WO2004009627A1 - Composes erythropoietiques pegyles - Google Patents

Composes erythropoietiques pegyles Download PDF

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Publication number
WO2004009627A1
WO2004009627A1 PCT/CA2003/001020 CA0301020W WO2004009627A1 WO 2004009627 A1 WO2004009627 A1 WO 2004009627A1 CA 0301020 W CA0301020 W CA 0301020W WO 2004009627 A1 WO2004009627 A1 WO 2004009627A1
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Prior art keywords
epo
glycosylated
nucleic acid
polymer
seq
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PCT/CA2003/001020
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English (en)
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John Douglas Cossar
Lawrence T. Malek
Donald I. H. Stewart
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Cangene Corporation
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Priority to AU2003246486A priority Critical patent/AU2003246486A1/en
Publication of WO2004009627A1 publication Critical patent/WO2004009627A1/fr

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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • 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

Definitions

  • the present invention relates to pegylated erythropoietic compounds, methods for their preparation and use, particularly in the treatment of conditions that benefit from stimulation of erythropoiesis, such as anemia. More specifically, the invention relates to polymer-derivatized, non- glycosylated erythropoietin (EPO) proteins that cause an increase in blood hematocrit when administered to a patient.
  • EPO polymer-derivatized, non- glycosylated erythropoietin
  • EPO BACKGROUND OF THE INVENTION
  • EPO is a hormone that is critical in the proliferation and differentiation of erythrocyte precursor cells.
  • the mature protein has 166 amino acids and the "precursor" form, which includes the leader peptide has 193 amino acids.
  • EPO is a highly glycosylated protein, having four carbohydrate groups attached at the nitrogen of asparagine at positions 24, 38 and 83 and at the oxygen of the serine at position 126
  • Non- or a-glycosylated versions of EPO produced respectively in bacterial systems or following treatment with glycosidase enzymes, are essentially inactive in vivo. This lack of in vivo activity of non-glycosylated EPO has been attributed to its rapid clearance from circulation (Spivak J. and Hogans, B. Blood, 1989, 73:90; Fukuda, M. et al. Blood, 1989, 73:84).
  • pegylated EPO is prepared from tresylmonomethoxypolyethylene glycol (TMPEG) and natural, unmodified glycosylated EPO, whereas in Beals J.M. et al., PEG-aldehdye is used to pegylate non-glycosylated EPO analogs in which positively charged amino acid residues have been added to wild-type non- glycosylated EPO.
  • TMPEG tresylmonomethoxypolyethylene glycol
  • PEG-aldehdye is used to pegylate non-glycosylated EPO analogs in which positively charged amino acid residues have been added to wild-type non- glycosylated EPO.
  • modified polymer derivatives of EPO described above do not have attached carbohydrate groups, they are less likely to be cleared from the circulation by natural glycosylation-mediated routes. These derivatives have increased size and mass, resulting in a decrease in rate of clearance through the kidney and thereby increased half-life.
  • Addition of polyethylene polymers to several different proteins has been shown to improve their pharmaceutical properties, yet, there are very few polymer-modified proteins that have been approved as therapeutics. Addition of polyethylene glycol groups to proteins has been problematic in that the coupling/activation step can cause substantial loss of biological activity.
  • the inability to control the coupling reaction has resulted in the addition of polymers at positions which cause steric hindrance and preclusion of protein-receptor binding.
  • US Patent No. 4,904,584 to Shaw describes the modification of one or more selected naturally occurring pegylation sites on a polypeptide, such as lysine residues replaced by a suitable amino acid, such as arginine to achieve a more homogeneous pegylated-peptide.
  • EPO is listed as one of the polypeptides that can be so modified, no examples are provided for EPO and no direction is provided in the patent to a person skilled in the art as to the pegylation sites of EPO or to the preferable sites for modification.
  • the expression system includes a novel DNA sequence encoding wildtype EPO that has been codon-optimized for expression in Streptomyces. Further, it has been found that the activity of pegylated-non-glycosylated EPO depends on the site that is pegylated. Accordingly, analogs of codon-optimized, wild-type non- glycosylated EPO were prepared by modifying the sites that are not desirable for pegylation, and the corresponding non-glycosylated pegylated EPO analogs prepared therefrom, were shown to have stability and bioactivity in vitro and in vivo. Accordingly, the present invention relates to a non-glycosylated EPO analog wherein one or more of the lysine resides available for pegylation is replaced with an amino acid that cannot be pegylated.
  • a modified non-glycosylated EPO protein selected from the group consisting of SEQ ID NO:1 ( Figure 1A), SEQ ID NO:2 ( Figure 1B) and SEQ ID NO:3 ( Figure 1C) or biologically active analogs or derivatives thereof.
  • the invention further relates to isolated, codon-optimized nucleic acid sequences encoding non-glycosylated EPO and the non-glycosylated EPO analogs of the invention or biologically active analogs or derivatives thereof.
  • the nucleic acid sequences encoding the non-glycosylated EPO analogs of the invention comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO:4 ( Figure 2A), SEQ ID NO:5 ( Figure 2B) and SEQ ID NO:6 ( Figure 2C).
  • the codon-optimized nucleic acid molecule encoding non-glycosylated EPO comprises SEQ ID NO:7 ( Figure 3).
  • the present invention further relates to chimeric nucleic acid molecules comprising the nucleic acid sequences of the invention for expression in host cells.
  • the chimeric molecules comprising the nucleic acid molecules of the invention comprise elements for the expression of the corresponding proteins in prokaryotic animals.
  • the present invention includes a chimeric nucleic acid molecule comprising in the 5' to 3' direction of transcription:
  • nucleic acid sequence capable of regulating transcription in said host cell operatively linked to;
  • a second nucleic acid sequence encoding a codon- optimized non-glycosylated EPO protein or a codon-optimized non- glycosylated EPO analog of the invention; and
  • a third nucleic acid sequence capable of terminating transcription in said host cell.
  • the present invention is further directed to host cells comprising the chimeric nucleic acid molecules of the invention.
  • the invention relates to polymer-derivatized, non- glycosylated EPO analogs having a protein portion and a polymer portion, wherein the protein portion is a non-glycosylated EPO analog wherein one or more of the lysine residues available for pegylation is replaced with an amino acid that cannot be pegylated and wherein the polymer portion consists of 1 or more polymer chains of polyethylene glycol.
  • the 1 or more polymer chains of ethylene glycol have the formula:
  • the present invention also includes pharmaceutical compositions comprising a polymer-derivatized, non-glycosylated EPO protein of the invention in admixture with a suitable diluent or carrier.
  • Non-derivitized non-glycosylated EPO proteins of the present invention do not have practical in vivo activity but are useful in the preparation of pegylated non-glycosylated EPO analogs.
  • PEGylation of non-glycosylated EPO proteins restores in vivo activity and may impart properties such as increased plasma half-life, reduced immunogenicity and antigenicity, improved solubility, reduced proteolytic susceptibility, improved bioavailability, reduced toxicity, reduced affinity to serum binding proteins, improved thermal and mechanical stability, as well as, improved compatibility with depot formulations compared to glycosylated erythropoietin and other polymer derivatives of non-glycosylated EPO.
  • the present invention further involves a method for preparing polymer- derivatized, non-glycosylated EPO proteins, comprising: a) adding an activated PEG compound to a solution containing a non-glycosylated EPO protein of the invention under conditions that permit the formation of a bond between an amino group of the non-glycosylated EPO analog and an activating group on PEG and b) isolating the polymer-derivatized, non- glycosylated EPO proteins.
  • a method for preparing polymer-derivatized, non-glycosylated EPO proteins comprising: a) adding pNPPEG to a solution containing a non-glycosylated EPO protein of the invention under conditions that permit the formation of an amide bond between an amino group of the non-glycosylated EPO analog and the carbonate of pNPPEG and b) isolating the polymer-derivatized, non- glycosylated EPO proteins.
  • the invention also involves a method for treating a condition that benefits from the stimulation of erythropoiesis comprising administering a therapeutically effective amount of a polymer-derivatized, non-glycosylated protein of the invention to a mammal in need thereof.
  • a mammal is human.
  • a polymer-derivatized, non- glycosylated protein of the invention to treat a condition that benefits from the stimulation of erythropoiesis as well as a use of a polymer-derivatized, non- glycosylated protein of the invention to prepare a medicament to treat a condition that benefits from the stimulation of erythropoiesis.
