Online citations, reference lists, and bibliographies.
← Back to Search

Enhancement Of Recombinant Human EPO Production And Glycosylation In Serum-free Suspension Culture Of CHO Cells Through Expression And Supplementation Of 30Kc19

J. Park, Zesong Wang, Hee-Jin Jeong, Hee Ho Park, B. Kim, W. Tan, S. Choi, T. Park
Published 2012 · Biology, Medicine

Cite This
Download PDF
Analyze on Scholarcy
Share
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 paper references
10.1093/GLYCOB/3.2.97
Biological roles of oligosaccharides: all of the theories are correct
A. Varki (1993)
10.1038/nrm2672
Molecular mechanisms of mTOR-mediated translational control
X. M. Ma (2009)
10.1016/J.AB.2006.11.010
Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry.
Milady Niñonuevo (2007)
10.1021/BI9513719
Influence of core fucosylation on the flexibility of a biantennary N-linked oligosaccharide.
H. Stubbs (1996)
10.1016/j.ab.2008.12.015
Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines.
Kyoung-Soon Jang (2009)
10.1007/BF02940260
Anti-apoptosis engineering
Eun Jeong Kim (2003)
10.1038/sj.cdd.4401821
Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system
J. Means (2006)
10.1023/A:1010977431061
Genetic engineering of CHO cells producing human interferon-γ by transfection of sialyltransferases
K. Fukuta (2004)
10.1083/jcb.200408054
The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells
L. Dorstyn (2004)
10.1016/J.TIBTECH.2004.02.004
Life and death in mammalian cell culture: strategies for apoptosis inhibition.
N. Arden (2004)
10.1021/bp020001q
Inhibition of Human Cell Apoptosis by Silkworm Hemolymph
S. Choi (2002)
10.1111/j.1748-1716.2009.01972.x
LKB1 and AMP‐activated protein kinase control of mTOR signalling and growth
R. Shaw (2009)
10.1038/nbt0910-917
Biopharmaceutical benchmarks 2010
G. Walsh (2010)
10.1016/J.PROCBIO.2011.10.022
Stabilization of enzymes by the recombinant 30Kc19 protein
J. Park (2012)
10.1006/BBRC.2000.2592
Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.
W. J. Rhee (2000)
10.1038/nbt1026
Production of recombinant protein therapeutics in cultivated mammalian cells
F. Wurm (2004)
10.1002/BIT.21023
Enhancement of recombinant protein production in Chinese hamster ovary cells through anti‐apoptosis engineering using 30Kc6 gene
S. Choi (2006)
10.1016/S0304-4165(00)00023-4
A new Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins.
A. Bragonzi (2000)
10.1016/J.PROCBIO.2008.09.018
Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-β in serum-free suspension culture of CHO cells
Tai Young Koo (2009)
10.1016/0167-4781(88)90086-3
Structures and expression of mRNAs coding for major plasma proteins of Bombyx mori.
N. Sakai (1988)
10.1002/BIT.20997
RNA interference of sialidase improves glycoprotein sialic acid content consistency
Frederyk Ngantung (2006)
10.1126/SCIENCE.291.5512.2364
Intracellular functions of N-linked glycans.
A. Helenius (2001)
10.1016/0141-0229(94)90149-X
Glycosylation of recombinant proteins: problems and prospects.
N. Jenkins (1994)
10.1038/NBT1103-1396A
Biopharmaceutical benchmarks—2003
G. Walsh (2003)
10.1002/BIT.20815
Enhancing recombinant glycoprotein sialylation through CMP‐sialic acid transporter over expression in Chinese hamster ovary cells
N. Wong (2006)
10.1016/j.cell.2006.08.019
Glycosylation in Cellular Mechanisms of Health and Disease
K. Ohtsubo (2006)
10.1007/S12257-009-0081-4
Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti-apoptotic effect in Sf9 cell
W. J. Rhee (2009)
10.1006/BBRC.2001.5148
Isolation and characterization of an apoptosis-inhibiting component from the hemolymph of Bombyx mori.
E. Kim (2001)
10.1016/S0006-291X(02)00746-5
Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells.
W. J. Rhee (2002)
10.1083/JCB.144.4.701
Altered Cytochrome c Display Precedes Apoptotic Cell Death in Drosophila
J. Varkey (1999)
10.1002/RCM.2079
Quantification of isomers from a mixture of twelve heparin and heparan sulfate disaccharides using tandem mass spectrometry.
J. Behr (2005)
10.1038/NBT0896-975
Getting the glycosylation right: Implications for the biotechnology industry
N. Jenkins (1996)
Appl Microbiol Biotechnol
(2012)
10.1002/bit.23091
Enhancement of recombinant human EPO production and sialylation in chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression
Zesong Wang (2011)
10.1007/S11814-010-0278-4
Cellular engineering for the high-level production of recombinant proteins in mammalian cell systems
J. Park (2010)
10.1093/GLYCOB/10.4.421
Remodeling of sugar chain structures of human interferon-gamma.
K. Fukuta (2000)
The two cytochromec species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells Remodeling of sugar chain structures of human interferon-γ
L Dorstyn (2000)
10.1038/sj.cdd.4401918
A cytochrome c-free fly apoptosome
L. Dorstyn (2006)
10.1083/jcb.200111107
The role of cytochrome c in caspase activation in Drosophila melanogaster cells
L. Dorstyn (2002)
10.1021/JA035660T
Elimination of oxidative degradation during the per-O-methylation of carbohydrates.
Ionel Ciucanu (2003)
10.1084/JEM.191.12.2171
The N-Glycans Determine the Differential Blood Clearance and Hepatic Uptake of Human Immunoglobulin (Ig)a1 and Iga2 Isotypes
A. Rifai (2000)
10.1093/GLYCOB/11.2.165
Ectopic expression of alpha1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase.
W. Wang (2001)
10.1016/J.PROCBIO.2010.03.029
Enhancement of therapeutic monoclonal antibody production in CHO cells using 30Kc6 gene
Zesong Wang (2010)



