CN116732066A - 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 - Google Patents
一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 Download PDFInfo
- Publication number
- CN116732066A CN116732066A CN202310417526.9A CN202310417526A CN116732066A CN 116732066 A CN116732066 A CN 116732066A CN 202310417526 A CN202310417526 A CN 202310417526A CN 116732066 A CN116732066 A CN 116732066A
- Authority
- CN
- China
- Prior art keywords
- factor
- mutant
- blood coagulation
- coagulation factor
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010076282 Factor IX Proteins 0.000 title claims abstract description 90
- 230000000694 effects Effects 0.000 title claims abstract description 43
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 19
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 239000013598 vector Substances 0.000 claims abstract description 29
- 238000001415 gene therapy Methods 0.000 claims abstract description 14
- 102000008300 Mutant Proteins Human genes 0.000 claims abstract description 11
- 108010021466 Mutant Proteins Proteins 0.000 claims abstract description 11
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 102100022641 Coagulation factor IX Human genes 0.000 claims description 67
- 229960004222 factor ix Drugs 0.000 claims description 44
- 208000009429 hemophilia B Diseases 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 11
- 102000037865 fusion proteins Human genes 0.000 claims description 10
- 208000031169 hemorrhagic disease Diseases 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 9
- 108020001507 fusion proteins Proteins 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 3
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 3
- 108060003951 Immunoglobulin Proteins 0.000 claims description 3
- 102000004338 Transferrin Human genes 0.000 claims description 3
- 108090000901 Transferrin Proteins 0.000 claims description 3
- 102000015395 alpha 1-Antitrypsin Human genes 0.000 claims description 3
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 claims description 3
- 229940024142 alpha 1-antitrypsin Drugs 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 102000018358 immunoglobulin Human genes 0.000 claims description 3
- 239000012581 transferrin Substances 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 6
- 238000004519 manufacturing process Methods 0.000 claims 6
- 229940105774 coagulation factor ix Drugs 0.000 abstract description 9