  • Conditions that benefit from the stimulation of erythropoiesis include, but are not limited to, anemia.
  • the invention also involves a method for increasing the hematocrit level in a mammal comprising administering a therapeutically effective amount of a polymer-derivatized, non-glycosylated proteinof the invention to a mammal in need thereof.
  • a mammal is human.
  • the present invention further provides a use of a polymer-derivatized, non-glycosylated protein of the invention to increase the hematocrit level in a mammal as well as a use of a polymer-derivatized, non-glycosylated protein of the invention to prepare a medicament to increase the hematocrit level in a mammal.
  • Figure 1A is the amino acid sequence for the K45R EPO analog (SEQ ID NO:1);
  • Figure 1 B is the amino acid sequence for the K116R EPO analog (SEQ ID NO:2)
  • Figure 1C is the amino acid sequence for the K45,116R EPO analog (SEQ ID NO:3);
  • Figure 2A is the nucleic acid sequence encoding the K45R EPO analog (SEQ ID NO:4);
  • Figure 2B is the nucleic acid sequence encoding the K116R EPO analog (SEQ ID NO:5);
  • Figure 2C is the nucleic acid sequence encoding the K45,116R EPO analog (SEQ ID NO:6);
  • Figure 3 is the codon optimized nucleic acid sequence of the protein- coding region for the wild type EPO (SEQ ID NO:7);
  • Figure 4 shows the nucleic acid sequences for the oligomers (SEQ ID NOS:8-23) used for gene construction of the nucleic acid molecule encoding the wild type EPO;
  • Figure 5 shows the nucleic acid sequence for the four synthetic oligomers (SEQ ID NOS:24-27) used to modify the EPO sequence to improve secretion efficiency;
  • Figure 6 shows the nucleic acid and amino acid sequence of the EPO expression fragment, including the promoter, modified protease B signal peptide and the nucleic acid sequence of the protein-coding region for the wild type EPO (SEQ ID NOS:28 and 29);
  • Figure 7 is a schematic showing the general mutation strategy for the production of the K45R EPO analog
  • Figure 8 is an SDS-PAGE showing protein purity after the third chromatography step using HiTrap Heparin (for the codon-optimized wild type EPO- Lane 2);
  • Figure 9 is an SDS-PAGE showing the fractions from a chromatographic fractionation of EPO+pNPPEG
  • Figure 10 is a graph showing the in vivo activity of non-pegylated, non- glycosylated EPO compared with 5 kDA pegylated EPO as determined using the polycythemic mouse assay (P.P. Dukes et al. J. Lab. Clin. Med. 74:250- 256 (1969));
  • Figure 11 is a graph showing the effect of PEG size on in vivo activity as determined using the polycythemic mouse assay (P.P. Dukes et al. J. Lab. Clin. Med. 74:250-256 (1969));
  • Figure 12 is a graph comparing the effect of pegylated non- glycosylated EPO (PEG WT), pegylated 45-arginine EPO (PEG K45R) and glycosylated EPO (Eprex) on the haematocrit levels in a rabbit; and
  • Figure 13 is a graph comparing the rate of clearance of pegylated non- glycosylated EPO (PEG WT), pegylated 45-arginine EPO (PEG K45R) and glycosylated EPO (Eprex) from circulation in the rabbit.
  • PEG WT pegylated non- glycosylated EPO
  • PEG K45R pegylated 45-arginine EPO
  • Eprex glycosylated EPO
  • EPO erythropoietin
  • EPO is a glycoprotein hormone that is secreted by the human kidney, that is found in human blood, and that stimulates formation of erythrocytes (erythropoiesis) in human bone marrow.
  • the amino acid sequence of the predominant allelic variant of the protein portion of erythropoietin is known (L. Owers-Narhi, et al., J. Biol. Chem. 266:23022- 23026 (1991)).
  • EPO consists of 166 amino acids, is comprised of about 40% carbohydrate, by mass, and has a total molecular weight of approximately 30.4 kDa.
  • the carbohydrate structure of EPO is heterogeneous, whereas the amino acid sequence of the predominant human allelic variant is not. Therefore, the term “erythropoietin” refers to a heterogeneous group of EPO molecules.
  • Non-glycosylated erythropoietin as used herein means human erythropoietin lacking attached glycosyl chains.
  • Non-glycosylated EPO has the amino acid sequence of EPO, but lacks N-linked glycosyl chains at positions 24, 38, and 83 and the O-linked glycosyl chain at position 126.
  • non-glycosylated EPO may lack the amino acid at position 166 or may have an arginine (Arg) at position 166. It has an apparent molecular weight of about 20 kDa.
  • Non-glycosylated EPO can be conveniently expressed in cell types that lack the ability to post-translationally attach glycosyl moieties to a protein, or can be produced by enzymatically removing the glycosyl chains from EPO.
  • Non-glycosylated EPO analogs as used herein means a non- glycosylated EPO analog wherein one or more of the lysines available for pegylation (or capable of being pegylated) in wild type non-glycosyated EPO is replaced with an amino acid that cannot be pegylated.
  • Non-glycosylated EPO analogs of the invention include non-glycosylated EPO analogs in which the amino acid at position 166 deleted.
  • non-glycosylated EPO analogs of the invention include those wherein the amino acid at position 166 is any amino acid.
  • the lysine that is replaced with an amino acid that cannot be pegylated is the lysine at position 45 and/or 116.
  • PEGylated non-glycosylated EPO refers to non- glycosylated EPO or a non-glycosylated EPO analog with 1 or more polymer chains of polyethylene glycol (PEG) attached thereto.
  • isolated DNA molecule refers to any DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location in genomic DNA.
  • isolated nucleic acid molecule refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location. All amino acid or protein sequences, unless otherwise designated, are written commencing with the amino-terminus ("N-terminus”) and concluding with the carboxy-terminus (“C-terminus”).
  • isolated amino acid sequence refers to any amino acid sequence, however, constructed or synthesized, which is locationally distinct from the naturally occurring sequence.
  • A Ala - alanine
  • R Arg - Arginine
  • N Asn - Asparagine
  • D Asp - Aspartic acid
  • C Cys - Cysteine
  • Q Gin - Glutamine
  • E Glu - Glutamic acid
  • G Gly - Glycine
  • H His - Histidine
  • I lie - Isoleucine
  • L Leu - Leucine
  • K Lys - Lysine
  • M Met - Methionine
  • F Phe - Phenyalanine
  • P Pro - Proline
  • S Ser - Serine
  • T Thr - Threonine
  • W Trp - Tryptophan
  • Y Tyr - Tyrosine
  • V Val - Valine
  • II Nucleic Acids and Proteins of the Invention
  • Streptomyces has been developed. The method involves the use of a novel, codon-optimized nucleic acid sequence encoding wild-type EPO. Further, it has been found that the activity of pegylated-non-glycosylated EPO can be affected by varying the number of sites in non-glycosylated EPO that can be pegylated. Wild-type non-glycosylated EPO therefore has been modified to replace one or more of the lysines that are capable of being pegylated with an amino acid that cannot be pegylated.
  • non-glycosylated EPO analogs as well as the non-glycosylated EPO produced as described herein, have been pegylated and the resulting pegylated non-glycosylated EPO proteins, tested for in vitro and in vivo activity in standard assays for erythropoietic activity. The results show that pegylation increases in vivo activity relative to non-glycosytated EPO.
  • the present invention provides a non-glycosylated EPO analog wherein one or more of the lysine residues that are capable of being pegylated is replaced with an amino acid that cannot be pegylated.
  • amino acid that cannot be pegylated refers to any amino acid that cannot be functionalized with a polyethylene glycol moiety using the PEGylation reaction conditions described herein.
  • amino acids include those nucleophilic functional group without a reactive (under the conditions described herein), such as arginine, glycine, alanine, phenylalanine and the like. It is suitable that the amino acid that cannot be pegylated is one that represents a conserved substitution for lysine. Examples of conservative substitutions for lysine include arginine and histidine.
  • lysine residues that are capable of being pegylated refers to one or more lysine residues in non- glycosylated EPO that are capable of being functionalized with a polyethylene glycol moiety using the reaction conditions described herein.
  • Certain lysines, for example lysine 152 have been shown by structural studies to lie within the hydrophobic core of the protein and therefore may not be accessible (or available) for pegylation (Cheetham et al. 1998, Nature Structural Biology, 5 (10), 861-866).
  • non-glycosylated EPO affects the in vivo activity of the pegylated EPO compounds. Therefore, in specific embodiments of the present invention, there is provided a non-glycosylated EPO analog wherein the lysine at position 45 and/or 116 has been replaced with an amino acid that cannot be pegylated such as arginine and histidine.
  • a protein having the amino acid sequence shown in SEQ ID NO:1 Figure 1A wherein the lysine at position 45 is replaced with arginine (K45R).
  • a protein having the amino acid sequence shown in SEQ ID NO:2 ( Figure 1 B) wherein the lysine at position 116 is replaced with arginine (K116R).
  • a protein having the amino acid sequence shown in SEQ ID NO:3 ( Figure 1C) wherein the lysines at positions 45 and 116 are replaced with arginine (K45,116R).
  • the invention also includes analogs and derivatives of the sequences shown in SEQ ID NOS:1-3.
  • analog in reference to SEQ ID NOS: 1-3 includes any protein or peptide having an amino acid residue sequence substantially identical SEQ ID NOS:1-3 in which one or more residues have been conservatively substituted with a functionally similar residue so that the resulting protein or peptide has the requisite activity.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine, for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine, for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid, for another.
  • the phrase "conservative substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such protein displays the requisite activity.
  • derivative in reference to SEQ ID NOS: 1-3 refers to a protein or peptide having one or more residues chemically derivatized by reaction of a functional side group provided that the requisite activity is retained.
  • derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O- alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine.
  • derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for serine; and ornithine may be substituted for lysine.
  • Non- glycosylated EPO analogs of the invention also include proteins having one or more additions and/or deletions or residues relative to the sequence of the non-glycosylated EPO analogs of the invention so long as the requisite activity is maintained.
  • non-glycosylated EPO proteins of the invention includes non-glycosylated EPO analogs of the invention and non-glycosylated EPO produced using the methods described herein.
  • the present invention further includes a novel, codon-optimized nucleic acid sequences encoding EPO for expression in Streptomyces.
  • the nucleic acid sequence comprises SEQ ID NO:7 ( Figure 3).
  • the present invention also includes an isolated nucleic acid sequence that encodes a non-glycosylated EPO analog wherein one or more of the lysines residues that are capable of being pegylated is replaced with an amino acid that cannot be pegylated.
  • an isolated nucleic acid sequence which encodes a non-glycosylated EPO analog wherein the lysine and position 45 and/or 116 has been replaced with an amino acid that cannot be pegylated.
  • the isolated nucleic acid sequence encodes a protein selected from the group consisting of SEQ ID NO:1 ( Figure 1A), SEQ ID NO:2 ( Figure 1B) and SEQ ID NO:3 ( Figure 1C) or an analog or derivative thereof.
  • nucleic acid sequence comprising: a) a nucleic acid sequence as shown in SEQ ID NO:4 ( Figure 2A) or SEQ ID NO:5 ( Figure 2B), SEQ ID NO:6 ( Figure 2C) or SEQ ID NO:7 ( Figure 3) wherein T can also be U; or b) a nucleic acid sequence that is complimentary to a nucleic acid sequence of (a).
  • the present invention further relates chimeric molecules comprising the nucleic acid sequences of the invention for expression in host cells.
  • the chimeric molecules comprising the nucleic acid sequences of the invention comprise elements for the expression of the corresponding proteins in prokaryotic animals.
  • the present invention includes a chimeric nucleic acid molecule comprising in the 5' to 3' direction of transcription:
  • the chimeric nucleic acid molecule comprises a nucleic acid sequence as shown in SEQ ID NO:28 ( Figure 6).
  • the proteins of the present invention may be produced by a variety of methods including recombinant DNA technology or well-known chemical procedures, such as solution or solid-phase peptide synthesis, or semi- synthesis in solution beginning with protein fragments coupled through conventional solution methods.
  • the non-glycosylated EPO proteins of the invention are prepared using recombinant DNA technology.
  • the present invention therefore relates to vectors that comprise the isolated nucleic acid molecules of the invention, host cells that are genetically engineered with the recombinant vectors, and methods of producing non- glycosylated EPO proteins by recombinant techniques.
  • the host cells are from a prokaryotic organism so that the recombinant EPO analogs are not glycosylated by the host.
  • isolated nucleic acid molecules of the invention means a codon optimized nucleic acid sequence capable of being expressed in a prokaryotic organism and encoding nonglycosylated EPO or a non-glycosylated EPO anolog of the invention. Accordingly, the present invention provides a method of producing a recombinant protein, comprising the steps of:
  • the nucleic acid sequence encoding non-glycosylated EPO comprises SEQ ID NO:7 ( Figure 3).
  • the nucleic acid sequence encoding a non-glycosylated EPO analog of the invention is a nucleic acid sequence encoding a non-glycosylated EPO analog wherein one or more of the lysines available for pegylation, is replaced with an amino acid that cannot be pegylated.
  • the nucleic acid sequence encoding a non-glycosylated EPO analog of the invention is a nucleic acid molecule encoding a protein selected from the group consisting of SEQ ID NO:1 ( Figure 1A), SEQ ID NO:2 ( Figure 1 B) and SEQ ID NO:3
  • the nucleic acid sequence encoding a non-glycosylated EPO analog of the invention comprises (a) a nucleic acid sequence as shown in SEQ ID NO:4 ( Figure
  • the chimeric nucleic acid sequence further comprises nucleic acid sequences coding for a secretion function, for example the modified protease B signal peptide.
  • the chimeric nucleic acid molecule comprises a nucleic acid sequence as shown in SEQ ID NO:28 ( Figure 6).
  • Nucleic acid sequences capable of regulating transcription of the nucleic acid molecules of the invention in said host cell may include an appropriate promoter, such as, for example, the Streptomyces aminoglycoside phosphotransferaase (Aph) promoter, phage lambda PL promoter, the E. coli lac, txp and tac promoters to name a few.
  • an appropriate promoter such as, for example, the Streptomyces aminoglycoside phosphotransferaase (Aph) promoter, phage lambda PL promoter, the E. coli lac, txp and tac promoters to name a few.
  • Other suitable promoters will be known to the skilled artisan. Such methods are well known in the art, for example, as described in US patent Nos. 5,580,734, 5,641 ,670, 5,733,746, and 5,733,761 , entirely incorporated herein by reference.
  • the chimeric molecules may will further contain sites for transcription initiation and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the chimeric molecules will preferably include a translation initiation codon (e.g., UAC) at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated.
  • a translation initiation codon e.g., UAC
  • a termination codon e.g., UAA, UGA or UAG
  • the chimeric molecules comprising the nucleic acid molecules encoding the non-glycosylated EPO proteins of the present invention can be joined to an expression vector for propagation in a host.
  • Expression vectors may optionally include at least one selectable marker.
  • markers include, for example, tetracycline, ampicillin, kanamycin, thiostrepton, or chloramphenicol resistance genes for culturing in Streptomyces lividans, E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces lividans, Bacillus. subtilis, Caulobacter crescentens, and Salmonella typhimurium cells.
  • the host is bacteria, more preferably Streptomyces lividans.
  • Vectors for use in bacteria include plJ680 available from John Innes Institute; pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNHSA, pNH16a, pNH18A, pNH46A, available from Stratagene; pET30 vectors from Novagen, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, and pUC8 available from Pharmacia.
  • Suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of a vector construct into a host cell can be effected by electroporation, transduction, infection, transformation or other methods. Such methods are described in many standard laboratory manuals, such as Ausubel, et al., ed., Current Protocols in Molecular Biology, Greene Publishing, NY, NY (1987-1998), Kieser, et al., Practical Streptomyces Genetics, John Innes Foundation, Norwich (2000) and Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, NY (1989).
  • the proteins of the present invention can be expressed in a modified form, such as a fusion protein, and can include, for example secretion signals and additional heterologous functional regions.
  • Signal peptides may be used to facilitate the extracellular discharge of proteins in both prokaryotic and eukaryotic environments. Alternate signal peptide sequences may function with heterologous coding sequences.
  • Signal peptides such as the Streptomyces griseus protease B signal peptide gene, can be incorporated into the EPO proteins of the present invention to facilitate extracellular translocation.
  • Additional heterologous functional regions may, for example, include a region of additional amino acids added to the N-terminus of an analog to improve stability and persistence in the host cell culture, during purification, or during subsequent handling and storage.
  • peptide moieties can be added to facilitate purification. Such regions can be removed prior to final preparation of an active protein. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.
