WO2019219048A1 - Conjugué polypeptidique fusionné ayant une durée de demi-vie prolongée - Google Patents

Conjugué polypeptidique fusionné ayant une durée de demi-vie prolongée Download PDF

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WO2019219048A1
WO2019219048A1 PCT/CN2019/087144 CN2019087144W WO2019219048A1 WO 2019219048 A1 WO2019219048 A1 WO 2019219048A1 CN 2019087144 W CN2019087144 W CN 2019087144W WO 2019219048 A1 WO2019219048 A1 WO 2019219048A1
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fusion protein
seq
mpeg
amino acid
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PCT/CN2019/087144
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Chinese (zh)
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王亚里
刘宾
王晓山
陈宪
李相�
朱鹿燕
王淑亚
王双
王文文
黄灵丽
王齐磊
胡海涛
张莉莉
高洁
任子甲
肖春峰
苏鸿声
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北京辅仁瑞辉生物医药研究院有限公司
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Priority to CN201980033065.7A priority Critical patent/CN113166271A/zh
Publication of WO2019219048A1 publication Critical patent/WO2019219048A1/fr

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    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

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  • the invention relates to the field of protein engineering, in particular to a fusion polypeptide conjugate having an extended half-life and a preparation method and application thereof.
  • Recombinant protein drugs are important types in biopharmaceuticals and are often administered clinically by intravenous or subcutaneous injection. However, after administration, protein drugs often degrade, resulting in decreased activity and reduced bioavailability. Repeated and multiple doses are often required to achieve the desired blood concentration and efficacy, reducing patient compliance. Therefore, it is clinically necessary to develop long-acting protein drugs.
  • the principles of the common method of increasing the half-life of a protein drug include increasing the molecular weight of the protein drug, reducing the glomerular filtration rate, or reducing the in vivo clearance of the protein.
  • Strategies for the implementation of conventional protein drug long-acting include glycosylation, PEGylation, albumin fusion, transferrin fusion, Fc fusion, and the like.
  • the above modifications or modifications have an effect on the activity of the protein itself, and such modification/modification often fails to achieve the desired effect.
  • a polypeptide is fused to a fusion partner capable of prolonging half-life (eg, Fc fragment, albumin, XTEN, or transferrin), and further with a hydrophilic polymer (eg, poly.
  • Alkyl diols are effective to increase the in vivo stability of biologically active polypeptides, particularly when hydrophilic polymers (eg, PEG) have a branched structure (or branched structure)
  • hydrophilic polymers eg, PEG
  • the molecular weight of the hydrophilic polymer is still within a certain range, for example, greater than or equal to a specific value, the in vivo stability of the biologically active polypeptide can be remarkably improved, thereby completing the present invention.
  • a biologically active polypeptide fusion protein conjugated to a polyalkylene glycol wherein the biologically active polypeptide moiety is directly linked to an extended half-life fusion partner or indirectly linked by a peptide linker, and the fusion protein is further The polyalkylene glycol is conjugated.
  • an activity selected from the group consisting of hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunoregulatory factors , interleukins, interferons, tumor necrosis factor, transforming growth factor, growth factor, colony stimulating factor, chemokine, neuropeptide, insulin, GLP-1, GLP-1 receptor agonist, growth hormone, erythropoiesis , G-CSF and GM-CSF.
  • fusion protein according to embodiment 1 or 2, wherein the fusion partner is: an immunoglobulin Fc fragment, albumin, XTEN or transferrin, the fusion partner is for example derived from a human; preferably an IgG Fc fragment
  • the IgG Fc fragment has a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to the FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:
  • fusion protein according to any one of embodiments 1 to 4, wherein the fusion protein is conjugated to a polyalkylene glycol which is random or site-directed, the conjugation position being selected from the group consisting of a free amino group and a thiol group. , a glycosyl group and/or a carboxyl group, preferably a free amino group.
  • the modifying agent may be a modified ester in the form of an activated ester, for example, the modifying agent is selected from the following formula (1) , (2) or (3):
  • m1 is preferably 5;
  • mPEG represents a mono-terminated polyethylene glycol group having a molecular weight of between 5 KD and 60 KD;
  • m2 is preferably 2; 0 ⁇ m3 ⁇ 6, m3 is preferably 1; mPEG represents a mono-terminated polyethylene glycol group having a molecular weight of 5KD-100KD Dalton, preferably 40KD , 50KD, 60KD, most preferably 40KD; or
  • 0 ⁇ m4 ⁇ 6, and m4 is preferably 2; mPEG represents a mono-terminated polyethylene glycol group having a molecular weight of 5KD-100KD.