- 238000004108 freeze drying Methods 0.000 abstract description 8
- 102200052251 rs199469629 Human genes 0.000 abstract description 7
- 239000004017 serum-free culture medium Substances 0.000 abstract description 7
- 230000035772 mutation Effects 0.000 abstract description 5
- 238000012258 culturing Methods 0.000 abstract description 4
- 238000001914 filtration Methods 0.000 abstract description 4
- 230000001939 inductive effect Effects 0.000 abstract description 4
- 230000010412 perfusion Effects 0.000 abstract description 4
- 230000001131 transforming effect Effects 0.000 abstract description 4
- 230000006378 damage Effects 0.000 abstract description 3
- 238000011049 filling Methods 0.000 abstract description 3
- 238000006467 substitution reaction Methods 0.000 abstract description 3
- 238000002560 therapeutic procedure Methods 0.000 abstract description 3
- 230000005856 abnormality Effects 0.000 abstract description 2
- 230000023555 blood coagulation Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 102000013831 Coagulation factor IX Human genes 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 102200063714 rs137852283 Human genes 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 206010053567 Coagulopathies Diseases 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 208000032843 Hemorrhage Diseases 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 230000000740 bleeding effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 102220643581 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1_R338L_mutation Human genes 0.000 description 3
- 239000013607 AAV vector Substances 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229940126585 therapeutic drug Drugs 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 2
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 208000031220 Hemophilia Diseases 0.000 description 2
- 208000009292 Hemophilia A Diseases 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000003114 blood coagulation factor Substances 0.000 description 2
- 230000035602 clotting Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 208000002109 Argyria Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102100037642 Elongation factor G, mitochondrial Human genes 0.000 description 1
- 101150039948 F9 gene Proteins 0.000 description 1
- 108010048049 Factor IXa Proteins 0.000 description 1
