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.

Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system.

Background: Optimization of chemically defined medium has been a critical way to produce monoclonal antibody. Usually, amount of glutamine was added into the feed medium, but a half of asparagine was added. Our study found that asparagine was important in the antibody production phase. Increasing the ratio of asparagine to glutamine in feed medium for enhancement of antibody production in CHO-DHFR cell culture would be an efficient way. Results:We optimized the total amount and the ratio of the two vital amino acids in feed medium to increase antibody production. In this work, we have demonstrated that feeding medium of high ratio between asparagine and glutamine (FB-H) can enhance the cell density after reaching the stationary phase. Moreover, FB-H was shown to improve cell maintenance, and increased the antibody production. The metabolic flux analysis proved that ratio of asparagine to glutamine had little influence on glycolysis. Furthermore, the TCA cycle of FB-H was enhanced by 20 % compared to that of low ratio of asparagine to glutamine (FB-L). And the energy metabolism of FB-H was 22.6 % higher than that of FB-L. For the later, lactate can be less produced in FB-H. Conclusions:We should improve the ratio between asparagine and glutamine in feed medium properly under the premise of no influence on cell growth to achieve high mAb producing goal.

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SEC-HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes.