WO2024087834A1 - Growth hormone fc-fusion protein injection and use thereof - Google Patents
Growth hormone fc-fusion protein injection and use thereof Download PDFInfo
- Publication number
- WO2024087834A1 WO2024087834A1 PCT/CN2023/114241 CN2023114241W WO2024087834A1 WO 2024087834 A1 WO2024087834 A1 WO 2024087834A1 CN 2023114241 W CN2023114241 W CN 2023114241W WO 2024087834 A1 WO2024087834 A1 WO 2024087834A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- growth hormone
- concentration
- fusion protein
- composition according
- injection
- Prior art date
Links
- 102000018997 Growth Hormone Human genes 0.000 title claims abstract description 188
- 108010051696 Growth Hormone Proteins 0.000 title claims abstract description 188
- 239000000122 growth hormone Substances 0.000 title claims abstract description 187
- 239000007924 injection Substances 0.000 title claims description 113
- 238000002347 injection Methods 0.000 title claims description 113
- 108091006020 Fc-tagged proteins Proteins 0.000 title claims description 26
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 128
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 128
- 239000000203 mixture Substances 0.000 claims abstract description 104
- 239000003223 protective agent Substances 0.000 claims abstract description 72
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 36
- 201000010099 disease Diseases 0.000 claims abstract description 35
- 239000003814 drug Substances 0.000 claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 26
- 229930006000 Sucrose Natural products 0.000 claims description 53
- 239000005720 sucrose Substances 0.000 claims description 53
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 39
- 239000000872 buffer Substances 0.000 claims description 38
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 34
- 239000003381 stabilizer Substances 0.000 claims description 33
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 31
- QZNNVYOVQUKYSC-JEDNCBNOSA-N (2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid;hydron;chloride Chemical compound Cl.OC(=O)[C@@H](N)CC1=CN=CN1 QZNNVYOVQUKYSC-JEDNCBNOSA-N 0.000 claims description 31
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 31
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 31
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 31
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 28
- 229960000281 trometamol Drugs 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 26
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 21
- 125000000185 sucrose group Chemical group 0.000 claims description 19
- 206010056438 Growth hormone deficiency Diseases 0.000 claims description 17
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical group [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 13
- 230000002159 abnormal effect Effects 0.000 claims description 13
- 239000002738 chelating agent Substances 0.000 claims description 12
- 229930182817 methionine Natural products 0.000 claims description 12
- 239000003963 antioxidant agent Substances 0.000 claims description 11
- 229910052751 metal Inorganic materials 0.000 claims description 11
- 239000002184 metal Substances 0.000 claims description 11
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 10
- 150000001413 amino acids Chemical group 0.000 claims description 10
- 230000003078 antioxidant effect Effects 0.000 claims description 9
- 239000004094 surface-active agent Substances 0.000 claims description 9
- 206010053759 Growth retardation Diseases 0.000 claims description 7
- 208000030507 AIDS Diseases 0.000 claims description 5
- 208000001362 Fetal Growth Retardation Diseases 0.000 claims description 5
- 206010070531 Foetal growth restriction Diseases 0.000 claims description 5
- 201000010769 Prader-Willi syndrome Diseases 0.000 claims description 5
- 208000001647 Renal Insufficiency Diseases 0.000 claims description 5
- 208000026928 Turner syndrome Diseases 0.000 claims description 5
- 238000002512 chemotherapy Methods 0.000 claims description 5
- 208000030941 fetal growth restriction Diseases 0.000 claims description 5
- 201000006370 kidney failure Diseases 0.000 claims description 5
- 239000001509 sodium citrate Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 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 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 229940124274 edetate disodium Drugs 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 229940073585 tromethamine hydrochloride Drugs 0.000 claims description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- 229940071643 prefilled syringe Drugs 0.000 claims description 2
- 239000000600 sorbitol Substances 0.000 claims description 2
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims 1
- 235000019799 monosodium phosphate Nutrition 0.000 claims 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims 1
- 229940069328 povidone Drugs 0.000 claims 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 23
- 230000005856 abnormality Effects 0.000 abstract description 3
- 239000006172 buffering agent Substances 0.000 abstract 1
- 230000002265 prevention Effects 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 73
- 108090000623 proteins and genes Proteins 0.000 description 51
- 102000004169 proteins and genes Human genes 0.000 description 51
- 239000000243 solution Substances 0.000 description 24
- 238000001514 detection method Methods 0.000 description 23
- 102000002265 Human Growth Hormone Human genes 0.000 description 22
- 108010000521 Human Growth Hormone Proteins 0.000 description 22
- 239000000854 Human Growth Hormone Substances 0.000 description 22
- 229920001184 polypeptide Polymers 0.000 description 17
- 108090000765 processed proteins & peptides Proteins 0.000 description 17
- 102000004196 processed proteins & peptides Human genes 0.000 description 17
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 17
- 238000013368 capillary electrophoresis sodium dodecyl sulfate analysis Methods 0.000 description 16
- 239000000047 product Substances 0.000 description 12
- 239000008176 lyophilized powder Substances 0.000 description 10
- 238000002156 mixing Methods 0.000 description 10
- 238000001542 size-exclusion chromatography Methods 0.000 description 9
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 8
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 8
- 230000008901 benefit Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 238000003860 storage Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000002144 chemical decomposition reaction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000007774 longterm Effects 0.000 description 6
- 238000000108 ultra-filtration Methods 0.000 description 6
- 230000006872 improvement Effects 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 102000007562 Serum Albumin Human genes 0.000 description 4
- 108010071390 Serum Albumin Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 230000002744 anti-aggregatory effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- 206010010356 Congenital anomaly Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 208000020221 Short stature Diseases 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 229960003589 arginine hydrochloride Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 150000005846 sugar alcohols Chemical class 0.000 description 2
- LWXNQQIVUUSCSL-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol trihydrochloride Chemical compound Cl.Cl.Cl.OCC(N)(CO)CO LWXNQQIVUUSCSL-UHFFFAOYSA-N 0.000 description 1
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 108010075254 C-Peptide Proteins 0.000 description 1
- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102100020948 Growth hormone receptor Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001121408 Homo sapiens L-amino-acid oxidase Proteins 0.000 description 1
- 101000827703 Homo sapiens Polyphosphoinositide phosphatase Proteins 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102100026388 L-amino-acid oxidase Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010027259 Meningitis tuberculous Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 102100023591 Polyphosphoinositide phosphatase Human genes 0.000 description 1
- 101100012902 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FIG2 gene Proteins 0.000 description 1
- 101100233916 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) KAR5 gene Proteins 0.000 description 1
- 108010068542 Somatotropin Receptors Proteins 0.000 description 1
- 208000022971 Tuberculous meningitis Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 210000004198 anterior pituitary gland Anatomy 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940069078 citric acid / sodium citrate Drugs 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000006240 deamidation Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000037824 growth disorder Diseases 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 208000001223 meningeal tuberculosis Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 229960000502 poloxamer Drugs 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- -1 that is Proteins 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/27—Growth hormone [GH], i.e. somatotropin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
Definitions
- the present invention belongs to the field of biomedicine. Specifically, the present invention relates to a growth hormone Fc fusion protein injection and its use. More specifically, the present invention relates to a composition and injection and its use.
- Growth hormone is an endogenous hormone secreted by the anterior pituitary gland, which plays an important role in maintaining human growth and metabolism.
- Human growth hormone (hGH) is widely used to treat short stature caused by GH deficiency (GHD) or insufficiency and other growth disorders. Due to the short half-life of GH, daily subcutaneous injection is required, which greatly increases the pain of the patient.
- long-acting protein drugs such as fusion protein drugs, polyethylene glycol (PEG) protein drugs
- PEG polyethylene glycol
- long-acting growth hormone drugs mainly include growth hormone fusion protein drugs and PEGylated growth hormone protein drugs. They are often in the form of lyophilized powder to maintain the stability of long-acting protein drugs. In the development of growth hormone fusion protein drugs, the dosage form disclosed in clinical trials is also mostly lyophilized powder. However, when using lyophilized powder for injection, there are many injection steps and repeated extraction is required, which increases the risk of glass debris particles and microorganisms. The injection of long-acting growth hormone drugs does not need to be repeatedly extracted and is highly safe.
- Long-acting growth hormone drugs are generally administered once every 7 days or even 14 days by subcutaneous injection. Administration once every 7 to 14 days means a significant increase in the single dose.
- the subcutaneous administration volume for children is usually limited to less than 1 ml, so long-acting growth hormone preparations need to be prepared at a higher concentration.
- Fc fusion proteins have average solubility, so the preparation of high-concentration, stable long-acting growth hormone injections is technically challenging.
- the present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent.
- the present invention provides a composition containing a growth hormone fusion protein, which can reduce or inhibit the generation of molecular fragments, improve the stability of the growth hormone fusion protein in the composition, and in particular can be prepared into an injection with high stability.
- the commonly used long-acting growth hormone protein drugs are mainly growth hormone fusion protein drugs, PEGylated growth hormone protein drugs, etc., which are often in the form of lyophilized powder to maintain the stability of long-acting protein drugs.
- patent CN101374536A discloses a drug in which PEG is covalently coupled to growth hormone, and the drug finally maintains the stability of PEGylated growth hormone protein in the form of lyophilized powder
- patent CN108498467A discloses a lyophilized powder injection of recombinant growth hormone, which adopts a protective agent, excipients, surfactants and other auxiliary materials, and the excipients replace water to bind to the protein during lyophilization, thereby providing a skeleton support effect, and surfactants are also added to prevent particle aggregation and form a hydrophilic structure around the molecule.
- lyophilized powder is currently achieved through freeze-drying technology.
- the preparation process of lyophilized powder of long-acting growth hormone protein drugs will change the original spatial structure of the growth hormone protein and increase the aggregates. Long-term use will easily lead to the production of antibodies.
- the injection of lyophilized powder requires more steps and repeated extraction, which increases the risk of glass fragments, particles and microorganisms in the lyophilized powder injection.
- the preparation method of the injection of long-acting growth hormone protein drug is simple, and no drying is required during the preparation process, which will not change the original spatial structure of the growth hormone protein, and no repeated extraction is required during injection, which is highly safe.
- the chemical degradation of short-acting growth hormone that is, growth hormone protein molecules not connected with PEG or Fc, is often reported to be the shedding or addition of some groups such as deamidation and oxidation.
- the inventors found that growth hormone Fc Fusion proteins are prone to truncation of molecular segments, and the truncation may occur at the growth hormone fragment or the linker or disulfide bond at the Fc end.
- Fc is an essential part of the growth hormone Fc fusion protein to extend the in vivo metabolic half-life and achieve long-term effect. Therefore, the generation of molecular fragments (molecular truncation) during the preparation or storage of growth hormone fusion protein will affect its long-term pharmacokinetic characteristics (referred to as long-term pharmacokinetic characteristics).
- the present invention provides a composition.
- the composition comprises: a growth hormone fusion protein, and at least one of a buffer and a protective agent.
- the composition of the present invention can reduce or inhibit the generation of molecular fragments, and improve the stability of the growth hormone fusion protein during the preparation and storage process.
- the present invention proposes an injection.
- the injection comprises: growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine and a protective agent; based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg/mL; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- the injection of the present invention can slow down or inhibit the generation of molecular fragments and has the advantage of high stability.
- the present invention proposes an injection.
- the injection includes growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, methionine and a protective agent; based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of methionine is 10-20 mM; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- the injection of the present invention can slow down or inhibit the generation of molecular fragments and has the advantage of high
- the present invention proposes an injection.
- the injection comprises: growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, disodium edetate and a protective agent; wherein, based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of disodium edetate is 0.15-1 mM; wherein, the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, and the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- the injection of the present invention can slow down or inhibit the
- the present invention provides a use of the aforementioned composition or the aforementioned injection in the preparation of a medicament for treating and/or preventing diseases related to abnormal growth hormone.
- the present invention provides a method for preventing and/or treating diseases related to abnormal growth hormone.
- the method comprises: administering a pharmaceutically acceptable amount of the aforementioned composition or the aforementioned injection to a subject.
- the method can effectively prevent or treat diseases related to abnormal growth hormone.
- the present invention provides an injection device or container.
- the injection device or container comprises: the aforementioned composition or the aforementioned injection solution.
- the injection device or container of the present invention is convenient for storing the aforementioned composition or the aforementioned injection solution.
- FIG1 is a polymer (% HMW) detection result of SEC-HPLC in Example 1 of the present invention.
- FIG2 is the CE-SDS detection result of the truncated fragment content (%LMW) in Example 1 of the present invention.
- FIG3 is the polymer (%HMW) detection result of SEC-HPLC in Example 2 of the present invention.
- FIG4 is the CE-SDS detection result of the truncated fragment content (%LMW) in Example 2 of the present invention.
- FIG5 is the polymer (%HMW) detection result of SEC-HPLC in Example 2 of the present invention.
- FIG6 is the CE-SDS detection result of the truncated fragment content (%LMW) in Example 2 of the present invention.
- FIG7 is the polymer (%HMW) detection result of SEC-HPLC in Example 4 of the present invention.
- FIG. 8 is the detection result of the truncated fragment content (%LMW) by CE-SDS in Example 4 of the present invention.
- first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality” is two or more.
- any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values.
- the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.
- the terms “optionally”, “optional” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
- growth hormone abnormality-related diseases generally refers to diseases caused by abnormal growth hormone, such as growth hormone deficiency or related diseases caused by alienation, including but not limited to, childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner Syndrome, Prader Willi Syndrome, intrauterine growth retardation, idiopathic short stature, renal failure, chemotherapy treatment and alienation during AIDS treatment.
- Growth hormone deficiency may include congenital or acquired deficiency. Regarding congenital defects, growth hormone deficiency may occur when the pituitary gland does not develop a disorder of growth hormone secretion. Acquired growth hormone deficiency may occur due to brain tissue damage caused by hypoxia due to difficulty in delivery.
- growth hormone deficiency causes include pituitary damage caused by radiation used to treat brain tumors or postnatal tuberculous meningitis.
- Growth hormone deficiency exhibits symptoms such as growth retardation and short stature, and congenital growth hormone deficiency exhibits low glucose symptoms, starting from newborns.
- children exhibit symptoms such as increased anxiety and reduced vitality.
- treatment refers to the use of agents for obtaining a desired pharmacological and/or physiological effect.
- the effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effects caused by the disease.
- Treatment covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease.
- Treatment covers any medication that administers a composition, injection, or medicament containing them to an individual to treat, cure, alleviate, improve, mitigate, or inhibit an individual's disease, including but not limited to administering a composition, injection, or medicament containing them as described herein. Give things to individuals in need.
- the terms “identity”, “homology” or “similarity” are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Day Hoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al.
- GAP Genetics Computing Group
- the term "at least 90% identity” refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.
- fragment refers to a target protein or polypeptide, and a target protein or polypeptide with N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and/or internal deletion.
- an Fc fragment includes a hinge region, a CH2 region, and a CH3 region, or a partial fragment thereof, such as only including a CH2 region and a CH3 region;
- a serum albumin fragment may be serum albumin or a truncated fragment of serum albumin;
- a growth hormone fragment may be growth hormone or a truncated fragment of growth hormone.
- the present invention provides a composition and an injection, and a preparation method and use thereof, which will be described in detail below.
- the present invention provides a composition.
- the composition comprises: a growth hormone fusion protein, and at least one of a buffer and a protective agent.
- the inventors have found through a large number of experiments that the addition of a buffer and/or a protective agent can reduce or inhibit the generation of molecular fragments, and improve the stability of the growth hormone fusion protein during the preparation and storage process.
