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Development of an automated capacitance-based, software-driven compact system allowing mammalian cells to be grown in sonoperfused submerged cultures under batch- (constant volume) or fedbatch (constant concentration) conditions with continuous monitoring of pH and dissolved oxygen at cell concentrations between 2- and 20 millions cells/ml. This system, together with its multiplexing capability (up to 4 bioreactors) and remote control via internet makes the following applications possible:
Measurement and control of viable cell density in a mammalian cell bioprocessing facility: Validation of the software
D. Sergeant, Awardee of a Walloon Region "Bourse de préactivité", Mons/Belgium; M. Moser, CILBIOTECH sa, Mons/Belgium; J.P. Carvell, Aber Instruments Ltd, Aberystwyth/United Kingdom
Because of the increased demand for cells growing in suspension (about 60-70% of all recombinant protein pharmaceuticals are produced in mammalian cells, most of which grow in continuously stirred tank reactor), ways are being sought to automate their production.
Many cell culture processes are based on perfusion or fed-batch bioreactor systems. Control of the feed or addition rates to maintain steady-state conditions in these bioreactors can be especially challenging due to high and fluctuating cell concentrations that can rapidly change environmental conditions. Simple control of perfusion bioreactors, based on infrequent daily sampling and estimation of the live cell concentration, will lead to large process deviations. Tight control of the perfusion or concentrate addition rate using real time, online cell concentration measurement allows the bioreactor to be operated under the optimum conditions for maximum recombinant protein production.
In this poster we report how a robust automatic perfusion rate control system based on on-line radio-frequency impedance probe has been used in perfused cytostat. The system operated in a completely closed loop i.e. no samples needed to be taken to obtain process information. In the control algorithm, a cell specific perfusion rate was specified and the cell density signal was converted into a perfusion flow rate. Example are shown of the actual time-dependent capacitance traces of a perfused Hela cell cultures evolving from batch (preset volume, increasing concentration) to fedbatch (increasing volume, preset cell concentration) with a preset cell concentration in the bioreactor.
Figure 1 presents the results of a 20-day culture of Hela cells inoculated at 2,5 x 106 cells/ml and ran automatically in batch mode until the pre-set concentration of 25x106 cells/ml is reached. As shown, the cell concentration then becomes automatically regulated and remains constant. The fluctuations in the volume of the culture represents daily sampling. Grown under such sonoperfused fedbatch ("cytostat") conditions, the only manipulations required consisted in changing regularly the feed-and waste bottles. Cell viability as measured by the trypan blue exclusion test averages 96%.
The computer-controlled growth of mammalian cells in sonoperfused cytostat mode presents the following characteristics:
The system lends itself ideally to be placed in a cGMP environment or confined in a restriction access barrier system.
Future developments consist in devising similar softwares specifically dedicated to the propagation of (genetically engineered) cell lines secreting therapeutic/prophylactic monoclonal antibodies, coagulation Factor VIII,…