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WO2005084711A1 - Erythropoietine recombinante pegylee a activite in vivo - Google Patents

Erythropoietine recombinante pegylee a activite in vivo Download PDF

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Publication number
WO2005084711A1
WO2005084711A1 PCT/CN2005/000254 CN2005000254W WO2005084711A1 WO 2005084711 A1 WO2005084711 A1 WO 2005084711A1 CN 2005000254 W CN2005000254 W CN 2005000254W WO 2005084711 A1 WO2005084711 A1 WO 2005084711A1
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epop
peg
epo
vivo
activity
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PCT/CN2005/000254
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English (en)
French (fr)
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Yonghong Ge
Zhiyong Chen
Xianwu Zeng
Lanjun Liu
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Chengdu Institute Of Biological Products
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Publication of WO2005084711A1 publication Critical patent/WO2005084711A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the present invention relates to a recombinant erythropoietin protein having no physiological activity in vivo by polyethylene glycol
  • hEPO Human Erythropoietin
  • hEPO Human Erythropoietin
  • Its physiological role is to stimulate the differentiation and proliferation of bone marrow erythrocyte precursor cells, play an important role in the process of red blood cell development and maturation, and is an endogenous regulator of red blood cell growth.
  • the curative effect is over 95%, the hemoglobin content (Hb) and hematocrit (Hct) are increased, the anemia is improved, the bleeding time is shortened, the quality of life is improved, the number of blood transfusions is reduced, and the patient who is performing kidney transplantation is also beneficial; (2) AIDS patients with anemia, especially patients treated with AZT are often associated with anemia, EPO treatment is effective; (3) cancer patients with anemia caused by chemotherapy, especially with cisplatin often with obvious anemia, EPO treatment is effective; (4) Chronic anemia, such as rheumatoid arthritis, cancer patients, when accompanied by anemia and need to transfusion to maintain treatment, EPO can reduce the number of blood transfusions in some patients; (5) anemia of hematopoietic stem cell diseases, such as myelodysplastic syndrome ( MDS;), patients with aplastic anemia, etc., if it is difficult to maintain blood transfusion, try EPO, about 25% ⁇ 30% of patients can improve anemia and reduce the number
  • EPO was started 3 weeks before the operation. When the increase in Hb was obvious, 400 ml of blood was collected and collected for 3 times to meet the needs of the patient. Use EPO to improve the quality of blood storage and reduce the amount of blood storage; (7) Prevention and treatment of anemia in premature infants: 50 ⁇ 100IU / kg, 3 times a week, starting after birth, for 4 weeks. It can be expected that EPO will be the first line of anemia treatment for a long period of time.
  • this mechanism greatly reduces the bioavailability of rhEPO, resulting in an increase in the amount of the drug, and the increase in the cost of treatment due to the expensive price of rhEPO; and due to the limitation of the number of EP0 receptors, The mechanism also limits the ability to increase the dose of the drug to achieve higher therapeutic effects.
  • the EPO molecule is a sialic acid-containing acidic glycoprotein that is stable to heat (invariance at 80 ° C) and acid-base (stable range between pH 3.5 and 10.0). Two pairs of disulfide bonds are formed between Cys 161 and Cys 7 , Cys 29 and Cys 33 in the protein peptide chain, wherein the disulfide bond between Cys 7 and Cys 161 is essential for the biological activity of EPO, if disulfide bonds Upon reduction, EPO will lose its biological activity.
  • the four glycosylation sites of the EPO molecule are on Asn 38 , Asn 24 , Asn 83 and Ser 126 , respectively, the first three being N-glycans and the latter being 0-sugar chains.
  • Sugar chains have different degrees of branching, with double, three or four tips, of which the four ends are the most common.
  • the results show that the degree of branching of the sugar chain has an effect on the biological activity of EPO in vivo, and the high branching of the N-terminal sugar chain is necessary for maintaining biological activity in vivo.
  • Desialic acid or deglycosylation does not affect the biological activity in vitro, but it greatly shortens the half-life in vivo (mainly by hepatocyte adsorption metabolism), and as a result, completely loses its biological activity in vivo. Only when the EPO molecule is fully glycosylated can it exhibit biological activity in the body. Therefore, the presence of a sugar chain structure is critical for the biological activity of EPO in vivo.
  • EPO expressed by eukaryotic cells is biologically active in vivo. Since the EPO glycosyl moiety accounts for 30 to 40% of the entire molecular weight and has a high degree of heterogeneity, the degree of glycan branching makes the molecular weight of EPO between 30 and 40 KD. OFiw/ey LC, Linderberg G, Fishman L , et al. The importance of N- and O- linked oligosaccharides for the biosynthesis and in vitro and vivo biological activities of erythropoietin. Blood, 1991, 77(3): 419-434. Numerous studies on EPO confirm the above conclusions.
  • rhEPO currently used in drug production uses eukaryotic cell expression systems, and studies on EPO characteristics and drug efficacy have also focused on rhEPO obtained using eukaryotic cell expression systems.
  • rhEPO obtained using eukaryotic cell expression systems.
  • One is to chemically modify the existing rhEPO.
  • a conjugate of EPO and PEG is disclosed, which is obtained by EPO sugar.
  • the protein is covalently linked to 1-3 lower decyloxy polyethylene glycol (PEG) groups for modification, which increases the circulating half-life and shelf life of EPO, and reduces EPO clearance and activity in vivo.
  • the other is to genetically engineer a portion of the amino acids in the EPO peptide chain to increase the glycosylation site and thereby increase the degree of glycosylation. See US Patent 5,856,298 and Amgen Inc. One Amgen Center Drive Thousand Oaks, California AranespTM Book.
  • EPO peptide protein reagent can also be used to immunize animal EPO antigen; or prepared into EP0 positive control standard, kit for EPO immunoassay, such as reverse blood coagulation, radioimmunoassay, enzyme-linked immunosorbent assay and the like. Summary of the invention
  • the present invention provides a PEG-EPOP conjugate, which uses a glycosylation-free and thus in vivo-active erythropoietin (EPO) protein (EPOP), such as: EPO obtained by the prokaryotic expression system without in vivo biological activity provided by the present invention
  • EPO erythropoietin
  • the protein is then chemically modified by PEG to obtain a product having physiological activity of erythropoiesis in vivo (PEG-EPOP).
  • the present invention also provides the use of a PEG-EPOP conjugate for the preparation of a medicament for treating anemia diseases and for raising red blood cells.
  • a PEG-EPOP conjugate of the formula (I) is provided - mPEG-X-EPOP
  • k is an integer from 100 to 1000, and the molecular weight is from 5,000 to 40,000 Daltons;
  • X is NH or 0, indicating the covalent attachment of PEG to EPOP chemical modification
  • EPOP is a recombinant EPO protein, which is an EPO protein without in vivo biological activity. Compared with recombinant EPO obtained by expression of natural EPO or eukaryotic expression system, it lacks a sugar chain moiety. In vivo, physiological activity of erythropoiesis is not observed in animal experiments. However, it has the in vitro cytological activity unique to EPO.
  • the invention has no physiological activity in vivo or no biological activity in vivo, and refers to the physiological effect of detecting the growth of red blood cells in the experimental animal by the currently known detection method according to the general therapeutic use dose, that is, EPOP is not It has the value of being used as an anemia correcting drug; of course, it is not excluded that the detection of a weaker in vivo activity can be detected by a larger dose or a higher sensitivity test.
  • EPOP having no physiological activity in vivo can be of various origins, and a homologous peptide chain having a peptide chain structure similar to that of natural EPO, whether synthetic or expressed by a prokaryotic system or a eukaryotic system, or even Modified, for example, a peptide chain of 166 amino acids or 167 amino acids expressed by a prokaryotic system in Examples 1 and 2 of the present invention can be used as a raw material of the PEG modification of the present invention because it lacks a sugar chain and does not have physiological activity in vivo. Thereby obtaining a product having physiological activity in vivo.
  • a recombinant EPO gene and an engineered bacteria which can be efficiently expressed by an Escherichia coli system are first constructed, and the expressed recombinant EPOP has no in vivo activity, and then Based on this EPOP, it was chemically modified with PEG 2 NHS-20K, and the modified PEG-EPOP conjugate showed significant in vivo physiological activity of erythropoiesis in animals.
