Development Of Cell Culture Process And Optimization Of Glycosylation Modification Of Omazumab

As a major type of Bronchial asthma,bronchial asthma is a chronic airway inflammation involving a variety of cells.According to incomplete statistics,about 80%of Chinese patients with bronchiolar asthma are allergic asthma,and most of them are IgE mediated.Omalizumab is a recombinant humanized anti-IGE monoclonal antibody that can treat moderate to severe allergic asthma mediated by IgE with significant efficacy and safety.Therefore,the development of domestic independent research and development of omazumab generic drugs is of great significance to the domestic pharmaceutical market.Firstly,CHO-K1 cells that could express omazumab provided by the customer were selected and a cell line that could stably express the antibody was selected for the experiment.Through different combinations of basic medium,supplementary medium,adding methods and culture temperature,a better culture process was selected,and the results were as follows:Under the combination of 5 different base culture-medium,4 different feeding culture-medium and their corresponding adding methods and 3 different temperatures,it was found that the base culture-medium CD CHO050 was paired with Feed008 and Feed009,and the adding time was Day3/5/7/9/11/13.When CD Feed 008/0094%/0.4%was added,the protein yield was the highest when Day5 was cooled to 32℃,and the results of protein CEX and SEC were similar to those of the original drug.Therefore,this culture condition was selected for follow-up experiments.However,the results of protein N-glycotype distribution under this condition were still different from that of the original study,specifically,the proportion of GOF and Man5 was about 25%and 50%higher than that of the original study,respectively,and the proportion of G1F was about 70%lower than that of the original study.The high proportion of Man5 sugar type will affect the quality of MAB drugs and increase the rate of drug clearance in the body.Therefore,in view of this situation,a single factor experiment was designed in combination with the literature,and the model was established through statistical analysis.The GraphPad Prism 9.0 software was used to identify the significance of the influence factors.It was found that these 7 kinds of uncommon sugars(raffinose,trisaccharide,trisaccharide,trehalose,lactulose,palagin sugar and alolone sugar)could reduce the proportion of Man5 sugar type,and raffinose had the best effect.The results of CEX,SEC,non-reduced CE-SDS,reduced CE-SDS and relative binding activity of binding proteins were obtained.Finally,the culture process of the cells was determined as follows:The basic medium CD CHO050 was supplemented with 1mM raffinose and supplemented with Feed008 and Feed009.The feeding time was Day3/5/7/9/11/13 and Feed 008/0094%/0.4%.The temperature was lowered to 32℃ on Day5.