  • an expression vector carrying a gene encoding a protein of the present invention is fransfected into a suitable host cell using standard methods, cells that contain the vector are propagated under conditions suitable for expression of the recombinant protein. For example, if the recombinant gene has been placed under the control of an inducible promoter, suitable growth conditions would incorporate the appropriate inducer.
  • the recombinantly produced protein may be purified from culture of transformed cells by any suitable means.
  • the non-glycosylated EPO proteins of the present invention can be purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, reversed-phase chromatography, hydroxylapatite chromatography, and size exclusion chromatography.
  • non-glycosylated EPO proteins of the present invention may be fused at the N-terminal or C-terminal end to several histidine residues.
  • This "histidine-tag” enables a single-step protein purification method referred to as “immobilized metal ion affinity chromatography” (IMAC) essentially as described in U.S. Patent 4,569,794, which hereby is incorporated by reference.
  • IMAC immobilized metal ion affinity chromatography
  • non-glycosylated EPO proteins containing N- terminal leader sequences in bacteria the proteins can be digested with an aminopeptidase such as a mono- or di-aminopeptidase, a serine protease such as trypsin, or even by chemical cleavage such as cleavage by cyanogen bromide.
  • an aminopeptidase such as a mono- or di-aminopeptidase, a serine protease such as trypsin, or even by chemical cleavage such as cleavage by cyanogen bromide.
  • an aminopeptidase such as a mono- or di-aminopeptidase, a serine protease such as trypsin, or even by chemical cleavage such as cleavage by cyanogen bromide.
  • Watson, et al., (1976) Methods Microb. 9:1-14 describe different aminopeptidases present in different bacteria including E.
  • coli and is entirely herein incorporated by reference, as is Canadian Patent Application 2,179,623 filed December 22, 1994, Laid open June 29, 1995, entitled "Proteases from Streptomyces and Use Thereof in Protein Expression Systems” that describes a family of proteases endogenous to Streptomyces cells and are suitable to degrade exogenous proteins secreted from Streptomyces host cells.
  • a nucleic acid molecule encoding the proteins of the invention were constructed from an ordered set of synthetic oligonucleotides SEQ ID NOS:8-23 ( Figure 4). These oligonucleotides were annealed and ligated in two reactions.
  • Gene fragments of the right size were purified from these mixtures and ligated to produce the EPO gene with compatible ends for cloning into the Streptomyces lividans/E.coli shuttle vector containing the Aph promoter, protease B signal peptide gene, BamHI-Hindlll cloning site, and Aph terminator region.
  • the APO.H vector was digested with BamHI and Hindlll restriction endonucleases and mixed with the synthetic EPO gene under appropriate conditions for ligation. Vectors isolated from the reaction mixture which showed a restriction digest pattern compatible with having the EPO gene correctly inserted were purified.
  • a set of four synthetic olignucleotides SEQ ID NOS:24-27 were used to replace the asparagines residue at position -2 (relative to the mature EPO protein) with an alanine and to introduce an additional proline residue at position -4.
  • the resulting expression fragment (APZ-EPO or pCAN42T, SEQ ID NO:28, shown in Figure 6) has the EPO gene downstream of the Aph promoter and fused directly at its N-terminus to the C-terminus of the gene for the modified Protease B signal peptide and upstream of the Aph terminator region.
  • the plasmids were transformed into protoplasts prepared from spores of Streptomyces lividans NCIMB 11416. Clones were collected and EPO expression verified by western blot analysis of culture supernatants. A single clone was selected and a seed bank prepared. Sequence of the EPO coding region was confirmed for the plasmid isolated from this clone. Genes for EPO analogs substituted at lysine residues 45 and/or 116 were prepared using synthetic oligonucleotides to change the specific codons for lysine (AAG) to that of another amino acid - e.g., that for arginine (CCG). The process is shown schematically in Figure 7 for the K45R mutant.
  • EPO analogues involves growth in simple media to late log phase of growth.
  • Cells are removed by filtration and the culture filtrate diluted and loaded directly onto a cation exchange chromatography column for capture of EPO protein.
  • a salt gradient is applied to elute protein bound to the column and those fractions containing EPO are pooled.
  • the pool is adjusted to high salt concentration for hydrophobic interaction chromatography.
  • Bound proteins are eluted from the HIC column by reducing salt concentration and those fractions containing EPO are pooled.
  • the final step of the purification process is a second cation exchange chromatography step.
  • the resulting EPO is substantially free from contaminating proteins (see Figure 8).
  • nucleic acid molecules of the invention While the expression of the nucleic acid molecules of the invention in prokaryotic hosts provides non-glycosylated EPO proteins, a person skilled in the art would undertand that the nucleic acid molecules of the invention may be expressed in eukaryotic hosts and the sugar part of the resulting glycosylated protein, removed using techniques known in the art, for example, using glycosidase enzymes. IV. Pegylation Methods
  • non-glycosylated EPO proteins of the present invention are appropriately expressed, refolded (depending on the expression system used), and purified, they can be modified. Proteins can be modified by covalently linking synthetic or natural macromolecules to the surface of the proteins. However, it has been difficult to endow delicate proteins with suitable new properties by attaching polymers without causing any loss of their functionality.
  • the present invention provides specific non-glycosylated EPO proteins which are modified by covalent attaching of polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the bioactivity of polymer modified proteins can be affected by factors such as: i) the size of the polymer; ii) the particular sites of attachment; iii) the degree of modification; iv) adverse coupling conditions; v) whether a linker is used for attachment or whether the polymer is directly attached; vi) generation of harmful co-products; vii) damage inflicted by the activated polymer; or viii) retention of charge.
  • factors such as: i) the size of the polymer; ii) the particular sites of attachment; iii) the degree of modification; iv) adverse coupling conditions; v) whether a linker is used for attachment or whether the polymer is directly attached; vi) generation of harmful co-products; vii) damage inflicted by the activated polymer; or viii) retention of charge.
  • polymer modification of cytokines in particular, has resulted in dramatic reductions in bioactivity (Francis, G.E. et al. Intl. J
  • the present invention provides methods for preparing non-glycosylated EPO proteins with polyethylene glycol polymers covalently attached, thereto.
  • the methods of this invention are used to directly attach polymers which vary in size.
  • the addition of polymers is controlled such that a bioactive population of non-glycosylated EPO derivatives can be purified for therapeutic use.
  • Polyethylene glycol or “PEG” refers to a hydrophilic polymer having the formula:
  • x is a number from about 70 to about 1200, preferably from about 450 to about 1200, even more preferably from about 450 to about 700.
  • Activated PEG as used herein means a polyethylene glycol derivative that comprises an activated group that reacts with a nucleophilic group to form a covalent bond.
  • PEG-aldehyde refers to a hydrophilic polymer having the formula:
  • Y is number from 1 to 4
  • x is a number from about 70 to about 1200, preferably from about 450 to about 1200, even more preferably from about 450 to about 700.
  • PEG-Propionaldehyde refers to a PEG-aldehyde hydrophilic polymer having the formula:
  • pNPPEG refers to PEG-p-nitrophenyl carbonate and is a hydrophilic polymer having the formula
  • x is a number from about 70 to about 1200, preferably from about 450 to about 1200, even more preferably from about 450 to about 700.
  • SPA-PEG refers to PEG-succinimidyl propionate and is a hydrophilic polymer having the formula
  • x is a number from about 70 to about 1200, preferably from about 450 to about 1200, even more preferably from about 450 to about 700.
  • C ⁇ -4 alkyl as used herein means straight and/or branched chain alkyl radicals containing from one to four carbon atoms and includes methyl, ethyl, propyl, isopropyl, t-butyl and the like.
  • the pegylated non-glycosylated EPO proteins of the present invention may be prepared using any known method.
  • the present invention therefore provides a method for preparing polymer-derivatized, non-glycosylated EPO proteins, comprising: a) adding an activated PEG compound to a solution containing a non-glycosylated EPO protein of the invention under conditions that permit the formation of a bond between an amino group of the non- glycosylated EPO protein and the activating group of PEG and b) isolating the polymer-derivatized, non-glycosylated EPO protein.
  • the activated PEG compound is selected from the group consisting of pNPPEG, TMPEG, PEG-aldehyde and SPA-PEG.
  • the size of PEG is 10kDa PEG.
  • a method used for preparing the non-glycosylated EPO polymer derivatives of the present invention involved the use of polyethylene glycol p- nitrophenyl carbonate (pNPPEG) to directly attach ethylene glycol groups to amino groups of available lysine residues.