  • fusion protein according to any one of embodiments 1 to 6, wherein the biologically active polypeptide moiety is linked to the fusion partner via a peptide linker comprising a flexible peptide linker and/or a rigid unit,
  • a peptide linker comprising a flexible peptide linker and/or a rigid unit
  • 1, 2, 3, 4, 5 or more of the rigid units may be included.
  • the flexible peptide linker comprises two or more amino acid residues selected from the group consisting of glycine, serine, alanine and threonine,
  • the flexible peptide linker has the sequence formula (GS) a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+ c+d ⁇ 1,
  • the flexible peptide linker has a sequence selected from the group consisting of:
  • the amino acid sequence of the terminal peptide has a consistency of 70%, 80%, 90%, 95% or higher; the rigid unit may comprise 1, 2 or more glycosylation sites;
  • the rigid unit comprises an amino acid sequence selected from the group consisting of:
  • SRLPGPSDTPILPQ SEQ ID NO: 14
  • the peptide linker comprises the sequence set forth in SEQ ID NO: 15.
  • a pharmaceutical composition comprising an effective amount of the fusion protein of any one of embodiments 1 to 9, and a pharmaceutically acceptable carrier.
  • a method for preventing and/or treating a disease which can be prevented and/or treated by the activity of a biologically active polypeptide which comprises administering the fusion protein according to any one of embodiments 1 to 9 or administering to a subject in need thereof.
  • a method of improving the half-life of a biologically active polypeptide wherein the biologically active polypeptide moiety is directly linked to a half-life enhancing fusion partner or indirectly linked to a peptide linker and further conjugated to a polyalkylene glycol.
  • the biologically active polypeptide moiety is capable of conferring the fusion protein an activity selected from the group consisting of hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunoregulatory factors, and leukocyte mediators. , interferon, tumor necrosis factor, transforming growth factor, growth factor, colony stimulating factor, chemokine, neuropeptide, insulin, GLP-1, GLP-1 receptor agonist, growth hormone, erythropoietin, G - CSF and GM-CSF.
  • an activity selected from the group consisting of hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunoregulatory factors, and leukocyte mediators. , interferon, tumor necrosis factor, transforming growth factor, growth factor, colony stimulating factor, chemokine, neuropeptide, insulin, GLP-1, GLP-1 receptor agonist, growth hormone, erythropoietin, G - CSF and GM-CSF.
  • the fusion partner is: an immunoglobulin Fc fragment, albumin, XTEN or transferrin, said fusion partner being for example derived from a human; preferably an IgG Fc fragment;
  • the IgG Fc fragment has a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to the FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:
  • the conjugation position is selected from the group consisting of a free amino group, a thiol group , a glycosyl group and/or a carboxyl group, preferably a free amino group.
  • the modifying agent may be a modified ester in the form of an activated ester and other types of modifying agents, more preferably
  • the modifier is selected from the following formula (1), (2) or (3):
  • m1 is preferably 5;
  • mPEG- represents a mono-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (1) is between 5KD and 60KD;
  • 0 ⁇ m2 ⁇ 6, m2 is preferably 2; 0 ⁇ m3 ⁇ 6, m3 is preferably 1; mPEG- represents a methoxy-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (2) 5KD-100KD, preferably 40KD, 50KD, 60KD, most preferably 40KD;
  • m4 is preferably 2; mPEG- represents a methoxy-mono-terminated polyethylene glycol group, and the modifier represented by formula (3) has a molecular weight of 5KD-100KD.
  • the flexible peptide linker comprises two or more amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine,
  • the flexible peptide linker has the sequence formula (GS) a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+ c+d ⁇ 1,
  • the flexible peptide linker has a sequence selected from the group consisting of:
  • the amino acid sequence of the terminal peptide has a consistency of 70%, 80%, 90%, 95% or higher; the rigid unit may comprise 1, 2 or more glycosylation sites;
  • the rigid unit comprises an amino acid sequence selected from the group consisting of:
  • SRLPGPSDTPILPQ SEQ ID NO: 14
  • the peptide linker comprises the sequence set forth in SEQ ID NO: 15.
  • Figure 1a shows the results of SEC-HPLC of FVIII-Fc (FF-0) without mPEG modification.
  • Figure 1b shows the results of SEC-HPLC of 5K molecular weight mPEG modified FVIII-Fc (FF-5L).