  • the invention adopts a prokaryotic expression system to obtain EPOP without in vivo biological activity, and then chemically modifies EPOP with PEG to obtain a PEG-EPOP conjugate, and observes in vivo activity in animal experiments. If the PEG-EPOP conjugate is used as a medicine for treating anemia diseases, the prokaryotic expression system is significantly reduced in production cost and scale cost, and has a great advantage over the eukaryotic expression system, which can significantly reduce the cost of treatment for patients. . DRAWINGS
  • Figure 1 is a plasmid construction diagram of the recombinant EPOP prokaryotic expression system.
  • Figure 2 PEG-EPO in vivo physiological activity test, using R-500 analyzer to determine the percentage of reticulocytes:
  • the ordinate is the percentage of reticulocytes
  • the abscissa is the blood collection time (1, 2, 3, 4 respectively) Represents blood collection on the fourth, fifth, sixth and seventh day after injection of the sample)
  • Figure 3 is a comparison of EPO protein expression of two amino acid sequences (167AA and 166AA);
  • the E. coli strain DH 5 a was purchased from the GIBCO DH 5 a standard strain.
  • restriction enzymes BamH I, EcoRI, T4 ligase and Taq DNA polymerase were purchased from Roche, and the CRISPR Plus Minipreps DNA Purification System was purchased from Promega.
  • the protein molecular weight marker was purchased from Huamei Company, and other reagents. All are domestic analytical pure reagents.
  • the EPO molecule expressed in human body is a glycosylated protein consisting of 166 amino acids. During the post-processing modification, its C-terminal Arg 166 position is cleaved to become a mature hEPO composed of 165 amino acids.
  • the cDNA sequence of hEPO is shown in SEQ ID NO. 1, and the amino acid sequence of the primary structure of the protein is SEQ ID NO. 2o, wherein the proportion of the rare codon of Escherichia coli accounts for 12%.
  • we optimize the use of codons by redesigning and synthesizing genes, eliminating rare codons. Replace most of the rare codons in hEPO with E.
  • the sequenced correct target gene was digested with restriction endonucleases BamH I and EcoR I to obtain the coding sequence (SEQ ID NO. 4), which was recovered after electrophoresis and doubled with restriction endonucleases BamH I and EcoR I.
  • the digested PBV 22C plasmid was ligated.
  • the ligation product was transformed into E. coli DH 5 ot, and an LB plate containing ampicillin was applied to obtain a transformant.
  • the transformant was picked, the plasmid was extracted, and the recombinant plasmid transformant containing the hEPO gene fragment was selected by double restriction analysis with restriction endonucleases BamH I and EcoR I.
  • the recombinant was inoculated into 5 ml of LB medium and cultured at 30 ° C, 180 rpm shaker for about 14 h. Then, it was transferred to LB medium at a ratio of 1:30, and cultured at 30 ° C for about 4 hours (at A 6 o Q 0.8). The culture was induced for 4 h at 42 °C. The culture was taken 1 ml, centrifuged, and the expressed protein was identified by SDS-PAGE and Western blot.
  • LB seed culture medium 10 g of peptone per litre, 5 g of yeast powder, 5 g of NaCl, pH 7.0, sterilized by high pressure, and ampicillin was added to 100 ug/ml when used.
  • Fermentation medium K 2 HP0 4 12g per KL, KH 2 P0 4 - 3H 2 0 20g, NaCl 20g,
  • Fermentation culture The preserved strains were plated, single colonies were picked, inoculated in LB seed medium, cultured for 14 to 16 hours, and then expanded for 12 hours at a ratio of 1:30. Then inoculated in NBS
  • the MPP-40 fermenter was cultured, fed once every hour during the culture, and the pH and oxygen solubility (DO) were adjusted according to the growth of the cells.
  • the fermentation product is isolated and purified according to methods known to those skilled in the art, such as the literature ⁇ to ⁇ mutations at three sites which are N-glycosylated in the mammalian protein decrease the aggregation of Escherichia coli-derived erythropoietin, Protein Engineering , Vol.14 no.2 pp.135-140, 2001, Linda O.Narhi etc. Recombination from prokaryotic expression systems
  • the present invention relates to the modification of EPOP with PEG2NHS-20K, and the chemical modification reaction is carried out as follows:
  • X is NH or 0, representing the covalent attachment of PEG to EPOP chemical modification, typically the side chain amino (NH) of lysine in the EPO peptide chain or the N-terminal amino group of EPO, or it may be in the EPO peptide chain Such as the side chain hydroxyl group (OH) of serine.
  • i and j are integers representing the length of the PEG carbon chain
  • n is an integer of 1 to 5, preferably 1;
  • the mPEG moiety has a molecular weight of from 5,000 to 40,000 Daltons, preferably from 10,000 to 20,000 Daltons.
  • the obtained recombinant EPO can be chemically modified by the following methods: 1. Exchange buffer system (ultrafiltration):
  • the recombinant human erythropoietin semi-finished product was concentrated with an ultrafilter previously treated with 0.5 mol/L NaOH depyrogenation, diafiltered 4 times with PH8.5, 50.0 mmol/L phosphate buffer, and the protein was adjusted with the same buffer.
  • the concentration is 1 mg/ml.
  • the protein concentration of the recombinant EPO sample may be 0.01 to 5 mg/ml, preferably 0.1 to 1 mg/ml, and most preferably 1 mg/ml in the present invention.
  • the molar ratio of the activated PEG ester to the modified protein is 1:1 to 1:10 or higher, and the weight ratio of PEG 2 NHS-20K is about 2:1 to 1:5, and the preferred weight ratio is 1 in the present invention. : 5.
  • Recombinant EPOP sample The purified EPO protein sample was expressed according to the method of Example 1. It contained N-terminal methionine and C-terminal arginine, and a total length of 167 amino acid peptide chain. The cell activity of UT-7 was 44000 IU. /ml;
  • PEG-EPOP conjugate sample PEG-modified PEG-EPOP conjugate sample prepared by the method of Example 2, the specific method in this experiment is: 5ML of the aforementioned recombinant EPOP sample is added with PEG2NHS-20K 50 mg, After the modification reaction was completed, it was not isolated and purified, and stored at 4 degrees for animal experiments.
  • mice Balb/C pure mice, weighing approximately 20 grams.
  • Example 3 Referring to Example 3, 0.2 ml was subcutaneously injected into each abdomen, and the percentage of reticulocytes was measured by R-500 analyzer instead of smear staining, and the results of the fourth, fifth, sixth, and seventh days after the injection were measured.
  • rhEPO positive control using Shanghai Fosun Technology Biological Cloning Company rhEPO product ⁇ , batch number 20040101, 2000 IU / bottle, 2ml physiological saline dissolved, take 200ul, diluted with physiological saline 4.8ml, concentration is 40IU / ml, There are 4 groups of 4 in each group.
  • 1.3. 2# Recombinant EPOP, unmodified control sample.
  • the purified EPO protein sample was expressed according to the method of Example 1.
  • the N-terminal methionine contained no C-terminal arginine, and the peptide chain of 166 amino acids was long.
  • the cell activity of UT-7 was 30,000 IU/ml, 100 times. Dilution, concentration 300 IU/ml. There are 4 groups of 4 in each group.
  • the peptide chain to which the present invention relates includes a peptide chain having a natural EPO amino acid sequence and a homologous peptide chain which is improved for various purposes, such as: 166 amino acids as proposed in the present invention (if the first methylthioamide from the N-terminus)
  • the acid MET should be 167 amino acids, and the examples of the present invention indicate that the EVP having the methionine still has the theoretically in vitro cytological activity and the in vivo physiological activity of the PEG modified physiological expression sequence and expression.
  • the natural peptide chain of Arg 166 is cut, or the peptide chain of a part of the amino acid is changed for its purpose, such as: reducing the glycosylation site to increase the stability of the peptide chain, etc., as long as the EPO homologous peptide chain is It is not within the scope of the present invention that the body can not exhibit or can only exhibit weak EPO physiological activity, and PEG modified to obtain strong physiological activity in vivo.
  • the modification reagent involved in the present invention is an activated PEG ester, and other methods can be used to covalently couple the PEG chain to the peptide chain of EPOP, including other kinds of activating groups, or activate the corresponding sites of the EPOP peptide chain.
  • mPEG of various structures of single-stranded, double-stranded or multi-stranded are also within the scope of the present invention.

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Description

聚乙二醇修饰后具有体内生理活性的重组促红细朐,牛成素 所属技术领域
本发明涉及以聚乙二醇对无体内生理活性的重组促红细胞生成素蛋白
(EPOP)进行修饰， 而获得的具有促红细胞生成作用的体内生理活性的结合物 (PEG-EPOP)。 背景技术
人红细胞生成素（human Erythropoietin, hEPO)是一种主要由肾脏合成并分 泌的糖蛋白激素， 在胚胎期 EPO产生于肝脏， 出生后 4个月转由肾脏产生。 其 生理作用是刺激骨髓红细胞前体细胞的分化与增殖，在红细胞发育成熟过程中起 着重要的作用， 是红细胞生长的内源性调节剂。
早年从再生障碍性贫血病人的尿中、 胎肾细胞的培养液中可获得较多的 hEPO, 但并不能满足临床需求。 1985年， 应用基因重组技术获得重组人 EPO (rhEPO),开始了工业化生产 EPO的历史，使 EPO在临床上广泛应用成为可能。 1989年 6月美国 Amgen公司生产的 rhEPO正式获得美国 FDA注册许可， 商品 名为 "EPOGEN® EPOETIN ALFA", 用于治疗慢性肾衰竭 (CRF)合并贫血症， 取 得了巨大的成功。 目前世界上已有多家公司开发生产 rhEPO,近十多年的临床应 用的结果表明 ΛΕΡΟ 的疗效显著， 副作用小， 是治疗各类贫血的特效药物， 也 是当前最为成功的基因工程治疗药物。 随着人们对 EPO的研究日益深入， 其临 床适应症将进一步扩大， 目前适用于： （1 )慢性肾衰合并贫血的治疗： 此类贫血 的一个主要原因是 EPO生成缺乏， EP0可谓替代治疗方法，其疗效达 95 %以上， 使血红蛋白含量 （Hb)及红细胞压积 （Hct)增高， 贫血改善， 出血时间缩短， 生活质量提高，输血次数减少，也有利于施行肾移植的病人；（2)艾滋病（AIDS) 病人伴贫血， 尤其应用 AZT治疗的病人常易伴发贫血， EPO治疗有效； （3)肿 瘤病人化疗所致贫血， 尤其使用顺铂常出现明显贫血， EPO治疗有效； （4) 慢 性病贫血，如类风湿性关节炎、肿瘤病人，当伴发贫血而需输血维持治疗者， EPO 可使部分病人减少输血的次数； （5)造血干细胞疾病的贫血， 如骨髓增生异常综 合征 （MDS；)、 再生障碍性贫血等的病人， 若需反复输血亦难以维持者， 可试用 EPO, 约 25 %〜30%病人可使贫血改善， 减少输血次数； （6) 自体供血输注： 美国血库协会规定， 择期手术的病人， 如矫形手术者宜取自血贮存， 以备手术时 输注， 术前 3周开始用 EPO， Hb上升明显时取血 400ml贮存， 采集 3次备用， 可以满足病人的需要。 使用 EPO可以提高贮血质量， 减少贮血数量； （7)早产 儿贫血的防治： 50〜100IU / kg， 每周 3次， 于出生后开始， 连用 4周。 可以预 计： EPO在一个相当长的时期内都将是贫血治疗的一线药物。
在釆用蛋白质药物治疗过程中，常因其较短的血浆半衰期而使其生物利用度 降低， 这在 EPO这样的激素类蛋白质上表现得尤为明显。 机体通过肾脏细胞上 的氧分压感受器调节 EPO的分泌量，通过肝细胞快速灭活释放到血浆中的 EPO， 由此建立维持红细胞压积相对恒定的灵敏调节机制：既可在贫血时大量促进红细 胞的释放， 又可防止过量释放红细胞造成血粘稠度过高等不利后果。 在使用 rhEPO对贫血疾病的治疗中， 此机制大大减低了 rhEPO的生物利用度， 造成用 药剂量的增加，而由于 rhEPO昂贵的价格也造成治疗费用的增加；同时由于 EP0 受体数量的限制，此机制也限制了加大用药剂量获得更高治疗效果的能力，在实
1
确 认本 际的治疗中，大多数情况下需采用每周 3剂的治疗方案，只在贫血症状纠正后的 维持治疗中可视情形降低用药剂量及用药频次至每周 2剂或每周 1剂。
EPO分子是一种含唾液酸的酸性糖蛋白， 对热 （在 80°C条件下不变性）和 酸碱（稳定范围在 PH 3.5〜10.0之间）较为稳定。 蛋白质肽链中 Cys161与 Cys7、 Cys29和 Cys33之间分别形成两对二硫键， 其中 Cys7和 Cys161之间的二硫键对于 EPO 的生物活性至关重要， 如果二硫键被还原， EPO将丧失其生物活性。 EPO 分子的四个糖基化位点分别在 Asn38、 Asn24、 Asn83和 Ser126上， 前三者为 N-糖 链， 后者为 0-糖链。 糖链的分枝程度不同， 有双末梢、 三末梢或四末梢， 其中 四末梢最为常见。 研究结果表明， 糖链的分枝程度对 EPO在体内的生物活性有 影响， N-端糖链的高度分枝对其维持生物体内生物活性是必要的。去唾液酸或去 糖基化不影响在体外的生物活性，但却大大缩短了在体内的半衰期（主要经肝细 胞吸附代谢)， 结果完全丧失了在体内的生物学活性。只有 EPO分子被充分糖基 化后， 才能在体内表现出生物活性。 因此糖链结构的存在对 EPO在体内的生物 活性至关重要。 也就是说只有真核细胞表达的 EPO在体内才具有生物活性。 由 于 EPO糖基结构部分占整个分子量的 30〜40%且有高度的不均一性， 其糖链分 枝程度的不同使 EPO 的分子量在 30〜40KD之间 OFiw/ey LC, Linderberg G, Fishman L, et al. The importance of N- and O- linked oligosaccharides for the biosynthesis and in vitro and vivo biological activities of erythropoietin. Blood, 1991, 77(3): 419-434. 众多关于 EPO的研究均证实了以上结论， 因此普遍的观点认 为重组 EPO必须以真核细胞表达， 如文献 ^ 。 促红细胞生成素及其临床用 途与市场前景。 《中国医药情报》 1999年第 5卷第 1期， ΡΠ λ
基于以上对自然 hEPO的认识，目前用于药物生产的 rhEPO均釆用真核细胞 表达系统， 而对 EPO特性及药效改进的研究也集中于采用真核细胞表达系统所 获得的 rhEPO上。 为增加 rhEPO的药效， 目前有两种较成功的方法， 一是对现 有的 rhEPO进行化学修饰， 如在 00898956专利申请中， 公开一种 EPO与 PEG 的偶联物， 其通过将 EPO糖蛋白共价连接 1〜3个低级浣氧基聚乙二醇 （PEG) 基团进行修饰， 而提高了 EPO的循环半衰期和血柴滞留时间， 并降低了 EPO的 清除率和提高其体内活性。 另一是通过基因工程的方法改变 EPO肽链中的部份 氨基酸以增加糖基化位点进而增加糖基化程度， 参见美国专利 US5856298 以及 Amgen Inc. One Amgen Center Drive Thousand Oaks, California产品 Aranesp™ 明书。
尽管以上改进取得了明显增加 rhEPO血浆半衰期的效果，但其依然是釆用现 行 rhEPO药品生产工艺所用的真核细胞表达系统， 生产成本应与现行产品相当。
在申请人的另一个较早的专利申请中 （申请号： 200410021859.7)， 通过对 已知的 hEPO基因的碱基序列进行重新设计， 消除大肠杆菌稀有密码子， 而代之 以大肠杆菌偏爱密码子， 并适量调整 GC含量， 成功地构建了可在原核系统（例 如大肠杆菌中）高效表达的重组基因和载体系统。虽然表达的重组人促红细胞生 成素蛋白 （EPOP ) 由于缺乏糖基化修饰而在体内不具有生理活性， 但原核系统 生产成本大大低于真核系统， 这样制备的无体内活性的 EPOP可用于例如制备 EPO肽链蛋白试剂、也可用于免疫动物的 EPO抗原； 或制备成 EP0阳性对照标 准品， 用于 EPO免疫检测的试剂盒， 如反相血凝、 放射免疫、 酶联免疫等方法。 发明内容
本发明提供一种 PEG-EP0P结合物， 采用无糖基化因而无体内活性的促红 细胞生存素（EPO)蛋白 （EPOP), 如： 本发明提供的由原核表达系统获得无体 内生物活性的 EPO蛋白， 再利用 PEG对 EPOP进行化学修饰， 获得具有体内促 红细胞生成作用生理活性的产物（PEG-EPOP)。
本发明还提供 PEG-EPOP结合物在制备治疗贫血性疾病药物及升高红细胞 药物中的用途。
根据本发明的一方面， 提供式 （I) 结构的 PEG-EPOP结合物- mPEG-X-EPOP
(I)
式（I) 中， mPEG的分子结构式为
Figure imgf000004_0001
其中 k为 100〜1000的整数， 分子量为 5000〜40000道尔顿；
X为 NH或 0， 表明 PEG对 EPOP化学修饰的共价连接方式；
EPOP为重组 EPO蛋白， 为无体内生物活性的 EPO蛋白， 与天然 EPO或真 核表达系统表达获得的重组 EPO相比缺少糖链部份， 在动物实验中观察不到促 红细胞生成的体内生理活性， 但是具有 EPO特有的体外细胞学活性。
本发明中所指无体内生理活性或无体内生物活性， 是指按一般治疗使用剂 量，在实验动物体内用目前已知的检测方法，检测不出明显的促红细胞生长的生 理作用， 即 EPOP不具备作为贫血纠正药物使用的价值； 当然， 不排除采用更大 剂量或更高灵敏度的检测方法可检测到较微弱体内活性的可能。
在本发明中， 无体内生理活性的 EPOP可以有多种来源， 凡肽链结构与天然 EPO相近的同源肽链， 无论是人工合成的， 还是通过原核系统或真核系统表达 的，甚至经过改造的，例如在本发明实施例 1、 2中通过原核系统表达的具有 166 个氨基酸或 167个氨基酸的肽链， 因为缺乏糖链而不具备体内生理活性，都可以 作为本发明 PEG修饰的原料， 从而获得具有体内生理活性的产物。
为了低成本地获得大量无体内活性的 EPO蛋白， 在本发明的一个实施方案 中， 首先构建可利用大肠杆菌系统高效表达的重组 EPO基因和工程菌， 其表达 的重组 EPOP无体内活性， 然后以此 EPOP为基础， 用 PEG2NHS-20K对其进行 化学修饰， 经修饰后的 PEG-EPOP结合物在动物体内表现出明显的促红细胞生 成的体内生理活性。
本发明采用原核表达系统获得无体内生物活性的 EPOP，再釆用 PEG对 EPOP 进行化学修饰， 获得 PEG-EPOP结合物， 并在动物实验中观察到具有体内活性。 如以此 PEG-EPOP结合物制成治疗贫血性疾病的药物， 因原核表达系统在生产 成本和规模成本上显著降低， 比真核表达系统具有的巨大优势，可明显地降低病 人的治疗用药费用。 附图说明
图 1为重组 EPOP原核表达系统质粒构建图。
图 2 PEG-EPO体内生理活性检测实验， 用 R— 500分析仪测定网织红细胞百分数： 图中： 纵坐标为网织红细胞百分数， 横坐标为采血时间 （1、 2、 3、 4分别 代表于注射样品后第四、 五、 六、 七日采血）
0#： 阴性对照
1#： rhEPO阳性对照
2#： 重组 EPOP, 未修饰对照
3#： PEG-EPOP结合物， 修饰后产物
图 3为两种氨基酸序列 （167AA和 166AA) 的 EPO蛋白表达对照图；
图中： 1 : 密码修改后， 表达的 167AA (含 167位 Arg)样品
2： 密码修改后， 表达的 166AA (不含 167位 Arg)样品 3： 1之诱导表达前对照样品
4： 2之诱导表达前对照样品 具体实施方式
下面结合附图，通过对本发明较佳实施方式的详细描述，说明但不限制本发 明。
【实施例 1】 由原核表达系统表达的重组 EPOP的制备
1. 菌株及质粒
质粒 PBV22Q全长 3.66Kb， 串联噬菌体？1^¾启动子， 下游为核糖体基因终止 信号， 氨苄青霉素抗性， 含 PLPR启动子的原核高效表达载体的组建 及其应用，病毒学报， 1990, 6 (2)： 11 ,该质粒由作者赠送。大肠杆菌菌株 DH5a 购自于 GIBCO公司 DH5a标准株。
2. 工具酶及试剂
限制性内切酶 BamH I、 EcoRI、 T4连接酶以及 Taq DNA聚合酶购于 Roche 公司， 质粒纯化试剂盒（Wizard Plus Minipreps DNA Purification System)购于 Promega公司， 蛋白分子量标记购于华美公司， 其它试剂均为国产分析纯试剂。
3. 人促红细胞生成素基因的合成
人体内表达的 EPO分子是由 166个氨基酸构成的糖基化蛋白质， 在后加工 修饰过程中其 C-末端 Arg166位被切除， 成为由 165个氨基酸组成的成熟 hEPO。 hEPO的 cDNA序列如 SEQID NO. 1所示， 其蛋白质一级结构的氨基酸序列如 SEQ ID N0. 2o其中大肠杆菌稀有密码子的比例占到 12%。为了让基因最佳化表 达， 我们通过对基因的重新设计和合成， 消除稀有密码子而利用最佳化密码子。 将 hEPO中的绝大部分稀有密码子替换成大肠杆菌偏爱密码子， 并适量调整 GC 含量， 同时在目的基因片段的两端增加 3个酶切位点： EcoR I: GAATTC; Nde l: CATATG; BamH I： GGATCC。密码替换前后对照如表 1中所示， 替换后合成的 序列中编码序列如 SEQ ID NO. 3所示。由于大肠杆菌基因表达以甲硫氨酸起始， 实际得到的肽链在 SEQ ID NO. 2序列 N端多一个甲硫氨酸， 共 167个氨基酸的 肽链， 蛋白一级结构没有其它改变。 表 1 hEPO基因密码替换前后对照表
替换前
替换后 GAATTCATATG
替换前 1 GCCCCACCACGCCT£ATCTGTGACAGCCGAGT£CTGGA^GaTACCTCIT 替换后 1 GCICCGCCGCGICTGATCTGTGArAGCCGIGTICTGGAiCGTTACCTGCT 替换前 51 GGA GCCAAGGAGGCCGAGAATATCACGACGGGCTGTGCTGAACACTGCA 替换后 51 GGA^GCrAA^GA^GCrGA^AACATCACGACGGGCTGTGCTGAACACTGCA 替换前 101 GCTTGAA1GAQAA1ATCACTGTCCCAGACACCAAAGTTAAITTCTATGCC 替换后 101 GCCTGAACGA4AACATCACTGTICCGGAIACCAAAGTTAACTTCTATGCI 替换前 151 TGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCT 替换后 151 TGGAA^CGrATGGA^GTrGGrCAGCAGGCrGTAGAAGT7TGGCAGGGCCT 替换前 201 GGCCCTGCTGTCGGAAGCTGT£CTGCGaGGCCAGGC£CTGlTGGTCAACT 替换后 201 GGCTCTGCTGTCGGAAGCTGTrCTGCGrGGCCAGGCrCTGCTGGTrAACT 替换前 251 CT^CCAGCCGTGGGAQCCCCTGCAGCTGCAIGTGGATAAAGCCGTCAGT 替换后 251 CT^GCCAGCCGTGGGA^CCCCTGCAGCTGCACGTGGATAAAGCrGTrAGT 替换前 301 GGCCT1CGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGA 替换后 301 GGCCTGCGIAGCCTGACCACTCTGCTGCGIGCTCTGGGAGCICAGAA^GA 替换前 351 AGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCA 替换后 351 AGCrATCiGCCCTCCGGATGCGGCTTCAGCTGCTCCGCTGCGIACCATCA 替换前 401 CTGCTGA£ACTTTCCG£AAACT£TTCCGAGT£TACIQCAA1TTCCT£CGQ 替换后 401 CTGCTGAIACTTTCCGIAAACTGTTCCGIGTrrACiGCAACTTCCTGCGI 替换前 451 GGAAA CTGAA CTGTACACAGGGGAQGCCTGCAGQACAGGGGACAGATG 替换后 451 GGAAA4CTGAA CTGTACACCGGIGA4GCrrGC GIACCGGIGA2 GrrG 替换前 501 A
替换后 501 AGGATCC
4. 序列合成方法：
方法参考 Jelenkovic G, Billings S' Chen Q,et al. Transformation of eggplant with synthetic ccrylllA gene produces a high level of resistance to the Colorado potato beetle. JAmer Soc Hort Sci, 1998.123(1) :19— 25, 釆用全基因合成的方法， 首先使 用设计软件设计长度约为 80nt 的 oligo , 这些 oligo之间互相形成约 18nt 的 overlap, 然后进行多轮 PCR扩增后得到全长基因， 电泳回收后进行克隆， 再测 序得到序列正确的克隆。
为了合成表达成熟 hEPO的 165个氨基酸（由 SEQ ID NO. 2所示的氨基酸 序列缺少第 166位精氨酸） 的基因序列， 设计如下两条引物， 用 PCR法从含有 经密码替换后的 EPO基因的质粒中重新扩增出缺少第 166位精氨酸密码（CGT) 的 EPO基因 (如 SEQ ID N0.4所示的表达 165个氨基酸的基因序列)， 按上述方 法同样获高效表达， 见图 3。 同样， 在大肠杆菌中表达的肽链为 N端增加一个甲 硫氨酸共 166个氨基酸的肽链。
引物： 5， GAG GAA TTC ATA TGG CTC CGC CGC GTC TG 3 '
5， C AG CTG GGA TCC TCA ATC ACC GGT ACG GCA 3 '
5. 重组质粒构建和筛选鉴定
将经测序正确的目的基因用限制性内切酶 BamH I和 EcoR I双酶切得到编码 序列 （SEQ ID NO. 4)， 电泳后回收后与用限制性内切酶 BamH I和 EcoR I同样 双酶切的 PBV22C质粒连接。连接产物转化大肠杆菌 DH5ot，涂布含氨苄青霉素的 LB平板得转化子。 挑取转化子， 提取质粒， 用限制性内切酶 BamH I和 EcoR I 双酶切分析， 筛选含有 hEPO基因片段的重组质粒转化子。
重组质粒的构建路线见图 1。
6. EPO基因在大肠杆菌中的表达
将重组子接种于 5ml LB培养基中， 在 30°C， 180rpm摇床培养 14h左右。 然 后以 1： 30的比例转入 LB培养基中， 30°C培养 4h左右 （A6oQ 0.8时）。 再以 42 °C诱导培养 4h。取培养物 lml， 离心后进行 SDS-PAGE及 Western blot鉴定所 表达的蛋白质。
7. 高密度发酵培养的培养基及培养条件：
7.1. LB种子培养基： 每立升含蛋白胨 10g， 酵母粉 5g, NaCl 5g, PH7.0,高压灭 菌， 使用时加入氨苄青霉素至 100ug/ml。
7.2. 发酵用培养基： 每 10L含 K2HP04 12g， KH2P04 - 3H20 20g, NaCl 20g,
MgS04 - 7H20 lOg, 葡萄糖 500g， 酵母粉 480g， 蛋白胨 430g， 高压灭菌 后使用。
7.3. 发酵培养： 将保存的菌种划平皿， 挑取单个菌落， 接种于 LB种子培养基 中， 培养 14〜16h后， 再以 1： 30的比例扩大培养 12h。 然后接种于 NBS
MPP-40发酵罐中培养，在培养过程中每间隔 1个小时补料一次， 并根据菌 体生长情况调整 PH和氧溶解度（DO)。
8. 纯化：
发酵产物按照本领域技术人员已知的方法分离、纯化蛋白， 如文献 ^ to ^ mutations at three sites which are N-glycosylated in the mammalian protein decrease the aggregation of Escherichia coli-derived erythropoietin, Protein Engineering , Vol.14 no.2 pp.135-140, 2001, Linda O.Narhi etc. 获得来自原核表达系统的重组
Figure imgf000008_0001
9. EPOP体外细胞学活性测定
9.1. 方法： 参 ΜΓΤ比色法与 EUSA法测定 rhEPO体外生物学活性的比较 [细 胞与分子免疫学杂志（J Cdl Mol Immunol) 2000;16(5)]韩蕾， 韩为跃, 收 获菌体以超声波破菌后离心。 上清液直接进行 UT7细胞活性检测， 沉淀以 6倍体积的 7M尿素溶解后进行 UT7细胞活性检测。 标准品为利血宝 （麒 麟鲲鹏公司） 3000IU/瓶 (2ml)
9.2. 结果： 对照的载体菌上清及沉淀溶解液中均未测得 UT7细胞活性。 构建的 工程菌破菌上清液测得 8IU/ml， 沉淀溶解液 400倍稀释后测得 700 IU/ml。 因此获得的重组 EPOP有维持 UT7细胞生长的活性，证明所获的重组 EPOP 肽链具有 EPO特有的体外细胞学活性。
【实施例 2】 PEG-EPOP结合物的制备
本发明涉及以 PEG2NHS-20K修饰 EPOP, 所进行的化学修饰反应式如下：
Figure imgf000008_0002
其中 X为 NH或 0， 代表 PEG对 EPOP化学修饰的共价连接方式， 一般为 EPO肽链中赖氨酸的侧链氨基（NH) 或 EPO的 N未端氨基， 也可以是 EPO肽 链中如丝氨酸的侧链羟基（OH)。
其中 i、 j为整数， 代表 PEG碳链的长度；
根据 PEG对 EPOP的修饰程度， n为 1〜5的整数， 优选为 1；
Figure imgf000008_0003
mPEG部分分子量 为 5000〜40000道尔顿， 优选 10000〜20000道尔顿。 可采用以下方法对获得的重组 EPO进行化学修饰: 1. 交换缓冲体系 （超滤法)：
用预先经 0.5mol/LNaOH除热原处理过的超滤器浓缩重组人促红细胞生成素 半成品， 并对 PH8.5、 50.0mmol/L磷酸盐缓冲液透滤 4次， 并用相同缓冲液调整 蛋白浓度为 lmg/ml。
2. 重组 EPO的化学修饰反应
取重组 EPO样品 （lmg/ml) 20ml, 加入 PEG2NHS-20K 100毫克（PEG2NHS 为 NEKTAR公司产品，产品使用说明见 Nektar Molecule Engingeering CATALOG 2003 ), 25°C缓慢搅拌下反应 30min， 加 Gly400毫克终止反应。
在化学修饰中，重组 EPO样品的蛋白浓度可以采用 0.01〜5 mg/ml，优选 0.1〜 1 mg/ml, 在本发明中最优选 lmg/ml。 活化 PEG酯与被修饰蛋白的摩尔比为 1： 1〜1： 10或更高， 如采用 PEG2NHS-20K重量比约为 2： 1〜1： 5， 在本发明 中优选重量比为 1： 5。
3. 分离及纯化
3.1. Superdex 200分子筛层析柱： 柱大小 (26cmX 100cm)
3.2. 清洗及平衡： 用无热原水洗层析柱， 流速 8〜10ml/min， 用 5倍柱床体积清 洗，然后用 5倍柱床体积的平衡缓冲液（0.2mol/LNaCl 20mmol/L柠檬酸缓 冲液 PH7.0)平衡柱。
3.3. 上样及洗脱： 将促红细胞生成素修饰反应混合物样品进样， 进样后用缓冲 液（0.2mol.dm-3 NaC1 20mmol.dm-3 柠檬酸缓冲液 PH7.0)洗脱， OD280nm 检测。 分别收集第一峰（PEG-EPOP结合物）和第二峰 （未反应 EPOP)。
【实施例 3】 PEG-EPOP结合物动物体内促红细胞生成活性的测定之一
1. 实验方法：参考《检测 EPO体内生物学活性的网织红细胞法的建立王箐舟、 程雅琴中国生物制品学杂志 1997年第 10卷第 1期》, 与文中略有不同的是 按作者推荐， 现采用仅注射一剂第四日釆血的方法。
1.1. 重组 EPOP样品： 依实施例 1方法表达制备的 EPO蛋白提纯样品， 含有 N 端的甲硫氨酸及 C端的精氨酸，共长 167个氨基酸的肽链， UT-7检测细胞 活性为 44000IU/ml;
1.2. PEG-EPOP结合物样品：参照实施例 2方法制备的 PEG修饰后的 PEG-EPOP 结合物样品， 在本次实验中的具体方法为： 5ML前述重组 EPOP样品中加 入 PEG2NHS-20K 50毫克， 修饰反应完全后未经分离及纯化， 置 4度保存 供动物实验用。
1.3. 实验动物： Balb/C纯系小鼠， 体重约 20克。
1.4. 样品以 20mM磷酸盐缓冲液 50倍稀释， 各样品组以背部皮下注射 0.2ml， 每组 3只， 给药后第四日眼眶静脉采血， 涂片染色， 测网织红百分数。 结果- 重组 EPOP: 2.6%、 3.4%、 3.7%, 均值为 3.2%
PEG-EPOP: 9.7%、 7.9%、 7.5%, 均值为 8.4%
阴性对照： 2.3%、 3.9%、 3.4%, 均值为 3.2% (历史值）
在动物实验中， 原核表达的重组 EPOP样品未能检测出促红细胞生成作用， 而同样量的 PEG修饰后 PEG-EPOP结合物样品有明显的促红细胞生成作用。 说 明重组 EPOP样品可以通过 PEG修饰获得原不具有的促红细胞生成素的体内活 性。 因此， 本实验的结果说明没有体内活性而有体外细胞活性的 EPO蛋白可以 通过 PEG修饰获得促红细胞生成素的体内活性。
【实施例 4】 PEG-EPOP结合物动物体内促红细胞生成活性的测定之二
1. 实验方法： 参考实施例 3， 每只腹部皮下注射 0.2ml， 以 R-500分析仪代替涂 片染色测网织红细胞百分数， 并测定注射后第四、 五、 六、 七日的结果。
1.1. 0#： 阴性对照共 4只， 注射生理盐水；
1.2. 1#： rhEPO阳性对照， 采用上海复星科技生物克隆公司 rhEPO产品贻宝， 批号 20040101， 2000IU/瓶， 2ml生理盐水溶解，取 200ul，加生理盐水 4.8ml 稀释， 浓度为 40IU/ml， 分 4组每组 4只。
1.3. 2#： 重组 EPOP, 未修饰对照样品。 依实施例 1方法表达制备的 EPO蛋白 提纯样品， 含有 N端的甲硫氨酸不含 C端的精氨酸， 共长 166个氨基酸的 肽链， UT-7检测细胞活性为 30000IU/ml， 100倍稀释， 浓度 300IU/ml。 分 4组每组 4只。
1.4. 3#： PEG-EPOP结合物， 前述 2#样品的修饰后产物， 修饰方法同实施例 3， 同 2#样品稀释分组。
2. 实验结果：
见图 2， 本次实验中重组 EPOP仅检测出微弱的体内活性， 而在 PEG-EPOP 结合物中测出明显的体内活性。 上述实施例表明， 无体内活性的 EPOP, 经过本发明方法修饰后的产物， 具 有良好的体内促红细胞生成的活性， 由于成本低廉， 应用前景好。
本发明涉及的肽链包括具有天然 EPO氨基酸顺序的肽链以及为各种目的改 进的同源肽链， 如： 按本发明中提出的具有 166个氨基酸 （如果从 N端的第一 位甲硫氨酸 MET起算应为 167个氨基酸， 本发明的实施例表明具有该甲硫氨酸 的 EPOP仍具有理论上应有的体外细胞学活性及 PEG修饰后的体内生理活性） 的天然表达序列以及表达的模拟后加工中切去 Arg166的天然肽链，或为它种目的 改变部分氨基酸的肽链， 如： 减少糖基化位点以增加肽链稳定性等等， 只要该 EPO同源肽链在体内不能表现或仅能表现微弱的 EPO生理活性，经 PEG修饰后 获得较强的体内生理活性， 均在本发明的范围内。
本发明涉及的修饰试剂为活化 PEG酯， 也可采用其它的方法将 PEG链与 EPOP的肽链共价偶联， 包括其它种类的活化基团， 或对 EPOP肽链的相应位点 也进行活化， 以及单链、 双链或多链的多种结构的 mPEG， 同样在本发明的范围 内。
很显然， 以上对本发明的详细描述并不限制本发明，本领域技术人员可以根 据本发明作出各种改变或变形，只要不脱离本发明的精神， 均应属于本发明所附 权利要求所定义的范围。

Claims

权利要求
1、 一种促红细胞生成素蛋白的聚乙二醇结合物， 其特征在于将无体内活性 的重组人促红细胞生成素蛋白经聚乙二醇化学修饰，获得具有体内促红细胞生成 活性的产物。
2、 权利要求 1所述的促红细胞生成素蛋白的聚乙二醇结合物， 其特征在于 其具有式 （I) 结构：
mPEG-X-EPOP (I)
式（I) 中， mPEG的分子结构式为 CH3 "CH2CH20 -fc"
其中 k为 100〜1000的整数，使结合物的 mPEG部分分子量为 5000〜40000 道尔顿；
X为 NH或 0， 代表 PEG对 EPOP化学修饰的共价连接方式；
EPOP代表无体内活性的重组人促红细胞生成素蛋白。
3、 权利要求 2所述的促红细胞生成素蛋白的聚乙二醇结合物， 其特征在于 其具有式 （II) 结构：
Figure imgf000011_0001
式（II) 中， n为 1〜5的整数， 代表 PEG对 EPOP的修饰程度；
mPEG的分子结构式为 CH3"H H2CH20
其中 lc为 100〜1000的整数， 为 i与 j之和， 使结合物的 mPEG部分分子量 为 5000〜40000道尔顿；
X为 NH或 0， 代表 PEG对 EPOP化学修饰的共价连接方式；
EPOP代表无体内活性的重组人促红细胞生成素蛋白。
4、 权利要求 2所述的促红细胞生成素蛋白的聚乙二醇结合物， 其特征在于 所述 EPOP是由原核表达系统表达的。
5、 权利要求 4所述的促红细胞生成素蛋白的聚乙二醇结合物， 其特征在于 所述 EPOP是由大肠杆菌表达系统表达的。
6、 权利要求 1所述的促红细胞生成素蛋白的聚乙二醇结合物在治疗贫血性 疾病药物中的应用。
7、 权利要求 1所述的促红细胞生成素蛋白的聚乙二醇结合物在制备升高红 细胞药物中的应用。
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WO2009094551A1 (en) 2008-01-25 2009-07-30 Amgen Inc. Ferroportin antibodies and methods of use
WO2010056981A2 (en) 2008-11-13 2010-05-20 Massachusetts General Hospital Methods and compositions for regulating iron homeostasis by modulation bmp-6
WO2011050333A1 (en) 2009-10-23 2011-04-28 Amgen Inc. Vial adapter and system
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WO2012135315A1 (en) 2011-03-31 2012-10-04 Amgen Inc. Vial adapter and system
US8383114B2 (en) 2007-09-27 2013-02-26 Amgen Inc. Pharmaceutical formulations
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WO2014144096A1 (en) 2013-03-15 2014-09-18 Amgen Inc. Drug cassette, autoinjector, and autoinjector system
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WO2015061389A1 (en) 2013-10-24 2015-04-30 Amgen Inc. Drug delivery system with temperature-sensitive control
WO2015061386A1 (en) 2013-10-24 2015-04-30 Amgen Inc. Injector and method of assembly
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WO2015171777A1 (en) 2014-05-07 2015-11-12 Amgen Inc. Autoinjector with shock reducing elements
WO2015187799A1 (en) 2014-06-03 2015-12-10 Amgen Inc. Systems and methods for remotely processing data collected by a drug delivery device
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WO2016100781A1 (en) 2014-12-19 2016-06-23 Amgen Inc. Drug delivery device with proximity sensor
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US9657098B2 (en) 2013-03-15 2017-05-23 Intrinsic Lifesciences, Llc Anti-hepcidin antibodies and uses thereof
WO2017100501A1 (en) 2015-12-09 2017-06-15 Amgen Inc. Auto-injector with signaling cap
WO2017120178A1 (en) 2016-01-06 2017-07-13 Amgen Inc. Auto-injector with signaling electronics
WO2017160799A1 (en) 2016-03-15 2017-09-21 Amgen Inc. Reducing probability of glass breakage in drug delivery devices
WO2017189089A1 (en) 2016-04-29 2017-11-02 Amgen Inc. Drug delivery device with messaging label
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WO2018151890A1 (en) 2017-02-17 2018-08-23 Amgen Inc. Drug delivery device with sterile fluid flowpath and related method of assembly
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WO2018172219A1 (en) 2017-03-20 2018-09-27 F. Hoffmann-La Roche Ag Method for in vitro glycoengineering of an erythropoiesis stimulating protein
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WO2020091981A1 (en) 2018-11-01 2020-05-07 Amgen Inc. Drug delivery devices with partial drug delivery member retraction
WO2020219482A1 (en) 2019-04-24 2020-10-29 Amgen Inc. Syringe sterilization verification assemblies and methods
WO2021041067A2 (en) 2019-08-23 2021-03-04 Amgen Inc. Drug delivery device with configurable needle shield engagement components and related methods
EP3981450A1 (en) 2015-02-27 2022-04-13 Amgen, Inc Drug delivery device having a needle guard mechanism with a tunable threshold of resistance to needle guard movement
WO2022246055A1 (en) 2021-05-21 2022-11-24 Amgen Inc. Method of optimizing a filling recipe for a drug container
WO2024094457A1 (en) 2022-11-02 2024-05-10 F. Hoffmann-La Roche Ag Method for producing glycoprotein compositions
WO2024186940A1 (en) * 2023-03-06 2024-09-12 Braidwell Labs Llc Codon optimization and methods of use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000032772A2 (en) * 1998-11-30 2000-06-08 Eli Lilly And Company Erythropoietic compounds
WO2004009627A1 (en) * 2002-07-19 2004-01-29 Cangene Corporation Pegylated erythropoietic compounds

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000032772A2 (en) * 1998-11-30 2000-06-08 Eli Lilly And Company Erythropoietic compounds
WO2004009627A1 (en) * 2002-07-19 2004-01-29 Cangene Corporation Pegylated erythropoietic compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FRANCIS G.E. ET AL: "PEGylation of cytokines and other therapeutics proteins and peptides: the importance of biological optimisation of coupling techniques.", INT.J. HEMATOL., vol. 68, no. 1, July 1998 (1998-07-01), pages 1 - 18, XP000907109 *

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EP3831427A1 (en) 2013-03-22 2021-06-09 Amgen Inc. Injector and method of assembly
WO2014149357A1 (en) 2013-03-22 2014-09-25 Amgen Inc. Injector and method of assembly
EP3421066A1 (en) 2013-10-24 2019-01-02 Amgen, Inc Injector and method of assembly
EP3957345A1 (en) 2013-10-24 2022-02-23 Amgen, Inc Drug delivery system with temperature-sensitive control
EP3501575A1 (en) 2013-10-24 2019-06-26 Amgen, Inc Drug delivery system with temperature-sensitive-control
WO2015061389A1 (en) 2013-10-24 2015-04-30 Amgen Inc. Drug delivery system with temperature-sensitive control
WO2015061386A1 (en) 2013-10-24 2015-04-30 Amgen Inc. Injector and method of assembly
EP3789064A1 (en) 2013-10-24 2021-03-10 Amgen, Inc Injector and method of assembly
WO2015119906A1 (en) 2014-02-05 2015-08-13 Amgen Inc. Drug delivery system with electromagnetic field generator
WO2015171777A1 (en) 2014-05-07 2015-11-12 Amgen Inc. Autoinjector with shock reducing elements
EP3785749A1 (en) 2014-05-07 2021-03-03 Amgen Inc. Autoinjector with shock reducing elements
US11213624B2 (en) 2014-06-03 2022-01-04 Amgen Inc. Controllable drug delivery system and method of use
WO2015187793A1 (en) 2014-06-03 2015-12-10 Amgen Inc. Drug delivery system and method of use
WO2015187797A1 (en) 2014-06-03 2015-12-10 Amgen Inc. Controllable drug delivery system and method of use
WO2015187799A1 (en) 2014-06-03 2015-12-10 Amgen Inc. Systems and methods for remotely processing data collected by a drug delivery device
US11992659B2 (en) 2014-06-03 2024-05-28 Amgen Inc. Controllable drug delivery system and method of use
EP4036924A1 (en) 2014-06-03 2022-08-03 Amgen, Inc Devices and methods for assisting a user of a drug delivery device