  • pNPPEG polyethylene glycol p- nitrophenyl carbonate
  • a method for preparing polymer- derivatized, non-glycosylated EPO proteins comprising: a) adding pNPPEG to a solution containing a non-glycosylated EPO protein of the invention under conditions that permit the formation of an amide bond between an amino group of the EPO and the carbonate of pNPPEG and b) isolating the polymer- derivatized, non-glycosylated EPO proteins.
  • Novel pegylated non-glycosylated EPO proteins have been prepared and shown to have erythropoietic activity in the polycythemic mouse assay
  • pegylated non-glycosylated EPO proteins of the invention are therefore useful for treating conditions which benefit from stimulation of erythropoiesis, for example anemia.
  • the present invention provides polymer-derivatized, non- glycosylated EPO protein having a protein portion and a polymer portion, wherein the protein portion is a non-glycosyated EPO protein of the invention and wherein the polymer portion consists of from 1 or more polymer chains of polyethylene glycol, or a pharmaceutically acceptable salt, hydrate and/or solvate thereof.
  • the 1 or more polymer chains of ethylene glycol have the formula:
  • pharmaceutically acceptable salt means an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.
  • solvate as used herein means a pegylated non-glycosylated
  • EPO protein wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate”. VI. Uses
  • novel polymer-derivatized, non- glycosylated EPO proteins have been prepared. Accordingly, the present invention includes all uses of the polymer-derivatized, non-glycosylated EPO proteins of the invention, including their use in therapeutic methods and compositions for stimulating erythropoiesis, their use in diagnostic assays and their use as research tools.
  • the polymer-derivatized, non-glycosylated EPO proteins of the invention have shown activity both in vitro and in vivo.
  • the present invention therefore provides a method for stimulating erythropoiesis comprising administering a therapeutically effective amount of a polymer-derivatized, non-glycosylated protein of the invention to a mammal in need thereof.
  • the mammal is human.
  • a use of a polymer-derivatized, non-glycosylated protein of the invention to stimulate erythropoiesis as well as a use of a polymer-derivatized, non-glycosylated protein of the invention to prepare a medicament to stimulate erythropoiesis.
  • Methods and compositions for stimulating erythropoiesis are useful in treating any condition that benefits from the stimulation of erythropoiesis. Conditions that benefit from the stimulation of erythropoiesis, include but are not limited to anemia.
  • Stimulating erythropoiesis generally refers to the ability of a compound to cause an increase in hemocrit levels from an established baseline. Therefore the invention also provides a method for increasing the hematocrit level in a mammal comprising administering a therapeutically effective amount of a polymer-derivatized, non-glycosylated protein of the invention to a mammal in need thereof.
  • the mammal is human.
  • the present invention further provides a use of a polymer-derivatized, non-glycosylated protein of the invention to increase the hematocrit level in a mammal as well as a use of a polymer-derivatized, non-glycosylated protein of the invention to prepare a medicament to increase the hematocrit level in a mammal.
  • polymer-derivatized, non- glycosylated EPO proteins of the invention would have therapeutic utility, for example as stimulators of erythropoiesis.
  • Proteins may be examined for their efficacy in stimulating erythropoiesis in vivo, for example, using the polycythemic mouse assay as described in P.P Dukes et al., J. Lab. Clin. Med. 4:250-256 (1969), and in vitro, using a 3 H-thymidine uptake assay in spleen cells as described in Beals, J.M. et al. WO 00/32772. Accordingly, the methods, uses and compositions of the invention are meant to include only those polymer-derivatized, non-glycosylated EPO proteins having the desired effect.
  • an "effective amount” or a "sufficient amount " of an agent as used herein is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an "effective amount” depends upon the context in which it is being applied.
  • an effective amount of an agent is, for example, an amount sufficient to achieve such a stimulation of erythropoiesis as compared to the response obtained without administration of the agent.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • “Palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
  • the polymer-derivatized, non-glycosylated EPO proteins of the invention are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising a polymer-derivatized, non-glycosylated EPO protein of the invention in admixture with a suitable diluent or carrier.
  • polymer-derivatized, non-glycosylated EPO proteins of the invention may be used in the form of the free base/acid, in the form of salts, solvates or hydrates. All forms are within the scope of the invention.
  • the described polymer-derivatized, non-glycosylated EPO proteins or salts, hydrates or solvates thereof may be administered to a mammal in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the polymer-derivatized, non-glycosylated EPO proteins and/or compositions of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly.
  • Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
  • the polymer-derivatized, non-glycosylated EPO proteins of the invention may be administered to an mammal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the polymer- derivatized, non-glycosylated EPO protein, chosen route of administration and standard pharmaceutical practice.
  • the dosage of the polymer-derivatized, non-glycosylated EPO proteins and/or compositions of the invention can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the animal to be treated.
  • One of skill in the art can determine the appropriate dosage based on the above factors.
  • the compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response.
  • the polymer-derivatized, non-glycosylated EPO proteins of the invention can be used alone or in combination with other agents that stimulate erythropoiesis or in combination with other types of treatment (which may or may not stimulate erythropoiesis) for the treatment and/or prevention of anemia or other disorders that benefit from stimulation of erythropoiesis.
  • polymer- derivatized, non-glycosylated EPO proteins of the invention are also useful in diagnostic assays, screening assays and as research tools.
  • the polymer-derivatized, non-glycosylated EPO proteins of the invention may be useful in identifying or detecting erythropoietic activity.
  • the polymer-derivatized, non- glycosylated EPO proteins of the invention may be radiolabelled and contacted with a population of cells. The presence of the radiolabel on the cells may indicate erythropoietic activity.
  • the polymer-derivatized, non-glycosylated EPO proteins of the invention may be used to identify other compounds that stimulate erythropoiesis.
  • the polymer-derivatized, non- glycosylated EPO proteins of the invention may be used in enzyme assays and assays to study the localization of erythropoietic activity.
  • the polymer-derivatized, non-glycosylated EPO proteins may also be radiolabelled.
  • Example 1 CANGENUSTM Expression of Non-glycosylated EPO Analogues
  • Streptomyces lividans transformed with vectors for expression of EPO and EPO analogues are grown in a 15 L fermenter with Tryptic Soy Broth (TSB,
  • Cultures are harvested at an optical density (OD 600 nm) of 4 and cells removed by filtration through two layers of Whatman #1 filter paper and 3 ⁇ m polysulphone cartridge filter. The filtrate is collected, sterile filtered (0.22 ⁇ m cartridge filter), diluted to 5.9 mS/cm, and adjusted to pH 6.8 by addition of NaOH. The correctly folded EPO is recovered from the culture supernatant by cation exchange chromatography (POROS 50 HS). The column (55 mL, 6 cm bed height) is first equilibrated in 20 mM phosphate buffer ph 6.8, 1 mM EDTA. The adjusted supernatant is loaded at 160 mL/min and the column washed with equilibration buffer.
  • EPO bound to the column is eluted with 350 mM NaCI in 20 mM phosphate buffer pH 6.8, 1 mM EDTA. Fractions containing EPO are pooled and adjusted to 2.5 M NaCI and 0.01% Brij 35. A 10 ml (5 cm bed height) Toyaperarl phenyl-HIC column is equilibrated with 20 mM PO 4 buffer, pH 6.8, 2.5 M NaCI, 1 mM EDTA, 0.01% (v/v) Brij 35. The EPO pool is loaded at 3.3 mL/min and the column washed with equilibration buffer.
  • EPO bound to the column is eluted with 0.5 M NaCI in 20 mM PO 4 buffer pH 6.8, 1 mM EDTA, 0.01 % Brij 35.
  • EPO contwining fractions are pooled diluted to a conductivity of 5.45 mS/cm.
  • a 1 mL (5 cm bed height) cation exchange column (POROS 50 HS) is equilibrated with 20 mM phosphate buffer pH 6.8, 1 mM EDTA, 0.01% Brij 35.
  • the EPO pool is loaded at 3.2 mL/min and the column washed with equilibration buffer.
  • EPO is eluted with 300 mM NaCI in 10 mM PO 4 buffer pH 6.8.
  • Resulting preparation is EPO at >95% purity by SDS-PAGE (see Figure 8, for example).
  • Example 2 Site-Directed Mutagenesis on EPO 45 th Amino Acid
  • Figure 7 illustrates the restriction map of EPO and the general cloning strategy of K45 EPO analog. A similar strategy was used to mutate the 116 site.
