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
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.