- the "growth hormone fusion protein" mentioned in the present invention generally refers to a protein obtained by fusing a growth hormone fragment having a natural growth hormone amino acid sequence with other protein fragments or polypeptide fragments, wherein the N-terminus of the growth hormone fragment is connected to the C-terminus of the protein fragment or polypeptide fragment, or the C-terminus of the growth hormone fragment is connected to the N-terminus of the protein fragment or polypeptide fragment; further, the growth hormone fragment and the protein fragment or polypeptide fragment can be connected by a connecting peptide.
- the protein fragment or polypeptide fragment is a fragment with certain functions, for example, the protein fragment or polypeptide fragment can increase the binding activity of the growth hormone fragment with the growth hormone receptor, or can reduce the ADCC effect of the growth hormone; wherein the protein fragment or polypeptide fragment includes but is not limited to the Fc fragment and the serum albumin fragment.
- the growth hormone fusion protein is a growth hormone F C fusion protein.
- the inventor unexpectedly discovered through experiments that the composition of the present invention has a good protective effect on the growth hormone F C fusion protein, and during the production and/or storage process of the growth hormone Fc fusion protein, it can slow down or inhibit the breakage of the growth hormone and Fc fragment, and the further chemical degradation of the growth hormone and Fc after the breakage, thereby reducing or avoiding the generation of molecular fragments, and preventing the excessive molecular fragments from affecting its long-acting pharmacokinetic characteristics.
- the "growth hormone Fc fusion protein" mentioned in the present invention generally refers to a protein obtained by fusing a growth hormone fragment with an Fc fragment.
- the Fc fragment can be derived from mouse antibodies, human antibodies, primate antibodies or their mutants.
- the Fc fragment refers to the Fc end of immunoglobulin IgG, which has different subtypes, such as IgG1, IgG2, IgG3, and IgG4.
- the Fc fragment can be a complete Fc fragment of a natural mouse, human or primate antibody (IgG), or an IgG complete Fc fragment inserted, replaced, or deleted by amino acid point mutation. Certain amino acids.
- the growth hormone F C fusion protein of the present invention may be the growth hormone F C fusion protein mentioned in patent application numbers 202210459224.3, 202210395423.2, and 202210395418.1.
- the buffer comprises at least one selected from the group consisting of histidine and histidine hydrochloride, sodium citrate and citric acid, and acetic acid/sodium acetate, thereby improving the stability of the growth hormone fusion protein.
- the buffer comprises histidine and histidine hydrochloride.
- the inventors have found through experiments that, compared with other buffer pairs (e.g., sodium citrate/citric acid, acetic acid/sodium acetate), the buffer pair of histidine and histidine hydrochloride can effectively reduce or avoid the generation of molecular fragments in the composition, improve the stability of the composition, and prevent excessive molecular fragments from affecting the long-acting pharmacokinetic characteristics of the composition.
- buffer pairs e.g., sodium citrate/citric acid, acetic acid/sodium acetate
- the molar concentration of the buffer is the total molar concentration of the buffer pair.
- the molar concentration of the buffer is the total molar concentration of histidine and histidine hydrochloride.
- histidine and histidine hydrochloride is synonymous with “histidine and histidine hydrochloride buffer pair”.
- the mass molar ratio of the growth hormone fusion protein to the buffer is (20-90) mg: (0.005-0.04) mmol, preferably (50-70) mg: (0.015-0.03) mmol.
- the stability of the growth hormone fusion protein can be further improved.
- the protective agent includes at least one selected from sucrose, trehalose, sorbitol and mannitol.
- sucrose trehalose
- sorbitol sorbitol
- mannitol mannitol
- the molar ratio of the growth hormone fusion protein to the protective agent is (20-90) mg: (20-90) mg, preferably (50-70) mg: (40-70) mg, and more preferably (50-70) mg: (42-64) mg.
- the protective agent is preferably sucrose.
- the protective agent includes sucrose and trehalose.
- sucrose and trehalose The inventors have found through experiments that the combination of sucrose and trehalose can prevent protein aggregation and further improve the stability of the growth hormone fusion protein.
- the mass ratio of the growth hormone fusion protein, sucrose and trehalose is (20-90) mg: (20-45) mg: (20-45) mg, preferably (40-90) mg: (15-35) mg: (15-35) mg.
- the stability of the growth hormone fusion protein in the composition can be further improved.
- the composition comprises a growth hormone fusion protein, a buffer and a protective agent, thereby further improving the stability of the growth hormone fusion protein.
- the mass molar ratio of the growth hormone fusion protein, the protective agent and the buffer is (20-90) mg: (20-90) mg: (0.015-0.03) mmol.
- the stability of the growth hormone fusion protein in the composition can be improved.
- the composition further comprises at least one of a stabilizer, a surfactant, an antioxidant and a metal chelator, thereby further improving the stability of the growth hormone fusion protein in the composition.
- the stabilizer includes at least one selected from tromethamine and tromethamine hydrochloride.
- tromethamine or tromethamine hydrochloride can form a non-covalently bonded spatial structure (such as a hydrogen bond) with a growth hormone molecule (such as an hGH molecule), thereby increasing the reaction energy and making the growth hormone fusion protein more stable, thereby further improving the stability of the growth hormone fusion protein.
- the stabilizer is tromethamine, thereby further improving the stability of the growth hormone fusion protein.
- the mass molar ratio of the growth hormone fusion protein to the stabilizer is (20-90) mg: (0.01-0.05) mmol, preferably (50-70) mg: (0.015-0.035) mmol.
- the stability of the growth hormone fusion protein can be further improved.
- the mass molar ratio of the growth hormone fusion protein to the antioxidant is (20-90) mg: (0.01-0.02) mmol.
- the stability of the growth hormone fusion protein in the composition can be further improved.
- the mass molar ratio of the growth hormone fusion protein to the metal chelator is (20-90) mg: (0.00015-0.01) mmol, preferably (40-90) mg: (0.00015-0.0003) mmol.
- the stability of the growth hormone fusion protein in the composition can be further improved.
- the antioxidant is selected from methionine.
- the antioxidant can competitively bind to dissolved oxygen in the injection solution to protect the growth hormone fusion protein from being oxidized.
- the metal chelator is selected from edetate disodium (also known as ETDA-2Na).
- ETDA-2Na edetate disodium
- the metal chelator can form a chelate with metal ions to prevent metal ions from catalyzing protein degradation.
- the mass ratio of the growth hormone fusion protein to the surfactant is (1-50): (0.1-1).
- the surfactant includes at least one selected from Tween 80, Tween 20 and Poloxamer P188.
- the composition comprises a growth hormone fusion protein, a buffer and a stabilizer, thereby further improving the stability of the growth hormone fusion protein.
- the mass molar ratio of the growth hormone fusion protein, the buffer and the stabilizer is (20-90) mg: (0.015-0.03) mmol: (0.015-0.035) mmol.
- the stability of the growth hormone fusion protein in the composition can be improved.
- the buffer comprises citricarb and histidine hydrochloride
- the stabilizer is tromethamine.
- the inventors have found through experiments that when the composition contains both histidine and histidine hydrochloride buffers and tromethamine, it can further slow down or inhibit the breakage between growth hormone and other protein fragments or polypeptide fragments, and the further chemical degradation of the growth hormone and protein fragments or polypeptide fragments after the breakage, reduce the generation of molecular fragments in the composition, and improve the protective effect on the growth hormone fusion protein.
- the composition comprises a growth hormone fusion protein, a protective agent and a stabilizer, thereby further improving the stability of the growth hormone fusion protein.
- the mass molar ratio of the growth hormone fusion protein, the protective agent and the stabilizer is (20-90) mg: (40-90) mg: (0.015-0.035) mmol, preferably (40-90) mg: (50-80) mg: (0.015-0.035) mmol, and more preferably (40-90) mg: (51-77) mg: (0.015-0.035) mmol.
- the stability of the growth hormone fusion protein in the composition can be improved.
- the stabilizer is tromethamine
- the protective agent includes sucrose, thereby further improving the stability of the growth hormone fusion protein.
- the stabilizer is tromethamine
- the protective agent includes sucrose and trehalose, thereby further improving the stability of the growth hormone fusion protein.
- the composition includes a growth hormone fusion protein, a buffer, a protective agent and a stabilizer.
- the inventor has found through a large number of experiments that the addition of a buffer, a protective agent and a stabilizer can effectively protect the growth hormone fusion protein, slow down or inhibit the generation of fractures between growth hormone fragments and other protein fragments or polypeptide fragments, and further chemical degradation of the growth hormone fragments and protein fragments or polypeptide fragments after the fracture, thereby reducing the generation of molecular fragments in the composition and improving the stability of the composition.
- the inventor has also found through a large number of experiments that due to the high stability of the growth hormone fusion protein in the composition, the composition does not require a complex preparation process and can be directly prepared into a highly stable injection, and the injection has good long-acting pharmacokinetic characteristics.
- the mass molar ratio of the growth hormone fusion protein, the protective agent, the stabilizer and the buffer is (40-90) mg: (20-90) mg: (0.01-0.05) mmol: (0.005-0.04) mmol.
- the stability of the growth hormone fusion protein in the composition can be improved.
- the mass molar ratio of the growth hormone fusion protein, the protective agent, the stabilizer and the buffer is (50-70) mg: (30-70) mg: (0.015-0.035) mmol: (0.015-0.03) mmol.
- the stability of the growth hormone fusion protein in the composition can be further improved.
- the mass molar ratio of the growth hormone fusion protein, tromethamine, histidine and histidine hydrochloride buffer pair, sucrose and trehalose is (50-70) mg: (0.015-0.035) mmol: (0.015-0.03) mmol: (20-45) mg: (20-45) mg.
- the cleavage between growth hormone and other protein fragments or polypeptide fragments, as well as the chemical degradation of growth hormone and other protein fragments or polypeptide fragments after cleavage can be slowed down or inhibited, and the generation of molecular fragments in the injection can be reduced, with the advantages of low molecular fragment content and high stability.
- the Nr-CE SDS and SEC purities of the composition under different storage conditions are 90% or above.
- the composition is an injection.
- the pH value of the composition is 6.1-7.2, preferably 6.4-7.2.
- the concentration of the growth hormone fusion protein is 20 to 90 mg/mL, preferably 50 to 70 mg/mL.
- the concentration of the protective agent is 20 to 90 mg/mL, preferably 30 to 70 mg/mL.
- the concentration of the buffer is 5-40 mM, preferably 15-30 mM.
- the concentration of the stabilizer is 10-50 mM, preferably 15-35 mM.
- the concentration of the antioxidant is 10-20 mM.
- the concentration of the chelating agent is 0.15-1 mM, preferably 0.15-0.3 mM.
- the growth hormone F C fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 4 or an amino acid sequence having at least 90% identity thereto.
- the present invention proposes an injection.
- the injection comprises: growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine and a protective agent; based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of the histidine and histidine hydrochloride is 15-30 mM, the concentration of the tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg/mL; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- the injection of the present invention can slow down or inhibit the breakage between growth hormone and other protein fragments or polypeptide fragments, and the chemical degradation of growth hormone and other protein fragments or polypeptide fragments after the breakage, reduce the generation of molecular fragments in the injection, and has the advantages of low molecular fragment content and high stability.
- the purity of Nr-CE SDS and SEC of this injection solution decreased more slowly under room temperature, light and pressure conditions such as 40°C, indicating that its long-term stability will be more stable when stored at the conventional 2-8°C for biological products in the dark.
- the growth hormone fusion protein is a growth hormone FC fusion protein.
- specific growth hormone FC fusion protein refer to the growth hormone FC fusion protein in the above composition.
- the present invention proposes an injection.
- the injection comprises growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, methionine and a protective agent; based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of methionine is 10-20 mM; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- the injection of the present invention further observed anti-aggregation improvement.
- the growth hormone fusion protein is a growth hormone FC fusion protein.
- specific growth hormone FC fusion protein refer to the growth hormone FC fusion protein in the above composition.
- the present invention proposes an injection.
- the injection comprises: growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, disodium edetate and a protective agent; wherein, based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of disodium edetate is 0.15-1 mM; wherein, the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, and the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- the injection of the present invention further observed significant improvements in anti
- the growth hormone fusion protein is a growth hormone FC fusion protein.
- specific growth hormone FC fusion protein refer to the growth hormone FC fusion protein in the above composition.
- the present invention provides a method for preparing the aforementioned composition.
- the method comprises: mixing a growth hormone fusion protein, a buffer, a protective agent and a stabilizer to obtain the composition; wherein the buffer comprises sodium citrate and citric acid.
- the method of the present invention can prepare the composition, and the preparation method is simple.
- the composition is an injection solution
- the mixing treatment is carried out by the following method: subjecting a first solution containing the growth hormone fusion protein to a first mixing treatment with a second solution containing the protective agent; subjecting the buffer and the stabilizer and optionally the antioxidant and/or the metal chelator to a second mixing treatment; subjecting the first mixing treatment product to a first ultrafiltration treatment, and subjecting the first ultrafiltration treatment product and the second mixing treatment product to a third mixing treatment to obtain the composition.
- the concentration of the growth hormone fusion protein in the first solution is 1-10 mg/mL.
- the first mixed treatment product before the first ultrafiltration treatment, is preliminarily subjected to a first concentration treatment to obtain
- the concentration of the growth hormone fusion protein is 10-20 mg/mL to obtain a first concentrated solution.
- the concentration of the growth hormone fusion protein in the third mixed treatment product is 10-20 mg/mL.
- the third mixing treatment product is subjected to a second concentration treatment to obtain a second concentrated solution having a concentration of the growth hormone fusion protein greater than 50 mg/mL.
- the method further comprises: after the second concentration process, diluting the second concentrated solution to obtain a composition having a concentration of the growth hormone fusion protein of 40 to 90 mg/mL.
- the present invention provides a use of the aforementioned composition, the aforementioned injection or the composition prepared according to the aforementioned method in preparing a drug for treating and/or preventing growth hormone abnormality-related diseases.
- the abnormal growth hormone-related diseases include at least one selected from childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation.
- the present invention provides a method for preventing and/or treating diseases related to abnormal growth hormone.
- the method comprises: administering a pharmaceutically acceptable amount of the aforementioned composition or the aforementioned injection to a subject.
- the method can effectively prevent or treat diseases related to abnormal growth hormone.
- the effective amount of the composition or injection of the present invention may vary depending on the mode of administration and the severity of the disease to be treated.
- the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials).
- the factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's body weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.
- the administration route of the method includes subcutaneous injection or intravenous injection.
- the abnormal growth hormone-related diseases include at least one selected from the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation.
- the present invention provides an injection device or container.
- the injection device or container comprises: the aforementioned composition or the aforementioned injection solution.
- the injection device or container of the present invention is convenient for storing the aforementioned composition or the aforementioned injection solution.
- the injection device or container of the present invention should be understood in a broad sense, and includes any product that can contain the composition or injection solution of the present invention.
- the injection device or container includes at least one selected from a container bottle and a syringe.
- the syringe includes at least one selected from an automatic syringe and a pre-filled syringe.
- HMW high molecular weight
- SEC size exclusion chromatography column
- % HMW refers to the percentage of the high molecular weight protein peak area relative to the sum of all detectable protein peaks in the preparation when detected by SEC-HPLC chromatography.
- LMW low molecular weight
- %HMW refers to the percentage of the high molecular weight protein peak area relative to the sum of all detectable protein peaks in the preparation when detected by SEC-HPLC chromatography.
- the content of HMW is used to indicate the degree of degradation of active protein molecules to produce truncated fragments.
- N/A in the examples herein means not added.