- 108010061932 Factor VIIIa Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010018852 Haematoma Diseases 0.000 description 1
- 101000880344 Homo sapiens Elongation factor G, mitochondrial Proteins 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 206010020608 Hypercoagulation Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940019700 blood coagulation factors Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000007820 coagulation assay Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 108010013935 factor IX-Padua Proteins 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 201000005665 thrombophilia Diseases 0.000 description 1
- 230000001732 thrombotic effect Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- PJVWKTKQMONHTI-UHFFFAOYSA-N warfarin Chemical compound OC=1C2=CC=CC=C2OC(=O)C=1C(CC(=O)C)C1=CC=CC=C1 PJVWKTKQMONHTI-UHFFFAOYSA-N 0.000 description 1
- 229960005080 warfarin Drugs 0.000 description 1
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/644—Coagulation factor IXa (3.4.21.22)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21022—Coagulation factor IXa (3.4.21.22)
-
- 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
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Epidemiology (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种高活性凝血因子IX突变体、重组蛋白与融合蛋白的制备与应用，突变体核苷酸序列如SEQ ID NO：1所示。制备方法包括如下步骤：(1)将凝血因子IX基因连入载体中，得到重组载体；(2)将上述重组载体转化宿主细胞，得到表达突变凝血因子IX蛋白细胞克隆；(3)于无血清培养基中连续灌流培养上述细胞克隆，诱导重组高活性凝血因子IX突变蛋白的表达；(4)分离纯化、过滤，最后灌装、冻干，即得。本发明证实了IX突变Arg384Gln确实导致凝血因子IX活性升高，而且没有导致除凝血机制异常之外的其他危害，对人体是安全的，打破了现有研究的技术偏见，具有很好的基因治疗和替代治疗前景。
Description
本申请是基于申请号为CN201610898732.6，申请日为2016年10月14日，发明名称为“一种高活性凝血因子IX突变体、重组蛋白与融合蛋白的制备与应用”的中国发明专利申请的分案申请。
技术领域
本发明属于血友病B领域，特别涉及一种高活性凝血因子IX突变体、重组蛋白与融合蛋白的制备与应用。
背景技术
血友病B是由于高活性凝血因子IX缺陷所导致的出血性疾病，重型病人的因子IX活性往往低于正常的1％，经常发生自发的出血导致肌肉血肿或者关节畸形。输注因子IX制剂(目前通常是体外重组表达的因子IX蛋白)补充患者体内的因子IX水平是目前唯一有效的治疗方法，但是需要频繁的给药。基因治疗是目前正在临床试验的治疗方法，通过病毒载体将正常的因子IX基因导入患者体内长期表达，从而达到提高因子IX水平，预防出血的目的。目前进行的基因治疗临床试验共三项。
2009年在意大利的padua的一个血栓性疾病的患者中发现一种突变的因子IX蛋白，Arg384Leu(R384L)，它的活性是正常人的7～9倍[X-Linked Thrombophilia with aMutant Factor IX(Factor IX Padua)，Paolo Simioni，The new england joumal ofmedicine]。随后的研究就把这种突变基因通过腺相关病毒载体(AAV)用于基因治疗(PCT/EP2009/061935)，临床试验获得了非常好的效果，病人的因子IX水平达到了正常的20％左右。
在专利中(PCT/EP2009/061935)，共提及了三种突变，均位于384位氨基酸，分别是Arg384Leu(r3841)，Arg384Asp(R384D)和Arg384Gln(R384Q)，其中，除Arg384Leu为自然发生外，其他两种为人工构建。体外表达的重组R384L的活性比正常的因子IX高7～9倍，R384D高4～5倍，而R384Q高13倍以上。因为Arg384Leu是在意大利的血栓患者中所发现的，除了形成血栓外，没有造成其他疾患，也没产生抗体，没有安全隐患，最终被专利持有人用于临床的基因治疗。选择Arg384Leu而不是活性更好的Arg384Gln的另外一条理由是担心活性更高导致血栓形成。
