High-level expression of recombinant IgG1 by CHO K1 platform

Ningning Xu, Jianfa Ou, Al-Karim (Al) Gilani, Lufang Zhou, Margaret Liu

PDF(382 KB)
PDF(382 KB)
Front. Chem. Sci. Eng. ›› 2015, Vol. 9 ›› Issue (3) : 376-380. DOI: 10.1007/s11705-015-1531-5
RESEARCH ARTICLE

High-level expression of recombinant IgG1 by CHO K1 platform

Author information +
History +

Abstract

The Chinese Hamster Ovary (CHO K1) cell was used to express a targeted anti-cancer monoclonal antibody by optimizing the platform of the construction of production cell line in this study. The adherent CHO K1 was first adapted to suspension culture in chemical defined medium. Then the glutamine synthetase (GS) vector was applied to construct a single plasmid to overexpress a monoclonal antibody IgG1. Post transfection, the production of cell pool was optimized by glutamine-free selection and amplification using various concentrations of methionine sulfoximine. The best cell pool of CHO K1/IgG1 was used to screen the top single clone using the limiting dilution cloning. Finally, a high IgG1 production of 780 mg/L was obtained from a batch culture. This study demonstrated that the construction of high producing cell line, from gene to clone, could be completed within six month and the gene amplification improved protein production greatly.

Graphical abstract

Keywords

Chinese hamster ovary (CHO) / monoclonal antibody / IgG1 / amplification / cell line development

Cite this article

Download citation ▾
Ningning Xu, Jianfa Ou, Al-Karim (Al) Gilani, Lufang Zhou, Margaret Liu. High-level expression of recombinant IgG1 by CHO K1 platform. Front. Chem. Sci. Eng., 2015, 9(3): 376‒380 https://doi.org/10.1007/s11705-015-1531-5

References

[1]
Leone-Bay A. Next-generation protein therapeutics summit conference report. Therapeutic Delivery, 2011, 2(10): 1233–1234
[2]
Leader B, Baca Q J, Golan D E. Protein therapeutics: A summary and pharmacological classification. National Review, 2008, 7(1): 21–39
[3]
Zhou L, Xu N, Sun Y, Liu X M. Targeted biopharmaceuticals for cancer treatment. Cancer Letters, 2014, 352(2): 145–151
[4]
Edelman G M. Antibody structure and molecular immunology. Science, 1973, 180(4088): 830–840
[5]
Chames P, Regenmortel M V, Weiss E, Baty D. Therapeutic antibodies: Successes, limitations and hopes for the future. British Journal of Pharmacology, 2009, 157: 220–233
[6]
Butler M, Spearman M. The choice of mammalian cell host and possibilities for glycosylation engineering. Current Opinion in Biotechnology, 2014, 30: 107–112
[7]
Zhang P, Chan K F, Haryadi R, Bardor M, Song Z. CHO glycosylation mutants as potential host cells to produce therapeutic proteins with enhanced efficacy. Advances in Biochemical Engineering/Biotechnology, 2013, 131: 63–87
[8]
Lai T, Yang Y, Ng S K. Advances in Mammalian cell line development technologies for recombinant protein production. Pharmaceuticals, 2013, 6(5): 579–603
[9]
Omasa T, Onitsuka M, Kim W D. Cell engineering and cultivation of Chinese Hamster Ovary (CHO) cells. Current Pharmaceutical Biotechnology, 2010, 11(3): 233–240
[10]
Lewis N E, Liu X, Li Y, Nagarajan H, Yerganian G, O’Brien E, Bordbar A, Roth A M, Rosenbloom J, Bian C, Xie M, Chen W, Li N, Baycin-Hizal D, Latif H, Forster J, Betenbaugh M J, Famili I, Xu X, Wang J, Palsson B O. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nature Biotechnology, 2013, 31(8): 759–765
[11]
Florian M. Wurm. CHO quasispecies—Implications for manufacturing processes. Processes, 2013, 1: 296–311
[12]
Cruz Edmonds M C, Tellers M, Chan C, Salmon P, Robinson D K, Markusen J. Development of transfection and high-producer screening protocols for the CHOK1SV cell system. Molecular Biotechnology, 2006, 34(2): 179–190
[13]
Xu X, Nagarajan H, Lewis N E, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W, Hammond S, Andersen M R, Neff N, Passarelli B, Koh W, Fan H C, Wang J, Gui Y, Lee K H, Betenbaugh M J, Quake S R, Famili I, Palsson B O, Wang J. The genomic sequence of the Chinese Hamster Ovary (CHO)-K1 cell line. Nature Biotechnology, 2011, 29(8): 735–741
[14]
Jerums M, Yang X. Optimization of cell culture media. BioProcess International, 2005, Supplement: 38–44
[15]
Dale L L. Mammalian expression cassette engineering for high-level protein production. BioProcess International, 2006, 4(5): 14–23
[16]
Kingston R E, Kaufman R J, Bebbington C R, Rolfe M R. Amplification using CHO cell expression vectors. Current Protocols in Molecular Biology. Hoboken: John Wiley & Sons, 2002
[17]
Porter AJ, Dickson AJ, Racher AJ. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing. Realizing the potential in bioreactors. Biotechnology Progress, 2010, 26(5): 1446–14554
[18]
Harraghy N, Regamey A, Girod P A, Mermod N. Using matrix attachment regions to improve recombinant protein production. Methods in Molecular Biology, 2012, 801: 93–110
[19]
Hou J J, Hughes B S, Smede M, Leung K M, Levine K, Rigby S, Gray P P, Munro T P. High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system. New Biotechnology, 2014, 31(3): 214–220
[20]
Nair A R, Jinger X, Hermiston T W. Effect of different UCOE-promoter combinations in creation of engineered cell lines for the production of Factor VIII. BMC Research Notes, 2011, 4(178): 1–8
[21]
Lequin R. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical Chemistry, 2005, 51(12): 2415–2418
[22]
Meng Y G, Liang J, Wong W L, Chisholm V. Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene, 2000, 242: 201–207
[23]
Clarke J, Porter A, Davis J M. Cloning. Animal cell culture. New York: John Wiley & Sons, 2011, 231–254

Acknowledgements

This work was supported by RGC fund from The University of Alabama and the BRIGE grant (No. 1342390 and IIP-1026648) from the National Science Foundation.

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg
AI Summary AI Mindmap
PDF(382 KB)

Accesses

Citations

Detail

Sections
Recommended

/