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1. 发明授权

KR100370942B1 재조합당단백호르몬의제조방법 有权
公开(公告)号：KR100370942B1
公开(公告)日：2003-12-24
申请号：KR1019980032997
申请日：1998-08-14
申请人： 동아쏘시오홀딩스 주식회사
发明人： 박장현 , 김현일 , 서광석 , 박세춘 , 이성희
IPC分类号： C12N15/85 , C12N15/19
摘要： PURPOSE: Preparation method of mass production of biologically active EPO from recombinant plasmid in CHO(chinese hamster ovary) cell is provided. CONSTITUTION: The invention is disclosed a mass production method from recombinant plasmid pDEP-173 consisted of zeocin resistance gene, EPO gene, isolated from human fetal liver cDNA library, under control of SRα promoter, AmvRNA4 enhancer, DHFR gene, and BGH polyadenylation signal. The plasmid is transfected into CHO cell and cultured with roller bottle in serum free medium. EPO is induced with n-butyric acid or methotrexate. Overexpressed EPO is purified with hydrophobic chromatography and anion exchange chromatography. The purified EPO only having N-acetyl neuraminic acid as N-terminal end of sialic acid has molecular weight of 34,000-47,000 and shows 7-8 isomer bands in isoelectric focusing around pH 3.7-5.2.
摘要翻译：目的：提供从CHO（中国仓鼠卵巢）细胞中重组质粒大量生产生物活性EPO的制备方法。构成：本发明公开了一种由SRα-1控制下从人胎肝cDNA文库中分离的由zeocin抗性基因，EPO基因组成的重组质粒pDEP-173的大量生产方法。启动子，AmvRNA4增强子，DHFR基因和BGH聚腺苷酸化信号。将质粒转染CHO细胞并在无血清培养基中用滚瓶培养。用正丁酸或甲氨蝶呤诱导EPO。过度表达的EPO用疏水色谱和阴离子交换色谱纯化。仅具有N-乙酰神经氨酸作为唾液酸N-末端的纯化EPO具有34,000-47,000的分子量，并且在等电聚焦约3.7-5.2的pH范围内显示出7-8个异构体带。

2. 发明授权

KR100394474B1 동물세포에서 재조합 당단백질의 연속식 대량생산 방법 有权
标题翻译： 동물세포에서재조합당단백질의연속식대량생산방
公开(公告)号：KR100394474B1
公开(公告)日：2003-08-09
申请号：KR1019990058878
申请日：1999-12-18
申请人： 동아쏘시오홀딩스 주식회사
发明人： 나규흠 , 박세춘 , 김현일 , 이태호 , 서광석 , 박장현 , 김병문 , 이성희 , 김원배
IPC分类号： C12N5/07
摘要： PURPOSE: Provided is a method for manufacturing recombinant glycoproteins in animal cells in a high yield. Thereby, the recombinant glycoprotein has high activity and cultured cells have high viability for a long time. CONSTITUTION: The method for manufacturing recombinant glycoproteins in animal cells in a high yield is comprised of the next steps of: i) cultivating and growing animal cells in medium including serum for 2-4 days; and ii) cultivating repeatedly the animal cells in medium having no serum for 2-3 days to obtain recombinant glycoproteins. And the recombinant glycoproteins include erythropoietin (EPO), tissue-type plasminogen activator (tPA), follicle stimulating hormone (FSH), luteinizing hormone (LH), interleukin-12 (IL-12), hCG (human chorionic gonadotropin), thrombopoietin (TPO), and Factor VIII.
摘要翻译：目的：提供一种以高产率在动物细胞中制造重组糖蛋白的方法。由此，重组糖蛋白具有高活性，并且培养的细胞长时间具有高生存力。构成：以高产率在动物细胞中制造重组糖蛋白的方法包括以下步骤：i）在包括血清的培养基中培养和生长动物细胞2-4天; 和ii）在不含血清的培养基中重复培养动物细胞2-3天以获得重组糖蛋白。重组糖蛋白包括促红细胞生成素（EPO），组织型纤溶酶原激活剂（tPA），促卵泡激素（FSH），黄体生成素（LH），白介素-12（IL-12），hCG（人绒毛膜促性腺激素），血小板生成素 TPO）和因子VIII。