1999年发表于j.biol.chem的论文[Protease and EGF1 Domains of Factor IXaPlay Distinct Roles in Binding to Factor VIIIa，Vol.274，No.26，Issue of June25，pp.18477-18486，1999]，也描述了因子IX突变Arg384Gln，文章作者在体外重组表达了这个蛋白，但是结果并没有显示高活性，仅为正常的(65％)。
发明内容
本发明所要解决的技术问题是提供一种高活性凝血因子IX突变体、重组蛋白与融合蛋白的制备与应用，该突变体在人体中发现证实了凝血因子IX突变Arg384Gln确实导致因子IX活性升高，而且没有导致除凝血机制异常之外的其他危害，对人体是安全的，打破了现有研究的技术偏见；经过试验可以发现正进行临床试验的Arg384Leu活性相仿，具有很好的基因治疗与重组蛋白替代治疗前景。
本发明提供了一种高活性凝血因子IX突变体，核苷酸序列如SEQ ID NO：1所示，位置1151位的核苷酸为A而非G。
凝血因子IX表达编码优化序列如SEQ ID NO：3所示。
本发明还提供了一种高活性凝血因子IX的突变蛋白，位置384的氨基酸为Q而非R。
具体的氨基酸序列如SEQ ID NO：2所示。
本发明还提供了一种编码突变蛋白的核酸，或与所述编码核酸长度相同且与所述编码核酸完全互补的核酸。
本发明还提供了一种表达突变蛋白(hFIXR384Q)进行基因治疗的载体rAAV-hFIXcoR384Q，其制备和检验包括如下步骤：
(1)利用三质粒方法制备AAV载体：将rAAV-hFIXcoR384Q载体质粒，包含AAV的Rep/Cap的辅助质粒及包含腺病毒辅助基因的辅助质粒转染293细胞，然后将rAAV-hFIXcoR384Q载体纯化及检测滴度。其它载体rAAV-hFIXcoR384L和rAAV-hFIXco利用同样的方法制备。
(2)将纯化的rAAV-hFIXR384Q、rAAV-hFIXcoR384L和rAAV-hFIXco载体注射B型血友病小鼠。选用4-8周的血友病小鼠，通过尾静脉将三种载体以4x1011病毒颗粒/毫升分别注射6-7只小鼠。PBS注射3只小鼠为阴性对照。
(3)半凝固法检测IX因子活性。在注射前和注射后第2周和第四周通过眼睛取血获得血浆，然后通过半凝固法检测血浆中的IX因子活性。
本发明还提供了一种高活性凝血因子IX的突变蛋白的制备方法，包括如下步骤：
(1)将人野生型或因子IXArg384Gln突变的人凝血因子IX基因连入载体中，得到重组载体；(2)将上述重组载体转化宿主细胞，得到重组细胞克隆；
(3)于无血清培养基中连续灌流培养上述重组细胞克隆，诱导重组高活性凝血因子IX的突变蛋白的表达；
(4)分离纯化、过滤，最后灌装、冻干，得到所表达的高活性凝血因子IX的突变蛋白。
所述步骤(3)中的无血清培养基为“SAFC Biosciences EX-CELLTM 302”(商品化的试剂)。
所述步骤(4)中的纯化包括初纯和精纯。
本发明还提供了一种高活性凝血因子IX的突变蛋白的应用，应用于制备基因治疗药物。
本发明还提供了一种高活性凝血因子IX的突变蛋白的应用，应用于制备血友病B或其他出血性疾病的重组蛋白治疗药物。
本发明还提供了一种高活性凝血因子IX的突变蛋白的应用，应用于制备融合蛋白延长凝血因子IX突变体半衰期，正常凝血因子IX在血浆中的半衰期为22小时，肝脏蛋白质水解以及受体介导的内吞作用也将同样清除外源引入的重组凝血因子IX自然突变体。与凝血因子IX自然突变体融合的蛋白包括但不局限于人白蛋白、免疫球蛋白Fc、转铁蛋白以及alpha1抗胰蛋白酶等。
方案1.一种高活性凝血因子IX突变体，其特征在于：核苷酸序列如SEQ ID NO：1所示，位置1151位的核苷酸为A而非G。
方案2.一种如方案1所述的高活性凝血因子IX的突变蛋白，其特征在于：氨基酸序列如SEQ ID NO：2所示，位置384的氨基酸为Q而非R。
方案3.编码如方案2中所述的高活性凝血因子IX的突变蛋白的核酸，或与所述编码核酸长度相同且与所述编码核酸完全互补的核酸。
方案4.一种表达如方案2中所述的高活性凝血因子IX的突变蛋白的载体。
方案5.根据方案4所述的一种高活性凝血因子IX的突变蛋白的载体，其特征在于：所述载体为重组腺相关病毒载体rAAV。
方案6.一种高活性凝血因子IX的突变蛋白的制备方法，包括如下步骤：
(1)将人野生型或凝血因子IXArg384Gln突变的人凝血因子IX基因连入载体中，得到重组载体；
(2)将上述重组载体转化宿主细胞，得到表达重组凝血因子IXArg384Gln突变细胞克隆；
(3)于无血清培养基中连续灌流培养上述重组细胞克隆，诱导重组高活性凝血因子IX的突变蛋白的表达；
(4)分离纯化、过滤，最后灌装、冻干，得到所表达的高活性凝血因子IX的突变蛋白。
方案7.一种如方案2所述的高活性凝血因子IX的突变蛋白的应用，其特征在于：应用于制备基因治疗药物。
方案8.一种如方案2所述的高活性凝血因了IX的突变蛋白的应用，其特征在于：应用于制备血友病B或其他出血性疾病的重组蛋白治疗药物。
方案9.一种如方案2所述的高活性凝血因子IX的突变蛋白的应用，其特征在于：应用于制备凝血因子IX自然突变体融合蛋白，并将其施用于血友病B或其他出血性疾病的重组蛋白治疗药物。
方案10.根据方案9所述的一种高活性凝血因子IX的突变蛋白的应用，其特征在于：所述融合蛋白为人白蛋白、免疫球蛋白Fc、转铁蛋白或alpha 1抗胰蛋白酶。
有益效果
