WO2004083242A1 - Conjugue de facteur stimulant les colonies de granulocytes humain presentant une stabilite accrue dans le sang et son procede de preparation - Google Patents

Conjugue de facteur stimulant les colonies de granulocytes humain presentant une stabilite accrue dans le sang et son procede de preparation Download PDF

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WO2004083242A1
WO2004083242A1 PCT/KR2004/000569 KR2004000569W WO2004083242A1 WO 2004083242 A1 WO2004083242 A1 WO 2004083242A1 KR 2004000569 W KR2004000569 W KR 2004000569W WO 2004083242 A1 WO2004083242 A1 WO 2004083242A1
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peg
conjugate
csf
csf derivative
derivative
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PCT/KR2004/000569
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English (en)
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Sung Youb Jung
Young Min Kim
Kyeong Bae Kim
Chang Ki Lim
Geun Hee Yang
Se Chang Kwon
Gwan Sun Lee
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Hanmi Pharm. Co. Ltd.
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Publication of WO2004083242A1 publication Critical patent/WO2004083242A1/fr

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    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21J—FIBREBOARD; MANUFACTURE OF ARTICLES FROM CELLULOSIC FIBROUS SUSPENSIONS OR FROM PAPIER-MACHE
    • D21J3/00—Manufacture of articles by pressing wet fibre pulp, or papier-mâché, between moulds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B30—PRESSES
    • B30B—PRESSES IN GENERAL
    • B30B3/00—Presses characterised by the use of rotary pressing members, e.g. rollers, rings, discs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B30—PRESSES
    • B30B—PRESSES IN GENERAL
    • B30B9/00—Presses specially adapted for particular purposes
    • B30B9/28—Presses specially adapted for particular purposes for forming shaped articles
    • G—PHYSICS
    • G09—EDUCATION; CRYPTOGRAPHY; DISPLAY; ADVERTISING; SEALS
    • G09B—EDUCATIONAL OR DEMONSTRATION APPLIANCES; APPLIANCES FOR TEACHING, OR COMMUNICATING WITH, THE BLIND, DEAF OR MUTE; MODELS; PLANETARIA; GLOBES; MAPS; DIAGRAMS
    • G09B1/00—Manually or mechanically operated educational appliances using elements forming, or bearing, symbols, signs, pictures, or the like which are arranged or adapted to be arranged in one or more particular ways
    • G09B1/32—Manually or mechanically operated educational appliances using elements forming, or bearing, symbols, signs, pictures, or the like which are arranged or adapted to be arranged in one or more particular ways comprising elements to be used without a special support
    • G09B1/36—Manually or mechanically operated educational appliances using elements forming, or bearing, symbols, signs, pictures, or the like which are arranged or adapted to be arranged in one or more particular ways comprising elements to be used without a special support the elements being connectible by corresponding projections and recesses

