Skip to main content
SpringerLink
Log in

Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Arden N, Betenbaugh MJ (2004) Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 22:174–180

    Article  CAS  Google Scholar 

  • Behr JR, Matsumoto Y, White FM, Sasisekharan R (2005) Quantification of isomers from a mixture of twelve heparin and heparan sulfate disaccharides using tandem mass spectrometry. Rapid Comm Mass Spectrom 19:2553–2562

    Article  CAS  Google Scholar 

  • Bragonzi A, Distefano G, Buckberry LD, Acerbis G, Foglieni C, Lamotte D, Campi G, Marc A, Soria MR, Jenkins N, Monaco L (2000) A new Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins. Biochim Biophys Acta 1474:273–282

    Article  CAS  Google Scholar 

  • Choi SS, Rhee WJ, Park TH (2002) Inhibition of human cell apoptosis by silkworm hemolymph. Biotechnol Prog 18:874–878

    Article  CAS  Google Scholar 

  • Choi SS, Rhee WJ, Kim EJ, Park TH (2006) Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene. Biotechnol Bioeng 95:459–467

    Article  CAS  Google Scholar 

  • Ciucanu I, Costello CE (2003) Elimination of oxidative degradation during the per-o-methylation of carbohydrates. J Am Chem Soc 125:16213–16219

    Article  CAS  Google Scholar 

  • Dorstyn L, Kumar S (2006) A cytochrome-c-free fly apoptosome. Cell Death Differ 13:1049–1051

    Article  CAS  Google Scholar 

  • Dorstyn L, Read S, Cakouros D, Huh JR, Hay BA, Kumar S (2002) The role of cytochrome-c in caspase activation in Drosophila melanogaster cells. J Cell Biol 156:1089–1098

    Article  CAS  Google Scholar 

  • Dorstyn L, Mills K, Lazebnik Y, Kumar S (2004) The two cytochrome-c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells. J Cell Biol 167:405–410

    Article  CAS  Google Scholar 

  • Fukuta K, Abe R, Yokomatsu T, Kono N, Asanagi M, Omae F, Minowa MT, Takeuchi M, Makino T (2000a) Remodeling of sugar chain structures of human interferon-γ. Glycobiology 10:421–430

    Article  CAS  Google Scholar 

  • Fukuta K, Yokomatsu T, Abe R, Asanagi M, Makino T (2000b) Genetic engineering of CHO cells producing human interferon-gamma by transfection of sialyltransferases. Glycoconj J 17:895–904

    Article  CAS  Google Scholar 

  • Helenius A, Aebi M (2001) Intracellular functions of N-linked glycans. Science 291:2364–2369

    Article  CAS  Google Scholar 

  • Jang KS, Kim YG, Gil GC, Park SH, Kim BG (2009) Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines. Anal Biochem 386:228–236

    Article  CAS  Google Scholar 

  • Jenkins N, Curling EM (1994) Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb Technol 16:354–364

    Article  CAS  Google Scholar 

  • Jenkins N, Parekh RB, James DC (1996) Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 14:975–981

    Article  CAS  Google Scholar 

  • Kim EJ, Park TH (2003) Anti-apoptosis engineering. Biotechnol Bioprocess Eng 8:76–82

    Article  CAS  Google Scholar 

  • Kim EJ, Rhee WJ, Park TH (2001) Isolation and characterization of an apoptosis-inhibiting component from the hemolymph of Bombyx mori. Biochem Biophys Res Commun 285:224–228

    Article  CAS  Google Scholar 

  • Koo TY, Park JH, Park HH, Park TH (2008) Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-β in serum-free suspension culture of CHO cells. Process Biochem 44:287–296

    Google Scholar 

  • Ma XM, Blenis J (2009) Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol 10:307–318

    Article  Google Scholar 

  • Means JC, Muro I, Clem RJ (2006) Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system. Cell Death Differ 13:1222–1234

    Article  CAS  Google Scholar 

  • Ngantung FA, Miller PG, Brushett FR, Tang GL, Wang DI (2006) RNA interference of sialidase improves glycoprotein sialic acid content consistency. Biotechnol Bioeng 95:106–119

    Article  CAS  Google Scholar 

  • Ninonuevo MR, Ward RE, LoCascio RG, German JB, Freeman SL, Barboza M, Mills DA, Lebrilla CB (2007) Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry. Anal Biochem 361:15–23

    Article  CAS  Google Scholar 

  • Ohtsubo K, Marth JD (2006) Glycosylation in cellular mechanisms of health and disease. Cell 126:855–867

    Article  CAS  Google Scholar 

  • Park JH, Park HH, Park TH (2010) Cellular engineering for the high-level production of recombinant proteins in mammalian cell systems. Kor J Chem Eng 27:1042–1048

