Characterization And Optimization Of Monoclonal Antibody Basic Charge Variants In CHO Cell Culture

Charge heterogeneity is a key property of antibody quality and has received great attention in the development and production of antibody drugs.Although many of the in vitro chemical mechanisms that initiate charge variants are clear,the underlying cause of charge variant formation is not yet due to multiple factors involved in cell culture,particularly the various media components and their interactions.Clearly,therefore,more research is urgently needed to clarify the reasons for the formation of antibody charge variants and related mechanisms.In the early development of CHO(Chinese hamster overy,CHO)cell serum-free medium,the addition of uridine significantly increased the maximum viable cell density and antibody expression.Based on the verification of this result,this study first analyzed the effects of uridine addition on the mass properties of antibody charge variants,glycosylation,polymer,fragmentation,hydrophobicity,etc.The study found that the addition of uridine did not improve.The problem of extremely high levels of basic charge variants of antibody.To this end,the composition of the basic charge variant in the antibody produced by adding uridine to the fed-batch medium was further examined,and it was found that although the content of the lysine variant was greatly reduced in the composition of the basic charge variant.However,the oxidative ratio of methionine and tryptophan in the Fab region of the antibody increases.It was confirmed by hydrogen peroxide and AAPH(2,2'-Azobis(2-methyl-propionamidine)dihydrochloride,AAPH)incubation that both methionine oxidation and tryptophan oxidation are important for the formation of basic charge variants of mAbl antibody.Subsequently,by adding an antioxidant such as taurine,ammonium ferric citrate,N-acetyl-L-cysteine,silymarin and lipoic acid to the cell culture medium,it was desired to reduce the content of the basic charge variant.It was found that taurine not only reduced the content of the basic charge variant of the antibody,but also increased the content of the main peak,and also increased the maximum viable cell density,the antibody yield was significantly increased.The addition of taurine had little effect on the quality properties of antibody such as glycosylation,polymerization and fragmentation.Real-time PCR and cell-free incubation experiments showed that taurine may reduce lysine variants by promoting expression of basic carboxypeptidase.The results of intracellular reactive oxygen species and redox potential of the culture medium showed that taurine may reduce the formation of oxidative variants by inhibiting the formation of reactive oxygen species and increasing the reducibility of the culture solution.The results of this study provide guidance for the future regulation of the generation of antibody charge heterogeneity in the production process and the improvement of the yield and quality of antibody drugs.