Bioprocess Development For The Production Of A Mouse-human Chimeric Anti-EGFRvⅢ Antibody C12 Expressed By CHO-C12 Cells In Suspension Culture

The mouse-human chimeric anti-EGFRvIII antibody C12 was expressed by recombinant CHO-C12 cells, which has potentials to be used as therapeutics solely or conbination with 5-fluorouracil(5-FU) for the treatment of EGFRvIII-positive Hepatocellular carcinoma (HCC). In this thesis,the adaption of rCHO-C12 cells growth to serum-free midium was carried out and culture characterastics of rCHO-C12 cells were investigated. Additionally, three bioprocesses including batch, fed-batch and perfusion bioprocesses have been developed for antibody C12 production in suspension culture.1) Direct adaption of rCHO-C12 cells growth to EX302 serum-free medium was established. The peak cell density of 6×106cells/mL was achieved when rCHO-C12 cells was cultured in EX302 serum-free medium. It was observed that the specific antibody C12 formation rate of rCHO-C12 cells cultivated in serum-free medium was slighly lower than that obtained with serum medium at exponential phase. The serum-free medium CHO-CD is better for rCHO-C12 cells with higher performances in viable cell density among the three test media CHO-CD (Gibco), EX302(JRH), SAF-CHO-G-001 (Qingdayouwei,China).Economically and experimentally, EX302 were more suitable for CHO-C12 cells large-scale culture in bioreactor.2) Culure performances like cell growth characterization and C12 production of rCHO-C12 cells cultured in shake flasks culutre and in a 5L bioreactor were investigated. The results showed that the maximum cell density and highest antibody concentration of rCHO-C12 cells batch-cultured in a 5L bioreactor were 4×106cells/mL and 190mg/L, respectively, which were approximately 70% of the values obtained in shake flasks culture. Also, the limited glucose and glutamine concentrations in bioreactor were 6mmol/L and 1mmol/L for rCHO-C12 cells growth, respectively. Regarding metabolic by-products lactate, ammonia and alanine accumulation in bioreator were remarkably lower than that in shake flasks. In addition, the specific consumption rate of the following consuming amino acids: Asp, Glu, Asn, Cys, Thr, Met, Trp, Ser, Ile, Leu in bioreactor culture were higher than that in shake flasks except to Ile and Leu. Especially, three amino acids including Asp,Glu,Asn was consumeded to depletion at early growth phase, resulting in limitation in cell growth and antibody production. Based on these results, a balanced amino-acid fed-batch bioprocess was developed.3) Fed-batch and perfusion bioprocesses in bioreator were successfully developed for antibody C12 production. The results showed that (1) compared with the batch culture mode, fed-batch and perfusion culture modes could effectively maintain higher cells viability so as to extend the cell culture duration; (2) The maximum mean yield coefficients like YVx/Gluc of 13.87×108cells/g,YVx/Glun of 291.94×108cells/g, YAb/gluc of 44.72mg/g and YAb/glun of 721.40 mg/g were achieved in perfusion culture mode with the highest total amount of antibody C12 of 1854mg, however, the maximum C12 Ab concentration of 282mg/L was reached in balanced amino acid fed-batch bioprocess. Besides, it was observed that the metabolic by-products lactic acid of 15mmol/L and ammonia of 3.8 mmol/L were lowest for perfusion culture among three developed culture processes. Therefore, perfusion culture mode could be the optimum bioprocess for antibody C12 production by rCHO-C12 cells.