WO2022063082A1 - 一种糖基化修饰的促红细胞生成素及其应用 - Google Patents

一种糖基化修饰的促红细胞生成素及其应用 Download PDF

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WO2022063082A1
WO2022063082A1 PCT/CN2021/119332 CN2021119332W WO2022063082A1 WO 2022063082 A1 WO2022063082 A1 WO 2022063082A1 CN 2021119332 W CN2021119332 W CN 2021119332W WO 2022063082 A1 WO2022063082 A1 WO 2022063082A1
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glycosylation
modified erythropoietin
erythropoietin
ratio
modified
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PCT/CN2021/119332
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English (en)
French (fr)
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朱建伟
江华
谢跃庆
杨开勇
肖晓东
王振玉
王曼
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美国杰科实验室有限公司
杰科（天津）生物医药有限公司
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Priority to EP21871452.5A priority Critical patent/EP4219537A1/en
Priority to US18/027,406 priority patent/US20230331798A1/en
Priority to CN202180064537.2A priority patent/CN116194585A/zh
Publication of WO2022063082A1 publication Critical patent/WO2022063082A1/zh

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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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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    • C12P21/005—Glycopeptides, glycoproteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
    • C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present application relates to the field of biomedicine, in particular to a glycosylation-modified erythropoietin and its application.
  • Erythropoietin is a glycoprotein hormone, which is one of the members of the hematopoietic cytokine superfamily. EPO exerts its biological effects by binding to the cell surface transmembrane receptor EPO receptor.
  • EPO When EPO binds to the EPO receptor, it can cause the conformation of the receptor to change, promote the adjacent two EPO receptors to approach each other to form a homodimer, and activate the JAK2 tyrosine kinase bound to the cytoplasmic near-membrane region of the receptor , resulting in the phosphorylation of multiple tyrosine residues of the receptor, and JAK2 can further phosphorylate STAT to initiate the expression of related genes, thereby activating multiple downstream signaling pathways and causing the proliferation and differentiation of the erythrocyte system.
  • Clinical experiments have proved that EPO has a therapeutic effect on a variety of diseases, mainly used for anemia caused by various reasons.
  • Some therapeutic erythropoietin drugs have the defects of short half-life and high frequency of clinical administration, which seriously affects the use of patients. The development of erythropoietin with a long half-life is imminent.
  • the present application provides a glycosylation-modified erythropoietin and an application thereof, including the glycosylation-modified erythropoietin and its preparation method, pharmaceutical composition, and simultaneously
  • the application in the preparation of a medicine for treating anemia and a method for prolonging the half-life of erythropoietin are provided.
  • the application provides a glycosylation-modified erythropoietin containing a glycan structure bound to an N-glycosylation site, the glycan structure comprising FA4G4L2S4, wherein F represents fucose and A represents N -Acetylglucosamine, G for galactose, L for lactose, S for sialic acid.
  • the ratio of the FA4G4L2S4 structure is 15% or more.
  • the glycan structure comprises FA4G4L1S4, wherein F represents fucose, A represents N-acetylglucosamine, G represents galactose, L represents lactose, and S represents sialic acid.
  • the ratio of FA4G4L1S4 is 20% or more.
  • the glycan structure comprises FA4G4S4, wherein F represents fucose, A represents N-acetylglucosamine, G represents galactose, and S represents sialic acid.
  • the ratio of FA4G4S4 is 10% or more.
  • the glycan structure comprises Neu5Gc, and the molar ratio of the Neu5Gc is 0.5% or less.
  • the glycosylation-modified erythropoietin comprises the amino acid sequence shown in SEQ ID NO.1.
  • the glycosylation-modified erythropoietin comprises the following N-glycosylation sites: N24, N30, N38, N83, and N88.
  • the present application provides a method for preparing the glycosylation-modified erythropoietin, comprising the following steps: under the condition of the glycosylation-modified erythropoietin, culturing the erythropoietin containing the CHO-S cells of the nucleic acid molecule of the amino acid sequence shown in SEQ ID NO: 1.
  • the present application provides a pharmaceutical composition comprising the glycosylation-modified erythropoietin and a pharmaceutically acceptable adjuvant.
  • the present application provides the use of the glycosylation-modified erythropoietin and/or the pharmaceutical composition for preparing a medicament for treating anemia.
  • the present application provides the use of the glycosylation-modified erythropoietin and/or the pharmaceutical composition in the preparation of a medicament for the treatment of anemia, and the anemia includes renal anemia , multiple myeloma anemia and/or cancer anemia.
  • the application provides a method of treating anemia comprising administering the glycosylation-modified erythropoietin and/or the pharmaceutical composition in a therapeutically effective amount to a subject in need thereof.
  • the application provides a method of treating anemia comprising administering the glycosylation-modified erythropoietin and/or the pharmaceutical composition to a subject in need thereof in a therapeutically effective amount, the anemia
  • the anemia include renal anemia, multiple myeloma anemia, and/or cancer anemia.
  • the present application provides the glycosylation-modified erythropoietin and/or the pharmaceutical composition for treating anemia.
  • the present application provides the glycosylation-modified erythropoietin and/or the pharmaceutical composition for treating anemia, the anemia including renal anemia, multiple myeloma anemia and/or or cancerous anemia.
  • the present application provides a method for prolonging the half-life of erythropoietin, comprising the steps of: administering the glycosylation-modified erythropoietin to a subject in need thereof.
  • the glycosylation-modified erythropoietin provided by the present application has the beneficial effect of increasing hemoglobin content, erythrocyte level, hematocrit and/or reticulocyte level by increasing the half-life.
  • Figure 1 shows a schematic structural diagram of the fragment of the glycosylation-modified erythropoietin expression vector described in the present application.
  • Figure 2 shows the immunoblot results of the glycosylation-modified erythropoietin described in the present application.
  • the first lane is a protein marker of known molecular weight
  • the second, fourth and sixth lanes are respectively the glycosylation-modified erythropoietin of the present application.
  • Three batches of purified erythropoietin products, lot#20190302-2, lot#20190303-2, lot#20190304-1, and lanes 3, 5, and 7 are the control group Darbepoetin (Darbe, lot#1078765A).
  • Figure 3 shows the IEF detection results of the glycosylation-modified erythropoietin described in the present application.
  • the first swimming lane is the control group Darbepoetin (Darbe, lot #1078765A), and the second, third and fourth swimming lanes are the sugars of the application.
  • Figure 4 shows the CZE detection results of the glycosylation-modified erythropoietin described in the present application.
  • the first row is the control group Darbepoetin (Darbe, lot #1078765A), and the second, third and fourth rows are the glycosyl groups of the present application.
  • Figure 5 shows the detection results of the N-linked glycosylation composition of the glycosylation-modified erythropoietin described in the present application, and the first, second, and third rows are respectively the glycosylation-modified erythropoietin of the present application.
  • Three batches of purified products were lot#20190302-2, lot#20190303-2, and lot#20190304-1, and the fourth row was the control group Darbepoetin (Darbe, lot#1078765A).
  • Figure 6 shows the results of the glycosylation-modified erythropoietin described in this application on the proliferation of TF-1 cells
  • 1 represents the control group Darbepoetin (Darbe, lot #1078765A)
  • 2, 3, and 4 represent the application, respectively
  • Figure 7 shows the results of the in vivo efficacy test of the glycosylation-modified erythropoietin described in the present application
  • 1 represents the blank group (vehicle group)
  • 2 represents the control group A (Darbepoetin, Darbe, lot #1078765A)
  • 3 Indicates control group B (EPO, Lixuebao, lot#17Y03B)
  • 4, 5, and 6 are the three batches of purified products of glycosylation-modified erythropoietin, lot#20190302-2, lot#20190303-2, respectively.
  • Figure 8 shows the half-life test results of the glycosylation-modified erythropoietin described in this application
  • 1 represents control group A (Darbepoetin, Darbe, lot #1078765A)
  • 2, 3, and 4 represent glycosylation of the application, respectively.
  • Three batches of purified products of modified erythropoietin, lot#20190302-2, lot#20190303-2, lot#20190304-1, and 5 represent control group B (EPO, Lixuebao, lot#17Y03B).
  • T 1/2 half-life
  • glycan structure generally refers to polysaccharides or oligosaccharides, ie polymeric compounds that upon acid hydrolysis yield multiple monosaccharides.
  • Glycosylation-modified proteins may comprise covalent coupling via asparagine or arginine ("N-linked glycosylation") or via serine or threonine ("O-linked glycosylation").
  • One or more glycan structures of the side groups of a polypeptide chain may comprise covalent coupling via asparagine or arginine ("N-linked glycosylation") or via serine or threonine ("O-linked glycosylation").
  • the FA4G4L2S4 glycan structure attached to the N-glycosylation site of erythropoietin can be the FA4G4L1S4 glycan structure attached to the N-glycosylation site of erythropoietin
  • it can be the FA4G4S4 glycan structure Attached to the N-glycosylation site of erythropoietin can be the glycan structure Neu5Gc attached to the N-glycosylation site of erythropoietin.
  • Glycan structures are classified into two-, three-, and four-antenna structures according to their branching (antenna) numbers.
  • the glycan structure is composed of various monosaccharides, according to Oxford nomenclature, including fucose (Fucose, referred to as Fuc or F), N-acetylglucosamine (N-Acetylglucosamine, referred to as GlcNAc, Gn or A), galactose ( Galactose, referred to as Gal or G), lactose (Lactose, referred to as Lac or L), mannose (Mannose, referred to as Man or M), N-acetylneuraminic acid (sialic acid, N-Acetylneuraminic, referred to as NANA, Neu5Ac or S) and/or N-glycolylneuraminic acid (N-Glycolylneuraminic, referred to as NGNA, Neu5Glc or Neu5Gc).
  • fucose Flucose, referred to as Fuc or F
  • the term “FA4G4L2S4" refers to a four-antennary glycan structure containing fucosylated, 4 N-acetylglucosamine, 4 galactose, 2 lactose, 4 sialic acids, where F represents a fucose-containing glycan structure Modification, A represents N-acetylglucosamine, G represents galactose, L represents lactose, S represents sialic acid, the number after A represents the number of N-acetylglucosamine on a glycan structure, the number after G The number represents the number of the galactose on a glycan structure, the number after L represents the number of the lactose on a glycan structure, and the number after S represents the number of the sialic acid on a glycan structure;
  • the term “FA4G4L1S4" refers to a four-antennary glycan structure containing fucosylated, 4 N
  • G represents galactose
  • S represents sialic acid
  • the number after A represents the number of N-acetylglucosamine on a glycan structure
  • the number after G represents the amount of galactose on a glycan structure.
  • Number, the number after S represents the number of said sialic acid on a glycan structure; the term "Neu5Gc" refers to N-glycolylneuraminic acid.
  • N-glycosylation site generally refers to a site on a glycosylation-modified protein that contains asparagine or arginine to covalently attach glycan structures, such as N-glycans
  • the sylation site can be an asparagine residue used to covalently link the glycan structure to the glycosylation-modified protein.
  • the N-glycosylation site can be the asparagine at positions 24, 30, 38, 83 and 88 on the glycosylated modified erythropoietin. , referred to as Asn or N) residues.
  • binding of the glycan structure to the N-glycosylation site generally refers to the connection between the glycan structure and the N-glycosylation site on the glycosylation-modified protein. physical or chemical interaction.
  • binding can be direct or indirect attachment or attachment, indirect attachment or attachment through another biomolecule or compound, direct covalent (eg, through chemical coupling) or non-covalent (such as ionic interactions, hydrophobic interactions, hydrogen bonding, etc.) binding or attachment.
  • the binding can be a covalent link between the glycan structure and the N-glycosylation site on the glycosylation-modified protein, or the binding can be aspartate between the monosaccharide of the glycan structure and the N-glycosylation site.
  • the free -NH2 group of the amide residue is covalently attached.