本发明证实了IX突变Arg384Gln确实导致因子IX活性升高，而且没有导致除凝血机制异常之外的其他危害，对人体是安全的，打破了现有研究的技术偏见；经过试验可以发现正进行临床试验的Arg338Leu活性相仿，具有很好的基因治疗和重组蛋白替代治疗前景。
附图说明
图1为本发明高活性凝血因子IX突变的示意图；
图2为本发明载体结构的示意图；
图3为小鼠血浆凝血因子IX活性。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
高活性凝血因子IX的突变蛋白的氨基酸序列如SEQ ID NO：2所示。
高活性凝血因子IX的突变蛋白的制备方法，包括如下步骤：
(1)将人野生型或因子IXArg384Gln突变的人凝血因子IX基因连入载体中，得到重组载体；
(2)将上述重组载体转化宿主细胞，得到重组菌株；
(3)于无血清培养基中连续灌流培养上述重组菌株，诱导重组高活性凝血因子IX的突变蛋白的表达；
无血清培养基为“SAFC Biosciences EX-CELLTM 302”(商品化的试剂)。为保证产品安全，防止血液来源制剂传播感染性疾病，故应用无血清培养基用于哺乳动物细胞培养、蛋白表达，细胞经对数期生长达到稳态后，将细胞密度维持在目标区间内，维持凝血因子IX高表达。
(4)分离纯化与冻干，从而得到所表达的凝血因子IX突变蛋白即相关融合蛋白。
在培养基收集后，经深层滤器澄清过滤并进一步分离纯化。纯化步骤分为初纯和精纯两个阶段，初纯：将过滤澄清的培养液在10倍超滤浓缩后通过有机溶剂/洗涤剂法灭活脂包膜病毒即HIV1/2、HCV以及HBV等；精纯：以离子交换(阴离子和阳离子)和分子筛等色谱层析方法进一步去除产品中残留以宿主细胞分泌的其他蛋白为主的杂质。纯化蛋白经过超滤换液、调节配方后再进行即20nm纳米膜除病毒过滤并冻干。冻干过程为速冻、淬火、冷冻、真空、主干燥、后干燥。冻干配方以甘氨酸、甘露醇、氯化钠、氯化钙等惰性糖类以及无机盐为主(组成为甘氨酸、甘露醇、氯化钠、氯化钙等；冻干时间为30小时)。
实施例2
高活性凝血因子IX的基因治疗AAV载体的制备方法，包括如下步骤：
(1)利用三质粒方法制备AAV载体。将rAAV-hFIXcoR384Q载体质粒，包含AAV的Rep/Cap的辅助质粒及包含腺病毒辅助基因的辅助质粒转染293细胞，然后将rAAV-hFIXcoR384Q载体进行氯化铯超速离心纯化及通过银染和定量PCR检测滴度。其它载体rAAV-hFIXcoR384L和rAAV-hFIXco利用同样的方法制备。
(2)将纯化的rAAV-hFIXR-384Q、rAAV-hFIXco-R384L和rAAV-hFIXco载体注射B型血友病小鼠。选用4-8周的血友病小鼠，通过尾静脉将三种载体以4x1011病毒颗粒/毫升分别注射6-7只小鼠。PBS注射3只小鼠为负对照。
(3)aPTT检测IX因子活性。在注射前和注射后第2周和第4周通过眼睛取血获得血浆，然后通过aPTT检测血浆中的IX因子活性。小鼠血浆凝血因子IX活性见图3。
凝血因子IX活性和抗原检测方法：
①凝固法检测凝血因子IX活性：
正常混合血浆用OV Buffer分别进行1∶10，1∶20，1∶40，1∶80，1∶160，1∶320稀释，血浆待测样本进行1∶10和1∶20稀释，细胞上清液不处理。取50微升稀释的正常混合血浆、待测血浆样本或转染凝血因子IX表达载体的细胞上清液，加入50微升凝血因子IX基质血浆，加入APTT试剂，37℃温育3分钟，再加入50微升氯化钙，ST4半自动血凝仪(Stago公司，法国)上记录凝固时间。以正常混合血浆1：10稀释的凝血因子IX活性为100％，以不同稀释度对应的凝固时间的log值和对应的活性得log值建立标准曲线，若相关系数R2大于0.95，则将待测样本的值带入计算，得到待测样本的凝血因子IX活性。
②双抗夹心法检测凝血因子IX的抗原：
用包被液(1.59g/L碳酸钠和2.94g/L碳酸氢钠，pH 9.6)将包被抗体(F9 ELISA试剂盒，Affinity Biologicals，EIA9-0035R1)1∶100稀释，加入稀释抗体100ul/孔，室温孵育2小时。重复洗涤3次。将正常混合血浆用样品稀释液(23.8g HEPES(free acid)/L，5.84g/LNaCI，3.72g/L Na2EDTA，10g/L BSA，0.1％吐温-20，Ph 7.2)分别1∶100对倍稀释至1∶3200。待测血浆样本以1∶200，1∶400和1∶800稀释，细胞上清液分别为原液、1∶10和1∶100稀释。每孔加入100ul稀释好的正混血浆或待测样品，室温放置90min。重复洗涤3次。将检测抗体用样品稀释液1∶100稀释，每孔加入100ul的稀释完的检测抗体，室温放置90min。重复洗涤3次。每孔加入100ul的OPD底物，待出现稳定的黄色之后(约5-10min)，每孔加入100ul的终止液。用酶标仪在450nm的波长下读取吸光度。建立标准曲线，并计算待测样本的抗原值。
③F9基因外显子扩增及测序引物和反应条件
1)PCR扩增
(1)引物：
F9 E1F CAACCTTAAGAArCTGACAGTAAAAA
F9 E1R TGCTGTCAAATCATGTAATCAAAA
F9 E2_3F GATTTTGGCTCCATGCCCTA
F9 E2_3R GTTCCCACACTGGCATAACC
F9 E4F CAGCTGGCTTCCAGGTCAGT
F9 E4R GCTTCTTGAACTCATATCCTGAAA
F9 E5F AACATGAATGCCCCCAATGT
F9 E5R TGATTTCAAAAGGAAGCAGATTCA
F9 E6F AGGATGGGCCTCAATCTCAA