  • PCAN042 EPO expression plasmid (Cangene Corp., Mississauga, Canada);
  • EPO SR 5' -GCC TGG CCG CGG AGG ACC G -3' (SEQ ID NO:31)
  • PCAN042 EPO expression vector
  • pKK223-3 plasmids were digested with Pstl and Hindlll. EPO insert and pKK223-3 backbone were ligated and transformed into JM109 cells. pKK-EPO positive clones were confirmed by restriction enzyme digestion and DNA sequence.
  • PCR products on pKK-EPO with EPO45F and EPOSR, EPOKF and EPO 45R primers, respectively, were mixed together. Over-lapping PCR products on this mixture with primers of Epo KF and Epo SR were then digested with Kpnl and SacII.. This DNA fragment containing mutation on 45 th aa was subcloned back to pKK-EPO. Positive clone containing expected mutation was named as pKK-EPO45.
  • Construct Pcan345 expression vector containing 45 th aa mutation PKK-EPO45 was digested with Pstl and Hindlll and the EPO45 insert was cloned back into Pcan042 backbone. Positive clone of PcanEPO45 was named as Pcan342 whose 45 th aa of EPO was mutated from Lys to Arg. Mutagenesis of the 116 site was performed in a similar fashion.
  • Example 3 PEGylation of Non-Glycosylated EPO Analogues
  • Purified EPO or EPO analog
  • EPO analog prepared as described above is reacted with activated 10 kDa PEG (e.g., TMPEG (PolyMASC or Sigma); ALD-PEG (Shearwater); SPA-PEG (Shearwater); PNP-PEG Carbonate (Cangene synthesis)) in 20 mM phosphate buffer pH 8.
  • the molar ratio of protein:PEG is 1 :200 and the reaction is conducted at ambient temperature for 2.5 hours.
  • the reaction mixture is adjusted to 4.5 mS/cm, pH 7.32 and loaded onto a cation exchange column (CIM disc-S, equilibrated with 10 mM PO 4 buffer pH 7.5, 0.01% Brij 35).
  • Mono-PEGylated EPO is eluted with a gradient from 0- 600 mM NaCI in 10 mM PO 4 buffer pH 7.5, 0.01% Brij 35.
  • the tendonsid fractions were run on an SDS-Page gel, shown in Figure 9.
  • Example 4 In vitro and In vivo Activity of Pegylated Non-glycosylated EPO Analogs
  • the pegylated non-glycosylated EPO analogs were tested in vitro in the anemic mouse spleen cell assay (G. Krystal, Exp. Hematol. 11 :649-660 (1983)) and in vivo in the polycythemic (hypoxic) mouse assay (P.P Dukes et al., J. Lab. Clin. Med. 74:250-256 (1969)).
  • the results are summarized in Table !
  • the in vivo activity of non-pegylated, non-glycosylated EPO compared with 5 kDA pegylated EPO as determined using the polycythemic mouse assay is shown in Figure 10.
  • the polycythemic mouse assay was also used to determine the effect of PEG-size on in vivo activity.
  • Cangene #1 is non-glycosylated, non-pegylated EPO produced as described herein
  • Cangene #2 is di-5 kDa PEG-EPO
  • Cangene #3 is mono-5 kDa PEG- EPO
  • Cangene #4 is mono-10 kDa PEG EPO.
  • the results show progressively increasing biological response with increasing PEG size and a preference for mono-PEGylation (i.e. one 10 kDa PEG group attached to the protein is better than two 5 kDa PEG groups attached to the protein).
  • Anemic mice were injected with equal amounts of the various EPO's based on activity determination from the in vitro mouse speen assay (Krystal, supra).
  • Example 5 Rabbit Haematocr ⁇ t and Clearance Assay The pegylated non-glycosylated EPO analogs were also tested in the rabbit to determine their effect on haematocrit levels and their clearance from circulation as described in A.J. Sytkowski et al. Proc. Nat. Acad. Sci. USA, 95(3):1184-1188 and A.J. Sytkowski et al. J. Biol. Chem. 274(35):24773- 24778 (1999). In the haematocrit study, in vivo responses of rabbits injected with 15,000 IU EPO (determined from in vitro assay of samples) were measured. The results are shown in Figure 12.
  • the PEG-EPO samples were 10 kDa PEG-EPO prepared using the PNP-PEG as described in Example 3.
  • the results show the equivalence of erythropoietic activity (increasing haematocrit) for all three samples (Eprex is a glycosylated EPO). Clearance of EPO for the same animals was determined using ELISA to measure the residual EPO in circulation in the rabbits. The results are shown in Figure 13. Once again the equivalence (on a U/U basis) of the PEG-EPOs of the present invention with glycosylated EPO is shown.

Abstract

La présente invention concerne des nouvelles protéines de l'érythropoïétine (EPO) non glycosylées, des acides nucléiques codant ces protéines, et des méthodes de préparation de ces protéines au moyen de la technologie d'ADN recombinant. Ladite invention a aussi pour objet de nouvelles protéines de l'érythropoïétine (EPO) non glycosylées dérivées de polymères, des méthodes de préparation et d'utilisation de ces composés dans le traitement d'un trouble qui bénéficie de la stimulation de l'érythropoïèse, par exemple, l'anémie. En outre, l'invention a trait à une méthode de pégylation d'une érythropoïétine (EPO) non glycosylée et à des analogues associés, par exemple, au moyen de p-nitrophényl polyéthylène glycol carbonate (pPNPEG).
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Cited By (93)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005084711A1 (fr) * 2004-03-02 2005-09-15 Chengdu Institute Of Biological Products Erythropoietine recombinante pegylee a activite in vivo
WO2006079155A1 (fr) * 2005-01-25 2006-08-03 Apollo Life Sciences Limited Molecules et leurs molecules chimeriques
WO2007010552A2 (fr) * 2005-03-17 2007-01-25 Serum Institute Of India Limited Conjugue d'erythropoietine peg n-terminal
WO2008019214A1 (fr) * 2006-08-04 2008-02-14 Prolong Pharmaceuticals, Inc. Erythropoïétine modifiée
WO2009010270A3 (fr) * 2007-07-17 2009-04-30 Hoffmann La Roche Purification de polypeptides polyéthylène-glycolés
WO2009094551A1 (fr) 2008-01-25 2009-07-30 Amgen Inc. Anticorps anti-ferroportine et procédés d'utilisation
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WO2011077067A1 (fr) * 2009-12-21 2011-06-30 Polytherics Limited Conjugués polymères d'érythropoïétine non glycosylée
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WO2019070472A1 (fr) 2017-10-04 2019-04-11 Amgen Inc. Adaptateur d'écoulement destiné à un dispositif d'administration de médicament
WO2019074579A1 (fr) 2017-10-09 2019-04-18 Amgen Inc. Dispositif d'administration de médicament comprenant un ensemble d'entraînement et procédé d'assemblage associé
WO2019089178A1 (fr) 2017-11-06 2019-05-09 Amgen Inc. Dispositif d'administration de médicament avec détection de positionnement et de débit
WO2019090086A1 (fr) 2017-11-03 2019-05-09 Amgen Inc. Systèmes et approches pour stériliser un dispositif d'administration de médicament
WO2019090303A1 (fr) 2017-11-06 2019-05-09 Amgen Inc. Ensembles de remplissage-finition et procédés associés
WO2019094138A1 (fr) 2017-11-10 2019-05-16 Amgen Inc. Pistons pour dispositifs d'administration de médicament
WO2019099324A1 (fr) 2017-11-16 2019-05-23 Amgen Inc. Mécanisme d'insertion d'aiguille pour dispositif d'administration de médicament
WO2019099322A1 (fr) 2017-11-16 2019-05-23 Amgen Inc. Auto-injecteur avec détection de décrochage et de point d'extrémité
EP3556411A1 (fr) 2015-02-17 2019-10-23 Amgen Inc. Dispositif d'administration de médicaments avec fixation et/ou de rétroaction assistée(s) sous vide
WO2019231582A1 (fr) 2018-05-30 2019-12-05 Amgen Inc. Mécanisme de libération thermique à ressort pour dispositif d'administration de médicament