3. 发明授权

KR100369788B1 재조합 인간 에리트로포이에틴의 제조방법 失效
标题翻译： 재조합인간에리트로포이에틴의제조방법
公开(公告)号：KR100369788B1
公开(公告)日：2003-01-29
申请号：KR1019990037463
申请日：1999-09-03
申请人： 동아쏘시오홀딩스 주식회사
发明人： 박장현 , 나규흠 , 김현일 , 서광석 , 박세춘 , 이태호 , 이성희 , 김원배 , 양재명
IPC分类号： C12N15/12 , C12N15/63
摘要： PURPOSE: A method for mass-producing recombinant erythropoietine(EPO) is provided for the treatment of anemia by regulating the red blood cell generation. CONSTITUTION: The method for mass-producing recombinant erythropoietine(EPO) comprises the steps of: preparing a recombinant plasmid pDEP-521 harboring Zeocin-resistant gene, Rous Sarcoma virus (RSV) promoter, Avian myeloblastosis virus RNA4(AmvRNA4) leader sequence, dihydrofolate reductase gene, human EPO genome gene, and bovine growth hormone polyadenylation signal sequence; producing a transformant baby hamster kidney(BHK) cell line DBHE318(KCLRF-BR-00024) by the transfection of host cells with the recombinant plasmid pDEP-521; and incubating the transformant to produce EPO, in which the produced EPO is characterized by 34,000 to 40,000 dalton of molecular weight, positive in western blotting, 7 to 8 isomer bands around pH 3.8 to 5.2 in isoelectrofocusing analysis, and similar in vivo activity of facilitating the red blood cell making in comparison to that of the international standard liquid.
摘要翻译：目的：通过调节红细胞生成来提供大量生产重组促红细胞生成素（EPO）的方法用于治疗贫血。构建：大量生产重组促红细胞生成素（EPO）的方法包括以下步骤：制备含有博莱霉素抗性基因，劳氏肉瘤病毒（RSV）启动子，禽成髓细胞瘤病毒RNA4（AmvRNA4）前导序列，二氢叶酸的重组质粒pDEP- 还原酶基因，人EPO基因组基因和牛生长激素聚腺苷酸化信号序列; 通过用重组质粒pDEP-521转染宿主细胞产生转化子幼仓鼠肾（BHK）细胞系DBHE318（KCLRF-BR-00024）; 并且将转化体温育以产生EPO，其中产生的EPO的特征在于分子量为34,000至40,000道尔顿，Western印迹阳性，等电聚焦分析中pH为3.8至5.2的7至8个异构体带，以及类似的促进促进红血球与国际标准液相比较。

4. 发明公开

KR1020000013877A 재조합당단백호르몬의제조방법 有权
标题翻译：来自CHO细胞的重组蛋白（EPO）的制备方法
公开(公告)号：KR1020000013877A
公开(公告)日：2000-03-06
申请号：KR1019980032997
申请日：1998-08-14
申请人： 동아쏘시오홀딩스 주식회사
发明人： 박장현 , 김현일 , 서광석 , 박세춘 , 이성희
IPC分类号： C12N15/85 , C12N15/19
CPC分类号： C12N15/8509 , A61K38/1709 , C07K14/575
摘要： PURPOSE: Preparation method of mass production of biologically active EPO from recombinant plasmid in CHO(chinese hamster ovary) cell is provided. CONSTITUTION: The invention is disclosed a mass production method from recombinant plasmid pDEP-173 consisted of zeocin resistance gene, EPO gene, isolated from human fetal liver cDNA library, under control of SRα promoter, AmvRNA4 enhancer, DHFR gene, and BGH polyadenylation signal. The plasmid is transfected into CHO cell and cultured with roller bottle in serum free medium. EPO is induced with n-butyric acid or methotrexate. Overexpressed EPO is purified with hydrophobic chromatography and anion exchange chromatography. The purified EPO only having N-acetyl neuraminic acid as N-terminal end of sialic acid has molecular weight of 34,000-47,000 and shows 7-8 isomer bands in isoelectric focusing around pH 3.7-5.2.
摘要翻译：目的：提供CHO（中国仓鼠卵巢）细胞中重组质粒生物活性EPO的大规模生产制备方法。构成：公开了在SRα启动子，AmvRNA4增强子，DHFR基因和BGH多聚腺苷酸化信号的控制下，从人胎肝cDNA文库中分离的zeocin抗性基因EPO基因的重组质粒pDEP-173的批量生产方法。将质粒转染到CHO细胞中，并用滚筒瓶在无血清培养基中培养。用正丁酸或甲氨蝶呤诱导EPO。用疏水层析和阴离子交换色谱纯化过表达的EPO。仅具有N-乙酰神经氨酸作为唾液酸N-末端的纯化EPO具有34,000-47,000的分子量，并且在pH 3.7-5.2附近显示7-8个等电聚焦异构体带。

