WO2012021088A1 - A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol - Google Patents

A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol Download PDF

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WO2012021088A1
WO2012021088A1 PCT/RU2011/000532 RU2011000532W WO2012021088A1 WO 2012021088 A1 WO2012021088 A1 WO 2012021088A1 RU 2011000532 W RU2011000532 W RU 2011000532W WO 2012021088 A1 WO2012021088 A1 WO 2012021088A1
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csf
conjugate
peg
stimulating factor
granulocyte colony
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PCT/RU2011/000532
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French (fr)
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Dmitriy Valentinovich Morozov
Tatyana Veniaminovna Chernovskaya
Lev Alexandrovich Denisov
Elena Georgievna Rudenko
Elena Leonidovna Morozova
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Closed Joint Stock Company "Biocad"
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Priority to SG2013004544A priority Critical patent/SG187572A1/en
Priority to MA35731A priority patent/MA34525B1/fr
Priority to KR1020137001860A priority patent/KR101549457B1/ko
Priority to CU20130012A priority patent/CU24139B1/es
Priority to CN201180044089.6A priority patent/CN103140499B/zh
Priority to RS20130094A priority patent/RS20130094A1/en
Publication of WO2012021088A1 publication Critical patent/WO2012021088A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • 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
    • 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

Definitions

  • the present invention is related to pharmaceuticals and medicine, namely, to new physiologically active conjugates of granulocyte colony-stimulating factor (G-CSF), in particular, to the new G-CSF conjugate with polyethylene glycol, suitable for medical use, for example, for the treatment of leukopenia, mainly various types of neutropenia in patients treated with myelosuppressive chemotherapy, patients undergoing hematopoietic stem cell transplantation, patients with chronic neutropenia, patients with AIDS and other infections.
  • G-CSF granulocyte colony-stimulating factor
  • G-CSF is a hematopoietic growth factor that stimulates proliferation, differentiation and maturation of granulocytes (Metcalf, 1992). Introduction of exogenous G-CSF causes rapid, specific and dose-dependent increase of neutrophils in peripheral blood (Welte et al, 1985; Hartung et al, 1995).
  • Human G-CSF gene was cloned and expressed in bacterial and mammalian cells; as a result, various variants of recombinant human G-CSF (rhG-CSF) have been developed and purified (Nagata et al., 1986; Souza et al., 1986; Komatsu et al., 1987).
  • Filgrastim consists of 175 amino acids, it is not glycosylated and has the same amino acid sequence as the natural protein, but contains an additional methionine residue at the N-terminus of the molecule.
  • Lenograstim consists of 174 amino acids, it is glycosylated and its amino acid sequence is fully consistent with that of natural human G-CSF.
  • rhG-CSF products which differ in glycosylation and/or protein sequence are available for clinical use for treatment of neutropenia occurring after chemotherapy, idiopathic, congenital and cyclic neutropenia; for the treatment of severe infections in combination with antibiotics and in neutropenic AIDS patients (Molineux, 2004).
  • rhG-CSF must be administered to patients by daily injection for optimal effectiveness. And daily monitoring of complete peripheral white blood cell counts must be performed.
  • G-CSF can be improved by using longer-acting medicinal forms, in which the native protein molecule is chemically bound to monomethoxy polyethylene glycol (mPEG).
  • mPEG monomethoxy polyethylene glycol
  • PEGylation of G-CSF leads to improved pharmacokinetics, increased half-life, reduced clearance, reduced fluctuations of concentration in the blood, reduction of immunogenicity and toxicity, increased activity in vivo and increased stability (Molineux, 2003; Molineux, 2004).
  • PEG-G-CSF conjugates are largely dependent on the molecular weight and type of the used activated mPEG.
  • the reactive functional groups of activated PEGs can then be attached to a specific site on protein, mainly on an amine, a sulfhydryl or other nucleophilic groups.
  • the preferred site of modification is the amino group of lysine and the N-terminus of polypeptide chain (Kinstler et al. 1996, Roberts et al., 2002).
