mode, the reactor was either run in perfusion or repeated-batch mode. Parallel to the cell growth inside the fixed bed the medium in the conditioning vessel was harvested permanently. A cross flow filtration module including a commercial asymmetric micro filtration membrane was set up parallel. Filtration was carried out either continuously or batch wise. To optimise the production and downstream processing of vector formation and concentration a holistic mathematical model including the complete cultivation and cross flow filtration process was developed and validated. The integrated optimisation with the aid of the extended model lead one to the conclusion that there is an optimal time to start the filtration process and that there are optimal filtration parameters to achieve the highest concentration of colony forming unit per ml (cfu/ml). Sensitivity analysis performed on the results of the optimisation showed that under optimal conditions the final concentration and the yield of the entire process is more sensitive to the parameters of the filtration than to the bioreaction due to the fast degradation of replication competent pseudo-type vectors.

High concentrated active vector supernatant can be produces in a semi-continuous way, combining continuous fix bed cultivation with the benefits of a cross flow filtration process. The cultivation and production process could be run stable for a long period of time such as 420h. The filtration as a part of the downstream process was successfully integrated into the cultivation system. Comparing the results of different culture systems 15 therapeutic doses of 100 ml with titer from 10^6 cfu ml^1 according to literature can be produced with 22 l medium after 420h in the fixed bed reactor combined with a cross flow filtration module, while in standard flask culture or roller bottles the required vector titer can only be reached after additional batch filtration. To produce the same quantity of active vector particles in flask culture a medium quantity of 40 to 50 l would be necessary. Moreover, scale up of the production process is more applicable for the fixed bed reactor system in comparison to culture flasks. This process setup might be an alternative to standard production procedures for the production of degradable bioactive products, because the product can be transferred from the culture medium into more stable conditions continuously. However, while testing a continuous filtration during the entire cultivation the process of cell growth was not stable enough to perform a long-term cultivation. To perform continuous filtration for 24 h and more the retention of nutrient need to be further investigate in order to avoid limitation of the cell culture.

The results of this study emphasises that not only optimisation of packaging cell lines but also optimisation of the cultivation and purification process is needed in order to achieve suitable concentrations for gene therapy applications such as treatment with retroviral particles.

Keywords: retrovirus, gene therapy, purification, membrane filtration, mathematical model, fixed bed reactor, continuous filtration