5. 发明公开

KR1020010064976A 재조합 인간 에리트로포이에틴의 정제 방법 有权
标题翻译：人类重组蛋白的纯化
公开(公告)号：KR1020010064976A
公开(公告)日：2001-07-11
申请号：KR1019990059466
申请日：1999-12-20
申请人： 동아쏘시오홀딩스 주식회사
发明人： 나규흠 , 서광석 , 김현일 , 이태호 , 박세춘 , 김승철 , 박장현 , 이성희 , 김원배
IPC分类号： C07K14/505
CPC分类号： C07K14/505 , C07K1/165 , C07K1/30
摘要： PURPOSE: Provided is the purification method of recombinant erythropoietin (rEPO) from animal cell culture medium. Erythropoietin is hematopoietic hormone excreted from kidney. CONSTITUTION: The purification process comprises the steps of: concentrating the animal cell culture medium by diafiltration through the membrane with 5-20 K mol. wt. cut-off (MWCO) capacity, the cell strain used being CHO (KCLRF-BP-00018) and BHK (KCLRF-BP-00024); precipitating the impurity by adding 20-120mM of divalent metal salts and reacting 1/2-2 hours, preferably, using 80mM of ZnCl2 and reacting at 4°C for 1 hour and centrifuging; adding the supernatant solution to the resin column and carrying out the anion exchange chromatography with 100mM, 250mM and 500mM NaCl step gradient solutions. the product being extracted by 100mM concentration fraction; going through hydrophobic chromatography with 0-70% ethanol linear gradient extraction, the product being extracted preferably by 0-30% concentration ethanol and finally going through anion exchange chromatography once more to collect the highly in vivo active erythropoietin in the isomer band of pI 3.5-4.5.
摘要翻译：目的：提供动物细胞培养基中重组红细胞生成素（rEPO）的纯化方法。红细胞生成素是从肾脏排泄的造血激素。构成：纯化方法包括以5-20Kmol浓缩通过膜渗滤的动物细胞培养基。重量。（KCLRF-BP-00018）和BHK（KCLRF-BP-00024）所使用的细胞株。通过加入20-120mM的二价金属盐沉淀杂质，并使其反应1 / 2-2小时，优选使用80mM的ZnCl 2，并在4℃下反应1小时并离心; 将上清液加入到树脂柱中，并用100mM，250mM和500mM NaCl步骤梯度溶液进行阴离子交换色谱。产物由100mM浓度级分提取; 通过0-70％乙醇线性梯度提取的疏水色谱法，优选用0-30％浓度的乙醇提取产物，最后通过阴离子交换色谱法再次收集pI 3.5异构体带内的高度体内活性促红细胞生成素 -4.5。

6. 发明公开

KR1020010105066A 인간 에리스로포이에틴을 고효율로 발현하는 재조합세포주 및 이의 제조방법 有权
标题翻译：可高效显示人类红细胞生成素（EPO）的重组细胞系及其生产方法
公开(公告)号：KR1020010105066A
公开(公告)日：2001-11-28
申请号：KR1020000026805
申请日：2000-05-18
申请人： 동아쏘시오홀딩스 주식회사
发明人： 나규흠 , 김현일 , 박장현 , 이성희 , 김원배 , 양재명
IPC分类号： C12N1/00
摘要： 본 발명은 인간 에리스로포이에틴(erythropoietin, EPO)을 고효율로 발현시키는 재조합 세포주 및 이의 제조방법에 관한 것으로, 구체적으로 5',3'-비번역서열(untranslated sequence)이 삭제된 EPO 게놈 유전자, SRα 프로모터, AMV(alfalfa mosaic virus) RNA4 리더서열, DHFR(dihydrofolate reductase) 유전자, 지오신-내성 유전자(zeocin-resistant gene)를 포함하는 pSRα-gEpo 발현벡터
(expression vector)를 BHK(Baby hamster kidney) 세포와 CHO(Chinese hamster ovary) 세포에 형질감염(transfection)시킨 후 메쏘트렉세이트(methotrexate, MTX)가 포함된 배지에서 선별배양함으로써 안정화시킨 인간 에리스로포이에틴 고발현 세포주 및 이의 제조방법에 관한 것이다. 본 발명의 인간 EPO 고발현 세포주에 의해 고역가로 대량생산된 EPO는 빈혈, 류마티스 관절염 또는 후천성 면역 결핍증 등의 치료제로 유용하게 사용될 수 있다.

