• Biomolecules & Therapeutics
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• 제6권1호
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• pp.50-55
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• 1998

Antigenic potential of a recombinant human erythropoietin (rhEPO) produced by Dong-A charm. Co. Ltd. was examined by active systemic anaphylaxis (ASA) test in guinea pigs, mouse-rat passive cutaneous anaphylaxis (PCA) reaction and passive hemagglutination (PHA) test. In ASA test, rhEPO induced the signs of restlessness, rubbing or licking nose, sneezing and coughing in the animals immunized with rhEPO 1000 lU/kg alone or rhEPO 1000 lU/kg incorporated into Freund\\\\`s complete adjuvant. In the mouse-rat PCA test, only one of six sera from the animals immunized with rhEPO 1000 lUng incorporated into Alum showed positive result. In the PHA test, rhEPO revealed negative results in all of the rhEPO-immunized groups. From these results, rhEPO was considered to produce IgE in guinea pigs and mice, but not IgG and/or IsM in mice. The results of this study were similar to those of the other recombinant human erythropoietin and these positive results were thought to be caused due to the fact that rhEPO were heterogeneous proteins to guinea pigs and mice. Considering the fact that rhErO has an identical structure with indigenous human erythropoietin, rhEPO is not thought to cause immunological problems in clinical use.

• Biomolecules & Therapeutics
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• 제8권2호
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• pp.171-178
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• 2000

To evaluate the pharmacokinetic properties and tissue distribution of a newly developed recombinant human erythropoietin (GC-rhEPO), we analyzed the plasma and tissue levels of erythropoietin by an ELISA after intravenous (IV) and subcutaneous (SC) adminstration to the male rats at the doses of 20, 100, 500 or 2,500 unit/kg. After single IV bolus injection of GC-rhEPO, the plasma concentration was rapidly increased and decreased with two phases with half-lives of 13.4 min and 2.94 hours. AUC was increased dose- dependently but plasma half-lives remained constant regardless of GC-rhEPO doses. Following SC administration, the plasma concentration increased slowly with half-life of 9.2 hours and reached peak at 8 hours. Mean residence time and bioavailability were 18.2 hours and 44%, respectively. After single IV dose of 100 unit/kg, tissue GC-rhEPO level was higher in bone marrow and spleen, while the depletion rate was slower in liver and bone marrow, indicating the higher affinity of GC-rhEPO to bone marrow. Taken together, the experimental results indicate that GC-rhEPO contained the typical pharmacokinetic properties and the tissue distribution patterns inherent to human erythropoietin.

• Biomolecules & Therapeutics
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• 제2권4호
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• pp.343-346
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• 1994

In vivo activity of recombinant human erythropoietin (rh-EPO) has been examined using polycythemic model in mice and acute hemorrhage model in rats. The number of reticulocytes in blood stream was increased after a single injection of rh-EPO depending on the dosage of rh-EPO in polycythemy model. It seemed that optimal dose of rh-EPO for polycythemic mice was around 1-10 U/kg. Rh-EPO also showed the effectiveness for increase of reticulocyte numbers both in male and female rats after bleeding. The number of reticulocytes and the change of hemoglobin concentration in the blood stream of normal rats has been examined after injection of rh-EPO. The maximum value of reticulocyte was observed on the 6th day of the injection in these normal rats. In addition, the increase of reticulocyte and the concentration of hemoglobin were dependent on the dosage of rh-EPO. The increase of hemoglobin concentration was continued to the 9th day after injection. In this study, the efficacy of rh-EPO was confirmed in both mice and rats.

• 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.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.17-17
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• 2003

The milk of transgenic animals may provide an attractive vehicle for large-scale production of hEPO. Since glycosylation is cell type specific, recombinant human EPO (rhEPO) produced in different host cells contain different patterns of oligosaccharides, which could affect the biological functions. However, there have been no reports on the characteristics of rhEPO derived from milk of transgenic animals. To address this objective, several transgenic mice by using pWAPhEPO and/or pBC1hEPO expression vector were produced. However, 2 lines of pWAPhEPO founder female mouse died during late gestational day (day 18) before offspring could be obtained. They showed a severe splenomegaly, Unlike those of pWAPhEPO, mammary gland epithelial cells from biopsies of lactating pBC1hEPO transgenic mice had marked immunoreactivity to EPO and any activity was not detected in other tissues. The expression level of rhEPO is about 0.7% of mammary gland cellular total soluble proteins and an amount of 300~500 mg/L rhEPO is secreted into milk. Furthermore, the pBC1hEPO transgenic mice transmitted this character to their progeny in mendelian manner. In order to determine the extent of glycosylation variation, N-linked oligosaccharide structures present in the milk-derived rhEPO were characterized. Most of milk-derived rhEPO is fully glycosylated. the biological activity of milk-derived rhEPO was comparable to that of purified CHO-derived rhEPO, and milk-derived rhEPO showed relatively stable after freezing and thawing. Taken together, the results illustrate the potential of transgenic animals in the large-scale production of biopharmaceuticals.

• KSBB Journal
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• 제32권2호
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• pp.90-95
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• 2017

Mammalian cell cultures have been used extensively to produce proteins for therapeutic agent because of their ability to perform post-translational modification including glycosylation. To produce recombinant protein, many factors and parameter are considered such as media composition, host cell type, and culture process. In this study, recombinant human erythropoietin (rhEPO) producing cell line was established by using glutamine synthetase system. To reduce serum concentration in media, we compared direct adaptation with step adaptation. Cell growth was faster in step adaptation. In low-level serum media, there were insufficient glucose for cell growth. Thus, we added glucose in low-level serum media from 2 g/L to 4.5 g/L. Titer of rhEPO was higher than other conditions at 4.5 g/L of glucose. Additionally, N-methyl-D-aspartate (NMDA), 13-cis-retinal, and pluronic F-68 (PF-68) were added to enhance productivity in CHO cell cultures. In conclusion, we applied CHO cell producing rhEPO to low-level of serum in media using step-adaptation. Also, we confirmed positive effect of NMDA, 13-cis-retinal, and PF-68.