  • the "ratio" of the glycan structure in the glycosylation-modified erythropoietin generally refers to the glycan structure in the glycosylation-modified erythropoietin
  • the molar ratio of the glycosylation-modified erythropoietin can be analyzed by mass spectrometry after enzymatic hydrolysis of the glycosylation-modified erythropoietin.
  • Exemplary mass spectrometry methods can include mass spectrometry coupled with HPLC.
  • the ratio of the FA4G4L2S4 structure can be more than 15%, the ratio of the FA4G4L1S4 can be more than 20%, the ratio of the FA4G4S4 can be more than 10%,
  • the molar ratio of Neu5Gc may be 0.5% or less.
  • the molar ratio can be the number of moles of FA4G4L2S4 structures/the number of moles of all glycan structures in the glycosylated erythropoietin, which can be the number of moles of FA4G4L1S4/the number of moles of all glycans in the glycosylated erythropoietin
  • the number of moles of structures which can be the number of moles of FA4G4S4/the number of moles of all glycan structures in the glycosylated erythropoietin, can be the number of moles of Neu5Gc/the number of moles of all glycans in the glycosylated erythropoietin
  • the number of moles of the structure can be the number of moles of the structure.
  • protein generally refers to a polymer of amino acid residues that is not limited to a minimum length. Polypeptides, peptides, oligopeptides, dimers, multimers and analogs are all included in this definition. Intact proteins and fragments thereof are also included in this definition. The term also includes post-expression modified forms of the protein including, but not limited to, glycosylation, acetylation, phosphorylation, and the like. For example, in the present application, protein may refer to glycosylation-modified erythropoietin and fragments thereof.
  • a cell "CHO-S cell” generally refers to a CHO-S Chinese hamster ovary cell into which a nucleic acid encoding a heterologous polypeptide, for example, can be introduced, eg, by transfection.
  • the CHO-S cells include variant progeny that have the same function or biological activity as screened in the original transfected cell.
  • erythropoietin and its abbreviation “EPO” generally refers to any erythropoietin polypeptide, including, but not limited to, recombinantly produced erythropoietin polypeptide, synthetically produced erythropoietin EPO polypeptides, native EPO polypeptides, erythropoietin polypeptides extracted from cells and tissues including but not limited to kidney, liver, urine and blood.
  • the erythropoietin can be erythropoietin having the amino acid sequence of SEQ ID No: 1.
  • erythropoietin also refers to a variant of the protein of SEQ ID No: 1 in which one or more amino acid residues have been altered, deleted or inserted, and which have the same biological activity as the unmodified protein, such as in As reported in EP 1 064 951 or US 6,583,272.
  • the biological activity resulting from the binding of erythropoietin to the EPO receptor may include: administration of erythropoietin to a subject by injection results in an increase in bone marrow cells of reticulocytes compared to a non-injected group or a control group of individuals and production of red blood cells.
  • glycosylation modification generally means that a carbohydrate moiety can be attached at one or more amino positions in a protein or polypeptide.
  • glycosylation-modified proteins or polypeptides contain one or more amino acid residues, such as arginine or asparagine, to which carbohydrate moieties are attached.
  • the glycosylation modification can be an N-linked glycoprotein.
  • N-linked glycoproteins can comprise glycan structures that bind to N-glycosylation sites, eg, glycan structures that are linked to N-glycosylation sites of asparagine residues in the protein.
  • glycoproteins include the following groups: glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and/or sialic acid.
  • the glycosylation modification of erythropoietin can be a FA4G4L2S4-containing glycan structure linked to the N-glycosylation site of erythropoietin, and can be a FA4G4L1S4-containing glycan structure linked to the N-glycosylation site of erythropoietin
  • the glycosylation site may be a FA4G4S4-containing glycan structure linked to the N-glycosylation site of erythropoietin, or a Neu5Gc-containing glycan structure linked to the N-glycosylation site of erythropoietin.
  • the ratio of the FA4G4L2S4 structure is 15% "above", which generally means that the ratio of the FA4G4L2S4 glycan structure can be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% glycosylation-modified erythropoietin.
  • the glycosylation-modified erythropoietin may comprise a ratio of FA4G4L2S4 glycan structures of at least 18.17%.
  • the ratio of the FA4G4L12S4 structure is 20% "above", which generally means that the ratio of the FA4G4L1S4 glycan structure can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80% %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% glycosylated erythropoietin.
  • a glycosylation-modified erythropoietin can be comprised of a ratio of FA4G4L1S4 glycan structures of at least 21.58%.
  • the ratio of the FA4G4S4 structure is 10% "above", which generally means that the ratio of the FA4G4S4 glycan structure can be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60% %, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% glycosylation-modified erythropoietin.
  • the glycosylation-modified erythropoietin may comprise a ratio of FA4G4S4 glycan structures of at least 14.02%.
  • the molar ratio of Neu5Gc is 0.5% "below", which generally means that the molar ratio of Neu5Gc can be at most 0.5%, at most 0.4%, at most 0.3%, at most 0.2%, at most 0.1%, at most 0.05%, Up to 0.02%, up to 0.01%, or 0% glycosylation-modified erythropoietin.
  • it may be erythropoietin whose glycan structure does not contain a glycosylation modification of Neu5Gc.
  • extending the half-life of erythropoietin generally means that the glycosylation-modified erythropoietin of the present application has increased resistance to proteases, which can cause the glycosylation-modified erythropoietin to be different from non-glycosylated erythropoietin.
  • EPO with a glycosylated form or glycosylated EPO with other glycan structures has an increased half-life compared to in vitro (eg, during production, purification, and storage) or in vivo (eg, after administration to a subject).
  • the glycosylation-modified erythropoietin of the present application exhibits an increase in half-life after administration to a subject, compared to unmodified EPO or glycosylated EPO with other glycan structures, the glycosylated erythropoietin of the present application exhibits an increase in half-life
  • the increased half-life of the sylation-modified erythropoietin can be increased by at least about or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%, 200%, 300%, 400%, 500% or more.
  • the increased half-life of the glycosylation-modified erythropoietins of the present application can be increased by at least about or at least 6-fold, 7-fold, 8-fold, compared to unmodified EPO or glycosylated EPO with other glycan structures times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more.
  • the present application provides a glycosylation-modified erythropoietin comprising 5 glycosylation sites: N24, N30, N38, N83 and N88. Glycosylation sites can be used to N-link the sugar chains of the glycan structures described herein.
  • the glycosylation-modified erythropoietin described in this application is expressed by transfecting a nucleic acid molecule encoding erythropoietin having an amino acid sequence such as SEQ ID No: 1 into CHO-S cells.
  • glycosylation-modified erythropoietin does not enhance the binding to the EPO receptor, and in some cases, the binding force to the EPO receptor may be reduced due to the sugar chain structure described in the present application.
  • the glycosylation-modified erythropoietin described in this application can increase half-life, thereby leading to increased biological activity in vivo, and increased hemoglobin content, erythrocyte level, hematocrit, and reticulocyte level.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 glycan structures.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 structures, and the ratio of the FA4G4L2S4 structures may be more than 15%.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% glycosylation-modified erythropoiesis White.
  • the ratio of glycan structures comprising FA4G4L2S4 may be at least 18.17% glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L1S4 glycan structures.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L1S4 structures, and the ratio of the FA4G4L1S4 may be more than 20%.
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% % or at least 99% glycosylation-modified erythropoietin.
  • the ratio of glycan structures comprising FA4G4L1S4 can be at least 21.58% glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4S4 glycan structures.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4S4 structures, and the ratio of the FA4G4S4 may be more than 10%.
  • the ratio of FA4G4S4 glycan structures can be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% glycosylation-modified erythropoietin.
  • the ratio of glycan structures comprising FA4G4S4 can be at least 14.02% glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may include Neu5Gc, and the molar ratio of the Neu5Gc may be 0.5% or less.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may include Neu5Gc, and the molar ratio of the Neu5Gc may be 0.5% or less.
  • the molar ratio of Neu5Gc can be at most 0.5%, at most 0.4%, at most 0.3%, at most 0.2%, at most 0.1%, at most 0.05%, at most 0.02%, at most 0.01%, or at most 0% glycosylated erythropoietic cells Generative.
  • it may be erythropoietin whose glycan structure does not contain a glycosylation modification of Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 glycan structures and one or more FA4G4L1S4 glycan structures.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 structures and one or more FA4G4L1S4 structures, the ratio of the FA4G4L2S4 structures may be more than 15% and the ratio of the FA4G4L1S4 structures may be more than 20%.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and the ratio of FA4G4L1S4 glycan structures Can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, At least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sugar base modified erythropo
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17%; and the ratio comprising FA4G4L1S4 glycan structures can be at least 21.58% glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structures may comprise one or more FA4G4L2S4 glycan structures and one or more FA4G4S4 glycan structures.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 structures and one or more FA4G4S4 structures, the ratio of the FA4G4L2S4 structures may be more than 15% and the ratio of the FA4G4S4 structures may be more than 10%.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and the ratio of FA4G4S4 glycan structures can be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% % or at least 99%
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17%; and the ratio comprising FA4G4S4 glycan structures can be at least 14.02% glycosylated erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L1S4 glycan structures and one or more FA4G4S4 glycan structures.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L1S4 structures and one or more FA4G4S4 structures, the ratio of the FA4G4L1S4 may be more than 20% and the ratio of the FA4G4S4 may be more than 10%.
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and the ratio of FA4G4S4 glycan structures may be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18% %, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, At least 95%, at least 96%, at least 97%
  • the ratio comprising FA4G4L1S4 glycan structures can be at least 21.58%; and the ratio comprising FA4G4S4 glycan structures can be at least 14.02% glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 glycan structures and one or more Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 structures and one or more Neu5Gcs, the ratio of the FA4G4L2S4 may be 15% or more and the molar ratio of the Neu5Gc may be 0.5% or less.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and the molar ratio of Neu5Gc may be Up to 0.5%, up to 0.4%, up to 0.3%, up to 0.2%, up to 0.1%, up to 0.05%, up to 0.02%, up to 0.01%, or 0% glycosylated modified erythropoietin.
  • the ratio of glycan structures comprising FA4G4L2S4 may be at least 18.17%; and the glycan structures may be glycosylated erythropoietins that do not comprise Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structures may comprise one or more FA4G4L1S4 glycan structures and one or more Neu5Gc glycan structures.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L1S4 structures and one or more Neu5Gc, the ratio of the FA4G4L1S4 may be 20% or more and the molar ratio of the Neu5Gc may be 0.5% or less.
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and the molar ratio of Neu5Gc may be at most 0.5%, at most 0.4%, at most 0.3%, at most 0.2%, at most 0.1%, at most 0.05%, at most 0.02%, at most 0.01% or 0% sugar Modified erythropoietin.
  • the ratio of glycan structures comprising FA4G4L1S4 may be at least 21.58%; and may be erythropoietin whose glycan structures do not comprise a glycosylation modification of Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structures may comprise one or more FA4G4L2S4 glycan structures, one or more FA4G4L1S4 glycan structures, and one or more FA4G4S4 glycan structures.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 structures, one or more FA4G4L1S4 structures, and one or more FA4G4S4 structures, and the ratio of the FA4G4L2S4 structures may be 15% or more, and the FA4G4L1S4 ratio may be 20% or more And the ratio of FA4G4S4 can be more than 10%.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%;
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and FA4G4S4
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17%, the ratio comprising FA4G4L1S4 glycan structures can be at least 21.58%; and the ratio comprising FA4G4S4 glycan structures can be at least 14.02% glycosylation-modified promoting Erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structures may comprise one or more FA4G4L2S4 glycan structures, one or more FA4G4L1S4 glycan structures, and one or more Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 structures, one or more FA4G4L1S4 structures, and one or more Neu5Gcs, the ratio of the FA4G4L2S4 structures may be more than 15%, the ratio of the FA4G4L1S4 may be more than 20%, and The molar ratio of Neu5Gc may be 0.5% or less.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%;
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and Neu5Gc The
  • the ratio of glycan structures comprising FA4G4L2S4 may be at least 18.17%, the ratio of glycan structures comprising FA4G4L1S4 may be at least 21.58%; and the glycan structures may be glycosylated erythropoietins that do not comprise Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structures may comprise one or more FA4G4L2S4 glycan structures, one or more FA4G4L1S4 glycan structures, one or more FA4G4S4 glycan structures, and one or more Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise one or more FA4G4L2S4 structures, one or more FA4G4L1S4 structures, one or more FA4G4S4 structures and one or more Neu5Gc, the ratio of the FA4G4L2S4 structures may be 15% or more, and the FA4G4L1S4
  • the ratio of FA4G4S4 may be more than 20%, the ratio of FA4G4S4 may be more than 10%, and the molar ratio of Neu5Gc may be less than 0.5%.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%;
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; FA4G4S4 poly
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17%, the ratio comprising FA4G4L1S4 glycan structures can be at least 21.58%; the ratio comprising FA4G4S4 glycan structures can be at least 14.02%; and can be glycan structures Glycosylation-modified erythropoietin that does not contain Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure can be bound directly or indirectly to the N-glycosylation site.