    Article  CAS  Google Scholar 

  • Park JH, Park HH, Choi SS, Park TH (2012) Stabilization of enzymes by the recombinant 30Kc19 protein. Process Biochem 47:164–169

    Article  CAS  Google Scholar 

  • Rhee WJ, Park TH (2000) Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis. Biochem Biophys Res Commun 271:186–190

    Article  CAS  Google Scholar 

  • Rhee WJ, Kim EJ, Park TH (2002) Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells. Biochem Biophys Res Commun 295:779–783

    Article  CAS  Google Scholar 

  • Rhee WJ, Lee EH, Park TH (2009) Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti-apoptotic effect in Sf9 cell. Biotechnol Bioprocess Eng 14:645–650

    Article  CAS  Google Scholar 

  • Rifai A, Fadden K, Morrison SL, Chintalacharuvu KR (2000) The N-glycans determine the differential blood clearance and hepatic uptake of human immunoglobulin (Ig)A1 and IgA2 isotypes. J Exp Med 191:2171–2182

    Article  CAS  Google Scholar 

  • Sakai N, Mori S, Izumi S, Haino-Fukushima K, Ogura T, Maekawa H, Tomino S (1988) Structures and expression of mRNAs coding for major plasma proteins of Bombyx mori. Biochim Biophys Acta 949:224–232

    Article  CAS  Google Scholar 

  • Shaw RJ (2009) LKB1 and AMP-activated protein kinase control of mTOR signalling and growth. Acta Physiol 196:65–80

    Article  CAS  Google Scholar 

  • Stubbs HJ, Lih JJ, Gustafson TL, Rice KG (1996) Influence of core fucosylation on the flexibility of a biantennary N-linked oligosaccharide. Biochemistry 35:937–947

    Article  CAS  Google Scholar 

  • Varkey J, Chen P, Jemmerson R, Abrams JM (1999) Altered cytochrome-c display precedes apoptotic cell death in Drosophila. J Cell Biol 144:701–710

    Article  CAS  Google Scholar 

  • Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3:97–130

    Article  CAS  Google Scholar 

  • Walsh G (2003) Biopharmaceutical benchmarks—2003. Nat Biotechnol 21:865–870

    Article  CAS  Google Scholar 

  • Wang W, Li W, Ikeda Y, Miyagawa JI, Taniguchi M, Miyoshi E, Sheng Y, Ekuni A, Ko JH, Yamamoto Y, Sugimoto T, Yamashita S, Matsuzawa Y, Grabowski GA, Honke K, Taniguchi N (2001) Ectopic expression of alpha1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase. Glycobiology 11:165–174

    Article  CAS  Google Scholar 

  • Wang ZS, Park JH, Park HH, Tan WS, Park TH (2010) Enhancement of therapeutic monoclonal antibody production in CHO cells using 30Kc6 gene. Process Biochem 45:1852–1856

    Article  CAS  Google Scholar 

  • Wang ZS, Park JH, Park HH, Tan WS, Park TH (2011) Enhancement of recombinant human EPO production and sialylation in Chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression. Biotechnol Bioeng 108:1634–1642

    Article  CAS  Google Scholar 

  • Wong NS, Yap MG, Wang DI (2006) Enhancing recombinant glycoprotein sialylation through CMP-sialic acid transporter over expression in Chinese hamster ovary cells. Biotechnol Bioeng 93:1005–1016

    Article  CAS  Google Scholar 

  • Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (nos. 2011-0000331, 2011K000682, and 2011-0020984). This work was also supported by the Pioneer Research Program and by Basic Science Research Program through the NRF grant funded by the Ministry of Education, Science, and Technology (nos. 2011-0001643 and 2010-0002520). This work was also supported by WCU (World Class University) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (R32-2010-000-10213-0) and by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (grant no. A103017).

Author information

Authors and Affiliations

  1. School of Chemical and Biological Engineering, Bio-MAX Institute, Seoul National University, Seoul, 151-744, Republic of Korea

    Ju Hyun Park, Zesong Wang, Hee-Jin Jeong, Hee Ho Park, Byung-Gee Kim & Tai Hyun Park

  2. The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China

    Zesong Wang & Wen-Song Tan

  3. Department of Food and Nutrition, Myongji University, Yongin, Gyeonggido, 449-728, Republic of Korea

    Shin Sik Choi

Authors
  1. Ju Hyun Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zesong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hee-Jin Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hee Ho Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Byung-Gee Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wen-Song Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shin Sik Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tai Hyun Park
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tai Hyun Park.

Additional information

Ju Hyun Park and Zesong Wang contributed equally to this study

Rights and permissions

Reprints and permissions

About this article

Cite this article

Park, J.H., Wang, Z., Jeong, HJ. et al. Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19. Appl Microbiol Biotechnol 96, 671–683 (2012). https://doi.org/10.1007/s00253-012-4203-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4203-0

Keywords

Search

Navigation