  • a wide range of mPEG derivatives has been used for amine PEGylation, for example: PEG- triazine, PEG-succinimidyl carbonate, PEG-succinimidyl succinate, PEG tresylate, PEG- aldehyde, PEG-N-Hydroxysuccinimide-activated esters, etc).
  • the patent US 2007/0014762 describes production of PEG-G-CSF conjugates using a variety of activated mPEGs with a molecular weight of 5000 Da, in particular mPEG- succinimidyl butanoate, mPEG-succinimidyl propionate, mPEG-succinimidyl a- methylbutanoate.
  • activated mPEGs with a molecular weight of 5000 Da
  • mPEG- succinimidyl butanoate mPEG-succinimidyl propionate
  • mPEG-succinimidyl a- methylbutanoate mPEG-succinimidyl a- methylbutanoate
  • the resulting PEG-G-CSF conjugate consisted of several positional isomers; each being conjugated with a PEG 5000 at a different site with epsilon-amino groups of lysine residues and alpha-amino group of the N-terminal amino acid.
  • the PEG-G-CSF conjugate was purified by ion exchange chromatography on a column of SP-Sepharose.
  • the patent EP 0401384 describes PEG-G-CSF conjugates, obtained using mPEG of linear and branched structures with a molecular weight of 4,500 - 10,000 Da, activated by different reactive groups (succinimydyl succinate, chloro-s-triazine, polyoxyethylenediamine). These PEG-G-CSF conjugates had a prolonged effect in vivo, and the degree of prolongation was dependent on the molecular weight of the conjugated PEG.
  • Half life of PEG-G-CSF conjugate with PEG 10 000 and unmodified G-CSF was 7.05 and 1.79 h, respectively
  • Succinimydyl succinate -PEG is prepared by reaction of mPEG with succinic anhydride, followed by activation of the carboxylic acid to the succinimidyl ester.
  • the polymer backbone contains a second ester linkage that remains after the conjugation reaction with a protein. This linkage is highly susceptible to hydrolysis after the polymer has been attached to the protein. Not only does this hydrolysis lead to loss of the benefits of PEG attachment, but the succinate tag that re mains on the protein after hydrolysis can act as a hapten and lead to immunogenicity of the remaining protein (Roberts, 2002).
  • the patent EP 0335423 describes G-CSF conjugates with triazine mPEG derivatives with a molecular weight of 300 - 30 000 Da. These PEG-G-CSF conjugates had a prolonged action, their specific activity ranged from 1 1 to 60% of the activity of unmodified G-G-CSF.
  • N-terminal PEGylation of rhG-CSF prepared in this manner with PEG 20 kDa was employed in the development of pegfilgrastim ("Neulasta") used in the treatment and prevention of different neutropenic states.
  • the mono-mPEG-G-CSF conjugate was isolated by ion- exchange chromatography using a SP Sepharose HP column.Biological activity of the resulting purified PEG-G-CSF conjugate was 68% of the native G-CSF activity. The content of unmodified G-CSF in the resulting conjugate was no more than 5%. Pharmacokinetic parameters of the conjugate were better than those of unmodified G-CSF. Animal experiments have shown that the introduction of PEG-G-CSF conjugate led to increased white blood cell count, and the PEG-G-CSF conjugate had more prolonged effect compared to unmodified G-CSF.
  • the content of white blood cells of animals reached a maximum after 1 day after injection, this level was maintained during the day and then the level of white blood cells decreased to baseline level 4 days after injection.
  • the maximum level of leukocytes in blood of animals was observed after 1 day after injection, then the content of leukocytes rapidly decreased.
  • the aim of the present invention was to obtain a new stable highly purified PEGylated G-CSF conjugate with high activity, with more prolonged effect, improved stability, improved pharmacokinetic parameters, with optimal combination of parameters of molecular weight of PEG and specific activity, with high degree of purity and suitable for medical use, as well as pharmaceutical compositions based on the claimed conjugate.