  • the glycan structure can be covalently bound directly to the N-glycosylation site.
  • the monosaccharide of the glycan structure can covalently bind directly to the free -NH2 group of the asparagine residue of the N-glycosylation site.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the ratio of the FA4G4L2S4 structure can be more than 15%.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the glycosylation modifications can be bound to the The N-glycosylation site of the glycosylation-modified erythropoietin.
  • a glycosylation modification comprising a ratio of FA4G4L2S4 glycan structures may be at least 18.17% may bind to the N-glycosylation site of the glycosylated modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L1S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L1S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the ratio of the FA4G4L1S4 can be more than 20%.
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% % or at least 99% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • a glycosylation modification comprising a ratio of FA4G4L1S4 glycan structures can be at least 21.58% can bind to the N-glycosylation site of the glycosylated modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the ratio of the FA4G4S4 can be more than 10%.
  • the ratio of FA4G4S4 glycan structures can be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • a glycosylation modification comprising a ratio of FA4G4S4 glycan structures can be at least 14.02% can bind to the N-glycosylation site of the glycosylated modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise Neu5Gc, and the glycan structure may bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the glycan structure may comprise Neu5Gc, and the glycan structure may bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, the The molar ratio of Neu5Gc may be 0.5% or less.
  • the molar ratio of Neu5Gc can be at most 0.5%, at most 0.4%, at most 0.3%, at most 0.2%, at most 0.1%, at most 0.05%, at most 0.02%, at most 0.01%, or 0% of the glycan structures can be bound to the The N-glycosylation site of the glycosylation-modified erythropoietin.
  • the molar ratio of Neu5Gc can be 0% and the glycan structure can bind to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure and the FA4G4L1S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure and the FA4G4L1S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83, and N88, and the ratio of the FA4G4L2S4 structure can be is 15% or more and the ratio of the FA4G4L1S4 can be 20% or more.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the glycosylation modifications can be bound to the N-glycosylation sites of the glycosylation-modified erythropoietin; and the ratio of FA4G4L1S4 glycan structures may be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25% %, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least at least
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylated modified erythropoietin; and the ratio comprising FA4G4L1S4 glycan structures
  • the glycosylation modification which may be at least 21.58%, may be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure and the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure and the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83, and N88, and the ratio of the FA4G4L2S4 structure can be is 15% or more and the ratio of the FA4G4S4 may be 10% or more.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the glycosylation modifications can be bound to the N-glycosylation sites of the glycosylation-modified erythropoietin; and the ratio of FA4G4S4 glycan structures may be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15% %, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%,
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylated modified erythropoietin; and the ratio comprising FA4G4S4 glycan structures
  • the glycosylation modification which may be at least 14.02%, may be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L1S4 structure and the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L1S4 structure and the FA4G4S4 structure may bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the ratio of the FA4G4L1S4 may be Above 20% and the ratio of the FA4G4S4 may be above 10%.
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% % or at least 99% of the glycosylation modifications may be bound to the N-glycosylation site of the glycosylation-modified erythropoietin; and the ratio of FA4G4S4 glycan structures may be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 20%
  • the ratio comprising FA4G4L1S4 glycan structures can be at least 21.58% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylated modified erythropoietin; and the ratio comprising FA4G4S4 glycan structures
  • the glycosylation modification which may be at least 14.02%, may be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the glycan structure can comprise Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the glycan structure can comprise Neu5Gc, the FA4G4L2S4 structure
  • the ratio of Neu5Gc may be 15% or more, and the molar ratio of Neu5Gc may be 0.5% or less.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the glycosylation modifications can be bound to the and the N-glycosylation site of the glycosylation-modified erythropoietin; and the molar ratio of Neu5Gc may be at most 0.5%, at most 0.4%, at most 0.3%, at most 0.2%, at most 0.1%, at most 0.05%, Up to 0.02%, up to 0.01%, or 0% of the glycan structure can be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • the ratio of glycan structures comprising FA4G4L2S4 can be at least 18.17% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylated modified erythropoietin; and the molar ratio of Neu5Gc can be 0 % of the glycan structure can be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L1S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the glycan structure can comprise Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L1S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the glycan structure can comprise Neu5Gc, the FA4G4L1S4 The ratio may be 20% or more, and the molar ratio of the Neu5Gc may be 0.5% or less.
  • the ratio of FA4G4L1S4 glycan structures can be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% % or at least 99% of the glycosylation modifications may be bound to the N-glycosylation site of the glycosylation-modified erythropoietin; and the molar ratio of Neu5Gc may be at most 0.5%, at most 0.4%, at most 0.3 %, at most 0.2%, at most 0.1%, at most 0.05%, at most 0.02%, at most 0.01%, or 0% of the glycan structures can be bound to the N-glycosylation site of the glyco
  • the ratio of glycan structures comprising FA4G4L1S4 can be at least 21.58% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylated modified erythropoietin; and the molar ratio of Neu5Gc can be 0 % of the glycan structure can be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure, the FA4G4L1S4 structure, and the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83, and N88.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure, the FA4G4L1S4 structure and the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, the The ratio of the FA4G4L2S4 structure may be 15% or more, the ratio of the FA4G4L1S4 may be 20% or more, and the ratio of the FA4G4S4 may be 10% or more.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the glycosylation modifications can be bound to the N-glycosylation sites of the glycosylation-modified erythropoietin; and the ratio of FA4G4L1S4 glycan structures may be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25% %, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least at least
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylated modified erythropoietin; and the ratio comprising FA4G4L1S4 glycan structures may be at least 21.58% glycosylation modification may be bound to the N-glycosylation site of said glycosylation-modified erythropoietin; and the ratio comprising FA4G4S4 glycan structures may be at least 14.02% glycosylation Modifications can be bound to the N-glycosylation site of the glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure and the FA4G4L1S4 structure may bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the glycan structure may comprise Neu5Gc .
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure and the FA4G4L1S4 structure may bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83 and N88, and the glycan structure may comprise Neu5Gc , the ratio of the FA4G4L2S4 structure may be more than 15%, the ratio of the FA4G4L1S4 may be more than 20%, and the molar ratio of the Neu5Gc may be less than 0.5%.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the glycosylation modifications can be bound to the N-glycosylation sites of the glycosylation-modified erythropoietin; and the ratio of FA4G4L1S4 glycan structures may be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25% %, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least at least
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylated modified erythropoietin; and the ratio comprising FA4G4L1S4 glycan structures At least 21.58% of the glycosylation modifications may be bound to the N-glycosylation site of the glycosylation-modified erythropoietin; and the molar ratio of Neu5Gc may be 0% of the glycan structures that may be bound to all The N-glycosylation site of the glycosylation-modified erythropoietin.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure, the FA4G4L1S4 structure, and the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83, and N88, and aggregate
  • the carbohydrate structure may comprise Neu5Gc.
  • the present application provides a glycosylation-modified erythropoietin glycan structure.
  • the FA4G4L2S4 structure, the FA4G4L1S4 structure, and the FA4G4S4 structure can bind to the N-glycosylation sites of the glycosylation-modified erythropoietin: N24, N30, N38, N83, and N88, and aggregate
  • the sugar structure may include Neu5Gc, the ratio of the FA4G4L2S4 structure may be 15% or more, the ratio of the FA4G4L1S4 may be 20% or more, and the ratio of the FA4G4S4 may be 10% or more.
  • the ratio of the FA4G4L2S4 structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the glycosylation modifications can be bound to the N-glycosylation sites of the glycosylation-modified erythropoietin; and the ratio of FA4G4L1S4 glycan structures may be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25% %, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least at least
  • the ratio comprising FA4G4L2S4 glycan structures can be at least 18.17% of the glycosylation modifications can be bound to the N-glycosylation site of the glycosylated modified erythropoietin; and the ratio comprising FA4G4L1S4 glycan structures may be at least 21.58% glycosylation modification may be bound to the N-glycosylation site of said glycosylation-modified erythropoietin; and the ratio comprising FA4G4S4 glycan structures may be at least 14.02% glycosylation A modification can bind to the N-glycosylation site of the glycosylation-modified erythropoietin; and the molar ratio of Neu5Gc can be 0% glycan structure can bind to the glycosylation-modified erythropoietin The N-glycosylation site of the prime.
  • the present application provides a method for preparing glycosylation-modified erythropoietin, comprising, under the condition of expressing the glycosylation-modified erythropoietin, culturing the erythropoietin encoding the glycosylation-modified erythropoietin.
  • Erythropoietin encoding an amino acid sequence such as SEQ ID No: 1 is inserted into an expression vector using a vector suitable for maintenance in mammalian host cells using standard techniques.
  • the vector typically contains the following elements suitable for use in mammalian host cells: a promoter and other "upstream" regulatory elements, an origin of replication, a ribosome binding site, a transcription termination site, a polylinker site, and a selectable marker.
  • the vector may also contain elements that also permit proliferation and maintenance in prokaryotic host cells.
  • suitable cells or cell lines include any cell or cell line of mammalian origin, including human origin, including Chinese hamster ovary cells CHO-S cells.
  • a nucleic acid molecule comprising a sequence encoding an amino acid sequence such as erythropoietin of SEQ ID No: 1 is introduced into a host cell using standard transformation or transfection techniques.
  • Culturing, amplifying and screening transformed host cells or transfected host cells are accomplished using publicly available methods (Gething et al., Nature 293, 620-625 (1981): Kaufman et al., Mol Cell. Biol. 5, 1750-1759 (1985) ); U.S. Patent No. 4,419,446).
  • the host cell containing the erythropoietin DNA coding sequence is cultured under conditions in which the analog is expressed.
  • the glycosylation-modified erythropoietin was recovered from the cell culture medium and purified using essentially the same method as previously described (WO 94/09257).
  • the purification method can isolate the glycosylation-modified erythropoietin described in this application.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the glycosylation-modified erythropoietin and pharmaceutically acceptable adjuvants such as diluents, carriers, solubilizers, emulsifiers, preservatives agents and/or adjuvants.
  • the pharmaceutical composition is suitable for a dosing regimen of less than three times a week.
  • the composition may be in liquid or lyophilized form, containing diluents (Tris, citrate, acetate or phosphate buffers), solubilizers such as Tween or polysorbate with varying pH and ionic strength Esters, carriers such as human serum albumin or gelatin, preservatives such as thimerosal, parabens or benzyl alcohol, antioxidants such as ascorbic acid or sodium metabisulfite, and other ingredients such as lysine or glycine.
  • diluents Tris, citrate, acetate or phosphate buffers
  • solubilizers such as Tween or polysorbate with varying pH and ionic strength Esters
  • carriers such as human serum albumin or gelatin
  • preservatives such as thimerosal, parabens or benzyl alcohol
  • antioxidants such as ascorbic acid or sodium metabisulfite
  • other ingredients such as lysine or glycine.
