(24b) Investigating the Effect of Critical Process Parameters and Media Additives on Site Specific N-Linked Glycosylation of Monoclonal Antibodies

Several therapeutic glycoproteins and viral proteins contain multiple N-linked glycosylation sites. The function of N-glycans at different sites can vary for different molecules. Recent advances in proteomics have revealed a large diversity in glycans present at these sites, revealing that about 20% of antibodies in the human body contain N-linked glycan sites in the Fab and Fc region [1]. VRC01 is an anti-HIV monoclonal antibody containing N-linked glycans in both regions. VRC01 producing CHO cells were cultured at three bioreactor pH conditions (6.75, 7 and 7.25). Site-specific glycan analysis was used to measure the fractions of different glycans on these two sites at different bioreactor pH conditions. The experimental data revealed major differences in glycan fractions across the two sites. Fc glycans were completely fucosylated and were not sialylated. Fab glycans were sialylated, highly galactosylated and contained limited fucosylation. Bioreactor pH had varying effects on the glycans present at these two different sites. A kinetic model for site specific N-linked glycosylation model was developed. The model parameters were used to quantify the differences in glycan structures across the two sites and provide mechanistic insights into why the differences were observed across the two sites. Changes in bioreactor culture time or pH leads to a shift in cellular metabolism or expression levels of N-linked glycosylation enzymes. The model was used to determine the enzyme activities that were validated using measurements of enzyme expression levels and intracellular nucleotide sugar concentrations. The site-specific N-linked glycosylation model has subsequently been used to guide experiments related to improving galactosylation of the VRC01 and study the effect of galactose addition on glycan fractions from both the sites. Widespread application of mathematical modeling in biopharma is hindered by the lack of studies that apply these models across multiple cell lines and molecules. Hence, the results obtained for VRC01 have been compared to the N-linked glycosylation fractions observed in Trastuzumab (i.e., Herceptin biosimilar) produced in another cell line under varying galactose concentrations and pH shift conditions [2]. The Fc region of VRC01 and Trastuzumab have similar N-linked glycosylation profiles (high fucosylation, moderate galactosylation, and very low sialylation). A model-based analysis on the effect of bioreactor pH and galactose addition on both these molecules has been performed.

Acknowledgements: This research was funded by US FOOD and DRUG ADMINISTRATION, grant number DHHS-FDA-R01FD006588, grant number U01FD007695A and NIIMBL PC 5.2-112.

References

1. Van De Bovenkamp, F.S., et al., The Emerging Importance of IgG Fab Glycosylation in Immunity. The Journal of Immunology, 2016. 196(4): p. 1435-1441.
2. Aron, G., et al., Temporal Effects of Galactose and Manganese Supplementation on Monoclonal Antibody N-Linked Glycosylation in Fed-Batch and Perfusion Bioreactor Operation. bioRxiv, 2023: p. 2023.04.15.535602.