Flux balance analysis and peptide mapping elucidate the impact of bioreactor pH on Chinese Hamster Ovary (CHO) cell metabolism and N-linked glycosylation in the Fab and Fc regions of the produced IgG

Chinese Hamster Ovary (CHO) cells were grown at different bioreactor pH conditions to detail how bioreactor pH affects cell metabolism and site-specific N-linked glycosylation of the produced broadly neutralizing anti-HIV IgG monoclonal antibody VRC01. The data show that pH affects cell growth, glucose/lactate metabolism, IgG production rates, nonessential amino acid metabolism and ammonia accumulation. Parsimonious Flux Balance Analysis (pFBA) and Flux Variability Analysis (FVA) provide insight into the effect of pH on core intracellular reactions at the different pH conditions and culture durations. pFBA revealed the contribution of sources for the production of the toxic metabolite ammonia and provided insights into the switch from ammonia production to consumption. It also documented that culture duration and pH alter the complex bimodal patterns (production/uptake) of several essential and non-essential amino acids. The VRC01 IgG has N-linked glycosylation sites in both the Fc region and the Fab region. Site- specific N-linked glycan analysis using glycopeptide mapping demonstrated that pH significantly affects the glycosylation profiles of the two IgG sites. The Fc region glycans were completely fucosylated but did not contain any sialylation. The Fab region glycans were not completely fucosylated but contained sialylated glycans. Bioreactor pH affected both the fucosylation and sialylation indexes in the Fab region and the galactosylation index of the Fc region. However, fucosylation in the Fc region was unaffected thus demonstrating that the effect of pH on site- specific N-linked glycosylation is complex.