  • glycosylation-modified erythropoietins described herein are formulated in liquid form with isotonic sodium chloride/sodium citrate buffer containing human albumin and, optionally, benzyl alcohol as a preservative.
  • the composition may contain analogs with 1, 2, 3, 4 or more additional sugar chains.
  • the pharmaceutical compositions of the present application can be administered by subcutaneous or intravenous injection. The final route of administration selected will depend on many factors, and the final route of administration can be determined by those skilled in the art.
  • the present application provides an application for treating anemia.
  • the glycosylation-modified erythropoietin provided by this application has a unique glycosylation form, and when it binds to the EPO receptor, it can cause the conformation of the EPO receptor to change, thereby activating multiple downstream signaling pathways and causing the proliferation of the erythrocyte system and differentiation administering the glycosylation-modified erythropoietin to a subject in need of treatment to stimulate erythropoiesis, increase hemoglobin levels, erythrocyte levels, hematocrit, reticulocyte levels, and alleviate anemia, such as renal anemia , multiple myeloma anemia and/or cancer anemia, such as renal anemia caused by chronic kidney disease and uremia, multiple myeloma anemia and cancer anemia caused by chemotherapy, etc.
  • the present application provides a glycosylation-modified erythropoietin and/or a pharmaceutical composition for treating anemia.
  • the glycosylation-modified erythropoietin is administered to a subject in need of treatment to stimulate erythropoiesis, increase hemoglobin levels, erythrocyte levels, hematocrit, reticulocyte levels, and alleviate anemia, such as renal anemia, multiple myeloma anemia and/or cancer anemia.
  • the present application provides a glycosylation-modified erythropoietin and/or a pharmaceutical composition for use in preparing a medicine for treating anemia.
  • the glycosylation-modified erythropoietin is administered to a subject in need of treatment to stimulate erythropoiesis, increase hemoglobin levels, erythrocyte levels, hematocrit, reticulocyte levels, and alleviate anemia, such as renal anemia, multiple myeloma anemia and/or cancer anemia.
  • the present application provides a method for prolonging the half-life of erythropoietin, when the glycosylation-modified erythropoietin described in the present application is administered to a subject in need of treatment, the glycosylation-modified erythropoietin Genitin was significantly reduced in the body clearance rate of the subjects, and the half-life in vivo was prolonged.
  • glycosylation-modified erythropoietin of the present application is a glycoprotein containing 165 amino acids. It contains 5 N glycosylation sites (N24, N30, N38, N83, N88).
  • amino acid sequence of the glycosylation-modified erythropoietin (as shown in SEQ ID NO: 1) is:
  • the expression vector plasmid pJY08301 was constructed on the backbone of the pIRES plasmid (ClonTech, Cat#631605, Lot 8061805A). De novo synthesis of the IL-2 signal peptide (SEQ ID No: 2) and the JL14001 gene (SEQ ID No: 3) were inserted into the MCS1 region of the pIRES plasmid.
  • the rat glutamine synthetase gene (rat GS) (SEQ ID No: 4) was partially isolated from pGSRK-1 (ATCC, Cat#63067, Lot#158451). The rat GS gene was de novo synthesized by integration into the 5' fragment and inserted into the MCS2 region of the pIRES plasmid (as shown in Figure 1).
  • CHO-S cells were transfected with pJY08301 using FuGene 6 transfection reagent.
  • the transfected CHO-S cells (transfectants) were placed in a continuous high-concentration methionine sulfoximine (MSX) culture environment, and high-yield clones were obtained by MSX pressure screening.
  • MSX methionine sulfoximine
  • the glycosylation-modified erythropoietin of the present application is obtained.
  • the transfected CHO-S cells were grown in CD CHO medium to a cell density of 3*10 6 cells/mL, and then entered the production stage. During the production phase, the incubation temperature was constant at 37°C, pH was maintained at 7.2, and dissolved oxygen was 50%.
  • the produced glycosylation-modified erythropoietin is separated and purified by an ion exchange purification column, and the obtained lot#20190302-2, lot#20190303-2, and lot#20190304-1 are the glycosylation-modified erythropoiesis of the application. Three batches of purified products.
  • the glycosylation-modified erythropoietin molecule of the present application was detected by Western-Blot. Pipette 100 ng of the purified sample and mix it with the loading buffer. Darbepoetin (Darbe, lot #1078765A) was used as a control, heated at 70 °C for 10 minutes, equilibrated to room temperature, and then centrifuged at 4000 rpm for 1 minute to collect the supernatant. . Add the sample to 4-12% Bis-Tris precast gel, add 1 ⁇ MOPS SDS electrophoresis buffer, set the power supply voltage and time parameters to 60V for 20 minutes and 180V for 50 minutes.
  • the molecular weight of the glycosylation-modified erythropoietin of the present application is different from that of Darbepoetin, and the molecular weight of the protein is higher than that of Darbepoetin.
  • the peptide map coverage of the glycosylation-modified erythropoietin of the present application was analyzed by peptide map analysis.
  • Darbe Darbepoetin
  • the column temperature is 60 °C
  • the detection wavelength is 214 nm
  • the sample loading is 10 microliters
  • the effective separation time is 2 to 60 minutes
  • the effective gradient is 5% to 50% of mobile phase B.
  • the mass spectrometer is in positive ion acquisition mode, MSE acquisition, ESI ion source, sample cone voltage is 40V, capillary voltage is 3kV, scanning range is 100-2000m/z, low-energy energy is 6eV, high-energy energy is 20-50eV, and scanning time is 0.5 seconds .
  • the results show that the glycosylation-modified erythropoietin and Darbepoetin (Darbe) peptide map coverage rate of the present application reaches more than 85%.
  • the isoelectric point of the glycosylation-modified erythropoietin of the present application was detected by isoelectric focusing electrophoresis (IEF). Place IEF gels in ampholyte buffer pH 2-6 and let stand overnight. Then, the IEF gel containing ampholyte was placed on an electrophoresis plate pre-cooled to 10°C, and pre-focused at 700V for 20 minutes to form a pH gradient field. 4.5 micrograms of purified samples were added to each well, Darbepoetin (Darbe, lot #1078765A) was used as a control, and the program was set to 500V for 20 minutes, 2000V for 120 minutes, and 2500V for 10 minutes.
  • IEF isoelectric focusing electrophoresis
  • lanes 1-4 are Darbepoetin and the three batches of purified products of the glycosylation-modified erythropoietin of the present application, lot#20190302-2, lot#20190303-2, and lot#20190304-1, respectively.
  • the results show that the number of bands of the purified product of the present application is consistent with that of Darbepoetin, but the purified product of the present application has a higher content of bands with a lower isoelectric point (pI).
  • the acid isomer ratio of the glycosylation-modified erythropoietin of the present application was detected by capillary zone electrophoresis (CZE).
  • CZE capillary zone electrophoresis
  • the purified samples were exchanged and concentrated into water with a concentration of 1.0 mg/ml, and Darbepoetin (Darbe, lot #1078765A) was used as a control.
  • CZE buffer 7M Urea, 0.01M NaCl, 0.01M sodium acetate, 0.01M tricine, 2.5mM 1,4-Diaminobutane. Turn on the PA800 plus instrument and install the capillary cartridge with an inner diameter of 50 microns and a total length of 110 cm.
  • the sialic acid content of the glycosylation-modified erythropoietin of the present application was analyzed by high performance liquid chromatography (HPLC). Pipette 25 microliters of the three batches of the glycosylation-modified erythropoietin purified products of the present application obtained in Example 1, lot#20190302-2, lot#20190303-2, lot#20190304-1, and 50 microliters of 200mM HCl. , 25 microliters of water, and mixed with Darbepoetin (Darbe, lot #1078765A) as a control, and heated at 80° C. for 2 hours.
  • Darbe Darbepoetin
  • Neu5Ac (Sigma, 19023-10MG) and Neu5Gc (USP, 1294284) standards were respectively taken and processed according to the above methods.
  • the final concentration of each point in the Neu5Ac standard curve was 10 ⁇ M, 25 ⁇ M, 50 ⁇ M, 75 ⁇ M and 100 ⁇ M; the Neu5Gc standard was formed.
  • the concentration of each point of the curve is 0.2 ⁇ M, 0.4 ⁇ M, 1 ⁇ M, 10 ⁇ M, 25 ⁇ M, 50 ⁇ M.
  • An aqueous solution containing only HCl was used as a negative control without the addition of the standard.
  • the N-linked glycosylation composition of the glycosylation-modified erythropoietin of the present application was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). Using Zeba spin column, 15 micrograms of the three batches of purified products of glycosylation-modified erythropoietin obtained in Example 1, lot#20190302-2, lot#20190303-2, and lot#20190304-1 of the present application were purified, and the solution was changed to water, rotary evaporated to dryness, then reconstituted to 2 mg/ml by adding HPLC water, Darbepoetin (Darbe, lot #1078765A) as a control.
  • HPLC-MS high performance liquid chromatography-mass spectrometry
  • the sugar mixture was extracted using a GlycoWorks HILIC ⁇ Elution cartridge, and the final sugar mixture was dissolved in 90 ⁇ l of SPE elution buffer, diluted with 100 ⁇ l of dimethylformamide (DMF) and 210 ⁇ l of acetonitrile.
  • Mobile phase A 50 mM ammonium formate, pH 4.4; mobile phase B: acetonitrile.
  • Chromatographic conditions sample loading 10 microliters; excitation light 265 nm, emission light 425 nm; column temperature 60°C; effective separation time 35 minutes, elution gradient 25%-46% mobile phase A.
  • the mass spectrometer was set to positive ion resolution mode, MS acquisition, cone voltage 50V, capillary voltage 3.0kV, desolvation temperature 250°C, scan range 400-3000m/z, scan time 0.5s.
  • the glycosylation-modified erythropoietin of the present application has more types and proportions of 4-branched sugars, and contains a higher proportion of N-acetyllactosamine.
  • the glycan structure ratios of FA4G4S4, FA4G4L1S4 and FA4G4L2S4 in Darbepoetin were 15.85%, 5.36% and 1.43%, respectively, and the glycan structure ratios of FA4G4S4, FA4G4L1S4 and FA4G4L2S4 in the purified samples of glycosylated erythropoietin were respectively 14.02%, 21.58% and 18.17%.
  • the affinity of the glycosylation-modified erythropoietin and EPO receptor was detected by a high-throughput molecular interaction analysis platform (GE, Biacore 8K). Set the flow cell temperature to 25 degrees, the sample chamber temperature to 25 degrees, and select the sensor chip as CM5.
  • GE high-throughput molecular interaction analysis platform
  • the EC 50 value of the purified sample of glycosylation-modified erythropoietin of the present application is higher than that of Darbepoetin, indicating that the affinity of the sample of the present application to the EPO receptor is lower than that of Darbepoetin.
  • tail vein blood was collected, left standing at room temperature for 2 hours, centrifuged at 1200g for 15 minutes, and the supernatant was taken and stored in - At 80°C, the blood sample content was detected by EPO Elisa kit.