  • n integers from 681 to 1 000;
  • JsTH-G-CSF - natural or recombinant polypeptide having the activity of G-CSF JsTH-G-CSF - natural or recombinant polypeptide having the activity of G-CSF.
  • Aldehyde derivatives of the activated mPEG (formula II) are used for production of the claimed PEG-G-CSF;
  • the molecular weight of the PEG-IFN conjugate is increased due to the weight of the PEG;
  • the level of the specific activity is higher;
  • the claimed PEG-G-CSF conjugate is highly purified, with a prolonged effect and is characterized by a high specific biological activity (at least 84-94% of the unmodified G-CSF), the purity of the protein >97%, high thermal stability, increased resistance to the action of protein-degrading enzymes, improved pharmacokinetic parameters.
  • compositions containing claimed PEG-G-CSF conjugate in an effective quantity as the active ingredient may also include pharmaceutically acceptable carriers, buffer agents (organic and inorganic acids and their salts, such as citrate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate buffers), stabilizers (sugar alcohols, amino acids, organic sugar or sugar alcohol, inositol, polyethylene glycol, polymers of amino acids, sulfur-reducing agent, such as urea, glutathione, thioglycerol etc., polypeptides with low molecular weight proteins such as human serum albumin, immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, monosaccharides such as mannose, xylose, fructose, glucose, disaccharides such as lactose, maltose, sucrose, trisaccharides such as raffinose, and polysaccharides such
  • the present invention also includes medicines on the basis of the claimed conjugate, in particular, for treatment of neutropenia.
  • the claimed PEGylated G-CSF as well as pharmaceutical compositions and medicinal products based on it can be used for the treatment of neutropenia occurring after chemotherapy, to improve tolerability of immunosuppressive medical drugs in bone marrow transplantation, to improve immune status patients suffering from AIDS and other infections, in particular systemic or invasive candidiasis.
  • Conjugate of the formula (I) with an optional pharmaceutically acceptable filler, diluents and / or pharmaceutically acceptable excipients is administered intravenously, subcutaneously, intramuscularly or by any other suitable means.
  • Route of administration vary depending, for example, on symptoms and age, frequency of administration and the interval between injections
  • an effective amount of the PEG-G-CSF agent is selected according to the abovementioned factors.
  • the conjugation reaction was conducted at a pH below 5.0 in the presence of a reducing agent at a temperature at or below 20°C.
  • the molar ratio of PEG/protein was 2,5-5/1.
  • the purified monoPEG-G-CSF was dialyzed against 10-50 itiM buffer solution at pH 4-5, added salt, or polysaccharides, or alcohols, or polyvinylpyrrolidone, or monosaccharides, and amino sugars, or proteins, or amino acids, and nonionic detergents and stored in plastic or glass bottles with siliconized surface at 4 ⁇ 2 °C.
  • Fig. 1 Kinetics of G-CSF PEGylation.
  • Fig. 2 Reverse phase high performance liquid chromatogram (RP-HPLC) of the PEG-G-CSF conjugate.
  • Fig. 3 Size exclusion high performance liquid chromatogram (SEC-HPLC)of the PEG-G-CSF conjugate.
  • Fig. 5 MALDI mass spectra of unmodified G-CSF (A) and PEG-G-CSF conjugate (B).
  • Fig. 6. Comparison of mass spectra of tryptic peptides of G-CSF (A) and PEG-G-CSF conjugate (B).
  • Fig. 7 Thermal stability of PEG-G-CSF conjugate (green line) and unmodified G-CSF (red line) at + (50 ⁇ 2) °C
  • Fig.8 Proteolytic stability of PEG-G-CSF conjugate (green line) and unmodified G-CSF (red line).
  • Fig. 10 Pharmacokinetics of the PEG-G-CSF conjugate (green line) and unmodified G-CSF (red line).