7. 发明授权

KR100389141B1 인간 에리스로포이에틴을 고효율로 발현하는 재조합세포주 및 이의 제조방법 有权
标题翻译：一种高效表达人促红细胞生成素的重组细胞系及其生产方法
公开(公告)号：KR100389141B1
公开(公告)日：2003-06-25
申请号：KR1020000026805
申请日：2000-05-18
申请人： 동아쏘시오홀딩스 주식회사
发明人： 나규흠 , 김현일 , 박장현 , 이성희 , 김원배 , 양재명
IPC分类号： C12N1/00
摘要： 본 발명은 인간 에리스로포이에틴(erythropoietin, EPO)을 고효율로 발현시키는 재조합 세포주 및 이의 제조방법에 관한 것으로, 구체적으로 5',3'-비번역서열(untranslated sequence)이 삭제된 EPO 게놈 유전자, SRα 프로모터, AMV(alfalfa mosaic virus) RNA4 리더서열, DHFR(dihydrofolate reductase) 유전자, 지오신-내성 유전자(zeocin-resistant gene)를 포함하는 pSRα-gEpo 발현벡터
(expression vector)를 BHK(Baby hamster kidney) 세포와 CHO(Chinese hamster ovary) 세포에 형질감염(transfection)시킨 후 메쏘트렉세이트(methotrexate, MTX)가 포함된 배지에서 선별배양함으로써 안정화시킨 인간 에리스로포이에틴 고발현 세포주 및 이의 제조방법에 관한 것이다. 본 발명의 인간 EPO 고발현 세포주에 의해 고역가로 대량생산된 EPO는 빈혈, 류마티스 관절염 또는 후천성 면역 결핍증 등의 치료제로 유용하게 사용될 수 있다.

8. 发明授权

KR100344059B1 재조합 인간 에리트로포이에틴의 정제 방법 有权
标题翻译：纯化重组人促红细胞生成素的方法
公开(公告)号：KR100344059B1
公开(公告)日：2002-07-24
申请号：KR1019990059466
申请日：1999-12-20
申请人： 동아쏘시오홀딩스 주식회사
发明人： 나규흠 , 서광석 , 김현일 , 이태호 , 박세춘 , 김승철 , 박장현 , 이성희 , 김원배
IPC分类号： C07K14/505
摘要： 본발명은재조합에리트로포이에틴(recombinant erythropoietin, rEPO)을생산하는동물세포의배양상등액으로부터재조합에리트로포이에틴을정제하는방법에관한것으로, 구체적으로본 발명의에리트로포이에틴정제방법은 2가금속양이온침전단계; 음이온교환크로마토그래피단계; 및소수성크로마토그래피단계등 보다단순화된공정만을포함하므로, 본발명의재조합에리트로포이에틴정제방법에따라재조합에리트로포이에틴이고순도·고효율로정제될수 있다.