Definitions

  • the present invention relates to a conjugate of a human granulocyte- colony stimulating factor ( G-CSF) derivative modified with a polyethylene glycol (PEG) and a method for the preparation thereof.
  • G-CSF human granulocyte- colony stimulating factor
  • PEG polyethylene glycol
  • G-CSFs Granulocyte-colony stimulating factors
  • cytokines that stimulate the expansion and differentiation of bone marrow progenitor cells and leukocytes. They also activate mature myeloid cells to mediate a variety of anti-microbial activities and inflammatory responses. They have been widely used in clinical transplantation which induces hematopoietic stem cell mobilization and increases myeloid progenitor cell population. It has been reported that the purified G-CSF is highly active in inducing granulocyte differentiation and suppresses stem cell self-replication in myeloid leukemia cells (Nicola et. al, JBC 258(14): 9017-9023, 1983).
  • G-CSF is a glycoprotein, consisting of 174 amino acids, which plays an important role in hematopoietic cell proliferation, differentiation of hematopoietic precursor cells, and activation of mature neutrophilic granulocytes.
  • hG-CSF gene transcribes into two kinds of mRNA consisting of 207 and 204 amino acids, respectively, that contain a leader sequence consisting of 30 amino acids for their secretion at the terminal amino residue (Nagata et al., Nature 319: 415-418, 1986; Nagata et al., EMBO J. 5(3): 575-581, 1986; Souza et al, Science 232: 61-65, 1986).
  • the difference in the two hG-CSFs is the presence or absence of three amino acids (Val-Ser-Glu) at the 35 th amino acid residue starting from the terminal amino residue. Accordingly, a mature hG- CSF protein has 174 or 177 amino acids.
  • hG-CSF consisting of 174 amino acids shows a 20-fold higher activity than hG-CSF consisting of 177 amino acids. However, whether both hG-CSFs are actually expressed in vivo is not elucidated at this time.
  • hG-CSF does not contain the amino acid sequences (Asn-X-Ser/Thr) relating to N-glycosylation, but has an O-glycosylation site at the 133 rd amino acid residue, threonine.
  • glycosylated hG-CSF glycoprotein is prepared from mammalian cells using a genomic DNA or cDAN encoding hG-CSF.
  • the glycosylated hG-CSF thus produced has O-glycosidic type sugar chain structures, but it has been known that the sugar moiety is not essential for hG-CSF activity (Lawrence, M. et al., Science 232: 61, 1986). Further, since these methods need to employ an expensive culture medium and complicated purification process to obtain the glycosylated hG-CSF, they are uneconomical.
  • hG-CSF non-glycosylated hG-CSF using a prokaryotic cell.
  • E. coli which produces hG- CSF consisting of 175 or 178 amino acids according to the characteristics of E. coli cell.
  • These products have an extra methionine residue added at the terminal amino residue by the action of the ATG codon existing at the site of initiation codon.
  • the additional methionine can trigger harmful immune response (EP Patent Publication No. 256,843).
  • most of the expressed hG-CSF accumulates in cytoplasm in the form of insoluble inclusion bodies and must be converted into an active form through refolding during purification.
  • hG-CSF exists partially in a reduced form, or forms an intermolecular disulfide coupling body or a defective disulfide coupling body. It is difficult to remove these by-products, which markedly lower the yield. In particular, it is extremely difficult to remove undesirable hG-CSF by-products such as misfolded hG-CSF.
  • hG-CSF has five cysteine residues, four of which participate in disulfide bonding, while the remaining cysteine in the unbound free form reduces the folding titer and stability in solution. Further, non-glycosylated hG-CSF expressed in E. coli is apt to aggregate at a high concentration because of its low solubility.
  • the present inventors have endeavored to develop a method for producing a soluble hG-CSF having no methionine residue added at the terminal amino residue thereof by using a microorganism.
  • the present inventors have previously generated a new signal peptide of E. coli thermostable enterotoxin II (Korean Patent Application No. 98-38061) and found that this new secretory signal peptide can be used for the mass-production of the native form of hG-CSF. Further, the present inventors have found that when an hG-CSF derivative obtained by replacing the 17 th cysteine residue with other amino acid ligated to the modified E.
  • thermostable enterotoxin II signal sequence is expressed, hG-CSF is produced in a wild-type form having no methionine residue added at its terminal amino residue in the periplasm of E. coli (Korean Patent No. 356140).
  • a protein when a protein has a small molecular weight, it may be lost in the process of clearing intestinal tract, kidney, spleen or liver through filteration, but such pegylation may reduce the clearance loss (Knauf, M. J. et al, J. Biol. Chem. 263: 15064, 1988). Many studies to prolong the in vivo half-life, increase the solubility or reduce the immune response by coupling PEG to a biologically active peptide or a protein have been actively progressed.
  • pegylation increases the solubility of peptide drug and prevents the hydrolysis thereof, thereby increasing the serum stability of the peptide drug without incurring any immune response due to its low antigenecity (Sade et al., J. Fermentation Bioengineering 71: 1370139, 1991).
  • pegylated polypeptides have the disadvantages of lowering both the activity and production yield of an active substance when the molecular weight of PEG increases.
  • PEG is covalently linked to one or more of free lysine residues existing at the surface of a protein, but if PEG binds to an active site directly involved in protein activity, the protein activity decreases. Further, since the binding of PEG to a lysine residue occurs in a random fashion, many kinds of pegylated protein complexes may co-exist as a mixture, and therefore, a purification process of a desired complex becomes more complicated and difficult (EP Patent Publication Nos: 0401384 and 0335423).
  • EP Patent Publication No: 0733067 has disclosed a method for preparing a uniform G-CSF composition which comprises a PEG-G-CSF complex prepared by a selective coupling of PEG to a terminal amino residue of G-CSF.
  • a fusion protein of PEG-G-CSF complex thus prepared shows enhanced stability, but this method does not use a wild-type G-CSF and produces an inactive protein having an extra methionine residue added at the terminal amino residue in the form of an inclusion body.
  • they have the problems of triggering harmful immune response, requiring a refolding process and a markedly low yield.
  • the present inventors have developed a PEG conjugate of an hG-CSF derivative having the 17 amino acid of the wild-type hG-CSF replaced with serine without an extra methionine residue added at the terminal amino residue.
  • the PEG-hG-CSF derivative conjugate of the present invention has a prolonged in vivo half-life without inducing an immune response in a subject, while minimizing the biological activity loss.
  • a primary object of the present invention is to provide a PEG conjugate of an hG-CSF derivative having a prolonged in vivo half-life and an improved biological activity without inducing an immune response in a subject; and a method for the preparation thereof.
  • Another object of the present invention is to provide a pharmaceutical composition comprising said PEG conjugate of hG-CSF derivative.
  • a further object of the present invention is to provide a method for enhancing the in vivo stability and prolonging the circulating half-life of hG- CSF, without sacrificing the activity thereof.
  • a PEG-hG-CSF derivative conjugate having a prolonged in vivo half-life which is prepared by modifying the terminal amino residue of hG-CSF derivative with one PEG molecule; and a method for the preparation thereof.
  • a pharmaceutical composition of hG-CSF having a prolonged in vivo half-life in comparison with unmodified hG-CSF which comprises said PEG- hG-CSF derivative conjugate and a pharmaceutically acceptable carrier.
  • a method for enhancing the in vivo half-life of hG-CSF which comprises specifically modifying the terminal amino residue of hG-CSF derivative with one PEG molecule.
  • Fig. 1 SDS-PAGE results of PEG-hG-CSF derivative conjugates
  • Fig. 2 a size exclusion chromatogram of PEG-hG-CSF derivative conjugates
  • Fig. 3 a result for comparing the peptide map of a PEG-hG-CSF derivative conjugate (B) with that of unmodified hG-CSF (A);
  • Fig. 4 in vivo activities of PEG-hG-CSF derivative conjugates;
  • Fig. 5 a pharmacokinetic graph showing the enhanced circulating half- life of a PEG-hG-CSF derivative conjugate.
  • the present invention relates to a conjugate of human G-CSF derivative (Korean Patent No: 356140) modified with PEG and a method for the preparation thereof.
  • the PEG conjugate of an hG-CSF derivative is a protein conjugate prepared by selectively modifying the terminal amino residue of an hG-CSF derivative with PEG, wherein the terminal amino residue does not necessarily participate in the binding of hG-CSF to a receptor thereof (Hill et, al., PNAS 90: 5167, 1993).
  • the hG-CSF derivative employed in the present invention may be either a natural one isolated from a mammal or a chemically synthesized recombinant. Further, it may also be obtained from prokaryotic or eukaryotic cells transformed with DNA coding the hG-CSF derivative by genetic engineering. For this purpose, E. coli, yeast (e.g., S. cerevisias) and mammalian cells (e.g., Chinese hamster ovary cell, CHO) can be employed as a host.
  • E. coli, yeast e.g., S. cerevisias
  • mammalian cells e.g., Chinese hamster ovary cell, CHO
  • the protein product expressed from hG-CSF derivative-transformed mammalian cells or eukaryotic cells may be glycosylated or non-glycosylated with a host cell-derived carbohydrate.
  • the protein product may include an incipient methionine residue at -1 position starting from the translation initiation codon.
  • a recombinant hG- CSF derivative obtained by replacing the 17 th amino acid residue of wild-type hG-CSF with serine according to the method described in Korean Patent No. 356140.
  • PEG employable in the present invention mat be any one of known soluble PEG polymers. In the process of modifying a protein with a PEG polymer having a reactive group, the PEG binding occurs at the ⁇ -amino group of lysine, cysteine or histidine residue depending on the nature of the reactive group of PEG. But, when such a PEG polymer having a reactive group is used, two more of PEG molecules may bind to a hG-CSF derivative, leading to the loss of its protein activity.
  • the binding between the hG-CSF derivative and PEG preferably occurs in a molar ratio of 1:1, and a suitable PEG polymer for this purpose is soluble in an aqueous environment such as a physiological environment and has a single reactive aldehyde group which specifically binds to the ⁇ -amino group of the terminal amino residue of hG-CSF derivative.
  • a PEG has the aldehyde reactive group at one end and an alkoxy group at the other end, and the terminal amino residue is threonine.
  • the molecular weight of said PEG is preferably, but not to be limited to, in the range of 2 to 100 kDa, more preferably, 10 to 40 kDa; and said PEG may be branched or not.
  • a pegylation reaction involving the amino group of the hG-CSF derivative and the aldehyde group of the PEG is carried out in the presence of a reducing agent.
  • the alkoxy-PEG-aldehyde is methoxy-PEG-aldehyde
  • the reducing agent is cyanoborohydride (NaCNBH 3 ), sodium borohydride, dimethylamine borate or pyridine borate.
  • the HPLC peak pattern of PEG-hG-CSF derivative conjugate differs from that of unmodified hG-CSF in specific ways due to the binding of PEG to one of protease cleavage fragments of PEG conjugate.
  • the protease may be, but not limited to, trypsin, subtilisin or endoproteinase Glu-C; and, more preferably, endoproteinase Glu-C.
  • the biological activity of the inventive PEG-hG-CSF derivative conjugate is measured according to the conventional methods well-known in the art (Baldwin et al., Ada Endocrinologica. 119: 326, 1988; Clark et al., J. Biol.
  • the present invention provides a pharmaceutical composition of hG-CSF having a prolonged in vivo half-life, which comprises the inventive PEG-hG-CSF derivative conjugate and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous and intramuscular introduction, and injection is more preferred.
  • the composition of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after it is administered to a patient, by employing any one of the procedures well-known in the art.
  • the formulation may be in the form of a tablet, pill, powder, sachet, elixer, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.
  • Suitable carriers, excipients or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, polyvinylpyrrolidone, cellulose, methylcellulose, microcrystalline magnesium stearate and mineral oil.
  • the formulation may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like.
  • a typical daily dose of the inventive PEG- hG-CSF derivative conjugate as an effective ingredient may range from about 0.01 to 100 g/kg body weight (calculating the mass of protein alone, without chemical modification). Owing to the enhanced stability of PEG-hG-CSF derivative conjugate of the invention, the total number and frequency of the administration of the formulation comprising the inventive PEG-G-CSF derivative conjugate can be considerably reduced.
  • HM10411 Recombinant hG-CSF derivative, HM10411 was prepared according to the method described in Korea Patent No: 356140.
  • the hG-CSF derivative thus prepared has the 17 th amino acid of wild-type G-CSF replaced with serine ( 17 S-G-CSF).
  • HM10411 was dissolved in 100 mM potassium phosphate buffer (pH 6.0) to a concentration of 5 mg/m.£, distributed into three vials, and then, adjusted to a final volume of 5 m-6.
  • An activated methoxy-PEG-aldehyde (Shearwater, USA) which has 20 kDa or 40 kDa of PEG was added to each of two vials in an amount corresponding to a HM10411 :methoxy-PEG-aldehyde molar ratio of 1 :4.
  • Sodium cyanoborohydride NaCNBH 3 , Sigma, USA
  • was added thereto to a final concentration of 20 mM and the reaction mixture was stirred at 4 ° C for 18 hrs.
  • ethanolamine was added thereto to a final concentration of 50 mM to inactivate unreacted PEG.
  • the reaction mixture was subjected to
  • the PEG modified HM10411 was further purified from the elution fraction obtained in Example 2 as follows.
  • the column effluent was subjected to size exclusion chromatography.
  • the concentrated effluent was loaded onto Superdex 200 (Pharmacia) column equilibrated with 10 mM sodium phosphate buffer (pH 7.0) and eluted with the same buffer at a flow rate of 1 m /min. Since the tri- and di-PEG-HM10411 conjugates which eluted earlier than the mono-PEG-HM10411 conjugate were removed to obtain the purified mono-PEG-HM10411 conjugate.
  • the mono-PEG-HM10411 conjugate was analyzed for the state of its modification and purity by SDS-PAGE using 15% criterion gel (Bio-Rad) stained with Coomassie blue, and also for the protein concentration by measuring absorbance at 280 nm using size exclusion chromatography according to Beer-Lambert theory (Bollag et al., Protein Methods Chapter 3, press in Wiley-Liss).
  • the respective appearance molecular weights of 20 kDa and 40 kDa mono-PEG-HM10411 conjugates were about 50 kDa and 120 kDa.
  • Fig. 1 shows the SDS-PAGE results of 20 kDa and 40 kDa mono-PEG-
  • HM10411 conjugates wherein lane 1 is 3.0 ⁇ g of a molecular weight marker (Invitron, bench marker, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 160, and 220 kDa starting from the bottom band); lane 2, 10 ⁇ g of unmodified HM10411; lane 3, 10 ⁇ g of 40 kDa mono-PEG-HM10411 conjugate; and lane 4, 10 ⁇ g of 20 kDa mono-PEG-HM10411 conjugate.
  • a band of PEG-HM10411 conjugate was observed at a position corresponding to a molecular weight calculated by adding the molecular weight of HM10411 to the molecular weight of PEG. Further, since there was no band containing other proteins besides the PEG-HM10411 conjugate, it was found that the purified PEG-HM10411 conjugate had a high purity.
  • Fig. 2 shows the results of size exclusion chromatography for each sample using HPLC (Hewlett-Packard, HPllOl).
  • HPLC Hewlett-Packard, HPllOl
  • the effluent was loaded onto an analyzing column (Biosep SEC-S3000, Phenomenex) equilibrated with the buffer mixed with a mixture of 100 mM sodium phosphate (pH 6.9) and 20% ethanol, eluted with the same buffer at a flow rate of 0.6 m ⁇ /min, and measured the absorbance at 214 nm.
  • the retention times of the unmodified HM10411, 20 kDa and 40 kDa mono-PEG-HM10411 conjugates were 11.832, 10.184 and 9.694 min, respectively. Further, it was found that the PEG modified HM10411 was observed as a single peak in a highly pure form.
  • Each of the protein cleavage fragments were injected into Vydac 238TP54 C4 column (4.6x 250 mm, 5 ⁇ of particle size, 300 A of small hole size) and the peptide map thereof was determined by an acetonitrile linear gradient method using 0.1% trifluoroacetic acid through HPLC.
  • HM10411 has seven Glu-C cleavage sites, eight peaks are theoretically expected to be detected, but actually Glu-C cleavage did not occur at all Glu-C cleavage sites and it was not possible to completely detect all peaks due to the insufficient resolution of the column.
  • the peak containing the terminal amino residue of HM10411 has been well defined in previous reports (Herman, et al., Pharm. Biotechnol. 9: 303, 1996), it should be possible to determine the amino acid sequences of the respective peaks obtained by Glu-c cleavage fractionation and to compare the peak patterns of unmodified HM10411 and PEG-HM10411 conjugate for the difference in their amino acid sequences.
  • HL-60 cells Human myelogenous originated cell line, HL-60 cells (ATCC CCL-240, Promyelocytic leukemia patient/36 yr old Caucasian female) were cultivated in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), and then, the number of cells were adjusted to about 2.2x 10 5 cells/ml DMSO (dimethylsulfoxide, culture grade, Sigma) was added to the cells to a final concentration of 1.25% (v/v).
  • FBS fetal bovine serum
  • DMSO treated culture solution having about 2x IO 4 cells per well was added to a 96-well plate (low evaporation 96 well plate, Corning) and incubated at 37 ° C for about 48 hrs in a 5% C0 2 incubator.
  • concentration of each sample was determined by using a G-CSF ELISA kit (Enzyme Linked Immunosorbent Assay, R&D systems), and each sample was diluted with RPMI1640 medium at an appropriate ratio to a concentration of 500 ng/m ⁇ .
  • the resulting solution was subjected to 10 cycles of sequential half dilution with RPMI 1640 medium.
  • Fig. 4 shows the results of measuring the in vivo activities. As can be seen from Fig. 4, all samples stimulated the growth of G-CSF sensitive cells, which suggesting that they exhibit in vivo activity. ⁇ Table 1>
  • the inventive PEG modified HM10411 conjugates showed a lower activity than the wild-type G-CSF (Filgrastim) or the unmodified HM10411.
  • 20 kDa PEG-HM10411 conjugate showed similar activity to that observed for 20 kDa PEG-G-CSF (Neulasta), and 40 kDa PEG-HM10411 conjugate, an activity lower than those observed for 20 kDa PEG modified proteins (Neulasta and 20 kDa PEG-HM10411).
  • mice Male Sprague Dawly (SD) rat groups (7-week old, average body weight 200-250 g) were used in the following experiments. Rats received subcutaneous injection of 100 g/kg of Filgrastim, unmodified HM10411 (control), Neulasta and 40 kDa PEG-HM 10411 conjugate, respectively. Blood samples were taken from the control group at 0.5, 1, 2, 4, 6, 12, 24, 30 and 48 hrs after the injection, and the samples of the test groups, 1, 6, 12, 24, 30, 48 and 72 hrs after the injection. Blood samples were collected in an eppendorf tube coated with heparin to prevent blood coagulation, and subjected to high- speed micro centrifugation at 4 ° C, 10,000* g for 5 min to remove cells. The protein concentration in sera was measured by ELISA using an antibody specific for G-CSF.
  • the half-life of 40 kDa PEG- HM10411 conjugate was longer than those of wild-type G-CSF and its derivative. Particularly, the half-life of 40 kDa PEG-HM 10411 conjugate was about 2.5-fold higher than that of Fligrastim and about 0.5-fold higher than that of Neulasta.
  • the increased in-blood half-life may prolong its stability and biological activity, and therefore, may reduce the number of injection to a patient.