  • the diluted reaction mixture prepared in accordance with Example 2 was loaded onto a column packed with 300 mL of CM-sepharose equilibrated with 10 mM sodium acetate buffer, pH 4.8 (buffer A) at flow rate of 10 ml / min. After loading of the material the column was washed to remove unbound material with 1500 ml of buffer A. A linear gradient 0-0,2 M of NaCI in buffer A (6000 mL) was used to elute the monoPEG-G-CSF from the column at flow rate of 5 ml / min. Fractions (50 mL) were collected and the absorbance at 280 and 260 ran was determined.
  • Fractions containing protein were analyzed by RP-HPLC and SDS-PAGE as described in Example 2.
  • Fractions containing monoPEGylated G-CSF with a purity of over 95%, were pooled, dialyzed against 10 volumes of 1.6 mM sodium acetate buffer, pH 4.0 ⁇ 0.2. Then sterile filtration was performed and samples were stored at 4 ⁇ 2 °C.
  • the purified PEG-G-CSF conjugate prepared in accordance with Example 3, was diluted with 20 mM sodium acetate buffer pH 5.0 to a protein concentration of 0.3 mg / ml.
  • Analytical RP HPLC was performed employing a gradient Waters 'Breeze' chromatograph on a C4 Symmetry column (4.6x150 mm) with UV detection at 214 nm. 100 microliters of the obtained sample was injected into the column.
  • the mono PEG-G-CSF is shown by RP_ HPLC (Fig. 2) eluted from the column as a single, symmetrical peak and to be highly pure, as judged by the presence of only minor impurities eluting before and after the main UV-absorbing peak (FIG. 2).
  • the impurities amount to less than 1 ,15% of the material eluting in the main peak.
  • the purified PEG-G-CSF conjugate prepared in accordance with Example 3, was diluted with 20 mM sodium acetate buffer pH 5.0 to a protein concentration of 0.3 mg / ml.
  • Analytical SEC HPLC was performed employing a Waters 'Breeze' chromatograph on a TSK G3000SWX column (30cm x 7.8mm). with UV detection at 214 nm. 100 microliters of the obtained sample was injected into the column.
  • the results of SEC-HPLC are presented on Fig. 3. It is seen that the sample of PEG-G-CSF eluted from the column as one distinct symmetrical peak, constituting 98.47%. From the results presented in Fig.
  • the claimed PEG-G-CSF conjugate does not contain free modified G-CSF.
  • the content of high-molecular weight aggregates in the claimed conjugate does not exceed 1.53%.
  • Purity of the claimed PEG- G-CSF conjugate according to the SEC HPLC is more than 98%. 11 000532
  • LAL Limulus Amebocyte Lysate
  • the results presented in Table 1 show that the BE content in the PEG-G-CSF conjugate sample is less than 3 EU (endotoxin units) per mg of protein, which is much lower than BE level allowable for drugs based on recombinant proteins.
  • the purified PEG-G-CSF conjugate sample prepared in accordance with the Example 3, was analyzed by, SDS-PAGE as described in Example 2. Samples containing 40 micrograms both PEG-G-CSF and unmodified G-CSF were loaded into each wells. The gel was stained with Coomassie R-250.
  • Figure 4 illustrates the SDS-PAGE profile of PEG-G-CSF (line 1) and unmodified G-CSF (line 2)..
  • the purified PEG-G-CSF appeared as single diffuse band with an apparent molecular weight higher than that in unmodified G-CSF.
  • MALDI-TOF MS Matrix assisted laser desorption time-of-fight mass spectrometry
  • MALDI-TOF mass spectra of unmodified G-CSF and PEG-conjugated G-CSF in the range m / z 10 000 - 50 000 are shown in Fig. 5.
  • Fig. 5A shows that the mass spectrum of G-CSF contains the main peak corresponding to a singly charged ion [M] + with a molecular weight of 18,816 Da.
  • the site of PEG conjugation with a G-CSF molecule was determined by comparing the mass spectra of tryptic peptides of the PEG-G-CSF conjugate and unmodified G-CSF. In the tryptic digest of the PEG-G-CSF conjugate there should be no peptide corresponding to the portion of the protein where a modification occurred.