WO2019231618A1 (fr) 2018-06-01 2019-12-05 Amgen Inc. Ensembles de trajet de fluide modulaires pour dispositifs d'administration de médicament
EP3593839A1 (fr) 2013-03-15 2020-01-15 Amgen Inc. Cassette de médicaments
WO2020023451A1 (fr) 2018-07-24 2020-01-30 Amgen Inc. Dispositifs d'administration pour l'administration de médicaments
WO2020023220A1 (fr) 2018-07-24 2020-01-30 Amgen Inc. Dispositifs d'administration de médicament hybrides dotés d'une partie de fixation collante à placer sur la peau et procédé de préparation associé
WO2020023444A1 (fr) 2018-07-24 2020-01-30 Amgen Inc. Dispositifs d'administration pour l'administration de médicaments
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WO2020028009A1 (fr) 2018-07-31 2020-02-06 Amgen Inc. Ensemble de trajet de fluide pour dispositif d'administration de médicament
WO2020068623A1 (fr) 2018-09-24 2020-04-02 Amgen Inc. Systèmes et procédés de dosage interventionnel
WO2020068476A1 (fr) 2018-09-28 2020-04-02 Amgen Inc. Ensemble d'activation d'échappement de fil de muscle pour un dispositif d'administration de médicament
WO2020072846A1 (fr) 2018-10-05 2020-04-09 Amgen Inc. Dispositif d'administration de médicament ayant un indicateur de dose
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WO2020081479A1 (fr) 2018-10-15 2020-04-23 Amgen Inc. Dispositif d'administration de médicament comprenant un mécanisme d'amortissement
WO2020081480A1 (fr) 2018-10-15 2020-04-23 Amgen Inc. Procédé d'assemblage de plate-forme pour dispositif d'administration de médicament
WO2020092056A1 (fr) 2018-11-01 2020-05-07 Amgen Inc. Dispositifs d'administration de médicament à rétraction d'aiguille partielle
WO2020091981A1 (fr) 2018-11-01 2020-05-07 Amgen Inc. Dispositifs d'administration de médicament à rétraction partielle d'élément d'administration de médicament
WO2020091956A1 (fr) 2018-11-01 2020-05-07 Amgen Inc. Dispositifs d'administration de médicament avec rétraction partielle de l'organe d'administration de médicament
WO2020219482A1 (fr) 2019-04-24 2020-10-29 Amgen Inc. Ensembles et procédés de vérification de stérilisation de seringue
WO2021041067A2 (fr) 2019-08-23 2021-03-04 Amgen Inc. Dispositif d'administration de médicament doté de composants configurables de mise en prise de protection d'aiguille et méthodes associées
WO2022033480A1 (fr) * 2020-08-11 2022-02-17 隆延生物科技（上海）有限公司 Préparation liquide et son application
EP3981450A1 (fr) 2015-02-27 2022-04-13 Amgen, Inc Dispositif d'administration de médicament ayant un mécanisme de protection d'aiguille présentant un seuil réglable de résistance au mouvement de l'élément de protection d'aiguille
EP4074355A1 (fr) 2011-04-20 2022-10-19 Amgen Inc. Appareil auto-injecteur
WO2022246055A1 (fr) 2021-05-21 2022-11-24 Amgen Inc. Procédé d'optimisation d'une recette de remplissage pour un récipient de médicament

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
WO1994024160A2 (fr) * 1993-04-21 1994-10-27 Brigham And Women's Hospital Erythropoïetines-muteines a activite renforcee
EP0640619A1 (fr) * 1993-08-17 1995-03-01 Amgen Inc. Analogues d'érythropoiétine avec des sites additionnels de glycosylation
WO2000024893A2 (fr) * 1998-10-23 2000-05-04 Amgen Inc. Methodes et compositions permettant de prevenir et de traiter l'anemie
WO2000032772A2 (fr) * 1998-11-30 2000-06-08 Eli Lilly And Company Composes erythropoietiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
WO1994024160A2 (fr) * 1993-04-21 1994-10-27 Brigham And Women's Hospital Erythropoïetines-muteines a activite renforcee
EP0640619A1 (fr) * 1993-08-17 1995-03-01 Amgen Inc. Analogues d'érythropoiétine avec des sites additionnels de glycosylation
WO2000024893A2 (fr) * 1998-10-23 2000-05-04 Amgen Inc. Methodes et compositions permettant de prevenir et de traiter l'anemie
WO2000032772A2 (fr) * 1998-11-30 2000-06-08 Eli Lilly And Company Composes erythropoietiques

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BINNIE C ET AL: "Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66.", PROTEIN EXPRESSION AND PURIFICATION. UNITED STATES DEC 1997, vol. 11, no. 3, December 1997 (1997-12-01), pages 271 - 278, XP004459932, ISSN: 1046-5928 *
FRANCIS G E ET AL: "PEGYLATION OF CYTOKINES AND OTHER THERAPEUTIC PROTEINS AND PEPTIDES: THE IMPORTANCE OF BIOLOGICAL OPTIMISATION OF COUPLING TECHNIQUES", INTERNATIONAL JOURNAL OF HEMATOLOGY, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 68, no. 1, July 1998 (1998-07-01), pages 1 - 18, XP000791226, ISSN: 0925-5710 *
LIN F K ET AL: "Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene.", GENE. NETHERLANDS 1986, vol. 44, no. 2-3, 1986, pages 201 - 209, XP009020931, ISSN: 0378-1119 *
WEN D ET AL: "ERYTHROPOIETIN STRUCTURE-FUNCTION RELATIONSHIPS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 269, no. 36, 9 September 1994 (1994-09-09), pages 22839 - 22846, XP000199310, ISSN: 0027-8424 *

Cited By (149)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005084711A1 (fr) * 2004-03-02 2005-09-15 Chengdu Institute Of Biological Products Erythropoietine recombinante pegylee a activite in vivo
WO2006079155A1 (fr) * 2005-01-25 2006-08-03 Apollo Life Sciences Limited Molecules et leurs molecules chimeriques
WO2007010552A2 (fr) * 2005-03-17 2007-01-25 Serum Institute Of India Limited Conjugue d'erythropoietine peg n-terminal
WO2007010552A3 (fr) * 2005-03-17 2007-07-12 Serum Inst India Ltd Conjugue d'erythropoietine peg n-terminal
WO2008019214A1 (fr) * 2006-08-04 2008-02-14 Prolong Pharmaceuticals, Inc. Erythropoïétine modifiée
US8765924B2 (en) 2006-08-04 2014-07-01 Prolong Pharmaceuticals, Inc. Modified erythropoietin
CN101889023B (zh) * 2007-07-17 2013-03-20 弗·哈夫曼-拉罗切有限公司 Peg化的多肽的纯化
RU2476439C2 (ru) * 2007-07-17 2013-02-27 Ф.Хоффманн-Ля Рош Аг Очистка пегилированных полипептидов
WO2009010270A3 (fr) * 2007-07-17 2009-04-30 Hoffmann La Roche Purification de polypeptides polyéthylène-glycolés
US8889837B2 (en) 2007-07-17 2014-11-18 Hoffman-La Roche Inc. Purification of pegylated polypeptides
US8795533B2 (en) 2007-07-17 2014-08-05 Hoffmann-La Roche Inc. Chromatographic methods
US8138317B2 (en) 2007-07-17 2012-03-20 Hoffmann-La Roche Inc. Purification of pegylated polypeptides
US8383114B2 (en) 2007-09-27 2013-02-26 Amgen Inc. Pharmaceutical formulations
US9320797B2 (en) 2007-09-27 2016-04-26 Amgen Inc. Pharmaceutical formulations
US10653781B2 (en) 2007-09-27 2020-05-19 Amgen Inc. Pharmaceutical formulations
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EP2803675A2 (fr) 2008-01-25 2014-11-19 Amgen, Inc Anticorps anti-ferroportine et procédés d'utilisation
EP2574628A1 (fr) 2008-01-25 2013-04-03 Amgen Inc. Anticorps de ferroportine et procédés d'utilisation
US9688759B2 (en) 2008-01-25 2017-06-27 Amgen, Inc. Ferroportin antibodies and methods of use
WO2009094551A1 (fr) 2008-01-25 2009-07-30 Amgen Inc. Anticorps anti-ferroportine et procédés d'utilisation
US9175078B2 (en) 2008-01-25 2015-11-03 Amgen Inc. Ferroportin antibodies and methods of use
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EP3693014A1 (fr) 2008-11-13 2020-08-12 The General Hospital Corporation Procédés et compositions pour la régulation de l'homéostasie du fer par modulation de la protéine bmp-6