- Example 1 Screening of buffer and pH value in human growth hormone Fc fusion protein injection
- fusion protein human growth hormone Fc fusion protein
- the preparation method of injection is as follows:
- the protein preconcentrate is maintained at a fixed volume, and the phosphate buffer of the protein preconcentrate is replaced with a buffer solution by ultrafiltration. Then, ultrafiltration and concentration are continued, and the target protein content is detected by ProteinA-HPLC.
- the concentration of human growth hormone Fc fusion protein should be concentrated to 20 mg/ml or above, such as about 20 mg/ml, 50 mg/ml, 70 mg/ml or 80 mg/ml, or up to about 90 mg/ml.
- step 5 Add a certain volume of buffer solution to the concentrated protein solution to dilute it to the planned human growth hormone Fc fusion protein concentration in the preparation (see Table 1 for details) to obtain the injection stock solution.
- the concentration of the concentrated human growth hormone Fc fusion protein is the same as the required concentration, the dilution treatment in step 4) is not required.
- SEC-HPLC separates sample molecules based on the pores of the filler. Large sample molecules cannot enter or can only enter part of the pores, while smaller molecules can enter most or all of the pores, thereby separating proteins of different molecular weights.
- the main parameters of the SEC-HPLC detection method described in this article are:
- Nr-CE-SDS detection uses AB SCIEX PA800plus capillary electrophoresis instrument and SDS gel buffer with the company's product number A30341.
- the main parameters of the equipment are:
- the protein aggregates (%HMW) detected by SEC-HPLC and the truncated fragments (%LMW) detected by CE-SDS are different in injection solutions with different buffer systems and pH conditions.
- %HMW of SEC-HPLC other systems such as the citric acid system (citric acid/sodium citrate) and the phosphate system (disodium hydrogen phosphate/sodium phosphate) increase with increasing pH, while the histidine system (histidine/histidine hydrochloride) decreases with increasing pH, indicating that the histidine system can reduce aggregates when the pH increases in the pH range of 6.1-7.0.
- the histidine system (histidine/histidine hydrochloride) changes less in the pH range of 6.1-7.0, while the citric acid system and the phosphate system increase with increasing pH, which will increase truncated fragments.
- the results of the histidine system are better than those of the citric acid system and the phosphate system in both SEC and CE. According to the trend of the histidine system, a higher pH, such as pH 7.2, can also be tried.
- Example 2 Screening of protective agents in human growth hormone Fc fusion protein injection
- fusion protein human growth hormone Fc fusion protein
- Table 3 The final concentration of human growth hormone Fc fusion protein (referred to as fusion protein) in the injection and the types and final concentrations of each excipient are specifically shown in Table 3, the preparation method and detection method of the injection are shown in Example 1, and the test results are shown in Table 4 and Figures 3-4.
- the results showed that the order of advantage in terms of thermal stability and anti-aggregation (% HMW of SEC) such as high temperature and freeze-thaw is: trehalose ⁇ sucrose> mannitol> arginine hydrochloride> sodium chloride.
- Arginine hydrochloride and sodium chloride are ionic salt protective agents, and the overall protection effect is not as good as other non-ionic protective agents of sugar alcohols.
- fusion protein human growth hormone Fc fusion protein
- Table 5 The preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Figures 5 to 6. Judging from the growth trend of CE-SDS and SEC for 30 days, sucrose and trehalose showed a synergistic effect, and the combination of sucrose and trehalose was more stable than that used alone.
- Example 3 Screening of stabilizers in human growth hormone Fc fusion protein injection
- Stabilizers are ingredients that are beneficial to the stability of active molecules.
- some stabilizers can non-covalently bind to active molecules through molecular interactions (such as hydrogen bonds, hydrophobic interactions), etc., to increase the energy required for their reaction.
- the inventors tested the thermal stability of the stabilizer on the growth hormone fusion protein and found that Tris or Tris-hydrochloride (tromethamine) has a similar effect. Tris has multiple hydrogen bond donors and receptors and can bind to protein molecules.
- the final concentration of human growth hormone Fc fusion protein (hereinafter referred to as fusion protein) in the injection and the types and final concentrations of each auxiliary material are specifically shown in Table 6, the preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Table 7. Due to the adjustment of the upstream fermentation and purification process, the stability of the provided protein is even worse, and significant aggregation and molecular truncation occur at 40°C, light and 5°C. However, in the same batch of stock solutions, the prescription samples in Table 6 were compared in parallel. The results showed that compared with the absence of stabilizer, the addition of Tris showed an improvement of 1 to 2 percentage points in CE-SDS detection of %LMW under light and 5°C.
- Example 4 Screening of antioxidants and metal chelators in human growth hormone Fc fusion protein injection
- the inventor screened antioxidants (methionine) and metal chelators (EDTA-2Na, also known as disodium edetate).
- methionine and disodium edetate have a certain stabilizing effect on the fusion protein, and the stability can be further improved by combining with stabilizers.
- fusion protein human growth hormone Fc fusion protein
- Table 8 the preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Figures 7 to 8 and Table 9.
- the results show that compared with not adding methionine and EDTA-2Na, methionine slightly improves HMW, and adding 0.15-0.3mM EDTA-2Na can significantly improve CE-SDS%LMW, and can also improve the aggregation (HMW) at high temperature of 40°C.
- Example 5 Screening of the amount of histidine buffer added to human growth hormone Fc fusion protein injection
- the final concentration of human growth hormone Fc fusion protein (abbreviated as fusion protein) in the injection and the types and final concentrations of each auxiliary material are specifically shown in Table 10. Among them, the preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Table 11.
- the %LMW of CE-SDS in the 10-30mM histidine system is lower than that in the 40mM histidine system. Therefore, the stability of the 10-30mM histidine system is relatively better.
- the sample preparation of this embodiment has accumulated 6 months of stability data under the actual shelf life storage conditions of 2-8°C. Judging from the SEC and CE-SDS results, the growth is slow, and it is expected to maintain a shelf life of 18-24 months.
- the SEC and CE-SDS purity is greater than 90%.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Endocrinology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- AIDS & HIV (AREA)
- Urology & Nephrology (AREA)
- Diabetes (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Reproductive Health (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention relates to a composition comprising a growth hormone fusion protein and a use thereof. The composition comprises: the growth hormone fusion protein and at least one of a buffering agent and a protective agent; a use of the described composition in the preparation of a drug for the treatment and/or prevention of growth hormone abnormality-related diseases.
Description
本发明属于生物医药领域，具体地，本发明涉及一种生长激素Fc融合蛋白注射液及其用途，更具体地，本发明涉及一种组合物和注射液及其用途。The present invention belongs to the field of biomedicine. Specifically, the present invention relates to a growth hormone Fc fusion protein injection and its use. More specifically, the present invention relates to a composition and injection and its use.
生长激素(GH)是一种脑下垂体前叶分泌的内源性激素，在维持人体生长代谢方面有重要作用。人类生长激素(hGH)广泛用于治疗因GH缺乏(GHD)或不足以及其他生长障碍导致的身材矮小。由于GH的半衰期短，需要每日皮下注射，大大增加了治疗者的痛苦。为了延长半衰期，长效化蛋白药物(例如融合蛋白药物、聚乙二醇(PEG)化蛋白药物)成为目前生物技术药物的研发主流。Growth hormone (GH) is an endogenous hormone secreted by the anterior pituitary gland, which plays an important role in maintaining human growth and metabolism. Human growth hormone (hGH) is widely used to treat short stature caused by GH deficiency (GHD) or insufficiency and other growth disorders. Due to the short half-life of GH, daily subcutaneous injection is required, which greatly increases the pain of the patient. In order to extend the half-life, long-acting protein drugs (such as fusion protein drugs, polyethylene glycol (PEG) protein drugs) have become the mainstream of biotechnology drug research and development.
目前常见的长效化生长激素药物主要有生长激素融合蛋白药物、PEG化生长激素蛋白药物，其常以冻干粉的形式保持长效化蛋白药物的稳定性，在开发的生长激素融合蛋白药物临床试验公开的剂型也以冻干粉居多。但采用冻干粉注射时，其注射步骤较多，需要反复抽取，增加了玻璃碎屑微粒和微生物的风险。而长效化生长激素药物的注射液无需反复抽取，安全性高。Currently, common long-acting growth hormone drugs mainly include growth hormone fusion protein drugs and PEGylated growth hormone protein drugs. They are often in the form of lyophilized powder to maintain the stability of long-acting protein drugs. In the development of growth hormone fusion protein drugs, the dosage form disclosed in clinical trials is also mostly lyophilized powder. However, when using lyophilized powder for injection, there are many injection steps and repeated extraction is required, which increases the risk of glass debris particles and microorganisms. The injection of long-acting growth hormone drugs does not need to be repeatedly extracted and is highly safe.
长效化生长激素药物一般7天，甚至14天给药一次，通过皮下注射。7～14天给药一次意味着单次给药剂量的显著增加，另外儿童皮下给药体积通常限制在1ml以内，所以长效化生长激素制剂需要制备成较高的浓度。Fc融合蛋白溶解度一般，所以制备高浓度的、稳定性好的长效化生长激素注射液在技术上具有较大挑战。Long-acting growth hormone drugs are generally administered once every 7 days or even 14 days by subcutaneous injection. Administration once every 7 to 14 days means a significant increase in the single dose. In addition, the subcutaneous administration volume for children is usually limited to less than 1 ml, so long-acting growth hormone preparations need to be prepared at a higher concentration. Fc fusion proteins have average solubility, so the preparation of high-concentration, stable long-acting growth hormone injections is technically challenging.
因此，亟需开发一种高浓度稳定性好的长效化生长激素药物的注射液。Therefore, there is an urgent need to develop an injection of a high-concentration, stable and long-acting growth hormone drug.
发明内容Summary of the invention
本发明旨在至少在一定程度上解决现有技术中存在的技术问题之一。为此，本发明提供了一种含有生长激素融合蛋白的组合物，本发明的组合物可减少或抑制分子碎片的产生，提高组合物中生长激素融合蛋白的稳定性，尤其是可制备成稳定性高的注射液。The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a composition containing a growth hormone fusion protein, which can reduce or inhibit the generation of molecular fragments, improve the stability of the growth hormone fusion protein in the composition, and in particular can be prepared into an injection with high stability.
本发明是基于发明人的下列发现而完成的：The present invention is accomplished based on the following findings of the inventors:
目前常用的长效化生长激素蛋白药物主要为生长激素融合蛋白药物、PEG化生长激素蛋白药物等，其常以冻干粉的形式保持长效化蛋白药物的稳定性。例如，专利CN101374536A公开了一种PEG共价偶联到生长激素的药物，该药物最终以冻干粉的形式保持PEG化生长激素蛋白的稳定；专利CN108498467A公开了一种重组生长激素的冻干粉针，其采用保护剂、赋形剂、表面活性剂等辅料配方，通过赋形剂在冻干时代替水与蛋白结合，从而提供骨架支撑的作用，同时还加入表面活性剂防止微粒聚集，在分子周围形成亲水结构。At present, the commonly used long-acting growth hormone protein drugs are mainly growth hormone fusion protein drugs, PEGylated growth hormone protein drugs, etc., which are often in the form of lyophilized powder to maintain the stability of long-acting protein drugs. For example, patent CN101374536A discloses a drug in which PEG is covalently coupled to growth hormone, and the drug finally maintains the stability of PEGylated growth hormone protein in the form of lyophilized powder; patent CN108498467A discloses a lyophilized powder injection of recombinant growth hormone, which adopts a protective agent, excipients, surfactants and other auxiliary materials, and the excipients replace water to bind to the protein during lyophilization, thereby providing a skeleton support effect, and surfactants are also added to prevent particle aggregation and form a hydrophilic structure around the molecule.
但目前冻干粉的制备是通过冷冻干燥技术获得的，针对长效化生长激素蛋白药物的冻干粉，在制备过程中会改变生长激素蛋白原有的空间结构，增加了聚合体，长期使用容易产生抗体，而且冻干粉针注射步骤较多，需要反复抽取，增加了冻干粉注射液中含有玻璃碎屑微粒和微生物的风险。However, the preparation of lyophilized powder is currently achieved through freeze-drying technology. The preparation process of lyophilized powder of long-acting growth hormone protein drugs will change the original spatial structure of the growth hormone protein and increase the aggregates. Long-term use will easily lead to the production of antibodies. In addition, the injection of lyophilized powder requires more steps and repeated extraction, which increases the risk of glass fragments, particles and microorganisms in the lyophilized powder injection.
长效化生长激素蛋白药物的注射液制备方法简单，制备过程中无需干燥，不会改变生长激素蛋白原有的空间结构，并且在注射时无需反复抽取，安全性高。短效生长激素，也即未与PEG或Fc等连接的生长激素蛋白分子常报道的化学降解是脱酰胺、氧化等部分基团的脱落或加成。发明人发现生长激素Fc
融合蛋白极易发生分子链段的截短，截短的位置可能是生长激素片段或者Fc端的连接子或二硫键处。Fc是生长激素Fc融合蛋白实现体内代谢半衰期延长，实现长效化的必不可少的部分，所以生长激素融合蛋白在制备或贮藏过程中存在产生分子碎片(分子截短)将影响其长效药代动力学特征(简称长效药代特征)。The preparation method of the injection of long-acting growth hormone protein drug is simple, and no drying is required during the preparation process, which will not change the original spatial structure of the growth hormone protein, and no repeated extraction is required during injection, which is highly safe. The chemical degradation of short-acting growth hormone, that is, growth hormone protein molecules not connected with PEG or Fc, is often reported to be the shedding or addition of some groups such as deamidation and oxidation. The inventors found that growth hormone Fc Fusion proteins are prone to truncation of molecular segments, and the truncation may occur at the growth hormone fragment or the linker or disulfide bond at the Fc end. Fc is an essential part of the growth hormone Fc fusion protein to extend the in vivo metabolic half-life and achieve long-term effect. Therefore, the generation of molecular fragments (molecular truncation) during the preparation or storage of growth hormone fusion protein will affect its long-term pharmacokinetic characteristics (referred to as long-term pharmacokinetic characteristics).
因此，在本发明的一个方面，本发明提出了一种组合物。根据本发明的实施例，所述组合物包括：生长激素融合蛋白，以及缓冲剂和保护剂中的至少之一。本发明的组合物可减少或抑制分子碎片的产生，在制备和贮藏过程中，提高了生长激素融合蛋白的稳定性。Therefore, in one aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises: a growth hormone fusion protein, and at least one of a buffer and a protective agent. The composition of the present invention can reduce or inhibit the generation of molecular fragments, and improve the stability of the growth hormone fusion protein during the preparation and storage process.
在本发明的另一方面，本发明提出了一种注射液。根据本发明的实施例，所述注射液包括：生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇和保护剂；以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL；其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。本发明的注射液可减缓或抑制分子碎片的产生，具有稳定性高的优点。In another aspect of the present invention, the present invention proposes an injection. According to an embodiment of the present invention, the injection comprises: growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine and a protective agent; based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg/mL; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL. The injection of the present invention can slow down or inhibit the generation of molecular fragments and has the advantage of high stability.
在本发明的又一方面，本发明提出了一种注射液。根据本发明的实施例，所述注射液包括生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇、甲硫氨酸和保护剂；以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL，所述甲硫氨酸的浓度为10～20mM；其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。本发明的注射液可减缓或抑制分子碎片的产生，具有稳定性高的优点。In another aspect of the present invention, the present invention proposes an injection. According to an embodiment of the present invention, the injection includes growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, methionine and a protective agent; based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of methionine is 10-20 mM; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL. The injection of the present invention can slow down or inhibit the generation of molecular fragments and has the advantage of high stability.
在本发明的又一方面，本发明提出了一种注射液。根据本发明的实施例，所述注射液包括：生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇、依地酸二钠和保护剂；其中，以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL，所述依地酸二钠的浓度为0.15～1mM；其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。本发明的注射液可减缓或抑制分子碎片的产生，具有稳定性高的优点。In another aspect of the present invention, the present invention proposes an injection. According to an embodiment of the present invention, the injection comprises: growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, disodium edetate and a protective agent; wherein, based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of disodium edetate is 0.15-1 mM; wherein, the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, and the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL. The injection of the present invention can slow down or inhibit the generation of molecular fragments and has the advantage of high stability.
在本发明的又一方面，本发明提出了一种前述的组合物或前述的注射液在制备药物中的用途，所述药物用于治疗和/或预防生长激素异常相关疾病。In another aspect of the present invention, the present invention provides a use of the aforementioned composition or the aforementioned injection in the preparation of a medicament for treating and/or preventing diseases related to abnormal growth hormone.
在本发明的又一方面，本发明提出了一种预防和/或治疗生长激素异常相关疾病的方法。根据本发明的实施例，所述方法包含：向受试者施用药学上可接受量的前述的组合物或前述的注射液。根据本发明的实施例，该方法可有效预防或治疗生长激素异常相关疾病。In another aspect of the present invention, the present invention provides a method for preventing and/or treating diseases related to abnormal growth hormone. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the aforementioned composition or the aforementioned injection to a subject. According to an embodiment of the present invention, the method can effectively prevent or treat diseases related to abnormal growth hormone.
在本发明的又一方面，本发明提出了一种注射装置或容器。根据本发明的实施例，所述注射装置或容器包括：前述的组合物或前述的注射液。本发明的注射装置或容器方便贮藏前述的组合物或前述的注射液。In another aspect of the present invention, the present invention provides an injection device or container. According to an embodiment of the present invention, the injection device or container comprises: the aforementioned composition or the aforementioned injection solution. The injection device or container of the present invention is convenient for storing the aforementioned composition or the aforementioned injection solution.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:
图1为本发明实施例1中的SEC-HPLC的聚体(％HMW)检测结果；FIG1 is a polymer (% HMW) detection result of SEC-HPLC in Example 1 of the present invention;
图2为本发明实施例1中的CE-SDS的截短片段含量(％LMW)检测结果；FIG2 is the CE-SDS detection result of the truncated fragment content (%LMW) in Example 1 of the present invention;
图3为本发明实施例2中的SEC-HPLC的聚体(％HMW)检测结果；FIG3 is the polymer (%HMW) detection result of SEC-HPLC in Example 2 of the present invention;
图4为本发明实施例2中的CE-SDS的截短片段含量(％LMW)检测结果；FIG4 is the CE-SDS detection result of the truncated fragment content (%LMW) in Example 2 of the present invention;
图5为本发明实施例2中的SEC-HPLC的聚体(％HMW)检测结果；FIG5 is the polymer (%HMW) detection result of SEC-HPLC in Example 2 of the present invention;
图6为本发明实施例2中的CE-SDS的截短片段含量(％LMW)检测结果；FIG6 is the CE-SDS detection result of the truncated fragment content (%LMW) in Example 2 of the present invention;
图7为本发明实施例4中的SEC-HPLC的聚体(％HMW)检测结果；FIG7 is the polymer (%HMW) detection result of SEC-HPLC in Example 4 of the present invention;
图8为本发明实施例4中的CE-SDS的截短片段含量(％LMW)检测结果。FIG. 8 is the detection result of the truncated fragment content (%LMW) by CE-SDS in Example 4 of the present invention.
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.
在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
在本文中，术语“生长激素异常相关疾病”通常是指由于生长激素异常引起的疾病，例如生长激素缺乏或异化状态引起的相关疾病，包括但不限于，儿童生长激素缺乏症、特发性体型矮小、成人生长激素缺乏、特纳氏综合征(TurnersSyndrome)、普拉德 威利综合征(Prader Willi Syndrome)、子宫内生长迟缓、特发性身材矮小、肾衰竭、化疗治疗和AIDS治疗期间的异化状态。生长激素缺乏可包括先天或获得性缺乏。关于先天缺陷，当垂体没有发展成生长激素分泌紊乱时，生长激素缺陷可能发生。获得性生长激素缺乏可能由于难于递送而导致缺氧导致脑组织损伤而发生。生长激素缺乏的其它原因包括由用于治疗脑瘤或出生后结核性脑膜炎的辐射引起的垂体损伤。生长激素缺乏表现出诸如生长迟缓和身材矮小的症状，并且先天生长激素缺乏表现出低葡萄糖症状，从新生儿开始。另外，儿童表现出诸如焦虑增加和活力降低的症状。Herein, the term "growth hormone abnormality-related diseases" generally refers to diseases caused by abnormal growth hormone, such as growth hormone deficiency or related diseases caused by alienation, including but not limited to, childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner Syndrome, Prader Willi Syndrome, intrauterine growth retardation, idiopathic short stature, renal failure, chemotherapy treatment and alienation during AIDS treatment. Growth hormone deficiency may include congenital or acquired deficiency. Regarding congenital defects, growth hormone deficiency may occur when the pituitary gland does not develop a disorder of growth hormone secretion. Acquired growth hormone deficiency may occur due to brain tissue damage caused by hypoxia due to difficulty in delivery. Other causes of growth hormone deficiency include pituitary damage caused by radiation used to treat brain tumors or postnatal tuberculous meningitis. Growth hormone deficiency exhibits symptoms such as growth retardation and short stature, and congenital growth hormone deficiency exhibits low glucose symptoms, starting from newborns. In addition, children exhibit symptoms such as increased anxiety and reduced vitality.
在本文中，术语“治疗”是指用于获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将组合物、注射液或含有它们的药物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述组合物、注射液或含有它们的药物的药
物给予有需要的个体。As used herein, the term "treatment" refers to the use of agents for obtaining a desired pharmacological and/or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a composition, injection, or medicament containing them to an individual to treat, cure, alleviate, improve, mitigate, or inhibit an individual's disease, including but not limited to administering a composition, injection, or medicament containing them as described herein. Give things to individuals in need.
在本文中，术语“同一性”、“同源性”或“相似相”均用于描述相对于参考序列的氨基酸序列或核酸序列时，采用通过常规的方法进行确定两个氨基酸序列或核酸序列之间的相同氨基酸或核苷酸的百分比，例如参见，Ausubel等，编著(1995)，Current Protocols in Molecular Biology，第19章(Greene Publishing and Wiley-Interscience，New York)；和ALIGN程序(Dayhoff(1978)，Atlas of Protein Sequence and Structure5：Suppl.3(National Biomedical Research Foundation，Washington，D.C.)。关于比对序列和测定序列同一性有很多算法，包括，Needleman等(1970)J.Mol.Biol.48：443的同源性比对算法；Smith等(1981)Adv.Appl.Math.2：482的局部同源性算法；Pearson等(1988)Proc.Natl.Acad.Sci.85：2444的相似性搜索方法；Smith-Waterman算法(Meth.Mol.Biol.70：173-187(1997)；和BLASTP，BLASTN，和BLASTX算法(参见Altschul等(1990)J.Mol.Biol.215：403-410)。利用这些算法的计算机程序也是可获得的，并且包括但不限于：ALIGN或Megalign(DNASTAR)软件，或者WU-BLAST-2(Altschul等，Meth.Enzym.，266：460-480(1996))；或者GAP，BESTFIT，BLAST Altschul等，上文，FASTA，和TFASTA，在Genetics Computing Group(GCG)包，8版，Madison，Wisconsin，USA中可获得；和Intelligenetics，Mountain View，California提供的PC/Gene程序中的CLUSTAL。As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Day Hoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482. ; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444 similarity search method; Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or W U-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC/Gene program provided by Intelligenetics, Mountain View, California.
在本文中，术语“至少90％同一性”指与各参考序列至少为90％，可为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％的同一性。As used herein, the term "at least 90% identity" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.
在本文中，术语“片段”是指目标蛋白或多肽，以及具有N-末端(N端)或C-末端(C端)截短、和/或内部删除的目标蛋白或多肽。例如，Fc片段包括铰链区、CH2区和CH3区或者其部分片段，例如只包含CH2区和CH3区；血清白蛋白片段可为血清白蛋白或者血清白蛋白的截短片段；生长激素片段可为生长激素或者生长激素的截短片段。In this article, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide with N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and/or internal deletion. For example, an Fc fragment includes a hinge region, a CH2 region, and a CH3 region, or a partial fragment thereof, such as only including a CH2 region and a CH3 region; a serum albumin fragment may be serum albumin or a truncated fragment of serum albumin; and a growth hormone fragment may be growth hormone or a truncated fragment of growth hormone.
本发明提出了一种组合物和注射液及其制备方法和用途，下面将分别对其进行详细描述。The present invention provides a composition and an injection, and a preparation method and use thereof, which will be described in detail below.
组合物combination
在本发明的一个方面，本发明提出了一种组合物。根据本发明的实施例，所述组合物包括：生长激素融合蛋白，以及缓冲剂和保护剂中的至少之一。发明人经过大量试验发现，缓冲剂和/或保护剂的添加可减少或抑制分子碎片的产生，在制备和贮藏过程中，提高了生长激素融合蛋白的稳定性。In one aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises: a growth hormone fusion protein, and at least one of a buffer and a protective agent. The inventors have found through a large number of experiments that the addition of a buffer and/or a protective agent can reduce or inhibit the generation of molecular fragments, and improve the stability of the growth hormone fusion protein during the preparation and storage process.
需要说明的是，本发明中提到的“生长激素融合蛋白”通常是指具有天然生长激素氨基酸序列的生长激素片段和其他蛋白片段或多肽片段融合得到的蛋白，其中，生长激素片段的N端与蛋白片段或多肽片段的C端相连、或者生长激素片段的C端与蛋白片段或多肽片段的N端相连；进一步地，生长激素片段和蛋白片段或多肽片段之间可通过连接肽相连。优选地，该蛋白片段或多肽片段为具有一定的功能的片段，例如该蛋白片段或多肽片段可提高生长激素片段与生长激素受体结合活性，或者可降低生长激素ADCC效应；其中，蛋白片段或多肽片段包括但不限于Fc片段和血清白蛋白片段。It should be noted that the "growth hormone fusion protein" mentioned in the present invention generally refers to a protein obtained by fusing a growth hormone fragment having a natural growth hormone amino acid sequence with other protein fragments or polypeptide fragments, wherein the N-terminus of the growth hormone fragment is connected to the C-terminus of the protein fragment or polypeptide fragment, or the C-terminus of the growth hormone fragment is connected to the N-terminus of the protein fragment or polypeptide fragment; further, the growth hormone fragment and the protein fragment or polypeptide fragment can be connected by a connecting peptide. Preferably, the protein fragment or polypeptide fragment is a fragment with certain functions, for example, the protein fragment or polypeptide fragment can increase the binding activity of the growth hormone fragment with the growth hormone receptor, or can reduce the ADCC effect of the growth hormone; wherein the protein fragment or polypeptide fragment includes but is not limited to the Fc fragment and the serum albumin fragment.
根据本发明的实施例，所述生长激素融合蛋白为生长激素FC融合蛋白。发明人经过试验意外的发现，本发明的组合物对生长激素FC融合蛋白具有较好的保护作用，在生长激素Fc融合蛋白在生产和/或贮藏过程中，可减缓或抑制生长激素及Fc片段发生断裂、以及断裂后生长激素和Fc进一步发生化学降解，从而减少或避免分子碎片的产生，防止分子碎片过高影响其长效药代特征。According to an embodiment of the present invention, the growth hormone fusion protein is a growth hormone F C fusion protein. The inventor unexpectedly discovered through experiments that the composition of the present invention has a good protective effect on the growth hormone F C fusion protein, and during the production and/or storage process of the growth hormone Fc fusion protein, it can slow down or inhibit the breakage of the growth hormone and Fc fragment, and the further chemical degradation of the growth hormone and Fc after the breakage, thereby reducing or avoiding the generation of molecular fragments, and preventing the excessive molecular fragments from affecting its long-acting pharmacokinetic characteristics.
需要说明的是，本发明中提到的“生长激素FC融合蛋白”通常是指生长激素片段与FC片段融合得到的蛋白。其中，Fc片段可自于鼠源抗体、人源抗体和灵长目源抗体或其突变体。Fc片段是指免疫球蛋白IgG的Fc端，其具有不同的亚型，例如IgG1、IgG2、IgG3、IgG4。Fc片段可以是天然的鼠源、人源或灵长源抗体(IgG)的完整Fc片段、或者在IgG完整Fc片段上通过氨基酸点突变插入、替换、删减
某些氨基酸。It should be noted that the "growth hormone Fc fusion protein" mentioned in the present invention generally refers to a protein obtained by fusing a growth hormone fragment with an Fc fragment. Among them, the Fc fragment can be derived from mouse antibodies, human antibodies, primate antibodies or their mutants. The Fc fragment refers to the Fc end of immunoglobulin IgG, which has different subtypes, such as IgG1, IgG2, IgG3, and IgG4. The Fc fragment can be a complete Fc fragment of a natural mouse, human or primate antibody (IgG), or an IgG complete Fc fragment inserted, replaced, or deleted by amino acid point mutation. Certain amino acids.
示例性地，本发明的生长激素FC融合蛋白可为专利申请号为202210459224.3、202210395423.2、202210395418.1中提到的生长激素FC融合蛋白。Illustratively, the growth hormone F C fusion protein of the present invention may be the growth hormone F C fusion protein mentioned in patent application numbers 202210459224.3, 202210395423.2, and 202210395418.1.
根据本发明的实施例，所述缓冲剂包括选自组氨酸和盐酸组氨酸、柠檬酸钠和柠檬酸、醋酸/醋酸钠中的至少之一。由此，可提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the buffer comprises at least one selected from the group consisting of histidine and histidine hydrochloride, sodium citrate and citric acid, and acetic acid/sodium acetate, thereby improving the stability of the growth hormone fusion protein.
根据本发明的实施例，所述缓冲剂包括组氨酸和盐酸组氨酸。发明人经过试验发现，相对于其他缓冲对(例如柠檬酸钠/柠檬酸、醋酸/醋酸钠)，组氨酸和盐酸组氨酸的缓冲对可有效减少或避免组合物中分子碎片的产生，提高组合物的稳定性，以及防止分子碎片过高影响组合物的长效药代特征。According to an embodiment of the present invention, the buffer comprises histidine and histidine hydrochloride. The inventors have found through experiments that, compared with other buffer pairs (e.g., sodium citrate/citric acid, acetic acid/sodium acetate), the buffer pair of histidine and histidine hydrochloride can effectively reduce or avoid the generation of molecular fragments in the composition, improve the stability of the composition, and prevent excessive molecular fragments from affecting the long-acting pharmacokinetic characteristics of the composition.
需要说明的是，在本文中，缓冲剂的摩尔浓度为缓冲对的总摩尔浓度。示例性地，缓冲剂的摩尔浓度为组氨酸和盐酸组氨酸的总摩尔浓度。其中，“组氨酸和盐酸组氨酸”与“组氨酸和盐酸组氨酸缓冲对”同义。It should be noted that, in this article, the molar concentration of the buffer is the total molar concentration of the buffer pair. Exemplarily, the molar concentration of the buffer is the total molar concentration of histidine and histidine hydrochloride. Wherein, "histidine and histidine hydrochloride" is synonymous with "histidine and histidine hydrochloride buffer pair".
根据本发明的实施例，所述生长激素融合蛋白和缓冲剂的质量摩尔比为(20～90)mg：(0.005～0.04)mmol，优选为(50～70)mg：(0.015～0.03)mmol。由此，可进一步提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein to the buffer is (20-90) mg: (0.005-0.04) mmol, preferably (50-70) mg: (0.015-0.03) mmol. Thus, the stability of the growth hormone fusion protein can be further improved.
