Abstract
The degree of sialylation of therapeutic glycoproteins affects its circulatory half-life and efficacy because incompletely sialylated glycoproteins are cleared from circulation by asialoglycoprotein receptors present in the liver cells. Mammalian expression systems, often employed in the production of these glycoprotein drugs, produce heterogeneously sialylated products. Here, we describe how to produce highly sialylated glycoproteins using a Chinese hamster ovary (CHO) cell glycosylation mutant called CHO-gmt4 with human erythropoietin (EPO) as a model glycoprotein. The protocol describes how to isolate and characterize the CHO glycosylation mutants and how to assess the sialylation of the recombinant protein using isoelectric focusing (IEF). It further describes how to inactivate the dihydrofolate reductase (DHFR) gene in these cells using zinc finger nuclease (ZFN) technology to enable gene amplification and the generation of stable cell lines producing highly sialylated EPO.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Morell AG, Gregoriadis G, Scheinberg IH et al (1971) The role of sialic acid in determining the survival of glycoproteins in the circulation. J Biol Chem 246:1461–1467
Grabenhorst E, Schlenke P, Pohl S et al (1999) Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj J 16:81–97
Hossler P, Khattak SF, Li ZJ (2009) Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 19:936–949
Goh JS, Zhang P, Chan KF et al (2010) RCA-I-resistant CHO mutant cells have dysfunctional GnT I and expression of normal GnT I in these mutants enhances sialylation of recombinant erythropoietin. Metab Eng 12:360–368
Goh JS, Liu Y, Liu H et al (2013) Highly sialylated recombinant human erythropoietin production in large-scale perfusion bioreactor utilizing CHO-gmt4 (JW152) with restored GnT I function. Biotechnol J 9:100–109
Rees S, Coote J, Stables J et al (1996) Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. Biotechniques 20:102–104, 106, 108–110
Ho SC, Bardor M, Feng H et al (2012) IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol 157:130–139
Schriebl K, Trummer E, Weik R et al (2006) A novel strategy for quantitative isoform detection directly performed from culture supernatant. J Pharm Biomed Anal 42:322–327
Santiago Y, Chan E, Liu PQ et al (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 105:5809–5814
Doyon Y, Vo TD, Mendel MC et al (2010) Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods 8:74–79
Urnov FD, Miller JC, Lee YL et al (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646–651
Acknowledgment
This work was funded by the Agency for Science, Technology and Research (A*STAR).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Goh, J.S.Y., Chan, K.F., Song, Z. (2015). Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants. In: Castilho, A. (eds) Glyco-Engineering. Methods in Molecular Biology, vol 1321. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2760-9_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2760-9_22
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2759-3
Online ISBN: 978-1-4939-2760-9
eBook Packages: Springer Protocols