  • Figure 1c shows the results of SEC-HPLC of 10K molecular weight mPEG modified FVIII-Fc (FF-10L).
  • Figure 1d shows the results of SEC-HPLC of 20K molecular weight mPEG modified FVIII-Fc (FF-20L).
  • Figure 1e shows the results of SEC-HPLC of 30K molecular weight mPEG modified FVIII-Fc (FF-30L).
  • Figure 1f shows the results of SEC-HPLC of 40K molecular weight mPEG modified FVIII-Fc (FF-40L).
  • Figure 2a shows the results of SEC-HPLC of the non-mPEG-modified FVIII-Linker1-Fc (FL1F-0) (purity >99%, polymer ⁇ 1%).
  • Figure 2b shows the results of SEC-HPLC of 20K molecular weight mPEG modified FVIII-L1-Fc (FL1F-20L) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 2c is a SEC-HPLC assay of linear, 30K molecular weight mPEG modified FVIII-L1-Fc (FL1F-30L) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 2d is a SEC-HPLC assay of linear, 40K molecular weight mPEG modified FVIII-L1-Fc (FL1F-40L) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 2e is a SEC-HPLC assay of linear 50 g molecular weight mPEG modified FVIII-L1-Fc (FL1F-50L) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 2f shows the results of SEC-HPLC of Y--40K molecular weight mPEG modified FVIII-L1-Fc (FL1F-40Y) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 3a shows the results of SDS-PAGE before and after liquid exchange of hFVIII-Fc (FF-0) stock solution G25 without modification of mPEG (H stands for reduction and F stands for non-reduction).
  • Figure 3b shows the results of SDS-PAGE (non-reduction) of hFVIII-Fc cross-linking with different molecular weights of mPEG (FF-5L to FF-40L).
  • Figure 3c shows the results of SDS-PAGE detection (reduction) of hFVIII-Fc cross-linked with different molecular weights mPEG (FF-5L to FF-40L).
  • polypeptide refers to a compound formed by dehydration condensation of a plurality of amino acid molecules.
  • polypeptide has the same meaning as “protein”, “protein” or “peptide” and can be used interchangeably;
  • biologically active polypeptide refers to a protein that is capable of exerting an effect on a particular physiological or pathological process of an organism, including: growth, development, apoptosis, death, catalysis, angiogenesis, pathology, tumorigenesis, Metastasis, signal transduction, coagulation, blood glucose and lipid regulation, etc., including but not limited to activities selected from the group consisting of hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunoregulatory factors, interleukins, interferons , tumor necrosis factor, transforming growth factor, growth factor, colony stimulating factor, chemokine, neuropeptide, insulin, GLP-1, GLP-1 receptor agonist, growth hormone, erythropoietin, G-CSF and GM -CSF.
  • fusion protein is also referred to as a fusion polypeptide, a chimeric polypeptide, or a chimeric protein, and is obtained by directly or two or more genes which independently encode different proteins, and which are obtained by translation and translation. A single protein of the functional properties of an original protein.
  • fusion partner refers to another polypeptide that is fused to a polypeptide of interest (ie, a polypeptide that is desired to extend its half-life) and an effect-enhancing variant thereof that is capable of altering the half-life of the fusion protein by a variety of different mechanisms.
  • the fusion partner delays in vivo clearance of the polypeptide of interest by interacting with a neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • the fusion partner is the Fc domain (Fc region) of an immunoglobulin, albumin, XTEN or transferrin or a portion thereof.
  • the IgG Fc domain is preferred due to the longer half-life of the IgG antibody.
  • the Fc domain may also be modified to improve other functions, such as complement binding and/or binding to certain Fc receptors. Mutations at positions 234, 235 and 237 in the IgG Fc domain will generally result in reduced binding to the Fc ⁇ RI receptor. It may also result in reduced binding to the Fc ⁇ RIIa and Fc ⁇ RIII receptors. These mutations do not alter binding to the FcRn receptor, which promotes long circulating half-life through the endocytic recycling pathway.
  • the modified IgG Fc domain of the fusion protein of the invention comprises one or more of the following mutations which will result in decreased affinity for certain Fc receptors (L234A, L235E and G237A) and C1q-mediated, respectively.
  • the guided complement binding is reduced (A330S and P331S).
  • polyalkylene glycol is a hydrophilic polymer which is conjugated to a specific position on a biologically active polypeptide and/or fusion partner in the present invention, and the polyalkylene glycol may be linear or Branched and may comprise one or more independently selected polymeric moieties.