根据本发明的实施例，所述保护剂包括选自蔗糖、海藻糖、山梨醇和甘露醇中的至少之一。由此，可调节组合物中的渗透压，进一步提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the protective agent includes at least one selected from sucrose, trehalose, sorbitol and mannitol. Thus, the osmotic pressure in the composition can be adjusted, and the stability of the growth hormone fusion protein in the composition can be further improved.
根据本发明的实施例，所述生长激素融合蛋白和保护剂的摩尔比为(20～90)mg：(20～90)mg，优选为(50～70)mg：(40～70)mg，更优选为(50～70)mg：(42～64)mg。由此，可进一步提高生长激素融合蛋白的稳定性。根据本发明的实施例，保护剂优选为蔗糖。According to an embodiment of the present invention, the molar ratio of the growth hormone fusion protein to the protective agent is (20-90) mg: (20-90) mg, preferably (50-70) mg: (40-70) mg, and more preferably (50-70) mg: (42-64) mg. Thus, the stability of the growth hormone fusion protein can be further improved. According to an embodiment of the present invention, the protective agent is preferably sucrose.
根据本发明的实施例，所述保护剂包括蔗糖和海藻糖。发明人经过试验发现，采用蔗糖与海藻糖的组合，可防止蛋白聚集，进一步提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the protective agent includes sucrose and trehalose. The inventors have found through experiments that the combination of sucrose and trehalose can prevent protein aggregation and further improve the stability of the growth hormone fusion protein.
根据本发明的实施例，所述生长激素融合蛋白、蔗糖和海藻糖的质量比为(20～90)mg：(20～45)mg：(20～45)mg，优选为(40～90)mg：(15～35)mg：(15～35)mg。由此，可进一步提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass ratio of the growth hormone fusion protein, sucrose and trehalose is (20-90) mg: (20-45) mg: (20-45) mg, preferably (40-90) mg: (15-35) mg: (15-35) mg. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.
根据本发明的实施例，所述组合物包括生长激素融合蛋白、缓冲剂和保护剂。由此，可进一步提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the composition comprises a growth hormone fusion protein, a buffer and a protective agent, thereby further improving the stability of the growth hormone fusion protein.
根据本发明的实施例，所述生长激素融合蛋白、保护剂和缓冲剂的质量摩尔比为(20～90)mg：(20～90)mg：(0.015～0.03)mmol。由此，可提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein, the protective agent and the buffer is (20-90) mg: (20-90) mg: (0.015-0.03) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be improved.
根据本发明的实施例，所述组合物进一步包括稳定剂、表面活性剂、抗氧化剂和金属螯合剂中的至少之一。由此，可进一步提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the composition further comprises at least one of a stabilizer, a surfactant, an antioxidant and a metal chelator, thereby further improving the stability of the growth hormone fusion protein in the composition.
根据本发明的实施例，所述稳定剂包括选自氨丁三醇和氨丁三醇盐酸盐中的至少之一。发明人发现，氨丁三醇或氨丁三醇盐酸盐可与生长激素分子(例如hGH分子)形成非共价健结合的空间结构(比如氢键)，提高反应能量，使生长激素融合蛋白更稳定，从而进一步提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the stabilizer includes at least one selected from tromethamine and tromethamine hydrochloride. The inventors have found that tromethamine or tromethamine hydrochloride can form a non-covalently bonded spatial structure (such as a hydrogen bond) with a growth hormone molecule (such as an hGH molecule), thereby increasing the reaction energy and making the growth hormone fusion protein more stable, thereby further improving the stability of the growth hormone fusion protein.
根据本发明的实施例，所述稳定剂为氨丁三醇。由此，可进一步提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the stabilizer is tromethamine, thereby further improving the stability of the growth hormone fusion protein.
根据本发明的实施例，所述生长激素融合蛋白和稳定剂的质量摩尔比为(20～90)mg：(0.01～0.05)mmol，优选为(50～70)mg：(0.015～0.035)mmol。由此，可进一步提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein to the stabilizer is (20-90) mg: (0.01-0.05) mmol, preferably (50-70) mg: (0.015-0.035) mmol. Thus, the stability of the growth hormone fusion protein can be further improved.
根据本发明的实施例，所述生长激素融合蛋白和抗氧化剂的质量摩尔比为(20～90)mg：(0.01～0.02)mmol。由此，可进一步提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein to the antioxidant is (20-90) mg: (0.01-0.02) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.
根据本发明的实施例，所述生长激素融合蛋白和金属螯合剂的质量摩尔比为(20～90)mg：(0.00015～0.01)mmol，优选为(40～90)mg：(0.00015～0.0003)mmol。由此，可进一步提高组合物中生长激素融合蛋白的稳定性。
According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein to the metal chelator is (20-90) mg: (0.00015-0.01) mmol, preferably (40-90) mg: (0.00015-0.0003) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.
根据本发明的实施例，所述抗氧化剂包括选自甲硫氨酸。由此，该抗氧化剂可竞争性地与注射液中溶解氧结合，保护生长激素融合蛋白避免被氧化。According to an embodiment of the present invention, the antioxidant is selected from methionine. Thus, the antioxidant can competitively bind to dissolved oxygen in the injection solution to protect the growth hormone fusion protein from being oxidized.
根据本发明的实施例，所述金属螯合剂包括选自依地酸二钠(又称ETDA-2Na)。由此，该金属螯合剂能与金属离子生成螯合物，防止金属离子催化蛋白降解。According to an embodiment of the present invention, the metal chelator is selected from edetate disodium (also known as ETDA-2Na). Thus, the metal chelator can form a chelate with metal ions to prevent metal ions from catalyzing protein degradation.
根据本发明的实施例，所述生长激素融合蛋白和表面活性剂的质量比为(1～50)：(0.1～1)。According to an embodiment of the present invention, the mass ratio of the growth hormone fusion protein to the surfactant is (1-50): (0.1-1).
根据本发明的实施例，所述表面活性剂包括选自吐温80、吐温20和泊洛沙姆P188中的至少之一。According to an embodiment of the present invention, the surfactant includes at least one selected from Tween 80, Tween 20 and Poloxamer P188.
根据本发明的实施例，所述组合物包括生长激素融合蛋白、缓冲剂和稳定剂。由此，可进一步提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the composition comprises a growth hormone fusion protein, a buffer and a stabilizer, thereby further improving the stability of the growth hormone fusion protein.
根据本发明的实施例，所述生长激素融合蛋白、缓冲剂和稳定剂的质量摩尔比为(20～90)mg：(0.015～0.03)mmol：(0.015～0.035)mmol。由此，可提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein, the buffer and the stabilizer is (20-90) mg: (0.015-0.03) mmol: (0.015-0.035) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be improved.
在本发明的一个优选实施例中，所述缓冲剂包括柠组氨酸和盐酸组氨酸，所述稳定剂为氨丁三醇。发明人经过试验发现，当组合物中同时含有组氨酸和盐酸组氨酸缓冲对及氨丁三醇时，可进一步减缓或抑制生长激素和其他蛋白片段或多肽片段之间产生断裂、以及断裂后的生长激素和蛋白片段或多肽片段进一步发生化学降解，减少组合物中分子碎片的产生，提高对生长激素融合蛋白的保护效果。In a preferred embodiment of the present invention, the buffer comprises citricarb and histidine hydrochloride, and the stabilizer is tromethamine. The inventors have found through experiments that when the composition contains both histidine and histidine hydrochloride buffers and tromethamine, it can further slow down or inhibit the breakage between growth hormone and other protein fragments or polypeptide fragments, and the further chemical degradation of the growth hormone and protein fragments or polypeptide fragments after the breakage, reduce the generation of molecular fragments in the composition, and improve the protective effect on the growth hormone fusion protein.
根据本发明的实施例，所述组合物包括生长激素融合蛋白、保护剂和稳定剂。由此，可进一步提高生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the composition comprises a growth hormone fusion protein, a protective agent and a stabilizer, thereby further improving the stability of the growth hormone fusion protein.
根据本发明的实施例，所述生长激素融合蛋白、保护剂和稳定剂的质量摩尔比为(20～90)mg：(40～90)mg：(0.015～0.035)mmol，优选为(40～90)mg：(50～80)mg：(0.015～0.035)mmol，更优选为(40～90)mg：(51～77)mg：(0.015～0.035)mmol。由此，可提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein, the protective agent and the stabilizer is (20-90) mg: (40-90) mg: (0.015-0.035) mmol, preferably (40-90) mg: (50-80) mg: (0.015-0.035) mmol, and more preferably (40-90) mg: (51-77) mg: (0.015-0.035) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be improved.
在本发明的一个优选实施例中，所述稳定剂为氨丁三醇，所述保护剂包括蔗糖。由此，可进一步提高生长激素融合蛋白的稳定性。In a preferred embodiment of the present invention, the stabilizer is tromethamine, and the protective agent includes sucrose, thereby further improving the stability of the growth hormone fusion protein.
在本发明的一个优选实施例中，所述稳定剂为氨丁三醇，所述保护剂包括蔗糖和海藻糖。由此，可进一步提高生长激素融合蛋白的稳定性。In a preferred embodiment of the present invention, the stabilizer is tromethamine, and the protective agent includes sucrose and trehalose, thereby further improving the stability of the growth hormone fusion protein.
根据本发明的实施例，所述组合物包括生长激素融合蛋白、缓冲剂、保护剂和稳定剂。发明人经过大量试验发现，缓冲剂、保护剂和稳定剂的添加可有效保护生长激素融合蛋白，减缓或抑制生长激素片段和其他蛋白片段或多肽片段之间产生断裂、以及断裂后的生长激素片段和蛋白片段或多肽片段进一步发生化学降解，从而减少组合物中分子碎片的产生，提高组合物的稳定性。并且，发明人经过大量试验还发现，由于该组合物中生长激素融合蛋白的稳定性高，该组合物无需复杂的制备工艺，可直接制备成高稳定性的注射液，且该注射液具有较好的长效药代特征。According to an embodiment of the present invention, the composition includes a growth hormone fusion protein, a buffer, a protective agent and a stabilizer. The inventor has found through a large number of experiments that the addition of a buffer, a protective agent and a stabilizer can effectively protect the growth hormone fusion protein, slow down or inhibit the generation of fractures between growth hormone fragments and other protein fragments or polypeptide fragments, and further chemical degradation of the growth hormone fragments and protein fragments or polypeptide fragments after the fracture, thereby reducing the generation of molecular fragments in the composition and improving the stability of the composition. In addition, the inventor has also found through a large number of experiments that due to the high stability of the growth hormone fusion protein in the composition, the composition does not require a complex preparation process and can be directly prepared into a highly stable injection, and the injection has good long-acting pharmacokinetic characteristics.
根据本发明的实施例，所述生长激素融合蛋白、保护剂、稳定剂和缓冲剂的质量摩尔比为(40～90)mg：(20～90)mg：(0.01～0.05)mmol：(0.005～0.04)mmol。由此，可提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein, the protective agent, the stabilizer and the buffer is (40-90) mg: (20-90) mg: (0.01-0.05) mmol: (0.005-0.04) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be improved.
根据本发明的实施例，所述生长激素融合蛋白、保护剂、稳定剂和缓冲剂的质量摩尔比为(50～70)mg：(30～70)mg：(0.015～0.035)mmol：(0.015～0.03)mmol。由此，可进一步提高组合物中生长激素融合蛋白的稳定性。According to an embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein, the protective agent, the stabilizer and the buffer is (50-70) mg: (30-70) mg: (0.015-0.035) mmol: (0.015-0.03) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.
在本发明的一个优选实施例中，所述生长激素融合蛋白、氨丁三醇、组氨酸和盐酸组氨酸缓冲对、蔗糖和海藻糖的质量摩尔比(50～70)mg：(0.015～0.035)mmol：(0.015～0.03)mmol：(20～45)mg：(20～45)mg。由此，可减缓或抑制生长激素与其他蛋白片段或多肽片段之间发生断裂、以及断裂后生长激素和其他蛋白片段或多肽片段发生化学降解，减少注射液中分子碎片的产生，具有分子碎片含量低、稳定性高等优点。尤其是该组合物在不同贮藏条件下的Nr-CE SDS和SEC纯度均在90％及以上。
In a preferred embodiment of the present invention, the mass molar ratio of the growth hormone fusion protein, tromethamine, histidine and histidine hydrochloride buffer pair, sucrose and trehalose is (50-70) mg: (0.015-0.035) mmol: (0.015-0.03) mmol: (20-45) mg: (20-45) mg. As a result, the cleavage between growth hormone and other protein fragments or polypeptide fragments, as well as the chemical degradation of growth hormone and other protein fragments or polypeptide fragments after cleavage, can be slowed down or inhibited, and the generation of molecular fragments in the injection can be reduced, with the advantages of low molecular fragment content and high stability. In particular, the Nr-CE SDS and SEC purities of the composition under different storage conditions are 90% or above.
根据本发明的实施例，所述组合物为注射液。According to an embodiment of the present invention, the composition is an injection.
根据本发明的实施例，所述组合物的pH值为6.1-7.2，优选为6.4～7.2。According to an embodiment of the present invention, the pH value of the composition is 6.1-7.2, preferably 6.4-7.2.
根据本发明的实施例，所述生长激素融合蛋白的浓度为20～90mg/mL，优选为50～70mg/mL。According to an embodiment of the present invention, the concentration of the growth hormone fusion protein is 20 to 90 mg/mL, preferably 50 to 70 mg/mL.
根据本发明的实施例，所述保护剂的浓度为20～90mg/mL，优选为30～70mg/mL。According to an embodiment of the present invention, the concentration of the protective agent is 20 to 90 mg/mL, preferably 30 to 70 mg/mL.
根据本发明的实施例，所述缓冲剂的浓度为5～40mM，优选为15～30mM。According to an embodiment of the present invention, the concentration of the buffer is 5-40 mM, preferably 15-30 mM.
根据本发明的实施例，所述稳定剂的浓度为10～50mM，优选为15～35mM。According to an embodiment of the present invention, the concentration of the stabilizer is 10-50 mM, preferably 15-35 mM.
根据本发明的实施例，所述抗氧剂的浓度为10～20mM。According to an embodiment of the present invention, the concentration of the antioxidant is 10-20 mM.
根据本发明的实施例，所述螯合剂的浓度为0.15～1mM，优选0.15～0.3mM。According to an embodiment of the present invention, the concentration of the chelating agent is 0.15-1 mM, preferably 0.15-0.3 mM.
根据本发明的实施例，生长激素FC融合蛋白具有如SEQ ID NO:1～4任一项所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列。
According to an embodiment of the present invention, the growth hormone F C fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 4 or an amino acid sequence having at least 90% identity thereto.
According to an embodiment of the present invention, the growth hormone F C fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 4 or an amino acid sequence having at least 90% identity thereto.
注射液
Injection
在本发明的另一方面，本发明提出了一种注射液。根据本发明的实施例，所述注射液包括：生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇和保护剂；以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL；其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。本发明的注射液在生产和/或贮藏过程中，可减缓或抑制生长激素与其他蛋白片段或多肽片段之间发生断裂、以及断裂后生长激素和其他蛋白片段或多肽片段发生化学降解，减少注射液中分子碎片的产生，具有分子碎片含量低、稳定性高等优点。并且，相比其他方案，该注射液在室温光照和40℃等压力条件下可见Nr-CE SDS和SEC纯度的下降变化更加缓慢，预示其在生物制品常规2～8℃，避光保存长期稳定性将更稳定。In another aspect of the present invention, the present invention proposes an injection. According to an embodiment of the present invention, the injection comprises: growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine and a protective agent; based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of the histidine and histidine hydrochloride is 15-30 mM, the concentration of the tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg/mL; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL. During the production and/or storage process, the injection of the present invention can slow down or inhibit the breakage between growth hormone and other protein fragments or polypeptide fragments, and the chemical degradation of growth hormone and other protein fragments or polypeptide fragments after the breakage, reduce the generation of molecular fragments in the injection, and has the advantages of low molecular fragment content and high stability. Moreover, compared with other solutions, the purity of Nr-CE SDS and SEC of this injection solution decreased more slowly under room temperature, light and pressure conditions such as 40°C, indicating that its long-term stability will be more stable when stored at the conventional 2-8°C for biological products in the dark.