F9 E6R GGAGGCCTTCTCACATTGGT
F9 E7F CATTCCATTTCTGCCAGCAC
F9 E7R TGACCCTTCTGCCTTTAGCC
F9 E8F GGTCAGTGGTCCCAAGTAGTCA
F9 E8R GGCTGGGCCCTTAGAAATG
(2)反应体系：包含1μl的10x HotStarTaq buffer，0.8μl的2.5mM dNTP，0.2μl的25mM Mg2+，0.06μl的5U/μl HotStarTaq polymerase，2μM的上下游引物各0.5μl，1μl的DNA模板，最后用H2O补足至10ul。
(3)反应条件：95℃15min；94℃15s，62℃40s(每个循环减0.5℃)，72℃1min，11个循环；94℃15s，56℃30s，72℃1min，24个循环；72℃2min。
2)测序
(1)测序引物
F9 E 1R TGCTGTCAAATCATGTAATCAAAA
F9 E2_3F GATTTTGGCTCCATGCCCTA
F9 E4F CAGCTGGCTTCCAGGTCAGT
F9 E5F AACATGAATGCCCCCAATGT
F9 E6R GGAGGCCTTCTCACATTGGT
F9 E7R TGACCCTTCTGCCTTTAGCC
F9 E8F GGTCAGTGGTCCCAAGTAGTCA
F9 E8R GGCTGGGCCCTTAGAAATG
(2)反应体系：3μl BigDye3.1 mIX，2μl浓度为1μM的测序引物，1-2μl纯化的PCR产物。
(3)反应条件：96℃1min；96℃10s，50℃5s，60℃4min，28个循环。患者及其家系成员凝血因子IX活性和抗原水平见表1：
表1
注：患者服用凝血因子IX合成抑制剂华法林治疗中，PT-INR 2.26。
Claims (10)
1.一种高活性凝血因子IX突变体，其特征在于：所述高活性凝血因子IX突变体的核苷酸序列如SEQ ID NO：1所示，位置1151位的核苷酸为A而非G。
2.如权利要求1所述的高活性凝血因子IX突变体在制备用于治疗血友病B或其他出血性疾病的基因治疗药物中的应用。
3.一种高活性凝血因子IX的突变蛋白，其特征在于：氨基酸序列如SEQ ID NO：2所示，位置384的氨基酸为Q而非R。
4.一种如权利要求3所述的凝血因子IX的突变蛋白在制备用于治疗血友病B或其他出血性疾病的药物中的应用。
5.一种凝血因子IX的重组蛋白在制备用于治疗血友病B或其他出血性疾病的药物中的应用，其特征在于，所述凝血因子IX的重组蛋白的氨基酸序列如SEQ ID NO：2所示，位置384的氨基酸为Q而非R。
6.一种凝血因子IX的突变蛋白的自然突变体融合蛋白在制备用于治疗血友病B或其他出血性疾病的药物中的应用，其特征在于，所述凝血因子IX的突变蛋白的氨基酸序列如SEQID NO：2所示，位置384的氨基酸为Q而非R。
7.根据权利要求6所述的应用，其特征在于，与所述凝血因子IX的突变蛋白的自然突变体融合的蛋白包括人白蛋白、免疫球蛋白Fc、转铁蛋白或alpha 1抗胰蛋白酶。
8.一种凝血因子IX突变体在制备用于治疗血友病B或其他出血性疾病的基因治疗药物中的应用，其特征在于，所述凝血因子IX突变体的核苷酸序列如SEQ ID NO：3所示。
9.一种rAAV-hFIXcoR384Q载体质粒，其特征在于，所述载体质粒的目的基因是凝血因子IX突变体，其核苷酸序列如SEQ ID NO：1所示。
10.如权利要求9所述的rAAV-hFIXcoR384Q载体质粒在制备用于治疗血友病B或其他出血性疾病的基因治疗药物中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310417526.9A CN116732066A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610898732.6A CN106497949A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
| CN202310417526.9A CN116732066A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610898732.6A Division CN106497949A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116732066A true CN116732066A (zh) | 2023-09-12 |
Family
ID=58294253
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310336787.8A Pending CN116355883A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
| CN202310417526.9A Pending CN116732066A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
