Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies' properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, 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 is 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 is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.
Background: Charge heterogeneity is one of the most critical quality attributes of antibodies, which has strong influence on drug's biological activity and safety. Finding out the key components that affecting charge variants is of great significance for establishing a competitive culture process. In this study, we first illustrated uridine's great impacts on antibody charge heterogeneity in CHO cell fed-batch cultures. Results:Uridine was beneficial to cell growth and the maintenance of cell viability, which made IVCC increased by 50% and the final titer improved by 64%. However, uridine had great influences on mAb's charge variants. In uridine added cultures, the acidic variant levels were about 9% lower than those in control cultures, while the basic variant levels were about 6% higher than those in control cultures. Further investigation found that the decrease of aggregates and glycated forms were responsible for the reduction of acidic variants. What's more, uridine decreased the lysine variant levels. Conclusions:Uridine's addition to fed-batch promoted cell growth and the final titer, in the meanwhile, uridine decreased the acidic variants dramatically. Therefore, feeding uridine is an efficient way to control the generation of acidic charge variants in up-stream process. These findings provide new ideas and guidance for the control and optimization of antibody charge heterogeneity in culture process developments. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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