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Abstract

一种糖基化修饰的促红细胞生成素，其含有结合到N-糖基化位点的聚糖结构，所述聚糖结构包含FA4G4L2S4。

Description

一种糖基化修饰的促红细胞生成素及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种糖基化修饰的促红细胞生成素及其应用。
背景技术
促红细胞生成素(EPO，erythropoietin)是一种糖蛋白类激素，属于造血细胞因子超家族成员之一，具有促进红细胞增殖和分化，提高机体血红蛋白浓度的功能。EPO通过与细胞表面跨膜受体EPO受体结合而发挥生物效应。当EPO与EPO受体结合时，可引起受体构象发生改变，促使相邻两个EPO受体相互靠近形成同源二聚体，激活与受体胞质近膜区结合的JAK2酪氨酸激酶，从而导致受体的多个酪氨酸残基被磷酸化，JAK2可进一步磷酸化STAT，启动相关基因表达，从而激活多个下游信号通路，引起红细胞系统的增殖和分化。临床实验证明，EPO对多种疾病有治疗作用，主要用于各种原因导致的贫血。
部分治疗性促红细胞生成素类药物存在半衰期短、临床给药频率高的缺陷，严重影响患者的使用。开发半衰期长的促红细胞生成素迫在眉睫。
发明内容
为了解决目前促红细胞生成素半衰期短的问题，本申请提供一种糖基化修饰的促红细胞生成素及其应用，包括糖基化修饰的促红细胞生成素及其制备方法、药物组合物，同时提供了在制备治疗贫血的药物中的应用和延长促红细胞生成素半衰期的方法。
本申请提供了一种糖基化修饰的促红细胞生成素，其含有结合到N-糖基化位点的聚糖结构，所述聚糖结构包含FA4G4L2S4，其中F代表岩藻糖，A代表N-乙酰葡糖胺，G代表半乳糖，L代表乳糖，S代表唾液酸。
在某些实施方式中，所述FA4G4L2S4结构的比率为15％以上。
在某些实施方式中，所述聚糖结构包含FA4G4L1S4，其中F代表岩藻糖，A代表N-乙酰葡糖胺，G代表半乳糖，L代表乳糖，S代表唾液酸。
在某些实施方式中，所述FA4G4L1S4的比率为20％以上。
在某些实施方式中，所述聚糖结构包含FA4G4S4，其中F代表岩藻糖，A代表N-乙酰葡糖胺，G代表半乳糖，S代表唾液酸。
在某些实施方式中，所述FA4G4S4的比率为10％以上。
在某些实施方式中，所述聚糖结构包含Neu5Gc，其所述Neu5Gc的摩尔比率为0.5％以下。
在某些实施方式中，糖基化修饰的促红细胞生成素包含SEQ ID NO.1所示的氨基酸序列。
在某些实施方式中，糖基化修饰的促红细胞生成素包含以下的N-糖基化位点：N24、N30、N38、N83和N88。
另一方面，本申请提供一种制备所述的糖基化修饰的促红细胞生成素的方法，其包括以下的步骤：在所述的糖基化修饰的促红细胞生成素条件下，培养包含编码SEQ ID NO:1所示的氨基酸序列的核酸分子的CHO-S细胞。
另一方面，本申请提供一种药物组合物，其包含所述的糖基化修饰的促红细胞生成素和药学上可接受的佐剂。
另一方面，本申请提供所述的糖基化修饰的促红细胞生成素和/或所述的药物组合物在制备药物中的用途，所述药物用于治疗贫血。
另一方面，本申请提供所述的糖基化修饰的促红细胞生成素和/或所述的药物组合物在制备药物中的用途，所述药物用于治疗贫血，所述贫血包括肾性贫血、多发性骨髓瘤贫血和/或癌性贫血。
另一方面，本申请提供了治疗贫血的方法，其包括以有效治疗量向需要其的对象施用的所述糖基化修饰的促红细胞生成素和/或所述的药物组合物。
另一方面，本申请提供了治疗贫血的方法，其包括以有效治疗量向需要其的对象施用的所述糖基化修饰的促红细胞生成素和/或所述的药物组合物，所述贫血包括肾性贫血、多发性骨髓瘤贫血和/或癌性贫血。
另一方面，本申请提供所述的糖基化修饰的促红细胞生成素和/或所述的药物组合物，其用于治疗贫血。
另一方面，本申请提供所述的糖基化修饰的促红细胞生成素和/或所述的药物组合物，其用于治疗贫血，所述贫血包括肾性贫血、多发性骨髓瘤贫血和/或癌性贫血。
另一方面，本申请提供一种延长促红细胞生成素半衰期的方法，其包括以下的步骤：向有需要的受试者施用所述的糖基化修饰的促红细胞生成素。
本申请提供的糖基化修饰的促红细胞生成素，通过提高半衰期，具有提高血红蛋白含量、红细胞水平、红细胞比容和/或网织红细胞水平的有益效果。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文 的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请所述糖基化修饰的促红细胞生成素表达载体片段结构示意图。
图2显示的是本申请所述糖基化修饰的促红细胞生成素的免疫印迹结果，第1泳道为已知分子量的蛋白marker，第2、4、6泳道分别为本申请糖基化修饰的促红细胞生成素的三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，第3、5、7泳道为对照组Darbepoetin(Darbe，lot#1078765A)。
图3显示的是本申请所述糖基化修饰的促红细胞生成素的IEF检测结果，第1泳道为对照组Darbepoetin(Darbe，lot#1078765A)，第2、3、4泳道分别为本申请糖基化修饰的促红细胞生成素的三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，第5泳道为空，第六泳道为已知pI的蛋白marker。
图4显示的是本申请所述糖基化修饰的促红细胞生成素的CZE检测结果，第1行为对照组Darbepoetin(Darbe，lot#1078765A)，第2、3、4行分别为本申请糖基化修饰的促红细胞生成素的三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1。
图5显示的是本申请所述糖基化修饰的促红细胞生成素的N-连接糖基化组成检测结果，第1、2、3行分别为本申请糖基化修饰的促红细胞生成素的三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，第4行为对照组Darbepoetin(Darbe，lot#1078765A)。
图6显示的是本申请所述糖基化修饰的促红细胞生成素对TF-1细胞增殖能力的结果，1表示对照组Darbepoetin(Darbe，lot#1078765A)，2、3、4分别表示本申请糖基化修饰的促红细胞生成素的三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1。
图7显示的是本申请所述糖基化修饰的促红细胞生成素的体内药效检测结果，1表示空白组(vehicle组)，2表示对照组A(Darbepoetin，Darbe，lot#1078765A)，3表示对照组B(EPO，利血宝，lot#17Y03B)，4、5、6分别为本申请糖基化修饰的促红细胞生成素的三批 纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1；与空白组相比显著性用*表示，****表示P<0.0001，***表示P<0.001，**表示P<0.01，*表示P<0.05；与对照组B相比显著性用#表示##表示P<0.01，#表示P<0.05。
图8显示的是本申请所述糖基化修饰的促红细胞生成素的半衰期检测结果，1表示对照组A(Darbepoetin，Darbe，lot#1078765A)，2、3、4分别为本申请糖基化修饰的促红细胞生成素的三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，5表示对照组B(EPO，利血宝，lot#17Y03B)。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“半衰期”或其缩写“T 1/2”，通常是指用于量化药物的一半剂量被受试者排泄所花费的时间。
在本申请中，术语“聚糖结构”，通常是指多糖或寡糖，即在酸水解之后产生多个单糖的多聚化合物。糖基化修饰的蛋白可以包含经由天冬酰胺或精氨酸(“N-连接的糖基化”)或经由丝氨酸或苏氨酸(“O-连接的糖基化”)共价偶联至多肽链的侧基的一个或多个聚糖结构。例如，可以是FA4G4L2S4聚糖结构连接到促红细胞生成素的N-糖基化位点，可以是FA4G4L1S4聚糖结构连接到促红细胞生成素的N-糖基化位点，可以是FA4G4S4聚糖结构连接到促红细胞生成素的N-糖基化位点，可以是聚糖结构Neu5Gc连接到促红细胞生成素的N-糖基化位点。聚糖结构根据其支化(天线)数目分为二天线、三天线和四天线结构。聚糖结构由各种单糖组成，按照Oxford命名法则，包括岩藻糖(Fucose，简称Fuc或F)、N-乙酰葡糖胺(N-Acetylglucosamine，简称GlcNAc、Gn或A)、半乳糖(Galactose，简称Gal或G)、乳糖(Lactose，简称Lac或L)、甘露糖(Mannose，简称Man或M)、N-乙酰神经氨酸(唾液酸、N-Acetylneuraminic，简称NANA、Neu5Ac或S)和/或N-羟乙酰神经氨酸(N-Glycolylneuraminic，简称NGNA、Neu5Glc或Neu5Gc)。例如，术语“FA4G4L2S4”是指含有岩藻糖化、4个N-乙酰葡糖胺、4个半乳糖、2个乳糖、4个唾液酸的四天线结构聚糖结构，其中F代表含有岩藻糖修饰，A代表N-乙酰葡糖胺，G代表半乳糖，L代表乳糖，S代表唾液酸，A后的数字代表一个聚糖结构上所述N-乙酰葡糖胺的个数，G后的数字代表 一个聚糖结构上所述半乳糖的个数，L后的数字代表一个聚糖结构上所述乳糖的个数，S后的数字代表一个聚糖结构上所述唾液酸的个数；术语“FA4G4L1S4”是指含有岩藻糖化、4个N-乙酰葡糖胺、4个半乳糖、1个乳糖、4个唾液酸的四天线结构聚糖结构，其中F代表含有岩藻糖修饰，A代表N-乙酰葡糖胺，G代表半乳糖，L代表乳糖，S代表唾液酸，A后的数字代表一个聚糖结构上所述N-乙酰葡糖胺的个数，G后的数字代表一个聚糖结构上所述半乳糖的个数，L后的数字代表一个聚糖结构上所述乳糖的个数，S后的数字代表一个聚糖结构上所述唾液酸的个数；术语“FA4G4S4”是指含有岩藻糖化、4个N-乙酰葡糖胺、4个半乳糖、4个唾液酸的四天线结构聚糖结构，其中F代表含有岩藻糖修饰，A代表N-乙酰葡糖胺，G代表半乳糖，S代表唾液酸，A后的数字代表一个聚糖结构上所述N-乙酰葡糖胺的个数，G后的数字代表一个聚糖结构上所述半乳糖的个数，S后的数字代表一个聚糖结构上所述唾液酸的个数；术语“Neu5Gc”是指N-羟乙酰神经氨酸。
在本申请中，术语“N-糖基化位点”，通常是指糖基化修饰的蛋白上包含天冬酰胺或精氨酸用以共价连接聚糖结构的位点，例如N-糖基化位点可以是用以共价连接聚糖结构至糖基化修饰的蛋白的天冬酰胺残基。例如，N-糖基化位点可以是所述的糖基化修饰的促红细胞生成素上第24位、第30位、第38位、第83位和第88位上的天冬酰胺(Asparagines，简称Asn或N)残基。
在本申请中，所述聚糖结构与所述N-糖基化位点的术语“结合”，通常是指聚糖结构与糖基化修饰的蛋白上N-糖基化位点之间的物理或化学相互作用。例如，结合可以是直接的或间接的连接或附着，间接的可以是通过另一生物分子或化合物的连接或附着，直接的可以是共价的(例如通过化学偶联)或非共价的(例如离子相互作用、疏水相互作用、氢键等)结合或附着。例如，结合可以是聚糖结构与糖基化修饰的蛋白上N-糖基化位点之间共价连接，结合也可以是聚糖结构的单糖与N-糖基化位点的天冬酰胺残基的的自由-NH2基通过共价连接。
在本申请中，所述聚糖结构在所述糖基化修饰的促红细胞生成素的所述“比率”，通常是指所述聚糖结构在所述糖基化修饰的促红细胞生成素中所占的摩尔比率，所述比率可以通过将所述糖基化修饰的促红细胞生成素酶解后并由质谱进行分析，示例性质谱分析方法可以包括与HPLC联用的质谱分析法。例如，所述的糖基化修饰的促红细胞生成素中，所述FA4G4L2S4结构的比率可以为15％以上，所述FA4G4L1S4的比率可以为20％以上，所述FA4G4S4的比率可以为10％以上，所述Neu5Gc的摩尔比率可以为0.5％以下。
在本申请中，术语“摩尔比率”通常作为所述糖蛋白经过糖苷酶酶解之后，释放所有聚糖结构，其中特定聚糖结构的摩尔比率＝其摩尔数目/蛋白质中所有聚糖结构摩尔数目进行计算且给出。例如摩尔比率，可以是FA4G4L2S4结构的摩尔数目/糖基化修饰的促红细胞生成素中所有聚糖结构的摩尔数目，可以是FA4G4L1S4的摩尔数目/糖基化修饰的促红细胞生成素中所有聚糖结构的摩尔数目，可以是FA4G4S4的摩尔数目/糖基化修饰的促红细胞生成素中所有聚糖结构的摩尔数目，可以是Neu5Gc的摩尔数目/糖基化修饰的促红细胞生成素中所有聚糖结构的摩尔数目。
在本申请中，术语“蛋白”通常是指不限于最小长度的氨基酸残基的聚合物。多肽、肽、寡肽、二聚体、多聚体和类似物均包括在该定义中。完整蛋白及其片段也都包括在此定义中。该术语也包括蛋白的表达后修饰形式，包括但不限于糖基化、乙酰化、磷酸化等。例如，本申请中，蛋白可以指糖基化修饰的促红细胞生成素及其片段。
在本申请中，细胞“CHO-S细胞”，通常是指可以通过例如转染导入例如编码异源多肽的核酸的CHO-S中国仓鼠卵巢细胞。所述CHO-S细胞包括原始转染细胞中筛选出来的功能或生物活性具有相同的功能或生物活性的变体后代。
在本申请中，术语“促红细胞生成素”和它的缩写“EPO”，通常是指任何促红细胞生成素多肽，包括但不限于，重组产生的促红细胞生成素多肽，合成产生的促红细胞生成素多肽，天然EPO多肽，从细胞以及组织提取的促红细胞生成素多肽，所述组织包括但不限于肾、肝、尿和血液。例如，促红细胞生成素可以是具有氨基酸序列SEQ ID No:1的促红细胞生成素。术语“促红细胞生成素”还指SEQ ID No:1的蛋白质的变体，其中一个或多个氨基酸残基被改变、删除或插入，并且其具有与未修饰的蛋白质相同的生物活性，例如在EP 1 064 951或US 6,583,272中报道的。促红细胞生成素与EPO受体结合后产生的所述生物活性可以包括：与未注射组或对照组个体相比，通过注射将促红细胞生成素给药至受试者致使骨髓细胞增加网织红细胞和红细胞的产生。
在本申请中，所述“糖基化修饰”通常是指，蛋白或多肽中可以在一个或多个氨基位置连接碳水化合物部分。通常糖基化修饰的蛋白或多肽含有一或多个氨基酸残基，例如精氨酸或天冬酰胺，以连接碳水化合物部分。例如，糖基化修饰可以是N-联的糖蛋白。N-联糖蛋白可以包含结合到N-糖基化位点的聚糖结构，例如连接至蛋白质中天冬酰胺残基N-糖基化位点的聚糖结构。在糖基化修饰的蛋白(糖蛋白)上发现的糖包括以下组：葡萄糖、半乳糖、甘露糖、岩藻糖、N-乙酰半乳糖胺(GalNAc)、N-乙酰葡糖胺(GlcNAc)和/或唾液酸。例如，促红细胞生成素的糖基化修饰，可以是含FA4G4L2S4聚糖结构连接到促红细胞生成素的N-糖基 化位点，可以是含FA4G4L1S4聚糖结构连接到促红细胞生成素的N-糖基化位点，可以是含FA4G4S4聚糖结构连接到促红细胞生成素的N-糖基化位点，可以是含Neu5Gc的糖结构连接到促红细胞生成素的N-糖基化位点。
在本申请中，所述FA4G4L2S4结构的比率为15％“以上”，通常是指包含FA4G4L2S4聚糖结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞生成素。例如，可以包含FA4G4L2S4聚糖结构的比率为至少18.17％的糖基化修饰的促红细胞生成素。
在本申请中，所述FA4G4L12S4结构的比率为20％“以上”，通常是指包含FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞生成素。例如，可以包含FA4G4L1S4聚糖结构的比率为至少21.58％的糖基化修饰的促红细胞生成素。
在本申请中，所述FA4G4S4结构的比率为10％“以上”，通常是指包含FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞生成素。例如，可以包含FA4G4S4聚糖结构的比率为至少14.02％的糖基化修饰的促红细胞生成素。
在本申请中所述Neu5Gc的摩尔比率为0.5％“以下”，通常是指Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的糖基化修饰的促红细胞生成素。例如，可以是聚糖结构不包含Neu5Gc的糖基化修饰的促红细胞生成素。
在本申请中，“延长”促红细胞生成素半衰期，通常是指本申请的糖基化修饰的促红细胞生成素对蛋白酶抗性增加，可以导致所述糖基化修饰的促红细胞生成素与不具有糖基化形式的EPO或具有其它聚糖结构的糖基化EPO相比在体外(例如在产生、纯化和储存期间)或者在体内(例如给予受试者之后)的半衰期增加。例如，在给予受试者本申请的糖基化修饰的促红细胞生成素之后，呈现出半衰期增加，在与未修饰的EPO或具有其它聚糖结构的糖 基化EPO相比，本申请的糖基化修饰的促红细胞生成素增加的半衰期可以增加至少大约或者至少1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、20％、30％、40％、50％、60％、70％、75％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％、200％、300％、400％、500％或更多。例如，在与未修饰的EPO或具有其它聚糖结构的糖基化EPO相比，本申请的糖基化修饰的促红细胞生成素增加的半衰期可以增加至少大约或者至少6倍、7倍、8倍、9倍、10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍、100倍或者更多倍。
发明详述
一方面，本申请提供一种糖基化修饰的促红细胞生成素，包含5个糖基化位点：N24、N30、N38、N83和N88。糖基化位点可用于N-联本申请所述聚糖结构的糖链。通过将编码氨基酸序列如SEQ ID No:1的促红细胞生成素的核酸分子转染入CHO-S细胞中，表达本申请所述的糖基化修饰的促红细胞生成素。虽然所述的糖基化修饰的促红细胞生成素与EPO受体的结合并没有增强，而且在某些情况下因为本申请所述糖链结构而可能降低与EPO受体的结合力。然而，本申请所述的糖基化修饰的促红细胞生成素可以提高半衰期，从而导致体内生物活性升高，提高血红蛋白含量、红细胞水平、红细胞比容、网织红细胞水平。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4聚糖结构。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4结构，所述FA4G4L2S4结构的比率可以为15％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的糖基化修饰的促红细胞生成素。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L1S4聚糖结构。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L1S4结构，所述FA4G4L1S4的比率可以为20％以上。例如，FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、 至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰的促红细胞生成素。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4S4聚糖结构。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4S4结构，所述FA4G4S4的比率可以为10％以上。例如，FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞生成素。例如，包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰的促红细胞生成素。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含Neu5Gc，其所述Neu5Gc的摩尔比率可以为0.5％以下。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含Neu5Gc，其所述Neu5Gc的摩尔比率可以为0.5％以下。例如，Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的糖基化修饰的促红细胞生成素。例如，可以是聚糖结构不包含Neu5Gc的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4聚糖结构和一个或多个FA4G4L1S4聚糖结构。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4结构和一个或多个FA4G4L1S4结构，所述FA4G4L2S4结构的比率可以为15％以上和所述FA4G4L1S4的比率可以为20％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；且FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少 98％或至少99％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的；且包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4聚糖结构和一个或多个FA4G4S4聚糖结构。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4结构和一个或多个FA4G4S4结构，所述FA4G4L2S4结构的比率可以为15％以上和所述FA4G4S4的比率可以为10％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；且FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的；且包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L1S4聚糖结构和一个或多个FA4G4S4聚糖结构。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L1S4结构和一个或多个FA4G4S4结构，所述FA4G4L1S4的比率可以为20％以上和所述FA4G4S4的比率可以为10％以上。例如，FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；且FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞 生成素。例如，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的；且包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4聚糖结构和一个或多个Neu5Gc。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4结构和一个或多个Neu5Gc，所述FA4G4L2S4的比率可以为15％以上和所述Neu5Gc的摩尔比率可以为0.5％以下。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；且Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的；且可以是聚糖结构不包含Neu5Gc的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L1S4聚糖结构和一个或多个Neu5Gc聚糖结构。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L1S4结构和一个或多个Neu5Gc，所述FA4G4L1S4的比率可以为20％以上和所述Neu5Gc的摩尔比率可以为0.5％以下。例如，FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；且Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的；且可以是聚糖结构不包含Neu5Gc的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4聚糖结构、一个或多个FA4G4L1S4聚糖结构和一个或多个FA4G4S4聚糖结构。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4结构、一个或多个FA4G4L1S4结构和一个或多个FA4G4S4结 构，所述FA4G4L2S4结构的比率可以为15％以上、所述FA4G4L1S4的比率可以为20％以上和所述FA4G4S4的比率可以为10％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；且FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的；且包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4聚糖结构、一个或多个FA4G4L1S4聚糖结构和一个或多个Neu5Gc。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4结构、一个或多个FA4G4L1S4结构和一个或多个Neu5Gc，所述FA4G4L2S4结构的比率可以为15％以上、所述FA4G4L1S4的比率可以为20％以上和所述Neu5Gc的摩尔比率可以为0.5％以下。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；且Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L2S4聚 糖结构的比率可以为至少18.17％的，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的；且可以是聚糖结构不包含Neu5Gc的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4聚糖结构、一个或多个FA4G4L1S4聚糖结构、一个或多个FA4G4S4聚糖结构和一个或多个Neu5Gc。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含一个或多个FA4G4L2S4结构、一个或多个FA4G4L1S4结构、一个或多个FA4G4S4结构和一个或多个Neu5Gc，所述FA4G4L2S4结构的比率可以为15％以上、和所述FA4G4L1S4的比率可以为20％以上、所述FA4G4S4的比率可以为10％以上和所述Neu5Gc的摩尔比率可以为0.5％以下。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的；且Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的糖基化修饰的促红细胞生成素。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的；包含FA4G4S4聚糖结构的比率可以为至少14.02％的；且可以是聚糖结构不包含Neu5Gc的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以直接或间接结合到N-糖基化位点。例如，所述聚糖结构可以通过共价作用直接结合到N-糖基化位点。例如，所述聚糖结构的单糖可以通过共价作用直接结合到N-糖基化位点的天冬酰胺残基的自由-NH 2基。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，所述FA4G4L2S4结构的比率可以为15％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L1S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L1S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，所述FA4G4L1S4的比率可以为20％以上。例如，FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，所述FA4G4S4的比率可以为10％以上。例如，FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含Neu5Gc，所述聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
在另一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述聚糖结构可以包含Neu5Gc，所述聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，所述Neu5Gc的摩尔比率可以为0.5％以下。例如，Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，Neu5Gc的摩尔比率可以为0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构和所述FA4G4L1S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构和所述FA4G4L1S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，所述FA4G4L2S4结构的比率可以为15％以上和所述FA4G4L1S4的比率可以为20％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少 22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构和所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构和所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，所述FA4G4L2S4结构的比率可以为15％以上和所述FA4G4S4的比率可以为10％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L1S4结构和所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L1S4结构和所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点： N24、N30、N38、N83和N88上，所述FA4G4L1S4的比率可以为20％以上和所述FA4G4S4的比率可以为10％以上。例如，FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，且聚糖结构可以包含Neu5Gc。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，且聚糖结构可以包含Neu5Gc，所述FA4G4L2S4结构的比率可以为15％以上，且所述Neu5Gc的摩尔比率可以为0.5％以下。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且Neu5Gc的摩尔比率可以为0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L1S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，且聚糖结构可以包含Neu5Gc。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L1S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，且聚糖结构可以包含Neu5Gc，所述FA4G4L1S4的比率可以为20％以上，且所述Neu5Gc的摩尔比率可以为0.5％以下。例如，FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且Neu5Gc的摩尔比率可以为0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构、所述FA4G4L1S4结构和所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构、所述FA4G4L1S4结构和所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，所述FA4G4L2S4结构的比率可以为15％以上、所述FA4G4L1S4的比率可以为20％以上，且所述FA4G4S4的比率可以为10％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、 至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构和所述FA4G4L1S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，且聚糖结构可以包含Neu5Gc。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构和所述FA4G4L1S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，且聚糖结构可以包含Neu5Gc，所述FA4G4L2S4结构的比率可以为15％以上、所述FA4G4L1S4的比率可以为20％以上，且所述Neu5Gc的摩尔比率可以为0.5％以下。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基 化位点；且包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且Neu5Gc的摩尔比率可以为0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构、所述FA4G4L1S4结构和所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，且聚糖结构可以包含Neu5Gc。
一方面，本申请提供一种糖基化修饰的促红细胞生成素的聚糖结构。所述FA4G4L2S4结构、所述FA4G4L1S4结构和所述FA4G4S4结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点：N24、N30、N38、N83和N88上，且聚糖结构可以包含Neu5Gc，所述FA4G4L2S4结构的比率可以为15％以上、所述FA4G4L1S4的比率可以为20％以上，且所述FA4G4S4的比率可以为10％以上。例如，FA4G4L2S4结构的比率可以为至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且FA4G4L1S4聚糖结构的比率可以为至少20％、至少21％、至少22％、至少23％、至少24％、至少25％、至少26％、至少27％、至少28％、至少29％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；FA4G4S4聚糖结构的比率可以为至少10％、至少11％、至少12％、至少13％、至少14％、至少15％、至少16％、至少17％、至少18％、至少19％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且Neu5Gc的摩尔比率可以为至多0.5％、至多0.4％、至多0.3％、至多0.2％、至多0.1％、至多0.05％、至多0.02％、至多0.01％或0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。例如，包含FA4G4L2S4聚糖结构的比率可以为至少18.17％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且包含FA4G4L1S4聚糖结构的比率可以为至少21.58％的糖基化修饰可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且包含FA4G4S4聚糖结构的比率可以为至少14.02％的糖基化修饰可以 结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点；且Neu5Gc的摩尔比率可以为0％的聚糖结构可以结合到所述糖基化修饰的促红细胞生成素的N-糖基化位点。
一方面，本申请提供一种制备糖基化修饰的促红细胞生成素的方法，包括在表达所述的糖基化修饰的促红细胞生成素条件下，培养包含编码所述的糖基化修饰的促红细胞生成素的核酸分子的CHO-S细胞。采用标准技术利用适合保持在哺乳动物宿主细胞中的载体将编码氨基酸序列如SEQ ID No:1的促红细胞生成素插入表达载体中。所述载体通常含有以下适用于哺乳动物宿主细胞的元件：启动子和其它“上游”调节元件、复制起点、核糖体结合位点、转录终止位点、多接头位点和选择标记。载体还可以含有同样允许在原核宿主细胞增殖和维持的元件。例如，合适的细胞或细胞系包括哺乳动物来源(包括人类来源)的任何细胞或细胞系，包括中国仓鼠卵巢细胞CHO-S细胞。用标准转化或转染技术将包含编码氨基酸序列如SEQ ID No:1的促红细胞生成素的序列的核酸分子导入宿主细胞中。利用公众可应用的方法完成培养、扩增和筛选转化宿主细胞或转染宿主细胞(Gething等，Nature 293，620-625(1981):Kaufman等，Mol Cell.Biol.5，1750-1759(1985)；美国专利第4，419，446号)。在可表达所述类似物的条件下培养含有促红细胞生成素DNA编码序列的宿主细胞。从所述细胞培养基回收所述糖基化修饰的促红细胞生成素，并采用基本上与以前所述(WO 94/09257)相同的方法进行纯化。所述纯化方法可分离本申请所述的糖基化修饰的促红细胞生成素。
一方面，本申请提供一种药用组合物，它包括治疗有效量所述糖基化修饰的促红细胞生成素和药学上可接受的佐剂如稀释剂、载体、增溶剂、乳化剂、防腐剂和/或辅助剂。所述药物组合物适用于每周低于三次的给药方案。所述组合物可以为液体或冻干形式，它含有具有不同pH值和离子强度的稀释剂(Tris、柠檬酸盐、乙酸盐或磷酸盐缓冲剂)、增溶剂如吐温或聚山梨酸酯、载体如人血清白蛋白或明胶、防腐剂如硫汞撒、对羟基苯甲酸酯类或苄醇、抗氧化剂如抗坏血酸或偏亚硫酸氢钠以及其它成分如赖氨酸或甘氨酸。对具体组合物的选择取决于许多因素，包括所治疗的病症、给药途径和需要的药代动力学参数。对适用于药用组合物的成分的更广泛的综述见Remington’s Pharmaceutical Sciences，第18版，A.R.Gennaro编辑，Mack，Easton，PA(1980)。例如，用含有人白蛋白和任选含有防腐剂苄醇的等渗氯化钠/柠檬酸钠缓冲液将本申请所述糖基化修饰的促红细胞生成素配制成液体形式。所述组合物可以含有具有1、2、3、4或更多个额外糖链的类似物。例如，可以通过皮下或静脉内注射给予本申请的药物组合物。最终选定的给药途径取决于许多因素，而本领域技术人员可确定最终给药途径。
一方面，本申请提供一种治疗贫血的应用。本申请提供的糖基化修饰的促红细胞生成素具有独特的糖基化形式，与EPO受体结合时，可引起EPO受体构象发生改变，从而激活多个下游信号通路，引起红细胞系统的增殖和分化将所述糖基化修饰的促红细胞生成素给予需要治疗的受试者以刺激红细胞生成，提高血红蛋白含量、红细胞水平、红细胞比容、网织红细胞水平，而减轻贫血，例如肾性贫血、多发性骨髓瘤贫血和/或癌性贫血，例如由慢性肾病和尿毒症导致的肾性贫血、多发性骨髓瘤贫血和化疗等引发的癌性贫血。
一方面，本申请提供一种糖基化修饰的促红细胞生成素和/或药物组合物，其用于治疗贫血的疾病。将所述糖基化修饰的促红细胞生成素给予需要治疗的受试者以刺激红细胞生成，提高血红蛋白含量、红细胞水平、红细胞比容、网织红细胞水平，而减轻贫血，例如肾性贫血、多发性骨髓瘤贫血和/或癌性贫血。
一方面，本申请提供一种糖基化修饰的促红细胞生成素和/或药物组合物，其在制备治疗贫血的药物中的应用。将所述糖基化修饰的促红细胞生成素给予需要治疗的受试者以刺激红细胞生成，提高血红蛋白含量、红细胞水平、红细胞比容、网织红细胞水平，而减轻贫血，例如肾性贫血、多发性骨髓瘤贫血和/或癌性贫血。
一方面，本申请提供一种延长促红细胞生成素半衰期的方法，当向需要治疗的受试者施用本申请所述糖基化修饰的促红细胞生成素后，所述糖基化修饰的促红细胞生成素在受试者体内清除速度明显降低，体内半衰期延长。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1糖基化修饰的促红细胞生成素的载体构建、转染和分离纯化
本申请的糖基化修饰的促红细胞生成素是一个含165个氨基酸的糖蛋白。其含有5个N糖基化位点(N24，N30，N38，N83，N88)。所述糖基化修饰的促红细胞生成素的氨基酸序列(如SEQ ID NO:1所示)为：
Figure PCTCN2021119332-appb-000001
表达载体质粒pJY08301是在pIRES质粒(ClonTech，Cat#631605，批号8061805A)骨 架上构建而成。从头合成IL-2信号肽(SEQ ID No:2)和JL14001基因(SEQ ID No:3)插入pIRES质粒中的MCS1区。大鼠谷氨酰胺合成酶基因(大鼠GS)(SEQ ID No:4)部分分离自pGSRK-1(ATCC，Cat#63067，Lot#158451)。大鼠GS基因通过整合完成从头合成5’片段，并以插入pIRES质粒中的MCS2区(如图1所示)。
使用FuGene 6转染试剂将pJY08301转染亲代CHO-S细胞。将转染后的CHO-S细胞(转染子)置于持续高浓度的蛋氨酸亚氨基代砜(methionine sulfoximine，MSX)培养环境下，通过MSX加压筛选，筛选获得高产量的克隆。
通过培养和纯化，得到了本申请的糖基化修饰的促红细胞生成素。将转染后的CHO-S细胞在CD CHO培养基中培养生长到3*10 6个/mL细胞密度，后进入生产阶段。生产阶段的培养温度恒定在37℃，pH维持在7.2，溶解氧为50％。生产获得的糖基化修饰的促红细胞生成素经过离子交换纯化柱分离并纯化，获得lot#20190302-2、lot#20190303-2、lot#20190304-1为本申请糖基化修饰的促红细胞生成素的三批纯化产物。
通过免疫印迹(Western-Blot)检测本申请糖基化修饰的促红细胞生成素的分子。吸取100纳克纯化所得样品与上样缓冲液混匀，Darbepoetin(Darbe，lot#1078765A)作为对照，70℃加热10分钟，平衡至室温，然后于4000转/分钟离心1分钟，收集上清液。将样品加入至4-12％Bis-Tris预制胶中，加入1×MOPS SDS电泳缓冲液，设置电源电压及时间参数为60V 20分钟，180V 50分钟。电泳结束后，将凝胶取出，放至含有PVDF膜的干转试剂盒中，设置干转仪参数为20V 1分钟、23V 4分钟、25V 2分钟进行转膜，结束后，利用10％脱脂奶粉溶液封闭此PVDF膜，然后加入1：1000配比的EOP一抗(Anti-Erythropoietin)工作液，室温孵育1小时，PBST洗涤PVDF膜，再加入1：2000二抗工作液(Goat Anti-Rabbit IgG)，室温孵育1小时，PBST洗涤PVDF膜，最后显色拍照。
结果如图2所示，本申请的糖基化修饰的促红细胞生成素的分子量与Darbepoetin不同，该蛋白的分子量比Darbepoetin高。