  • Table 2 shows the mass spectra of experimental tryptic peptides for unmodified G-CSF and PEG-G-CSF conjugate. The table shows that the mass spectra of tryptic peptides of the unmodified G-CSF almost coincide with those-of the PEG-G-CSF conjugate.
  • PEG-G-CSF conjugate prepared according to Example 3, diluted to a concentration of 0.2 ng / ml of RPMI medium and the serial dilutions of the samples to be tested were prepared in assay media.
  • Serial dilutions of the unmodified G-CSF and international reference standard of G-CSF activity (1-st International Standard 88/502, granulocyte colony stimulating factor, human, rDNA-derived, 10000 IU/amp) were analyzed in parallel. 100 microliters of the diluted protein samples were added to the test wells of a flat-bottom 96-well tissue culture plate.
  • the M-NFS cells were washed and resuspended at a concentration of 1,5 xl0 5 /ml in RPMI media. 100 microliters of the cell suspension were added to each well and the plates incubated at 37 °C in a 5% C0 2 tissue culture incubator for 50 hrs. Then, in each well 20 microliters of dye Alamar Blue (or similar, for example, MTT) were added and plates incubated under the same conditions for 14 hours.
  • dye Alamar Blue or similar, for example, MTT
  • the increase in the number of cells was determined using a microplate reader by an increase of optical density at a wavelength of 545 nm and 630 nm.
  • the specific biological activity of the tested samples (B 0 ) in IU / mg was calculated using the formula:
  • Bo-— where, B 0 - specific biological activity of PEG-G-CSF or G-CSF (IU / mg);
  • Example 1 Subsamples (100 microliters) were taken every 60 min and 20 microliters 10% SDS was added to stop the tryptic reaction and SDS-PAGE was performed, as described in Example 1.. After electrophoresis gels were stained with Coomassie-R-250 and densitometric analysis of stained gels was performed. The area of the main band was determined by a computer program GelPro Analyser. The area of the main band in the initial (untreated with trypsin) sample was taken as 100%.
  • Fig. 8 presents data on the sensitivity of the PEG-G-CSF conjugate and G-CSF to the treatment by trypsin.
  • PEG-G-CSF conjugate was determined by antibody binding activity using a modification of the ELISA assay with the use of a set of reagents ProCon G-CSF ("Protein contour" LLC).
  • PEG-G-CSF conjugate, prepared in accordance with example 3 was diluted in 10 mM Tris-HCl buffer, pH 7.2, containing 140 mM NaCI and 1% bovine serum albumin to a concentration of 1 mkg/ ml. Then 100 microliters of the obtained samples were added into the wells of the microtiter plate previously coated with monoclonal antibodies against G-CSF.
  • the serum samples for measuring either native or monoPEGylated G-CSF were used in the ELISA assay with mouse monoclonal immunoglobulins to different epitopes of human G- CSF.
  • a wells of the microtiter plate were coated with monoclonal antibodies against G-CSF (250 ng/well in 100 microliters of 20 mM Tris-HCl buffer (TB), pH 9.0. After an overnight incubation at 4 °C, 200 microliters of TB, pH 7.4, containing 1% bovine serum albumin (BSA) and 0.05% Tween-20 were added into the wells to block of nonspecific binding sites.
  • G-CSF 250 ng/well in 100 microliters of 20 mM Tris-HCl buffer (TB), pH 9.0. After an overnight incubation at 4 °C, 200 microliters of TB, pH 7.4, containing 1% bovine serum albumin (BSA) and 0.05% Tween-20 were added into the wells to block of nonspecific binding sites.
  • BSA bovine serum albumin
  • PEG-G-CSF PEGylated recombinant granulocyte colony-stimulating human factor
  • Active ingredient PEGylated recombinant granulocyte colony-stimulating human factor (PEG-G-CSF), - 0,6 - 3,0 mg
  • Packaging of the finished product for example, an aqueous solution containing
  • a set containing the medical drug for example, an aqueous solution containing PEG-G-CSF, obtained according to the Example 3 as an active ingredient.