  • the polyalkylene glycol is polyethylene glycol including m-PEG, polypropylene glycol including m-PPG, and the like.
  • the polyalkylene glycol in the present invention may be polyethylene glycol (PEG), and the main chain may be linear or branched.
  • Branched polymer backbones are well known in the art.
  • a branched polymer has a central branched core portion and one or more linear polymer chains attached to the central branched core. It is preferred in the present invention to use PEG in branched form.
  • the branched polyethylene glycol can be represented by the formula R(-PEG-OH)m, wherein R represents a core moiety, such as glycerol or pentaerythritol, and m represents the number of arms.
  • the number of branches in the branched PEG or mPEG is 2, also referred to herein as "Y-type" PEG or mPEG, ie, a branched PEG comprising two PEG or linear methoxy PEG .
  • polystyrene resin examples include, but are not limited to, other polyalkylene glycols (e.g., polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, etc.), polyoxyethylated polyols, olefmic alcohols , polyvinylpyrrolidone, polyhydroxypropylmethacrylamide, poly([ ⁇ ]-hydroxy acid), polyvinyl alcohol, polyphosphazene, polyoxazoline, poly-N-acryloylmorpholine and copolymers thereof, Meta-copolymers and mixtures.
  • PPG polypropylene glycol
  • copolymers of ethylene glycol and propylene glycol etc.
  • polyoxyethylated polyols examples include, but are not limited to, other polyalkylene glycols (e.g., polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, etc.), polyoxyeth
  • PEG modification is used, more preferably mPEG modification, wherein the modification is a random modification or a site-directed modification, the position of the modification comprising a free amino group, a thiol group, a glycosyl group, and/or a carboxyl group.
  • the modifier for the random modification of the free amino group of mPEG may be selected from the group consisting of: mPEG-SS (methoxypolyethylene glycol-succinimide succinate), mPEG-SC (methoxypolyethylene glycol-succinimide carbonate), mPEG-SPA (methoxypolyethylene glycol-succinimidyl propionate) and mPEG-SG (methoxypolyethylene glycol- Succinimide glutarate) and the like.
  • mPEG-SS methoxypolyethylene glycol-succinimide succinate
  • mPEG-SC methoxypolyethylene glycol-succinimide carbonate
  • mPEG-SPA methoxypolyethylene glycol-succinimidyl propionate
  • mPEG-SG methoxypolyethylene glycol- Succinimide glutarate
  • the N-terminal modifiers are: mPEG-ALD (methoxy polyethylene glycol-acetaldehyde), mPEG-pALD (methoxy polyethylene glycol-propionaldehyde) and mPEG-bALD (methoxy polyethylene glycol) -butyraldehyde) and the like.
  • the modifiers mPEG-SS, mPEG-SC, mPEG-SPA, mPEG-SG, mPEG-ALD, mPEG-pALD, mPEG-bALD are linear or branched in shape.
  • the modifier used in the random thiol-based modification is mPEG-mal (methoxypolyethylene glycol-maleimide), mPEG-OPSS (methoxypolyethylene) One of alcohol-o-dithiopyridine), mPEG-Vinylsulfone (methoxypolyethylene glycol-vinylsulfone), and mPEG-Thiol (methoxypolyethylene glycol-thiol).
  • the modifier used in the random modification of the glycosyl and/or carboxyl groups is mPEG-ZH (methoxypolyethylene glycol-hydrazide).
  • the structure of the mPEG-modified modifier is as shown in formula (1):
  • m1 is preferably 5; mPEG- represents a mono-terminated polyethylene glycol group, and the modifier represented by formula (1) has a molecular weight of 5 kD-60 kD (kD, thousand Daoer) Preferably, it is 40 kD.
  • the mPEG random modification of the free amino group is carried out using the modifying agent represented by the formula (1).
  • the structure of the mPEG-modified modifier is as shown in formula (2):
  • m2 is preferably 2; 0 ⁇ m3 ⁇ 6, m3 is preferably 1; mPEG- represents a methoxy-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (2) It is 5 kD to 60 kD, preferably 40 kD.
  • the mPEG random modification of the free amino group is carried out using the modifier shown in formula (2).
  • the structure of the mPEG-modified modifier is as shown in formula (3):
  • m4 is preferably 2; mPEG- represents a methoxy-mono-terminated polyethylene glycol group, and the modifier represented by the formula (3) has a molecular weight of 5 kD to 60 kD, preferably 40 kD.