9. 发明公开

KR1020010057107A 동물세포에서 재조합 당단백질의 연속식 대량생산 방법 有权
标题翻译：在动物细胞中大量制备重组蛋白的方法
公开(公告)号：KR1020010057107A
公开(公告)日：2001-07-04
申请号：KR1019990058878
申请日：1999-12-18
申请人： 동아쏘시오홀딩스 주식회사
发明人： 나규흠 , 박세춘 , 김현일 , 이태호 , 서광석 , 박장현 , 김병문 , 이성희 , 김원배
IPC分类号： C12N5/07
CPC分类号： C12N5/0602 , C07K14/435
摘要： PURPOSE: Provided is a method for manufacturing recombinant glycoproteins in animal cells in a high yield. Thereby, the recombinant glycoprotein has high activity and cultured cells have high viability for a long time. CONSTITUTION: The method for manufacturing recombinant glycoproteins in animal cells in a high yield is comprised of the next steps of: i) cultivating and growing animal cells in medium including serum for 2-4 days; and ii) cultivating repeatedly the animal cells in medium having no serum for 2-3 days to obtain recombinant glycoproteins. And the recombinant glycoproteins include erythropoietin (EPO), tissue-type plasminogen activator (tPA), follicle stimulating hormone (FSH), luteinizing hormone (LH), interleukin-12 (IL-12), hCG (human chorionic gonadotropin), thrombopoietin (TPO), and Factor VIII.
摘要翻译：目的：提供以高产率在动物细胞中制备重组糖蛋白的方法。因此，重组糖蛋白具有高活性，培养细胞长时间具有高活力。构成：以高产量在动物细胞中制备重组糖蛋白的方法包括以下步骤：i）在含有血清的培养基中培养和培养动物细胞2-4天; 和ii）在没有血清的培养基中重复培养动物细胞2-3天以获得重组糖蛋白。重组糖蛋白包括红细胞生成素（EPO），组织型纤溶酶原激活物（tPA），促卵泡激素（FSH），促黄体激素（LH），白细胞介素-12（IL-12），hCG（人绒毛膜促性腺激素），血小板生成素 TPO）和因子VIII。

10. 发明公开

KR1020010026239A 재조합 인간 에리트로포이에틴의 제조방법 失效
标题翻译：生产重组蛋白激酶（EPO）的方法
公开(公告)号：KR1020010026239A
公开(公告)日：2001-04-06
申请号：KR1019990037463
申请日：1999-09-03
申请人： 동아쏘시오홀딩스 주식회사
发明人： 박장현 , 나규흠 , 김현일 , 서광석 , 박세춘 , 이태호 , 이성희 , 김원배 , 양재명
IPC分类号： C12N15/12 , C12N15/63
CPC分类号： C12N15/85 , A61K38/00 , C07K14/47
摘要： PURPOSE: A method for mass-producing recombinant erythropoietine(EPO) is provided for the treatment of anemia by regulating the red blood cell generation. CONSTITUTION: The method for mass-producing recombinant erythropoietine(EPO) comprises the steps of: preparing a recombinant plasmid pDEP-521 harboring Zeocin-resistant gene, Rous Sarcoma virus (RSV) promoter, Avian myeloblastosis virus RNA4(AmvRNA4) leader sequence, dihydrofolate reductase gene, human EPO genome gene, and bovine growth hormone polyadenylation signal sequence; producing a transformant baby hamster kidney(BHK) cell line DBHE318(KCLRF-BR-00024) by the transfection of host cells with the recombinant plasmid pDEP-521; and incubating the transformant to produce EPO, in which the produced EPO is characterized by 34,000 to 40,000 dalton of molecular weight, positive in western blotting, 7 to 8 isomer bands around pH 3.8 to 5.2 in isoelectrofocusing analysis, and similar in vivo activity of facilitating the red blood cell making in comparison to that of the international standard liquid.
摘要翻译：目的：提供重组红细胞生成素（EPO）的制备方法，用于通过调节红细胞生成来治疗贫血。构成：重组红细胞生成素（EPO）的制备方法包括以下步骤：制备含有Zeocin抗性基因的重组质粒pDEP-521，Rous肉瘤病毒（RSV）启动子，禽成髓细胞病毒病毒RNA4（AmvRNA4）前导序列，二氢叶酸还原酶基因，人EPO基因组基因和牛生长激素多聚腺苷酸化信号序列; 通过用重组质粒pDEP-521转染宿主细胞，产生转化子仓鼠肾（BHK）细胞系DBHE318（KCLRF-BR-00024）; 并培养转化子以产生EPO，其中所产生的EPO的特征在于分子量为34,000至40,000道尔顿，Western印迹为阳性，在等电聚焦分析中为约3.8至5.2的7至8个异构体带，并且类似的体内活性促进红细胞制作与国际标准液相比。

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