Desenvolvimento de processo de produção de fator VIII recombinante em biorreator.

Abstract

The deficiency of activity of the coagulation fator VIII (FVIII) is associated to a cronical disease, heamophilia A, which is controlled by the reposition of this protein in the blood. Nowadays, in Brazil, all the factor VIII consumed is produced abroad and has blood plasma origin. However, the recombinant factor VIII (rFVIII) is a safer regarding contamination risks. Aiming to obtain the proper activity and to avoid immunogenic reactions, it is desirable to produce a rFVIII in post-translation modifications similar to those observed at the normal protein. The objective of this work was to establish a process for rFVIII production in a bench bioreactor, bubble-free, using two human cell lines transfected by Hemocentro de Ribeirão Preto -rHeLa e rSKHep. When cultured in DMEM medium with 10% of fetal bovine serum (FBS), these cell lines showed similar behaviors regarding cell growth and metabolism. Both had an average cell production (X) of 1,47x10 6 cells/mL (CV=4%) and average specific cell growth rate ( X,MÁX ) of 0,0284 h -1 (CV=2%). Results for Y X/GLC and Y LAC/GLC were also similar, with average values of 3,65x10 8 cell/g (CV=14%) and 0,734 g/g (CV=2%), respectively. The main difference was observed in the Y NH4/X value that was 4,15 times higher in the rHeLa cell line than in SKHep. The rHeLa cells were easily adapted to suspension and to serum-free, animal-component-free and chemically defined media. Preliminary studies to evaluate the best conditions to cell growth and metabolism were developed using this cell line. were developed. However, analytical techniques implemented during the project showed that rHeLa cells had lost its ability to produce the rFVIII. The rSKHep cell line couldn't be successfully adapted to suspension growth or to serum free media, so it was necessary to adopt microcarrier (mic). The studies to idenitify the best conditions for the culture system (3 gmic/L and 3 cells/mic) were performed using previous results from the rHeLa studies. Studies of rFVIII stability showed that it degradates 65% when exposed to 37 °C for 48 hours. Therefore, the perfusion mode, with and internal spinfilter, was an alternative to produce continuously a protein which activity wouldn't be seriously affected due to its low exposure time to high temperatures. The culture in perfusion mode was started with 3 cells/mic and it was used 3 g mic/L. The perfusion mode started before the interruption of the exponential growth phase, with a hydraulic residence time of 24 hours and independent glucose and glutamine feeds aiming to maintain the concentrations of these substrates at 1 and 0,35 g/L, respectively. The controlled variables (cell concentration, glucose and glutamine) were kept constant for three hydraulic residence times. The maximum rFVIII concentration obtained was similar to the one from the batch culture. However, the total amount of rFVIII produced at the perfusion mode (16,156.27 UI) was 5,5 times higher than in batch mode, resulting in a productivity of 2,5 times higher.