  • a free thiol-based mPEG random modification is carried out using a modifying agent represented by formula (3).
  • the size of the polymer backbone can vary, but polymers (e.g., PEG, mPEG, PPG, or mPPG) typically range from about 0.5 KD to about 160 KD, such as from about 1 KD to about 100 KD. More specifically, the size of each of the conjugated hydrophilic polymers of the present invention varies primarily in the range of from about 1 KD to about 80 KD, from about 2 KD to about 70 KD; from about 5 KD to about 70 KD; from about 10 KD to about 60 KD, about 20KD to about 50KD; about 30KD to about 50KD or about 30KD-40KD. It should be understood that these sizes represent approximate values and are not accurate measurements, which is recognized in the art.
  • the size of the PEG or mPEG used in the present invention is 35 KD or more (ie, not less than 35 KD), preferably not less than 40 KD, not less than 45 KD, not less than 50 KD, not less than 55 KD, not less than 60 KD, Not less than 65KD or not less than 70KD, for example, the molecular weight is specifically 40KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD, 110KD, 120KD, 130KD, 140KD, 150KD or 160KD
  • modified circulating half-life The molecule of the invention has an altered circulating half-life, preferably an increased circulating half-life, as compared to a wild-type factor biologically active polypeptide.
  • the cyclic half-life is preferably increased by at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 100%, more preferably At least 125%, more preferably at least 150%, more preferably at least 175%, more preferably at least 200%, and most preferably at least 250% or 300%. Even more preferably, the molecule has an increase in circulating half-life of at least 400%, 500%
  • pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present application can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present application can also be formulated into a sustained release preparation.
  • hFVIII fusion protein expression plasmids were constructed according to molecular cloning techniques well known to those skilled in the art, and the expression plasmids were transfected into DHFR-deficient CHO cells, respectively (see U.S. Patent No. 4,818,679), to express each hFVIII. Fusion protein (Table 1). The specific preparation procedure for the fusion protein is described in Chinese Patent No. ZL201610692838.0, which is incorporated herein in its entirety by reference.
  • BDD FVIII BDD FVIII, consisting of 90kD A1-A2 heavy chain and 80kD light chain;
  • **SEQ ID NO. 7 - SEQ ID NO. 12 indicates that the adaptor is ligated from the rigid unit set forth in SEQ ID NO. 12 to the C-terminus of the flexible peptide linker set forth in SEQ ID NO. 7;
  • SEQ ID NO. - SEQ ID NO. 11 shows that the linker is formed by the rigid unit shown by SEQ ID NO. 11 linked to the C-terminus of the flexible peptide linker shown by SEQ ID NO.
  • each 1.1.1 fusion protein is subjected to centrifugation and filtration, followed by Affinity chromatography/Hydrophobic Interaction Chromatography/Ion-Exchange chromatography. / Size exclusion chromatography, respectively obtained five hFVIII fusion proteins FF-0, FL1F-0, FL2F-0, F (full length) L1F'-0 and F (full length) L2F" -0, SEC-HPLC detected the polymer ⁇ 5%.
  • the five hFVIII fusion proteins were separately prepared as a protein concentration of 0.95mg / ml hFVIII fusion protein stock;
  • Buffer preparation 20 mM Hepes, 0.1 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8.0, pH 7.0;
  • mPEG-SC weigh mPEG-SC according to the ratio of hFVIII fusion protein and mPEG-SC (purchased from Beijing Keykai Technology Co., Ltd.) molar ratio (1:1):100 (molecular weight 5kD, 10kD respectively) , 20kD, 30kD, 40kD structure such as the linear L-shaped mPEG-SC shown in formula (1), and the molecular weight of 40kD structure as shown in formula (2), branched-chain Y-shaped mPEG-SC), added 1.1.
  • Buffer preparation 20 mM histidine, 0.1 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8.0, pH 7.0;
  • Binding buffer 20 mM histidine, 0.1 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8.0, pH 7.0; elution buffer: 20 mM histidine, 2.0 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8. 0, pH 7.0; CIP: 0.5 M NaOH;
  • test results of FF-0 to FF-40L are shown in Figures 1a-1f.
  • results of FL1F-0 to FL1F-50L and FL1F-40Y are shown in Figures 2a-2f.
  • the results show that the purity of FL1F-0 to FL1F-60L is >95%, the polymer is ⁇ 5%, and the uncrosslinked ⁇ 1%.