根据本发明的实施例，所述生长激素融合蛋白为生长激素FC融合蛋白，具体生长激素FC融合蛋白参见上述组合物中的生长激素FC融合蛋白。According to an embodiment of the present invention, the growth hormone fusion protein is a growth hormone FC fusion protein. For specific growth hormone FC fusion protein, refer to the growth hormone FC fusion protein in the above composition.
在本发明的又一方面，本发明提出了一种注射液。根据本发明的实施例，所述注射液包括生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇、甲硫氨酸和保护剂；以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL，所述甲硫氨酸的浓度为10～20mM；其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。本发明的注射液更进一步观察到抗聚集改善。In another aspect of the present invention, the present invention proposes an injection. According to an embodiment of the present invention, the injection comprises growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, methionine and a protective agent; based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of methionine is 10-20 mM; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL. The injection of the present invention further observed anti-aggregation improvement.
根据本发明的实施例，所述生长激素融合蛋白为生长激素FC融合蛋白，具体生长激素FC融合蛋白参见上述组合物中的生长激素FC融合蛋白。According to an embodiment of the present invention, the growth hormone fusion protein is a growth hormone FC fusion protein. For specific growth hormone FC fusion protein, refer to the growth hormone FC fusion protein in the above composition.
在本发明的又一方面，本发明提出了一种注射液。根据本发明的实施例，所述注射液包括：生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇、依地酸二钠和保护剂；其中，以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL，所述依地酸二钠的浓度为0.15～1mM；其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。本发明的注射液更进一步观察到抗聚集和分子截断的显著改善，从而达到2-8℃长期的产品稳定要求。In another aspect of the present invention, the present invention proposes an injection. According to an embodiment of the present invention, the injection comprises: growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, disodium edetate and a protective agent; wherein, based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of disodium edetate is 0.15-1 mM; wherein, the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, and the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL. The injection of the present invention further observed significant improvements in anti-aggregation and molecular truncation, thereby achieving the long-term product stability requirements of 2-8°C.
根据本发明的实施例，所述生长激素融合蛋白为生长激素FC融合蛋白，具体生长激素FC融合蛋白参见上述组合物中的生长激素FC融合蛋白。According to an embodiment of the present invention, the growth hormone fusion protein is a growth hormone FC fusion protein. For specific growth hormone FC fusion protein, refer to the growth hormone FC fusion protein in the above composition.
制备组合物和注射液的方法Methods for preparing compositions and injections
在本发明的又一方面，本发明提出了一种制备前述的组合物的方法。根据本发明的实施例，所述方法包括：将生长激素融合蛋白、缓冲剂、保护剂和稳定剂进行混合处理，以便获得所述组合物；其中，所述缓冲剂包括柠檬酸钠和柠檬酸。本发明的方法可制备得到组合物，且制备方法简单。In another aspect of the present invention, the present invention provides a method for preparing the aforementioned composition. According to an embodiment of the present invention, the method comprises: mixing a growth hormone fusion protein, a buffer, a protective agent and a stabilizer to obtain the composition; wherein the buffer comprises sodium citrate and citric acid. The method of the present invention can prepare the composition, and the preparation method is simple.
根据本发明的实施例，所述组合物为注射液，所述混合处理是通过如下方法进行的：将含有所述生长激素融合蛋白的第一溶液与含有所述保护剂的第二溶液进行第一混合处理；将所述缓冲液和稳定剂以及任选地抗氧化剂和/或金属螯合剂进行第二混合处理；将第一混合处理产物进行第一超滤处理，将第一超滤处理产物和所述第二混合处理产物进行第三混合处理，以便得到所述组合物。According to an embodiment of the present invention, the composition is an injection solution, and the mixing treatment is carried out by the following method: subjecting a first solution containing the growth hormone fusion protein to a first mixing treatment with a second solution containing the protective agent; subjecting the buffer and the stabilizer and optionally the antioxidant and/or the metal chelator to a second mixing treatment; subjecting the first mixing treatment product to a first ultrafiltration treatment, and subjecting the first ultrafiltration treatment product and the second mixing treatment product to a third mixing treatment to obtain the composition.
根据本发明的实施例，所述第一溶液中所述生长激素融合蛋白的浓度为1～10mg/mL。According to an embodiment of the present invention, the concentration of the growth hormone fusion protein in the first solution is 1-10 mg/mL.
根据本发明的实施例，在所述第一超滤处理之前，预先将第一混合处理产物进行第一浓缩处理，得
到所述生长激素融合蛋白的浓度为10～20mg/mL的第一浓缩液。According to an embodiment of the present invention, before the first ultrafiltration treatment, the first mixed treatment product is preliminarily subjected to a first concentration treatment to obtain The concentration of the growth hormone fusion protein is 10-20 mg/mL to obtain a first concentrated solution.
根据本发明的实施例，所述第三混合处理产物中所述生长激素融合蛋白的浓度为10～20mg/mL。According to an embodiment of the present invention, the concentration of the growth hormone fusion protein in the third mixed treatment product is 10-20 mg/mL.
根据本发明的实施例，在所述第三混合处理之后，将第三混合处理产物进行第二浓缩处理，得到所述生长激素融合蛋白的浓度大于50mg/mL的第二浓缩液。According to an embodiment of the present invention, after the third mixing treatment, the third mixing treatment product is subjected to a second concentration treatment to obtain a second concentrated solution having a concentration of the growth hormone fusion protein greater than 50 mg/mL.
根据本发明的实施例，所述方法进一步包括：在所述第二浓缩处理之后，将所述第二浓缩液进行稀释处理，以便得到所述生长激素融合蛋白的浓度为40～90mg/mL的组合物。According to an embodiment of the present invention, the method further comprises: after the second concentration process, diluting the second concentrated solution to obtain a composition having a concentration of the growth hormone fusion protein of 40 to 90 mg/mL.
用途use
在本发明的又一方面，本发明提出了一种前述的组合物、前述的注射液或依据前述的方法制备的组合物在制备药物中的用途，所述药物用于治疗和/或预防生长激素异常相关疾病。In another aspect of the present invention, the present invention provides a use of the aforementioned composition, the aforementioned injection or the composition prepared according to the aforementioned method in preparing a drug for treating and/or preventing growth hormone abnormality-related diseases.
根据本发明的实施例，所述生长激素异常相关疾病包括选自儿童生长激素缺乏症、特发性体型矮小、成人生长激素缺乏、特纳氏综合征、普拉德 威利综合征、肾衰竭、化疗治疗和AIDS治疗期间的异化状态引起的疾病和子宫内生长迟缓中的至少之一。According to an embodiment of the present invention, the abnormal growth hormone-related diseases include at least one selected from childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation.
治疗和/或预防生长激素异常相关疾病的方法Method for treating and/or preventing diseases related to abnormal growth hormone
在本发明的又一方面，本发明提出了一种预防和/或治疗生长激素异常相关疾病的方法。根据本发明的实施例，所述方法包含：向受试者施用药学上可接受量的前述的组合物或前述的注射液。根据本发明的实施例，该方法可有效预防或治疗生长激素异常相关疾病。In another aspect of the present invention, the present invention provides a method for preventing and/or treating diseases related to abnormal growth hormone. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the aforementioned composition or the aforementioned injection to a subject. According to an embodiment of the present invention, the method can effectively prevent or treat diseases related to abnormal growth hormone.
本发明所述的组合物或注射液的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the composition or injection of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's body weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.
根据本发明的实施例，所述方法的给药途径包括皮下注射或静脉注射。According to an embodiment of the present invention, the administration route of the method includes subcutaneous injection or intravenous injection.
根据本发明的实施例，所述生长激素异常相关疾病至少包括选自下列之一：儿童生长激素缺乏症、特发性体型矮小、成人生长激素缺乏、特纳氏综合征、普拉德 威利综合征、肾衰竭、化疗治疗和AIDS治疗期间的异化状态引起的疾病、子宫内生长迟缓。According to an embodiment of the present invention, the abnormal growth hormone-related diseases include at least one selected from the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation.
注射装置或容器Injection device or container
在本发明的又一方面，本发明提出了一种注射装置或容器。根据本发明的实施例，所述注射装置或容器包括：前述的组合物或前述的注射液。本发明的注射装置或容器方便贮藏前述的组合物或前述的注射液。In another aspect of the present invention, the present invention provides an injection device or container. According to an embodiment of the present invention, the injection device or container comprises: the aforementioned composition or the aforementioned injection solution. The injection device or container of the present invention is convenient for storing the aforementioned composition or the aforementioned injection solution.
在本文中，本发明的注射装置或容器应作广义理解，其包含任何可容纳本发明的组合物或注射液的产品。Herein, the injection device or container of the present invention should be understood in a broad sense, and includes any product that can contain the composition or injection solution of the present invention.
根据本发明的实施例，所述注射装置或容器包括选自容纳瓶和注射器中的至少之一。According to an embodiment of the present invention, the injection device or container includes at least one selected from a container bottle and a syringe.
根据本发明的实施例，所述注射器包括选自自动注射器和预填充注射器中的至少之一。According to an embodiment of the present invention, the syringe includes at least one selected from an automatic syringe and a pre-filled syringe.
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得
的常规产品。The scheme of the present invention will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or in accordance with the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they can be obtained commercially. conventional products.
本文提及的制剂的“高分子量”(HMW)种类的相关蛋白是指从尺寸排阻色谱柱(SEC)洗脱下来的任何出峰时间早于主峰的相关蛋白的含量，HMW的含量用于指示蛋白的聚集程度。％HMW是指SEC-HPLC色谱检测时高分子量蛋白峰面积相对于制剂中所有可检测蛋白峰总和的百分比。The "high molecular weight" (HMW) related proteins of the preparations mentioned herein refer to the content of any related proteins eluted from the size exclusion chromatography column (SEC) earlier than the main peak, and the content of HMW is used to indicate the degree of aggregation of the protein. % HMW refers to the percentage of the high molecular weight protein peak area relative to the sum of all detectable protein peaks in the preparation when detected by SEC-HPLC chromatography.
本文提及的“低分子量”(LMW)种类的相关蛋白是从非还原毛细管电泳(nrCE-SDS检)洗脱下来的任何出峰时间早于主峰的相关蛋白的含量。％HMW是指SEC-HPLC色谱检测时高分子量蛋白峰面积相对于制剂中所有可检测蛋白峰总和的百分比，HMW的含量用于指示活性蛋白分子降解产生截短碎片的程度。The "low molecular weight" (LMW) related proteins mentioned in this article are any related proteins that elute from non-reducing capillary electrophoresis (nrCE-SDS detection) earlier than the main peak. %HMW refers to the percentage of the high molecular weight protein peak area relative to the sum of all detectable protein peaks in the preparation when detected by SEC-HPLC chromatography. The content of HMW is used to indicate the degree of degradation of active protein molecules to produce truncated fragments.
本文实施例中的N/A指未添加。N/A in the examples herein means not added.
需要说明的是，本申请实施例中采用的人生长激素Fc融合蛋白具体氨基酸序列如SEQ ID NO:1，其通过常规的方法制备得到；其余辅料均通过市购获得。It should be noted that the specific amino acid sequence of the human growth hormone Fc fusion protein used in the examples of the present application is shown in SEQ ID NO: 1, which is prepared by conventional methods; the remaining auxiliary materials are purchased from the market.
实施例1：人生长激素Fc融合蛋白注射液中缓冲液和pH值的筛选Example 1: Screening of buffer and pH value in human growth hormone Fc fusion protein injection
1、注射液中人生长激素Fc融合蛋白(简称融合蛋白)的终浓度和各辅料的种类及其终浓度具体参见表1。1. The final concentration of human growth hormone Fc fusion protein (hereinafter referred to as fusion protein) in the injection and the types of excipients and their final concentrations are specifically shown in Table 1.
2、注射液的制备方法如下所示：2. The preparation method of injection is as follows:
1)取一定体积的含磷酸缓冲液的蛋白液，该蛋白液中人生长激素Fc融合蛋白的浓度为2-3mg/mL，按30mg/mL量加入蔗糖溶解。然后用0.45μm过滤器过滤除颗粒，得到过滤液。1) Take a certain volume of protein solution containing phosphate buffer, in which the concentration of human growth hormone Fc fusion protein is 2-3 mg/mL, add sucrose at 30 mg/mL to dissolve, and then filter with a 0.45 μm filter to remove particles to obtain a filtrate.
2)使用milipore pellicon2 30KD超滤膜包对过滤液进行预浓缩处理，预浓缩至人生长激素Fc融合蛋白的浓度为10-20mg/mL，得到蛋白预浓缩液。2) Use milipore pellicon2 30KD ultrafiltration membrane package to pre-concentrate the filtrate to a concentration of 10-20 mg/mL of human growth hormone Fc fusion protein to obtain a protein pre-concentrate.
3)按照表1配方，将人生长激素Fc融合蛋白和蔗糖之外的其他辅料配制成buffer液，用盐酸或氢氧化钠将buffer液的pH值调节至规定值(具体参见表1)。3) According to the formula in Table 1, human growth hormone Fc fusion protein and other auxiliary materials except sucrose are prepared into a buffer solution, and the pH value of the buffer solution is adjusted to a specified value with hydrochloric acid or sodium hydroxide (see Table 1 for details).
4)蛋白预浓缩液在维持固定体积下，通过超滤将蛋白预浓缩液的磷酸缓冲液置换成buffer液。然后继续超滤浓缩，ProteinA-HPLC检测目标蛋白含量，应浓缩至人生长激素Fc融合蛋白的浓度为至20mg/ml及以上，例如大约20mg/ml、50mg/ml、70mg/ml或80mg/ml或最高可以达到大约90mg/ml。4) The protein preconcentrate is maintained at a fixed volume, and the phosphate buffer of the protein preconcentrate is replaced with a buffer solution by ultrafiltration. Then, ultrafiltration and concentration are continued, and the target protein content is detected by ProteinA-HPLC. The concentration of human growth hormone Fc fusion protein should be concentrated to 20 mg/ml or above, such as about 20 mg/ml, 50 mg/ml, 70 mg/ml or 80 mg/ml, or up to about 90 mg/ml.
5)向浓缩结束的蛋白液加入一定体积的buffer液稀释至制剂中拟定的人生长激素Fc融合蛋白浓度(具体参见表1)，得到注射液原液，当浓缩的人生长激素Fc融合蛋白浓度和所需浓度相同，无需进行第4)的稀释处理。5) Add a certain volume of buffer solution to the concentrated protein solution to dilute it to the planned human growth hormone Fc fusion protein concentration in the preparation (see Table 1 for details) to obtain the injection stock solution. When the concentration of the concentrated human growth hormone Fc fusion protein is the same as the required concentration, the dilution treatment in step 4) is not required.
6)根据注射液原液保存条件，经必要的解冻、混匀、0.2μm除菌滤器过滤后分装至西林瓶，密封即得注射液。6) According to the storage conditions of the injection stock solution, after necessary thawing, mixing, filtering through a 0.2 μm sterilizing filter, dispense into vials and seal to obtain the injection solution.
