(511e) Insights into the Effect of Bioreactor pH on Chinese Hamster Ovary Cell Metabolism and Site-Specific N-Linked Glycosylation of VRC01 Mab

Chinese Hamster Ovary (CHO) cells are used to produce 60% of the monoclonal antibodies (CHO) on the market [1]. CHO cell process development involves cell line development and media optimization. This is followed by bioreactor studies to determine the pH, dissolved oxygen and temperature setpoints. It is widespread practice in the industry to work on temperature shifts in the culture to maximize production of mAbs [2, 3]. However, studies have also used pH shifts in the culture to maximize the titer and control product quality [4, 5]. Scaling up the production to large scale bioreactors shows that gradients in pH within the bioreactor can significantly affect the metabolism and N-linked glycosylation [6, 7]. Although there exist studies in the literature that have reported on the effect of bioreactor pH on the uptake/production of various metabolites [8], the literature studies reporting daily measurements of concentrations of glucose, lactate, ammonia, amino acids and titer at different pH conditions in a fed-batch bioreactor is scarce. About 20% of IgG molecules in human plasma were determined to have N-linked glycosylation sites in both the Fc and Fab regions [9]. Fab glycans can also influence mAb affinity, activity, half-life and aggregation [10]. The mAb produced in this study is the VRC01 molecule. This is a broadly neutralizing antibody used to treat HIV. The VRC01 molecule contains glycans in the Fc and Fab region. The effect of cell culture parameters on Fab region glycosylation of mAbs has been poorly studied in the literature. This study was performed to better understand the effect of pH on CHO cell metabolism and site specific N-linked glycosylation.

To study the effect of bioreactor pH on metabolism and N-linked glycosylation, VRC01 producing CHO cells were grown at pH of 6.75, 7 and 7.25 in fed-batch mode in a 1 L Eppendorf bioflo 120 bioreactor system. Daily measurements of concentrations of glucose, lactate, ammonia, titer, amino acid and CHO cells were taken. Samples to measure site specific N-linked glycosylation were taken every two days, starting from day 4 of the culture. Increased glucose uptake rate, increased lactate production rates, increased titer production rates, and reduced ammonia production rates were observed when bioreactor pH was increased. Significant difference in amino acid uptake rates were also observed. This shows that changes in cell culture media could prevent overfeeding or depletion of nutrients at different pH conditions. The differences in glycan structures on the two sites were measured by denaturing and digesting the mAb using trypsin. This was followed by intact glycopeptide analysis by using a LC-MS [11]. The experimental results showed that glycans in the Fc region were fully fucosylated and were not sialylated. Less than 25% of the glycans in the Fab region were fucosylated. Nearly 70% of the glycans in the Fab region were sialylated. Increase in pH led to a decrease in fucosylation in the Fab region but did not affect fucosylation in the Fc region. Increase in pH also led to increase in sialylation in the Fab region. Reduction in pH also led to decrease in galactosylation in both regions. The use of a mathematical model could provide further insight into the activity of N-linked glycosylation enzymes at the various pH conditions.

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