| CN201610898732.6A Pending CN106497949A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310336787.8A Pending CN116355883A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610898732.6A Pending CN106497949A (zh) | 2016-10-14 | 2016-10-14 | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN116355883A (zh) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201420139D0 (en) | 2014-11-12 | 2014-12-24 | Ucl Business Plc | Factor IX gene therapy |
| EP3630974A1 (en) * | 2017-05-22 | 2020-04-08 | Baxalta Incorporated | Viral vectors encoding recombinant fix with increased expression for gene therapy of hemophilia b |
| MX2021001901A (es) * | 2018-08-20 | 2021-05-27 | Ucl Business Ltd | Nucleotidos de codificacion del factor ix. |
| US10842885B2 (en) | 2018-08-20 | 2020-11-24 | Ucl Business Ltd | Factor IX encoding nucleotides |
| GB2576508A (en) * | 2018-08-20 | 2020-02-26 | Ucl Business Plc | Factor IX encoding nucleotides |
| CN109439679A (zh) * | 2018-10-09 | 2019-03-08 | 上海市儿童医院 | 一种高活性重组人凝血因子ix融合蛋白及其制备方法 |
| CN117126832B (zh) * | 2023-10-26 | 2024-01-30 | 四川维亚本苑生物科技有限公司 | 一种重组人凝血因子ix融合蛋白及其制备方法 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4219547A3 (en) * | 2008-09-15 | 2023-10-18 | uniQure biopharma B.V. | Factor ix polypeptide mutant, its uses and a method for its production |
| TWI557135B (zh) * | 2010-11-03 | 2016-11-11 | 介控生化科技公司 | 經修飾之第九因子多胜肽及其用途 |
-
2016
- 2016-10-14 CN CN202310336787.8A patent/CN116355883A/zh active Pending
- 2016-10-14 CN CN202310417526.9A patent/CN116732066A/zh active Pending
- 2016-10-14 CN CN201610898732.6A patent/CN106497949A/zh active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN106497949A (zh) | 2017-03-15 |
| CN116355883A (zh) | 2023-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN116732066A (zh) | 一种高活性凝血因子ix突变体、重组蛋白与融合蛋白的制备与应用 | |
| US9982248B2 (en) | Factor IX polypeptide mutant, its uses and method for its production | |
| CN112126636B (zh) | 一种高活性凝血因子XI突变体Ala570Thr | |
| CN108220274B (zh) | 一种高活性凝血因子xi突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用 | |
| Faulkner et al. | Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 2. The isolation, crystallization and chemical characterization of mouse encephalomyocarditis virus | |
| WO2017028558A1 (zh) | sDR5-Fc融合蛋白突变体及其应用 | |
| CN110257358A (zh) | 一种高纯人凝血因子ix制剂的生产方法 | |
| CN109260462B (zh) | 一种凝血酶原突变体蛋白及其编码核酸的应用 | |
| CN116590262A (zh) | 一种凝血因子ix突变体及其应用 | |
| CN117126832B (zh) | 一种重组人凝血因子ix融合蛋白及其制备方法 | |
| JPS61103895A (ja) | ＨＢｓＡｇの精製方法 | |
| SIMIONI et al. | Patent 2737094 Summary | |
| JPH04182436A (ja) | 医薬組成物 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |