Development Of Production Process Of Monoclonal Antibody By Perfusion Culture In Recombinant CHO Cell Reactor Based On Scale Down Model

Monoclonal antibodies have become the leading products in the biopharmaceutical market because of their high specificity and low side effects.For antibody products with different quality characteristics,in order to improve product quality and expression,reduce production cost and increase process flexibility,many kinds of culture processes for mammalian cells have emerged.Among them,perfusion culture process has the advantages of improving antibody productivity and product quality,and is expected to become one of the important production methods in cell culture process in the future.Aiming at humanized monoclonal antibody products,taking Chinese hamster ovary cell(CHO)as the research object,using design of experiment(DOE)as the development tool,the scale-down model of antibody production by perfusion culture and the corresponding production process were established.Firstly,the frequency of fluid exchange and the concentration of culture medium were determined by shake flask culture.Based on this,a reduced model of semi perfusion culture was established.The static perfusion culture was studied by releasing cell suspension,and the dynamic perfusion culture strategy was determined.The antibody expression was further increased by screening the cooling temperature and cooling time.Subsequently,glucose was added into the perfusion medium in advance to determine the appropriate sugar content of the perfusion medium.The reduced model and production process of antibody production by perfusion culture were established under the condition of shake flask culture.Using the central composite experimental design,the partial reduction model was determined as follows:the initial upper tank medium was the original concentration basic medium,the perfusion medium was 1.2 times the concentration basic medium,and the liquid exchange interval was 16 h+8 h;The cooling strategy was determined by using Box-Behnken experimental design,in which the cooling temperature was 35℃ and the density of live cells was 37.5 × 106 cells/mL;The glucose content of perfusion medium was determined to be 19 g/L by single factor experimental design.Finally,the perfusion culture process was verified in a 5 L reactor.Compared with the mature 5 L reactor fed culture process,the reactor scale perfusion culture process established in this study increased the culture days by 1.4 times and the protein expression by 2.5 times,and had good consistency in product quality(protein purity,charge heterogeneity and glycosylation),A highly efficient antibody reactor perfusion culture process was successfully established by using the shaking flask model of recombinant CHO cells for antibody production.This study provides an efficient and low-cost development method for the perfusion culture process of other antibody products,which has important application value.