• BMB Reports
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• 제30권5호
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• pp.315-319
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• 1997

It was discovered that monoclonal anti-erythropoietin (EPO) antibody CFC-6 can neutralize EPO-induced cell activation. To know the binding site of CFC-6, recombinant human erythropoietin (rhEPO) was digested with Glu-C, followed by a separation using high performance liquid chromato graphy (HPLC). Each HPLC fraction was blotted on the nitrocellulose membrane and the membrane was treated with anti-EPO antibody CFC-6 and anti-mouse antibody which is modified with peroxidase. Only one spot showed the color and the fraction was sequenced by Edman degradation. The results suggest that CFC-6 recognizes amino acid sequence V63-W-Q-G-L-A-L-L-S-E72 which is a part of helix II of the EPO molecule. Binding of CFC-6 to EPO may inhibit EPO binding to its receptor, which implies that the antibody binding site and the receptor binding site are close or overlapping.

• Biomolecules & Therapeutics
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• 제6권1호
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• pp.56-62
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• 1998

Mutagenicity of recombinant human erythropoietin (rhEPO) was examined in the reverse mutation test on bacteria, in the chromosomal aberration test on cultured mammalian cells and in the micronucleus test on mice. The reverse mutation test was performed by a plate incorporation method with or wothout a metabolic activation system (59 Mix) using Salmonella typhimurium strain TA100, TA1535, TA98 and TA 1537. The rhEPO did not significantly increase revertant colonies in any of the test strains under any conditions at dose levels ranging from 1000 H/ml to 62.5 lu/plate, compared with the vehicle control. In the chromosomal aberration test using cultured Chinese Hamster Lung (CHL) cells, the number of aberrant cells was not increased in the presence or absence of 59 Mix at concentrations of 1000 lU/ml to 250 lU/ml, compared with the vehicle control. In the micronucleus test, male ICR mice were given rhEPO intraperitoneally at a dose level of 25000, 12500 and 6250 lU/kg. The incidence of bone marrow micronucleated polychromatic erythrocytes was not different from that of the vehicle control. From these results, rhEPO is considered to be non-mutagenic under the present test conditions.

• 한국미생물·생명공학회지
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• 제32권2호
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• pp.142-148
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• 2004

에리스로포이에틴은 인간 적혈구분화의 조절인자로 작용한다. 유전자 재조합 인간에리스로포이에틴(rhEPO)은 동물세포에서 생산되고 있는 재조합 당단백질의 하나이며 당쇄부분이 전체 분자량의 40％를 차지한다. 시알산 함량은 체내약물 투여 지속기간과 직접적인 연관이 있어 시알산 함량은 의약용 당단백질의 중요한 성질로 여겨진다. 본 연구에서는 CHO세포 배양액에 시알산 생합성 전구물질인 N-acetylmannosamine(ManNAc)과 sialidase 저해제인 2-deoxy-2,3-hyo-N-acetylneuraminic acid(NeuAc2en)를 첨가하여 rhEPO의 sialic acid함량을 증가시킬 수 있었다. 특히, 배양액에 20 mM ManNAc/0.5 mM NeuAc2en를 첨가할 때 대조구에 비하여 약 10배의 시알산 함량이 증가하였으며 세포성장이나 배양액의 rhEPO생산량에는 영향이 없었다. rhEPO의 정제시 시알산 함량이 11∼15％인 다당쇄 rhEPO분획을 얻었으며, 배양액 내에 20 mM ManNAc와 0.5 mM NeuAc2en를 동시에 첨가함으로 대조구에 비하여 시말산함량이 높은다당쇄 rhEPO의 생산성이 50％ 증가하였다.

• 약학회지
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• 제47권6호
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• pp.422-431
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• 2003

The use of recombinant human erythropoietin (rhEPO), a stimulator of erythropoiesis, banned in sports because of the medical risk associated with thrombosis. Due to analytical difficulties to differentiate between natural human EPO (hEPO) and rhEPO, blood parameters of erythropoiesis such as contents of hemoglobin (cut-off value <17.5 g/d l for man, and < 16.0 g/dl for women), hematocrit and reticulocytes (cut-off value <2.0%) were measured to focus the misuse of rhEPO. We conducted anti-doping test for 122 blood samples of the World Cup athletes. The mean values of key parameters are as follows; 14.5$\pm$1.0 g/dl for hemoglobin, 41.7$\pm$2.8% for hematocrit, and 1.3$\pm$0.4% for reticulocyte. Blood sample was found to be stable up to 8 hours for the reticulocyte measurement. In addition, the soluble transferrin receptor and ferritin levels were measured by immunoassay methods using plasma samples (n=28) in which the mean value was 0.8$\pm$0.5 $\mu\textrm{g}$/$m\ell$ and 54.6$\pm$33.7 ng/$m\ell$, respectively. The results indicate that all samples tested were negative for the blood parameters of indirect anti-doping test for hEPO misuse. The statistical evaluation suggest that several other parameters such as red blood cell, mean corpuscular hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin and white blood cell could be considered as factors influencing hEPO function in addition to five parameters mentioned.