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.
Bibliographical noteFunding Information:
C.S.S., M.G.M., and C.M.O. contributed equally to this work. C.S.S. and M.G.M. designed and built gene expression constructs and generated the cell lines used in the study. C.M.O., T.S.L., and P.D. were involved in modeling the impact of overexpression of genes involved in glycosylation. K.L. and H.-Y.F. generated the parental cell line, CHO-2C10, used in this study. X.C. and L.Z. analyzed the glycan profiles of the samples generated from the various cell lines. All authors discussed the results. W.-S.H., M.J.S., C.S.S., M.G.M., C.M.O., T.S.L., and N.S. prepared the manuscript. C.S.S. and M.G.M. are supported in part by a grant from the University of Minnesota Office of the Vice President of Research. M.G.M. is supported in part by the NIGMS Biotechnology Training Program (T32GM008347–22). M.J.S. is supported by The Defense Advanced Research Projects Agency (Grant No. D17AP00028). The views, opinions, and findings contained in this article are those of the authors and should not be interpreted as representing the official views or policies, either expressed or implied, of the Defense Advanced Research Projects Agency or the Department of Defense. The authors declare no competing financial interest.
© 2019 American Chemical Society.
- CHO cells
- DNA assembly
- IgG glycosylation
- post-translational modification
- systems-level modeling