通过肽图分析对本申请糖基化修饰的促红细胞生成素的肽图覆盖率进行分析。吸取180微升纯化所得样品与20微升10×糖蛋白变性缓冲液混匀，Darbepoetin(Darbe)作为对照，100℃加热10分钟，然后加入20微升10×GlycoBuffer、40微升10％NP-40、1微升PNGase F、119微升水，37℃孵育2小时。然后样品与40微升盐酸胍、8微升DTT混匀，56℃静置40分钟。加入碘乙酰胺，室温避光放置50分钟，然后将混合溶液置换至碳酸氢铵中。按照1:50(w/w)比例分别向溶液中加入内切酶Trpsin、LysC、GluC，37℃静置21小时。向各样品中加入15％甲酸溶液终止反应。流动相A：0.1％甲酸水溶液；流动相B：0.1％甲酸的乙腈溶液。 柱温60℃，检测波长214nm，上样量为10微升，有效分离时间为2～60分钟，有效梯度为5％～50％流动相B。质谱为正离子采集模式，MSE采集，ESI离子源，样品锥孔电压为40V，毛细管电压3kV，扫描范围100～2000m/z，低能能量为6eV，高能能量为20～50eV，扫描时间为0.5秒。结果显示，本申请的糖基化修饰的促红细胞生成素与Darbepoetin(Darbe)肽图覆盖率达85％以上。
通过等电聚焦电泳检测(IEF)检测本申请糖基化修饰的促红细胞生成素的等电点。将IEF预制胶放入pH 2-6的两性电解质缓冲液中，静置过夜。然后将含有两性电解质的IEF胶放在预冷至10℃的电泳平板上，700V预聚焦20分钟，形成pH梯度场。向每孔中加入4.5微克纯化所得样品，Darbepoetin(Darbe，lot#1078765A)作为对照，设置程序为500V 20分钟，2000V 120分钟，2500V 10分钟。电泳结束后，将IEF胶放于固定液(TCA 6克，5-Sulfosalicylic acid 2.04克，加水至50毫升)中1小时。用水冲洗干净后，放入染色液中过夜，水溶液脱色后，拍照分析。
如图3所示，泳道1-4分别为Darbepoetin和本申请的糖基化修饰的促红细胞生成素三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1。结果显示，本申请的纯化产物的条带个数与Darbepoetin的条带个数一致，但本申请的纯化产物低等电点(pI)的条带含量较多。
通过毛细管区带电泳(CZE)检测本申请糖基化修饰的促红细胞生成素的酸性异构体比例。利用3KDa超滤浓缩管将纯化所得样品换液浓缩至水中，浓度为1.0毫克/毫升，Darbepoetin(Darbe，lot#1078765A)作为对照。配制CZE缓冲液：7M Urea、0.01M NaCl、0.01M sodium acetate、0.01M tricine、2.5mM 1,4-Diaminobutane。打开PA800 plus仪器，安装毛细管卡盒，毛细管内径为50微米，总长度为110厘米。设置程序为：20.0psi，水，清洗15分钟；20.0psi，0.1M NaOH，清洗5分钟；20.0psi，CZE缓冲液，浸润15分钟；0.7psi，真空进样30秒；20.0KV，分离样品240分钟。放入样品瓶和废液瓶，设置卡盒温度为10℃，毛细管运行温度为35℃，检测波长为214nm，检测时长为214分钟。
如图4所示，从上到下分别为Darbepoetin及本申请的糖基化修饰的促红细胞生成素三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1。结果显示，本申请的纯化产物与Darbepoetin相比，主峰右侧峰的比例要高，表明本申请的纯化产物中酸性异构体的比例要高于Darbepoetin。
实施例2糖基化修饰的促红细胞生成素的糖基化结构分析
通过高效液相色谱法(HPLC)分析本申请糖基化修饰的促红细胞生成素的唾液酸含量。 吸取25微升实施例1中纯化所得本申请的糖基化修饰的促红细胞生成素三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，与50微升200mM HCl、25微升水混匀，Darbepoetin(Darbe，lot#1078765A)作为对照，于80℃加热2小时。分别取Neu5Ac(Sigma,19023-10MG)和Neu5Gc(USP,1294284)标准品，按照上述方法处理，最终配制的组成Neu5Ac标准曲线的各个点浓度为10μM、25μM、50μM、75μM、100μM；组成Neu5Gc标准曲线的各个点浓度为0.2μM、0.4μM、1μM、10μM、25μM、50μM。以不加标准品，只含有HCl的水溶液为阴性对照。荧光标记液制备：0.87毫克DMB、44.3微升HAc、29.1微升2-Mercaptoethanol、39.6微升Na 2S 2O 4，加入至162.3微升水中。等体积的样品与荧光标记液混匀，于50℃加热3小时，然后加水终止反应。流动相：70毫升甲醇、90毫升乙腈，加入840毫升水，混匀。设置HPLC仪器流速为0.5毫升/分钟，时间60分钟，上样量为10微升，激发光373nm，发射光448nm。收集色谱结果并绘制相应的标准曲线，计算本申请糖基化修饰的促红细胞生成素的纯化产物中Neu5Ac和Neu5Gc含量，如下表1所示。
表1样品中Neu5Ac和Neu5Gc含量
样品 Neu5Ac含量(mol/mol) Neu5Gc含量(mol/mol)
Darbe 21.293 0.069
lot#20190302-2 21.214 0.420
lot#20190303-2 20.530 0.332
lot#20190304-1 20.947 0.343
通过高效液相色谱质谱联用法(HPLC-MS)分析本申请糖基化修饰的促红细胞生成素的N-连接糖基化组成。利用Zeba离心柱将15微克实施例1中纯化所得本申请的糖基化修饰的促红细胞生成素三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，换液至水中，旋转蒸干，然后加入HPLC水复溶至2毫克/毫升，Darbepoetin(Darbe，lot#1078765A)作为对照。向样品中加入6微升5％RapiGest SF溶液和15.3微升水，混匀，并置于95℃加热5分钟，静置至室温。加入1.2微升Rapid PNGase F，50℃加热15分钟，室温静置冷却。向上述糖基释放混合物中加入12微升RapiFlour-MS溶液，混匀，室温标记10分钟，然后加入358微升乙腈。利用GlycoWorks HILIC μElution小柱提取糖混合物，最终糖混合物溶于90微升SPE洗脱缓冲液中，加入100微升二甲基甲酰胺(DMF)和210微升乙腈稀释洗脱液。流动相A：50mM甲酸铵，pH 4.4；流动相B：乙腈。色谱条件：上样量10微升；激发光265nm，发射光425nm；柱温60℃；有效分离时间35分钟，洗脱梯度为25％—46％流动相A。质谱仪设置为正离子分辨率模式，MS采集，锥孔电压50V，毛细管电压3.0kV，脱溶剂温度250℃， 扫描范围400-3000m/z，扫描时间0.5秒。
结果如图5所示，本申请糖基化修饰的促红细胞生成素与Darbepoetin相比，4分支的糖种类多且占比例大，而且所含N-乙酰乳糖胺的比例更高。Darbepoetin中FA4G4S4、FA4G4L1S4和FA4G4L2S4的聚糖结构比率分别为15.85％、5.36％和1.43％，本申请糖基化修饰的促红细胞生成素的纯化样品中FA4G4S4、FA4G4L1S4和FA4G4L2S4的聚糖结构比率分别为14.02％、21.58％和18.17％。
实施例3糖基化修饰的促红细胞生成素的亲和力分析
通过高通量分子相互作用分析平台(GE，Biacore 8K)对本申请糖基化修饰的促红细胞生成素与EPO受体的亲和力进行检测。设置流动池温度为25度，样品室温度为25度，选用传感器芯片为CM5。将实施例1中纯化所得本申请的糖基化修饰的促红细胞生成素三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，与EPO受体(Sinobiological，10707-H08H)分配于微盘中，Darbepoetin(Darbe，lot#1078765A)作为对照，运行仪器进行检测。与EPO受体的亲和力检测如下表2所示，本申请糖基化修饰的促红细胞生成素的纯化样品受体亲和力比Darbepoetin的亲和力低。
表2样品与EPO受体的亲和力
Figure PCTCN2021119332-appb-000002
实施例4糖基化修饰的促红细胞生成素的细胞活性分析
复苏人血液白血病细胞TF-1(
Figure PCTCN2021119332-appb-000003
CRL-2003 TM)并传代2次。梯度稀释实施例1中纯化所得本申请的糖基化修饰的促红细胞生成素三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，Darbepoetin(Darbe，lot#1078765A)作为对照，稀释范围从5000纳克/毫升至0.03纳克/毫升。分别取50微升稀释后的样品加入至96孔板中，然后置于37℃、5％CO 2培养箱中。将已复苏的TF-1细胞转移至离心管中，800转/分钟离心收集细胞，利用基础培养基(由RPMI-1640加10％FBS)重悬细胞，将制备的细胞悬浊液加入至含 有样品的96孔板中，1×10 4细胞每孔，混合均匀后将96孔板置于37℃、5％CO 2培养箱中培养3天，加入MTS(Promega，G3580)显色，最终检测490nm处各孔的吸光值，对所得结果进行分析。
结果如图6所示，表3所示TF-1细胞增殖结果，本申请糖基化修饰的促红细胞生成素的纯化样品在0.52ng/mL-800ng/mL范围内，体外活性随浓度的升高而增强，表现出明显的量效关系，样品相对效价＝Darbepoetin的EC 50值/样品EC 50值。而且，本申请糖基化修饰的促红细胞生成素的纯化样品的EC 50值比Darbepoetin要高，表明本申请的样品与EPO受体的亲和力比Darbepoetin要低。
表3样品对TF-1细胞增殖的效果(N/A表示无数据)
Figure PCTCN2021119332-appb-000004
实施例5糖基化修饰的促红细胞生成素的体内药效检测
准备9周龄雌性免疫缺陷小鼠CD-1(体重约为25克)，采用皮下注射、单次给药方式，给予小鼠实施例1中纯化所得本申请的糖基化修饰的促红细胞生成素三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，PBS作为空白组(vehicle组)，Darbepoetin(Darbe，lot#1078765A)作为对照组A和EPO(利血宝，lot#17Y03B)作为对照组B，给药 剂量为15微克/千克。在给药后7天，心脏取血，血液样品加入EDTA-Na 2，置于4℃保存。利用血液分析仪检测小鼠血红蛋白含量、红细胞水平、红细胞比容、网织红细胞水平的结果。
结果如图7显示，给药7天后，注射本申请的样品的小鼠血红蛋白含量、红细胞水平、红细胞比容、网织红细胞水平明显高于空白组小鼠，注射本申请的样品的升高作用强于注射Darbepoetin和EPO。
实施例6糖基化修饰的促红细胞生成素的体内半衰期分析
准备10周龄雄性SD大鼠(体重约为300克)，采用皮下注射、单次给药方式，给予大鼠实施例1中纯化所得本申请的糖基化修饰的促红细胞生成素三批纯化产物lot#20190302-2、lot#20190303-2、lot#20190304-1，Darbepoetin(Darbe，lot#1078765A)作为对照组A和EPO(利血宝，lot#17Y03B)作为对照组B，给药剂量为2微克/千克。在给药后分别于4小时、10小时、24小时、32小时、48小时、72小时、96小时尾静脉取血0.3毫升，室温静置2小时，1200g离心15分钟，取上清保存于-80℃，采用EPO Elisa试剂盒检测血液中样品含量。
结果如图8，表4所示，本申请的样品的血药浓度随时间变化曲线及结果，与EPO和Darbepoetin相比，本申请的样品体内清除速度明显降低，体内半衰期延长，本申请的样品三批纯化样品的皮下注射后大鼠半衰期分别为19.91小时，19.15小时和20.21小时，本申请的样品的消除半衰期比Darbepoetin长30％。
表4样品在体内半衰期
Figure PCTCN2021119332-appb-000005
Figure PCTCN2021119332-appb-000006
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (14)

  1. 一种糖基化修饰的促红细胞生成素，其含有结合到N-糖基化位点的聚糖结构，所述聚糖结构包含FA4G4L2S4。
  2. 根据权利要求1所述的糖基化修饰的促红细胞生成素，其中，所述FA4G4L2S4结构的比率为15％以上。
  3. 根据权利要求1-2中任一项所述的糖基化修饰的促红细胞生成素，其中所述聚糖结构包含FA4G4L1S4。
  4. 根据权利要求3所述的糖基化修饰的促红细胞生成素，其中所述FA4G4L1S4的比率为20％以上。
  5. 根据权利要求1-4中任一项所述的糖基化修饰的促红细胞生成素，其中所述聚糖结构包含FA4G4S4。
  6. 根据权利要求5所述的糖基化修饰的促红细胞生成素，其中所述FA4G4S4的比率为10％以上。
  7. 根据权利要求1-6中任一项所述的糖基化修饰的促红细胞生成素，其中所述聚糖结构包含Neu5Gc，其所述Neu5Gc的摩尔比率为0.5％以下。
  8. 根据权利要求1-7中任一项所述的糖基化修饰的促红细胞生成素，其包含SEQ ID NO.1所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的糖基化修饰的促红细胞生成素，其包含以下的N-糖基化位点：N24、N30、N38、N83和N88。
  10. 制备权利要求1-9中任一项所述的糖基化修饰的促红细胞生成素的方法，其包括以下的步骤：在表达权利要求1-9中任一项所述的糖基化修饰的促红细胞生成素条件下，培养包含编码SEQ ID NO:1所示的氨基酸序列的核酸分子的CHO-S细胞。
  11. 药物组合物，其包含权利要求1-9中任一项所述的糖基化修饰的促红细胞生成素和药学上可接受的佐剂。
  12. 权利要求1-9中任一项所述的糖基化修饰的促红细胞生成素，和/或权利要求11所述的药物组合物在制备药物中的用途，所述药物用于治疗贫血。
  13. 根据权利要求12所述的用途，其中所述贫血包括肾性贫血、多发性骨髓瘤贫血和/或癌性贫血。
  14. 延长促红细胞生成素半衰期的方法，其包括以下的步骤：向有需要的受试者施用权利要求1-9中任一项所述的糖基化修饰的促红细胞生成素。
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