Effects Of Uridine On Charge Heterogeneity Of Monoclonal Antibody During RCHO Cells Fed Batch Cultures

With the wide application of therapeutic monoclonal antibody in various disease treatment,more and more manufacturers focused on the development of CHO cells culture process.Charge heterogeneity is one of the most critical quality attributes of antibodies,and has strong influence on drug's biological activity and safety.For antibody producing process development,the effective control of the charge variants generated in fed-batch culture is very important,which determines the stability and economy of the process.In this paper,the charge heterogeneity characteristic of a monoclonal antibody(mAb)produced by an rCHO cell line was firstly investigated This study investigated culture parameters' effects on charge heterogeneity based by a monoclonal antibody(mAb)produced by a rCHO cell line,and found that uridine is a key component that strongly affects the charge heterogeneity of antibody,and its mechanism of action was investigated.First,uridine was found to have an important influence on cell growth,antibody producing,and charge heterogeneity by factorial experiments of uridine,manganese,and galactose.Uridine was beneficial to the maintenance of cell viability,which made IVCD increased by 50%,and the final titer improved by 64%.What's more,the acidic and basic variants contents in no uridine fed cultures is 30.3%and 11.8%,while in uridine fed cultures,acidic variants contents decreased to 20.1%,and basic variants increased to 25.2%.Then,based on the significant effect of uridine on acidic and basic variants,post-translational modifications such as glycoforms,aggregates,and glycation levels were investigated by normal phase-HPLC,size exclusion chromatography and boronate affinity chromatoraphy.The results showed that the decrease of aggregates and glycated level are responsible for the reduction of acidic variants,and the increase of basic variants comes from non-lysine variants.Then,the mechanism of uridine's effects on charge variants was further explored from the perspective of pH and redox balance.The addition of uridine lead intracellular and extracellular pH significantly increased compared with control cultures.At the same time,the intracellular ROS content decreased,and the extracellular redox potential decreased,indicating that uridine fed cultures presents more reducing condition.The increase of pH can enhance the catalytic activity of carboxypeptidase and PAM enzymes,which caused the increase of basic variants.The enhancement of reducing power can inhibit the formation of aggregates,which caused a decrease of acidic variants.Through extracellular cell-free experiments,the extracellular changes of acid and basic charge variants were investigated.It was found that uridine feeding decreased excellular degradation rate of basic variants and the decrease in cell viability accelerated the generation of acidic variants and the degradation of basic variants.Based on the significant effect of uridine on antibody charge heterogeneity,the effects of the concentration of uridine feeding and the time of feeding on the antibody charge variant content were investigated.The result shows that when uridine feed concentration was 0.1×,the contents of acidic variants and basic variants were 27.1%and 14.1%,respectively,indicated that the effect on charge variants was smaller than amount of 1 x concetration.Feed time also influences charge variants,the basic variants contents can be controlled when feed uridine just from day 6 to day 12.The acidic variants and basic variants can be controled to 22.1%and 17.6%using 1× uridine concetration and feed from day 6 to day 12.This feeding strategy dereased the excessive contents of charge variants.Through the research of this paper,uridine was found to be a key component influencing the antibody charge heterogeneity in fed-batch medium,and its influence mechanism was discussed.These findings provides new ideas and guidance for the development of other monoclonal antibody processes.