Model-Driven Engineering of N‑Linked Glycosylation in Chinese Hamster Ovary Cells

Chinese hamster ovary
(CHO) cells are used for industrial production
of protein-based therapeutics (i.e., “biologics”). Here
we describe a method for combining systems-level kinetic models with
a synthetic biology platform for multigene overexpression to rationally
perturb N-linked glycosylation. Specifically, we sought to increase
galactose incorporation on a secreted Immunoglobulin G (IgG) protein.
We rationally design, build, and test a total of 23 transgenic cell
pools that express single or three-gene glycoengineering cassettes
comprising a total of 100 kilobases of engineered DNA sequence. Through
iterative engineering and model refinement, we rationally increase
the fraction of bigalactosylated glycans five-fold from 11.9% to 61.9%
and simultaneously decrease the glycan heterogeneity on the secreted
IgG. Our approach allows for rapid hypothesis testing and identification
of synergistic behavior from genetic perturbations by bridging systems
and synthetic biology.