Cultivos de células animais visando a altas concentrações celulares: processo em perfusão e suplementação com aminoácidos.

Abstract

Animal cells have been under research as a platform for the expression of recombinant proteins, ranging from veterinary vaccines to blood coagulation factors for treating hemophilia. Examples include insect Drosophila melanogaster S2 and hamster BHK-21 cells, currently being studied for the production of rabies virus glycoprotein. Regardless of the cultivation strategy, high cell concentrations are usually associated to a higher protein production. Thus, the aim of this research was to investigate animal cell cultivation strategies that would allow higher cell concentrations than those previously reported. Cells of Drosophila melanogaster S2 expressing the rabies virus glycoprotein (S2AcGPV) were cultivated in shake flasks at 100 rpm and 28 ℃, in SF 900 II serum-free medium supplemented with the following amino acids: asparagine, cysteine, proline, and serine. The addition of the four amino acids to the medium increased the maximum cell concentration (X V MÁX) in 16%. When only cysteine was added to the medium, the maximum specific growth rate (µ MÁX) was 56% higher. In this condition, the cell yield on glucose (Y X/GLC) was 47% higher, indicating a more efficient glucose metabolism. These results show that cysteine is likely a limiting substrate of S2AcGPV cells growing in SF 900 II medium. In turn, baby hamster kidney cells (BHK-21/C13), adapted to growth in suspension culture, were cultivated in perfusion, a continuous process with cell retention that allows higher cell concentration than batch or continuous cultures without cell retention. A stirred tank bioreactor with a working volume of 1.5 L was used, with an internal spin-filter with 10 µm diameter pores attached to the impeller shaft. Temperature was controlled at 37 ℃, pH at 7.2, agitation at 80 rpm and dissolved oxygen at 50% of air saturation. The maximum cell concentration reached 15.7 x 10 6 cells mL-1 , much higher than the cell concentration achieved in a standard batch cultivation (5 x 10 6 cells mL-1). Cell viability was above 90% during the 48-day cultivation period. During the batch phase of the perfusion cultivation, specific rates of glucose (q GLC) and glutamine (q GLN) consumption were 84% and 32% higher, respectively, when compared to the batch cultivation. Similarly, the specific rates of lactate (q LAC) and ammonium (q NH4) formation were 78% and 102% higher, respectively. During perfusion, the cell maintenance coefficient was not negligible and represented 83% of total glucose consumption. These data indicate that the presence of an internal spin-filter may be associated to cell stress. In perfusion, cell concentration was about 3 times higher than that in continuous culture without cell recycle. In conclusion, it was proved that suspension-adapted BHK-21 cells can be cultivated in a laboratory-scale bioreactor with an internal spin-filter, in order to achieve high cell concentrations.