EP4362039A2 (en) 2014-06-03 2024-05-01 Amgen Inc. Controllable drug delivery system and method of use
US11738146B2 (en) 2014-06-03 2023-08-29 Amgen Inc. Drug delivery system and method of use
WO2016049036A1 (en) 2014-09-22 2016-03-31 Intrinsic Lifesciences Llc Humanized anti-hepcidin antibodies and uses thereof
US10323088B2 (en) 2014-09-22 2019-06-18 Intrinsic Lifesciences Llc Humanized anti-hepcidin antibodies and uses thereof
WO2016061220A2 (en) 2014-10-14 2016-04-21 Amgen Inc. Drug injection device with visual and audio indicators
EP3943135A2 (en) 2014-10-14 2022-01-26 Amgen Inc. Drug injection device with visual and audible indicators
EP3689394A1 (en) 2014-12-19 2020-08-05 Amgen Inc. Drug delivery device with live button or user interface field
US11357916B2 (en) 2014-12-19 2022-06-14 Amgen Inc. Drug delivery device with live button or user interface field
WO2016100781A1 (en) 2014-12-19 2016-06-23 Amgen Inc. Drug delivery device with proximity sensor
WO2016100055A1 (en) 2014-12-19 2016-06-23 Amgen Inc. Drug delivery device with live button or user interface field
US11944794B2 (en) 2014-12-19 2024-04-02 Amgen Inc. Drug delivery device with proximity sensor
US10765801B2 (en) 2014-12-19 2020-09-08 Amgen Inc. Drug delivery device with proximity sensor
US10799630B2 (en) 2014-12-19 2020-10-13 Amgen Inc. Drug delivery device with proximity sensor
EP3848072A1 (en) 2014-12-19 2021-07-14 Amgen Inc. Drug delivery device with proximity sensor
EP3556411A1 (en) 2015-02-17 2019-10-23 Amgen Inc. Drug delivery device with vacuum assisted securement and/or feedback
EP3981450A1 (en) 2015-02-27 2022-04-13 Amgen, Inc Drug delivery device having a needle guard mechanism with a tunable threshold of resistance to needle guard movement
WO2017039786A1 (en) 2015-09-02 2017-03-09 Amgen Inc. Syringe assembly adapter for a syringe
WO2017100501A1 (en) 2015-12-09 2017-06-15 Amgen Inc. Auto-injector with signaling cap
WO2017120178A1 (en) 2016-01-06 2017-07-13 Amgen Inc. Auto-injector with signaling electronics
WO2017160799A1 (en) 2016-03-15 2017-09-21 Amgen Inc. Reducing probability of glass breakage in drug delivery devices
EP3721922A1 (en) 2016-03-15 2020-10-14 Amgen Inc. Reducing probability of glass breakage in drug delivery devices
EP4035711A1 (en) 2016-03-15 2022-08-03 Amgen Inc. Reducing probability of glass breakage in drug delivery devices
WO2017189089A1 (en) 2016-04-29 2017-11-02 Amgen Inc. Drug delivery device with messaging label
WO2017192287A1 (en) 2016-05-02 2017-11-09 Amgen Inc. Syringe adapter and guide for filling an on-body injector
WO2017197222A1 (en) 2016-05-13 2017-11-16 Amgen Inc. Vial sleeve assembly
WO2017200989A1 (en) 2016-05-16 2017-11-23 Amgen Inc. Data encryption in medical devices with limited computational capability
WO2017209899A1 (en) 2016-06-03 2017-12-07 Amgen Inc. Impact testing apparatuses and methods for drug delivery devices
WO2018004842A1 (en) 2016-07-01 2018-01-04 Amgen Inc. Drug delivery device having minimized risk of component fracture upon impact events
WO2018034784A1 (en) 2016-08-17 2018-02-22 Amgen Inc. Drug delivery device with placement detection
WO2018081234A1 (en) 2016-10-25 2018-05-03 Amgen Inc. On-body injector
WO2018136398A1 (en) 2017-01-17 2018-07-26 Amgen Inc. Injection devices and related methods of use and assembly
WO2018152073A1 (en) 2017-02-17 2018-08-23 Amgen Inc. Insertion mechanism for drug delivery device
WO2018151890A1 (en) 2017-02-17 2018-08-23 Amgen Inc. Drug delivery device with sterile fluid flowpath and related method of assembly
WO2018165143A1 (en) 2017-03-06 2018-09-13 Amgen Inc. Drug delivery device with activation prevention feature
WO2018164829A1 (en) 2017-03-07 2018-09-13 Amgen Inc. Needle insertion by overpressure
WO2018165499A1 (en) 2017-03-09 2018-09-13 Amgen Inc. Insertion mechanism for drug delivery device
WO2018172219A1 (en) 2017-03-20 2018-09-27 F. Hoffmann-La Roche Ag Method for in vitro glycoengineering of an erythropoiesis stimulating protein
WO2018183039A1 (en) 2017-03-28 2018-10-04 Amgen Inc. Plunger rod and syringe assembly system and method
EP4512445A2 (en) 2017-03-28 2025-02-26 Amgen Inc. Plunger rod and syringe assembly system
EP4241807A2 (en) 2017-03-28 2023-09-13 Amgen Inc. Plunger rod and syringe assembly system and method
WO2018226565A1 (en) 2017-06-08 2018-12-13 Amgen Inc. Torque driven drug delivery device
WO2018226515A1 (en) 2017-06-08 2018-12-13 Amgen Inc. Syringe assembly for a drug delivery device and method of assembly
WO2018236619A1 (en) 2017-06-22 2018-12-27 Amgen Inc. REDUCING THE IMPACTS / IMPACTS OF ACTIVATION OF A DEVICE
WO2018237225A1 (en) 2017-06-23 2018-12-27 Amgen Inc. ELECTRONIC DRUG DELIVERY DEVICE COMPRISING A CAP ACTIVATED BY A SWITCH ASSEMBLY
