Process Optimization Based On The Structural Characterization Of Acidic Charge Variants For CHO Cells In Fed-batch Culture

With the increasingly stringent supervision on the safety of biological drug by FDA or CFDA,the research on quality analysis and control of antibody drug has become the hot spot.Charge variant,which can affect antibodies' properties in vitro or vivo,becomes a critical quality attribute and a routine product lot release indicator in the bio-pharmaceutical industry.However,the understanding of charge heterogeneity is not in-depth,and the charge variant contents are still difficult to achieve precise regulation in the large-scale production of antibody drugs.During the early upstream process(in tubes)development of GS-CHO cells expressing IgG1 antibody,the acidic charge variant contents of antibody were 18.4%,which were 1.47 times higher than that of the original drugs(12.5%).According to the concept of "quality by design",a comprehensive structural characterization of acidic charge variant,including N-linked glycoforms,disulfide bond reduction,aggregation,fragmentation,asparagine deamidation and secondary structure,is critical to solve the above problem.To understand the multiple sources of the acidic charge variant completely,the major charge forms of IgGl antibody were firstly isolated and then analyzed by a battery of characterization tools.It was found that various degrees of disulfide bond reduction,deamination of HC-T8 Asn84 and HC-T35 Asn388,and aggregation account for the majority of acidic charge heterogeneity and the terminal galactosylation content was in relation to the acidic charge heterogeneity.The relationship between acidic charge heterogeneity and galactosylation content was further explored by weak cation exchange chromatography with the use of ?-1-4 galactosidase digestion experiment.The results showed that galactosylation was not the source of acidic charge variants per se.Next,to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism,cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography.It was found that acidic charge variant contents were significantly decreased by pH downshift.Then,to reveal the mechanism by which the content of acidic charge variants was reduced under pH downshift condition,the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored.Several analysis experiments including size exclusion chromatography,capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions,tryptic peptide mapping,and reduced antibody mass were applied.The results showed that the mechanism by which the content of acidic charge variants was reduced was that the contents of disulfide bond reduction,galactosylation,and asparagine deamination of the HC-T35 Asn388 in the Fc domain were reduced by pH downshift.What's more,it was found that amino acids and vitamins had positive effects on the formation of acidic charge variants using the cell-free antibody incubation experiment.Besides,carbohydrates and metal ions had no effect on the contents of acidic cahrge variants.Through the univariate cell experiment,it was found that vitamin V concentration(1×?50×)had no effect on the acidic charge variant contents of the antibody.Meanwhile,the acidic charge variant contents were increased by increasing the amino acid C concentration(1×?40×).Its mechanism was then further explored from two aspects,which were the oxidative reduction potential of the culture supernatant and the mRNA level of thioredoxin and glutaredoxin.The results showed that the oxidative reduction potential was decreased by increasing amino acid C concentration,which led to the increase of the disulfide bond reduction rate,and then resulted in an increase of acidic charge variant contents.Based on the above study results,a cell culture process with low acidic charge variant contents was designed.Finally,the acidic cahrge variant contents were reduced to 14.5%from 24.5%in the 2-L bioreactor scale.Through this research,we can not only deepen the understanding of the acidic charge variants of the antibody,but also provide guidance for the industrial production of antibodies with low acidic charge variant contents.