RESUMEN
Two murine hybridoma cell lines named 1D12 and 3B1 secreting antibody against human blood group B were developed. Effect of medium composition and hyperosmotic stress on cell growth, viability and monoclonal antibody (MAb) production was examined during down-stream process in suspension culture. Both of the cell lines secreted MAb of higher titre if the basal medium was supplemented with 2.5 g/l L-glucose and 419 mg/l L-glutamine. Additional nutrients like transferrine, amino acids, ethanolamine, selenium were added to promote cell growth and viability. In case of cell line 1D12 enhancement of MAb production was observed using hyper-osmotic medium as well. Having data of these preliminary experiments we optimized the large scale production of MAbs in bioreactor. These MAbs serve as a base of the anti-B blood group reagent registered in Hungary.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/inmunología , Anticuerpos Monoclonales/biosíntesis , Animales , División Celular , Supervivencia Celular , Medios de Cultivo , Humanos , Hibridomas , Ratones , Células Tumorales CultivadasRESUMEN
A human/mouse heterohybridoma cell line secreting anti-Rh(D) antibody was established by the fusion of Epstein-Barr Virus (EBV) transformed human B lymphocytes and human/mouse heteromyelomas. During scaling-up the cell line was adapted to suspension culture, then medium composition was gradually altered into a more economical one. The adapted cell line has kept its multiplication and antibody secreting characteristics in the new medium. Parameters of large scale batch cultivation in bioreactors were optimized in working volume of 3.6 litre and production was carried out under these parameters in working volume of 24 litre. Optimized parameters were the follows: (i) the cell line is highly sensitive to dissolved oxygen (DO) level, its optimal DO is 10%; (ii) optimal inoculum cell count is 3 x 10(5) cell/ml; (iii) Marine-blade impeller was chosen for supporting the requested oxygen transfer. Using the optimized parameters HUMAN Co. Ltd. produces the Monoclonal Anti-D (IgM) reagent, which has been registered in 1995 in Hungary and now it is on the market.