• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1945-1952
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• 2008

Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human ${\alpha}2$,3-sialyltransferase (${\alpha}2$,3-ST) and ${\beta}1$,4-galactosyltransferase (${\beta}1$,4-GT) are responsible for terminal sialylation and galactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When ${\alpha}2$,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1 %. When ${\alpha}2$,3-ST and ${\beta}1$,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT is more effective than the expression of ${\alpha}2$,3-ST alone. Coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.

• Biomolecules & Therapeutics
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• 제6권2호
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• pp.171-181
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• 1998

DA-3585, a biosynthetic recombinant human erythropoietin has been developed as a treatment for anemia associated with chronic renal failure in Dong-A pharmaceutical Co. Ltd. This study was carried out to assess its acute and subacute toxicities in rabbits. DA-3585 was intravenously administered to rabbits at dose levels of 6250, 12500 or 25000 lU/kg for single dose toxicity study and at dose levels of 100, 500 or 2500 lU/kg/day for 4-week repeated dose toxicity study. In the acute toxicity study, dose up to 25000 lU/kg had no adverse effect on the behavior or body weight gain. Pathological examinations revealed no abnormal gross lesions related to DA-3585. In the subacute toxicity study, all animals survived until termination of treatment. DA-3585 had no influence on clinical signs, food and water intake or on body weight changes. Hematological examination showed increases in the number of RBC, hemoglobin contents and hematocrit values with a dose dependent manner in the animals treated with DA-3585. Histopathological examination revealed erythroid hyperplasia in the bone marrow and extramedullary hematopoiesis in the liver. The changes detected in the hematological and histopathological examination presumably represent exaggerated pharmacological effects of erythropoietin. The NOAEL (no-observed-adverse-effect-level) of DA-3585 was estimated to be 100 lU/kg/ day under this study condition.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.338.1-338.1
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• 2002

The efficient Erythropoietin(EPO)-expression system in mammalian cells is required for massive production for therapeutic use. Ammonium ion is a major problem in the production of useful proteins by cultured animal cells and therefore it is of importance to devise a system by which a high productivity of human therapeutic recombinant protein can be maintained or enhanced under low ammonium concentration. (omitted)

• Reproductive and Developmental Biology
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• 제34권2호
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• pp.111-115
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• 2010

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. In this study, we analyzed by immunohistochemical methods adaptive mechanisms to excessive erythrocytosis in transgenic (tg) mice expressing dimeric human erythropoietin (dHuEPO) gene. Splenomegaly was observed over 11~21 times in the tg mice. The 2,672 candidate spleen-derived genes were identified through the microarray analysis method, and decreased genes were higher than increased genes in the spleen. The specific proteins in the increased and decreased genes were analyzed by immunohistochemical methods. Our results demonstrate that problems of abnormal splenomegaly would solve in tg mice overexpressing dHuEPO gene.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.221-224
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• 2001

Effect of hyperosmotic pressure on growth of recombinant Chinese hamster 。 vary cells and Erythropoietin (EPO) production was investigated. Cells were cultivated in batch modes at various osmolalities. When the osmolality increased from 314 to 463mOsm/Kg, specific EPO productivity (qp) was increased up to 1.6-fold but cell growth was inhibited. EPO has a complex oligosaccharide structure that plays an important role in biological activity in vivo. To investigate the influence of hypoerosmotic pressure on the glycosylation, structural analysis of oligosaccharide was calTied out. Recombinant human EPO was produced by CHO cells grown under various osmotic pressure and purified from culture supernatants by heparin-sepharose affinity column and immunoaffinity column. N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were labeled with fluorescent dye, 2-aminobenzamide and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of GU (glucose unit) value. Glycan analysis by HPLC showed that neutral (asialo) oligosaccharide was increased slightly with an increase in osmolality. In portion of sialylated glycan, total relative amount of mono- and di-sialyated glycan was increased but that of tri- and tetra-sialylated glycan decreased as osmolality was increased.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.170.3-171
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• 2003

