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

Christopher S. Stach; Meghan G. McCann; Conor M. O'Brien; Tung S. Le; Nikunj Somia; Xinning Chen; Kyoungho Lee; Hsu Yuan Fu; Prodromos Daoutidis; Liang Zhao; Wei Shou Hu; Michael Smanski

doi:10.1021/acssynbio.9b00215

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

Christopher S. Stach, Meghan G. McCann, Conor M. O'Brien, Tung S. Le, Nikunj Somia, Xinning Chen, Kyoungho Lee, Hsu Yuan Fu, Prodromos Daoutidis, Liang Zhao, Wei Shou Hu, Michael Smanski

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Abstract

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.

Original language	English (US)
Pages (from-to)	2524-2535
Number of pages	12
Journal	ACS Synthetic Biology
Volume	8
Issue number	11
DOIs	https://doi.org/10.1021/acssynbio.9b00215
State	Published - Nov 15 2019

Bibliographical note

Funding 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.

Publisher Copyright:
© 2019 American Chemical Society.

Keywords

CHO cells
DNA assembly
IgG glycosylation
post-translational modification
systems-level modeling

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title = "Model-Driven Engineering of N-Linked Glycosylation in Chinese Hamster Ovary Cells",

abstract = "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.",

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note = "Funding 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. Publisher Copyright: {\textcopyright} 2019 American Chemical Society.",

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AU - Le, Tung S.

AU - Somia, Nikunj

AU - Chen, Xinning

AU - Lee, Kyoungho

AU - Fu, Hsu Yuan

AU - Daoutidis, Prodromos

AU - Zhao, Liang

AU - Hu, Wei Shou

AU - Smanski, Michael

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N2 - 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.

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

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Keywords

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