3、然后将各样品得到的注射液在40℃条件下进行SEC-HPLC和nrCE-SDS检测，具体检测结果参见表2和图1～2。3. The injection solutions obtained from each sample were then subjected to SEC-HPLC and nrCE-SDS detection at 40°C. For specific test results, see Table 2 and Figures 1-2.
SEC-HPLC和nrCE-SDS检测如下所示：SEC-HPLC and nrCE-SDS detection are as follows:
SEC-HPLC是根据填料孔隙而分离样品分子，样品大分子不能进入或只能进入部分孔隙，而较小分子则能进入大多数或全部孔隙，从而使不同分子量的蛋白分离。本文所述的SEC-HPLC检测方法主要参数为：
SEC-HPLC separates sample molecules based on the pores of the filler. Large sample molecules cannot enter or can only enter part of the pores, while smaller molecules can enter most or all of the pores, thereby separating proteins of different molecular weights. The main parameters of the SEC-HPLC detection method described in this article are:
SEC-HPLC separates sample molecules based on the pores of the filler. Large sample molecules cannot enter or can only enter part of the pores, while smaller molecules can enter most or all of the pores, thereby separating proteins of different molecular weights. The main parameters of the SEC-HPLC detection method described in this article are:
Nr-CE-SDS检测使用AB SCIEX公司PA800plus型毛细管电泳仪及该公司货号为A30341的SDS凝胶缓冲液，设备主要参数为：
Nr-CE-SDS detection uses AB SCIEX PA800plus capillary electrophoresis instrument and SDS gel buffer with the company's product number A30341. The main parameters of the equipment are:
Nr-CE-SDS detection uses AB SCIEX PA800plus capillary electrophoresis instrument and SDS gel buffer with the company's product number A30341. The main parameters of the equipment are:
表1：样品1～8的注射液中各成分的配比
Table 1: Proportions of the components in the injection solutions of samples 1 to 8
Table 1: Proportions of the components in the injection solutions of samples 1 to 8
表2：样品1～8中SEC-HPLC和nrCE-SDS的检测结果
Table 2: SEC-HPLC and nrCE-SDS test results for samples 1 to 8
Table 2: SEC-HPLC and nrCE-SDS test results for samples 1 to 8
由表1～2和图1～2可知，不同缓冲体系和pH值条件的注射液中，SEC-HPLC检测到的蛋白聚体(％HMW)和CE-SDS检测的截短碎片含量(％LMW)不同。就SEC-HPLC的％HMW而言，其他如柠檬酸体系(柠檬酸/柠檬酸钠)和磷酸体系(磷酸氢二钠/磷酸钠)随pH升高而增加，而组氨酸体系(组氨酸/盐酸组氨酸)随pH升高而减少，说明组氨酸体系在pH值为6.1～7.0范围升高pH时可以降低聚集体。就CE-SDS的％LMW而言，组氨酸体系(组氨酸/盐酸组氨酸)在pH值为6.1-7.0范围变化较小，柠檬酸体系和磷酸体系则随pH升高而增加，将增加截短碎片。综合比较，组氨酸体系无论SEC还是CE的结果都优于柠檬酸体系、磷酸体系。根据组氨酸体系的趋势，还可尝试再高些的pH，例如pH值为7.2。As shown in Tables 1-2 and Figures 1-2, the protein aggregates (%HMW) detected by SEC-HPLC and the truncated fragments (%LMW) detected by CE-SDS are different in injection solutions with different buffer systems and pH conditions. As for the %HMW of SEC-HPLC, other systems such as the citric acid system (citric acid/sodium citrate) and the phosphate system (disodium hydrogen phosphate/sodium phosphate) increase with increasing pH, while the histidine system (histidine/histidine hydrochloride) decreases with increasing pH, indicating that the histidine system can reduce aggregates when the pH increases in the pH range of 6.1-7.0. As for the %LMW of CE-SDS, the histidine system (histidine/histidine hydrochloride) changes less in the pH range of 6.1-7.0, while the citric acid system and the phosphate system increase with increasing pH, which will increase truncated fragments. In a comprehensive comparison, the results of the histidine system are better than those of the citric acid system and the phosphate system in both SEC and CE. According to the trend of the histidine system, a higher pH, such as pH 7.2, can also be tried.
实施例2：人生长激素Fc融合蛋白注射液中保护剂的筛选Example 2: Screening of protective agents in human growth hormone Fc fusion protein injection
发明人为了筛选出较适的保护剂，测试了5种保护剂对生长激素融合蛋白的热稳定性和冻融保护。注射液中人生长激素Fc融合蛋白(简称融合蛋白)的终浓度和各辅料的种类及其终浓度具体参见表3，注射液的制备方法和检测方法参见实施例1，检测结果参见表4和图3～4。结果发现，在高温和冻融等热稳定性抗聚集(SEC的％HMW)方面的优势顺序为：海藻糖≈蔗糖>甘露醇>盐酸精氨酸>氯化钠。盐酸精氨酸和氯化钠属于离子型盐类保护剂，整体保护效果不如其他糖醇类的非离子型保护剂。In order to screen out a more suitable protective agent, the inventor tested the thermal stability and freeze-thaw protection of 5 kinds of protective agents on growth hormone fusion protein. The final concentration of human growth hormone Fc fusion protein (referred to as fusion protein) in the injection and the types and final concentrations of each excipient are specifically shown in Table 3, the preparation method and detection method of the injection are shown in Example 1, and the test results are shown in Table 4 and Figures 3-4. The results showed that the order of advantage in terms of thermal stability and anti-aggregation (% HMW of SEC) such as high temperature and freeze-thaw is: trehalose ≈ sucrose> mannitol> arginine hydrochloride> sodium chloride. Arginine hydrochloride and sodium chloride are ionic salt protective agents, and the overall protection effect is not as good as other non-ionic protective agents of sugar alcohols.
表3：样品9～13的注射液中各成分的配比
Table 3: Proportions of the components in the injection solutions of samples 9 to 13
Table 3: Proportions of the components in the injection solutions of samples 9 to 13
表4：样品9～13中SEC-HPLC和nrCE-SDS的检测结果
Table 4: SEC-HPLC and nrCE-SDS test results for samples 9 to 13
Table 4: SEC-HPLC and nrCE-SDS test results for samples 9 to 13
进一步地，发明人测试了非离子型糖醇保护剂组合与单独保护剂的差异。注射液中人生长激素Fc融合蛋白(简称融合蛋白)的终浓度和各辅料的种类及其终浓度具体参见表5。注射液的制备方法和检测方法参见实施例1，检测结果参见图5～6。综合CE-SDS和SEC 30天的增长变化趋势来评判，蔗糖与海藻糖显现了协同效应，蔗糖与海藻糖组合使用较单独使用的稳定性更优。Furthermore, the inventors tested the difference between the combination of non-ionic sugar alcohol protective agents and a single protective agent. The final concentration of human growth hormone Fc fusion protein (referred to as fusion protein) in the injection and the types and final concentrations of each excipient are specifically shown in Table 5. The preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Figures 5 to 6. Judging from the growth trend of CE-SDS and SEC for 30 days, sucrose and trehalose showed a synergistic effect, and the combination of sucrose and trehalose was more stable than that used alone.
表5：样品14～18的注射液中各成分的配比
Table 5: Proportions of the components in the injection solutions of samples 14 to 18
Table 5: Proportions of the components in the injection solutions of samples 14 to 18
实施例3：人生长激素Fc融合蛋白注射液中稳定剂的筛选Example 3: Screening of stabilizers in human growth hormone Fc fusion protein injection
稳定剂是对活性分子稳定有益的成分，例如某些稳定剂可以通过分子相互作用(如氢键、疏水作用)等与活性分子非共价结合，提高其反应所需的能量。发明人为了筛选出最适的稳定剂，测试了稳定剂对生长激素融合蛋白的热稳定性，发现Tris或Tris-盐酸盐(氨基丁三醇)就有类似作用，Tris具有多个氢键供体和受体，可与蛋白分子结合。Stabilizers are ingredients that are beneficial to the stability of active molecules. For example, some stabilizers can non-covalently bind to active molecules through molecular interactions (such as hydrogen bonds, hydrophobic interactions), etc., to increase the energy required for their reaction. In order to screen out the most suitable stabilizer, the inventors tested the thermal stability of the stabilizer on the growth hormone fusion protein and found that Tris or Tris-hydrochloride (tromethamine) has a similar effect. Tris has multiple hydrogen bond donors and receptors and can bind to protein molecules.
本实施例中，注射液中人生长激素Fc融合蛋白(简称融合蛋白)的终浓度和各辅料的种类及其终浓度具体参见表6，注射液的制备方法和检测方法参见实施例1，检测结果参见表7。由于上游发酵提纯工艺进行调整，提供的蛋白的稳定性情况更加糟糕，在40℃、光照及5℃等都发生显著的聚集和分子截短现象。但在相同批次原液平行比较表6处方样品，结果表明相较于不添加稳定剂，添加Tris在光照和5℃下CE-SDS检测％LMW显现了1～2百分点的改善，尽管不太显著，但对于人生长激素Fc融合蛋白所面临的极易产生LMW而言，该细微的有益改善也具有非凡价值的；并且，发明人进一步对稳定剂的添加量进行探究，Tris的量在15-25mM随用量增加，CE-SDS检测的％LMW改善相应增加。
In this embodiment, the final concentration of human growth hormone Fc fusion protein (hereinafter referred to as fusion protein) in the injection and the types and final concentrations of each auxiliary material are specifically shown in Table 6, the preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Table 7. Due to the adjustment of the upstream fermentation and purification process, the stability of the provided protein is even worse, and significant aggregation and molecular truncation occur at 40°C, light and 5°C. However, in the same batch of stock solutions, the prescription samples in Table 6 were compared in parallel. The results showed that compared with the absence of stabilizer, the addition of Tris showed an improvement of 1 to 2 percentage points in CE-SDS detection of %LMW under light and 5°C. Although not very significant, for the human growth hormone Fc fusion protein, which is prone to LMW, this subtle beneficial improvement is also of extraordinary value; and the inventor further explored the amount of stabilizer added. The amount of Tris increased with the dosage at 15-25mM, and the %LMW improvement detected by CE-SDS increased accordingly.
表6：样品19～23的注射液中各成分的配比
Table 6: Proportions of the components in the injection solutions of samples 19 to 23
Table 6: Proportions of the components in the injection solutions of samples 19 to 23
表7：样品19～23中SEC-HPLC和nrCE-SDS的检测结果
Table 7: SEC-HPLC and nrCE-SDS test results for samples 19 to 23
Table 7: SEC-HPLC and nrCE-SDS test results for samples 19 to 23
实施例4：人生长激素Fc融合蛋白注射液中抗氧化剂和金属螯合剂的筛选
Example 4: Screening of antioxidants and metal chelators in human growth hormone Fc fusion protein injection
发明人为了进一步提高注射液的稳定性，对抗氧化剂(甲硫氨酸)和金属螯合剂(EDTA-2Na，又称依地酸二钠)进行筛选。发明人发现，甲硫氨酸、依地酸二钠对融合蛋白具有一定的稳定作用，通过与稳定剂组合可进一步的提高稳定性。In order to further improve the stability of the injection, the inventor screened antioxidants (methionine) and metal chelators (EDTA-2Na, also known as disodium edetate). The inventor found that methionine and disodium edetate have a certain stabilizing effect on the fusion protein, and the stability can be further improved by combining with stabilizers.
本实施例中，注射液中人生长激素Fc融合蛋白(简称融合蛋白)的终浓度和各辅料的种类及其终浓度具体参见表8，注射液的制备方法和检测方法参见实施例1，检测结果参见图7～8和表9。结果发现，相较于不添加甲硫氨酸和EDTA-2Na，甲硫氨酸则有轻微改善HMW，添加0.15-0.3mM的EDTA-2Na可显著改善CE-SDS％LMW，同时还能改善高温40℃的聚集(HMW)。In this example, the final concentration of human growth hormone Fc fusion protein (hereinafter referred to as fusion protein) in the injection and the types and final concentrations of each auxiliary material are specifically shown in Table 8, the preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Figures 7 to 8 and Table 9. The results show that compared with not adding methionine and EDTA-2Na, methionine slightly improves HMW, and adding 0.15-0.3mM EDTA-2Na can significantly improve CE-SDS%LMW, and can also improve the aggregation (HMW) at high temperature of 40°C.
表8：样品24～28的注射液中各成分的配比
Table 8: Proportions of the components in the injection solutions of samples 24 to 28
Table 8: Proportions of the components in the injection solutions of samples 24 to 28
表9：样品24、26和28中SEC-HPLC和nrCE-SDS的检测结果
Table 9: SEC-HPLC and nrCE-SDS test results for samples 24, 26 and 28
Table 9: SEC-HPLC and nrCE-SDS test results for samples 24, 26 and 28
实施例5：人生长激素Fc融合蛋白注射液中组氨酸缓冲液添加量的筛选Example 5: Screening of the amount of histidine buffer added to human growth hormone Fc fusion protein injection
注射液中人生长激素Fc融合蛋白(简称融合蛋白)的终浓度和各辅料的种类及其终浓度具体参见表10。其中，注射液的制备方法和检测方法参见实施例1，检测结果参见表11。The final concentration of human growth hormone Fc fusion protein (abbreviated as fusion protein) in the injection and the types and final concentrations of each auxiliary material are specifically shown in Table 10. Among them, the preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Table 11.
表10：样品29～32的注射液中各成分的配比
Table 10: Proportions of the components in the injection solutions of samples 29 to 32
Table 10: Proportions of the components in the injection solutions of samples 29 to 32
表11：样品29～32中SEC-HPLC和nrCE-SDS的检测结果
Table 11: SEC-HPLC and nrCE-SDS test results for samples 29 to 32
Table 11: SEC-HPLC and nrCE-SDS test results for samples 29 to 32
由表10～11可知，相较于40mM的组氨酸体系，10～30mM的组氨酸体系中CE-SDS的％LMW较低。因此，10～30mM的组氨酸体系的稳定性相对更优。最终在合适的离子强度及相应的保护剂、稳定剂的共同作用下，本实施例的样品制剂在真实货架期保存条件2-8℃下已积累6个月的稳定性数据，从SEC和CE-SDS结果看增长较慢，预期能维持货架期18-24个月内SEC和CE-SDS纯度大于90％。As shown in Tables 10 to 11, the %LMW of CE-SDS in the 10-30mM histidine system is lower than that in the 40mM histidine system. Therefore, the stability of the 10-30mM histidine system is relatively better. Finally, under the combined action of appropriate ionic strength and corresponding protective agents and stabilizers, the sample preparation of this embodiment has accumulated 6 months of stability data under the actual shelf life storage conditions of 2-8°C. Judging from the SEC and CE-SDS results, the growth is slow, and it is expected to maintain a shelf life of 18-24 months. The SEC and CE-SDS purity is greater than 90%.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.
Claims (44)
- 一种组合物，其特征在于，包括：A composition, characterized in that it comprises:生长激素融合蛋白，以及缓冲剂和保护剂中的至少之一。Growth hormone fusion protein, and at least one of a buffer and a protective agent.
- 根据权利要求1所述的组合物，其特征在于，所述生长激素融合蛋白为生长激素Fc融合蛋白。The composition according to claim 1, characterized in that the growth hormone fusion protein is a growth hormone Fc fusion protein.
- 根据权利要求1所述的组合物，其特征在于，所述缓冲剂包括选自组氨酸和盐酸组氨酸、柠檬酸钠和柠檬酸、磷酸二氢钠和磷酸氢二钠中的至少之一。The composition according to claim 1, characterized in that the buffer comprises at least one selected from the group consisting of histidine and histidine hydrochloride, sodium citrate and citric acid, sodium dihydrogen phosphate and disodium hydrogen phosphate.