An efficient Erythropoietin (EPO)-expression system in mammalian cells is required for massive production for therapeutic use. Ammonium ion is a major problem in the production of valuable recombinant proteins in cultured animal cells. Therefore, it is of importance to devise a system by which a high productivity of human therapeutic recombinant protein can be maintained or enhanced under low ammonium concentration. To reduce the ammonium ion accumulated in EPO producing Chinese Hamster Ovary (CHO) ceels, IBE, we introduced the first two genes of the urea cycle, carbamoyl phosphate synthetase (CPSI) and arnithine transcarbamoylase (OTC), into IBE using a stable transfection method. (omitted)

• KSBB Journal
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• 제13권3호
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• pp.295-302
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• 1998

Human Erythropoietin (EPO) gene is cloned in quail fibrosarcoma cell, QT35. Because molecular weight of EPO is similar to that of serum albumin, cell culture with serum containing medium makes purification of EPO very difficult. Using fractional factorial study, we have developed serum free medium for the recombinant QT35 cell lines, QT N4D4 and QT SY-IMP, which have cloned EPO with glutamine synthetase (GS) gene amplification system and with puromycin selective marker, respectively. Among the seven frequently used medium components, fibronectin, BSA, and EGF were the most important for EPO production. However, sufficient fibronectin supplement to the medium did not make any good attachment of QT35 to culture plate over 3 days. Therefore, to maximize EPO production, we attempted a medium-shift at confluence from serum containing medium to serum free medium(QT SFM6). Using the medium-shift protocol with QT SFM6, nearly the same productivity of EPO was achieved comparing with that without medium-shift. This result was true in both QT35 cell lines in three types of culture, i.e. T flask, microcarrier and roller bottle cultures.

• Biomolecules & Therapeutics
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• 제8권2호
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• pp.184-193
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• 2000

To evaluate GC-rhEPO, human erythropoietin produced by recombinant DNA technique, its general pharmacological properties were investigated in experimental animals administering intravenously and in vitro test system. GC-rhEPO at doses of 70,700 and 7,000 IU/kg body weight had no influence on general behavior, spontaneous motor activity, thiopental-inducted sleeping time, writhing syndrome induced by acetic acid, strychnine-induced convulsions, charchoal meal propulsion in mice, and body temperature, gastric juice secretion, urine and electrolyte excretion in rats. In anesthetized rabbits, GC-rhEPO (70, 700 and 7,000 lU/kg, i.v.) did not alter respiratory rate, blood pressure, heat rate. In in vitro experiments, GC-rhEPO did not affect the contractions of the isolated ileum of guinea pigs and the muscle twitchs of isolated neuromuscular junction of the rats. In addition, GC-rhEPO did not affect the blood coagulation time and ADP-induced platelet aggregation in plasma of rabbits. Taken together, these results indicate that GC-rhEPO does not induce any adverse effects in the experimental animals.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1087-1092
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• 2001

The effect of Sodium Butyrate (NaBu) on the N-linked oligosaccharide structure of Erythropoietin (EPO) was investigated. Recombinant human EPO was produced by CHO cells grown in an $MEM{\alpha}$ medium with or without 5 mM NaBu, and purified from the culture supernatants using a heparin-sepharose affinity column and immunoaffinity column. The N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were then labeled with a fluorescent dye, 2-aminobenzamide, and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of a GU (glucose unit) vague. A glycan analysis by HPLC showed that the most significant characteristic effect of NaBu was a reduction in the proportion of glycans with Sri-and tetrasialylated oligodaccharides from $21.30\%$ (tri-) and $14.86\%$ (tetra-) in the control cultures (without NaBu) to $8.72\%$ (tri-) and $1.25\%$ (tetra-) in the NaBu-treated cultures, respectively. It was also found that the proportion of asialo-glycan increased from $12.54\%\;to\;23.6\%$ when treated with NaBu.

• 대한임상약리학회:학술대회논문집
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• 대한임상약리학회 1992년도 정기총회 및 추계학술대회
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• pp.44-44
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• 1992