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WO2011050333A1 (fr) 2009-10-23 2011-04-28 Amgen Inc. Adaptateur de fiole et système
WO2011077067A1 (fr) * 2009-12-21 2011-06-30 Polytherics Limited Conjugués polymères d'érythropoïétine non glycosylée
WO2011156373A1 (fr) 2010-06-07 2011-12-15 Amgen Inc. Dispositif d'administration de médicament
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US11458247B2 (en) 2012-11-21 2022-10-04 Amgen Inc. Drug delivery device
WO2014081780A1 (fr) 2012-11-21 2014-05-30 Amgen Inc. Dispositif d'administration de médicament
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US10682474B2 (en) 2012-11-21 2020-06-16 Amgen Inc. Drug delivery device
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US11439745B2 (en) 2012-11-21 2022-09-13 Amgen Inc. Drug delivery device
US11344681B2 (en) 2012-11-21 2022-05-31 Amgen Inc. Drug delivery device
US9657098B2 (en) 2013-03-15 2017-05-23 Intrinsic Lifesciences, Llc Anti-hepcidin antibodies and uses thereof
WO2014144096A1 (fr) 2013-03-15 2014-09-18 Amgen Inc. Cartouche à médicament, auto-injecteur et système d'auto-injection
US10239941B2 (en) 2013-03-15 2019-03-26 Intrinsic Lifesciences Llc Anti-hepcidin antibodies and uses thereof
US9803011B2 (en) 2013-03-15 2017-10-31 Intrinsic Lifesciences Llc Anti-hepcidin antibodies and uses thereof
EP3593839A1 (fr) 2013-03-15 2020-01-15 Amgen Inc. Cassette de médicaments
EP3831427A1 (fr) 2013-03-22 2021-06-09 Amgen Inc. Injecteur et procédé d'assemblage
WO2014149357A1 (fr) 2013-03-22 2014-09-25 Amgen Inc. Injecteur et procédé d'assemblage
WO2015061389A1 (fr) 2013-10-24 2015-04-30 Amgen Inc. Système de distribution de médicaments équipé d'un dispositif de commande sensible à la température
WO2015061386A1 (fr) 2013-10-24 2015-04-30 Amgen Inc. Injecteur et procédé d'assemblage
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WO2015119906A1 (fr) 2014-02-05 2015-08-13 Amgen Inc. Système d'administration de médicament doté d'un générateur de champ électromagnétique
EP3785749A1 (fr) 2014-05-07 2021-03-03 Amgen Inc. Auto-injecteur doté d'éléments de réduction de choc
WO2015171777A1 (fr) 2014-05-07 2015-11-12 Amgen Inc. Auto-injecteur comprenant des éléments de réduction de choc
US11738146B2 (en) 2014-06-03 2023-08-29 Amgen Inc. Drug delivery system and method of use
EP4036924A1 (fr) 2014-06-03 2022-08-03 Amgen, Inc Dispositifs et procédés destinés à aider un utilisateur d'un dispositif d'administration de médicaments
WO2015187793A1 (fr) 2014-06-03 2015-12-10 Amgen Inc. Système d'administration de médicament et son procédé d'utilisation
WO2015187797A1 (fr) 2014-06-03 2015-12-10 Amgen Inc. Système d'administration de médicament pouvant être commandé et son procédé d'utilisation
WO2015187799A1 (fr) 2014-06-03 2015-12-10 Amgen Inc. Systèmes et procédés pour traiter à distance des données collectées par un dispositif d'administration de médicament
US11213624B2 (en) 2014-06-03 2022-01-04 Amgen Inc. Controllable drug delivery system and method of use
WO2016049036A1 (fr) 2014-09-22 2016-03-31 Intrinsic Lifesciences Llc Anticorps anti-hepcidine humanisés et utilisations de ceux-ci
US10323088B2 (en) 2014-09-22 2019-06-18 Intrinsic Lifesciences Llc Humanized anti-hepcidin antibodies and uses thereof
WO2016061220A2 (fr) 2014-10-14 2016-04-21 Amgen Inc. Dispositif d'injection de médicament comportant des témoins visuels et sonores
EP3943135A2 (fr) 2014-10-14 2022-01-26 Amgen Inc. Dispositif d'injection de médicament avec indicateurs visuels et audibles
WO2016100781A1 (fr) 2014-12-19 2016-06-23 Amgen Inc. Dispositif d'administration de médicament doté d'un capteur de proximité
US11357916B2 (en) 2014-12-19 2022-06-14 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
EP3689394A1 (fr) 2014-12-19 2020-08-05 Amgen Inc. Dispositif d'administration de médicaments avec bouton mobile ou panneau d'interface utilisateur
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
WO2016100055A1 (fr) 2014-12-19 2016-06-23 Amgen Inc. Dispositif d'administration de médicament ayant un bouton direct ou un champ d'interface utilisateur
EP3848072A1 (fr) 2014-12-19 2021-07-14 Amgen Inc. Dispositif d'administration de médicament comportant un capteur de proximité
EP3556411A1 (fr) 2015-02-17 2019-10-23 Amgen Inc. Dispositif d'administration de médicaments avec fixation et/ou de rétroaction assistée(s) sous vide
EP3981450A1 (fr) 2015-02-27 2022-04-13 Amgen, Inc Dispositif d'administration de médicament ayant un mécanisme de protection d'aiguille présentant un seuil réglable de résistance au mouvement de l'élément de protection d'aiguille
WO2017039786A1 (fr) 2015-09-02 2017-03-09 Amgen Inc. Adaptateur d'ensemble de seringue pour une seringue
WO2017100501A1 (fr) 2015-12-09 2017-06-15 Amgen Inc. Auto-injecteur avec capuchon de signalisation
WO2017120178A1 (fr) 2016-01-06 2017-07-13 Amgen Inc. Auto-injecteur pourvu d'une électronique de signalisation
WO2017160799A1 (fr) 2016-03-15 2017-09-21 Amgen Inc. Réduction de la probabilité de casse du verre dans des dispositifs d'administration de médicament
EP4035711A1 (fr) 2016-03-15 2022-08-03 Amgen Inc. Réduction de la probabilité de rupture de verre dans des dispositifs d'administration de médicament
EP3721922A1 (fr) 2016-03-15 2020-10-14 Amgen Inc. Réduction de la probabilité de rupture de verre dans des dispositifs d'administration de médicament
WO2017189089A1 (fr) 2016-04-29 2017-11-02 Amgen Inc. Dispositif d'administration de médicament avec étiquette de messagerie
WO2017192287A1 (fr) 2016-05-02 2017-11-09 Amgen Inc. Adaptateur de seringue et guide pour remplir un injecteur sur le corps
WO2017197222A1 (fr) 2016-05-13 2017-11-16 Amgen Inc. Ensemble manchon de flacon
WO2017200989A1 (fr) 2016-05-16 2017-11-23 Amgen Inc. Chiffrement de données dans des dispositifs médicaux à capacité de calcul limitée
WO2017209899A1 (fr) 2016-06-03 2017-12-07 Amgen Inc. Appareils et procédés d'essai au choc destinés aux dispositifs d'administration de médicaments
WO2018004842A1 (fr) 2016-07-01 2018-01-04 Amgen Inc. Dispositif d'administration de médicament présentant un risque réduit au minimum de fracture de composant lors d'événements d'impact
WO2018034784A1 (fr) 2016-08-17 2018-02-22 Amgen Inc. Dispositif d'administration de médicament avec détection de positionnement.
WO2018081234A1 (fr) 2016-10-25 2018-05-03 Amgen Inc. Injecteur porté sur le corps
WO2018136398A1 (fr) 2017-01-17 2018-07-26 Amgen Inc. Dispositifs d'injection et procédés d'utilisation et d'assemblage associés
WO2018151890A1 (fr) 2017-02-17 2018-08-23 Amgen Inc. Dispositif d'administration de médicament à trajet d'écoulement de fluide stérile et procédé d'assemblage associé
WO2018152073A1 (fr) 2017-02-17 2018-08-23 Amgen Inc. Mécanisme d'insertion pour dispositif d'administration de médicament
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WO2020092056A1 (fr) 2018-11-01 2020-05-07 Amgen Inc. Dispositifs d'administration de médicament à rétraction d'aiguille partielle
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WO2022033480A1 (fr) * 2020-08-11 2022-02-17 隆延生物科技（上海）有限公司 Préparation liquide et son application
WO2022246055A1 (fr) 2021-05-21 2022-11-24 Amgen Inc. Procédé d'optimisation d'une recette de remplissage pour un récipient de médicament

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