- 根据权利要求1所述的组合物，其特征在于，所述生长激素融合蛋白和缓冲剂的质量摩尔比为(20～90)mg：(0.005～0.04)mmol。The composition according to claim 1, characterized in that the mass molar ratio of the growth hormone fusion protein to the buffer is (20-90) mg: (0.005-0.04) mmol.
- 根据权利要求4所述的组合物，其特征在于，所述生长激素融合蛋白和缓冲剂的质量摩尔比为(50～70)mg：(0.015～0.03)mmol。The composition according to claim 4, characterized in that the mass molar ratio of the growth hormone fusion protein and the buffer is (50-70) mg: (0.015-0.03) mmol.
- 根据权利要求1所述的组合物，其特征在于，所述保护剂包括选自蔗糖、海藻糖、山梨醇和甘露醇中的至少之一。The composition according to claim 1, characterized in that the protective agent comprises at least one selected from sucrose, trehalose, sorbitol and mannitol.
- 根据权利要求1所述的组合物，其特征在于，所述生长激素融合蛋白和保护剂的摩尔比为(20～90)mg：(20～90)mg。The composition according to claim 1 is characterized in that the molar ratio of the growth hormone fusion protein to the protective agent is (20-90) mg: (20-90) mg.
- 根据权利要求1～7任一项所述的组合物，其特征在于，所述缓冲剂包括组氨酸和盐酸组氨酸。The composition according to any one of claims 1 to 7, characterized in that the buffer comprises histidine and histidine hydrochloride.
- 根据权利要求1～7任一项所述的组合物，其特征在于，所述保护剂包括蔗糖和海藻糖。The composition according to any one of claims 1 to 7, characterized in that the protective agent comprises sucrose and trehalose.
- 根据权利要求9所述的组合物，其特征在于，所述生长激素融合蛋白、蔗糖和海藻糖的质量比为(20～90)mg：(20～45)mg：(20～45)mg。The composition according to claim 9 is characterized in that the mass ratio of the growth hormone fusion protein, sucrose and trehalose is (20-90) mg: (20-45) mg: (20-45) mg.
- 根据权利要求10所述的组合物，其特征在于，所述生长激素融合蛋白、蔗糖和海藻糖的质量比为(50～70)mg：(15～35)mg：(15～35)mg。The composition according to claim 10 is characterized in that the mass ratio of the growth hormone fusion protein, sucrose and trehalose is (50-70) mg: (15-35) mg: (15-35) mg.
- 根据权利要求1～7任一项所述的组合物，其特征在于，进一步包括稳定剂、表面活性剂、抗氧化剂和金属螯合剂中的至少之一。The composition according to any one of claims 1 to 7, further comprising at least one of a stabilizer, a surfactant, an antioxidant and a metal chelating agent.
- 根据权利要求12所述的组合物，其特征在于，所述稳定剂包括选自氨丁三醇和氨丁三醇盐酸盐中的至少之一。The composition according to claim 12, characterized in that the stabilizer comprises at least one selected from tromethamine and tromethamine hydrochloride.
- 根据权利要求12所述的组合物，其特征在于，所述生长激素融合蛋白和稳定剂的质量摩尔比为(20～90)mg：(0.01～0.05)mmol。The composition according to claim 12, characterized in that the mass molar ratio of the growth hormone fusion protein and the stabilizer is (20-90) mg: (0.01-0.05) mmol.
- 根据权利要求12所述的组合物，其特征在于，所述生长激素融合蛋白和稳定剂的质量摩尔比为(50～70)mg：(0.015～0.035)mmol。The composition according to claim 12, characterized in that the mass molar ratio of the growth hormone fusion protein and the stabilizer is (50-70) mg: (0.015-0.035) mmol.
- 根据权利要求12所述的组合物，其特征在于，所述生长激素融合蛋白和抗氧化剂的质量摩尔比为(20～90)mg：(0.01～0.02)mmol。The composition according to claim 12, characterized in that the mass molar ratio of the growth hormone fusion protein to the antioxidant is (20-90) mg: (0.01-0.02) mmol.
- 根据权利要求12所述的组合物，其特征在于，所述生长激素融合蛋白和金属螯合剂的质量摩尔比为(20～90)mg：(0.00015～0.01)mmol。The composition according to claim 12, characterized in that the mass molar ratio of the growth hormone fusion protein to the metal chelator is (20-90) mg: (0.00015-0.01) mmol.
- 根据权利要求12所述的组合物，其特征在于，所述生长激素融合蛋白和金属螯合剂的质量摩尔比为(50～70)mg：(0.00015～0.0003)mmol。The composition according to claim 12, characterized in that the mass molar ratio of the growth hormone fusion protein to the metal chelator is (50-70) mg: (0.00015-0.0003) mmol.
- 根据权利要求12所述的组合物，其特征在于，所述抗氧化剂为甲硫氨酸。The composition according to claim 12, characterized in that the antioxidant is methionine.
- 根据权利要求12所述的组合物，其特征在于，所述金属螯合剂为依地酸二钠。The composition according to claim 12, characterized in that the metal chelator is disodium edetate.
- 根据权利要求12所述的组合物，其特征在于，所述生长激素融合蛋白和表面活性剂的质量比为(1～50)：(0.1～1)。The composition according to claim 12, characterized in that the mass ratio of the growth hormone fusion protein to the surfactant is (1-50): (0.1-1).
- 根据权利要求12所述的组合物，其特征在于，所述表面活性剂包括选自吐温80、吐温20和泊 洛沙姆P188中的至少之一。The composition according to claim 12, characterized in that the surfactant comprises a surfactant selected from Tween 80, Tween 20 and Povidone At least one of the Losham P188.
- 根据权利要求12所述的组合物，其特征在于，所述组合物为注射液。The composition according to claim 12, characterized in that the composition is an injection.
- 根据权利要求23所述的组合物，其特征在于，所述组合物的pH值为6.1-7.2。The composition according to claim 23, characterized in that the pH value of the composition is 6.1-7.2.
- 根据权利要求23所述的组合物，其特征在于，所述组合物的pH值为6.4～7.2。The composition according to claim 23, characterized in that the pH value of the composition is 6.4 to 7.2.
- 根据权利要求23所述的组合物，其特征在于，所述生长激素融合蛋白的浓度为20～90mg/mL。The composition according to claim 23, characterized in that the concentration of the growth hormone fusion protein is 20 to 90 mg/mL.
- 根据权利要求26所述的组合物，其特征在于，所述生长激素融合蛋白的浓度为50～70mg/mL。The composition according to claim 26, characterized in that the concentration of the growth hormone fusion protein is 50-70 mg/mL.
- 根据权利要求23所述的组合物，其特征在于，所述保护剂的浓度为20～90mg/mL。The composition according to claim 23, characterized in that the concentration of the protective agent is 20 to 90 mg/mL.
- 根据权利要求28所述的组合物，其特征在于，所述保护剂的浓度为30～70mg/mL。The composition according to claim 28, characterized in that the concentration of the protective agent is 30 to 70 mg/mL.
- 根据权利要求23所述的组合物，其特征在于，所述缓冲剂的浓度为5～40mM。The composition according to claim 23, characterized in that the concentration of the buffer is 5 to 40 mM.
- 根据权利要求30所述的组合物，其特征在于，所述缓冲剂的浓度为15～30mM。The composition according to claim 30, characterized in that the concentration of the buffer is 15 to 30 mM.
- 根据权利要求23所述的组合物，其特征在于，所述稳定剂的浓度为10～50mM。The composition according to claim 23, characterized in that the concentration of the stabilizer is 10 to 50 mM.
- 根据权利要求32所述的组合物，其特征在于，所述稳定剂的浓度为15～35mM。The composition according to claim 32, characterized in that the concentration of the stabilizer is 15 to 35 mM.
- 根据权利要求1所述的组合物，其特征在于，所述生长激素融合蛋白具有如SEQ ID NO:1～4任一项所示的氨基酸序列。The composition according to claim 1 is characterized in that the growth hormone fusion protein has an amino acid sequence as shown in any one of SEQ ID NO: 1 to 4.
- 一种注射液，其特征在于，包括：An injection, characterized in that it comprises:生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇和保护剂；Growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine and a protective agent;以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL；Based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg/mL;其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。Wherein, the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, and the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- 一种注射液，其特征在于，包括：An injection, characterized in that it comprises:生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇、甲硫氨酸和保护剂；Growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, methionine and a protective agent;以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL，所述甲硫氨酸的浓度为10～20mM；Based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of methionine is 10-20 mM;其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。Wherein, the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, and the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- 一种注射液，其特征在于，包括：An injection, characterized in that it comprises:生长激素融合蛋白、组氨酸、盐酸组氨酸、氨丁三醇、依地酸二钠和保护剂；Growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, edetate disodium and protective agent;以所述注射液总体积计，所述生长激素融合蛋白的浓度为50～70mg/mL，所述组氨酸和盐酸组氨酸的总浓度为15～30mM，所述氨丁三醇的浓度为15～35mM，所述保护剂的浓度为40～90mg/mL，所述依地酸二钠的浓度为0.15～1mM；Based on the total volume of the injection, the concentration of the growth hormone fusion protein is 50-70 mg/mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg/mL, and the concentration of edetate disodium is 0.15-1 mM;其中，所述保护剂为蔗糖，所述蔗糖的浓度为40～90mg/mL；或者，所述保护剂为蔗糖和海藻糖，所述蔗糖的浓度为20～45mg/mL，所述海藻糖的浓度为20～45mg/mL。Wherein, the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg/mL; or, the protective agent is sucrose and trehalose, and the concentration of the sucrose is 20-45 mg/mL, and the concentration of the trehalose is 20-45 mg/mL.
- 权利要求1～34任一项所述的组合物或权利要求35～37任一项所述的注射液在制备药物中的用途，所述药物用于治疗和/或预防生长激素异常相关疾病。Use of the composition according to any one of claims 1 to 34 or the injection according to any one of claims 35 to 37 in the preparation of a medicament for treating and/or preventing diseases related to abnormal growth hormone.
- 根据权利要求38所述的用途，其特征在于，所述生长激素异常相关疾病包括选自儿童生长激素缺乏症、特发性体型矮小、成人生长激素缺乏、特纳氏综合征、普拉德 威利综合征、肾衰竭、化疗治疗和AIDS治疗期间的异化状态引起的疾病和子宫内生长迟缓中的至少之一。The use according to claim 38 is characterized in that the diseases related to abnormal growth hormone include at least one selected from childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation.
- 一种治疗和/或预防生长激素异常相关疾病的方法，其特征在于，包括： A method for treating and/or preventing diseases related to abnormal growth hormone, comprising:向受试者施用药学上可接受量的权利要求1～34任一项所述的组合物或权利要求35～37任一项所述的注射液。A pharmaceutically acceptable amount of the composition according to any one of claims 1 to 34 or the injection according to any one of claims 35 to 37 is administered to a subject.
- 根据权利要求40所述的方法，其特征在于，所述生长激素异常相关疾病包括选自儿童生长激素缺乏症、特发性体型矮小、成人生长激素缺乏、特纳氏综合征、普拉德 威利综合征、肾衰竭、化疗治疗和AIDS治疗期间的异化状态引起的疾病和子宫内生长迟缓中的至少之一。The method according to claim 40 is characterized in that the abnormal growth hormone-related diseases include at least one selected from childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by alienation during chemotherapy and AIDS treatment, and intrauterine growth retardation.
- 一种注射装置或容器，其特征在于，包括：An injection device or container, characterized in that it comprises:权利要求1～34任一项所述的组合物或权利要求35～37任一项所述的注射液。The composition according to any one of claims 1 to 34 or the injection according to any one of claims 35 to 37.
- 根据权利要求42所述的注射装置或容器，其特征在于，所述注射装置或容器包括选自容纳瓶和注射器中的至少之一。The injection device or container according to claim 42 is characterized in that the injection device or container includes at least one selected from a container bottle and a syringe.
- 根据权利要求43所述的注射装置或容器，其特征在于，所述注射器包括选自自动注射器和预填充注射器中的至少之一。 The injection device or container according to claim 43, characterized in that the syringe includes at least one selected from an automatic injector and a pre-filled syringe.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211338540.1A CN115845032A (en) | 2022-10-28 | 2022-10-28 | Growth hormone F C Fusion protein injection and application thereof |
| CN202211338540.1 | 2022-10-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024087834A1 true WO2024087834A1 (en) | 2024-05-02 |
Family
ID=85662066
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2023/114241 WO2024087834A1 (en) | 2022-10-28 | 2023-08-22 | Growth hormone fc-fusion protein injection and use thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN115845032A (en) |
| WO (1) | WO2024087834A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115845032A (en) * | 2022-10-28 | 2023-03-28 | 深圳科兴药业有限公司 | Growth hormone F C Fusion protein injection and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105848645A (en) * | 2013-09-27 | 2016-08-10 | 韩美药品株式会社 | Sustained type human growth hormone preparation |
| US20180127478A1 (en) * | 2016-09-16 | 2018-05-10 | Wei-Chiang Shen | SINGLE CHAIN Fc-DIMER-HUMAN GROWTH HORMONE FUSION PROTEIN FOR IMPROVED DRUG DELIVERY |
| CN108136043A (en) * | 2015-10-01 | 2018-06-08 | 诺和诺德股份有限公司 | Protein conjugate |
| US20190224281A1 (en) * | 2016-08-30 | 2019-07-25 | Genexine, Inc. | PHARMACEUTICAL COMPOSITION FOR TREATING GROWTH HORMONE DEFICIENCY CONTAINING hGH FUSION PROTEIN |
| WO2022207798A1 (en) * | 2021-04-01 | 2022-10-06 | Ascendis Pharma A/S | Use of long-acting growth hormone for treating inflammation-induced diseases |
| CN115845032A (en) * | 2022-10-28 | 2023-03-28 | 深圳科兴药业有限公司 | Growth hormone F C Fusion protein injection and application thereof |
-
2022
- 2022-10-28 CN CN202211338540.1A patent/CN115845032A/en active Pending
-
2023
- 2023-08-22 WO PCT/CN2023/114241 patent/WO2024087834A1/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105848645A (en) * | 2013-09-27 | 2016-08-10 | 韩美药品株式会社 | Sustained type human growth hormone preparation |
| CN108136043A (en) * | 2015-10-01 | 2018-06-08 | 诺和诺德股份有限公司 | Protein conjugate |
| US20190224281A1 (en) * | 2016-08-30 | 2019-07-25 | Genexine, Inc. | PHARMACEUTICAL COMPOSITION FOR TREATING GROWTH HORMONE DEFICIENCY CONTAINING hGH FUSION PROTEIN |
| US20180127478A1 (en) * | 2016-09-16 | 2018-05-10 | Wei-Chiang Shen | SINGLE CHAIN Fc-DIMER-HUMAN GROWTH HORMONE FUSION PROTEIN FOR IMPROVED DRUG DELIVERY |
| WO2022207798A1 (en) * | 2021-04-01 | 2022-10-06 | Ascendis Pharma A/S | Use of long-acting growth hormone for treating inflammation-induced diseases |
| CN115845032A (en) * | 2022-10-28 | 2023-03-28 | 深圳科兴药业有限公司 | Growth hormone F C Fusion protein injection and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115845032A (en) | 2023-03-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10888619B2 (en) | Stable aqueous etanercept composition | |
| US11000588B2 (en) | Etanercept formulations stabilized with sodium chloride | |
| WO2024087834A1 (en) | Growth hormone fc-fusion protein injection and use thereof | |
| WO2024120458A1 (en) | Taci-fc fusion protein liquid pharmaceutical preparation |