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A novel, site-specific N-linked glycosylation model provides mechanistic insights into the process-condition dependent distinct Fab and Fc glycosylation of an IgG1 monoclonal antibody produced by CHO VRC01 cells
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  • Jayanth Venkatarama Reddy,
  • Thomas Leibiger,
  • Sumit K. Singh,
  • Kelvin Lee,
  • Eleftherios Papoutsakis,
  • Marianthi Ierapetritou
Jayanth Venkatarama Reddy
University of Delaware Department of Chemical & Biomolecular Engineering
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Thomas Leibiger
University of Delaware Department of Chemical & Biomolecular Engineering
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Sumit K. Singh
University of Delaware Department of Chemical & Biomolecular Engineering
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Kelvin Lee
University of Delaware Department of Chemical & Biomolecular Engineering
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Eleftherios Papoutsakis
University of Delaware Department of Chemical & Biomolecular Engineering

Corresponding Author:[email protected]

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Marianthi Ierapetritou
University of Delaware Department of Chemical & Biomolecular Engineering
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Abstract

The CHO VRC01 cell line produces an anti-HIV IgG1 monoclonal antibody containing N-linked glycans on both the Fab (variable) and Fc (constant) regions. Site-specific glycan analysis was used to measure the complex effects of cell culture process conditions on Fab and Fc glycosylation. Experimental data revealed major differences in glycan fractions across the two sites. Bioreactor pH was found to influence fucosylation, galactosylation, and sialylation in the Fab region and galactosylation in the Fc region. To understand the complex effects of process conditions on site-specific N-linked glycosylation, a kinetic model of site-specific N-linked glycosylation was developed. The model parameters provided mechanistic insights into the differences in glycan fractions observed in the Fc and Fab regions. Enzyme activities calculated from the model provided insights into the effect of bioreactor pH on site-specific N-linked glycosylation. Model predictions were experimentally tested by measuring glycosyltransferase-enzyme mRNA-levels and intracellular nucleotide sugar concentrations. The model was used to demonstrate the effect of increasing galactosyltransferase activity on site-specific N-linked glycan fractions. Experiments involving galactose and MnCl 2 supplementation were used to test model predictions. The model is capable of providing insights into experimentally measured data and also of making predictions that can be used to design media supplementation strategies.
13 Sep 2024Submitted to Biotechnology and Bioengineering
16 Sep 2024Submission Checks Completed
16 Sep 2024Assigned to Editor
16 Sep 2024Review(s) Completed, Editorial Evaluation Pending
22 Sep 2024Reviewer(s) Assigned
25 Oct 2024Editorial Decision: Revise Major