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PCT/RU2011/000532 2010-08-13 2011-07-19 A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol WO2012021088A1 (en)

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SG2013004544A SG187572A1 (en) 2010-08-13 2011-07-19 A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol
MA35731A MA34525B1 (fr) 2010-08-13 2011-07-19 Nouveau conjugué de facteur de croissance hématopoïétique g-csf et de polyéthylène glycol
KR1020137001860A KR101549457B1 (ko) 2010-08-13 2011-07-19 폴리에틸렌글리콜과 과립구 콜로니 자극인자(g-csf)의 신규 접합체
CU20130012A CU24139B1 (es) 2010-08-13 2011-07-19 Conjugado de factor estimulante de colonia de granulocito (g-csf)con polietileno glicol
CN201180044089.6A CN103140499B (zh) 2010-08-13 2011-07-19 一种粒细胞集落刺激因子与聚乙二醇的共轭物
RS20130094A RS20130094A1 (en) 2010-08-13 2011-07-19 NEW CONJUGATE OF FACTORS WHICH STIMULATES THE COLONY OF GRANULOCYTES (G-CSF) WITH POLYETHYLENE GLYCOL

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RU2010133875/10A RU2446173C1 (ru) 2010-08-13 2010-08-13 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
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WO2016009451A2 (en) 2014-07-14 2016-01-21 Gennova Biopharmaceuticals Limited A novel process for purification of rhu-gcsf
US10251814B2 (en) 2011-02-15 2019-04-09 Cis Pharma Ag Cefuroxime safety delivery system
EP3705492A4 (en) * 2017-10-30 2021-08-18 Hankook Korus Pharmaceutical Co. Ltd. PROCESS FOR PRODUCING HIGH YIELD CONJUGATES IN WHICH A COLONY-STIMULATING FACTOR AND A POLYOL ARE CONJUGATED
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RU2535002C2 (ru) * 2013-04-04 2014-12-10 Федеральное государственное бюджетное учреждение "Научно-исследовательский институт фармакологии" Сибирского отделения Российской академии медицинских наук Средство коррекции отдаленных последствий нарушений сперматогенеза, вызванных цитостатическим воздействием
EP3107533A4 (en) 2014-02-18 2017-10-18 Children's Hospital Los Angeles Compositions and methods for treating neutropenia
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US10251814B2 (en) 2011-02-15 2019-04-09 Cis Pharma Ag Cefuroxime safety delivery system
CN103908427A (zh) * 2013-01-05 2014-07-09 石药集团百克(山东)生物制药有限公司 一种聚乙二醇修饰的rhG-CSF注射液及其制备方法
CN103908427B (zh) * 2013-01-05 2014-12-17 石药集团百克(山东)生物制药有限公司 一种聚乙二醇修饰的rhG-CSF注射液及其制备方法
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EP3705492A4 (en) * 2017-10-30 2021-08-18 Hankook Korus Pharmaceutical Co. Ltd. PROCESS FOR PRODUCING HIGH YIELD CONJUGATES IN WHICH A COLONY-STIMULATING FACTOR AND A POLYOL ARE CONJUGATED
WO2021185921A1 (en) * 2020-03-17 2021-09-23 Drugrecure Aps Liquid formulation of gm-csf for inhalation

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MA34525B1 (fr) 2013-09-02
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RU2446173C1 (ru) 2012-03-27
CL2013000400A1 (es) 2013-07-26
CO6670557A2 (es) 2013-05-15
SG187572A1 (en) 2013-03-28
NI201300007A (es) 2014-05-26
KR20130043167A (ko) 2013-04-29
ECSP13012399A (es) 2013-05-31
CU20130012A7 (es) 2013-04-19
MY160732A (en) 2017-03-15
CU24139B1 (es) 2015-12-23
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