WO2019014014A1 (en) 2017-07-14 2019-01-17 Amgen Inc. NEEDLE INSERTION-RETRACTING SYSTEM HAVING DOUBLE TORSION SPRING SYSTEM
EP4292576A2 (en) 2017-07-21 2023-12-20 Amgen Inc. Gas permeable sealing member for drug container and methods of assembly
WO2019018169A1 (en) 2017-07-21 2019-01-24 Amgen Inc. PERMEABLE GAS SEALING ELEMENT FOR MEDICINE CONTAINER AND METHODS OF ASSEMBLY
EP4085942A1 (en) 2017-07-25 2022-11-09 Amgen Inc. Drug delivery device with gear module and related method of assembly
WO2019022951A1 (en) 2017-07-25 2019-01-31 Amgen Inc. DRUG DELIVERY DEVICE WITH GEAR MODULE AND ASSEMBLY METHOD THEREOF
WO2019022950A1 (en) 2017-07-25 2019-01-31 Amgen Inc. DRUG DELIVERY DEVICE WITH CONTAINER ACCESS SYSTEM AND ASSEMBLY METHOD THEREOF
WO2019032482A2 (en) 2017-08-09 2019-02-14 Amgen Inc. HYDRAULIC-PNEUMATIC PRESSURE CHAMBER DELIVERY SYSTEM
WO2019036181A1 (en) 2017-08-18 2019-02-21 Amgen Inc. BODY INJECTOR WITH STERILE ADHESIVE PATCH
WO2019040548A1 (en) 2017-08-22 2019-02-28 Amgen Inc. NEEDLE INSERTION MECHANISM FOR DRUG DELIVERY DEVICE
WO2019070472A1 (en) 2017-10-04 2019-04-11 Amgen Inc. FLOW ADAPTER FOR MEDICATION DELIVERY DEVICE
WO2019070552A1 (en) 2017-10-06 2019-04-11 Amgen Inc. DRUG DELIVERY DEVICE COMPRISING A LOCKOUT ASSEMBLY AND ASSOCIATED ASSEMBLY METHOD
EP4257164A2 (en) 2017-10-06 2023-10-11 Amgen Inc. Drug delivery device with interlock assembly and related method of assembly
WO2019074579A1 (en) 2017-10-09 2019-04-18 Amgen Inc. DRUG DELIVERY DEVICE COMPRISING A DRIVE ASSEMBLY AND ASSEMBLY METHOD THEREOF
WO2019090086A1 (en) 2017-11-03 2019-05-09 Amgen Inc. Systems and approaches for sterilizing a drug delivery device
WO2019090079A1 (en) 2017-11-03 2019-05-09 Amgen Inc. System and approaches for sterilizing a drug delivery device
WO2019090303A1 (en) 2017-11-06 2019-05-09 Amgen Inc. Fill-finish assemblies and related methods
WO2019089178A1 (en) 2017-11-06 2019-05-09 Amgen Inc. Drug delivery device with placement and flow sensing
WO2019094138A1 (en) 2017-11-10 2019-05-16 Amgen Inc. Plungers for drug delivery devices
WO2019099324A1 (en) 2017-11-16 2019-05-23 Amgen Inc. Door latch mechanism for drug delivery device
WO2019099322A1 (en) 2017-11-16 2019-05-23 Amgen Inc. Autoinjector with stall and end point detection
WO2019231582A1 (en) 2018-05-30 2019-12-05 Amgen Inc. Thermal spring release mechanism for a drug delivery device
WO2019231618A1 (en) 2018-06-01 2019-12-05 Amgen Inc. Modular fluid path assemblies for drug delivery devices
WO2020023220A1 (en) 2018-07-24 2020-01-30 Amgen Inc. Hybrid drug delivery devices with tacky skin attachment portion and related method of preparation
WO2020023451A1 (en) 2018-07-24 2020-01-30 Amgen Inc. Delivery devices for administering drugs
WO2020023444A1 (en) 2018-07-24 2020-01-30 Amgen Inc. Delivery devices for administering drugs
WO2020023336A1 (en) 2018-07-24 2020-01-30 Amgen Inc. Hybrid drug delivery devices with grip portion
WO2020028009A1 (en) 2018-07-31 2020-02-06 Amgen Inc. Fluid path assembly for a drug delivery device
WO2020068623A1 (en) 2018-09-24 2020-04-02 Amgen Inc. Interventional dosing systems and methods
WO2020068476A1 (en) 2018-09-28 2020-04-02 Amgen Inc. Muscle wire escapement activation assembly for a drug delivery device
WO2020072577A1 (en) 2018-10-02 2020-04-09 Amgen Inc. Injection systems for drug delivery with internal force transmission
WO2020072846A1 (en) 2018-10-05 2020-04-09 Amgen Inc. Drug delivery device having dose indicator
WO2020081479A1 (en) 2018-10-15 2020-04-23 Amgen Inc. Drug delivery device having damping mechanism
WO2020081480A1 (en) 2018-10-15 2020-04-23 Amgen Inc. Platform assembly process for drug delivery device
WO2020092056A1 (en) 2018-11-01 2020-05-07 Amgen Inc. Drug delivery devices with partial needle retraction
WO2020091956A1 (en) 2018-11-01 2020-05-07 Amgen Inc. Drug delivery devices with partial drug delivery member retraction
WO2020091981A1 (en) 2018-11-01 2020-05-07 Amgen Inc. Drug delivery devices with partial drug delivery member retraction
WO2020219482A1 (en) 2019-04-24 2020-10-29 Amgen Inc. Syringe sterilization verification assemblies and methods
WO2021041067A2 (en) 2019-08-23 2021-03-04 Amgen Inc. Drug delivery device with configurable needle shield engagement components and related methods
WO2022246055A1 (en) 2021-05-21 2022-11-24 Amgen Inc. Method of optimizing a filling recipe for a drug container
WO2024094457A1 (en) 2022-11-02 2024-05-10 F. Hoffmann-La Roche Ag Method for producing glycoprotein compositions
WO2024186940A1 (en) * 2023-03-06 2024-09-12 Braidwell Labs Llc Codon optimization and methods of use thereof

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