This paper is referenced by
Enhancement of L-Asparaginase Activity in L5178Y Mouse Lymphoma Cells by Conjugating 30Kc19 Protein and L-Asparaginase by Cleavable Linker
S. J. Yang (2016)
10.1038/s41598-017-05641-1
Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation
D. Falck (2017)
Application of functional genomics in the production of viral biopharmaceuticals: vaccines and vectors for gene therapy
A. S. F. Oliveira (2012)
10.1007/s10719-018-9835-6
Sialylation and sialyltransferase in insects
S. Ghosh (2018)
10.1038/s41598-019-53391-z
A human expression system based on HEK293 for the stable production of recombinant erythropoietin
C. Chin (2019)
10.1016/J.PROCBIO.2013.06.018
Anti-apoptotic mechanism of silkworm hemolymph in HeLa cell apoptosis
W. J. Rhee (2013)
10.1002/biot.201600040
Protein‐stabilizing and cell‐penetrating properties of α‐helix domain of 30Kc19 protein
Jina Ryu (2016)
10.1002/biot.201400253
Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration
Hee Ho Park (2014)
10.1080/10826076.2016.1247713
DNA removal on different chromatography supports used for EPO purification by spike studies
Lili Hernández (2016)
10.1002/mas.21471
Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2011-2012.
D. Harvey (2017)
10.1002/(issn)1860-7314
Biotechnology Journal
Mathew M. Jessop (2019)
10.1007/s10529-019-02681-7
Improved recombinant protein production by regulation of transcription and protein transport in Chinese hamster ovary cells
Kaiming Chen (2019)
10.1007/s11814-013-0108-6
Anti-inflammatory effects of silkworm hemolymph on lipopolysaccharide-stimulated macrophages
Mi-Ra Chang (2013)
10.1080/09168451.2017.1350563
A short synthetic chimeric sequence harboring matrix attachment region/PSAR2 increases transgene expression in Chinese hamster ovary cells
Q. Li (2017)
10.1002/bit.26549
Inhibition of apoptosis using exosomes in Chinese hamster ovary cell culture
Seora Han (2018)
10.1016/j.jbiotec.2014.02.001
Novel human renal proximal tubular cell line for the production of complex proteins.
Lukas Fliedl (2014)
10.1016/J.PROCBIO.2015.03.008
Enhancement of human erythropoietin production in Chinese hamster ovary cells through supplementation of 30Kc19-30Kc6 fusion protein
Hee Ho Park (2015)
10.1007/s12257-015-0152-7
Inhibition of apoptosis in HeLa cell by silkworm storage protein 1, SP1
J. Lee (2015)
10.1007/s00253-014-6045-4
Stabilization of cellular mitochondrial enzyme complex and sialyltransferase activity through supplementation of 30Kc19 protein
J. Park (2014)
10.1007/978-3-319-10320-4_9
Glycosylation in Cell Culture
M. Spearman (2015)
10.1016/j.biomaterials.2013.11.001
Enzyme delivery using the 30Kc19 protein and human serum albumin nanoparticles.
Hong Jai Lee (2014)
Utilización de elementos reguladores en "cis" y "trans" para la mejora de vectores baculovirus
J. Vidal (2014)
10.1080/07388551.2017.1416577
Impact of host cell line choice on glycan profile
Justin Bryan Goh (2018)
10.1007/978-1-4939-6972-2_2
Glycoengineering of CHO Cells to Improve Product Quality.
Q. Wang (2017)
Semantic Scholar Logo Some data provided by SemanticScholar