Cell Culture Process Development And Optimization For The Anti-CD20 Monoclonal Antibody

Non-Hodgkin's lymphoma(NHL)which is a type of malignant lymphoma ranked fifth in the most commonly diagnosed cancer.Rituximab(anti CD20 monoclonal antibody)which has played a very important role in treatment of NHL is a new and effective drug in inhibiting tumor cell growth,and the importance in the treatment of NHL is gradually enhanced.Optimization of animal cell culture for improving the quality and consistent glycosylation of anti-CD20 monoclonal antibody in the industrial production process is critical for implementing industrialization and enhancing competitiveness in domestic and international markets.First of all,the basal and feed medium was determined by screening experiment.The detail results were as follows:in this experiment,the protein qualities(SEC,CEX and glycosylation)were not significantly impacted by different basal and feed medium.Which combined SFM4with 2x103A(containing 30 g/L YM001)and BR7 as feed medium has the highest titer.However,the glycosylation of protein from four shake flasks were very different from the standard,G0F proportion was about 23%higher than standard,G1F and G2F proportion were about 17%and 5%lower than standard respectively.To deal with higher G0F proportion and lower G1F and G2F proportion,Taking full factorial experiment design to determine How manganese chloride,uridine and galactose effected the glycosylation of protein.It was found that besides manganese chloride,other two additions significantly impacted on the protein quantity and glycosylation.By JMP software analysis:the basal medium was SFM4 added 4.5 mM uridine and4.5 mM galactose in it,feed medium was 2x103A(containing 30 g/L YM001)and BR7.Finally,two parallel 3 L bioreactors were used to validate the previous glycosylation optimization results,bioreactor cultures were seeded at 0.50~0.70×10~6 cells/mL in 3L bioreactors with 1.5 L working volume under conditions of 36.5?,pH 6.9±0.2,40%dissolved oxygen,250 rpm agitation.the basal medium was SFM4 added 4.5 mM uridine and4.5 mM galactose in it,feed medium was 2x103A(containing 30 g/L YM001)and BR7.Culture duration for 14 days,which achieved titer of2.4 g/L,the protein qualities(SEC?CEX and glycosylation)were quilt consistent with the standard.