  • the product obtained in the item "1.2.2” is subjected to SDS-PAGE detection, and includes the following steps:
  • Glue 1 ⁇ Tris-glycine electrophoresis buffer: SDS 0.4 g, Tris base 1.21 g, glycine 7.5 g, and double distilled water to 400 mL.
  • sample preparation the sample to be tested is mixed with an equal volume of loading buffer, and an equal volume of 0.1 mg/mL of 2-mercaptoethanol is added to the sample by reduction SDS-PAGE; non-reducing SDS-PAGE is not added 2- Mercaptoethanol. After the sample was mixed with the loading buffer, it was bathed in boiling water for 10 minutes.
  • Electrophoresis 10 ⁇ l of the sample to be tested and the protein Marker were sequentially added to the spotting hole, and concentrated electrophoresis was carried out at a voltage of 60 V. It was observed that the bromophenol blue dye was concentrated to the separation gel and the voltage was increased to 120 V. Separate and electrophoresis until the bromophenol blue dye reaches the bottom of the separation gel and turn off the power.
  • Example 2 Indirect determination of in vitro activity of mPEG-modified hFVIII fusion protein by chromogenic substrate method
  • the activity of the mPEG-modified hFVIII fusion protein prepared in Example 1 was determined using a chromogenic substrate assay.
  • the Chromogenix Coatest SP FVIII kit (Chromogenix, Ref. K824086) was used to determine the principle: when activated by thrombin, FVIIIa binds to FIXa in the presence of phospholipids and calcium ions to form an enzyme complex, which in turn activates factor X transformation. Into its active form Xa. Activation of the formed factor Xa can then cleave its specific chromogenic substrate (S-2765), releasing the chromophoric group pNA.
  • Eloctate is a recombinant Factor VIII Fc fusion protein already marketed by Bioverativ, which has not been modified by mPEG.
  • the human coagulation factor VIII titer used in the present invention is also referred to as the first-stage method.
  • One-stage assay for FVIII biological activity was performed by correcting the ability of FVIII-deficient plasma to cause prolonged clotting time.
  • a kit Coagulation Factor VIII Deficient Plasma (Cat. No. OTXW17) manufactured by the German company Siemens was used. The method comprises: first, diluting a known potency FVIII activity standard, WHO International Standard 8th International Standard Factor VIII Concentrate (Cat. No.
  • mice After stopping bleeding (no significant blood outflow at the incision), the rat tail was removed from the saline tube, and the mouse was placed on a 37 ° C heating pad to maintain its body temperature without touching the wound. After the mice were awakened, they were placed in a rat cage padded with A4 white paper, kept in a single cage, and replaced with white paper or a mouse cage after each observation to determine the degree of bleeding. After the tail was counted, the survival rate of the mice within 48 hours and the number of rebleeding within 12 hours after the tailings were counted (12 hours in total, and the number of bleedings within one hour was counted once). The results are shown in Table 5.
  • the rate of re-bleeding rate is the proportion of mice with re-bleeding during the statistical period.
  • the severe bleeding rate is the phenomenon of severe bleeding (+++) or multiple moderate hemorrhage (++) in the re-bleeding statistics within 12. The proportion of mice.
  • moderate bleeding means: A4 white paper has a lot of blood, covering an area of not less than 30%, and the blood mark is medium in color, but there is no large area of blood beach (area > 3cm2); severe bleeding (++ +) means: A4 white paper has a lot of blood on the surface, the coverage area is not less than 30%, the blood mark is heavy, and there is a large area of blood beach; even if the coverage area is less than small, it can be regarded as severe bleeding (large blood loss in mice) The range of activity is reduced, and the blood is heavily wetted with white paper).

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Abstract

La présente invention concerne une protéine fusionnée polypeptidique biologiquement active conjuguée à un polyalkylène glycol et ayant une durée de demi-vie cyclique prolongée, un procédé de préparation correspondant et une utilisation associée. Une fraction polypeptidique biologiquement active et un partenaire de fusion sont directement ou indirectement liés par un lieur peptidique, et ladite protéine fusionnée est en outre conjuguée à un polyalkylène glycol. La période de demi-vie est améliorée de manière significative par rapport à une protéine fusionnée polypeptidique biologiquement active non modifiée par polyalkylène glycol.
PCT/CN2019/087144 2018-05-18 2019-05-16 Conjugué polypeptidique fusionné ayant une durée de demi-vie prolongée WO2019219048A1 (fr)

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