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Abstract

L'invention concerne un conjugué d'un dérivé de hG-CSF modifié par PEG présentant une stabilité in vivo accrue et une demi-vie prolongée dans le sang, tout en réduisant la possibilité d'induire une réponse immune.
PCT/KR2004/000569 2003-03-21 2004-03-17 Conjugue de facteur stimulant les colonies de granulocytes humain presentant une stabilite accrue dans le sang et son procede de preparation WO2004083242A1 (fr)

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KR1020030017867A KR20040083268A (ko) 2003-03-21 2003-03-21 안정성이 증가된 인간 과립구 콜로니 자극인자 융합체 및이의 제조방법
KR10-2003-0017867 2003-03-21

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WO2006094530A1 (fr) * 2005-03-11 2006-09-14 Siegfried Ltd. Conjugues de proteines et de dipolymeres et leurs procedes de preparation
EP1834963A1 (fr) * 2006-03-13 2007-09-19 Siegfried Ltd. Conjugues de proteines et de dipolymeres et leurs procedes de preparation
WO2008060002A1 (fr) * 2006-11-17 2008-05-22 Dong-A Pharm. Co., Ltd. Conjugué polyéthylène glycol-g-csf
WO2012021088A1 (fr) * 2010-08-13 2012-02-16 Closed Joint Stock Company "Biocad" Nouveau conjugué de facteur de croissance hématopoïétique g-csf et de polyéthylène glycol
CN102485742A (zh) * 2010-12-02 2012-06-06 山东新时代药业有限公司 一种聚乙二醇单修饰的重组人粒细胞集落刺激因子的制备及分离纯化方法

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US7737260B2 (en) 2003-11-13 2010-06-15 Hanmi Pharm. Co., Ltd Protein complex using an immunoglobulin fragment and method for the preparation thereof
KR100735784B1 (ko) * 2005-07-20 2007-07-06 재단법인 목암생명공학연구소 인간 과립구콜로니자극인자 변이체 및 이의 화학적 접합물

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006094530A1 (fr) * 2005-03-11 2006-09-14 Siegfried Ltd. Conjugues de proteines et de dipolymeres et leurs procedes de preparation
WO2006095029A2 (fr) * 2005-03-11 2006-09-14 Siegfried Ltd. Conjugues di-polymere-proteine et procedes de preparation de ceux-ci
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RU2446173C1 (ru) * 2010-08-13 2012-03-27 Зао "Биокад" Новый функционально активный, высокоочищенный стабильный конъюгат гранулоцитарного колониестимулирующего фактора (г-ксф) с полиэтиленгликолем с пролонгированным биологическим действием, пригодный для медицинского применения, и иммунобиологическое средство на его основе
EA019043B1 (ru) * 2010-08-13 2013-12-30 Закрытое Акционерное Общество "Биокад" Новый конъюгат гранулоцитарного колониестимулирующего фактора (г-ксф) с полиэтиленгликолем
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